CN115475164A - Protein degradation targeting chimera capable of degrading PDGFR-beta and preparation method and application thereof - Google Patents
Protein degradation targeting chimera capable of degrading PDGFR-beta and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a protein degradation targeting chimera capable of degrading PDGFR-beta, a preparation method and application thereof.A compound with an active reaction group carboxyl is obtained by hydrolyzing sorafenib serving as an anti-tumor drug, and then the compound is connected with E3 ubiquitin ligase ligands of different types through different types of linkers through an amide condensation reaction to obtain a protein degradation targeting chimeric molecule. The protein degradation targeting chimera has the advantages of simple preparation method, easy realization and higher yield, and can be used for preparing medicaments for treating or preventing cancers, particularly antitumor medicaments with PDGFR-beta targets.
Description
Technical Field
The invention belongs to the technical field of drug synthesis, and relates to a protein degradation targeting chimera capable of degrading PDGFR-beta, and 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: the tumor cell growth inhibitor can directly inhibit the proliferation of tumor cells by blocking a cell signaling pathway mediated by RAF/MEK/ERK, and can indirectly inhibit the growth of tumor cells by inhibiting Vascular Endothelial Growth Factor Receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR), but has severe adverse reactions in clinical treatment and is easy to generate drug resistance after long-term application.
The protein degradation Targeting Chimera (PROTAC) is a bifunctional molecule capable of simultaneously combining target protein and E3 ubiquitin ligase, and the distance between the target protein and the E3 ligase is shortened by simultaneously combining the target protein and the E3 ubiquitin ligase, so that ubiquitination of the target protein is induced, the ubiquitinated target protein can be recognized and degraded by 26S proteasome, and the purpose of thoroughly eliminating disease-related protein is achieved. Compared with small molecular inhibitors, PROTAC has the advantages of small dosage, difficult generation of drug resistance and the like, so the PROTAC presents a vigorous development situation in the field of new drug research and development.
Traditional small molecule inhibitors adopt an 'occupancy-driven' action mechanism, rely on close binding 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 chimera capable of degrading PDGFR-beta, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a protein degradation targeting chimera capable of degrading PDGFR-beta has the following structure:
a preparation method of the protein degradation targeting chimera capable of degrading PDGFR-beta comprises the following steps:
the Sorafenib is hydrolyzed to obtain a compound with an active reaction group carboxyl, and then the compound with the active reaction group carboxyl and an E3 ubiquitin ligase ligand are subjected to amide condensation reaction through a linker to obtain the protein degradation targeting chimera capable of degrading PDGFR-beta.
Further, the method comprises the following steps:
1) Under the protection of nitrogen, sorafenib, sodium hydroxide and absolute ethyl alcohol are subjected to reflux reaction to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing the tetrahydrofuran in ice water bath, adding a sodium hydroxide solution, then dropwise adding a 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 the ice bath condition, stirring, adding VH032, and obtaining a compound 3 after the reaction is finished, wherein the structural formula is as follows:
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 HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under an ice bath condition, and obtaining a protein degradation targeting chimera capable of degrading 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, sorafenib, sodium hydroxide and absolute ethyl alcohol are subjected to reflux reaction to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing the tetrahydrofuran in an ice water bath, adding a sodium hydroxide solution, then dropwise adding a tetrahydrofuran solution of di-tert-butyl dicarbonate, and stirring to obtain a compound 2, wherein the structural formula is as follows:
3) Dissolving the compound 2 and HATU in DMF, dropwise adding triethylamine under the ice bath condition, stirring, adding lenalidomide, and reacting to obtain a compound 5, wherein the structural formula is as follows:
4) Dissolving the compound 5 in ethyl acetate solution containing hydrogen chloride, and reacting to obtain a compound 6, wherein the structural formula is as follows:
5) Dissolving the compound 1 and HATU in DMF, dropwise adding triethylamine under an ice bath condition, stirring until active ester is generated, then adding the compound 6, and reacting to obtain a protein degradation targeting chimera capable of degrading PDGFR-beta, wherein the structural formula is as follows:
further, the method comprises the following steps:
1) Under the protection of nitrogen, sorafenib, sodium hydroxide and absolute ethyl alcohol are subjected to reflux reaction 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 the ice bath condition, adding 1, 4-butanediamine, and reacting to obtain a compound 7, wherein the structural formula is as follows:
3) Heating 4-fluorothalidomide, glycine tert-butyl ester hydrochloride, DIPEA and DMF for reaction to obtain a compound 8, wherein the structural formula is as follows:
4) Dissolving the compound 8 in dry dichloromethane, and dropwise adding trifluoroacetic acid under an ice bath condition to react to obtain a compound 9, wherein the structural formula is as follows:
5) Dissolving the compound 9 and HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under the ice bath condition, stirring until active ester is generated, then adding the compound 7, and reacting to obtain a protein degradation targeting chimera capable of degrading PDGFR-beta, wherein the structural formula is as follows:
the application of the protein degradation targeting chimera capable of degrading PDGFR-beta in preparing antitumor drugs.
