WO2014013495A1

WO2014013495A1 - Membrane-targeting aminoglycoside-based cationic amphiphiles and therapeutic uses

Info

Publication number: WO2014013495A1
Application number: PCT/IL2013/050612
Authority: WO
Inventors: Micha Fridman; Ido M. HERZOG; Mark Feldman; Yifat BERKOV-ZRIHEN
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2012-07-19
Filing date: 2013-07-18
Publication date: 2014-01-23

Abstract

The present invention relates to amphiphilic aminoglycoside (AG) derivatives, especially derivatives of tobramycin, kanamycin A and paromomycin comprising hydrophobic side chains linked to the AG moiety by various linkers. These novel derivatives are useful as broad spectrum antibacterial agents targeting bacterial cell membranes, while having minimal toxicity due to non-selective targeting of eukaryotic cell membranes. The present invention further relates to methods for preparing the novel derivatives, pharmaceutical compositions including such compounds, and methods of using these compounds and compositions, especially as membrane-targeting antibacterial agents for treating bacterial infections, and for inhibiting bio film growth.

Description

MEMBRANE-TARGETING AMINOGLYCOSIDE-BASED CATIONIC AMPHIPHILES AND THERAPEUTIC USES

FIELD OF THE INVENTION

The present invention relates to bacterial membrane-targeting aminoglycoside (i.e., tobramycin, kanamycin or paromomycin) based cationic amphiphiles having potent antibacterial and reduced hemolytic activity, methods for their preparation, pharmaceutical compositions including such compounds, and methods of using these compounds and compositions, especially as antibacterial agents having broad antibacterial activity against both Gram-positive and Gram-negative bacteria (e.g., for treating soft tissue and skin infections while having reduced hemolytic activity).

BACKGROUND OF THE INVENTION

Membranes and cell walls are essential constituents required for the viability of all bacteria and therefore serve as attractive targets for the development of antibiotics. Amongst cell-wall-targeting antibiotics, several families of peptidoglycan biosynthesis inhibitors, including 2-lactams that irreversibly inhibit the activity of the peptidoglycan trans-peptidation biosynthetic step (Chung, 2009; Fisher, 2005); glyco-peptide antibiotics such as vancomycin that competitively inhibit the trans-peptidation step, and the glyco-lipid antibiotic moenomycin A inhibits the peptidoglycan trans-glycosylation step (Healy, 2000; Ge, 1999; Yuan, 2008; Fuse, 2010). To date, disruption of the bacterial membrane bi-layer has been poorly exploited as a strategy for the development of antibiotics.

Bacterial membrane-disrupting-antibiotics offer several advantages over antimicrobial agents that target intracellular bacterial targets. First, membrane disruption is not dependent on the bacterial cell cycle state and is therefore a promising strategy for the eradication of dormant bacteria and treatment of persistent infections (Hurdle, 2011). Second, antimicrobial agents that act in the extracellular bacterial environment evade intracellular resistance mechanisms and are expected to maintain prolonged clinical efficacy. Finally, cell permeability consideration, which is often a significant challenge for drug designers, is not necessary for the design of membrane-targeting antibiotics. Although peptidoglycan exists solely in bacteria, membranes

SUBSTITUTE SEIET(RULE 26) composed of lipid bi-layers are common to all cells; therefore, avoiding cytotoxicity to eukaryotic cells through non-selective membrane disruption is a major challenge. In contrast to most eukaryotic cell membranes, both Gram-positive and Gram-negative bacteria membranes are highly negatively charged due to a high content of anionic lipids such as cardiolipins and phosphatidylglycerol (Weghuber, 2011 ; Epand, 2007). Gram-negative bacteria membranes also have the negatively charged core of the lipopolysaccharide (LPS), while negatively charged techoic acids are major constituents of Gram-positive bacteria cell walls (Silhavy, 2010; Swoboda, 2010).

Hence, both Gram-positive and Gram-negative bacteria membranes attract positively charged organic compounds through ionic interactions. LPS that compose the Gram-negative outer membrane leaflet is unique to bacteria and serves as a target for the polymixin family of antibiotics (Figure 1). Polymixins composed of a cyclic cationic decapeptide with an N- terminal hydrophobic residue are highly potent and clinically used antibiotics that bind to the negatively charge LPS core and disrupt the outer membrane of Gram negative bacteria (Tsubery, 2002; Tsubery, 2000). The potency and broad-spectrum activity of polymixins against Gram-negative bacteria demonstrates the potential that lies in the development of membrane-targeting antibiotics and the need to develop such antimicrobials that will target Gram-positive bacteria as well.

Bacterial skin infections, including chronic infections related to diabetes, venous stasis, or arterial insufficiency account for a significant percentage of infectious diseases (Landis, 2008; Lipsky, 2004; Abdulrazak, 2005). Patients with infected wounds are frequently treated with systemic antibiotics and, in addition, topical antibiotic treatments (Schwartz, 2010; Lipsky, 2009). There is a large repertoire of potent antimicrobial agents that are unfit for internal use due to their toxicity but that are tolerated topically (Gelmetti, 2008). These can be used in treatment of skin infections caused by multidrug-resistant organisms that are unaffected by systemic antibiotic treatment.

Amongst the frequently used topical antimicrobial agents are gramicidins, polymixins, and aminoglycosides such as neomycin B (Schwartz, 2010, Lipsky, 2009; Gelmetti, 2008; Hancock, 1999). The heterogeneous oligo-peptide mixture of gramicidins is effective mainly against Gram positive bacteria but not against most Gram positive bacilli. Gramicidins are also highly hemolytic, making them extremely toxic when taken internally (Gelmetti, 2008; Hartmann, 2010, Mogi, 2009). The membrane targeting cyclic lipo-peptides polymixins are active against several Gram negative pathogens, but polymixins are not active against Gram positive bacteria and are also highly toxic when used systemically (Mogi, 2009; Zavascki, 2007). The clinical efficacy of the aminoglycoside neomycin B is continuously reduced as an ever increasing number of bacterial strains that acquire resistance to this aminoglycoside antibiotic (Glupczynski, 1999; Houghton, 2010). Topical use of neomycin B may be accompanied by undesired contact dermatitis side effects (Gehrig, 2008). Aminoglycosides are also highly nephrotoxic and ototoxic when used internally (Huth, 2011), Mingeot-Leclercq, 1999). Although topical antibiotic treatment is tolerated with gramicidins, polymixins and neomycin B, side effect occur when these antibiotics are used internally, or if high doses of these toxic antimicrobial agents can make their way into the blood system through open wounds or highly damaged external tissue. Therefore, there is a constant need for topical antibiotics that are effective against a wide spectrum of bacteria that exhibit minimal toxic side effects.

In recent years, several studies have demonstrated the potential of positively charged aminoglycosides (AGs) as scaffolds for the development of membrane-targeting cationic amphiphilic antimicrobial agents by the attachment of hydrophobic residues to one or more positions on the AG (Ouberai, 2011 ; Bera, 2010(a); Bera, 2010 (b); Baussanne, 2010; Hanessian, 2010). It was recently demonstrated that the attachment of aliphatic chains to the 6"-position of the aminoglycoside tobramycin (Figure 1, compound 1) resulted in potent antimicrobial agents and provided evidence for their membrane-disruption activity (Herzog, 2012). Several lines of evidence support the hypothesis that these cationic amphiphiles act by dirupting bacterial membranes (Herzog, 2012, Bera, 2010(a); Bera 2010 (b); Ouberai, 2011).

WO 2010/004433 discloses hydrophobically enhanced aminoglycosides, such as aminoglycoside-lipid conjugates, and their use as antibacterial agents.

There is an unmet need in the art for new antibacterial agents that are active against both gram-positive and gram-negative bacteria on the one hand, and that have reduced hemolytic activity on the other hand.

SUMMARY OF THE INVENTION

The present invention relates to amphiphilic aminoglycoside (AG) derivatives, especially derivatives of tobramycin, kanamycin A and paromomycin comprising hydrophobic side chains linked to the AG moiety by various linkers as described herein. These novel derivatives are useful as broad spectrum antibacterial agents by targeting bacterial cell membranes, while having minimal toxicity due to non-selective targeting of eukaryotic cell membranes. The present invention further relates to methods for preparing the novel derivatives, pharmaceutical compositions including such compounds, and methods of using these compounds and compositions, especially as membrane-targeting antibacterial agents, to combat bacteria that cause, e.g., soft tissue and skin infections, and for inhibiting biofilm growth.

The present invention is based on the discovery of novel aminoglycoside-based cationic amphiphiles differing in the chemical bond linking their hydrophobic and hydrophilic parts. In some embodiments, the novel compounds are based on the scaffold of aminoglycosides such as tobramycin and kanamycin A, linked to hydrophobic aliphatic chains via a linker such as a thioether, sulfone, sulfonyl, amide or triazolyl. In other embodiments, the novel compounds are paromomycin-based cationic amphiphiles that are based on the scaffold of paromomycin, linked at the two primary hydroxy sites to hydrophobic aliphatic chains via a linker such as a thioether, sulfone, sulfonyl, amide or triazolyl. Several compounds of the invention demonstrate potent antimicrobial and biofilm growth inhibition properties (e.g., they combat bacteria that cause soft tissue and skin infection), while exhibiting a dramatic reduction in red blood cells hemolysis, thus demonstrating that it is possible to maintain the antimicrobial potency of such molecules while reducing their undesired hemolytic effect.

Compared to mammalian cell membranes, bacterial membranes contain a high percentage of anionic lipids (Weghuber, 2011 ; Epand, 2007). It was therefore hypothesized that optimization of the interactions between the positively charged amines on the aminoglycoside segment of the cationic amphiphile and the negatively charged bacterial membrane surface may improve the antimicrobial activity of these compounds and their selectivity for bacterial membranes. In some embodiments, anchoring of the positively- charged aminoglycoside to the bacterial membrane was enhanced by attaching aliphatic chains on two different positions on the aminoglycoside scaffold.

The amphiphilic aminoglyoside analogues of the present invention include four structural motifs that affect their antibacterial activity as well as their specificity towards bacterial membranes: (1) the sugar backbone; (2) the length of the aliphatic chain (or chains); (3) the type of chemical bond that links the hydrophobic aliphatic chain and the pseudo- oligo saccharide; and (4) the number of positive charges on the molecule. Tobramycin and Kanamycin A Derivatives:

In some embodiments, the compounds of the present invention are based on tobramycin (1) and kanamycin A (6) (Figure 1), modified with lipophillic linear alkyl chains on the 6"- primary alcohol and/or the 4'-primary alcohol of each aminoglycoside. While kanamycin A and tobramycin share high strutural similarity, these two pseudo-oligosaccharides differ in the number of amine groups (four for kanamycin A and five for tobramycin) and therfore, under physiologial conditions, the overall positive charge of tobramycin is higher than that of kanamycin A. Tobramycin (1) based cationic amphiphiles are particularly interesting since similar to polymixins, this AG also contains five primary amines which are positively charged under physiological conditions.

Without wishing to be bound by any particular mechanism or theory, it is contemplated that the selectivity towards bacterial membranes is based on ionic interactions of the positively charged aminoglyco side-based cationic amphiphiles with the negatively charged bacterial membranes. A decrease in the overall positive charge of the cationic amphiphiles by decreasing the number of amines on the amino glyoside scaffold (e.g., by using kannamycin A analogs vs. tobramycin analogs), may not dramatically affect the affinity to bacterial membranes which are highly negatively charged, yet on the other hand it may significantly reduce the affinity of the kanamycin A amphiphiphiles towards eukaryotic cell membranes which are far less negatively charged.

Moreover, selectivity of the amphiphilic aminoglycoside analogues towards bacterial membranes may also be achieved based on the high overall negative charge of bacterial membranes, by significantly enhancing the net positive charge by increasing the number of ammonium groups on these analogues. This may be accomplished by creating amphiphilic dimeric structures of the amphiphilic aminoglycosides, as demonstrated hereinbelow (e.g., compounds XII and 10), therefore doubling the net positive charge of these molecules compared to analogues with a single aminoglycoside scaffold.

Selectivity towards bacterial cells may be demonstrated by comparing the hemolytic activity and eukariotic cell toxicity of the kanamycin A and tobramycin analogues, as described herein.

The linear alkyl chains typically range from 12 to 16 carbons in length which were found to be the optimal lenghths leading to the most potent and broad spectrum antibacterial activity (Herzog, 2012). When two alkyl chains are present, the length of each chain is preferably between 4 and 8 carbon atoms. The total number of carbon atoms (whether originating from one alkyl or two alkyl chains) is preferably from 12 to 16. The linker can be any functional group that links the aminoglycoside to the lipophillic alkyl group. Non-limiting examples include thioether, sulfone and sulfonyl ester-bond based compounds, amide bond based compounds and triazole analogues, as further described hereinbelow.

Thus, in one embodiment, the present invention relates to a tobramycin or kanamycin A derivative represented by the structure of formula I:

wherein

2 2

A is OH or -L -R , wherein:

(i) when A is OH, L¹ is selected from the group consisting of -OSO₂-, triazolyl, and -NH-C(=0)-; and R¹ is a Ci₂-Ci₆ linear alkyl;

(ii) when A is -L²-R², each of L¹ and L² is selected from the group consisting of - S-, -SO₂-, -OSO₂-, triazolyl and -NH-C(=0)-; and each of R¹ and R² is a linear C4-C8 alkyl; and

R³ and R⁴ are each OH; or R³ is NH₂ and R⁴ is H;

including salts, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

In one embodiment of formula I, R³ is N¾ and R⁴ is H. In accordance with this embodiment, the compound is a tobramycin derivative, represented by the structure of formula II:

II

In another embodiment of formula I, R³ and R⁴ are each OH. In accordance with this embodiment, the compound is a kanamycin A derivative represented by the structure of formula III:

III

The compounds of the present invention have, in some embodiments, different functional groups linking their hydrophobic (alkyl) and hydrophilic (aminoglycoside) components.

In some embodiments, A in the compounds of formula I-III is OH, in which case the linker L¹ is selected from a sulfonyl ester, a triazolyl and an amide. Preferably, A is in the equatorial position. Non-limiting examples of such compounds are depicted in compounds of Formula IV, V and VI, respectively, with each possibility representing a separate embodiment of the present invention. For each of compounds IV, V and VI, R³ and R⁴ may each be OH (kanamycin A derivative) or R³ is NH₂ and R⁴ is H (tobramycin derivative), with each possibility representing a separate embodiment of the present invention. The structures of compounds IV, V and VI are depicted in the detailed description hereinbelow.

As contemplated herein, it has been discovered that linkage of aminoglycosides of formula I wherein A is OH to lipophilic hydrocarbon chains, and in particular linear alkyl chains of 12 to 16 carbons in length, lead to potent and broad spectrum antibacterial activity. Thus, in some embodiments, R¹ in any of the compounds of formula I- VI is a linear alkyl of 12 to 16 carbons, examples of which include -(CH₂)nCH₃, -(CH₂)i₃CH₃ and -(CH₂)i5CH₃. Each possibility represents a separate embodiment of the present invention. In some embodiments, A in the compounds of formula I-III is -L²-R² wherein R² and L² are as defined above. In this case, each of the linkers L¹ and L² is selected from a thioether, a sulfone, a sulfonyl ester, a triazolyl and an amide. Preferably, A is in the axial position. Non- limiting examples of such compounds are depicted in compounds of Formula VII, VIII, IX, X and XI, respectively, with each possibility representing a separate embodiment of the present invention. For each of compounds VII, VIII, IX, X and XI, R³ and R⁴ may each be OH (kanamycin A derivative) or R³ is NH₂ and R⁴ is H (tobramycin derivative), with each possibility representing a separate embodiment of the present invention. A currently preferred compound is compound (17). The structures of compounds (17), VII, VIII, IX, X and XI, are depicted in the detailed description hereinbelow.

As contemplated herein, it has further been discovered that linkage of aminoglycosides

2 2

of formula I wherein A is -L -R to lipophilic hydrocarbon chains, and in particular linear alkyl chains of 4 to 8 carbons in length, lead to potent and broad spectrum antibacterial activity. Thus, in some embodiments, each of R¹ and R² in any of the compounds of formula VII-XII is a linear alkyl of 4 to 8 carbons, preferably 6 to 8 carbon atoms. Without wishing to be bound by any particular mechanism or theory, it is contemplated that the sum of the carbons of the two alkyl groups in such compounds is preferably between 12 and 16 carbons, which has been found to be a preferred length for the monoalkylated derivatives described above.

In another embodiment, the present invention relates to a dimeric compound represented by the structure of formula XII:

wherein

R¹ is a C₁₂-C16 linear alkyl;

L¹ is selected from the group consisting of -S-, -SO₂-, -OSO₂-, triazolyl and -NH- C(=0)-; R³ and R⁴ are each OH; or R³ is NH₂ and R⁴ is H; and

B is selected from an unsubstituted or substituted alkyl, aryl, aryloxy, alkyloxy and amide, preferably wherein B is phenoxy;

An example of such compound is compound 10 (e.g., compounds 10a and 10b).

10a: R³ = NH₂, R⁴ = H

10b: R³, R⁴ = OH

Paromomycin Derivatives:

In additional embodiments, the compounds of the present invention are based on dialkylated derivatives of paromomycin. For the preparation of di-alkylated aminoglycosides the pseudo-tetrasaccharide paromomycin (1A) was chosen (Figure 1) for two reasons: Like tobramycin (Figure 1), this aminoglycoside scaffold has five amine functionalities that are positively charged under physiological conditions. However, unlike tobramycin, paromomycin has two primary alcohols therefore making it possible to readily and chemo-selectively alkylate these two alcohols in the presence of the six secondary alcohols of this aminoglycoside. As demonstrated herein, with tobramycin-based cationic amphiphiles, the optimal antimicrobial activity was obtained by attaching C12, C14, and Ci6 alkyl chains to the aminoglycoside. A substantial drop in antimicrobial activity was observed for tobramycin-based amphiphiles with shorter or longer alkyl chains. To maintain the hydrophobicity/hydrophilicity ratio that was optimal in the case of the potent tobramycin cationic amphiphiles, Ce, C₇, and Cs alkyl chains were chosen for the preparation of the di-alkylated paromomycin derivatives.

A collection of paromomycin-based di-alkylated cationic amphiphiles differing in the lengths of their aliphatic chain residues were thus designed, synthesized, and evaluated against 14 Gram positive pathogens that are known to cause skin infections. Paromomycin derivatives that were di-alkylated with C7 and Cs linear aliphatic chains had improved antimicrobial activities relative to the parent aminoglycoside as well as to the clinically used membrane- targeting antibiotic gramicidin D; several novel derivatives were at least 16 fold more potent than the parent aminoglycoside paromomycin. A comparison between a di-alkylated and a mono -alkylated paromomycin indicated that, for this sugar backbone, the di-alkylation strategy leads to both an improvement in antimicrobial activity and to a dramatic reduction in undesired red blood cell hemolysis caused by many aminoglycoside-based cationic amphiphiles. Scanning electron microscopy provided evidence for cell surface damage by the novel di- alkylated paromomycin derivatives.

Selectivity towards bacterial cells may be demonstrated by comparing the hemolytic activity and eukariotic cell toxicity of the paromomycin analogues, as described herein.

