CN111170997A

CN111170997A - Carbazole compound and preparation method and application thereof

Info

Publication number: CN111170997A
Application number: CN201911415794.7A
Authority: CN
Inventors: 刘寿平; 林水木; 刘家勇
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-05-19

Abstract

The invention relates to a carbazole compoundA method for preparing the same and application thereof. The carbazole compound has the following structural general formula, can be used for preparing antibacterial drugs, can effectively slow down or overcome the generation of bacterial drug resistance, and can effectively avoid cross drug resistance with the traditional antibacterial drugs.

Description

Carbazole compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to carbazole compounds and a preparation method and application thereof.

Background

In recent years, diseases caused by drug-resistant bacterial infections are seriously threatening the health of human beings all over the world. The phenomenon of resistance of pathogens to antibacterial drugs is increasing due to abuse and misuse of antibacterial drugs and a large decrease in approved new antibacterial drugs. The problem of antimicrobial drug resistance has been listed by the World Health Organization (WHO) as one of the ten major threats to global health in 2019.

Currently, clinically effective treatment regimens for drug resistant bacterial infections are very limited and have met with significant challenges. In addition, most conventional antibacterial drugs contain the same molecular skeleton. Since the early 90 s of the last century, antibacterial drugs with New Molecular Entities (NMEs) in clinical studies have been seriously lacking. Most of the currently approved antibacterial drugs are still based on the traditional antibacterial molecular skeleton, such as macrolides, quinolones, tetracyclines and cephalosporins, etc. In general, antibacterial drugs having similar mechanisms of action or molecular backbones are more susceptible to cross-resistance.

Therefore, the development of highly effective and low toxic antibacterial agents based on new molecular entities to cope with the problem of bacterial resistance has been imminent.

Disclosure of Invention

Based on this, there is a need for providing carbazole-based compounds. The carbazole compound can be used for preparing antibacterial drugs, can effectively slow down or overcome the generation of bacterial drug resistance, and can effectively avoid cross drug resistance with the traditional antibacterial drugs.

The specific technical scheme is as follows:

carbazole compounds with the following structural general formula or pharmaceutically acceptable salts thereof or stereoisomers thereof or solvates thereof or prodrug molecules thereof:

wherein R is₁Selected from: at least one R_aSubstituted or unsubstituted C1-C10 straight or branched chain alkyl, at least one R_aSubstituted or unsubstituted C3-C8 straight or branched chain alkenyl;

R_aeach independently selected from: H. halogen;

R₂is a cationic group selected from: at least one R_bSubstituted or unsubstituted C2-C5 cycloalkoxy, at least one R_bA substituted or unsubstituted C1-C5 alkylamine;

R_beach independently selected from: H. OH, carbonyl, C1-C15 linear or branched alkyl, C1-C15 linear or branched alkyl alcohol, C1-C15 linear or branched alkylmercapto, C1-C10 alkylamine, C5-C10 nitrogen-containing heteroaryl, C1-C5 aminoiminoalkyl, or a combination of the foregoing, which, when each two of the foregoing groups are attached to the same carbon atom, are linked to each other to form a ring or not.

The invention also provides a preparation method of the carbazole compound or the pharmaceutically acceptable salt thereof or the stereoisomer thereof or the solvate thereof or the prodrug molecule thereof, which adopts one of the following synthetic routes (I) to (III) to prepare:

scheme (I):

scheme (II):

scheme (III):

wherein the starting material can be prepared by the synthetic route (II).

The invention also provides application of the carbazole compound or pharmaceutically acceptable salt thereof or stereoisomer thereof or solvate thereof or prodrug molecule thereof in preparing a medicament with antibacterial effect.

The invention also provides the application of the carbazole compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the solvate thereof, or the prodrug molecule thereof, and the composition of zinc ions in preparing the medicine with antibacterial effect.

The principle and advantages of the invention are as follows:

currently, clinical drugs containing a carbazole skeleton have been commercialized including antihypertensive drugs carvedilol and carbazolol, and anticancer drugs ellipticine and illicit. However, there is no carbazole-based antibacterial drug approved for clinical use. Carbazole has special molecular structure characteristics, and various functional groups can be easily introduced into the carbazole skeleton. Therefore, carbazole can be used as an advantageous framework for antimicrobial drug development, and has the potential of avoiding cross-drug resistance with traditional antimicrobial drugs.

In this work, we designed and synthesized a series of carbazole-like small molecules that mimic antimicrobial peptides. And respectively introducing a hydrophobic lipid chain and a cation module into the carbazole parent nucleus to form a cationic amphiphilic structure. The introduced cationic groups (such as aliphatic amine and basic amino acid) can promote the interaction between the carbazole derivative and the negatively charged bacterial membrane through electrostatic attraction, and the introduced hydrophobic aliphatic chains have strong hydrophobic interaction with the bacterial phospholipid bilayer, so that the permeability of the bacterial membrane is modified and even broken, the leakage of the bacterial cell content is triggered, and the bacterial cell death is caused. The cell membrane of bacteria is negatively charged because the phospholipid components of the bacterial cell membrane mainly include electronegative Phosphatidylglycerol (PG) and Cardiolipin (CL) and electroneutral Phosphatidylethanolamine (PE). The eukaryotic cell membrane is electrically neutral, because the phospholipid component of the eukaryotic cell membrane mainly includes electrically neutral Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingomyelin (SM), and the like. Therefore, the carbazole-based compound can selectively act on bacteria and effectively resist drug-resistant bacterial infection.

The preparation method takes 4-epoxypropane oxy carbazole as a starting raw material, the starting raw material is an intermediate for synthesizing antihypertensive drugs carvedilol and carbazol Xinan, and the preparation method is common and cheap, so that the preparation cost is low, and the steps are simple.

Drawings

FIG. 1 is a graph showing the results of the drug resistance test for Compound 29 and norfloxacin-induced bacteria (Staphylococcus aureus ATCC 29213);

FIG. 2 is a graph showing the results of an antibacterial experiment of Compound 29 and vancomycin in a mouse keratitis model caused by Staphylococcus aureus ATCC 29213.

Detailed Description

The carbazole-based compounds of the present invention, the preparation method thereof, and the use thereof will be described in further detail with reference to specific examples.

The invention provides carbazole compounds with the following structural general formula or pharmaceutically acceptable salts thereof or stereoisomers thereof or solvates thereof or prodrug molecules thereof:

R_aeach independently selected from: H. halogen;

Specifically, the relevant schemes for the pharmaceutically acceptable salts of the above carbazole-based compounds are as follows: if the compound is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, sulfuric, succinic, tartaric, p-toluenesulfonic acid and the like. Particular embodiments include citric acid, hydrobromic acid, hydrochloric acid, phosphoric acid, sulfuric acid, maleic acid, tartaric acid. Other exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, phosphate, acid phosphate, isonicotinic acid, lactic acid, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, fumarate, maleate, gentisate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methylsulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.

In one specific embodiment, R₂Selected from: C2-C5 cycloalkoxy or a group of the general structural formula:

wherein m + n is 1-5.

In one particular embodiment, n ≠ 0.

In one specific embodiment, R_bEach independently selected from: H. OH, carbonyl, C1-C10 linear or branched alkyl, C1-C5 linear or branched alkyl alcohol, C1-C5 linear or branched alkylmercapto, C2-C6 alkylamine, C5-C8 nitrogen-containing heteroaryl, C1-C2 aminoiminoalkyl, or a combination of the foregoing, which, when each two of the foregoing groups are attached to the same carbon atom, are linked to each other to form a ring or not.

