Method for preparing nitrogen-containing heterocyclic compounds

Abstract

A method for preparing a nitrogen-containing heterocyclic compound represented by formula (I) is disclosed. The method involves carrying out a coupling reaction between compound 1A and compound 2a, followed by a deprotection reaction, an amidation reaction and a deprotection reaction, to obtain the target compound in a total of four steps. This method has a short reaction path, mild conditions, simple operation, convenient post-treatment, high yield, high purity, and is suitable for industrial amplification production. TIFF2025506307000021.tif75170

Description

[Technical field]

[0001] The present invention relates to a method for preparing a nitrogen-containing heterocyclic compound.

[0002] Dipeptidyl peptidase 1 (DPP1), also known as cathepsin C, is a lysosomal papain family cysteinyl protease involved in intracellular protein degradation. During the maturation process of neutrophils, DPP1 activates neutrophil serine proteases (NSPs), such as neutrophil elastase (NE), proteinase 3 (Pr3), and cathepsin G (CatG), by cleaving the N-terminal dipeptide of target proteins. DPP1 is involved in various inflammatory diseases, such as Wegener's granulomatosis, rheumatoid arthritis, pneumonia, and viral infections. Previous studies have shown that inhibition of DPP1 shows good therapeutic effects against neutrophil-induced highly inflammatory lung diseases, such as bronchiectasis, chronic obstructive pulmonary disease (COPD), and acute lung injury. Therefore, targeting DPP1 to suppress the overactivation of NSPs is expected to have a therapeutic effect on bronchiectasis.

[0003] In Patent Document 1, the formula (I) [ka] A small molecule DPP1 inhibitor has been prepared. The compound exhibits relatively high DPP1 inhibitory activity, has excellent bioavailability and pharmacokinetic properties, and has the advantages of low toxicity and high safety. It is intended for use in the treatment of lung diseases such as non-cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease (COPD), acute lung injury, and cystic fibrosis bronchiectasis. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] PCT / CN2020 / 114500 Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the preparation method described in Patent Document 1, purification of the intermediate is difficult, and multiple steps of column purification are required, and there are many reaction conditions that are unfavorable for large-scale production, and there are also drawbacks such as low yield and high cost. Therefore, it is necessary to improve the preparation process to facilitate large-scale industrial production. [Means for solving the problem]

[0006] The present invention relates to a compound represented by formula (I) [ka] The present invention provides a method for preparing a compound represented by the formula: Step 1: Compound 2a and compound 1A are subjected to a coupling reaction to obtain intermediate 1B; [ka] and Step 2: Intermediate 1B is deprotected to give intermediate 1C or a salt thereof. [ka]

[0007] The starting compounds 2a and 1A can be synthesized by referring to the methods described in existing chemical textbooks, literature, patents, etc., or can be purchased as commercially available products. For example, the preparation method of compound 1A is described in WO 2015 / 110826A1 (page 50), and the preparation method of compound 2a is described in WO 2016 / 139355A1 (page 52).

[0008] The coupling reaction in step 1 can be typically carried out under alkaline conditions using a catalyst such as palladium or nickel, including, but not limited to, one or more of NiCl2, NiCl2-diglyme (diglycol dimethyl ether nickel dichloride), Ni(COD)2, Pd(OAc)2, PdCl2, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(PPh3)4, Pd2(dba)3, Pd(PhCN)2Cl2, PEPPSI-iPr, PdCl2[P(Cy)3]2, or [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (Pd(dppf)Cl2·CH2Cl2); The alkaline reagent for creating the alkaline condition is selected from, but is not limited to, one or more of K3PO4, K2HPO4, K2CO3, KHCO3, Cs2CO3, Na2CO3, NaHCO3, KF, sodium acetate, potassium acetate, pyridine, triethylamine, or N,N-diisopropylethylamine, or an aqueous solution thereof; In some specific embodiments, step 1 comprises dissolving compound 2a and compound 1A in an organic solvent, where the organic solvent is an organic solvent capable of dissolving the reactants, including, but not limited to, one or more of acetonitrile, toluene, dichloromethane, ethyl acetate, acetone, methanol, ethanol, isopropanol, 2-methyltetrahydrofuran, tetrahydrofuran, and 1,4-dioxane, and then reacting with K3PO4, K2HPO4, K2CO3, KHCO3, Cs2CO3, Na2CO3, NaHCO3, KF, sodium acetate, potassium acetate, pyridine, triethylamine, or N,N- Diisopropylethylamine or one or more of their aqueous solutions are added, followed by the addition of one or more catalysts selected from NiCl2, NiCl2-diglyme, Ni(COD)2, Pd(OAc)2, PdCl2, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(PPh3)4, Pd2(dba)3, Pd(PhCN)2Cl2, PEPPSI-iPr, PdCl2[P(Cy)3]2, and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (Pd(dppf)Cl2·CH2Cl2), and warming the mixture to 80°C ± 5°C under nitrogen protection to complete the reaction. The mixture is cooled to 80°C ± 5°C for a complete reaction), and then purified water or ethanol is added to the reaction solution, or the mixture is directly cooled to 5°C to 55°C, filtered and dried to obtain intermediate 1B. Unless otherwise specified, the alcohol solvent includes, but is not limited to, methanol, ethanol, propanol, isopropanol, n-butanol, etc. Under the reaction conditions, the reaction in the step has a fast reaction rate, a high conversion rate, simple post-treatment, and high product purity.

[0009] In some specific embodiments, in step 1, the reaction system is subjected to a nitrogen purge prior to the addition of the catalyst, after which another nitrogen purge is performed to substantially remove oxygen from the reaction system in order to avoid deactivation of the catalyst.

