EA045519B1 - ANTIBACTERIAL DERIVATIVES OF AMINOGLICOSIDES - Google Patents

Description

This application claims priority based on the following applications:

Application number CN 201910463155.1, filed May 30, 2019; and Application number CN 202010299506.2, filed April 16, 2020.

Field of technology

The present invention relates to the field of medicine, in particular to a new class of aminoglycoside derivatives, their pharmaceutically acceptable salts or isomers, their pharmaceutically acceptable compositions and their use in the preparation of a medicament for the treatment of diseases associated with bacterial infection.

State of the art

Of particular interest in modern drug discovery is the development of new small molecule, orally bioavailable drugs that act by binding to RNA. RNA, which serves as an intermediary between DNA and proteins, was considered a completely flexible molecule without significant structural complexity. Recent research has revealed the surprising complexity of RNA structure. RNA has a structural complexity that rivals that of proteins rather than simple motifs such as DNA. Genome sequencing reveals both the sequences of proteins and the mRNAs that encode them. Since proteins are synthesized using an RNA template, such proteins can be inhibited by preventing their production in the first place by interfering with mRNA translation. Since both proteins and RNA are potential drug targeting sites, the number of targets identified through genome sequencing efforts is effectively doubling. These observations open up a new world of opportunities for the pharmaceutical industry to target RNA with small molecules.

Modern research in the field of biochemistry and molecular biology has shown that the binding of the 30S subunit of the bacterial ribosome to tRNA is one of the key stages of protein synthesis. To date, crystal structures of the 30S ribosomal subunit have been successfully described in at least two bacteria (Thermus thermophiles and Escherichia coli). From the crystal structures, three sites that bind to tRNA can be clearly identified: the aminoacyl site A, the peptide site P, and the E (exit) site. Aminoglycoside drugs specifically bind to site A of the 16S rRNA decoding site of the 30S subunit of the bacterial ribosome, causing an error in mRNA translation, resulting in interference with protein synthesis to kill pathogenic bacteria. Aminoglycoside drugs are highly effective broad-spectrum antibiotics and are the most commonly used anti-infective drugs. Most aminoglycoside drugs have expected pharmacokinetic properties and exhibit synergistic effects with other anti-infective drugs, making them excellent options for the treatment of life-threatening infections. Over the past few decades, many variations of this type of antibiotic have gained popularity in clinical practice.

The history of aminoglycoside drugs began with the discovery of streptomycin in 1944. Later, a number of landmark compounds (kanamycin, gentamicin, tobramycin) were successfully released, and the status of aminoglycoside drugs in the treatment of gram-negative bacterial infections was established. From the 1970s to the 1990s, the semisynthetic aminoglycoside antibiotics dibekacin, amikacin, netilmicin, isepamycin, and etimicin appeared one after another, indicating that aminoglycoside antibiotics that are effective against bacteria resistant to early antibiotics and have low level of adverse reactions can be successfully obtained by semisynthetic routes, but the development of aminoglycoside antibiotics has slowed down. Meanwhile, specialists have conducted extensive basic and clinical research on aminoglycoside drugs, in particular their bactericidal and drug resistance mechanisms, which not only give specialists a deeper understanding of this type of antibiotics, but also provide a theoretical basis for our clinical rational use of drugs, reducing the number of drug-resistant bacteria and developing new aminoglycoside drugs against drug-resistant bacteria using the results of these studies.

Aminoglycoside drugs are glycosides formed by combining amino sugars and aminocyclic alcohols through oxygen bridges. There are streptomycin produced by Streptomyces, natural aminoglycoside drugs such as gentamicin produced by Micromonospora, and semisynthetic aminoglycoside drugs such as etimicin and amikacin, all of which are broad-spectrum antibacterial drugs. Aminoglycoside drugs are primarily used for systemic infections caused by susceptible aerobic gram-negative bacteria. Although various cephalosporins and quinolones have been widely used in clinical practice in recent years, aminoglycoside drugs are still used to treat serious infections caused by aerobic gram-negative bacteria because they have a longer post-antibiotic effect (PAE) against common infections.

- 1,045,519 monegative bacteria such as Pseudomonas aeruginosa, Klebsiella Pneumoniae and Escherichia coli.

With the long-term and large-scale use of aminoglycoside drugs in clinical practice, this class of drugs inevitably faces serious problems of drug resistance. At the same time, common side effects of aminoglycoside drugs, such as ototoxicity and nephrotoxicity, also limit the use of aminoglycoside drugs. In recent years, some drug molecules have emerged that can address the problem of resistance to traditional antibiotics, such as Achaogen's recently developed plazomycin (WO 2009067692), which has completed phase 3 clinical trials.

The present invention is aimed at solving the problems of severe drug resistance due to inactivating enzymes and the presence of ototoxicity and nephrotoxicity for traditional antibiotics such as etimicin, amikacin, gentamicin and the like. A class of new aminoglycoside drugs with a broader antibacterial spectrum and better activity are obtained by a simpler synthetic method compared to compounds known from the prior art.

Brief description of the invention

The present invention provides a compound represented by the following formula:

or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition containing as an active ingredient a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof and the above pharmaceutical composition for the preparation of a medicament for the treatment of diseases associated with bacterial infection; and in some embodiments, the above bacterial infection is a carbapenem-resistant Enterobacteriaceae infection.

Technical results

The present invention synthesizes the compound of formula (II) and its isomers using a simpler preparation method and provides a new class of aminoglycoside antibiotics to combat drug-resistant bacterial infection caused by superbugs such as CRE (carbapenem-resistant Enterobacteriaceae), thereby solving the problems of drug resistance due to enzyme inactivation and the presence of ototoxicity and nephrotoxicity for traditional antibiotics. Meanwhile, the compound of the present invention has a wider antibacterial spectrum, better activity and no cytotoxicity.

Definitions and Descriptions

Unless otherwise specified, the following terms and phrases as used herein are intended to have the following meanings. A specific term or phrase should not be considered vague or unclear without specific definition, but should be taken in its ordinary meaning. Where a trademark appears herein, it is intended to identify the relevant product or its active ingredient.

The term pharmaceutically acceptable as used herein refers to compounds, materials, compositions and/or dosage forms that have been determined by good medical judgment to be suitable for use in contact with tissues of humans and animals without undue toxicity, irritation, allergic reactions or other problems or complications, and appropriate reasonable benefit/risk ratio.

The term pharmaceutically acceptable salt refers to a salt of a compound of the present invention that is prepared from a compound of the present invention using a specific substituent(s) and a relatively non-toxic acid or base. In the case where the compound of the present invention contains a relatively acidic functional group, a base addition salt can be prepared by bringing the neutral form of the compound into contact with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. In the case where the compound of the present invention contains a relatively basic functional group, an acid addition salt can be prepared by bringing the neutral form of the compound into contact with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include:

- 2 045519 include salts of inorganic acids and salts of organic acids. The inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc. The organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like. Examples of pharmaceutically acceptable acid addition salts also include salts of amino acids (such as arginine, etc.) and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups such that they can be converted into any base or acid addition salt.

The pharmaceutically acceptable salt of the present invention can be synthesized from a starting compound containing an acidic or basic radical by conventional chemical methods. Typically, such salts are prepared by reacting said compounds in free acid or free base form with stoichiometric amounts of a suitable base or acid in water or an organic solvent or a mixture thereof.

