Steroidal compounds for the treatment of central nervous system disorders, methods of making, uses and pharmaceutical compositions

Abstract

The present application provides a steroid compound for treating central nervous system diseases, having a 9β, 10α structure and having the following structural formula: which acts as a GABA modulator, prolongs the opening time of chloride ion channel activated by GABA, modulates the excitability of central nervous system (CNS), treats and prevents CNS related disorders; and the 9β, 10α steroid compound of the present application has a sedative effect and can be used as a sedative.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, specifically to steroidal compounds for treating central nervous system diseases, their preparation methods, uses, and pharmaceutical compositions. Background Technology

[0002] Brain excitability is defined as the arousal level in animals, a continuum ranging from coma to seizures, and regulated by various neurotransmitters. GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the vertebrate central nervous system, with up to 40% of neurons in the brain utilizing GABA as a neurotransmitter. GABA receptors are a class of intracellular receptors that respond to the neurotransmitter GABA. There are three subtypes of GABA receptors: GABAA receptors, GABAB receptors, and GABAC receptors.

[0003] The term "neurosteroids" was coined by E. Baulieu of France in 1981. Neurosteroids can regulate neural function and alter brain excitability by binding to different neural receptors (such as GABAA receptors), thereby treating or alleviating CNS (central nervous system) related diseases. Neurosteroids exert various physiological and pathological effects through receptor binding, primarily through ligand-gated ion channels. GABAA receptors are ligand-gated ion channels; upon activation, they open chloride ion channels, triggering chloride ion influx, causing cell hyperpolarization, and thus inhibiting neurotransmission and reducing central nervous system excitability. The higher the chloride ion concentration in neurons, the lower the brain excitability (arousal level).

[0004] Known neurosteroid drugs include allogeneic brexanolone and pregnenolone (PREG). Among them, allogeneic brexanolone is a highly effective positive allosteric modulator of synaptic and exosynaptic GABAA receptors, which can prolong the opening time of GABA-induced chloride ion channels.

[0005] However, these neurosteroids have some issues regarding the breadth of indications, drug efficacy, toxicity, bioavailability, and metabolic stability. There is a need for new and improved neuroactive steroids as modulators of brain excitability, as well as drugs for the prevention and treatment of CNS-related diseases. Summary of the Invention

[0006] This invention provides a steroidal compound or a pharmaceutically acceptable salt thereof, said steroidal compound having a 9β,10α structure and having the following structural formula:

[0007]

[0008] R1 is selected from H, substituted or unsubstituted C. 1～6Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 carbonyl group, or -CH2OR 1a , where R 1a Selected from substituted or unsubstituted C 1～6 Alkyl, or substituted or unsubstituted C 2～6 alkenyl;

[0009] Where R 2a R 2b Each is independently selected from H, halogen, substituted or unsubstituted C. 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 carbon cyclo group or OR 2c , where R 2c Selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, or substituted or unsubstituted C 3～6 carbon cyclo group;

[0010] R 3a R 3b Each is independently selected from H or OR 3c , where R 3c Selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, or substituted or unsubstituted C 3～6 carbonyl group, or R 3a and R 3b Combination forms = O;

[0011] R 4a R 4b Each is independently selected from H, halogen, substituted or unsubstituted C. 1～6 alkyl;

[0012] R1′ is selected from H, or substituted or unsubstituted heteroaryl groups;

[0013] Indicates a single bond or a double bond;

[0014] When a certain When it is a double bond, the adjacent It is a single key.

[0015] In some embodiments of the present invention, the steroidal compound has the following structural formula:

[0016]

[0017] In this ring, ring A is a substituted or unsubstituted heteroaryl group, and the heteroatom is N, with the number of heteroatoms N ranging from 1 to 4.

[0018] In some embodiments of the present invention, ring A is selected from the following groups.

[0019]

[0020] The above groups are either unsubstituted or substituted with groups selected from the following: H, halogens, -NO2, -CN, -OR′, -N(R′)2, -C(=O)R′, -C(=O)OR′, -OC(=O)R′ or -OC(=O)OR′, wherein R′ is selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 Carbocyclic, substituted or unsubstituted C 3～6 Heterocyclic group, substituted or unsubstituted aryl group, or substituted or unsubstituted heteroaryl group.

[0021] In some embodiments of the present invention, the steroid compound has the following structural formula.

[0022]

[0023] R5, R6, and R7 are each independently selected from H, halogens, -NO2, -CN, -OR′, -N(R′)2, -C(=O)R′, -C(=O)OR′, -OC(=O)R′, or -OC(=O)OR′, where R′ is selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 Carbocyclic, substituted or unsubstituted C 3～6 Heterocyclic group, substituted or unsubstituted aryl group, or substituted or unsubstituted heteroaryl group.

[0024] In some embodiments of the present invention, the steroidal compound is selected from the following structural formulas.

[0025]

[0026] In some embodiments of the present invention, the steroidal compound is selected from the following structural formulas.

[0027]

[0028] In some embodiments of the present invention, the steroid compound has the following structural formula.

[0029]

[0030] R5′, R6′, and R7′ are each independently selected from H, halogens, -NO2, -CN, -OR′, -N(R′)2, -C(=O)R′, -C(=O)OR′, -OC(=O)R′, or -OC(=O)OR′, where R′ is selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 Carbocyclic, substituted or unsubstituted C 3～6 Heterocyclic group, substituted or unsubstituted aryl group, or substituted or unsubstituted heteroaryl group.

[0031] In some embodiments of the present invention, the steroid compound has the following structural formula.

[0032]

[0033] This invention provides a method for preparing steroidal compounds having the following structural formula:

[0034]

[0035] The steroidal compound is prepared by a nucleophilic substitution reaction of a compound of the following formula.

[0036]

[0037] Where X is a halogen.

[0038] This invention provides a method for preparing steroidal compounds having the following structural formula:

[0039]

[0040] The steroidal compound is prepared from dydrogesterone via reduction and / or addition reactions.

[0041] The present invention also provides an intermediate compound for preparing the steroidal compound of the present invention, said intermediate compound having the following structural formula:

[0042]

[0043] Where X is a halogen.

[0044] The present invention also provides the use of the steroidal compounds of the present invention or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating central nervous system-related disorders.

[0045] In some embodiments of the present invention, the central nervous system-related conditions are selected from: sleep disorders, mood disorders, schizophrenia spectrum disorders, spastic disorders, memory disorders and / or cognitive disorders, motor disorders, personality disorders, autism spectrum disorders, pain, and traumatic brain injury.

[0046] In some embodiments of the present invention, the central nervous system-related conditions are selected from: insomnia, depression, anxiety, epilepsy, dementia, neuropathic pain, or tremor.

[0047] In some embodiments of the present invention, the dementia is Alzheimer's disease.

[0048] The present invention also provides a pharmaceutical composition comprising a steroidal compound or a pharmaceutically acceptable salt thereof as described in the present invention, and a pharmaceutically acceptable excipient.

[0049] Compared with the prior art, the present invention achieves at least the following beneficial technical effects:

[0050] The 9β,10α steroidal compounds of this invention act as GABA modulators, prolonging the opening time of GABA-activated chloride ion channels, regulating the excitability of the central nervous system (CNS), and treating and preventing CNS-related diseases. Furthermore, the 9β,10α steroidal compounds of this invention have a sedative effect and can be used as sedatives, with a stronger sedative effect in male animals and men.

[0051] Unlike traditional steroid compounds, the methyl group at position C-10 of the compound of this invention is in the α configuration (while the H at position 9 is in the β configuration), resulting in an overall conformational change. Compared to the β configuration at position C-10, the compound of this invention exhibits better efficacy, pharmacokinetic properties, bioavailability, stability, and safety.

[0052] Compared to traditional steroidal compounds with a β configuration at the C-10 position, the 9β,10α steroidal compounds of this invention exhibit stronger binding specificity and higher activity to GABAA receptors; their binding to GABAA receptors is more selective, reducing binding to non-target receptors, such as hormone receptors, resulting in less steroid hormone activity and fewer side effects.

[0053] Meanwhile, compared with compounds with a β configuration at the C-10 position, the 9β,10α steroidal compound of the present invention is insensitive to enzymes in vivo, has significantly reduced reactivity with enzymes in vivo, is not easily degraded by enzymes, is metabolized more slowly in vivo, and has a longer duration of efficacy.

[0054] Furthermore, the drug composition prepared using the 9β,10α steroidal compound of the present invention has a more flexible administration method, as it can be taken orally and is convenient to use. Attached Figure Description

[0055] Figure 1 The current-time detection spectrum of GABA receptor at 15 μM is shown.

[0056] Figure 2 The current-time detection spectrum of the GABA receptor in TM 17 with 15 μM GABA + 0.1 μM is shown.

[0057] Figure 3 The current-time detection spectrum of the GABA receptor for TM 17 with 15 μM + 1 μM GABA is shown.

[0058] Figure 4 The calculation results for EC50 of TM 17 are shown.

[0059] Figure 5 The calculated EC50 for TM 18 is shown. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated herein.

[0062] definition

[0063] In this invention, the term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1-8 carbon atoms (denoted as C). 1-8 Alkyl groups. In some embodiments, the alkyl group has 1-6 carbon atoms (C66-C66). 1-6 Alkyl group). In some embodiments, the alkyl group has 1-3 carbon atoms (C60-C62). 1-3 Alkyl). C 1-6Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, and their various branched isomers. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, carbocyclic, alkoxy, halogen, hydroxyl, oxo, amino, amine, acyl, acyloxy, or ester groups.

