Water-soluble allopregnanolone derivatives, their production method and use

Abstract

The present invention relates to the pharmaceutical field, specifically to a compound represented by formula I, its racemate, stereoisomer, tautomer, solvate, crystalline polymorph or a pharma- ceutically acceptable salt thereof, in which R2 and R4 are each independently selected from H or D, R1 and R3 are each independently selected from CH3, CH2D, CHD2 or CD3, and the compound of formula I contains at least one deuterium atom. The present invention provides an allopregnanolone derivative having good physical / chemical stability and suitable for oral administration by structurally modifying the hydroxyl group of allopregnanolone, on the premise of retaining the pharmacological activity of allopregnanolone. [Case 1] JPEG2024535126000023.jpg37169

Description

Detailed Description of the Invention

[0001] This application claims priority from a prior application, bearing application number 202111084187.4 and entitled "Water-soluble allopregnanolone derivatives and their preparation and use," filed with the State Intellectual Property Office of China on September 14, 2021. The above prior application is incorporated herein by reference in its entirety. [Technical field] The present invention relates to the pharmaceutical field, and more particularly to a water-soluble allopregnanolone derivative suitable for oral administration, a method for producing the same, uses thereof, a pharmaceutical composition containing the same, and uses thereof for the prevention or treatment of central nervous system disorders and sedative-hypnotic disorders, the treatment of Alzheimer's disease, the treatment of epilepsy, or the treatment of depression, particularly postpartum depression. [Background technology] Neuroactive steroids are steroids active on nervous tissue that play an important regulatory role in the human body. Neuroactive steroids mainly include progesterone, pregnenolone, and the progesterone metabolite pregnanolone. Neuroactive steroids play an important physiological role in the human body, and impaired synthesis in the human body can cause various neurological (CN 104736158 A) or psychiatric disorders (Expert Opin Ther Targets. 2014, 18(6):679-90).

[0002] Pregnanolone-based substances are now generally considered to include the compounds pregnanolone, allopregnanolone, epipregnanolone, and epialopregnanolone (see Table 1).

[0003] [Table 1]

[0004] Patent document CN1300219A discloses that different pregnanolone-based substances have different mechanisms of action in regulating the central nervous system and have different physiological effects on the central nervous system. Allopregnanolone (3α-OH-5α-pregnan-20-one) is an important GABAA (γ-aminobutyric acid A) receptor and has antiepileptic, hypnotic, antimigraine, and anti-anxiety effects. Epipregnanolone (3β-OH-5α-pregnan-20-one) blocks and antagonizes the effects of allopregnanolone, allowing for appropriate control and termination of anesthesia induced by allopregnanolone, etc.

[0005] Allopregnanolone (3α-OH-5α-pregnan-20-one) has been a hot topic of research in recent years, and in 1986 it was identified as a positive modulator of the GABAA receptor. Allopregnanolone primarily binds to the α and β subunits of the GABAA receptor, increasing the frequency of chloride ion channel opening in the receptor and reducing neuronal excitability, which may result in sedative and anxiolytic effects.

[0006] It has been reported that the levels of progesterone and its metabolites in the body vary during different stages of the menstrual cycle. A decrease in progesterone and its metabolite levels before the onset of menstruation can lead to premenstrual syndrome (PMS), which is characterized by recurring symptoms such as stress, anxiety, and migraines before the onset of the menstrual cycle, which disappear after menstruation (Dalton, K., Premenstrual Syndrome and Progesterone Therapy, 2nd ed., Chicago Yearbook, Chicago (1984)). Postpartum depression is also associated with abnormal progesterone and its metabolite levels. As pregnancy progresses, plasma allopregnanolone levels in healthy pregnant women increase, and then decrease rapidly after delivery. Research has shown that a decrease in allopregnanolone content is closely correlated with the onset and progression of many psychiatric disorders, such as anxiety, depression, and tremors, and exogenous administration of allopregnanolone can significantly improve the above-mentioned psychiatric symptoms.

[0007] However, conventional allopregnanolone has low water solubility, poor oral bioavailability, a human plasma half-life of approximately 45 minutes, and is rapidly metabolized, making it unsuitable for oral formulation.

[0008] Patent document CN104736158A discloses a method for treating epilepsy or persistent epilepsy by intravenously administering a composition of allopregnanolone and cyclodextrin, in which the cyclodextrin content in the composition is 1-30%, the blood drug concentration is 50-2300 nM, and the treatment period is long, at least 24 hours. However, cyclodextrin is a polymeric compound and carries a certain risk of nephrotoxicity (Safety Research on Cyclodextrin Derivatives, a Drug Supramolecular Material, China Materials Science, Technology and Equipment, Vol. 5, 2009, pp. 1-3), posing safety risks.

[0009] In 2019, SAGE Therapeutics' allopregnanolone injection, under the trade name ZULRESSO, was approved for sale in the United States. Allopregnanolone injection is a sterile, clear, colorless, preservative-free intravenous injection in which allopregnanolone and sulfobutyl beta-cyclodextrin sodium form a clathrate compound, improving the solubility of allopregnanolone. Allopregnanolone injection can produce stable physiological concentrations of allopregnanolone via intravenous injection to achieve therapeutic effects, but requires a long intravenous infusion of up to 60 hours, a complex infusion method, and specialized medical staff must continuously monitor the infusion and provide necessary interventions on-site. This leads to poor patient compliance when injecting allopregnanolone, making it extremely inconvenient for medical staff to administer. Allopregnanolone injection should be administered at a rate of 30 μg / kg / h from 0 to 4 hours, increasing to 60 μg / kg / h from 4 to 24 hours, increasing to 90 μg / kg / h from 24 to 52 hours (for patients who cannot tolerate 90 μg / kg / h, the dose can be set to 60 μg / kg / h), decreasing to 60 μg / kg / h from 52 to 56 hours, and decreasing to 30 μg / kg / h from 56 to 60 hours. [Summary of the Invention] The object of the present invention is to provide an allopregnanolone derivative that is suitable for oral administration, has high bioavailability, takes effect quickly, and can maintain a stable physiological concentration in the body for a long period of time.