In one embodiment, the present invention relates to a paromomycin derivative represented by the structure of formula I-A:

wherein

L¹ and L² are each independently selected from the group consisting of -S-, -SO₂, -OSO₂-, triazolyl and -NH-C(=0)-; and

1 2

R and R are each independently a linear C4 to Cs alkyl;

The paromomycin derivatives of the present invention comprise different functional groups (linkers L¹ and L²) linking their hydrophobic (alkyl) and hydrophilic (aminoglycoside) components. The linkers are selected from thioethers (-S-), sulfonyls (-SO₂), sulfonyl esters (- OSO₂-), triazolyl and amides (-NH-C(=0)-). Non-limiting examples of such compounds are depicted in compounds of Formula II-A, III-A, IV-A, V-A and VI-A, respectively, with each possibility representing a separate embodiment of the present invention. Currently preferred compounds are represented by formula 22, 23 or 24, with each possibility representing a separate embodiment of the present invention. The structures of compounds II-A, III-A, IV-A, V-A, VI-A, 22, 23 and 24 are depicted in the detailed description hereinbelow.

As contemplated herein, it has been discovered that linkage of aminoglycosides of formula I-A to lipophilic hydrocarbon chains, and in particular linear alkyl chains of 4 to 8 carbons in length, lead to potent and broad spectrum antibacterial activity. Thus, in some

1 2

embodiments, each of R and R in any of the compounds of formula I-A to VI-A is a linear alkyl of 4 to 8 carbons, preferably 6 to 8 carbon atoms (CI¾(CH₂)5-, CI¾(CH₂)6-, and CI¾(CH₂)₇-). Without wishing to be bound by any particular mechanism or theory, it is contemplated that the sum of the carbons of the two alkyl groups in such compounds is preferably between 12 and 16 carbons, which has been found to be a preferred length for conferring potent antibacterial on the one end, and minimal hemolytic potential on the other.

In another aspect, the present invention relates to an anti-bacterial pharmaceutical composition comprising a compound according to any of formulae I to XII, or I-A to VI-A as described herein, or any compound covered by said formulae, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions of the invention may be a form suitable for oral administration, intravenous administration by injection, topical administration, administration by inhalation, or administration via a suppository.

In another aspect, the present invention relates to a method of combating bacteria, or treating bacterial infections, comprising the step of administering to a subject in need thereof a compound of any of formulae I to XII, or I-A to VI-A as described herein, or any compound covered by said formulae, or a pharmaceutical composition comprising such compound. The bacteria may be Gram-positive or Gram-negative, with each possibility representing a separate embodiment of the present invention. In some embodiments, the bacteria cause skin infections. In other embodiments, the bacteria cause soft tissue (e.g., throat) infections.

In another aspect, the present invention relates to a method of combating bacteria, comprising the step of contacting the bacteria with a compound of any of formulae I to XII, or I-A to VI-A as described herein, or any compound covered by said formulae, or a composition comprising such compound.

In another aspect the present invention relates to the use of a compound of any of formulae I to XII, or I-A to VI-A as described herein, or any compound covered by said formulae, or a pharmaceutical composition comprising such compound, for the manufacture of a medicament for combating bacteria or treating bacterial infections.

In another aspect, the present invention relates to a compound of any of formulae I to XII, or I-A to VI-A as described herein, or any compound covered by said formulae, or to a pharmaceutical composition comprising such compound, for use in combating bacteria or treating bacterial infections.

In another embodiment, the present invention relates to a method of inhibiting biofilm growth, comprising the step of contacting the biofilm or a surface comprising the biofilm with a compound of any of formulae I to XII, or I-A to VI-A as described herein, or any compound covered by said formulae, or a pharmaceutical composition comprising such compound.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Structures of the Gram-negative bacteria targeting polymixins and bacterial ribosome targeting aminoglycoside antibiotics tobramycin, kanamycin A, and paromomycin.

Figure 2: : Laboratory rat RBC hemolysis assay. Amphiphilic tobramycin analogues were incubated with RBCs isolated from a laboratory rat at concentrations of (A) 32 μg/mL, (B) 64 μg/mL, and (C) 128 μg/mL for 1 hour at 37°C. All experiments were performed in triplicate, and results are the average from two different sets of experiments using blood samples from two laboratory rats.

Figure 3: Rat RBCs were incubated with tested compounds for 1 hour at 37°C: 22 (X), 23 (A), 24 (·), 25 (—♦—), gramicidin D (■). All experiments were performed in triplicate, and results are the average of at least two different sets of experiments using blood samples from different laboratory rats. Figure 4: Scanning electron microscopic (SEM) images of S. epidermidis ATCC12228 with and without drug: (a) Untreated control bacteria cells, (b) Cells after 1 hour of incubation at 37 °C with 1 ^ug/mL paromomycin 1A. (c) Cells after 1 hour of incubation at 37 °C with 1 μg/mh of compound 24.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to amphophilic aminoglycoside (AG) derivatives, especially derivatives of tobramycin, kanamycin A and paromomycin comprising hydrophobic side chains linked to the AG moiety by various linkers. These novel derivatives are useful as broad spectrum antibacterial agents targeting bacterial cell membranes, while having minimal toxicity due to non-selective targeting of eukaryotic cell membranes. The present invention further relates to methods for preparing the novel derivatives, pharmaceutical compositions including such compounds, and methods of using these compounds and compositions, especially as membrane-targeting antibacterial agents to combat bacteria that cause, e.g., soft tissue and skin infections, and for inhibiting biofilm growth.

Tobramycin and Kanamycin A Derivatives:

In one embodiment, the present invention relates to a tobramycin or kanamycin A derivative represented by the structure of formula I:

wherein

A is OH or -L²-R², wherein:

(i) when A is OH, L¹ is selected from the group consisting of -OSO₂-, triazolyl, and -NH-C(=0)-; and R¹ is a C12-C16 linear alkyl; (ii) when A is -L²-R², each of L¹ and L² is selected from the group consisting of - S-, -SO₂-, -OSO₂-, triazolyl and -NH-C(=0)-; and each of R¹ and R² is a linear C4-C8 alkyl; and

R³ and R⁴ are each OH; or R³ is NH₂ and R⁴ is H;

including salts, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Several non-limiting embodiments of the compounds of formula I are compounds of formulae II, III, IV, V, VI, VII, VIII, IX, X and XI, or any of the exemplified compounds of formula (4a), (4b), (4c), (5a), (5b), (5c), (6a), (6b), (6c), (14a), (14b), (14c), (15a), (15b), (15c), (16a), (16b), (16c) and (17), which are described hereinbelow. Dimeric structures of any of such compounds, as represented by Formula XII (e.g., compound 10) may also be prepared and are encompassed by the present invention. Each possibility represents a separate embodiment of the present invention.

In some embodiments, A in the compounds of formula I-III is OH, in which case the linker L¹ is selected from a sulfonyl ester, a triazolyl and an amide. Preferably, A is in the equatorial position. Non-limiting examples of such compounds are depicted in compounds of Formula IV, V and VI, respectively, with each possibility representing a separate embodiment of the present invention. For each of compounds IV, V and VI, R³ and R⁴ may each be OH (kanamycin A derivative) or R³ is N¾ and R⁴ is H (tobramycin derivative), with each possibility representing a separate embodiment of the present invention.

IV

V

VI

In some embodiments, A in the compounds of formula I-III is -L²-R² wherein R² and L² are as defined above. In this case, each of the linkers L¹ and L² is selected from a thioether, a sulfone, a sulfonyl ester, a triazolyl and an amide. Preferably, A is in the axial position. Non- limiting examples of such compounds are depicted in compounds of Formula VII, VIII, IX, X and XI, respectively, with each possibility representing a separate embodiment of the present invention. For each of compounds VII, VIII, IX, X and XI, R³ and R⁴ may each be OH (kanamycin A derivative) or R³ is NH₂ and R⁴ is H (tobramycin derivative), with each possibility representing a separate embodiment of the present invention.

IX

XI

According to some embodiments, the compound is a tobramycin or kanamycin A derivative selected from the group consisting of the following structures, with each possibility representing a separate embodiment of the present invention:

In an additional preferred embodiment, the compound is represented by the structure of formula 17:

Paromomycin Derivatives:

In one embodiment, the present invention relates to a paromomycin derivative represented by the structure of formula I- A:

wherein

1 2

R and R are each independently a linear C4 to Cs alkyl;

Several non-limiting embodiments of the compounds of formula I are compounds of formulae II-A, III-A, IV-A, V-A and VI-A, or any of the exemplified compounds of formula (22), (23), (24), (26a), (26b), (26c), (27a), (27b), (27c), (28a), (28b), (28c), (29a), (29b) and (29c), which are described hereinbelow. Each possibility represents a separate embodiment of the present invention. In some embodiments, each of the linkers L¹ and L² is selected from a thioether, a sulfone, a sulfonyl ester, a triazolyl and an amide.

In one particular embodiment, L¹ and L² are each a thioether of the formula -S-, and the compound is represented by the structure of formula II-A:

In another particular embodiment, L¹ and L² are each a sulfonyl of the formula -SO₂-, and the compound is represented by the structure of formula III-A:

In another particular embodiment, L¹ and \ are each a sulfonyl ester of the formula -OSO2-, and the compound is represented by the structure of formula IV-A:

In another particular embodiment, L¹ and L² are each a triazolyl,

compound is represented by the structure of formula V-A:

In another particular embodiment, L¹ and L² are each an amide of the formula - NH-C(=0)-, and the compound is represented by the structure of formula VI-A:

In some preferred embodiments, the compound is represented by the structure of formula 22, 23 or 24, with each possibility representing a separate embodiment of the present invention.

According to other embodiments, the compound is a paromomycin derivative selected from the group consisting of the following structures, with each possibility representing a separate embodiment of the present invention:

Chemical Definitions:

The term "alkyl", used herein alone or as part of another group denotes linear and branched, saturated groups having from 1 to 20 carbon atoms. Some preferred alkyl groups for use in the compounds of the present invention include linear alkyl groups having from 12 to 16 carbon atoms. Other preferred alkyl groups for use in the compounds of the present invention include linear alkyl groups having from 4 to 8 carbon atoms. The alkyl group may be unsubstituted or substituted. An alkyloxy is an alkyl that is bonded to an oxygen atom (alkyl- O). In one embodiment, the alkyl is a linear alkyl of 6 carbon atoms (CH₃(CH₂)5-). In one embodiment, the alkyl is a linear alkyl of 7 carbon atoms (CH₃(CH₂)₆-). In one embodiment, the alkyl is a linear alkyl of 8 carbon atoms (CH₃(CH₂)₇-). In general, for monalkylated derivatives, the length of the alkyl group is preferably from 12 to 16 carbon atoms, and for dialkylated derivatives, the length of the alkyl group is preferably from 6 to 8 carbon atoms.

The term "aryl" used herein alone or as part of another group denotes an aromatic ring system containing from 6-14 ring carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic and the like. Non-limiting examples of aryl groups are phenyl, naphthyl including 1- naphthyl and 2-naphthyl, and the like. The aryl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl. An aryloxy is an aryl that is bonded to an oxygen atom (aryl-O, e.g., phenoxy).

The term "amino protecting group" as used herein refers to a readily cleavable group bonded to an amino group. The nature of the amino protecting group is not critical so long as the derivatized amino group is stable. Examples of amino-protecting groups include t- butoxycarbonyl (BOC), benzyloxycarbonyl, acetyl, phenylcarbonyl, or a silyl group, which can be substituted with alkyl (trialkylsilyl), with an aryl (triarylsilyl) or a combination thereof (e.g., dialkylphenylsilyl), e.g., trimethylsilyl (TMS) or t-butyldimethyl silyl (TBDMS). Other suitable amino-protecting agents and amino-protecting groups, as well as methods of protection and deprotection, have been described in, e.g., T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2^nd Ed., John Wiley and Sons (1991) and A. J. Pearson and W. R. Roush, Activating Agents and Protecting Groups, John Wiley and Sons (1999), each of which is incorporated herein by reference.

All stereoisomers of the above compounds are contemplated, either in admixture or in pure or substantially pure form. The compounds of the present invention can have asymmetric centers at any of the atoms. Consequently, the compounds can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The present invention contemplates the use of any racemates (i.e. mixtures containing equal amounts of each enantiomers), enantiomerically enriched mixtures (i.e., mixtures enriched for one enantiomer), pure enantiomers or diastereomers, or any mixtures thereof. The chiral centers can be designated as R or S or R,S or d,D, 1,L or d,l, D,L. The sugar residues include residues of D-sugars, L-sugars, or racemic derivatives of sugars.

One or more of the compounds of the invention, may be present as a salt. The term "salt" encompasses both basic and acid addition salts, including but not limited to salts with amine nitrogens, and include salts formed with the organic and inorganic anions discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic or inorganic acids. Such acids include, but are not limited to, hydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.

The present invention also includes solvates of the compounds of the present invention and salts thereof. "Solvate" means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates and the like. "Hydrate" is a solvate wherein the solvent molecule is water.

The present invention also includes polymorphs of the compounds of the present invention and salts thereof. The term "polymorph" refers to a particular crystalline state of a substance, which can be characterized by particular physical properties such as X-ray diffraction, IR spectra, melting point, and the like.

Therapeutic Uses

As contemplated herein, the present invention is based on the finding that tobramycin or kanamycin A derivatives of formula I, or compounds of formula II-XI or any compounds encompassed by such formulae, or dimeric structures according to formula XII as described above, are active as antibacterial agents against both Gram-positive and Gram-negative bacteria, targeting their membranes selectively, while at the same time exhibiting minimal toxicity due to non-selective targeting of eukaryotic cell membranes. In other embodiments, the present invention is based on the finding that paromomycin derivatives of formula I-A, or compounds of formula II-A to VI-A or any compounds encompassed by such formulae as described above, are active as antibacterial agents against both Gram-positive and Gram- negative bacteria, targeting their membranes selectively, while at the same time exhibiting minimal toxicity due to non-selective targeting of eukaryotic cell membranes.

The antibacterial compositions of the invention may be used for medicinal purposes and in such a case the composition is a pharmaceutical composition for the treatment of bacterial infections, e.g., bacteria that cause skin infections or soft tissue (e.g., throat) infections, or for inhibiting of biofilm growth.

Thus, in one, the present invention relates to a method of combating bacteria, or treating bacterial infections, comprising the step of administering to a subject in need thereof (1) a tobramycin or kanamycin A derivative of formula I or a compound of any of formulae II- XI or any compounds encompassed by such formulae; (2) a paromomycin derivative of formula I-A or a compound of any of formulae II-A to VI-A or any compounds encompassed by such formulae; or (3) dimeric structures according to formula XII as described herein, or a pharmaceutical composition comprising any of the aforementioned compounds. Each possibility represents a separate embodiment of the present invention. In another aspect the present invention relates to the use of (1) a tobramycin or kanamycin A derivative of formula I or compounds of formula II-XI or any compounds encompassed by such formulae; (2) a paromomycin derivative of formula I-A or a compound of any of formulae II-A to VI-A or any compounds encompassed by such formulae; or (3) dimeric structures according to formula XII as described above, or a pharmaceutical composition comprising any of the aforementioned compounds, for the manufacture of a medicament for combating bacteria or treating bacterial infections. Each possibility represents a separate embodiment of the present invention.

In another aspect, the present invention relates to (1) a tobramycin or kanamycin A derivative of formula I or compounds of formula II-XI or any compounds encompassed by such formulae; (2) a paromomycin derivative of formula I-A or a compound of any of formulae II-A to VI-A or any compounds encompassed by such formulae; or (3) dimeric structures according to formula XII as described above, or to a pharmaceutical composition comprising any of the aforementioned compounds, for use in combating bacteria or treating bacterial infections. Each possibility represents a separate embodiment of the present invention.

The anti bacterial composition may also be used for disinfecting purposes for example of surfaces, devices (including medical devices), cultures of eukaryotic cells or tissue, water pipes and water filters, food and agricultural products. The anti bacterial compositions may also be used to inhibit biofilm growth. The present invention further concerns a method for combating bacteria by contacting the bacteria with an effective amount of (1) a tobramycin or kanamycin A derivative of formula I or compounds of formula II-XI or any compounds encompassed by such formulae; (2) a paromomycin derivative of formula I-A or a compound of any of formulae II-A to VI-A or any compounds encompassed by such formulae; or (3) dimeric structures according to formula XII as described above, or to a pharmaceutical composition comprising any of the aforementioned compounds.

The contact may be ex vivo on a surface, on a device, in cell/tissue culture dish, in food, water, agricultural product as described above. Alternatively the contact may be in the body of a human or non human subject.

The term "anti-bacterial" may refer to one or more of the following effects: killing the bacteria (bacteriocide), causing halt of growth of bacteria (bacteriostatic), prevention of bacterial infection, prevention of bio-film formation and disintegration of a formed biofilm, and decrease in bacterial virulence. Examples of bacterial strain that can be treated/disinfected by the composition of the invention (both as a disinfecting composition and as a pharmaceutical composition) are all Gram-negative and Gram-positive bacteria and in particular pathogenic gram negative and gram positive bacteria.

The term "combating bacteria" or "treating bacterial infection" may refer to one of the following: decrease in the number of bacteria, killing or eliminating the bacteria, inhibition of bacterial growth (stasis), inhibition of bacterial infestation, inhibition of biofilm formation, disintegration of existing biofilm, or decrease in bacterial virulence.

The compounds of the present invention are effective against a wide variety of Gram- positive and Gram-negative cells, non-limiting examples of which include

Gram-positive: Streptococcus pyogenes M12 (strain MGAS9429), Methicillin- resistant Staphylococcus aureus (MRSA), Streptococcus mutans UA 159, vancomycin- resistant enterococci (VRE), Enterococcus faecalis ATCC 29212, Staphylococcus aureus (Oxford strain ATCC9144), Staphylococcus epidermidis ATCC 35984, and Staphylococcus epidermidis ATCC 12228, S. aureus oxford NCTC6571 ; S. aureus Cowan ATCC12598; G, S. pyogenes MlTl ; H, S. pyogenes M2; I, S. pyogenes M3; J, S. pyogenes M5; K, S. pyogenes M24; L, S. pyogenes JRS75; M, S. pyogenes glossy; and N, S. pyogenes T5.

Gram- negative: Pseudomonas aeruginosa ATCC33347, Shigella sonnei clinical isolate 6831(0-antigen positive), and Shigella sonnei clinical isolate 6831 (O-antigen negative).

The methods of the invention both ex-vivo and in the body of the subject may further comprise co administration of at least one additional anti-bacterial agent such as state of the art antibiotics.

As used herein, the term "administering" refers to bringing in contact with a compound of the present invention. Administration can be accomplished to cells or tissue cultures, or to living organisms, for example humans. In one embodiment, the present invention encompasses administering the compounds of the present invention to a human subject.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs. A "therapeutically effective amount" is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. Pharmaceutical Compositions

Although the compounds of the present invention can be administered alone, it is contemplated that such compounds will be administered in pharmaceutical compositions further containing at least one pharmaceutically acceptable carrier or excipient. Where combination treatments are used, each of the components can be administered in a separate pharmaceutical composition, or the combination can be administered in one pharmaceutical composition.

Thus, in one embodiment, the present invention contemplates pharmaceutical compositions that comprise a compound of formula I, and a pharmaceutically acceptable excipient. In one embodiment, the compound is represented by the structure of formula II. In another embodiment, the compound is represented by the structure of formula III. In another embodiment, the compound is represented by the structure of formula IV. In another embodiment, the compound is represented by the structure of formula V. In another embodiment, the compound is represented by the structure of formula VI. In another embodiment, the compound is represented by the structure of formula VII. In another embodiment, the compound is represented by the structure of formula VIII. In another embodiment, the compound is represented by the structure of formula IX. In another embodiment, the compound is represented by the structure of formula X. In another embodiment, the compound is represented by the structure of formula XI. In another embodiment, the compound is a dimeric structure represented by the structure of formula XII.

In another embodiment, the present invention contemplates pharmaceutical compositions that comprise a compound of formula IA, and a pharmaceutically acceptable excipient. In another embodiment, the compound is represented by the structure of formula II-A. In another embodiment, the compound is represented by the structure of formula III-A. In another embodiment, the compound is represented by the structure of formula IV-A. In another embodiment, the compound is represented by the structure of formula V-A. In another embodiment, the compound is represented by the structure of formula VI-A. Each possibility represents a separate embodiment of the present invention.

The pharmaceutical compositions of the present invention can be formulated for administration by a variety of routes including oral, rectal, transdermal, parenteral (subcutaneous, intraperitoneal, intravenous, intra-arterial, transdermal and intramuscular), topical, intranasal, or via a suppository. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise as an active ingredient at least one compound of the present invention as described hereinabove, and a pharmaceutically acceptable excipient or a carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and, more particularly, in humans.

During the preparation of the pharmaceutical compositions according to the present invention the active ingredient is usually mixed with a carrier or excipient, which may be a solid, semi-solid, or liquid material. The compositions can be in the form of tablets, pills, capsules, pellets, granules, powders, lozenges, sachets, cachets, elixirs, suspensions, dispersions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

The carriers may be any of those conventionally used and are limited only by chemical- physical considerations, such as solubility and lack of reactivity with the compound of the invention, and by the route of administration. The choice of carrier will be determined by the particular method used to administer the pharmaceutical composition. Some examples of suitable carriers include lactose, glucose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water and methylcellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents, surfactants, emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; flavoring agents, colorants, buffering agents (e.g., acetates, citrates or phosphates), disintegrating agents, moistening agents, antibacterial agents, antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), and agents for the adjustment of tonicity such as sodium chloride. Other pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

In one embodiment, in the pharmaceutical composition the active ingredient is dissolved in any acceptable lipid carrier (e.g., fatty acids, oils to form, for example, a micelle or a liposome).

In some embodiments, nanocarriers are used to effectuate intracellular uptake or transcellular transport of the compounds of the invention. Nanocarriers are miniature devices or particles that can readily interact with biomolecules on cell surfaces and within cells. Pharmaceutical nanocarriers such as viral vectors, polymeric nanoparticles, and liposomes are advantageous for delivering pharmaceutically active agents more selectively to target cells. Nanocarriers also effectively enhance the delivery of poorly-soluble therapeutics and control the release rate of encapsulated compounds. Many nanocarriers are natural or synthetic polymers that have defined physical and chemical characteristics. As a consequence, a person skilled in the art can engineer desired properties, such as target selectivity, biodegradability, biocompatibility, and responsiveness to environmental factors (e.g., pH or temperature changes), into nanocarriers to improve performance. Any nanocarriers can be used in the context of the present invention.

For preparing solid compositions such as tablets, the principal active ingredient(s) is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, from about 0.1 mg to about 2000 mg, from about 0.1 mg to about 500 mg, from about 1 mg to about 100 mg, from about 100 mg to about 250 mg, etc. of the active ingredient(s) of the present invention.

Any method can be used to prepare the pharmaceutical compositions. Solid dosage forms can be prepared by wet granulation, dry granulation, direct compression and the like. The solid dosage forms of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention may be incorporated, for administration orally or by injection, include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

Another formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art.

In yet another embodiment, the composition is prepared for topical administration, e.g. as an ointment, a gel a drop or a cream. For topical administration to body surfaces using, for example, creams, gels, drops, ointments and the like, the compounds of the present invention can be prepared and applied in a physiologically acceptable diluent with or without a pharmaceutical carrier. The present invention may be used topically or transdermally bacterial infections. Adjuvants for topical or gel base forms may include, for example, sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol and wood wax alcohols.

Alternative formulations include nasal sprays, liposomal formulations, slow-release formulations, pumps delivering the drugs into the body (including mechanical or osmotic pumps) controlled-release formulations and the like, as are known in the art.

The compositions are preferably formulated in a unit dosage form. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

In preparing a formulation, it may be necessary to mill the active ingredient to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active ingredient is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

It may be desirable to administer the pharmaceutical composition of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, infusion to the liver via feeding blood vessels with or without surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material.

The compounds may also be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. It is preferred that administration is localized, but it may be systemic. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

A compound of the present invention can be delivered in an immediate release or in a controlled release system. In one embodiment, a compound of the invention is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the compound over a controlled period of time at a selected site. Examples of preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.

Furthermore, at times, the pharmaceutical compositions may be formulated for parenteral administration (subcutaneous, intravenous, intraarterial, transdermal, intraperitoneal or intramuscular injection) and may include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Oils such as petroleum, animal, vegetable, or synthetic oils and soaps such as fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents may also be used for parenteral administration. Further, in order to minimize or eliminate irritation at the site of injection, the compositions may contain one or more nonionic surfactants. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described and known in the art.

Doses and Dosing Schedules

The amount of a compound of the invention that will be effective in the treatment of a particular bacterial infection will depend on the nature of the nature of the infection, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. A preferred dosage will be within the range of 0.01-1000 mg/kg of body weight, more preferably, 0.1 mg/kg to 100 mg/kg and even more preferably 1 mg/kg to lOmg/kg. Effective doses may be extrapolated from dose -response curves derived from in vitro or animal model test bioassays or systems.

The administration schedule will depend on several factors such as the bacterial infection being treated, the severity and progression, the patient population, age, weight etc. For example, the compositions of the invention can be taken once-daily, twice-daily, thrice daily, etc.

EXAMPLES

The following are non-limiting examples for the preparation of AE -based compounds attached to amino sugars and amino carba-sugars. It is apparent to a person of skill in the art that the present invention is not limited to the currently recited synthetic schemes and that variations in reaction conditions and reagents are possible and are encompassed by the broad scope of the present invention.

Example 1 - Preparation of Amphiphilic Tobramycin and Kanamycin A Aminoglycoside Derivatives

Scheme 1 generally represents various embodiments of the amphiphilic tobramycin and kanamycin A aminoglycoside derivatives of the present invention. Such derivatives may be prepared in accordance with the processes as described herein. Some currently preferred embodiments are described hereinbelow, while it is understood that various modifications in the synthetic scheme such as the choice of protecting groups, reagents and solvents are encompassed within the scope of the present invention.

Scheme 1: Synthesis of amphiphilic tobramycin analogues: Reagents and conditions: (a) R'SH, Cs₂C0₃, DMF, 25-60°C, 63-92%; (b) neat TFA, rt, 97%-quantitative yields; (c) mCPBA (3 equiv), CHCI3, r.t. 70-99%; (d) NaN₃, DMF, 60°C, 12 h, 91 %; (e) R'CCH, CuS0₄- 5H₂0 (0.1 equiv), sodium ascorbate (0.2 equiv), DMF, microwave irradiation, 87-94%; (f) PMe₃ (1M in THF), aqueous NaOH, rt, 80%; (g) R'COOH, HBTU, DIEA, DMF, 71-86%.

It is noted that other nitrogen protecting groups as defined herein may be used instead of the BOC protecting group described hereinabove, with suitable protection and deprotection methods being utilized as known in the art.

The most potent and broad-spectrum antimicrobial activity was observed for thioether analogues containing C12-, C14-, and Ci6-linear alkyl chains (Scheme 1A, 2a-c). The aliphatic chain length affected not only antimicrobial activity but also the level of undesired red blood cell (RBC) hemolysis; the C12 analogue had the least hemolytic activity. It was hypothesized that the aliphatic alkyl chains and the AG scaffold are required for optimal antimicrobial activity but that altering the link between these two segments should not have a dramatic effect on the antimicrobial performance, yet may affect the specificity of these compounds towards different membranes. It was further contemplated that linking the AG scaffold to the aliphatic chain through different chemical groups may affect the binding interactions with specific membrane components, therefore affecting the specificity of these compounds towards bacterial and RBC membranes. To test this using the tobramycin scaffold as an exemplary scaffold, several types of chemical bonds between the aliphatic chain and tobramycin were evaluated. Thioether-linked analogues (Scheme 1A, 2a-c) were compared to sulfone- linked analogues (3a-c), triazole ring-linked analogues (Scheme IB, 4a-c), and amide bond- linked analogues (Scheme 1C, 5a-c). The thioethers 2a-c were prepared from penta NH-Boc protected-6"-0-trisyl tobramycin (Scheme 1 A, la) as previously reported.¹⁸' ¹⁹ Oxidation of the protected thioether analogues (lb-d) using raCPBA (70-99% yield) followed by the removal of the NH-Boc protecting groups in neat TFA, gave the sulfone analogues (3a-c). The 6"-0-trisyl group of la was replaced by an azide to yield compound le,²⁰ which served as a precursor for the preparation of the triazole analogues (Scheme IB, 4a-c). Microwave-heated click reaction using le and terminal alkynyl aliphatic chains, followed by the removal of the NH-Boc groups, gave the desired triazole analogues 4a-c. Reduction of the 6"-azido group of le using the Staudinger reaction conditions resulted in superior yields (80%) of the 6"-amino tobramycin analogue If (Scheme 1C) compared to the reduction of the azide under catalytic hydrogenation conditions (H₂, Pd/C, MeOH), although both methods can be used to prepare this compound. Compound If served as the precursor for the preparation of the amide analogues (Scheme 1C); If was coupled to linear aliphatic carboxylic acids using HBTU (71-86% yield), and the NH- Boc groups were removed to yield the amide-linked analogues (Scheme 1C, 5a-c).

It is understood that, although Scheme 1 is described with respect to the synthesis of tobramycin analogs, similar compounds and reactions can be obtained using the kanamycin A scaffold. Scheme 2 generally represents various aminoglycosides of the present invention that are based on the kanamycin A scaffold.

Scheme 2: Synthesis of amphiphilic kannamycin A analogues: Reagents and conditions: (a) R'SH, Cs₂C0₃, DMF, 25-60°C; (b) neat TFA, rt; (c) mCPBA (3 equiv), CHCI3, r.t.; (d) NaN₃, DMF, 60°C, 12 h; (e) R'CCH, CuS0₄-5H₂0 (0.1 equiv), sodium ascorbate (0.2 equiv), DMF, microwave irradiation; (f) PMe₃ (1M in THF), aqueous NaOH, rt,; (g) R'COOH, HBTU, DIEA, DMF.

Example 2 - Results: Tobramycin Derivatives

The minimum inhibitory concentrations (MICs) of the semi-synthetic tobramycin amphiphiles were determined for 11 Gram-positive and Gram- negative strains (Table 1) (see Example 4 for experimental conditions). Amongst the Gram-positive bacteria were pathogenic strains such as Streptococcus pyogenes M12 (strain A), a hospital isolate of methicillin- resistant Staphylococcus aureus (MRSA; strain B), and vancomycin-resistant Enterococcus (VRE; strain D) with high levels of resistance to tobramycin (MIC = 64 μg/mL for strain A and >128 μg/mL for strains B and D). Amongst the Gram-negative isolates were the pathogenic and highly tobramycin resistant (MIC > 128 μg/mL) Pseudomonas aeruginosa (ATCC 33347; strain I) and Shigella sonnei, which is responsible for the severe foodborne disease shigellosis. Two types of S. sonnei were tested: O-antigen positive (strain J), and O-antigen negative (strain K) (Shepherd, 2000). In general, analogues with a C14 linear aliphatic chain (2b, 3b, 4b, and 5b) exhibited the most potent antimicrobial activity, which was in most cases one to two double dilutions more potent than the activity of the corresponding C₁₂ and Ci₆ linear aliphatic chain analogues. The chemical links between the AG and the aliphatic chain did not have significant effects on MIC values against the tested strains with the exception of the sulfone linked analogues 3a-c. These analogues were somewhat less potent than the corresponding un- oxidized thioether analogues 2a-c. Some of the amphophilic tobramycin analogues demonstrated high potency against strains that were highly tobramycin resistant: The MIC of tobramycin against S. pyogenes M12 (strain A) was 64 μg/mL; the thioether 2b, triazole 4b, and the amide analogue 5b were 32 and 16 times more potent against this strain, respectively. A significant improvement in antimicrobial activity of the semi-synthetic analogues compared to that of tobramycin was also observed in the case of S. mutans UA159 and S. epidermis ATCC35984 (strains C and G, respectively). Although most of the synthetic analogues were not active against the tested P. aeruginosa (strain I), the C₁₂ chain triazole analogue 4a and amide analogue 5a demonstrated improved antimicrobial activity against this strain relative to tobramycin (MICs = 64 and 32 μg/mL, respectively, and MIC >128 μg/mL for tobramycin). The antibacterial activity of tobramycin and six out of the twelve synthetic analogues was better against O-antigen positive S. sonnei (strain J) than against the corresponding O-antigen negative strain K. This difference may be explained by the higher overall negative charge of the membrane of the O-antigen positive S. sonnei, which contains the negatively charged 2- acetamido-2-deoxy-L-altruronic acid (Liu, 2010).

Table 1: MIC values ^g/mL) of tobramycin and its amphiphilic analogues

MICs ^g/ml) for tested bacterial strains⁶

AG" A B C D E F G H I J K

1 64 >128 128 >128 >128 16 128 <1 >128 16 32

2(a) 8 64 8 64 128 16 8 8 128 32 64

2(b) 2 32 2 16 64 8 4 4 128 16 32

2(c) 4 64 4 64 128 16 8 4 >128 32 64

3(a) 32 >128 16 64 >128 64 64 128 >128 >128 >128

3(b) 4 64 4 32 64 16 16 16 128 32 64 3(c) 8 32 8 32 64 8 16 16 >128 32 128

4(a) 8 128 4 32 128 16 8 16 64 64 64

4(b) 4 64 4 32 128 8 8 4 128 32 32

4(c) 16 32 8 16 128 16 8 4 >128 128 64

5(a) 16 128 4 64 128 16 8 8 32 128 128

5(b) 4 32 4 32 64 8 4 4 128 16 32

5(c) 8 32 4 32 128 8 4 4 128 32 32

^a AG = Aminoglycoside. ^b MIC values were determined against Gram-positive bacterial strains: A, S. pyogenes serotype M12 (strain MGAS9429); B, MRSA; C, S. mutans UA159; D, VRE; E, E. faecalis ATCC29212; F, S. aureus ATCC9144; G, S. epidermis ATCC35984; H, S. epidermis ATCC12228 and Gram-negative bacterial strains: I, P. aeruginosa ATCC33347; J, S. sonnei clinical isolate 6831 (O-antigen positive); and K, S. sonnei clinical isolate 6831 (O-antigen negative). All strains were tested by using the double-dilution method (from a starting concentration of 128 μg/mL). All experiments were performed in triplicate, and results were obtained from two different sets of experiments.

It was previously demonstrated that low micromolar concentrations of saturated fatty acids inhibit the formation of biofilms formed by S. aureus and Listeria monocytogenes strains (Nguyen, 2012). This activity may be explained by the fact that the amphiphilic structures of fatty acids destabilize interactions between protein and polysaccharide components that form biofilm matrices. Interestingly, the most potent inhibitors were C12-C14 aliphatic chain carboxylic acids. The applicants have therefore determined the minimal biofilm inhibition concentration (MBIC) values for each of the C12 and C14 chain tobramycin analogues (Table 2) (see Example 5 for experimental conditions). MBIC tests were performed using S. mutans UA 159 and S. epidermidis ATCC 35984 grown under biofilm-forming conditions. Compared to tobramycin (MBIC range of 64-128 μg/mL), the tested analogues demonstrated improved biofilm growth inhibition properties (MBIC range of 4-32 μg/mL) against the tested strains (Table 2). Table 2: Biofilm growth inhibition. MBIC values ^g/mL) of the amphiphilic tobramycin analogues and tobramycin (1).

AG^a

Strain" 1 2a 2b 3a 3b 4a 4b 5a 5b

C 64 4 4 16 4 4 4 8 4

G 128 16 8 32 8 16 16 8 8

a AG = Aminoglycoside. ^h S. mutans UA159; C, S. epidermis ATCC35984; G. All strains were tested by using the double-dilution method (starting form 128 μg/mL). S.mutans biofilm was grown in BHI+Sucrose 2%, at final dilution 1 : 100. S.epidermidis biofilm was grown in TSB+Glucose 1 %, at final dilution 1 :100. Bio films were stained using crystal violet. All experiments were performed in triplicates and results were obtained from two different sets of experiments.

Finally, the hemolytic activity was determined using a hemolysis assay using laboratory rat RBCs (Figure 2A-C) (see Example 6 for experimental details).¹⁸ The MIC and MBIC values were significantly lower than the concentrations required for 100% hemolysis for all analogues (Figure 2). In most cases, the MIC range of analogues with the C14 aliphatic chain was 2-32 μg/mL; these analogues caused significant hemolysis (-23-43%) at 32μg/mL. All of the Ci₄ aliphatic chain analogues caused extensive hemolysis (74.4+5.5%- 100%) at a concentration of 64 μg/mL. The Ci6 aliphatic chain analogues also caused high levels of hemolysis at 64 μg/mL 37.9+5.1%- 81.8+2.3%).

No direct correlation between the antibacterial potency and the hemolytic activity was detected for the thioether, triazole, or amide analogues. As initially hypothesized, the hemolytic activity of the different tobramycin analogues was affected by the type of bond between the aliphatic chain and the AG scaffold. The biggest effect was observed for the C12 aliphatic chain analogues. At 64 μg/mL the triazole C12 aliphatic chain analogue 4a demonstrated the highest hemolytic effect (26.3+2.7%) of the C12 aliphatic chain tobramycin analogues. The C12 aliphatic chain amide analogue 5a caused almost no hemolysis at the same concentration (0.0 + 0.4%). At 128μg/mL, the triazole analogue 4a caused extensive hemolysis (89.1+1.6%), the thioether 2a caused 71.6+8.3% hemolysis, yet the amide analogue 5a caused significantly less hemolysis (10.2+0.8%). The lowest hemolytic activity at all of the tested concentrations was observed for the C12 sulfone analogue 3a, however, this compound had poor antimicrobial activity against the tested strains. In contrast, while the C12 amide analogue 5a was potent against several of the tested bacterial strains, and was the most potent analogue against the tested P. aeruginosa (strain I), it caused the lowest levels of hemolysis at a concentration which was 16-32 times higher than the MIC values of this compound against several of the tested strains.

In conclusion, twelve 6"-aliphatic chain tobramycin analogues differing in the chemical linkage between the AG and the hydrophobic chain (thioether, sulfone, sulfonyl, triazole, and amide bonds) and in the length of their hydrophobic linear aliphatic chain (C12, C14, and C½ chains) were synthesized and evaluated for their antimicrobial activity against eleven bacterial strains. Of the three chain lengths tested, the C14 aliphatic chain analogues were the most potent antimicrobial agents, and were in most cases one or two double dilutions more potent than corresponding C12 and Ci6 chain analogues. In some cases, the antimicrobial activity was at least 32 fold more potent than that of the parent AG tobramycin. MBIC tests indicated that some of the compounds possessed biofilm growth inhibition properties that were significantly more potent than that of tobramycin. Finally, RBC hemolysis tests revealed that there was not a linear correlation between the antimicrobial potency and the hemolytic activity of the amphiphilic tobramycin analogues. Both the aliphatic chain length and the type of chemical linkage between the hydrophilic and hydrophobic parts of the molecule affect the specificity towards bacterial membranes. The C12 linear aliphatic chain 6"-amide analogue 5a is of particular interest. This analogue was significantly more potent than tobramycin and caused little measurable hemolysis of laboratory rat RBCs at concentrations up to 32 times higher than the MIC values of this compound against some of the tested strains. The results of this study demonstrate that the choice of the hydrophobic segment and of the chemical group that links the hydrophobic region to the AG are important factors in the design of such membrane targeting antibiotics.

Example 3: Synthetic Methods: Tobramycin Derivatives

General information.

lH NMR spectra (including 1D-TOCSY) were recorded on a Bruker Avance™ 400 spectrometer, and chemical shifts reported (in ppm) were calibrated to CD₃OD (δ = 3.31) with CD₃OD as the solvent, and to HOD (δ =4.63) with D₂0 as the solvent. ¹³C NMR spectra were recorded on a Bruker Avance™ 400 spectrometer at 100.6 MHz. Multiplicities are reported using the following abbreviations: br = broad, s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, t = triplet, dt = doublet of triplets, app. q = appears quartet, m = multiplet, and td = triplet of doublets. Coupling constants (J) are given in Hertz. Low-resolution electron spray ionization (LR-ESI) mass spectra were measured on a Waters 3100 mass detector. High-resolution electron spray ionization (HR-ESI) mass spectra were measured on a Waters Synapt instrument. Chemical reactions were monitored by TLC (Merck, Silica gel 60 F254). Visualization was achieved using a cerium-molybdate stain ((NH4)2Ce(N0₃)6 (5 g), (NIDeMo Oz HzO (120 g), H₂S0₄ (80 mL), H₂0 (720 mL)). All reactions were carried out under an argon atmosphere with anhydrous solvents, unless otherwise noted. All chemicals unless otherwise stated, were obtained from commercial sources. Tobramycin was bought from Tzamal D-Chem Laboratories Ltd. Compound la was prepared as previously described (Michael, 1999).

Compounds lb, lc, Id, 3a and 3b were prepared as previously reported (Herzog, 2012). Compounds were purified flash chromatography (S1O₂, Merck, Kieselgel 60). S. pyogenes serotype M12 (strain MGAS9429) (A), methicillin-resistant S. aureus (MRSA) (B), and vancomycin-resistant Enterococcus (VRE) (D) were a gift from Prof. Itzhak Ofek (Faculty of Medicine, Tel Aviv University). S. mutans UA159 (C) was a gift from Prof. Doron Steinberg (Faculty of Dental Medicine, The Hebrew University of Jerusalem). E. faecalis ATCC29212 (E), S.aureus ATCC9144 Oxford strain (F), S. epidermidis ATCC35984 (G), S. epidermidis ATCC12228 (H), and P. aeruginosa ATCC33347 (I) were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). S. sonnei clinical isolate 6831(0- antigen positive) (J), and S. sonnei clinical isolate 6831 (O-antigen negative) (K) were a gift from Prof. Dani Cohen (School of Public Health, Tel Aviv University).

Scheme 3: Tobramycin-based precursors and numbering system demonstrated for compound la.

(CH₂)₁₃CH₃ (1c) (CH₂)₁₃CH₃ (3b) (CH₂)₁₅CH₃ (1d)

2. Synthetic procedures

Compound le: Compound le was prepared as previously reported (Disney, 2007) with the following changes: Compound la (2.3 gr, 1.9 mmol) in DMF (12 mL) was added sodium azide (197 mg, 3.0 mmol) and

the reaction was stirred under argon at 75°C overnight. Propagation of the reaction was monitored by TLC (EtO Ac/petroleum ether: 7/3). Upon completion, the solvent was removed by evaporation under reduced pressure; the crude was re- dissolved in EtOAc (80 mL), washed twice with brine (2x60 mL), dried over anhydrous MgS0₄ and concentrated under reduced pressure. Purification by flash column chromatography (S1O₂, EtO Ac: petroleum ether) afforded compound le as a white solid (1.68, 91 %); LR-ESI m/z calc'd for C₄₃H76N₈Oi₈ 992.53, found 994.07 [M+H]⁺.

Compound If: Compound le (470 mg, 0.47 mmol) was dissolved in THF (2 mL), NaOH 0.01M (100 μί) and stirred at ambient temperature for 10 minutes after which PMe₃ (1M solution in THF, 0.5 mL, 0.50 mmol) was

added to the reaction mixture. Propagation of the reaction was monitored by TLC (EtO Ac/petroleum ether: 7/3). Upon completion, the solvent was removed by evaporation under reduced pressure, and the crude was purified by flash column chromatography (S1O2, MeOH: CH2CI2) to afford compound If (366 mg, 80%) as a white solid: ^lU NMR (400 MHz, D₂0) (Fig. SI) δ 5.10 (br s, 2H, Η-Γ, H-l"), 3.95 (ddd, J_x = 9.8 Hz, = 7.0 Hz, h = 3.0 Hz, 1H, H-5"), 3.69 (t, J = 10.2Hz, 1H, H-3"), 3.65-3.35 (m, 11H, H-l , H-3, H-4, H-5, H-6, H-2', H-4', H-5', H-6'(2H), H-2"), 3.18 (t, J = 9.8 Hz, 1H, H-4"), 3.02 (br d, J = 13.0 Hz, 1H, H-6"), 2.70 (dd, = 13.5 Hz, J₂ = 6.9 Hz, 1H, H-6"), 2.10 (br d, J = 10.6 Hz, 1H, H-2eq), 2.01 (m, 1H, H-3'eq), 1.65 (app. q, J_{l =} J₂ = J₃ = 12.1 Hz, 1 H, H-3'ax), 1.52- 1.40 (m, 46H, H-2ax, 5xC0₂C(CH₃)₃); ¹³C NMR (100 MHz, CD₃OD) (Fig. S2) δ 159.5, 159.3, 157.9, 157.8, 99.4 (anomeric C), 99.3 (anomeric C), 82.8, 80.7, 80.5, 80.4, 80.2, 76.9, 74.1 , 73.5, 72.1, 71.5, 66.4, 57.2, 51.6, 51.1 , 51.0, 43.5, 42.0, 35.9, 34.3, 28.9, 28.8 ; LR-ESI m/z calc'd for C₄₃H₇₈N₆Oi₈ 966.54, found 967.94 [M+H]⁺

Compound 3c: Compound Id (100 mg, 0.08 mmol) in CHCI₃ (3 mL) was treated with ra-chloroperbenzoic acid (70-75%) (54 mg, -0.31 mmol), and stirred at ambient temperature under argon atmosphere. Propagation of the

reaction was monitored by ESI-MS by following the disappearance of the starting material ([M+H]⁺, m/z 1209.43) and the formation of the corresponding sulfone ([M+H]⁺, m/z 1241.15). Upon completion, the reaction mixture was diluted with CHCI₃ (15 mL), washed twice with a 1 M water solution of KOH (5 mL), concentrated under reduced pressure, and treated with 99% TFA (1.0 mL) for 3 min after which reaction mixture was evaporated under reduced pressure. The product was dissolved in a minimal volume of H₂0 and freeze-dried to afford the TFA salt of compound 3c as a white foam (89 mg, 82%). ^lU NMR (400 MHz, D₂0) (Fig. S3) δ 5.57 (d, J = 3.4 Hz, 1H, Η- Γ), 4.97 (d, J = 3.2 Hz, 1H, H- l "), 4.28 (t, J = 9.2 Hz, 1H, H-5"), 3.91-3.76 (m, 4H, H-4, H-5 , H-5', H- 2"), 3.67 (t, J = 9.0 Hz, 1H, H-6), 3.60-3.35 (m, 7H, H- l , H-3, H-2', H-4', H-3", H-4", H-6"), 3.33-3.23 (m, 2H, H-6', H-6"), 3.08 (m, 2H, SCbCH^CH^CHs), 3.02 (dd, J_x = 13.5 Hz, J₂ = 7.8 Hz, 1H, H-6'), 2.43 (m, 1H, H-2eq), 2.16 (m, 1 H, H-3'eq), 1.86 (m, 2H, H-2ax, H-3'ax), 1.62 (m, 2H, SOzCH^CH^wCHs), 1.26 (m, 2H, SOzCHz CH^wCHs), 1.22-1.07 (m, 24H, S0₂CH₂(CH₂)i₄CH₃), 0.74 (t, 3H, J = 6.9 Hz, SOzCHz CHz^CHs); ¹³C NMR (100 MHz, D₂0) (Fig. S4) δ 162.8 (q, J = 35 Hz, CF₃C0₂H), 116.4 (q, J = 290 Hz, CF₃C0₂H), 100.6 (anomeric C), 94.8 (anomeric C), 84.4, 77.6, 74.6, 70.2, 67.9, 67.7, 64.9, 54.52, 54.46, 52.4, 49.3, 48.2, 48.0, 40.2, 31.7, 29.9, 29.52, 29.49, 29.3, 29.2, 29.1, 28.7, 28.5, 27.9, 27.8, 22.5, 21.7, 13.7; HRESI-MS m/z calc'd for

762.4663, found 762.4659 [M+Na]⁺. NHBoc-protected 4a: Compound le (151 mg, 0.15 mmol), in DMF (1 mL), was added CuS0₄- 5H₂0 (3.6 mg, 0.01 mmol), sodium ascorbate (5.0 mg, 0.03 mmol) and 1-tetradecyne 90% (75 μΐ,, 0.30

mmol). The reaction mixture was irradiated by a microwave for 4 min. Propagation of the reaction was monitored by TLC (EtOAc/petroleum ether: 7/3) and upon completion, reaction mixture was diluted with EtOAc (10 mL) and the organic layer was washed twice with brine (2x20 mL). The aqueous layer was extracted again with EtOAc (10 mL) and the combined organic layers were dried over anhydrous MgSC and concentrated under reduced pressure. Purification by flash column chromatography (S1O₂, EtOAc: petroleum ether) afforded the corresponding NHBoc-protected 4a as a white solid (171 mg, 95%). ^lU NMR (400 MHz, CD₃OD) δ 7.75 (s, 1H, triazole ring), 5.11 (d, J = 3.3 Hz, 1H, H-l "), 5.06 (br s, 1 H, Η- Γ), 4.68 (br d, J = 14.0 Hz, 1 H, H-6"), 4.55 (m, 1H, H-6"), 4.42 (m, 1H, H-5"), 3.74 (t, J = 10.0 Hz, H-3"), 3.64-3.27 (m, 11H, H-l , H-3, H-4, H-5, H-6, H-2', H-4', H-5', H-6' (2H), H-2"), 2.99 (t, J = 9.8 Hz, 1H, H-4"), 2.68 (t, J = 7.7 Hz, 2H, C₂HN₃CH₂(CH₂)ioCH₃), 2.04 (m, 2H, H-2eq, H-3'eq), 1.66 (m, 3H, H-3'ax, C₂HN₃CH₂(CH₂)ioCH3 ), 1.47-1.28 (m, 64H, H-2ax, 5xC0₂C(CH₃)₃,

0.90 (t, J = 7.0 Hz, 3H, C₂HN₃CH₂(CH₂)ioCH3); ¹³C NMR (100 MHz, CD₃OD) δ 159.4, 159.2, 157.9, 157.7, 149.0 (triazole ring), 124.6 (triazole ring), 99.8 (anomeric C), 98.8 (anomeric C),

83.2, 81.7, 80.7, 80.5, 80.4, 80.2, 76.5 , 73.6, 71.8, 71.7, 70.4, 66.5, 57.1 , 51.7, 51.3, 42.1 , 36.1 ,

34.3, 33.1 , 31.8, 30.8, 30.6, 30.5, 30.4, 28.9, 28.84, 28.80, 26.4, 23.7, 14.4; LR-ESI m/z calc'd for C₅₇Hio₂N₈Oi₈ 1 186.73, found 1188.27 [M+H]⁺.

NHBoc-protected 4b: Compound le (200 mg, 0.20 mmol), CuS0₄- 5H₂0 (5 mg, 0.02 mmol), sodium ascorbate (9 mg, 0.05 mmol), DMF (1.5 mL), 1 - hexadecyne 90% (120 0.43 mmol). Purification

by flash column chromatography (S1O₂, EtOAc:petroleum ether) afforded the corresponding NHBoc-protected 4b as a white solid (212 mg, 87%). ^XH NMR (400 MHz, CD₃OD) δ 7.75 (s, 1H, triazole ring), 5.11 (d, J = 3.3 Hz, 1H, H-l "), 5.06 (br s, 1 H, H- l'), 4.69 (br d, J = 13.9 Hz, 1 H, H-6"), 4.55 (m, 1H, H-6"), 4.42 (m, 1H, H-5"), 3.74 (t, J = 9.9 Hz, H-3"), 3.64-3.27 (m, 11H, H- l , H-3, H-4, H-5 , H-6, H-2', H-4', Η-5', H-6' (2H), H-2"), 2.99 (t, J = 9.8 Hz, 1H, H-4"), 2.68 (t, J = 7.8 Hz, 2H, C₂HN₃CH₂(CH₂)i₂CH₃), 2.04 (m, 2H, H-2eq, H-3'eq), 1.66 (m, 3H, H-3'ax, C₂HN₃CH₂(CH₂)i₂CH₃ ), 1.47-1.28 (m, 68H, H-2ax, 5xC0₂C(CH₃)₃, C₂HN₃CH₂(CH₂)i₂CH₃), 0.90 (t, J = 7.0 Hz, 3H, C₂HN₃CH₂(CH₂)i₂CH₃); ¹³C NMR (100 MHz, CD₃OD) δ 159.4, 159.2, 157.8, 157.7, 149.0 (triazole ring), 124.6 (triazole ring), 99.8 (anomeric C), 98.8 (anomeric C), 83.1 , 81.8, 80.7, 80.4, 80.3, 80.2, 76.5 , 73.6, 71.8, 71.6, 70.4, 66.5, 57.0, 51.7, 51.2, 42.1 , 36.0, 34.3, 33.0, 31.8, 30.8, 30.6, 30.5, 30.4, 30.3, 28.9, 28.84, 28.80, 26.4, 23.7, 14.5 ; LR-ESI m/z calc'd for CsgHioeNsOis 1214.76, found 1216.27 [M+H]⁺.

NHBoc-protected 4c: Compound le (150 mg, 0.15 mmol), CuS0₄- 5H₂0 (2 mg, 0.01 mmol), sodium ascorbate (3 mg, 0.02 mmol), DMF (1 mL), 1- hexadecyne >95% (100 0.32 mmol). Purification

by flash column chromatography (Si0₂,

EtOAc:petroleum ether) afforded the corresponding NHBoc-protected 4c as a white solid (170 mg, 90%). ^lU NMR (400 MHz, CD₃OD) δ 7.75 (s, 1H, triazole ring), 5.11 (d, J = 3.3 Hz, 1H, H-l "), 5.06 (br s, 1H, Η-Γ), 4.69 (br d, J = 13.9 Hz, 1H, H-6"), 4.55 (m, 1H, H-6"), 4.42 (m, 1H, H-5"), 3.74 (t, J = 10.0 Hz, H-3"), 3.66-3.26 (m, 11H, H-l , H-3, H-4, H-5, H-6, H-2', H-4', H-5', H-6' (2H), H-2"), 2.99 (t, J = 9.9 Hz, 1H, H-4"), 2.68 (t, J = 7.8 Hz, 2H, C₂HN₃CH₂(CH₂)i₄CH₃), 2.04 (m, 2H, H-2eq, H-3'eq), 1.66 (m, 3H, H-3'ax, C₂HN₃CH₂(CH₂)i₄CH₃), 1.48-1.26 (m, 72H, H-2ax, 5xC0₂C(CH₃)₃, C₂HN₃CH₂(CH₂)i₄CH₃), 0.90 (t, J = 7.0 Hz, 3H, C₂HN₃CH₂(CH₂)i₄CH₃); ¹³C NMR (100 MHz, CD₃OD) δ 159.4, 159.2, 157.8, 157.7, 148.9 (triazole ring), 124.6 (triazole ring), 99.8 (anomeric C), 98.8 (anomeric C), 83.1 , 81.8, 80.7, 80.4, 80.3, 80.2, 76.5 , 73.6, 71.8, 71.6, 70.4, 66.5, 57.0, 51.7, 51.2, 42.0, 36.0, 34.3, 33.0, 31.8, 30.8, 30.6, 30.5, 30.4, 30.3, 28.9, 28.84, 28.80, 26.4, 23.7, 14.5 ; LR-ESI m/z calc'd for C₆iHnoN₈Oi₈ 1242.79, found 1243.89 [M+H]⁺.

NHBoc-protected 5a: Tridecanoic acid 98% (36 mg, 0.17 mmol) and NN-diisopropylethylamine (DIEA) (83 μί, 0.50 mmol) in dry DMF (2 mL), was added HBTU (77 mg, 0.20 mmol) and stirred at ambient

temperature for 15 min under argon atmosphere. The mixture was then cooled in an ice-bath, added with compound If (81 mg, 0.08 mmol) and allowed to reach ambient temperature. Propagation of the reaction was monitored by TLC (EtOAc: petroleum ether/7 :3), and upon completion, the reaction mixture was diluted with EtOAc (50 mL) and the organic layer was washed three times with brine (3x40 mL). The aqueous layer was extracted again with EtOAc (50 mL) and the combined organic layers were dried over anhydrous MgSC and concentrated under reduced pressure. Purification by flash column chromatography (Si0₂, EtOAc:petroleum ether) gave the corresponding NHBoc-protected 5a (84 mg, 86%) as a white solid. : Η NMR (400 MHz, CD₃OD) δ 5.13 (br s, 1H, Η-Γ), 5.04 (br d, J = 3.4 Hz, 1H, H-l "), 4.04 (m, 1 H, H- 5"), 3.69-3.31 (m, 14H, H- l , H-3, H-4, H-5, H-6, H-2', H-4', H-5', H-6' (2H), H-2", H-3", H-6" (2H)), 3.16 (t, J = 9.9 Hz, 1H, H-4"), 2.21 (t, J = 7.7 Hz, 2H, NHCOCH₂(CH₂)ioCH₃), 1.98- 2.12 (m, 2H, H-2eq, H-3'eq), 1.65 (app. q, J = 12.1 Hz, 1H, H-3'ax), 1.62-1.25 (m, 66H, H-2ax, 5xC0₂C(CH3)₃, NHCOCH₂(CH₂)i₀CH₃), 0.90 (t, J = 7.0 Hz, 3H, NHCOCH₂(CH₂)ioCH3); ¹³C NMR (100 MHz, CD₃OD) δ 177.0 (NHCO), 159.3, 159.2, 158.0, 157.8, 157.6, 99.8 (anomeric C), 99.4 (anomeric C), 83.2, 82.6, 80.7, 80.5, 80.3, 80.1 , 77.0, 73.5, 72.6, 72.1, 70.7, 66.5, 57.0, 51.7, 51.0, 42.1 , 41.5, 37.1, 34.3, 33.1, 31.8 , 30.8, 30.7, 30.5, 28.8, 27.0, 23.7, 14.5 ; LR- ESI m/z calc'd for C₅6Hio₂N₆Oi₉ 1 162.72, found 1163.76 [M+H]⁺.

NHBoc-protected 5b: Pentadecanoic acid 99% (40 mg, 0.17 mmol), DIEA (82 μί, 0.49 mmol), dry DMF (2 mL), HBTU (75 mg, 0.20 mmol), If (80 mg, 0.08 mmol). Purification by flash column chromatography

(Si0₂, EtOAc:petroleum ether) gave the corresponding NHBoc-protected 5b as a white solid (77 mg, 78%). ^lU NMR (400 MHz, CD₃OD) δ 5.13 (br s, 1H, H-l '), 5.04 (br d, J = 3.6 Hz, 1H, H-l "), 4.03 (m, 1H, H-5"), 3.67-3.30 (m, 14H, H-l , H-3, H-4, H-5, H-6, H-2', H-4', H-5', H-6' (2H), H-2", H-3", H-6" (2H)), 3.16 (t, J = 9.9 Hz, 1 H, H- 4"), 2.21 (t, J = 7.8 Hz, 2H, NHCOCH₂(CH₂)i₂CH₃), 2.13-1.98 (m, 2H, H-2eq, H-3'eq), 1.66 (app. q, J = 12.2 Hz, 1H, H-3'ax), 1.62- 1.28 (m, 70H, H-2ax, 5xC0₂C(CH₃)₃, NHCOCH₂(CH₂)i₂CH₃), 0.90 (t, J = 7.0 Hz, 3H, NHCOCH₂(CH₂)i₂CH3); ¹³C NMR (100 MHz, CD₃OD) δ 177.0 (NHCO), 159.3, 159.2, 158.0, 157.8, 157.6, 99.9 (anomeric C), 99.4 (anomeric C), 83.2, 82.7, 80.7, 80.5, 80.4, 80.2, 77.0, 73.5, 72.7, 72.1 , 70.7, 66.6, 57.0, 51.8, 51.1 , 42.0, 41.6, 37.1 , 35.9, 34.3, 33.1, 31.8, 30.8, 30.7, 30.5, 28.9, 28.83, 28.81 , 27.0, 23.7, 14.4; LR-ESI m/z calc'd for CssHioeNeOig 1 190.75, found 1191.83 [M+H]⁺.

NHBoc-protected 5c. Heptadecanoic acid >98% (45 mg, 0.17 mmol), DIEA (83 μΐ,, 0.50 mmol), dry DMF (2 mL), HBTU (75 mg, 0.20 mmol), If (80 mg, 0.08 mmol). Purification by flash column chromatography

(Si0₂, EtOAc:petroleum ether) gave the corresponding NHBoc-protected 5c as a white solid (72 mg, 71 %). ^lU NMR (400 MHz, CD₃OD) δ 5.13 (br s, 1H, Η-Γ), 5.04 (br d, J = 3.6 Hz, 1H, H-l "), 4.03 (m, 1H, H-5"), 3.69-3.30 (m, 14H, H-l , H-3, H-4, H-5, H-6, H-2', H-4', H-5', H-6' (2H), H-2", H-3", H-6" (2H)), 3.16 (t, J = 9.9 Hz, 1 H, H- 4"), 2.21 (t, J = 7.8 Hz, 2H, NHCOCH₂(CH₂)i₄CH₃), 2.13-1.98 (m, 2H, H-2eq, H-3'eq), 1.66 (app. q, J = 12.2 Hz, 1H, H-3'ax), 1.62- 1.27 (m, 74H, H-2ax, 5xC0₂C(CH₃)₃, NHCOCH₂(CH₂)i4CH₃), 0.90 (t, J = 7.0 Hz, 3H, NHCOCH₂(CH₂)i₄CH3); ¹³C NMR (100 MHz, CD₃OD) δ 177.0 (NHCO), 159.3, 159.2, 158.0, 157.8, 157.6 99.9 (anomeric C), 99.4 (anomeric C), 83.2, 82.7, 80.7, 80.5, 80.4, 80.1 , 77.0, 73.5, 72.7, 72.1 , 70.7, 66.6, 57.0, 51.8, 51.1 , 42.0, 41.6, 37.1, 34.3, 33.1 , 31.8 , 30.8, 30.5, 28.9, 28.8, 27.0, 23.7, 14.4; LR-ESI m/z calc'd for C₆oHiioN₆Oi₉ 1218.78, found 1219.71 [M+H]⁺.

Compound 4a: NHBoc-protected 4a (42 mg, 0.04 mmol) was treated with 99% TFA (2mL) at ambient temperature for 3 min. The TFA was then removed under reduced pressure, the product was re-dissolved

in a minimal volume of H₂0 and freeze-dried to afford 4a as a white foam (42 mg, 94%). ^XH NMR (400 MHz, D₂0) (Fig. S5) δ 7.60 (s, 1H, triazole ring), 5.69 (d, J = 2.9 Hz, 1H, H-l '), 4.91 (d, J = 3.5 Hz, 1H, H- l "), 4.62 (m, 1H, H- 6"), 4.46 (dd, Ji = 13.8 Hz, J₂ = 7.0 Hz, 1H, H-6"), 4.21 (ddd, Λ = 9.1 Hz, J₂ = 7.0 Hz, h = 2.1 Hz, 1H, H-5"), 3.84 (t, 7 = 9.8 Hz, 1H, H-4), 3.79-3.72 (m, 2H, H-5', H-2"), 3.65 (t, J = 8.9 Hz, 1H, H-5), 3.63-3.52 (m, 3H, H-6, H-2', H-4'), 3.47-3.28 (m, 5H, H-l , H-3, H-6', H-3", H-4"), 3.08 (m, 1H, H-6'), 2.53 (t, J = 6.3 Hz, 1H, C₂HN₃CH₂(CH₂)i₀CH₃), 2.40 (dt, J_l = 12.6 Hz, J₂ = J₃ = 4.1 Hz, 1H, H-2eq), 2.20 (dt, J_x = 12.1 Hz, J₂ = Js = 4.4 Hz, 1H, H-3'eq), 1.88 (app. q, J_x = J₂ = J₃ = 11.6 Hz, 1H, H-3'ax), 1.78 (app. q, J_{l =} J₂ = J₃ = 12.6 Hz, 1H, H-2ax), 1.43 (m, 2H, C₂HN₃CH₂(CH₂)ioCH₃), 1.18-1.06 (m, 18H, CzHNsCHzCCH^hoCHs), 0.65 (t, 3H, J = 6.8 Hz, C₂HN₃CH₂(CH₂)ioCH₃); ¹³C NMR (100 MHz, D₂0) (Fig. S6) δ 162.9 (q, J = 35 Hz, CF₃CO₂H), 148.6 (triazole ring), 124.9 (triazole ring), 1 16.4 (q, 7 = 290 Hz, CF₃CO₂H), 100.9 (anomeric C), 93.8 (anomeric C), 84.0, 76.7, 74.4, 70.9, 70.2, 67.9, 66.6, 64.7, 54.8, 50.3, 49.6, 48.4, 47.8, 40.1 , 31.3, 29.9, 29.6, 29.4, 28.9, 28.7, 28.6, 28.3, 27.7, 24.5, 22.1 , 13.5 ; HRESI- MS m/z calc'd for C₃₂H₆₂N₈0₈Na 709.4588, found 709.4583 [M+Na]⁺.

Compound 4b: NHBoc-protected 4b (59 mg, 0.05 mmol) was treated with 99% TFA (2mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, the product was re- dissolved in a minimal volume of H₂0 and freeze- dried to afford 4b as a white foam (58 mg, 93%). H NMR (400 MHz, D₂0) (Fig. S7) δ 7.47 (s, 1H,

triazole ring), 5.72 (d, J = 3.2 Hz, 1H, Η-Γ), 4.89 (d, J = 3.1 Hz, 1H, H- l "), 4.58(m, 1H, H-6"), 4.40 (dd, Ji = 14.5 Hz, J₂ = 5.8 Hz, 1H, H-6"), 4.21 (m, 1H, H-5"), 3.87 (t, J = 9.6 Hz, 1 H, H- 4), 3.77 (td, Ji = J₂ = 8.6 Hz, J₃ = 3.4 Hz, 1H, H-5'), 3.71 (dd, = 10.7 Hz, J₂ = 3.1 Hz, 1 H, H- 2"), 3.68-3.56 (m, 2H, H-5, H-6), 3.55-3.24 (m, 7H, H- l , H-3, H-2', H-4', H-6', H-3", H-4"), 3.01 (dd, Ji = 13.4 Hz, J₂ = 7.8 Hz, 1H, H-6'), 2.48 (t, J = 7.4 Hz, 2H, C₂HN3CH₂(CH₂)i₂CH₃), 2.40 (dd, = 12.2 Hz, J₂ = 3.6 Hz, 1H, H-2eq), 2.19 (dd, J_x = 7.3 Hz, J₂ = 4.5 Hz, 1H, H-3'eq), 1.91 (app. q, J_{l =} J₂ = J₃ = 11.4 Hz, 1H, H-3'ax), 1.83 (app. q, J_{l =} J₂ = J₃ = 12.6 Hz, 1H, H- 2ax), 1.44 (m, 2H, CzHNsC^CHz^CHs), 1.56-1.04 (m, 22H, CzHNsCHz CHz^CHs), 0.68 (t, 3H, J = 6.8 Hz, C₂HN3CH₂(CH₂)i₂CH₃); ¹³C NMR (100 MHz, D₂0) (Fig. S8) δ 162.7 (q, J = 35 Hz, CF₃CO₂H), 148.5 (triazole ring), 124.4 (triazole ring), 116.4 (q, J = 290 Hz, CF₃CO₂H), 100.9 (anomeric C), 93.7 (anomeric C), 84.1 , 76.7, 74.4, 70.7, 70.2, 67.9, 66.5, 64.9, 54.8, 50.2, 49.7, 48.4, 47.9, 40.3 , 31.7, 30.7, 29.8, 29.6, 29.4, 29.3, 29.1, 29.0, 28.8, 27.7, 24.7, 22.4, 13.6; HRESI-MS m/z calc'd for C₃4H₆7N₈0₈ 715.5082, found 715.5089 [M+H]⁺.

Compound 4c: NHBoc-protected 4c (46 mg, 0.04 mmol) was treated with 99% TFA (2mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, the product was re- dissolved in a minimal volume of H₂0 and freeze-

dried to afford 4c as a white foam (48 mg, 99%). ¾ NMR (400 MHz, D₂0) (Fig. S9) δ 7.43 (s, 1H, triazole ring), 5.73 (d, J = 3.2 Hz, 1H, H-1 '), 4.88 (d, J = 3.0 Hz, 1H, H- 1 "), 4.57 (m, 1H, H-6"), 4.38 (dd, J_t = 14.2 Hz, J₂ = 5.4 Hz, 1H, H-6"), 4.21 (m, 1H, H-5"), 3.88 (t, J = 9.6 Hz, 1H, H-4), 3.77 (td, J_\ = J₂ = 8.5 Hz, J₃ = 3.4 Hz, 1H, H-5'), 3.71 (dd, J_x = 10.7 Hz, J₂ = 3.1 Hz, 1H, H-2"), 3.68-3.56 (m, 2H, H-5, H-6), 3.54-3.28 (m, 6H, H-1 , H-3, H-2', H-4', H-6', H-3"), 3.25 (t, J = 9.9 Hz, 1H, H-4"), 2.99 (dd, J_x = 13.3 Hz, J₂ = 7.9 Hz, 1H, H-6'), 2.47 (t, J = 7.2 Hz, 2H, C₂HN₃CH₂(CH₂)i₄CH₃), 2.40 (dd, = 8.2 Hz, J₂ = 3.6 Hz, 1H, H-2eq), 2.19 (dd, Λ = 7.4 Hz, h = 4.4 Hz, 1H, H-3'eq), 1.92 (app. q, J_{l =} J₂ = J₃ = 11.5 Hz, 1H, H-3'ax), 1.84 (app. q, J_{l =} J₂ = J₃ = 12.6 Hz, 1H, H-2ax), 1.43 (m, 2H, C₂HN₃CH₂(CH₂)i₄CH₃), 1.19-1.03 (m, 26H, C₂HN₃CH₂(CH₂)i₄CH₃), 0.69 (t, 3H, J = 6.9 Hz, C₂HN₃CH₂(CH₂)i₄CH₃); ¹³C NMR (100 MHz, D₂0) (Fig. S10) δ 162.7 (q, J = 35 Hz, CF₃C0₂H), 148.5 (triazole ring), 124.2 (triazole ring), 116.4 (q, J = 290 Hz, CF₃C0₂H), 100.9 (anomeric C), 93.7 (anomeric C), 84.1 , 76.7, 74.4, 70.7, 70.2, 67.9, 66.5, 65.0, 54.9, 50.2, 49.7, 48.4, 47.9, 40.3, 31.8, 30.8, 29.8, 29.6, 29.4, 29.3, 29.2, 29.0, 28.9, 27.7, 24.8, 22.5, 13.7 ; HRESI-MS m/z calc'd for C₃6H₇oN₈0₈Na 765.5214, found 765.5216 [M+Na]⁺.

Compound 5a: NHBoc-protected 5a (23.8 mg, 0.02 mmol) was treated with 99% TFA (lmL) at ambient temperature for 3 min. The TFA was then removed under reduced pressure, the residue was re-dissolved in

a minimal volume of H₂0 and freeze-dried to afford 5a as a white foam (21.5 mg, 85%). H NMR (400 MHz, D₂0) (Fig. S l l) δ 5.67 (d, J = 3.5 Hz, 1H, H-1 '), 4.91 (d, J = 3.5 Hz, 1 H, H- 1 "), 3.88-3.73 (m, 4H, H-4, H-5', H-2", H-5"), 3.70 (t, J = 9.0 Hz, 1H, H-5), 3.65-3.26 (m, 10H, H-1 , H-3, H-6, H-2', H-4', H-6', H-3", H-4", H- 6"(2H)), 3.12 (dd, Λ =13.6 Hz, J₂ = 7.0 Hz, 1H, H-6'), 2.40 (dt, = 12.5 Hz, J₂ = J₃ = 4.2 Hz, 1H, H-2eq), 2.17 (m, 1H, H-3'eq), 2.1 1 (t, J = 7.5 Hz, 2H, NHCOCH₂(CH₂)i₀CH₃), 1.89 (app. q, J_{l =} J₂ = J₃ = 11.0 Hz, 1H, H-3'ax), 1.79 (app. q, J_{l =} J₂ = J₃ = 12.6 Hz, 1H, H-2ax), 1.43 (m, 2H, NHCOCH₂(CH₂)ioCH₃), 1.18-1.06 (m, 18H, NHCOCH₂(CH₂)i₀CH₃), 0.70 (t, 3H, J = 7.0 Hz, NHCOCH₂(CH₂)ioCH₃); ¹³C NMR (100 MHz, D₂0) (Fig. S 12) δ 178.2 (NHCO), 162.9 (q, J = 35 Hz, CF₃C0₂H), 116.4 (q, J = 290 Hz, CF₃C0₂H), 100.9 (anomeric C), 94.1 (anomeric C), 83.6, 77.3 , 74.2, 71.0, 70.4, 68.1 , 66.6, 64.4, 54.7, 49.8, 48.4, 47.8, 39.8, 38.9, 35.8, 31.2, 29.3, 28.8, 28.7, 28.6, 28.5, 28.4, 28.3, 27.8, 25.4, 22.0, 13.4; HRESI-MS m/z calc'd for CsiHesNeOg 663.4657, found 663.4648 [M+H]⁺.

Compound 5b: NHBoc-protected 5b (24.0 mg, 0.02 mmol) was treated with 99% TFA (lmL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, the residue was dissolved in a

minimal volume of H₂0 and freeze-dried to afford 5b as a white foam (24.9 mg, 98%). H NMR (400 MHz, D₂0) (Fig. S13) δ 5.66 (d, J = 3.5 Hz, 1H, H-1'), 4.92 (d, J = 3.6 Hz, 1H, H-1 "), 3.86-3.73 (m, 4H, H-4, H-5', H-2", H-5"), 3.69 (t, J = 9.0 Hz, 1H, H-5), 3.65-3.26 (m, 10H, H-1 , H-3, H-6, H-2', H-4', H-6', H-3", H-4", H-6"(2H)), 3.11 (dd, 7i = 13.6 Hz, J₂ = 7.1 Hz, 1H, H-6'), 2.39 (dt, J_x = 12.5 Hz, J₂ = J₃ = 4.2 Hz, 1H, H-2eq), 2.19-2.09 (m, 3H, H-3'eq, NHCOCH₂(CH₂)i₂CH₃), 1.89 (app. q, J_{l =} J₂ = J₃ = 12.0 Hz, 1H, H-3'ax), 1.78 (app. q, Ji = h = = 12.6 Hz, 1H, H-2ax), 1.43 (m, 2H, NHCOCH₂(CH₂)i₂CH₃), 1.17-1.08 (m, 22H, NHCOCH₂(CH₂)i₂CH₃), 0.71 (t, 3H, J = 7.0 Hz, NHCOCH₂(CH₂)i₂CH₃); ¹³C NMR (100 MHz, D₂0) (Fig. S14) δ 178.1 (NHCO), 162.9 (q, J = 35 Hz, CF₃CO₂H), 116.4 (q, J = 290 Hz, CF₃CO₂H), 100.9 (anomeric C), 94.0 (anomeric C), 83.7, 77.6, 74.2, 71.0, 70.3, 68.1 , 66.6, 64.5, 54.7, 49.8, 48.4, 47.8, 39.9, 38.9, 35.8, 31.3, 29.8, 29.4, 28.9, 28.7, 28.6, 28.5, 28.4, 28.0, 25.4, 22.1 , 13.5; HRESI-MS m/z calc'd for C₃₃H₆₇N₆0₉ 691.4970, found 691.4963 [M+H]⁺.

Compound 5c: NHBoc-protected 5c (33.2 mg, 0.03 mmol) was treated with 99% TFA (1.3mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, the product was re-dissolved in a

minimal volume of H₂0 and freeze-dried to afford 5c as a white foam (33.8 mg, 96%). H NMR (400 MHz, D₂0) (Fig. S15) δ 5.67 (d, J = 3.1 Hz, 1H, H-1'), 4.90 (d, J = 2.8 Hz, 1H, H-1 "), 3.86 (t, J = 9.3 Hz, 1H, H-4), 3.82-3.75 (m, 3H, H-5', H- 2", H-5"), 3.68 (t, J = 8.8 Hz, 1H, H-5), 3.62 (t, J = 9.1 Hz, 1H, H-6), 3.57-3.20 (m, 9H, H-1 , H-3, H-2', H-4', H-6', H-3", H-4", H-6"(2H)), 3.04 (dd, = 13.4 Hz, J₂ = 7.7 Hz, 1H, H-6'), 2.38 (m, 1H, H-2eq), 2.18-2.04 (m, 3H, H-3'eq, NHCOCH₂(CH₂)i₄CH₃), 1.89 (app. q, Ji = J₂ = h = 11.9 Hz, 1H, H-3'ax), 1.81 (app. q, h = J₂ = h = 12.4 Hz, 1H, H-2ax), 1.40 (m, 2H, NHCOCH₂(CH₂)i₄CH₃), 1.18-1.06 (m, 26H, NHCOCH₂(CH₂)i₄CH₃), 0.71 (t, 3H, J = 6.8 Hz, NHCOCH₂(CH₂)i₄CH₃); ¹³C NMR (100 MHz, D₂0) (Fig. S16) δ 177.2 (NHCO), 162.5 (q, J = 35 Hz, CF₃C0₂H), 116.0 (q, J = 290 Hz, CF₃C0₂H), 100.5 (anomeric C), 93.5 (anomeric C), 83.3, 77.0, 73.8, 70.8, 70.3, 67.8, 66.1 , 64.4, 54.2, 49.5, 48.0, 47.5, 39.7, 38.5, 35.5, 31.3, 29.4, 29.1 , 28.9, 28.8, 28.6, 28.5, 27.5, 25.1, 22.1, 13.3; HRESI-MS m/z calc'd for C₃5H₇oN₆0₉Na 741.5102 found 741.5101 [M+Na]⁺.

Example 4: Minimal Inhibitory Concentration (MIC) test.

Starter cultures were incubated for 24 hours (37 °C, 5% C0₂, aerobic conditions), and diluted in fresh broth medium to obtain an optical density of 0.008 (OD₆55). All strains were tested using a double-dilution method starting at 128 μg/mL in 96-well plates (Sarstedt, Newton, NC). After 24 hours of incubation, MTT reagent (50 μΕ of a 1 mg/mL solution in H₂0) was added to each well followed by additional incubation at 37°C for 30 min. MIC values ^g/mL) were determined as the lowest concentration at which no bacterial growth was observed. Results were obtained from two independent experiments and each experiment was done in triplicate. Tested strains: Gram-positive: Streptococcus pyogenes M12 (strain MGAS9429) (A), Methicillin-resistant Staphylococcus aureus (MRSA) (B), Streptococcus mutans UA 159 (C), vancomycin-resistant enterococci (VRE) (D), Enterococcus faecalis ATCC 29212 (E), Staphylococcus aureus (Oxford strain ATCC9144) (F), Staphylococcus epidermidis ATCC 35984, and Staphylococcus epidermidis ATCC 12228. Gram-negative: Pseudomonas aeruginosa ATCC33347 (I), Shigella sonnei clinical isolate 6831(0-antigen positive) (J), and Shigella sonnei clinical isolate 6831 (O-antigen negative) (K). All strains were grown in Brain Heart Infusion broth (BHI) (BBL Microbiology Systems, Cockeysville, MD) with the exception of Shigella sonnei that was grown in Trypticase Soy Broth (TSB).

Example 5: Minimal Biofilm Inhibitory Concentration (MBIC) test

S. epidermis ATCC35984 (G) and S. mutans UA 159 (C) were grown in biofilms in Trypticase Soy Broth (TSB) (BBL Microbiology Systems, Cockeysville, MD) supplemented with glucose 1%, and in BHI supplemented with sucrose 2%, respectively with presence of the tested analogues. Strains were tested using a double-dilution starting at 128 μg/mL, aerobically at 37 °C, 5% C0₂, 96-well plates. After 24 hours of growth, the plates were vigorously washed three times with phosphate-buffered saline (PBS) to remove any unattached bacteria and then dried for 1 hour at 60 °C. The air-dried wells were stained with 0.1 % crystal violet (200 μΐ_^) for 30 min, and the plate was rinsed with PBS. The air-dried plates were added with 200 μΐ_^ of acetic acid 30%, and the OD at 570 nm was measured by microtiter plate reader (Tecan). Experiments were performed in triplicate.

Example 6: Red blood cells (RBCs) hemolysis assay

Rat RBC solution (2% w/w) was incubated with 6"-tobramycin analogues using the double dilution method starting at concentration of 256 μg/mL for 1 h at 37 °C, 5% CO₂. Negative control was PBS and positive control was 1 % w/v solution of Triton X100 (100% lysis). Following centrifugation (2,000 rpm, 10 min, ambient temperature), the supernatant was drawn off and its absorbance measured at 550 nm using a microplate reader (Genios, TECAN). The results were expressed as percentage of hemoglobin released relative to the positive control (Triton X100).

This experiment was performed in triplicate, and the results are an average of two different sets of blood.

Example 7 - Design and Synthesis of Di-Alkylated Vara mo my c in- Has ed Cationic amphiphiles

Scheme 4 generally represents various embodiments of the amphiphilic paromomycin- based aminoglycoside derivatives of the present invention. Such derivatives may be prepared in accordance with the processes as described herein. Some currently preferred embodiments are described hereinbelow, while it is understood that various modifications in the synthetic scheme such as the choice of protecting groups, reagents and solvents are encompassed within the scope of the present invention.

Scheme 4: Synthesis of mono- and di-alkylated paromomycin-based cationic amphiphiles.

The five amine groups of commercially available paromomycin were protected with Boc groups to afford the penta-NHBoc protected paromomycin derivative 21a in 85% yield (Scheme 4) (Michael, 1999; Pathak, 2005). Selective conversion of the two primary alcohols at positions C-6' and C-5" of 21a to the corresponding O-trisyl leaving groups using 30 equivalents of 2,4,6-triisopropylbenzene-sulfonyl chloride gave compound 21b in 86%. Compound 21b was reacted with l-«-hexanethiol, l-«-heptanethiol, or l-«-octanethiol resulting in the NHBoc-protected di-alkylated paromomycin derivatives 22a, 23a, and 24a in yields ranging from 76 to 80%. Removal of the NHBoc protecting groups in neat TFA gave the penta TFA salts of the 6', 5" dithioether paromomycin derivatives 22, 23, and 24 with no need for further purification. When the penta-NHBoc paromomycin 21a was reacted with 20 equivalents of 2,4,6-triisopropylbenzene-sulfonyl chloride, a mixture of the di-trisylated product 21b and of the mono-trisylated product 25a was obtained (43% and 33% isolated yields, respectively, Scheme 4). NMR characterization of 25a confirmed that the O-trisylation took place on the C-6' primary alcohol of paromomycin (confirmed by ID-TOCSY NMR performed on a sample of 25a after the removal of the NHBoc groups). The 6'-0-trisyl of 25a was displaced by l-«-hexadecanethiol resulting in compound 25b; 25b was treated with TFA to yield the Ci₆ aliphatic chain thioether 25. Compound 25 was prepared as the single aliphatic chain anchor analogue of the di-Cs aliphatic chain paromomycin analogue 24. It is noted that other nitrogen protecting groups as defined herein may be used instead of the BOC protecting group described hereinabove, with suitable protection and deprotection methods being utilized as known in the art.

Example 8 - Results: Paromomycin-based cationic amphiphiles

A. Antibacterial Activity Tests Against Skin Injection Causing Bacteria

Compounds 22-25 were screened against 14 bacterial strains known to cause skin infections, and their minimum inhibitory concentrations (MICs) were determined using the double dilution protocol (Table 3).

Table 3. Antibacterial activity: MIC values ^g/mL) of mono- and di-alkylated amphiphiles (22-25), the parent drug paromomycin (1A), and the membrane targeting antibiotic gramicidin D. ^[a] MIC values were determined against: A, S. aureus oxford NCTC6571 ; B, MRSA; C, S. epidermidis ATCC12228 (biofilm negative); D, S. epidermidis ATCC35984/RP62A (biofilm positive); E, S. aureus Cowan ATCC12598 ; F, S. pyogenes serotype M12 (strain MGAS9429); G, S. pyogenes M 1T1 ; H, S. pyogenes M2; I, S. pyogenes M3 ; J, S. pyogenes M5; K, S. pyogenes M24; L, S. pyogenes JRS75; M, S. pyogenes glossy; and N, S. pyogenes T5.

These studies focused on strains belonging to two major families of Gram positive bacteria: Staphylococci and streptococci. Of the strains belonging to the Staphylococci genus were pathogens such as methicillin-resistant Staphylococcus aureus (MRSA, strain B) (Bearden, 2008), Staphylococcus aureus (Cowan, Strain E) (Loffler, 2010), which causes skin infections in patients with compromised immune systems such as HIV carriers, and two strains of Staphylococcus epidermidis (strains C and D) (Otto, 2009) that were once regarded as harmless human skin colonizing bacteria but are now recognized as major opportunistic pathogens. Also tested were nine strains of Streptococcus pyogenes. These bacteria colonize mainly the throat and skin of humans and are often the cause of skin and soft-tissue infections (Johansson, 2010).

Of the tested di-alkylated paromomycin derivatives, the C alkyl chain derivative 22 was the least active against all of the tested strains. Compound 22 was less potent than the parent paromomycin 1A against the five tested Staphylococci strains A-E. The di-C6 alkylated derivative 22 had superior activity compared to paromomycin 1A against all nine Streptococcus pyogenes strains F-N (MIC range from 4 to 16 //g/mL for compound 22, and 16 to >64 /g/mL for paromomycin 1A). The di-C7 alkyl chain derivative 23 was superior to compound 22 against most of the 14 tested strains (MIC range from 2 to 16 /g/mL). The most potent antimicrobial activity was observed for the di-Cs alkyl chain derivative 24 with MICs ranging from 2 to 8 //g/mL against all tested strains. The 6'-Ci₆ linear aliphatic chain paromomycin derivative 25, designed as the mono-alkyl chain anchor analogue of the di-Cs alkyl chain paromomycin derivative 24, had poor antimicrobial activity against all of the tested bacterial strains (MIC range from 8 to >64 //g/mL). In addition to comparing the antimicrobial activity of the alkylated paromomycins to that of the ribosome targeting parent antibiotic paromomycin 1A, their activity was evaluated against that of the membrane targeting oligopeptide mixture gramicidin D (Koo, 2001). With the exceptions of the two Staphylococcus epidermidis strains (C and D), gramicidin D was ineffective against the tested Staphylococci strains (Table 3). Compounds 23 and 24 were significantly more potent than gramicidin D against the tested Staphylococci strains A, B, and E with MIC values comparable to or better than those of gramicidin D against strains C and D. In contrast to the lack of activity against Staphylococci strains, gramicidin D demonstrated very good antimicrobial activity against seven of the nine tested Streptococci strains (for strains G-L, MICs < 0.5 //g/mL, and for strain N, MIC = 2 //g/mL). Although more potent than compounds 22, 23, and 24 against seven of the tested Streptococci strains, gramicidin D demonstrated very poor activity against S. pyogenes serotype M12 (strain F) and only moderate activity against S. pyogenes glossy (strain M; MIC > 64 //g/mL, and MIC = 16 //g/mL, respectively); compounds 22-24 were more potent than gramicidin D against these Streptococci strains. The antimicrobial activity tests demonstrated that the di-alkylated paromomycin amphiphiles have a broader spectrum of antimicrobial activity than the parent aminoglycoside 1A or the membrane targeting antibiotic gramicidin D. The significantly more potent antimicrobial activity of the di- Cs alkyl chain paromomycin derivative 24 compared to that of the mono Ci₆ linear aliphatic chain paromomycin derivative 25 demonstrates the favorable effect that may result from the attachment of more than one linear aliphatic chain to the aminoglycoside scaffold. B. Red blood cell hemolysis test

The selectivity of all of the amphiphilic paromomycin derivatives 22-25 for bacterial membranes was studied by testing the hemolytic activity of these compounds on red blood cells (RBCs) isolated from laboratory rats. The percentage of hemolysis was determined after one hour of incubation with increasing concentrations of the tested compounds (up to 256 /g/mL) at 37 °C. The membrane targeting gramicidin D caused hemolysis at low concentrations close to the MIC range of this antimicrobial agent (2.4+1.4% at 2 /g/mL), and a steep concentration-dependent elevation in the percentage of hemolysis was observed for gramicidin D (Figure 3). In contrast, even at concentrations of 128 //g/mL, compounds 22, 23, and 24 were significantly less hemolytic then gramicidin D (Figure 3). The aliphatic chain length affected the percentage of hemolysis with the lowest hemolysis caused by the di-C6 alkyl chain paromomycin derivative 22 (3.4+1.2% hemolysis at a concentration of 256 //g/mL) and the highest by the di-Cs alkyl chains paromomycin derivative 24 (62.5+7.9% at 256

Whereas the mono Ci₆ linear aliphatic chain paromomycin derivative 25 demonstrated poor antimicrobial activity against the tested bacterial strains, this compound caused drastic RBC hemolysis at concentrations that were lower than its MIC range. At a concentration of 16//g/mL, compound 25 already caused 33.8+5.2% hemolysis of rat RBCs; at the same concentration compounds 22, 23, and 24 caused almost no measurable hemolysis (0% for 22 and 23, and 0.8+0.3% for 24). Moreover, at a concentration of 32 //g/mL compound 25 caused almost a 100% hemolysis; the di-alkylayed paromomycin derivative 24 and the highly hemolytic gramicidin D caused no more than 60% hemolysis even at a concentration of 256 //g/mL. Hemolysis experiments demonstrated that compound 25 had no selectivity for bacterial membranes and acted in a manner similar to that of non-specific membrane disrupting detergents.

At a concentration of 128 //g/mL, the di-C6 alkyl chain paromomycin derivative 22 did not cause any measurable hemolysis; this compound was also the least potent antimicrobial agent against the 14 tested bacterial strains. The high end of the MIC range of derivative 23 against the 14 tested bacterial strains was 16 //g/mL; for 11 of the tested strains the MIC of compound 23 was not higher than 4 //g/mL. However, at 32 //g/mL which is two to eight times higher than the MICs, this compound caused almost no measurable hemolysis (3.6+1.9%). The di-Cs alkyl chain paromomycin derivative 24 demonstrated the most potent antimicrobial activity against all of the tested bacterial strains (MIC range from 2 to 8 μg/ h). The paromomycin derivative with the di-C₇ alkyl chain, 23, was either as active or one double dilution less active than compound 24 against the tested bacterial strains and caused significantly less RBC hemolysis than compound 24. At 256 //g/mL, compound 23 caused 17.6+4.3% hemolysis; compound 24 caused 62.5+7.9% hemolysis at the same concentration. The high level of hemolysis caused by compound 24 at 256 /g/mL was similar to that caused by gramicidin D. Hence, of the three di-alkylated paromomycins, the di-C₇ alkyl chain derivative 23 that demonstrated potent antimicrobial activities against all 14 bacterial strains and was dramatically less hemolytic then both Gramicidin D and di-Cs alkyl chain paromomycin derivative 24. Therefore, in terms of the ratio of hemolysis to antimicrobial activity, compound 23 is the most potent of the di-alkylated paromomycins that were studied.

C. Scanning electron microscopy (SEM) evidence for bacterial cell damage

In order to visualize bacterial cell morphological changes that result from exposure to the synthetic di-alkylated paromomycin-based cationic amphiphiles, we incubated a culture of S. epidermidis ATCC12228, which was susceptible to both the parent aminoglycoside paromomycin (1A) and to the most potent of the di-alkylated paromomycins, compound 24 at a concentration of 1 /g/mL; this concentration is half of the MIC of the two selected antimicrobial agents against this bacterial strain.

Under these experimental conditions, untreated bacterial cells had a smooth surface morphology (Figure 4a). After 1 hour of incubation with paromomycin 1A, moderate wrinkling of the bacterial cell surface was observed (Figure 4b). As paromomycin 1A is a protein synthesis inhibitor, the accumulation of damaged and non-functional membrane proteins may explain the observed wrinkled cell surfaces. Bacterial cells that were treated with the di-Cs alkyl chain paromomycin derivative 24 were severely damaged (Figure 4c). These cells had severe surface wrinkling and roughening that resulted from irregularly- shaped cell walls and damaged membranes. At higher concentrations of compound 24, mainly severely damaged cells and cellular debris were observed. The percentage of genes in the bacterial genome that encode bacterial membrane proteins averages between 20 and 30% depending on the strain (Wallin, 1998). Unlike the minor cell surface damage caused by paromomycin 1A that presumably results from impaired membrane protein synthesis, the severe cell surface damage caused by compound 24 can be rationalized by direct and rapid membrane disrupting effects of this compound.

D. Conclusions

In conclusion, three 5",6'-di-alkylated paromomycin-based cationic amphiphiles differing in the length of the linear hydrophobic chain (C , C₇, and Cs chains) were synthesized and evaluated for their antimicrobial activity against 14 bacterial pathogens known to cause skin infections. Of the three chain lengths tested, the di-Cs aliphatic chain paromomycin derivative 24 was the most potent antimicrobial agent against all of the tested strains and was at least 16 times more potent than the membrane targeting antibiotic gramicidin D and the parent ribo some-targeting aminoglycoside paromomycin 1A against most of the tested Staphylococci strains and some of the tested Streptococci strains.

RBC hemolysis tests indicated that both the aliphatic chain length and number of chains affected the level of undesired hemolysis of red blood cells by the reported paromomycin- based cationic amphiphiles. The Ce chain-based paromomycin derivative 22 was the least hemolytic; however, this compound was also the least potent antimicrobial agent of the three di-alkylated paromomycins tested. The di-C7 aliphatic chain paromomycin analogue 23 is of particular interest. This compound was either as effective or one double dilution less potent than the di-Cs aliphatic chain paromomycin analogue 24 against all of the tested bacteria, yet this compound caused considerably less hemolysis of laboratory rat RBCs compared to both compound 24 and the clinically used gramicidin D. Moreover, the mono Ci₆ linear aliphatic chain paromomycin derivative 25, which was synthesized as the mono aliphatic chain analogue of the di-Cs chain paromomycin analogue 24, demonstrated poor antimicrobial activity against all tested bacterial strains and caused almost 100% hemolysis of RBCs even at a concentrations close to the MIC.

Scanning electron microscopy (SEM) experiments revealed that bacterial cells that were incubated with the di-Cs aliphatic chain paromomycin analogue 24 had extensive cell surface damage compared to that caused by the parent ribosome-targeting aminoglycoside 1A. This extensive damage caused by analogue 24 is attributed to the disruption of the membranes of the treated bacteria and further supports the suggestion that these aminoglycoside-based cationic amphiphiles exert their biological activity through disruption of the bacterial cell membrane. The results of this study demonstrate that di-n-alkylation of paromomycin results in potent antimicrobial agents that are effective against a broad spectrum of Gram positive pathogens. The newly synthesized compounds caused significantly less hemolysis compared to membrane targeting antibiotics such as gramicidin D. This study also demonstrates that the di- «-alkylation approach may be more favorable than the mono-n-alkylation approach in designing aminoglyco side-based cationic amphiphiles both in terms of enhancing the antimicrobial activity and in reducing the undesired hemolytic effect.

Example 9: Synthetic Methods: Paromomycin Derivatives

A. General methods and instrumentation

Methods and instrumentation for these experiments are as described above in Example 3.

All chemicals, unless otherwise stated, were obtained from commercial sources. Compounds were purified flash chromatography (S1O₂, Merck, Kieselgel 60).

B. Synthetic procedures

Compound 21a. Paromomycin sulfate (1.0 g, 1.2 mmol) was dissolved in a mixture of dioxane/water (2:1, 15 ml), and triethylamine (1.7 ml, 12 mmol) was added. The solution was sonicated for 10 min and then B0C₂O (2.6 g, 12 mmol) was added, and the mixture was stirred at ambient temperature for 12 hours. Reaction progress was monitored by TLC (9% methanol in dichloro methane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (150mL), washed with brine, dried over MgSC , and concentrated under reduced pressure. The crude was purified by flash column chromatography over silica gel (7.5% methanol/dichloromethane) to afford compound 21a as a white solid (1.15 g, 85%).

Compound 21b. A solution of 21a (1.5 g, 1.34 mmol) in pyridine (20 mL) was treated with 2,4,6-triisopropylbenzenesulfonyl chloride (12.22 g, 40.35 mmol, 30.0 equiv). The reaction mixture was stirred at 23 °C for 12 h. Reaction progress was monitored by TLC (3% methanol in dichloro methane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (100 mL), washed with brine, dried over MgSC , and concentrated under reduced pressure. The concentrated crude was purified by flash column chromatography over silica gel (2.5% methanol in dichloromethane) to afford the desired product 21b as a white solid (1.9 g, 86%): ^lU NMR (500 MHz, methanol-d4) δ 7.32 (s, 2H, SOsCeEbXCHMCIDe), 7.31 (s, 2H, S0₃C₆H₂(CH)3(CH3)6), 5.47 (s, 1H, Η-Γ), 5.19 (s, 1H, H-l "), 4.85 (m, 1H, Η-Γ"), 4.40 (m, 1H, Η-6'), 4.38 - 4.30 (m, 2H, H-2", Η-6'), 4.25-4.14 (m, 4H, H-3", H-5"(2H), H-4"), 4.01 (m, 1H, H-5'), 3.91 (t, J = 3.1 Hz, 1H, H-4'"), 3.81 (t, J = 7.2 Hz, 1H, H-5'"), 3.77 (m, 1 H, H-3'"), 3.62 (dd, J = 10.3, 4.0 Hz, 1H, H-4'), 3.56 - 3.26 (m, 12H, H- l , H-3, H-4, H-5 , H-6, H-2', H-3', H- 2"', S0₃C6H₂(CH)₃(CH₃)₆(4H)), 3.20 (m, 1H, H-6'"), 2.99 (m, 3H, S0₃C6H₂(CH)₃(CH₃)₆(2H), H-6'"), 1.97 (m, 1H, H-2eq), 1.47 - 1.43 (m, 12H, 46H, 5XC0₂C(CH₃)₃, H-2ax), 1.33 - 1.23 (m, 36H, SQ₃C_fiH9(CH)₃(CHQ_fi). ¹³C NMR (100 MHz, methanol-d4) δ 157.5 , 157.1 , 156.8 , 156.7 , 156.3 , 154.0 , 150.8 , 149.4 , 148.0 , 129.3 , 129.1 , 123.6 , 122.0 , 109.2 (anomeric), 98.8 (anomeric), 97.3 (anomeric) , 85.0 , 79.5 , 79.2 , 79.1 , 78.8 , 77.9 , 77.1 , 74.0 , 73.2 , 73.0 , 72.5 , 70.0 , 69.9 , 68.5 , 67.1 , 54.6 , 52.0 , 50.9 , 49.5 , 39.8 , 34.1 , 34.0 , 33.9 , 29.4 ,

29.4 , 29.3 , 29.0 , 27.5 , 27.4 , 27.3 , 24.0 , 23.8 , 22.8 , 22.5 . LR-ESI m/z calc'd for C₇₈H₁₂₈N5O₂₈S₂ 1646.83 , found 1646.01 [M-H]^".

Compound 22a. To a solution of compound 21b (250 mg, 0.15 mmol) and CS₂CO₃ (198 mg, 0.6mmol) in dry DMF (4 mL) was added 1 -hexanethiol (0.30 mL, 2.12 mmol), and the mixture was stirred at 23 °C overnight. Reaction progress was monitored by TLC (5% methanol in dichlorome thane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (100 mL), washed with brine, dried over MgSC , and concentrated under reduced pressure. The concentrated crude was purified by flash column chromatography over silica gel (4% methanol/dichloromethane) to afford compound 22a (159 mg, 80%) as a white solid: ^XH NMR (400 MHz, methanol-d4) δ 5.44 (bs, 1H, H-l '), 5.13 (d, J = 2.7 Hz, 1H, H- l"), 4.92 (d, J = 1.8 Hz, H- l'"), 4.25 (m, 2H, H-2", H-4"), 4.09 (m, 1H, H-3"), 3.91 (m, 3H, H-5', H-3'", H- 5"'), 3.76 (m, 1H, H-4'"), 3.66 - 3.42 (m, 6H, H-4, H-5, H-6, H-2', H-3', H-2'"), 3.33-3.30 (m, 5H, H- l , H-3, H-4', H-6"'(2H)), 2.99 (dd, 1H, J = 13.8, 2.7 Hz, H-6'), 2.85 (m, 2H, H-5"), 2.71 (m, 1 H, H-6'), 2.69 - 2.57 (m, 4H, 2xSCH_?CH₉(CH₉)₃C¾), 2.05 - 1.94 (m, 1H, H-2eq), 1.67 - 1.53 (m, 4H, 2xSCH₂CH₂(CH₂)₃CH₃), 1.53 - 1.37 (m, 58H, 5xC0₂C(CH₃)₃, 2xSCH₂CH₂(CH₂)₃CH₃, H-2eq), 0.99 - 0.82 (m, 6H, 2x SCH₂CH₂(CH₂)₃CH₃). ¹³C NMR (125 MHz, methanol-d4) δ 157.4 , 157.0 , 156.8 , 156.7 , 156.3 , 109.9 (anomeric), 98.9 (anomeric) ,

97.5 (anomeric), 85.9 , 81.2 , 79.7 , 79.4 , 79.2 , 79.1 , 78.8 , 74.5 , 73.9 , 73.0 , 72.8 , 72.3 , 72.2 , 70.1 , 67.4 , 55.2 , 52.1 , 40.3 , 34.2 , 33.5 , 33.3 , 32.3 , 31.3 , 31.2 , 29.6 , 29.4 , 28.3 , 28.2 , 27.6 , 22.3 , 22.3 , 13.1 , 13.0 .LR-ESI m/z calc'd for C60H₁₁0N5O₂₂S₂ 1316.70, found 1316.58 [M+H]⁺. Compound 23a. To a solution of compound 21b (250 mg, 0.15 mmol) and CS₂CO₃ (198 mg, 0.6mmol) in dry DMF (4 mL) was added 1 -heptanethiol (0.33 mL, 2.12 mmol), and the reaction was stirred at 23 °C overnight. Reaction progress was monitored by TLC (5% methanol in dichloromethane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (100 mL), washed with brine, dried over MgS0₄, and concentrated under reduced pressure. The concentrated crude was purified by flash column chromatography over silica gel (4% methanol/dichloromethane) to afford compound 23a (155 mg, 76%) as a white solid: H NMR (500 MHz, methanol-d4) δ 5.47 (bs, 1H, Η- Γ), 5.15 (d, J = 3.6 Hz, 4H, H-l "), 4.93 (bs, 1H, Η-Γ"), 4.39 - 4.22 (m, 2H, H-4", H-2"), 4.10 (q, J = 5.2 Hz, 1H, H-3"), 4.02 - 3.87 (m, 3H, H-5', H-3'", H-5'"), 3.79 (bs, 1H, H-4'"), 3.66 - 3.42 (m, 6H, H-4, H-5 , H-6, H-2', H-3', H-2'"), 3.38 - 3.27 (m, 5H, H-l , H-3, H-4', H-6"'(2H)), 3.09 - 2.96 (m, 1H, H-6'), 2.98 - 2.84 (m, 2H, H-5"), 2.73 (m, 1H, H-6'), 2.70-2.62 (m, 4H, 2xSCH₂CH₂(CH₂)₄CH₃), 2.02 (dd, J = 13.5, 4.8 Hz, 1H, H-2eq), 1.66-1.59 (m, 4H, 2xSCH₂CH₂(CH₂)₄CH₃), 1.57 - 1.15 (m, 62H, 5xC0₂C(CH₃)₃, 2xSCH₂CH₂(CH₂)₄CH₃, H-2ax), 0.94- 0.91 (m, 6H, 2xSCH₂CH₂(CH₂)₅CH₃). ¹³C NMR (125 MHz, methanol-d4) δ 157.4 , 157.1 , 156.7 , 156.3 , 109.9 (anomeric) , 98.8 (anomeric) , 97.5 (anomeric) , 86.0 , 81.2 , 79.6 , 79.4 , 79.2 , 79.1 , 78.8 , 74.5 , 73.9 , 73.0 , 72.9 , 72.3 , 70.2 , 67.4 , 55.3 , 52.1 , 50.9 , 50.1 , 48.4 , 40.4 , 34.2 , 33.6 , 33.3 , 32.3 , 31.6 , 31.6 , 29.6 , 29.5 , 28.8 , 28.7 , 28.6 , 28.5 , 27.6 , 27.4 , 27.4 , 22.3 , 22.3 , 13.1 .LR-ESI m/z calc'd for C₆₂Hii₄N₅0₂₂S₂ 1344.73, found 1344.67 [M+H]⁺.

Compound 24a. To a solution of compound 21b (250 mg, 0.15 mmol) and Cs₂C0₃ (198 mg, 0.6mmol) in dry DMF (4 mL) was added 1-octanethiol (0.36 mL, 2.12 mmol), and the mixture was stirred at 23 °C overnight. Reaction progress was monitored by TLC (5% methanol in dichloromethane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (100 mL), washed with brine, dried over MgSC , and concentrated under reduced pressure. The concentrated crude was purified by flash column chromatography over silica gel (4% methanol/dichloromethane) to afford compound 24a (162 mg, 78%) as a white solid: ^lH NMR (500 MHz, methanol-d4) δ 5.48 (bs, 1H, H-l'), 5.15 (d, J = 2.8 Hz, 1 H, H- l"), 4.94 (bs, 1H, H-l '"), 4.46 - 4.22 (m, 2H, H-2", H-4"), 4.11 (q, J = 5.6 Hz, 1H, H-3"), 3.93 (m, 3H, H-5', H-3'", H-5'"), 3.79 (m, 1H, H-4'"), 3.70 - 3.57 (m, 6H, H-4, H-5, H-6, H-2', H-3', H-2'"), 3.39 - 3.25 (m, 5H, H-l , H-3, H-4', H-6"'(2H)), 3.01 (dd, 7 = 13.9, 2.7 Hz, 1H, H-6'), 2.87 (m, 2H, H- 5"), 2.72 (dd, J = 13.8, 7.3 Hz, 1H, H-6'), 2.70 - 2.61 (m, 4H, 2xSCH₂CH₂(CH₂)₅CH₃), 2.02 (m, 1H, H-2eq), 1.62 (m, 4H, 2xSCH₂CH₂(CH₂)₅CH₃), 1.53 - 1.41 (m, 66H, 5xC0₂C(CH₃)₃, 2xSCH₂CH₂(CH₂)₅CH₃, H-2ax), 0.94 - 0.91 (m, 6H, 2xSCH₂CH₂(CH₂)_sCH₃). ¹³C NMR (125 MHz, methanol-d4) δ 157.4 , 157.0 , 156.8 , 156.7 , 156.3 , 109.9 (anomeric), 98.9 (anomeric) , 97.5 (anomeric), 86.0 , 81.2 , 79.7 , 79.4 , 79.2 , 79.1 , 78.8 , 74.5 , 73.9 , 73.0 , 72.9 , 72.3 , 70.1 , 67.4 , 55.2, 52.1 , 50.9 , 50.1 , 48.4 , 40.3 , 34.4 , 34.2 , 33.6 , 33.3 , 32.3 , 31.7 , 31.6 , 29.6 , 29.5 , 29.1 , 29.1 , 29.0 , 28.7 , 28.6 , 27.6 , 27.4 , 27.3 , 22.3 , 13.11 .LR- ESI mJz calc'd for C₆4Hii₈N₅0₂₂S₂ 1372.76, found 1372.54 [M+H]⁺.

Compound 22. Compound 22a (20 mg, 0.014 mmol) was treated with 95% TFA (0.7 mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, and the residue was dissolved in a minimal volume of H₂0 and freeze-dried to afford compound 22 (21 mg, quantitative yield) as a white foam: H NMR (400 MHz, methanol-d4) δ 5.73 (d, J = 3.9 Hz, 1H, Η-Γ), 5.36 (d, J = 3.7 Hz, 1H, H-l"), 5.34 (d, J = 1.7 Hz, 1H, Η-Γ"), 4.45 (t, J = 4.7 Hz, 1H, H-3"), 4.40 (m, 1H, H-2"), 4.33 (dt, J = 8.4, 4.2 Hz, 1H, H-4"), 4.29 (ddd, J = 7.2, 3.9, 1.5 Hz, 1H, H-5'"), 4.15 (t, J = 3.2 Hz, 1H, H-3'"), 3.99 (t, J = 9.6 Hz, 1H, H-4), 3.96 - 3.85 (m, 2H, H-3', H-5'), 3.81 (t, J = 9.0 Hz, 1H, H-5), 3.69 (m, 1H, H-4'"), 3.63 (m, 1H, H-6), 3.57 - 3.33 (m, 5H, H-3, H-2', H-4', H-2'", H-6'"), 3.29-3.22 (m, 2H, H-l , H-6'"), 3.10-3.04 (m, 2H, H- 5", H-6'), 2.83 (dd, J = 13.0, 8.0 Hz, 1H, H-5"), 2.75 (dd, J = 14.3, 6.7 Hz, 1H, H-6'), 2.66 - 2.56 (m, 4H, 2xSCH₂CH₂(CH₂)₃CH₃), 2.48 (dt, 7 = 12.4, 4.2 Hz, 1H, H-2eq), 1.92 (q, J = 12.6 Hz, 1H, H-2ax), 1.67 - 1.54 (m, 4H, 2xSCH₂CH₂(CH₂)₃CH₃), 1.50-1.24 (m, 12H, 2xSCH₂CH₂(CH₂)₃CH₃), 0.90 (m, 6H, 2xSCH₂CH₂(CH₂)₃CH₃). ¹³C NMR (100 MHz, methanol-d4) δ 161.7 (q, J = 34.6 Hz, CF₃C0₂H), 116.7 (q, J = 292.8 Hz, CF₃C0₂H), 110.7 (anomeric) , 96.2 (anomeric) , 95.5 (anomeric) , 85.2 , 80.7 , 78.4 , 78.0 , 74.3 , 73.9 , 72.4 , 71.82 , 70.6 , 68.8 , 67.7, 54.3 , 51.5 , 49.6 , 49.2 , 40.2 , 35.1 , 32.8 , 32.6 , 32.2 , 31.2 , 31.1 , 29.4 , 29.3 , 28.3 , 28.1 , 22.2 , 22.2 , 13.0 , 12.9. HRESI-MS m/z calc'd for C₃₅H7oN₅Oi₂S₂ 816.4462 found 816.4460 [M+H]⁺.

Compound 23. Compound 23a (15 mg, 0.011 mmol) was treated with 95% TFA (0.7 mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, and the residue was dissolved in a minimal volume of H₂0 and freeze-dried to afford compound 23 (15.6 mg, quantitative yield) as a white foam: H NMR (400 MHz, methanol-d4) δ 5.70 (d, J = 3.9 Hz, 1H, H-l'), 5.35 (d, J = 3.8 Hz, 1H, H-l"), 5.34 (d, J = 2.0 Hz, 1H, H-l'"), 4.45 (t, J = 4.6 Hz, 1H, H-3"), 4.41 (t, J = 4.2 Hz, 1H, H-2"), 4.33 (m, 1H, H-4"), 4.29 (ddd, J = 7.1 , 3.8, 1.5 Hz, 1H, Η-5'"), 4.14 (t, 7 = 3.2 Hz, 1H, H-3'"), 3.98 - 3.84 (m, 3H, H-4, H-3', H-5'), 3.80 (t, 7 = 9.0 Hz, 1H, H-5), 3.70 (m, 1H, H-4'"), 3.61 (t, 7 = 9.7 Hz, 1H, H-6), 3.50 - 3.34 (m, 5H, H-3, H-2', H-4', H-2", H-6'"), 3.29 - 3.19 (m, 2H, H-6'", H-l), 3.07 (m, 2H, H-5", H-6'), 2.83 (dd, 7 = 13.0, 8.1 Hz, 1H, H-5"), 2.75 (dd, 7 = 14.2, 6.9 Hz, 1H, H-6'), 2.69 - 2.55 (m, 4H, 2xSCH₂CH₂(CH₂)₄CH₃), 2.44 (dt, 7 = 12.5, 4.2 Hz, 1H, H-2eq), 1.87 (q, 7 = 12.6 Hz, 1H, H- 2ax), 1.62 (m, 4H, 2xSC¾C¾(C¾)4C¾), 1.50 - 1.19 (m, 16H, 2xSCH₂CH₂(CH₂)₄CH₃), 0.98 - 0.78 (m, 6H, 2xSCH₂CH₂(CH₂)₃CH₃). ¹³C NMR (125 MHz, methanol-d4) δ 161.7 (q, 7 = 34.6 Hz, CF₃CO₂H), 116.7 (q, 7 = 292.7 Hz, CF₃C0₂H), 110.7 (anomeric) , 96.2 (anomeric), 95.5 (anomeric) , 85.4 , 80.7 , 78.4 , 74.3 , 73.9 , 72.5 , 71.8 , 70.7 , 68.8 , 67.8 , 67.7 , 54.4 , 51.5 , 49.7 , 49.2 , 40.2 , 35.1 , 32.8, 32.7, 32.2, 31.58 , 31.5 , 29.5 , 29.4 , 28.7 , 28.6 , 28.4 , 22.3 , 22.2 , 13.0. HRESI-MS m/z calc'd for C₃₇H₇₄N5O₁₂S₂ 844.4775 found 844.4770 [M+H]⁺.

