WO2017152755A1 - Substituted biphenyl compound and pharmaceutical composition thereof - Google Patents

Abstract

The present invention provides a substituted biphenyl compound and a pharmaceutical composition thereof, the substituted biphenyl compound being a compound represented by formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvent compound thereof. The compound in the present invention has a better neprilysin inhibitory activity, better performance in pharmacodynamics/pharmacokinetics, good applicability and high safety, can be used in the preparation of drugs for treating diseases related to neprilysin, and has a good market development prospect.

Description

Substituted biphenyl compound and pharmaceutical composition thereof Technical field

The invention belongs to the technical field of medicine, and in particular relates to a substituted biphenyl compound and a pharmaceutical composition thereof, which can be used for treating enkephalinase-mediated related diseases.

Background technique

Enkephalinase (NEP) is an endothelial membrane-bound Zn ²⁺ metalpeptidase found in many organs and tissues, including the brain, kidney, lung, gastrointestinal tract, heart and peripheral vascular structures. NEP makes many Degradation and inactivation of the peptide, such as enkephalin, circulating bradykinin, angiotensin peptide, and natriuretic peptide, wherein the natriuretic peptide has Several effects include, for example, vasodilation and urinary sodium excretion/diuresis, as well as inhibition of cardiac hypertrophy and ventricular fibrosis. Therefore, NEP plays an important role in constant blood pressure and cardiovascular health.

Enkephalinase is involved in the breakdown of a variety of biologically active oligopeptides, cleavage of peptide bonds on the amino side of hydrophobic amino acid residues. Metabolized peptides include atrial natriuretic peptide (ANP), bombesin, bradykinin, calcitonin gene-related peptide, endothelin, enkephalin, neurotensin, substance P, and vasoactive intestinal peptide . Some of these peptides have potent vasodilation and neurohormonal function, diuretic and natriuretic activity or modulate behavioral effects.

Studies have shown that probable enkephalinase (sNEP) may have prognostic value and dynamic changes in acute heart failure. The levels of soluble enkephalinase (mean 0.67 ng/mL) at admission were examined in 350 hospitalized patients with acute heart failure. Both short-term (2 months) and long-term (1.2 years) follow-up results showed that levels of soluble enkephalinase were associated with a composite endpoint of cardiovascular death and hospitalization for heart failure. The concentration of soluble enkephalinase at admission was clinically indicative of the trend toward the composite end point at 2 months, and was significantly associated with various clinical variables and NT-proBNP concentrations in the additional analysis at the end of follow-up. The association. The concentration of soluble enkephalinase at discharge was reduced from 0.70 ng/mL to 0.52 ng/mL (J. Lupón et. Al., JACC Heart Fail, 2015, 3, 641-644).

China is rapidly entering an aging society. At the end of 2013, the population aged 65 or older was 132 million. It is estimated that by 2050, the population of >60 years old will reach 1/3. Aging may lead to a surge in heart failure patients and an increase in hospitalization. According to the meta-analysis data of 2013, the prevalence of heart failure in China was 1.3%, an increase of 0.4% over 2003. Community surveys show that the prevalence of heart failure increases roughly every doubling in age by 10 years. The incidence of heart failure in Chinese population is 0.7-0.9 per 1,000 people, and 500,000 new cases of heart failure occur each year. The prognosis of patients with heart failure is very poor, and the 5-year mortality rate is above 50%. Heart failure mortality remained high. A study of 10,714 heart failure patients in 42 hospitals in different cities showed that the mortality rates of heart failure in 1980, 1990, and 2000 were 39.9%, 37.7%, and 41.1%, respectively.

Therefore, there is still a need in the art to develop compounds having inhibitory activity or better pharmacodynamic properties for enkephalinase.

Summary of the invention

In view of the above technical problems, the present invention discloses an enkephalinase inhibitor, a pharmaceutical composition and an application thereof, It has better enkephalinase inhibitory activity and/or has better pharmacodynamic/pharmacokinetic properties.

In this regard, the technical solution adopted by the present invention is:

An enkephalinase inhibitor, a biphenyl compound substituted as shown in formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound,

Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And at least one of R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ is deuterated or deuterated.

As a further improvement of the present invention, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹⁵ , R ¹⁶ , R ¹⁷ and R ¹⁸ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹⁹ , R ²⁰ , R ²¹ and R ²² are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, the compound may be selected from the following compounds or a pharmaceutically acceptable salt thereof, but is not limited to the following compounds:

With this technical solution, the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.

Preferably, the strontium isotope content of the strontium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), It is preferably more than 30%, more preferably more than 50%, more preferably more than 75%, more preferably more than 95%, more preferably more than 99%.

Specifically, in the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , The osmium isotope content of each of the R ⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ is at least 5%. More preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, still more preferably greater than 40%, More preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably more than 70%, more preferably more than 75%, more Preferably, it is greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, and even more preferably greater than 99%.

In another preferred embodiment, the compound does not include a non-deuterated compound.

The invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the enkephalinase inhibitor as described above, or a crystalline form, a pharmaceutically acceptable salt, a hydrate or a solvent thereof A pharmaceutical composition of a compound, stereoisomer, prodrug or isotopic variation.

As a further improvement of the present invention, the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant At least one of a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.

As a further improvement of the present invention, the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.

Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.

As a further improvement of the present invention, it further comprises other active compounds, which may be selected from the group consisting of: HMG-Co-A reductase inhibitors, angiotensin receptor blockers, angiotensin converting enzyme inhibitors, calcium Channel blockers, endothelin antagonists, renin inhibitors, diuretics, ApoA-I mimics, antidiabetic drugs, diet pills, aldosterone receptor blockers, endothelin receptor blockers, aldosterone synthase Inhibitors, CETP inhibitors and type 5 phosphodiesterase inhibitors.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: administering a pharmaceutically acceptable carrier to an enkephalinase inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, or a hydrate thereof Or the solvate is mixed to form a pharmaceutical composition.

The compound of the present invention has enkephalinase enzyme inhibitory activity, and thus it is expected to be useful for treating a disease by inhibiting the brain A therapeutic agent for a patient or a disease or condition that is treated by increasing its peptide substrate content. Accordingly, one aspect of the invention relates to a method of treating a patient suffering from a disease or condition which is treated by inhibition of enkephalinase, comprising administering to the patient a therapeutically effective amount of a compound of the invention. Another aspect of the invention relates to a method of treating a cardiovascular disease comprising administering to a patient a therapeutically effective amount of a compound of the invention. Another aspect of the invention relates to a method of treating hyperenvironia involving inhibition of enkephalinase in a mammal comprising administering to the mammal an enkephalinase inhibiting amount of a compound of the invention.

The invention also discloses the use of a substituted biphenyl enkephalinase inhibitor as described above, i.e., the compounds of the invention are advantageously useful as therapeutic agents for the treatment of conditions such as hypertension and heart failure.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Herein, "halogen" means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.

As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".

As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).

Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium). Salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl A base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed of morpholine, piperazine, and lysine, respectively.

The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.

