Diketopiperazine methylated derivative as well as preparation method and application thereof

Abstract

The invention discloses a diketopiperazine methylated derivative as well as a preparation method and application thereof, and belongs to the technical field of medicines. The technical problem to be solved is to provide a diketopiperazine methylated derivative with a new structure and a preparation method thereof. The key point of the technical scheme is that the diketopiperazine methylated derivative is a compound as shown in the following formula I, an isomer, a prodrug, a stable isotope derivative, a pharmaceutically acceptable salt or a mixture thereof, and in the formula I, R1 and R2 are independently selected from H and substituted or unsubstituted alkyl; l1 and L2 are respectively and independently selected from substituted or unsubstituted alkylene; x1 and X2 are respectively and independently selected from N, O or S; and L3 is independently selected from an alkenylene group and a C5-C8 bridged ring group. Formula I

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to diketopiperazine methylated derivatives, their preparation methods, and applications. Background Technology

[0002] For understanding the technical content of this invention: Drug delivery systems have always been an important area of ​​research in pharmaceutical formulation. Traditional methods are affected by the pH environment of the digestive tract and various enzymes, leading to the rapid destruction or inactivation of some bioactive substances, such as calcitonin, insulin, and mucopolysaccharides, in the gastrointestinal tract. Furthermore, due to the inherent physicochemical properties of drugs, some sensitive drugs are prone to degradation, and poorly soluble drugs have low bioavailability. Developing efficient delivery systems can enhance drug efficacy and reduce toxic side effects. Heterocyclic compounds have always been one of the most important classes of organic compounds. In organisms, they are the main components of living cells and participate in important biochemical processes. For this reason, heterocyclic compounds possess a wide range of pharmacological activities; nearly half of the currently available therapeutic drugs contain heterocyclic structures. Piperazine (PP) is an important class of biologically active N-heterocyclic natural products, composed of a six-membered nitrogen-containing heterocycle with two nitrogen atoms in opposite positions on the ring. Piperazine is one of the most widely found structures in drugs and has promising applications in biomedical fields such as drug design. This is because the nitrogen atom at the 4-position of piperazine can act as a basic amine, while the nitrogen atom at the 1-position can introduce hydrogen bond acceptors or hydrophobic groups through reactions with other compounds, thereby regulating drug interactions and hydrophilicity / hydrophobicity. Many piperazine compounds possess important pharmacological properties and show broad application prospects in antitumor, antibacterial, anti-inflammatory, and antioxidant activities. In addition, piperazine compounds have the ability to open tight junctions between cells, making them a highly efficient and low-toxicity penetration enhancer widely used in drug delivery.

[0003] Diketopiperazine compounds are a novel type of material that can be used to form drug-loaded microspheres by loading or adsorbing drugs onto their surfaces, thereby improving drug stability and permeability. Drug-loaded microspheres using diketopiperazine compounds as carriers have small diameters, large surface areas, high drug loading capacities, and are simple to manufacture. They are suitable for the preparation of various types of drugs, such as cationic drugs with API molecular weights of 500-140,000 Da, anionic drugs, hydrophilic / lipophilic drugs, peptides, and proteins.

[0004] Technosphere® technology, which uses fumarodiketopiperazine (FDKP, CAS: 176738-91-3) as a carrier to prepare microspheres, is a novel drug delivery technology. Mannkind's Afrezza rapid-acting insulin, produced using this technology, was approved by the FDA in 2014 and is currently the only inhaled insulin formulation on the market, fully demonstrating the transmucosal permeation-enhancing function of diketopiperazine compounds as drug carriers.

[0005]

[0006] Relevant patent documents retrieved: This document, published in China (CN115991678A) on April 21, 2023, discloses a diketopiperazine compound, its preparation method, and its applications. This invention provides a diketopiperazine compound as shown in the formula, or a pharmaceutically acceptable salt thereof, which can be used as self-assembly loaded drug microspheres to achieve efficient drug delivery.

[0007]

[0008] Relevant non-patent literature retrieved: The journal or book title is "Doctoral Dissertation of South China University of Technology," and the article title is "Application Research of Polymer Carriers Constructed Based on Piperazine Derivatives in Intracellular Protein Delivery." The publication date is April 2023. This article discloses a series of cationic polymer carriers based on piperazine and its derivatives, and explores their effectiveness and structure-activity relationship in intracellular protein delivery. Based on the modification of polymers with piperazine and its derivatives, a series of efficient and safe intracellular protein delivery carriers were obtained. Through the integration of piperazine molecules with other functional elements, a series of protein delivery systems meeting different application requirements were obtained.

