CN109096278B - Fluoroquinolone-azole hybrid derivative, preparation method and application thereof - Google Patents

Abstract

The invention discloses a fluoroquinolone-azole derivative shown as a formula I, wherein the derivatives connect pharmacophores of fluoroquinolone and azole medicaments with each other through a proper connecting structure, and biological activity experiments prove that the compounds have dual activities of inhibiting bacteria and fungi and have good application prospects.

Description

Fluoroquinolone-azole hybrid derivative, preparation method and application thereof

Technical Field

The invention relates to a compound for treating bacterial and/or fungal infection, in particular to ten series of fluoroquinolone-azole hybrid derivatives.

Background

With the wide application of broad-spectrum antibiotics, hormones and immunosuppressants, the spread of AIDS, and the popularization and application of organ transplantation and interventional technology, the incidence rate of microbial infections such as fungi and bacteria is on the rise, reports on drug-resistant strains are gradually increased, and the treatment of fungal and bacterial infections faces a serious challenge.

The azole antifungal medicine is the main clinical fungus infection treating medicine at present, and the medicine has the advantages of stable metabolism, oral taking, injection, curative effect on both superficial fungi and deep fungi, etc. The aromatic ethyl cyclic ketal compounds such as ketoconazole and the like are the first choice drugs for treating superficial fungal infection, and the third-generation antifungal drugs represented by fluconazole and itraconazole are the first choice drugs for treating deep fungal infection. However, the azole antifungal drug can act with a plurality of cytochrome P450 proteins of human bodies, so that the azole antifungal drug has serious adverse reactions. The fluconazole has relatively narrow antibacterial spectrum, low activity on fungi such as aspergillus, and the like, is easy to generate drug resistance, and has the problem of instable oral bioavailability and the like.

The fluoroquinolone medicines are continuously marketed in the 80 th of the 20 th century, have the advantages of wide antibacterial spectrum, high antibacterial activity, strong tissue penetrability, high bioavailability, long half-life period, high blood concentration, wide tissue distribution and the like, can be used by a single medicine, show good antibacterial activity on gram-positive bacteria and gram-negative bacteria, and are widely used for treating infectious diseases of a urogenital system, a respiratory system and a digestive system. However, as the clinical unreasonable application of antibacterial drugs is increased, the drug resistance problem is increasingly highlighted.

In response to the above-mentioned drawbacks of antifungal and bacterial drugs, scientists have been continuously searching for new compounds with antifungal and bacterial activity. For example, CN108368053A discloses a class of benzazepine compounds directed against gram-negative bacteria UDP-3-O- (R-3-hydroxymyristoyl) -N-acetylglucosamine deacetylase (LpxC) targets, thereby circumventing the currently common bacterial resistance mechanism. CN108368102A discloses a diazine compound, which is designed aiming at a beta euglucan synthase (echinocandin) which is a less antifungal target to be researched, and can be used as a substitute drug when polyene and azole antifungal drugs are resistant. Although new achievements are generated in the research and development of antifungal and bacterial drugs, the current research and development direction is based on single target point research, and reports of drugs capable of acting on both fungal and bacterial target points are not found yet. With the development of proteomics, genomics and other disciplines, the understanding of the in vivo action mechanism of drugs has been gradually deepened from the cellular level to the molecular level, and for some diseases, even if the target selection is very accurate, the activity and selectivity of the designed compound are strong, but finally the drug effect is not satisfactory because the drugs are influenced by various factors in vivo. Therefore, the research of multi-target drugs for disease causes is becoming a hot spot. The most common design method of multi-target drugs is called a backbone combination method, which uses two molecules targeting different targets as starting compounds, and after the main structure including pharmacophores is reserved, backbone integration is performed on the two. However, a number of practices have shown that, although the backbone integration method is theoretically feasible, it is unpredictable whether the affinity for the original target can be retained due to the fact that the structure and physicochemical properties of the compound after integration may be very different from those before integration.

Disclosure of Invention

The invention carries out heterozygous reaction on the pharmacophores of the azole antifungal drugs and the fluoroquinolone drugs by a linker to obtain a fluoroquinolone-azole heterozygous compound with antifungal and bacterial activities, and the specific technical scheme of the invention is as follows:

a compound of formula I, racemates, stereoisomers, tautomers, nitroxides or pharmaceutically acceptable salts thereof:

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-R₁；R₁Is selected from H; C1-C6 alkoxy or halogen;

q is selected from CH or N;

r' is selected from halogen;

y is selected from:

wherein n is selected from 1,2 or 3; r₂Selected from H, halogen or C1-C6 alkyl; r' is selected from hydrogen or C1-C6 alkyl; one side of the mark indicates the connection end with L;

l represents a linking structure.

In the above technical scheme, L is preferably the following group:

wherein R is₃、R₄Independently selected from H, halogen, hydroxyl, amino or C1-C6 alkyl; m is selected from 0, 1,2 or 3; one side of the label is used to indicate the connection with Y.

