Steroid compound, application and preparation method thereof and pharmaceutical composition containing steroid compound

Abstract

The invention relates to the field of compounds, and discloses a steroidal compound with a structure of a general formula I, application and a preparation method thereof and a pharmaceutical composition containing the steroidal compound, the steroidal compound has a relatively strong positive regulation effect on a GABAA receptor, can be used for preparing oral medicines for resisting severe depression and postpartum depression, and has the characteristic of high efficiency.

Description

Technical Field

[0001] The present invention relates to the field of compounds, and in particular to a new steroid compound, a preparation method, and the use of the steroid compound as a drug preparation, as well as a pharmaceutical composition containing the steroid compound and the use of the pharmaceutical composition as a drug preparation. Background Art

[0002] Depression is usually a mood disorder with low mood as the main clinical manifestation. It is manifested as three lows: low mood, decreased will, and slow thinking. Pessimism, despair, delusion, loss of appetite, functional impairment, serious suicide attempts and even suicidal behavior may occur, which will pose a serious threat to human health and must be taken seriously and treated in time.

[0003] At present, the drugs used to treat depression can be divided into five categories according to their mechanism of action: A. tricyclic antidepressants; B. monoamine oxidase inhibitors (MAOI); C. selective serotonin reuptake inhibitors (SSRI); D. selective norepinephrine (NA) reuptake inhibitors; E. serotonin and norepinephrine reuptake inhibitors. Traditional antidepressants do not have ideal therapeutic effects on severe depression and postpartum depression. Instead, they have very obvious toxic side effects of varying degrees, such as anorexia, diarrhea, nausea, etc., and may also cause headaches, fatigue, insomnia, anxiety, tremors, sexual dysfunction (including impotence, decreased libido), drowsiness, etc.

[0004] On March 19, 2019, the U.S. FDA approved Brexanolone (Brexanolone, trade name Zulresso) injection for the treatment of postpartum depression in adult women. This is the first drug in history specifically for postpartum depression (PPD). Brexanolone is a synthetic allopregnanolone analog that can enhance the expression of recombinant GABA A The current mediated by GABA in mammalian cells with receptors. However, the administration of brexanolone is continuous intravenous infusion for more than 60 hours. During the two and a half days of brexanolone infusion, medical staff must be on site to continuously monitor the patient, which is particularly inconvenient.

[0005] Therefore, due to the low oral bioavailability of existing drugs such as brexanolone, there is an urgent need to develop the next generation of synthetic oral allopregnanolone analogs for the treatment of major depression and postpartum depression. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a steroid compound having a structure of general formula I and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds,

[0007]

[0008] in,

[0009] R1, R2, R3, R4, R5, R x are respectively selected from hydrogen, fluorine, chlorine, bromine, iodine, carbon, nitrogen, oxygen, sulfur, methyl, monofluoromethyl, difluoromethyl, trifluoromethyl, cyano, acyl, formaldehyde oxime, formaldehyde oxime ether, nitro, and carboxyl, wherein the acyl is selected from alkanoyl and halogen substituents thereof, alkenoyl and halogen substituents thereof, alkynyl and halogen substituents thereof, alkoxycarbonyl and halogen substituents thereof, alkenyloxycarbonyl and halogen substituents thereof, alkynyloxycarbonyl and halogen substituents thereof, aryloxycarbonyl and halogen substituents thereof, heteroaryloxycarbonyl and halogen substituents thereof, alkylaminocarbonyl and halogen substituents thereof, enaminocarbonyl and halogen substituents thereof, alkynylaminocarbonyl and halogen substituents thereof, arylaminocarbonyl and halogen substituents thereof, and heteroarylaminocarbonyl and halogen substituents thereof;

[0010] C1-C6 alkanes and halogen-substituted products thereof, C1-C6 ethers and halogen-substituted products thereof, C1-C6 alkoxy fatty chains and halogen-substituted products thereof, C1-C6 alkylthiol fatty chains and halogen-substituted products thereof, C1-C6 alkylamino fatty chains and halogen-substituted products thereof, C2-C6 olefins and halogen-substituted products thereof, C2-C6 alkynes and halogen-substituted products thereof;

[0011] Five-membered ring aromatic hydrocarbon group, six-membered ring aromatic hydrocarbon group, C3-C7 cycloalkane group;

[0012] A five-membered heterocyclic aromatic hydrocarbon group containing nitrogen, oxygen or sulfur;

[0013] Six-membered nitrogen heterocyclic aromatic hydrocarbon group;

[0014] Heterocycloalkane containing nitrogen, oxygen or sulfur;

[0015] Hydroxyl, amino, thiol;

[0016] A hydroxyl group, an amino group, a thiol group connected to a five-membered aromatic hydrocarbon, a six-membered aromatic hydrocarbon, a C3-C7 cycloalkane, a five-membered heteroaromatic hydrocarbon containing nitrogen, oxygen or sulfur, a six-membered nitrogen heteroaromatic hydrocarbon, or a heterocycloalkane containing nitrogen, oxygen or sulfur,

[0017] Protective groups such as esters and amides formed by inorganic acids or C1-C6 organic acids with hydroxyl groups and amino groups;

[0018] M is selected from carbon, nitrogen, oxygen, sulfur, and phosphorus;

[0019] When M is nitrogen, one of R2 or R3 is absent, and a nitrogen oxide may be formed; or both R2 and R3 are present, and a quaternary ammonium salt may be formed;

[0020] wherein, when M is oxygen, R2 or R3 is absent;

[0021] Wherein, when M is sulfur, both R2 and R3 are present to form a sulfone;

[0022] or, one of R2 or R3 is absent, forming a sulfoxide,

[0023] Or, neither R2 nor R3 exists.

[0024] According to one embodiment of the present invention, a steroid compound having a structure of general formula I and a stereoisomer, a pharmaceutically acceptable salt, a hydrate, a solvate or an isotope compound thereof, R x The plane refers to the plane or approximate plane of the heterocyclic ring containing M and N.

[0025] According to one embodiment of the present invention, a steroid compound having a structure of general formula I and a stereoisomer, a pharmaceutically acceptable salt, a hydrate, a solvate or an isotope compound thereof, R x Facing outward.

[0026] According to one embodiment of the present invention, a steroid compound having a structure of the general formula I and a stereoisomer thereof, a pharmaceutically acceptable salt, a hydrate, a solvate or an isotope compound, R2 and R3 are connected together to form a C8-C12 spiro ring, a C8-C12 azaspiro ring, a C8-C12 oxaspiro ring, or R2, R x Connected together to form a C8-C12 bridge ring, a C8-C12 nitrogen bridge ring, and a C8-C12 oxygen bridge ring.

[0027] According to one embodiment of the present invention, in the steroid compound having the structure of general formula I and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds, R1, R2, R3, R4, R5 are not hydrogen.

[0028] According to one embodiment of the present invention, in the steroid compound having the structure of general formula I and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds, R1, R2, R3, R4, R5 has and only one is hydrogen; or, R1, R2, R3, R4, R5 has and only two are hydrogen; or, R1, R2, R3, R4, R5 has and only three are hydrogen; or R1, R2, R3, R4, R5 has and only four are hydrogen; or, R1, R2, R3, R4, R5 are all hydrogen.

