HK1170664A - Treatment of urinary tract infections with antibacterial aminoglycoside compounds - Google Patents

Description

Treatment of urinary tract infections using antibacterial aminoglycoside compounds

Reference to related applications

The present application claims priority from united states provisional patent application No. 61/178,469 filed 5/14/2009 and united states provisional patent application No. 61/305,460 filed 2/17/2010, in accordance with united states code volume 35, clause 119 (u.s.c. § 119 (e)). The foregoing application is incorporated by reference herein in its entirety.

Background

FIELD

The present invention relates to methods of treating urinary tract infections, in particular to methods of treating complicated urinary tract infections, using antibacterial aminoglycoside compounds.

Description of the Related Art

National Institutes of Health (NIH), the consultation Committee for renal and urinary Diseases (KUDAB) estimates nearly 1200 million Urinary Tract Infections (UTI) are reported annually in the United states (U.S.) (see: great TL. Urnary trade Infection in Women. editor Litwin MS, sample CS. urological Diseases in America. DHHS, PHS, NIH, NIDDK. Washington, DC: GPO; 2007.NIH publication 07-5512: 587. Data 619; and National center of health statistics: National center of health Discharge Suvel: 2004 Annun sum Diagnosis and Procedure 2006 Data (National Hospital Discharge Survey: Annual Diagnosis and Procedure Data 2004. disease control and prevention center).

Enterobacteria (Enterobacteriaceae) are currently the most prominent microorganisms associated with acute infections of the lower urinary tract. In a posterior analysis of six well-controlled trials (meta-analysis) associated with women with acute cystitis, the most common pathogens isolated from urine medium were E.coli (E.coli) (78.6%), followed by Staphylococcus saprophyticus (4.4%), Klebsiella pneumoniae (4.3%) and Proteus mirabilis (3.7%) (see Echols RM, Tosiello RL, Haverstock DC, tissue AD. Demographic, Clinical, and Treatment Parameters infection of the Outrather microorganisms. Clin Inf 1999; 29 (July): 113): 119). Over 75% of Nosocomial urinary tract Infections, mainly complex urinary tract Infections (cUTI) and pyelonephritis Caused by gram-Negative microorganisms including enterobacter and pseudomonas aeruginosa (see Gaynes R, Edwards JR. overview of Nosocomial Infections used by byGram-Negative Bacillus coli Dis 2005 (September); 41: 848-. The most commonly reported pathogen isolated from patients with UTI hospitalized in intensive care units is escherichia coli. In addition, a significant increase in the rate of Klebsiella pneumoniae (Staphylococcus saprophyticus) was reported for these patients between 1986 and 2004 (see Gaynes R, Edwards JR. national Hospital infection supervision System, Overview of nosocomial infections used by Gram-Negative Bacilli) Clin Inf Dis 2005 (September); 41: 848-854). Coli accounts for 80% or more of cases of non-complex pyelonephritis and is the most common microorganism isolated from Patients with complex pyelonephritis (see Talan DA, Krishnaadasan A, Abrahamiana FM, Stamm WE, Moran GJ.Presence and Risk factory Analysis of Trimethoprim-Sulfamethoxazole-and Fluoroquinone-Resistant Escherichia coli Infection expression deletion Department Patients with pyrosphere stuck, 2008; 47: 1150-.

Of particular interest are the recently emerging antibiotic-resistant strains of escherichia coli isolated from the urinary tract of emergency room patients diagnosed with pyelonephritis. The drug resistance of these isolates (isolates) to trimethoprim-Sulfamethoxazole is reported to be 20% or higher and to increase resistance to fluoroquinolones (see Talan DA, Krishnaadasan A, Abrahamian FM, Stamm WE, Moran GJ.Presence and Risk factory Analysis of Trimethoprim-Sulfamethoxazole-and fluoroquinolone-resistant Escherichia coli infection expression adjuvant pathogen depletion region, CID 2008; 47: 1150-. In addition, resistance to conventional treatment of acute urinary tract infections is often increased. In the united states in 2001, more than 286,000 clinical isolates collected from women's outpatient patients with UTI increased in total to 17% of sulfamethoxazole resistance between 1995 to 2001. Also in 2001, resistance to ciprofloxacin increased 3-fold, from 0.7% to 2.5% (see Karlowsky JA, Kelly LJ, Thornsberry C, Jones ME, Sahm DF. trends in an anti-microbial resistance array in the United states. anti-microbial Agents Chemother 2002, October 46 (8): 2540-. Even more alarming in this year is the reported proportion of multidrug resistant (MDR) e.coli of more than 12%. Multidrug resistance is defined as resistance to at least one drug from a different class (see Karlowsky JA, Kelly LJ, Thornsberry C, Jones ME, Sahm DF. trends in antimicrobial resistance and amino transfer in infectious diseases isolates of Escherichia coli from infectious diseases in the United states. antimicrobial Agents Chemothers.2002; 46 (8): 2540-. By 2005, more than 50% of fluoroquinolone-resistant E.coli isolates from female outpatients from 40 regions of the North America Federation of urinary Tract Infection showed resistance to at least two other classes of antibiotics (see Karlowsky JA, Hoban DJ, Decorby MR, Lang NM, Zhanel GG. Fluoroquinolone-resistant urea isolates of Escherichia coli from microbial soil organism, antibiotic resistant, nutrient from the North America urinary Tract Infection collagen colloidal alkaline-Quinolone resistance, antibiotic Agents Chemotherapy.2006; 50 (6): 2251-4). In a European supervised study, nearly 1% of the Spanish-derived urine isolates of E.coli are MDRs, which exhibit resistance to at least seven different antibiotics (see Kahlmeter G, Menday P. Cross-resistance and associated resistance in2478 Escherichia coli from the Pan-European ECO. SENS project evaluating the antimicrobial activity of the pathogens free of compounded urine extracts 2003 July, 52 (1): 128-31). The proportion of Trimethoprim-Sulfamethoxazole resistance in E.coli urinary isolates from the American emergency room is currently reported to be 20% or higher (see TalanDA, Krishnaadasan A, Abrahamian FM, Stamm WE, Moran GJ. Prevalienceand Risk Factor Analysis of Trimethoprim-Sulfamethoxazole-and Fluoroquinone-Resistant Escherichia coli Infection expression vector department Patients with pyrosphere intermediate 2008; 47: 1150-.

Thus, despite advances in the art, there remains a need for new antibacterial agents and methods for treating urinary tract infections, particularly complicated urinary tract infections. The present invention fulfills these needs and further provides related advantages.

Brief description of the drawings

Briefly, the present invention relates to methods of treating urinary tract infections, particularly complicated urinary tract infections, using antibacterial aminoglycoside compounds.

In one embodiment, a method is provided for treating a urinary tract infection in a mammal in need thereof, comprising administering to the mammal an effective amount of an antibacterial aminoglycoside compound.

In further embodiments, the antibacterial aminoglycoside compound is amikacin, gentamicin, tobramycin, lidomycin (netromycin), apramycin (apramycin), streptomycin, kanamycin, dibekacin, arbekacin, sisomicin, paromomycin, xanthomycin (kirromycin), thiostrepton, neomycin, netilmicin (netilmicin), or a modified derivative of any of the foregoing, or an antibacterial aminoglycoside compound having the following structure (I):

or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof,

wherein:

Q₁is hydrogen,

Q₂Is hydrogen, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -C (═ NH) NR₄R₅、-(CR₁₀R₁₁)_pR₁₂、

Q₃Is hydrogen, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -C (═ NH) NR₄R₅、-(CR₁₀R₁₁)_pR₁₂、

Each R is₁、R₂、R₃、R₄、R₅、R₈And R₁₀Independently is hydrogen or C₁-C₆A hydrocarbon group, or R₁And R₂Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R₂And R₃Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R₁And R₃Together with the atoms to which they are attached can form a carbocyclic ring having from 4 to 6 ring atoms, or R₄And R₅Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R is₆And R₇Independently hydrogen, hydroxy, amino or C₁-C₆A hydrocarbon group, or R₆And R₇Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R is₉Independently hydrogen or methyl;

Each R is₁₁Independently hydrogen, hydroxy, amino or C₁-C₆A hydrocarbyl group;

each R is₁₂Independently is hydroxy or amino;

each n is independently an integer from 0 to 4;

each m is independently an integer from 0 to 4; and

each p is independently an integer from 1 to 5, an

Wherein (i) Q₁、Q₂And Q₃Is not hydrogen, and (ii) if Q₁Is hydrogen, then Q₂And Q₃At least one of which is-C (═ NH) NR₄R₅。

These and other aspects of the invention will become apparent upon reference to the following detailed description.

Brief Description of Drawings

Figure 1 is a graph showing the dose response of example 1, gentamicin, ciprofloxacin and imipenem (positive control) to the AG-resistant e.coli clinical isolate (AECO 1003) in a mouse neutropenic strand model. Log of CFU/strand after 24 hours of antibiotic treatment versus CFU/strand just before antibiotic treatment (2 hours post infection)₁₀The difference indicates the activity. Total dose per 24 hours is shown; the dose was q12 hours. Each group had 6 mice. Inoculum 1.5X 10 ═ m³CFU。

Figure 2 is a graph showing the dose response of example 1, gentamicin and imipenem (positive control) to the AG-resistant clinical isolate of klebsiella pneumoniae (AKPN1073) in a mouse neutropenic strand model. Log of CFU/strand after 24 hours of antibiotic treatment versus CFU/strand just before antibiotic treatment (2 hours post infection) ₁₀The difference indicates the activity. Total dose per 24 hours is shown; the dose is q 12 hours. Each group had 6 mice. Inoculum 1.3 × 10⁴CFU。

FIG. 3 is a graph showing example 1, imipenem and Imipenem in a mouse neutropenic strand modelA graph of the dose response of ciprofloxacin to KPC expression of a clinical isolate expressing klebsiella pneumoniae (AKPN 1109). Log of CFU/strand after 24 hours of antibiotic treatment versus CFU/strand just before antibiotic treatment (2 hours post infection)₁₀The difference indicates the activity. Total dose per 24 hours is shown; q 12 hours was administered. Each group had 6 mice. Inoculum of 8.3 × 10⁵CFU。

FIG. 4 is a graph showing the dose response of example 1, arbekacin, gentamicin, vancomycin and daptomycin to MRSA (ATCC 33591) in a mouse neutropenic strand model. Log of CFU/strand after 24 hours of antibiotic treatment versus just before antibiotic treatment (2 hours post infection)₁₀The difference indicates the activity. Total dose per 24 hours is shown; the dose is q 12 hours. Each group had 6 mice. Inoculum 1.2 × 10³CFU。

Detailed Description

In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these details.

Throughout this specification and claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to have an open, inclusive meaning, such as "including but not limited to".

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present application. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used in the specification and the appended claims, the following terms have the meanings given below, unless the contrary is specified.

"amino" means-NH₂A group.

"cyano" refers to the group-CN.

"hydroxy" or "hydroxyl" refers to an-OH group.

"imino" means an ═ NH substituent.

"nitro" means-NO ₂A group.

"oxo" refers to an ═ O substituent.

"thio" means ═ S substituent.

"hydrocarbyl" refers to a saturated or unsaturated, straight or branched hydrocarbon chain radical (i.e., containing one or more double and/or triple bonds) consisting solely of carbon and hydrogen atoms, having from one to twelve carbon atoms (C)₁-C₁₂Hydrocarbyl group), preferably one to eight carbon atoms (C)₁-C₈Hydrocarbyl) or one to six carbon atoms (C)₁-C₆Hydrocarbyl) and is linked to the remainder of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1, 4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless the specification otherwise specifically states, the hydrocarbon group may be optionally substituted.

"alkylene" or "alkylene chain" refers to a saturated or unsaturated (i.e., containing one or more double and/or triple bonds) straight or branched divalent hydrocarbon chain consisting only of carbon and hydrogen, with the remainder of the molecule attached to the group, and having from one to twelve carbon atoms, e.g., methylene, ethenyl, propenyl, n-butenyl, ethenylene, propenyl, n-butenylene, propynyl, n-butynyl, and the like. The alkylene chain is connected to the rest of the molecule by a single or double bond and to the group by a single or double bond. The point of attachment of the alkylene chain to the rest of the molecule and to the group can be through one or any two carbons in the chain. Unless the specification otherwise specifically states, the alkylene chain may be optionally substituted.

"hydrocarbyloxy" means a compound of the formula-OR_aWherein R is_aIs a hydrocarbyl group as defined above containing from one to twelve carbon atoms. Unless the specification otherwise specifically states, the hydrocarbyloxy group may be optionally substituted.

"hydrocarbylamino" refers to the formula-NHR_aor-NR_aR_aWherein each R is_aIndependently a hydrocarbyl group as defined above containing from one to twelve carbon atoms. Unless the specification otherwise specifically states, an alkylamino group may be optionally substituted.

"Thioalkyl" means a compound of the formula-SR_aWherein R is_aIs a hydrocarbyl group as defined above containing from one to twelve carbon atoms. Unless the specification otherwise specifically states, the thioalkyl group may be optionally substituted.

"aryl" means a hydrocarbon ring system comprising hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For the purposes of the present invention, aryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may comprise fused or bridged ring systems. Aryl groups include, but are not limited to, those derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, phenanthrylene, anthracene,Aryl groups of fluoranthene, fluorene, asymmetric indacene, symmetric indacene, indane, indene, naphthalene, phenalene, phenanthrene, obsidian, pyrene and triphenylene. Unless the specification otherwise specifically states, the term "aryl" or the prefix "aryl" (for example in "aryl" is intended to include optionally substituted aryl.

"aryl" refers to the formula-R_b-R_cWherein R is_bIs that the above-mentionedA defined alkylene chain and R_cIs one or more aryl groups as defined above, such as benzyl, benzhydryl, and the like. Unless the specification otherwise specifically states, the aromatic hydrocarbon group may be optionally substituted.

"cycloalkyl" or "carbocyclic" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, which may contain fused or bridged ring systems, and having three to fifteen carbon atoms, preferably three to ten carbon atoms, and which is saturated or unsaturated and is attached to the remainder of the molecule by a single bond. Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7-dimethyl-bicyclo [2.2.1] heptyl, and the like. Unless the specification otherwise specifically states, the cycloalkyl group may be optionally substituted.

"cycloalkyl-hydrocarbyl" means a compound of the formula-R_bR_dWherein R is_dIs a hydrocarbylene chain as defined above and R_gIs a cyclic hydrocarbon group as defined above. Unless the specification otherwise specifically states, the cycloalkyl hydrocarbon group may be optionally substituted.

By "fused" is meant any ring structure described herein that is fused to an existing ring structure in a compound disclosed herein. When the fused ring is a heterocyclic or heteroaryl ring, any carbon atom on the existing ring structure that is part of the fused heterocyclic or fused heteroaryl ring may be substituted with a nitrogen atom.

"halo" or "halogen" refers to bromo, chloro, fluoro, or iodo.

"haloalkyl" refers to a hydrocarbyl group as defined above substituted with one or more halogens as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2, 2, 2-trifluoroethyl, 1, 2-difluoroethyl, 3-bromo-2-fluoropropyl, 1, 2-dibromoethyl, and the like. Unless the specification otherwise specifically states, the halogenated hydrocarbon group may be optionally substituted.

"Heterocyclyl" or "heterocycle" refers to a stable 3-to 18-membered non-aromatic ring group consisting of two to twelve carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur. Unless the specification otherwise specifically states, the heterocyclic group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclic group may be optionally oxidized; the nitrogen atoms may be optionally quaternized; and the heterocyclic group may be partially or fully saturated. Examples of such heterocyclic groups include, but are not limited to, dioxolanyl (dioxolanyl), thienyl [1, 3] dithianyl (thienyl [1, 3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl (trithianyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxo-thiomorpholinyl, and 1, 1-dioxo-thiomorpholinyl. Unless the specification specifically states otherwise, the heterocyclic group may be optionally substituted.

"N-heterocyclyl" means a heterocyclic group as defined above that contains at least one nitrogen, and wherein the point of attachment of the heterocyclic group to the remainder of the molecule is through a nitrogen atom in the heterocyclic group. Unless the specification otherwise specifically states, the N-heterocyclyl group may be optionally substituted.

"Heterocyclylalkyl" means a compound of the formula-R_bR_eWherein R is_bIs a hydrocarbylene chain as defined above and R_eIs a heterocyclic group as defined above, and if the heterocyclic ring is a nitrogen-containing heterocyclic group, the heterocyclic ring may be attached to the hydrocarbon group at the nitrogen atom position. Unless the specification otherwise specifically states, the heterocyclylalkyl group may be optionally substituted.

"heteroaryl" refers to a 5-to 14-membered ring system group containing hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, heteroaryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which may contain fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group may be optionally oxidized; the nitrogen atoms may be optionally quaternized. Examples include, but are not limited to, azanyl (azepinyl), acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl (benzothiazodiazolyl), benzo [ b ] [1, 4] dioxepinyl (benzo [ b ] [1, 4] dioxepinyl), 1, 4-benzodioxoyl (1, 4-benzodioxolyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxyhexenyl, benzopyranyl, benzopyranonyl, benzofuranonyl (benzothiophenyl), benzotriazolyl, benzo [4, 6] imidazo [1, 2-a ] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, furanyl, benzofuranyl, benzothiophenyl (benzothiophenyl), benzonaphthofuranyl, benzonaphtho [1, 6] imidazo [1, 2-a ] pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, furanyl, benzofuranyl, cinnolinyl, benzothiophenyl, benzothi, Furanone group, isothiazole group, imidazole group, indazole group, indolyl group, indazole group, isoindolyl group, indolinyl group, isoindolinyl group, isoquinolyl group, indolizinyl group, isoxazolyl group, 1, 5-naphthyridine group, oxadiazolyl group, 2-oxoazepinyl group, oxazolyl group, epoxyethyl group, 1-oxidopyridinyl group (1-oxidopyridinyl group), 1-oxidopyrimidinyl group, 1-oxidopyridazinyl group, 1-phenyl-1H-pyrrolyl group, phenazine group, phenothiazinyl group, phenoxazinyl group, phthalazine group, pteridinyl group, purinyl group, pyrrolyl group, pyrazolyl group, pyridyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, quinazolinyl group, quinoxalyl group, quinolyl group, quinuclidinyl group, isoquinolyl group, tetrahydroquinolyl group, thiazolyl group, thiadiazolyl group, triazolyl group, tetrazolyl group, Triazinyl and thienyl (i.e., thienyl). Unless the specification otherwise specifically states, heteroaryl groups may be optionally substituted.

"N-heteroaryl" refers to a heteroaryl group as defined above containing at least one nitrogen, wherein the point of attachment of the heteroaryl group to the remainder of the molecule is through the nitrogen atom in the heteroaryl group. Unless the specification otherwise specifically states, the N-heteroaryl group may be optionally substituted.

"Heteroarylalkyl" means a compound of the formula-R_bR_fWherein R is_bIs a hydrocarbylene chain as defined above and R_fIs heteroaryl as defined above. Unless the specification otherwise specifically states, the heteroarylalkyl group may be optionally substituted.

The term "substituted" as used herein refers to any of the above groups (i.e., hydrocarbyl, hydrocarbylene, hydrocarbyloxy, hydrocarbylamino, thioalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, halohydrocarbyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and/or heteroarylalkyl) in which at least one hydrogen atom is replaced by a bond other than a hydrogen atom, such as, but not limited to, a halogen atom such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, hydrocarbyloxy groups, and ester groups; sulfur atoms in groups such as thiol groups, hydrocarbon thio groups, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, hydrocarbyl amines, dialkyl amines, aryl amines, hydrocarbyl aryl amines, diaryl amines, N-oxides, imines, and enamines; silicon atoms in groups such as trihydrocarbylsilyl, dihydrocarbylarylsilyl, hydrocarbyl diarylsilyl, and triarylsilyl groups; and other heteroatoms in various other groups. "substituted" also refers to any of the above groups in which one or more hydrogen atoms are replaced by a higher order bond (e.g., double or triple bond) of a heteroatom, such as oxygen in the group of oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes where one or more hydrogen atoms is replaced by-NR _gR_h、-NR_gC(＝O)R_h、-NR_gC(＝O)NR_gR_h、-NR_gC(＝O)OR_h、-NR_gSO₂R_h、-OC(＝O)NR_gR_h、-OR_g、-SR_g、-SOR_g、-SO₂R_g、-OSO₂R_g、-SO₂OR_g、＝NSO₂R_gand-SO₂NR_gR_hSubstituted of the above groupsEither of them. "substituted" also refers to those in which one or more hydrogen atoms is replaced by — C (═ O) R_g、-C(＝O)OR_g、-C(＝O)NR_gR_h、-CH₂SO₂R_g、-CH₂SO₂NR_gR_hAny of the above groups substituted. In the foregoing, R_gAnd R_hAre the same or different and are independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "substituted" also refers to any of the foregoing groups in which one or more hydrogen atoms are replaced by a bond to amino, cyano, hydroxy, imino, nitro, oxo, thio, halogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and/or heteroarylalkyl. Further, each of the foregoing substituents may be optionally substituted with one or more of the above substituents.

By "prodrug" is meant a compound that is convertible to a biologically active compound under physiological conditions or by solvolysis. Thus, the term "prodrug" refers to a metabolic precursor of a pharmaceutically acceptable compound. Prodrugs can be inert when administered to a subject in need thereof, but convert to the active compound in vivo. Typically, the prodrug is rapidly converted in vivo to yield the parent compound, for example, by hydrolysis in blood. Prodrug compounds often offer the advantage of solubility, histocompatibility or sustained release in the mammalian body (see Design of produgs (1985), p.7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, t.et al, a.c.s.symposium Series (american society of chemical society, attorney), volume 14 and Bioreversible Carriers in Drug Design, editors Edward b.roche, american pharmaceutical Association and Pergamon Press (american society of pharmaceuticals and pegman Press, 1987).

The term "prodrug" is also intended to include any covalently bonded carriers that release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of compounds can be prepared in such a way that functional groups present in the compounds are modified by either routine manipulation or by cleaving the modification into the parent compound in vivo. Prodrugs include compounds wherein a hydroxy, amino, or mercapto group is bonded to any group that cleaves to form a free hydroxy, free amino, or free mercapto group, respectively, when a prodrug of such compounds is administered to a mammalian subject. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohols or amide derivatives of amine functional groups among these compounds, and the like.

The invention disclosed herein is also intended to include the use of all the pharmaceutically acceptable compounds disclosed herein which are isotopically labeled by one or more atoms replaced by atoms having a different atomic mass or mass number. Examples of isotopes that can be mixed with the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, respectively, for example ²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、³⁶Cl、¹²³I and¹²⁵I. these radiolabeled compounds can be used to help determine or detect the effectiveness of a compound by, for example, characterization of the location or pattern of action, or binding affinity to pharmacologically important locations of action. Certain isotopically-labeled compounds, such as those mixed with radioisotopes, are useful in drug and/or matrix tissue distribution studies. Radioisotope tritium, i.e.³H and carbon-14 i.e¹⁴C is particularly suitable for this purpose due to its ease of mixing and the well-established detection means.

Using compounds such as deuterium²Heavier isotope substitutions of H may provide specific therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosageAre desirable and therefore preferred in some circumstances.

Using means such as¹¹C、¹⁸F、¹⁵O and¹³n-emitting positron isotope substitution transducers are used in Positron Emission Tomography (PET) studies for detecting substrate receptor occupancy. Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the preparations and examples set forth below, using a suitable isotopically labeled reagent in place of the non-labeled reagent used previously.

The invention disclosed herein is also intended to include the use of in vivo metabolites of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, primarily from enzymatic processes. Accordingly, the present invention includes compounds prepared by a method comprising administering a compound disclosed herein to a mammal for a time sufficient to produce a metabolite thereof, and the present invention includes such compounds. Typically, such products are identified by administering a detectable dose of a radiolabeled compound to an animal or human, such as rat, mouse, guinea pig, monkey, for a time sufficient for metabolism to occur, and isolating the conversion products from urine, blood or other biological samples.

By "stable compound" and "stable structure" is meant that the compound is sufficiently stable to be present in effective purity after isolation from the reaction mixture and formulation as an effective therapeutic agent.

"mammal" includes humans and two domestic animals such as laboratory animals and domestic pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), as well as non-domestic animals such as wild animals, and the like.

"any" or "optionally" means that the subsequently described event may or may not occur, and that the description includes instances where said event or circumstance occurs or instances where it does not. For example, "optionally substituted aryl" means that the aryl group may or may not be substituted, and that the description includes both substituted aryl groups and aryl groups without substituents.

A "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, colorant/colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the U.S. food and drug administration for acceptable use in humans or livestock.

"pharmaceutically acceptable salts" include both acid addition salts and base addition salts.

"pharmaceutically acceptable acid addition salts" refers to those salts which retain biological effectiveness and the properties of the free base, which are not biologically or otherwise undesirable, and which are formed from inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, as well as inorganic acids such as, but not limited to, acetic, 2-dichloroacetic, fatty, alginic, ascorbic, aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, camphoric, camphor-10-sulfonic, decanoic, hexanoic, octanoic, carbonic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1, 2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, mucic, gentisic, heptoic, gluconic, glucuronic, glutamic, glutaric, 2-oxo-glutaric, 2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, mucic, gentisic, heptoic, gluconic, glucuronic, glutamic, glutaric, 2-oxo-glutaric, Glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

"pharmaceutically acceptable base addition salts" refers to those salts that retain biological effectiveness and the properties of the free acid, which are not biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and substituted amines of basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzylamine (benethamine), benzathine (benzathine), ethylenediamine, grape-vine, meglumine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyurethane, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

Crystallization often produces solvates of the compounds. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of a compound and one or more molecules of a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds may exist in the form of hydrates, including monohydrates, dihydrate, hemihydrate, sesquihydrates, trihydrate, tetrahydrate, and the like, as well as the corresponding solvate forms. The compound may be a true solvate, while in other cases the compound may retain only adventitious water or may be a mixture of water with some adventitious solvent.

"pharmaceutical composition" refers to a formulation of a compound and a vehicle generally acceptable in the art for delivering a biologically active compound to a mammal, such as a human. Such media therefore include all pharmaceutically acceptable carriers, diluents or excipients.

An "effective amount" or "therapeutically effective amount" refers to an amount of a compound, as defined below, that is sufficient to effect treatment of a urinary tract infection in a mammal, preferably a human, when administered to the mammal, preferably a human. The amount of the compound that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and this disclosure.

As used herein, "treatment" or "treatment" includes treatment of a related disease or disease state in a mammal, preferably a human, having the related disease or disease state, and includes:

(i) preventing the occurrence of a disease or disease state in a mammal, particularly when such mammal is susceptible to, but has not yet been diagnosed with, said disease state;

(ii) inhibiting, i.e., arresting the development of, the disease or disease state;

(iii) alleviating, i.e., causing regression of, the disease or disease state; or

(iv) Alleviating the symptoms resulting from the disease or condition, i.e., alleviating pain, without addressing the underlying disease or condition. As used herein, the terms "disease" and "disease state" may be used interchangeably or may be different in that a particular disease or disease state may not have a known pathogen (and therefore the cause has not been determined), and therefore has not been considered a disease but only an undesirable disease state or syndrome, where a clinician has identified a series of more or less specific syndromes.

"urinary tract infection" refers to a bacterial infection of the lower or upper urinary tract. Acute infections of the urinary tract can be classified as uncomplicated or complex. Lower urinary tract infections, including cystitis and urethritis, generally belong to a non-complex group. However, a lower urinary tract infection is considered to be a complication if the patient develops an infection of any one of the following: 1) indwelling catheters, 2) volume remaining after voiding, 3) neurological bladder dysfunction, 4) signs of obstructive urinary tract disease, 5) azotemia resulting from renal disease, or 6) male urinary retention resulting from benign prostatic hypertrophy. Upper urinary tract infections are characterized by signs and symptoms of an ascending infection and generally fall into a category of complexity. Acute Pyelonephritis requiring hospitalization generally belongs to the complexity classification because the disease state is similar to the Treatment and management of complex Urinary Tract Infections, often requiring IV antibiotic Treatment (see Stamm we. united tray Infections, Harrison's Principles of Internal Medicine, 15 th edition. copyright 2001, editor Braunwald, Fauci, Kasper, Hauser, Longo, Jameson, chapter 280: 1620. dona 1625; Food and Drug Administration, Center for Drug Evaluation and Research (CDER), complex urine Tract infection and drinking of piezoelectric devices-development-of Antimicrobial Drugs for Treatment, draw guidfue (american society of health and human services, industrial guidelines, complex Treatment of infection and development of Drugs for treating Pyelonephritis, western Medicine of renal infection, 1998; western Medicine of western Medicine, western Medicine of health and Medicine of health, Research, western Medicine of health and human services, japanese society of diseases, western Medicine of diseases, Research and Research of diseases), october, Clin Infect Dis.1999; 29(4): 745-758). Thus, "complicated urinary tract infection" as used herein refers to a bacterial infection of the lower or upper urinary tract in the presence of anatomical abnormalities, or abnormalities in catheter function.

The antibacterial aminoglycoside compounds disclosed herein or pharmaceutically acceptable salts thereof may contain one or more asymmetric centers and may thus give rise to enantiomeric, diastereomeric and other stereoisomeric forms, which may be defined as (R) -or (S) -or (D) -or (L) -for amino acids, according to absolute stereochemistry. The present application is intended to include the use of all such possible isomers, as well as racemates and optically pure forms thereof. Optically active (+) and (-), (R) -and (S) -or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents or using conventional resolution techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation/separation of the individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemates (or racemates of salts or derivatives) using, for example, chiral High Pressure Liquid Chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, it is intended that the compounds contain both E and Z geometric isomers, unless otherwise specified. Likewise, all tautomeric forms are also intended to be included.

"stereoisomers" refers to compounds consisting of the same atoms joined by the same bond but having different three-dimensional structures that are not interchangeable. The present application encompasses various stereoisomers and mixtures thereof and includes "enantiomers," which refers to two stereoisomers whose molecules are not superimposable as mirror images of each other.

"tautomer" refers to the migration of a proton from one atom of a molecule to another atom of the same molecule. The present application includes tautomers of any of the compounds.

As noted above, in one embodiment, a method is provided for treating a urinary tract infection in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an antibacterial aminoglycoside compound.

In further embodiments, the urinary tract infection is a complex urinary tract infection.

In another embodiment, the antibacterial aminoglycoside compound is amikacin, gentamicin, tobramycin, lidoxine, apramycin, streptomycin, kanamycin, dibekacin, arbekacin, sisomicin, paromomycin, xanthomycin, thiostreptocin, neomycin, netilmicin, or a modified derivative of any of the foregoing.

In another embodiment, the antibacterial aminoglycoside compound has the following structure (I):

or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof,

wherein:

Q₁is hydrogen,

Q₂Is hydrogen, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -C (═ NH) NR ₄R₅、-(CR₁₀R₁₁)_pR₁₂、

Q₃Is hydrogen, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -C (═ NH) NR₄R₅、-(CR₁₀R₁₁)_pR₁₂、

Each R is₁、R₂、R₃、R₄、R₅、R₈And R₁₀Independently is hydrogen or C₁-C₆A hydrocarbon group, or R₁And R₂Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R₂And R₃Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R₁And R₃Together with the atoms to which they are attached can form a carbocyclic ring having from 4 to 6 ring atoms, or R₄And R₅Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R is₆And R₇Independently hydrogen, hydroxy, amino or C₁-C₆A hydrocarbon group, or R₆And R₇Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R is₉Independently hydrogen or methyl;

each R is₁₁Independently hydrogen, hydroxy, amino or C₁-C₆A hydrocarbyl group;

each R is₁₂Independently is hydroxy or amino;

each n is independently an integer from 0 to 4;

each m is independently an integer from 0 to 4; and

each p is independently an integer from 1 to 5, an

Wherein (i) Q ₁、Q₂And Q₃Is not hydrogen, and (ii) if Q₁Is hydrogen, then Q₂And Q₃At least one of which is-C (═ NH) NR₄R₅。

The compounds of structure (I) are novel antibacterial aminoglycoside compounds disclosed in co-pending international PCT patent application No. US2008/084399 entitled "antibacterial aminoglycoside analogs" filed on 21.11.2008 (which application claims priority to U.S. provisional patent application No. 60/989,645 filed on 21.11.2007) (which aforementioned application is incorporated herein by reference in its entirety). Thus, in further embodiments of the present application, other embodiments of the following structure (I) disclosed in the aforementioned co-pending applications may be used.

More specifically, in additional embodiments of the compounds of structure (I), R₈Is hydrogen.

In other embodiments, each R is₉Is methyl.

In further embodiments, Q₁And Q₂Is not hydrogen. In certain of the foregoing embodiments, Q₃Is hydrogen.

