CN115475164B - Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof - Google Patents

Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof Download PDF

Info

Publication number
CN115475164B
CN115475164B CN202211008631.9A CN202211008631A CN115475164B CN 115475164 B CN115475164 B CN 115475164B CN 202211008631 A CN202211008631 A CN 202211008631A CN 115475164 B CN115475164 B CN 115475164B
Authority
CN
China
Prior art keywords
compound
protein degradation
structural formula
pdgfr
beta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202211008631.9A
Other languages
Chinese (zh)
Other versions
CN115475164A (en
Inventor
张�杰
司茹
潘晓艳
卢闻
单媛媛
王瑾
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xian Jiaotong University
Original Assignee
Xian Jiaotong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xian Jiaotong University filed Critical Xian Jiaotong University
Priority to CN202211008631.9A priority Critical patent/CN115475164B/en
Publication of CN115475164A publication Critical patent/CN115475164A/en
Application granted granted Critical
Publication of CN115475164B publication Critical patent/CN115475164B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a protein degradation targeting chimeric of degradable PDGFR-beta, a preparation method and application thereof, wherein an antitumor drug sorafenib is hydrolyzed to obtain a compound with an active reactive group carboxyl, and then the compound is connected with different types of E3 ubiquitin ligase ligands through different types of linker through amide condensation reaction to obtain a protein degradation targeting chimeric molecule. The preparation method of the protein degradation targeting chimeric agent is simple, easy to realize and high in yield, and can be used for preparing medicines for treating or preventing cancers, in particular antitumor medicines with PDGFR-beta targets.

