WO2008083543A1

WO2008083543A1 - Nitrogen-containing heterocyclic modified compounds and uses thereof

Info

Publication number: WO2008083543A1
Application number: PCT/CN2007/002985
Authority: WO
Inventors: Yanmei Li; Weihui Wu; Peng Lei; Xiaoyang Su; Jia Hu
Original assignee: Tsinghua University
Priority date: 2007-01-11
Filing date: 2007-10-18
Publication date: 2008-07-17

Abstract

A nitrogen-containing heterocyclic modified compounds and their uses in preparing the medicaments of preventing and treating the Alzheimer's disease, Parkinson's disease and other degenerative nervous system disease or type two diabetes. Studies show that the provided compounds can inhibit aggregation of Aβ, also can make the Aβ aggregated depolymerize, and will eventually hydrolysic eliminate the Aβ protein, inhibit and remove the Aβ toxicity, the said compounds has broad application prospects in the field of medicine.

Description

Compound with nitrogen-containing heterocyclic modification and its application

The present invention relates to compounds and their use, and more particularly to compounds having nitrogen-containing heterocyclic modifications and their use in the manufacture of a medicament for the treatment and prevention of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes.

Background technique

Nervous system degenerative diseases such as Alzheimer's disease and Parkinson's disease and type 2 diabetes are protein conformations. Among them, Alzheimer's disease (AD), also known as Alzheimer's disease, is a central nervous system degenerative disease, clinical manifestations of cognitive impairment, memory loss, eventually loss of thinking ability, movement disorders, life Can't take care of themselves. Pathological features are mainly senile plaque (SP), neurofibrillary tangles (NFT), and selective neuronal and synaptic loss. With the aging of the global population, Alzheimer's disease has become an important cause of human death. For many years, the main drug for clinical treatment of this disease is cholinesterase inhibitor, which acts on the cholinergic nervous system and has a mitigating effect on the disease, but it cannot prevent the occurrence and development of the disease. At present, the pathogenesis of Alzheimer's disease is not very clear, but it is generally believed that the key cause of the disease is the accumulation of excess β-amyloid peptide (β-amyloid peptide, Αβ). The main component) has a detrimental effect on cell membranes, synapses and axons, and leads to neurofibrillary tangles. Therefore, preventing the aggregation of Αβ or degrading Αβ can inhibit and eliminate the toxicity of Αβ, thereby achieving the purpose of treating and preventing Alzheimer's disease.

Invention disclosure

SUMMARY OF THE INVENTION An object of the present invention is to provide a compound having a nitrogen-containing heterocyclic ring modification which has an inhibitory effect on the aggregation of Αβ, and which has a depolymerization effect on Αβ aggregates and finally degrades Αβ.

The nitrogen-containing heterocyclic modified compound provided by the present invention includes a polyphenol compound, a polypeptide compound, and a styryl compound.

The nitrogen-containing heterocyclic modified polyphenolic compound provided by the present invention has a structural formula of the formula - (. 3⁄4) „(3⁄4) „^ (Formula I) wherein, any nitrogen-containing heterocyclic hydrocarbon An amino group or an acyclic alkaneamino group having 4 or more nitrogen atoms; η = 1-5 _; an arbitrary natural, unnatural amino acid residue or a corresponding amino acid residue having a modifying group, m=0- 5; ^ for any polyphenols.

The preferred one is Cyclen (N-linked-1,4,7,10-tetraazacyclododecane) or Trpn (N-linked-indole, Ν'-bis(3-aminopropyl)propane-1,3- Diamine); η is preferably 1; 3⁄4 is preferably one of 20 natural amino acid residues or a corresponding amino acid residue having a modifying group (more preferably a methyl modified amino acid residue), m is preferably 0 -3 (more preferably 1); Yi is preferably curcumin (Cur), nordihydroguaiaretic acid (NDGA) or rosmarinic acid (RA). The chemical structural formula of Cyclen is as shown in Formula IV: (Formula IV) The chemical structural formula of the Tpn is as shown in Formula V

(Formula V) The structural formula of Cur is as shown in Formula VI:

Formula VI) The structural formula of the NDGA is as shown in Formula VII:

(Formula VII) The structural formula of the RA is as

(Formula IX) The polyphenol compound of the formula I is more preferably a glycine residue, a leucine residue, an isoleucine, an alanine residue, a proline residue, or a styrene-butene. A tyrosine residue or a tyrosine residue; particularly preferably a glycine residue, a leucine residue, an isoleucine, or an alanine residue.

The amino acid residue constituting the polyphenolic compound may be a D form (dextrorotatory), an L form (left-handed) conformation, preferably a D-type conformation.

The nitrogen-containing heterocyclic modified polypeptide compound provided by the present invention has the structural formula of the formula:

_{_{_{R 2 (CH 2) n (}}} X 2) m ( Formula _[pi) wherein, R ₂ is any amino group or a nitrogen-containing heterocyclic noncyclic embankment alkane hydrocarbon group having 4 or more than 4 nitrogen atoms; n = 1-5 X ₂ is any natural, non-natural amino acid residue or a corresponding amino acid residue having a modifying group, m-2-ΙΟ; the C-terminus of the polypeptide compound is a carboxyl group (a COOH) or an amide group ( A CONH ₂ ) is either a hydroxyl group after the reduction (mono-OH:), an amino group (-NH ₂ ) or an alkyl group.

R ₂ in the formula II is preferably Cyclen (N-linked-1,4,7,10-tetraazacyclotetradecene) or Trpn (3, 3- ', 3"-triaminotripropylamine); n is preferably 1; X ₂ is preferably one of 20 natural amino acid residues or a corresponding amino modification (preferably methyl modified), m preferably 3 -5 (more preferably 5) The chemical structural formula of Cyclen is as shown in Formula IV; the chemical structural formula of the Trpii is as shown in Formula V.

More preferably, X ₂ is a lysine residue, a leucine residue, a proline residue, a phenylalanine residue, a tyrosine residue or an aspartic acid residue. X _{2 is} particularly preferably a leucine residue, a proline residue, an aspartic acid residue or an amino acid residue constituting the polypeptide compound, which may be D-form (dextrorotatory), L-form (left-handed) conformation, It is preferably a D-type conformation.

The nitrogen-containing heterocyclic modified styrene compound provided by the present invention has the structural formula of the formula:

R ₃ (CH ₂ ) „(X ₃ ) _m Y ₃ (Formula ^{1 n)} wherein, is any nitrogen-containing heterocyclic hydrocarbon amino group or acyclic a hydrocarbylene amino group having 4 or more nitrogen atoms; η = 1 -5; X is any natural, non-natural amino acid residue or a corresponding amino acid residue having a modifying group, m = 0 - 5; Y ₃ is any compound having a styryl structure.

The R ₃ is preferably Cyclen (N-linked-1,4,7,10-tetraazacyclododecane) or Trpn (N-linked-oxime, Ν'-bis(3-aminopropyl)propanoid-1 , 3-diamine); η is preferably 1; Χ ₃ is preferably one of 20 natural amino acid residues or a corresponding amino acid residue having a modifying group (preferably methyl modified), m preferably 0- 3 (more preferably 1); Y ₃ is preferably Congo red (CR) or Benzoic acid (3,3 '-[1 ,4-phenylenedi-(lE)-2,l -ethenediyl] bis [6-hydroxy-], BA). The chemical structural formula of the Cyclen is as shown in Formula IV; the chemical structural formula of the Trpn is as shown in Formula V.

The structural formula of the CR is as shown in the formula X:

(Formula X) The structural formula of the BA is as

(Formula XI) X in the structural formula (Formula III) of the above styryl compound is more preferably a glycine residue, a leucine residue, an isoleucine, an alanine residue, a proline residue, or a benzene. An alanine residue or a tyrosine residue; X is particularly preferably a glycine residue, a leucine residue, an isoleucine or an alanine residue.

The amino acid residue constituting the styryl compound may be D type (dextrorotatory), L type (left-handed) conformation, preferably L-shaped conformation.

The polyphenols, polypeptides and styrenated foods provided by the present invention can be synthesized by various conventional chemical synthesis methods, for example, by the method of ester formation or amide formation in the presence of a coupling agent or other methods for synthesizing Formula I and a compound of the formula III; a compound of the formula II can be synthesized by solid phase synthesis, including t-Boc chemistry and 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, preferably 9 -芴methoxycarbonyl Chemical law.