In more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₁Is hydrogen; r₂Is hydrogen; and each R₃Is hydrogen. For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₁Is hydrogen; and R₂And R ₃Together with the atoms to which they are attached form a compound having 4 toA heterocycle of 6 ring atoms.

For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₃Is hydrogen; and R₁And R₂Together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms.

For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₂Is hydrogen; and R₁And R₃Together with the atoms to which they are attached form a carbocyclic ring having from 4 to 6 ring atoms.

For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₂Is hydrogen; and each R₃Is hydrogen.

In other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₂Is hydrogen; and each R₃Is hydrogen.

In other more particular embodiments of the foregoing, Q₂Is- (CR)₁₀R₁₁)_pR₁₂. In certain embodiments, each R is₁₀Is hydrogen. In certain embodiments, each R is₁₁Is hydrogen.

In other more particular embodiments of the foregoing, Q₂Is an optionally substituted cycloalkyl group. In certain embodiments, Q₂Is unsubstituted. In certain embodiments, Q₂Substituted by hydroxy or amino groups

In other more particular embodiments of the foregoing, Q₂Is an optionally substituted heterocyclylalkyl group. In certain embodiments, Q ₂Is unsubstituted. In certain embodiments, Q₂Substituted by hydroxy or amino.

In other embodiments, Q₁And Q₃Is not hydrogen. In certain embodiments, Q₂Is hydrogen.

In more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₁Is hydrogen; r₂Is hydrogen; and each R₃Is hydrogen. For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₁Is hydrogen; and R₂And R₃Together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms.

For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₃Is hydrogen; and R₁And R₂Together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms.

For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₂Is hydrogen; and R₁And R₃Together with the atoms to which they are attached form a carbocyclic ring having from 4 to 6 ring atoms.

For example, Q₁Can be as follows:

in other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₂Is hydrogen; and each R₃Is hydrogen.

In other more particular embodiments of the foregoing, Q₁Comprises the following steps:

wherein: r₂Is hydrogen; and each R₃Is hydrogen.

In other more particular embodiments of the foregoing, Q ₃Is- (CR)₁₀R₁₁)_pR₁₂. In certain embodiments, each R is₁₀Is hydrogen. In certain embodiments, each R is₁₁Is hydrogen.

In other more particular embodiments of the foregoing, Q₃Is an optionally substituted cycloalkyl group. In certain embodiments, Q₃Is unsubstituted. In certain embodiments, Q₃Substituted by hydroxy or amino groups

In other more particular embodiments of the foregoing, Q₃Is an optionally substituted heterocyclylalkyl group. In certain embodiments, Q₃Is unsubstituted. In certain embodiments, Q₃Substituted by hydroxy or amino.

In other more particular embodiments of the foregoing, Q₃Is an optionally substituted heterocyclic group. In certain embodiments, Q₃Is unsubstituted. In certain embodiments, Q₃Substituted by hydroxy or amino.

In other more particular embodiments of the foregoing, Q₃is-C (═ NH) NH₂。

In other embodiments, Q₂And Q₃Is not hydrogen. In certain embodiments, Q₁Is hydrogen.

In more particular embodiments of the foregoing, Q₂is-C (═ NH) NH₂。

In other more particular embodiments of the foregoing, Q₃is-C (═ NH) NH₂。

It is to be understood that Q is in any embodiment of and in the compound of structure (I) as set forth above ₁、Q₂、Q₃、R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁Or R₁₂Any particular substituent set forth herein may be independently combined with substituents of other embodiments and/or compounds of structure (I) to form embodiments not specifically set forth above. Furthermore, where a series of substituents is recited for any particular substituent in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the particular embodiment and/or claim and the remaining substituents are deemed to be included within the scope of the present invention.

For administration purposes, the antibacterial aminoglycoside compounds disclosed herein can be administered as crude chemicals or can be formulated as pharmaceutical compositions. Such pharmaceutical compositions comprise an antibacterial aminoglycoside compound disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. The antibacterial aminoglycoside compound is present in the composition in an amount effective to treat the particular disease or condition of interest, i.e., an amount sufficient to treat urinary tract infections and preferably having acceptable toxicity to the patient. One skilled in the art can determine the antibacterial activity of the antibacterial aminoglycoside compounds disclosed herein, for example, as described in the examples below. Suitable concentrations and dosages can be readily determined by those skilled in the art.

The antibacterial aminoglycoside compounds disclosed herein have spectral antibacterial activity against gram-positive and gram-negative bacteria as well as enteric and anaerobic bacteria. Representative susceptible organisms generally include those gram positive and gram negative, aerobic and anaerobic organisms capable of being inhibited from growth by the antibacterial aminoglycoside compounds disclosed herein, such as staphylococcus, lactobacillus, streptococcus, sarcina, escherichia, enterobacter, klebsiella, pseudomonas, acinetobacter, mycobacterium, proteus, campylobacter, citrobacter, neisseria, bacillus, bacteroides, peptococcus, clostridium, salmonella, shigella, serratia, haemophilus, bacillus and other organisms. For example, representative bacterial infections that may be treated according to the methods of the present invention include, but are not limited to, Bacillus anthracis (Bacillus autotrans), Enterococcus faecalis (Enterococcus faecalis), Corynebacterium (Corynebacterium), Diptheria ((Diptheriae), Escherichia coli ((Escherichia coli), Streptomyces coelicolor ((Streptococcus coelicolor), Streptococcus pyogenes ((Streptococcus pyenes), Streptococcus candidus ((Streptococcus monophilus), Streptococcus agalactiae ((Streptococcus agalactiae), Streptococcus pneumoniae ((Streptococcus pneumoniae), Salmonella typhi), Salmonella paratyphi ((Salmonella paratyphi), Salmonella ((Salmonella pneumoniae), Salmonella pneumoniae ((Streptococcus), Staphylococcus aureus ((Streptococcus pneumoniae), Staphylococcus aureus ((Streptococcus (Staphylococcus aureus), Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Staphylococcus aureus), Escherichia coli (Escherichia coli), Escherichia coli (Staphylococcus aureus), Escherichia coli (Escherichia coli), Staphylococcus aureus), Escherichia coli (Escherichia coli), Escherichia coli (Staphylococcus aureus), Staphylococcus (Escherichia coli), Escherichia coli (Escherichia coli), Staphylococcus, Mycoplasma pneumoniae ((Mycoplasma pneumonii), Mycobacterium tuberculosis ((Yersinianthercica), Yersinia pestis ((Yersinia pestis), cholera ((Vibrio cholera), Vibrio parahaemolyticus ((Vibrio parahaemolyticus), Rickettsia prototheca ((Rickettsia wazekii), Rickettsia rickettsii ((Rickettsia Rickettsia rickettsii), Rickettsia minor (Rickettsia aktatarii), Bacillus dentiger ((Clostridiphilium), Clostridium tetani ((Clostridianum), Clostridium perfringens ((Clostridia), Clostridium parahaemophilus ((Clostridium parahaemophilus), Clostridium parahaemophilus (Clostridium parahaemophilus), Clostridium (Clostridium parahaemophilus), Clostridium difficicus, Clostridium (Clostridium parahaemophilus), Clostridium difficile (Clostridium difficile), Clostridium difficile, bordetella pertussis ((Bordetella pertussis), Shigella flexneri ((Shigella spp.), Campylobacter jejuni ((Campylobacter jejuni), Proteus ((Proteus spp.), Citrobacter freundii ((Citrobacter spp.), Enterobacter ((Enterobacter spp.), Pseudomonas aeruginosa), Propionibacterium (Propionibacterium spp.), Bacillus ((Bacillus santhracus), Pseudomonas syringae (Pseudomonas syringae), Spirospira ((Spirlulinus), Neisseria meningitidis ((Neisseria meningitidis), Listeria monocytogenes ((Listeria monocytogenes), Neisseria gonorrhoeae ((Neisseria norrha), Borrelia (Borrelia neralis), Borrelia ((Borrelia burgeriella), Borrelia ((Borrelia burgdorferi), Borrelia ((Listeria monocytogenes), Borrelia burghia), Borrelia burgdorferi), Borrelia ((Listeria monocytogenes), Borrelia monocytogenes ((Listeria monocytogenes), Borrelia (Borrelia), Borrelia ((Listeria monocytogenes), Borrelia (Borrelia), Borrelia ((Neisseria monocytogenes), Bor, Infection with Mycobacterium avium (Mycobacterium avium), Mycobacterium smegmatis (Mycobacterium smegmatis), Methicillin-resistant staphylococcus aureus (Methicillin-resistant staphylococcus aureus), Vancomycin-resistant enterococci (Vancomycin-resistant enterococcus), and multi-drug resistant bacteria (e.g., bacteria resistant to more than 1, more than 2, more than 3, or more than 4 different drugs).

The antibacterial aminoglycoside compounds disclosed herein or a pharmaceutically acceptable salt thereof can be administered in a single form or in a suitable pharmaceutical composition by any acceptable mode of administration for agents which serve a similar purpose. The pharmaceutical compositions of the present invention can be prepared by combining the antibacterial aminoglycoside compound disclosed herein with suitable pharmaceutically acceptable carriers, diluents or excipients and can be formulated into solid, semisolid, liquid or gaseous forms of formulations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration for such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a patient. The composition to be administered to an individual or patient takes the form of one or more dosage units, where, for example, a tablet may be one dosage unit and a container of the compound in aerosol form may hold a plurality of dosage units. The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: the science and Practice of Pharmacy, 20 th edition (Philadelphia institute of Pharmacy, 2000). In any event, the compositions to be administered comprise a therapeutically effective amount of an antibacterial aminoglycoside compound disclosed herein, or a pharmaceutically acceptable salt thereof, for the treatment of urinary tract infections, in accordance with the teachings of the present invention.

The pharmaceutical compositions of the present invention may be in solid or liquid form. In one aspect, the carrier is granular such that the composition is in the form of, for example, a tablet or a powder. The carrier can be a liquid, while the composition is, for example, an oral syrup, an injectable liquid, or an aerosol, which is useful, for example, in administration by inhalation.

When intended for oral administration, the pharmaceutical composition is preferably in a solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included within the scope of solid or liquid forms contemplated herein.

As a solid composition for oral administration, the pharmaceutical composition may be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, implants and the like. Such solid compositions typically comprise one or more inert diluents or edible carriers. Furthermore, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth or gelatin; excipients such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring and coloring agents such as peppermint, methyl salicylate, or orange flavoring.

When the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or an oil.

The pharmaceutical compositions may be in the form of liquids, for example, elixirs, syrups, solutions, emulsions or suspensions. As two examples, the liquid may be for oral administration or for delivery by injection. When intended for oral administration, preferred compositions comprise, in addition to the antibacterial aminoglycoside compound, one or more of a sweetener, a preservative, a coloring agent/agent and a taste enhancer. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizing agents, and isotonic agents may be included.

The liquid pharmaceutical compositions of the present invention, whether they be solutions, suspensions or other similar forms, may contain one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, ringer's solution, isotonic sodium chloride; non-volatile oils such as synthetic mono-or diglycerides which may serve as a solvent or suspending medium, polyethylene glycol, glycerol, propylene glycol or other solvent; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting the osmotic pressure such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

Liquid pharmaceutical compositions of the present invention intended for parenteral or oral administration should contain an amount of the antibacterial aminoglycoside compound disclosed herein in order to obtain a suitable dosage.

The pharmaceutical compositions of the present invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The matrix may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be included in pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoretic device.

The pharmaceutical compositions of the present invention may be intended for rectal administration in the form of suppositories, which dissolve in the rectum and release the drug. Compositions for rectal administration may comprise an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycols.

The pharmaceutical compositions of the present invention may comprise a variety of materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may comprise a material that forms an envelope around the active ingredient. The material forming the coating is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be packaged in gelatin capsules.

The pharmaceutical compositions of the present invention in solid or liquid form may comprise agents that bind the antibacterial aminoglycoside compounds disclosed herein and thereby aid in the delivery of the compounds. Suitable agents capable of performing this function include monoclonal or polyclonal antibodies, proteins or liposomes.

The pharmaceutical compositions of the present invention may be comprised of dosage units that can be administered in aerosol form. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to those consisting of sealed packages. Delivery may be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. The aerosol formulations of antibacterial aminoglycoside compounds disclosed herein can be delivered in a single phase, two phase or three phase system to deliver the active ingredient. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. One skilled in the art can determine preferred aerosols without undue experimentation.

The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. For example, pharmaceutical compositions intended for administration by injection can be prepared by combining the antibacterial aminoglycoside compounds disclosed herein with sterile, dilute water to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the antibacterial aminoglycoside compound to facilitate dissolution or uniform suspension of the compound in an aqueous delivery system.

The antibacterial aminoglycoside compound disclosed herein or a pharmaceutically acceptable salt thereof is administered in a therapeutically effective amount, which varies depending on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, health condition, sex and diet of the patient, the rate of excretion, drug combination, the severity of the particular condition or disease state, and the individual being treated.

The antibacterial aminoglycoside compound disclosed herein or a pharmaceutically acceptable derivative thereof may also be administered concurrently with, prior to, or subsequent to the administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage form containing the antibacterial aminoglycoside compound disclosed herein and one or more additional active agents, as well as administration of the antibacterial aminoglycoside compound, with each active agent in its own separate pharmaceutical dosage form. For example, the antibacterial aminoglycoside compound and the other active agent can be administered to the patient in combination with a single orally administered dosage composition, such as a tablet or capsule, or the agents can be administered as separate oral dosage forms. Where separate dosage forms are used, the antibacterial compound disclosed herein and one or more additional active agents can be administered substantially simultaneously, i.e., simultaneously, or separately at staggered times, i.e., sequentially; combination therapy is understood to include all such regimens.

It is to be understood that in the present description, combinations of substituents and/or variations of the formula described are possible as long as such contribution results in a stable compound.

It will be appreciated by those skilled in the art that in the synthetic methods described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butyloxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for mercapto include-C (O) -R "(where R" is hydrocarbyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acids include hydrocarbyl, aryl or arylalkylhydrocarbyl esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting Groups is described in detail in Green, T.W. and P.G.M.Wutz (Protective Groups in organic Synthesis (1999), 3 rd edition, Wiley.) it is understood by those skilled in the art that protecting Groups may also be polymeric resins such as Wang resin, Rink resin or 2-chlorotrityl 1-chloro resin.

It is also understood by those skilled in the art that although the protected derivatives of the antibacterial aminoglycoside compounds disclosed herein may not possess pharmacological activity, they may be administered to a mammal and thereafter metabolized in vivo to form an antibacterial aminoglycoside compound having pharmacological activity. Thus, such derivatives may be described as "prodrugs". All prodrugs of the antibacterial aminoglycoside compounds disclosed herein are included within the scope of the present invention.

Furthermore, all antibacterial aminoglycoside compounds disclosed herein in free base or acid form can be converted into pharmaceutically acceptable salts thereof by methods known to those skilled in the art by treatment with a suitable inorganic or organic base or acid. The salts of the antibacterial aminoglycoside compounds disclosed herein can be converted to their free base or acid forms by standard techniques.

The following examples illustrate various methods for preparing antibacterial aminoglycoside compounds of structure (I):

(I)

wherein Q₁、Q₂、Q₃、R₈And R₉As defined herein. It is understood that one skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It will also be appreciated that other compounds of structure (I), not specifically exemplified below, can be prepared by those skilled in the art in a similar manner to that described below, by using appropriate starting components and varying the synthesis parameters as required. In general, the starting components can be obtained from sources such as SigmaAldrich, Lancaster Synthesis, Inc., Maybrid, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those of skill in the art (see, e.g., Advanced Organic Chemistry: Reactions, mechanics, and Structure, 5 th edition (Wiley, 12 months 2000)) or prepared as described herein.

The following examples are provided for the purpose of illustration and not limitation.

Examples

General synthetic procedure

Method A: to a stirred solution of sisomicin derivative (0.06mmol) in MeOH (2mL) was added aldehyde (0.068mmol), silica-supported cyanoborohydride (0.1g, 1.0mmol/g) and the reaction mixture was heated to 100 ℃ by microwave irradiation (100 watt power) for 15 minutes. The completion of the reaction was checked by MS and after completion all solvents were removed by rotary evaporation. The resulting residue was dissolved in EtOAc (20mL) and used5% NaHCO₃(2X 5mL), followed by brine (5 mL). The organic phase is then washed with Na₂SO₄Dried, filtered and the solvent removed by rotary evaporation.

Method B: to a solution of the sisomicin derivative (0.078mmol) in DMF (1ml) was addedMolecular sieves (15-20) followed by aldehyde (0.15mmol) and shaking the reaction was carried out for 2.5 hours. The completion of the reaction was checked by MS and more aldehyde (0.5 eq) was added if needed. The reaction mixture was then added dropwise to the stirred NaBH at 0 deg.C₄(0.78mmol) in MeOH (2mL) and the reaction stirred for 1 hour. By H₂The reaction was diluted with O (2mL) and EtOAc (2 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3X 3 mL). In Na ₂SO₄The combined organic layers were dried, filtered and concentrated to dryness.

Step 2: deprotection of para-nitrobenzyloxycarbonyl (PNZ)

To a stirred p-nitrobenzyloxycarbonyl protected sisomicin derivative (0.054mmol) in EtOH (1.5mL) and H₂O (1mL) solution 1N NaOH (0.3mL) was added followed by Na₂S₂O₄(0.315mmol) and the reaction mixture was heated at 70 ℃ for 12 h. The progress of the reaction was monitored by MS. After completion, with H₂The reaction mixture was diluted with O (5mL) and extracted with EtOAc (2X 10 mL). By H₂The combined organic layers were washed with O (2X 5mL), brine (5mL) and washed with Na₂SO₄Dried, filtered and concentrated to dryness.

And step 3: deprotection of tert-Butoxycarbonyl (Boc)(removal of the tert-butyldimethylsilyl protecting group under these conditions)

Of importance: prior to deprotection of the t-butyloxycarbonyl group, the sample must be thoroughly dried by drawing water under high vacuum for 3 hours.

Method A: to a stirred solution of tert-butoxycarbonyl protected sisomicin (0.054mmol) in DCM (1mL) was addedMolecular sieves (4-6) and trifluoroacetic acid (0.6 mL). The reaction was stirred at room temperature for 1 hour and checked for completion by MS. After completion, the reaction mixture was diluted with ether (15mL) to induce precipitation. The vial was centrifuged and the supernatant decanted. The precipitate was washed with diethyl ether (2X 15ml), decanted and dried in vacuo.

Method B: to a stirred solution of tert-butoxycarbonyl-protected sisomicin derivative (0.078mmol) in DCM (1.5mL) at 0 deg.C was added trifluoroacetic acid (1.5 mL). The reaction was stirred for 45 minutes and checked for completion by MS. After completion, the reaction was diluted with dichloroethane (10ml) and concentrated to dryness. The final dilution/concentration step was repeated twice.

And 4, step 4: BOP and PyBOP coupling

Method A: to a stirred solution of the sisomicin derivative (0.078mmol) in DMF (1mL) was added the acid (0.16mmol) followed by PyBOP (0.16mmol) and DIPEA (0.31mmol) and the reaction was stirred overnight. With EtOAc (3mL) and H₂The reaction mixture was diluted O (3mL) and the aqueous layer was separated and extracted with EtOAc (3X 3 mL). In Na above₂SO₄The combined organic layers were dried, filtered and concentrated to dryness.

Method B: to a stirred solution of the sisomicin derivative (0.073mmol) in DMF (1mL) was added a solution of the acid (0.102mmol), DIPEA (0.43mmol) and BOP (0.102mmol) in DMF (1mL) and the reaction was stirred for 4 hours while its progress was monitored by MS. The reaction mixture was diluted with water (8mL) and extracted with EtOAc (2X 10 mL). With 5% NaHCO₃The combined organic layers were washed with (2X 3mL) aqueous solution and brine (3mL) over Na₂SO₄Dried, filtered and concentrated to dryness.

And 5: epoxide opening

To a stirred solution of sisomicin derivative (0.06mmol) in MeOH (2mL) was added epoxide (0.07mmol), LiClO₄(0.15mmol) and the reaction mixture was heated to 100 ℃ by microwave irradiation for 90 minutes. The progress of the reaction was monitored by MS. After completion, the solvent was removed by rotary evaporation. The resulting residue was dissolved in EtOAc (20mL) and washed with H₂O (2X 5mL) and brine (5mL) in Na₂SO₄Dried, filtered and concentrated to dryness.

Step 6: deprotection of phthalimido group

To a stirred solution of phthalimido-protected sisomicin (0.064mmol) in EtOH (3mL) was added hydrazine (0.32mmol) and the reaction mixture was heated to reflux for 2 hours. The progress of the reaction was monitored by MS. After cooling to room temperature, the cyclic by-product precipitated and was removed by filtration. The filtrate was concentrated to dryness to give a residue, which was dissolved in EtOAc (20mL) with 5% NaHCO₃(2X 5mL) and brine (5mL) in Na₂SO₄Dried, filtered and concentrated to dryness.

And 7: addition of guanidine group

To a stirred solution of the sisomicin derivative (0.063mmol) in DMF (1mL) was added l H-pyrazole-1-carboxamidine hydrochloride (0.09mmol) followed by DIPEA (0.862mL) and the reaction mixture was heated to 80 ℃ and stirred overnight. The progress of the reaction was monitored by MS. After completion, the reaction mixture was cooled to room temperature and diluted with water (3 mL). The aqueous phase was separated and extracted with EtOAc (2X 5mL), and the combined organics were washed with brine (5mL) over Na ₂SO₄Dried, filtered and concentrated to dryness.

And 8: sulfonylation of Nitrobenzene (nosylation)

To a stirred solution of sisomicin derivative (0.23mmol) in DCM (20mL) was added 2-nitrobenzenesulfonyl chloride (0.25mmol) and DIPEA (0.3mmol), and the reaction was stirred for 3 hours. The progress of the reaction was monitored by MS. After completion, byDCM was removed by rotary evaporation and the resulting residue was dissolved in ethyl acetate (50mL) and then 5% NaHCO was used₃(2X 10mL) and brine (10 mL). The combined organic layers were then washed with Na₂SO₄Dried, filtered and concentrated to dryness.

And step 9: deprotection of Nitrobenzenesulfonyl (nosyl)

To a stirred solution of the nitrobenzenesulfonyl protected sisomicin derivative (0.056mmol) in DMF (1.5mL) was added thiophenol (0.224mmol), K₂CO₃(1.12mmol) and the reaction mixture was stirred for 2 hours, the progress of which was monitored by MS. Upon completion, the reaction mixture was diluted with water (5mL) and extracted with ethyl acetate (2X 10 mL). The combined organic layers were washed with water (2X 5mL) and brine (5mL) over Na₂SO₄Dried, filtered and concentrated to dryness.

Step 10: removal of p-nitrobenzyloxycarbonyl by hydrogenolysis

To a stirred solution of the sisomicin derivative (0.41mmol) in EtOH (60mL) was added AcOH (0.14mL) followed by Pd/C (30% by weight). The reaction vessel is evacuated and filled with H ₂(1 atmosphere) and the reaction mixture was stirred for 6 hours. The reaction vessel was then evacuated and filled with nitrogen. The solid was removed by filtration through a pad of celite and washed with MeOH (10 mL). Evaporation of the solvent yielded the desired product.

Step 11: mono-alkylation

To a stirred solution of nitrobenzenesulfonyl-protected sisomicin derivative (0.072mmol) in DMF (1.5mL) was added the halogenated alkane (0.144mmol), K₂CO₃(0.216mmol) and the reaction mixture was heated to 80 ℃ and its progress was monitored by MS. Upon completion, the reaction mixture was diluted with water (2mL) and extracted with ethyl acetate (2X 5 mL). The combined organic layers were washed with brine (1.5mL) over Na₂SO₄Dried, filtered and concentrated to dryness.

Step 12: sulphonylation

To a stirred solution of sisomicin skeleton (0.067mmol) in DCM (3mL) was added DIPEA (0.128mol) and sulfonyl chloride (0.07 mmol). The reaction mixture was stirred at room temperature and its progress monitored by MS. Upon completion, the solvent was removed by rotary evaporation and the residue was dissolved in ethyl acetate (20mL) with 5% NaHCO₃(2X 5mL) and brine (5mL) in Na₂SO₄Dried, filtered and concentrated to dryness.

Step 13: N-Boc protection

To a stirred solution of the amine (4.64mmol) in THF (10mL) was added 1N NaOH (10mL) followed by tert-butoxycarbonyl-anhydride (5.57mmol) and the progress of the reaction was checked by MS. Upon completion, THF was removed by rotary evaporation and water (40mL) was added. Separate the aqueous phase and use Et ₂O (2X 30ml) extraction. By addition of diluted H₃PO₄The aqueous phase was acidified to pH 3 and then extracted with EtOAc (2 × 60 ml). By H₂The combined organic layers were washed with O (2X 30mL) and brine (30mL) over Na₂SO₄Dried, filtered and concentrated to dryness.

Step 14: synthesis of epoxides

To a stirred solution of olefin (5.16mmol) in chloroform (20mL) at 0 deg.C was added m-chloroperoxybenzoic acid (8.0mmol) and the reaction mixture was stirred at 0 deg.C for 30 minutes, then allowed to warm to room temperature. The progress of the reaction was monitored by MS and TLC and additional portions of meta-CPBA were added as needed. After completion, the reaction mixture was diluted with chloroform (50mL) and 10% Na₂SO₃(2X 30mL) aqueous solution, 10% NaHCO₃(2X 50mL) and brine (50 mL). In Na₂SO₄The organic layer was dried, filtered and concentrated to give the crude product, which was purified by flash chromatography (silica gel/n-hexane: ethyl acetate 0-25%).

Step 15: general procedure for the Synthesis of alpha-hydroxycarboxylic acids

Step # 1O- (trimethylsilyl) cyanohydrin: A50-mL flask equipped with a magnetic stir bar and drying tube was filled with ketone or aldehyde (0.010mmol), followed by THF (50mL), trimethylsilylcyanide (1.39g, 14mmol), and zinc iodide (0.090g, 0.28mmol), and the reaction mixture was stirred at room temperature for 24 hours. Evaporation of the solvent gave a residue which was dissolved in EtOAc (60mL) with 5% NaHCO ₃(2X 30mL) aqueous solution, H₂O (30mL) and brine (30mL) in Na₂SO₄Dried, filtered and concentrated to dryness to give the crude product, which was taken to the next step without further purification.

Step #2 acid hydrolysis to alpha-hydroxycarboxylic acids: AcOH (25ml) and concentrated HCl (25ml) were added to the unpurified material from step #1 and the reaction mixture was refluxed for 2-3 hours. The reaction mixture was then concentrated to dryness to yield a white solid, which was carried on to the next step without further purification.

Step #3. Boc protection: to a stirred solution of the solid from step #2 in 2M NaOH (20mL) and IsoPrOH (20mL) was added the tert-butoxycarbonyl portion at 0 deg.C₂O (6.6g, 3mmol) and the reaction mixture was allowed to warm to room temperature for 4 hours. Then, the iso-PrOH is evaporated and H is added₂O (50mL), then the aqueous phase was separated and Et₂O (2X 30ml) extraction. By addition of diluted H₃PO₄The aqueous layer was acidified to pH 3 and extracted with EtOAc (2 × 60 ml). By H₂The combined organic layers were washed with O (2X 30mL) and brine (30mL) over Na₂SO₄Dried, filtered and concentrated to give the target N-t-butoxycarbonyl- α -hydroxycarboxylic acid in 56-72% yield.

Aldehydes and ketones used:n-tert-butoxycarbonyl-3-pyrrolidone, N-tert-butoxycarbonyl-3-azetidinone, N-tert-butoxycarbonyl-4-piperidone and N-tert-butoxycarbonyl-3-azetidinecarboxaldehyde.

Step 16: fluorenylmethoxycarbonyl (fluorenylmethoxycarbonyl) group pairProtection of amines

To a stirred solution of amine (0.049mol) in DCM (100mL) was added DIPEA (16mL, 0.099mol) and the reaction mixture was cooled to 0 ℃. Fmoc-Cl (12.8g, 0.049mol) was then added in portions over several minutes and the reaction was allowed to warm to room temperature over 2 hours. The organic layer was washed with water (2X 50mL) and brine (50mL) over Na₂SO₄Dried, filtered and concentrated to dryness to give the fluorenylmethoxycarbonyl protected amine (90-95% yield).

And step 17: mitsunobu alkylation

To a stirred solution of the nitrobenzenesulfonylated sisomicin derivative (0.087mmol) in toluene (2.5mL) was added the alcohol (0.174mmol), triphenylphosphine (0.174mmol) and the reaction mixture was cooled in a refrigerator at 4 ℃ for 10 minutes. Then a cooled solution of DEAD (0.174mmol of 2mL of anhydrous toluene) was added and the reaction was shaken overnight. The progress of the reaction was monitored by MS and additional alcohol and triphenylphosphine were added if required. Upon completion, ethyl acetate (30mL) was added and treated with 5% NaHCO₃The organic phase was washed with (2X 5mL) aqueous solution and brine (5mL) over Na₂SO₄Dried, filtered and concentrated to dryness.

Step 18: aldehyde synthesis by TEMPO/bleach oxidation

To a vigorously stirred solution of alcohol (1.54mmol) in DCM (4mL) were added TEMPO (0.007g, 0.045mmol, 0.03 mol%) and 2M aqueous KBr (75mL, 0.15mmol, 10 mol%) and the reaction mixture was cooled to-10 ℃. In a separate flask, NaHCO₃(0.5g, 9.5mmol) was dissolved in bleach (25mL, Chlorox 6.0% NaOCl) to give a 0.78M solution of buffer NaOCl. This freshly prepared 0.78M NaOCl solution (2.3mL, 1.8mmol, 117 mol%) was added to the reaction mixture for 5 minutes and the reaction was stirred at 0 ℃ for an additional 30 minutes. The organic phase was separated and the aqueous layer was extracted with dichloromethane (2X 4 mL). Using 10% of Na₂S₂O₃(4mL) aqueous, saturated NaHCO₃(2X 4mL) aqueous solution, brineThe combined organic layers were washed (5mL) over Na₂SO₄Dried and concentrated to dryness.

Step 19: alcohol synthesis by borane reduction

To a stirred solution of the acid (1.5mmol) in THF (5mL) at-10 deg.C was slowly added 1.0M BH₃THF (2.98mL, 2.98 mmol). And the reaction mixture was stirred vigorously at-10 ℃ for another 3 minutes, then allowed to warm to room temperature overnight. By dropwise addition of HOAc/H₂A solution of O (1: 1v/v, 2.0mL) quenched the reaction. TH was removed by rotary evaporation and saturated NaHCO was added₃(15mL) aqueous solution. The aqueous layer was extracted with DCM (3X 5mL) and saturated aqueous NaHCO ₃The combined organic layers were washed (2X 5mL), brine (10mL) and washed with Na₂SO₄Dried, filtered and concentrated to dryness.

Step 20: EDC coupling

To a stirred solution of sisomicin derivative (0.048mmol) in DMF (0.3mL) and THF (0.6mL) was added EDC (0.058mmol) followed by HONb (0.062mmol) and acid (0.058mmol) and the reaction was allowed to stir overnight. By H₂The reaction was quenched with O (2mL) and EtOAc (4mL) was added. With saturated NaHCO₃Aqueous solution, saturated NH₄The organic layer was washed with aqueous Cl solution over Na₂SO₄Dried, filtered and concentrated to dryness.

General purification procedure

Method # 1: purification by alkaline conditions

Mobile phase:

a-contains 10mM NH₄Water of OH

B-contains 10mM NH₄Acetonitrile of OH

Column:

a: Waters-XTerra preparation column MS C18OBD

19×100mm，5μm

Gradient: at a flow rate of 20 ml/min, at 0% for 20 minutes, then 0-20% for 200 minutes

B: Waters-XTerra preparation of MS C18OBD column

50×100mm，5μm

Gradient: at a flow rate of 20 ml/min, at 0% for 20 minutes, then 0-20% for 200 minutes

Collection was triggered by MS signal using Waters-XTerra. The collected fractions were dried by lyophilization and analyzed by LC/MS/ELSD. Pure fractions were combined and analyzed by LC/MS/ELSD for final purity check. Quantification was performed by LC/MS/CLND system.