In one particular embodiment, the C5-C8 nitrogen-containing heteroaryl group has the structure:

wherein w is 0, 1, 2 or 3 and V is independently at each occurrence C or N.

wherein w is 0, 1, 2 or 3 and V is independently at each occurrence C or N.

In one specific embodiment, R₁Selected from: at least one R_aSubstituted or unsubstituted C1-C10 straight chain alkyl, at least one R_aSubstituted or unsubstituted C3-C6 branched alkenyl.

In one specific embodiment, the carbazole-based compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the solvate thereof, or the prodrug molecule thereof is selected from the following compounds:

scheme (I):

scheme (II):

scheme (III):

wherein the starting material can be prepared by the synthetic route (II).

Specific examples of the preparation of the compounds are provided below.

The synthetic route is as follows:

(A)

(II)

(III)

(IV)

The preparation method of the compound comprises the following steps:

synthesis of Compound 2

4-Oxypropyleneoxy carbazole (100mg, 0.42mmol) was dissolved in a DMF (10mL) solution, methyl iodide (52. mu.L, 0.84mmol) and NaOH (33.43mg, 0.84mmol) were then added, and the mixture was stirred at 60 ℃ for 2 hours. After completion of the reaction, the mixture was diluted with ethyl acetate and extracted 3 times with water. The organic layer was concentrated under reduced pressure, and the resulting crude product was purified by silica gel chromatography (ethyl acetate/petroleum ether ═ 1/4) to give compound 2 as a pale yellow solid (86.2mg, 81%).¹H NMR(400MHz,CDCl₃)δ8.36–8.33(m,1H),7.47–7.41(m,1H),7.38–7.33(m,2H),7.25–7.22(m,1H),7.03(d,J＝8.2Hz,1H),6.65(d,J＝8.0Hz,1H),4.48–4.21(m,2H),3.81(s,3H),3.56–3.50(m,1H),2.99–2.83(m,2H).¹³C NMR(100MHz,CDCl₃)δ155.09,142.65,140.36,126.44,124.98,123.27,122.07,119.29,112.17,107.98,102.10,101.06,68.89,50.47,44.96,29.38.HRMS(ESI+):calculated for C₁₆H₁₆NO₂[M+H]⁺254.1181,found 254.1169。

Synthesis of Compound 3

Following the synthesis of compound 2, starting with 4-epoxypropyleneoxycarbazole (73.1mg, 0.31mmol), NaOH (24.4mg, 0.61mmol) and 1-iodopropane (59 μ L, 0.61mmol), compound 3 was prepared as a pale yellow solid (79.8mg, 93%).¹H NMR(400MHz,CDCl₃)δ8.37–8.33(m,1H),7.45–7.33(m,3H),7.25–7.20(m,1H),7.04(d,J＝8.2Hz,1H),6.65(d,J＝7.9Hz,1H),4.50–4.19(m,4H),3.57–3.52(m,1H),3.02–2.84(m,2H),1.96–1.83(m,2H),0.95(t,J＝7.4Hz,3H).¹³C NMR(100MHz,CDCl₃)δ155.17,142.19,139.89,126.33,124.89,123.35,122.13,119.18,112.22,108.28,102.42,100.90,68.89,50.48,44.96,44.89,22.42,11.85.HRMS(ESI+):calculated for C₁₈H₂₀NO₂[M+H]⁺282.1494,found 282.1490。

Synthesis of Compound 4

Following the synthesis of compound 2, starting with 4-epoxypropyleneoxycarbazole (104.2mg, 0.44mmol), NaOH (33.4mg, 0.84mmol) and 1-iodopentane (114 μ L, 0.87mmol), compound 4 was prepared as a pale yellow solid (102.5mg, 76%).¹H NMR(400MHz,CD₃OD)δ8.28(d,J＝7.9Hz,1H),7.45–7.29(m,3H),7.20–7.14(m,1H),7.10–7.06(m,1H),6.72–6.68(m,1H),4.60–4.49(m,1H),4.35–4.25(m,2H),4.15–4.08(m,1H),3.55–3.50(m,1H),2.97–2.85(m,2H),1.87–1.77(m,2H),1.38–1.28(m,4H),0.89–0.79(m,3H).¹³C NMR(100MHz,CD₃OD)δ156.43,143.29,141.05,127.45,125.77,124.08,123.28,119.89,113.14,109.35,103.39,101.96,70.13,51.52,45.09,43.80,30.35,29.73,23.50,14.29.HRMS(ESI+):calculated for C₂₀H₂₄NO₂[M+H]⁺310.1807,found310.1794。

Synthesis of Compound 5

Following the synthesis of compound 2, starting with 4-epoxypropyleneoxycarbazole (100mg, 0.42mmol), NaOH (33.43mg, 0.84mmol) and 1-iodoheptane (137 μ L, 0.84mmol), compound 5 was prepared as a pale yellow solid (106.7mg, 76%).¹H NMR(400MHz,CD₃OD)δ8.28(d,J＝7.8Hz,1H),7.43–7.29(m,3H),7.20–7.14(m,1H),7.07(d,J＝8.2Hz,1H),6.69(d,J＝7.9Hz,1H),4.56–4.48(m,1H),4.29(t,J＝7.1Hz,2H),4.15–4.06(m,1H),3.54–3.48(m,1H),3.00–2.81(m,2H),1.87–1.73(m,2H),1.32–1.18(m,8H),0.84(t,J＝6.4Hz,3H).¹³C NMR(100MHz,CD₃OD)δ156.42,143.28,141.04,127.45,125.76,124.09,123.28,119.89,113.14,109.36,103.40,101.95,70.11,51.52,45.09,43.81,32.86,30.16,30.01,28.12,23.56,14.35.HRMS(ESI+):calculatedfor C₂₂H₂₈NO₂[M+H]⁺338.2120,found 338.2106。

Synthesis of Compound 6

Following the synthesis of compound 2, starting with 4-epoxypropyleneoxycarbazole (80mg, 0.33mmol), NaOH (26.4mg, 0.66mmol) and 1-iodononane (125 μ L, 0.67mmol), compound 6 was prepared as a pale yellow solid (82.3mg, 70%).¹H NMR(400MHz,CD₃OD)δ8.28(d,J＝7.8Hz,1H),7.45–7.36(m,2H),7.34(t,J＝8.1Hz,1H),7.20–7.14(m,1H),7.08(d,J＝8.2Hz,1H),6.71(d,J＝7.9Hz,1H),4.57–4.51(m,1H),4.31(t,J＝7.1Hz,2H),4.17–4.10(m,1H),3.56–3.50(m,1H),2.99–2.86(m,2H),1.88–1.76(m,2H),1.34–1.19(m,12H),0.86(t,J＝7.0Hz,3H).¹³C NMR(100MHz,CD₃OD)δ156.46,143.32,141.07,127.45,125.77,124.10,123.30,119.90,113.17,109.38,103.43,101.98,70.17,51.54,45.11,43.84,32.95,30.53,30.46,30.26,29.98,28.12,23.66,14.39.HRMS(ESI+):calculated for C₂₄H₃₂NO₂[M+H]⁺366.2433,found 366.2421。