[0010] Another object of the present invention is to provide a composition containing an allopregnanolone derivative and the use of said allopregnanolone derivative in the manufacture of a drug for treating diseases caused by abnormalities of the central nervous system.

[0011] The object of the present invention is achieved by the following technical solutions: According to a first aspect, the present invention provides a compound of general formula I, its racemate, stereoisomer, tautomer, solvate, crystalline polymorph or a pharmaceutically acceptable salt thereof,

[0012] [ka]

[0013] Among them, R2 and R4 are each independently selected from H (hydrogen) or D (deuterium); R1 and R3 are each independently selected from CH3, CH2D, CHD2, or CD3; Provided that the compounds of formula I contain at least one deuterium atom.

[0014] In some embodiments of the invention, the compound of Formula I contains 1 to 8 deuterium atoms, or the compound of Formula I contains 1 to 7 deuterium atoms, or the compound of Formula I contains 1 to 6 deuterium atoms, specifically, the compound of Formula I contains at least 1, 2, 3, 4, 5, 6, 7, or 8 deuterium atoms.

[0015] In some embodiments of the invention, R2 is D.

[0016] In some embodiments of the invention, R2 is D, and R1 and R3 are CH3.

[0017] In some embodiments of the invention, R2 is D, and R1 and R3 are CD3.

[0018] According to an embodiment of the present invention, the deuteration ratio of each D is at least 3500-fold (52.5%), preferably at least 4000-fold (60%), preferably at least 4500-fold (67.5%), more preferably at least 5000-fold (75%), more preferably at least 5500-fold (82.5%), more preferably at least 6000-fold (90%), more preferably at least 6333.3-fold (95%), even more preferably at least 6466.7-fold (97%), even more preferably at least 6566.7-fold (98.5%), even more preferably at least 6600-fold (99%), even more preferably at least 6633.3-fold (99.5%).

[0019] Preferably, any atom not designated as deuterium is present at its natural isotopic abundance.

[0020] In specific embodiments of the invention, the compound of formula I is a compound of formula Ia or formula Ib:

[0021] [ka]

[0022] wherein R1, R2, R3, and R4 are as defined above.

[0023] In some preferred embodiments of the present invention, examples of compounds according to Formula I are as follows:

[0024] [ka]

[0025] Where compounds of the invention can exist in tautomeric forms, the present invention includes all tautomeric forms.

[0026] The compounds of the present invention can exist in stereoisomeric forms (enantiomers, diastereoisomers). The present invention therefore includes enantiomers or diastereoisomers and their respective mixtures. From this mixture of enantiomers and / or diastereoisomers, homogeneous compositions of stereoisomers can be separated by known methods.

[0027] The present invention further provides a method for preparing a compound according to formula I above, comprising the steps of:

[0028] [ka]

[0029] wherein R1 to R4 are as defined above, and R5 is a protecting group such as an amino protecting group, e.g., tert-butoxycarbonyl (Boc), The compound of formula II is reacted with the compound of formula III, followed by removal of the protecting group to give the compound of formula I.

[0030] The present invention further provides a pharmaceutical composition comprising at least one of a compound of Formula I, its racemate, stereoisomer, tautomer, solvate, crystalline polymorph, or a pharmaceutically acceptable salt thereof.

[0031] According to the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants.

[0032] According to the present invention, the pharmaceutical composition is used for oral administration, and the pharmaceutical composition may be in the form of tablets, pills, lozenges, sugar-coated tablets, capsules, etc.

[0033] The pharmaceutical compositions of the present invention can be prepared by methods well known in the art, such as conventional mixing, granulation, and sugar-coating granulation. For example, conventional mixing, filling, or tabletting methods can be used to prepare solid oral compositions. For example, the active compound can be mixed with a solid excipient, optionally pulverized, and optionally added with other suitable excipients, and then the mixture can be granulated to obtain tablet or sugar-coated cores. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0034] The present invention provides the use of a compound of formula I of the present invention, its racemate, stereoisomer, tautomer, solvate, crystalline polymorph or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention in the manufacture of a medicament for use in the prevention or treatment of central nervous system disorders, sedative-hypnotic, treatment of Alzheimer's disease, treatment of epilepsy or treatment of depression, particularly postpartum depression.

[0035] The present invention provides a method for the prevention or treatment of central nervous system disorders, sedation-hypnosis, treatment of Alzheimer's disease, treatment of epilepsy or treatment of depression, particularly postpartum depression, which comprises administering to an individual in need thereof a prophylactically or therapeutically effective amount of a compound of formula I of the present invention, its racemate, stereoisomer, tautomer, solvate, crystalline polymorph or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention.

[0036] According to the present invention, the central nervous system disease is, for example, traumatic brain injury, essential tremor, epilepsy (including refractory persistent epilepsy and rare genetic epilepsy (such as Dravet syndrome and Rett syndrome)), depression (including postpartum depression), and Alzheimer's disease. The central nervous system disease is, for example, selected from essential tremor, epilepsy, clinical depression, postpartum or postpartum depression, atypical depression, psychotic major depression, catatonic depression, seasonal affective disorder, dysthymia, bipolar depression, depressive personality disorder, recurrent transient depression, mild depressive disorder, bipolar disorder or manic-depressive disorder, posttraumatic stress disorder, depression due to a chronic medical condition, treatment-resistant depression, treatment-refractory depression, suicidal tendencies, suicidal ideation, or suicidal behavior.

