Pharmaceutical application of sulfanyl acetamide compound

Abstract

The invention discloses an application of a sulfanyl acetamide compound with a structure shown in a formula I in preparation of a medicine for preventing and / or treating tumors and idiopathic pulmonary fibrosis. The compound can be stably combined with key amino acid residues of DDR1 protein, has a remarkable inhibition effect on phosphorylation activation of the DDR1 protein and various tumor cells, plays an anti-tumor role by effectively inhibiting protein activity, and provides an effective lead compound for subsequent drug design. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a pharmaceutical application of a sulfanylacetamide compound, and in particular to an application of a sulfanylacetamide compound in preparing a drug for preventing and / or treating tumors and idiopathic pulmonary fibrosis. Background Art

[0002] Discoidin Domain Receptor 1 (DDR1) is a collagen receptor with tyrosine kinase activity. Unlike other receptor tyrosine kinases, DDR1 has a unique discoidin domain at the extracellular end, which can make the extracellular end of the cell highly ordered. Corresponding to tumor cells, DDR1 is like wrapping a layer of "barbed wire" around the tumor, which plays a role in hindering the infiltration of immune cells and killing tumor cells. Studies have shown that DDR1 is abnormally upregulated in a variety of tumors, such as breast cancer, liver cancer, non-small cell lung cancer, etc., and is associated with poor prognosis. In addition, studies have found that inhibiting the expression of DDR1 can reduce the immunosuppressive effect in the tumor microenvironment. Therefore, DDR1 is not only considered to be one of the targets of molecular targeted therapy, but also a potential target for immunotherapy.

[0003] Currently developed DDR1 inhibitors can be divided into two categories: one is a monoclonal antibody that acts on the extracellular domain, and the other is a small molecule inhibitor that targets the intracellular domain. Due to the structural similarity between the intracellular region of DDR1 and the kinase domain of other RTKs, some small molecule tyrosine kinase inhibitors (TKIs) such as dasatinib, nilotinib and imatinib can also block the activity of DDR1 kinase. However, due to the existence of multiple targets, non-selective inhibitors will cause unnecessary side effects such as hypertension, proteinuria and rash. Summary of the invention

[0004] Purpose of the invention: The present invention aims to provide an application of a sulfanylacetamide compound in the preparation of a drug specifically targeting DDR1 protein.

[0005] Technical solution: The sulfanyl acetamide compound having a structure of formula I or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or a mixture thereof of the present invention is used in the preparation of a drug for preventing and / or treating tumors and idiopathic pulmonary fibrosis.

[0006]

[0007] Wherein, L and M are selected from C(O) or CR 1 R 2 ; R 1 , R 2 Selected from hydrogen, halogen, hydroxyl, amino, C 1 -C4 Alkyl, C 1 -C 4 Alkoxy, C 1 -C 4 Haloalkyl, C 1 -C 4 Haloalkoxy, or R 1 , R 2 Together with the connected C ring, it forms a 3-6 membered cycloalkyl.

[0008] Preferably, the pharmaceutically acceptable salts are salts of the compounds of the invention, prepared from compounds with specific substituents found in the invention and relatively non-toxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, Citric acid, tartaric acid and methanesulfonic acid and similar acids; organic acid salts also include amino acids (such as arginine, etc.), glucuronic acid and other organic acid salts. Certain specific compounds of the present invention contain basic and acidic functional groups, and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0009] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0010] Preferably, in the structure of formula I, L and M are selected from C(O) or CR 1 R 2; R 1 , R 2 is selected from hydrogen, methyl, ethyl, fluorine, chlorine, trifluoromethyl, trifluoromethoxy, or R 1 , R 2 It can be combined with the connected C ring to form cyclopropyl and cyclobutyl.

[0011] Further preferably, in the structure of formula I, L is selected from C(O), M is selected from CR 1 R 2 ; R 1 , R 2 is selected from hydrogen, methyl, ethyl, fluorine, chlorine, trifluoromethyl, trifluoromethoxy, or R 1 , R 2 It can be combined with the connected C ring to form cyclopropyl and cyclobutyl.

[0012] Preferably, the drug has the following chemical structure:

[0013]

[0014] Preferably, the drug is a drug that inhibits tumor growth or metastasis.

[0015] Preferably, the drug is a drug for preventing and / or treating breast cancer, liver cancer, non-small cell lung cancer, tongue squamous cell carcinoma, ovarian cancer, and esophageal squamous cell carcinoma.

