Isotope-labeled and unlabeled gentamicin C congeners

Abstract

The present invention generally relates to the field of isotopically labeled and unlabeled gentamicin congeners and to methods for synthesizing such congeners. In particular, the present invention relates to at least one isotopically labeled gentamicin C or its salts, solvates, or derivatives. 13 C, D and / or 15 Gentamicin C containing an N atom, and gentamicin C or its salts, solvates, or derivatives, in particular, at least one 13 C, D and / or 15 This invention relates to a method for preparing gentamicin C containing a nitrogen atom, or its salts, solvates, or derivatives.

Description

Technical Field

[0001] The present invention generally relates to the field of isotopically labeled and unlabeled gentamicin congeners and synthetic methods for preparing such congeners. In particular, the present invention relates to isotopically labeled gentamicin C or a salt or solvate or derivative thereof, at least one 13 C, D and / or 15 N atom-containing gentamicin C, and a method for preparing gentamicin C or a salt or solvate or derivative thereof, particularly one containing at least one 13 C, D and / or 15 N atom-containing gentamicin C or a salt or solvate or derivative thereof.

Background Art

[0002] Gentamicin C is a potent and most commonly prescribed aminoglycoside antibiotic with broad antibiotic activity but a very narrow therapeutic margin. Gentamicin C is not a homogeneous compound but is composed of and used as a mixture of five congeners (C1, C1a, C2, C2a, and C2b) with varying degrees of antibacterial efficacy.

[0003] Gentamicin C has the following general structure,

Chemical Formula

[0004] As shown in structure (1), gentamicin C contains 2-deoxystreptamine (ring A) and two amino sugar moieties, namely galosamine (ring C) and purprosamine (ring B). Galosamine is glycosidically bonded to the C-6 hydroxyl group of 2-deoxystreptamine, thereby forming the pseudodisaccharide galamine (ring A+C). Purprosamine (ring B) is glycosidically bonded to the C-4 of galamine.

[0005] The main drawbacks of gentamicin C treatment are nephrotoxic and ototoxic side effects, with each of these congeners exhibiting a different ability to cause nephrotoxicity. (A Non-Nephrotoxic Gentamicin Congener That Retains Antimicrobial Efficacy, JASN October 2006, 17(10)2697-2705; DOI: https: / / doi.org / 10.1681 / ASN.2005101124). For example, gentamicin congener C2 was isolated from natural gentamicin and shown to retain its normal bactericidal properties without inducing cytotoxicity or nephrotoxicity in a rat model of gentamicin toxicity. Due to limited access to pure gentamicin congeners, there are still few studies focusing on the different toxic effects of each of these compounds. Furthermore, methods for preparing pure congeners in high yield and high purity would be advantageous, especially on an industrial scale, to reduce side effects.

[0006] Bulman et al. (American Society for Microbiology, Antimicrobial Agents and Chemotherapy, Volume 64, Issue 9, 20 August 2020) showed substantial variability in the amount of gentamicin congeners in clinical formulations of gentamicin and commercially available gentamicin discs used to test for antimicrobial susceptibility. This study measured the relative abundances of four major congeners contained in commercially available antibiotic susceptibility test (AST) discs and found that the amount of gentamicin congeners varied by up to 4.1 times. Furthermore, the potency of congeners against strains possessing a common aminoglycoside modifying enzyme (AME) differed by up to 128 times, and the nephrotoxicity of individual gentamicin congeners also differed significantly in cell-based and repeated-dose rat nephrotoxicity studies. The authors concluded that because the microbiological activity of congeners can differ, unexpected gentamicin treatment failures may occur if the ratios of gentamicin congeners present in susceptibility test products and clinically administered formulations are not the same.

[0007] Therefore, there is still a need for a favorable approach to provide a certain reliable purity of gentamicin congeners. Furthermore, there is a need for a reliable method to determine the amount of each gentamicin congener in a sample, such as a patient-derived sample, and to determine the composition of a clinical formulation in relation to the ratio of congeners present therein.

[0008] Some derivatives of aminoglycoside antibiotics have been published and can be obtained by modifying existing end compounds. Furthermore, the synthesis of several gentamicin derivatives and other aminoglycosides has recently been published: International Publication No. 2019 / 079706.

[0009] Furthermore, Rajasekaran and Crich (Org. Lett. 2020, 22, 3850-3854) recently published a synthetic approach to the synthesis of gentamicin B1 and gentamicin X2 (both starting from shisomycin). In the synthesis of gentamicin B1, a protected glycosyl acceptor is reacted with a protected galamine-based 6-azido-6,7-dideoxy-D-glycero-D-glucoheptopyranosyl acceptor. However, the resulting intermediates are not suitable for the synthesis of gentamicin C analogs. Moreover, the synthesis of isotopically labeled compounds is not described.

[0010] Therefore, a favorable synthetic approach to obtain pure gentamicin C congeners is still needed. Furthermore, synthetic approaches to produce novel gentamicin derivatives are still needed in the search for new antibiotics to combat microbial resistance and / or reduce the side effects of known aminoglycoside antibiotics. Moreover, to date, no sources or synthetic routes for specific isotope-labeled gentamicin congeners are known. Such pure isotope-labeled congeners would be highly advantageous, for example, as calibration standards or internal standards for determining the amount of at least one gentamicin C congener present in a given sample, for example, by mass spectrometry. [Overview of the project]

[0011] The solution to this objective is achieved by providing embodiments characterized in the claims.

[0012] Surprisingly, each gentamicin C congener, particularly C1, C1a, C2, C2a, and C2b, can be advantageously prepared in high purity by chemical synthesis, and preferably, each does not contain significant amounts of the other congeners as contaminants. In particular, this approach also enables the synthesis of isotopically labeled gentamicin C congeners, which can be advantageously used as calibration standards to determine, for example, the amount of at least one gentamicin C congener present in a given sample. Specifically, this involves the use of mass spectrometry.

[0013] The synthetic approach of the present invention and the gentamicin C congener according to the present invention are described in more detail below.

[0014] According to a preferred embodiment, the present invention relates to isotope-labeled gentamicin C or its salts, solvates, or derivatives, wherein gentamicin C comprises at least one 13 C, D and / or 15 Contains N atoms

[0015] Furthermore, the present invention relates to the compound of formula I*). [ka] Preferably, the use of a compound of formula (I) is preferred. [ka] During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD- and- 13 Selected from the group consisting of CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. The term "orthogonal protecting group" refers to a protecting group that attaches to a molecular structure and is cleaved from the molecular structure without affecting other protecting groups present in the molecular structure.

[0016] Furthermore, the present invention relates to the compound of formula (I-1*). [ka] Preferably, the compound of formula (I-1), [ka] During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are selected independently from the following groups: -CH-, -CD-, - 13 CD-or- 13 CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG5, and PG6 are appropriate protecting groups.

[0017] Furthermore, the present invention relates to a compound of formula (I*), preferably formula (I), for the preparation of gentamicin C, preferably isotope-labeled gentamicin C. [ka] Preferably, the use of a compound of formula (I) is preferred. [ka] During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. This method preferably involves removing the protecting group PG4 to obtain the compound of formula (I-1*). [ka] Preferably the compound of formula (I) [ka] This further includes obtaining.

[0018] Furthermore, the present invention relates to a method for preparing gentamicin C or its salts, solvates, or derivatives, preferably isotope-labeled gentamicin C or its salts, solvates, or derivatives. This method uses the glycosylated acceptor (A) in the following formula [ka] The donor building block (B*) in the following formula [ka] And preferably, a donor building block (B) of the following formula [ka] It reacts with the following structure [ka] This includes obtaining a compound having the following characteristics: During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, -13 CD- or - 13 selected from the group consisting of CH- R 011 is -CH2-, -CD2-, -CHD-, - 13 CD2-, - 13 CH2-, - 13 CHD-, and -R 11 (R1R2)-, wherein R 1 and R 2 are independently of each other -H, -D, -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 CD3, -CD2H, -CDH2, and -CD3, wherein R 1 or R 2 at least one of which is -H or -D, wherein R 11 is C or 13 C, wherein PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6, the method preferably further comprises removing the protecting group PG4 to obtain a compound of formula (I-1*

Chemical formula

Chemical formula

[0019] Furthermore, the present invention relates to a method for preparing the above gentamicin C or a salt or solvate or derivative thereof, wherein the gentamicin C or a salt or solvate or derivative thereof is an isotope-labeled gentamicin C having the following structure,

Chemical formula

[0020] Furthermore, the present invention relates to a method for preparing gentamicin C or its salts, solvates, or derivatives, wherein gentamicin C or its salts, solvates, or derivatives are isotope-labeled gentamicin C. [ka] More preferably, having a structure according to formula (3), [ka] In the formula, R 1 , R 2 and R 3 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, R 9 and R 10 At least one of them is -C(DH)-, or the isotope-labeled gentamicin C more preferably has the following structure. [ka]

[0021] Furthermore, the present invention relates to gentamicin C or its salts or solvates obtained or obtainable by the above method, preferably at least one 13 C, D and / or 15 This relates to isotope-labeled gentamicin C containing a nitrogen atom, or its salts or solvates.

[0022] Furthermore, the present invention relates to a pharmaceutical composition comprising the above-mentioned gentamicin C and a pharmaceutically acceptable excipient.

[0023] Furthermore, the present invention relates to a kit comprising the above-described isotope-labeled gentamicin C or its salt or solvate, or gentamicin C obtained or obtainable by the above-described method, and a container.

[0024] Furthermore, the present invention relates to a method for treating a bacterial infection in a subject requiring treatment for a bacterial infection, comprising administering the above-described isotope-labeled gentamicin C or its salt or solvate, or gentamicin C obtained or obtainable by the above-described method, or the above-described pharmaceutical composition to the subject.

[0025] Furthermore, the present invention relates to the use of the above-mentioned at least one isotope-labeled gentamicin C, or its salts, solvates, or derivatives, as a calibration standard or internal standard for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample.

[0026] Furthermore, the present invention relates to a diagnostic composition comprising the above-mentioned gentamicin C and a suitable excipient. This may be a preservative such as sodium azide, such as a protein-based stabilizer, a pH buffer salt such as sodium citrate, sodium phosphate, or sodium bicarbonate, or an organic solvent such as acetonitrile or ethanol that modifies the solubility of gentamicin C. As used herein, the term “diagnostic composition” refers to a composition for identifying the presence or absence of at least one gentamicin C congener, and comprising at least isotope-labeled gentamicin C.

[0027] Furthermore, the present invention relates to at least one isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, for use as a calibration standard or internal standard for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample. It should be understood that one isotope-labeled gentamicin C may be used as the calibration standard or internal standard, or a mixture of at least two isotope-labeled gentamicin C congeners may be used.

[0028] Furthermore, the present invention relates to a method for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, wherein the method is: (a) Mix the sample with a known amount of at least one of the above-mentioned isotope-labeled gentamicin C, or its salt, solvate, or derivative. (b) Analyzing the sample by mass spectrometry. (c) Comparing the peak area of ​​at least one analyte of interest to a standard curve, wherein the standard curve is prepared using a standard containing at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. This includes determining the amount of at least one target analyte in the sample.

[0029] Furthermore, the present invention also relates to a computer-implemented method for evaluating a sample containing at least one gentamicin analog, comprising the following steps: (aa) Mix the sample with a known amount of at least one isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, or mix the sample with a known amount of a diagnostic composition containing the at least one isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, and receive the peak area value of the isotope-labeled gentamicin C in the sample. (bb) A step of receiving the peak area value of at least one gentamicin C congener present in the sample. (cc) A step of comparing the peak area values ​​of at least one isotope-labeled gentamicin C and at least one gentamicin C congener, and a step of receiving the value of the amount of at least one gentamicin C congener; and (dd) Includes the step of evaluating the sample for comparison and / or calculations performed in step (cc).

[0030] Furthermore, the present invention relates to a diagnostic system, preferably a clinical diagnostic system, suitable for carrying out a method for determining the presence and / or amount of at least one analyte of interest, preferably at least one gentamicin C congener, present in a sample, the method comprising steps (a), (b), and (c) as described above.

[0031] Furthermore, the present invention relates to the use of the above-described diagnostic system for determining the presence or absence or quantity of at least one target analyte in a sample.

[0032] Isotope-labeled gentamicin C As used in this invention, the term "isotopically labeled gentamicin C" means that gentamicin C contains at least one isotope-labeled gentamicin C. 13 C, D and / or 15 This implies that N is included.

[0033] According to a preferred embodiment, this relates to isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, wherein gentamicin C is at least one 13 C, D and / or 15 Contains a N atom. Preferably, the present invention relates to isotope-labeled gentamicin C or a salt thereof, wherein gentamicin C contains at least one 13 C, D and / or 15 Contains N atoms

[0034] Preferably, the gentamicin is an isotope-labeled gentamicin selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1, and gentamicin C1a.

[0035] Preferred salts include those prepared by the reaction of gentamicin C with a mineral acid or organic acid. When the above-mentioned pharmaceutical composition containing gentamicin C also contains a salt of gentamicin C, a pharmaceutically acceptable salt is used. Such salts are known to those skilled in the art.

[0036] Acids commonly used to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, trifluoroacetic acid, and acetic acid. Examples of such pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, bromides, iodides, trifluoroacetates, acetates, propionates, decanoates, caprylates, acrylates, formates, hydrochlorides, isobutyrates, caproates, heptanoates, propiolates, oxalates, malons, succinates, suberates, sebacinates, fumarates, maleates, and bronates. Examples include tin-1,4-dioate, hexyn-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylene sulfonates, phenyl acetates, phenylpropionates, phenyl butyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, as well as those formed with organic acids such as maleic acid and methanesulfonic acid. Salts of amine groups may also include quaternary ammonium salts in which the amino nitrogen has a suitable organic group such as an alkyl, alkenyl, alkynyl, or aralkyl moiety. It should be recognized that certain counterions forming part of any salt of the present invention are not usually important properties, for example in pharmaceutical compositions or when used in analytical methods according to the present invention, such as when used as a calibration standard, or in methods including mass spectrometry of the whole salt, unless the counterions contribute to undesirable qualities or properties. Preferred salts are, for example, salts formed by reaction with sulfuric acid or trifluoroacetic acid, and therefore sulfate salts or trifluoroacetate salts. More preferably, isotope-labeled gentamicin C exists as a trifluoroacetate salt.More preferably, the isotope-labeled gentamicin C obtained or obtainable by the above method exists as a trifluoroacetate.

[0037] The term “pharmaceutically acceptable solvate” also encompasses suitable solvates of gentamicin C in the present invention, which, when combined with a solvent such as water, methanol, ethanol, DMSO, acetonitrile, or a mixture thereof, form suitable solvates such as the corresponding hydrate, methanol salt, ethanol salt, DMSO solvate, or acetonitrile salt.

[0038] Preferably, the isotope-labeled gentamicin C according to the present invention, or the isotope-labeled gentamicin C obtained or obtainable by the above method, is labeled at least, preferably only, in the purpurosamine moiety (ring B), in other words, the purpurosamine moiety (ring B) is preferably labeled in the ring or within the substituents of the ring 13 C, D and / or 15 It contains an N atom. In other words, in the purpurosamine portion of structure (1), at least one C is 13 Substituted by C, and / or at least one H is substituted by D, and / or at least one N is 15 It is substituted with N. More preferably, only the purprosamine portion is labeled.

[0039] Preferably, the isotope-labeled gentamicin C described above, or the isotope-labeled gentamicin C obtained or obtainable by the method described above, is substantially free of other congeners.

[0040] Within the scope of the present invention, the term "substantially free of other congeners" means that each gentamicin C contains, more preferably substantially free of, impurities of each other gentamicin C congener in amounts less than 1% by weight, preferably less than 0.5% by weight, more preferably less than 0.1% by weight, more preferably less than 0.05% by weight, more preferably less than 0.01% by weight, more preferably essentially free, and more preferably not free, based on the total weight of gentamicin C that is substantially free of other congeners as determined by HPLC, HPLC-MS, and LCMS methods. Typically, the amount of other congeners present or the absence of other congeners is determined by a spiking assay in an LCMS instrument. Such spiking assays are known to those skilled in the art. Typically, when the amount (or absence) of an analyte is determined by a spiking assay, a known amount (spike) of the analyte is added to a sample, and the spiked sample is then analyzed. The amount of analyte corresponds to the total amount of analyte (analyte in the sample + added spike) minus the added spike of the analyte. In this context, the term "other gentamicin C congeners" means unlabeled or differently isotope-labeled gentamicin congeners, including congeners that have the same molecular structure but are isotope-labeled at different atomic positions or are not isotope-labeled at atomic positions.

[0041] Preferably, the isotope-labeled gentamicin C described above, or the isotope-labeled gentamicin C obtained or obtainable by the method described above, is substantially pure.

[0042] Within the scope of the present invention, the term "substantially pure" means that each gentamicin C or its salt or solvate contains less than 5% by weight, preferably less than 4% by weight, more preferably less than 3% by weight, more preferably less than 2% by weight, more preferably less than 1% by weight, more preferably less than 0.5% by weight, more preferably less than 0.1% by weight, more preferably less than 0.05% by weight, more preferably less than 0.01% by weight, and more preferably essentially no impurities, and the term impurities includes all additional components such as additional solvents other than (other) gentamicin C congeners.

[0043] According to a preferred embodiment, isotope-labeled gentamicin C having the general structure (1) is an isotope-labeled version of gentamicin C1, and therefore R 1 -CH3, R 2 -H and R 3 -CH3, and isotope-labeled gentamicin C1 is preferably labeled at least, more preferably only, the purprosamine moiety. Therefore, residue R 1 , R 2 and R 3 In the purprosamine portion of structure (1) including, at least one C is 13 Substituted by C, and / or at least one H is substituted by D, and / or at least one N is 15 It is substituted with N. Preferably, the isotope-labeled gentamicin C1 is substantially free of other congeners. Furthermore, the isotope-labeled gentamicin C1 is preferably substantially pure. More preferably, the gentamicin C1 is substantially free of other congeners and is substantially pure.

[0044] According to a more preferred embodiment, isotope-labeled gentamicin C having the general structure (1) is an isotope-labeled version of gentamicin C2, and therefore R 1 -CH3, R 2 -H and R 3-H, and isotope-labeled gentamicin C2 is preferably labeled at least, more preferably only, the purprosamine moiety. Therefore, residue R 1 , R 2 and R 3 In the purprosamine portion of structure (1) including, at least one C is 13 Substituted by C, and / or at least one H is substituted by D, and / or at least one N is 15 It is substituted with N. Preferably, the isotope-labeled gentamicin C2 is substantially free of other congeners. Furthermore, the isotope-labeled gentamicin C2 is preferably substantially pure. More preferably, the gentamicin C2 is substantially free of other congeners and substantially pure.

[0045] According to a more preferred embodiment, isotope-labeled gentamicin C having the general structure (1) is an isotope-labeled version of gentamicin C1a, and therefore R 1 -H, R 2 -H and R 3 -H, and isotope-labeled gentamicin C1a is preferably labeled at least, more preferably only, the purprosamine moiety. Therefore, preferably, residue R 1 , R 2 and R 3 In the purprosamine portion of structure (1) including, at least one C is 13 Substituted by C, and / or at least one H is substituted by D, and / or at least one N is 15 It is substituted with N. Preferably, the isotope-labeled gentamicin C1a is substantially free of other congeners. Furthermore, the isotope-labeled gentamicin C1a is preferably substantially pure. More preferably, the gentamicin C1a is substantially free of other congeners and substantially pure.

[0046] According to a more preferred embodiment, isotope-labeled gentamicin C having the general structure (1) is an isotope-labeled version of gentamicin C2a, and therefore R 1 -H, R2 -CH3 and R 3 -H, and isotope-labeled gentamicin C2a is preferably labeled at least, more preferably only, the purprosamine moiety. Therefore, residue R 1 , R 2 and R 3 In the purprosamine portion of structure (1) including, at least one C is 13 Substituted by C, and / or at least one H is substituted by D, and / or at least one N is 15 It is substituted with N. Preferably, the isotope-labeled gentamicin C2a is substantially free of other congeners. Furthermore, the isotope-labeled gentamicin C2a is preferably substantially pure. More preferably, the gentamicin C2a is substantially free of other congeners and substantially pure.

[0047] According to a more preferred embodiment, isotope-labeled gentamicin C having general structure (1) is an isotope-labeled version of gentamicin C2b, and therefore R 1 -H, R 2 -H and R 3 -CH3, and isotope-labeled gentamicin C2b is preferably labeled at least, more preferably only, the purprosamine moiety. Therefore, residue R 1 , R 2 and R 3 In the purprosamine portion of structure (1) including, at least one C is 13 Substituted by C, and / or at least one H is substituted by D, and / or at least one N is 15 It is substituted with N. Preferably, the isotope-labeled gentamicin C2b is substantially free of other congeners. Furthermore, the isotope-labeled gentamicin C2b is preferably substantially pure. More preferably, the gentamicin C2b is substantially free of other congeners and substantially pure.

[0048] Preferably, isotope-labeled gentamicin C has structure (2), [ka] It comprises a salt or solvate thereof, in which R 1 , R 2 and R 3 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, R 4 -NH-, -ND-, - 15 ND- and - 15 Selected from the group consisting of NH-, R 5 -NH2, -ND2, - 15 ND 2、 - 15 NDH and - 15 Selected from the group consisting of NH2, R 6 , R 7 and R 8 They are independent of each other: -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R 9 and R 10 They are independent of each other: -CH2-, -CDH-, -CD2-, - 13 CDH-, - 13 CD2- and- 13 Selected from the group consisting of CH2-, R 11 is C or 13 It is C.

[0049] More preferably, in these cases, R 5 is -NH2, and R 6 , R 7 and R 8 It is -CH-, and isotope-labeled gentamicin C has structure (3) in particular. [ka]

[0050] According to one preferred embodiment, isotope-labeled gentamicin C has the structure shown in formula (3), where R 9 and R 10 At least one of them is -C(DH)-, and more preferably has the following structure. [ka]

[0051] Therefore, preferably, isotope-labeled gentamicin C has a structure selected from the group consisting of the following: [ka] The isotope-labeled gentamicin has structure (4a) and mixtures thereof.

[0052] According to a more preferred embodiment, R 9 and R 10 Both are -C(H2)-, and labeled gentamicin has structure (5). [ka]

[0053] According to a preferred embodiment, isotope-labeled gentamicin C has a general structure (2), preferably structure (3), more preferably structure (4) or (5), and is isotope-labeled gentamicin C2, and R 1 -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 1 is -CD3, R 2 and R 3 These are mutually independent of each other: -H or -D.

[0054] In a more preferred embodiment, isotope-labeled gentamicin C has a general structure (2), preferably structure (3), more preferably structure (4) or (5), and is isotope-labeled gentamicin C1, R 1 -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 1 is -CD3, R 2 is -H or -D, R 3 -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 3 It is -CD3.

[0055] In a more preferred embodiment, isotope-labeled gentamicin C has a general structure (2), preferably structure (3), more preferably structure (4) (for example, a structure selected from the group consisting of (4a), (4b), (4c), and (4d)) or structure (5), and is isotope-labeled gentamicin C1a, R 1 is -H or -D, R 2 and R 3 It is also -H or -D, R 4 -NH-, -ND-, - 15 ND- and - 15 It is selected from the group consisting of NH-.

[0056] In a more preferred embodiment, isotope-labeled gentamicin C has a general structure (2), preferably structure (3), more preferably structure (4) or (5), and is isotope-labeled gentamicin C2a, R 1 is -H or -D, R 3 is -H or -D, R 2 is -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, -13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 2 It is -CD3.

[0057] In a more preferred embodiment, isotope-labeled gentamicin C has a general structure (2), preferably structure (3), more preferably structure (4) or (5), and is isotope-labeled gentamicin C2b, and R 1 is -H or -D, R 2 is -H or -D, R 3 is -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 3 It is -CD3.

[0058] Preferably, in structures (4) and (5), R 11 C is C.

[0059] The following isotope-labeled compounds or their salts or solvates are particularly preferred. [Table 1]

[0060] Therefore, the present invention also relates to isotope-labeled gentamicin C2 having the structure of formula (5), wherein R 1 is -CD3, R 2 is H, and the base -R 4 -R 3 is -NH2, and R 11 It is preferably C.

[0061] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having the structure of formula (5), wherein R 1 is -CD3, R 2 H is H, and -R 4 teeth- 15 NH- and -R3 teeth- 13 CH3, R 11 It is preferably C.

[0062] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having the structure of formula (5), wherein R 1 is -CD3, R 2 H is R 4 is -NH-, and R 3 is -CH3, R 11 It is preferably C.

[0063] Furthermore, the present invention also relates to isotope-labeled gentamicin C2a having the structure of formula (5), wherein R 1 H is R 2 is -CD3, R 4 is -NH-, and R 3 is -H, and R 11 It is preferably C.

[0064] Furthermore, the present invention also relates to isotope-labeled gentamicin C2b having the structure of formula (5), wherein R 1 H is R 2 is -H, and R 4 is -NH-, and R 3 is -CD3, R 11 It is preferably C.

[0065] Therefore, the present invention also relates to isotope-labeled gentamicin C2 having a structure according to formula (4), preferably according to formula (4a), wherein R 1 is -CD3, R 2 is H, and the base -R 4 -R 3 is -NH2, and R 11 It is preferably C.

[0066] Furthermore, the present invention also relates to isotope-labeled gentamicin C2 having a structure according to formula (4), preferably according to formula (4a), wherein R 1 is -CD3, R 2is H, and the base -R 4 -R 3 teeth 15 NH2, R 11 It is preferably C.

[0067] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having a structure according to formula (4), preferably according to formula (4a), wherein R 1 is -CD3, R 2 H is H, and -R 4 teeth- 15 NH- and -R 3 teeth- 13 CH3, R 11 It is preferably C.

[0068] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having a structure according to formula (4), preferably according to formula (4a), wherein R 1 is -CD3, R 2 H is R 4 is -NH-, and R 3 is -CH3, R 11 It is preferably C.

[0069] Furthermore, the present invention also relates to isotope-labeled gentamicin C2a having a structure according to formula (4), preferably formula (4a), wherein R 1 H is R 2 is -CD3, R 4 is -NH-, and R 3 is -H, and R 11 It is preferably C.

[0070] Furthermore, the present invention also relates to isotope-labeled gentamicin C2b having a structure according to formula (4), preferably formula (4a), wherein R 1 H is R 2 is -H, and R 4 is -NH-, and R 3 is -CD3, R 11 It is preferably C.

