CN119684369A - Ruthenium complex, preparation method and use in preparing medicine for preventing or treating neurodegenerative diseases - Google Patents
Ruthenium complex, preparation method and use in preparing medicine for preventing or treating neurodegenerative diseases Download PDFInfo
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Abstract
The invention provides a ruthenium complex, wherein the structure of the ruthenium complex is shown as a formula (I): (I) Wherein R 1、R2、R3、R4 is independently selected from H, hydroxyl, methyl, C2-C6 linear or branched alkyl, C3-C6 cycloalkyl, C2-C6 linear or branched unsaturated hydrocarbon, and at least one of R 1、R2、R3、R4 is hydroxyl. Also provides a preparation method of the ruthenium complex, application of the ruthenium complex in preparing medicaments for preventing or treating neurodegenerative diseases and a pharmaceutical composition for preventing or treating the neurodegenerative diseases.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a ruthenium complex, a preparation method and application thereof in preparing medicines for preventing or treating neurodegenerative diseases.
Background
Neurodegenerative diseases are a class of diseases characterized by neuronal dysfunction and loss, including Alzheimer's Disease (AD), parkinson's Disease (PD), and the like. One of the key features of these diseases is misfolding and pathological aggregation of proteins. For example, in alzheimer's disease, amyloid Precursor Protein (APP) produces amyloid β (aβ) via the action of a range of enzymes, and particularly the aβ42 subtype is closely related to the formation of aβ plaques. The deposition of these plaques in the brain is a pathological feature of AD, for example in Parkinson's disease, an alpha-synuclein is a neuronal protein that is predominantly located at the presynaptic terminal, and is involved in regulating the stability of the neural membrane, affecting presynaptic membrane signaling and cell membrane transport, and the misfolding of alpha-synuclein forms lewy bodies (Lewy bodies) and lewy neurites (Lewy neurites), one of the major pathological markers of PD.
Current clinical drugs are used in a variety of ways to treat parkinson's disease, increasing dopaminergic stimulation through biochemical pathways involved in dopamine synthesis and metabolism. There are still few drugs that can efficiently deagglomerate aggregated alpha-synuclein fibers. There is therefore a need for a compound capable of deaggregating or inhibiting alpha-synuclein fibers.
Disclosure of Invention
In view of this, the present invention provides a ruthenium complex, a preparation method and an application in preparing a medicament for preventing or treating neurodegenerative diseases in order to at least partially solve at least one of the above-mentioned technical problems.
According to an embodiment of one aspect of the present invention, there is provided a ruthenium complex, wherein,
The ruthenium complex has a structure shown in a formula (I):
(I);
Wherein, R 1、R2、R3、R4 is independently selected from H, hydroxyl, methyl, C2-C6 straight chain or branched chain alkyl, C3-C6 cycloalkyl, C2-C6 straight chain or branched chain unsaturated alkyl, at least one of R 1、R2、R3、R4 is hydroxyl, X is halogen atom, and M + is cation.
According to an embodiment of another aspect of the present invention, there is provided a method for preparing a ruthenium complex, the method comprising:
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 under heating and stirring.
According to an embodiment of a further aspect of the present invention there is provided the use of a ruthenium complex in the manufacture of a medicament for the prevention or treatment of neurodegenerative diseases.
According to an embodiment of a further aspect 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.
According to the embodiment of the invention, ruthenium atoms in the ruthenium complex can be coordinated and combined with partial amino acids such as methionine and histidine in the synuclein, phenolic hydroxyl groups have the effect of chelating metal ions, can chelate metal ions such as iron ions and the like, can remove active oxygen, and benzene rings have better affinity to the synuclein, so that the ruthenium complex has stronger binding effect with the alpha-synuclein, thereby inhibiting aggregation of the alpha-synuclein or depolymerizing protein fibers, protecting nerve cells from toxicity of alpha-synuclein oligomers, and further relieving symptoms of Parkinson's disease.
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The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the present invention with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing the results of inhibition of alpha-synuclein aggregation by ruthenium complexes Ru1, ru2, ru3 and NAMI-A according to an embodiment of the invention;
FIG. 2 is a graph showing the inhibition of alpha-synuclein aggregation by ruthenium complex Ru2 according to an embodiment of the invention over time;
FIG. 3 is a graph showing the results of a depolymerization test of an α -synuclein fiber by a ruthenium complex Ru2 according to an embodiment of the present invention;
FIG. 4 is a graph showing the results of biotoxicity tests of ruthenium complexes Ru1, ru2, and Ru3 according to the examples of the invention;
FIG. 5 is a graph showing the results of oxidative stress test of Ru2 on SH-SY5Y cells according to an embodiment of the present invention;
FIG. 6 is a graph showing the results of an interaction test of the ruthenium complex Ru2 according to the embodiment of the invention for disrupting alpha-synuclein and cell membrane;
FIG. 7 is a graph showing the results of a rotating rod test of a ruthenium complex Ru2 for a Parkinson model mouse according to an embodiment of the invention;
FIG. 8 is a graph showing the results of mass spectrometry detection of ruthenium complex Ru2 bound to alpha-synuclein in an embodiment of the invention.
