Ciprofloxacin derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and provides a ciprofloxacin derivative as well as a preparation method and application thereof. The ciprofloxacin derivative provided by the invention has a structure as shown in a formula I, an electrostatic effect sequence (amino acid part) with positive charges is connected to a lipid part, and then a series of amphiphilic ciprofloxacin derivatives are formed through conjugation of Linker and ciprofloxacin. The electrostatic effect sequence with positive charges can enhance the interaction with a bacterial cell membrane with negative charges through electrostatic interaction, then the lipid part can be inserted into a phospholipid bilayer of the bacterial cell membrane, so that the integrity of the bacterial cell membrane is damaged, the ciprofloxacin derivative enters the cell, and the ciprofloxacin derivative is degraded. Finally, bacterial cell content leakage, bacterial chromosome breakage and bacterial cell death are caused. According to the amphiphilic ciprofloxacin derivative disclosed by the invention, the amphiphilic structure and the membrane destruction mechanism of the antibacterial peptide are simulated, so that the ciprofloxacin derivative shows the unique advantage that the drug resistance is not easy to generate. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a ciprofloxacin derivative and a preparation method and application thereof. Background Art

[0002] The third-generation fluoroquinolone ciprofloxacin has excellent antibacterial activity and pharmacokinetic properties with few side effects and has been used clinically for the treatment of various bacterial infections for about 30 years. They inhibit bacterial nucleic acid synthesis by destroying topoisomerase IV and DNA gyrase and causing bacterial chromosome breakage, thereby exerting their antibacterial effects.

[0003] Ciprofloxacin is becoming increasingly ineffective due to the urgent and widespread spread of drug-resistant pathogens. The main mechanisms leading to resistance to quinolones are gene mutation (topoisomerase variation; changes in bacterial cell membrane permeability; bacterial biofilm formation) and acquisition of resistance plasmids (plasmid-mediated resistance; bacterial active drug efflux mechanism). How to reverse the problem of ciprofloxacin's high tendency to develop resistance has long plagued researchers in this field, and there is no effective strategy to date. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a ciprofloxacin derivative and a preparation method and application thereof. The ciprofloxacin derivative provided by the present invention is not easy to produce drug resistance and has high bactericidal activity in mice.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a ciprofloxacin derivative having a structure shown in Formula I:

[0007]

[0008] In Formula I, the lipid moiety is an alkyl group, a benzyl group or a substituted benzyl group, and the number of carbon atoms in the alkyl group is 5 to 16;

[0009] The types of amino acids in the amino acid part include one or more of lysine, histidine and arginine, and the number of amino acids in the amino acid part is 1 to 4;

[0010] The lipid portion and the amino acid portion are bonded via an amide bond;

[0011] The amino acid portion and the linker are bonded via an amide bond;

[0012] The linker and the ciprofloxacin moiety are bonded via an amide bond.

[0013] The invention provides a ciprofloxacin derivative.

[0014] The ciprofloxacin derivative provided by the present invention connects a positively charged electrostatic effect sequence (amino acid part) to a lipid part, and then conjugates with ciprofloxacin through a linker to form a series of amphiphilic ciprofloxacin derivatives. The positively charged electrostatic effect sequence can enhance the interaction with the negatively charged bacterial cell membrane through electrostatic action, and then the lipid part can be inserted into the phospholipid bilayer of the bacterial cell membrane, resulting in the destruction of the integrity of the bacterial cell membrane, and the ciprofloxacin derivative enters the cell, eventually leading to leakage of bacterial cell contents, bacterial chromosome breakage and bacterial cell death. The ciprofloxacin derivative of the present invention simulates the amphiphilicity and membrane destruction mechanism of AMPs (antimicrobial peptides, AMP) so that the ciprofloxacin derivative exhibits a unique advantage of not being easy to produce drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The hemolytic toxicity of compounds Cip, 16u, 22e, 22g, and 28b on rat red blood cells;

[0016] Figure 2 The cytotoxicity results of compounds Cip, 16u, 22e, 22g, and 28b on L929 cells at different concentrations;

[0017] Figure 3 The membrane destruction results of drugs at different concentrations;

[0018] Figure 4 The results of the evaluation of the tendency of compounds Cip and 28b to induce drug resistance in MRSA (a) and E.coil (b);

[0019] Figure 5 In vivo antibacterial activity of compound 28b and ciprofloxacin in subcutaneous infection in mice. DETAILED DESCRIPTION

[0020] The present invention provides a ciprofloxacin derivative having a structure shown in Formula I:

[0021]

[0022] In the present invention, in Formula I, the lipid moiety is an alkyl group, a benzyl group or a substituted benzyl group, and the number of carbon atoms of the alkyl group is 5 to 16. In the present invention, the alkyl group is preferably a straight-chain alkyl group, and the number of carbon atoms of the straight-chain alkyl group is preferably 5 to 16, and specifically preferably 5, 6, 8, 9, 10, 11, 12, 14 or 16. In the present invention, the substituted benzyl group is preferably a monosubstituted benzyl group or a disubstituted benzyl group, the monosubstituted benzyl group is preferably a 2-substituted benzyl group or a 4-substituted benzyl group, and the disubstituted benzyl group is preferably a 2,6-disubstituted benzyl group. In the present invention, the substituents in the substituted benzyl group preferably include one or more of fluorine, chlorine, bromine and phenyl. In the present invention, the structural formula of the lipid moiety is specifically shown as follows:

[0023]

[0024] In the present invention, in Formula I, the types of amino acids in the amino acid part include one or more of lysine (Lys), histidine (His) and arginine (Arg), and the number of amino acids in the amino acid part is 1 to 4, preferably 1, 2, 3 or 4. In the present invention, when the number of amino acids in the amino acid part is multiple, the types of multiple amino acids are preferably multiple combinations, and the same amino acid can also be repeated. In the present invention, the structural formula of the amino acid part is preferably as follows:

[0025]

[0026] In the present invention, the Linker is preferably

[0027] The * portion in the Linker is connected to the ciprofloxacin portion.

[0028] In the present invention, the lipid part and the amino acid part are bonded via an amide bond. In the present invention, the acyl group in the amide bond between the lipid part and the amino acid part is preferably derived from the amino acid part, and the amino group is preferably derived from the lipid part.

[0029] In the present invention, the amino acid part and the linker are bonded via an amide bond. In the present invention, the acyl group in the amide bond between the amino acid part and the linker is preferably from the linker, and the amino group is preferably from the amino acid part.

[0030] In the present invention, the linker and ciprofloxacin are bonded via an amide bond. In the present invention, in the amide bond between the linker and ciprofloxacin, the acyl group is preferably from the linker, and the amino group is preferably from ciprofloxacin.

[0031] In the present invention, the ciprofloxacin derivative preferably has any of the following structures:

[0032]

[0033] In 16a-u, when n is 1, R4 is

[0034] When n is 2, R4 is

[0035] When n is 3, R4 is

[0036] In 22a-g, R5 is The * portion in R5 is connected to the ciprofloxacin portion.

[0037] In 28a-e, R6 is

[0038] In a specific embodiment of the present invention, the ciprofloxacin derivative preferably has the following structure:

[0039]

[0040]

[0041]

[0042]

[0043] The present invention also provides a method for preparing the ciprofloxacin derivative described in the above technical solution, comprising the following steps:

[0044] The lipid part, the amino acid part, the linker and ciprofloxacin are bonded to obtain the ciprofloxacin derivative.

[0045] The following is based on The structural division is described in detail for the preparation methods.

[0046] In the present invention, the preparation method of the ciprofloxacin derivative having the structure shown in Formula 16a-u (hereinafter referred to as the first preparation method) comprises the following steps:

[0047] Swelling 2-chlorotrityl chloride resin to obtain a swollen resin; mixing the swollen resin, Fmoc-Lys(Boc)-OH shown in Formula 10 and N,N-diisopropylethylamine (DIPEA), and sequentially performing solid phase synthesis reaction, end-capping reaction and Fmoc removal to obtain a structural compound shown in Formula 11, wherein the structural compound shown in Formula 11 is an amino acid grafted compound containing one amino acid;

[0048] When n is 2 in the structure shown in Formula 16a-u, the present invention mixes the structural compound shown in Formula 11, Fmoc-Lys(Boc)-OH shown in Formula 10, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF), and sequentially performs solid phase synthesis reaction and removes Fmoc to obtain an amino acid grafted compound containing two amino acids; when n is 3 in the structure shown in Formula 16a-u, Fmoc-Lys(Boc)-OH shown in Formula 10, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are then added and mixed, and sequentially performs solid phase synthesis reaction and removes Fmoc to obtain an amino acid grafted compound containing three amino acids;

[0049] The amino acid grafted compound, terephthalic acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF) are mixed and subjected to a solid phase synthesis reaction to obtain a structural compound shown in Formula 12;

[0050] The solution of ciprofloxacin methyl ester is added dropwise to the solution of the compound of the structure shown in Formula 12, and solid phase synthesis reaction and cleavage reaction are sequentially performed to obtain the compound of the structure shown in Formula 13; the solution of the compound of the structure shown in Formula 12 comprises the compound of the structure shown in Formula 12, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and the solution of ciprofloxacin methyl ester comprises ciprofloxacin methyl ester, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF);

[0051] The solution of the compound of formula 13 and the solution of R4-H are mixed, and an amide condensation reaction is performed to obtain a compound of formula 14; the solution of the compound of formula 13 comprises the compound of formula 13, 1,2-dichloroethane (EDC), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and the solution of R4-H comprises R4-H, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF);

[0052] The compound of the structure represented by Formula 14 is subjected to a hydrolysis reaction to obtain a compound of the structure represented by Formula 15;

[0053] Remove Boc from the compound of the structure represented by Formula 15 to obtain the compound of the structure represented by Formula 16a-u;

[0054]

[0055] The present invention swells 2-chlorotrityl chloride resin to obtain a swollen resin; the swollen resin, Fmoc-Lys(Boc)-OH shown in Formula 10 and DIPEA are mixed, and solid phase synthesis reaction, end-capping reaction and Fmoc removal are sequentially performed to obtain a structural compound shown in Formula 11, wherein the structural compound shown in Formula 11 is an amino acid grafted compound containing one amino acid. In the present invention, the swelling reagent is preferably dichloromethane. In the present invention, the amount ratio of the 2-chlorotrityl chloride resin and dichloromethane is preferably 0.22mmol:6mL. In the present invention, the swelling temperature is preferably room temperature, and the time is preferably 15min. In the present invention, the molar ratio of the 2-chlorotrityl chloride resin and Fmoc-Lys(Boc)-OH is preferably 1:2, and the molar ratio of Fmoc-Lys(Boc)-OH and DIPEA is preferably 0.44:0.79. In the present invention, the temperature of the solid phase synthesis reaction is preferably room temperature, the time is preferably 1 to 5 hours, the solid phase synthesis reaction is preferably carried out under shaking conditions, and the solid phase synthesis reaction is preferably carried out in a solid phase synthesis tube. After the solid phase synthesis reaction, the present invention preferably further comprises: discharging the reaction solution and washing the resin, the washing comprising sequentially performing DMF washing and DCM washing, the number of DMF washing is preferably 3 times, and the number of DCM washing is preferably 3 times. In the present invention, the reagent for the end-capping reaction is preferably a DIPEA / MeOH / DCM mixed solution, in which the volume ratio of DIPEA, MeOH and DCM is preferably 1:2:17, the amount ratio of the 2-chlorotrityl chloride resin and the DIPEA / MeOH / DCM mixed solution is preferably 0.22mmol:5mL, the temperature of the end-capping reaction is preferably room temperature, and the time is preferably 30min. After the end-capping reaction, the present invention preferably further comprises: washing the end-capped resin, and the washing preferably comprises washing with DCM and washing with DMF in sequence, the number of washings with DCM is preferably 3 times, and the number of washings with DMF is preferably 3 times. In the present invention, the reagent for removing Fmoc is preferably a piperidine / DMF mixed solution, and the volume fraction of piperidine in the piperidine / DMF mixed solution is preferably 20%. In the present invention, the time for removing Fmoc is preferably 10min, and the number of times is preferably 2 times.

