A mitochondrial protease targeting chimera, preparation method and use

Abstract

The present invention discloses a mitochondrial protease targeting chimera, a preparation method and a use thereof, and belongs to the technical field of pharmaceutical chemistry. The mitochondrial protease targeting chimera of the present invention comprises a AAA+ protease family binding structure in mitochondria and a binding structure of a protein in mitochondria, and the binding structure of the AAA+ protease family in mitochondria and the binding structure of the protein in mitochondria are linked by a linker. The mitochondrial protease targeting chimera of the present invention is constructed by using an enzyme of the AAA+ protease family in mitochondria, and the protein is degraded by the AAA+ protease in mitochondria, which is the first method to achieve targeted degradation of protein targets in organelles, and can be used to prepare AAA+ protease-based drugs for protein degradation in eukaryotic cells.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a mitochondrial protease targeting chimera, a preparation method and use thereof. Background Art

[0002] Targeted protein degradation (TPD) is a new concept for drug development by using bifunctional chemical small molecules to interact with target proteins and protein degrading enzymes at the same time. At present, specific and efficient degradation of disease-related proteins can be achieved through two natural protein degradation systems in cells, namely the ubiquitin proteasome system (UPS) and the lysosomal degradation pathway, thereby achieving the effect of treating diseases. Classical methods in the drug discovery process usually aim to identify high-affinity small molecules that regulate the activity of target proteins. This occurs in an occupancy-driven manner, where inhibitors bind to their targets and occupy the binding site, thereby inhibiting the target function. This strategy has been very successful for many targets that contain tractable active or allosteric sites, such as enzymes or receptors. However, most proteins remain difficult to address, and 80-85% of the human proteome is undruggable due to the lack of available binding pockets and suitable chemicals. The biggest advantage of TPD compared to traditional small molecule inhibitors is that only binders are needed to recruit target proteins for degradation, rather than high-affinity inhibitors, which greatly solves the problem of protein undruggability. Currently, the most widely used method for degrading proteins through the ubiquitin proteasome system is PROTAC bifunctional small molecules: protein degradation targeted chimeras (PROTACs) include three parts, namely an E3 ubiquitin ligase ligand, a target protein ligand and a specially designed linker, and the two active ligands are connected together through the linker structure. After the ligand binds to the E3 ubiquitin ligase and the target protein to bring them closer, the protein is ubiquitinated and the target protein is degraded through the ubiquitination process. Due to the high dependence of the ubiquitin protein system, PROTACs technology is mainly used to degrade cytoplasmic and nuclear protein targets. Subsequently, many technologies that utilize lysosomal pathways, including endocytosis-lysosomal pathways and autophagy-lysosomal pathways, have emerged to degrade proteins, such as LYTACs, ATTECs, AUTACs, etc., which have expanded the range of degradable targets.

[0003] However, targeted degradation of protein targets in organelles has not been achieved so far. Protein abnormalities in organelles are associated with many diseases, the most common of which is protein abnormalities in mitochondria. Abnormal protein homeostasis in mitochondria is an important feature and driver of a series of human diseases, such as Leber hereditary optic neuropathy, maternal hereditary deafness, type II diabetes, Parkinson's disease and various cancers. In addition, when cells are stimulated by stress, a large number of denatured proteins in the cytoplasm will also be transported to the mitochondria and degraded by AAA+ proteases. LRPPRC protein is a newly discovered tumor marker for lung adenocarcinoma: it is a leucine-rich triangular pentapeptide repeat motif protein) and is a mitochondrial protein. The small molecule GAA was obtained through high-throughput screening, which greatly reduced the stability of LRPPRC protein after binding to it, and then LRPPRC protein was recognized and hydrolyzed by proteases. Although this is also a method of protein degradation, we cannot modularly design a new protein hydrolysis system. Although proteins located in the outer membrane of mitochondria can still be effectively degraded through the traditional ubiquitin-proteasome pathway, they cannot be degraded through the ubiquitin-proteasome pathway because there are no ubiquitination system proteins inside mitochondria. Summary of the invention

[0004] In a first aspect, the present invention aims to provide a mitochondrial protease targeting chimera that can target and degrade mitochondrial matrix proteins, which can be used to prepare AAA+ protease-based drugs for protein degradation in eukaryotic cells.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A mitochondrial protease targeting chimera includes a binding structure of the AAA+ protease family in mitochondria and a binding structure of a protein in mitochondria, wherein the binding structure of the AAA+ protease family in mitochondria and the binding structure of the protein in mitochondria are linked by a linker. The mitochondrial protease targeting chimera (MtPTAC) of the present invention is constructed by using the AAA+ protease family in mitochondria, and degrades proteins by using the AAA+ protease in mitochondria. It is the first method to achieve targeted degradation of protein targets in organelles, and can be used to prepare AAA+ protease-based drugs for protein degradation in eukaryotic cells.

[0007] In one embodiment, the binding structure of the AAA+ protease family in mitochondria is selected from ONC201 derivatives, which are compounds shown in the structure of formula E; ; In formula E, X selects a halogen. Targeted protein degradation (TPD) is an emerging protein regulation technology. At present, all TPDs developed in eukaryotic cells rely on the ubiquitin-proteasome or lysosome system, and therefore are powerless against target proteins in membrane organelles (such as mitochondria) that lack proteasomes and lysosomes. The mitochondrial targeted chimera (MtPTAC) of the present invention can solve the above problems and can effectively degrade mitochondrial proteins, which are closely related to various diseases. This is of great significance to subsequent drug development and may become a new direction.

[0008] In one embodiment, the binding structure of the mitochondrial protein is selected from IMT or 6,8-bis(benzylthio)octanoic acid; IMT is a compound as shown in the structure of formula 2F; linker is a PEG compound; .

[0009] In one embodiment, the mitochondrial protease targeting chimera is a compound as shown in the structure of Formula 3B or Formula 3C, or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof;

[0010] ; ; In Formula 3B or Formula 3C, n is any natural number from 1 to 10.

[0011] In one embodiment, in Formula 3B or Formula 3C, n is any natural number from 1 to 5.

[0012] In one embodiment, in Formula 3B or Formula 3C, n is any natural number from 1 to 3.

[0013] In one embodiment, the mitochondrial protease targeting chimera is a compound as shown in the structure of formula 3B-1, or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof; . The MtPTAC is a bifunctional small molecule, one end of which can bind to the mitochondrial casein hydrolyzing protease P (ClpP), and the other end can bind to the target protein (mitochondrial RNA polymerase POLRMT), which can activate the hydrolase activity of ClpP, and at the same time make the target protein close to ClpP, specifically hydrolyze the target protein in the mitochondria, and achieve the purpose of targeted degradation of mitochondrial matrix proteins. The effect of the mitochondrial precursor targeting chimera (MtPTAC) of the present invention on the degradation of proteins in cells (degradation exceeds 80% after 24 hours of treatment) and specificity have been verified, and a significant decrease in mtDNA has been detected; in animals, the tumors in the group treated with the mitochondrial precursor targeting chimera (MtPTAC) of the present invention were significantly smaller than those in the placebo control group, and the target protein and mtDNA levels were also detected to be decreased. Therefore, the mitochondrial precursor targeting chimera (MtPTAC) of the present invention can effectively degrade mitochondrial matrix proteins, and mitochondrial proteins are closely related to various diseases, which is of great significance to subsequent drug development and may become a new direction; the mitochondrial precursor targeting chimera (MtPTAC) of the present invention also has anti-tumor application prospects.

[0014] In one embodiment, the mitochondrial protease targeting chimera is a compound as shown in the structure of formula 3C-2, or a pharmaceutically acceptable salt, hydrate, solvate or polycrystal thereof, or, the mitochondrial protease targeting chimera is a compound as shown in the structure of formula 3C-3, or a pharmaceutically acceptable salt, hydrate, solvate or polycrystal thereof;

[0015] ; This MtPTAC has significant protein degradation ability.

[0016] In a second aspect, the present invention aims to provide a use of an ONC201 derivative as shown in the structure of formula E in preparing a mitochondrial protease targeting chimera. The ONC201 derivative of the present invention has an active site that can provide a ligation reaction, and can be linked to the binding structure of a mitochondrial protein through a linker to prepare a mitochondrial protease targeting chimera.