Specific geometric isomers or stereoisomers of the compounds of the present invention may exist. The present invention contemplates all such compounds, including tautomers, cis-isomers and trans-isomers, (-)-enantiomers and (+)-enantiomers, (R)-enantiomers and (S)enantiomers, diastereomers, (D)-isomers, (L) -isomers, racemic mixtures thereof and other mixtures such as enantiomers or diastereomeric enriched mixtures. All of these mixtures are included within the scope of the present invention. Additional asymmetric carbon atoms may be present on substituents such as alkyl. All of these isomers and mixtures thereof are included within the scope of the present invention.

Unless otherwise noted, the term enantiomer or optical isomer refers to stereoisomers that are mirror images of each other.

Unless otherwise noted, the term cis-, trans-isomer or geometric isomer is a consequence of the inability to rotate freely due to the double bonds or single bonds of the ring-forming carbon atoms.

Unless otherwise specified, the term diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are not mirror images of each other.

Unless otherwise noted, (D) or (+) means dextrorotatory, (L) or (-) means left-handed, and (DL) or (±) means racemic.

Unless otherwise noted, the solid wedge link (X*) and the dashed wedge link (-''') are used to represent the absolute configuration of the stereocenter, the straight solid line link (x*) and the straight dash line link (-*'”' ) are used to represent the relative configuration of the stereocenter, and a wavy line (X) is used to represent a wedge-shaped solid line relationship (X*) or a wedge-shaped dashed line relationship (·'''), or a wavy line (X) is used to represent a straight solid line relationship (X) and connections with a straight dashed line (.>'''').

The compound of the present invention may be specific. Unless otherwise specified, the term tautomer or tautomeric form means that at room temperature, isomers of different functional groups are in dynamic equilibrium and can rapidly convert into each other. If tautomers are possible (for example, in solution), chemical equilibrium of the tautomers can be achieved. For example, in the case of a protic tautomer (also called a prototropic tautomer), interconversion occurs by proton migration, such as keto-enol tautomerization and imino-enamine tautomerization. In the case of a valence tautomer, interconversion occurs by recombination of some of the bonding electrons. A specific example of keto-enol tautomerization is the tautomerization of two tautomers of pentan-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise specified, the terms single-isomer-rich, isomer-rich, single-enantiomer-rich, or enantiomer-rich refer to a content of one of the isomers or enantiomers that is less than 100% and is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6 % or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

Unless otherwise noted, the term isomeric purity or enantiomeric purity refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomeric or enantiomeric purity (EP value) is 80%.

Optically active (R)- and (S)-isomers, as well as D- and L-isomers, can be prepared by chiral synthesis or by using chiral reagents or other conventional methods. If not

- 3 045519 bypassing the enantiomer of the compound of the present invention, it can be obtained by asymmetric synthesis or derivatization using chiral auxiliaries, in which the resulting mixture of diastereomers is separated and the auxiliary groups are removed to obtain the desired pure enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with a suitable optically active acid or base, after which the diastereoisomers are separated in a conventional manner known in the art , and restore pure enantiomers. In addition, separation of enantiomers and diastereomers is typically accomplished by chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (eg, carbamate formation from amines). The compounds of the present invention may contain non-naturally occurring atomic isotope ratios of one or more of the atoms constituting the compound. For example, compounds can be labeled with radioisotopes such as tritium ( ^3H ), iodine-125 ( ^125I ), or C-14 ( ^14C ). As another example, deuterated drugs can be formed by replacing hydrogen with deuterium. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reducing toxic side effects, increasing drug stability, increasing efficacy, and prolonging the biological half-life of drugs. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention. Optional or optional means that the event or condition described below may, but is not required to, occur, and the present description includes a situation where the event or condition occurs and a situation where the event or condition does not occur.

For drugs or pharmacologically active agents, the term effective amount or therapeutically effective amount refers to a sufficient amount of the drug or agent that is non-toxic but can provide the desired effect. In the case of an oral dosage form according to the present invention, the effective amount of one active substance in the composition refers to the amount necessary to achieve the desired effect in combination with another active substance in the composition. The determination of the effective amount varies from person to person depending on the age and general condition of the recipient, as well as the specific active substance. A suitable effective amount in the case can be determined by those skilled in the art in accordance with routine experimentation.

The terms active ingredient, therapeutic agent, active substance or active agent refer to a chemical compound that can provide effective treatment for the target disorder, disease or condition.

The term substituted means that any one or more hydrogen atoms on a particular atom are replaced by substituents and may include deuterium and hydrogen variants, provided that the valency of the particular atom is normal and the substituted compound is stable. In the case where the substituent is oxygen (ie, =O), this means that two hydrogen atoms are replaced. Substitution with oxygen does not occur in aromatic groups. The term optionally substituted means that it may be substituted or unsubstituted. Unless otherwise indicated, the type and number of substituents can be chosen arbitrarily based on the fact that they can be implemented chemically.

When any variable (such as R) occurs more than once in the composition or structure of a compound, its definition in each situation is independent. Thus, for example, if a group is substituted with 0-2 R's, said group may optionally be substituted with no more than two R's, and R has independent variations in each situation. In addition, combinations of substituents and/or variants thereof are only permitted if such combinations will result in stable compounds.

When the index of a linking group is 0, for example -(CRR) ₀ -, this indicates that the linking group is a single bond.

In the case where one of the variables is selected from a single link, this means that the two groups connected by it are directly linked. For example, when the L in A-L-Z represents a single bond, it means that the structure indicated is actually an A-Z.

In the case where a substitute is vacant, this means that the substitute is absent. For example, when X in A-X is vacant, this means that the specified structure is actually A. When it is not specified which atom of the specified substituent is bonded to a substituted group, such substituent can be bonded through any atom. For example, a pyridyl group as a substituent can be attached to a substituted group through any of the carbon atoms of the pyridine ring.

When the direction of connection of the specified linking group is not specified, the direction of connection is 4 045519

tion is arbitrary. For example, in the linking group L is

-MW-, and -MW- can connect ring A and ring B in the direction corresponding to the reading order from left to right, obtaining

and can also connect ring A and ring B in the direction opposite to the reading order from left to right, resulting in C A V W-μΎ B).Combinations of linking groups, substituents and/or variants thereof are permitted only if such combination will result in to stable connections.

Unless otherwise specified, the term C _1-6 alkyl is used to designate a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. C _1-6 alkyl includes C _1-5 , Cm, C _1-3 , C _1-2 , C _2-6 , C _2-4 , C ₆ and C ₅ alkyl and the like. It can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine). Examples of C _1-6 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl and tert-butyl) , pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.

Unless otherwise specified, the term C _1-3 alkyl is used to designate a linear or branched saturated hydrocarbon group containing from 1 to 3 carbon atoms. C _1-3 alkyl includes C _1-2 and C _2-3 alkyl and the like. It can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine). Examples of C _1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

The term leaving group refers to a functional group or atom that can be replaced by another functional group or atom as a result of a substitution reaction (eg, nucleophilic substitution). For example, typical leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-tosylate, etc., acyloxy groups such as acetoxy, trifluoroacetoxy, etc.

