A ciprofloxacin derivative, its preparation method and application

Abstract

The application belongs to the technical field of biological medicine, and provides a ciprofloxacin derivative, a preparation method and application thereof. The ciprofloxacin derivative provided by the application has a structure shown in formula I, a positively charged electrostatic effect sequence (an amino acid part) is connected to a lipid part, then is conjugated with ciprofloxacin through a linker, and a series of amphipathic ciprofloxacin derivatives are formed. The positively charged electrostatic effect sequence can enhance the interaction with a negatively charged bacterial cell membrane through electrostatic action, then the lipid part can be inserted into a phospholipid bilayer of the bacterial cell membrane, leading to the integrity of the bacterial cell membrane being destroyed, the ciprofloxacin derivative entering the cell, and finally leading to the leakage of the bacterial cell content, the breakage of the bacterial chromosome and the death of the bacterial cell. The amphipathic ciprofloxacin derivative of the application simulates the amphipathic structure and membrane destruction mechanism of an antibacterial peptide, so that the ciprofloxacin derivative exhibits a unique advantage of not being prone to drug resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a ciprofloxacin derivative, a preparation method and application thereof. BACKGROUND

[0002] The third generation of fluoroquinolone drugs, ciprofloxacin, has excellent antibacterial activity and pharmacokinetic characteristics, and has small side effects, and has been used in clinical treatment of various bacterial infections for about 30 years. They inhibit bacterial nucleic acid synthesis by destroying topoisomerase IV and DNA gyrase, and cause bacterial chromosome breakage, thereby exerting antibacterial effect.

[0003] Due to the emergency and extensive spread of drug-resistant pathogens, ciprofloxacin becomes more and more ineffective. The main mechanisms leading to quinolone drug resistance are gene mutation (topoisomerase variation; bacterial cell membrane permeability change; bacterial biofilm formation) and acquisition of resistance plasmid (plasmid-mediated drug resistance; bacterial active drug efflux mechanism). How to reverse the problem that ciprofloxacin is prone to drug resistance has long plagued researchers in this field, and so far there is no effective strategy. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a ciprofloxacin derivative, a preparation method and application thereof. The ciprofloxacin derivative provided by the present application is not prone to drug resistance, and has high bactericidal activity in mice.

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

[0006] The present application provides a ciprofloxacin derivative having the 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 of the alkyl group is 5-16;

[0009] The type of amino acid in the amino acid moiety includes one or more of lysine, histidine and arginine, and the number of amino acids in the amino acid moiety is 1-4;

[0010] The lipid moiety and the amino acid moiety are bonded by an amide bond;

[0011] The amino acid moiety and the linker are bonded by an amide bond;

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

[0013] The present application provides a ciprofloxacin derivative.

[0014] The ciprofloxacin derivative provided by the present application 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 ciprofloxacin derivatives with amphiphilicity. The positively charged electrostatic effect sequence can enhance the interaction with the negatively charged bacterial cell membrane through electrostatic effect, and then the lipid part can insert into the phospholipid bilayer of the bacterial cell membrane, leading to the integrity of the bacterial cell membrane being destroyed, the ciprofloxacin derivative entering the cell, and finally leading to the leakage of the bacterial cell content, the breakage of the bacterial chromosome and the death of the bacterial cell. The ciprofloxacin derivative of the present application simulates the amphiphilicity and membrane destruction mechanism of AMPs (Antimicrobial peptides, AMP), so that the ciprofloxacin derivative exhibits a unique advantage of not being prone to drug resistance. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0017] Figure 3 Membrane destruction of drugs at different concentrations;

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

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

[0020] The present application provides a ciprofloxacin derivative having the structure shown in formula I:

[0021]

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

[0023]

[0024] In the present application, in the formula I, the kind of amino acid in the amino acid moiety includes one or more of lysine (Lys), histidine (His) and arginine (Arg), and the number of amino acids in the amino acid moiety is 1 to 4, and preferably 1, 2, 3 or 4. In the present application, when the number of amino acids in the amino acid moiety is plural, the kinds of the plural amino acids are preferably a plurality of combinations, and the same amino acid can also be repeated. In the present application, the structural formula of the amino acid moiety is preferably as shown below:

[0025]

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

[0027] The * site in the Linker is connected to the moieties of ciprofloxacin.

[0028] In the present application, the lipid moiety and the amino acid moiety are bonded by an amide bond. In the present application, the acyl group in the amide bond by which the lipid moiety and the amino acid moiety are bonded is preferably from the amino acid moiety, and the amino group is preferably from the lipid moiety.

[0029] In the present application, the amino acid moiety and the Linker are bonded by an amide bond. In the present application, the acyl group in the amide bond by which the amino acid moiety and the Linker are bonded is preferably from the Linker, and the amino group is preferably from the amino acid moiety.

[0030] In the present application, the Linker and the moieties of ciprofloxacin are bonded by an amide bond. In the present application, the acyl group in the amide bond by which the Linker and the moieties of ciprofloxacin are bonded is preferably from the Linker, and the amino group is preferably from the moieties of ciprofloxacin.

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

[0032]

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

[0034] when n is 2, R4 is

[0035] when n is 3, R4 is

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

[0037] 28a-e, R6 is

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

[0039]

[0040]

[0041]

[0042]

[0043] The present application also provides a preparation method of the ciprofloxacin derivative described in the above technical solution, comprising the following steps:

[0044] bonding the lipid moiety, the amino acid moiety, the linker and the ciprofloxacin to obtain the ciprofloxacin derivative.

[0045] The preparation method will be described in detail below according to the structure division of .

[0046] In the present application, the preparation method of the ciprofloxacin derivative represented by the structure of formula 16a-u (hereinafter referred to as the first preparation method) comprises the following steps:

[0047] The 2-chlorotrityl chloride resin is swelled to obtain a swelled resin; the swelled resin, Fmoc-Lys(Boc)-OH shown in formula 10, and N,N-diisopropylethylamine (DIPEA) are mixed to sequentially perform solid phase synthesis reaction, capping reaction, and Fmoc removal to obtain a compound shown in formula 11, which is an amino acid grafted compound containing one amino acid;

[0048] When n in the structure shown in formula 16a-u is 2, the 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) are mixed to sequentially perform solid phase synthesis reaction and Fmoc removal to obtain an amino acid grafted compound containing two amino acids; when n in the structure shown in formula 16a-u is 3, Fmoc-Lys(Boc)-OH shown in formula 10, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), and N,N-dimethylformamide (DMF) are further added to sequentially perform solid phase synthesis reaction and Fmoc removal 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 to perform solid phase synthesis reaction to obtain a compound shown in formula 12;

[0050] A solution of ciprofloxacin methyl ester is added dropwise to a solution of the compound shown in formula 12 to sequentially perform solid phase synthesis reaction and cleavage reaction to obtain a compound shown in formula 13; the solution of the compound shown in formula 12 includes the compound 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 includes ciprofloxacin methyl ester, N,N-diisopropylethylamine (DIPEA), and N,N-dimethylformamide (DMF);

[0051] mixing a solution of a compound of formula 13 and a solution of R4-H, to perform an amide condensation reaction, 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] performing a hydrolysis reaction on the compound of formula 14, to obtain a compound of formula 15;