Synthesis of Compound 7

4-Oxypropyleneoxy carbazole (2.01g, 6.54mmol) was dissolved in DMF (30mL) solution, followed by the addition of potassium tert-butoxide (2.83g, 25.20mmol) and potassium iodide (2.09g, 12.60mmol), followed by the slow dropwise addition of 1-bromo-3-methyl-2-butene (2.89mL, 25.20 mmol). The reaction mixture was stirred at 50 ℃ for 30 minutes. After completion of the reaction, the mixture was diluted with ethyl acetate and extracted 3 times with water. The organic layer was concentrated under reduced pressure and the resulting crude product was purified by silica gel chromatography (ethyl acetate/petroleum ether) to afford compound 7 as a white solid (1.46g, 57%).¹H NMR(400MHz,CDCl₃)δ8.36(d,J＝7.8Hz,1H),7.46–7.40(m,1H),7.35(t,J＝8.0Hz,2H),7.26–7.22(m,1H),7.03(d,J＝8.2Hz,1H),6.65(d,J＝8.0Hz,1H),5.31–5.22(m,1H),4.87(d,J＝6.4Hz,2H),4.48–4.24(m,2H),3.57–3.52(m,1H),3.00–2.87(m,2H),1.92(s,3H),1.70(s,3H).¹³C NMR(100MHz,CDCl₃)δ156.66,143.12,140.87,136.17,127.47,125.67,124.14,123.50,121.20,119.92,113.28,109.37,103.21,101.86,75.31,70.28,59.53,41.94,25.71,18.20.HRMS(ESI+):calculated for C₂₀H₂₂NO₂[M+H]⁺308.1651,found 308.1642。

Synthesis of Compound 8

Compound 2(54.3mg, 0.33mmol) was dissolved in MeOH (5mL) and dimethylamine (0.5mL) was added. The mixture was stirred at 65 ℃ for 6 hours. After completion of the reaction, the organic solvent in the reaction mixture was evaporated under reduced pressure. The crude product obtained was purified by silica gel chromatography (ethyl acetate/petroleum ether ═ 1/1) to afford compound 8 as a pale yellow solid (58.8mg, 92%).¹H NMR(400MHz,CDCl₃)δ8.32(d,J＝7.7Hz,1H),7.48–7.43(m,1H),7.41–7.36(m,2H),7.27–7.23(m,1H),7.04(d,J＝8.0Hz,1H),6.70(d,J＝7.8Hz,1H),4.35–4.19(m,3H),3.84(s,3H),2.79–2.58(m,2H),2.40(s,6H).¹³C NMR(100MHz,CDCl₃)δ155.27,142.61,140.35,126.56,124.88,123.01,122.13,119.20,112.04,108.03,101.87,100.98,70.42,66.42,62.58,45.71(2×CH₃),29.38.HRMS(ESI+):calculated for C₁₈H₂₃N₂O₂[M+H]⁺299.1760,found 299.1746。

Synthesis of Compound 9

Following the synthesis of compound 8, starting with 3(37.6mg, 0.13mmol) and dimethylamine (0.5mL), compound 9 was prepared as a pale yellow solid (38.9mg,89％)。¹H NMR(400MHz,CDCl₃)δ8.34–8.31(m,1H),7.47–7.35(m,3H),7.26–7.22(m,1H),7.05(d,J＝7.9Hz,1H),6.69(d,J＝7.9Hz,1H),4.34–4.21(m,5H),2.77–2.58(m,2H),2.39(s,6H),1.97–1.85(m,2H),0.97(t,J＝7.4Hz,3H).¹³CNMR(100MHz,CDCl₃)δ155.41,142.13,139.86,126.42,124.76,123.12,122.20,119.08,112.10,108.30,102.14,100.79,70.49,66.52,62.56,45.80(2×CH₃),44.88,22.41,11.85.HRMS(ESI+):calculated for C₂₀H₂₇N₂O₂[M+H]⁺327.2073,found 327.2069。

synthesis of Compound 10

Following the synthetic procedure for compound 8, starting from 4(65.7mg, 0.21mmol) and dimethylamine (0.5mL), compound 10 was prepared as a pale yellow solid (60.7mg, 81%).¹H NMR(400MHz,CDCl₃)δ8.32(d,J＝7.8Hz,1H),7.46–7.34(m,3H),7.26–7.21(m,1H),7.04(d,J＝8.2Hz,1H),6.69(d,J＝8.0Hz,1H),4.35–4.20(m,5H),2.79–2.58(m,2H),2.40(s,6H),1.91–1.81(m,2H),1.39–1.33(m,4H),0.88(t,J＝7.0Hz,3H).¹³C NMR(100MHz,CDCl₃)δ155.41,142.06,139.79,126.42,124.76,123.12,122.20,119.06,112.10,108.25,102.09,100.77,70.48,66.50,62.55,45.79(2×CH₃),43.32,29.46,28.78,22.56,14.03.HRMS(ESI+):calculated for C₂₂H₃₁N₂O₂[M+H]⁺355.2386,found355.2373。

Synthesis of Compound 11

Following the synthetic procedure for compound 8, starting from 5(61.9mg, 0.18mmol) and dimethylamine (0.5mL), compound 11 was prepared as a pale yellow solid (65.3mg, 93%).¹H NMR(400MHz,CDCl₃)δ8.33–8.28(m,1H),7.45–7.33(m,3H),7.25–7.20(m,1H),7.02(d,J＝7.9Hz,1H),6.67(d,J＝7.7Hz,1H),4.34–4.20(m,5H),2.77–2.57(m,2H),2.38(s,6H),1.91–1.78(m,2H),1.35–1.21(m,8H),0.85(t,J＝7.0Hz,3H).¹³C NMR(100MHz,CDCl₃)δ155.41,142.05,139.78,126.42,124.75,123.12,122.20,119.06,112.09,108.25,102.10,100.76,70.48,66.53,62.55,45.81(2×CH₃),43.35,31.80,29.16,29.09,27.32,22.67,14.13.HRMS(ESI+):calculated for C₂₄H₃₅N₂O₂[M+H]⁺383.2699,found 383.2689。

Synthesis of Compound 12

Following the synthetic procedure for compound 8, starting from 6(53.7mg, 0.18mmol) and dimethylamine (0.5mL), compound 12 was prepared as a pale yellow solid (53.4mg, 88%).¹H NMR(400MHz,CDCl₃)δ8.32(d,J＝7.7Hz,1H),7.46–7.34(m,3H),7.26–7.21(m,1H),7.04(d,J＝8.2Hz,1H),6.69(d,J＝8.0Hz,1H),4.37–4.20(m,5H),2.79–2.58(m,2H),2.39(s,6H),1.92–1.76(m,2H),1.39–1.23(m,12H),0.87(t,J＝6.9Hz,3H).¹³C NMR(100MHz,CDCl₃)δ155.41,142.05,139.78,126.41,124.75,123.12,122.20,119.05,112.10,108.25,102.10,100.76,70.48,66.51,62.54,45.80(2×CH₃),43.34,31.89,29.54,29.49,29.31,29.06,27.35,22.70,14.16.HRMS(ESI+):calculated for C₂₆H₃₉N₂O₂[M+H]⁺411.3012,found 411.3003。

Synthesis of Compound 13

According to the synthesis of compound 8, starting with 7(41.5mg,0.14mmol) and dimethylamine (0.5mL), compound 13 was prepared as a white solid (38.6mg, 81%).¹H NMR(400MHz,CD₃OD)δ8.35–8.31(m,1H),7.38(d,J＝0.9Hz,1H),7.37–7.36(m,1H),7.33(t,J＝8.1Hz,1H),7.20–7.13(m,1H),7.03(d,J＝8.2Hz,1H),6.71(d,J＝7.9Hz,1H),5.25–5.18(m,1H),4.88(d,J＝6.5Hz,2H),4.32–4.25(m,1H),4.21–4.17(m,2H),2.78–2.59(m,2H),2.35(s,6H),1.91(s,3H),1.71–1.68(m,3H).¹³C NMR(100MHz,)δ156.58,143.09,140.84,136.14,127.47,125.69,124.14,123.46,121.16,119.90,113.24,109.41,103.27,101.88,71.72,68.68,63.55,46.11(2×CH₃),41.90,25.71,18.19.HRMS(ESI+):calculated for C₂₂H₂₉N₂O₂[M+H]⁺353.2229,found353.2227。

