CN107827815B

CN107827815B - Fluoroquinolone amino derivatives and application thereof in preventing and treating citrus diseases

Info

Publication number: CN107827815B
Application number: CN201711102891.1A
Authority: CN
Inventors: 胡军华; 杨大成; 范莉; 龙艳玲; 冯计周; 黄敏; 王帆; 刘�文; 刘洁; 陈菲菲; 刘晋宇; 张泽朝; 杨真; 张金坤; 谌阳; 陈思雅
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2017-11-10
Filing date: 2017-11-10
Publication date: 2021-08-03
Anticipated expiration: 2037-11-10
Also published as: CN107827815A

Abstract

The citrus canker and the brown spot are common diseases of citrus, and at present, a few medicines capable of preventing and treating the two diseases have certain defects. According to the invention, the compound shown as the formula I or the formula II is obtained by connecting the 7-site amino group of the fluoroquinolone medicine with the active fragment through a connecting structure, and experiments show that the compound has the effects of preventing and treating citrus canker and brown spot and has a very good application prospect.

Description

Fluoroquinolone amino derivatives and application thereof in preventing and treating citrus diseases

Technical Field

The invention relates to fluoroquinolone amino derivatives and application thereof, in particular to application of the fluoroquinolone amino derivatives in preventing and treating citrus canker and citrus brown spot.

Background

Citrus, as the first fruit in the world, has yield and economic value that determine its dominance throughout the fruit tree industry. In recent years, the citrus industry in our country has developed rapidly and has gradually become an important post industry for promoting the development of national rural economy and guaranteeing the income of farmers. However, the citrus fruits are often vulnerable to diseases and insect pests during the growth, storage, transportation and other processes, various biotoxins are generated and accumulated, the yield and the quality of the citrus are seriously affected, and the economic value of the citrus is greatly reduced.

Citrus canker (Citrus canker) is a broad bacterial disease caused by the Xanthomonas citri (Xanthomonas axonopodis pv. citri, Xac) which affects Citrus growing worldwide, mainly infesting Citrus, Citrus and Citrus plants of the rutaceae family. The Xac mainly infects overground parts of citrus leaves, treetops, fruits, skin spines, trunks and the like, so that ulcer surrounded by withered rings and necrosis of the fruits or the surfaces of the leaves can be caused, water-immersed lesions on the leaves are 2-5 mm in diameter, the leaves are suberized, rough in surface and grey brown, and are cracked in a crater shape; in addition to leaf symptoms, it can also lead to fruit abscission, fruit tree aging, and loss of commodity value of infected fruits.

Citrus brown spot (Citrus brown spot) is a fungal disease caused by a pathogenic type of Alternaria alternata orange (Alternaria alternata pathotype). The alternaria alternate mainly infects citrus leaves, twigs and fruits, which not only affects the growth and development of citrus and reduces the fruit yield, but also reduces the fruit quality and seriously affects the production of citrus. In recent years, citrus brown spot disease occurs in successive citrus production areas such as Yunnan, Chongqing, Zhejiang and Hunan in China, the damage is aggravated year by year, and high attention is paid to all aspects. China is an important original production center of citrus, the cultivation area and annual yield of citrus are the first in the world at present, citrus varieties are numerous and have high disease incidence, tangerine, tribute citrus and ponkan citrus in citrus are susceptible to diseases, and the proportion of the varieties in citrus in China is high, so that the potential danger of brown spot to the broad-skin citrus in China is not ignored.

For citrus canker, because the cost for preventing and eradicating citrus canker is very high and the eradication effect is not obvious, at present, the prevention is mainly performed abroad and domestically, seedlings are grown in non-citrus canker areas, and copper preparations and agricultural antibiotics are sprayed to prevent and reduce the spread of the canker. The copper preparation has the defects of easy generation of drug resistance, heavy metal tolerance caused by horizontal transfer among germs, accumulation of copper ions in soil as potential phytotoxin and influence on the environment after long-term use, and the like, and is also a hazard to fruits, particularly under the dry and hot environment. Recently, the use of streptomycin for agricultural use has been prohibited, and the control agents effective against ulcer germs are seriously lacking, and new compounds are urgently required for replacement.

The citrus brown spot belongs to new diseases in China, corresponding technical reserves and systematic researches are lacked, and especially, the research and accumulation of pathogenic types, infection epidemic laws, effective prevention and treatment technologies, efficient prevention and treatment agents and the like causing diseases in different production areas are lacked, so that no effective and reliable prevention and treatment technology exists at present. Therefore, the development of high-efficiency control agents for controlling alternaria alternata is important and urgent.

Disclosure of Invention

The invention aims to provide a series of compounds with quinolone structures, racemates, stereoisomers, tautomers, nitric oxides or pharmaceutically acceptable salts thereof, and application of the compounds in preventing and treating citrus canker and brown spot. The structure of the compound shown in the formula I is as follows:

wherein X is selected from: C1-C6 alkyl; C3-C6 cycloalkyl; a substituted or unsubstituted C6-C10 aryl group, the substituents on the aryl group being one or more independently selected from: halogen; an amino group; a hydroxyl group; C1-C6 alkyl; C3-C6 cycloalkyl;

z is selected from: n or C-R₇；R₇Is selected from H; C1-C6 alkoxy or halogen;

R₁and R₂Each independently selected from: h; C1-C6 alkyl; C1-C6 haloalkyl; C1-C6 alkoxy; halogen; a hydroxyl group; amino or cyano;

Q₁is selected from H; halogen;

R₃or R₄Each is independently selected from H; C1-C6 alkyl, said C1-C6 alkyl being optionally substituted by hydroxy; an amino group; halogen; a cyano group; a urea group; thiourea group substitution;

or R₃，R₄Taken together with N to form a 5-8 membered heterocyclic ring, which heterocyclic ring may be optionally substituted with one or more of the following substituents:

an amino group; a hydroxyl group; a tert-butoxycarbonyl group; benzyl or C1-C6 alkyl, said C1-C6 alkyl being optionally substituted by hydroxy; an amino group;

halogen; C1-C4 alkoxy;

A₁，A₂，A₃，A₄independently selected from C-R₅Or N, and A₁，A₂At least one of which is N;

R₅is selected from H; C1-C4 alkyl; C1-C4 alkoxy; an amino group; an amido group; a hydroxyl group; halogen; phenyl or benzyl;

R₆is 1-4, independently selected from H, C1-C6 alkyl, said C1-C6 alkyl being optionally substituted by hydroxy; an amino group; halogen or

Cyano substitution; C1-C6 alkoxy, which C1-C6 alkoxy may be optionally substituted by halogen;

B₁selected from S or NH;

het is a 5-7 membered heteroaromatic ring containing at least 1 nitrogen atom;

R₈selected from H, C1-C6 alkyl;

n and k are each an integer of 0 to 3.

The invention also provides a compound with a structure shown in the formula II:

R₈selected from H, C1-C6 alkyl;

Q₂is selected from

Or halogen;

het is a 5-7 membered heteroaromatic ring containing at least 1 nitrogen atom;

n and k are each an integer of 0 to 3;

m is 1 or 2.

