An antitumor compound, composition and application thereof

Abstract

The present invention provides a tumor suppressor compound targeting LDHC, and provides a novel candidate compound or a precursor molecule thereof for the research and development of drugs targeting LDHC.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor compounds, and in particular to a tumor suppressor compound using LDHC as a target. Background Art

[0002] LDHC has been confirmed to be an important and unique member of the LDH family and a new potential anti-tumor target. (1) LDHC has a unique composition and is the only active tetramer composed of C subunits. It preferentially catalyzes the conversion of pyruvate to lactate. (2) LDHC protein is re-expressed in various malignant tissues, making its expression highly tumor-specific, while its expression in normal somatic tissues is strictly controlled and is only specifically present in mature testes and sperm of mammals. In contrast, LDHA and LDHB are widely expressed in normal human somatic tissues. (3) LDHC has a wider optimal pH, better thermal stability and higher catalytic efficiency, and is more adaptable to the complex pH changes and efficient glycolysis requirements in tumor cells, playing an important role in the energy metabolism of tumor cells. (4) The promoting role of LDHC in tumors has been gradually revealed, and its expression is upregulated in a variety of human malignant tumor tissues such as lung cancer, breast cancer, and melanoma. LDHC is highly expressed in lung adenocarcinoma tissues and mediates EMT-driven cancer cell migration and invasion by activating the PI3K / Akt / GSK-3β signaling pathway. LDHC is highly expressed in renal cancer tissues, and degrades basement membrane and extracellular matrix components by inducing EMT and high expression of MMP9, promoting tumor cell migration and invasion. LDHC can be used as a biomarker for early diagnosis, efficacy evaluation, and recurrence prediction of liver and breast cancer. In addition, the applicant's previous research results also confirmed that LDHC is significantly highly expressed in human lung cancer tissues, which can promote the proliferation and growth of A549 subcutaneous tumors, and downregulating LDHC expression can significantly inhibit the proliferation of A549 cells. 4

Tan, H., et al. Identification of human LDHC4 as a potential target for anticancer drug discovery. Acta Pharm Sin B 12, 2348-2357 (2022)

[0003] Currently, no high-quality LDHC inhibitors or their precursor molecules have been reported. It is necessary to screen compounds with novel skeletons that can bind to LDHC protein with high affinity, providing candidate compounds or their precursor molecules for the development of drugs targeting LDHC. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention first provides a 3,6,7-substituted-pentapyrimidone skeleton compound, a representative compound as shown in Formula I

[0005]

[0006] Wherein R1 and R3 are alkyl, aryl, aralkyl, substituted aryl;

[0007] R2 is N,N-diformyl or thio;

[0008] R4 is an alkyl group or R4 is absent;

[0009] X and Y are taken from C, N or S respectively.

[0010] In a preferred embodiment, R1 or R3 is selected from One of them.

[0011] In a preferred embodiment, R1 of the compound is selected from One of them.

[0012] In a preferred embodiment, R3 of the compound is selected from One of them.

[0013] In a preferred embodiment, R4 is an alkyl group, preferably -CH3;

[0014] In a preferred embodiment, the R4 group is absent.

[0015] In a preferred embodiment, when X is S, then Y is N, R2 is S, R1 and R3 are aryl or substituted aryl, and R4 is absent.

[0016] In a preferred embodiment, when X is S, Y is N, R2 is S, R1 and R3 are aryl groups or aryl groups substituted with halogen elements, and R4 does not exist.

[0017] In a preferred embodiment, when X is S, Y is N, R2 is S, and R1 and R3 are In one case, R4 does not exist.

[0018] In a preferred embodiment, when Y is S, X is C, and R2 is R1 is R4 is -CH3.

[0019] In a preferred embodiment, the compound is any one of the following compounds 1 to 3, preferably compound 3

[0020]

[0021] The present invention also provides an anti-tumor composition, which contains any one of the aforementioned compounds or a pharmaceutically acceptable salt of the compound.

[0022] Furthermore, the composition also includes other cytotoxic agents.

[0023] In a preferred embodiment, the cytotoxic agent comprises gefitinib, erlotinib, afatinib, osimertinib, carboplatin, paclitaxel, gemcitabine or an anti-tumor antibody.