[0009] Common protein delivery carriers in existing technologies include cationic liposomes, dendritic macromolecules, cell-penetrating peptides, and commercial reagents (such as Lipofectamine). Piperazine / DKP-based polymeric carriers may offer the following potential advantages: biodegradability (avoiding long-term toxicity), good water solubility, ease of structural modification through chemical synthesis, and tunable stimulus-responsive release behavior.

[0010] In summary, developing a novel diketopiperazine methylated derivative that can be better applied to drug delivery systems is a key research focus for researchers in this field.

[0011] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: 1. The challenge of balancing vector biosafety and transfection efficiency: While high-density cationic groups (such as protonated nitrogen atoms in piperazine) can effectively compress negatively charged proteins and promote cellular uptake, excessive positive charge can also lead to significant cytotoxicity and may trigger strong nonspecific serum protein adsorption and immune responses. How to minimize vector cytotoxicity while ensuring high-efficiency intracellular delivery is one of the core challenges of this invention.

[0012] 2. Insufficient endosome escape efficiency: After protein drugs are stored in cells, the vast majority become trapped in acidic endosomes and are eventually degraded, rendering them ineffective. Designing a delivery vector structure with efficient endosome escape capabilities (such as through the "proton sponge effect") is crucial for successful delivery. A single piperazine structure may not be sufficient for efficient escape in all cell types; precise structural modifications of piperazine derivatives are needed to optimize their buffering capacity.

[0013] 3. The contradiction between the stability of the carrier-protein complex and its dissociation kinetics: The carrier needs to form a stable complex with the protein to protect it from degradation in the extracellular environment. However, once inside the cytoplasm, the complex must be able to dissociate promptly and completely to release the active protein. Designing a carrier structure that can intelligently respond to the intracellular environment (such as pH, reduced glutathione) to achieve controlled release is extremely challenging.

[0014] 4. Synthetic Challenges of Diketopiperazine (DKP) Skeleton Functionalization: Synthesizing novel diketopiperazine methylated derivatives from piperazines often involves cumbersome synthetic routes, low yields, or the use of expensive and toxic reagents, making large-scale production difficult. Ensuring controllable molecular weight distribution and well-defined structure of the final polymer is also a significant technical challenge.

[0015] 5. The contradiction between universality and specificity: Developing a "universal" vector to deliver proteins with different isoelectric points, molecular weights, and functions is an ideal goal. However, in reality, for a specific protein (such as a CRISPR-Cas9 nuclease or antibody), it may be necessary to adjust the vector's structural parameters (such as hydrophilicity and charge density) to achieve optimal results. Establishing a clear "structure-activity relationship" and designing accordingly requires extensive experimental screening and validation. Summary of the Invention

[0016] The purpose of this invention is to provide: A novel diketopiperazine methylated derivative, its preparation method and application, and related technologies, to solve technical problems such as providing a novel diketopiperazine methylated derivative or a combination thereof.

[0017] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0018] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0019] The definition of standard chemical terms can be found in the reference "Basic Organic Chemistry (Volumes 1 & 2), Xing Qiyi, Higher Education Press, 3rd Edition, 2005-06."

[0020] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0021] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic alkali or acid. When the compounds of the present invention are acidic, their corresponding salts can be readily prepared from inorganic or organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (copper and cuprous), iron, ferrous, lithium, magnesium, manganese (manganese and manganese), potassium, sodium, zinc, etc. Preferred are ammonium, calcium, magnesium, potassium, and sodium salts. Salts prepared from organic bases include primary, secondary, and tertiary amines derived from natural and synthetic sources. Pharmaceutically acceptable non-toxic organic bases that can form salts include arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, dicyclohexylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When the compounds of the present invention are basic, their corresponding salts can be readily prepared from inorganic or organic acids. Such acids include, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyethylsulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, viscous acid, nitric acid, pyric acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc.

[0022] As used herein, the term "prodrug" refers to any compound or conjugate that releases an active parent drug when administered to an animal or human subject. Prodrugs can be prepared by modifying functional groups present in a compound in a manner that allows the modification to be cleaved by the parent compound under normal operating conditions or in vivo. Prodrugs include compounds in which a hydroxyl, amino, thiosulfate, or carboxyl group is bonded to any group such that when administered to a mammalian subject, it can be cleaved to form a free hydroxyl, amino, thiosulfate, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of compounds containing alcohol and amine functional groups. The preparation and use of prodrugs are discussed in Thiguchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Proceedings of the American Chemical Society (ACS) Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated in full.