Combinations of the above polyphenols, polypeptides or styrenic compounds with transition metals such as copper, cobalt, nickel, palladium, zinc or iron are also within the scope of the invention.

Another object of the present invention is to provide a medicament for the treatment and prevention of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes.

The active ingredient of the medicament provided by the present invention is a polyphenol, a polypeptide, a styrene compound, a complex of the above compound with a transition metal copper, cobalt, nickel, palladium, zinc or iron, and a salt of the above compound. One or several.

Further, the polyphenols, polypeptides and styrenic compounds may also be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include those derived from organic or inorganic bases, organic or inorganic acids. Salts obtained from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts and zinc salts, etc., effects Preferred are ammonium salts, potassium salts, calcium salts, lithium salts, magnesium salts and sodium salts; pharmaceutically acceptable non-toxic organic base salts include primary, secondary and tertiary amine salts. The acid to be reacted with the polyphenols, polypeptides and styrenic compounds provided by the present invention includes hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like.

When necessary, one or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs, including conventional diluents, excipients, protective agents, absorption enhancers, fillers, binders, humectants in the pharmaceutical field. , disintegrants, surfactants, etc. The excipient may be carboxymethyl starch, gelatin, cellulose, sodium hydrogencarbonate, sodium chloride or polyethylene glycol; the protective agent may be EDTA or benzylammonium; the absorption enhancer may be poly Sorbate-80, azone, carboxymethylcellulose or 9-dodecyl ether, and the like.

In addition to being encapsulated, the medicament of the present invention can also be formulated into a plurality of pharmaceutical forms such as tablets, injections, solutions, granules and the like. The above various dosage forms of the drug can be prepared according to a conventional method in the pharmaceutical field.

The oral dose of the drug is generally 0.05-50 mg/kg, which can be used one or more times for 1 to 6 months.

DRAWINGS

Figure 1 shows the experimental results of the inhibition of Αβ aggregation by polyphenols by ThT fluorescence. Figure 2 is the experimental results of the depolymerization of phenolic aggregates by polyphenols.

Figure 3 shows the results of inhibition of Αβ aggregation by peptides detected by ThT fluorescence assay.

Figure 4 shows the results of the depolymerization of Αβ aggregates by peptide compounds.

Figure 5 is an experimental result of the inhibition of Αβ aggregation by styrene-based compounds by ThT fluorescence. Figure 6 is the experimental result of depolymerization of stilbene-based compounds to Αβ aggregates.

Figure 7 shows the results of electron microscopic observation on the inhibition of Αβ aggregation by peptide compounds.

Figure 8 shows the results of electron microscopic observation of the depolymerization of peptides on Αβ aggregates.

Figure 9 is an electron microscopic observation of the inhibitory effect of polyphenols on Αβ aggregation.

Figure 10 is an electron microscopic observation of the inhibitory effect of styryl compounds on Αβ aggregation.

Figure 11 shows the results of MALDI-TOF-MS analysis of the hydrolysis of Αβ by polyphenols. Figure 12 shows the results of MALDI-TOF-MS analysis of the cleavage of Αβ by peptides. 13 is a polyphenol compound produced results Η _₂ 0 ₂ Inhibition of copper-mediated Αβ

FIG 14 is a peptide compounds produced inhibition results ¾0 ₂ of copper-mediated Αβ

Figure 15 is an analysis result of the inhibition effect of styrene-based compounds on copper-mediated Αβ production Η _{2 2} ₂ Figure 16 is the result of analysis of protective effects of polyphenols on rat hippocampal neurons by inhibiting Αβ toxicity

Figure 17 is the analysis of the protective effect of peptide compounds on rat hippocampal neurons by inhibiting Αβ toxicity.

Figure 18 shows the results of analysis of the protective effect of styryl compounds on rat hippocampal neurons by inhibiting Αβ toxicity.

The best way to implement the invention

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1. Preparation of polyphenolic compounds of the formula XII

The synthesis of the polyphenolic compound of the formula XII of the present invention comprises the following steps:

a. Synthesis of Cyclen-containing monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclodene-1-yl]acetic acid: with Cyclen (purchased from Acros, USA) Company) as raw material, first reacted with Boc20 (di-tert-butyl dicarbonate, purchased from Shanghai Jill Biochemical Company) to obtain 1,4,7-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclo Second House (3B _0C -Cyclen) (see Eiichi Kimura, Shin Aoki, Tohru Koike, and Motoo Shiro, A Tris(ZnII-1,4,7, 10-tetraazacyclododecane) Complex as a New Receptor for Phosphate Dianions in Aqueous Solution, Journal of American Chemical Society, 1997, Vol 119, 3068-3076), and reacting the product with ethyl bromoacetate (see Joong Won Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh) , Protein-Cleaving Catalyst Selective for Protein Substrate, Organic Letters, 2002, Vol 4, 4155-4158) to give 1,4,7-tri-tert-butoxycarbonyl-10-(2-ethoxy-2-oxoethyl)- 1,4,7,10-tetraazacyclotetradecyl, finally obtained by hydrolysis with NaOH (see Joong Won Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Jung) Hun Suh, Protei-Cleaving

Catalyst Selective for Protein Substrate, Organic Letters, 2002, Vol 4, 4155-4158), to obtain Cyclen-containing monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclotetradecene Second hospital small base] acetic acid.

b. Curcumin (purchased from Acros, USA) and Cyclen-containing monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclodene-1-yl Acetic acid coupling, the coupling conditions are coupling agent EDC (purchased from Shanghai Yansheng Biochemical Company), HOBt (purchased from Shanghai Jill Biochemical Company) and catalyst DMAP (Acros, USA) (mixing ratio: 4 times the amount of Cyclen-containing single) The objective product was obtained by reacting 1 part of curcumin, 4 times the amount of coupling agent EDC, 4 times the amount of HOBt and catalyst DMAP) at room temperature for 15 hours.

c After the reaction of the step b, the crude product was separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether (1: 4)), and the obtained product was removed with a cleavage reagent trifluoroacetic acid (TFA, purchased from Shanghai Jill Biochemical Co., Ltd.). The Boc protecting group gives a crude polyphenolic compound.

d. The crude polyphenolic compound was subjected to HPLC method (column: C18 packed column (purchased from Agilent, USA); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 mL/min _; detection wavelength: 380 nm _; column temperature: Room Warm) Separation of pure polyphenolic compounds.

After testing, the polyphenolic compound prepared by the above method has a correct structure, and its structural formula is as shown in Formula XII, and the purity is over 95%.

(Formula XII)

Example 2 Preparation of a polyphenolic compound of the formula XIII

The synthesis of the polyphenolic compound of the formula XIII of the present invention comprises the following steps: a. glycine methyl ester (purchased from Shanghai Jill Biochemical Co., Ltd.) and Cyclen containing monomer [4,7,10-tri-tert-butoxycarbonyl-1, 4,7,10-tetraazacyclotetradecyl-1-yl]acetic acid coupling, the coupling condition is the coupling agent HBTU (purchased from Shanghai Yansheng Biochemical Company), HOBt (purchased from Shanghai Jill Biochemical Company), 1 times The amount of the Tpnn-containing monomer, 1 time amount of glycine methyl ester, 1 time amount of coupling agent HBTU and 1 time amount of HOBt were reacted at room temperature for 12 hours to obtain a coupling product.

b. After the reaction of the step a, the crude product is separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether (1: 4)) to obtain a glycine methyl ester containing a cyclen monomer, and then hydrolyzed with NaOH (see Joong Won).

Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-Cleaving Catalyst Selective for Protein Substrate, Organic Letters, 2002, Vol 4, 4155-4158) to obtain glycine containing Cyclen monomer linkage .

c. The product obtained in step b is coupled with curcumin under the conditions of coupling agent EDC (purchased from Shanghai Yansheng Biochemical Co., Ltd.), HOBt (purchased from Shanghai Jill Biochemical Co., Ltd.) and catalyst DMAP (mixing ratio: 4 times the amount) Glycine containing Cyclen monomer, 1x amount of curcumin, 4 times the amount of coupling agent EDC and 4 times the amount of HOBt) were reacted at room temperature for 15 hours to obtain a coupled product.

d. After the reaction of the step c, the crude product was separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether (1: 4)), and the obtained product was subjected to a cleavage reagent, trifluoroacetic acid (TFA, purchased from Shanghai Jill Biochemical Co., Ltd.). The Boc protecting group is removed to give a crude polyphenolic compound.

e. The crude polyphenolic compound was subjected to HPLC method (column column: C18 packed column (purchased from Agilent, USA); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 mL/min; detection wavelength: 380 nm; column temperature : Room temperature) Separation of pure polyphenolic compounds.