Method # 2: purification by acidic conditions

Mobile phase:

A-Water containing 0.1% TFA

B-acetonitrile containing 0.1% TFA

Column:

A：Microsorb BDS Dynamax

21.4×250mm，10μm，

gradient: 0-100%, flow rate 25 ml/min

B：Microsorb BDS Dynamax

41.4×250mm，10μm，

Gradient: 0-100%, flow rate 45 ml/min

Method # 3: hydrophilic interaction chromatography (HILIC) purification

Buffering agent:

buffer A-3400ml of acetonitrile

600ml of water

15ml of acetic acid

15ml of TEA

Buffer B-4000ml of water

100ml of TEA

100ml of acetic acid

Column: poly C-polyhydroxyethyl A

150×21mm，5μm

Gradient: 20-70%, 10ml/35 min

The ELSD signal is used to trigger collection. The fractions were dried by lyophilization and analyzed by LC/MS/ELSD. The pure fractions were then combined, diluted with water and lyophilized. The dried fraction was redissolved in water and lyophilized three times to ensure complete removal of TEA. Any sample that showed trace amounts of TEA was subjected to additional drying. For delivery, the pure compound was dissolved to a concentration of > 10 mg/ml. The final purity was checked by LC/MS/ELSD and quantified by LC/MS/CLND.

Common intermediate

Sisomicin

Amberlite (Amberlite) IRA-400(OH form) (200g) was washed with MeOH (3X 200 ml). To a stirred suspension of washed resin in MeOH (150mL) was added sisomicin sulfate (20.0g, 0.029mol) and the mixture was stirred overnight. The resin was then filtered and washed with MeOH (100mL), and the combined organic layers were concentrated to dryness to give the target sisomicin (11.57g, 0.026 mol) Yield 89.6%): MS M/e [ M + H ]]⁺448.3 is calculated, 448.1 is obtained.

(N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -4-nitro-benzoic acid ester

To a stirred solution of 4-nitrobenzyl chloroformate (5.0g, 0.023mol) in THF (90mL) at 0 deg.C was added N-hydroxy-5-norbornene-2, 3-dicarboximide (4.16g, 0.023mol) followed by dropwise addition of dissolved Et₃N (3.2mL, 0.02mol) in THF (50mL) and the reaction stirred for 4 hours while gradually warming to room temperature. The reaction vessel was then placed in a refrigerator (-5 ℃) for 1 hour to induce precipitation of triethylamine hydrochloride, which was removed by filtration. The filtrate was concentrated to dryness to give a residue which was vigorously stirred in MeOH (80mL) for 1 hour, then filtered to give (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -4-nitro-benzoate (7.98g, 0.022mol, 96% yield) as a white solid: TLC (n-Hexane: EtOAc v/v 1: 1) R_f＝0.35。

2, 5-dioxo-pyrrolidin-1-yl-4-nitrobenzyl carbonate (p-nitrobenzyloxycarbonyl-succinimide)

To a stirred solution of N-hydroxysuccinimide (5.35g, 46.5mmol) in anhydrous THF (100mL) was added p-nitrobenzyl chloroformate (10.0g, 46.5mmol), and the solution was cooled in an ice bath. Triethylamine (6.5mL, 4.89g, 46.5mmol) was added over 10 minutes, after 30 minutes, the reaction mixture was allowed to warm to room temperature and stirred overnight. The slurry was cooled in an ice bath and filtered, followed by washing with ethyl acetate. The filtrate was concentrated in vacuo and the residue triturated with methanol. The solid was isolated by filtration to give 2, 5-dioxopyrrolidin-1-yl-4-nitrobenzyl carbonate.

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin

To a stirred solution of sisomicin (30.1g, 0.067mol) in MeOH (700mL) was added zinc acetate (37.07g, 0.202mol) followed by a slow addition of S-ethyl trifluoroacetoacetate (9.37mL, 0.074mol) in MeOH (100mL) and the reaction was allowed to proceed in N₂Stirring was continued overnight. A solution of triethylamine (37.5mL, 0.27mol) and p-nitrobenzyloxycarbonyl-succinimide (64.2g, 0.179mol) in THF (1L) was then added dropwise and the reaction stirred for 3 hours. Evaporation of the solvent gave a crude product, which was dissolved in DCM (2L) and concentrated NH₄OH∶H₂O (3: 1v/v, 2X 800mL) and brine (800mL) and washed over MgSO₄Dried, filtered and concentrated to dryness. The residue was dissolved in ethyl acetate (1L) and washed with AcOH: H₂O (1/9v/v 1L). The aqueous layer was washed with ethyl acetate (2 × 1L), basified with 10N NaOH to pH 12, and extracted with ethyl acetate (2 × 1L). The organic layer was washed with brine (500mL) over MgSO₄Dried, filtered and concentrated to give a residue. The crude product was dissolved in ethyl acetate (500mL) and the solution was allowed to stand overnight. The precipitated solid was removed by filtration and the remaining filtrate was concentrated to give the crude product, which was purified by reverse phase HPLC method 2-column B to give the target 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin (MS M/e [ M + H) ]⁺Calculate 902.3, find 902.2).

6 ' -trifluoroacetyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1-acetyl-3 ' -tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin (0.7g, 0.77mmol) in MeOH (7mL) at 0 deg.C was added acetic anhydride (0.095mL, 1.01mmol) slowly and the reaction allowed to warm to room temperature overnight. By MS-chase reaction, it was determined that the intermediate 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1-acetyl-sisomicin (MS M/e [ M + H ]]⁺944.3 is calculated, 944.2, [ M + Na ] is obtained]⁺966.3) is fully formed. The reaction mixture was then cooled to 0 ℃ and DIPEA (0.54mL, 3.11mmol) was added followed by tert-butoxycarbonyl anhydride (0.53mL, 2.33mmol) and the reaction was stirred for 6 hours while the progress was followed by MS. Using glycine (0.29g, 3.88mmol) and K₂CO₃The reaction was quenched (0.54g, 3.88mmol) and stirred overnight. After evaporation of the solvent, the residue is taken up in H₂The layers were separated between O (10mL) and EtOAc (10 mL). The aqueous layer was separated and further extracted with EtOAc (3X 10mL) and washed with Na₂SO₄The combined organic layers were dried, filtered and concentrated to dryness to give the target 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1-acetyl-3 "-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ]) ]⁺1044.4 is calculated, 1044.0, [ M + Na ] is obtained]⁺1066.3) which was carried on to the next step without further purification.

2 ', 3-di-p-nitrobenzyloxycarbonyl-1-acetyl-3' -tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1-acetyl-3 "-tert-butoxycarbonyl-sisomicin (0.77mmol) in MeOH (5mL) was added concentrated NH₄OH (8.2mL) and the reaction was stirred overnight. Evaporation of the solvent gave a crude product which was purified by preparative HPLC method 2-column B to give the target 2', 3-di-p-nitrobenzyloxycarbonyl-1-acetyl-3 "-tert-butoxycarbonyl-sisomicin (0.35g, 0.36mmol, 46.7% yield)Purity > 95%): MSm/e [ M + H]⁺948.4 is calculated, 948.2 is obtained.

N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyric acid

To a stirred solution of 4-amino-2 (S) -hydroxybutyric acid (5.0g, 0.041mol) in dioxane: H₂O (200mL, 1: 1v/v) solution was added K₂CO₃(11.6g, 0.084mol) followed by p-nitrobenzyl chloroformate (9.23g, 0.043mol) and the reaction mixture was stirred overnight. The resulting precipitate was removed by filtration and the organic solvent was removed by rotary evaporation. The resulting aqueous solution was acidified to pH 1 by addition of 1MHCl (100 mL). After ethyl acetate (100mL) was added to the aqueous layer, the product precipitated and was collected by filtration. The filtrate was added to a separatory funnel and the organic layer was separated. After addition of ethyl acetate (100mL) to the aqueous layer, a second precipitation occurred, the product was collected by filtration and the process was repeated once more. The combined organic layers were then left overnight at-5 ℃ to induce precipitation of the product, which was collected by filtration. The target N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyric acid (9.3g, 0.031mol, 75% yield, 90% purity) was subjected to the next step without further purification. MS M/e [ M + H ] ]⁺299.1 is calculated, 298.9 is obtained.

(N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butanoic acid ester

To a stirred solution of N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyric acid (8.95g, 30.0mmol) in THF (200mL) at 0 deg.C was slowly added DCC (6.8g, 33.0mmol) and the reaction stirred for 30 min. Then dropwise addingA solution of N-hydroxy-5-norbornene-2, 3-dicarboxylic acid imide (6.45g, 36.0mmol) in THF (100mL) for 1 hour. The precipitated urea was removed by filtration and the remaining filtrate was concentrated to dryness. The residue was dissolved in ethyl acetate (200mL) and washed with H₂O (150mL) over MgSO₄Dried, filtered and concentrated to dryness. The product was recrystallized from ethyl acetate/ether to give the target N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butanoate (10.0g, 21.78mmol, 72.6% yield). MS M/e [ M + H ]]⁺482.1 was calculated and 482.2 was obtained.

(N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -benzoyl-butyric acid ester

To a stirred solution of (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyrate (6.4g, 0.014mol) in THF (65mL) was added triphenylphosphine (4.0g, 0.015mmol) followed by benzoic acid (1.9g, 0.015mmol) and the reaction mixture was cooled to 0 ℃. DIAD (3.0mL, 0.015mol) was then added dropwise and the reaction mixture was stirred for an additional 50 minutes. Evaporation of the solvent gave the crude product, which was purified by flash chromatography (silica gel/N-hexane: ethyl acetate 20-100%) to give the target (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -benzoyl-butyrate (2.3g, 4.08mmol, 29.1% yield) with light contamination of triphenoxyphosphine: ¹HNMR(400MHz，CDCl₃)δ8.17(d，2H)，7.98(d，2H)，7.44-7.70(m，5H)，5.96-6.18(m，2H)，5.41-5.55(m，1H)，5.10(s，2H)，3.40-3.58(m，2H)，3.21-3.39(m，4H)，2.10-2.22(m，2H)，1.44-1.60(m，2H)。

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -O-benzoyl-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin (2.5g, 2.77mmol) in DMF (50mL) was added (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -benzoyl-butyrate (2.3g, 4.08mmol) and the reaction was stirred for 24 hours. DIPEA (2.5mL, 0.014mol) was then added followed by tert-butoxycarbonyl acid anhydride (2.5mL, 0.011mol) and the reaction mixture was stirred for an additional 2 hours. The amino acetic acid (2.5g, 0.033mol) and K are then added in portions₂CO₃(4.6g, 0.033mol) H₂O (50mL) solution for 5 min and the reaction mixture was stirred for 1 h. The reaction mixture was diluted with ethyl acetate (300mL) and the aqueous layer was separated. With 1M citric acid (150mL), saturated NaHCO₃The organic layer was washed with (30mL) aqueous solution, brine (30mL) and MgSO₄Dried, filtered and concentrated to dryness to give the crude product, which was purified by reverse phase HPLC method 2-column B to give the target 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -O-benzoyl-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (1.6g, 1.15mmol, 41.5% yield).

2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 ' -trifluoroacetyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -O-benzoyl-butyryl) -3 ' -tert-butoxycarbonyl-sisomicin (1.6g, 1.15 m)mol) in MeOH (30mL) concentrated NH was added₄OH (3mL) and the reaction was stirred for 3 days. Ethyl acetate (30mL) was then added and the aqueous layer was separated. The organic layer was washed with 1M NaOH (20mL), brine (20mL) over MgSO₄Dried on and concentrated to dryness to give 2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (1.4g, MS M/e [ M + H]⁺1186.4 is calculated, 1186.2, [ M + Na ] is obtained]⁺1208.3) which is subjected to a step without further purification.

(R) -Ethyl 3-azido-2-hydroxypropionate

Ethyl- (2R) -2, 3-epoxypropionate (0.5g, 4.3mmol), ammonium chloride (0.253g, 4.73mmol) and sodium azide (0.336g, 5.17mmol) were mixed in DMF (8mL) and the mixture was heated at 75 ℃ for 14 h. The reaction was cooled to room temperature and partitioned between water and diethyl ether/n-hexane (1: 1 v/v). The phases were separated and the organic phase was washed once with water, brine, respectively, over MgSO ₄Dried, filtered and concentrated to an oil which was purified by flash chromatography (silica gel/n-hexane: 10% ethyl acetate) to give (R) -ethyl-3-azido-2-hydroxypropionate (0.47g, 2.97mmol, 69% yield) as a clear oil. R_f0.27 (n-hexane: 10% EtOAc, v/v, p-anisaldehyde); MSm/e [ M + Na ]]⁺The calculation is carried out at 182.1, and 182.0 is obtained.

(R) -3- (tert-Butoxycarbonylamino) -2-hydroxypropionic acid

Step 1) to a stirred solution of (R) -ethyl-3-azido-2-hydroxypropionate (159mg, 1.0mmol) in ethanol (4mL) was added acetic acid (0.10mL) followed by 5% Pd/C (25mg) after replacing the flask with nitrogen. The flask was equipped with a hydrogen balloon and stirred for 1 hour. The flask was then replaced with nitrogen, the mixture was filtered through celite and the pad was washed with ethanol (4 mL).

Step 2) to the filtrate was added 1M NaOH (3mL), followed by tert-butyloxycarbonyl₂O (0.28mL, 0.27g, 1.2mmol), and the solution was stirred at room temperature for 2 days. The solution was then partitioned between ether and water and the phases were separated. The aqueous phase was washed twice with diethyl ether and 1M NaHSO₄Acidified and extracted with ethyl acetate. The ethyl acetate phase was washed with brine over MgSO₄Dry above, filter and concentrate to an oil which solidifies to give (R) -3- (tert-butoxycarbonylamino) -2-hydroxypropionic acid (117mg, 57% yield): r _f＝0.22(CHCl₃: 10% IPA, 1% AcOH, ninhydrin).

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonylamino) -2-hydroxy-propionyl ] -sisomicin

(R) -3- (tert-Butoxycarbonylamino) -2-hydroxypropionic acid (1.3g, 6.3mmol) and HONB (1.35g, 7.5mmol) were dissolved in THF (40mL), the solution was cooled to 0 deg.C and EDC (1.33g, 6.9mmol) was added. After 20 minutes, the reaction was allowed to warm to room temperature. After 6 hours, a solution of 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin (5.23g, 5.8mmol) in DMF (25mL) was added and the solution was allowed to stir overnight. The reaction was concentrated to remove THF and partitioned between water and ethyl acetate. Separating phase and using water and saturated NaHCO respectively₃The ethyl acetate phase was washed once with water and brine. Then the ethyl acetate phase is placed over Na₂SO₄Dried, filtered and concentrated to a residue. The residue was chromatographed by reverse phase HPLC method 2-column B to give 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonyl) as a pale white foamPhenylamino) -2-hydroxy-propionyl]Sisomicin (1.64g, 1.51mmol, 24% yield): MS M/e [ M + H ]]⁺1089.4 was calculated to obtain 1089.2.

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonylamino) -2-hydroxy-propionyl ] -3 "-tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonylamino) -2-hydroxy-propionyl group]-sisomicin (1.52g, 1.39mmol) in THF (10mL) and methanol (5mL) was added Boc₂O (0.65mL, 0.62g, 2.8 mmol). After three hours, glycine (312mg, 4.17mmol) and 0.5M K were added₂CO₃(24mL) and the reaction was stirred vigorously for one hour. Then, the mixture was partitioned between ethyl acetate and water, and the phases were separated. The ethyl acetate phase was washed once with water and brine, respectively, over MgSO₄Drying, filtering and concentrating to dryness to give 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonylamino) -2-hydroxy-propionyl group as a solid]-3 "-tert-butyloxycarbonyl-sisomicin, which is subjected to the next step without further purification. MS M/e [ M-tert-butoxycarbonyl group]⁺1089.4 was calculated to obtain 1089.2.

2 ', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butyloxycarbonylamino) -2-hydroxy-propionyl ] -3' -tert-butyloxycarbonyl-sisomicin

To 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonylamino) -2-hydroxy-propionyl]-3 "-tert-butyloxycarbonyl-sisomicin(1.39mmol) in methanol (45mL) was added concentrated ammonium hydroxide (45mL, ca. 12M). The solution was allowed to stand at ambient temperature for 18 hours and then concentrated in vacuo. The residue was partitioned between ethyl acetate and water and the phases were separated. The aqueous phase was back extracted once with ethyl acetate. The combined ethyl acetate phases were concentrated to give a residue which was dissolved in a 1: 1v/v mixture of methanol/acetic acid/water and purified by reverse phase HPLC method 2-column B. The pure fractions were combined and washed with 1M Na₂CO₃Basified and concentrated in vacuo to remove acetonitrile. The mixture was then extracted twice with ethyl acetate. The final ethyl acetate phases were combined, washed with brine, over MgSO₄Dried, filtered and concentrated to give 2', 3-di-p-nitrobenzyloxycarbonyl-1- [ (R) -3- (tert-butoxycarbonylamino) -2-hydroxy-propionyl as a white solid]-3 "-tert-Butoxycarbonyl-sisomicin (316mg, 30% yield). MS M/e [ M + H ]]⁺1093.4 is calculated, 1093.3 is obtained.

N-Boc-3-amino-2 (S) -hydroxy-propionic acid

Stirring of S-isoserine (4.0g, 0.038mol) dioxane: H at 0 deg.C ₂O (100mL, 1: 1v/v) solution was added N-methylmorpholine (4.77mL, 0.043mol) followed by tert-butoxycarbonyl₂O (11.28mL, 0.049mol) and the reaction was stirred overnight while gradually warming to room temperature. Glycine (1.0g, 0.013mol) was then added and the reaction stirred for 20 min. The reaction was cooled to 0 ℃ and saturated NaHCO was added₃(75mL) of an aqueous solution. The aqueous layer was washed with ethyl acetate (2X 60mL) and then NaHSO was used₄Acidified to pH 1. The solution was then extracted with ethyl acetate (3X 70mL) and washed with Na₂SO₄The combined organic layers were dried, filtered and concentrated to dryness to give the target N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionic acid (6.30g, 0.031mmol, 81.5% yield):¹H NMR(400MHz，CDCl₃)δ7.45(bs，1H)，5.28(bs，1H)，4.26(m，1H)，3.40-3.62(m，2H)，2.09(s，1H)，1.42(s，9H)；¹³C NMR(100MHz，CDCl₃)δ174.72，158.17，82，71.85，44.28，28.45。

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

When MS showed completion of the reactive ester (MSm/e [ M + Na ]]⁺389.1 calculation, 389.1) formation was determined by slowly adding HONB (1.14g, 6.34mmol) and EDC (1.21g, 6.34mmol) to a stirred solution of N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionic acid (1.30g, 6.34mmol) in DMF (14ml) and stirring the reaction mixture for 2 hours. Then 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin (4.76g, 5.28mmol) was added and the reaction was allowed to stir overnight. With saturated NaHCO ₃The reaction was quenched with aqueous (10mL) and extracted with EtOAc (5X 15 mL). In Na₂SO₄The combined organic layers were dried, filtered and evaporated to dryness to give the crude product, which was purified by reverse phase HPLC method 2-column B to give the target 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (1.66g, 1.52mmol, 29% yield, > 95% purity): MS M/e [ M + H ]]⁺1089.4 was calculated to obtain 1089.2, [ M + Na ]]⁺1111.3。

6 ' -trifluoroacetyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin

6' -trifluoroacetyl radical stirred at 0 deg.CA suspension of the yl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (1.66g, 1.52mmol) in MeOH (20mL) was added DIPEA (0.53mL, 3.05mmol) followed by tert-butoxycarbonyl-anhydride (0.52mL, 2.29mmol) and the reaction was allowed to warm to room temperature. After 2 hours each material went into solution. The reaction was cooled to 0 ℃ and quenched with glycine (0.5g, 6.66mmol) and saturated NaHCO₃And (4) quenching the aqueous solution. The reaction was extracted with EtOAc (3X 20mL) and washed with Na ₂SO₄The combined organic layers were dried, filtered and evaporated to dryness to give 6 ' -trifluoroacetyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺1189.4 is calculated, 1188.8, [ M + Na ] is obtained]⁺1211.3) was used in the next step without further purification.

2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butoxycarbonyl-sisomicin

6 ' -trifluoroacetyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin (1.52mmol) was dissolved in MeOH (12mL) and concentrated NH was added₄OH (20mL), and the reaction was stirred overnight. Evaporation of the solvent gave the crude product, which was purified by reverse phase HPLC method 2-column B to give the target 2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.96g, 0.79mmol, 51.9% yield, > 95% purity): MS M/e [ M + H ]]⁺1093.4 is calculated, 1093.2, [ M + Na ] is obtained]⁺1115.3。

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyric acid (1.47g, 4.9mmol) in DMF (50ml) was slowly added HONB (0.884g, 4.9mmol) and EDC (0.945g, 4.9mmol) and the reaction mixture was stirred for 2 h. Then 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-sisomicin (3.42g, 3.8mmol) was added and the reaction was allowed to stir overnight. With saturated NaHCO₃The reaction was quenched with aqueous (30ml) and extracted with EtOAc (5 × 50 mL). Over MgSO₄The combined organic layers were dried, filtered and concentrated to give the target 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-3-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ])]⁺1182.4, found 1182.4), which was carried on to the next step without further purification.

6 ' -trifluoroacetyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 ' -tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-3-amino-2 (S) -hydroxy-butyryl) -sisomicin (4.9mmol) in MeOH (50mL) at 0 deg.C was added DIPEA (1.70mL, 9.8mmol) followed by tert-butoxycarbonyl acid anhydride (1.6g, 7.35mmol) and the reaction was allowed to warm to room temperature. The reaction was then cooled to 0 ℃ and quenched with glycine (1.10g, 14.7mmol) and saturated aqueous NaHCO ₃And (4) quenching. The reaction was extracted with EtOAc (3X 50mL) and over MgSO₄The combined organic layers were dried, filtered and evaporated to dryness to give 6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2)(S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin, which was used in the next step without further purification.

2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin

6 '-trifluoroacetyl-2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (4.9mmol) was dissolved in MeOH (30mL) and concentrated NH was added₄OH (50mL), the reaction was stirred overnight. Evaporation of the solvent gave the crude product, which was purified by reverse phase HPLC method 2-column B to give the target product 2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin. MS M/e [ M + H ]]⁺1186.4 is calculated, 1186.3 is obtained.

6' -p-nitrobenzyloxycarbonyl sisomicin

To a stirred solution of sisomicin (19.1g, 42.65mmol) in MeOH (300mL) was added Zn (OAc) ₂(23.5g, 0.128mol) and the reaction mixture was stirred for 1 hour until all the zinc had come into solution. Then a solution of (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -4-nitro-benzoate (15.28g, 42.65mmol) in DCM (150mL) was added dropwise over 3 h and the reaction was allowed to stir overnight. The reaction was then concentrated to dryness to give the crude product, which was slowly added to 10% NH with vigorous stirring₄Aqueous OH (480mL) and DCM (180 mL). The aqueous layer was separated, washed with DCM (3X 160mL) and diluted with brine (250 mL). Use DCM: IPA (7: 3v/v, 4 in a production line)160mL) the aqueous layer was extracted. With 10% NH₄The combined organic layers were washed with aqueous OH: brine (7: 3v/v, 200mL) over MgSO₄Dried, filtered and concentrated to give the target 6' -p-nitrobenzyloxycarbonyl-sisomicin: MS M/e [ M + H ]]⁺627.3 is calculated, 627.2 is obtained; CLND 95% purity.

(N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -tert-butyl-carbonate

To a stirred solution of N-hydroxy-5-norbornene-2, 3-dicarboximide (20.0g, 0.112mol) in THF (200mL) at 0 deg.C was added triethylamine (0.65mL, 4.8mmol), followed by dropwise addition of tert-butoxycarbonyl₂O (29.23g, 0.134mol) in THF (30mL) and the reaction was allowed to stir overnight while gradually warming to room temperature. A precipitate formed which was filtered and washed with cold THF (200 mL). The crude solid was then stirred vigorously in MeOH (100mL) for 1 hour, washed with MeOH (50mL) and dried in vacuo before filtration to give the target (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -tert-butyl carbonate (28.0g, 0.1mol, 89.3% yield) as a white solid: TLC (n-hexane: ethyl acetate, 1: 1v/v), R _f＝0.44；NMR(400MHz，DMSO-d₆)δ6.10(bs，2H)，3.48(bs，2H)，3.29-3.32(m，2H)，1.58-1.62(m，1H)，1.50-1.55(m，1H)，1.47(s，9H)。

6 '-p-nitrobenzyloxycarbonyl-2', 3-di-tert-butoxycarbonyl-sisomicin

To a stirred solution of 6' -p-nitrobenzyloxycarbonyl-sisomicin (5.86g, 9.35mmol) in MeOH (100mL) was added Zn (OAc)₂(5.15g, 28.05mmol) and the reaction mixture was stirred1 hour until all solids dissolved. A solution of (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -tert-butyl carbonate (4.96g, 17.77mmol) in THF (48mL) was added dropwise over 4 hours and the reaction mixture was allowed to stir overnight. Triethylamine (2.61mL, 18.7mmol) was then added followed by a solution of (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -tert-butyl carbonate (1.31g, 4.68mmol) in THF (12mL) and the reaction mixture was stirred for an additional 24 h. The reaction was quenched by the addition of glycine (2.81g, 37.4 mmol). The solvent was removed by rotary evaporation to give a residue, which was dissolved in DCM (200mL) and washed with H₂Concentrated NH to O₄OH (7: 3v/v, 3X 50mL) wash. Over MgSO₄The organic layer was dried, filtered and concentrated to dryness. The solid was dissolved in 0.1M aqueous AcOH (2.0L) and washed with ethyl acetate: diethyl ether (9: 1v/v, 4X 1.0L). Then, with concentrated NH₄The aqueous layer was basified to pH 10 with OH, treated with salt and extracted with ethyl acetate (3 × 30 mL). Over MgSO ₄The combined organic layers were dried, filtered and concentrated to give 6 '-p-nitrobenzyloxycarbonyl-2', 3-di-tert-butoxycarbonyl-sisomicin (4.1g, 4.96mmol, 53.0% yield, 92% purity): MS M/e [ M + H ]]⁺827.4 was calculated and 827.2 was obtained.

(N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -9-fluorene-acetate

To a stirred solution of N-hydroxy-5-norbornene-2, 3-dicarboximide (7.38g, 0.041mol) in THF (200mL) was added N-methylmorpholine (4.53mL, 0.041mol) at 0 deg.C, followed by dropwise addition of a solution of 9-fluorenylmethylchloroformate (10.15g, 0.039mol) in THF (50mL), and the reaction was stirred overnight while gradually warming to room temperature. Then, the flask was cooled to 0 ℃ and the precipitated salts were removed by filtration. The filtrate was concentrated in vacuo to give a waxy residue which was precipitated from methanol to give (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -9-fluorene-acetate (9.9g, 0.025 g)mol, yield 61.0%), which was carried on to the next step without further purification: TLC (n-hexane: ethyl acetate 3: 1v/v), R_f＝0.28。

6 ' -p-nitrobenzyloxycarbonyl-2 ', 3, 3 ' -tri-tert-butoxycarbonyl-1-fluorenylmethoxycarbonyl-sisomicin

To a stirred solution of 6 '-p-nitrobenzyloxycarbonyl-2', 3-bis-BOC-sisomicin (7.38g, 8.93mmol) in THF (200mL) was added (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -9-fluorene-acetate (2.51g, 6.25mmol) and the reaction was allowed to react for 1 hour while monitoring the progress by HPLC and MS (MS M/e [ M + H ]) ]⁺Calculation 1049.5, found 1049.4). Additional (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -9-fluorene-acetate (0.05 eq) was added and the reaction stirred for 1.5 hours. Then N-methylmorpholine (0.98ml, 8.93mmol) was added followed by tert-butoxycarbonyl acid anhydride (3.94g, 17.85mmol) and the reaction stirred for 3 hours. The reaction was quenched by the addition of glycine (7.51g, 40.18mmol) and allowed to stir overnight. The precipitated salt was filtered and the resulting solution was concentrated to dryness to give a residue, which was dissolved in DCM (150mL) and saturated NaHCO₃(3X 80mL) aqueous solution, 1M citric acid (3X 80mL), H₂O∶NaHCO₃(1: 1v/v, 80mL), brine (40mL) and over MgSO₄And drying. Filtration and evaporation of the solvent gave the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1-fluorenylmethoxycarbonyl-sisomicin (MS M/e [ M + Na ]]⁺Calculation 1171.5, found 1171.3), which was carried on to the next step without further purification.

6 ' -p-nitrobenzyloxycarbonyl-2 ', 3, 3 ' -tri-tert-butoxycarbonyl-sisomicin

To a stirred solution of 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1-fluorenylmethoxycarbonyl-sisomicin (8.93mmol) in DCM (150mL) was added tris (2-aminoethyl) amine (13.37mL, 89.27mmol) slowly and the reaction was stirred for 45 min. Then, the mixture was diluted with saline (3 × 100mL), a phosphate buffer solution (2 × 500mL, 1 × 100mL) having a pH of 5.5, and H ₂O (100mL), aqueous saturated NaHCO₃The reaction mixture was washed (100mL) and brine (100 mL). The organic phase was concentrated to give the crude product, which was purified by reverse phase HPLC method 2-column B to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (2.77g, 2.99mmol, 33.5% yield, 93% purity): MS M/e [ M + H ]]⁺927.4 is calculated, 927.2 is obtained.

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

To a stirred solution of N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionic acid (0.93g, 4.53mmol) in DMF (8ml) was slowly added HONB (0.82g, 4.53mmol) and EDC (0.87g, 4.53mmol) and the reaction mixture was stirred for 2 h. Then, 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (3.0g, 3.23mmol) was added and the reaction was allowed to stir overnight. By H₂The reaction was quenched with O (10mL) and extracted with EtOAc (5X 15 mL). In Na₂SO₄The combined organic layers were dried, filtered and concentrated to dryness to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ]) ]⁺1114.5 is calculated, 1113.9, [ M + Na ] is obtained]⁺1136.3) which was carried on to the next step without further purification.

2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Subjecting 6 ' -p-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (3.23mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to give 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (2.0g, 2.14mmol, 66.2% yield, > 65%) to: MSm/e [ M + H]⁺935.5, obtain 935.3, [ M + Na ]]⁺957.3。

N-Boc-4-amino-2 (S)) -hydroxy-butyric acid

To a stirred stream of S-4-amino-2-hydroxy-butyric acid (51.98g, 0.44mol) in dioxane: H₂O (2L, 1: 1v/v) solution was added K₂CO₃(106g, 0.91mol), followed by a solution of tert-butoxycarbonyl-anhydride (100g, 0.46mol) in dioxane (100mL) and the reaction was stirred overnight. The reaction was washed with DCM (2X 300mL) and H₃PO₄The aqueous layer was acidified to pH 2. The aqueous layer was extracted with DCM (2X 300mL) and over MgSO₄The combined organic layers were dried, filtered and concentrated to dryness to give the target N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxybutyric acid (48.2g, 50% yield).

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyric acid (1.35g, 6.17mmol) in DMF (12ml) was slowly added HONB (1.11g, 6.17mmol) and EDC (1.18g, 6.17 mmol). Then, a solution of 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (4.4g, 4.75mmol) in DMF (13mL) was added slowly and the reaction was allowed to stir overnight. The reaction was cooled to 0 ℃ and saturated NaHCO was used₃Aqueous (20mL) was quenched and extracted with Et0Ac (50 mL). With saturated NaHCO₃The combined organic layers were washed with aqueous (2X 20mL), brine (25mL) and over MgSO₄Dried, filtered and concentrated to dryness to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H + ] -M)]⁺1128.5, found 1129.4), which was carried on to the next step without further purification.

2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 6 ' -p-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (4.75mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to give 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ] ]⁺949.5 is calculated, 949.1, [ M + Na ] is obtained]⁺971.4。

6 ', 2' -di-p-nitrobenzyloxycarbonyl sisomicin

Sisomicin (12.9g, 28.9mmol) and nickel (II) acetate (29g, 115.6mmol) were dissolved in methanol (900ml) and the green solution was cooled in an ice-water bath. To this solution was added 2, 4-dioxo-3-azabicyclo [3.2.1 ] in solid form]Oct-6-en-3-yl 4-nitrobenzyl carbonate (16.6g, 46.2 mmol). The mixture was allowed to slowly warm to room temperature and stirred overnight. The solution was concentrated in vacuo to a green oil, and the oil was partitioned between concentrated ammonium hydroxide (ca. 12M) and ethyl acetate. The phases were separated and the violet aqueous phase was back-extracted once with ethyl acetate. The combined ethyl acetate phases were washed once with brine, diluted with 10% by volume of isopropanol and extracted three times with 5% aqueous acetic acid. The combined acetic acid phases were basified to pH > 11 with 6M NaOH and then extracted twice with ethyl acetate. The final two ethyl acetate phases were combined and washed once with brine over Na₂SO₄Dried, filtered and concentrated in vacuo to 1/2 volumes. The product precipitated during concentration and was isolated by filtration to give 6 ', 2' -di-p-nitrobenzyloxycarbonyl-sisomicin (12.1g, 65% yield) as a white solid. MSm/e [ M + H ]⁺806.3 is calculated, and 806.2 is obtained.