[0071] Furthermore, the present invention also relates to isotope-labeled gentamicin C1a having a structure according to formula (4), preferably formula (4a), wherein R 1 H is R 2 is -H, and R 4 teeth- 15 NH- and R 3 is -H, and R 11 It is preferably C.

[0072] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having a structure according to formula (4), preferably according to formula (4a), wherein R 1 is -CD3, R 2 H is R 4 It is -NH- and -R 3 is -CD3, R 11 It is preferably C.

[0073] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having a structure according to formula (4), preferably according to formula (4a), wherein R 1 is -CD3, R 2 H is H, and -R 4 teeth- 15 NH- and -R 3 is -CD3, R 11 It is preferably C.

[0074] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having the structure of formula (5), wherein R 1 is -CD3, R 2 H is H, and -R 4 It is -NH- and -R 3 is -CD3, R 11 It is preferably C.

[0075] Furthermore, the present invention also relates to isotope-labeled gentamicin C1 having the structure of formula (5), wherein R 1 is -CD3, R 2 H is H, and -R 4 teeth- 15 NH- and -R 3is -CD3, R 11 It is preferably C.

[0076] Furthermore, the present invention also relates to isotope-labeled gentamicin C2a having the structure of formula (5), wherein R 1 H is R 2 is -CH3, R 4 teeth- 15 NH- and R 3 is -H, and R 11 It is preferably C.

[0077] Furthermore, the present invention also relates to isotope-labeled gentamicin C2a having the structure of formula (5), wherein R 1 H is R 2 is -CD3, R 4 teeth- 15 NH- and R 3 is -H, and R 11 It is preferably C.

[0078] Furthermore, the present invention also relates to isotope-labeled gentamicin C2a having a structure according to formula (4), preferably formula (4a), wherein R 1 H is R 2 is -CD3, R 4 teeth- 15 NH- and R 3 is -H, and R 11 It is preferably C.

[0079] Furthermore, the present invention also relates to isotope-labeled gentamicin C2b having the structure of formula (5), wherein R 1 H is R 2 is -H, and R 4 teeth- 15 NH- and R 3 is -CH3, R 11 It is preferably C.

[0080] Furthermore, the present invention also relates to isotope-labeled gentamicin C2b having the structure of formula (5), wherein R 1 H is R2 is -H, and R 4 teeth- 15 NH- and R 3 is -CD3, R 11 It is preferably C.

[0081] Furthermore, the present invention also relates to isotope-labeled gentamicin C2b having a structure according to formula (4), preferably formula (4a), wherein R 1 H is R 2 is -H, and R 4 teeth- 15 NH- and R 3 is -CD3, R 11 It is preferably C.

[0082] Therefore, the present invention also relates to isotope-labeled gentamicin C2 having the structure of formula (5), wherein R 1 is -CD3, R 2 is H, and the base -R 4 -R 3 teeth- 15 NH2, R 11 It is preferably C.

[0083] Preferably, the above and below isotope-labeled gentamicin C congeners exist as trifluoroacetates.

[0084] Method according to the present invention: As outlined above, the present invention further relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably a method for preparing the isotope-labeled gentamicin C or a salt, solvate, or derivative thereof. Preferably, the gentamicin C or a salt, solvate, or derivative thereof prepared by the method according to the present invention has at least one 13 C, D and / or 15This is isotope-labeled gentamicin C containing a nitrogen atom. In the method of the present invention, a precursor of galamine building block AC is reacted with a precursor of purprosamine building block B*, preferably B, thereby bonding the precursor of purprosamine building block B*, preferably B, to carbon C-4 of galamine precursor AC, particularly via a glycosidic bond.

[0085] Furthermore, the present invention also relates to gentamicin C homologues that can be obtained or obtained by the method described herein. Preferably, the isotope-labeled gentamicin C described above can be obtained or obtained by the method of the present invention.

[0086] In the method of the present invention, each gentamicin can be synthesized substantially free of other congeners. Therefore, preferably, gentamicin C, or isotope-labeled gentamicin C obtained or obtainable by the method described above, is substantially free of other congeners. Furthermore, gentamicin C, or isotope-labeled gentamicin C obtained or obtainable by the method described above, is substantially pure and substantially free of other congeners.

[0087] See the definitions of the terms "substantially free from other congeners" and "substantially pure" above.

[0088] In the sense of the present invention, the term “precursor” means a suitable modified and / or protected building block that can be converted in a further reaction step into the respective final building blocks present in each gentamicin C or each isotope-labeled gentamicin C according to the present invention.

[0089] This advantageous method provides access to all gentamicin C congeners by chemical synthesis, and as a result, each congener is accessible without significant amounts of other congeners acting as contaminants. Therefore, each synthetic gentamicin C congener is preferably substantially free of other congeners. Furthermore, the approach of the present invention is advantageous, preferably, in the purporosamine moiety, 13 C and 15 N-isotope substitution enables the synthesis of isotopically labeled gentamicin C congeners. Furthermore, this method allows for the introduction of modifications to the compound structure at specific positions.

[0090] When preparing isotope-labeled gentamicin C, at least one isotope label may already be present in one of the building blocks, or it may be introduced in a further method reaction step. If at least one isotope label is already present in one of the building blocks, the label is preferably present in the purprosamine building block B*, preferably in the precursor of B.

[0091] Preferably, the galamine precursor building blocks A to C are glycosylated acceptors (A) having the following structure: [ka] In the formula, PG1, PG2, and PG3 are appropriate protecting groups.

[0092] Preferably, the purpurosamine building block is a glycosylated donor (B*) of the following formula [ka] More preferably, formula (B), [ka] And, In the formula, R 6 , R 7 , R 8 , R 91 and R101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, PG4, PG5 and PG6 are suitable protecting groups, PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6, and R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, R 11 is C or 13 It is C.

[0093] The term "orthogonal" is used to mean that PG4 can be bonded and cleaved without affecting the protecting groups PG1, PG2, PG3, PG5, and PG6. Therefore, PG4 can be removed without removing PG1, PG2, PG3, PG5, and PG6.

[0094] The term "suitable protecting group" refers to any organic moiety that readily bonds to a group to be protected, such as an amino group or a hydroxyl group, and, upon bonding to the respective group, inactivates the resulting protecting group for reaction conditions carried out in the rest of the compound, and can be removed at an appropriate time to regenerate the respective functional group. PG1 is preferably a suitable amine protecting group, while PG2, PG3, PG4, PG5, and PG6 are suitable hydroxyl protecting groups. Such protecting groups are known to those skilled in the art and are selected in particular as follows:

[0095] Preferably, PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and more preferably PG3 is Bn.

[0096] Preferably, PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. Preferably, PG2 and PG3 are the same.

[0097] According to a preferred embodiment, PG2 is Bn.

[0098] Preferably, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. According to one preferred embodiment, PG1 is para-trifluoromethylbenzoyl.

[0099] Preferably, PG4 is a protecting group selected from the group consisting of a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). More preferably, PG4 is benzoyl (Bz).

[0100] Preferably, PG5 and PG6 together form a cyclic group, and more preferably, PG5 and PG6 together form a diacetal protecting group. Such protecting groups are known to those skilled in the art and are described, for example, in Synlett 1996, 8, 793-795 and J. Chem. Soc., Perkin Trans. 1, 1997, 2023-2032.

[0101] Preferably, PG5 and PG6 together form the following group. [ka]

[0102] In particular, the present invention relates to the above method, wherein the method is a glycosylated acceptor (A) of the following formula [ka] The glycosylated donor (B*) in the following formula, [ka] More preferably, equation (B) [ka] It reacts with the following structure [ka] Compounds having formula (I), preferably formula (I) [ka] The process involves obtaining a compound of the following: In the formula, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, In the formula, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are suitable protecting groups, PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6, and more preferably PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably a protecting group selected from the group consisting of benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), more preferably PG4 is benzoyl (Bz), PG5 and PG6 together form a cyclic group, and more preferably PG5 and PG6 together form a diacetal protecting group, particularly the following groups. [ka]

[0103] Furthermore, the present invention also relates to gentamicin C homologues that can be obtained or obtained by the method described herein. Preferably, the isotope-labeled gentamicin C described above can be obtained or obtained by the method of the present invention.

[0104] Compound of formula (I): Furthermore, the present invention relates to a compound of the following formula (I*), [ka] Preferably the compound is (I) [ka] Regarding In the formula, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, R 11 is C or 13 C is the appropriate protective device, and PG1, PG2, PG3, PG4, PG5, and PG6 are suitable protective devices.

[0105] Preferably, the compound has structure (Ia), [ka] In the formula, PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6, and more preferably PG4 is a silyl protecting group, a pivaloyl group (Piv) or a benzoyl protecting group, preferably a protecting group selected from the group consisting of benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl (TIPS), more preferably PG4 is benzoyl (Bz), and PG5 and PG6 together form a cyclic group, and more preferably PG5 and PG6 together form a diacetal protecting group, particularly the following groups. [ka]

[0106] In particular, the present invention also relates to compounds of formula (I*), more preferably compounds of formula (I), and more preferably compounds of formula (Ia), wherein PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2, PG2 is a silyl protecting group, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and a benzyl protecting group, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-D PG3 is a protecting group selected from the group consisting of MPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1, PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, PG4 is a protecting group selected from the group consisting of silyl protecting groups, pivaloyl (Piv) or benzoyl protecting groups, preferably benzoyl (Bz), 2,4The protecting group is selected from the group consisting of 6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and PG5 and PG6 preferably together form a cyclic group, and more preferably together form a diacetal protecting group, particularly the following groups. [ka]

[0107] In some preferred embodiments, the present invention relates to a compound of formula (I*), [ka] Preferably, the compound of formula (I), [ka] During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. especially, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the compound has structure (Ia), [ka] The compound of formula (I*), the compound of formula (I), and the compound of formula (Ia) each contain at least one 13 C, D and / or 15 Contains N atoms

[0108] More preferably, the present invention also relates to compounds of formula (Ib), more preferably to compounds of formula (Ic): [ka] Preferably, it has the following structure. [ka]

[0109] In another preferred embodiment, in a compound of formula (I*), more preferably a compound of formula (I), and more preferably a compound of formula (Ia), PG1 together with PG2 forms a cyclic protecting group, particularly a cyclic urethane protecting group. More preferably, the compound has structure (Id), [ka] More preferably, structure (Ie), [ka] In particular, structure (If) [ka] It has.

[0110] The present invention also relates to the use of the above-mentioned compound of formula (I*), preferably formula (I), preferably formula (Ia), for preparing gentamicin C or a salt or solvate thereof, wherein gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1 and gentamicin C1a, and gentamicin is more preferably isotope-labeled gentamicin.

[0111] According to one particular preferred embodiment, the present invention relates to the use of the above-mentioned compound of formula (Ib), preferably formula (Ic), for preparing gentamicin C or a salt or solvate thereof, wherein gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1 and gentamicin C1a, and gentamicin is more preferably isotope-labeled gentamicin.

[0112] According to another specific preferred embodiment, the present invention relates to the use of the above-mentioned compound of formula (Id), preferably formula (Ie), more preferably (If), for preparing gentamicin C or a salt or solvate thereof, wherein gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1 and gentamicin C1a, and gentamicin is more preferably isotope-labeled gentamicin.

[0113] Provision of glycosylated acceptor (A) The galamine precursor building blocks A-C are referred to as glycosylated acceptors (A) below and above.

[0114] The glycosylated acceptor (A) described above and used in the method according to the present invention preferably has the following structure: [ka] In the formula, PG1, PG2, and PG3 are appropriate protecting groups as described above.

[0115] According to one preferred embodiment, the glycosylated acceptor (A) has structure (A1). [ka]

[0116] According to another preferred embodiment, the glycosylated acceptor A has structure (A2). [ka]

[0117] Preferably, the method according to the present invention further includes providing a glycosylated acceptor (A).

[0118] The provision of building block (A) is not particularly limited and includes, for example, any possible synthesis of building block (A). Preferably, the provision is carried out by a semi-synthetic approach starting from sisomycin.

[0119] Cisomycin is an aminoglycoside antibiotic, isolated, for example, from the fermentation broth of a novel species of the genus Micromonospora inositola (see MJ Weinstein et al., J Antibiot(Tokyo)) Antibiot(Tokyo) 1970 Nov;23(11):551-4, and has the following structure. [ka]

[0120] It may be obtained, for example, as a sulfate, from Merck, for example.

[0121] Preferably, the method of the present invention comprises the following steps: (a) further comprising the step of providing a glycosylated acceptor (A), wherein the glycosylated acceptor (A) preferably has the following structure. [ka]

[0122] Preferably, the provision includes the conversion of the primary amine functional group of shisomecin to an azide group, appropriate protection of the hydroxyl group and appropriate protection of the secondary amine group, to obtain a compound of formula (a3). [ka] This is transformed to obtain the compound of formula (A).

[0123] More preferably, step (a) includes at least the following steps: (a1) A step of providing a compound of formula (a1) or a salt thereof, [ka] (a2) A step of converting the primary amine group of the compound of formula (a1) to an azide group. The process of appropriately protecting the hydroxyl group to obtain the following compounds. [ka] In the formula, PG2 and PG3 are suitable protecting groups as described above, and preferably PG2 and PG3 are the same. (a3) A step to obtain the compound of formula (a3) ​​by appropriately protecting the Me-NH- group with the protecting group PG1. [ka] In the formula, PG1 is a suitable protecting group as described above. (a4) and the step of converting the compound of formula (a3) ​​to compound (A), The glycosylated acceptor of formula (A) preferably has structure (A1) or (A2).

[0124] It should be understood that the provision in step (a1) includes, for example, obtaining a compound as a salt such as a sulfate from Merck KGaA, and optionally converting the salt to a free base.

[0125] According to a first preferred embodiment, the glycosylated acceptor of formula (A) has structure (A1).

[0126] According to a second preferred embodiment, the glycosylated acceptor of formula (A) has structure (A2).

[0127] Preferably, step (a4) is carried out under acidic conditions, for example, using H2SO4 in MeOH.

[0128] For example, the synthesis of glycosylated acceptors can be carried out as shown in Figure 8 and Example A2.

[0129] Donor Building Block The purpurosamine precursor building block is referred to below and above as donor building block (B*), or, according to a preferred embodiment, as donor building block (B).

[0130] The donor building block described above and used in the method according to the present invention preferably has structure (B*), [ka] More preferably, the donor building block has structure (B), [ka] In the formula, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13CD-or- 13 Selected from the group consisting of CH-, PG4, PG5, and PG6 are appropriate protective devices, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, R 11 is C or 13 C. As described above, PG5 and PG6 preferably form a cyclic group together, and more preferably PG5 and PG6 form a diacetal protecting group together.

[0131] According to a preferred embodiment, the donor building block described above and used in the method according to the present invention has structure (B1), [ka] PG4 is more preferably Bz.

[0132] If the donor building block is not isotope-labeled, the donor building block (B) preferably has structure (B2), [ka] PG4 is even more preferably Bz.

[0133] Preferably, the method according to the present invention further comprises providing a glycosylated donor (B).

[0134] Therefore, preferably, the method of the present invention comprises the following steps: (a) A step of providing a glycosylated donor (B*), preferably (B), wherein the glycosylated donor preferably has the following structure [ka] More preferably, the process further includes having structure (B). [ka]

[0135] The provision of glycosylated donors (B*), preferably (B), is not particularly limited and includes, for example, any possible synthesis of glycosylated donors.

[0136] For example, the provision of glycosylated donors (B*) and (B) can be carried out as shown in Figure 7 and Example A1, respectively.

[0137] As outlined above, the donor building block preferably has structure (B), while the glycosylated acceptor of formula (A) preferably has structure (A1) or (A2).

[0138] Accordingly, the present invention also relates to a method in which the donor building block preferably has structure (B) and the glycosylated acceptor formula (A) preferably has structure (A1) or (A2), as described above.

[0139] Preferably, the method according to the present invention removes the protecting group PG4 to obtain the compound of formula (I-1*), [ka] Preferably a compound of formula (I-1), [ka] Preferably, this further includes obtaining a compound of formula (Ia-1). [ka]

[0140] Therefore, the present invention also relates to the above method and gentamicin C obtained or obtainable by the method, the method comprising the following steps: (i) Glycosylated acceptor A of the following formula [ka] The donor building block (B*) in the following formula [ka] And preferably, the donor building block (B*) of the following formula [ka] By reacting with it, a compound having the following structure is obtained. [ka] Preferably (I) [ka] A process to obtain, In the formula, R 6 , R 7 , R 8 , R 91 and R 101 These are, independently of each other, -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, in the formula, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, R 1 or R 2 At least one of them is -H or -D, and R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, process (ii) Remove the protecting group PG4 to obtain the compound of formula (I-1*), [ka] Preferably a compound of formula (I-1), [ka] This includes the process of obtaining [something].

[0141] In some preferred embodiments, a method for preparing gentamicin C or its salts, solvates, or derivatives, wherein gentamicin C or its salts, solvates, or derivatives are prepared in a manner in which at least one 13 C, D and / or 15 It is an isotope-labeled gentamicin C containing a N atom, (i) Glycosylated acceptor (A) of the following formula [ka] The glycosylated donor (B*) in the following formula [ka] And preferably, formula (B) [ka] By reacting with it, a compound having structure (I*) is produced. [ka] Preferably, the compound of formula (I) [ka] Including seeing, During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. especially, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the donor building block has the following structure: [ka] When a glycosylated donor (B) is reacted with a glycosylated acceptor (A), structure (Ia) is formed. [ka] A compound having the following characteristics is obtained; Each of the compounds of formula (I*), formula (I), and formula (Ia) contains at least one 13 C, D and / or 15 Contains N atoms

[0142] In some preferred embodiments, this method (ii) Remove the protecting group PG4 to obtain the compound of formula (I-1*), [ka] Preferably a compound of formula (I-1), [ka] More preferably, a compound of formula (Ia-1). [ka] This further includes obtaining.

[0143] The method for removing the protecting group PG4 depends on the protecting group used. Suitable methods are known to those skilled in the art. Preferably, when the protecting group PG4 is a Bz protecting group, the removal of PG4 is carried out under basic hydrolysis conditions, for example, using NaOMe, KOH, or K2CO3, preferably NaOMe. The removal of the PG4 group can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent, for example, a solvent selected from the group consisting of methanol, ethanol, iPrOH, or nPrOH, preferably in MeOH, and in a mixture of two or more thereof. The compound is preferably reacted for a time in the range of 2 minutes to 24 hours, for example, overnight.

[0144] Preferably, by removal, a compound of formula (I-1*), more preferably a compound of formula (I-1), More preferably, a compound of formula (Ia-1). [ka] This can be obtained.

[0145] Furthermore, the present invention also relates to a compound of formula (I-1*), or a compound obtained or obtainable by the method of the present invention, having the following structure: [ka] Preferably structure (I-1), [ka] The present invention relates more to compounds having the structure of formula (Ia-1). [ka]

[0146] Next, depending on the desired homologue, compound (I-1*), preferably (I-1), more preferably (Ia-1) is further modified to obtain the final gentamicin C homologue, preferably the above and below isotope-labeled gentamicin C homologues. Advantageously, starting from compound (I*), compound (I), or compound (Ia), respectively, those skilled in the art can prepare any desired gentamicin C homologue by appropriately modifying the primary hydroxyl group (or group-OPG4).

[0147] Therefore, this chemical pathway may be used to obtain all gentamicin C congeners, for example, as shown in Figures 1-5.

[0148] The following describes a specific preferred method.

[0149] Method A - Preferably using gentamicin C2, C2a, or C1, for example. According to the first preferred embodiment A, the primary hydroxyl group of compound (I-1), preferably compound (Ia-1), is subjected to oxidative conditions such that the hydroxyl group is converted to an aldehyde group. This step is carried out particularly, optionally, when isotope-labeled gentamicin C2, C2a, or C1 or its derivatives are to be prepared.

[0150] Therefore, the method according to the present invention preferably involves the following steps (iii)(A) ​​A compound of formula (I-1), preferably (Ia-1), is reacted with an oxidizing agent to form formula (I-2A), [ka] Preferably (Ia-2A) [ka] The further method includes obtaining the compound.

[0151] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, wherein gentamicin C is preferably gentamicin C2, C2a, or C1, and further comprises step (iii)(A).

[0152] Suitable oxidation methods for primary hydroxyl groups are known to those skilled in the art. Suitable oxidation methods include, but are not limited to, Swan oxidation (DMSO, oxalyl chloride and TEA), Fitzner-Moffat oxidation (DCC / DMSO), Dess-Martin periodinane oxidation, oxidation using TEMPO and co-oxidants, or the use of supervalent iodide reagents such as 2-iodoxybenzoic acid (IBX), TPAP / NMO (tetrapropylammonium perruthenate / N-methylmorpholine N-oxide), or modifications thereof.

[0153] In a preferred embodiment, the oxidation reaction is Dess-Martin oxidation.

[0154] Preferably, the reaction in step (iii)(A) ​​is carried out in an anhydrous solvent or a mixture of solvents such as dichloromethane (DCM), acetone, tetrahydrofuran (THF), or a mixture thereof; more preferably, the reaction solvent is DCM. The reaction in step (iii)(A) ​​is carried out at a temperature preferably in the range of -80°C to the reflux temperature of the solvent used, and preferably in the range of -20°C to rt.

[0155] When preparing arbitrarily isotope-labeled gentamicin C2, C2a, or C1 or its derivatives, the compound of formula (I-2A), preferably the compound of formula (Ia-2A), is further modified by an addition reaction with a nucleophile at the (electrophilic) carbon atom of the aldehyde group, thereby forming the group R 12 Preferably, a methyl group or an isotope-labeled methyl group, particularly -CH3 or -CD3, is introduced.

[0156] Preferably, this reaction is carried out in the presence of a basic organometallic reagent, preferably an alkyllithium compound, alkylmagnesium compound, alkylcopper compound, alkylaluminum compound, or alkylzinc compound, such as a Grignard reagent, more preferably a Grignard reagent, and especially in the presence of CD3MgI or CH3MgI.

[0157] Therefore, the method according to the present invention preferably involves the following steps (iv)(A) Modify the carbon atom of the aldehyde group with a nucleophile, thereby forming the group R 12 This is bonded to a carbon atom, preferably a methyl group or an isotope-labeled methyl group, particularly -CH3 or -CD3, thereby forming the compound of formula (I-3A). [ka] Preferably, the process further includes the step of forming a compound of (Ia-3A), [ka] In the formula, R 12The is -CH3 or -CD3, preferably -CD3. Accordingly, the present invention also relates to a method for preparing the above-mentioned gentamicin C or a salt or solvate or derivative thereof, preferably isotope-labeled gentamicin C or a salt or solvate or derivative thereof, and to gentamicin obtained or obtainable by the method, wherein gentamicin C is preferably gentamicin C2, C2a or C1, and further comprises steps (vi)(A) ​​and (iv)(A).

[0158] Depending on the further process, R 12 R is in the final gentamicin C obtained or obtainable by the present invention (see, for example, Figure 6). 1 or R 2 It corresponds to one of the following. For gentamicin C1 and C2, for example, R 12 is R 1 Corresponding to, in the case of gentamicin C2a, R 12 is R 2 In response to this, preferably, the reaction is carried out in the presence of a basic organometallic reagent, preferably an alkyllithium compound, alkylmagnesium compound, alkylcopper compound, alkylaluminum compound, or alkylzinc compound, such as a Grignard reagent, more preferably a Grignard reagent, and especially in the presence of CD3MgI or CH3MgI.

[0159] Method A1 - Gentamicin C2 is preferred. When optionally isotope-labeled gentamicin C2 or its derivatives are prepared, this method involves converting the primary hydroxyl group of compound (I-3A), preferably compound (Ia-3A), into a leaving group, and preferably the compound thus obtained to nitrogen or 15 The method further comprises reacting with a nucleophile containing a nitrogen atom, such as an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide.

[0160] As used in this context of the present invention, the term "leaving group" refers to the bonded center of the substrate and the nucleophile, such as an amine group, ammonia, hydrazine, hydrazide, 15 This indicates a molecular fragment that, upon reaction with NH3 or an azide ion, detaches a pair of electrons from the reaction substrate via heterolytic bond cleavage. Examples of leaving groups include, among others, halogens, sulfonic acid esters (e.g., particularly mesyl and tosyl or triflate groups).

[0161] Therefore, the method according to the present invention preferably involves the following steps (v)(A1) Convert the primary hydroxyl group to a leaving group -L to obtain the compound of formula (I-4A1), [ka] Preferably, the process further includes a step to obtain the compound (Ia-4A1), [ka] The leaving group is preferably a sulfonic acid ester, and step (v)(A1) includes a reaction with preferably a sulfonic acid halide, preferably a chloride.

[0162] More preferably, L is -O-tosyl or -O-mesyl.

[0163] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, wherein gentamicin C is preferably gentamicin C2, and further comprises step (v)(A1). Preferably, the reaction is carried out in pyridine.

[0164] The compound of formula (I)(A1) is preferably reacted with a nucleophile in the presence of a suitable base.

[0165] Therefore, the method according to the present invention preferably comprises the following steps: (vi)(A1) A compound of formula (I-4A1), preferably (Ia-4A1), is reacted with a nucleophile, for example, an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide to obtain a compound of formula (I-5A1), [ka] Preferably, the process further includes a step to obtain the compound (Ia-5A1), [ka] In the formula, Nu is a bound nucleophile, preferably Nu is -N3, -NH2, -NH-NH2, -NH-N(R H ) 2、 -NH-NH(R H ) 、 - 15 NH-NH2, - 15 NH- 15 NH2, - 15 NH- 15 N(R H ) 2、 - 15 NH- 15 NH(R H ), - 15 NH-N(R H ) 2、 - 15 NH-NH(R H ), 15 N-labeled-N3, -NH-Me, -NH 13 CH3, - 15 NH 13 CH3, - 15 NH-Me, and - 15 Selected from the group consisting of NH2, R H is an aryl or alkyl, preferably an alkyl, The reaction is preferably carried out in the presence of a suitable base.

[0166] Suitable bases are preferably amino groups containing a base, and most preferably non-nucleophilic or small nucleophilic bases selected from the group consisting of diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N-methylimidazole, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, colidine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), and 1,5-diazabicyclo(4.3.0)nona-5-ene (DBN).

[0167] Preferably, the reaction is carried out in an organic solvent such as methanol or DMF.