After testing, the polyphenolic compound prepared by the above method has a correct structure, and its structural formula is as shown in Formula XIII, and the purity is over 95%.

(Formula XIII)

Example 3 Preparation of polyphenolic compounds of the formula IX The synthesis of the polyphenolic compound of the formula IX of the present invention comprises the following steps: a. Trpn-containing monomer [3(3',3"-di-tert-butoxycarbonyl-3',3"-diamino)aminopropyl. Preparation of amino]acetic acid: The synthesis method is as follows: using Trpn (purchased from Acros, USA) as raw material, first reacted with Boc20 (di-tert-butyl dicarbonate, purchased from Shanghai Jill Biochemical Co., Ltd.) to obtain 3', 3"-two uncle Butoxycarbonyl-3,3',3"-triaminopropylamine (2Boc-Trpn) (See Eiichi Kimura, Shin Aoki, Tohru Koike, and Motoo Shiro, A Tris (ZnII-1, 4, 7, 10- Tetraazacyclododecane) Complex as a New Receptor for Phosphate Dianions in Aqueous Solution, Journal of American Chemical Society, 1997, Vol 119, 3068-3076), and reacting the product with ethyl bromoacetate (see Joong Won Jeon, Sang Jun Son, Chang) Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-Cleaving Catalyst Selective for Protein Substrate, Organic Letters, 2002, Vol 4, 4155-4158) [3(3', 3"-di-tert-butoxycarbonyl) -3', 3''-Diamino)aminopropylamino]acetate, finally hydrolyzed with NaOH

(See Joong Won Jeon, Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-Cleaving Catalyst Selective for Protein Substrate, Organic Letters, 2002, Vol 4, 4155-4158). Monomeric [3 (3', 3"-di-tert-butoxycarbonyl-3',3''-diamino)aminopropylamino]acetic acid.

b. coupling curcumin with a Trp-containing monomer [3 (3', 3"-di-tert-butoxycarbonyl-3', 3''-diamino)aminopropylamino]acetic acid, the coupling condition is a coupling agent EDC (purchased from Shanghai Yansheng Biochemical Company), HOBt (purchased from Shanghai Jill Biochemical Co., Ltd.) and catalyst DMAP (mixing ratio: 4 times the amount of Tripn-containing monomer, 1 times the amount of curcumin, 4 times the amount of even The mixture EDC was reacted with 4 times the amount of HOBt) at room temperature for 15 hours to obtain the objective product.

c After the reaction of the step b, the crude product was separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether (1: 4)), and the obtained product was removed with a cleavage reagent trifluoroacetic acid (TFA, purchased from Shanghai Jill Biochemical Co., Ltd.). The protecting group is obtained to obtain a crude polyphenol compound.

d. The crude polyphenolic compound was subjected to HPLC method (column column: C18 packed column (purchased from Agilent, USA); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 niL/min _; detection wavelength: 380 nm _; column temperature : Room temperature) Separation of pure polyphenolic compounds.

After testing, the polyphenolic compound prepared by the above method has a correct structure, and its structural formula is as shown in Formula IX, and the purity is over 95%.

(Formula IX)

Example 4 Preparation of a polypeptide compound of the formula XV

The synthesis of the polypeptide compound of the formula XV of the present invention comprises the following steps:

a. The amino acids (phenylalanine, phenylalanine, valine, leucine, lysine) were linked one by one according to the Fmoc solid phase synthesis strategy on Wang resin (purchased from Shanghai Jill Biochemical Co., Ltd.). It is said that the C terminal of the compound is a carboxyl group (a COOH), a Wang resin is used, and the C terminal is an amide (a CONH ₂ ) ruthenium using a Rink resin).

b. Cyclen-containing monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclodene-1-yl]B The acid is linked to the small peptide containing the Wang resin obtained in the step a by the method of linking the amino acids in the step a, and the coupling condition is the coupling agent HBTU (purchased from Shanghai Yansheng Biochemical Co., Ltd.) and HOBt (purchased from Shanghai Jill Biochemical Co., Ltd.) in the presence of Ratio: 4 times the amount of the Cyclen-containing monomer, 1 time of the small resin containing the Wang resin, 4 times the amount of the coupling agent HBTU and 4 times the amount of HOBt) were reacted at room temperature for 3 hours to obtain the objective product.

c using the cleavage reagent trifluoroacetic acid (TFA, purchased from Shanghai Jill Biochemical Co., Ltd.) used in the synthesis of the polypeptide, the polypeptide portion of the polypeptide-linked resin obtained by the reaction of the step c is cleaved off with the resin, and the protective group is removed, and filtered. The TFA was removed to obtain a crude polypeptide compound.

d. The crude peptide compound was subjected to HPLC method (column column: C18 packed column (purchased from Agilent, USA); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 mL/min _; detection wavelength: 215 nm _; column temperature: The pure peptide compound of the present invention is isolated at room temperature.

After testing, the polypeptide compound prepared by the above method has the correct structure, and its structural formula is as shown in the formula XV, and the purity is over 95%.

Example 5 Preparation of a polypeptide compound of the formula XVI

The synthesis of the polypeptide compound of the formula XVI of the present invention comprises the following steps: a. In the same manner as in Example 4, the amino acid (phenylalanine, phenylalanine, valine, leucine, lysine) , glycine) are attached one by one to Wang resin.

b. The Cyclen-containing monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclodene-1-yl]acetic acid and the Wang-containing resin obtained in the step a The small peptides are linked, and the coupling conditions are in the presence of coupling agent HBTU and HOBt (mixing ratio: 4 times the amount of Cyclen-containing monomer, 1 times the amount of small peptide containing Wang resin, 4 times the amount of coupling agent HBTU and 4 times The amount of HOBt) was reacted at room temperature for 3 hours to obtain the objective product.

c The polypeptide moiety in the polypeptide-attached resin obtained by the reaction of the step b is cleaved from the resin by the cleavage reagent trifluoroacetic acid (TFA) used in the synthesis of the polypeptide, and the protective group is removed to obtain a crude polypeptide compound.

d. The crude compound of the formula II is isolated by HPLC (isolation conditions are the same as in Example 4) to obtain a pure peptide compound.

After testing, the polypeptide compound prepared by the above method has the correct structure, and its structural formula is as shown in Formula XVI, and the purity is over 95%.

Example 6. Preparation of a polypeptide compound of the formula XVII

The synthesis of the polypeptide compound of the formula XVII of the present invention comprises the following steps:

a. The amino acids (aspartic acid, phenylalanine, valine, leucine) were each attached to Wang resin in the same manner as in Example 4.

c The polypeptide moiety in the polypeptide-attached resin obtained by the reaction of the step b is separated from the resin by the cleavage reagent trifluoroacetic acid (TFA) used in the synthesis of the polypeptide, and the protective group is removed to obtain a crude polypeptide compound.

d. The crude product was separated by HPLC (isolation conditions as in Example 4) to obtain a pure peptide compound. After testing, the polypeptide compound prepared by the above method has the correct structure, and its structural formula is as shown in Formula XVII, and the purity is over 95%.

Example 7. Preparation of a polypeptide compound of the formula XVIII

The synthesis of the polypeptide compound of the formula XVIII of the present invention comprises the following steps: a. In the same manner as in Example 4, the amino acid (phenylalanine, phenylalanine, valine, leucine, lysine) ) Connected one by one on Wang resin.

b. The Trnpn-containing monomer [3 (3''3''-di-tert-butoxycarbonyl-3''3''-diamino)aminopropylamino]acetic acid and the small Wang resin-containing resin obtained in the step a Peptide-linked, in the presence of coupling agents HBTU and HOBt

(Mixing ratio: 4 times the amount of the monomer containing Cyclen, 1 time of the small peptide containing Wang resin, 4 times the amount of the coupling agent HBTU and 4 times the amount of HOBt) were reacted at room temperature for 3 hours to obtain the objective product.

c The cleavage reagent trifluoroacetic acid (TFA) used in the synthesis of the polypeptide is cleaved from the resin in the polypeptide-attached resin obtained by the reaction of the step c, and the protective group is removed to obtain a crude polypeptide compound. d. The crude polypeptide compound was isolated by HPLC method (isolation conditions were the same as in Example 4) to obtain a pure peptide compound.