Detailed Description
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the invention. It may be evident, however, that one or more embodiments may be practiced without these specific details. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "comprising" as used herein indicates the presence of a feature, step, operation, but does not preclude the presence or addition of one or more other features.
Where a convention analogous to "at least one of A, B and C, etc." is used, in general such a convention should be interpreted in accordance with the meaning of one of skill in the art having generally understood the convention (e.g., "a system having at least one of A, B and C" would include, but not be limited to, systems having a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B, C together, etc.). Where a formulation similar to at least one of "A, B or C, etc." is used, in general such a formulation should be interpreted in accordance with the ordinary understanding of one skilled in the art (e.g. "a system with at least one of A, B or C" would include but not be limited to systems with a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B, C together, etc.).
In the present invention, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
In the present application, "pharmaceutical composition" refers to a formulation of a compound of the present application with a medium commonly accepted in the art for delivery of biologically active compounds to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote the administration of organisms, facilitate the absorption of active ingredients and further exert biological activity.
In the present 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, dispersing agent, suspending agent, stabilizer, isotonizing agent, solvent, or emulsifying agent that is approved by the relevant government regulatory agency as acceptable for human or livestock use.
In the present invention, "independently" means that the subject has a plurality of subjects, which may be the same or different from each other.
In the present invention, unless otherwise specified, the expression of chemical elements generally includes the concept of isotopes having the same chemical properties, for example, the expression of "hydrogen (H)" includes the concept of 1H (protium or H), 2H (deuterium or D), and the expression of carbon (C) includes 12C, 13C, etc., and will not be described again.
In the present invention, ca to Cb are expressed in such a manner that the group has a carbon number of a to b, and unless otherwise specified, the carbon number generally excludes the carbon number of the substituent.
In the present invention, the term "alkyl" may include a branched or straight-chain saturated aliphatic monovalent hydrocarbon group having a prescribed number of carbon atoms. Examples of the C1-C6 alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, and hexyl.
In the present invention, the term "quaternary ammonium cation" may be represented by the formula NL 4 +, wherein each L is independently selected from alkyl, aryl, alkylaryl, and wherein each of alkyl, aryl, and alkylaryl is optionally substituted with one or more substituents each independently selected from-NO 2, -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 C 1-C6 alkyl, -NH 2、-NL'2-NH(C1-C6 alkyl), -N (C 1-C6 alkyl) 2、C1-C6 alkoxy, C 1-C6 haloalkoxy, Hydroxy (C 1-C6 alkyl), hydroxy (C 1-C6 alkoxy), alkoxy (C 1-C6 alkyl), alkoxy (C 1-C6 alkoxy), C 1-C6 alkyl-NL ' 2、C1-C6 alkyl-SL ', -CONH (C 1-C6 alkyl), -CON (C 1-C6 alkyl) 2、-CO2H、-CO2 L ', -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 the group consisting of optionally substituted C 1-C10 alkyl, optionally substituted C 3-C10 cycloalkyl, optionally substituted C 3-C10 heterocyclyl, Optionally substituted heteroaryl, optionally substituted aryl, hydrocarbyl, amino, -NH 2、-NL'2-NH(C1-C6 alkyl), -N (C 1-C6 alkyl) 2、C1-C6 alkoxy, C 1-C6 haloalkoxy, Hydrocarbyl (C 1-C6 alkyl), hydrocarbyl (C 1-C6 alkoxy), alkoxy (C 1-C6 alkyl), alkoxy (C 1-C6 alkoxy), C 1-C6 alkyl-NL '2、C1-C6 alkyl-SL' or a combination thereof.
In the present invention, the term "linear or branched hydrocarbon group" may include a linear or branched saturated aliphatic monovalent hydrocarbon group or an unsaturated aliphatic monovalent hydrocarbon group having a prescribed number of carbon atoms. The unsaturated hydrocarbon group having a linear or branched chain of 2 to 6 carbon atoms may be a saturated hydrocarbon group having a linear or branched chain of 2 to 6 carbon atoms, in which one or more single bonds are replaced with double bonds, or the like.
The term "treatment" refers to contacting (e.g., administering) a subject with a medicament, composition, etc., based on the present invention after suffering from a disease, such that the symptoms of the disease are reduced compared to when not contacted, and does not mean that the symptoms of the disease must be completely inhibited. Suffering from a disease refers to the appearance of symptoms of the disease in the body.
The term "preventing" refers to the alleviation of symptoms after a disease by contacting (e.g., administering) a subject with a medicament, composition, etc., according to the present invention, prior to the disease, as compared to when not contacted, and does not mean that complete inhibition of the disease is necessary.