[0023] The term "isomer" as used in this article refers to stereoisomers, geometric isomers, tautomers, etc. The term "stereoisomer" refers to any of the various stereoisomer configurations (e.g., enantiomers, diastereomers, and racemates) of an asymmetric compound (e.g., a compound having one or more asymmetricly substituted carbon atoms or an "asymmetric center"). Compounds of this disclosure containing an asymmetric center can be separated by optical activity (enantiomers or diastereomers) or optical inactivation (racemate). The term "enantiomer" includes stereoisomer pairs that are not mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemate mixture". The term "diastereomer" refers to a stereoisomer having at least two asymmetric atoms that are not mirror images of each other. Certain compounds containing one or more asymmetric centers can produce enantiomers, diastereomers, or other stereoisomers, which can be defined as (R)- or (S)- with respect to absolute configuration at each asymmetric center according to the Cahn-Ingold-Prelog RS system. The term "or" can be used to indicate the absolute configuration of a resolved compound at the asymmetric center. Methods for preparing optically active forms from racemic mixtures are known in the art, such as by HPLC resolution or stereoselective synthesis.

[0024] The terms “geometric isomers” or “cis and trans isomers” refer to compounds that have the same formula but whose functional groups are rotated to different orientations in three-dimensional space.

[0025] The term "tautomer" encompasses proton transfer tautomers in isoprotonated states of compounds having the same formula and total charge. Examples of proton transfer tautomers include, but are not limited to, keto-enol pairs, amide-imine pairs, lactam-lactamimide pairs, enamine-imine pairs, and cyclic forms in which a proton may occupy two or more positions in a heterocyclic system, such as 1H-imidazole and 3H-imidazole, 1H-1,2,4-triazole, 2H-1,2,4-triazole and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. Tautomers may be in equilibrium or spatially locked into one form by appropriate substitution. Unless otherwise stated, the compounds disclosed herein identified as a particular tautomer form by name or structure are intended to include other tautomer forms.

[0026] The term "alkylene" itself, or as part of another substituent, refers to a divalent (di-residue) alkyl group. Examples of "alkylene" are, but not limited to, -CH2CH2CH2CH2-. Typically, the "alkylene" group is C1-C. 24 Alkylenes, and C1-C 10Alkylene, C1-C8 alkylene, C1-C6 alkylene, or C1-C4 alkylene. The “lower alkylene” group is C1-C6 alkylene, as well as C1-C4 alkylene. Where the alkylene is optionally substituted with one or more substituents, the one or more is typically 1, 2, 3, 4, or 5, and 1, 2, 3, or 4, and 1, 2, or 3, and 1 or 2, or 1, wherein the multiple substituents are chosen independently unless otherwise indicated. It should be understood that any substitution of the alkylene or alkylene substituted on other parts is linked at any available atom to provide a stable compound.

[0027] The term "alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group with a specific number of carbon atoms, including all isomers. Common abbreviations for alkyl groups include methyl ("Me" or CH3), ethyl ("Et" or CH2CH3), propyl ("Pr" or CH2CH2CH3), and butyl ("Bu" or CH2CH2CH2CH3), etc. For example, "C..." 1-6 Alkyl (or "C1-C6 alkyl") refers to a straight-chain or branched alkyl group having a specific number of carbon atoms, including all isomers.

[0028] In a first aspect, the present invention provides: a diketopiperazine methylated derivative, said diketopiperazine methylated derivative being a compound represented by Formula I, an isomer thereof, a prodrug, a stable isotopic derivative thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof:

[0029] Formula I R1 and R2 are each independently selected from H, substituted or unsubstituted alkyl groups; L1 and L2 are each independently selected from substituted or unsubstituted alkylene groups; X1 and X2 are each independently selected from N, O or S; L3 is independently selected from alkenyl groups and C5-C8 bridged cyclic groups.

[0030] Preferably, R1 and R2 are each independently selected from C1-C6 alkyl groups; L1 and L2 are each independently selected from C1-C6 alkylene groups; X1 and X2 are each independently selected from N or O; L3 is independently selected from alkenyl and C5-C7 bridged cyclic groups.

[0031] More preferably, R1 and R2 are each independently selected from methyl groups; L1 and L2 are each independently selected from C1-C5 alkylene groups; X1 and X2 are each independently selected from N; L3 is independently selected from alkenyl and C5-bridged cycloyl groups.

[0032] Preferably, the structural formula of the compound is: ; L3 and L4 are as described above.

[0033] More preferably, the compound is selected from the following structures: , , , .