Preferably, in any of the above embodiments, Q is selected from N; r' is selected from fluorine;

l is selected from:

preferably, in any of the above embodiments, X is selected from: methyl, ethyl, propyl, cyclopropyl, substituted or unsubstituted phenyl, the substituents on said phenyl being one or more independently selected from: halogen; an amino group; hydroxy or C1-C3 alkyl;

z is selected from: n or C-R₁；R₁Is selected from H; methoxy, ethoxy or halogen.

The invention also provides compounds shown as TM1-TM10 and partial intermediates in the specific embodiment, racemates, stereoisomers, tautomers, nitrogen oxides or pharmaceutically acceptable salts thereof.

The invention also claims a pharmaceutical composition comprising any of the above compounds, racemates, stereoisomers, tautomers, nitric oxides or pharmaceutically acceptable salts thereof.

Preferably, any compound, racemate, stereoisomer, tautomer, nitrogen oxide or pharmaceutically acceptable salt thereof can be prepared into any pharmaceutically acceptable dosage form, namely the pharmaceutical composition of the invention can also comprise pharmaceutically acceptable carriers and/or auxiliary agents.

Preferably, one or more other active ingredients and any compound, racemate, stereoisomer, tautomer, nitrogen oxide or pharmaceutically acceptable salt thereof can be prepared into a compound medicament according to the requirements.

The invention also claims the application of any compound, racemate, stereoisomer, tautomer, nitric oxide or pharmaceutically acceptable salt thereof in preparing medicines for treating diseases caused by fungal infection and/or bacterial infection.

Fungal infections described herein include, but are not limited to, Aspergillus fumigatus, Candida tropicalis, Candida albicans, and Candida parapsilosis infections; particular preference is given to Aspergillus fumigatus infections. The bacterial infections described herein include, but are not limited to, infections with Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Salmonella, Acinetobacter, drug-resistant Citrobacter, drug-resistant Staphylococcus aureus, and drug-resistant Klebsiella pneumoniae.

Terms definition and interpretation:

unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. 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.

When "the present compound" or "the present compound" is used herein, unless otherwise specified, at least the compound represented by formula I, its racemate, stereoisomer, tautomer, nitrogen oxide, or a pharmaceutically acceptable salt thereof is intended to be encompassed.

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.

The term "C6-C10 aryl" is understood to mean preferably an aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon having 6 to 10 carbon atoms, in particular a ring having 6 carbon atoms ("C6 aryl"), for example phenyl; or biphenyl, or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl.

The pharmaceutically acceptable salts of the compounds of the present invention may be acidic salts or basic salts. Such as inorganic acid salts, organic base salts, or inorganic base salts.

The compounds of the invention are preferably obtained in unmodified form or by means of conventional techniques with the adjuvants conventionally used in formulations in the art, for example liquid formulations (e.g. sprays, emulsions, suspensions, solutions, emulsifiable concentrates, solution concentrates), semisolid formulations (e.g. creams, ointments, patches, gels, liposomal formulations) and solid formulations (e.g. powders, granules, tablets, etc.).

The term pharmaceutically acceptable carrier and/or adjuvant includes, but is not limited to: sugars such as lactose, sucrose, mannitol, and sorbitol; starches, such as corn starch, tapioca starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; calcium phosphates such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic, cationic and anionic surfactants; polyethylene glycol; fatty alcohols; and grain hydrolyzed solids and other nontoxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, and the like, which are commonly used in pharmaceutical formulations.

Detailed Description

EXAMPLE I Synthesis of TM1 series of Compounds

The general route is as follows:

to a 250mL round bottom flask was added 2',4' -difluoro-2- [1- (1H-1,2, 4-triazolyl)]Acetophenone (1.115g,5mmol) and 10mL methanol were stirred at room temperature and KBH added in portions under ice-bath conditions₄(0.405g,7.5mmol), after the addition, the ice bath was removed, the mixture was stirred at 20-40 ℃ and the progress of the reaction was checked by TLC. After the reaction is finished, removing the solvent by rotary evaporation, adding 15mL of water, adjusting the pH value to 1-3 by concentrated hydrochloric acid, stirring for 1h at room temperature, and stirring with 10% K₂CO₃The pH was adjusted to 8 to 9, and the mixture was left to stand, filtered under suction, and washed with water (10 mL. times.3) to give IM1 as a white solid.

A100 mL round bottom flask was charged with IM1(0.672g,3mmol), succinic anhydride (0.603g,6mmol), acetone 6mL, Et₃N0.15 mL, with stirring, was warmed to reflux and the progress of the reaction was monitored by TLC. After the reaction, 10mL of water was added to the reaction solution, followed by crystallization with stirring, filtration to collect the solid, and recrystallization with ethanol to obtain a white solid IM 1-2.

Adding IM1-2(1.0mmol), HBTU (1.2mmol), floxacin (1.1mmol) and dry DCM 10mL in turn into a 100mL round-bottom flask, stirring at room temperature, adding DIPEA or Et under ice bath condition₃N (2.0mmol), the ice bath was removed and the reaction continued at room temperature, and the progress of the reaction was monitored by TLC. After the reaction was complete, the stirring was stopped. Suction filtration, filter cake washing with DCM (10 mL. times.3); the filtrate was collected, in turn, with saturated NaHCO₃The resulting mixture was washed with a solution (15 mL. times.2), a 1.5N diluted hydrochloric acid aqueous solution (15 mL. times.2), and a saturated NaCl solution (15 mL. times.2), and then allowed to stand for separation, and then dried over anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure, and purifying by column chromatography (DCM/CH)₃OH ═ 80/1), the eluate was collected and evaporated to dryness under reduced pressure; drying under vacuum gave TM1, the results of the relevant experiments are shown in Table 1.