[0010] In some specific embodiments, the feed ratio range of the reaction in step 1 is as follows: 2a:1A:palladium reagent=1:1.0-3.0:0.001-0.2, preferably 2a:1A:palladium reagent=1:0.95-1.20:0.001-0.03, and potassium carbonate is added in the following amount range: 2a:K2CO3=1.0:1.5-8.0, preferably 2a:K2CO3=1.0:2.0-3.0.

[0011] In some specific embodiments, step 2 includes: Add intermediate 1B and an acid to an organic solvent, and fully reacting the mixture while controlling the temperature at 20°C to 55°C, and then filter it to obtain intermediate 1C or a salt thereof. Optionally, the filter cake is added to an organic solvent, the mixture is heated to 80°C±5°C, stirred for 1-5 hours, cooled to 20°C±5°C, stirred, filtered and dried to obtain intermediate 1C or a salt thereof. wherein the acid is selected from one or more of sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, benzenesulfonic acid, methanesulfonic acid, and p-toluenesulfonic acid monohydrate; the organic solvent is selected from one or more of methanol, ethanol, isopropanol, isopropyl acetate, ethyl acetate, toluene, acetone, methyl isobutyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, and acetonitrile; The Boc deprotection reaction is generally carried out at a controlled temperature after dissolving the intermediate and an acid in an organic solvent, such as, but not limited to, sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid monohydrate, and the like, and the organic solvent, such as, but not limited to, methanol, ethanol, isopropanol, isopropyl acetate, ethyl acetate, toluene, acetone, methyl isobutyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, acetonitrile, and the like, at a temperature of about 0° C. to 80° C.

[0012] In some specific embodiments of the invention, step 2 comprises: Intermediate 1B and p-toluenesulfonic acid monohydrate are added to acetonitrile, and the mixture is reacted completely while controlling the temperature at 25°C±5°C. Then, it is filtered, and the filter cake is added to acetonitrile, and the mixture is heated to 80°C±5°C and stirred for 1 to 5 hours, and cooled to 20°C±5°C, and stirred, filtered and dried to obtain Intermediate 1C or a salt thereof.

[0013] The reaction of step 2 of the present invention is preferably carried out in acetonitrile using p-toluenesulfonic acid monohydrate as the acid. Its advantages are that the reaction rate is fast, the product is directly precipitated at the end of the reaction and can be obtained by filtration, and the reaction operation is simple. In the post-treatment, the product is further stirred in hot acetonitrile. This has the advantages of being excellent in removing impurities after slurrying with heating, and the product is non-hygroscopic.

[0014] In some specific embodiments, the feed molar ratio of the reaction in step 2 is 1B:acid=1:1.0-5.0, and in some embodiments, the ratio is preferably 1B:acid=1:1.5-3.0.

[0015] In some specific embodiments, the preparation method of formula (I) of the present invention further includes: Step 3: intermediate 1C or a salt thereof and compound INT-3 or a salt thereof are subjected to an amidation reaction to obtain intermediate 1D; [ka] or / and Step 4: Intermediate 1D is deprotected under acidic conditions to give compound I or a salt thereof. [ka]

[0016] The amidation reaction in step 3 is generally a dehydration reaction carried out in a condensing reagent under alkaline conditions.

[0017] In some specific embodiments, step 3 includes: Add intermediate 1C or its salt and compound INT-3 or its salt to an organic solvent, add organic amine, then add acid-amine condensation reagent under nitrogen protection while controlling the temperature at 5℃-20℃, then react the mixture completely while maintaining the temperature at 25℃±5℃, then wash the reaction liquid with sodium chloride aqueous solution, carry out liquid phase separation, add medicinal carbon or activated carbon to the organic phase, stir and filter the mixture, add anhydrous sodium sulfate to dry the organic phase, and carry out filtration and concentration to obtain intermediate 1D; wherein the organic solvent is selected from one or more of tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, dichloromethane, acetone, methyl isobutyl ketone, isopropyl acetate, and ethyl acetate; the organic amine is selected from one or more of triethylamine, 2,6-dimethylpyridine, pyridine, DBU, and N,N-diisopropylethylamine; and The acid-amine condensing reagent is selected from one or more of HATU, COMU, EDCI, BOP, and propylphosphonic anhydride.

[0018] In some specific embodiments, the range of the material ratio in step 3 is 1C:INT-3=1:0.9-3.0, preferably 1C:INT-3=1:1.0-1.5, and the condensation reagent is generally added in the following amount range: 1C:condensation reagent=1:1.0-5.0, preferably 1C:condensation reagent=1:1.2-2.0.

[0019] In some specific embodiments, step 3 comprises washing the reaction solution sequentially with an alkaline aqueous solution and an acidic aqueous solution before washing with an aqueous sodium chloride solution; Here, the alkaline aqueous solution is selected from one or more of an aqueous potassium phosphate solution, an aqueous potassium carbonate solution, an aqueous potassium hydrogen carbonate solution, an aqueous sodium carbonate solution, and an aqueous sodium hydrogen carbonate solution. The washing steps are intended to remove unreacted 1C or INT-3 by acid and alkaline washing.

[0020] The deprotection reaction in step 4 is generally carried out in an organic solvent under acidic conditions.

[0021] In some specific embodiments, step 4 includes: Add intermediate 1D and an acidic reagent to an organic solvent, then keep the temperature at 25°C±5°C while the mixture is reacted completely, then add base dropwise while controlling the material temperature of the reaction solution below 25°C, cool the resulting mixture to 10°C±5°C for crystallization, filter and dry to obtain compound I; Optionally, after filtration, the filter cake is washed with purified water, and then the filter cake is added to ethanol, the mixture is stirred at 20°C±5°C, filtered and dried to obtain compound I or a salt thereof.

[0022] The advantage of using the above conditions in the reaction of step 4 is that the work-up is simple; once the deprotection is complete, the product can be directly precipitated by adding a base and obtained by filtration.