Compared with the prior art, the beneficial effects of the invention are: the compound of the invention has excellent inhibition to enkephalinase; the technology of deuteration changes the metabolism of the compound in the organism, so that the compound has better Pharmacokinetic Kinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability; the substitution of a hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, and improves the therapeutic effect of the drug; Substituting a hydrogen atom in a compound inhibits certain metabolites and increases the safety of the compound.

detailed description

The preparation of the structural compound of the formula (I) of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

Example 1 Preparation of 4-((2S,4R)-1-(1,1'-biphenyl-4-yl)-5-ethoxy-4-methyl-3,4-d2-5-oxo Pentane-2-yl)amino)-4-oxobutanoic acid (Compound S-1), the specific synthetic steps are as follows:

Step 1 Synthesis of Boc-L-tyrosine methyl ester (Compound 2).

L-Tyrosine methyl ester (10 g, 50 mmol) was added to the reaction flask, dissolved in 250 mL of dichloromethane, and triethylamine (13.3 g, 100 mmol) was added to the reaction flask, and di-tert-butyl dicarbonate was added (12.3). g, 56 mmol, stirred at 30 ° C for 3 hours. The reaction of the starting material was completed by TLC. The reaction mixture was concentrated and purified by column chromatography to give 12 g of compound 2, yield 82%, LC-MS (APCI): m/z = 196.3 (M -100+1) ⁺ .

Step 2 Synthesis of (R)-3-trifluoromethanesulfonic acid-phenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid methyl ester (Compound 3).

Compound 2 (12 g, 40.6 mmol) was added to the reaction flask, dissolved in 100 mL of dichloromethane, and pyridine (8.1 g, 1.02 mmol) was added at room temperature, cooled to -15 ° C, and trifluoromethanesulfonic anhydride (14.4 g, 51 mmol). Stir at -15 ° C for 30 minutes. LC-MS detection of starting material the reaction was complete, diluted with water 100ml and extracted with ethyl acetate (150ml x 2), the combined organic phases were washed with saturated brine, dried Na ₂ SO _4, filtered and concentrated, purified by column chromatography to give 4.4g compound 3, yield 25.4%, LC-MS (APCI): m/z = 428.3 (M + 1) ⁺ .

Step 3 Synthesis of (R)-3-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid methyl ester (Compound 5).

To the reaction flask was added tetrakistriphenylphosphine palladium (38.8 mg, 0.03 mmol), compound 3 (480 mg, 1.12 mmol), phenylboronic acid (270 mg, 2.24 mmol) and potassium carbonate (232 mg, 1.6 mmol). 10 mL of toluene was heated to 80 ° C for 3 hours, and the compound 3 was completely reacted by TLC. The reaction mixture was concentrated and purified by column chromatography to yield 340 mg of Compound 5, yield 85%, LC-MS (APCI): m/z = 356 (M +1) ⁺ .

Step 4 Synthesis of (R)-3-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid (Compound 6).

Compound 5 (340 mg, 0.95 mmol) was added to the reaction flask, and 10 mL of tetrahydrofuran was added to dissolve. 1 mL of 1 M sodium hydroxide solution was added, and the reaction was stirred at room temperature for 2 hours. The reaction of the starting material was completely confirmed by TLC, and the reaction mixture was concentrated in a reaction flask. After adding 10 mL of water and 10 mL of ethyl acetate, the mixture was diluted with EtOAc (EtOAc) (EtOAc) (EtOAc) compound 6, yield 67.6%, LC-MS (APCI ): m / z = 340 (m-1) -.

Step 5 Synthesis of (R)-3-biphenyl-4-yl-2-tert-butoxycarbonyl-amino-methoxymethylaminopropionic acid (Compound 7).

Compound 6 (4.65 g, 13.63 mmol), methoxymethylamine (1.6 g, 25.89 mmol), EDCI (5.22 g, 27.26 mmol), triethylamine (1.625 g, 16.35 mmol), 1 Hydroxybenzotriazole (HOBT, 2.76 g, 20.44 mmol), 60 mL of a solvent dichloromethane was added, and the reaction was stirred at room temperature for 16 hours. The TLC was used to detect the reaction of the starting material. The reaction mixture was concentrated and purified by column chromatography to give 4.51 g of Compound 7 (yield: 83.3%, LC-MS (APCI): m/z = 401 (M+1) ⁺ .

Step 6 Synthesis of (R)-3-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropanal (Compound 8).

Under a nitrogen atmosphere, lithium aluminum hydride (386 mg, 10.19 mmol) was added to the reaction flask, and 100 mL of tetrahydrofuran was added. After cooling to 0 ° C, compound 7 (2.72 g, 6.79 mmol) was added, and the reaction was stirred at 0 ° C for 1 hour, quenched with aqueous potassium hydrogen sulfate solution, 20 mL of 1 M hydrochloric acid solution, and extracted with ethyl acetate three times, and the organic phase was combined. , washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give compound 8 2.3g, yield ~ 100%, LC-MS ( APCI): m / z = 326 (m + 1) +.

Step 7 Synthesis of (R)-3-biphenyl-4-yl-tert-butoxycarbonyl-amino-2-chi-2-methyl-pentanoic acid ethyl ester (Compound 10)

Compound 8 (2.1 g, 6.45 mmol) was added to the reaction flask, dissolved in 50 mL of dichloromethane, and ethoxycarbonyltriphenylphosphine (4.68 g, 12.92 mmol) was added, and the reaction was stirred at room temperature for 18 hours, TLC detection The starting material was completely reacted, and the reaction mixture was concentrated and purified by column chromatography to give 2.4 g of Compound 10 (yield: 90.9%), LC-MS (APCI): m/z=410 (M+1) ⁺ .

Step 8 Synthesis of (R)-3-biphenyl-4-yl-tert-butoxycarbonyl-amino-3,4-d2-2-methyl-pentanoic acid ethyl ester (Compound 11).

Compound 10 (0.3 g, 0.732 mmol) was added to the reaction flask, dissolved in 8 mL of deuterated methanol, and 10% palladium carbon (91 mg) was added to the reaction flask, and the reaction was carried out under hydrogen (50 psi) for 24 hours. After completion, the mixture was filtered through Celite, washed with 40 mL of methanol, and the organic phase was concentrated to give 0.3 g of Compound 11 in a yield of 99.6%, LC-MS (APCI): m/z = 414 (M+1) ⁺ .

Step 9 Synthesis of (R)-3-biphenyl-4-yl-2-amino-3,4-d2-2-methyl-pentanoic acid (Compound 12).

Compound 11 (300 mg, 0.755 mmol) was added to the reaction flask, and 50 mL of ethyl acetate was added to dissolve. 4 mL of 1 M hydrochloric acid dioxane was added at room temperature, and the reaction was carried out for 3 hours at room temperature. The starting material was completely reacted by TLC, and the solvent was concentrated to give 300 mg of compound. 12, used directly in the next step without purification.

Step 10 4-(4R)-1-(1,1'-Biphenyl-4-yl)-5-ethoxy-4-methyl-3,4-d2-5-oxopentan-2- Synthesis of amide)amino)-4-oxobutyric acid (Compound 13).

Compound 12 (300 mg, 0.946 mmol), succinic anhydride (0.143 mg, 1.417 mmol) was added to a reaction flask, and dissolved in 6 mL of dichloromethane and 6 mL of pyridine, and allowed to react at room temperature for 24 hours. TLC detection of the reaction of the starting material was complete, the reaction solution was concentrated to remove pyridine and dichloromethane, water and ethyl acetate were added, 1M hydrochloric acid solution was washed three times, saturated sodium chloride solution was washed three times, dried over anhydrous sodium sulfate, concentrated, purified by column chromatography compound 13 to 340mg, yield 86.7%, LC-MS (APCI ): m / z = 414 (m-1) -.

