阅读说明：本技术 用于治疗新型冠状病毒肺炎继发细菌感染性疾病的截短侧耳素类化合物 (Pleuromutilin compounds for treating novel bacterial infectious diseases secondary to coronavirus pneumonia ) 是由 袁莉莉 李少春 于 2020-04-20 设计创作，主要内容包括：本发明涉及新型截短侧耳素类化合物、它们的药物组合物及使用方法。此外,本发明还涉及用于治疗细菌感染的治疗方法,包括耐药性微生物造成的感染,包括多重耐药性微生物。本发明特别涉及用于治疗新型冠状病毒(COVID-19或SARS-Cov-2)肺炎继发细菌感染性疾病的方法和化合物及其药用组合物,为打赢新冠肺炎疫情防控阻击战提供科技支撑。(The present invention relates to novel pleuromutilin compounds, their pharmaceutical compositions and methods of use. In addition, the invention relates to therapeutic methods for treating bacterial infections, including infections caused by drug-resistant microorganisms, including multidrug-resistant microorganisms. The invention particularly relates to a method and a compound for treating a novel bacterial infectious disease secondary to coronavirus (COVID-19 or SARS-Cov-2) pneumonia and a medicinal composition thereof, and provides scientific support for winning new coronary pneumonia epidemic situation prevention and control fight.)

1. The present invention provides a compound of formula I, or a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate, or crystalline form thereof:

wherein the content of the first and second substances,

n is an integer of 0 to 4;

q is-CH₂-,

R¹Is hydroxy, hydrogen or deuterium;

R²is hydroxy, hydrogen, deuterium, halogen;

R^2‘is hydroxy, hydrogen, deuterium, halogen;

R³is hydroxyHydrogen, deuterium, halogen;

R⁴is hydrogen, deuterium, halogen, hydroxy, amino, C₁-C₆Alkyl or cycloalkyl;

R^4‘is hydrogen, deuterium, halogen, hydroxy, amino, C₁-C₆Alkyl or cycloalkyl radicals, R^4‘And R⁴May form a ring of 3 to 6 carbon atoms, R^4‘And R⁴May form a 3-6 heteroatom ring with O, S, N;

R⁵is hydrogen, one or more halogen, hydroxy, amino substituted C₁-C₆Alkyl or cycloalkyl;

R⁶is hydrogen, deuterium, C₁-C₆Alkyl or cycloalkyl.

2. The present invention provides a compound of formula II, or a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate, or crystalline form thereof:

wherein the content of the first and second substances,

n is an integer of 0 to 4;

R¹is hydroxy, hydrogen or deuterium;

R²is hydroxy, hydrogen, deuterium, halogen;

R^2‘is hydroxy, hydrogen, deuterium, halogen;

R³is hydroxy, hydrogen, deuterium, halogen;

R⁴is hydrogen, deuterium, halogen, hydroxy, amino, C₁-C₆Alkyl or cycloalkyl;

R⁵is hydrogen, one or more halogen, hydroxy, amino substituted C₁-C₆Alkyl or cycloalkyl;

R⁶is hydrogen, deuterium, C₁-C₆Alkyl or cycloalkyl.

3. A compound as provided in claims 1-2, or a stereoisomer, prodrug, active metabolite, or pharmaceutically acceptable salt, solvate, or crystalline form thereof, wherein the compound is represented by any one of the following structural formulae:

4. a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 3 in unit dosage form and one or more pharmaceutically acceptable carriers.

5. A combination comprising a therapeutically effective amount of a compound according to any one of claims 1 to 3 and another agent having therapeutic activity against a pathogenic microorganism-mediated disease.

6. Use of a compound of any one of claims 1 to 3, an N-oxide thereof, or a prodrug thereof, for the manufacture of a medicament for the treatment of a condition mediated by a pathogenic microorganism.

7. The use of claim 6, wherein the microorganism-mediated disorder is selected from the group consisting of systemic infectious diseases, ocular infectious diseases, human sexually transmitted diseases, upper respiratory infectious diseases, lower respiratory infectious diseases, skin and soft tissue infectious diseases, acute sinusitis, chronic bronchitis, community-acquired pneumonia, hospital-acquired pneumonia, coronavirus pneumonia, and novel coronavirus pneumonia (COVID-19).

Technical Field

The present invention relates to novel pleuromutilin compounds, their pharmaceutical compositions and methods of use. In addition, the invention relates to therapeutic methods for treating bacterial infections, including infections caused by drug-resistant microorganisms, including multidrug-resistant microorganisms. The invention particularly relates to a method and a compound for treating bacterial infectious diseases secondary to novel coronavirus (COVID-19 or SARS-Cov-2) pneumonia and a medicinal composition thereof, and provides scientific support for fighting against winning epidemic situations, controlling and fighting against warfare.

Technical Field

Novel coronavirus pneumonia is often followed by bacterial infections, the most closely related bacteria including Streptococcus pneumoniae, Haemophilus influenzae, as well as Mycoplasma pneumoniae, Chlamydia and Moraxella catarrhalis. The drugs with more clinical application in pneumonia comprise moxifloxacin, azithromycin, ceftriaxone and cefuroxime, but with the abuse of antibiotics, drug-resistant bacteria continuously appear, and a novel antibiotic is urgently needed in clinic. According to the novel coronavirus lung diagnosis and treatment principle, although more moxifloxacin and azithromycin are used for secondary bacterial infection, the effect still needs to be further confirmed, and the existing global antibiotics can not be developed to meet the requirements of clinical anti-infective drugs.