Description

Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof
Technical Field
The invention belongs to the technical field of drug synthesis, and relates to a protein degradation targeting chimera of degradable PDGFR-beta, a preparation method and application thereof.
Background
Sorafenib (Sorafenib) is a novel multi-target antitumor drug, can act on tumor cells and tumor blood vessels simultaneously, and has dual antitumor effects: can directly inhibit the proliferation of tumor cells by blocking a cell signaling pathway mediated by RAF/MEK/ERK, can indirectly inhibit the growth of tumor cells by blocking the formation of tumor neovascularization by inhibiting vascular endothelial growth factor receptor VEGFR and platelet derived growth factor receptor PDGFR, but has serious adverse reaction in clinical treatment and is extremely easy to generate drug resistance after long-term application.
The protein degradation targeting chimeric body (Proteolysis TARGETING CHIMERA, PROTAC) is a bifunctional molecule capable of simultaneously combining a target protein and E3 ubiquitin ligase, and the target protein and the E3 ubiquitin ligase are simultaneously combined to draw the distance between the target protein and the E3 ligase, so that ubiquitination of the target protein is induced, and the ubiquitinated target protein can be recognized and degraded by a 26S proteasome, so that the aim of thoroughly removing disease-related proteins is fulfilled. Compared with small molecular inhibitors, PROTAC has the advantages of small dosage, difficult generation of drug resistance and the like, so that the development situation is vigorous in the field of new drug development.
Traditional small molecule inhibitors adopt an 'occupation driving' action mechanism, rely on tight combination with target proteins, and are easy to cause drug resistance after long-term application.
Disclosure of Invention
The invention aims to provide a protein degradation targeting chimeric body capable of degrading PDGFR-beta, a preparation method and application thereof, and the protein degradation targeting chimeric body has the function of inducing PDGFR-beta protein degradation and can be used for preparing antitumor drugs.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a protein degradation targeting chimera of degradable PDGFR- β, the chimera having the structure:
A method for preparing a protein degradation targeting chimera of a degradable PDGFR-beta as described above, comprising the steps of:
The sorafenib is hydrolyzed to obtain a compound with an active reactive group carboxyl, and then the compound with the active reactive group carboxyl is subjected to an amide condensation reaction with an E3 ubiquitin ligase ligand through a linker to obtain the protein degradation targeting chimeric of the degradable PDGFR-beta.
Further, the method comprises the following steps:
1) Under the protection of nitrogen, carrying out reflux reaction on sorafenib, sodium hydroxide and absolute ethyl alcohol to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing in an ice-water bath, adding sodium hydroxide solution, then dropwise adding tetrahydrofuran solution of di-tert-butyl dicarbonate, stirring, and obtaining a compound 2 after the reaction is finished, wherein the structural formula is as follows:
3) Dissolving the compound 2 and HATU in dry dichloromethane, dropwise adding DIPEA under ice bath condition, stirring, adding VH032, and after the reaction is finished, obtaining a compound 3 with the following structural formula:
4) Dissolving the compound 3 in ethyl acetate solution containing hydrogen chloride, and obtaining a compound 4 after the reaction is finished, wherein the structural formula is as follows:
5) Dissolving the compound 1, the compound 4 and the HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under ice bath condition, and obtaining the protein degradation targeting chimeric of the degradable PDGFR-beta after the reaction is finished, wherein the structural formula is as follows:
further, the method comprises the following steps:
1) Under the protection of nitrogen, carrying out reflux reaction on sorafenib, sodium hydroxide and absolute ethyl alcohol to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing in an ice-water bath, adding sodium hydroxide solution, then dropwise adding tetrahydrofuran solution of di-tert-butyl dicarbonate, and stirring to obtain a compound 2 with the following structural formula:
3) Dissolving the compound 2 and HATU in DMF, dropwise adding triethylamine under ice bath condition, stirring, adding lenalidomide, and reacting to obtain a compound 5 with the following structural formula:
4) Dissolving the compound 5 in ethyl acetate solution containing hydrogen chloride, and reacting to obtain a compound 6 with the following structural formula:
5) Dissolving the compound 1 and HATU in DMF, dropwise adding triethylamine under ice bath condition, stirring to generate active ester, then adding the compound 6, and reacting to obtain the protein degradation targeting chimera of the degradable PDGFR-beta, wherein the structural formula is as follows:
further, the method comprises the following steps:
1) Under the protection of nitrogen, carrying out reflux reaction on sorafenib, sodium hydroxide and absolute ethyl alcohol to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving the compound 1 and PyBop in dry dichloromethane, adding DMF, dropwise adding triethylamine under ice bath condition, and then adding 1, 4-butanediamine for reaction to obtain a compound 7 with the following structural formula:
3) Heating 4-fluoro thalidomide, glycine tert-butyl ester hydrochloride, DIPEA and DMF to react to obtain a compound 8, wherein the structural formula is as follows:
4) Dissolving the compound 8 in dry dichloromethane, dropwise adding trifluoroacetic acid under ice bath condition, and reacting to obtain a compound 9 with the following structural formula:
5) Dissolving the compound 9 and HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under ice bath condition, stirring to generate active ester, then adding the compound 7, and reacting to obtain the protein degradation targeting chimeric of degradable PDGFR-beta, wherein the structural formula is as follows:
The application of the protein degradation targeting chimera of the degradable PDGFR-beta in preparing antitumor drugs.
Further, the antitumor drug is a drug taking PDGFR-beta kinase as a target point.
Further, the antitumor drug is an anti-glioma drug.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, a multi-target small molecule inhibitor sorafenib is connected with different E3 ubiquitin ligase ligands, so that the protein degradation targeting chimeric is obtained. The protein degradation targeting chimera (PROTACs) can selectively induce the degradation of PDGFR-beta protein. The preparation method of the protein degradation targeting chimeric agent is simple, easy to realize and high in yield.
