Ruthenium complex, preparation method and application in preparation of medicine for preventing or treating neurodegenerative diseases

Abstract

The invention provides a ruthenium complex, and the structure of the ruthenium complex is as shown in a formula (I): # imgabs0 # (I), wherein R1, R2, R3 and R4 are independently selected from H, hydroxyl, methyl, C2-C6 linear chain or branched chain alkyl, C3-C6 cycloalkyl and C2-C6 linear chain or branched chain unsaturated alkyl, and at least one of R1, R2, R3 and R4 is hydroxyl. The invention also provides a preparation method of the ruthenium complex, application of the ruthenium complex in preparation of drugs for preventing or treating neurodegenerative diseases and a pharmaceutical composition for preventing or treating neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a ruthenium complex, a preparation method and an application thereof in preparing a medicine for preventing or treating neurodegenerative diseases. Background Art

[0002] Neurodegenerative diseases are a class of diseases characterized by neuronal dysfunction and loss, including Alzheimer's disease (AD), Parkinson's disease (PD), etc. One of the key features of these diseases is the misfolding and pathological aggregation of proteins. For example, in Alzheimer's disease, amyloid precursor protein (APP) is converted into amyloid β (Aβ) through the action of a series of enzymes, especially the Aβ42 subtype, which is closely related to the formation of Aβ plaques. The deposition of these plaques in the brain is one of the pathological characteristics of AD; for example, in Parkinson's disease, α-synuclein is a neuronal protein mainly localized in the presynaptic terminal, which is related to regulating the stability of the neural membrane, affecting presynaptic membrane signal transduction and cell membrane transport. After misfolding, α-synuclein forms Lewy bodies and Lewy neurites, which is one of the main pathological markers of PD.

[0003] Current clinical drugs are used to treat Parkinson's disease in a variety of ways, increasing dopaminergic stimulation by participating in the biochemical pathways of dopamine synthesis and metabolism. However, there are still few drugs that can effectively disaggregate aggregated α-synuclein fibers. Therefore, there is a need for a compound that can disaggregate aggregated α-synuclein fibers or inhibit α-synuclein. Summary of the invention

[0004] In view of this, in order to at least partially solve at least one of the above-mentioned technical problems, the present invention provides a ruthenium complex, a preparation method and an application in preparing a drug for preventing or treating neurodegenerative diseases.

[0005] According to an embodiment of one aspect of the present invention, a ruthenium complex is provided, wherein:

[0006] The structure of the ruthenium complex is shown in formula (I):

[0007] (I);

[0008] Wherein, R1, R2, R3, and R4 are independently selected from H, hydroxyl, methyl, C2-C6 straight or branched alkyl, C3-C6 cycloalkyl, and C2-C6 straight or branched unsaturated hydrocarbon group, and at least one of R1, R2, R3, and R4 is hydroxyl; X is a halogen atom; M + It is a cation.

[0009] According to another embodiment of the present invention, a method for preparing a ruthenium complex is provided, the method comprising:

[0010] A solution of ruthenium trichloride in an organic solvent, dihydroxyphenylalanine and a substance selected from sodium chloride, potassium chloride and water are reacted in the presence of an acid with heating and stirring.

[0011] According to an embodiment of yet another aspect of the present invention, there is provided a use of a ruthenium complex in the preparation of a medicament for preventing or treating a neurodegenerative disease.

[0012] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a ruthenium complex and a pharmaceutically acceptable carrier.

[0013] According to the embodiments of the present invention, the ruthenium atoms in the ruthenium complex can coordinate and bind with some amino acids in synuclein, such as methionine and histidine, and the phenolic hydroxyl group has the function of chelating metal ions, can chelate metal ions such as iron ions, and can remove reactive oxygen, and the benzene ring has a good affinity for synuclein, so it has a strong binding effect with α-synuclein, thereby being able to inhibit the aggregation or disaggregation of α-synuclein protein fibers, and can protect nerve cells from the toxicity of α-synuclein oligomers, thereby being able to alleviate the symptoms of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above and other objects, features and advantages of the present invention will become more apparent through the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:

[0015] Figure 1 This is a graph showing the results of the ruthenium complexes Ru1, Ru2, Ru3 and NAMI-A inhibiting α-synuclein aggregation in an embodiment of the present invention;

[0016] Figure 2 This is a graph showing the inhibitory effect of the ruthenium complex Ru2 on α-synuclein aggregation over time in an embodiment of the present invention;

[0017] Figure 3 This is a test result diagram of the depolymerization effect of the ruthenium complex Ru2 on α-synuclein fibers in an embodiment of the present invention;

[0018] Figure 4 The figure is a biological toxicity test result diagram of ruthenium complexes Ru1, Ru2 and Ru3 according to the embodiment of the present invention;

[0019] Figure 5 This is a graph showing the test results of the ruthenium complex Ru2 on oxidative stress of SH-SY5Y cells in an embodiment of the present invention;

[0020] Figure 6This is a test result diagram of the ruthenium complex Ru2 in an embodiment of the present invention destroying the interaction between α-synuclein and cell membrane;

[0021] Figure 7 This is a graph showing the results of the rotarod test of Parkinson's model mice using the ruthenium complex Ru2 of an embodiment of the present invention;

[0022] Figure 8 This is a diagram showing the results of mass spectrometry detection of ruthenium complex Ru2 binding to α-synuclein in an embodiment of the present invention. DETAILED DESCRIPTION

[0023] Below, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of embodiments of the present invention. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion of concepts of the present invention.

