Bacprotacs targeting dihydropteroate synthase and preparation method and application thereof

Abstract

This invention discloses BacPROTACs targeting dihydropteranoic acid synthase, their preparation method, and their applications, belonging to the fields of medicinal chemistry and anti-infective drug technology. The BacPROTACs have the structure shown in Formula I, II, or III, and are composed of a DHPS inhibitor fragment, a linker group, and a phosphorylated arginine (pArg) fragment. The pArg fragment, as a functional group capable of interacting with the bacterial ClpCP protease system, is coupled to a DHPS inhibitor through linkers of different types and chain lengths, resulting in bifunctional molecules possessing both DHPS binding and ClpCP recruitment capabilities. These compounds can recruit the ClpCP protease system while binding to DHPS, thereby inducing or promoting DHPS degradation, reducing target protein levels, and expanding the pharmacodynamic mechanism of DHPS-targeted drugs. Compared with existing DHPS inhibitors that mainly rely on reversible inhibition, this invention provides a new technical solution for the development of antibacterial drugs through a design strategy combining inhibition and targeted degradation. These compounds can be used to prepare antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and anti-infective drug technology, specifically relating to a BacPROTAC (Bacterial Proteolysis-Targeting Chimeras) that targets dihydropteroate synthase, its preparation method, and its application. Background Technology

[0002] Bacterial infectious diseases have always been a significant public health problem threatening human health. Although the discovery and use of antibiotics have greatly reduced the incidence and mortality of infectious diseases over the past century, the problem of bacterial resistance has become increasingly serious due to the widespread use and even abuse of antibiotics. The prevalence of drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenem-resistant Enterobacteriaceae (CRE), and multidrug-resistant Pseudomonas aeruginosa (MDRPA) has become a major challenge in clinical treatment. The World Health Organization has listed antibiotic resistance as one of the three major threats to global public health in the 21st century. Therefore, the development of antimicrobial drugs with novel mechanisms of action to address the increasingly serious problem of bacterial resistance has become an urgent need in the field of drug development.

[0003] Dihydropteroate synthase (DHPS) is a key enzyme in the bacterial de novo biosynthesis of folic acid, catalyzing the condensation of p-aminobenzoic acid (pABA) with dihydropteridine pyrophosphate to form dihydropteroic acid. Dihydropteroic acid is further converted into dihydrofolate and tetrahydrofolate. Tetrahydrofolate, as a one-carbon unit carrier, participates in the biosynthesis of purines, thymine nucleotides, and various amino acids, and is crucial for bacterial DNA replication, cell proliferation, and survival. Since humans lack a de novo folic acid synthesis pathway and rely on exogenous folic acid intake, DHPS, as a bacterial-specific enzyme, has become an ideal target for antibacterial drug development. Currently, drug development strategies targeting DHPS primarily focus on enzyme activity inhibition, with sulfonamide antibiotics being representative examples. As structural analogs of p-aminobenzoic acid, sulfonamides typically inhibit folic acid synthesis by occupying its active site or interfering with substrate binding and catalytic processes, thereby inhibiting bacterial growth. Most of these drugs are site-driven inhibitors, and their efficacy usually depends on maintaining sufficient exposure levels and sustained target occupancy in vivo. These modes of action are still limited to temporary inhibition of target enzyme activity, and have limitations such as a relatively simple mechanism of action, easy recovery of target function, and a high risk of drug resistance.

[0004] In recent years, the rapid development of targeted protein degradation (TPD) technology has provided a novel approach to drug development. Protein degradation-targeting chimeras (PROTACs), as bifunctional molecules, consist of a target protein ligand, a linker group, and an E3 ubiquitin ligase ligand. They can recruit E3 ligases while binding to the target protein, inducing ubiquitination and degradation by the proteasome, thereby achieving functional depletion of the target protein. Compared with traditional small molecule inhibitors, PROTACs have advantages such as catalytic action, overcoming drug resistance potential, and targeting difficult-to-drug targets. However, PROTAC technology is mainly applied to eukaryotic cells, and its action depends on the ubiquitin-proteasome system within eukaryotic cells. Bacteria, as prokaryotes, lack the classic ubiquitin-proteasome degradation system, therefore traditional PROTAC technology cannot be directly applied to bacterial targets. In recent years, researchers have discovered a similar protein degradation system in bacteria—the Clp protease complex (Caseinolytic protease complex), in which ClpCP is the main proteolytic system in Gram-positive bacteria such as Bacillus and Mycobacteria. The ClpCP system, composed of the ClpC ATPase and the ClpP protease subunit, recognizes and degrades substrate proteins with specific tag sequences (such as phosphorylated arginine residues, pArg). This discovery provides a theoretical basis for the development of bacterial proteolysis-targeting chimeras (BacPROTACs). Previous studies have reported the successful induction of target protein degradation and inhibition of bacterial growth by constructing BacPROTACs targeting mycobacterial ClpC1 or other essential bacterial proteins using phosphorylated arginine (pArg) as the ClpCP recognition ligand. However, there are currently no reports of applying BacPROTAC technology to target DHPS, nor are there any technical solutions for conjugating DHPS inhibitors with pArg fragments to construct bifunctional molecules to induce DHPS degradation.

[0005] In summary, developing a novel bifunctional molecule that can target DHPS and induce its degradation by recruiting the bacterial ClpCP protease system is expected to overcome the limitations of traditional DHPS inhibitors, such as reversible inhibition, easy drug resistance, and single mechanism of action, and provide a new technical path for the development of antibacterial infection drugs. Summary of the Invention

[0006] Addressing the limitations of existing DHPS-targeting drugs, which rely solely on reversible enzyme activity inhibition, leading to easy recovery of target function, significant drug resistance issues, and a lack of DHPS-targeting protein degraders, this invention aims to provide BacPROTACs (Bacterial Proteolysis-Targeting Chimeras) targeting dihydropteroate synthase, their preparation method, and their applications. This invention represents the first application of BacPROTACs technology to DHPS targets, achieving a breakthrough in mechanism from "activity inhibition" to "target clearance."

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses a BacPROTAC targeting dihydropteroate synthase, which is a compound of formula I, II, or III or a pharmaceutically acceptable salt thereof, as well as the compound of formula I, II, or III or a pharmaceutically acceptable salt thereof, a solvent compound, an enantiomer, a diastereomer, or a racemic mixture thereof; the structural formula of the compound of formula I, II, or III is as follows:

[0008] or

[0009] or

[0010] Among them, Linker is n = 0, 2, 4, 6, 8.