[0064] The term "alkenyl" refers to an alkyl group as defined above, consisting of at least two carbon atoms and at least one carbon-carbon double bond. In some embodiments, the alkenyl group is C0. 2-8 Alkenyl group. In some embodiments, the alkenyl group is C0. 2-6 Alkenyl group. In some embodiments, the alkenyl group is C0. 2-4 Alkenyl. The carbon-carbon double bond can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). C 2–6 Non-limiting examples of alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc. Alkenyl groups can be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups, independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, alkoxy, halogen, hydroxyl, oxo, amino, amine, acyl, acyloxy, or ester groups.

[0065] The term "alkynyl" refers to an alkyl group as defined above, consisting of at least two carbon atoms and at least one carbon-carbon triple bond. In some embodiments, the alkynyl group is C0. 2-8 Alkyne group. In some embodiments, the alkynyl group is C. 2-6 Alkyne group. In some embodiments, the alkynyl group is C. 2-4 Alkynyl. Non-limiting examples include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, etc. The alkynyl group can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, alkoxy, halogen, hydroxyl, oxo, amino, amine, acyl, acyloxy, or ester.

[0066] The term "carbocyclic group" refers to a saturated or partially unsaturated all-carbon non-aromatic cyclic hydrocarbon group. A saturated carbocyclic group is called a "cycloalkyl group." In some embodiments, the carbocyclic group comprises 3-6 ring carbon atoms. In some embodiments, the carbocyclic group comprises 5 or 6 ring carbon atoms. 3-6Non-limiting examples of carbocyclic groups include cyclopropyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc. The carbocyclic group can be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, haloalkyl, alkenyl, alkynyl, carbocyclic, alkoxy, halogen, hydroxyl, oxo, amino, amine, acyl, acyloxy, or ester groups.

[0067] The term "heterocyclic group" refers to a saturated or partially unsaturated cyclic hydrocarbon substituent, wherein one or more ring atoms are heteroatoms selected from nitrogen or oxygen, and the remaining ring atoms are carbon. In some embodiments, the heterocyclic group comprises 3 to 12 ring atoms, of which 1 to 4 are heteroatoms. Non-limiting examples of heterocyclic groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, dihydroimidazoyl, dihydrofuranyl, piperidinyl, piperazineyl, pyranyl, etc. Heterocyclic groups can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, haloalkyl, alkoxy, halogen, hydroxy, or amino groups.

[0068] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system. In some embodiments, the aryl group has 6-10 cyclic carbon atoms, such as phenyl, 1-naphthyl, 2-naphthyl, etc. The aryl group can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, haloalkyl, alkoxy, halogen, hydroxy, or amino groups.

[0069] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen and nitrogen, preferably nitrogen. The heteroaryl group is preferably 5 to 10-membered, such as imidazolyl, pyrazolyl, pyrroleyl, triazolyl, tetrazolyl, pyridyl, pyrazinyl, etc. The heteroaryl ring may be fused to an aryl ring, wherein the ring connected to the parent structure is the heteroaryl ring, and non-limiting examples include:

[0070]

[0071] The heteroaryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, haloalkyl, halogen or amino groups.

[0072] "Alkoxy" refers to -O- (alkyl) and -O- (cycloalkyl), where alkyl and carbocyclic groups are defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopentoxy, cyclohexoxy, etc.

[0073] "Acyl" refers to the group -C(O)R a , where R aIt is hydrogen, or an alkyl, alkenyl, alkynyl, carbocyclic, or aryl group as defined herein. Non-limiting examples include acetyl (-C(O)CH3), cyclohexylcarbonyl, benzoyl (-C(O)Ph), etc.

[0074] "Acyloxy group" refers to the group -OC(O)R b , where R b It is hydrogen, or alkyl, alkenyl, alkynyl, carbocyclic or aryl as defined herein.

[0075] "Ester group" refers to the group -COOR c , where R c It is an alkyl, alkenyl, ynyl, carbocyclic or aryl group as defined herein.

[0076] "Amino" refers to the group -NR'R", where R' and R" are hydrogen or alkyl as defined herein, and at least one of R' and R" is not hydrogen.

[0077] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0078] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0079] "Hydroxy" refers to the -OH group.

[0080] "O" refers to =O.

[0081] "Amino" refers to -NH2.

[0082] "Carboxyl group" refers to -C(O)OH.

[0083] When listing a range of values, the intention is to include every value within that range and its subranges. For example, "C" 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0084] "Multiple" refers to two or more, such as 2-5, 2-3, 2, 3, 4 or 5, etc.

[0085] The different terms "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B or C", and "X is A, B and C" all express the same meaning, that is, X can be any one or more of A, B, and C.

[0086] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted aryl" means that the alkyl group may but does not have to be present, and the description includes cases where the aryl group is substituted with an alkyl group and cases where the aryl group is not substituted with an alkyl group.

[0087] "Substitution" means that one or more hydrogen atoms in a group are independently replaced by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort.

[0088] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Specifically, such salt is non-toxic and may be an organic or inorganic acid addition salt and a base addition salt.

[0089] The steroidal compounds of the present invention

[0090] For ease of understanding, the carbon atom labels of steroid compounds are shown below. For example, the 10th carbon atom can be denoted as C-10. The four rings are labeled A, B, C, and D from left to right.

[0091]

[0092] The compounds described herein may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be individual enantiomers, diastereomers, or geometric isomers, or may be mixtures of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. The invention further includes the compounds described herein as individual isomers substantially free of other isomers, or as mixtures of multiple isomers.

[0093] The general formula compounds (including at least two compounds) with an α-methyl group at the C-10 position involved in this invention, and the specific compounds (compounds in the examples, including intermediates) have the following configurations:

[0094]

[0095] Besides the groups whose configurations are already indicated in the above formula, other groups or H connected to the steroid ring can be α or β configurations. In the examples, TM17-20 and TM25-28 have the following configurations:

[0096]

[0097] The 9β,10α steroidal compounds of the present invention have stereoconformities that differ from those of conventional steroidal compounds. Taking the 5α series as an example, the AB ring conformations of the 9β,10α steroidal compounds of the present invention and conventional 9α,10β steroidal compounds are shown below, respectively.

[0098]

[0099] In an embodiment of the present invention, a steroidal compound or a pharmaceutically acceptable salt thereof is provided, said steroidal compound having a 9β,10α structure and having the following structural formula:

[0100]

[0101] R1 is selected from H, substituted or unsubstituted C. 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 carbonyl group, or -CH2OR 1a , where R 1a Selected from substituted or unsubstituted C 1～6 Alkyl, or substituted or unsubstituted C 2～6 alkenyl;

[0102] Where R 2a R 2b Each is independently selected from H, halogen, substituted or unsubstituted C. 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 carbon cyclo group or OR 2c , where R 2c Selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, or substituted or unsubstituted C 3～6 carbon cyclo group;

[0103] R 3a R 3b Each is independently selected from H or OR3c , where R 3c Selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, or substituted or unsubstituted C 3～6 carbonyl group, or R 3a and R 3b Combination forms = O;

[0104] R 4a R 4b Each is independently selected from H, halogen, substituted or unsubstituted C. 1～6 alkyl;

[0105] R1′ is selected from H, or substituted or unsubstituted heteroaryl groups;

[0106] Indicates a single bond or a double bond;

[0107] When a certain When it is a double bond, the adjacent It is a single key.

[0108] In some implementations, R1 is selected from H and C. 1～3 Alkyl, C 1～3 Halogenated alkyl or -CH2OR 1a , where R 1a Selected from C 1～3 alkyl.

[0109] In some implementations, R1 is selected from H.

[0110] In some embodiments, R1 is selected from methyl.

[0111] In some implementations, R1 is selected from -CH2OCH3.

[0112] In some embodiments, the R1 configuration is α-type or β-type, and correspondingly, the -OH configuration at the C-3 position is β-OH or α-OH.

[0113] In some embodiments, the R1 configuration is β-type, and the C-3 position is α-OH.

[0114] In some implementations, the R 2a R 2b R 3a R 3b R 4a R 4b All are H.

[0115] In some implementations, the It is a single bond, with β-H at C-8 and α-H at C-14.

[0116] First aspect (R1′ represents a substituted or unsubstituted heteroaryl group)

[0117] In some embodiments, when R1′ is selected from substituted or unsubstituted heteroaryl groups, the heteroatom therein is N.

[0118] In some embodiments, the steroid compound has the following structural formula:

[0119]

[0120] In this ring, ring A is a substituted or unsubstituted heteroaryl group, and the heteroatom is N, with the number of heteroatoms N ranging from 1 to 4.

[0121] In some implementations, ring A is a five-membered ring, and the number of heteroatoms N is 2 or 3.