The small molecular protein degradation targeting chimera can selectively carry out ubiquitination marking on PDGFR-beta protein, induce protein degradation and has better anti-tumor effect than a PDGFR-beta protein inhibitor. Inhibition of PDGFR-beta protein often requires maintenance of the drug at a high concentration for a long period of time, possibly causing serious side effects; and only a small amount of compounds are needed for inducing protein degradation, the process is similar to catalytic reaction, and an equimolar amount of drugs is not needed, so that the use of the targeting chimera for degrading the protein with small molecules can reduce the use dosage of the drugs and reduce toxic and side effects. The small molecular protein degradation targeting chimeric can be used for preparing medicines for treating or preventing cancers, in particular to antitumor medicines taking PDGFR-beta kinase as a target point.
Unlike the "occupancy-driven" mechanism of action of conventional small molecule inhibitors, the "event-driven" mode of action of the protein degradation targeting chimera, therefore the small molecule protein degradation targeting chimera of the present invention is independent of tight binding to the target protein, and is capable of counteracting resistance due to point mutations. The small molecule protein degradation targeting chimera of the invention can selectively reduce the level of target protein in patient cells by recognizing and ubiquitinating the target protein, and then recognizing and degrading the ubiquitinated target protein by a proteasome.
Drawings
FIG. 1 is a protein degradation effect investigation result of a protein degradation targeting chimeric constructed in the invention on U87 cells;
FIG. 2 shows the results of investigation of the protein degradation effect of the protein degradation targeting chimera constructed in the invention on U87 cells;
FIG. 3 shows the results of investigation of the time dependence of protein degradation of U87 cells by the protein degradation targeting chimera SV constructed in the invention.
Detailed Description
The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.
Unlike the "occupancy-driven" mechanism of action of traditional small molecule inhibitors, the "event-driven" mode of action of protein degradation targeting chimeras, independent of tight binding to the target protein, is able to resist resistance due to point mutations. The protein degradation targeting chimeric can selectively reduce the level of the target protein in cells of a patient by recognizing and ubiquitinating the target protein, and then recognizing and degrading the ubiquitinated target protein by a proteasome, thereby achieving the aim of treating some diseases.
The current research is mature and uses more ligand VH032 which targets only CRBN (thalidomide and its analogues) and VHL.
Therefore, the invention selects sorafenib as a small molecule inhibitor of a target protein, VH032 and thalidomide analogues as ligands of targeted E3 ubiquitin ligase, and constructs a multi-target protein degradation targeting chimeric molecule capable of degrading VEGFR-2 and PDGFR-beta.
The invention obtains the protein degradation targeting chimeric body by using the multi-target small molecule inhibitor sorafenib and different E3 ubiquitin ligase ligand to connect. The protein degradation targeting chimeric (PROTACs) can selectively induce the degradation of PDGFR-beta protein, and can be applied to the preparation of medicines for treating cancers.
The structure of the protein degradation targeting chimeric constructed based on the multi-target antitumor drug sorafenib and the E3 ubiquitin ligase ligand is as follows:
The preparation method of the protein degradation targeting chimeric constructed by taking sorafenib and E3 ubiquitin ligase ligand as target heads comprises the following synthesis steps:
The antitumor drug sorafenib is hydrolyzed to obtain a compound with an active reactive group carboxyl, and then the compound is connected with different types of E3 ubiquitin ligase ligands through amide condensation reaction by different types of Linker, so as to obtain a series of protein degradation targeting chimeras.
The application of the protein degradation targeting chimera based on the multi-target antitumor drug sorafenib in preparing antitumor drugs with PDGFR-beta kinase as a target.
The protein degradation targeting chimeric has anti-tumor activity in vitro, and can be applied to the preparation of anti-tumor drugs.
Wherein the antitumor drug is an anti-glioma drug.
Overnight in the present invention, i.e., 12h.
Example 1
A preparation method of a protein degradation targeting chimeric SV constructed by taking an E3 ubiquitin ligase ligand VH032 as a target head comprises the following synthesis steps:
1) Under the protection of nitrogen, 1.9mmol of Sorafenib, 29mmol of sodium hydroxide and 20mL of absolute ethanol are reacted at 80 ℃ under reflux for 10h. After the TLC detection reaction is finished, absolute ethyl alcohol is removed by decompression, a small amount of water is added, the pH is regulated to 3 by using 2mol/L hydrochloric acid, yellow solid is separated out, suction filtration is carried out, a filter cake is dried, and yellow brown powder is obtained, namely the compound 1, the structural formula is shown as below, 0.8g is obtained, and the yield is 91.45%. LC-MS (ESI, m/z): 452.00[ M+H ] +,450.00[M-H]-.
2) 48.5Mmol of gamma-aminobutyric acid is dissolved in 80mL of tetrahydrofuran and placed in an ice-water bath, 80mL of 1mol/L sodium hydroxide solution is added, then 53.3mmol of di-tert-butyl dicarbonate tetrahydrofuran solution is dropwise added, stirring is carried out at room temperature, ninhydrin is used for detecting the reaction progress, after the reaction is finished, the volatile solvent is removed by decompression, 1mol/L hydrochloric acid is used for adjusting to 2-3, ethyl acetate extraction is carried out, saturated sodium chloride is used for washing the organic phase, anhydrous sodium sulfate is used for drying, suction filtration is carried out, and the solvent is removed by decompression, thus obtaining pale yellow compound 2 (8.24 g), the structural formula is shown in the specification, the yield is 83.65%, and LC-MS (ESI, m/z) 204.30[ M+H ] +,202.10[M+H]+.