TABLE 1 Synthesis results and characterization of TM1 series of compounds

EXAMPLE II Synthesis of TM2 series of Compounds

The general route is as follows:

NaH (0.813g,20mmol) and THF (15 mL) were added to a 250mL three-necked flask and stirred at room temperature; a solution of IM1(2.245g,10mmol) dissolved in 10mL of THF is slowly dropped by a constant pressure dropping funnel, and the solution is transferred into an oil bath at 80 ℃ for reaction for 30min after the addition; a solution of methyl 2-bromoacetate (1.735g,15mmol) in 10mL of THF was slowly added dropwise. TLC monitored the progress of the reaction. The stirring was stopped, the reaction mixture was concentrated under reduced pressure, poured into cold water (30mL) and extracted with dichloromethane (2X 20 mL). And combining the organic extracts, washing with saturated saline (2X 20mL), drying with anhydrous sodium sulfate, evaporating to dryness under reduced pressure, purifying by column chromatography (PE/EA is 1/2), collecting eluent, evaporating to dryness under reduced pressure, and drying in vacuum to obtain the pure product IM 2-2.

A100 mL round-bottom flask was charged with a mixed solvent of IM2-2(1.930g,6.5mmol), methanol 9mL and water 5mL in this order, stirred magnetically, and charged with LiOH. H₂O (0.819g,19.5mmol), stirring was continued and the progress of the reaction was monitored by TLC. And after the reaction is finished, evaporating methanol under reduced pressure, cooling, adjusting the pH value to 2-3, separating out a light yellow solid, performing suction filtration, washing a filter cake with water (2X 10mL), and collecting the filter cake to obtain a light yellow solid IM 2-3.

Adding IM2-3(1.0mmol), HBTU (1.2mmol), floxacin (1.0mmol) and dried DCM15mL in turn into a 100mL round-bottom flask, stirring, cooling in ice bath, adding DIPEA or Et₃N (2.0mmol), the ice bath was removed and the reaction continued at room temperature. TLC monitored the progress of the reaction and when the product point was essentially unchanged, the stirring was stopped. Suction filtration, filter cake washing with DCM (10 mL. times.3); the filtrate was collected, in turn, with saturated NaHCO₃The solution (15 mL. times.2), 1.5N diluted hydrochloric acid aqueous solution (15 mL. times.2), saturated NaCl solution (15 mL. times.2) were washed with anhydrous Na₂SO₄Drying, drying by reduced pressure,column chromatography purification (DCM/CH)₃OH-120/1), collecting the eluate, evaporating to dryness under reduced pressure, and vacuum drying to obtain pure TM2, the results of the relevant experiments are shown in table 2.

TABLE 2 Synthesis results and characterization of TM2 series of compounds

EXAMPLE III Synthesis of TM3 series of Compounds

The general route is as follows:

a100 mL round bottom flask was charged with Sasa (2mmol), DCM 5mL and base (NaHCO)₃Or Et₃N, 3mmol), stirring in ice bath, dropwise adding 5mL of 1mmol of phosgene (BTC) in DCM, stirring at room temperature, and monitoring the progress of the reaction by TLC. After the reaction, 15mL of saturated saline was added, the pH was adjusted to 4-5, and the mixture was separated and washed with saturated saline (10 mL. times.3). Separating, drying with anhydrous sodium sulfate, and removing the solvent by rotary evaporation to obtain intermediates IM 3-1-IM 3-8.

A100 mL round-bottom flask was charged with IM1(1.2mmol), DCM 10mL, Et₃N (1.5mmol) and DMAP (0.05mmol) were stirred in an ice bath, and then IM3-X (X ═ 1-8) (1mmol) was added thereto, and the mixture was warmed to room temperature, stirred further, and the progress of the reaction was monitored by TLC. After completion of the reaction, 15mL of DCM was added, and the mixture was washed with an acidic saturated saline solution (25 mL. times.3) and anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure, and purifying by column chromatography (DCM/CH)₃OH — 150/1), and the eluate was collected and evaporated to dryness under reduced pressure to give pure TM 3. By using the method, 8 target compounds of a TM3 series are synthesized, and the related experimental results are shown in Table 3.

TABLE 3 Synthesis results and characterization of TM3 series of compounds

EXAMPLE IV Synthesis of TM4 series of Compounds

The general route is as follows:

a100 mL round bottom flask was charged with IM1(0.5mmol), base (1mmol) and 6mL DCM in that order, stirred for 20min, then chloroacetyl chloride (0.75mmol) was added dropwise as a solution, and the reaction was continued at-5 deg.C and monitored by TLC for completion. Removing solvent by rotary evaporation under reduced pressure, adding 15mL of water, extracting with EA (15 mL. times.3), collecting organic phase, washing with saturated saline (15 mL. times.3), and removing anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure, purifying by column chromatography (PE/EA is 1/1), collecting eluate, evaporating to dryness under reduced pressure, standing at room temperature for one day, and vacuum drying to obtain intermediate IM 4-2.