[0017] In a second aspect, the present invention aims to provide a method for preparing the above-mentioned mitochondrial protease targeting chimera.

[0018] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0019] The method for preparing a mitochondrial protease targeting chimera comprises the following steps:

[0020] Step 1), linking the ONC201 derivative and the PEG compound to obtain intermediate 3A, which is a compound shown in the structure of formula 3A;

[0021] Step 2), linking the intermediate 3A and the inhibitor to obtain a mitochondrial protease targeting chimera;

[0022] ; In formula 3A, n is any natural number from 1 to 10.

[0023] In one embodiment, the PEG compound is , n is any natural number from 1 to 10.

[0024] In a preferred embodiment, in the PEG compound, n is any natural number from 1 to 5.

[0025] In a more preferred embodiment, in the PEG compound, n is any natural number from 1 to 3.

[0026] In a preferred embodiment, in Formula 3A, n is any natural number from 1 to 5.

[0027] In a more preferred embodiment, in Formula 3A, n is any natural number from 1 to 3.

[0028] In a third aspect, the present invention aims to provide a compound as shown in Formula 3A. The compound as shown in Formula 3A is a template for mitochondrial protease targeting chimera, and the mitochondrial protease targeting chimera template can be used to modularly design a MtPTAC system suitable for mitochondrial protein degradation.

[0029] In a fourth aspect, the present invention aims to provide a use of a compound as shown in the structure of formula 3A in the preparation of a mitochondrial protease targeting chimera.

[0030] In a fifth aspect, the present invention aims to provide a use of the above-mentioned mitochondrial protease targeting chimera in the preparation of a drug for protein degradation, wherein the protein is a protein in a eukaryotic cell.

[0031] In one embodiment, the degradation of proteins in eukaryotic cells is based on AAA+ proteases.

[0032] In one embodiment, the mitochondrial protease targeting chimera is selected from the compound shown in the structure of formula 3B-1 and / or the compound shown in the structure of formula 3C-2 and / or the compound shown in the structure of formula 3C-3.

[0033] In a sixth aspect, the present invention aims to provide a use of the above-mentioned mitochondrial protease targeting chimera in the preparation of drugs for treating mitochondrial dysfunction and / or anti-tumor drugs.

[0034] In one embodiment, a mitochondrial protease targeting chimera is used in the preparation of a medicament for targeted degradation of mitochondrial matrix proteins.

[0035] In one embodiment, the mitochondrial protease targeting chimera is selected from the compound shown in the structure of formula 3B-1 and / or the compound shown in the structure of formula 3C-2 and / or the compound shown in the structure of formula 3C-3.

[0036] In one embodiment, the mitochondrial protease targeting chimera is used in the preparation of an anti-tumor drug, and the mitochondrial protease targeting chimera is a compound as shown in the structure of formula 3B-1.

[0037] In a preferred embodiment, the mitochondrial protease targeting chimera is used in the preparation of an anti-lung cancer drug, and the mitochondrial protease targeting chimera is a compound as shown in the structure of formula 3B-1.

[0038] In a sixth aspect, the present invention aims to provide a pharmaceutical composition comprising the above-mentioned mitochondrial protease targeting chimera.

[0039] In one embodiment, the mitochondrial protease targeting chimera is selected from the compound shown in the structure of formula 3B-1 and / or the compound shown in the structure of formula 3C-2 and / or the compound shown in the structure of formula 3C-3.

[0040] The present invention has the following beneficial effects: the mitochondrial targeted chimera (MtPTAC) of the present invention is constructed by utilizing the AAA+ protease family in mitochondria, and the AAA+ protease family in mitochondria degrades proteins, thereby realizing the first targeted degradation of protein targets in organelles; the mitochondrial targeted chimera (MtPTAC) of the present invention can effectively degrade mitochondrial matrix proteins, and can be used to prepare drugs for treating mitochondrial dysfunction; the mitochondrial precursor targeted chimera (MtPTAC 3B-1) of the present invention has an anti-tumor effect, and can be used to prepare anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the sequence diagram of ClpP and POLRMT used for ITC;

[0042] Figure 2 is the electropherogram of ClpP and POLRMT;

[0043] Figure 3 The target protein is hydrolyzed by MtPTAC in vitro; (a) is the immunoblot of POLRMT in A549 cells treated with different concentrations of MtPTAC 3B-1, MtPTAC 3B-2, and MtPTAC 3B-3; (b) is the immunoblot of POLRMT in A549 cells after treatment with different concentrations (0 μM, 5 μM, 10 μM, and 20 μM) of MtPTAC 3B-1 for 72 hours;

[0044] Figure 4is the immunoblot of POLRMT in different cell lines treated with different concentrations of MtPTAC 3B-1;

[0045] Figure 5 Immunoblotting of POLRMT in PC9 and A549 cells treated with 40 µM MtPTAC 3B-1 at different times;

[0046] Figure 6 qRT-PCR analysis of POLRMT mRNA levels in A549 cells and PC9 cells treated with different concentrations of MtPTAC 3B-1;

[0047] Figure 7 Proteomic analysis of the effect of 40 μM MtPTAC 3B-1 treatment on mitochondrial proteins; the heat map shows the fold change (log2FC) of mitochondrial protein abundance after 6 hours (a) and 12 hours (b) of MtPTAC 3B-1 treatment compared with DMSO treatment. The mitochondrial protein types were provided by the MitoCarta3.0 database;

[0048] Figure 8 Proteomic analysis of the effect of 40 μM MtPTAC 3B-1 treatment on mitochondrial proteins; heat map showing the fold change (log2FC) in mitochondrial protein abundance after 6 h (a) and 12 h (b) treatment with MtPTAC 3B-1 compared with DMSO treatment, mitochondrial protein species were provided by the UniProt database; (c) Immunoblotting of mitochondrial protein ATP5A in A549 and PC9 cells treated with different concentrations of MtPTAC 3B-1 for 24 h;

[0049] Fig. 9 LC-MS detection of MtPTAC 3B-1 in mitochondria; after A549 cells were treated with 20 μM MtPTAC 3B-1 for 0 h (a), 8 h (b), 16 h (c) and 24 h (d), mitochondria were extracted and the signal of MtPTAC3B-1 was analyzed by LC-MS; (e) is the signal of the MtPTAC 3B-1 standard sample; by normalizing the peak area of ​​MtPTAC3B-1 in HPLC with the protein amount in each sample (f), the relative level of MtPTAC 3B-1 in mitochondria during the MtPTAC 3B-1 treatment time was obtained, and the LC-MS results showed that MtPTAC3B-1 entered the mitochondria in a time-dependent manner;

[0050] Fig.10 To measure the binding of small molecule MtPTAC 3B-1 to ClpP by ITC;

[0051] Fig.11To measure the binding of the small molecule MtPTAC 3B-1 to POLRMT by ITC;

[0052] Fig.12 MtPTAC 3B-1 induces POLRMT degradation by bringing POLRMT into proximity with ClpP; (a) Schematic diagram of FLIM-FRET monitoring the proximity of POLRMT and ClpP, and representative lifetime heat maps of HEK293 cells treated with the corresponding constructs and MtPTAC 3B-1 or IL; When FRET occurs between mNeonGreen and mScarlet-I, the lifetime of mNeonGreens is shortened, (b) scatter plot of lifetime data for each group; immunoblot of POLRMT in A549 cells treated with AL plus free IMT (c) or IL plus free ONC201 (d);

[0053] Fig.13 Molecular dynamics simulation of the formation of the POLRMT / MtPTAC 3B-1 / ClpP ternary complex; (a) The last 20 ns trajectory of the molecular dynamics simulation of the ternary structure at 310.15 K. The structure is plotted every 2 ns. The color represents the time step and changes in the form of red-white-blue. The trajectory simulation results prove that the ClpP / MtPTAC 3B-1 / POLRMT complex is stable; (b) The statistics of the RMSD of the protein in the ternary structure relative to its representative structure in the last 20 ns. All conformations show small RMSD.