[0056] After obtaining the compound of structure shown in formula 11, when n in the structure shown in formula 16a-u is 2, the present invention mixes the compound of structure shown in formula 11, Fmoc-Lys(Boc)-OH, DIC, HOBt and DMF shown in formula 10, and sequentially performs solid phase synthesis reaction and removes Fmoc to obtain an amino acid grafted compound containing two amino acids; when n in the structure shown in formula 16a-u is 3, then Fmoc-Lys(Boc)-OH, DIC, HOBt and DMF shown in formula 10 are added and mixed, and sequentially performs solid phase synthesis reaction and removes Fmoc to obtain an amino acid grafted compound containing three amino acids.

[0057] In the present invention, the molar ratio of the 2-chlorotrityl chloride resin to Fmoc-Lys(Boc)-OH is preferably 1:2, the molar ratio of the 2-chlorotrityl chloride resin to DIC is preferably 1:4, the molar ratio of DIC to HOBt is preferably 1:1, and the amount ratio of the 2-chlorotrityl chloride resin to DMF is preferably 0.22mmol:3mL. In the present invention, the parameters and process of the solid phase synthesis reaction and the removal of Fmoc are consistent with the solid phase synthesis reaction and the removal of Fmoc in the process of preparing compound 11, and are not repeated here.

[0058] After obtaining the amino acid grafted compound, the present invention mixes the amino acid grafted compound, terephthalic acid, HCTU, DIPEA and DMF, and performs a solid phase synthesis reaction to obtain a structural compound shown in Formula 12.

[0059] In the present invention, the molar ratio of the 2-chlorotrityl chloride resin to terephthalic acid is preferably 1:5, the molar ratio of terephthalic acid to HCTU is preferably 10:9, the molar ratio of HCTU to DIPEA is preferably 1:2, and the amount ratio of 2-chlorotrityl chloride resin to DMF is preferably 0.22mmol:3mL. In the present invention, the temperature of the solid phase synthesis reaction is preferably room temperature, the time is preferably 1 to 5h, and the solid phase synthesis reaction is preferably carried out under shaking conditions. After the solid phase synthesis reaction, the present invention preferably further comprises: removing the solution and washing the resin, the washing comprising sequentially washing with DMF and washing with DCM, the number of DMF washings is preferably 3 times, and the number of DCM washings is preferably 3 times.

[0060] After obtaining the compound of structure shown in Formula 12, the solution of ciprofloxacin methyl ester is added dropwise to the solution of the compound of structure shown in Formula 12, and solid phase synthesis reaction and cleavage reaction are sequentially performed to obtain the compound of structure shown in Formula 13; the solution of the compound of structure shown in Formula 12 comprises the compound of structure shown in Formula 12, HCTU, DIPEA and DMF, and the solution of ciprofloxacin methyl ester comprises ciprofloxacin methyl ester, DIPEA and DMF.

[0061] In the present invention, in the solution of the structural compound shown in Formula 12, the molar ratio of 2-chlorotrityl chloride resin and HCTU is preferably 0.22:0.40, the molar ratio of HCTU and DIPEA is preferably 0.40:0.79, and the dosage ratio of DIPEA and DMF is preferably 0.79mmol:3mL. In the present invention, in the solution of ciprofloxacin methyl ester, the molar ratio of ciprofloxacin methyl ester and DIPEA is preferably 1:1, and the dosage ratio of ciprofloxacin methyl ester and DMF is preferably 0.44mmol:3mL. In the present invention, the molar ratio of 2-chlorotrityl chloride resin and ciprofloxacin methyl ester is preferably 1:2. In the present invention, the temperature of the solid phase synthesis reaction is preferably room temperature, and the time is preferably 1 to 5h. After the solid phase synthesis reaction, the present invention preferably further comprises: removing the solution, washing the resin with DMF and DCM in sequence, the number of DMF washings is preferably 3 times, and the number of DCM washings is preferably 3 times. In the present invention, the cleavage solution of the cleavage reaction is preferably a HFIP / DCM mixed solution, in which the volume ratio of HFIP and DCM is preferably 1:4, and the dosage ratio of the 2-chlorotrityl chloride resin and the cleavage solution is preferably 0.22mmol:8mL. In the present invention, the time of the cleavage reaction is preferably 2h. After the cleavage reaction, the present invention preferably further comprises: collecting the cleavage solution; washing the cleaved resin to obtain a washing solution; combining the cleavage solution and the washing solution to obtain a combined solution; performing reduced pressure distillation on the combined solution to obtain a crude compound; after dissolving the crude compound, filtering, and purifying the obtained filtrate by preparative HPLC and freeze-drying to obtain a structural compound shown in Formula 13. In the present invention, the washing reagent is preferably the cleavage solution, and the number of washings is preferably 3 times. In the present invention, the dissolving reagent is preferably a methanol aqueous solution, in which the volume ratio of methanol and water is preferably 1:1, and the filtering device is preferably a Whatman filter, and the pore size of the Whatman filter is preferably 0.22μm.

[0062] After obtaining the compound of structure shown in Formula 13, the present invention mixes the solution of the compound of structure shown in Formula 13 and the solution of R4-H, and performs an amide condensation reaction to obtain the compound of structure shown in Formula 14, wherein the solution of the compound of structure shown in Formula 13 includes the compound of structure shown in Formula 13, EDC, HOBt, DIPEA and DMF, and the solution of R4-H includes R4-H, DIPEA and DMF.

[0063] In the present invention, in the solution of the compound of the structure shown in Formula 13, the molar ratio of the compound of the structure shown in Formula 13 to EDC is preferably 0.17:0.26, the molar ratio of EDC to HOBt is preferably 1:1, the dosage ratio of the compound of the structure shown in Formula 13 to DMF is preferably 0.17mmol:8mL, and the molar ratio of the compound of the structure shown in Formula 13 to DIPEA is preferably 1:2. In the present invention, in the solution of R4-H, the molar ratio of R4-H to DIPEA is preferably 1:1, and the molar ratio of the compound of the structure shown in Formula 13 to R4-H is preferably 1:2. In the present invention, the temperature of the amide condensation reaction is preferably room temperature, the time is preferably 1 to 5h, and the amide condensation reaction is preferably carried out under stirring. After the amide condensation reaction is completed, the present invention preferably further comprises: quenching the reaction, extracting the obtained reaction liquid, washing, drying and distilling the obtained organic phase in sequence to obtain a structural compound shown in Formula 14; the reagent for the quenching reaction is preferably 1 mol / L hydrochloric acid, the extractant for the extraction is preferably DCM, the number of extractions is preferably 3 times, the washing reagent is preferably saturated NaCl washing, and the drying reagent is preferably anhydrous Na2SO4.

[0064] After obtaining the compound of structure shown in Formula 14, the present invention hydrolyzes the compound of structure shown in Formula 14 to obtain the compound of structure shown in Formula 15. In the present invention, the solvent of the hydrolysis reaction preferably includes methanol aqueous solution, the volume ratio of methanol and water in the methanol aqueous solution is preferably 5:1, and the amount ratio of the compound of structure shown in Formula 14 to the methanol aqueous solution is preferably 0.17mmol:6mL. In the present invention, the reagent of the hydrolysis reaction preferably also includes lithium hydroxide, and the molar ratio of the compound of structure shown in Formula 14 to lithium hydroxide is preferably 1:10. In the present invention, the temperature of the hydrolysis reaction is preferably room temperature, the time is preferably 2 to 4h, and the hydrolysis reaction is preferably carried out under stirring. After the hydrolysis reaction, the present invention preferably further includes: quenching the reaction, extracting the obtained reaction liquid, and washing, drying and reducing pressure distillation of the obtained organic phase in turn to obtain the compound of structure shown in Formula 15; the reagent of the quenching reaction is preferably 1mol / L hydrochloric acid; the extractant of the extraction is preferably DCM, the number of extractions is preferably 3 times, the washing reagent is preferably saturated NaCl, and the drying reagent is preferably anhydrous Na2SO4.

[0065] After obtaining the structural compound shown in Formula 15, the present invention removes Boc from the structural compound shown in Formula 15 to obtain the structural compound shown in Formula 16a-u. In the present invention, the reagent for removing Boc preferably includes trifluoroacetic acid dichloromethane solution (TFA / DCM solution). In the present invention, in the TFA / DCM solution, the volume ratio of TFA and DCM is preferably 19:1, and the amount ratio of the structural compound shown in Formula 15 and the TFA / DCM solution is preferably 0.17mmol:5mL. In the present invention, the temperature for removing Boc is preferably room temperature, the time is preferably 2h, and the removal of Boc is preferably carried out under stirring. After the removal of Boc is completed, the present invention preferably further comprises: performing reduced pressure distillation on the obtained feed liquid to obtain a crude product, dissolving the crude product and filtering it, and sequentially preparing HPLC purification and freeze-drying treatment on the obtained filtrate to obtain the structural compound shown in 16a-u; the dissolving reagent is preferably a methanol aqueous solution, in which the volume ratio of methanol to water is preferably 1:1, and the filtering equipment is preferably a Whatman filter, and the pore size of the Whatman filter is preferably 0.22 μm.