[0024] In a preferred embodiment, the composition further comprises a tumor stem cell differentiation agent.

[0025] In a preferred embodiment, the tumor stem cell differentiation agent is selected from: retinoic acid, BRD7552, noggin, niacinamide, dexamethasone, and cytarabine.

[0026] Furthermore, the composition also includes a pharmaceutically acceptable carrier or excipient.

[0027] In a preferred embodiment, the pharmaceutically acceptable carrier or excipient includes one or more of starch, dextrin, microcrystalline cellulose, inorganic salts and powdered sugar.

[0028] The present invention also provides a LDHC inhibitor, wherein the composition contains any one of the aforementioned compounds or a pharmaceutically acceptable salt of the compound.

[0029] Furthermore, the composition also includes a pharmaceutically acceptable carrier or excipient.

[0030] In a preferred embodiment, the pharmaceutically acceptable carrier or excipient includes one or more of starch, dextrin, microcrystalline cellulose, inorganic salts and powdered sugar.

[0031] Furthermore, the composition also includes a pharmaceutically acceptable nanotechnology carrier delivery system.

[0032] In a preferred embodiment, the pharmaceutically acceptable nanotechnology carrier delivery system includes nanocapsules, liposome nanocarriers, polymer nanocarriers, silicon-based nanocarriers, ferritin nanoparticles and other nanocarriers.

[0033] The present invention also provides a targeted protein degrader (Targeted Protein Degraders) precursor compound for targeting LDHC protein degradation, wherein the precursor compound contains any of the aforementioned compounds or a pharmaceutically acceptable salt of the compound.

[0034] In a preferred case, the targeted protein degrader precursor compound is a target protein ligand of a protein hydrolysis targeting chimera (PROTAC, Proteolysis Targeting Chimeras).

[0035] Furthermore, the protein hydrolysis targeting chimera also contains an E3 ubiquitin ligase ligand and a linker.

[0036] In another preferred embodiment, the targeted protein degradation agent is molecular glue.

[0037] The present invention also provides the use of the above-mentioned compound or composition in the preparation of anti-tumor drugs.

[0038] In a preferred embodiment, the tumor is selected from tumors with high expression of LDHC protein.

[0039] In a preferred embodiment, the tumor is selected from breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, and ovarian cancer.

[0040] The present invention also provides the use of the above-mentioned compound or composition in the preparation of LDHC inhibitors.

[0041] The present invention also provides the use of the aforementioned compound or composition in the preparation of targeted protein degraders (Targeted Protein Degraders) for targeted LDHC protein degradation.

[0042] In a preferred case, the targeted protein degrader is a proteolysis targeting chimera (PROTAC).

[0043] In another preferred embodiment, the targeted protein degradation agent is molecular glue.

[0044] The compounds, compositions and applications provided by the embodiments of the present invention have the following beneficial effects:

[0045] 1. Provides a new compound with the potential of high-quality LDHC inhibitor or its precursor molecule.

[0046] 2. A new compound is provided, which has the potential to be used as a precursor molecule for preparing a targeted protein degrader for targeting LDHC protein degradation.

[0047] 3. Provided is a compound that has the ability to inhibit tumor cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0049] Figure 1 DSF screening principle (a) and screening results (be).

[0050] Figure 2 : SPR principle (a) and SPR results of compound 1 (b), compound 2 (c) and compound 3 (d) combined with LDHC.

[0051] Figure 3 :The survival rates of non-small cell lung cancer A549 cells and normal cells treated with compounds 1, 2 and 3 were measured (repetition number n=3). (a) is the survival rate of cells treated with compound 1, (b) is the survival rate of cells treated with compound 2, (c) is the survival rate of cells treated with compound 3, and (d) is the survival rate of normal cells treated with compounds 1, 2, and 3.

[0052] Figure 4 : (a) LDHC protein information (red: alpha-helices; blue: beta-strands); (b) LDHC secondary structure element (SSE) distribution; (c) protein secondary structure element composition of each trajectory frame during the 100ns simulation; (d) each residual and its SSE distribution over time.

