The invention relates to the field of chemical derivatization. In particular, it relates to modified aminoglycosides, such as aminoglycosidic antibiotics, and methods for producing them.

Aminoglycosides represent one of the largest classes of antibacterials with activity against both Gram negative and Gram positive bacteria. These antibiotics exert their antibacterial activity by binding to the decoding site (A-site) in 16S rRNA. However, the increased bacterial resistance against aminoglycoside antibiotics gained the interest in modification of these to obtain new active compounds since four decades. Especially the neamine moiety of these antibiotics received major attention, since the main resistance is caused by enzymatic modifications at ring I and II. The introduction of negative charge at ring II by 3'-phosphotranferase (APH-3'), and erasing positive charges at ring II and I by acetyltransferases (ACCs) reduces their biological activity ⁸ . Thus, chemical derivatization of the hydroxy group at 3-position of ring II and transformations of the amino group in 1-position of ring I are approaches to overcome bacterial resistance ⁹ > ¹⁰ . For example the modification of the N-1 position of the 2-desoxy-streptamine ring (2-DOS, ring I) enabled the development of the semisynthetic antibiotic amikacin. However, most regioselective modifications of aminoglycoside antibiotics require multi-step synthesis, ⁹ ' ^11-13 due to the presence of multiple hydroxy- and amine group having a similar reactivity.

Therefore, interest is raised in literature to establish methods enabling facile regioselective introduction of functionalities in structurally complex molecules. These methods are generally based on non-covalent protective group strategies ^{14 15} or enzymatic approaches. However, the modification of the N-3 position at the 2-DOS ring is still limited to enzymatic approaches. ^{16 17} The application of extensive synthetic routes were so far enabled for the neamine 4 with low overall yields. ^{11 18}

In view of the fact that modification of aminoglycoside is becoming a promising tool to overcome antibacterial resistance, the present inventors set out to develop a simple, non-enzymatic method for regioselective derivatization of the 3-N position of the 2-desoxy-streptamine ring. Preferably, the method is cost-efficient, high scalable method and does not require any protection- or deprotection steps. Also, the reaction is preferably performed in an aqueous solution. It was surprisingly found that most, if not all, of these goals could be met the provision of a single step regioselective diazo-transfer reaction at the 3-N position of diverse neamine antibiotics in aqueous buffer solution at neutral pH using the diazo-transfer reagent imidazole- 1-sulfonyl azide. The method is reliable, fast and economically attractive since it does not require any prior protection- or deprotection steps after modification.

Accordingly, the invention relates to a method for the regioselective diazotation of a desoxy-streptamine-substituted aminoglycoside, comprising contacting the aminoglycoside with imidazole- 1-sulfonyl azide hydrochloride under neutral pH conditions to allow for the conversion of the single amine group at the 3-C position of the desoxy-streptamine ring into an azide group.

Azides have proven to be useful precursors to amines in organic syntheses. The diazotransfer reaction followed by regioselective azide reduction of compounds containing multiple azides has been disclosed in the art. Nyffeler et at. (J. Am Chem. Soc. 2002, 124, 10773-10778) reported the use of a specific ratio of solvents and zinc chloride as a catalyst, resulting in a more efficient diazotransfer reaction capable of delivering >90% conversion per amine with shorter reaction times than those previously reported. Azides were then reduced with good regioselectivity in moderate yields by a modification of the Staudinger reaction using trimethylphosphine at low temperatures. Thus, according to the method of Nyffeller, all amines were converted to an azide and only the subsequent step of azide reduction was found to be regioselective. In contrast, a method of the invention allows for regioselective diazotation.

The diazo-transfer reagent used in the present invention has been recently introduced as a shelf-stable, non-explosive and water soluble reagent. ²⁰ A further advantage of this reagent is that is has got a good functional group tolerance and is even reactive without catalyst, such as Cu(II), Ni(II) or Zn(II). This reagent has proven to be a straightforward way to convert free amines into azides via an aqueous diazo-transfer (Scheme la). Hence, in one embodiment, the diazotation is performed in an aqueous solution.

Scheme 1. Introduction of azides using imidazole- 1-sulfonyl azide hydrochloride as diazo-transfer reagent.

Schoffelen et al. (Chem. Sci., 2001, 2, 701) disclose the regioselective diazotation of proteins using imidazole- 1-sulfonylazide at pH 11 and pH 8.5. In contrast, in a method according to the invention, the diazotation reaction is performed at neutral pH, i.e.. pH 6.5-7.5, preferably pH 6.8-7.3. It was found that a neutral pH range was essential to allow for regioselective modification of the single amine group at the 3-C position of the desoxy-streptamine ring.

The other reaction conditions for the diazotation can be determined by routine optimization. Any suitable solvent may be used. Preferably, it is water or an aqueous buffer, like a phosphate buffer. The diazotransfer reagent imidazole- 1-sulfonyl azide hydrochloride can be added as an aqueous solution, e.g. 10- 100 mM, preferably adjusted to about pH 7.5- 8.5. The diazotransfer reagent may be prepared by methods known in the art, see Goddard-Borger, E. D., Stick, R. V. Org. Lett. 2007, 9, 3797. For example, it comprises treating sulfuryl chloride with sodium azide in acetonitrile, followed by the addition of excess imidazole. The hydrochloride salt can be precipitated using ethanolic hydrochloric acid. Typically, an excess amount of the diazotransfer reagent, like 5-20 equivalents, are used relative to the aminoglycoside in a method of the invention. The reaction is suitably performed at room temperature. However, any temperature in the range of about 10-40 °C, preferably 15 -30°C can be used. Incubation periods can vary between about 6 and 48 hours. The reaction mixture is preferably stirred. Good results are obtained with overnight (about 14- 18h) incubations. The reaction may thereafter be quenched, e.g. by adding an aqueous ethylamine solution.

It was surprisingly found that, in absence of Cu(II) and at neutral pH, exclusively a single amine is transformed reaching conversion of 82%. Therefore, diazotation is preferably performed in the absence of a Cu(II) catalyst, more in particular in the absence of a Cu(II) sulphate catalyst.

The invention provides a method for the regioselective diazotation of the amine at the 3-C position of the desoxy-streptamine ring of any aminoglycoside. An aminoglycoside is a molecule or a portion of a molecule composed of amino-modified sugars. Several aminoglycosides function as antibiotics that are effective against certain types of bacteria. Hence, in one embodiment the invention provides a method for the regioselective diazotation of an aminoglycoside antibiotic. They include amikacin, arbekacin, neomycin, paromomycin, and apramycin. In a preferred embodiment, the aminoglycoside is a neamine-based aminoglycoside antibiotic, for instance selected from the group consisting of neomycin, apramycin, neamin, amikacin, paromomycin, ribostamycin, framycetin and isepamecin.

Also provided herein are neamin-based aminoglycosides wherein the amine at the 3-C position of the desoxy-streptamine ring is transformed into an azide, obtainable by a method of the invention. An azide -derivative of a neamine-based aminoglycoside is characterized by an azide at 3-C position of the desoxy-streptamine ring and the absence of any protective groups at the other amine-moieties. Provided is an azide- derivative according to Formula I:

Formula I wherein Ri, R2 = H, or any carbohydrate conjugated at the C-1 position;

R3 = H and R4 = acyl residue, preferably a-hydroxy,y-amino butyrate

Exemplary compounds include C-3-azido neomycin B, C-3-azido paromomycin, C-3- azido ribostamycin, C-3-azido neamine, C-3-azido amikacin and C-3-azido apramycin.

