Application of steroid compound and pharmaceutical composition

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a steroid compound and a pharmaceutical composition. The steroid compound can significantly inhibit excitability of primary cerebral cortex neurons and effectively relieve stimulation of an epilepsy inducer 4-aminopyridine (4-AP) to neurons, and is expected to become an important candidate drug for treating neuron injury and related nervous system diseases.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an application of a steroid compound and a pharmaceutical composition. Background Art

[0002] Spontaneous calcium oscillation (SCO) is a calcium signal fluctuation phenomenon in cells without external stimulation or exogenous regulators, which is manifested as periodic regular changes in cytoplasmic calcium levels within a certain range. This oscillation is particularly important in neurons. As a key indicator of neuronal excitability, its occurrence is affected by the release of neurotransmitters, the generation of neuronal action potentials, and the activity of multiple ion channels such as NMDAR, AMPAR, RyR and IP3R.

[0003] In supraoptic neurons, SCO is regulated by Ca 2+ Channels and Na + / Ca 2+ The exchanger mediates the removal of extracellular Na + concentrated or by using Na + / Ca 2+ The exchange inhibitor KB-R7943 can effectively block calcium oscillations. Glutamate, as one of the main neurotransmitters, plays a central role in regulating SCO. It not only triggers Ca 2+ in neurodegenerative diseases caused by inflammation, but also plays a key role in regulating SCO. 2+ Glutamate can mediate neurotoxicity and enhance calcium oscillations by increasing the level of nicotinic acid adenine dinucleotide phosphate (NAADP) in neurons. Abnormal uptake and transport of glutamate are often associated with a variety of diseases, including Huntington's disease, Alzheimer's disease, Tourette syndrome, epilepsy, stroke, demyelinating disease, hyperammonemia, myocardial ischemia and cancer.

[0004] Dopamine, another important neurotransmitter, is able to inhibit glutamate-induced NMDA receptor calcium signals and currents, suggesting a potential protective role in regulating SCO. The synchronization of SCO may be affected in diseases associated with dysfunction of dopamine metabolism and signaling, such as schizophrenia, addiction, attention deficit hyperactivity disorder, and Parkinson's disease.

[0005] Chronic amyloidosis significantly suppresses spontaneous functional Ca 2+ activity in primary hippocampal neurons in Alzheimer's disease 2+ Activity, suggesting that abnormalities in SCO may be involved in the progression of the disease. In recent years, the role of SCO in the regulation of neuropathic pain has received increasing attention. Spontaneous persistent pain, hyperalgesia, and pain paresthesia caused by nerve damage are closely related to abnormalities in primary afferent neurons, and these neurons exhibit spontaneous Ca 2+The oscillations are synchronized with the spontaneous firing of neurons and reflect the excitatory state of the neurons.

[0006] Given the core role of SCO in a variety of neurological diseases and the limitations of existing treatments, drug discovery strategies based on SCO regulatory mechanisms have shown great potential. This not only provides a new perspective for understanding neuronal activity, but also opens the way for the development of a new generation of highly effective and low-toxic neurological drugs, which has far-reaching scientific significance and clinical application prospects.

[0007] In this context, the present invention aims to explore and utilize the regulatory mechanism of SCO in order to discover innovative treatments, reveal new therapeutic targets, promote the development of new drugs, and address the challenges in the current treatment of neurological diseases. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide an application and pharmaceutical composition of a steroid compound, which can be used for regulating or protecting neuronal function, and can be used to treat diseases related to neuronal function regulation, including epilepsy, depression, Alzheimer's disease, Huntington's disease, Tourette's disease, demyelinating diseases, schizophrenia, addiction, attention deficit hyperactivity disorder, Parkinson's disease and neuropathic pain.

[0009] Specifically, the present invention provides a steroid compound as shown in formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer or polymorph thereof in the preparation of a neuron function modulator.

[0010]

[0011] Among them, R 1 Selected from H or -OSO 3 Na;

[0012] R 2 Selected from OH or -OSO 3 Na;

[0013] R 3 Select from H or R 3 With R 4 together is a double bond;

[0014] R 4 Selected from H or -OSO 3 Na;

[0015] R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0016] In some specific embodiments of the present invention, R 5 The biological activity is higher when a branched alkyl group is used. The substituent on the branched alkyl group may be methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl and tert-butyl, and preferably methyl or tert-butyl.