The term protecting group includes, but is not limited to, an amino protecting group, a hydroxy protecting group, or a sulfhydryl protecting group. The term amino protecting group refers to a protecting group that is suitable for preventing side reactions at the nitrogen center of an amino group. Typical amino protecting groups include, but are not limited to, formyl; acyl such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethyloxycarbonyl such as benzyloxycarbonyl (Cbz) and 9fluorenylmethyloxycarbonyl (Fmoc); arylmethyl such as benzyl (Bn), trityl (Tr), 1,1-di(4'methoxyphenyl)methyl; silyl such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and so on. The term hydroxy protecting group refers to a protecting group that is suitable for preventing adverse reactions of a hydroxyl group. Typical hydroxy protecting groups include, but are not limited to, alkyl such as methyl, ethyl and tert-butyl; acyl such as alkanoyl (such as acetyl); arylmethyl such as benzyl (Bn), p-methyloxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (DPM); silyl such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and so on. The compounds of the present invention can be prepared by various synthetic routes well known to those skilled in the art, including the specific embodiments listed below, embodiments prepared by combination thereof with other chemical synthesis routes, and equivalent alternatives well known to those skilled in the art. . Preferred embodiments include, but are not limited to, examples according to the present invention.

The solvent used in the present invention is commercially available.

In the present invention, the following abbreviations are used: CFU means the number of colonies; Boc means tert-butoxycarbonyl; MIC means minimum inhibitory concentration.

Brief description of drawings

In fig. 1 shows in vivo efficacy data for Compound 1 (at a dose of 30 mg/kg) and plazomycin (at a dose of 30 mg/kg) in a mouse thigh muscle model (Enterobacteria ATCC-25922).

In fig. Figure 2 shows in vivo efficacy data for Compound 1 (at a dose of 10 mg/kg and 30 mg/kg), plazomycin (at a dose of 10 mg/kg and 30 mg/kg) and meropenem (at a dose of 100 mg/kg) in a pneumonia model mice (Klebsiella Pneumoniae ATCC-BAA-1705).

In fig. Figure 3 shows changes in the amplitude of the action potential of the compound: changes in the amplitude of the PDS of compound 1, gentamicin and plazomycin at different intensities when the frequency was fixed at 32 kHz.

In fig. Figure 4 shows changes in the amplitude of the action potential of the compound: changes in the amplitude of the PDS of compound 1, gentamicin and plazomycin at different intensities when the frequency was fixed at 16 kHz.

In fig. Figure 5 shows changes in the amplitude of the compound action potential: changes in value

- 5 045519 amplitudes of the PDS of compound 1, gentamicin and plazomycin at different intensities with a short sound (click).

In fig. 6 shows damage to cochlear hair cells: A illustrates damage to inner hair cells caused by Compound 1, gentamicin and plazomycin; and B illustrates damage to outer hair cells caused by Compound 1, gentamicin and plazomycin.

In fig. Figure 7 shows changes in spiral ganglion neuron density induced by Compound 1, gentamicin, and plazomycin.

In fig. Figure 8 shows the regression curve of plazomycin toxicity on HK-2 cells.

In fig. 9 shows the toxicity regression curve of Compound 1 on HK-2 cells.

In fig. Figure 10 shows the regression curve of netilmicin toxicity on HK-2 cells.

In fig. Figure 11 shows the regression curve of amikacin toxicity on HK-2 cells.

Detailed implementation options

The present invention is described in detail with the help of the following examples, which are not intended to imply any unfavorable limitation of the present invention. Although the present invention has been described in detail herein with specific embodiments thereof disclosed, various changes and improvements that can be made thereto will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Example 1. Connection 1.

Stage 1.

Diphenylphosphinylhydroxylamine (10 g, 42.88 mmol, 1 eq.), compound 1-1 (13.65 g, 128.64 mmol, 12.19 mL, 3 eq.) and sodium tert-butoxide (4.95 g, 51.46 mmol, 1.2 eq.) was dissolved in tetrahydrofuran (100 ml), stirred and reacted at 5-15°C for 16 hours. The reaction liquid was filtered and the filtrate was concentrated to obtain compound 1-2.

Stage 2.

Compound 1-2 (5.19 g, 42.84 mmol, 1 eq.) obtained in the previous step in tetrahydrofuran (100 ml) and N,N-di-BOC-1H-pyrazole-1-carboxamidine (13, 30 g, 42.84 mmol, 1 eq.) was stirred and reacted at 66°C for 16 hours. The reaction liquid was cooled to room temperature and extracted with ethyl acetate (100 mlx2) after adding water (300 ml). The organic phases were combined, dried over sodium sulfate and filtered. The filtrate was concentrated to obtain the crude product, and compound 1-3 was obtained by column chromatography (silica, petroleum ether/ethyl acetate=20/1, 1/1 (v/v)).

- 6 045519

Stage 3.

Compound 1-3 (1 g, 2.75 mmol, 1 eq.) and 2-iodoxybenzoic acid (847.59 mg, 3.03 mmol, 1.1 eq.) were dissolved in dimethyl sulfoxide (10 ml), and the reaction liquid stirred at 40°C for 1 hour reaction. The reaction liquid was filtered, and the filtrate was subjected to extraction with tert-butyl methyl ether (20 mlx2 times) after adding water (40 ml). The organic phases were combined and washed with saturated sodium thiosulfate (10 ml). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give compound 1-4.

Stage 4.

Amberlite (ion exchange resin) IRA-402(OH) (500 g) was added to methanol (500 ml), and the resulting solution was stirred at 20°C for 1 hour. The mixture was then filtered, the filter residue was added to methanol (500 ml) , and then Compound 1-5 was added to the mixture. The mixture was stirred at 20°C for 11 hours. During the reaction, compound 1-5 dissolved. The reaction liquid was filtered, and the filtrate was concentrated to obtain compound 1-6. LCMS (ESI) m/z: 448.4 (M+1).

Stage 5.

Compound 1-6 (15 g, 33.52 mmol, 1 eq.) was dissolved in methanol (150 ml), and then S-ethyl-2,2,2-trifluoroethylthioether (4.24 g, 26.82 mmol, 0.8 eq.) in methanol (150 ml). The mixed solution was stirred at 20°C for 16 hours. Zinc acetate (14.72 g, 80.44 mmol, 2.4 eq.) was then added to the solution, and then (N-hydroxy- 5-norbornene-2,3-dicarboxylimido)-tert-butyl ester (16.85 g, 60.33 mmol, 1.8 eq.) and triethylamine (10.17 g, 100.55 mmol, 14.00 ml , 3 eq.) in tetrahydrofuran (170 ml). The reaction liquid was stirred at 20°C for 30 hours, then quenched with glycine (7 g) and then concentrated. The concentrated liquid was diluted with dichloromethane (1000 ml), and then washed twice with aqueous ammonia (300 ml) (water: ammonia=7:3). The organic phase was concentrated. The crude product was purified by column chromatography (silica, dichloromethane/methanol=50/1-5/1 (v/v), containing a small amount of ammonia water) to give compound 1-7. LCMS (ESI) m/z: 744.3 (M+1).

Stage 6.

(2S)-4-(tert-Butyloxycarbonylamino)-2-hydroxybutyric acid (6.85 g, 31.26 mmol, 1.5 eq.) was dissolved in N,N-dimethylformamide (150 ml), and to the resulting solution was added N-Hydroxy-5norbornene-2,3-dicarboximide (5.60 g, 31.26 mmol, 1.5 eq) and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide (4.85 g, 31.26 mmol, 5 .53 ml, 1.5 eq.). The reaction liquid was stirred at 20°C for 2 hours, followed by addition of compound 1-7 (15.5 g, 20.84 mmol, 1 eq.) thereto. The reaction liquid was stirred at 20°C for 16 hours, then diluted with water (200 ml) and extracted with ethyl acetate (50 ml x 3). The combined organic phases were washed with brine (100 ml), then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to obtain a mixture. The mixture was purified by column chromatography (silica, dichloromethane/methanol=50/1-10/1 (v/v)) to give compound 18. LCMS (ESI) m/z: 945.5 (M+1 ).