In the compounds of formula I or formula II above, the substituents may be preferably selected as follows, independently in free combination:

het is selected from

The 5-8 membered heterocyclic ring is selected from:

x is selected from: a methyl group; an ethyl group; cyclopropyl or 4-fluorophenyl; the claim part does not have this sentence and the next sentence

Z is selected from: n or C-R₇；R₇Is selected from H; a methoxy group; fluorine or chlorine; the present invention also provides compounds of the structure:

the invention also provides a pharmaceutical composition, which comprises the compound, racemate, stereoisomer, tautomer, nitric oxide or pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical preparation, which comprises the compound, racemate, stereoisomer, tautomer, nitrogen oxide or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or auxiliary agent.

The invention also provides a compound medicine which is characterized by comprising the compound, the racemate, the stereoisomer, the tautomer, the nitric oxide or the pharmaceutically acceptable salts thereof and other active ingredients.

The invention also provides application of the compound, racemate, stereoisomer, tautomer, nitrogen oxide or pharmaceutically acceptable salt thereof in preparing a medicament for preventing and treating citrus-related diseases. The citrus-related disease is preferably citrus canker or citrus brown patch.

The present invention also provides a method for treating citrus-related diseases comprising applying to a citrus fruit tree and/or fruit an agent comprising a compound of the present invention, racemates, stereoisomers, tautomers, nitric oxides or pharmaceutically acceptable salts thereof. Terms definition and interpretation:

unless otherwise indicated, the groups and term definitions set forth in the specification and claims of this application may be combined with each other in any combination and permutation. The definitions of the groups and the structures of the compounds in such combinations and after the combination are within the scope of the present specification.

The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenol the enol type predominates. The present invention encompasses all tautomeric forms of the compounds.

The term "halogen" refers to F, Cl, Br and I. In other words, F, Cl, Br, and I may be described as "halogen" in the present specification.

The term "C1-C6" is understood to mean preferably a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 6 carbon atoms, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, and the like, or isomers thereof.

Where a range of numerical values is recited in the specification and claims herein, and where the range of numerical values is defined as an "integer," it is understood that the two endpoints of the range are recited and each integer within the range is recited. For example, "any integer from 0 to 4" should be understood to recite each integer from 0, 1,2,3, 4.

The pharmaceutically acceptable salts of the compounds of the present invention may be acidic salts or basic salts. Pharmaceutically acceptable salts may be acid addition salts of the compounds of the invention having sufficient basicity, for example having a nitrogen atom in the chain or ring, for example with the following inorganic acids: for example hydrochloric, hydrofluoric, hydrobromic, hydroiodic, sulfuric, pyrosulfuric, phosphoric or nitric acid, or hydrogen sulfates, or the acid addition salts with the following organic acids: such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, gluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, and mixtures thereof, Mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

In addition, another suitable pharmaceutically acceptable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt (e.g., sodium or potassium salt), an alkaline earth metal salt (e.g., calcium or magnesium salt), an ammonium salt, or a salt with an organic base which affords a physiologically acceptable cation, such as a salt with: sodium ions, potassium ions, N-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexanediamine, ethanolamine, glucosamine, meglumine, sarcosine, serinol, trihydroxymethylaminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol.

Detailed Description

Example 1

To a 100mL round bottom flask were added sequentially the starting materials Gatifloxacin (GAT)1mmol and NaHCO₃2.5mmol and 5mL of DCM, and a mixed solution of 2mmol of chloroacetyl chloride and 2mL of DCM is added dropwise in an ice bath. After the addition, the reaction was stirred continuously in ice bath and the progress of the reaction was monitored by TLC. After the reaction is finished, adding a little ice-cold saturated NaCl solution to dissolve the solid, adjusting the pH to 4-5 by using ice-cold 2N HCl solution, stirring uniformly, transferring into a separating funnel for separating liquid, extracting twice by DCM, combining organic phases, washing by using saturated NaCl solution, and adding anhydrous Na₂SO₄Drying and removing the solvent by rotary evaporation. Recrystallizing or performing column chromatography to obtain pure product, [ M + H ], drying, and weighing]⁺453.1342。

Example 2

Adding the raw materials GAT 1mmol and NaHCO into a 100mL round-bottom flask in turn₃2.5mmol and 5mL of DCM, and a mixed solution of 2mmol of chloropropionyl chloride and 2mL of DCM is added dropwise in an ice bath. After the addition, the reaction was stirred continuously in ice bath and the progress of the reaction was monitored by TLC. After the reaction is finished, adding a little ice-cold saturated NaCl solution to dissolve the solid, adjusting the pH to 4-5 by using ice-cold 2N HCl solution, stirring uniformly, transferring into a separating funnel for separating liquid, extracting twice by DCM, combining organic phases, washing by using saturated NaCl solution, and adding anhydrous Na₂SO₄Drying and removing the solvent by rotary evaporation. Recrystallizing or performing column chromatography to obtain pure product, [ M + H ], drying, and weighing]⁺467.1453。

Example 3

Adding the raw materials GAT 1mmol and NaHCO into a 100mL round-bottom flask in turn₃2.5mmol and 4mL of DCM, and a mixed solution of 2.5mmol of 4-chlorobutyryl chloride and 2mL of DCM is added dropwise in an ice bath. After the addition, the reaction was stirred continuously in ice bath and the progress of the reaction was monitored by TLC. After the reaction is finished, adding a little ice-cold saturated NaCl solution to dissolve the solid, adjusting the pH to 4-5 by using ice-cold 2N HCl solution, stirring uniformly, transferring into a separating funnel for separating liquid, extracting twice by DCM, combining organic phases, washing by using saturated NaCl solution, and adding anhydrous Na₂SO₄Drying and removing the solvent by rotary evaporation. Recrystallizing to obtain pure product, [ M + H ], drying, weighing]⁺481.1679。

Example 4

A100 mL round-bottom flask was charged with 1.880g (5mmol) of GAT, 20mL of DCM, and 0.601g (6mmol) of succinic anhydride in that order. Adding Na after 30min₂CO₃0.635g (6 mmol). The reaction was stirred at room temperature and monitored by TLC for progress of the reaction. After the reaction is finished, a large amount of yellow oily matter is arranged at the bottom of the reaction bottle. Solvent DCM was removed by rotary evaporation and H was added₂O20 mL, insoluble matter was dissolved. Adjusting pH to 2 with 2N HCl solution, collecting a large amount of yellow oily substance at the bottom of the bottle, refrigerating in refrigerator to obtain solid, and vacuum filtering to obtain yellow solid. Naturally drying to obtain product, [ M + H]⁺476.1834。

Example 5

Adding 1mmol of sarafloxacin and 2mL of DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, and adding 3mmol of NaHCO₃After 20min, 2.5mmol of 4-chlorobutyryl chloride in DCM (2 m) are added dropwise from a constant-pressure dropping funnelL) solution (the dropping speed is about 1d/2s), the reaction is continued in ice bath after the dropping is finished, and the TLC tracking is monitored until the reaction is finished. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring through O15mL and 20mL of DCM, standing and filtering if solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺491.1238。

