CN117362391A - Polypeptide coupled protein degradation targeting chimeric compound for targeted degradation of BRD4, intermediate, preparation method and application - Google Patents
Polypeptide coupled protein degradation targeting chimeric compound for targeted degradation of BRD4, intermediate, preparation method and application Download PDFInfo
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- CN117362391A CN117362391A CN202311161473.5A CN202311161473A CN117362391A CN 117362391 A CN117362391 A CN 117362391A CN 202311161473 A CN202311161473 A CN 202311161473A CN 117362391 A CN117362391 A CN 117362391A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention provides a polypeptide coupling protein degradation targeting chimeric compound for targeted degradation of BRD4, an intermediate thereof, a preparation method and application. The compound has the structure of formula (1), compared with the conventional compoundSystemic PROTAC drugs, which can be substituted by alpha V β 3 The integrin receptor is accurately identified, has stronger water solubility, tumor targeting and penetrability in tumor tissues, and can express alpha in high degree V β 3 The cancer cells of the receptor show good curative effect on resisting breast cancer. In addition, studies have shown that the compounds of the present invention have better anti-breast cancer effects in animal models and patient-derived organoids than traditional PROTAC drugs.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide coupling protein degradation targeting chimeric compound for targeted degradation of BRD4, an intermediate thereof, a preparation method and application.
Background
The Integrin (Integrin) receptor family is a heterodimeric transmembrane protein consisting of an alpha subunit and a beta subunit, which is widely present on the cell surface and is involved in a variety of physiological and pathological processes including cell adhesion, proliferation, migration, vascular leakage, inflammation, and the like. Many integrin protein subtypes are closely related to pathological processes such as tumor development, progression, invasion, metastasis, and neovascularization, e.g., α 1-6 β 1 、α 6 β 4 、α v β 3 Alpha and alpha v β 5 . In 1984, the arginine-glycine-aspartic acid (RGD) tripeptide sequence in fibronectin was found to be able to interact with the alpha of tumor vessel surface V β 3 Integrin receptor recognition, thus alpha v β 3 Cancer cells with high integrin expression allow polypeptides containing the RGD integrin recognition motif to be targeted for recognition. Currently, targeted delivery based on RGD polypeptides has been successfully used to deliver drugs, biologies and viruses to tumor vessels.
Protein degradation targeting chimeras (proteolysis targeting chimera, PROTAC) are heterobifunctional molecules consisting of a target protein ligand, a linker, and an E3 ligase ligand. Protein degradation targeting chimeras coordinate the formation of ternary complexes by recruiting E3 ligase near the target protein, thereby inducing ubiquitination of the target protein, resulting in final degradation of the target protein by the proteasome. Compared with the traditional small molecule inhibitor, the PROTAC has remarkable advantages in the aspects of targeting non-drug-resistant proteins, enhancing selectivity, overcoming drug resistance and the like. However, PROTAC also has inherent limitations. Relatively large molecular weight PROTAC molecules tend to result in poor water solubility and cell membrane permeability. In addition, the inability to achieve tissue-selective degradation of PROTAC, overcoming elevated interstitial pressure in tumor tissue and the difficulty of penetrating vascular barriers into tumors, is also a major challenge in drug research based on protein degradation targeting chimeras.
The difficulty in penetrating the vascular barrier, poor water solubility and insufficient targeting are one of the major problems faced by the PROTAC drugs. The invention discovers that the tripeptide sequence containing arginine-glycine-aspartic acid (RGD) can be combined with alpha on the surface of tumor blood vessels on the basis of consulting literature V β 3 Integrin receptor recognition, polypeptide coupled drugs constructed based on polypeptides containing RGD tripeptide sequences can enter tumor cells more easily, and have better targeting effect. The invention is therefore based mainly on the reported ability to be alpha v β 3 The polypeptide identified by integrin receptor is targeted therapeutic carrier, and several kinds of polypeptide capable of being synthesized by alpha are designed and synthesized v β 3 Polypeptide coupled protein degradation targeting chimeric (PPCs) drugs identified by integrin receptors, and research on in-vitro and in-vivo antitumor activity and action mechanism of the drugs. With respect to the present invention, the base can be alpha v β 3 The PPCs medicine which is constructed by the polypeptide identified by the integrin receptor and achieves the anti-tumor effect by degrading bromodomain-containing protein 4 (bromodomain-containing protein, BRD 4) is not reported except the team at present.
Disclosure of Invention
The first object of the present invention is to overcome the defects of the prior PROTAC drugs and provide a base for overcoming the defects in the prior artCan then be alpha v β 3 And PPCs medicine with antitumor effect is achieved by degrading BRD 4.
A second object of the invention is to provide a method based on the fact that it is capable of being α v β 3 A preparation method of PPCs medicine which is constructed by integrin receptor-identified polypeptide and achieves the anti-tumor effect by degrading BRD 4.
A third object of the present invention is to provide a method based on the ability to be alpha v β 3 Application of PPCs medicine constructed by integrin receptor-identified polypeptide and achieving anti-tumor effect by degrading BRD 4.