6 ', 2 ' -di-p-nitrobenzyloxycarbonyl-1, 3, 3 ' -tri-tert-butoxycarbonyl-sisomicin

To a flask containing a stirred solution of 6 ', 2' -di-p-nitrobenzyloxycarbonyl-sisomicin (4.1g, 5.09mmol) in THF (70mL) and methanol (70mL) placed in a water bath was added di-tert-butyl-dicarbonate (5.8mL, 5.51g, 25.5 mmol). After 2 hours, glycine (1.9g, 25.5mmol), water (70mL) and 1M sodium carbonate (15mL) were added and the mixture was stirred vigorously for 12 hours. The mixture was concentrated to remove THF and methanol, and water (100mL) was added to suspend the solids. The solid was isolated by filtration, washed with water and dried to give 6' as a white solid,2' -di-p-nitrobenzyloxycarbonyl-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (5.41g, 96% yield). R_f＝0.15(CHCl₃：5％IPA v/v，UV)MS m/e[M-B℃]⁺1006.5 is calculated, 1006.4 is obtained.

1, 3, 3' -tri-tert-butoxycarbonyl-sisomicin

In a flask 6 ', 2' -di-p-nitrobenzyloxycarbonyl-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (4.84g, 4.38mmol) and sodium hyposulfite (7.6g, 44mmol) were combined with ethanol (70mL) and water (70 mL). The flask was equipped with a condenser and the mixture was heated at 60 ℃ for 12 hours. The mixture was then heated at 65 ℃ for an additional three hours, followed by cooling to room temperature. The mixture was partitioned between 0.2M NaOH and ethyl acetate and the phases were separated. The aqueous phase was back extracted once with ethyl acetate. The combined organic phases were washed once with brine over Na ₂SO₄Dry, filter and concentrate to an oil. The oil was triturated with ether and the solid was isolated by filtration to give 6 ', 2' -di-p-nitrobenzyloxycarbonyl-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (2.71g, 83% yield) as a white solid. R_f＝0.23(IPA∶CHCl₃4: 1, 2% NH₃UV, ninhydrin); MSm/e [ M + H]⁺748.4 is calculated, 748.3 is obtained.

6 '-p-nitrobenzyloxycarbonyl-1, 3, 3' -tri-tert-butoxycarbonyl-sisomicin

1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (8.5g, 11.4mmol) was dissolved in methanol (212mL) and cooled in an ice-water bath, followed by addition of triethylamine (1.75mL, 12).5 mmol). Adding 2, 4-dioxo-3-azabicyclo [3.2.1 ] in solid form]Oct-6-en-3-yl 4-nitrobenzyl carbonate (4.08g, 11.4 mmol). After 1 hour, the reaction was concentrated to a residue, which was partitioned between ether/ethyl acetate (1: 1v/v) and water. The phases were separated and the organic phase was washed once with 5% aqueous acetic acid to remove the remaining starting material. The organic phase is then diluted with 1/3 volumes of n-hexane and extracted three times with 5% aqueous acetic acid. The last three aqueous phases were combined, treated with salt to about 10% saturated NaCl, and extracted twice with ethyl acetate. The last two ethyl acetate phases were combined and washed once with 1M NaOH and brine, respectively, over Na ₂SO₄Dried, filtered and concentrated. The resulting residue was triturated with ether/n-hexane and the solid was isolated by filtration to give 6' -p-nitrobenzyloxycarbonyl-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (6.2g, 61% yield) as a white solid. Unreacted starting material in the starting aqueous phase can be recovered by simply basifying the solution, extracting it into ethyl acetate over Na₂SO₄Dried and concentrated. MS M/e [ M + H ]]⁺927.4 is calculated, 927.4 is obtained.

6 ', 2' -di-p-nitrobenzyloxycarbonyl-3-tert-butoxycarbonyl-sisomicin

6 ', 2' -di-p-nitrobenzyloxycarbonyl-sisomicin (5.5g, 6.8mmol) and zinc acetate (4.5g, 20.4mmol) were dissolved in methanol (200mL) and the solution was cooled in an ice-water bath. Adding tert-butyl-2, 4-dioxo-3-azabicyclo [3.2.1 ]]Oct-6-en-3-ylcarbonate (1.9g, 6.8mmol, tert-butoxycarbonyl-ONb) and the reaction was allowed to slowly warm to room temperature and stirred overnight. Adding tert-butyl-2, 4-dioxo-3-azabicyclo [3.2.1 ]]Oct-6-en-3-yl carbonate (500mg,. about.1.7 mmol), and the solution was stirred for four hours. Adding another part of tert-butyl-2, 4-dioxo-3-azabicyclo [3.2.1 ]]Oct-6-en-3-ylcarbonate (500mg) and The reaction was stirred for another four hours. The reaction was then concentrated to an oil, which was partitioned between concentrated ammonium hydroxide (ca. 12M) and ethyl acetate and the phases separated. The ethyl acetate phase was washed once with concentrated ammonium hydroxide and water, respectively, and then twice with 20% saturated 5% aqueous acetic acid containing NaCl. Then, the ethyl acetate phase was diluted with 20% by volume of n-hexane and extracted with 5% aqueous acetic acid. The final acetic acid phase was basified to pH > 11 with 6m naoh and extracted once with fresh ethyl acetate. The final ethyl acetate phase was washed once with brine over Na₂SO₄Dry, filter and concentrate to an oil. The oil was dissolved in ethyl acetate (16mL) and added dropwise to diethyl ether (200mL) to precipitate the product. The solid was isolated by filtration and washed with ether to give 6 ', 2' -di-p-nitrobenzyloxycarbonyl-3-tert-butoxycarbonyl-sisomicin (3.82g, 62% yield) as a white solid. MS M/e [ M + H ]]⁺906.4 is calculated to obtain 906.3.

6 ', 2' -di-p-nitrobenzyloxycarbonyl-3-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of 6 ', 2' -di-p-nitrobenzyloxycarbonyl-3-tert-butoxycarbonyl-sisomicin (10.0g, 11.0mmol) in DMF (100mL) was added N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyric acid (3.15g, 14.4mmol) and the reaction was cooled to-40 ℃ and stirred for 30 min. Then PyBOP (6.9g, 13.2mmol) followed by DIPEA (7.7mL, 40.4mmol) was added and the reaction stirred at-40 ℃ for 3 h. The reaction was diluted with EtOAc (200mL) and washed with water (2X 100 mL). The aqueous layer was separated and extracted with EtOAc (100 mL). In Na ₂SO₄The combined organic layers were dried, filtered and concentrated to give 6 ', 2' -di-p-nitrobenzyloxycarbonyl-3-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin as an orange-yellow solid (67% HPLC purity), which was purifiedProceed to the next step without further purification.

6 ', 2 ' -di-p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of 6 ', 2' -di-p-nitrobenzyloxycarbonyl-3-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (11.0mmol) in THF (100mL) was added N-methylmorpholine (2.44mL, 22.1mmol) followed by tert-butoxycarbonyl-anhydride (4.82g, 22.1mmol) at 0 deg.C and the reaction mixture was stirred for 18 h. The reaction mixture was concentrated to dryness to give a crude product, which was purified by flash chromatography (silica gel/dichloromethane: methanol 0-7%) to give the target 6 ', 2' -di-p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (10.47g, 9.46mmol, 86.0% yield, 85% analytical HPLC purity): MSm/e [ M + Na ] ]⁺1229.5 is calculated, 1229.4 is obtained.

3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred mixture of 6 ', 2' -di-p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (10.5g, 8.71mmol) in EtOH (100mL) and H₂O (50mL) solution was added 1M NaOH (34.8mL, 34.8mmol) followed by Na₂S₂O₄(12.1g, 69.6mmol) and the reaction mixture was heated at 70 ℃ for 18 h. After cooling, a precipitate formed which was removed by filtration and washed with MeOH(25mL) washing. Removal of organic solvent by rotary evaporation, followed by addition of H₂O (100mL) and acetic acid (200mL) to obtain an acidic solution (pH 4), which was washed with EtOAc (2X 100 mL). Then, with concentrated NH₄The aqueous layer was basified to pH 12 with OH (20mL), salted with NaCl (6.0g) and extracted with EtOAc (2 × 200 mL). In Na₂SO₄The combined organic layers were dried, filtered and concentrated to give the target 3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (4.78g, 5.45mmol, 62.6% yield, MSm/e [ M + H ] M]⁺849.5 is calculated, 849.3, [ M + Na ] is obtained]⁺871.3) which was carried on to the next step without further purification.

6 '-para-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of 3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (4.78g, 5.45mmol) in Me0H (75mL) was added DIPEA (0.95mL, 5.45mmol) followed by (N-hydroxy-5-norbornene-2, 3-dicarboxy-imino) -4-nitro-benzyl carbonate (HONB-p-nitrobenzyloxycarbonyl, 1.75g, 4.90mmol) and the reaction mixture was stirred for 1 hour. Evaporation of the solvent gave an oily residue which was dissolved in EtOAc (100mL) with H₂O (2X 100mL) and Et₂O (75mL) and n-hexane (50 mL). Then, the organic layer was extracted with 5% aqueous AcOH (100mL) and the aqueous layer was separated, salted with NaCl (3.0g) and extracted with EtOAc (3X 100 mL). In Na₂SO₄The combined organic layers were dried, filtered and concentrated to give the target 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (3.08g, 3.32mmol, 60.9% yield; MS M/e [ M + H ])]⁺Calculating 1028.5 to obtain 1028.3; HPLC purity90.0%) which was carried on to the next step without further purification.

Example 1

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Treatment of 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.10g, 0.105mmol) with tert-butyldimethylsilyloxyacetaldehyde according to step 1-method A to give 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3" -tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin target (MSm/e [ M + H ] -)]⁺Calculation 1107.6, found 1107.4), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (2-tert-Butyldimethylsilyloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.105mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (1-105 mmol) Somicin: MSm/e [ M + H]⁺593.3 is calculated, 593.2, [ M + Na ] is obtained]⁺615.3, respectively; the purity of CLND was 97.5%.

Example 2

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6 '- (2-hydroxy-ethyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

To a stirred solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin (0.075g, 0.063mmol) in DMF (2mL) was added glycolaldehyde dimer (0.015g, 0.125mmol) and the reaction mixture was stirred for 6 hours. Then adding NaCNBH₃(0.070g, 1.11mmol) and AcOH (0.145mL) in MeOH (6mL) and the reaction mixture stirred for an additional 5 minutes. The reaction was diluted with EtOAc (10mL) and washed with H₂O (10mL) over MgSO₄Dried on top, filtered and concentrated to dryness to give the target 6 '- (2-hydroxy-ethyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (MSm/e [ M + H + H)]⁺Calculation 1230.5, found 1230.3), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butyloxycarbonyl-sisomicin

6 ' - (2-hydroxy-ethyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.063mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to give the crude product, which was purified by method 2-column A to give 6 ' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (0.016g, 0.023mmol, 36.5% yield).

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.016g, 0.023mmol) was treated with 90% trifluoroacetic acid (0.5mL) for 25 min. By addition of H₂The reaction was quenched by O (5mL) and the aqueous layer was lyophilized to give the crude product, which was purified by method 1-column A to give the target 6' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺593.3 is calculated, 5933.2, [ M + Na ] is obtained]⁺615.4, respectively; CLND: purity 98.2%).

Example 3

6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

To a stirred solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin (0.075g, 0.063mmol) in DMF (2mL) was added glyceraldehyde dimer (0.023g, 0.126mmol) and the reaction mixture was stirred for 6 hours. Then adding NaCNBH₃(0.070g, 1.11mmol) and AcOH (0.145mL) in MeOH (6mL) and the reaction mixture stirred for an additional 5 minutes. The reaction was diluted with EtOAc (10mL) and washed with H₂O (10mL) over MgSO₄Dried, filtered and concentrated to dryness to give the target 6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H)]⁺Computing]260.5, 1260.3) was obtained and was carried on to the next step without further purification.

6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butyloxycarbonyl-sisomicin

6 ' - (2-hydroxy-propanol) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.063mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield the crude product, which was purified by method 2-column A to yield 6 ' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (0.016g, 0.022mmol, 34.9%) as a crude product: MS M/e [ M + H ] ]⁺723.4 is calculated, 723.3, [ M + Na ] is obtained]⁺745.4。

6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.016g, 0.022mmol) was treated with 90% aqueous trifluoroacetic acid (0.5mL) for 25 minutes. By addition of H₂O (5mL) quench the reaction and lyophilize the aqueous layer to yield the crude product, which is purified by method 1-column A to yield the target 6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺623.3 is calculated, 623.3, [ M + Na ] is obtained]⁺645.4, respectively; CLND: purity 99.0%).

Example 4

6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl sisomicin

To a stirred solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin (0.100g, 0.084mmol) in DMF (2mL) was added N-tert-butoxycarbonyl-piperidine-4-carbaldehyde (0.036g, 0.168mmol) and the reaction mixture was stirred for 6 hours. Then adding NaCNBH ₃(0.070g, 1.11mmol) and AcOH (0.145mL) in MeOH (6mL) and the reaction mixture stirred for an additional 5 minutes. The reaction was diluted with EtOAc (10mL) and washed with H₂O (10mL) over MgSO₄Dried, filtered and concentrated to dryness to give the crude product, which is purified by method 2-column a to give the target 6 '- (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitro) carbaldehydeBenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.037g, 0.027mmol, yield 32.1%): MS M/e [ M + H ]]⁺1383.6 is calculated, 1383.4 is obtained.

6' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.037g, 0.027mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield the crude product, which was purified by method 2-column A to yield 6 ' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (0.005g, 0.006mmol, 22.2% yield): MS M/e [ M + H ] ]⁺846.5 is calculated, 846.4, [ M + Na ] is obtained]⁺868.5。

6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.015g, 0.018mmol) was treated with 90% aqueous trifluoroacetic acid (0.5mL) for 25 minutes. By addition of H₂O (5mL) quench the reaction and lyophilize the aqueous layer to yield the crude product, which is purified by method 1-column A to yield the target 6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺646.4 is calculated, 646.3, [ M + Na ] is obtained]⁺668.4；CLND: the purity was 99.2%.

Example 5

6' - (methyl-cyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6 '- (methyl-cyclopropyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

To a stirred solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin (0.100g, 0.084mmol) in DMF (2mL) was added cyclopropanecarboxaldehyde (0.012mL, 0.168mmol) and the reaction mixture was stirred for 6 hours. Then adding NaCNBH ₃(0.070g, 1.11mmol) and AcOH (0.145mL) in MeOH (6mL) and the reaction mixture stirred for an additional 5 minutes. The reaction was diluted with EtOAc (10mL) and washed with H₂O (10mL) extraction over MgSO₄Dried on top, filtered and concentrated to dryness to give the target 6 '- (methylcyclopropyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (MSm/e [ M + H + H)]⁺Calculation 1240.5, found 1240.4), which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

Reacting 6 '- (methyl-cyclopropyl) -2', 3-di-p-nitroBenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.084mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield 6' - (methylcyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺703.4 is calculated, 703.3, [ M + Na ] is obtained]⁺725.4) which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6' - (methyl-cyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.084mmol) was treated with 90% aqueous trifluoroacetic acid (0.5mL) for 25 minutes. By addition of H₂The reaction was quenched O (5mL) and the aqueous layer was lyophilized to give the crude product, which was purified by method 1-column a to give the target 6' - (methyl-cyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin (0.0014g, 0.0023mmol, 2.7% yield): MSm/e [ M + H]⁺603.4 was calculated, and 603.2, [ M + Na ] was obtained]⁺625.4, respectively; CLND: the purity was 98.3%.

Example 6

6' - (3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

Boc-3-amino-propionaldehyde

To a stirred solution of 3- (tert-butoxycarbonyl-amino) -1-propanol (25mL, 0.144mol) in saturated DCM (1).0L) of aqueous solution Dess-Martin reagent (99.2g, 233.9mmol) was added and the reaction mixture was stirred for 1 hour. The reaction was then diluted with diethyl ether (1.0L), followed by Na₂S₂O₃(250g) 80% NaHCO₃(450g of 1.0L of H₂O) solution. The reaction was stirred vigorously for 30 minutes until two layers formed, the upper layer being clear. The reaction was filtered to remove precipitated solids and the aqueous layer was extracted with diethyl ether (1.0L). With saturated NaHCO₃(1.0L)、H₂The organic layer was washed with O (1.0L) and brine (1L) over Na ₂SO₄Dried and concentrated to a clear oil. The crude oil was dissolved in EtOAc: N-hexane (1: 1v/v, 1.0L) and filtered through a short silica gel column to give the target N-tert-butoxycarbonyl-3-amino-propionaldehyde (21.7g, 0.125mol, 85.6% yield):¹HNMR(400MHz，CDCl₃)δ9.77(s，1H，CHO)，4.85(bs，1H，NH)，3.36-3.42(m，2H，CH₂)，2.67(t，2H，CH₂)，1.39(s，9H，(CH₃)₃)。

6 '- (N-Boc-3-amino-propyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-Boc-sisomicin

To a stirred solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin (0.150g, 0.126mmol) in DMF (2mL) was added N-tert-butoxycarbonyl-propionaldehyde (0.043g, 0.252mmol) and the reaction mixture was stirred for 6 hours. Then adding NaCNBH₃(0.070g, 1.11mmol) and AcOH (0.145mL) in MeOH (6mL) and the reaction mixture stirred for an additional 5 minutes. The reaction was diluted with EtOAc (10mL) and washed with H₂O (10mL) over MgSO₄Dried, filtered and concentrated to dryness to give the target 6 '- (N-tert-butoxycarbonyl-3-amino-propyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxy)Carbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (MSm/e [ M + H) ]⁺Calculation 1343.5, found 1343.4), which was carried on to the next step without further purification.

6' - (N-Boc-3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-Boc-sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-amino-propyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.126mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to give 6 ' - (N-tert-butoxycarbonyl-3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (MSm/e [ M + H ])]⁺806.5 is calculated, 806.4, [ M + Na ] is obtained]⁺828.4) which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin

6' - (N-tert-Butoxycarbonyl-3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.126mmol) was treated with 90% trifluoroacetic acid (0.5mL) in water for 25 min. By addition of H₂O (5mL) quench the reaction and lyophilize the aqueous layer to yield the crude product, which is purified by method 1-column A to yield the target 6' - (3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H) ]⁺Calculating 606.4 to obtain 606.3; CLND: purity 99.4%).

Example 7

6' -methyl-cyclopropyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 ' -methyl-cyclopropyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 ' -tert-butyloxycarbonyl sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was treated with cyclopropanecarboxaldehyde to give the target 6 ' -methylcyclopropyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin, which was taken to the next step without further purification.

6 '-methyl-cyclopropyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3' -tert-butyloxycarbonyl sisomicin

Crude 6 ' -methylcyclopropyl-2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonylsisomicin (0.078mmol) was subjected to step 10 to give 6 ' -methylcyclopropyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonylsisomicin, which was subjected to the next step without further purification.

6' -methyl-cyclopropyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '-methyl-cyclopropyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl sisomicin (0.078mmol) was subjected to step 3-method B to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield the target 6' -methylcyclopropyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin: MS M/e [ M + H ]]⁺Calculating 589.3 to obtain 589.3; the purity of CLND was 99.5%.

Example 8

6' -methyl-piperidinyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidinyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.055mmol) was treated with N-tert-butoxycarbonyl-piperidine-4-carbaldehyde to give the corresponding 6 ' - (methyl-N-tert-butoxycarbonyl-piperidinyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonyl sisomicin, which was taken to the next step without further purification.

6' - (methyl-N-tert-Butoxycarbonyl-piperidinyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-piperidinyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonylsisomicin (0.055mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (methyl-N-tert-butoxycarbonyl-piperidinyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonylsisomicin, which was subjected to the next step without further purification.

6' -methyl-piperidinyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidinyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl sisomicin (0.055mmol) was subjected to step 3-method B to give the crude product, which was purified by reverse phase HPLC method 1-column A to give the target 6' -methylpiperidinyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin: MS M/e [ M + H ]]⁺Calculating 632.4 to obtain 632.4; the purity of CLND was 99.0%.

Example 9

6' - (2-hydroxy-ethyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-ethyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "tert-butyloxycarbonyl sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.055mmol) was treated with glycolaldehyde dimer and AcOH (0.005ml) to give the target 6 ' - (2-hydroxy-ethyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin, which was taken to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin

6 ' - (2-hydroxy-ethyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonylsisomicin (0.055mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (2-hydroxy-ethyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonylsisomicin (MS M/e [ M + H + C/])]⁺Calculation 779.4, found 779.4), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-ethyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonylsisomicin (0.055mmol) was subjected to step 3-method B to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 6'- (2-hydroxy-ethyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin: MS M/e [ M + H ]]⁺Calculating 579.3 to obtain 579.3; the purity of CLND was 99.0%.

Example 10

6' - (2-hydroxy-propanol) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was treated with glyceraldehyde dimer and AcOH (0.005ml) to give the corresponding 6 ' - (2-hydroxy-propanol) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonyl sisomicin, which was taken to the next step without further purification.

6 '- (2-hydroxy-propanol) -1- (3-amino-2 (R) -hydroxy-propionyl) -3' -tert-butyloxycarbonyl sisomicin

6 ' - (2-hydroxy-propanol) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonylsisomicin (0.078mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to give 6 ' - (2-hydroxy-propanol) -1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3"-tert-Butoxycarbonyloximecin (MS M/e [ M + H)]⁺Calculation 809.4, found 809.4), which was carried on to the next step without further purification.

6' - (2-hydroxy-propanol) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-propanol) -1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl sisomicin (0.078mmol) was subjected to step 3-method B to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield the target 6' - (2-hydroxy-propanol) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin: MS M/e [ M + H ]]⁺609.3 is calculated to obtain 609.2, [ M + Na ]]⁺631.2, respectively; the purity of CLND was 98.2%.

Example 11

6' - (3-amino-propyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 ' - (N-Boc-3-aminopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-Boc-3-amino-2 (R) -hydroxy-propionyl) -3 ' -Boc-sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was treated with N-tert-butoxycarbonyl-3-amino-propionaldehyde to give the corresponding 6 ' - (N-tert-butoxycarbonyl-3-amino-propyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butoxycarbonyl sisomicin, which was subjected to the next step without further purification.

6 '- (N-Boc-3-aminopropyl) -1- (N-Boc-3-amino-2 (R) -hydroxy-propionyl) -3' -Boc sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-aminopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin (0.078mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to give 6 ' - (N-tert-butyloxycarbonyl-3-aminopropyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3" -tert-butyloxycarbonyl sisomicin (MSm/e [ M + H + ]) ]⁺Calculation 892.5, found 892.3), which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin

6 '- (N-tert-Butoxycarbonyl-3-amino-propyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (R) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin (0.078mmol) was subjected to step 3-method B and purified by reverse phase HPLC method 1-column A to yield the target 6' - (3-aminopropyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin: MS M/e [ M + H ]]⁺593.4 is calculated, 593.3, [ M + Na ] is obtained]⁺614.3, respectively; the purity of CLND was 92.8%.

Example 12

6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.17mmol) was treated with N-tert-butoxycarbonyl-piperidine-4-carbaldehyde to give the corresponding 6 ' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3" -tert-butoxycarbonyl sisomicin, which was subjected to the next step without further purification.

6' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

Subjecting 6 ' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.17mmol) to step 10 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin: MS M/e [ M + H ]]⁺846.5 is calculated, 846.4 is obtained.

6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.17mmol) was subjected to step 3-method B to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield the target 6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ]]⁺646.4 is calculated, 646.3, [ M + Na ] is obtained]⁺668.4; the purity of CLND was 97.8%.

Example 13

6' - (methyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (methyl-cyclopropyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was treated with cyclopropanecarboxaldehyde to give the target 6 ' - (methyl-cyclopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MSm/e [ M + H ] M]⁺Calculation 1147.5, found 1147.4), which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin

Step 2 was performed with 6 ' - (methyl-cyclopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) to give 6 ' - (methyl-cyclopropyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ] /)]⁺789.4 is calculated, 789.4, [ M + Na ] is obtained ]⁺811.3) which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (methyl-cyclopropyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was subjected to step 3-method B to give the crude product, which was purified by reverse phase HPLC method 1-column A to give the target 6' - (methyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0008g, 0.0014mmol, 1.8% yield): MS M/e [ M + H ]]⁺Calculating 589.3, obtaining 589.3, [ M + Na ]]⁺611.4, respectively; the purity of CLND was 98.9%.

Example 14

6' - (2-hydroxy-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was treated with glyceraldehyde dimer and AcOH (0.005ml) to give the corresponding 6 ' - (2-hydroxy-propanol) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MSm/e [ M + H ] /) ]⁺1167.5 is calculated, 1167.3, [ M + Na ] is obtained]⁺1189.4) which was carried on to the next step without further purification.

6 '- (2-hydroxy-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butyloxycarbonyl-sisomicin

6 ' - (2-hydroxy-propanol) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to give 6 ' - (2-hydroxy-propanol) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺809.4 is calculated, 809.3, [ M + Na ] is obtained]⁺831.3) which was carried on to the next step without further purification.

6' - (2-hydroxy-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Reacting 6' - (2-hydroxy-propanol) -1- (C)N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.078mmol) was subjected to step 3-method B to give the crude product, which was purified by reverse phase HPLC method 1-column a to give the target 6' - (2-hydroxy-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.00137g, 0.0022mmol, yield 2.8%): MS M/e [ M + H ] ]⁺609.3 is calculated, 609.3, [ M + Na ] is obtained]⁺631.4, respectively; the purity of CLND was 97.9%.

Example 15

6' - (methyl-piperidin-4-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N-tert-Butoxycarbonyl-piperidin-4-yl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' tert-butyloxycarbonyl-sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.082mmol) was treated with N-tert-butoxycarbonyl-piperidine-4-carbaldehyde followed by purification by reverse phase HPLC method 2-column a to give the corresponding 6 ' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (0.021g, 0.017mmol, 20.7%): MSm/e [ M + H]⁺1290.6 is calculated, 1290.3, [ M + Na ] is obtained]⁺1312.5)。

6' - (methyl-N-tert-Butoxycarbonyl-piperidin-4-yl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.021g, 0.017mmol) was performed to remove the p-nitrobenzyloxycarbonyl group to give 6' - (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H). ]⁺932.5 is calculated, 932.4, [ M + Na ] is obtained]⁺954.5) which was carried on to the next step without further purification.

6' - (methyl-piperidin-4-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-piperidin-4-yl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.017mmol) was subjected to step 3-method B to give the crude product, which was purified by reverse phase HPLC method 1-column A to give the target 6' - (methyl-piperidin-4-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.003g, 0.0047mmol, 27.6% yield): MS M/e [ M + H ]]⁺632.4 is calculated, 632.3, [ M + Na ] is obtained]⁺654.4; the purity of CLND was 96.9%.

Example 16

6' - (2-hydroxy-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-ethyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.5g, 0.41mmol) was treated with glycolaldehyde dimer and AcOH (0.005ml) to give 6 ' - (2-hydroxy-ethyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + Na ]/] ]⁺Calculation 1159.5, found 1159.4), which was carried on to the next step without further purification.

6 '- (2-hydroxy-ethyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butoxycarbonyl-sisomicin

Subjecting the crude mixture of 6 ' - (2-hydroxy-ethyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (2-hydroxy-ethyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MSm/e [ M + H ]/]]⁺779.4 was calculated to find 779.3), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Reacting 6' - (2-hydroxy-ethyl) -1- (N-tert-butoxy)The crude mixture of carbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin was subjected to step 3-method B to give the crude product, which was purified by reverse phase HPLC method 1-column a to give 6' - (2-hydroxy-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0142g, 0.0245mmol, 5.9% yield): MS M/e [ M + H ] ]⁺Calculation 579.3 to obtain 579.2, [ M + Na ]]⁺601.3; the purity of CLND was 94.5%.

Example 17

6' - (3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 '' -tert-butyloxycarbonyl-sisomicin

To a solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butoxycarbonyl-sisomicin (0.176g, 0.15mmol) in DMF (2mL) was added 3-phthalimido-propionaldehyde (0.06g, 0.29mmol) andmolecular sieves (15-20) and the reaction was shaken for 2 hours. Then adding NaCNBH₃(0.018g, 0.29mmol) in MeOH (4mL) and the reaction stirred overnight. The reaction was diluted with EtOAc (5mL) and saturated NaHCO₃The organic layer was washed with aqueous solution (3mL) and brine (3mL) over Na₂SO₄Dried, filtered and concentrated to give 6 ' - (N-phthalimido-3-aminopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺Computing1280.5, found 1280.3), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.15mmol) was subjected to step 6 for removal of phthalimido to give 6 '- (3-amino-propyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ] M]⁺Calculation 1150.5, found 1150.4), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butyloxycarbonyl-sisomicin

6 ' - (3-amino-propyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.15mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (3-amino-propyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ]) ]⁺Calculation 792.5, found 792.4), which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (3-amino-propyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.15mmol) was subjected to step 3-method B to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield the target 6' - (3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0021g, 0.0034mmol, 2.3% yield): MSm/e [ M + H]⁺592.4 is calculated, 592.2, [ M + Na ] is obtained]⁺614.3, respectively; the purity of CLND was 91.6%.

Example 18

6' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl-cyclopropyl) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

Following step 1, method B, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.084mmol) was treated with cyclopropanecarboxaldehyde to give the target 6 ' - (methyl-cyclopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ]/] ]⁺1240.5 is calculated, 1240.4, [ M + Na ] is obtained]⁺1262.4) which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

6 ' - (methyl-cyclopropyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.084mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺703.4 is calculated, 703.3, [ M + Na ] is obtained]⁺725.4) which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.084mmol) was treated with 90% aqueous trifluoroacetic acid (0.5mL) for 25 minutes. By addition of H₂The reaction was quenched by O (5mL) and the aqueous layer was lyophilized to give the crude product, which was purified by method 1-column a to give the target 6' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H) ]⁺603.4 was calculated, and 603.2, [ M + Na ] was obtained]⁺625.4, respectively; CLND purity 98.3%).

Example 19

6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin

To a stirred solution of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (R) -hydroxy-butyryl) -3' -tert-butoxycarbonyl-sisomicin trifluoroacetate (0.110g, 0.085mmol) in DMF (1mL) was added DIPEA (0.019mL, 0.11mmol) followed by glyceraldehyde dimer (0.032g, 0.17mmol) and the reaction mixture was stirred for 6 hours. Then adding NaCNBH₃(0.070g, 1.11mmol) and AcOH (0.145mL) in MeOH (6mL) and the reaction mixture stirred for an additional 5 minutes. The reaction was diluted with EtOAc (10mL) and washed with H₂O (10mL) extraction over MgSO₄Dried, filtered and concentrated to dryness to give the target 6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin, which was carried on to the next step without further purification. MS M/e [ M + H ] ]⁺1260.5 is calculated, 1260.3 is obtained.

6' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butyloxycarbonyl-sisomicin

6 ' - (2-hydroxy-propanol) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-p-nitrobenzyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.085mmol) was subjected to step 10 for removal of p-nitrobenzyloxycarbonyl to yield the crude product, which was purified by method 2-column A to yield 6 ' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -3" -tert-butyloxycarbonylButoxycarbonyl-sisomicin (0.009g, 0.011mmol, yield 13.4%). MS M/e [ M + H ]]⁺723.4 is calculated, 723.3 is obtained.

6' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -3 "-tert-butoxycarbonyl-sisomicin (0.009g, 0.011mmol) was treated with 90% aqueous trifluoroacetic acid (0.5mL) for 25 minutes. By addition of H₂O (5mL) quench reaction and lyophilize aqueous layer to give crude product, which is purified by method 1-column A to give target 6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ])]⁺623.3 is calculated, 623.3, [ M + Na ] is obtained ]⁺645.4, respectively; the purity of CLND was 96.6%.

Example 20

6' - (3-amino-2-hydroxy-propionyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-Boc-3-amino-2-hydroxy-propionyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-Boc-3-amino-2 (S) -hydroxy-propionyl) -3 ' -Boc-sisomicin

Following step 4, method A, 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin (0.078mmol) was treated with N-tert-butoxycarbonyl-3-amino-2-hydroxy-propionic acid to give the corresponding 6 ' - (N-tert-butoxycarbonyl)Carbonyl-3-amino-2-hydroxy-propionyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butoxycarbonyl sisomicin (MS M/e [ M + Na ]]⁺Calculation 1302.5, found 1302.4), which was carried on to the next step without further purification.