[0034] Secondly, the present invention provides a method for preparing the diketopiperazine methylated derivatives described above, characterized in that the reaction equation is as follows:

[0035] S1: 2-Amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluorohexanoic acid (SM) reacts with m-cresol in the presence of a dehydrating agent and a solvent to undergo a dehydration and ring-closure reaction, yielding compound 1; S2: Compound 1 undergoes a deprotection reaction (deprotection of Cbz) under palladium-catalyzed hydrogenation to give compound 2; S3: Compound 2 undergoes a condensation reaction with an acid in the presence of a condensing agent to give compound 3; S4: Compound 3 undergoes a hydrolysis reaction to give compound 4.

[0036] Preferably, in step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to m-cresol is 1:3-10.

[0037] More preferably, in step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to m-cresol is 1:4-5.

[0038] Preferably, in step S1, the dehydrating agent is selected from at least one of P2O5, concentrated sulfuric acid, or calcium chloride.

[0039] More preferably, in step S1, the dehydrating agent is P2O5.

[0040] Preferably, in step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to the dehydrating agent is 1:0.1-1.0.

[0041] More preferably, in step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to the dehydrating agent is 1:0.3-0.5.

[0042] Preferably, in step S1, the temperature of the dehydration and ring-closing reaction is 100-200℃.

[0043] More preferably, in step S1, the temperature of the dehydration and ring-closing reaction is 150-180°C.

[0044] Preferably, in step S1, the solvent is selected from at least one of N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran.

[0045] More preferably, in step S1, the solvent is N-methylpyrrolidone.

[0046] Preferably, in step S2, the mass ratio of compound 1 to palladium on carbon is 1:0.01-0.2.

[0047] More preferably, in step S2, the mass ratio of compound 1 to palladium on carbon is 1:0.08-0.15.

[0048] Preferably, in step S2, the temperature of the deprotection reaction is 10-60°C.

[0049] More preferably, in step S2, the temperature of the deprotection reaction is 20-40°C.

[0050] Preferably, in step S3, the molar ratio of compound 2 to acid is 1:1.8-4.

[0051] More preferably, in step S3, the molar ratio of compound 2 to acid is 1:2-3.

[0052] Preferably, in step S3, the condensing agent is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), O-azabenzotriazole tetramethylurea hexafluorophosphate (HATU), or 1-hydroxybenzotriazole (HOBt).

[0053] More preferably, in step S3, the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).

[0054] Preferably, in step S3, the equivalent ratio of the acid to the condensing agent is 1.8-6:1.

[0055] More preferably, in step S3, the equivalent ratio of the acid to the condensing agent is 2-4:1.

[0056] Preferably, in step S4, the temperature of the hydrolysis reaction is 20-60°C.

[0057] More preferably, in step S4, the temperature of the hydrolysis reaction is 25-40°C.

[0058] Thirdly, the present invention provides the use of the diketopiperazine methylated derivatives described above in the preparation of pharmaceuticals for drug delivery.

[0059] Preferably, the drug comprises a compound represented by Formula I as a penetration enhancer, an isomer thereof, a prodrug, a stable isotopic derivative, a pharmaceutically acceptable salt or a mixture thereof, and a pharmaceutically acceptable carrier and active ingredient.

[0060] Preferably, the pharmaceutical composition comprises a composition suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any particular case depends on the specific subject, the nature and severity of the condition to which the active ingredient is administered. The pharmaceutical composition can be prepared by any method known in the field of pharmaceutics.

[0061] The active ingredient can be administered orally in solid or liquid dosage forms, such as capsules, tablets, lozenges, sugar lozenges, granules, and powders, and in liquid forms such as elixirs, syrups, emulsions, dispersions, and suspensions. The active ingredient can also be administered parenterally in sterile liquid dosage forms such as dispersions, suspensions, or solutions. Other dosage forms that can be used to administer the active ingredient include ointments, creams, drops, transdermal patches, or powders for topical administration; ophthalmic solutions or suspensions for ocular administration, i.e., eye drops; sprays or powder compositions for inhalation or intranasal administration; or creams, ointments, sprays, or suppositories for rectal or vaginal administration. Gelatin capsules contain the active ingredient and a powdered carrier, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products to provide sustained release of the drug over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from air, or they can be enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorants and flavoring agents to increase patient acceptability. Generally, water, suitable oils, saline solutions, aqueous solutions of dextran (glucose), and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain water-soluble salts of the active ingredient, suitable stabilizers, and buffering agents as needed. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts, and sodium EDTA may also be used. Furthermore, parenteral solutions may contain preservatives such as benzalkonium chloride, methylparaben, or propylparaben, and chlorobutanol. For inhalation administration, the compounds of the present invention can be conveniently delivered in aerosol form from pressurized packaging or a nebulizer. The compound can also be delivered in a formulated powder form, which can be inhaled with the aid of a powder inhaler device. A preferred delivery system for inhalation is a metered-dose inhaler (MDI) aerosol, which can be formulated as a suspension or solution of the compound of Formula I in a suitable propellant, such as a fluorocarbon or hydrocarbon. For ophthalmic administration, ophthalmic formulations can be formulated as a solution or suspension of the compound of Formula I in a suitable ophthalmic carrier at an appropriate weight percentage, thereby maintaining sufficient contact time between the compound and the ocular surface to allow the compound to penetrate into the cornea and internal regions of the eye.

