A TRADD inhibitor derivative and its preparation method and application

Abstract

The present invention discloses a TRADD inhibitor derivative, its preparation method, and application. The general structural formula of the TRADD inhibitor derivative is: #imgabs0#. Based on structural biology, the present invention designs and obtains a series of TRADD inhibitor derivatives with high biological activity. The TRADD inhibitors provided by the present invention have similar activity to commercially available TNF-α biological inhibitors, but exhibit significantly lower adverse reactions, improved safety, and lower cost. Overall, they are superior to TNF-α biological inhibitors and can be used to treat acute and chronic kidney damage, with broad clinical application prospects.

Description

Technical Field

[0001] The present invention relates to a TRADD inhibitor derivative and a preparation method and application thereof. Background Art

[0002] Acute kidney injury (AKI) is a clinical syndrome caused by a short-term acute decrease or loss of renal filtration function due to various reasons. In recent years, the incidence of AKI has been on the rise. There are approximately 13 million AKI patients worldwide each year, of which about 1.7 million patients die from AKI and its complications. [1] Although AKI is a reversible disease, some severe patients still cannot repair their kidneys on their own. Over time, the kidneys will show fibrosis lesions, which will gradually evolve into chronic kidney disease and even end-stage renal disease. They have to receive renal replacement therapy, which brings a heavy economic burden to society and families. [2] There are many causes of AKI, including acute injuries caused by nephrotoxic drugs (methotrexate, vancomycin, cisplatin, etc.), infectious diseases (sepsis, bacterial endocarditis, etc.), acute injuries related to renal parenchymal or renal vascular diseases, acute injuries caused by urethral obstruction, etc. [3]. At present, there is a lack of broad-spectrum, specific, safe, efficient and well-tolerated therapeutic drugs. Prevention and early diagnosis remain the most important links in the prevention and treatment of AKI, and symptomatic treatment is also the main mode of intervention. However, although the main protective measures used in clinical practice have a certain protective effect, the effect is not ideal and their use is limited. [4-5] .

[0003] Acute tubular necrosis, interstitial nephritis, and chronic renal fibrosis are the most common acute and chronic kidney injuries. [6] . Renal tubular epithelial cells (RTECs), as the most important functional cells of the renal tubules, play an important role in mediating the secretion of endogenous and exogenous metabolic waste products by the renal tubules. Due to the urine concentrating function of the renal tubules, the endogenous and exogenous metabolic waste products taken up by RTECs are dozens of times more than those of the rest of the kidney tissues. Therefore, RTECs are extremely sensitive to environmental harmful stimuli such as chemical poisons, ischemia and hypoxia, and irreversibly undergo various forms of cell regulated death such as cell necrosis and ferroptosis. The RTECs cell membrane ruptures, and organelles and endogenous factors (proteins, RNA, DNA, etc.) are released into the renal tissue, turning on damage-related molecular patterns, causing tissue toxic pro-inflammatory responses, and attracting peripheral inflammatory immune cells to enter the damaged tissues. Through the tumor necrosis factor-α (TNF-α) released by immune cells, the tumor necrosis factor receptors of RTECs are recognized, further promoting programmed cell necrosis and apoptosis. [7] This vicious cycle of necrosis-inflammation-necrosis is one of the main pathological mechanisms of acute and chronic renal tubular injury. Therefore, blocking the key targets of this cycle through exogenous drugs can terminate the cycle and promote kidney self-repair.

[0004] TNF-α is a multifunctional cytokine secreted primarily by activated macrophages. TNF-α acts on RTECs to induce apoptosis and necrosis, inhibiting renal tubular secretion. It also activates the nuclear transcription factor NF-κB in RTECs, inducing the secretion of a variety of chemokines and inflammatory cytokines that participate in tissue inflammation. [8-9] . TNF-α binds to its receptor TNFR to exert its biological effects. TNFR has two subtypes, type I receptor (TNFR1) and type II receptor (TNFR2), which trigger different signaling pathways and biological effects after activation. Activated TNFR1 recruits and binds to proteins containing death domains, including tumor necrosis factor receptor-associated death domain protein (TRADD), receptor-interacting protein kinase 1 (RIPK1), tumor necrosis factor receptor-associated factor 2 (TRAF2), cell inhibitor of apoptosis protein (cIAP) and linear ubiquitin chain assembly complexes, forming protein complex I on the cell membrane. At this time, depending on the ubiquitination status of RIPK1, it can activate the downstream NF-κB signaling pathway and promote inflammatory immune responses. It can also dissociate through the K63 ubiquitin chain, promote the dissociation of RIPK1 and TRADD and other proteins from TNFR1 into the cytoplasm, recruit and bind to Fas-associated death domain protein (FADD) and RIPK3 to form complex II, activate downstream programmed apoptosis and necrosis signals, and induce cell death. [9] Functionally, TNFR1 is crucial for inducing cytotoxicity, proinflammatory reactions, and immune responses, while TNFR2 is mainly involved in immune regulation and protecting tissue function. Currently available TNF-α biological inhibitors are mainly divided into anti-TNF-α monoclonal antibodies and fusion proteins formed by TNFR and immunoglobulin IgG, including infliximab, adalimumab, golimumab, becelimumab, and etanercept. They exert therapeutic effects by clearing or blocking TNF-α in the patient's body and are used to treat various chronic inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ankylosing spondylitis, and psoriasis. [8] Although these biological agents have shown clear efficacy in clinically approved indications, TNF-α inhibitors block both TNFR1 and TNFR2 signaling pathways, and serious adverse reactions have occurred during their use, including severe infections and increased risk of malignant tumors. [10-11] Furthermore, TNF-α biological inhibitors are expensive, with monthly treatment costs of several thousand yuan still a significant expense for most families (see Table 1 below). Therefore, the development of novel small-molecule chemotherapeutic drugs that selectively inhibit TNFR1 signaling without affecting TNFR2 physiological signaling, while maintaining efficacy while reducing the risk of serious adverse reactions, is clearly novel, creative, and practical.

