Immunosuppressant based on protide prodrug modification as well as preparation method and application of immunosuppressant

Abstract

The invention discloses an immunosuppressive agent based on protide prodrug modification and a preparation method and application thereof, the immunosuppressive agent adopts Protide technology to carry out structure improvement on everolimus, rapamycin and cyclosporin, side effects are effectively reduced, and enrichment of drugs at diseased regions is increased.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a class of protides of everolimus, rapamycin and cyclosporin, as well as preparation methods and applications thereof. Compared with everolimus, rapamycin and cyclosporin, these protides can release the original drugs in a targeted manner, thus having high specificity and producing less toxic side effects. Background Art

[0002] Protide technology is a prodrug approach designed to achieve efficient intracellular delivery of nucleoside analog monophosphates and monophosphonates. The design concept utilizes aromatic groups and amino acid ester moieties to mask the hydroxyl group of the monophosphate or monophosphonate group, forming prodrug molecules. Once these prodrug molecules enter cells, intracellular enzymes cleave off the aromatic and amino acid ester moieties, releasing the free nucleoside monophosphate or monophosphonate, thereby exerting their biological activity. Although this technology was initially used to discover nucleoside protides for antiviral and anticancer drugs, it has been increasingly applied to other disease areas in recent years. Furthermore, the protide prodrug approach has garnered increasing attention as a prodrug strategy for the intracellular delivery of monophosphorylated non-nucleoside compounds, such as glucosamine, sphingosine 1-phosphate (S1P), 4-phospho-D-erythrohydroxamic acid, and 5-phospho-erythrohydroxamic acid.

[0003] Rapamycin and everolimus are mammalian target of rapamycin (mTOR) inhibitors and are widely used clinically to treat advanced renal cell carcinoma, locally advanced or metastatic progressive pancreatic neuroendocrine tumors, advanced drug-resistant breast cancer, and giant cell astrocytoma. Furthermore, both drugs can be used in combination with other immunosuppressants in organ transplantation procedures, such as kidney and heart transplants, to prevent rejection after organ transplantation. Despite this, both drugs are associated with numerous adverse reactions, including increased risk of infection, stomatitis, rash, diarrhea, metabolic abnormalities, and fatigue. These adverse reactions are primarily due to the over-inhibition of mTOR-related pathways by rapamycin or everolimus, as mTOR inhibitors. The mTOR signaling pathway plays a key role in the immune system, oral mucosal cells, skin cells, intestinal function, and metabolic processes. Drug inhibition of this pathway can weaken immune cell function, leading to an increased risk of infection, and disrupt insulin signaling and fat and sugar metabolism, leading to metabolic abnormalities and other adverse reactions.

[0004] Cyclosporine, a calcineurin inhibitor, is a first-line immunosuppressant for preventing rejection after solid organ transplantation (eg, kidney, liver, and heart transplantation). It also plays an important role in the treatment of autoimmune diseases. However, cyclosporine is associated with a range of adverse reactions, including nephrotoxicity, hypertension, neurotoxicity, hirsutism, gingival hyperplasia, metabolic disorders, and an increased risk of opportunistic infections. These adverse reactions are primarily due to cyclosporine blocking T lymphocyte activation (particularly IL-2 transcription). However, this effect lacks cell specificity and may inadvertently damage other normal cells or systems.

[0005] Although everolimus, rapamycin, and cyclosporine have significant clinical application value, their side effects limit their development. Based on the principle of protide prodrugs, we have developed protide prodrugs for the treatment of tumors, organ transplantation, and autoimmune diseases. Compared to traditional everolimus, rapamycin, and cyclosporine, protides are protected from degradation and metabolism by enzymes in the body, extending the drug's half-life and improving its pharmacokinetic properties. Furthermore, by adjusting the structure and properties of protide prodrugs and selecting appropriate aromatic groups and amino acid ester moieties, we can increase drug accumulation at the site of disease, enhance the drug's selectivity for target cells, and increase drug distribution to the tissues and organs in need of treatment, reducing the number of dosing times while minimizing side effects on normal tissues and improving patient compliance.

[0006] The present inventors have discovered, through experiments, a prodrug of everolimus, rapamycin and cyclosporine protide, as well as methods and uses of the protide for treating tumors, organ transplantation and autoimmune diseases. Summary of the Invention

[0007] The present invention provides an immunosuppressant based on protide prodrug modification, a preparation method and application thereof, and adopts protide technology to improve the structure of everolimus, rapamycin and cyclosporine, effectively reducing side effects and increasing the enrichment of drugs in lesion sites.

[0008] The technical solution of the present invention is as follows: A compound of formula (1), formula (2) and formula (3) can be used to treat tumors and organ transplantation

[0009] Compound of formula (1)

[0010]

[0011] or a pharmaceutically acceptable salt, racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof;

[0012] in:

[0013] R 1 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl.;

[0014] R 2 Selected from C 1-6 Alkyl, hydrogen;

[0015] R 3 Selected from C 1-6 Alkyl, phenyl, benzyl, which are optionally substituted by zero, one or more substituents selected from the following: halogen, amino, hydroxyl, -OC 1-6 Alkyl, C 1-6 alkyl.;

[0016] L is selected from C2~C 12 At least one of an aliphatic chain or a polyethylene glycol chain, optionally interrupted by zero, one or more substituents selected from the group consisting of: -O-, -COO-, -CONH-, triazole, including but not limited to any one of the following structures