[0011] The compound is selected from the following compounds or their pharmaceutically acceptable salts:

[0012]

[0013] Preferably, the pharmaceutically acceptable salt is a salt formed by BacPROTACs targeting dihydropteroate synthase with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

[0014] Secondly, the present invention also discloses a method for preparing the above-mentioned BacPROTACs targeting dihydropteridine synthase, comprising the following steps: (1) Using (benzyloxycarbonyl)-L-arginine as a raw material, benzyl bromide was used to protect the carboxyl group to obtain compound a1; then it was reacted with bis(2,2,2-trichloroethyl)phosphoryl chloride to obtain compound a2; compound a2 was deprotected in an acidic solvent by Pd / C and hydrogen to obtain compound a3; compound a3 was introduced with Fmoc protecting group to obtain intermediate compound a4; Among them, compound a1 is Compound a2 is Compound a3 is Compound a4 is ; (2) Using 4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzoic acid as a raw material, acid-amine condensation reaction was carried out with alkylamines of different lengths to obtain the corresponding compounds b1-b5; Among them, compounds b1-b5 are n = 0, 2, 4, 6, 8; Alternatively, with 2-((2-amino-6-oxo-6,9-dihydro-1) H Using purine-8-yl)thioacetic acid as a raw material, acid-amine condensation reaction was carried out with alkylamines of different lengths to obtain the corresponding compounds b6-b10; Among them, compounds b6-b10 are n = 0, 2, 4, 6, 8; Or, with ( R )-2-((2-amino-6-oxo-6,9-dihydro-1 H Using purine-8-yl)thio)-2-phenylacetic acid as a raw material, an acid-amine condensation reaction was carried out with alkylamines of different lengths to obtain the corresponding compounds b1-b15; Compounds b1-b15 are ; (3) Compound b1-compound b15 loses the Boc group under the action of TFA to generate compound c1-compound c15; Among them, compounds c1-c5 are n=0, 2, 4, 6, 8; compounds c6-c10 are n = 0, 2, 4, 6, 8; compounds c11-c15 are n = 0, 2, 4, 6, 8; (4) Compound a4 reacts with compound c1-compound c15 to form compound d1-compound d15; in, The structural formulas of compounds d1-d5 are as follows: n = 0, 2, 4, 6, 8; Compounds d6-d10 are n=0, 2, 4, 6, 8; the structural formulas of compounds d11-d15 are as follows: n = 0, 2, 4, 6, 8; (5) Compound d1-compound d15 loses the 9-fluorenylmethoxycarbonyl protecting group under the action of a 20% piperidine and DMF mixed solution to generate compound e1-compound e15; Among them, compounds e1-e5 are n=0, 2, 4, 6, 8; compounds e6-e10 are n=0, 2, 4, 6, 8; compounds e11-e15 are n = 0, 2, 4, 6, 8; (6) Compound e1-compound e15 is introduced with acetamide under the action of acetic anhydride and DIEA to generate compound f1-compound f15; Among them, compounds f1-f5 are n=0, 2, 4, 6, 8; compounds f6-f10 are n = 0, 2, 4, 6, 8; compounds f11-f15 are n = 0, 2, 4, 6, 8; (7) Dissolve compound f1-compound f15 in ammonium carbonate buffer solution, add ethanol and palladium on carbon catalyst, and under hydrogen atmosphere, compound f1-compound f15 removes 2,2,2-trichloroethyl group under the action of the reaction system to generate target compound 1-compound 15; Among them, compounds 1-5 are n=0, 2, 4, 6, 8; compounds 6-10 are n=0, 2, 4, 6, 8; compounds 11-15 are n = 0, 2, 4, 6, 8.

[0015] In step (1), the molar ratio of benzyloxycarbonyl-L-arginine to benzyl bromide is 1:(1~2), and the molar ratio of compound a1 to bis(2,2,2-trichloroethyl)phosphoryl chloride is 1:(1~2); in step (2), 4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzoic acid, 2-((2-amino-6-oxo-6,9-dihydro-1 H -purine-8-yl)thio)acetic acid, ( R )-2-((2-amino-6-oxo-6,9-dihydro-1 HThe molar ratio of purine-8-yl)thio)-2-phenylacetic acid to alkylamine chains of different lengths is 2:1; in step (4), the molar ratio of compound a4 to compound c1-compound c15 is (1~2):1.

[0016] Preferably, in step (1), the molar ratio of benzyloxycarbonyl-L-arginine to benzyl bromide is 1:1.1, and the molar ratio of compound a1 to bis(2,2,2-trichloroethyl)phosphoryl chloride is 1:1.3; in step (2), 4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzoic acid, 2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)acetic acid, ( R The molar ratio of 2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)-2-phenylacetic acid to alkylamine chains of different lengths is 2:1; in step (4), the molar ratio of compound a4 to compound c1-compound c15 is 1.2:1.

[0017] Preferably, in step (1), the reaction temperature of compound a2 is room temperature, the solvent used is acetonitrile, and the reaction time is 7h; in steps (2) and (4), in the synthesis of compounds b1-b15 and d1-d15, the carboxylic acid is activated with HATU and then reacted with Linker of different lengths, and the solvent used is N,N-dimethylformamide; in step (5), the solvent used is a mixed solution of 20% piperidine and DMF, which is used to remove the 9-fluorenylmethoxycarbonyl protecting group; in step (6), acetylation reaction is carried out with acetic anhydride, and the solvent used is NMP solution.

[0018] Thirdly, the present invention also discloses the application of the above-mentioned BacPROTACs targeting dihydropteroate synthase in the preparation of antibacterial infection drugs.

[0019] The bacteria include Gram-positive bacteria, Gram-negative bacteria, and drug-resistant bacteria.

[0020] Preferably, the drug is a preparation for use alone or in combination with other antibacterial drugs, and the preparation is an oral tablet, capsule, granule, syrup, premix or micro-pellet, or a liniment or injection for non-oral administration.

[0021] Fourthly, the present invention also discloses a pharmaceutical composition comprising the above-mentioned BacPROTACs targeting dihydropteroate synthase as active ingredients, and pharmaceutically acceptable excipients or diluents.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides BacPROTACs targeting dihydropteranoic acid synthase (DHPS), developing a strategy for BacPROTACs targeting DHPS. While retaining the targeting recognition ability of DHPS inhibitors, a functional group that can be recognized or recruited by the bacterial protein degradation system ClpCP protease complex is introduced. These two groups are coupled through a linker group, constructing a bifunctional molecule that combines target binding and degradation system recruitment. This molecule can recruit the ClpCP protease complex while binding to DHPS, thereby inducing DHPS degradation, reducing target protein levels, and achieving a pharmacodynamic mechanism that expands from traditional "activity inhibition" to "target clearance." This enhances the inhibitory effect on bacterial growth and provides a new molecular design approach for addressing bacterial resistance.

[0023] The BacPROTACs targeting DHPS constructed in this invention contain a bifunctional molecular backbone of a DHPS inhibitor fragment-Linker-pArg fragment. They can bind to and inhibit DHPS while recruiting the bacterial ClpCP protease system to induce or promote DHPS degradation, thereby reducing the level of the target protein and achieving sustained functional exhaustion. This overcomes the limitations of existing DHPS inhibitors, which mainly rely on reversible inhibition and have relatively simple action pathways.

[0024] Furthermore, this invention introduces pArg as a ClpCP recognition ligand and uses linkers of different chain lengths for coupling to obtain a series of compounds that can effectively target target proteins and simultaneously possess inhibitory and degradation activities, thereby overcoming the problems of existing technologies such as single structural type, only inhibitory effect, and limited drug efficacy pathways.

[0025] This invention provides a method for preparing BacPROTACs targeting bacterial dihydropteroate synthase. This method uses various commercially available synthetic building blocks as raw materials and obtains the target product in a high yield through common chemical reactions. All reactions avoid the use of high temperature, high pressure and highly toxic reagents, and can be carried out under relatively mild conditions. It has low requirements for reaction equipment and low environmental pollution. At the same time, it has high atom economy and is suitable for industrial production. Attached Figure Description

[0026] Figure 1 The results show the minimum inhibitory concentration (MIC) determination of compound 13 against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300.

[0027] Figure 2 The results show the MIC determination of compound 13 against multidrug-resistant Staphylococcus aureus (MDRSA) MDRSA-171.