[0029] According to one embodiment of the present invention, the steroid compound having the general formula I is synthesized by the following route:

[0030] The compound B1 is subjected to a catalytic hydrogenation reaction to obtain a compound B2;

[0031] Methylating compound B2 to obtain compound B3;

[0032] Compound B3 is subjected to ylide reaction to obtain compound B4;

[0033] Compound B4 is subjected to a hydroboration oxidation reaction to obtain compound B5;

[0034] The compound B5 is subjected to an oxidation reaction to obtain a compound B6;

[0035] Compound B6 is subjected to a haloform reaction to obtain compound B;

[0036] Compound B is subjected to condensation reaction with fragment Q to obtain a steroid compound of the general formula I.

[0037] Among them, compound B1 is 19-nor-4-androstenedione, compound B is (3R, 5R, 8R, 9R, 10S, 13S, 14S, 17S)-3-hydroxy-3, 13-dimethylestrone-17-oic acid, and compound Q is a nitrogen-containing six-membered heterocyclic ring substituted with M at the para position.

[0038]

[0039] The term "heteroaryl", "heterocyclic aromatic hydrocarbon group" as used herein refers to an aromatic ring group containing a monocyclic or multiple condensed (fused) rings (e.g., containing 2 or 3 rings) of 5 to 14 ring atoms in the ring, wherein in addition to carbon atoms, the ring atoms also contain at least one heteroatom selected from oxygen, nitrogen and / or sulfur. If the ring is aromatic, the sulfur atom and the nitrogen atom may also exist in oxidized form. Multiple condensed (fused) ring heteroaryl is a monocyclic heteroaryl as defined above fused with one or more rings selected from the following to form a multiple condensed ring system: heteroaryl (to form, for example, naphthyridinyl, such as 1,8-naphthyridinyl), heterocyclic (for example, to form 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocyclic (to form, for example, 5,6,7,8-tetrahydroquinolinyl) and aryl (to form, for example, indazolyl). Such multiple fused ring systems can be optionally substituted with one or more (e.g., 1, 2, 3, or 4) oxo groups on the carbocyclic or heterocyclic portions of the fused rings. When valence requirements permit, the rings of the multiple fused ring systems can be interconnected by fusion, spirocyclic, and bridging bonds. It should be understood that the individual rings of the multiple fused ring systems can be connected to each other in any order. It should also be understood that the attachment point of the multiple fused ring system can be at any position of the multiple fused ring system, including heteroaryl, heterocyclic, aryl, or carbocyclic portions of the multiple fused system. It should also be understood that the attachment point of the heteroaryl group can be on any suitable atom of the heteroaryl group, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryl groups include, but are not limited to, pyridinyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzimidazolyl, thiaindenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 3b,4,4a,5-tetrahydro-1H-cyclopropane[3,4]cyclopenta[1,2-c]pyrazolyl.

[0040] As used herein, the terms "heterocyclyl" and "heterocycloalkane" refer to monocyclic saturated or partially unsaturated groups having 3 to 8-membered monocyclic rings or multiple condensed (fused) rings or bridged rings having 3 to 14 ring atoms in the ring, wherein in addition to carbon atoms, the ring atoms also contain at least one heteroatom selected from oxygen, nitrogen and / or sulfur. Examples of heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, morpholinyl, piperidinyl, piperazinyl, dihydropyridinyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl, benzo[d]imidazolyl, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl, aziridine, azetidine, tetrahydropyrrolyl, azepanyl, azooctanyl, Oxirane, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepanyl, oxocanyl, thiirane, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, thiocanyl, tetrahydroimidazolyl, tetrahydropyrazolyl, tetrahydrooxazolyl, tetrahydroisoxazolyl, tetrahydrothiazolyl, tetrahydroisothiazolyl, dioxanyl, thioxanyl, dithianyl and the like.

[0041] The term "stereoisomer" as used herein refers to a compound having the same chemical composition and connectivity, but whose atoms have different orientations in space that cannot be interchanged by single bond rotation. "Stereoisomer" includes "diastereomers" and "enantiomers". "Diastereomers" refers to stereoisomers with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral characteristics and reactivity. Diastereomeric mixtures can be separated under high-resolution analytical procedures (such as crystallization, electrophoresis and chromatography). "Enantiomers" refers to two stereoisomers of compounds that are non-overlapping mirror images of each other.

[0042] As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ only in the position and electron distribution of one (or more) mobile atoms, such as keto-enol tautomers.

[0043] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0044] Any formula or structure given herein, including Formula I or any formula disclosed herein, is also intended to represent unlabeled forms of the compound as well as isotopically labeled forms. These forms of the compound may also be referred to as "isotopically labeled" or "isotopically enriched analogs." An isotopically labeled compound has a structure as depicted herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as, but not limited to, 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 Cl, 123 I and 125 I. Such compounds can be synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced with deuterium.

[0045] As used herein, the terms "solvate" and "solvate" refer to an association or complex of one or more solvent molecules and a compound of the invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0046] The term "pharmaceutically acceptable salt" as used herein refers to salts which are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and which are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1 19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed of amino groups with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts formed using methods used in the art, such as ion exchange methods. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C1-C4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts and amine cations formed using counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates, if appropriate.

[0047] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a steroid compound having the structure of general formula I and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds as active ingredients.

[0048] According to one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent.

[0049] According to another aspect of the present invention, the aforementioned pharmaceutical composition is used as a drug for preparing a drug for treating central nervous system-related diseases, and central nervous system-related diseases include: postpartum or postpartum depression, clinical depression, atypical depression, major depressive disorder, catatonic depression, dysthymia, double depression, depressive personality disorder, recurrent brief depression, mild depressive disorder, bipolar disorder, manic-depressive disorder, mood disorder, anxiety, post-traumatic stress disorder (PTSD), premenstrual dysphoric disorder (PMDD), depression caused by chronic medical conditions, difficult Treatment of depression, suicidal tendencies, suicidal ideation or behavior, premenstrual syndrome, generalized anxiety disorder, seasonal affective disorder (SAD), social anxiety disorder, memory loss, stress intolerance, Niemann-Pick disease type C or related neurological or physical symptoms, epilepsy, essential tremor, epileptiform disorders, NMDA hypofunction, migraine, status epilepticus, sleep disorders, fragile X syndrome, 5-alpha reductase inhibitor-induced depression, PCDH19 female pediatric epilepsy, sexual dysfunction, cognitive impairment, Parkinson's disease or Alzheimer's disease.

[0050] According to another aspect of the present invention, the aforementioned steroid compound having the structure of general formula I and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds are used as a drug for preparing a drug for treating central nervous system-related diseases, and central nervous system-related diseases include: postpartum or postpartum depression, clinical depression, atypical depression, major depressive disorder, tension depression, dysthymia, double depression, depressive personality disorder, recurrent short-term depression, mild depressive disorder, bipolar disorder, manic-depressive disorder, mood disorder, anxiety, post-traumatic stress disorder (PTSD), premenstrual dysphoria (PMDD), depression due to a chronic medical condition, treatment-resistant depression, suicidal tendencies, suicidal ideation or behavior, premenstrual syndrome, generalized anxiety disorder, seasonal affective disorder (SAD), social anxiety disorder, memory loss, stress intolerance, Niemann-Pick disease type C or related neurological or physical symptoms, epilepsy, essential tremor, epileptiform disorder, NMDA hypofunction, migraine, status epilepticus, sleep disturbance, fragile X syndrome, 5-alpha reductase inhibitor-induced depression, PCDH19 female pediatric epilepsy, sexual dysfunction, cognitive impairment, Parkinson's disease or Alzheimer's disease.