[0017] Compound of formula (2)

[0018]

[0019] or a pharmaceutically acceptable salt, racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof;

[0020] in:

[0021] R 1 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl;

[0022] R 2 Selected from C 1-6 Alkyl, hydrogen;

[0023] R 3 Selected from C 1-6 Alkyl, phenyl, benzyl, which are optionally substituted by zero, one or more substituents selected from the following: halogen, amino, hydroxyl, -OC 1-6 Alkyl, C 1-6 alkyl;

[0024] L is selected from C2~C 12At least one of an aliphatic chain or a polyethylene glycol chain, optionally interrupted by zero, one or more substituents selected from the group consisting of: -O-, -COO-, -CONH-, triazole, including but not limited to any one of the following structures

[0025] Compound of formula (3)

[0026]

[0027] or a pharmaceutically acceptable salt, racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof;

[0028] in:

[0029] R 1 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl;

[0030] R 2 Selected from C 1-6 Alkyl, hydrogen;

[0031] R 3 Selected from C 1-6 Alkyl, phenyl, benzyl, which are optionally substituted by zero, one or more substituents selected from the following: halogen, amino, hydroxyl, -OC 1-6 Alkyl, C 1-6 alkyl;

[0032] L is selected from C2~C 12 At least one of an aliphatic chain or a polyethylene glycol chain, optionally interrupted by zero, one or more substituents selected from the group consisting of: -O-, -COO-, -CONH-, triazole, including but not limited to any one of the following structures

[0033] In the above formula, Indicates a replacement position.

[0034] Furthermore, the compounds of formula (1), formula (2), and formula (3) can be selected from any of the following compounds:

[0035]

[0036]

[0037] The present invention also relates to pharmaceutical compositions comprising the above compounds or pharmaceutically acceptable salts, solvates, esters, acids, metabolites or prodrugs thereof, as well as methods and uses of the compounds or pharmaceutical compositions for inhibiting mTOR kinase activity and calcineurin activity, and methods and uses of the compounds or pharmaceutical compositions for treating, preventing or ameliorating diseases, disorders or conditions regulated by or affected by mTOR kinase activity and calcineurin activity or involving mTOR kinase and calcineurin activity.

[0038] The compounds provided herein may exhibit tautomerism, structural isomerism, and stereoisomerism. The present invention encompasses any tautomeric, structural, or stereoisomer forms thereof, and mixtures thereof, which have the ability to modulate kinase activity, and this ability is not limited to any one isomer or mixture thereof.

[0039] The present invention also provides a pharmaceutical composition comprising the compound of formula (1), formula (2), formula (3) or a pharmaceutically acceptable salt thereof, a racemic mixture, a hydrate, a solvate, a prodrug, an enantiomer, a diastereomer, a tautomer, and one or more pharmaceutically acceptable carriers, diluents, and excipients.

[0040] The compounds described herein can be made and / or used as pharmaceutically acceptable salts. The types of pharmaceutically acceptable salts include, but are not limited to: (1) acid-forming salts formed by reacting the free base form of the compound with a pharmaceutically acceptable inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid, or the like; or by reacting the free base form of the compound with an organic acid, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, malic acid, citric acid, succinic acid, maleic acid, tartaric acid, fumaric acid, trifluoroacetic acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 4-methylbicyclo-[2.2.2]octanedio ... 2-ene-1-carboxylic acid, 2-naphthalenesulfonic acid, tert-butylacetic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, salicylic acid, hydroxynaphthoic acid, stearic acid, muconic acid, etc.; (2) forming salts with bases, which are formed when the acidic protons in the parent compound are replaced by metal ions, such as alkali metal ions (such as lithium, sodium, potassium), alkaline earth metal ions (such as magnesium or calcium) or aluminum ions; or coordinated with organic bases or inorganic bases, acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, etc.; acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

[0041] A pharmaceutically acceptable carrier (i.e., a pharmaceutically acceptable carrier) is a carrier that is compatible with the active ingredient in the composition (in some embodiments, stabilizes the active ingredient) and is not harmful to the subject being treated. Pharmaceutically acceptable carriers and / or excipients can be selected from diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants, flavorings, buffers, stabilizers, solubilizers, and combinations thereof.

[0042] The pharmaceutical compositions comprising the compounds of formula (1), formula (2), formula (3) and / or their pharmaceutically acceptable salts described herein can be administered in various known ways, such as orally, topically, rectally, parenterally, by inhalation or implantation.

[0043] Depending on the therapeutic purpose, the pharmaceutical composition can be prepared into various types of dosage unit forms, such as tablets, pills, powders, liquid preparations, suspensions, emulsions, granules, capsules, elixirs, tinctures, suppositories and injections (solutions and suspensions).

[0044] To form the pharmaceutical composition into tablet form, any excipient known and widely used in the art may be used. For example, carriers such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silicic acid; binders such as water, ethanol, propanol, ordinary syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, and the like; disintegrants such as dry starch, sodium alginate, agar powder, kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyethylene sorbitan, sodium lauryl sulfate, monoglyceride of stearate, starch, and lactose; disintegration inhibitors such as white sugar, glyceryl tristearate, coconut oil, and hydrogenated oil; adsorption promoters such as quaternary ammonium hydroxide and sodium lauryl sulfate; wetting agents such as glycerol and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; and lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol. Ordinary coating materials can be selected as needed to make sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double-layer film tablets and multi-layer tablets.