[0005] Table 1 Monthly treatment costs of five TNF-α inhibitors

[0006]

[0007] Data source: https: / / www.sohu.com / a / 453402225_120055892

[0008] TRADD is an intracellular molecular adaptor protein that interacts with TNFR1, TRAF2, FADD, and RIPK. TRADD mediates multiple signaling pathways, including complex I-mediated NF-κB pro-inflammatory signaling and complex II-mediated apoptosis and necrosis signaling pathways. [9] TNFR2 does not contain a death domain and does not interact with TRADD protein. Therefore, TRADD is a key target molecule for selective inhibition of TNFR1 signaling. [9] (See Figure 1 ) and is also a potential drug target for preventing and treating acute and chronic kidney damage. ICCB-19 is a currently known small molecule compound that inhibits TRADD, but its administration method is single, via injection, and its biological activity is relatively mild, resulting in weak drugability. Therefore, there are currently no marketed drugs that specifically target TRADD. The present invention proposes for the first time a drug for protecting against kidney damage whose primary pharmacological mechanism of action is to block the binding of TRADD to related functional proteins. The TRADD inhibitor obtained in the present invention has high bioavailability, strong activity, and can be taken orally, thus having strong drugability and greater translational and application value.

[0009] References

[0010] [1] Lewington AJ, CerdáJ, Mehta RL. Raising awareness of acute kidneyinjury: a global perspective of a silent killer. Kidney Int. 2013;84(3):457-67.

[0011] [2]Grams ME, Sang Y, Coresh J, Ballew SH, Matsushita K, Levey AS, GreeneTH, Molnar MZ, Szabo Z, Kalantar-Zadeh K, Kovesdy CP.Candidate Surrogate EndPoints for ESRD after AKI.JAm Soc Nephrol.2016;27(9):2851-9.

[0012] [3]Ronco C,Bellomo R,Kellum JA.Acute kidney injury.Lancet.2019;394(10212):1949-1964.

[0013] [4] BG, MS,Cider A.Is hydrotherapy an appropriate form ofexercise for elderly patients with biventricular systolic heart failure?JGeriatr Cardiol.2012;9(4):408-410.

[0014] [5]Singh VK,Seed TM.The efficacy and safety of amifostine for theacute radiation syndrome.Expert Opin Drug Saf.2019;18(11):1077-1090.

[0015] [6]Belavgeni A,Meyer C,Stumpf J,Hugo C,Linkermann A.Ferroptosis andNecroptosis in the Kidney.Cell Chem Biol.2020Apr 16;27(4):448-462.doi:10.1016 / j.chembiol.2020.03.016.PMID:32302582.

[0016] [7]Dvoriantchikova G,Lypka KR,Adis EV,Ivanov D.Multiple typesofprogrammed necrosis such as necroptosis,pyroptosis,oxytosis / ferroptosis,andparthanatos contribute simultaneously to retinal damage after ischemia-reperfusion.Sci Rep.2022;12(1):17152.

[0017] [8]Jang DI,Lee AH,Shin HY,Song HR,Park JH,Kang TB,Lee SR,Yang SH.TheRole ofTumor Necrosis Factor Alpha(TNF-α)in Autoimmune Disease and CurrentTNF-αInhibitors in Therapeutics.Int J Mol Sci.2021;22(5):2719.

[0018] [9]Lousa I,Reis F,Santos-Silva A,Belo L.The Signaling Pathway of TNFReceptors:Linking Animal Models ofRenal Disease to Human CKD.Int J MolSci.2022;23(6):3284.

[0019]

[10] Shivaji UN,Sharratt CL,Thomas T,Smith SCL,Iacucci M,Moran GW,Ghosh S,Bhala N.Review article:managing the adverse events caused by anti-TNFtherapy in inflammatory bowel disease.Aliment Pharmacol Ther.2019;49(6):664-680.