Step 11 4-((2S,4R)-1-(1,1'-Biphenyl-4-yl)-5-ethoxy-4-methyl-3,4-d2-5-oxopentane Synthesis of tert-butyl 2-yl)amino)-4-oxobutanoate (Compound 14)

Under a nitrogen atmosphere, a compound 13 (340 mg, 0.819 mmol) was added to a reaction flask, and 5 mL of toluene was added thereto to dissolve, and N,N-dimethylformamide di-tert-butyl acetal (681 mg, 3.33 mmol) was added. The reaction was carried out at 80 ° C for 3 hours, and the reaction mixture was purified by TLC. EtOAc was evaporated. to give 91mg of compound 14, yield 29.9%, LC-MS (APCI ): m / z = 470.6 (m + 1) +.

Step 12 4-((2S,4R)-1-(1,1'-Biphenyl-4-yl)-5-ethoxy-4-methyl-3,4-d2-5-oxopentane -2-yl) ammonia Synthesis of 4-oxobutanoic acid (Compound S-1).

Add compound 14 (91mg, 0.194mmol) to the reaction flask, add 2mL of dichloromethane to dissolve, add trifluoroacetic acid (89mg, 0.776mmol) at room temperature, react at room temperature for 5 hours, complete the reaction of TLC to complete the reaction, concentrate the reaction solution, column layer Purification afforded 46 mg of compound S-1 in a yield of 57.5%, LC-MS (APCI): m/z = 414 (M+1) ^{+ 1} H NMR (300 MHz, CDCl ₃ ) δ 7.61 - 7.55 (m, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.33 (dd, J = 8.4, 6.1 Hz, 1H), 7.28-7.20 (m, 2H), 5.90 (t, J = 10.1 Hz, 1H), 4.23 (d, J = 7.1 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 2.83 (m1H), 2.64 (t, J = 6.5 Hz, 2H), 2.48-2.32 (m, 2H), 1.35-1.18 (m, 6H).

Example 2 Preparation of 4-((2S,4R)-1-(1,1'-biphenyl-4-yl)-5-(d5-ethoxy)-4-methyl-5-oxopentane 2-yl)amino)-4-oxobutanoic acid (Compound S-2), the specific synthetic steps are as follows:

Step 1 Synthesis of (R)-3-biphenyl-4-yl-tert-butoxycarbonyl-amino-2-methyl-pentanoic acid ethyl ester (Compound 15).

Compound 10 (0.3 g, 0.732 mmol) was added to the reaction flask, dissolved in 8 mL of methanol, added with 10% palladium on carbon 90 mg, and reacted with hydrogen (50 psi) for 3 hours. The reaction of the starting material was completely confirmed by TLC, and filtered with celite. The filter cake was washed with 40 mL of methanol, and the organic phase was concentrated to give 0.3 g of Compound 15 (yield: 99.6%, LC-MS (APCI): m/z = 412 (M+1) ⁺ .

Step 2 Synthesis of (R)-3-biphenyl-4-yl-2-amino-2-methyl-pentanoic acid (Compound 16).

Compound 11 (870 mg, 2.19 mmol) was added to a reaction mixture, which was dissolved in 20 mL of methanol, and potassium hydroxide (368 mg, 6.57 mmol) was added at room temperature for 5 hours. The TLC was used to detect the complete reaction of the starting material, and the pH was acidified with 1M hydrochloric acid, and extracted with ethyl acetate (50 mL×3). The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate 68.1%, LC-MS (APCI): m/z = 368 (M-1) ^- .

Step 3 Synthesis of (R)-3-biphenyl-4-yl-2-amino-2-methyl-d5-pentanoic acid ethyl ester (Compound 17)

Compound 16 (250 mg, 0.676 mmol) was added to the reaction flask, and dissolved in 1 mL of deuterated ethanol. To the reaction mixture was added 1.25 mL of 4 M hydrochloric acid dioxane, and the mixture was heated to 60 ° C for 3 hours, and the reaction of the starting material was completely confirmed by TLC. 6 mL of water was added to the reaction mixture, the pH was made basic with aqueous sodium carbonate, and extracted with ethyl acetate (30 mL×4). The organic phase was combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate The product 17 to give 230mg, yield 100%, LC-MS (APCI ): m / z = 317 (m + 1) +.

Step 4 4-(4R)-1-(1,1'-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentan-2-yl)amino) Synthesis of 4-oxobutyric acid (Compound 18).

Compound 17 (230 mg, 0.726 mmol), succinic anhydride (11 mg, 1.09 mmol) was added to the reaction mixture, which was dissolved in 6 mL of dichloromethane and 6 mL of pyridine, and allowed to react at room temperature for 24 hours. TLC detected the reaction of the starting material completely, the reaction solution was concentrated to remove pyridine and dichloromethane, diluted with ethyl acetate and water, washed three times with 1M hydrochloric acid solution, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated to give 197 mg of compound. 18, used directly in the next reaction.

Step 5 4-((2S,4R)-1-(1,1'-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentan-2-yl Synthesis of tert-butyl amino)-4-oxobutanoate (Compound 19).

Compound 18 (197 mg, 0.470 mmol) was added to a reaction flask under a nitrogen atmosphere and dissolved with 5 mL of toluene. N,N-Dimethylformamide di-tert-butyl acetal (395 mg, 1.94 mmol) was added. The reaction was carried out at 80 ° C for 2 hours, and the reaction of the starting material was completed by LC-MS. The mixture was cooled to room temperature, then 1M hydrochloric acid was added, and the mixture was extracted with ethyl acetate three times, washed with saturated sodium chloride solution, and the organic phase was combined, dried over anhydrous sodium sulfate and concentrated. compound 19 was purified to give 67mg, yield 49.7%, LC-MS (APCI ): m / z = 475 (m + 1) +.

Step 6 4-((2S,4R)-1-(1,1'-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentan-2-yl Synthesis of amino)-4-oxobutyric acid (S-2).

Compound 19 (67 mg, 0.14 mmol) was added to the reaction flask, which was dissolved in dichloromethane (2 mL), trifluoroacetic acid (64 mg, 0.564 mmol) was added and reacted at room temperature for 5 hours. The reaction of the starting material was completely confirmed by TLC. Purification afforded 23 mg of compound S-2 in a yield of 39.6%, LC-MS (APCI): m/z = 417 (M + 1) ⁺ ; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.55 (m, J = 15.7 , 7.7 Hz, 4H), 7.43 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.28-7.18 (m, 2H), 5.89 (d, J = 8.6 Hz, 2H) ), 4.24 (d, J = 3.3 Hz, 2H), 2.93 - 2.76 (m, 2H), 2.59 (m, 3H), 2.42 (t, J = 6.3 Hz, 2H), 1.94 (m1H), 1.52 (m1H) ), 1.16 (d, J = 7.1 Hz, 3H).

Example 3 Preparation of 4-((2S,4R)-1-(1,1'-biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl Amino)-4-d4-oxobutyric acid (Compound S-3), the specific synthetic steps are as follows:

Step 1 4-(4R)-1-(1,1'-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4 Synthesis of -d4-oxobutyric acid (Compound 20).