[0045] In order to shape the pharmaceutical composition into a pill form, any excipient known and widely used in the art can be used, for example, carriers such as lactose, starch, coconut oil, hardened vegetable oil, kaolin and talc; binders such as gum arabic powder, tragacanth powder, gelatin and ethanol; disintegrants such as agar and kelp powder;

[0046] In order to shape the pharmaceutical composition into a suppository form, any excipient known and widely used in the art may be used, for example, polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin and semi-synthetic glycerides and the like.

[0047] To prepare a pharmaceutical composition in the form of an injection, the solution or suspension can be sterilized (preferably by adding an appropriate amount of sodium chloride, glucose, or glycerol) and prepared into an injection with an osmotic pressure equal to that of blood. Any commonly used carrier in the art can be used in the preparation of the injection, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acid esters of polyethylene sorbitan. In addition, conventional solvents, buffers, and analgesics can also be added.

[0048] In the present invention, the method of administration of the pharmaceutical composition is not particularly limited. Various dosage forms can be selected for administration based on the patient's age, gender, and other conditions and symptoms. For example, tablets, pills, solutions, suspensions, emulsions, granules, or capsules can be administered orally; injections can be administered alone or mixed with an injectable delivery fluid (such as a glucose solution or an amino acid solution) for intravenous injection; and suppositories are administered rectally.

[0049] On the other hand, the present invention also provides a method for inhibiting the activity of mTOR and calcineurin in vivo or in vitro, which comprises contacting mTOR and calcineurin with an effective amount of a compound of formula (1), formula (2), formula (3) and / or a pharmaceutically acceptable salt thereof.

[0050] On the other hand, the present invention also provides a method for inhibiting the activity of mTOR and calcineurin in vivo or in vitro, which comprises contacting mTOR and calcineurin with a pharmaceutical composition in an amount effective to inhibit the activity of mTOR and calcineurin, wherein the pharmaceutical composition comprises a compound of formula (1), formula (2), or formula (3) (e.g., any compound herein) and / or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0051] On the other hand, the present invention also provides a method for treating a disease in an individual that responds to the inhibition of mTOR and calcineurin, which comprises administering to an individual in need thereof a compound of formula (1), formula (2), or formula (3) and / or a pharmaceutically acceptable salt thereof in an amount that can effectively inhibit mTOR and calcineurin in the individual.

[0052] On the other hand, the present invention also provides a method for treating a disease in an individual that responds to the inhibition of mTOR and calcineurin, comprising administering to an individual in need thereof a pharmaceutical composition in an amount that can effectively inhibit mTOR and calcineurin in the individual, wherein the pharmaceutical composition comprises a compound of formula (1), formula (2), or formula (3) and / or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0053] The present invention also provides the use of the compounds of formula (1), formula (2), formula (3) or their pharmaceutically acceptable salts, racemic mixtures, hydrates, solvates, prodrugs, enantiomers, diastereomers, tautomers, or pharmaceutical compositions in the preparation of mTOR inhibitors and calcineurin inhibitors.

[0054] The present invention also provides the use of the compounds of formula (1), formula (2), and formula (3), or pharmaceutically acceptable salts thereof, racemic mixtures, hydrates, solvates, prodrugs, enantiomers, diastereomers, tautomers thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for treating diseases responsive to inhibition of mTOR or calcineurin. The diseases include inflammatory diseases, autoimmune diseases, cancer, and organ transplantation.

[0055] Inflammatory diseases refer to pathological conditions that result in an inflammatory response, particularly due to neutrophil chemotaxis. Examples of such diseases include inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); ischemia-reperfusion injury, including tissue reperfusion injury caused by surgery, myocardial ischemia such as myocardial infarction, cardiac arrest, postoperative reperfusion after cardiac surgery, and abnormal coronary vasoconstriction after percutaneous transluminal coronary angioplasty; tissue reperfusion injury after stroke and abdominal aortic aneurysm surgery; cerebral edema secondary to stroke; cranial trauma; hemorrhagic shock; ventricular asphyxia; adult respiratory distress syndrome; acute lung injury; Behçet's disease; dermatomyositis; polymyositis; multiple sclerosis; dermatitis; meningitis Inflammation; encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoid arthritis; Sjögren's syndrome; vasculitis; diseases involving leukocytic infiltration; inflammatory diseases of the central nervous system secondary to sepsis or trauma, multiple organ injury syndrome: alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerulonephritis, sepsis, sarcoidosis; immunopathological reactions caused by tissue / organ transplantation; lung inflammation, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis, and cystic fibrosis.

[0056] Autoimmune diseases are diseases or conditions caused by an immune response to self-antigens, resulting in damage to the body's own tissues or organs. Examples of autoimmune diseases include, but are not limited to, chronic obstructive pulmonary disease, allergic rhinitis, lupus erythematosus, myasthenia gravis, multiple sclerosis (MS), rheumatoid arthritis, psoriasis, inflammatory bowel disease, asthma, idiopathic thrombocytopenic purpura, and myeloproliferative disorders such as myelofibrosis and polycythemia vera / essential thrombocythemia myelofibrosis.

[0057] The inflammatory diseases and autoimmune diseases include rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), systemic vasculitis, allergic rhinitis, asthma, systemic lupus erythematosus, Sjögren's syndrome, pemphigus, multiple sclerosis, psoriasis, Hashimoto's thyroiditis, type I diabetes, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, scleroderma, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, autoimmune hepatitis, thyroid autoimmune disease, idiopathic thrombocytopenic purpura, etc.