Synthesis of Compound 14

According to the procedure for the synthesis of compound 8, starting from 7(100mg, 0.32mmol) and diethylamine (0.5mL), compound 14 was prepared as a white solid (97.3mg, 79%).¹H NMR(400MHz,CD₃OD)δ8.35(d,J＝7.7Hz,1H),7.43–7.38(m,2H),7.36(t,J＝7.8Hz,1H),7.18(t,J＝6.5Hz,1H),7.06(d,J＝8.2Hz,1H),6.74(d,J＝8.0Hz,1H),5.24(t,J＝6.2Hz,1H),4.91(d,J＝6.6Hz,2H),4.35–4.27(m,1H),4.25(d,J＝4.7Hz,2H),3.06–2.73(m,6H),1.93(s,3H),1.72(s,3H),1.15(t,J＝7.2Hz,6H).¹³C NMR(100MHz,CD₃OD)δ156.57,143.14,140.88,136.25,127.51,125.72,124.13,123.45,121.17,119.88,113.24,109.44,103.30,101.85,71.48,71.46,68.45,56.88,41.97(2×CH₂),25.73,18.21,11.32(2×CH₃).HRMS(ESI+):calculated for C₂₄H₃₃N₂O₂[M+H]⁺381.2542,found 381.2537。

Synthesis of Compound 15

According to the synthesis of compound 8, starting from 7(80.39mg, 0.26mmol) and dipropylamine (0.5mL), compound 15 was prepared as a white solid (80.6mg, 75%).¹H NMR(400MHz,CD₃OD)δ8.34(d,J＝7.8Hz,1H),7.39–7.29(m,3H),7.18–7.12(m,1H),7.06–6.98(m,1H),6.75–6.64(m,1H),5.25–5.17(m,1H),4.92–4.86(m,2H),4.29–4.15(m,3H),2.91–2.65(m,2H),2.54–2.44(m,4H),1.93–1.87(m,3H),1.69(s,3H),1.54–1.45(m,4H),0.88–0.83(m,6H).¹³C NMR(100MHz,CD₃OD)δ156.73,143.12,140.86,136.13,127.46,125.65,124.22,123.53,121.21,119.84,113.30,109.37,103.15,101.83,71.37,69.13,58.42,58.08(2×CH₂),41.94,25.72,21.14(2×CH₂),18.20,12.18(2×CH₃).HRMS(ESI+):calculated for C₂₆H₃₇N₂O₂[M+H]⁺409.2855,found 409.2851。

Synthesis of Compound 16

Following the synthetic procedure for compound 8, starting from 7(80.35mg, 0.26mmol) and dibutylamine (0.5mL), compound 16 was prepared as a white solid (81.4mg, 71%).¹H NMR(400MHz,CD₃OD)δ8.38–8.31(m,1H),7.39–7.36(m,2H),7.33(t,J＝8.1Hz,1H),7.19–7.11(m,1H),7.03(d,J＝8.2Hz,1H),6.71(d,J＝8.0Hz,1H),5.25–5.17(m,1H),4.89(d,J＝6.4Hz,2H),4.29–4.22(m,2H),4.22–4.15(m,1H),2.94–2.65(m,2H),2.55–2.48(m,4H),1.91(s,3H),1.70(s,3H),1.48–1.40(m,4H),1.29–1.21(m,4H),0.83(t,J＝7.3Hz,6H).¹³C NMR(100MHz,CD₃OD)δ156.73,143.14,140.88,136.12,127.47,125.64,124.22,123.56,121.23,119.86,113.33,109.38,103.14,101.82,71.22,69.14,58.31,55.92(2×CH₂),41.95,30.28(2×CH₂),25.73,21.68(2×CH₂),18.21,14.34(2×CH₃).HRMS(ESI+):calculated for C₂₈H₄₁N₂O₂[M+H]⁺437.3168,found 437.3164。

Synthesis of Compound 17

According to the synthesis of compound 8, starting from 7(41.5mg,0.14mmol) and tetrahydropyrrole (0.5mL)Starting material, compound 17 was prepared as a white solid (49.3mg, 84%).¹H NMR(400MHz,CD₃OD)δ8.31(d,J＝7.8Hz,1H),7.41–7.37(m,2H),7.34(t,J＝8.1Hz,1H),7.23–7.15(m,1H),7.05(d,J＝8.1Hz,1H),6.71(d,J＝7.8Hz,1H),5.25–5.16(m,1H),4.47–4.37(m,1H),4.30–4.17(m,2H),3.30–3.25(m,2H),3.24–3.16(m,4H),2.03–1.97(m,4H),1.90(s,3H),1.69(s,3H).¹³C NMR(100MHz,CD₃OD)δ156.37,143.21,140.97,136.37,127.58,125.89,124.15,123.39,121.13,120.08,113.30,109.59,103.64,102.07,71.37,67.70,59.50,55.69(2×CH₂),42.02,25.77,24.03(2×CH₂),18.26.HRMS(ESI+):calculated for C₂₄H₃₁N₂O₂[M+H]⁺379.2386,found379.2382。

Synthesis of Compound 18

According to the procedure for the synthesis of compound 8, starting from 7(57.2mg, 0.19mmol) and piperidine (0.5mL), compound 18 was prepared as a white solid (61.8mg, 76%).¹H NMR(400MHz,CDCl₃)δ8.32(d,J＝7.7Hz,1H),7.45–7.40(m,1H),7.38(d,J＝3.8Hz,1H),7.36–7.34(m,1H),7.26–7.21(m,1H),7.02(d,J＝8.1Hz,1H),6.69(d,J＝7.9Hz,1H),5.30–5.23(m,1H),4.88(d,J＝6.4Hz,2H),4.35–4.26(m,2H),4.25–4.17(m,1H),2.73–2.63(m,4H),2.50–2.39(m,2H),1.94–1.90(m,3H),1.72–1.69(m,3H),1.68–1.59(m,4H),1.53–1.45(m,2H).¹³C NMR(100MHz,CDCl₃)δ155.49,141.90,139.64,135.11,126.43,124.75,123.20,122.38,120.10,119.15,112.26,108.32,102.11,100.89,70.63,65.69,61.87,54.96,41.37(2×CH₂),26.23(2×CH₂),25.67,24.35,18.29.HRMS(ESI+):calculated for C₂₅H₃₃N₂O₂[M+H]⁺393.2542,found 393.2537。

Synthesis of Compound 19

According to the synthesis of compound 8, starting from 7(51mg, 0.17mmol) and N-methylpiperazine (0.5mL), compound 19 was obtained as a yellow oil (46.5mg, 64%).¹H NMR(400MHz,CD₃OD)δ8.32(d,J＝7.7Hz,1H),7.38–7.35(m,2H),7.32(t,J＝8.1Hz,1H),7.21–7.12(m,1H),7.02(d,J＝8.2Hz,1H),6.69(d,J＝8.0Hz,1H),5.19(t,J＝6.5Hz,1H),4.85(d,J＝6.6Hz,2H),4.31–4.24(m,1H),4.21(d,J＝4.9Hz,2H),2.97–2.69(m,10H),2.55(s,3H),1.89(s,3H),1.68(s,3H).¹³C NMR(100MHz,CD₃OD)δ156.62,143.13,140.88,136.26,127.53,125.75,124.11,123.44,121.13,119.95,113.25,109.48,103.31,101.95,71.50,68.45,61.64,54.95(2×CH₂),52.85(2×CH₂),44.46,41.95,25.73,18.21.HRMS(ESI+):calculated for C₂₅H₃₄N₃O₂[M+H]⁺408.2651,found 408.2648。