In practicing the inventive concept, it was found that the drugs for treating parkinson's disease are classified as (a) dopamine substitutes and levodopa decarboxylase inhibitors for blocking peripheral levodopa decarboxylation because dopamine does not cross the blood brain barrier itself, such as levodopa, (B) dopamine agonists for activating dopamine receptors, such as bromocriptine mesylate and ropinirole hydrochloride, (c) catechol-O-methyltransferase inhibitors for blocking the metabolism of dopamine substitutes, such as entacapone, (d) monoamine oxidase B inhibitors for blocking the metabolism of dopamine, such as selegiline hydrochloride, rasagiline, and saphenolamide, etc.
One key pathological hallmark of parkinson's disease is the presence of intracellular protein inclusions called lewy bodies and lewy neurites, the major component of which is misfolded and aggregated protein α -synuclein. Abnormal accumulation and spread of toxic α -synuclein aggregates, a major molecular event in the pathogenesis of parkinson's disease, can lead to a variety of cellular dysfunctions including impaired mitochondrial function, endoplasmic reticulum stress, disruption of the autophagy-lysosomal pathway, and synaptic and nuclear disorders. Thus, targeting α -synuclein and inhibiting its aggregation is a potential therapeutic strategy. Related art as Anle b strongly blocks oligomer accumulation, neuronal degeneration and disease progression in animal models of parkinson's disease by inhibiting alpha-synuclein aggregation, and also, e.g., the polyphenol flavonoids EGCG extracted from green tea leaves, inhibit the formation of toxic alpha-synuclein oligomers in vitro and are capable of converting alpha-synuclein oligomers into non-toxic species. Even though there are many current therapeutic studies targeting α -synuclein, there are still few drugs that can efficiently deagglomerate aggregated α -synuclein fibers.
Unlike the small organic molecule drugs described above, metal complexes also show potential in the treatment of neurodegenerative diseases. Ruthenium complexes are widely explored for their various biological applications because of their lower toxicity than platinum-based complexes. The ruthenium complex NAMI-A in the related technology can effectively inhibit aggregation of alpha-synuclein and depolymerize aggregated alpha-synuclein fibers, and has potential of developing medicines for relieving symptoms of Parkinson's disease and other neurodegenerative diseases.
Wherein the NAMI-A has the structure shown in the following formula (III):
(III)。
Specifically, according to an embodiment of one aspect of the present invention, there is provided a ruthenium complex having the structure shown in the following formula (I):
(I);
Wherein, R 1、R2、R3、R4 is independently selected from H, hydroxyl, C1-C6 straight chain or branched chain alkyl, C3-C6 cycloalkyl, C2-C6 straight chain or branched chain alkyl, at least one of R 1、R2、R3、R4 is hydroxyl, preferably, at least two of R 1、R2、R3、R4 are hydroxyl;
further preferably, any two of R 1、R2、R3、R4 are hydroxyl groups and the remaining two are H;
Further preferably, R 1 and R 2 are hydroxy and R 3 and R 4 are H, or R 2 and R 3 are hydroxy and R 1 and R 4 are H, or R 3 and R 4 are hydroxy and R 1 and R 2 are H;
More preferably, R 1 and R 2 are hydroxy, R 3 and R 4 are H and X + is Na +, or R 2 and R 3 are hydroxy, R 1 and R 4 are H and X + is H +, or R 3 and R 4 are hydroxy, R 1 and R 2 are H and X + is K +.
X is a halogen atom, preferably F, cl, br or I, more preferably Cl.
In the case where any two of R 1、R2、R3、R4 are hydroxyl groups, it is preferable that these two hydroxyl groups are adjacent.
M + is a cation, preferably a monovalent cation, more preferably H +、Na+、K+, an ammonium ion, a quaternary ammonium cation or an imidazolium, further preferably H +、Na+ or K +.
According to the embodiment of the invention, ruthenium atoms in the ruthenium complex can be coordinated and combined with partial amino acids such as methionine and histidine in the synuclein, phenolic hydroxyl groups have the effect of chelating metal ions, can chelate metal ions such as iron ions and the like, can remove active oxygen, and benzene rings have better affinity to the synuclein, so that the ruthenium complex has stronger binding effect with the alpha-synuclein, thereby inhibiting aggregation of the alpha-synuclein or depolymerizing protein fibers, protecting nerve cells from toxicity of alpha-synuclein oligomers, and further relieving symptoms of Parkinson's disease.
As an embodiment of another aspect of the present invention, there is provided a method for preparing a ruthenium complex, the method comprising:
Reacting a solution of RuX 3 in an organic solvent, a compound of formula (II) below and MX in the presence of an acidic aqueous solution under heating and stirring to obtain a compound of formula (I);
(II)。
In some embodiments of the invention, the method of preparing the ruthenium complex comprises:
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 under heating and stirring.
In some embodiments of the invention, the solution of ruthenium trichloride in the organic solvent is obtained by dissolving ruthenium trichloride in the organic solvent and then heating and refluxing the solution.
In some embodiments of the invention, the method further comprises centrifuging the mixture obtained after the reaction, and steaming the supernatant to obtain the ruthenium complex with the structure shown in the formula (1).