Furthermore, the anti-tumor drug is a drug taking PDGFR-beta kinase as a target.
Furthermore, the anti-tumor drug is an anti-glioma drug.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains a protein degradation target chimera by connecting a multi-target small molecule inhibitor sorafenib with different E3 ubiquitin ligase ligands. The protein degradation target chimeras (PROTACs) can selectively induce the degradation of PDGFR-beta protein. The protein degradation targeting chimera of the invention has simple preparation method, easy realization and higher yield.
The small-molecule protein degradation targeting chimera can selectively carry out ubiquitination marking on PDGFR-beta protein, induces protein degradation, and has better anti-tumor effect than PDGFR-beta protein inhibitors. Inhibition of PDGFR- β protein often requires maintenance of the drug at higher concentrations for long periods of time, potentially causing serious side effects; only a small amount of compounds are needed for inducing protein degradation, the process is similar to catalytic reaction, and equimolar amount of medicine is not needed, so that the dosage of the medicine can be reduced by using the small molecular protein degradation targeting chimera, and toxic and side effects are reduced. The small-molecule protein degradation targeting chimera can be used for preparing medicines for treating or preventing cancers, and particularly can be used for preparing antitumor medicines taking PDGFR-beta kinase as a target.
Unlike the traditional 'occupancy-driven' action mechanism of small molecule inhibitors, the 'event-driven' action mode of the protein degradation targeting chimera is adopted, so that the small molecule protein degradation targeting chimera of the invention does not depend on the tight combination with the target protein and can resist the drug resistance caused by point mutation. The small molecule protein degradation targeting chimera of the present invention can selectively reduce the level of target protein in patient's cells by recognizing and ubiquitinating the target protein, followed by recognition and degradation of the ubiquitinated target protein by proteasome.
Drawings
FIG. 1 is a result of investigating the protein degradation effect of the protein degradation targeting chimera constructed according to the present invention on U87 cells;
FIG. 2 is the result of the investigation of the protein degradation effect of the protein degradation targeting chimera constructed according to the present invention on U87 cells;
FIG. 3 shows the result of investigation on the protein degradation time dependence of the protein degradation targeting chimera SV constructed by the invention on U87 cells.
Detailed Description
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Unlike the "occupancy-driven" mechanism of action of traditional small molecule inhibitors, the "event-driven" mode of action of proteolytic degradation-targeted chimeras, independent of tight binding to the target protein, is able to counteract drug resistance due to point mutations. The protein degradation target chimera can selectively reduce the level of the target protein in the cells of the patient by identifying and ubiquitinating the target protein and then identifying and degrading the ubiquitinated target protein by a proteasome, thereby achieving the purpose of treating some diseases.
Currently, the only ligands that target CRBN (thalidomide and its analogs) and VHL, VH032, are more mature and more widely used.