[0053] Figure 5 :(a) RMSD of protein (light blue) and RMSD of small molecule (pink) in the complex of protein LDHC and compound 3 in 100 ns molecular dynamics. (b) Superposition of the conformation of the complex of protein LDHC (light blue) and compound 3 (pink) in the last 100 frames (90-100 ns) of the molecular dynamics trajectory.

[0054] Figure 6 :(a) RMSF of protein LDHC molecular dynamics simulation in 40-100ns (green positions indicate amino acids that interact with compound molecules and proteins). (b) Coloring according to RMSF (red indicates high flexibility, blue indicates low flexibility).

[0055] Figure 7:(a) Contribution of amino acids in the protein LDHC binding site to the binding of compound 3 in the 50-100ns molecular dynamics simulation. (b) 2D schematic diagram of the binding mode of compound 3 and protein LDHC in the 50-100ns molecular dynamics simulation. (c) Schematic diagram of the interaction mode between protein LDHC and compound 3 in the final optimized conformation of molecular dynamics.

[0056] Figure 8 :(a) RMSD of protein (light blue) and RMSD of small molecule (dark red) in the complex of protein LDHC and coenzyme NADH in 100 ns molecular dynamics. (b) Superposition of the conformation of the complex of protein LDHC (light blue) and compound 3 (light blue) in the last 100 frames (90-100 ns) of the molecular dynamics trajectory. (c) Superposition of the crystal structure of the complex of protein LDHC (light yellow) and coenzyme NADH (light green) with the molecular dynamics optimized conformation of the complex of protein LDHC (light blue) and coenzyme NADH (dark red). (d) Superposition of the crystal structure of the complex of protein LDHC (light yellow) and coenzyme NADH (light green) with the molecular dynamics optimized conformation of the complex of protein LDHC (light blue) and compound 3 (pink). (e) Binding free energy between small molecule A5 (pink) and coenzyme NADH (dark red) and protein NADH, respectively, in the last 10 ns of the trajectory (1000 frames in total). DETAILED DESCRIPTION

[0057] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention in this specification are only for the purpose of describing specific embodiments and are not used to limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0058] Example 1 Characterization of protein-ligand interactions by differential scanning fluorimetry

[0059] Differential Scanning Fluorimetry (DSF) can monitor the changes in protein conformation during the heating process by using fluorescent dyes or endogenous fluorescent signals of proteins to calculate its melting temperature Tm (the temperature at which the folded protein and unfolded protein are equal). The most commonly used dye DSF is SYPRO Orange dye, which is an environmentally sensitive hydrophobic dye. When the temperature rises, the protein unfolds and the hydrophobic part is exposed. The dye specifically binds to the hydrophobic part of the protein and the fluorescence is enhanced. If the compound can bind to the LDHC protein, it will lead to an increase in the stability of the LDHC protein, which is further manifested as an increase in the melting temperature of the LDHC protein. The advantages of this method are (1) preliminary or large-scale detection of whether there is binding force between the protein and the compound; (2) low sample consumption; (3) high data accuracy and a wide temperature change range; (4) it is expected to discover inhibitors that can bind to new sites on the LDHC.

[0060] The LDHC protein sequence is shown in SEQ ID NO.1

[0061] MSTVKEQLIEKLIEDDENSQCKITIVGTGAVGMACAISILLKDLADELALVDVALDKLKGEMMDLQHGSLFFSTSKITSGKDYSVSANSRIVIVTAGARQQEGETRLALVQRNVAIMKSIIPAIVHYSPDCKILVVSNPVDILTYIVWKISGLPVTRVIGSGCNLD SARFRYLIGEKLGVHPTSCHGWIIGEHGDSSVPLWSGVNVAGVALKTLDPKLGTDSDKEHWKNIHKQVIQSAYEIIKLKGYTSWAIGLSVMDLVGSILKNLRRVHPVSTMVKGLYGIKEELFLSIPCVLGRNGVSDVVKINLNSEEEALFKKSAETLWNIQKDLIF

[0062] DSF Experimental Procedure

[0063] (1) Instrument preparation: DSF mainly uses a fluorescent quantitative PCR instrument to complete parallel detection on a 96-well or 384-well PCR plate to achieve high-throughput screening of target proteins and compounds.