[0051] Pharmaceutical compositions and administration

[0052] The compound or its pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate and pharmaceutically acceptable salt provided by the present invention are usually administered in the form of a pharmaceutical composition. The present invention provides a pharmaceutical composition, which comprises a compound or its pharmaceutically acceptable salt, stereoisomer, tautomer, prodrug, solvate and pharmaceutically acceptable salt provided by the present invention as an active ingredient and one or more pharmaceutically acceptable carriers. The pharmaceutical composition can be administered alone or in combination with other therapeutic agents. Such compositions are prepared in a manner well known in the art, such as Reminton's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (GS Banker & C.T. Rhodes, Eds.).

[0053] Pharmaceutically acceptable carriers can be solid or liquid. Wherein, solid carriers can be one or more substances used as excipients, diluents, sweeteners, solubilizers, lubricants, adhesives, tablet disintegrating agents, stabilizers, preservatives or encapsulating materials. Liquid carriers can be solvents or liquid dispersion media. Suitable solid carriers include, but are not limited to, for example, cellulose, glucose, lactose, mannitol, magnesium stearate, magnesium carbonate, sodium carbonate, saccharin sodium, sucrose, dextrin, talc, starch, pectin, gelatin, tragacanth gum, gum arabic, sodium alginate, parabens, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter, etc. Suitable liquid carriers include, but are not limited to, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil), glycerides, agar, pyrogen-free water, isotonic saline, Ringer's solution and mixtures thereof.

[0054] The pharmaceutical composition according to the present invention can be in any form suitable for the intended method of administration. For example, when used for oral use, it can be prepared into tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs, solutions, sprays. The composition for oral use can be prepared according to any method known in the art for making pharmaceutical compositions. The pharmaceutical composition of the present invention can also be prepared into preparations suitable for intrapulmonary or intranasal administration, such as preparations, nasal drops or nasal sprays for aerosol or dry powder administration. The pharmaceutical composition of the present invention can also be prepared into suppositories suitable for rectal administration. The pharmaceutical composition of the present invention can also be prepared into transdermal preparations for topical administration or eye drops suitable for ocular administration.

[0055] The pharmaceutical composition of the present invention can be administered parenterally, intramuscularly, subcutaneously, orally, topically in single doses or multiple doses.

[0056] The effective dose of the compounds of the present invention depends at least on the nature of the condition being treated, toxicity, delivery method and pharmaceutical formulation, and will be determined by a clinician using conventional dose escalation studies. It can be expected that about 0.0001 to about 100 mg per kilogram of body weight per day; generally about 0.01 to about 10 mg per kilogram of body weight per day; more typically, about 0.01 to about 5 mg per kilogram of body weight per day; most typically about 0.05 to about 0.5 mg per kilogram of body weight per day. For example, the daily candidate dose for an adult of about 70 kg body weight will be in the range of 1 mg to 1000 mg, preferably in the range of 5 mg to 500 mg, and can be in the form of a single dose or multiple doses.

[0057] The steroid compound, stereoisomer, pharmaceutically acceptable salt, hydrate, solvate or isotope compound of the general formula I structure of the present invention can simultaneously act on multiple GABA on the synaptic surface through allosteric regulation. A Ion channel receptors promote the flow of chloride ions into cells along the electrochemical gradient of the GABA receptor complex (GRC). The increase in the level of this anion in the cell leads to hyperpolarization of the transmembrane potential, making neurons less sensitive to excitatory input, that is, brain excitability is reduced. The compound of the present invention not only has an efficient process of action, but also can solve the problem of inconvenience caused to patients by long-term intravenous drip. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The trend of the remaining percentage of each compound is shown. DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0059] It should be understood that the terms "include" and "comprising" used in the description and claims of the present invention indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0060] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the claims, the singular forms of "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise. It should also be further understood that the term "and / or" used in the specification of the present invention and the claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes these combinations.

[0061] Table 1 shows preferred compounds of the present invention.

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Before synthesizing the above compounds, it is necessary to first prepare intermediates.

[0069] Preparation and synthesis of intermediate B: Compound B1 is commercially available 19-nor-4-androstenedione.

[0070]

[0071] in,

[0072] The first step is the synthesis of compound B2

[0073] In a three-necked flask, add compound B1, add tetrahydrofuran to dissolve, then add 10% palladium carbon and hydrobromic acid (48% content), replace the atmosphere with nitrogen, replace the atmosphere with hydrogen, stir at room temperature under a hydrogen environment, leave overnight, monitor the completion of the reaction by TLC, filter through celite, concentrate under reduced pressure, remove the solvent, crystallize with acetone, filter, and obtain B2.

[0074] Step 2 Synthesis of Compound B3

[0075] In a three-necked flask, add 2,6-di-tert-butyl-p-cresol, add tetrahydrofuran to dissolve, cool to -78°C, then add 2M trimethylaluminum toluene solution, after the addition is complete, warm the reaction solution to room temperature, react for 1.5 hours, nitrogen protection, then cool the reaction solution to -78°C, add the toluene solution of compound B2, keep warm for 1.5 hours, then drop 3M methylmagnesium bromide tetrahydrofuran solution to the reaction solution, control the temperature below -75°C, after the drop is complete, keep warm for 1.5 hours, warm to room temperature, stir and react overnight. TLC monitoring, drop saturated ammonium chloride solution, control the temperature not higher than 5°C, the reaction solution has bubbles, and a large amount of white solid precipitates, filter, take the mother liquor, wash with water, dry, concentrate under reduced pressure, and purify by column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain oil product B3.

[0076] Step 3 Synthesis of Compound B4

[0077] Add ethyl triphenylphosphine bromide to a three-necked flask, add tetrahydrofuran to dissolve, cool to 0°C, then dissolve potassium tert-butoxide in tetrahydrofuran and add to the reaction solution. After completion, heat to 60°C, add B3 in tetrahydrofuran, react at 60°C, monitor by TLC, cool to room temperature, add saturated ammonium chloride solution, concentrate under reduced pressure, add ethyl acetate, separate the organic phase, and wash the organic phase with water. The organic phase is distilled under reduced pressure, and the residue is separated by column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain product B4.

[0078] Step 4 Synthesis of Compound B5

[0079] Add compound B4 to a three-necked flask, add tetrahydrofuran to dissolve, cool to 0°C, add 10M borane dimethyl sulfide complex dimethyl sulfide solution under nitrogen protection, control the temperature not higher than 10°C, complete the dropwise reaction, and react at room temperature for 3h to produce a white precipitate. Monitor by TLC, add sodium hydroxide aqueous solution, wait for the reaction solution to become translucent, add 30% hydrogen peroxide aqueous solution, and produce a white precipitate, control the temperature not higher than 10°C throughout the process, add sodium thiosulfate solution after the reaction is completed, quench, add ethyl acetate, stir, separate, wash the organic phase with water, and concentrate under reduced pressure to obtain product B5.