Upon examination, the polypeptide compound prepared by the above method has a correct structure, and its structural formula is as shown in Formula XVIII, and the purity is over 95%.

Example 8. Preparation of a Styryl Compound of the Formula XIX

The synthesis of the styryl compound of the formula XIX of the present invention comprises the following steps: a. Congo red (purchased from Sigma, USA) and containing. (^11 monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclotetradecyl]-acetic acid coupling, the coupling condition is the coupling agent EDC (purchased from Shanghai Prolonged biochemical company), HOBt (purchased from Shanghai Jill Biochemical Co., Ltd.) and catalytic amount DMAP (Acros, USA) in the presence of (mixing ratio: 4 times the amount of Cyclen-containing monomer, 1 times the amount of Congo red, 4 times the amount The coupling agent EDC, 4 times the amount of HOBt and a catalytic amount of DMAP) were reacted at room temperature for 15 hours to obtain the desired product.

b. After the reaction of the step b, the crude product was separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether (1: 4)), and the obtained product was subjected to a cleavage reagent, trifluoroacetic acid (TFA, purchased from Shanghai Jill Biochemical Co., Ltd.). Removal of the protecting group gives a crude styrene-based compound.

c The crude product was subjected to HPLC method (column: C18 packed column (purchased from Agilent, USA); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 mL/min; detection wavelength: 215 nm; column temperature: room temperature) separation A pure product of a styryl compound is obtained.

After testing, the styrene-based compound prepared by the above method has a correct structure, and its structural formula is as shown in the formula XIX, and the purity is over 95%.

Example 9. Preparation of Styryl Compounds of the Formula XX Structure

The synthesis of the styryl compound of the formula XX of the present invention comprises the following steps:

a. benzoic acid (purchased from Sigma, USA) and Cyclen-containing monomer [4,7,10-tri-tert-butoxycarbonyl-1,4,7,10-tetraazacyclotetradecyl]acetic acid Coupling, the connection condition is the coupling agent EDC (purchased from Shanghai Yan Long biochemical company), HOBt (purchased from Shanghai Jill Biochemical Company) and DMAP in the presence of (mixing ratio: 4 times the amount of Cyclen containing monomer, 1 times the amount of benzoic acid, 4 times the amount of coupling agent EDC and 4 times The amount of HOBt) was reacted at room temperature for 15 hours to obtain the objective product.

c The crude product was subjected to HPLC method (column: C18 packed column (purchased from Agilent, USA, please provide the type of column and its purchase); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 mL/min; detection Wavelength: 215mn; column temperature: 20 ° C) The pure product of the styryl compound was isolated.

After testing, the styrene-based compound prepared by the above method has the correct structure, and its structural formula is as shown in Formula XX, and the purity is over 95%.

(Formula XX)

Example 10 Preparation of a Styryl Compound of the Formula XXI

The synthesis of the styryl compound of the formula XXI of the present invention comprises the following steps:

a. The benzoic acid (purchased from Sigma, USA) and the monomer containing Trpn [3(3"3"-di-tert-butoxycarbonyl-3-

"3"-Diamino)aminopropylamino]acetic acid coupling, the coupling conditions were the coupling agent EDC (purchased from Shanghai Yansheng Biochemical Company), HOBt (purchased from Shanghai Jill Biochemical Company) and the presence of DMAP (mixing ratio: 4 A multiple amount of the Tpnpn-containing monomer, 1 time amount of the benzoic acid, 4 times the amount of the coupling agent EDC and 4 times the amount of HOBt) were reacted at room temperature for 15 hours to obtain the objective product.

b. After the reaction of step b, the crude product is separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether)

(1: 4)), the obtained product was subjected to removal of a protective group by a cleavage reagent trifluoroacetic acid (TFA, purchased from Shanghai Jill Biochemical Co., Ltd.) to obtain a crude styrene-based compound.

c The crude styrene-based compound was subjected to HPLC method (column: C18 packed column (purchased from Agilent, USA, please provide the type of column and its purchase); mobile phase: acetonitrile/water mixed system; flow rate - 2.8mL /min _; detection wavelength: 215 nm; column temperature: room temperature) The pure product of the styryl compound was isolated.

After testing, the styrene-based compound prepared by the above method has a correct structure, and its structural formula is as shown in the formula XXI, and the purity is over 95%.

(Formula XXI) Example 11 Preparation of a Styryl Compound of the Formula XXII

The synthesis of the stupid vinyl compound of the formula XXII of the present invention comprises the following steps _: a. Glycine methyl ester (purchased from Shanghai Jill Biochemical Co., Ltd.) and Trpn-containing monomer [3(3''3''-di-tert-butoxycarbonyl-3''3''-diamino)aminopropylamino] Acetic acid (according to the embodiment 3a) coupling, the connection conditions are the coupling agent HBTU (purchased from Shanghai Yansheng Biochemical Company), HOBt (purchased from Shanghai Jill Biochemical Company), 1 times the amount of TRP-containing monomer, 1 time The amount of glycine methyl ester, 1 time amount of coupling agent HBTU and 1 time amount of HOBt were reacted at room temperature for 12 hours to obtain a coupled product.

b. After the reaction of the step a, the crude product is separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether (1: 4)) to obtain a glycine methyl ester containing a Trpn monomer and then hydrolyzed with NaOH (see Joong Won). Jeon,

Sang Jun Son, Chang Eun Yoo, In Seok Hong, Jung Bae Song, and Junghun Suh, Protein-Cleaving Catalyst Selective for Protein Substrate, Organic Letters, 2002, Vol 4, 4155-4158) A glycine containing a Trpn monomer linkage is obtained.

c. Coupling the product obtained in step b with benzoic acid under the conditions of coupling agent EDC (purchased from Shanghai Yansheng Biochemical Co., Ltd.), HOBt (purchased from Shanghai Jill Biochemical Co., Ltd.) and catalyst DMAP (mixing ratio: 4 times the amount) The Tpn-containing monomer, 1 time amount of benzoic acid, 4 times the amount of coupling agent EDC and 4 times the amount of HOBt) were reacted at room temperature for 15 hours to obtain a coupled product.

d. After the reaction of step c, the crude product is separated by a silica gel column (column separation conditions: ethyl acetate: petroleum ether)

e. The crude styrene-based compound was subjected to HPLC method (column: C18 packed column (purchased from Agilent, USA, please provide the type of column and its purchase); mobile phase: acetonitrile/water mixed system; flow rate: 2.8 mL/min; detection wavelength: 215nm; column temperature: room temperature) Separation of pure styrene-based compounds.

After testing, the styrene-based compound prepared by the above method has the correct structure, and its structural formula is as shown in formula XXII, and the purity is over 95%.

(Formula XXII) Example 12. ThT fluorescence detection of polyphenolic compounds for inhibition of Αβ aggregation and depolymerization of Αβ aggregates

Αβ is aggregated into fibers under near physiological conditions in vitro. The fibers can be combined with Thioflavin T (ThT) to exhibit specific fluorescence at an excitation wavelength of 440 nm and an emission wavelength of 485 nm. The intensity can quantitatively reflect the number of fibers, so that the quantitative determination of the degree of Αβ aggregation can be achieved. The ThT fluorescence method is now used to detect the inhibition of Αβ aggregation by the polyphenolic compounds provided by the present invention and the depolymerization of Αβ aggregates. The experimental methods are as follows:

Αβ42 stock solution preparation: Add hexafluoroisopropanol (HFIP, Acros, USA) to Αβ42 (purchased from American Peptide Company), make the peptide concentration Img/mL, shake gently for 12 hours at room temperature, blow dry with nitrogen, add The 200 μΜ & ΟΗ solution was prepared into a ΙΟΟμΜ Αβ42 stock solution and stored in a -80 ° C refrigerator. Preparation of polyphenolic compound stock solution: The compounds synthesized in Examples 1, 2, and 3 were separately added to PBS with pH 7.4 (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g). Prepare a 1.92 mM stock solution by adding water to 1000 mL) and store in a -80 °C freezer.