Example 6

Adding 1mmol clinafloxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 2-chloroacetyl chloride in DCM (2mL) was added dropwise from a constant pressure addition funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracking was performed until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring through O15mL and 20mL of DCM, standing and filtering if solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺443.0635。

Example 7

Adding 1mmol clinafloxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 3-chloropropionyl chloride in DCM (2mL) is added dropwise through a constant pressure dropping funnel (the dropping speed is about 1d/2s), the reaction is continued in ice bath after the dropwise addition is finished, and the TLC tracking monitoring is carried out until the reaction is finishedAnd (6) ending. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring by using 15mL of O and 20mL of DCM, standing and filtering if a solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺457.0814。

Example 8

Adding 1mmol clinafloxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 4-chlorobutyryl chloride in DCM (2mL) was added dropwise from a constant pressure dropping funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracing was carried out until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring through O15mL and 20mL of DCM, standing and filtering if solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺471.0927。

Example 9

Adding 1mmol norfloxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 2-chloroacetyl chloride in DCM (2mL) was added dropwise from a constant pressure addition funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracking was performed until the reaction was completed. Stopping stirring, adding H₂O15mL and DCM20mL, stirred with 1N HCl solutionAdjusting the pH to 3-4, standing and filtering if solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺397.1021。

Example 10

Adding 1mmol of balofloxacin and 2mL of DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, and adding 3mmol of NaHCO₃After 20min, 2.5mmol of 2-chloroacetyl chloride in DCM (2mL) was added dropwise from a constant pressure addition funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracking was performed until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring through O15mL and 20mL of DCM, standing and filtering if solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺467.1428。

Example 11

Adding 1mmol of sarafloxacin and 2mL of DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, and adding 3mmol of NaHCO₃After 20min, a constant pressure dropping funnel is used for dropwise adding 2.5mmol of 3-chloropropionyl chloride solution in DCM (2mL) (the dropping speed is about 1d/2s), the reaction is continued in ice bath after the dropwise adding is finished, and TLC tracking monitoring is carried out until the reaction is finished. Stopping stirring, adding H₂O15mL and DCM20mL, adjusting pH to 3-4 with 1N HCl solution under stirring, standing if there is a solid, suction filtering, washing the filter cake with DCM 3 times, the filter cake is left to go throughPurifying by one step; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺477.1173。

Example 12

To a 100mL round-bottom flask was added sequentially

2mmol、CHCl₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of the intermediate compound of example 1 was added and transferred to a 35 ℃ water bath for reaction and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value of the O10 mL and 1N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺521.2406。

Example 13

To a 100mL round-bottom flask were added 1mmol of 2-aminoethanol and CHCl in that order₃3mL, 1.2mmol of the compound from example 3 was added and the reaction was refluxed in a 60 ℃ water bath and monitored by TLC for progress. The reaction was stopped when the compound of example 3 was no longer reduced. Rotary evaporating to remove solvent, and performing column chromatography to obtain pure product, [ M + H ]]⁺505.2466。

Example 14

To a 100mL round-bottom flask was added sequentially

1mmol、CHCl₃3mL, 1.2mmol of the compound from example 3 was added and the reaction was refluxed in a 60 ℃ water bath and monitored by TLC for progress. The reaction was stopped when the compound of example 3 was no longer reduced. Rotary evaporating to remove solvent, and performing column chromatography to obtain pure product, [ M + H ]]⁺549.2726。

Example 15

2-amino acetic acid (50mmol) and 100mL of methanol are added into a reaction bottle, and SOCl is slowly dropped under ice bath₂(125mmol) and after dropping, the reaction solution is transferred into a water bath at 60 ℃ and stirred for reflux reaction, and TLC monitoring is carried out until the reaction is finished. Reduced pressure rotary evaporation to remove methanol and most SOCl₂20mL of methanol was added and the mixture was re-rotary evaporated to remove SOCl as much as possible₂Vacuum drying to obtain corresponding 2-amino methyl acetate hydrochloride.

Adding INA (isonicotinic acid 20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) in sequence into a reaction bottle, stirring for 10min, and adding Et₃N (60mmol), after 0.5-1 h, methyl 2-aminoacetate hydrochloride (22mmol) was added and TLC was monitored until the reaction was complete. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1h, spin-drying by a rotary evaporator, and carrying out column chromatography to obtain the 4-pyridyl carbonyl methyl aminoacetate.

4-pyridylcarbonyl-amino-acetic acid methyl ester (15mmol) and CH are added into a reaction bottle in sequence₃OH-H₂O(V _CH3OH:V _H2O(3: 1) solution (15mL), LiOH · H was added under ice-bath₂O (45mmol), stirred and monitored by TLC to the end of the reaction. Rotating evaporator to remove CH₃Removing OH, adjusting pH to about 3 with 4N HCl solution in ice bath, precipitating a large amount of white solid, vacuum filtering, washing filter cake with filtrate for 2 times, washing with acetone for 2 times, and oven drying at 50 deg.C to obtain 4-pyridyl carbonyl amino acetic acid.

4-Pyridylcarbonylaminoacetic acid (1mmol), HBTU/TBTU (1.2mmol), DCM (3mL) and Et are added into a reaction bottle in sequence₃N (3mmol), clinafloxacin (1mmol),stirring is carried out under controlled temperature, and TLC is carried out until the reaction is finished. Add DCM20mL, move to separatory funnel, saturate Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺528.1445。

Example 16

A100 mL round-bottom flask was charged with 1mmol of the compound of formula 9, 5mL of toluene, and 2mmol of Et₃And N, stirring for 20-30min, adding 2mmol of INA, moving to an oil bath kettle at 110-120 ℃ for reflux reaction after 10min, and monitoring by TLC until the reaction is complete. Spin-drying the reaction solution with rotary evaporator, adding 30mL DCM, stirring for dissolving, vacuum filtering, washing the filter cake with DCM for 3 times, washing the filtrate with 10% citric acid solution (15mL × 1), collecting the organic phase, and collecting anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, [ M + H ]]⁺483.1642。

Example 17

Adding 1mmol norfloxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 4-chlorobutyryl chloride in DCM (2mL) was added dropwise from a constant pressure dropping funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracing was carried out until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring through O15mL and 20mL of DCM, standing and filtering if solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. And (4) carrying out spin drying on a rotary evaporator to obtain a crude product, carrying out column chromatography to obtain a pure product, and carrying out vacuum drying to obtain an intermediate.