[0016] More preferably, the drug is a drug for preventing and / or treating breast cancer, liver cancer, non-small cell lung cancer, tongue squamous cell carcinoma, ovarian cancer, and esophageal squamous cell carcinoma with high expression of discoidin domain receptor 1 protein.

[0017] More preferably, the drug is a drug that inhibits discoidin domain receptor 1 protein.

[0018] Still more preferably, the drug is a drug that inhibits discoidin domain receptor 1 protein in a concentration-dependent manner.

[0019] Still more preferably, the drug is a drug that specifically targets and inhibits the discoidin domain receptor 1 protein.

[0020] More preferably, the drug is a drug that downregulates the phosphorylation level of discoidin domain receptor 1 protein.

[0021] More preferably, the drug is a drug that reduces immunosuppression in the tumor microenvironment.

[0022] Preferably, the compounds of the present invention are used in combination with other drugs having discoidin domain receptor 1 protein inhibitory activity.

[0023] More preferably, the combined administration is selected from simultaneous administration or sequential administration.

[0024] More preferably, when the simultaneous administration is adopted, pharmaceutical preparations containing different single active ingredients or compound pharmaceutical preparations containing different active ingredients are administered.

[0025] More preferably, when sequential administration is adopted, the order of administration of different drugs is not particularly limited.

[0026] The pharmaceutical composition of the present invention, which uses the compound of formula I or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or mixture thereof as an active ingredient, is used in the preparation of drugs for preventing and / or treating tumors and idiopathic pulmonary fibrosis.

[0027] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable carrier.

[0028] Further preferably, the pharmaceutically acceptable carrier can be an adjuvant widely used in the field of drug production. The adjuvant is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical adjuvant can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical adjuvant can include one or more of the following adjuvants: adhesives, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0029] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0030] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] The compounds screened out by the present invention can stably bind to the key amino acid residues of the DDR1 protein, have a significant inhibitory effect on a variety of tumor cells at the cellular level, and also have a significant inhibitory effect on the phosphorylation activation of the DDR1 protein at the molecular level. They exert anti-tumor effects by effectively inhibiting protein activity, and provide effective lead compounds for subsequent drug design. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the simulated docking of compound G621-0569 and DDR1 protein;

[0034] Figure 2 This is the affinity fitting diagram of compound G621-0569 binding to DDR1 protein;

[0035] Figure 3 The results show the inhibitory effect of compound G621-0569 on the proliferation of five tumor cells;

[0036] Figure 4 This is the result of the down-regulation of the phosphorylation level of DDR1 protein by compound G621-0569. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below in conjunction with embodiments.

[0038] Example 1: High-throughput virtual screening to obtain candidate compounds

[0039] Virtual Screening (VS) is a drug discovery method used to analyze large databases or compound collections to identify potential lead compounds. Compared with traditional drug screening methods, this method can greatly reduce the number of experimental screening compounds, shorten the drug development cycle and save development costs. Structure-based virtual screening starts with the three-dimensional structure of the target protein and uses a computer program to dock the target protein with an available library of drug-like compounds. The binding ability between the compound and the protein can be evaluated based on docking scores and binding free energy calculations.

[0040] 1. Experimental methods

[0041] (1) Compound collection and pretreatment

[0042] Based on the Chemdiv database, 3 million drug-like compounds and their 2D sdf structure files were obtained. The LigPrep module in the Schrödinger software was used to pre-process the compounds and generate all 3D chiral conformations.

[0043] (2) ADME / T evaluation

[0044] QikProp module was used to predict all ADME / T parameter scores of ligand compounds, and a total of 51 type parameters were obtained, among which Lipinski's five principles were mainly used for evaluation: mol_MW<500, QPlogPo / w<5, donorHB≤5, accptHB≤10.

[0045] (3) Protein pretreatment

[0046] The crystal structure of DDR1 protein was obtained from the RCSB PDB database (PDB ID: 7FEH). The protein crystals were processed by protein preparation wizard module of Schrödinger software, including protein preprocessing, regenerate states of native ligand, H-bond assignment optimization, protein energy minimization, and removal of waters.

[0047] (4) Active site identification

[0048] The Receptor Grid Generation module in Schrödinger was used to set the most appropriate Enclosing box to perfectly encapsulate the binding site of the natural ligand, and on this basis, the active site of the protein was obtained.

[0049] (5) Molecular docking

[0050] Each processed ligand in the compound library was molecularly docked with the active site of DDR1 protein in turn (HTVS, SP and XP were used respectively, and the docking accuracy was gradually improved). The lower the score, the lower the binding free energy of the compound with the protein and the higher the binding stability.