[0064] (2) Sample preparation: The protein sample needs to be of high purity (>75%), and the purity of the compound is checked by LC-MS quality control;

[0065] (3) Reaction system configuration: According to the experimental design, the protein and ligand are configured into systems with different concentrations. The sample volume is 20-25 μL and placed in a 96-well plate. The protein buffer is used as a blank control. Each test group has three replicates. The buffer can be replaced according to actual needs. The DSF reaction system mainly includes protein samples, reaction buffer, fluorescent dyes and candidate compound ligands, each test well.

[0066] (4) On-line: Place the above-configured system into a fluorescence quantitative PCR instrument, adjust the excitation and emission wavelengths to the corresponding wavelengths of the fluorescent dye, and set the heating rate to 1°C / 10s. The temperature range is generally from 25°C to 75°C, and the fluorescence intensity is measured every 1 to 3°C.

[0067] (5) Data analysis: The fluorescence intensity versus temperature curve was plotted using data processing software, and the Tm value and related parameters of the protein sample were calculated by fitting the Boltzmann transport equation (BTE).

[0068] DSF Experiment Results

[0069] We screened LDHC inhibitors using differential fluorescence scanning. So far, we have screened about 2,000 solid compounds and found three compounds that can change the thermal melting temperature of LDHC ( Figure 1 ), named compounds 1, 2 and 3 respectively. The Tm value of protein LDHC in the DSF experiment was 70.4℃. After compound 1 reacted with LDHC protein, its Tm showed an obvious rightward shift of 1.4℃, and the Tm value was 71.8℃, indicating that the binding of compound 1 can increase the stability of protein LDHC. After compound 2 reacted with protein LDHC, its Tm showed an obvious leftward shift, and the Tm value was 68.7℃, indicating that compound 2 can bind to LDHC, but this binding may reduce the stability of LDHC protein. After compound 3 binds to LDHC, the Tm value shifts to the right, indicating that the binding of compound 3 can improve the stability of protein LDHC. In short, the DSF results show that compounds 1, 2 and 3 can all bind to protein LDHC, among which compounds 1 and 3 can significantly improve the stability of LDHC protein.

[0070] The molecular formula of compound 1 is C20H17N3OS3, the molecular formula of compound 2 is C18H11ClFN3OS3, and the molecular formula of compound 3 is C18H19N3O2S2. The structural formulas of the three compounds are as follows:

[0071]

[0072] Example 2 Surface Plasmon Resonance (SPR) Determination of Binding Affinity of Compounds to LDHC

[0073] Principle: A layer of biomolecule recognition membrane is fixed on the surface of the chip, and then the sample to be tested flows over the chip surface. If there are molecules in the sample that can interact with the biomolecule recognition membrane on the chip surface, it will cause the refractive index of the gold film surface to change, and eventually lead to a change in the SPR angle. By monitoring the change in the SPR angle, the binding constant Ka, dissociation constant Kd or affinity constant KD between molecules can be obtained ( Figure 2 (a) in the figure). Advantages of SPR: (1) It can monitor the dynamic process of the reaction in real time and continuously; (2) It can be detected quickly and conveniently; (3) It has a very wide range of applications; (4) It does not require labeling of the analyte; (5) It is suitable for use in turbid, opaque or colored solutions. Through SPR, we can effectively evaluate the binding affinity of compounds 1, 2 and 3 with LDHC.

[0074] The specific steps of the SPR-based compound-LDHC binding affinity test are as follows:

[0075] (1) Calculation of coupling amount: Calculate the coupling amount of LDHC according to the formula Rmax = MW compound / MW LDHC × R ligand coupling level × S stoichiometric ratio;

[0076] (2) Coupling: NHS and EDC mixed solution, coupling protein and ethanolamine HCl solution were injected to make the coupling response value of CM5 chip reach about 13000RU;

[0077] (3) Injection: dilute the compound with PBS (final DMSO content is 5%), with 50 μM as the highest concentration, make a 2-fold gradient dilution and centrifuge, then inject at a speed of 100 uL / 30 seconds;

[0078] (4) Data analysis: After deducting the blank (buffer: 1xPBST+5% DMSO) using the analysis software Tracedrawer 1.8, enter the affinity evaluation and set the fitting parameters: select local for Bmax and local for BI. The binding constant Ka, dissociation constant Kd, and affinity KD of LDHC and the compound are obtained by fitting.