Compound 15 (617 mg, 1.98 mmol), deuterated succinic anhydride (288 mg, 2.77 mmol) was added to the reaction flask, dissolved in 6 mL of dichloromethane and 6 mL of pyridine, and then allowed to react at room temperature for 6 hours. TLC detected the reaction of the starting material was complete, the reaction mixture was concentrated to remove pyridine and dichloromethane, ethyl acetate was added to the reaction flask, washed three times with 1 M hydrochloric acid solution, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated. The crude product of Compound 20 was obtained 409 mg, which was directly used for the next reaction.

Step 2 4-((2S,4R)-1-(1,1'-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino Synthesis of tert-butyl 4-d 4-oxobutanoate (Compound 21).

Under a nitrogen atmosphere, a compound 20 (409 mg, 1.992 mmol) was added to a reaction flask, and 20 mL of toluene was added to dissolve. Then N,N-dimethylformamide di-tert-butyl acetal (0.84 g, 3.97 mmol) was added. The reaction was carried out at 80 ° C for 3.5 hours, and the reaction was completed by TLC. The mixture was cooled to room temperature, then 1M hydrochloric acid was added, and the mixture was extracted three times with ethyl acetate. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated. compound 21, yield 33.1%, LC-MS (APCI ): m / z = 465 (m + 1) +.

Step 3 4-((2S,4R)-1-(1,1'-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino Synthesis of 4-d4-oxobutyric acid (Compound S-3).

Compound 21 (78 mg, 0.165 mmol) was added to the reaction flask, and 8 mL of dichloromethane was added to dissolve. Then, trifluoroacetic acid (78 mg, 0.66 mmol) was added to the reaction flask, and the reaction was carried out for 5 hours at room temperature. The reaction of the starting material was completely confirmed by TLC. Purification by column chromatography to give 55 mg of compound S-3 (yield: 80.8%), LC-MS (APCI): m/z = 416 (M+1) ⁺ ; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.61 7.55 (m, 2H), 7.52 (d, J = 8.1 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.23 (d, J = 8.1) Hz, 2H), 5.97 (d, J = 8.7 Hz, 1H), 4.24 (m, J = 8.9, 5.7 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 2.88-2.76 (m, 2H) ), 2.62-2.47 (m, 1H), 1.94 (m, J = 13.8, 11.2, 6.4 Hz, 1H), 1.52 (m, J = 14.2, 9.9, 4.2 Hz, 1H), 1.30-1.19 (m, 3H) ), 1.16 (d, J = 7.1 Hz, 3H).

Example 4 Preparation of 4-((2S,4R)-1-(1,1'-d2-biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentane-2 -amino)-4-oxobutanoic acid (Compound S-4), the specific synthetic steps are as follows:

Step 1 L-d2-Synthesis of Tyrosine (Compound 22).

Compound 1 (1 g, 15 mmol), sodium hydroxide (104 mg, 2.6 mmol) was added to a microwave reaction flask, and 15 mL of heavy water was added. The mixture was heated to 180 ° C for 1 hour in the microwave, cooled to room temperature, and solid precipitated. After filtration, the filter cake was washed 3 times with water, and the filter cake was dried in a vacuum oven below 50 ° C to obtain 0.66 g of compound 22, yield 66%, LC. -MS(APCI): m/z = 182 (M-1) ^- ; ¹ H NMR (300 MHz, DMSO) δ 9.48 (d, J = 9.4 Hz, 1H), 7.08-6.92 (m, 2H), 3.65 (s, 3H), 3.02 (qd, J = 14.1, 5.3 Hz, 2H).

Step 2 Synthesis of L-d2-tyrosine methyl ester (Compound 23).

22 (2 g, 10 mmol) was added to the reaction flask, dissolved in methanol (40 mL), cooled to 0 ° C, and thionyl chloride (2.7 g, 20 mmol) was added dropwise to the reaction at 0 ° C. After stirring to room temperature for 18 hours, the reaction mixture was concentrated, and excess thionyl chloride was removed to obtain 2.45 g of Compound 23, which was directly used for the next reaction.

Step 3 Synthesis of L-d2-Boc-tyrosine methyl ester (Compound 24).

The crude product (2.45 g, 8.24 mmol) was added to a reaction flask, dissolved in 60 ml of dichloromethane, and triethylamine (3.35 g, 33.1 mmol) was added, and the mixture was warmed to 30 ° C, and Boc anhydride (3.07 g, 14.08 mmol) was added. After stirring for 3 hours, the material was completely reacted by TLC. The reaction mixture was concentrated and purified by column chromatography to yield 2.8 g of compound 24 as a yellow solid, yield 87.5%, LC-MS (APCI): m/z = 298 (M+1) ) ⁺ .

Step 4 Synthesis of (R)-3-trifluoromethanesulfonic acid-d2-phenyl-4-yl-2-tert-butoxycarbonyl-aminopropanoic acid methyl ester (Compound 25).

Compound 24 (2.9 g, 9.35 mmol) was added to a reaction flask, dissolved in 50 mL of dichloromethane, and pyridine (2 g, 25 mmol) was added at room temperature. Cool to -15 ° C and add trifluoromethanesulfonic anhydride (3.4 g, 12.05 mmol). The reaction solution was stirred at this temperature for 30 minutes. LC-MS was used to detect the reaction of the starting material. The reaction mixture was diluted with water (100 mL). The mixture was diluted with ethyl acetate (100 mL×2), and the organic phase was combined, washed with brine, dried over anhydrous sodium sulfate compound 25, yield 95.4%, LC-MS (APCI ): m / z = 430.3 (m + 1) +.

Step 5 Synthesis of (R)-3-d2-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid methyl ester (Compound 26).

Tetrakistriphenylphosphine palladium (322 mg, 0.279 mmol), compound 25 (4 g, 9.32 mmol), phenylboronic acid (2.27 g, 18.63 mmol) and potassium carbonate (1.93 g, 13.97 mmol) were added to the reaction flask under nitrogen. After adding 80 mL of toluene, the reaction was heated to 80 ° C for 3 hours, and the compound 25 was completely reacted by LC-MS. The reaction mixture was concentrated and purified by column chromatography ( petroleum ether: ethyl acetate = 6:1) to afford 75.1%, LC-MS (APCI): m/z = 358 (M + 1) ⁺ .

Step 6 Synthesis of (R)-3-d2-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid (Compound 27).

Compound 26 (2.5 g, 6.99 mmol) was added to the reaction flask, dissolved in 30 mL of tetrahydrofuran, and then 28 mL of 1 M sodium hydroxide solution was added thereto, and the reaction was stirred at room temperature for 4 hours. The reaction of the starting material was completely confirmed by TLC, and the reaction mixture was concentrated. 30 mL of water and 30 mL of ethyl acetate were added to the reaction flask, and the pH was made acidic with a 1 M hydrochloric acid solution, extracted with ethyl acetate (100 mL×3), and the organic phase was combined and dried over anhydrous sodium sulfate compound 27, as a yellow oil, yield 92.9%, LC-MS (APCI ): m / z = 342 (m-1) -.