In a specific embodiment, the invention provides a compound according to any one of the following structures,

C-3-azido Neamine C-3-azido Neomycin B C-3-azido Paromomycin

derivative derivative derivative

C-3-azido Amikacin C-3-azido Kanamycin A C-3-azido Kanamycin B derivative derivative derivative

C-3-azido Gentamicin C1 a C-3-azido Gentamicin C1 C-3-azido Gentamicin C2 derivative derivative derivative

C-3-azido NB54 C-3-azido NB84

derivative derivative

C-3-azido G418 C-3-azido ACHN490 derivative derivative

wherein X = is selected from the group consisting of CH2, C=0, CHOH(ax),

CHOH(eq), CH-NH2(ax) and CH-NH2(eq); R ¹ = H, 2-L-hydroxy-gamma-amino butyryl or gamma-amino butyryl; and R ² = NH2 or OH.

An azide- derivative according to the invention is advantageously used in the manufacture of an aminoglycoside antibiotic having antimicrobial activity and showing increased resistance against bacterial aminoglycoside modifying enzymes (AMEs). In particular, it can serve as intermediate in an approach to provide an analog which is protected against a bacterial enzyme having aminoglycoside acetyltransferase (AAC) activity and capable of N-acetylation, hence erasing a positive charge at the 3-position of ring I. Selective transformation of the amino group at the 3-position of ring I into an azide, followed by azide modification into a moiety which can not be N-acetylated by AAC is a powerful tool to protect against AMEs.

The invention therefore also provides for the regioselective diazotation of the amine at the 3-C position of the desoxy-streptamine ring of an aminoglycoside, comprising contacting the aminoglycoside with imidazole- 1-sulfonyl azide

hydrochloride under neutral pH (pH 6.5-7.5) conditions to allow for the conversion of a single amine group into an azide group, further comprising chemical modification of the azide group.

For example, provided is an aminoglycoside antibiotic derivative wherein the 3-C position of the desoxy-streptamine ring carries a moiety which is not recognized as a substrate by a bacterial acetyl transferase (AAC) enzyme, i.e. wherein the moiety is an alkylated amine, a quaternized amine or amide, obtainable by a method of the invention.

In one aspect, azide modification comprises reduction to amine, followed by alkylation. When the amine is alkylated, the AAC enzymes are not able to acylate this position anymore and with this antibacterial resistance can be overcome. Preferably, the azide is converted into a NR2 or Ν1¾ ⁺ moiety, wherein R is C1-C5 alkyl, preferably C1-C3 alkyl. The alkyl can be linear, branched, substituted or unsubstituted. Linear, unsubstituted alkyls are preferred. For example, R is methyl, ethyl or propyl. In another aspect, the azide is converted into a carbamate, a primary amine or amide.

In a further preferred embodiment, the azide is converted to a substituted or unsubstituted triazole moiety, referring to either one of a pair of isomeric chemical compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms. Substituted 1,2,3-triazoles can be produced using click chemistry, e.g. via the azide alkyne Huisgen cyclo addition, in which an azide and an alkyne undergo a 1,3-dipolar cycloaddition reaction.

The invention also provides a novel antibacterial aminoglycoside compound according to the general formula II, Formula III, Formula IV, or a salt thereof.

Formula II Formula III

wherein R = a linear or branched lower alkyl, preferably C1-C5 alkyl, such as C1-C.3 alkyl;

Ri, R2 = H, or any carbohydrate conjugates at CI position;

R4 = acyl residue, preferably a-hydroxy,y-amino butyrate.

In one embodiment, a compound according to any one of the following structures is provided:

Neamine Neomycin B Paromomycin derivative derivative derivative

Gentamicin C1 a Gentamicin C1 Gentamicin C2 derivative derivative derivative

NB54 derivative NB84 derivative

Neamine Neomycin B Paromomycin derivative derivative derivative

Gentamicin C1a Gentamicin C1 Gentamicin C2 derivative derivative derivative

NB54 derivative NB84 derivative

Neamine Neomycin B Paromomycin derivative derivative derivative

Ribostamycin Apramycin Tobramycin derivative derivative derivative

Amikacin Kanamycin A Kanamycin B derivative derivative derivative

Gentamicin C1 a Gentamicin C1 Gentamicin C2 derivative derivative derivative

NB54 derivative NB84 derivative

G418 derivative ACHN490 derivative

wherein X is selected from the group consisting of CH2, C=0, CHOH(ax), CHOH(eq),

CH-NH ₂ (ax), and CH-NH ₂ (eq);

R is methyl, ethyl, propyl, butyl, pentyl or hexyl;

R ¹ = H, 2-L-hydroxy-gamma-amino butyryl or gamma-amino butyryl; and

In one embodiment, Formula III above depicts the desoxy-streptamine ring of a neamine antibiotic such that the invention provides a di- or tri-alkylated neamine antibiotic. Exemplary compounds include 3-N,N-di-alkyl neomycin B, 3-N,N-tri-alkyl neomycin B, 3-N,N-di-alkyl paromomycin, 3-N,N-tri-alkyl paromomycin, 3-N,N-di- alkyl ribostamycin, 3-N,N-tri-alkyl ribostamycin, 3-N,N-di-alkyl neamine, 3-N,N-tri- alkyl neamine, 3-N,N-di-alkyl amikacin, 3-N,N-tri-alkyl amikacin, 3-N,N-di-alkyl apramycin and 3-N,N-tri-alkyl apramycin. Preferably, alkyl is methyl, ethyl or propyl. In still a further embodiment, the invention provides a compound of any of the following structures:

Amikacin Kanamycin A Kanamycin B derivative derivative derivative

Gentamicin C1 a Gentamicin C1 Gentamicin C2 derivative derivative derivative

NB54 derivative NB84 derivative

G418 derivative ACHN490 derivative wherein:

X is CH ₂ , C=0, CHOH(ax), CHOH(eq), CH-NH ₂ (ax) or CH-NH ₂ (eq)

R is methyl, ethyl, propyl, butyl, pentyl or hexyl

R ¹ is H, 2-L-hydroxy-gamma-amino butyryl or gamma-amino butyryl

R ² = NH ₂ or OH Y = a hydrogen bond acceptor or donor, preferably wherein the (acceptor is NH2, hydrazine, aldehyde, ketone, acid or NO2, or wherein the donor is hydroxyl, amine or ammonium;.

n = 1-5.