[0017] In some specific embodiments of the present invention, the C 3 ~C 18 The branched alkyl group is selected from: C 3 ~C 12 Alkyl, or C 3 ~C 11 alkyl;

[0018] The C 3 ~C 18 The branched alkenyl group is selected from C 3 ~C 12 Alkenyl, C having at least one tert-butyl substitution 3 ~C 11 Alkenyl.

[0019] In some specific embodiments of the present invention, R 5 The present invention has found that when R 5 When the carbon chain is extended to 6 carbon atoms and the number of substituents is further increased, its biological activity is significantly improved.

[0020] In some specific embodiments of the present invention, the C6-C18 branched alkyl group is selected from: C6-C12 straight-chain alkyl groups having at least 3 methyl substitutions; i.e., straight-chain alkyl groups of 9 to 12 carbon atoms having at least 3 methyl substitutions, and the total number of carbon atoms does not exceed 18, and the positions of the multiple methyl groups can be arranged at the positions of R 5 The 2nd to 4th carbon atom and the last carbon atom from the end of the substituent;

[0021] Or, the C6-C18 branched alkyl group is selected from: a straight-chain alkyl group of 4-11 carbon atoms having at least one tert-butyl group and at least one carbon atom substituted, and the total number of carbon atoms does not exceed 18, and the position of the tert-butyl group can be set to R 5 The position of the methyl group on the 2nd to 4th carbon atom from the end of the substituent group includes the position of the methyl group on the carbon atom with R 5 The last carbon atom starting from the end of the substituent.

[0022] In some specific embodiments of the present invention, R 5The branched alkenyl group is selected from C9 to C16, and C9 to C16. Preferably, the C9 to C16 branched alkenyl group is selected from: C6 to C11 straight-chain alkenyl groups substituted with 3 to 5 methyl groups, i.e., straight-chain alkenyl groups of 9 to 11 carbon atoms substituted with 3 to 5 methyl groups, and the total number of carbon atoms does not exceed 16, and the positions of the multiple methyl groups are preferably arranged at the position of R 5 The 2nd to 4th carbon atom and the last carbon atom from the end of the substituent;

[0023] Or, the C9-C16 branched alkenyl group is selected from: C6-C9 straight-chain alkenyl groups having one tert-butyl group and 1-2 methyl groups as substitutions, and the total number of carbon atoms does not exceed 16, and the position of the tert-butyl group can be set to R 5 The position of the methyl group on the 2nd to 4th carbon atom from the end of the substituent group includes the position of the methyl group on the carbon atom with R 5 The last carbon atom starting from the end of the substituent.

[0024] In some specific embodiments of the present invention, when R 1 When selected from H, R 2 Selected from OH;

[0025] Or, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na;

[0026] Or, when R 3 When selected from H, R 4 Selected from -OSO 3 Na;

[0027] Or, when R 3 With R 4 When they form a double bond, R 4 Selected from H.

[0028] In some specific embodiments of the present invention, R 5 Select from the following substituents:

[0029]

[0030] In some specific embodiments of the present invention, when R 1 When selected from H, R 2 is selected from OH; the general structural formula at this time is shown in Formula IA:

[0031]

[0032] Among them, R 3 Select from H or R 3 With R 4 together is a double bond;

[0033] R 4 Selected from H or -OSO 3 Na;

[0034] R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0035] In some specific embodiments of the present invention, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na; the general structure at this time is shown in Formula IB:

[0036]

[0037] Among them, R 3 Select from H or R 3 With R 4 together is a double bond;

[0038] R 4 Selected from H or -OSO 3 Na;

[0039] R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0040] In some specific embodiments of the present invention, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na, R 3 When selected from H, R 4 Selected from -OSO 3 Na; the general structural formula is further shown in Formula IBa:

[0041]

[0042] Among them, R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0043] In some specific embodiments of the present invention, R1 When selected from H, R 2 Selected from OH, R 3 With R 4 Together they form a double bond, R 4 is selected from H, and the general structure is further shown in Formula IAa:

[0044]

[0045] Among them, R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0046] In some specific embodiments of the present invention, R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na, R 3 With R 4 Together they form a double bond, R 4 is selected from H, and the general structure is further shown in Formula IBb:

[0047]

[0048] Among them, R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0049] In some specific embodiments of the present invention, the structure of the steroid compound represented by formula (I) is one of the following structures:

[0050]

[0051]

[0052] In some specific embodiments of the present invention, the neuron function modulator can achieve regulation or protection of neuron function through at least one of the following mechanisms:

[0053] ① Promote the normalization of neuronal function by regulating the spontaneous calcium oscillation of neurons;

[0054] ② It protects neurons by relieving abnormal neuronal stimulation caused by the epilepsy-inducing agent 4-aminopyridine (4-AP).