Synthesis of Compound 20

According to the synthesis of compound 8, starting with 7(53.6mg, 0.17mmol) and diethanolamine (0.5mL), compound 20 was prepared as a white solid (64.8mg, 87%).¹H NMR(400MHz,CDCl₃)δ8.28(d,J＝7.7Hz,1H),7.43–7.37(m,1H),7.33(t,J＝8.0Hz,2H),7.24–7.19(m,1H),6.99(d,J＝8.1Hz,1H),6.61(d,J＝7.9Hz,1H),5.28–5.22(m,1H),4.84(d,J＝6.4Hz,2H),4.38–4.29(m,1H),4.25–4.15(m,2H),3.83–3.74(m,2H),3.67–3.60(m,2H),2.92–2.76(m,4H),2.62–2.54(m,2H),1.91(s,3H),1.72–1.67(m,3H).¹³C NMR(100MHz,CDCl₃)δ155.23,141.85,139.59,135.12,126.47,124.81,123.11,122.24,120.05,119.14,112.09,108.37,102.17,100.83,70.04,67.62,59.52,58.71,57.44,50.81,41.32(2×CH₂),25.65,18.26.HRMS(ESI+):calculatedfor C₂₄H₃₃N₂O₄[M+H]⁺367.2368,found 367.2381。

Synthesis of Compound 21

According to the synthetic method of compound 8, starting from 7(62.5mg, 0.20mmol) and thiomorpholine (0.5mL), compound 21 was obtained as a white solid (80.5mg, 91%).¹H NMR(400MHz,CDCl₃)δ8.31–8.24(m,1H),7.44–7.39(m,1H),7.38–7.32(m,2H),7.24–7.20(m,1H),7.01(d,J＝8.2Hz,1H),6.67(d,J＝8.0Hz,1H),5.28–5.22(m,1H),4.87(d,J＝6.4Hz,2H),4.33–4.25(m,2H),4.24–4.17(m,1H),3.03–2.94(m,2H),2.82–2.73(m,4H),2.73–2.63(m,4H),1.91(s,3H),1.69(s,3H).¹³CNMR(100MHz,CDCl₃)δ155.32,141.91,139.65,135.19,126.44,124.83,123.05,122.28,120.03,119.18,112.22,108.42,102.27,100.88,70.31,65.82,61.88,55.54(2×CH₂),41.39,28.12(2×CH₂),25.67,18.29.HRMS(ESI+):calculated for C₂₄H₃₁N₂O₂S[M+H]⁺411.2106,found 411.2102。

Synthesis of Compound 22

Following the synthetic procedure for compound 8, starting with 7(50mg, 0.17mmol) and ethylamine (0.5mL), compound 22 was prepared as a white solid (38.8mg, 68%).¹H NMR(400MHz,CD₃OD)δ8.30(d,J＝7.7Hz,1H),7.42–7.38(m,2H),7.35(t,J＝8.1Hz,1H),7.22–7.16(m,1H),7.07(d,J＝8.2Hz,1H),6.74(d,J＝8.0Hz,1H),5.27–5.16(m,1H),4.94–4.91(m,2H),4.51–4.18(m,3H),3.43–3.35(m,1H),3.30–3.23(m,1H),3.14(q,J＝7.0Hz,2H),1.92(s,3H),1.70(s,3H),1.34(t,J＝7.2Hz,3H).¹³C NMR(100MHz,CD₃OD)δ156.17,143.15,140.91,136.32,127.52,125.85,124.00,123.28,121.06,120.03,113.19,109.56,103.66,101.97,70.90,66.90,51.16,44.19,41.95,25.72,18.21,11.42.HRMS(ESI+):calculated for C₂₂H₂₉N₂O₂[M+H]⁺353.2229,found 353.2224。

Synthesis of Compound 23

Following the synthetic procedure for compound 8, starting from 7(50mg, 0.16mmol) and n-propylamine (0.5mL), compound 23 was prepared as a white solid (40.5mg, 68%).¹H NMR(400MHz,CD₃OD)δ8.29(d,J＝7.6Hz,1H),7.44–7.32(m,3H),7.23–7.14(m,1H),7.08(d,J＝8.2Hz,1H),6.74(d,J＝8.0Hz,1H),5.27–5.16(m,1H),4.98–4.90(m,2H),4.53–4.17(m,3H),3.43–3.34(m,1H),3.29–3.22(m,1H),3.08–2.94(m,2H),1.92(s,3H),1.80–1.65(m,5H),1.02(t,J＝7.1Hz,3H).¹³C NMR(100MHz,CD₃OD)δ156.18,143.19,140.95,136.36,127.52,125.87,123.98,123.29,121.08,120.02,113.23,109.58,103.69,101.99,70.95,66.92,51.57,50.69,41.99,25.72,20.59,18.21,11.24.HRMS(ESI+):calculated for C₂₃H₃₁N₂O₂[M+H]⁺367.2386,found 367.2380。

Synthesis of Compound 24

Following the synthetic procedure for compound 8, starting from 7(56.7mg,0.18mmol) and isopropylamine (0.5mL), compound 24 was prepared as a white solid (58.2mg, 87%).¹H NMR(400MHz,CDCl₃)δ8.31(d,J＝7.8Hz,1H),7.46–7.40(m,1H),7.39–7.34(m,2H),7.26–7.21(m,1H),7.02(d,J＝8.2Hz,1H),6.68(d,J＝8.0Hz,1H),5.30–5.24(m,1H),4.88(d,J＝6.4Hz,2H),4.34–4.20(m,3H),3.10–3.04(m,1H),2.97–2.86(m,2H),2.63(br,2H),1.92(s,3H),1.71(s,3H),1.15–1.11(m,6H).¹³C NMR(100MHz,CDCl₃)δ155.30,141.90,139.63,135.17,126.45,124.82,123.08,122.27,120.04,119.19,112.20,108.39,102.25,100.92,70.55,68.63,49.61,49.13,41.38,25.65,23.09,22.92,18.28.HRMS(ESI+):calculated for C₂₃H₃₁N₂O₂[M+H]⁺367.2368,found 367.2381。

Synthesis of Compound 25

According to the method for synthesizing compound 8, starting from 7(53.7mg,0.17mmol) and n-pentylamine (0.5mL), compound 25 was obtained as a white solid (64.6mg, 93%).¹H NMR(400MHz,CDCl₃)δ8.30(d,J＝7.7Hz,1H),7.45–7.41(m,1H),7.39–7.34(m,2H),7.26–7.21(m,1H),7.02(d,J＝8.2Hz,1H),6.68(d,J＝8.0Hz,1H),5.30–5.23(m,1H),4.88(d,J＝6.3Hz,2H),4.35–4.19(m,3H),3.08–2.90(m,2H),2.75–2.63(m,2H),2.45(br,2H),1.92(s,3H),1.71(s,3H),1.36–1.25(m,6H),0.90(t,J＝6.7Hz,3H).¹³C NMR(100MHz,CDCl₃)δ155.28,141.90,139.63,135.17,126.45,124.83,123.07,122.26,120.04,119.21,112.19,108.39,102.25,100.92,70.53,68.31,52.19,49.96,41.37,29.64,29.49,25.65,22.64,18.28,14.12.HRMS(ESI+):calculated forC₂₅H₃₅N₂O₂[M+H]⁺395.2699,found 395.2698。