[0023] In some specific embodiments, the acidic reagent in step 4 is selected from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, benzenesulfonic acid, methanesulfonic acid, and p-toluenesulfonic acid monohydrate; The organic solvent is selected from one or more of acetonitrile, methanol, ethanol, isopropanol, isopropyl acetate, ethyl acetate, acetone, methyl isobutyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran, and methyl tert-butyl ether, and the base is selected from one or more of dilute aqueous ammonia, LiOH, NaOH, KOH, K3PO4, K2CO3, KHCO3, Cs2CO3, Na2CO3, and NaHCO3.

[0024] In some specific embodiments, the range of material ratio in step 4 is as follows: 1D:acid=1.0:1.0-6.0, preferably 1D:acid=1.0:2.0-4.0, and the base is added dropwise in the following amount: 1D:base=1.0:2.0-10.0, preferably 1D:base=1.0:3.0-6.0.

[0025] In some specific embodiments, step 4 further comprises: Purification step: subjecting the obtained compound I to recrystallization. Recrystallization reagents that can be used include, but are not limited to, acetonitrile, methanol, ethanol, isopropanol, isopropyl acetate, ethyl acetate, acetone, methyl isobutyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran, n-heptane, methyl tert-butyl ether, dimethyl sulfoxide, N-methylpyrrolidone, and mixed solvents thereof.

[0026] In some specific embodiments, the purification step of step 4 comprises: The obtained compound I is added to a mixed solution of absolute ethanol-water, acetonitrile-water, acetonitrile-methanol, acetonitrile-isopropanol, acetonitrile-tert-butanol, acetonitrile-n-butanol, or acetonitrile-absolute ethanol, the mixture is heated to 75°C±5°C, stirred to dissolve and clarify, optionally hot filtered, the filtrate is cooled under stirring to crystallize, filtered and dried to obtain purified compound I.

[0027] In some specific embodiments, the material to liquid ratio of the crude product to the solvent (by volume) in the refining step is in the range of 1.0:10.0 to 40.0, preferably 1.0:12.0 to 20.0.

[0028] In some specific embodiments, cooling the filtrate under stirring for crystallization in step 4 is carried out in two steps: first, cooling the filtrate to 35°C ± 5°C, and carrying out crystallization while maintaining the temperature constant for 20 minutes to 1.5 hours, and then cooling the filtrate to 5°C ± 5°C, and carrying out crystallization while maintaining the temperature constant for 1 to 3 hours. The advantages of stepwise crystallization are excellent impurity removal and high yield.

[0029] In the present invention, the terms "fully reacting" and "a complete reaction" refer to a reaction state in which the content of the main raw material is ≦1.0% as determined by sampling for HPLC monitoring.

[0030] The salt of the present invention refers to a salt formed between an acidic compound and an organic base or an inorganic base, or a salt formed between a basic compound and an organic acid or an inorganic acid, and examples thereof include triethanolamine salts, diethanolamine salts, monoethanolamine salts, carbonates, bicarbonates, hydrobromides, hydrochlorides, sulfates, malates, fumarates, tartrates, oxalates, citrates, benzenesulfonates, and p-toluenesulfonates.

[0031] The preparation of salts is well known in the art. For example, a compound is first reacted with an acid or base in a solvent, and then separated by precipitation, filtration, etc. to obtain the corresponding salt. Similarly, the use of salts as a substitute for free acids or free bases in chemical reactions is also well known in the art. When the present invention describes a compound that undergoes a certain type of reaction to produce another substance, it includes the case where the salt form is used to participate in the reaction and the product is obtained in the form of a salt. When the product is a salt, the salt can be dissolved in a solvent and subjected to a neutralization reaction by adding a corresponding acid or base to liberate the compound, and then the free compound can be obtained by normal post-treatment.

[0032] In the preparation method of the present invention, the operation of the whole process route is very simple, and the yield of each intermediate synthesis step is high, so that the overall yield is greatly improved, and as a result, the production cost is reduced. The intermediate products of the whole synthesis process can be crystallized and purified, and have high purity and yield, which is suitable for large-scale industrial production.

[0033] Unless otherwise indicated, the operations of the present invention are carried out according to conventional operations in the art. The literature and monographs in the art provide detailed syntheses of reactants that can be used to prepare the compounds described herein or provide articles describing the preparations for reference. The following literature and monographs are referenced: “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., New York; S.R.Sandler et al.,“Organic Functional Group Preparations,”2nd Ed.,Academic Press,New York,1983; H.O.House,“Modern Synthetic Reactions”,2nd Ed.,W.A.Benjamin,Inc.Menlo Park,Calif. 1972; T.L.Gilchrist,“Heterocyclic Chemistry”,2nd Ed.,John Wiley & Sons,New York,1992; J.March,“Advanced Organic Chemistry:Reactions,Mechanisms and Structure”,4th Ed.,Wiley-Interscience,New York,1992; Fuhrhop,J. and Penzlin G.“Organic Synthesis:Concepts,Methods, Starting Materials”,Second,Revised and Enlarged Edition(1994)John Wiley & Sons ISBN:3-527-29074-5; Hoffman,R.V.“Organic Chemistry,An Intermediate Text”(1996)Oxford University Press,ISBN 0-19-509618-5; Larock,R.C.“Comprehensive Organic Transformations:A Guide to Functional Group Preparations”2nd Edition(1999)Wiley-VCH,ISBN:0-471-19031-4; March,J.“Advanced Organic Chemistry:Reactions,Mechanisms, and Structure”4th Edition(1992)John Wiley & Sons,ISBN:0-471-60180-2; Otera, J. (editor) “Modern Carbonyl Chemistry” (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. “Patai’s 1992 Guide to the Chemistry of Functional Groups” (1992) Interscience ISBN: 0-471-93022-9; Solomons, T.W.G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J.C., “Intermediate Organic Chemistry” 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; “Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann’s Encyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; “Organic Reactions” (1942 - 2000) John Wiley & Sons, in over 55 volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in 73 volumes.