Therefore, the invention selects the small molecule inhibitor taking sorafenib as a target protein, and the VH032 and thalidomide analogs as ligands of the target E3 ubiquitin ligase to construct a multi-target protein degradation target chimeric molecule capable of degrading VEGFR-2 and PDGFR-beta.
The protein degradation target chimera is obtained by connecting a multi-target small molecule inhibitor sorafenib and different E3 ubiquitin ligase ligands. The protein degradation target chimeras (PROTACs) can selectively induce the degradation of PDGFR-beta protein, and can be applied to the preparation of medicaments for treating cancers.
A protein degradation targeting chimera constructed based on multi-target antitumor drugs sorafenib and an E3 ubiquitin ligase ligand has the following structure:
a preparation method of a protein degradation targeting chimera constructed by taking sorafenib and an E3 ubiquitin ligase ligand as a target head comprises the following synthetic steps:
the antitumor drug sorafenib is hydrolyzed to obtain a compound with active reaction group carboxyl, and then is connected with different types of E3 ubiquitin ligase ligands through amide condensation reaction by different types of linkers (Linker) to obtain a series of protein degradation target chimeras.
The application of the protein degradation targeting chimera based on the multi-target antitumor drug sorafenib in preparing an antitumor drug taking PDGFR-beta kinase as a target.
The protein degradation targeting chimera has antitumor activity in vitro, and can be used for preparing antitumor drugs.
Wherein the antitumor drug is an anti-glioma drug.
In the invention, the time is 12 hours.
Example 1
A preparation method of a protein degradation targeting chimera 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 ethyl alcohol are refluxed and reacted for 10 hours at 80 ℃. After TLC detection reaction is finished, decompressing and removing absolute ethyl alcohol, adding a small amount of water, adjusting pH to 3 by using 2mol/L hydrochloric acid, separating out yellow solid, performing suction filtration, and drying a filter cake to obtain yellow brown powder, namely the compound 1, wherein the formula is as follows, 0.8g is collected, and the yield is 91.45%. LC-MS (ESI, m/z) 452.00[ 2 ], [ M + H ]] + ，450.00[M-H] - 。
2) Dissolving 48.5mmol of gamma-aminobutyric acid in 80mL of tetrahydrofuran, placing the solution in an ice water bath, adding 80mL of 1mol/L sodium hydroxide solution, dropwise adding 53.3mmol of di-tert-butyl dicarbonate solution in the tetrahydrofuran solution, stirring at room temperature, detecting the reaction process by ninhydrin, after the reaction is finished, performing reduced pressure rotary removal on the volatile solvent, adjusting the volatile solvent to 2-3 by using 1mol/L hydrochloric acid, extracting by using ethyl acetate, washing an organic phase by using saturated sodium chloride, drying by using anhydrous sodium sulfate, performing suction filtration, and performing reduced pressure rotary removal on the solvent to obtain a light yellow compound 2 (8.24 g), wherein the structural formula is as follows, the yield is 83.65%, and LC-MS (ESI, m/z): 204.30M + H (m/z)] + ，202.10[M+H] + 。
3) 0.46mmol of the compound 2,0.46mmol of HATU (2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, the English name 2- (7-Azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluoro phosphate) was dissolved in 20mL of dry dichloromethane, 0.93mmol of DIPEA (diisopropylethylamine) was added dropwise under ice-bath conditions, stirring was carried out at room temperature for 15min, and 0.23mmol of VH032 (recommended for feeding toNamed) was stirred at room temperature overnight (i.e., 12 h), after the reaction was completed, water was added, dichloromethane was extracted 3 times (i.e., 3 ×), the organic phase was washed with saturated sodium chloride, and anhydrous Na was added 2 SO 4 And (5) drying. The drying agent is removed by suction filtration, and the solvent is removed by rotary removal under reduced pressure. Separating by column chromatography to obtain transparent oily substance, compound 3 (0.11 g), with the structural formula as follows, yield 76.97%, LC-MS (ESI, m/z): 616.40[ M ] +H] + 。