[0020]

[11] Peluso R,Cafaro G,Di Minno A,Iervolino S,Ambrosino P,Lupoli G,DiMinno MN.Side effects ofTNF-αblockers in patients with psoriatic arthritis:evidences from literature studies.Clin Rheumatol.2013;32(6):743-53. Summary of the Invention

[0021] my country is rich in natural drug resources, from which small molecule compounds can be screened as lead compounds. Based on structural biology, chemical biology and other technical means, the structure of the lead compound can be rationally optimized, and small molecules with clear pharmacological targets, good biological activity, few adverse reactions and high bioavailability can be further screened out, which have important transformation and application prospects. Paeonol, an active ingredient in the authentic medicinal material Paeony Bark in Anhui Province, has multiple pharmacological activities such as anti-tumor, antibacterial and antioxidant, but its effect is weak and its clinical application is greatly limited. The present invention uses paeonol as the basic structure and computer-assisted molecular docking technology to design TRADD inhibitors. According to the docking results, the structure is optimized and specific new small molecule compounds are synthesized, aiming to obtain a batch of new TRADD inhibitors with high safety, few adverse reactions and strong activity, which can selectively inhibit the TNFR1 signaling pathway and are used to improve acute and chronic kidney damage.

[0022] The TRADD inhibitor derivatives of the present invention have the following general structural formula:

[0023]

[0024] Where:

[0025] R is selected from phenylbutyryl, 2-5-dimethoxyphenylsulfonyl, 3-cyclopentylpropionyl, 4-methoxyphenylacetyl,

[0026] (1S)-4,7,7-trimethyl-3-oxo-2-oxobicyclo[2.2.1]heptane-1-carbonyl, 3-cyclohexylpropionyl, ((1R,4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methanesulfonyl, 1-adamantanyl, 2,5-dimethylphenylacetyl, 2-thiopheneacetyl, phenoxyacetyl, phenylpropionyl, morpholinopropionyl, 2-[(diphenylmethyl)mercapto]acetyl, 2-((phenylthiocarbonyl)thio)acetyl, 2-(4-chlorophenyl)thioacetyl, pyrrole One of propionyl, 4-oxo-4-phenylbutyryl, 2-(benzothiazolylthio)acetyl, cyclohexylacetyl, phenoxyacetyl, 3-amino-3-phenylpropionyl, benzyloxyacetyl, 2-(2-cyanophenyl)acetyl, 3-(4-bromophenyl)propionyl, 2-methoxyphenylsulfonyl, phenylthioacetyl, [4-(trifluoromethyl)phenylthio]acetyl, 2-(4-methylphenylcarbonylthiothiothio)acetyl, (R)-2-hydroxy-2-phenylpropionyl, and 5-mercapto-1,3,4-thiadiazol-2-ylthio)acetyl.

[0027] Specifically, the TRADD inhibitor derivatives of the present invention are selected from the compounds with the following structures:

[0028]

[0029]

[0030]

[0031]

[0032] One of the methods for preparing the TRADD inhibitor derivatives of the present invention comprises the following steps:

[0033] Paeonol is dissolved in approximately 40 parts by weight of dichloromethane, and a halogenated alkane solution of an acylating agent is added dropwise in the presence of an acid-binding agent and a catalyst. The mixture is stirred and reacted in the dark for 24 hours at room temperature under anhydrous and oxygen-free conditions. After recrystallization from petroleum ether, the mixture is extracted and washed multiple times with saturated brine, saturated sodium bicarbonate solution, and ethyl acetate. The organic phase is collected, dried over anhydrous sodium sulfate, concentrated, recrystallized, and filtered to obtain the finished product.

[0034] The catalyst is DMAP (4-dimethylaminopyridine), and the added amount is 3-15% of the amount of paeonol.

[0035] The acid binding agent selected is preferably triethylamine (Et3N).

[0036] The acylating agent is selected from phenylbutyryl chloride, 2,5-dimethylphenylacetyl chloride, 3-cyclopentylpropionyl chloride, 4-methoxyphenylacetyl chloride, (1S)-4,7,7-trimethyl-3-oxo-2-oxobicyclo[2.2.1]heptane-1-carbonyl chloride, 3-cyclohexylpropionyl chloride, ((1R,4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methanesulfonyl chloride, 1-adamantanecarbonyl chloride, 2-thiopheneacetyl chloride, phenoxyacetyl chloride, phenylpropionyl chloride, benzyloxyacetyl chloride, 2-methoxybenzenesulfonyl chloride, and 2-5-dimethoxybenzenesulfonyl chloride. The molar ratio of paeonol to the acylating agent is 1:1.5-1:2.

[0037] The halogenated alkane is selected from dichloromethane, chloroform, 1,1-dichloroethane, 1,2-dichloroethane or 1,3-dichloroethane.