Stage 7.

Compound 1-8 (16.40 g, 17.35 mmol, 1 eq), di-tert-butyl dicarbonate (4.55 g, 20.83 mmol, 4.78 mL, 1.2 eq), DIEA ( 2.69 g, 20.83 mmol, 3.6 mL, 1.2 eq.) was dissolved in tetrahydrofuran (170 mL). Nitrogen replacement was performed three times. The reaction liquid was stirred at 20°C for 16 hours. The reaction liquid was diluted with water (200 ml), and then subjected to extraction with dichloromethane (100 mlx2). The combined organic phases were washed successively with 0.1 M hydrochloric acid (20 ml) and brine (60 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to obtain a solid mixture. The mixture was purified by column chromatography (silica, petroleum ether/ethyl acetate=15/1-0/1 (v/v)) to give the target compound 1-9. LCMS (ESI) m/z: 1045.3 (M+1).

Stage 8.

Compound 1-9 (15.00 g, 14.35 mmol, 1 eq.) and ammonia water (63.70 g, 1.82 mol, 70 mL, 126.62 eq.) were dissolved in methanol (80 mL), and the resulting mixture was stirred at 20°C for 16 hours. The reaction liquid was concentrated to remove the solvent, diluted with water (100 ml) and extracted with dichloromethane (100 ml x 3 times). The combined organic phases were washed with brine (200 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the concentrated mixture was purified by column chromatography (silica, first petroleum ether/ethyl acetate = 10/1-0/1 (v/v), then dichloromethane/methanol = 6/1 (v/v). ), the eluent contained a small amount of ammonia water) to obtain compound 1-10. LCMS (ESI) m/z: 949.3 (M+1).

Stage 9.

Compound 1-4 (50.63 mg, 0.15 mmol) and compound 1-10 (145.00 mg, 0.15 mmol) were dissolved in methanol (5.00 ml), and then a 4A molecular sieve (0. 5 g). The mixture was stirred for 0.5 h

- 7 045519 at 18°C in a nitrogen atmosphere. Sodium cyanoborohydride (19.20 mg, 0.30 mmol) was then added and stirred for 1 hour. Completion of the reaction was determined by LC-MS. The reaction liquid was filtered, concentrated and separated using preparative HPLC: Phenomenex Synergi C18 150x25 mmx10 µm; mobile phase: [water (0.225% formic acid)-acetonitrile]; % acetonitrile: 60%-90% for 10 min; to obtain compound 1-11. LCMS (ESI) m/z: 1294.7 (M+1).

Stage 10.

Compound 1-11 (91.00 mg, 71.97 µmol) was dissolved in anhydrous dichloromethane (2.00 ml), cooled to 0°C under nitrogen atmosphere. Trifluoroacetic acid (1.54 g, 13.51 mmol) was added and the reaction liquid was stirred at 0-19°C for 9 hours, concentrated at room temperature, suspended with acetonitrile/methyl tert-butyl ether (4 ml, 1/3), filtered and concentrated to obtain compound 1.

^1H NMR (400 MHz, D2O) δ (ppm): 5.62 (s, 1H), 5.24 (s, 1H), 5.08 (s, 1H), 4.09-4.06 (m, 1H), 3.98-3.96 (m, 2H), 3.94-3.93 (m, 5H), 3.77-3.73 (m, 4H), 3.24 (s, 1H), 3.22-3.10 (m, 6H), 2.83 (s, 3H), 2.61-2.14 (m, 2H), 2.04-2.14 (m, 6H), 1.26 (s, 3H); LCMS (ESI) m/z: 664.5 (M+1).

Example 2. Compound 2.

Stage 1.

Compound 2-1 (1 g, 12.19 mmol, 71.94 µl, 1.2 eq), N,N-bis-BOC-1H-pyrazole-1-carboxamidine (3.15 g, 10.16 mmol , 1 eq.) and triphenylphosphine (3.20 g, 12.19 mmol, 1.2 eq.) were dissolved in tetrahydrofuran (40 ml), DIAD (2.46 g, 12.19 mmol, 2.37 ml, 1 .2 eq.) was added dropwise at 0°C. The resulting mixture was then heated to 20°C and stirred for 12 hours. Water (100 ml) was added to the reaction solution, which was then extracted with ethyl acetate (50 ml, 3 times). The combined organic phases were washed with water (30 ml, 3 times), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography (silica, petroleum ether/ethyl acetate = 50/1 to 20/1 (v/v)) to give compound 2-2.

Stage 2.

Compound 2-2 (3.25 g, 8.67 mmol, 0.5 eq.) was added to compound 1-2 (2.1 g, 17.34 mmol, 1 eq.) in tetrahydrofuran (50 ml) at 20 °C, and the reaction liquid was stirred at 67°C for 12 hours. Water (100 ml) was added to the reaction liquid, which was then extracted using ethyl acetate (50 ml x 3). The combined organic phases were washed with water (30 ml, 3 times), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product, which was purified by column chromatography (silica, petroleum ether/ethyl acetate = 10/1 to 1/1 (v/v)) to obtain compound 2-3.

- 8 045519

Stage 3.

2-Iodoxybenzoic acid (108.09 mg, 386.02 µmol, 1.1 eq.) was added to compound 2-3 (150 mg, 350.93 µmol, 1 eq.) in dimethyl sulfoxide (3 ml) at 40°C . The reaction liquid was stirred at 40°C for 2 hours. Saturated sodium bicarbonate/sodium thiosulfate (30 ml (v/v)) was added to the reaction liquid, and the reaction liquid was extracted with ethyl acetate (20 ml x 2 ). The combined organic phases were washed with saturated sodium bicarbonate/sodium thiosulfate (10 ml x 3 times (1/1, v/v)), dried over anhydrous sodium sulfate, filtered and concentrated to give compound 2-4.

Stage 4.

Compound 2-5 (118 mg, 277.37 µmol, 1.1 eq.) and molecular sieve 4A (300 mg) were added to compound 1-10 (239.32 mg, 252.16 µmol, 1 eq.) in 1 ,2-dichloroethane (2 ml) at 20°C. The resulting mixture was stirred for 1 hour, and then sodium acetate borohydride (64.13 mg, 302.59 μmol, 1.2 eq.) was added. The reaction liquid was stirred at 20°C for 12 hours. Water (20 ml) was added to the reaction liquid, which was then extracted with dichloromethane (20 ml x 3 ). The combined organic phases were washed with water (10 ml x 3), dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product, which was then purified using preparative HPLC (column: Phenomenex Synergi C18 150 x 25 mm x 10 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; % acetonitrile: 35%-56%, 7 min) to obtain compound 2-6. LCMS (ESI) m/z: 1358.7 (M+1).

Stage 5.