[0037] In all administration methods of the compound of general formula I described herein, the daily dose is 0.01 to 200 mg / kg body weight.

[0038] Dosage regimens can be adjusted to provide the optimum desired response. For example, a single oral administration can be used, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the needs of the therapeutic situation. It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated and may include single or multiple doses. It should further be understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition.

[0039] Definitions and Explanations Unless otherwise specified, all technical and scientific terms used herein have the same meaning as understood by a person skilled in the art to which this invention belongs. All patents and published publications related to this invention are incorporated herein by reference in their entirety.

[0040] As used herein, the following definitions apply unless otherwise stated: When a trade name is mentioned in this application, it is intended to refer to the corresponding product or its active ingredient.

[0041] As used herein, the terms "comprises," "includes," "has," "contains," or "relate to," and other variations thereof herein, are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0042] The term "prophylaxis or treatment" means administering a compound or formulation according to the present invention to prevent, ameliorate or eliminate a disease or one or more symptoms associated with a disease, and includes: (i) preventing a disease or disease state from occurring in a mammal, particularly where the mammal is susceptible to the disease state but has not yet been diagnosed as suffering from the disease state; (ii) inhibiting the disease or disease state, i.e., inhibiting its progression; (iii) Relieving the disease or disease state, i.e., alleviating the disease or disease state.

[0043] The term "deuteration ratio" refers to the ratio of the amount of a synthesized isotope to the amount of a naturally occurring isotope. Unless otherwise specified, when a position in a structure is defined as H, i.e., hydrogen (H-1), that position contains only the naturally occurring isotope amount. When a position in a structure is defined as D, i.e., deuterium (H-2), that position contains an isotope amount that is at least 3340 times greater (i.e., at least 50.1% deuterium isotopes) than the naturally occurring isotope amount (0.015%).

[0044] The term "therapeutically effective amount" refers to an amount of a compound of the present invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the method of administration, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art from their knowledge and this disclosure.

[0045] The term "pharmaceutically acceptable" means that the compound, material, composition, and / or dosage form is, within the best of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / hazard ratio.

[0046] Pharmaceutically acceptable salts of the compounds of the present invention include salts formed with pharmaceutically acceptable acids and those formed with pharmaceutically acceptable bases.

[0047] As used herein, the term "pharmaceutically acceptable acid" refers to any acid that is medicinally acceptable, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, formic acid, acetic acid, acetoacetic acid, trifluoroacetic acid, propionic acid, pyruvic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, stearic acid, palmitic acid, oxalic acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanedisulfonic acid, isethionic acid, 1,5-naphthalenedisulfonic acid, 2,5-dihydroxybenzoic ... -naphthalenesulfonic acid, camphorsulfonic acid, sulfamic acid, lactic acid, benzenesulfonic acid, p-toluenesulfonic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, lactic acid, tartaric acid, citric acid, malic acid, benzoic acid, salicylic acid, cinnamic acid, naphthoic acid, pamoic acid, nicotinic acid, orotic acid, methylsulfuric acid, dodecylsulfuric acid, glutamic acid, aspartic acid, gluconic acid, glucuronic acid, or any combination thereof.

[0048] As used herein, the term "pharmaceutically acceptable base" refers to a medicinal base, for example, an inorganic base (such as an alkali metal hydroxide or alkaline earth metal hydroxide) or an organic base (such as an amine (primary, secondary, or tertiary amine)). Examples of suitable salts include, but are not limited to, organic salts derived from amino acids, ammonia, primary, secondary, and tertiary amines, and cyclic amines (e.g., diethylamine salts, piperidine salts, morpholine salts, piperazine salts, choline salts, meglumine salts, tromethamine salts, etc.), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0049] The term "solvate" refers to a formulation formed by coordination of a compound of the present invention with a solvent molecule in its solid or liquid state. Hydrates are a specific form of solvates, in which the coordination is with water.

[0050] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their salts with pharmaceutically acceptable auxiliaries. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present invention to an organism.

[0051] The term "pharmaceutically acceptable adjuvant" refers to an adjuvant that does not have a significant irritating effect on the organism and does not impair the biological activity and performance of the active compound. Suitable adjuvants are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0052] The pharmaceutical compositions of the present invention can be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable adjuvants, and for oral administration, the pharmaceutical compositions can be prepared by mixing the active compounds with pharmaceutically acceptable adjuvants well known in the art, which can be used to formulate the compounds of the present invention into tablets, pills, lozenges, dragees, capsules, etc., and administer them orally to patients.

[0053] As used herein, an "individual" includes a human or a non-human animal. Exemplary human individuals include human individuals (referred to as patients) suffering from a disease (e.g., a disease described herein) or normal individuals. "Non-human animals" in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and / or farm animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0054] Beneficial effects The present invention provides an allopregnanolone derivative represented by Formula I, which has good physical / chemical stability and is suitable for oral administration, obtained by structurally modifying the hydroxyl group of allopregnanolone while retaining the pharmacological activity of allopregnanolone. The allopregnanolone derivative can exert its pharmacological effect by releasing the active drug into the body. The allopregnanolone derivative of the present invention has stable metabolism, good oral bioavailability, little toxicity or side effects, rapid onset of action after oral administration, and the ability to maintain stable physiological concentrations of allopregnanolone in the body for long periods of time. It can be formulated into an appropriate oral formulation, which can improve drug safety and patient compliance and administration convenience. [Brief description of the drawing] FIG. 1 shows the pharmacokinetic curve of allopregnanolone in the plasma of male rats orally administered with the compound of the present invention. [Figure 2] Pharmacokinetic curve of allopregnanolone in plasma of beagle dogs orally administered with the compound of the present invention. [Mode for Carrying Out the Invention] The compounds of the general formula of the present invention and their preparation methods and applications will be described in more detail below with reference to specific examples. It should be understood that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the claims of the present invention. Any technology realized based on the above content of the present invention is included in the scope of the claims of the present invention.