The invention provides a polypeptide coupled protein degradation targeting chimeric compound for targeting and degrading BRD4, which has a structure shown in a formula (1):
wherein X is any polypeptide having RGD tripeptide sequence capable of being alpha v β 3 Linear polypeptides, cyclic peptides or polypeptide analogs recognized by integrin receptors, including but not limited to,
linear RGD polypeptide:
A1：H-H-G-R-G-D
A2：R-G-D
A3：C-R-G-D-K
cyclic RGD polypeptide:
A4：c(C-R-G-D-F-V)
A5：c(C-R-G-D-F-K)
A6：c(C-R-G-D-F-E)
A7：cyclo-(Arg-Gly-Asp- D -Phe-Val)
A8：cyclo-(Arg-Gly-Asp- D -Phe-Lys)
A9：cyclo-(Arg-Gly-Asp- D -Tyr-Glu)
iRGD：c(C-R-G-D-K-G-P-D-C)
cilengitide (cinengitide):
Cyclo(L-arginylglycyl-L-aspartyl-D-phenylalanyl-N-methyl-L-valyl)。
y is PROTAC (hereinafter referred to as PRO) with BRD4 degradation capability, and has the following structure,
the PRO compound is characterized in that: the compound is formed by connecting JQ1 and VHL ligand through an alkyl chain with three PEG, and the VHL ligand part of the compound is provided with unprotected hydroxyl and can be connected with one carboxyl end of a connector.
The linker is an alkyl chain with ester bond or disulfide bond connecting the two groups of X and Y together, and the two ends of the linker are provided with carboxyl groups which can be respectively connected with the terminal amino (or side chain amino) of X and the hydroxyl on Y, including but not limited to the following structures,
B1：
B2：
in another aspect, in certain embodiments, the present invention also provides a method of preparing a compound of formula (1).
In another aspect, the invention also relates to a pharmaceutical composition comprising a compound of formula (1) as described above, or a stereoisomer or a pharmaceutically acceptable salt thereof.
In another aspect, the present application also relates to the use of a compound of formula (1), or a stereoisomer or a pharmaceutically acceptable salt thereof, as described above, in the manufacture of a medicament for the prevention and/or treatment of a disease associated with BRD4 protein.
In another aspect, the present application also relates to a method of preventing and/or treating a disease associated with BRD4 protein, comprising administering to a subject in need thereof a compound of formula (1) or a stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.
In another aspect of the present invention,the application also relates to the use of the compound of formula (1) or a stereoisomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition for the prevention and/or treatment of alpha v β 3 Use of a disease associated with integrin receptor expression.
In certain embodiments, the sum α v β 3 Diseases associated with integrin receptor expression include breast cancer.
The invention has the beneficial effects that:
the invention provides three compounds obtained by connecting polypeptide containing RGD sequence with PROTAC drug through linker for the first time. Wherein, the S-RPR and the iPR adopt the linker B2 as a Glutathione (GSH) reactive linker, and the linker can generate disulfide bond rupture around tumor cells to release therapeutic drugs. Of these three compounds, the ip was optimal for tumor targeting and tumor permeability, and its water solubility was also enhanced relative to the proto-PROTAC drug PRO. For alpha V β 3 IC of MDA-MB-231 cell with high expression and iPR 50 IC with a value of 88nM, ratio PRO 50 The value was smaller than 90nM. In the aspect of reducing BRD4, iPR has high expression alpha v β 3 Integrin receptor cells have similar degradability to PRO, and degrade at 0.11. Mu.M, but iPR acts earlier than PRO (16 h;24 h), showing the advantage of easy uptake. iPR in low expression alpha v β 3 BRD4 degradation in integrin receptor cells is weaker than PRO, showing that the binding is based on the ability to be alpha-coated v β 3 Accurate targeting and selectivity of PPCs medicine constructed by integrin receptor-recognized polypeptide. Cell migration resistance experiments show that at the same concentration, iPR has stronger effect than PRO. Animal experiment results show that in MDA-MB-231 nude mice transplanted tumor model, the in vivo anti-tumor activity of iPR is stronger, and the TGI values of iPR and PRO are 62.3% and 36.3% respectively when the drug is administered at the same concentration. Mechanism experiments prove that H is respectively carried out on tumor sections of the iPR and PRO treatment groups&E staining, obviously damaging tumor tissue blood vessels of the iPR treatment group, collapsing tissue tissues outside the blood vessels, slightly changing the PRO treatment group, showing that the iPR changes the permeability of the tumor blood vessels, and enhancing the protein degradation targeting chimeric drug in the tumor groupPenetration and diffusion in the weave. In addition, the invention also uses MDA-MB-231 organoid model to verify the anti-tumor activity of iPR and PRO, and the result shows that the iPR has better organoid proliferation inhibiting activity than PRO, and the IC 50 The values were 0.95. Mu.M and 2.1. Mu.M, respectively.
Drawings
FIG. 1 is a synthetic scheme for compound intermediate S4 of the present invention.
FIG. 2 is a synthetic scheme for the compound L-RPR of the present invention.
FIG. 3 is a synthetic scheme for compound intermediate S10 of the present invention.
FIG. 4 is a synthetic scheme for the compound S-RPR of the present invention.
Fig. 5 is a synthetic route diagram of the compound iPR of the present invention.
FIG. 6 shows the results of an in vitro stability test of the compound represented by formula (1) of the present invention.
FIG. 7 shows the results of GSH-responsive drug release test for the compound of formula (1).
FIG. 8 is alpha v β 3 Expression in normal breast cells and breast cancer cells.
FIG. 9 (A) shows antiproliferative activity of PRO and the compounds of the invention iPR, PRO on MDA-MB-231 cells.
FIG. 9 (B) shows antiproliferative activity of PRO and the compounds iPR and PRO of the present invention on L02 cells.
FIG. 10 shows the degradation of BRD4 protein in MDA-MB-231 cells by PRO and the compounds of the present invention iPR, S-RPR.
FIG. 11 shows the degradation of BRD4 protein in MCF-7 cells and L02 cells by PRO and the compounds of the invention iPR and S-RPR.