[0055] In some specific embodiments of the present invention, the neuron function modulator can be applied to diseases related to neuron function regulation, including epilepsy, depression, Alzheimer's disease, Huntington's disease, Tourette's disease, demyelinating disease, schizophrenia, addiction, attention deficit hyperactivity disorder, Parkinson's disease and neuropathic pain.

[0056] In some specific embodiments of the present invention, the neuronal function modulator is a spontaneous calcium oscillation inhibitor and an inhibitor of 4-aminopyridine-induced neuronal calcium oscillation.

[0057] The present invention also provides a pharmaceutical composition for preventing or treating diseases related to neuronal function regulation, characterized in that:

[0058] It comprises a steroidal compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer or polymorph thereof,

[0059]

[0060] Among them, R 1 Selected from H or -OSO 3 Na;

[0061] R 2 Selected from OH or -OSO 3 Na;

[0062] R 3 Select from H or R 3 With R 4 together is a double bond;

[0063] R 4 Selected from H or -OSO 3 Na;

[0064] R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group;

[0065] It also contains pharmaceutically acceptable carriers or excipients.

[0066] In some specific embodiments of the present invention, R 5 The biological activity is higher when a branched alkyl group is used. The substituent on the branched alkyl group may be methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl and tert-butyl, and preferably methyl or tert-butyl.

[0067] In some specific embodiments of the present invention, the C 3 ~C18 The branched alkyl group is selected from: C 3 ~C 12 Alkyl, or C 3 ~C 11 alkyl;

[0068] The C 3 ~C 18 The branched alkenyl group is selected from C 3 ~C 12 Alkenyl, C having at least one tert-butyl substitution 3 ~C 11 Alkenyl.

[0069] In some specific embodiments of the present invention, R 5 The present invention has found that when R 5 When the carbon chain is extended to 6 carbon atoms and the number of substituents is further increased, its biological activity is significantly improved.

[0070] In some specific embodiments of the present invention, the C6-C18 branched alkyl group is selected from: C6-C12 straight-chain alkyl groups having at least 3 methyl substitutions; i.e., straight-chain alkyl groups of 9 to 12 carbon atoms having at least 3 methyl substitutions, and the total number of carbon atoms does not exceed 18, and the positions of the multiple methyl groups can be arranged at the positions of R 5 The 2nd to 4th carbon atom and the last carbon atom from the end of the substituent;

[0071] Or, the C6-C18 branched alkyl group is selected from: a straight-chain alkyl group of 4-11 carbon atoms having at least one tert-butyl group and at least one carbon atom substituted, and the total number of carbon atoms does not exceed 18, and the position of the tert-butyl group can be set to R 5 The position of the methyl group on the 2nd to 4th carbon atom from the end of the substituent group includes the position of the methyl group on the carbon atom with R 5 The last carbon atom starting from the end of the substituent.

[0072] In some specific embodiments of the present invention, R 5 The branched alkenyl group is selected from C9 to C16, and C9 to C16. Preferably, the C9 to C16 branched alkenyl group is selected from: C6 to C11 straight-chain alkenyl groups substituted with 3 to 5 methyl groups, i.e., straight-chain alkenyl groups of 9 to 11 carbon atoms substituted with 3 to 5 methyl groups, and the total number of carbon atoms does not exceed 16, and the positions of the multiple methyl groups are preferably arranged at the position of R 5 The 2nd to 4th carbon atom and the last carbon atom from the end of the substituent;

[0073] Or, the C9-C16 branched alkenyl group is selected from: C6-C9 straight-chain alkenyl groups having one tert-butyl group and 1-2 methyl groups as substitutions, and the total number of carbon atoms does not exceed 16, and the position of the tert-butyl group can be set to R 5 The position of the methyl group on the 2nd to 4th carbon atom from the end of the substituent group includes the position of the methyl group on the carbon atom with R 5 The last carbon atom starting from the end of the substituent.