[0055] The intermediate compounds according to the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternative methods known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0056] The chemical reactions of the specific embodiments of the present invention are completed in a suitable solvent, which must be suitable for the chemical reactions of the present invention and the necessary reagents and materials. To obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic steps or reaction processes based on the existing embodiments.

[0057] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0058] In the following examples, experimental methods for which no specific conditions are specified generally follow conventional conditions or conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight, and the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0059] In the present invention, the following abbreviations are used: aq represents aqueous solution, DMSO represents dimethyl sulfoxide, EtOAc represents ethyl acetate, EtOH represents ethanol, TFA represents trifluoroacetic acid, i-PrOH represents isopropanol, ECS represents extracellular fluid, ICS represents intracellular fluid, and MI001 represents allopregnanolone.

[0060] Comparative Example 1: Preparation of Comparative Compound 1 (C1) Hydrochloride

[0061] [ka]

[0062] Step 1: A 1000 mL three-neck round-bottom flask was charged with MI001 (50.0 g, 157.0 mmol, 1.0 eq), Boc-L-Val-OH (tert-butoxycarbonyl-L-valine) (40.9 g, 188.2 mmol), 4-dimethylaminopyridine (1.9 g, 15.5 mmol), and 500 mL of dichloromethane. The reaction mixture was stirred and cooled to -5 to 10 °C under nitrogen gas protection. A solution of dicyclohexylcarbodiimide (38.9 g, 188.5 mmol) in 80 mL of dichloromethane was added dropwise. The reaction mixture was then incubated at this temperature for 3 h. The reaction was monitored by thin-layer chromatography (TLC) until completion, at which point the reaction was stopped. The reaction mixture was filtered, and the filter cake was washed with 100 mL of dichloromethane. The filtrate was concentrated under reduced pressure, and the crude product was purified by 100-200 mesh silica gel sand column chromatography (petroleum ether (60-90) / ethyl acetate = 20:1-10:1) to give an off-white waxy solid (78.2 g, yield 96.2%).

[0063] Step 2: A 1000 mL three-neck round-bottom flask was charged with the product from Step 1 (78 g, 150.6 mmol, 1.0 eq) and dichloromethane (320 mL). Under nitrogen gas protection and magnetic stirring, the system was cooled to 0-10 °C and trifluoroacetic acid (171.8 g, 1506 mmol) was rapidly added dropwise. The reaction was then allowed to proceed at 15-25 °C for 3 h to quench the reaction. The reaction mixture was poured into a solution of sodium bicarbonate (164.5 g, 1958 mmol), quenched with water (780 mL), and dichloromethane (700 mL) was added. The mixture was stirred and allowed to stand for separation to obtain the organic phase. The organic phase was further washed with 500 mL of purified water and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to give an off-white solid (59.5 g, 94.6% yield).

[0064] Step 3: 0.5 g of the above product was taken, and isopropanol (0.5 mL) and isopropyl acetate (7.5 mL) were added. The mixture was dissolved at room temperature until clear. Then, a solution of hydrochloric acid in ethyl acetate (0.6 mL, 2.0 M hydrochloric acid in ethyl acetate) was added dropwise. The temperature was lowered to 5-10°C, and a large amount of solid precipitated. The solid was suction filtered and dried to obtain the product (0.36 g, yield 66.5%, purity 99.90% by HPLC).

[0065] 1 H NMR(400 MHz,CDCl3)δ 8.83(brs,3H),5.23-5.14(m,1H),4.00-3.88(m,1H),2.52(t,J=8.7 Hz,2H),2.22-2.08(m,1H),2.11(s,3H),2.06-1.96(m,1H),1.86-1.08(m,18H),1.18(m,3H),1.17 (m,3H),1.04-0.88(m,1H),0.86-0.71(m,1H),0.80(s,3H),0.61(s,3H).MS m / z:418.3[M+H] + Example 1 Synthesis of Compound 1 and its Hydrochloride

[0066] [ka]

[0067] Synthesis of intermediate 1a: A 100 mL single-neck flask was charged with Boc-L-Val-OH-3-d (1.20 g, 5.5 mmol), MI001 (1.91 g, 6.0 mmol), 4-dimethylaminopyridine (0.07 g, 0.6 mmol), and dichloromethane (15 mL). A solution of dicyclohexylcarbodiimide (1.24 g, 6.0 mmol) in dichloromethane (5 mL) was added dropwise at 20 °C and stirred overnight. The dicyclohexylurea was removed by filtration, and the filtrate was concentrated. The resulting mixture was purified by column chromatography (petroleum ether / ethyl acetate = 20:1 to 7:1) to afford 2.3 g of Intermediate 1a (80% yield) as a colorless oil.

[0068] 1 H NMR(400 MHz,CDCl3)δ 5.10(m,1H),5.07(d,J=9.5 Hz,1H),4.23(d,J=9.1 Hz,1H),2.53(t,J=8.8 Hz,1H),2.26-2.08(m,1H),2.11(s,3H),2.06-1.95(m,1H),1.85-0.71(m,20H),1.46(s,9H),0.98 (s,3H),0.90(s,3H),0.80(s,3H),0.61(s,3H). Synthesis of Compound 1: Intermediate 1a (2.3 g, 4.4 mmol) and dichloromethane (15 mL) were added to a 100 mL single-neck flask and dissolved with stirring at 20 °C. Trifluoroacetic acid (5.02 g, 44.0 mmol) was then added dropwise. The temperature was controlled at 15-25 °C and the reaction was continued with stirring for 3-4 h, after which dichloromethane (20 mL) was added. The reaction mixture was gradually poured into an aqueous sodium bicarbonate solution (15 g / 50 mL) with stirring, and the mixture was stirred for 5-15 min. The mixture was then allowed to stand and separated. The organic phase was washed with purified water (50 mL), separated, and dried over anhydrous sodium sulfate. After filtration and concentration, compound 1 (1.73 g) was obtained.

