CN115417910A

CN115417910A - Compound for targeted degradation of HSP90 protein based on tripterine, and preparation method and application thereof

Info

Publication number: CN115417910A
Application number: CN202211168825.5A
Authority: CN
Inventors: 甘学兰; 于超; 王帆
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2022-09-24
Filing date: 2022-09-24
Publication date: 2022-12-02

Abstract

The invention discloses a compound capable of targeted degradation of HSP90 protein synthesized based on tripterine, and a preparation method and application thereof, belonging to the field of biological medicine. The compound provided by the invention can be used as a protein hydrolysis targeting chimera, can directly degrade HSP90 protein in cancer cells, influence the function of client protein and block downstream signals thereof, so that the proliferation of the cancer cells is inhibited, the apoptosis of the cancer cells is promoted, and the compound is expected to be used for preparing novel antitumor drugs; the invention also provides a method for synthesizing the series of compound molecules based on the tripterine.

Description

Compound for targeted degradation of HSP90 protein based on tripterine, and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, and particularly relates to a compound for targeted degradation of HSP90 protein, and a preparation method and application thereof

Background

HSPs are important chaperones conserved in all organisms, the content of which is doubled compared to normal under heat stress, also called stress proteins, which are used as chaperones in the body to fold client proteins correctly, maintain the normal structure of proteins, and play an important role in regulating the balance of protein synthesis and degradation and protein localization, HSPs are classified into HSP100, HSP90, HSP70, HSP60 and small heat shock proteins according to homology differences and molecular weights, HSP90 is an important member of the HSPs family, and has functions of regulating the stability of substrate proteins, regulating the cell cycle, regulating apoptosis, coordinating hormone signaling, etc. in vivo, HSP90 is expressed in tumor cells far higher than that of normal cells, and its "client proteins" such as epidermal growth factor receptor 2, BCR/ABL fusion gene, protein kinase B, C-Raf, cyclin-dependent kinase 4, polp-like kinase 1, mutant p53, hypoxia-inducible factor 1, steroid hormone receptor, survivin and telomerase, reverse transcriptase and the like are mutated or highly expressed in tumors, and these "client proteins" play a critical role in tumor cell proliferation, anti-apoptosis, migration and invasion-related pathways, and thus can induce degradation of their "client proteins" by inhibiting the function of HSP90 and down-regulate the levels of various downstream proteins, thereby inhibiting the development of tumors by inhibiting various signal pathways, HSP90 has become an important target for tumor therapy, and HSP90 inhibitors have become a hotspot and frontier for anti-tumor research.

There are four main classes of HSP90 inhibitors currently available: geldanamycin (GA) and derivatives thereof, radicicol (radicico 1), neomycin (novobiocin) and a class of purine structure-based compounds have the problems of poor treatment effect, serious toxic reaction and the like, and off-target toxicity is easy to occur at high dose, so that the clinical development of geldanamycin is hindered. Since the first HSP90 inhibitor entered clinical research in the 90 th 20 th century, many pharmaceutical companies have been eagerly on the rise, researchers have adopted various development strategies such as combination, single drug or adaptive switching, and there are over 30 candidate drugs that entered clinical trials in 30 years, but unfortunately because of their high toxicity, no HSP90 protein inhibitor has sufficient efficacy to obtain FDA approval, and over 20 candidates have declared clinical failure. By far, no HSP90 inhibitor has been approved for the market worldwide.

The targeted degradation of proteins using proteolytic targeting chimeras (PROTACs) has become an emerging therapeutic approach. The ProTACs are heterogeneous bifunctional small molecules, two ends of which contain different ligands, one end is a ligand of E3 ligase, the other end is a ligand of intracellular target protein, and the two ligands are connected by a linker. The PROTAC can bind to both the target protein and the E3 ubiquitin ligase, recruit the target protein to the vicinity of the E3 ubiquitin ligase, and utilize an intracellular ubiquitin-proteasome system (UPS) to realize polyubiquitination of the target protein, which finally leads to recognition and degradation of the target protein by a proteasome. The PROTACs are used as a great technical innovation, have the advantages of changing the target from 'non-druggability' to 'druggability', small effective dosage, low toxicity, high selectivity, avoidance of drug resistance caused by overexpression and the like, and are a hotspot mode of the current tumor treatment. Therefore, it is very necessary to develop a corresponding PROTAC molecule against HSP90 protein.

Disclosure of Invention

In order to solve the problems in the background art, the invention provides a compound for targeted induction of HSP90 protein degradation based on tripterine, and a preparation method and application thereof. The compound can play a role in various cancer cells by directly degrading HSP90 protein, has stronger antitumor activity and is expected to be used for preparing antitumor drugs.