[0079] SPR experimental results:

[0080] In order to further verify whether the small molecule compounds obtained by DSF can directly bind to LDHC and deeply understand the kinetic characteristics of the compounds binding to LDHC, we used the gold standard SPR for molecular interactions to test the affinity of the compounds binding to LDHC. Figure 2As shown in (b), the binding of LDHC to compounds 1, 2 and 3 is concentration-dependent, and the binding affinities kD are 76.3 μM, 45.8 μM and 3.31 nM, respectively (micromolar to millimolar levels are often considered to be weak binding affinities, nM is usually defined as high affinity, and the higher the affinity means the stronger the interaction between the compound and the target protein).

[0081] In order to further understand the binding characteristics of compounds 1, 2 and 3 with LDHC, we conducted an in-depth analysis of the binding rates (Ka) and dissociation rates (Kd) of different compounds with LDHC. Figure 2 (b)- Figure 2 As shown in (d), compound 3 binds to LDHC protein in a unique "fast binding, fast dissociation" manner, suggesting that low doses of compound 3 can reach saturation, but multiple doses are required. For compounds 1 and 2, they mainly interact with LDHC in a "slow binding, slow dissociation" manner, suggesting that the above compounds require high doses to achieve saturation, but the drug effect lasts for a long time.

[0082] Example 3 Effect of Compounds on LDHC-Overexpressing Tumor Cell Line A549 (A549 LDHC )

[0083] In order to test whether the above compounds have the activity of killing tumor cells, we treated A549 with different concentrations (500 μM, 50 μM, 5 μM and 0.5 μM) of the compounds. LDHC The cells were treated for 72 hours, and then CCK8 assay was performed to determine the effect of the compound on tumor cell growth by calculating the cell survival rate. Figure 3 As shown, under the intervention of compound 1 at a concentration of 500 μM, A549 LDHC The cell survival rate was only 19.0%; under 50μM intervention, A549 LDHC The cell survival rate was 42.9%; under 5μM intervention, A549 LDHC The cell survival rate was 80.8% ( Figure 3 (a)). Under the intervention of compound 2 at a concentration of 500 μM, A549 LDHC The cell survival rate was only 13.4% under 50μM intervention. LDHC The cell survival rate was 37.6%, and 5 μM intervention had no significant inhibitory effect ( Figure 3 (b)). Under the intervention of compound 3 at a concentration of 500 μM, A549 LDHC The cell survival rate was only 19.9% ​​under 50 μM intervention. LDHC The cell survival rate was 25.8%, and 5 μM intervention had no significant inhibitory effect ( Figure 3 (c) in the figure.

[0084] As a control, we selected compounds 1, 2 and 3 at a higher concentration of 200 μM to treat three normal cells, 293T (human kidney epithelial cell line), Het-1a (esophageal epithelial cells), NP460 (nasopharyngeal epithelial cells), and none of the three compounds showed significant inhibition of normal cell viability ( Figure 3 (d)), and the overall survival rate was higher than that of tumor cells treated with 50 μM compounds 1, 2, and 3. This indicates that compounds 1, 2, and 3 have tumor cell selectivity and can be used as candidate tumor-suppressing drugs, or as candidate compounds for preparing tumor-suppressing drugs.

[0085] Example 4 Kinetic simulation of compound 3 and LDHC

[0086] In some special cases, when protein crystals cannot be obtained or the protein structure cannot be analyzed by X-ray diffraction, molecular docking can be used to analyze the interaction between the target and the ligand. This study used the LDHC:NAD:EAA complex structure as a template and used AutoDock Vina software to simulate the binding mode of LDHC and compound 3, providing important structural information for the subsequent structural optimization and modification of compound 3.