Compound 2-8 (40 mg, 29.44 µmol, 1 eq.) was dissolved in dichloromethane (1 ml) and trifluoroacetic acid (1.54 g, 13.51 mmol, 1 ml, 458.70 eq.) was added at 0 °C. The reaction liquid was heated to 20°C and stirred for 2 hours, and then cooled to 0°C. Methyl tert-butyl ether (15 ml) was added and the resulting mixture was filtered, washed with methyl tert-butyl ether (2 ml x 3) and dried with an oil pump at 40°C to give compound 2.

¹ H NMR (400 MHz, D2O) δ = 6.18-5.87 (m, 1H), 5.63 (s, 1H), 5.28-5.22 (m, 1H), 5.08 (d, J= 3.8 Hz, 1H), 4.23- 4.16 (m, 1H), 4.09-4.03 (m, 3H), 3.98-3.89 (m, 2H), 3.83 (br t, J=5.2 Hz, 1H), 3.79-3.68 (m, 8H), 3.46-3.38 (m, 1H), 3.33 (br d, J=13.0 Hz, 1H), 3.26-3.22 (m, 2H), 3.16-3.07 (m, 2H), 2.83 (s, 3H), 2.69- 2.55 (m, 1H), 2.42-2.28 (m, 1H), 2.19-2.06 (m, 2H), 1.95-1.85 (m, 1H), 1.83-1.71 (m, 1H), 1.29-1.23 (m, 3H). LCMS (ESI) m/z: 758.3 (M+1).

Example 3. Compound 3.

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h

Stage 1.

Compound 3-1 (16.00 g, 115.12 mmol) was dissolved in acetonitrile (200.00 mL) and N-BOC-hydroxylamine (15.33 g, 115.12 mmol) and DBU (19.28 g) were added sequentially , 126.63 mmol). The resulting mixture was reacted at 11-25°C for 16 hours and then concentrated. The residue was diluted with ethyl acetate (350 ml), washed with water (100 ml x 3), washed once with brine (100 ml), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography (filler: silica gel powder, eluent: ethyl acetate/petroleum ether = 0-1/1 (v/v)) to obtain compound 3-2.

Stage 2.

Compound 3-2 (1.00 g, 5.23 mmol) and a solution of hydrogen chloride in dioxane (10 ml, 4 mmol) were mixed and stirred at 20°C for 16 hours and then concentrated under reduced pressure to give compound 3 -3.

Stage 3.

Compound 3-3 (581.27 mg, 6.38 mmol) and N,N-bis-Boc-1-guanylpyrazole (1.80 g, 5.8 mmol) were dissolved in tetrahydrofuran (20 ml) and triethylamine (1 ml). The solution was stirred at 80°C for 16 h, and the completion of the reaction was determined using LC-MS. The mixture was poured into water (100 ml) and extracted with ethyl acetate (100 ml x 3). The combined organic phases were washed with brine (30 ml), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography (filler: silica gel powder, eluent: ethyl acetate/petroleum ether = 50/1-20/1 (v/v)) to obtain compound 3-4.

Stage 4.

2-Iodoxybenzoic acid (0.34 g, 1.2 mmol) was added to compound 3-4 (0.40 g, 1.50 mmol) in dimethyl sulfoxide (5.00 ml), and the resulting mixture was stirred at 40°C in for 3 hours in a nitrogen atmosphere. The reaction liquid was diluted with ethyl acetate (100 ml), washed with water (50 mlx2) and brine (50 ml) and concentrated. The residue was separated by column chromatography (filler: silica gel powder, eluent: ethyl acetate/petroleum ether = 0-1/1) to give compound 3-5.

Stage 5.

Molecular sieve 4A (0.5 g) was added to compound 3-5 (50.63 mg, 0.15 mmol) and compound 110 (145.00 mg, 0.15 mmol) in methanol (5.00 ml). The resulting mixture was stirred for 0.5 h at 18°C under a nitrogen atmosphere, and then stirred for another 1 h after adding sodium cyanoborohydride (19.20 mg, 0.30 mmol). Completion of the reaction was determined using LC-MS. The reaction liquid was filtered, concentrated and separated using preparative HPLC: Phenomenex Synergi C18 150x25x10 µm; mobile phase: [water (0.225% formic acid)-acetonitrile]; % acetonitrile: 60%-90% for 10 min; to obtain compound 3-7.

Stage 6.

Compound 3-7 (91.00 mg, 71.97 µmol) was dissolved in anhydrous dichloromethane (2.00 ml) and cooled

- 10 045519 to 0°C in a nitrogen atmosphere. Trifluoroacetic acid (1.54 g, 13.51 mmol) was added and the reaction liquid was stirred at 0-19°C for 9 hours, concentrated at room temperature, and the residue was washed with acetonitrile/methyl tert-butyl ether (4 ml, 1/3) to obtain compound 3.

¹ H NMR (400 MHz, D2O) δ(ppm): 5.62 (s, 1H), 5.24 (s, 1H), 5.08 (s, 1H), 4.09-4.06 (m, 1H), 3.98-3.96 (m, 2H), 3.94-3.93 (m, 5H), 3.77-3.73 (m, 4H), 3.24 (s, 1H), 3.22-3.10 (m, 6H), 2.83 (s, 3H), 2.61-2.14 (m, 2H), 2.04-2.14 (m, 6H), 1.26 (s, 3H); LCMS (ESI) m/z: 664.5(M+1).

Analysis of biological activity.

Experimental Example 1: Detection of the antibacterial effect of the compound (MIC).

Three strains of Enterobacteriaceae E. coli ATCC 25922, E. coli ATCC BAA-2523, K. pneumonia ATCC BAA-1705 were used to determine the minimum inhibitory concentration (MIC) of each compound by microfluidic dilution method in accordance with the requirements of the Clinical and Laboratory Standards Institute (CLSI) . Compounds subjected to a series of 2-fold dilutions (with a final concentration range of 0.125 μg/ml -128 μg/ml) were added to a 96-well round bottom plate (cat. no. 3788, Corning). One clone of fresh bacteria on a Mueller Hinton II agar plate (MHA, cat. no. 211438, BD BBL™) after overnight culture was collected and suspended in sterile saline to adjust the concentration to 1 x 108 CFU/ml, and then diluted to 5x105 CFU/mL with Mueller-Hinton II Broth (MHC, Cat. No. 212332, BD BBL™), 100 μL of which was added to a 96-well round bottom plate containing the drug. The plate was inverted and incubated at 37°C for 20–24 h, and the MIC value was read. The lowest drug concentration that inhibits bacterial growth was determined as the MIC. The results are shown in table. 1.

Table 1

Antibacterial effect detection data (MIC) of compounds according to the present invention

Strains MIC(µM) K. pneumoniae ATCC VAA-1705 E. coP ATCC VAA-2523 E. coP ATCC 25922 Connection 1 4 2 2 Connection 2 4 4 2 Connection 3 0.25 1 0.5

Conclusion: The compounds of the present invention have good antibacterial activity in vitro.

Experimental example 2. Evaluation of pharmacokinetics in rats.

Purpose of the experiment.

Test the pharmacokinetic parameters of the compound of the present invention in rats.

Experiment protocol:

1) Experimental drug: Compound 1;

2) Experimental animals: 3 male SD rats aged 7-9 weeks;

3) Drug preparation: An appropriate amount of drug was weighed and dissolved in saline to obtain a 60 mg/ml solution.

Progress of the experiment.