Pleuromutilin (pleuromutilin), a compound of the formula,

antibiotics which are naturally occurring, are produced, for example, by The basidiomycetes pleuromutilis (Basidomycetes Pleurotusmutilus) and Pleurotus palustris (P.passecerrianus), see The Merck lndex, 13 th edition, item 7617. Many other pleuromutilins have been developed as antimicrobial agents, having the main ring structure of pleuromutilin and being substituted on the side chain primary alcoholic hydroxyl group. Due to their pronounced antimicrobial activity, a series of pleuromutilin derivatives, such as the amino-hydroxy-substituted cyclohexylsulfanylacetamidelines disclosed in WO2008/113089, have been found to have superior antimicrobial activity. The 14-O- { [ (4-amino-2-hydroxy-cyclohexyl) sulfanyl ] acetyl) -thimerosides disclosed in WO2008/113089 show good application prospects, they have good inhibitory activity against gram-positive and gram-negative pathogens, which are associated with pathogenic bacteria associated with infections of the respiratory tract and the skin and skin structures.

These disclosed pleuromutilin derivatives are simple substitutions on the side chain hydroxyl groups and are not modified on the main structural parent ring. Therefore, the prior side chain modified pleuromutilin derivatives have weak activity against gram-negative bacteria, poor in vivo stability, low oral absorption and utilization rate, side effect of diarrhea and even cardiotoxicity.

The present invention provides novel pleuromutilin derivatives. They have the advantages of strong activity against gram-negative bacteria, high in-vivo stability, good oral absorption availability, good lung targeting property, low cardiac toxicity and no diarrhea side effect. In particular provides a new choice for a novel medicine for treating bacterial infection secondary to coronavirus pneumonia.

Detailed Description

I. Compound (I)

Briefly, the present invention relates to novel pleuromutilin derivatives. The compounds of the present invention have the following structural formula (I), formula (II) or are stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof:

in one aspect, the invention relates to compounds of formula (I),

wherein the content of the first and second substances,

n is an integer of 0 to 4;

q is-CH₂-,

R¹Is hydroxy, hydrogen or deuterium;

R²is hydroxy, hydrogen, deuterium, halogen;

R^2‘is hydroxy, hydrogen, deuterium, halogen;

R³is hydroxy, hydrogen, deuterium, halogen;

R⁴is hydrogen, deuterium, halogen, hydroxy, amino, C₁-C₆Alkyl or cycloalkyl;

R^4‘is hydrogen, deuterium, halogen, hydroxy, amino, C₁-C₆Alkyl or cycloalkyl radicals, R^4‘And R⁴May form a ring of 3 to 6 carbon atoms, R^4‘And R⁴May form a 3-6 heteroatom ring with O, S, N;

R⁵is hydrogen, one or more halogen, hydroxy, amino substituted C₁-C₆Alkyl or cycloalkyl;

R⁶is hydrogen, deuterium, C₁-C₆Alkyl or cycloalkyl.

In another aspect, the invention relates to a compound of formula (II),

wherein the content of the first and second substances,

n is an integer of 0 to 4;

R¹is hydroxy, hydrogen or deuterium;

R²is hydroxy, hydrogen, deuterium, halogen;

R^2‘is hydroxy, hydrogen, deuterium, halogen;

R³is hydroxy, hydrogen, deuterium, halogen;

R⁴is hydrogen, deuterium, halogen, hydroxy, amino, C₁-C₆Alkyl or cycloalkyl;

R⁵is hydrogen, one or more halogen, hydroxy, amino substituted C₁-C₆Alkyl or cycloalkyl;

R⁶is hydrogen, deuterium, C₁-C₆Alkyl or cycloalkyl.

In yet another aspect, the pleuromutilin derivatives of formula (I) or formula (II), stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof, according to the present invention are represented by any one of the following structural formulae:

the compounds of the invention are generally used in the form of the free acid or free base. Alternatively, the compounds of the present invention may be used in the form of acid or base salts. Acid addition salts of the free amino compounds of the present invention may be prepared by methods well known in the art and may be prepared from organic and inorganic acids. Suitable acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, phenylacetic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, benzoic acid, adipic acid, benzoic acid, benzenesulfonic acid, butyric acid, camphoric acid, camphorsulfonic acid, cyclohexylsulfamic acid, ethanesulfonic acid, fumaric acid, glutamic acid, glycolate salts, hemisulfuric acid, 2-hydroxyethanesulfonic acid, heptanoic acid, hexanoic acid, hydroxymaleic acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, phenylacetic acid, diphosphonic acid, picric acid, trimethylacetic acid, propionic acid, quinic acid, salicylic acid, stearic acid, succinic acid, sulfamic acid, malic acid, maleic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, oxalic acid, pamoic acid, picric, Sulfanilic acid, tartaric acid, toluene sulfonic acid (p-toluene sulfonic acid), trifluoroacetic acid, and undecanoic acid. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, hydrogen carbonate, hydroiodic, persulfuric, and hydrogen sulfate. Base salts include salts with carboxylate anions and include salts with anions such as those selected from alkali metal salts such as sodium, lithium and potassium; alkaline earth metal salts such as aluminum, calcium, and magnesium salts; salts with organic bases such as choline, meglumine, piperazine, dicyclohexylamine salts, and N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, ornithine, etc. Thus, the term "pharmaceutically acceptable salt" of formula (I) or formula (II) shall include and all acceptable salt forms.

Basic amino-containing groups can be quaternized with such agents as: lower alkyl halides, such as methyl, ethyl, propyl and butyl halides: dialkyl sulfates such as dimethyl, diethyl, dibutyl, diamyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl halides; arylalkyl halides such as benzyl bromide and others. Non-toxic physiologically acceptable salts are preferred, although other salts may be used, for example for isolating or purifying the product. The salt may be formed by conventional means, for example by reacting the product in free base form with one or more equivalents of a suitable acid in a solvent or medium in which the salt is insoluble, removing the solvent in vacuo or by freeze-drying, or by exchanging the anion of an existing salt for another anion on a suitable ion exchange resin.

