WO2011056545A2

WO2011056545A2 - Antimicrobial ionic liquids

Info

Publication number: WO2011056545A2
Application number: PCT/US2010/054048
Authority: WO
Inventors: Mark W. Grinstaff; Michel Wathier; George A. O'toole
Original assignee: Boston University
Current assignee: Boston University
Priority date: 2009-10-26
Filing date: 2010-10-26
Publication date: 2011-05-12
Anticipated expiration: 2012-04-26
Also published as: WO2011056545A3

Abstract

Certain aspects of the present invention relate to compositions, formulations, and use of ionic liquids as antimicrobial agent (e.g. as antibacterial and/or antifungal agents). In certain embodiments, the ionic liquids can be pared with one or more singly charge species or with one or more multi-charged species.

Description

Atty. Docket No. BUX-008.25

Antimicrobial Ionic Liquids

RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent Application serial number 61/254,957, filed October 26, 2009; which is hereby

incorporated by reference in its entirety.

BACKGROUND

One of the most disturbing trends in recent years is the growth of resistant strains of bacteria with the simultaneous dearth of new antimicrobial agents. There have been relatively few new antimicrobial agents in recent years, and those that have been approved, including Cubicin (daptomycin) and Zyvox (linezolid), specifically target Gram-positive bacteria. Therefore, there is a need for new broad-spectrum antimicrobial agents and for new classes of antibiotics that are effective versus current resistant strains.

Resistant organisms such as S. aureus and Enterococcus continue to be a threat in the context of the clinic. S. aureus is a well-known bacterial pathogen with a troubling propensity to develop resistance to a broad range of therapeutically important antibiotics. For example, up to 50% of S. aureus strains are resistant to methicillin (so-called MRS A), and recent studies indicate the appearance of S. aureus strains resistant to vancomycin, typically considered a treatment of last resort. Similarly, vancomycin resistant

Enterococcus (VRE) strains have also been reported, and such VRE strains can account for up to 30% of enteroccal infections. Furthermore, even for the recently introduced Cubicin (daptomycin) and Zyvox (linezolid), reports of resistance to these compounds appeared within 6 months of their introduction to the clinic. In addition to antibiotic resistant strains of S. aureus, there are increasing reports of S. aureus infections caused by methicillin resistant strains contracted by otherwise healthy individuals in a community setting, so called community acquired (CA)-MRSA.

Gram-negative pathogens are also on the rise. Multidrug-resistant P. aeruginosa and Acinetobacter strains are a growing concern in hospital settings, as are Escherichia coli and Klebsiella, which produce extended-spectrum β-lactamases (ESBL). These ESBL are isolated from a number of diseased sites, including urinary tract and blood infections, thus they present a danger to patients with a wide number of infections. As is the case with

B3806550.2 Atty. Docket No. BUX-008.25

MRSA, ESBL-producing organisms have been found in both hospital and community settings. Thus, while resistant Gram-positive organisms have received a great deal of attention, it is also clear that antibiotic resistance among Gram-negative organisms is also an emerging threat. Thus the discovery of new class of broad-spectrum antimicrobial agents would provide a significant advance to treating these resistant Gram-negative infections in the clinic.

Fungi are also important pathogens. Invasive fungal infections pose a serious threat to individuals with underlying medical issues such as HIV. As is the case with bacteria, fungal resistance to frontline antifungal compounds, such as azoles, is also a significant problem. Furthermore, some current antifungals have limitations in regards to their systemic use or their activity versus pathogens such as Aspergillus. Thus, the need to develop new antifungal compounds is also urgent. The increasing concern about resistance is reflected in such documents as the Public Health Action Plan to Combat Antimicrobial Resistance issued by the CDC. Despite the pressing need for new antimicrobial compounds, there are few new broad spectrum and Gram-negative-targeted compounds in the pipeline.

SUMMARY

Certain aspects of the present invention relate to compositions, formulations, and use of ionic liquids as antimicrobial agent (e.g. as antibacterial and/or antifungal agents). In certain embodiments, the ionic liquids can be paired with one or more singly charge species or with one or more multi-charged species.

In certain embodiments, the method involves the use of one or more ionic liquids as an antimicrobial agent. In certain embodiments, the one or more ionic liquids may be used in combination with one or more non-ionic liquid antimicrobial agents. In certain embodiments, the ionic liquids can be delivery to a patient via oral, iv, ip, or topical treatment. Another aspect of the invention relates to the use of the ionic liquids to coat the surface of a medical implant or device and thus prevent a microbial infection and/or biofilm formation.

Additional features and advantages of the present invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may

B3806550.2 - 2 - Atty. Docket No. BUX-008.25 be learned by practice of the present invention. The objectives and other advantages of the present invention will be realized and obtained by means of the elements and combinations particularly pointed out in the written description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a table of ionic liquids.

Figure 2 depicts a scheme showing one approach to the synthesis of an ionic liquid.

Figure 3 depicts a scheme showing one approach to the synthesis of an ionic liquid.

Figure 4 depicts additional [A] multi-cationic and [B] multi-anionic architectures of the invention.

Figure 5 depicts selected approaches to forming cationic and anionic components of the invention.

Figures 6A-6C depicts cytotoxicity test results of Dex C-10- and PBS-treated corneas in mouse keratitis model.

Figures 7A-7C shows the efficacy of Dex C-10 against P. aeruginosa in mouse keratitis mo del .

Figure 8 shows the efficacy of Dex C-10 against S. Aureus in an interperitoneal mouse model.

Figure 9 shows the efficacy of Dex C-10 against P. aeruginosa in an interperitoneal mouse model.

DETAILED DESCRIPTION

The ionic liquids disclosed herein can find various applications in the

biotechnology, pharmaceutical and medical fields. For example, the ionic liquids of the present invention can be used in the treatment of microbial infections. Accordingly, one aspect of the present invention relates to methods which generally include administration of an effective amount of an ionic liquid, or a pharmaceutical composition thereof, to an individual in need thereof. In another embodiment, the ionic liquids can also be used with medical devices to prevent implant infections. They can also be used in non-medical applications for example in marine applications to prevent biofouling of ships, boats, or

B3806550.2 Atty. Docket No. BUX-008.25 other structures. In certain embodiments, the ionic liquids are multicationic or

multianionic.