[0168] The reaction temperature is preferably in the range of 0 to 120°C, more preferably in the range of 20 to 85°C, and the temperature may preferably be varied within the above given range or kept essentially constant.

[0169] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, and to gentamicin C obtained or obtainable by such method, wherein gentamicin C is preferably gentamicin C2, and further comprises steps (v)(A1) and (vi)(A1).

[0170] Therefore, preferably, the method further comprises step (v)(A1). In this step, the protecting groups PG5 and PG6 of the compound of formula (I-5A1), preferably (Ia-5A1), are preferably removed, and the resulting diol is preferably converted to an alkene group, thereby forming formula (I-6A1), [ka] Preferably, the compound (Ia-6A1) is formed. [ka]

[0171] Methods for removing the protecting groups PG5 and PG6, and for converting the resulting diol into an alkene, are known to those skilled in the art and are not particularly limited.

[0172] These methods include, but are not limited to, methods known as Corey-Winter olefin synthesis using, for example, thiocarbonyldiimidazole or thiophosgene and trimethylphosphite, or the conditions disclosed in Org. Lett. 2000, 2, 25, 4029-4031. Furthermore, the diol may be converted to the corresponding epoxide and then deoxygenated (see, for example, Org. Synth. 1981, 60, 29).

[0173] The method for removing the protecting groups PG5 and PG6 depends on the protecting groups used. Suitable methods are known to those skilled in the art. Preferably, PG5 and PG6 together form a cyclic group, more preferably a diacetal protecting group, more preferably the following groups, [ka] Deprotection is preferably carried out under acidic conditions, for example, using a strong mineral acid or organic acid, such as HCl or TFA. The removal of these protecting groups can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent optionally mixed with water, more preferably in a solvent selected from the group consisting of methanol, ethanol, trifluoroethanol (TFE), dichloromethane, 1,2-dichloroethane, DMF, DMSO, NMP, methanol, ethanol, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl terbutyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, optionally mixed with water, most preferably in dichloromethane and water. Preferably, the removal is carried out at a temperature in the range of 0 to 40°C, more preferably in the range of 10 to 30°C, and more preferably at room temperature. During the reaction, the temperature may be varied or kept essentially constant.

[0174] A suitable method for converting a diol to an alkene group is known to those skilled in the art and is described above.

[0175] Preferably, the free hydroxyl group is converted to a leaving group (L), such as a sulfonic acid ester, such as a mesyl group, tosyl group, or triflate group, preferably a triflate group, and then removed in a further step.

[0176] Such removal is preferably carried out using Na2S2O3 in combination with NaI, and the removal can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent selected from the group consisting of acetone, acetonitrile, γ-butyrolactone, DMSO, dichloromethane, DMF, DMSO, NMP, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tart-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, preferably in acetone.

[0177] Preferably, the method according to this preferred embodiment includes the following steps: (vii)(A1)-N3, -NH2, -NH-NH2, -NH-N(R H ) 2、 -NH-NH(R H ) 、 - 15 NH-NH2, - 15 NH- 15 NH2, - 15 NH- 15 N(R H ) 2、 - 15 NH- 15 NH(R H ), - 15 NH-N(R H ) 2、 - 15 NH-NH(R H ), 15 N label-N 3、 and - 15 Nu(R) is selected from the group consisting of NH2. H This involves converting the azide group present in formula (I-6A1), preferably (Ia-6A1), which includes an aryl or alkyl group (preferably alkyl), to a primary amine group, thereby forming the compound of formula (I-7A1). [ka] Preferably, the process further includes a step to obtain the compound (Ia-7A1), [ka] R 12 is -CH3 or -CD3, preferably -CD3, and Nu* is -NH2 or - 15 The substance is NH2. Preferably, this step is carried out in the presence of a phosphine, such as an alkylphosphine or arylphosphine, particularly PPh3, TCEP, or PMe3, preferably PMe3.

[0178] Preferably, in a further step, the double bond is reduced. Preferably, in this step, conditions are applied such that PG3 and PG2 are removed in the same step. Alternatively, the method includes a step of reducing the alkene group and at least one step of removing PG3 and PG2.

[0179] Reduction of the alkene group and optional removal of PG2 and PG3 can optionally yield isotope-labeled PG1-protected gentamicin C2.

[0180] The reduction is preferably carried out in the presence of hydrogen or deuterium using a suitable catalyst. This allows for the advantageous removal of protecting groups, such as PG3 and PG2, that may be removed under such reduction conditions.

[0181] Therefore, preferably, the method according to this second preferred embodiment B comprises the following steps: (viii)(A1) The further step includes reducing the compound of formula (I-7A1), preferably (Ia-7A1), using a suitable catalyst, preferably in the presence of hydrogen or deuterium, thereby preferably removing PG2 and PG3 as well.

[0182] PG2 and PG3 are removed in particular when both of them are Bn groups.

[0183] Reduction is preferably carried out in the presence of hydrogen using a suitable catalyst. If deuteration is desired, D2 may be used. The catalyst may be homogeneous or heterogeneous. If a heterogeneous catalyst is used, the catalyst is preferably Pd, Pd / C, Pt or Pt / C, Rh / Al2O3, Pd / Al2O3, Pt / Al2O3, Pd(OH)2, PtO2, PdO*(H2O) x The catalyst is selected from the group consisting of Pd / CaCO3, Pd / BaSO4, Rh / Al2O3, and Ru / Al2O3, and more preferably the catalyst is a palladium-containing catalyst, more preferably Pd / C.

[0184] In this case, the reduction of the double bond may be carried out at a temperature of approximately 0°C to 100°C for approximately 1 hour to 48 hours, for example, overnight.

[0185] The amount of catalyst used in this reaction relative to the total amount of the compound of formula (I-7A1) is preferably in the range of 0.1 mol% to 100 mol%, preferably 5 mol% to 10 mol%, based on the total amount of the compound of formula (I-7A1) in [mol]. Regarding the solvent used in step (vi)(B), any suitable organic solvent known to those skilled in the art may be used. Preferably, the solvent is selected from the group consisting of acetic acid and water.

[0186] After reduction and removal of PG2 and PG3, PG1 is preferably removed to obtain the final isotope-labeled gentamicin C2. This removal is preferably carried out under basic conditions, using, for example, KOH, NaOMe, NaOH, or Ba(OH)2, at a temperature from about rt to about 100°C for about 1 hour to about 48 hours, for example, overnight.

[0187] Each of the obtained gentamicin C or gentamicin C derivatives may be subjected to appropriate post-treatment. Such post-treatment may preferably include one or more steps, at least one of which is purification such as extraction and / or precipitation and / or filtration and / or chromatography.

[0188] Method A2 - For example, preferred with gentamicin C1. When optionally isotope-labeled gentamicin C1 or its derivatives are prepared, Method A further comprises converting the primary azide group of compound (I-3A), preferably compound (Ia-3A), to a primary amine group.

[0189] Therefore, this method involves the following steps: (v)(A2) The primary azide group of compound (I-3A), preferably compound (Ia-3A), is converted to a primary amine group to obtain the compound of formula (I-4A2), [ka] Preferably, the process further includes a step of obtaining the compound (Ia-4A2). [ka]

[0190] Preferably, this step is carried out in the presence of a phosphine, such as an alkylphosphine or arylphosphine, particularly PPh3, TCEP, or PMe3, preferably PMe3.

[0191] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, wherein gentamicin C is preferably gentamicin C1, and further comprises step (v)(A2).

[0192] Preferably, in a further step, the primary amine group is protected with a suitable protecting group PG7 to obtain a compound of formula (I-5A2), for example, a compound of formula (Ia-5A2). Preferred protecting groups are known to those skilled in the art. In particular, PG7 is a Cbz group.

[0193] Therefore, the method according to the present invention preferably involves the following steps (vi)(A2) Protect the primary amine group to form the compound of formula (I-5A2), [ka] Preferably, the process further includes a step of obtaining the compound (Ia-5A2). [ka]

[0194] Preferably, in a subsequent step, the method converts the secondary hydroxyl group of compound (I-5A2), preferably compound (Ia-5A2), into a leaving group, and preferably the compound thus obtained is exposed to nitrogen or 15This method involves reacting a nucleophile containing a nitrogen atom, such as an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide.

[0195] As used in this context of the present invention, the term "leaving group" refers to the bonded center of the substrate and the nucleophile, such as an amine group, ammonia, hydrazine, hydrazide, 15 This indicates a molecular fragment that, upon reaction with NH3 or an azide ion, detaches a pair of electrons from the reaction substrate via heterolytic bond cleavage. Examples of leaving groups include, among others, halogens, sulfonic acid esters (e.g., particularly mesyl and tosyl or triflate groups).

[0196] Therefore, the method according to the present invention preferably involves the following steps (vii)(A2) Convert the primary hydroxyl group to a leaving group -L to obtain the compound of formula (I-6A2), [ka] Preferably, the process further includes a step to obtain the compound (Ia-6A2), [ka] The leaving group is preferably a sulfonic acid ester, and the process involves a reaction with a sulfonic acid halide, preferably a chloride.

[0197] More preferably, L is -O-tosyl or -O-mesyl.

[0198] Next, it is preferable to react the compound of formula (I-6A2), preferably (Ia-6A2), with a nucleophile in the presence of a suitable base.

[0199] Therefore, the method according to the present invention preferably comprises the following steps: (viii)(A2) A compound of formula (I-6A2), preferably (Ia-6A2), is reacted with a nucleophile, for example, an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide to obtain a compound of formula (I-7A2). [ka] Preferably, the process further includes a step to obtain the compound (Ia-7A2), [ka] In the formula, Nu is a bound nucleophile, preferably Nu is -N3, -NH2, -NH-NH2, -NH-N(R H ) 2、 -NH-NH(R H ) 、 - 15 NH-NH2, - 15 NH- 15 NH2, - 15 NH- 15 N(R H ) 2、 - 15 NH- 15 NH(R H ), - 15 NH-N(R H ) 2、 - 15 NH-NH(R H ), 15 N label-N 3、 -NH-Me, -NH 13 CH3, - 15 NH 13 CH3, - 15 NH-Me and - 15 Selected from the group consisting of NH2, R H The compound is aryl or alkyl, preferably alkyl, and the reaction is preferably carried out in the presence of a suitable base. Preferably Nu is 15 This is N-labeled azide.

[0200] Suitable bases are preferably amino groups containing a base, and most preferably non-nucleophilic or small nucleophilic bases selected from the group consisting of diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N-methylimidazole, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, colidine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), and 1,5-diazabicyclo(4.3.0)nona-5-ene (DBN).

[0201] Preferably, the reaction is carried out in an organic solvent such as methanol or DMF.

[0202] The reaction temperature is preferably in the range of 0 to 120°C, more preferably in the range of 20 to 85°C, and the temperature may preferably be varied within the above given range or kept essentially constant.

[0203] Preferably, the method further comprises step (ix)(A2). In this step, the protecting groups PG5 and PG6 of the compound of formula (I-7A2), preferably (Ia-7A2), are preferably removed, and the resulting diol is preferably converted to an alkene group, thereby forming formula (I-8A2), [ka] Preferably, the compound (Ia-8A2) is formed. [ka]

[0204] Methods for removing the protecting groups PG5 and PG6, and for converting the resulting diol into an alkene, are known to those skilled in the art and are not particularly limited.

[0205] These methods include, but are not limited to, methods known as Corey-Winter olefin synthesis using, for example, thiocarbonyldiimidazole or thiophosgene and trimethylphosphite, or the conditions disclosed in Org. Lett. 2000, 2, 25, 4029-4031. Furthermore, the diol may be converted to the corresponding epoxide and then deoxygenated (see, for example, Org. Synth. 1981, 60, 29).

[0206] The method for removing the protecting groups PG5 and PG6 depends on the protecting groups used. Suitable methods are known to those skilled in the art. Preferably, PG5 and PG6 together form a cyclic group, more preferably a diacetal protecting group, more preferably the following groups, [ka] Deprotection is preferably carried out under acidic conditions, for example, using a strong mineral acid or organic acid, such as HCl or TFA. The removal of these protecting groups can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent optionally mixed with water, more preferably in a solvent selected from the group consisting of methanol, ethanol, trifluoroethanol (TFE), dichloromethane, 1,2-dichloroethane, DMF, DMSO, NMP, methanol, ethanol, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl terbutyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, optionally mixed with water, most preferably in dichloromethane and water. Preferably, the removal is carried out at a temperature in the range of 0 to 40°C, more preferably in the range of 10 to 30°C, and more preferably at room temperature. During the reaction, the temperature may be varied or kept essentially constant.

[0207] A suitable method for converting a diol to an alkene group is known to those skilled in the art and is described above.

[0208] Preferably, the hydroxyl group is converted to a leaving group (L), such as a sulfonic acid ester, such as a mesyl group, tosyl group, or triflate group, preferably a triflate group, and then removed in a further step.

[0209] Such removal is preferably carried out using Na2S2O3 in combination with NaI. The removal can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent selected from the group consisting of acetone, acetonitrile, γ-butyrolactone, DMSO, dichloromethane, DMF, DMSO, NMP, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tart-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, preferably in acetone.

[0210] Nu in the compound of formula I-8A2, preferably (Ia-8A2), is -N3 or 15 N label-N 3で In some cases, the method preferably further comprises step (xA2), namely the reduction of the azide and the conversion of the primary amine to a tertiary amine, and the compound has structure (I-9A2), [ka] Preferably, it has structure (Ia-9A2). [ka]

[0211] Step (xA2) is preferably carried out in the presence of benzaldehyde and NaCNBH3, and further in the presence of paraformaldehyde and NaCNBH3.

[0212] Furthermore, this method preferably includes the removal of PG2, PG3, and PG7. In this context, please refer to the respective conditions described above and below.

[0213] PG2, PG3, and PG7 are particularly preferably Bn and / or Cbz groups. Removal is preferably carried out by reduction. In this case, it is also preferable that any additional Bn groups on the secondary amine are removed.

[0214] Reduction is preferably carried out in the presence of hydrogen using a suitable catalyst. If deuteration is desired, D2 may be used. The catalyst may be homogeneous or heterogeneous. If a heterogeneous catalyst is used, the catalyst is preferably Pd, Pd / C, Pt or Pt / C, Rh / Al2O3, Pd / Al2O3, Pt / Al2O3, Pd(OH)2, PtO2, PdO*(H2O) x The catalyst is selected from the group consisting of Pd / CaCO3, Pd / BaSO4, Rh / Al2O3, and Ru / Al2O3, and more preferably the catalyst is a palladium-containing catalyst, more preferably Pd / C. Suitable conditions have already been described above.

[0215] Preferably, PG1 is then removed to obtain final gentamicin C1 or isotope-labeled gentamicin C1. This removal is preferably carried out under basic conditions, using, for example, KOH, NaOMe, NaOH, or Ba(OH)2, at a temperature from about RT to about 100°C for about 1 hour to about 48 hours, for example, overnight.

[0216] Each of the obtained gentamicin C or gentamicin C derivatives may be subjected to appropriate post-treatment. Such post-treatment may preferably include one or more steps, at least one of which is purification such as extraction and / or precipitation and / or filtration and / or chromatography.

[0217] Method A3 - For example, preferred gentamicin C2a When optionally isotope-labeled gentamicin C2a or its derivatives are prepared, Method A further comprises converting the primary azide group of compound (I-3A), preferably compound (Ia-3A), to a primary amine group.

[0218] Therefore, this method involves the following steps: (v)(A3) The primary azide group of compound (I-3A3), preferably compound (Ia-3A3), is converted to a primary amine group to obtain the compound of formula (I-4A3), [ka] Preferably, the process further includes a step of obtaining the compound (Ia-4A3). [ka]

[0219] Preferably, this step is carried out in the presence of a phosphine, such as an alkylphosphine or arylphosphine, particularly PPh3, TCEP, or PMe3, preferably PMe3.

[0220] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, wherein gentamicin C is preferably gentamicin C2a, and further comprises step (v)(A3).

[0221] Preferably, in a further step, the primary amine group is protected with a suitable protecting group PG7 to obtain a compound of formula (I-5A3), for example, a compound of formula (Ia-5A3). Preferred protecting groups are known to those skilled in the art. In particular, PG7 is a Cbz group.

[0222] Therefore, the method according to the present invention preferably involves the following steps (vi)(A3) Protect the primary amine group to form the compound of formula (I-5A3), [ka] Preferably, the process further includes a step of obtaining the compound (Ia-5A3). [ka]

[0223] Preferably, PG7 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, and trifluoroacetyl. According to one preferred embodiment, PG7 is Cbz.

[0224] Preferably, in a further step, the primary alcohol is reacted via the Mitsunobu reaction to form a compound of formula (I-6A3), [ka] Preferably, the compound (Ia-6A3) is obtained. [ka]

[0225] Preferably, the reaction is carried out in the presence of triphenylphosphine and an azodicarboxylate salt such as diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD). Typically, the reaction is carried out first at a temperature in the range of -30°C to 0°C, preferably about -10°C, typically in THF or toluene, and then heated under reflux.

[0226] Preferably, in a subsequent step, the method converts the primary hydroxyl group of compound (I-6A3), preferably compound (Ia-6A3), into a leaving group, and preferably the compound thus obtained is exposed to nitrogen or 15 This method involves reacting a nucleophile containing a nitrogen atom, such as an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide.

[0227] As used in this context of the present invention, the term "leaving group" refers to the bonded center of the substrate and the nucleophile, such as an amine group, ammonia, hydrazine, hydrazide, 15This indicates a molecular fragment that, upon reaction with NH3 or an azide ion, detaches a pair of electrons from the reaction substrate via heterolytic bond cleavage. Examples of leaving groups include, among others, halogens, sulfonic acid esters (e.g., particularly mesyl and tosyl or triflate groups).

[0228] Therefore, the method according to the present invention preferably involves the following steps (vii)(A3) Convert the primary hydroxyl group to a leaving group -L to obtain the compound of formula (I-7A3), [ka] Preferably, the process further includes a step to obtain the compound (Ia-7A3), [ka] The leaving group is preferably a sulfonic acid ester, and step (v)(A1) includes a reaction with preferably a sulfonic acid halide, preferably a chloride.

[0229] More preferably, L is -O-tosyl or -O-mesyl.

[0230] Next, it is preferable to react the compound of formula (I-7A3), preferably (Ia-7A3), with a nucleophile in the presence of a suitable base.

[0231] Therefore, the method according to the present invention preferably involves the following steps (viii)(A3) A compound of formula (I-7A3), preferably (Ia-7A3), is reacted with a nucleophile, for example, an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide to obtain a compound of formula (I-8A3), [ka] Preferably, the process further includes a step to obtain the compound (Ia-8A3), [ka] In the formula, Nu is a bound nucleophile, preferably Nu is -N3, -NH2, -NH-NH2, -NH-N(R H ) 2、 -NH-NH(R H ) 、 - 15 NH-NH2, - 15 NH- 15 NH2, - 15 NH- 15 N(R H ) 2、 - 15 NH- 15 NH(R H ), - 15 NH-N(R H ) 2、 - 15 NH-NH(R H ), 15 N label-N 3、 -NH-Me, -NH 13 CH3, - 15 NH 13 CH3, - 15 NH-Me and - 15 Selected from the group consisting of NH2, R H The base is aryl or alkyl, preferably alkyl, and the reaction is preferably carried out in the presence of a suitable base.

[0232] Suitable bases are preferably amino groups containing a base, and most preferably non-nucleophilic or small nucleophilic bases selected from the group consisting of diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N-methylimidazole, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, colidine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), and 1,5-diazabicyclo-(4.3.0)nona-5-ene (DBN).

[0233] Preferably, the reaction is carried out in an organic solvent such as methanol or DMF.

[0234] The reaction temperature is preferably in the range of 0 to 120°C, more preferably in the range of 20 to 85°C, and the temperature may preferably be varied within the above given range or kept essentially constant.

[0235] Therefore, preferably, the method further comprises step (ix)(A3). In this step, the protecting groups PG5 and PG6 of the compound of formula (I-8A3), preferably (Ia-8A3), are preferably removed, and the resulting diol is preferably converted to an alkene group, thereby forming formula (I-9A3), [ka] Preferably, the compound (Ia-9A3) is formed. [ka]

[0236] Methods for removing the protecting groups PG5 and PG6, and for converting the resulting diol into an alkene, are known to those skilled in the art and are not particularly limited.

[0237] These methods include, but are not limited to, methods known as Corey-Winter olefin synthesis using, for example, thiocarbonyldiimidazole or thiophosgene and trimethylphosphite, or the conditions disclosed in Org. Lett. 2000, 2, 25, 4029-4031. Furthermore, the diol may be converted to the corresponding epoxide and then deoxygenated (see, for example, Org. Synth. 1981, 60, 29).

[0238] The method for removing the protecting groups PG5 and PG6 depends on the protecting groups used. Suitable methods are known to those skilled in the art. Preferably, PG5 and PG6 together form a cyclic group, more preferably a diacetal protecting group, more preferably the following groups, [ka] Deprotection is preferably carried out under acidic conditions, for example, using a strong mineral acid or organic acid, such as HCl or TFA. The removal of these protecting groups can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent optionally mixed with water, more preferably in a solvent selected from the group consisting of methanol, ethanol, trifluoroethanol (TFE), dichloromethane, 1,2-dichloroethane, DMF, DMSO, NMP, methanol, ethanol, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl terbutyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, optionally mixed with water, most preferably in dichloromethane and water. Preferably, the removal is carried out at a temperature in the range of 0 to 40°C, more preferably in the range of 10 to 30°C, and more preferably at room temperature. During the reaction, the temperature may be varied or kept essentially constant.

[0239] Preferably, the hydroxyl group is converted to a leaving group (L), such as a sulfonic acid ester, such as a mesyl group, tosyl group, or triflate group, preferably a triflate group, and then removed in a further step.

[0240] Such removal is preferably carried out using Na2S2O3 in combination with NaI. The removal can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent selected from the group consisting of acetone, acetonitrile, γ-butyrolactone, DMSO, dichloromethane, DMF, DMSO, NMP, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tart-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, preferably in acetone.

[0241] Preferably, the method further includes reduction of a double bond. Preferably, in this step, conditions are applied such that PG3, PG2, and PG7 are removed in the same step. Alternatively, the method includes a step of reducing an alkene group and at least one step of removing PG3, PG2, and PG7.

[0242] Reduction of the alkene group and optional removal of PG2, PG3, and PG7 can optionally yield isotopically labeled and PG1-protected gentamicin C2a.

[0243] The reduction is preferably carried out in the presence of hydrogen using a suitable catalyst. This allows for the advantageous removal of protecting groups that may be removed under such reducing conditions.

[0244] Therefore, preferably, the method according to this second preferred embodiment B comprises the following steps: (x)(A3) The further step includes reducing the compound of formula (I-9A3), preferably (Ia-9A3), using a suitable catalyst, preferably in the presence of hydrogen, thereby preferably removing PG2, PG3, and PG7.

[0245] PG2, PG3, and PG7 are removed in particular if they are Bn and / or Cbz groups.

[0246] Reduction is preferably carried out in the presence of hydrogen using a suitable catalyst. If deuteration is desired, D2 may be used. The catalyst may be homogeneous or heterogeneous. If a heterogeneous catalyst is used, the catalyst is preferably Pd, Pd / C, Pt or Pt / C, Rh / Al2O3, Pd / Al2O3, Pt / Al2O3, Pd(OH)2, PtO2, PdO*(H2O) x The catalyst is selected from the group consisting of Pd / CaCO3, Pd / BaSO4, Rh / Al2O3, and Ru / Al2O3, and more preferably the catalyst is a palladium-containing catalyst, more preferably Pd / C. Suitable conditions have already been described above.

[0247] After reduction and removal of PG2, PG3, and PG7, PG1 is preferably removed to obtain the final isotope-labeled gentamicin C2a. This removal is preferably carried out under basic conditions such as KOH, NaOMe, NaOH, or Ba(OH)2.

[0248] Each of the obtained gentamicin C or gentamicin C derivatives may be subjected to appropriate post-treatment. Such post-treatment may preferably include one or more steps, at least one of which is purification such as extraction and / or precipitation and / or filtration and / or chromatography.

[0249] Method A4 - Preferred alternative synthesis of gentamicin C1 In a more preferred embodiment A4, the primary hydroxyl group of compound (I-3A) [ka] Preferably compound (Ia-3A) [ka] The compound is protected with a suitable protecting group PG4', and then preferably the protecting groups PG5 and PG6 are removed. The protecting group PG4' is orthogonal to other protecting groups present in compound (I-3A) and compound (Ia-3A), and is preferably selected from the group consisting of SEM, TBS, triethylsilyl (TES), TBDPS, TIPS, and allyl. Method 4A is particularly preferred when optionally isotope-labeled gentamicin C1 or its derivatives are prepared.

[0250] Therefore, the method according to the present invention preferably involves the following steps (v)(A4) A step of protecting the hydroxyl group of a compound of formula (I-3A), preferably compound (Ia-3A), with a protecting group PG4' selected from the group consisting of SEM, TBS, triethylsilyl (TES), TBDPS, TIPS, and allyl, wherein PG4' is preferably allyl, and the protecting group is preferably introduced by the reaction of compound (I-3A), preferably compound (Ia-3A), with NaH and allyl bromide. Compound of formula (I-4A4), [ka] Preferably, the process further includes a step of obtaining the compound (Ia-4A4). [ka]

[0251] Therefore, preferably, the method further comprises step (vi)(A4). In this step, the protecting groups PG5 and PG6 of the compound of formula (I-4A4), preferably (Ia-4A4), are preferably removed, and the resulting diol is preferably converted to an alkene group, thereby forming formula (I-5A4). [ka] Preferably, the compound (Ia-5A4) is formed. [ka]

[0252] Methods for removing the protecting groups PG5 and PG6, and for converting the resulting diol into an alkene, are known to those skilled in the art and are not particularly limited.

[0253] These methods include, but are not limited to, methods known as Corey-Winter olefin synthesis using, for example, thiocarbonyldiimidazole or thiophosgene and trimethylphosphite, or the conditions disclosed in Org. Lett. 2000, 2, 25, 4029-4031. Furthermore, the diol may be converted to the corresponding epoxide and then deoxygenated (see, for example, Org. Synth. 1981, 60, 29).