I. Inhibition of the inhibition of Αβ aggregation by the polyphenolic compounds of the invention

Take the 42β42 stock solution, the above polyphenolic compound stock solution and copper chloride to prepare a solution containing 20 μΜ Αβ42, 0 to 160 μΜ of the above polyphenolic compound and 0.4 to 128 μΜ Ο ι ²⁺ , adjusted to pH 7.4 at 37 ° C After 3 days of incubation, the Αβ42 stock solution incubated under the same conditions was used as a control. The ThT measurement method was as follows: 20 ul of the incubation sample solution was placed in a 700 ul ΙΟμΜ ThT (pH 7.4) solution, and the mixture was uniformly mixed, and the fluorescence absorption at an excitation wavelength of 440 nm and an emission wavelength of 485 nm was measured.

The detection result is shown in FIG. 1 , and the degree of aggregation of the blank sample of Αβ42 is 100%, and is added to the embodiment of the present invention.

The aggregation of Αβ after 1, 2, 3 synthesized compounds was significantly inhibited (see curve I in Figure 1 (compound prepared in Example 1); curve 11 (compound prepared in Example 2); curve ΙΠ (prepared in Example 3) The compound)), demonstrating that the polyphenolic compound provided by the present invention can significantly inhibit the aggregation of Aβ.

2. Detection of the depolymerization of Αβ aggregates by the polyphenolic compounds of the present invention

The β42 stock solution was taken, diluted to a solution containing 20 μM Αβ42, adjusted to pH 7.4, and then incubated at 37 °C. Two days later, ThT fluorescence and electron microscopy (TEM) were performed on the incubation samples, and it was found that Αβ42 was aggregated, and then the polyphenol compound stock solution and copper chloride synthesized by the present invention were separately added to prepare 20, 40, 80 μm, respectively. The phenolic compound and the solution of 16, 32, 64 μΜ Ο ι ²⁺ (ρΗ 7.4) were further incubated at 37 ° C for 8 days, and the ThT fluorescence absorption of the Αβ42 solution was measured in the same manner as in the first step, and the continuous detection was carried out for 8 days. .

Among them, different concentrations of the compound prepared in Example 1 for the depolymerization of Αβ aggregates are shown in Figure 2 as curve II (20 μΜ polyphenolic compound), III (40 μΜ polyphenolic compound), IV (80 μΜ polyphenol). The compound of the formula) indicates that the polyphenolic compound of the present invention has a good depolymerization effect on the aggregated Αβ42 aggregate (curve I). In addition, the compounds synthesized in Examples 2 and 3 also have a good depolymerization effect on the aggregated Αβ42 aggregates, and it is proved that the polyphenolic compounds of the present invention also have a good depolymerization effect on the Αβ42 aggregates in which bismuth is aggregated.

Example 13. ThT fluorescence detection of the inhibition of Αβ aggregation by the polypeptide compounds of the present invention and the depolymerization of Αβ aggregates

Αβ will aggregate into fibers under near physiological conditions in vitro. The fibers can be combined with thio yellow pigment TXThioflavin T, ThT). At excitation wavelength 440nm, emission wavelength (emission) 485nm, specific fluorescence, fluorescence intensity can be Quantitatively reflects the number of fibers, so that the degree of aggregation of Αβ can be quantitatively determined. The ThT fluorescence method is now used to detect the inhibition of Αβ aggregation by the polypeptide compounds of the present invention and the depolymerization of Αβ aggregates. The experimental method is as follows - formulating Αβ42 stock solution: adding hexafluoroiso to Αβ42 (purchased from American Peptide Company) Propanol (HFIP, purchased from Acros, USA), the peptide concentration was 1 mg/mL, shaken at room temperature for 12 hours, blown dry with nitrogen, and added to a solution of ΜΝμΜ Αβ42, which was placed in a -80 Ό refrigerator. save.

Formulating a stock solution of the polypeptide compound of the present invention: dividing the polypeptide compound synthesized in Examples 4, 5, 6, and 7 2985 was mixed with pH 7.4 PBS (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, added water to 1000 mL, pH 7.4) to prepare a stock solution of 1.92 mM peptide compound. Store in a -80 ° C refrigerator.

I. Inhibition of the inhibition of Αβ aggregation by the compounds of the present invention

Taking the 42β42 stock solution, the above-mentioned polypeptide compound stock solution and copper chloride were formulated into a solution containing 20 uM Αβ42, 0 to 160 uM of the above polypeptide compound and 0.4 to 128 μΜ Cu ²⁺ , adjusted to pH 7.4, and incubated at 37 ° C. For 3 days, the Αβ42 stock solution incubated under the same conditions was used as a control. The ThT measurement method is as follows: 20 ul of the incubation sample solution is placed in a 700 ul lOuM ThT (pH 7.4) solution, and the mixture is homogenized to measure the fluorescence absorption at an excitation wavelength of 440 nm and an emission wavelength of 485 nm.

As shown in Fig. 3, the degree of aggregation with the blank sample of Αβ42 was 100%, and the aggregation of Αβ was significantly inhibited by the addition of the polypeptide compound of the present invention synthesized in Examples 4, 5, 6, and 7 (see the curve in Fig. 3). I (polypeptide compound prepared in Example 1), II (polypeptide compound prepared in Example 5), III (polypeptide compound prepared in Example 6), IV (polypeptide compound prepared in Example 7), demonstrating the polypeptide of the present invention The compounds can significantly inhibit the aggregation of Aβ.

2. Detection of the depolymerization of Αβ aggregates by compounds

The β42 stock solution was taken, diluted to a solution containing 20 uM Αβ42, adjusted to pH 7.4, and then incubated at 37 °C. Two days later, ThT fluorescence and electron microscopy (TEM) were performed on the incubation samples, and it was found that Αβ42 was aggregated, and then the peptide compound compound solution and copper chloride synthesized by the present invention were separately added to prepare polypeptides containing 20, 40, and 80 μM, respectively. The compound and the solution of 16, 32, 64 μΜ Ο ι ²⁺ (ρΗ 7.4) were further incubated at 37 ° C for 10 days, and the ThT fluorescence absorption of the Αβ42 solution was determined by the same method as in the first step, and the detection was continued until 10 day.

Wherein, the detection results of the polypeptide compounds prepared in different concentrations in Example 4 are as shown in the curve II (20 μΜ polypeptide compound), the curve III (40 μΜ polypeptide compound), and the curve IV (80 μΜ polypeptide compound) in FIG. 4, indicating The polypeptide compounds of the invention have a good depolymerization effect on the aggregated Αβ42 aggregates (curve I). In addition, different concentrations of the polypeptide compounds synthesized in Examples 5, 6, and 7 also have a good depolymerization effect on the aggregated Aβ42 aggregates, demonstrating that the polypeptide compounds of the present invention also have aggregated Aβ42 aggregates. Very good depolymerization.

Example 14. ThT fluorescence method for the inhibition of Αβ aggregation by the styryl compounds of the present invention and the depolymerization of Αβ aggregates

Αβ is aggregated into fibers under near physiological conditions in vitro. The fibers can be combined with Thioflavin T (ThT) to exhibit specific fluorescence at an excitation wavelength of 440 nm and an emission wavelength of 485 nm. The intensity can quantitatively reflect the number of fibers, so that the quantitative determination of the degree of Αβ aggregation can be achieved. The inhibitory effect of the compound of the present invention on Αβ aggregation and the depolymerization of Αβ aggregates by ΊΊΪΓfluorescence method are now examined. The experimental methods are as follows:

Αβ42 stock solution preparation: Add hexafluoroisopropanol (HFIP) to Αβ42 (purchased from American Peptide Company), make the peptide concentration lmg/mL, shake gently for 12 hours at room temperature, blow dry with nitrogen, add 200μΜΝ & ΟΗ solution, prepare A 100 μΜΑ β42 stock solution was stored in a -80 ° C refrigerator.

Styrene-based compound stock preparation: The styryl groups synthesized in Examples 8, 9, 10, and 11 were combined. Add PBS (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, add water to 1000 mL) of pH 7.4 to prepare a 1.92 mM stock solution, and place it in a refrigerator at -80 °C. Saved in. ·

I. Inhibition of the inhibition of Αβ aggregation by the styryl compounds of the present invention

Taking the 42β42 stock solution, the above styrene-based compound stock solution and copper chloride are formulated into a solution containing 20 ΜΑαΜΑβ42, 0 to 160 uM of the above styryl compound and 0.4 to 128 μΜ Cu ²⁺ to adjust the pH to 7.4 at 37 ° C. The cells were incubated for 3 days under the same conditions, and the Αβ42 stock solution incubated under the same conditions was used as a control. The ThT measurement method is as follows: 20 ul of the incubation sample solution is placed in a 700 ul lOuM ThT (pH 7.4) solution, and the mixture is homogenized to measure the fluorescence absorption at an excitation wavelength of 440 nm and an emission wavelength of 485 nm.