[0051] (6) MM-GBSA analysis

[0052] The lowest-scoring ligand and the active site of DDR1 protein were subjected to MM-GBSA calculation analysis. MM-GBSAdG Bind can approximately represent the binding free energy of small molecules and proteins. The lower the binding free energy, the higher the binding stability of the ligand and DDR1 protein.

[0053] 2. Experimental results

[0054] There are 2,107,089 compounds in the ChemDiv compound library. After pre-processing the ligands of these compounds, a total of 3,044,650 compound conformations were obtained. These ligand compounds were subjected to pharmacophore virtual screening, ADME / T evaluation, HTVS docking, SP docking, and XP docking. Finally, the top 10% of the compounds (1,733 compounds) were taken for MM-GBSA analysis. According to the MM-GBSA analysis results and referring to the XP Gscore docking score, the TOP10 compounds were obtained. Among them, the docking score of the candidate compound G621-0569 with the DDR1 protein is -11.711, and the MM-GBSA binding free energy is -59.14 kcal / mol. Both the docking score and the binding free energy are very low, indicating that G621-0569 binds stably to the DDR1 protein. Figure 1 As shown, G612-0569 forms two hydrogen bonds and a salt bridge with DDR1 protein residue ASP784, a π-π bond with residue PHE762, and a salt bridge with residue GLU672. In addition, residues VAL654, ALA653, VAL624, LEU773, LEU687, etc. of DDR1 protein form hydrophobic forces on G612-0569.

[0055] Example 2: Surface Plasmon Resonance (SPR) Experiment to Detect the Affinity of Candidate Compounds to DDR1 Protein

[0056] 1. Experimental methods

[0057] The experiment uses Biacore S200 optical biosensor and CM5 chip to determine the binding of ligand compounds to target proteins. First, pre-enrichment is performed. The DDR1 protein is diluted to 20μg / ml with sodium acetate buffer of 4 different pH (4.0, 4.5, 5.0, 5.5), and then flows through the blank chip surface to determine the optimal coupling pH. Under this pH condition, the DDR1 protein is bound to the chip surface using an amino coupling kit. Then, the test compound is prepared into a 10mM stock solution with DMSO, and then the stock solution is diluted 20 times with 1.05x phosphate buffer (PBS-P). After that, the analyte concentration is diluted to 200μM as the highest injection concentration with the prepared 5% DMSO running buffer, and then 6 concentration gradients are diluted in half. At the same time, 4.5% and 5.8% DMSO are prepared for solvent correction. After the program runs, the 1:1 kinetic binding model in Biacore S200Evaluation Software is used to fit the experimental data.

[0058] 2. Experimental results

[0059] The affinity between the candidate compounds obtained by virtual screening and the DDR1 protein is verified by surface plasmon resonance (SPR) technology, which can eliminate false positive results caused by scoring. The candidate compounds after eliminating false positives are tested for biological activity through experimental research, and the targeted specific DDR1 receptor antagonist small molecules are retained, providing options for the development of new drugs for tumor treatment.

[0060] KD value at 10E -3 and 10E -6 The KD value of compound G621-0569 for DDR1 is 2.358E -4 , indicating that G621-0569 can effectively bind to DDR1 protein ( Figure 2 ). In addition, G621-0569 performed well in a concentration-dependent binding response, that is, the binding response value increased with the increase of drug concentration.

[0061] Example 3: Cytotoxicity (CCK-8) assay to detect the inhibitory effect of candidate compounds on tumor cell proliferation

[0062] 1. Experimental methods

[0063] Five tumor cells (Cal-27, SCC7, HepG 2, MDA-MB-231, A549) in logarithmic growth and good condition were counted and inoculated into 96-well plates at a certain density, 100 μL per well. After the cells adhered overnight, the candidate compound solutions with concentrations of 0 μM, 10 μM, 20 μM, 50 μM, and 100 μM were added to intervene in the cells. After 48 hours of treatment, CCK-8 reagent was added, 10 μL per well. After incubation for 1 to 2 hours, the culture plate was removed and detected at a wavelength of 450 nm on an ELISA reader.

[0064] 2. Experimental results

[0065] G621-0569 can significantly inhibit the proliferation of tumor cells, especially Cal-27 cells and A549 cells ( Figure 3 ). Therefore, compound G621-0569 not only has a strong affinity with DDR1, but also has a good tumor inhibitory effect, and is a promising lead compound for DDR1 inhibitors.