[0038] The second method for preparing the TRADD inhibitor derivative of the present invention comprises the following steps:

[0039] Paeonol and an acylating agent are mixed and dissolved in 40 parts by weight of dichloromethane, reacted at room temperature for 48 hours in the presence of an acid binder, a dehydrating agent and a catalyst, recrystallized with petroleum ether after the reaction, extracted and washed with saturated brine and ethyl acetate, the organic phase is separated and collected, concentrated, eluted with an equal proportion gradient through a silica gel chromatography column, and the eluate is collected, and then concentrated and purified to obtain a finished product; the eluate is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 12:1-3:1.

[0040] The catalyst is DMAP, the acid-binding agent is Et3N, and the dehydrating agent is EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (EDCI.HCL) or DCC (dicyclohexylcarbodiimide).

[0041] The acylating agent is selected from morpholinepropionic acid, 2-[(benzhydryl)mercapto]acetic acid, 2-((phenylthioformyl)thio)acetic acid, 2-(4-chlorophenyl)thioacetic acid, pyrrole-1-propionic acid, 4-oxo-4-phenylbutyric acid, 2-(benzothiazolylthio)acetic acid, cyclohexylacetic acid, phenoxyacetic acid, 3-amino-3-phenylpropionic acid, 2-(2-cyanophenyl)acetic acid, 3-(4-bromophenyl)propionic acid, phenylthioacetic acid, [4-(trifluoromethyl)phenylthio]acetic acid, 2-(4-methylphenylcarbonylthiothiothio)acetic acid, (R)-2-hydroxy-2-phenylpropionic acid, 5-mercapto-1,3,4-thiadiazol-2-ylthioacetic acid, and the molar ratio of paeonol to the acylating agent is 1:1.5-1:2.

[0042] The invention relates to the use of the TRADD inhibitor derivative in the preparation of a drug for treating acute and / or chronic kidney injury.

[0043] The invention relates to an application of the TRADD inhibitor derivative in the preparation of a drug for treating acute kidney injury caused by drug-induced and / or infectious diseases.

[0044] The invention relates to an application of the TRADD inhibitor derivative in the preparation of a drug for treating chronic kidney damage and / or renal fibrosis caused by pharmaceutical and / or infectious diseases.

[0045] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0046] 1. This invention targets the biomolecule TRADD, using structural biology as a foundation and computational molecular docking technology to design and obtain highly active TRADD inhibitors. The bioactivity and renal protective effects of TRADD inhibitors are similar to those of currently commercially available TNF-α biological inhibitors, but with enhanced safety and significantly fewer adverse reactions than TNF-α biological inhibitors. This addresses the significant adverse reactions associated with the use of TNF-α biological inhibitors and the lack of specific treatments for acute and chronic kidney damage.

[0047] ICCB-19 is a currently known small molecule compound that inhibits TRADD, but its administration route is limited to injection, and its biological activity and renal protective effects are weaker than those of the related inhibitors of the present invention, resulting in weak drugability. Paeonol's unstable structure, volatility, and ease of metabolism in the human body, as well as its weak effects, have limited its clinical application. In contrast, the TRADD inhibitor obtained in the present invention has high bioavailability, strong activity, and can be taken orally, thus having strong drugability and greater translational and application value.

[0048] 3. The TRADD inhibitor obtained by the present invention is simple to prepare, has a mature process route, and is suitable for industrial production. The raw material paeonol is inexpensive, and its cost price is much lower than that of TNF-α biological inhibitors. This solves the problem of high prices of TNF-α biological inhibitors and reduces the financial pressure on patients. Therefore, it has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the TNFR signaling pathway and the effects of TRADD inhibitors.

[0050] Figure 2 Absolute bioavailability and tissue distribution of TRADD inhibitors ( * P<0.05, ** P < 0.01 vs. paeonol. n = 6).

[0051] Figure 3 The protective effect of TRADD inhibitor on cisplatin-induced acute kidney injury in mice ( ## P<0.01 vs. normal group;

[0052] * P<0.05, ** P < 0.01 vs. AKI group; ΔΔ P < 0.01 vs. ICCB-19 group; δδ P < 0.01 vs. paeonol group, n = 6).

[0053] Figure 4 The effect of TRADD inhibitor on the pathological changes of acute and chronic injury tissues in mice induced by cisplatin. a: normal group; b: model group; c: compound 3; d: compound 14; e: compound 15; f: compound 17; g: compound 29; h: ICCB-19; i: paeonol; j: infliximab; No. 1: HE staining; No. 2: Masson staining; (200×).

[0054] Figure 5 It is a TRADD inhibitor that selectively inhibits the TNFR1 signaling pathway.