[0024] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The term "comprising" used herein indicates the existence of features, steps, operations, but does not exclude the existence or addition of one or more other features.

[0025] In the case of using expressions such as "at least one of A, B, and C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, or C" should include but is not limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.).

[0026] In the present invention, the term "pharmaceutically acceptable" refers to compounds, substances, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0027] In this application, "pharmaceutical composition" refers to a preparation of the compound of the present invention and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of the organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0028] In this application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or livestock use.

[0029] In the present invention, “independently” means that when there are multiple subjects, they may be the same as or different from each other.

[0030] In the present invention, the expression of chemical elements, unless otherwise specified, generally includes the concept of isotopes with the same chemical properties. For example, the expression "hydrogen (H)" also includes the concepts of 1H (protium or H) and 2H (deuterium or D) with the same chemical properties; carbon (C) includes 12C, 13C, etc., which will not be repeated.

[0031] In the present invention, the expression of Ca to Cb represents that the number of carbon atoms in the group is a to b. Unless otherwise specified, the number of carbon atoms in the group generally does not include the number of carbon atoms in the substituent.

[0032] In the present invention, the term "alkyl" may include a branched or straight chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. For example, examples of C1 to C6 alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, etc.

[0033] In the present invention, the term "quaternary ammonium cation" can be represented by the formula NL4 +wherein each L is independently selected from alkyl, aryl, alkaryl, and wherein each of the alkyl, aryl and alkaryl groups is optionally substituted with one or more substituents each independently selected from the group consisting of -NO2, -CN, -OH, oxo, imino, -CONH2, -CONL'2, -CNNL'2, -CSNL'2, -CONH-OH, -CONH-NH2, -NHCOL', -NHCSL', -NHCNL', -NC(=O)OL', -NC(=O)NL', -NC(=S)OL', -NC(=S)NL', -SO2L', -SOL', -SL', -SO2OL', -SO2N(L')2, -NHNL'2, -NNL', C1-C6 haloalkyl, optionally substituted C1-C6 C1-C6 alkyl, -NH2, -NL'2-NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NL'2, C1-C6 alkyl-SL', -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, -CO2L', -OCOL, -OCOL', -OC(=O)OL', -OC(=O)NL', -OC(=S)OL', -OC(=S)NL' or a combination thereof; wherein each L' independently represents hydrogen, or is selected from a group comprising optionally substituted C1-C 10 Alkyl, optionally substituted C3-C 10 Cycloalkyl, optionally substituted C3-C 10 Heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, alkyl, amino, -NH2, -NL'2-NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, alkyl(C1-C6 alkyl), alkyl(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NL'2, C1-C6 alkyl-SL' or a combination thereof.

[0034] In the present invention, the term "straight or branched hydrocarbon group" may include a straight or branched saturated aliphatic monovalent hydrocarbon group or an unsaturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The unsaturated hydrocarbon group as a C2-C6 straight or branched chain may be a C2-C6 straight or branched chain saturated hydrocarbon group in which one or more single bonds are replaced with double bonds.

[0035] The term "treatment" means that after a subject has a disease, the subject is exposed to (e.g., administered) a drug, composition, etc. according to the present invention, thereby alleviating the symptoms of the disease compared to when the subject has not been exposed to the drug, composition, etc., and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

[0036] The term "prevention" means that before a subject develops a disease, the symptoms after developing the disease are alleviated compared to when the subject does not develop the disease by contacting (eg administering) the drug, composition, etc. according to the present invention, but does not necessarily mean that the disease must be completely suppressed.

[0037] In the process of realizing the concept of the present invention, it was found that drugs for treating Parkinson's disease are classified into: (a) dopamine substitutes and levodopa decarboxylase inhibitors, which are used to block peripheral levodopa decarboxylation because dopamine itself does not cross the blood-brain barrier, such as levodopa; (b) dopamine agonists, which are used to activate dopamine receptors, such as bromocriptine mesylate and ropinirole hydrochloride; (c) catechol-O-methyltransferase inhibitors, which are used to block the metabolism of dopamine substitutes, such as entacapone; (d) monoamine oxidase B inhibitors, which are used to block the metabolism of dopamine, such as selegiline hydrochloride, rasagiline and safinamide, etc.

[0038] A key pathological hallmark of Parkinson's disease is the presence of intracellular protein inclusions called Lewy bodies and Lewy neurites, the main components of which are misfolded and aggregated proteins, α-synuclein. The abnormal accumulation and spread of toxic α-synuclein aggregates are the main molecular events in the pathogenesis of Parkinson's disease. α-synuclein aggregates can lead to various cellular dysfunctions, including impaired mitochondrial function, endoplasmic reticulum stress, disruption of the autophagy-lysosomal pathway, and synaptic and nuclear dysregulation. Therefore, targeting α-synuclein and inhibiting its aggregation is a potential therapeutic strategy. In related technologies, such as Anle138b, by inhibiting α-synuclein aggregation, strongly blocked oligomer accumulation, neuronal degeneration and disease progression in animal models of Parkinson's disease; and EGCG, a polyphenolic flavonoid extracted from green tea leaves, inhibited the formation of toxic α-synuclein oligomers in vitro and was able to convert α-synuclein oligomers into non-toxic species. Even though there are many therapeutic studies targeting α-synuclein, there are still few drugs that can effectively disaggregate aggregated α-synuclein fibers.