As shown in Fig. 5, the degree of aggregation of the blank sample of Αβ42 was 100%, and the aggregation of Αβ was significantly inhibited after the addition of the styryl compound of the present invention in Examples 8, 9, 10, 11 (see curve I in Fig. 5). (Compound prepared in Example 8; curve II (compound prepared in Example 9); curve III (compound prepared in Example 10); curve IV (compound prepared in Example 11)), demonstrating the styryl group of the present invention The compound significantly inhibits the aggregation of Aβ.

2. Detection of the depolymerization of Αβ aggregates by compounds

The β42 stock solution was taken, diluted to a solution containing 20 μM Αβ42, adjusted to pH 7.4, and then incubated at 37 °C. Two days later, ThT fluorescence and electron microscopy (TEM) were performed on the incubation samples, and it was found that Αβ42 was aggregated, and then the styrene-based compound storage solution and copper chloride synthesized by the present invention were separately added to prepare 20, 40, 80 μ, respectively. The styryl compound and the solution of 20, 32, 64 μΜ Cu ²⁺ (pH 7.4) were further incubated at 37 ° C for 8 days, and the ThT fluorescence absorption of the Αβ42 solution was determined by the same method as in the first step, and the continuous detection was performed. 8 days.

Among them, different concentrations of the styryl compound prepared in Example 9 for the depolymerization of Αβ aggregates are shown in Fig. 6 as curve II (20 μΜ styryl compound), III (40 μΜ styryl compound), ΐν (80 μΜ styryl compound), indicating that the styryl compound of the present invention has a good depolymerization effect on the aggregated Αβ42 aggregate (curve I). In addition, the styryl compounds synthesized in Examples 8, 10, and 11 also have a good depolymerization effect on the agglomerated Αβ42 aggregates, demonstrating that the styrene-based compounds of the present invention have aggregated Αβ42 aggregates. There is also a good depolymerization effect.

Example 15. Electron microscopic observation of the inhibitory effect of compounds on Αβ aggregation and depolymerization of Αβ aggregates Αβ aggregates can be observed under electron microscopy. The morphology of Αβ42 samples on copper network is observed directly by electron microscopy. To further detect the inhibitory effect of the polypeptide compound of the present invention on Αβ aggregation and the depolymerization of Αβ aggregates, the detection method is as follows:

Peptide compound stock solution: The polypeptide compounds synthesized in Examples 4, 5, 6, and 7 were separately added to PBS pH 7.4 (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, Add 1.92 mM stock solution to water (100 mL) and store in a -80 ° C freezer.

1. Electron microscopic observation of the inhibitory effect of compounds on Αβ aggregation

Take Αβ42 stock solution (see Example 12 for formulation), store the peptide compound, and prepare copper chloride solution containing 20μΜΑβ42, 40μΜ peptide compound and 32μΜ Cu ²⁺ to adjust the pH to 7.4 at 37 °C. After 7 days of incubation as a sample solution, the Αβ42 stock solution incubated under the same conditions was used as a control; then the sample solution was taken 8 μl. Place on a copper wire coated with carbon film, let stand for 1 minute, use a filter paper to remove excess liquid, wash the copper mesh twice with water, wait until the copper mesh is slightly dry, add ΙΟμΙ 1-2% phosphotungstic acid dyed, and let stand 1 Minutes, the liquid was removed by filter paper, and the copper mesh was placed in a drier for 12-24 hours. Finally, the copper mesh of the loaded sample was placed under an electron microscope, and the sample morphology was observed under the conditions of a voltage of 100 kV and a magnification of 50,000.

Among them, the electron microscope observation results of Αβ42 and the polypeptide compound prepared in Example 4 after 3 days of incubation are shown in Fig. 7

In the B map (scale: lOOnm), compared with the control (Fig. 7 in Fig. 7), the polypeptide compound significantly inhibited the aggregation of Αβ. Further, the polypeptide compounds synthesized in Examples 5, 6, and 7 also significantly inhibited the aggregation of Aβ, indicating that the polypeptide compound of the present invention can significantly inhibit the aggregation of Aβ.

For the polyphenols and styryl compounds synthesized in the present invention, the above method can also be employed to observe the inhibitory effect of the compound on Αβ aggregation. The results of electron microscopic observation of the polyphenol compound synthesized in Example 1 are shown in Fig. 9; the results of electron microscopic observation of the styryl compound synthesized in Example 9 are shown in Fig. 10. As can be seen from the figure, the polyphenols and styryl compounds synthesized by the present invention can significantly inhibit the aggregation of Αβ.

2. Electron microscopic observation of the depolymerization of Αβ aggregates by compounds

The β42 stock solution was taken, diluted to a solution containing 20 μM β42, adjusted to 7.4, and then incubated at 37 °C. Two days later, ThT fluorescence and electron microscopy were performed on the incubation samples, and it was found that Αβ42 was aggregated, and then a solution of the polypeptide compound and copper chloride were separately added to prepare a solution containing 40 μΜ of the polypeptide compound and 32 μΜ of Cu ²⁺ (pH 7.4). Incubation was continued for 5 days at 37 ° C, and electron microscopic observation was carried out, and Αβ42 incubated under the same conditions was used as a control.

The SEM observation results of the polypeptide compound prepared in Example 4 after incubation for 2 days after Αβ42 incubation for 2 days are shown in Fig. 8 (scale: lOOnm), and the control is shown in Fig. 8, which indicates the polypeptide. The compounds have a good depolymerization effect on the aggregated Αβ aggregates, and the samples in which the peptide compounds function are fundamentally changed in morphology from the Αβ aggregates. In addition, the polypeptide compounds synthesized in Examples 5, 6, and 7 also have good depolymerization effect on the aggregated Αβ aggregates, indicating that the polypeptide compounds prepared by the present invention have good Αβ aggregates which have accumulated. Depolymerization.

Example 16. MALDI-TOF-MS analysis of the hydrolysis of Αβ by the compounds of the present invention

The Αβ and its aggregates are hydrolyzed into small fragments by the action of the polyphenols or polypeptide compounds of the present invention, thereby achieving the action of removing Αβ. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is used to analyze the Αβ hydrolysis and removal. The polyphenols are used as an example. The specific methods are as follows:

Preparation of polyphenolic compound stock solution: The polyphenolic compounds synthesized in Examples 1, 2, and 3 were separately added to pH 7.4; PBS (Na ₂ HP0 ₄ -123⁄40 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, Add 1.92 mM stock solution to water (100 mL) and store in a -80 ° C freezer.

Take Αβ42 stock solution (see Example 12 for formulation), prepare polyphenol compound stock solution and copper chloride to prepare a solution containing 20μΜ Αβ42, 160μΜ polyphenolic compound and 128μΜ Cu ²⁺ to adjust the pH to 7.4 at 37 °C. Incubate for 5 days as a sample solution. 20 μL of the sample solution was placed in a small centrifuge tube, and desalted by a Ziptip C18 purification column (Eppendorf), and then eluted with 80% acetonitrile, followed by MALDI-TOF-MS analysis.

The results of MALDI-TOF-MS analysis of the polyphenolic compound synthesized in Example 1 are shown in Fig. 11, and the polyphenol compound synthesized in Example 1 (see Figure B in Fig. 11) was used for the treatment of Αβ42 in the sample after 5 days. Minute The ion peak disappears and the small fragment molecular ion peak appears and enhances, indicating that the polyphenolic compound of the present invention is indeed

Αβ acts as a hydrolysis scavenging agent, whereas Αβ42 without polyphenolic compounds in the control group has a distinct molecular ion front (see Figure 中 in Figure 11). Further, the polyphenol compounds synthesized in Examples 2 and 3 can also have a hydrolysis scavenging action on Αβ, and it is proved that the polyphenol compound of the present invention has a hydrolysis scavenging effect on Αβ.

The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to analyze the hydrolysis and scavenging effects of peptide compounds on Αβ and its aggregates. The results are the same as above. Among them, the peptide compounds synthesized in Example 5 are used to remove Αβ hydrolysis. The results of the MALDI-TOF-MS analysis of the effect are shown in the diagram of Fig. 12; indicating that the polyphenols and polypeptide compounds prepared by the present invention have a hydrolysis scavenging effect on both Αβ and its aggregates.