[0080] Step 5 Synthesis of Compound B6

[0081] In a 1L three-necked flask, add compound B5, add dichloromethane to dissolve, weigh pyridinium chlorochromate, add to the reaction solution, stir at room temperature, and react overnight. Monitor by TLC, add sodium sulfite solution, quench, separate, retain the organic phase, add dichloromethane to the aqueous phase for extraction, wash the organic phase with water, distill under reduced pressure, and purify by column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain product B6.

[0082] Synthesis of intermediate B

[0083] In a three-necked flask, add sodium hydroxide solution, cool to 0°C, drop bromine, dissolve completely, add dioxane, stir at room temperature, react for 2h, dissolve compound B6 in dioxane and water, add the reaction solution, stir at room temperature, overnight. Monitor by TLC, add sodium sulfite solution to quench, heat to 80°C to react for 3h, adjust pH to 2-3 with 1M dilute sulfuric acid aqueous solution at room temperature, and white solid precipitates, filter and dry to obtain product B.

[0084] Example 1

[0085] Synthesis of compound 1

[0086]

[0087] Compound B was added to the reaction flask, DMF was added to dissolve, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added, the reaction was stirred at room temperature for 20 minutes, methylpiperazine was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, water and ethyl acetate were added, the layers were stirred, the organic phase was washed with water, distilled under reduced pressure, and column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) was performed to obtain an off-white solid 1.

[0088] 1 H NMR (400MHz, chloroform-d) δ3.77-3.68(m,4H),2.71-2.62(m,5H),2.48(s,3H),2.38-2.28(m,2H),1.85-1.7 6(m,4H),1.71-1.61(m,3H),1.49-1.40(m,5H),1.34-1.23(m,10H),1.13-1.04(m,3H),0.71(s,3H).

[0089] MS: m / z[M+H] + 403.35.

[0090] Example 2

[0091] Synthesis of compound 4

[0092]

[0093] Add compound B and DMF to the reaction bottle to dissolve, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), stir at room temperature, react for 20 minutes, add morpholine, and react at room temperature overnight. After the reaction, add water and ethyl acetate, stir and separate, wash the organic phase with water, distill under reduced pressure, and column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain an off-white solid 4.

[0094] 1 H NMR (400MHz, chloroform-d) δ3.63-3.58(m,8H), 2.69-2.65(t,J=8.0Hz,1H), 2.33(q,J=2 0.0,12.0Hz,1H),1.85-1.77(m,4H),1.70-1.62(m,4H),1.49-1.40(m,5H),1.35 -1.26(m,10H),1.13 -1.07(m,3H),0.73(s,3H).

[0095] MS: m / z[M+H] + 390.5.

[0096] Example 3

[0097] Synthesis of compound 5

[0098]

[0099] Add compound B and DMF to the reaction bottle to dissolve, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), stir at room temperature, react for 20 minutes, add (S)-2-methylmorpholine and N,N-diisopropylethylamine (DIEA), and react at room temperature overnight. After the reaction, add water and ethyl acetate, stir and separate, wash the organic phase with water, distill under reduced pressure, and column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain an off-white solid 5.

[0100] 1 H NMR (400 MHz, chloroform-d) δ 3.89 (s, 1H), 3.50-3.45 (m, 4H), 2.98 (s, 3H), 2.68 (m, 1H), 2.35 (q, J = 12.0, 9.0 Hz, 1H), 1.86-1.68 (m, 8H), 1.49-1.45 (m, 5H), 1.42-1.39 (m, 10H), 1.27-1.25 (m, 3H), 1.19-1.05 (m, 2H), 0.73 (s, 3H).

[0101] MS: m / z[M+H] + 404.30.

[0102] Example 4

[0103] Synthesis of compound 6

[0104]

[0105] Add compound B and DMF to the reaction bottle, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), react at room temperature for 20 minutes, add (R)-2-methylmorpholine, react at room temperature overnight. Add water and ethyl acetate, stir and separate, wash the organic phase with water, distill under reduced pressure, and column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain an off-white solid 6.

[0106] 1 H NMR (400 MHz, chloroform-d) δ4.14-4.09(m,1H),3.92-3.80(m,4H),3.53-3.42(m,2H),3.06(s,3H),2.68(br,1H),2.35(q,J=20.0,12.0 Hz,1H),1.86-1.61(m,8H),1.49(s,1H),1.45-1.41(m,4H),1.39-1.31(m,7H),1.27-1.25(m,3H),1.20-1.05(m,2H),0.68(s,3H).

[0107] MS: m / z[M+H] + 404.2.

[0108] Example 5

[0109] Synthesis of compound 10

[0110]

[0111] Compound B and DMF were added to the reaction flask, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the mixture was reacted at room temperature for 20 min. Compound 10a was added and the mixture was reacted at room temperature overnight. Water and ethyl acetate were added, and the mixture was stirred to separate into layers. The organic phase was washed with water, evaporated under reduced pressure, and subjected to column chromatography (petroleum ether:ethyl acetate = 1:1, volume ratio) to obtain an off-white solid 10.

[0112] 1 H NMR (400 MHz, chloroform-d) δ 3.97 (d, 1H), 3.56-3.43 (m, 4H), 3.39 (s, 3H), 3.00-2.92 (m, 1H), 2.71-2.67 (m, 1H), 2.54-2.35 (m, 2H), 2.32-2.27 (m, 1H), 1.86-1.68 (m, 8H), 1.45-1.27 (m, 18H), 0.72 (s, 3H).

[0113] MS: m / z[M+H] + 434.4

[0114] Example 6

[0115] Synthesis of compound 11

[0116]

[0117] Add compound B and DMF to the reaction flask, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), react at room temperature for 20 minutes, add compound 11a, react at room temperature overnight. Add water and ethyl acetate, stir to separate, wash the organic phase with water, evaporate under reduced pressure, and perform column chromatography (petroleum ether: ethyl acetate = 1:1, volume ratio) to obtain an off-white solid 11.

[0118] 1 H NMR (400 MHz, chloroform-d) δ 3.98 (d, 1H), 3.57-3.43 (m, 4H), 3.39 (s, 3H), 3.00-2.92 (m, 1H), 2.71-2.67 (m, 1H), 2.54-2.35 (m, 2H), 2.32-2.27 (m, 1H), 1.86-1.68 (m, 8H), 1.46-1.27 (m, 18H), 0.71 (s, 3H).

[0119] MS: m / z[M+H] + 434.4

[0120] Example 7

[0121] Synthesis of compound 22

[0122]

[0123] Compound B and DMF were added to the reaction flask, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the mixture was reacted at room temperature for 20 min. Compound 22a was added and the mixture was reacted at room temperature overnight. Water and ethyl acetate were added, and the mixture was stirred to separate into layers. The organic phase was washed with water, evaporated under reduced pressure, and subjected to column chromatography (petroleum ether:ethyl acetate = 4:1, volume ratio) to obtain an off-white solid 22.

[0124] 1H NMR (400MHz, chloroform-d) δ4.21(s,1H),2.90-2.75(m,1H),2.55-2.45(m,1H),2.40-2.23(m,1H),1.89-1.75(m,6H),1.73 -1.63(m,6H),1.47-1.37(m,6H),1.35-1.31(m,2H),1.29-1.25(m,9H),1.15-1.05(m,4H),0.93(d,3H)0.76(s,3H).