[0069] Synthesis of the hydrochloride salt of compound 1 (1'): The obtained compound 1 (1.59 g, 3.8 mmol) was dissolved in ethyl acetate (20 mL) and isopropanol (1.3 mL), and a solution of hydrogen chloride in ethyl acetate (2.4 M, 1.6 mL, 3.8 mmol) was added dropwise. The mixture was stirred at 20 °C for 1 h, filtered, and the filter cake was washed with ethyl acetate (20 mL). The mixture was dried at 40 °C under oil pump vacuum (P ≦ −0.09 MPa) for 4 h to give a white solid (1.18 g, 68% yield).

[0070] 1H NMR(400 MHz,CDCl3)δ 8.83(brs,3H),5.23-5.14(m,1H),4.00-3.88(m,1H),2.52(t,J=8.7 Hz,1H),2.22-2.08(m,1H),2.11(s,3H),2.06-1.96(m,1H),1.86-1.08(m,18H),1.18(s,3H),1.17 (s,3H),1.04-0.88(m,1H),0.86-0.71(m,1H),0.80(s,3H),0.61(s,3H).MS m / z:419.28[M+H] + . Example 2 Synthesis of Compound 2 and its Hydrochloride Salt

[0071] [ka]

[0072] Synthesis of intermediate 2a: Under nitrogen gas protection, a 100 mL three-neck flask was charged with Boc-L-Val-OH-d6 (2 g, 8.95 mmol), dichloromethane (30 g), MI001 (2.85 g, 8.95 mmol), and 4-dimethylaminopyridine (0.11 g, 0.90 mmol). The mixture was cooled to -5 to 5 °C with stirring, and a solution of dicyclohexylcarbodiimide (2.1 g, 10 mmol) in dichloromethane (7.5 g) was added dropwise. The mixture was incubated at 15 to 25 °C for 3 h. The reaction was monitored by TLC until completion. The reaction mixture was washed with water, stirred, and separated. The resulting organic phase was dried over anhydrous sodium sulfate, concentrated, and then purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to give Intermediate 2a (3.2 g, 68.5% yield) as a colorless oil.

[0073] Synthesis of compound 2: The product 2a obtained in the previous step was dissolved in dichloromethane (16 mL), trifluoroacetic acid (10.7 g) was added, and the mixture was stirred at 15-25°C for 3-4 h. The reaction of the raw materials was monitored by TLC until completion. The reaction mixture was added to aqueous sodium bicarbonate (40 mL), the pH was adjusted to 7-8, dichloromethane (10 mL) was added, and the organic phase was separated and retained. The aqueous phase was further extracted once with dichloromethane (20 mL). The organic phases were combined and washed with water (5 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then concentrated to dryness until a solid was obtained. Acetonitrile (8 mL) was added to form a slurry, and the mixture was filtered to obtain compound 2 (1.2 g).

[0074] Synthesis of the hydrochloride salt of compound 2 (2'): Compound 2 (0.6 g) was taken, and isopropanol (0.6 mL) and isopropyl acetate (9 mL) were added. After dissolving at room temperature, a solution of hydrochloric acid in ethyl acetate (0.7 mL, 2.0 M hydrochloric acid in ethyl acetate) was added dropwise. The temperature was lowered to 5-10°C, and a large amount of solid precipitated. The solid was suction filtered and dried to obtain the product (0.3 g, yield 46.2%, purity HPLC 99.7%).

[0075] 1 H NMR(400 MHz,CDCl3)δ8.85(brs,3H),5.22(s,1H),3.95(brs,1H),2.54(t,J=8.7 Hz,1H),2.49 (s,1H),2.19-2.14(m,1H),2.14(s,3H),2.05-2.02(m,1H),1.84-1.72(m,5H),1.61-1.4 1(m,7H),1.32-1.17(m,6H),1.04-0.88(m,1H),0.85(m,1H),0.82(s,3H),0.63(s,3H).MS m / z:424.39[M+H] + . Example 3 Synthesis of Compound 3 and its Hydrochloride

[0076] [ka]

[0077] Synthesis of intermediate 3a: A 100 mL single-neck flask was charged with Boc-Val-OH-2-d (1.0 g, 4.6 mmol), MI001 (1.46 g, 4.6 mmol), 4-dimethylaminopyridine (0.06 g, 0.5 mmol), and dichloromethane (15 mL). A solution of dicyclohexylcarbodiimide (0.95 g, 4.6 mmol) in dichloromethane (5 mL) was added dropwise at 20 °C and stirred overnight. The mixture was filtered and the filtrate was concentrated. Column chromatography (petroleum ether / ethyl acetate = 20:1 to 7:1) afforded Intermediate 3a (1.6 g, 56% yield) as a colorless oil.

[0078] Synthesis of compound 3: The product, intermediate 3a, obtained in the previous step was dissolved in dichloromethane (8 g), trifluoroacetic acid (4.5 g) was added, and the mixture was stirred at 15-25°C for 3-4 h. The reaction of the raw materials was monitored by TLC until completion. The reaction mixture was added to 20 mL of aqueous sodium bicarbonate solution, the pH was adjusted to 7-8, dichloromethane (5 mL) was added, and the organic phase was separated and retained. The aqueous phase was further extracted once with dichloromethane (10 mL). The organic phases were combined and washed with water (5 mL x 3). The organic phase was dried over anhydrous sodium sulfate and then concentrated to dryness until a solid was obtained. 4 mL of acetonitrile was added to form a slurry, which was then filtered to obtain the free base (0.6 g).