In some embodiments of the present invention, the heating and refluxing time may be 3-5 hours, 3.5 hours, 4 hours, 5 hours, but is not limited to the recited values, and other non-recited values in the range are equally applicable, and the heating and stirring time is 3-6 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, but is not limited to the recited values, and other non-recited values in the range are equally applicable, and the temperature includes 60-100 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, but is not limited to the recited values, and other non-recited values in the range are equally applicable.
In some embodiments of the invention, the molar ratio of ruthenium trichloride to dihydroxyphenylalanine is 1 (2-5), preferably 1 (2-3), and may be 1:2, 1:3, 1:4, 1:5, but is not limited to the recited values, and other non-recited values within the range are equally applicable.
According to the embodiment of the invention, the ruthenium complex can be simply and quickly prepared by ruthenium trichloride, dihydroxyphenylalanine and a cationic solution, the reaction raw materials are easy to obtain, the cost is low, and the prepared product is separated out in a crystal form, so that the ruthenium complex with higher purity can be conveniently and quickly obtained.
As an embodiment of another aspect of the present invention, there is provided use of a ruthenium complex for preparing a medicament for preventing or treating a neurodegenerative disease.
According to embodiments of the invention, neurodegenerative diseases are caused by abnormal aggregation or misfolding of alpha-synuclein and amyloid deposition.
According to the embodiment of the invention, the ruthenium complex can limit aggregation and toxicity of the ruthenium complex by forming stable interaction with amyloid, or can be combined with alpha-synuclein which can form amyloid-like fiber structure, so that the aggregation and toxicity of the ruthenium complex are limited, even the aggregated alpha-synuclein fiber can be depolymerized due to the strong binding capacity of ruthenium ligand to the alpha-synuclein, and the membrane-induced protein conformational change of the alpha-synuclein can be prevented, and the action of alpha-synuclein oligomer and nerve cell membrane can be inhibited, so that the toxic action of the alpha-synuclein can be inhibited. In vitro experiments, the ruthenium complex can effectively inhibit aggregation of alpha-synuclein and depolymerize alpha-synuclein fibers, can inhibit toxicity of alpha-synuclein oligomer to cells, can relieve symptoms of parkinsonism in animal models, and has good application prospects.
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, amyotrophic lateral sclerosis.
According to embodiments of the present invention, the deposition of misfolded alpha-synuclein in Parkinson's disease in the central and peripheral nervous systems results in the occurrence of Parkinson's disease, alzheimer's disease is characterized by extracellular accumulation of amyloid beta (Abeta) as senile plaques and intracellular aggregation of tau to form neurofibrillary tangles (NFT), co-aggregation of alpha-synuclein with amyloid-beta can stabilize beta-sheet-rich oligomers and enhance the association between beta-barrel-forming misfolded alpha-synuclein aggregates and in vivo Alzheimer's disease pathology, huntington's disease is also associated with abnormal aggregation of alpha-synuclein, alpha-synuclein aggregates are typical pathologies of Lewy body dementia, exogenously produced alpha-synuclein fibrils can destroy endogenous complement of alpha-synuclein a prion-like manner to form cytotoxic aggregates, amyotrophic lateral sclerosis is mainly associated with abnormal aggregation of TDP-43 protein, and TDP-43 protein can form amyloid in neurodegenerative diseases. Ruthenium complexes inhibit parkinson's disease, alzheimer's disease, huntington's disease, dementia with lewy bodies, or amyotrophic lateral sclerosis by inhibiting abnormal aggregation or misfolding of alpha-synuclein and amyloid deposition.
According to the embodiment of the invention, the preparation formulation of the medicine is at least one selected from the group consisting of emulsion, oil, powder, water, suspending agent, tablet, granule, capsule and nano-preparation.
According to the embodiment of the invention, the dosage forms of the multiple medicaments meet different treatment requirements, improve the compliance of patients, optimize the curative effect and safety of the medicaments, can meet different clinical requirements and patient preferences, and adapt to different administration routes.
As an embodiment of another aspect 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.
According to embodiments of the invention, degenerative diseases are caused by abnormal aggregation or misfolding of alpha-synuclein and amyloid deposition.
According to an embodiment of the present invention, the neurodegenerative disease comprises at least one of parkinson's disease, alzheimer's disease, huntington's disease, lewy body dementia, amyotrophic lateral sclerosis.
In some embodiments of the present invention, the amount of ruthenium complex used in the drug for preventing or treating degenerative diseases is 0 to 1mol/L, the amount may be 0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L, or 1mol/L, but is not limited to the values recited, and other values not recited in the range are equally applicable.
According to an embodiment of the invention, the medicament further comprises a pharmaceutically acceptable carrier.
According to the embodiment of the invention, the drug carrier can improve the oral absorption of the poorly soluble drug, can improve the solubility and bioavailability of the drug by wrapping the drug in the carrier, and can avoid the rapid capture of reticuloendothelial system in vivo by the drug carrier, so as to prolong the exposure time of the drug in the systemic circulation and enhance the curative effect of the drug.