Examples

[0034] Detailed Description of the Embodiments: The contents of the present invention will be described in detail below with reference to the following examples. If no specific conditions are given in the examples, conventional conditions are used in the experimental methods. The examples listed are intended to better illustrate the contents of the present invention, but should not be construed as limiting the contents of the present invention. According to the above-mentioned contents of the present invention, those skilled in the art can make insubstantial modifications or adjustments to the embodiments, which still fall within the scope of protection of the present invention.

[0035] Detection Method : The structures of the compounds are determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are given in units of 10-6 (ppm). NMR was measured on (Bruker Avance III 400 and Bruker Avance 300) NMR instruments, the measurement solvents were deuterated dimethylsulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), the internal standard was tetramethylsilane (TMS), MS was measured on Agilent 6120B (ESI) and Agilent 6120B (APCI), and HPLC was measured on an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18 100×4.6 mm, 3.5 μM).

[0036] Preparation of compounds : [ka] In the present invention, the target compound is prepared through four steps: coupling reaction of compound 1A with compound 2a, followed by deprotection reaction, amidation reaction, and deprotection reaction. This method has a short route, mild conditions, simple operation, convenient post-treatment, high yield and purity, and is suitable for industrial-scale production. The target compound I has high activity, high bioavailability, low toxicity and side effects, and has potential as a pharmaceutical.

[0037] Example 1 : tert-Butyl (S)-(1-cyano-2-(2-fluoro-4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)ethyl)carbamate (1B) [ka]

[0038] Method I : In a 50L reaction vessel, 1,4-dioxane (10.005 kg), compound 1A (1.600 kg, see the method in WO 2015 / 110826 A1) and compound 2a (2.000 kg, see the method in WO 2016 / 139355 A1) were added under stirring, followed by potassium carbonate aqueous solution (6.605 kg, which was prepared by dissolving potassium carbonate (1.600 kg) in purified water (5.005 kg)). After the addition, the mixture was purged with nitrogen three times. [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (100.0 g) was added, and the mixture was purged with nitrogen once. Under nitrogen protection, the reaction solution was heated to 80°C ± 5°C and reacted for about 2 hours. Then, samples were taken and monitored by HPLC, and the reaction was terminated when the target value of the in-process control, ie, the content of compound 2a, reached ≦1.0%.

[0039] Purified water (5.000 kg) was added to the reaction liquid, and the mixture was cooled to 10°C ± 5°C. Purified water (10.005 kg) was added, and the resulting mixture was stirred at 10°C ± 5°C for about 1 hour, crystallized, and filtered. The filter cake was washed twice with purified water (2.500 kg x 2) and collected. Anhydrous ethanol (12.605 kg) and the filter cake were added to a 50L reactor, and the mixture was stirred at 20°C ± 5°C for about 0.5 hours, filtered, and then the filter cake was washed twice with anhydrous ethanol (1.000 kg x 2) and collected.

[0040] Drying : The filter cake was dried at 55° C.±5° C. under vacuum at ≦−0.07 MPa for about 16 hours and recovered to give Intermediate 1B (2.143 kg, yield: 89.4%, HPLC: 95.91%).

[0041] 1 H NMR (400MHz, DMSO) δ7.90(s,1H), 7.72-7.30(m,6H), 4.72(s,1H), 3.41(d,3H), 3.09-3.21(m,2H), 1.37(s,9H).

[0042] LCMS m / z=356.1[M-56+H] + .

[0043] Method II : Acetonitrile (100 g), compound 1A (20.15 g) and compound 2a (16.02 g) were added to a 1 L reaction flask under stirring, followed by potassium carbonate aqueous solution (16.57 g, prepared by dissolving potassium carbonate (16.53 g) in purified water (40 g). After the addition, the mixture was purged with nitrogen three times. [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (0.83 g) was added. Under nitrogen protection, the reaction solution was heated to 80°C ± 5°C and reacted for about 2 hours, after which the reaction was terminated.

[0044] The reaction mixture was cooled to 50°C ± 5°C and absolute ethanol (80 g) was added to induce crystallization for about 1 hour. The mixture was filtered and the filter cake was washed with absolute ethanol (20 g) and collected.

[0045] Drying : The filter cake was dried at 55° C.±5° C. under vacuum at ≦−0.07 MPa for about 16 hours and recovered to give Intermediate 1B (24.41 g, yield: 83.0%).

[0046] Method III : In a 20L reaction vessel, 1,4-dioxane (5.5 kg), compound 1A (0.551 kg) and compound 2a (0.434 kg) were added under stirring, followed by potassium carbonate aqueous solution (1.744 kg, prepared by dissolving potassium carbonate (0.444 kg) in purified water (1.30 kg). After the addition, the mixture was purged with nitrogen three times. [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (208 g) was added. Under nitrogen protection, the reaction solution was heated to 80°C ± 5°C and reacted for about 3 hours, after which the reaction was terminated.

[0047] The reaction solution was cooled to 20°C±5°C, stirred for about 1 hour to allow crystallization, and filtered. The filter cake was washed twice with purified water (1.0 kg×2) and collected. Anhydrous ethanol (3.2 kg) and the filter cake were added to a 20 L reactor, the mixture was heated to 70°C±5°C, stirred for about 1.5 hours, cooled to room temperature, filtered, and the filter cake was washed with anhydrous ethanol (1.20 kg) and collected.

[0048] Drying : The filter cake was dried at 55° C.±5° C. under vacuum at ≦−0.07 MPa for about 18 hours and recovered to give Intermediate 1B (0.435 kg, yield: 74.0%).