[0122] In some embodiments, ring A is selected from the following groups

[0123]

[0124] The above groups are either unsubstituted or substituted with groups selected from the following: H, halogens, -NO2, -CN, -OR′, -N(R′)2, -C(=O)R′, -C(=O)OR′, -OC(=O)R′ or -OC(=O)OR′, wherein R′ is selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 Carbocyclic, substituted or unsubstituted C 3～6 Heterocyclic group, substituted or unsubstituted aryl group, or substituted or unsubstituted heteroaryl group. (1.1)

[0126] In some embodiments, the steroid compound has the following structural formula

[0127]

[0128] R5, R6, and R7 are each independently selected from H, halogens, -NO2, -CN, -OR′, -N(R′)2, -C(=O)R′, -C(=O)OR′, -OC(=O)R′, or -OC(=O)OR′, where R′ is selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6Carbocyclic, substituted or unsubstituted C 3～6 Heterocyclic group, substituted or unsubstituted aryl group, or substituted or unsubstituted heteroaryl group.

[0129] In some implementations, one of R5, R6, and R7 is -CN, and the rest are H.

[0130] In some implementations, R1 is selected from H and C. 1～3 Alkyl or C 1～3 Haloalkyl, R 2a R 2b R 3a R 3b R 4a R 4b All are H.

[0131] In some embodiments, the steroid compound is selected from the following structural formulas.

[0132]

[0133] In some embodiments, the steroid compound is selected from the following structural formulas.

[0134]

[0135] In some embodiments, R1 is selected from methyl or ethyl. (1.2)

[0137] In some embodiments, the steroid compound has the following structural formula

[0138]

[0139] R5′, R6′, and R7′ are each independently selected from H, halogens, -NO2, -CN, -OR′, -N(R′)2, -C(=O)R′, -C(=O)OR′, -OC(=O)R′, or -OC(=O)OR′, where R′ is selected from H, substituted or unsubstituted C 1～6 Alkyl, substituted or unsubstituted C 2～6 alkenyl, substituted or unsubstituted C 2～6 Alkyne group, substituted or unsubstituted C 3～6 Carbocyclic, substituted or unsubstituted C 3～6 Heterocyclic group, substituted or unsubstituted aryl group, or substituted or unsubstituted heteroaryl group.

[0140] In some implementations, R5′, R6′, and R7′ are all H.

[0141] In some implementations, R1 is selected from H and C. 1～3 Alkyl or C1～3 Haloalkyl, R 2a R 2b R 3a R 3b R 4a R 4b All are H. It is a single key.

[0142] In some embodiments, the steroid compound is selected from the following structural formulas.

[0143]

[0144] Second aspect (R1′ is H)

[0145] In some embodiments, the steroid compound has the following structural formula

[0146]

[0147] In some embodiments, the steroid compound has the following structural formula

[0148]

[0149] In some embodiments, the steroid compound is selected from the following structural formulas.

[0150]

[0151] In some embodiments, the steroid compound is selected from the following structural formulas.

[0152]

[0153] Uses of steroid compounds

[0154] The steroidal compounds of the present invention, or pharmaceutically acceptable salts thereof, can modulate GABA for the treatment and prevention of CNS-related disorders. Exemplary CNS disorders associated with GABA regulation include, but are not limited to: sleep disorders [e.g., insomnia], mood disorders [e.g., depression, postpartum depression, depressive disorders (e.g., mild depression), bipolar disorder (e.g., I and / or II), anxiety disorders (e.g., generalized anxiety disorder (GAD), social anxiety disorder), stress response, post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (e.g., obsessive-compulsive disorder (OCD))], schizophrenia spectrum disorders [e.g., schizophrenia, schizophrenic affective disorder], and spastic / convulsive disorders [e.g., epilepsy]. (e.g., status epilepticus (SE)), seizures), memory impairment and / or cognitive impairment [e.g., attention deficit hyperactivity disorder (ADHD), dementia (e.g., Alzheimer's disease, Lewis body type dementia, vascular dementia), movement disorders [e.g., Huntington's disease, Parkinson's disease], personality disorders [e.g., antisocial personality disorder, obsessive-compulsive personality disorder], autism spectrum disorders (ASD) [e.g., autism, single-cause autism], tremor (e.g., essential tremor), pain [e.g., neuropathic pain], traumatic brain injury (TBI).

[0155] Clinical depression, also known as major depressive disorder (MDD), unipolar depression, or recurrent depression, is a mental disorder characterized by a pervasive and persistent low mood, accompanied by low self-esteem and loss of interest or pleasure in normally enjoyable activities. Symptoms and remissions of depression can be determined by a physician or psychologist (e.g., through a mental status examination).

[0156] "Treatment" for any disease or condition means improving said disease or condition (i.e., preventing (suppressing) said disease or reducing at least one manifestation, extent, or severity of its clinical symptoms). In another embodiment, "treatment" means improving at least one physical parameter, which may not be identifiable to the subject. In yet another embodiment, "treatment" means physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical parameters), or both, regulating a disease or condition. In yet another embodiment, "treatment" involves slowing the progression of a disease.

[0157] "Effective amount" means the amount of a compound that is sufficient to achieve such treatment when administered to a subject for the treatment of a disease.

[0158] Pharmaceutical Composition

[0159] The present invention provides a pharmaceutical composition comprising a steroidal compound or a pharmaceutically acceptable salt thereof as described in the present invention, and pharmaceutically acceptable excipients.

[0160] The dosage forms of the pharmaceutical compositions of this invention include: tablets, capsules, granules, powders, aerosols, powder sprays, suspensions, solutions, emulsions, injections, etc. Depending on the characteristics of their respective dosage forms, the routes of administration include oral, sublingual, injection (intravenous / intramuscular), pulmonary / tracheal, etc.

[0161] In some embodiments, the pharmaceutical composition provided by the present invention is an oral solid dosage form, preferably a tablet. In addition to the active ingredient, the oral solid dosage form also contains pharmaceutical excipients. These pharmaceutical excipients are all conventional pharmaceutical excipients in the art, including fillers (also known as diluents), disintegrants, binders or wetting agents, lubricants (including flow aids), etc. The amount of pharmaceutical excipients used can be in the conventional dosage.

[0162] The fillers typically include lactose, microcrystalline cellulose, mannitol, pregelatinized starch, starch, sucrose, dextrin, sorbitol, calcium carbonate, calcium bicarbonate, hydroxypropyl methylcellulose, and ethyl cellulose. They can be used alone or in combination.

[0163] The disintegrants generally include starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl starch, croscarmellose sodium, croscarmellose, and low-substituted hydroxypropyl cellulose, etc. They can be used alone or in combination.

[0164] The adhesives or wetting agents mentioned generally include polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, polyethylene glycol, starch paste, water, and ethanol solutions of various concentrations. They can be used alone or in combination.

[0165] The lubricants generally include zinc stearate, magnesium stearate, calcium stearate, sodium stearate fumarate, talc, fatty acid sucrose esters, micronized silica gel, stearic acid, and solid polyethylene glycol, etc. They can be used alone or in combination.

[0166] If necessary, other excipients may be added to the above composition, such as sweeteners (e.g., aspartame, stevioside, etc.), colorants (e.g., yellow iron oxide, red iron oxide, etc.), stabilizers (e.g., citric acid, lactic acid, malic acid, etc.), and pH adjusters (e.g., sodium bicarbonate, fumaric acid, citric acid, etc.).

[0167] If desired, the above composition may also contain other suitable active ingredients.

[0168] The preparation of the aforementioned oral solid dosage form can be carried out according to conventional methods for preparing oral solid dosage forms in this technical field. For example, tablets can be prepared by wet granulation and tableting, dry granulation and tableting, fluidized bed granulation and tableting, direct compression of powder mixtures, etc. When the oral solid dosage form is a tablet, it can be further coated as needed to produce film-coated tablets or sugar-coated tablets. The coating materials include cellulose, acrylic resins, and sugars, such as hydroxypropyl methylcellulose and sucrose, etc., and plasticizers, anti-adhesives, and light-blocking agents may also be added.

[0169] The dosage of the above composition is adjusted according to the nature and severity of the patient's condition, the route of administration, and the patient's age and weight.

[0170] Preparation of steroidal compounds of the present invention

[0171] First aspect

[0172] In an embodiment of the present invention, when the steroid compound has the following structural formula...

[0173]

[0174] Wherein ring A is a substituted or unsubstituted heteroaryl group, the heteroatom is N, and the number of heteroatoms N is 1 to 4; the steroidal compound is prepared by a nucleophilic substitution reaction of a compound of the following formula.

[0175]

[0176] Where X is a halogen.

[0177] Accordingly, the present invention provides an intermediate compound for preparing the steroidal compound, the intermediate compound having the following structural formula.

[0178]

[0179] Where X is a halogen.

[0180] Second aspect

[0181] In an embodiment of the present invention, when the steroid compound has the following structural formula...

[0182]

[0183] The steroidal compound is prepared from dydrogesterone via reduction and / or addition reactions.

[0184] The compounds of this invention can be prepared using a variety of synthetic routes and methods. As examples, some compounds of this invention can be synthesized via routes L1 or L2 as follows.

[0185] Route L1

[0186] Using dydrogesterone as a raw material, TM1 and TM2 were first prepared through the following process route, then TM9-TM12 were prepared using TM1 and TM2, and then TM25-TM28 were prepared from TM9-TM12.