[0087] Molecular simulation methods:

[0088] (1) Molecular docking: The 3D structure of compound 3 was drawn by ChemDraw and converted using Chem3D. The small molecule was preprocessed using Autodock Tools and saved as a PDBQT file. The LDHC (https: / / www.rcsb.org / structure / 7EPM) crystal structure was retrieved from the PDB database (https: / / www.rcsb.org / ) and preprocessed using Autodock Tools to remove redundant molecules and water molecules, merge polar hydrogen atoms, and save as a PDBQT file. Finally, the small molecule was docked to the active site of LDHC using Autodock Vina.

[0089] (2) Molecular dynamics: In order to further optimize the binding mode of compound 3 and LDHC, conventional molecular dynamics simulations were performed on the docked protein-small molecule complex using the Desmond program. The opls2005 force field was used to parameterize the protein and small molecule, and TIP3P was used as the solvation water model. The protein-small molecule complex was placed in a cubic water box and solvated, and then the charge of the system was neutralized by adding 0.150M chloride ions and sodium ions. The energy of the system was first minimized for 50,000 steps using the steepest descent minimization method. Then, the positions of heavy atoms were restricted for NVT equilibrium and NPT equilibrium, and each equilibrium step was run for 50,000 steps. The system temperature was maintained at 300K and the system pressure was maintained at 1 bar. After completing the two equilibrium stages, an unrestricted simulation of 100ns was performed. The energy and coordinate system of the trajectory were saved every 10ps, and a total of 10,000 frames of trajectory were saved. After completing the simulation trajectory, the interaction pattern was plotted and the binding free energy of the last 10ns trajectory was calculated.

[0090] Molecular dynamics simulation optimization of LDHC-compound 3 complex:

[0091] In order to verify the hypothesis at the molecular level, we first analyzed the LDHC protein, then docked compound 3 to LDHC and optimized the LDHC-compound 3 complex by molecular dynamics simulation for 100 ns. After 100 ns of molecular dynamics, the molecular dynamics trajectory was analyzed.

[0092] Analysis of LDHC secondary structure composition:

[0093] Analysis of the LDHC protein revealed that the LDHC protein contained 39.48% α-Helix, 14.38% β-Strand, and the secondary structure accounted for 53.86% of the entire protein ( Figure 4 Subsequently, we analyzed the composition of protein secondary structure elements in each trajectory frame during the 100 ns simulation and found that the composition remained stable at around 54% ( Figure 4 Each residual and its SSE distribution over time also show a stable state ( Figure 4 (d) in the figure ensures the reliability of subsequent analysis results.

[0094] RMSD analysis of LDHC-compound 3 complex

[0095] In order to obtain a more detailed interaction pattern between the lead compound 3 and LDHC, we used Gromacs to perform 100ns MD on the complex structure. Before analyzing the MD trajectory and energy, this project calculated the RMSD of LDHC protein and compound 3 during the MD process and superimposed the conformation of the crystal structure and the dynamic conformation to verify the accuracy and feasibility of this MD. The results are as follows Figure 5 As shown in (a) in the 100ns molecular dynamics process, the RMSD of the small molecule and the protein are in a relatively stable state after 40ns, that is, the structure of the complex formed by the small molecule and the protein is in a stable state. Therefore, we can analyze the trajectory of 40-100ns. Figure 5 As shown in (b), the molecular dynamics trajectory conformations of the LDHC-compound 3 complex have a good superposition. It is worth noting that compound 3 does not bind to the catalytic site but binds near the Active-site Loop.

[0096] RMSF analysis of LDHC-compound 3 complex

[0097] Through trajectory analysis, we can get Figure 6 The RMSF of the LDHC amino acid residues shown in (a) is shown in Figure 1, and the structure is colored and plotted according to the size of the RMSF. The protein LDHC structure has three main flexible regions, such as Figure 6 In (a), a, b and c regions, where a and b are flexible loops and c is the C-terminus of the LDHC protein, compound 3 mainly interacts with the amino acids in region a and binds to the flexible loop a region, i.e., the active-site loop ( Figure 6 (b)). This result is consistent with the structural characteristics of other members of the LDH family.