Ionic Liquids

Ionic liquids are "salt-like" materials that are liquid at relatively low temperatures, e.g., less than 400 °C. Today most scientists consider an ionic liquid a salt that is a liquid below 100 °C. Many of the common ionic liquids have melting points at room temperature or below. The first ionic liquids prepared were corrosive materials that had limited utility. In the early 1990s, Wilkes reported a less corrosive air-stable ionic material; this result has provided the impetus for much of the research and development focused on ionic liquids.

Ionic liquids are typically composed of a mono-cationic organic compound, such as a compound based on the structure of a imidazolium, pyridinium, pyrrolidinium, phosphonium, ammonium or sulfonium, and an inorganic or organic anion, such as a alkyl sulfate, tosylate, methansulfonate, hexafluorophosphate, tetrafluoroborate, halide, or carboxylic acid. For example, the prototypical ionic liquid of l-ethyl-3-methylimidazolium ethyl sulfate has a melting point of less than -20 °C; whereas, sodium chloride has a melting point of 801 °C. The strong ionic interaction between these mono-cations and mono-anions results in low vapor pressure, non-flammable materials with high thermal, mechanical, and electrochemical stability. More recently, a dicationic organic compound with two mono- anions has been reported.

Ionic liquids have found uses in a wide range of applications including, but not limited to, lubricants, MADLI-TOF matrices, protein crystallization matrices, solvents for heterogeneous catalysis, solvents for homogeneous catalysis, solvent for organic synthesis, solvents for desulfurization, liquid crystals, thermal fluids, fuel cells, sensors, metal finishers, materials for gas separations, distillation fluids, extraction mediums, and membrane technology.

The phosphonium based ionic liquids such as tetradecyl(tributyl)phosphonium chloride, see Figure 3, are of wide-spread interest for applications given their favorable material properties such as low vapor pressure and high thermal, mechanical, and electrochemical stability. These ionic liquids are prepared by nucleophilic addition of tertiary phosphines to haloalkanes (Figure 3, eqn. 1) and as such a number of mono and di- substituted alkyl compounds have been prepared of that general structure {Green Chemistry

B3806550.2 - 4 - Atty. Docket No. BUX-008.25

2003, 5, 143; Chem. Materials 2007, 19, 5848-5850; and J. Am. Chem. Soc. 2008, 130, 9648-9649).

The ionic liquid described below can have compositions that possess single cation and single anion as well as multi-cationic and multi-anionic centers, such as those described in International Patent Application Publication No. WO 07/124397 to Grinstaff et al. ; hereby incorporated by reference in its entirety.

For example, in certain embodiments, the ionic liquids of the invention include a plurality of organic cations that contain independently for each occurrence a heterocycle selected from the group consisting of azathiozoles, pyrazoles, thiazoles, isothiazoles, oxothiazoles, oxazines, oxazo lines, oxazoboroles, dithioazoles, triazoles, selenozoles, oxaphopholes, pyrroles, boroles, furans, thiophenes, phospholes, pentazoles, indoles, indolines, oxazoles, isoozazoles, isotriazoles, tetrazoles, benzofurans, dibenzofurans, benzothiophenes, dibenzothiophenes, thiadiazoles, pyrimidines, pyrazines, pyridazines, piperazines, pipidines, morpholenes, pyrans, annolines, phthalzines, quinazolines, quinoxalines, quino lines, isoquinolines, thazines, oxazines, and azaannulenes. Acyclic organic cations are also included in the invention; for example, amines such as amidines, imines, guanidines, phosphines such as phosphinimines, arsines, stibines, ethers, thioethers, and selenoethers.

Likewise, in certain embodiments, the ionic liquids of the invention include a plurality of organic and inorganic anions that contain independently for each occurrence a carboxylic acid, sulfonic acid, tetrafluoroborate, hexafluorophosphate, bis- trifluoromethane-sulfonimide, and derivatives thereof. Additional anionic species of the invention include borates, phosphates, nitrates, sulfates, triflates, antimonates,

phosphoniums, carboranes, poly-oxo metallates, and metalloboranes.

One aspect of the invention relates to the ionic liquids formed via the ionic liquid reagents as described herein. For example, one aspect of the invention relates to an ionic liquid comprising an anionic component and a cationic component, wherein:

said anionic component is Y or X-[Y]_m;

m is 1-100 inclusive;

B3806550.2 - 5 - Atty. Docket No. BUX-008.25

X is selected from the group consisting of linear or branched alkanes, linear or branched alkenes, linear or branched alkynes, monocyclic or polycyclic aromatics, monocyclic or polycyclic heteroaromatics, linear or branched polysilanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyalkylene oxides, linear or branched polysiloxanes, linear or branched polyacrylates, linear or branched polyacetals, linear or branched acrylics, linear or branched cellulosics, linear or branched polyethers, linear or branched halogenated polyethers, linear or branched halocarbons, linear or branched polyamides, linear or branched polycarbonates, linear or branched polyethylenes, linear or branched polypropylenes, linear or branched polystyrenes, and linear or branched polyurethanes, or a combination thereof;

Y is selected, independently for each occurrence, from the group consisting of carboxylic acids, sulfonic acids, tetrafiuoroborates, hexafluorophosphates,

bis-trifluoromethane-sulfonimides, borates, phosphates, nitrates, sulfates, triflates, antimonates, phosphoniums, carboranes, poly-oxo metallates, and metalloboranes;

said cationic component is W-[Z]_n;

n is 1-100 inclusive;

W is absent, or selected from the group consisting of linear or branched alkanes, linear or branched alkenes, linear or branched alkynes, monocyclic or polycyclic aromatics, monocyclic or polycyclic heteroaromatics, linear or branched polysilanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyalkylene oxides, linear or branched polysiloxanes, linear or branched polyacrylates, linear or branched polyacetals, linear or branched acrylics, linear or branched cellulosics, linear or branched polyethers, linear or branched halogenated polyethers, linear or branched halocarbons, linear or branched polyamides, linear or branched polycarbonates, linear or branched polyethylenes, linear or branched polypropylenes, linear or branched polystyrenes, and linear or branched polyurethanes, or a combination thereof;

Z is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formyl, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl,

B3806550.2 - 6 - Atty. Docket No. BUX-008.25 hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl,

heterocyclyl, silyl,

R-N- provided that at least one Z is selected from the group consisting of R

Β3806550.2 - 7 - Atty. Docket No. BUX-008.25

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formyl, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R^B-R^c; any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7- membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

R^B is -C(=0)-, -C(=0)N(H , -C(=0)0-, -C(=0)S-, arylene, heteroarylene or heterocyclylene;

R is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl or a macromolecule;

provided that exactly one R is a bond to W; and at least one R is alkylene-R -R .