[0058] The cancer includes, but is not limited to, solid tumors or hematological malignancies, including cancers of the skin, tissues, organs, bones, cartilage, blood, and blood vessels, including both primary cancers and metastatic cancers. Non-limiting examples of solid tumors include pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen-independent prostate cancer; kidney cancer, including, for example, metastatic renal cell carcinoma; hepatocellular carcinoma; lung cancer, including, for example, non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and lung adenocarcinoma; ovarian cancer, including, for example, progressive epithelial carcinoma or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, for example, head and neck squamous cell carcinoma; skin cancer, including, for example, malignant melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, for example, gliomas, anaplastic oligodendrogliomas, adult glioblastoma multiforme, and adult anaplastic astrocytomas; bone cancer; soft tissue sarcomas; and thyroid cancer.

[0059] The organ transplant includes but is not limited to kidney transplant, liver transplant, heart transplant, lung transplant, pancreas transplant, small intestine transplant, upper limb transplant, face transplant, uterus transplant, cornea transplant, skin transplant, bone transplant, blood vessel transplant, bone marrow transplant, testicle transplant, and ovary transplant.

[0060] In addition, the compounds of formula (1), formula (2), and formula (3) described herein and / or their pharmaceutically acceptable salts can be used in combination with other active ingredients for the treatment of inflammatory diseases, autoimmune diseases, cancer, and organ transplantation. The compounds of formula (1), formula (2), and formula (3) and / or their pharmaceutically acceptable salts can be used separately from other active ingredients or prepared into a compound preparation. Other active ingredients are those known to be effective in treating mTOR and calcineurin-mediated diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The drug concentrations in different tissues (liver, spleen, lung, kidney, pancreas, and peripheral blood) in the immunosuppressive activity test were measured;

[0062] Figure 2 The results of routine blood tests in the immunosuppressive activity test;

[0063] Figure 3 These are the biochemical test results in the immunosuppressive activity test. DETAILED DESCRIPTION

[0064] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The operating methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0065] Example 1: Preparation of Everolimus-43-O-(2-(4-(2-((((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetyl)1

[0066]

[0067] Synthesis of A1: Dissolve isopropyl alanine hydrochloride (1.67 g, 10.0 mmol) and phenylphosphine dichloride (2.09 g, 10.0 mmol) in 20 ml of anhydrous dichloromethane. Protect the reaction flask with argon and place it in a cold trap at -30°C. Then, slowly add 1.5 ml of triethylamine dropwise. A large amount of white solid precipitates. After the addition is complete, bring the reaction to room temperature and stir for 30 min. Then, add 3-butene-1-ol (0.7 g, 10.0 mmol) to the reaction flask, followed by 4-dimethylaminopyridine (1.22 g, 10.0 mmol) dissolved in 5 ml of anhydrous dichloromethane. Stir at room temperature for 30 min. The reaction solution is diluted with dichloromethane, washed with 1 M hydrochloric acid solution, dried over anhydrous sodium sulfate, and concentrated by column chromatography using PE / EA (v / v, 2:1) to obtain A (2.5 g, 74%) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ7.36–7.28(m,2H),7.26–7.19(m,2H),7.19–7.12(m,1H),5.08–4.94(m,1H),4.24–4.14(m,2H) ,4.05–3.92(m,1H),3.80–3.66(m,1H),2.65–2.53(m,2H),2.05–1.99(m,1H),1.42–1.33(m,3H),1.28–1.17(m,6H). 13C NMR (101MHz, CDCl3) δ172.9,150.6,129.5,124.8,120.1,79.5,70.3,69.1,64.5,50.3,50.2,21.6,21.5,20.9.

[0068] Synthesis of A3: Dissolve A2 (250 mg, 0.26 mmol) in 5 ml of anhydrous dichloromethane, add 0.2 ml of triethylamine, and place the reaction tube under argon in an ice bath. Dissolve chloroacetyl chloride (60 mg, 0.54 mmol) in anhydrous dichloromethane and slowly add dropwise to the reaction tube. After the addition is complete, bring the reaction tube to room temperature and stir for 30 minutes. The reaction solution is diluted with dichloromethane, washed with water, dried over sodium sulfate, and concentrated by column chromatography to separate PE / EA (v / v, 1:1) to yield a white solid (170 mg, 57%). 1 H NMR (500MHz, CDCl3) δ6.43–6.23(m,1H),6.22–6.09(m,1H),6.05–5.86(m,1H),5.58–5.51(m,1H),5.50–5.39(m,1H),5.32–5.23(m,1H),5. 21–5.13(m,1H),4.38–4.27(m,2H),4.24–4.05(m,4H),3.90–3.76(m,3H),3.74(d,J=5.8Hz,1H),3.72–3.63(m,1H),3.62–3.53(m,1H),3.4 8–3.38(m,5H),3.38–3.28(m,3H),3.21–3.08(m,4H),3.08–2.99(m,1 H),2.77–2.68(m,1H),2.63–2.55(m,1H),2.37–2.25(m,2H),2.04–1. 91(m,4H),1.85(m,1H),1.80–1.69(m,6H),1.68–1.58(m,6H),1.56–1 .40(m,4H),1.38–1.17(m,8H),1.17–0.82(m,16H),0.76–0.64(m,1H). 13C NMR (126MHz, CDCl3) δ208.2,169.2,167.7,166.7,140.1,136.0,133.6,130.1,129.5, 126.6,126.4,98.5,84.8,84.3,83.2,77.1,76.8,75.6,67.6,67.2,65.8,60.4,59.3, 57.8,55.8,51.2,46.5,44.2,41.4,40.8,40.2,38.3,36.3,35.1,33.7,33.1,32.9,31.7,31.2,30.1,27.2,27.0,25.3,21.5,20.6,16.2,16.0,15.8,14.2,13.7,13.2,10.1.