Representative examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups of compounds of formula (I) or formula (II). Further, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, propyl ester, and the like may be included. In the case of hydroxyl groups, mixed anhydrides such as methoxy, ethoxy, propoxy, tert-butoxy, and the like may be included. The effect of long-acting is achieved by the compounds of formula (I) and their metabolites forming chain fatty ethers, long-chain fatty esters or long-chain fatty amides via hydroxyl or amino groups with long-chain fatty alcohols or acids, such as pentanol or acid, hexanol or acid, heptanol or acid, octanol or acid, nonanol or acid, decanol or acid, lauryl alcohol or acid, stearyl alcohol or acid, and the like.

The compounds of formula (I) or formula (II) have one or more chiral centres, it being understood that the invention includes all such stereoisomers, including enantiomers and diastereomers. It is therefore to be understood that the present invention includes within its definition any such optically active or racemic form having the above-mentioned activity, provided that certain compounds of formula (I) or formula (II) may exist in optically active or racemic form due to one or more asymmetric carbon atoms. The present invention includes all such stereoisomers having activity as defined herein.

The synthesis of the optically active form can be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials, or by resolution of the racemic form. The racemates can be separated into the individual enantiomers using known methods (cf., for example, Advanced Organic Chemistry: third edition: Author J March, p. 104-107). Suitable methods include the formation of diastereomeric derivatives by reaction of the racemic material with a chiral auxiliary, followed by separation of the enantiomers, for example by chromatography, followed by cleavage of the auxiliary species. Similarly, the above activities can be assessed using standard laboratory techniques as described below.

Thus, throughout the specification, when reference is made to a compound of formula (I) or formula (II), it is to be understood that the term compound includes isomers, mixtures of isomers and stereoisomers that are derivatives of pleuromutilin.

The configuration of the substituents attached to the asymmetric carbon atom of the thimerosal-tricycles (tricyclus) is preferably the same as the configuration of the native pleuromutilins.

Stereoisomers may be separated using conventional techniques, for example chromatography or fractional crystallisation. The enantiomers may be separated, for example, by separation of the racemates by fractional crystallization, resolution or HPLC. Diastereomers may be separated by separation by different physical properties of the diastereomers, for example, by fractional crystallization, HPLC, or flash chromatography. Specific stereoisomers may alternatively be made by chiral synthesis from chiral starting materials or by derivatization using chiral reagents under conditions that do not cause racemisation or epimerisation.

When a particular stereoisomer is provided (whether by separation, by chiral synthesis, or by other means), it may be advantageous to provide the stereoisomer substantially separately from other stereoisomers of the same compound. In one aspect, a mixture containing a particular stereoisomer of a compound of formula (I) or formula (II) can contain less than 30% by weight, specifically less than 20% by weight, and more specifically less than 10% by weight of other stereoisomers of the same compound. In another aspect, a mixture containing a particular stereoisomer of a compound of formula (I) or formula (II) may contain less than 6% by weight, particularly less than 3% by weight, and more particularly less than 2% by weight, of other stereoisomers of that compound. In another aspect, a mixture containing a particular stereoisomer of a compound of formula (I) or formula (II) can contain less than 1% by weight, specifically less than 0.5% by weight, and more specifically less than 0.1% by weight of other stereoisomers of the compound.

It is to be understood that the present invention includes within its definition any such tautomeric form having the aforementioned activity, provided that certain compounds of formula (I) or formula (II) as hereinbefore defined may exist in tautomeric form. Thus, the present invention relates to all tautomeric forms of the compounds of formula (I) or formula (II), whether or not explicitly detailed in the specification.

It is also understood that certain compounds of formula (I) or formula (II) and pharmaceutically acceptable salts thereof may exist in solvated as well as unsolvated forms such as hydrated forms. It is to be understood that the invention encompasses all such solvated forms. For the sake of clarity, this includes solvated (e.g. hydrated) forms of the free form of the compound, as well as solvated (e.g. hydrated) forms of the salt of the compound.

For the sake of clarity, it is to be understood that the atom of the compound of formula (I) or formula (II) and any examples or embodiments disclosed herein is intended to encompass all isotopes of that atom. For example, H (or hydrogen) includes any isotopic form of hydrogen, including 1H,²H, (D) and³h (T); c includes any isotopic form of carbon, including¹²C、¹³C and¹⁴c; o includes any isotopic form of oxygen, including¹⁶O、¹⁷O and¹⁸o; n includes any isotopic form of nitrogen, including¹³N、¹⁴N and¹⁵n; p includes any isotopic form of phosphorus, including³¹P and³²p; s includes any isotopic form of sulfur, including³²S and³⁵s; f includes any isotopic form of fluorine, including¹⁹F and¹⁸f; cl includes any isotopic form of chlorine, including³⁵Cl、³⁷Cl and³⁶cl, and the like. In one aspect, the compound of formula (I) or formula (II) comprises an isotope of an atom encompassed therein in an amount corresponding to its naturally occurring abundance. However, in certain instances, it is desirable to have atoms enriched in one or more particular isotopes that are typically naturally present in lower abundance. For example,¹h is typically present in an abundance of greater than 99.98%, however, in one aspect, the compounds of the invention may be enriched at one or more positions where H is present¹H or³H. In another aspect, when the compounds of the invention are enriched in radioisotopes, for example³H and¹⁴c, the compounds are useful in drug and/or substrate tissue distribution assays. It is to be understood that the present invention encompasses all such isotopic forms which are useful in the treatment of bacterial infections.

II. Application method

The compounds of the present invention have the general formula (I) or formula (II) or are stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof, which exhibit activity against pathogenic microorganisms.