Synthesis of Compound 26

According to the procedure for the synthesis of compound 8, starting from 7(59.2mg, 0.19mmol) and n-octylamine (0.5mL), compound 26 was prepared as a white solid (77.5mg, 92%).¹H NMR(400MHz,CDCl₃)δ8.30(d,J＝7.7Hz,1H),7.45–7.33(m,3H),7.24(t,J＝7.4Hz,1H),7.03(d,J＝8.1Hz,1H),6.69(d,J＝7.9Hz,1H),5.31–5.22(m,1H),4.88(d,J＝6.1Hz,2H),4.33–4.19(m,3H),3.07–2.90(m,2H),2.77–2.61(m,2H),2.33(br,2H),1.92(s,3H),1.71(s,3H),1.35–1.23(m,12H),0.91–0.85(m,3H).¹³CNMR(100MHz,CDCl₃)δ155.30,141.90,139.63,135.17,126.44,124.82,123.06,122.26,120.04,119.20,112.19,108.38,102.24,100.92,70.56,68.44,52.21,50.07,41.38,31.92,30.17,29.59,29.35,27.36,25.65,22.74,18.28,14.18.HRMS(ESI+):calculatedfor C₂₈H₄₁N₂O₂[M+H]⁺437.3168,found 437.3168。

Synthesis of Compound 27

According to the method for synthesizing compound 8, starting from 7(50.3mg,0.16mmol) and n-nonanamine (0.5mL), compound 27 was obtained as a white solid (60.8mg, 82%).¹H NMR(400MHz,CDCl₃)δ8.29(d,J＝7.8Hz,1H),7.45–7.40(m,1H),7.39–7.33(m,2H),7.25–7.21(m,1H),7.02(d,J＝8.2Hz,1H),6.68(d,J＝8.0Hz,1H),5.30–5.22(m,1H),4.88(d,J＝6.4Hz,2H),4.33–4.20(m,3H),3.07–2.89(m,2H),2.77–2.60(m,2H),1.92(s,3H),1.70(s,3H),1.34–1.23(m,16H),0.88(t,J＝6.8Hz,3H).¹³C NMR(100MHz,CDCl₃)δ155.31,141.90,139.63,135.16,126.44,124.82,123.06,122.27,120.04,119.20,112.19,108.38,102.24,100.92,70.58,68.50,52.23,50.10,41.38,31.99,30.24,29.71,29.67,29.65,29.42,27.37,25.65,22.77,18.28,14.21.HRMS(ESI+):calculated for C₃₀H₄₅N₂O₂[M+H]⁺465.3481,found 465.3471。

Synthesis of Compound 28

According to the synthesis of compound 8, starting from 7(50mg, 0.17mmol) and ammonia (2mL), compound 28 was obtained as a white solid (26.5mg, 50%).¹H NMR(400MHz,(CD₃)₂SO)δ8.32–8.21(m,1H),7.49(d,J＝7.9Hz,1H),7.43–7.29(m,2H),7.19(t,J＝7.4Hz,1H),7.12(d,J＝8.1Hz,1H),6.75(d,J＝7.9Hz,1H),5.23–5.06(m,1H),4.95(d,J＝6.1Hz,2H),4.22–4.07(m,2H),4.03–3.93(m,1H),2.97–2.72(m,2H),1.90(s,3H),1.66(s,3H).¹³C NMR(100MHz,(CD₃)₂SO)δ155.09,141.31,139.08,134.80,126.66,124.69,122.68,121.65,119.96,118.90,111.26,108.78,102.20,100.95,79.24,70.12,70.02,44.63,25.37,18.07.HRMS(ESI+):calculated forC₂₀H₂₅N₂O₂[M+H]⁺325.1916,found 325.1911。

Synthesis of Compound 29

Compound 28(200mg, 0.62mmol) was dissolved in DMF (15mL) solution and 1H-pyrazole-1-carboxamidine hydrochloride (225.90mg, 1.54mmol) and N, N-diisopropylethylamine (254.72. mu.L, 1.54mmol) were added. The mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was diluted with 1-butanol and extracted three times with water. The organic layers were combined and concentrated under vacuum. The resulting crude product was purified by HPLC to afford compound 29 as a white solid (116.5mg, 52%).¹H NMR(400MHz,CD₃OD)δ8.30(d,J＝7.7Hz,1H),7.42–7.32(m,3H),7.21–7.15(m,1H),7.07(d,J＝8.2Hz,1H),6.74(d,J＝8.0Hz,1H),5.22(t,J＝6.3Hz,1H),4.91(d,J＝6.5Hz,2H),4.38–4.18(m,3H),3.70–3.48(m,2H),1.92(s,3H),1.70(s,3H).¹³C NMR(100MHz,CD₃OD)δ159.78,156.34,143.18,140.92,136.32,127.50,125.80,124.04,123.35,121.11,120.03,113.23,109.50,103.55,101.92,70.43,69.90,46.09,41.99,25.72,18.21.HRMS(ESI+):calculated for C₂₁H₂₇N₄O₂[M+H]⁺367.2134,found 367.2133。

Synthesis of Compound 30

14(51mg, 0.13mmol) was dissolved in MeOH (10mL) and iodomethane (0.5mL) was added. The mixture was stirred at room temperature for 4 hours. After the reaction was completed, the organic solvent in the reaction mixture was evaporated under reduced pressure. The resulting crude product was purified by HPLC to afford compound 30 as a white solid (48.4mg, 69%).¹H NMR(400MHz,CD₃OD)δ8.26(d,J＝7.8Hz,1H),7.38–7.29(m,3H),7.20–7.13(m,1H),7.04(t,J＝7.6Hz,1H),6.76–6.68(m,1H),5.18–5.13(m,1H),4.84(d,J＝6.4Hz,2H),4.72–4.62(m,1H),4.33–4.17(m,2H),3.63–3.31(m,6H),3.08(s,3H),1.87(s,3H),1.66(s,3H),1.30(t,J＝7.3Hz,6H).¹³C NMR(100MHz,CD₃OD)δ155.95,143.22,140.95,136.43,127.60,125.98,123.89,123.18,120.99,120.05,113.21,109.71,103.88,102.14,71.33,70.96,65.57,64.25,59.15,58.83,41.99,25.73,18.22,9.05,8.24.HRMS(ESI+):calculated for C₂₅H₃₅IN₂O₂[M-I]⁺395.2699,found 395.2695。

Synthesis of Compound 31

According to the procedure for the synthesis of compound 30, starting from 16(60.0mg, 0.14mmol) and iodomethane (0.5mL), compound 31 was prepared as a white solid (58.1mg, 64%).¹H NMR(400MHz,CD₃OD)δ8.23(d,J＝7.8Hz,1H),7.37–7.28(m,3H),7.14(t,J＝6.2Hz,1H),7.01(d,J＝8.2Hz,1H),6.74(t,J＝6.7Hz,1H),5.22–5.08(m,1H),4.81(d,J＝5.1Hz,2H),4.66–4.57(m,1H),4.39–4.11(m,2H),3.65–3.47(m,2H),3.40–3.31(m,2H),3.24(t,J＝7.9Hz,2H),3.12–3.03(m,3H),1.89–1.81(m,3H),1.71–1.50(m,7H),1.30–1.16(m,4H),0.82(t,J＝7.3Hz,6H).¹³C NMR(100MHz,CD₃OD)δ155.70,143.28,141.00,136.44,127.65,126.03,123.86,123.19,120.99,120.13,113.25,109.76,103.92,102.13,70.82,65.16,65.12,64.32,63.91,42.01(2×CH₂),25.75,25.23,25.20,20.64,20.61,18.23,13.85,13.83.HRMS(ESI+):calculated for C₂₉H₄₃IN₂O₂[M-I]⁺451.3319,found451.3323。

