RU2809634C2 - Compound with methyllactam ring and its use in pharmaceutics - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: group of inventions includes a compound of formula [I], its pharmaceutically acceptable salt, use, a pharmaceutical composition containing it, a method of inhibiting SGLT1 and a method of treating diabetes.

EFFECT: compound of formula [I] with SGLT1-inhibiting activity used for the treatment of diabetes.

11 cl, 2 dwg, 7 tbl, 8 ex

Description

Field of technology

The present invention relates to a compound with a methyl lactam ring having SGLT1 inhibitory activity, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same compound, and its use in pharmaceuticals.

State of the art

SGLT1, i.e. Sodium-dependent glucose cotransporter 1 is known to play a major role in the absorption of glucose and galactose in the small intestine. Glucose-galactose malabsorption syndrome has been reported to occur in patients with SGLT1 deficiency. In addition, the expression of SGLT1 in the small intestine is confirmed to be increased in patients with diabetes, and it is believed that the increase in sugar absorption in patients with diabetes is caused by the high level of SGLT1 expression in the small intestine.

Based on these data, an SGLT1 inhibitor is expected to normalize blood glucose levels by blocking glucose absorption in the small intestine. Therefore, SGLT1 inhibitor is considered as an effective treatment against diabetes and diabetes complications associated with hyperglycemia. It is also considered as an effective anti-obesity agent by inhibiting the entry of glucose into the body (Non-Patent Documents 1 and 2).

Voglibose (generic name) is a medicinal product approved for manufacture and sale under the Japan Pharmaceutical Affairs Act, Art. 14 (Permit number: 21600AMZ00368). Voglibose reduces elevated postprandial glucose levels by inhibiting disaccharidase, α-glucosidase, which breaks down disaccharides present in the intestinal mucosa into monosaccharides, and inhibiting or slowing the digestion and absorption of carbohydrates in the gastrointestinal tract. This pharmacological action is known to be effective against type 2 diabetes with delayed development and impaired glucose tolerance.

Based on the available evidence, inhibiting the absorption of sugars in the small intestine with an SGLT1 inhibitor and thereby reducing elevated postprandial blood sugar levels is considered an effective treatment for delayed onset type 2 diabetes with impaired glucose tolerance.

The expression of SGLT1 in cardiomyocytes was also confirmed. It is known that GLUT1 (glucose transporter type 1) and GLUT4 (glucose transporter type 4) usually play a role in the entry of glucose into cardiomyocytes, and the contribution of SGLT1 is less. However, SGLT1 expression is induced in cardiac muscle in mice injected with mutated PRKAG2 genes (AMPK-activated protein kinase gamma 2 subunit), which is the gene responsible for hereditary hypertrophic cardiomyopathy (glycogen storage myocardosis), or in mice undergoing treatment from myocardial ischemia, and SGLT1 has been reported to be involved in glucose uptake by cardiomyocytes in these pathologies. It is believed that glucose metabolized by SGLT1 is excessively accumulated or metabolized in cardiomyocytes and destabilizes these cells. In past mouse models, it has been reported that glycogen accumulation in cardiac muscle is indeed inhibited by exposure to the nonselective SGLT inhibitor, phlorizin.

Based on available data, the SGLT1 inhibitor is believed to be effective against hypertrophic cardiomyopathy and coronary artery disease by inhibiting excess glucose uptake by cardiomyocytes (Non-Patent Documents 3 and 4).

SGLT1 is stabilized in cancer cells by epidermal growth factor receptors, i.e. proteins on the surface of many types of cancer cells. It is known that transporters of glucose, lactic acid, amino acids, etc. are involved in the supply of nutrients to cancer cells, and this is especially true for glucose transport, because SGLT1 and GLUT1 continuously supply glucose to cancer cells. When glucose is not supplied for a long time, cells are destroyed due to autophagy.

Based on the available data, it is believed that the SGLT1 inhibitor inhibits the supply of glucose to cancer cells and has an anti-cancer effect (Non-Patent Documents 5 and 6).

Because carbohydrates from food are broken down into monosaccharides in the gastrointestinal tract and absorbed in the upper gastrointestinal tract, many sugars never reach the lower gastrointestinal tract. However, when drugs that slow or inhibit glucose absorption are administered, or when large amounts of stable polysaccharides are ingested, undigested sugars enter and remain in the lower gastrointestinal tract, causing osmotic diarrhea.

The SGLT1 inhibitor inhibits glucose absorption and increases the amount of monosaccharides in the lower gastrointestinal tract. Therefore, it is believed that an SGLT1 inhibitor will be effective against constipation.

Diabetes is caused by elevated blood glucose levels due to insufficient action of insulin, and persistently elevated blood glucose levels can cause diabetic complications (eg, retinopathy, nephropathy, and neuropathy, which are collectively known as microangiopathy; and cerebrovascular disease, coronary artery disease, and occlusive disease). arteriosclerosis of the lower extremities, which are generally known as macroangiopathy). Other diseases associated with high blood glucose include obesity.

Diabetes is divided into type 1 diabetes and type 2 diabetes. It is believed that type 1 diabetes develops due to insufficient insulin action caused by the destruction of pancreatic β-cells that secrete insulin, and type 2 diabetes develops due to exposure to environmental factors such as overeating, lack of physical activity, obesity, stress , aging, in addition to a number of genetic factors including decreased insulin production and insulin resistance. The diagnosis of diabetes is divided into three types, namely normal, borderline and diabetic type, according to the level of glucose in the blood. When any of the following points (1) – (4) are detected:

(1) blood glucose level 126 mg/dL or more in the morning on an empty stomach,

(2) two-hour value of 200 mg/dL or more in 75 g OGTT (oral glucose tolerance test),

(3) blood glucose level of 200 mg/dL or more when measured at a random time point,

(4) 6.5% or more HbA1cg,

then the subject is classified as a diabetic type and is diagnosed with diabetes or suspected diabetes (Non-Patent Document 7).

The OGTT used in point (2) above is one of the methods for diagnosing diabetes. In general, when a person is administered a solution containing 75 g of glucose on an empty stomach, and a certain time after taking the glucose, the blood glucose level is 200 mg/dL or more, the person is diagnosed with diabetes (Non-Patent Document 7). Thus, the OGTT is a tool for diagnosing diabetes, and a compound that lowers blood glucose levels in subjects in an OGTT glucose challenge test is considered effective against diabetes.

Non-patent documents

[Non-Patent Document 1] Am J Physiol Gastrointest Liver Physiol. 2002; 282(2):G241-8

[Non-Patent Document 2] Nature. 1991; 350(6316):354-6

[Non-Patent Document 3] J Mol Cell Cardiol. 2010; 49(4):683-92

[Non-Patent Document 4] Cardiovasc Res. 2009; 84(1):111-8

[Non-Patent Document 5] Cancer Cell. 2008, 13: 385-93

[Non-Patent Document 6] Pharmacol Ther. 2009, 121: 29-40

[Non-Patent Document 7] Treatment Guide for Diabetes 2016-2017

Brief description of the invention

A compound with a methyl lactam ring is described that has SGLT1 inhibitory activity and can be used as a drug, or a pharmaceutically acceptable salt thereof; a pharmaceutical composition containing it; and its application in pharmaceuticals.

After extensive research, the inventors of the present invention discovered a specific compound with a methyl lactam ring, resulting in the present invention.

In one embodiment, a compound having formula [I], or a pharmaceutically acceptable salt thereof, is described, as well as its use in pharmaceuticals.

The compound having formula [I] or a pharmaceutically acceptable salt thereof has SGLT1 inhibitory activity and therefore can be used for the treatment and/or prevention of various diseases or pathological conditions that are expected to improve by regulating SGLT1 activity. The compound having formula [I] or a pharmaceutically acceptable salt thereof may also be used for the treatment and/or prevention of various diseases or pathological conditions that may be caused by elevated blood glucose levels.

Brief description of drawings

In fig. 1 shows that the compound of Example 1 (hereinafter referred to as “compound 1”) significantly reduces blood glucose levels in SD rats with a glucose load in the OGTT, compared to rats receiving vehicle alone.

In fig. Figure 2 shows that of all the compounds tested, only compound 1 significantly reduced blood glucose levels in SD rats loaded with glucose in the OGTT, compared to rats receiving vehicle alone.

Description of Embodiments

The present invention includes the embodiments illustrated below.

Clause 1. Compound having formula [I]:

or a pharmaceutically acceptable salt thereof.

Claim 2. A pharmaceutical composition containing the compound of Claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Claim 3. An SGLT1 inhibitor containing the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

Claim 4. A therapeutic or prophylactic agent for diabetes containing the compound of Claim 1 or a pharmaceutically acceptable salt thereof.

Claim 5. The therapeutic or prophylactic agent of Claim 4, wherein the diabetes is type 2 diabetes.

Claim 6. A method of inhibiting SGLT1, comprising administering a therapeutically effective amount of a compound of Claim 1 or a pharmaceutically acceptable salt thereof to a mammal.

Claim 7. A method of treating or preventing diabetes, comprising administering a therapeutically effective amount of a compound of Claim 1 or a pharmaceutically acceptable salt thereof to a mammal.

Claim 8. The method of Claim 7, wherein the diabetes is type 2 diabetes.

Claim 9. Use of a compound of Claim 1 or a pharmaceutically acceptable salt thereof for the production of an SGLT1 inhibitor.

Claim 10. Use of the compound of Claim 1 or a pharmaceutically acceptable salt thereof for the production of a therapeutic or prophylactic agent against diabetes.

Clause 11. Use as per Clause 10, wherein the diabetes is type 2 diabetes.

Claim 12. The compound of Claim 1 or a pharmaceutically acceptable salt thereof for use in inhibiting SGLT1.

Claim 13. The compound of Claim 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of diabetes.

Claim 14. The compound of Claim 13 or a pharmaceutically acceptable salt thereof, wherein the diabetes is type 2 diabetes.

Item 15. A commercial package containing the composition of Item 2 and written instructions that the composition can or should be used for the treatment and/or prevention of diabetes.

Clause 16. A kit containing the composition of Clause 2 and written instructions that the composition can or should be used for the treatment and/or prevention of diabetes.

The term “pharmaceutically acceptable salt” includes any salts known in the art that are not associated with excessive toxicity. Such a pharmaceutically acceptable salt includes, but is not limited to, inorganic acid salts, organic acid salts, inorganic base salts, and organic base salts. Various forms of pharmaceutically acceptable salts are well known in the art and are described, for example, in the following sources:

(a) Berge et al., J. Pharm. Sci., 66, p1-19 (1977),

(b) Stahl et al., “Handbook of Pharmaceutical Salt: Properties, Selection, and Use” (Wiley-VCH, Weinheim, Germany, 2002),

(c) Paulekuhn et al., J. Med. Chem., 50, p6665-6672 (2007).

The compound having formula [I] can be reacted with an inorganic acid, an organic acid, an inorganic base or an organic base according to known techniques to obtain its corresponding pharmaceutically acceptable salt.

Such inorganic acid salt includes salt with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid and sulfuric acid. Such a salt preferably includes a salt with hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid and hydrobromic acid.