FIG. 12 shows the anti-migration of PRO and iPR compounds of the invention against MDA-MB-231 cells at various concentrations.
FIG. 13 shows the anti-cell migration of PRO and the compounds S-RPR, iPR of the present invention against MCF-7 cells and L02 cells at the same concentration.
FIG. 14 shows the change in tumor volume in nude mice after (A) administration of PRO and the compound of the invention iPR at different concentrations in a model of nude mice MDA-MB-231 transplantation tumor; (B) change in body weight of nude mice following administration; (C) tumor size after the end of the treatment cycle; (D) change in organ weight in nude mice after administration.
FIG. 15 is an evaluation of BRD4 levels in tumor tissues of nude mice after administration of PRO and the compound of the invention iPR at various concentrations in a model of nude mice MDA-MB-231 transplantation tumor.
FIG. 16 is H & E staining of tumor vascular tissue sections of nude mice after administration of PRO and the compound of the invention iPR at different concentrations in a model of nude mice MDA-MB-231 graft.
Fig. 17 shows the cytotoxicity test results of PRO and iPR of the present invention on triple negative breast cancer organoids.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to the accompanying drawings and specific embodiments, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
Example 1
(1) Synthetic compound intermediate S4:
referring to fig. 1, starting from the Boc-protected VHL ligand (S1), intermediate S3 was obtained by replacing the Boc protecting group with an Fmoc protecting group. Finally, the intermediate S3 reacts with succinic anhydride under the catalysis of DMAP to generate ester, and a product S4 is generated. The structural formulas of S1, S2, S3 and S4 in the synthetic route are as follows:
(2) The compound of formula (1), the compound prepared in this example, referred to as L-RPR, has the structure A1-B1-PRO, was prepared as follows:
referring to FIG. 2, standard Fmoc solid phase polypeptide synthesis (solid phase peptide synthesis, SPPS) methods were used.
1) 2-chlorotrityl chloride resin and amino acid with Fmoc protecting group (2.0 eq.) were dissolved in DMF, HCTU and DIPEA were added as coupling agents, and reacted at 30 ℃ for 1 hour, amino acid connection order, i.e. polypeptide sequence order (A1: H-H-G-R-G-D), and verifying the completion of each coupling step by using an ninhydrin detection method, removing amino protecting groups of amino acids by using a 20% anhydrous piperidine DMF (v/v) solution after the completion of each amino acid connection, and ensuring that the next amino acid can be connected until the connection of the last amino acid is successful.
2) 2-chlorotrityl chloride resin (intermediate 3 of fig. 2) and intermediate (2.0 eq.) of formula (S4) after all amino acids are connected are dissolved in DMF solution, HCTU and DIPEA are added as coupling agents, and reacted at 30 ℃ for 1 hour to prepare intermediate 4 of fig. 2;
3) Intermediate 4 of fig. 2 was reacted in 20% piperidine DMF (v/v) solution to remove Fmoc protecting groups, then an amino-protected PEG linker (2.0 eq.) was added, HCTU and DIPEA were added as coupling agents, and reacted at 30 ℃ for 1 hour to prepare intermediate 5 of fig. 2;
4) Intermediate 5 of fig. 2 was reacted in 20% piperidine DMF (v/v) solution to remove Fmoc protecting group, then JQ1-COOH (2.0 eq.) was added, HCTU and DIPEA were added as coupling agents, and reacted at 30 ℃ for 1 hour to prepare intermediate 6 of fig. 2;
5) Adding trifluoroacetic acid mixed solution (95% TFA,2.5% phenol and 2.5% methane sulfonic acid) into the intermediate 6 in the figure 2, cutting for 20 minutes, filtering, pouring the filtrate into cold diethyl ether, generating a large amount of dark green precipitate, centrifugally collecting the precipitate, and washing the precipitate with the cold diethyl ether for 2-3 times to obtain a crude product;
6) The crude product is further purified by preparative high performance liquid chromatography (high performance liquid chromatography, HPLC) and freeze-dried to obtain the L-RPR pure product, the structural formula of which is shown as formula (7).
The structural formulas of the intermediates 2,3,4,5,6 and the product L-RPR in the above synthetic route are as follows:
wherein, the 'small black balls' in the structural formula represent 2-chlorotrityl chloride, the structural formula of R is as follows,representing the position of the connection to the linker:
example 2
(1) Synthesis of Compound intermediate S10:
referring to FIG. 3, starting from Boc protected VHL ligand (S1), reaction with 4-nitrobenzoate forms intermediate S5. Intermediate S5 was reacted with 2,2' -dithiodiethanol under DMAP catalysis followed by removal of the Boc protecting group using trifluoroacetic acid to give intermediate S6. Intermediate S6 is condensed with 5,8, 11-trioxa-2-aza-tridecanedioic acid-1-tert-butyl ester to obtain intermediate S7, and Boc protection is removed to obtain intermediate S8. Condensing S8 with JQ1-COOH to obtain an intermediate S9. Finally, the intermediate S9 reacts with succinic anhydride under the catalysis of DMAP to obtain a product S10.
In the above synthetic route, the structural formulas of S5, S6, S7, S8, S9, S10 are as follows:
wherein R in the structural formula 1 Is that Representing the location of the connection to the linker.
(2) The compound of formula (1), the compound prepared in this example, referred to as S-RPR, has the structure A2-B2-PRO, was prepared as follows:
referring to FIG. 4, the synthesis of PPCs S-RPR is similar to the L-RPR method. The difference is that the synthesized polypeptide sequence is (A2: R-G-D), and the coupled compound is changed into S10 after the polypeptide is synthesized. After completion of the polypeptide synthesis on the resin, compound S10 (2.0 eq.) was coupled to the synthesized polypeptide using a similar method to the amino acid linkage.