Example 17. TCEP-DTNB method for detecting the inhibitory effect of the compound of the present invention on the production of hydrogen peroxide by Αβ. Αβ can form hydrogen peroxide (Η ₂ 0 ₂ ) mediated by copper. Tris-(2-carboxyethyl)phosphine, hydrochloride

(tris(2-carboxyethyl)phosphine hydrochloride, TCEP, purchased from Alfa Aesar, USA) can be quantitatively reacted with hydrogen peroxide. Unreacted TCEP can be combined with probe 5,5'-bisthio-(2-nitrobenzene). (5,5,-dithiobis (2-nitrobenzoic acid, DTNB, purchased from Sigma, USA) reacts to produce 2-nitro-5-thiobenzoate (NTB), NTB at wavelength There is UV-visible absorption near 410nm. Therefore, by measuring the UV absorption intensity, the concentration of hydrogen peroxide generated by Αβ can be quantitatively reflected. The stronger the UV absorption, the more the DTNB reacts with TCEP, which means the reaction with hydrogen peroxide. The smaller the TCEP, the less the hydrogen peroxide produced by Αβ is. The TCEP-DTNB method is used to detect the inhibition of hydrogen peroxide by 本β by the compound of the present invention. Taking polyphenols as an example, the experimental methods are as follows:

TCEP stock solution preparation: TCEP was dissolved in PBS (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, added with water to 1000 mL) at pH 7.4 to obtain a jlO mM stock solution.

DTNB stock solution preparation: DTNB was dissolved in PBS (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, added with water to 1000 mL) at pH 7.4 to obtain a 10 mM stock solution.

Gly-Cu stock solution configuration: 10 mM CuCl ₂ and 60 mMGly (purchased from Gil Biochemical) were dissolved in water and shaken at room temperature for 24 hours.

The mixture of polyphenols synthesized in Examples 1, 2, and 3 containing ΙΟμΜΑβ, ΙμΜ Gly-Cu solution, 50 μΜ ΤΌΕΡ and 50 μΜ was prepared, incubated at 37 60 for 60 min, and then 50 μM DTNB was added to measure the UV absorption at 412 nm. A mixture of catalase (Catalase, purchased from Sigma) was used as a control (hydrogen peroxide which can be decomposed by catalase, which is basically considered to be a blank system of hydrogen peroxide).

The test results are shown in Fig. 13 (the ordinate indicates the relative amount of hydrogen peroxide; the abscissa indicates different experimental groups, II is the polyphenol compound prepared in Example 1, 12 is the polyphenol compound prepared in Example 2, 13 is The polyphenol compound prepared in Example 3, the Αβ group is a control group to which the polyphenol compound of the present invention is not added, and compared with the control group, the polyphenol compound of the present invention can significantly inhibit the catalytic generation of hydrogen peroxide by Aβ. Thereby weakening the toxic effect of Aβ.

For the polypeptide compound and the styrene compound, the results are the same as above. The detection results of the inhibitory effect of the polypeptide compound synthesized by the present invention on the production of hydrogen peroxide by Aβ are shown in FIG. 14, wherein II is the polypeptide compound prepared in Example 4, and 12 is the polypeptide compound prepared in Example 5, 13 For the polypeptide compound prepared in Example 6, 14 is the polypeptide compound prepared in Example 7, and the Αβ group is not added with the styrene of the present invention. Control group of compounds). The results of the detection of hydrogen peroxide by the styrene compound synthesized by the present invention for Αβ are shown in Fig. 15, wherein II is the styryl compound prepared in Example 8, and 12 is the styrene group prepared in Example 9. The compound, 13 is a styrene-based compound prepared in Example 10, 14 is a styrene-based compound prepared in Example 11, and the Αβ-group is a control group to which no styry-based compound of the present invention is added. Compared with the control group, it was shown that the polypeptides and styrene compounds prepared by the present invention can significantly inhibit the catalytic generation of hydrogen peroxide by Aβ, thereby weakening the toxic effect of Aβ.

Example 18. ΜΤΤ (thiazole blue) method for detecting the protective effect of the compound of the present invention on nerve cells The guanidine method is a tetramethylazozolium salt microenzyme reaction colorimetric method. Rhodium is a thiazole salt, chemical name 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide, and the aqueous solution is yellow-orange. Rat hippocampal neuron cells undergo proliferation and activation after ConA action, and their intracellular mitochondrial succinate dehydrogenase activity increases accordingly. MTT acts as a substrate to participate in the reaction, forming blue formazan particles deposited on cells. Inside or around the cells, after dissolving in DMSO, it is a blue-violet solution. The OD value of the cell culture can be measured by an enzyme labeling instrument at a wavelength of 570 nm. The degree of cell proliferation in the reaction system was calculated based on the magnitude of the OD value. The protective effect of the compound of the present invention on nerve cells is now detected by the MTT method, and the structural compound of the formula I is taken as an example. The experimental method is as follows - preparation of hippocampal neuron cells: rat neurons refer to the literature (Isabella A. Graef, Paul G .

Mermelstein, Kryn Stankunas, Joel R. Neilson, Karl Deisseroth, Richard W. Tsien, Gerald R. Crabtree, L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons, Nature, 1999, Vol 401 Prepared by the method in 703-708).

Preparation of Compound Stock Solution: The compounds synthesized in Examples 1, 2, and 3 were separately added to PBS (Na ₂ HP0 ₄ -12H ₂ 0 3.73 g, KH ₂ P0 ₄ 0.43 g, NaCl 7.2 g, added with water to 1000 mL) at pH 7.4. Prepare a stock solution of 1.92 mM and store in a -80 ° C freezer.

Take the 42β42 stock solution (see Example 12 for formulation), prepare the polyphenolic compound stock solution and copper chloride of the present invention to prepare a solution containing 40 μΜΑβ42, 40 μΜ of the polyphenolic compound solution of the present invention and 32 μΜ Cu ²⁺ to adjust the pH to 7.4. It was incubated at 37 ° C for 3 days as a sample solution. Then, the cells were diluted with the cell culture medium to a cultured neuron cell at a ratio of 1:4, and the neuron cells were further cultured at 37 ° C, 5% CO ₂ for two days, and then purchased according to the MTT product specification (purchased from the United States). Sigma) Performs MTT experiments.

The test results are shown in Fig. 16 (the ordinate indicates the cell survival rate, and the abscissa indicates the different experimental groups, wherein II is the polyphenol compound prepared in Example 1, 12 is the polyphenol compound prepared in Example 2, and 13 is the preparation of Example 3. The polyphenol compound, the Αβ group is a control group in which the polyphenol compound of the present invention is not added, the blank group is a blank control group in which the polyphenol compound of the present invention and the Αβ42 stock solution are not added, and the polyphenols of the present invention are not added. Compared with the control group of the compound, the polyphenol compound synthesized by the present invention can significantly inhibit the cytotoxicity of Aβ.

For the polypeptides and styrenic compounds synthesized in the present invention, the results are the same as above. The results of the detection of neuropeptide protection by the polypeptide compound are shown in FIG. 17, wherein the ruthenium group is the polypeptide compound prepared in Example 4, the 12 groups are the polypeptide compound prepared in Example 5, and the 13 groups are the embodiment 6. The prepared polypeptide compound, 14 groups were the polypeptide compounds prepared in Example 7, the Αβ group was the control group without the addition of the polypeptide compound of the present invention, and the blank group was the blank control of the polypeptide compound of the present invention and the Αβ42 stock solution. group). The results of the detection of cytoprotective effects of styrene compounds are shown in Fig. 18, wherein II is prepared in Example 8. Styrene based compound, 12 is the styryl compound prepared in Example 9, 13 is the styryl compound prepared in Example 10, 14 is the styryl compound prepared in Example 11, and the Αβ group is not In the control group to which the styryl compound of the present invention was added, the blank group was a blank control group in which the styryl compound and the Αβ42 stock solution of the present invention were not added. Compared with the control group, it was shown that the polypeptides and styrene compounds prepared by the present invention can significantly inhibit the cytotoxicity of Aβ, thereby protecting the nerve cells and making the cell survival rate higher. .