[0053] performing Boc removal on the compound of formula 15, to obtain the compound of formula 16a-u;

[0054]

[0055] The present application swells 2-chlorotrityl chloride resin to obtain a swollen resin; mixes the swollen resin, Fmoc-Lys(Boc)-OH shown in formula 10 and DIPEA, and sequentially performs solid phase synthesis reaction, capping reaction and Fmoc removal to obtain a compound shown in formula 11, which is an amino acid grafted compound containing one amino acid. In the present application, the swelling agent is preferably dichloromethane. In the present application, the ratio of the amount of 2-chlorotrityl chloride resin to dichloromethane is preferably 0.22 mmol:6 mL. In the present application, the swelling temperature is preferably room temperature, and the time is preferably 15 min. In the present application, the molar ratio of 2-chlorotrityl chloride resin to Fmoc-Lys(Boc)-OH is preferably 1:2, and the molar ratio of Fmoc-Lys(Boc)-OH to DIPEA is preferably 0.44:0.79. In the present application, the temperature of the solid phase synthesis reaction is preferably room temperature, and the time is preferably 1-5 h, and the solid phase synthesis reaction is preferably performed under oscillation, and is preferably performed in a solid phase synthesis tube. After the solid phase synthesis reaction, the present application preferably further comprises: discharging the reaction solution, and washing the resin, which comprises sequentially performing DMF washing and DCM washing, and the number of DMF washing is preferably 3 times, and the number of DCM washing is preferably 3 times. In the present application, the reagent for the capping reaction is preferably a DIPEA / MeOH / DCM mixed solution, and in the DIPEA / MeOH / DCM mixed solution, the volume ratio of DIPEA, MeOH and DCM is preferably 1:2:17, and the ratio of the amount of 2-chlorotrityl chloride resin to the DIPEA / MeOH / DCM mixed solution is preferably 0.22 mmol:5 mL, and the temperature of the capping reaction is preferably room temperature, and the time is preferably 30 min, and after the capping reaction, the present application preferably further comprises: washing the capped resin, which preferably comprises sequentially performing DCM washing and DMF washing, and the number of DCM washing is preferably 3 times, and the number of DMF washing is preferably 3 times. In the present application, the reagent for Fmoc removal 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 application, the time for Fmoc removal is preferably 10 min, and the number of times is preferably 2 times.

[0056] After obtaining the compound shown in formula 11, when n in the structure shown in formula 16a-u is 2, the present application mixes the compound shown in formula 11, Fmoc-Lys(Boc)-OH shown in formula 10, DIC, HOBt and DMF, and sequentially carries out solid phase synthesis reaction and Fmoc removal 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 shown in formula 10, DIC, HOBt and DMF are added, and sequentially carries out solid phase synthesis reaction and Fmoc removal to obtain an amino acid grafted compound containing three amino acids.

[0057] In the present application, the molar ratio of the 2-chlorotrityl chloride resin and Fmoc-Lys(Boc)-OH is preferably 1:2, the molar ratio of the 2-chlorotrityl chloride resin and DIC is preferably 1:4, the molar ratio of DIC and HOBt is preferably 1:1, and the usage ratio of the 2-chlorotrityl chloride resin and DMF is preferably 0.22 mmol:3 mL. In the present application, the parameters and process of the solid phase synthesis reaction and Fmoc removal are consistent with those in the preparation of compound 11, which will not be described here.

[0058] After obtaining the amino acid grafted compound, the present application mixes the amino acid grafted compound, terephthalic acid, HCTU, DIPEA and DMF, and carries out solid phase synthesis reaction to obtain a compound shown in formula 12.

[0059] In the present application, the molar ratio of the 2-chlorotrityl chloride resin 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 usage ratio of the 2-chlorotrityl chloride resin and DMF is preferably 0.22 mmol:3 mL. In the present application, the temperature of the solid phase synthesis reaction is preferably room temperature, and the time is preferably 1-5 h, and the solid phase synthesis reaction is preferably carried out under oscillation. After the solid phase synthesis reaction, the present application preferably further comprises: removing the solution, washing the resin, and the washing comprises sequentially carrying out DMF washing and DCM washing, the number of times of the DMF washing is preferably 3 times, and the number of times of the DCM washing is preferably 3 times.

[0060] After obtaining the compound shown in formula 12, a solution of ciprofloxacin methyl ester is added dropwise to a solution of the compound shown in formula 12, and sequentially carries out solid phase synthesis reaction and cleavage reaction to obtain a compound shown in formula 13; the solution of the compound shown in formula 12 comprises the compound 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 application, the molar ratio of 2-chlorotritylchloride resin and HCTU in the solution of the compound of formula 12 is preferably 0.22:0.40, the molar ratio of HCTU and DIPEA is preferably 0.40:0.79, and the ratio of the amount of DIPEA and DMF is preferably 0.79 mmol:3 mL. In the present application, the molar ratio of ciprofloxacin methyl ester and DIPEA in the solution of ciprofloxacin methyl ester is preferably 1:1, and the ratio of the amount of ciprofloxacin methyl ester and DMF is preferably 0.44 mmol:3 mL. In the present application, the molar ratio of 2-chlorotritylchloride resin and ciprofloxacin methyl ester is preferably 1:2. In the present application, the temperature of the solid-phase synthesis reaction is preferably room temperature, and the time is preferably 1-5 h. After the solid-phase synthesis reaction, the present application preferably further comprises removing the solution, and sequentially washing the resin with DMF and DCM, the number of times of DMF washing is preferably 3, and the number of times of DCM washing is preferably 3. In the present application, the cleavage solution of the cleavage reaction is preferably a HFIP / DCM mixed solution, the volume ratio of HFIP and DCM in the HFIP / DCM mixed solution is preferably 1:4, and the ratio of the amount of 2-chlorotritylchloride resin and cleavage solution is preferably 0.22 mmol:8 mL. In the present application, the time of the cleavage reaction is preferably 2 h, and after the cleavage reaction, the present application 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, subjecting the combined solution to vacuum distillation to obtain a crude compound, dissolving the crude compound, filtering the obtained filtrate, purifying the filtrate by preparative HPLC, and freeze-drying to obtain the compound of formula 13.

[0062] After obtaining the compound of formula 13, the present application mixes the solution of the compound of formula 13 and the solution of R4-H to perform an amide condensation reaction, to obtain the compound of formula 14, the solution of the compound of formula 13 comprising the compound of formula 13, EDC, HOBt, DIPEA and DMF, and the solution of R4-H comprising R4-H, DIPEA and DMF.

[0063] In the present application, the molar ratio of the compound of formula 13 to EDC in the solution of the compound of formula 13 is preferably 0.17:0.26, the molar ratio of EDC to HOBt is preferably 1:1, the ratio of the amount of use of the compound of formula 13 to DMF is preferably 0.17 mmol:8 mL, and the molar ratio of the compound of formula 13 to DIPEA is preferably 1:2. In the present application, the molar ratio of R4-H to DIPEA in the solution of R4-H is preferably 1:1, and the molar ratio of the compound of formula 13 to R4-H is preferably 1:2. In the present application, the temperature of the amide condensation reaction is preferably room temperature, and the time is preferably 1-5 h, and the amide condensation reaction is preferably carried out under stirring. After the amide condensation reaction, the present application preferably further comprises: quenching reaction, the obtained reaction liquid is extracted, the obtained organic phase is sequentially washed, dried, and distilled under reduced pressure to obtain the compound of formula 14; the reagent of the quenching reaction is preferably 1 mol / L hydrochloric acid, the extracting agent of the extraction is preferably DCM, the number of times of the extraction is preferably 3, the reagent of the washing is preferably saturated NaCl washing, and the reagent of the drying is preferably anhydrous Na2SO4.