6' - (N-Boc-3-amino-2-hydroxy-propionyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-amino-2-hydroxy-propionyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin (0.078mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to give 6 ' - (N-tert-butyloxycarbonyl-3-amino-2-hydroxy-propionyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butyloxycarbonyl sisomicin (MS M/e [ M + H). ]⁺922.5 was calculated to obtain 922.3, [ M + Na ]]⁺944.4) which was carried on to the next step without further purification.

6' - (3-amino-2-hydroxy-propionyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (N-tert-Butoxycarbonyl-3-amino-2-hydroxy-propionyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl sisomicin (0.078mmol) was subjected to step 3-method B to give the crude product, which was purified by reverse phase HPLC method 1-column A to give the target 6' - (3-amino-2-hydroxy-propionyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0076g, 0.012mmol, 15.4% yield): MS m-e[M+H]⁺622.3 is calculated, and 622.3, [ M + Na ] is obtained]⁺644.4, respectively; the purity of CLND was 99.5%.

Example 21

6' - (2-hydroxy-3-propionamide) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (2-hydroxy-3-propionamide) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin

Treatment of 2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin (0.15mmol) with epoxypropionamide according to step 5 gave 6 ' - (2-hydroxy-3-propionamide) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 ' -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ] M ]⁺Calculation 1180.5, found 1180.8), which was carried on to the next step without further purification.

6' - (2-hydroxy-3-propionamide) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin

Subjecting the crude mixture of 6 ' - (2-hydroxy-3-propionamide) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 6 ' - (2-hydroxy-3-propionamide) -1- (N-tert-butoxycarbonyl-3-amino-2 (S)) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (MSm/e [ M + H)]⁺Calculation 822.4, found 822.3), which was carried on to the next step without further purification.

6' - (2-hydroxy-3-propionamide) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

The crude mixture of 6 '- (2-hydroxy-3-propionamide) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin was subjected to step 3-method B for removal of tert-butoxycarbonyl, followed by purification by reverse phase HPLC method 1-column a to give 6' - (2-hydroxy-3-propionamide) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0093g, 0.015mmol, 10% yield): MS M/e [ M + H ] ]⁺622.3 was calculated and 622.2, [ M + Na ] was obtained]⁺644.3; the purity of CLND was 96.2%.

Example 22

6' - (3-amino-2-hydroxy-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-Boc-3-amino-2-hydroxy-propyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-Boc-3-amino-2 (S) -hydroxy-propionyl) -3 ' -Boc-sisomicin

2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.15mmol) was treated with N-tert-butoxycarbonyl-oxiran-2-yl-methylamine to give the corresponding 6 ' according to step 5 '- (N-tert-Butoxycarbonyl-3-amino-2-hydroxy-propyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3' -tert-butyloxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺Calculation 1266.6, found 1266.7), which was carried on to the next step without further purification.

6 '- (N-Boc-3-amino-2-hydroxy-propyl) -1- (N-Boc-3-amino-2 (S) -hydroxy-propionyl) -3' -Boc-sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-amino-2-hydroxy-propyl) -2 ', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butoxycarbonyl-sisomicin (0.15mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to give 6 ' - (N-tert-butoxycarbonyl-3-amino-2-hydroxy-propyl) -1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3" -tert-butoxycarbonyl-sisomicin (MS M/e [ M + H). ]⁺Calculation 908.5, found 908.4), which was carried on to the next step without further purification.

6' - (3-amino-2-hydroxy-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (N-tert-Butoxycarbonyl-3-amino-2-hydroxy-propyl) -1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -3 "-tert-butyloxycarbonyl-sisomicin (0.15mmol) was subjected to step 3-method B for removal of the tert-butyloxycarbonyl group followed by purification by reverse phase HPLC method 1-column A to give 6' - (3-amino-2-hydroxy-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0044g, 0.0072mmol, 4.8% yield): MS M/e [ M + H ]]⁺Calculation 608.3, 608.2, [ M + Na ] was obtained]⁺630.3, respectively; the purity of CLND was 91%.

Example 23

6' - (2-hydroxy-propanol) -1- (2-hydroxy-acetyl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin

Following step 4, method B, 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.075g, 0.081mmol) was treated with glycolic acid to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ]) ]⁺Calculation 985.5, found 985.9), which was carried on to the next step without further purification.

2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 2 for removing p-nitrobenzyloxycarbonyl to give 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H +)]⁺Calculation 806.4, found 806.9), which was carried on to the next step without further purification.

6 '- (2-hydroxy-propanol) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin

Following step 1-method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with DL-glyceraldehyde to give the target 6 ' - (2-hydroxy-propanol) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 880.5, found 880.9), which was carried on to the next step without further purification.

6' - (2-hydroxy-propanol) -1- (2-hydroxy-acetyl) -sisomicin

6 ' - (2-hydroxy-propanol) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-hydroxy-propanol) -1- (2-hydroxy-acetyl) -sisomicin (0.0058g, 0.010mmol, 12.3% yield): MSm/e [ M + H ]⁺Calculating 580.3 to obtain 580.6; the purity of CLND was 89.3%.

Example 24

6' - (3-amino-propyl) -1- (2-hydroxy-acetyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with N-phthalimido-propionaldehyde to yield the target 6 ' - (N-phthalimido-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 993.5, found 993.9), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 6 for phthalimide deprotection to yield 6 '- (3-amino-propyl) -2', 3, 3" -tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H)]⁺Calculation 863.5, found 864.1), which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (2-hydroxy-acetyl) -sisomicin

6 ' - (3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (3-amino-propyl) -1- (2-hydroxy-acetyl) -sisomicin (0.0035g, 0.0062mmol, 7.6% yield): MS M/e [ M + H ]]⁺Computing563.3, obtaining 563.2; the purity of CLND was 88.9%.

Example 25

6' - (2-hydroxy-ethyl) -1- (2-hydroxy-acetyl) -sisomicin

6 '- (2-tert-Butyldimethylsiloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with tert-butyl-dimethylsiloxy-acetaldehyde to give target 6 ' - (2-tert-butyldimethylsiloxy-ethyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H)]⁺Calculation 964.6, found 964.9), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (2-hydroxy-acetyl) -sisomicin

6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-hydroxy-acetyl) -sisomicin (0.081mmoL)) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-hydroxy-ethyl) -1- (2-hydroxy-acetyl) -sisomicin (0.0152g, 0.028mmoL, 34.6%) as: MSm/e [ M + H]⁺Calculating 550.3 to obtain 550.5; the purity of CLND was 90.7%.

Example 26

6' - (3-amino-propyl) -1- (2-amino-ethylsulfonamide) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ', 3, 3 ' -tri-tert-butoxycarbonyl-1- (N-phthalimido-2-amino-ethylsulfonamide) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.075g, 0.081mmol) was treated with N-phthalimido-ethanesulfonyl chloride to give the target 6 '-para-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-phthalimido-2-amino-ethylsulfonamide) -sisomicin (MSm/e [ M + H ] M]⁺Calculation 1164.5, found 1164.6), which was carried on to the next step without further purification.

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-amino-ethylsulfonamide) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-phthalimido-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 6 for phthalimido deprotection to give 6 '-para-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (2-amino-ethylsulfonamide) -sisomicin (MSm/e [ M + H ])]⁺Calculation 1034.5, found 1035.2), which was carried on to the next step without further purification.

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin

6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 13 for N-tert-butoxycarbonyl protection to give 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (MSm/e [ M + H ])]⁺Calculation 1134.5, found 1135.0), which was carried on to the next step without further purification.

2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-Butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to give 2 ', 3, 3" -Tri-tert-Butoxycarbonyl-1- (N-tert-Butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 955.5, found 956.2), which was carried on to the next step without further purification.

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was treated with N-phthalimido-propionaldehyde to yield the target 6 ' - (N-phthalimido-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1142.6, found 1143.5), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 6 for phthalimido deprotection to give 6 '- (3-amino-propyl) -2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (MSm/e [ M + H ] M]⁺Calculate 1012.5, find 1012.9), which is carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (2-amino-ethylsulfonamide) -sisomicin

6 ' - (3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 3-method A for removing the tert-butoxycarbonyl group to give a crude product, which was purified by reverse phase HPLC method 3 to give 6 ' - (3-amino-propyl) -1- (2-amino-ethylsulfonamide) -sisomicin (0.0029g, 0.0047mmol, 5.8% yield): MS M/e [ M + H ]]⁺612.3 is calculated, and 612.4 is obtained; the purity of CLND was 84.7%.

Example 27

6' - (2-hydroxy-propanol) -1- (2-amino-ethylsulfonamide) -sisomicin

6 '- (2-hydroxy-propanol) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081) was treated with DL-glyceraldehyde to give the target 6 ' - (2-hydroxy-propanol) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1029.5, found 1030.0), which was carried on to the next step without further purification.

6' - (2-hydroxy-propanol) -1- (2-amino-ethylsulfonamide) -sisomicin

6 ' - (2-hydroxy-propanol) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-hydroxy-propanol) -1- (2-amino-ethylsulfonamide) -sisomicin (0.0031g, 0.0049mmol, 6.0% yield): MSm/e [ M +H]⁺Calculating 629.3 to obtain 629.2; the purity of CLND was 88.2%.

Example 28

6' - (2(S) -hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl- (S) -1- (2, 2-dimethyl-1, 3-dioxolan-4-yl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.078mmol) was treated with (R) -2, 2-dimethyl-1, 3-dioxolane-4-carbaldehyde to give the corresponding 6 ' - (methyl- (S) -1- (2, 2-dimethyl-1, 3-dioxolan-4-yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1063.6, found 1063.4), which was carried on to the next step without further purification.

6' - (2(S) -hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Step 3-method B was performed on 6 ' - (2(S) -hydroxy-propanol) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.078mmol) to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield the target 6 ' - (2(S) -hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (l-B)Somicin: MSm/e [ M + H]⁺623.3 is calculated, 623.4, [ M + Na ] is obtained]⁺645.3; the purity of CLND was 97.9%.

Example 29

6' - (2-hydroxy-ethyl) -1- (2-amino-ethylsulfonamide) -sisomicin

6 '- (2-tert-Butyldimethylsiloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-2-amino-ethylsulfonamide) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081) was treated with tert-butyldimethylsiloxyacetal to give target 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3" -tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (MSm/e [ M + H ] M]⁺Calculation 1113.6, found 1114.2), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (2-amino-ethylsulfonamide) -sisomicin

6 ' - (2-tert-Butyldimethylsiloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-amino-ethylsulfonamide) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-hydroxy-ethyl) -1- (2-amino-ethylsulfonamide)Sisomicin (0.00l9g, 0.0032mmol, 3.9% yield): MS M/e [ M + H ]]⁺Calculating 599.3 to obtain 599.2; the purity of CLND was 90.5%.

Example 30

6' - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (N-Boc-2, 2-dimethyl-1, 3-oxazolidine-methyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.079mmol) was treated with N-tert-butoxycarbonyl-4-formyl-2, 2-dimethyl-1, 3-oxazolidine to give target 6 ' - (N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidine-methyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1162.7, found 1163.1), which was carried on to the next step without further purification.

6, - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 6 '- (N-tert-Butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidine-methyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) to a step for removing tert-butoxycarbonylStep 3-method a to give the crude product, which was purified by reverse phase HPLC method 3 to give 6' - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0082g, 0.013mmol, 16.4% yield): MS M/e [ M + H ] ]⁺Calculating 622.4 to obtain 622.6; the purity of CLND was 75.5%.

Example 31

6' - (4-hydroxy-piperidin-4-yl) -methyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

N-Boc-1-oxa-6-azaspiro [2.5] octane

4-methylene-piperidine (0.222g, 1.12mmol) was subjected to step 14 to form the target N-tert-butyloxycarbonyl-1-oxa-6-azaspiro [2.5]]Octane (0.215g, 1.01mmol, 90.2% yield):¹H NMR(250MHz，DMS0-d₆)δ3.29-3.61(m，6H)，1.56-1.70(m，2H)，1.30-1.54(m，11H)。

6 '- (4-hydroxy-N-tert-butoxycarbonyl-piperidin-4-yl) -methyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

According to step 5, N-tert-butyloxycarbonyl-1-oxa-6-azaspiro [2.5]]Octane treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.079mmol) to give the target 6 ' - (4-hydroxy-N-tert-butoxycarbonyl-piperidin-4-yl) -methyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2(S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺Calculation 1162.7, found 1163.2), which was carried on to the next step without further purification.

6' - (4-hydroxy-piperidin-4-yl) -methyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (4-hydroxy-N-tert-butoxycarbonyl-piperidin-4-yl) -methyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (4-hydroxy-piperidin-4-yl) -methyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0023g, 0.0035mmol, 4.4%) as a crude product: MSm/e [ M + H]⁺Calculating 662.4 to obtain 662.8; the purity of CLND was 94.5%.

Example 32

6' - (2-hydroxy-5-amino-pentyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2- (pent-4-enyl) -isoindoline-1, 3-dione

To a stirred solution of 5-bromo-pentene (6.0g, 0.040mol) in DMF (30mL) was added K₂CO₃(4.7g, 0.034mol) and potassium phthalimide (6.21g, 0.033mmol) and heating the reaction mixture at 100 ℃ for 1 hour. The reaction mixture was cooled to room temperature and water (50mL) was added. The aqueous layer was then extracted with ethyl acetate (2X 50mL) and 5% NaHCO₃The combined organic layers were washed with (2X 20mL) aqueous solution, brine (30mL) and washed with Na₂SO₄And drying. Filtration and evaporation of the solvent gave an oil which was purified by flash chromatography (silica gel/n-hexane: ethyl acetate 0-35%) to give the target 2- (pent-4-enyl) -isoindoline-1, 3-dione as a solid (6.36g, 0.029mmol, 72.5% yield): MS M/e [ M + H ] ]⁺Calculating 216.1 to obtain 216.1; NMR (250MHz, DMSO-d)₆)δ7.79-7.95(m，4H)，5.70-5.91(m，1H)，4.90-5.11(m，2H)，3.58(t，2H)，1.98-2.10(m，2H)，1.59-1.78(m，2H)。

2- (3- (Oxiran-2-yl) -propyl) -isoindoline-1, 3-dione

2- (pent-4-enyl) -isoindoline-1, 3-dione (6.36g, 0.029mmol) is subjected to step 14 for epoxide formation to give 2- (3- (oxiran-2-yl) -propyl-isoindoline-1, 3-dione (5.8g, 0.025mmol, 86.2% yield) MS M/e [ M + H ])]⁺Calculating 232.1 to obtain 232.1;¹H NMR(250MHz，DMSO-d₆)δ7.75-7.90(m，4H，Ar)，3.52(t，2H，CH₂)，2.87-2.96(m，1H，CH)，2.70(t，1H)，2.30-2.45(m，1H)，1.36-1.80(m，4H)。

6 '- (N-phthalimido-2-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-2', 3" -yl-2- (3-oxido-2-yl) propyl) -isoindoline-1, 3-dione was treated according to step 5-butyryl) -sisomicin (0.075g, 0.079mmol) to yield target 6 '- (N-phthalimido-2-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 1180.6, found 1181.1), which was carried on to the next step without further purification.

6 '- (2-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (N-phthalimido-2-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 6 for removal of phthalimido to yield 6 '- (2-hydroxy-5-amino-pentyl) -2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] -)]⁺Calculation 1050.6 to obtain 1051.3), which was carried on to the next step without further purification.

6' - (2-hydroxy-5-amino-pentyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (2-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 3 to yield 6 '- (2-hydroxy-5-amino-pentyl) -l- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0024g, 0.0037mmol, yielding 6' - (2-hydroxy-5-amino-pentyl) -l- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0024g, 0.0037mmol, yield)The rate is 4.7%): MS M/e [ M + H ]]⁺Calculating 650.4 to obtain 650.8; the purity of CLND was 95.3%.

Example 33

6' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (1.0g, 1.05mmol) was treated with trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl-carbaldehyde to give target 6 ' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 1132.6, found 1133.0), which was carried on to the next step without further purification.

6' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (1.05mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product byReverse phase HPLC method 1-column B purified it to give 6' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.110g, 0.174mmol, 16.6% yield): MS M/e [ M + H ] ]⁺632.4 is calculated, 632.8 is obtained; the purity of CLND was 96.1%.

Example 34

6' - (2-hydroxy-ethyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Boc-3-hydroxypyrrolidine-3-carboxylic acid

N-tert-Butoxycarbonyl-3-pyrrolidone (0.010mmol) was subjected to step 15 to yield the target N-tert-butyloxycarbonyl-3-hydroxy-pyrrolidine-3-carboxylic acid.

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Following step 4, method B, 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.075g, 0.081mmol) was treated with N-tert-butoxycarbonyl-3-hydroxy-pyrrolidine-3-carboxylic acid to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1140.6, found 1141.4), which was carried on to the next step without further purification.

2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to yield 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ]) ]⁺Calculation 961.5, found 961.8), which was carried on to the next step without further purification.

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was treated with tert-butyldimethylsiloxyacetal to give target 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3" -tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1119.6, found 1119.9), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-hydroxy-ethyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.008g, 0.013mmol, 16.0% yield): MS M/e [ M + H ] ]⁺Calculating 605.3 to obtain 605.8; the purity of CLND was 92.2%.

Example 35

6' - (2-hydroxy-4-amino-butyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

N-Boc-1-amino-but-3-ene

3-buten-1-amine (4.93g, 0.069mol) was subjected to step 13 for protection of tert-butoxycarbonyl group to give a crude product, which was purified by flash chromatography (silica gel/N-hexane: ethyl acetate 0-30%) to give N-tert-butoxycarbonyl-1-amino-but-3-ene (6.47g, 0.038mol, yield 55.1%).

Boc-2- (oxiran-2-yl) -carbamic acid ethyl ester

N-tert-Butoxycarbonyl-1-amino-but-3-ene (6.47g, 0.038mol) was subjected to step 14 for epoxide formation to give a crude product which was purified by flash chromatography (silica gel/N-hexane: ethyl acetate 0-45%) to yield N-tert-butoxycarbonyl-2- (oxiran-2-yl) -carbamic acid ethyl ester (6.0g, 0.032mol, 84.2% yield):¹H NMR(250MHz，DMSO-d₆)δ2.98-3.09(m，2H)，2.83-2.92(m，1H)，2.65(t，1H)，2.42(dd，1H)，1.44-1.66(m，2H)，1.36(s，9H，(CH₃)₃)。

6 '- (N-Boc-2-hydroxy-4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) with N-tert-butoxycarbonyl-2- (oxiran-2-yl) -carbamic acid ethyl ester as per step 5 gave 6 ' - (N-tert-butoxycarbonyl-2-hydroxy-4-amino-butyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ]/] - ]⁺Calculation 1148.6, found 1149.1), which was carried on to the next step without further purification.

6' - (2-hydroxy-4-amino-butyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-2-hydroxy-4-amino-butyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-hydroxy-4-amino-butyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl)Yl) -sisomicin (0.0015g, 0.0023mmol, yield 2.8%): MSm/e [ M + H]⁺Calculating 648.4 to obtain 648.4; the purity of CLND was 87.1%.

Example 36

6' - (methyl-cyclopropyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

N-tert-Butoxycarbonyl-3-hydroxy-azetidine-3-carboxylic acid

N-tert-butoxycarbonyl-3-azetidinone (21.9g, 0.128mol) was subjected to step 15 to give the target N-tert-butoxycarbonyl-3-hydroxy-azetidine-3-carboxylic acid (18.7g, 0.086mol, 67.0% yield): MS M/e [ M + H ]]⁺218.1 is calculated, and 218.2 is obtained.

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

Following step 4, method B, 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.075g, 0.081mmol) was treated with N-tert-butoxycarbonyl-3-hydroxy-azetidine-3-carboxylic acid to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin, which was subjected to the next step without further purification.

2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to yield 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 947.5 to obtain 948.0), which was carried on to the next step without further purification.

6 '- (methyl-cyclopropyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) was treated with cyclopropanecarboxaldehyde to give the target 6 ' - (methyl-cyclopropyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] /)]⁺Calculation 1001.6, found 1101.9), which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' - (methyl-cyclopropyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (methyl-cyclopropyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.0041g, 0.0068mmol, 8.4% yield): MS M/e [ M + H ]]⁺Calculating 601.3 to obtain 601.6; the purity of CLND was 88.2%.

Example 37

6' - (2-hydroxy-ethyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) was treated with tert-butyldimethylsiloxyacetal to give target 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3" -tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1105.6, found 1106.0), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (2-hydroxy-ethyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.0039g, 0.0066mmol, 8.1% yield): MS M/e [ M + H ] ]⁺591.3 is calculated, and 591.4 is obtained; the purity of CLND was 94.7%.

Example 38

6' - (2-amino-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (N-Boc-2-amino-ethyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.079mmol) was treated with N-tert-butoxycarbonyl-2-aminoacetaldehyde to give target 6 ' - (N-tert-butoxycarbonyl-2-amino-ethyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ]/[ M + H ])]⁺Calculation 1092.6, found 1093.0), which was carried on to the next step without further purification.

6' - (2-amino-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-2-amino-ethyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of the tert-butyloxycarbonyl group to give the crude product, which was purified by reverse phase HPLC method 3 to give 6 ' - (2-amino-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0048g, 0.0081mmol, 10.2%) as: MS M/e [ M + H ] ]⁺592.4 is calculated, 592.6 is obtained; the purity of CLND was 77.1%.

Example 39

6' - (methyl- (1-hydroxy-3-methylamino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

3-methylene-1-methylamino-cyclobutane

To a stirred solution of 3-methylene-1-cyano-cyclobutane (2.5g, 0.026mol) in THF (35ml) at 0 deg.C was slowly added 2M LiAlH₄(22mL, 0.044mmol) and the reaction was allowed to warm to room temperature. Then, by adding saturated NH₄The reaction was quenched with aqueous Cl (10mL) and THF (10 mL). The organic layer was separated and concentrated to dryness to give a residue, which was dissolved in ethyl acetate (100 mL). 5% NaHCO was used₃The organic layer was washed (2X 20mL) with brine (20mL) and washed with Na₂SO₄Dried, filtered and concentrated to give the target 3-methylene-1-methylamino-cyclobutane as an oil, which was taken to the next step without further purification.

3-methylene-1-N-tert-butoxycarbonyl-methylamino-cyclobutane

To a stirred solution of 3-methylene-1-methylamino-cyclobutane (2.52g, 0.026mol) in 1N NaOH (15mL) and THF (15mL) was added tert-butoxycarbonyl₂O (6.7g, 0.030mol) and the reaction mixture was stirred overnight. THF was evaporated and the aqueous layer extracted with ethyl acetate (2X 40 mL). With 5% NaHCO ₃The combined organic layers were washed (2X 20mL), brine (20mL) and washed with Na₂SO₄Dried, filtered and concentrated to dryness to give the crude product, which was purified by flash chromatography (silica gel/N-hexane: ethyl acetate 0% -60%) to give the target 3-methylene-1-N-tert-butoxycarbonyl-methylamino-cyclobutane (1.9g, 0.0096mol, 36.9% yield):¹HNMR(250MHz，DMSO-d₆)δ6.88(bs，1H)，4.72(s，2H)，2.95-3.05(m，2H)，2.56-2.71(m，2H)，2.21-2.40(m，3H)，1.20(s，9H)。

N-tert-Butoxycarbonyl-1-oxaspiro [2.3] hexan-5-yl-methylamine

3-methylene-1-N-tert-Butoxycarbonyl-methylamino-cyclobutane (1.9g, 0.0096mol) was subjected to step 14 for epoxide formation to give N-tert-butoxycarbonyl-1-oxaspiro [2.3]Hexane-5-yl-methylamine (1.34g, 6.27mol, 65.3% yield):¹H NMR(250MHz，DMSO-d₆)δ2.99-3.10(m，2H)，2.60-2.66(m，2H)，1.99-2.47(m，5H)，1.40(s，9H)。

6 '- (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-methylamino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

According to step 5, N-tert-butyloxycarbonyl-1-oxaspiro [2.3] is used]Hexane-5-Yl-methylamine treatment of 2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-Butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.079mmol) to give the target 6 ' - (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-methylamino-cyclobutyl) -2 ', 3, 3" -Tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] -) ]⁺Calculation 1162.7, found 1163.0), which was carried on to the next step without further purification.

6' - (methyl- (1-hydroxy-3-methylamino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-methylamino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removing tert-butoxycarbonyl to yield a crude product, it was purified by reverse phase HPLC method 3 to give 6' - (methyl- (1-hydroxy-3-methylamino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0037g, 0.0056mmol, 7.1% yield): MS M/e [ M + H.]⁺Calculating 662.4 to obtain 662.0; the purity of CLND was 82.5%.

Example 40

6' - (3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was treated with N-phthalimidopropional to yield target 6 ' - (N-phthalimido-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M ]⁺Calculation 1148.6, found 1148.8), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 6 for phthalimido deprotection to give 6 '- (3-amino-propyl) -2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin, which was subjected to the next step without further purification.

6' - (3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' - (3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.0023g, 0.0037mmol, 4.6%) as: MS M/e [ M + H ] ]⁺Calculating 618.4 to obtain 618.8; the purity of CLND was 93.1%.

EXAMPLE 41

6' - (methyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 '- (methyl-cyclopropyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was treated with cyclopropanecarboxaldehyde to give target 6 ' - (methyl-cyclopropyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] /)]⁺Calculation 1015.6, found 1015.6), which was carried on to the next step without further purification.

6' - (methyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' - (methyl-cyclopropyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (methyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.0021g, 0.0034mmol, 4.2%) as: MS M/e [ M + H ] ]⁺Calculating 615.4 to obtain 615.2; the purity of CLND was 96.5%.

Example 42

6' - (2-hydroxy-3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 '- (N-Boc-2-hydroxy-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) with N-tert-butoxycarbonyl-oxiran-2-yl-methylamine as per step 5 to give target 6 ' - (N-tert-butoxycarbonyl-2-hydroxy-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1134.6, found 1134.9), which was carried on to the next step without further purification.

6' - (2-hydroxy-3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Subjecting 6 ' - (N-tert-butoxycarbonyl-2-hydroxy-3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) to step 3-method a of removing tert-butoxycarbonyl to give a crude product which was purified by reverse phase HPLC method 3 to give 6 ' - (2-hydroxy-3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.003g, 0.0047mmol, 5.8%) as a crude product: MSm/e [ M + H ]⁺Calculating 634.4 to obtain 634.4; the purity of CLND was 95.1%.

Example 43

6' - (4-amino-butyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Fmoc-4-amino-diethyl butyral

Following step 16, 4-amino-diethylbutyral (8.0g, 0.050mol) fluorenylmethoxycarbonyl was protected to give the target N-fluorenylmethoxycarbonyl-4-amino-diethylbutyral (22.08g, MSm/e [ M + Na ]]⁺Calculate 406.2, find 406.1), proceed to the next step without further purification.

Fmoc-4-amino-butyraldehyde

To a stirred solution of Fmoc-4-amino-diethylbutyral (0.050mmol) in 1, 4-dioxane (100mL) was added aqueous HCl (100mL, 11v/v，H₂O: concentrated HCl) and the progress of the reaction was monitored by MS. After completion, the organic solvent was removed by rotary evaporation and the aqueous layer was extracted with ethyl acetate (2 × 200 mL). With 5% NaHCO₃The combined organic layers were washed (2X 75mL), brine (75mL) over Na₂SO₄Dried, filtered and concentrated to dryness to give the target N-fluorenylmethoxycarbonyl-4-amino-butyraldehyde (15.35g, 0.049mol, 90.0% yield), which was subjected to the next step without further purification: MS M/e [ M + Na ]]⁺The calculation was carried out at 332.1, and 332.0 was obtained.

6 '- (N-fluorenylmethoxycarbonyl-4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.079mmol) was treated with N-fluorenylmethoxycarbonyl-4-amino-butyraldehyde to give target 6 ' - (N-fluorenylmethoxycarbonyl-4-amino-butyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ]/]]⁺Calculation 1242.7, found 1242.9), which was carried on to the next step without further purification.

6 '- (4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of 6 '- (N-fluorenylmethoxycarbonyl-4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was addedPiperidine (0.3mmol) was added to a solution of DMF (1.5mL) and the reaction mixture was stirred for 2 hours. The reaction mixture was then diluted with water (5mL) and extracted with ethyl acetate (2X 10 mL). The combined organic layers were washed with water (2X 5mL), brine (5mL) and washed with Na₂SO₄Dried, filtered and concentrated to dryness to give 6 '- (4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ]) ]⁺1020.6 was calculated to obtain 1020.9), which was carried on to the next step without further purification.

6' - (4-amino-butyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (4-amino-butyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (4-amino-butyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.010g, 0.016mmol, 20.2%) as follows: MS M/e [ M + H ]]⁺Calculating 620.4 to obtain 620.8; the purity of CLND was 93.4%.

Example 44

6' - (5-amino-pentyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-Nitrobenzenesulfonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.079mmol) was subjected to step 8 for M-nitrobenzenesulfonylation to give the target 6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -Tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ]) ]⁺Calculation 1134.5, found 1134.8), which was carried on to the next step without further purification.

6 ' -Nitrobenzenesulfonyl-6 ' - (N-tert-butoxycarbonyl-5-amino-pentyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Treatment of 6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) with N-tert-butoxycarbonyl-5-amino-pentanol according to step 17 gave 6 ' -nitrobenzenesulfonyl-6 ' - (N-tert-butoxycarbonyl-5-amino-pentyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1319.6, found 1319.9), which was carried on to the next step without further purification.

6 '- (N-Boc-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Reacting 6 ' -nitrobenzenesulfonyl-6 ' - (N-tert-butoxycarbonyl-5-amino-pentyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl)-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 9 for removal of nitrobenzenesulfonyl to yield 6 '- (N-tert-butoxycarbonyl-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H) -N-tert-butoxycarbonyl-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e) ]⁺Calculation 1134.7, found 1135.0), which was carried on to the next step without further purification.

6' - (5-amino-pentyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-5-amino-pentyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to give the crude product, which was purified by reverse phase HPLC method 3 to give 6 ' - (5-amino-pentyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.009g, 0.014mmol, 17.7% yield): MS M/e [ M + H ]]⁺Calculating 634.4 to obtain 634.6; the purity of CLND was 82.6%.

Example 45

6' - (Ethyl-2- (1-methylpiperazin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2- (4-Boc-1-methylpiperazin-2-yl) -ethanol

2- (1-Methylpiperazin-2-yl) -ethanol (0.5g, 3.47mmol) was subjected to t-butyloxycarbonyl protection according to step 13 to give 2- (4-t-butyloxycarbonyl-1-methylpiperazineOxazin-2-yl) -ethanol (0.75g, 3.08mmol, 88.7% yield): MS M/e [ M + H ]]⁺245.2 was calculated, and 245.1 was obtained.

6' - (Ethyl-2- (4-tert-butoxycarbonyl-1-methylpiperazin-2-yl) -1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Treatment of 6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) with 2- (4-tert-butoxycarbonyl-1-methylpiperazin-2-yl) -ethanol according to step 17 to give 6 ' -nitrobenzenesulfonyl-6 ' - (ethyl-2- (4-tert-butoxycarbonyl-1-methylpiperazin-2-yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1360.7, found 1360.8), which was carried on to the next step without further purification.

6 '- (Ethyl-2- (4-tert-butyloxycarbonyl-1-methylpiperazin-2-yl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Reacting 6 ' -nitrobenzenesulfonyl-6 ' - (ethyl-2- (4-tert-butoxycarbonyl-1-methylpiperazin-2-yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 9 for removing nitrobenzenesulfonyl to give 6 '- (ethyl-2- (4-tert-butoxycarbonyl-1-methylpiperazin-2-yl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H). ]⁺1175.7 was calculated, 1176.0) was obtained, and this was carried out to the next stepWithout further purification.

6' - (Ethyl-2- (1-methylpiperazin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (Ethyl-2- (4-tert-butoxycarbonyl-1-methylpiperazin-2-yl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removing tert-butoxycarbonyl to give a crude product, it was purified by reverse phase HPLC method 3 to give 6' - (ethyl-2- (1-methylpiperazin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.010g, 0.015mmol, 18.9% yield): MS M/e [ M + H.]⁺675.4 is calculated, and 675.4 is obtained; the purity of CLND was 93.0%.