[0054] Fig.14 For MtPTAC 3B-1 reduction COX-1 and COX-2 mRNA levels of A549 cells treated with MtPTAC 3B-1 were detected by real-time PCR COX-1 and COX-2 mRNA, GAPDH The mRNA level of was used as an internal reference;

[0055] Fig.15 To investigate the reduction of mitochondrial DNA levels by MtPTAC 3B-1, A549 cells (a) and PC9 cells (b) were treated with 40 μM MtPTAC 3B-1 for different time periods, and mitochondrial DNA was detected by real-time PCR. The DNA level of DNA methyltransferase 1 (DNMT1, a gene in the nuclear genome) was used as an internal reference;

[0056] Fig.16 Representative proliferation curves of A549 (a) and PC9 (b) cells treated with different concentrations of MtPTAC 3B-1;

[0057] Fig.17Representative clone images of A549 and PC9 cells treated with different concentrations of MtPTAC 3B-1 (a), and quantification of the number of cell clones in each group (b);

[0058] Fig.18 Flow cytometry of A549 cells treated with 40 μM MtPTAC 3B-1;

[0059] Fig.19 Flow cytometry of HEK293 cells treated with 40 μM MtPTAC 3B-1;

[0060] Fig. 20 The effect of MtPTAC 3B-1 on HEK293 cell viability;

[0061] Fig.21 Effects of MtPTAC 3B-1 or MtPTAC 3B-2 on A549 cell viability (a), effects of MtPTAC 3B-1 or IMT on A549 cell viability (b);

[0062] Fig. 22 Comparison of MtPTAC 3B-1 and MtPTAC 3B-2 in in vivo tumor growth, (a) images of subcutaneous tumors isolated from mice treated with MtPTAC3B-1, MtPTAC 3B-2, or placebo for 14 days, (b) quantification of tumor weights obtained from mice treated with either MtPTAC3B-1, MtPTAC 3B-2, or placebo;

[0063] Fig.23 The weight changes of mice before and after MtPTAC 3B-1 treatment;

[0064] Fig.24 Immunoblotting of POLRMT in 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 in the MtPTAC 3B-1 group;

[0065] Fig.25 is an immunoblot of oxidative phosphorylation complexes in tumor tissues from mice treated with placebo or MtPTAC 3B-1;

[0066] Fig.26 This is an immunoblot of A549 cells treated with different concentrations of protein degraders MtPTAC 3C-1, MtPTAC 3C-2, and MtPTAC 3C-3 for 24 hours. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0068] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0069] Embodiment 1:

[0070] Synthesis and purification of the mitochondrial protease targeting chimera (MtPTAC 3B)

[0071] 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 400 MHz spectrometer. Unless otherwise stated, spectra were measured at 298 K and referenced to solvent chemical shifts. NMR chemical shifts ( δ ) is expressed in ppm, the coupling constant ( J ) are expressed in Hz. Data are reported as follows: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) polymorph, (br) broad. ESI(+) high-resolution mass spectra were acquired 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.

[0072] The chemicals used in this example were obtained from suppliers (as shown in Table 1) without further purification. Flash chromatography was performed using silica gel (200-300 mesh) as the stationary phase.

[0073] Table 1 Chemicals

[0074]

[0075] 1. Synthesis of ONC201 derivatives

[0076] Synthetic routes of ONC201 derivatives

[0077]

[0078] Reaction conditions: (a) methyl 3-bromopropionate, potassium carbonate, acetonitrile, 2 hours; (b) sodium methoxide, xylene, reflux, overnight; (c) iodomethane, methanol, room temperature, overnight; (d) p-bromobenzylamine, 1,4-dioxane, reflux, overnight; (e) sodium methoxide, methanol, reflux, overnight.

[0079] 1.1. Synthesis of dimethyl 3,3'-(benzyl azodiyl) dipropionate (1A)

[0080] Aniline (2.14 g, 20 mmol, 1 eq) and methyl 3-bromopropionate (7.5 g, 44 mmol, 2.2 eq) were dissolved in acetonitrile (50 ml), triethylamine (4.5 g, 44 mmol, 2.2 eq) was added and stirred at room temperature overnight. The mixture was then diluted with water (200 ml), the aqueous layer was extracted with dichloromethane (200 ml x 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 1A (2.7 g, 48.3% yield). 1 H NMR (400 MHz, CDCl3- d ) δ 7.26-7.18(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, CDCl3- d ) δ 173.2, 140.1, 128.4, 128.1, 127.0, 53.7, 51.6, 44.4, 34.5. Theoretical calculated value C 15 H 22 NO4 + [M+H] + =280.1543, HRMS (ESI) actual calculated value: 280.1545.

[0081] 1.2. Synthesis of 1-benzyl-4-oxopiperidine-3-carboxylic acid methyl ester (1B)

[0082] 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 was refluxed overnight. The mixture was then diluted with ice water (100 ml), the aqueous layer was extracted with dichloromethane (100 ml x 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) δ 7.45-7.39(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) δ 186.0, 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 NO3 + [M+H] + =248.1281, HRMS (ESI) actual calculated value: 248.1284.

[0083] 1.3, 2-(methylthio)-4,5-dihydro-1 H -Synthesis of Imidazole Hydroiodide (1C)

[0084] 4,5-dihydro-1 H -imidazole-2-thiol (1.2 g, 10 mmol, 1 eq) was dissolved in methanol, iodomethane (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) δ 9.98 (s, 1H), 3.36 (s, 4H), 2.63 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ 170.9, 45.7, 14.0. Theoretical calculation value C4H9N2S + [M+H] + =117.0481, HRMS (ESI) actual calculated value: 117.0481.

[0085] 1.4、 N -(4-bromobenzyl)-4,5-dihydro-1 H -Synthesis of Imidazol-2-amine (1D)

[0086] 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 was 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) δ 7.58-7.49(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) δ 135.6, 131.6, 128.9, 121.4, 45.2, 44.3, 42.8. Theoretical calculated value C 10 H 13 BrN3 + [M+H] + =254.0287, HRMS (ESI) actual calculated value: 254.0289.

[0087] 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 )-Keto(1E)

[0088] 1B (243 mg, 1 mmol, 1 eq) and 1D (247 mg, 1 mmol, 1 eq) were dissolved in anhydrous methanol, sodium methoxide (112 mg, 3 mmol, 3 eq) was added, and the mixture was refluxed overnight. The mixture was then diluted with ice water (20 ml), the aqueous layer was extracted with dichloromethane (20 ml x 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) δ 7.50-7.45(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) δ 161.1, 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 value C 23 H 24 BrN4O + [M+H] + =451.1128, HRMS (ESI) actual calculated value: 451.1126.

[0089] 2. Synthesis of IMT

[0090] Synthesis route of IMT

[0091]

[0092] Reaction conditions: (a) sodium hydride, diethyl carbonate, tetrahydrofuran, reflux, overnight; (b) resorcinol, trifluoroacetic acid, reflux, overnight; (c) cesium carbonate, N , N -dimethylformamide, ethyl 2-bromopropionate, 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-piperidinate, 40°C, 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 , N -Diisopropylethylamine, tert-butyl 2-(2-(2-aminoethoxy)ethoxyethylcarbamate, 40°C, overnight.

[0093] 2.1. Synthesis of ethyl 3-oxo-3-(o-tolyl)propionate (2A)

[0094] Will O-Methylbenzophenone (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 was refluxed overnight. The mixture was then diluted with ice water (200 ml), the aqueous layer was extracted with dichloromethane (200 ml x 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) δ 7.82(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) δ 197.1, 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 NaO3 + [M+Na] + =229.0835, HRMS (ESI) actual calculated value: 229.0837.

[0095] 2.2, 7-hydroxy-4-(o-tolyl)-2 H -Synthesis of amino-2-ketone (2B)

[0096] 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 was 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) δ 161.9, 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 value C 16 H 11 O3 - [MH] - =251.0714, HRMS (ESI) actual calculated value: 251.0713.

[0097] 2.3, 2-((2-oxo-4-(o-tolyl)-2 H Synthesis of ethyl (2C)-(7-methen-1-yl)oxy)propanoate

[0098] 2B (100 mg, 0.40 mmol, 1 eq) was dissolved in N , N -Dimethylformamide, ethyl 2-bromopropionate (145 mg, 0.80 mmol, 2 eq) and cesium carbonate (390 mg, 1.20 mmol, 3 eq) were added to room temperature overnight. The mixture was then diluted with water (20 ml), water was extracted with dichloromethane (20 ml x 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) δ 7.44-7.27(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 (100 MHz, MeOD- d 4) δ 171.5, 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 value C 21 H 21 O5 + [M+H] + =353.1384, HRMS (ESI) actual calculated value: 353.1376.