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 cosolvent, a diluent, a stabilizer, a suspending agent, a pigment-free agent, and a flavoring agent;
in the case of an injectable formulation, the carrier comprises at least one of a preservative, a solubilizing agent, and a stabilizer;
In the case of a topical formulation, the carrier comprises at least one of a matrix, a diluent, a lubricant, and a preservative.
According to the embodiment of the invention, in the oral preparation, the adhesive, the lubricant, the disintegrating agent, the cosolvent, the diluent, the stabilizer, the suspending agent, the pigment-free agent and the flavoring agent play a key role, so that the physicochemical property of the medicine is improved, the curative effect and the safety of the medicine are improved, the acceptability of the preparation is also increased, and different clinical demands and patient preferences are met. In the injection, the preservative, the solubilizer and the stabilizer can ensure the safety, the effectiveness and the stability of the medicine, and improve the medication compliance of patients. In a topical formulation, the matrix, diluent, lubricant, preservative aid in the stable release and effective delivery of the drug, and also improve the safety of the formulation. The dosage form of the medicine can be optimized and the treatment effect can be improved by reasonably selecting and using the auxiliary materials.
According to an embodiment of the present invention, the administration mode of the drug includes at least one of oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, and topical administration.
The technical scheme of the invention is further described below by means of specific embodiments and with reference to the accompanying drawings. It should be noted that the following specific examples are given by way of illustration only and the scope of the present invention is not limited thereto. The drugs or reagents used in the examples below were obtained commercially or were prepared by a known preparation method. Methods used in the following examples, such as Image J, are well known in the art, and may be described in textbooks or related documents, and are not repeated.
EXAMPLE 1 preparation of ruthenium complexes
The synthetic route is as follows:
Ruthenium complex Ru1:
Step S1, weighing 3 mmol ruthenium trichloride, placing the ruthenium trichloride in a round-bottom flask, adding 10 mL ethanol, heating to reflux for reaction for 5 hours, and changing the solution into dark green after the reaction is finished. The solution was filtered and concentrated to 1 mL on a rotary evaporator.
Step S2, 50 mu L of concentrated hydrochloric acid, 2mL of 2 mM NaCl aqueous solution and 9 mmol L-3, 4-dihydroxyphenylalanine are added into the reaction solution obtained in the step S1, heated to 60 ℃, heated and stirred for reaction for 6h. And spin-drying the solution by using a rotary evaporator, washing the solution for 3 times by using acetone, taking out the solid, drying the solid to finally obtain the ruthenium complex Ru1, and characterizing the ruthenium complex Ru1 to prove that the ruthenium complex Ru1 is successfully prepared.
Spectrogram 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).
The mass spectrum characterization result of the ruthenium complex Ru1 comprises ESR-MS, calculated value, m/z= 587.9616 (Ru 1-H) +,609.9435 (Ru1-Na)+ and experimental value, m/z= 587.6834 (Ru 1-H) +,609.6294 (Ru1-Na)+.
Ruthenium complex Ru2:
Step S1-step S2 are repeated, except that 50 mu L of concentrated hydrochloric acid, 2mL of water and 6mmol of levodopa are added into the reaction solution obtained in step S1, the mixture is heated to 80 ℃, stirred and reacted for 3 hours, and finally ruthenium complex Ru2 is obtained, and the ruthenium complex Ru2 is characterized to indicate that the ruthenium complex Ru2 is successfully prepared.
Spectrogram 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).
The mass spectrum characterization result of the ruthenium complex Ru2 comprises ESR-MS, calculated value, m/z= 565.9796 (Ru 2-H) + and experimental value, m/z= 565.8375 (Ru 2-H) +.
Ruthenium complex Ru3:
Step S1-step S2 are repeated, except that 20 mu L of concentrated hydrochloric acid, 2 mL of 2 mM KCl aqueous solution and 15 mmol of 3-hydroxy-D-tyrosine are added into the reaction solution obtained in step S1, the mixture is heated to 100 ℃, stirred and reacted for 5 hours, and finally ruthenium complex Ru3 is obtained, and the ruthenium complex Ru3 is characterized to indicate that the ruthenium complex Ru3 is successfully prepared.
Spectrogram information of ruthenium complex Ru3 NMR 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).
The mass spectrum characterization result of the ruthenium complex Ru3 comprises ESR-MS, calculated value, m/z= 603.9355 (Ru 1-H) +,641.8914 (Ru1-K)+ and experimental value, m/z= 603.7528 (Ru 1-H) +,6641.6839 (Ru1-K)+.
EXAMPLE 2 inhibition test of the ruthenium complexes Ru1, ru2 and Ru3 on alpha-synuclein aggregation
The α -synuclein monomer was dissolved in Phosphate Buffered Saline (PBS) to prepare a 70 μm concentration solution, and 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 the final concentration of 0 μm, 7 μm, 21 μm, 35 μm, 70 μm, respectively, of α -synuclein solution.