Adding Sasa (1mmol) and Et in turn into a 100mL round-bottom flask₃N (1.5mmol) and 5mL DMF, stirring for 20min, adding IM4-2(1.2mmol), moving to 60 deg.C oil bath to stir the reaction, and monitoring by TLC until the reaction is complete. 30mL of ice water and 15mL of DCM were added, the pH was adjusted to about 3, the mixture was separated, washed with saturated brine (20 mL. times.5), and anhydrous Na was added₂SO₄Drying, evaporating to dryness under reduced pressure, and purifying by column chromatography (DCM/CH)₃OH-150/1), collecting the eluate, evaporating to dryness under reduced pressure, and vacuum drying to obtain pure TM4, the results of which are shown in table 4.

TABLE 4 Synthesis results and characterization of TM4 series of compounds

EXAMPLE five Synthesis of TM5 series of Compounds

The general route is as follows:

a100 mL round-bottom flask was charged with IM1(0.5mmol), 5mL DCM, and 1.0mmol base, the flask was moved to a low temperature reactor at 0 deg.C for reaction, 3-chloropropionyl chloride (1.0mmol) was added dropwise, DMAP (0.1mmol) was added, and monitored by TLC. After completion of the reaction, the solvent was removed by rotary evaporation, 10mL of water was added, EA extraction (5 mL. times.3) was performed, and the mixture was washed with saturated brine (10 mL. times.3) and anhydrous Na₂SO₄Drying, reduced pressure evaporation to dryness to obtain IM5-2, which is directly used in the next reaction.

IM5-2(0.6mmol) and 4mL DMF were added to a 100mL reaction flask, stirred to dissolve, then added with fleroxacin (0.5mmol) and base (1.5mmol), stirred at 65-100 deg.C for reaction, and the progress of the reaction was monitored by TLC. After the reaction, 20mL of water was added, 30mL of DCM was added to adjust the pH to 2-3, the layers were separated and the organic phase was washed 4 times (15mL of water +2mL of 1.5N HCl), anhydrous Na₂SO₄Drying, rotary steaming, and purifying by column chromatography (DCM/CH)₃OH ═ 130/1), the eluate was collected, evaporated to dryness under reduced pressure and dried in vacuo to give TM5, and the results of the relevant experiments are shown in table 5.

TABLE 5 TM5 Synthesis results and characterization

EXAMPLE six Synthesis of TM6 series of Compounds

The general route is as follows:

a100 mL round bottom flask was charged with IM1(0.113g,0.5mmol), DCM 4mL, and base (1.0mmol), the flask was transferred to a low temperature reactor and reacted at 0 deg.C, 4-chlorobutyryl chloride (0.141g,1.0mmol) was added dropwise, stirring, and monitored by TLC. After completion of the reaction, the solvent was removed by rotary evaporation, 10mL of water was added, EA extraction (5 mL. times.3) was performed, and the mixture was washed with saturated brine (10 mL. times.3) and anhydrous Na₂SO₄Drying, and evaporating to dryness under reduced pressure to obtain IM 6-2.

To a 100mL round bottom flask were added sequentially IM6-2(1.2mmol), floxacin (1mmol) and 4mL DMF, Et₃N (1.2mmol), the reaction was stirred at 70-95 ℃ and monitored by TLC until the reaction was complete. 30mL of ice water and 25mL of DCM were added, the pH was adjusted to about 3, the mixture was separated, washed with saturated brine (20 mL. times.5), and anhydrous Na was added₂SO₄Drying, evaporating to dryness under reduced pressure, and purifying by column chromatography (DCM/CH)₃OH-150/1), the eluate was collected and evaporated to dryness under reduced pressure to obtain TM6, and the results of the relevant experiments are shown in table 6.

TABLE 6 TM6 Synthesis results and characterization

EXAMPLE seven Synthesis of TM7 series of Compounds

The general route is as follows:

ph is added into a 100mL round-bottom flask in turn₃P (1mmol), 3mL of toluene and 2-bromoacetic acid methyl ester (1.2mmol) are stirred for 10min at room temperature, transferred into an oil bath at 60-80 ℃ for heating reaction, the temperature is raised to 120 ℃ after 6h for reflux, the reflux is carried out for 3h, the room temperature is cooled, white solid is separated out, the filtration is carried out, and a filter cake is washed by ether (2mL multiplied by 5) to obtain white solid IM 7-1.

1.5mL of DMF and NaH (0.03g,0.75mmol) are sequentially added to a 100mL three-necked flask, stirred at room temperature for 0.5H, then a solution of IM7-1(0.311g,0.75mmol) in DMF (3mL) is added dropwise under the protection of nitrogen, stirred at room temperature for 2H, and 1mL of 2',4' -difluoro-2- [1- (1H-1,2, 4-triazolyl) is added dropwise]Acetophenone (0.113g,0.5mmol) in DMF was stirred at room temperature and monitored by TLC. After the reaction is finished, water is added and stirred, a part of triphenylphosphine oxide is removed by suction filtration, filtrate is extracted by EA until the water phase has no product, the organic phases are combined, washed by water (20mL multiplied by 2), washed by saturated salt solution (20mL multiplied by 3), and anhydrous Na₂SO₄Drying, decompressing and evaporating to dryness, and purifying by column chromatography to obtain a pure product IM 7-2.