[0187]

[0188]

[0189] Synthesis of TM1 and TM2

[0190] TM1 and TM2 were obtained from dydrogesterone via catalytic hydrogenation. The catalysts were metal catalysts such as palladium on carbon or platinum on carbon, and the solvents were tetrahydrofuran, ethyl acetate, methanol, or mixtures thereof. The reaction temperature ranged from room temperature to 50°C, and the reaction time was 4–16 h. Using methanol as a solvent resulted in the formation of a low-polarity ketal byproduct at the 3-position, which needed to be decomposed by acid during post-treatment. TM1 and TM2 had very similar polarities, with TM1 being slightly less polar than TM2. The reaction solution was filtered to obtain a mixed solution of TM1 and TM2, with TM1 being the predominant product. TM1 was obtained by recrystallization from isopropyl ether, and TM2 was obtained by column chromatography.

[0191] Synthesis of TM9-TM12

[0192] Based on the preparation of TM1 and TM2, the carbonyl group at the 3-position was selectively reduced to obtain TM9, TM10, TM11, and TM12 via the Meerwein–Ponndorf–Verley reduction reaction. The catalyst was aluminum isopropoxide or aluminum tert-butoxide, the solvent was chloroform or toluene, the reducing agent was isopropanol or cyclohexanol, the reaction temperature was 60–100 °C, and the reaction time was 1–8 h. The proportions of the reaction products TM9, TM10, TM11, and TM12 were related to the reaction time. A small amount of byproducts from the reduction of the carbonyl group at the 20-position were produced. TM10 and TM11 were the main products corresponding to TM2 and TM1, respectively. The four products TM12, TM9, TM11, and TM10 could be obtained sequentially by column chromatography.

[0193] Synthesis of TM25-TM28

[0194] Based on the preparation of TM9, TM10, TM11 and TM12, a bromination reaction is first carried out, followed by a substitution reaction, to obtain the compounds TM25-TM28 of the present invention, namely TM25, TM26, TM27 and TM28.

[0195] Specifically, based on the preparation of TM9, TM10, TM11, and TM12, the corresponding methyl group at position 22 is first brominated, followed by a substitution reaction with 4-cyanopyrazole to generate the target product. The bromination reaction uses bromine as the brominating agent, hydrobromic acid as the catalyst, methanol or ethanol as the solvent, and a reaction time of 0.5-2 hours at a temperature of 20℃-40℃. The reaction can be quenched by adding sodium bicarbonate aqueous solution, followed by extraction, separation, and drying. The product can be directly added to the next step without purification. The substitution reaction uses potassium carbonate as the base, acetone or tetrahydrofuran as the solvent, and a reaction temperature of 25℃-40℃ for 0.5-3 hours. Extraction with water and dichloromethane, separation, drying, and column chromatography yield products TM25-TM28, namely TM25, TM26, TM27, and TM28.

[0196] Route L2

[0197] Using dydrogesterone as a raw material, TM1 and TM2 were first prepared through the following process route, then TM13-TM16 were prepared using TM1 and TM2, and then TM17-TM20 were prepared from TM13-TM16.

[0198]

[0199]

[0200] The synthesis of TM1 and TM2 is the same as that of route L1.

[0201] Synthesis of TM13-TM16

[0202] Based on the preparation of TM1 and TM2, selective addition of a carbonyl group at the 3-position to TM1 or TM2 yields TM13, TM14, TM15, and TM16. The reagents used in the addition reaction are methylmagnesium chloride or methylmagnesium bromide, the solvent is toluene or tetrahydrofuran, the reaction temperature is -70°C to -30°C, the reaction time is 15 min to 1 h, and the amount of Grignard reagent added is 1.2 to 2 times the amount of the starting material. The reaction produces the target product and a 3,20-position double addition byproduct. Column chromatography yields TM13 and TM14, and TM15 and TM16, respectively.

[0203] Synthesis of TM17-TM20

[0204] Based on the preparation of TM13, TM14, TM15 and TM16, a bromination reaction is first carried out, followed by a substitution reaction, to obtain the compounds TM17-TM20 of the present invention, namely TM17, TM18, TM19 and TM20.

[0205] Specifically, based on the preparation of TM13, TM14, TM15, and TM16, the corresponding methyl group at position 22 is first brominated, followed by a substitution reaction with 4-cyanopyrazole to generate the target product. The bromination reaction uses bromine as the brominating agent, hydrobromic acid as the catalyst, methanol or ethanol as the solvent, and a reaction time of 0.5-2 hours at a temperature of 20°C-40°C. The reaction can be quenched by adding sodium bicarbonate aqueous solution, followed by extraction, separation, and drying. The product can be directly added to the next step without purification. The substitution reaction uses potassium carbonate as the base, acetone or tetrahydrofuran as the solvent, and a reaction temperature of 25°C-40°C for 0.5-3 hours. Extraction with water and dichloromethane, followed by separation, drying, and column chromatography, yields products TM17-TM20, namely TM17, TM18, TM19, and TM20.

[0206] In the preparation of the compounds of the present invention, the protection or deprotection reaction of the functional groups is carried out according to known methods, such as those described in the following literature: Protective Groups in Organic Synthesis, 4th Edition (Theodora W. Greene, Peter GMWuts), Wiley-Interscience (2007).

[0207] Examples of alcohol hydroxyl protecting groups include: ether-type protecting groups such as methoxymethyl ether (-OMOM), trimethylsilyl ether (-OTMS), tetrahydropyranyl ether, etc.; carboxylic acid ester-type protecting groups such as acetate (-OAc), etc.; and sulfonate-type protecting groups such as p-toluenesulfonate (-OTs), etc.

[0208] Examples of ketone carbonyl protecting groups include: ketal protecting groups such as dimethyl ketal; cyclic ketal protecting groups such as 1,3-dioxolane and 1,3-dioxane; oxime protecting groups such as O-methyloxime; and hydrazone protecting groups such as N,N-dimethylhydrazone.

[0209] The present invention will be further described below through specific embodiments.

[0210] Example 1: Synthesis of TM9-TM12

[0211] Weigh 10.0 g of dydrogesterone, add 100 mL of methanol, 100 mL of THF, and 1.0 g of wet palladium on carbon (5% purity). React overnight at room temperature. TLC (P:E = 2:1) shows the disappearance of fluorescence, indicating the reaction is complete. Filter to remove palladium on carbon, add 10 mL of water, then add 5 mL of hydrochloric acid, stir. TLC shows complete decomposition of the ketal at position 3. Rotate the solvent to dryness, add 50 mL of isopropyl ether and homogenize to obtain 4.5 g of TM1. Rotate the mother liquor to dryness and pass it through a column (PE:EA = 8:1) to obtain 0.8 g of TM2, and a mixture of most of TM1 and TM2.

[0212] Weigh 5.0 g of the hydrogenated products of dydrogesterone—a mixture of TM1 and TM2—from the above synthesis, dissolve in 150 mL of chloroform, add 5.0 g of aluminum isopropoxide and 7.5 mL of cyclohexanol, and stir under reflux for 6 h. TLC (P:E = 2:1) showed that most of the starting material had been converted, with multiple product spots showing increased polarity. Column chromatography yielded 0.15 g TM9, 0.78 g TM10, 2.55 g TM11, and 0.10 g TM12.

[0213] The test results are as follows:

[0214] TM9 : 1 H NMR (400MHz, CDCl3) δ4.07(d,J=3.2Hz,1H),2.55(t,J=9.2Hz,1H),2.15(m,1H),2.11(s,3H),1.95-1.84(m,2H),1.84-1.72(m, 6H),1.72-1.57(m,6H),1.55-1.45(m,2H),1.45-1.31(m,3H),1.30-1.21(m,1H),1.20-1.15(m,1H),1.12(s,3H),0.65(s,3H).

[0215] 13 C NMR (100MHz, CDCl3) δ209.62,77.24,66.92,65.02,61.92,49.73,43.97,37.89 ,36.80,35.78,34.34,34.16,31.74,31.36,29.06,25.56,23.68,22.31,21.23.

[0216] TM10 : 1 H NMR (400MHz, CDCl3) δ3.61 (ddd, J=16.1, 10.8, 5.0Hz, 1H), 2.57 (s, 1H), 2.11 (s, 3H),1.92-1.83(m,2H),1.74-1.82(s,4H),1.74-1.66(m,3H),1.65-1.61(m,1H) ,1.54-1.45(m,2H),1.45-1.40(m,1H),1.37-1.30(m,3H),1.30-1.27(m,1H),1. 15-1.08(m,3H),1.01(s,3H),0.97-0.90(m,1H),0.87-0.81(m,1H),0.63(s,3H).

[0217] 13 C NMR(100MHz,CDCl3)δ209.65,71.32,64.80,48.68,45.33,44.81,44.33,38.85,37.68,37.60,37.42,35.49,31.46,30.96,29.70,29.19,25.40,25.27,22.19,21.93,16.43,12.85.

[0218] TM11 : 1 H NMR(400MHz,CDCl3)δ3.69-3.57(m,1H),2.56(t,J=9.2Hz,1H),2.18-2.12(m,1H),2.11(s,4H),2.03(t,J=5.2Hz,1H),1.95(dt,J=14.6,3.5Hz,1H),1.92-1.83(m,2H),1.83-1.74(m,4H),1.73-1.65(m,4H),1.63-1.58(m,2H),1.54-1.45(m,1H),1.44-1.24(m,4H),1.20-1.12(m,1H),1.10(s,3H),0.91(td,J=14.2,3.7Hz,1H),0.63(s,3H).

[0219] 13 C NMR(100MHz,CDCl3)δ209.68,71.87,64.87,48.72,44.32,42.18,37.72,37.21,36.57,35.62,34.56,31.47,31.38,31.12,29.69,25.45,23.33,22.18,22.16,21.58,12.65.