Such organic acid salt includes salt with acetic acid, adipic acid, alginic acid, 4-aminosalicylic acid, dehydromethylene citric acid, benzoic acid, benzenesulfonic acid, calcium edetate, camphoric acid, camphor-10-sulfonic acid, carbonic acid, citric acid, edetic acid , ethane-1.2-disulfonic acid, dodecyl sulfuric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glucuronic acid, glucoheptonic acid, glycollylarsanilic acid, hexylresorcinic acid, hydroxynaphthoic acid, 2-hydroxy-1-ethanesulfonic acid, lactic acid, lacto bionic acid, malic acid, maleic acid, mandelic acid, methanesulfonic acid, methylsulfuric acid, methylnitric acid, methylenebis(salicylic acid), galactaric acid, naphthalene-2-sulfonic acid, 2-naphthoic acid, 1,5-naphthalene disulfonic acid, oleic acid, oxalic acid, pamoic acid acid, pantotenic acid, pectic acid, picrinic acid, propionic acid, polygalaxic acid, salicylic acid, stearic acid, amber acid, gall-dubious acid, vigoric acid, texlinic acid, thythocyanic acid, triftoomasic acid, porelululsuluscous acid, and uniform acidic acid. , aspartamic acid and glutamic acid. Such a salt is preferably a salt with oxalic acid, maleic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid, trifluoroacetic acid, benzoic acid, glucuronic acid, oleic acid, pamoic acid, methanesulfonic acid , benzenesulfonic acid, p-toluenesulfonic acid and 2-hydroxy-1-ethanesulfonic acid.

Such inorganic base salt includes lithium, sodium, potassium, magnesium, calcium, barium, aluminum, zinc, bismuth and ammonium salt. Such a salt preferably includes a sodium, potassium, calcium, magnesium and zinc salt.

Such organic base salt includes those with arecoline, betaine, choline, clemizole, ethylenediamine, N-methylglucamine, N-benzylphenethylamine, tris(hydroxymethyl)methylamine, arginine and lysine. Such a salt preferably includes a salt with tris(hydroxymethyl)methylamine, N-methylglucamine and lysine.

The compound having formula [I] or a pharmaceutically acceptable salt thereof may exist in the form of a solvate. The term “solvate” means a compound in which a solvent molecule is coordinated to a compound having formula [I], or a pharmaceutically acceptable salt thereof, and includes a hydrate. The solvate is preferably a pharmaceutically acceptable solvate; and includes, for example, the hydrate, ethanolate and dimethyl sulfoxide solvate of the compound having formula [I], or a pharmaceutically acceptable salt thereof. Such solvate particularly includes the hemihydrate, monohydrate, dihydrate and monoethanolate of the compound having formula [I]; and the sodium salt monohydrate of the compound having formula [I], and the 2/3 ethanolate of its dihydrochloride salt. These solvates can be prepared by any known methods.

A compound having formula [I] may be labeled with an isotope such as ² H, ³ H, ¹⁴ C and ³⁵ S.

The compound having formula [I] or a pharmaceutically acceptable salt thereof is preferably a substantially purified compound having formula [I] or a pharmaceutically acceptable salt thereof, and more preferably the compound having formula [I] or a pharmaceutically acceptable salt thereof , having a purity of 80% or higher.

The compound having formula [I], or a pharmaceutically acceptable salt thereof, has SGLT1 inhibitory activity, and therefore can be used for the treatment and/or prevention of various diseases or pathological conditions that are expected to improve by regulating SGLT1 activity, such as diabetes (e.g., diabetes type 1 and type 2 diabetes), obesity, complications of diabetes (eg, retinopathy, nephropathy, and neuropathy, which are collectively known as microangiopathy; and cerebrovascular disease, coronary artery disease, and arteriosclerosis obliterans of the lower extremities, which are collectively known as macroangiopathy), hypertrophic cardiomyopathy, coronary heart disease, cancer and constipation.

The term “SGLT1 inhibition” means that the function of SGLT1 is inhibited until its activity disappears or decreases; and, for example, it means that the function of SGLT1 is inhibited according to the results of Test Example 1 described below. The term “SGLT1 inhibition” preferably means “human SGLT1 inhibition”. Inhibition of function or disappearance or reduction of activity is preferably performed when clinically indicated in humans.

The term “SGLT1 inhibitor” means any substance that inhibits SGLT1 and includes small molecules, nucleic acids, polypeptides, proteins, antibodies and vaccines. The term “SGLT1 inhibitor” preferably means “human SGLT1 inhibitor”.

The compound having formula [I] or a pharmaceutically acceptable salt thereof may also be useful for the treatment and/or prevention of various diseases or conditions that may be caused by elevated blood glucose levels.

The term “various diseases or conditions that may be caused by elevated blood glucose levels” includes, for example, diabetes (eg, type 1 diabetes and type 2 diabetes), obesity, and complications of diabetes (eg, retinopathy, nephropathy and neuropathy, which are collectively known as microangiopathy; and cerebrovascular disease, coronary heart disease and arteriosclerosis obliterans of the lower extremities, which are collectively known as macroangiopathy).

The term “treatment” includes alleviating the condition, preventing worsening, maintaining remission, preventing exacerbation, and preventing relapse.

The term “prevention” or “prophylaxis” includes delaying the onset of pathological conditions. For example, “preventing diabetes” includes slowing the onset of type 2 diabetes when glucose intolerance is impaired.

The pharmaceutical composition of the present invention can be prepared from a therapeutically effective amount of a compound having formula [I], or a pharmaceutically acceptable salt thereof, and at least one or more pharmaceutically acceptable carriers, optionally followed by mixing, according to known techniques in the field of medicinal products. The amount of a compound having formula [I] or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition varies depending on factors such as the type of dosage form and the dosage size, and is in the range, for example, from 0.1 to 100 wt. % of the total amount of the composition.

The dosage form, which can be formulated based on the compound having formula [I], or a pharmaceutically acceptable salt thereof, includes oral preparations such as tablets, capsules, granules, powders, cast tablets, syrups, emulsions and suspensions; and parenteral medications such as topical medications, suppositories, injections, eye drops, nasal medications, and pulmonary medications.

The term “pharmaceutically acceptable carrier” includes various organic or inorganic substances that are commonly used as a component of the formulation. Such substances include, for example, excipients, disintegrants, binders, flow improvers, and lubricants for solid preparations; solvents, solubilizers, suspending agents, tonicity agents, buffering agents, and soothing agents for liquid preparations; as well as bases, emulsifiers, wetting agents, stabilizers, dispersants, plasticizers, pH adjusters, absorption stimulants, gelling agents, antiseptic agents, fillers, solubilizers, and suspending agents for semi-solid preparations. If necessary, additional additives such as preservatives, antioxidants, colors and sweeteners can be added.

Such “excipient” includes, for example, lactose, white soft sugar, D-mannitol, D-sorbitol, corn starch, dextrin, microcrystalline cellulose, crystalline cellulose, carmellose, carmellose calcium, sodium carboxymethyl starch, low-substituted hydroxypropylcellulose and gum arabic.

Such “disintegrant” includes, for example, carmellose, calcium carmellose, sodium carmellose, sodium carboxymethyl starch, croscarmellose sodium, crospovidone, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose and crystalline cellulose.

Such “binder” includes, for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone, crystalline cellulose, white soft sugar, dextrin, starch, gelatin, sodium carmellose and acacia.

Such “flow improver” includes, for example, light anhydrous silica gel and magnesium stearate.

Such “lubricant” includes, for example, magnesium stearate, calcium stearate and talc.

Such “solvent” includes, for example, purified water, ethanol, propylene glycol, macrogol, sesame oil, corn oil and olive oil.

Such “solubilizer” includes, for example, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate and sodium citrate.

Such “suspending agent” includes, for example, benzalkonium chloride, carmellose, hydroxypropylcellulose, propylene glycol, povidone, methylcellulose and glycerol monostearate.

Such “tonicity agent” includes, for example, glucose, D-sorbitol, sodium chloride and D-mannitol.

Such “buffer material” includes, for example, disodium hydrogen phosphate, sodium acetate, sodium carbonate and sodium citrate.

Such a “sedative” includes, for example, benzyl alcohol.

Such “base” includes, for example, water, animal or vegetable oils such as olive oil, corn oil, peanut oil, sesame oil and castor oil, lower alcohols such as ethanol, propanol, propylene glycol, 1,3-butylene glycol and phenol, higher fatty acids and their esters, waxes, higher alcohols, polyhydric alcohols, hydrocarbons such as white petrolatum, liquid paraffin and paraffin, hydrophilic petrolatum, purified lanolin, absorbent ointment, hydrated lanolin, hydrophilic ointment, starch, pullulan, acacia , gum tragacanth, gelatin, dextran, cellulose derivatives such as methylcellulose, carboxymethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose, synthetic polymers such as carboxyvinyl polymer, sodium polyacrylate, polyvinyl alcohol and polyvinylpyrrolidone, propylene glycol, macrogol such as Macrogol 200 to 600, and a combination of two or more of the above.

Such a “preservative” includes, for example, ethyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, sodium dehydroacetate and sorbic acid.

Such “antioxidant” includes, for example, sodium sulfite and ascorbic acid.

Such “color” includes, for example, food colors (eg, Food Red No. 2 or No. 3, Food Yellow No. 4 or No. 5) and β-carotene.

Such “sweetener” includes, for example, sodium saccharin, dipotassium glycyrrhizinate and aspartame.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, topically, rectally, intravenously, intramuscularly and subcutaneously) to humans, as well as non-human mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, cows, horses, sheep and monkeys. Dosage varies depending on the subject being administered, the diseases, pathological conditions, dosage forms and route of administration. For example, a daily dose for oral administration to an adult patient will typically range from about 0.01 to about 1 g of active ingredient, i.e. a compound having the formula [I]. This daily dose can be administered at one time or in several doses.

A kit such as an administration, treatment and/or prophylactic kit, a package such as packaged articles, and a complex and/or drug box that includes a pharmaceutical composition containing a compound of formula [I] or its a pharmaceutically acceptable salt as the active substance or active agent, and written material about the composition indicating that the composition can or should be used for treatment and/or prophylaxis. Such a kit, package and drug complex may contain one or more containers filled with a pharmaceutical composition or one or more active substances and other drugs (or ingredients) used in the composition. Examples of such kits, packaging and drug complexes include commercial kits, commercial packaging and commercial medical complexes for appropriate use in the treatment and/or prevention of certain diseases. The written material contained in such kit, package or unit includes a warning or insert in a form prescribed by the governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, stating the governmental agency's approval to manufacture, use or sell the products for administration to humans . Such a kit, package or medical assembly may include packaged products, as well as devices for corresponding administration operations, designed to provide the most preferred medical treatment and/or prophylaxis, including the treatment and/or prophylaxis of certain diseases.

A method for preparing a compound having formula [I] or a pharmaceutically acceptable salt thereof is illustrated below. However, the method for producing the compound having formula [I] or a pharmaceutically acceptable salt thereof is not limited to only those described below.

Each compound obtained in each step can optionally be isolated and/or purified according to any of the known methods such as distillation, recrystallization and column chromatography, or, optionally, the subsequent step can be carried out without isolation and/or purification.

As used herein, the term “room temperature” means a temperature that is not under control and includes, in one embodiment, a temperature between 1°C and 40°C.

Method of obtaining A

The compound having formula [I] can be obtained according to Preparation Method A1 or A2, as shown in the diagram below.

Method of obtaining A1

(Stage A1-1)

A compound having formula [I] can be prepared by reacting a compound having formula [1] or a salt thereof with a compound having formula [2] or a salt thereof in the presence of a condensing agent and an additive in a solvent.