[0099] 2.4, 2-((2-oxo-4-(o-tolyl)-2 H Synthesis of 2-(2-methen-7-yl)oxy)propionic acid (2D)

[0100] 2C (81 mg, 0.23 mmol) was dissolved in 1N sodium hydroxide in methanol and water and stirred at room temperature overnight. The mixture was concentrated on a rotary evaporator and acidified to pH 2 with hydrochloric acid, and the aqueous layer was 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 was purified by flash column chromatography to give 2D (52 mg, 70% yield). 1 H NMR (400 MHz, MeOD- d 4) δ 7.43-7.27(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 value C 19 H 15 O5 - [MH] - =323.0925, HRMS (ESI) actual calculated value: 323.0925.

[0101] 2.5, 1-(2-((2-oxo-4-(o-tolyl)-2 H Synthesis of ethyl (2-methen-7-yl)oxy)propionyl)piperidine-3-carboxylate (2E)

[0102] 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 , N -diisopropylethylamine (62 mg, 0.48 mmol, 3 eq.), ethyl 3-piperidinylcarboxylate (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 was purified by flash column chromatography to give 2E (68 mg, 77% yield). 1 H NMR (400 MHz, MeOD- d 4) δ 7.44-7.25 (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) δ 173.1, 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 value C 27 H 30 NO6 + [M+H] + =464.2068, HRMS (ESI) actual calculated value: 464.2070.

[0103] 2.6, 1-(2-((2-oxo-4-(o-tolyl)-2 H Synthesis of 2-(2-methen-7-yl)oxy)propionyl)piperidine-3-carboxylic acid (2F)

[0104] 2E (78 mg, 0.18 mmol) was dissolved in 1N sodium hydroxide in methanol and water and stirred at room temperature overnight. 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 x 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) δ 7.44-7.25 (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 CNMR (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 value C 25 H 24 NO6 - [MH] - =434.1609, HRMS (ESI) actual calculated value: 434.1609.

[0105] 2.7, tert-butyl (2-(2-(1-(2-((2-oxo-4-(o-tolyl)-2 H Synthesis of (1-(2-(4-(4-(4-(4-(4-(4-(4-(4-(4-(4-(4-(4-piperidine-3-carboxamide)ethoxy)ethoxyethyl)carbamate (IL))

[0106] 2F (43.5 mg, 0.1 mmol, 1 eq) was dissolved in N , N - dimethylformamide, and add N,N,N ' , N '-Tetramethyl- O -(7-Azabenzotriazol-1-yl)urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq), N , N -diisopropylethylamine (39 mg, 0.3 mmol, 3 eq) and tert-butyl 2-(2-aminoethoxy)ethoxycarbamate (49.6 mg, 0.2 mmol, 2 eq) were added and the mixture was stirred at 40°C overnight. It 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 was purified by flash column chromatography to give IL (34 mg, 51.1% yield). 1 H NMR (400 MHz, MeOD- d 4) δ 7.35-7.22 (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) δ 174.2, 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 value C 36 H 48 N3O9 + [M+H] + =666.3358, HRMS (ESI) actual calculated value: 665.3356.

[0107] 3. Synthesis of mitochondrial protease targeting chimera (MtPTAC 3B)

[0108] Synthetic route of MtPTAC 3B

[0109]

[0110] Reaction conditions: (a) palladium acetate, potassium carbonate, tert-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 , N -Diisopropylethylamine, 2F, 40°C, overnight.

[0111] 3.1 Synthesis of Intermediate 3A

[0112] 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 Synthesis of (4-(2-amino)-1,4-dimethyl)-1,4-diphenyl ...

[0113] 1E (100 mg, 0.22 mmol, 1 eq) was dissolved in tert-butyl alcohol, and tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (67.32 mg, 0.33 mmol, 1.5 eq) was added for freezing and 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) were weighed for deoxygenation, and the solution was transferred to the container and reacted at 90 ° C overnight. The reaction mixture was filtered to obtain a solution, and then the solvent was removed on a rotary evaporator, and the residue was purified by flash column chromatography to obtain 3A-1 (60 mg, 47.5% yield). 1 H MNR (400MHz, MeOD- d 4) δ 7.37-7.30 (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) δ 161.7, 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 43N6O4 + [M+H] + =575.3341, HRMS (ESI) actual calculated value: 575.3346.

[0114] 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 Synthesis of (4-(2-amino)-1,2-dimethyl ...

[0115] 1E (100 mg, 0.22 mmol, 1 eq) was dissolved in tert-butyl alcohol and tert-butyl 2-(2-(2-aminoethoxy)ethoxy)carbamate (81.84 mg, 0.33 mmol, 1.5 eq) was added for freezing and 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) were weighed for deoxygenation, and the solution was transferred to the container and reacted at 90 ° C overnight. The reaction mixture was filtered to obtain a solution, and then the solvent was removed on a rotary evaporator, and the residue was purified by flash column chromatography to obtain 3A-2 (65 mg, 47.8% yield). 1 H NMR (400 MHz, CDCl3- d ) δ 7.43-7.11(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, CDCl3- d ) δ 160.5, 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 O5N6 + [M+H]+ =619.3603, HRMS (ESI) actual calculated value: 619.3602.

[0116] 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 Synthesis of (4-(2-amino)-1,2-dimethyl ...

[0117] 1E (100 mg, 0.22 mmol, 1 eq) was dissolved in tert-butyl alcohol, and tert-butyl (2-(2-(2-(2-(-2-(2-aminoethoxy)ethoxyethyl)carbamate) (95 mg, 0.33 mmol, 1.5 eq) was added for cryo-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 1,4-dihydro-1,4-dihydro-2-nitropropene (2,4-dihydro-1,4-dihydro- ... 1 H NMR (400 MHz, MeOD- d 4) δ 7.37-7.30 (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 (100 MHz, 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 O6N6 + [M+H] + =663.3865, HRMS (ESI) actual calculated value: 663.3863.

[0118] 3.2 Synthesis of MtPTAC 3B

[0119] 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 )-(2-(2-oxo-4-(o-tolyl)-2- H Synthesis of 2-(4-(2-formyl-7-oxy)propionyl)piperidine-3-carboxamide (3B-1)

[0120] 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 h, then concentrated by rotary evaporation. 2F (43.5 mg, 0.1 mmol, 1 eq) was dissolved in N , N -dimethylformamide, and N,N,N ' ,N '-Tetramethyl- O -(7-azabenzotriazol-1-yl)urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq) and N , N -Diisopropylethylamine (39 mg, 0.3 mmol, 3 eq) was added and reacted at 40°C overnight. Then it was diluted with ethyl acetate (20 ml) and washed three times with saturated ammonium chloride solution, and the combined organic layer was 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) δ 174.1, 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 O7N7 + [M+H] + =892.4392, HRMS (ESI) actual calculated value: 892.4386.

[0121] 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 )-((2-oxo-4-(o-tolyl)-2- H Synthesis of 2-(4-(2-(4-(-formyl-7-yl)oxy)propionyl)piperidine-3-carboxamide (MtPTAC 3B-2)

[0122] 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 h, then concentrated by rotary evaporation. 2F (43.5 mg, 0.1 mmol, 1 eq) was dissolved in N , N -dimethylformamide, and N,N,N ' ,N '-Tetramethyl- O -(7-azabenzotriazol-1-yl)urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq) and N , N -Diisopropylethylamine (39 mg, 0.3 mmol, 3 eq) was added and reacted overnight at 40 ° C. The mixture was then diluted with ethyl acetate (20 ml) and then 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) δ 7.42-7.20 (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, 11 1.9, 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 O8N7 + [M+H] + =936.4655, HRMS (ESI) actual calculated value: 936.4654.

[0123] 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 )-((2-oxo-4-(o-tolyl)-2- H Synthesis of 2-(4-(2-(4-(-formyl-7-yl)oxy)propionyl)piperidine-3-carboxamide (MtPTAC 3B-3)

[0124] 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 h, then concentrated by rotary evaporation. 2F (43.5 mg, 0.1 mmol, 1 eq) was dissolved in N,N-dimethylformamide and N,N,N ' ,N '-Tetramethyl- O -(7-azabenzotriazole-1-yl)urea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq) and N,N-diisopropylethylamine (39 mg, 0.3 mmol, 3 eq) were added and reacted overnight at 40 ° C. The mixture was then diluted with ethyl acetate (20 ml) and then 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) δ 7.54-6.95 (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) δ 174.2, 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 O9N7 + [M+H] + =980.4917, HRMS (ESI) actual calculated value: 980.4919.