3) 0.46Mmol of Compound 2,0.46mmol of HATU (2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, english name 2- (7-Azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) was dissolved in 20mL of dry dichloromethane, 0.93mmol of DIPEA (diisopropylethylamine) was added dropwise under ice bath conditions, stirred at room temperature for 15min, 0.23mmol of VH032 (suggested name) was added, stirred at room temperature overnight (i.e. 12 h), after the reaction was completed, water was added, dichloromethane was extracted 3 times (i.e. 3X), the organic phase was washed with saturated sodium chloride and dried over anhydrous Na 2SO4. The drying agent is removed by suction filtration, and the solvent is removed by spin-on under reduced pressure. The clear oily substance, compound 3 (0.11 g), was isolated by column chromatography and had the following structural formula, yield 76.97%, LC-MS (ESI, m/z): 616.40[ M+H ] +.
4) 0.158Mmol of compound 3 was dissolved in 2mol/L of ethyl acetate solution containing hydrogen chloride, stirred at room temperature overnight, and the filter cake (white solid) obtained by suction filtration was compound 4 (0.081 g), having the following structural formula, yield 99.45%, LC-MS (ESI, m/z): 516.40[ M+H ] +.
5) 0.24Mmol of Compound 1,0.24mmol of Compound 4,0.37mmol of HATU was dissolved in 15mL of dry dichloromethane, 1mL of DMF (N, N-dimethylformamide) was added to assist in the dissolution, and 0.97mmol of DIPEA (diisopropylethylamine) was added dropwise under ice-bath conditions and stirred at room temperature. TLC checked the progress of the reaction, after the reaction was completed, an appropriate amount of water was added, extracted with ethyl acetate (3X), washed with saturated NaCl, and dried over anhydrous Na 2SO4. The drying agent is removed by suction filtration, the solvent is removed by decompression, and the white product, namely the protein degradation targeting chimeric SV (0.065 g) is obtained by column chromatography, the structural formula is shown as follows, the yield is 28.26 percent, LC-MS (ESI, m/z): 949.35[ M+H ] +,947.45[M-H]-.
Example 2
A preparation method of a protein degradation targeting chimeric SL constructed by taking E3 ubiquitin ligase ligand Lenalidomide as a target head comprises the following synthesis steps:
1) Under the protection of nitrogen, 1.9mmol of Sorafenib, 29mmol of sodium hydroxide and 20mL of absolute ethanol are reacted at 80 ℃ under reflux for 10h. After the TLC detection reaction is finished, absolute ethyl alcohol is removed by decompression, a small amount of water is added, the pH is regulated to 3 by using 2mol/L hydrochloric acid, yellow solid is separated out, suction filtration is carried out, a filter cake is dried, and yellow brown powder is obtained, namely the compound 1, the structural formula is shown as below, 0.8g is obtained, and the yield is 91.45%. LC-MS (ESI, m/z): 452.00[ M+H ] +,450.00[M-H]-.
2) 48.5Mmol of gamma-aminobutyric acid is dissolved in 80mL of tetrahydrofuran and placed in an ice-water bath, 80mL of 1mol/L sodium hydroxide solution is added, then 53.3mmol of di-tert-butyl dicarbonate tetrahydrofuran solution is dropwise added, stirring is carried out at room temperature, ninhydrin is used for detecting the reaction progress, after the reaction is finished, the volatile solvent is removed by decompression, 1mol/L hydrochloric acid is used for adjusting to 2-3, ethyl acetate extraction is carried out, saturated sodium chloride is used for washing the organic phase, anhydrous sodium sulfate is used for drying, suction filtration is carried out, and the solvent is removed by decompression, thus obtaining pale yellow compound 2 (8.24 g), the structural formula is shown in the specification, the yield is 83.65%, and LC-MS (ESI, m/z) 204.30[ M+H ] +,202.10[M+H]+.
3) 0.85Mmol of Compound 2, HATU was dissolved in DMF, triethylamine was added dropwise under ice bath conditions, stirring was carried out at room temperature for 15min, 0.77mmol of Lenalidomide (Lenalidomide) was added, stirring was carried out at room temperature overnight, water was added, extraction was carried out with ethyl acetate, the organic phase was washed with saturated sodium chloride, dried over anhydrous Na 2SO4, the drying agent was removed by suction filtration, the solvent was removed by spin-drying under reduced pressure, and a transparent oily substance, compound 5 (0.42 g) was obtained by column chromatography, having the following structural formula, yield was 97.66%, LC-MS (ESI, m/z): 467.10[ M+Na ] +,443.05[M-H]-.
4) 0.94Mmol of compound 5 was dissolved in 2mol/L of ethyl acetate solution containing hydrogen chloride, stirred at room temperature for 2 hours, and the filter cake (white solid) obtained by suction filtration was compound 6 (0.32 g) having the following structural formula, yield 99.76%, LC-MS (ESI, m/z): 345.05[ M+H ] +,342.90[M-H]-.
5) 0.24Mmol of Compound 1,0.48mmol of HATU was dissolved in 20mL of DMF, and under ice-bath conditions, 0.93mmol of triethylamine was added dropwise, followed by stirring for 30min by TLC to determine if active ester was formed, followed by 0.24mmol of Compound 6, stirring at room temperature. TLC checked the progress of the reaction, after the reaction was completed, an appropriate amount of water was added, extracted with ethyl acetate (3X), washed with saturated NaCl, and dried over anhydrous Na 2SO4. The drying agent is removed by suction filtration, the solvent is removed by decompression, and the white product, namely the protein degradation targeting chimeric SL (0.12 g) is obtained by column chromatography (ethyl acetate), the structural formula is shown in the specification, the yield is 64.59 percent, and the LC-MS (ESI, m/z) is 800.05[ M+Na ] +,776.10[M-H]-.
Example 3
The preparation method of the protein degradation targeting chimeric SF constructed by taking the E3 ubiquitin ligase ligand Thalidomide fluoride as a target head comprises the following synthesis steps:
1) Under the protection of nitrogen, 1.9mmol of Sorafenib, 29mmol of sodium hydroxide and 20mL of absolute ethanol are reacted at 80 ℃ under reflux for 10h. After the TLC detection reaction is finished, absolute ethyl alcohol is removed by decompression, a small amount of water is added, the pH is regulated to 3 by using 2mol/L hydrochloric acid, yellow solid is separated out, suction filtration is carried out, a filter cake is dried, and yellow brown powder is obtained, namely the compound 1, the structural formula is shown as below, 0.8g is obtained, and the yield is 91.45%. LC-MS (ESI, m/z): 452.00[ M+H ] +,450.00[M-H]-.