Example 46

6' - (methyl- (1-hydroxy-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

3-methylene-cyclobutanecarboxylic acid

To stirred KOH (70.0g, 1.25mol) in EtOH/H₂A solution of O (500mL, 1: 1v/v) was added 3-methylenecyclobutanecarbonitrile (25.0g, 0.26mol) and the reaction mixture was refluxed for 6 hours. The progress of the reaction was monitored by TLC and after completion, the mixture was cooled and acidified to pH 3-4 using HCl. Evaporate ethanol and use Et ₂The remaining aqueous layer was O (200 mL). The organic layer was washed with water (2X 20mL), brine (30mL) over Na₂SO₄Drying, filtering and concentrating toDrying to give 3-methylene-cyclobutanecarboxylic acid, which is carried on to the next step without further purification:¹H NMR(250MHz，CDCl₃)δ10.75(bs，1H)，4.80(s，2H)，2.85-3.26(m，5H)。

boc-3-methylene-cyclobutylamine

To a stirred solution of 3-methylene-cyclobutanecarboxylic acid (1.0g, 8.9mmol) in THF (90mL) was added NaN₃(2.0g, 31.1mmol) followed by tetrabutylammonium bromide (0.48g, 1.5mmol) and Zn (OTf)₂(0.1g, 0.3mmol) and the reaction mixture was heated to 40 ℃. Then immediately adding Boc₂O (tert-butyloxycarbonyl)₂O) (2.1g, 9.8mmol) and the reaction was heated at 45 ℃ overnight. The reaction was then cooled to 0 ℃ and treated with 10% NaNO₂Aqueous solution (180mL) was quenched. THF was evaporated and the aqueous layer extracted with EtOAc (180 mL). With 5% NaHCO₃The organic layer was washed with aqueous solution (2X 20mL), brine (30mL) and washed with Na₂SO₄Dried, filtered and concentrated to dryness to give the crude product, which is purified by flash chromatography (silica gel/N-hexane: ethyl acetate: 0-90%) to give the target N-tert-butoxycarbonyl-3-methylene-cyclobutylamine (0.57g, 3.1mmol, 34.9% yield):¹H NMR(250MHz，CDCl₃)δ4.83(s，2H)，4.79(bs，1H)，4.05-4.23(m，1H)，2.92-3.11(m，2H)，2.50-2.65(m，2H)，1.44(s，9H)。

N-Boc-1-oxaspiro [2.3] hexan-5-amine

N-tert-Butoxycarbonyl-3-methylene-cyclobutylamine (1.65g, 9.0mmol) was subjected to step 14 for epoxide formation to give N-tert-butyloxycarbonyl1-oxaspiro [2.3 ] yl]Hexane-5-amine (1.46g, 7.33mmol, 81.5% yield):¹H NMR(250MHz，CDCl₃)δ4.79(bs，1H)，4.13-4.31(m，1H)，2.66-2.83(m，4H)，2.31-2.47(m，2H)，1.45(s，9H)。

6' - (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

According to step 5, N-tert-butyloxycarbonyl-1-oxaspiro [2.3 ] is used]Hexane-5-amine treatment of 2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-Butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) to give 6 ' - (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2 ', 3, 3" -Tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1148.6, found 1148.6), which was carried on to the next step without further purification.

6' - (methyl- (1-hydroxy-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removing tert-butoxycarbonyl to yield a crude product, it was purified by reverse phase HPLC method 3 to give 6' - (methyl- (1-hydroxy-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0098g, 0.015mmol, 18.9% yield): MS M/e [ M + H. ]⁺Calculating 648.4 to obtain 648.4; CLND purity of82.0％。

Example 47

6' - (methyl- (1-hydroxy-3-amino-cyclobutyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '- (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

According to step 5, N-tert-butyloxycarbonyl-1-oxaspiro [2.3 ] is used]Hexane-5-amine treatment of 2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-Butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) to give 6 ' - (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2 ', 3, 3" -Tri-tert-Butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1146.6, found 1147.0), which was carried on to the next step without further purification.

6' - (methyl- (1-hydroxy-3-amino-cyclobutyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '- (methyl- (1-hydroxy-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column A The reaction was performed to give 6' - (methyl- (1-hydroxy-3-amino-cyclobutyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.0089g, 0.014mmol, 17.3% yield) MS M/e [ M + H ] -%)]⁺646.4 is calculated, and 646.6 is obtained; the purity of CLND was 95.7%.

Example 48

6' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was treated with N-phthalimidopropional to yield target 6 ' - (N-phthalimido-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] /)]⁺Calculation 1136.6, found 1136.7), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

The 6 ' - (N-phthalimido-3-amino-propyl) -2 ', 3, 3 ' -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy- Butyryl) -sisomicin (0.079mmol) was subjected to step 6 for phthalimido deprotection to give 6 '- (3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺Calculation 1006.6, found 1007.1), which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.010g, 0.016mmol, 20.2%) as follows: MS M/e [ M + H ]]⁺Calculating 606.4 to obtain 606.4; the purity of CLND was 95.8%.

Example 49

6' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '- (methyl-N-tert-butyloxycarbonyl-pyrrolidin-2-yl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butoxycarbonyl) aldehyde treatment with N-tert-butyloxycarbonyl-DL-prolinaldehyde according to step 1-method AButoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) to yield target 6 '- (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] -N-tert-butoxycarbonyl-2 (S) -hydroxy-butyryl)]⁺Calculation 1132.6, found 1133.0), which was carried on to the next step without further purification.

6' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.010g, 0.016mmol, 20.2% yield): MS M/e [ M + H ]]⁺632.4 is calculated, 632.8 is obtained; the purity of CLND was 90.9%.

Example 50

6' - (2(S) -hydroxy-3-propanoic acid) -1- (4-amino-2 (S) -hydroxy-butanoyl) -sisomicin

6 '- (2(S) -hydroxy-3-methyl-propanoate) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butanoyl) -sisomicin

Treatment of 2 with methyl-2- (R) -glycerate according to step 5', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) to give the target 6 ' - (2(S) -hydroxy-3-methyl-propionate) -2 ', 3, 3" -Tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1051.6, found 1052.2), which was carried on to the next step without further purification.

6' - (2(S) -hydroxy-3-propanoic acid) -1- (4-amino-2 (S) -hydroxy-butanoyl) -sisomicin

6 ' - (2(S) -hydroxy-3-methyl-propionate) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.079mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and ester hydrolysis to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2(S) -hydroxy-3-propionic acid) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0028g, 0.0044mmol, 5.6%) as a crude product: MS M/e [ M + H ] ]⁺Calculating 637.3 to obtain 637.6; the purity of CLND was 89.8%.

Example 51

6' - (2, 2-dimethyl-3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N-Boc-2, 2-dimethyl-3-amino-propionaldehyde

N-tert-Butoxycarbonyl-2, 2-dimethylpropanol (0.415g, 2.04mmol) was subjected to step 18 to give N-tert-butyloxycarbonyl-2, 2-dimethyl-3-amino-propanal (0.39g, 1.94mmol, 95.1% yield):¹H NMR(250MHz，CDCl₃)δ9.42(s，1H)，4.80(bs，1H)，3.11(d，2H)，1.39(s，9H)，1.06(s，6H)。

6 '- (N-Boc-2, 2-dimethyl-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N-tert-butoxycarbonyl-2, 2-dimethyl-3-amino-propionaldehyde to give the target 6 ' - (N-tert-butoxycarbonyl-2, 2-dimethyl-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin, which was subjected to the next step without further purification.

6' - (2, 2-dimethyl-3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-2, 2-dimethyl-3-amino-propyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to give the crude product, which was purified by reverse phase HPLC method 3 to give 6 ' - (2, 2-dimethyl-3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0057g, 0.0092mmol, yield 11.5%): MSm/e [ M + H]⁺Calculating 620.4 to obtain 620.8; the purity of CLND was 97.4%.

Example 52

6' - (3-amino-3-cyclopropyl-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N-Boc-3-amino-3-cyclopropylpropanal

N-tert-Butoxycarbonyl-3-amino-propanol (0.130g, 0.60mmol) was subjected to step 18 for oxidation to the corresponding N-tert-butyloxycarbonyl-3-amino-3-cyclopropylpropanal, which was carried on to the next step without further purification.

6 '- (N-Boc-3-amino-3-cyclopropyl-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N-tert-butoxycarbonyl-3-cyclopropylpropanal to give 6 ' - (N-tert-butoxycarbonyl-3-amino-3-cyclopropyl-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (r), which was taken to the next step without further purification.

6' - (3-amino-3-cyclopropyl-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-amino-3-cyclopropyl-propyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to give the crude product, which was purified by reverse phase HPLC method 3 to give 6 ' - (3-amino-3-cyclopropyl-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0067g, 0.010mmol, 12.5%) as: MS M/e [ M + H ]]⁺632.4 is calculated, 632.8 is obtained; the purity of CLND was 96.7%.

Example 53

6' - (methyl-4 (S) -hydroxy-pyrrolidin-2 (R) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

4(S) -tert-butyldimethylsilyloxy-N-tert-butyloxycarbonyl-pyrrolidine-2 (R) -carbaldehyde

4(S) -tert-Butyldimethylsilyloxy-N-tert-butyloxycarbonyl-pyrrolidine-2 (R) -methanol (0.50g, 1.50mmol) was subjected to step 18 for oxidation to the corresponding 4(S) -tert-butyldimethylsilyloxy-N-tert-butyloxycarbonyl-pyrrolidine-2 (R) -carbaldehyde, which was carried on to the next step without further purification.

6 '- (methyl-N-tert-Butoxycarbonyl-4 (S) -tert-butyldimethylsilyloxy-2 (R) -pyrrolidin-2 (R) -yl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with 4(S) -tert-butyldimethylsilyloxy-N-tert-butoxycarbonyl-pyrrolidine-2 (R) -carbaldehyde to give the target 6 ' - (methyl-N-tert-butoxycarbonyl-4 (S) -tert-butyldimethylsilyloxy-pyrrolidin-2 (R) -yl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1248.7, found 1248.8), which was carried on to the next step without further purification.

6' - (methyl-4 (S) -hydroxy-pyrrolidin-2 (R) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-4 (S) -tert-butyldimethylsilyloxy-pyrrolidin-2 (R) -yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (methyl-4 (S) -hydroxy-pyrrolidin-2 (R) -yl-methyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0022g, 0.0035mmol, 4.4% yield): MS M/e [ M + H ] ]⁺Calculating 634.4 to obtain 634.6; the purity of CLND was 98.0%.

Example 54

6' - (3-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

3-tert-butyldimethylsilyloxy-propionaldehyde

3-tert-Butyldimethylsilyloxy-propanol (0.50g, 2.62mmol) was subjected to step 18 for oxidation to the corresponding 3-tert-butyldimethylsilyloxy-propionaldehyde, which was carried on to the next step without further purification.

6 '- (3-tert-Butyldimethylsilyloxy-propanol) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with 3-tert-butyldimethylsilyloxy-propionaldehyde to give 6 ' - (3-tert-butyldimethylsilyloxy-propanol) -2 ', 3, 3" -tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ]/]]⁺Calculation 1107.6 to find 1107.9) which was carried on to the next step without further purification.

6' - (3-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (3-tert-Butyldimethylsilyloxy-propanol) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl and TBS to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (3-propanol)) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.011g, 0.018mmol, 22.5% yield): MS M/e [ M + H ]]⁺593.3 is calculated, 593.8 is obtained; the purity of CLND was 98.4%.

Example 55

6' - (2-methyl-2-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

2-methyl-N-tert-butoxycarbonyl-2-amino-propionaldehyde

2-methyl-N-tert-butoxycarbonyl-2-amino-propanol (0.83g, 4.38mmol) was subjected to step 18 for oxidation to the corresponding 2-methyl-N-tert-butoxycarbonyl-2-amino-propionaldehyde (0.706g, 3.77mmol, 86.1% yield):¹H NMR(250MHz，CDCl₃)δ9.40(s，1H)，1.57(s，1H)，1.41(s，9H)，1.30(s，6H)。

6 '- (2-methyl-N-tert-butyloxycarbonyl-2-amino-propyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with 2-methyl-N-tert-butoxycarbonyl-2-amino-propionaldehyde to give 6 ' - (2-methyl-N-tert-butoxycarbonyl-2-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ] -N-tert-butoxycarbonyl-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e) ]⁺Calculation 1106.6, find 1107.0), advance itThe next step was carried out without further purification.

6' - (2-methyl-2-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (2-methyl-N-tert-butoxycarbonyl-2-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (2-methyl-2-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.010g, 0.016mmol, 20.0%) as a crude product: MS M/e [ M + H ]]⁺Calculating 606.4 to obtain 606.4; the purity of CLND was 99.2%.

Example 56

6' - (methyl-1-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N-Boc-1-amino-cyclobutanecarboxylic acid

Ethyl 1-amino-cyclobutanecarboxylate (1.0g, 6.28mmol) was dissolved in 1N HCl (10mL) and the reaction was heated to reflux for 2 hours. The reaction mixture was then concentrated to dryness to give a crude product, which was subjected to step 13 for t-butyloxycarbonyl protection to give the target N-t-butyloxycarbonyl-1-amino-cyclobutanecarboxylic acid.

N-Boc-1-amino-cyclobutyl-methanol

N-tert-Butoxycarbonyl-1-amino-cyclobutanecarboxylic acid (6.28mmol) was subjected to step 19 for reduction to the corresponding N-tert-butoxycarbonyl-1-amino-cyclobutyl-methanol.

N-Boc-1-amino-cyclobutanecarboxaldehyde

N-tert-butoxycarbonyl-1-amino-cyclobutyl-methanol (0.25g, 1.24mmol) was subjected to step 18 to give the corresponding N-tert-butoxycarbonyl-1-amino-cyclobutanecarboxaldehyde (0.24g, 1.20mmol, 96.8% yield):¹H NMR(250MHz，CDCl₃)δ9.0(s，1H)，4.91(bs，1H)，3.74(bs，2H)，1.71-2.20(m，4H)，1.42(s，9H)。

6 '- (N-Boc-methyl-1-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N-tert-butoxycarbonyl-1-amino-cyclobutanecarboxaldehyde to give 6 ' - (N-tert-butoxycarbonyl-methyl-1-amino-cyclobutyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1118.6, found 1118.9), which was carried on to the next step without further purification.

6' - (methyl-1-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-methyl-1-amino-cyclobutyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to give the crude product, which was purified by reverse phase HPLC method 1-column A to give 6 ' - (methyl-1-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.002g, 0.0032mmol, 4.0%) as: MS M/e [ M + H ]]⁺Calculating 618.4 to obtain 619.0; the purity of CLND was 69.4%.

Example 57

6' - (3-amino-propyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '- (N-Boc-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.49g, 0.46mmol) was treated with N-tert-butoxycarbonyl-3-amino-propionaldehyde to give 6 ' - (N-tert-butoxycarbonyl-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M ]⁺The calculation 1104.6 is carried out in such a way that,1104.6) was obtained and was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-amino-propyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.46mmol) was subjected to step 3-method B for removal of the tert-butyloxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column B to yield 6 ' - (3-amino-propyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin: MS M/e [ M + H ]]⁺604.4 is calculated, 604.2 is obtained; the purity of CLND was 92.4%.

Example 58

6' - (3-amino-propyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

N-tert-butyloxycarbonyl-3-amino-cyclobutanone

To a vigorously stirred mixture of N-tert-butoxycarbonyl-3-methylene-cyclobutylamine (9.8g, 53.5mmol) in DCM (160mL) and H₂O (160mL) solution was added K₂CO₃(3g, 21.7mmol) followed by NaClO₄(35g, 163.5mmol), tetrabutylammonium chloride (0.2g, 0.72mmol) and RuCl₃(0.6g, 7.6 mmol). During the reaction, the organic solution turned dark brown, the catalyst turned black, and the upper aqueous layer turned white. The reaction was monitored by TLC and, after completion, passed through a pad of celite The reaction mixture was filtered. The filtrate was transferred to a separatory funnel and the aqueous layer was extracted with DCM (2X 50 mL). With 5% NaHCO₃The combined organic layers were washed (2X 30mL), brine (30mL) and washed with Na₂SO₄Dried, filtered and evaporated to dryness to give the crude product, which was purified by flash chromatography (silica gel/N-hexane: ethyl acetate 0-60%) to give the target N-tert-butoxycarbonyl-3-amino-cyclobutanone (7.13g, 38.53mmol, 72% yield): NMR (250MHz, CDCl)₃)δ4.88(bs，1H)，4.13-4.29(m，1H)，3.23-3.41(m，2H)，2.9-3.05(m，2H)，1.39(s，9H)。

N-Boc-1-hydroxy-3-amino-cyclobutyl-carboxylic acid

N-tert-Butoxycarbonyl-3-amino-cyclobutanone (7.13g, 38.53mmol) is subjected to step 15 to yield the target N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-carboxylic acid (MS M/e [ M + H ])]⁺The calculation was carried out at 232.1 to obtain 232.2.

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

Treatment of 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.87mmol) with N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-carboxylic acid according to step 4-method a gave the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin, which was subjected to the next step without further purification.

2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.87mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to yield 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (MS M/e [ M + H +/e)]⁺Calculation 961.5 to find 961.3), which was carried on to the next step without further purification.

6 '- (N-Boc-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.87mmol) was treated with N-tert-butoxycarbonyl-3-amino-propionaldehyde to give target 6 ' - (N-tert-butoxycarbonyl-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (MS M/e [ M + H ]/] ]⁺Calculation 1118.6, found 1118.6), which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-3-amino-propyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.87mmol) was subjected to step 3-method B for removal of the tert-butyloxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column B to yield 6 ' - (3-amino-propyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin: MS M/e [ M + H ]]⁺Calculating 618.4 to obtain 618.2; the purity of CLND was 84.2%.

Example 59

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (N-Boc-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (1.0g, 1.07mmol) was treated with N-tert-butoxycarbonyl-3-trans-amino-cyclobutyl-carbaldehyde to give 6 ' - (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ] -N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e) ]⁺Calculation 1118.6, found 1118.5), which was carried on to the next step without further purification.

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (1.07mmol) was subjected to step 3-method B for removal of the tert-butyloxycarbonyl group to give the crude product, which was purified by reverse phase HPLC method 1-column B to give 6 ' - (methyl-trans-3-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.033g, 0.053mmol, 4.9%) as: MS M/e [ M + H ]]⁺618.4 is calculated, 618.3, [ M + Na ] is obtained]⁺640.3; the purity of CLND was 96.5%.

Example 60

6' - (methyl-trans-3-amino-cyclobutyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 '- (N-Boc-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (1.0g, 1.042mmol) was treated with N-tert-butoxycarbonyl-3-trans-amino-cyclobutyl-carbaldehyde to give the target 6 ' - (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (MS M/e [ M + H). ]⁺Calculation 1144.6, found 1144.5), which was carried on to the next step without further purification.

6' - (methyl-trans-3-amino-cyclobutyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (1.042mmol) was subjected to step 3-method B for removal of the tert-butyloxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column B to yield 6 ' - (methyl-trans-3-amino-cyclobutyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.033g, 0.051mmol, 4.9%) as a crude product: MS M/e [ M + H ]]⁺644.4 is calculated, 644.3 is obtained; the purity of CLND was 94.5%.

Example 61

6' -methyl-1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '-Nitrobenzenesulfonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (1.0g, 1.06mmol) was subjected to step 8 for nitrobenzenesulfonyl to give 6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -Tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ]) ]⁺Calculation 1132.5, find 1132.8), advance itThe next step was carried out without further purification.

6 ' -methyl-6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' -Nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (1.06mmol) was treated with MeI according to step 11 to give 6 ' -methyl-6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1146.5, found 1147.0), which was carried on to the next step without further purification.

6 '-methyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' -methyl-6 ' -Nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (1.06mmol) was subjected to step 9 for nitrobenzenesulfonyl deprotection to give 6 ' -methyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M ]⁺Calculation 961.5, found 961.8), which was carried on to the next step without further purification.

6' -methyl-1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 ' -methyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (1.06mmol) was subjected to step 3-method A for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column B to yield 6 ' -methyl-1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.247g, 0.441mmol, 41.6% yield): MS M/e [ M + H ]]⁺561.3 is calculated, 561.2 is obtained; the purity of CLND was 96.7%.

Example 62

6' - (2-hydroxy-ethyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.65g, 0.67mmol) was treated with tert-butyldimethylsilyloxyacetaldehyde to give 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3" -tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin target (MS M/e [ M + H ], [ M + H ]) ]⁺Calculation 1119.6, found 1119.9), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' - (2-tert-Butyldimethylsilyloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.67mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield the crude product, which was purified by reverse phase HPLC method 1-column B to yield 6 ' - (2-hydroxy-ethyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.067g, 0.111mmol, 16.6% yield): MS M/e [ M + H ]]⁺Calculating 605.3 to obtain 605.6; the purity of CLND was 97.5%.

Example 63

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '- (N-Boc-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

Following step 1, method B, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-azetidin-3-yl-acetyl) -sisomicin (1.0g, 1.06mmol) was treated with N-tert-butoxycarbonyl-3-trans-amino-cyclobutyl-carbaldehyde to give target 6 ' - (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H). ]⁺1130.6 is calculated and 1130 is obtained.5) It was carried on to the next step without further purification.

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin

6 '- (N-tert-Butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (1.06mmol) was subjected to step 3-method B for removal of tert-butyloxycarbonyl to give a crude product, it was purified by reverse phase HPLC method 1-column B to give 6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin (0.018g, 0.029mmol, 2.7% yield): MS M/e [ M + H ]]⁺Calculating 630.4 to obtain 630.3; the purity of CLND was 75.6%.

Example 64

6' -methyl-1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 '-Nitrobenzenesulfonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

2 ', 3, 3 "-Tri-tert-Butoxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (1.0g, 1.04mmol) was subjected to step 8 for nitrobenzenesulfonyl to give 6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -Tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-1-hydroxy-3-amino-cyclobutyl-ethan-ethyl Acyl) -sisomicin (MS M/e [ M + H)]⁺Calculation 1146.5, found 1147.0), which was carried on to the next step without further purification.

6 ' -methyl-6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' -Nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (1.04mmol) was treated with MeI according to step 11 to give 6 ' -methyl-6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1160.5, found 1161.1), which was carried on to the next step without further purification.

6 '-methyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' -methyl-6 ' -Nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (1.04mmol) was subjected to step 9 for nitrobenzenesulfonyl deprotection to give 6 ' -methyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (MS M/e [ M + H ] /) ]⁺Calculation 975.5, found 975.9), which was carried on to the next step without further purification.

6' -methyl-1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin

6 ' -methyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (1.04mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 1-column B to yield 6 ' -methyl-1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin (0.098g, 0.170mmol, 16.3% yield): MS M/e [ M + H ]]⁺Calculating 575.3 to obtain 575.3; the purity of CLND was 98.5%.

Example 65

6' - (methyl-4 (S) -amino-pyrrolidin-2 (S) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N, N-di-tert-butoxycarbonyl-4 (S) -amino-2 (S) -methanol-pyrrolidine

N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidine-2 (S) -carboxylic acid (1.03g, 3.12mmol) was subjected to step 19 to give the corresponding N, N-di-tert-butoxycarbonyl-4 (S) -amino-2 (S) -methanol pyrrolidine (0.605g, 1.91mmol, 61.2% yield), which was taken to the next step without further purification.

N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidine-2 (S) -carbaldehyde

N, N-di-tert-butoxycarbonyl-4 (S) -amino-2 (S) -methanol pyrrolidine (0.486g, 1.53mmol) was subjected to step 18 for oxidation to the corresponding N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidine-2 (S) -carbaldehyde, which was taken to the next step without further purification.

6 '- (methyl-N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidin-2 (S) -yl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidine-2 (S) -carbaldehyde to give the target 6 ' - (methyl-N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidin-2 (S) -yl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1233.7, found 1234.0), which was carried on to the next step without further purification.

6' - (methyl-4 (S) -amino-pyrrolidin-2 (S) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N, N-di-tert-butoxycarbonyl-4 (S) -amino-pyrrolidin-2 (S) -yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (methyl-4 (S) -amino-pyrrolidin-2 (S) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0006g, 0.0009mmol, 1.1% yield): MS M/e [ M + H ]]⁺Calculating 633.4 to obtain 633.4; the purity of CLND was 81.7%.

Example 66

6' - (methyl-1-aminomethyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N-Boc-1-aminomethyl-cyclopropyl-methanol

N-tert-Butoxycarbonyl-1-aminomethyl-cyclopropanecarboxylic acid (1.0g, 4.64mmol) was subjected to step 19 to give the corresponding N-tert-butyloxycarbonyl-1-aminomethyl-cyclopropyl-methanol (0.99g, MS M/e [ M + H ])]⁺Calculated 202.1, found 202.1), which is carried on to the next step without further purification.

N-Boc-1-aminomethyl-cyclopropanecarboxaldehyde

N-tert-Butoxycarbonyl-1-aminomethyl-cyclopropyl-methanol (0.87g, 4.32mmol) was subjected to step 18 for oxidation to the corresponding N-tert-butyloxycarbonyl-1-aminomethyl-cyclopropanecarboxaldehyde, which was carried on to the next step without further purification.

6 '- (methyl-N-tert-butyloxycarbonyl-1-aminomethyl-cyclopropyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N-tert-butoxycarbonyl-1-aminomethyl-cyclopropanecarboxaldehyde to give target 6 ' - (methyl-N-tert-butoxycarbonyl-1-aminomethyl-cyclopropyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MSm/e [ M + H ] -N-tert-butoxycarbonyl-1-amino-2 (S) -hydroxy-propionyl) -sisomicin (MSm/e ]⁺Calculation 1118.6, found 1118.8), which was carried on to the next step without further purification.

6' - (methyl-1-aminomethyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-1-aminomethyl-cyclopropyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (methyl-1-aminomethyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0033g, 0.0053mmol, 6.6%) as a crude product: MS M/e [ M + H ]]⁺Calculating 618.4 to obtain 618.4; the purity of CLND was 94.5%.

Example 67

6' - (methyl-1-amino-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N-Boc-1-amino-cyclopropyl-methanol

N-tert-Butoxycarbonyl-1-amino-cyclopropanecarboxylic acid (0.25g, 1.24mmol) was subjected to step 19 to give the corresponding N-tert-butyloxycarbonyl-1-amino-cyclopropyl-methanol (0.051g, 0.27mmol, 21.8% yield), which was taken to the next step without further purification.

N-Boc-1-amino-cyclopropanecarboxaldehyde

N-tert-Butoxycarbonyl-1-amino-cyclopropyl-methanol (0.051g, 0.27mmol) was subjected to step 18 for oxidation to the corresponding N-tert-butyloxycarbonyl-1-amino-cyclopropanecarboxaldehyde, which was carried on to the next step without further purification.

6 '- (methyl-N-tert-butyloxycarbonyl-1-amino-cyclopropyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N-tert-butoxycarbonyl-1-amino-cyclopropanecarboxaldehyde to give 6 ' - (methyl-N-tert-butoxycarbonyl-1-amino-cyclopropyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1104.6, found 1105.2), which was carried on to the next step without further purification.

6' - (methyl-1-amino-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-1-amino-cyclopropyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (methyl-1-amino-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0042g, 0.0069mmol, 8.6% yield): MS M/e [ M + H ] ]⁺Calculating 604.4 to obtain 604.6; the purity of CLND was 95.4%.

Example 68

6' - (2-hydroxy-4-amino-butyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (N-Boc-2-hydroxy-4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) with ethyl N-tert-butoxycarbonyl-2- (oxiran-2-yl) -carbamate according to step 5 to give 6 ' - (N-tert-butoxycarbonyl-2-hydroxy-4-amino-butyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ] -N-tert-butoxycarbonyl-2- (oxiran-2-yl) -sisomicin (MS M/e)]⁺1122.6 was calculated, 1122.9) was obtained, and it was subjected to the next step toNo further purification was required.

6' - (2-hydroxy-4-amino-butyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-2-hydroxy-4-amino-butyl) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to give the crude product, which was purified by reverse phase HPLC method 3 to give 6 ' - (2-hydroxy-4-amino-butyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0024g, 0.0038mmol, 4.7%) as: MSm/e [ M + H ]⁺Calculating 622.4 to obtain 622.6; the purity of CLND was 93.2%.

Example 69

6' - (methyl-1 (R) -amino-2 (S) -hydroxy-cyclopent-4 (S) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N-Boc-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopentane-4 (S) -carboxylic acid

To a stirred solution of methyl N-tert-butoxycarbonyl-1 (R) -amino-2 (S) -hydroxy-cyclopentane-4 (S) -carboxylate (0.622g, 2.40mmol) in DCM (1.9mL) were added imidazole (0.164g, 2.41mmol), DMAP (0.047g, 0.35 mmol) and TBSCl (0.363g, 2.40mmol), and the reaction was stirred at room temperature for 18 hours and subsequently heated at 40 ℃ for 1 hour. The reaction mixture was cooled to room temperature and washed with H₂O (3mL) quench. The organic layer was separated and concentrated to dryness toA residue was generated, which was dissolved in isopropanol (6mL) and 1M NaOH (2.9mL), and the reaction was heated at 60 ℃ for 1 hour. The reaction was cooled to 0 ℃ and slowly acidified to pH 3 with 1M HCl (3 mL). After addition of chloroform (18mL), the organic layer was separated and washed with Na₂SO₄Dried and concentrated to dryness to give the target acid (0.75g, 2.09mmol, 87.1% yield).

N-Boc-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-4 (S) -hydroxymethyl-cyclopentane

N-tert-Butoxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopentane-4 (S) -carboxylic acid (0.53g, 1.47mmol) was subjected to step 19 for reduction to the corresponding N-tert-butyloxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-4 (S) -hydroxymethyl-cyclopentane (0.44g, 1.27mmol, 86.4% yield):¹H NMR(250MHz，CDCl₃)δ4.69-4.79(m，1H)，4.08-4.13(m，1H)，3.88(bs，1H)，3.52-3.61(m，2H)，2.16-2.30(m，2H)，1.96-2.14(m，2H)，1.48-1.53(m，2H)，1.47(s，9H)，0.91(s，9H)，0.09(s，6H)。

N-Boc-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopentane-4 (S) -carbaldehyde

N-tert-Butoxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-4 (S) -hydroxymethyl-cyclopentane (0.44g, 1.27mmol) was subjected to step 18 for oxidation to the corresponding N-tert-butyloxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopentane-4 (S) -carbaldehyde (0.42g, 1.22mmol, 96.1% yield).

6 '- (methyl-N-tert-Butoxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopent-4 (S) -yl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was treated with N-tert-butoxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopentane-4 (S) -carbaldehyde to give the target 6 ' - (methyl-N-tert-butoxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopent-4 (S) -yl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) Sisomicin (MS M/e [ M + H) ]⁺Calculation 1262.7, found 1263.2), which was carried on to the next step without further purification.

6' - (methyl-1 (R) -amino-2 (S) -hydroxy-cyclopent-4 (S) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-1 (R) -amino-2 (S) -tert-butyldimethylsilyloxy-cyclopent-4 (S) -yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield crude product, which was purified by reverse phase HPLC method 3 to yield 6 ' - (methyl-1 (R) -amino-2 (S) -hydroxy-cyclopent-4 (S) -yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0-0039g, 0.0060mmol, yield 7.5%): MS M/e [ M + H ]]⁺Calculating 648.4 to obtain 648.4; the purity of CLND was 91.6%.

Example 70

6' - (Ethyl-2- (3-hydroxy-azetidin-3-yl)) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Tert-butyl-2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) acetate

To a stirred solution of N-tert-butoxycarbonyl-3-azetidinone (0.45g, 2.64mmol) in THF (5mL) was slowly added 0.5M Et of 2-tert-butoxy-2-oxyethyl-zinc chloride ₂O (10mL, 5.0mmol) solution and the reaction mixture was stirred for 5 hours. Then, with saturated NH₄The reaction was quenched with aqueous Cl (10mL), and the aqueous layer was separated and extracted with ethyl acetate (2X 30 mL). With 5% NaHCO₃The combined organic layers were washed with aqueous (2X 10mL), brine (15mL) and washed with Na₂SO₄Dried, filtered and concentrated to dryness to give tert-butyl-2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetate (MS M/e [ M + H)]⁺Calculation 288.2, found 287.7).

2- (N-Boc-3-hydroxy-azetidin-3-yl) -acetic acid

To a stirred solution of tert-butyl-2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetate (0.86g, 2.99mmol) in dioxane (18mL) was added 3M HCl (5mL) and the mixture was heated at 70 ℃ for 1 h. The reaction mixture was then cooled to 0 ℃ and basified with 2M NaOH (8mL), followed by the addition of tert-butyloxycarbonyl₂O (1.0g, 4.6 mmol). Reacting the mixtureWarmed to room temperature for 2 hours and then concentrated on a rotary evaporator to half its total volume. Then, isopropanol (3mL) and chloroform (12mL) were added and the mixture was cooled to 0 ℃ and slowly acidified to pH 3 with 1M HCl. The organic layer was then separated over Na ₂SO₄Dried and concentrated to dryness to give 2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetic acid (0.65g, 2.81mmol, 94.0% yield).