[0062] Useful pharmaceutical dosage forms for administering the compounds of the present invention include, but are not limited to, hard and soft gelatin capsules, tablets, parenteral injections, and oral suspensions.

[0063] When the compounds of the present invention are administered stepwise or in combination with other therapeutic agents, the same dosage forms as described above may be used. When the drugs are administered in physical combinations, the dosage form and route of administration should be selected based on the compatibility of the combined drugs.

[0064] The present invention has at least the following beneficial effects: This invention provides a novel diketopiperazine methylated derivative and its preparation method, which can be widely used in the field of drug delivery. Detailed Implementation

[0065] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0066] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0067] Basic Example 1:

[0068] Step 1: Dehydration cyclization reaction: ( S 2-Amino-6-(((benzyloxy)carbonyl)amino)-2-methylhexanoic acid (SM-1, 10 g, 34 mmol) and m-cresol (16.7 g, 154 mmol) were dissolved in NMP (50 mL), and 1.67 g of P2O5 was added. The mixture was heated to 160-165 °C and reacted for 10 h. After cooling to room temperature, the solution was slowly added dropwise to 100 mL of ice water, precipitating a yellow solid. After filtration and drying, 10.2 g of compound 1 was obtained, with a yield of 54.3% and a purity of 98%.

[0069] 1 H NMR (500 MHz, DMSO, ppm): 8.10 (s, 2H, NH), 7.33-7.37 (m, 8H),7.22 (t, 2H), 2.97 (dd, 4H, NCH2), 1.74(s, 6H, CH3),1.61-1.68 (m, 4H, CH2),1.29-1.40 (m, 8H, CH2CH2).

[0070] ESI MS: 553.27 (M+H).

[0071] Step 2: Hydrogenation: Compound 1 (10.0 g, 18.57 mmol) was suspended in a 1:1 mixture of methanol and acetic acid (50 mL) replaced with N2, and 10% Pd / C (1.0 g) was added. The apparatus was connected to a hydrogen balloon system, and the needle was suspended above the liquid surface after a brief bubbling of hydrogen gas. The reaction was stirred overnight and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure. The resulting viscous substance was resuspended in a minimal amount of dichloromethane, washed with ethyl acetate to precipitate a white solid, and then filtered under vacuum to obtain compound 2 (3.8 g, yield 75.7%, purity 98%).

[0072] Optical rotation: [α] 20 _D +25.5° (c 0.1, methanol).

[0073] 1 H NMR (500 MHz, D2O, ppm): 4.14 (t, 2H, COCH), 2.99 (t, 4H, NCH2), 1.84-1.88 (m, 4H, CH2), 1.67-1.71 (m, 4H, CH2), 1.39-1.52 (m, 4H, CH2).

[0074] ESI MS: 285.19 (M+H).

[0075] Basic Implementation Example 2

[0076] Step 1: Dehydration cyclization reaction: ( R 2-Amino-6-(((benzyloxy)carbonyl)amino)-2-methylhexanoic acid (SM-2, 20 g, 68 mmol) and m-cresol (33.4 g, 308 mmol) were dissolved in NMP (100 mL), and 3.34 g of P2O5 was added. The mixture was heated to 160-165 °C and reacted for 10 h. After cooling to room temperature, the mixture was slowly added dropwise to 200 mL of ice water, precipitating a yellow solid. After filtration and drying, 22.4 g of compound 6 was obtained, with a yield of 59.4% and a purity of 98%.

[0077] 1 H NMR (500 MHz, DMSO, ppm): 8.13 (s, 2H, NH), 7.32-7.38 (m, 8H),7.24 (t, 2H), 2.99 (dd, 4H, NCH2), 1.76(s, 6H, CH3),1.62-1.69 (m, 4H, CH2),1.28-1.1 (m, 8H, CH2CH2).

[0078] ESI MS: 553.28 (M+H).

[0079] Step 2: Hydrogenation: Compound 6 (12.0 g, 22.28 mmol) was suspended in a 1:1 mixture of methanol and acetic acid (N2-replaced) (60 mL), and 10% Pd / C (1.2 g) was added. The apparatus was connected to a hydrogen balloon system, stirred overnight at 25-30 °C, and then filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure. The resulting viscous substance was resuspended in 10 mL of dichloromethane, washed with ethyl acetate to precipitate a white solid, and then filtered under vacuum to obtain compound 7 (4.9 g, yield 81.3%, purity 98.5%).