[0079] Synthesis of the hydrochloride salt of compound 3 (3'): The above product, Compound 3 (0.5 g), was taken, and isopropanol (0.5 mL) and isopropyl acetate (7.5 mL) were added. After dissolving at room temperature, a solution of hydrogen chloride in ethyl acetate (0.6 mL, 2.0 M hydrogen chloride in ethyl acetate) was added dropwise. The temperature was lowered to 5-10°C, and a large amount of solid precipitated. The solid was suction filtered and dried to obtain the product (0.36 g, yield 66.5%, purity 99.70% by HPLC).

[0080] 1H NMR(400 MHz,CDCl3)δ8.84(brs,3H),5.21(s,1H),2.56-2.48(m,2H),2.21-2.17(m,1H),2.13 (s,3H),2.05-2.02(m,1H),1.83-1.36(m,12H),1.32-1.19(m,12H),1.04-0.95(m,1H),0.90-0.84(m,1H), 0.82(s,3H),0.63(s,3H).MS m / z:419.25 [M+H] + . Example 4 Synthesis of Compound 4 and its Hydrochloride

[0081] [ka]

[0082] Compound 4 and the hydrochloride salt (4') of compound 4 were prepared according to the synthesis method of Example 1 using MI001 and Boc-D-Val-OH-3-d as starting materials.

[0083] Hydrochloride salt of compound 4 (4'), a white solid. 1 H NMR(400 MHz,CDCl3) δ 8.76(brs,3H),5.16(brs,1H),4.00-3.88(m,1H),2.52(t,J=8.7 Hz,1H),2.22-2.08(m,1H),2.08(s,3H),2.06-1.96(m,1H),1.86-1.08(m,18H),1.18(s,3H),1.17(s,3H),1.04-0.88(m, 1H),0.86-0.71(m,1H),0.79(s,3H),0.61(s,3H).MS m / z:419.35[M+H] + . Example 5 Synthesis of Compound 5 and its Hydrochloride

[0084] [ka]

[0085] Compound 5 and the hydrochloride salt (5') of compound 5 were prepared according to the synthesis method of Example 2 using MI001 and Boc-D-Val-OH-d7 as starting materials.

[0086] Compound 5 hydrochloride (5'), a white solid. 1 H NMR(400 MHz,CDCl3) δ8.85(brs,3H),5.22(s,1H),3.95(brs,1H),2.54(t,J=8.7 Hz,1H),2.19-2.14(m,1H),2.14(s,3H),2.05-2.02(m,1H),1.84-1.72(m,5H),1.61-1.4 1(m,7H),1.32-1.17(m,6H),1.04-0.88(m,1H),0.85(m,1H),0.82(s,3H),0.63(s,3H).MS m / z:425.39 [M+H] + . Test example 1: Compound solubility experiment 1. Sample Preparation Preparation of external standard solution: 50 mg of the compound to be measured was precisely weighed and placed in a 10 mL volumetric flask. An appropriate amount of pure water was added and dissolved by ultrasonication. The solution was diluted to the mark and then mixed uniformly to obtain an external standard solution with a concentration of 5.0 mg / mL.

[0087] Preparation of solution for measurement: 1.0 g of the compound to be measured was precisely weighed and dissolved in 20 mL of pure water. The solution was dissolved by stirring at 25 °C for 24 hours, centrifuged, and the supernatant was removed and filtered through a 0.45 μm filter membrane to obtain a filtrate. 1 mL of the above filtrate was precisely weighed and placed in a 5 mL measuring flask. Diluted to the mark with pure water and mixed uniformly to obtain a solution for measurement.

[0088] 2. Measurement of saturated solubility by external standard method Chromatography conditions: Column: Waters XBridge C8 3.5 μm 4.6 × 100 mm NRT2019-21#, column temperature: 45 °C, detection wavelength: 205 nm.

[0089] Mobile phase A: 10 mM / L (NH4)2HPO4 solution, mobile phase B: acetonitrile, isocratic elution, A:B = 40:60, flow rate: 1.0 mL / min.

[0090] Sampling injection volume: 10 μL, run time: 10 min.

[0091] Using pure water as a blank control solution, the peak areas of the external standard solution and the standard solution were measured by high performance liquid chromatography with a UV detector. 外部標準 , A 測定待ち The saturated solubility C of the compound to be measured was calculated using the following formula: C=A 測定待ち / A 外部標準 ×5 mg / mL × 5. The specific results are shown in Table 2 below.

[0092] [Table 2]

[0093] Measurement example 2: Patch clamp experiment results The manual patch clamp method was used to detect the effects of compounds on hERG potassium channel currents in stably expressing Chinese hamster ovary cells. Drug inhibition of cardiac hERG potassium ion channel is the main cause of prolonged myocardial repolarization, and the IC of hERG was significantly increased. 50 Higher values ​​indicated lower cardiotoxicity.

[0094] Experimental materials: experimental compounds (compound 1 prepared by the method of the present invention, comparative compound 1), dimethyl sulfoxide (Sigma-Aldrich (Shanghai) Tracing Co., Ltd.), cisapride (positive control, commercially available product), Chinese hamster ovary (CHO) cell line, CHO-hERG cells (Sophion Biotechnology Co., Ltd.).