[0066] In the present invention, the preparation formula of the first preparation method is as follows:

[0067]

[0068] In the present invention, the preparation method of the ciprofloxacin derivative having the structure shown in Formula 22a-g (hereinafter referred to as the second preparation method) comprises the following steps:

[0069] The structural compound represented by Formula 11, Fmoc-Lys(Boc)-OH, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are mixed, and solid phase synthesis reaction, end-capping reaction and Fmoc removal are sequentially performed, and then the corresponding amino acid, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are added and mixed, and the solid phase synthesis reaction and Fmoc removal are repeated to obtain the structural compound represented by Formula 17;

[0070] When R5 is When the compound of the structure represented by Formula 17, N,N-diisopropylethylamine (DIPEA), succinic anhydride and N,N-dimethylformamide (DMF) are mixed, and a solid phase synthesis reaction is performed to obtain a compound of the structure represented by Formula 18;

[0071] When R5 is When the compound of the structure shown in Formula 17, Fmoc-R5-COOH, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are mixed, and solid phase synthesis reaction and Fmoc removal are carried out in sequence; the obtained Fmoc-removed feed solution, succinic anhydride, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF) are mixed, and solid phase synthesis reaction is carried out to obtain the compound of the structure shown in Formula 18; the Fmoc-R5-COOH has the following structure:

[0072] A solution of the compound of formula 18 and a solution of ciprofloxacin methyl ester are mixed, and solid phase synthesis reaction and cleavage reaction are sequentially performed to obtain a compound of formula 19; the solution of the compound of formula 18 comprises the compound of formula 18, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and the solution of ciprofloxacin methyl ester comprises ciprofloxacin methyl ester, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF);

[0073] A solution of a compound of Formula 19 and a solution of nonylamine are mixed to perform an amide condensation reaction to obtain a compound of Formula 20, wherein the solution of the compound of Formula 19 comprises the compound of Formula 19, 1,2-dichloroethane (EDC), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and the solution of nonylamine comprises nonylamine, DIPEA and N,N-dimethylformamide (DMF);

[0074] The compound of the structure represented by Formula 20 is subjected to a hydrolysis reaction to obtain a compound of the structure represented by Formula 21;

[0075] The compound of the structure represented by Formula 21 is subjected to Boc removal to obtain the compound of the structure represented by Formula 22a-g;

[0076]

[0077] The present invention mixes the structural compound shown in Formula 11, Fmoc-Lys(Boc)-OH, DIC, HOBt and DMF, performs solid phase synthesis reaction, end-capping reaction and removal of Fmoc in sequence, continues to add the corresponding amino acid, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) to mix, repeats the solid phase synthesis reaction and removal of Fmoc, and obtains the structural compound shown in Formula 17. In the present invention, the preparation method of the structural compound shown in Formula 11 is consistent with the above technical scheme, which is not repeated here. In the present invention, the molar ratio of the structural compound shown in Formula 11 and Fmoc-Lys(Boc)-OH is preferably 1:2, the molar ratio of Fmoc-Lys(Boc)-OH and DIC is preferably 1:2, the molar ratio of DIC and HOBt is preferably 1:1, and the amount ratio of HOBt and DMF is preferably 0.88mmol:3mL. In the present invention, the parameters of the solid phase synthesis reaction, the end-capping reaction and the removal of Fmoc and the subsequent post-treatment are consistent with those in the above technical solution and will not be described in detail herein.

[0078] After obtaining the structural compound shown in formula 17, when R5 is In the present invention, the compound of the structure shown in Formula 17, N,N-diisopropylethylamine (DIPEA), succinic anhydride and N,N-dimethylformamide (DMF) are mixed to perform a solid phase synthesis reaction to obtain a compound of the structure shown in Formula 18. In the present invention, the molar ratio of the compound of the structure shown in Formula 17 to N,N-diisopropylethylamine is preferably 0.22:1.76, and the molar ratio of the compound of the structure shown in Formula 17 to succinic anhydride is preferably 1:4. In the present invention, the temperature, time and post-treatment of the solid phase synthesis reaction are consistent with the above technical solution, which will not be repeated here.

[0079] After obtaining the structural compound shown in formula 17, when R5 is

[0080] In the present invention, the compound of the structure shown in Formula 17, Fmoc-R5-COOH, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are mixed, and solid phase synthesis reaction and Fmoc removal are carried out in sequence; the obtained Fmoc-removed feed solution, succinic anhydride, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF) are mixed, and solid phase synthesis reaction is carried out to obtain the compound of the structure shown in Formula 18; the Fmoc-R5-COOH has the following structure:

[0081] In the present invention, the molar ratio of the compound of formula 17 to Fmoc-R5-COOH is preferably 1:2, the molar ratio of the compound of formula 17 to DIC is preferably 1:4, the molar ratio of DIC to HOBt is preferably 1:1, and the amount ratio of the compound of formula 17 to DMF is preferably 0.22mmol:3mL. In the present invention, the solid phase synthesis reaction and the removal of Fmoc and subsequent post-treatment are consistent with the above technical solution, which will not be repeated here.

[0082] After obtaining the compound of structure shown in Formula 18, the present invention mixes the solution of the compound of structure shown in Formula 18 and the solution of ciprofloxacin methyl ester, and sequentially performs solid phase synthesis reaction and cleavage reaction to obtain the compound of structure shown in Formula 19, wherein the solution of the compound of structure shown in Formula 18 comprises the compound of structure shown in Formula 18, HCTU, DIPEA and DMF, and the solution of ciprofloxacin methyl ester comprises ciprofloxacin methyl ester, DIPEA and DMF.

[0083] In the present invention, in the solution of the structural compound shown in Formula 18, the molar ratio of the structural compound shown in Formula 18 to HCTU is preferably 0.22:0.40, the molar ratio of HCTU to DIPEA is preferably 0.4:0.79, and the amount ratio of the structural compound shown in Formula 18 to DMF is preferably 0.22mmol:3mL. In the present invention, in the solution of ciprofloxacin methyl ester, the molar ratio of ciprofloxacin methyl ester to DIPEA is preferably 1:1, and the amount ratio of DIPEA to DMF is preferably 0.44mmol:3mL. In the present invention, the molar ratio of the structural compound shown in Formula 18 to ciprofloxacin methyl ester is preferably 1:2. In the present invention, the solid phase synthesis reaction and the cleavage reaction are preferably consistent with the above-mentioned technical scheme, which will not be repeated here.

[0084] After obtaining the compound of structure shown in Formula 19, the present invention mixes the solution of the compound of structure shown in Formula 19 and the solution of nonylamine, and performs an amide condensation reaction to obtain the compound of structure shown in Formula 20, wherein the solution of the compound of structure shown in Formula 19 comprises the compound of structure shown in Formula 19, EDC, HOBt, DIPEA and DMF, and the solution of nonylamine comprises nonylamine, DIPEA and DMF.

[0085] In the present invention, in the solution of the compound of the structure shown in Formula 19, the molar ratio of the compound of the structure shown in Formula 19 and EDC is preferably 0.17:0.26, the molar ratio of EDC and HOBt is preferably 1:1, the dosage ratio of the compound of the structure shown in Formula 19 and DMF is preferably 0.17mmol:8mL, and the molar ratio of the compound of the structure shown in Formula 19 and DIPEA is preferably 1:2. In the present invention, in the solution of nonylamine, the molar ratio of nonylamine and DIPEA is preferably 1:1. In the present invention, the molar ratio of the compound of the structure shown in Formula 19 and nonylamine is preferably 1:2. In the present invention, the conditions of the amide condensation reaction and its subsequent treatment are consistent with the above-mentioned technical scheme, and will not be repeated here.

[0086] After obtaining the compound of structure shown in Formula 20, the present invention performs a hydrolysis reaction on the compound of structure shown in Formula 20 to obtain a compound of structure shown in Formula 21. In the present invention, the parameters of the hydrolysis reaction are consistent with the above technical solution and will not be repeated here.

[0087] After obtaining the compound of structure shown in Formula 21, the present invention removes Boc from the compound of structure shown in Formula 21 to obtain the compound of structure shown in Formula 22a-g. In the present invention, the reagent and step of removing Boc are preferably consistent with the above technical solution, which will not be repeated here.

[0088] In the present invention, the preparation formula of the second preparation method is as follows:

[0089]

[0090] In the present invention, the preparation method of the ciprofloxacin derivative having the structure shown in Formula 28a-e (hereinafter referred to as the third preparation method) comprises the following steps:

[0091] Swelling 2-chlorotrityl chloride resin to obtain a swollen resin; mixing the swollen resin, corresponding amino acid and N,N-diisopropylethylamine (DIPEA), sequentially performing solid phase synthesis reaction, end-capping reaction and removing Fomc; adding corresponding amino acid, repeating solid phase synthesis reaction and removing Fomc sequentially, to obtain a compound with a structure shown in Formula 23;

[0092] The compound of the structure shown in Formula 23, terephthalic acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF) are mixed and subjected to a solid phase synthesis reaction to obtain a compound of the structure shown in Formula 24;

[0093] The solution of the compound of formula 24 and the solution of ciprofloxacin methyl ester are mixed, and solid phase synthesis reaction and cleavage reaction are sequentially performed to obtain the compound of formula 25, wherein the solution of the compound of formula 24 comprises the compound of formula 24, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and the solution of ciprofloxacin methyl ester comprises ciprofloxacin methyl ester, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide;

[0094] A solution of a compound of formula 25 and a solution of nonylamine are mixed to perform an amide condensation reaction to obtain a compound of formula 26; the solution of the compound of formula 25 comprises the compound of formula 25, 1,2-dichloroethane (EDC), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF); and the solution of nonylamine comprises nonylamine, N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF);

[0095] The compound of the structure represented by Formula 26 is subjected to a hydrolysis reaction to obtain a compound of the structure represented by Formula 27;

[0096] The compound of the structure represented by Formula 27 is subjected to Boc removal to obtain the compound of the structure represented by Formula 28a-e;

[0097]

[0098]

[0099] The present invention swells 2-chlorotrityl chloride resin to obtain a swollen resin; the swollen resin, the corresponding amino acid and DIPEA are mixed, and solid phase synthesis reaction, end-capping reaction and Fomc removal are sequentially performed; the corresponding amino acid is added, and solid phase synthesis reaction, end-capping reaction and Fomc removal are repeated in sequence to obtain a structural compound shown in Formula 23. In the present invention, the swelling, solid phase synthesis reaction, end-capping reaction and Fomc removal are consistent with the above technical solution, and are not repeated here. In the present invention, the corresponding amino acid contains a protective group.

[0100] After obtaining the compound of structure shown in Formula 23, the present invention mixes the compound of structure shown in Formula 23, terephthalic acid, HCTU, DMF and DIPEA, and performs a solid phase synthesis reaction to obtain the compound of structure shown in Formula 24. In the present invention, the molar ratio of the compound of structure shown in Formula 23 and terephthalic acid is preferably 1:5, the molar ratio of terephthalic acid and HCTU is preferably 10:9, the molar ratio of HCTU and DIPEA is preferably 1:2, and the amount ratio of the compound of structure shown in Formula 23 and DMF is preferably 0.22mmol:3mL. In the present invention, the solid phase synthesis reaction and its subsequent post-treatment are preferably consistent with the above technical solution, which will not be repeated here.

[0101] After obtaining the compound of structure shown in Formula 24, the present invention mixes the solution of the compound of structure shown in Formula 24 and the solution of ciprofloxacin methyl ester, and sequentially performs solid phase synthesis reaction and cleavage reaction to obtain the compound of structure shown in Formula 25, wherein the solution of the compound of structure shown in Formula 24 comprises the compound of structure shown in Formula 24, HCTU, DIPEA and DMF, and the solution of ciprofloxacin methyl ester comprises ciprofloxacin methyl ester, DIPEA and DMF.