[0049] Example 2 (S)-2-Amino-3-(2-fluoro-4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)propannitrile 4-Methylbenzenesulfonic acid (1C 4-Methylbenzenesulfonic acid) [ka]

[0050] Method I : Acetonitrile (16.785 kg), intermediate 1B (2.1381 kg) and p-toluenesulfonic acid monohydrate (2.950 kg) were added to a 50 L glass reactor under stirring. After the addition, the temperature was controlled at 25°C ± 5°C, and the mixture was reacted for about 2 hours. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the target value of the in-process control, i.e., the content of intermediate 1B was ≦1.0%, was reached.

[0051] The reaction mixture was filtered and the filter cake was washed with acetonitrile (1.670 kg) and collected. Acetonitrile (2.1381 kg) and the filter cake were added to the reaction kettle and the mixture was warmed to 80°C ± 5°C and stirred for 3 hours. The resulting mixture was cooled to 20°C ± 5°C and stirred for 1 hour. The reaction mixture was filtered and the filter cake was washed with acetonitrile (1.670 kg) and collected.

[0052] Drying : The filter cake was dried at 55° C.±5° C. under a vacuum of ≦−0.07 MPa for about 16 hours and recovered to give intermediate 1C 4-methylbenzenesulfonic acid (2.141 kg, yield: 85.5%, HPLC: 99.43%).

[0053] 1 H NMR (400MHz, DMSO) δ9.04(s,3H), 7.70-7.37(m,8H), 7.13(d,2H), 4.90(dd,1H), 3.41(s,3H), 3.29(t,2H), 2.29(s,3H).

[0054] LCMS m / z=312.2[M-172+H] + .

[0055] Method II : Acetonitrile (400 g), intermediate 1B (50.30 g) and p-toluenesulfonic acid monohydrate (69.30 g) were added to a 1 L reaction flask under stirring. After the addition, the temperature was controlled at 50° C.±5° C., and the mixture was reacted for about 1 hour. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the in-process control content of intermediate 1B was ≦1.0%. The reaction mixture was filtered, and the filter cake was washed with acetonitrile (10 g) and collected.

[0056] Drying : The filter cake was dried at 55° C.±5° C. under a vacuum of ≦−0.07 MPa for about 24 hours to give intermediate 1C 4-methylbenzenesulfonic acid (55.70 g, yield: 95.0%).

[0057] Method III : Acetonitrile (160 g), intermediate 1B (20.3 g) and concentrated hydrochloric acid (17.8 g) were added to a 0.5 L reaction flask under stirring. After the addition, the temperature was controlled at 25° C.±5° C., and the mixture was reacted for about 2 hours. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the content of the in-process control of intermediate 1B was ≦1.0%. The reaction mixture was filtered, and the filter cake was washed with acetonitrile (10 g) and collected.

[0058] Drying : The filter cake was dried at 55° C.±5° C. under a vacuum of ≦−0.07 MPa for about 18 hours and recovered to give the hydrochloride salt of intermediate 1C (14.4 g, yield: 85.0%, hygroscopic).

[0059] Example 3 tert-Butyl (S)-2-(((S)-1-cyano-2-(2-fluoro-4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)ethyl)carbamoyl)-1,4-oxazepane-4-carboxylate (1D) [ka]

[0060] Method I : In a 50L reactor, ethyl acetate (19.060 kg), intermediate 1C (2.1413 kg) and INT-3 (1.1300 kg, purchased from Pharmablock Sciences Inc, Nanjing) were added under stirring, followed by N,N-diisopropylethylamine (1.725 kg). After addition, under nitrogen protection, the reaction solution was cooled to 5°C ± 5°C, and propylphosphonic anhydride (4.240 kg) was added dropwise with temperature control at 10°C ± 5°C. After addition, the temperature was maintained at 25°C ± 5°C, and the mixture was reacted for about 2 hours. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the target value of the in-process control, i.e., the content of intermediate 1C, reached ≦1.0%.

[0061] The reaction mixture was washed successively with aqueous sodium bicarbonate (prepared by dissolving 1.070 kg of sodium bicarbonate in 20.350 kg of water), aqueous citric acid (prepared by dissolving 2.150 kg of citric acid monohydrate in 19.275 kg of water), and sodium chloride (prepared by dissolving 4.300 kg of sodium chloride in 17.135 kg of water). Medicinal charcoal (0.210 kg) was added to the organic phase and the mixture was stirred for about 0.5 hours. The resulting mixture was filtered through a pad of Celite (0.540 kg) and the filter cake was washed with ethyl acetate (1.905 kg). Anhydrous sodium sulfate (1.070 kg) was added to the organic phase and dried for about 0.5 hours. The mixture was filtered, the filter cake was washed with ethyl acetate (1.905 kg), and the filtrates were combined.

[0062] The resulting filtrate was concentrated under reduced pressure at 50°C ± 5°C until no clear fractions remained to give intermediate 1D (2.385 kg, overweight; amount calculated based on 100% yield), which was used as is in the next reaction (HPLC: 99.09%).

[0063] 1H NMR (400MHz, CDCl3) δ7.54-7.01(m,7H), 5.18(s,1H), 4.25-3.94(m,3H), 3. 54(dd,2H), 3.46(s,3H), 3.39-3.04(m,4H), 1.99(d,2H), 1.54-1.39(m,9H).

[0064] LCMS m / z=483.2[M-56+1] + .

[0065] Method II : To a 2 L reaction flask, dichloromethane (1.3 kg), Intermediate 1C (120.03 g) and INT-3 (108.76 g) were added under stirring, followed by N,N-diisopropylethylamine (149.55 g). After addition, the reaction was cooled to 15° C.±5° C. and HATU (190.47 g) was added in one portion. After addition, the mixture was reacted for about 16 hours while maintaining the temperature at 25° C.±5° C., after which the reaction was terminated.