[0095] Manual Patch Clamp Test Method: Exponentially growing CHO-hERG cells were harvested and resuspended in extracellular solution (ECS) for use. The cells were seeded into a recording cell and placed on an inverted microscope stage. Randomly selected cells in the recording cell were then tested. A perfusion system was attached to the inverted microscope stage, and the cells were continuously perfused with ECS.

[0096] Manual patch clamp recording microelectrodes were prepared using glass capillaries filled with intracellular solution. On the day of patch clamp testing, electrodes were prepared using borosilicate glass tubing (GC150TF-10, Harvard Apparatus Co., UK). After filling the electrodes with ICS, the resistance was between 2 and 5 MΩ.

[0097] The clamp voltage was -80 mV, and the initial step was depolarized to +60 mV and maintained for 850 ms to open the hERG channels. The voltage was then set to -50 mV and maintained for 1275 ms, generating a repulsive current, or tail current. The peak value of the tail current was measured and used for analysis. Finally, the voltage was returned to the clamp voltage (-80 mV). During the initial recording phase of perfusion with the solvent control working solution, the tail current peak was monitored until three or more scan curves were stable. Then, the test sample / positive control working solution was perfused until the inhibitory effect of the test sample / positive control working solution on the hERG current peak reached a steady state.

[0098] hERG currents were recorded using the whole-cell patch clamp technique at room temperature. The output signal of the patch clamp amplifier was digital-to-analog converted and low-pass filtered at 2.9 kHz. Data were collected and recorded using Patchmaster Pro software, and processed using Origin 8E software to obtain the IC of hERG. 50 The values ​​were calculated and the experimental data results are shown in Table 3 below.

[0099] [Table 3]

[0100] Drug inhibition of the cardiac hERG potassium ion channel is the main cause of prolonged myocardial repolarization, and the inhibition of cardiac repolarization was observed at half the inhibitory concentration (IC) of Compound 1 against hERG. 50 ) value was 1.93 μM. Compared with the comparative compound 1, the compound 1 of the present invention had lower hERG inhibitory activity, and lower cardiac toxicity and side effects.

[0101] Testing example 3: Metabolic research The metabolic stability of the compounds of the examples and the rate of production of the active substance allopregnanolone were determined in vitro (human liver microsome incubation system).

[0102] Experimental materials and reagents: Human liver microsomes (Corning, catalog number 452117), testosterone (Jiuding Chemical Co., Ltd.), propafenone (Anpu Co., Ltd.), diclofenac, tolbutamide, acetonitrile, and DMSO were obtained from Sigma; NADPH (reduced coenzyme II) was obtained from Chem-Impex International; 0.1 M pH 7.4 PBS (phosphate buffer, self-made); all other reagents were analytical reagents.

[0103] Instrumentation, conditions, and parameters: Liquid chromatography-mass spectrometry (LC / MS / MS, Shimadzu LC 30-AD, MS API 4000) with an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm column, Part No. 186002350), mobile phase acetonitrile-water-formic acid (50:50:0.1), flow rate 0.7 mL / min, sample injection volume 5 μL, column temperature room temperature. Electrospray ionization (ESI) source, spray voltage 4.8 KV, capillary temperature (TEM) 300 °C, sheath gas N2, flow rate 10 psi, assist gas N2, flow rate 1 psi, collision gas (CID) Ar, pressure 1.5 mTorr. Mass spectrometry scanning method was mass spectrometric multiple reaction monitoring (MRM) with positive ion detection. The internal standard was a solution of tolbutamide at 0.2 μg / mL in acetonitrile, with a minimum quantitation limit of 5 ng / mL and a correlation coefficient of >0.99.

[0104] In vitro metabolism study method: Testosterone, propafenone, or diclofenac was used as the reference validation detection system, and allopregnanolone (compound MI001) and comparative compound 1 were used as references. In vitro tests were conducted in a human liver microsome incubation system to observe the concentration decrease rate of the compounds of the examples and the production rate of MI001, and the in vitro metabolic stability of each compound of the examples and its ability to maintain the concentration of MI001 in liver microsomes were evaluated.

[0105] Approximately 10 mg of each sample to be measured was precisely weighed, dissolved in 0.1 mL of DMSO, and serially diluted with pure water to 10 μM and 1 μM standard stock solutions. The detection incubation system was prepared in an ice bath according to Table 3. NADPH was added to the incubation system (see Table 4 for its composition) to initiate the reaction. 50 μL was immediately transferred to 150 μL of acetonitrile to serve as the zero time point sample and 1 μM standard curve sample. The 1 μM standard stock solution was then added to the incubation system, and 50 μL was immediately transferred to 150 μL of acetonitrile to serve as the 0.1 μM standard curve sample. The remaining system was placed in a 37°C water bath, and 50 μL was transferred to 150 μL of acetonitrile at 5 min, 15 min, 30 min, 1 h, and 2 h. Each sample was shaken and centrifuged at 18,000 g for 10 min. The supernatant was collected and analyzed by LC / MS / MS. Experimental data results for some example compounds are shown in Table 5 below.

[0106] [Table 4]

[0107] [Table 5]

[0108] The metabolism of testosterone, propafenone, or diclofenac demonstrated that the detection system was normal. The results of comparative compound 1 and the example compounds indicated that the compounds of the present invention have good metabolic stability in human liver microsomes. The concentration of allopregnanolone metabolized from the example compounds in the liver microsome system could rapidly reach a stabilized level, while comparative compound 1 could not reach a stabilized level.

[0109] Testing example 4: Study of pharmacokinetic properties 1. Pharmacokinetic study in SD rats The purpose of this experiment was to study the single oral administration of the compound of the present invention solution and allopregnanolone solution in SD rats, detect allopregnanolone, the active ingredient in plasma, and evaluate the pharmacokinetic (PK) characteristics in SD rats.