[0102] In the present invention, in the solution of the structural compound shown in Formula 24, the molar ratio of the structural compound shown in Formula 24 to HCTU is preferably 0.22:0.4, the molar ratio of the structural compound shown in Formula 24 to DIPEA is preferably 0.22:0.79, and the amount ratio of the structural compound shown in Formula 24 to DMF is preferably 0.22mmol:3mL. In the present invention, in the solution of ciprofloxacin methyl ester, the molar ratio of ciprofloxacin methyl ester to DIPEA is preferably 1:1, and the amount ratio of ciprofloxacin methyl ester to DMF is preferably 0.44mmol:3mL. In the present invention, the molar ratio of the structural compound shown in Formula 24 to ciprofloxacin methyl ester is preferably 1:2. In the present invention, the conditions of the solid phase synthesis reaction and the cleavage reaction and the subsequent post-treatment are preferably consistent with the above-mentioned technical scheme, and will not be repeated here.

[0103] After obtaining the compound of structure shown in Formula 25, the present invention mixes the solution of the compound of structure shown in Formula 25 with the solution of nonylamine, and performs an amide condensation reaction to obtain the compound of structure shown in Formula 26, wherein the solution of the compound of structure shown in Formula 25 comprises the compound of structure shown in Formula 25, EDC, HOBt, DIPEA and DMF, and the solution of nonylamine comprises nonylamine, DIPEA and DMF. In the present invention, in the solution of the compound of structure shown in Formula 25, the molar ratio of the compound of structure shown in Formula 25 and EDC is preferably 0.17:0.26, the molar ratio of EDC and HOBt is preferably 1:1, the amount ratio of the compound of structure shown in Formula 25 and DMF is preferably 0.17mmol:8mL, and the molar ratio of the compound of structure shown in Formula 25 and DIPEA is preferably 1:2. In the present invention, in the solution of nonylamine, the molar ratio of nonylamine and DIPEA is preferably 1:1. In the present invention, the molar ratio of the compound of structure shown in Formula 25 and nonylamine is preferably 1:2. In the present invention, the amide condensation reaction is preferably consistent with the above technical solution, which will not be described in detail here.

[0104] After obtaining the compound of structure shown in Formula 25, the present invention hydrolyzes the compound of structure shown in Formula 26 to obtain the compound of structure shown in Formula 27. In the present invention, the hydrolysis reaction is preferably consistent with the above technical solution, which will not be described in detail here.

[0105] After obtaining the compound of structure shown in Formula 27, the present invention removes Boc from the compound of structure shown in Formula 27 to obtain the compound of structure shown in Formula 28a-e. In the present invention, the removal of Boc is preferably consistent with the above technical solution, which will not be repeated here.

[0106] In the present invention, the preparation formula of the third preparation method is as follows:

[0107]

[0108] The present invention also provides the use of the ciprofloxacin derivatives described in the above technical solution or the ciprofloxacin derivatives prepared by the preparation method described in the above technical solution in the preparation of antibacterial drugs. The present invention does not specifically limit the application mode, and the operation familiar to those skilled in the art can be used.

[0109] In the present invention, the antibacterial agent preferably includes anti-Gram-positive bacteria and / or anti-Gram-negative bacteria.

[0110] The ciprofloxacin derivatives provided by the present invention and their preparation methods and applications are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0111] Example

[0112] Compounds 16a-16u were prepared as follows:

[0113] 2-Chlorotrityl chloride (CTC) resin (300 mg, 0.22 mmol) was swollen in dichloromethane (6 mL) for 15 min. Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol) and DIPEA (140 μL, 0.79 mmol) were added to a solid phase synthesis tube and shaken at room temperature for 2 h to carry out solid phase synthesis reaction to connect the first amino acid to the resin. The end-capping reaction was carried out with 5 mL of DIPEA / MeOH / DCM (1 / 2 / 17, v / v / v) for 30 min, and the resin was washed with DCM (4 mL×3) and DMF (4 mL×3). At room temperature, the resin was treated with 20% piperidine / DMF (v / v) solution for 10 min (×2) to remove Fmoc and obtain compound 11, which is an amino acid grafted compound containing one amino acid.

[0114] When n is 2 in the structure shown in Formula 16a-u, Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol), DIC (120 μL, 0.88 mmol) and HOBt (121 mg, 0.88 mmol) were then added and premixed in DMF (3 mL) for 5 min. The mixture was then transferred to a solid phase synthesis tube and shaken at room temperature for 2 h for solid phase synthesis reaction. The solution was removed and washed with DMF (3 mL×3) and DCM (3 mL×3). The resin was then treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (×2) to remove Fmoc. After the reaction was completed, the solution was drained and the resin was washed with DCM (3 mL×3) and DMF (3 mL×3) to obtain an amino acid grafted compound containing two amino acids. When n is 3 in the structure shown in Formula 16a-u, Fmoc-Lys(Boc)-OH, DIC, HOBt and DMF shown in Formula 10 are then added and mixed, and solid phase synthesis reaction and Fmoc removal are carried out in sequence to obtain an amino acid grafted compound containing three amino acids.

[0115] Then terephthalic acid (185 mg, 1.1 mmol), HCTU (415 mg, 0.99 mmol) and DIPEA (349 μL, 1.98 mmol) were premixed in DMF (3 mL) for 5 min and transferred to a solid phase synthesis tube. After shaking at room temperature for 2 h, the solution was removed and washed with DMF (3 mL×3) and DCM (3 mL×3) to obtain compounds 12a-12u.

[0116] A DMF (3 mL) solution of HCTU (166 mg, 0.40 mmol) and DIPEA (140 μL, 0.79 mmol) was added to a solid phase synthesis tube for 5 min, and then a pre-mixed DMF (3 mL) solution of ciprofloxacin methyl ester (154 mg, 0.44 mmol) and DIPEA (78 μL, 0.44 mmol) was added, and the mixture was reacted at room temperature for 5 h, and then the solution was removed, washed with DMF (4 mL × 3) and DCM (4 mL × 3), and then cleaved with 8 mL of cleavage solution (HFIP / DCM 1:4, v / v) for 2 h. Intermediates 13a-13u were cleaved from the resin, and the cleavage solution was collected. The resin was washed three times with 4 mL of cleavage solution to obtain a washing solution; the cleavage solution and the washing solution were combined and distilled under reduced pressure. The crude compounds of 13a-13u were dissolved in a water / methanol (1:1) solution (10 mL), filtered with a Whatman filter (0.22 μm), and then analyzed by analytical HPLC. After purification by preparative HPLC and freeze-drying, pure 13a-13u white powders were obtained.

[0117] 13a-13u (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) were dissolved in anhydrous DMF (8 mL), and DIPEA (60 μL, 0.34 mmol) was added. The reaction solution was stirred at 0°C for 5 min, and then a pre-mixed DMF mixed solution of R4-H (0.34 mmol) and DIPEA (60 μL, 0.34 mmol) was added. The reaction was stirred at room temperature for 5 h. After the reaction was completed, it was quenched with 1 mol / L HCl. The organic phase was extracted with DCM (×3) and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude products 14a-14u were used directly without further purification.

[0118] LiOH (73 mg, 1.7 mmol) was partially added to a solution of compound 14a-14u (0.17 mmol) in 6 mL of MeOH / H2O (5:1, v / v). The reaction mixture was stirred at room temperature for 2 h, quenched with 1 mol / L HCl after completion of the reaction, the organic phase was extracted with DCM (×3) and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude products 15a-15u were used directly without further purification.

[0119] Compounds 15a-15u (0.17 mmol) were dissolved in 5 mL of TFA / DCM solution (19 / 1, v / v). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the crude product compounds 16a-16u were dissolved in water / methanol (1:1) solution (10 mL) by vacuum distillation, filtered through a Whatman filter (0.22 μm), and detected by analytical HPLC. After preparative HPLC purification and lyophilization, 16a-16u were obtained as white powders.

[0120]

[0121] 1 H NMR(500MHz,MeOD)δ8.73(s,1H),7.99(d,J=7.5Hz,2H),7.84(d,J=13.0Hz,1H),7. 59(d,J=7.6Hz,3H),4.55(dd,J=8.9,5.5Hz,1H),4.02(m,2H),3.68(m,3H),3.50(m, 2H),3.38(m,2H),3.21(t,2H),2.94(s,2H),1.93(m,1H),1.84m,1H),1.72(m,2H),1 .53(m,4H),1.40(m,2H),1.33(m,4H),1.22(m,2H),0.90(t,J=6.9Hz,3H).ESI-HRMS calcdfor C 36 H 45 FN6O6[M+H] + :677.3385; found 677.3475.

[0122]

[0123] 1 H NMR(500MHz,MeOD)δ8.72(s,1H),7.99(d,J=7.5Hz,2H),7.81(d,J=13.0Hz,1H),7.6 0(d,J=7.4Hz,3H),4.56(dd,J=8.8,5.6Hz,1H),4.08-3.91(m,2H),3.69(m,3H),3.4 5(m,4H),3.22(t,2H),2.94(t,2H),1.95(m,1H),1.89-1.80(m,1H),1.73(m,2H),1. 52(m,4H),1.41(m,2H),1.37-1.29(m,6H),1.23(m,2H),0.94-0.87(t,3H).ESI-HRMS calcdfor C37 H 47 FN6O6[M+H] + :691.3541;found691.3637.

[0124]

[0125] 1 H NMR(500MHz,MeOD)δ8.74(s,1H),8.00(s,2H),7.84(d,J=12.6Hz,1H),7.61(s,3H),4.55(t,1H),4.04(m,2H),3.70(m,3H),3.61-3.33(m,4H),3.22(t,J=7.0Hz,2H),2.96(m,2H),1.95(m,1H),1.86(m,1H),1.74(m,2H),1.58-1.49(m,4H),1.42(m,1H),1.37-1.24(m,13H),0.92-0.88(t,3H).ESI-HRMS calcd for C 39 H 51 FN6O6[M+H] + :719.3854;found719.3944.

[0126]

[0127] 1 H NMR(500 MHz,MeOD)δ8.72(s,1H),7.97(d,J=7.7 Hz,2H),7.82(d,J=13.0Hz,1H),7.58(d,J=8.0 Hz,3H),4.53(dd,J=8.9,5.6 Hz,1H),4.01(m,2H),3.67(m,3H),3.43(m,4H),3.19(t,2H),2.92(m,2H),1.96-1.88(m,1H),1.84(m,1H),1.71(m,2H),1.50(m,4H),1.39(m,2H),1.33-1.24(m,13H),1.20(m,2H),0.88(t,2H).ESI-HRMS calcd forC 40 H 53 FN6O6[M+H] + :733.4011;found 733.4087.