[0064] After obtaining the compound of formula 14, the present application carries out hydrolysis reaction on the compound of formula 14 to obtain the compound of formula 15. In the present application, the solvent of the hydrolysis reaction preferably comprises methanol aqueous solution, the volume ratio of methanol to water in the methanol aqueous solution is preferably 5:1, and the ratio of the amount of use of the compound of formula 14 to the methanol aqueous solution is preferably 0.17 mmol:6 mL. In the present application, the reagent of the hydrolysis reaction further preferably comprises lithium hydroxide, and the molar ratio of the compound of formula 14 to lithium hydroxide is preferably 1:10. In the present application, the temperature of the hydrolysis reaction is preferably room temperature, and the time is preferably 2-4 h, and the hydrolysis reaction is preferably carried out under stirring. After the hydrolysis reaction, the present application preferably further comprises: quenching reaction, the obtained reaction liquid is extracted, the obtained organic phase is sequentially washed, dried, and distilled under reduced pressure to obtain the compound of formula 15; the reagent of the quenching reaction is preferably 1 mol / L hydrochloric acid; the extracting agent of the extraction is preferably DCM, the number of times of the extraction is preferably 3, the reagent of the washing is preferably saturated NaCl, and the reagent of the drying is preferably anhydrous Na2SO4.

[0065] After obtaining the compound of formula 15, the present application removes Boc from the compound of formula 15 to obtain the compound of formula 16a-u. In the present application, the reagent for removing Boc preferably includes trifluoroacetic acid dichloromethane solution (TFA / DCM solution). In the present application, the volume ratio of TFA and DCM in the TFA / DCM solution is preferably 19:1, and the use ratio of the compound of formula 15 and TFA / DCM solution is preferably 0.17 mmol:5 mL. In the present application, the temperature for removing Boc is preferably room temperature, and the time is preferably 2 h. The removal of Boc is preferably performed under stirring. After the removal of Boc is completed, the present application preferably further includes: performing vacuum distillation on the obtained solution to obtain a crude product, dissolving the crude product and filtering, and sequentially performing HPLC purification and freeze-drying treatment on the obtained filtrate to obtain the compound of formula 16a-u. The dissolving reagent is preferably methanol aqueous solution, the volume ratio of methanol and water in the methanol aqueous solution is preferably 1:1, and the filtering device is preferably Whatman filter with a pore size of 0.22 μm.

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

[0067]

[0068] In the present application, the preparation method of the cyclopropyl ciprofloxacin derivative of formula 22a-g (hereinafter referred to as the second preparation method) includes the following steps:

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

[0070] When R5 is The compound of formula 17, N,N-diisopropyl ethylamine (DIPEA), succinic anhydride and N,N-dimethylformamide (DMF) are mixed, and solid-phase synthesis reaction is performed to obtain the compound of formula 18;

[0071] When R5 is mixing the compound represented by formula 18, succinic anhydride, N,N- diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and performing a solid-phase synthesis reaction to obtain a compound represented by formula 18; the Fmoc-R5-COOH has the following structure:

[0072] mixing a solution of the compound represented by formula 18 and a solution of ciprofloxacin methyl ester, sequentially performing a solid-phase synthesis reaction and a cleavage reaction to obtain a compound represented by formula 19; the solution of the compound represented by formula 18 includes the compound represented by formula 18, 6-chlorobenzotriazol-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA) and N,N-dimethylformamide (DMF), and the solution of ciprofloxacin methyl ester includes ciprofloxacin methyl ester, DIPEA and DMF;

[0073] mixing a solution of the compound represented by formula 19 and a solution of nonylamine, performing an amide condensation reaction to obtain a compound represented by formula 20; the solution of the compound represented by formula 19 includes the compound represented by formula 19, 1,2-dichloroethane (EDC), 1-hydroxybenzotriazole (HOBt), DIPEA and DMF, and the solution of nonylamine includes nonylamine, DIPEA and DMF;

[0074] performing a hydrolysis reaction on the compound represented by formula 20 to obtain a compound represented by formula 21;

[0075] performing Boc removal on the compound represented by formula 21 to obtain a compound represented by formula 22a-g;

[0076]

[0077] The compound shown in formula 11, Fmoc-Lys(Boc)-OH, DIC, HOBt and DMF are mixed, and solid-phase synthesis reaction, capping reaction and Fmoc removal are sequentially carried out, and then the corresponding amino acid, N,N'-diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are added, and the solid-phase synthesis reaction and Fmoc removal are repeated to obtain a compound shown in formula 17. In the present application, the preparation method of the compound shown in formula 11 is consistent with the above technical solution, and will not be repeated here. In the present application, the molar ratio of the 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 0.88 mmol:3 mL. In the present application, the parameters of the solid-phase synthesis reaction, capping reaction and Fmoc removal and the subsequent post-treatment are consistent with the above technical solution, and will not be repeated here.

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

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

[0080] , the compound shown in formula 17, Fmoc-R5-COOH, N,N'-diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and N,N-dimethylformamide (DMF) are mixed, and solid-phase synthesis reaction and Fmoc removal are sequentially carried out; the obtained Fmoc removal liquid, succinic anhydride, N,N-diisopropyl ethylamine (DIPEA) and N,N-dimethylformamide (DMF) are mixed, and solid-phase synthesis reaction is carried out to obtain a compound shown in formula 18; the Fmoc-R5-COOH has the following structure:

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

[0082] After obtaining the compound shown in formula 18, the present application mixes a solution of the compound shown in formula 18 and a solution of ciprofloxacin methyl ester, and sequentially performs a solid-phase synthesis reaction and a cleavage reaction to obtain a compound shown in formula 19, wherein the solution of the compound shown in formula 18 comprises the compound 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 application, in the solution of the compound shown in formula 18, the molar ratio of the 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 usage ratio of the compound shown in formula 18 to DMF is preferably 0.22 mmol:3 mL. In the present application, in the solution of ciprofloxacin methyl ester, the molar ratio of ciprofloxacin methyl ester to DIPEA is preferably 1:1, and the usage ratio of DIPEA to DMF is preferably 0.44 mmol:3 mL. In the present application, the molar ratio of the compound shown in formula 18 to ciprofloxacin methyl ester is preferably 1:2. In the present application, the solid-phase synthesis reaction and the cleavage reaction are preferably consistent with the above technical solutions, and will not be described here again.

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

[0085] In the present application, the molar ratio of the compound of formula 19 to EDC in the solution of the compound of formula 19 is preferably 0.17:0.26, the molar ratio of EDC to HOBt is preferably 1:1, the ratio of the amount of the compound of formula 19 to DMF is preferably 0.17 mmol:8 mL, and the molar ratio of the compound of formula 19 to DIPEA is preferably 1:2. In the present application, the molar ratio of nonylamine to DIPEA in the solution of nonylamine is preferably 1:1. In the present application, the molar ratio of the compound of formula 19 to nonylamine is preferably 1:2. In the present application, the conditions of the amide condensation reaction and the post-treatment thereof are consistent with the above technical solution, and will not be described here.