[0125] Embodiment 2:

[0126] Performance testing of the mitochondrial protease targeting chimera (MtPTAC 3B)

[0127] 1. Experimental methods

[0128] 1.1 Western Blot

[0129] The cells were seeded in a 6-well plate and treated with drugs (different concentrations) for 24 hours after 24 hours. The cells were lysed using 100 μL of RIPA lysis buffer with MtPTAC added and placed in 1.5 mL centrifuge tubes respectively. The protein content was quantified and normalized using the BCA kit. Finally, the protein loading buffer was added and boiled in a 95°C metal bath for 5 minutes. The precast gel was used to determine the loading amount according to the normalization treatment for gel electrophoresis (90V, 120min), and then transferred to a PVDF membrane (activated with methanol in advance). The membrane was blocked with milk at a mass concentration of 5% for 1-2 hours and incubated with antibodies overnight (dilution ratio 1:2000, diluent mass concentration 5% milk). After the incubation, the primary antibody was recovered and the PDVF membrane was washed several times with PBST (PBS solution containing 0.1% Tween 20), and then the corresponding species secondary antibody was used for incubation for 1 hour to continue washing the bands and imaging in a gel chromatograph using an ultrasensitive developer (medium). The antibodies used are shown in Table 2. Numbers show the relative expression levels of POLRMT (normalized with Tubulin or GADPH).

[0130] Table 2 Antibodies

[0131]

[0132] 1.2 Proteomic methods

[0133] Cells were lysed with an EDTA-free lysis buffer and MtPTAC mixture containing 50 mM PBS and 500 mM NaCl. Protein concentration was quantified by BCA assay with 50 μg of total protein per sample, using in-solution enzyme digestion. Each 100 μL of lysate was reduced with 10 mM DTT, followed by alkylation with 20 mM IAA, and then the protein sample was digested with trypsin (10 μg / mL) at 37 ° C overnight. The digest was collected by centrifugation, and the filter device was rinsed with 50 μL 0.5 M NaCl and then centrifuged again. The resulting solution was combined and acidified with 10% trifluoroacetic acid. Acidified peptides were desalted using a C18 tip (Pierce, Thermo Scientific) according to the manufacturer's instructions. The desalted peptides were eluted with 50 μL 50% MeCN, and the eluted product was dried under vacuum and resuspended in 30 μL 0.1% formic acid (FA). Desalted peptides were subjected to an OrbitrapExploris 480 (Thermo Fisher Scientific) mass spectrometer in data-dependent acquisition (DDA) mode to acquire MS data. All raw files were searched against the Uniprtot human protein sequence database (UP000005640, 74349 forward entry, 2019) in the Proteome Discoverer (PD) software (version 2.4). The mass tolerance for precursors or peptides was 10 ppm, the mass tolerance for fragment ions was 0.02 Da, two missed trypsin cleavages were allowed, and cysteine ​​carbamidomethylation was set as a fixed modification. Each group contained four biological replicates, and proteins that could be detected in at least three samples in each group were included for subsequent differential expression analysis.

[0134] 1.3. Mitochondria Extraction Method Using Mitochondria Kit

[0135] 1) 2 × 10 71. Pellet cells. 2. Add 800 µL of Mitochondria Isolation Reagent A. Vortex at medium speed for 5 seconds and incubate the tube on ice for exactly 2 minutes. 3. Transfer the cell suspension to a Dounce tissue grinder. 4. Homogenize the cells on ice, performing enough strokes to effectively lyse the cells. 5. Return the lysed cells to the original tube and add 800 µL of Mitochondria Isolation Reagent C. 6. Rinse the Dounce tissue grinder with 200 µL of Mitochondria Isolation Reagent A and add it to the tube containing the sample. 7. Invert the tube several times to mix (do not vortex). 8. Centrifuge the tube at 700 × g for 10 minutes at 4°C. 9. Transfer the supernatant to a new 2.0 mL tube and centrifuge at 12,000 × g for 15 minutes at 4°C. 10. Transfer the supernatant (cytosol fraction) to a new tube and the pellet contains the isolated mitochondria. 11) Add 500 µL of Mitochondria Isolation Reagent C to the pellet and centrifuge at 12,000 × g for 5 min. Discard the supernatant. 12) Keep the mitochondrial pellet on ice prior to downstream processing. Freezing and thawing may compromise mitochondrial integrity.

[0136] 1.4 LC-MS method

[0137] The extracted mitochondria were lysed with 50 μL of lysis solution, and 1 μL of each sample was taken and injected onto a BEH C18 (2.1*50mm1.7μm) column. The solvent delivery module was used to flow a HPLC gradient of 30-95% B (A solvent phase: 0.1% formic acid in water, B solvent: 0.1% formic acid in acetonitrile) at a flow rate of 0.3 mL / min in 7 minutes. The sample eluting the column flowed through a UV detector at a wavelength of 254 nm. Mass spectrometry detection was performed using an Agilent LCMS-6495C quadrupole mass spectrometer and anelec-trospray ionization (ESI) interface. The ESI source was set to positive ionization mode, and the mass selective detector was used in multiple reaction monitoring (MRM) mode to obtain the highest possible selectivity and sensitivity. The instrument scanned in the m / z range of 400–1500, and quantification was performed by peak area. Data acquisition and processing were completed using the Agilent LC-MS Solution Software LCMS-6495C system and Graphpad 7. First, the retention time and fragmentation peak of the pure MtPTAC 3B-1 were determined by liquid chromatography-mass spectrometry (LC / MS), and then the above mitochondrial lysate was tested by LC / MS, and a signal with the same retention time and fragmentation peak as MtPTAC 3B-1 was found. It is worth noting that the concentration of MtPTAC3B-1 in mitochondria increases with the increase of treatment time, indicating that the entry of MtPTAC 3B-1 into mitochondria is a relatively slow process.

[0138] 1.5. siRNA sequences targeting ClpP

[0139] The siRNA sequences targeting ClpP are shown in Table 3.

[0140] Table 3 siRNA sequences targeting ClpP

[0141]

[0142] 1.6. Design, purification and isothermal titration calorimetry (ITC) of recombinant proteins

[0143] First, recombinant POLRMT and ClpP need to be purified. The expression and purification methods of ClpP and POLRMT are as follows: The sequence diagram of ClpP and POLRMT used for ITC is shown in Figure 1 The optimized ORF sequences were synthesized by Genscript and inserted into pET23b between the NdeI and XhoI sites, respectively, and then confirmed by sequencing. Figure 2 BL21(DE3) and the corresponding plasmids were cultured in LB at 37°C. 600 When the value reached 0.8, isopropyl-1-thio-BD-pyranogalactoside was added at a final concentration of 0.1mM, and the bacteria were cultured at 37°C for 4 hours to express ClpP, or at 16°C for another 18 hours to express POLRMT. The bacteria were harvested by centrifugation, resuspended in binding buffer (20mM Tris-HCl, 500mM NaCl, 5mM imidazole, 5% glycerol, pH 7.8), and lysed by ultrasound. After centrifugation (10000rpm, 30 minutes), the supernatant was passed through a nickel-nitrilotriacetic acid agarose column pre-equilibrated with binding buffer, and then washed with 50mM imidazole binding buffer. Finally, ClpP and POLRMT were eluted with 400mM imidazole and concentrated by centrifugal ultrafiltration.

[0144] 1.7 Molecular dynamics (MD) simulation

[0145] The structure of the ternary complex ClpP / 3B-1 / POLRMT was studied by MD simulation. The MD simulation of ClpP / 3B-1 / POLRMT was divided into three parts: 1) calculation of POLRMT of ligand 2G; 2) calculation of ClpP using ONC201 derivative 1E; 3) combining these two parts to obtain the complex ClpP / 3B-1 / POLRMT.