B3806550.2 - 8 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein said anionic component is Y.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Y is selected from the group consisting of [CH3CO2]^"1, [CI]^"1, [CIO4]^"1, [Br]

¹, [PFe] ¹, [BF4]^"1, [B(CN)₄] ¹, [AICL,]^"1, [AI2CI7]^"1, [CuClz]^"1, [Cu₂Cl₃] [ZnCl₃] [ZnCU] ²,

[Fed,]^"1, [FeCU]^"1, [FeaCl,]^-1, [TiCls]^"1, [TiCl₆]^"2, [SnCl,] ¹, [SnCls] ¹, [SnCle]^"2, and

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein said anionic component is X-[Y]_m.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Y is selected, independently for each occurrence, from the group consisting of -[SO,]^"1, -[S0₄]-², -[SbFs] ¹, -[N(CN)] ¹, -[CF₂S(0)₂NS(0)₂CF₃] ¹, -[PF5] ¹, - [BiCN^ and - BF,]-¹.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Y is -[C0₂]^_1.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein m is 2-20 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein m is 2-10 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, where m is 2, 3 or 4.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein X is selected, independently for each occurrence, from the group consisting of linear or branched alkanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyethers, linear or branched polyacrylic acids, linear or branched polyamides, and polyether triamines, or a combination thereof.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein X is A, AO(AO)_pA, or ANH(ANH)_PA; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

B3806550.2 - 9 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein X is C(R')(A)₃, C(R)[(AO)_pA]₃, C(R)[(ANH)_PA]₃, N(A-)₃, N[(AO)_pA]₃, or N[(ANH)_PA]₃; A is, independently for each occurrence, -(0(¾)_ρ-; Q is, independently for each occurrence, hydrogen or methyl; p is, independently for each occurrence, 0-40 inclusive; and R' is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein X is C(ACH₂)₄, C[(AO)_pA]₄, C[(ANH)_PA]₄, (A)₂N(AO)_pAN(A)₂, or (A)₂N(ANH)_PAN(A)₂; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein W is selected, independently for each occurrence, from the group consisting of linear or branched alkanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyethers, linear or branched polyacrylic acids, linear or branched polyamides, polyether triamines, or a combination thereof.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein W is A, AO(AO)_pA, or ANH(ANH)_PA; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein W is C(R')(A)₃, C(R)[(AO)_pA]₃, C(R)[(ANH)_PA]₃, N(A)₃, N[(AO)_pA]₃, or N[(ANH)_PA]₃; A is, independently for each occurrence, -(0(¾)_ρ-; Q is, independently for each occurrence, hydrogen or methyl; p is, independently for each occurrence, 0-40

B3806550.₂ - 10 - Atty. Docket No. BUX-008.25 inclusive; and R' is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein W is C(ACH₂)₄, C[(AO)_pA]₄, C[(ANH)_pA]₄, (A)₂N(AO)_pAN(A)₂, or (A)₂N(ANH)_PAN(A)₂; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein X and W are the same.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein n is 2-20 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein n is 2-10 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, where n is 2, 3, or 4.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Z is selected, independently for each occurrence, from the group consisting

B3806550.₂ - 11 - Atty. Docket No. BUX-008.25

ΑΗ, ΑΟΗ, -CH(OAH)(AOH), -(p-AH)Ph, and -Si(OH)(AH) A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and r is, independently for each occurrence, 0-40 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Z is selected, independently

for each occurrence, from the group consisting of

the present invention

relates to the aforementioned ionic liquid, wherein Z is

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl or alkylene-R^B-R^c.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R^B is -C(=0)-, -C(=0)N(H)-, -C(=0)0-, or -C(=0)S-. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R^B is C(=0)N(H)-. In certain embodiments, the present invention relates to the

aforementioned ionic liquid, wherein R^B is -C(=0)S-. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R^B is heterocycylene. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R^B is ^ .

B3806550.2 - 12 - Atty. Docket No. BUX-008.25

In certain embodiments R is alkyl, aralkyl, aryl, heteroaryl or a macromolecule. In certain embodiments R^c is alkyl. In certain embodiments R^c is aralkyl. In certain embodiments R is an enzyme. In certain embodiments R is an enzyme with one or more pendant amines, thiols and/or alcohols. In certain embodiments R is an enzyme. In certain embodiments R^c is an enzyme with one or more pendant amines. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R^c is lysozyme.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 1-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein alkylene is selected from the group consisting of -[CH₂]k-; wherein k is 10- 30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein alkylene is selected from the group consisting of -CH₂-, -CH₂CH₂-,

-CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, and

-CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-.

Another aspect of the invention relates to an ionic liquid comprising an anionic component and a cationic component, wherein:

said anionic component is Y;

Y is selected, independently for each occurrence, from the group consisting of carboxylic acids, sulfonic acids, tetrafluoroborates, hexafluorophosphates,

said cationic component is Z;

Z is selected, independently for each occurrence, from the group consisting of

B3806550.₂ - 13 - Atty. Docket No. BUX-008.25

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succimmidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R -R ; any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7- membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

R^B is oxo, arylene, heteroarylene or heterocyclylene;

R^c is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succimmidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl;

B3806550.2 - 14 - Atty. Docket No. BUX-008.25 provided that at least one is alkylene-R -R .

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Y is selected from the group consisting of [CH₃CO₂]^"1, [CI]^"1, [CIO₄]^"1, [Br] \ [I]^"1, [SO4]^"2, [CH3SO3]^"1, [SbF₆]-\ [N(CN)₂]-\ [CF₃S(0)2NS(0)₂CF₃]-¹, [PF₆] \ [BF4] ¹, [B(CN)₄]-¹, [AICI₄]-¹, [A Clv]-¹, [CuCl₂]^_1, [CujClj]-¹, [ZnCy ¹, [ZnCU]^"2,

[FeC ]^"1, [FeCL,]^"1, [FezCly]^"1, [TiCls] ¹, [TiCl₆]^"2, [SnClj] ¹, [SnCls] ¹, [SnCl₆]^"2, and In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Z is selected, independently for each occurrence, from the group consisting

. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein Z is R- %P-R

R .