[0039] Different from the above-mentioned organic small molecule drugs, metal complexes also show potential in the treatment of neurodegenerative diseases. Ruthenium complexes are widely explored for their various biological applications because they are less toxic than platinum complexes. In the related art, the ruthenium complex NAMI-A can effectively inhibit the aggregation of α-synuclein and disaggregate the aggregated α-synuclein fibers, and has the potential to be developed as a drug to alleviate the symptoms of Parkinson's disease and other neurodegenerative diseases.

[0040] The structure of NAMI-A is shown in the following formula (III):

[0041] (III).

[0042] Specifically, according to an embodiment of one aspect of the present invention, a ruthenium complex is provided, the structure of which is shown in the following formula (I):

[0043] (I);

[0044] Wherein, R1, R2, R3, and R4 are independently selected from H, hydroxyl, C1-C6 straight or branched alkyl, C3-C6 cycloalkyl, and C2-C6 straight or branched hydrocarbon, and at least one of R1, R2, R3, and R4 is a hydroxyl group; preferably, at least two of R1, R2, R3, and R4 are hydroxyl groups;

[0045] Further preferably, any two of R1, R2, R3, and R4 are hydroxyl groups and the remaining two are H;

[0046] Further preferably, R1 and R2 are hydroxyl groups and R3 and R4 are H, or R2 and R3 are hydroxyl groups and R1 and R4 are H, or R3 and R4 are hydroxyl groups and R1 and R2 are H;

[0047] More preferably, R1 and R2 are hydroxyl groups, R3 and R4 are H and X + for Na + , or R2 and R3 are hydroxyl groups, R1 and R4 are H and X + H + , or R3 and R4 are hydroxyl groups, R1 and R2 are H and X + K + .

[0048] X is a halogen atom, preferably F, Cl, Br or I, more preferably Cl.

[0049] When any two of R1, R2, R3, and R4 are hydroxyl groups, the two hydroxyl groups are preferably adjacent to each other.

[0050] M + is a cation, preferably a monovalent cation, more preferably H + 、Na + , K + , ammonium ion, quaternary ammonium cation or imidazolium, further preferably H + 、Na + or K + .

[0051] According to the embodiments of the present invention, the ruthenium atoms in the ruthenium complex can coordinate and bind with some amino acids in synuclein, such as methionine and histidine, and the phenolic hydroxyl group has the function of chelating metal ions, can chelate metal ions such as iron ions, and can remove reactive oxygen, and the benzene ring has a good affinity for synuclein, so it has a strong binding effect with α-synuclein, thereby being able to inhibit the aggregation or disaggregation of α-synuclein protein fibers, and can protect nerve cells from the toxicity of α-synuclein oligomers, thereby being able to alleviate the symptoms of Parkinson's disease.

[0052] As an embodiment of another aspect of the present invention, a method for preparing a ruthenium complex is provided, the method comprising:

[0053] A solution of RuX3 in an organic solvent, a compound of the following formula (II) and MX are reacted in the presence of an acidic aqueous solution under heating and stirring to obtain a compound of formula (I);

[0054] (II).

[0055] In some specific embodiments of the present invention, the method for preparing the ruthenium complex comprises:

[0056] A solution of ruthenium trichloride in an organic solvent, dihydroxyphenylalanine and a substance selected from sodium chloride, potassium chloride and water are reacted in the presence of an acid with heating and stirring.

[0057] In some specific embodiments of the present invention, the solution of ruthenium trichloride in an organic solvent is obtained by dissolving ruthenium trichloride in an organic solvent and then heating the solution under reflux.

[0058] In some specific embodiments of the present invention, the process further comprises centrifuging the mixture obtained after the reaction and rotary evaporating the supernatant to obtain a ruthenium complex having a structure as shown in formula (1).

[0059] In some specific embodiments of the present invention, the heating reflux time is 3 to 5 hours, which can be 3 hours, 3.5 hours, 4 hours, or 5 hours, but is not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable; the heating stirring time is 3 to 6 hours, which can be 3 hours, 3.5 hours, 4 hours, 5 hours, or 6 hours, but is not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable; the temperature includes 60 to 100°C, which can be 60°C, 70°C, 80°C, 90°C, or 100°C, but is not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable.

[0060] In some specific embodiments of the present invention, the molar ratio of ruthenium trichloride to dihydroxyphenylalanine is 1:(2-5), preferably 1:(2-3), and can be 1:2, 1:3, 1:4, 1:5, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0061] According to an embodiment of the present invention, a ruthenium complex can be simply and quickly prepared by using ruthenium trichloride, dihydroxyphenylalanine and a cationic solution, and the reaction raw materials are easily available and low in cost. The prepared product is precipitated in the form of crystals, and a ruthenium complex with high purity can be obtained quickly and conveniently.

[0062] As an embodiment of another aspect of the present invention, there is provided a use of a ruthenium complex in the preparation of a drug for preventing or treating a neurodegenerative disease.

[0063] According to an embodiment of the present invention, the neurodegenerative disease is caused by abnormal aggregation or misfolding of α-synuclein and amyloid deposition.

[0064] According to the embodiments of the present invention, the ruthenium complex can limit its aggregation and toxicity by forming a stable interaction with amyloid, or bind to α-synuclein that can form an amyloid-like fiber structure to limit its aggregation and toxicity, and even due to the strong binding ability of the ruthenium ligand to α-synuclein, it can disaggregate the already aggregated α-synuclein fibers, and can also prevent the membrane-induced protein conformational changes of α-synuclein, inhibit the interaction between α-synuclein oligomers and nerve cell membranes, thereby inhibiting the toxic effects of α-synuclein. In in vitro experiments, the ruthenium complex can effectively inhibit the aggregation of α-synuclein and disaggregate α-synuclein fibers, while being able to inhibit the toxicity of α-synuclein oligomers to cells, and can alleviate the symptoms of Parkinson's disease in animal models, and has good application prospects.