1) 2-chlorotrityl chloride resin and amino acid with Fmoc protecting group (2.0 eq.) were dissolved in DMF, HCTU and DIPEA were added as coupling agents, and reacted at 30 ℃ for 1 hour, amino acid connection order, i.e. polypeptide sequence order (A2: R-G-D), verifying the completion of each coupling step by using an ninhydrin detection method, and removing an amino protecting group of the amino acid by using a 20% anhydrous piperidine DMF (v/v) solution after the completion of the connection of each amino acid, so as to ensure that the connection of the next amino acid can be completed until the connection of the last amino acid is successful;
2) 2-chlorotrityl chloride resin (intermediate 8 of FIG. 4) and intermediate (2.0 eq.) of formula (S10) after all amino acids are connected are dissolved in DMF solution, HCTU and DIPEA are added as coupling agents and reacted at 30℃for 1 hour to prepare intermediate 9 of FIG. 4;
3) Adding trifluoroacetic acid mixed solution (95% TFA,2.5% phenol and 2.5% methane sulfonic acid) into the intermediate 9 in the figure 4, cutting for 20 minutes, filtering, pouring the filtrate into cold diethyl ether, generating a large amount of dark green precipitate, centrifugally collecting the precipitate, and washing the precipitate with the cold diethyl ether for 2-3 times to obtain a crude product;
4) The crude product is further purified by preparative high performance liquid chromatography and freeze-dried to obtain the S-RPR pure product, the structural formula of which is shown as formula (10).
The structural formulas of intermediates 8,9 and product S-RPR in the above synthetic route are as follows:
wherein, the 'small black balls' in the structural formula represent 2-chlorotrityl chloride, the structural formula of R is as follows,representing the position of the connection to the linker:
example 3
The compound of formula (1), the compound prepared in this example, termed iPR, has the structure iRGD-B2-PRO, was prepared as follows:
referring to FIG. 5, the synthesis of the PCCs drug iPR is similar to the S-RPR method.
1) Dissolving 2-chlorotrityl chloride resin and amino acid (2.0 eq.) with Fmoc protecting group in DMF, adding HCTU and DIPEA as coupling agent, reacting at 30deg.C for 1 hour, wherein the amino acid connection sequence is polypeptide sequence (C-R-G-D-K-G-P-D-C), wherein the side chain amino protecting group of lysine (K) is ivDde group, the amino protecting group of last cysteine is Boc group, verifying the completion of each coupling step by ninhydrin detection method, removing amino protecting group of amino acid by 20% anhydrous piperidine DMF (v/v) solution after each amino acid connection is completed, ensuring that the next amino acid can be connected until the last amino acid connection is successful;
2) Reacting 2-chlorotrityl chloride resin (intermediate 12 in fig. 5) with all amino acids in 5% hydrazine hydrate DMF (v/v) solution at 30 ℃ for 30 minutes, and removing ivDde protecting groups on side chain amino groups of lysine (K) to prepare intermediate 13 in fig. 5;
3) Intermediate 13 of fig. 5 and intermediate (2.0 eq.) of formula (S10) were dissolved in DMF solution, HCTU and DIPEA were added as coupling agents and reacted at 30 ℃ for 1 hour to prepare intermediate 14 of fig. 5;
4) Adding trifluoroethylene into the intermediate 14 in fig. 5, cutting for 20 minutes, filtering, pouring the filtrate into cold diethyl ether, generating a large amount of dark green precipitate, centrifugally collecting the precipitate, and washing the precipitate with the cold diethyl ether for 2-3 times to obtain a crude product;
5) The crude product is further purified by preparative high performance liquid chromatography and freeze-dried to obtain intermediate 15 of FIG. 5;
6) Intermediate 15 of fig. 5 was dissolved in an aqueous solution containing 40% dmso (v/v) and stirred overnight to promote cyclization and disulfide bond formation;
7) The crude product is further purified by preparative high performance liquid chromatography and freeze-dried to obtain an iPR pure product, and the structural formula of the iPR pure product is shown as formula (16).
The structural formulas of intermediates 12, 13, 14, 15 and product iPR in the above synthetic route are as follows:
wherein, the 'small black balls' in the structural formula represent 2-chlorotrityl chloride, the structural formula of R is as follows,representing the position of the connection to the linker: />
Experimental example 1 in vitro stability test (HPLC test) of the Compound of the invention
Step a: dissolving L-RPR, S-RPR and iPR in PBS solution containing 5% serum to form 50 μm solution;
step b: incubating the prepared solution at 37deg.C, taking 20 μl of the solution at different time intervals, quantitatively detecting by HPLC, and using a chromatographic column as C18 analytical column under mobile phase conditions of MeCN (0.1% CF) 3 COOH)：H 2 O(0.1％CF 3 COOH) =10:90-90:10, stability of the compound was determined by calculating the ratio of the peak area of the prototype compound at different time points to the zero time peak area.
The experimental results are shown in FIG. 6, and after 72h incubation, the L-RPR, S-RPR and iPR contents were 10.2%,94.6% and 89.9%, respectively, indicating poor in vitro stability of L-RPR and good in vitro stability of S-RPR and iPR.