Step 7 Synthesis of (R)-3-d2-biphenyl-4-yl-2-tert-butoxycarbonyl-amino-methoxymethylaminopropionic acid (Compound 28).

Compound 27 (2.23 g, 6.50 mmol), methoxymethylamine (637 mg, 12.32 mmol), carbodiimide (EDCI, 2.48 g, 13.14 mmol), triethylamine (0.773 g, 7.78) were added to the reaction flask. Mmol), HOBT (1.313 g, 9.7 mmol), 40 mL of dichloromethane was added and stirred at room temperature for 18 hours. The reaction of the starting material was completed by TLC, and the reaction mixture was concentrated and purified by column chromatography to yield 2.04 g of Compound 28, yield 78.1%, LC-MS (APCI): m/z = 403 (M+1) ⁺ .

Step 8: Synthesis of R-3-d2-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropanal (Compound 29).

Under a nitrogen atmosphere, lithium aluminum hydride (290 mg, 7.6 mmol) was added to the reaction flask, and 50 mL of tetrahydrofuran was added. After cooling to 0 ° C, the compound 28 (2.04 g, 5.07 mmol) was added, and the mixture was stirred at 0 ° C for 1 hour, quenched with potassium hydrogen sulfate, and then, 1 M hydrochloric acid solution 20 mL, ethyl acetate was extracted three times, then the organic phase was combined. washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to give compound 29 1.838g, yield ~ 100%, LC-MS ( APCI): m / z = 328 (m + 1) +.

Step 9 Synthesis of (R)-3-d2-biphenyl-4-yl-tert-butoxycarbonyl-amino-2py-2-methyl-pentanoic acid ethyl ester (Compound 30).

Compound 29 (1.83 g, 5.5 mmol) was added to the reaction flask, which was dissolved in 40 mL of dichloromethane, and ethoxycarbonylethyltriphenylphosphine (4.46 g, 12.3 mmol) was added to the reaction flask and stirred at room temperature. The reaction of the starting material was completed by TLC, and the reaction mixture was concentrated and purified by column chromatography to yield 1.71 g of Compound 31, yield 74.3%, LC-MS (APCI): m/z = 412.5 (M+1) ⁺ .

Step 10 Synthesis of (R)-3-d2-biphenyl-4-yl-tert-butoxycarbonyl-amino-2-methyl-pentanoic acid ethyl ester (Compound 31).

Compound 30 (0.7 g, 1.70 mmol) was added to the reaction flask, dissolved in 10 mL of methanol, and 100 mg of palladium carbon was added to the reaction flask, and the reaction was carried out under hydrogen (50 psi) for 3 hours. The reaction of the starting material was completely confirmed by TLC, and filtered using celite. , the filter cake washed with 40mL of methanol, 0.77g organic phase was concentrated to give compound 31, yield 100% LC-MS (APCI) : m / z = 414 (m + 1) +.

Step 11 Synthesis of (R)-3-d2-biphenyl-4-yl-2-amino-2-methyl-pentanoic acid ethyl ester (Compound 32).

Compound 32 (790 mg, 1.92 mmol) was added to the reaction flask, and 10 mL of ethyl acetate was added thereto, and then 4 mL of 4 M hydrochloric acid dioxane was added to the reaction mixture, and the mixture was reacted at room temperature for 4 hours. The reaction of the starting material was completed by LCMS, and the reaction mixture was concentrated to give a crude compound (yield: 0.714 g). LC-MS (APCI): m / z = 314 (M + 1) +.

Step 12 4-(4R)-1-(1,1'-d2-biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino) Synthesis of -4-oxobutyric acid (Compound 33)

Compound 32 (700 mg, 2.23 mmol), succinic anhydride (3.35 mg, 3.35 mmol) was added to the reaction flask, and 15 mL of dichloromethane and 15 mL of pyridine were added and dissolved, and the mixture was reacted at room temperature for 6 hours. The reaction of the starting material was completed by LC-MS. The reaction mixture was concentrated to remove pyridine and dichloromethane. Ethyl acetate was added to the reaction flask, washed three times with 1 M hydrochloric acid solution, three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated. After that, 0.982 g of Compound 33 was obtained, which was directly used for the next reaction without further purification.

Step 13 4-((2S,4R)-1-(1,1'-d2-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl Synthesis of tert-butyl amino)-4-oxobutanoate (Compound 34).

Compound 33 (982 mg, 9.51 mmol) was added to the reaction flask under a nitrogen atmosphere, and toluene (20 mL) was dissolved. N,N-Dimethylformamide di-tert-butyl acetal (2.01 mg, 9.51 mmol) was added. The mixture was heated to 80 ° C for 1 hour, and the reaction was completed by TLC. The mixture was cooled to room temperature. 1M hydrochloric acid was added and extracted with ethyl acetate three times. The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. Analysis of compound 34 to give 284mg, yield 34.5%, LC-MS (APCI ): m / z = 470 (m + 1) +.

Step 14 4-((2S,4R)-1-(1,1'-d2-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl Synthesis of amino)-4-oxobutyric acid (Compound S-4).

Compound 34 (284 mg, 0.6 mmol) was added to the reaction flask, and 4 mL of dichloromethane was added to dissolve. Then, trifluoroacetic acid (4 mL) was added to the reaction flask, and the reaction was carried out for 5 hours at room temperature. The reaction of the starting material was completely confirmed by TLC, and the reaction mixture was concentrated. Chromatography to give 157 mg of compound S-4, yield 62.8%, LC-MS (APCI): m/z = 414 (M+1) ⁺ ; ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.62-7.55 (m) , 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.34 (m, J = 8.4, 6.2 Hz, 1H), 7.23 (d, J = 7.0 Hz, 2H), 5.79 (d, J = 12.6 Hz) , 1H), 4.10 (m, J = 16.9, 9.7 Hz, 2H), 2.85 (d, J = 1.7 Hz, 2H), 2.68-2.59 (m, 2H), 2.55 (m, J = 14.2, 8.2, 5.0 Hz, 1H), 2.47-2.35 (m, 2H), 1.93 (dd, J = 14.2, 9.5 Hz, 1H), 1.60-1.48 (m, 1H), 1.29-1.20 (m, 3H), 1.16 (d, J = 7.1 Hz, 3H).

Example 5 Preparation of 4-((2S,4R)-1-(1,1'-d5-biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentane-2 -amino)-4-oxobutanoic acid (Compound S-5), the specific synthetic steps are as follows:

Step 1d5-Synthesis of bromobenzene (Compound 36).

Concentrated sulfuric acid (11.89g, 6.5mL) was added to the reaction flask, diluted with water 24mL, cooled to 0 ° C, deuterated benzene (3g, 0.035mol) was added dropwise. After the addition was completed, bromic acid was added twice at 0 °C. Sodium (5.92 g, 0.039 mol), which was reacted for 18 hours at room temperature, 50 mL of ice water was added, and the mixture was extracted three times with n-hexane, and the organic phases were combined, washed with saturated sodium hydrogen carbonate, saturated sodium chloride and dried over anhydrous sodium sulfate. Concentration at 30 ° C gave 2.38 g of compound 36 in a yield of 41.8%.

Step 2d5 - Synthesis of phenylboronic acid (Compound 37).