4) Dissolving 0.158mmol of compound 3 in 2mol/L ethyl acetate solution containing hydrogen chloride, stirring overnight at room temperature, and filtering to obtain filter cake (white solid) which is compound 4 (0.081 g), with the structural formula as follows, yield 99.45%, LC-MS (ESI, m/z): 516.40[ m ] +H] + 。
5) 0.24mmol of 1,0.24mmol of the compound 4,0.37mmol of HATU was dissolved in 15mL of dry dichloromethane, and 1mL of DMF (N, N-dimethylformamide) was added for solubilization, and 0.97mmol of DIPEA (diisopropylethylamine) was added dropwise under ice bath conditions, and stirred at room temperature. TLC detection reaction process, after the reaction is finished, adding a proper amount of water, extracting with ethyl acetate (3X), washing with saturated NaCl, and anhydrous Na 2 SO 4 And (5) drying. Suction filtering to remove desiccant, rotary removing solvent under reduced pressure, and separating by column chromatography to obtain white product, i.e. protein degradation targeting chimera SV (0.065 g), with structural formula as follows, yield 28.26%, LC-MS (ESI, m/z): 949.35[ M ] +H] + ，947.45[M-H] - 。
Example 2
A preparation method of a protein degradation targeting chimera SL constructed by taking an E3 ubiquitin ligase ligand Lenalidomide (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 ethyl alcohol are refluxed and reacted for 10 hours at 80 ℃. After TLC detection reaction is finished, decompressing and removing absolute ethyl alcohol, adding a small amount of water, adjusting pH to 3 by using 2mol/L hydrochloric acid, separating out yellow solid, performing suction filtration, and drying a filter cake to obtain yellow brown powder, namely the compound 1, wherein the formula is as follows, 0.8g is collected, and the yield is 91.45%. LC-MS (ESI, m/z) 452.00[ 2 ] M + H] + ，450.00[M-H] - 。
2) Dissolving 48.5mmol of gamma-aminobutyric acid in 80mL of tetrahydrofuran, placing the solution in an ice water bath, adding 80mL of 1mol/L sodium hydroxide solution, dropwise adding 53.3mmol of di-tert-butyl dicarbonate solution in the tetrahydrofuran solution, stirring at room temperature, detecting the reaction process by ninhydrin, after the reaction is finished, performing reduced pressure rotary removal on the volatile solvent, adjusting the volatile solvent to 2-3 by using 1mol/L hydrochloric acid, extracting by using ethyl acetate, washing an organic phase by using saturated sodium chloride, drying by using anhydrous sodium sulfate, performing suction filtration, and performing reduced pressure rotary removal on the solvent to obtain a light yellow compound 2 (8.24 g), wherein the structural formula is as follows, the yield is 83.65%, and LC-MS (ESI, m/z): 204.30M + H (m/z)] + ，202.10[M+H] + 。
3) Dissolving 0.85mmol of compound 2, HATU in DMF, adding triethylamine dropwise under ice bath condition, stirring at room temperature for 15min, adding 0.77mmol of Lenalidomide (Lenalidomide), stirring at room temperature overnight, adding water, extracting with ethyl acetate, washing the organic phase with saturated sodium chloride, and adding anhydrous Na 2 SO 4 Drying, vacuum filtering to remove desiccant, removing solvent under reduced pressure, and separating by column chromatography to obtain transparent oily substanceCompound 5 (0.42 g), having the formula shown below, is obtained in 97.66% yield, LC-MS (ESI, m/z): 467.10[ M ] +Na] + ，443.05[M-H] - 。
4) Dissolving 0.94mmol of compound 5 in 2mol/L ethyl acetate solution containing hydrogen chloride, stirring at room temperature for 2h, and vacuum filtering to obtain filter cake (white solid) which is compound 6 (0.32 g), with the structural formula as follows, yield of 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, 0.93mmol of triethylamine was added dropwise under ice-bath conditions, and after stirring for 30min, TLC was performed to detect whether an active ester was formed, followed by addition of 0.24mmol of compound 6 and stirring at room temperature. TLC detecting reaction progress, adding appropriate amount of water after reaction, extracting with ethyl acetate (3X), washing with saturated NaCl, anhydrous Na 2 SO 4 And (5) drying. Vacuum filtering to remove desiccant, rotary removing solvent under reduced pressure, and separating with column chromatography (ethyl acetate) to obtain white product, i.e. protein degradation targeting chimera SL (0.12 g), with structural formula as follows, yield 64.59%, LC-MS (ESI, m/z): 800.05[ M ] +Na + ]] + ，776.10[M-H] - 。