For example, the following antibacterial activities: against gram-positive bacteria, for example, coagulase-positive Staphylococci (Staphyloccci) such as Staphylococcus aureus (Staphyloccocus aureus), coagulase-negative Staphylococci such as Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus haemolyticus (Staphyloccocus haemolyticus), and Streptococcus (Streptococcus) such as Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus pneumoniae (Streptococcus pneumoniae), Enterococci (Enterococci) such as Enterococcus faecium (Enterococcus faecium), and Listeria monocytogenes (Listeria monocytogenes); and anti-gram-negative bacteria such as Moraxella (Moraxella) such as Moraxella catarrhalis (Moraxella catarrha1is), and Haemophilus such as Haemophilus influenzae (Haemophilus influenzae), and Legionella such as Legionella pneumophila (Legionella), Neisseriaceae such as Neisseria gonorrhoeae (Neisseria gonorrhoeae); and anti-mycoplasmas (mycoplasmas), Chlamydia (Chlamydia), and obligate anaerobes such as Bacteroides fragilis (Bacteroides fragilis), Clostridium difficile (Clostridium difficile), Clostridium (fusobacterium spp.), and Propionibacterium (Propionibacterium spp.).

In vitro Activity against aerobic bacteria by agar dilution test or microdilution test according to the Clinical Laboratory Standards Committee (CLSI, formerly NCCLS) document No. M7-A7Vo1.26, No.2: "Methods for dilution of Antimicrobial Succinity Tests for bacteria that Grow aerobic-applied Standard; measurement was performed in the seventh edition (2006) ": anti-anaerobe assays were performed according to the clinical laboratory Standard Committee (CLSI, Pre-NCCLS) document No. Mll-A6, Vo1.24, No.2: "Methods for anti-microbial Suceptibility Testing of Antimicrobial Bacteria-Approved Standard; sixth edition (2004) ": and in vivo activity was tested against staphylococcus aureus in a mouse model of septicemia.

The compounds of the invention are therefore suitable for the treatment and prophylaxis of diseases which are mediated by microorganisms, for example bacteria. Diseases which can be treated also include, for example, Helicobacter (Helicobacter pylori) mediated diseases and Mycobacterium tuberculosis (Mycobacterium tuberculosis) mediated diseases. Diseases that can be treated also include inflammatory diseases in general, where the inflammation, including acne for example, is mediated by microorganisms.

In another aspect, the present invention provides a compound of the invention for use as a medicament, preferably as an antimicrobial such as an antibiotic and e.g. an anti-anaerobic medicament.

In another aspect, the invention provides a compound of the invention for use in the treatment of acne.

In a further aspect, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of a disease mediated by a microorganism, such as a bacterium, for example a disease mediated by a bacterium, for example selected from staphylococci, streptococci, enterococci; diseases mediated by bacteria, for example selected from moraxella, haemophilus, legionella, neisseriaceae; diseases mediated by helicobacter; diseases mediated by mycobacterium tuberculosis; diseases mediated by mycoplasma, chlamydia and obligate anaerobes.

The use of a compound of the invention having the general formula (I) or formula (II) or being a stereoisomer, prodrug, active metabolite or a pharmaceutically acceptable salt, solvate or crystalline form thereof, in the manufacture of a medicament for the treatment of a human disease condition mediated by a bacterial infection.

The compounds of the invention are also suitable for use as veterinary, e.g. veterinary active compounds, e.g. for the prevention and treatment of microbial, e.g. bacterial, diseases in animals, e.g. poultry, pigs and cattle: and dilution liquids such as are used in artificial insemination and egg soaking techniques. In another aspect, the present invention provides a compound of the invention for use as a veterinary drug. In a further aspect, the present invention provides a compound of the invention for use in the preparation of a veterinary composition for use as a veterinary drug. In another aspect, the invention provides a veterinary method of prevention and treatment of microbial, e.g. bacterial, diseases, which comprises administering to a subject in need of such treatment an effective amount of a compound of the invention, e.g. in the form of a veterinary composition.

In one aspect, the terms "infection" and "bacterial infection" may refer to a gynecological infection. In another aspect, the terms "infection" and "bacterial infection" may refer to a Respiratory Tract Infection (RTI). In yet another aspect, the terms "infection" and "bacterial infection" may refer to a sexually transmitted disease. In yet another aspect, the terms "infection" and "bacterial infection" may refer to Urinary Tract Infection (UTI). In a further aspect, the terms "infection" and "bacterial infection" may refer to an acute episode of chronic bronchitis (ACEB). In a still further aspect, the terms "infection" and "bacterial infection" may refer to acute otitis media. In one aspect, the terms "infection" and "bacterial infection" may refer to acute sinusitis. In another aspect, the terms "infection" and "bacterial infection" may refer to an infection by a drug-resistant bacterium. In yet another aspect, the terms "infection" and "bacterial infection" may refer to catheter-related sepsis. In yet another aspect, the terms "infection" and "bacterial infection" may refer to chancroid. In a further aspect, the terms "infection" and "bacterial infection" may refer to chlamydia. In a still further aspect, the terms "infection" and "bacterial infection" may refer to community-acquired pneumonia (CAP). In still a further aspect, the terms "infection" and "bacterial infection" may refer to complex skin and skin structure infections. In one aspect, the terms "infection" and "bacterial infection" may refer to uncomplicated skin and skin structure infections. In another aspect, the terms "infection" and "bacterial infection" may refer to endocarditis. In yet another aspect, the terms "infection" and "bacterial infection" may refer to febrile neutropenia. In yet another aspect, the terms "infection" and "bacterial infection" may refer to gonococcal meningitis. In a further aspect, the terms "infection" and "bacterial infection" may refer to gonococcal urethritis. In a still further aspect, the terms "infection" and "bacterial infection" may refer to nosocomial pneumonia (HAP). In yet another aspect, the terms "infection" and "bacterial infection" may refer to osteomyelitis. In a further aspect, the terms "infection" and "bacterial infection" may refer to sepsis. In a still further aspect, the terms "infection" and "bacterial infection" may refer to syphilis. In a further aspect, the terms "infection" and "bacterial infection" may refer to intra-abdominal infection (IAI). In a further aspect, the terms "infection" and "bacterial infection" may refer to a bacterial infection secondary to influenza. In a further aspect, the terms "infection" and "bacterial infection" may refer to a bacterial infection secondary to coronary viral pneumonia. In a further aspect, the terms "infection" and "bacterial infection" may refer to a secondary bacterial infection of a novel coronavirus pneumonia.