[0074] In some specific embodiments of the present invention, when R 1 When selected from H, R 2 Selected from OH;

[0075] Or, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na;

[0076] Or, when R 3 When selected from H, R 4 Selected from -OSO 3 Na;

[0077] Or, when R 3 With R 4 When they form a double bond, R 4 Selected from H.

[0078] In some specific embodiments of the present invention, R 5 Select from the following substituents:

[0079]

[0080] In some specific embodiments of the present invention, when R 1 When selected from H, R 2 is selected from OH; the general structural formula at this time is shown in Formula IA:

[0081]

[0082] Among them, R 3 Select from H or R 3 With R 4 together is a double bond;

[0083] R 4 Selected from H or -OSO 3 Na;

[0084] R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0085] In some specific embodiments of the present invention, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na; the general structure at this time is shown in Formula IB:

[0086]

[0087] Among them, R 3 Select from H or R 3 With R 4 together is a double bond;

[0088] R 4 Selected from H or -OSO 3 Na;

[0089] R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0090] In some specific embodiments of the present invention, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na, R 3 When selected from H, R 4 Selected from -OSO 3 Na; the general structural formula is further shown in Formula IBa:

[0091]

[0092] Among them, R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0093] In some specific embodiments of the present invention, R 1 When selected from H, R 2 Selected from OH, R 3 With R 4 Together they form a double bond, R 4 is selected from H, and the general structure is further shown in Formula IAa:

[0094]

[0095] Among them, R 5 Selected from C3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0096] In some specific embodiments of the present invention, R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na, R 3 With R 4 Together they form a double bond, R 4 is selected from H, and the general structure is further shown in Formula IBb:

[0097]

[0098] Among them, R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

[0099] In some specific embodiments of the present invention, the structure of the steroid compound represented by formula (I) is one of the following structures:

[0100]

[0101] In some specific embodiments of the present invention, the pharmaceutical composition can achieve regulation or protection of neuronal function through at least one of the following mechanisms:

[0102] ① Promote the normalization of neuronal function by regulating the spontaneous calcium oscillation of neurons;

[0103] ② It protects neurons by relieving abnormal neuronal stimulation caused by the epilepsy-inducing agent 4-aminopyridine (4-AP).

[0104] In some specific embodiments of the present invention, the diseases associated with neuronal function regulation include epilepsy, depression, Alzheimer's disease, Huntington's disease, Tourette's disease, demyelinating diseases, schizophrenia, addiction, attention deficit hyperactivity disorder, Parkinson's disease and neuropathic pain.

[0105] Compared with the prior art, the present invention proposes steroid compounds that can be used to regulate or protect neuronal functions and can be used to treat diseases related to neuronal function regulation. Such steroid compounds are an important class of natural organic compounds that are widely present in biological tissues, especially in marine organisms, where they are widely distributed and have high content, which is conducive to the development and utilization of marine medicinal resources. DETAILED DESCRIPTION

[0106] 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 and should not be used to limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0107] Example 1

[0108] Preparation of steroidal compounds 1 to 6 of the present invention

[0109] The sponge Topsentiasp. was mechanically crushed, ultrasonically extracted several times with dichloromethane and methanol (volume ratio 1:1), the extracts were combined, and concentrated under reduced pressure to obtain 148.0 g of crude extract. The extract was suspended in a 90% methanol aqueous solution, extracted 4 times with an equal volume of petroleum ether, and concentrated under reduced pressure to obtain 6.7 g of petroleum ether layer extract; then the methanol solution was adjusted to a volume ratio of 60%, extracted 4 times with an equal volume of dichloromethane, and concentrated under reduced pressure to obtain 7.0 g of dichloromethane layer extract. The dichloromethane layer extract (about 6.0 g) was separated by normal phase silica gel (PE / EtOAc 40:1~1:1, CH 2 Cl 2 / MeOH 50:1~0:1) to obtain Fr.1~Fr.11. Fr.4 group was separated by repeated positive and reverse phase silica gel separation, Sephadex LH-20 gel separation (CH 2 Cl 2 / MeOH2:1) and HPLC purification (MeOH / H 2 O19:1) to obtain compound 1; the n-butanol layer (about 20.0 g) was subjected to MCI separation (MeOH / H 2 O1:9~9:1) to obtain 5 components Fr.1~Fr.5, and Fr.4 was separated by Sephadex LH-20 gel (CH 2 Cl 2 / MeOH 3:7), purified by HPLC (MeOH / H 2 O7:3) to obtain compounds 2 to 6.

