CN111285923B

CN111285923B - PSD-95 inhibitor

Info

Publication number: CN111285923B
Application number: CN202010147529.1A
Authority: CN
Inventors: 周述靓; 王鹏; 邓岚
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2020-03-05
Filing date: 2020-03-05
Publication date: 2023-02-03
Anticipated expiration: 2040-03-05
Also published as: CN111285923A

Abstract

The invention relates to the field of medicine synthesis, and discloses a PSD-95 inhibitor. The PSD-95 inhibitor is used for preparing a pharmaceutical composition for treating diseases, and an application of the pharmaceutical composition in preparing a neuroprotective medicament after ischemic brain injury.

Description

PSD-95 inhibitor

Technical Field

The invention relates to a PSD-95 inhibitor and application thereof.

Background

Acute ischemic stroke is one of the diseases with the highest fatality rate and disability rate all over the world, and an effective treatment method is lacking clinically at present. Cerebral ischemia causes a cascade of energy deprivation to cell death, with early events mainly involving excitotoxicity and oxidative stress, and late events dominated by inflammatory responses and apoptosis. At present, a large number of basic and clinical studies are devoted to the intervention of signaling molecules associated with stroke pathology in order to develop effective neuroprotective therapies. Excitotoxicity is used as the initial link of stroke pathology and is the primary target of stroke treatment. After cerebral ischemia, excitatory glutamate is excessively released and can continuously act on N-methyl-D-aspartate (NMDA) receptors, so that a series of downstream harmful signal pathways are started, such as intracellular Ca & lt 2+ & gt overload, free radical generation and the like, and finally, nerve cells are killed. Recent studies have shown that PSD-95 proteins in the postsynaptic dense region (PSD) bridge NMDA receptors to a variety of toxic signaling molecules within cells. The PSD-95 is inhibited to lead the NMDA receptor and PSD-95 protein to be dissociated, thereby relieving excitotoxicity and not influencing the activity and synaptic function of the NMDA receptor. The inflammatory response traverses the entire pathological course of post-stroke brain injury and has a major impact on neuronal cell death and neural remodeling.

Disclosure of Invention

The invention provides a novel PSD-95 inhibitor and application thereof.

To achieve the above objects, the present invention provides, in a first aspect, a compound of structure I, a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, a prodrug based on the compound, or any mixture thereof.

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-

Ser-Ser-Ile-Glu-Ser-Asp-Val-AA1(R1)-AA2(R2)-AA3

Structure I

AA1 in structure I is Lys, or Dap, or Orn, or Dab, or Dah;

AA2 in structure I is Cys, or is absent;

AA3 in Structure I is NH ₂ Or is OH;

r1 in structure I is 2-cholesterol butanoic acid, or 2-cholesterol isobutyric acid, or 3-cholesterol butanoic acid, or 3-cholesterol isobutyric acid, 4-cholesterol butanoic acid, or 2-cholesterol pentanoic acid, or 2-cholesterol isovaleric acid, or 3-cholesterol pentanoic acid, or 5-cholesterol pentanoic acid, or 2-cholesterol hexanoic acid, or 6-cholesterol hexanoic acid, or 2-cholesterol heptanoic acid, or 7-cholesterol heptanoic acid, or 2-cholesterol octanoic acid, or 8-cholesterol octanoic acid, or CH ₃ (CH ₂ ) _n1 CO-(γGlu) _n2 -, or is absent;

wherein: n1 is an integer of 10 to 20;

n2 is an integer of 1 to 5;

n3 is an integer of 1 to 30;

n4 is an integer of 1 to 5;

n5 is an integer of 1 to 5.