[0110] Experimental materials: Male SD rats (body weight 180 - 220 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK(Beijing)2016 - 0006), experimental compound (manufactured according to the method of the examples of the present invention), purified water (self - made).

[0111] Experimental method: Male SD rats were randomly grouped (3 rats per group). During the test period, they were allowed to freely ingest water, fasted for more than 12 h before administration, and fed 4 h after administration. Forced oral administration was performed, and each group of SD rats was administered a 5% Tween aqueous solution of the experimental compound at a dose of 20 mg / kg (based on the amount of allopregnanolone).

[0112] Blood samples were collected into K2EDTA anticoagulant tubes at 0 min before administration, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h after administration, and temporarily stored on ice until centrifugation.

[0113] Plasma was centrifuged within 60 min after blood collection (centrifuged at 8000 rpm for 5 min under the condition of 2 - 8°C). After centrifugation, the plasma was transferred to a 96 - well plate or a centrifuge tube, transported in an ice case, and stored at ≤ - 15°C until LC - MS / MS detection. The drug concentration in SD rat plasma was measured by the LC - MS / MS biological analysis method. Using the non - compartmental model, WinNonlinTM (Version 8.3, Certara, USA) was used to analyze the blood drug concentration - time data, evaluate the pharmacokinetic (PK) characteristics in SD rats. The data are shown in Table 6, and the pharmacokinetic curve is shown in Figure 1.

[0114]

Table 6

[0115] 2. Pharmacokinetic study in beagle dogs The present inventors have found through heterologous liver microsome stability experiments that the metabolism of the compounds of the present invention in heterologous liver microsomes is essentially similar, and that the metabolic behavior in beagle dog liver microsomes is closest to that in human liver microsomes. The purpose of this experiment was to study the single oral administration of each solution of the compounds of the present invention to beagle dogs, detect the active ingredient allopregnanolone in plasma, and evaluate the drug metabolism kinetics (PK) characteristics in beagle dogs.

[0116] Test materials: male beagle dogs (weight 6-15 kg, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), experimental compounds (prepared according to the methods of the examples of the present invention), purified water (self-prepared).

[0117] Experimental method: Male beagle dogs were randomly divided into groups (3 dogs per group). They were allowed free access to water during the test period, fasted for at least 12 hours before administration, and fed 4 hours after administration. Each group was given a 5% Tween solution of the experimental compound at a dose of 10 mg / kg (based on the amount of allopregnanolone) by oral gavage.

[0118] Blood samples were collected into K2EDTA anticoagulant tubes at 0 min before administration, and at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h after administration, and temporarily stored on ice until centrifugation.

[0119] Plasma was centrifuged within 30 min after blood collection (10 min at 3200 rpm at 2-8°C). After centrifugation, the plasma was transferred to a 96-well plate or centrifuge tubes, transported in ice cases, and stored at ≤-60°C until detection by LC-MS / MS. Drug concentrations in beagle dog plasma were measured using LC-MS / MS bioanalysis. Blood drug concentration-time data were analyzed using WinNonlin (Version 6.3 or later) using a non-compartmental model to evaluate the drug's metabolic kinetics (PK) characteristics in beagle dogs. The data are shown in Table 7, and the pharmacokinetic curves are shown in Figure 2.

[0120] [Table 7]

[0121] The above results show that the compounds of the present invention can significantly improve the pharmacokinetic properties, especially the AUC and C max All of these were significantly improved, demonstrating that the compound of the present invention can be administered orally, which can largely overcome the shortcomings of intravenous allopregnanolone formulations, such as the long administration time and the need for constant attention by medical staff, and can greatly improve patient compliance and the convenience of administration for medical staff.

[0122] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention. [Brief explanation of the drawings]

[0123] [Figure 1] 1 is a pharmacokinetic curve of allopregnanolone in plasma of male rats orally administered with a compound of the present invention. [Figure 2] 1 is a pharmacokinetic curve of allopregnanolone in plasma of beagle dogs orally administered with a compound of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: 【Chemical 1】 Among them, the compound of formula I is a compound of formula Ia: 【Chemistry 2】 Among them, R 2 , R 4 are each independently selected from H or D (deuterium); R 1 , R 3 are each independently CH 3 , C.H. 2 D, CHD 2 or CD 3 Selected from R 1 , R 2 , R 3 , and R 4 each contain at least one deuterium atom; A compound of formula I or a pharmaceutically acceptable salt thereof.

2. The compounds according to formula I contain 1, 2, 3, 4, 5, 6, 7 or 8 deuterium atoms, The compound according to claim 1 .

3. R 2 is D, The compound according to claim 1 .

4. The compound according to formula I is selected from the following structures: The compound according to claim 1 . 【Chemistry 3】

5. A method for producing the compound according to any one of claims 1 to 4, comprising the steps of: reacting a compound of formula II with a compound of formula III and removing the protecting group to obtain a compound of formula I; 【Chemistry 4】 Among them, R 1 ~R 4 is as defined in any one of claims 1 to 4, and R 5 is a protecting group.

6. A pharmaceutical composition comprising at least one compound of formula I according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants; The adjuvant is a binder, diluent, disintegrant, lubricant, glidant, sweetener or flavoring agent. The pharmaceutical composition according to claim 6.

8. The pharmaceutical composition is a tablet, pill, lozenge, sugar-coated tablet, or capsule. The pharmaceutical composition according to claim 6.

9. Use of a compound of formula I according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the manufacture of a drug for use in the prevention or treatment of central nervous system diseases or for sedation-hypnosis.

10. The central nervous system diseases are traumatic brain injury, essential tremor, epilepsy, depression and Alzheimer's disease.

10. The use according to claim 9.

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