[0098] Example 5 Analysis of Binding Mode of LDHC-Compound 3 Complex

[0099] Analysis of LDHC amino acid residues involved in binding compound 3

[0100] We then further analyzed the binding mode between compound 3 and protein LDHC. We first analyzed the amino acids that play an important role in the binding of compound 3 in the stable range of the molecular dynamics trajectory (40-100ns). The results are as follows Figure 7 As shown in (a), the amino acids that play an important role in small molecule binding are mainly Gly30, Asp53, Val54, Arg100, Ile117 and Tyr248, and their main functions are hydrogen bonds, water bridges and hydrophobic interactions.

[0101] Analysis of compound groups involved in binding to LDHC

[0102] Then the occupancy rate of the interaction formed between 40 and 100 ns was counted (number of frames forming interaction / total number of frames). Figure 7 As shown in (b), the binding of small molecules to proteins mainly depends on the two carbonyl structures on compound 3. The carbonyl on the amide can form hydrogen bonds with Arg100 indirectly or directly through a water molecule. In addition, the carbonyl on the aromatic ring can also form hydrogen bonds with Asp53 and Gly30 indirectly through water molecules. Finally, the binding mode of the final optimized conformation of molecular dynamics is analyzed, as shown in Figure 7 As shown in (c), Gly30, Asp53 and Arg100 interact with compound 3 through hydrogen bonds or water bridges. In addition, there is a hydrophobic cavity under the benzene ring of compound 3 molecule. Compound 3 can be modified later to increase the binding force between compound 3 and LDHC, so that it can competitively bind to LDHC and play a role in inhibiting enzyme activity.

[0103] Example 6 Comparative Analysis of Binding Modes of LDHC-Compound 3 Complex and LDHC-NADH Complex

[0104] LDHC-NADH molecular dynamics simulation

[0105] In order to compare the binding mode of compound 3 with LDHC and verify the feasibility of molecular dynamics, we also performed molecular dynamics simulation on the crystal structure of the complex between protein LDHC and coenzyme NADH (PDB ID: 7EPM).

[0106] After 100 ns of molecular dynamics, the molecular dynamics trajectory was analyzed. First, the RMSD of the trajectory protein LDHC and the coenzyme NADH were extracted separately, and the conformations of the last 5 ns were extracted for superposition, such as Figure 8 As shown in (a) in Figure 3, the RMSD of the coenzyme NADH and the protein are both in a relatively stable state after 40 ns, so we can analyze the trajectory from 40 to 100 ns. Figure 8 As shown in (b), the molecular dynamics trajectory conformation of the LDHC-NADH complex has a good superposition. Then we superimpose the crystal structure of the complex of protein LDHC and coenzyme NADH with the molecular dynamics optimized conformation of the complex of protein LDHC and coenzyme NADH. Figure 8 As shown in (c), both the protein LDHC and the coenzyme NADH have good superposition, indicating that the kinetic simulation can accurately predict the binding mode of the coenzyme and LDHC, and also confirms the accuracy of the binding mode of compound 3 and LDHC.

[0107] Comparison of binding modes between LDHC and compound 3 / cofactor NADH

[0108] In addition, we also superimposed the crystal structure of the complex of protein LDHC and coenzyme NADH with the molecular dynamics optimized conformation of the complex of protein LDHC and compound 3. Figure 8 As shown in (d), the binding position of the small molecule and the coenzyme are the same position. In addition, after the binding of compound 3, the loop a and b of LDHC have a significant change compared with the binding mode of NADH ( Figure 8 (d)) in the figure shows that the binding with coenzyme NADH is different, and the structure of LDHC undergoes significant changes after the binding of compound 3.

[0109] Comparison of binding free energy between LDHC and compound 3 / cofactor NADH

[0110] When compound 3 exerts its inhibitory effect, it needs to competitively bind to the binding site of NADH. Therefore, in order to further explore the binding strength of compound 3, coenzyme NADH and protein LDHC, we extracted the 1000-frame trajectory of the last 10ns of the molecular dynamics of compound 3, coenzyme NADH and protein LDHC and calculated the binding free energy of compound 3, coenzyme NADH and protein LDHC. The lower the binding free energy of the compound and the protein, the more stable the binding between the two, and the binding energy <-5.0 kcal / mol indicates that the compound binds well to the target. The results are as follows Figure 8 As shown in (e), the binding free energy of compound 3 and coenzyme NADH is approximately -50 and -90 kcal / mol, respectively, indicating that the binding of coenzyme NADH is greater than that of compound 3. This explains why compound 3 can bind to LDHC in the SPR experiment without the participation of coenzyme NADH, but does not show enzyme inhibitory activity in the enzyme activity experiment with the participation of coenzyme NADH. In view of this, we will further modify the hydrophobic cavity near compound 3 in the future to improve the affinity so that it can competitively bind to LDHC to inhibit enzyme activity.