The condensing agent used in this case includes, for example, dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), diisopropylcarbodiimide, 1,1'-carbonyldiimidazole (CDI), O-(7 -azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate (COMU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate ( PyBOP), diphenylphosphoryl azide and propylphosphonic anhydride.

The additive used in this case includes, for example, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), 4-dimethylaminopyridine and 1-methylimidazole.

The solvent used herein includes, for example, halogenated hydrocarbon solvents such as chloroform; ether solvents such as tetrahydrofuran; polar solvents such as pyridine, acetonitrile, N,N-dimethylformamide; and a mixture of any of the above solvents.

The reaction temperature in such a case is in the range, for example, from 0°C to 100°C.

When a salt of a compound having the formula [1] is used, the reaction can be carried out in the presence of a base. Such a base includes, for example, organic bases such as triethylamine and alkali metal salts such as sodium carbonate.

The compound having formula [I] can also be prepared by converting the compound having formula [2] with a halogenating agent into the corresponding carboxylic acid halide in a solvent, and then reacting with the compound having formula [1] in the presence of a base.

The halogenating agent used in this reaction includes, for example, oxalyl chloride and thionyl chloride. The preferred halogenating agent is oxalyl chloride.

The base used in this reaction includes, for example, organic bases such as pyridine, triethylamine and N,N-diisopropylethylamine; and alkali metal salts such as sodium bicarbonate and sodium carbonate. The preferred base is pyridine.

The solvent used herein includes, for example, halogenated hydrocarbon solvents such as chloroform; ether solvents such as cyclopentyl methyl ether and tetrahydrofuran; hydrocarbon solvents such as toluene; and a mixture of any of the above solvents with water. The preferred solvent is chloroform.

The reaction temperature in such a case is in the range, for example, from 0°C to 80°C, preferably from 0°C to 60°C.

When preparing carboxylic acid halide, N,N-dimethylformamide can be added as an additive.

Method for obtaining A2

In this diagram, P ^N1 represents a protecting group for the amino group. P ^N1 is preferably a 2,4-dimethoxybenzyl group.

Stage A2-1

The compound having the formula [1] or a salt thereof and the compound having the formula [3] or a salt thereof can be reacted according to Preparation Method A1 Step A1-1 to obtain a compound having the formula [4].

Stage A2-2

The compound having the formula [I] or a salt thereof can be obtained by removing P ^N1 from the compound having the formula [4] in a deprotection reaction. The deprotection reaction can be carried out under suitable conditions depending on P ^N1 .

For example, when P ^N1 represents 2,4-dimethoxybenzyl, the compound having formula [I] or a salt thereof can be prepared by reaction with an acid in the presence of an additive in a solvent.

The acid used in this case includes, for example, methanesulfonic acid, p-toluenesulfonic acid and trifluoroacetic acid. The preferred acid is trifluoroacetic acid.

The additive used in this case includes, for example, anisole and triethylsilane. The preferred additive is anisole.

The solvent used herein includes, for example, halogenated hydrocarbon solvents such as dichloromethane, hydrocarbon solvents such as toluene, water, and a mixture of any of the foregoing. An organic acid such as trifluoroacetic acid can also be used as a solvent.

The reaction temperature in such a case is in the range, for example, from 0°C to 130°C, preferably from 25°C to 80°C.

When an acid is used in this step, a compound having the formula [5] is obtained:

or its salt. The compound having formula [I] or a salt thereof can be prepared by converting the hydroxyl group in the compound having formula [5] or a salt thereof into a tert-butoxy group according to any known method.

For example, a compound having formula [I] or a salt thereof can be prepared by reacting a compound having formula [5] or a salt thereof with di-tert-butyl dicarbonate in the presence of magnesium perchlorate.

The solvent used herein includes, for example, halogenated hydrocarbon solvents such as chloroform and ether solvents such as tetrahydrofuran. The preferred solvent is chloroform.

The reaction temperature in such a case is in the range, for example, from 0°C to 100°C, preferably from room temperature to 70°C.

Method of obtaining B

The compound having the formula [1] can be obtained according to Preparation Method B1 as shown in the diagram below.

Method for obtaining B1

In this diagram, L ¹ represents the leaving group. L ¹ is preferably chlorine, bromine or iodine. P ^N2 each independently represents an amine protecting group. The two P ^N2 groups are preferably combined with the nitrogen atom to which they are attached to form 2,5-dimethylpyrrole.

Stage B1-1

The compound having the formula [7] or a salt thereof can be prepared by introducing P ^N2 into the amino group in the compound having the formula [6] or a salt thereof according to any of the known methods. The introduction of the protecting group can be carried out under suitable conditions depending on P ^N2 . For example, when two P ^N2 groups are combined with the nitrogen atom to which they are attached to form 2,5-dimethylpyrrole, a compound having formula [7] can be prepared by reacting a compound having formula [6] with 2,5-hexanedione in a solvent under acidic conditions.

The acid used in this case includes, for example, concentrated hydrochloric acid, concentrated sulfuric acid, amidosulfuric acid, p-toluenesulfacid and acetic acid. The preferred acid is acetic acid.

The solvent used herein includes, for example, alcohol solvents such as ethanol, ether solvents such as tetrahydrofuran, hydrocarbon solvents such as toluene, polar solvents such as N,N-dimethylformamide, halogenated hydrocarbon solvents such as dichloroethane, and a mixture of any of the listed solvents. An organic acid such as acetic acid can also be used as a solvent.

The reaction temperature in such a case is in the range, for example, from room temperature to 150°C, preferably from 80°C to 140°C.

Stage B1-2

The compound having the formula [8] can be prepared, for example, by a method comprising:

Step (a): reacting a compound having the formula [7] with dibromodifluoromethane in the presence of a base and a catalyst in a solvent, and

Step (b): fluorination of the resulting product in the presence of tetramethylammonium fluoride or silver (I) tetrafluoroborate in a solvent.

The base used in Step (a) includes, for example, sodium hydride and potassium tert-butoxide. The preferred base is sodium hydride.

The catalyst used in Step (a) includes, for example, tetrabutylammonium bromide and zinc. The preferred catalyst is tetrabutylammonium bromide.

The solvent used in Step (a) includes, for example, ether solvents such as tetrahydrofuran and polar solvents such as N,N-dimethylformamide. The preferred solvent is N,N-dimethylformamide.

The reaction temperature in Step (a) is in the range, for example, from 0°C to 40°C, preferably from 0°C to room temperature.

When tetramethylammonium fluoride is used in Step (b), the solvent used includes, for example, ether solvents such as 1,4-dioxane and polar solvents such as sulfolane. The preferred solvent is sulfolane. When silver(I) tetrafluoroborate is used in Step (b), the solvent used therein includes, for example, halogenated hydrocarbon solvents such as dichloromethane. The preferred solvent is dichloromethane.

When tetramethylammonium fluoride is used in Step (b), the reaction temperature is then in the range of, for example, 80°C to 180°C, preferably 100°C to 140°C. When silver(I) tetrafluoroborate is used in Step (b), the reaction temperature is then in the range of, for example, -78°C to 50°C, preferably -78°C to room temperature.

Stage B1-3

A compound having formula [9] can be prepared by introducing L ¹ into a compound having formula [8] in the presence of a base in a solvent. For example, when L ¹ represents iodine, a compound having formula [9] can be prepared by iodinating a compound having formula [8] in the presence of a base in a solvent.

The base used herein includes, for example, n-butyllithium, lithium diisopropylamide, lithium hexamethyldisilazide and lithium tetramethylpiperidide. The preferred base is n-butyllithium.

The iodinating agent used in this case includes, for example, iodine, iodine monochloride, N-iodosuccinimide and 1-chloro-2-iodoethane. The preferred iodinating agent is iodine.

The solvent used herein includes, for example, ether solvents such as tetrahydrofuran, hydrocarbon solvents such as toluene, and a mixture of any of the foregoing. The preferred solvent is tetrahydrofuran.

The reaction temperature in such a case is in the range, for example, from -100°C to 40°C, preferably from -78°C to 20°C.

Stage B1-4

The compound having the formula [10] or a salt thereof can be prepared by removing P ^N2 from the compound having the formula [9] in a deprotection reaction. The deprotection reaction can be carried out under suitable conditions depending on P ^N2 . For example, when two P ^N2 are combined with the nitrogen atom to which they are attached to form 2,5-dimethylpyrrole, the compound having formula [10] or a salt thereof can be prepared by reacting the compound having formula [9] with hydroxylamine in a solvent .

The solvent used herein includes, for example, alcohol solvents such as ethanol, water, and a mixture of any of the above. The preferred solvent is a mixture of alcohol solvents and water.

The reaction temperature in such a case is in the range, for example, from 40°C to 150°C, preferably from 80°C to 130°C.

Hydroxylamine hydrochloride can be used instead of hydroxylamine. In this case, the reaction can be carried out in the presence of a base. The base used herein includes, for example, organic bases such as triethylamine and alkali metal salts such as sodium carbonate. The preferred base is triethylamine.

Stage B1-5

A compound having formula [1] or a salt thereof can be prepared by a Suzuki reaction between a compound having formula [10] or a salt thereof and a compound having formula [11]. For example, a compound having formula [1] or a salt thereof can be prepared by reacting a compound having formula [10] or a salt thereof with a compound having formula [11] in the presence of a base and a palladium catalyst in a solvent.

The palladium catalyst used in this reaction includes, for example, tetrakis(triphenylphosphine)palladium, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-dichloromethane adduct, [1,1'-bis(di-tert- butylphosphino)ferrocene]dichloropalladium(II) and a mixture of palladium(II) acetate and tricyclohexylphosphine, 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl or 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl. The preferred palladium catalyst is the [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-dichloromethane adduct.

The base used in this reaction includes, for example, tripotassium phosphate, cesium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium carbonate and triethylamine. The preferred base is tripotassium phosphate, cesium carbonate or sodium carbonate.

The solvent herein includes, for example, ether solvents such as 1,4-dioxane, tetrahydrofuran, diethyl ether and 1,2-dimethoxyethane; alcohol solvents such as methanol, ethanol, 1-propanol and 2-propanol; hydrocarbon solvents such as toluene, n-hexane and xylene; polar solvents such as N,N-dimethylformamide, dimethyl sulfoxide and acetonitrile; and a mixture of any of the above solvents with water. The preferred solvent is 1,2-dimethoxyethane, toluene, dimethyl sulfoxide, or a mixture of any of these solvents with water.

The reaction temperature in such a case is in the range, for example, from 20°C to 150°C, preferably from 80°C to 130°C.

The compound having the formula [11] can be obtained according to any of the known methods. The corresponding boronic acid ester can be used instead of the compound having formula [11] in the reaction in step B1-5. For example, such a boronic acid ester can be produced according to Production Method B2, as shown in the diagram below.

Method for obtaining B2

In this diagram, R ¹ represents fluorine or a hydroxyl group. L ² represents a leaving group. L ² preferably represents chlorine, bromine, iodo, p-toluenesulfonyloxy, methanesulfonyloxy or trifluoromethanesulfonyloxy group. B(OR ² ) ₂ is a boronic acid ester. R ² is each independently, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl, or alternatively, OR ² can be combined with the boron atom to which they are attached, with formation of a cyclic boronic acid ester. B(OR ² ) ₂ is preferably pinacol boronic acid ester.