100mL circleA bottom flask was charged with 1mmol of the above intermediate, 5mL of toluene, and 2mmol of Et₃And N, stirring for 20-30min, adding 2mmol of INA, moving to an oil bath kettle at 110-120 ℃ for reflux reaction after 10min, and monitoring by TLC until the reaction is complete. Spin-drying the reaction solution with rotary evaporator, adding 30mL DCM, stirring for dissolving, vacuum filtering, washing the filter cake with DCM for 3 times, washing the filtrate with 10% citric acid solution (15mL × 1), collecting the organic phase, and collecting anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, [ M + H ]]⁺411.1956。

Example 18

Adding 1mmol enoxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 2-chloroacetyl chloride in DCM (2mL) was added dropwise from a constant pressure addition funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracking was performed until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring by using 15mL of O and 20mL of DCM, standing and filtering if a solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, anhydrous Na₂SO₄And (5) drying. And (4) carrying out spin drying on a rotary evaporator to obtain a crude product, carrying out column chromatography to obtain a pure product, and carrying out vacuum drying to obtain an intermediate.

A100 mL round-bottom flask was charged with 1mmol of the above intermediate, 5mL of toluene, and 2mmol of Et₃And N, stirring for 20-30min, adding 2mmol of INA, moving to an oil bath kettle at 110-120 ℃ for reflux reaction after 10min, and monitoring by TLC until the reaction is complete. Spin-drying the reaction solution with rotary evaporator, adding 30mL DCM, stirring for dissolving, vacuum filtering, washing the filter cake with DCM for 3 times, washing the filtrate with 10% citric acid solution (15mL × 1), collecting the organic phase, and collecting anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, [ M + H ]]⁺484.1616。

Example 19

Adding 1mmol enoxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, 2.5mmol of 4-chlorobutyryl chloride in DCM (2mL) was added dropwise from a constant pressure dropping funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracing was carried out until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring by using 15mL of O and 20mL of DCM, standing and filtering if a solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. And (4) carrying out spin drying on a rotary evaporator to obtain a crude product, carrying out column chromatography to obtain a pure product, and carrying out vacuum drying to obtain an intermediate.

A100 mL round-bottom flask was charged with 1mmol of the above intermediate, 5mL of toluene, and 2mmol of Et₃And N, stirring for 20-30min, adding 2mmol of pyrazinecarboxylic acid (POA), moving to an oil bath kettle at 110-120 ℃ for reflux reaction after 10min, and monitoring by TLC until the reaction is complete. Spin-drying the reaction solution with rotary evaporator, adding 30mL DCM, stirring for dissolving, vacuum filtering, washing the filter cake with DCM for 3 times, washing the filtrate with 10% citric acid solution (15mL × 1), collecting the organic phase, and collecting anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, [ M + H ]]⁺513.1892。

Example 20

A100 mL round-bottom flask was charged with 1mmol of the compound of example 10, 5mL of toluene, and 2mmol of Et₃And N, stirring for 20-30min, adding 2mmol of pyrazinecarboxylic acid (POA), moving to an oil bath kettle at 110-120 ℃ for reflux reaction after 10min, and monitoring by TLC until the reaction is complete. Spin-drying the reaction solution with rotary evaporator, adding 30mL DCM, stirring for dissolving, vacuum filtering, washing the filter cake with DCM for 3 times, and filtering to obtain filtrate 1Washing with 0% citric acid solution (15 mL. times.1), collecting organic phase, anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, [ M + H ]]⁺554.2046。

Example 21

Adding 1mmol of balofloxacin and 2mL of DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, and adding 3mmol of NaHCO₃After 20min, 2.5mmol of 4-chlorobutyryl chloride in DCM (2mL) was added dropwise from a constant pressure dropping funnel (dropping speed about 1d/2s), the reaction was continued in ice after dropping, and TLC tracing was carried out until the reaction was completed. Stopping stirring, adding H₂Adjusting the pH value to 3-4 by using a 1N HCl solution under stirring by using 15mL of O and 20mL of DCM, standing and filtering if a solid exists, washing a filter cake for 3 times by using DCM, and reserving the filter cake for further purification; the filtrate was transferred to a separatory funnel, separated, the aqueous phase was extracted with DCM (15 mL. times.1), the organic phases were combined, washed with saturated NaCl solution (15 mL. times.1), the organic phase was collected, and anhydrous Na was added₂SO₄And (5) drying. And (4) carrying out spin drying on a rotary evaporator to obtain a crude product, carrying out column chromatography to obtain a pure product, and carrying out vacuum drying to obtain an intermediate.

A100 mL round-bottom flask was charged with 1mmol of the above intermediate, 5mL of toluene, and 2mmol of Et₃And N, stirring for 20-30min, adding 2mmol of pyrazinecarboxylic acid (POA), moving to an oil bath kettle at 110-120 ℃ for reflux reaction after 10min, and monitoring by TLC until the reaction is complete. Spin-drying the reaction solution with rotary evaporator, adding 30mL DCM, stirring for dissolving, vacuum filtering, washing the filter cake with DCM for 3 times, washing the filtrate with 10% citric acid solution (15mL × 1), collecting the organic phase, and collecting anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, [ M + H ]]⁺582.2359。

Example 22

A100 mL round-bottom flask was sequentially added with 2mmol isoniazid and 4mmol NaHCO₃And 5mL DCM was added, stirred at-5 ℃ for 20min, and then constant pressure was applied3mmol of chloroacetyl chloride solution is added into the dropping funnel dropwise (the dropping speed is about 1d/2s), the reaction is continued at the temperature of 5 ℃, and the TLC tracking monitoring is carried out until the reaction is finished. With stirring, DCM-CH was added₃OH solution (V)_DCM:V_CH3OH2: 1) standing until the solid in the reaction flask does not decrease, vacuum filtering, and adding DCM-CH to the filter cake₃OH solution was washed 3 times, and the filtrate was washed with anhydrous Na₂SO₄Drying, spin-drying with a rotary evaporator to obtain a crude product, adding 5mL of DCM, stirring for 20min, carrying out suction filtration, washing a filter cake with DCM for 3 times, and carrying out vacuum drying to obtain an intermediate for later use.

1mmol of norfloxacin and 4mmol of Et are sequentially added into a 100mL round-bottom flask₃N and 2mL DMF, stirring for 20min, adding the intermediate, moving to 60 ℃ after 10min, stirring for reaction, and monitoring by TLC until the reaction is finished. Adding 10mL of ice-cold saturated aqueous salt solution while stirring, adjusting pH to about 8 with 1N HCl solution, suction filtering, extracting the filtrate with DCM (10 mL. times.2), combining the organic phases, and adding anhydrous Na₂SO₄Drying, rotary drying with rotary evaporator, and purifying with filter cake by column chromatography to obtain [ M + H ]]⁺497.1943。

Example 23

Alanine (50mmol) and 100mL of methanol were added to a reaction flask, and SOCl was slowly added dropwise under ice bath₂(125mmol) and after dropping, the reaction solution is transferred into a water bath at 60 ℃ and stirred for reflux reaction, and TLC monitoring is carried out until the reaction is finished. Reduced pressure rotary evaporation to remove methanol and most SOCl₂20mL of methanol was added and the mixture was re-rotary evaporated to remove SOCl as much as possible₂Vacuum drying to obtain the corresponding alanine methyl ester hydrochloride.