Experimental example 2 GSH-reactive drug Release test (HPLC test) of the Compound of the invention
Step a: S-RPR and iPR are respectively dissolved in PBS solution to form 50 mu M solution;
step b: adding GSH with equal concentration, simulating high GSH environment in tumor cells, and incubating the prepared solution at 37 ℃;
step c: quantitative determination of 20. Mu.L of the solution by HPLC was performed at various time intervals, using a chromatographic column as C18 analytical column with mobile phase conditions of MeCN (0.1% CF 3 COOH)：H 2 O(0.1％CF 3 COOH) =10:90-90:10, the release capacity of the compound was determined by calculating the ratio of peak area to zero peak area at different time points of the parent compound and the concentration of the released PROTAC compound at different time periods.
The experimental results are shown in FIG. 7, wherein the drug release of the compound of the invention is increased in a time-dependent manner, and the S-RPR is completely released within 2 hours, and the release rate is 90.3%; the release rate of iPR within 2h is 82.5% which is slightly lower than S-RPR.
Experimental example 3 different cell lines αvβ3 integrin receptor expression (flow cytometry detection method)
Step a: 3×10 cells of different cell types in logarithmic growth phase per well 5 Density of each was inoculated in 6-well plates with 2mL of DMEM medium at 37℃and 5% CO per well 2 Culturing for 24h;
step b: sucking the culture medium away, rinsing with PBS for 3 times, adding donkey serum, and incubating for 1h to block the heterologous protein;
step c: after aspiration of the blocked serum, the cells were washed 3 times with PBS and incubated with primary antibody for 30min at 4 ℃;
step d: recovering the primary antibody, washing the cells again, and incubating with the secondary antibody for 1h at room temperature;
step e: recovering the secondary antibody, washing cells with PBS, and then digesting with trypsin;
step f: collecting the cells into a centrifuge tube, discarding the original solution, and re-dispersing the cells with PBS;
step g: flow cytometry detection was performed.
Experimental results: as a result, as shown in FIG. 8, among the four cells, normal MCF-10A cells, L02 cells and MCF-7 cells were low in expression of alpha v β 3 Integrin receptor, MDA-MB-231 cell high expression alpha v β 3 Integrin receptors.
Experimental example 4 in vitro antitumor Activity test of the Compounds of the invention against different cell lines (CCK 8 method)
Step a: inoculation of 5X 10 3 Individual/well cells (100 μl) were plated in 96-well plates, with 100 μl of PBS added around them;
step b: placing in a cell incubator at 37deg.C and 5% CO 2 After culturing for 24 hours under the condition, adding 100 mu L of the compound to be tested with different concentrations, and continuously placing the compound into a cell incubator for culturing for 72 hours, wherein three compound holes are formed in each concentration;
step c: removing the liquid medicine, and adding basic culture containing 10% of CCK 8;
step d: after 30min of treatment at 37 ℃ under dark condition, the OD value at 452nm is measured by an enzyme-labeled instrument, and the data is imported into GraphPad software to map and simulate IC 50 。
The experimental results are shown in FIG. 9 and Table 1, for a high expression of alpha v β 3 IC of iPR in MDA-MB-231 breast cancer cells of integrin receptor 50 IC of value and PRO 50 The values were comparable, 88nM and 90nM, respectively, the S-RPR activity was poor, IC 50 A value of 190nM; in low expression of alpha v β 3 PRO activity in MCF-7 breast cancer cells of integrin receptor is significantly better than iPR and S-RPR, IC 50 The values were 30nM,260nM and 190nM, respectively. Furthermore, alpha is expressed at the same low level v β 3 The in vitro anti-proliferation results of the test drug in L02 breast cancer cells of the integrin receptor are also consistent with those in MCF-7 breast cancer cells. This result shows that iPr and S-RPR obtained by design synthesis are highly expressed for alpha v β 3 Cells of the integrin receptor have good targeting ability.
Table 1 antiproliferative activity of the compounds of the invention on different cells.
Experimental example 5 Targeted degradation of BRD4 by the inventive Compounds in different cells (Western Blot method)
Step a: : the cell lines of different kinds in logarithmic growth phase are pressed to 3X 10 5 Density of each well was inoculated into 6-well plates, 2mL of DMEM medium was added to each well, 37℃and 5% CO 2 Culturing for 24h;
step b: sucking the culture medium away, and washing the cells 2 times with PBS;
step c: in a concentration-dependent degradation activity experiment, treating cells with compounds to be tested with different concentrations, and placing the cells in a cell incubator for 24 hours; in time-dependent degradation activity experiments, cells were treated with different concentrations of compound (300 nM) and placed in cell incubators for corresponding times (4, 8, 12, 16, 20, 24 h);
step d: discarding the compound solution, placing the 6-well plate in an ice bath, washing the cells with PBS and treating the cells with a cell lysis buffer containing a phosphatase inhibitor (1%), a proteolytic enzyme inhibitor (1%), and incubating the cells in the ice bath for 0.5h;
step e: collecting cell lysate in a centrifuge tube after grinding, swirling for 1 time every 5min, repeating for 3 times;
step f: centrifuging the tube containing the cell lysate in a centrifuge (4 ℃ C., 1.2X10) 4 rpm，15min)；
Step g: PBS (18 mu L), cell supernatant (2 mu L), newly prepared BCA test solution (200 mu L) and a microplate reader are added to each well of the 96-well plate to test the protein concentration;
step h: taking supernatant (60 mu L) into a centrifuge tube, adding protein loading buffer (15 mu L), and heating in a metal bath to denature protein (100 ℃ for 15 min);
step i: electrophoresis (90V, 2 h) was performed according to total protein content (30. Mu.g) as determined by protein concentration test;
step j: a rapid film transfer instrument performs film transfer (25V, 7 min);
step k: the rapid sealing liquid is sealed for 0.5h at room temperature, and TBST is washed for 3 times;
step l: incubating GPC-3 primary antibody (1:1000) at 2-8deg.C overnight;
step m: recovering primary antibody, washing with TBST for 3 times, each time for 10min;
step n: incubating the secondary antibody for 1.5h at room temperature;
step o: recovering the secondary antibody, and washing with TBST for 3 times, each time for 10min;
step p: the Odyssey double-color infrared laser imaging system scans and develops, and the internal reference is beta-Tubulin.