Example 3
A preparation method of a protein degradation targeting chimera SF constructed by taking 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 ethyl alcohol are refluxed and reacted for 10 hours at 80 ℃. After the TLC detection reaction is finished, decompressing and removing absolute ethyl alcohol by spinning, adding a small amount of water, adjusting the pH to 3 by using 2mol/L hydrochloric acid, separating out yellow solid, performing suction filtration, and drying a filter cake to obtain tawny powder, namely a compound 1, wherein the structural formula is as follows, the yield is 0.8g, and the yield is 91.45%. LC-MS (ESI, m/z) 452.00[ 2 ] M + H] + ，450.00[M-H] - 。
2) 1.33mmol of compound 1,2.66mmol of PyBop (benzotriazole-1-yl-oxytripyrrolidinophosphonium Hexafluorophosphate, england name: benzotriazol-1-yl-oxytripyridolidino-phosphonium hexafluophorophosphate) dissolved in 30mL of dry dichloromethane was dissolved, 1mL of DMF was added for solubilization, 5.31mmol of triethylamine was added dropwise under ice bath conditions, after stirring for 15min, 2.66mmol of 1, 4-butanediamine or 1, 8-octanediamine was added, stirring at room temperature was carried out, after TLC detection reaction was completed, the solvent was removed under reduced pressure, water was added, ethyl acetate was extracted (3X), washed with saturated sodium chloride, and anhydrous Na was added 2 SO 4 And (5) drying. The drying agent was removed by suction filtration and the product was isolated by column chromatography as compound 7 (0.61 g) in the following formula in 88.52% yield. LC-MS (ESI, m/z) 522.25[ 2 ], [ M + H ]] + 。
3) Dissolving 1.81mmol of compound Thalidomide fluoride,2.00mmol of glycine tert-butyl ester hydrochloride, 2.72mmol of DIPEA and 4mL of DMF in a 100mL eggplant-shaped bottle, placing the bottle in a microwave reactor, reacting at 85 ℃ for 50min, adding water and ethyl acetate for extraction after the reaction is finished, washing with saturated sodium chloride, and reacting with anhydrous Na 2 SO 4 And (5) drying. Vacuum filtering to remove desiccant, removing solvent under reduced pressure, and separating by column chromatography to obtain yellow fluorescent substance (compound 8 (0.48 g), with the following structural formula, yield of 68.57%, and LC-MS (ESI, m-z):410.05[M+Na] + ，386.00[M-H] - 。
4) Dissolving 0.93mmol of compound 8 in dry dichloromethane, dropwise adding trifluoroacetic acid under ice bath condition, stirring overnight at room temperature, drying under reduced pressure, and separating by column chromatography to obtain yellow fluorescent product, i.e. compound 9 (0.11 g), with the following structural formula and yield of 35.48%. LC-MS (ESI, m/z) 331.00[ 2 ], [ M + H ]] + ，329.90[M-H] - 。
5) 0.30mmol of compound 9,0.45mmol of HATU is dissolved in 10mL of dry dichloromethane, 1mL of DMF is added for dissolution assistance, 1.20mmol of DIPEA is added dropwise under ice bath conditions, stirring is carried out for 15min, TLC is carried out to detect whether active ester is generated, and then 0.30mmol of compound 7 is added, and stirring is carried out at room temperature. TLC detecting reaction progress, adding appropriate amount of water after reaction, extracting with dichloromethane (3X), washing with saturated NaCl, anhydrous Na 2 SO 4 And (5) drying. Suction filtering to remove desiccant, rotary removing solvent under reduced pressure, and separating by column chromatography to obtain yellow green fluorescent product, i.e. protein degradation targeting chimera SF (0.03 g), with structural formula as follows, yield of 11.95%, LC-MS (ESI, m/z): 835.15[ M ] +H +] + ，833.10[M-H] - 。
Example 4
And (3) measuring the proliferation inhibition activity of the protein degradation targeting chimeric cells.