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein alkylene is selected from the group consisting of -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -CH₂CFI₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, and

-CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-.

B3806550.₂ - 15 - Atty. Docket No. BUX-008.25

said anionic component is Y;

said cationic component is Z;

Z is

;

R¹ is alkyl;

R² is alkyl;

R³ is alkyl;

R⁴ is alkylene-R⁵;

alcohol, CN, and

R⁶ is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl or a macromolecule.

\ [I]^"1, [S0₄]-², [CH3SO3]-¹, [SbFs]^"1, [N(CN)₂]^_1, [CFaS^ S^aCFa]-¹, [PF₆] \ [BF4]^"1,

B3806550.2 - 16 - Atty. Docket No. BUX-008.25

[B(CN)₄]^"1, [AICI4]^"1, [AIZCIT]^"1,

[CU.CIJ] ¹, [ZnCy ¹, [ZnCl₄]^"2,

[FeC ]^"1, [FeCU]^"1, [FeaCl,]^-1, [TiCls]^"1, [TiCle]^"2, [SnCy ¹, [SnClj]^"1, [SnCle]^"2, and

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R¹ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R¹ is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R² is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R² is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R³ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R³ is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R⁴ is selected from the group consisting of -[CH2]kR-5; wherein k is 1 -30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R⁴ is selected from the group consisting of -[CH₂]_kR⁵; wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein alkylene is selected from the group consisting of -[CH₂]_kR⁵; wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R⁴ is -CH₂R⁵, -CH₂CH₂R⁵, -CH₂CH₂CH₂R⁵, -CH₂CH₂CH₂CH₂R⁵,

-CH2CH2CH2CH2CH2R⁵, -CHzCHjCHzCHzCHzCHzR⁵, -CH2CH2CH2CH2CH2CH2CH2R⁵ , -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂R⁵, or -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂R⁵. In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R⁴ is -CH₂CH₂CH₂R⁵.

B3806550.₂ - 17 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic

O

liquid, wherein R⁵ is ^ . In certain embodiments, the present invention relates to

O

the aforementioned ionic liquid, wherein ⁵ is ^ ^ ; and R⁶ is alkyl.

In certain e present invention relates to the aforementioned ionic liquid, wherein R

In certain embodiments, the present invention relates to

O the aforementioned ionic liquid, wherein R is ^H ; and R is alkyl, aralkyl and aryl.

NsN

,N-_R6

liquid, wherein R is . In certain embodiments, the present invention relates

,N~_R6

to the aforementioned ionic liquid, wherein R is ; and R is alkyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein R⁶ is alkyl, aralkyl or aryl.

B3806550.2 - 18 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic liquid, wherein the ionic liquid reagent is

II ^' P⁺(CH₂CH₂CH2CH3)3 C|-

O , CM-

Methods for Preparing Ionic Liquids

As described above, an ionic liquid is a small molecule compound that can be easily coupled to another small molecule or macromolecule (such as an enzyme) to afford an ionic liquid or ionic viscoelastic. In certain embodiments, the reaction between the ionic liquid and the small molecule or macromolecule is well-defined and high yielding (e.g. greater than about 60%); is thermodynamically favored; can be performed in one-step; and

B3806550.2 - 19 - Atty. Docket No. BUX-008.25 comprises an ionic liquid which is stable enough to be stored (and shipped) prior to use. As such the type of coupling chemistry that can be used for the ionic liquid are limited.

One example of coupling chemistry which can be used with ionic liquids is click chemistry as described in Kolb et al. (Angew. Chem. Int. Ed. Engl. 2001, 40, 2004-2021). One example of a click chemistry reaction is the Azide-Alkyne Huisgen Cycloaddition, which is a 1 ,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1 ,2,3-triazole. To demonstrate the generality of this synthetic approach for creating new ionic liquids, an ionic liquid bearing a terminal alkyne (phosphonium (trihexyl) pentyne) was reacted with an azide-containing small molecule ((S)-3-azidopropane-l,2- diol) to form a triazole-containing ionic liquid.

One example of coupling chemistry which can be used with ionic liquids is amide bond formation, such as via an active N-hydroxysuccinimide (NHS) ester or acyl chloride. For example, in certain embodiments the ionic liquid can possess one or more electrophilic groups (e.g., NHS, acyl chloride, isocynate) for coupling with a nucleophilic small molecule or macromolecule.

Amide bond formation using an N-hydroxysuccinimide ester was first reported in 1963 by Callahan for peptide synthesis (J. Am. Chem. Soc. 1963, 85; J. Am. Chem. Soc. 1964, 86, 1839-1841). Since then, this active ester has been used extensively as it reacts with primary amines to afford the stable, irreversible amide bond in good yield (typically 60-99%). This activated ester is relatively stable in weakly basic solutions and the reaction rates are dependent on the basicity of the amine as well as sterics with primary amines reacting faster then secondary amines (J. Am. Chem. Soc. 1987, 109, 3087-3091).

Moreover, NHS activated esters are stable enough to be purified and stored for future reactions facilitating wide-spread use of NHS activated ester "reagents" by many types of scientists. The success of this reaction is exemplified through its application in

biomacromolecule modification and the impact this reaction has had on the bioconjugate field.

To demonstrate the generality of this synthetic approach for creating ionic liquids, an ionic liquid bearing an NHS activate ester (Figure 4; 2, IL-OSu) was prepared and reacted with a series of electronically and structurally different nucleophiles including alkyl, benzyl, and aromatic amines, an alkyl thiol, and lysozyme. As such, we are able to

B3806550.2 - 20 - Atty. Docket No. BUX-008.25 evaluate the effect of amine pKa, amine vs. thiol reactivity, and a protein on this reaction. IL-OSu was prepared by first reacting tributyl phosphine with chlorobutyric acid neat under microwave conditions at 60 °C for 15 minutes to obtain compound 3. The reaction was quantitative. In comparison a 40% yield was obtained after two weeks in refluxing toluene. The use of microwaves to accelerate and increase yields has been reported for

imadozilinium based ionic liquids but not yet for phosphonium ionic liquids. Next, N- hydroxysuccinimide was reacted with 3 in the presence of DCC to afford 2 in 87 % yield. The complete synthetic reaction details and characterization data including ^ΧΗ NMR, ¹ C NHR and elemental analysis can be found below.