[0080] Optical rotation: [α] 20 _D -24.8° (c 0.1, methanol).

[0081] 1 H NMR (500 MHz, DMSO- d6 , ppm): 4.15 (t, 2H, COCH), 2.94 (t, 4H,NCH2), 1.85-1.89 (m, 4H, CH2), 1.65-1.70 (m, 8H, CH2, NH2), 1.37-1.50 (m, 10H,CH2).

[0082] ESI MS: 285.19 (M+H).

[0083] Example 1

[0084] Step 1: Compound 2 (5.0 g, 19.5 mmol), compound 3 (6.63 g, 39 mmol), EDCI (9.08 g, 30.6 mmol), and triethylamine (7.99 g, 78 mmol) were dissolved in 80 mL of anhydrous THF. The reaction mixture was reacted overnight at room temperature, and the reaction solution was poured into ice water. The organic layer was separated and extracted once with 50 mL of ethyl acetate in the aqueous phase. The organic layers were combined and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH = 10:1-5:1) to obtain 8.72 g of white foamy solid compound 4, with a yield of 76%.

[0085] Step 2: Dissolve compound 4 (8.0 g, 13.5 mmol) in 80 mL of a methanol:water (20:1) mixture, adjust the pH to 12 with 10% sodium hydroxide aqueous solution, react for 2 h, adjust the pH to 1-2 with 6N hydrochloric acid, precipitate a solid, filter, wash with water until neutral, dry to obtain a white solid compound 5 in an amount of 6.5 g, yield: 85.3%, purity: 95%.

[0086] Optical rotation: [α] 20 _D +5.7° (c 0.1, methanol).

[0087] 1 H NMR (500 MHz, DMSO- d6 , ppm): 8.02 (s, 2H, NH), 3.11 (t, 4H, NCH2), 1.90-1.95 (m, 8H, CH2), 1.83 (s, 6H, CH3), 1.72-1.75 (m, 8H, CH2), 1.45-1.56 (m, 10H, CH2); MS: 561.28 (M+H) Example 2

[0088] Step 1: Compound 2 (6.0 g, 23.4 mmol), compound 10 (6.08 g, 46.8 mmol), EDCI (10.9 g, 36.7 mmol), and triethylamine (9.6 g, 93.6 mmol) were dissolved in 100 mL of anhydrous THF. The reaction mixture was reacted overnight at 25-30 °C. The reaction solution was then poured into 200 mL of ice water. The organic layer was separated and extracted once with 60 mL of ethyl acetate in the aqueous phase. The organic layers were combined and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH = 10:1-5:1) to obtain 9.2 g of white foamy solid compound 11, with a yield of 77.3% and a purity of 96%.

[0089] 1 H NMR (500 MHz, DMSO- d 6, ppm): 6.25 (2H, d, J =12Hz), 5.94 (2H, d, J =12.2Hz), 4.15 (s, 2H, CONH), 3.99 (s, 2H, NCH2), 3.65 (s, 6H, CH3), 1.81-1.89(m, 4H, CH2), 1.76(s, 6H, CH3), 1.65-1.73 (m, 8H, CH2), 1.39-1.52 (m, 4H, CH2).

[0090] ESI MS: 509.25 (M+H).

[0091] Step 2: Dissolve compound 11 (8.0 g, 15.7 mmol) in 100 mL of a methanol:water (20:1) mixture, adjust the pH to 12 with 10% sodium hydroxide aqueous solution, react for 2 h, adjust the pH to 1-2 with 6 N hydrochloric acid, precipitate a solid, filter, wash with water until neutral, and dry to obtain a white solid of 6.4 g of compound 12, yield: 84.8%, purity: 95%.

[0092] Optical rotation: [α] 20 _D +10.5° (c 0.1, methanol).

[0093] 1 H NMR (500 MHz, DMSO- d 6, ppm): 6.24 (2H, d, J =12Hz), 5.92 (2H, d, J =12.2Hz), 4.14 (s, 2H, CONH), 3.99 (s, 2H, NCH2), 1.84-1.88 (m, 4H, CH2), 1.75 (s, 6H, CH3), 1.67-1.71 (m, 8H, CH2), 1.39-1.52 (m, 4H, CH2).

[0094] ESI MS: 481.22 (M+H).