In one embodiment of the present invention, the terms "infection" and "bacterial infection" refer to an infection by a gram-negative bacterium, also referred to as a "gram-negative infection". In one aspect of this embodiment, the gram-negative infection is an infection that is resistant to one or more antibiotics. In one aspect of this embodiment, the gram-negative infection is an infection that is resistant to multiple drugs.

In one embodiment of the invention, the terms "infection" and "bacterial infection" refer to an infection by a gram-positive bacterium, also referred to as a "gram-positive infection". In one aspect of this embodiment, the gram-positive infection is an infection that is resistant to one or more antibiotics. In one aspect of this embodiment, the gram-positive infection is an infection that is resistant to multiple drugs.

The use of a compound of the invention having the general formula (I) or formula (II) or being a stereoisomer, prodrug, active metabolite or pharmaceutically acceptable salt, solvate or crystalline form thereof, an N-oxide thereof or a prodrug thereof, for the manufacture of a medicament for the treatment of a condition mediated by a pathogenic microorganism. Wherein the microorganism-mediated disorder is selected from the group consisting of systemic infectious diseases, ocular infectious diseases, human sexually transmitted diseases, upper respiratory infectious diseases, lower respiratory infectious diseases, skin and soft tissue infectious diseases, acute sinusitis, chronic bronchitis, community-acquired pneumonia, hospital-acquired pneumonia, coronavirus pneumonia, and novel coronavirus pneumonia (COVID-19).

Human sexually transmitted diseases include vaginitis, cervicitis, and salpingitis, and nongonococcal urethritis caused by ureaplasma urealyticum.

The compounds of the present invention having the general formula (I) or formula (II) or being stereoisomers, prodrugs, active metabolites or pharmaceutically acceptable salts, solvates or crystalline forms thereof may be administered in combination with any antibiotic belonging to, but not limited to, the clavams (clavams), carbapenems, macrolides, penicillins and/or cephalosporins. In one aspect of the invention, the compounds of the invention are combined with one or more of the following: penicillin, methicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin, amoxicillin, alloline, carbenicillin, ticarine, melorarine, piperacillin, cephalexin, cephalothin, CXA-101, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefotaxin, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefixime, ceftaroline, cefepime, cefpirome, ceftaroline, imipenem, meropenem, ertapenem, faropenem, thiopenem, doripenem, PZ-601(Protez Pharmaceutica), ME1036(Forestlabs), BAL30072, MC-1, tomopenem (tomopenem), tebipenem, aztreonam, tigemonam, nocardiosin A or toboxinine) -beta-lactam.

In one aspect of the invention, the compounds of the invention are combined with one or more of the following: aminoglycosides, macrolides, ketolides, streptogramins, oxazolidinones, tetracyclines, fluoroquinolones, coumarin antibiotics, glycopeptides, lipoglycopeptides, nitroimidazoles, ansamycins, lichol, mupirocin (mupirocyn), fosfomycin, tobramycin, linezolid, daptomycin, vancomycin, tazobactam, avibactam, clavulanic acid, LK-157, LK-176, SA-1-204, SA-2-13, BLI-489(Pfizer/Wyeth), BAL0029880(Baselea) and/or relebactam (MK 7655).

III、Preparation

Pure forms or suitable pharmaceutical compositions of the compounds of the present invention or pharmaceutically acceptable salts thereof may be administered by any acceptable mode of administration of agents that serve similar utilities. The pharmaceutical compositions of the present invention may be prepared by combining a compound of the present invention with a suitable pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration for the pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, the term parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. One or more dosage units in the composition to be administered to a subject or patient, wherein, for example, a capsule can be a single dosage unit and a container containing a compound of the invention in aerosol form can hold a plurality of dosage units. The actual methods of preparing the dosage forms are known, or will be known, to those skilled in the art. The compositions to be administered will contain, in any event, a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, in order to treat the disease or condition of interest in accordance with the teachings of the present invention.

Pharmaceutical compositions

The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is a microparticle, such that the composition is in the form of, for example, a tablet or powder. The carrier can be a liquid and the composition can be, for example, an oral syrup, an injectable liquid, or an aerosol suitable for administration, for example, by inhalation. When intended for oral administration, the pharmaceutical composition is preferably selected from solid or liquid forms, wherein semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as solid or liquid. For oral solid compositions, the pharmaceutical compositions can be formulated in the form of powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, xanthan gum or gelatin; excipients, such as starch, lactose or dextrin; disintegrating agents, such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants, such as magnesium stearate or hydrogenated vegetable oil (Sterotex); glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavoring agent, such as peppermint, methyl salicylate, or sweet orange flavoring; and a colorant.

When the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier, for example polyethylene glycol or an oil. The pharmaceutical composition may be in liquid form, such as a tincture, syrup, solution, emulsion, or suspension. Such liquids may be administered orally, or delivered by injection, as two examples. When intended for oral administration, the compositions preferably contain, in addition to a compound of the invention, one or more of sweetening agents, preserving agents, dyes/colorants and flavor enhancers. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents and isotonicity agents may be included.

The liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may include one or more adjuvants selected from sterile diluents such as water for injection, physiological saline solutions, preferably physiological saline, Ringer's solution, isotonic sodium chloride, non-volatile oils (e.g., synthetic mono-or diglycerides which may be used as a solvent or suspending medium), solvents such as polyethylene glycol, glycerol, propylene glycol, and the like; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium oxide or dextrose. Parenteral formulations can be packaged in ampules, disposable syringes or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

Liquid pharmaceutical compositions of the invention intended for parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage is obtained. The pharmaceutical compositions of the invention may be intended for topical administration, in which case the carrier preferably comprises a solution, emulsion, ointment or gel base. For example, the base may comprise one or more of paraffin oil, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (e.g., water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in the pharmaceutical compositions for topical administration. If transdermal administration is intended, the composition may comprise a transdermal patch or an iontophoretic device.