[0065] According to an embodiment of the present invention, the neurodegenerative disease is at least one of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, and amyotrophic lateral sclerosis.

[0066] According to an embodiment of the present invention, Parkinson's disease is caused by the deposition of misfolded α-synuclein in the central nervous system and peripheral nervous system; Alzheimer's disease is characterized by the extracellular accumulation of amyloid β (Aβ) as senile plaques and intracellular aggregation of tau to form neurofibrillary tangles (NFTs), and the co-aggregation of α-synuclein and amyloid-β can stabilize β-fold-rich oligomers and enhance the formation of β-barrels. There is a correlation between misfolded α-synuclein aggregates and Alzheimer's disease pathology in vivo; Huntington's disease is also associated with abnormal aggregation of α-synuclein; aggregation of α-synuclein is a typical pathology of Lewy body dementia, and exogenously produced α-synuclein fibrils can destroy the endogenous complement of α-synuclein into cytotoxic aggregates in a prion-like manner; amyotrophic lateral sclerosis is mainly related to the abnormal aggregation of TDP-43 protein, which can form amyloid aggregates in neurodegenerative diseases. Ruthenium complexes inhibit Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia or amyotrophic lateral sclerosis by inhibiting the abnormal aggregation or misfolding of α-synuclein and amyloid protein deposition.

[0067] According to an embodiment of the present invention, the dosage form of the drug is selected from at least one of the following: emulsion, oil, powder, aqueous solution, suspension, tablet, granule, capsule, nano preparation.

[0068] According to the embodiments of the present invention, the dosage forms of various drugs meet different treatment needs, improve patient compliance, optimize drug efficacy and safety, can meet different clinical needs and patient preferences, and adapt to different administration routes.

[0069] As an embodiment of another aspect of the present invention, a pharmaceutical composition for preventing or treating neurodegenerative diseases is provided, comprising a ruthenium complex and a pharmaceutically acceptable carrier.

[0070] According to an embodiment of the present invention, the neurodegenerative disease is caused by abnormal aggregation or misfolding of α-synuclein and amyloid deposition.

[0071] According to an embodiment of the present invention, the neurodegenerative disease includes at least one of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, and amyotrophic lateral sclerosis.

[0072] In some specific embodiments of the present invention, the dosage of ruthenium complex in the drug for preventing or treating degenerative diseases is 0~1 mol / L, which can be 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, 1 mol / L, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0073] According to an embodiment of the present invention, the drug further includes a pharmaceutically acceptable carrier.

[0074] According to an embodiment of the present invention, the drug carrier can improve the oral absorption of poorly soluble drugs. By encapsulating the drug in the carrier, the solubility and bioavailability of the drug can be increased. At the same time, the drug carrier can escape the rapid capture of the reticuloendothelial system in the body, prolong the exposure time of the drug in the systemic circulation, and enhance the efficacy of the drug.

[0075] According to an embodiment of the present invention, in the case where the drug is an oral preparation, the carrier includes at least one of a binder, a lubricant, a disintegrant, a solubilizer, a diluent, a stabilizer, a suspending agent, a pigment, and a flavoring agent;

[0076] In the case where the drug is an injectable preparation, the carrier includes at least one of a preservative, a solubilizer, and a stabilizer;

[0077] In the case where the drug is a topical preparation, the carrier includes at least one of a base, a diluent, a lubricant, and a preservative.

[0078] According to embodiments of the present invention, in oral preparations, adhesives, lubricants, disintegrants, cosolvents, diluents, stabilizers, suspending agents, no pigment, flavoring agents play a key role, they not only improve the physicochemical properties of the drug, improve the efficacy and safety of the drug, but also increase the acceptability of the preparation, meet different clinical needs and patient preferences. In injections, preservatives, solubilizers, stabilizers can ensure the safety, effectiveness and stability of the drug, while improving the medication compliance of patients. In topical preparations, matrix, diluents, lubricants, preservatives contribute to the stable release and effective delivery of the drug, and also improve the safety of the preparation. By rationally selecting and using these adjuvants, the dosage form of the drug can be optimized to improve the therapeutic effect.

[0079] According to an embodiment of the present invention, the administration method of the drug includes at least one of oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and local administration.

[0080] The technical solution of the present invention is further described below by specific examples in combination with the accompanying drawings. It should be noted that the following specific examples are only used as examples, and the protection scope of the present invention is not limited thereto. The drugs or reagents used in the following examples are all commercially available or homemade by known preparation methods. The methods used in the following examples, such as Image J, are all well-known methods in the art and can be carried out by description in textbooks or relevant literature, and will not be repeated here.

[0081] Example 1 Preparation of ruthenium complex

[0082] The synthesis path is as follows:

[0083]

[0084] Ruthenium complex Ru1:

[0085] Step S1: Weigh 3 mmol of ruthenium trichloride into a round-bottom flask, add 10 mL of ethanol, heat to reflux for 5 hours, and the solution turns dark green after the reaction. Filter the solution and concentrate it to 1 mL using a rotary evaporator.