[0125] MS: m / z[M+H] + 402.4

[0126] Example 8

[0127] Synthesis of compound 29

[0128]

[0129] Compound B and DMF were added to the reaction flask, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the mixture was reacted at room temperature for 20 min. Compound 29a was added and the mixture was reacted at room temperature overnight. Water and ethyl acetate were added, and the mixture was stirred to separate into layers. The organic phase was washed with water, evaporated under reduced pressure, and subjected to column chromatography (petroleum ether:ethyl acetate = 2:1, volume ratio) to obtain an off-white solid 29.

[0130] 1 H NMR (400MHz, DMSO-d6) δ4.21-4.31(m,1H), δ4.18-4.07(m,1H), δ3.96-3.75 (m,2H),δ3.72-3.63(m,3H),δ3.55-3.40(m,2H),δ3.35-3.20(m,2H),2.90-2 .80(m,1H),2.16-2.07(m,1H),1.74-1.68(m,3H),1.65-1.57(m,4H),1.42-1 .34(m,4H),1.30-1.22(m,8H),1.12(s,4H),1.05-0.98(m,2H),0.67(s,3H).

[0131] MS: m / z[M+H] + 448.4

[0132] Example 9

[0133] Synthesis of compound 41

[0134]

[0135] Compound B and DMF were added to the reaction flask, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the mixture was reacted at room temperature for 20 min. Compound 41a was added and the mixture was reacted at room temperature overnight. Water and ethyl acetate were added, and the mixture was stirred to separate layers. The organic phase was washed with water, evaporated under reduced pressure, and subjected to column chromatography (petroleum ether:ethyl acetate = 4:1, volume ratio) to obtain an off-white solid 41.

[0136] 1 H NMR (400MHz, DMSO-d6) δ5.15-4.40(m,4H), δ4.09(t,1H), δ3.95-3.65(m,2H), δ3.44-3.33(m,1H),2.81-2.69(m,1 H), 2.38-2.26(m,4H),2.22-2.10(m,4H),1.98-1.76(m,10H),1.74-1.65(m,5H),1.63-1.50(m,3H),1.16(s,3H).

[0137] MS: m / z[M+H] + 458.2

[0138] Example 10

[0139] Synthesis of compound 44

[0140]

[0141] Synthesis of intermediate 44a

[0142]

[0143] Weigh material A into a reaction flask, add dichloromethane to dissolve, then add triethylamine and di-tert-butyl dicarbonate, and stir at room temperature for 12 hours. Take a sample for TLC detection. If the raw material reacts completely, the reaction is terminated. Add water to the reaction system and extract with dichloromethane three times. Collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain crude product 44a;

[0144] Synthesis of intermediate 44b

[0145]

[0146] Weigh the crude product 44a in a reaction flask, add dichloromethane to dissolve, replace the gas in the reaction flask with nitrogen, place it in an ice bath and stir, add Dess-Martin reagent, slowly warm it to room temperature and stir for 4 hours. Sample TLC detection, the raw material reaction is complete, and the reaction is terminated. Add saturated sodium bicarbonate aqueous solution to the reaction system, stir and quench, extract with dichloromethane three times, then backwash the organic phase with purified water, collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product 44b;

[0147] Synthesis of intermediate 44c

[0148]

[0149] Weigh the crude product 44b into a reaction flask, add dichloromethane to dissolve, replace the gas in the reaction flask with nitrogen, place it in an ice bath and stir, add diethylaminosulfur trifluoride (DAST), slowly warm it to room temperature and stir for 12 hours. Sample TLC detection, the raw material reaction is complete, and the reaction is terminated. Add purified water to the reaction system and extract with dichloromethane three times. Collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product 44c;

[0150] Synthesis of intermediate 44d

[0151]

[0152] Weigh the crude product 44c into a reaction flask, add 4M HCl 1,4-dioxane solution, and stir at room temperature for 2 hours. Take a sample for TLC detection. If the raw material reacts completely, the reaction is terminated. Concentrate the reaction system under reduced pressure to obtain the crude product 44d.

[0153] Synthesis of compound 44

[0154]

[0155] Compound B and DMF were added to the reaction flask, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and the mixture was reacted at room temperature for 20 min. Compound 44d was added and the mixture was reacted at room temperature overnight. Water and ethyl acetate were added, and the mixture was stirred to separate into layers. The organic phase was washed with water, evaporated under reduced pressure, and subjected to column chromatography (petroleum ether:ethyl acetate = 5:1, volume ratio) to obtain an off-white solid 44.

[0156] 1 H NMR (400MHz, chloroform-d) δ5.90-5.59(m,1H), δ4.95-4.38(m,1H), δ4.13-3.87(m,2H), δ3.70-3.48(m,2H), δ3.30-3.05(m,1H), 2.85-2.65(m,1H), 2.38-2.20(m,1H),1.87-1.77(m,4H),1.75-1.63(m,4H),1.57(m,5H), 1.47-1.39(m,4H),1.31-1.24(m,7H),1.14-0.86(m,3H),0.73(s,3H).

[0157] MS: m / z[M+H] + 458.2

[0158] Embodiment 11

[0159] Synthesis of compound 49

[0160]

[0161] Add compound B and DMF to the reaction bottle to dissolve, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), stir at room temperature, react for 20 minutes, add (S)-2-ethylmorpholine and N,N-diisopropylethylamine (DIEA), and react at room temperature overnight. After the reaction, add water and ethyl acetate, stir and separate, wash the organic phase with water, distill under reduced pressure, and column chromatography (petroleum ether: ethyl acetate = 3:1, volume ratio) to obtain an off-white solid 49.

[0162] 1 H NMR (400 MHz, chloroform-d) δ 3.89 (s, 1H), 3.50-3.45 (m, 4H), 2.98-2.78 (m, 2H), 2.68 (m, 1H), 2.35 (q, J = 12.0, 9.0 Hz, 1H), 1.86-1.68 (m, 8H), 1.49-1.45 (m, 5H), 1.42-1.39 (m, 13H), 1.27-1.25 (m, 3H), 1.19-1.05 (m, 2H), 0.73 (s, 3H).

[0163] MS: m / z[M+H] + 428.3

[0164] Example 12

[0165] Synthesis of Compounds 52 and 53

[0166]

[0167] Compound B and DMF were added to the reaction flask for dissolution, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added, the mixture was stirred at room temperature for 20 min, 2-cyclopropylmorpholine and N,N-diisopropylethylamine (DIEA) were added, and the mixture was reacted at room temperature overnight. After the reaction, water and ethyl acetate were added, the mixture was stirred and layered, the organic phase was washed with water, distilled under reduced pressure, and column chromatography (petroleum ether: ethyl acetate = 2:1, volume ratio) was performed to obtain an off-white solid 52a, which was separated by chiral column (mobile phase acetonitrile: 5% trifluoroacetic acid aqueous solution = 60:40, isocratic elution, flow rate 1 ml / min, wavelength 200 nm) to obtain white solid 52 and white solid 53.