R2 in structure I is 2-cholesteryl acetate, or is 2-cholesteryl propionate, or is 3-cholesteryl propionate, 2-cholesteryl butyrate, or is 2-cholesteryl isobutyrate, or is 3-cholesteryl butyrate, or is 3-cholesteryl isobutyrate, 4-cholesteryl butyrate, or is 2-cholesteryl valerate, or is 2-cholesteryl isovalerate, or is 3-cholesteryl valerate, or is 5-cholesteryl valerate, or is 2-cholesteryl hexanoate, or is 6-cholesteryl hexanoate, or is 2-cholesteryl heptanoate, or is 7-cholesteryl heptanoate, or is 2-cholesteryl octanoate, or is 8-cholesteryl octanoate, or is absent.

The invention also provides pharmaceutical compositions comprising a compound according to the invention and the use of a pharmaceutical composition comprising a compound of the invention for the preparation of a medicament for the treatment of a disease.

Preferably, the pharmaceutical composition is used for preparing neuroprotective drugs after ischemic brain injury.

Further details of the invention are set forth below, or some may be appreciated in embodiments of the invention.

Unless otherwise indicated, the quantities of the various ingredients, reaction conditions, and so forth used herein are to be construed in any case as "substantially" and "approximately". Accordingly, unless expressly stated otherwise, all numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the standard deviation found in the respective experimental conditions.

Herein, when a chemical structural formula and a chemical name of a compound are ambiguous or ambiguous, the compound is exactly defined by the chemical structural formula. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description, whether partial or complete, including similar structures as described above, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer), and any mixtures of such stereoisomers. Mixtures of these racemates and stereoisomers may also be further resolved into the enantiomers or stereoisomers of their constituent members by those skilled in the art using non-stop separation techniques or methods of chiral molecular synthesis.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above case, a single enantiomer or diastereomer, such as an optical isomer, can be obtained by asymmetric synthesis or racemate resolution. Resolution of the racemates can be accomplished by various methods, such as conventional recrystallization from resolution-assisting reagents, or by chromatographic methods. In addition, the compounds of formula I also include cis and/or trans isomers with double bonds.

The compounds of the present invention include, but are not limited to, the compounds of formula I and all of their pharmaceutically acceptable different forms. The pharmaceutically acceptable different forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above and any mixtures of these forms.

The prodrug comprises an ester or amide derivative of the compound shown as the structural formula I contained in the compound.

The compound shown in the structure I provided by the invention has stable property, is a long-acting PSD-95 inhibitor, and has a remarkable protection effect on nerves after ischemic brain injury.

Detailed Description

The invention discloses a PSD-95 inhibitor and application thereof, and a person skilled in the art can appropriately improve related parameters by referring to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the compounds and processes described herein, as well as other changes and combinations of the foregoing, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The Chinese names corresponding to the English abbreviations related in the invention are shown in the following table:

EXAMPLE 1 preparation of Compound 1

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-

Glu-Ser-Asp-Val-Lys(Pal-γGlu)-NH ₂

The preparation method comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, carrying out acidolysis on the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to sequentially insert corresponding protective amino acids or fragments in the following sequences on a carrier resin by the solid-phase coupling synthesis method to prepare the peptide resin:

in the preparation method, the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2 to 6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.

In the above production method, the substitution value of the carrier resin is 0.2 to 1.0mmol/g resin, and preferably 0.3 to 0.5mmol/g resin.

In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The deprotection time of the Fmoc protection removal is 10 to 60 minutes, and preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

The coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from one of DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate, 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3,3-tetramethylurea hexafluorophosphate, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar consumption of the condensation reagent is 1.2 to 6 times of the total molar number of the amino in the amino resin, and preferably 2.5 to 3.5 times.

The coupling reaction needs to add an activating reagent, wherein the activating reagent is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, and 1-hydroxybenzotriazole is preferred. The amount of the activating agent is 1.2 to 6 times, preferably 2.5 to 3.5 times the total molar number of the amino groups in the amino resin.

In a preferred embodiment of the present invention, the Fmoc deprotection reagent is a mixed solution of PIP/DMF (piperidine/N, N-dimethylformamide) and the mixed solution contains 10-30% (V) piperidine. The dosage of the Fmoc-removing protective reagent is 5-15 mL per gram of amino resin, and is preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acidolysis while removing the resin and side chain protecting groups to obtain a crude product:

more preferably, the acidolysis agent used in the acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL per gram of the peptide resin; preferably, 7 to 10mL of acid hydrolysis agent per gram of peptide resin is required.