The pharmaceutical compositions of the invention may be intended for rectal administration, in the form of suppositories, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. The base includes, but is not limited to, lanolin, cocoa butter, and polyethylene glycols.

The pharmaceutical compositions of the present invention may include a variety of substances that alter the physical form of the solid or liquid dosage unit. For example, the composition may include a substance that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

The pharmaceutical compositions of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby aids in the delivery of the compound. Suitable agents with this capability include monoclonal or polyclonal antibodies, proteins or liposomes.

The pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from the jelly-like variety to systems consisting of pressurized packs. Delivery may be by liquefied or compressed gas, or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds of the invention may be delivered as single, two, or three phase systems to deliver the active ingredient. The delivery side of the aerosol comprises the necessary containers, actuators, valves, sub-containers, etc., which together may form a kit. The preferred aerosol formulations can be determined by those skilled in the art without undue experimentation.

The pharmaceutical compositions of the present invention can be prepared by methods well known in the pharmaceutical arts. For example, pharmaceutical compositions to be administered by injection may be prepared by combining a compound of the invention with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compounds of the present invention, thereby facilitating dissolution or uniform suspension of the compounds in an aqueous delivery system.

For the carrier of the injectable preparation, for example, distilled water, or a physiological salt solution, a glucose solution, other isotonic solution is used. The carrier for capsules, powders, granules or tablets is used in admixture with known pharmaceutical excipients (for example, starch, maltose, sucrose, calcium carbonate or calcium phosphate), binders (for example, starch, gum arabic, carboxymethylcellulose, hydroxypropylcellulose or crystalline cellulose), lubricants (for example, magnesium stearate, talc) and disintegrants (for example, carboxymethylcellulose and talc). Thus, the pharmaceutical and antibacterial compositions for use in this specification may contain formula (I), formula (II) alone or in stereoisomer, prodrug, active metabolite or pharmaceutically acceptable salt, solvate or crystalline form thereof, or may contain a carrier as described above, or may contain an appropriate amount of any other suitable antibacterial compound.

The compounds of the present invention, or pharmaceutically acceptable salts thereof, are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular compound employed; metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the patient; mode and time of administration; the rate of excretion; a pharmaceutical composition; the severity of the particular disorder or condition; and individuals undergoing therapy.

The compounds of the present invention or pharmaceutically acceptable derivatives thereof can also be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapy includes the administration of a single pharmaceutical dosage formulation containing a compound of the present invention and one or more other active agents, as well as the administration of separate pharmaceutical dosage formulations of a compound of the present invention with each active agent itself. For example, the compound of the present invention and another active agent can be administered to a patient in a single orally administered composition (e.g., a tablet or capsule), or each agent can be administered as a separate orally administered formulation. In the case of separate administration formulations, the compound of the invention and the one or more additional active agents may be administered substantially at the same time (i.e., simultaneously) or at separately staggered times (i.e., sequentially); combination therapy is understood to include all such regimens.

The dosage of the pharmaceutical combination of the present invention can be adjusted depending on the disease state, the administration route, and the age or weight of the patient. For oral administration to adults, the dosage is usually 0.1-100 mg/kg/day, preferably 1-60 mg/kg/day. The appropriate dose of the present invention should be set in consideration of age, body weight, condition, administration route, etc. of the patient, and the oral administration is usually in the range of 0.5 to 100 mg/kg/day, preferably 1 to 60 mg/kg/day. For parenteral administration, the dosage of the pharmaceutical composition of the present invention varies widely depending on the route of administration, but is usually in the range of 0.05 to 50 mg/kg/day, preferably 1 to 20 mg/kg/day.

The dose of the compound of the present invention varies depending on the administration method, age, body weight, condition of the patient and the kind of the disease, but in general, in the case of oral administration, the dose is about 0.5mg to 3000mg, preferably about 100mg to 2000mg per 1 day for an adult. Can be administered separately as required. In addition, in the case of parenteral administration, the dose is about 1mg to 10000mg, preferably about 10mg to 1000mg per 1 day for an adult. It is to be understood that in the present invention, combinations of substituents and/or variables of the formulas are permissible only if they result in stable compounds.

V. examples

The colloquial name refers to the IUPAC system name (1S, 2R, 3S, 4S, 6R, 7R, 8R, 14R) -3, 6-dihydroxy-2, 4, 7, 14-tetramethyl-4-vinyl-tricyclo [5.4.3.0 ]^1,8]Tetradecan-9-one. In these examples, pleuromutilin derivatives, such as BC-3781, are numbered analogously to the numbering system for Muslim forest described in H.Berner (Berner, H.; Schulz, G.; Schneider H.tetrahedron 1980, 36, 1807-1811.):

14-O- { [ (1R,2R,4R) -4-amino-2-hydroxycyclohexyl-sulfanyl ] acetyl } -mordenin (BC-3781) was prepared according to Chinese patent CN201410406131. X.

Certain preferred embodiment aspects of the invention are shown by way of illustration in the following non-limiting examples. The reagents are commercially available or prepared according to literature methods.

The novel pleuromutilin derivatives disclosed herein may be prepared by microbial transformation or by methods well known in the art of organic chemistry. If desired, the reaction product is optionally purified using conventional techniques including, but not limited to, filtration, distillation, crystallization, chromatographic separation and purification. Such materials are optionally characterized by conventional means including physical constants and spectral data.

Certain preferred embodiment aspects of the invention are shown by way of illustration in the following non-limiting examples. The reagents are commercially available or prepared according to literature procedures.