Example 19, toxicity test '

1) General toxicity test

Cat and rat test models (purchased from the Academy of Military Medical Sciences) were used, and cats (24, female, male and half, weighing 2.4-3.7 kg, divided into 4 groups, 6 in each group, with doses of 0.05, 50, respectively). , 120mg/kg) test, observe the effects of the peptide compounds prepared in Examples 4-7 on blood pressure, respiratory rate, heart rate, etc.; using mice (40, female, male and half, weighing 18-20g, divided into 4 groups, Each group of 10, with doses of 0.05, 30, 50 mg/kg, respectively, was tested to observe the effect of the polypeptide compounds prepared in Examples 4-7 on the spontaneous activity of rats. The test results showed that the three doses of the polypeptide compound of the present invention had no significant effect on blood pressure, respiratory rate and amplitude, heart rate and heart rhythm of the cat, and had no significant effect on the number of spontaneous activities of the rats. The above general toxicity test was carried out by using the polyphenols and styrene compounds synthesized by the present invention under the same experimental conditions as above, and the results were identical to those of the polypeptide compounds, indicating that the polyphenols and styrene compounds synthesized by the present invention exert blood pressure on the cats. There was no significant effect on respiratory rate and amplitude, heart rate, and heart rate, and there was no significant effect on the number of spontaneous activities of rats.

2) Acute toxicity test

The polyphenol compound prepared in Example 1 of the present invention, the polypeptide compound prepared in Example 4, and the styryl compound prepared in Example 8 were tested for acute toxicity by the following method: The experimental model (purchased from the Academy of Military Medical Sciences), Kunming mice (40, divided into blank control group and the preparation group of the present invention, 20 in each group), the maximum oral administration rate of 10.63 g/kg , equivalent to 2834 times the clinical dose (3.75mg/kg), observed for 28 days, including the weight gain and loss rate, activity and death of the mice, the test results are shown in Table 1:

Table 1 Results of acute toxicity test of the compound of the present invention on mice

The results showed that the maximum dose of oral administration was 10.63 g/lg, which was equivalent to 2834 times of the clinical dose. However, no acute toxicity occurred in the test animals. Only male mice showed a higher growth rate than female mice. , 07 002985 illustrates that the polyphenols, polypeptides and styryl compounds provided by the present invention are less toxic.

3) Long-term toxicity test

Using a rat model, SPF-grade Wistar rats (purchased from the Academy of Military Medical Sciences, weighing 90-100 g) were divided into a blank control group and three dose groups of the polypeptide compounds of the present invention prepared in Examples 1-4 (93.75, 187.5 and 375mg/kg), a total of 13 groups, 44 rats in each group, the blank control group was given the same dose of normal saline, once daily intragastric administration, even for 6 months, the observation indicators include the general physiological indicators of the test animals and Peripheral blood phase, blood biochemistry, liver function, renal function and other major organ index and histopathological changes, the test results show that the rats orally administered different doses (93.75, 187.5 and 375 mg / kg) of the polypeptide compounds of the present invention 25, 50, and 100 times of clinical drug use, continuous administration for 6 months, no abnormalities of various indicators, no significant difference between different dose groups (p>0.05), indicating long-term use of the polypeptide of the present invention The compound is safe and has no cumulative effect of toxicity. For the polyphenols and styrenic compounds synthesized in the present invention, the test results obtained under the same experimental conditions are the same as above, indicating that the polyphenols and styryl compounds of the present invention are safe and have no cumulative effect of toxicity.

Industrial application

The present invention provides a class of compounds having nitrogen-containing heterocyclic modifications, including polyphenolic compounds, polypeptide-based compounds, and styryl-based compounds. Studies have shown that this type of compound not only inhibits the aggregation of Aβ, but also depolymerizes the aggregated Aβ aggregates, and finally hydrolyzes and removes Aβ, inhibiting and eliminating the toxicity of Aβ. A drug containing such a compound as an active ingredient can be used for the treatment and prevention of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes.

Claims

Rights request

1. A compound having a nitrogen-containing heterocyclic ring modification capable of degrading and removing Αβ.

The compound according to claim 1, wherein the compound is a polyphenol compound having a nitrogen-containing heterocyclic ring modification, and has a structural formula of the formula I:

(Formula I) wherein: any nitrogen-containing heterocycloalkaneamino group or 4 or more nitrogen-containing acyclic hydrocarbylamino groups; η = 1-5 _{; 1} is any natural, unnatural amino acid residue or corresponding Amino acid residue with a modifying group, m=0-5; ¥ ₁ is any polyphenol.

3. A compound according to claim 2, characterized in that: said Cyclen or Trpn.

4. A compound according to claim 2 wherein: Is one of the 20 natural amino acid residues or the corresponding amino acid residue with a modifying group, m = 0-3.

The compound according to claim 4, wherein the modifying group is a methyl group; the glycine residue, the leucine residue, the isoleucine, the alanine residue, and the hydrazine; A residue, a phenylalanine residue or a tyrosine residue.

6. A compound according to claim 2 wherein: said Cur, NDGA or RA.

The compound according to claim 1, wherein the compound is a polypeptide compound having a nitrogen-containing heterocyclic modification, and has a structural formula of the formula II -

R ₂ (CH ₂ ) _n (X ₂ ) _m (Formula II) wherein R ₂ is any nitrogen-containing heterocycloalkaneamino group or acyclic a hydrocarbylene amino group having 4 or more nitrogen atoms; n=1-5 _; X ₂ is any natural or non-natural amino acid residue corresponding modifying group having an amino acid residue, m = 2-10; C-terminus of the polypeptide or compound is an amido group or a carboxyl group is reduced after Hydroxyl, amino, or hospital base.

The compound according to claim 7, wherein the R ₂ is Cyclen or Trpn.

9. The compound according to claim 7, wherein: X ₂ is one of 20 natural amino acid residues or a corresponding amino acid residue having a modifying group, m = 3-5.

10. The compound according to claim 9, wherein: the modifying group is a methyl group; the X ₂ is a lysine residue, a leucine residue, a proline residue, a phenylalanine An acid residue, a tyrosine residue or an aspartic acid residue.

The compound according to claim 1, wherein the compound is a styrene compound having a nitrogen-containing heterocyclic ring modification, and has a structural formula of the formula III:

R ₃ (CH ₂ ) _n (X ₃ ) _m Y ₃ (wherein R ₃ is any nitrogen-containing heterocyclic hydrocarbon amino group or acyclic a hydrocarbon amino group having 4 or more nitrogen atoms; n= L-5; X ₃ is any natural, unnatural amino acid residue or corresponding amino acid residue having a modifying group, m=0-5 _; Y ₃ is any compound having a styryl structure.

The compound according to claim 11, wherein: said Cyclen or Trpn.

13. The compound according to claim 11, wherein: X ₃ is 20 natural amino groups One of the acid residues or the corresponding amino acid residue with a modifying group, m=0-3.

The compound according to claim 13, wherein: the modifying group is a methyl group; and the X ₃ is a glycine residue, a leucine residue, an isoleucine, an alanine residue, Proline residue, phenylalanine residue or tyrosine residue.

The compound according to claim 11, wherein the Y ₃ is CR or BA.

16. A composite of a compound according to any of claims 1-15 and a transition metal; said transition metal being copper, cobalt, nickel, palladium, zinc or iron.

17. A salt of a compound according to any one of claims 1-15.

The salt according to claim 17, wherein the salt is a sodium salt, a potassium salt, an ammonium salt, a calcium salt, a copper salt, an iron salt, a ferrous salt, a lithium salt, a magnesium salt, a manganese salt, Aluminum salt, manganese salt, zinc salt, primary amine salt, secondary amine salt, tertiary amine salt, hydrochloride salt, phosphate salt, acetate salt, oxalate salt or tartrate salt.

A medicament for treating and preventing a neurodegenerative disease, which comprises the compound according to any one of claims 1 to 15 as an active ingredient.

The medicine according to claim 19, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or type 2 diabetes.

A medicament for treating and preventing a neurodegenerative disease by using the complex of claim 16 as an active ingredient.

The medicine according to claim 21, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or type 2 diabetes.

A medicament for treating and preventing a neurodegenerative disease by using the salt according to claim 17 or 18 as an active ingredient.

The medicine according to claim 23, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or type 2 diabetes.

25. Use of a compound according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment and prevention of a neurodegenerative disease.

26. The use according to claim 25, wherein: the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or type 2 diabetes.

27. Use of the complex of claim 16 for the manufacture of a medicament for the treatment and prevention of a neurodegenerative disease.

28. The use according to claim 27, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or type 2 diabetes.

29. Use of a salt according to claim 17 or 18 for the manufacture of a medicament for the treatment and prevention of a neurodegenerative disease.

30. The use according to claim 29, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease or type 2 diabetes.