[0128]

[0129] 1 H NMR(500 MHz,MeOD)δ8.76(s,1H),8.00(d,J=7.8 Hz,2H),7.95(d,J=8.1Hz,1H),7.87(d,J=13.1 Hz,1H),7.61(d,J=7.8 Hz,3H),4.55(d,J=10.1,5.1 Hz,1H),4.03(m,2H),3.76(m,1H),3.69(m,2H),3.51(m,2H),3.39(m,2H),3.22(m,2H),2.97-2.91(m,2H),1.99-1.92(m,1H),1.86(m,1H),1.73(m,2H),1.57-1.48(m,4H),1.42(m,2H),1.29(m,18H),1.23(m,2H),0.91-0.88(t,3H).ESI-HRMS calcd for C 43 H 59 FN6O6[M+H] + :775.4480;found 775.4573.

[0130]

[0131] 1 H NMR(500 MHz,MeOD)δ8.74(s,1H),7.99(d,J=7.8 Hz,2H),7.85(d,J=13.0Hz,1H),7.60(d,J=7.8 Hz,3H),4.55(dd,J=8.9,5.6 Hz,1H),4.02(m,2H),3.72(m,3H),3.50(m,2H),3.38(m,2H),3.21(m,2H),2.94(t,2H),1.94(m,1H),1.85(m,1H),1.72(m,2H),1.59-1.46(m,4H),1.41(m,2H),1.34-1.26(m,22H),1.22(m,2H),0.89(t,3H).ESI-HRMScalcd for C 45 H 63 FN6O6[M+H] + :803.4793;found 803.4876.

[0132]

[0133] 1H NMR(500 MHz,MeOD)δ8.76(s,1H),8.00(s,2H),7.87(d,J=12.8 Hz,1H),7.72-7.48(m,3H),4.56(dd,J=8.8,5.5 Hz,1H),4.04(m,2H),3.70(m,2H),3.52(m,2H),3.40(m,2H),3.32(m,1H),3.23(t,2H),2.96(s,2H),1.91(m,2H),1.74(m,2H),1.53(m,4H),1.42(m,2H),1.28(m,26H),1.23(m,2H),0.90(t,J=6.9 Hz,3H).ESI-HRMS calcd forC 47 H 67 FN6O6[M+H] + :831.5106;found 831.5189.

[0134]

[0135] 1 H NMR(500 MHz,MeOD)δ8.70(s,1H),7.99(s,2H),7.80(d,J=12.7 Hz,1H),7.58(s,3H),7.29(m,4H),7.22(m,1H),4.61(dd,J=8.8,5.4 Hz,1H),4.41(s,2H),3.99(m,2H),3.67(m,3H),3.58-3.33(m,4H),2.92(m,2H),1.98(m,1H),1.88(m,1H),1.71(m,2H),1.51(m,J=30.3 Hz,2H),1.40(m,2H),1.26-1.15(m,2H).ESI-HRMS calcd for C 38 H 41 FN6O6[M+H] + :697.3072;found 697.3159.

[0136]

[0137] 1H NMR(500 MHz,MeOD)δ8.74(s,1H),8.01(s,2H),7.85(d,J=12.7 Hz,1H),7.60(s,3H),7.44-7.36(m,2H),7.31-7.23(m,2H),4.64(t,1H),4.51(s,2H),4.03(m,2H),3.69(m,3H),3.45(m,4H),2.94(m,2H),1.99(m,1H),1.90(m,1H),1.74(m,2H),1.55(m,2H),1.41(m,2H),1.24(m,2H).ESI-HRMS calcd for C 38 H 40 ClFN6O6[M+H] + :731.2682;found731.2763.

[0138]

[0139] 1 H NMR(500 MHz,MeOD)δ8.72(s,1H),7.96(d,J=7.5 Hz,2H),7.81(d,J=12.9Hz,1H),7.68-7.49(m,3H),7.41(d,J=8.0 Hz,2H),7.32-7.27(m,1H),4.76(m,1H),4.66(m,1H),4.61(m,1H),4.02(m,2H),3.68(m,3H),3.51(m,2H),3.37(m,2H),2.92(m,2H),1.97-1.81(m,2H),1.71(m,2H),1.51(m,2H),1.45-1.36(m,2H),1.22(m,2H).ESI-HRMScalcd for C 38 H 39 Cl2FN6O6[M+H] + :765.2292;found 765.2374.

[0140]

[0141] 1H NMR(500 MHz,MeOD)δ8.74(s,1H),7.96(d,J=7.2 Hz,2H),7.84(d,J=12.8Hz,1H),7.59(s,3H),7.33 m,1H),7.27(d,J=8.1 Hz,1H),7.11(m,1H),4.64(dd,J=14.0,1.6 Hz,1H),4.59(dd,J=8.5,5.7 Hz,1H),4.54(dd,J=14.1,1.5 Hz,1H),4.01(m,2H),3.69(m,3H),3.45(m,4H),2.91(m,2H),1.92(m,1H),1.84(m,1H),1.70(m,2H),1.44(m,4H),1.27-1.17(m,2H).ESI-HRMS calcd forC 38 H 39 ClF2N6O6[M+H] + :749.2588;found 749.2674.

[0142]

[0143] 1 H NMR(500 MHz,MeOD)δ8.73(s,1H),8.00(s,2H),7.85(d,J=12.9 Hz,1H),7.56(dd,J=7.7,4.5 Hz,7H),7.39(m,4H),7.30(t,1H),4.64(dd,J=8.0 Hz,1H),4.46(s,2H),4.01(m,2H),3.66(m,3H),3.48(m,2H),3.42-3.31(m,2H),2.94(m,2H),2.05-1.97(m,1H),1.91(m,1H),1.73(m,2H),1.61-1.47(m,2H),1.39(m,2H),1.20(m,2H).ESI-HRMS calcdfor C 44 H 45 FN6O6[M+H] + :773.3385;found 773.3446.

[0144]

[0145] 1H NMR(500 MHz,MeOD)δ8.67(s,1H),8.00(d,J=8.3 Hz,2H),7.77-7.68(m,1H),7.60(d,J=8.2 Hz,2H),7.54(d,J=6.2 Hz,1H),4.52(dd,J=8.4,6.2 Hz,1H),4.36(dd,J=9.4,5.0 Hz,1H),4.02(s,2H),3.71(m,3H),3.48(m,2H),3.38(m,2H),3.18(m,2H),2.95(m,4H),1.99-1.82(m,3H),1.78-1.67(m,5H),1.58-1.48(m,5H),1.41(m,2H),1.34-1.25(m,13H),1.21(m,2H),0.92-0.87(t,3H).LC-MS calcd for C 46 H 65 FN8O7[M+H] + :861.50;found 861.51.

[0146]

[0147] 1 H NMR(500 MHz,MeOD)δ8.63(d,J=2.4 Hz,1H),8.07-7.97(m,2H),7.69-7.65(m,1H),7.59(d,J=5.7 Hz,2H),7.55-7.49(m,1H),4.52(m,1H),4.41-4.33(m,1H),4.01(m,2H),3.70(m,3H),3.49(m,2H),3.37(m,2H),3.23-3.14(m,2H),3.02-2.91(m,4H),2.01-1.88(m,3H),1.85(m,1H),1.78-1.73(m,3H),1.71-1.66(m,2H),1.59(m,2H),1.51(m,4H),1.43-1.38(m,3H),1.28(m,10H),1.24(m,2H),1.21(m,2H),0.88(t,3H).LC-MS calcd forC 47 H 67 FN8O7[M+H] + :875.51;found 875.52.

[0148]

[0149] 1 H NMR(500 MHz,MeOD)δ8.76(s,1H),8.00(s,2H),7.86(d,J=12.7 Hz,1H),7.61(s,3H),4.52(m,1H),4.37(dd,J=9.4,4.9 Hz,1H),4.04(m,2H),3.70(m,3H),3.53(m,1H),3.41(m,3H),3.19(m,2H),2.95(m,4H),1.98-1.83(m,3H),1.72(m,5H),1.60-1.48(m,6H),1.42(m,2H),1.31(m,18H),1.26(m,2H),0.90(t,3H).ESI-HRMS calcd for C 49 H 71 FN8O7[M+H] + :903.5430;found 903.5499.

[0150]

[0151] 1 H NMR(500 MHz,DMSO-d6)δ8.65(dd,J=12.5,7.2 Hz,2H),8.12(d,J=6.8 Hz,1H),7.99(d,J=7.8 Hz,2H),7.90(m,10H),7.56(d,J=8.2 Hz,3H),4.43(dd,J=8.3 Hz,1H),4.25(dd,J=8.0 Hz,1H),4.18(dd,J=7.0 Hz,1H),3.87(m,3H),3.56(m,2H),3.44(m,2H),3.33(m,2H),,3.07(m,3H),2.99(m,1H),2.78(m,4H),1.84-1.74(m,2H),1.67(m,2H),1.61-1.51(m,6H),1.48-1.42(m,2H),1.34(m,7H),1.22(m,13H),1.16(m,2H),0.83(t,3H).LC-MS calcd for C 51 H 75 FN 10 O8[M+H] + :975.57;found 975.59.

[0152]

[0153] 1H NMR(500 MHz,MeOD)δ8.89-8.62(m,1H),7.95(d,J=63.0 Hz,3H),7.78(s,1H),7.61(s,2H),4.49(m,1H),4.32(m,2H),4.19-3.90(m,2H),3.76(m,2H),3.57-3.37(m,3H),3.17(m,3H),3.07-2.83(m,7H),1.92(m,2H),1.73(m,10H),1.51(m,8H),1.29(m,16H),0.89(t,J=6.8 Hz,3H).ESI-HRMS calcd for C 52 H 77 FN 10 O8[M+H] + :989.5910;found989.5965.

[0154]

[0155] 1 H NMR(500 MHz,DMSO-d6)δ8.67(d,J=7.1 Hz,1H),8.64(s,1H),8.11(d,J=7.4Hz,1H),7.98(d,J=8.1 Hz,2H),7.88(m,9H),7.86(m,1H),7.58-7.55(m,2H),4.42(dd,J=8.1 Hz,1H),4.24(dd,J=7.9 Hz,1H),4.15(dd,J=7.7 Hz,1H),3.87(m,2H),3.82-3.76(m,3H),3.44(m,2H),3.33(m,2H),3.06(m,1H),2.98(m,1H),2.81-2.72(m,6H),1.83-1.74(m,2H),1.71(m,2H),1.62-1.50(m,10H),1.39-1.30(m,9H),1.22(m,13H),1.16(m,2H),0.83(t,3H).ESI-HRMS calcd for C 53 H 79 FN 10 O8[M+H] + :1003.6066;found 1003.6124.

[0156]

[0157] 1H NMR(500 MHz,MeOD)δ8.75(s,1H),8.01(d,J=7.8 Hz,2H),7.90-7.82(m,1H),7.60(d,J=7.8 Hz,3H),4.50(dd,J=8.7,6.0 Hz,1H),4.35(dd,J=9.1,5.2 Hz,1H),4.29(dd,J=9.2,5.2 Hz,1H),4.03(m,2H),3.72(m,3H),3.51(m,2H),3.39(m,2H),3.24-3.10(m,2H),3.01-2.89(m,6H),2.00-1.86(m,3H),1.79-1.61(m,9H),1.52(m,6H),1.4-1.38(m,3H),1.34-1.25(m,19H),1.22(m,2H),0.91-0.88(t,3H).ESI-HRMS calcd for C 55 H 83 FN 10 O8[M+H] + :1031.6379;found 1031.6473.