[0095] Example 3

[0096] Step 1: Compound 7 (6.0 g, 23.4 mmol), compound 3 (7.96 g, 46.8 mmol), EDCI (10.9 g, 36.7 mmol), and triethylamine (9.6 g, 93.6 mmol) were dissolved in 100 mL of anhydrous THF. The reaction mixture was reacted overnight at 25-30 °C. The reaction solution was then poured into 200 mL of ice water. The organic layer was separated and extracted once with 60 mL of ethyl acetate in the aqueous phase. The organic layers were combined and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH = 10:1-5:1) to obtain 9.0 g of white foamy solid compound 8, with a yield of 78.4% and a purity of 96%.

[0097] 1 H NMR (500 MHz, DMSO- d6, ppm): 8.09 (s, 2H, NH), 3.65 (s, 6H, CH3), 3.17 (t, 4H, NCH2), 1.92-1.99 (m, 8H, CH2), 1.87 (s, 6H, CH3), 1.72-1.75 (m, 10H,CH2), 1.44-1.58 (m, 8H, CH2); MS: 589.32 (M+H).

[0098] Step 2: Dissolve compound 8 (8.5 g, 14.3 mmol) in 100 mL of a methanol:water (20:1) mixture, adjust the pH to 12 with 10% sodium hydroxide aqueous solution, react for 2 h, adjust the pH to 1-2 with 6N hydrochloric acid, precipitate a solid, filter, wash with water until neutral, and dry to obtain 6.5 g of off-white solid compound 9, yield: 78.7%, purity: 95%.

[0099] Optical rotation: [α] 20 _D -4.5° (c 0.1, methanol).

[0100] 1 H NMR (500 MHz, DMSO- d6 , ppm): 8.08 (s, 2H, NH), 3.16 (t, 4H, NCH2), 1.91-1.97 (m, 8H, CH2), 1.85 (s, 6H, CH3), 1.71-1.73 (m, 10H, CH2), 1.43-1.57 (m, 8H, CH2); MS: 561.28 (M+H).

[0101] Example 4

[0102] Step 1: Compound 7 (3.0 g, 11.7 mmol), compound 10 (3.04 g, 23.4 mmol), EDCI (5.5 g, 18.8 mmol), and triethylamine (4.8 g, 46.8 mmol) were dissolved in 60 mL of anhydrous THF. The reaction mixture was reacted overnight at 25-30 °C. The reaction solution was then poured into 100 mL of ice water. The organic layer was separated and extracted once with 40 mL of ethyl acetate in the aqueous phase. The organic layers were combined and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH = 10:1-5:1) to obtain 5.0 g of white foamy solid compound 13, with a yield of 84.0% and a purity of 96%.

[0103] 1H NMR (500 MHz, DMSO- d 6, ppm): 6.23 (2H, d, J =12.5Hz), 5.94 (2H, d, J =12.7Hz), 4.13 (s, 2H, CONH), 3.93 (s, 2H, NCH2), 3.65 (s, 6H, CH3), 1.81-1.88(m, 4H, CH2), 1.76 (s, 6H, CH3), 1.63-1.75(m, 8H, CH2), 1.38-1.56 (m, 4H, CH2).

[0104] ESI MS: 509.25 (M+H).

[0105] Step 2: Compound 13 (4.0 g, 7.8 mmol) was dissolved in 500 mL of a methanol:water (20:1) mixture. The pH was adjusted to 12 with 10% sodium hydroxide aqueous solution. After reacting for 2 h, the pH was adjusted to 1-2 with 6 N hydrochloric acid. A solid precipitated out. The solid was filtered, washed with water until neutral, and dried to obtain 3.0 g of off-white solid compound 14. The yield was 79.5% and the purity was 96%.

[0106] Optical rotation: [α] 20 _D -9.8° (c 0.1, methanol).

[0107] 1 H NMR (500 MHz, DMSO- d 6, ppm): 6.25 (2H, d, J =12.5Hz), 5.96 (2H, d, J =12.7Hz), 4.15 (s, 2H, CONH), 3.95 (s, 2H, NCH2), 1.82-1.89 (m, 4H, CH2), 1.78(s, 6H, CH3), 1.68-1.72 (m, 8H, CH2), 1.38-1.55 (m, 4H, CH2).

[0108] ESI MS: 481.25 (M+H).

[0109] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A diketopiperazine methylated derivative, characterized in that, The diketopiperazine methylated derivative is a compound of Formula I, its isomers, prodrugs, stable isotopic derivatives, pharmaceutically acceptable salts, or mixtures thereof: Formula I R1 and R2 are each independently selected from H, substituted or unsubstituted alkyl groups; L1 and L2 are each independently selected from substituted or unsubstituted alkylene groups; X1 and X2 are each independently selected from N, O or S; L3 is independently selected from alkenyl groups and C5-C8 bridged cyclic groups.