Stage B2-1

A compound having formula [13] can be prepared by converting R ¹ to a tert-butoxy group in a compound having formula [12]. The reaction can be carried out according to any of the known methods.

When R ¹ represents fluorine, the compound having the formula [13] can be prepared, for example, by reacting the compound having the formula [12] with sodium tert-butoxide or potassium tert-butoxide in a solvent. The solvent used herein includes, for example, ether solvents such as tetrahydrofuran; and polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide. The preferred solvent is N,N-dimethylformamide. The reaction temperature in such a case is in the range, for example, from 0°C to 100°C, preferably from room temperature to 85°C.

When R ¹ represents a hydroxyl group, the compound having the formula [13] can be obtained, for example, by the method for preparing the compound having the formula [I] or a salt thereof from the compound having the formula [5] or a salt thereof described in the Method receiving A2 Stage A2-2.

Stage B2-2

The compound having the formula [14] can be prepared by reacting the compound having the formula [13] with a boron-containing compound in the presence of a palladium catalyst, an organophosphorus compound and a base in a solvent.

The palladium catalyst herein includes, for example, palladium(II) acetate, palladium(II) chloride and tris(dibenzylideneacetone)dipalladium(0).

The organophosphorus compound in this case includes, for example, triphenylphosphine, tricyclohexylphosphine, 1,1'-bis(diphenylphosphino)ferrocene, 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl and 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl.

Instead of a palladium catalyst and an organophosphorus compound, tetrakis(triphenylphosphine)palladium, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-dichloromethane or [1,1'-bis(di-tert-butylphosphino)ferrocene] adduct can be used. dichloropalladium(II).

The base in this case includes, for example, potassium acetate, sodium carbonate, cesium carbonate and potassium carbonate. The preferred base is potassium acetate.

The boron-containing compound in this case includes, for example, bis(pinacolato)diborone.

The solvent herein includes, for example, ether solvents such as 1,4-dioxane, tetrahydrofuran and 1,2-dimethoxyethane; hydrocarbon solvents such as toluene; and polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide. The preferred solvent is dimethyl sulfoxide.

The reaction temperature in such a case is in the range, for example, from room temperature to 150°C, preferably from 70°C to 110°C.

Method for obtaining C

The compound having the formula [2] or a salt thereof and the compound having the formula [3] or a salt thereof can be produced according to Preparation Method C1, as shown in the diagram below.

Method for obtaining C1

In this diagram, P ^C1 and P ^C2 each independently represent a protecting group for a carboxy group. Preferably, P ^C1 and P ^C2 are each independently methyl, ethyl, tert-butyl or benzyl. R ³ is each independently methoxy or ethoxy. L ³ represents a leaving group. L ³ is preferably bromine or chlorine. The remaining characters have the same meanings as above.

Stage C1-1

A compound having formula [17] can be prepared by reacting a compound having formula [15] with a compound having formula [16] in the presence of a base in a solvent.

The base used in this reaction includes, for example, potassium tert-butoxide, sodium methoxide, sodium ethoxide, lithium diisopropylamide, potassium hexamethyldisilazane, potassium carbonate, cesium carbonate and sodium hydride. The preferred base is potassium tert-butoxide.

The solvent herein includes, for example, ether solvents such as tetrahydrofuran; alcohol solvents such as methanol and ethanol; polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide. The preferred solvent is tetrahydrofuran.

The reaction temperature in such a case is in the range, for example, from -78°C to 100°C, preferably from 0°C to 70°C.

Stage C1-2

The compound having the formula [18] can be prepared by reacting the compound having the formula [17] with formaldehyde (preferably a formaldehyde solution) in the presence of a base in a solvent.

The base used in this reaction includes, for example, potassium tert-butoxide, sodium methoxide, sodium ethoxide, lithium diisopropylamide, potassium hexamethyldisilazane, potassium carbonate, cesium carbonate and sodium hydride. The preferred base is potassium carbonate.

The solvent herein includes, for example, ether solvents such as tetrahydrofuran; alcohol solvents such as methanol and ethanol; and polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide. The preferred solvent is tetrahydrofuran.

The reaction temperature in such a case is in the range, for example, from -78°C to 100°C, preferably from 0°C to 70°C.

Stage C1-3

A compound having formula [20] can be prepared by reacting a compound having formula [18] with a compound having formula [19] in a solvent.

The solvent herein includes, for example, hydrocarbon solvents such as toluene; alcohol solvents such as methanol and ethanol; and a mixture of any of the above solvents. The preferred solvent is toluene.

The reaction temperature in such a case is in the range, for example, from 20°C to 150°C, preferably from 80°C to 130°C.

Stage C1-4

The compound having the formula [21] or a salt thereof can be prepared by removing ^PCl from the compound having the formula [20] in a deprotection reaction. The deprotection reaction can be carried out under suitable conditions depending on P ^C1 . For example, when P ^Cl represents ethyl, the compound having the formula [21] or a salt thereof can be obtained by hydrolyzing the compound having the formula [20] in the presence of a base in a solvent.

The base used in this reaction includes, for example, lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium ethoxide. The preferred base is sodium ethoxide.

The solvent herein includes, for example, alcohol solvents such as ethanol, ether solvents such as tetrahydrofuran, water, and a mixture of any of the foregoing. The preferred solvent is a mixture of ethanol and water.

The reaction temperature in such a case is in the range, for example, from 0°C to 100°C, preferably from 0°C to 40°C.

Stage C1-5

The compound having the formula [3] or a salt thereof can be obtained by isolating from the compound having the formula [21] or a salt thereof. Isolation of a compound having formula [3] or a salt thereof can be carried out under conditions suitable for separation according to methods well known in the art. For example, a compound having formula [3] or a salt thereof can be prepared by isolating its diastereomeric salt with a basic optically active reagent, followed by treating the salt with an acid.

The main optically active reagent in this case includes, for example, (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol.

The solvent used in preparing the diastereomeric salt includes, for example, alcohol solvents such as 2-propanol, ether solvents such as 1,2-dimethoxyethane, polar solvents such as acetonitrile, and a mixture of any of these solvents with water. The preferred solvent is acetonitrile, 1,2-dimethoxyethane, or a mixture of any of these solvents with water.

The optical purity of the diastereomeric salt can be improved by recrystallization. The solvent used in the recrystallization includes, for example, ether solvents such as 1,2-dimethoxyethane, polar solvents such as acetonitrile, and a mixture of any of these solvents with water. The preferred solvent is a mixture of acetonitrile and water.

The acid used to treat the diastereomeric salt includes, for example, hydrochloric acid, sulfuric acid and potassium hydrogen sulfate. The preferred acid is hydrochloric acid.

The solvent used in treating the diastereomeric salt includes, for example, ester solvents such as ethyl acetate, ether solvents such as tetrahydrofuran, water, and a mixture of any of the foregoing. The preferred solvent is a mixture of ethyl acetate and water.

Stage C1-6

The compound having the formula [2] or a salt thereof can be prepared by removing P ^N1 from the compound having the formula [3] or a salt thereof in a deprotection reaction. The deprotection reaction can be carried out under suitable conditions depending on P ^N1 . For example, when P ^N1 is 2,4-dimethoxybenzyl, the compound having the formula [2] or a salt thereof can be produced according to Production Method A2 Step A2-2.

Examples

The present invention is illustrated in more detail using Syntheses, Examples, Comparative Examples, Test Examples and Preparation Examples, which are given below, but do not limit the scope of the present invention.

The abbreviations used in this text have the following meanings:

DMF: N,N-dimethylformamide

DMSO: dimethyl sulfoxide

THF: tetrahydrofuran

CPME: cyclopentyl methyl ether

¹ H-NMR spectra were recorded in CDCl ₃ or DMSO-d ₆ with tetramethylsilane as internal standard, and all δ values are given in ppm. Spectra were recorded on an NMR spectrometer with an operating frequency of 400 MHz, unless otherwise stated.

The symbols used in the Examples have the following meanings:

s: singlet

d: doublet

t: triplet

kv: quartet

dd: doublet of doublets

ddd: doublet of doublets

wide with: wide singlet

m: multiplet

J: spin-spin coupling constant.

Synthesis 1. Preparation of 2-(3-(tert-butoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

Stage 1. Preparation of 1-bromo-3-(tert-butoxy)-5-fluorobenzene

Di-tert-butyl dicarbonate (1.14 g) and magnesium perchlorate (58 mg) were successively added to 3-bromo-5-fluorophenol (500 mg) at room temperature under a flow of argon. The reaction mixture was stirred at 50°C for 1 hour 20 minutes. Di-tert-butyl dicarbonate was added to the reaction mixture at 50°C. The reaction mixture was stirred at 50°C for 1 hour and further stirred at 65°C for 1 hour, and then cooled to room temperature. Di-tert-butyl dicarbonate was added to the reaction mixture at room temperature. The reaction mixture was stirred at 65°C for 3 hours. The reaction mixture was cooled to room temperature and a mixture of n-hexane/ethyl acetate (1/1) was added. The reaction mixture was washed successively with 3N. hydrochloric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, then dried over sodium sulfate and evaporated. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 1/0 to 20/1) to give the title compound (437 mg) in 68% yield.

¹ H-NMR (CDCl ₃ ) δ: 1.35 (s, 9H), 6.62-6.66 (m, 1H), 6.92-6.98 (m, 2H).

Step 2. Preparation of 2-(3-(tert-butoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

To a solution of 1-bromo-3-(tert-butoxy)-5-fluorobenzene (437 mg) obtained in Step 1 in DMSO (5 ml), potassium acetate (434 mg), bis(pinacolato)diborone (898 mg) were added sequentially ) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-dichloromethane adduct (144 mg) under argon at room temperature. The reaction mixture was stirred at 90°C for 2.5 hours. The reaction mixture was cooled to room temperature. A mixture of n-hexane/ethyl acetate (1/1) and water were sequentially added to the reaction mixture. The mixture was stirred at room temperature for 50 minutes and left overnight. A mixture of n-hexane/ethyl acetate (1/1), water, silica gel and celite were successively added to the mixture. The reaction mixture was stirred, then the undissolved particles were filtered off and washed with a mixture of n-hexane/ethyl acetate (1/1). The filtrate was extracted with a mixture of n-hexane/ethyl acetate (1/1). The organic layer was washed successively with water twice and with a saturated aqueous sodium chloride solution, dried over sodium sulfate and evaporated. The residue was purified by silica gel thin layer chromatography (eluent: n-hexane/ethyl acetate = 10/1) to give the title compound (443 mg) in 85% yield.

¹ H-NMR (CDCl ₃ ) δ: 1.33 (s, 12H), 1.36 (s, 9H), 6.77-6.82 (m, 1H), 7.18-7.23 (m , 2H).