Synthesis of Compound 34

Compound 28(100mg, 0.308mmol) was dissolved in DMF (10mL) and then Fmoc-Lys (Fmoc) -OH (455.2mg, 0.77mmol), HATU (292.99mg, 0.77mmol) and N, N-diisopropylethylamine (254.72. mu.L, 1.54mmol) were added and the mixture was stirred at room temperature overnight. After the reaction is completed, mixingThe compound was diluted with ethyl acetate and extracted three times with water. The organic layer was concentrated under reduced pressure to give crude product 32, which was used in the next reaction without further purification. The resulting crude product 32 was added to a 20% piperidine/DMF (v/v) solution (5mL) at room temperature, and the reaction was stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was diluted with 1-butanol and extracted three times with water. The combined organic layers were concentrated under reduced pressure. The crude product was purified by HPLC to give 21.1mg of compound 34 as a white solid, 22% total yield over the two steps.¹HNMR(400MHz,CD₃OD)δ8.40–8.31(m,1H),7.43–7.33(m,3H),7.20–7.15(m,1H),7.07(d,J＝8.2Hz,1H),6.73(d,J＝7.9Hz,1H),5.27–5.19(m,1H),4.93(d,J＝6.4Hz,2H),4.35–4.18(m,3H),3.84–3.69(m,2H),3.67–3.47(m,1H),2.92–2.80(m,2H),1.94(s,3H),1.89–1.76(m,2H),1.71(s,3H),1.68–1.61(m,2H),1.52–1.42(m,2H).¹³C NMR(100MHz,CD₃OD)δ172.00,156.59,143.15,140.89,136.30,127.50,125.75,124.29,123.42,121.13,120.03,113.26,109.42,103.39,101.85,70.96,69.99,54.54,44.06,41.98,40.20,32.73,28.13,25.74,22.99,18.24.HRMS(ESI+):calculated for C₂₆H₃₇N₄O₃[M+H]⁺453.2688,found453.2865。

Synthesis of Compound 35

Compound 28(100mg, 0.308mmol) was dissolved in DMF (10mL) and then Fmoc-Arg-OH (305.5mg, 0.77mmol), HATU (292.99mg, 0.77mmol) and N, N-diisopropylethylamine (254.72. mu.L, 1.54mmol) were added and the mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was diluted with ethyl acetate and extracted three times with water. The organic layer was concentrated under reduced pressure to give crude product 32, which was used in the next reaction without further purification. The resulting crude product 32 was added to a 20% piperidine/DMF (v/v) solution (5mL) at room temperature, and the reaction was stirred at room temperature for 30 minutes. After completion of the reaction, the mixture was diluted with 1-butanol and extracted three times with water. The combined organic layers were concentrated under reduced pressure. What is needed isThe crude product was purified by HPLC to give 42.8mg of compound 35 as a white solid in 30% total yield over the two steps.¹HNMR(400MHz,CD₃OD)δ8.39–8.31(m,1H),7.42–7.31(m,3H),7.20–7.14(m,1H),7.05(d,J＝8.2Hz,1H),6.72(d,J＝7.2Hz,1H),5.21(t,J＝6.4Hz,1H),4.95–4.90(m,2H),4.35–4.19(m,3H),3.93–3.68(m,2H),3.64–3.48(m,1H),3.22–3.11(m,2H),1.97–1.81(m,5H),1.76–1.61(m,5H).¹³C NMR(100MHz,CD₃OD)δ158.79,156.54,143.19,140.93,136.32,127.53,125.78,124.28,124.26,123.46,121.19,120.04,113.29,109.46,103.43,101.87,70.99,69.99,54.31,44.03,42.02,41.77,30.18,25.77,25.44,18.25.HRMS(ESI+):calculatedfor C₂₆H₃₇N₆O₃[M+H]⁺481.2927,found 481.2927。

Synthesis of Compound 36

According to the procedure for the synthesis of compound 8, starting from 7(52.7mg, 0.17mmol) and lutidine amine (0.5mL), compound 36 was prepared as a dark green oil (23.5mg, 27%).¹H NMR(400MHz,CDCl₃)δ8.58–8.54(m,2H),8.16(d,J＝7.8Hz,1H),7.61–7.54(m,2H),7.42–7.31(m,5H),7.16–7.07(m,3H),6.99(d,J＝8.1Hz,1H),6.65(d,J＝8.0Hz,1H),5.28–5.22(m,1H),4.86(d,J＝6.4Hz,2H),4.43–4.35(m,1H),4.34–4.16(m,2H),4.13–3.96(m,4H),3.27–2.98(m,2H),1.94–1.89(m,3H),1.71–1.67(m,3H).¹³C NMR(100MHz,CDCl₃)δ158.91(2×C),155.43,148.86(2×CH),141.85,139.57,136.93(2×CH),135.08,126.41(2×C),124.62,123.38(2×CH),123.16,122.35(2×CH),120.09,119.10,112.17,108.21,101.97,100.79,70.13,68.03,60.44,58.54,41.35,25.65,18.28.HRMS(ESI+):calculated for C₃₂H₃₅N₄O₂[M+H]⁺507.2760,found 507.2761。

The above compounds were tested for activity:

the test method comprises the following steps:

antibacterial activity (most preferred)Low inhibitory concentration) assay

Minimum Inhibitory Concentration (MIC) determination was determined by the two-fold broth dilution method, according to american Clinical and Laboratory Standards Institute (CLSI) standards. The samples were first dissolved in DMSO/H₂O to prepare a stock of samples of 1000. mu.g/mL (final DMSO concentration. ltoreq.2.5%). The stock was serially diluted with Muller Hinton Broth (MHB) medium. Bacteria were incubated at 37 ℃ for 24 hours on MHA plates and then the bacterial concentration was adjusted to about 10⁶CFU/mL. The bacterial suspension (100 μ L) was mixed with two-fold serial dilutions of the sample (100 μ L) in a 96-well plate (final sample concentrations of 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 μ g/mL). After incubation at 37 ℃ for 24 hours, the mixture was read for absorption wavelength at 600nm using a microplate reader. The lowest compound concentration at which no significant growth of bacteria was observed was the MIC value.

Determination of hemolytic Activity

Rabbit Red Blood Cells (RBCs) were centrifuged at 2500rpm for 3 minutes and washed twice with PBS, and then resuspended in PBS to make an 8% v/v suspension of RBCs. RBCs suspensions (100 μ L) were incubated with equal volumes of two-fold serial dilutions of the compound (100 μ L) for 1 hour at 37 ℃. The mixture was then centrifuged at 2500rpm for 5 minutes, and the supernatant (100 μ L) was transferred to a 96-well plate and read for absorbance at 576nm using a microplate reader. A2% Triton X-100 solution-treated group was used as a positive control, and a 0.5% DMSO-treated group was used as a negative control. Hemolytic activity was calculated by the following formula: hemolytic activity [ (% Abs)_{Sample (I)}–Abs_{Positive control})/(Abs_{Positive control}–Abs_{Negative control})]×100％。

Induced bacterial drug resistance test

The initial MIC of the compound against staphylococcus aureus ATCC29213 was obtained according to the MIC determination method described above. Bacterial suspensions were then prepared using bacteria from wells at 0.5 × MIC for the next MIC value determination. The experiment was repeated daily for 21 days.