The time for the cleavage with the acidolysis agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

Further, the crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure product, and the specific method comprises the following steps:

adding water into the crude product, stirring, adjusting pH value to completely dissolve, filtering the solution with 0.45 μm mixed microporous membrane, and purifying;

purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the flow rate of 77mm 250mm chromatographic column is 90mL/min, eluting by gradient system, purifying by circulating sample injection, loading the crude product solution into chromatographic column, starting mobile phase elution, collecting main peak, evaporating acetonitrile, and obtaining purified intermediate concentrated solution;

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use;

performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, and the chromatographic column for purification has 10 μm reversed phase C18, 77mm 250mm chromatographic column flow rate of 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); loading the sample into a chromatographic column by adopting a gradient elution and circulating sample loading method, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain a pure acetic acid aqueous solution, and freeze-drying to obtain a pure product.

1. Synthesis of peptide resins

Rink Amide BHHA resin is used as carrier resin, and is coupled with protected amino acid shown in the following table in sequence through Fmoc protection removal and coupling reaction to prepare peptide resin. The protected amino acids used in this example correspond to the protected amino acids shown below:

(1) 1 st protected amino acid inserted into main chain

Dissolving 0.03mol of the 1 st protected amino acid and 0.03mol of HOBt in a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.

0.01mol of Rink amide MBHA resin (substitution value: about 0.4 mmol/g) was removed by PIP/DMF 20% for 25 minutes, and washed and filtered to obtain Fmoc-removed resin.

And adding the activated 1 st protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 60-300 minutes, filtering and washing to obtain the resin containing 1 protected amino acid.

(2) 2-21 protected amino acids connected to main chain

And sequentially inoculating the corresponding 2 nd to 21 st protected amino acids by the same method for inoculating the 1 st protected amino acid of the main chain to obtain the resin containing 21 amino acids of the main chain.

(3) Side chain insertion of the 1 st protected amino acid

Taking 0.03mol of side chain 1 st protected amino acid and 0.03mol of HOBt, and dissolving with a proper amount of DMF; and adding 0.03mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.

2.5mmol of tetratriphenylphosphine palladium and 25mmol of phenylsilane are dissolved by using a proper amount of dichloromethane, deprotection is carried out for 4 hours, and the solution is filtered and washed to obtain a resin with Alloc removed for later use.

Adding the activated side chain No. 1 protected amino acid solution into the Alloc-removed resin, performing coupling reaction for 60-300 minutes, filtering and washing to obtain the side chain No. 1 protected amino acid-containing resin.

(4) Side chain insertion of the 2 nd protected amino acid

And sequentially inoculating the 2 nd protective amino acid and the mono-protective fatty acid corresponding to the side chain by adopting the same method for inoculating the 1 st protective amino acid into the main chain to obtain the peptide resin.

2. Preparation of crude product

Adding a cleavage reagent (10 mL of cleavage reagent/g of resin) with a volume ratio of TFA/water/EDT = 95/5 into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting a filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with anhydrous ether for 3 times, and drying to obtain a white-like powder which is a crude product.

3. Preparation of the pure product

Dissolving the crude product in water under stirring, filtering the solution with 0.45 μm mixed microporous membrane, and purifying. Purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is 10 μm reversed phase C18, the mobile phase system is 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the flow rate of the chromatographic column of 30mm 250mm is 20mL/min, eluting by a gradient system, purifying by circulating sample injection, sampling the crude product solution in the chromatographic column, starting the mobile phase elution, collecting the main peak, evaporating off acetonitrile, and obtaining a purified intermediate concentrated solution;

filtering the purified intermediate concentrated solution with 0.45 μm filter membrane for use, and performing salt exchange by high performance liquid chromatography with 1% acetic acid/water solution-acetonitrile as mobile phase system, and 10 μm reversed phase C18 as purification chromatographic packing, and with flow rate of 20mL/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications); adopting gradient elution and circulation sample loading method, loading sample in chromatographic column, starting mobile phase elution, collecting atlas, observing change of absorbance, collecting main peak of salt exchange and analyzing liquid phase to detect purity, combining main peak solutions of salt exchange, concentrating under reduced pressure to obtain pure acetic acid water solution, and freeze drying to obtain pure acetic acid solution with purity of 96.3% and total yield of 22.1%. Molecular weight is 3031.7 (100% M + H).