Synthesis 2. Preparation of (3R,4R)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid

Stage 1. Preparation of diethyl 2-methyl-3-methylene succinate

THF (2.55 L) was added to potassium tert-butoxide (180 g) at room temperature under a stream of nitrogen. Triethyl phosphonoacetate (314 g) was added dropwise to the resulting mixture under ice-cooling for 13 minutes. The addition funnel used was washed with tetrahydrofuran (511 ml) and the washing solutions were added to the reaction mixture. The reaction mixture was stirred for 2 hours and 9 minutes while cooling with ice. Ethyl 2-bromopropionate (247 g) was added dropwise to the mixture over 20 minutes under ice-cooling. The addition funnel used was washed with tetrahydrofuran (79 ml) and the washing solutions were added to the reaction mixture. The reaction mixture was stirred at room temperature for 22 hours 45 minutes. Potassium carbonate (188 g) was added to the reaction mixture over 1 minute while cooling with ice. A 37% aqueous solution of formaldehyde (152 ml) was added dropwise to the reaction mixture over 10 minutes while cooling with ice. The reaction mixture was stirred at room temperature for 19 hours 44 minutes. Water (1.57 L) was added to the reaction mixture at room temperature over 1 minute. The reaction mixture was stirred at room temperature for 1 hour 48 minutes. The reaction mixture was separated. The separated aqueous layer was extracted with tetrahydrofuran (200 ml) twice. The resulting organic layers were combined and evaporated. Toluene (471 ml) and saturated aqueous sodium chloride (471 ml) were added to the residue. The reaction mixture was stirred and separated. The organic layer was dried over sodium sulfate (63 g). Sodium sulfate was filtered off. Separately, the same reaction was carried out with triethyl phosphonoacetate (300 g) to obtain a filtrate, which was then combined with the filtrate obtained above to obtain a solution of the title compound (equivalent to 2.66 mol) in toluene (approximately 921 ml). The resulting solution of the title compound in toluene was considered to be obtained in 100% yield and was used in the next step. The formation of the title compound was confirmed by HPLC analysis.

The following instruments and HPLC analysis conditions were used:

Instrument: HPLC system from Shimadzu Corporation, High-Performance Liquid Chromatograph Prominence

Registration conditions:

Column: Kinetex C18: 2.6 µm, 50 mm x 2.1 mm (Phenomenex)

Column temperature: 40°C

Flow rate: 0.4 ml/min.

Analysis time: 10 min.

Detector wavelength: UV (220 nm)

Mobile phase: (Solution A) water, (Solution B) acetonitrile.

Mobile phase gradient: Maintain the ratio (Solution A/Solution B (vol%)) between Solution A and Solution B 80/20 from 0 minutes to 0.01 minutes after injection, linearly changed from 80/20 to 10/90 from 0 .01 minutes to 7 minutes, maintained 10/90 from 7 minutes to 8 minutes, ramped from 10/90 to 80/20 from 8 minutes to 9 minutes, and maintained 80/20 from 9 minutes to 10 minutes.

The retention time of the title compound was about 3.7 minutes under the HPLC analysis conditions used.

Step 2. Preparation of a mixture of ethyl (cis)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylate and ethyl (trans)-1-(2,4-dimethoxybenzyl)-4-methyl -5-oxopyrrolidine-3-carboxylate

To a solution of diethyl 2-methyl-3-methylene succinate (equivalent to 2.66 mol) obtained in Step 1 in toluene (about 921 ml), 2,4-dimethoxybenzylamine (468 g) was added dropwise over 2 minutes at room temperature in nitrogen flow. The reaction mixture was stirred at 120°C for 5 hours 45 minutes. The reaction mixture was left to stand over the weekend at room temperature. The reaction mixture was cooled with ice to an internal temperature of about 15°C. 2N was added dropwise to the reaction mixture. hydrochloric acid solution (1.33 L), and the resulting mixture was stirred. The reaction mixture was separated. The aqueous layer was extracted with toluene (150 ml). The organic layers were combined, washed with a mixture of saturated aqueous sodium chloride and water (600 ml, saturated aqueous sodium chloride/water = 1/1), dried over sodium sulfate (120 g), evaporated, and dried under reduced pressure at room temperature in overnight to give the crude title compound (790 g; cis/trans = about 1/1, including 5.5 wt.% toluene). The formation of the title compound was confirmed by HPLC analysis.

The following instruments and HPLC analysis conditions were used:

Instrument: HPLC system from Shimadzu Corporation, High-Performance Liquid Chromatograph Prominence

Analysis conditions:

Column: Atlantis T3: 5 µm, 150 mm x 4.6 mm (Waters)

Column temperature: 40°C

Flow rate: 1.15 ml/min.

Analysis time: 18 min.

Detector wavelength: UV (220 nm)

Mobile phase: (Solution A) 10 mM (sodium) phosphate buffer (pH = 2.6), (Solution B) acetonitrile.

Mobile phase gradient: Maintain the ratio (Solution A/Solution B (vol%)) between Solution A and Solution B 60/40 from 0 minutes to 0.5 minutes after injection, linearly changed from 60/40 to 10/90 from 0 .5 minutes to 8 minutes, maintained 10/90 from 8 minutes to 12.5 minutes, linearly changed from 10/90 to 60/40 from 12.5 minutes to 13.5 minutes, and maintained the 60/40 ratio from 13. 5 minutes to 18 minutes.

Retention times were about 6.6 minutes for ethyl (cis)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylate and about 6.9 minutes for ethyl (trans)-1-( 2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylate under the conditions used in HPLC analysis.

Step 3. Preparation of (trans)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid

In a crude mixture (790 g, including 5.5 wt.% toluene) ethyl (cis)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylate and ethyl (trans)-1- (2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylate obtained in Step 2, ethanol (1.15 L) was added at room temperature under a stream of nitrogen. Sodium ethoxide (20 wt% solution in ethanol, 1.15 L) was added dropwise to the reaction mixture at room temperature over 31 minutes. The reaction mixture was stirred at room temperature for 2 hours 57 minutes. The reaction mixture was cooled with ice and water (1.84 L) was added dropwise over 33 minutes. CPME (1.8 L) and toluene (1.8 L) were added to the reaction mixture at room temperature, and the resulting mixture was separated (organic layer 1). CPME (1.8 L) was added to the aqueous layer, and the resulting mixture was separated (organic layer 2). The solvent (1.8 L) was removed from the aqueous layer by evaporation. 6N was added dropwise to the aqueous layer. hydrochloric acid solution (110 ml) while cooling with ice, and then ethyl acetate (1.8 L) was added. 6N was added dropwise to the resulting mixture. hydrochloric acid solution (300 ml) under ice-cooling, and the resulting mixture was stirred for about 10 minutes. Water (2.2 L), 6 N. was successively added to the mixture. hydrochloric acid solution (50 ml), water (1.0 l), 10% sodium hydrogen sulfate solution (300 ml) and ethanol (300 ml) with ice cooling. The mixture was stirred at room temperature overnight. Ethyl acetate (600 ml) was added to the resulting mixture, and the resulting mixture was separated. The aqueous layer was extracted with ethyl acetate (600 ml) twice. The organic layers were combined (except organic layer 1 and organic layer 2) and washed with a mixture of saturated aqueous sodium chloride and water (1 L, saturated aqueous sodium chloride/water = 1/1). Sodium sulfate (120 g) and activated carbon (30 g) were added to the resulting organic layer, and the mixture was stirred at room temperature for 1 hour. The mixture was filtered through celite to remove undissolved particles. Undissolved particles were washed with ethyl acetate (3 L). The filtrates were combined and evaporated, dried under reduced pressure at room temperature for 3 hours to obtain the crude title compound (561 g).

Organic layer 1 and organic layer 2 described above were separately combined and evaporated. Toluene (450 ml) and water (450 ml) were added to the residue, and the resulting mixture was separated. The aqueous layer was washed with toluene (450 ml) twice. Ethyl acetate (450 ml) was added to the aqueous layer. 6N was added dropwise to the resulting mixture. hydrochloric acid solution (70 ml) with ice cooling. Ethyl acetate (300 ml) was added to the resulting mixture, and the mixture was separated. The aqueous layer was extracted with ethyl acetate (150 ml). The organic ethyl acetate layers were combined and washed with a mixture of saturated aqueous sodium chloride and water (225 ml, saturated aqueous sodium chloride/water = 1/1). Sodium sulfate (30 g) and activated carbon (7.5 g) were added to the organic layer, and the mixture was stirred at room temperature for 1 hour. The resulting mixture was filtered to remove undissolved particles. Undissolved particles were washed with ethyl acetate (750 ml). The filtrates were combined and evaporated, and dried under reduced pressure at room temperature for 3 hours to obtain the crude title compound (87.3 g).

This crude product was combined with the crude title compound described above, and then CPME (3 L) was added under a stream of nitrogen. The mixture was stirred at 120°C. The resulting mixture was slowly cooled to room temperature with stirring over 17 hours 34 minutes. The mixture was cooled with ice and stirred at an internal temperature of approximately 1°C for 3 hours. The precipitate was filtered off and washed with cold CPME (900 ml). The residue was dried under reduced pressure at 50°C overnight to give the title compound (585 g) with a total yield of 75% in 3 steps. The formation of the title compound was confirmed by HPLC analysis and NMR.

The apparatus and HPLC analysis conditions were the same as in Step 2. The retention time of the title compound was about 3.1 minutes under the specified HPLC analysis conditions.

^1H -NMR ( _CDCl3 ) δ: 1.33 (d, 3H, J = 6.5 Hz), 2.68-2.85 (m, 2H), 3.33-3.48 (m, 2H ), 3.80 (s, 6H), 4.43 (s, 2H), 6.42-6.46 (m, 2H), 7.11-7.15 (m, 1H).

Step 4. Preparation of diastereomeric salt of (3R,4R)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid with (1R,2R)-(-)-2-amino-1 -(4-nitrophenyl)-1,3-propanediol

To (trans)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid (585 g) obtained in Step 3 was added acetonitrile (2.9 L) at room temperature under flow nitrogen. The mixture was stirred at 85°C. (1R,2R)-(-)-2-amino-1-(4-nitrophenyl)-1,3-propanediol (254 g) was added to the resulting mixture over 14 minutes at 85°C. The reaction mixture was stirred at 90°C for 2 hours 48 minutes. The reaction mixture was cooled to room temperature with stirring overnight. The precipitate was filtered off and washed with acetonitrile (2.4 L). The residue was dried under normal pressure for 8.5 hours at room temperature to obtain crude crystals of the title compound (516 g). Acetonitrile (2.5 L) and water (0.5 L) were added to the resulting crude crystals at room temperature under a nitrogen stream. The resulting mixture was stirred at 100°C for 1 hour 14 minutes. Acetonitrile (1.5 L) was added dropwise to the mixture at 100°C for 1 hour 7 minutes. The mixture was stirred at 100°C for 10 minutes. The mixture was cooled to room temperature with stirring over 21 hours 10 minutes. The mixture was stirred for 3 hours 54 minutes while cooling with ice. The precipitate was separated by filtration and washed with acetonitrile (1.5 L). The residue was dried under atmospheric pressure at room temperature for 4 hours to give the title compound (448 g, 99.8% de) in 45% yield. The formation of the title compound was confirmed by HPLC analysis.

The following instruments and HPLC analysis conditions were used:

Instrument: HPLC system from Shimadzu Corporation, High-Performance Liquid Chromatograph Prominence

Registration conditions:

Column: CHIRAL PAK AD-3R: 3 µm, 150 mm x 4.6 mm (Daicel)

Column temperature: 40°C

Flow rate: 0.50 ml/min.

Analysis time: 10 min.

Detector wavelength: UV (220 nm)

Mobile phase: (Solution A) 10 mM (sodium) phosphate buffer (pH = 2.6), (Solution B) acetonitrile.