[0086] After obtaining the compound of formula 20, the present application performs a hydrolysis reaction on the compound of formula 20 to obtain a compound of formula 21. In the present application, the parameters of the hydrolysis reaction are consistent with the above technical solution, and will not be described here.

[0087] After obtaining the compound of formula 21, the present application removes Boc from the compound of formula 21 to obtain a compound of formula 22a-g. In the present application, the reagent and steps for removing Boc are preferably consistent with the above technical solution, and will not be described here.

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

[0089]

[0090] In the present application, the preparation method of the cyclofloxacin derivative of formula 28a-e (hereinafter referred to as the third preparation method) includes the following steps:

[0091] The 2-chlorotrityl chloride resin is swelled to obtain a swelled resin; the swelled resin, a corresponding amino acid, and N,N-diisopropylethylamine (DIPEA) are mixed to sequentially perform a solid-phase synthesis reaction, a capping reaction, and Boc removal on the swelled resin; a corresponding amino acid is added, and the solid-phase synthesis reaction and Boc removal are repeatedly performed in sequence to obtain a compound of formula 23;

[0092] The compound of formula 23, terephthalic acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyl uronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA), and N,N-dimethylformamide (DMF) are mixed to perform a solid-phase synthesis reaction to obtain a compound of formula 24;

[0093] Mixing a solution of a compound shown in formula 24 and a solution of ciprofloxacin methyl ester, sequentially performing solid phase synthesis reaction and cleavage reaction, to obtain a compound shown in formula 25, the solution of the compound shown in formula 24 comprising the compound shown in formula 24, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropyl ethylamine (DIPEA) and N,N-dimethyl formamide (DMF), the solution of the ciprofloxacin methyl ester comprising ciprofloxacin methyl ester, N,N-diisopropyl ethylamine (DIPEA) and N,N-dimethyl formamide;

[0094] Mixing a solution of a compound shown in formula 25 and a solution of nonylamine, performing amide condensation reaction, to obtain a compound shown in formula 26; the solution of the compound shown in formula 25 comprising the compound shown in formula 25, 1,2-dichloroethane (EDC), 1-hydroxybenzotriazole (HOBt), N,N-diisopropyl ethylamine (DIPEA) and N,N-dimethyl formamide (DMF), the solution of the nonylamine comprising nonylamine, N,N-diisopropyl ethylamine (DIPEA) and N,N-dimethyl formamide (DMF);

[0095] Performing hydrolysis reaction on the compound shown in formula 26, to obtain a compound shown in formula 27;

[0096] Performing Boc removal on the compound shown in formula 27, to obtain a compound shown in formula 28a-e;

[0097]

[0098]

[0099] The present application performs swelling on 2-chlorotrityl chloride resin, to obtain swollen resin; mixing the swollen resin, corresponding amino acid and DIPEA, sequentially performing solid phase synthesis reaction, capping reaction and Fomc removal; adding corresponding amino acid, repeatedly sequentially performing solid phase synthesis reaction, capping reaction and Fomc removal, to obtain a compound shown in formula 23. In the present application, the swelling, solid phase synthesis reaction, capping reaction and Fomc removal are consistent with the above technical solution, which will not be repeated here. In the present application, the corresponding amino acid contains a protecting group.

[0100] After obtaining the compound shown in formula 23, the present application mixes the compound shown in formula 23, terephthalic acid, HCTU, DMF and DIPEA to carry out a solid-phase synthesis reaction to obtain a compound shown in formula 24. In the present application, the molar ratio of the compound shown in formula 23 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 usage ratio of the compound shown in formula 23 to DMF is preferably 0.22 mmol:3 mL. In the present application, the solid-phase synthesis reaction and the subsequent post-treatment are preferably consistent with the above technical solutions, and will not be repeated here.

[0101] After obtaining the compound shown in formula 24, the present application mixes a solution of the compound shown in formula 24 and a solution of ciprofloxacin methyl ester, and sequentially carries out a solid-phase synthesis reaction and a cleavage reaction to obtain a compound shown in formula 25, wherein the solution of the compound shown in formula 24 comprises the compound 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 application, in the solution of the compound shown in formula 24, the molar ratio of the compound shown in formula 24 to HCTU is preferably 0.22:0.4, the molar ratio of the compound shown in formula 24 to DIPEA is preferably 0.22:0.79, and the usage ratio of the compound shown in formula 24 to DMF is preferably 0.22 mmol:3 mL. In the present application, in the solution of ciprofloxacin methyl ester, the molar ratio of ciprofloxacin methyl ester to DIPEA is preferably 1:1, and the usage ratio of ciprofloxacin methyl ester to DMF is preferably 0.44 mmol:3 mL. In the present application, the molar ratio of the compound shown in formula 24 to ciprofloxacin methyl ester is preferably 1:2. In the present application, the conditions of the solid-phase synthesis reaction and the cleavage reaction and the subsequent post-treatment are preferably consistent with the above technical solutions, and will not be repeated here.

[0103] After obtaining the compound shown in formula 25, the present application mixes a solution of the compound shown in formula 25 and a solution of nonylamine to carry out an amide condensation reaction, to obtain a compound shown in formula 26, the solution of the compound shown in formula 25 comprising the compound shown in formula 25, EDC, HOBt, DIPEA and DMF, the solution of nonylamine comprising nonylamine, DIPEA and DMF. In the present application, in the solution of the compound shown in formula 25, the molar ratio of the compound shown in formula 25 to EDC is preferably 0.17:0.26, the molar ratio of EDC to HOBt is preferably 1:1, the usage ratio of the compound shown in formula 25 to DMF is preferably 0.17 mmol:8 mL, and the molar ratio of the compound shown in formula 25 to DIPEA is preferably 1:2. In the present application, in the solution of nonylamine, the molar ratio of nonylamine to DIPEA is preferably 1:1. In the present application, the molar ratio of the compound shown in formula 25 to nonylamine is preferably 1:2. In the present application, the amide condensation reaction is preferably consistent with the above technical solutions, which will not be repeated here.

[0104] After obtaining the compound shown in formula 25, the present application carries out a hydrolysis reaction on the compound shown in formula 26 to obtain a compound shown in formula 27. In the present application, the hydrolysis reaction is preferably consistent with the above technical solutions, which will not be repeated here.

[0105] After obtaining the compound shown in formula 27, the present application carries out Boc removal on the compound shown in formula 27 to obtain a compound shown in formula 28a-e. In the present application, the Boc removal is preferably consistent with the above technical solutions, which will not be repeated here.

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

[0107]

[0108] The present application also provides the use of the cyclopropyl ciprofloxacin derivative of the above technical solutions or the cyclopropyl ciprofloxacin derivative prepared by the preparation method of the above technical solutions in the preparation of antibacterial drugs. The present application does not make specific limitations on the use mode, which can be operated by those skilled in the art.

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

[0110] The cyclopropyl ciprofloxacin derivative, the preparation method and the application thereof provided by the present application will be described in detail below in combination with examples, but they should not be understood as limitations on the protection scope of the present application.