2) 1.33Mmol of compound 1,2.66mmol of PyBop (benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, english name: benzotriazol-1-yl-oxytripyrrolidino-phosphonium Hexafluorophosphate) is dissolved in 30mL of dry dichloromethane, 1mL of DMF is added for assisting in dissolving, 5.31mmol of triethylamine is dropwise added under ice bath condition, after stirring for 15min, 2.66mmol of 1, 4-butanediamine or 1, 8-octanediamine is added, stirring is carried out at room temperature, after TLC detection reaction is finished, the solvent is removed by spin reduction, water is added, ethyl acetate extraction (3×), saturated sodium chloride washing is carried out, and anhydrous Na 2SO4 is dried. The drying agent was removed by suction filtration, and the product was isolated as a brown yellow oil by column chromatography, compound 7 (0.61 g), having the following structural formula, in 88.52% yield. LC-MS (ESI, m/z): 522.25[ M+H ] +.
3) 1.81Mmol of compound Thalidomide fluoride,2.00mmol of glycine tert-butyl ester hydrochloride and 2.72mmol DIPEA,4mL DMF are mixed and dissolved in a 100mL eggplant-shaped bottle, and the mixture is placed in a microwave reactor for reaction at 85 ℃ for 50min, and after the reaction is finished, water is added, ethyl acetate is used for extraction, saturated sodium chloride is used for washing, and anhydrous Na 2SO4 is used for drying. The drying agent is removed by suction filtration, the solvent is removed by decompression, and the yellow fluorescent substance is obtained by column chromatography separation, namely the compound 8 (0.48 g), the structural formula is shown as follows, the yield is 68.57%, and LC-MS (ESI, m/z) is 410.05[ M+Na ] +,386.00[M-H]-.
4) 0.93Mmol of compound 8 is dissolved in dry dichloromethane, trifluoroacetic acid is added dropwise under ice bath condition, stirring is carried out at room temperature overnight, drying is carried out under reduced pressure, and yellow fluorescent product is obtained through column chromatography separation, namely compound 9 (0.11 g), the structural formula is as follows, and the yield is 35.48%. LC-MS (ESI, m/z): 331.00[ M+H ] +,329.90[M-H]-.
5) 0.30Mmol of Compound 9,0.45mmol of HATU was dissolved in 10mL of dry dichloromethane, 1mL of DMF was added to assist in dissolution, 1.20mmol of DIPEA was added dropwise under ice-bath conditions, stirring was carried out for 15min, TLC was used to determine whether an active ester was formed, then 0.30mmol of Compound 7 was added, and stirring was carried out at room temperature. TLC checked the progress of the reaction, after the reaction was completed, an appropriate amount of water was added, extracted with dichloromethane (3X), washed with saturated NaCl, and dried over anhydrous Na 2SO4. The drying agent is removed by suction filtration, the solvent is removed by decompression, and the yellow-green fluorescent product, namely the protein degradation targeting chimera SF (0.03 g) is obtained by column chromatography, the structural formula is shown as follows, the yield is 11.95 percent, and LC-MS (ESI, m/z): 835.15[ M+H ] +,833.10[M-H]-.
Example 4
Protein degradation targeting chimeric cell proliferation inhibition activity assay.
The activity detection of the protein degradation targeting chimeric cell level adopts an MTT detection method. Ea.hy926 cells, hepG2 cells and U87 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare single cell suspensions, which were plated in 96-well plates (3000 cells/well) with 180 μl per well. Culturing in a constant temperature incubator with 5% CO 2 at 37deg.C for 24 hr, and adding medicine after cell adhesion. Each group is provided with 3 compound holes, 20 mu L/hole serum-free culture medium is added into each of the negative control group and the blank group, 20 mu L/hole medicines with different concentrations are added into each experimental group (the medicines are diluted by the serum-free culture medium), and the experimental groups are placed into a constant temperature incubator with 5% CO 2 at 37 ℃ for continuous culture. After 48h of drug action, 22. Mu.L/well MTT solution (final concentration 0.5 mg/mL) was added, incubated at 37℃for 4h, the supernatant was carefully aspirated, 150. Mu.L/well DMSO was added, and the mixture was thoroughly shaken on a decolorizing shaker for 10min. The absorbance (OD) values of each well were measured at 490nm using an ELISA.
Numerical value processing: inhibition = (OD Negative group -OD administration group )/(OD Negative group -OD Blank group ) x 100%;
TABLE 1 cell proliferation inhibiting Activity of protein degradation targeting chimeras
As can be seen from Table 1, the protein degradation targeting chimera prepared by the present invention has reduced proliferation inhibitory activity on various cells as compared with the parent compound, but still retains a certain proliferation inhibitory activity.
Example 5
And (5) examining the degradation effect of the protein degradation agent on the target protein.
Ea.hy926 cells or U87 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare single cell suspensions, which were plated in 6-well plates (5×10 5/well) with 2mL per well. Culturing in a constant temperature incubator with 5% CO 2 at 37deg.C for 24 hr, and adding medicine after cell adhesion. The cell is treated by the protein degradation targeting chimeric body with different concentrations, the cell is placed in a constant temperature incubator with 5% CO 2 for incubation for 72 hours, then protein extraction is carried out, and the relevant protein level is detected by adopting a Western Blot immunoblotting method, so that the effect of the constructed intracellular self-assembly type protein degradation agent on U87 cells is obviously better than that of EA.hy926 cells as shown in the results of figures 1 and 2, and all the protein degradation targeting chimeric body can obviously degrade PDGFR-beta protein at the concentration of 10 mu M, thus indicating that the constructed protein degradation agent has good application prospect and can be used for preparing antitumor drugs.
Example 6
Time dependence investigation of protein degradation agent on target protein degradation.
U87 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare single cell suspensions, which were plated in 6-well plates (5×10 5/well) with 2mL per well. Culturing in a constant temperature incubator with 5% CO 2 at 37deg.C for 24 hr, and adding medicine after cell adhesion. The protein degradation targeting chimeric SV treated cells were given 10 mu M, incubated in a constant temperature incubator at 37℃with 5% CO 2, then sampled at different time points (4 h,8h,12h,16h,24h,48h,72 h) for protein extraction, and the relevant protein levels were detected by Western Blot immunoblotting, as shown in FIG. 3, the protein degradation effect on PDGFR-beta protein could be achieved by treating the cells for 4h with the protein degradation agent SV, the 16h degradation effect was optimal, the protein degradation and protein synthesis could be in a dynamic equilibrium state with the effect time at the later stage, and the protein degradation effect remained substantially unchanged.