EXAMPLE 2 preparation of Compound 2

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-

Glu-Ser-Asp-Val-Lys (2-Cholesterol butanoic acid) -NH ₂

The procedure is as in example 1, using the protected amino acids as in the following table:

7.2g of pure product is obtained, the purity is 95.8 percent, and the total yield is 23.2 percent. Molecular weight is 3100.8 (100% M + H).

EXAMPLE 3 preparation of Compound 3

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-

Glu-Ser-Asp-Val-Cys (cholesteryl acetate) -NH ₂

1. Preparation of peptide resins

2. preparation of crude product

Adding a cleavage reagent (10 mL of the cleavage reagent/gram of resin) with a volume ratio of TFA: water: EDT = 95: 5 into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous diethyl ether for precipitation, washing the precipitate with anhydrous diethyl ether for 3 times, drying to obtain white-like powder, dissolving the white-like powder in pure DMSO, adding equal mol of trifluoroacetic acid solution of bromoacetic acid cholesteryl ester, adding pure diisopropylethylamine for adjusting to be alkaline, carrying out RP-HPLC tracking reaction, and obtaining a crude product solution after the reaction is finished.

3. Preparation of the pure product

The procedure was as in example 1 and was followed,

4.9g of pure product is obtained, the purity is 96.7 percent, and the total yield is 15.9 percent. Molecular weight is 3072.9 (100% M + H).

Example 4 in vivo Activity assay

In SD rats of 6-10 weeks, central cerebral artery occlusion (MCAO) focal cerebral ischemia of the rats is caused by a wire-embolism method, and the wire-embolism is pulled out 2 hours after ischemia to realize reperfusion. Reperfusion was given immediately, i.v., a single administration, and the solvent control group was given an equivalent amount of physiological saline.

1. Cerebral infarct volume

3 days after MCAO reperfusion, groups of surviving rats were dissected, hearts perfused with pre-cooled PBS, brain tissue was taken, stained with 1% red tetrazolium (TTC), and percent infarct volume was calculated.

Volume% infarct = volume infarct right/volume brain right x 100%.

Volume percent of cerebral infarction

EXAMPLE 5 determination of Primary pharmacokinetic Properties

Each compound was divided into two dosing groups: SD rats, 4 males per group, 8 in total.

Tail vein intravenous injection group: the dose is 1mg/kg, rat orbital veins are respectively bled before (0 h) and 30min, 1h, 2h, 4h, 8h, 24h, 48h, 96h and 144h after administration, and plasma samples are centrifugally separated.

Subcutaneous administration group: the dose is 1mg/kg, rat orbital veins are respectively bled before (0 h) and 1h, 2h, 3h, 4h, 8h, 24h, 48h, 96h and 144h after administration, and plasma samples are separated by centrifugation.

Plasma concentrations of the corresponding compounds were determined in plasma samples of SD rats by the combined liquid chromatography and mass spectrometry, and the half-lives of the compounds after intravenous and subcutaneous administration in SD rats (SC) were as follows:

compound (I)	t _1/2 (h)
		Compound 1	6.1
Compound 2	8.2
		Compound 3	7.5

Claims

1. A compound represented by structure I:

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val-Lys(Pal-γGlu)-NH ₂

（I）。

2. a pharmaceutically acceptable salt or solvate of a compound of claim 1.

3. Use of a compound of claim 1 for the preparation of a neuroprotective medicament to treat ischemic brain injury.