A100 mL round-bottom flask was charged with a mixed solvent of IM7-2(2.1g,7.5mmol), 10mL methanol and 5mL water in this order, and LiOH. H was added₂O (1.26g,30mmol), stirred in an ice bath and the progress of the reaction monitored by TLC. After the reaction is finished, removing the methanol by reduced pressure distillation, adjusting the pH value to 2-3, extracting by EA (10mL multiplied by 3), combining organic phases and anhydrous Na₂SO₄Drying, and evaporating to dryness under reduced pressure to obtain IM 7-3.

To a 100mL round bottom flask were added sequentially IM7-3(0.294g,1.1mmol), HBTU (0.455g,1.2mmol), Floxacin (1.0mmol) and 8mL dry DCM, stirred at room temperature, and DIPEA or Et was added₃N (2.0mmol), the reaction was continued at room temperature, the progress of the reaction was monitored by TLC, and stirring was stopped when the product spot was essentially unchanged. Suction filtration was performed, the filter cake was washed with DCM (5 mL. times.3), and the filtrate was successively washed with saturated NaHCO₃The solution (10 mL. times.2), 1.0N HCl aqueous solution (15 mL. times.2), saturated NaCl solution (15 mL. times.2) were washed with anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure, purifying by column chromatography, and evaporating to dryness under reduced pressure to obtain pure TM 7. By using the method, 8 target compounds of TM7 series are synthesized, and the related experimental results are shown in Table 7.

TABLE 7 results of synthesis and characterization of TM7

EXAMPLE eight Synthesis of TM8 series of Compounds

The general route is as follows:

to a 100mL round-bottomed flask were added, in order, IM1(10mmol), acetonitrile 15mL, 4, 6-dichloropyrimidine (12mmol), and K₂CO₃(23mmol), the reaction was stirred in a 90 ℃ oil bath and monitored by TLC. After the reaction, the solvent was removed by rotary evaporation, 30mL of DCM and 15mL of water were added, the mixture was magnetically stirred, and the mixture was allowed to stand, separated, and the aqueous phase was back-extracted with DCM (10 mL. times.2), combined organic phases, washed with saturated brine (30 mL. times.3), and anhydrous Na₂SO₄Drying, reduced pressure evaporation, column chromatography purification (PE/EA: 6/1), collecting eluent, reduced pressure evaporation to dryness, IM 8-2.

To a 100mL round-bottom flask were added sequentially IM8-2(1.1mmol), floxacin (1mmol), DMF 3.0mL, K₂CO₃(1.5mmol), stirring magnetically in an oil bath at 90 ℃ and monitoring by TLC. After completion of the reaction, 30mL of ice water and DCM15mL were added to adjust the pH to about 4, followed by liquid separation, washing with saturated brine (20 mL. times.5), and anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure, and purifying by column chromatography (DCM/CH)₃OH ═ 120/1), the eluate was collected, evaporated to dryness under reduced pressure and dried in vacuo to give TM8, and the results of the relevant experiments are shown in table 8.

TABLE 8 Synthesis results and characterization of TM8

EXAMPLE nine Synthesis of TM9 and TM10 series of Compounds

The general route is as follows:

adding IM1(2.25g,10mmol), 20mL ethanol and 2.5mL water in turn into a 100mL round-bottom flask, stirring for 20min at 9-11 ℃, adding NaOH (1.20g,30mmol) and TBAB (0.31g,0.1mmol), moving a reaction bottle into a 30 ℃ water bath for heating, dropwise adding (S) -epichlorohydrin or (R) -epichlorohydrin (9.21g,100mmol), raising the reaction temperature to 50 ℃ for reaction, and monitoring the reaction process by TLC. After completion of the reaction, ethanol was distilled off under reduced pressure, 15mL of water was added, EA (25 mL. times.2) was extracted, and the mixture was washed with saturated brine (20 mL. times.3) and anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure to obtain yellow oily substance, and performing column chromatography to obtain yellowish oily substance IM9-2 (from S-epichlorohydrin) or IM10-2 (from R-epichlorohydrin).

1mmol of IM9-2 (or IM10-2), 3mL of absolute ethyl alcohol, 1.1mmol of floxacin and NaHCO are added into a 100mL round-bottom flask in sequence₃(1.5mmol), the reaction was stirred in an oil bath at 85 ℃ and monitored by TLC until the reaction was complete. Removing the solvent by rotary evaporation, adding 15mL of water and 25mL of DCM, stirring, adjusting the pH to 5-6, standing, separating, washing with saturated saline (20mL multiplied by 3), and anhydrous Na₂SO₄Drying, evaporating to dryness under reduced pressure to obtain crude product, performing column chromatography to obtain pure TM9 or TM10 series target compounds, vacuum drying, and storing at low temperature, wherein the related experimental results are shown in Table 9.