[0111] Examples

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

[0113] The 2-chlorotrityl chloride (CTC) resin (300 mg, 0.22 mmol) was swelled 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 the solid-phase synthesis tube for the solid-phase synthesis reaction at room temperature for 2 h to attach the first amino acid to the resin. The resin was capped with 5 mL of DIPEA / MeOH / DCM (1 / 2 / 17, v / v / v) for 30 min, and then washed with DCM (4 mL x 3) and DMF (4 mL x 3). The Fmoc group was removed by treating the resin with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2) to give compound 11, which is an amino acid grafted compound containing one amino acid.

[0114] When n is 2 in the structure of 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 pre-mixed in DMF (3 mL) for 5 min, and then transferred to the solid-phase synthesis tube for the solid-phase synthesis reaction at room temperature for 2 h. The solution was removed, and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3). The Fmoc group was removed again by treating the resin with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2). After the reaction was completed, the solution was drained, and the resin was washed with DCM (3 mL x 3) and DMF (3 mL x 3) to give an amino acid grafted compound containing two amino acids. When n is 3 in the structure of formula 16a-u, Fmoc-Lys(Boc)-OH, DIC, HOBt and DMF of formula 10 were mixed, and the solid-phase synthesis reaction and the removal of Fmoc were sequentially performed to give 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 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, the solution was removed, and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3) to give compounds 12a-12u.

[0116] To the solid-phase synthesis tube was added HCTU (166 mg, 0.40 mmol) and DIPEA (140 μL, 0.79 mmol) in DMF (3 mL) for 5 min, then the 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 reaction was allowed to proceed at room temperature for 5 h. The solution was removed, and the tube was washed with DMF (4 mL x 3) and DCM (4 mL x 3), and then cleaved with 8 mL of cleavage solution (HFIP / DCM 1 :4, v / v) for 2 h. The intermediate 13a-13u was cleaved from the resin, and the cleavage solution was collected. The resin was washed with 4 mL of cleavage solution three times to obtain the washing solution. The cleavage solution and the washing solution were combined and distilled under reduced pressure. The crude compound of 13a-13u was dissolved in water / methanol (1 :1) solution (10 mL), filtered with Whatman filter (0.22 μm), and then analyzed by HPLC analysis. The pure 13a-13u was obtained as a white powder after purification by preparative HPLC and lyophilization.

[0117] To the solution of 13a-13u (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) in dry DMF (8 mL) was added DIPEA (60 μL, 0.34 mmol), and the reaction was stirred at 0 °C for 5 min. Then the pre-mixed solution of R4-H (0.34 mmol) and DIPEA (60 μL, 0.34 mmol) in DMF was added, and the reaction was stirred at room temperature for 5 h. After the reaction was completed, the reaction was quenched with 1 mol / L HC1, and the organic phase was extracted with DCM (x 3) and washed with brine. The organic phase was dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product 14a-14u was used directly without further purification.

[0118] To the solution of 13a-13u (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) in dry DMF (8 mL) was added DIPEA (60 μL, 0.34 mmol), and the reaction was stirred at 0 °C for 5 min. Then the pre-mixed solution of R4-H (0.34 mmol) and DIPEA (60 μL, 0.34 mmol) in DMF was added, and the reaction was stirred at room temperature for 5 h. After the reaction was completed, the reaction was quenched with 1 mol / L HC1, and the organic phase was extracted with DCM (x 3) and washed with brine. The organic phase was dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product 14a-14u was used directly without further purification.

[0119] Compound 15a-15u (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 was distilled under reduced pressure, dissolved in water / methanol (1:1) solution (10 mL), filtered through Whatman filter (0.22 pm), and detected by analytical HPLC. After purification by preparative HPLC and lyophilization, 16a-16u were obtained as white powder.

[0120]

[0121] 1 H NMR (500 MHz, MeOD) d 8.73 (s, 1H), 7.99 (d, J = 7.5 Hz, 2H), 7.84 (d, J = 13.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 3H), 4.55 (dd, J = 8.9, 5.5 Hz, 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), 1.84 m, 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 = 6.9 Hz, 3H). ESI-HRMS calcd for C 36 H 45 FN6O6[M+H] + : 677.3385; found 677.3475.

[0122]

[0123] 1 H NMR (500 MHz, MeOD) d 8.72 (s, 1H), 7.99 (d, J = 7.5 Hz, 2H), 7.81 (d, J = 13.0 Hz, 1H), 7.60 (d, J = 7.4 Hz, 3H), 4.56 (dd, J = 8.8, 5.6 Hz, 1H), 4.08 - 3.91 (m, 2H), 3.69 (m, 3H), 3.45 (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 calcd for C37 H 47 FN6O6[M+H] + :691.3541; found 691.3637.

[0124]

[0125] 1 H NMR (500 MHz, MeOD) δ 8.74 (s, 1H), 8.00 (s, 2H), 7.84 (d, J = 12.6 Hz, 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 = 7.0 Hz, 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; found 719.3944.

[0126]

[0127] 1 H NMR (500 MHz, MeOD) δ 8.72 (s, 1H), 7.97 (d, J = 7.7 Hz, 2H), 7.82 (d, J = 13.0 Hz, 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 for C 40 H 53 FN6O6[M+H] + :733.4011; found 733.4087.

[0128]

[0129] 1 H NMR (500 MHz, MeOD) δ 8.76 (s, 1H), 8.00 (d, J = 7.8 Hz, 2H), 7.95 (d, J = 8.1 Hz, 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) δ 8.74 (s, 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 13.0 Hz, 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-HRMS calcd for C 45 H 63 FN6O6[M+H] + :803.4793; found 803.4876.

[0132]

[0133] 1H NMR (500 MHz, MeOD) δ 8.76 (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 for C 47 H 67 FN6O6[M+H] + :831.5106; found 831.5189.

[0134]

[0135] 1 H NMR (500 MHz, MeOD) δ 8.70 (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) δ 8.74 (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; found 731.2763.

[0138]

[0139] 1 H NMR (500 MHz, MeOD) δ 8.72 (s, 1H), 7.96 (d, J = 7.5 Hz, 2H), 7.81 (d, J = 12.9 Hz, 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-HRMS calcd for C 38 H 39 Cl2FN6O6[M+H] + :765.2292; found 765.2374.

[0140]

[0141] 1H NMR (500 MHz, MeOD) δ 8.74 (s, 1H), 7.96 (d, J = 7.2 Hz, 2H), 7.84 (d, J = 12.8 Hz, 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 for C 38 H 39 ClF2N6O6[M+H] + : 749.2588; found 749.2674.

[0142]

[0143] 1 H NMR (500 MHz, MeOD) δ 8.73 (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 calcd for C 44 H 45 FN6O6[M+H] + : 773.3385; found 773.3446.

[0144]

[0145] 1H NMR (500 MHz, MeOD) δ 8.67 (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) δ 8.63 (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 for C 47 H 67 FN8O7[M+H] + :875.51; found 875.52.