Compound 24. Compound 24a (30 mg, 0.021 mmol) was treated with 95% TFA (0.7 mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, and the residue was dissolved in a minimal volume of H₂O and freeze-dried to afford compound 24 (31.5 mg, quantitative yield) as a white foam: ^lR NMR (400 MHz, methanol-d4) δ 5.73 (d, 7 = 3.9 Hz, 1H, H-l'), 5.36 (d, 7 = 3.7 Hz, 1H, H-l"), 5.34 (d, 7 = 1.7 Hz, 1H, H-l'"), 4.45 (t, 7 = 4.7 Hz, 1H, H-3"), 4.40 (m, 1H, H-2"), 4.33 (dt, 7 = 8.4, 4.2 Hz, 1H, H-4"), 4.29 (ddd, 7 = 7.2, 3.9, 1.5 Hz, 1H, H-5'"), 4.15 (t, 7 = 3.2 Hz, 1H, H-3'"), 3.99 (t, 7 = 9.5 Hz, 1H, H-4), 3.88 (m, 2H, H-3', H-5'), 3.81 (t, 7 = 9.0 Hz, 1H, H-5), 3.69 (m, 1H, H-4'"), 3.63 (dd, 7 = 10.4, 9.0 Hz, 1H, H-6), 3.56 - 3.33 (m, 5H, H-3, H-2', H-4', H-2", H-6'"), 3.30 - 3.19 (m, 2H, H-l , H-6'"), 3.07 (m, 2H, H-6', H-5"), 2.83 (dd, 7 = 13.1, 8.0 Hz, 1H, H-5"), 2.76 (dd, 7 = 14.3, 6.7 Hz, 1H, H-6'), 2.67 - 2.56 (m, 4H, 2xSCH₂CH₂(CH₂)5CH₃), 2.47 (dt, 7 = 12.5, 4.3 Hz, 1H, H-2eq), 1.92 (q, 7 = 12.5 Hz, 1H, H-2ax), 1.72 - 1.51 (m, 4H, 2xSCH₂CH₂(CH₂)5CH₃), 1.48 - 1.17 (m, 20H, 2xSCH₂CH₂(CH₂)₅CH₃), 0.97 - 0.67 (m, 6H, 2xSCH₂CH₂(CH₂)₅CH₃). ¹³C NMR (100 MHz, methanol-d4) δ 161.7 (q, 7 = 34.7 Hz, CF₃C0₂H), 116.7 (q, 7 = 292.7 Hz, CF₃C0₂H ), 110.7 (anomeric) , 96.1 (anomeric) , 95.5 (anomeric) , 85.3 , 80.7 , 78.4 , 78.0 , 74.4 , 73.9 , 72.4 , 71.7 , 70.6 , 68.8 , 67.8 , 54.3 , 51.5 , 49.6 , 49.2 , 40.2 , 35.7 , 32.8 , 32.6 , 32.2 , 31.6 , 31.5 , 29.5 , 29.4 , 28.7 , 28.4 , 28.1 , 22.3 , 13.0. HRESI-MS m/z calc'd for C₃₉H₇₈N₅Oi₂S₂ 872.5088 found 872.5087 [M+H]⁺. Compound 25a. A solution of 21a (0.5 g, 0.44 mmol) in pyridine (10 mL) was treated with 2,4,6-triisopropylbenzenesulfonyl chloride (2.71 g, 8.96 mmol, 20.0 equiv). The reaction mixture was stirred at 23 °C for 12 h. Reaction progress was monitored by TLC (3% methanol in dichloro methane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (lOOmL), washed with brine, dried over MgSC , and concentrated under reduced pressure. The concentrated crude was purified by flash column chromatography over silica gel (3% methanol in dichloromethane) to afford 21b (0.31 , 43 %) and the desired product 25a as a white solid (0.2 g, 33%): H NMR (400 MHz, methanol-d4) δ 7.29 (s, 2H, S0₃C₆H2(CH)3(CH3)6), 5.43 (bs, 1H, H- 1 '), 5.12 (d, 1H, J = 2.2 Hz, H- l "), 4.89 (bs, 1 H, Η- Γ"), 4.35 (m, 3H, H-6'(2H), H-5'), 4.18 (m, 2H, H-2", H-3"), 4.01 - 3.86 (m, 4H, H-4", H-4', H-3'", H-5'"), 3.83 - 3.73 (m, 2H, H-2'", H-5"), 3.64 (dd, J = 12.2, 6.7 Hz, 1H, H-5"), 3.61 - 3.21 (m, 12H, H-l , H-3, H-4, H-5, H-6, H-2', H-3', H-4'", H-6"'(2H), SOsCeHz CHMCHs^ (2H)), 2.95 (hep, J = 6.9 Hz, 1 H, SChG,H9(CH CH _ft), 1.93 (dt, J = 12.1, 4.3 Hz, 1H, H-2eq), 1.51-1.38 (m, 46H, 5xC0₂C(CH₃)₃, H-2ax), 1.32-1.22 (m, 18H, SOsCeH^CHMCH^e). ¹³C NMR (100 MHz, methanol-d4) δ 159.7 , 159.4 , 159.0 , 159.3, 158.7 , 156.2 , 153.0 , 131.6 , 125.8 , 111.9 (anomeric) , 100.9 (anomeric) , 100.0 (anomeric) , 88.9 , 84.1 , 81.6 , 81.5 , 81.3 , 81.0 , 79.7 , 79.3 , 76.8 , 76.4 , 75.22 , 73.9 , 72.4 , 72.3 , 71.9 , 70.3 , 69.6 , 65.1 , 57.4 , 54.4 , 53.3 , 52.21 , 42.5 , 36.4 , 31.7 , 29.8 , 29.7 , 29.6 , 26.0 , 24.8. LR-ESI m/z calc'd for GaHioeNsCfeeS 1380.69, found 1380.58 [M-H]^".

Compound 25b. To a solution of compound 25a (80 mg, 0.057 mmol) and CS₂CO₃ (mg, 0.115 mmol) in dry DMF (1 mL) was added 1 - hexadecanethiol (0.12 mL, 0.4 mmol), and the mixture was stirred at 23 °C overnight. Reaction progress was monitored by TLC (5% methanol in dichloromethane). Upon completion, the reaction mixture was concentrated, diluted with ethyl acetate (100 mL), washed with brine, dried over MgS0₄, and concentrated under reduced pressure. The concentrated crude was purified by flash column chromatography over silica gel (4% methanol / dichloromethane) to afford compound 25b (60 mg, 77%) as a white solid: ^XH NMR (400 MHz, methanol-d4) δ 5.32 (bs, 1H, H- l '), 5.14 (d, J = 2.6 Hz, 1H, H-l "), 4.89 (s, 1H, H-l '"), 4.22 (m, 1H, H-2"), 4.17 (m,lH, H-3"), 3.97 - 3.79 (m, 5H, H-5', H- 4", H-5", H-3'", H-5'"), 3.76 (m, 1 H, H-4'"), 3.68 (dd, J = 12.2, 6.4 Hz, 1H, H-5"), 3.63 - 3.19 (m, 11H, H-l , H-3, H-4, H-5, H-6, H-2', H-3', H-4', H-2'", H-6"'(2H)), 2.99 (dd, J = 13.8, 2.7 Hz, 1H, H-6'), 2.72 (dd, J = 13.8, 7.0 Hz, 1H, H-6'), 2.64 (t, J = 7.2 Hz, 2H, SCH₂CH₂(CH₂)i₂CH₃), 1.98 (dt, J = 13.0, 4.0 Hz, 1 H, H-2eq), 1.59 (m, 2H, SCH₂CH₂(CH₂)i₂CH₃), 1.53 - 1.39 (m, 46H, 5xC0₂C(CH₃)₃, H-2ax), 1.30- 1.29 (m, 24H, SCH₂CH₂(CH₂)i₂CH₃), 0.97 - 0.70 (m, 3H, SCH₂CH₂(CH₂)i₂CH₃). ¹³C NMR (100 MHz, methanol-d4) δ 157.5 , 157.1 , 156.8 , 156.3 , 109.6 (anomeric) , 98.9 (anomeric), 97.8 (anomeric), 86.4 , 82.0 , 79.3 , 79.2 , 79.0 , 76.9 , 74.6 , 74.1 , 73.0 , 72.6 , 72.3 , 71.6 , 70.2 , 67.5 , 62.4 , 55.5 , 52.1 , 40.4 , 34.2 , 33.7 , 33.2 , 31.6 , 29.6 , 29.3 , 29.0 , 29.0 , 28.5 , 27.5 , 27.4 , 27.3 , 22.3 , 13.0 . LR-ESI mJz calc'd for C₆4Hii₈N₅0₂3S 1356.79, found 1356.34 [M+H]⁺.

Compound 25. Compound 25b (20 mg, 0.014 mmol) was treated with 95% TFA (0.7 mL) at ambient temperature for 3 min. The TFA was removed under reduced pressure, and the residue was dissolved in a minimal volume of H₂0 and freeze-dried to afford compound 25 (21 mg, quantitative yield) as a white foam: H NMR (400 MHz, methanol-d4) δ 5.57 (d, J = 3.8 Hz, 1H, Η-Γ), 5.35 (d, J = 3.1 Hz, 1H, H-l "), 5.30 (d, J = 1.7 Hz, 1H, Η- Γ"), 4.52 (t, J = 5.5 Hz, 1H, H-3"), 4.36 - 4.24 (m, 2H, H-5'", H-2"), 4.17 (m, 1 H, H-4"), 4.14 (m, 1H, H-3'"), 3.95 (t, J = 9.8 Hz, 1 H, H-4), 3.92 - 3.73 (m, 5H, H-5, H-3', H-5', H-5"(2H)), 3.68 (m, 1H, H-4'"), 3.61 (m, 1H, H-6), 3.54 (td, J = 12.6, 10.2, 4.3 Hz, 1H, H-3), 3.44 (m, 1H, H-2'"), 3.42 - 3.21 (m, 5H, H- l , H-2', H-4', H-6"'(2H)), 3.09 (dd, J = 14.1 , 2.2 Hz, 1H, H-6'), 2.68 (dd, J = 14.3, 8.0 Hz, 1H, H-6'), 2.59 (m, 2H, SCH₂CH₂(CH₂)i₂CH₃), 2.48 (dt, J = 12.6, 4.5 Hz, 1H, H-2eq), 1.90 (q, J = 12.6 Hz, 1H, H-2ax), 1.71 - 1.52 (m, 2H, SCH₂CH₂(CH₂)i₂CH₃), 1.41 -1.25 (m, 24H, SCH₂CH₂(CH₂)i₂CH₃), 0.90-0.85 (m, 3H, SCH₂CH₂(CH₂)₁₂CH₃). ¹³C NMR (100 MHz, methanol-d4) δ 161.7 (q, J = 34.8 Hz, CF₃C0₂H ), 116.7 (q, J = 292.4, CF₃C0₂H), 110.3 (anomeric) , 97.3 (anomeric) , 95.6 (anomeric) , 84.2 , 82.0 , 79.6 , 75.5 , 74.6 , 73.9 , 72.5 , 72.2 , 70.6 , 68.9 , 67.8 , 67.7 , 59.6 , 54.2 , 51.5 , 49.7 , 49.4 , 40.2 , 32.7 , 32.6 , 31.6 , 29.4 , 29.3, 29.0 , 28.9 , 22.3 , 13.01. HRESI-MS m/z calc'd for C₃₉H78N₅Oi₃S 856.5317 found 856.5318 [M+H]⁺.

C. Biological assays

1. Minimal inhibitory concentration protocol

Starter cultures were incubated for 24 hours (37 °C, 5% C0₂, aerobic conditions) and then diluted in fresh medium to obtain an optical density of 0.004 (ΟΌ οο)- All strains were tested using the double-dilution method starting at 128 /ig/mL in 96-well plates (Sarstedt). After 24 hours of incubation, MTT (50 μΐ_^ of a 1 mg/niL solution in H₂0) was added to each well followed by additional incubation at 37°C for 2 hours. MIC values ^g/mL) were determined as the lowest concentration at which no bacterial growth was observed. Results were obtained from two independent experiments, and each experiment was done in triplicate.

2. Red blood cell hemolysis protocol

A sample of rat RBCs (2% w/w) were incubated with each of the tested compounds for 1 hour at 37 °C, 5 % CO₂ using the double dilution method starting at concentration of 256 μg/ h. Negative control was PBS and positive control was 1 % w/v solution of Triton X100 (100% hemolysis). Following centrifugation (2,000rpm, 10 min, ambient temperature), the supernatant was removed and its absorbance measured at 550 nm using a microplate reader (Genios, TECAN). The results are expressed as percentage of hemoglobin released relative to the positive control (Triton X100). Experiments were performed in triplicate, and the results are an average of experiments in blood samples taken from at least two rats.

3. Scanning electron microscopy protocol

Cultures of S. epidermidis ATCC12228 were incubated for 4 hours at 37 °C under aerobic conditions to obtain an optical density of 0.2 (OD₆₀₀) and then harvested by centrifugation at 4,000 rpm for 10 min at 4 °C. The bacterial pellet was washed twice with PBS (pH 7.4) and resuspended in PBS (pH 7.4) to obtain an optical density of 1.0 (ΟΌ οο)- The bacterial suspension was diluted two fold after treatment with the tested compound at a concentration of 1 μg/mL at 37 °C for 1 hour. The cells were then spun down at 6,000 rpm for 4 min at 4 °C, washed with PBS (pH 7.4) three times, and fixed with 2.5% glutar aldehyde/PBS buffer overnight at 4 °C. The cells were then washed three times in 0.1 M PB (pH 7.4), dehydrated in series of graded ethanol solutions (30% to 100%), and dried in vacuum desiccation. Finally, the samples were coated with palladium-gold and viewed via scanning electron microscopy (Quanta 200FEG ESEM).

Abbreviations

BHI, brain heart infusion; BOC, teri-butoxycarbonyl; 1 -D-TOCSY, Total Correlation Spectroscopy; DIEA, N,N-Diisopropylethylamine; DMF, dimethylformamide; EtOAc, ethyl acetate; HBTU, 0-(Benzotriazol- l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HR-ESI- High resolution electron spray ionization; LR-ESI, Low resolution electron spray ionization MIC, minimum inhibitory concentration; MBIC, minimal biofilm inhibitory concentration; MTT, thiazolyl blue tetrazolium bromide; PBS, phosphate buffered saline; RBC, red blood cells; rt, room temperature; SEM, Scanning electron microscopic; TIBSCl, 2,4,6-triisopropylbenzenesulfonyl chloride; TFA, trifluoro acetic acid; TLC, thin layer chromatography; TSB, trypticase soy broth.

The work leading to this invention has received funding from the European Community's Seventh Framework Programme (FP7/2007-2012) under grant agreement No. 246673.

The contents of each of the references cited are incorporated by reference herein in their entirety as if fully set forth herein.

It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described herein above. Rather the scope of the invention is defined by the claims that follow.

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Claims

1. A tobramycin or kanamycin A derivative represented by the structure of formula I, or a paromomycin derivative represented by the structure of formula I-A:

wherein, in formula I:

A is OH or -L²-R², wherein:

(i) when A is OH, L¹ is selected from the group consisting of -OSO₂-, triazolyl, and -NH-C(=0)-; and R¹ is a C₁₂-C₁6 linear alkyl;

(ii) when A is -L²-R², each of L¹ and L² is selected from the group consisting of -S-, -SO₂-, -OSO₂-, triazolyl and -NH-C(=0)-; and each of R¹ and R² is a linear C₄-C8 alkyl; and

R³ and R⁴ are each OH; or R³ is NH₂ and R⁴ is H;

wherein, in formula I-A:

1 2

R and R" are each independently a linear C₄ to Cs alkyl; including salts, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

2. A tobramycin or kanamycin A derivative represented by the structure of formula I according to claim 1.

3. The compound according to claim 2, wherein R³ is N¾ and R⁴ is H, and the compound is a tobramycin derivative, represented by the structure of formula II:

II

4. The compound according to claim 2, wherein R³ and R⁴ are each OH, and the compound is a kanamycin A derivative represented by the structure of formula

III:

The compound according to any of claims 2 to 4, wherein A is OH in the equatorial position.

The compound according to claim 5, wherein L¹ is a sulfonyl ester of the formula -OSO₂-, and the compound is represented by the structure of formula IV:

IV The compound according to claim 5 , wherein L¹ is a triazolyl, and the compound is represented by the structure of formula V:

V

8. The compound according to claim 5, wherein L¹ is an amide of the formula -NH- C(=0)-, and the compound is represented by the structure of formula VI:

VI

9. The compound according to any of claims 2 to 8, wherein R¹ is selected from the group consisting of -(CH₂)nCH₃, -(CH₂)i₃CH₃ and -(CH₂)i₅CH₃.

10. The compound according to claim 5 , which is selected from the group consisting of compound (4a), (4b), (4c), (5a), (5b), (5c), (6a), (6b), (6c), (14a), (14b), (14c), (15a), (15b), (15c), (16a), (16b) and (16c).

11. The compound according to any of claims 2 to 4, wherein A is -L²-R².

12. The compound according to claim 11 , wherein A is in the axial position.

13. The compound according to claim 11 , wherein L¹ and L² are each a thioether of the formula -S-, and the compound is represented by the structure of formula VII:

VII

14. The compound according to claim 11 , wherein L¹ and L² are each a sulfonyl of the formula -SO₂-, and the compound is represented by the structure of formula VIII:

VIII

15. The compound according to claim 11 , wherein L¹ and L² are each a sulfonyl ester of the formula -OSO₂-, and the compound is represented by the structure of formula IX:

IX

16. The compound according to claim 11 , wherein L¹ and L² are each a triazolyl, and the compound is represented by the structure of formula X:

17. The compound according to claim 11 , wherein L¹ and L² are each an amide, and the compound is represented by the structure of formula XI:

XI

18. The compound according to any of claims 11 to 17, wherein R¹ and R² are each a C6-Cs linear alkyl.

19. The compound according to any of claims 11 to 13, which is represented by the structure of formula 17:

17

20. A paromomycin derivative represented by the structure of formula I-A according to claim 1.

21. The compound according to claim 20, wherein each of R¹ and R² is independently selected from the group consisting of -(0¾)5θ¾, -(0¾)6θ¾ and -(CH₂)₇CH₃.

22. The compound according to claim 20, wherein L¹ and L² are each a thioether of the formula -S-, and the compound is represented by the structure of formula II- A:

23. The compound according to claim 20, wherein L¹ and L² are each a sulfonyl of the formula -SO₂-, and the compound is represented by the structure of formula III- A:

24. The compound according to claim 20, wherein L¹ and L² are each a sulfonyl ester of the formula -OSO₂-, and the compound is represented by the structure of formula IV-A:

25. The compound according to claim 20, wherein L¹ and L² are each a triazolyl, and the compound is represented by the structure of formula V-A:

26. The compound according to claim 20, wherein L¹ and L² are each an amide of the formula -NH-C(=0)-, and the compound is represented by the structure of formula VI-A:

27. The compound according to claim 20, which is represented by the structure of formula 22, 23 or 24:

28. The compound according to claim 20, which is selected from the group consisting of formula (26a), (26b), (26c), (27a), (27b), (27c) (28a), (28b), (28c), (29a), (29b) and (29c).

29. A dimeric compound represented by the structure of formula XII:

wherein

R¹ is a C12-C16 linear alkyl;

L¹ is selected from the group consisting of -S-, -SO₂-, -OSO₂-, triazolyl and -NH- C(=0)-;

R and R are each OH; or R is NH₂ and R is H; and

B is selected from an unsubstituted or substituted alkyl, aryl, aryloxy, alkyloxy and amide;

30. The compound according to claim 29, which is represented by the structure of formula 10:

31. An anti-bacterial pharmaceutical composition comprising a compound according to any of claims 1 to 30, and a pharmaceutically acceptable carrier or excipient.

32. The pharmaceutical composition of claim 31, wherein the composition is in a form suitable for oral administration, intravenous administration by injection, topical administration, administration by inhalation, or administration via a suppository.

33. A method of combating bacteria, or treating a bacterial infection, comprising the step of administering to a subject in need thereof a compound according to any one of claims 1-30, or a pharmaceutical composition according to claim 31 or 32.

34. Use of a compound according to any one of claims 1 -30 or a pharmaceutical composition according to claim 31 or 32, for the manufacture of a medicament for combating bacteria or treating a bacterial infection.

35. A compound according to any one of claims 1-30 or a pharmaceutical composition according to claim 3 lor 32, for use in combating bacteria or treating a bacterial infection.

36. A method for combating bacteria or treating a bacterial infection, comprising the step of contacting the bacteria with a compound according to any one of claims 1-30, or a pharmaceutical composition according to claim 31 or 32.

37. The method, use or compound according to any one of claims 33 to 36, wherein the bacteria is a Gram-positive bacteria.

38. The method, use or compound according to any one of claims 33 to 36, wherein the bacteria is a Gram-negative bacteria.

39. The method, use or compound according to any one of claims 33 to 36, wherein the bacteria causes skin infections or soft tissue infections.

40. A method of inhibiting biofilm growth, comprising the step of contacting the biofilm or a surface comprising the biofilm with a compound according to any one of claims 1-30, or a pharmaceutical composition according to claim 31 or 32.