[0066] The reaction mixture was washed successively with an aqueous solution of sodium bicarbonate, an aqueous solution of citric acid, and sodium chloride. Anhydrous sodium sulfate (1.0 kg) was added to the organic phase and dried for about 0.5 hours. The mixture was filtered, the filter cake was washed with dichloromethane (1.905 kg), and the filtrates were combined.

[0067] The resulting filtrate was concentrated under reduced pressure at 50° C.±5° C. until no visible fractions remained, to give intermediate 1D (221.30 g, overweight; amount calculated based on 100% yield), which was used as is in the next reaction.

[0068] Method III : In a 1L reaction flask, ethyl acetate (800g), intermediate 1C (100.0g) and INT-3 (53.26g) were added under stirring, followed by N,N-diisopropylethylamine (80.11g). After the addition, under nitrogen protection, the reaction solution was cooled to 5°C ± 5°C, and propylphosphonic anhydride (197.66g) was added dropwise while controlling the temperature at 10°C ± 5°C. After the addition, the temperature was maintained at 25°C ± 5°C, and the mixture was allowed to react for about 2 hours. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the target value of the in-process control, i.e., the content of intermediate 1C was ≦1.0%, was reached.

[0069] The reaction mixture was washed successively with an aqueous solution of sodium bicarbonate, an aqueous solution of citric acid, and sodium chloride. Anhydrous sodium sulfate (200 g) was added to the organic phase and dried for about 0.5 hours. The mixture was filtered, the filter cake was washed with ethyl acetate (50 g), and the filtrates were combined.

[0070] The resulting filtrate was concentrated under reduced pressure at 50° C.±5° C. until no obvious fractions remained, to give intermediate 1D (115.08 g, overweight, amount calculated based on 100% yield), which was used as is in the next reaction.

[0071] Example 4 (S)-N-((S)-1-cyano-2-(2-fluoro-4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)ethyl)-1,4-oxazepane-2-carboxamide (Compound I) [ka]

[0072] Method I : Acetonitrile (9.305 kg) and p-toluenesulfonic acid monohydrate (2.530 kg) were added to a 50 L double-walled glass reactor containing the intermediate 1D concentrate (method I of Example 3) under stirring. After the addition, the temperature was maintained at 25°C ± 5°C, and the mixture was reacted for about 2 hours. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the target value of the in-process control, i.e., the content of intermediate 1D was ≦1.0%, was reached.

[0073] The reaction solution was cooled to 10°C±5°C, and dilute aqueous ammonia (a mixture of 1.075 kg aqueous ammonia and 36.000 kg purified water) was added dropwise while controlling the raw material temperature below 25°C. After the addition, the mixture was cooled to 10°C±5°C and crystallized for 2 hours. The resulting mixture was filtered, and the filter cake was washed with purified water (11.930 kg). The filter cake and ethanol (14.890 kg) were added to a 50L double-walled glass reaction kettle, and the mixture was stirred at 20°C±5°C for 0.5 hours and filtered. The filter cake was washed with ethanol (1.860 kg) and collected. The filter cake was dried under vacuum (vacuum level ≦-0.07MPa) at 55°C±5°C for about 13 hours and collected to obtain crude compound I (1.6627 kg, yield: 85.6%).

[0074] purification : Acetonitrile (9.100 kg), absolute ethanol (9.220 kg) and crude compound I (1.6627 kg) were added to a 100 L reactor under stirring, the mixture was heated to an internal temperature of 75°C ± 5°C, stirred to dissolve and clarify, and filtered while hot. The filtrate was transferred to a 100 L reactor (if the product precipitates from the filtrate, heat the filtrate until the solution becomes clear) and cooled to 35°C ± 5°C under stirring. The temperature was kept constant until solids precipitated, and then the mixture was stirred for about 0.5 hours while keeping the temperature constant. The resulting mixture was cooled to 5°C ± 5°C and crystallized for 2 hours while keeping the temperature constant. The mixture was filtered, and the filter cake was washed with ethanol (1.300 kg) and collected.

[0075] Drying : The filter cake was dried at 55° C.±5° C. under vacuum at ≦−0.07 MPa for about 25 hours to give compound I (1.4060 kg, yield: 84.6%, total yield: 55.0% (calculated based on compound 2a), HPLC: 99.93%).

[0076] 1 H NMR(400MHz,DMSO)δ8.69(d,1H), 7.64(d,1H), 7.61-7.51(m,2H), 7.46(t,2H), 7.39(d,1H), 5.06(q,1H), 4.01(dd,1H), 3.87(ddd,1H), 3.7 3(ddd,1H), 3.40(s,3H), 3.34-3.28(m,1H), 3.20(dd,1H), 3.06(dd,1H), 2.78(ddd,1H), 2.69-2.54(m,2H), 2.21(s,1H), 1.84-1.63(m,2H).

[0077] LCMS m / z=439.2[M+1] + .

[0078] Method II : Acetonitrile (19.26 kg), p-toluenesulfonic acid monohydrate (3.712 kg) and intermediate 1D (3.500 kg) were added to a 100 L double-walled glass reactor under stirring. After addition, the temperature was maintained at 25°C ± 5°C and the mixture was reacted for about 2 hours. Then, a sample was taken and monitored by HPLC, and the reaction was terminated when the target value of the in-process control, i.e., the content of intermediate 1D was ≦1.0%, was reached.

[0079] The reaction solution was concentrated to a reaction volume of about 20 L and cooled to 10° C.±5° C., followed by the addition of dilute aqueous ammonia (a mixture of 4.0 kg aqueous ammonia and 56.00 kg purified water). After addition, the mixture was cooled to 10° C.±5° C. and allowed to crystallize for 0.5 hours. The mixture was filtered, and the filter cake was washed with purified water (5 kg) and collected. The filter cake was dried at 55° C.±5° C. under vacuum (vacuum≦−0.07 MPa) for about 72 hours and collected to obtain crude compound I (2.552 kg).