[0028] Figure 3The results show the MIC determination of compound 13 against carbapenem-resistant Escherichia coli (CR-EC-361). Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0031] The present invention will now be described in further detail with reference to the accompanying drawings: This invention provides a BacPROTAC targeting dihydropteroate synthase, comprising compounds of formulas I, II, and III or pharmaceutically acceptable salts thereof, and solvent compounds, enantiomers, diastereomers, or mixtures thereof in any proportion thereof, including racemic mixtures; the structural formulas of the compounds of formulas I, II, and III are as follows:

[0032] or

[0033] or

[0034] Linker is one of the following linking groups:

[0035] Where n = 0, 2, 4, 6, 8.

[0036] The method for preparing BacPROTACs targeting dihydropteridine synthase provided by this invention is as follows: (1) Using (benzyloxycarbonyl)-L-arginine as a starting material, benzyl bromide was used to protect the carboxyl group to obtain compound a1; then it was reacted with bis(2,2,2-trichloroethyl)phosphoryl chloride to obtain compound a2; compound a2 was deprotected in an acidic solvent by Pd / C and hydrogen to obtain compound a3; subsequently, Fmoc protecting group was introduced into compound a3 to obtain compound a4; the reaction formula is as follows:

[0037] (2) 4-(((2-amino-4-hydroxypterin-6-yl)methyl)amino)benzoic acid, 2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)acetic acid, ( R )-2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)-2-phenylacetic acid undergoes amide condensation reaction with alkylamine chains of different lengths to obtain the corresponding compounds b1-b15;

[0038] (3) Compound b1-compound b15 loses its Boc group under the action of TFA to generate compound c1-compound c15, as shown in the following reaction formula:

[0039] (4) Using compound a4 as a starting material, an amide condensation reaction is carried out with compound c1-compound c15 to generate compound d1-compound d15, as shown in the following reaction formula;

[0040] (5) Compounds d1-d15 lose the 9-fluorenylmethoxycarbonyl protecting group under the action of a 20% piperidine and DMF mixed solution to generate e1-e15, as shown in the following reaction formula;

[0041] (6) Compound e1-compound e15 undergoes an acetylation reaction with acetic anhydride to generate compound f1-compound f15, as shown in the following reaction formula:

[0042] (7) Dissolve compound f1-compound f15 in ammonium carbonate buffer solution, add ethanol and palladium on carbon catalyst, and under hydrogen atmosphere, compound f1-compound f15 removes 2,2,2-trichloroethyl group under the action of this reaction system to generate target compound 1-compound 15; the reaction formula is as follows;

[0043] 1. Specific examples of the synthesis of compounds 1-15 The structural formulas of representative compounds of this invention are shown below:

[0044]

[0045] The following describes the synthesis of the above compounds.

[0046] I. Examples of Preparation of BacPROTACs Compounds Targeting Dihydropteridine Synthase Example 1: Preparation of Compound 1 This embodiment provides compound (S)-(N-(4-acetamido-5-(((4-((((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)methyl)amino)5-oxopentyl)carbamoyl)phosphatidyl (compound 1), and the specific preparation process is as follows: 1. Synthesis of intermediate compound a4

[0047] (1) Synthesis of compound a1 Benzyloxycarbonyl-L-arginine (5.55 g, 18 mmol) was placed in a reactor, and 100 mL of N-methylpyrrolidone was added and stirred to dissolve it. Then, benzyl bromide (2.34 mL, 19.8 mmol) was added, and the reaction was carried out at room temperature in the dark with stirring for 8 hours. The reaction progress was monitored by TLC (developing solvent: dichloromethane: methanol = 20:1). After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent. The resulting solid residue was washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate (3 × 50 mL), and the organic phase was collected. The residue was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol = 20:1). The target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain compound a1 (6.41 g, white solid), with a yield of 89.37%.

[0048] (2) Synthesis of compound a2 Compound a1 (5.98 g, 15 mmol) and bis(2,2,2-trichloroethyl)phosphoryl chloride (8.53 g, 22.5 mmol) were placed in a reactor and dissolved in 100 mL of acetonitrile. The reactor was cooled to 0 °C in an ice-water bath, and triethylamine (4.17 mL, 30 mmol) was slowly added. The reactor was stirred at room temperature for 7 h, and the reaction progress was monitored by TLC (evolving solvent: dichloromethane: methanol = 10:1). After the reaction was completed, most of the solvent was removed under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol = 10:1). The target fraction was collected, concentrated under reduced pressure, and dried under vacuum to obtain compound a2 (8.74 g, pale yellow solid), with a yield of 78.61%.

[0049] (3) Synthesis of compound a3 The obtained compound a2 (7.78 g, 10.5 mmol) was placed in a reactor, and 60 mL of trifluoroacetic acid (TFA), 60 mL of AcOH and 1.8 g of Pd / C catalyst were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The degree of reaction was monitored by TLC. After the reaction was completed, the reaction mixture was filtered to remove Pd / C. The filtered reaction solution was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 8:1). The target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain compound a3 (4.99 g, white solid), with a yield of 91.93%.

[0050] (4) Synthesis of compound a4 Compound a3 (4.65 g, 9 mmol), 9-fluorenylmethyl-N-succinimide carbonate (3.64 g, 10.8 mmol), and triethylamine (2.50 mL, 18 mmol) were placed in a reactor and dissolved in 80 mL of acetonitrile. The mixture was heated and stirred overnight at 60 °C, and the reaction was monitored by TLC. After the reaction was completed, the resulting reaction solution was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 10:1). The purified solution was dried to give compound a4 (4.37 g, white solid), with a yield of 65.69%.

[0051] 2. Preparation of intermediate compound d1

[0052] (1) Synthesis of compound b1 4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzoic acid (7.49 g, 24 mmol), HATU (10.95 g, 28.8 mmol), and DIEA (5.02 mL, 28.8 mmol) were dissolved in 50 mL of N,N-dimethylformamide (DMF) and reacted at room temperature for 0.5 h to activate the carboxylic acid. Then, tert-butyl (aminomethyl)carbamate (1.75 g, 12 mmol) was added, and the reaction was carried out at room temperature for 8 h, monitored by TLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the mixture was purified by column chromatography (dichloromethane:methanol = 10:1). The purified compound was dried to give compound b1 (4.85 g), with a yield of 91.76%.

[0053] (2) Synthesis of compound c1 Compound b1 (3.96 g, 9 mmol) was dissolved in 30 mL of trifluoroacetic acid (TFA) and 10 mL of dichloromethane. The mixture was stirred overnight at room temperature and monitored by TLC. After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the mixture was purified by column chromatography (dichloromethane:methanol = 8:1). After drying, compound c1 (2.28 g) was obtained, with a yield of 74.43%.

[0054] (3) Synthesis of compound d1 Compound a4 (4.44 g, 6 mmol), HATU (2.74 g, 7.2 mmol), and DIEA (1.25 mL, 7.2 mmol) were placed in a reactor and dissolved in 40 mL of DMF. The reaction was carried out at room temperature for 0.5 h to activate the carboxylic acid. Then, compound c1 (1.70 g, 5 mmol) was added and the reaction was monitored by TLC. After the reaction was completed, the resulting reaction solution was washed with saturated sodium chloride aqueous solution, extracted with ethyl acetate, and the organic phase was collected. The organic phase was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 10:1). The purified organic phase was dried to obtain compound d1 (4.29 g) in 80.83% yield.