The mixing apparatus 900 rpm was continuously mixed and incubated at 37℃for 48 hours, and fibril formation was detected by fluorescence of the dye thioflavin (ThT). 200. Mu.L of the culture broth was mixed with 1.5. Mu.L of 20 mM thT and incubated 20 min in a 37℃oven. The fluorescence intensity of ThT was measured on a fluorescence spectrophotometer, the excitation wavelength of ThT was 450 nm, and the emission wavelength was 490 nm, and the results were shown in fig. 1.
FIG. 1 is a graph showing the results of inhibition of alpha-synuclein aggregation by Ru1, ru2, ru3 and NAMI-A of ruthenium complexes according to embodiments of the invention.
As can be seen from fig. 1, as the concentration of ruthenium complex increases, thT fluorescence intensity decreases with decreasing α -synuclein aggregation, and the decrease in α -synuclein aggregation with addition of ruthenium complexes Ru1, ru2 and Ru3 is greater than that with addition of NAMI-a, indicating that ruthenium complexes Ru1, ru2 and Ru3 are effective α -synuclein aggregation inhibitors, capable of inhibiting α -synuclein aggregation in vitro, and superior in aggregation inhibition effect to NAMI-a.
EXAMPLE 3 test of inhibition of alpha-synuclein aggregation by ruthenium Complex Ru2
The α -synuclein monomer was dissolved in PBS to prepare a 70 μm concentration solution, and the ruthenium complex Ru2 prepared in example 1 and the control NAMI-A were added, respectively, to obtain an α -synuclein solution having a final concentration of 21. Mu.M for the ruthenium complex Ru2 and NAMI-A.
The 37 ℃ mixing instrument 900 rpm is used for continuous mixed culture for 0h, 12h, 24h and 48 h respectively. The formation of fibrils was detected by fluorescence of the dye thioflavin (ThT) in the same manner as in example 2, and the results are shown in figure 2.
FIG. 2 is a graph showing the inhibition of alpha-synuclein aggregation by ruthenium complex Ru2 according to an embodiment of the invention over time.
As can be seen from FIG. 2, the addition of the ruthenium complex Ru2 reduced the fluorescence intensity of ThT, the extent of aggregation of alpha-synuclein was reduced, and the change in the fluorescence intensity of ThT within 48 h was small, indicating that the ruthenium complex was able to permanently inhibit aggregation of alpha-synuclein. The ThT fluorescence intensity of the ruthenium complex Ru2 is lower than that of NAMI-A, which shows that the capability of the ruthenium complex Ru2 for inhibiting alpha-synuclein aggregation is better than that of NAMI-A.
EXAMPLE 4 depolymerization test of ruthenium Complex Ru2 on alpha-synuclein fibers
The alpha-synuclein monomer is dissolved in PBS to prepare a solution with the concentration of 70 mu M, and the solution is continuously mixed and cultured for 48 hours by a 37 ℃ mixing instrument 900 rpm, so that the alpha-synuclein fiber is obtained.
The ruthenium complex Ru2 prepared in example 1 was added to pre-formulated alpha-synuclein fibres to give alpha-synuclein fibre solutions with final concentrations of ruthenium complex Ru2 of 0. Mu.M, 42. Mu.M, 70. Mu.M, 140. Mu.M, and incubated for 2h in a 37℃oven. Degradation of fibrils was detected by fluorescence of the dye thioflavin (ThT) in the same manner as in example 2, and the results are shown in figure 3.
FIG. 3 is a graph showing the results of a depolymerization test of alpha-synuclein fiber by ruthenium complex Ru2 according to an embodiment of the present invention.
As can be seen from fig. 3, as the concentration of the ruthenium complex Ru2 increases, thT fluorescence intensity decreases with increasing degree of degradation of the α -synuclein fiber, indicating that the ruthenium complex Ru2 is an effective depolymerizing agent for α -synuclein fiber.
EXAMPLE 5 cytotoxicity test of ruthenium complexes Ru1, ru2 and Ru3 after Co-cultivation with alpha-synuclein oligomers
The cells used were neuroblastoma cells (SH-SY 5Y), which are widely used in the study of neurodegenerative diseases, cultured in DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. SH-SY5Y cells were digested by adding a solution containing 0.25% pancreatin to the flask, and the cells were diluted with DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at an appropriate concentration, and added to 96-well plates at a cell concentration of 5X 10 3 cells/well, 100. Mu.L per well. 5% CO 2, 37℃culture 24 h.
Preparing an alpha-synuclein oligomer solution, namely dissolving an alpha-synuclein monomer in PBS to prepare a solution with the concentration of 400 mu M, continuously mixing and culturing the solution for 12 h by a 37 ℃ mixing and homogenizing instrument 900rpm, and passing the cultured protein solution through a 0.22 mu M membrane to obtain the alpha-synuclein oligomer solution.