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding alanine methyl ester hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1h, spin-drying by a rotary evaporator, and carrying out column chromatography to obtain an intermediate.

The intermediate (15mmol) and CH are added into a reaction bottle in sequence₃OH-H₂O(V _CH3OH:V _H2O(3: 1) solution (15mL), LiOH · H was added under ice-bath₂O (45mmol), stirred and monitored by TLC to the end of the reaction. Rotating evaporator to remove CH₃Removing OH, adjusting pH to about 3 with 4N HCl solution in ice bath, precipitating a large amount of white solid, vacuum filtering, washing filter cake with filtrate for 2 times, washing with acetone for 2 times, and oven drying at 50 deg.C to obtain intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) were added in sequence to a reaction flask, DIPEA3mmol and gatifloxacin (1mmol) were added in ice bath, stirring was carried out under controlled temperature, and TLC was carried out until the reaction was completed. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺552.2217。

Example 24

3-aminopropionic acid (50mmol) and 100mL of methanol were added to a reaction flask, and SOCl was slowly added dropwise under ice bath₂(125mmol) and after dropping, the reaction solution is transferred into a water bath at 60 ℃ and stirred for reflux reaction, and TLC monitoring is carried out until the reaction is finished. Reduced pressure rotary evaporation to remove methanol and most SOCl₂20mL of methanol was added and the mixture was re-rotary evaporated to remove SOCl as much as possible₂And vacuum drying to obtain the corresponding 3-amino methyl propionate hydrochloride.

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding 3-methyl aminopropionate hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1h, spin-drying by a rotary evaporator, and carrying out column chromatography to obtain an intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) were added in sequence to a reaction flask, DIPEA3mmol and gatifloxacin (1mmol) were added in ice bath, stirring was carried out under controlled temperature, and TLC was carried out until the reaction was completed. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺552.2253。

Example 25

4-aminobutyric acid (50mmol) and 100mL of methanol are added into a reaction bottle, and SOCl is slowly added dropwise under ice bath₂(125mmol) and after dropping, the reaction solution is transferred into a water bath at 60 ℃ and stirred for reflux reaction, and TLC monitoring is carried out until the reaction is finished. Reduced pressure rotary evaporation to remove methanol and most SOCl₂20mL of methanol was added and the mixture was re-rotary evaporated to remove SOCl as much as possible₂Vacuum drying to obtain the corresponding 4-amino butyric acid methyl ester hydrochloride.

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding 4-aminobutyric acid methyl ester hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄DryingAnd (5) carrying out spin-drying on a rotary evaporator for 1 hour, and carrying out column chromatography to obtain an intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) were added in sequence to a reaction flask, DIPEA3mmol and gatifloxacin (1mmol) were added in ice bath, stirring was carried out under controlled temperature, and TLC was carried out until the reaction was completed. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺566.2409。

Example 26

Glycine (50mmol) and 100mL of methanol were added to a reaction flask, and SOCl was slowly added dropwise under ice bath₂(125mmol) and after dropping, the reaction solution is transferred into a water bath at 60 ℃ and stirred for reflux reaction, and TLC monitoring is carried out until the reaction is finished. Reduced pressure rotary evaporation to remove methanol and most SOCl₂20mL of methanol was added and the mixture was re-rotary evaporated to remove SOCl as much as possible₂And vacuum drying to obtain the corresponding glycine methyl ester hydrochloride.

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding glycine methyl ester hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1h, and spin-drying with rotary evaporatorAnd carrying out column chromatography to obtain an intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol), DCM (3mL) and Et were added sequentially to the reaction flask₃N3 mmol, norfloxacin (1mmol), stirring under controlled temperature, and monitoring by TLC until the reaction is finished. Add DCM20mL, move to separatory funnel, saturate Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺482.1853。

Example 27

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding alanine methyl ester hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1 hr, rotary drying with rotary evaporator, and performing column chromatography to obtain the final productAnd (3) an intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) were added in sequence to a reaction flask, DIPEA3mmol and norfloxacin (1mmol) were added in ice bath, stirring was performed under controlled temperature, and TLC was performed until the reaction was completed. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺496.2015。

Example 28

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) were added in sequence to a reaction flask, DIPEA3mmol and norfloxacin (1mmol) were added in ice bath, stirring was performed under controlled temperature, and TLC was performed until the reaction was completed. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺496.2064。

Example 29

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding 4-aminobutyric acid methyl ester hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1h, spin-drying by a rotary evaporator, and carrying out column chromatography to obtain an intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) were added in sequence to a reaction flask, DIPEA3mmol and norfloxacin (1mmol) were added in ice bath, stirring was performed under controlled temperature, and TLC was performed until the reaction was completed. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺510.2148。

Example 30

Adding INA (20mmol), HOBt (24mmol), DCC (24mmol) and DCM (20mL) into a reaction bottle in sequence, stirring for 10min, adding DIPEA under ice bath, adding glycine methyl ester hydrochloride (22mmol) after 0.5-1 h, and monitoring by TLC until the reaction is finished. Freezing in refrigerator for 2 hr, filtering, washing filter cake with DCM, and filtering to obtain filtrate with saturated Na₂CO₃Aqueous (20 mL. times.2), aqueous phase extracted with DCM (30 mL. times.3), combined organic phases, anhydrous Na₂SO₄Drying for 1h, spin-drying by a rotary evaporator, and carrying out column chromatography to obtain an intermediate.

The intermediate compound (1mmol), HBTU/TBTU (1.2mmol), DCM (3mL) and Et were added sequentially to the reaction flask₃N3 mmol, enoxacin (1mmol), stirring at controlled temperature, and monitoring by TLC until the reaction is finished. Add DCM20mL, move to separatory funnel, saturate Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺483.1754。

Example 31

In a reaction flaskAdding the intermediate (15mmol) and CH₃OH-H₂O(V _CH3OH:V _H2O(3: 1) solution (15mL), LiOH · H was added under ice-bath₂O (45mmol), stirred and monitored by TLC to the end of the reaction. Rotating evaporator to remove CH₃Removing OH, adjusting pH to about 3 with 4N HCl solution in ice bath, precipitating a large amount of white solid, vacuum filtering, washing filter cake with filtrate for 2 times, washing with acetone for 2 times, and oven drying at 50 deg.C to obtain intermediate.

And sequentially adding the intermediate compound (1mmol), HBTU/TBTU (1.2mmol) and DCM (3mL) into a reaction bottle, adding DIPEA3mmol and lomefloxacin (1mmol) under ice bath, stirring at controlled temperature, and monitoring by TLC until the reaction is finished. DCM20mL was added, the mixture was transferred to a separatory funnel and saturated with Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying for 1H, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H]⁺528.2053。

Example 32

To a 100mL round bottom flask were added in sequence GAT 5mmol, DCM 15mL, NaHCO₃7.5mmol, stirring, and slowly adding a solution of 2.5mmol of phosgene solid in 5mL of Dichloromethane (DCM) under ice bath. After the dropwise addition, the reaction mixture was transferred to room temperature and stirred, and the progress of the reaction was monitored by TLC. After the reaction, 10mL of ice-cold saturated NaCl solution and 2N HCl solution were added to adjust the pH to 4-5, and the solutions were separated. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, and removing the solvent by rotary evaporation to obtain an intermediate.