[0111] Example 7 Synthesis of Compounds

[0112] Synthesis of compound 1

[0113] Compound 1a and o-toluene isothiocyanate were used as raw materials and reacted at room temperature for 24 hours to generate thiourea intermediate 1b. Then, the formic acid group of 1b underwent esterification in the presence of sulfuric acid to generate intermediate 1c. 1c was cyclized into a six-membered heterocycle in the presence of sodium hydroxide and water to obtain the target compound 1.

[0114]

[0115] Scheme 1Synthesis of compound 1. Reagents and conditions: (i) saturated Na 2 CO 3 ,MeCN,RT24h,HCl,filter.(ii)H 2 SO 4 ,EtOH,reflux,24h.(iii)NaOH / H 2 O,reflux,3h.

[0116] Synthesis of compound 2

[0117] Compound 2a was used as the raw material and di(tri-tert-butylphosphine)palladium was used as the ligand to generate intermediate 2b under the action of iodomethane and zinc. Then 2b was reacted with o-fluorophenyl isothiocyanate at 100°C for 18 hours to generate intermediate 2c. 2c was cyclized in the presence of sodium methoxide and methanol to generate the final product, a six-membered heterocyclic compound 2.

[0118]

[0119] Scheme 2 Synthesis of compound 2. Reagents and conditions: (i): MeI, Zinc, THF, Pd(t-Bu 3 P) 2 , 65℃, 2h. (ii) DMAP, MeCN, 100℃, 18h. (iii) MeONa / MeOH, 60℃, 6h.

[0120] Synthesis of compound 3

[0121] Compound 3a and methyl cyanoacetate were reacted at 50°C for 28 hours to generate intermediate compound 3b, 3b was reacted at 100°C for 18 hours to generate thiourea compound 3c, and then 3c was reacted in the presence of hydrochloric acid and ethanol to generate the final product, six-membered heterocyclic compound 3.

[0122]

[0123] Scheme 3 Synthesis of compound 3. Reagents and conditions: (i): TEA, Sulfur, DMF 50℃, 28h. (ii) 1,4-Dioxane, reflux, 12h. (iii) HCl, EtOH.

[0124] The above examples show that compounds 1, 2, and 3 have the ability to bind to the target LDHC and inhibit the activity of LDHC, and also show that compounds 1, 2, and 3 have the effect of inhibiting the growth of tumor cells without affecting the growth of normal cells. Therefore, compounds 1, 2, and 3 have the potential to inhibit LDHC and can be used as candidate tumor suppressor drugs or candidate compounds for preparing tumor suppressor drugs. At the same time, LDHC can also be used as a precursor compound of a targeted degrader (such as PROTAC or molecular glue) targeting LDHC.

[0125] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An antitumor composition, characterized in that: The composition contains any one of the compounds represented by formula (I) to formula (III) or a pharmaceutically acceptable salt of the compound, and a pharmaceutically acceptable carrier or excipient. The structures of formula (I) to formula (III) are shown below: 。 2. The composition according to claim 1, characterized in that The composition may also include an additional cytotoxic agent.

3. The composition according to claim 1, characterized in that The composition also includes a tumor stem cell differentiation agent.

4. Use of compounds represented by formula (I) to (III) in the preparation of anti-tumor drugs, the structures of formula (I) to (III) are shown below: 。 5. The application according to claim 4, characterized in that: The anti-tumor drug is an LDCH inhibitor.

6. The use according to claim 5, characterized in that: The LDCH inhibitor is a targeted protein degrader that targets LDHC protein degradation.

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