The experimental results are shown in fig. 10 and 11, with iPR at α v β 3 The MDA-MB-231 cells with high expression of integrin receptor have similar degradation capacity to the PRO, and can be degraded at 0.11 mu M, but iPR has the advantage of being easy to be taken up because of earlier onset than PRO (16 h;24 h). iPR in low expression alpha v β 3 BRD 4-degrading ability in integrin receptor-based cells (MCF-7 cells, L02 cells) is weaker than PRO, showing a basis for alpha v β 3 Accurate targeting and selectivity of PPCs medicament constructed by integrin receptor recognition sequence polypeptide.
Experimental example 6Transwell experiments to examine the Effect of the Compounds of the invention on tumor cell migration
Step a: cells in the logarithmic growth phase were digested with pancreatin. The cells were diluted to single cell suspension with medium (dmem+10% fbs+1% diabody) to adjust the cell density to 5×10 4 Inoculating the strain into a Transwell upper chamber;
step b: test compounds were added to the lower chamber in the presence of 20% FBS medium, and then the cell culture plates were placed at 37℃in 5% CO 2 Culturing for 48 hours in a cell culture box;
step c: after the incubation, the upper chamber was washed with PBS solution and fixed with 4% paraformaldehyde for 10min;
step d: staining the cells with 10% crystal violet solution for 10min, washing the cells with PBS;
step e: the migrated cells were observed under a microscope under different fields of view.
The experimental results are shown in FIGS. 12 and 13 at α v β 3 In MDA-MB-231 cells with high integrin receptor expression, the anti-cell migration capability of iPR is stronger than that of PRO; at alpha v β 3 PRO has a stronger ability to resist cell migration in MCF-7 cells and L02 cells, which have low integrin receptor expression.
Experimental example 7 in vivo antitumor Activity test of the Compound iPr of the invention
Step a: MDA-MB-231 cells were grown at 6X 10 6 The concentration of each single is implanted under the waist of a female nude mouse with the age of 5-6 weeks, when the average tumor volume reaches 100mm 3 In vivo experiments were started at this time;
step b: nude mice were randomly divided into 4 groups. Three groups of PBS solutions (containing 0.5% dimethyl sulfoxide, 2% poloxamer, 0.5% Tween 80 and ddH 2O) were intravenously injected with PRO (5. Mu.M/kg), iPR (5. Mu.M/kg) and iPR (8. Mu.M/kg), respectively, 1 time a day, and the Control group (Control) was intravenously injected with the same volume of PBS;
step c: measuring the tumor size by a vernier caliper, and measuring the weight of the mice every four days;
step d: mice were sacrificed 22 days after drug treatment;
step e: dissecting, collecting and weighing heart, lung, liver, spleen and kidney as main viscera; tumor of the mice was excised and photographed; (using the formula volume=ab 2 Calculating tumor volume, wherein A, B represents tumor long diameter and tumor wide diameter respectively, and data adopts single factor analysis of variance to obtain P<A difference of 0.05 is statistically significant);
step f: slicing the tumor, respectively performing immunofluorescence and immunohistochemical staining, and evaluating the degradation condition of BRD4 in the tissue after drug treatment;
step g: tumor sections were H & E stained to assess whether iPR penetrated tumor vessels.
As shown in fig. 14, 15 and 16, in the nude mice MDA-MB-231 transplantation tumor model, the compound iPR of the present invention showed excellent anti-tumor drug effect (tgi=62.3%, 5 μm; tgi=81.5%, 8 μm), significantly better than PRO group (tgi=36.3%, 5 μm), and no significant change in major organ tissue weight, and no significant toxicity; immunofluorescence and immunohistochemical staining results show that the BRD4 level of the iPR treatment group is lower, indicating that the treatment effect of iPR is better than PRO; h & E tumor tissue staining results show that tumor tissue blood vessels of the iPR treatment group are obviously damaged, tissue tissues outside the blood vessels collapse, and PRO treatment group is only slightly changed, which shows that iPR changes the permeability of the tumor blood vessels and enhances the permeation and diffusion of protein degradation targeting chimeric drugs in the tumor tissues.
EXAMPLE 8 anti-tumor Activity test of the inventive Compound iPR in patient-derived MDA-MB-231 organoid model (CCK 8 assay)
Step a: adding a common culture medium with different concentrations of the medicaments to be detected into 100 mu L of prepared human breast cancer organoid culture medium, inoculating the culture medium into a 96-well plate, and setting 3 multiple wells;
step b: these organoids were then exposed to 5% CO at 37℃ 2 Culturing in a cell incubator for 3 to 5 days;
step c: mu.L of CCK8 reagent was added to each well and 5% CO at 37℃ 2 Incubating for 4 hours under the condition;
step d: cell viability was assessed by measuring absorbance at 450 nm. Cell viability and inhibition were calculated As cell viability= [ (As-Ab)/(Ac-Ab) ] ×100%, cell inhibition = [ (Ac-As)/(Ac-Ab) ] ×100% (As represents absorbance values of treatment wells of different drug concentrations, ac represents absorbance values of control wells, ab represents absorbance values of blank wells).