[0148]

[0149] 1 H NMR (500 MHz, MeOD) δ 8.76 (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) δ 8.65 (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) δ 8.75 (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 (500 MHz, MeOD) δ 8.77 (s, 1H), 8.01 (s, 2H), 7.88 (d, J = 12.6 Hz, 1H), 7.61 (s, 3H), 4.50 (t, 1H), 4.37 (t, 1H), 4.30 (dd, J = 9.0, 5.1 Hz, 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 (500 MHz, DMSO-de) d 8.69 (d, J = 6.8 Hz, 1H), 8.63 (m, 1H), 8.53 (t, 1H), 8.15 (d, J = 6.7 Hz, 1H), 8.02 (dd, J = 13.7, 8.0 Hz, 3H), 7.90 (m, 8H), 7.65 - 7.55 (m, 7H), 7.45 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 8.5 Hz, 3H), 4.48 - 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; found 1029.5335.

[0162] Compounds 22a-g were prepared as follows:

[0163] The 2-chlorotrityl chloride (CTC) resin (300 mg, 0.22 mmol) was swelled in dichloromethane (6 mL) for 15 min. Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol) and DIPEA (140 μί, 0.79 mmol) were added to the solid-phase synthesis tube to perform the solid-phase synthesis reaction at room temperature for 2 h to link the first amino acid to the resin. The resin was capped with 5 mL of DIPEA / MeOH / DCM (1 / 2 / 17, v / v / v) for 30 min, and then washed with DCM (4 mL x 3) and DMF (4 mL x 3). The Fmoc group was removed by treating the resin with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2) to give compound 11.

[0164] To the solid-phase synthesis tube was added Fmoc-Lys(Boc)-OH (208 mg, 0.44 mmol), DIC (120 μL, 0.88 mmol) and HOBt (121 mg, 0.88 mmol) pre-mixed in DMF (3 mL) for 5 min, then transferred to the solid-phase synthesis tube. After the solid-phase synthesis reaction at room temperature for 2 h, the solution was removed and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3). Fmoc group was removed again by treating the resin with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2). After the reaction was completed, the solution was drained and the resin was washed with DCM (3 mL x 3) and DMF (3 mL x 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 pre-mixed in DMF (3 mL) for 5 min, then transferred to the solid-phase synthesis tube. The solid-phase synthesis reaction was carried out at room temperature for 2 h. The solution was removed and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3). Fmoc protecting group was removed by treating the resin with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2) to give compound 17.

[0165] Next, DIPEA (306 μL, 1.76 mmol), succinic anhydride (88 mg, 0.88 mmol) and DMF were added to the solid-phase synthesis tube. The solid-phase synthesis reaction was carried out at room temperature for 1 h. The solution was removed and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3) to give 18a.

[0166] Next, Fmoc-R5-COOH (0.44 mmol), DIC (120 μL, 0.88 mmol) and HOBt (121 mg, 0.88 mmol) were pre-mixed in DMF (3 mL) for 5 min, then transferred to the solid-phase synthesis tube. After the solid-phase synthesis reaction at room temperature for 2 h, the solution was removed and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3). Fmoc group was removed again by treating the resin with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2). After the removal of Fmoc was completed, the solution was drained and the resin was washed with DCM (3 mL x 3) and DMF (3 mL x 3). Next, succinic anhydride (88 mg, 0.88 mmol) and DIPEA (306 μL, 1.76 mmol) were added to the solid-phase synthesis tube. The solid-phase synthesis reaction was carried out at room temperature for 1 h. The solution was removed and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3) to give 18b-18g.

[0167] To the solid phase synthesis tube was added HCTU (166 mg, 0.40 mmol) and a solution of DIPEA (140 μL, 0.79 mmol) in DMF (3 mL) and mixed for 5 min, ciprofloxacin methyl ester (154 mg, 0.44 mmol), DIPEA (78 μL, 0.44 mmol) and DMF (3 mL) were mixed to give a solution of ciprofloxacin methyl ester and the reaction was allowed to proceed at room temperature for 5 h. The solution was removed and the resin was washed with DMF (4 mL x 3) and DCM (4 mL x 3) and cleaved with 8 mL of cleavage solution (HFIP / DCM 1 :4, v / v) for 2 h. The cleavage solution was collected and the resin was washed with 4 mL of cleavage solution three times to give a wash solution; the cleavage solution and wash solution were combined and evaporated under reduced pressure. The crude 19a-19g was dissolved in water / methanol (1 :1) solution (10 mL) and filtered through a Whatman filter (0.22 μm) and then analyzed by HPLC analysis. The pure 19a-19g was obtained as a white powder after purification by preparative HPLC and lyophilization.

[0168] To a solution of 19a-19g (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) in dry DMF (8 mL) was added DIPEA (60 μL, 0.34 mmol) and the reaction was stirred at 0 °C for 5 min. A pre-mixed solution of nonylamine (63 μL, 0.34 mmol) and DIPEA (60 μL, 0.34 mmol) in DMF was added and the reaction was stirred at room temperature for 5 h. The reaction was quenched with 1 mol / L HCI and the organic phase was extracted with DCM (x 3) and washed with brine, dried over anhydrous Na2SO4and concentrated under reduced pressure. The crude product 20a-20g was used directly without further purification.

[0169] To a solution of 20a-20g (0.17 mmol) in 6 mL of MeOH / H2O (5:1, v / v) was added LiOH (73 mg, 1.7 mmol) portionwise. The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with 1 mol / L HCI and the organic phase was extracted with DCM (x 3) and washed with brine, dried over anhydrous Na2SO4and 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 was distilled under reduced pressure, dissolved in water / methanol (1:1) solution (10 mL), filtered through Whatman filter (0.22 pm), and detected by analytical HPLC. After purification by preparative HPLC and lyophilization, 22a-22g were obtained as white powder.

[0171]

[0172] 1 H NMR (500 MHz, DMSO-d6) d 8.64 (s, 1H), 8.29 (d, J = 7.1 Hz, 1H), 7.97 (m, 7H), 7.87 (d, J = 13.1 Hz, 1H), 7.78-7.72 (m, 2H), 7.56 (d, J = 5.8 Hz, 1H), 4.17-4.10 (m, 3H), 3.82 (m, 2H), 3.74-3.67 (m, 3H), 3.64 (m, 1H), 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 = 6.8 Hz, 3H). ESI-HRMS calcd for C 51 H 83 FN 10 O8[M+H] + :983.6379; found 983.6461.

[0173]

[0174] 1H NMR (500 MHz, MeOD) δ 8.83 - 8.64 (m, 1H), 8.00 - 7.80 (m, 1H), 7.64 - 7.42 (m, 1H), 4.24 (dd, J = 9.0, 4.7 Hz, 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, 11.6 Hz, 3H), 2.43 (m, 1H), 1.88 (d, J = 15.3 Hz, 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 = 1.1 Hz, 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) δ 8.52 (s, 1H), 7.85 (s, 1H), 7.53 (d, J = 13.1 Hz, 1H), 7.39 (d, J = 6.3 Hz, 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 for C 57 H 93 FN 12 O 10 [M+H] + :1125.7122; found 1125.7215.

[0177]

[0178] 1 H NMR (500 MHz, DMSO-de) d 8.66 (s, 1H), 8.32 (d, J = 6.6 Hz, 1H), 8.09 (d, J = 7.6 Hz, 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) d 8.76 (s, 1H), 7.86 (d, J = 13.1 Hz, 1H), 7.59 (d, J = 7.3 Hz, 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) δ 8.68 (s, 1H), 7.73 (d, J = 13.0 Hz, 1H), 7.53 (d, J = 7.0 Hz, 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 = 6.4 Hz, 2H), 0.88 (t, 3H). LC-MS calcd for C 58 H 96 FN 11 O 11 [M+H] + :1142.72; found 1142.73.