[0254] The method for removing the protecting groups PG5 and PG6 depends on the protecting groups used. Suitable methods are known to those skilled in the art. Preferably, PG5 and PG6 together form a cyclic group, more preferably a diacetal protecting group, more preferably the following groups, [ka] Deprotection is preferably carried out under acidic conditions, for example, using a strong mineral acid or organic acid, such as HCl or TFA (optionally containing formic acid). The removal of these protecting groups can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent optionally mixed with water, more preferably in a solvent selected from the group consisting of methanol, ethanol, trifluoroethanol (TFE), dichloromethane, 1,2-dichloroethane, DMF, DMSO, NMP, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl terbutyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile and mixtures of two or more thereof, optionally mixed with water, most preferably in dichloromethane and water. Preferably, the removal is carried out at a temperature in the range of 0 to 40°C, more preferably in the range of 10 to 30°C, and more preferably at room temperature. The temperature may be varied or kept essentially constant during the reaction.

[0255] Preferably, the hydroxyl group is converted to a leaving group (L), such as a sulfonic acid ester, such as a mesyl group, tosyl group, or triflate group, preferably a triflate group, and then removed in a further step.

[0256] Such removal is preferably carried out using Na2S2O3 in combination with NaI. The removal can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent selected from the group consisting of acetone, acetonitrile, γ-butyrolactone, DMSO, dichloromethane, DMF, DMSO, NMP, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tart-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, preferably in acetone.

[0257] Preferably, in a further step (vii)(A4), the protecting group PG4' is removed to obtain the respective free hydroxyl groups. Deprotection methods are known to those skilled in the art and are selected according to each protecting group to be removed. When PG4' is an allyl group, the following deprotection methods are considered to be, for example, deprotection with trihaloborane, deprotection with tert-butyllithium, or transition metal catalysts such as PdCl2 or Pd catalysts.

[0258] Preferably, the double bond is reduced thereafter or in the same reaction step. Preferably, in this step, conditions are applied such that other protecting groups are not removed and the azide functional group remains unchanged.

[0259] Advantageously, the reaction is carried out under dry and non-acidic conditions and preferably involves a reduction method to produce and / or manipulate diimide as an H2-supplied hydrogenating reagent. Preferably, any aryl- or heteroarylsulfonyl hydrazide is used in a dry solvent, preferably in a temperature range of 60 to 180°C. More preferably, a benzenesulfonyl hydrazide is used, preferably at 100 to 130°C, more preferably in dry xylene.

[0260] Therefore, preferably, the method according to this second preferred embodiment B comprises the following steps: (vii) Remove (A4)-PG4' and reduce the alkene group, Compound of formula (I-6A4), [ka] Preferably, the process further includes a step of obtaining the compound (Ia-6A4). [ka]

[0261] It should be understood that step (vii)(A4) may include multiple steps and optionally post-processing steps. For example, preferably, PG4' is removed before reduction of the double bond in the presence of PdCl2 in MeOH.

[0262] Preferably, in a further step, the hydroxyl group is converted to a leaving group, and then preferably to nitrogen or 15 A nucleophile containing a nitrogen atom, and, for example, an optionally isotope-labeled amine, preferably MeNH2, 13 CH3NH2, 13 CH3 15 NH2 or Me 15 React with NH2.

[0263] As used in this context of the present invention, the term "leaving group" refers to a molecular fragment that, upon reaction between the substrate's bound center and a nucleophile, detaches the reacting substrate along with a pair of electrons through heterolytic bond cleavage. Examples of leaving groups include, among others, halogens, sulfonic acid esters (e.g., particularly mesyl and tosyl or triflate groups).

[0264] Therefore, the method according to the present invention preferably involves the following steps (viii)(A4) Convert the primary hydroxyl group to a leaving group -L to obtain the compound of formula (I-7A4), [ka] Preferably, the process further includes a step to obtain the compound (Ia-7A4), [ka] The leaving group is preferably a sulfonic acid ester, and (viii)(A4) involves a reaction with a sulfonic acid halide, preferably a chloride.

[0265] More preferably, L is -O-tosyl or -O-mesyl.

[0266] The resulting compound is preferably subsequently reacted with a nucleophile in the presence of a suitable base.

[0267] Therefore, the method according to the present invention preferably involves the following steps (ix)(A4) A compound of formula (I-7A4), preferably (Ia-7A4), as a nucleophile, more preferably MeNH2, 13 CH3NH2, 13 CH3 15 NH2 or Me 15 When reacted with NH2, the compound of formula (I-8A4) is obtained. [ka] Preferably, the process further includes a step to obtain the compound (Ia-8A4), [ka] Nu is MeNH2, 13 CH3NH2, 13 CH3 15 NH2 or Me 15The solvent is selected from the group consisting of NH2, preferably MeNH2. Preferably, the reaction is carried out in a solvent selected from the group consisting of water, methanol, ethanol, trifluoroethanol (TFE), dichloromethane, DMF, DMSO, NMP, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures thereof.

[0268] The reaction temperature is preferably in the range of 0 to 120°C, more preferably in the range of 20 to 85°C, and the temperature may preferably be varied within the above given range or kept essentially constant.

[0269] Preferably, the method further comprises converting azidogrose to an amine. Refer to the respective conditions described above in this context. Furthermore, the method preferably comprises the removal of PG2 and PG3. Refer to the respective conditions described above in this context.

[0270] Preferably, PG1 is then removed to obtain the final isotope-labeled gentamicin C1. This removal is preferably carried out under basic conditions such as KOH, NaOMe, NaOH, or Ba(OH)2. Refer to the respective conditions described above in this context.

[0271] Each of the obtained gentamicin C or gentamicin C derivatives may be subjected to appropriate post-treatment. Such post-treatment may preferably include one or more steps, at least one of which is purification such as extraction and / or precipitation and / or filtration and / or chromatography.

[0272] Method B-gentamicin C1a preferred synthesis According to the second preferred embodiment B, the primary hydroxyl group of compound (I-1), preferably compound (Ia-1), is converted to a leaving group, which is then preferably nitrogen or 15 The reaction is carried out with a nucleophile containing a nitrogen atom, such as an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide. Method B is particularly preferred when optionally isotope-labeled gentamicin C1a or a derivative thereof is prepared.

[0273] As used in this context of the present invention, the term "leaving group" refers to the bonded center of the substrate and the nucleophile, such as an amine group, hydrazine, hydrazide, ammonia, 15 This indicates a molecular fragment that, upon reaction with NH3 or an azido anion, detaches a pair of electrons from the reaction substrate through heterolytic bond cleavage. Examples of leaving groups include, among others, halogens, sulfonic acid esters (e.g., particularly mesyl and tosyl or triflate groups).

[0274] Therefore, the method according to the present invention preferably involves the following steps (iii)(B) Convert the primary hydroxyl group to a leaving group -L to obtain the compound of formula (I-2B), [ka] Preferably, the process further includes the step of obtaining the compound (Ia-2B), [ka] The leaving group is preferably a sulfonic acid ester, and (iii)(B) involves a reaction with a sulfonic acid halide, preferably a chloride.

[0275] More preferably, L is -O-tosyl or -O-mesyl.

[0276] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, wherein gentamicin C is preferably gentamicin C1a, and further comprises step (iii)(B). Preferably, the reaction is carried out in pyridine.

[0277] The compound of formula (I-2B) is preferably reacted with a nucleophile in the presence of a suitable base.

[0278] Therefore, the method according to the present invention preferably involves the following steps (iv)(B) A compound of formula (I-2B), preferably (Ia-2B), is reacted with a nucleophile, for example, an optionally isotope-labeled amine, optionally isotope-labeled ammonia, optionally isotope-labeled hydrazine, optionally isotope-labeled hydrazide, or optionally isotope-labeled azide to obtain a compound of formula (I-3B). [ka] Preferably, the process further includes a step to obtain the compound (Ia-3B), [ka] In the formula, Nu is a bound nucleophile, preferably Nu is -N3, -NH2, -NH-NH2, -NH-N(R H ) 2、 -NH-NH(R H ) 、 - 15 NH-NH2, - 15 NH- 15 NH2, - 15 NH- 15 N(R H ) 2、 - 15 NH- 15 NH(R H ), - 15 NH-N(R H ) 2、 - 15 NH-NH(R H ),15 N label-N 3、 -NH-Me, -NH 13 CH3, - 15 NH 13 CH3, - 15 NH-Me and - 15 Selected from the group consisting of NH2, R H The base is aryl or alkyl, preferably alkyl, and the reaction is preferably carried out in the presence of a suitable base.

[0279] A suitable base is preferably an amino group containing a base, and most preferably a base selected from the group consisting of diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine, N-methylimidazole, 1,4-diazabicyclo[2.2.2]octane (DABCO), N-methylpiperidine, N-methylpyrrolidine, 2,6-lutidine, colidine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), and 1,5-diazabicyclo-(4.3.0)nona-5-ene (DBN).

[0280] Preferably, the reaction is carried out in an organic solvent such as water, methanol, ethanol, trifluoroethanol (TFE), dichloromethane, DMF, DMSO, NMP, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, or mixtures thereof.

[0281] The reaction temperature is preferably in the range of 0 to 120°C, more preferably in the range of 20 to 85°C, and the temperature may preferably be varied within the above given range or kept essentially constant.

[0282] Accordingly, the present invention also relates to a method for preparing gentamicin C or a salt, solvate, or derivative thereof, preferably isotope-labeled gentamicin C or a salt, solvate, or derivative thereof, and to gentamicin C obtained or obtainable by such method, wherein gentamicin C is preferably gentamicin C1a, and further comprises steps (iii)(B) and (iii)(B1).

[0283] If Nu is an azide, formula (I-3B), preferably (Ia-3B), is further reacted directly, preferably in step (iv)(B1). Nu is preferably -NH2 or - 15 If the compound is an amine such as NH2, the amine is preferably first converted to an azide. Such methods are known to those skilled in the art and preferably include the reaction of imidazole-1-sulfonyl azide hydrochloride, CuSO4, and K2CO3 in a methanol diazotransfer reaction, as disclosed in Org. Lett. 2007, 9(19), 3797-3800. Furthermore, reactions with ZnCl2, triethylamine, trifluoromethanesulfonyl chloride, and NaN3 are preferred. Such reactions are preferably carried out in water. Preferably, in this reaction, NaN3 and trifluoromethanesulfonyl chloride react with five trifluoromethanesulfonyl azides before adding this azide to the compound to be reacted with Nu.

[0284] Therefore, preferably, the method further includes step (iv)(B1). In this step, Nu is - 15 N=N², - 15 N= 15 The protecting groups PG5 and PG6 of the compound of formula (I-3B), preferably (Ia-3B), which is N2 or -N3, are preferably removed, and the resulting diol is preferably converted to an alkene group, thereby forming formula (I-4B). [ka] Preferably, the compound (Ia-4B) is formed. [ka]

[0285] Methods for removing the protecting groups PG5 and PG6, and for converting the resulting diol into an alkene, are known to those skilled in the art and are not particularly limited.

[0286] These methods include, but are not limited to, methods known as Corey-Winter olefin synthesis using, for example, thiocarbonyldiimidazole or thiophosgene and trimethylphosphite, or the conditions disclosed in Org. Lett. 2000, 2, 25, 4029-4031. Furthermore, the diol may be converted to the corresponding epoxide and then deoxygenated (see, for example, Org. Synth. 1981, 60, 29).

[0287] The method for removing the protecting groups PG5 and PG6 depends on the protecting groups used. Suitable methods are known to those skilled in the art. Preferably, PG5 and PG6 together form a cyclic group, more preferably a diacetal protecting group, more preferably the following groups, [ka] Deprotection is preferably carried out under acidic conditions, for example, using a strong mineral acid or organic acid, such as HCl or TFA. The removal of these protecting groups can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent optionally mixed with water, more preferably in a solvent selected from the group consisting of methanol, ethanol, trifluoroethanol (TFE), dichloromethane, 1,2-dichloroethane, DMF, DMSO, NMP, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl terbutyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, optionally mixed with water, most preferably in dichloromethane and water. Preferably, the removal is carried out at a temperature in the range of 0 to 40°C, more preferably in the range of 10 to 30°C, and more preferably at room temperature. During the reaction, the temperature may be varied or kept essentially constant.

[0288] Suitable methods for converting diols to alkene groups are known to those skilled in the art. Please refer to the examples disclosed above and below.

[0289] Preferably, the hydroxyl group is converted to a leaving group (L), such as a sulfonic acid ester, such as a mesyl group, tosyl group, or triflate group, preferably a triflate group, and then removed in a further step.

[0290] Such removal is preferably carried out using Na2S2O3 in combination with NaI. Preferably, the reaction is carried out in an organic solvent selected from the group consisting of acetone, acetonitrile, γ-butyrolactone, DMSO, dichloromethane, DMF, DMSO, NMP, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tart-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, preferably in acetone.

[0291] Preferably, the method according to this second preferred embodiment B comprises the following steps: (v)(B)- 15 N=N², - 15 N= 15 The azide group present in formula (I-4B), preferably (Ia-4B), which contains a group Nu selected from the group consisting of N2 or -N3, is converted to a primary amine group, thereby forming the compound of formula (I-5B). [ka] Preferably, the process further includes the step of obtaining the compound (Ia-5B), [ka] R 3 H is R 4 teeth- 15 Selected from NH- and -NH-.

[0292] Preferably, this step is carried out in the presence of a phosphine, such as an alkylphosphine or arylphosphine, particularly PPh3, TCEP, or PMe3, preferably PMe3.

[0293] Preferably, in a further step, the double bond is reduced. Preferably, in this step, conditions are applied such that PG3 and PG2 are removed in the same step. Alternatively, the method includes a step of reducing the alkene group and at least one step of removing PG3 and PG2.

[0294] Reduction of the alkene group and optional removal of PG2 and PG3 can optionally yield isotope-labeled PG1-protected gentamicin C1a.

[0295] Reduction is preferably carried out in the presence of hydrogen using a suitable catalyst. If deuteration is desired, D2 may be used. This allows for the advantageous removal of protecting groups that may be removed under such reducing conditions, such as PG3 and PG2.

[0296] Therefore, preferably, the method according to this second preferred embodiment B comprises the following steps: (vi)(B) The process further comprises reducing the compound of formula (I-5B), preferably (Ia-5B), using a suitable catalyst, preferably in the presence of hydrogen, thereby preferably removing PG2 and PG3. If deuteration is desired, D2 may be used.

[0297] PG2 and PG3 are removed in particular when both of them are Bn groups.

[0298] Reduction is preferably carried out in the presence of hydrogen using a suitable catalyst. If deuteration is desired, D2 may be used. The catalyst may be homogeneous or heterogeneous. If a heterogeneous catalyst is used, the catalyst is preferably Pd, Pd / C, Pt or Pt / C, Rh / Al2O3, Pd / Al2O3, Pt / Al2O3, Pd(OH)2, PtO2, PdO*(H2O) x The catalyst is selected from the group consisting of Pd / CaCO3, Pd / BaSO4, Rh / Al2O3, and Ru / Al2O3, and more preferably the catalyst is a palladium-containing catalyst, more preferably Pd / C.

[0299] In this case, the reduction of the double bond may be carried out at a temperature of approximately 0°C to 100°C for approximately 1 hour to 48 hours, for example, overnight.

[0300] The amount of catalyst used in this reaction relative to the total amount of the compound of formula (I-5B) is preferably in the range of 0.1 mol% to 100 mol%, preferably 5 mol% to 10 mol%, based on the total amount of the compound of formula (I-5B) in [mol]. Regarding the solvent used in step (vi)(B), any suitable organic solvent known to those skilled in the art may be used. In particular, the solvent is selected from the group consisting of acetic acid and water. If deuteration is desired, acetic acid-D4 and D2O may be used.

[0301] After reduction and removal of PG2 and PG3, PG1 is preferably removed to obtain the final isotope-labeled gentamicin C1a. This removal is preferably carried out under basic conditions such as KOH, NaOMe, NaOH, or Ba(OH)2. Refer to the respective conditions described above in this context.

[0302] The obtained gentamicin C or gentamicin derivative may be subjected to appropriate post-treatment. Such post-treatment may preferably include one or more steps, at least one of which is purification such as extraction and / or precipitation and / or filtration and / or chromatography.

[0303] Method C - Preferred synthesis of, for example, gentamicin C2b In a more preferred embodiment C, the primary hydroxyl group of compound (I-1), preferably compound (Ia-1), is protected with a suitable protecting group PG4', and preferably thereafter the protecting groups PG5 and PG6 are removed. The protecting group PG4' is orthogonal to other protecting groups present in compound (I-1) and compound (Ia-1), and is preferably selected from the group consisting of SEM, TBS, triethylsilyl (TES), TBDPS, TIPS, and allyl. Method C is particularly preferred when optionally isotope-labeled gentamicin C2b or its derivatives are prepared.

[0304] Therefore, the method according to the present invention preferably involves the following steps (iii)(C) A step of protecting the hydroxyl group of a compound of formula (I-1), preferably compound (Ia-1), with a protecting group PG4' selected from the group consisting of SEM, TBS, triethylsilyl (TES), TBDPS, TIPS, and allyl, wherein PG4' is preferably allyl, and the protecting group is preferably introduced by a reaction between compound (I-1) and NaH and allyl bromide. Compounds of formula (IC), [ka] Preferably, the process further includes the step of obtaining a compound of (Ia-C). [ka]

[0305] Therefore, preferably, the method further comprises step (iv)(C). In this step, the protecting groups PG5 and PG6 of the compound of formula (IC), preferably (Ia-C), are preferably removed, and the resulting diol is preferably converted to an alkene group, thereby forming the compound of formula (I-1C), [ka] Preferably, the process further includes a step of obtaining a compound of (Ia-1C). [ka]

[0306] Methods for removing the protecting groups PG5 and PG6, and for converting the resulting diol into an alkene, are known to those skilled in the art and are not particularly limited.

[0307] These methods include, but are not limited to, methods known as Corey-Winter olefin synthesis using, for example, thiocarbonyldiimidazole or thiophosgene and trimethylphosphite, or the conditions disclosed in Org. Lett. 2000, 2, 25, 4029-4031. Furthermore, the diol may be converted to the corresponding epoxide and then deoxygenated (see, for example, Org. Synth. 1981, 60, 29).

[0308] The method for removing the protecting groups PG5 and PG6 depends on the protecting groups used. Suitable methods are known to those skilled in the art. Preferably, PG5 and PG6 together form a cyclic group, more preferably a diacetal protecting group, more preferably the following groups, [ka] Deprotection is preferably carried out under acidic conditions, for example, using a strong mineral acid or organic acid, such as HCl or TFA (optionally containing formic acid). The removal of these protecting groups can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent optionally mixed with water, more preferably in a solvent selected from the group consisting of methanol, ethanol, trifluoroethanol (TFE), dichloromethane, 1,2-dichloroethane, DMF, DMSO, NMP, propanol, isopropanol, butanol, s-butanol, t-butanol, tetrahydrofuran, 2-methyltetrahydrofuran, methyl terbutyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile and mixtures of two or more thereof, optionally mixed with water, most preferably in dichloromethane and water. Preferably, the removal is carried out at a temperature in the range of 0 to 40°C, more preferably in the range of 10 to 30°C, and more preferably at room temperature. The temperature may be varied or kept essentially constant during the reaction.

[0309] Suitable methods for converting diols to alkene groups are known to those skilled in the art. Please refer to the examples disclosed above and below.

[0310] Preferably, the hydroxyl group is converted to a leaving group (L), such as a sulfonic acid ester, such as a mesyl group, tosyl group, or triflate group, preferably a triflate group, and then removed in a further step.

[0311] Such removal is preferably carried out using Na2S2O3 in combination with NaI. The removal can be carried out in any suitable solvent known to those skilled in the art. Preferably, the reaction is carried out in an organic solvent selected from the group consisting of acetone, acetonitrile, γ-butyrolactone, DMSO, dichloromethane, DMF, DMSO, NMP, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tart-butyl ether, diethyl ether, diisopropyl ether, toluene, acetonitrile, and mixtures of two or more thereof, preferably in acetone.

[0312] Preferably, in a further step (v)(C), the protecting group PG4' is removed to obtain the respective free hydroxyl groups. Deprotection methods are known to those skilled in the art and are selected according to each protecting group to be removed. When PG4' is an allyl group, the following deprotection methods are, for example, deprotection with trihaloborane, deprotection with tert-butyllithium, and PdC l It is thought to be a transition metal catalyst such as a Pd catalyst, as indicated by 2.

[0313] Preferably, the double bond is reduced thereafter or in the same reaction step. Preferably, in this step, conditions are applied such that other protecting groups are not removed and the azide functional group remains unchanged. Advantageously, the reaction is carried out under dry and non-acidic conditions and preferably includes a reduction method for producing and / or manipulating the diimide as an H2 supply hydrogenating reagent. Preferably, any aryl- or heteroarylsulfonyl hydrazide is used in a dry solvent, preferably in a temperature range of 60 to 180°C. More preferably, a benzenesulfonyl hydrazide is used, preferably in dry xylene, preferably at 100 to 130°C.

[0314] Therefore, preferably, the method according to this second preferred embodiment B comprises the following steps: (v)(C)-PG4' is removed, and the alkene group is reduced, Compound of formula (I-2C), [ka] Preferably, the process further includes the step of obtaining a compound of (Ia-2C). [ka]

[0315] Preferably, this method involves converting the group -CH2-OH to the group R 11 (R 1 R 2 )-R 4 -R 3The process further includes the steps (vi)(C) to convert to R 11 is C, preferably -R 11 (R 1 R 2 )- is -CH2-, and R3 and R4 are as described above. In this step, the primary hydroxyl group of compound (I-2C), preferably compound (Ia-2C), is converted to a leaving group, and the compound thus obtained is preferably a nucleophile HR 4 -R 3 Preferably H-NH-R 3 or H- 15 NH-R 3 And R reacted, 3 is more comfortable -CH3, - 13 CH3, - 13 It is CD3 or -CD3.

[0316] As used in this context of the present invention, the term "leaving group" refers to the bonded center of the substrate and the nucleophile, such as an amine group, ammonia, hydrazine, hydrazide, 15 This indicates a molecular fragment that, upon reaction with NH3 or an azido anion, detaches a pair of electrons from the reaction substrate through heterolytic bond cleavage. Examples of leaving groups include, among others, halogens, sulfonic acid esters (e.g., particularly mesyl and tosyl or triflate groups).

[0317] In step (vi)(C), preferably a compound of formula (I-3C), [ka] More preferably, the compound (Ia-3C) [ka] This yields -C(R 1 R 2 )- is -CH2-. Therefore, in this step, the primary hydroxyl group of compound (I-C2), preferably compound (Ia-C2), becomes -R 4 -R 3 Converted to -R 4 -R3 Preferably H-NH-R 3 or H- 15 NH-R 3 And R 3 is more comfortable -CH3, - 13 CH3, - 13 It is CD3 or -CD3.

[0318] Preferably, the method according to this third preferred embodiment C is (vi)(C) Convert the azide group present in formula (I-C3), preferably (Ia-C3), to a primary amine group to obtain a compound of formula (I-4C), preferably (Ia-4C), and remove the remaining protecting group. Compound of formula (I-5C), [ka] Preferably, this further includes obtaining a compound of (Ia-5C), [ka] -R 4 -R 3 Preferably H-NH-R 3 or H- 15 NH-R 3 And R 3 is more comfortable -CH3, - 13 CH3, - 13 It is CD3 or -CD3.

[0319] Methods for converting azides to amine groups and deprotecting them are known to those skilled in the art and are not particularly limited. It should be understood that (vi)(C) can be carried out in one or more steps. Preferably, the azide is first converted to an amine, then PG1 and PG2 are removed, and finally PG1 is removed. Thus, preferred methods have already been described in preferred embodiments A and B above and may also be applied to step (vi)(C).

[0320] Use of compositions containing gentamicin C (a) Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising gentamicin C and a pharmaceutically acceptable excipient according to the present invention.

[0321] As used herein, the term “pharmaceutical composition” refers to a composition applied to a medical use. Such composition comprises gentamicin C according to the present invention and pharmaceutically acceptable excipients. Depending on the intended medical use, such composition may contain further components.

[0322] The pharmaceutical composition of the present invention, upon administration to a subject, provides a therapeutically effective dose of gentamicin C according to the present invention. This can be achieved as a result of a bolus administration, i.e., a single dose, or as a result of separate or sequential doses. The pharmaceutical composition is preferably for topical or systemic administration. Conventionally, pharmaceutical compositions can be administered orally, intramuscularly, intravascularly, or subcutaneously. However, depending on the properties and the desired therapeutic effect and mode of action, the pharmaceutical composition may also be administered by other routes.

[0323] Pharmaceutical compositions are preferably administered in conventional dosage forms prepared by combining the components with standard pharmaceutically acceptable excipients according to conventional procedures. These procedures may involve appropriately mixing or dissolving the components in the desired preparation. Preferably, a solution is assumed. It will be understood that the form and characteristics of the pharmaceutically acceptable excipients are determined by the amount of the active ingredient combined, the route of administration, and other well-known variables.

[0324] Excipients must be compatible with the other components of the formulation and acceptable in the sense that they are not harmful to the recipient. Pharmaceutically acceptable excipients used may include solid, gel, or liquid carriers. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers include phosphate-buffered saline, syrup, oil, water, emulsion, various wetting agents, distilled water, saline, Ringer's solution, dextrose solution, and Hanks' solution. Similarly, carriers may contain time-delaying materials well known in the art, such as glyceryl monostearate or glyceryl distearate, either alone or with wax. Suitable carriers include those described above and others well known in the art; see, for example, Remington's Pharmaceutical Sciences, 23rd Edition - October 30, 2020, Mack Publishing Company, Easton, Pennsylvania.

[0325] Furthermore, the pharmaceutical composition may also contain other components such as adjuvants or non-toxic, non-therapeutic, and non-immunogenic stabilizers.

[0326] The therapeutically effective dose refers to the amount of gentamicin C used in a pharmaceutical composition that induces the desired therapeutic effect, for example, to cure a bacterial infection in the subject. The therapeutic efficacy and toxicity of a compound can be determined by standard pharmaceutical procedures, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal in 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. The administration regimen is determined by the attending physician and other clinical factors. As is well known in the medical field, the dose for one patient depends on many factors, including the patient's physique, body surface area, age, the specific compound being administered, sex, time and route of administration, general condition, and other drugs being administered concurrently. Progress can be monitored by periodic evaluations.

[0327] The pharmaceutical composition according to the present invention may also contain drugs or other components that are added to the pharmaceutical in its formulation.