[0086] Step S2: Add 50 μL of concentrated hydrochloric acid, 2 mL of 2 mM NaCl aqueous solution and 9 mmol of L-3,4-dihydroxyphenylalanine to the reaction solution obtained in step S1, heat to 60°C, and heat and stir to react for 6 hours. Then spin dry the solution using a rotary evaporator, wash with acetone 3 times, take out the solid and dry it, and finally obtain the ruthenium complex Ru1, and characterize the ruthenium complex Ru1 to show that the ruthenium complex Ru1 has been successfully prepared.

[0087] Spectral information of ruthenium complex Ru1 nuclear magnetic resonance hydrogen spectrum (1H NMR): 1H NMR (500 MHz, D2O) δ6.79 (d, J=6.7Hz, 1H), δ6.68 (d, J=6.3, 1H), δ6.60 (d,J=6.8, 1H), δ3.92 (dd, J=15.6 Hz, 11.4Hz, 1H), δ3.42 (dd, J=15.1 Hz,11.0 Hz, 2H).

[0088] Mass spectrometry characterization results of ruthenium complex Ru1: ESR-MS: calculated value, m / z=587.9616 (Ru1-H) + ,609.9435(Ru1-Na) + ; Exp. value, m / z=587.6834 (Ru1-H) + ,609.6294 (Ru1-Na) + .

[0089] Ruthenium complex Ru2:

[0090] Repeat steps S1 to S2, except that 50 μL of concentrated hydrochloric acid, 2 mL of water and 6 mmol of levodopa are added to the reaction solution obtained in step S1, heated to 80°C, stirred and reacted for 3 hours, and finally a ruthenium complex Ru2 is obtained. The ruthenium complex Ru2 is characterized to indicate that the ruthenium complex Ru2 is successfully prepared.

[0091] Spectral information of ruthenium complex Ru2 nuclear magnetic resonance hydrogen spectrum (1H NMR): 1H NMR (500 MHz, D2O) δ6.86 (d, J=6.8Hz, 1H), δ6.73 (s, 1H), δ6.68 (d, J=6.9, 1H), δ3.84 (dd, J=15.3 Hz,11.5Hz, 1H), δ3.30 (dd, J=14.7 Hz, 10.7 Hz, 2H).

[0092] Mass spectrometry characterization results of ruthenium complex Ru2: ESR-MS: calculated value, m / z=565.9796 (Ru2-H) + ; Exp. value, m / z=565.8375 (Ru2-H) + .

[0093] Ruthenium complex Ru3:

[0094] Repeat steps S1 to S2, except that 20 μL of concentrated hydrochloric acid, 2 mL of 2 mM KCl aqueous solution and 15 mmol of 3-hydroxy-D-tyrosine are added to the reaction solution obtained in step S1, heated to 100°C, stirred and reacted for 5 hours, and finally a ruthenium complex Ru3 is obtained, and the ruthenium complex Ru3 is characterized to indicate that the ruthenium complex Ru3 is successfully prepared.

[0095] Spectral information of ruthenium complex Ru3 nuclear magnetic resonance hydrogen spectrum (1H NMR): 1H NMR (500 MHz, D2O) δ6.86 (s, 1H), δ6.75 (d, J=6.6, 1H), δ6.68 (d,J=6.8, 1H), δ3.78 (dd, J=15.5 Hz,11.4Hz, 1H), δ3.29 (dd, J=14.9 Hz,10.6 Hz, 2H).

[0096] Mass spectrometry characterization results of ruthenium complex Ru3: ESR-MS: calculated value, m / z=603.9355 (Ru1-H) + ,641.8914(Ru1-K) + ; Exp. value, m / z=603.7528 (Ru1-H) + ,6641.6839 (Ru1-K) + .

[0097] Example 2 Test of the inhibitory effect of ruthenium complexes Ru1, Ru2 and Ru3 on α-synuclein aggregation

[0098] The α-synuclein monomer was dissolved in phosphate buffered saline (PBS) to prepare a solution with a concentration of 70 μM. At the same time, the ruthenium complexes Ru1, Ru2 and Ru3 prepared in Example 1 and the control group NAMI-A were added to the α-synuclein monomer solution to obtain α-synuclein solutions with final concentrations of 0 μM, 7 μM, 21 μM, 35 μM and 70 μM, respectively.

[0099] The culture was continuously mixed and cultured at 37°C at 900 rpm for 48 h, and the formation of fibrils was detected by the fluorescence of the dye thioflavin (ThT). 200 μL of culture medium was mixed with 1.5 μL of 20 mM ThT and incubated in a 37°C oven for 20 min. The fluorescence intensity of ThT was detected on a fluorescence spectrophotometer. The excitation wavelength of ThT was 450 nm and the emission wavelength was 490 nm. The results were as follows: Figure 1 shown.

[0100] Figure 1 This is a graph showing the results of the ruthenium complexes Ru1, Ru2, Ru3 and NAMI-A inhibiting α-synuclein aggregation in an embodiment of the present invention.

[0101] according to Figure 1 It can be seen that with the increase of ruthenium complex concentration, the ThT fluorescence intensity decreases, the aggregation degree of α-synuclein decreases, and the degree of reduction in α-synuclein aggregation with the addition of ruthenium complexes Ru1, Ru2 and Ru3 is greater than the degree of reduction in α-synuclein aggregation with the addition of NAMI-A, indicating that ruthenium complexes Ru1, Ru2 and Ru3 are effective α-synuclein aggregation inhibitors, which can inhibit the aggregation of α-synuclein in vitro, and the aggregation inhibition effect is better than NAMI-A.