[0055] 3. Preparation of target compound 1

[0056] (1) Synthesis of compound e1 Compound d1 (4.25 g, 4 mmol) was placed in a reactor and dissolved in 50 mL of a mixed solution of 20% piperidine and DMF. The mixture was heated and stirred overnight at 45 °C, and monitored by TLC. After the reaction was completed, the resulting reaction solution was extracted with water and ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by column chromatography (dichloromethane:methanol = 5:1). The target fraction was collected, concentrated under reduced pressure, and dried under vacuum to obtain compound e1 (1.91 g), with a yield of 56.89%.

[0057] (2) Synthesis of compound f1 Compound e1 (1.26 g, 1.5 mmol), acetic anhydride (170.15 μL, 1.8 mmol), and DIEA (313.52 μL, 1.8 mmol) were placed in a reactor and dissolved in 30 mL of NMP. The mixture was heated and stirred overnight at 70 °C, and monitored by TLC. After the reaction was completed, the resulting reaction solution was washed with saturated sodium bicarbonate and sodium chloride aqueous solutions, extracted with ethyl acetate, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 8:1), and the target component was collected, concentrated under reduced pressure, and dried under vacuum to obtain compound f1 (1.07 g), with a yield of 80.94%.

[0058] (3) Synthesis of Compound 1 Compound f1 (881.31 mg, 1 mmol) was dissolved in 10 mL of ammonium carbonate buffer solution, and 15 mL of ethanol and 1 g of Pd / C were added. The reaction system was filled with hydrogen gas and stirred vigorously for 4 h. Compound f1 lost 2,2,2-trichloroethyl under the action of this reaction system. After the reaction was completed, Pd / C was removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol = 2:1). After drying, compound 1 (248.36 mg) was obtained, with a yield of 40.15%.

[0059] The structural identification data of compound 1 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 11.56 (s, 1H),9.53 (s, 2H), 8.61 (d, J = 3.3 Hz, 1H), 8.42 (s, 1H), 7.77 (ddd, J = 8.4, 2.8,1.4 Hz, 3H), 7.46 (d, J = 10.4 Hz, 1H), 7.05 – 6.99 (m, 1H), 6.92 (t, J= 5.0 Hz,1H), 6.88 – 6.82 (m, 2H), 6.57 (s, 1H), 6.18 (t, J = 3.3 Hz, 1H), 4.68 (s, 1H), 4.56 (dd, J = 6.0, 0.7 Hz, 2H), 4.24 (dt, J = 8.4, 5.7 Hz, 1H), 3.31 – 3.13 (m,3H), 1.94 (s, 3H), 1.81 – 1.69 (m, 2H), 1.69 – 1.44 (m, 4H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.92, 169.43, 168.34, 164.49, 161.58,159.23, 156.68, 152.04, 149.24, 148.79, 129.88, 128.13, 125.27, 113.13,52.71, 45.61, 45.04, 42.01, 28.80, 25.62, 22.51. Example 2 This embodiment provides the preparation of (S)-(N-(4-acetamido-5-((4-(4-((((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)butyl)amino)-5-oxopentyl)carbamoyl)phosphatonic acid (compound 2). The preparation method is the same as in Example 1, only the corresponding starting materials need to be changed. The yield of compound 2 is 35.23%.

[0060] The structural identification data of compound 2 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 11.49 (s, 1H),9.42 (s, 2H), 8.55 (d, J = 3.4 Hz, 1H), 8.41 (s, 1H), 7.71 (ddd, J = 8.5, 2.9,1.6 Hz, 3H), 7.44 (d, J = 10.8 Hz, 1H), 7.02 – 6.97 (m, 1H), 6.91 (t, J = 5.5 Hz,1H), 6.86 – 6.81 (m, 2H), 6.52 (s, 1H), 6.14 (t,J = 3.1 Hz, 1H), 4.66 (s, 1H), 4.52 (dd, J = 6.2, 0.5 Hz, 2H), 4.21 (dt, J = 8.6, 5.4 Hz, 1H), 3.28 – 3.12 (m,7H), 1.92 (s, 3H), 1.77 (d, J = 5.5 Hz, 2H), 1.76 – 1.66 (m, 2H), 1.64 – 1.42 (m, 4H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.93, 169.44, 168.35, 164.42, 161.59,159.24, 156.69, 152.05, 149.25, 148.80, 129.89, 128.14, 125.28, 113.14,57.21, 52.72, 45.62, 42.05, 38.22, 37.77, 28.81, 28.29, 25.63, 22.52. Example 3 This embodiment provides the preparation of (S)-(N-(4-acetamido-5-((6-(4-((((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)hexyl)amino)5-oxopentyl)carbamoyl)phosphatidyl (compound 3). The preparation method is the same as in Example 1, only the corresponding starting materials need to be changed. The yield of compound 3 is 30.76%.

[0061] The structural identification data of compound 3 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 11.43 (s, 1H), 9.42 (s, 2H), 8.54 (d, J = 4.2 Hz, 1H), 8.45 (s, 1H), 7.79 (ddd, J = 8.0, 2.9,1.5 Hz, 3H), 7.54 (s, 2H), 7.44 (d, J = 10.1 Hz, 1H), 7.06 – 6.97 (m, 1H), 6.96(t, J= 5.3 Hz, 1H), 6.84 – 6.81 (m, 2H), 6.53 (s, 1H), 6.12 (t, J = 3.9 Hz, 1H), 4.65 (s, 1H), 4.59 (dd, J = 6.1, 0.8 Hz, 2H), 4.26 (dt, J = 8.2, 5.8 Hz, 1H), 3.31 – 3.13 (m, 7H), 1.93 (s, 3H), 1.81 – 1.69 (m, 4H), 1.68 – 1.44 (m, 6H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.91, 169.41, 168.31, 164.42, 161.55,159.24, 156.66, 152.02, 149.21, 148.76, 145.75, 129.84, 128.12, 125.25,113.11, 57.34, 52.77, 45.66, 42.08, 38.11, 37.23, 36.75, 28.82, 28.25, 27.52,25.67, 22.58. Example 4 This embodiment provides the preparation of (S)-(N-(4-acetamido-5-((8-(4-((((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)octyl)amino)5-oxopentyl)carbamoyl)phosphatonic acid (compound 4), using the same method as in Example 1, only requiring the replacement of the corresponding starting materials. The yield of compound 4 was 36.55%.

[0062] The structural identification data of compound 4 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 11.48 (s, 1H),9.51 (s, 2H), 8.52 (d, J = 3.2 Hz, 1H), 8.41 (s, 1H), 7.73 (ddd, J = 8.2, 2.9,1.3 Hz, 3H), 7.47 (d, J = 10.6 Hz, 1H), 7.05 – 6.94 (m, 1H), 6.90 (t, J= 5.2 Hz,1H), 6.83 – 6.82 (m, 2H), 6.61 (s, 1H), 6.54 (s, 2H), 6.12 (t, J = 3.1 Hz, 1H), 4.67 (s, 1H), 4.51 (dd, J = 6.5, 0.6 Hz, 2H), 4.21 (dt, J = 8.3, 5.8 Hz, 1H), 3.34 – 3.16 (m, 7H), 1.90 (s, 3H), 1.83 – 1.72 (m, 6H), 1.68 – 1.43 (m, 8H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.94, 169.46, 168.35, 167.42, 164.43,161.52, 159.26, 156.64, 155.27, 152.12, 149.23, 148.75, 144.06, 129.83,128.12, 125.24, 122.37, 113.12, 52.74, 45.66, 42.02, 38.21, 37.27, 36.73,28.86, 28.23, 25.65, 22.57. Example 5 This embodiment provides the preparation of (S)-(N-(4-acetamido-5-((10-(4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)decyl)amino)5-oxopentyl)carbamoyl)phosphatidyl acid (compound 5), using the same method as in Example 1, only requiring the replacement of the corresponding starting materials. The yield of compound 5 was 21.18%.