[0220] TM12 : 1H NMR (400MHz, CDCl3) δ4.03 (t, J = 2.4Hz 1H),2.56(t,J=9.2Hz,1H),2.18–2.07(m,1H),2.11(s,3H),1.87–1.75(m,4H),1.75–1.63(m,5H),1.63–1.58(m,3H),1.53(s,1H),1 .52–1.42(m,3H),1.42-1.35(m,1H),1.25–1.19(m,1H),1.11(dd,J=11.6,6.2Hz,1H),1.07–1.00(m,1H),0.98(s,3H),0.63(s,3H).

[0221] 13 C NMR (100MHz, CDCl3) δ209.67,66.52,64.85,48.76,45.28,44.39,39.24,38.05,37.65 ,35.65,35.50,34.12,31.46,29.06,28.70,25.41,24.91,22.19,21.46,15.35,12.81.

[0222] Example 2: Synthesis of TM26 and TM27, and TM25 and TM28

[0223] Synthesis of TM26

[0224] Weigh 0.36 g of TM10, dissolve it in 15 mL of methanol, add 3 drops of hydrobromic acid (48%) and 0.5 g of bromine, stir and react at 28 °C for 1 h. TLC showed complete conversion. Quench the reaction with sodium bicarbonate aqueous solution, then extract with 30 mL of DCM, separate the layers, dry, and evaporate to dryness to obtain the brominated product. Add 20 mL of acetone, 0.4 g of potassium carbonate and 0.2 g of 4-cyanopyrazole to the brominated product, react at 28 °C for 1 h. TLC (PE:EA = 1:1) showed the formation of a product with increased polarity. Extract with water and dichloromethane, separate the layers, dry, and pass through a column to obtain the product. Pulp with water and isopropyl ether to obtain 0.16 g of product TM26.

[0225] The test results are as follows:

[0226] TM26: 1H NMR(400MHz, CDCl3)δ7.86(s,1H),7.80(s,1H),4.95(dd,J=43.2,17.9Hz,2H),3.71–3.51(m,1H),2.65(t,J=8.9Hz,1H),2.17(m,1H),1.9 6–1.63(m,13H),1.62–1.43(m,4H),1.42–1.34(m,2H),1.34–1.28(m, 2H),1.21–1.08(m,3H),1.02(s,3H),0.98–0.76(m,4H),0.68(s,3H).

[0227] 13 C NMR (100MHz, CDCl3) δ202.02,142.35,136.08,113.24,93.10,71.20,61.94,61.58,48.88,45.39,45.1 3,44.74,38.80,37.65,37.60,37.42,35.45,30.90,29.15,25.43,25.20,22.37,21.87,16.50,13.25.

[0228] Synthesis of TM27

[0229] Weigh 1.21 g of TM11, dissolve it in 30 mL of methanol, add 10 drops of hydrobromic acid (48%) and 1.80 g of bromine, stir the reaction at 25 °C for 1.5 h, and TLC shows complete conversion. Quench the reaction with sodium bicarbonate aqueous solution, then add 60 mL of DCM for extraction, separate the layers, dry, and evaporate to dryness to obtain the brominated product. Add 40 mL of acetone, 1.8 g of potassium carbonate and 0.6 g of 4-cyanopyrazole to the brominated product, and react at 28 °C for 1 h. TLC (PE:EA = 1:1) shows the formation of a product with increased polarity. Extract with water and dichloromethane, separate the layers, dry, and pass through a column to obtain the product. Pulp with water and isopropyl ether to obtain 0.83 g of product TM27.

[0230] Synthesis of TM25 and TM28

[0231] Using similar process steps as those used to synthesize TM26 and TM27, TM25 and TM28 were synthesized.

[0232] Example 3: Synthesis of TM13, TM14, TM15 and TM16

[0233] Synthesis of TM13 and TM14

[0234] Weigh 3.16 g TM1, dissolve it in 63 mL of toluene, cool to -40 °C, and add 9.3 mL of tetrahydrofuran solution of 1.6 M methyl magnesium chloride dropwise. After the addition is complete, continue the reaction for 20 min. TLC (PE:DCM:EA = 4:2:1) shows that most of the starting material has reacted completely, and four product spots are formed. Add methanol to quench the reaction, add ethyl acetate and ammonium chloride aqueous solution, extract, separate, dry, and pass through column chromatography to obtain 0.52 g TM13 and 0.25 g TM14.

[0235] The test results are as follows.

[0236] TM13 : 1 H NMR (400MHz, CDCl3) δ2.57(t,J=9.2Hz,1H),2.18-2.12(m,1H),2.10(s,3H),1.95-1.83(m,3H),1.82-1.70(m,6H),1.70-1.58(m,4H ),1.46-1.34(m,3H),1.34-1.28(m,1H),1.28-1.25(m,1H),1.22(s,3H),1.19(m,1H),1.15(s,3H),1.14-1.04(m,2H),0.62(s,3H).

[0237] 13 C NMR (100MHz, CDCl3) δ209.64,70.11,64.84,48.68,44.36,39.41,38.27,37.72,35.53,34 .47,34.33,33.72,31.67,31.48,30.29,29.93,25.49,22.80,22.15,21.98,21.64,12.59.

[0238] TM14 : 1 H NMR (400MHz, CDCl3) δ2.56(t,J=9.2Hz,1H),2.10(s,3H),2.04(t,J=5.9Hz,1H),1.94–1.85(m,3H),1.85–1.69(m,7H),1.68–1.54 (m,4H),1.51(dd,J=13.6,3.3Hz,1H),1.45–1.34(m,3H),1.25(s,3H),1.20(m,1H),1.12(s,3H),1.04–0.94(m,1H),0.63(s,3H).

[0239] 13C NMR (100MHz, CDCl3) δ209.63,71.97,64.87,48.74,44.31,41.25,41.21,37.74,36.47,36 .02,35.95,34.46,31.45,30.80,29.84,26.06,25.44,23.22,22.15,22.06,21.55,12.68.

[0240] Synthesis of TM15 and TM16

[0241] Weigh 1.0 g TM2, dissolve it in 30 mL of toluene, cool to -60 °C, and add 2.5 mL of tetrahydrofuran solution of methyl magnesium chloride (1.6 M). After the addition is complete, continue the reaction for 30 min. TLC (PE:DCM:EA = 4:2:1) shows that most of the starting material has reacted and four product spots have been formed. Add methanol to quench the reaction, add ethyl acetate and ammonium chloride aqueous solution, extract, separate, dry, and pass through column chromatography to obtain 0.18 g TM15 and 0.53 g TM16.

[0242] The test results are as follows.

[0243] TM16: 1 H NMR (400MHz, CDCl3) δ2.57(t,J=9.2Hz,1H),2.18–2.07(m,1H),2.11(s,3H),1.90–1.74(m,5H),1.74-1.65(m,4H),1.65–1.59( m,2H),1.54-1.47(m,3H),1.38(t,J=5.4Hz,1H),1.34–1.27(m,2H),1.26(s,4H),1.18–1.05(m,3H),1.01(s,3H),0.63(s,3H).

[0244] 13 C NMR (100MHz, CDCl3) δ209.67,71.46,64.79,48.76,45.45,44.33,44.20,42.92,38.38,37 .97,37.58,35.96,35.50,31.47,29.14,27.05,25.44,25.25,22.20,21.86,15.94,12.87.

[0245] Example 4: Synthesis of TM17, TM18, TM19 and TM20

[0246] Synthesis of TM17

[0247] Weigh 0.66 g of TM13, dissolve it in 20 mL of methanol, add 6 drops of hydrobromic acid (48%) and 0.96 g of bromine, stir at 25 °C for 2 h, and TLC (PE:DCM:EA = 4:2:1) shows complete conversion. Quench the reaction with sodium bicarbonate aqueous solution, then add 50 mL of DCM for extraction, separate the contents, dry, and evaporate to dryness to obtain the brominated product. Add 30 mL of acetone, 1.2 g of potassium carbonate and 0.5 g of 4-cyanopyrazole to the brominated product, and react at 28 °C for 1.5 h. TLC (PE:EA = 1:1) shows the formation of a product with increased polarity. Extract with water and dichloromethane, separate the contents, dry, and pass through a column to obtain the product. Pulp with water and isopropyl ether to obtain 0.37 g of product TM17.

[0248] The test results are as follows.

[0249] TM17: 1 H NMR (400MHz, CDCl3) δ7.85(s,1H),7.80(s,1H),4.95(dd,J=45.5,17.9Hz,2H),2.65(t,J=9.0Hz,1H),2.24-2.13(m,1H),2.03-1 .90(m,2H),1.89–1.59(m,11H),1.50–1.36(m,3H),1.35–1.28(m,2H),1.23(s,3H),1.21-1.17(m,2H),1.15(s,3H),0.68(s,3H).

[0250] 13 C NMR (100MHz, CDCl3) δ201.97,142.36,136.02,113.21,93.16,70.13,62.01,61.62,48.91,45.45,39.38,3 8.23,37.82,35.56,34.48,34.34,33.70,31.70,30.22,29.95,25.53,22.75,22.35,21.98,21.61,13.01.