[0146] The parameters were set as follows: throughout the calculation steps, the amber force field (FF14SB) was used for the protein and the GAFF force field was used for the ligand. MD was performed using the Gromacs program (version 2021.5). The leapfrog integration algorithm, the stochastic velocity rescaling (Bussi-Parinello) thermostat, and the stochastic cell rescaling barometer were used for all MD processes (always the NPT ensemble). The step size was set to 2 fs. The cutoff radius for vdW interactions was set to 10 Å, and the spME algorithm was used for electrostatic interaction calculations. The system setup was done using the pdb2gmx tool in Gromacs 2021.5 and the Antecomber tool in the Ambertools21 suite, where periodic boundary conditions were used; the box shape was a cube with an absolute size of 12 Å larger on each side than the maximum size of the system, the RESP2 (0.5) atomic partial charges of the ligands were calculated by the Multiwfn 3.8 (dev) program, and the wave functions at the B3LYP-D3 (BJ) / def2 TZVP level were calculated by the ORCA 5.0.2 software. Explicit TIP3P water was added, and the system was neutralized with potassium and chloride ions and salted to 0.02 M ionic strength. The ion concentrations refer to the experimental results of the mitochondrial matrix in previous reports, as sodium and chloride ions were added at a concentration of 0.005 M.

[0147] For the complex of POLRMT / IMT, the starting coordinates of POLRMT were derived from the crystal structure downloaded from the Protein Data Bank (PDB) portal. To replace the ligand in this structure with IMT, the ligand in PDB entry 7A8P was removed and then the PDB file was processed on the Swiss Model website to add hydrogen atoms, construct missing loops, and assign protonation states of ionizable groups by assuming a pH of 7.8, which is consistent with the mitochondrial matrix. Molecular docking of the prepared protein with 2G has been performed by the AutodockVina 1.2.3 program using the Vina scoring function. Then, MD was performed by a three-stage process: (1) energy minimization of the complex by the steepest descent method for 1000 steps; (2) simulation of 400 ps of annealing from 0 K to 310.15 K with a harmonic potential to suppress the backbone; and (3) simulation of 50 ns to relax the loops and side chains.

[0148] For complex ClpP / 1E, the starting coordinates of human mitochondrial ClpP were taken from PDB entry 6DL79. MD simulations were performed using the same procedure as described above.

[0149] 1.8. Drawing of cell proliferation curve

[0150] The growth curves of A549 and PC9 were detected 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 data were continuously recorded from 0 to 196 hours, and then the growth curves of the cells in each well were plotted.

[0151] 1.9 Cell cloning and proliferation experiments

[0152] The cells were seeded in a 12-well plate at a density of 300 cells / well and then cultured in a medium containing a specific drug for 2 weeks until the cells grew into colonies (more than 100 cells), during which the medium was replaced every 3 days. After washing with PBS, ethanol was added for fixation, and a crystal violet staining solution was added for 5-10 minutes. The 12-well plate was then rinsed with a large amount of water and dried. A clone picture of each well was obtained, and the number of clones in each well was calculated.

[0153] 1.10 Cell viability assay

[0154] The relative cell viability of different cell lines was assessed using a cell counting kit 8 (CCK-8) according to the manufacturer's instructions. Cell viability was normalized using the same volume of DMSO-treated cells and untreated cells as controls.

[0155] 1.11. Flow cytometry

[0156] A549 cells and HEK293 cells were seeded in 6-well plates and treated with 40 μM 3B-1 for 24 hours and 72 hours, respectively. Equal volumes of DMSO-treated and untreated cells were used as controls. Annexin V-FITC apoptosis detection kit (Beyotime, 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 at 37°C for 15 minutes. The samples were then analyzed by flow cytometer SP6800 spectrometer.

[0157] 1.12. Mouse Animal Experiment

[0158] All studies involving mice were approved by the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences. BALB / c nude mice (Vitron Lever) aged 4–6 weeks were used for mouse experiments. A549 cell suspension (1 × 10 7 / mL, 200μL) to establish a cancer xenograft mouse model. When the tumor size reached about 100mm 3At the same time, mice were randomly divided into MtPTAC 3B-1 treatment group, MtPTAC 3B-2 treatment group and placebo treatment group. 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, the nude mice were sacrificed and the subcutaneous tumors were peeled off to measure the tumor weight. The subcutaneous tumors were further used for immunoblotting. In addition, the body weight of all mice was recorded before and after the experiment.

[0159] 2. Experimental results

[0160] 2.1. Mitochondrial proteolysis test

[0161] The protein degradation ability of MtPTAC (3B-1, 3B-2, 3B-3) was tested in the A549 cell line: A549 cells were treated with four different concentrations of MtPTAC (0, 0.1, 10 and 40 μM) for 24 hours, and the changes in their levels were analyzed by western blotting (see Figure 3 a), MtPTAC 3B-1 can degrade 80% of POLRMT, while MtPTAC 3B-2 and MtPTAC 3B-3 cannot significantly reduce the protein level of POLRMT. Extending the treatment time of MtPTAC 3B-1 to 72 hours (see Figure 3 b), 10 μM MtPTAC3B-1 can significantly degrade POLRMT.

[0162] The protein degradation ability of MtPTAC 3B-1 was tested in other cell lines (see Figure 4 ), in the selected four cell lines (A549, H1299, H460 and PC9), after 24 hours of treatment with 40μM MtPTAC 3B-1, significant protein degradation was consistently observed. In addition, when A549 and PC9 cell lines were treated with 40μM MtPTAC 3B-1 for different time periods (0-24h), a significant time dependence was observed (see Figure 5 ).

[0163] The RNA level of POLRMT was detected before and after the addition of MtPTAC 3B-1, and the quantitative PCR (qRT-PCR) experiment with reverse transcription showed that (see Figure 6 ): Cells were treated with 40 μM MtPTAC 3B-1 for 24 hours, and the mRNA level of POLRMT did not change significantly when the protein level decreased significantly, which confirmed that MtPTAC 3B-1 directly affects the level of the target protein POLRMT. Therefore, MtPTAC 3B-1 can effectively and directly induce the degradation of target proteins in a variety of cell lines, and the degradation efficiency is closely related to the length of the polyethylene glycol linker and is time-dependent.

[0164] 2.2. Mitochondrial proteolysis specificity test

[0165] Quantitative analysis using mitochondrial protein information provided by the MitoCarta3.0 database showed that none of the mitochondrial proteins decreased significantly (log2FC < -1, p < 0.001) after 6 h of treatment (see Figure 7 a), while after 12 hours of treatment, only POLRMT was significantly downregulated (log2FC=-1.093, p=0.000883) (see Figure 7 b). Similar trends were observed using mitochondrial protein information provided by the UniProt database (see Figure 8 a and Figure 8 b). The level of ATP5A protein in mitochondria was also tested (see Figure 8 c), immunoblotting showed that its levels were not affected. Therefore, MtPTAC 3B-1 induced the specific degradation of POLRMT in mitochondria, rather than the non-specific degradation of mitochondrial proteins as the ClpP ligand ONC201 does.

[0166] 2.3. Research on the mechanism of mitochondrial proteolysis

[0167] A549 cell lines were treated with 20 μM MtPTAC 3B-1 for 0, 8, 16 and 24 hours, and their mitochondria were extracted using a mitochondrial kit. First, the retention time and fragmentation peaks of the pure MtPTAC 3B-1 were determined by liquid chromatography-mass spectrometry (LC / MS), and then the above mitochondrial lysate was tested by LC / MS. The results are shown in Fig. 9 , a signal with the same retention time and fragmentation peak as MtPTAC 3B-1 was found. It is worth noting that the concentration of MtPTAC 3B-1 in mitochondria increases with the increase of treatment time, which indicates that the entry of MtPTAC 3B-1 into mitochondria is a relatively slow process.

[0168] Three siRNAs were designed to knock down ClpP in A549 cells (see Table 3 for the sequences of siRNAs), and MtPTAC 3B-1 was added for treatment. Even when 40 μM MtPTAC 3B-1 was used to treat A549 cells in which ClpP was knocked down by siNRA, the total protein amount and molecular weight of POLRMT in A549 cells did not change significantly. MG132 was used to block the proteolytic activity of the proteasome. The results of immunoblotting of cells treated with 40 μM MtPTAC 3B-1 showed that the inhibition of ubiquitin proteasome activity by MG132 could not prevent MtPTAC 3B-1 from degrading POLRMT in cells. Therefore, the degradation of POLRMT induced by MtPTAC3B-1 is ClpP-dependent rather than ubiquitin-proteasome-dependent.