Mass spectrum: electrospray mass spectra were recorded on a Waters single quadrupole mass spectrometer using flow injection in alternating positive and negative ion mode. The mass range was 120 ℃ 2000Da, the capillary voltage was set at 4500V, and nitrogen was used for atomization.

LC-MS spectrometer detector, Waters, PDA (200 and 320nm), Mass Detector: ZQ

Eluent: a is acetonitrile containing 0.05 percent of trifluoroacetic acid; acetonitrile/water-1/9 (v/v) containing 0.05% trifluoroacetic acid.

Example 1.14-O- { [ (1R,2R,4R) -4- ((S) -4-amino-2-hydroxybutyryl) -amino-2-hydroxycyclohexyl-sulfanyl ] -acetyl } -Muslim line

The synthetic route is as follows:

compound 1: 14-O- { [ (1R,2R,4R) -4- ((S) -4-amino-2-hydroxybutyryl) -amino-2-hydroxycyclohexyl-sulfanyl ] -acetyl } -Musomalin

At room temperature, 9g of intermediate 9 was dissolved in 100ml of DCM, 18ml of TFA was added and the mixture was stirred for 2 hours. After TLC detection reaction is completed, adding saturated sodium bicarbonate solution, adjusting pH to 7-8, and extracting. The organic layer was washed 1 time with water and 2 times with saturated sodium bicarbonate solution. The organic layer was concentrated under reduced pressure. The crude product was obtained and recrystallized from a mixed solvent of THF/MTBE to give a white solid as example Compound 1.

LC-Ms ESI+：609.6[M+H]⁺。

¹H-NMR (DMSO-d6,400MHz, partial characteristic peaks) 0.62-0.64(3H, d), 0.85-0.86(3H, d),1.25-1.49(12H, m),1.79-1.89(3H, m),2.00-2.20(7H, m),2.43(1H, m),3.04-3.06(2H, m),3.33-3.38 (2H, m),4.16-4.20(1H, m),5.10-5.21(2H, m),5.60-5.63(1H, d),6.24-6.35(1H, m).

Intermediate 2: (R) - (3-Cyclohexenyl) -carbamic acid tert-butyl ester

(R) - (3-cyclohexenyl) -carbamic acid tert-butyl ester is a compound known in the literature and an overview of the preparation is given. 61g of (R) -3-cyclohexenecarboxylic acid (available from Sahn's chemical technology, Shanghai, Ltd.) and 600mL of toluene were added to the reaction flask and stirred at room temperature. 53.8g of triethylamine is added dropwise and stirred for 30min after the dropwise addition. Then, 140g of DPPA (diphenylphosphoryl azide) was added dropwise, the temperature was slowly raised to 95 ℃ and the stirring was maintained for 15min, followed by heating and refluxing. TLC detection of the disappearance of the starting material, the reaction solution was cooled to 80 ℃ and 180g of tert-butanol was added dropwise, followed by 1.6g of CuCl. All mixtures were then warmed to 100 ℃ and stirred for an additional 50min, and the reaction was complete by TLC. The reaction solution was cooled, and 4L of a saturated aqueous solution of sodium hydrogencarbonate was added. The mixture was stirred for 2 hr. Filtering, extracting mother liquor, washing the organic layer with water for 2 times, and evaporating the mother liquor under reduced pressure to obtain a crude product. Purifying the crude product by flash silica gel column chromatography, and eluting the crude product with an eluent: ethyl acetate/petroleum ether to yield 81.1g of a solid.

Intermediate 3: (1R,3R, 6S) - (7-oxa-bicyclo [4.1.0] hept-3-yl) -carbamic acid tert-butyl ester

135g m-CPBA (m-chloroperoxybenzoic acid) and 730mL of methylene chloride were added to the reactor and the mixture was cooled to 10-15 ℃. Slowly adding 90g (R) - (3-cyclohexenyl) -carbamic acid tert-butyl ester solution in dichloromethane (150ml) dropwise, maintaining the reaction system at below 25 ℃, adding 45ml dichloromethane, stirring for 50min after the addition is finished, then heating to reflux, carrying out reflux reaction for 2hr, and detecting by TLC that the reaction is complete. Cooling the reaction solution to-5-0 deg.C, stirring for 2hr, filtering, washing the filtrate with 10% sodium thiosulfate solution for 2 times, washing with 10% sodium bicarbonate solution for 2 times, washing with water for 2 times, concentrating under reduced pressure to minimum volume, adding toluene, distilling under reduced pressure again, repeating the operation for 1 time to obtain toluene solution (content: 75%) of intermediate 3.

Intermediate 4 { (1R,2R,4R) -4- [ (tert-Butoxycarbonyl) -amino ] -2-hydroxy-cyclohexyl } -phenylthiocarbamate

{ (1R,2R,4R) -4- [ (tert-butoxycarbonyl) -amino ] -2-hydroxy-cyclohexyl } -benzenethiocarbamate is a known compound in the literature and gives an overview of the preparation. 54.6g of a toluene solution (content: 75%) of the above intermediate 3 was added with 60ml of toluene, and stirred at room temperature. Below 25 c, a solution of 2.9ml of thiobenzoic acid in 30ml of toluene is added dropwise. Adding 1.7g tetrabutylammonium chloride monohydrate, heating to 40-45 deg.C, stirring for reaction for 4hr, and detecting by TLC to obtain the final product. Cooling the reaction solution to 15 ℃, dropwise adding a 5% sodium bicarbonate aqueous solution into the reaction solution, carrying out extraction and washing for 2 times under the condition that the pH value is 6-7. The organic layer was concentrated to give an oil, which was recrystallized from toluene/heptane and dried in vacuo to give intermediate 4.

Intermediate 5[ (1R,3R,4R) -3-hydroxy-4-mercaptocyclohexyl ] -carbamic acid tert-butyl ester

[ (1R,3R,4R) -3-hydroxy-4-mercaptocyclohexyl ] -carbamic acid tert-butyl ester is a known compound in the literature and gives an overview of the preparation. 19.7g of intermediate 4 and 180ml of DCM are mixed and stirred at 20 ℃. Adding 2g of 1, 4-dithio DL-threitol and 10ml of DCM for washing, then adding 4.2g of hydrazine hydrate, stirring the reaction solution at 20 ℃ for reacting for 3 hours, and detecting the reaction completion by HPLC. After completion of the reaction, 195ml of a 1M phosphoric acid solution was added thereto, and the mixture was stirred at room temperature. Extracting, washing the water layer with 1M phosphoric acid solution, and washing with saturated sodium chloride. The organic layer was concentrated to dryness under reduced pressure to give intermediate 5 as a white solid.

Intermediate 6: 22-O-p-toluenesulfonyl pleuromutilin

22-O-p-toluenesulfonyl pleuromutilin is a compound known in the literature and an overview of the preparation is given.

39g of pleuromutilin (from Jiangsu Fengyuan bioengineering Co., Ltd.) was dissolved in 125ml of DCM, 19.7g of p-toluenesulfonyl chloride was added, the temperature was reduced to 10-15 ℃ and 27.3ml of 5.7M aqueous NaOH solution was slowly added. The reaction solution is heated and refluxed for 20 hours, and the reaction is detected to be complete by HPLC. The reaction solution was cooled to room temperature, dichloromethane and water were added, extraction was performed, the organic layer was washed with water 3 times, the organic layer was concentrated, DCM was added for azeotropy, 120ml heptane was slowly added to the concentrate to obtain a white suspension, which was stirred for 2 hours, filtered, and the filter cake was washed with heptane. The solid was dried to give 48.5g of a white solid, 22-O-p-toluenesulfonylstyrocidin.

Intermediate 7-O- { [ (1R,2R,4R) -4-tert-Butoxycarbonylamino-2-hydroxycyclohexyl-sulfanyl ] acetyl } -me lin

14-O- { [ (1R,2R,4R) -4-tert-Butoxycarbonylamino-2-hydroxycyclohexyl-sulfanyl ] acetyl } -morelin is a known compound in the literature and gives an overview of the preparation.

9.5g of 22-O-p-toluenesulfonylstyromutilin were suspended in 90ml of MTBE, and 0.61g of benzyl-tri-n-butylammonium chloride was added to the mixed solution. Then 5.7g of intermediate 4 were added and finally 40ml of 1M aqueous NaOH solution were added dropwise. The resulting mixture was stirred for 3 hours at 20 ℃ and HPLC checked for complete reaction, extraction was carried out, a DCM layer was retained, the organic layer was washed successively with 1M aqueous NaOH, 0.1M aqueous phosphoric acid, and saturated sodium bicarbonate. The organic layer was concentrated under reduced pressure, and the resulting concentrate was dissolved in isopropanol and concentrated again. 12.4g of a white foamy solid, 14-O- { [ (1R,2R,4R) -4-tert-butoxycarbonylamino-2-hydroxycyclohexyl-sulfanyl]Acetyl } -somalene. LC-Ms ESI +: 608.4[ M + H]⁺

BC-3781: 14-O- { [ (1R,2R,4R) -4-amino-2-hydroxycyclohexyl-sulfanyl ] acetyl } -mullin

14-O- { [ (1R,2R,4R) -4-amino-2-hydroxycyclohexyl-sulfanyl ] acetyl } -methoprene, a compound known in the literature, is a summary of the preparation.

18g of intermediate 7 was dissolved in 180ml of DCM at room temperature, 36ml of TFA was added and the mixture was stirred for 2 hours. After TLC detection reaction is completed, adding saturated sodium bicarbonate solution, adjusting pH to 7-8, and extracting. The organic layer was washed with water 1 time and with saturated sodium bicarbonate solution 2 times. The organic layer was concentrated under reduced pressure. The crude product was obtained and recrystallized from a THF/MTBE mixed solvent to give 13.1gBC-3781, LC-Ms ESI +: 508.3[ M + H]⁺

Intermediate 8: (S) -4-Boc-amino-2-hydroxybutyric acid

(S) -4-amino-2-hydroxybutyric acid (10g) was dissolved in dioxane: to a mixed solvent of water (400mL, 1: 1v/v) were added potassium carbonate (23.2g) and a solution of Boc anhydride (19.2g) in dioxane (50mL) in this order, and the mixture was stirred at room temperature overnight. The dioxane was removed by rotary evaporation, the aqueous phase was washed with dichloromethane (100mL x 2), then the pH of the aqueous phase was adjusted to 2-3 with phosphoric acid, dichloromethane was extracted (100mL x 2), the organic phases were combined, dried and distilled under reduced pressure to give (S) -4-Boc-amino-2-hydroxybutyric acid (12g, 65%).

Intermediate 9: 14-O- { [ (1R,2R,4R) -4- ((S) -4-Boc-amino-2-hydroxybutyryl) -amino-2-hydroxycyclohexyl-sulfanyl ] -acetyl } -Musomalin

10g (119.7mmol) of BC-3781 are dissolved in 100ml of DMF, 4g (29.54mmol) of HOBt is added for activation for 30min, then 6.5g (29.54mmol) of (S) -4-Boc-amino-2-hydroxybutyric acid and 5.7g (29.54mmol) of EDCI are added, 3g (29.54mmol) of triethylamine are added dropwise for reaction at room temperature for 2h, TLC detection shows complete reaction, the mixture is poured into 300ml of ice water, stirred for 30min, filtered with suction and dried to obtain a white solid, namely the intermediate 9. LC-Ms ESI +: 709.9[ M + H]⁺；LC-Ms ESI-:707.8[M-H]^-， 753.9[M+HCOO-]^-。