[0328] Finally, please understand that the formulation of pharmaceutical compositions is carried out under GMP standardization conditions, etc., to ensure the quality, pharmaceutical safety, and efficacy of the pharmaceuticals.

[0329] The pharmaceutical compositions of the present invention can be therapeutically applied in various medical fields, particularly to treat bacterial infections. Typical medical use cases are described in more detail elsewhere in this specification.

[0330] The present invention further relates to a kit comprising gentamicin C and a container according to the present invention.

[0331] As used herein, the term “kit” refers to the set of components described above provided in a container. The container also typically includes instructions for using gentamicin C according to the present invention. Furthermore, the kit may typically include additional components, such as drugs, necessary for administering the gentamicin C in any use described herein.

[0332] The present invention also relates to a method for treating bacterial infections in subjects requiring treatment of bacterial infections, comprising administering gentamicin C or a pharmaceutical composition of the present invention to the subject.

[0333] Furthermore, it will be understood that the present invention also relates to isotope-labeled gentamicin C or its salts, solvates, or derivatives according to the present invention, or to pharmaceutical compositions of the present invention for use in the treatment of bacterial infections in subjects requiring treatment of bacterial infections.

[0334] As used herein, the term “bacterial infection” refers to the process by which bacteria enter the body of an individual, subsequently multiply, and typically cause an inflammatory response within the body. Bacteria can enter the body through different routes, including the respiratory, pharyngeal, wound, or bloodstream routes, including the gastrointestinal route. Typically, bacterial infections may be associated with a variety of symptoms, such as fatigue, fever, swollen lymph nodes, headache, respiratory problems, gastrointestinal problems, nausea, and / or vomiting. Bacterial infections can be caused by pathogenic bacteria. Pathogenic bacteria are specially adapted and possess mechanisms to overcome the body's normal defense mechanisms, allowing them to invade parts of the body where bacteria are not normally found, such as the blood. Pathogenic bacteria can also invade the surface epithelium, skin, or mucous membranes, and many can migrate to other parts of the body. Rarely, pathogenic bacteria can infect a perfectly healthy person, but infections usually only occur when the body's defense mechanisms are compromised. Such compromises can be caused by trauma or underlying debilitating diseases, such as wounds, poisoning, chills, fatigue, and malnutrition. Pathogenic bacteria can typically be grown in cultures and identified using Gram staining techniques. Bacteria are further subdivided into Gram-positive and Gram-negative bacteria according to this technique.

[0335] Preferably, the bacterial infection according to the present invention is an infection caused by Gram-negative bacteria. More preferably, the Gram-negative bacteria are selected from the group consisting of: Haemophilus influenzae, Shigella sp., Escherichia coli, Enterobacter, Klebsiella, Proteus, Pseudomonas aeruginosa, Citrobacter, Serratia, and Yersinia enterocolitica.

[0336] As used herein, the term “to treat” refers to any improvement or enhancement of a bacterial infection or its symptoms as referred herein. More preferably, however, “to treat” means that the bacterial infection is cured. It will be understood that treatment may not occur in 100% of the subjects to which the composition is administered. However, this term requires that treatment occurs in a statistically significant portion of the subjects (e.g., a cohort in a cohort study). Whether a portion is statistically significant can be further easily determined by those skilled in the art using various well-known statistical assessment tools, e.g., determining confidence intervals, determining p-values, Student's t-tests, Mann-Whitney tests, etc. Further details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. P-values ​​are preferably 0.05, 0.01, 0.005, 0.001, or 0.0001.

[0337] As used herein, the term “subject” refers to animals, including rodents, pets, domesticated animals, or laboratory animals such as primates. More typically, the subjects referred to herein are mammals, preferably humans. Preferably, the subjects suffer from renal failure. As used herein, renal failure refers to impaired renal function in the subject. Typically, renal failure is characterized by renal function being 15% or less of that of a healthy control. Renal function is typically investigated by measuring the glomerular filtration rate (GFR). Renal failure as understood herein typically has serious consequences for the subject suffering from it, such as overvolt, uremia, and high potassium levels in the blood. Renal failure can be acute or chronic.

[0338] As used herein, the term “administer” means applying non-isotope-labeled gentamicin C or its salts, solvates, or derivatives to a target being treated so that it can exert its therapeutic effect. For this purpose, non- or isotope-labeled gentamicin C or its salts, solvates, or derivatives are typically introduced into the body either systemically or topically. Routes of administration are well known to those skilled in the art and include those mentioned elsewhere herein. Preferably, the non- or isotope-labeled gentamicin C or its salts, solvates, or derivatives should be administered systemically. Also preferably, the non- or isotope-labeled gentamicin C or its salts, solvates, or derivatives should be administered topically, preferably for ocular administration.

[0339] (b) Diagnostic compositions Furthermore, the present invention also relates to a diagnostic composition comprising isotope-labeled gentamicin C and a suitable excipient according to the present invention. As used herein, the term “diagnostic composition” means a composition for identifying the presence or absence of at least one gentamicin C congener, comprising at least one isotope-labeled gentamicin C.

[0340] Furthermore, the present invention also provides the use of at least one isotope-labeled gentamicin C or a salt, solvate, or derivative according to the present invention as a calibration standard for determining the amount or presence of at least one analyte of interest, preferably at least one gentamicin C congener, present in a sample.

[0341] As used in this context, the term “sample” refers to any portion of material that contains or is suspected of containing at least one gentamicin C congener. Such a sample may typically be a liquid sample from a solution containing or suspected of containing such at least one gentamicin C congener. Such a solution may typically be prepared or arise as an artificial sample, for example, during a manufacturing process, or as a solid biological or clinical sample, including but not limited to fluid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph, or as dried blood spots and tissue extracts. Further examples of biological or clinical samples are cell cultures or tissue cultures.

[0342] Preferably, the sample is obtained from a biological or clinical sample that includes, but is not limited to, a fluid sample such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph, or a solid biological or clinical sample such as dried blood spots and tissue extracts.

[0343] As used in this context, the term “determine” refers to determining the presence or absence and / or quantity of at least one gentamicin C congener. Furthermore, the term refers to determining the quantity of at least one present gentamicin C congener. Thus, as used herein, “determine” encompasses both qualitative and quantitative determinations.

[0344] Quantitative determination includes determining absolute and relative amounts. In principle, any detection method can be used to determine the amount of at least one gentamicin C congener. Such detection techniques are well known in the art. Therefore, the determination of amounts in the method of the present invention can be carried out by any technique that enables the detection of the presence or absence or amount of each congener. Preferably, the amounts are determined by mass spectrometry (MS), more preferably by MRM-based MS.

[0345] Furthermore, the present invention relates to at least one isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, for use as a calibration standard or internal standard for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample.

[0346] At least one internal standard can be added to the sample. Typically, the “internal standard” (ISTD) is a known amount of substance that, when applied to the mass spectral detection workflow, exhibits similar properties to the analyte of interest (i.e., including any pretreatment, concentration, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is distinctly distinguishable from it. For example, during ion mobility separation, the ISTD has the same ion size as the analyte of interest but a different m / z ratio. Preferably, the internal standard has an ion size indistinguishable from the analyte but a different m / z ratio. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows for the distinction of ions from the ISTD and ions from the analyte by mass spectrometry using different mass / charge (m / z) ratios. Both are subjected to fragmentation to obtain daughter ions. These daughter ions can be distinguished by their respective m / z ratios and their respective parent ions. As a result, separate determination and quantification of signals from the ISTD and the analyte can be performed. Since the ISTD is added in a known amount, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for a relative comparison of the amount of detected analyte and enables the clear identification and quantification of the target analyte(s) present in the sample when the analyte(s) reach the mass spectrometer. Typically, though not always, the ISTD used within the scope of the present invention is at least one isotope-labeled gentamicin C, which is added to the sample and determined (i.e., the amount of the internal standard compound is determined). Therefore, the internal standard is preferably not naturally present in the sample being tested. The internal standard can be dissolved in a suitable solvent.

[0347] Furthermore, the present invention relates to a method for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, wherein the method is: (a) Mix the sample with a known amount of at least one of the above-mentioned isotope-labeled gentamicin C, or its salt, solvate, or derivative. (b) Analyzing the sample by mass spectrometry. (c) Comparing the peak area of ​​at least one analyte of interest to a standard curve, wherein the standard curve is prepared using a standard containing at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. This includes determining the amount of at least one target analyte in the sample. Preferably, before analyzing the sample by mass spectrometry, the sample according to (a) is mixed with a predetermined amount of isotope-labeled gentamicin C according to (b).

[0348] In a preferred embodiment, step (c) is: (c1) Analyze the sample mixed with ISTD by mass spectrometry, and (c2) Comparing the peak area of ​​at least one analyte of interest with a standard curve, wherein the standard curve is prepared using a standard containing at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. (c3) including calculating the amount of at least one target analyte in the sample.

[0349] Preferably, the mass spectrometry is MRM-based (multiple reaction monitoring) mass spectrometry.

[0350] Furthermore, the present invention also relates to a computer-implemented method for evaluating a sample containing at least one gentamicin analog, comprising the following steps: (aa) A step of mixing the sample with a known amount of at least one of the above-mentioned isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, and a step of receiving the peak area value of the isotope-labeled gentamicin C in the sample. (bb) A step of receiving the peak area value of at least one gentamicin C congener present in the sample. (cc) A step of comparing the peak area values ​​of at least one isotope-labeled gentamicin C and at least one gentamicin C congener, and a step of receiving the value of the amount of at least one gentamicin C congener; and (dd) Includes the step of evaluating the sample for comparison and / or calculations performed in step (cc).

[0351] As used herein, the term “computer implementation” means that the method or any of its individual steps is performed in an automated manner on a data processing unit, typically contained in a data processing device such as a computer. The data processing unit shall receive a value for the amount of the analyte of interest, which is a gentamicin C congener, present in the sample. Such a value may be a volume, a relative volume, or any other calculated value reflecting a volume as described in detail elsewhere herein.

[0352] As used herein, the term “compare” encompasses comparing a determined quantity of at least one congener referred to herein with isotope-labeled gentamicin C as a reference. It should be understood that as used herein, comparison refers to any type of comparison made between a selected value for a quantity and a reference. Comparisons may be performed manually or with computer assistance. Quantity and reference values ​​can, for example, be compared with one another, and such comparisons may be performed automatically by a computer program that executes an algorithm for comparison. The computer program performing the assessment provides the desired assessment in an appropriate output format.

[0353] The present invention also, in principle, envisions a computer program, a computer program product, or a computer-readable storage medium in which such a computer program is tangibly incorporated, the computer program, when executed on a data processing device or computer, includes instructions for performing the methods of the present invention as specified above.

[0354] Furthermore, the present invention relates to a diagnostic system, preferably a clinical diagnostic system, suitable for carrying out a method for determining the presence or amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, the method comprising steps (a), (b), and (c) as described above.

[0355] Furthermore, the present invention relates to the use of the above-described diagnostic system for determining the presence or amount of at least one target analyte in a sample.

[0356] In summary, the following embodiments are preferred based on the findings of the present invention.

[0357] 1. Isotope-labeled gentamicin C or its salt, solvate, or derivative, wherein gentamicin C is at least one 13 C, D and / or 15 Contains N atoms

[0358] 2. The isotopic-labeled gentamicin C according to Embodiment 1, wherein gentamicin C is an isotopic-labeled gentamicin C selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1, and gentamicin C1a.

[0359] 3. The isotope-labeled gentamicin C described in Embodiment 1, having the following structure, [ka] or a salt or solvate thereof, During the ceremony, R 1 , R 2and R 3 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and -CD3, R 1 or R 2 At least one of them is -H or -D, R 4 -NH-, -ND-, - 15 ND- and - 15 Selected from the group consisting of NH-, R 5 -NH2, -ND2, - 15 ND 2、 - 15 NDH and - 15 Selected from the group consisting of NH2, R 6 , R 7 and R 8 They are selected independently from the following groups: -CH-, -CD-, - 13 CD-or- 13 CH-, R 9 and R 10 They are selected independently from the following groups: -CH2-, -CDH-, -CD2-, - 13 CDH-, - 13 CD2- and- 13 CH2- R 11 is C or 13 Isotope-labeled gentamicin C, or its salts or solvates.

[0360] 4.R 1 However, -CH3,- 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 1The isotope-labeled gentamicin C according to Embodiment 3, wherein is -CD3.

[0361] 5.R 2 and R 3 However, the isotope-labeled gentamicin C according to Embodiment 4 is -H or -D independently of each other.

[0362] 6.R 2 However, it is -H or -D, and R 3 However, -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 3 The isotope-labeled gentamicin C according to Embodiment 4, wherein is -CD3.

[0363] 7.R 1 The isotope-labeled gentamicin C according to Embodiment 3, wherein is -H or -D.

[0364] 8.R 2 and R 3 However, the isotope-labeled gentamicin C according to Embodiment 7 is -H or -D independently of each other.

[0365] 9.R 2 However, it is -H or -D, and R 3 However, -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 3 The isotope-labeled gentamicin C according to Embodiment 7, wherein is -CD3.

[0366] 10.R 3 However, it is -H or -D, and R 2 However, -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13Selected from the group consisting of CD3, -CDH2, -CD2H, and CD3, preferably R 2 The isotope-labeled gentamicin C according to Embodiment 7, wherein is -CD3.

[0367] 11. The isotope-labeled gentamicin C according to Embodiment 3, having the following structure. [ka]

[0368] 12.R 11 The isotope-labeled gentamicin C described in Embodiment 11, wherein C is C.

[0369] 13.R 1 One of them is -CD3, and R 2 H is the base -R 4 -R 3 is -NH2 (in other words, R 4 is -NH_, and R 3 Isotope-labeled gentamicin C according to Embodiment 11 or 12 (where H is present).

[0370] 14.R 1 One of them is -CD3, and R 2 H is -R 4 but- 15 NH- and -R 3 but- 13 The isotope-labeled gentamicin C described in Embodiment 11 or 12 is CH3.

[0371] 15.R 1 One of them is -CD3, and R 2 H is R 4 is -NH-, and R 3 The isotope-labeled gentamicin C according to embodiment 11 or 12, wherein is -CH3.

[0372] 16.R 1 One of them is H, and R 2 is -CD3, R 4 is -NH-, and R 3The isotope-labeled gentamicin C according to embodiment 11 or 12, wherein is -H.

[0373] 17.R 1 One of them is -H, and R 2 is -H, and R 4 is -NH-, and R 3 The isotope-labeled gentamicin C according to embodiment 11 or 12, wherein is -CD3.

[0374] 18. The isotope-labeled gentamicin C according to Embodiment 4, having the following structure. [ka]

[0375] 19.R 11 The isotope-labeled gentamicin C described in Embodiment 18, wherein C is C.

[0376] 20.R 10 is -CDH-, and R 9 The isotope-labeled gentamicin C according to embodiment 18 or 19, wherein is -CDH-.

[0377] 21. An isotope-labeled gentamicin C according to any one of embodiments 18 to 20, [ka] A structure selected from the following group [ka] [ka] [ka] [ka] and mixtures thereof, Preferably, the structure is isotope-labeled gentamicin C, as shown below. [Chemical formula]

[0378] 22.R 1 and R 2 are both -H, -R 4 is - 15 NH-, -R 3 is -H, the isotopically labeled gentamicin C according to any one of Embodiments 18 to 21.

[0379] 23. The isotopically labeled gentamicin C according to any one of Embodiments 1 to 22, wherein the gentamicin C exists as trifluoroacetate.

[0380] 24. The isotopically labeled gentamicin C according to any one of Embodiments 1 to 23 is a substantially pure homolog containing less than 1.0% by weight of other gentamicin C homologs.

[0381] 25. The compound of formula (I*), [Chemical formula] preferably the compound of formula (I), [Chemical formula] wherein, R 6 , R 7 , R 8 , R 91 and R 101 are each independently selected from the group consisting of -CH-, -CD-, - 13 CD- or - 13 CH-, R 011 is -CH2-, -CD2-, -CHD-, - 13 CD2-, - 13 CH2-, - 13 CHD-, and -R 11 (R1R2)-, wherein R 1 and R 2These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. especially, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the compound has structure (Ia). [ka]

[0382] 26. Compounds of formula (I*), [ka] Preferably a compound of formula (I), [ka] During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, - 13 CDH2, - 13 CD2H, -13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. especially, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the compound has structure (Ia) [ka] It has, The compound of formula (I*), the compound of formula (I), and the compound of formula (Ia) each contain at least one 13 C, D and / or 15 A compound containing a nitrogen atom.

[0383] 27. The compound according to Embodiment 25 or 26, wherein PG4 is benzoyl (Bz).

[0384] 28. PG5 and PG6 together form the following base [ka] A compound according to any one of embodiments 25 to 27, which forms [a specific compound].

[0385] 29. The compound according to any one of embodiments 25 to 28, wherein PG3 is Bn.

[0386] 30. The compound according to any one of embodiments 25 to 29, wherein PG2 is Bn.

[0387] 31. The compound according to any one of Embodiments 25 to 30, wherein PG1 is para-trifluoromethylbenzoyl.

[0388] 32. A compound according to Embodiment 25 or Embodiment 26, having the following structure, [ka] Preferably, the following structure, [ka] A compound having the following properties.

[0389] 33. A compound according to any one of embodiments 25 to 29, wherein PG1 and PG2 together form a cyclic urethane group, preferably with structure (Id), [ka] More preferably, structure (Ie), [ka] A compound having the following properties.

[0390] 34. A compound described in Embodiment 33, with structure (If) [ka] A compound having the following properties.

[0391] 35. Use of a compound according to any one of Embodiments 25 to 34 for preparing gentamicin C or a salt or solvate thereof, wherein gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1 and gentamicin C1a, and more preferably gentamicin C according to any one of Embodiments 1 to 26.

[0392] 36. A method for preparing gentamicin C or its salts, solvates, or derivatives, wherein gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1, and gentamicin C1a, and gentamicin C is preferably isotope-labeled, and the method is: (i) Glycosylated acceptor (A) of the following formula [ka] The glycosylated donor (B*) in the following formula [ka] And preferably, formula (B) [ka] By reacting with it, a compound having structure (I*) is produced. [ka] Preferably the compound of formula (I) [ka] Including seeing, During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are -CH-, -CD-, - 13 CD-or- 13 Selected from the group consisting of CH-, R 011 -CH2-, -CD2-, -CHD-, - 13 CD2-,- 13 CH2-, - 13 CHD-, and -R 11 Selected from the group consisting of (R1R2)-, where R 1 and R 2 These are -H, -D, -CH3, - independently of each other. 13 CH3, -13 CDH2, - 13 CD2H, - 13 Selected from the group consisting of CD3, -CD2H, -CDH2, and -CD3, in the formula, R 1 or R 2 At least one of them is -H or -D, where R 11 is C or 13 C is, In the formula, PG1, PG2, PG3, PG4, PG5, and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5, and PG6. especially, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethylbenzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the donor building block has the following structure [ka] It has the structure (Ia) obtained when the glycosylated donor (B) is reacted with the glycosylated acceptor (A). [ka] A method for obtaining a compound having [a certain characteristic].

[0393] 37. A method for preparing gentamicin C or its salts, solvates, or derivatives, wherein gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1, and gentamicin C1a, and gentamicin C is isotope-labeled, and at least one 13 C, D and / or 15 The method includes N atoms, (ii) Glycosylated acceptor (A) of the following formula [ka] The glycosylated donor (B*) in the following formula [ka] And preferably, formula (B) [ka] react with to obtain a compound having the structure (I*), [Chemical formula] preferably a compound of formula (I) [Chemical formula] including obtaining wherein R 6 , R 7 , R 8 , R 91 and R 101 are, independently of each other, selected from the group consisting of -CH-, -CD-, - 13 CD- or - 13 CH-, R 011 is selected from the group consisting of -CH2-, -CD2-, -CHD-, - 13 CD2-, - 13 CH2-, - 13 CHD-, and -R 11 (R1R2)-, wherein R 1 and R 2 are, independently of each other, selected from the group consisting of -H, -D, -CH3, - 13 CH3, - 13 CDH2, - 13 CD2H, - 13 CD3, -CD2H, -CDH2, and -CD3, wherein at least one of R 1 or R 2 is -H or -D, wherein R 11 is C or 13 C, wherein PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6, In particular, PG1 is a protecting group selected from the group consisting of benzyloxycarbonyl (Cbz), benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and a cyclic protecting group that forms a cyclic group together with PG2, PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the donor building block has the following structure [ka] It has the structure (Ia) obtained when the glycosylated donor (B) is reacted with the glycosylated acceptor (A). [ka] A method for obtaining a compound having [a certain characteristic].

[0394] 38. The method according to embodiment 36 or 37, wherein PG4 is benzoyl (Bz).

[0395] 39. The method according to any one of embodiments 36 to 38, wherein PG5 and PG6 together form the following group. [ka]

[0396] 40. The method according to any one of embodiments 36 to 39, wherein PG3 is Bn and PG2 is Bn.

[0397] 41. The method according to any one of Embodiments 36 to 40, wherein PG1 is para-trifluoromethylbenzoyl.

[0398] 42. The method according to Embodiment 36 or 37, wherein the glycosylated acceptor (A) has the following structure: [ka] Glycosylated donor (B) has structure (B1), [ka] More preferably, structure (B2) [ka] It has, PG4 is even more preferably Bz, in this method.

[0399] 43. The method according to Embodiment 42, wherein the glycosylated donor (B) has structure (B2) and the compound of formula (I) has the following structure. [ka]

[0400] 44. The method according to Embodiment 36 or 37, wherein the glycosylated acceptor (A) has the following structure: [ka] Glycosylated donor (B) has structure (B1), [ka] More preferably, structure (B2) [ka] It has, PG4 is preferably Bz, The glycosylated donor (B) preferably has structure (B2), PG4 is Bz, and the compound of formula (I) has the following structure [ka] A method having

[0401] 45. A method according to any one of embodiments 36 to 44, The method is (ii) Remove the protecting group PG4 to obtain the compound of formula (I-1*), [ka] Preferably a compound of formula (I-1), [ka] More preferably, a compound of formula (Ia-1). [ka] A method that further includes obtaining

[0402] 46. ​​A method according to any one of embodiments 36 to 44, The method is (iii) Remove the protecting group PG4 to obtain the compound of formula (I-1*), [ka] Preferably a compound of formula (I-1), [ka] More preferably, a compound of formula (Ia-1). [ka] Including obtaining; Equations (I-1*), (I-1), and (Ia-1) each have at least one 13 C, D and / or 15 A method having an N atom.

[0403] 47. The method according to Embodiment 45 or 46, (iii)(A) ​​A compound of formula (I-1*), preferably formula (I-1), preferably formula (Ia-1), is reacted with an oxidizing agent to form formula (I-2A), [ka] Preferably (Ia-2A) [ka] A method further comprising obtaining a compound of the above.

[0404] 48. The method described in Embodiment 47, (iii) The carbon atom of the aldehyde group of the (A1) structure (I-2A) or more preferably (Ia-2A) is modified with a nucleophile, thereby forming the group R 12 This is bonded to a carbon atom, preferably a methyl group or an isotope-labeled methyl group, particularly -CH3 or -CD3, thereby forming the compound of formula (I-3A). [ka] Preferably a compound of type (Ia-3A) [ka] A method further comprising the step of forming.

[0405] 49. The method described in Embodiment 45 or 46, (iii)(B) Convert the primary hydroxyl group to a leaving group -L to obtain the compound of formula (I-2B), [ka] Preferably the compound of type (Ia-2B) [ka] This further includes obtaining, The leaving group is preferably a halogen or a sulfonic acid ester, and (iii)(B) is a method comprising the reaction with a sulfonic acid halide, preferably a chloride.

[0406] 50. The method described in Embodiment 49, (iii)(B1) A compound of formula (I-2B), preferably (Ia-2B), is an amine, ammonia, 15 Reacting NH3 with a nucleophile such as hydrazine, hydrazide, or azide yields the compound of formula (I-3B). [ka] Preferably a compound of type (Ia-3B) [ka] This further includes obtaining, In the formula, Nu is a bound nucleophile, preferably Nu is -N3, -NH2, -NH-NH2, -NH-N(R H ) 2、 -NH-NH(R H ) 、 - 15 NH-NH2, - 15 NH- 15 NH2, - 15 NH- 15 N(RH ) 2、 - 15 NH- 15 NH(R H ), - 15 NH-N(R H ) 2、 - 15 NH-NH(R H ), 15 N-labeled-N3, -NH-Me, -NH 13 CH3, - 15 NH 13 CH3, - 15 NH-Me, and - 15 Selected from the group consisting of NH2, R H is an aryl or alkyl, preferably an alkyl, and The reaction is carried out preferably in the presence of a suitable base.

[0407] 51. The method according to Embodiment 45 or 46, (iii)(C) A step of protecting the hydroxyl group of a compound of formula (I-1), preferably compound (Ia-1), with a protecting group PG4' selected from the group consisting of SEM, TBS, triethylsilyl (TES), TBDPS, TIPS, and allyl, wherein PG4' is preferably allyl, and the protecting group is preferably introduced by a reaction between compound (I-1) and NaH and allyl bromide. Compound of formula (I-2B), [ka] Preferably the compound of type (Ia-2B) [ka] A method that further includes obtaining

[0408] 52. Gentamicin C is obtained or can be obtained by the method described in any one of Embodiments 36 to 51, gentamicin C or a salt or solvate thereof, preferably at least one 13 C, D and / or 15Isotope-labeled gentamicin C containing a nitrogen atom, or its salts or solvates.

[0409] 53. A pharmaceutical composition comprising gentamicin C according to any one of Embodiments 1 to 24 and a pharmaceutically acceptable excipient.

[0410] 54. A kit comprising gentamicin C as described in any one of Embodiments 1 to 24 and a container.

[0411] 55. A method for treating a bacterial infection in a subject requiring treatment for a bacterial infection, comprising administering gentamicin C according to any one of Embodiments 1 to 24 or the pharmaceutical composition according to Embodiment 54 to the subject.

[0412] 56. The method according to claim 55, wherein the subject is suffering from renal failure.

[0413] 57. An isotope-labeled gentamicin C according to any one of Embodiments 1 to 24 or a salt, solvate, or derivative thereof, or a pharmaceutical composition according to Embodiment 53, for use in the treatment of bacterial infections in subjects requiring treatment for bacterial infections.

[0414] 58. The isotope-labeled gentamicin C or its salt, solvate, or derivative according to Embodiment 57, wherein the subject is suffering from renal failure.