[0168] Structural characterization of compound 52:

[0169] 1 H NMR(400MHz, DMSO-d6)δ4.39-4.23(m,1H),4.01(d,J=13.2Hz,1H),3.82(q,J= 13.9,11.6Hz,2H),3.35-3.15(m,2H),3.10-2.84(m,2H),2.76-2.54(m,2H),2. 49-2.43(m,1H),2.22-2.05(m,1H),1.76-1.53(m,8H),1.44-0.94(m,18H),0. 84(dd,J=13.0,5.7Hz,2H),0.59(s,3H),0.49-0.40(m,2H),0.36-0.17(m,2H).

[0170] MS: m / z[M+H] + 430.3

[0171] Structural characterization of compound 53:

[0172] 1 H NMR (400MHz, DMSO-d6) δ4.36(d,J=10.4Hz,1H),4.18(d,J=13.4Hz,1H),3.94(d,J=12.5Hz,1H),3 .88-3.72(m,1H),3.35-3.07(m,2H),2.98-2.77(m,2H),2.62(td,J=14.7,12.8,8.8Hz,2H),2.20 -2.09(m,1H),1.81-1.53(m,8H),1.47-0.94(m,19H),0.85(q,J=7.4,6.9Hz,2H),0.59(s,3H),0.54-0.42(m,2H),0.40-0.08(m,2H).

[0173] MS: m / z[M+H] + 430.3

[0174] In the present invention, although only the synthesis of compounds 1, 4, 5, 6, 10, 11, 22, 29, 41, 44, 49, 52, and 53 is used as examples, those skilled in the art can understand that the compounds listed in the general formula of the present invention can be prepared by referring to the above-mentioned synthesis route.

[0175] Biological evaluation

[0176] Electrophysiological test:

[0177] Shuranolone (SAGE-217) is a novel synthetic neuroactive steroid GABA approved for marketing.A The receptor positive allosteric modulator has good oral pharmacokinetic properties and can support oral daily doses. The present invention uses the same test method to test the activity of the compound with SAGE-217 as a comparison.

[0178] Test method: Using a CHO cell line transiently expressing GABA (α4β3δ) receptors, the effects of compounds on GABA (α4β3δ) receptors were studied, and the half-maximal effect concentration (EC 50 ) value.

[0179] The experiment used a manual patch clamp system HEKA EPC 10 signal amplifier and a digital conversion system for whole-cell current recording of the chloride ion current of the GABA channel. The experiments were all carried out at normal room temperature.

[0180] After the whole cell seal was formed, the cell membrane voltage was clamped at -70 mV. The peak current was recorded in the gap-free mode after GABA 5 nM, a mixture of the compound of the present invention and GABA 5 nM from low to high concentrations, and GABA 10 μM were sequentially injected onto the cell surface.

[0181] Dosing method of the test substance: GABA5nM and each concentration of the compound of the present invention were mixed and administered 1-2 times, followed by washing with extracellular fluid for 1 min before testing the next concentration. The test data were collected by EPC 10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0182] Current recording: When administering, place the coverslip with cells in a recording bath under an inverted microscope, and flow the blank control external solution and the working solution of the compound of the present invention through the recording bath from low concentration to high concentration by gravity perfusion to act on the cells. During the recording, a peristaltic pump is used for liquid exchange. At least two cells are used for each concentration and the test is repeated twice independently.

[0183] The peak current of GABA (α4β3δ) receptor after the mixed solution of each concentration of the compound of the present invention and GABAECx was acted on GABAECX+Compound ) and the peak current of GABA ECx-induced receptors GABA ECX ) was normalized to calculate the PAM effect of the compound of the present invention (% Normalized to GABA EC X ),

[0184] Right now, by The mean, standard deviation (SD) and standard error (SE) were calculated for each concentration-effect ratio, and the data were expressed as mean±SE.

[0185] Y=Bottom+(Top-Bottom) / (1+10^((LogEC 50 -X)×HillSlope))

[0186] The EC of the compounds of the present invention was calculated by the above equation: 50 The dose-dependent effect was fitted nonlinearly, where EC 50 is the half-maximal effect concentration. EC 50 The calculation and curve fitting were completed using GraphPad Prism software.

[0187] The test results are shown in Table 2.

[0188] Table 2.

[0189]

[0190] EC 50 (nM) Range:

[0191] A:EC 50 <300nM;

[0192] B:300nM≤EC 50 <500nM;

[0193] C:500nM≤EC 50 <1000nM;

[0194] D:EC 50 ≥1000nM.

[0195] As shown in Table 2, the activity of the compounds (EC 50 The smaller the size, the stronger the activity) 2>1>3, 5>4>6, 8>7>9, 10>11, 12>13, 14>15, 16>17, 18>19, 20>21, that is, the biological activity data prove that among the compounds selected by the present invention, R x The activity of compounds oriented inward (e.g. compounds 2, 5, 8) is stronger than that of compounds without chiral substitution (e.g. compounds 1, 4, 7), and is more obvious than that of R x The activity of compounds facing outward (e.g. compounds 3, 6, 9) proves that the activity of compounds facing inward (R) x In GABA A At the same time, compounds 5, 10, 14, 45, 52, and 53 of the present invention have a very critical effect on the activity of GABA (α4β3δ) EC50 All of them were less than 300 nM, indicating that the compounds of the present invention have an effect on GABA A The receptor has a high positive regulation efficiency.

[0196] Liver microsome stability test:

[0197] The liver microsomal metabolic stability test is the most widely used in vitro model for studying the metabolic conversion process of drugs under the action of phase I metabolic enzymes (such as cytochrome P450 enzymes). There are species differences in the metabolism of drugs, so the liver microsomal metabolic stability test is usually conducted in multiple species. The most commonly used species include humans for predicting clinical doses, rats for toxicity studies, mice for specific efficacy models, and dogs and monkeys for large animal toxicology studies.

[0198] In this study, a commercially available female SD rat liver phase I metabolic stability kit (containing liver microsomes and reagents used in the study) was used for in vitro metabolism studies.

[0199] The microsomes were stored in a refrigerator (below -60.0°C) before use. Detailed microsome information is shown in the table below.

[0200] Species strain gender Number of donors source Part Number batch number Rat SD female 300 IPHASE 0121D1.01 23F013

[0201] Reagents and consumables:

[0202]

[0203]

[0204] instrument:

[0205] Water bath (HH-42, Guohua, Changzhou, China)

[0206] Liquid handling station (iPPA2-96-500(E), Apricot, Covina, USA)

[0207] Liquid handling station (iPPA2-96-125(E), Apricot, Covina, USA)

[0208] Centrifuge (5910R, Eppendorf, Hamburg, Germany)

[0209] Test steps:

[0210] Prepare 8 96-well incubation plates, named T0, T5, T15, T30, T45, T60, Blank60 and NCF60. The reaction time points corresponding to the first 6 incubation plates are 0, 5, 15, 30, 45 and 60 min, respectively. No test or control compound is added to the Blank60 plate and samples are taken after 60 min of incubation. In the NCF60 plate, potassium phosphate buffer is used instead of NADPH regeneration system solution for incubation for 60 min. All time point samples are three replicates.

[0211] Add 2.00 μL of test or control working solution and 100 μL of microsome working solution (liver microsomal protein concentration is 1.00 mg / mL) to T0, T5, T15, T30, T45, T60 and NCF60 plates, respectively, and only add microsome working solution to Blank60 plate. Then, place the incubation plates Blank60, T5, T15, T30, T45 and T60 except T0 and NCF60 in a 37.0℃ water bath for preincubation for about 10 min.