As shown in FIG. 17, iPR has better activity of inhibiting organoid proliferation than PRO, and IC 50 The values were 0.95. Mu.M and 2.1. Mu.M, respectively.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (15)
1. A polypeptide coupled protein degradation targeting chimeric compound for targeting and degrading BRD4 has a structural general formula as shown in formula (1),
wherein X is any polypeptide having RGD tripeptide sequence capable of being alpha v β 3 Linear polypeptides, cyclic peptides or polypeptide analogs that are recognized by integrin receptors;
the linker is an alkyl chain with an ester bond or a disulfide bond which connects two groups of X and Y together, and two ends of the linker are provided with carboxyl groups which can be respectively connected with the terminal amino (or side chain amino) of X and the hydroxyl on Y;
y is PROTAC with the capability of degrading BRD4 and has the following structure,
the PROTAC is linked by a JQ1 and VHL ligand through an alkyl chain with three PEGs, and the VHL ligand moiety has an unprotected hydroxyl group, which can be linked to one side of the linker at the carboxyl terminus.
2. The BRD4 targeted degradation polypeptide conjugated protein degradation targeting chimeric compound according to claim 1, wherein the linear polypeptide, cyclic peptide or polypeptide analogue has an RGD tripeptide sequence capable of being alpha-substituted v β 3 Integrin receptor recognition includes, but is not limited to, the following structures: H-H-G-R-G-D, R-G-D, C-R-G-D-K, C (C-R-G-D-F-V), C (C-R-G-D-K-G-P-D-C), C (C-R-G-D-F-K), C (C-R-G-D-F-E), cyclo- (Arg-Gly-Asp- D -Phe-Val)、cyclo-(Arg-Gly-Asp- D -Phe-Lys)、cyclo-(Arg-Gly-Asp- D -Tyr-Glu) or
Cyclo(L-arginylglycyl-L-aspartyl-D-phenylalanyl-N-methyl-L-valyl)。
3. Polypeptide-coupled protein degradation targeted to degrade BRD4 according to claim 1A untargeting chimeric compound, characterized in that said linker is selected from the group consisting of
4. The BRD4 targeted degradation polypeptide coupled protein degradation targeting chimeric compound according to claim 1, wherein X is H-G-R-G-D and the linker isThe structural formula of the compound of the formula (1) is
Wherein R is
5. The BRD4 targeted degradation polypeptide coupled protein degradation targeting chimeric compound according to claim 1, wherein X is R-G-D and the linker isThe structural formula of the compound of the formula (1) is
Wherein R is
6. The BRD 4-targeted, degradation-targeted polypeptide-coupled protein degradation targeting chimeric compound of claim 1, which is specificCharacterized in that X is C (C-R-G-D-K-G-P-D-C), the linker isThe structural formula of the compound of the formula (1) is
Wherein R is
7. A compound intermediate for synthesizing the polypeptide coupled protein degradation targeting chimeric compound for targeting to degrade BRD4 according to claim 4, wherein the intermediate has a structure shown in a formula (S4),
8. a compound intermediate for synthesizing the polypeptide coupled protein degradation targeting chimeric compound for targeting to degrade BRD4 according to claim 5 or 6, wherein the intermediate has a structure shown in formula (S10),
wherein R is 1 Is that
9. A process for preparing the intermediate of claim 7, comprising:
starting with a compound of formula (S1), wherein the compound of formula (S1) has a Boc protecting group;
replacing the Boc protecting group of the compound of formula (S1) with an Fmoc protecting group to obtain a compound of formula (S3);
reacting a compound of formula (S3) with succinic anhydride under the catalysis of DMAP to generate ester, and obtaining the intermediate.
10. A process for preparing the intermediate of claim 8, comprising:
starting with a compound of formula (S1), wherein the compound of formula (S1) has a Boc protecting group;
reacting a compound of formula (S1) with 4-nitrobenzoate salt to produce a compound of formula (S5);
reacting a compound of formula (S5) with 2,2' -dithiodiethanol under DMAP catalysis, followed by removal of the Boc protecting group using trifluoroacetic acid to give a compound of formula (S6);
condensing a compound of formula (S6) with 1-tert-butyl 5,8, 11-trioxa-2-azatridecanedioate to obtain a compound of formula (S7), and removing Boc protection to obtain a compound of formula (S8);
condensing a compound of formula (S8) with JQ1-COOH to obtain a compound of formula (S9);
reacting a compound of formula (S9) with succinic anhydride under DMAP catalysis to produce the intermediate.