[0415] 59. The isotopically labeled gentamicin C or its salt, solvate, or derivative according to Embodiment 57 or 58, wherein the isotopically labeled gentamicin C or its salt, solvate, or derivative is to be administered systemically.

[0416] 60. The isotope-labeled gentamicin C or its salt, solvate, or derivative according to Embodiment 57 or 58, wherein the isotope-labeled gentamicin C or its salt, solvate, or derivative is preferably administered topically for ocular administration.

[0417] 61. The isotope-labeled gentamicin C or a salt, solvate, or derivative thereof according to any one of Embodiments 57 to 60, wherein the bacterial infection is caused by Gram-negative bacteria.

[0418] 62. The Gram-negative bacteria are selected from the group consisting of the following: isotope-labeled gentamicin C or its salt, solvate, or derivative according to Embodiment 61: Haemophilus influenzae, Shigella sp., Escherichia coli, Enterobacter, Klebsiella, Proteus, Pseudomonas aeruginosa, Citrobacter, Serratia, and Yersinia enterocolitica.

[0419] 63. A diagnostic composition comprising at least one isotope-labeled gentamicin C described in any one of Embodiments 1 to 24, or an isotope-labeled gentamicin C or its salt or solvate described in Embodiment 52, and a suitable excipient.

[0420] 64. Use of at least one isotopically labeled gentamicin C or a salt, solvate, or derivative described in any one of Embodiments 1 to 24, or use of the isotopically labeled gentamicin C or a salt, solvate, or derivative described in Embodiment 52, as a calibration standard for determining the amount of at least one gentamicin C congener present in a sample.

[0421] 65. The use according to Embodiment 64, wherein the sample is obtained from a biological or clinical sample including, but not limited to, a fluid sample such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph, or a solid biological or clinical sample such as dried blood spots and tissue extracts.

[0422] 66. The use according to Embodiment 64, wherein the sample is obtained from a cell culture or tissue culture.

[0423] 67. A method for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, (a) The sample is mixed with at least one known amount of isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, as described in any one of Embodiments 1 to 24, or at least one 13 C, D and / or 15 Mix the sample with the isotope-labeled gentamicin C of Embodiment 52 containing an N atom or a salt or solvate thereof, or mix the sample with a known amount of the diagnostic composition described in Embodiment 63 containing a known amount of at least one isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof. (b) Analyzing the sample by mass spectrometry. (c) Comparing the peak area of ​​at least one analyte of interest to a standard curve, wherein the standard curve is prepared using a standard containing at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. A method comprising determining the amount of at least one target analyte in a sample.

[0424] 68. The method according to Embodiment 67, wherein, before analyzing the sample by mass spectrometry, the sample according to (a) is mixed with a predetermined amount of isotope-labeled gentamicin C according to (b).

[0425] 69. The method according to Embodiment 68, wherein step (c) is (c1) Analyze the sample mixed with ISTD by mass spectrometry, and (c2) Comparing the peak area of ​​at least one analyte of interest with a standard curve, wherein the standard curve is prepared using a standard containing at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. (c3) A method comprising calculating the amount of at least one analyte of interest in a sample.

[0426] 70. The method according to any one of embodiments 67 to 69, wherein the mass spectrometry is MRM-based mass spectrometry.

[0427] 71. The method according to any one of embodiments 67 to 69, wherein the method is automated.

[0428] 72. A kit for quantifying the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, comprising at least one isotope-labeled gentamicin C or a salt, solvate, or derivative described in any one of Embodiments 1 to 24, or at least one 13 C, D and / or 15 A kit comprising, as a calibration standard or internal standard, isotopically labeled gentamicin C or its salts or solvates as described in Embodiment 52, which contains an N atom, for example, one isotopically labeled gentamicin C or its salts, solvates, or derivatives, or a mixture of two, three, four, or five isotopically labeled gentamicin C congeners or their salts, solvates, or derivatives.

[0429] 73. For use as a calibration standard or internal standard for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, as described in any one of Embodiments 1 to 24, or at least one 13 C, D and / or 15 An isotope-labeled gentamicin C or its salt or solvate according to Embodiment 52, which contains a nitrogen atom.

[0430] 74. A diagnostic system, preferably a clinical diagnostic system, suitable for carrying out a method for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, wherein the method is (a) The sample is mixed with at least one known amount of isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, as described in any one of Embodiments 1 to 24, or at least one 13 C, D and / or 15 Mix the sample with the isotope-labeled gentamicin C of Embodiment 52 containing an N atom or a salt or solvate thereof, or mix the sample with a known amount of the diagnostic composition described in Embodiment 63 containing a known amount of at least one isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof. (b) Analyzing the sample by mass spectrometry. (c) Comparing the peak area of ​​at least one analyte of interest to a standard curve, wherein the standard curve is prepared using a standard containing at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. A diagnostic system that includes determining the amount of at least one target analyte in a sample.

[0431] 75. Use of the diagnostic system according to Embodiment 74 to determine the presence or amount of at least one target analyte in a sample.

[0432] 76. A computer-implemented method for evaluating a sample containing at least one gentamicin analog, comprising the following steps: (aa) The sample is mixed with at least one known amount of isotope-labeled gentamicin C, or a salt, solvate, or derivative thereof, as described in any one of Embodiments 1 to 24, or at least one 13 C, D and / or 15A step of mixing the sample with isotopically labeled gentamicin C of Embodiment 52 containing an N atom or a salt or solvate thereof, or a step of mixing the sample with a known amount of a diagnostic composition according to Embodiment 63 containing a known amount of at least one isotopically labeled gentamicin C, or a salt, solvate, or derivative thereof, and a step of receiving the peak area value of the isotopically labeled gentamicin C in the sample. (bb) A step of receiving the peak area value of at least one gentamicin C congener present in the sample. (cc) A step of comparing the peak area values ​​of at least one isotope-labeled gentamicin C and at least one gentamicin C congener, and a step of receiving the value of the amount of at least one gentamicin C congener; and A computer implementation method comprising the step of evaluating a sample for comparison and / or calculations performed in step (dd) (cc).

[0433] 77.R 1 is -CD3, R 2 H is the base -R 4 -R 3 but- 15 NH2, R 11 The isotope-labeled gentamicin C according to embodiment 11 or 12, wherein is preferably C. [Brief explanation of the drawing]

[0434] [Figure 1] The synthesis of gentamicin C2-D3 from OBz-shisomycin (1) carried out in Example 1 is shown. [Figure 2] The synthesis of gentamicin C1a-15N-D2 from OBz-shisomycin (1) carried out in Example 2 is shown. [Figure 3-1] The synthesis of gentamicin C1-D3 from OBz-shisomycin (1) carried out in Example 3 is shown. [Figure 3-2] The synthesis of gentamicin C1-D3 from OBz-shisomycin (1) carried out in Example 3 is shown. [Figure 4]This shows the synthesis of gentamicin C2a-D3 from OBz-shisomycin (1). [Figure 5] This shows the synthesis of gentamicin C2b-D3 from OBz-shisomycin (1). [Figure 6] This shows the structure of a naturally occurring gentamicin C congener. [Figure 7] The synthesis of glycosylated donor (B) performed in reference example A1 is shown. [Figure 8] This shows the synthesis of glycosylated acceptor (A) starting from shisomecin, as performed in Reference Example A2. [Figure 9] The synthesis of compound 1 OBz-shisomycin, as performed in Reference Example A, is shown. [Figure 10-1] The alternative synthesis of gentamicin C1-D3 from OBz-shisomycin (1) performed in Example 4 is shown. [Figure 10-2] The alternative synthesis of gentamicin C1-D3 from OBz-shisomycin (1) performed in Example 4 is shown. [Figure 11-B1] The chromatograms of samples Cal 1 and Cal 6 for analyte gentamicin C1, using gentamicin C1-D3 as an isotopic internal standard, are shown. [Figure 11-B2] The chromatograms of samples Cal 1 and Cal 6 for the analyte gentamicin C1a, using gentamicin C1a-15N-D2 as the isotope-labeled internal standard, are shown. [Figure 11-B3] The chromatograms of samples Cal 1 and Cal 6 for analytes gentamicin C2, C2a, and C2b, using gentamicin C2-D3 as an isotopic internal standard, are shown. [Examples]

[0435] The following embodiments are merely illustrative of the present invention and should not be construed in any way as limiting the scope of the invention.

[0436] Example A: Synthesis of compound 1 OBz-Cisomycin (1) [ka] A.1 Synthesis of Donor Compounds [ka] Compound A1-a was obtained according to the references: Chem.Commun.,2019,55,13291-13294 and Angew.Chem.2003,115,4389-4292.

[0437] [ka] Compound A1-a (97.25 g, 0.26 mol) was dissolved in DMF (400 mL) at room temperature. Then, hydrazine acetate (24 g, 0.26 mol) was added. After 2 hours at room temperature, an additional hydrazine acetate (2 g) was added. After 1 hour, the reaction mixture was concentrated, dissolved in isopropyl acetate, and washed with KHCO3, H2O, and NaHCO3. The organic layer was dried over Na2SO4, filtered, and dried to obtain 88.3 g of product A1-b.

[0438] 1 ¹H NMR confirmed that compound A1-b was obtained.

[0439] 1 H-NMR(400MHz,288 k,cdcl3):δ=5.22(dd,j=10.4,10.5Hz,1H),5.39(t,j=3.5Hz,1h)5.09-4.97(m,2h),4.73(dd, j=7.9Hz,0.6H),4.31-4.19(m,2.6H).4.16-4.08(m,2.2H),3.76-3.68(m,0.6H),3.64-3.60(br m,1H),3.52-3.45(m,0.6H),3.42(ddd,1H),2.09(s,3H),2.08(s,3H),2.08(s,1.8H),2.04(s,3H),2.02(s,1.8H),2.01(s,1.8H)ppm.

[0440] [ka] Compound A1-b (88.3 g, 0.26 mol) was dissolved in DCM (500 mL) at room temperature. Then, imidazole (21.78 g, 0.26 mol) was added. The reaction mixture was cooled in an ice bath, and then TBDMS-Cl (44.19 g, 0.29 mol) was added. The reaction mixture was left to stir at room temperature overnight. The organic layer was then washed with H2O and citrate, dried over MgSO4, filtered, and concentrated under vacuum to obtain 108 g of product A1-c (91%).

[0441] 1 ¹H NMR confirmed that A1-c was obtained.

[0442] 1 H-NMR(400MHz,288 K,CDCl3):δ=4.98-4.91(m,2H),4.61(d,1H),4.17(dd,1H),4.09(dd,1H),3.70-3.61(m,1H ),3.45-3.36(m,1H),2.06(s,3H),2.05(s,3H),2.00(s,3H),0.92(s,9H),0.15(d,6H)ppm.

[0443] [ka] Compound A1-c (108.58 g, 0.24 mol) was dissolved in MeOH (350 mL) at room temperature. Then, 7 M ammonia (350 mL) was added to the MeOH. The reaction mixture was stirred at room temperature for 20 hours. After the conversion was complete, the reaction mixture was concentrated at 45°C to obtain crude compound A1-d, which was purified by flash column chromatography (column: 330 g silica, n-heptane with 10% acetone) to obtain 72.69 g of compound d (yield 93%).

[0444] 1 ¹H NMR confirmed that compound A1-d was obtained.

[0445] 1 H-NMR (400MHz, 288 K, CDCl3): δ=4.58(d,1H),4.29(br d,1H),4.12(d,1H),3.85(br t,2H),3.62(td,1H),3.36(td,1H),3.32-3.27(m,1H),3.24(dd,1H),2.81(br t,1H),0.94(s,9H),0.16(d,6H)ppm.

[0446] [ka] Compound A1-d (72.19 g, 0.25 mol) was dissolved in DCM (1.3 L) at room temperature. Subsequently, tetramethoxybutane (45.68 g, 0.25 mol) was added. The reaction mixture was cooled in an ice bath, and then BF3Et2O (27.9 mL, 0.26 mol) was added. The mixture was then stirred at room temperature. After the reaction was complete, the reaction mixture was washed with saturated NaHCO3 aqueous solution, the organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by flash column chromatography to obtain 65.6 g of compound A1-e (67%).

[0447] Column: 750g silica gel, toluene / DCM = 1%~6%.

[0448] LC-MS confirmed the acquisition of compound A1-e. M=456(M+Na) +

[0449] 1 H-NMR(400MHz,288 K,CDCl3):δ=4.57(d,1H),3.84(ddd,1H),3.75-3.67(m,2H),3.55(t,1H),3.47(ddd,1H),3.34(dd,1 H),3.30(s,3H),3.25(s,3H),1.79(dd,1H),1.34(s,3H),1.28(s,3H),0.93(s,9H),0.15(d,6H)ppm.

[0450] [ka] Compound A1-e (66.05 g, 0.15 mol) was dissolved in ACN (350 mL) at room temperature. Then, DIPEA (33.90 mL, 0.198 mol), DMAP (18.61 g, 0.15 mol), and benzoyl chloride (21.20 mL, 25.59 mol) were added. The mixture was stirred at room temperature. After the reaction was complete, the reaction mixture was diluted with methyl tert-butyl ether and washed with H2O, citrate, and NaHCO3. The organic phase was then dried over Na2SO4, filtered, and concentrated under vacuum. 82.19 g of the crude product, compound A1-f (quantitative), was purified by flash column chromatography and then reacted further.

[0451] Compound A1-f was confirmed to have been obtained by LC-MS. M=456(M+Na) +

[0452] [ka] Compound A1-f (35 g, 0.065 mol) was dissolved in THF (350 mL) at room temperature. The reaction mixture was cooled to -20°C, and then AcOH (3.72 mL, 0.065 mol) and TBAF (65.1 mL, 0.065 mol) were added. The mixture was stirred at -10°C for approximately 25 minutes. After the reaction was complete, the reaction mixture was diluted with methyl tert-butyl ether and washed with citrate and NaHCO3. The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. Crude product A1-g 26.48 g was obtained in 96.1% yield.

[0453] LCMS and 1 ¹H-NMR confirmed the acquisition of compound A1-g. M=446(M+Na) +

[0454] A mixture of α- and β-isomers: 1H-NMR (400MHz, 288 K, CDCl3): δ 8.07-8.01(m,2H),7.53-7.59(m,1H),7.46-7.40(m,2H),5.33(br t,0.8H),4.67(d,0.2H),4.62(dd,0.2H),4.59(dd,0.8H),4.49(dd,0.8H),4.46(dd,0.2H),4.38-4.29(m,1.6H),3.9 3-3.82(m,1H),3.83-3.77(ddd,0.2H),3.37-3.65(dd,0.4H),3.52-3.45(m,1H),3.38(s,2.4H),3.33(0.6H),3.21(br ppm.

[0455] [ka] Compound A1-g (26.45 g, 0.0625 mol) was dissolved in DCM (350 mL) at room temperature. The reaction mixture was cooled in an ice bath, and then DBU (2.80 mL, 0.019 mmol) and trichloroacetonitrile (56.4 mL, 0.562 mmol) were added. The mixture was stirred for approximately 1 hour. After the reaction was complete, the reaction mixture was concentrated under vacuum. The crude product A1-h(B) was purified by flash column chromatography to obtain 27.23 g.

[0456] 1¹H-NMR confirmed the acquisition of compound A1-h. ¹H-NMR (400MHz, 288 K, CDCl3):δ 8.77-8.70 (m, 0.9H), 8.05-7.97 (m, 2H), 7.59-7.52 (m, 1H), 7.46-7.38 (m, 2H), 6.45 (d, 0.7H), 5.70-5.66 (m, 2H), 4.62-4.44 (m, 2H), 4.40-4.25 (m, 1.6H), 4.01 -3.88(m,1.0H),3.75(dd,1.0H),3.37(s,2.4H),3.32(s,0.6H),3.16(s,2.4H), 3.14(s,0.6H)1.37(s,2.4H),1.35(s,0.6H),1.32(s,2.4H),1.28(s,0.6H)ppm.

[0457] A.2 Provision of acceptor compounds [ka] 4.60 g, 0.053 mol, 1 equivalent, obtained from Merck KGaA) was dissolved in 180 mL of H2O at room temperature. Subsequently, 0.87 g, 0.006 mol of ZnCl2 and 74 mL, 0.531 mol of NEt3 were added. The mixture was cooled in an ice bath. In a separate flask, 17.27 g, 0.27 mol of NaN3 was suspended in 160 mL of acetonitrile at a low temperature (2-8°C). Then, trifluoromethanesulfonyl chloride (44.76 g, 0.27 mol) was added to the azide solution. The previously prepared shisomycin solution was slowly added to the resulting TfN3 suspension. The temperature was slowly raised to room temperature. After the reaction was complete, methanol (10 mL) was added and the suspension was concentrated. H2O was added to the concentrated mixture, and the resulting solution was extracted with phenylethylamine. The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by flash column chromatography to obtain 18.27 g of psisomecin N3 (62.4%).

[0458] Column: 330g silica, ethyl acetate with 0%~20% MeOH.

[0459] LC-MS confirmed the presence of sisomycin N3. M=552,2(M+H) +

[0460] 1 H-NMR(400MHz,288 K,CDCl3):δ=5.89(d,1H),4.99(d,1H),4.95(dd,1H),3.80-3.52(m,8H),3.45-3.35(m,2H),3.3 2-3.26(m,1H),2.61(s,3H),2.51-2.40(m,2H),2.36-2.25(m,2H),1.48(q,1H),1.18(s,3H)ppm.

[0461] [ka] 16.05 g, 0.028 mol, 1 equivalent of psisomecin N3 was dissolved in 180 mL of distilled mineral water (DCM) at room temperature. The resulting mixture was cooled in an ice bath. Then, 90 mL of H2O and 7.86 g of K2CO3 were added. 7.26 g of 4-(trifluoromethyl)benzoyl chloride in 60 mL of DCM was added to this mixture. The mixture was then stirred at ice bath temperature for 1 hour, followed by heating to 15°C. After the reaction was complete, the mixture was extracted with DCM and washed with H2O. The organic layer was dried over MgSO4, filtered, and concentrated to obtain the dry product, which was purified by flash chromatography to obtain 17.75 g of pure psisomecin N3CF3 (87% yield).

[0462] Column: 330g Buchi(registered trademark) silica column (toluene / Â=6:4)

[0463] LC-MS confirmed the acquisition of sisomycin N3CF3. M=724,4(M+H) +

[0464] 1H-NMR (400MHz, 288 K, CDCl3): δ=7.75-7.65(m,2.2H),7.64-7.50(m,2.2H),7.28-7.13(m,1.8H),6.96(br d,0.2H),5.92-5.79(m,1H),5.25-5.19(m,0.8H),5.08(d,0.1H),5.01-4.93(m,1.2H),4.15-4.08(0.9H),4.00(br d,0.1H)3.84-3.54(m,6H),3.54-3.28(m,4H),3.25(s,0.4H),3.22-3.15(m,0.3H),3.09(s,0.5H),3.08(s,2H),2. 94(d,0.2H),2.80(d,0.2H),2.70(d,0.6H),2.54-2.42(m,1H),2.42-2.24(m,3H).2.11-2.00(m,1H),1.60-1.46(2x q,1H),1.31(s,0.6H),1.28(s,2H),1.10(s,0.4H)ppm.

[0465] [ka] 17.68 g, 0.024 mol of cysomicin N3CF3 was dissolved in 170 mL of DMF and cooled in an ice bath. Then, 8.93 g, 0.024 mol of TBAI, 5.69 g, 0.142 mol of NaH (60% by weight dispersion in mineral oil), and 17.4 mL, 0.142 mol of BnBr were added. The reaction mixture was stirred at room temperature for approximately 3 hours. Subsequently, the reaction mixture was poured into ice-cold H2O and extracted with phenylethylamine. The organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0466] Crude shisomecin N3Bn was purified by crystallization and subsequent column chromatography to obtain 20.82 g of pure shisomecin N3Bn (yield 88.4%).

[0467] LC-MS confirmed the acquisition of shisomecin N3Bn. M = 10¹⁶ (M + Na) +

[0468] 1H-NMR(400MHz,288 K, CDCl3):δ=7.75-7.67(m,2.3H),7.60(d,0.9H),7.44(d,0.9H),7.40-7.14(m,14H), 6.90(d,1.2H),6.04(d,0.4H),5.94(d,0.6H),5.67(d,0.4H),5.37(d,0.7H),5.33(br s,0.3H),5.15(dd,0.9H),4.94(ddd,1H),4.89(d,1H),4.54(dd,1.6H),4.35(dd,1H),4.28-4.18(m, 2H),4.12(q,0.6H),4.00(d,0.6H),3.92(t,0.4H),3.87-3.75(m,2H),3.75-3.55(m,4H),3.52(d,0. 5H),3.48-3.38(m,1.2H),3.35(d,0.9H),3.33-3.24(m,1H),3.22(q,0.9H),3.05(s,1.7H),2.70(s, 1.3H),2.45-2.31(m,2H),2.25-2.10(m,1H),1.65-1.50(m,1.2H),0.58(s,1.3H),0.39(s,1.7H)ppm.

[0469] [ka] 21.3 g, 0.021 mol of cysomicin N3Bn was dissolved in 160 mL of MeOH and cooled in an ice bath. Then, concentrated H2SO4 (8.9 mL, 0.16 mol) was added. The reaction mixture was stirred at room temperature. After the reaction was complete, the mixture was cooled in an ice bath and diluted with toluene and MTBE. The organic layer was washed with NaHCO3, dried over MgSO4, filtered, and concentrated. Crude acceptor (A) was purified by flash column chromatography to obtain 12.86 g of pure acceptor (A) (yield 75.8%).

[0470] Column: 330g Buchi(registered trademark) silica column (toluene / Â=8:2).

[0471] LCMS confirmed that the desired acceptor (A) could be obtained. M = 838(M + Na)+

[0472] 1 The acceptance of acceptor (A) was confirmed by 1H NMR.

[0473] 1 H-NMR(400MHz,288 K,CDCl3):δ=7.73-7.64(m,2H),7.60(d,1H),7.40-7.13(m,14H),6.94(d,1H),6.02(d,0.5H),5.88(d,0.5H),5.2 9(d,0.5H),5.20(d,0.5H),4.97-4.86(m,1.5H),4-61-4.50(dd,1H),4.46-4.34(m,1H),4.34-4.22(m,2.5H),4.07 (d,0.5H),3.93(d,0.5H),3.76(t,0.5H),3.67-3.54(m,3H),3.52-3.38(m,3H),3.07(s,1.5H),3.00(t,0.5H),2.7 6(t,1.5H),2.53(dd,0.8H),2.36(s,1.5H),2.35-2.26(m,1H),1.63-1.48(m,1H),0.94(s,3H),0.63(s,1.5H)ppm.

[0474] A.3 Glycosylation of acceptor and donor compounds [ka] A suspension of acceptor (A) (500 mg, 0.613 mmol), donor A1-h (B) (497 mg, 1.4 equivalents, 0.858 mmol), and ground molecular sieve 4 Å (100 mg) in dry DCM (15 mL) was stirred at room temperature under an argon atmosphere for 30 minutes. The reaction mixture was cooled to -60°C, and diluted TMSOTf (0.3 equivalents, 50 μL in 1.0 mL DCM) was added dropwise. The reaction mixture was slowly warmed to -10°C. After 5 hours, the reaction mixture was diluted in DCM, and the reaction was stopped by filtering through Celite into a stirred solution of NaHCO3 (saturated, aqueous solution). The organic layer was washed with saturated NaHCO3, dried over NaSO4, filtered, and concentrated. Trisaccharide compound 1 was purified by flash column chromatography (DCM / MeOH = 150 / 1 to 100 / 1) to obtain 549 mg of pure compound 1 as a white solid (yield 73%).

[0475] Compound 1 was confirmed to have been obtained by LC-MS. M = 1243 (M + Na) +

[0476] 1 H-NMR (400MHz, 288 K, CDCl3):δ=ppm 0.02-0.08(m,3H)0.37(s,2H)0.51-0.57(m,2H)1.19-1.38(m,10H)1.60-1.77(m,1H)2.29-2.44(m,1H)2.67-2.71(m,2H)3.0 0-3.39(m,14H)3.42-3.83(m,11H)3.98(d,J=11.04Hz,1H)4.14-4.37(m,6H)4.40-4.65(m,6H)4.88(dd,J=11.55,3.51Hz,2H )5.12-5.25(m,2H)5.29(s,4H)5.54(d,J=4.02Hz,1H)5.66-5.87(m,2H)5.92(d,J=3.51Hz,1H)6.03(d,J=3.51Hz,1H)6.47-6 .72(m,2H)6.83-6.93(m,2H)7.12-7.28(m,9H)7.30-7.47(m,14H)7.51-7.61(m,3H)7.63-7.85(m,4H)7.99-8.13(m,3H)ppm.

[0477] Example 1: Synthesis of gentamicin C2-D3 from OBz-shisomycin (1) [ka] 1.1 OBz-Cisomycin (1) to OH-Cisomycin (2) [ka] OBz-shisomycin 1 (4.60 g, 3.77 mmol, 1 equivalent) was dissolved in MeOH (65 mL) at room temperature. Then, 25 mol% NaOMe (200 μL) in the MeOH was added dropwise until the pH reached 11. Next, the mixture was stirred at room temperature for 6 hours (TLC n-Hex / Â=3 / 1). If the conversion was not complete, the pH was adjusted back to 11 and the reaction mixture was stirred overnight. Once the conversion was complete, the reaction mixture was neutralized with concentrated HCl to pH 5-6 (approximately 250 μL), concentrated under vacuum to obtain crude OH-shisomycin 2, which was purified by flash column chromatography, column: PF-30SIHP-F0120 g (n-Hex / Â, elution at 50%) to obtain 4.10 g of OH-shisomycin 2 (yield 97%).

[0478] LC-MS confirmed the acquisition of OH-Cisomycin 2. M=1117(M+H) +

[0479] 1H-NMR(400MHz,288 K,CDCl3):δ=0.33-0.47(m,1H)0.57(s,1H)0.78-1.01(m,1H)1.21-1.38(m,5H)1.55-1.62(m,1H)1.65-1.81(m,1H)1.97 -2.07(m,1H)2.31-2.48(m,1H)2.61-2.79(m,1H)2.98-3.13(m,1H)3.16-3.25(m,2H)3.28-3.51(m,4H)3.55-3.68(m,2H) 3.69-3.90(m,2H)3.96-4.06(m,1H)4.08-4.27(m,2H)4.29-4.39(m,1H)4.44-4.66(m,1H)4.68-4.92(m,1H)5.11-5.25(m ,1H)5.25-5.39(m,1H)6.04(d,J=3.51Hz,1H)6.38(s,1H)6.85(d,J=7.53Hz,1H)7.13-7.39(m,7H)7.63-7.76(m,1H)ppm.