[0055] Figure 6 Safety analysis of TRADD inhibitors and TNF-α biological inhibitors ( ** P < 0.01 vs. infliximab; ΔΔ P<0.01 vs. adalimumab group; $$ P < 0.01 vs. Yisaipu group, N = 8). DETAILED DESCRIPTION

[0056] The technical solutions of the present invention are further described in detail by the following examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0057] Example 1: Synthesis of 2-acetyl-5-methoxyphenyl 2-[(5-sulfanyl-1,3,4-thiadiazol-2-yl)sulfanyl]acetate

[0058]

[0059] Weigh 3g of 5-mercapto-1,3,4-thiadiazol-2-ylthioacetic acid solid powder and 2g of paeonol and add them to a 250ml three-necked flask. Then weigh 3.2g of EDCI and 300mg of dimethylaminopyridine and add them to the three-necked flask. Install the exhaust joint and the anhydrous oxygen-free device to pump and aerate. After completion, add 40ml of dichloromethane solution to the three-necked flask and mix thoroughly. Mix and stir under a magnetic stirrer for 48 hours. After completion, add saturated potassium bisulfate solution and ethyl acetate to extract twice, combine the organic phases, dry over anhydrous sodium sulfate, and recover the solvent under reduced pressure to dryness to obtain a light yellow oil. Use petroleum ether and ethyl acetate as eluents for chromatography column separation and purification to obtain 1.1g of a light yellow solid with a yield of 37%. 1 H NMR (300 MHz, CDCl3 / DMSO) δ7.75 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.53 (dd, J = 8.5, 2.1 Hz, 1H), 4.27-4.07 (m, 2H), 3.84 (s, 3H), 2.62 (s, 3H). Molecular formula: C 13 H 12 N2O4S3;ESI-MS m / z:357.43[M + +H].

[0060] Example 2: Synthesis of 2-acetyl-5-methoxyphenyl (1S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylate

[0061]

[0062] Weigh 1.662g of paeonol and 61mg of dimethylaminopyridine into a 250ml three-necked flask. Install a dropper and a vacuum connector, then install an anhydrous and oxygen-free device. Vacuum and aerate. Add 40ml of dichloromethane (DCM) to the flask and mix thoroughly. Then, add 2ml of triethylamine and mix thoroughly. Add 3.29g of (1S)-4,7,7-trimethyl-3-oxo-2-oxobicyclo[2.2.1]heptane-1-carbonyl chloride to a 100ml two-necked eggplant-shaped flask. Add approximately 10ml of a mixed solvent of dichloromethane and tetrahydrofuran and shake thoroughly to mix. Then, inject the mixture into the dropper with a syringe and slowly add the above mixture dropwise while stirring with a magnetic stirrer. Stir at room temperature for 20-24 hours. The original solution was first dried using a rotary evaporator, 40 ml of petroleum ether was added and dried again, and then extracted with saturated brine or a mixed solution of saturated sodium bicarbonate solution and ethyl acetate. After extraction twice, the organic phases were combined, concentrated under reduced pressure, and then filtered and dried, and vacuum filtered to obtain about 2.5 g of a white solid. 1 HNMR (300 MHz, CDCl3 / DMSO) δ7.74 (d, J = 8.5 Hz, 1H), 6.86 (d, J = 2.1 Hz, 1H), 6.50 (dd, J = 8.5, 2.1 Hz, 1H), 3.84 (s, 3H), 2.61 (s, 3H), 2.41-2.01 (m, 5H), 1.25 (s, 3H), 1.11 (d, J = 15.1 Hz, 6H). Molecular formula: C 19 H 22 O6; ESI-MS m / z:347.38[M + +H].

[0063] Example 3: Synthesis of 2-acetyl-5-methoxyphenyl {7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl} methanesulfonate

[0064]

[0065] Weigh 1.662g of paeonol and 61mg of dimethylaminopyridine into a 250ml three-necked flask, install a dropper and a vacuum connector, install an anhydrous and oxygen-free device, and perform vacuuming and aeration. After completion, add 40ml of dichloromethane (DCM) to the three-necked flask and mix thoroughly. Then add 2ml of triethylamine to the three-necked flask and mix thoroughly. Add 3.29g of (7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-yl)methanesulfonyl chloride to a 100ml two-necked eggplant-shaped flask, add 10ml of a mixed solvent of dichloromethane and tetrahydrofuran, and shake thoroughly to mix. Then, use a syringe to inject into the dropper and slowly add the above mixture dropwise while stirring with a magnetic stirrer. Stir at room temperature in the dark for 20-24 hours. The stock solution was first dried using a rotary evaporator, and 40 ml of petroleum ether was added and dried again. The product was then extracted twice with saturated brine or a mixture of saturated sodium bicarbonate solution and ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain approximately 3 g of a crude white solid. The product was then filtered, dried, and vacuum filtered to obtain approximately 2.5 g of a white solid. The yield was 45.6%. 1 HNMR (300 MHz, CDCl3 / DMSO) δ7.77 (d, J = 8.5 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.56 (dd, J = 8.5, 2.1 Hz, 1H), 3.85-3.67 (m, 5H), 2.61 (s, 3H), 2.48-2.27 (m, 2H), 2.17-1.90 (m, 3H), 1.83-1.56 (m, 2H), 1.02 (dd, J = 15.0, 1.5 Hz, 6H). Molecular formula: C 19 H 24 O6S; ESI-MS m / z:381.46[M + +H].