[0110] Compound 1:

[0111]

[0112] White powder, [α] 20D =-48.9(c0.11, CH 2 Cl 2 ); ESI-MS m / z 427.4 [M+H] +, molecular formula is C 30 H 50 O. Its 1 H-NMR and 13 C-NMR data can be found in the literature (Calderón GJ, Castellanos L, Duque C, et al. Ophirasterol, a new C31sterol from the marine sponge Topsentiaophiraphidites [J]. Steroids, 2004, 69: 93–100.).

[0113] Compound 2:

[0114]

[0115] Colorless oil; [α] 20D = +20.1(c0.20,CH 3 OH); ESI-MS m / z 759.3 [M-Na] - , molecular formula C 31 H 53 O 12 S 3 Na 3 .That 1 H-NMR and 13 C-NMR data can be found in literature reports (GunasekeraSP, SennettSH, Kelly-BorgesM, et al. Ophirapstanoltrisulfate, anewbiologicallyactivesteroidsulfatefromthedeepwatermarinespongeTopsentiaphira phidites[J]. Journal of Natural Products, 1994, 57(12):1751-1754.).

[0116] Compound 3:

[0117]

[0118] Colorless oil; [α] 20D = +13.9 (c0.15, CH 3 OH); ESI-MS m / z 633.2 [M-Na] - , molecular formula C 22 H 35 O 12 S 3 Na 3 .That 1H-NMR and 13 C-NMR data are reported in the literature (Makarieva TN, Stonik VA, Dmitrenok AS, et al. New polarsteroids from the sponges Trachyopsis halichondroides and Cymbastelacorallio phila [J]. Steriods, 1995, 60: 316-320.).

[0119] Compound 4:

[0120]

[0121] Colorless oil; [α] 20D = +32.3 (c0.40, CH 3 OH); ESI-MS m / z 757.1 [M-Na] - , molecular formula C 31 H 51 O 12 S 3 Na 3 .That 1 H-NMR and 13 C-NMR data can be found in literature reports (Makarieva TN, Shubina LK, Kalinovsky AI, et al. Steroids in Porifera Ⅱ. Steroid derivatives from two sponges of the family Halichondriiade. Sokotrasterolsulfate, amarine steroid with a new pattern of side chain alkylation [J]. Steroids, 1983, 42 (3): 267-281.).

[0122] Compound 5:

[0123]

[0124] Colorless oil; [α] 20D = +29.2 (c0.10, CH 3 OH); ESI-MS m / z 717.3 [M-Na] - , molecular formula C 28 H 47 O 12 S 3 Na 3 .That 1 H-NMR and13 C-NMR data can be found in the literature (Bifulco G, Bruno I, Minale L, et al. Novel HIV-inhibitory Halistanol sulfates F-H from amarine sponge, Pseudoaxin is a digitata [J]. Journal of Natural Products, 1994, 57 (1): 164-167.).

[0125] Compound 6:

[0126]

[0127] Colorless oil; [α] 20D = +13.9 (c0.15, CH 3 OH); ESI-MS m / z 633.2 [M-Na] - , molecular formula C 22 H 35 O 12 S 3 Na 3 .That 1 H-NMR and 13 C-NMR data can be found in the literature (Sperry S, Crews P. Haliclostanone sulfate and halistanol sulfate from an Indo-Pacific Haliclonasponge [J]. Journal of Natural Products, 1997, 60: 29-32.).

[0128] Preparation of Comparative Example Compounds

[0129] The sponge Iotrochotasp. sample was mechanically crushed, ultrasonically extracted several times with dichloromethane and methanol (volume ratio 1:1), the extracts were combined, and concentrated under reduced pressure to obtain 102.0g of crude extract. The extract was suspended in a 90% methanol aqueous solution, extracted 4 times with an equal volume of petroleum ether, and concentrated under reduced pressure to obtain 48.4g of petroleum ether layer extract; then the methanol solution was adjusted to a volume ratio of 60%, extracted 3 times with an equal volume of dichloromethane, and concentrated under reduced pressure to obtain 4.6g of dichloromethane layer extract. The dichloromethane layer extract (about 4.0g) was separated by normal phase silica gel (PE / EtOAc20:1~1:1, CH 2 Cl 2 / MeOH 50:1~0:1) to obtain Fr.1~Fr.11. Fr.4 was separated by repeated positive and reverse phase silica gel separation and Sephadex LH-20 gel separation (CH 2 Cl 2 / MeOH 2:1), HPLC purification (MeOH / H 2 O24:1) to obtain compounds D1~D3.