A series of different ionic liquids were synthesized as shown in Figure 2 by reacting

2 with the corresponding nucleophile. The general coupling procedure involved dissolving compound 2 (1.2 eq.) in dry dichloromethane followed by the addition of triethylamine (1.2 eq.) and the appropriate amine or thiol nucleophile (1 eq.) under nitrogen. The mixture was stirred for 12 hrs at room temperature, concentrated, dried and analyzed by NMR and HR-mass spectrometry. The primary alkyl and benzyl amines reacted with 2 in good to high yield (greater than about 80%). As expected, a lowering of the amine pKa either through para substitution of benzyl amine with fluorine or the use of aniline instead of benzyl amine reduced the coupling yield. The reaction was not limited to amines, an alkyl thiol reacted readily with 2 in high yield to afford the thioester linkage and the

corresponding ionic liquid (Figure 2; entry 6).

One aspect of the invention relates to a method of preparing an ionic liquid comprising the step of:

reacting an ionic liquid reagent containing a first chemical group with a small molecule or macromolecule containing a second chemical group to form an ionic liquid comprising the ionic liquid reagent covalently bound to the small molecule,

wherein the first chemical group and the second chemical group react to form a bond;

the ionic liquid reagent comprises an anionic component and a cationic component; said anionic component is Y or X-[Y]_m;

m is 1-100 inclusive;

B3806550.2 - 21 - Atty. Docket No. BUX-008.25

said cationic component is W-[Z]_n;

n is 1-100 inclusive;

Z is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl,

B3806550.2 - 22 - Atty. Docket No. BUX-008.25 hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl,

heterocyclyl, silyl,

R-N- provided that at least one Z is selected from the group consisting of R

Β3806550.2 - 23 - Atty. Docket No. BUX-008.25

R , and

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R^A; any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7- membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

R^A comprises at least one electrophile, nucleophile, dipolarophile, 1,3-dipole, diene or dieneophile;

provided that exactly one R is a bond to W; and at least one R is alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned method, wherein said anionic component is Y.

In certain embodiments, the present invention relates to the aforementioned method wherein Y is selected from the group consisting of [CH3CO2]^"1, [CI]^"1,

[Br]^"1, [I]^"1, [S0₄r², [CH3SO3]^"1,

[BF₄]^_1,

[B(CN)₄]^"1, [AICI4]^"1, [AbCl,]^"1,

[CuzCla]-¹, [ZnClaj^"1, [ZnCl₄]^"2, [Zn₂Cl₅]^"1,

¹, [SnClj] ¹, [SnCl₆]^"2, and

[CiCL,] .

B3806550.₂ - 24 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned method, wherein said anionic component is X-[Y]_m.

In certain embodiments, the present invention relates to the aforementioned method, wherein Y is selected, independently for each occurrence, from the group consisting of -[CO2]-¹, -[SO3]-¹, -[S0₄]-², -[SbFs]^"1, -[N(CN)]-¹,

-[PF5]^"1, - [BiCN^. and -tBFaT¹.

In certain embodiments, the present invention relates to the aforementioned method, wherein Y is -[C0₂]^_1.

In certain embodiments, the present invention relates to the aforementioned method, wherein m is 2-20 inclusive. In certain embodiments, the present invention relates to the aforementioned method, wherein m is 2-10 inclusive In certain embodiments, the present invention relates to the aforementioned method, where m is 2, 3 or 4.

In certain embodiments, the present invention relates to the aforementioned method, wherein X is selected, independently for each occurrence, from the group consisting of linear or branched alkanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyethers, linear or branched polyacrylic acids, linear or branched polyamides, and polyether triamines, or a combination thereof.

In certain embodiments, the present invention relates to the aforementioned method, wherein X is A, AO(AO)_pA, or ANH(ANH)pA; A is, independently for each occurrence, -(CQ₂)p-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned method, wherein X is C(R')(A)₃, C(R')[(AO)_pA]₃, C(R')[(ANH)_PA]₃, N(A-)₃, N[(AO)_pA]₃, or

N[(ANH)pA]₃; A is, independently for each occurrence, -(CQ2)_P-; Q is, independently for each occurrence, hydrogen or methyl; p is, independently for each occurrence, 0-40 inclusive; and R' is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl,

B3806550.2 - 25 - Atty. Docket No. BUX-008.25 alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

In certain embodiments, the present invention relates to the aforementioned method, wherein X is C(ACH₂)₄, C[(AO)_pA]₄, C[(ANH)pA]₄, (A)₂N(AO)_pAN(A)₂, or

(A)₂N(ANH)_PAN(A)₂; A is, independently for each occurrence, -(CQ₂)_P-; Q is,

independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned method, wherein W is selected, independently for each occurrence, from the group consisting of linear or branched alkanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyethers, linear or branched polyacrylic acids, linear or branched polyamides, polyether triamines, or a combination thereof.

In certain embodiments, the present invention relates to the aforementioned method, wherein W is A, AO(AO)_pA, or ANH(ANH)_pA; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned method, wherein W is C(R)(A)₃, C(R)[(AO)_pA]₃, C(R)[(ANH)_PA]₃, N(A)₃, N[(AO)_pA]₃, or N[(ANH)_PA]₃; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; p is, independently for each occurrence, 0-40 inclusive; and R is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

B3806550.₂ - 26 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned method, wherein W is C(ACH₂)₄, C[(AO)_pA]₄, C[(ANH)pA]₄, (A)₂N(AO)_pAN(A)₂, or

(A)₂N(ANH)_PAN(A)₂; A is, independently for each occurrence, -(0(¾)_ρ-; Q is,

In certain embodiments, the present invention relates to the aforementioned method, wherein X and W are the same.

In certain embodiments, the present invention relates to the aforementioned method, wherein n is 2-20 inclusive. In certain embodiments, the present invention relates to the aforementioned method, wherein n is 2-10 inclusive In certain embodiments, the present invention relates to the aforementioned method, where n is 2, 3, or 4.