Claims (6)

1. A protein degradation targeting chimera of degradable PDGFR-beta, which is characterized in that the chimera has the following structure:
、
Or (b)
。
2. A method of preparing a protein degradation targeting chimera of a degradable PDGFR- β according to claim 1, comprising the steps of:
The sorafenib is hydrolyzed to obtain a compound with an active reactive group carboxyl, and then the compound with the active reactive group carboxyl is subjected to an amide condensation reaction with an E3 ubiquitin ligase ligand through a linker to obtain the protein degradation targeting chimeric of the degradable PDGFR-beta.
3. The method for preparing the protein degradation targeting chimera of the degradable PDGFR-beta according to claim 2, which comprises the following steps:
1) Under the protection of nitrogen, carrying out reflux reaction on sorafenib, sodium hydroxide and absolute ethyl alcohol to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing in an ice-water bath, adding sodium hydroxide solution, then dropwise adding tetrahydrofuran solution of di-tert-butyl dicarbonate, stirring, and obtaining a compound 2 after the reaction is finished, wherein the structural formula is as follows:
3) Dissolving the compound 2 and HATU in dry dichloromethane, dropwise adding DIPEA under ice bath condition, stirring, adding VH032, and after the reaction is finished, obtaining a compound 3 with the following structural formula:
4) Dissolving the compound 3 in ethyl acetate solution containing hydrogen chloride, and obtaining a compound 4 after the reaction is finished, wherein the structural formula is as follows:
5) Dissolving the compound 1, the compound 4 and the HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under ice bath condition, and obtaining the protein degradation targeting chimeric of the degradable PDGFR-beta after the reaction is finished, wherein the structural formula is as follows:
。
4. The method for preparing the protein degradation targeting chimera of the degradable PDGFR-beta according to claim 2, which comprises the following steps:
1) Under the protection of nitrogen, carrying out reflux reaction on sorafenib, sodium hydroxide and absolute ethyl alcohol to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing in an ice-water bath, adding sodium hydroxide solution, then dropwise adding tetrahydrofuran solution of di-tert-butyl dicarbonate, and stirring to obtain a compound 2 with the following structural formula:
3) Dissolving the compound 2 and HATU in DMF, dropwise adding triethylamine under ice bath condition, stirring, adding lenalidomide, and reacting to obtain a compound 5 with the following structural formula:
4) Dissolving the compound 5 in ethyl acetate solution containing hydrogen chloride, and reacting to obtain a compound 6 with the following structural formula:
5) Dissolving the compound 1 and HATU in DMF, dropwise adding triethylamine under ice bath condition, stirring to generate active ester, then adding the compound 6, and reacting to obtain the protein degradation targeting chimera of the degradable PDGFR-beta, wherein the structural formula is as follows:
。
5. The method for preparing the protein degradation targeting chimera of the degradable PDGFR-beta according to claim 2, which comprises the following steps:
1) Under the protection of nitrogen, carrying out reflux reaction on sorafenib, sodium hydroxide and absolute ethyl alcohol to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving the compound 1 and PyBop in dry dichloromethane, adding DMF, dropwise adding triethylamine under ice bath condition, and then adding 1, 4-butanediamine for reaction to obtain a compound 7 with the following structural formula:
3) Heating 4-fluoro thalidomide, glycine tert-butyl ester hydrochloride, DIPEA and DMF to react to obtain a compound 8, wherein the structural formula is as follows:
4) Dissolving the compound 8 in dry dichloromethane, dropwise adding trifluoroacetic acid under ice bath condition, and reacting to obtain a compound 9 with the following structural formula:
5) Dissolving the compound 9 and HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under ice bath condition, stirring to generate active ester, then adding the compound 7, and reacting to obtain the protein degradation targeting chimeric of degradable PDGFR-beta, wherein the structural formula is as follows:
。
6. use of the protein degradation targeting chimera of the degradable PDGFR-beta according to claim 1 for preparing an anti-glioma drug.
CN202211008631.9A 2022-08-22 2022-08-22 Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof Active CN115475164B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211008631.9A CN115475164B (en) 2022-08-22 2022-08-22 Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211008631.9A CN115475164B (en) 2022-08-22 2022-08-22 Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115475164A CN115475164A (en) 2022-12-16
CN115475164B true CN115475164B (en) 2024-06-04