[0158]

[0159] 1 H NMR(500MHz,MeOD)δ8.77(s,1H),8.01(s,2H),7.88(d,J=12.6Hz,1H),7.61(s,3H),4.50(t,1H),4.37(t,1H),4.30(dd,J=9.0,5.1Hz,1H),4.04(m,2H),3.85-3.62(m,3H),3.49(m,2H),3.35(m,2H),3.18(m,2H),2.97(m,6H),1.91(m,3H),1.73(m,9H),1.56-1.48(m,5H),1.41(m,3H),1.33-1.24(m,30H),0.90(t,3H).LC-MS calcd for C 59 H 91 FN 10 O8[M+H] + :1087.70;found 1087.70.

[0160]

[0161] 1H NMR (500MHz, DMSO-d6) δ8.69(d,J=6.8Hz,1H),8.63(m,1H),8.53(t,1H),8.15(d,J=6.7Hz,1H),8.02(d d,J=13.7,8.0Hz,3H),7.90(m,8H),7.65-7.55(m,7H),7.45(t,J=7.6Hz,2H),7.34(t,J=8.5Hz,3H),4.4 8-4.43(m,2H),4.33(m,2H),4.29(m,2H),3.89(m,2H),3.79(m,1H),3.56(m,2H),3.45(m,2H),3.33(m, 2H),2.81-2.74(m,5H),1.84-1.69(m,4H),1.65-1.50(m,9H),1.42-1.29(m,7H),1.17(m,2H).ESI-HRMS calcd for C 56 H 69 FN 10 O8[M+H] + :1029.5284; found1029.5335.

[0162] Compound 22a-g was prepared as follows:

[0163] 2-Chlorotrityl chloride (CTC) resin (300 mg, 0.22 mmol) was swollen in dichloromethane (6 mL) for 15 min. Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol) and DIPEA (140 μL, 0.79 mmol) were added to a solid phase synthesis tube and shaken at room temperature for 2 h to carry out solid phase synthesis reaction to connect the first amino acid to the resin. The end-capping reaction was carried out with 5 mL of DIPEA / MeOH / DCM (1 / 2 / 17, v / v / v) for 30 min, and the resin was washed with DCM (4 mL×3) and DMF (4 mL×3). At room temperature, the resin was treated with 20% piperidine / DMF (v / v) solution for 10 min (×2) to remove Fmoc and obtain compound 11.

[0164] Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol), DIC (120 μL, 0.88 mmol) and HOBt (121 mg, 0.88 mmol) were added to a solid phase synthesis tube and premixed in DMF (3 mL) for 5 min, then transferred to a solid phase synthesis tube, shaken at room temperature for 2 h, the solution was removed, washed with DMF (3 mL × 3) and DCM (3 mL × 3), and then the resin was treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (× 2) to remove the Fmoc group. After the reaction was completed, the solution was drained and the resin was washed with DCM (3 mL × 3) and DMF (3 mL × 3). Next, Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol), DIC (121 μL, 0.88 mmol) and HOBt (120 mg, 0.88 mmol) were premixed in DMF (3 mL) for 5 min, then transferred to a solid phase synthesis tube, shaken at room temperature for 2 h, the solution was drained, and then washed with DMF (3 mL×3) and DCM (3 mL×3). The resin was treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (×2) to remove the Fmoc protecting group to obtain compound 17.

[0165] Then, DIPEA (306 μL, 1.76 mmol), succinic anhydride (88 mg, 0.88 mmol) and DMF were added to the solid phase synthesis tube and the reaction was shaken at room temperature for 1 h. The solution was discharged and then washed with DMF (3 mL×3) and DCM (3 mL×3) to obtain 18a.

[0166] Then, Fmoc-R5-COOH (0.44 mmol), DIC (120 μL, 0.88 mmol) and HOBt (121 mg, 0.88 mmol) were added to the solid phase synthesis tube and pre-mixed in DMF (3 mL) for 5 min, and then transferred to the solid phase synthesis tube. After shaking at room temperature for 2 h for solid phase synthesis reaction, the solution was removed, washed with DMF (3 mL × 3) and DCM (3 mL × 3), and then the resin was treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (× 2) to remove Fmoc. After the removal of Fmoc, the solution was drained and the resin was washed with DCM (3 mL × 3) and DMF (3 mL × 3). Then, succinic anhydride (88 mg, 0.88 mmol) and DIPEA (306 μL, 1.76 mmol) in DMF (3 mL) were added to a solid phase synthesis tube and the reaction was shaken at room temperature for 1 h. The solution was discharged and then washed with DMF (3 mL×3) and DCM (3 mL×3) to obtain 18b-18g.

[0167] Add HCTU (166 mg, 0.40 mmol) and DIPEA (140 μL, 0.79 mmol) in DMF (3 mL) to a solid phase synthesis tube and mix for 5 min. Mix ciprofloxacin methyl ester (154 mg, 0.44 mmol), DIPEA (78 μL, 0.44 mmol) and DMF (3 mL) to obtain a ciprofloxacin methyl ester solution. React at room temperature for 5 h, then remove the solution, wash with DMF (4 mL × 3) and DCM (4 mL × 3), and then cleave with 8 mL of cleavage solution (HFIP / DCM 1:4, v / v) for 2 h. 19a-19g was cleaved from the resin, and the cleavage solution was collected. The resin was washed three times with 4 mL of cleavage solution to obtain a washing solution. The cleavage solution and the washing solution were combined and distilled under reduced pressure. The crude compound 19a-19g was dissolved in a water / methanol (1:1) solution (10 mL), filtered with a Whatman filter (0.22 μm), and then analyzed by analytical HPLC. After purification by preparative HPLC and freeze-drying, pure 19a-19g white powder was obtained.

[0168] 19a-19g (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) were dissolved in anhydrous DMF (8 mL), and DIPEA (60 μL, 0.34 mmol) was added. The reaction solution was stirred at 0°C for 5 min, and then a pre-mixed DMF mixed solution of nonylamine (63 μL, 0.34 mmol) and DIPEA (60 μL, 0.34 mmol) was added. The reaction was stirred at room temperature for 5 h. After the reaction was completed, it was quenched with 1 mol / L HCl, and the organic phase was extracted with DCM (×3) and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product 20a-20g was used directly without further purification.

[0169] To a solution of compound 20a-20g (0.17 mmol) in 6 mL of MeOH / H2O (5:1, v / v) was partially added LiOH (73 mg, 1.7 mmol). The reaction mixture was stirred at room temperature for 2 h, and after completion of the reaction, it was quenched with 1 mol / L HCl, the organic phase was extracted with DCM (×3) and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product 21a-21g was used directly without further purification.

[0170] Compound 21a-21g (0.17 mmol) was dissolved in 5 mL of TFA / DCM solution (19 / 1, v / v). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the crude product compound 22a-22g was dissolved in water / methanol (1:1) solution (10 mL) by vacuum distillation, filtered through a Whatman filter (0.22 μm), and detected by analytical HPLC. After preparative HPLC purification and lyophilization, 22a-22g was obtained as a white powder.

[0171]

[0172] 1 H NMR (500MHz, DMSO-d6) δ8.64(s,1H),8.29(d,J=7.1Hz,1H),7.97(m,7H),7.87(d,J=13.1Hz,1H),7. 78-7.72(m,2H),7.56(d,J=5.8Hz,1H),4.17-4.10(m,3H),3.82(m,2H),3.74-3.67(m,3H),3.64(m,1 H),3.38(m,4H),3.00(m,2H),2.80-2.74(m,6H),2.69-2.62(m,2H),2.44(m,1H),1.77-1.68(m,3H) ,1.58-1.50(m,9H),1.39-1.31(m,10H),1.20(m,18H),1.12(m,2H),0.83(t,J=6.8Hz,3H).ESI-HRMS calcd for C 51 H 83 FN 10 O8[M+H] + :983.6379; found 983.6461.

[0173]

[0174] 1H NMR(500MHz,MeOD)δ8.83-8.64(m,1H),8.00-7.80(m,1H),7.64-7.42(m,1H),4.24(dd,J=9.0,4.7Hz,2H),4.20-4.16(m,1H),3.77(m,4H),3.49-3.35(m,5H),3.16(m,3H),2.93(m,7H),2.68(m,2H),2.54(dd,J=30.5,11.6Hz,3H),2.43(m,1H),1.88(d,J=15.3Hz,4H),1.70(m,8H),1.56(m,2H),1.51-1.46(m,4H),1.44-1.41(m,2H),1.32-1.26(m,20H),1.23(d,J=1.1Hz,2H),0.89(t,3H).ESI-HRMS calcd for C 54 H 88 FN 11 O9[M+H] + :1054.6751;found 1054.6823.

[0175]

[0176] 1 H NMR(500 MHz,MeOD)δ8.52(s,1H),7.85(s,1H),7.53(d,J=13.1 Hz,1H),7.39(d,J=6.3Hz,1H),4.15(m,2H),4.08(dd,J=9.2,4.8 Hz,1H),3.75-3.67(m,4H),3.63(m,1H),3.41-3.26(m,8H),3.06(m,2H),2.86(d,J=7.6 Hz,6H),2.61(t,2H),2.54-2.42(m,3H),2.35-2.25(m,3H),1.84-1.75(m,4H),1.62(m,8H),1.43-1.36(m,5H),1.34-1.29(m,3H),1.17(d,J=12.9 Hz,20H),1.12(m,2H),0.78(t,3H).ESI-HRMS calcd forC 57 H 93 FN 12 O 10 [M+H] + :1125.7122;found 1125.7215.

[0177]

[0178] 1 H NMR(500 MHz,DMSO-d6)δ8.66(s,1H),8.32(d,J=6.6 Hz,1H),8.09(d,J=7.6Hz,1H),8.04-8.01(m,1H),7.91(m,7H),7.67-7.62(m,2H),7.58(s,1H),4.14-4.07(m,3H),3.69(m,4H),3.44(m,4H),3.33(d,J=25.5 Hz,2H),3.01(m,4H),2.77(m,5H),2.39-2.35(m,4H),1.75-1.64(m,4H),1.60-1.47(m,12H),1.36(d,J=13.7 Hz,9H),1.30-1.27(m,5H),1.23(m,18H),1.19(m,2H),0.85(t,J=6.8 Hz,3H).ESI-HRMS calcd for C 58 H 96 FN 11 O9[M+H] + :1110.7377;found 1110.7430.