[0066] Example 4: Synthesis of 2-acetyl-5-methoxyphenyl-2-(benzyloxy)acetate

[0067]

[0068] Weigh 1.662g paeonol and the dimethylaminopyridine of 61mg and join in the three-necked flask of 250ml, install point liquid drop bucket and pumping joint, install anhydrous oxygen-free device, pump and inflate, after completion, add 40ml dichloromethane DCM and fully mix in three-necked flask, then add the triethylamine of 2ml to three-necked flask, fully mix.Point liquid drop bucket will add dichloromethane and tetrahydrofuran mixed solvent of about 10ml, finally the benzyloxyacetyl chloride of 3.29g is directly joined in the solvent of 10ml and mix, under magnetic stirring apparatus, slowly drip said mixture, lucifuge is stirred at room temperature 20-24 hour.Take stoste and first be spin-dried with Rotary Evaporators, add 40ml petroleum ether and be spin-dried again, extracted by saturated aqueous common salt or saturated sodium bicarbonate solution and ethyl acetate mixed solution, after extraction twice, merge organic phase, after concentrating under reduced pressure, carry out the separation and purification of silica gel column chromatography, finally obtain white solid 2.31g or so. Yield: 69.9%. 1 HNMR (300 MHz, CDCl3 / DMSO) δ7.75 (d, J = 8.6 Hz, 1H), 7.41-7.24 (m, 5H), 6.87 (d, J = 2.2 Hz, 1H), 6.50 (dd, J = 8.5, 2.1 Hz, 1H), 4.49 (t, J = 0.9 Hz, 2H), 4.31 (s, 2H), 3.84 (s, 3H), 2.62 (s, 3H). Molecular formula: C 18 H 18 O5; ESI-MS m / z:315.34[M + +H].

[0069] Example 5: Synthesis of 2-acetyl-5-methoxyphenyl-2-phenoxyacetate

[0070]

[0071] Get the paeonol of 1.662g and the dimethylaminopyridine of 61mg and join in the three-necked flask of 250ml, install point liquid drop bucket and exhaust joint, install anhydrous oxygen-free device, carry out exhaust inflation, after completion, add 40ml dichloromethane DCM in three-necked flask and fully mix, then add the triethylamine of 2ml to three-necked flask, fully mix.Point liquid drop bucket will add dichloromethane and tetrahydrofuran mixed solvent of about 10ml, finally the phenoxyacetyl chloride of 3.29g is directly joined in the solvent of 10ml and mix, under magnetic stirring apparatus, slowly drip said mixture, lucifuge is stirred at room temperature 20-24 hour.Get stoste and first use rotary evaporator to be spin-dried for, add 40ml petroleum ether and be spin-dried for again, by saturated aqueous common salt or saturated sodium bicarbonate solution and ethyl acetate mixed solution, extract, after extraction twice, merge organic phase, after concentrating under reduced pressure, carry out the separation and purification of silica gel column chromatography, finally obtain white solid 1.3g or so. Yield 40%.1 H NMR (300 MHz, CDCl3 / DMSO) δ7.75 (d, J = 8.6 Hz, 1H), 7.36-7.23 (m, 2H), 7.11-7.00 (m, 1H), 7.00-6.90 (m, 2H), 6.87 (d, J = 2.1 Hz, 1H), 6.50 (dd, J = 8.5, 2.1 Hz, 1H), 4.77 (s, 2H), 3.84 (s, 3H), 2.62 (s, 3H). Molecular formula: C 17 H 16 O5; ESI-MS m / z: 301.31[M + +H].

[0072] The present invention conducted tests on acute toxicity, bioactivity, bioavailability, adverse reactions, etc. using some of the above derivative samples, and the results are as follows.

[0073] 1. Acute toxicity study of TRADD inhibitors

[0074] Kunming mice were gavaged with a TRADD inhibitor (5g / kg) and observed for two weeks with no deaths or abnormal toxic reactions. During the observation period, mice in all groups had smooth fur, normal movements, no spasms, ataxia, or other symptoms, and no abnormal secretions were observed in the eyes, nose, or mouth. All groups showed normal ocular findings, including normal pupil constriction or dilation, and proptosis. At the end of the observation period, mice in all groups were sacrificed, and macroscopic observation revealed no significant lesions in major organs, including the heart, liver, spleen, lungs, and kidneys, in any of the treated groups. Histological staining results showed that the liver, kidneys, spleen, heart, and lungs in all groups were structurally normal, with no significant lesions or abnormalities.

[0075] 2. In vitro activity test of TRADD inhibitors

[0076] Humanized renal tubular epithelial cell lines (HK-2) were seeded in 96-well plates and treated with a series of compound working solutions (25 μM) for 48 hours. Cell viability was assessed using CCK8 assays. As shown in Table 2, HK-2 cell viability after 24 hours of stimulation with the various compounds remained unchanged compared to the control group, with no statistically significant differences between the groups. This suggests that the 25 μM concentration of each small molecule compound had no significant effect on HK-2 cells.