It will be understood that modification at one or more additional positions of ring I and/or II of the neamine antibiotic is also within the scope of the invention. In one embodiment, the N- l position of ring I is modified. In another embodiment, the hydroxyl- group at the 3-position of ring II is (be it chemically or enzymatically) derivatized to protect against introduction of a positive charge by a bacterial 3'- phosp ho transferase (APH-3') activity. Preferably, the additional derivatization comprises selective oxidation of the hydroxyl- group at the 3-position of ring II by contacting the aminoglycoside in the presence of a transition metal catalyst complex with an oxidizing agent. The transition metal catalyst complex may comprise Pd2+, Rh3+, Ir3+, Ru3+, Pt2+ or Cu2+, preferably palladium. In one specific aspect, the transition metal catalyst complex is a palladium phenanthroline complex

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that, whereas the invention discloses regioselective diazotation of a single amino group without the use of protecting groups, the synthetic processes described herein for providing antimicrobial compounds do not exclude the use of suitable protecting groups and, optionally, de-protecting steps. Such functional groups to be protected include hydroxyl, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxyl include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t- butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include - C(0)-R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein.

It will also be appreciated by those skilled in the art, although a protected derivative of an antibacterial aminoglycoside compound disclosed herein may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form an antibacterial aminoglycoside compound which is pharmacologically active. Such derivatives may therefore be described as "prodrugs". All prodrugs of antibacterial aminoglycoside compounds disclosed herein are included within the scope of the invention.

Furthermore, all antibacterial aminoglycoside compounds disclosed herein which exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the antibacterial aminoglycoside compounds disclosed herein can be converted to their free base or acid form by standard techniques.

Still further useful modifications are those disclosed in WO2012/004684, wherein at least one guanidino group is attached to a primary or secondary carbon atom of the aminoglycoside group and wherein at least one lipid group attached through a bond or a linker to a branched carbon atom of the aminoglycoside; or a salt thereof. In certain embodiments, the aminoglycoside-lipid conjugate comprises at least two guanidino groups attached to a primary or secondary carbon atom of the aminoglycoside group.

A further aspect of the invention relates to drug discovery, more specifically to the screening for novel antibacterial compounds that are based on or derived from an azide- derivative obtained by regioselective diazotation. In one embodiment, the invention provides a method for identifying an aminoglycoside antibiotic, comprising the steps of (i) providing at least one candidate compound using the regioselective diazotation of the amine at the 3-C position of the desoxy-streptamine ring of an aminoglycoside according to the invention, (ii) determining whether the candidate compound has antimicrobial activity. Step (i) preferably comprising providing a desoxy-streptamine-substituted aminoglycoside wherein the amine at the C-3 position is transformed into an azide by contacting the aminoglycoside with imidazole- 1- sulfonyl azide hydrochloride at a pH in the range of 6.5-7.5 under neutral pH conditions, and wherein the azide is further chemically modified, preferably by reduction to amine followed by alkylation and acylation. For example, acylation is suitably used to provide a compound according to Formula IV.

Most preferably, one or more candidate compounds are provided wherein the C-3 azide is converted into a NR2 or NR3 ⁺ moiety, wherein each of R is independently Ci- C5 alkyl, e.g. C1-C3 alkyl. to allow for the conversion of the single amine group at the 3-C position of the desoxy-streptamine ring into an azide group. In one embodiment, the candidate compound is obtained by chemical modification of C-3-azido neomycin B, C-3-azido paromomycin, C-3-azido ribostamycin, C-3-azido neamine, C-3-azido amikacin or C-3-azido apramycin. Antimicrobial activity is suitably assessed using methods known in the art, e.g. in a double dilution method against E. coli and B. subtilis (Green et al. Antimicr. Agents and Chemother. 2011, p. 3207-3213).

To compare microbiological activity of the test compounds, minimum inhibitory concentrations (MICs) can be determined. Antibiotic MICs can be obtained using the broth microdilution method according to approved guidelines. Candidate compounds can be evaluated using a panel of strains that do not possess known aminoglycoside resistance mechanisms, for example ATCC strains of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Klebsiella pneumonia (ATCC 43816),

Pseudomonas aeruginosa (ATCC 27853) and Acinetobacter baumannii (ATCC 19606). These strains are routinely used as a reference to provide the quality control for antibiotic ranges. Activity of the test compounds is preferably determined using QC strains and strains with known aminoglycoside resistance mechanisms.

Preferably, the identification method also comprises determining resistance of the aminoglycoside against N-acylation, for instance using recombinant AAC enzyme, preferably AAC(3)-IV. The enzyme may be part of a cell lysate or it may be (partially) purified.

Aminoglycosides have several potential antibiotic mechanisms, some as protein synthesis inhibitors, although their exact mechanism of action is not fully known. They interfere with the proofreading process, causing increased rate of error in synthesis with premature termination. Also, there is evidence of inhibition of ribosomal translocation where the peptidyl-tRNA moves from the A-site to the P-site. They can also disrupt the integrity of bacterial cell membrane and they bind to the bacterial 30S ribosomal subunit.

Hence, the present invention also contemplates pharmaceutical compositions. A pharmaceutical composition may comprise, for example, an aminoglycoside-analog according to the invention in a pharmaceutically acceptable formulation. The aminoglycoside-analog as disclosed herein may be used in methods of treatment. In some aspects, the invention provides a method of treating a bacterial infection in a subject comprising administering to the subject an effective amount of an

aminoglycoside analog. The bacterium causing the bacterial infection may be a multidrug resistant bacterium.

The bacterial infection may be caused by, for example, a Gram-positive bacterium. Non-limiting examples of Gram-positive bacteria include Staphylococcus aureus methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, methicillin-resistant S. epidermidis (MRSE), Enterococcus faecalis, Enterococcus faecium, and Streptococcus pneumoniae. Alternatively, the bacterial infection may be caused by, for example, a Gram-negative bacterium. Non-limiting examples of Gram-negative bacteria include E. coli, gentamicin-resistant E. coli or amikacin-resistant E. coli, P. aeruginosa or gentamicin-resistant P. aeruginosa. In certain embodiments regarding treatment of a bacterial infection using a

aminoglycoside-analog of the present invention, the minimum inhibitory

concentration of the aminoglycoside antibiotic-analog (MIC) is < 64 μg/mL. Certain methods contemplate an additional step comprising administration of a second antibacterial agent.

In one aspect, the bacterial infection is a urinary tract infection.

In certain embodiments, methods of the present invention may further comprise diagnosing a subject as needing treatment for a bacterial infection prior to

administering an aminoglycoside antibiotic-analog. In other embodiments, methods of the present invention may further comprise administering treatment to a subject who has been identified as needing treatment for a bacterial infection.

Also contemplated are methods of preventing a bacterial infection in a subject comprising administering to the subject an effective amount of an aminoglycoside antibiotic analog. Such methods may further comprise diagnosing the subject as needing preventative treatment for the bacterial infection prior to administering the aminoglycoside antibiotic.

Further embodiments include administering a combination of compounds to a subject in need thereof. A combination can include an aminoglycoside compound, composition, pharmaceutical composition described herein with an additional medicament. Examples of additional medicaments include an antibacterial agent, antifungal agent, an antiviral agent, an anti-inflammatory agent and an anti-allergic agent.

Examples of additional antibacterial agents include chloramphenicol, tetracyclines, synthetic and semi- synthetic penicillins, beta-lactams, quinolones, fluoroquinolnes, macrolide antibiotics, peptide antibiotics, and cyclosporines.