To a 100mL round bottom flask was added piperidine 2mmol, CHCl₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of intermediate is added, the mixture is moved to 35 ℃ water bath for reaction, and the progress of the reaction is monitored by TLC. After the reaction is finished, if the product is easily dissolved in water, filtering, and using DCM and CH to make filter cake₃And (5) washing with an OH mixed solution. Adjusting the pH value of the filtrate to 7-8 with HCl-EA solution, and adjusting the pH value to Na₂SO₄Drying; if the product is less water soluble, 15mL DCM, 10mL H are added₂O，And adjusting the pH value of the 2N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying; removing solvent by rotary evaporation to obtain crude product, and performing column chromatography (DCM/CH)₃OH) to obtain pure product, [ M + Na]⁺509.2171。

Example 33

To a 100mL round-bottom flask were added 2mmol of 1-methylpiperazine and CHCl in that order₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of intermediate is added, the mixture is moved to 35 ℃ water bath for reaction, and the progress of the reaction is monitored by TLC. After the reaction is finished, if the product is easily dissolved in water, filtering, and using DCM and CH to make filter cake₃And (5) washing with an OH mixed solution. Adjusting the pH value of the filtrate to 7-8 with HCl-EA solution, and adjusting the pH value to Na₂SO₄Drying; if the product is less water soluble, 15mL DCM, 10mL H are added₂And adjusting the pH value of the O, 2N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying; removing solvent by rotary evaporation to obtain crude product, and performing column chromatography (DCM/CH)₃OH) to obtain pure product, [ M + H ]]⁺502.2460。

Example 34

To a 100mL round bottom flask were added in sequence GAT 5mmol, DCM 15mL, NaHCO₃7.5mmol, stirring, and slowly adding a solution of 2.5mmol of phosgene in DCM 5mL under ice bath. After the dropwise addition, the mixture is transferred to room temperature to be stirred and reacted, TLAnd C, monitoring the reaction process. After the reaction, 10mL of ice-cold saturated NaCl solution and 2N HCl solution were added to adjust the pH to 4-5, and the solutions were separated. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, and removing the solvent by rotary evaporation to obtain an intermediate.

To a 100mL round-bottomed flask were added 2mmol of thiosemicarbazide and CHCl in that order₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of intermediate is added, the mixture is moved to 35 ℃ water bath for reaction, and the progress of the reaction is monitored by TLC. After the reaction is finished, if the product is easily dissolved in water, filtering, and using DCM and CH to make filter cake₃And (5) washing with an OH mixed solution. Adjusting the pH value of the filtrate to 7-8 with HCl-EA solution, and adjusting the pH value to Na₂SO₄Drying; if the product is less water soluble, 15mL DCM, 10mL H are added₂And adjusting the pH value of the O, 2N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying; removing solvent by rotary evaporation to obtain crude product, and performing column chromatography (DCM/CH)₃OH) to obtain pure product, [ M + H ]]⁺493.1673。

Example 35

To a 100mL round bottom flask were added in sequence GAT 5mmol, DCM 15mL, NaHCO₃7.5mmol, stirring, and slowly adding a solution of 2.5mmol of phosgene in DCM 5mL under ice bath. After the dropwise addition, the reaction mixture was transferred to room temperature and stirred, and the progress of the reaction was monitored by TLC. After the reaction, 10mL of ice-cold saturated NaCl solution and 2N HCl solution were added to adjust the pH to 4-5, and the solutions were separated. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, and removing the solvent by rotary evaporation to obtain an intermediate.

To a 100mL round-bottom flask were added 2mmol of 4-benzylpiperazine and CHCl in that order₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of intermediate is added, the mixture is moved to 35 ℃ water bath for reaction, and the progress of the reaction is monitored by TLC. After the reaction is finished, if the product is easily dissolved in water, filtering, and using DCM and CH to make filter cake₃And (5) washing with an OH mixed solution. Adjusting the pH value of the filtrate to 7-8 with HCl-EA solution, and adjusting the pH value to Na₂SO₄Drying; water of the productLess soluble, add 15mL DCM, 10mL H₂And adjusting the pH value of the O, 2N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying; removing solvent by rotary evaporation to obtain crude product, and performing column chromatography (DCM/CH)₃OH) to obtain pure product, [ M + H ]]⁺578.2773。

Example 36

2mmol of 2-aminoethanol and CHCl were added in this order to a 100mL round-bottomed flask₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 1 was added and the mixture was transferred to a 35 ℃ water bath for reaction and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value of the O10 mL and 1N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺477.2144。

Example 37

To a 100mL round-bottom flask was added sequentially

2mmol、CHCl₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 1 was added and the mixture was transferred to a 35 ℃ water bath for reaction and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value of the O10 mL and 1N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺521.2406。

Example 38

To a 100mL round bottom flask was added 2- (methylamino) ethyl groups in sequence2mmol of (E) -1-ol and CHCl₃ 3mL、K₂CO₃2mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 1 was added and the mixture was transferred to a 35 ℃ water bath for reaction and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value of the O10 mL and 1N HCl solution to 7-8, and separating the liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and obtaining an organic phase of anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺491.2300。

Example 39

To a 100mL round-bottom flask was added sequentially

2mmol、、CHCl₃ 3mL、NaHCO₃5mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 2 was added and the mixture was transferred to a water bath at 50 ℃ and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value to 7-8 by using a 2N HCl solution in an amount of 10mL of O, and separating liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and removing anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺535.2563。

Example 40

To a 100mL round-bottom flask were added 2mmol of 1-methylpiperazine, 2mmol of CHCl₃ 3mL、NaHCO₃5mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 2 was added and the mixture was transferred to a water bath at 50 ℃ and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value to 7-8 by using a 2N HCl solution in an amount of 10mL of O, and separating liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and removing anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺530.2773。

EXAMPLE 41

To a 100mL round bottom flask was added piperidine 2mmol, CHCl₃ 3mL、NaHCO₃5mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 2 was added and the mixture was transferred to a water bath at 50 ℃ and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value to 7-8 by using a 2N HCl solution in an amount of 10mL of O, and separating liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and removing anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺543.2613。

Example 42

To a 100mL round-bottom flask were added 2mmol of 4-benzylpiperidine and CHCl in that order₃ 3mL、NaHCO₃5mmol, stirring at room temperature for 30 min. 1mmol of the compound of example 2 was added and the mixture was transferred to a water bath at 50 ℃ and the progress of the reaction was monitored by TLC. After the reaction was complete, 15mL of DCM and H were added₂Adjusting the pH value to 7-8 by using a 2N HCl solution in an amount of 10mL of O, and separating liquid. Washing with 10mL of saturated NaCl solution, separating liquid, and removing anhydrous Na₂SO₄Drying, rotary steaming to obtain crude product, and performing column chromatography to obtain pure product, [ M + H ]]⁺634.3035。