[0169] ITC using recombinant purified POLRMT and ClpP proteins: ITC measurements were performed at 298K using MicroCal VP-ITC. All proteins and small molecules were dissolved in an aqueous solution containing 5% DMSO. To demonstrate the binding affinity between IMT and ClpP, MtPTAC 3B-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, MtPTAC 3B-1 was dissolved at a final concentration of 10 μM and titrated into a POLRMT solution (1 μM). The plotted ITC curves demonstrated that MtPTAC 3B-1 can directly bind to POLRMT and ClpP (see Fig.10 and Fig.11 ). In addition, with POLRMT(K d =44.5 μM) and 3B-1 with ClpP (K d =794 nM) of 3B-1 and POLRMT (K d =33.8μM) and ClpP (K d =269 nM)) with similar binding constants to IMT. These results indicate that MtPTAC 3B-1 has the same order of magnitude binding force to both ClpP and POLRMT as the original ligand, indicating that the structural design of MtPTAC 3B-1 is very reasonable. It is worth noting that immunoblotting results showed that the protein was effectively degraded after 72 hours of treatment with 10 μM MtPTAC 3B-1, which is lower than the Kd value of the MtPTAC3B-1 / POLRMT complex. This indicates that MtPTAC 3B-1 can be repeatedly used for the binding and degradation of target proteins.

[0170] To explore whether MtPTAC 3B-1 can bring POLRMT and ClpP closer, fluorescence resonance energy transfer (FRET) experiments were performed using living cells. Fig.12 a, POLRMT was expressed in cells by fusion with mNeonGreen, while ClpP was expressed by fusion with mScarlet-I. In order to capture the ClpP / 3B-1 / POLRMT ternary complex in a longer time window, and to facilitate observation and prevent false positives in the narrow space of mitochondria, the mitochondrial localization sequences of ClpP and POLRMT were removed so that ClpP-mScarlet-I and POLRMT-mNeonGreen stayed in the larger space of the cytoplasm instead of mitochondria. Since mNeonGreen acts as a donor fluorophore to excite the acceptor fluorophore mScarlet-I, the reduction in the spatial distance between the two fluorophores can be reflected in the reduction of the fluorescence lifetime of mNeonGreen. Cells expressing the mNeon-Green-mScarlet-I fusion protein showed that the fluorescence lifetime of mNeonGreen was significantly shorter than those co-expressing unfused mNeonGreen-mScarlet-I, indicating that FRET can be generated in this cellular environment. In cells co-transfected with ClpP-mScarlet-I and POLRMT-mNeonGreen constructs, addition of MtPTAC 3B-1 significantly shortened the lifetime of mNeonGreen, and a similar degree of reduction was observed in the positive control mNeon-Green-mScarlet-I. In contrast, IL lacking the ClpP binding ligand was unable to reduce the fluorescence lifetime of mNeonGreen (see Fig.12 b). In addition, immunoblotting experiments showed that all cells treated with IL molecules plus free ONC201 or AL molecules plus free IMT failed to significantly reduce the protein level of POLRMT (see Fig.12 c and Fig.12 d) The above FRET and immunoblotting experiments show that MtPTAC 3B-1 induces POLRMT degradation by bringing POLRMT into proximity with ClpP.

[0171] The possible structure of the ternary complex ClpP / 3B-1 / POLRMT was investigated by MD simulation. The simulation analysis after the MD run was performed as follows: the trajectory frames of the last 20 nanoseconds were extracted and then clustered using the Gromacs method. Any two trajectory frames were overlaid and the distance between them was the root mean square deviation (RMSD) of the protein backbone atoms, with the cutoff distance set to 2.5Å. Trajectory frames with a distance lower than this distance were considered as a cluster. The representative structure of the largest cluster of the MD simulation was selected as the representative architecture of the simulation run. The stability of ClpP / 3B-1 / POLRMT was then further confirmed in a 100ns simulation: an overlay of the trajectory frames was plotted, and quantitative statistics of the trajectories were performed to examine the changes in the ternary complex geometry. The global average root mean square deviation (RMSD) was only about 2.0Å, and the maximum RMSD in the simulation was below 3.0Å, indicating that the ternary complex ClpP / 3B-1 / POLRMT can be obtained (see Fig.13 ).

[0172] 2.4. Inhibition of tumor cell proliferation by MtPTAC 3B-1

[0173] The effect of MtPTAC 3B-1 on mitochondrial gene mRNA levels was tested by qRT-PCR. The results are shown in Fig.14 It can be seen that after MtPTAC 3B-1 treatment of cells, the representative mitochondrial DNA encoding genes in the cells ( COX-1 and COX-2 ) mRNA levels were significantly reduced. The effect of MtPTAC 3B-1 on mitochondrial DNA levels was tested by qRT-PCR. The results are shown in Fig.15 , it can be seen that the copy number of mitochondrial DNA in cells treated with MtPTAC 3B-1 ( ND1 , ND2 , ND5 , COX3 and ATPA6 The reduction of mitochondrial gene mRNA expression and DNA copy number indicated that MtPTAC 3B-1 effectively inhibited the biological function of POLRMT.

[0174] Proliferation curves of cells treated with MtPTAC 3B-1 were plotted. Fig.16 As can be seen, real-time cell proliferation assays of different cancer cell lines (A549 and PC9) showed a dose-dependent inhibition of cell proliferation by MtPTAC 3B-1 (0-40 μM). The results of the cloning assay of cells treated with MtPTAC 3B-1 are shown in Fig.17, it can be seen that MtPTAC 3B-1 significantly reduced the number of cancer cell clones in a dose-dependent manner (from 0-40 μM). In short, at the cellular level, MtPTAC 3B-1 can significantly inhibit tumor cell proliferation.

[0175] A549 cells and HEK293 cells were treated with 40 μM MtPTAC 3B-1 for 24 or 72 h, and apoptotic cells were detected by flow cytometry using the AnnexinV-FITC / PI apoptosis detection kit. Figure 18-19 , among which, Annexin V-FITC and PI double negative cells are living cells, Annexin V-FITC and PI double positive cells are late apoptotic cells, Annexin V-FITC negative cells and PI positive cells are necrotic cells, and Annexin V-FITC positive cells and PI negative cells are early apoptotic cells. It can be seen that after treatment with MtPTAC 3B-1 at a final concentration of 40 μM, the number of apoptotic cells did not increase significantly compared with the DMSO treatment group, whether in the long-term (72 hours) or short-term (24 hours) treatment group, that is, regardless of the cell type and treatment time, MtPTAC3B-1 does not induce cell apoptosis. Highly confluent (80%) H293K cells were seeded into culture dishes and treated with MtPTAC 3B-1 for 96 hours. 0-40 μM MtPTAC 3B-1 did not significantly inhibit cell activity (see Fig. 20 ). The above results confirmed that 3B-1 can significantly inhibit the growth of tumor cells and has no cytotoxicity.

[0176] Low confluence (20%) A549 cells were treated with 40 μM MtPTAC 3B-1, MtPTAC 3B-2, and IMT for 96 h, and cell viability was determined by CCK8. Fig.21 , it can be seen that MtPTAC 3B-1, MtPTAC3B-2 and IMT all have certain cell growth inhibitory abilities; at the same concentration, MtPTAC 3B-1 exhibits a stronger cell inhibitory effect than MtPTAC 3B-2 and IMT. This difference may be caused by different factors. On the one hand, the degradation of POLRMT can more thoroughly inhibit its enzymatic activity; on the other hand, the degradation of POLRMT can inhibit its other biological functions independent of transcriptional activity, such as mitochondrial ribosome generation.

[0177] The above results indicate the importance of POLRMT degradation in the cell inhibition caused by MtPTAC 3B-1.

[0178] 2.5. Inhibition of cell proliferation by MtPTAC 3B-1 in animals

[0179] The proliferation of tumors in mice Fig. 22 It can be seen that after two weeks of treatment, the MtPTAC 3B-1 group showed a significantly lower tumor weight than the placebo group; the tumor proliferation of the MtPTAC 3B-2 group was not inhibited, and there was no significant difference from the placebo group; this shows that the degradation of MtPTAC 3B-1 plays a key role in the inhibition of animal tumor proliferation, and MtPTAC3B-1 has no significant off-target effects. Fig.23 , it can be seen that after two weeks of treatment, there was no significant change in the body weight of mice in the MtPTAC3B-1 group and the placebo group; this indicates that the safety of MtPTAC3B-1 has been satisfactorily verified.