Examples of antifungal agents include azoles, diazoles, triazoles, miconazole, fluconazole, ketoconazole, clotrimazole, itraconazole griseofulvin, ciclopirox, amorolfine, terbinafine, amphotericin B, potassium iodide, and flucytosine (5FC). Examples of antifungal agents include vidarabine, acyclovir, gancyclovir, nucleoside- analog reverse transcriptase inhibitors, AZT (zidovudine), ddl (didanosine), ddC (zalcitabine), d4T (stavudine), 3TC (lamivudine), non-nucleoside reverse transcriptase inhibitors, nevirapine, delavirdine, protease Inhibitors, saquinavir, ritonavir, indinavir, nelfinavir, ribavirin, amantadine, rimantadine and interferon Examples of anti-inflammatory and/or anti-allergic agents include corticosteroids, non-steroidal antiinflammatory drugs, anti- histamines, immunomodulating agents, and immuno suppressants.

Administration of the antibacterial aminoglycoside compounds disclosed herein, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical

compositions of the invention can be prepared by combining an antibacterial aminoglycoside compound disclosed herein with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, 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. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon

administration of the composition to a patient. Compositions that will be

administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. The composition to be administered will, in any event, contain a therapeutically effective amount of an antibacterial aminoglycoside compounds disclosed herein, or a pharmaceutically acceptable salt thereof, for treatment of a urinary tract infection in accordance with the teachings of this invention. A pharmaceutical composition of the invention may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory

administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents 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; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. 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 oil.

The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to an antibacterial aminoglycoside compound, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they be solutions, suspensions or other like form, may include 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, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; 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 the adjustment of tonicity 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. An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for either parenteral or oral administration should contain an amount of an antibacterial aminoglycoside compound disclosed herein such that a suitable dosage will be obtained. The pharmaceutical composition of the invention may be intended for topical

administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

The pharmaceutical composition of the invention may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.

Alternatively, the active ingredients may be encased in a gelatin capsule.

The pharmaceutical composition of the invention in solid or liquid form may include an agent that binds to an antibacterial aminoglycoside compound disclosed herein and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome.

The pharmaceutical composition of the invention may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of antibacterial aminoglycoside compounds disclosed herein may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols. EXPERIMENTAL SECTION General

Ή-NMR- and heteronuclear single- quantum correlation (HSQC) spectra as well as attached proton test (ATP) were recorded on a Varian Unity Inova (500 MHz and 600 MHz) NMR spectrometer at 25 °C. High resolution mass spectrometry (HRMS) was carried out on a LTQ ORBITRAP XL instrument (Thermo Scientific) employing electron impact ionization in positive ion mode (EI+). Chromatographic separations were carried out on a Shimadzu VP series high performance liquid chromatography (HPLC) modular system (DGU- 14A3 Online Vacuum-Degasser, two LC-20 AT pumps, SIL-20A auto sampler, CTP-20 A column oven, RID- 10 refractive detector, FRC-10 A fraction collector and Shimadzu LCsolution software). HPLC purification was performed with a Waters Spherisorb ODS-2 Cis analytical (250 x 4.6 mm) and semi- preparative column (250 x 10mm) (spherical particles of 5 μπι and 80 A pore size) using isocratic elution at 40 °C. A pH-meter (Hanna Instruments pH 209) equipped with a glass combination electrode was used for pH adjustments of the reaction buffers.

Materials

All chemicals and reagents were purchased from commercial suppliers and used without further purification, unless otherwise noted. Neomycin B trisulfate x hydrate (VETRANAL ^® ), paromomycin sulfate salt (98%), ribostamycin sulfate salt, aparmycin sulfate, amikacin sulfate, sulfuryl chloride (97%), sodium azide (95%), acetonitril (99.8%), imodazole (99%) and methanolic 3N HC1 solution were purchased from Sigma Aldrich and used as received. For HPLC purification heptafluorobutyric acid (HFBA) (Fluka, puriss. p. a., for ion chromatography) and acetone {Sigma- Aldrich, HPLC grade) were used. Ultrapure water (specific resistance > 18.4 ΜΩ cm) was obtained by Milli-Q water purification system (Sartorius ^® ) . Neamine hydrochloride was synthesized according procedure from literature. General procedures

Synthesis of diazotransfer reagent Imidazole-l-sulfonyl Azide Hydrochloride

HCI

Sulfuryl chloride (1.6 mL, 20 mmol) was added dropwise to an ice-cooled suspension of sodium azide (1.3 g, 20 mmol) in acetonitril (20 mL) and the mixture was stirred overnight at room temperature. Imidazole (2.6g, 38 mmol) was added portion-wise to the ice-cooled mixture and the resulting slurry was stirred for additional 3h at room temperature. The mixture was diluted with ethyl acetate (40 mL), washed with water (2 x 40 mL) and then with saturated aqueous sodium hydrogen carbonate (2 x 40 mL), dried over MgS04 and filtered. The filtrate was cooled in an ice-batch and a 3M HCI methanolic solution (lOmL) was added dropwise to precipitate the product. Finally, the filer cake was washed with EtOAc (3 x 10 mL) to obtain 7 as colourless

hydrochloride salt. Yield: 1.9 g (9.1 mmol, 45% yield). Ή-NMR (D ₂ 0, 400MHz) δ (p.p.m.) 9.53 (s, IH, H-2), 8.07 (s, IH, H-5), 7.67 (s, IH, H-4). ^C-NMR (D2O, 400MHz) δ (p.p.m.) 137.6, 122.6, 120.18. HRMS (EI+) (m/z): found 174.0078 [M-C1] ⁺ , calc.

174.0080 [M-C1] ⁺ .

Synthesis of 3-C-azide neamine antibiotics.

10 mM sodium phosphate buffer

(pH = 7.3), r.t., 30 min

Scheme 1: Regioselective transformation of 3-C position of 2-desoxy streptamine ring in neamine antibiotics. This Example describes three procedures that were applied for different antibiotics. The reaction step is the same in all of them; they differ only in the purification step. a. Synthesis of antibiotic derivatives 8 and 9

After 11 μηιοΐ aminoglycoside antibiotic was dissolved in 7.5 mL of 10 mM sodium phosphate buffer (pH 7.3). 3.7 mL of a 48 mM aqueous solution of diazo-transfer reagent, which was adjusted to pH 8 by adding 2 M NaOH solution, was added into the solution of the antibiotic and the reaction mixture was stirred overnight at room temperature. The reaction was quenched by adding 0.9 mL aqueous 7 wt% ethylamine solution. After incubating at r.t. for 30 min the reaction mixture was freeze dried and the crude mixture was dissolved in 1.5 mL water. Each 30 μΕ fraction was purified by HPLC using a Waters Spherisorb ODS-2Cis analytic column (water/acetone 1:0.9 containing 12.1 mM HFBA) at a flow rate of 1 ml/min at 40°C to afford the antibiotic derivatives 8 and 9. b. Synthesis of antibiotic derivatives 10 and 11.