[0066] Example 4: Western Blot experiment to verify the effect of candidate compounds on DDR1 protein phosphorylation level

[0067] 1. Experimental methods

[0068] Take cells in good growth state and in the logarithmic growth phase for plating, and add different concentrations of drugs for intervention after overnight attachment. After incubation for a certain period of time, collect the cells, lyse them with RIPA lysis buffer containing protease inhibitors for 30 minutes, centrifuge them at 11000g for 10 minutes in a 4℃ low-temperature centrifuge after lysis, carefully transfer the supernatant to a new centrifuge tube, add 5*Loading Buffer, mix well and heat at 100℃ for 5 minutes to denature them. Subsequently, separate the proteins with 10% sodium dodecyl sulfate (SDS-PAGE) and transfer them to nitrocellulose membranes. After the transfer is completed, put them in 5% skim milk powder for blocking at room temperature for 2 hours. Cut the membrane according to the marker position, soak them in the corresponding primary antibody solution, and keep them in a refrigerator at 4℃ overnight. After washing with an appropriate amount of TBST solution, soak the membrane in the secondary antibody solution and incubate it on a shaker at room temperature for 2 hours. After washing again, mix the developer A and B in a ratio of 1:1 and drop them onto the membrane for development.

[0069] 2. Experimental results

[0070] Compared with the control group, the phosphorylation level of DDR1 protein in collagen-induced Cal-27 cells was significantly increased, while the total protein level did not change significantly. After pretreatment with compound G621-0569, the phosphorylation level of DDR1 decreased in a concentration-dependent manner, and the difference was statistically significant compared with the model group at 20 μM (P < 0.05) ( Figure 4 ). The results showed that G621-0569 could inhibit the protein activity of DDR1 by binding to DDR1 and downregulating its phosphorylation level.

[0071] In summary, the present invention conducts drug screening based on 3 million drug-like compounds in the Chemdiv database to find new DDR1 lead compounds. Subsequently, the top 10 compounds are selected as candidate DDR1 small molecule inhibitors, and the candidate compounds are verified by surface plasmon resonance technology, cytotoxicity and other experiments. Among them, the compound with the best comprehensive affinity with DDR1 and cell killing ability is G621-0569. The compounds of the present invention have obvious DDR1 inhibitory activity, which provides a new choice for the development and application of DDR1-related tissue fibrosis lesions and tumor disease therapeutic drugs. This type of compound can be further structurally optimized as a lead compound of DDR1 inhibitors, and has good application prospects.

Claims

1. Use of a sulfanylacetamide compound or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or mixture thereof in the preparation of a medicament for preventing and / or treating tumors or idiopathic pulmonary fibrosis, wherein the compound has a structure of formula I, In the structure, L and M are selected from C(O) or CR1R2; R1 and R2 are selected from hydrogen, halogen, hydroxyl, amino, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 haloalkoxy, or R1 and R2 form a 3-6 membered cycloalkyl with the connected C ring.

2. The use according to claim 1, characterized in that: In the structure of formula I, L and M are selected from C(O) or CR1R2; R1 and R2 are selected from hydrogen, methyl, ethyl, fluorine, chlorine, trifluoromethyl, trifluoromethoxy, or R1 and R2 and the connected C ring form cyclopropyl or cyclobutyl.

3. The use according to claim 1, characterized in that: The drug has the following chemical structure:

4. The use according to claim 1, characterized in that: The medicine is a medicine for inhibiting tumor growth or metastasis.

5. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and / or treating breast cancer, liver cancer, non-small cell lung cancer, tongue squamous cell carcinoma, ovarian cancer, and esophageal squamous cell carcinoma.

6. The use according to claim 5, characterized in that: The medicine is a medicine for preventing and / or treating breast cancer, liver cancer, non-small cell lung cancer, tongue squamous cell carcinoma, ovarian cancer and esophageal squamous cell carcinoma with high expression of discoidin domain receptor 1 protein.

7. The use according to claim 6, characterized in that: The drug is a drug that inhibits discoidin domain receptor 1 protein.

8. The use according to claim 7, characterized in that: The drug is a drug that specifically targets and inhibits the discoidin domain receptor 1 protein.

9. The use according to claim 6, characterized in that: The drug is a drug for down-regulating the phosphorylation level of discoidin domain receptor 1 protein.

10. The use according to claim 6, characterized in that: The drug is a drug that reduces immunosuppression in the tumor microenvironment.

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