[0180] Mouse tumor tissue was homogenized to obtain tissue proteins, and immunoblotting was performed. Fig.24 It can be seen that in mice treated with MtPTAC 3B-1, the amount of intact POLRMT was significantly reduced, while the amount of cleaved POLRMT was significantly increased. The molecular weight of POLRMT in mice treated with MtPTAC 3B-1 was further reduced to about 70kD, which is much smaller than the molecular weight observed in cell lines; this indicates that MtPTAC3B-1 can achieve tumor suppression in vivo by degrading POLRMT. Immunoblotting of proteins related to oxidative phosphorylation complexes in tissue protein samples is shown in Figure 2. Fig.25 , it can be seen that the protein level of NDUFB8 (complex I, whose expression is controlled by POLRMT) in the MtPTAC 3B-1 treated group was significantly reduced, while ATP5A and UQCRC2 (complex V and complex III, whose expression is not regulated by POLRMT) remained unchanged, indicating that the degradation of POLRMT by MtPTAC 3B-1 plays an important role in the cell phenotype.

[0181] The above results indicate that MtPTAC 3B-1 can induce POLRMT degradation in vivo and exhibit antitumor activity.

[0182] Embodiment 3:

[0183] Synthesis and purification of the mitochondrial protease targeting chimera (MtPTAC 3B)

[0184] 1. Synthesis route of CPI613

[0185]

[0186] Reaction conditions: (a) NaOH solution, NaBH4, 40°C; (b) benzyl bromide, NaOH, acidified with hydrochloric acid.

[0187] 1.1. Synthesis of (+ / -)-dihydrolipoic acid (B)

[0188] Weigh 2.06 g (0.01 mol, 1 eq) of lipoic acid (A) and dissolve it in 20 mL of NaOH (1 N) solution, heat to 40 ° C and stir to react until completely dissolved. Then add 567 mg NaBH4 (0.015 mol, 1.5 eq) in 3 batches and slowly add and cool to room temperature to react overnight. LC-MS monitors the raw material and product. When the raw material peak is not detected, the reaction is considered to be complete, and the product can be directly used for the next step without purification.

[0189] 1.2, 6,8-Bis(benzylthio)octanoic acid (CPI-613, 4C)

[0190] In the above untreated reaction mixture, 800 mg of NaOH (0.02 mol, 2 eq) was added, and then 2.05 g of benzyl bromide (0.012 mol, 1.2 eq) was slowly dripped and reacted overnight. The reaction solution was acidified with hydrochloric acid and extracted with ethyl acetate (20 mL × 3). The organic phase was further washed with dilute hydrochloric acid, dried with anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was recrystallized and dried from a petroleum ether-ethyl acetate system to obtain 2.6 g of a white powder (yield 67%), and its nuclear magnetic resonance spectrum data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 7.27-7.19 (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 = 7.4Hz, 2H), 1.70-1.62(m, 2H), 1.43 (dt, J = 22.9, 7.4 Hz, 4H), 1.33-1.20 (m, 2H).

[0191] 2. Synthesis of mitochondrial protease targeting chimera (MtPTAC 3C)

[0192] 2.1 Synthesis route of MtPTAC 3C

[0193]

[0194] Reaction conditions: (a) DCM, TFA, room temperature overnight; (b) HATU, DIPEA, DMF, CPI613, room temperature overnight.

[0195] According to the different lengths of the PEG chains, they are named MtPTAC 3C-1 (n=1), MtPTAC 3C-2 (n=2), and MtPTAC3C-3 (n=3). The synthesis methods of MtPTAC 3C-1, MtPTAC 3C-2, and MtPTAC3C-3 are the same, and the steps are as follows:

[0196] a) 57.4 mg (0.1 mmol, 1 eq) of 3A was dissolved in 20 mL of DCM, 5 mL of TFA was added and the mixture was allowed to react overnight at room temperature. After the reaction was complete, the solvent and TFA were removed by vacuum evaporation;

[0197] b) The reactant of step a) was dissolved in 5 mL of DMF, and 57 mg HATU (0.15 mmol, 1.5 eq) and 39 mg DIPEA (0.3 mmol, 3 eq) and 39 mg CPI613 (0.1 mmol, 1 eq) were added and left 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 a silica gel column to obtain MtPTAC3C-1 (32 mg, yield 38%), MtPTAC 3C-2 (35 mg, yield 39%), and MtPTAC3C-3 (29 mg, yield 31%).

[0198] 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 ) δ 7.56-7.20 (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).

[0199] 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 )-(4-(4-(2-methyl-1-thio)-1-[4-(4-(2-methyl-1-thio)-1-yl)methyl)phenyl)amino)ethoxy)ethoxy)ethoxy)ethyl)ethyl)-6,8-bis(benzylthio)octanamide (MtPTAC 3C-2), 1 H NMR (400 MHz, Chloroform- d ) δ 7.35-7.20 (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).

[0200] N -(2-(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)ethoxy)ethoxy)ethyl)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).

[0201] Embodiment 4:

[0202] Performance testing of the mitochondrial protease targeting chimera (MtPTAC 3C)

[0203] 1. Mitochondrial proteolysis test

[0204] The protein degradation ability of MtPTAC (3C-1, 3C-2, 3C-3) was tested in the A549 cell line (the test method was the same as in Example 2). A549 cells were treated with four different concentrations (0, 10, 20 and 40 μM) of MtPTAC (3C-1, 3C-2, 3C-3) for 24 hours, and their level changes were analyzed by western blotting. The results are shown in Fig.26 , it can be seen that MtPTAC 3C-2 (40 μM) and MtPTAC3C-3 (20 μM and 40 μM) can significantly reduce the level of PDH protein, and MtPTAC 3C-3 has better protein degradation ability.

[0205] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0206] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A mitochondrial protease targeting chimera, comprising a binding structure of a mitochondrial AAA+ protease family and a binding structure of a mitochondrial protein, wherein the binding structure of the mitochondrial AAA+ protease family and the binding structure of the mitochondrial protein are linked by a linker; The binding structure of the AAA+ protease family in mitochondria is selected from ONC201 derivatives, which are compounds shown in the structure of formula E; ; In formula E, X is selected from halogen; The binding structure of the mitochondrial protein is selected from IMT or 6,8-bis(benzylthio)octanoic acid; the IMT is a compound as shown in the structure of formula 2F, ; The mitochondrial protease targeting chimera is a compound as shown in the structure of Formula 3B or Formula 3C, ; ; In Formula 3B or Formula 3C, n is any natural number from 1 to 10; The linker is a PEG compound.

2. Use of the ONC201 derivative as shown in the structure of formula E in the preparation of the mitochondrial protease targeting chimera as claimed in claim 1, , wherein X is selected from halogen.

3. The method for preparing the mitochondrial protease targeting chimera according to claim 1, comprising the following steps: Step 1), linking the ONC201 derivative and the PEG compound to obtain intermediate 3A, which is a compound shown in the structure of formula 3A; Step 2), linking the intermediate 3A and the binding structure of the mitochondrial protein to obtain a mitochondrial protease targeting chimera; the binding structure of the mitochondrial protein is selected from IMT or 6,8-bis(benzylthio)octanoic acid; ; In formula 3A, n is any natural number from 1 to 10.

4. A compound as shown in formula 3A, ; In formula 3A, n is any natural number from 1 to 10.

5. Use of the compound represented by the structure of formula 3A as claimed in claim 4 in preparing the mitochondrial protease targeting chimera as claimed in claim 1.

6. Use of the mitochondrial protease targeting chimera according to claim 1 in the preparation of a drug for protein degradation, wherein the protein is a protein in a eukaryotic cell, and the degradation of the protein in the eukaryotic cell is based on AAA+ protease.

7. Use of the mitochondrial protease targeting chimera according to claim 1 in the preparation of drugs for treating mitochondrial dysfunction and / or anti-tumor drugs. 8 . A pharmaceutical composition comprising the mitochondrial protease targeting chimera according to claim 1 .

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