N-Boc-3- (2-hydroxy-ethyl) -azetidin-3-ol

2- (N-tert-Butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetic acid (0.44g, 1.90mmol) was subjected to step 19 for reduction to give the corresponding N-tert-butoxycarbonyl-3- (2-hydroxy-ethyl) -azetidin-3-ol (0.29g, 1.33mmol, 70.0% yield).

2- (N-Boc-3-hydroxy-azetidin-3-yl) -acetaldehyde

N-tert-Butoxycarbonyl-3- (2-hydroxy-ethyl) -azetidin-3-ol (0.29g, 1.33mmol) was subjected to step 18 for oxidation to the corresponding 2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetaldehyde, which was carried on to the next step without further purification.

6 '- (Ethyl-2- (N-tert-butyloxycarbonyl-3-hydroxy-azetidin-3-yl)) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetaldehyde was treated with 2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl) -acetaldehyde to give 6 ' - (ethyl-2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl)) -2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H). ]⁺Calculation 1134.6, found 1135.1), which was carried on to the next step without further purification.

6' - (Ethyl-2- (3-hydroxy-azetidin-3-yl)) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (Ethyl-2- (N-tert-butoxycarbonyl-3-hydroxy-azetidin-3-yl)) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (Ethyl-2- (3-hydroxy-azetidin-3-yl)) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0098g, 0.015mmol, 18.7% yield): MS M/e [ M + H ]]⁺Calculating 634.4 to obtain 634.8; the purity of CLND was 92.4%.

Example 71

6' -methylcyclopropyl-1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

N-tert-Butoxycarbonyl-3-hydroxymethyl-azetidine

N-tert-Butoxycarbonyl-azetidine-3-carboxylic acid (1.94g, 9.64mmol) was subjected to step 19 for reduction to the corresponding N-tert-butoxycarbonyl-3-hydroxymethyl-azetidine, which was carried on to the next step without further purification.

N-Boc-azetidine-3-carbaldehyde

N-tert-Butoxycarbonyl-3-hydroxymethyl-azetidine (9.64mmol) was subjected to step 18 for oxidation to the target N-tert-butyloxycarbonyl-azetidine-3-carbaldehyde, which was taken to the next step without further purification.

2- (N-Boc-azetidin-3-yl) -2-hydroxy-acetic acid

N-tert-Butoxycarbonyl-azetidine-3-carbaldehyde (1.60g, 8.64mmol) was subjected to step 15 to give the target 2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetic acid (MSm/e [ M + H ])]⁺Calculate 232.1, find 231.8).

6 '-para-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Following step 4, method B, 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.075g, 0.081mmol) was treated with 2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetic acid to give the target 6 '-p-nitrobenzyloxycarbonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 1140.5, found 1140.8), which was carried on to the next step without further purification.

2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 961.5, found 962.0), which was carried on to the next step without further purification.

6 '-methylcyclopropyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-2-azetidin-3-yl-2-hydroxy-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with cyclopropanecarboxaldehyde to give the target 6 ' -methylcyclopropyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-Butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ]]⁺Calculation 1015.6, found 1015.8), which was carried on to the next step without further purification.

6' -methylcyclopropyl-1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' -methylcyclopropyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' -methylcyclopropyl-1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0033g, 0.0054mmol, 6.7% yield): MS M/e [ M + H ]]⁺Calculating 615.4 to obtain 615.5; the purity of CLND was 77.4%.

Example 72

6' - (methyl-trans-3-amino-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (N-Boc-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Following step 1, method B, 2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl) -formaldehyde was treated with N-tert-butoxycarbonyl-trans-3-amino-cyclobutyl-formaldehyde-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) to give 6 '- (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H) -sisomicin (MS M/e) ]⁺Calculation 1144.6, found 1145.0), which was carried on to the next step without further purification.

6' - (methyl-trans-3-amino-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' - (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (methyl-trans-3-amino-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0053g, 0.0082mmol, 10.1% yield): MS M/e [ M + H ]]⁺Calculating 644.4 to obtain 644.4; the purity of CLND was 86.0%.

Example 73

6' - (methyl-azetidin-3-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (methyl-N-tert-butyloxycarbonyl-azetidin-3-yl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.9g, 0.96mmol) was treated with N-tert-butoxycarbonyl-azetidine-3-carbaldehyde to give 6 ' - (methyl-N-tert-butoxycarbonyl-azetidin-3-yl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ] M ]⁺Calculation 1104.6, found 1105.1), which was carried on to the next step without further purification.

6' - (methyl-azetidin-3-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-azetidin-3-yl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.96mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to give a crude product, which was purified by reverse phase HPLC method 1-column B to give 6 ' - (methyl-azetidin-3-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0082g, 0.014mmol, 1.46% yield): MSm/e [ M + H]⁺Calculating 604.4 to obtain 604.6; the purity of CLND was 86.3%.

Example 74

6' - (methyl-1-aminomethyl-cyclopropyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (methyl-N-tert-butyloxycarbonyl-1-aminomethyl-cyclopropyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (2- (N-tert-butyloxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Following step 1, method a, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with N-tert-butoxycarbonyl-l-aminomethyl-cyclopropanecarboxaldehyde to give 6 ' - (methyl-N-tert-butoxycarbonyl-1-aminomethyl-cyclopropyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H). ]⁺Calculation 1144.6, found 1144.8), which was carried on to the next step without further purification.

6' - (methyl-1-aminomethyl-cyclopropyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-1-aminomethyl-cyclopropyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removing tert-butoxycarbonyl to give a crude product, it was purified by reverse phase HPLC method 1-column a to give 6' - (methyl-1-aminomethyl-cyclopropyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0005g, 0.0008mmol, 0.9% yield): MS M/e [ M + H ]]⁺Calculating 644.4 to obtain 644.6; the purity of CLND was 79.8%.

Example 75

6' - (2-hydroxy-ethyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (2- (N-tert-butyloxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butyloxycarbonyl-1- (2- (N-tert-butyloxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with tert-butyldimethylsilyloxyacetaldehyde to give target 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ]/] ]⁺Calculation 1119.6, found 1119.8), which was carried on to the next step without further purification.

6' - (2-hydroxy-ethyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' - (2-tert-Butyldimethylsilyloxy-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to give a crude product, which was purified by reverse phase HPLC method 1-column A to give 6 ' - (2-hydroxy-ethyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0037g, 0.0061mmol, yield 7.5%): MS M/e [ M + H ]]⁺Calculating 605.3 to obtain 605.7; the purity of CLND was 82.4%.

Example 76

6' - (3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was treated with N-phthalimidopropional to give target 6 ' - (N-phthalimido-3-amino-propyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ]/] ]⁺Calculation 1148.6, found 1148.8), which was carried on to the next step without further purification.

6 '- (3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (N-phthalimido-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 6 for phthalimido deprotection to give 6 '- (3-amino-propyl) -2', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 1018.6, found 1018.9), which was carried on to the next step without further purification.

6' - (3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' - (3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of the tert-butoxycarbonyl group to give the crude product, which was purified by reverse phase HPLC method 1-column A to give 6 ' - (3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.003g, 0.0048mmol, 5.9% yield): MSm/e [ M + H ]⁺Calculating 618.4 to obtain 618.8; the purity of CLND was 87.5%.

Example 77

6' - (2-hydroxy-4-amino-butyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (N-Boc-2-hydroxy-4-amino-butyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) with N-tert-butoxycarbonyl-2- (oxiran-2-yl) -carbamic acid ethyl ester as per step 5 gave 6 ' - (N-tert-butoxycarbonyl-2-hydroxy-4-amino-butyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azacyclo-aza-cyclo-star) as title compound (i) and (ii) as a suspensionButan-3-yl) -2-hydroxy-acetyl-sisomicin (MS M/e [ M + H)]⁺Calculated 1148.6, found 1148.9), which was carried on to the next step without further purification.

6' - (2-hydroxy-4-amino-butyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-2-hydroxy-4-amino-butyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (2-hydroxy-4-amino-butyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0013g, 0.002mmol, yield 2.5%): MS M/e [ M + H ] ]⁺Calculating 648.4 to obtain 648.4; the purity of CLND was 80.8%.

Example 78

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 '- (N-Boc-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-carbonyl) was treated with N-tert-butoxycarbonyl-trans-3-amino-cyclobutyl-carbaldehydeYl-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) to yield 6 '- (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] -as target]⁺Calculation 1144.6, found 1145.1), which was carried on to the next step without further purification.

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' - (N-tert-butoxycarbonyl-methyl-trans-3-amino-cyclobutyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.0025g, 0.0039mmol, 4.8%): MS M/e [ M + H ] ]⁺Calculating 644.4 to obtain 644.4; the purity of CLND was 93.9%.

Example 79

6' - (methyl-1-aminomethyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 '- (methyl-N-tert-butyloxycarbonyl-1-aminomethyl-cyclopropyl) -2', 3, 3 "-tri-tert-butyloxycarbonyl-1- (N-tert-butyloxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was treated with N-tert-butoxycarbonyl-1-aminomethyl-cyclopropanecarboxaldehyde to yield 6 ' - (methyl-N-tert-butoxycarbonyl-1-aminomethyl-cyclopropyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1144.6, found 1145.0), which was carried on to the next step without further purification.

6' - (methyl-1-aminomethyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-1-aminomethyl-cyclopropyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (methyl-1-aminomethyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin (0.0018g, 0.0028mmol, 3.5%) as a crude product: MS M/e [ M + H ] ]⁺Calculating 644.4 to obtain 644.6; the purity of CLND was 80.2%.

Example 80

6' - (4-hydroxy-5-amino-pentyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '-Nitrobenzenesulfonyl-2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) was subjected to step 8 for nitrobenzenesulfonyl to give 6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 1120.5, found 1120.9), which was carried on to the next step without further purification.

6 ' - (4, 5-epoxy-pentyl) -6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Treatment of 6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) with 5-bromo-1, 2-cyclopentane according to step 11 gave 6 ' - (4, 5-epoxy-pentyl) -6 ' -nitrobenzenesulfonyl-2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ]/] ]⁺Calculation 1204.5, found 1204.6), which was carried on to the next step without further purification.

6 ' - (4-hydroxy-5-amino-pentyl) -6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

According to step 5, 27% aqueous NH solution was used₃Treatment of 6 '- (4, 5-epoxy-pentyl) -6' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) to give 6' - (4-hydroxy-5-amino-pentyl) -6 '-nitrobenzenesulfonyl-2', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ]/]]⁺Calculation 1221.6, found 1222.2), which was carried on to the next step without further purification.

6 '- (4-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (4-hydroxy-5-amino-pentyl) -6 ' -nitrobenzenesulfonyl-2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 9 for nitrobenzenesulfonyl deprotection to give 6 ' - (4-hydroxy-5-amino-pentyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (MS M/e [ M + H ]/] ]⁺Calculation 1036.6, found 1037.1), which was carried on to the next step without further purification.

6' - (4-hydroxy-5-amino-pentyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 '- (4-hydroxy-5-amino-pentyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield crude product, which was purified by reverse phase HPLC method 1-column ATo give 6' - (4-hydroxy-5-amino-pentyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0020g, 0.0031mmol, 3.9% yield): MSm/e [ M + H]⁺Calculating 636.4 to obtain 636.4; the purity of CLND was 94.5%.

Example 81

6' - (N- (azetidin-3-yl) -2-amino-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

N- (N-Boc-azetidin-3-yl) -2-amino-ethanol

Following step 1, method a, N-tert-butoxycarbonyl-3-azetidinone (1.0g, 5.84mmol) was treated with ethanolamine to give N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-ethanol (0.75g, 3.46mmol, 62.3% yield): MS M/e [ M + H ]]⁺217.1 is calculated, and 217.2 is obtained.

N-Boc-N- (N-Boc-azetidin-3-yl) -2-amino-ethanol

N- (N-tert-Butoxycarbonyl-azetidin-3-yl) -2-amino-ethanol (0.75g, 3.46mmol) was subjected to step 13 for tert-butyloxycarbonyl protection to give a crude product, which was purified by flash chromatography (silica gel/N-hexane: ethyl acetate 0-100%) to give N-tert-butyloxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-ethanol (MS M/e [ M + H ])]⁺Calculate 317.2, find 317.4).

N-Boc-N- (N-Boc-azetidin-3-yl) -2-amino-acetaldehyde

N-tert-butoxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-ethanol was subjected to step 18 for oxidation to N-tert-butoxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-acetaldehyde, which was carried on to the next step without further purification.

6 '- (N-Boc-N- (N-Boc-azetidin-3-yl) -2-amino-ethyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin

Following step 1, method A, 2 ', 3, 3 "-tri-tert-butoxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-acetaldehyde was treated with N-tert-butoxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-acetaldehyde to give the corresponding 6 ' - (N-tert-butoxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-ethyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.075g, 0.080mmol) (MS M/e [ M + H ] ]⁺Calculation 1233.7, found 1233.9), which was carried on to the next step without further purification.

6' - (N- (azetidin-3-yl) -2-amino-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-N- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-amino-ethyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.080mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to give a crude product, which was purified by reverse phase HPLC method 1-column A to give 6 ' - (N- (azetidin-3-yl) -2-amino-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin (0.0069g, 0.011mmol, yield 13.7%): MS M/e [ M + H ]]⁺Calculating 633.4 to obtain 633.4; the purity of CLND was 85.5%.

Example 82

6' - (2-hydroxy-3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (N-Boc-2-hydroxy-3-amino-propyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

Treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) with N-tert-butyl- (2-oxiranyl-methyl) carbamate according to step 5 to give the target 6 ' - (N-tert-butoxycarbonyl-2-hydroxy-3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H). ]⁺Calculation 1134.6, found 1135.1), which was carried on to the next step without further purification.

6' - (2-hydroxy-3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' - (N-tert-Butoxycarbonyl-2-hydroxy-3-amino-propyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (2-hydroxy-3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0012g, 0.0018mmol, yield 2.3%): MS M/e [ M + H ]]⁺Calculating 634.4 to obtain 634.6; the purity of CLND was 82.5%.

Example 83

6' - (methyl-3-amino-1-hydroxy-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 '- (methyl-N-tert-butoxycarbonyl-3-amino-1-hydroxy-cyclobutyl) -2', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

According to step 5, N-tert-butyloxycarbonyl-1-oxaspiro [2.3 ] is used]Hexane-5-amine treatment of 2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmo1) to yield target 6 ' - (methyl-N-tert-butoxycarbonyl-3-amino-1-hydroxy-cyclobutyl) -2 ', 3, 3" -tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (MS M/e [ M + H ]/[ M +/e ]/] ]⁺Calculation 1160.6, found 1161.0), it is carried out to the next step without further oneAnd (5) purifying.

6' - (methyl-3-amino-1-hydroxy-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin

6 ' - (methyl-N-tert-butoxycarbonyl-3-amino-1-hydroxy-cyclobutyl) -2 ', 3, 3 "-tri-tert-butoxycarbonyl-1- (2- (N-tert-butoxycarbonyl-azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column A to yield 6 ' - (methyl-3-amino-1-hydroxy-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin (0.0013g, 0.0019mmol, yield 2.3%): MS M/e [ M + H ]]⁺Calculating 660.4 to obtain 660.4; the purity of CLND was 94.3%.

Example 84

2' - (methyl-pyrrolidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method B, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) was treated with N-tert-butoxycarbonyl-3-pyrrolidinecarboxaldehyde to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-3-yl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-3-yl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (methyl-pyrrolidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 2 '- (methyl-N-tert-butoxycarbonyl-pyrrolidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (methyl-pyrrolidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ]]⁺632.4 is calculated, 632.3, [ M + Na ] is obtained ]⁺654.4; the purity of CLND was 93.7%.

Example 85

2' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method B, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) was treated with N-tert-butoxycarbonyl-prolal to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H). ]⁺Computing1032.6, found 1032.5), which was carried on to the next step without further purification.

2' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 2 '- (methyl-N-tert-butoxycarbonyl-pyrrolidin-2-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ]]⁺632.4 is calculated, 632.3, [ M + Na ] is obtained]⁺654.4; the purity of CLND was 97.6%.

Example 86

2' - (N-methyl-amino-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-N-methyl-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.060g, 0.06mmol) was treated with N-tert-butoxycarbonyl-sarcosine to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-N-methyl-amino-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification, as per step 20.

2' - (N-Boc-N-methyl-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-N-methyl-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to give 2 ' - (N-tert-butoxycarbonyl-N-methyl-amino-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺1020.6 was calculated to obtain 1020.4), which was carried on to the next step without further purification.

2' - (N-methyl-amino-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 2 '- (N-tert-butoxycarbonyl-N-methyl-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (N-methyl-amino-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ] ]⁺Calculating 620.3 to obtain 620.3, [ M + Na [ ]]⁺642.3, respectively; the purity of CLND was 97.6%.

Example 87

2' - (2-amino-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-2-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.060g, 0.06mmol) was treated with N-tert-butoxycarbonyl-glycine according to step 20 to give the target 6 ' -para-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-2-amino-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (N-Boc-2-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-2-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to give 2 ' - (N-tert-butoxycarbonyl-2-amino-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (2-amino-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 2 '- (N-tert-butoxycarbonyl-2-amino-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) to step 3-method B for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (2-amino-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ]]⁺606.3 is calculated, 606.3, [ M + Na ] is obtained]⁺628.2, respectively; the purity of CLND was 97.4%.

Example 88

2' - (2-amino-propionyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-2-amino-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 4, method A, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.060g, 0.06mmol) was treated with N-tert-butoxycarbonyl-alanine to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-2-amino-propionyl) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl ) Sisomicin (MS M/e [ M + H)]⁺1199.6 is calculated, 1199.2, [ M + Na ] is obtained]⁺1221.4) which was carried on to the next step without further purification.

2' - (N-Boc-2-amino-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-2-amino-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to give 2 ' - (N-tert-butoxycarbonyl-2-amino-propionyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺1020.6, 1020.4, [ M + Na ] was obtained]⁺1042.4) which was carried on to the next step without further purification.

2' - (2-amino-propionyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 2 '- (N-tert-butoxycarbonyl-2-amino-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) to step 3-method B for removal of tert-butoxycarbonyl to give a crude product, which was purified by reverse phase HPLC method 1-column a to give 2' - (2-amino-propionyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0092g, 0.0148mmol, 24.7% yield): MS M/e [ M + H ] ]⁺Calculating 620.3 to obtain 620.2, [ M + Na ]]⁺642.4; the purity of CLND was 97.5%.

Example 89

2' - (3-amino-2-hydroxy-propionyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-3-amino-2-hydroxy-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 4, method A, 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.065g, 0.06mmol) to yield the target 6 '-p-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-3-amino-2-hydroxy-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺1215.6 is calculated, 1215.0, [ M + Na ] is obtained]⁺1237.3) which was carried on to the next step without further purification.

2' - (N-Boc-3-amino-2-hydroxy-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-3-amino-2-hydroxy-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to give 2 ' - (N-butylbenzyloxycarbonyl) -tert-Butoxycarbonyl-3-amino-2-hydroxy-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ]]⁺1036.6 is calculated, 1036.3, [ M + Na ] is obtained]⁺1058.4) which was carried on to the next step without further purification.

2' - (3-amino-2-hydroxy-propionyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting '- (N-tert-butoxycarbonyl-3-amino-2-hydroxy-propionyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) to step 3-method B for removal of tert-butoxycarbonyl to give a crude product, which was purified by reverse phase HPLC method 1-column a to give 2' - (3-amino 2-hydroxy-propionyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.005g, 0.008mmol, 13.3% yield): MS M/e [ M + H ]]⁺Calculate 636.3, find 636.2, [ M + Na ]]⁺658.3, respectively; the purity of CLND was 97.5%.

Example 90

2' - (pyrrolidin-2-yl-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-pyrrolidin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.060g, 0.06mmol) was treated with N-tert-butoxycarbonyl-proline to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-pyrrolidin-2-yl-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin according to step 20, which was subjected to the next step without further purification.

2' - (N-Boc-pyrrolidin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-pyrrolidin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (N-tert-butoxycarbonyl-pyrrolidin-2-yl-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (pyrrolidin-2-yl-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (N-tert-Butoxycarbonyl-pyrrolidin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.06mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column A to yield 2' - (pyrrolidin-2-yl-acetyl) -1- (4-amino-2-yl) to yield(S) -hydroxy-butyryl) -sisomicin: MS M/e [ M + H ]]⁺646.4 is calculated, 646.3, [ M + Na ] is obtained]⁺668.2, respectively; the purity of CLND was 78.0%.

Example 91

2' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-phthalimido-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a solution of 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.105g, 0.102mmol) in DMF (1mL) was added 3-phthalimido-propionaldehyde (0.041g, 0.204mmol) andmolecular sieves (10-15) and the reaction was shaken for 2 hours. Then adding NaCNBH₃(0.013g, 0.204mmol) in MeOH (3mL) and the reaction stirred overnight. The reaction was diluted with EtOAc (5mL) and saturated NH ₄Aqueous Cl solution, saturated NaHCO₃The organic layer was washed with aqueous solution (3mL) and brine (3mL) over Na₂SO₄Dried, filtered and concentrated to dryness to give 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-phthalimido-3-amino-propyl) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺1215.6 is calculated, 1215.3, [ M + Na ] is obtained]⁺1237.3) which was carried on to the next step without further purification.

6 '-para-nitrobenzyloxycarbonyl-2' - (3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-phthalimido-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.102mmol) was subjected to step 6 for removal of phthalimido to yield 6 '-para-nitrobenzyloxycarbonyl-2' - (3-amino-propyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺1085.5 is calculated, 1085.4, [ M + Na ] is obtained]⁺1107.4) which was carried on to the next step without further purification.

2' - (3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' - (3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.102mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to yield 2 ' - (3-amino-propyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 906.5, found 906.2), which was carried on to the next step without further purification.

2' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2' - (3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.102mmol) was subjected to a procedure for removal of tert-butoxycarbonyl group

Step 3-method B to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0021g, 0.0035mmol, 3.4% yield): MS M/e [ M + H ]]⁺606.4 is calculated, 606.2, [ M + Na ] is obtained]⁺628.3, respectively; the purity of CLND was 94.0%.

Example 92

2' - (morpholin-2-yl-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-morpholin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 4, method a, 6' -p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) to yield the target 6 '-p-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-morpholin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1255.6, found 1255.8), which was carried on to the next step without further purification.

2' - (N-Boc-morpholin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-morpholin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (N-tert-butoxycarbonyl-morpholin-2-yl-acetyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H). ]⁺1076.6 is calculated, 1076.3, [ M + Na ] is obtained]⁺1098.4) which was carried on to the next step without further purification.

2' - (morpholin-2-yl-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (N-tert-butoxycarbonyl-morpholin-2-yl-acetyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 2' - (morpholin-2-yl-acetyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0051g, 0.0075mmol, 10.3% yield): MSm/e [ M + H]⁺676.4 is calculated, 676.2, [ M + Na ] is obtained]⁺698.4, respectively; the purity of CLND was 96.2%.

Example 93

2' - (2-amino-ethyl-sulfonamide) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-phthalimido-2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

To a stirred solution of 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.108g, 0.105mmol) in DMF (1mL) at 0 deg.C was added DIPEA (0.054mL, 0.31mmol) followed by N-phthalimido-2-amino-ethanesulfonyl chloride (0.048g, 0.175mmol) and the reaction was allowed to warm to room temperature. The reaction was diluted with EtOAc (4mL) and washed with H ₂O (3X 4mL) wash. In Na₂SO₄The combined organic layers were dried, filtered and concentrated to give 6 '-p-nitrobenzyloxycarbonyl-2' - (N-phthalimido-2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺1265.5 is calculated, 1265.3, [ M + Na ] is obtained]⁺1287.2) which was carried on to the next step without further purification.

6 '-para-nitrobenzyloxycarbonyl-2' - (2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Reacting 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-phthalimido-2-amino-ethylsulfonamide) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (S)0.105mmol) was performed for the removal of phthalimido group step 6 to yield 6 '-p-nitrobenzyloxycarbonyl-2' - (2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H)]⁺Calculation 1135.5, found 1134.9), which was carried on to the next step without further purification.

2' - (2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' - (2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.105mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to yield 2 ' - (2-amino-ethylsulfonamide) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺956.5 is calculated, 956.2, [ M + Na ] is obtained]⁺978.3) which was carried on to the next step without further purification.

2' - (2-amino-ethylsulfonamide) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (2-amino-ethylsulfonamide) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.105mmol) was subjected to step 3-method B for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by reverse phase HPLC method 1-column A to yield 2' - (2-amino-ethylsulfonamide) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.016g, 0.0244mmol,yield 23.2%): MS M/e [ M + H ]]⁺656.3 is calculated to obtain 656.1, [ M + Na ]]⁺678.3; the purity of CLND was 92.3%.

Example 94

2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.200g, 0.195mmol) was treated with N, N-dimethyl-2, 2-dimethyl-3-amino-propionaldehyde (0.033g, 0.25mmol) to give the target 6 '-p-nitrobenzyloxycarbonyl-2' - (N), n-dimethyl-2, 2-dimethyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1141.6, found 1141.5), which was carried on to the next step without further purification.

2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Reacting 6 '-p-nitrobenzyloxycarbonyl-2' - (N, N-dimethyl-2, 2-dimethyl-3)-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.195mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H) -sisomicin (MS M/e) ]⁺962.6 is calculated, 962.4, [ M + Na ] is obtained]⁺984.4) which was carried on to the next step without further purification.

2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.195mmol) was subjected to step 3 for removal of tert-butoxycarbonyl-method B to give a crude product, it was purified by reverse phase HPLC method 1-column a to give 2' - (N, N-dimethyl-2, 2-dimethyl-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.00069g, 0.001mmol, 0.5% yield): MS M/e [ M + H ]]⁺662.4 is calculated, 662.3, [ M + Na ] is obtained]⁺684.3, respectively; the purity of CLND was 86.2%.

Example 95

2' - (2(S) -amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-2 (S) -amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.200g, 0.195mmol) was treated with N-tert-butoxycarbonyl-2 (S) -amino-propionaldehyde to give the target 6 ' -para-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-2 (S) -amino-propyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification, according to step 1-method A.

2' - (N-Boc-2 (S) -amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

(iv) subjecting 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-2 (S) -amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.195mol) to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (N-tert-butoxycarbonyl-2 (S) -amino-propyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 1006.6, found 1007.1), which was carried on to the next step without further purification.

2' - (2(S) -amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (N-tert-Butoxycarbonyl-2 (S) -amino-propyl) -3, 3' -di-tert-butyloxycarbonylYl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.195mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (2(S) -amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0035g, 0.0058mmol, 3.0% yield): MS M/e [ M + H ] ]⁺Calculating 606.4 to obtain 606.3; the purity of CLND was 89.4%.

Example 96

2' - (azetidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-azetidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.200g, 0.195mmol) to yield the target 6 '-p-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-azetidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MSm/e [ M + H).]⁺Calculation 1183.6, found 1184.3), which was carried on to the next step without further purification.

2' - (N-Boc-azetidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-azetidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.195mmol) was subjected to step 2 for removal of para-nitrobenzyloxycarbonyl to yield 2 ' - (N-tert-butoxycarbonyl-azetidin-3-yl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] -) ]⁺Calculation 1004.6, found 1005.1), which was carried on to the next step without further purification.

2' - (azetidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 2 '- (N-tert-butoxycarbonyl-azetidin-3-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.195mmol) to step 3-method B for removal of the tert-butoxycarbonyl group to yield a crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (azetidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0144g, 0.024mmol, 12.3%) as: MS M/e [ M + H ]]⁺604.4 is calculated, and 604.2, [ M + Na ] is obtained]⁺626.3, respectively; the purity of CLND was 99.2%.

Example 97

2' - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (methyl-N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidin-4-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.100g, 0.097mmol) was treated with N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidin-4-carbaldehyde (0.026g, 0.12mmol) to produce the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (methyl-N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidin-4-yl) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) - Sisomicin (MS M/e [ M + H ] ]⁺Calculation 1241.6, found 1242.1), which was carried on to the next step without further purification.

2' - (methyl-N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidin-4-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (methyl-N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidin-4-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.097mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to produce 2 ' - (methyl-N-tert-butoxycarbonyl-2, 2-dimethyl-1, 3-oxazolidin-4-yl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS m |) e [ M + H]⁺Calculation 1062.6, found 1063.3), which was carried on to the next step without further purification.

2' - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (methyl-N-tert-butoxycarbonyl-2, 2-methyl-1, 3-oxazolidin-4-yl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.097mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield a crude product, which was purified by reverse phase HPLC method 1-column a to yield 2' - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0042g, 0.0067mmol, 6.9%) as a crude product: MSm/e [ M + H ]⁺622.4 was calculated, and 622.3, [ M + Na ] was obtained]⁺644.4, respectively; the purity of CLND was 93.9%.

Example 98

2' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (2-tert-butyldimethylsilyloxy-ethyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) was treated with tert-butyldimethylsilyloxyacetaldehyde to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (2-tert-butyldimethylsilyloxy-ethyl) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ] M]⁺Calculation 1186.6, found 1187.1), it is carried out to the next step without the need forAnd (5) further purifying.

2' - (2-tert-Butyldimethylsilyloxy-ethyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (2-tert-butyldimethylsilyloxy-ethyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for the removal of p-nitrobenzyloxycarbonyl to give 2 ' - (2-tert-butyldimethylsilyloxy-ethyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (2-tert-butyldimethylsilyloxy-ethyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3-method a for removal of tert-butoxycarbonyl to yield the crude product, which was purified by method 3 to yield 2' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0107g, 0.018mmol, 24.6% yield): MSm/e [ M + H]⁺593.3 is calculated, 593.8 is obtained; the purity of CLND was 95.9%.

Example 99

2' - (2, 5-diamino-pentanoyl) -1- (4-amino-2 (S) -hydroxy-butanoyl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl, N-tert-butoxycarbonyl-2, 5-diamino-pentanoyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 4, method B, 6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) was treated with tert-butoxycarbonyl-DL-ORN (tert-butoxycarbonyl) -OH to give the target 6 '-p-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl), N-Boc-2, 5-diamino-pentanoyl) -3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MSm/e [ M + H). ]⁺Calculation 1342.7, found 1342.7), which was carried on to the next step without further purification.

2 '- (N-Boc, N-Boc-2, 5-diamino-pentanoyl) -3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl, N-tert-butoxycarbonyl-2, 5-diamino-pentanoyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to produce 2 ' - (N-tert-butoxycarbonyl, N-tert-butoxycarbonyl-2, 5-diamino-pentanoyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, it was carried on to the next step without further purification.

2' - (2, 5-diamino-pentanoyl) -1- (4-amino-2 (S) -hydroxy-butanoyl) -sisomicin

2 '- (N-tert-Butoxycarbonyl, N-tert-butoxycarbonyl-2, 5-diamino-pentanoyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3 for removal of tert-butoxycarbonyl group-method A to yield the crude product, which was purified by method 3 to yield 2' - (2, 5-diamino-pentanoyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0075g, 0.0113mmol, 15.5%) as a crude product: MS M/e [ M + H ] ]⁺Calculating 663.4 to obtain 663.4; the purity of CLND was 94.8%.

Example 100

2' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (2-hydroxy-propanol) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method A, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) was treated with DL-glyceraldehyde dimer to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (2-hydroxy-propanol) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (3: (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (S, N-tert-butoxycarbonyl-2-hydroxy-propanol))MSm/e[M+H]⁺Calculated 1102.5 to obtain 1103.2), which was carried on to the next step without further purification.

2' - (2-hydroxy-propanol) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-Nitrobenzyloxycarbonyl-2 ' - (2-hydroxy-propanol) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for the removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (2-hydroxy-propanol) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (2-hydroxy-propanol) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3-method a for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by method 3 to yield 2' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0008g, 0.00128mmol, 1.75% yield): MSm/e [ M + H]⁺Calculating 623.3 to obtain 623.8; the purity of CLND was 94.7%.

Example 101

2' - (2-hydroxy-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (2-hydroxy-N-tert-butoxycarbonyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-p-nitrobenzyloxycarbonyl-3, 3' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) to yield the target 6 '-p-nitrobenzyloxycarbonyl-2' - (2-hydroxy-N-tert-butoxycarbonyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MSm/e [ M + H). ]⁺Calculation 1201.6, found 1201.6), which was carried on to the next step without further purification.

2' - (2-hydroxy-N-tert-butoxycarbonyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (2-hydroxy-N-tert-butoxycarbonyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' - (2-hydroxy-N-tert-butoxycarbonyl-3-amino-propyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MSm/e [ M + H).]⁺Calculation 1022.6, found 1023.1), which was carried on to the next step without further purification.

2' - (2-hydroxy-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (2-hydroxy-N-tert-butoxycarbonyl-3-amino-propyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3-method a for removal of tert-butoxycarbonyl to yield the crude product, which was purified by method 3 to yield 2' - (2-hydroxy-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0112g, 0.018mmol, 24.6%) by: MSm/e [ M + H ]⁺Calculating 622.4 to obtain 622.6; the purity of CLND was 88.3%.