The activity of protein degradation targeting chimeric cell level is detected by 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 seeded in 96-well plates (3000 cells/well) eachWells 180. Mu.L. Put at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator, and adding the medicine after the cells adhere to the wall after 24 hours. Setting 3 multiple wells per group, adding 20 μ L/well serum-free culture medium to the negative control group and blank group, adding 20 μ L/well of drug with different concentrations to the experimental group (diluting drug with serum-free culture medium), adding into 37 deg.C, and 5% 2 Continuously culturing in a constant temperature incubator. After 48h of drug action, 22. Mu.L/well of MTT solution (final concentration 0.5 mg/mL) was added, after incubation at 37 ℃ for 4h, the supernatant was carefully aspirated off, DMSO was added at 150. Mu.L/well and shaken well on a destaining shaker for 10min. The absorbance (OD) of each well was measured at 490nm using an enzyme-linked immunosorbent assay.
Numerical value processing: inhibition rate = (OD) Negative group -OD Administration set )/(OD Negative group -OD Blank group )×100％；
TABLE 1 cell proliferation inhibitory Activity of protein degradation targeting chimeras
As can be seen from table 1, the protein degradation targeting chimeras prepared according to the present invention have a decreased proliferation inhibitory activity against various cells compared to the parent compound, but still retain a certain proliferation inhibitory activity.
Example 5
And (3) investigating the degradation effect of the protein degradation agent on the target protein.
Subjecting EA.hy926 cells or U87 cells in logarithmic growth phase to digestion with 0.25% trypsin to obtain single cell suspension, and inoculating the single cell suspension to 6-well plate (5X 10) 5 One/well), 2mL per well. Put at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator, and adding the medicine after the cells adhere to the wall after 24 hours. Administration of different concentrations of protein degrading targeting chimera treated cells at 37 ℃ 5% 2 Incubating in a constant temperature incubator for 72h, then extracting protein, detecting the level of related protein by using a Western Blot immunoblotting method, and obtaining the result as shown in figures 1 and 2, wherein the effect of the constructed intracellular self-assembled protein degradation agent on U87 cells is obviously better than that of EA.hy926 cells,all the protein degradation targeting chimeras can obviously degrade PDGFR-beta protein at the concentration of 10 mu M, and the protein degradation agent constructed by the invention has good application prospect and can be used for preparing anti-tumor drugs.
Example 6
Time-dependent investigation of the degradation of a target protein by a protein degrading agent.
U87 cells in logarithmic growth phase were digested with 0.25% trypsin to prepare a single cell suspension, which was inoculated into 6-well plates (5X 10) 5 One/well), 2mL per well. Charging at 37 deg.C, 5% 2 Culturing in a constant temperature incubator, and adding medicine after the cells adhere to the wall after 24 hours. Administration of 10 μ M of protein degradation targeting chimera SV treated cells, placed at 37 ℃,5% CO 2 Incubation is carried out in a constant temperature incubator, then sampling is carried out at different time points (4h, 8h,12h, 169h, 24h,48h and 72h) for protein extraction, and then the Western Blot immunoblotting is adopted to detect the related protein level, so that the result is shown in figure 3, the PDGFR-beta protein can be degraded by treating cells for 4h by using a protein degrading agent SV, the 16h degradation effect is optimal, the protein degradation and the protein synthesis can be in a dynamic balance state with the prolonging of the action time in the later period, and the protein degradation effect is basically kept unchanged.