11. A method of synthesizing a BRD 4-targeted, degradation-targeted chimeric compound of a polypeptide-coupled protein of claim 4, comprising:
1) The process of claim 9 for preparing a compound intermediate having the structure of formula (S4);
2) 2-chlorotrityl chloride resin and amino acid with Fmoc protecting group (2.0 eq.) were dissolved in DMF, HCTU and DIPEA were added as coupling agents, and reacted at 30 ℃ for 1 hour, amino acid connection order, i.e. polypeptide sequence order (A1: H-H-G-R-G-D), verifying the completion of each coupling step by using an ninhydrin detection method, and removing amino protecting groups of amino acids by using 20% anhydrous piperidine DMF (v/v) solution after the completion of the connection of each amino acid, so as to ensure that the connection of the next amino acid can be completed until the connection of the last amino acid is successful, thereby preparing the compound of the formula (3);
3) Dissolving a compound of formula (3) and an intermediate (2.0 eq.) of formula (S4) in DMF solution, adding HCTU and DIPEA as coupling agents, and reacting at 30 ℃ for 1 hour to obtain a compound of formula (4);
4) Dissolving a compound of formula (4) in 20% piperidine DMF (v/v) solution to remove Fmoc protecting group, then adding PEG linker (2.0 eq.) with amino protection, adding HCTU and DIPEA as coupling agent, and reacting at 30 ℃ for 1 hour to obtain a compound of formula (5);
5) Removing Fmoc protecting group from compound of formula (5) in 20% piperidine DMF (v/v) solution, then adding JQ1-COOH (2.0 eq.) and HCTU and DIPEA as coupling agent, reacting for 1 hour at 30 ℃ to obtain compound of formula (6);
6) Adding trifluoroacetic acid mixed solution (95% TFA,2.5% phenol and 2.5% methane sulfonic acid) into the compound of the formula (6), cutting for 20 minutes, filtering, pouring the filtrate into cold diethyl ether, generating a large amount of dark green precipitate, centrifugally collecting the precipitate, and washing the precipitate with the cold diethyl ether for 2-3 times to obtain a crude product;
7) Further purifying the crude product by preparative high performance liquid chromatography, and freeze-drying to obtain a compound of formula (1);
12. a method of synthesizing a BRD 4-targeted, degradation-targeted chimeric compound of the polypeptide-coupled protein of claim 5, comprising:
1) The process of claim 10 for preparing a compound intermediate having the structure of formula (S10);
2) 2-chlorotrityl chloride resin and amino acid with Fmoc protecting group (2.0 eq.) were dissolved in DMF, HCTU and DIPEA were added as coupling agents, and reacted at 30 ℃ for 1 hour, amino acid connection order, i.e. polypeptide sequence order (A2: R-G-D), verifying the completion of each coupling step by using an ninhydrin detection method, and removing amino protecting groups of amino acids by using a 20% anhydrous piperidine DMF (v/v) solution after the completion of the connection of each amino acid, so as to ensure that the connection of the next amino acid can be completed until the connection of the last amino acid is successful, thereby preparing the compound of the formula (8).
3) Dissolving a compound of formula (8) and an intermediate (2.0 eq.) of formula (S10) in DMF solution, adding HCTU and DIPEA as coupling agents, and reacting at 30 ℃ for 1 hour to obtain a compound of formula (9);
4) Adding trifluoroacetic acid mixed solution (95% TFA,2.5% phenol and 2.5% methane sulfonic acid) into the compound of the formula (9), cutting for 20 minutes, filtering, pouring the filtrate into cold diethyl ether, generating a large amount of dark green precipitate, centrifugally collecting the precipitate, and washing the precipitate with the cold diethyl ether for 2-3 times to obtain a crude product;
5) Further purifying the crude product by preparative high performance liquid chromatography, and freeze-drying to obtain a compound of formula (1);
13. a method of synthesizing a BRD 4-targeted, degradation-targeted chimeric compound of the polypeptide-coupled protein of claim 6, comprising:
1) The process of claim 10 for preparing a compound intermediate having the structure of formula (S10);
2) Dissolving 2-chlorotrityl chloride resin and amino acid (2.0 eq.) with Fmoc protecting group in DMF, adding HCTU and DIPEA as coupling agent, reacting at 30deg.C for 1 hour, wherein the amino acid connection sequence is polypeptide sequence (C-R-G-D-K-G-P-D-C), wherein the side chain amino protecting group of lysine (K) is ivDde group, the amino protecting group of last cysteine is Boc group, verifying the completion of each coupling step by ninhydrin detection method, removing amino protecting group of amino acid by 20% anhydrous piperidine DMF (v/v) solution after each amino acid connection is completed, ensuring that the next amino acid can be connected until the last amino acid connection is successful, and preparing compound of formula (12);
3) Reacting the compound of formula (12) in 5% hydrazine hydrate DMF (v/v) solution at 30 ℃ for 30 minutes, and removing the ivDde protecting group on the side chain amino group of lysine (K) to prepare a compound of formula (13);
4) Dissolving a compound of formula (13) and an intermediate (2.0 eq.) of formula (S10) in DMF solution, adding HCTU and DIPEA as coupling agents, and reacting at 30 ℃ for 1 hour to obtain a compound of formula (14);
5) Adding trifluoroethylene into a compound of a formula (14), cutting for 20 minutes, filtering, pouring the filtrate into cold diethyl ether, generating a large amount of dark green precipitate, centrifugally collecting the precipitate, and washing the precipitate with the cold diethyl ether for 2-3 times to obtain a crude product;
6) Further purifying the crude product by preparative high performance liquid chromatography, and freeze-drying to obtain a compound of formula (15);
7) Dissolving a compound of formula (15) in an aqueous solution containing 40% dmso (v/v), stirring overnight to promote cyclization and disulfide bond formation;
8) Further purifying the crude product by preparative high performance liquid chromatography, and freeze-drying to obtain a compound of formula (1);
14. a pharmaceutical composition comprising a BRD4 targeted degradation polypeptide coupled protein degradation targeting chimeric compound of any one of claims 1-6, or a stereoisomer or pharmaceutically acceptable salt thereof.
15. For preventing and/or treating alpha v β 3 A BRD 4-targeted, degradation-degrading polypeptide-coupled protein degradation-targeted chimeric compound according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 14, for a disease associated with integrin receptor expression;
preferably, the said sum alpha v β 3 A disease associated with integrin receptor expression is selected from the group consisting of neoplastic diseases;
preferably, the neoplastic disease is selected from breast cancer.
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