[0102] Example 3 Test of the inhibitory effect of ruthenium complex Ru2 on α-synuclein aggregation

[0103] The α-synuclein monomer was dissolved in PBS to prepare a solution with a concentration of 70 μM, and the ruthenium complex Ru2 prepared in Example 1 and the control group NAMI-A were added respectively to obtain an α-synuclein solution with a final concentration of 21 μM of the ruthenium complex Ru2 and NAMI-A.

[0104] The mixture was mixed and cultured continuously at 37°C and 900 rpm for 0 h, 12 h, 24 h, and 48 h. The formation of protofibrils was detected by fluorescence of the dye thioflavin (ThT). The detection method was the same as that of Example 2. The results were as follows: Figure 2 shown.

[0105] Figure 2This is a graph showing how the inhibitory effect of the ruthenium complex Ru2 on α-synuclein aggregation changes over time in an example of the present invention.

[0106] according to Figure 2 It can be seen that the addition of ruthenium complex Ru2 reduces the fluorescence intensity of ThT, the aggregation degree of α-synuclein is reduced, and the ThT fluorescence intensity changes very little within 48 hours, indicating that the ruthenium complex can permanently inhibit the aggregation of α-synuclein. The ThT fluorescence intensity after adding ruthenium complex Ru2 is lower than the fluorescence intensity after adding NAMI-A, indicating that the ability of ruthenium complex Ru2 to inhibit the aggregation of α-synuclein is better than NAMI-A.

[0107] Example 4 Testing of the Depolymerization Effect of Ruthenium Complex Ru2 on α-Synuclein Fibers

[0108] The α-synuclein monomer was dissolved in PBS to prepare a solution with a concentration of 70 μM, and the solution was continuously mixed and cultured at 37° C. and 900 rpm in a homogenizer for 48 hours to obtain α-synuclein fibers.

[0109] The ruthenium complex Ru2 prepared in Example 1 was added to the pre-prepared α-synuclein fibers to obtain α-synuclein fiber solutions with final concentrations of 0 μM, 42 μM, 70 μM, and 140 μM of the ruthenium complex Ru2, and incubated in a 37°C oven for 2 h. The degradation of the fibrils was detected by fluorescence of the dye thioflavin (ThT), and the detection method was the same as that of Example 2, and the results were as follows: Figure 3 shown.

[0110] Figure 3 This is a graph showing the test results of the depolymerization effect of the ruthenium complex Ru2 on α-synuclein fibers according to an embodiment of the present invention.

[0111] according to Figure 3 It can be seen that as the concentration of the ruthenium complex Ru2 increases, the ThT fluorescence intensity decreases and the degree of degradation of α-synuclein fibers increases, indicating that the ruthenium complex Ru2 is an effective depolymerizing agent for α-synuclein fibers.

[0112] Example 5 Cytotoxicity test after co-culture of ruthenium complexes Ru1, Ru2 and Ru3 with α-synuclein oligomers

[0113] The cells used were neuroblastoma cells (SH-SY5Y), which are widely used in the study of neurodegenerative diseases. They were cultured in DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. A solution containing 0.25% trypsin was added to the culture flask to digest the SH-SY5Y cells. The cells were then diluted to an appropriate concentration in DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. 5×10 3 Cells were added to a 96-well plate at a concentration of 100 μL per well and cultured at 37°C in an atmosphere of 5% CO2 for 24 h.

[0114] Preparation of α-synuclein oligomer solution: Dissolve α-synuclein monomer in PBS to prepare a solution with a concentration of 400 μM, and continuously culture with a mixer at 37°C at 900 rpm for 12 h. Pass the cultured protein solution through a 0.22 μm membrane to obtain an α-synuclein oligomer solution.

[0115] The above-mentioned α-synuclein oligomer solution was dissolved in PBS to prepare a solution with a concentration of 300 μM, and the ruthenium complex Ru1, Ru2 or Ru3 prepared in Example 1 was added at the same time to obtain α-synuclein oligomer solutions with final concentrations of ruthenium complexes Ru1, Ru2 and Ru3 of 0 μM, 150 μM, 300 μM, 600 μM and 1500 μM, respectively, and cultured at 37°C for 2 h.

[0116] The above-cultured α-synuclein oligomer solution containing ruthenium complexes Ru1, Ru2 and Ru3 was added to a 96-well plate containing SH-SY5Y cells that had been cultured for 24 hours, and DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to make each well finally have 100 μL of liquid. The final concentrations of ruthenium complexes Ru1, Ru2 or Ru3 in the drug-added wells were 0 μM, 5 μM, 10 μM, 20 μM, and 50 μM, respectively, and the final concentration of α-synuclein oligomers was 10 μM.

[0117] Set up blank wells, do not add ruthenium-complexed α-synuclein oligomer solution, only add culture medium. After the cells are cultured in an incubator with 5% CO2 and 37°C for 24 hours, remove the liquid in the wells and add 100μL 1 mg / mL MTT solution to each well. 5% CO2, 37°C for 4 hours. Remove the liquid in the wells, add 120μL DMSO to each well, shake and incubate on a shaker for 15 minutes, and detect the absorbance at 570nm. The cell activity of the wells in the blank control group culture medium that do not contain ruthenium complexes and α-synuclein oligomers is recorded as 100%, and then it is used as a control to calculate the cell survival rate of the drug-added group.

[0118] Figure 4 This is a graph showing the biological toxicity test results of the ruthenium complexes Ru1, Ru2 and Ru3 according to the embodiments of the present invention.