[0179]

[0180] 1 H NMR(500 MHz,MeOD)δ8.76(s,1H),7.86(d,J=13.1 Hz,1H),7.59(d,J=7.3Hz,1H),4.35-4.21(m,3H),3.83(s,4H),3.79-3.74(m,1H),3.50-3.43(m,2H),3.41-3.36(m,2H),3.23-3.13(m,4H),2.98-2.92(m,6H),2.76(t,2H),2.54(t,2H),2.30-2.21(m,2H),1.84(m,3H),1.72(m,9H),1.62-1.56(m,2H),1.54-1.47(m,8H),1.45-1.41(m,3H),1.30(d,J=10.4 Hz,33H),1.23(m,2H),0.90(t,3H).LC-MS calcd for C 63 H 106 FN 11 O9[M+H] +:1180.81;found 1180.82.

[0181]

[0182] 1 H NMR(500 MHz,MeOD)δ8.68(s,1H),7.73(d,J=13.0 Hz,1H),7.53(d,J=7.0Hz,1H),4.39(d,J=4.9 Hz,2H),4.25(m,2H),4.17(m,1H),3.84(m,2H),3.78-3.71(m,5H),3.70-3.66(m,2H),3.56(m,2H),3.46-3.39(m,4H),3.37(d,J=5.6 Hz,1H),3.15(m,2H),3.01-2.91(m,7H),2.70-2.61(m,1H),2.59-2.53(m,2H),2.44(m,1H),1.94-1.85(m,4H),1.78-1.68(m,8H),1.62-1.53(m,3H),1.52-1.46(m,4H),1.44-1.41(m,3H),1.26(m,18H),1.23(d,J=6.4Hz,2H),0.88(t,3H).LC-MS calcd for C 58 H 96 FN 11 O 11 [M+H] + :1142.72;found1142.73.

[0183]

[0184] 1HNMR(500MHz,MeOD)δ8.72(s,1H),7.79(d,J=13.1Hz,1H),7.55(d,J=6.1Hz,1H),4.28(m,4H),3.96(dd ,J=11.0,5.0Hz,1H),3.91-3.71(m,6H),3.49-3.38(m,3H),3.23-3.12(m,2H),2.95(d,J=6.6Hz,7H),2 .88-2.80(m,2H),2.68(dd,J=15.1,6.7Hz,1H),2.54(m,1H),1.96(m,1H),1.91-1.81(m,3H),1.73(m,9 H),1.54-1.46(m,6H),1.45-1.40(m,3H),1.28(d,J=10.5Hz,18H),1.23(m,2H),0.89(t,3H).ESI-HRMS calcd forC 54 H 88 FN 11 O 10 [M+H] + :1070.6700; found 1070.6757.

[0185] Compound 28a-g was prepared as follows:

[0186] 2-Chlorotrityl chloride (CTC) resin (300 mg, 0.22 mmol) was swollen in dichloromethane (6 mL) for 15 min, and the corresponding amino acid (0.44 mmol) and DIPEA (140 μL, 0.79 mmol) were added to the solid phase synthesis tube, and the solid phase synthesis reaction was carried out by shaking at room temperature for 2 h, and then the reaction solution was discharged and washed with DMF (4 mL × 3) and DCM (4 mL × 3) to achieve the connection between the first amino acid and the resin. Then, 6 mL of DIPEA / MeOH / DCM (1 / 2 / 17, v / v / v) was used for end-capping reaction for 30 min. The resin was washed with DCM (4 mL × 3) and DMF (4 mL × 3). At room temperature, the resin was treated with 20% piperidine / DMF (v / v) solution for 10 min (× 2) to remove Fmoc. Next, the corresponding amino acid (0.44 mmol), DIC (121 μL, 0.88 mmol) and HOBt (120 mg, 0.88 mmol) were premixed in DMF (3 mL) for 5 min and then transferred to a solid phase synthesis tube. The reaction was shaken at room temperature for 2 h, the solution was discharged, and then washed with DMF (3 mL × 3) and DCM (3 mL × 3). The resin was treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (× 2) to remove Fmoc, and the above experimental operation was repeated to access the corresponding amino acids to obtain compounds 23a-23e.

[0187] Then terephthalic acid (185 mg, 1.1 mmol), HCTU (415 mg, 0.99 mmol) and DIPEA (349 μL, 1.98 mmol) were premixed in DMF (3 mL) for 5 min and transferred to a solid phase synthesis tube. After shaking at room temperature for 2 h, the solution was removed and washed with DMF (3 mL×3) and DCM (3 mL×3) to obtain compounds 24a-24e.

[0188] A solution of HCTU (166 mg, 0.40 mmol) and DIPEA (140 μL, 0.79 mmol) in DMF (3 mL) was added to the reaction vessel for 5 min, and then a pre-mixed solution of ciprofloxacin methyl ester (154 mg, 0.44 mmol) and DIPEA (78 μL, 0.44 mmol) in DMF (3 mL) was added, and the mixture was reacted at room temperature for 5 h, and then the solution was removed, washed with DMF (4 mL×3) and DCM (4 mL×3), and then cleaved with 8 mL of cleavage solution (HFIP / DCM 1:4, v / v) for 2 h. 25a-25e was cleaved from the resin, and the cleavage solution was collected. The resin was washed three times with 4 mL of cleavage solution to obtain a washing solution; the cleavage solution and the washing solution were combined and distilled under reduced pressure. The crude compounds of 25a-25e were dissolved in water / methanol (1:1) solution (10 mL), filtered with a Whatman filter (0.22 μm), and then analyzed by analytical HPLC. After purification by preparative HPLC and lyophilization, pure 25a-25e g of white powder were obtained.

[0189] 25a-25e (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) were dissolved in anhydrous DMF (8 mL), and DIPEA (60 μL, 0.34 mmol) was added. The reaction solution was stirred at 0°C for 5 min, and then a pre-mixed DMF mixed solution of nonylamine (63 μL, 0.34 mmol) and DIPEA (60 μL, 0.34 mmol) was added. The reaction was stirred at room temperature for 5 h. After the reaction was completed, it was quenched with 1 mol / L HCl. The organic phase was extracted with DCM (×3) and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude products 26a-26e were used directly without further purification.

[0190] To a solution of compound 26a-26e (0.17 mmol) in 6 mL of MeOH / H2O (5:1, v / v) was partially added LiOH (73 mg, 1.7 mmol). The reaction mixture was stirred at room temperature for 2 h, and after completion of the reaction, it was quenched with 1 mol / L HCl, the organic phase was extracted with DCM (×3) and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude products 27a-27e were used directly without further purification.

[0191] Compounds 27a-27e (0.17 mmol) were dissolved in 5 mL of TFA / DCM solution (19 / 1, v / v). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the crude product compounds 28a-28e were dissolved in water / methanol (1:1) solution (10 mL) by vacuum distillation, filtered through a Whatman filter (0.22 μm), and detected by analytical HPLC. After preparative HPLC purification and lyophilization, 28a-28e were obtained as white powders.

[0192]

[0193] 1 H NMR(500MHz,MeOD)δ8.66(s,1H),7.91(dd,J=7.8,4.1Hz,2H),7.79(m,1H),7.49(d,J=7.7Hz,3H),4.4 6-4.36(m,1H),4.23(m,2H),3.92(m,2H),3.65(m,1H),3.58(m,2H),3.40(m,2H),3.26(d,J=12.2Hz,2 H),3.17-3.07(m,3H),3.06-3.03(m,1H),2.87(m,4H),1.91-1.74(m,4H),1.63(m,7H),1.55-1.49(m, 2H),1.42(m,5H),1.31(m,2H),1.19(m,12H),1.09(d,J=14.5Hz,2H),0.78(q,J=7.0Hz,3H).ESI-HRMS calcd for C 52 H 77 FN 12 O8[M+H] + :1017.5971; found 1017.6033.

[0194]

[0195] 1H NMR(500MHz,MeOD)δ8.69(s,1H),8.01(d,J=7.4Hz,2H),7.76(d,J=13.0Hz,1H),7.60(m,3H),4.51(dd,J=7.7Hz,1H),4.38(dd,J=8.3,5.2Hz,1H),4.30(dd,J=9.1,5.2Hz,1H),4.02(m,2H),3.68(m,3H),3.51(m,2H),3.37(d,J=16.7Hz,2H),3.24-3.15(m,4H),2.98(m,2H),2.90(t,2H),2.00-1.90(m,3H),1.73(m,12H),1.54-1.48(m,3H),1.41(m,2H),1.29(m,12H),1.22(m,2H),0.89(t,3H).ESI-HRMS calcd for C 52 H 77 FN 12 O8[M+H] + :1017.5971;found 1017.6047.

[0196]

[0197] 1 H NMR(500MHz,MeOD)δ8.70(s,1H),8.01(d,J=8.1Hz,2H),7.76(d,J=13.0Hz,1H),7.60(d,J=7.9Hz,2H),7.56(d,J=6.2Hz,1H),4.51(dd,J=13.6,8.1Hz,1H),4.36(dd,J=9.1,5.3Hz,1H),4.30(dd,J=9.2,4.4Hz,1H),4.02(m,2H),3.74(m,1H),3.68(m,2H),3.50(m,2H),3.38(m,2H),3.27(t,2H),3.22-3.12(m,2H),2.92(m,4H),2.04-1.89(m,3H),1.79(m,4H),1.73-1.63(m,5H),1.56-1.46(m,5H),1.42(m,3H),1.30(m,12H),1.22(m,2H),0.89(t,3H).ESI-HRMS calcd for C 52 H 77 FN 12 O8[M+H] + :1017.5971;found 1017.6041.

[0198]

[0199] 1 H NMR(500MHz,DMSO-d6)δ8.65(s,2H),8.06(s,1H),8.01-7.85(m,5H),7.60-7.45(m,6H),7.29(m,4H),6.98-6.69(m,5H),4.45(m,5H),4.29(m,3H),4.17(m,2H),3.84(d,J=28.9Hz,3H),3.56(m,1H),3.08(m,5H),2.98(m,1H),1.82(m,1H),1.74(d,J=10.4Hz,2H),1.61(m,2H),1.53(m,4H),1.35(m,3H),1.22(m,16H),1.16(m,2H),0.84(t,3H).ESI-HRMScalcd for C 52 H 77 FN 16 O8[M+H] + :1073.6564;found 1073.6621.

[0200]

[0201] 1 H NMR(500MHz,MeOD)δ8.81(s,1H),8.66(s,1H),8.02(d,J=8.2Hz,2H),7.69(d,J=13.0Hz,1H),7.61(d,J=8.2Hz,2H),7.54(d,J=7.0Hz,1H),7.36(s,1H),4.69(dd,J=8.7,5.4Hz,1H),4.53(dd,J=8.5,6.0Hz,1H),4.33(dd,J=9.2,5.2Hz,1H),4.03(m,2H),3.72(m,3H),3.51(m,2H),3.39(m,2H),3.32-3.26(m,1H),3.23-3.13(m,3H),3.02(t,2H),2.96(t,2H),1.97(m,2H),1.86(m,1H),1.81-1.75(m,3H),1.73-1.68(m,2H),1.62(m,2H),1.53-1.48(m,3H),1.43(m,2H),1.34-1.27(m,13H),1.23(m,2H),0.90(t,3H).ESI-HRMScalcd for C52 H 72 FN 11 O8[M+H] + :998.5549; found998.5614.