[0063] The structural identification data of compound 5 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 11.42 (s, 1H),9.42 (s, 2H), 8.53 (d, J = 4.0 Hz, 1H), 8.47 (s, 1H), 7.79 (dd, J = 8.4, 2.9 Hz, 3H), 7.49 (d, J = 10.6 Hz, 1H), 7.03 – 6.92 (m, 1H), 6.87 (t, J= 5.1 Hz, 1H), 6.81 – 6.73 (m, 2H), 6.53 (s, 1H), 6.14 (t, J = 3.3 Hz, 1H), 4.62 (s, 1H), 4.46(dd, J = 6.2, 0.7 Hz, 2H), 4.21 (dt, J = 8.4, 5.3 Hz, 1H), 3.36 – 3.12 (m, 7H), 2.38 – 2.01 (m, 2H), 1.91 (d, J = 5.1 Hz, 2H), 1.83 (s, 3H), 1.51 – 1.41 (m, 6H), 1.40 – 1.26 (m, 10H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.94, 171.72, 170.11, 169.46, 168.35,164.41, 161.55, 159.21, 158.56, 156.64, 152.01, 149.22, 148.76, 144.31,129.86, 128.11, 125.26, 122.26, 121.23, 113.15, 57.32, 52.76, 47.83, 45.66,42.05, 37.73, 28.85, 28.26, 25.65, 22.53. Example 6 This embodiment provides the preparation of (S)-(N-(4-acetamido-5-(((2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)acetamido)methyl)amino)-5-oxopentyl)carbamoyl)phosphatidyl (compound 6), which is prepared by the same method as in Example 1, except that the corresponding raw materials are changed. The yield of compound 6 is 19.53%.

[0064] The structural identification data for compound 6 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.08 (s, 1H),8.13 (s, 2H), 7.96 (t, J = 3.8 Hz, 1H), 7.71 (t, J = 3.4 Hz, 1H), 7.38 (d, J= 8.2Hz, 1H), 6.31 (s, 1H), 6.12 (s, 2H), 5.59 (t, J = 3.6 Hz, 1H), 4.77 (s, 1H), 4.46 – 4.26 (m, 3H), 3.88 (s, 2H), 3.19 (tdd, J = 5.3, 3.4, 1.2 Hz, 2H), 1.94(s, 3H), 1.77 – 1.49 (m, 5H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.36, 171.38, 170.46, 158.45, 157.48,153.25, 150.87, 148.75, 116.46, 57.74, 42.08, 37.35, 35.03, 28.88, 25.45,22.03. Example 7: This embodiment provides the preparation of (S)-(N-(4-acetamido-5-((4-(2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)acetamido)butyl)amino)-5-oxopentyl)carbamoyl)phosphatonic acid (compound 7). The preparation method is the same as in Example 1, only the corresponding raw materials need to be changed. The yield of compound 7 is 15.87%.

[0065] The structural identification data of compound 7 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.09 (s, 1H), 8.29 (s, 1H), 8.15 (s, 2H), 7.33 (s, 1H), 7.14 (t, J = 4.9 Hz, 1H), 7.04 (t, J =5.4 Hz, 1H), 6.31 (s, 1H), 6.12 (s, 2H), 5.56 (t, J = 3.9 Hz, 1H), 4.72 (s, 1H), 4.33 – 4.25 (m, 1H), 3.87 (s, 2H), 3.24 – 3.08 (m, 6H), 1.92 (s, 3H), 1.89 – 1.53 (m, 8H). 13 C NMR (101 MHz, DMSO- d6) δ 173.35, 171.32, 170.47, 158.45, 157.47,153.27, 150.83, 148.76, 116.43, 57.77, 42.02, 38.76, 35.24, 28.83, 27.52,27.01, 25.26, 24.27, 22.88. Example 8 This embodiment provides the preparation of (S)-(N-(4-acetamido-5-((6-(2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)acetamido)hexyl)amino)-5-oxopentyl)carbamoyl)phosphatidyl (compound 8). The preparation method is the same as in Example 1, only the corresponding raw materials need to be changed. The yield of compound 8 is 13.87%.

[0066] The structural identification data for compound 8 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.13 (s, 1H), 8.31 (s, 1H), 8.12 (s, 2H), 7.30 (s, 1H), 7.16 (t, J = 4.4 Hz, 1H), 7.07 (t, J =5.6 Hz, 1H), 6.33 (s, 1H), 6.15 (s, 2H), 5.57 (t, J = 3.2 Hz, 1H), 4.77 (s, 1H), 4.40 – 4.20 (m, 1H), 3.82 (s, 2H), 3.34 – 2.97 (m, 6H), 1.89 (s, 3H), 1.85 – 1.39 (m, 12H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.32, 171.33, 170.43, 158.49, 157.41,153.29, 150.89, 148.70, 116.44, 57.71, 41.04, 38.72, 35.21, 30.85, 30.02,28.89, 27.98, 27.17, 25.23, 24.22, 22.81. Example 9 This embodiment provides the preparation method of (S)-(N-(4-acetamido-5-((8-(2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)acetamido)octyl)amino)-5-oxopentyl)carbamoyl)phosphatic acid (compound 9) as in Example 1, only requiring the replacement of the corresponding raw materials. The yield of compound 9 is 13.87%.

[0067] The structural identification data of compound 9 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.09 (s, 1H), 8.31 (s, 1H), 8.09 (s, 2H), 7.21 (s, 1H), 7.15 (t, J = 4.2 Hz, 1H), 7.01 (t, J =5.4 Hz, 1H), 6.29 (s, 1H), 6.11 (s, 2H), 5.52 (t, J = 3.6 Hz, 1H), 4.70 (s, 1H), 4.31 – 4.22 (m, 1H), 3.80 (s, 2H), 3.31 – 3.03 (m, 6H), 1.87 (s, 3H), 1.81 – 1.25 (m, 16H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.36, 171.35, 170.43, 158.47, 157.45,153.27, 150.84, 148.76, 116.43, 57.77, 41.03, 38.76, 35.72, 30.94, 30.02,28.48, 28.01, 27.91, 27.18, 26.33, 25.23, 24.26, 22.82. Example 10 This embodiment provides a method for preparing (S)-(N-(4-acetamido-5-((10-(2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)acetamido)decyl)amino)-5-oxopentyl)carbamoyl)phosphatidyl acid (compound 10) as in Example 1, only requiring the replacement of the corresponding raw materials. The yield of compound 10 is 21.45%.