Example 43

Adding 3mmol of 1H-1,2, 4-triazole and 2mL of DMF (dimethyl formamide) in sequence into a 100mL round-bottom flask, stirring at room temperature, adding 4mmol of NaH, stirring at room temperature for 30min, adding 1mmol of the compound of example 2, and moving in a water bath at 80 ℃ for reaction. TLC monitored the progress of the reaction. After the reaction is finished, 20mL of ice-cold saturated NaCl solution is added, and 2N HCl solution is added to adjust the pH value to be 5-6, so that a large amount of solid is separated out. Filtering, washing the filter cake with ice-cold saturated NaCl solution, washing with ice water twice, drying to obtain crude product, performing column chromatography and thin layer chromatography to obtain pure product, [ M + Na ]]⁺521.19192。

Example 44

Adding 3mmol of 1H-1,2, 4-triazole-3-amine and 2mL of DMF (dimethyl formamide) in turn into a 100mL round-bottom flask, stirring at room temperature, adding 4mmol of NaH, stirring at room temperature for 30min, adding 1mmol of the compound of example 2, and moving in a water bath at 80 ℃ for reaction. TLC monitored the progress of the reaction. After the reaction is finished, 20mL of ice-cold saturated NaCl solution is added, and 2N HCl solution is added to adjust the pH value to be 5-6, so that a large amount of solid is separated out. Filtering, washing the filter cake with ice-cold saturated NaCl solution, washing with ice water twice, drying to obtain crude product, performing column chromatography and thin layer chromatography to obtain pure product, [ M + Na ]]⁺536.20282。

Example 45

Adding 3mmol of 1H-tetrazole-5-amine and 2mL of DMF (dimethyl formamide) in sequence into a 100mL round-bottom flask, stirring at room temperature, adding 4mmol of NaH, stirring at room temperature for 30min, adding 1mmol of the compound of example 2, and moving in a water bath at 80 ℃ for reaction. TLC monitored the progress of the reaction. After the reaction is finished, 20mL of ice-cold saturated NaCl solution is added, and 2N HCl solution is added to adjust the pH value to be 5-6, so that a large amount of solid is separated out. Suction filtering, washing the filter cake with ice-cold saturated NaCl solution, washing with ice water twice, drying to obtain crude product, column chromatography and thin-layer chromatography to obtain pure product, [ M + H ]]⁺515.2135。

Example 46

To a 100mL round bottom flask was added 5- (difluoromethoxy) -1H-benzo [ d ] in sequence]3mmol of imidazole-2-thiol and 2mL of DMF are stirred at room temperature, NaH 4mmol is added, stirring is carried out at room temperature for 30min, 1mmol of the compound of example 2 is added, and the mixture is transferred to a water bath at 80 ℃ for reaction. TLC monitored the progress of the reaction. After the reaction is finished, 20mL of ice-cold saturated NaCl solution is added, and 2N HCl solution is added to adjust the pH value to be 5-6, so that a large amount of solid is separated out. Suction filtering, washing the filter cake with ice-cold saturated NaCl solution, washing with ice water twice, drying to obtain crude product, column chromatography and thin-layer chromatography to obtain pure product, [ M + H ]]⁺646.1942。

Example 47

To a 100mL round bottom flask was added sequentially benzo [ d ]]3mmol of thiazole-2-thiol and 2mL of DMF are stirred at room temperature, NaH 4mmol is added, stirring is carried out at room temperature for 30min, 1mmol of the compound of example 2 is added, and the mixture is transferred to a water bath at 80 ℃ for reaction. TLC monitored the progress of the reaction. After the reaction is finished, 20mL of ice-cold saturated NaCl solution is added, and 2N HCl solution is added to adjust the pH value to be 5-6, so that a large amount of solid is separated out. Suction filtering, washing the filter cake with ice-cold saturated NaCl solution, washing with ice water twice, drying to obtain crude product, column chromatography and thin-layer chromatography to obtain pure product, [ M + H ]]⁺597.1636。

Example 48

5-methyl-1, 3, 4-thiadiazole-2-thiol 3mmol and DMF 2mL are sequentially added into a 100mL round-bottom flask, stirred at room temperature, added with NaH 4mmol, stirred at room temperature for 30min, added with the compound 1mmol of the example 2, and transferred to a water bath at 80 ℃ for reaction. TLC monitored the progress of the reaction. After the reaction is finished, 20mL of ice-cold saturated NaCl solution is added, and 2N HCl solution is added to adjust the pH value to be 5-6, so that a large amount of solid is separated out. Filtering, washing the filter cake with ice-cold saturated NaCl solution, washing with ice water twice, drying to obtain crude product, performing column chromatography and thin layer chromatography to obtain pure product, [ M + Na ]]⁺584.1408。

Example 49

A100 mL round-bottom flask was charged with 0.204g (3mmol) of 1H-imidazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 3. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + H [ ]]⁺512.2304。

Example 50

A100 mL round-bottom flask was charged with 3mmol of 2-methyl-1H-imidazole, 10mL of toluene, and example 3Compound 0.484g (1 mmol). Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + H [ ]]⁺526.2460。

Example 51

A100 mL round-bottom flask was charged with 3mmol of 1H-pyrazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 3. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺534.2123。

Example 52

A100 mL round-bottomed flask was charged with 3mmol of 3, 5-dimethyl-1H-pyrazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 3. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + H [ ]]⁺540.2617。

Example 53

A100 mL round-bottom flask was charged with 1H-benzo [ d ]]Imidazole 3mmol, toluene 10mL, example 5 compound 0.484g (1 mmol). Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + H [ ]]⁺562.2460。

Example 54

A100 mL round-bottom flask was charged with 3mmol of 1H-1,2, 3-triazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. Reaction ofAfter completion, the solvent toluene is removed by rotary evaporation, and the mixture is subjected to solid column chromatography and thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]+535.2076。

Example 55

A100 mL round-bottom flask was charged with 3mmol of 1H-tetrazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺536.2028。

Example 56

A100 mL round-bottom flask was charged with 3mmol of 5-methyl-1H-tetrazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺550.2185。

Example 57

A100 mL round-bottom flask was charged with 3mmol of 5-amino-1H-tetrazole, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺551.2137。

Example 58

A100 mL round bottom flask was charged with 5- (difluoromethoxy) -1H-benzo [ d ]]3mmol of imidazole-2-thiol, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, removing the solvent toluene by rotary evaporation, and carrying out solid column chromatographyAnd thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺682.1918。

Example 59

A100 mL round-bottomed flask was charged with 3mmol of 1-methyl-1H-tetrazole-5-thiol, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺582.1905。

Example 60

To a 100mL round bottom flask was added benzo [ d ]]3mmol of thiazole-2-thiol, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + Na%]⁺633.1612。