[0251] Synthesis of TM20

[0252] Weigh 0.36 g of TM14, dissolve it in 15 mL of methanol, add 3 drops of hydrobromic acid (48%) and 0.50 g of bromine, stir and react at 25 °C for 2 h. TLC (PE:DCM:EA = 4:2:1) shows complete conversion. Quench the reaction with sodium bicarbonate aqueous solution, then add 50 mL of DCM for extraction, separate the contents, dry, and evaporate to dryness to obtain the brominated product. Add 20 mL of acetone, 0.5 g of potassium carbonate and 0.2 g of 4-cyanopyrazole to the brominated product, react at 28 °C for 1.5 h, TLC (PE:EA = 1:1) shows the formation of a product with increased polarity. Extract with water and dichloromethane, separate the contents, dry, and pass through a column to obtain the product. Pulp with water and isopropyl ether to obtain 0.15 g of product TM20.

[0253] The test results are as follows.

[0254] TM20: 1 H NMR (400MHz, CDCl3) δ7.86 (s, 1H), 7.80 (s, 1H), 4.94 (dd, J = 42.0, 17.8Hz, 2 H),2.64(t,J=9.0Hz,1H),2.25–2.10(m,1H),2.07(t,J=6.0Hz,1H),1.98–1 .65(m,11H),1.51(dd,J=13.6,3.5Hz,1H),1.46–1.34(m,4H),1.25(s,4H), 1.23-1.15(m,2H),1.13(s,3H),1.01(td,J=14.3,4.0Hz,1H),0.69(s,3H).

[0255] 13 C NMR (100MHz, CDCl3) δ201.94,142.36,136.03,113.21,93.15,71.97,62.05,61.59,48.97,45.38,41.19,4 1.17,37.83,36.44,36.01,35.96,34.45,30.75,29.85,26.12,25.48,23.18,22.35,22.07,21.52,13.11.

[0256] Synthesis of TM19

[0257] Weigh 0.19 g of TM15, dissolve it in 10 mL of methanol, add 2 drops of hydrobromic acid (48%) and 0.35 g of bromine, stir and react at 25 °C for 2 h. TLC (PE:DCM:EA = 4:2:1) shows complete conversion. Quench the reaction with sodium bicarbonate aqueous solution, then add 30 mL of DCM for extraction, separate the contents, dry, and evaporate to dryness to obtain the brominated product. Add 20 mL of acetone, 0.3 g of potassium carbonate and 0.15 g of 4-cyanopyrazole to the brominated product, react at 28 °C for 2 h, TLC (PE:EA = 1:1) shows the formation of a product with increased polarity. Extract with water and dichloromethane, separate the contents, dry, and pass through a column to obtain the product. Pulp with water and isopropyl ether to obtain 0.05 g of product TM19.

[0258] The test results are as follows.

[0259] TM19: 1 H NMR (400MHz, CDCl3) δ7.83 (d, J=20.7Hz, 2H), 4.95 (dd, J=42.4, 17.9Hz, 2H), 2.64 (t, J=9.0Hz, 1H), 2.17 (ddd, J=13.6, 11.6, 5.8Hz, 1H), 1.96–1.78 (m,5H),1.78–1.61(m,6H),1.56–1.45(m,3H),1.41(t,J=13.2Hz,2H),1.3 0-1.22(m,4H),1.21(s,4H),1.11–1.02(m,1H),0.94(s,3H),0.69(s,3H).

[0260] 13 C NMR (100MHz, CDCl3) δ202.00,142.36,136.03,113.21,93.15,69.80,62.01,61.59,48.99,45.44,45.00,4 1.53,41.06,37.73,37.55,35.88,35.50,34.56,31.66,29.02,25.45,24.81,22.37,21.62,15.41,13.25.

[0261] Synthesis of TM18

[0262] Weigh 0.51 g of TM16, dissolve it in 20 mL of methanol, add 5 drops of hydrobromic acid (48%) and 0.74 g of bromine, stir and react at 28 °C for 1 h. TLC (PE:DCM:EA = 4:2:1) shows complete conversion. Quench the reaction with sodium bicarbonate aqueous solution, then add 50 mL of DCM for extraction, separate the contents, dry, and evaporate to dryness to obtain the brominated product. Add 30 mL of acetone, 1.2 g of potassium carbonate and 0.3 g of 4-cyanopyrazole to the brominated product, react at 32 °C for 1 h, TLC (PE:EA = 1:1) shows the formation of a product with increased polarity. Extract with water and dichloromethane, separate the contents, dry, and pass through a column to obtain the product. Pulp with water and isopropyl ether to obtain 0.18 g of product TM18.

[0263] The test results are as follows:

[0264] TM18: 1 H NMR (400MHz, CDCl3) δ7.86 (s, 1H), 7.80 (s, 1H), 4.95 (dd, J = 44.8, 17.9Hz, 2 H),2.65(t,J=9.0Hz,1H),2.23–2.07(m,1H),1.95–1.73(m,7H),1.73–1.65( m,3H),1.64-1.57(m,2H),1.54–1.48(m,2H),1.41(dd,J=6.9,5.1Hz,1H),1 .35–1.28(m,2H),1.26(s,4H),1.15–1.08(m,2H),1.02(s,3H),0.69(s,3H).

[0265] 13 C NMR (100MHz, CDCl3) δ201.99,142.36,136.04,113.22,93.14,71.39,61.94,61.58,48.96,45.39,45.26,4 4.13,42.84,38.34,37.98,37.65,35.91,35.47,29.10,27.06,25.47,25.17,22.39,21.81,16.01,13.28.

[0266] Example 5

[0267] I. Simulation of compound docking with GABAA receptor and hormone receptor molecules

[0268] The following compounds were scored using the docking software MOE (Molecular Operating Environment, a comprehensive software system for pharmaceuticals and life sciences developed by Chemical Computing Group ULC in Canada). MOE scores are calculated based on parameters such as electrostatics, hydrogen bonding, molecular attraction, and molecular orbitals between molecules. A more negative score indicates a lower free energy, more stable conformation, and better bonding.

[0269] The following compound (which differs from TM17 in that the methyl group at C10 is in the unflipped β configuration), TM17, and the GABAA receptor were docked and scored. The docking scores were -5.39 and -5.72, respectively, indicating that the 10α configuration compound TM17 obtained after the flipping binds better to the GABAA receptor and is expected to have higher activity.

[0270]

[0271] Furthermore, the binding scores of TM17 to androgen receptors and estrogen receptors were calculated separately. The results showed that the binding score of TM17 to androgen receptors was -5.30, and the binding score of TM17 to estrogen receptors was -4.97, both lower than the binding score of -5.72 to GABAA receptors. This indicates that the binding of TM17 to GABAA receptors is more selective, thereby reducing side effects on non-target receptors.

[0272] II. Pharmacological Experiments: Effects of the Compounds in the Examples of this Invention on Chloride Ion Channels and Acute Depression in Mice 1. Experimental Objective

[0273] A chloride ion channel model was constructed using human glioma cells, and an acute depression model was established using mice. The effects of neurosteroidal compounds on chloride ion channels and the acute depression mouse model were comprehensively evaluated.

[0274] 2. Test materials

[0275] Samples: TM17, TM18, TM19, TM20; Reference standard: Solvent reference standard - edible oil.

[0276] Experimental cells: Human glioma cells U251, provided by the Cancer Institute of Central South University, cultured under high glucose DMEM + 10% FBS conditions.

[0277] Experimental animals: 60 ICR mice, half male and half female, with females weighing 14.55–19.22g and males weighing 14.84–17.44g, provided by Hunan Silek Jingda Experimental Animal Co., Ltd.

[0278] Rearing Environment: Environment Level: General; Temperature: 22-26℃; Relative Humidity: 40%-70%; Cage Size: 465×300×200mm 3 Feed and drinking water: Complete pelleted SPF rat feed, 10kg / bag, bottled purified water; Bedding: Corn cob bedding.

[0279] 3. Test methods

[0280] 3.1. Cell Experiment Methods

[0281] MQAE is a chloride ion fluorescent probe with bromide ions as its paired anion. Its maximum excitation wavelength is approximately 355 nm, and its maximum emission wavelength is approximately 460 nm. When the intracellular chloride ion concentration increases, the fluorescence intensity of MQAE decreases proportionally to the chloride ion concentration; that is, the fluorescence intensity is inversely proportional to the chloride ion concentration. MQAE is prepared using Krebs-HEPES buffer, which contains a high concentration of chloride ions. Incubation of U251 cells with MQAE increases the intracellular chloride ion concentration. Using a chloride-free buffer to prepare the sample, after mixing, a chloride ion concentration gradient is created between the intracellular and extracellular spaces. Activation of GABAa receptors with γ-aminobutyric acid (GABA) opens chloride ion channels, causing intracellular chloride ion efflux and enhancing the cell's fluorescence intensity. The fluorescence intensity reflects the degree of chloride ion channel opening, allowing for the detection of the effect of neurosteroidal compounds on chloride ion channels.

[0282] Reagent preparation

[0283] Chlorine-free buffer: 20mM HEPES, 2.4mM K2HPO4, 1mM KH2PO4, 1mM CaSO4, 130mM NaNO3, 10mM Glucose.

[0284] Chlorine-containing buffer: 20mM HEPES, 2.4mM K2HPO4, 1mM KH2PO4, 1mM CaSO4, 130mM NaCl, 10mM M glucose.

[0285] Krebs-HEPES buffer: 20mM HEPES, 128mM NaCl, 2.5mM KCl, 2.7mM CaCl2, 1mM MgCl2, 16mM glucose.