[0069] Synthesis of A4: A3 (170 mg, 0.16 mmol) was dissolved in 5 mL of DMF, and sodium azide (30 mg, 0.46 mmol) and potassium iodide (1 mg, 0.01 mmol) were added. The mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with water, extracted with ethyl acetate, dried over sodium sulfate, concentrated, and then separated by column chromatography using PE / EA (v / v, 1:1) to afford a white solid (160 mg, 93%). 1 HNMR(500MHz, CDCl3)δ6.43–6.28(m,1H),6.27–6.05(m,1H),6.04–5.86(m,1H),5.59–5.48(m,1H),5.47–5.37 (m,1H),5.37–5.09(m,2H),4.77(s,1H),4.51–4.20(m,4H),4.23–4.10(m,1H),3.94–3.78(m,4H),3.78–3.63( m,1H),3.61–3.53(m,1H),3.51–3.27(m,7H),3.24–2.94(m,4H),2.77–2.68(m,1H),2.64–2.55(m,1H),2.39–2 .25(m,2H),2.11–1.92(m,4H),1.91–1.40(m,26H),1.40–1.17(m,7H),1.13–0.79(m,12H),0.76–0.64(m,1H). 13C NMR (126MHz, CDCl3) δ208.2,169.2,168.3,166.7,140.1,136.0,133.6,130.2,1 29.5,126.7,126.4,98.5,84.3,83.2,75.6,67.7,67.2,65.4,59.4,57.8,55.9, 51.2,50.3,46.5,44.2,41.4,40.6,40.2,38.9,38.3,36.2,35.1,33.7,33.0,31.7,31.2,30.1,27.2,27.0,25.3,21.5,20.6,16.2,16.0,15.9,13.7,13.1,10.2.

[0070] Synthesis of compound 1: A4 (160 mg, 0.15 mmol) was dissolved in 5 ml of acetonitrile, and A1 (100 mg, 0.29 mmol) was dissolved in 1 ml of acetonitrile and then injected into a reaction tube. Cuprous iodide (10 mg, 0.05 mmol) was added to the reaction tube, followed by argon protection. 0.2 ml of triethylamine was added to the reaction solution and stirred at room temperature for 30 min. The reaction solution was directly concentrated, mixed with silica gel powder, and then separated by column chromatography using DCM / MeOH (v / v, 50:1) to obtain a white solid (150 mg, 37%). 1H NMR(500MHz, CDCl3)δ7.47–7.39(m,1H),7.34–7.28(m,2H),7.23–7.12(m,3H) ,6.43–6.26(m,1H),6.22–6.09(m,1H),6.07–5.87(m,1H),5.59–5.48(m,1H),5 .47–5.34(m,1H),5.33–5.23(m,1H),5.21–5.09(m,3H),5.04–4.95(m,1H),4.7 4(s,1H),4.47–4.26(m,4H),4.24–4.14(m,1H),4.00–3.90(m,1H),3.90–3.77( m,2H),3.77–3.53(m,3H),3.50–3.37(m,5H),3.36–3.29(m,1H),3.23–2.99(m, 7H),2.88–2.65(m,2H),2.64–2.54(m,1H),2.38–2.25(m,1H),2.24–2.12(m,1H ),2.11–1.91(m,4H),1.89–1.56(m,16H),1.54–1.40(m,4H),1.39–1.16(m,17H ),1.16–1.02(m,6H),1.01–0.93(m,4H),0.93–0.83(m,8H),0.77–0.64(m,1H). 13 C NMR (126MHz, CDCl3) δ169.2,129.6,126.4,124.8,123.4,120.4,120.3,98. 5,84.8,83.2,77.1,69.2,69.1,67.5,67.2,65.8,65.6,59.3,57.7,55.8,51 .3,50.7,50.4,50.3,46.5,41.4,33.1,32.0,31.5,30.1,27.1,27.0,25.2,22.6,21.7,21.6,21.5,21.0,20.9,16.2,16.0,15.8,14.1,13.7,13.2,10.2.

[0071] Example 2: Everolimus-43-O-(2-(4-(2-((((S)-1-methoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetyl)2

[0072]

[0073] The preparation method is similar to that of Example 1, except that the reactants are replaced.

[0074] White solid; 1 H NMR (400 MHz, CDCl3) δ 7.47–7.38 (m, 1H), 7.34–7.27 (m, 2H), 7.22–7.12 (m, 3H), 6.43–6.21 (m, 1H), 6.19–6.08 (m, 1H), 5.99–5.86 (m, 1H), 5.57–5.45 (m, 1H), 5.43–5.37 (m, 1H), 5.30–5.23 (m, 1H), 5.19–5.09 (m, 3H), 4.74 (s, 1H), 4.45–4.27 (m, 4H), 4.23–4.15 (m, 1H), 4.05–3.94 (m, 1H), 3.91–3.77 (m, 2H), 3.76–3.53 (m, 3H), 3.48–3.36 (m, 6H), 3.35–3.27 (m, 4H), 3.21–3.10 (m, 5H), 3.09–2.99 (m, 1H), 2.76–2.64 (m, 2H), 2.61–2.53 (m, 1H), 2.37–2.28 (m, 2H), 2.10–1.90 (m, 2H), 1.89–1.72 (m, 2H), 1.71–1.67 (m, 5H), 1.66–1.56 (m, 7H), 1.54–1.40 (m, 3H), 1.38–1.29 (m, 5H), 1.28–1.17 (m, 2H), 1.16–1.01 (m, 10H), 1.00–0.81 (m, 17H), 0.76–0.64 (m, 1H). 13 C NMR (101 MHz, CD3OD) δ 212.6, 207.8, 169.6, 167.9, 167.0, 139.4, 137.9, 137.0, 132.7, 131.0, 129.4, 127.7, 127.1, 124.7, 124.4, 120.2, 120.2, 99.3, 85.8, 83.4, 83.1, 83.0, 76.2, 74.1, 67.2, 65.3, 57.0, 56.8, 55.0, 51.4, 51.3, 50.4, 50.2, 48.3, 48.1, 47.8, 47.6, 47.4, 47.2, 47.0, 46.5, 45.9, 44.1, 40.4, 40.1, 38.7, 36.1, 35.8, 35.1, 34.3, 33.7, 32.8, 31.1, 30.0, 26.4, 24.8, 24.7, 20.7, 20.5, 19.1, 14.7, 14.2, 12.8, 12.6, 9.6, 7.9.

[0075] Example 3: Everolimus-43-O-(2-(4-(2-((((S)-1-tert-butoxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetyl)3

[0076]

[0077] The preparation method is similar to that of Example 1, except that the reactants are replaced.

[0078] White solid; 1 H NMR (400MHz, CDCl3) δ7.35–7.28(m,3H),7.25–7.12(m,3H),6.43–6.26(m,1H),6.19–6.09(m,1H),6.00–5.87(m,1H),5.58–5.47(m,1H),5.44–5 .38(m,1H),5.31–5.25(m,1H),5.20–5.10(m,3H),4.75(s,1H),4.43–4. 28(m,2H),4.24–4.13(m,3H),3.96–3.78(m,4H),3.76–3.63(m,1H),3.6 2–3.53(m,2H),3.47–3.37(m,4H),3.36–3.29(m,2H),3.19–3.01(m,4H) ,2.76–2.68(m,1H),2.63–2.54(m,3H),2.38–2.28(m,1H),2.06–1.95(m ,2H),1.82–1.68(m,6H),1.67–1.57(m,7H),1.38–1.29(m,12H),1.28–1 .19(m,3H),1.17–1.02(m,10H),1.01–0.82(m,24H),0.76–0.67(m,1H). 13C NMR (101MHz, CD3OD) δ212.6,207.8,198.46,172.8,169.6,167.9,167.0,150.9,139.4,137.9,137.0,132.7,131.0,129.7,129.4,129.4,1 27.7,127.1,126.2,124.7,120.2,120.1,120.0,99.4,85.8,83.4,83 .0,81.2,79.2,76.2,74.1,70.2,70.1,67.2,67.1,65.3,64.8,64.7,5 7.0,56.8,55.0,51.3,50.9,50.7,48.3,48.1,47.9,47.7,47.4,47.2,47.0,45.9,44.1,40.4,40.1,39.8,38.7,36.1,35.8,35.1,34.4,34.3,33.7,32.8,31.1,30.0,29.7,26.9,26.8,26.5,24.9,24.7,20.8,20.5,19.9,19.8,19.7,19.3,19.2,14.8,14.7,14.3,12.9,12.6,9.6.

[0079] Example 4: Everolimus-43-O-(2-(4-(2-((((S)-1-benzyloxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetyl)4

[0080]

[0081] The preparation method is similar to that of Example 1, except that the reactants are replaced.

[0082] White solid; 1H NMR(400MHz,CDCl3)δ7.38–7.27(m,7H),7.23–7.11(m,3H),6.43–6.26(m,1H),6.19–6.09(m,1H),6.00–5.87(m,1H),5.58–5.48(m,1H),5.44–5.38(m,1H),5.32–5.24(m,1H),5.20–5.07(m,5H),4.75(s,1H),4.40–4.25(m,4H),4.21–3.99(m,1H),3.90–3.73(m,3H),3.71–3.53(m,2H),3.47–3.28(m,10H),3.18–3.00(m,6H),2.76–2.68(m,2H),2.62–2.49(m,1H),2.38–2.28(m,2H),2.07–1.93(m,1H),1.81–1.68(m,5H),1.67–1.54(m,5H),1.53–1.44(m,2H)1.43–1.30(m,6H),1.29–1.19(m,2H),1.18–1.02(m,11H),1.01–0.82(m,20H),0.76–0.64(m,1H). 13 C NMR(101MHz,CD3OD)δ212.6,207.8,198.5,169.6,167.9,167.0,139.4,137.9,137.0,136.0,132.7,131.0,129.4,128.3,128.0,127.7,127.1,126.2,124.7,120.2,99.4,85.8,83.4,83.1,83.0,76.2,74.1,70.2,67.2,66.6,65.3,57.0,56.8,55.0,51.3,50.2,48.3,48.1,47.9,47.6,47.4,47.2,47.0,45.9,44.1,40.4,40.1,39.8,38.7,36.1,35.8,35.1,34.3,33.7,32.8,31.4,31.1,30.0,29.7,28.8,26.4,24.8,24.7,22.4,21.6,20.7,20.5,19.0,14.7,14.6,14.3,13.1,12.9,12.6,10.4,9.6.

[0083] Example 5: Cyclosporin-O-(2-(4-(2-((((S)-1-benzyloxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetyl)5

[0084]

[0085] The preparation method is similar to that of Example 1, except that the reactants are replaced.

[0086] White solid; 1 H NMR (400MHz, CDCl3) δ8.00–7.94(m,1H),7.73–7.45(m,1H),7.37–7.30(m,2H ),7.27–7.21(m,3H),7.20–7.14(m,3H),5.74–5.68(m,1H),5.50–5.46(m,1H) ,5.39–5.32(m,2H),5.17–4.98(m,5H),4.88–4.80(m,1H),4.78–4.63(m,2H), 4.57–4.48(m,1H),4.25–4.15(m,3H),4.06–3.95(m,1H),3.90–3.78(m,2H),3 .69–3.59(m,1H),3.55–3.49(m,3H),3.44–3.37(m,3H),3.31–3.18(m,3H),3 .16–3.08(m,6H),2.82–2.69(m,6H),2.65–2.56(m,3H),2.48–2.38(m,2H),2. 19–1.96(m,6H),1.82–1.58(m,6H),1.54–1.34(m,9H),1.31–1.20(m,12H),1. 13–1.00(m,12H),0.99–0.93(m,12H),0.92–0.80(m,18H),0.77–0.70(m,3H). 13C NMR (101MHz, CDCl3) δ173.5,171.6,171.2,170.4,170.1,129.7,126.3,124.9,120.3,79.6,77.4,77 .1,76.8,74.8,70.4,69.3,64.6,58.8,57.9,57.6,55.4,50.4,48.8,48.6,48.3,45.2,40.7,39.5,3 9.0,37.4,36.0,35.7,34.0,31.6,31.2,29.8,29.6,29.1,25.4,25.0,24.9,24.7,24.5,23.9,23.8,23.7,23.5,23.4,21.9,21.8,21.7,21.2,20.7,20.3,19.9,18.8,18.4,18.2,18.0,16.8,16.0,9.9.

[0087] Example 6: Rapamycin-43-O-(2-(4-(2-((((S)-1-benzyloxy-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)ethyl)-1H-1,2,3-triazol-1-yl)acetyl)6

[0088]

[0089] The preparation method is similar to that of Example 1, except that the reactants are replaced.

[0090] White solid; 1H NMR (400MHz, CDCl3) δ7.35–7.27(m,3H),7.24–7.11(m,3H),6.43–6.25(m,1H),6.19–6.09(m,1H),6.00–5.86(m,1H),5.58–5.45(m,1H),5.44–5 .38(m,1H),5.32–5.24(m,2H),5.20–5.10(m,1H),5.05–4.95(m,1H),4. 83–4.72(m,1H),4.30–4.15(m,1H),4.00–3.82(m,1H),3.78–3.62(m,1H) ,3.61–3.53(m,1H),3.48–3.28(m,9H),3.19–3.10(m,5H),2.78–2.66(m ,2H),2.63–2.52(m,1H),2.38–2.28(m,2H),2.16–1.92(m,3H),1.91–1.8 2(m,1H),1.81–1.68(m,9H),1.67–1.57(m,9H),1.56–1.41(m,3H),1.40 –1.19(m,6H),1.18–1.02(m,8H),1.01–0.93(m,9H),0.92–0.81(m,15H). 13 C NMR (101MHz, CD3OD) δ150.9,131.0,129.4,124.8,120.3,80.7,68.8,68.7,54.9,48.3,48.1,47.9,47.7,47.6,47. 5,47.4,47.3,47.2,47.0,40.2,38.8,35.2,34.4,33.2,32.6,29.3,26.6,21.2,20.7,19.2,14.8,14.4,13.3,12.8.

[0091] The beneficial effects of the present invention are demonstrated by the following test examples.

[0092] Immunosuppressive activity test

[0093] 1. Experimental Method 1: Drug Efficacy Evaluation Experiment

[0094] Pharmacokinetic experiments were conducted by taking everolimus (EVE) as the control group and compound 1 (M-EVE) prepared in the example as the experimental group.

[0095] Pharmacokinetic study methods:

[0096] Six mice (half male and half female) were administered intraperitoneally at a dose of 1 mg / kg / day. Blood samples were collected from various tissues (liver, spleen, lung, kidney, pancreas, and peripheral blood) at 0.5, 1, 2, 4, 12, 24, and 48 hours after administration. Immediately after blood collection, the tubes were gently inverted at least five times to ensure thorough mixing and then placed on ice. Blood was anticoagulated with heparin and centrifuged at 8000 rpm for 5 minutes to separate serum from red blood cells. Serum was aspirated and transferred to a 2 ml polypropylene tube using a pipette and stored at -40°C until analysis by LC-MS. High-concentration samples were diluted with blank mouse plasma. For analysis, 50 μl of plasma was added with 2.5 μl of Meth-H2O and 5 μl of internal standard solution (20 ng / ml). Protein was precipitated with 150 μl of methanol and centrifuged at 13000 rpm. The upper organic phase was collected and filtered through a microporous membrane, and 2 μl of the sample was injected into LC-MS for analysis.

[0097] Blood routine test method:

[0098] The orbital venous plexus blood collection method was adopted. When collecting blood from the orbit, the mouse was anesthetized and fixed, and the venous plexus was pierced from the inner canthus with a capillary glass tube. The blood naturally flowed into the blood collection tube. After blood collection, pressure was applied to stop bleeding for 30 seconds. Immediately after blood collection, the blood was injected into the blood collection tube containing EDTA-K2 anticoagulant and gently inverted 8-10 times to avoid hemolysis. A fully automatic blood cell analyzer was used for detection, and matching calibration materials were used to calibrate parameters such as white blood cells, red blood cells, and platelets. The sample was returned to room temperature before detection, and after mixing again, 85μl of whole blood was drawn into the injection port for detection. The detection parameters include total white blood cell count (WBC), absolute lymphocyte count (Lymphocytes), platelet count (Platelets), red blood cell count (RBC), and hemoglobin concentration (HGB).

[0099] Biochemical detection methods:

[0100] Blood was collected using the orbital blood sampling method and placed in a standard vacuum tube. After standing at room temperature for 30 minutes, it was centrifuged at 3000 rpm for 10 minutes to separate the serum. Serum was analyzed using a fully automated biochemical analyzer and accompanying reagents. Test parameters included albumin (ALB), total protein (Total Protein), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), glucose (Glucose), and creatinine (Cr).

[0101] The efficacy of the drug is evaluated through indicators such as liver / kidney function, glucose and lipid metabolism, and blood routine.

[0102] 2. Experimental results 1:

[0103] Figure 1is the drug concentration in different tissues (liver, spleen, lung, kidney, pancreas, peripheral blood), Figure 2 The results of routine blood tests are: Figure 3 For biochemical test results.

[0104] Depend on Figure 1 It can be seen that compared with the EVE group, M-EVE showed an obvious liver-targeting trend, with higher concentrations in the liver and lower drug concentrations in other tissues.

[0105] Depend on Figure 2 It can be seen that both EVE and M-EVE groups reduced the proportion of white blood cells and lymphocytes, and the degree of inhibition in the M-EVE group was relatively mild.

[0106] Depend on Figure 3 It can be seen that after 2 weeks of administration, the levels of ALT, TC, GLU, and CR in the EVE group changed significantly, while the changes in the M-EVE group were smaller, indicating that M-EVE has lower toxic and side effects.

[0107] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An immunosuppressant based on protide prodrug modification, characterized in that: One of the following compounds: Compound of formula (1) or a pharmaceutically acceptable salt, racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof; in: R 1 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl; R 2 Selected from C 1-6 Alkyl, hydrogen; R 3 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl; L is selected from C2~C 12 At least one of an aliphatic chain or a polyethylene glycol chain, which is optionally interrupted by zero, one or more substituents selected from the group consisting of: -O-, -COO-, -CONH-, triazolyl; Compound of formula (2) or a pharmaceutically acceptable salt, racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof; in: R 1 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl; R 2 Selected from C 1-6 Alkyl, hydrogen; R 3 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl; L is selected from C2~C 12 At least one of an aliphatic chain or a polyethylene glycol chain, which is optionally interrupted by zero, one or more substituents selected from the group consisting of: -O-, -COO-, -CONH-, triazolyl; Compound of formula (3) or a pharmaceutically acceptable salt, racemic mixture, hydrate, solvate, prodrug, enantiomer, diastereomer, or tautomer thereof; in: R 1 Selected from C 1-6 Alkyl, phenyl, benzyl, which is optionally substituted by zero, one or more substituents selected from the group consisting of halogen, amino, hydroxy, -OC 1-6 Alkyl, C 1-6 alkyl.; R 2 Selected from C 1-6 Alkyl, hydrogen; R 3 Selected from C 1-6 Alkyl, phenyl, benzyl, which are optionally substituted by zero, one or more substituents selected from the following: halogen, amino, hydroxyl, -OC 1-6 Alkyl, C 1-6 alkyl; L is selected from C2~C 12 At least one of an aliphatic chain or a polyethylene glycol chain, which is optionally interrupted by zero, one or more substituents selected from the group consisting of: -O-, -COO-, -CONH-, and triazolyl.

2. The immunosuppressant based on protide prodrug modification according to claim 1, characterized in that: In formulas (1) to (3), L is selected from any one of the following structures:

3. The immunosuppressant based on protide prodrug modification according to claim 1, characterized in that: The compounds of formula (1), formula (2) and formula (3) are selected from any one of the following compounds:

4. A pharmaceutical composition, characterized in that The invention comprises a compound of formula (1), formula (2), or formula (3) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, a racemic mixture, a hydrate, a solvate, a prodrug, an enantiomer, a diastereomer, or a tautomer thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

5. Use of the protide-based prodrug-modified immunosuppressant according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 3 in the preparation of a drug.

6. The use according to claim 5, characterized in that The drug is used to treat diseases that respond to everolimus, rapamycin, and cyclosporine.

7. The use according to claim 6, characterized in that The disease is one or more of renal cancer, liver cancer, pancreatic neuroendocrine tumor, breast cancer, giant cell astrocytoma, renal angiomyolipoma, kidney transplantation, liver transplantation, heart transplantation, lung transplantation rejection, autoimmune disease, graft-versus-host disease, metabolic disease, inflammatory disease, and nephrotic syndrome.

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