[0480] 1.2 OH-Cisomycin 2 to Aldehyde 3 [ka] OH-Cisomycin 2 (4.13 g, 3.70 mmol, 1 equivalent) was dissolved in dry DCM (120 mL) at room temperature. To this suspension, 4 Å (300 mg) of ground molecular sieves and NaHCO3 (3.11 g, 37.02 mmol, 10 equivalents) were added. The reaction mixture was cooled in an ice bath, and then Dess Martin periodinane (2.36 g, 5.56 mmol, 1.5 equivalents) was added in two portions. After 5 minutes, the ice bath was removed, and the reaction mixture was warmed to room temperature. The reaction mixture was stirred for 2 hours. n-Hexane (60 mL) was added, and the mixture was stirred for 30 minutes. The mixture was then filtered through Celite, washed with DCM, and dried over MgSO4. After filtration, the solution was concentrated to dryness. Aldehyde 3 was used in the next reaction without further purification.

[0481] LC-MS confirmed the acquisition of aldehyde 3. M=1115(M+H) +

[0482] 1.3 Aldehydes 3 to Alcohol 4 [ka] Methylmagnesium iodide D3 (1M, 3 equivalents, 11.03 mL) was added to dry diethyl ether (30 mL) and cooled to -50°C. Aldehyde 3 (4.10 g, 3.68 mmol, 1 equivalent) dissolved in dry diethyl ether (100 mL) was added dropwise to this solution (white / yellow suspension). The reaction mixture was stirred at -50°C for 30 minutes, then diluted with diethyl ether, quenched with saturated NH4Cl, dried over MgSO4, filtered, and concentrated to dryness.

[0483] Crude alcohol 4 was purified by flash column chromatography, column: PF-30SIHP-F0330g (n-Hex / siRNA, elution at 40%), yielding 3.17 g of alcohol 4 (76% yield in 2 steps).

[0484] LC-MS confirmed the acquisition of alcohol 4. M=1134(M+H) +

[0485] 1.4 Alcohol 4 to Mesylate 5 [ka] Alcohol 4 (1.00 g, 0.88 mmol) was dissolved in dry pyridine (20 mL), and then MsCl (2.64 mmol, 3 equivalents) was added dropwise. The reaction mixture was stirred at room temperature for approximately 16 hours. The reaction mixture was then diluted with DCM, washed with NaHCO3, the organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0486] Crude mesylate 5 was purified by flash column chromatography, column: PF-30SIHP-F0040g (n-Hex / Âde, elution at 60%), to obtain 962 mg of mesylate 5 (yield 90%).

[0487] LC-MS confirmed the acquisition of mesylate 5. M=1212(M+H) +

[0488] 1.5 Mesiraht 5 to Azid 6 [ka] Mesylate 5 (950 mg, 0.78 mmol) was dissolved in dry DMF (15 mL), and then NaN3 (509.6 mg, 7.84 mmol, 10 equivalents) was added at room temperature. The resulting suspension was heated to 90°C and stirred overnight. Once the reaction was deemed complete, the reaction mixture was cooled to room temperature, diluted with ethyl acetate, and washed with H2O. The organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0489] Crude azide 6 was used in the next step without further purification.

[0490] LC-MS confirmed the presence of azide 6. M=1159(M+H) +

[0491] 1.6 Azid 6 to Diol 7 [ka] Crude azide 6 (0.78 mmol) from the previous reaction was dissolved in 7 mL of DCM. Then, 0.5 mL of H2O and 10 mL of TFA were added. The reaction mixture turned yellow. Stirring was continued at room temperature for 30 minutes. After that, the reaction mixture was diluted with DCM, and 10 mL of saturated NaHCO3 aqueous solution was added dropwise. After extraction, the organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0492] Crude diol 7 was purified by flash column chromatography, column: PF-30SIHP-F0080g (interchim column) (n-Hex / Â, elution at 55%), yielding 800 mg of diol 7 (98% yield in 2 steps).

[0493] 1.7 Diol 7 to Triflate 8 [ka] Diol 7 (800 mg, 0.77 ml) was dissolved in dry DCM (12 mL) under an argon atmosphere, and then dry pyridine (30 equivalents, 1.85 mL) was added. The reaction mixture was cooled to 0°C, and trifluoromethanesulfonic anhydride (3.83 mmol, 5 equivalents) was added. The reaction mixture turned yellow. Stirring was continued at room temperature for 30 minutes. After another 1.5 hours, the reaction was not complete, so more trifluoromethanesulfonic anhydride (300 μL, 5 equivalents) was added. Once the reaction was complete (IPK by LC-MS), the reaction mixture was diluted with DCM, quenched with saturated NaHCO3 aqueous solution, and washed with brine. The organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0494] Crude triflate 8 was used in the next step without further purification.

[0495] 1.8 Triflate 8 to Alken 9 [ka] Crude triflate 8 (0.77 mmol) from the previous reaction was dissolved in 10 mL of dry acetone. Subsequently, Na2S2O3 (3.08 mmol, 4 equivalents) and NaI (2.31 mmol, 3 equivalents) were added at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was diluted with SiO2 and washed with 10% Na2S2O3, saturated NaHCO3 aqueous solution, and brine. The organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0496] Crude alkene 9 was purified by flash column chromatography, column: PF-30SIHP-F0080g (interchim column) (n-Hex / Â, elution at 30%), yielding 640 mg of alkene 9 (82% yield in 2 steps).

[0497] Alkene 9 was confirmed to have been obtained by LC-MS. M=10¹¹(M+H) +

[0498] 1.9 Alkenes 9 to Amines 10 [ka] Alkene 9 (630 mg, 0.62 mmol) was dissolved in THF (10 mL), and then 2 mL of NaOH (0.1 M, aqueous solution) was added. Subsequently, PMe3 (1 M, 3.74 mmol, 6 equivalents in THF) was added. The reaction mixture was heated at 50°C for 2 hours. The reaction mixture was then transferred to a round-bottom flask and concentrated to dryness.

[0499] Crude amine 10 was purified by HPLC using a YMC Actus C18-S column (ACN / H2O, eluting at 80%) to obtain 575 mg of amine 10 (as TFA salt, yield 68%).

[0500] LC-MS confirmed the acquisition of amine 10. M = 907(M + H) +

[0501] 1 ¹H NMR confirmed that amine 10 was obtained.

[0502] 1¹H NMR (400MHz, methanol-d4): δ=1.08 (s,1H) 1.20-1.42 (m,3H) 2.17 (br d,J=12.05Hz,1H) 2.30 (q,J=12.59Hz,1H) 2.47-2.64 (m,3H) 2.66 (br s,1H)3.03-3.28(m,1H)3.31-3.42(m,1H)3.43-3.69(m,3H)3.78-3.95(m,2H)3.95-4.19(m,2H)4.31-4.53(m,4H)4.56-4.68 (m,2H)4.69-4.82(m,2H)4.90-5.10(m,1H)5.27(dd,J=14.87,11.36Hz,2H)5.34-5.47(m,1H)5.52(d,J=2.76Hz,1H)5.58(br s,1H)5.75-6.02(m,2H)6.03-6.17(m,1H)7.21-7.46(m,11H)7.55(br d,J=8.28Hz,2H)7.66-7.89(m,2H)ppm.

[0503] Amine 10 to Compound 11 [ka] Amine 10 (575 mg, 0.63 mmol) was dissolved in 5 mL of acetic acid and 15 mL of H2O. 10 wt% Pd / C (20 mol%) was added to this solution, and the reaction was pressurized under H2 (1 atm). The reaction was stirred overnight at room temperature, then filtered through Celite, washed with MeOH and H2O, and concentrated to dryness.

[0504] Crude compound 11 was used in the next step without further purification.

[0505] Compound 11 was confirmed by LC-MS. M=639(M+H) +

[0506] 1.10 From compound 11 to gentamicin C2-D3(12) [ka] Crude compound 11 from the previous reaction was dissolved in 2 mL of H2O, and 2 mL of EtOH was added. Then, 10 mL of KOH (1.5 M aqueous solution) was added. Stirring was continued at 55°C for 4 hours. After the reaction was complete, the solution was neutralized to pH=8 with TFA, concentrated, and freeze-dried.

[0507] The obtained crude gentamicin C2-D3 (12) was purified by HPLC using a YMC Actus C18-S column (H2O + 0,1% TFA elution), and gentamicin C2-D3 was provided in 72.5 mg (as TFA salt).

[0508] Subsequently, crude product 12 was further purified by chromatography of SepPak C18 (elution with H2O) to provide 64.8 mg of gentamicin C2-D3.

[0509] 1 ¹H NMR confirmed the acquisition of gentamicin 2-D3.

[0510] LC-MS confirmed the acquisition of gentamicin C2-D3. M=467(M+H) + .

[0511] 1 H NMR(500MHz,D2O)δ=1.33(s,3H)1.49-1.62(m,1H)1.76-2.07(m,4H)2.49-2.57(m,1H)2.91(s,3H)3.42-3.62(m,6H)3.75-3.8 8(m,2H)3.92-4.02(m,2H)4.05-4.12(m,1H)4.22(dd,J=10.88,3.63Hz,1H)5.08(d,J=3.78Hz,1H)5.80(d,J=3.47Hz,1H)ppm.

[0512] Example 2: Gentamicin C1a- from OH-Cisomycin 2 15 N-D2 synthesis 2.1 OH-Cisomycin 2 to Tosylate 3 [ka] OH-Cisomycin 2 (1.5 g, 1.34 mmol, 1 equivalent) was dissolved in pyridine (25 mL) at room temperature. Then, TsCl (640 mg, 2.5 equivalents) was added. The reaction mixture was stirred continuously at room temperature for 20 hours. After the reaction was complete, the mixture was concentrated to dryness.

[0513] Tosylate 3 was purified by flash column chromatography, column: Interchim:PF-30SIHP-F0120g (n-Hex / RINKAN, elution at 30%), yielding 1.35 g (80%) of tosylate 3.

[0514] LC-MS confirmed the acquisition of tosylate 3. M=1271(M+H) + .

[0515] 2.2 From Toshirate 3 15 N-amine 4 [ka] Tosylate 3 (1.3 g, 1.02 mmol, 1 equivalent) was dissolved in dry NMP (19 mL) under argon at room temperature in a suitable pressure tube. The mixture was cooled to -30°C. 15 NH3(g) was slowly bubbling into the mixture until the volume increased by approximately 2 mL. The pressure tube was sealed and kept warm at room temperature. It was then heated to 85°C and stirred at this temperature for 2 days. The reaction mixture was then concentrated to dryness.

[0516] The rough obtained in this way 15 N-amine 4 was purified by flash column chromatography, column: (n-Hex / siRNA + 1% MeOH, elution at 1 / 1, 1 / 2, 1 / 3), and 1.19 g was obtained. 15 N-amine 4 was obtained as an oily substance.

[0517] LCMS 15 We confirmed that N-amine 4 was obtained. M=1117(M+H) +

[0518] 2.3 15 N-amine 4 to azid 5 [ka] 15 N-amine 4 (1.1 g, 0.98 mmol) was dissolved in 50 mL of dry MeOH, and then K2CO3 (680 mg, 4.9 mmol, 5 equivalents) was added. The reaction mixture was cooled in an ice bath, and then imidazole-1-sulfonyl azide HCl (629 mg, 2.95 mmol, 3 equivalents) and CuSO4*(H2O)5 (25 mg, 0.098 mmol, 0.1 equivalent) were added. After 10 minutes, the ice bath was removed, and stirring was continued overnight at room temperature. After the reaction was complete, the mixture was concentrated to dryness, the residue was redissolved in DCM, and washed with saturated NaHCO3 aqueous solution. The organic phase was dried over MgSO4, filtered, and concentrated.

[0519] Crude azide 5 was used in the next step without further purification.

[0520] LC-MS confirmed the acquisition of azide 5. M=1143(M+H) +

[0521] 2.4 Azide 5 to Diol 6 [ka] Crude azide 5 from the previous reaction was dissolved in 7 mL of DCM. Then, 0.5 mL of H2O and 10 mL of TFA were added. The resulting reaction mixture turned yellow. Stirring was continued at room temperature for 30 minutes. After that, the reaction mixture was diluted with DCM, and 10 mL of saturated NaHCO3 aqueous solution was added dropwise. After extraction, the organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0522] Crude diol 6 was purified by flash column chromatography, column: PF-30SIHP-F0080 g (n-Hex / Â, elution at 50%), yielding 613 mg of diol 6 (59% in two steps).

[0523] LC-MS confirmed the acquisition of diol 6. M = 1029(M + H) +

[0524] 2.5 Diol 6 to Triflate 7 [ka] Diol 6 (380 mg, 0.37 ml) was dissolved in dry DCM (3 mL) under an argon atmosphere, and then dry pyridine (15 equivalents, 450 μL) was added. The reaction mixture was cooled to 0°C, and trifluoromethanesulfonic anhydride (1.85 mmol, 5 equivalents) was added. The reaction mixture turned yellow. Stirring was continued at room temperature for 30 minutes. After another 1.5 hours, the reaction was not complete, so more trifluoromethanesulfonic anhydride (30 μL) was added. Once the reaction was complete, the reaction mixture was diluted with DCM, quenched with saturated NaHCO3 aqueous solution, and washed with brine. The organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0525] Crude triflate 7 was used in the next step without further purification.

[0526] LC-MS confirmed the presence of triflate 7. M=1293(M+H) +

[0527] 2.6 Triflate 7 to Alken 8 [ka] Crude triflate 7 (0.36 mmol) from the previous reaction was dissolved in 7 mL of dry acetone. Subsequently, Na2S2O3 (1.45 mmol, 4 equivalents) and NaI (1.27 mmol, 3.5 equivalents) were added at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was diluted with SiO2 and washed with 10% by weight aqueous solution of Na2S2O3, saturated aqueous solution of NaHCO3, and brine. After extraction, the organic layer was dried over MgSO4, filtered, and concentrated to dryness.

[0528] Crude alkene 8 was purified by flash column chromatography using column interchim:PF-30SIHP-F0040 g (n-Hex / Â, elution at 40%) to obtain 208 mg of alkene 8 (57% in two steps).

[0529] Alkene 8 was confirmed to have been obtained by LC-MS. M=995(M+H) + .

[0530] 2.7 Alkenes 8 to Amines 9 [ka] Alkene 8 (208 mg, 0.21 mmol) was dissolved in THF (1 mL), and then 0.2 mL of NaOH (0.1 M aqueous solution) was added. Subsequently, PMe3 (1.0 M, 1.25 mmol, 6 equivalents in THF) was added. The reaction mixture was heated at 50°C for 2 hours, then transferred to a suitable round-bottom flask and concentrated to dryness.

[0531] Crude amine 9 was purified by HPLC using a YMC Actus C18-S column (MeCN + 0,1% TFA / H2O + 0,1% TF, elution at 80%) to obtain 211 mg of amine 9 (as TFA salt, 75%).

[0532] LC-MS confirmed the acquisition of amine 9. M = 891(M + H) +

[0533] 2.8 Amine 9 to D2-genta 10 [ka] Amine 9 (211 mg as TFA salt) was dissolved in 2 mL of acetic acid-D4 and 1 mL of D2O. In a separate flask, Pd / C (10 wt% Pd, 1 spatula), 2 mL of acetic acid-D4, and 1 mL of D2O were added and flushed with D2 for 10 minutes. The prepared mixture of amine 9 was added to this suspension and set under D2 pressure (1 atm). The reaction mixture was left at room temperature overnight, then filtered through a Celite plug, washed with MeOH and H2O, and the filtrate was concentrated to dryness.

[0534] Crude D2-genta 10 was used in the next step without further purification.

[0535] LC-MS confirmed the acquisition of D2-genta 10. M=625(M+H) +

[0536] 2.9 D2-genta 10 to gentamicin C1a- 15 N-D211 [ka] Crude D2-genta 10 from the previous reaction was dissolved in 2 mL of H2O. Then, 10 mL of NaOH (1 M aqueous solution) was added. The mixture was stirred at 60°C for 4 hours. After the reaction was complete, the mixture was neutralized to pH=5 with TFA and freeze-dried. The dried product was dissolved in 30 mL of H2O, purified with Sep-Pak C18 (see, for example, www.flinnsci.com “Sep-Pak(R)C18 Cartridge for Column Chromatography”), and eluted with H2O and 50% ACN.

[0537] The combined product containing the H2O fraction was freeze-dried and purified by HPLC using a YMC Actus C18-S column (H2O + 0.1% TFA elution), yielding 77.2 mg of gentamicin C1α- 15 N-D211 was obtained as a TFA salt.

[0538] LCMS revealed that gentamicin C1α-15 It was confirmed that N-D211 was obtained. M=453(M+H) + M=475(M+Na) +

[0539] 1 ¹H NMR revealed gentamicin C1α- 15 We confirmed that N-D211 was obtained.

[0540] 1 H NMR(500MHz,D2O):δ=1.36(s,3H)1.58-1.63(m,1H)1.88-2.07(m,4H)2.54-2. 60(m,1H)2.93(s,3H)3.09-3.15(m,1H)3.24-3.27(m,1H)3.27-3.30(m,1H)3. 49-3.55(m,2H)3.55-3.65(m,3H)3.77-3.88(m,2H)3.98-4.05(m,2H)4.16-4. 20(m,1H)4.22-4.26(m,1H)5.11(d,J=3.56Hz,1H)5.81(d,J=3.56Hz,1H)ppm.

[0541] 3. Example 3: Experiments with Gentamicin C1-D3 - By preferred synthesis of a substitute for Gentamicin C1 (Method A4): 3.1 Alcohol 4 to Allyl Ether C1-4 [ka] Sodium hydride (60% by weight in mineral oil, 140 mg, 3.50 mmol, 1.80 equivalents) was suspended in dry N,N-dimethylformamide (5.0 mL) at room temperature under an inert gas atmosphere. The resulting mixture was cooled to 0°C. Alcohol 4 (2.20 g, 1.94 mmol, 1.00 equivalent) dissolved in dry N,N-dimethylformamide (5.0 mL) was slowly added, and the reaction mixture was stirred for 30 minutes. Then, allyl bromide (0.67 mL, 7.77 mmol, 4.00 equivalents) was added dropwise, and the reaction mixture was heated to room temperature and stirred overnight. The brownish reaction mixture was then concentrated under vacuum. The crude product was purified by normal-phase MPLC (silica, gradient n-hexane / siRNA = 100:0 to 30:70). When dried in a vacuum, allyl ether C1-4 (1.97 g, 1.68 mmol, 86%) was obtained as a colorless solid. TIFF2026511288000258.tif15164

[0542] 3.2 Allyl ether C1-4 to diol C1-5 [ka] Allyl ether C1-4 (1.97 g, 1.68 mmol) was dissolved in dichloromethane (10.0 mL) at room temperature. To this, dest. H2O (2 drops) and trifluoroacetic acid (20.0 mL) were added. The reaction mixture then turned yellow and was stirred for 30 minutes until substance C1-4 was completely consumed. The reaction mixture was then diluted with dichloromethane (40.0 mL) and neutralized by slowly adding saturated bicarbonate aqueous solution. The organic layer was separated, and the aqueous phase was extracted with dichloromethane (2 × 50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated to dryness. The crude product was purified by normal-phase MPLC (silica, gradient n-hexane / acetone = 75:25~0:100). Upon drying in vacuum, diol C1-5 (1.77 g, 1.49 mmol, 89%) was obtained as a colorless solid. TIFF2026511288000260.tif15164

[0543] 3.3 Diol C1-5 to Bistriflote C1-6 [ka] Diol C1-5 (900 mg, 0.85 mmol, 1.00 equivalent) was dissolved in dry dichloromethane (18.0 mL) at room temperature under an inert gas atmosphere. Then, dry pyridine (2.06 mL, 25.5 mmol, 30.0 equivalent) was added with stirring, and the reaction mixture was cooled to 0°C. Trifluoromethanesulfonic anhydride (1.08 mL, 6.79 mmol, 8.00 equivalent) was added, and stirring was continued at room temperature for 2 hours. The reaction mixture first became turbid, then changed to a brownish solution. Once the diol C1-5 was consumed, the resulting solution was diluted with dichloromethane (50 mL) and then rapidly washed with aqueous HCl (0.1 M, 2 × 50 mL) and saturated aqueous bicarbonate (50 mL). The aqueous phase was re-extracted with dichloromethane (2 × 50 mL), and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated under vacuum. The resulting crude orange solid (1.08 g) was used in further reactions without further purification. TIFF2026511288000262.tif15164

[0544] 3.4 Bistriflate C1-6 to Alken C1-7 [ka] Crude bistriflate C1-6 (assuming 0.85 mmol, 1.00 equivalent) from conversion 3.3 was dissolved in dry acetone (13.5 mL) at room temperature under an inert gas atmosphere. Subsequently, Na2S2O3 (537 mg, 3.40 mmol, 4.00 equivalent) and NaI (382 mg, 2.55 mmol, 3.00 equivalent) were added. The mixture was stirred at room temperature for 27 hours until substance C1-6 was completely consumed. The reaction was then diluted with dichloromethane (150 mL) and washed with aqueous Na2S2O3 solution (10 wt%, 50 mL), saturated bicarbonate solution (50 mL), and brine (50 mL). The aqueous phase was re-extracted with dichloromethane (2 × 50 mL), and the combined organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The crude brown wax obtained in this manner was redissolved in acetonitrile and purified by reverse-phase HPLC (C-18 reverse-phase silica, gradient MeCN / H2O = 20:80~95:5). The product-containing fraction was recovered and freeze-dried to obtain alkene C1-7 (540 mg, 0.53 mmol, 62%, 2 steps) as a colorless foam. TIFF2026511288000264.tif15164

[0545] 3.5 Alkene C1-7 to Alcohol C1-8 [ka] Alkene C1-7 (540 mg, 0.53 mmol, 1.00 equivalent) and PdCl2 (37.4 mg, 0.21 mmol, 0.40 equivalent) were dissolved in dry methanol (16.2 mL) at room temperature under an inert gas atmosphere. The resulting mixture was stirred for 40 hours until the starting material (C1-7) was completely consumed, and then filtered through a short Celite® plug (methanol washing). The resulting yellow wax was purified by reverse-phase HPLC (C-18 reverse-phase silica, gradient MeCN / H2O = 20:80~95:5). The product-containing fraction was collected and freeze-dried to obtain alcohol C1-8 (494 mg, 0.50 mmol, 95%) as a colorless foam. TIFF2026511288000266.tif15164

[0546] 3.6 Alcohol C1-8 to Triazide C1-9 [ka] Alcohol C1-8 (50.0 mg, 50.7 μmol, 1.00 equivalent) and pre-dried benzenesulfonyl hydrazide (51.7 mg, 0.30 mmol, 6.00 equivalent) were suspended in dry p-xylene (2.80 mL) by sonication in a suitable pressure vessel under an inert gas atmosphere. The reaction mixture was heated to 145°C for 30 minutes, and a fresh batch of pre-dried benzenesulfonyl hydrazide (51.7 mg, 0.30 mmol, 6.00 equivalent) was added. The reaction mixture was again heated to 145°C, and the cycle described was repeated every 30 minutes until a reaction time of 2 hours had elapsed, adding a total of approximately 24.0 equivalents of benzenesulfonyl hydrazide to completely consume substance C1-8. The reaction mixture was then diluted with dichloromethane (50 mL) and washed with saturated aqueous bicarbonate (40 mL). The aqueous phase was extracted with dichloromethane (2 × 30 mL), and the combined organic layer was dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude orange product was redissolved in acetonitrile and purified by reverse-phase HPLC (C-18 reverse-phase silica, gradient MeCN / H₂O = 20:80~95:5). The product-containing fraction was recovered and freeze-dried to obtain triazide C1-9 (33.9 mg, 34.3 μmol, 67%) as a colorless foam. TIFF2026511288000268.tif15164

[0547] 3.7 Triazide C1-9 to Mesylate C1-10 [ka] Triazide C-19 (150 mg, 0.15 mmol, 1.00 equivalent) was dissolved in dry pyridine (3.40 mL) at room temperature under an inert gas atmosphere. The stirred mixture was cooled to 0°C, and mesyl chloride (94.1 μL, 1.20 mmol, 8.00 equivalent) was added dropwise. After 10 minutes, the reaction mixture was warmed to room temperature and stirred for 3 hours. Anhydrous ethanol (100 μL) was added at 0°C to quench the reaction, and stirring was continued for a further 10 minutes. The resulting solution was then diluted with dichloromethane (50 mL) and rapidly washed with aqueous HCl (0.2 M, 2 × 50 mL) and aqueous bicarbonate (7.0 wt%, 50 mL). The aqueous phase was re-extracted with dichloromethane (2 × 50 mL), and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude product (C1-10) was obtained as a colorless foamy substance (211 mg) and used in further reactions without further purification. TIFF2026511288000270.tif15164

[0548] 3.8 Mesylate C1-10 to Methylamine C1-11 [ka] Crude mesylate C1-10 (assuming 0.15 mmol) was suspended in an aqueous solution of methylamine (44 wt%, 6.00 mL) in a suitable pressure vessel under an inert gas atmosphere at room temperature. The reaction mixture was heated to 105°C with vigorous stirring and allowed to react for 48 hours. Reaction control by LMCS showed complete conversion at this point. The resulting solution was then diluted with saturated aqueous bicarbonate (50 mL) and extracted with dichloromethane (4 × 50 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under vacuum. The resulting yellowish foamy substance (216 mg) was redissolved in acetonitrile and purified by reverse-phase HPLC (C-18 reverse-phase silica, gradient MeCN / H₂O / TFA = 20:80:0.05~95:5:0.05). The product-containing fraction was recovered and freeze-dried to obtain methylamine C1-11 (141 mg, 126 μmol, 83%) as a colorless solid with a dr ratio of 1:2, which does not indicate the stereochemistry shown above at the introduced methylamine residue. TIFF2026511288000272.tif10164