Family

ID=84422470

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211008631.9A Active CN115475164B (en) 2022-08-22 2022-08-22 Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN115475164B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116143758B (en) * 2022-12-21 2024-04-23 天津科技大学 Azaflavonoid targeting protein chimera and application thereof in preparation of antitumor drugs

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104736569A (en) * 2012-01-12 2015-06-24 耶鲁大学 Compounds & methods for the enhanced degradation of targeted proteins & other polypeptides by an e3 ubiquitin ligase
WO2016197114A1 (en) * 2015-06-05 2016-12-08 Arvinas, Inc. Tank-binding kinase-1 protacs and associated methods of use
CN109999035A (en) * 2019-05-05 2019-07-12 西安交通大学 Sorafenib derivative application in preparation of anti-tumor drugs
CN110545853A (en) * 2017-04-28 2019-12-06 石英治疗有限公司 RAF degrading coupled compounds
CN110642849A (en) * 2019-10-08 2020-01-03 中南大学湘雅医院 Protein degradation targeting chimera and preparation method and application thereof
CN112592331A (en) * 2020-12-17 2021-04-02 武汉大学 Oseltamivir PROTAC compound, preparation method thereof and application thereof in anti-influenza virus drugs
CN113614076A (en) * 2019-01-03 2021-11-05 密执安大学评议会 Estrogen receptor protein degradation agent
CN114573570A (en) * 2022-02-21 2022-06-03 中山大学 Alpha-synuclein targeted compound and preparation method and application thereof
CN114736264A (en) * 2022-04-14 2022-07-12 华东师范大学 Tau protein visual PROTAC degradation compound and preparation method and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10584101B2 (en) * 2016-10-11 2020-03-10 Arvinas, Inc. Compounds and methods for the targeted degradation of androgen receptor