After 11 μηιοΐ aminoglycoside antibiotic was dissolved in 7.5 mL of 10 mM sodium phosphate buffer (pH 7.3) 3.7 mL of a 48 mM aqueous solution of diazotransfer reagent, which was adjusted to pH 8 by adding 2 M NaOH solution, was added into the solution of the antibiotic and the reaction mixture was stirred overnight at room temperature. The reaction was quenched by adding 0.9 mL aqueous 7 wt% ethylamine solution. After incubating at r.t. for 30 min the reaction mixture is freeze dried and the crude mixture is purified by column chromatography using the upper layer of CH3Cl/MeOH/17%NH32: l: l mixture as eluent. After evaporation at reduced pressure and freeze-drying the antibiotic derivatives was dissolved in 1.5 mL water. Each 30 μΕ fraction was purified by HPLC using a Waters Spherisorb ODS-2Cis analytic column (water/acetone 1:0.9 containing 12.1 mM HFBA) at a flow rate of 1 ml/min at 40°C to afford the antibiotic derivatives 10, 11. c. Synthesis of antibiotic derivatives 12 and 13 After 11 μηιοΐ aminoglycoside antibiotic was dissolved in 7.5 mL of 10 mM sodium phosphate buffer (pH 7.3). 3.7 mL of a 48 mM aqueous solution of diazo-transfer reagent, which was adjusted to pH 8 by adding 2 M NaOH solution, was added into the solution of the antibiotic and the reaction mixture was stirred overnight at room temperature. By addition of 1M aqueous HC1 solution a pH of 2 was adjusted and each 100 fraction was purified by HPLC using a Waters Spherisorb ODS-2Cis analytic column (water/acetone 1:0.9 containing 12.1 mM HFBA) at a flow rate of 1 ml/min at 40°C to afford the antibiotic derivatives 12 and 13.

Analytical Data

C-3-azido neomycin B x 5 HFBA (8). The title compound was prepared according to the general procedure described above. Derivative 8 was obtained as a white solid. For the measurement of regioselectivity and the characterization of the compound Ή-NMR,

HSQC and ¹³ C-NMR spectra were recorded and electrospray ionization (ESI)-MS was employed. The yield was determined by HPLC using a 1.1 mM aqueous neomycin B solution as reference: Rt = 7.7 min, 60% yield. TLC: R _f = 0.57 (CH ₃ Cl/MeOH/17%NH32: l: l). Ή- NMR (D ₂ 0, 500 MHz) δ (p.p.m.) 5.72 (d, J = 4 Hz, 1H, 1-H'), 5.33 (d, J = 1.5 Hz, 1H, 1- H"), 5.26 (s, 1H, 1-H'"), 4.40 (t, J = 5.5 Hz, 1H, 3-H"), 4.37 (dd, J = 4.5 Hz, J = 2 Hz, 1H, 2-H"), 4.28 (t, J = 5 Hz, 1H, 5-H'"), 4.27 (m, 1H, 5-H'), 4.21 (m, 2H, 3-H'", 4-H"), 3.91-3.85 (m, 2H, 5-H _a ", 3-H'), 3.80-3.75 (m, 3H, 4-H'", 5-H, 3-H), 3.72-3.67 (m, 2H, 4-H, 5-H _b "), 3.58 (t, J = 10 Hz, 1H, 6-H), 3.56 (s(br), 1H, 2-H'"), 3.47-3.33 (m, 5H, 6- H _a ', 6-H _a '", 6-H _b '", 4-H', 2-H'), 3.26 (dt, J = 11.25 Hz, J = 3.5 Hz, 1H, 1-H), 3.19 (dd, J = 13.5 Hz, J = 8 Hz, 1H, 6-H _b '), 2.47 (dt, J = 13 Hz, J = 4 Hz, 1H , 2-H _e ), 1.70 (dd, J = 12.3 Hz, 1H, 2-Ha). ¹³ C-NMR(D ₂ 0, 500 MHz) δ (p.p.m.) 109.84 (1-C"), 94.51 (1-C"), 94.30 (1-C), 84.89 (5-C), 80.48 (4-C"), 76.26 (4-C), 74.86 (3-C"), 72.84 (2-C"), 71.72 (6-C), 69.81 (4-C), 69.05 (5-C"), 68.00 (3-C), 67.68 (5-C), 66.77 (3-C"), 66.72 (4- C"), 59.88 (5-C"), 58.14 (3-C), 53.01 (2-C), 50.15 (2-C"), 49.21 (1-C), 39.67 (6-C"), 39.55 (6-C), 28.72 (2-C). M = C23H44N8O13; HRMS (EI+) (m/z): found 641.30652

[M+H] ⁺ , calc. 657.31006 [M+H] ⁺ . C-3-azido paromomycin x 4 HFBA (9). The title

compound was prepared according to the general procedure described above. Derivative 9 was obtained as a white solid. For the measurement of regioselectivity and the characterization of the compound Ή-NMR, HSQC and 1 ³ C-NMR spectra were recorded and electrospray ionization (ESI)-MS was employed. The yield was determined by HPLC using a 1.1 mM aqueous paromomycin solution as reference: Rt = 5.9 min, 47% yield. TLC: R _f = 0.64 (CH ₃ Cl/MeOH/17%NH32: l: l). Ή-NMR (D ₂ 0, 500 MHz) δ (p.p.m.) 5.72 (d, J = 3 Hz, 1H, 1-H'), 5.33 (s, 1H, 1-H"), 5.26 (s, 1H, 1-H"), 4.48 (t, J = 5.5 Hz, 1H, 3-H"), 4.38 (d, J = 4 Hz, 1H, 2-H"), 4.28 (m, 1H, 5-H'"), 4.22- 4.16 (m, 2H, 3-H'", 4-H"), 4.02 (d, J = 9.5 Hz, 1H, 5-H'), 3.92-3.84 (m, 2H, 5-H _a ", 3- H'), 3.82-3.75 (m, 4H, 4-H'", 6-H _a ', 6-Ht/, 5H), 3.73-3.63 (m, 3H, 5-H _b ", 4-H, 3-H), 3.57 (t, J = 10 Hz, 1H, 6-H), 3.56 (s(br), 1H, 2-H'"), 3.50 (t, J = 9.75 Hz, 1H 4-H'), 3.44 (dd, J = 17.5 Hz, J = 9.5 Hz, 1H, 6-H _a "'), 3.39-3.34 (m, 2H, 6-Ht/", 2-H'), 3.25 (dt, J = 11.5 Hz, J = 3.5 Hz, 1H , 1-H), 2.44 (dt, J = 12.5 Hz, J = 4Hz, 1H , 2-H _e ), 1.67 ( dd, J = 12.15 Hz, 1H, 2-Ha). ¹³ C-NMR(D ₂ 0, 500 MHz) δ (p.p.m.) 110.33 (C-l"), 95.11 (C-l'), 94.88 (1-C ^" ), 85.29 (5-H), 80.69 (4-C ^" ), 76.81 (4-C), 74.87 (3-C ^" ), 73.07 (2-C ^" ), 72.45 (5-C), 72.22 (6-C), 69.88 (5-C"), 69.07 (3-C), 68.68 (4-C), 67.30 (3-C"), 66.96 (4- C ^" ), 60.01 (5-C ^" ), 59.62 (6-C), 58.37 (3-C), 53.81 (2-Η ^' ), 50.52 (2-H ^'" ), 49.55 (C-l), 40.03 (6-C"), 29.13 (2-C). C23H43N7O14; HRMS (EI+) (m/z): found 642.29346 [M+H] ⁺ , calc. 642.29408 [M+H] ⁺ .

C-3-azido ribostamycin x 3 HFBA (10). The title compound was prepared according to the general procedure described above. Derivative 10 was obtained as a white solid. For the measurement of regioselectivity and the

characterization of the compound Ή-NMR, HSQC and ¹³ C- NMR spectra were recorded and electrospray ionization

(ESI)-MS was employed, The yield was determined by HPLC using a 1.1 mM aqueous ribostamycin solution as reference: Rt = 4.1 min, 44% yield. TLC: Rf = 0.73 (CH ₃ Cl MeOH/17%NH32: l: l). Ή-NMR (D ₂ 0, 500 MHz) δ (p.p.m.) 5.77 (d, J = 3.5 Hz, 1H, 1-H'), 5.31 (d, J = 1.5 Hz, 1H, 1-H"), 4.28 (dt, J = 9 Hz, J = 3 Hz, 1H, 5-H'), 4.21 (dd, J = 4.5 Hz, J = 3Hz, 1H, 2-H"), 4.15 (t, J = 7.5 Hz, J = 4.5 Hz, 1H, 3-H"), 4.05 (dt, J = 6.5 Hz, J = 2.5 Hz, 1H, 4-H"), 3.93-3.88 (m, 2H, 5-H _a ", 3-H'), 3.82-3.72 (m, 3H, 3- H, 4-H, 4-H), 3.65 (dd, H = 12.5 Hz, J = 6.5 Hz, 1H, 5-H _b "), 3.60 (t, J = 9.8 Hz, 1H, 6- H), 3.48-3.43 (m, 2H, 4-H', 6-H _a '), 3.39 (dd, J = 10.5 Hz, J= 4.0 Hz, 1H, 2-H'), 3.29 (dt, J = 11.5 Hz, J = 4.0 Hz, 1H, 1-H), 3.23 (dd, J = 13.5 Hz, J = 7.5 Hz, 1H 6-H _b '), 2.50 (dt, 12.5 Hz, J = 4.0 Hz, 1H, 2-H _e ), 1.73 (dd, J = 12.5 Hz, 1H, 2-H _a ). ¹³ C-NMR(D ₂ 0, 500 MHz) δ (p.p.m.) 110.64 (1-C"), 95.01 (1-C), 85.50 (5-C), 82.12 (4-C"), 76.71 (4-C), 74.97 (2-C"), 72.29 (6-C), 70.29 (4-C), 68.94 (3-C"), 68.80 (3-C), 68.32 (5-C), 60.85 (5-C ^" ), 58.44 (3-C), 53.54 (2-C), 49.53 (1-C), 39.90 (6-C), 29.10 (2-C). C17H32N6O10; HRMS (EI+) (m/z): found 481.22470 [M+H] ⁺ , calc. 481.22470 [M+H] ⁺ .

C-3-azido neamine x 3 HFBA (11). The title compound was prepared according to the general procedure described above. Derivative 11 was obtained as a white solid. For the

measurement of regioselectivity and the characterization of the compound Ή-NMR, HSQC and ¹³ C-NMR spectra were recorded and electrospray ionization (ESI)-MS was employed. The yield was determined by HPLC using a 1.1 mM aqueous neamine solution as reference: Rt = 2.3 min, 48% yield. TLC: Rf = 0.71 (CH3Cl/MeOH/17%NH ₃ 2: l: l). Ή-NMR (D2O, 500 MHz) δ (p.p.m.) 5.69 (d, J = 3.5 Hz, 1H, 1-H'), 4.23 (t, J = 9.0 Hz, 1H, 5-H'), 3.93 (t, J = 10.0 Hz, 1H, 3-H'), 3.80 (dt, J = 10.8 Hz, J = 5.0 Hz, 1H, 3-H), 3.68 (t, J = 9.3 Hz, 1H, 5-H), 3.63 (t, J = 9.0 Hz, 1H, 4- H), 3.53 (t, J = 9.8 Hz, 1H, 6-H), 3.50-3.46 (m, 2H, 4-H', 6-H _a '), 3.43 (dd, J = 11.0 Hz, J = 3.5 Hz, 1H, 2-H'), 3.32-3.24 (dt, 2H, 1-H, 6-Hb'), 2.51 (dt, J = 13.0 Hz, J = 4.5 Hz, 1H, 2-He), 1.77 (dd, J = 12.5 Hz, 1H, 2-H _a ). ¹³ C-NMR(D ₂ 0, 500 MHz) δ (p.p.m.) 95.36 (1-C), 78.80 (5-C), 74.77 (4-C), 72.23 (6-C), 70.26 (4-C), 68.55 (3-C), 68.16 (5-C), 58.18 (3-C), 53.31 (2-C), 49.36 (1-C), 39.69 (6-C), 29.07 (2-C). CiaHsaNeOe HRMS (EI+) (m/z): found 349.18066 [M+H] ⁺ , calc. 349.18301 [M+H] ⁺ . C-3-azido amikacin x 3 HFBA (12). The title compound was prepared according to the general procedure described above. Derivative 12 was obtained as a white solid. For the measurement of regioselectivity and the characterization of the

compound Ή-NMR, HSQC and ¹³ C-NMR spectra were recorded and electrospray ionization (ESI)-MS was employed. The yield was determined by HPLC using a 1.1 mM aqueous amikacin solution as reference: Rt = 3.7 min, 65% yield. TLC: R _f = 0.34 (CH ₃ Cl/MeOH/17%NH32: l: l). Ή-NMR (D ₂ 0, 500 MHz) δ (p.p.m.) 5.50 (d, J = 4 Hz, 1H, 1-H'), 5.14 (d, J = 3.5 Hz, 1H, 1-H"), 4.26 (dd, J = 9 Hz, J = 3.5 Hz, 2H, a-H _a , a-H _b ), 4.17 (dt, J = 8.8 Hz, J = 3 Hz, 1H, 5-H'), 4.09-4.04 (m, 2H, 1-H, 3-H'), 3.80 (s, 2H, 6-H _a , 6-H _b ), 3.78-3.71 (m, 4H, 6-H, 5-H, 5-H", 2-H"), 3.69-3.66 (m, 2H, 4-H", 3-H), 3.62-3.55 (m, 2H, 2-H', 4-H), 3.44-3.35 (m, 6-H _a ', 3-H", 4-H ^" ), 3.23-3.16 (m, 3H, , 6-Hb ^' , y-H _a , γ-Hb), 2.22-2.13 (m, 2H, 2-H _e , 6-H _a ), 1.95 (m, 1H, 6-Hb), 1.66 (dd, J = 12.7 Hz, 1H, 2-H _a ). ¹³ C-NMR(D ₂ 0, 500 MHz) δ (p.p.m.) 101.16 (C- l'), 100.31 (C- l"), 83.56 (C-4), 82.03 (C-6), 77.48 (C-5), 74.84 (C-5"), 74.24 (C-3'), 73.71 (C-2'), 73.23 (C-4'), 71.99 (C-a), 70.59 (C-5'), 70.56 (C-2"), 67.81 (C-4"), 67.87 (C-6"), 61.73 (C-3), 57.71 (C-3"), 51.29 (C- l), 42.83 (C-6'), 39.28 (C-γ), 34.23 (C-2), 33.17 (C-β). M = C22H41N7O13; HRMS (EI+) (m/z): found 612.27999 [M+H] ⁺ , calc.