The above object of the present invention is achieved by the following technical solutions;

an HSP 90-targeted PROTAC compound has the following chemical structural formula:

where n =3,4,5,6.

Preferably, n =3.

Preferably, the compound is:

the synthesis method of the PROTAC molecule comprises the following synthetic route:

compared with the prior art, the invention has the beneficial effects that:

the compound provided by the invention takes tripterine as a target protein ligand, thalidomide as an E3 ligase ligand and carbon chains with different lengths as linkers to construct PROTACs. In-vitro anti-tumor activity test and in-vitro protein degradation experiments show that the compound provided by the invention shows good anti-tumor activity and excellent HSP90 protein degradation effect, can be used for preparing medicaments for treating or preventing tumors, and has great scientific and social values; the invention also provides a method for synthesizing the series of PROTAC molecules based on the tripterine.

Drawings

FIG. 1 is a scheme for the synthesis of compounds of the present invention;

FIG. 2A is the NMR spectrum of Compound 1 a;

FIG. 2B is a nuclear magnetic spectrum of Compound 1B;

FIG. 2C is the nuclear magnetic spectrum of Compound 1C;

FIG. 2D is the nuclear magnetic spectrum of Compound 1D;

FIG. 3A is a graph showing the results of an investigation of the concentration gradient degradation of HSP90 protein in 4T1 cells by Compound 1 a;

FIG. 3B is a graph showing the results of investigation of the concentration gradient degradation of HSP90 protein in U87 cells by Compound 1 a;

FIG. 3C is a study of the time-gradient degradation of HSP90 protein in 4T1 cells by Compound 1 a;

FIG. 3D is a study of the time-gradient degradation of HSP90 protein in U87 cells by Compound 1 a;

Detailed Description

The following examples of the present invention are described in further detail, and are intended to be illustrative, but not limiting, of the present invention.

The experimental procedures used in the following examples are, unless otherwise specified, all conventional procedures well known in the art.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Preparation of compound for targeted degradation of HSP90 protein based on tripterine

The synthetic route is as follows:

(1) Synthesis of Compound 4

Dissolving compound 2 (552mg, 2mmol), glycine tert-butyl ester (324mg, 2.4mmol) and N, N-diisopropylethylamine (660 ul, 4 mmol) in 5 mLN-methyl pyrrolidone, and carrying out reflux reaction at 90 ℃ for 12-18 h; adding purified water to quench and react after the reaction is finished, extracting with ethyl acetate for three times, combining organic phases, and washing the organic phases once with saturated salt water; the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product, which was purified by column chromatography to give compound 3.

Compound 3 (503mg, 1.3mmol) was dissolved in 20mL of dichloromethane, 20mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2h. Spin-dry under reduced pressure and remove the solvent to give compound 4.

(2) Synthesis of Compound 1a

Dissolving compound 5 (180mg, 0.4mmol) in 5mL of anhydrous N, N-dimethylformamide, adding sodium bicarbonate (100 mg, 1.2 mmol), stirring at room temperature for 20-30 min, then slowly dropping a solution of 1,3-dibromopropane (162mg, 0.8mmol) and 2.5mL of acetonitrile (dropping over 10 min), reacting at room temperature for 12-24 h (TLC detection), at which time the raw materials are completely reacted; adding purified water to quench and react, extracting the reaction solution with ethyl acetate for three times, combining organic phases, and purifying and washing the organic phases twice; the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product which was purified by column chromatography to give compound 6a.

Compound 4 (33.2mg, 0.1mmol) was dissolved in 10mLN, sodium carbonate (22mg, 0.2mmol) was added to N-dimethylformamide, and stirred at room temperature for 30min, then compound 6a (46mg, 0.08mmol) was added to the reaction flask, and the temperature was raised to 80 ℃ under nitrogen protection for about 4h. The organic phase was removed by concentration under reduced pressure to give a crude product, which was then purified on a thin layer plate to give compound 1a.

(3) Synthesis of Compound 1b

Referring to a preparation process flow chart and a preparation method of the

compound

1a, 1,3-dibromopropane is replaced by 1,4-dibromobutane (172.8mg, 0.08mmol), and the rest of the synthesis steps and the reaction conditions are the same, so that the compound 1b is obtained.

(4) Synthesis of Compound 1c

Referring to the preparation process scheme and the preparation method of

compound

1a, 1,3-dibromopropane was replaced with 1,5-dibromopentane (184mg, 0.08mmol), and the rest of the synthesis steps and reaction conditions were the same, giving compound 1c.

(5) Synthesis of Compound 1d

Referring to the preparation process flow diagram and the preparation method of

compound

1a, 1,3-dibromopropane was replaced by 1,6-dibromohexane (172.8mg, 0.08mmol), and the rest of the synthesis steps and reaction conditions were the same, giving compound 1d.