The alpha-synuclein oligomer solution is dissolved in PBS to prepare a solution with the concentration of 300 mu M, meanwhile, ruthenium complexes Ru1, ru2 or Ru3 prepared in the embodiment 1 are added to obtain alpha-synuclein oligomer solutions with the final concentrations of 0 mu M, 150 mu M, 300 mu M, 600 mu M and 1500 mu M of the ruthenium complexes Ru1, ru2 and Ru3 respectively, and the solution is cultured at 37 ℃ for 2 h.
The above-cultured solution of the α -synuclein oligomer containing ruthenium complexes Ru1, ru2 and Ru3 was added to a 96-well plate containing SH-SY5Y cells which had been cultured for 24 h, and DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added to give a final 100. Mu.L of liquid per well. The final concentration of the ruthenium complex Ru1, ru2 or Ru3 in the drug-adding hole is 0 mu M, 5 mu M, 10 mu M, 20 mu M and 50 mu M respectively, wherein the final concentration of the alpha-synuclein oligomer is 10 mu M.
Blank wells were set, without addition of ruthenium-containing complexed alpha-synuclein oligomer solution, only with medium. After culturing the cells in an incubator at 5% CO 2, 37℃for 24h, the liquid in the wells was removed and 100. Mu.L of 1/mg/mL MTT solution was added to each well. 4h was incubated at 37℃with 5% CO 2. The wells were removed, 120 μl DMSO was added to each well, incubated for 15min with shaking, and absorbance at 570 nm was measured. The cell viability of the dosed group was calculated as a control, with cells in the blank medium without wells of ruthenium complex and alpha-synuclein oligomer, recorded as 100%.
FIG. 4 is a graph showing the results of biotoxicity tests of ruthenium complexes Ru1, ru2, and Ru3 according to the examples of the invention.
As can be seen from fig. 4, the cell viability was less than 60% when the α -synuclein oligomer was alone. The cell survival rate is improved after the ruthenium complexes Ru1, ru2 and Ru3 are added, and the cell survival rate is improved along with the improvement of the concentration of the ruthenium complexes, which shows that the ruthenium complexes Ru1, ru2 and Ru3 can effectively inhibit cytotoxicity generated by alpha-synuclein oligomer.
EXAMPLE 6 oxidative stress test of ruthenium Complex Ru2 on SH-SY5Y cells
The cells used were neuroblastoma cells (SH-SY 5Y) and the culture method was the same as in example 5. SH-SY5Y cells were digested by adding a solution containing 0.25% pancreatin to the flask, and 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 3X 105 cells/well, 2 mL per well, 5% CO 2, 37℃for 24 h.
Preparing an alpha-synuclein oligomer solution, namely dissolving an alpha-synuclein monomer in PBS to prepare a solution with the concentration of 70 mu M, and simultaneously adding the ruthenium complex Ru2 prepared in the example 1 to obtain the alpha-synuclein solution with the final concentration of 0 mu M and 70 mu M of the ruthenium complex Ru 2. The 37 ℃ mixing instrument 900 rpm continuously mixes and cultures 48 h.
The above-cultured solution of the ruthenium complex Ru 2-containing alpha-synuclein oligomer was added to a 6-well plate containing SH-SY5Y cells which had been cultured for 24 h, and DMEM-F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was added so that each well was eventually filled with 2 mL liquid, and the final concentration of the ruthenium complex Ru2 in the dosing well was 0. Mu.M and 10. Mu.M, respectively, wherein the final concentrations of the alpha-synuclein oligomer were all 10. Mu.M.
Blank wells were set, without addition of ruthenium-containing complexed alpha-synuclein oligomer solution, only with medium. After cells were incubated in an incubator at 5% CO 2, 37℃for 24h, the liquid in the wells was removed, washed three times with PBS, ROS probe 2',7' -dichlorofluorescein diacetate (DCFH-DA) was added to the wells, and diluted with DMEM-F12 medium without fetal bovine serum to give a liquid per well of 1 mL and a final concentration of DCFH-DA of 10. Mu.M, 37℃for incubation of 30 min. The liquid in the wells was removed and washed three times with PBS, and 1 mL of DMEM-F12 medium without fetal calf serum was added to each well. Cells were photographed using a fluorescent inverted microscope and excited with light having a wavelength of 488 nm. The intensity of green fluorescence generated by the active oxygen probe DCFH-DA in the cells was quantified using imageJ software to obtain digitized fluorescence intensity data, the results of which are shown in FIG. 5.
FIG. 5 is a graph showing the results of oxidative stress test of Ru2 on SH-SY5Y cells according to an embodiment of the present invention.
As can be seen from fig. 5, the fluorescence intensity increased significantly after the addition of the α -synuclein oligomer, while the addition of the ruthenium complex Ru2 reduced the fluorescence intensity significantly, indicating that the ruthenium complex Ru2 was able to reduce the intracellular oxidative stress level.