Compound 36 (2.379 g, 14.68 mmol), pinacol borate (5.5 g, 21 mmol), Pd(dppf)Cl ₂ (1.04 g, 4.4 mmol) and potassium acetate (4.2 g, were added to the dried reaction flask. 43.5mmol), 100mL anhydrous dioxane was added under nitrogen protection, heated to 88 ° C and stirred for 6 hours, TLC detection of the reaction of the starting material was complete, diluted with ethyl acetate 100mL, concentrated to give an oily liquid, purified by column chromatography to obtain 1.5g Compound 37, yield 81.5%.

Step 3 Synthesis of (R)-3-d5-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid methyl ester (Compound 38).

To the reaction flask was added tetrakistriphenylphosphine palladium (212 mg, 0.16 mmol), compound 3 (2.63 g, 6.13 mmol), phenylboronic acid (1.48 g, 12.27 mmol) and potassium carbonate (12.71 g, 8.8 mmol). 60 mL of toluene was added, the temperature was raised to 80 ° C, and the reaction was stirred for 5 hours. The reaction of Compound 3 was completed by TLC. The reaction mixture was concentrated and purified by column chromatography to obtain 1.82 g of Compound 38, yield 81.9%, LC-MS (APCI): m/z =361(M+1) ⁺ .

Step 4 Synthesis of (R)-3-d5-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropionic acid (Compound 39).

Add compound 38 (1.82g, 5.05mmol) to the reaction flask, add 30mL of tetrahydrofuran to dissolve, then add 15mL of 1M sodium hydroxide solution, the reaction solution is stirred at room temperature for 2 hours, TLC detection of the raw material reaction is complete, concentrate the reaction solution To the reaction flask, 20 mL of water and 20 mL of ethyl acetate were added, and the pH was adjusted to ～2 with a 1 M hydrochloric acid solution, and extracted with ethyl acetate (50 mL×3). 1.75g to give compound 39 as a yellow oil, a yield of 100%, LC-MS (APCI ): m / z = 365 (m-1) -.

Step 5 Synthesis of (R)-3-d5-biphenyl-4-yl-2-tert-butoxycarbonyl-amino-methoxymethylaminopropionic acid (Compound 40)

Compound 39 (1.75 g, 5.05 mmol), methoxymethylamine (0.59 g, 9.59 mmol), EDCI (1.93 g, 10.09 mmol), triethylamine (0.6 g, 6.05 mmol), HOBT (1.02 g, 7.57 mmol), 40 mL of dichloromethane was added, and the reaction was stirred at room temperature for 16 hours. The reaction of the starting material was completed by LC-MS. The reaction mixture was concentrated and purified by column chromatography to yield 1.25 g of Compound 40, yield 60.9%, LC-MS (APCI): m/z = 406 (M+1) ⁺ .

Step 6 Synthesis of (R)-3-d5-biphenyl-4-yl-2-tert-butoxycarbonyl-aminopropanal (Compound 41).

Under a nitrogen atmosphere, lithium aluminum hydride (180 mg, 4.63 mmol) was added to the reaction flask, and 50 mL of tetrahydrofuran was added. After cooling to 0 ° C, the compound 40 (1.25 g, 3.08 mmol) was added, and the mixture was stirred at 0 ° C for 1 hour, and the potassium hydrogen sulfate solution was quenched, 10 mL of 1 M hydrochloric acid solution was added, and the mixture was extracted three times with ethyl acetate. washed with saturated sodium chloride, dried over anhydrous sodium sulfate and dried to give 0.962g of compound 41, yield is about 94.3%, LC-MS (APCI ): m / z = 331 (m + 1) +.

Step 7 Synthesis of (R)-3-d5-biphenyl-4-yl-tert-butoxycarbonyl-amino-2py-2-methyl-pentanoic acid ethyl ester (Compound 42).

Compound 41 (0.962 g, 2.91 mmol) was added to the reaction flask, which was dissolved in dichloromethane (30 mL), and then ethoxycarbonylethyltriphenylphosphine (2.11 g, 5.84 mmol) was added to the reaction flask and stirred at room temperature. After reacting for 18 hours, the reaction of the starting material was completed by TLC. The reaction mixture was concentrated and purified by column chromatography to give the compound (yield: <^RTIgt;</RTI&^gt;

Step 8 Synthesis of (R)-3-d5-biphenyl-4-yl-tert-butoxycarbonyl-amino-2-methyl-pentanoic acid ethyl ester (Compound 43).

Compound 42 (0.7 g, 1.69 mmol) was added to the reaction flask, and dissolved in 15 mL of methanol. Then, 100 mg of palladium carbon was added to the reaction flask, and the reaction was carried out under hydrogen (50 psi) for 3 hours. The reaction of the starting material was completely determined by TLC using celite. filtered, the filter cake washed with 40mL of methanol, 0.69g organic phase was concentrated to give compound 43, yield 98.1%, LC-MS (APCI ): m / z = 417 (m + 1) +.

Step 9 Synthesis of (R)-3-d5-biphenyl-4-yl-2-amino-2-methyl-pentanoic acid ethyl ester (Compound 44).

Compound 44 (0.96 mg, 2.30 mmol) was added to the reaction flask, and 50 mL of ethyl acetate was added thereto to dissolve. To the reaction mixture was added 2 mL of dioxane 4M 2 mL, and the mixture was reacted at room temperature for 3 hours, and the mixture was completely reacted by TLC to remove the solvent. 562 mg of compound 44 were obtained which was used directly in the next step without further purification.

Step 10 4-(4R)-1-(1,1'-d5-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino) Synthesis of -4-oxobutyric acid (Compound 45)

Compound 44 (562 mg, 1.77 mmol), succinic anhydride (243 mg, 2.43 mmol) was added to a reaction flask, and dissolved in 8 mL of dichloromethane and 8 mL of pyridine, and then reacted at room temperature for 24 hours. TLC detected the reaction of the starting material was complete, the reaction mixture was concentrated to remove pyridine and dichloromethane, ethyl acetate was added to the reaction flask, washed three times with 1 M hydrochloric acid solution, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated. After that, a crude product of 0.829 mg of Compound 45 was obtained, which was directly used for the next reaction.

Step 11 4-((2S,4R)-1-(1,1'-d5-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl Synthesis of tert-butyl amino)-4-oxobutanoate (Compound 46)

Compound 45 (829 mg, 1.992 mmol) was added to a reaction flask under a nitrogen atmosphere and dissolved with 20 mL of toluene. N,N-Dimethylformamide di-tert-butyl acetal (1.7 g, 8.03 mmol) was added. The reaction was carried out at 80 ° C for 3 hours, and the reaction was completed by TLC. EtOAc was evaporated. yield 37.5%, LC-MS (APCI ): m / z = 473.6 (m + 1) +.