Example 61

A100 mL round-bottomed flask was charged with 3mmol of 5-amino-1, 3, 4-thiadiazole-2-thiol, 10mL of toluene, and 0.484g (1mmol) of the compound of example 5. Controlling the temperature to react, and detecting the reaction progress by TLC. After the reaction is finished, the solvent toluene is removed by rotary evaporation, and the solid column chromatography and the thin layer chromatography (DCM: CH)₃OH 30:1) to obtain pure product, [ M + H [ ]]⁺577.1698。

Example 62

Adding 1mmol clinafloxacin and 2mL DCM into a 100mL round-bottom flask, cooling in an ice bath, magnetically stirring, adding 3mmol NaHCO₃After 20min, a solution of 2.5mmol of acetyl chloride in DCM (2mL) was added dropwise from a constant pressure addition funnel at a rate of about 1d/2s, and the ice bath was repeatedThe reaction was continued and monitored by TLC tracking to the end of the reaction. Stopping stirring, adding H₂O15mL and DCM20mL, pH 3-4 adjusted with 1N HCl solution under stirring, liquid separation, aqueous phase extraction with DCM (15mL × 1), organic phase combination, saturated NaCl solution washing (15mL × 1), organic phase collection, anhydrous Na₂SO₄And (5) drying. Rotary drying with rotary evaporator to obtain crude product, performing column chromatography to obtain pure product, and vacuum drying to obtain product, [ M + H ]]⁺409.1119。

Example 63

Sequentially adding CF into the reaction bottle₃COOH (1.2mmol), HBTU/TBTU (1.44mmol), DCM (3mL), Et was added₃N3 mmol and clinafloxacin (1mmol), stirring at controlled temperature, and monitoring by TLC until the reaction is finished. Add DCM20mL, move to separatory funnel, saturate Na₂CO₃Washing with an aqueous solution (20 mL. times.1), washing with a 0.5N HCl solution (20 mL. times.1), washing with a saturated saline solution (20 mL. times.1), and anhydrous Na₂SO₄Drying, rotary drying with rotary evaporator, and performing column chromatography to obtain pure product, [ M + H ]]⁺462.0845。

Determination of bacteriostatic activity of citrus canker pathogen

The determination method comprises the following steps: 1mg of the sample was dissolved in 50. mu.L of DMSO, and the volume was adjusted to 550. mu.L with ultrapure water as a sample stock solution (1.82 mg/mL). mu.L of the mother liquor was taken as a sample solution a (0.0182mg/mL) in 1mL of ultrapure water (0.02% Tween), and then sample solutions b (0.0091mg/mL), c (0.00455mg/mL), d (0.002275mg/mL), e (0.0011375mg/mL), and f (0.00056875mg/mL) were prepared in this order by the double dilution method.

Washing the ulcer germs cultured for 3 days on the PDA culture medium with 5mL of LB liquid culture medium, adding into 195mL of LB liquid culture medium, and shaking and mixing for later use. 450 mu L of the bacterial liquid of the citrus canker pathogen and 50 mu L of the sample solutions with different concentrations (a to F) are respectively added into each 2mL centrifuge tube, so that the final concentrations of the samples in each mixed bacterial liquid are respectively A (0.00182mg/mL), B (0.00091mg/mL), C (0.000455mg/mL), D (0.0002275mg/mL), E (0.00011375mg/mL), F (0.000056875mg/mL), 28 ℃, 200 r.min^-1After constant temperature shaking culture for 14hDetermination of OD₆₀₀The OD value of each mixed bacterial solution was calculated and the inhibition ratio was calculated (inhibition ratio%_{Blank space}-OD_{Sample (I)})/OD_{Blank space}X 100%). Each treatment was replicated three times.

TABLE 1 inhibitory Effect of Compounds on Citrus canker

The PBT data processing system is used for carrying out statistical analysis on the test results of all the medicaments to obtain the inhibition rate, the toxicity regression equation and the EC of all the medicaments on the citrus canker pathogenic bacteria₅₀、EC₉₀And a correlation coefficient (r).

TABLE 2 inhibitory Effect of Compounds on Citrus canker

Determination of bacteriostatic activity of citrus brown spot pathogen

The determination method comprises the following steps: washing conidia of the brown spot germs cultured for 7 days on the PDA culture medium by using 0.05 percent Tween 80, and filtering four layers of sterile lens wiping paper for later use; a1 mg sample of the compound was weighed out and dissolved in 50. mu.L DMSO, and the volume was adjusted to 550. mu.L with ultrapure water as a sample stock solution (1.82 mg/mL). mu.L of the mother solution was taken as a sample solution a (0.00364mg/mL) in 1mL of PDA (0.02% Tween), and a sample solution b (0.00182mg/mL) was prepared in order by the double dilution method. After adding 0.5mL of the sample solution and 1.5mL of LPDA medium to each well of the 48-well plate to give final concentrations of A (0.000910mg/mL) and B (0.000455mg/mL), respectively, 2. mu.L of the conidia suspension was inoculated and cultured with light at 28 ℃ for 3 days to measure the colony diameter. Using the colony inoculated on the PDA culture medium without sample solution as a blank control, calculating the inhibition ratio (the inhibition ratio is%_{Blank space}Diameter of colonies_{Sample (I)}) Colony diameter_{Blank space}×100％)。

TABLE 3 results of the anti-Phosphaerella citrina activity of the compounds

From the above table, it can be seen that even at a relatively low concentration (0.00182mg/mL), the compounds of the examples of the present invention still exhibit excellent bacteriostatic activity against citrus canker pathogens, and at the same time, the compounds of the present invention also have a certain bacteriostatic activity against brown spot pathogen, and play a certain role in preventing and treating citrus canker pathogens.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof:

wherein X is selected from: an ethyl group; a C3 cycloalkyl group;

z is selected from: n or C-R₇；R₇Is selected from H; methoxy, fluoro or chloro;

R₁and R₂Each independently selected from: h; a methyl group;

Q₁is selected from

R₃Or R₄Each is independently selected from H; methyl, hydroxyethyl; the R is₃And R₄Not H at the same time;

or R₃，R₄Taken together with N to form a 5-8 membered heterocyclic ring, said 5-8 membered heterocyclic ring selected from:

said heterocycle may be optionally substituted with one methyl or benzyl;

R₅is selected from H; methyl or amino;

R₆is 1, independently selected from H, difluoromethoxy;

B₁selected from S or NH;

the het is selected from

R₈Selected from H, methyl;

n is an integer of 1 to 3;

k is 0 or 1.

2. A compound of the structure:

3. a pharmaceutical composition comprising a compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical formulation comprising a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and/or adjuvant.

5. A combination comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof and other active ingredients.

6. Use of a compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for controlling citrus-related diseases.

7. Use according to claim 6, wherein the citrus-related disease is citrus canker or citrus brown spot.

8. A method of treating citrus-related diseases comprising applying to a citrus tree and/or fruit an agent comprising a compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.