[0068] The structural identification data of compound 10 are as follows: 1 H NMR (400 MHz, DMSO-d6 ) δ 12.09 (s, 1H), 8.26 (s, 1H), 8.10 (s, 2H), 7.31 (s, 1H), 7.16 (t, J = 4.8 Hz, 1H), 7.09 (t, J =5.1 Hz, 1H), 6.37 (s, 1H), 6.18 (s, 2H), 5.52 (t, J = 3.3 Hz, 1H), 4.71 (s, 1H), 4.45 – 4.16 (m, 1H), 3.82 (s, 2H), 3.33 – 2.97 (m, 6H), 1.84 (s, 3H), 1.82 – 1.21 (m, 20H). 13 C NMR (101 MHz, DMSO- d6 ) δ 173.29, 171.34, 170.48, 158.43, 157.48,153.23, 150.82, 148.78, 116.47, 57.73, 41.08, 38.23, 35.24, 30.96, 30.16,29.67, 29.32, 29.01, 28.88, 27.49, 26.14, 26.12, 25.21, 24.27, 22.84. Example 11 This embodiment provides the preparation of (N-((S)-4-acetamido-5-((((R)-2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-2-phenylacetamido)methyl)amino)-5-oxopentyl)carbamoyl)phosphatidyl (compound 11). The preparation method is the same as in Example 1, only the corresponding raw materials need to be changed. The yield of compound 11 is 16.27%.

[0069] The structural identification data of compound 11 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.09 (s, 1H), 8.81 (s, 1H), 8.13 (s, 2H), 7.59 (t, J = 3.2 Hz, 1H), 7.54 (t, J = 3.3 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.35 (d, J= 4.8 Hz, 4H), 7.31 – 7.25 (m, 1H), 6.31 (s,1H), 6.12 (s, 2H), 5.59 (t, J = 3.3 Hz, 1H), 5.24 (s, 1H), 4.77 (s, 1H), 4.62(dt, J = 14.5, 3.2 Hz, 1H), 4.41 (dt, J = 14.5, 3.2 Hz, 1H), 4.30 (dt, J = 8.4, 5.7Hz, 1H), 3.19 (tdd, J = 5.2, 3.3, 1.0 Hz, 2H), 1.82 (s, 3H), 1.77 – 1.29 (m, 4H). 13 C NMR (101 MHz, DMSO- d6 ) δ 171.96, 170.42, 158.85, 157.83, 153.11,150.06, 140.26, 134.58, 129.64, 128.97, 128.42, 127.54, 116.86, 56.15, 54.24,52.75, 41.28, 28.77, 24.63, 22.51. Example 12 This embodiment provides the preparation of (N-((S)-4-acetamido-5-((4-((R)-2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-2-phenylacetamido)butyl)amino)-5-oxopentyl)carbamoyl)phosphatidyl (compound 12). The preparation method is the same as in Example 1, only the corresponding raw materials need to be changed. The yield of compound 12 is 18.79%.

[0070] The structural identification data of compound 12 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.03 (s, 1H), 8.86 (s, 1H), 8.15 (s, 2H), 7.39 – 7.32 (m, 5H), 7.30 – 7.23 (m, 2H), 7.04(t, J = 5.7 Hz, 1H), 6.32 (s, 1H), 6.15 (s, 2H), 5.54 (t, J= 3.6 Hz, 1H), 5.17(d, J = 4.3 Hz, 1H), 4.79 (s, 1H), 4.40 – 4.17 (m, 1H), 3.36 – 2.93 (m, 6H), 1.93 (s, 3H), 1.87 – 1.28 (m, 8H). 13 C NMR (101 MHz, DMSO- d6 ) δ 171.95, 170.98, 170.44, 158.88, 157.84,153.17, 150.34, 140.27, 134.54, 129.68, 128.93, 128.15, 127.53, 116.89,56.16, 54.23, 41.29, 38.94, 28.74, 27.98, 27.14, 24.62, 22.57. Example 13 This embodiment provides the preparation of (N-((S)-4-acetamido-5-((6-(((R)-2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-2-phenylacetamido)hexyl)amino)-5-oxopentyl)carbamoyl)phosphatidyl (compound 13), using the same method as in Example 1, only requiring the replacement of the corresponding starting materials. The yield of compound 13 was 19.29%.

[0071] The structural identification data of compound 13 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.05 (s, 1H), 8.71 (s, 1H), 8.18 (s, 2H), 7.38 – 7.31 (m, 5H), 7.29 – 7.20 (m, 2H), 7.01(t, J = 5.6 Hz, 1H), 6.31 (s, 1H), 6.10 (s, 2H), 5.58 (t, J = 3.6 Hz, 1H), 5.14(d, J = 3.6 Hz, 1H), 4.71 (s, 1H), 4.46 – 4.21 (m, 1H), 3.42 – 2.90 (m, 6H), 1.92 (s, 3H), 1.86 – 1.19 (m, 12H). 13C NMR (101 MHz, DMSO- d6 ) δ 171.98, 170.94, 170.43, 158.84, 157.87,153.14, 150.08, 140.25, 134.54, 129.67, 128.93, 128.47, 127.57, 116.82,56.18, 54.22, 42.29, 38.85, 37.93, 30.05, 28.79, 27.16, 27.12, 24.68, 22.53. Example 14 This embodiment provides the preparation of (N-((S)-4-acetamido-5-((8-((R)-2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)-2-phenylacetamido)octyl)amino)-5-oxopentyl)carbamoyl)phosphatic acid (compound 14). The preparation method is the same as in Example 1, only the corresponding raw materials need to be changed. The yield of compound 14 is 19.31%.

[0072] The structural identification data of compound 14 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.11 (s, 1H), 8.82 (s, 1H), 8.15 (s, 2H), 7.41 – 7.30 (m, 5H), 7.29 – 7.23 (m, 2H), 7.02(t, J = 5.4 Hz, 1H), 6.34 (s, 1H), 6.19 (s, 2H), 5.62 (t, J = 3.2 Hz, 1H), 5.15(d, J = 4.2 Hz, 1H), 4.79 (s, 1H), 4.52 – 4.20 (m, 1H), 3.43 – 3.08 (m, 6H), 1.97 (s, 3H), 1.84 – 1.07 (m, 16H). 13 C NMR (101 MHz, DMSO- d6) δ 171.99, 170.95, 170.48, 158.84, 157.83,153.17, 150.02, 140.28, 134.53, 129.65, 128.98, 128.48, 127.57, 116.81,56.18, 54.23, 42.21, 38.82, 37.98, 30.09, 29.38, 29.34, 28.75, 27.67, 27.13,24.68, 22.56. Example 15 This embodiment provides the preparation of (N-((S)-4-acetamido-5-((10-((R)-2-((2-amino-6-oxo-6,9-dihydro-1H-purine-8-yl)thio)-2-phenylacetamido)decyl)amino)5-oxopentyl)carbamoyl)phosphatidyl (compound 15), which is prepared by the same method as in Example 1, except that the corresponding raw materials are changed. The yield of compound 15 is 17.26%.

[0073] The structural identification data of compound 15 are as follows: 1 H NMR (400 MHz, DMSO- d6 ) δ 12.04 (s, 1H), 8.73 (s, 1H), 8.14 (s, 2H), 7.43 – 7.32 (m, 5H), 7.28 (d, J = 4.7 Hz, 2H), 7.06(t, J = 5.1 Hz, 1H), 6.29 (s, 1H), 6.09 (s, 2H), 5.54 (t, J = 3.3 Hz, 1H), 5.12(d, J = 3.4 Hz, 1H), 4.72 (s, 1H), 4.39 – 4.18 (m, 1H), 3.29 – 2.93 (m, 6H), 1.95 (s, 3H), 1.86 – 1.04 (m, 20H). 13 C NMR (101 MHz, DMSO- d6) δ 171.95, 170.91, 170.45, 158.88, 157.83,153.17, 150.02, 140.27, 134.54, 129.67, 128.92, 128.45, 127.58, 116.82,56.18, 54.23, 42.26, 38.82, 37.57, 32.59, 30.23, 29.93, 29.28, 29.07, 28.72,27.93, 27.17, 24.64, 22.55. II. Bioactivity Assay Based on Examples 1-15, the in vitro antibacterial activity of BacPROTACs compounds 1-15, which target dihydropteroate synthase prepared in this invention, was determined to verify their inhibitory effect on different bacterial strains, so as to evaluate their potential application value as anti-infective drugs.