In certain embodiments, the present invention relates to the aforementioned method, wherein Z is selected, independently for each occurrence, from the group consisting of

CH(OAH)(AOH), -AH)Ph, and -Si(OH)(AH) A is, independently for each occurrence,

-(CQ₂)_p-; Q is, independently for each occurrence, hydrogen or methyl; and r is, independently for each occurrence, 0-40 inclusive. In certain embodiments, the present invention relates to the aforementioned method, wherein Z is selected, independently for

B3806550.2 - 27 - Atty. Docket No. BUX-008.25

each occurrence, from the group consisting of

. In certain embodiments, the present invention

R-P-R

relates to the aforementioned method, wherein Z is R

In certain embodiments, the present invention relates to the aforementioned method, wherein R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl or alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an electrophile. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is a carboxyl-containing electrophile. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an N-hydroxysuccinimide (NHS) ester, acyl chloride or isocynate. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an N- hydroxysuccinimide (NHS) ester.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is a nucleophile. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an amine or thiol. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an alkyl amine. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an alkyl thiol. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aralkyl amine. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aralkyl thiol. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aryl amine. In certain embodiments, the present invention relates to the aforementioned method, wherein R is an aryl thiol.

B3806550.2 - 28 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an enzyme. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an enzyme with one or more pendant amines. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is lysozyme.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is a dipolarophile. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is alkynyl.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is a 1,3-dipole. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is azidyl. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is acyl azidyl.

In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_k-; wherein k is 1-30 inclusive. In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the

aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]k-; wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂-,

-CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, and

-CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-.

Another aspect of the invention relates to a method of preparing an ionic liquid comprising the step of:

B3806550.2 - 29 - Atty. Docket No. BUX-008.25 wherein the first chemical group and the second chemical group react to form a bond;

the ionic liquid reagent comprises an anionic component and a cationic component; said anionic component is Y;

said cationic component is Z;

Z is selected, independently for each occurrence, from the group consisting of

is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl,

B3806550.2 - 30 - Atty. Docket No. BUX-008.25 alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R^A; any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7- membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

provided that at least one R is alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned method, wherein Y is selected from the group consisting of [CH₃CO₂]^"1, [CI]^"1,

[Br]^"1, [I]^"1, [S0₄]^"2, [CH₃SO₃]^"1,

[CF₃S(0)₂NS(0)₂CF₃] ¹, [PFe] ¹, [BF₄]^"1,

[B(CN)₄]^"1, [AICI4]^"1, [AbCl,]^"1, [CuCy¹, [CU2C13]^"1, [ZnCy ¹, [ZnCUr², [Z^Cls]^"1, [FeCbr¹, [FeCU]-¹, [Feffi^"1, [T1CI₅] ¹, [TiCl₆]^"2, [SnCy ¹, [SnClj] ¹, [SnCl₆]^"2, and

In certain embodiments, the present invention relates to the aforementioned method, wherein Z is R-P-R

R .

In certain embodiments, the present invention relates to the aforementioned method, wherein R is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl or alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an electrophile. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is a carboxyl-containing electrophile. In certain

B3806550.2 - 31 - Atty. Docket No. BUX-008.25 embodiments, the present invention relates to the aforementioned method, wherein R^A is an N-hydroxysuccinimide (NHS) ester, acyl chloride or isocynate. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an N- hydroxysuccinimide (NHS) ester.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is a nucleophile. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an amine or thiol. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an alkyl amine. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an alkyl thiol. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aralkyl amine. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aralkyl thiol. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aryl amine. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an aryl thiol.

In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an enzyme. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is an enzyme with one or more pendant amines, thiols and/or alcohols. In certain embodiments, the present invention relates to the

aforementioned method, wherein R^A is an enzyme with one or more pendant amines. In certain embodiments, the present invention relates to the aforementioned method, wherein R^A is lysozyme.

In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_k-; wherein k is 1-30

B3806550.2 - 32 - Atty. Docket No. BUX-008.25 inclusive. In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the

aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_k-; wherein k is 20-30 inclusive.

-CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-.

bis-trifiuoromethane-sulfonimides, borates, phosphates, nitrates, sulfates, trifiates, antimonates, phosphoniums, carboranes, poly-oxo metallates, and metalloboranes;

said cationic component is Z;

R is alkyl;

R² is alkyl;

B3806550.2 - 33 - Atty. Docket No. BUX-008.25

R³ is alkyl;

R⁴ is alkylene-R⁵;

c*° ^

or ¾ ; CI, Br, I, thiol, alcohol, CN, and

R⁶ is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

In certain embodiments, the present invention relates to the aforementioned method, wherein Y is selected from the group consisting of [CH₃CO₂]^"1, [CI]^"1, [CIO₄]^"1, [Br]^"1, [I]^"1, [S0₄]^"2, [CH3S03]^"1, [SbF₆]^"\ [N(CN)₂]-\ [CF₃S(0)₂NS(0)₂CF₃]-¹, [PF₆] \ [BF4]^"1,

[B(CN)₄]-¹, [AICI4]-¹, [A Clv]-¹,

[FeCls]^"1, [Fed,]^"1, p^Cl,]^"1, [TiCls] ¹, [TiCle]^"2, [SnClj] ¹, [SnCls] ¹, [SnCy², and In certain embodiments, the present invention relates to the aforementioned method, wherein R¹ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned method, wherein R¹ is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned method, wherein R² is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned method, wherein R² is butyl or hexyl.

B3806550.2 - 34 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned method, wherein R³ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned method, wherein R³ is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_kR⁵; wherein k is 1-30 inclusive. In certain embodiments, the present invention relates to the aforementioned method, wherein alkylene is selected from the group consisting of -[CH₂]_kR⁵; wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the

aforementioned method, wherein alkylene is selected from the group consisting of - [CH₂]_kR⁵; wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned method, wherein R⁴ is -CH₂R⁵, -CH₂CH₂R⁵, -CH₂CH₂CH₂R⁵, -CH₂CH₂CH₂CH₂R⁵,

-CH₂CH₂CH₂CH₂CH₂R^"!, -CH₂CH₂CH₂CH₂CH₂CH₂R^"!, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂R^"!, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂R⁵, or -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂R⁵. In certain embodiments, the present invention relates to the aforementioned method, wherein R⁴ is -CH₂CH₂CH₂R⁵.