TABLE 9 Synthesis and characterization of TM9 and TM10

EXAMPLE ten test of inhibitory Activity against fungi, bacteria and drug-resistant bacteria

(I), universal sample, culture medium and reagent

1. Fluoroquinolone active compound, control and derivative samples thereof

Gatifloxacin (GAT), Enoxacin (ENX), Lomefloxacin (LOM), Balofloxacin (BAL) (shanghai dary fine chemical ltd, AR); ciprofloxacin (CIP), Norfloxacin (NOR), Sarafloxacin (SAR), Clinafloxacin (CLX) (zhengzhou clinita biochemical technology ltd. > 95%); fluconazole (Fuz) (Tianjin pharmaceutical group); the fluoroquinolone azole hybrid molecules are prepared by self.

2. Culture medium and reagent

Beef extract peptone medium: 0.3% of beef extract, 1% of peptone, 0.5% of sodium chloride, 1% of agar powder and deionized water. Adjusting the pH value to 7.0-7.2, subpackaging in 500mL conical flasks, sterilizing for later use at 121 ℃ for 20 minutes by high-pressure steam;

ordinary liquid medium: 0.3% of beef extract, 1% of peptone, 0.5% of sodium chloride and deionized water. Adjusting the pH value to 7.0-7.2, subpackaging in 500mL conical flasks, sterilizing for later use at 121 ℃ for 20 minutes by high-pressure steam;

0.9% physiological saline, 1M Na₂HPO₄Solution, 1M NaH₂PO₄The solution is prepared at 121 ℃ for 30 minutes and sterilized by high-pressure steam for standby.

(II) in vitro fungus inhibitory Activity test

1. Test strains

Aspergillus fumigatus; candida tropicalis; candida albicans; candida albicans ATCC 90023; candida parapsilosis ATCC22019 (the above strain is provided by clinical laboratory of medical academy of sciences of Sichuan province, national Hospital of Sichuan province).

2. Minimum Inhibitory Concentration (MIC) determination method

2.1 preparation of the test solution

According to the titer or content of the substance to be detected and the required volume, the required amount of the substance to be detected is calculated, various required antibacterial substances to be detected are accurately weighed, the substance to be detected is diluted to the required concentration by using a proper solvent and a diluent (the mass of a sample is 3.2mg, a mother solution is prepared from 3.2mg/mL to 3200 mu g/mL, 320 mu L of stock solution is absorbed, and the stock solution is diluted to 1024 mu g/mL by broth to obtain a liquid A to be detected).

2.2 preparation of the bacterial suspension

Inoculating the preserved strain into a common liquid culture medium, and placing the strain in a constant temperature shaking table at 37 ℃ for activation culture for 17 hours. After activation, the cells were diluted to 10 with brain heart infusion Broth (BHI) respectively⁵CFU/mL of bacterial suspension is ready for use.

2.3 sample application and culture

Under the aseptic condition, adding 100 mu L of prepared solution to be detected (with the concentration of 1024 mu g/mL) into the first hole of each row, then diluting the substance to be detected twice, namely adding the solution to be detected into the first hole, fully blowing the solution by using a liquid transfer gun (at least three times or more) to fully and uniformly mix the substance to be detected and broth, then sucking 100 mu L of the solution to be detected, adding the solution to the second hole, fully blowing the solution to be detected and fully and uniformly mix the solution with the broth, repeating the steps until reaching the tenth hole, sucking 100 mu L of the solution and discarding; at the moment, the concentration of the analyte in each hole is 512,256,128,64,32,16,8,4,2 and 1 mu g/mL from left to right in sequence. The last 2 wells contained no test substance and served as bacterial growth control and negative control, respectively. Then 100 mu L of diluted bacterial liquid is added into 1-11 holes, and the concentration of the substance to be detected in each hole, namely the final substance to be detected, is 256,128,64,32,16,8,4,2,1 and 0.5 mu g/mL from left to right in sequence; the last well was not inoculated with the bacterial solution and was used as a negative control. And putting the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for 24 hours.

2.4 determination of results

After the completion of the culture, the 96-well plate was taken out from the incubator, and the growth of bacteria in the well was observed. Before the results are determined, the results are meaningful only when the bacteria in the growth control wells grow normally and the negative control wells do not grow. The concentration of the drug in the wells with no bacterial growth was visually observed as the MIC of the drug against the bacteria. If the hole jumping phenomenon occurs, repeated tests are needed for verification.

3. MIC value results and discussion

The results of MIC values of the novel fluoroquinolone azole hybrid molecule on Aspergillus fumigatus, Candida tropicalis, Candida albicans ATCC90023 and Candida parapsilosis ATCC22019 are shown in Table 10.

TABLE 10 MIC determination of 5 fungi for the test substances (in. mu.g/mL)

As can be seen from Table 10, the compounds of TM1-TM10 series of the present invention have significant inhibitory activity against fungi, and particularly have significantly better inhibitory activity against Aspergillus fumigatus than the positive control drugs.