[0080] purification : In a 100L reactor, isopropanol (19.63 kg) and crude compound I (1.6627 kg) were added under stirring, the mixture was heated to an internal temperature of 75°C ± 5°C, stirred for about 2 hours, then cooled to 25°C ± 5°C, and crystallized for about 16 hours while maintaining the temperature constant. The mixture was filtered, and the filter cake was washed with isopropanol (1.300 kg) and collected.

[0081] Drying : The filter cake was dried at 55° C.±5° C. under vacuum at ≦−0.07 MPa for about 24 hours to give Compound I (2.3442 kg, yield: 82.3%).

[0082] Biological testing : 1. In vitro DPP1 enzyme activity assay : Recombinant human DPP1 enzyme (R&D System, Cat. No. 1071-CY) at a final concentration of 100 μg / mL and recombinant human cathepsin L (R&D System, Cat. No. 952-CY) at a final concentration of 20 μg / mL were mixed and incubated at room temperature for 1 hour to activate the DPP1 enzyme. The activated DPP1 enzyme was diluted 100 times, and 5 μL of different concentrations of compounds and 5 μL of diluted DPP1 enzyme were added to a 384-well plate and incubated at room temperature for 30 minutes. 10 μL of substrate Gly-Arg-AMC (bachem, Cat. No. I-1215) was added at a concentration of 20 μM, and the incubation was continued at room temperature for 60 minutes, and the fluorescence intensity was detected by a microplate reader (excitation wavelength = 380 nm, emission wavelength = 460 nm). IC 50 The values ​​were calculated using the DosResp function in Origin2019 software.

[0083] [Table 1]

[0084] conclusion : The compounds of the present invention exhibit relatively high inhibitory activity against the DPP1 receptor.

[0085] 2. Pharmacokinetics test in rats : 1.1 Experimental animals : Male SD rats, approximately 220 g, 6 to 8 weeks old, 6 rats / compound. Purchased from Chengdu Senwell Experimental Animals Co., Ltd.

[0086] 1.2 Experimental design : On the day of the experiment, six SD rats were randomly assigned to groups based on body weight. The animals were fasted for 12 to 14 hours before administration, allowed to have water, and fed 4 hours after administration.

[0087] [Table 2]

[0088] Vehicle for intravenous administration: 5% DMA + 5% Solutol + 90% saline, vehicle for intragastric administration: 0.5% MC, control compound INS1007, i.e. compound 2 of WO 2015 / 110826, was prepared according to the method of the patent.

[0089] Before and after administration, 0.1 ml of blood was collected from the rat's orbit under isoflurane anesthesia and placed in an EDTAK2 centrifuge tube. Plasma was collected by centrifugation at 5000 rpm and 4°C for 10 minutes. Blood collection times for the intravenous administration group: 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h, and blood collection times for the intragastric administration group: 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h. All samples were stored at -80°C before analysis and detection.

[0090] [Table 3]

[0091] conclusion : The compounds of the present invention have relatively good bioavailability and pharmacokinetic properties.

[0092] 3. 14-day repeated dose oral toxicity test in rats : According to body weight, SD rats were randomly divided into the following groups: vehicle control group (0.5% MC), INS1007 group (30, 100 and 300 mg / kg) and compound I group (30, 100 and 300 mg / kg). For the treatment groups, each group contained 16 rats, and for the vehicle control group, each group contained 10 rats, including the same number of male and female rats. The rats were orally administered with the corresponding concentration of drug or vehicle every day for 14 consecutive days, with a 7-day recovery period in between. During the treatment period, general symptoms were observed, and the body weight and food intake of each group were measured. At the end of the treatment period and the end of the recovery period, hematological examination, serum biochemistry examination and gross dissection were performed separately for the rats of each group.

[0093] conclusion : The compounds of the present invention are less toxic than INS1007 and therefore safer at the same dose.

Claims

1. Formula (I) 【Chemistry 1】 1. A method for preparing a compound represented by the formula: Step 1: Compound 2a and compound 1A are subjected to a coupling reaction to obtain intermediate 1B. 【Chemistry 2】 and, Step 2: Add intermediate 1B and an acid to an organic solvent, and react the mixture completely while controlling the temperature at 20°C to 55°C, and then filter it to obtain intermediate 1C or a salt thereof. Optionally, adding the filter cake to an organic solvent, warming the mixture to 80°C ± 5°C, stirring for 1 to 5 hours, cooling it to 20°C ± 5°C, stirring, filtering and drying to obtain intermediate 1C or a salt thereof. 【Chemistry 2】 wherein the acid is selected from one or more of sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, benzenesulfonic acid, methanesulfonic acid, and p-toluenesulfonic acid monohydrate; The organic solvent is selected from one or more of methanol, ethanol, isopropanol, acetonitrile, isopropyl acetate, ethyl acetate, toluene, acetone, methyl isobutyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether and acetonitrile.

2. 2. The method of claim 1, wherein step 1 comprises: The compound 2a and the compound 1A are dissolved in an organic solvent, a base is added, and then a catalyst is added under nitrogen protection, and the mixture is heated to 80°C ± 5°C to complete the reaction, and then the reaction mixture is cooled to 5°C - 55°C, and the mixture is stirred to crystallize, and then filtered and dried to obtain intermediate 1B; Optionally, after filtration, the filter cake is added to an alcohol solvent, and the mixture is stirred for 10 minutes to 2 hours, followed by filtration and drying to obtain intermediate 1B. wherein the organic solvent is selected from one or more of acetonitrile, toluene, dichloromethane, ethyl acetate, acetone, methanol, ethanol, isopropanol, 2-methyltetrahydrofuran, tetrahydrofuran, and 1,4-dioxane; The base is K ３ P.O. ４ , K. ２ HPO ４ , K. ２ CO ３ , KHCO ３ , Cs ２ CO ３ , Na ２ CO ３ , NaHCO ３ , KF, sodium acetate, potassium acetate, pyridine, triethylamine or N,N-diisopropylethylamine or an aqueous solution thereof; The catalyst is NiCl ２ , NiCl ２ - Diglyme, Ni(COD) ２ , Pd(OAc) ２ , PdCl ２ , Pd(PPh ３ ) ２ Cl ２ , Pd(dppf)Cl ２ , Pd(PPh ３ ) ４ , Pd ２ (dba) ３ , Pd(PhCN) ２ Cl ２ , PEPPSI-iPr, PdCl ２ [P(Cy) ３ ] ２ and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex.