[0212] T0 plate samples were first added with 600 μL of stop solution (the stop solution for the test and control samples was an acetonitrile solution containing 250 nM tolbutamide) and then the NADPH regeneration system working solution was added. On the NCF60 plate, 98.0 μL of potassium phosphate buffer was added to each well and incubated for 60 min. After the pre-incubation of the Blank60, T5, T15, T30, T45 and T60 plates was completed, 98.0 μL of NADPH regeneration system working solution was added to each sample well to start the reaction.

[0213] After incubation for an appropriate time (e.g., 5, 15, 30, 45, and 60 min), 600 μL of stop solution was added to each well of the test sample and the control sample on Blank60, T5, T15, T30, T45, T60, and NCF60 plates to terminate the reaction. All sample plates were shaken and centrifuged at 3220 × g for 20 min. 100 μL of the test sample supernatant was diluted into 300 μL of pure water containing 0.3% formic acid and 50% acetonitrile for LC-MS / MS analysis, and 100 μL of the control supernatant was diluted into 300 μL of pure water for LC-MS / MS analysis. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The retention time of the analyte and internal standard, chromatogram acquisition, and chromatogram integration were processed using the software Analyst (Sciex, Framingham, MA, USA).

[0214] Data analysis

[0215] The in vitro elimination rate constant k of the test and control compounds was obtained by converting the ratio of the peak area of ​​the compound to the internal standard into the residual percentage in the following formula:e :

[0216]

[0217]

[0218] when

[0219]

[0220] By k e In vitro hepatic microsomal intrinsic clearance (CL int(mic) ) and hepatic intrinsic clearance (CL (liver) ).

[0221] CL int(mic) =0.693 / T 1 / 2 / Microsomal protein content (microsomal concentration during incubation mg / mL).

[0222] CL int(liver) =CL int(mic) × amount of microsomal protein in the liver (mg / g) × liver weight to body weight ratio.

[0223] Based on the well-stirred model, the in vivo hepatic clearance (CL (liver) ):

[0224] CL (liver) =(CL int(liver) ×f u ×Q h ) / (CL int(liver) ×f u +Q h ), default f u (free fraction in blood) is equal to 1. The parameters used in the formula are as follows:

[0225]

[0226] The test results of the concentration data of the six compounds and the positive drug SAGE-217 (Female Rat) at each time point are summarized in Table 3:

[0227] Table 3.

[0228]

[0229]

[0230] The residual percentage trend of each compound in Table 3 is as follows Figure 1 As shown, the horizontal axis is time and the vertical axis is the remaining percentage.

[0231] The in vitro half-life and intrinsic clearance of each compound in Table 3 are summarized in Table 4:

[0232] Table 4.

[0233]

[0234] Data Analysis and Discussion:

[0235] From Table 3, Figure 1 As can be seen from the data in Table 4, the liver microsome stability of compound 10 is significantly better than that of SAGE-217, the liver microsome stability of compound 52 is similar to that of SAGE-217, and the liver microsome stability of compound 45 is slightly worse than that of SAGE-217.

[0236] In vivo pharmacokinetic studies:

[0237] Study purpose: To investigate the pharmacokinetics of the target compound in female SD rats after a single oral gavage and a single tail vein administration, with a focus on obtaining its C max 、T 1 / 2 and AUC; record in detail the status and behavioral performance of the animals at different time points after administration; and calculate its oral bioavailability based on the pharmacokinetic data results of tail vein administration.

[0238] Trial Drugs:

[0239] Compounds 10, 45, and 52: 0.6 mg / ml aqueous solution (30% SBECD as solvent, oral administration);

[0240] Compounds 10, 45, and 52: 0.2 mg / ml aqueous solution (30% SBECD as solvent, intravenous administration).

[0241] Test method:

[0242] Oral gavage administration in rats: Three female SD rats were orally gavaged with compound X once, with a dose of 6 mg / kg (about 2 ml of drug solution). Blood was collected from the orbital venous plexus at 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration. The blood was anticoagulated with heparin and centrifuged to separate the plasma for testing.

[0243] Rat tail vein administration: Three female SD rats were administered compound X once through the tail vein, with a dose of 2 mg / kg (about 2 ml of drug solution). Blood was collected from the orbital venous plexus at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration. The blood was anticoagulated with heparin and centrifuged to separate the plasma for testing.

[0244] Plasma sample processing: accurately measure 50 μL of plasma sample, add 150 μL of protein precipitant (5 ng / mL testosterone-d3, 5 ng / mL Terfenadine, 5 ng / mL Buspirone in acetonitrile solution), vortex mix for 1 min, centrifuge (4000 rpm, 4°C) for 10 min, take 120 μL of supernatant, add 40 μL of 50% methanol aqueous solution, vortex mix for 1 min, and detect by UPLC-MS / MS.

[0245] Data calculation and statistics:

[0246] Drug concentrations were directly read out by UPLC-MS / MS, and the mean, standard deviation, and coefficient of variation were calculated using Excel. max , C max , AUC and other pharmacokinetic parameters were calculated by DAS2.0 software.

[0247] The pharmacokinetic parameters of compounds 10, 45 and 52 are shown in Table 5:

[0248] Table 5.

[0249]

[0250] Data Analysis:

[0251] From the PK parameters in female SD rats, it was found that compounds 10 and 52 had good metabolic stability in female SD rats, good oral absorption, high blood drug concentration and exposure, and high bioavailability.

[0252] The present invention provides a novel structure, high efficiency, oral, anti-severe depression and postpartum depression drug, which can simultaneously act on multiple GABA on the synaptic surface through allosteric regulation. A Ion channel receptors promote the influx of chloride ions into cells along the electrochemical gradient of the GABA receptor complex (GRC). Increased levels of this anion in cells lead to hyperpolarization of the transmembrane potential, making neurons less sensitive to excitatory inputs, i.e., reduced brain excitability. The compounds of the present invention have an effect on GABA A The receptor has a strong positive regulatory effect, which is beneficial to improve the bioavailability of drugs.

[0253] Although multiple embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art may conceive of many modifications, changes, and alternatives without departing from the ideas and spirit of the present invention. It should be understood that in the practice of the present invention, various alternatives to the embodiments of the present invention described herein may be adopted. The appended claims are intended to define the scope of protection of the present invention and therefore cover equivalents or alternatives within the scope of these claims.