612.28351 [M+H] ⁺ .

ido apramycin x 4 HFBA (13).

The title compound was prepared according to the general procedure described above. Derivative 13 was obtained as a white solid. For the

measurement of regioselectivity and the characterization of the compound Ή-NMR, HSQC and ¹³ C-NMR spectra were recorded and electrospray ionization (ESI)-MS was employed. The yield was determined by HPLC using a 1.1 mM aqueous apramycin solution as reference: Rt = 5.8 min, 49% yield. TLC: Rf = 0.89 (CH ₃ Cl/MeOH/17%NH ₃ 2: l: l). Ή-NMR (D2O, 500 MHz) δ (p.p.m.) 5.52 (d, J = 4 Hz, 1H, 1-H"), 5.51 (d, J = 4 Hz, 1H, 1-H'), 5.25 (d, J = 8.5 Hz, 1H, 8-H'), 4.54 (s, 1H, 6-H'), 3.04-3.93 (m, 4H, 3-H", 5-H", 4-H', 5-H'), 3.87 (dd, J = 12.5 Hz, J = 3 Hz, 1H, 6-H _a "), 3.81 (dd, J = 12.5 Hz, J = 4.5 Hz, 1H, 6-H _b "), 3.73 (dd, J = 10 Hz, J = 4 Hz, 1H, 2-H"), 3.68-3.62 (m, 3H, 4-H, 3-H, 2-H'), 3.60 (t, J = 9 Hz, 1H, 5-H), 3.51 (t, J = 9.8 Hz, 1H, 6-H), 3.40 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H, 7- H'), 3.82 (t, J = 10.3 Hz, 1H, 4-H"), 3.26 (dt, J = 11.5 Hz, J = 4 Hz, 1H, 1-H), 2.82 (s, 3H, CH ₃ ), 2.48 (dt, J = 13 Hz, J = 4.3 Hz, 1H, 2-H _e ), 2.39 (dt, J = 11 Hz, J = 4.3 Hz, 1H, 3-He'), 2.04 (dd, J = 12 Hz, 1H, 3-H _a '), 1.73 (dd, J = 12.3 Hz, 1H, 2-H _a ). ¹³ C-NMR(D ₂ 0, 500 MHz) δ (p.p.m.) 98.75 (C- 1'), 97.57 (C- 1"), 95.78 (C-8'), 83.19 (C-4), 77.89 (C-5), 75.11 (C-6), 73.00 (C-2"), 72.10 (C-5'), 71.92 (C-3"), 70.90 (C-5"), 68.64 (C-4'), 65.47 (C-6'), 62.84 (C-6"), 62.31 (C-7'), 61.13 (C-3), 54.59 (C-4"), 52.43 (C- 1), 50.59 (C-2'), 32.36 (C-CH ₃ ), 32.10 (C-2), 29.19 (C-3'). M = C21H39N7O11; HRMS (EI+) (m/z): found 566.27484 [M+H] ⁺ , calc. 566.27803 [M+H] ⁺ .

3-N,N-di-methyl neomycin B (1). Neomycin B derivative 1 was obtained as a white solid. For the the characterization of the compound Ή-NMR and HSQC spectra were recorded. NMR analysis of antibiotic acetic acid salt: Ή-NMR (400 MHz, D2O, 25 °C, TMS): δ (ppm) = 5.78 (d, J = 3.2 Hz, 1H, 1- H'), 5.40 (s, 1H, 1-H"), 5.28 (s, 1H, 1-H'"), 4.51 (t, J = 5.6 Hz, 1H, 3-H"), 4.41 (m, 1H, 2-H"), 4.30 (t, J = 4.8 Hz, 1H, 5-H'"),

4.21 (m, 3H, 5-H', 3-H'", 4-H"), 4.14 (t, J = 9.6 Hz, 1H, 4-H),

3.97 (t, J = 9.8 Hz, 1H, 3-H'), 3.92-3.86 (m, 2H, 5-H _a ", 5-H), 3.81-3.72 (m, 2H, 4-H'", 5-Hb"), 3.63 (t, J = 9.8 Hz, 1H, 6-H), 3.57 (s(br), 1H, 2-H'"), 3.48 (t, J = 4.8 Hz, 1H, 4- H'), 3.44-3.20 (m, 7H, 6-H _a ', 6-H _b ', 6-H _a "', 6-H _b '", 1-H, 2-H', 3-H), 2.54 (s, 6H, CH ₃ ), 2.32 (dt, J = 12.8 Hz, J = 3.6 Hz, 1H , 2-H _eq ), 1.90 (s, 18H, CH3-COO), 1.67 (dd, J = 12.3 Hz, 1H, 2-H _ax ). ¹³ C-NMR (100.6 MHz, D2O, 25 °C, TMS): δ (ppm) = 108.9 (C- 1"), 95.3 (C- 1'"), 94.7 (C- 1'), 84.4 (C-5), 81.3 (C-4"), 75.2 (C-3"), 74.5 (C-4), 73.6 (C-2"), 72.9 (C-6), 70.0 (C-5'"), 69.8 (C-4'), 68.8 (C-3'), 68.6 (C-5'), 67.5 (C-3'"), 67.1 (C-4'"), 60.1 (C-5"), 59.8 (C-3), 53.3 (C-2'), 50.7 (C-2'"), 50.3 (C- 1), 40.2, 39.2 (2C, C-6', C-6'"), 38.8 (2C, N(CH ₃ ) ₂ ), 22.9 (6C, CH ₃ ) 21.0 (C-2). 3-N-Y-amino butyrate neomycin B (2a). Neomycin B derivative 2a was obtained as a white solid. For the the characterization of the compound Ή-NMR and HSQC spectra were recorded and. NMR analysis of antibiotic acetic acid salt: Ή-NMR (400 MHz, D ₂ 0, 25 °C, TMS): δ (ppm) = 5.74 (d, J = 2.8 Hz, 1H, 1-H'), 5.40 (d, J = 2.4 Hz, 1H, 1-H"), 5.09 (s, 1H, 1-H'"), 4.23 (t, J = 5.4 Hz, 1H, 3-

H"), 4.09 (m, 2H, 2-H", 5-H'"), 4.02-3.90 (m, 3H, 3-H, 3-

H'", 4-H"), 3.73-3.64 (m, 2H, 5-H _a ", 5-H), 3.59-3.43 (m, 6H, 4-H, 4-H'", 5-H _b ", 6-H, 3-H', 5-H'), 3.34 (t, J = 9.6 Hz, 1H, 2-H'"), 3.25-2.95 (m, 7H, 6-H _a ', 6-H _b ', 6-H _a '", 6- Hb'", 1-H, 2-H', 4-H'), 2.82 (t, J = 7.2 Hz, 2H, y-CH ₂ ), 2.28 (t, J = 7.0 Hz, 2H, a-CH ₂ ), 1.79 (m, 21H, 6-CH ₂ , CHs-COO, 2-H _eq ), 1.52 (dd, J = 12.3 Hz, 1H, 2-H _ax ). ¹³ C-NMR (100.6 MHz, D ₂ 0, 25 °C, TMS): δ (ppm) = 109.8 (C-1"), 95.1 (C- 1'"), 94.2 (C- 1'), 85.2 (C-5), 81.0 (C-4"), 75.6 (C-4), 75.1 (C-3"), 72.9 (C-2"), 71.9 (C-6), 69.5 (C-5'"), 69.2 (C-4'), 68.5 (C-3'), 68.2 (C-5'), 67.0 (C-3'"), 66.7 (C-4'"), 60.1 (C-5"), 53.1 (C-2'), 50.2 (C-2'"), 49.5 (C-1), 46.7 (C-3), 39.7, 38.9 (2C, C-6', C-6'"), 38.1 (y-CH ₂ ), 31.9 (a-CH ₂ ), 29.7 (C-2). 22.4 (6C, CH ₃ ), 21.9 (6-CH ₂ ).