[0119] according to Figure 4 It can be seen that when α-synuclein oligomers exist alone, the cell survival rate is less than 60%. After adding ruthenium complexes Ru1, Ru2 and Ru3, the cell survival rate increases, and as the concentration of ruthenium complexes increases, the cell survival rate increases accordingly, indicating that ruthenium complexes Ru1, Ru2 and Ru3 can effectively inhibit the cytotoxicity produced by α-synuclein oligomers.

[0120] Example 6 Oxidative stress test of ruthenium complex Ru2 on SH-SY5Y cells

[0121] The cells used were neuroblastoma cells (SH-SY5Y), and the culture method was the same as that of Example 5. A solution containing 0.25% trypsin was added to the culture flask to digest the SH-SY5Y cells, and then the cells were diluted with DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at an appropriate concentration, and added to a 6-well plate at a cell concentration of 3×105 cells / well, 2 mL per well, 5% CO2, and cultured at 37°C for 24 h.

[0122] Preparation of α-synuclein oligomer solution: Dissolve α-synuclein monomer in PBS to prepare a solution with a concentration of 70 μM, and add the ruthenium complex Ru2 prepared in Example 1 to obtain α-synuclein solutions with a final concentration of 0 μM and 70 μM of the ruthenium complex Ru2. Mix and culture continuously at 37°C at 900 rpm for 48 h.

[0123] The above-cultured α-synuclein oligomer solution containing the ruthenium complex Ru2 was added to a 6-well plate containing SH-SY5Y cells that had been cultured for 24 h, and DMEM-F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to finally have 2 mL of liquid in each well. The final concentrations of the ruthenium complex Ru2 in the drug-added wells were 0 μM and 10 μM, respectively, and the final concentration of α-synuclein oligomers was 10 μM.

[0124] A blank well was set up, and no ruthenium-complexed α-synuclein oligomer solution was added, and only culture medium was added. After the cells were cultured in an incubator with 5% CO2 and 37°C for 24 hours, the liquid in the wells was removed, and the cells were washed three times with PBS. The ROS probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to the wells and diluted with DMEM-F12 culture medium without fetal bovine serum so that the liquid in each well was 1 mL, and the final concentration of DCFH-DA was 10μM. Incubate at 37°C for 30 min. The liquid in the wells was removed, and the cells were washed three times with PBS. 1 mL of DMEM-F12 culture medium without fetal bovine serum was added to each well. The cells were photographed using an inverted fluorescence microscope and excited with light of a wavelength of 488 nm. Using ImageJ software, the intensity of the green fluorescence generated by the reactive oxygen probe DCFH-DA in the cells was quantified to obtain digital fluorescence intensity data. The results are shown in Figure 2. Figure 5 shown.

[0125] Figure 5 This is a graph showing the test results of the oxidative stress of SH-SY5Y cells by the ruthenium complex Ru2 according to an embodiment of the present invention.

[0126] according to Figure 5 It can be seen that the fluorescence intensity increased significantly after the addition of α-synuclein oligomers, while the addition of the ruthenium complex Ru2 significantly reduced the fluorescence intensity, indicating that the ruthenium complex Ru2 can reduce the level of oxidative stress in cells.

[0127] Example 7 Test of the Ruthenium Complex Ru2 Disrupting the Interaction between α-Synuclein and Cell Membrane

[0128] The cells used were HeLa cells, which were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. A solution containing 0.25% trypsin was added to the culture flask to digest the HeLa cells, and then the cells were diluted with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at an appropriate concentration, and 8×10 4 Cells were added to the confocal dish at a concentration of 100 cells / dish, 1 mL per well. Incubate at 5% CO2, 37°C for 24 h. Remove the liquid in the dish, add FITC-labeled α-synuclein, and dilute with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, so that the liquid in each dish is 1 mL, and the final concentration of FITC-labeled α-synuclein is 10 μM. Incubate at 5% CO2, 37°C for 6 h.

[0129] Remove the liquid in the dish, add the ruthenium complex Ru2 prepared in Example 1, and dilute with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, so that each dish has 1 mL of liquid and the final concentration of the ruthenium complex Ru2 is 10 μM. In addition, set up a control group, without adding the ruthenium complex Ru2, but only adding the medium. Incubate at 5% CO2, 37°C for 2 h.

[0130] Remove the liquid in the dish, wash three times with PBS, record cell fluorescence imaging with confocal microscopy, and excite with light at a wavelength of 488 nm. Use ImageJ software to quantify the intensity of green fluorescence generated by FITC to obtain digital fluorescence intensity data. The results are as follows: Figure 6 shown.

[0131] Figure 6 This is a test result diagram of the ruthenium complex Ru2 in an embodiment of the present invention that destroys the interaction between α-synuclein and cell membrane.

[0132] according to Figure 6 It can be seen that there is obvious fluorescence when the ruthenium complex Ru2 is not added, and the fluorescence disappears significantly after the ruthenium complex Ru2 is added, indicating that the ruthenium complex Ru2 can destroy the interaction between α-synuclein and cell membrane.

[0133] Example 8: Ruthenium complex Ru2 used in the rotarod test of Parkinson's disease model mice

[0134] C57BL / 6 mice were selected for animal experiments. All mice were female, and each weighed about 20 g. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was selected to induce a mouse Parkinson's disease model. The specific modeling method was: each mouse was intraperitoneally injected once a day, with 100 μL of 5 mg / mL MPTP injected each time, for 5 consecutive days. In addition, a control group was set up, which was not injected with MPTP but only with PBS.