The animals were administered the drug at a dose of 150 mg/kg and a concentration of 60 mg/ml by a single intravenous drip infusion through the tail vein for 30 minutes. Plasma samples were collected from animals at 0, 0.0333, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postadministration. LC-MS/MS method was used to determine the drug concentration in the plasma sample, and the kinetic parameters of the tested drug are given in Table. 2.

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table 2

Results of evaluating the pharmacokinetics of the compound according to the present invention in rats

Connection Clearance speed Maximum concentration Volume distributed AND I Half-life AND I Area under the curve CI (ml/kg/m and N) Stach (nM) Vd (l/kg) T1/2 (H) AUC (nM"h) Connection 1 9.48 315667 1.50 3.62 440373

Conclusion: The compound of the present invention has good pharmacokinetic properties in rats.

Experimental Example 3: Pharmacokinetics Study in Mice.

Purpose of the experiment.

The purpose of this experiment is to evaluate the pharmacokinetic behavior of the compound after a single intravenous injection and intragastric administration, as well as to study the bioavailability after intragastric administration.

Progress of the experiment.

Male CD-1 mice between 7 and 10 weeks of age were selected and treated by intravenous administration at a dose of 1 mg/kg. Mice were fasted for at least 12 hours before administration and fed again 4 hours after administration. The mice were able to drink throughout the experiment.

On the day of the experiment, animals in the intravenous group were administered the corresponding compound by a single injection through the tail vein with an injection volume of 5 ml/kg. Animals were weighed before administration, and the volume of administration was calculated based on body weight. Sample collection times were: 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours. Approximately 30 μl of whole blood was collected through the saphenous vein at each time point to obtain plasma for high-performance liquid chromatography with tandem mass. spectrometry (LC-MS/MS) to determine concentration. All animals were euthanized under CO2 anesthesia after PK samples were collected at the last time point. The noncompartmental model of pharmacokinetic software WinNonlin™ version 6.3 (Pharsight, Mountain View, CA) was used to process plasma concentration data, and the linear-log trapezoidal method was used to calculate pharmacokinetic parameters.

Experiment results.

The results of assessing PK properties in mice are shown in Table. 3.

Table 3

Evaluation of the pharmacokinetic properties of the compound of the present invention in mice

Connection Speed and ground clearance Maximum concentration Volume distributed AND I Half-life AND I Area under the curve CI (ml/kg/mi N) Stach (nM) Vd (l/kg) T1/2 (h) AUC (ηΜ 4) Connection 1 15.1 7088 0.369 0.396 1924

Conclusion: The compound of the present invention has good pharmacokinetic properties in mice.

Experimental example 4. Experimental evaluation of the effectiveness of the drug in mice (mouse thigh muscle model).

female CD-1 mice were divided into 4 cages, 3 mice per cage, and were injected i.d.

- 12045519 peritoneal injection of the immunosuppressant cyclophosphamide (150 mg/kg).

After 24 h, 4-cage mice were again treated by intraperitoneal injection with the immunosuppressant cyclophosphamide (100 mg/kg). E. coli strain ATCC-25922 (Enterobacteria ATCC-25922) was isolated on an MNA plate. The isolated colonies were collected and dissolved in saline to obtain a bacterial solution of E. coli ATCC-25922 at a concentration of 1.00E+07 CFU/ml for subsequent use in infecting mouse thigh muscle. The amount of bacterial solution administered by injection into the thigh muscle of experimental mice was 100 μl/mouse, that is, the inoculation amount was 1.00E+06 CFU/mouse. 2 h after infection, thigh muscle tissue from mice in the control group was collected and placed in 10 ml of saline, homogenized, and spot-plated on a gradient dilution plate.

Specific administration to mice was carried out as follows.

(1) 2 hours after infection: After 2 hours of infection, the thigh muscle tissue of mice in the first cage was collected and placed in 10 ml of saline, homogenized and spot plated on a gradient dilution plate, in duplicate for each mouse. The number of bacteria introduced into the mouse thigh muscle tissue was counted. Mice in the third and fourth cages were treated by subcutaneous injection with 30 mg/kg plazomycin and compound 1, respectively.

(2) 10 hours after infection: Mice in the third and fourth cages were subcutaneously injected with 30 mg/kg plazomycin and compound 1, respectively. After 24 hours of infection, the thigh muscle tissue of mice in the second to fourth cages was collected and placed in 10 ml of physiological solution, homogenized and spot-placed on a plate with a gradient dilution, in duplicate for each mouse. The number of bacteria introduced into the mouse thigh muscle tissue was counted, and the results of the experiment were summarized and shown in FIG. 1.

Conclusion: The results shown in Fig. 1 indicate that compound 1 at 30 mg/kg has better in vivo efficacy than plazomycin.

Experimental example 5. Experimental assessment of the effectiveness of the drug in mice (mouse pneumonia model).

CD-1 mice were divided into 7 cages, 3 mice per cage, and given the immunosuppressant cyclophosphamide (150 mg/kg) by intraperitoneal injection on day 4.

On day 1, 7-cage mice were again treated by intraperitoneal injection with the immunosuppressant cyclophosphamide (100 mg/kg). Strain Kpn ATCC-BAA-1705 (Klebsiella Pneumoniae ATCC-VAA1705) was isolated on a plate with MNA. The isolated colonies were collected and dissolved in physiological solution to obtain a bacterial solution Kpn ATCC-BAA-1705 with a concentration of 4.00E+08 CFU/ml for subsequent use for infection of mouse lungs. The amount of bacterial solution with which the lungs of experimental mice were infected was 50 μl/mouse, that is, the inoculation amount was 2.00E+07 CFU/mouse. At 2 h and 24 h postinfection, lung tissue from mice in the control group was collected and placed in 5 ml of saline, homogenized, and spot-plated on a gradient dilution plate.

Specific administration to mice was carried out as follows.

(1) 2 h postinfection: At 2 h postinfection, lung tissue from mice in the first cage was collected and placed in 5 ml saline, homogenized, and spot plated on a gradient dilution plate in duplicate for each mouse. The number of bacteria introduced into the mouse lung tissue was counted. Mice in the third and fourth cages were administered by subcutaneous injection, respectively, 30 mg/kg and 10 mg/kg of compound plazomycin, mice in the fifth and sixth cages were administered by subcutaneous injection, respectively, 30 mg/kg and 10 mg/kg of compound 1, and mice in the seventh cage were given 100 mg/kg meropenem by subcutaneous injection.

(2) 10 hours after infection: Mice in the third and fourth cages were administered by subcutaneous injection, respectively, 30 mg/kg and 10 mg/kg plazomycin, mice in the fifth and sixth cages were administered by subcutaneous injection, respectively, 30 mg/kg and 10 mg/kg of compound 1, and mice in the seventh cage were administered by subcutaneous injection of 100 mg/kg of meropenem. After 24 hours of infection, the lung tissue of mice in the second to seventh cages was collected and placed in 5 ml of saline, homogenized and spot-plated on a gradient dilution plate, in duplicate for each mouse. The number of bacteria carried in mouse lung tissue was counted, and the results of the experiment were summarized and shown in FIG. 2.

Conclusion: in Fig. 2 shows that plazomycin and compound 1 have good in vivo activity in a model of lung infection with Klebsiella Pneumoniae strain 1705. At the same time, the effectiveness of compound 1 is better than that of plazomycin, and the effectiveness of compound 1 at a dose of 10 mg/kg is equivalent to the effectiveness of plazomycin at a dose of 30 mg/kg.