3. 3. The method of claim 1 or 2, further comprising: Step 3: Intermediate 1C or a salt thereof and compound INT-3 or a salt thereof are subjected to an amidation reaction to obtain intermediate 1D. 【Transformation 3】 or / and Step 4: Intermediate 1D is deprotected under acidic conditions to give compound I or a salt thereof. 【Chemistry 4】

4. The method of claim 3, wherein step 3 comprises: Intermediate 1C or a salt thereof and compound INT-3 or a salt thereof are added to an organic solvent, an organic amine is added, and then an acid-amine condensation reagent is added dropwise under nitrogen protection while controlling the temperature at 5°C to 20°C, and then the mixture is allowed to react completely while maintaining the temperature at 25°C ± 5°C, and then the reaction liquid is washed with an aqueous sodium chloride solution, and liquid phase separation is carried out, activated carbon or medicinal carbon is added to the organic phase, the mixture is stirred and filtered, anhydrous sodium sulfate is added to dry the organic phase, and then filtering and concentration are carried out to obtain intermediate 1D or a salt thereof. wherein the organic solvent is selected from one or more of tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, dichloromethane, acetone, methyl isobutyl ketone, isopropyl acetate, and ethyl acetate; the organic amine is selected from one or more of triethylamine, 2,6-dimethylpyridine, pyridine, DBU, and N,N-diisopropylethylamine; and said acid-amine condensing reagent is selected from one or more of HATU, COMU, EDCI, BOP, and propylphosphonic anhydride.

5. Step 3 includes washing the reaction solution with an alkaline aqueous solution and an acidic aqueous solution in sequence before washing the reaction solution with an aqueous sodium chloride solution; wherein the alkaline aqueous solution is selected from one or more of a potassium phosphate aqueous solution, a potassium carbonate aqueous solution, a potassium bicarbonate aqueous solution, a sodium carbonate aqueous solution, and a sodium bicarbonate aqueous solution; and the acidic aqueous solution is selected from one or more of aqueous hydrochloric acid, aqueous sulfuric acid, aqueous phosphoric acid, and aqueous citric acid.

6. The method of claim 3, wherein step 4 comprises: Add intermediate 1D and an acidic reagent to an organic solvent, and allow the mixture to react completely while maintaining the temperature at 25°C ± 5°C. Then, add a base dropwise while controlling the temperature of the reaction mixture to 25°C or less. Cool the resulting mixture to 10°C ± 5°C to crystallize, and filter and dry to obtain compound I. Optionally, after filtration, the filter cake is washed with purified water, and then the filter cake is added to the alcohol reagent, and the mixture is stirred at 20°C ± 5°C, filtered and dried to obtain Compound I or a salt thereof.

7. the acidic reagent in step 4 is selected from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, benzenesulfonic acid, methanesulfonic acid, and p-toluenesulfonic acid monohydrate; the organic solvent is selected from one or more of acetonitrile, methanol, ethanol, isopropanol, isopropyl acetate, ethyl acetate, acetone, methyl isobutyl ketone, tetrahydrofuran, 2-methyltetrahydrofuran, and methyl tert-butyl ether; and The base may be dilute aqueous ammonia, LiOH, NaOH, KOH, K ３ P.O. ４ , K. ２ CO ３ , KHCO ３ , Cs ２ CO ３ , Na ２ CO ３ , NaHCO ３ or aqueous solutions thereof.

8. The preparation method according to claim 6, further comprising the following purification step: The obtained compound I is added to a solution of absolute ethanol-water, acetonitrile-water, acetonitrile-absolute ethanol, acetonitrile-methanol, acetonitrile-isopropanol, acetonitrile-tert-butanol or acetonitrile-n-butanol, the mixture is heated to 75°C ± 5°C, stirred to dissolve and clarify, optionally hot filtered, the filtrate is cooled under stirring to crystallize, filtered and dried to obtain purified compound I.

9. 9. The method according to claim 8, wherein the cooling of the filtrate under stirring for crystallization is carried out in two steps: First, the filtrate is cooled to 35°C ± 5°C and crystallization is carried out while maintaining the temperature constant for 20 minutes to 1.5 hours, then the filtrate is cooled to 5°C ± 5°C and crystallization is carried out while maintaining the temperature constant for 1 to 3 hours.

10. The method of claim 7, further comprising the following purification step: The obtained compound I is added to a solution of absolute ethanol-water, acetonitrile-water, acetonitrile-absolute ethanol, acetonitrile-methanol, acetonitrile-isopropanol, acetonitrile-tert-butanol or acetonitrile-n-butanol, the mixture is heated to 75°C ± 5°C, stirred to dissolve and clarify, optionally hot filtered, the filtrate is cooled under stirring to crystallize, filtered and dried to obtain purified compound I.

11. The method of claim 10, wherein the cooling of the filtrate under stirring for crystallization is carried out in the following two steps: First, the filtrate is cooled to 35°C ± 5°C and crystallization is carried out while maintaining the temperature constant for 20 minutes to 1.5 hours, then the filtrate is cooled to 5°C ± 5°C and crystallization is carried out while maintaining the temperature constant for 1 to 3 hours.

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