Mobile phase supply mode: The ratio (Solution A/Solution B (vol%)) between Solution A and Solution B was maintained at 60/40.

Retention times were about 5.6 minutes for (3R,4R)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid and about 6.5 minutes for (3S,4S)- 1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid under the specified HPLC analysis conditions.

The conformation of the title compound was determined by X-ray crystallography on its single crystal obtained by recrystallization from methyl isobutyl ketone.

The diastereomeric excess was determined from the HPLC peak areas and was ((3R,4R)/(3S,4S) = 99.886%/0.114%).

Step 5. Preparation of (3R,4R)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid

To the diastereomeric salt of (3R,4R)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid with (1R,2R)-(-)-2-amino-1-(4 -nitrophenyl)-1,3-propanediol (448 g) obtained in Step 4, ethyl acetate (1.8 L) and water (1.34 L) were added at room temperature. 6N was added dropwise to the resulting mixture. hydrochloric acid solution (168 ml) at room temperature for 16 minutes. The mixture was separated. The aqueous layer was extracted with ethyl acetate (450 ml) three times. The organic layers were combined and washed successively with 2N. hydrochloric acid solution (224 ml) and saturated aqueous sodium chloride solution (224 ml), and then dried over sodium sulfate (90 g) and evaporated. Toluene (220 ml) was added to the residue and the mixture was evaporated. The residue was dried under reduced pressure at room temperature to give the title compound (254 g) in 98% yield.

^1H -NMR (DMSO- _D6 ) δ: 1.15 (d, 3H, J = 7.2 Hz), 2.50-2.58 (m, 1H), 2.73-2.83 (m , 1H), 3.18-3.25 (m, 1H), 3.30-3.38 (m, 1H), 3.75 (s, 3H), 3.77 (s, 3H), 4, 19-4.35 (m, 2H), 6.48 (dd, 1H, J = 8.4, 2.3 Hz), 6.56 (d, 1H, J = 2.3 Hz), 7.00 (d, 1H, J = 8.4 Hz), 12.61 (brs, 1H).

Example 1. Synthesis of (3R,4R)-N-(5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-4-methyl-5- oxopyrrolidine-3-carboxamide

Step 1. Preparation of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazole

Acetic acid (1 L) was added to 1H-pyrazol-3-amine (100 g) at room temperature, and the resulting mixture was stirred for 5 minutes. 2,5-hexanedione (148 ml) was added to the resulting mixture at room temperature, and the mixture was stirred for 5 minutes. The reaction mixture was stirred at 120°C for 2.5 hours and cooled to room temperature. Water (1 L) was added to the reaction mixture at room temperature. The reaction mixture was stirred at room temperature for 50 minutes. The solid precipitate was filtered off and washed with water (1 L). The resulting wet residue was dried under atmospheric pressure at room temperature overnight, and then dried under reduced pressure at 65°C for 3 days and 8.5 hours to obtain the title compound (172.47 g) in 89% yield.

¹ H-NMR (CDCl ₃ ) δ: 2.11 (s, 6H), 5.90 (s, 2H), 6.25 (d, 1H, J = 2.4 Hz), 7.51 (d, 1H, J = 2.4 Hz).

Step 2. Preparation of a mixture of 1-(bromodifluoromethyl)-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazole and 1-(bromodifluoromethyl)-5-(2,5-dimethyl-1H- pyrrol-1-yl)-1H-pyrazole

DMF (100 ml) was added to sodium hydride (14.9 g) under argon under ice cooling. A suspension of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazole (40 g) obtained in Step 1 in DMF (150 ml) was added dropwise to the resulting mixture under ice-cooling for 20 minutes. The addition funnel used was washed with DMF (50 ml) and the washing solutions were added to the reaction mixture. The reaction mixture was stirred while cooling with water for 1.5 hours. Tetrabutylammonium bromide (0.80 g) was added to the reaction mixture under ice-cooling. The reaction mixture was stirred under ice cooling for 15 minutes. A solution of dibromodifluoromethane (45 ml) in DMF (50 ml) was added dropwise to the reaction mixture under ice-cooling for 15 minutes. The reaction mixture was stirred while cooling with water for 2 hours and 10 minutes. Dibromodifluoromethane (20 ml) was added dropwise to the mixture under argon atmosphere while cooling with water. The reaction mixture was stirred while cooling with water for 40 minutes and left overnight. A saturated aqueous ammonium chloride solution (200 ml) was added to the reaction mixture under ice-cooling. Ethyl acetate and water were added to the reaction mixture. The reaction mixture was filtered through celite and the filtrate was separated. The aqueous layer was extracted with ethyl acetate. The organic layers were combined and a saturated aqueous sodium chloride solution was added. The mixture was filtered through celite and the filtrate was separated. The aqueous layer was extracted with ethyl acetate. The organic layers were combined, then dried over sodium sulfate and evaporated. Toluene (250 ml) was added to the residue and the resulting mixture was evaporated. This procedure was repeated. Ethyl acetate (about 150 ml) was added to the residue, and undissolved particles were filtered off. These undissolved particles were washed with ethyl acetate. The filtrates were combined and evaporated. The residue was dried under reduced pressure with stirring at room temperature for 10 minutes. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 30/1 to 20/1) to give the title compound (40.6 g, including 3.7 wt.% hexane, 1-(bromodifluoromethyl )-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazole: 1-(bromodifluoromethyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1H- pyrazole = about 3:1) with a yield of 54%.

¹ H-NMR (CDCl ₃ ) δ: 2.03 (s, 1.5H), 2.18 (s, 4.5H), 5.89 (s, 1.5H), 5.91 (s, 0 ,5H), 6.39-6.41 (m, 1H), 7.86-7.88 (m, 1H).

Step 3. Preparation of a mixture of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(trifluoromethyl)-1H-pyrazole and 5-(2,5-dimethyl-1H-pyrrol-1-yl) -1-(trifluoromethyl)-1H-pyrazole

Into a solution of a mixture of 1-(bromodifluoromethyl)-3-(2,5-dimethyl-1H-pyrrol-1-yl)-1H-pyrazole and 1-(bromodifluoromethyl)-5-(2,5-dimethyl-1H-pyrrol- 1-yl)-1H-pyrazole (40.6 g, including 3.7 wt.% hexane) obtained in Step 2, in sulfolane (400 ml) was added tetramethylammonium fluoride (13.0 g) at room temperature under argon . The reaction mixture was stirred at 100°C for 1 hour. Tetramethylammonium fluoride (9.4 g) was added to the reaction mixture at 100°C. The reaction mixture was stirred at 100°C for 1 hour 15 minutes. Tetramethylammonium fluoride (10 g) was added to the mixture at 100°C. The reaction mixture was stirred at 100°C for 40 minutes. Tetramethylammonium fluoride (5 g) was further added to the mixture at 100°C. The reaction mixture was stirred at 100°C for 2 hours 5 minutes and then cooled to room temperature. Water (400 ml) and saturated aqueous sodium hydrogen carbonate (200 ml) were slowly and successively added to the mixture under ice-cooling. n-hexane/ethyl acetate (2/3) (400 ml) was added to the reaction mixture. The mixture was filtered through celite and the filtrate was separated. The organic layer was washed with a saturated aqueous sodium chloride solution. The aqueous layers were combined and extracted with n-hexane/ethyl acetate (2/3) (300 ml). The organic layer was washed with a saturated aqueous sodium chloride solution. The organic layers were combined, dried over sodium sulfate and evaporated. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 30/1 to 25/1) to give the title compound (21.85 g, 3-(2,5-dimethyl-1H-pyrrole- 1-yl)-1-(trifluoromethyl)-1H-pyrazole: 5-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(trifluoromethyl)-1H-pyrazole = about 6:1, including 24 .4 wt.% n-hexane) with a yield of 51%.

¹ H-NMR (CDCl ₃ ) δ: 2.00 (s, 0.86H), 2.16 (s, 5.1H), 5.89 (s, 1.7H), 5.91 (s, 0 ,29H), 6.40 (d, 0.86H, J = 2.8 Hz), 6.42 (d, 0.14H, J = 1.6 Hz), 7.83 (d, 0.14H, J = 1.6 Hz), 7.87 (d, 0.86H, J = 2.8 Hz).

Step 4. Preparation of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-iodo-1-(trifluoromethyl)-1H-pyrazole

Into a solution of a mixture of 3-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(trifluoromethyl)-1H-pyrazole and 5-(2,5-dimethyl-1H-pyrrol-1-yl)-1 -(trifluoromethyl)-1H-pyrazole (21.85 g, including 24.4 wt.% n-hexane) obtained in Step 3 in THF (180 ml) was added dropwise a solution of n-butyllithium in n-hexane (1 .55M, 51.1 ml) at -70°C for 5 minutes under argon. The reaction mixture was stirred at -70°C for 25 minutes. A solution of iodine (18.3 g) in THF (50 ml) was added dropwise to the reaction mixture at -70°C over 5 minutes. The addition funnel used was washed with tetrahydrofuran (10 ml) and the washing solutions were added to the reaction mixture. The reaction mixture was stirred at -70°C for 30 minutes. Iodine (0.90 g) was added to the mixture at -70°C. The reaction mixture was stirred at -70°C for 0.5 hour. Water (250 ml) and ethyl acetate (250 ml) were sequentially added to the mixture at -70°C. The reaction mixture was stirred at room temperature and separated. The organic layer was washed successively with 10% aqueous sodium hydrogen sulfate (250 ml) and saturated aqueous sodium chloride (150 ml), dried over sodium sulfate and evaporated. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 50/1 to 30/1). Fractions containing the title compound were collected and evaporated. n-hexane was added to the residue. The mixture was evaporated until the residue weighed 27.5 g. n-hexane (20 ml) was added to the residue. The resulting suspension was stirred at room temperature for 10 minutes. The precipitate was filtered, washed with n-hexane (30 ml) and dried under reduced pressure to give the title compound (17.14 g) in 67% yield. This filtrate was then evaporated. The residue was crystallized from n-hexane to give the title compound (1.63 g) in 6.4% yield.

¹ H-NMR (CDCl ₃ ) δ: 2.15 (s, 6H), 5.88 (s, 2H), 6.60 (s, 1H).

Step 5. Preparation of 5-iodo-1-(trifluoromethyl)-1H-pyrazol-3-amine

To 3-(2,5-dimethyl-1H-pyrrol-1-yl)-5-iodo-1-(trifluoromethyl)-1H-pyrazole (18.77 g) obtained in Step 4, a mixture of ethanol and water was added successively (ethanol/water = 2/1, 480 ml), hydroxylamine hydrochloride (73.5 g) and triethylamine (14.7 ml) at room temperature. The reaction mixture was stirred at 100°C for 38 hours 20 minutes. The reaction mixture was cooled to room temperature and ethanol was removed by evaporation. A solution of sodium hydroxide (42.3 g) in water (130 ml) was slowly added to the reaction mixture, followed by ethyl acetate (200 ml) while cooling with ice. The reaction mixture was stirred and separated. The aqueous layer was extracted with ethyl acetate (200 ml). The organic layers were combined, washed with saturated aqueous sodium chloride, dried over sodium sulfate and evaporated. Ethyl acetate (30 ml) and n-hexane (30 ml) were added to the residue, and insoluble particles were filtered off. The filtrate was evaporated. The residue was purified by silica gel column chromatography (eluent: n-hexane/ethyl acetate = 4/1 to 3/1) to give the title compound (16.27 g, including 14 wt% ethyl acetate) in 96% yield.