[0130] The sponge Topsentiasp. was mechanically crushed, ultrasonically extracted several times with dichloromethane and methanol (volume ratio 1:1), the extracts were combined, and concentrated under reduced pressure to obtain 148.0 g of crude extract. The extract was suspended in a 90% methanol aqueous solution, extracted 4 times with an equal volume of petroleum ether, and concentrated under reduced pressure to obtain 6.7 g of petroleum ether layer extract; then the methanol solution was adjusted to a volume ratio of 60%, extracted 4 times with an equal volume of dichloromethane, and concentrated under reduced pressure to obtain 7.0 g of dichloromethane layer extract. The dichloromethane layer extract (about 6.0 g) was separated by normal phase silica gel (PE / EtOAc 40:1~1:1, CH 2 Cl 2 / MeOH 50:1~0:1) to obtain Fr.1~Fr.11. Fr.4 component was separated by repeated positive and reverse phase silica gel separation and Sephadex LH-20 gel separation (CH 2 Cl 2 / MeOH2:1) and HPLC purification (MeOH / H 2 O19:1) to obtain compounds D4 and D5;

[0131] Comparative Example Compound Structure Identification:

[0132] Compound D1:

[0133]

[0134] White powder; [α] 20D =-33.4(c0.20,CH 2 Cl 2 ); ESI-MS m / z 413.3 [M+H] + , molecular formula C 29 H 48 O. Its 1 H-NMR and 13 C-NMR data are reported in the literature (Jung HA, Jung HJ, Jeong HY, et al.

[0135] Anti-adipogenicactivityoftheediblebrownalgaEckloniastoloniferaanditsconstituentfuc osterolin3T3-L1adipocytes[J].ArchivesofPharmacalResearch,2014,37:713-720.)

[0136] Compound D2:

[0137]

[0138] White powder; [α] 20D =-30.5(c0.14, CH 2 Cl 2 ); ESI-MS m / z 413.4 [M+H] + , molecular formula is C 29 H 48 O. Its 1 H-NMR and 13 C-NMR data can be found in the literature (Zhang Xiaoqi, Qi Jin, Ye Wencai, et al. Study on chemical components of Xanthium sibiricum stems [J]. Journal of China Pharmaceutical University, 2004, 35(5): 404-405.).

[0139] Compound D3:

[0140]

[0141] White powder; [α] 20D =-29.0(c0.20,CH 2 Cl 2 ); ESI-MS m / z 415.2 [M+H] + , molecular formula is C 29 H 50 O. Its 1 H-NMR and 13 C-NMR data can be found in the literature (Bousetla A, Zellagui A, Derouiche K. et al. Chemical constructs of the roots of Algerian Bunium in crassatuman devaluation of its anti microbial activity [J]. Arabian Journal of Chemistry, 2015, 8: 313-316.).

[0142] Compound D4:

[0143]

[0144] White powder, [α] 20D =-30.5(c0.11, CH 2 Cl 2 ); ESI-MS m / z 427.4 [M+H] + , molecular formula C 30 H 52 O. Its1 H-NMR and 13 C-NMR data can be found in the literature (Calderón GJ, Castellanos L, Duque C, et al. Ophirasterol, a new C31sterol from the marine sponge Topsentiaophiraphidites [J]. Steroids, 2004, 69: 93–100.).

[0145] Compound D5:

[0146]

[0147] White powder, [α] 20D =-29.9(c0.17, CH 2 Cl 2 ); ESI-MS m / z 441.2 [M+H] + , molecular formula is C 31 H 52 O. Its 1 H-NMR and 13 C-NMR data can be found in the literature (Calderón GJ, Castellanos L, Duque C, et al. Ophirasterol, a new C31sterol from the marine sponge Topsentiaophiraphidites [J]. Steroids, 2004, 69: 93–100.).

[0148] Example 2

[0149] The inhibitory activities of compounds 1-6 and D1-D5 prepared in Example 1 on spontaneous calcium oscillations and 4-AP-induced calcium oscillations in mouse neurons were tested in vitro.