[0074] (1) Experimental materials Test compounds: Compounds 1-15 of the BacPROTACs targeting dihydropteroate synthase prepared in Examples 1-15 of this invention were dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) to prepare a stock solution of 5.12 mg / mL. After sterilization by filtration through a 0.22 μm microporous membrane, the solutions were dispensed into sterile EP tubes and stored at -20°C for later use. Before use, the solutions were diluted with MHB medium to the required concentration, ensuring that the final DMSO concentration did not exceed 1% (v / v) to eliminate the influence of the solvent on bacterial growth.

[0075] Positive control drug: Moxifloxacin (purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd., product number: BD43888) was used as a positive control according to the Clinical and Laboratory Standards Institute (CLSI) method. It was dissolved in an appropriate amount of DMSO to prepare a stock solution of 5.12 mg / mL, filtered sterilized using the same method, and stored at -20℃ for later use.

[0076] The strains used in this experiment included standard quality control strains and clinically isolated drug-resistant strains: Gram-positive bacteria: Staphylococcus aureus ( Staphylococcus aureus ATCC 25923, Bacillus subtilis ( Bacillus subtilis ATCC 6633, Staphylococcus epidermidis ( Staphylococcus epidermidis ATCC 12228, methicillin-resistant Staphylococcus aureus (ATCC 12228, methicillin-resistant Staphylococcus aureus) Methicillin-resistant Staphylococcus aureus Multidrug-resistant Staphylococcus aureus MRSA) ATCC 43300, multidrug-resistant Staphylococcus aureus (MRSA) Vancomycin-resistant Enterococcus faecalis Acinetobacter baumanniiMDRSA) MDRSA-171 (clinical isolate), vancomycin-resistant Enterococcus faecalis ( Escherichia coli VRE) VREfs-80 (clinical isolate); Gram-negative bacteria: Acinetobacter baumannii ( Pseudomonas aeruginosa ATCC 19606, Escherichia coli ( Salmonella enterica serovar Typhimurium ATCC 25922, Pseudomonas aeruginosa ( Multidrug-resistant Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ( Carbapenem-resistant Escherichia coli ATCC 14028, Multidrug-resistant Pseudomonas aeruginosa ( Multidrug-resistant Acinetobacter baumannii MDRPA) MDRPA-264 (clinical isolate), carbapenem-resistant Escherichia coli ( Figure 1 CR-EC) CR-EC-361 (clinical isolate), multidrug-resistant Acinetobacter baumannii ( Figure 2 MDRAB-183 (clinical isolate).

[0077] The standard control strains mentioned above were purchased from the American Type Culture Collection (ATCC). The remaining strains were clinically isolated drug-resistant strains, which were provided by the Institute of Antibiotics, Huashan Hospital, Fudan University, and were used after being identified using routine microbiological methods.

[0078] (2) Experimental methods The minimum inhibitory concentration (MIC) of BacPROTACs targeting dihydropteranoic acid synthase was determined using the microbroth dilution method; the specific procedures are as follows: (1) Culture medium preparation: Prepare 1 L of MHB culture medium (MHB culture medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.) according to the product label prescription, dispense it into Erlenmeyer flasks, autoclave at 121℃ for 15 min, and cool for later use; (2) Sample solution preparation: Weigh the BacPROTACs targeting dihydropteroate synthase and the positive control moxifloxacin of the test sample respectively, dissolve them in a mixed solvent of water / DMSO, and prepare a 5.12 mg / mL stock solution for later use. (3) Strain culture and preparation of bacterial suspension: Under aseptic conditions, the experimental strain was inoculated into MHB medium and cultured overnight at 37°C and 200 rpm with shaking. The next day, it was transferred to fresh MHB medium at a ratio of 1:50 and cultured at 37°C until the logarithmic growth phase. Subsequently, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland units using MHB medium, and then diluted 100 times to obtain a concentration of 1×10⁻⁶. 6CFU / mL bacterial suspension, for later use; (4) Determination of MIC using the micro-broth two-fold dilution method: Take a sterile 96-well plate, add 10 μL of the test compound to each well, and perform serial dilution using the two-fold dilution method. Simultaneously, set up a positive control with the drug and a growth control group without the drug. Then, add 190 μL of diluted bacterial solution to each well to achieve a final bacterial concentration of 5 × 10⁻⁶. 5 CFU / mL, and incubate the 96-well plate in a 37°C constant temperature and humidity incubator for 20 h.

[0079] (5) Endpoint color interpretation: After incubation, add 20 μL of 0.01% resazurin solution to each well and continue incubation in a constant temperature and humidity chamber at 37℃ for 1-2 h. When the growth control well turns from blue to pink, the results can be observed. The lowest drug concentration that does not change color (remains blue) is the MIC value of the compound.

[0080] Table 1: Minimum inhibitory concentration (MIC, μg / mL) of the test drug against standard control strains

[0081] Table 2: Minimum inhibitory concentration (MIC, μg / mL) of the test drug against drug-resistant strains

[0082] As shown in Tables 1 and 2, the BacPROTACs 1-15 targeting dihydropteroate synthase of this invention exhibit varying degrees of antibacterial activity against both tested Gram-positive and Gram-negative bacteria, with MICs ranging from 0.06 to 128 μg / mL. Compound 13 showed a more pronounced advantage against drug-resistant Gram-positive bacteria, with an MIC of 1 μg / mL against MRSA ATCC 43300, and MICs of 2 and 4 μg / mL against MDRSA-171 and VREfs-80, respectively. Its overall activity was significantly higher than that of the control drug moxifloxacin (2, 4, and 8 μg / mL, respectively), indicating stronger antibacterial potential in drug-resistant Staphylococcus aureus and drug-resistant Enterococcus systems. Regarding drug-resistant Gram-negative bacteria, compound 13 showed a MIC of 4 μg / mL against CR-EC-361, superior to the control drug's 8 μg / mL. Its MICs against MDRPA-264 and MDRAB-183 were 4 and 8 μg / mL, respectively, comparable to the control drug moxifloxacin, indicating that the degradation mechanism antibiotics of this invention can effectively combat bacterial resistance. Compound 13 also showed slightly higher MICs against non-resistant Escherichia coli ATCC 25922 and non-resistant Pseudomonas aeruginosa ATCC 27853 than the control drug. Overall, compound 13 exhibited relatively strong antibacterial potential across various strains, particularly showing outstanding activity against drug-resistant Gram-positive bacteria. In conclusion, BacPROTACs containing structures targeting dihydropteroate synthase demonstrate good potential in enhancing antibacterial activity against drug-resistant Gram-positive bacteria (including MRSA) and maintaining antibacterial activity against various drug-resistant Gram-negative bacteria, supporting their status as a candidate compound for combating drug-resistant bacteria.