[0077] HK-2 cells were treated with various concentrations of small molecule compounds and cisplatin for 48 hours. The results of cell viability assay using the CCK8 assay are shown in Table 2. All synthesized small molecule compounds effectively inhibited cisplatin-induced cytotoxicity. Twenty-two of these compounds exhibited half-maximal inhibitory concentrations (IC50) values ​​lower than those of the positive control drug ICCB-19. The top eight compounds (Compound 3, Compound 4, Compound 14, Compound 15, Compound 17, Compound 18, Compound 19, and Compound 29) were selected from the highest to lowest bioactivity for subsequent experiments.

[0078] Table 2. TRADD inhibitors inhibit the cytotoxic effect of cisplatin on HK-2 cells

[0079]

[0080] Note: Compound 3: 2-acetyl-5-methoxyphenyl-2-(2,5-dimethylphenyl) acetate; Compound 4: 2-acetyl-5-methoxyphenyl-2-methoxybenzene-1-sulfonate; Compound 14: 2-acetyl-5-methoxyphenyl-2,5-dimethoxybenzene-1-sulfonate; Compound 15: 2-acetyl-5-methoxyphenyl-2-(phenylthio) acetate; Compound 17: 2-acetyl-5-methoxyphenyl-2-(benzyloxy) acetate; Compound 18: 2-acetyl-5-methoxyphenyl-2-(4-methoxyphenylthioformylthio) acetate; Compound 19: 2-acetyl-5-methoxyphenyl 4-oxo-4-phenylbutyrate; Compound 29: 2-acetyl-5-methoxyphenyl-2-phenoxy acetate;

[0081] 3. Absolute bioavailability and tissue distribution of TRADD inhibitors after single oral administration

[0082] At 50 mg kg -1 The rats were gavaged with TRADD inhibitor (ig) and blood samples were collected at 5, 10, 15, 30, 60, 90, 120, 180, 240, 360, and 720 min before and after administration. -1The rats were injected with TRADD inhibitors at the tail vein at a dose of 100 mg / dL, and then blood was collected at 5, 10, 15, 30, 60, 90, 120, 240, 360, and 720 min before and after administration. The plasma samples were processed, injected and analyzed by UPLC-MS / MS, and converted into blood drug concentrations. The DAS2.0 software was used to identify the compartment model, and the pharmacokinetic parameter area under the drug-time curve (AUC) was calculated. The absolute bioavailability was obtained by calculation. Among them, the absolute bioavailability of compound 3, compound 14, compound 15, compound 17, compound 18, compound 19, and compound 29 was higher than that of paeonol (P < 0.05). The results are shown in the figure. Figure 2 . At 50mg·kg -1 Rats were orally gavaged with TRADD inhibitors for 7 days. The rats were killed 60 min, 180 min, and 360 min after the last gavage. Kidney tissues were collected and homogenized. The supernatant of the homogenate was extracted and injected and analyzed by UPLC-MS / MS to measure the concentration of target compounds in tissues. The tissue concentrations of compounds 3, 14, 15, 17, and 29 were significantly higher than those of paeonol (P < 0.01). Figure 2 .

[0083] 4. Renal protective effect of TRADD inhibitors

[0084] An AKI model was induced in C57BL / 6J mice by a single intraperitoneal injection of cisplatin (20 mg / kg). The mice were randomly divided into 10 groups: normal group, model group, compound 3 (50 mg / kg, gavage), compound 14 (50 mg / kg, gavage), compound 15 (50 mg / kg, gavage), compound 17 (50 mg / kg, gavage), compound 29 (50 mg / kg, gavage), ICCB-19 (5 mg / kg, ip), paeonol group (50 mg / kg, gavage), and infliximab group (3.5 mg / kg, ip). Except for the infliximab group, all treatment groups were administered with the drug starting 5 days before modeling. After 7 consecutive days of treatment, the mice were sacrificed, and bilateral kidney tissue was isolated. Partial tissue from one kidney was prepared for paraffin sectioning, and the renal cortex of the remaining kidney was frozen at -80°C for later use. The normal and model groups were administered with equal amounts of vehicle.

[0085] The results of renal function biochemical index test were as follows Figure 3As shown. Compared with the normal group, the blood SCr and BUN levels of AKI mice were significantly increased, and the difference was statistically significant (P<0.01), indicating that AKI renal function was significantly abnormal. Compared with the AKI model group, compound 3, compound 14, compound 15, compound 17, compound 29, ICCB-19, and infliximab administration can significantly reduce the blood SCr and BUN levels of rats with renal injury, and the difference is statistically significant (P<0.05). Compared with ICCB-19, the SCr and BUN levels in the compound 3, compound 14, compound 15, and compound 17 groups were significantly reduced (P<0.05). Compared with the paeonol group, the blood creatinine and urea nitrogen in the compound 3, compound 14, compound 15, and compound 17 groups were significantly reduced (P<0.05). There was no significant difference in the improvement of renal function indicators between compound 3, compound 14, compound 15, and compound 17 and infliximab.