3-N- -amino propionyl neomycin B (2b). Neomycin B derivative 2b was obtained as a white solid. For the the characterization of the compound Ή-NMR and HSQC spectra were recorded and. NMR analysis of antibiotic acetic acid salt: Ή-NMR (400 MHz, D ₂ 0, 25 °C, TMS): δ (ppm) = 5.72 (d, J = 3.6 Hz, 1H, 1-H'), 5.18 (d, J = 3.6 Hz, 1H, 1-H"), 5.08 (s, 1H, 1-H'"), 4.22 (t, J = 5.4 Hz, 1H, 3-

H"), 4.11 (m, 2H, 2-H", 5-H'"), 4.01 (m, 2H, 3-H'", 4-H"),

3.93 (dt, J = 11. Hz, J = 4 Hz, 1H, 3-H), 3.73-3.64 (m, 7H, 5-H _a ", 5-H, 4-H, 4-H'", 5- H _b ", 3-H', 5-H'), 3.42 (t, J = 10 Hz, 1H, 6-H), 3.38 (s(br), 1H, 2-H'"), 3.23 (t, J = 9.6 Hz, 1H, 4-H'), 3.21-3.01 (m, 8H, 6-H _a ', 6-H _b ', 6-H _a "', 6-H _b '", 1-H, 2-H', 6-CH ₂ ), 2.49 (m, 2H, a-CH ₂ ), 2.00 (dt, 1H, J = 12.8 Hz, J = 4.8 Hz, 1H, 2-Heq), 1.79 (s, 18H, CH ₃ - COO), 1.47 (dd, J = 12.7 Hz, 1H, 2-H _ax ). ¹³ C-NMR (100.6 MHz, D ₂ 0, 25 °C, TMS): δ (ppm) = 109.7 (C- 1"), 95.0 (C- 1'"), 93.8 (C-1'), 85.3 (C-5), 81.0 (C-4"), 75.2 (C-4), 75.1 (C-3"), 72.8 (C-2"), 71.7 (C-6), 69.4 (C-5'"), 69.2 (C-4'), 68.2, 68.1 (2C, C-3', C-5'), 66.9 (C-3'"), 66.6 (C-4'"), 60.2 (C-5"), 53.0 (C-2'), 50.0 (C-2'"), 49.5 (C- 1), 46.5 (C-3), 39.7, 38.9 (2C, C-6', C-6'"), 34.6 (6-CH ₂ ), 31.3 (a-CH ₂ ), 29.5 (C-2). 20.9 (6C, CH ₃ ).

3-N-a-amino acetyl neomycin B (2c). Neomycin B derivative 2c was obtained as a white solid. For the the characterization of the compound Ή-NMR and HSQC spectra were recorded and. NMR analysis of antibiotic acetic acid salt: Ή-NMR (400 MHz, D ₂ 0, 25 °C, TMS): δ (ppm) = 5.75 (d, J = 3.2 Hz, 1H, 1-H'), 5.18 (d, J = 2.4 Hz, 1H, 1-H"), 5.06 (s, 1H, 1-H'"), 4.18 (t, J = 5.4 Hz, 1H, 3-H"), 4.07 (m,

2H, 2-H", 5-H'"), 3.99 (m, 3H, 3-H'", 4-H", 3-H), 3.70-3.43

(m, 10H, 5-Ha", 5-H, 4-H, 4-H'", 5-H _b ", 3-H', 5-H', 6-H, a-CH ₂ ), 3.35 (t, J = 5.0 Hz, 1H, 2-H'"), 3.22-2.82 (m, 7H, 6-H _a ', 6-H _b ', 6-H _a '", 6-H _b '", 1-H, 2-H', 4-H'), 1.92 (m, 1H, 2-He _q ), 1.76 (s, 18H, CHs-COO), 1.52 (dd, J = 12.7 Hz, 1H, 2-H _ax ). ¹³ C-NMR (100.6 MHz, D ₂ 0, 25 °C, TMS): δ (ppm) = 109.7 (C- 1"), 95.0 (C- 1'"), 93.7 (C-1'), 85.1 (C-5), 80.9 (C-4"), 75.1 (C-4), 75.0 (C-3"), 72.7 (C-2"), 71.5 (C-6), 69.4 (C-5'"), 69.2 (C-4'), 68.1, (2C, C-3', C-5'), 66.9 (C-3'"), 66.6 (C-4'"), 60.1 (C-5"), 53.0 (C-2'), 50.1 (C-2'"), 49.5 (C-1), 47.1 (C-3), 40.0 (a-CH ₂ ), 39.6, 38.9 (2C, C-6', C-6'"), 29.5 (C-2). 21.7 (6C, CH ₃ ).

d. Minimal Inhibitory Concentration (MIC) Test

The minimal inhibitory concentrations (MICs) of neomycin B and derivatives 1, 2a, 2b and 2c were determined in an E. coli strain without resistance, as well as in E. coli strain ACC harbouring the resistance causing enzyme acetyltransferase ACC-3.

Enzyme ACC-3 catalyses the acetylation reaction of the amino group in C-3 position at the 2-desoxystrepamine (2-DOS) ring in aminoglycoside antibiotics resulting in deactivation of the drug in resistant bacteria. As shown in Table 1, all synthesized derivatives show antibacterial activity in resistant and not resistant E. coli strains. However, these compounds exhibit lower antibacterial activity compared to the unmodified antibiotic neomycin B against the E. coli strain not expressing enzyme ACC-3. The modification at the C-3 position results in lower affinity to the ribosomal unit 30S in E. coli bacteria.

In contrast, when ACC-3 is expressed in E. coli compound 2b exhibits similar activity as neomycin B. As shown in Table 1, neomycin B and 2b exhibit a MIC value of 0.016 mM. Moreover, the dimethylated antibiotic derivative 1 shows even a higher antibacterial activity in resistant bacteria than neomycin B with MIC values of 0.008 mM and 0.016 mM, respectively (Table 1). This result clearly demonstrates that a modification at the C-3 position is highly advantageous to overcome antibacterial resistance caused by the acetyltransferase ACC-3.

Table 1: minimal inhibitory concentration test

compound MIC MIC (mM)

(mM) E.coli ACC- E.coli 3

n n ^e omycin o.oos 0.0 16

15

1 0.25 0.008

2a 0. .-) 0.25

2b 0.25 0.016

2c 0.25 0.03 1

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