Experimental Example 6: Report of a Study on the Hearing Safety of New Aminoglycoside Antibiotics.

Objectives of the study.

To evaluate the effects of compound 1 and the existing antibiotic plazomycin on auditory function in guinea pigs and to assess the hearing toxicity of compound 1.

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Research method.

Healthy adult guinea pigs (150-250 g) were used as study subjects and were randomly divided into saline control group, gentamicin group, plazomycin compound group and compound 1 group with 8 animals in each group.

Subcutaneous administration was used, and the following tests were performed during and after administration for 14 consecutive days.

1. To analyze the effect of various drugs on the auditory function of guinea pigs, the compound action potential (APP) was recorded in animals on the 14th day (29th day, that is, 4 weeks) after administration. The results were analyzed and changes in threshold shift, amplitude, latency and other parameters were compared among different treatment groups.

2. After processing different groups of animals and collecting data on their auditory function, the cochlea of the animals was removed for fixation and staining. On one side, the basilar membrane surface of the cochlea was prepared to count hair cell loss to map the cochlea, and on the other side, the cochlea was decalcified and frozen sections were taken. The density of spiral ganglion neurons was counted and compared between groups.

Research results.

1. Method of administration and processing.

Gentamicin from Dalian Meilun Biotechnology Co., Ltd. was used. and plazomycin and compound 1 from WuXi AppTec (WuHan) Co., Ltd., solutions of which were prepared each time immediately before use using saline solution to dissolve to a concentration of 50 mg/ml, and the injection dose was 100 mg/kg body weight. Method: Subcutaneous injection and confirm that there is no fluid leakage after each injection.

2. Analysis of the compound action potential (APP).

Compound action potential (CAP) was tested and recorded when clicks and different frequencies of pure tones (1 kHz-32 kHz) were applied to each group of animals, and changes in amplitude and latency were mainly compared when clicks and mid- and high-frequency pure tones were applied ( 16, 32 kHz). The magnitude of the amplitude reflects the sensitivity of the hair cells and auditory nerves. The greater the amplitude and the greater the slope of the input/output (I/O) curve, the better the sensitivity and the better the function. In addition, the cochlea from the apical helix to the basal helix responded to low-frequency to high-frequency sounds, respectively, which is the frequency correspondence of the basilar membrane of the cochlea. Functional changes at different frequencies correspond to different structural and functional changes in the cochlea from the apical helix to the basal helix. The magnitude of latency has also been related to hair cell function and auditory nerve response. In general, increasing the threshold value when the cochlea is damaged will inevitably lead to an increase in latency. In addition, the increase in latency when the threshold value did not change significantly also reflected a decrease in the synchrony of discharges in the auditory nerve. In other words, the increase in latency reflected a decrease in response function. Previously, the ototoxicity of aminoglycoside antibiotics was mainly concentrated in the high-frequency region. In this study, the gentamicin group was consistent with previous results, as summarized below.

Compound 1 only caused a decrease in the amplitude of the experimental group's PDS at high frequency (32 kHz), indicating hearing damage in the high-frequency region, but the amplitude was still higher than that of the gentamicin and plazomycin groups. Damage in the plazomycin group occurred over a wider range, damage occurred at both 16 kHz and 32 kHz, and damage at 32 kHz was greater than that of compound 1 but significantly less than that of the gentamicin group. The damage in the experimental group caused by gentamicin was concentrated in the high frequency (32 kHz) region, the threshold shift at 32 kHz was obvious, and the damage was more severe than that of the drugs in the other two groups (see Figs. 3-5).

1) The results for amplitude at 16 kHz showed that compound 1 was no different from the control group, while the plazomycin group had an impairment of 25.9%.

2) The results for the 32 kHz PDS amplitude showed that compound 1, plazomycin and gentamicin caused hearing damage at 32 kHz of 34.7%, 48.2% and 74.3%, respectively, that is, compound 1 still caused hearing damage at 32 kHz, which, however, was reduced by 13.5% and 39.6%, respectively, compared with plazomycin and gentamicin.

3) The click PDS amplitude indicated that both compound 1 and gentamicin were consistent with the control group, while plazomycin caused hearing damage.

The specific meanings were as follows.

1) PDS amplitude at 16 kHz: Two-factor analysis of variance (Holm-Sidak method) showed that there were differences between the four groups of animals, F3, 570 = 7.858, p < 0.001. Among them, there was no statistical difference in hearing between animals in the compound 1 group, control group and gentamicin group. The hearing of animals in the plazomycin group was lower than that of the control group (t=4.566, p<0.001), the gentamicin group (t=4.099, p<0.001) and the compound 1 group (t=2.799,

- 14 045519 p=0.021), respectively. *: p<0.05. The response of each group was maximum at 90 dB, at which the hearing of animals in the compound 1 group (381.646 ± 20.895 μV) was significantly higher than in the plazomycin group (282.058 ± 22.569 μV, t = 2.799, p = 0.021) and was not significantly different from the control group (383.130 ± 19.545) and the gentamicin group (373.329 ± 15.332 μV).

2) PDS amplitude at 32 kHz: Two-factor analysis of variance (Holm-Sidak method) showed that there were differences between the four groups of animals, F3, 570 = 100.611, p < 0.001. The hearing of animals in the compound 1 group was lower than the hearing in the control group (t=5.019, p<0.001), higher than in the plazomycin group (t=3.128, p=0.002) and in the gentamicin group (t=10.484, p< 0.001). The response of each group was maximum at a value of 90 dB, at which the hearing of animals in the compound 1 group (79.420 ± 7.000 μV) was lower than in the control group (121.608 ± 6.548 μV, t = 4.401, p < 0.001), higher than in the gentamicin group (31.272±5.137 μV, t=5.545, p<0.001) and the plazomycin group (62.982±7.561 μV, t=1.595, p=0.111).

3) PDS amplitude at click: Two-factor analysis of variance (Holm-Sidak method) showed differences between the four groups of animals, F3, 570 = 6.751, p < 0.001. The hearing of animals in the compound 1 group was significantly better than in the plazomycin group (t=3.493, p=0.003), and did not differ significantly between the control group and the gentamicin group.

Overall, the results for the amplitude of the PDS proved that the hearing damage in experimental animals caused by compound 1 was significantly less than the hearing damage caused by gentamicin and plazomycin.

3. Changes in the number of hair cells.

To compare the effects of different drugs on hair cells, hair cell staining and counting were performed on the entire basilar membrane of the cochlea. The results showed that in the gentamicin group, there was a loss of outer hair cells of 12-67.7% in the mid- and high-frequency region (60-100% of the apical helix), and the loss was more obvious in the high-frequency region. In the plazomycin group, there was 11.2-28.1% outer hair cell loss in the high-frequency region (70-100% of the apical turn) and 16.7-24.2% outer hair cell loss at the onset of the apical turn. curl (10-20%). However, in the compound 1 group, outer hair cell loss occurred only in the low frequency region (from the upper helix to 40%), in which the rate of outer hair cell loss was approximately 2.5-11%, and outer hair cells were relatively intact in the high frequency region ( see Fig. 6 B). Specific values are shown in table. 4.

In the compound 1 group, the inner hair cells were almost undamaged. In both the plazomycin and gentamicin groups, there was a loss of inner hair cells near the end of the basal helix (100% of the apical helix) of 3.5 ± 3.0% and 9.3 ± 4.1%, respectively (see Table 1). Fig. 6A). Specific values are shown in table. 5.