[0549] 3.9 Methylamine C1-11 to tetraamine C1-12 [ka] Methylamine C1-11 (141 mg, 126 μmol, 1.00 equivalent) was dissolved in dry tetrahydrofuran (3.0 mL) at room temperature under an inert gas atmosphere. Then, trimethylphosphine (1 M in THF, 0.76 mL, 0.76 mmol, 6.00 equivalent) was added with stirring, and the reaction mixture was heated at 50°C for 3 hours. After cooling to room temperature, the resulting mixture was concentrated under vacuum and then redissolved in a mixture of water, TFA, and methanol (v / v = 4:0.04:1, 1.0 mL), and purified by reverse-phase HPLC (Chromolith® C-18 reverse-phase silica, gradient MeCN / H2O / TFA = 10:90:0.1~98:2:0.1). The product-containing fraction was recovered and freeze-dried, yielding tetraamine C1-12 (153 mg, 111 μmol, 88%) as a colorless solid with a dr ratio of 1:2, which does not support the stereochemistry shown above at the deuterium-labeled methyl group. TIFF2026511288000274.tif10164

[0550] 3.10 Tetraamine C1-12 to Triol C1-13 [ka] Tetraamine C1-12 TFA salt (153 mg, 111 μmol, 1.00 equivalent) was dissolved in methanol (4.33 mL) and aqueous HCl (0.1 M, 6.50 mL) at room temperature under an inert gas atmosphere. Pearlman's catalyst (20 wt% Pd, 234 mg, 333 μmol, 3.00 equivalent) was added, and the reaction mixture was vigorously stirred and flushed with H2 (1 atm, 3 times). The mixture was hydrogenated for 22 hours and then filtered through a Celite® plug (methanol washing). The resulting solution was concentrated under vacuum to obtain a colorless solid residue, which was then redissolved in a mixture of water, TFA, and methanol (v / v = 4:0.04:1, 1.0 mL). Purification was performed by reverse-phase HPLC (C-18 reverse-phase silica, gradient MeCN / H2O / TFA = 5:95:0.05~90:10:0.05). The product-containing fraction was recovered and freeze-dried to obtain triol C1-13 (103 mg, 92.9 μmol, 84%) as a colorless solid with a dr ratio of 1:2, which does not support the stereochemistry shown above at the deuterium-labeled methyl group. TIFF2026511288000276.tif10164

[0551] 3.11 Triol C1-13 to Gentamicin C1-D3 [ka] The TFA salt of triol C1-13 (102 mg, 92.0 μmol, 1.00 equivalent) was dissolved in aqueous sodium hydroxide solution (1.5 M, 4.11 mmol, 67.0 equivalents) at room temperature under an inert gas atmosphere. The reaction mixture was heated at 60°C for 7 hours with stirring until C1-13 was completely consumed. The pH was adjusted to approximately 6.0 by adding a mixture of H2O and TFA (v / v=4:1), and the resulting mixture was concentrated under vacuum. The resulting yellowish wax was redissolved in a mixture of H2O and TFA (v / v=98:2) and purified by reverse-phase HPLC-MS (C-18 reverse-phase silica, gradient MeCN / H2O / TFA=0:100:0.2~10:90:0.2). The product-containing fraction was recovered and freeze-dried to obtain gentamicin C1-D3 (25.5 mg, 24.3 μmol, 26%) as its TFA salt, as a colorless solid, and as a single isomer. TIFF2026511288000278.tif10164

[0552] 1 H NMR(d2o,dcl,dds,500MHz):δ=5.86(j=3.51Hz,1h),5.12(d,j=3.66Hz,1h),4.24(dd,j=11.00,4.00H z),4.17(dt,j=12.28,2.63Hz,1h),4.07-3.97(m,2h),3.87(t,j=8.85Hz,1h),3.81(t,j=9.77Hz,1h) ,3.67-3.55(m,3h),3.53(dd,j=12.97,6.71Hz,2h),3.49(d,j=2.90Hz,1h),2.94(s,3h),2.76(s,3h) ,2.57(dt,j=12.59,4.16Hz,1h),2.12-1.94(m,4h),1.58(dq,j=13.40,4.30Hz,1h),1.37(s,3h)ppm. 13 C NMR(D2O,DCl,DDS,125MHz):δ=104.0,98.3,86.5,79.7,72.6,72.1,70.6,69.0,66.0,60.3,52.3,51.5,51.1,37.2,34.0,30.7,25.0,23.7,23.3 ppm. 19F NMR(D2O,DCl,DDS,470MHz):δ=75.59(s)ppm.

[0553] 4. Example 4: Experiments with Gentamicin C1-D3 - By preferred synthesis of Gentamicin C1 (Method A2): 4.1 Alcohol 4 to Triamine C1-14 [ka] Alcohol 4 was dissolved in THF (16 mL / mmol) and treated with 0.1 M NaOH and PMe3 (1 M, 4 equivalents in THF). The mixture was stirred at room temperature until strong gas generation ceased, and then heated to 50°C for 30 minutes. The solvent was removed under reduced pressure, and the crude product was purified by RP-MPLC (25 → 100% MeCN) to obtain the desired product C1-14 as a yellow / beige solid. TIFF2026511288000280.tif10164

[0554] 4.2 Triamine C1-14 to alcohol C1-15 [ka] Triamine C1-14 was dissolved in a mixture of THF / saturated NaHCO3 (v:v=1:1, 80 mL / mmol), and CbzCl (4 equivalents) was added. The mixture was thoroughly stirred at room temperature for 1 hour. After UHPLC analysis showed complete conversion to the desired product, the layers were separated, and the aqueous phase was re-extracted twice with ethyl acetate. The combined organic fraction was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. Purification by flash chromatography (25 → 75% ethyl acetate in cyclohexane) yielded the desired product C1-15 as a yellow foam. TIFF2026511288000282.tif10164

[0555] 4.3 Alcohol C1-15 to Mesylate C1-16 [ka] Alcohol C1-15 was dissolved in anhydrous pyridine (20 mL / mmol), and methanesulfonyl chloride (3 equivalents) was added. The mixture was stirred at room temperature for 16 hours. After complete conversion was observed by UHPLC analysis, the mixture was quenched by adding saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane. After phase separation, the aqueous phase was re-extracted twice with dichloromethane. The combined organic fraction was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. Purification by flash chromatography (25 → 70% dimethyl cyclohexane) yielded mesylate C1-16 as a beige foam. TIFF2026511288000284.tif10164

[0556] 4.4 Mesiraht C1-16 to Azid C1-17 [ka] Mesylate C1-16 was dissolved in anhydrous DMF (16 mL / mmol), and sodium azide (10 equivalents) was added. The mixture was heated to 80°C and stirred at this temperature for 16 hours. After UHPLC analysis showed complete conversion, the mixture was quenched by adding water, and the mixture was extracted with ethyl acetate. After phase separation, the aqueous phase was re-extracted twice with ethyl acetate. The combined organic fraction was washed with saturated sodium bicarbonate solution, dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product C1-17 (a brownish-beige foamy substance) was used directly in the next step. TIFF2026511288000286.tif10164

[0557] 4.5 Azide C1-17 to Dior C1-18 [ka] Compound C1-17 was dissolved in dichloromethane / water (v:v=15:1, 9 mL / mmol), and TFA (55 equivalents) was added. The mixture was stirred for 2 hours, and then carefully quenched by adding saturated sodium bicarbonate solution until gas generation ceased. The layers were separated, and the aqueous phase was re-extracted twice with dichloromethane. The combined organic fraction was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by RP-MPLC (60-100% MeCN) yielded the desired product C1-18 as a white foam. TIFF2026511288000288.tif9164

[0558] 4.6 Dior C1-18 to Bistriflart C1-19 [ka] Diol C1-18 was dissolved in dichloromethane (5.5 mL / mmol), and pyridine anhydride (30 equivalents) was added under argon. The solution was cooled to 0°C, and trifluoromethanesulfonic anhydride (10 equivalents) was added. The mixture was stirred at 0°C for 24-48 hours (until UHPLC analysis indicated completion of the reaction). The reaction mixture was diluted with dichloromethane and carefully quenched with saturated NaHCO3 (water) solution. The aqueous phase was extracted with dichloromethane (twice), washed with 10 wt% citric acid (aqueous solution), dried over sodium sulfate, and the solvent was removed under reduced pressure. Bistriflate C1-19 was used in further reactions without further purification. TIFF2026511288000290.tif10164

[0559] 4.7 Bistriflart C1-19 to Alken C1-20 [ka] Bistriflate C1-19 was dissolved in anhydrous acetone (20 mL / mmol), and sodium thiosulfate (6 equivalents, dry) and sodium iodide (4.5 equivalents, dry) were added. The mixture was stirred at room temperature for 72 hours. The orange-brown reaction mixture was diluted with ethyl acetate and washed with 10 wt% sodium thiosulfate solution, saturated sodium bicarbonate solution, and brine. The organic phase was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by RP-MPLC (65-100% MeCN) to obtain olefin C1-20 as a yellow foam. TIFF2026511288000292.tif10164

[0560] 4.8 Alkene C1-20 to Amine C1-21 [ka] Alkene C1-20 was dissolved in THF (25 mL / mmol) and treated with NaOH (0.1 M aqueous solution) and PMe3 (1 M in THF, 1.5 equivalents). The mixture was stirred at room temperature for 16 hours. After the reaction was complete (as shown by UHPLC), acetic acid (20 equivalents) was added and the solvent was removed under reduced pressure. The crude product was purified by RP-MPLC (50 → 100% MeCN) to obtain the desired product C1-21 as an orange solid. TIFF2026511288000294.tif10164

[0561] 4.9 Amine C1-21 to Methylbenzylamine C1-22 [ka] Free amine C1-21 in MeOH (6 mL / mmol) was mixed with benzaldehyde (1.1 equivalents), sodium borohydride cyanohydride (1.15 equivalents), and acetic acid (1.0 equivalent). The mixture was stirred at room temperature until the starting material was consumed. Paraformaldehyde (5 equivalents), a 3 Å molecular sieve, and acetic acid (10 equivalents) were added, and the mixture was stirred at room temperature for 4 hours. Then, sodium borohydride cyanohydride (2.5 equivalents) was added, and the mixture was stirred for a further 16 hours. The mixture was quenched by adding saturated sodium bicarbonate solution and extracted with ethyl acetate (3 times). The combined organic layers were dried, filtered, and the solvent was removed under reduced pressure. The crude product was purified by RP-MPLC (50 → 100% MeCN) to obtain the desired product C1-22 as a beige solid. TIFF2026511288000296.tif10164

[0562] 4.10 Methylbenzylamine C1-22 to Methylamine C1-23 [ka] Compound C1-22 was suspended in AcOH / H2O (v:v=2:1, 30 mL / mmol) and Pd / C (10%, 1 equivalent) was added. The atmosphere was changed to hydrogen (four times), and the mixture was stirred at room temperature until the starting materials and all intermediates were converted to the desired product. The atmosphere was changed to argon, and the mixture was filtered through a Celite pad and thoroughly washed with water. The combined fraction was freeze-dried or the solvent was removed by azeotropic distillation with MeOH. The crude product was purified by RP-MPLC (0 → 50% MeCN) to obtain the desired product C1-23 as a TFA salt, which was an off-white solid. TIFF2026511288000298.tif10164

[0563] 4.11 Methylamine C1-23 to Gentamicin C1-D3 [ka] Methylamine C1-23 was dissolved in a solution of saturated barium hydroxide (35 equivalents) and heated to 60°C. After the starting material was completely consumed, dry ice was added until the pH reached 7. The mixture was filtered, and the solid precipitate was washed with water. TFA (20 equivalents) was added to the aqueous phase, and the newly formed white precipitate was removed by filtering. The filter cake was washed with water, and the combined aqueous phase was frozen in a dry ice / iPrOH bath and freeze-dried. The crude product was purified via RP-HPLC to obtain gentamicin C1-D3 as the TFA salt, which was an off-white solid. The analytical data recorded for the compounds obtained by the described reaction sequence were found to be identical to the data already described above in Chapter 3.11 of the Examples section.

[0564] Example B: Gentamicin C1-D3 and Gentamicin C1a- as internal standards in MRM-based LCMS analysis of gentamicin. 15 Use of N D2 and gentamicin C2-D3. Methods: To quantify both the total amount of gentamicin C as a mixture of congeners in the sample and the content of the mixture of congeners, which are single congeners gentamicin C1 and C1a, as well as gentamicin C2, C2a, and C2b, an LC-MS / MS method was devised that included MRM transitions prepared for all compounds. A SeQuant Zic-cHILIC, 3 μm, 100 Å, 2.1 mm × 100 mm column (Merck KgaA; batch: FC098347; serial number: 913801) with a flow rate of 0.5 mL / min was used with an Agilent Infinity II equipped with a PAL autosampler connected to an AB Sciex 6500+MS, having solvent A: water containing 1.0% HCOOH and 50 mM NH4Ac and solvent B: CH3CN containing 1.0% HCOOH. Gentamicin C1-D3, Gentamicin C1a- 15 For N D2 and gentamicin C2-D3, two MRM transitions were used. Natural gentamicin C, as a mixture of congeners included in the measurement, was quantitatively analyzed in terms of its congeneral composition. 1The samples were pre-analyzed by 1H NMR spectroscopy (gentamicin C1 28.5%, gentamicin C1a 28.5%, gentamicin C2, C2a, and C2b 42.8%).

[0565] [Table 2]

[0566] Calibration curve: A calibration curve was recorded by plotting the area ratio (area analyte / area internal standard) against the analyte concentration, using a mixture of natural gentamicin C congeners of known composition (see above) and the MRM transition settings described in Table B1. The measured samples were internal standard gentamicin C1-D3 (2 μg / mL), gentamicin 1a- 15 The samples contained N-D2 (2 μg / mL) and gentamicin C2-D3 (2 μg / mL), as well as a mixture of natural gentamicin C congeners at the respective concentrations shown in Table B2 (samples Cal1-Cal6). The solvent mixture for all calibration and quality control (QC) samples was 100 mM formic acid in water. Three samples (QC1-QC3) with unknown concentrations of the mixture of natural gentamicin C congeners were measured to access the mean recovery (%) and CV recovery (standard deviation / mean * 100) of the established calibration curve. The results are also shown in Table B2.

[0567] [Table 3]

[0568] Figures 12-B1 to 12-B3 show example chromatograms of samples Cal1 and Cal6 for determining the content of gentamicin C1, gentamicin C2, and gentamicin C1a, respectively.

[0569] The results above illustrate the applicability of using isotopically labeled gentamicin C congeners in a substantially pure form, substantially free of each other congener, mixed with other isotopically labeled gentamicin C congeners of similar grade to determine the content of gentamicin C congeners, and thus the total gentamicin C content, in in vitro samples of unknown concentrations via MRM-based mass spectrometry connected to an LC system. Furthermore, the concentration range of the analytes shown is suitable for quantifying the amount of gentamicin C typically found in human serum and plasma. Therefore, the isotopically labeled gentamicin C congeners used can be used to develop diagnostic methods for analyzing known volumes of human samples according to their gentamicin C content or the content of different gentamicin C congeners.

[0570] TIFF2026511288000302.tif155164

Claims

1. Isotope-labeled gentamicin C or its salt or solvate, wherein at least one 13 C, D and / or 15 Contains N atoms; The isotope-labeled gentamincin C has the following structure 【Chemistry 1】 It has, During the ceremony, R 1 、 R 2 and R 3 are, independently of each other, -H, -D, -CH 3 , - 13 CH 3 , - 13 CDH 2 , - 13 CD 2 H, - 13 CD 3 , -CDH 2 , -CD 2 H and -CD 3 selected from the group consisting of R 1 or R 2 At least one of them is -H or -D, R 4 is -NH-, -ND-, - 15 ND- and - 15 Selected from the group consisting of NH-, R 5 is, -NH 2 , -ND 2 , - 15 ND 2、 - 15 NDH and - 15 NH 2 Selected from the group consisting of, R 6 , R 7 and R 8 These are, independently of each other, -CH-, -CD-, - 13 CD - or - 13 Selected from the group consisting of CH-, R 9 and R 10 These are, independently of each other, -CH 2 -, -CDH-, -CD 2 -, - 13 CDH-,- 13 CD 2 - and - 13 CH 2 - Selected from the group consisting of, R 11 is C or 13 C is Isotope-labeled gentamicin C or its salts or solvates.

2. The isotope-labeled gentamicin C according to claim 1, wherein the isotope-labeled gentamicin C contains less than 1% by weight of each other gentamicin C congener based on the total amount of the isotope-labeled gentamicin C.

3. Compound of formula (I*) 【Chemistry 2】 Preferably a compound of formula (I). 【Transformation 3】 And, During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are, independently of each other, -CH-, -CD-, - 13 CD - or - 13 Selected from the group consisting of CH-, R 011 is, -CH 2 -, -CD 2 -, -CHD-, - 13 CD 2 -, - 13 CH 2 -, - 13 CHD- and -R 11 (R 1 R 2 Selected from the group consisting of ) - R 1 and R 2 These are -H, -D, and -CH, which are independent of each other. 3 , - 13 CH 3 , - 13 CDH 2 , - 13 CD 2 H, - 13 CD 3 , -CD 2 H, -CDH 2 , and -CD 3 Selected from the group consisting of R 1 or R 2 At least one of them is -H or -D, R 11 is C or 13 It is C, During the ceremony, PG1, PG2, PG3, PG4, PG5 and PG6 are suitable protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6. especially, -PG1 is a protecting group selected from the group consisting of Cbz, benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. -PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. -PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. -PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). -PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the compound has structure (Ia) 【Chemistry 4】 It has, In the formula, PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6. especially, -PG1 is a protecting group selected from the group consisting of Cbz, benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. -PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. -PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. -PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). - A compound in which PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group.

4. Compound of formula (I*) 【Transformation 5】 Preferably a compound of formula (I). 【Transformation 6】 And, During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are, independently of each other, -CH-, -CD-, - 13 CD - or - 13 Selected from the group consisting of CH-, R 011 is selected from the group consisting of -CH 2 -, -CD 2 -, -CHD-, - 13 CD 2 -, - 13 CH 2 -, - 13 CHD-, and -R 11 (R 1 R 2 ) - and is selected from the group consisting of R 1 and R 2 are, independently of each other, -H, -D, -CH 3 (-), - 13 CH 3 (-), - 13 CDH 2 (-), - 13 CD 2 H, - 13 CD 3 (-), -CD 2 H, -CDH 2 (-), and -CD 3 and is selected from the group consisting of R 1 or R 2 at least one of which is -H or -D, and R 11 is C or 13 C In the formula, PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6. especially, -PG1 is a protecting group selected from the group consisting of Cbz, benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. -PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. -PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. -PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). -PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group. Preferably, the compound has structure (Ia) 【Transformation 7】 It has, In the formula, PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6. especially, -PG1 is a protecting group selected from the group consisting of Cbz, benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. -PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. -PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. -PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). -PG5 and PG6 together form a cyclic group, preferably a diacetal protecting group; The compound of formula (I*), the compound of formula (I), and the compound of formula (Ia) each have at least one 13 C, D and / or 15 A compound containing a nitrogen atom.

5. A method for preparing gentamicin C or its salts, solvates, or derivatives, wherein the gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1, and gentamicin C1a, and the gentamicin C is preferably isotope-labeled, and the method is (i) Glycosylated acceptor (A) of the following formula 【Transformation 8】 The glycosylated donor (B*) in the following formula 【Chemistry 9】 Preferably, formula (B) 【Chemistry 10】 Reacting with a glycosylated donor, the structure (I*) 【Chemistry 11】 A compound having formula (I), preferably formula (I) 【Chemistry 12】 This includes obtaining a compound having the following characteristics: During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are, independently of each other, -CH-, -CD-, - 13 CD - or - 13 Selected from the group consisting of CH-, R 011 is, -CH 2 -, -CD 2 -, -CHD-, - 13 CD 2 -, - 13 CH 2 -, - 13 CHD- and -R 11 (R 1 R 2 ) - Selected from the group consisting of R1 and R 2 These are -H, -D, and -CH, which are independent of each other. 3 , - 13 CH 3 , - 13 CDH 2 , - 13 CD 2 H, - 13 CD 3 , -CD 2 H, -CDH 2 , and -CD 3 Selected from the group consisting of R 1 or R 2 At least one of them is -H or -D, R 11 is C or 13 It is C, In the formula, PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6. especially, PG1 is a protecting group selected from the group consisting of Cbz, benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic protecting group, preferably a diacetal protecting group. The above method is preferably, (ii) Remove the protective group PG4 and obtain formula (I-1*) 【Chemistry 13】 A compound of formula (I-1), preferably of formula (I-1) 【Chemistry 14】 A method further comprising obtaining a compound of the above.

6. A method for preparing gentamicin C or its salts, solvates, or derivatives, wherein the gentamicin C is preferably selected from the group consisting of gentamicin C2, gentamicin C2a, gentamicin C2b, gentamicin C1, and gentamicin C1a, and the gentamicin C is isotope-labeled, and at least one 13 C, D and / or 15 The method includes N atoms, (ii) Glycosylated acceptor (A) in the following formula 【Chemistry 15】 The glycosylated donor (B*) in the following formula, 【Chemistry 16】 Preferably, formula (B) 【Chemistry 17】 Reacting with a glycosylated donor, the structure (I*) [Chemistry 18] A compound having formula (I), preferably formula (I) 【Chemistry 19】 This includes obtaining a compound having the following characteristics: During the ceremony, R 6 , R 7 , R 8 , R 91 and R 101 These are, independently of each other, -CH-, -CD-, - 13 CD - or - 13 Selected from the group consisting of CH-, R 011 is, -CH 2 -, -CD 2 -, -CHD-, - 13 CD 2 -, - 13 CH 2 -, - 13 CHD- and -R 11 (R 1 R 2 ) - Selected from the group consisting of R1 and R 2 These are -H, -D, and -CH, which are independent of each other. 3 , - 13 CH 3 , - 13 CDH 2 , - 13 CD 2 H, - 13 CD 3 , -CD 2 H, -CDH 2 , and -CD 3 Selected from the group consisting of R 1 or R 2 At least one of them is -H or -D, R 11 is C or 13 It is C, In the formula, PG1, PG2, PG3, PG4, PG5 and PG6 are appropriate protecting groups, and PG4 is orthogonal to PG1, PG2, PG3, PG5 and PG6. especially, PG1 is a protecting group selected from the group consisting of Cbz, benzoyl (Bz), acetyl, trifluoromethyl-benzoyl, trifluoroacetyl, and cyclic protecting groups that form a cyclic group together with PG2. PG2 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), as well as benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM) and 4-(3,4-dimethoxyphenyl)benzyl, and cyclic protecting groups that form a cyclic group together with PG1. PG3 is a protecting group selected from the group consisting of silyl protecting groups, preferably 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS), and benzyl protecting groups, preferably benzyl (Bn), para-methoxybenzyl (PMB), dimethoxybenzyl (3,4-DMPM, 3,5-DMPM, 2,5-DMPM, 2,6-DMPM, and 2,3-DMPM), and 4-(3,4-dimethoxyphenyl)benzyl. PG4 is a silyl protecting group, a pivaloyl group (Piv), or a benzoyl protecting group, preferably benzoyl (Bz), 2,4,6-trimethylbenzoyl, para-phenyl-benzoyl, para-bromobenzoyl, trifluoromethyl-benzoyl, or 2-(trimethylsilyl)ethoxymethyl (SEM), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS). PG5 and PG6 together form a cyclic protecting group, preferably a diacetal protecting group. The above method is preferably, (iii) Remove the protective group PG4 and obtain formula (I-1*) 【Chemistry 20】 A compound of formula (I-1), preferably of formula (I-1) 【Chemistry 21】 A method further comprising obtaining a compound of the above.

7. The method according to claim 5 or 6, wherein the glycosylated acceptor (A) has the following structure 【Chemistry 22】 It has, The glycosylated donor (B) has the following structure 【Chemistry 23】 It has, The compound of formula (I) has the following structure 【Chemistry 24】 A method having.

8. The method according to claim 5 or 6, wherein the glycosylated acceptor (A) has the following structure 【Chemistry 25】 It has, The glycosylated donor (B) has the following structure 【Chemistry 26】 It has, The compound of formula (I) has the following structure 【Chemistry 27】 A method having.

9. At least one isotope-labeled gentamicin C according to any one of claims 1 to 4, or at least one 13 C, D and / or 15 A diagnostic composition comprising at least one isotope-labeled gentamicin C containing an N atom, or a salt or solvate thereof, or gentamicin C obtained or obtainable by the method described in any one of claims 5 to 8, and a suitable excipient.

10. A calibration standard for determining the amount of at least one gentamicin C congener present in a sample, which is at least one isotope-labeled gentamicin C according to any one of claims 1 to 4, or at least one 13 C, D and / or 15 Use of gentamicin C, or a salt or solvate thereof, that is labeled with at least one isotope containing an N atom, or gentamicin C obtained or obtainable by the method described in any one of claims 5 to 8.

11. A method for determining the amount of at least one target analyte, preferably at least one gentamicin C congener, present in a sample, (a) The sample is given a known amount of at least one isotope-labeled gentamicin C according to any one of claims 1 to 4, or at least one 13 C, D and / or 15 Mixing with gentamicin C, or a salt or solvate thereof, which contains at least one isotope-labeled gentamicin C containing an N atom, or gentamicin C obtained or obtainable by the method described in any one of claims 5 to 8. (b) Analyze the sample by mass spectrometry. (c) Comparing the peak area of ​​at least one analyte of interest with a standard curve, wherein the standard curve is prepared using a standard containing the at least one isotope-labeled gentamicin C or its salt or solvate or derivative, and at least one analyte of interest. A method comprising determining the amount of at least the target analyte in the sample.

12. A computer-implemented method for evaluating a sample containing at least one gentamicin homolog, comprising the following steps: (aa) The sample is given a known amount of at least one isotope-labeled gentamicin C according to any one of claims 1 to 4, or at least one 13 C, D and / or 15 A step of mixing the sample with at least one isotope-labeled gentamicin C containing an N atom, or a salt or solvate thereof, or gentamicin C obtained or obtainable by the method described in any one of claims 5 to 8, or mixing the sample with a known amount of the diagnostic composition described in claim 9, and receiving the peak area value of the isotope-labeled gentamicin C in the sample. (bb) A step of receiving the peak area value of at least one gentamicin C congener present in the sample. (cc) A step of comparing the peak area values ​​of the at least one isotope-labeled gentamicin C with the at least one gentamicin C congener, and a step of receiving the value of the amount of the at least one gentamicin C congener; and A computer implementation method comprising the step of evaluating the sample with respect to the comparison and / or calculation performed in step (dd) (cc).

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