Claims (8)
1. A protein degradation targeting chimera capable of degrading PDGFR-beta is characterized in that the chimera has the following structure:
2. a method for preparing the protein degradation targeting chimera of claim 1, which degrades PDGFR- β, comprising the steps of:
hydrolyzing sorafenib to obtain a compound with an active reaction group carboxyl, and then carrying out amide condensation reaction on the compound with the active reaction group carboxyl and an E3 ubiquitin ligase ligand through a linker to obtain the protein degradation targeting chimera capable of degrading PDGFR-beta.
3. The method for preparing the PDGFR-beta degradable protein degradation targeting chimera according to claim 2, comprising the following steps:
1) Under the protection of nitrogen, sorafenib, sodium hydroxide and absolute ethyl alcohol are subjected to reflux reaction to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing the tetrahydrofuran in ice water bath, adding a sodium hydroxide solution, then dropwise adding a 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 the ice bath condition, stirring, adding VH032, and obtaining a compound 3 after the reaction is finished, wherein the structural formula is as follows:
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 HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under an ice bath condition, and obtaining a protein degradation targeting chimera capable of degrading PDGFR-beta after the reaction is finished, wherein the structural formula is as follows:
4. the method for preparing the PDGFR-beta degradable protein degradation targeting chimera according to claim 2, comprising the following steps:
1) Under the protection of nitrogen, sorafenib, sodium hydroxide and absolute ethyl alcohol are subjected to reflux reaction to obtain a compound 1, wherein the structural formula is as follows:
2) Dissolving gamma-aminobutyric acid in tetrahydrofuran, placing the tetrahydrofuran in an ice water bath, adding a sodium hydroxide solution, then dropwise adding a tetrahydrofuran solution of di-tert-butyl dicarbonate, and stirring to obtain a compound 2, wherein the structural formula is as follows:
3) Dissolving the compound 2 and HATU in DMF, dropwise adding triethylamine under the ice bath condition, stirring, adding lenalidomide, and reacting to obtain a compound 5, wherein the structural formula is as follows:
4) Dissolving the compound 5 in ethyl acetate solution containing hydrogen chloride, and reacting to obtain a compound 6, wherein the structural formula is as follows:
5) Dissolving the compound 1 and HATU in DMF, dropwise adding triethylamine under an ice bath condition, stirring until active ester is generated, then adding the compound 6, and reacting to obtain a protein degradation targeting chimera capable of degrading PDGFR-beta, wherein the structural formula is as follows:
5. the method for preparing the PDGFR-beta degradable protein degradation targeting chimera according to claim 2, comprising the following steps:
1) Under the protection of nitrogen, sorafenib, sodium hydroxide and absolute ethyl alcohol are subjected to reflux reaction 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 the ice bath condition, adding 1, 4-butanediamine, and reacting to obtain a compound 7, wherein the structural formula is as follows:
3) Heating 4-fluorothalidomide, glycine tert-butyl ester hydrochloride, DIPEA and DMF for reaction to obtain a compound 8, wherein the structural formula is as follows:
4) Dissolving the compound 8 in dry dichloromethane, and dropwise adding trifluoroacetic acid under an ice bath condition to react to obtain a compound 9, wherein the structural formula is as follows:
5) Dissolving the compound 9 and HATU in dry dichloromethane, adding DMF, dropwise adding DIPEA under the ice bath condition, stirring until active ester is generated, then adding the compound 7, and reacting to obtain a protein degradation targeting chimera capable of degrading PDGFR-beta, wherein the structural formula is as follows:
6. use of the protein degradation targeting chimera of claim 1 that degrades PDGFR-beta in preparation of anti-tumor drugs.
7. The use of claim 6, wherein the anti-tumor drug is a drug targeting PDGFR-beta kinase.
8. The use according to claim 6, wherein the antineoplastic drug is an anti-glioma drug.
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