[0183]

[0184] 1HNMR (500 MHz, MeOD) δ 8.72 (s, 1H), 7.79 (d, J = 13.1 Hz, 1H), 7.55 (d, J = 6.1 Hz, 1H), 4.28 (m, 4H), 3.96 (dd, J = 11.0, 5.0 Hz, 1H), 3.91-3.71 (m, 6H), 3.49-3.38 (m, 3H), 3.23-3.12 (m, 2H), 2.95 (d, J = 6.6 Hz, 7H), 2.88-2.80 (m, 2H), 2.68 (dd, J = 15.1, 6.7 Hz, 1H), 2.54 (m, 1H), 1.96 (m, 1H), 1.91-1.81 (m, 3H), 1.73 (m, 9H), 1.54-1.46 (m, 6H), 1.45-1.40 (m, 3H), 1.28 (d, J = 10.5 Hz, 18H), 1.23 (m, 2H), 0.89 (t, 3H). ESI-HRMS calcd for C 54 H 88 FN 11 O 10 [M+H] + :1070.6700; found 1070.6757.

[0185] Compounds 28a-g were prepared as follows:

[0186] The 2-chlorotrityl chloride (CTC) resin (300 mg, 0.22 mmol) was swelled in dichloromethane (6 mL) for 15 min, 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 the reaction at room temperature for 2 h, then the reaction solution was drained, and washed with DMF (4 mL x 3) and DCM (4 mL x 3) to achieve the attachment of the first amino acid to the resin. The resin was then capped with 6 mL of DIPEA / MeOH / DCM (1 / 2 / 17, v / v / v) for 30 min. The resin was washed with DCM (4 mL x 3) and DMF (4 mL x 3). The resin was treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2) to remove the Fmoc. Next, the corresponding amino acid (0.44 mmol), DIC (121 μL, 0.88 mmol) and HOBt (120 mg, 0.88 mmol) were pre-mixed in DMF (3 mL) for 5 min, and then transferred to the solid-phase synthesis tube. The reaction was carried out by shaking at room temperature for 2 h, the solution was drained, and then washed with DMF (3 mL x 3) and DCM (3 mL x 3). The resin was treated with 20% piperidine / DMF (v / v) solution at room temperature for 10 min (x 2) to remove the Fmoc, and the above procedure was repeated to incorporate the corresponding amino acid to obtain compounds 23a-23e.

[0187] The terephthalic acid (185 mg, 1.1 mmol), HCTU (415 mg, 0.99 mmol) and DIPEA (349 μL, 1.98 mmol) were 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, the solution was removed, and washed with DMF (3 mL x 3) and DCM (3 mL x 3) to obtain compounds 24a-24e.

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

[0189] To a solution of 25a-25e (0.17 mmol), EDC (50 mg, 0.26 mmol) and HOBt (36 mg, 0.26 mmol) in dry DMF (8 mL) was added DIPEA (60 μL, 0.34 mmol) and the reaction was stirred at 0 °C for 5 min. A pre-mixed solution of nonylamine (63 μL, 0.34 mmol) and DIPEA (60 μL, 0.34 mmol) in DMF was added and the reaction was stirred at room temperature for 5 h. The reaction was quenched with 1 mol / L HCI and the organic phase was extracted with DCM (x 3) and washed with brine, dried over anhydrous Na2SO4and concentrated under reduced pressure. The crude product 26a-26e was 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 added LiOH (73 mg, 1.7 mmol) portion-wise. The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with 1 mol / L HCI and the organic phase was extracted with DCM (x 3) and washed with brine, dried over anhydrous Na2SO4and concentrated under reduced pressure. The crude product 27a-27e was used directly without further purification.

[0191] Compound 27a-27e (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 28a-28e was dissolved in water / methanol (1:1) solution (10 mL), filtered through Whatman filter (0.22 pm), and detected by analytical HPLC. After purification by preparative HPLC and lyophilization, 28a-28e were obtained as white powder.

[0192]

[0193] 1 H NMR (500 MHz, MeOD) d 8.66 (s, 1H), 7.91 (dd, J = 7.8, 4.1 Hz, 2H), 7.79 (m, 1H), 7.49 (d, J = 7.7 Hz, 3H), 4.46-4.36 (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 = 12.2 Hz, 2H), 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 = 14.5 Hz, 2H), 0.78 (q, J = 7.0 Hz, 3H). ESI-HRMS calcd for C 52 H 77 FN 12 O8[M+H] + :1017.5971; found 1017.6033.

[0194]

[0195] 1H NMR (500 MHz, MeOD) δ 8.69 (s, 1H), 8.01 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 13.0 Hz, 1H), 7.60 (m, 3H), 4.51 (dd, J = 7.7 Hz, 1H), 4.38 (dd, J = 8.3, 5.2 Hz, 1H), 4.30 (dd, J = 9.1, 5.2 Hz, 1H), 4.02 (m, 2H), 3.68 (m, 3H), 3.51 (m, 2H), 3.37 (d, J = 16.7 Hz, 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 (500 MHz, MeOD) δ 8.70 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 13.0 Hz, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7.56 (d, J = 6.2 Hz, 1H), 4.51 (dd, J = 13.6, 8.1 Hz, 1H), 4.36 (dd, J = 9.1, 5.3 Hz, 1H), 4.30 (dd, J = 9.2, 4.4 Hz, 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 (500 MHz, DMSO-d6) δ 8.65 (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 = 28.9 Hz, 3H), 3.56 (m, 1H), 3.08 (m, 5H), 2.98 (m, 1H), 1.82 (m, 1H), 1.74 (d, J = 10.4 Hz, 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- HRMS calcd for C 52 H 77 FN 16 O8[M+H] + : 1073.6564; found 1073.6621.

[0200]

[0201] 1 H NMR (500 MHz, MeOD) δ 8.81 (s, 1H), 8.66 (s, 1H), 8.02 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 13.0 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 7.0 Hz, 1H), 7.36 (s, 1H), 4.69 (dd, J = 8.7, 5.4 Hz, 1H), 4.53 (dd, J = 8.5, 6.0 Hz, 1H), 4.33 (dd, J = 9.2, 5.2 Hz, 1H), 4.03 (m, 2H), 3.72 (m, 3H), 3.51 (m, 2H), 3.39 (m, 2H), 3.32 - 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-HRMS calcd for C52 H 72 FN 11 O8[M+H] + :998.5549; found998.5614.

[0202] Performance testing

[0203] (I) Determination of minimum inhibitory concentration

[0204] To investigate the antibacterial effect of ciprofloxacin derivatives, the minimum inhibitory concentration (MIC) was determined. First, *S. aureus* strain (purchased from the China Industrial Microbial Culture Collection Center) was used. The compound concentration was initially diluted sequentially from 50 μg / mL. After administration, the mixture was incubated at 37℃ for 16 h. The OD value of the bacterial culture was measured using a microplate reader. 600 The MIC of the antimicrobial peptide was determined by the concentration of the peptide that inhibits 99.9% of bacterial growth. Ciprofloxacin (Cip), a known drug, 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 the China Industrial Microbial Culture Collection Center) and Gram-negative bacteria (E. coil ATCC 25922, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606, purchased from the China Center for Type Culture Collection, Wuhan University). The results are shown in Table 2.