[0286] Sample preparation matrix (containing 5 μM GABA): Take 100 mL of chloride-free buffer and mix it with 100 mL of serum-free DMEM at a ratio of 1:1.

[0287] Preparation of MQAE dye: Take one vial of MQAE dye (20mg): The relative molecular weight of MQAE is 326.19. Add 61.31μL of Krebs-HEPES buffer to prepare a 1M solution. Take 30μL and prepare 6mL of the solution with Krebs-HEPES buffer to make the MQAE dye working solution. The final concentration is 5mM.

[0288] Preparation of working solution for test sample

[0289] TM17, TM18, TM19, and TM20 were prepared to a working concentration of 20 μM using the sample preparation matrix.

[0290] Cell preparation and viability testing group settings

[0291] U251 cells were collected via routine digestion and seeded into 96-well plates at a density of 1 × 10⁶ cells / well. 5 Cells were placed in each well and cultured overnight in an incubator. After the cells adhered, they were washed three times with chlorine-free buffer and then incubated with 100 μL of MQAE working solution per well under the following conditions: protected from light, 37°C, for 30 min.

[0292] TM17 to TM20 are the test samples. All samples were prepared into working solutions with a final concentration of 20 μM using chlorine-free buffer. A chlorine-free buffer solution without the sample was set as a blank control. The group settings are shown in Table 1 below.

[0293] Table 1: Samples used for testing

[0294] sample Concentration (μM) MQAE incubation time Excitation light color Emitted light color Number of duplicate holes Blank control -- 30min blue green 6 TM17 10 30min blue green 6 TM18 10 30min blue green 6 TM19 10 30min blue green 6 TM20 10 30min blue green 6

[0295] Detection

[0296] Turn on the fluorescence microscope and the fluorescence light source. Turn off the external light source. Adjust the B / G intensity of the fluorescence light source to 85 and the UV intensity to 0. After the emitted light intensity stabilizes (approximately 15 minutes), take the 96-well culture plate that has finished incubating in the dark. Add 100 μL of the prepared working solution containing the sample to each well. After 1 minute, observe under the fluorescence microscope. Take pictures of the field of view where cell growth is relatively uniform under a 10x objective lens. Take 6 pictures for each sample. Use ImageJ software to perform semi-quantitative analysis on the average fluorescence intensity of the captured field of view. The results are expressed as mean ± standard deviation.

[0297] 3.2. Animal testing methods

[0298] Grouping and Identification of Animals

[0299] After a 3-day acclimatization period, the experimental animals were administered drugs according to their assigned groups. The experimental groups were a model group, a compound TM1 group, and a compound TM2 group. Edible oil was used as the solvent for all test compound groups, and the administration route was intraperitoneal injection. The solvent control group received an equal volume of edible oil via intraperitoneal injection. The total administration volume for all groups was 20 mL / kg.

[0300] The experimental dosage and grouping are detailed in Table 2.

[0301] Dosing cycle settings

[0302] Brexanolone is used to treat postpartum depression. The dosage is 100 mg every 12 hours via continuous intravenous injection over 60 hours. At the start of administration, the dosing rate needs to be gradually increased, and at the end, it needs to be gradually decreased. Therefore, the total dosage for the treatment period is 450 mg. Referring to the Brexanolone treatment cycle, a 3-day dosing cycle is set, with one dose administered daily in the morning.

[0303] The clinical dosage of Brexanolone is approximately 200 mg / day. For an adult weighing 70 kg, the clinical dose of Brexanolone is 2.857 mg / kg / day. The equivalent dose conversion factor for humans and mice based on body surface area is 0.0026. Assuming a mouse weight of 20 g, the converted mouse dose is: 2.857 mg / kg / day × 70 kg × 0.0026 / 0.02 kg = 25.999 mg / kg / day, which is approximately 26.0 mg / kg / day.

[0304] This study is for efficacy verification and uses a single-dose test. The dosing cycle and clinical dosage of the compound of this invention are the same as those of Brexanolone described above.

[0305] Table 2 Experimental Dosage and Group Design

[0306]

[0307] Testing indicators and testing methods

[0308] The detection index was the behavioral changes of mice in the last 4 minutes of the 6-minute period, including the time of first immobility and the duration of immobility; where immobility refers to the animal giving up active struggle and being in a state of no struggle.

[0309] Testing Method: 24 hours after drug administration, each group of animals was placed in the laboratory for 30 minutes to 1 hour to reduce their anxiety about the new environment. A camera was installed in front of the tail suspension box, and the corresponding parameters were set in the software, recording animal numbers, status, and other information. The experimental animals were removed, and a mark was made 3 mm from the tail tip with a marker. The tail was then secured with medical tape. After all animals in the same group were secured with tape, the mice in the same group were quickly suspended in the tail suspension box. Behavioral data was recorded for 6 minutes using behavioral software, and behavioral changes were analyzed in the following 4 minutes.

[0310] After the experiment, the tape was removed from the mouse's tail and the animal was returned to its cage.

[0311] 4. Test Results

[0312] 4.1. Effects of TM series samples on chloride ion channels in U251 cells

[0313] GABA activates chloride ion channels via GABAa receptors on the neuronal cell membrane, leading to an intracellular influx of chloride ions and the generation of heterogeneous postsynaptic potentials that inhibit neuronal excitation. In this experiment, U251 cells were incubated with an MQAE probe prepared in Krebs-HEPES buffer containing a high concentration of chloride ions. Once the intracellular and extracellular chloride ion concentrations reached equilibrium, a high-chloride-ion cell model was established. GABA was then used to activate chloride GABAa receptors, opening chloride ion channels.

[0314] The TM series compounds are neurosteroids with positive allosteric activity against GABAa receptors, prolonging the time for GABA to activate GABAa receptors and extending the opening time of chloride channels. When TM series samples prepared with a chloride-free matrix were added to U251 cells, the extracellular chloride ion concentration decreased, indicating that the TM series samples prolonged the chloride channel opening time, resulting in continuous chloride ion efflux. MQAE fluorescence intensity showed an inverse correlation with intracellular chloride ion concentration; therefore, intracellular fluorescence intensity reflects the duration of chloride channel opening, indirectly reflecting the activity of the TM series samples.

[0315] The average fluorescence intensity of the solvent control group was 42.543±4.202, while the average fluorescence intensities of TM17, TM18, TM19, and TM20 were 47.499±2.240, 48.621±1.006, 51.201±3.371, and 47.562±2.659, respectively. Compared with the solvent control group, the average fluorescence intensity of each sample group increased significantly, and the difference was statistically significant (P<0.05), indicating that the TM series samples can significantly prolong the opening time of GABA-activated chloride ion channels.

[0316] The mean fluorescence intensity analysis of the TM series samples after intervention in U251 cells is detailed in Table 3.

[0317] Table 3. Fluorescence Results Analysis (n=6)

[0318] Group Mean fluorescence intensity (mean ± standard deviation) p-value Solvent control group 42.543±4.202 -- TM17 47.499±2.240 0.004** TM18 48.621±1.006 0.001** TM19 51.201±3.371 0.000** TM20 47.562±2.659 0.003**

[0319] Note: "*" indicates that compared with the solvent control group, P < 0.05; "**" indicates that compared with the solvent control group, P < 0.01.

[0320] 4.2. Effects of TM series samples on a mouse model of acute depression

[0321] When a mouse is suspended upside down by its head, it initially struggles violently to escape. After failing to escape, it remains motionless, a state considered a sign of "despair." The duration of immobility in the tail-suspended mouse test is used to detect despair behavior and is commonly used in initial screening tests for antidepressants.

[0322] In this experiment, among female animals, the immobility time of mice in the TM17 and TM20 groups was prolonged by more than 10% compared with the model control group, but there was no statistically significant difference (P > 0.05), possibly due to large individual differences within the groups. Among male animals, the immobility time of mice in all sample groups was prolonged by more than 10%, with the TM18 and TM20 groups showing a prolongation of approximately 20%, which was statistically significant (P < 0.05). This indicates that after three consecutive days of injection of the TM series samples, the sedation effect varied by sex, and a stronger sedative effect was observed in male mice.

[0323] Table 4 details the duration of immobility in mice after prophylactic administration of TM series samples in the tail suspension test.

[0324] Table 4. Duration of immobile state in tail suspension test animals (n=5)

[0325]

[0326] Note: "*" indicates that compared with the model control group, P < 0.05; "**" indicates that compared with the model control group, P < 0.01.

[0327] 5. Experimental Conclusions

[0328] By constructing an intracellular hyperchloride ion model, and using TM17–TM20 series samples to treat cells with hyperchloride ion levels, fluorescence microscopy observation and analysis revealed that intracellular fluorescence intensity increased after intervention with the TM series samples. This indicates that the TM17–TM20 series samples can prolong the opening time of GABA-activated chloride ion channels.

[0329] By establishing a mouse model of acute depression, prophylactic administration of TM17-TM20 series samples showed no significant difference in immobility time in female animals, but significantly prolonged immobility time in male animals. This indicates that the TM17-TM20 series samples have a sedative effect on mice with acute depression.

[0330] Based on the results of in vivo and in vitro experiments, it is speculated that the TM17-TM20 series samples have a therapeutic effect on depression.