[0135] The ruthenium complex Ru2 prepared in Example 1 was dissolved in water to obtain a ruthenium complex Ru2 aqueous solution with a final concentration of 2 mg / mL. The ruthenium complex solution was injected into the tail vein of Parkinson's disease model mice, 100 μL each time, for 5 consecutive days. In addition, an MPTP modeling group was set up, in which no ruthenium complex solution was injected, but only PBS was injected.

[0136] All mice were subjected to the rotating rod test. During the experiment, when the animals were placed on the rotating rod in the center of the drum, in order to avoid slipping, the mice adjusted their balance by themselves and ran as the rotating rod rotated. The movement coordination and sense of balance of the mice were assessed by measuring the time the mice walked on the drum. The mice were trained for two days before the formal experiment. In the formal experiment, three mice in each experimental group were placed in the center of the drum at the same time, and the starting speed was set to 15 rpm, the ending speed was 50 rpm, the acceleration time was 30s, and the duration was 10min. The time each mouse stayed on the rotating rod was recorded. Each mouse repeated the experiment three times, and the interval between each time was 1h as the fatigue recovery time of the mouse.

[0137] Figure 7 This is a graph showing the results of the rotarod test on Parkinson's model mice using the ruthenium complex Ru2 of an embodiment of the present invention.

[0138] according to Figure 7 It can be seen that the time that healthy mice without modeling can stay on the rotarod can be maintained for more than 200 seconds, while the time that Parkinson's model mice after MPTP modeling stay on the rotarod is less than 50 seconds. The time that Parkinson's model mice injected with ruthenium complex Ru2 stay on the rotarod increases significantly, indicating that ruthenium complex Ru2 can effectively alleviate the motor symptoms of Parkinson's model mice.

[0139] Example 9 Mass Spectrometry Detection of Ruthenium Complex Ru2 Coordinating with α-Synuclein

[0140] The α-synuclein monomer was dissolved in an ammonium acetate solution (20 mM) to prepare a solution with a concentration of 25 μM. At the same time, the ruthenium complex Ru2 prepared in Example 1 was added to obtain an α-synuclein solution with a final concentration of 75 μM of the ruthenium complex Ru2. The solution was incubated in an oven at 37°C for 3 hours. The incubated α-synuclein solution containing the ruthenium complex Ru2 was detected by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode. The results are as follows: Figure 8 shown.

[0141] Figure 8 This is a diagram showing the results of mass spectrometry detection of ruthenium complex Ru2 binding to α-synuclein in an embodiment of the present invention.

[0142] according to Figure 8 It can be seen that after the addition of ruthenium complex Ru2, peaks of ruthenium binding to α-synuclein (m / z=1637.8074, 1842.3669) appeared (m / z=1648.9158, 1650.9186, 1652.7985, 1854.8599, 1856.9838, 1859.2217), indicating that the ruthenium complex Ru2 can coordinate and bind to α-synuclein.

[0143] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A ruthenium complex, wherein: The structure of the ruthenium complex is shown in formula (I): （I）； Wherein, R1, R2, R3, and R4 are independently selected from H, hydroxyl, methyl, C2-C6 straight-chain or branched alkyl, C3-C6 cycloalkyl, and C2-C6 straight-chain or branched unsaturated hydrocarbon, and at least one of R1, R2, R3, and R4 is hydroxyl; X is a halogen atom; M + It is a monovalent cation.

2. The ruthenium complex according to claim 1, wherein M + Selected from H + 、Na + , K + , ammonium ion, quaternary ammonium cation or imidazolium; X is selected from F, Cl, Br or I, preferably, X is Cl; At least two of R1, R2, R3 and R4 are hydroxyl groups.

3. The ruthenium complex according to claim 1 or 2, wherein Any two of R1, R2, R3, and R4 are hydroxyl groups and the remaining two are H; preferably, R1 and R2 are hydroxyl groups and R3 and R4 are H, or R2 and R3 are hydroxyl groups and R1 and R4 are H, or R3 and R4 are hydroxyl groups and R1 and R2 are H; More preferably, R1 and R2 are hydroxyl groups, R3 and R4 are H and X + for Na + , or R2 and R3 are hydroxyl groups, R1 and R4 are H and X + H + , or R3 and R4 are hydroxyl groups, R1 and R2 are H and X + K + .

4. A method for preparing the ruthenium complex according to any one of claims 1 to 3, the method comprising: A solution of RuX3 in an organic solvent, a compound of the following formula (II) and MX are reacted under heating and stirring in the presence of an acidic aqueous solution; (II); wherein R1, R2, R3, R4, M and X are as defined in claim 1.

5. Use of the ruthenium complex according to any one of claims 1 to 3 in the preparation of a medicament for preventing or treating neurodegenerative diseases.

6. The use according to claim 5, wherein: The neurodegenerative diseases are caused by abnormal aggregation or misfolding of α-synuclein and amyloid deposition.

7. The use according to claim 5, wherein: The neurodegenerative disease is at least one of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, and amyotrophic lateral sclerosis.

8. A pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising the ruthenium complex according to claim 1 or 2 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, wherein The neurodegenerative diseases are caused by abnormal aggregation or misfolding of α-synuclein and amyloid deposition.

10. The pharmaceutical composition according to claim 8, wherein The neurodegenerative disease includes at least one of Parkinson's disease, Alzheimer's disease, Huntington's disease, Lewy body dementia, and amyotrophic lateral sclerosis.

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