[0150] 1. Experimental Methods

[0151] Primary neuron culture of cerebral cortex: C57 suckling mice born 0-1 days old were taken, decapitated, and the brain was removed. The cerebral cortex was separated under a dissecting microscope, the meninges were carefully peeled off, and the cortex was prepared into a single cell suspension. The cell suspension was centrifuged and inoculated on a 96-well plate and cultured at 37°C and 5% CO. 2 In a cell culture incubator with a humidity of 95%, the medium was changed every 4 and 7 days, and the plates were tested every 8 days.

[0152] Intracellular Ca 2+ Concentration determination: Aspirate the medium in the 96-well plate, add 60 μL dye loading buffer to each well, and incubate at 37°C and 5% CO 2Incubate for 1 hour in an environment with 95% humidity, then wash 5 times with Lockes' buffer and place the cells in FLIPR for plate reading. After 300 seconds of plate reading, add 25 μL of compound solution with a concentration of 8 times the final concentration, continue plate reading for 900 seconds, add 25 μL of 4-AP, continue plate reading, and repeat two wells for each compound. Observe intracellular Ca 2+ Level changes.

[0153] 2. Experimental Results

[0154] The in vitro activity of the steroid compound prepared in Example 1 was tested. The steroid compound prepared in Example 1 showed different degrees of inhibitory activity. 50 The values ​​are shown in Table 1.

[0155] Table 1. EC values ​​of compounds on the amplitude and frequency of spontaneous calcium oscillations and 4-AP-induced calcium oscillations 50 value

[0156]

[0157] Note: “N” stands for EC 50 Value ≥ 30 μM; “N / T” means activity not measured.

[0158] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a steroidal compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer or polymorph thereof in the preparation of a neuron function modulator, in, R 1 Selected from H or -OSO 3 Na; R 2 Selected from OH or -OSO 3 Na; R 3 Select from H or R 3 With R 4 together is a double bond; R 4 Selected from H or -OSO 3 Na; R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group.

2. The use according to claim 1, Features: The C 3 ~C 18 The branched alkyl group is selected from: C 3 ~C 12 Alkyl, or C 3 ~C 11 alkyl; The C 3 ~C 18 The branched alkenyl group is selected from C 3 ~C 12 Alkenyl, C having at least one tert-butyl substitution 3 ~C 11 Alkenyl.

3. The use according to claim 2, Features: R 5 Select from the following substituents:

4. The use according to claim 1, Features: When R 1 When selected from H, R 2 Selected from OH; Or, when R 1 Selected from -OSO 3 Na, R 2 Selected from -OSO 3 Na; Or, when R 3 When selected from H, R 4 Selected from -OSO 3 Na; Or, when R 3 With R 4 When they form a double bond, R 4 Selected from H.

5. The use according to claim 1, It is characterized in that The structure of the steroid compound represented by formula (I) is one of the following structures:

6. The use according to any one of claims 1 to 5, Features: The neuron function modulator can achieve regulation or protection of neuron function through at least one of the following mechanisms: ① Promote the normalization of neuronal function by regulating the spontaneous calcium oscillation of neurons; ② It protects neurons by relieving abnormal neuronal stimulation caused by the epilepsy-inducing agent 4-aminopyridine.

7. The use according to claim 6, Features: The neuron function regulator can be applied to diseases related to neuron function regulation, including epilepsy, depression, Alzheimer's disease, Huntington's disease, Tourette's disease, demyelinating disease, schizophrenia, addiction, attention deficit hyperactivity disorder, Parkinson's disease and neuropathic pain.

8. A pharmaceutical composition for preventing or treating diseases related to neuronal function regulation, Features: It comprises a steroidal compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer or polymorph thereof, Among them, R 1 Selected from H or -OSO 3 Na; R 2 Selected from OH or -OSO 3 Na; R 3 Select from H or R 3 With R 4 together is a double bond; R 4 Selected from H or -OSO 3 Na; R 5 Selected from C 3 ~C 18 Straight or branched alkyl, C 3 ~C 18 A straight chain or branched alkenyl group; It also contains pharmaceutically acceptable carriers or excipients.

9. The pharmaceutical composition according to claim 8, Features: The structure of the steroid compound represented by formula (I) is one of the following structures:

10. The pharmaceutical composition according to any one of claims 8 to 9, Features: The diseases related to the neuronal function regulation include epilepsy, depression, Alzheimer's disease, Huntington's disease, Tourette's disease, demyelinating diseases, schizophrenia, addiction, attention deficit hyperactivity disorder, Parkinson's disease and neuropathic pain.

Images