(III) in vitro bacterial inhibitory Activity test

A part of fluoroquinolone-azole hybrid molecules are selected, and the inhibition of staphylococcus aureus ATCC6538, escherichia coli ATCC25922, vibrio parahaemolyticus ATCC17802, pseudomonas aeruginosa ATCC27853, salmonella ATCC13076 and acinetobacter ATCC19606 are determined by a broth dilution method recommended by NCCLS, so that corresponding MIC values are obtained and compared with fluoroquinolone (positive control).

1. Test strains

Staphylococcus aureus ATCC6538, Escherichia coli ATCC25922, Vibrio parahaemolyticus ATCC17802, Pseudomonas aeruginosa ATCC27853, Salmonella ATCC13076, Acinetobacter ATCC19606 (all strains are provided by the inspection and quarantine office of Chongqing City)

2. Minimum Inhibitory Concentration (MIC) determination

(1) Preparation of test solution

The required amount of the test substance is calculated according to the titer or content of the test substance and the required volume, various required antibacterial test substances are accurately weighed, the test substance is diluted to the required concentration by using a proper solvent and a diluent (the sample mass is 1mg, 1mg/0.195 mL-5.12 mg/mL-5120 mu g/mL of mother solution is prepared first, then 100 mu L of stock solution is absorbed, and the stock solution is diluted to 512 mu g/mL by using broth to obtain the test solution A).

(2) Preparation of the bacterial suspension

Inoculating the preserved strain into a common liquid culture medium, and placing the strain in a constant temperature shaking table at 37 ℃ for activation culture for 17 hours. After activation, the mixture is diluted to 10 respectively by brain heart infusion Broth (BHI) culture medium⁵CFU/mL of bacterial suspension is ready for use.

(3) Sample application and culture

Under sterile conditions, 100 μ L of blank broth was added to each well of a 96-well plate. Adding 100 mu L of prepared solution to be tested (with the concentration of 512 mu g/mL) into the first hole of each row, then diluting the substance to be tested twice, namely adding the solution to be tested into the first hole, fully blowing the solution (at least three times) by using a liquid transfer gun to fully mix the substance to be tested with broth, sucking 100 mu L of the solution to be tested with the broth to be added into the second hole, fully blowing the solution to be mixed with the broth to be fully mixed, repeating the steps until reaching the tenth hole, sucking 100 mu L of the solution to be discarded; at this time, the concentration of the analyte in each well is 256,128,64,32,16,8,4,2,1, 0.5. mu.g/mL from left to right, and finally 2 wells contain no analyte, one is used as a bacterial growth control and one is used as a negative control. Then 100 mu L of diluted bacterium liquid is added into the holes 1-11, and the concentration of the substance to be detected in each hole, namely the final substance to be detected, is 128,64,32,16,8,4,2,1,0.5, 0.25 and 0 mu g/mL from left to right in sequence; the last well was not inoculated with the bacterial solution and was used as a negative control. And putting the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 16-20 h.

(4) Determination of results

After the completion of the culture, the 96-well plate was taken out from the incubator, and the growth of bacteria in the well was observed. Before the results are determined, the results are meaningful only when the bacteria in the growth control wells grow normally and the negative control wells do not grow. The concentration of the drug in the wells with no bacterial growth was visually observed as the MIC of the drug against the bacteria. If the hole jumping phenomenon occurs, repeated tests are needed for verification.

TABLE 11 MIC values (in. mu.g/mL) for six bacteria for the test substances

As can be seen from Table 11, some of the compounds of TM1-TM10 of the present invention have strong inhibitory effect on six tested strains of bacteria.

(IV) in vitro drug-resistant bacterium inhibitory Activity test

The MIC of a test object to sensitive bacteria escherichia coli, pseudomonas aeruginosa PA01, staphylococcus aureus ATCC25129 and staphylococcus aureus ATCC33591 is firstly determined by adopting a micro broth dilution method recommended by NCCLS, and the antibacterial activity of all target molecules is obtained. Selecting compounds with MIC less than or equal to 4 mu g/mL for carrying out MIC value determination of drug-resistant bacteria.

1 test strains

Sensitive bacteria: e.coli; pseudomonas aeruginosa PA 01; staphylococcus aureus ATCC 25129; staphylococcus aureus ATCC33591 (this strain is provided by the laboratory of the Xunhuan teacher, college of medicine, southwest).

Drug-resistant bacteria: drug-resistant staphylococcus aureus (strain numbers 806S and 817S); drug-resistant klebsiella pneumoniae (strain numbers 747K and 810K); drug-resistant Citrobacter (strain No. 822N) (this strain is available from third-military medical science).

2 Minimum Inhibitory Concentration (MIC) determination

(1) Preparing a solution to be detected: according to the titer or content of the substance to be detected and the required volume, the required amount of the substance to be detected is calculated, and various required antibacterial substances to be detected are accurately weighed, and the substance to be detected is diluted to the required concentration by using a proper solvent and a diluent (the sample mass is 3.2mg, a mother solution is prepared by first preparing 3.2mg/1 mL-3.2 mg/mL-3200 mug/mL, then 50 muL of stock solution is absorbed, and the stock solution is diluted by using sterile water to 500 muL of 32 mug/mL to be detected, namely liquid A to be detected).