Step 12 4-((2S,4R)-1-(1,1'-d5-Biphenyl-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl Synthesis of amino)-4-oxobutyric acid (S-5)

Compound 46 (264 mg, 0.558 mmol) was added to the reaction flask, and dissolved in 8 mL of dichloromethane. Trifluoroacetic acid (256 mg, 2.25 mmol) was added to the reaction flask, and reacted at room temperature for 5 hours. The reaction of the starting material was completely confirmed by TLC. Purification by column chromatography gave 178 mg of compound S-5, yield 76.7%, LC-MS (APCI): m/z = 417 (M+1) ^{+ 1} H NMR (300 MHz, CDCl ₃ ) δ 7.54 (d , J=8.2Hz, 2H), 7.24 (d, J=8.2Hz, 2H), 5.77(d, J=8.7Hz, 1H), 4.26(d, J=8.8Hz, 1H), 4.13(qd,J = 7.2, 3.0 Hz, 2H), 2.86 (d, J = 6.6 Hz, 2H), 2.71-2.59 (m, 2H), 2.60-2.50 (m, 1H), 2.49-2.38 (m, 2H), 1.99- 1.88 (m, 1H), 1.56 (m, J = 14.2, 10.0, 4.3 Hz, 1H), 1.33-1.24 (m, 3H), 1.17 (d, J = 7.1 Hz, 3H).

Example 6 Preparation of 4-((2S,4R)-1-(1,1'-d5-biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentane 2-yl)amino)-4-oxobutyric acid (Compound S-6), the specific synthetic steps are as follows:

Step 1 Synthesis of (R)-3-d5-biphenyl-4-yl-2-amino-2-methyl-pentanoic acid (Compound 47).

Compound 43 (300 mg, 0.745 mmol) was added to a reaction flask, and dissolved in 10 mL of methanol, and then potassium hydroxide (125 mg, 2.24 mmol) was added to the reaction mixture for 6 hours at room temperature. The TLC was used to detect the reaction of the starting material, and the pH was adjusted to be acidic with 1M hydrochloric acid, and extracted with ethyl acetate (60 mL×3). The organic phase was washed with saturated sodium chloride and dried over anhydrous sodium sulfate. LC-MS (APCI): m / z = 373 (m-1) -.

Step 2 Synthesis of (R)-3-d5-biphenyl-4-yl-2-amino-2-methyl-d5-pentanoic acid ethyl ester (Compound 48).

Compound 47 (214 mg, 0.571 mmol) was added to the reaction flask, and 1.5 mL of deuterated ethanol was added to dissolve. To the reaction mixture was added 4 mL of dioxane hydrochloride 1.5 mL, and the mixture was heated to 60 ° C for 3 hours. The reaction of TLC was complete. 6 mL of water was added to the reaction mixture, the pH was made alkaline with aqueous sodium carbonate solution, extracted with ethyl acetate (30 mL×4), and purified by column chromatography to give 186 mg of compound 48, yield 100%, LC-MS (APCI) :m/z=322(M+1) ⁺ .

Step 3 4-(4R)-1-(1,1'-d5-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentan-2-yl) Synthesis of amino)-4-oxobutanoic acid (Compound 49).

Compound 48 (186 mg, 0.578 mmol), succinic anhydride (88 mg, 0.867 mmol) was added to the reaction flask. It was dissolved by adding 5 mL of dichloromethane and 5 mL of pyridine, and reacted at room temperature for 24 hours. The reaction of the starting material was completed by LC-MS. The reaction mixture was concentrated to remove pyridine and dichloromethane. Ethyl acetate was added to the reaction flask, washed three times with 1 M hydrochloric acid solution, washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After concentration, 290 mg of the crude product of Compound 49 was obtained, which was directly used for the next reaction.

Step 4 4-((2S,4R)-1-(1,1'-d5-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentane-2 Synthesis of tert-butyl (meth)amino)-4-oxobutanoate (Compound 50)

Compound 56 (290 mg, 0.684 mmol) was added to a reaction flask under nitrogen and dissolved in 5 mL of toluene. Then N,N-dimethylformamide di-tert-butyl acetal (580 mg, 2.82 mmol) was added. The reaction was carried out at 80 ° C for 3 hours, and the reaction was completed by LC-MS. The mixture was evaporated to room temperature, then 1M hydrochloric acid was added, and the mixture was extracted three times with ethyl acetate. The organic phase was combined and dried over anhydrous sodium sulfate. compound 50, yield 50.6%, LC-MS (APCI ): m / z = 480 (m + 1) +.

Step 5 4-((2S,4R)-1-(1,1'-d5-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentane-2 Synthesis of -amino)amino)-4-oxobutanoic acid (Compound S-6)

Compound 57 (109 mg, 0.227 mmol) was added to the reaction flask and dissolved in 2 mL of dichloromethane. Then, trifluoroacetic acid (105 mg, 0.91 mmol) was added to the reaction flask, and reacted at room temperature for 5 hours. The reaction of the starting material was completely confirmed by TLC. After purification by column chromatography, 89 mg of compound S-6 was obtained in a yield of 93.6%, LC-MS (APCI): m/z = 422 (M+1) ^{+ 1} H NMR (300 MHz, CDCl ₃ ) δ 7.62 -7.43 (m, 2H), 7.34 - 7.13 (m, 2H), 5.92 (d, J = 8.7 Hz, 1H), 4.22 (m, J = 30.2, 15.1 Hz, 1H), 2.92 - 2.76 (m, 2H) ), 2.62 (dd, J = 13.1, 6.6 Hz, 2H), 2.54 (m, J = 14.3, 8.3, 5.0 Hz, 1H), 2.42 (t, J = 6.4 Hz, 2H), 1.93 (m, J = 13.8, 9.5, 4.1 Hz, 1H), 1.53 (m, J = 14.1, 9.9, 4.2 Hz, 1H), 1.16 (d, J = 7.1 Hz, 3H).

Example 7 Preparation of 4-((2S,4R)-1-(1,1'-d2-biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentane 2-yl)amino)-4-oxobutanoic acid (Compound S-7), the specific synthetic steps are as follows:

Step 1 Synthesis of (R)-3-d2-biphenyl-4-yl-2-amino-2-methyl-pentanoic acid (Compound 51).

Compound 31 (500 mg, 1.25 mmol) was added to the reaction mixture, which was dissolved in 20 mL of methanol, and then potassium hydroxide (210 mg, 3.75 mmol) was added to the reaction mixture for 5 hours at room temperature. The TLC was used to detect the reaction of the starting material, and the pH was acidified with 1M hydrochloric acid, and extracted with ethyl acetate (50 mL×3). The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. yield 53.0%, LC-MS (APCI ): m / z = 370 (m-1) -.

Step 2 Synthesis of (R)-3-d2-biphenyl-4-yl-2-amino-2-methyl-d5-pentanoic acid ethyl ester (Compound 52).

Compound 51 (246 mg, 0.662 mmol) was added to the reaction flask, and dissolved in 1.5 mL of deuterated ethanol. Then, 1.25 mL of 4 M hydrochloric acid dioxane was added to the reaction mixture, and the mixture was heated to 60 ° C for 3 hours, and the reaction of the raw materials was detected by TLC. Completely, 6 mL of water was added to the reaction mixture, the pH was made basic with aqueous sodium carbonate, and extracted with ethyl acetate (30 mL×4). The organic phase was combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate after give 221mg compound 52, yield 100%, LC-MS (APCI ): m / z = 319 (m + 1) +.

Step 3 4-(4R)-1-(1,1'-d2-biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentan-2-yl) Synthesis of amino)-4-oxobutyric acid (Compound 53).