[0086] The results of renal tissue pathomorphological analysis were as follows Figure 4 As shown. HE staining showed that the renal tubular structure of the normal group of mice was clear and complete, with normal morphology, intact epithelial cells, intact basement membrane, obvious tubular structure, and no obvious inflammatory cell infiltration in the renal interstitium. The renal tubular epithelial cell structure of the mice in the cisplatin-induced AKI group was swollen, the tubular lumen was reduced, and some epithelial cells showed obvious edema, vacuolar degeneration and cell necrosis. At the same time, some renal tubular epithelial cells were shed. Each drug-treated group, including compound 3, compound 14, compound 15, compound 17, compound 29, ICCB-19, and infliximab, can improve the above-mentioned histopathological manifestations to varying degrees.

[0087] C57BL / 6J male mice aged 6-8 weeks were selected and housed in an SPF animal room for adaptive feeding for 1 week. After the adaptation period, the mice were randomly divided into groups. Except for the normal group, all mice in each group were intraperitoneally injected with 8 mg / kg cisplatin solution prepared in advance with normal saline, once a week for 4 times. 72 hours after the last injection of cisplatin, the mice were killed and the kidneys of each mouse were collected and immersed in 4% paraformaldehyde fixative. The sections were then prepared for Masson staining. Staining showed that the normal group mice had fewer collagen fibers in the renal tissue, while the model group mice had significantly more collagen fibers. Compound 3, compound 14, compound 15, compound 17, compound 29, ICCB-19, and infliximab can improve the above-mentioned fibrosis pathological manifestations to varying degrees.

[0088] 5. TRADD inhibitors selectively inhibit the TNFR1 signaling pathway

[0089] The IP experimental results are shown in Figure 5TNF-α stimulation of the renal tubular epithelial cell line NRK-52E can significantly promote the interaction between TRADD and FADD, and TRADD and TRAF2. Compounds 15 and 17 significantly reduced the interaction of the above proteins, suggesting that they effectively blocked the TNFR1 downstream signaling pathway. In addition, TNF-α stimulation can significantly activate and upregulate the expression of Akt, a downstream molecule of TNFR2. On this basis, after intervention with compounds 15, 17 and ICCB-19, Akt expression did not change significantly, suggesting that compounds 15, 17 and ICCB-19 selectively inhibit the TNFR1 downstream signaling pathway and have no obvious effect on the TNFR2 downstream signaling.

[0090] 6. Comparative analysis of adverse reactions between TRADD inhibitors and TNF-α biological inhibitors

[0091] C57BL / 6J mice were injected with a single dose of Gram-positive Staphylococcus aureus via the tail vein to establish a mouse infection model. A normal group, a model group, compound 3 (50 mg / kg), compound 14 (50 mg / kg), compound 15 (50 mg / kg), compound 17 (50 mg / kg), compound 29 (50 mg / kg), ICCB-19 (50 mg / kg), a paeonol group (50 mg / kg), an infliximab group (3.5 mg / kg), an adalimumab group (3.5 mg / kg), and a tadalafil group (3.5 mg / kg). TNF-α biological inhibitors were administered intraperitoneally every other day. The remaining treatment groups were administered for 18 consecutive days. The mice were then sacrificed, and lung, liver, brain, and spleen tissues were collected for analysis. There was no death in the normal group of mice during the entire experimental period. The survival rate of the control group infection model was 65% during the experimental period, while the survival rates of the mice in the compound 3, compound 14, compound 15, compound 17, compound 29, and ICCB-19 groups were similar to or slightly higher than those in the control group. The survival rates of the mice in the TNF-α biological inhibitor infliximab, adalimumab, and ysaipu groups were all lower than 20%, which were significantly lower than those in the TRADD inhibitor and control groups. Compared with the control group, the bacterial replication numbers in the brain tissue, lung tissue, and liver tissue of the mice in each TRADD inhibitor group were similar, but the bacterial replication numbers in the TNF-α biological inhibitor infliximab, adalimumab, and ysaipu groups were significantly increased. The results are shown in Figure 3. Figure 6 Administration of TNF-α biological inhibitors aggravated the infection severity of model animals. The TRADD inhibitor of the present invention has significant advantages over TNF-α biological inhibitors.

Claims

1. A TRADD inhibitor derivative, characterized in that A compound selected from the following structures: 、 、 、 、 。 2. Use of the TRADD inhibitor derivative according to claim 1 in the preparation of a drug for treating acute and / or chronic kidney injury.

3. Use of the TRADD inhibitor derivative according to claim 1 in the preparation of a drug for treating acute kidney injury caused by drug-induced and / or infectious diseases.

4. Use of the TRADD inhibitor derivative according to claim 1 in the preparation of a drug for treating chronic kidney damage and / or renal fibrosis caused by pharmaceutical and / or infectious diseases.

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