Example 102

2' - (4-amino-butyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' -nitrobenzenesulfonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) was treated with 2-nitrobenzenesulfonyl chloride according to step 8 to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' -nitrobenzenesulfonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

6 ' -para-nitrobenzyloxycarbonyl-2 ' -nitrobenzenesulfonyl-2 ' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' -nitrobenzenesulfonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was treated with N-tert-butoxycarbonyl-4-amino-1-butanol according to step 17 to give the target 6 ' -para-nitrobenzyloxycarbonyl-2 ' -nitrobenzenesulfonyl-2 ' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H). ]⁺Calculation 1384.6, found 1384.2), which was carried on to the next step without further purification.

6 '-para-nitrobenzyloxycarbonyl-2' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' -nitrobenzenesulfonyl-2 ' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 9 for nitrobenzenesulfonyl deprotection to yield the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MSm/e [ M + H).]⁺Calculation 1199.6, found 1200.1), which was carried on to the next step without further purification.

2' - (N-Boc-4-amino-butyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-nitrobenzyloxycarbonyl-2 ' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 2 for the removal of p-nitrobenzyloxycarbonyl to yield the target 2 ' - (N-tert-butoxycarbonyl-4-amino-butyl) -3, 3" -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (4-amino-butyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (N-tert-Butoxycarbonyl-4-amino-butyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3-method A for removal of tert-butyloxycarbonyl to yield the crude product, which was purified by method 3 to yield 2' - (4-amino-butyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.00065g, 0.001mmol, 1.37%) as: MSm/e [ M + H]⁺Calculating 620.4 to obtain 620.8; the purity of CLND was 85.6%.

Example 103

2' -guanidine-1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' -guanidine-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 7, 6 ' -p-nitrobenzyloxycarbonyl-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.7g, 0.68mmol) was treated with 1H-pyrazole-1-carboxamidine hydrochloride (0.142g, 0.96mmol) to give the target 6 ' -p-nitrobenzyloxycarbonyl-2 ' -guanidine-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MSm/e [ M + H ] M ]⁺Calculation 1070.5, found 1070.8), which was carried on to the next step without further purification.

2' -guanidine-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 ' -p-Nitrobenzyloxycarbonyl-2 ' -guanidine-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.68mmol) was subjected to step 2 for removal of p-nitrobenzyloxycarbonyl to yield 2 ' -guanidine-3, 3 ' -di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H ])]⁺Calculation 891.5, found 891.9), which was carried on to the next step without further purification.

2' -guanidine-1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '-guanidine-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.68mmol) was subjected to step 3-method B for removal of tert-butoxycarbonyl to yield the crude product, which was purified by reverse phase HPLC method 1-column B to yield 2' -guanidine-1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.110g, 0.186mmol, 27.4% yield): MS M/e [ M + H ]]⁺Calculating 591.3 to obtain 591.6; the purity of CLND was 97.5%.

Example 104

2' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

6 '-para-nitrobenzyloxycarbonyl-2' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Following step 1, method a, 6' -p-nitrobenzyloxycarbonyl-3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-3-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.075g, 0.073mmol) to yield the target 6 '-p-nitrobenzyloxycarbonyl-2' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (MS M/e [ M + H).]⁺Calculation 1211.6, found 1212.0), which was carried on to the next step without further purification.

2' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin

Subjecting 6 '-p-nitrobenzyloxycarbonyl-2' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) to step 2 for removal of p-nitrobenzyloxycarbonyl to yield

2' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin, which was subjected to the next step without further purification.

2' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin

2 '- (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -3, 3 "-di-tert-butoxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.073mmol) was subjected to step 3-method a for removal of tert-butoxycarbonyl to yield the crude product, which was purified by method 3 to yield 2' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin (0.0103g, 0.016mmol, 21.9%) as a crude product: MSm/e [ M + H]⁺632.4 is calculated, 632.8 is obtained; the purity of CLND was 90.4%.

Example 105

6 ', 2' -biguanide-sisomicin

6 ', 2' -biguanide-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin

1, 3, 3 ' -Tri-tert-Butoxycarbonyl-sisomicin (0.075g, 0.100mmol) was treated with 1H-pyrazole-1-carboxamidine hydrochloride (0.037g, 0.25mmol) to give the target 6 ', 2 ' -biguanide-1, 3, 3 "-Tri-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ]) ]⁺832.5 was calculated to obtain 832.8), which was carried on to the next step without further purification.

6 ', 2' -biguanide-sisomicin

6 ', 2' -biguanide-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.100mmol) was subjected to step 3-method a for removal of tert-butoxycarbonyl to yield the crude product, which was purified by method 3 to yield 6 ', 2' -biguanide-sisomicin (0.0017g, 0.0032mmol, 3.2% yield): MS M/e [ M + H ]]⁺532.3 is calculated, 532.6 is obtained; the purity of CLND was 92.2%.

Example 106

6 '- (2-hydroxy-ethyl) -2' -guanidine-sisomicin

6 ' -para-nitrobenzyloxycarbonyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 ' -tri-tert-butoxycarbonyl-sisomicin

According to step 7, N-di-tert-butyl is usedTreatment of 6 ' -p-nitrobenzyloxycarbonyl-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.075g, 0.081mmol) with butoxycarbonyl-1H-pyrazole-1-carboxamidine to give the target 6 ' -p-nitrobenzyloxycarbonyl, 2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺Calculated 1169.6, found 1170.1), which was carried on to the next step without further purification.

2 '-N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3' -tri-tert-butoxycarbonyl-sisomicin

6 ' -p-nitrobenzyloxycarbonyl, 2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was subjected to the step 10 for removal of p-nitrobenzyloxycarbonyl to yield the target 2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin (MSm/e [ M + H ])]⁺Calculation 990.5, found 990.9), which was carried on to the next step without further purification.

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin

Following step 1, method A, 2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was treated with tert-butyldimethylsilyloxyacetaldehyde to yield the target 6 ' - (2-tert-butyldimethylsilyloxy-ethyl) -2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin (MSm/e [ M + H ])]⁺Calculation 1148.7, found 1149.1), which was carried on to the next step without further purification.

6 '- (2-hydroxy-ethyl) -2' -guanidine-sisomicin

6 '- (2-tert-Butyldimethylsilyloxy-ethyl) -2' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was subjected to step 3-method A for removal of tert-butoxycarbonyl and TBS to yield the crude product, which was purified by method 1-column A to yield 6 '- (2-hydroxy-ethyl) -2' -guanidine-sisomicin (0.00096g, 0.0018mmol, 2.2% yield): MS M/e [ M + H ] ]⁺534.3 is calculated, 534.2 is obtained; the purity of CLND was 84.4%.

Example 107

6 '- (methyl-trans-3-amino-cyclobutyl) -2' -guanidine-sisomicin

6 '- (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin

Following step 1, method A, 2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was treated with N-tert-butoxycarbonyl-trans-3-amino-cyclobutyl-carbaldehyde to give target 6 ' - (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ] M]⁺Calculation 1173.7, found 1174.1), which was carried on to the next step without further purification.

6 '- (methyl-trans-3-amino-cyclobutyl) -2' -guanidine-sisomicin

6 '- (methyl-trans-N-tert-butoxycarbonyl-3-amino-cyclobutyl) -2' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was subjected to step 3-method A for removal of the tert-butoxycarbonyl group to yield the crude product, which was purified by method 1-column A to yield 6 '- (methyl-trans-3-amino-cyclobutyl) -2' -guanidine-sisomicin (0.001g, 0.0017mmol, 2.1% yield): MSm/e [ M + H ]⁺573.4 is calculated to obtain 573.1; the purity of CLND was 86.8%.

Example 108

6 '-methyl-2' -guanidine-sisomicin

6 ' -Nitrobenzenesulfonyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 ' -tri-tert-butoxycarbonyl-sisomicin

2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was treated with 2-nitrobenzenesulfonyl chloride according to step 8 to give the target 6 ' -nitrobenzenesulfonyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin, which was subjected to the next step without further purification.

6 '-Nitrobenzenesulfonyl-6' -methyl-2 '-N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3' -tri-tert-butoxycarbonyl-sisomicin

6 ' -Nitrobenzenesulfonyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was treated with iodomethane according to step 11 to give the target 6 ' -nitrobenzenesulfonyl-6 ' -methyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H +/M /)]⁺Calculate 1189.5, find 1190.0), proceed to the next step without further purification.

6 ' -methyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 ' -tri-tert-butoxycarbonyl-sisomicin

6 ' -Nitrobenzenesulfonyl-6 ' -methyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was subjected to step 9 for nitrobenzenesulfonyl deprotection to give the target 6 ' -methyl-2 ' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3" -tri-tert-butoxycarbonyl-sisomicin (MS M/e [ M + H ])]⁺Calculation 1004.6, found 1005.1), which was carried on to the next step without further purification.

6 '-methyl-2' -guanidine-sisomicin

6 '-methyl-2' -N, N-di-tert-butoxycarbonyl-guanidine-1, 3, 3 "-tri-tert-butoxycarbonyl-sisomicin (0.081mmol) was subjected to step 3 for removal of tert-butoxycarbonyl-method A to yield the crude product, which was purified by method 1-column A to yield 6 '-methyl-2' -guanidine-sisomicin (0.0029g, 0.0058mmol, 7.1% yield): MS M/e [ M + H ]]⁺Calculating 504.3 to obtain 504.4;the purity of CLND was 94.3%.

Example 109

Wherein at least one R can be prepared by the general synthetic and purification procedures described above₉A compound of structure (I) wherein the group is hydrogen:

for example, in the syntheses of examples 1-108, the corresponding 3 "and 4" demethylated compounds were prepared and purified from the crude product using method 1 or method 3 of the general purification steps described above.

Example 110

MIC test protocol

Per M7-A7[2006 ] by reference to the Clinical and Laboratory Standardization Institute (CLSI) liquid Medium microdilution method]The Minimum Inhibitory Concentration (MIC) was determined. Quality control ranges using E.coli ATCC25922, P.aeruginosa ATCC27853 and Staphylococcus aureus (S.aureus) ATCC 29213 and the interpretation criteria of the comparison reagents are described in CLSIM100-S17[2007]Is published in (1). Briefly, serial dilutions of two-fold of test compound were prepared at 2X concentration in Mueller Hinton broth. Dilutions of compounds were mixed with bacterial inoculum in a 1: 1 ratio in 96-well assay plates. The inoculum was prepared by suspension of colonies from agar plates prepared the previous day. Bacteria were suspended in sterile saline and added to each assay plate to obtain 5 × 10⁵Final concentration of CFU/mL. The plates were incubated at 35 ℃ for 20 hours in ambient air. MIC was determined as the lowest concentration of test compound that did not cause visible bacterial growth compared to untreated controls. Shown in Table 1 belowData are presented for some representative compounds.

TABLE 1

Example #	AEC0001	APAE001
			1	A	B
2	B	B
			3	B	C
4	B	B
			5	A	B
6	B	B
			7	A	B
8	A	B
			9	B	C
10	B	B
			11	A	B
12	B	B
			13	B	C
14	B	B
			15	A	B
16	A	B
			17	A	B
18	A	B

19	A	B
			20	C	C
21	B	B
			22	B	B
23	C	C
			24	B	B
25	B	B
			26	B	B
27	B	C
			28	B	B
29	B	C
			30	A	B
31	B	B
			32	A	B
33	A	B
			34	A	B
35	A	B
			36	A	B
37	A	B
			38	A	B
39	A	B
			40	B	B
41	A	B
			42	B	B
43	A	A
			44	A	B
45	A	B
			46	A	B
47	B	B
			48	A	B
49	A	B
			50	C	C
51	A	C
			52	A	B
53	B	C
			54	A	B
55	B	C
			56	A	C
57	A	A
			58	A	B
59	A	B
			60	A	B
61	A	B
			62	A	B
63	A	B
			64	A	B
65	B	B
			66	A	B
67	B	B
			68	B	B
69	A	B
			70	B	C
71	B	C
			72	B	B
73	B	B
			74	B	C

75	B	C
			76	B	B
77	B	B
			78	A	B
79	B	C
			80	A	A
81	B	C
			82	B	C
83	B	C
			84	A	B
85	A	B
			86	B	B
87	B	B
			88	B	B
89	A	B
			90	A	B
91	A	A
			92	A	C
93	A	B
			94	B	C
95	A	C
			96	A	B
97	A	B
			98	B	B
99	B	B
			100	B	C
101	A	B
			102	A	B
103	A	A
			104	A	B
105	C	C
			106	A	B
107	B	A
			108	A	B

AECO001 is ATCC25922 and APAE001 is ATCC 27853.

MIC example:

MIC 1.0. mu.g/mL or less ═ A

MIC 1.0 μ g/mL to 16.0 μ g/mL ═ B

MIC greater than 16.0 [ mu ] g/mL ═ C

Example 111

In vivo efficacy model

As shown in table 2 below, some representative compounds and some known aminoglycosides (i.e., gentamicin and amikacin) were tested for in vivo efficacy in a mouse sepsis model of infection. Two models were run against each compound using escherichia coli and pseudomonas aeruginosa QC bacterial strains. The same design was used for both studies. Male CD-1(CRL) -derived mice (each weighing 24. + -. 2 grams) were inoculated with a 2 XDD 90-100 dose of E.coli ATCC 25922 (4.5X 105 CFU/mouse) in 0.5mL of BHI broth containing 5% mucin or 2 XDD 90-100 dose of Pseudomonas aeruginosa ATCC 27853 (5.8X 104CFU/0.5 mL/mouse) in BHI broth containing 5% mucin with IP. 1 hour after bacterial challenge, mice received a single SC or IV dose of vehicle or test matrix to evaluate anti-infective activity in vivo. Mortality was recorded once daily for 7 days after bacterial inoculation. As shown in table 2, all single IV or SC doses of test compound increased survival in a dose-dependent manner in both studies.

TABLE 2

MIC example:

MIC 1.0. mu.g/mL or less ═ A

MIC 1.0 μ g/mL to 16.0 μ g/mL ═ B

MIC greater than 16.0 [ mu ] g/mL ═ C

ED50 value of mg/kg

Example 112

As shown in table 3 below, certain disubstituted sisomicin derivatives, certain polysubstituted sisomicin derivatives, and sisomicin were tested for a defined resistance mechanism comprising covalent modification of the 6' -amino group in a number of aminoglycosides against QC and aminoglycoside resistant bacterial strains. These MIC tests were performed according to the same protocol as set forth in example 110. As shown, substituted sisomicin derivatives having a non-methyl group at the 6 'position have increased activity against strains expressing the AAC 6' -modified enzyme. Furthermore, disubstituted sisomicin derivatives exhibit higher activity on those strains expressing the AAC 6' -modified enzyme relative to the monosubstituted derivatives.

TABLE 3

Test compounds	AECO001	AECO040	ASMA003	AACA005
					Sisomicin	0.5	32	8	32
Monosubstituted compounds 1	1	＞64	1	2
					Monosubstituted compounds 2	1	1	0.5	4
Monosubstituted compounds 3	0.5	0.25	1	0.5
					Monosubstituted compounds 4	2	16	1	1
Monosubstituted compounds 5	0.5	8	2	32
					Monosubstituted compounds 6	0.5	4	4	16
Monosubstituted compounds 7	1	4	16	32
					Example 1	0.5	0.5	2	2
Example 12	1	0.5	4	2
					Example 13	1	0.125	2	2
Example 16	1	1	2	2
					Example 17	1	0.5	2	2
Example 18	1	0.25	4	2
					Example 48	1	0.5	2	2
Example 61	1	16	4	2

Diagram x:

comparative compounds:

example 113

To determine the activity of representative antibacterial aminoglycoside compounds against bacterial species that commonly cause complicated urinary tract infections worldwide, isolates were collected from urine or blood from bacteremia sources documented as urinary tract infections at 47 medical centers in the united states and europe (10 countries). The strains were tested for susceptibility to gentamicin, amikacin, example 1 and 10 of the comparisons by CLSI broth microdilution. These MIC tests were performed according to the same protocol as set forth in example 110 below. Pathogens (total 169) include escherichia coli (e.coli), Klebsiella spp, Enterobacter spp, Citrobacter spp, proteus mirabilis, morganella spp, pseudomonas aeruginosa, staphylococcus aureus (s.aureus), and staphylococcus saprophyticus (s.saprophyticus).

MIC of example 1 among E.coli, Klebsiella, Enterobacter and Citrobacter, as shown in Table 4 below₉₀Values of 0.5 to 2. mu.g/ml. Higher MIC was observed for Proteus mirabilis and Morganella ₉₀Values (4-8. mu.g/ml) and highest are Pseudomonas aeruginosa (MIC)₉₀16. mu.g/ml). Example 1 MIC for a peptide with ≦ 0.25/0.5 μ g/ml_50/90The saprophytic staphylococci activity of value was very strong. Staphylococcus aureus isolates containing 43.5% oxacillin-resistant strains had a MIC of 2 μ g/ml₉₀. The total susceptibility rates of gentamicin and amikacin are 86.4% and 94.1% respectively; and example 1 inhibited 90.5% and 98.8% of the strains at ≤ 4 μ g/ml and ≤ 16 μ g/ml, respectively. The susceptibility of staphylococci to ciprofloxacin, piperacillin/tazobactam (pip/tazo), nitrofurantoin and trimethoprim/sulfanilamide (trim/sulfa) was 61.9%, 73.8%, 33.3% and 100%, respectively. The susceptibility rates of ciprofloxacin, piperacillin/tazobactam and trimethoprim/sulfonamides to enteric pathogens are 77.8%, 88.0% and 68.5%, respectively. The susceptibility rate of pseudomonas aeruginosa isolates to ciprofloxacin was only 52.6% and the susceptibility rate to piperacillin/tazobactam was 73.7%.

Example 1 is active against gram negative pathogens, staphylococcus aureus and staphylococcus saprophyticus causing cases of complicated urinary tract infections, even in the presence of antibiotic resistance causing oxacillin resistance up to the current first line.

TABLE 4

Example 114

In vivo efficacy of novel glycosides (Neoglycosides) on Enterobacteriaceae and MRSA

In vivo activity of example 1 was determined in a mouse granulocyte-reduction strand model against seven bacterial strains including susceptible escherichia coli and klebsiella pneumoniae (klebsiella pneumoniae); multi-drug resistant (MDR) clinical isolates of escherichia coli and klebsiella pneumoniae that exhibit resistance to a variety of antibiotics, including AG; MRSA; and two Klebsiella Pneumoniae Carbapenemases (KPC) expressing the strains (see Table 5) (Andes and Craig. Antimicrob Agents Chemother.2002, 46: 1665-1670). For this efficacy model, groups of six CD-1 mice were granulocytopenic by two intraperitoneal injections of cyclophosphamide. The first injection was 150mg/kg three days before infection (day 4) and the second injection was 100mg/kg one day before infection (day 1). In the study on day 0, animals were inoculated intramuscularly (0.1ml) with known amounts of Colony Forming Units (CFU) of specific bacterial strains (ATCC 25922, AECO 1003, ATCC 43816, AKPN 1073, AKPN 1109, ATCC 33591 or ASMA 1030), quantified as toxicity to maximum bacterial load of each strain in the model while avoiding mortality in untreated controls. Following bacterial challenge, antibiotics were given by subcutaneous injection at 2 and 14 hours. At 26 hours, infected femoral tissues were harvested, homogenized and plated to calculate CFU. Untreated control animals were harvested at 2 hours post-infection to evaluate initial bacterial load and growth without antibiotic treatment was detected at 26 hours post-infection.

Example 1 performed well on all 7 strains including gram-negative MDR strains and MRSA, reducing bacterial titers (bacterial titers) in each case to or below the initial bacterial load (i.e. static level). Table 5 shows MIC, ED of the tested bacterial strains₅₀And ED₅₀The ratio/MIC. In vivo efficacy and in vitro Activity (ED) of example 1₅₀MIC) was the same as gentamicin, which indicates that example 1 maintained the favorable pharmacokinetic/pharmacodynamic properties of currently marketed Aminoglycosides (AGs). In contrast to strains susceptible to gentamicin, example l showed the same in vivo Efficacy (ED) as gentamicin₅₀). However, gentamicin was ineffective when gentamicin resistant strains were used (ED)₅₀> 64mg/kg) and example 1 was effective.

Example 1 the efficacy dose response against MDR strains of e.coli (figure 1), two Klebsiella strains (Klebsiella) strains (figures 2 and 3) and MRSA strain (figure 4) can be compared to other antibiotics. Example 1 gentamicin, ciprofloxacin and imipenem (positive control) vs 1.5X 10³The invasion activity of CFU escherichia coli (AECO 1003) resistant to AG clinical isolates was comparable (fig. 1). After 24 hours of treatment with the highest dose of example 1, the bacterial titer dropped below the initial bacterial load determined 2 hours after inoculation.

Similarly, example 1, gentamicin and imipenem (positive control) vs. 1.3 × 10⁴The invasive activity of AG-resistant clinical isolates of CFU klebsiella pneumoniae (k. pneumoconiae) (AKPN 1073) was comparable (fig. 2). After 24 hours of treatment with the two highest doses of example 1, the bacterial load was reduced to a level below the initial bacterial load determined 2 hours after inoculation.

Example 1 Gentamicin, Imipenem and ciprofloxacin 8.3X 10⁵The KPC expression of CFU klebsiella pneumoniae (AKPN 1109) was comparable in the invasive activity of the clinical isolates (fig. 3). After 24 hours of treatment with the highest test dose of example 1, the bacterial load was reduced to a level below the initial bacterial load determined 2 hours after inoculation.

Also, example 1, arbekacin, gentamicin, vancomycin and daptomycin pairs 1.2X 10³The invasive activity of CFU MRSA (ATCC 33591) was comparable (fig. 4). After 24 hours of treatment with the two highest doses of test example 1, the bacterial load decreased to a level below the initial bacterial load determined 2 hours after inoculation.

These results indicate that example 1 can meet the increasing unmet medical needs for a number of indications, the main pathogen being the drug-resistant gram-negative pathogen of enterobacter. In addition, it is highly advantageous to have bactericidal properties against MRSA. Example 1 demonstrates good in vivo activity against susceptible and MDR bacterial strains tested in this model. These results provide in vivo evidence of the in vitro activity of example 1 against gram-negative bacterial strains including those expressing multidrug resistance mechanisms.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications included in this specification are incorporated herein by reference, in their entirety, consistent with this specification.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (58)

1. A method for treating a urinary tract infection in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an antibacterial aminoglycoside compound.

2. The method of claim 1, wherein the urinary tract infection is a complicated urinary tract infection.

3. The method of claim 1 or 2, wherein the antibacterial aminoglycoside compound is amikacin, gentamicin, tobramycin, lidoxine, apramycin, streptomycin, kanamycin, dibekacin, arbekacin, sisomicin, paromomycin, xanthomycin, thiostrepton, neomycin, netilmicin, or a modified derivative of any of the foregoing.

4. The method of claim 1 or 2, wherein the antibacterial aminoglycoside compound has the following structure (I):

or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof,

wherein:

Q₁is hydrogen,

Q₂Is hydrogen, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -C (═ NH) NR ₄R₅、-(CR₁₀R₁₁)_pR₁₂、

Q₃Is hydrogen, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -C (═ NH) NR₄R₅、-(CR₁₀R₁₁)_pR₁₂、

Each R is₁、R₂、R₃、R₄、R₅、R₈And R₁₀Independently is hydrogen or C₁-C₆A hydrocarbon group, or R₁And R₂Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R₂And R₃Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms, or R₁And R₃Together with the atoms to which they are attached can form a carbocyclic ring having from 4 to 6 ring atoms, or R₄And R₅Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R is₆And R₇Independently hydrogen, hydroxy, amino or C₁-C₆A hydrocarbon group, or R₆And R₇Together with the atoms to which they are attached can form a heterocyclic ring having from 4 to 6 ring atoms;

each R is₉Independently hydrogen or methyl;

each R is₁₁Independently hydrogen, hydroxy, amino or C₁-C₆A hydrocarbyl group;

each R is₁₂Independently is hydroxy or amino;

each n is independently an integer from 0 to 4;

each m is independently an integer from 0 to 4; and

each p is independently an integer from 1 to 5, an

Wherein (i) Q ₁、Q₂And Q₃Is not hydrogen, and (ii) if Q₁Is hydrogen, then Q₂And Q₃At least one of which is-C (═ NH) NR₄R₅。

5. The method of claim 4, wherein R₈Is hydrogen.

6. The method of claim 4 or 5, wherein each R is₉Is methyl.

7. The method of any one of claims 4-6, wherein Q₁And Q₂Is not hydrogen.

8. The method of claim 7, wherein Q₃Is hydrogen.

9. The method of claim 7 or 8, wherein Q₁Comprises the following steps:

wherein:

R₁is hydrogen;

R₂is hydrogen; and

each R is₃Is hydrogen.

10. The method of claim 9, wherein Q₁Comprises the following steps:

11. the method of claim 7 or 8, wherein Q₁Comprises the following steps:

wherein:

R₁is hydrogen; and

R₂and R₃Connected theretoThe atoms together form a heterocyclic ring having from 4 to 6 ring atoms.

12. The method of claim 11, wherein Q₁Comprises the following steps:

13. the method of claim 7 or 8, wherein Q₁Comprises the following steps:

wherein:

R₃is hydrogen; and

R₁and R₂Together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms.

14. The method of claim 13, wherein Q₁Comprises the following steps:

15. the method of claim 7 or 8, wherein Q₁Comprises the following steps:

wherein:

R₂is hydrogen; and

R₁And R₃Together with the atoms to which they are attached form a carbocyclic ring having from 4 to 6 ring atoms.

16. The method of claim 15, wherein Q₁Comprises the following steps:

17. the method of claim 7 or 8, wherein Q₁Comprises the following steps:

wherein:

R₂is hydrogen; and

each R is₃Is hydrogen.

18. The method of claim 7 or 8, wherein Q₁Comprises the following steps:

wherein:

R₂is hydrogen; and

each R is₃Is hydrogen.

19. The method of any one of claims 7-18, wherein Q is₂Is- (CR)₁₀R₁₁)_pR₁₂。

20. The method of claim 19, wherein each R is₁₀Is hydrogen.

21. The method of claim 20, wherein each R is₁₁Is hydrogen.

22. The method of any one of claims 7-18, wherein Q is₂Is an optionally substituted cycloalkyl group.

23. The method of claim 22, wherein Q₂Is unsubstituted.

24. The method of claim 22, wherein Q₂Substituted by hydroxy or amino.

25. The method of any one of claims 7-18, wherein Q is₂Is an optionally substituted heterocyclylalkyl group.

26. The method of claim 25, wherein Q is₂Is unsubstituted.

27. The method of claim 25, wherein Q is₂Substituted by hydroxy or amino.

28. The method of claim 7, wherein the compound is:

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin;

6' - (2-hydroxy-propanol) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin;

6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin;

6' - (methyl-cyclopropyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin;

6' - (3-amino-propyl) -1- (4-amino-2 (R) -hydroxy-butyryl) -sisomicin;

6' -methyl-cyclopropyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin;

6' -methyl-piperidinyl-1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin;

6' - (2-hydroxy-propanol) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin;

6' - (3-amino-propyl) -1- (3-amino-2 (R) -hydroxy-propionyl) -sisomicin;

6' - (methyl-piperidin-4-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (methyl-cyclopropyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (2-hydroxy-propanol) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (methyl-piperidin-4-yl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (3-amino-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (methyl-cyclopropyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6 '- (2-hydroxy-propanol) -2', 3-di-p-nitrobenzyloxycarbonyl-1- (N-tert-butoxycarbonyl-4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (3-amino-2-hydroxy-propyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (2-hydroxy-acetyl) -sisomicin;

6' - (3-amino-propyl) -1- (2-amino-ethylsulfonamide) -sisomicin;

6' - (2-hydroxy-propanol) -1- (2-amino-ethylsulfonamide) -sisomicin;

6' - (2(S) -hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (2-amino-ethylsulfonamide) -sisomicin;

6' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin;

6' - (2-hydroxy-4-amino-butyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin;

6' - (methyl-cyclopropyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin;

6' - (methyl- (1-hydroxy-3-methylamino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin;

6' - (methyl-cyclopropyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin;

6' - (2-hydroxy-3-amino-propyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin;

6' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (methyl-pyrrolidin-2-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

6' - (3-amino-propyl) -1- (3-hydroxy-azetidin-3-yl-acetyl) -sisomicin;

6' - (3-amino-propyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin;

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-amino-2 (S) -hydroxy-propionyl) -sisomicin;

6' - (methyl-trans-3-amino-cyclobutyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (1-hydroxy-3-amino-cyclobutyl-acetyl) -sisomicin;

6' -methylcyclopropyl-1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin;

6' - (methyl-trans-3-amino-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin;

6' - (2-hydroxy-ethyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin;

6' - (3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin;

6' - (methyl-trans-3-amino-cyclobutyl) -1- (3-hydroxy-pyrrolidin-3-yl-acetyl) -sisomicin;

6' - (2-hydroxy-3-amino-propyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin; or

6' - (methyl-3-amino-1-hydroxy-cyclobutyl) -1- (2- (azetidin-3-yl) -2-hydroxy-acetyl) -sisomicin.

29. The method of any one of claims 4-6, wherein Q₁And Q₃Is not hydrogen.

30. The method of claim 29, wherein Q is₂Is hydrogen.

31. The method of claim 29 or 30, wherein Q is₁Comprises the following steps:

wherein:

R₁is hydrogen;

R₂is hydrogen; and

each R is₃Is hydrogen.

32. The method of claim 31, wherein Q₁Comprises the following steps:

33. the method of claim 29 or 30, wherein Q is ₁Comprises the following steps:

wherein:

R₁is hydrogen; and

R₂and R₃Together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms.

34. The method of claim 33, wherein Q₁Comprises the following steps:

35. the method of claim 29 or 30, wherein Q is₁Comprises the following steps:

wherein:

R₃is hydrogen; and

R₁and R₂Together with the atoms to which they are attached form a heterocyclic ring having from 4 to 6 ring atoms.

36. The method of claim 35, wherein Q₁Comprises the following steps:

37. the method of claim 29 or 30, wherein Q is₁Comprises the following steps:

wherein:

R₂is hydrogen; and

R₁and R₃Together with the atoms to which they are attached form a carbocyclic ring having from 4 to 6 ring atoms.

38. The method of claim 37, wherein Q₁Comprises the following steps:

39. the method of claim 29 or 30, wherein Q is₁Comprises the following steps:

wherein:

R₂is hydrogen; and

each R is₃Is hydrogen.

40. The method of claim 29 or 30, wherein Q is₁Comprises the following steps:

wherein:

R₂is hydrogen; and

each R is₃Is hydrogen.

41. The method of any one of claims 29-40, wherein Q₃Is- (CR)₁₀R₁₁)_pR₁₂。

42. The method of claim 41, wherein each R is₁₀Is hydrogen.

43. The method of claim 42, wherein each R is₁₁Is hydrogen.

44. The method of any one of claims 29-40, wherein Q ₃Is an optionally substituted cycloalkyl group.

45. The method of claim 44, wherein Q₃Is unsubstituted.

46. The method of claim 44, wherein Q₃Substituted by hydroxy or amino.

47. The method of any one of claims 29-40, wherein Q₃Is an optionally substituted heterocyclylalkyl group.

48. The method of claim 47, wherein Q₃Is unsubstituted.

49. The method of claim 47In which Q is₃Substituted by hydroxy or amino.

50. The method of any one of claims 29-40, wherein Q₃Is an optionally substituted heterocyclic group.

51. The method of claim 50, wherein Q₃Is unsubstituted.

52. The method of claim 50, wherein Q₃Substituted by hydroxy or amino.

53. The method of any one of claims 29-40, wherein Q₃is-C (═ NH) NH₂。

54. The method of claim 29, wherein the compound is:

2' - (3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' - (2(S) -amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' - (azetidin-3-yl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' - (2-amino-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' - (2-hydroxy-ethyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' - (2-hydroxy-propanol) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' - (2-hydroxy-3-amino-propyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin;

2' -guanidino-1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin; or

2' - (methyl-trans-3-amino-cyclobutyl) -1- (4-amino-2 (S) -hydroxy-butyryl) -sisomicin.

55. The method of any one of claims 4-6, wherein Q₂And Q₃Is not hydrogen.

56. The method of claim 55 wherein Q₁Is hydrogen.

57. The method of claim 55 or 56, wherein Q₂is-C (═ NH) NH₂。

58. The method of any one of claims 55-57, wherein Q₃is-C (═ NH) NH₂。