Claims

1. A steroid compound having a structure of general formula I and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotopic compounds, characterized in that: in, R1, R2, R3, R4, R5, R x Selected from: Hydrogen, fluorine, chlorine, bromine, iodine, carbon, nitrogen, oxygen, sulfur, methyl, monofluoromethyl, difluoromethyl, trifluoromethyl, cyano, acyl, formaldehyde oxime, formaldehyde oxime ether, nitro, carboxyl; C1-C6 alkane groups and halogen substitutes thereof, C1-C6 aliphatic chain substituents containing oxygen heteroatoms and halogen substitutes thereof, C1-C6 aliphatic chain substituents containing sulfur heteroatoms and halogen substitutes thereof, C1-C6 aliphatic chain substituents containing nitrogen heteroatoms and halogen substitutes thereof, C2-C6 olefin groups and halogen substitutes thereof, C2-C6 alkynyl groups and halogen substitutes thereof; Five-membered ring aromatic hydrocarbon group, six-membered ring aromatic hydrocarbon group, C3-C7 cycloalkane group; A five-membered heterocyclic aromatic hydrocarbon group containing nitrogen, oxygen or sulfur; Six-membered nitrogen heterocyclic aromatic hydrocarbon group; Heterocycloalkane containing nitrogen, oxygen or sulfur; Hydroxyl, amino, thiol; A hydroxyl group, an amino group or a thiol group connected to a five-membered aromatic hydrocarbon, a six-membered aromatic hydrocarbon, a C3-C7 cycloalkane, a five-membered heteroaromatic hydrocarbon containing nitrogen, oxygen or sulfur, a six-membered nitrogen heteroaromatic hydrocarbon, or a heterocycloalkane containing nitrogen, oxygen or sulfur; Esters and amides formed by inorganic acids, C1-C6 organic acids, hydroxyl groups and amino groups; in, M is selected from carbon, nitrogen, oxygen, sulfur, phosphorus, in, When M is nitrogen, one of R2 or R3 is absent; or, when M is nitrogen, both R2 and R3 are present; When M is oxygen, R2 or R3 is absent; When M is sulfur, both R2 and R3 are present to form a sulfone; or, when M is sulfur, one of R2 or R3 is absent, forming a sulfoxide; Alternatively, when M is sulfur, neither R2 nor R3 is present.

2. The steroid compound having the general formula I structure according to claim 1, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds, characterized in that: The acyl group is selected from the group consisting of alkanoyl and halogen substituents thereof, alkenoyl and halogen substituents thereof, alkynoyl and halogen substituents thereof, alkoxycarbonyl and halogen substituents thereof, alkenyloxycarbonyl and halogen substituents thereof, alkynyloxycarbonyl and halogen substituents thereof, aryloxycarbonyl and halogen substituents thereof, heteroaryloxycarbonyl and halogen substituents thereof, alkylaminocarbonyl and halogen substituents thereof, enaminocarbonyl and halogen substituents thereof, alkynylaminocarbonyl and halogen substituents thereof, arylaminocarbonyl and halogen substituents thereof, and heteroarylaminocarbonyl and halogen substituents thereof.

3. The steroid compound having the general formula I structure according to claim 1, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds, characterized in that: R x Towards the inside of the face.

4. The steroid compound having the general formula I structure according to claim 1, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds, characterized in that: R x Facing outward.

5. The steroid compound having the general formula I structure according to any one of claims 1 to 4, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotopic compounds, characterized in that: R2 and R3 are connected to form a C8-C12 spiro ring, a C8-C12 azaspiro ring, or a C8-C12 oxaspiro ring; Or, R2, R x Connected to form a C8-C12 bridge ring, a C8-C12 nitrogen bridge ring, and a C8-C12 oxygen bridge ring.

6. The steroid compound having the general formula I structure according to any one of claims 1 to 4, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotopic compounds, characterized in that: R1, R2, R3, R4 and R5 are not hydrogen.

7. The steroid compound having the general formula I structure according to any one of claims 1 to 4, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotopic compounds, characterized in that: R1, R2, R3, R4, R5, and only one of them is hydrogen; or, Only two of R1, R2, R3, R4 and R5 are hydrogen; or, Only three of R1, R2, R3, R4 and R5 are hydrogen; or, Only four of R1, R2, R3, R4 and R5 are hydrogen; or, R1, R2, R3, R4 and R5 are all hydrogen.

8. The steroid compound having the general formula I structure according to claim 1, and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds, characterized in that: The steroid compound is selected from:

9. A pharmaceutical composition, characterized in that The steroid compound having the structure of general formula I as claimed in any one of claims 1 to 8 and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotope compounds are contained as active ingredients.

10. The pharmaceutical composition according to claim 9, characterized in that A pharmaceutically acceptable carrier or diluent is also included.

11. Use of the pharmaceutical composition according to claim 9 as a drug for treating central nervous system-related diseases, The central nervous system related disorders include: Postpartum or postpartum depression, clinical depression, atypical depression, major depressive disorder, catatonic depression, dysthymia, double depression, depressive personality disorder, recurrent brief depression, minor depressive disorder, bipolar disorder, manic-depressive disorder, mood disorder, anxiety, post-traumatic stress disorder (PTSD), premenstrual dysphoric disorder (PMDD), depression due to a chronic medical condition, treatment-resistant depression, suicidal tendencies, suicidal ideation or behavior, premenstrual syndrome, generalized anxiety disorder, seasonal affective disorder (SAD), social anxiety disorder, memory loss, stress intolerance, Niemann-Pick disease type C or related neurological or physical symptoms, epilepsy, essential tremor, epileptiform disorder, NMDA hypofunction, migraine, status epilepticus, sleep disorder, fragile X syndrome, 5α-reductase inhibitor-induced depression, PCDH19 female pediatric epilepsy, sexual dysfunction, cognitive impairment, Parkinson's disease or Alzheimer's disease.

12. Use of the steroid compound having the general formula I structure as claimed in any one of claims 1 to 8 and its stereoisomers, pharmaceutically acceptable salts, hydrates, solvates or isotopic compounds as a drug for the treatment of central nervous system-related diseases, The central nervous system related disorders include: Postpartum or postpartum depression, clinical depression, atypical depression, major depressive disorder, catatonic depression, dysthymia, double depression, depressive personality disorder, recurrent brief depression, minor depressive disorder, bipolar disorder, manic-depressive disorder, mood disorder, anxiety, post-traumatic stress disorder (PTSD), premenstrual dysphoric disorder (PMDD), depression due to a chronic medical condition, treatment-resistant depression, suicidal tendencies, suicidal ideation or behavior, premenstrual syndrome, generalized anxiety disorder, seasonal affective disorder (SAD), social anxiety disorder, memory loss, stress intolerance, Niemann-Pick disease type C or related neurological or physical symptoms, epilepsy, essential tremor, epileptiform disorder, NMDA hypofunction, migraine, status epilepticus, sleep disorder, fragile X syndrome, 5α-reductase inhibitor-induced depression, PCDH19 female pediatric epilepsy, sexual dysfunction, cognitive impairment, Parkinson's disease or Alzheimer's disease.

13. The method for preparing a steroid compound having the general formula I according to any one of claims 1 to 8, characterized in that: The preparation route is as follows: The compound B1 is subjected to a catalytic hydrogenation reaction to obtain a compound B2; Methylating compound B2 to obtain compound B3; Compound B3 is subjected to ylide reaction to obtain compound B4; Compound B4 is subjected to a hydroboration oxidation reaction to obtain compound B5; The compound B5 is subjected to an oxidation reaction to obtain a compound B6; Compound B6 is subjected to a haloform reaction to obtain compound B; Compound B is subjected to condensation reaction with fragment Q to obtain a steroid compound of the general formula I. Wherein, compound B1 is 19-nor-4-androstenedione, compound B is 3-hydroxy-3,13-dimethylestrone-17-acid, and compound Q is a nitrogen-containing six-membered heterocyclic ring substituted with M at the para position.

Images