In certain embodiments, the present invention relates to the aforementioned method,

In certain emb present invention relates to the aforementioned method, wherein R⁵ is

, the present invention relates to the aforementioned method,

B3806550.₂ - 35 - Atty. Docket No. BUX-008.25

wherein the Z is

or

P⁺(CH2CH2CH2CH2CH2CH3)3

wherein the ionic liquid reagent is

or

P⁺(CH₂CH2CH₂CH2CH2CH3)3 CI

In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the second chemical group is an amine. In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the second chemical group is a thiol.

In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the ionic liquid reagent is reacted with a small molecule. In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the ionic liquid reagent is reacted with a small molecule; and the small molecule is an alkyl amine, aryl amine, aralkyl amine, alkyl thiol, aryl thiol or aralkyl thiol.

In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the ionic liquid reagent is reacted with a

macromolecule. In certain embodiments, the present invention relates to the

aforementioned method and the attendant definitions, wherein the ionic liquid reagent is reacted with a macromolecule; and the macromolecule is an enzyme. In certain

embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the ionic liquid reagent is reacted with a macromolecule; and the macromolecule is lysozyme.

In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the reaction is acid-catalyzed or base-catalyzed.

B3806550.2 - 36 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the reaction is selected from the group consisting of SN2 displacement reactions, 1 ,2- or 1 ,4-addition reactions, 1 ,3-dipolar cycloaddition reactions, Diels- Alder reactions, condensation reactions (such as esterification and amidation), acylation reactions, ring-forming reactions, and metathesis reactions. In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the reaction is a condensation reaction. In certain embodiments, the present invention relates to the aforementioned method and the attendant definitions, wherein the reaction is a 1 ,3-dipolar cycloaddition.

Ionic Liquid Reagents

Certain aspects of the invention relate to ionic liquid reagents, as used in the methods described above. For example, one aspect of the invention relates to an ionic liquid reagent comprising an anionic component and a cationic component, wherein:

said anionic component is Y or X-[Y]_m;

m is 1 - 100 inclusive;

said cationic component is W-[Z]_N;

B3806550.2 - 37 - Atty. Docket No. BUX-008.25 n is 1-100 inclusive;

Z is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl,

B3806550.2 - 38 - Atty. Docket No. BUX-008.25

^R©

R-N-R provided that at least one Z is selected from the group consisting of R ,

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R^A; any two adjacent R, taken together with the

B3806550.2 - 39 - Atty. Docket No. BUX-008.25 atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7- membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

provided that exactly one R is a bond to W; and at least one R is alkylene-R^A .

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein said anionic component is Y.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Y is selected from the group consisting of [CH₃CO₂]^"1, [CI]^"1, [CIO4]^"1, [Br]^"1, [I]^"1, [SO4]^"2, [CH3SO3]^"1, [SbFg] ¹, [NCC ^] ¹, [CFsS^NSCO^CFs] ¹, [PF₆]-\ [BF4]^"1, [B(CN)₄]-\ [AICI4]-¹, [Alffl-¹, [CuCy¹, [C112CI3]-¹, [ZnCy¹, [ZnCl₄]-², [Z^Cls]-¹, [FeC ]^"1, [FeCU]^"1, ^Cl,]^"1, [T1CI5] ¹, [TiCl₆]^"2, [SnCls] ¹, [SnClj] ¹, [SnCl₆]^"2, and [CrCL,]^-1.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein said anionic component is X-[Y]_m.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Y is selected, independently for each occurrence, from the group consisting of -[C02]-¹, -[SO3]^"1, -[S0₄]^"2, -[SbFj]^"1, -[N(CN)] ¹, -[CFzSCO^NSCO^] ¹, -[PF₅]^"1, -[BCC ^] ¹, and -[BF₃]^"1.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Y is -[CO2]^"1.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein m is 2-20 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein m is 2-10 inclusive In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, where m is 2, 3 or 4.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein X is selected, independently for each occurrence, from the group consisting of linear or branched alkanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyethers, linear or branched

B3806550.2 - 40 - Atty. Docket No. BUX-008.25 polyacrylic acids, linear or branched polyamides, and polyether triamines, or a combination thereof.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein X is A, AO(AO)_pA, or ANH(ANH)_PA; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein X is C(R')(A)₃, C(R')[(AO)pA]₃, C(R')[(ANH)_PA]₃, N(A-)₃, N[(AO)_pA]₃, or N[(ANH)_PA]₃; A is, independently for each occurrence, -(C h ; Q i^s' independently for each occurrence, hydrogen or methyl; p is, independently for each occurrence, 0-40 inclusive; and R' is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl,

isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein X is C(ACH₂)₄, C[(AO)pA]₄, C[(ANH)_PA]₄, (A)₂N(AO)_pAN(A)₂, or (A)₂N(ANH)_PAN(A)₂; A is, independently for each occurrence, -(CQ₂)_P-; Q is,

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein W is selected, independently for each occurrence, from the group consisting of linear or branched alkanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyethers, linear or branched polyacrylic acids, linear or branched polyamides, polyether triamines, or a combination thereof.

B3806550.₂ - 41 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein W is A, AO(AO)_pA, or ANH(ANH)_PA; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein W is C(R')(A)₃, C(R')[(AO)_pA]₃, C(R')[(ANH)_PA]₃, N(A)₃, N[(AO)_pA]₃, or N[(ANH)_PA]₃; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; p is, independently for each occurrence, 0-40 inclusive; and R' is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein W is C(ACH₂) , C[(AO)_pA]₄, C[(ANH)_PA]₄, (A)₂N(AO)_pAN(A)₂, or (A)₂N(ANH)_PAN(A)₂; A is, independently for each occurrence, -(CQ₂)_P-; Q is, independently for each occurrence, hydrogen or methyl; and p is, independently for each occurrence, 0-40 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein X and W are the same.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein n is 2-20 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein n is 2- 10 inclusive In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, where n is 2, 3, or 4.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Z is selected, independently for each occurrence, from the group