2. The diketopiperazine methylated derivative according to claim 1, characterized in that, in, R1 and R2 are each independently selected from C1-C6 alkyl groups; L1 and L2 are each independently selected from C1-C6 alkylene groups; X1 and X2 are each independently selected from N or O; L3 is independently selected from alkenyl and C5-C7 bridged cyclic groups.

3. The diketopiperazine methylated derivative according to claim 2, characterized in that, in, R1 and R2 are each independently selected from methyl groups; L1 and L2 are each independently selected from C1-C5 alkylene groups; X1 and X2 are each independently selected from N; L3 is independently selected from alkenyl and C5-bridged cycloyl groups.

4. The diketopiperazine methylated derivative according to claim 3, characterized in that, The compound is selected from the following structures: ； L3 and L4 are as described in any one of claims 1-3.

5. The diketopiperazine methylated derivative according to claim 4, characterized in that, The compound is selected from the following structures: 、 、 、 。 6. The method for preparing the diketopiperazine methylated derivative according to any one of claims 1-5, characterized in that, The reaction equation is shown below: S1: 2-Amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid reacts with m-cresol in the presence of a dehydrating agent and a solvent to give compound 1; S2: Compound 1 undergoes a deprotection reaction under palladium-catalyzed hydrogenation to give compound 2; S3: Compound 2 undergoes a condensation reaction with an acid in the presence of a condensing agent to give compound 3; S4: Compound 3 undergoes a hydrolysis reaction to give compound 4.

7. The preparation method according to claim 6, characterized in that, In step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to m-cresol is 1:3-10.

8. The preparation method according to claim 7, characterized in that, In step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to m-cresol is 1:4-5.

9. The preparation method according to claim 6, characterized in that, In step S1, the dehydrating agent is selected from at least one of P2O5, concentrated sulfuric acid, or calcium chloride.

10. The preparation method according to claim 9, characterized in that, In step S1, the dehydrating agent is P2O5.

11. The preparation method according to claim 6, characterized in that, In step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to the dehydrating agent is 1:0.1-1.

0.

12. The preparation method according to claim 11, characterized in that, In step S1, the molar ratio of 2-amino-6-(((benzyloxy)carbonyl)amino)-5,5-difluoro-hexanoic acid to the dehydrating agent is 1:0.3-0.

5.

13. The preparation method according to claim 6, characterized in that, In step S1, the temperature of the dehydration and ring-closing reaction is 100-200℃.

14. The preparation method according to claim 13, characterized in that, In step S1, the temperature of the dehydration and ring-closing reaction is 150-180℃.

15. The preparation method according to claim 6, characterized in that, In step S1, the solvent is selected from at least one of N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran.

16. The preparation method according to claim 15, characterized in that, In step S1, the solvent is N-methylpyrrolidone.

17. The preparation method according to claim 6, characterized in that, In step S2, the mass ratio of compound 1 to palladium on carbon is 1:0.01-0.

2.

18. The preparation method according to claim 17, characterized in that, In step S2, the mass ratio of compound 1 to palladium on carbon is 1:0.08-0.

15.

19. The preparation method according to claim 6, characterized in that, In step S2, the temperature of the deprotection reaction is 10-60℃.

20. The preparation method according to claim 6, characterized in that, In step S3, the molar ratio of compound 2 to acid is 1:1.8-4.

21. The preparation method according to claim 20, characterized in that, In step S3, the molar ratio of compound 2 to acid is 1:2-3.

22. The preparation method according to claim 6, characterized in that, In step S3, the condensing agent is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, O-azabenzotriazole tetramethylurea hexafluorophosphate, or 1-hydroxybenzotriazole.

23. The preparation method according to claim 22, characterized in that, In step S3, the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

24. The preparation method according to claim 6, characterized in that, In step S3, the equivalent ratio of the acid to the condensing agent is 1.8-6:

1.

25. The preparation method according to claim 24, characterized in that, In step S3, the equivalent ratio of the acid to the condensing agent is 2-4:

1.

26. The preparation method according to claim 6, characterized in that, In step S4, the temperature of the hydrolysis reaction is 20-60℃.

27. Use of the diketopiperazine methylated derivative according to any one of claims 1-5 in the preparation of a medicament for drug delivery.

28. The application according to claim 27, characterized in that, The drug comprises a compound represented by Formula I as a penetration enhancer, its isomers, prodrugs, stable isotopic derivatives, pharmaceutically acceptable salts or mixtures thereof, and pharmaceutically acceptable carriers and active ingredients.

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