¹ H-NMR (CDCl ₃ ) δ: 3.93 (b.s, 2H), 6.09 (s, 1H).

Step 6. Preparation of 5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-amine

2-(3-( tert-butoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (127 mg), obtained from Step 2 of Synthesis 1, palladium(II) acetate (6.5 mg) and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (20 mg) at room temperature under argon. The reaction mixture was stirred at room temperature for 4 minutes. A 2M aqueous solution of tripotassium phosphate (1.5 ml) was added to the reaction mixture at room temperature. The reaction mixture was stirred at 90°C for 47 minutes. The mixture was cooled to room temperature. Ethyl acetate and a saturated aqueous solution of sodium hydrogen carbonate were added to the reaction mixture. The reaction mixture was filtered through cotton and extracted with ethyl acetate. The organic layer was washed successively with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution, dried over sodium sulfate and evaporated. The residue was combined with a portion of the title compound (15 mg) separately prepared analogously to this step using 5-iodo-1-(trifluoromethyl)-1H-pyrazol-3-amine (70 mg, including 14 wt.% ethyl acetate) obtained in Step 5, and the resulting mixture was purified by silica gel thin layer chromatography (eluent: n-hexane/ethyl acetate = 3/1) to obtain the title compound (108 mg).

¹ H-NMR (CDCl ₃ ) δ: 1.36 (s, 9H), 3.93 (b.s, 2H), 5.83 (s, 1H), 6.75-6.85 (m, 3H ).

Step 7. Preparation of (3R,4R)-N-(5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-1-(2,4 -dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxamide

Into a solution of (3R,4R)-1-(2,4-dimethoxybenzyl)-4-methyl-5-oxopyrrolidine-3-carboxylic acid (55 mg) prepared analogously to Step 5 of Synthesis 2 in chloroform (0.55 ml), DMF (1 μL) and oxalyl chloride (33 μL) were added sequentially under ice-cooling under argon. The reaction mixture was stirred under ice cooling for 50 minutes. The mixture was evaporated and dried under reduced pressure. To the residue were added sequentially chloroform (0.4 ml) and 5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-amine (40 mg) obtained in Step 6 , in an argon atmosphere with ice cooling. Pyridine (50 μL) was added to the reaction mixture under ice cooling. The reaction mixture was stirred under ice cooling for 5 minutes and at room temperature for 35 minutes. A saturated aqueous sodium hydrogen carbonate solution was added to the mixture at room temperature, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride, dried over sodium sulfate and evaporated. The residue was purified by silica gel thin layer chromatography (eluent: n-hexane/ethyl acetate = 1/1) to give the title compound (60 mg) in 80% yield. The formation of the title compound was confirmed by thin layer chromatography (eluent: n-hexane/ethyl acetate = 2/1, Rf: 0.19).

Step 8. Preparation of (3R,4R)-N-(5-(3-fluoro-5-hydroxyphenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-4-methyl-5-oxopyrrolidine-3- carboxamide

K (3R,4R)-N-(5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-1-(2,4-dimethoxybenzyl) To the -4-methyl-5-oxopyrrolidine-3-carboxamide (60 mg) obtained in Step 7, anisole (58 μl) and trifluoroacetic acid (2 ml) were added at room temperature. The reaction mixture was stirred at 80°C for 1 hour 20 minutes. The mixture was evaporated. A saturated aqueous sodium hydrogen carbonate solution was added to the residue, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride, dried over sodium sulfate and evaporated. The residue was purified by silica gel thin layer chromatography (eluent: chloroform/ethyl acetate = 1/1) to give the title compound (29.9 mg) in 76% yield.

^1H -NMR (DMSO- _d6 ) δ: 1.06 (d, 3H, J = 7.2 Hz), 2.50-2.53 (m, 1H), 2.96-3.04 (m , 1H), 3.17-3.23 (m, 1H), 3.40-3.46 (m, 1H), 6.67-6.81 (m, 3H), 6.96 (s, 1H ), 7.67 (s, 1H), 10.34 (s, 1H), 11.26 (s, 1H).

Step 9. Preparation of (3R,4R)-N-(5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-4-methyl-5- oxopyrrolidine-3-carboxamide

To (3R,4R)-N-(5-(3-fluoro-5-hydroxyphenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-4-methyl-5-oxopyrrolidine-3-carboxamide (30 mg) obtained in Step 8, di-tert-butyl dicarbonate, chloroform (1 ml) and magnesium perchlorate were added sequentially at room temperature. The reaction mixture was stirred at 55°C for 0.5 hour. Magnesium perchlorate was added to the reaction mixture at 55°C. The reaction mixture was stirred at 55°C for 1 hour 10 minutes. More magnesium perchlorate was added to the mixture at 55°C. The reaction mixture was stirred at 55°C for 20 minutes. The mixture was cooled to room temperature and ethyl acetate was added. The reaction mixture was washed successively with 1N. a solution of hydrochloric acid and a saturated aqueous solution of sodium chloride, dried over sodium sulfate and evaporated. The residue was purified by silica gel thin layer chromatography (eluent: chloroform/methanol = 15/1) to give the title compound (19.2 mg) in 56% yield.

^1H -NMR (DMSO- _d6 ) δ: 1.08 (d, 3H, J = 7.2 Hz), 1.34 (s, 9H), 2.51-2.55 (m, 1H), 2.98-3.06 (m, 1H), 3.19-3.25 (m, 1H), 3.42-3.48 (m, 1H), 6.95 (s, 1H), 7. 00-7.07 (m, 2H), 7.11-7.17 (m, 1H), 7.68 (s, 1H), 11.28 (s, 1H).

MS (M+H) 443, MS (M-H) 441.

Step 10. Preparation of crystals of (3R,4R)-N-(5-(3-(tert-butoxy)-5-fluorophenyl)-1-(trifluoromethyl)-1H-pyrazol-3-yl)-4-methyl-5 -oxopyrrolidine-3-carboxamide

The title compound (100 mg) was stirred in ethanol (0.4 ml) at 65°C for 8 minutes until dissolved. Water (0.4 ml) was added dropwise to the solution at 65°C for 2 minutes. The mixture was stirred at 65°C for 10 minutes. The mixture was cooled to 25°C with stirring for 2 hours. The mixture was then stirred at room temperature for 2 hours. The precipitate that formed was separated by filtration. The resulting solid was washed with ethanol/water (=1/1) and dried under reduced pressure at 60°C to obtain crystals of the title compound (87.8 mg) in 88% yield.

Comparative example

Compound A, Compound B and Compound C, all of which are shown in the table below, were prepared as described in WO 2013/031922.

Connection A Connection B Connection C

Metabolite 1 (ie the metabolite of Compound 1) and Metabolite C (ie the metabolite of Compound C), both of which are shown in the table below, were prepared according to Example 1 described above and the description in WO 2013/031922.

Metabolite 1 Metabolite C

Test Example 1

The activity of test compounds as SGLT1 inhibitors (IC ₅₀ values) was calculated from the cellular uptake of labeled α-methyl-D-glucopyranoside ( ¹⁴ C-AMG) carried by SGLT1.

1) Formation of a plasmid expressing human SGLT1

A DNA fragment containing the human SGLT1 gene was amplified by PCR (polymerase chain reaction) using pCMV6-hSGLT1 (OriGene) as a template. A NheI recognition and cleavage sequence was added to human SGLT1 upstream of the Kozac consensus sequence from the vector, and a stop codon, TAG, and SalI recognition and cleavage sequence were added immediately downstream of the protein translation region of human SGLT1. The purified DNA fragment was digested with the restriction enzymes NheI and SalI, then ligated with pcDNA3.1 (+), which was digested with NheI and XhoI, thereby forming a plasmid expressing human SGLT1. The human SGLT1 nucleic acid sequence inserted into the vector was completely identical to the protein translation region of the human SGLT1 sequence (Accession number NM_000343) registered in GenBank, and the sequence of the region connected to the vector was as expected.

2) Formation of cell lines stably expressing human SGLT1

The human SGLT expression plasmid, pcDNA-hSGLT1, was transfected into each CHO-K1 cell using Lipofectamine 2000 (Invitrogen) and cultured in the presence of G418 (Nacalai Tesque) to select for drug-resistant cell lines. The cell line having the highest ratio (S/B ratio) of the amount of cellular uptake of ¹⁴ C-AMG per cell to the amount of cellular uptake of ¹⁴ C-AMG after exposure to the SGLT inhibitor, phlorizin, was selected as the cell line stably expressing human SGLT1 from among the lines drug-resistant cells.

3) Assessment of SGLT1 inhibitory activity

Cells stably expressing human SGLT1 were seeded at 5 x 10 ⁴ cells per well in BioCoat ^TM Poly-D-Lysine 96-well plates with lid (Becton, Dickinson and Company) and cultured at 37°C in an atmosphere of 5% CO ₂ during the night. The medium was replaced with 100 μl/well Na(-) buffer (140 mM choline chloride, 2 mM KCl, 1 mM _MgCl2 , 1 mM _CaCl2 , 10 mM HEPES, 5 mM Tris, pH 7.4), and the mixture was left at 37°C in an atmosphere of 5% CO ₂ for 20 minutes. After removing the Na(-) buffer, 40 μl/well of a test compound solution prepared in Na(+) buffer (140 mM NaCl, 2 mM KCl, 1 mM _MgCl2 , 1 mM _CaCl2 , 10 mM HEPES, 5 mM Tris) was added , pH 7.4) containing BSA (bovine serum albumin). Then 40 μl/well of Na(+) buffer containing 8 kBq ¹⁴ C-AMG and 2 mM AMG was added and the resulting mixture was mixed well. In the control sample, 40 μl/well of Na(-) buffer containing BSA was added, and in addition, 40 μl/well of Na(-) buffer containing 8 kBq of ¹⁴ C-AMG and 2 mM AMG was added, and the resulting mixture was mixed well. After incubation for 1 hour at 37°C in an atmosphere of 5% CO _{2 ,} cells were washed twice with 100 μl per well of ice-cold wash buffer (100 mM AMG, 140 mM choline chloride, 2 mM KCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ , 10 mM HEPES, 5 mM Tris, pH 7.4) to stop the reaction. Cell lysate was prepared by adding 50 μl/well of 0.2N. aqueous NaOH solution. To assess ¹⁴ C-AMG uptake capacity, the entire cell lysate was transferred to an OptiPlate 96 (Perkin-Elmer) with 100 μl/well MicroScint-40 (Perkin-Elmer), and the ¹⁴ C CPM (counts per minute) was measured using TOPCOUNT. NXT (Perkin-Elmer).

The result was calculated by subtracting the average CPM value for the control sample from the average CPM value for each treated cell. The degree of inhibition for each compound tested at each concentration was calculated using the following equation:

[(A-B)/A] x 100

where A is the result for the control sample, and B is the result when treated with each test compound.

Each _IC50 value (50% inhibitory concentration) for each test compound was calculated from the two concentrations above and below the 50% inhibition, and from the inhibition value. In this study, compound 1 was confirmed to have SGLT1 inhibitory activity.