EXAMPLE 7 interaction test of ruthenium Complex Ru2 to disrupt alpha-synuclein and cell membrane
The cells used were sea-Lawster cells (Hela) and were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. HeLa cells were digested by adding a solution containing 0.25% pancreatin to the flask, and the cells were diluted with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at appropriate concentrations and added to confocal dishes at a cell concentration of 8X 10 4 cells/dish, 1mL per well. 5% CO 2, 37℃culture 24 h. The liquid in the dish was removed, FITC-labeled alpha-synuclein was added and diluted with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to give 1mL liquid per dish and a final concentration of FITC-labeled alpha-synuclein of 10. Mu.M. Incubate 6h at 5% CO 2, 37 ℃.
The liquid in the dish was removed, ruthenium complex Ru2 prepared in example 1 was added and diluted with DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin to give 1 mL liquid per dish and a final concentration of ruthenium complex Ru2 of 10. Mu.M. In addition, a control group was set, and the ruthenium complex Ru2 was not added, but only the medium was added. Incubate 2h at 37 ℃ with 5% co 2.
The dish was removed from the liquid, washed three times with PBS, and fluorescence imaging of the cells was recorded by confocal microscopy, and excitation was performed with light having a wavelength of 488 nm. The intensity of the green fluorescence generated by FITC was quantified using ImageJ software to yield digitized fluorescence intensity data, the results of which are shown in fig. 6.
FIG. 6 is a graph showing the results of an interaction test of the ruthenium complex Ru2 according to the embodiment of the invention for disrupting alpha-synuclein and cell membrane.
As can be seen from fig. 6, there is a significant fluorescence when the ruthenium complex Ru2 is not added, and the fluorescence is significantly lost after the ruthenium complex Ru2 is added, which indicates that the ruthenium complex Ru2 can destroy the interaction between the α -synuclein and the cell membrane.
EXAMPLE 8 ruthenium Complex Ru2 for parkinsonism model mouse rod-rotating test
For animal experiments, C57BL/6 mice were selected, which were female, each about 20 g. A model of 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) induced parkinson's disease in mice was selected, and the specific modeling method was that each mouse was intraperitoneally injected once per day, with 100 μl of 5mg/mL MPTP per injection for 5 days. In addition, a control group was set, and no MPTP was injected, but only PBS was injected.
The ruthenium complex Ru2 prepared in example 1 was dissolved in water to give an aqueous ruthenium complex Ru2 solution having a final concentration of 2 mg/mL. The mice in the parkinsonism model were injected with the ruthenium complex solution by tail vein, 100 μl each, for 5 consecutive days. In addition, MPTP molding was set, and the ruthenium complex solution was not injected, but only PBS was injected.
All mice were subjected to a stick-turning experiment during which, when the animals were placed on a stick-turning at the center of the drum, the mice were allowed to self-adjust the balance to avoid slipping, run as the stick-turning was turned, and the motor coordination and balance sensation of the mice were assessed by measuring the time the mice were walked on the drum. Mice were trained for two days prior to the formal experiments. In the formal experiment, three mice of each experiment group are simultaneously placed in the center of a roller, the starting rotating speed is set to be 15 rpm, the stopping rotating speed is set to be 50 rpm, the acceleration time is set to be 30s, the duration time is set to be 10min, the residence time of each mouse on a rotating rod is recorded, the experiment is repeated for three times for each mouse, and the interval between each time is set to be 1h as the fatigue recovery time of the mouse.
FIG. 7 is a graph showing the results of a rotating rod test of a ruthenium complex Ru2 for a Parkinson model mouse according to an embodiment of the invention.
As can be seen from fig. 7, the residence time of healthy mice without modeling in the rotating stick can be maintained at 200s or more, while the residence time of parkinson model mice after MPTP modeling in the rotating stick is less than 50s. The residence time of the parkinsonism model mice injected with the ruthenium complex Ru2 on the rotating rod is obviously increased, which proves that the ruthenium complex Ru2 can effectively relieve the movement symptoms of the parkinsonism model mice.
Example 9 Mass Spectrometry detection of ruthenium Complex Ru2 coordination binding alpha-synuclein
The α -synuclein monomer was dissolved in ammonium acetate solution (20 mM) to prepare a 25 μm concentration solution, and simultaneously the ruthenium complex Ru2 prepared in example 1 was added to obtain a 75 μm final concentration α -synuclein solution of the ruthenium complex Ru2, and incubated in an oven at 37 ℃ for 3h. The detection was carried out by electrospray ionization mass spectrometry (ESI-MS) and the result was shown in FIG. 8, using a positive ion mode, for the cultured solution of the ruthenium complex Ru2 in the alpha-synuclein.
FIG. 8 is a graph showing the results of mass spectrometry detection of ruthenium complex Ru2 bound to alpha-synuclein in an embodiment of the invention.
As can be seen from fig. 8, after addition of ruthenium complex Ru2, peaks (m/z= 1648.9158, 1650.9186, 1652.7985, 1854.8599, 1856.9838, 1859.2217) of ruthenium binding to α -synuclein (m/z= 1637.8074, 1842.3669) appear, indicating that ruthenium complex Ru2 is able to coordinate to α -synuclein.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
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