In vivo antimicrobial Activity assay

All animal experiments were approved by the laboratory animal center of southern China university of agriculture and were conducted according to the policy of the department of health. Female mice (6-8 weeks, average body weight 20 g) were used in this study. Mice were given peritoneal injections of cyclophosphamide (100mg/kg)3 times 5 days before infection to prepare mouse immunosuppressive models. After anesthetizing the mice, the cornea of the right eye of the mice was scarred with a sterile needle. Then 15. mu.L of the bacteria (Staphylococcus aureus ATCC29213) were dropped onto the injured cornea. These mice were randomly divided into 3 groups (n ═ 5). Treatment was started one day after infection. The medicine is dropped once every two hours, four times a day, and the medicine is continuously administrated for three days. The third day after dosing, mice were sacrificed and the eyeballs were removed. Viable cell counts on the cornea were counted by standard plate (MHA plate) counting method.

Second, test results

2.1 results of in vitro antibacterial and hemolytic activities are shown in tables 1 and 2.

TABLE 1 antibacterial Activity of carbazole derivatives 1-35 against gram-positive bacteria and hemolytic Activity against Rabbit Red blood cells

TABLE 2 antibacterial Activity of carbazole derivative 36 in combination with Zinc ion (6.25. mu.g/mL) against gram-negative and gram-positive bacteria

Wherein the MIC values of the compounds 29 and 36 to gram-positive bacteria are 0.78-3.125. mu.g/mL, both of which show very excellent antibacterial activity, and the MIC value of the compound 36 to 6.25. mu.g/mL of zinc ion combined with the compound also shows very excellent antibacterial activity to gram-negative bacteria is 3.13. mu.g/mL. Furthermore, both compounds show very low hemolytic toxicity to rabbit erythrocytes.

2.2 results of the induced bacterial drug resistance test

The greatest advantage of membrane active antibacterial drugs is that they have a very low probability of inducing resistance. To assess the propensity of compound 29 to induce development of bacterial resistance, the present inventors conducted a laboratory simulation study of resistance development on compound 29. As shown in fig. 1, the MIC value of compound 29 was constant over the 21 day test period, indicating that compound 29 was effective in avoiding the development of resistance to staphylococcus aureus. In contrast, after 10 passages of norfloxacin, the MIC increased 128-fold, indicating that norfloxacin was highly likely to induce the development of bacterial resistance. This result indicates that compound 29 is effective in slowing down or even overcoming the development of bacterial resistance.

2.3 evaluation results of antibacterial efficacy in animal bodies

Compound 29 was evaluated for in vivo efficacy by topical administration in a mouse model of corneal infection induced by staphylococcus aureus ATCC 29213. Each group of mice (n ═ 5) was treated with topical administration of 0.5% of

compound

29, 5% of vancomycin (used as positive control) or 5% of glucose (used as negative control), respectively, daily for 24 hours after infection of the mouse cornea with staphylococcus aureus ATCC29213, 4 times a day for three consecutive days. As shown in fig. 2, compound 29 (0.5%) and vancomycin (5%) were able to reduce the total number of bacterial colonies by 4.17 and 3.99 logs, respectively, with the therapeutic effect of compound 29 being comparable to that of vancomycin (5%). These results indicate that compound 29 still maintains excellent antibacterial activity in animals and is effective in curing corneal infection in mice induced by gram-positive bacteria (staphylococcus aureus ATCC 29213).

In conclusion, through a series of structure optimization, the invention obtains a plurality of high-efficiency and low-toxicity antibacterial candidate compounds. Wherein the compound 29 has excellent antibacterial activity (MIC of 0.78-1.56 μ g/mL) against gram-positive bacteria, low toxicity to mammalian cells, and very low hemolytic activity (HC)₅₀>200. mu.g/mL). The compound 29 can destroy the bacterial cell membrane to cause bacterial death, has quick bactericidal performance, and can effectively overcome the generation of bacterial drug resistance in the laboratory drug resistance simulation research. More importantly, compound 29 caused bacterial keratitis in mice in staphylococcus aureus ATCC29213Excellent in vivo antibacterial activity was also shown in the model.

In addition, compound 36 exhibited excellent antibacterial activity against gram-positive bacteria and very low hemolytic toxicity. Compared with the single application, the MIC value of the compound 36 and 6.25 mu g/mL of zinc ions for gram-positive bacteria is reduced by one time, the antibacterial activity for gram-negative bacteria is greatly enhanced, and the MIC value is reduced from more than 50 mu g/mL to 3.125 mu g/mL. The results indicate that compound 36, when used in combination with zinc ions, exhibits excellent and broad-spectrum antibacterial activity.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Carbazole compounds with the following structural general formula or pharmaceutically acceptable salts thereof or stereoisomers thereof or solvates thereof or prodrug molecules thereof:

R_aeach independently selected from: H. halogen;

R₂is a cationA group selected from: at least one R_bSubstituted or unsubstituted C2-C5 cycloalkoxy, at least one R_bA substituted or unsubstituted C1-C5 alkylamine;

2. The carbazole-based compound according to claim 1, wherein R is R, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a solvate thereof, or a prodrug molecule thereof₂Selected from: C2-C5 cycloalkoxy or a group of the general structural formula:

wherein m + n is 1-5.

3. The carbazole-based compound according to claim 1 or 2, wherein R is R, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a solvate thereof, or a prodrug molecule thereof_bEach independently selected from: H. OH, carbonyl, C1-C10 linear or branched alkyl, C1-C5 linear or branched alkyl alcohol, C1-C5 linear or branched alkylmercapto, C2-C6 alkylamine, C5-C8 nitrogen-containing heteroaryl, C1-C2 aminoiminoalkyl, or a combination of the foregoing, which, when each two of the foregoing groups are attached to the same carbon atom, are linked to each other to form a ring or not.

4. The carbazole-based compound according to claim 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug molecule thereof, wherein the C5-C8 nitrogen-containing heteroaryl group has the following structure:

wherein w is 0, 1, 2 or 3 and V is independently at each occurrence C or N.

5. The carbazole-based compound according to claim 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug molecule thereof, wherein the C5-C8 nitrogen-containing heteroaryl group has the following structure:

wherein w is 0, 1, 2 or 3 and V is independently at each occurrence C or N.

6. The carbazole-based compound according to claim 1 or 2, wherein R is R, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a solvate thereof, or a prodrug molecule thereof₁Selected from: at least one R_aSubstituted or unsubstituted C1-C10 straight chain alkyl, at least one R_aSubstituted or unsubstituted C3-C6 branched alkenyl.

7. The carbazole-based compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug molecule thereof, wherein the carbazole-based compound is selected from the group consisting of:

8. the process for preparing carbazole-based compounds according to any one of claims 1 to 7, wherein the carbazole-based compounds are prepared according to one of the following schemes (I) to (III):

scheme (I):

scheme (II):

scheme (III):

wherein the raw materials are prepared by adopting a synthetic route (II).

9. Use of a carbazole-based compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug molecule thereof, for the preparation of a medicament having antibacterial efficacy.

10. Use of a composition of a carbazole-based compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug molecule thereof, and zinc ion for the preparation of a medicament having antibacterial efficacy.