Example 2

CCK-8 tests the inhibitory activity of the compounds of the invention on different tumor cells:

inoculating different tumor cells (4 T1, U87, A549, hepg2, MDA-MB-231) into T25 flasks, placing at 37 ℃ with DMEM medium containing 10% FBS, 5% CO ₂ When the cell density reached 80% or more, the medium in the flask was discarded, the flask was washed twice with PBS, 1ml of cells was digested with trypsin, after the cells had fallen off, 10% FBS-containing DMEM medium was added to stop the digestion, the cells were transferred to a centrifuge tube and centrifuged for 3min, after the centrifugation was completed, the supernatant was discarded, 1ml of 10% FBS-containing DMEM medium was added to resuspend the cells, the cell number in the cell suspension was counted with a cell counting plate, and the cell suspension was then cultured in 2X 10 cells per well ⁴ Cells were seeded in 96-well plates at a density of 100. Mu.l per well. The next day, medium was discarded, 100. Mu.l of fresh medium was added to the control group, and 5% CO at 37 ℃ with different concentrations of drug-containing medium added to the experimental group, each group having 5 parallel wells ₂ The culture was continued for 72 hours, 10. Mu.l of CCK-8 solution was added to each well, and the culture was continued for 1 to 4 hours, and the absorbance was measured at 450nm with a microplate reader. Cell viability was calculated from absorbance and IC was calculated ₅₀ Values, cell viability (%) = (control OD-experimental OD)/control OD × 100%, experiments were repeated 3 times and averaged. The in vitro antitumor activity of the compounds of the invention is shown in table 1.

TABLE 1 inhibition of the proliferative Activity of Compounds 1a-d and Tripterine on different tumor cells (Compound treatment for 72h, IC of Compound) ₅₀ (μM))

As can be seen from Table 1, five tumor cells of Compound 1a all have lower IC ₅₀ The inhibition of tumor cells was better and the sensitivity of different tumor cells to the drug was different, with 4T1 cells and U87 cells being most sensitive to the compound 1a. Therefore, the compound has obvious effect of resisting tumor cell proliferation, is effective to various tumor cells, and is expected to become a new anti-tumor medicament.

Example 3

WesternBlot test for degradation of HSP90 protein by compound 1a

1, cell treatment: taking 4T1 cells and U87 cells in the logarithmic growth phase into a 6-well plate, treating the cells with the compound 1a at the corresponding concentration after the cells are attached to the wall, and collecting the cells after incubation for corresponding time;

2, extracting cell whole protein: the medium in the 6-well plate was discarded, washed three times with PBS, 100. Mu.l of 1% PMSF-containing RIPA lysate was added to each well after discarding PBS, the cells were lysed on ice for 15min, the lysate was collected, centrifuged at 4 ℃ at 12000g for 20min, and the supernatant was taken for protein quantification using BCA kit. Diluting the protein to the same concentration by PBS after the quantification is finished, adding 5 XLoadingBuffer, fully shaking and uniformly mixing, denaturing at 100 ℃ for 5 minutes, and storing at-20 ℃ after uniform mixing or directly using for WesternBlot detection;

3, preparing separation gel and concentrated gel with proper concentration: the concentration of the separation gel is 10 percent, and the concentration of the concentrated gel is 5 percent.

4, loading: protein samples were centrifuged and mixed well and loaded onto SDS-PAGE gel wells. And adjusting the sample loading volume according to the protein quantification result. The loading amount of the protein is about 20 mug;

5, electrophoresis: carrying out electrophoresis at 80V after electrophoresis is switched on, carrying out electrophoresis at 120V after a protein sample enters separation gel, and stopping electrophoresis when bromophenol blue is about to run out of the separation gel;

(6) and (3) membrane rotation, namely removing the rubber plate after electrophoresis is finished, slightly prying the glass plate, and scraping off the concentrated glue. And (3) assembling the cut glue, the PVDF film soaked with methanol and the filter paper in a transfer clamp in sequence to ensure that no bubbles exist between every two layers. The assembly sequence is as follows: transfer clip black side (negative electrode) -sponge pad-filter paper-glue-PVDF membrane-filter paper-sponge pad-transfer clip transparent side (positive electrode). Inserting the transfer clip into an electrotransfer groove, adding a film transfer liquid, inserting into an ice box, and placing in an ice bath for 90min at 250mA for film transfer;

(7) and (3) sealing: after the membrane is transferred, the power supply is cut off, the PVDF membrane is taken out and soaked in 5 percent of skimmed milk powder, and the PVDF membrane is sealed for 1 hour at room temperature;

(8) incubating the primary antibody: the primary antibody was diluted to the appropriate concentration with the primary antibody dilution and incubated with the blocked PVDF membrane overnight at 4 ℃. After incubation, the membrane was washed on a decolouring shaker at room temperature for 3 times with TBST, 10min each time;

(9) incubation of secondary antibody: and (3) diluting the secondary antibody to a proper concentration by using a secondary antibody diluent, and incubating the secondary antibody and the PVDF membrane incubated with the primary antibody on a decoloration shaking table at room temperature for 1h. After incubation, the membrane was washed on a decolouring shaker at room temperature for 3 times with TBST, 10min each time;

exposure to r: and uniformly mixing the two reagents A and B of the ECL in an equal volume in an EP tube, uniformly dripping the mixture on the protein surface of the PVDF membrane, and exposing and imaging to obtain a protein band result.

The degradation activity of compound 1a of the present example on HSP90 protein is as follows: in 4T1 cells and U87 cells, the degradation of HSP90 protein by compound 1a was time-dependent and concentration-dependent, the more HSP90 protein was degraded by compound 1a with time, and the more HSP90 protein was degraded by a higher concentration of compound 1a in the same time, and the results are shown in fig. 3.

Claims

1. A compound that targets HSP90 protein degradation, or a pharmaceutically acceptable salt thereof, having the chemical structural formula:

wherein n is any natural number from 3 to 6.

2. The compound or pharmaceutically acceptable salt thereof targeted for degradation of HSP90 proteins according to claim 1, wherein n =3.

3. The method for synthesizing the HSP90 protein targeted degradation compound of claim 1, wherein the synthetic route is as follows:

the method comprises the following steps:

(1) Synthesis of Compound 4:

(1) dissolving a compound 2, glycine tert-butyl ester and N, N-diisopropylethylamine in N-methylpyrrolidone, and carrying out reflux reaction at 90 ℃ for 12-18 h; adding purified water to quench and react after the reaction is finished, extracting for three times by using ethyl acetate, combining organic phases, and washing the organic phases once by using saturated saline solution; drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the organic phase under reduced pressure to obtain a crude product, and purifying by column chromatography to obtain a compound 3;

(2) dissolving compound 3 in a mixed solvent of dichloromethane and trifluoroacetic acid (dichloromethane: trifluoroacetic acid = 1:1), and stirring at room temperature for 2h; after the reaction is finished, performing reduced pressure spin drying, and removing the solvent to obtain a compound 4;

(2) Synthesis of Compound 1:

(1) dissolving the compound 5 in anhydrous N, N-dimethylformamide, adding sodium bicarbonate, stirring at room temperature for 20-30 min, slowly dropping 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane, 1,6-dibromohexane and acetonitrile solution, and reacting at room temperature for 12-24 h; adding purified water to quench the reaction after the reaction is finished, extracting the reaction product for three times by using ethyl acetate, combining organic phases, and washing the organic phases twice by using purified water; drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the organic phase under reduced pressure to obtain a crude product, and purifying by column chromatography to obtain compounds 6a-6d;

(2) dissolving the compound 4 in anhydrous N, N-dimethylformamide, adding sodium carbonate, stirring at room temperature for 30min, adding the compound 6a or 6b or 6c or 6d into a reaction bottle, and heating to 80 ℃ under the protection of nitrogen to react for about 4h; after the reaction was completed, the reaction mixture was cooled to room temperature, and the organic phase was removed by concentration under reduced pressure to obtain a crude product, which was then purified using a thin layer plate to obtain compounds 1a to 1d.

4. The production method according to claim 3, characterized in that: in the step (1), the molar ratio of the compound 2 to the tert-butyl glycinate and the N, N-diisopropylethylamine is 1:1.2:2.

5. the production method according to claim 3, characterized in that: in the step (2), the molar ratio of the compound 5 to sodium bicarbonate and 1,3-dibromopropane or 1,4-dibromobutane or 1,5-dibromopentane or 1,6-dibromohexane is 1:3:2;

the mol ratio of the compound 4 to the sodium carbonate and the compound 6a or 6b or 6c or 6d is 5:10:4.

6. a pharmaceutical composition characterized by: the HSP90 protein targeted degradation compound or the pharmaceutically acceptable salt thereof as claimed in claim 1 is used as an active ingredient or a main active ingredient, and is prepared into a pharmaceutically acceptable dosage form together with a pharmaceutically acceptable carrier.

7. Use of the HSP90 protein targeted degrading compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a targeted HSP90 degrading agent.

8. Use of the HSP90 protein targeted degrading compound of claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention or treatment of tumors.