Compound 52 (211 mg, 0.662 mmol) and succinic anhydride (86 mg, 0.86 mmol) were added to the reaction mixture, which was dissolved in 5 mL of dichloromethane and 5 mL of pyridine, and the mixture was stirred at room temperature for 24 hours. The TLC was used to detect the reaction of the starting material. The reaction mixture was concentrated, and the solvent was removed. Ethyl acetate was added to the reaction flask, washed three times with 1 M hydrochloric acid solution, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated to give compound The crude product of 236 mg of 53 was used directly in the next reaction.

Step 4 4-((2S,4R)-1-(1,1'-d2-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentane-2 Synthesis of tert-butyl ester of -amino)-4-oxobutanoic acid (Compound 54)

Compound 53 (276 mg, 0.657 mmol) was added to a reaction flask under a nitrogen atmosphere, and dissolved in 5 mL of toluene. Then N,N-dimethylformamide di-tert-butyl acetal (552 mg, 2.7 mmol) was added. The reaction was carried out at 80 ° C for 2 hours, and the reaction was completed by TLC. The mixture was evaporated to room temperature. 1M hydrochloric acid was added, and the mixture was extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. Yield 48.3%, LC-MS (APCI): m/z = 475 (M+1) ⁺

Step 5 4-((2S,4R)-1-(1,1'-d2-Biphenyl-4-yl)-5-d5-ethoxy-4-methyl-5-oxopentane-2 Synthesis of -amino)amino)-4-oxobutanoic acid (Compound S-7)

Compound 54 (111 mg, 0.232 mmol) was added to the reaction flask, and dissolved in 2 mL of dichloromethane. Then, 2 mL of trifluoroacetic acid was added to the reaction flask, and the reaction was carried out for 5 hours at room temperature. The reaction of the starting material was completely confirmed by TLC, and the reaction mixture was concentrated. Purification afforded 72 mg of compound S-7 in a yield of 75%, LC-MS (APCI): m/z = 419 (M+1) ^{+ 1} H NMR (300 MHz, CDCl ₃ ) δ 7.61 - 7.55 (m, 2H ), 7.43 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.28-7.21 (m, 2H), 5.88 (d, J = 12.6 Hz, 1H), 2.89-2.74 (m, 2H), 2.62 (m, J = 12.9, 6.2 Hz, 2H), 2.54 (m, J = 14.2, 8.2, 4.9 Hz, 1H), 2.42 (t, J = 6.4 Hz, 2H), 1.97- 1.82 (m, 1H), 1.62-1.46 (m, 1H), 1.17 (t, J = 6.8 Hz, 3H).

Biological activity test.

Metabolic stability evaluation.

Microsomal experiments: human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).

Preparation of stock solution: A certain amount of the compound powder of the example was accurately weighed and dissolved to 5 mM with DMSO.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

A solution of NADPH regeneration system (containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride) was prepared and placed on wet ice before use.

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

Table 1 Evaluation of Hepatic Particle Metabolism of the Example Compounds

As shown in the above table, the compounds of the present invention exhibited excellent metabolic stability in both human liver microsome experiments and rat liver microsome experiments compared to Shakupit.

Rat pharmacokinetics experiment

EXPERIMENTAL OBJECTIVE: To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of Sacubitril (Sacurbit) and the compounds of the examples.

Experimental animals:

Type and strain: SD rat grade: SPF grade

Gender and quantity: male, 6

Weight range: 180 ~ 220g (actual weight range is 187 ~ 197g)

Source: Shanghai Xipuer Bikai Experimental Animal Co., Ltd.

experiment procedure:

Before the blood sample was collected, 20 L of 2M sodium fluoride solution (esterase inhibitor) was previously added to the EDTA-K2 anticoagulation tube, dried in an 80 degree oven, and stored in a 4 degree refrigerator.

Rats, males, weighing 187-197 g, were randomly divided into 2 groups and started fasting one afternoon before the experiment. At night, you can drink water freely and give food 4 hours after administration. Group A was given Sacubitril 3 mg/kg, and group B was given 3 mg/kg of the compound of the example. Blood was taken from the orbital vein of the rat for about 100-200 L at 15 min, 30 min, 1, 2, 3, 5, 8, and 10 h after administration. Place in an EDTA-K2 anticoagulated 0.5 mL Eppendorf tube and mix immediately. After anticoagulation, gently invert the tube 5-6 times as soon as possible, place the blood in the ice box, within 30 minutes. The blood samples were centrifuged at 4000 rpm, 10 min, 4 ° C, and all plasma was collected and immediately stored at -20 ° C. Plasma concentrations in plasma at each time point were determined after sample collection at all time points.

According to the average blood drug concentration-time data obtained after the above, the male SD rats were subjected to ig-administered Sacubitril (3 mg/kg) and the compound of the example (3 mg/kg) according to the non-compartmental statistical moment theory using Winnonin software. Post-pharmacokinetic related parameters.

Experiments have shown that the compound of the present invention has superior activity to Sacubitril and has superior pharmacokinetic properties as compared with Sacubitril, and thus is more suitable as a compound for inhibiting enkephalinase, and is therefore suitable for preparing a medicament for treating heart failure.

It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples are generally in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (11)

1. An enkephalinase inhibitor characterized by a biphenyl compound represented by formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvent Compound,

   

   Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

   Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And at least one of R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ is deuterated or deuterated.
2. The enkephalinase inhibitor according to claim 1, wherein each of R ¹ , R ² , R ³ , R ⁴ and R ^{5 is} independently hydrazine or hydrogen.
3. The enkephalinase inhibitor according to claim 1, wherein R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are each independently anthracene or hydrogen.
4. The enkephalinase inhibitor according to claim 1, wherein each of R ¹⁵ , R ¹⁶ , R ¹⁷ and R ^{18 is} independently hydrazine or hydrogen.
5. The enkephalinase inhibitor according to claim 1, wherein each of R ¹⁹ , R ²⁰ , R ²¹ and R ^{22 is} independently hydrazine or hydrogen.
6. The enkephalinase inhibitor according to claim 1, wherein each of R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ^{27 is} independently hydrazine or hydrogen.
7. The enkephalinase inhibitor according to claim 1, wherein the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof:

   

   

   
8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an enkephalinase inhibitor according to any one of claims 1 to 7, or a crystalline form thereof, a pharmaceutically acceptable salt, A pharmaceutical composition of a hydrate or solvate, stereoisomer, prodrug or isotopic variation.
9. The pharmaceutical composition according to claim 8, which further comprises other active compounds, which may be selected from the group consisting of: HMG-Co-A reductase inhibitors, angiotensin receptor blockers, blood vessels Angiotensin-converting enzyme inhibitors, calcium channel blockers, endothelin antagonists, renin inhibitors, diuretics, ApoA-I mimics, antidiabetic drugs, diet pills, aldosterone receptor blockers, endothelin receptors Body blockers, aldosterone synthase inhibitors, CETP inhibitors, and type 5 phosphodiesterase inhibitors.
10. Use of an enkephalinase inhibitor according to any one of claims 1 to 9 for the preparation of a medicament for treating an enkephalinase-mediated disease, such as heart failure, hypertension, lung hypertension.
11. A method of treating and/or preventing a disease associated with enkephalinase in a subject, the method comprising administering to the subject a formula according to any one of claims 1 to 7 ( I) A compound or a polymorphic form thereof, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, an isotope variant, a hydrate or a solvent compound, or a pharmaceutical composition according to any one of claims 8 or 9.