[0331] III. GABAA receptor patch-clamp electrophysiological assay (primary cells)

[0332] 1. Fluids used for electrophysiological recording

[0333] Extracellular fluid (GABA-001-1)

[0334] 140mM NaCl, 5mM CsCl, 2mM CaCl2·2H2O, 1mM MgCl2·6H2O, 5mM HEPES, 10mM D-Glucose, and NaOH to adjust pH to 7.4.

[0335] Intracellular fluid (GABA-001-2)

[0336] 130mM CsCl, 0.1mM CaCl2·2H2O, 2mM MgCl2·6H2O, 1.1mM EGTA, 5mM Na2-ATP, 10mM HEPES, CsOH adjust pH=7.2.

[0337] Extracellular fluid was stored for 2 weeks. Intracellular fluid, after preparation, was aliquoted into 1 mL tubes and stored at -20°C. Freshly thawed intracellular fluid was used daily for experiments. All intracellular fluid was used within three months. After three months, the old intracellular fluid was discarded and freshly prepared.

[0338] 2. Patch-clamp detection of primary hippocampal neurons in SD neonatal rats

[0339] The voltage stimulation protocol for whole-cell patch-clamp recording of GABA receptor currents is as follows: After whole-cell sealing, the cell membrane voltage is clamped at -70 mV. GABA receptor currents are recorded in gap-free mode (for primary hippocampal neurons from SD rats, 50 μM D-AP5, 20 μM DNQX, 10 μM Strychnine, and 300 nM TTX are added to the extracellular fluid to block NMDA, AMPA, Glycine, and Nav receptors). The cell surface is sequentially sprayed with low concentrations of GABA (15 μM), mixtures of low concentrations of GABA and test substances from low to high concentrations (GABA 15 μM + TM17 0.1 μM, GABA 15 μM + TM17 1 μM), and 300 μM GABA, recording the peak current. Test substance administration: Each concentration of test substance is administered 1-2 times, followed by rinsing with extracellular fluid for 2-3 min before measuring the next concentration. Finally, 300 μM GABA is administered as a control. The test data were acquired by the EPC-10 amplifier (HEKA) and stored in the PatchMaster (HEKA) software.

[0340] A capillary glass tube was drawn into a recording electrode using a microelectrode drawing instrument. Under an inverted microscope, the microelectrode manipulator was used to bring the recording electrode into contact with the cell, and negative pressure was applied to aspirate and form a GΩ seal. After forming the GΩ seal, rapid capacitance compensation (pF) was performed, and then negative pressure was continued to rupture the cell membrane, forming a whole-cell recording mode. Slow capacitance compensation was then performed, and the membrane capacitance (pF) and series resistance were recorded. No leakage compensation was applied.

[0341] A coverslip containing cells was placed in the recording bath of an inverted microscope. The working solution of the test substance and the external solution without the compound were sequentially flowed through the recording bath from low to high concentration using gravity perfusion, thus acting on the cells. A vacuum pump was used for fluid exchange during recording. All electrophysiological experiments were performed at room temperature, with three parallel experiments. The results are shown in Table 5. Figure 1-3 As shown, where Figure 1 , Figure 2 and Figure 3 The detection spectra of TM 17 with GABA 15μM, GABA 15μM+0.1μM and GABA 15μM+1μM are shown respectively.

[0342] Table 5: Detection Results of TM 17

[0343]

[0344] The same method was used to test TM18, and the results are shown in Table 6 below.

[0345] Table 6: Detection Results of TM 18

[0346]

[0347] The above experimental results show that under the action of TM17 and TM18, the inward current (chloride ion current) of GABAA receptors is significantly increased, indicating that TM17 and TM18 are good positive regulators of GABAA receptors.

[0348] IV. GABAA receptor patch-clamp electrophysiological assay (stable cells)

[0349] (I) Stable cell lines and cell culture

[0350] 1. Using GABA stably expressed in HEK293 cells A The (α1β2γ2) cell line has the following gene information: GABA α1 (NP_000797.2), GABA β2 (NP_000804.1), and GABAγ2 (NP_000807.2).

[0351] 2. Cell preparation:

[0352] 1) Seed cells in culture medium (containing 0.5 μg / mL puromycin and 10% fetal bovine serum) in a 6 cm cell culture dish. When the cell confluence is 30%-70%, remove the old culture medium, add 1 ml PBS, gently rinse the cells and discard the medium.

[0353] 2) Add 1 ml of TrypLe™ Express Gently shake the culture dish to cover the bottom of the cell culture dish. Then place it in a 37°C CO2 incubator for 2-3 minutes. After removing it, gently pipette the cells to suspend them.

[0354] 3) Place the adherent cells into a sterile centrifuge tube and centrifuge at 1000 rpm for 3 min, then adjust the cell concentration to 3 × 10⁻⁶. 4 Cells / mL were seeded into 500 μL of cell culture in 24-well cell slides. After the cells adhered well, electrophysiological experiments were performed overnight.

[0355] (II) Intracellular and extracellular fluids

[0356] 1) Extracellular fluid (mM): 137 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 Glucose, 10 HEPES. Adjust the pH to 7.4 with NaOH.

[0357] 2) Intracellular fluid (mM): 140 CsCl, 5 EGTA, 10 HEPES. Adjust pH to 7.2 with CsOH.

[0358] (III) Preparation of the test sample

[0359] 1) Dilute the test sample with dimethyl sulfoxide (DMSO) to prepare a 30 mM stock solution.

[0360] 2) Dilute the test sample stock solution sequentially with DMSO to obtain secondary stock solutions of 10 mM, 3 mM, 1 mM, 0.3 mM and 0.1 mM.

[0361] 3) Before the experiment, take 5 μL of the mother solution and the secondary mother solution and dilute them into 5 mL of extracellular fluid containing 3 μM GABA. After 1,000-fold dilution, the final concentrations of the test sample are 10 μM, 3 μM, 1 μM, and 0.1 μM. These concentrations are used to detect the EC50 of the test sample using patch clamp.

[0362] (iv) Whole-cell testing procedure

[0363] 1) Glass recording electrodes were drawn using a Sutter P-1000 microelectrode drawing instrument.

[0364] 2) Place the cell coverslip into the extracellular fluid bath and observe the cells using a 10x objective lens.

[0365] 3) Use a micromanipulator to slowly bring the glass electrode close to the cell. When the field of view at the tip of the glass electrode is close to the same horizontal plane as the field of view of the cell, adjust the objective lens to 40x and slowly contact the cell surface.

[0366] 4) When the glass electrode just touches the cell surface, the liquid ingress resistance increases slightly. Carefully continue to move the glass electrode downward until the liquid ingress resistance increases to 1 / 3–2 / 3 of the initial liquid ingress resistance. Stop moving when the glass electrode stops moving and quickly and gently draw it from the side hole of the holder to form a gigaohm seal.

[0367] 5) Slowly apply negative pressure to break the membrane and form a whole-cell recording mode.

[0368] 6) The cells were clamped at -70mV, and the peak GABA current was recorded. The cells were sprayed onto the cell surface for 10-15 seconds using gravity dosing and cumulative dosing methods. The same cell was cumulatively dosed with negative (0.1% DMSO), 3μM GABA, and a mixture of 3μM GABA and the test sample (from low to high).

[0369] (V) Data Processing

[0370] 1) Data quality control

[0371] Valid data in the experiment must meet the following criteria:

[0372] Initial sealing resistance > 1 GΩ; series resistance (Ra) < 15 MΩ; film resistance Rm > 200 MΩ;

[0373] 2) Data Analysis

[0374] Data conforming to the above GABA current quality standards will be further analyzed using the following steps:

[0375] 2.1) Standardize the current of the test sample and the GABA EC10 (3μM) current.

[0376]

[0377] 2.2) The half-activation concentration of each compound was calculated using the following equation:

[0378] Y=Bottom+(Top-Bottom) / (1+10^((LogEC50-X)×HillSlope))

[0379] The dose-dependent effect was nonlinearly fitted using the above equation, where EC50 represents the half-activation concentration and Hillslope represents the Hill coefficient. Curve fitting and EC50 calculation were performed using GraphPad Prism 5 software, and the results are shown below. Figure 4 and Figure 5 middle.

[0380] Compared to the control GABA with an EC50 of 10.82 μM, the EC50 for TM17 was 8.708 μM, and for TM18 it was 1.646 μM. This also indicates that under the action of TM17 and TM18, the inward current (chloride ion current) of GABAA receptors is significantly increased, further confirming that TM17 and TM18 are good positive regulators of GABAA receptors.

[0381] The above description is merely an embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A steroidal compound or a pharmaceutically acceptable salt thereof, characterized in that, The steroid compound has a 9β,10α structure and the following structural formula: 。 2. A method for preparing the steroidal compound of claim 1, characterized in that, The steroidal compound is prepared by a nucleophilic substitution reaction of a compound of the following formula. ， Where X is bromine.

3. An intermediate compound for preparing the steroidal compound of claim 1, characterized in that, The intermediate compound has the following structural formula. ， Where X is bromine.

4. Use of the steroidal compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating central nervous system-related disorders.

5. The use according to claim 4, wherein the central nervous system-related condition is selected from: sleep disorders, schizophrenia spectrum disorders, memory disorders and / or cognitive disorders.

6. The use according to claim 5, wherein the central nervous system-related condition is selected from: insomnia, depression, anxiety, and epilepsy.

7. A pharmaceutical composition comprising the steroidal compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

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