[0202] Performance Testing

[0203] (I) Determination of minimum inhibitory concentration

[0204] In order to study the antibacterial effect of ciprofloxacin derivatives, the minimum inhibitory concentration (MIC) was determined. First, the S. aureus strain (purchased from the China Industrial Microbiological Culture Collection) was used, and the compound concentration was diluted in equal multiples starting from 50 μg / mL. After administration, it was incubated in a 37°C constant temperature incubator for 16 hours, and the bacterial solution was measured by an enzyme marker at OD 600 The MIC of the antimicrobial peptide was the concentration of peptide that could inhibit 99.9% bacterial growth. The known drug ciprofloxacin (Cip) was used as a positive control in the experiment.

[0205] Table 1 Structure-activity relationship of ciprofloxacin derivatives

[0206]

[0207] The antibacterial effects of compounds 16u, 22e, 22g and 28b against Gram-positive bacteria (E. gallinarum ATCC 49573, MRSA ATCC 43300, E. faecalis ATCC 19433, purchased from China Industrial Microbiological Culture Collection Center) and Gram-negative bacteria (E. coil ATCC 25922, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606, purchased from China Type Culture Collection Center, Wuhan University) were further evaluated. The results are shown in Table 2.

[0208] Table 2 MIC of ciprofloxacin derivatives against Gram-positive and Gram-negative bacteria

[0209]

[0210]

[0211] (II) Hemolytic toxicity study

[0212] 1.1 Experimental steps:

[0213] The hemolytic effect of the drug was determined by measuring the light absorption value after the drug was co-incubated with rat venous blood, observing the color change of the red blood cell solution and calculating the hemolysis rate.

[0214] (1) Collect venous blood from SD rats into a venous blood collection tube containing sodium heparin anticoagulant. Mix well during blood collection to prevent blood coagulation. Centrifuge the collected blood at 800g for 5 min, discard the serum, and wash with PBS three times to obtain red blood cells, which are then diluted to 8% (V / V) with PBS and added to a 96-well plate (100 μL / well);

[0215] (2) The drug was prepared into a solution with a concentration of 200 μg / mL to 6.25 μg / mL by two-fold dilution method. The drug was then added to a 96-well plate with red blood cells (100 μL / well) and incubated at 37°C for 1 h.

[0216] (3) After incubation for 1 h, the 96-well plate was photographed and centrifuged at 1200 g for 15 min. 80 μL of the supernatant was placed in a new 96-well plate and the absorbance (OD) was measured at 490 nm using a multifunctional microplate reader. 490 PBS was used as a negative control, and 2% TritonX-100 was used as a positive control.

[0217] (4) Calculate the hemolysis rate of the drug according to formula 1;

[0218]

[0219] 1.2 Experimental results: The results are as follows Figure 1 As shown, Figure 1 The hemolytic toxicity of compounds Cip, 16u, 22e, 22g, and 28b on rat red blood cells is shown in Table 1. Figure 1 It can be seen that compounds Cip (ciprofloxacin), 16u and 28b hardly produce hemolysis at concentrations of 200 μg / mL and below.

[0220] (III) Cytotoxicity assay

[0221] 1.1 Experimental steps:

[0222] The cytotoxicity of drug solution to mammalian cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Mouse fibroblast L929 cells were used in this experiment.

[0223] (1) L929 cells were cultured in DMEM medium containing 10% FBS at a density of 8×10 3 / well was placed in an incubator with an atmosphere of 95% air and 5% CO2 at 37°C for 24 h;

[0224] (2) 200 μL per well of drug was added to cells at final concentrations ranging from 200 μg / mL to 6.25 μg / mL;

[0225] (3) After incubation for 24 h, 20 μL of 5 mg / mL MTT solution was added and incubated at 37 °C for another 4 h;

[0226] (4) The culture medium in each well was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve formazan.

[0227] (5) OD measurement with multifunctional microplate reader 570 .

[0228] 1.2 Experimental results, see Figure 2 , Figure 2 The cytotoxicity results of compounds Cip, 16u, 22e, 22g, and 28b on L929 cells at different concentrations; Figure 2 It can be seen that compounds Cip, 16u, 22e, 22g and 28b all exhibit certain cytotoxicity on L929 cells. At a concentration of 25 μg / mL, the survival rate of compounds Cip, 16u and 28b on L929 cells is above 80%.

[0229] (IV) Fluorescence staining and flow cytometry determination

[0230] The membrane-breaking effect of the drug was monitored by measuring the percentage of PI-stained positive bacteria after co-incubation of the drug and bacteria using flow cytometry.

[0231] 1.1 Experimental steps:

[0232] (1) After the bacterial solution grew to the logarithmic phase, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash with PBS, and dilute to 10 6 -10 7 CFU / mL for future use;

[0233] (2) Compound 28b was diluted with PBS to 8×MIC, 4×MIC, and 2×MIC. The positive control group was polymyxin B and compound Cip (8×MIC), and the negative control group was PBS;

[0234] (3) Incubate 100 μL of bacterial solution and 100 μL of drug solution on a shaker at 37°C for 1 h;

[0235] (4) Add PI to the mixed dye and incubate for 10 min in dark conditions. Add the mixed solution into the flow cytometer for detection.

[0236] 1.2 Experimental results Figure 3 , Figure 3 The membrane destruction results of drugs at different concentrations are shown in Table 1. Figure 3It can be seen that compared with the PBS group, the proportion of PI-positive bacteria of compound 28b at 4×MIC and 8×MIC was 16.60% and 57.22%, respectively, indicating that the bacterial membrane has been destroyed by compound 28b.

[0237] (V) Evaluation of in vitro induced drug resistance

[0238] The first generation MIC data of compound 28b and ciprofloxacin against MRSA (ATCC 33591) were obtained as described in the above MIC study. Then, the MRSA in the well next to the last clear well was diluted 10 6 CFU / mL, and the MIC was measured again after culturing at 37°C for 24 h. This step was repeated until the 25th generation. The same method was used to detect the drug resistance of compound 28b and ciprofloxacin to Escherichia coli (ATCC 25922). The results are shown in Figure 4 , Figure 4 The results of the evaluation of the tendency of compounds Cip and 28b to induce drug resistance in MRSA (a) and E.coil (b) are shown in Table 1. Figure 4 It can be seen that the MIC value of compound 28b against MRSA did not change after 25 passages, while the MIC value of ciprofloxacin increased by 32 times. Similar results were observed for E. coli. Therefore, this result shows that this type of antibiotic is difficult to make bacteria resistant.

[0239] (VI) Study on the treatment of subcutaneous infection in mice

[0240] Healthy female ICR mice (16-20 g each) were randomly divided into 4 groups, including normal group, control group, ciprofloxacin group, and compound 28b group. The concentration of ciprofloxacin hydrochloride solution was 5 mg / mL, while compound 28b was dissolved in sterile PBS at a concentration of 5 mg / mL. After the mice were anesthetized, the back hair was removed with a razor and depilatory cream. Subsequently, 100 μL of MRSA (1×10 8 CFU / mL), forming a subcutaneous abscess. After 30 minutes, 100 μL of sterile PBS, ciprofloxacin hydrochloride solution or compound 28b solution was injected into the bacterial infection site. After 48 hours, the mice were euthanized and the infected skin was collected. To evaluate the antibacterial effect, the bacteria in the abscess were counted using the standard plate counting method, and the bacterial colonies visible on the plate were imaged. The infected skin was fixed with 4% paraformaldehyde, embedded in paraffin, and stained with H&E to evaluate the antibacterial effects of various drugs. The IL-6 and TNF-α contents in the skin homogenate solution were determined using a commercial ELISA kit (Dakoway Company, Shenzhen, China) according to the manufacturer's instructions. The results are shown in Figure 5 , Figure 5In vivo antibacterial activity of compound 28b and ciprofloxacin in subcutaneous infection in mice, Figure 5 In the figure, a is a schematic diagram of the MRSA-induced subcutaneous infection model in mice, b is a photograph of the mouse skin taken 48 hours after infection after different treatments, c is the H&E staining, Masson staining and Giemsa staining images of different skin samples, d is a representative photograph of the MRSA colonies cultured from the skin homogenate of each group, e is a schematic diagram of the level of TNF-α in the skin homogenate of each group, f is a schematic diagram of the level of IL-6(f) in the skin homogenate of each group, g is the bacterial load and bacterial survival rate, h is the bacterial survival rate of each group 48 hours after injection of MRSA, Figure 5 It can be seen that the dorsal skin surface of the mice in the control group was obviously swollen, and skin abscesses appeared in the subcutaneous tissue, while the subcutaneous tissue of the mice treated with ciprofloxacin still had obvious abscesses or erythema, while the subcutaneous tissue of the mice treated with compound 28b was normal. Compared with the control group, both ciprofloxacin and compound 28b treatment significantly reduced the number of bacteria remaining on the skin and the production of MRSA-induced proinflammatory cytokines including TNF-α and IL-6.

[0241] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ciprofloxacin derivative, characterized in that: It has the structure shown in formula I: In Formula I, the lipid moiety is an alkyl group, a benzyl group or a substituted benzyl group, and the number of carbon atoms in the alkyl group is 5 to 16; The types of amino acids in the amino acid part include one or more of lysine, histidine and arginine, and the number of amino acids in the amino acid part is 1 to 4; The lipid portion and the amino acid portion are bonded via an amide bond; The amino acid portion and the linker are bonded via an amide bond; The linker and the ciprofloxacin moiety are bonded via an amide bond.

2. The ciprofloxacin derivative according to claim 1, characterized in that The substituted benzyl group is a monosubstituted benzyl group or a disubstituted benzyl group, the monosubstituted benzyl group is a 2-substituted benzyl group or a 4-substituted benzyl group, and the disubstituted benzyl group is a 2,6-disubstituted benzyl group.

3. The ciprofloxacin derivative according to claim 1 or 2, characterized in that: The substituents in the substituted benzyl group include one or more of fluorine, chlorine, bromine and phenyl.

4. The ciprofloxacin derivative according to claim 1, characterized in that In the amide bond between the lipid part and the amino acid part, the acyl group comes from the amino acid part, and the amino group comes from the lipid part.

5. The ciprofloxacin derivative according to claim 1 or 4, characterized in that The structural formula of the lipid part is shown below:

6. The ciprofloxacin derivative according to claim 1 or 4, characterized in that: The structural formula of the amino acid part is as follows:

7. The ciprofloxacin derivative according to claim 1, characterized in that The Linker is The * portion in the Linker is connected to the ciprofloxacin portion.

8. The ciprofloxacin derivative according to claim 1, characterized in that It has the following structure:

9. The method for preparing a ciprofloxacin derivative according to any one of claims 1 to 8, characterized in that: The following steps are involved: The lipid part, the amino acid part, the linker and ciprofloxacin are bonded to obtain the ciprofloxacin derivative.

10. Use of the ciprofloxacin derivative according to any one of claims 1 to 8 or the ciprofloxacin derivative prepared by the preparation method according to claim 9 in the preparation of antibacterial drugs.

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