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

[0209]

[0210]

[0211] (II) Hemolytic toxicity studies

[0212] 1.1 Experimental Procedure:

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

[0214] (1) Collect the venous blood of SD rats in the venous blood collection tube containing heparin sodium anticoagulant, pay attention to mix evenly during the blood collection process to prevent blood clotting. Centrifuge the collected blood at 800g for 5 min, discard the serum, and wash the red blood cells with PBS for 3 times, then dilute them to 8% (V / V) with PBS, and add them to the 96-well plate (100 μL / well);

[0215] (2) Prepare the drug solution with a concentration of 200 μg / mL to 6.25 μg / mL by using the double dilution method. Then add the drug to the 96-well plate with red blood cells (100 μL / well), and incubate at 37°C for 1 h;

[0216] (3) After co-incubation for 1 h, take a photo of the 96-well plate, centrifuge at 1200g for 15 min, take 80 μL of the supernatant to a new 96-well plate, and measure the absorbance (OD 490 ) at 490 nm with a multifunctional enzyme label instrument. PBS is used as the negative control, and 2% Triton X-100 is used as the positive control.

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

[0218]

[0219] 1.2 Experimental results: The results are shown in Figure 1 , Figure 1 The hemolytic toxicity of compounds Cip, 16u, 22e, 22g, and 28b on rat red blood cells can be seen from Figure 1 : Compounds Cip (ciprofloxacin), 16u, and 28b almost do not produce hemolysis at a concentration of 200 μg / mL or below.

[0220] (Three) Cytotoxicity determination

[0221] 1.1 Experimental steps:

[0222] Determine the cytotoxicity of drug solutions on mammalian cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In this experiment, mouse fibroblast L929 cells were used.

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

[0224] (2) Add 200 μL of drug solution per well with a final concentration of 200 μg / mL to 6.25 μg / mL to the cells;

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

[0226] (4) Discard the medium in each well, add 150μL dimethyl sulfoxide (DMSO) to dissolve the formazan.

[0227] (5) Measure OD 570 .

[0228] 1.2 Experimental results, see Figure 2 , Figure 2 Table 2 for the cytotoxicity results of compounds Cip, 16u, 22e, 22g, 28b on L929 cells at different concentrations; it can be seen from Figure 2 that compounds Cip, 16u, 22e, 22g and 28b all showed certain cytotoxicity on L929 cells, and at a concentration of 25μg / mL, the survival rate of L929 cells treated with compounds Cip, 16u and 28b was more than 80%.

[0229] (Four), fluorescence staining and flow cytometry determination

[0230] The membrane breaking effect of the drug was monitored by flow cytometry by determining the proportion of PI-stained positive bacteria after co-incubation of the drug with bacteria.

[0231] 1.1 Experimental steps:

[0232] (1) After centrifugation of the bacterial solution grown to the logarithmic phase at 8000rpm for 10min, the supernatant was discarded, and the solution was diluted to 10 6 -10 7 CFU / mL for standby;

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

[0234] (3) 100μL of bacterial solution was incubated with 100μL of drug solution on a 37℃ shaking table for 1h;

[0235] (4) Add PI mixed dye and incubate in the dark for 10min, then add the mixed solution to the flow tube for detection.

[0236] 1.2 Experimental results see Figure 3 , Figure 3 Table 2 for the membrane damage results of the drug at different concentrations, from Figure 3As can be seen, the percentage of PI positive bacteria was 16.60% and 57.22% at 4xMIC and 8xMIC of compound 28b, respectively, compared with PBS. This indicates that the bacterial membrane has been destroyed by compound 28b.

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

[0238] The first generation MIC data of compound 28b and ciprofloxacin against MRSA (ATCC 33591) were obtained as described in the MIC study above. Then, MRSA in the last transparent well was diluted 10 6 times into the next well, and the MIC was determined again after 24 h incubation at 37°C. This step was repeated until the 25th generation. The drug resistance of compound 28b and ciprofloxacin against E. coli (ATCC 25922) was determined in the same way. The results are shown in Table 2. Figure 4 Figure 4 Table 2. MIC of compound 28b and ciprofloxacin against MRSA (a) and E. coli (b) at the 25th generation Figure 4 As can be seen, the MIC value of compound 28b against MRSA did not change after 25 generations, while the MIC value of ciprofloxacin increased by 32 times. Similar results were observed for E. coli. Therefore, this result indicates that it is difficult for this type of antibiotic to induce drug resistance in bacteria.

[0239] (VI), therapeutic study 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 with a concentration of 5 mg / mL. After the mice were anesthetized, the back hair was removed with a razor and depilatory cream. Then, 100 μL of MRSA (1 x 10 8 CFU / mL) was injected subcutaneously in the middle of the back of the mice to form a subcutaneous abscess. After 30 min, 100 μL of sterile PBS, ciprofloxacin hydrochloride solution, or compound 28b solution was injected into the bacterial infection site. After 48 h, the mice were euthanized, and the infected skin was collected. To evaluate the antibacterial effect, the bacteria in the abscess were counted using standard plate counting method, and the bacterial colonies visible on the plate were imaged. The infected skin was fixed with 4% paraformaldehyde and embedded in paraffin, and stained with H&E to evaluate the antibacterial effect of various drugs. The IL-6 and TNF-α contents in the skin homogenate solution were determined using commercial ELISA kits (Dakewe, Shenzhen, China) according to the manufacturer's instructions. The results are shown in Table 3. Figure 5 Figure 5 ​​In vivo antibacterial activity of compound 28b and ciprofloxacin in mice subcutaneous infection, Figure 5 In the figure, a is a schematic diagram of MRSA-induced mouse subcutaneous infection model, b is a photograph of the skin of mice taken at 48 hours after infection after different treatments, c is H&E staining, Masson staining and Giemsa staining images of different skin samples, d is a representative photograph of 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 bacterial load and bacterial survival rate, h is the bacterial survival rate of each group 48 hours after injection of MRSA, and i is a schematic diagram of the level of IL-10 in the skin homogenate of each group. Figure 5 It can be seen that the dorsal skin surface of the control group mice was obviously swollen, and skin abscess appeared in the subcutaneous tissue, while the subcutaneous tissue of the mice treated with ciprofloxacin was still observed to have obvious abscess or erythema, and the subcutaneous tissue of the mice treated with compound 28b was normal. Compared with the control group, both ciprofloxacin and compound 28b treatment can significantly reduce the number of bacteria remaining on the skin and the production of pro-inflammatory cytokines induced by MRSA, including TNF-α and IL-6.

[0241] The above is only the preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A ciprofloxacin derivative, characterized by, having the structure of Formula I: The ciprofloxacin derivative has any one of the following structures:

2. The process for the preparation of ciprofloxacin derivatives according to claim 1, characterized in that, comprising the steps of: bonding the lipid moiety, the amino acid moiety, the linker and the ciprofloxacin to obtain the ciprofloxacin derivative.

3. Use of the ciprofloxacin derivative of claim 1 or the ciprofloxacin derivative prepared by the method of claim 2 in the preparation of an antibacterial medicament.

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