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104736569A (en) * 2012-01-12 2015-06-24 耶鲁大学 Compounds & methods for the enhanced degradation of targeted proteins & other polypeptides by an e3 ubiquitin ligase
WO2016197114A1 (en) * 2015-06-05 2016-12-08 Arvinas, Inc. Tank-binding kinase-1 protacs and associated methods of use
CN110545853A (en) * 2017-04-28 2019-12-06 石英治疗有限公司 RAF degrading coupled compounds
CN113614076A (en) * 2019-01-03 2021-11-05 密执安大学评议会 Estrogen receptor protein degradation agent
CN109999035A (en) * 2019-05-05 2019-07-12 西安交通大学 Sorafenib derivative application in preparation of anti-tumor drugs
CN110642849A (en) * 2019-10-08 2020-01-03 中南大学湘雅医院 Protein degradation targeting chimera and preparation method and application thereof
CN112592331A (en) * 2020-12-17 2021-04-02 武汉大学 Oseltamivir PROTAC compound, preparation method thereof and application thereof in anti-influenza virus drugs
CN114573570A (en) * 2022-02-21 2022-06-03 中山大学 Alpha-synuclein targeted compound and preparation method and application thereof
CN114736264A (en) * 2022-04-14 2022-07-12 华东师范大学 Tau protein visual PROTAC degradation compound and preparation method and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Siegelin MD等.Sorafenib exerts anti-glioma activity in vitro and in vivo.《Neurosci Lett》.2010,第478卷(第3期),第165-170页. *
Sorafenib exerts anti-glioma activity in vitro and in vivo;Siegelin MD等;《Neurosci Lett》;第478卷(第3期);第165-170页 *
池肇春主编.《消化系统疾病鉴别诊断与治疗学 第二版》.山东科学技术出版社,2017,第659页. *
神经胶质瘤血管生成的研究进展;郭天瑶;程婷婷;詹显全;;中国医师杂志(第06期);全文 *
索拉菲尼对人神经胶质瘤细胞增殖和凋亡影响;魏亮等;《解剖科学进展》;20160430;第22卷(第02期);172-175 *
索拉菲尼对人神经胶质瘤细胞增殖和凋亡的影响;魏亮;张军峰;孟喜君;;解剖科学进展(第02期);全文 *

Also Published As

Publication number Publication date
CN115475164A (en) 2022-12-16

Similar Documents

Publication Publication Date Title
EP1650205A1 (en) Cyclohexanecarboxylic acid compound
CN115475164B (en) Protein degradation targeting chimeric capable of degrading PDGFR-beta, preparation method and application thereof
CN106866572B (en) Nitric oxide donator type β elemene derivatives and its production and use
WO2024040768A1 (en) 5-pyridine-1h-indazole compound, pharmaceutical composition, and use
CN113861213B (en) Toosendanin PROTAC compound with STAT3 degradation activity and preparation method and application thereof
CN115403561B (en) Intracellular self-assembled protein degradation agent based on thalidomide analogue, and preparation method and application thereof
CN104211759B (en) The formamido group acid derivative of dimethoxy hydroxyphenyl β carbolines 3, it is prepared, nanostructured, activity and application
CN115385859A (en) Protein degradation agent capable of realizing intracellular self-assembly and preparation method and application thereof
CN115286687A (en) Intracellular self-assembly degradation agent based on bioorthogonal reaction and preparation method and application thereof
CN106883277B (en) A kind of furazan class NO donator type scutellarin derivative with anti-tumor activity and its preparation method and application
CN115160326A (en) Phthalocyanine complex of targeted IDO enzyme and preparation method and application thereof
CN107573322A (en) Imatinib dinitrogen oxide, preparation method and use
CN117263936B (en) Imidazo [1,2-a ] pyridine derivative, preparation method thereof and application thereof in drug for inhibiting central nervous system penetrating HDAC6
CN110041239B (en) N- (benzoyl) -L-cysteine methyl ester derivative and preparation method and application thereof
CN102485735B (en) 6-fructosamine-4-arylamidoquinazoline derivative and purpose thereof
CN113929639B (en) Antitumor compound with GSTP1 as target spot, and preparation method and application thereof
CN115260161B (en) Self-assembled protein degradation agent with tumor cell specificity and preparation method and application thereof
CN116143758B (en) Azaflavonoid targeting protein chimera and application thereof in preparation of antitumor drugs
CN106928224B (en) Indoles Sophoridine derivative and preparation method thereof
CN115340591A (en) Intracellular self-assembled protein degradation agent based on VH032 and preparation method and application thereof
CN116574081B (en) Chlorogenic acid-Huang Qinsu conjugate and preparation method and application thereof
CN115557890B (en) Polysubstituted acridone alkyl derivative and preparation method and application thereof
CN109400597B (en) VEGFR-2 inhibitor ABT-869-based protein degradation targeting chimera and preparation method and application thereof
CN117567392A (en) Pterocarpus stilbene paeonol acetic acid compound, and preparation method and medical application thereof
CN115368333A (en) Compound containing ibuprofen-coumarin skeleton and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant