CN116751199A

CN116751199A - Mitochondrial protease targeted chimera, preparation method and application

Info

Publication number: CN116751199A
Application number: CN202310646457.9A
Authority: CN
Inventors: 方晓红; 王大驰; 王文溪; 周卫; 甘绍举
Original assignee: Institute Of Basic Medicine And Oncology Chinese Academy Of Sciences Preparatory
Current assignee: Institute Of Basic Medicine And Oncology Chinese Academy Of Sciences Preparatory
Priority date: 2023-06-02
Filing date: 2023-06-02
Publication date: 2023-09-15

Abstract

The invention discloses a mitochondrial protease targeted chimeric, a preparation method and application thereof, and belongs to the technical field of pharmaceutical chemistry. The mitochondrial protease targeting chimera comprises an intra-mitochondrial AAA+ protease family binding structure and an intra-mitochondrial protein binding structure, wherein the intra-mitochondrial AAA+ protease family binding structure and the intra-mitochondrial protein binding structure are linked through a linker. The mitochondrial protease targeting chimera is constructed by utilizing AAA+ protease family enzymes in mitochondria, and the protein is degraded by the AAA+ protease in the mitochondria, so that the targeting degradation means of the protein target in organelles is realized for the first time, and the mitochondrial protease targeting chimera can be used for preparing eukaryotic cell protein degradation medicines based on AAA+ protease.

Description

Mitochondrial protease targeted chimera, preparation method and application

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a mitochondrial protease targeting chimeric, a preparation method and application thereof.

Background

The interaction of the difunctional chemical small molecules with the target protein and the protein degrading enzyme simultaneously is utilized to realize the Target Protein Degradation (TPD), thereby bringing a new concept for drug research and development. At present, through two natural protein degradation systems in cells, namely ubiquitin-proteasome system (UPS) and lysosome degradation pathway, specific and efficient degradation of related proteins of diseases can be realized, thereby achieving the effect of treating the diseases. Classical methods in drug discovery processes are generally aimed at identifying high affinity small molecules that modulate the activity of target proteins. This occurs in an occupied driving manner, with the inhibitor binding to its target and occupying the binding site, thereby inhibiting the target function. This strategy has been very successful for many targets that contain manageable active or allosteric sites (e.g., enzymes or receptors). However, most proteins remain problematic, resulting in 80-85% of human proteomes being unable to be formulated due to the lack of available binding pockets and suitable chemicals. The greatest advantage of TPD over traditional small molecule inhibitors is that only binding agents are required to recruit the protein of interest for degradation, rather than high affinity inhibitors, which greatly solves the problem of protein non-patentability. The most widely used protein degradation by ubiquitin protease system is PROTAC bifunctional small molecule: the protein degradation targeting chimeras (PROTACs) comprise three parts, namely an E3 ubiquitin ligase ligand, a target protein ligand and a specially designed linker, wherein the two active ligands are connected together through the linker structure. After binding the E3 ubiquitin ligase and the target protein by their ligand, the protein is tagged with a ubiquitination tag and the target protein is degraded by the ubiquitination process. Due to the high dependency of ubiquitin protein systems, the PROTACs technology is mainly used to degrade cytoplasmic and nuclear protein targets. Subsequently, many techniques for degrading proteins using lysosomal pathways including endocytic-lysosomal pathway and autophagic-lysosomal pathway, etc., such as LYTACs, ATTECs, AUTACs, etc., have emerged, expanding the scope of degradable targets.

However, targeted degradation of protein targets in organelles has not been achieved to date. Protein abnormalities in organelles are associated with a number of diseases, most commonly in mitochondria. Abnormalities in mitochondrial protein homeostasis are important features and driving factors for a range of human diseases, such as Leber hereditary optic neuropathy, maternal deafness, type II diabetes, parkinson's disease, and various cancers, among others. In addition, when cells are stimulated by stress, a large number of variable proteins in the cytoplasm are also transported into mitochondria and degraded by aaa+ proteases. LRPPRC protein is a newly discovered marker for lung adenocarcinoma tumor: is a leucine-rich triangular pentapeptide repetitive motif protein) is a mitochondrial protein. Small molecule GAA is obtained by high throughput screening, which binds to LRPPRC protein, resulting in a substantial decrease in its stability, followed by proteolytic recognition of the LRPPRC protein. Although this is also a method of protein degradation, we cannot design new proteolytic systems modularly therewith. While proteins located on the outer mitochondrial membrane can still be efficiently degraded by the traditional ubiquitin-proteasome pathway, the mitochondrial interior is not degraded by the ubiquitination-proteasome pathway due to the absence of ubiquitination system proteins.

Disclosure of Invention

In a first aspect, the present invention aims to provide a mitochondrial protease targeting chimera capable of targeting degradation of mitochondrial matrix proteins, which can be used for preparing a eukaryotic intracellular protein degradation drug based on AAA+ proteases.

The technical scheme adopted by the invention for achieving the purpose is as follows:

a mitochondrial protease targeting chimera comprising a binding structure of an intra-mitochondrial aaa+ protease family and a binding structure of an intra-mitochondrial protein, the binding structure of the intra-mitochondrial aaa+ protease family linked to the binding structure of the intra-mitochondrial protein by a linker. The mitochondrial protease targeting chimera (MtPTAC) is constructed by utilizing AAA+ protease family in mitochondria, and the target degradation means of protein targets in organelles is realized for the first time by degrading proteins by AAA+ protease in mitochondria, so that the mitochondrial protease targeting chimera (MtPTAC) can be used for preparing medicines for degrading proteins in eukaryotic cells based on AAA+ protease.

In one embodiment, the binding structure of the intramitochondrial aaa+ protease family is selected from ONC201 derivatives, which are compounds represented by the structure of formula E;the method comprises the steps of carrying out a first treatment on the surface of the In formula E, X is selected from halogen. Targeting Protein Degradation (TPD) is an emerging protein regulatory technology. Currently, all TPDs developed in eukaryotic cells rely on ubiquitin-proteasome or lysosomal systems and therefore are unable to target proteins in membrane organelles (e.g., mitochondria) that lack proteasome and lysosomes. The mitochondria targeting chimera (MtPTAC) can solve the problems, can effectively degrade mitochondrial proteins which are closely related to various diseases, has important significance for subsequent drug development, and can become a new direction.

In one embodiment, the binding structure of the intramitochondrial protein is selected from IMT or 6, 8-bis (benzylthio) octanoic acid; IMT is a compound represented by the structure of formula 2F; linker is PEG compound;。

in one embodiment, the mitochondrial protease targeted chimera is a compound represented by the structure of formula 3B or formula 3C, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof;

；

the method comprises the steps of carrying out a first treatment on the surface of the In formula 3B or formula 3C, n is any natural number from 1 to 10.

In one embodiment, in formula 3B or formula 3C, n is any natural number from 1 to 5.

In one embodiment, in formula 3B or formula 3C, n is any natural number from 1 to 3.

In one embodiment, the mitochondrial protease targeted chimera is a compound represented by the structure of formula 3B-1, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof;

. The MtPTAC is a difunctional small molecule, one end of the MtPTAC can be combined with mitochondrial casein hydrolase P (ClpP), the other end of the MtPTAC can be combined with target protein (mitochondrial RNA polymerase POLRMT), the hydrolase activity of the ClpP can be activated, and meanwhile, the target protein is close to the ClpP, and the target protein in mitochondria is specifically hydrolyzed, so that the aim of degrading mitochondrial matrix protein in a targeted way is fulfilled. The effect (more than 80% of degradation in 24 hours of treatment) and the specificity of the mitochondrial precursor targeting chimera (MtPTAC) of the invention in intracellular degradation of proteins have been verified, and a significant decrease in mtDNA was detected; in animals, the tumors in the treatment group treated with the mitochondrial precursor targeted chimera (MtPTAC) of the invention were significantly smaller than those in the placebo control group, as well as decreased levels of target protein and mtDNA were detected. Therefore, the mitochondrial precursor targeting chimera (MtPTAC) can effectively degrade mitochondrial matrix proteins which are closely related to various diseases, has important significance for subsequent drug development and can become a new direction; the mitochondrial precursor targeting chimera (MtPTAC) also has an anti-tumor application prospect.

In one embodiment, the mitochondrial protease targeted chimera is a compound represented by the structure of formula 3C-2, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, or the mitochondrial protease targeted chimera is a compound represented by the structure of formula 3C-3, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof;

；

. The method comprisesMtPTAC has a significant proteolytic capacity.

In a second aspect, the present invention aims to provide an application of an ONC201 derivative shown in a formula E structure in preparing a mitochondrial protease targeting chimera. The ONC201 derivative provided by the invention has an active site capable of providing a connection reaction, and can be linked with a binding structure of an intra-mitochondrial protein through a connector for preparing a mitochondrial protease targeting chimera.

In a second aspect, the present invention aims to provide a method for preparing the above-mentioned mitochondrial protease-targeted chimera.

a method for preparing a mitochondrial protease targeted chimera comprising the steps of:

step 1), linking an ONC201 derivative and a PEG compound to obtain an intermediate 3A, wherein the intermediate is a compound shown in a structure of a formula 3A;

Step 2), linking the intermediate 3A with an inhibitor to obtain a mitochondrial protease targeting chimeric body;

the method comprises the steps of carrying out a first treatment on the surface of the In formula 3A, n is any natural number from 1 to 10.

In one embodiment, the PEG compound isN is any natural number from 1 to 10.

In a preferred embodiment, in the PEG compound, n is any natural number from 1 to 5.

In a more preferred embodiment, in the PEG compound, n is any natural number from 1 to 3.

In a preferred embodiment, in formula 3A, n is any natural number from 1 to 5.

In a more preferred embodiment, in formula 3A, n is any natural number from 1 to 3.

In a third aspect, the present invention is directed to a compound represented by the structure of formula 3A. The compound shown in the structure of the formula 3A is a template for a mitochondrial protease targeted chimera, and the mitochondrial protease targeted chimera template can be used for modularly designing an MtPTAC system suitable for mitochondrial protein degradation.

In a fourth aspect, the present invention is directed to the use of a compound represented by the structure of formula 3A for the preparation of a mitochondrial protease targeted chimera.

In a fifth aspect, the present invention provides an application of the above-mentioned mitochondrial protease targeting chimera in preparing a drug for protein degradation, wherein the protein is eukaryotic intracellular protein.

In one embodiment, the degradation of the protein in eukaryotic cells is based on aaa+ proteases.

In one embodiment, the mitochondrial protease targeting chimera is selected from the group consisting of a compound represented by the structure of formula 3B-1 and/or a compound represented by the structure of formula 3C-2 and/or a compound represented by the structure of formula 3C-3.

In a sixth aspect, the present invention aims to provide an application of the above-mentioned mitochondrial protease targeting chimera in preparing medicines for treating abnormal mitochondrial function and/or antitumor medicines.

In one embodiment, the use of a mitochondrial protease targeting chimera in the manufacture of a medicament for targeted degradation of a mitochondrial matrix protein.

In one embodiment, the use of a mitochondrial protease targeted chimera in the manufacture of an antitumor drug, the mitochondrial protease targeted chimera being a compound represented by the structure of formula 3B-1.

In a preferred embodiment, the use of a mitochondrial protease targeted chimera in the manufacture of an anti-lung cancer medicament, the mitochondrial protease targeted chimera being a compound represented by the structure of formula 3B-1.

In a sixth aspect, the present invention is directed to a pharmaceutical composition comprising the above-described mitochondrial protease targeting chimera.

The invention has the following beneficial effects: the mitochondrion targeting chimera (MtPTAC) is constructed by utilizing an AAA+ protease family in mitochondria, and the targeted degradation of a protein target point in a organelle is realized through the degradation of proteins by the AAA+ protease family in mitochondria for the first time; the mitochondria targeting chimera (MtPTAC) can effectively degrade mitochondrial matrix proteins, and can be used for preparing medicines for treating abnormal mitochondrial functions; the mitochondrial precursor targeting chimera (MtPTAC 3B-1) has an anti-tumor effect and can be used for preparing anti-tumor drugs.

Drawings

FIG. 1 is a sequence diagram of ClpP and POLRMT for ITC;

FIG. 2 is an electrophoretogram of ClpP and POLRMT;

FIG. 3 is an in vitro hydrolysis of a target protein by MtPTAC; (a) Immunoblots for POLRMT in A549 cells treated with different concentrations of MtPTAC 3B-1, mtPTAC 3B-2, mtPTAC 3B-3; (b) Immunoblots of POLRMT in a549 cells after 72 hours of treatment with MtPTAC 3B-1 at different concentrations (0 μΜ, 5 μΜ, 10 μΜ and 20 μΜ);

FIG. 4 is an immunoblot of POLRMT in different cell lines treated with different concentrations of MtPTAC 3B-1;

FIG. 5 is an immunoblot of POLRMT in PC9 and A549 cells treated with 40. Mu.M MtPTAC3B-1 at different times;

FIG. 6 is a qRT-PCR analysis of POLRMT mRNA levels in A549 cells and PC9 cells treated with different concentrations of MtPTAC 3B-1;

FIG. 7 is a proteomic analysis of the effect of 40. Mu.M MtPTAC3B-1 treatment on mitochondrial proteins; the thermal plot shows fold change in mitochondrial protein abundance (log) after 6 hours (a) and 12 hours (B) of MtPTAC3B-1 treatment compared to DMSO treatment ₂ FC), mitochondrial protein species were provided by the mitocarta3.0 database;

FIG. 8 is a proteomics of the effect of 40. Mu.M MtPTAC3B-1 treatment on mitochondrial proteinsAnalyzing; the thermal plot shows fold change in mitochondrial protein abundance (log) after 6 hours (a) and 12 hours (B) of MtPTAC3B-1 treatment compared to DMSO treatment ₂ FC), mitochondrial protein species are provided by UniProt database; (c) Immunoblots of mitochondrial protein ATP5A in a549 and PC9 cells treated with different concentrations of MtPTAC3B-1 for 24 hours;

FIG. 9 is an LC-MS assay for MtPTAC3B-1 in mitochondria; after treatment of A549 cells with 20. Mu.M MtPTAC3B-1 for 0h (a), 8h (B), 16h (c) and 24h (d), mitochondria were extracted and analyzed for signals of MtPTAC3B-1 by LC-MS; (e) signal for MtPTAC3B-1 standard; by normalizing the peak area of MtPTAC3B-1 in HPLC to the amount of protein in each sample (f), the relative level of MtPTAC3B-1 in mitochondria during the MtPTAC3B-1 treatment time was obtained, LC-MS results showed that MtPTAC3B-1 entered mitochondria in a time dependent manner;

FIG. 10 is a graph showing measurement of binding of small molecule MtPTAC 3B-1 to ClpP by ITC;

FIG. 11 is a graph showing the measurement of binding of small molecule MtPTAC 3B-1 to POLRMT by ITC;

FIG. 12 is a graph of MtPTAC 3B-1 inducing POLRMT degradation by bringing POLRMT close to ClpP; (a) FLIM-FRET schematic to monitor the proximity of POLRMT and ClpP, representative life heat maps of HEK293 cells with corresponding constructs and MtPTAC 3B-1 or IL treatment; when FRET occurs between the mneon green and mstarlet-I, the lifetime of the mneon green is shortened, (b) a scatter plot of lifetime data per set; immunoblotting of POLRMT in a549 cells treated with AL plus free IMT (c) or IL plus free ONC201 (d);

FIG. 13 is a molecular dynamics simulation of the formation of a POLRMT/MtPTAC 3B-1/ClpP ternary complex; (a) The last 20ns track of ternary structure molecular dynamics simulation under 310.15K, drawing a structure every 2ns, representing a time step by color, and changing in red-white-blue form, and the track simulation result proves that the ClpP/MtPTAC 3B-1/POLRMT complex is stable; (b) Proteins in ternary structures showed smaller RMSDs in all conformations relative to statistics of RMSDs for their representative structures in the past 20 ns;

FIG. 14 shows a decrease in MtPTAC 3B-1 COX-1AndCOX-2a549 cells were treated with MtPTAC3B-1 with real-time PCR detectionCOX-1AndCOX-2is used for the preparation of the mRNA of (A),GAPDHis used as an internal reference;

FIG. 15 shows that MtPTAC3B-1 reduced mitochondrial DNA levels, A549 cells (a) and PC9 cells (B) were treated with 40. Mu.M MtPTAC3B-1 at different times, mitochondrial DNA was detected by real-time PCR, and DNA levels of DNA methyltransferase 1 (DNMT 1, a gene in the nuclear genome) were used as internal references;

FIG. 16 is a representative proliferation curve of A549 (a) and PC9 (B) cells treated with different concentrations of MtPTAC 3B-1;

FIG. 17 is a representative clone image (a) of A549 and PC9 cells treated at different concentrations of MtPTAC3B-1, quantifying the number of cell clones in each group (B);

FIG. 18 is a flow cytometer of treating A549 cells with 40. Mu.M MtPTAC 3B-1;

FIG. 19 is a flow cytometry plot of HEK293 cells treated with 40 μM MtPTAC 3B-1;

FIG. 20 is a graph showing the effect of MtPTAC3B-1 on HEK293 cell viability;

FIG. 21 is a graph showing the effect of MtPTAC3B-1 or MtPTAC 3B-2 on A549 cell viability (a), the effect of MtPTAC3B-1 or IMT on A549 cell viability (B);

FIG. 22 is a comparison of MtPTAC3B-1 and MtPTAC 3B-2 in tumor growth in vivo, (a) images of subcutaneous tumors isolated from mice treated with MtPTAC3B-1, mtPTAC 3B-2 or placebo for 14 days, (B) quantification of tumor weight obtained from mice treated with either MtPTAC3B-1, or MtPTAC 3B-2 or placebo;

FIG. 23 is a graph showing changes in body weight of mice before and after MtPTAC 3B-1 treatment;

FIG. 24 is an immunoblot of POLRMTs from tumors obtained from mice treated with MtPTAC 3B-1 or control placebo, one full length POLRMT (F-POLRMT) and two cleaved POLRMTs (C-POLRMT) were detected by the MtPTAC 3B-1 group;

FIG. 25 is an immunoblot of oxidative phosphorylation complexes in tumor tissue from mice treated with placebo or MtPTAC 3B-1;

FIG. 26 is an immunoblot of A549 cells treated with different concentrations of protein degrading agents MtPTAC 3C-1, mtPTAC 3C-2, mtPTAC 3C-3 for 24 h.

Detailed Description

The present invention will be further described in detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1:

synthesis and purification of mitochondrial protease targeted chimeras (MtPTAC 3B)

All synthetic intermediates and final products in this example were verified by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). Nuclear Magnetic Resonance (NMR) analysis was performed on a Bruker 400MHz spectrometer. Unless otherwise indicated, spectra were measured at 298K and referenced to solvent chemical shift. NMR chemical shift [ ] δ) Expressed in ppm, the coupling constant [ ]J) Expressed in Hz. The data are reported as follows: (s) singlet, (d) singlet, (t) triplet, (q) quadbate, (m) polymorphism, (br) broad. ESI (+) high resolution mass spectra were obtained on a Thermo Scientific Orbitrap Exploris 480 quadrupole time-of-flight mass spectrometer equipped with a Z-spray interface with a mass range of 100-1200 Da.

This example was obtained from a commercial supplier (as shown in table 1) with chemicals without further purification. Flash chromatography was performed using silica gel (200-300 mesh) as stationary phase.

TABLE 1 chemical

1. Synthesis of ONC201 derivatives

Synthetic route for ONC201 derivatives

Reaction conditions: (a) methyl 3-bromopropionate, potassium carbonate, acetonitrile, 2 hours; (b) sodium methoxide, xylene, reflux, overnight; (c) methyl iodide, methanol, at room temperature overnight; (d) P-bromobenzylamine, 1, 4-dioxane, reflux overnight; (e) sodium methoxide, methanol, reflux overnight.

1.1 Synthesis of dimethyl 3,3' - (Benzylazediyl) dipropionate (1A)

Aniline (2.14 g, 20 mmol, 1 eq.) and methyl 3-bromopropionate (7.5 g, 44 mmol, 2.2 eq.) are dissolved in acetonitrile (50 ml), triethylamine (4.5 g, 44 mmol, 2.2 eq.) is added and stirred overnight at room temperature. The mixture was then diluted with water (200 ml), the aqueous layer was extracted with dichloromethane (200 ml×2), and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue purified by flash column chromatography to give 1A (2.7 g, 48.3% yield). ¹ H NMR(400 MHz，CDCl ₃ -d)δ7.26-7.18(m，3H)，7.17-7.12(m，2H)，3.70(s，2H)，3.57(s，6H)，3.00-2.65(t，J= 6.5 Hz，4H)，2.43(t，J= 6.5 Hz，4H)。 ¹³ C NMR(100 MHz，CDCl ₃ -d)δ173.2, 140.1, 128.4, 128.1, 127.0, 53.7, 51.6, 44.4, 34.5. Theoretical calculated value C ₁₅ H ₂₂ NO ₄ ⁺ [M+H] ⁺ =280.1543, hrms (ESI) actual calculated: 280.1545.

synthesis of methyl 1, 2, 1-benzyl-4-oxopiperidine-3-carboxylate (1B)

1A (5.32 g, 20 mmol, 1 eq.) was dissolved in anhydrous xylene, sodium methoxide (1.2 g, 22 mmol, 1.1 eq.) was added and the reaction mixture refluxed overnight. The mixture was then diluted with ice water (100 ml), the aqueous layer was extracted with dichloromethane (100 ml×2), and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 1B (3.2 g, 64.8% yield). ¹ H NMR(400 MHz，DMSO-d ₆ )δ7.45-7.39(m，2H)，7.36(dq，J= 7.7，2.1 Hz，3H)，4.73(s，2H)，3.58(s，3H)，3.51(s，1H)，3.44(t，J= 7.6 Hz，2H)，2.31(dd，J= 8.2，7.1 Hz，2H)，1.99(dt，J= 12.1，7.1 Hz)。 ¹³ C NMR(100 MHz，DMSO-d ₆ )δ186.0，165.3，160.0，136.0，129.4，128.6，128.4，99.1，70.3，59.6, 50.7, 46.1, 36.1. Theoretical calculated value C ₁₄ H ₁₈ NO ₃ ⁺ [M+H] ⁺ =248.1281, hrms (ESI) actual calculated: 248.1284.

1.3, 2- (methylthio) -4, 5-dihydro-1HSynthesis of imidazole hydroiodide (1C)

4, 5-dihydro-1HImidazole-2-thiol (1.2 g, 10 mmol, 1 eq) was dissolved in methanol, methyl iodide (1.6 g, 11 mmol, 1.1 eq) was added and the mixture was stirred at room temperature overnight. The solvent was removed on a rotary evaporator and the solid was washed with petroleum ether to give 1C (2.6 g, 96.3% yield). ¹ H NMR(400 MHz，DMSO-d ₆ )δ9.98(s，1H)，3.36(s，4H)，2.63(s，3H)。 ¹³ C NMR(100 MHz，DMSO-d ₆ )δ170.9, 45.7, 14.0. Theoretical calculated value C ₄ H ₉ N ₂ S ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =117.0481: 117.0481.

1.4、N- (4-bromobenzyl) -4, 5-dihydro-1HSynthesis of imidazol-2-amine (1D)

1C (2.3 g, 10 mmol, 1 eq.) was dissolved in 1, 4-dioxane, p-bromobenzylamine (1.86 g, 10 mmol, 1 eq.) was added and the mixture refluxed overnight. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 1D (0.45 g, 17.7% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.58-7.49(m，2H)，7.30-7.14(m，1H)，4.38(s，2H)，3.73(s，4H)，3.65(s，1H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ135.6, 131.6, 128.9, 121.4, 45.2, 44.3, 42.8. Theoretical calculated value C ₁₀ H ₁₃ BrN ₃ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =254.0287: 254.0289.

1.5, 7-benzyl-4- (4-bromobenzyl) -2,4,6,7,8, 9-hexahydroimidazo [1,2 ]a]Pyrido [3,4 ]e]Pyrimidine-5 (1)H) Synthesis of ketone (1E)

1B (243 mg, 1 mmol, 1 eq.) and 1D (247 mg, 1 mmol, 1 eq.) were dissolved in anhydrous methanol and sodium methoxide (112 mg3 mmoles, 3 eq.) the mixture was refluxed overnight. The mixture was then diluted with ice water (20 ml), the aqueous layer was extracted with dichloromethane (20 ml×2) and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 1E (145 mg, 32% yield). ¹ H NMR(400 MHz，DMSO-d ₆ )δ7.50-7.45(m，2H)，7.36-7.29(m，4H)，7.29-7.22(m，3H)，4.86(s，2H)，3.93(t，J= 9.1 Hz，2H)，3.70(dd，J= 9.8，8.3Hz，2H)，3.61 (s，2H)，3.02 (s，2H)，2.62 (t，J= 5.7 Hz，2H)，2.55-2.51(m，2H)。 ¹³ C NMR(100 MHz，DMSO-d ₆ )δ161.1, 152.4, 147.8, 138.7, 137.2, 131.5, 130.5, 129.2, 128.7, 127.5, 120.6, 99.7, 61.8, 50.5, 49.2, 48.8, 46.8, 44.4, 26.3. Theoretical calculated value C ₂₃ H ₂₄ BrN ₄ O ⁺ [M+H] ⁺ =451.1128, hrms (ESI) actual calculated: 451.1126.

2. synthesis of IMT

Synthetic route for IMT

Reaction conditions: (a) Sodium hydride, diethyl carbonate, tetrahydrofuran, reflux, overnight; (b) resorcinol, trifluoroacetic acid, reflux, overnight; (c) Cesium carbonate and a salt of cesium carbonate,N，N-dimethylformamide, ethyl 2-bromopropionate, at room temperature overnight; (d) 1N sodium hydroxide, water: methanol=1:2, room temperature, overnight; (e) 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-hydroxybenzotriazole,N，N-diisopropylethylamine, ethyl 3-piperidinecarboxylate, 40 ℃ overnight; (f) 1N sodium hydroxide, water: methanol=1:2, room temperature, overnight; (g)N，N，N′，N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate,N，Ndiisopropylethylamine, tert-butyl 2- (2- (2-aminoethoxy) ethoxyethylcarbamate, at 40℃overnight.

Synthesis of ethyl 1, 3-oxo-3- (o-tolyl) propionate (2A)

Will beOMethylbenzophenone (4 g, 29.9 mmol, 1 eq.) was dissolved in anhydrous tetrahydrofuran, diethyl carbonate (4.4 g, 36 mmol, 1.2 eq.) and sodium hydride (1.44 g, 36 mmol, 1.2 eq.) were added and the mixture refluxed overnight. The mixture was then diluted with ice water (200 ml), the aqueous layer was extracted with dichloromethane (200 ml×2), and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 2A (4.1 g, 66.4% yield). ¹ H NMR(400 MHz，DMSO-d ₆ )δ7.82(dd，J= 7.7，1.3 Hz，1H)，7.47(td，J= 7.5，1.3 Hz，1H)，7.34(td，J= 8.2，7.5，1.7 Hz，2H)，4.13-4.05(m，4H)，2.43(s，3H)，1.15(t，J= 7.1 Hz，3H)。 ¹³ C NMR(100 MHz，DMSO-d ₆ )δ197.1, 168.1, 138.4, 136.8, 132.5, 132.3, 129.9, 126.4, 61.0, 48.5, 21.3, 14.4. Theoretical calculated value C ₁₂ H ₁₄ NaO ₃ ⁺ [M+Na] ⁺ =229.0835, hrms (ESI) actual calculated: 229.0837.

2.2, 7-hydroxy-4- (o-tolyl) -2HSynthesis of amino-2-ones (2B)

2A (4 g, 19.4 mmol, 1 eq.) was dissolved in trifluoroacetic acid, resorcinol (4.3 g, 38.8 mmol, 2 eq.) was added and the mixture refluxed overnight. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 2B (2.2 g, 45% yield). ¹ H NMR(400 MHz，DMSO-d ₆ )δ10.66(s，1H)，7.49-7.27(m，3H)，7.24-7.17(m，1H)，6.87-6.75(m，2H)，6.73-6.68(m，1H)，6.10(s，1H)，2.11(s，3H)。 ¹³ C NMR(100 MHz，DMSO-d ₆ )δ161.9, 160.7, 156.2, 155.7, 135.4, 135.3, 130.8, 129.5, 128.8, 128.4, 126.6, 113.8, 111.7, 111.4, 103.0, 19.8. Theoretical calculated value C ₁₆ H ₁₁ O ₃ ^- [M-H] ^- =251.0714, hrms (ESI) actual calculated: 251.0713.

2.3, 2- ((2-oxo-4- (o-methyl)Phenyl) -2H-methyl-7-yl) oxy) ethyl propionate (2C) synthesis

2B (100 mg, 0.40 mmol, 1 eq.) was dissolved inN,NTo dimethylformamide were added ethyl 2-bromopropionate (145 mg, 0.80 mmol, 2 eq.) and cesium carbonate (390 mg, 1.20 mmol, 3 eq.) overnight at room temperature. The mixture was then diluted with water (20 ml), the water was extracted with dichloromethane (20 ml×2) and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 2C (81 mg, 58% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.44-7.27(m，3H)，7.20-7.13(m，1H)，6.96(d，J= 8.8 Hz，1H)，6.89(dd，J= 2.5，0.6 Hz，1H)，6.81 (dd，J= 8.9，2.5 Hz，1H)，6.12 (s，1H)，5.01 (q，J= 6.7 Hz，1H)，4.21(q，J= 7.1 Hz，2H)，2.13 (d，J= 1.9 Hz，3H)，1.61 (d，J= 6.8 Hz，3H)，1.25(t，J= 7.1 Hz，3H)。 ¹³ C NMR(100MHz，MeOD-d ₄ )δ171.5, 161.6, 161.1, 156.8, 155.2, 135.1, 134.9, 130.2, 129.1, 128.1, 127.9, 125.9, 113.4, 112.9, 112.0, 101.9, 72.5, 61.3, 18.4, 17.2, 13.1. Theoretical calculated value C ₂₁ H ₂₁ O ₅ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =353.1384: 353.1376.

2.4, 2- ((2-oxo-4- (o-tolyl) -2H-methyl-7-yl) oxy) propanoic acid (2D) synthesis

2C (81 mg, 0.23 mmol) was dissolved in 1N sodium hydroxide in methanol and water and stirred overnight at room temperature. The mixture was concentrated on a rotary evaporator and acidified to pH 2 with hydrochloric acid and the aqueous layer extracted with dichloromethane (20 ml x 2). The combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue purified by flash column chromatography to give 2D (52 mg, 70% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.43-7.27(m，3H)，7.18(dd，J= 7.4，1.6 Hz，1H)，6.97-6.87(m，2H)，6.82(dd，J= 8.8，2.1 Hz，1H)，6.11(d，J= 1.7 Hz，1H)，4.91(d，J= 8.3 Hz，1H)，2.13(s，3H)，1.62(d，J= 6.6 Hz，3H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ161.7, 161.4, 156.9, 155.2, 135.0, 134.9, 130.2, 129.0, 128.1, 127.8, 125.9, 113.2, 112.9, 112.9, 111.8, 101.9, 101.9, 18.4, 17.4. Theoretical calculated value C ₁₉ H ₁₅ O ₅ ^- [M-H] ^- Actual calculated hrms (ESI) =323.0925: 323.0925.

2.5, 1- (2- ((2-oxo-4- (o-tolyl) -2)H-methyl-7-yl) oxy) propionyl) piperidine-3-carboxylic acid ethyl ester (2E) synthesis

2D (52 mg, 0.16 mmol, 1 eq), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (46 mg, 0.24 mmol, 1.5 eq), 1-hydroxybenzotriazole (32.5 mg, 0.24 mmol, 1.5 eq), N,NDiisopropylethylamine (62 mg, 0.48 mmol, 3 eq.) and ethyl 3-piperidinecarboxylate (38 mg, 0.24 mmol, 1.5 eq.). The mixture was then diluted with ethyl acetate (20 ml) and washed 3 times with saturated ammonium chloride solution, and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue purified by flash column chromatography to give 2E (68 mg, 77% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.44-7.25 (m，3H)，7.17 (dd，J= 7.5，1.5 Hz，1H)，7.05-6.75 (m，3H)，6.17-6.01 (m，1H)，5.35 (d，J= 1.2 Hz，1H)，4.37-4.12(m，1H)，4.12-3.99 (m，3H)，3.79(m，1H)，3.43 (t，J= 6.6 Hz，1H)，2.71-2.43 (m，1H)，2.12 (s，3H)，1.88-1.68 (m，2H)，1.60-1.55 (m，3H)，1.30-1.15 (m，5H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ173.1, 171.6, 169.7, 161.5, 161.0, 156.8, 155.3, 135.0, 130.2, 129.1, 128.1, 125.9, 113.0, 111.9, 102.0, 72.0, 60.2, 45.3, 42.5, 40.6, 26.4, 24.2, 19.5, 18.5, 17.0, 13.1. Theoretical calculated value C ₂₇ H ₃₀ NO ₆ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =464.2068: 464.2070.

2.6、1- (2- ((2-oxo-4- (o-tolyl) -2)H-methyl-7-yl) oxy) propionyl) piperidine-3-carboxylic acid (2F) synthesis

2E (78 mg, 0.18 mmol) was dissolved in 1N sodium hydroxide in methanol and water and stirred overnight at room temperature. The mixture was concentrated on a rotary evaporator and acidified to pH 2 with hydrochloric acid. The aqueous layer was extracted with dichloromethane (20 ml×2). The combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give 2F (31 mg, 40.0% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.44-7.25(m，3H)，7.19(d，J=7.5 Hz，1H)，7.04-6.74(m，3H)，6.12 (d，J= 6.0 Hz，1H)，5.58-5.29 (m，1H)，4.46-3.94 (m，1H)，3.80 (t，J= 4.2 Hz，1H)，3.55 (dq，J= 8.0，3.7，2.7Hz，1H)，3.28-2.96 (m，1H)，2.53-2.34(m，1H)，2.14 (s，3H)，2.11-1.89(m，2H)，1.87-1.61 (m，2H)，1.60-1.55(m，3H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ169.9, 161.9, 160.9, 157.1, 155.3, 135.1, 130.2, 129.0, 128.1, 125.9, 112.8, 111.6, 102.3, 71.7, 45.5, 44.6, 42.5, 29.1, 27.4, 26.7, 25.2, 23.4, 18.4, 17.0, 16.6. Theoretical calculated value C ₂₅ H ₂₄ NO ₆ ^- [M-H] ^- =434.1609, hrms (ESI) actual calculated: 434.1609.

2.7, tert-butyl (2- (2- (1- (2- ((2-oxo-4- (o-tolyl) -2)H-formyl-7-yl) oxy) propionyl) piperidine-3-carboxamide) ethoxy) ethoxyethyl) carbamate (IL)

2F (43.5 mg, 0.1 mmol, 1 eq.) was dissolved inN,N-dimethylformamide and addingN,N,N′, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq),N，NDiisopropylethylamine (39 mg, 0.3 mmol, 3 eq) and tert-butyl 2- (2-aminoethoxy) ethoxycarbamate (49.6 mg, 0.2 mmol, 2 eq) and the mixture was stirred at 40 ℃ overnight. Then diluted with ethyl acetate (20 ml) and dissolved with saturated ammonium chlorideThe solution was washed 3 times, and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give IL (34 mg, 51.1% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.35-7.22 (m，3H)，7.11 (d，J= 7.5 Hz，1H)，6.93-6.70(m，3H)，6.04 (d，J= 7.6 Hz, 1H)，5.38-5.28 (m，1H)，4.39-3.78 (m，2H)，3.60-3.18 (m，11H)，3.17-3.02 (m，3H)，2.36-2.24 (m, 1H)，2.05 (s，3H)，1.96-1.55(m，4H)，1.52-1.43 (m，3H)，1.33(s，9H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ174.2, 173.9, 169.7, 161.6, 161.0, 156.9, 155.3, 135.0, 130.2, 129.1, 128.1, 125.9, 113.3, 111.9, 102.2, 101.7, 71.6, 69.9, 69.1, 45.4, 44.6, 42.9, 42.4, 39.8, 38.8, 37.5, 27.4, 25.2, 23.5, 18.4, 17.0, 16.6. Theoretical calculated value C ₃₆ H ₄₈ N ₃ O ₉ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =666.3358: 665.3356.

3. synthesis of mitochondrial protease-targeted chimeras (MtPTAC 3B)

Synthetic route to MtPTAC 3B

Reaction conditions: (a) Palladium acetate, potassium carbonate, t-butanol, dicyclohexyl [3, 6-dimethoxy-2 ',4',6 '-triisopropyl [1,1' -biphenyl ]]-2-yl]Phosphine, reflux, overnight; (b) trifluoroacetic acid, dichloromethane, room temperature, 2h; (c)N,N,N′,N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate,N,Ndiisopropylethylamine, 2f,40 ℃ overnight.

3.1 Synthesis of intermediate 3A

3.1.1. tert-butyl (2- ((4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-)a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -yl) methyl) phenyl) amino) ethoxy) ethyl) carbamate (3A-1) (AL) synthesis

1E (100 mg, 0.22 mmol, 1 gAn amount) in t-butanol and adding [2- (2-aminoethoxy) ethyl ]]Tert-butyl carbamate (67.32 mg, 0.33 mmol, 1.5 eq.) was freeze-deoxygenated. Palladium acetate (10 mg, 0.044 mmol, 0.2 eq.) dicyclohexyl [3, 6-dimethoxy-2 ',4',6 '-triisopropyl [1,1' -biphenyl ] ]-2-yl]Phosphine (48 mg, 0.088 mmol, 0.4 eq) and potassium carbonate (72 mg, 0.528 mmol, 2.4 eq) were weighed to remove oxygen and the solution was transferred to the vessel and reacted overnight at 90 ℃. The reaction mixture was filtered to obtain a solution, then the solvent was removed on a rotary evaporator, and the residue was purified by flash column chromatography to give 3A-1 (60 mg, 47.5% yield). ¹ H MNR(400 MHz，MeOD-d ₄ )δ7.37-7.30 (m，4H)，7.29-7.23 (m，1H)，7.17-7.12 (m，2H)，6.64-6.55 (m，2H)，4.86 (s，2H)，4.02-3.92 (m，2H)，3.84 (tt，J= 8.5，1.6 Hz，2H)，3.72-3.54 (m，6H)，3.47 (t，J= 5.5 Hz，2H)，3.20(m，6H)，2.71 (t，J= 5.8 Hz，2H)，2.54 (t，J= 5.8 Hz，2H)，1.42 (s，9H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ161.7, 157.1, 157.1, 153.3, 148.2, 137.0, 129.2, 153.3, 148.2, 148.2, 137.0, 129.2, 128.9, 128.1, 127.2, 125.0, 125.0, 112.7, 101.1, 78.7, 70.2, 70.2, 69.6, 69.0, 61.7, 49.3, 48.6, 46.6, 46.2, 44.6, 43.4, 39.9, 27.4, 25.6. Theoretical calculation C ₃₂ H ₄₃ N ₆ O ₄ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =575.3341: 575.3346.

3.1.2. tert-butyl (2- ((4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-)a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -group) methyl) phenyl) amino) ethoxy) ethyl) carbamate (3A-2) synthesis

1E (100 mg, 0.22 mmol, 1 eq.) was dissolved in t-butanol and t-butyl 2- (2- (2-aminoethoxy) ethoxy) carbamate (81.84 mg, 0.33 mmol, 1.5 eq.) was added for freeze deoxygenation. Palladium acetate (10 mg, 0.044 mmol, 0.2 eq.) dicyclohexyl [3, 6-dimethoxy-2 ',4',6 '-triisopropyl [1,1' -biphenyl ] ]-2-yl]Phosphine (48 mg, 0.088 mmol)0.4 eq) and potassium carbonate (72 mg, 0.528 mmol, 2.4 eq) and oxygen were weighed out and the solution was transferred to the vessel and reacted overnight at 90 ℃. The reaction mixture was filtered to obtain a solution, then the solvent was removed on a rotary evaporator, and the residue was purified by flash column chromatography to give 3A-2 (65 mg, 47.8% yield). ¹ H NMR(400 MHz，CDCl ₃ -d)δ7.43-7.11(m，8H)，6.52-6.32(m，1H)，4.86(s，2H)，3.91-3.68(m，4H)，3.69-3.36(m，12H)，3.28-3.09(m，4H)，2.57(m，2H)，2.41-2.28(m，2H)、1.37(s，9H)。 ¹³ C NMR(100 MHz，CDCl ₃ -d)δ160.5, 155.0, 152.1, 146.5, 144.2, 136.6, 129.3, 128.1, 127.4, 126.3, 111.7, 101.1, 69.2, 68.6, 61.2, 53.7, 49.4, 48.5, 47.2, 45.7, 43.9, 42.5, 39.3, 30.9, 28.7, 27.4, 25.6. Theoretical calculation C ₃₄ H ₄₇ O ₅ N ₆ ⁺ [M+H] ⁺ =619.3603, hrms (ESI) actual calculated: 619.3602.

3.1.3, tert-butyl (2- (2- ((4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-)a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -group) methyl) phenyl) amino) ethoxy) ethoxyethyl) carbamate (3A-3) synthesis

1E (100 mg, 0.22 mmol, 1 eq.) was dissolved in t-butanol and t-butyl (2- (2- (2- (-2- (2-aminoethoxy) ethoxyethyl) carbamate) (95 mg, 0.33 mmol, 1.5 eq.) was added to freeze deoxygenated palladium acetate (10 mg, 0.044 mmol, 0.2 eq.) dicyclohexyl [3, 6-dimethoxy-2 ',4',6 '-triisopropyl [1,1' -biphenyl) ]-2-yl]Phosphine (48 mg, 0.088 mmol, 0.4 eq) and potassium carbonate (72 mg, 0.528 mmol, 2.4 eq) were weighed to remove oxygen and the solution was transferred thereto and reacted overnight at 90 ℃. The reaction solution was filtered to obtain a liquid, then the solvent was removed on a rotary evaporator, and the residue was purified by flash column chromatography to give 3A-3 (52 mg,35.7% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.37-7.30 (m，4H)，7.29-7.26 (m，1H)，7.19-7.11 (m，2H)，6.66-6.51 (m，2H)，4.86 (s，2H)，3.98 (t，J= 9.0 Hz，2H)，3.87-3.78(m，2H)，3.68 (d，J= 2.8 Hz，2H)，3.65-3.56 (m，12H)，3.52-3.35(m，2H)，3.25-3.13 (m，6H)，2.70 (t，J= 5.8 Hz，2H)，2.54 (t，J= 6.0 Hz，2H)，1.41(s，9H)。 ¹³ C NMR(100MHz，MeOD-d ₄ )δ161.6, 157.0, 153.3, 148.1, 147.4, 137.0, 129.2, 128.9, 128.1, 127.2, 124.8, 112.6, 101.1, 78.7, 70.2, 69.9, 69.7, 69.2, 61.7, 48.6, 46.2, 44.6, 43.3, 39.9, 27.4, 25.6. Theoretical calculation C ₃₆ H ₅₁ O ₆ N ₆ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =663.3865: 663.3863.

3.2 Synthesis of MtPTAC 3B

3.2.1、N- (2- ((4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-)a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -yl) phenyl) amino ethoxy) ethyl) -1- (2- (2-oxo-4- (o-tolyl) -2H-formyl-7-oxo-propionyl) piperidine-3-carboxamide (3B-1) synthesis

3A-1 (57.4 mg, 0.1 mmol, 1 eq.) was dissolved in 25% trifluoroacetic acid in dichloromethane and reacted at room temperature for 2 hours, then concentrated by rotary evaporation. 2F (43.5 mg, 0.1 mmol, 1 eq.) was dissolved in N,NIn dimethylformamide and willN,N,N′,N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq.) andN，Ndiisopropylethylamine (39 mg, 0.3 mmol, 3 eq.) was added and reacted at 40 ℃ overnight. Then diluted with ethyl acetate (20 ml) and washed 3 times with saturated ammonium chloride solution, and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give MtPTAC 3B-1 (23 mg, 25.8% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.47-7.20 (m，9H)，7.19-7.02 (m，3H)，7.00-6.74 (m，3H)，6.52 (dd，J= 8.6，2.9 Hz，2H)，6.22-6.00 (m，1H)，5.44-5.26 (m，1H)，4.81 (s，2H)，4.70-4.26 (m，2H)，4.06-3.74 (m，6H)，3.70-3.31 (m，8H)，3.24-3.05 (m，5H)，2.67 (q，J= 5.9 Hz，2H)，2.51(q，J= 5.6 Hz，2H)，2.42-2.27(m，1H)，2.22-2.03 (m，3H)，1.94-1.71 (m，2H)，1.70-1.47 (m，5H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ174.1, 169.7, 161.6, 161.0, 156.8, 155.3, 153.2, 148.2, 147.3, 137.1, 135.0, 130.2, 129.2, 128.9, 127.9, 127.2, 125.9, 124.9, 113.1, 112.6, 111.9, 102.2, 101.8, 101.1, 71.2, 70.2, 69.1, 69.0, 61.6, 49.2, 48.0, 46.8, 46.2, 45.4, 44.6, 43.3, 42, 42.4, 38.9, 29.5, 29.0, 27.4, 25.6, 25.1, 23.4, 18.5, 17.0, 16.7. Theoretical calculation C ₅₂ H ₅₈ O ₇ N ₇ ⁺ [M+H] ⁺ =892.4392, hrms (ESI) actual calculated: 892.4386.

3.2.2、N- (2- (2- ((4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-))a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -methyl) phenyl) amino) ethoxy ethyl) -1- ((2-oxo-4- (o-tolyl) -2 H-formyl-7-yl) oxy) propionyl) piperidine-3-carboxamide (MtPTAC 3B-2) synthesis

3A-2 (61.8 mg, 0.1 mmol, 1 eq.) was dissolved in 25% trifluoroacetic acid in dichloromethane and reacted at room temperature for 2 hours, then concentrated by rotary evaporation. 2F (43.5 mg, 0.1 mmol, 1 eq.) was dissolved inN,NIn dimethylformamide and willN,N,N′,N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq.) andN，Ndiisopropylethylamine (39 mg, 0.3 mmol, 3 eq.) was added and reacted at 40 ℃ overnight. The mixture was then diluted with ethyl acetate (20 ml) and washed 3 times with saturated ammonium chloride solution. The combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give MtPTAC 3B-2 (33 mg, 36.2% yield). ¹ H NMR(400 MHz，MeOD-d ₄ )δ7.42-7.20 (m，10H)，7.19-7.08 (m，2H)，7.00-6.75 (m，3H)，6.57-6.43 (m，1H)，6.14-6.04 (m，1H)，5.46-5.29 (m，1H)，4.87-4.76 (m，2H)，4.48-4.10 (m，2H)，4.11-3.83 (m，2H)，3.82 -3.46 (m，13H)，3.25-3.00 (m，4H)，3.00-2.74 (m，3H)，2.74-2.28 (m，4H)，2.26-2.15 (m，1H)，2.14-2.06 (m，3H)，1.97-1.66 (m，4H)，1.57-1.52 (m，3H)。 ¹³ C NMR(100 MHz，MeOD-d ₄ )δ174.1, 173.2, 169.7, 161.5, 161.0, 156.8, 155.2, 153.7, 153.27, 148.3, 138.7, 138.2, 136.9, 135.0, 130.2, 129.1, 128.7, 128.2, 128.1, 127.4, 126.9, 125.9, 112.8, 112.4, 111.9, 71.7, 70.1, 69.3, 61.5, 50.0, 49.4, 48.6, 45.4, 44.6, 43.2, 42.8, 42.5, 38.8, 37.5, 35.8, 35.2, 32.3, 31.7, 29.4, 27.4, 26.7, 25.6, 25.2, 23.5, 22.4,8.5, 17.0, 16.7, 13.1. Theoretical calculation C ₅₄ H ₆₂ O ₈ N ₇ ⁺ [M+H] ⁺ Actual calculated hrms (ESI) =936.4655: 936.4654.

3.2.3、N- (2- (2- ((4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-))a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -methyl) phenyl) amino) ethoxy ethyl) -1- ((2-oxo-4- (o-tolyl) -2H-formyl-7-yl) oxy) propionyl) piperidine-3-carboxamide (MtPTAC 3B-3) synthesis

3A-3 (66.2 mg, 0.1 mmol, 1 eq.) was dissolved in 25% trifluoroacetic acid in dichloromethane and reacted at room temperature for 2 hours, then concentrated by rotary evaporation. 2F (43.5 mg, 0.1 mmol, 1 eq.) was dissolved in N, N-dimethylformamide andN,N,N′,N' -tetramethyl-OUrea- (7-azabenzotriazol-1-yl) hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq.) and N, N-diisopropylethylamine (39 mg, 0.3 mmol, 3 eq.) were added and reacted at 40 ℃ overnight. The mixture was then diluted with ethyl acetate (20 ml) and washed 3 times with saturated ammonium chloride solution. The combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and the residue was purified by flash column chromatography to give MtPTAC 3B-3 (31 mg, 35.1% yield). ¹ H NMR (400 MHz，MeOD-d ₄ )δ7.54-6.95 (m，12H)，6.92-6.66 (m，3H)，6.50-6.40 (m，1H)，6.07-5.96 (m，1H)，5.46-5.23 (m，1H)，4.71 (s，2H)，4.40-3.68 (m，6H)，3.57-3.39 (m，20H)，3.17-2.90 (m，3H)，2.87-2.59 (m，2H)，2.53-2.24 (m，2H)，2.14 (dd，J= 15.4，7.9 Hz，1H)，2.08-1.98 (m，3H)，1.96-1.58 (m，4H)，1.52-1.42 (m，3H)。 ¹³ C NMR (100 MHz，MeOD-d ₄ )δ174.2, 173.3, 167.0, 155.3, 153.7, 138.7, 138.2, 135.0, 130.2, 129.5, 129.0, 128.7, 128.2, 128.1, 128.0, 127.4, 127.0, 126.9, 125.9, 113.2, 112.6, 111.9, 70.2, 70.0, 69.8, 69.1, 68.3, 63.0, 61.6, 60.7, 49.5, 46.4, 45.4, 44.9, 44.6, 43.2, 42.8, 39.3, 38.9, 37.5, 35.8, 35.1, 32.3, 31.7, 30.4, 29.4, 29.1, 26.7, 25.5, 25.2, 23.5, 22.4, 18.4, 16.7, 13.1. Theoretical calculation C ₅₆ H ₆₆ O ₉ N ₇ ⁺ [M+H] ⁺ Actual hrms (ESI) calculated = 980.4917: 980.4919.

example 2:

performance test of mitochondrial protease targeted chimeras (MtPTAC 3B)

1. Experimental method

1.1 Western blotting

Cells were seeded in 6-well plates and treated 24h after dosing (different concentrations). The protein content normalization was quantified with BCA kit using 100 μl of RIPA lysate with MtPTAC added to lyse cells and placed in 1.5mL centrifuge tubes, respectively. Finally, adding protein Loading Buff, boiling in a metal bath at 95 ℃ for 5min, determining Loading amount according to normalization treatment by using a pre-prepared gel, performing gel electrophoresis (90V, 120 min), and transferring to a PVDF membrane (activated by methanol in advance). Blocking for 1-2h with milk at 5% by mass, incubating overnight with antibody (dilution ratio 1:2000, milk at 5% by mass of dilution). After incubation was completed, primary antibodies were recovered and the PDVF membrane was washed several times with PBST (PBS solution containing 0.1% tween 20), then the strips were washed continuously with the corresponding species secondary antibodies incubated for 1h and imaged in a gel chromatograph using a hypersensitive developer (medium), the antibodies used are shown in table 2. The numbers show the relative expression levels of polrbt (normalized with Tubulin or GADPH).

TABLE 2 antibodies

1.2 proteomics methods

Cells were lysed with lysis buffer without EDTA and MtPTAC mixture containing 50mM PBS and 500mM NaCl. Protein concentration was quantified by BCA assay, 50 μg of total protein per sample, using enzymatic digestion in solution. Every 100. Mu.L of lysate was reduced with 10mM DTT, followed by alkylation with 20mM IAA, and then protein samples were digested with trypsin (10. Mu.g/mL) at 37℃overnight. The digestate was collected by centrifugation and the filter was rinsed with 50 μl 0.5M NaCl and then centrifuged again. The resulting solutions were combined and acidified with 10% trifluoroacetic acid. The acidified peptide was desalted using a C18 tip (Pierce, thermo Scientific) according to the manufacturer's instructions. The desalted peptide was eluted using 50 μl of 50% MeCN and the eluted product was dried under vacuum and resuspended in 30 μl of 0.1% Formic Acid (FA). Desalted peptides were subjected to an orbitrapepexploris 480 (Thermo Fisher Scientific) mass spectrometer in a Data Dependent Acquisition (DDA) mode to obtain MS data. All original files were searched against the Uniprtot human protein sequence database (UP 000005640, 74349 forward entry, 2019) in the proteome finder (PD) software (version 2.4). The mass tolerance of the precursor or peptide was 10ppm and the mass tolerance of the fragment ion was 0.02Da, allowing the cleavage of the twice omitted trypsin, the cysteine carbamoylmethylation was set as the fixed modification. Each group contains four biological replicates and proteins detectable in at least three samples in each group are included for subsequent differential expression analysis.

1.3 mitochondrial extraction method using mitochondrial kit

1) The harvested cell suspension was centrifuged at 850 Xg in a 2.0mL microcentrifuge tube for 2 minutes to give a 2X 10 suspension ⁷ The cells are granulated. 2) 800 μl of mitochondrial separation reagent a was added. The tubes were incubated on ice for exactly 2 minutes with medium speed vortexing for 5 seconds. 3. The cell suspension was transferred to a Dounce tissue mill. 4) Cells were homogenized on ice and a sufficient stroke was performed to effectively lyse the cells. 5) Cells to be lysedReturning to the original test tube, and adding 800 [ mu ] L of mitochondrial separation reagent C. 6) The Dounce tissue grinder was rinsed with 200 μl mitochondrial isolation reagent A and added to the tube containing the sample. 7) The tube is turned over for several times to mix (without vortexing). 8) The tube was centrifuged at 700 Xg for 10 minutes at 4 ℃. 9) The supernatant was transferred to a fresh 2.0mL tube and centrifuged at 12000 Xg for 15 min at 4 ℃.10 The supernatant (cytosol fraction) was transferred to a new tube, with particles containing isolated mitochondria. 11 Add 500 μl of mitochondrial separation reagent C to the pellet and centrifuge at 12000×g for 5 min, discard the supernatant. 12 Before downstream processing, the mitochondrial particles are kept on ice, and freezing and thawing can cause damage to mitochondrial integrity.

1.4 LC-MS method

Extracted mitochondria were lysed with 50 μl of lysis solution, 1 μl of each sample was taken and injected onto a BEH C18 (2.1 x 50mm 1.7 μm) column. The solvent delivery module was used to flow through an HPLC gradient of 30-95% B (solvent phase A: 0.1% formic acid in water, B solvent: 0.1% formic acid in acetonitrile) at a flow rate of 0.3mL/min over 7 minutes. The sample from the elution column was passed through a UV detector at 254 nm. Mass spectrometry detection was performed using an Agilent LCMS-6495C quadrupole mass spectrometer and an anelec-trospiy ionization (ESI) interface. The ESI source is set to positive ionization mode and the mass selective detector is used in Multiple Reaction Monitoring (MRM) mode to obtain as high a selectivity and sensitivity as possible. The instrument was scanned in the m/z range of 400-1500 and quantified by peak area. Data acquisition and processing was done using agilent LC-MS solution software LCMS-6495C system and Graphpad 7. The retention time and the peak of fragments were first determined using liquid chromatography-mass spectrometry (LC/MS) for MtPTAC3B-1 pure, and then the mitochondrial lysate above was passed through LC/MS testing, finding a signal consistent with the retention time of MtPTAC3B-1 and the peak of fragments. Notably, the increasing concentration of MtPTAC3B-1 in the mitochondria with increasing treatment time suggests that MtPTAC3B-1 enters the mitochondria as a slower process.

1.5 siRNA sequences against ClpP

The siRNA sequences against ClpP are shown in table 3.

TABLE 3 siRNA sequences against ClpP

1.6 design purification of recombinant proteins and Isothermal Titration Calorimetry (ITC)

First, purification of recombinant POLRMT and ClpP was required, and the expression and purification methods of ClpP and POLRMT were as follows: the sequence diagrams of ClpP and POLRMT for ITC are shown in FIG. 1. The optimized ORF sequence was synthesized by Genscript and inserted into pET23b between NdeI and XhoI sites, respectively, and then confirmed to be correct by sequencing, as shown in FIG. 2.BL21 (DE 3) and the corresponding plasmids were cultivated in LB at 37 ℃. When the OD of the bacterial liquid ₆₀₀ When the value reached 0.8, isopropyl-1-thio-B-D-galactopyranoside, which had a final concentration of 0.1mM, was added, and the bacteria were either cultured at 37 ℃ for 4 hours to express ClpP, or at 16 ℃ for 18 hours to express POLRMT. The bacteria were harvested by centrifugation, resuspended in binding buffer (20 mM Tris-HCl, 500mM NaCl, 5mM imidazole, 5% glycerol, pH 7.8) and lysed by sonication. After centrifugation (10000 rpm,30 minutes), the supernatant was passed through a nickel-nitrilotriacetic acid agarose column pre-equilibrated with binding buffer, followed by washing with 50mM imidazole binding buffer. Finally ClpP and POLRMT were eluted with 400mM imidazole and concentrated by centrifugal ultrafiltration.

1.7 Molecular Dynamics (MD) simulation

The structure of ternary complex ClpP/3B-1/POLRMT was studied by MD simulation, which was divided into three parts: 1) Calculation of the polrbt of ligand 2G; 2) Calculating ClpP with ONC201 derivative 1E; 3) Combining these two parts gives the complex ClpP/3B-1/POLRMT.

The setting parameters are as follows: throughout the calculation step, the amber force field (FF 14 SB) was used for the protein, the GAFF force field for the ligand, and MD was performed using the Gromacs program (version 2021.5). All MD processes (always NPT ensemble) used a leapfrog integration algorithm, a random speed recalibration (Bussi-Parinello) thermostat, and a random unit recalibration barometer. The step size is set to 2fs. The cutoff radius for vdW interaction was set to 10 a and electrostatic interaction calculations were performed using the sPME algorithm. The system setup was done using the pdb2gmx tool in Gromacs 2021.5 and the Antecomber tool in Ambertools21 suite, where cycle boundary conditions were used; the box is a cube with an absolute dimension on each side 12 a greater than the maximum dimension of the system, the rest 2 (0.5) atomic partial charge of the ligand is calculated by the multisfn 3.8 (dev) program, and the wave function of the B3LYP-D3 (BJ)/def 2 TZVP level is calculated by ORCA 5.0.2 software. Explicit TIP3P water was added and the system was neutralized with potassium and chloride ions and salified to 0.02M ionic strength. The ion concentration refers to the experimental results of the mitochondrial matrix in the previous report, as sodium ions and chloride ions were added at a concentration of 0.005M.

For the complex of POLRMT/IMT, the start coordinates of POLRMT are from the crystal structure downloaded from the Protein Database (PDB) portal. To replace the ligand in this structure with IMT, the ligand in PDB entry 7A8P was removed, then the PDB file was subjected to addition of hydrogen atoms on the Swiss Model website, construction of a deletion loop, partitioning of the protonated state of the ionizable group by assuming pH 7.8, which conforms to the mitochondrial matrix. Molecular docking of the prepared protein with 2G has been performed by the autopock Vina 1.2.3 program by the Vina scoring function. MD is then performed by a three-stage process: (1) Energy minimization of the composite by 1000 steps by the steepest descent method; (2) 400 picoseconds of annealing from 0K to 310.15K were simulated, with harmonic potentials suppressing the stem; (3) 50ns was simulated to relax the ring and side chains.

For complex ClpP/1E, the starting coordinates for human mitochondrial ClpP are from PDB entry 6DL79.MD simulation was performed using the same procedure as described above.

1.8 drawing of cell proliferation Curve

The growth curves of a549 and PC9 were examined using the xCELLigence RTCA MP system (agilent). 2000 cells were seeded in the wells of an E-plate 16 well plate (agilent), and the index of adherent cells was measured every 1 hour. The cells were continuously recorded for 0-196 hours and then the growth curves of the cells in each well were plotted.

1.9 cell cloning proliferation experiments

Cells were seeded at a density of 300 cells/well in 12-well plates and then cultured in medium containing the specific drug for 2 weeks until cells grew into colonies (over 100 cells), during which time the medium was changed every 3 days. After washing with PBS, ethanol was added for fixation, and crystal violet staining solution was added for 5-10 minutes. The 12-well plate is then rinsed with a large amount of water and dried. A clone picture was obtained for each well and the number of clones in each well was calculated.

1.10 measurement of cell Activity

The relative cell viability of the different cell lines was assessed using cell counting kit 8 (CCK-8) according to the manufacturer's instructions. Cell activity was normalized using the same volume of DMSO-treated cells and untreated cells as controls.

1.11, cell flow

A549 cells and HEK293 cells were seeded in 6-well plates and treated with 40 μm of 3B-1 for 24 hours and 72 hours, respectively, and treated with equal volumes of DMSO and untreated cells served as controls. An Annexin V-FITC apoptosis detection kit (Beyotide, C1062S) was used for apoptosis detection. A549 and HEK293 cells were digested with EDTA-free trypsin and incubated with propidium iodide and annexin V-FITC for 15 min at 37 ℃. The samples were then analyzed by a flow cytometer SP6800 spectrum analyzer.

1.12 experiments with mice and animals

All studies related to mice were approved by the Hangzhou medical institute of China academy of sciences. Mice experiments were performed using 4-6 week old BALB/c nude mice (Vitron Lever). Subcutaneous injection of A549 cell suspension (1×10) ⁷ /mL,200 μl), a cancer xenogenic tumor mouse model was established. When the tumor size reaches about 100mm ³ At this time, mice were randomized into the MtPTAC 3B-1, mtPTAC 3B-2 and placebo treatment groups. MtPTAC 3B-1 or MtPTAC 3B-2 was dissolved in 1% castor oil solution and administered by intratumoral injection (5 mg/kg, once every two days). Two weeks later, nude mice were sacrificed and subcutaneous tumors were peeled off to measure tumor weight. Subcutaneous tumors were further used for immunoblotting. In addition, body weights of all mice were recorded before and after the experiment.

2. Experimental results

2.1 mitochondrial proteolysis assay

Protein degradation ability of MtPTAC (3B-1, 3B-2, 3B-3) was tested in A549 cell line: a549 cells were treated with four different concentrations (0, 0.1, 10 and 40 μm) of MtPTAC, respectively, for 24 hours, whose level changes were analyzed by western blotting (see fig. 3 a), mtPTAC 3B-1 could degrade 80% of polrbt, mtPTAC 3B-2 and MtPTAC 3B-3 could not significantly reduce the protein level of polrbt. Extending the time for which MtPTAC 3B-1 treated cells to 72 hours (see fig. 3B), 10 μm of MtPTAC 3B-1 could significantly degrade POLRMT.

The ability of MtPTAC 3B-1 to degrade protein in other cell lines was tested (see fig. 4), with a consistent appearance of significant degradation of protein after 24 hours of treatment with MtPTAC 3B-1 at a concentration of 40 μm in the 4 cell lines selected (a 549, H1299, H460 and PC 9). Furthermore, treatment of a549 and PC9 cell lines with MtPTAC 3B-1 at a concentration of 40 μm at different times (0-24 h) exhibited significant time dependence (see fig. 5).

RNA levels of POLRMT before and after addition of MtPTAC 3B-1 were detected, and quantitative PCR with reverse transcription (qRT-PCR) experiments showed (see FIG. 6): cells were treated with 40. Mu.M MtPTAC 3B-1 for 24 hours, and there was no significant change in the mRNA level of POLRMT when protein levels were significantly reduced, confirming that MtPTAC 3B-1 directly affected the level of the target protein POLRMT. Thus, mtPTAC 3B-1 can effectively induce target protein degradation directly in a variety of cell lines, and degradation efficiency is closely related to polyethylene glycol linker length and is time dependent.

2.2 mitochondrial proteolysis specificity test

Quantitative analysis using mitochondrial protein information provided by the mitocarta3.0 database showed that no mitochondrial protein was significantly reduced (log) after 6 hours of treatment ₂ FC < -1 >, p < 0.001) (see FIG. 7 a), whereas only POLRMT was significantly down-regulated after 12 hours of treatment (log) ₂ Fc= -1.093, p= 0.000883) (see fig. 7 b). Similar trends were observed using mitochondrial protein information provided by the UniProt database (see fig. 8a and 8 b). At the same time, the level of ATP5A protein in mitochondria was tested (see fig. 8 c), and immunoblots showed that its level was not affected. Thus, mtPTAC 3B-1 induced POLRM in mitochondriaSpecific degradation of T, rather than non-specific degradation of mitochondrial proteins as would be caused by ClpP ligand ONC 201.

2.3 mitochondrial proteolysis mechanism Studies

A549 cell lines 0, 8, 16 and 24 hours were treated with 20 μm MtPTAC 3B-1, respectively, and their mitochondria were extracted by mitochondrial kit, respectively. The retention time and the peak of fragments of the MtPTAC 3B-1 pure were first determined using liquid chromatography-mass spectrometry (LC/MS), and then the above mitochondrial lysate was subjected to LC/MS testing, and the results are shown in fig. 9, which show that the signal consistent with the retention time and the peak of fragments of MtPTAC 3B-1 was found. Notably, the concentration of MtPTAC 3B-1 in the mitochondria increased with increasing treatment time, indicating that MtPTAC 3B-1 entered the mitochondria as a slower process.

ClpP (sequences of siRNA are shown in Table 3) in 3 siRNA knockdown A549 cells was designed, and MtPTAC3B-1 was added for treatment. Even with 40 μm MtPTAC3B-1 treatment in a549 cells with siNRA knocked out ClpP, the total protein amount and molecular weight of polrbt in a549 cells did not change significantly. The immunoblotting results of cells treated with 40. Mu.M MtPTAC3B-1, using MG132 to block the proteolytic activity of the proteasome, showed that: inhibition of ubiquitin-proteasome activity by MG132 does not prevent MtPTAC3B-1 from degrading POLRMT in cells. Thus, mtPTAC3B-1 induced POLRMT degradation is ClpP dependent and not ubiquitin-proteasome dependent.

ITC using recombinant purified POLRMT and ClpP proteins: ITC measurements were performed with MicroCal VP-ITC at 298K. All proteins and small molecules were dissolved in an aqueous solution containing 5% DMSO. To demonstrate the binding affinity between IMT and ClpP, mtPTAC3B-1 was dissolved in a solution with a final concentration of 40 μm and titrated into a ClpP solution (4 μm). To determine the binding affinity between ONC201 and POLRMT, mtPTAC3B-1 was solubilized at a final concentration of 10 μm and titrated into POLRMT solution (1 μm). The plotted ITC curve demonstrates that MtPTAC3B-1 can bind directly to POLRMT and ClpP (see FIGS. 10 and 11). Furthermore, it has POLRMT (K _d 3B-1 of =44.5 μm) and with ClpP (K) _d Binding constants (Kd) of 3B-1 of =794 nM) are respectively equivalent to those of the binding constant having POLRMT (K) _d =33.8μM)And has ClpP (K) _d =269 nM). These results indicate that MtPTAC3B-1 has binding force of the same order of magnitude as the original ligands of ClpP and POLRMT at the same time, indicating that the structural design of MtPTAC3B-1 is quite reasonable. Notably, immunoblotting results showed that the protein was efficiently degraded after 72 hours of treatment with 10. Mu.M MtPTAC3B-1, which was below the Kd value of the MtPTAC3B-1/POLRMT complex. This suggests that MtPTAC3B-1 can be reused for binding and degradation of target proteins.

To explore whether MtPTAC3B-1 could bring POLRMT and ClpP closer together, fluorescence Resonance Energy Transfer (FRET) experiments were performed with living cells, and the results are shown in FIG. 12a, in which POLRMT was expressed in cells by fusion with mNarGreen, and ClpP was expressed by fusion with mScarlet-I. To capture the ClpP/3B-1/polrbt ternary complex over a longer time window, while to facilitate observation to prevent false positives in small spaces of mitochondria, the mitochondrial localization sequences of ClpP and polrbt were removed to allow ClpP-mstarlet-I and polrbt-mneon green to reside in the spatially larger cytoplasm rather than in mitochondria. Since mNarGreen acts as a donor fluorophore to excite the acceptor fluorophore mScarlet-I, the decrease in spatial distance between these two fluorophores can be reflected in a decrease in mNarGreen fluorescence lifetime. Cells expressing mNannGreen-mScarlet-I fusion protein showed a significantly shorter fluorescence lifetime of mNannGreen than those cells coexpressing unfused mNannGreen-mScarlet-I, indicating that FRET can be generated in the environment of the cells. In cells co-transfected with ClpP-mScarlet-I and POLRMT-mNarEonGreen constructs, addition of MtPTAC3B-1 significantly shortened the lifetime of mNarEonGreen, and similar degrees of reduction were observed in the positive control mNarEon-Green-mScarlet-I. In contrast, IL lacking ClpP binding ligand did not decrease the fluorescence lifetime of mNanGreen (see FIG. 12 b). Furthermore, immunoblotting experiments showed that none of the cells treated with IL molecules plus free ONC201 or AL molecules plus free IMT significantly reduced the protein level of POLRMT (see fig. 12c and 12 d). FRET and immunoblot experiments described above indicate that MtPTAC3B-1 induces degradation of POLRMT by bringing POLRMT into proximity with ClpP.

The possible structure of ternary complex ClpP/3B-1/POLRMT was studied by MD simulation. Simulation analysis after MD operation was performed as follows: the last 20 nanosecond track frame is extracted, and then clustering analysis is performed by using a Gromacs method. Any two trace boxes are covered, the distance between them is the Root Mean Square Deviation (RMSD) of the protein backbone atoms, and the cut-off distance is set to 2.5 a. Track frames below this distance are considered as one cluster. A representative structure of the largest cluster of MD simulations is selected as a representative architecture for simulation run. The stability of ClpP/3B-1/POLRMT was further demonstrated in the 100ns simulation below: an overlay of the track frame is drawn and quantitative statistics are performed on the tracks to check for changes in the ternary complex geometry. The global mean Root Mean Square Deviation (RMSD) is only about 2.0 a, with the maximum RMSD in the simulation being below 3.0 a, indicating that the ternary complex ClpP/3B-1/polrbt can be obtained (see fig. 13).

2.4 inhibition of tumor cell proliferation by MtPTAC 3B-1

The effect of MtPTAC 3B-1 on the mRNA level of mitochondrial gene was tested by qRT-PCR, and the results are shown in FIG. 14, which shows that representative mitochondrial DNA encoding gene in cells after the cells were treated with MtPTAC 3B-1 COX-1AndCOX-2) Is significantly reduced. The effect of MtPTAC3B-1 on mitochondrial DNA level was tested using qRT-PCR, and the results are shown in FIG. 15, which shows that the copy number of mitochondrial DNA in cells after MtPTAC3B-1 treatment [ (]ND1、ND2、ND5、COX3AndATPA6) And also significantly reduced. Both mRNA expression and DNA copy number reduction of mitochondrial genes indicate that MtPTAC3B-1 effectively inhibited the biological function of POLRMT.

The proliferation curve of MtPTAC3B-1 treated cells was plotted, see fig. 16, and it can be seen that the real-time cell proliferation assay of the different cancer cell lines (a 549 and PC 9) exhibited a dose-dependent inhibition of cell proliferation of MtPTAC3B-1 (0-40 μm). Cloning experiments of MtPTAC3B-1 treated cells see fig. 17, it can be seen that MtPTAC3B-1 significantly reduced the number of clones of cancer cells (from 0-40 μm) in a dose dependent manner. In conclusion, mtPTAC3B-1 can significantly inhibit tumor cell proliferation at the cellular level.

A549 cells and HEK293 cells were treated with 40. Mu.M MtPTAC3B-1 for 24 or 72 hours and apoptotic cells were detected by Annexin V-FITC/PI apoptosis detection kit using flow cytometry, the results are shown in FIGS. 18-19, wherein Annexin V-FITC and PI double negative cells were living cells, annexin V-FITC and PI double positive cells were late apoptotic cells, annexin V-FITC negative cells and PI positive cells were necrotic cells, and Annexin V-FITC positive cells and PI negative cells were early apoptotic cells. It can be seen that after a final concentration of 40 μm MtPTAC3B-1 treatment, neither long-term (72 hours) nor short-term (24 hours) treatment groups, the number of apoptotic cells did not significantly increase compared to DMSO treatment groups, i.e., mtPTAC3B-1 did not induce apoptosis regardless of cell type and treatment time. High confluence (80%) H293K cells were seeded into petri dishes and treated with MtPTAC3B-1 for 96 hours, 0-40. Mu.M MtPTAC3B-1 did not significantly inhibit cell activity (see FIG. 20). The above results demonstrate that 3B-1 can significantly inhibit tumor cell growth and is not cytotoxic.

Low confluence (20%) A549 cells were treated with 40. Mu.M MtPTAC3B-1, mtPTAC3B-2 and IMT for 96 hours and cell viability was determined by CCK8, as seen in FIG. 21, mtPTAC3B-1, mtPTAC3B-2 and IMT all had some cell growth inhibition capacity; at the same concentration, mtPTAC3B-1 showed stronger cell inhibition than MtPTAC3B-2 and IMT, and this difference may be caused by different factors, on the one hand, degradation of POLRMT may inhibit its enzymatic activity more thoroughly, and on the other hand, degradation of POLRMT may inhibit its other biological functions independent of transcriptional activity, such as mitochondrial ribosome production, etc.

The above results indicate the importance of POLRMT degradation in MtPTAC3B-1 induced cell inhibition.

2.5 inhibition of cell proliferation in animals by MtPTAC3B-1

Proliferation of tumors in mice see figure 22, it can be seen that MtPTAC3B-1 group showed significantly lower tumor weight than placebo group after two weeks of treatment; tumor proliferation was not inhibited in the MtPTAC3B-2 group and was not significantly different from the placebo group; this suggests that the degradation of MtPTAC3B-1 plays a key role in tumor proliferation inhibition in animals, and that MtPTAC3B-1 has no significant off-target effect. Body weights of mice are seen in fig. 23, and it can be seen that there was no significant change in body weights of MtPTAC3B-1 and placebo group mice after two weeks of treatment; this indicates that the security of MtPTAC3B-1 was satisfactorily verified.

Mouse tumor tissue was homogenized to obtain histones and immunoblotted, resulting in fig. 24, which shows that in MtPTAC3B-1 treated mice, the amount of intact POLRMT was significantly reduced, while the amount of lysed POLRMT was significantly increased. The molecular weight of POLRMT in MtPTAC3B-1 treated mice was further reduced to about 70kD, much less than that observed in the cell line; this suggests that MtPTAC3B-1 can achieve tumor inhibition in vivo by degrading POLRMT. Immunoblots of oxidative phosphorylation complex associated proteins in tissue protein samples are shown in fig. 25, and it can be seen that NDUFB8 (complex I, whose expression is controlled by POLRMT) protein levels in MtPTAC3B-1 treatment group were significantly reduced, ATP5A and uqrc 2 (complex V and complex III, whose expression was not regulated by POLRMT) remained unchanged, suggesting that MtPTAC3B-1 degradation of POLRMT plays an important role in cell phenotype.

The above results indicate that MtPTAC3B-1 can induce degradation of polrbt in vivo and exhibit antitumor activity.

Example 3:

1. Synthetic route for CPI613

Reaction conditions: (a) NaOH solution, naBH4, 40 ℃; (b) acidification of benzyl bromide, naOH and hydrochloric acid.

1.1 Synthesis of (+/-) -dihydrolipoic acid (B)

Lipoic acid (A) 2.06g (0.01 mol,1 eq) was weighed into 20mL NaOH (1N) solution, heated to 40℃and stirred to react until complete dissolution. Then 567mg NaBH is added ₄ (0.015 mol,1.5 eq) was added slowly in 3 portions and allowed to react overnight at room temperature. LC-MS monitors the raw materials and the products, and considers the reaction to be completed when no raw material peak is detected, and the products can be directly subjected to the next reaction without purification。

1.2, 6, 8-bis (benzylthio) octanoic acid (CPI-613,4C)

To the untreated reaction mixture, 800mg of NaOH (0.02 mol,2 eq) was added, and then 2.05g of benzyl bromide (0.012 mol,1.2 eq) was slowly added dropwise to react overnight. The reaction solution was acidified by addition of hydrochloric acid and extracted with ethyl acetate (20 ml×3), the organic phase was washed successively with dilute hydrochloric acid, dried over anhydrous sodium sulfate and concentrated to give a crude product. The crude product was recrystallized from a petroleum ether-ethyl acetate system and dried to give 2.6g (67% yield) of white powder whose nmr spectrum data is as follows: ¹ H NMR (400 MHz，Chloroform-d)δ7.27-7.19 (m，8H)，7.19-7.12(m，2H)，3.59 (d，J= 10.1 Hz，4H)，2.49 (q，J= 6.4 Hz，1H)，2.46-2.39 (m，2H)，2.22 (t，J= 7.4 Hz，2H)，1.70-1.62(m，2H)，1.43 (dt，J= 22.9，7.4 Hz，4H)，1.33-1.20 (m，2H)。

2. synthesis of mitochondrial protease targeted chimeras (MtPTAC 3C)

2.1 synthetic route to MtPTAC 3C

Reaction conditions: (a) DCM, TFA, room temperature overnight; (b) HATU, DIPEA, DMF, CPI613, overnight at room temperature.

The different chain lengths of PEG are respectively named as MtPTAC3C-1 (n=1), mtPTAC 3C-2 (n=2), and MtPTAC3C-3 (n=3). The synthesis method of the MtPTAC3C-1, the MtPTAC 3C-2 and the MtPTAC3C-3 is the same, and the steps are as follows:

a) 57.4mg (0.1 mmol,1 eq) of 3A are dissolved in 20mL of DCM, 5mL of TFA are added overnight at room temperature, and after completion of the reaction the solvent and TFA are removed by evaporation in vacuo;

b) The reaction of step a) was dissolved in 5mL of DMF, and 57mg of HATU (0.15 mmol,1.5 eq) and 39mg of DIPEA (0.3 mmol,3 eq) and 39mg of CPI613 (0.1 mmol,1 eq) were added overnight at room temperature. After the reaction was completed, the reaction mixture was diluted with EA, and the organic phase was washed with a saturated sodium bicarbonate solution and a saturated ammonium chloride solution. Finally, the organic phase was dried over anhydrous sodium sulfate, and concentrated by vacuum evaporation and purified by silica gel column to give MtPTAC3C-1 (32 mg, yield 38%), mtPTAC 3C-2 (35 mg, yield 39%), mtPTAC3C-3 (29 mg, yield 31%), respectively.

N- (2- (4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-)a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -methyl) phenyl) amino) ethoxy) ethyl) -6, 8-bis (benzylthio) octanamide (MtPTAC 3C-1), ¹ H NMR (400 MHz，Chloroform-d)δ7.56-7.20 (m，13H)，7.19-6.83 (m，5H)，6.55 (d，J= 8.4 Hz，1H)，4.21-3.69(m，8H)，3.58 (d，J= 1.2 Hz，2H)，3.56 (d，J= 1.7 Hz，2H)，3.52-3.11 (m，9H)，2.69 (s，4H)，2.50 (t，J= 6.5 Hz，2H)，2.42(tt，J= 7.5，2.3 Hz，2H)，2.07 (q，J= 7.7 Hz，2H)，1.70-1.61(m，2H)，1.50-1.35 (m，6H)。

N- (2- (2- (4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-) a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -yl) methyl) phenyl) amino) ethoxy) ethyl) -6, 8-bis (benzylthio) octanamide (MtPTAC 3C-2), ¹ H NMR (400 MHz，Chloroform-d)δ7.35-7.20 (m，13H)，7.19-6.83 (m，5H)，6.55 (d，J= 8.4 Hz，1H)，4.37-4.09(m，2H)，3.94 (dd，J= 18.5，9.2 Hz，2H)，3.80 (d，J= 17.4 Hz，4H)，3.65-3.56(m，13H)，3.50-3.41 (m，4H)，2.91(d，J= 24.1 Hz，2H)，2.64 (d，J= 16.6 Hz，2H)，2.51 (d，J= 6.7 Hz，2H)，2.43(td，J= 6.4，5.6，2.1 Hz，2H)，2.03-1.86 (m，4H)，1.66 (dd，J= 9.7，5.7 Hz，6H)。

N- (2- (2- (4- ((7-benzyl-5-oxo-1,2,6,7,8,9-hexahydroimidazo [1, 2-)a]Pyrido [3,4 ]e]Pyrimidine-4 (5)H) -methyl) phenyl) amino) ethoxy) ethyl) -6, 8-bis (benzylthio) octanamide (MtPTAC 3C-3), ¹ H NMR (400 MHz，Chloroform-d)δ7.36-7.20 (m，13H)，7.18-6.88 (m，5H)，6.52-6.42 (m，1H)，4.19 (d，J= 10.0 Hz，2H)，3.97-3.86(m，2H)，3.74 (dd，J= 15.0，5.4 Hz，4H)，3.61-3.35 (m，21H)，2.83 (dd，J= 19.8，7.7 Hz，2H)，2.59 (d，J= 16.0 Hz，2H)，2.49(q，J= 7.5，7.0 Hz，2H)，2.42 (td，J= 7.6，2.1 Hz，2H)，1.97 (td，J= 11.1，10.2，5.2Hz，2H)，1.65 (qd，J= 7.1，3.2 Hz，2H)，1.50-1.35 (m，6H)。

example 4:

performance test of mitochondrial protease targeted chimeras (MtPTAC 3C)

1. Mitochondrial proteolysis assay

Protein degradation ability of MtPTAC (3C-1, 3C-2, 3C-3) was tested in A549 cell line (test method was the same as in example 2), A549 cells were treated with four different concentrations (0, 10, 20 and 40. Mu.M) of MtPTAC (3C-1, 3C-2, 3C-3) respectively for 24 hours, and the results were shown in FIG. 26, in which it was seen that MtPTAC 3C-2 (40. Mu.M) and MtPTAC3C-3 (20. Mu.M and 40. Mu.M) could significantly reduce the level of PDH protein, and MtPTAC3C-3 had better protein degradation ability.

Conventional operations in the operation steps of the present invention are well known to those skilled in the art, and are not described herein.

While the foregoing embodiments have been described in detail in connection with the embodiments of the invention, it should be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like made without departing from the spirit and scope of the invention.

Claims

1. A mitochondrial protease targeting chimera comprising a binding structure of an intra-mitochondrial aaa+ protease family and a binding structure of an intra-mitochondrial protein, the binding structure of the intra-mitochondrial aaa+ protease family linked to the binding structure of the intra-mitochondrial protein by a linker.

2. A mitochondrial protease targeted chimera according to claim 1, characterized in that: the wireThe binding structure of the intragranular aaa+ protease family is selected from ONC201 derivatives, which are compounds represented by the structure of formula E;the method comprises the steps of carrying out a first treatment on the surface of the In formula E, X is selected from halogen.

3. A mitochondrial protease targeted chimera according to claim 1 or 2, characterized in that: the binding structure of the intramitochondrial protein is selected from IMT or 6, 8-bis (benzylthio) octanoic acid; the IMT is a compound shown as a structure of a formula 2F; the linker is a PEG compound;。

4. a mitochondrial protease targeted chimera according to claim 1, characterized in that: the mitochondrial protease targeting chimera is a compound shown in a structure of a formula 3B or a formula 3C, or pharmaceutically acceptable salt, hydrate, solvate or polycrystal thereof;

；

5. Use of an ONC201 derivative represented by the structure of formula E in the preparation of a mitochondrial protease targeting chimera.

6. A method of preparing the mitochondrial protease targeted chimera of any one of claims 1-4, comprising the steps of:

7. A compound shown in the structure of formula 3A.

8. Use of a compound represented by the structure of formula 3A in the preparation of a mitochondrial protease targeting chimera.

9. Use of the mitochondrial protease targeted chimera of any one of claims 1-4 in the manufacture of a medicament for the degradation of a protein, said protein being an eukaryotic intracellular protein.

10. Use according to claim 9, characterized in that: the protein is eukaryotic cell internal protein, and the degradation of the eukaryotic cell internal protein is performed based on AAA+ protease.

11. Use of the mitochondrial protease targeted chimera according to any one of claims 1-4 for the preparation of a medicament for the treatment of mitochondrial dysfunction and/or an anti-tumor medicament.

12. A pharmaceutical composition comprising the mitochondrial protease targeted chimera of any one of claims 1-4.