(2) Preparation of bacterial suspension: inoculating the preserved strain into a common liquid culture medium, and placing the strain in a constant temperature shaking table at 37 ℃ for activation culture for 17 hours. After activation, the cells were diluted to 10 with brain heart infusion Broth (BHI) respectively⁵CFU/mL of bacterial suspension is ready for use.

(3) Sample adding and culturing: the first well of each column of the 96-well plate was filled with 80. mu.L of blank broth, and the remaining wells were filled with 50. mu.L of blank broth. 20. mu.L of the prepared test solution (with a concentration of 32. mu.g/mL) was added to the first well of each column, and then the test substance was diluted twice. Adding the solution to be tested into the first hole, fully blowing (at least three times) the solution by using a liquid transfer gun to fully mix the substance to be tested with the broth, sucking 50 mu L of the solution, adding the solution into the second hole, fully blowing the solution to be tested to fully mix the solution with the broth, repeating the steps until reaching the eighth hole, sucking 50 mu L of the solution, and discarding; at the moment, the concentration of the substance to be detected in each hole is 64,32,16,8,4,2,1 and 0.5 mu g/mL from left to right in sequence, the last two columns of each plate are used as controls, two columns do not contain the substance to be detected, one column is used as a bacteria growth control to add bacteria liquid, and the other column is used as a negative control to not add bacteria liquid. And adding 50 mu L of diluted bacterium solution into the 1-8 holes, wherein the concentration of the substance to be detected in each hole, namely the final substance to be detected, is 32,16,8,4,2,1,0.5 and 0.25 mu g/mL from left to right in sequence. And (3) putting the inoculated 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 20-24h, and then observing and recording the result.

(4) And (4) judging a result: after the completion of the culture, the 96-well plate was taken out from the incubator, and the growth of bacteria in the well was observed. Before the results are determined, the results are meaningful only when the bacteria in the growth control wells grow normally and the negative control wells do not grow. The concentration of the drug in the wells with no bacterial growth was visually observed as the MIC of the drug against the bacteria. Each substance to be tested is subjected to 2 parallel tests on each strain at the same time, if a plurality of jumping holes occur, the result is not reported, and the test needs to be repeated.

3 results

3.1 MIC value determination for certain sensitive strains

MIC values of target molecules for Escherichia coli, Pseudomonas aeruginosa PA01, Staphylococcus aureus ATCC25129 and Staphylococcus aureus ATCC33591 were determined in the laboratory of the xuanheng teacher of the institute of medicine, university in southwest, and the results are shown in Table 12 below.

TABLE 12 MIC values (μ g/mL) of test substances against sensitive bacteria

As can be seen from Table 12, the compounds of TM1-TM10 series of the present invention have inhibitory activity against sensitive bacteria, and the bacteriostatic activity of some of the compounds is significantly better than that of the positive control drugs.

3.2 MIC value determination for partially drug-resistant strains

MIC values of the compounds selected from Table 12 and having MIC of 4. mu.g/mL or less were determined for drug-resistant Citrobacter (822N), drug-resistant Staphylococcus aureus (806S and 817S) and drug-resistant Klebsiella pneumoniae (747K and 810K), and the results are shown in Table 13.

TABLE 13 MIC values (μ g/mL) for drug-resistant Citrobacter, Staphylococcus aureus and Klebsiella pneumoniae of the test substances

As can be seen from Table 13, the compounds of TM1-TM10 series of the present invention have inhibitory activities against drug-resistant Citrobacter, drug-resistant Staphylococcus aureus and drug-resistant Klebsiella pneumoniae, and particularly, the inhibitory activity against drug-resistant Citrobacter is significantly superior to that of positive control drugs.

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 (8)

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

wherein X is selected from: ethyl, cyclopropyl, F substituted phenyl;

z is selected from: n or C-R₁；R₁Is selected from H; methoxy or halogen;

q is selected from N;

r' is selected from halogen;

y is selected from:

wherein n is selected from 1,2 or 3; r₂Selected from H, halogen or C1-C6 alkyl; r' is selected from hydrogen or C1-C6 alkyl; one side of the mark indicates the connection end with L;

l represents a linking structure;

wherein L is selected from:

wherein R is₃、R₄Independently selected from H, halogen, hydroxyl, amino or C1-C6 alkyl; m is selected from 0, 1,2 or 3; one side of the label is used to indicate the connection with Y.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof: wherein R' is selected from fluorine.

3. A compound represented by:

4. a pharmaceutical composition comprising a compound of any of claims 1-3 or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition further comprises: a) pharmaceutically acceptable carriers and/or adjuvants; and/or b) one or more suitable further active ingredients.

6. Use of a compound according to any of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a disease caused by a fungal and/or bacterial infection.

7. The use according to claim 6, wherein the fungal infection is an infection caused by Aspergillus fumigatus, Candida tropicalis, Candida albicans or Candida parapsilosis.

8. The use of claim 6, wherein the bacterial infection is an infection caused by Staphylococcus aureus, Escherichia coli, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Salmonella, Acinetobacter, drug-resistant Citrobacter, drug-resistant Staphylococcus aureus, and drug-resistant Klebsiella pneumoniae.