[0083] Figure 3 The results of the MIC determination of compound 13 against MRSA ATCC 43300 are shown: In wells with a concentration of 1 μg / mL or higher, the medium remained blue, indicating that bacterial growth was inhibited; in wells with a concentration of 0.5 μg / mL or lower, the medium turned pink, indicating that bacterial growth was not inhibited. Therefore, the MIC of compound 13 against MRSA ATCC 43300 is 1 μg / mL. ​ The MIC determination results for compound 13 against multidrug-resistant Staphylococcus aureus MDRSA-171 are shown: the medium remained blue in wells with concentrations of 2 μg / mL and above. Therefore, the MIC of compound 13 against MDRSA-171 is 2 μg / mL. ​The MIC determination results for compound 13 against carbapenem-resistant Escherichia coli CR-EC-361 were shown: the medium remained blue in wells with a concentration of 4 μg / mL or higher; the medium turned pink in wells with a concentration of 2 μg / mL or lower. Therefore, the MIC of compound 13 against CR-EC-361 is 4 μg / mL.

[0084] In summary, the BacPROTACs targeting dihydropteroate synthase prepared in this invention have good in vitro antibacterial activity. In particular, compound 13 has better activity against drug-resistant Gram-positive bacteria than the existing clinical drug moxifloxacin, and has the potential to be further developed into an anti-infective drug.

[0085] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A type of BacPROTAC targeting dihydropteridine synthase, characterized in that, The compounds are those represented by Formula I, Formula II, or Formula III, or pharmaceutically acceptable salts thereof, as well as solvent compounds, enantiomers, diastereomers, or mixtures thereof in any proportion, including racemic mixtures, of the compounds represented by Formula I, Formula II, or Formula III, or pharmaceutically acceptable salts thereof. The structural formula of the compound of formula I, formula II or formula III is: or or Linker is one of the following linking groups: Where n = 0, 2, 4, 6, 8.

2. The BacPROTACs targeting dihydropteridine synthase according to claim 1, characterized in that, The compound is selected from the following compounds or their pharmaceutically acceptable salts: 。 3. The BacPROTACs targeting dihydropteridine synthase according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by BacPROTACs targeting dihydropteroate synthase with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, oxalic acid, malonic acid, succinic acid, citric acid, malic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, glutamic acid, or aspartic acid.

4. A method for preparing BacPROTACs targeting dihydropteridine synthase as described in any one of claims 1-3, characterized in that, The following steps are included: (1) Using (benzyloxycarbonyl)-L-arginine as a raw material, benzyl bromide was used to protect the carboxyl group to obtain compound a1; then it was reacted with bis(2,2,2-trichloroethyl)phosphoryl chloride to obtain compound a2; Compound a2 was deprotected in an acidic solvent by Pd / C and hydrogen to give compound a3; compound a3 was then introduced with an Fmoc protecting group to give intermediate compound a4. Among them, compound a1 is Compound a2 is Compound a3 is Compound a4 is ; (2) Using 4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzoic acid as a raw material, acid-amine condensation reaction was carried out with alkylamines of different lengths to obtain the corresponding compounds b1-b5; Among them, compounds b1-b5 are n = 0, 2, 4, 6, 8; Alternatively, with 2-((2-amino-6-oxo-6,9-dihydro-1) H Using purine-8-yl)thioacetic acid as a raw material, acid-amine condensation reaction was carried out with alkylamines of different lengths to obtain the corresponding compounds b6-b10; Among them, compounds b6-b10 are n = 0, 2, 4, 6, 8; Or, with ( R )-2-((2-amino-6-oxo-6,9-dihydro-1 H Using purine-8-yl)thio)-2-phenylacetic acid as a raw material, an acid-amine condensation reaction was carried out with alkylamines of different lengths to obtain the corresponding compounds b1-b15; Among them, compounds b11-b15 are ; (3) Compound b1-compound b15 loses the Boc group under the action of TFA to generate compound c1-compound c15; Among them, compounds c1-c5 are n=0, 2, 4, 6, 8; compounds c6-c10 are n = 0, 2, 4, 6, 8; compounds c11-c15 are n = 0, 2, 4, 6, 8; (4) Compound a4 reacts with compound c1-compound c15 to form compound d1-compound d15; The structural formulas of compounds d1-d5 are as follows: n = 0, 2, 4, 6, 8; d6-d10 are n=0, 2, 4, 6, 8; the structural formulas of compounds d11-d15 are as follows: n = 0, 2, 4, 6, 8; (5) Compound d1-compound d15 loses the 9-fluorenylmethoxycarbonyl protecting group under the action of a 20% piperidine and DMF mixed solution to generate compound e1-compound e15; Among them, compounds e1-e5 are ，n=0、2、4、6、8； Compounds e6-e10 are ，n=0、2、4、6、8； Compounds e11-e15 are n = 0, 2, 4, 6, 8; (6) Compound e1-compound e15 is introduced with acetamide under the action of acetic anhydride and DIEA to generate compound f1-compound f15; Among them, compounds f1-f5 are n = 0, 2, 4, 6, 8; Compounds f6-f10 are n = 0, 2, 4, 6, 8; Compounds f11-f15 are n = 0, 2, 4, 6, 8; (7) Dissolve compound f1-compound f15 in ammonium carbonate buffer solution, add ethanol and palladium on carbon catalyst, and under hydrogen atmosphere, compound f1-compound f15 removes 2,2,2-trichloroethyl group under the action of the reaction system to generate target compound 1-compound 15; Among them, compounds 1-5 are n = 0, 2, 4, 6, 8; Compounds 6-10 are n=0, 2, 4, 6, 8; compounds 11-15 are n = 0, 2, 4, 6, 8.

5. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio of benzyloxycarbonyl-L-arginine to benzyl bromide is 1:(1~2), and the molar ratio of compound a1 to bis(2,2,2-trichloroethyl)phosphoryl chloride is 1:(1~2); in step (2), 4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzoic acid, 2-((2-amino-6-oxo-6,9-dihydro-1 H -purine-8-yl)thio)acetic acid, ( R )-2-((2-amino-6-oxo-6,9-dihydro-1 H The molar ratio of purine-8-yl)thio)-2-phenylacetic acid to alkylamine chains of different lengths is 2:1; in step (4), the molar ratio of compound a4 to compound c1-compound c15 is (1~2):

1.

6. The preparation method according to claim 4, characterized in that, In step (1), the reaction temperature of compound a2 is room temperature, the solvent used is acetonitrile, and the reaction time is 7h. In steps (2) and (4), in the synthesis of compounds b1-b15 and compounds d1-d15, the carboxylic acid is activated with HATU and then reacted with Linker of different lengths. The solvent used is N,N-dimethylformamide. In step (5), the solvent used is a mixed solution of 20% piperidine and DMF to remove the 9-fluorenylmethoxycarbonyl protecting group. In step (6), acetylation is carried out with acetic anhydride. The solvent used is NMP solution.

7. The use of a BacPROTAC targeting dihydropteridine synthase as described in any one of claims 1-3 in the preparation of an antibacterial drug.

8. The application according to claim 7, characterized in that, The bacteria include Gram-positive bacteria, Gram-negative bacteria, and drug-resistant bacteria.

9. The application according to claim 7, characterized in that, The drug is a preparation for use alone or in combination with other antibacterial drugs, and the preparation is an oral tablet, capsule, granule, syrup, premix or micro-pellet, or a liniment or injection for non-oral administration.

10. A pharmaceutical composition, characterized in that, The active ingredient comprises BacPROTACs targeting dihydropteroate synthase as described in any one of claims 1-3, and pharmaceutically acceptable excipients or diluents.

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