Example Test 2

OGTT (Oral glucose tolerance test)

Vehicle (0.5% methylcellulose solution) or compound 1 (1, 3 or 10 mg/kg) suspended in 0.5% methylcellulose solution was administered orally at 5 ml/kg to fasted animals for approximately 4 hours. male SD rats (8 weeks old, Nihon Charles River K.K., 6 animals in each group). After 16 hours, glucose loading was carried out by oral administration of 0.4 g/ml glucose solution in an amount of 5 ml/kg. Blood was taken from the tail vein immediately before the glucose load, as well as 30, 60 and 120 minutes after the glucose load; and blood glucose levels were measured using an automatic chemistry analyzer (HITACHI, Model No. 7180).

The results are presented in Fig. 1. Shown are the mean values ± standard deviation for the ratio of the area under the curve (ΔAUC) of blood glucose levels for the 120-minute glucose load group to those for the control group (% of Vehicle). Statistical analysis was performed using Steele's test. The significance level was two-sided 5%. The results show that Compound 1 significantly reduced blood glucose levels after glucose loading compared to the control group.

Example Test 3

OGTT (Oral glucose tolerance test)

Vehicle (0.5% methylcellulose solution) or Compound 1, Compound A or Compound B (3 mg/kg in each case) suspended in 0.5% methylcellulose solution was administered orally in an amount of 5 ml/kg to the contained without food for about 4 hours in male SD rats (8 weeks old, Nihon Charles River K.K., 5 animals in each group). After 16 hours, glucose loading was carried out by oral administration of 0.4 g/ml glucose solution in an amount of 5 ml/kg. Blood was taken from the tail vein immediately before the glucose load, as well as 30, 60 and 120 minutes after the glucose load; and blood glucose levels were measured using an automatic chemistry analyzer (HITACHI, Model No. 7180).

The results are presented in Fig. 2. Shown are the mean values ± standard deviation for the ratio of the area under the curve (ΔAUC) of blood glucose levels for the 120-minute glucose load group to those for the control group (% of Vehicle). Statistical analysis was performed using Dunnett's test. The significance level was two-sided 5%. The results show that only compound 1 significantly reduced blood glucose levels after a glucose load, compared to the control group.

Example Test 4

Ames test (reverse mutation test)

This test examined Metabolite 1 and Metabolite C. The purpose of this test was to evaluate the potential of each metabolite to cause back mutations in standard lines of Salmonella typhimurium (TA98, TA1537, TA100 and TA1535) and Escherichia coli (WP2uvrA), in the presence or absence of a metabolic activation system rat liver (S9 mix).

The solvent used in this case was dimethyl sulfoxide (DMSO, 100 μl/well).

The test was carried out by pre-incubation with or without S9 mix. When the test was performed without S9 mix, sodium phosphate buffer solution (pH 7.4) was added.

0.5 ml of S9 mix or 0.5 ml of 0.1 mol/l sodium phosphate buffer solution (pH 7.4), and 0.1 ml of bacterial culture solution were added to a test tube containing 0.1 ml of negative control (only DMSO), metabolite or positive control. The mixtures were pre-incubated at 37°C for 20 minutes with shaking. After pre-incubation, 2 ml of top layer agar was added and the mixtures were vortexed and distributed into plates. Two plates were used for each test sample. The plates were incubated at 37 ± 1°C for 48 hours or more, and the number of revertant colonies was counted. The average number of revertant colonies for each test sample was calculated. The presence or absence of growth inhibition due to the antibacterial action of the test samples, as well as precipitation, were assessed visually or using a stereomicroscope. Results were scored as positive if the average number of revertant colonies showed a dose-dependent increase of up to 2-fold over the negative control at one or more dosages. The assessment was carried out using average values without using statistical comparison.

The test results are shown in the following tables (Tables 1-4 and Tables 5-7). In conclusion, Metabolite 1 does not have the potential to cause back mutations in any of the bacterial lines tested, while Metabolite C has the potential to cause back mutations in the bacterial line TA98 with S9 mix and in TA100 with S9 mix.

Table 1

Test sample Dose
(µg/well) S9 Mix Number of revertant colonies TA98 TA100 DMSO (0.1 ml) + 36 133 Metabolite 1 2.3 + 35 120 6.9 + 31 119 21 + 35 117 62 + 28 104 185 + 16 * 78 * 556 † + 15 * 59 * 1667 † + 13 * 50 * 5000 † + 13 * 52 * B[a]P 5.0 + 455 1069 +: S9 mix present *: Growth inhibition †: Sediment DMSO: Dimethyl sulfoxide B[a]P: Benzo[a]pyrene The number of revertant colonies shows the average value for each plate.

table 2

Test sample Dose
(µg/well) S9 Mix Number of revertant colonies TA1537 TA1535 WP2uvrA DMSO (0.1 ml) + 13 102 25 Metabolite 1 2.3 + eleven 13 31 6.9 + 10 7 31 21 + 9 6 32 62 + 6 8 40 185 + 2 * 5 * 16 * 556 + 0 * 4 * 18 * 1667 + 0 * 4 * 9 * 5000 † + 0 * 2 * 0 * 2AA 2.0 + -- 223 -- 10.0 + -- -- 818 B[a]P 5.0 + 119 -- -- +: S9 mix present *: Growth inhibition †: Sediment --: Not tested DMSO: Dimethyl sulfoxide 2AA: 2-aminoanthracene B[a]P: Benzo[a]pyrene The number of revertant colonies shows the average value for each plate.

Table 3

Test sample Dose
(µg/well) S9 Mix Number of revertant colonies WP2uvrA DMSO (0.1 ml) + 31 Metabolite 1 6.9 + 31 12 + 28 21 + 25 36 + 34 62 + 35 107 + 25 185 + 9 * 2AA 10.0 + 740 +: S9 mix present *: Growth inhibition DMSO: Dimethyl sulfoxide 2AA: 2-aminoanthracene The number of revertant colonies shows the average value for each plate.

Table 4

Test sample Dose
(µg/
hole) S9 Mix Number of revertant colonies TA98 TA1537 TA100 TA1535 WP2uvrA DMSO (0.1 ml) - 18 8 100 8 26 Metabolite 1 2.3 - 14 7 99 6 32 6.9 - 16 10 113 9 27 21 - 14 9 124 8 31 62 - 21 9 88 8 24 185 - 9 * 0 * 38 * 0 * 15 * 556 - 0 * 0 * 0 * 0 * 8 * 1667 - 0 * 0 * 0 * 0 * 5 * 5000 † - 0 * 0 * 0 * 0 * 0 * AF-2 0.01 - -- -- 633 -- 69 0.1 - 341 -- -- -- -- ICR-191 1.0 - -- 1170 -- -- -- S.A. 0.5 - -- -- -- 217 -- --: Not tested *: Growth inhibition †: Sediment DMSO: Dimethyl sulfoxide AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide SA: Sodium azide ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine dihydrochloride The number of revertant colonies shows the average value for each plate.

Table 5

Test sample Dose
(µg/well) S9 Mix Number of revertant colonies TA98 TA100 DMSO (0.1 ml) + 28 117 Metabolite C 2.34 + 38 526 # 4.69 + 36 778 # 9.38 + 73 # 1210 # 18.8 + 107 # 1745 # 37.5 + 133 # 2049 # 75 + 153 # 2147 # 150 + 133 # 2043 # 300 + 138 * 1412 * B[a]P 5.0 + 404 1078 #: Results were assessed as positive if the average number of revertant colonies showed a dose-dependent increase that reached 2-fold compared to the negative control. *: Growth inhibition DMSO: Dimethyl sulfoxide B[a]P: Benzo[a]pyrene The number of revertant colonies shows the average value for each plate.

Table 6

Test sample Dose
(µg/well) S9 Mix Number of revertant colonies TA1537 TA1535 WP2uvrA DMSO (0.1 ml) + 6 5 21 Metabolite C 2.3 + 6 8 28 6.9 + 7 8 23 21 + 7 5 21 62 + 9 4 26 185 + 9 * 5 * 17 556 † + 4 * 4 * 8 * 1667 † + 4 * 5 * 12 * 5000 † + 5 * 4 * 16 * 2AA 2.0 + -- 250 -- 10.0 + -- -- 685 B[a]P 5.0 + 80 -- -- --: Not tested *: Growth inhibition †: Sediment DMSO: Dimethyl sulfoxide 2AA: 2-aminoanthracene B[a]P: Benzo[a]pyrene The number of revertant colonies shows the average value for each plate.

Table 7

Test sample Dose
(µg/
hole) S9 Mix Number of revertant colonies TA98 TA1537 TA100 TA1535 WP2uvrA DMSO (0.1 ml) - 17 6 86 6 18 Metabolite C 2.3 - 14 3 87 6 15 6.9 - 15 1 * 99 5 * 16 21 - 17 3 * 48 * 6 * 17 62 - 8 3 * 41 * 3 * 13 185 - 8 * 2 * 45 * 4 * 13 556 † - 8 * 0 * 33 * 0 * 13 * 1667 † - 8 * 0 * 25 * 1 * 10 * 5000 † - 0 * 0 * 35 * 0 * eleven * AF-2 0.01 - -- -- 542 -- 74 0.1 - 317 -- -- -- -- ICR-191 1.0 - -- 1131 -- -- -- S.A. 0.5 - -- -- -- 222 -- --: Not tested *: Growth inhibition †: Sediment DMSO: Dimethyl sulfoxide AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide SA: Sodium azide ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine dihydrochloride The number of revertant colonies shows the average value for each plate.

Examples of drugs

Examples of preparations containing the compound of the present invention include, for example, the preparations described below. However, the present invention is not limited to these exemplary preparations.

Example of drug 1 (capsule)

(1) Connection 1 30 mg (2) Microcrystalline cellulose 10 mg (3) Lactose 19 mg (4) Magnesium stearate 1 mg

Ingredients (1), (2), (3) and (4) are mixed for subsequent filling into a gelatin capsule.

Example of drug 2 (tablet)

(1) Connection 1 10 g (2) Lactose 50 g (3) Corn starch 15 g (4) Calcium carmellose 44 g (5) Magnesium stearate 1 g

The entire quantity of ingredients (1), (2) and (3) and 30 g of ingredient (4) are combined with water, vacuum dried and then granulated. The resulting granules are mixed with 14 g of ingredient (4) and 1 g of ingredient (5), and tabletted on a tablet press. In this way, 1000 tablets are obtained, containing 10 mg of compound 1 in each tablet.

Industrial applicability

The compound having formula [I] or a pharmaceutically acceptable salt thereof has SGLT1 inhibitory activity and therefore can be used for the treatment and/or prevention of various diseases or pathological conditions that are expected to improve by regulating SGLT1 activity. The compound having formula [I] or a pharmaceutically acceptable salt thereof can also be used for the treatment and/or prevention of various diseases or pathological conditions that may be caused by elevated blood glucose levels.

Claims (13)

1. A compound having the formula [I]: or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition capable of inhibiting SGLT1, containing a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 3. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof as an SGLT1 inhibitor. 4. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof as a therapeutic agent against diabetes. 5. Use according to claim 4, wherein the diabetes is type 2 diabetes. 6. A method of inhibiting SGLT1, comprising administering to a mammal a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof. 7. A method of treating diabetes, comprising administering to a mammal a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof. 8. The method of claim 7, wherein the diabetes is type 2 diabetes. 9. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the production of an SGLT1 inhibitor. 10. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the production of a therapeutic agent against diabetes. 11. Use according to claim 10, wherein the diabetes is type 2 diabetes.