Thus, in the compound group, only a small loss of outer hair cells in the apical helix was observed, and the rest, in particular the basal helix and inner hair cells, remained intact. The toxicity of compound 1 to hair cells was significantly lower than that of gentamicin and plazomycin.

4. Changes in neurons of the spiral ganglion.

The guinea pig cochlea was defined as helix 1, helix 2, helix 3, and helix 4 from the basal to the apical helix. Spiral ganglion neurons (SGNs) were stained using TuJ on frozen sections, and their area-specific density was counted and compared between groups. There was no significant difference in SGN density in each helix between the compound 1 group and the control group, that is, there was no damage to spiral ganglion neurons. In the gentamicin group, there was obvious damage in every helix. The plazomycin group showed a decrease in SGN density near the apical helix, but it was better than the gentamicin group. Two-way ANOVA revealed a significant difference between groups, F(3, 74)=35.43, p<0.0001 (see Figure 7). Specific values are shown in table. 6.

Conclusion: Based on the analysis of compound action potentials between different groups, it was confirmed that in the compound 1 group, hearing damage was observed only at high frequency (32 kHz), which was better than that in the plazomycin and gentamicin groups. Observation of hair cells and neurons of the spiral ganglion confirmed that, with the exception of 2.5-11% loss of outer hair cells near the apical helix, compound 1 did not cause obvious damage to outer hair cells in other areas, and the number of inner hair cells and the number of neurons spiral ganglion were not affected, which was significantly better than in the gentamicin and plazomycin groups. Therefore, compound 1 was administered subcutaneously to animals (guinea pigs) for 14 consecutive days, and its ototoxicity was less than that of plazomycin and gentamicin after another 14 days. Based on the results of this study, it was confirmed that Compound 1 prepared according to the present invention was better than plazomycin and gentamicin in terms of auditory toxicity.

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Table 4

Outer ear hair cell damage at day 29 (%, percentage)

Damage percentage(%) Connection 1 Plazomycin Gentamicin 10 11.0±1.3 24.4±13.5 9.5±2.9 20 6.3±0.9 16.7±13.4 4.1±0.7 thirty 6.2±1.1 10.5±6.5 2.7±0.7 40 2.5±1.4 7.8±4.0 3.2±1.0 50 1.1±0.8 7.2±2.0 3.2±1.5 60 1.4±1.5 5.7±5.1 12.6+5.6 70 0.7±0.3 11.2±11.8 13.6+6.1 80 0.5±0.3 19.9±15.7 12.0+5.4 90 0.2±0.3 20.7±7.9 42.8+8.2 100 0.9±0.7 28.1±10.1 67.7+9.8

Table 5

Inner hair cell damage at day 29 (%, percentage)

Damage percentage (%) Connection 1 Plazomycin Gentamicin 10 0.3±0.3 0.5±0.4 0 20 0 0.3±0.3 0 thirty 0 0.5±0.5 0 40 0.1±0.1 0.2±0.2 0 50 0 0 0 60 0 0.8±0.8 0.4±0.4 70 0.3±0.3 1.3±1.3 0.6±0.6 80 0 0 0.2±0.2 90 0 0 2.5±1.7 100 0.7±0.5 3.5±3.0 9.3±4.1

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Table 6

Change in the density of neurons of the spiral ganglion on the 29th day (p/10000 µm ² )

Curl Gentamicin Plazomycin Connection 1 Control Curl 1 4.407±0.517 6.816±0.852 8.230±0.500 7.548±0.534 Curl 2 5.540±0.757 5.876±0.536 8.282±0.254 7.695±0.298 Curl 3 4.604±0.598 5.553±0.793 8.136±0.247 7.935±0.292 Curl 4 5.259±0.280 5.641±0.767 7.469±0.772 6.936±0.205

Experimental Example 7: Toxicity test of compounds of the present invention on NK-2 cells.

Cell preparation.

On the day of the experiment, when the NK-2 cells in the culture flask reached 80-90% confluency, the culture medium was removed, the cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS) and digested for 1-2 min using 3 ml of trypsin (in the flask for T150 cell cultures), and 9 ml of complete medium (RPMI-1640+10% FBS) was immediately added to complete the digestion. Once completed, a single cell suspension was obtained by gentle pipetting and centrifuged at 1000 rpm for 5 min. The supernatant was removed, fresh complete medium was added, and the cells were pipetted evenly. The actual cell density was measured according to the cell counter and the cell suspension was adjusted to 2.5 x 10 ⁵ cells/ml. 80 µl of cell suspension was collected using a row pipettor and added to a 96-well black-bottom plate ( ^2x104 cells/well), which was designated as a cell plate, and then incubated in a CO2 incubator for 4. 5 hours

Receiving connections:

a) a stock solution of the compound was prepared according to the following table using complete medium as a solvent;

Table 7

Connection information

Compound Weight (mg) Purity (%) Concentration (mg/ml) Volume (µl) Plazomycin 5.88 99.65 50 117.19 Compound 1 5.51 95 50 104.69 Netilmicin 5.00 - 50 100.00 Amikacin 5.74 - 50 114.80

b) 50 microliters of complete medium was added to columns 3-11 of a 96-well V-bottom plate;

c) 75 microliters of test compound (50 mg/ml) and positive control were added to the second column of a 96-well V-bottom plate;

d) 25 microliters of compound was withdrawn from the second column and added to the third column, blown and aspirated several times using a multichannel pipette, then 25 microliters of liquid was withdrawn from the third column and added to the fourth column, and then subjected to a 3-fold serial dilution to 10 th columns. From column 2 to column 11, the compound concentration was 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02, 0.008, 0 mg/ml;

f) 20 μl of the resulting compound solutions with different concentrations were transferred using a multi-channel pipette into the corresponding wells of the cell plate, which was defined as the test plate.

Claims (4)

Culturing the test plate. All plates were incubated in an incubator at 37°C, 5% CO2 for 43 hours. Reading. After incubation, 10 microliters of Alma Blue was added to the test plate. The test plate was immediately incubated in an incubator at 37°C, 5% CO2 for 3 hours. The fluorescence value of each well of the test plate was then read using a microplate reader (wavelength Ex 540 nm/Et 585 nm). The curve was then simulated using Prism software to calculate the _CC50 value. Research results. Table 8 Experimental results and predicted CC ₅₀ Compound CC ₅ o for NK-2 cell (mg/ml) Maximum degree of inhibition (%) of CC ₅ o compound for cells predicted using software (Prism) (mg/ml) Plazomycin >10 35.98 19.53 Compound 1 >10 21.96 113.9 Netilmicin 10.64 48.73 10.64 Amikacin 8,157 68.40 8,156 Conclusion: The toxicity of compound 1 and plazomycin to HK-2 cells was significantly lower than that of netilmicin and amikacin. Combined with the toxicity regression curve and software prediction (Figures 8-11), the toxicity of Compound 1 to HK-2 cells was lower than that of plazomycin. CLAIM 1. A compound represented by the following formula: or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition containing as an active ingredient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable carrier. 3. Use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 or a pharmaceutical composition according to claim 2 for the preparation of a medicament for the treatment of diseases associated with bacterial infection. 4. Use according to claim 3, wherein the bacterial infection is a carbapenem-resistant infection caused by Enterobacteriaceae. -