B3806550.₂ - 42 - Atty. Docket No. BUX-008.25

AH, AOH, -CH(OAH)(AOH), -(p-AH)Ph, and -Si(OH)(AH) A is, independently for each occurrence, -(0<¾)_ρ-; Q is, independently for each occurrence, hydrogen or methyl; and r is, independently for each occurrence, 0-40 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Z is selected,

independently for each occurrence, from the group consisting of

In certain embodiments, the present

^R©

R-P-R

invention relates to the aforementioned ionic liquid reagent, wherein Z is R

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl or alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an electrophile. In certain embodiments, the present invention

B3806550.2 - 43 - Atty. Docket No. BUX-008.25 relates to the aforementioned ionic liquid reagent, wherein R^A is a carboxyl-containing electrophile. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an N-hydroxysuccinimide (NHS) ester, acyl chloride or isocynate. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an N-hydroxysuccinimide (NHS) ester.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is a nucleophile. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an amine or thiol. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an alkyl amine. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an alkyl thiol. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an aralkyl amine. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an aralkyl thiol. In certain

embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an aryl amine. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an aryl thiol.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an enzyme. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an enzyme with one or more pendant amines, thiols and/or alcohols. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an enzyme with one or more pendant amines. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is lysozyme.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is a dipolarophile. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is alkynyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is a 1,3-dipole. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is azidyl. In certain

B3806550.2 - 44 - Atty. Docket No. BUX-008.25 embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is acyl azidyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]k-; wherein k is 1-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CI¾]_k-; wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -CH₂-, -CH₂CH₂-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, and

-CH₂CH₂CH2CH₂CH₂CH₂CH₂CH₂CH2-.

Another aspect of the invention relates to an ionic liquid reagent comprising an anionic component and a cationic component, wherein:

said anionic component is Y;

bis-trifiuoromethane-sulfonimides, borates, phosphates, nitrates, sulfates, triflates, antimonates, phosphoniums, carboranes, poly-oxo metallates, and metalloboranes;

said cationic component is Z;

Z is selected, independently for each occurrence, from the group consisting of

B3806550.₂ - 45 - Atty. Docket No. BUX-008.25

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R^A (alkylene-R^B-R^c); any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6- membered, or 7-membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and R^A comprises at least one electrophile, nucleophile, dipolarophile, 1,3-dipole, diene or dieneophile;

provided that at least one R is alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Y is selected from the group consisting of [CH₃CO2]^"1, [CI]^"1, [CIO4]^"1, [Br!¹, [I]^"1, [S0₄] ², [CH3SO3]^"1, [SbF₆] [N(CN)₂] [CF₃S(0)₂NS(0)₂CF₃] \ [PFe]^"1, [BF4]^"1, [B(CN)₄]-\ [AICI4]^"1, [AkCl,]^"1, [CuCfe]^"1, [CuzClaj^"1, [ZnC ]^"1, [ZnCl₄]^"2,

[TiClj] ¹, [TiCl₆]^"2, [SnCy ¹, [SnClj] ¹, [SnCl₆]^"2, and [CiCL,]^"1.

B3806550.₂ - 46 - Atty. Docket No. BUX-008.25

consisting of

In certain embodime present invention relates to the aforementioned ionic liquid reagent, wherein Z is

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl or alkylene-R^A.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an electrophile. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is a carboxyl-containing electrophile. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an N-hydroxysuccinimide (NHS) ester, acyl chloride or isocynate. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is an N-hydroxysuccinimide (NHS) ester.

B3806550.2 - 47 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is a 1,3-dipole. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is azidyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R^A is acyl azidyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]_k-; wherein k is 1-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CI¾]_k-; wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-, and

-CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂-.

said anionic component is Y;

B3806550.₂ - 48 - Atty. Docket No. BUX-008.25

said cationic component is Z;

R is alkyl;

R² is alkyl;

R³ is alkyl;

R⁴ is alkylene-R⁵;

or ; CI, Br, I, thiol, alcohol, CN, and

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein Y is selected from the group consisting of [CH₃CO2]^"1, [CI]^"1, [CIO4]-¹, [Br]^"1, [I]^"1, [SO4]-², [CH3SO3]-¹, [SbFs] ¹, [NCC ^] ¹, [CFsS^NS^CFs] ¹, [PFe]^"1, [BF4]^"1,

[A1CU]^"1, [AI2CI7]-¹, [CuCy¹, [CuzCy¹, [ZnCla]^"1, [ZnC ]^"2,

B3806550.2 - 49 - Atty. Docket No. BUX-008.25

[TiClj] ¹, [Tide]^"2, [SnClj] ¹, [SnClj] ¹, [SnCl₆]^"2, and [CrC r¹.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R¹ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R¹ is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R² is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R² is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R³ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R³ is butyl or hexyl.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 1-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 10-30 inclusive. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein alkylene is selected from the group consisting of -[CH₂]_kS wherein k is 20-30 inclusive.

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R⁴ is -CH₂R⁵, -CH₂CH₂R⁵, -CH₂CH₂CH₂R⁵, -CH₂CH₂CH₂CH₂R⁵, -CH₂CH₂CH₂CH₂CH₂R^"!, -CH₂CH₂CH₂CH₂CH₂CH₂R^"!, -CH₂CH₂CH₂CH₂CH₂CH₂CH₂R^"!, -CH2CH2CH2CH2CH2CH2CH2CH2R⁵, or -CHzCHzCHzCHzCHzCHjCHzCHzCHaR⁵. In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R⁴ is -CH₂CH₂CH₂R⁵.

O

liquid reagent, wherein R⁵ is 0 ; and R⁶ is succinimidyl.

B3806550.₂ - 50 - Atty. Docket No. BUX-008.25

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein R 5 is ^ ^N

In certain embodime present invention relates to the aforementioned ionic liquid reagent, wherein R is

liquid reagent, wherein the Z is

or

P⁺(CH₂CH₂CH₂CH₂CH₂CH3)3

In certain embodiments, the present invention relates to the aforementioned ionic liquid reagent, wherein the ionic liquid reagent is

P⁺(CH₂CH₂CH₂CH₂CH₂CH3)3 CI

Treating and/or Preventing Microbial Infections

One aspect of the invention relates to a method of treating or preventing a microbial infection comprising the step of administering an effective amount of any of the aforementioned ionic liquids, or a pharmaceutical composition thereof, to a patient in need thereof.

In a method of treatment of the present invention, any one or more of the aforementioned ionic liquids, or a pharmaceutical composition thereof, will generally be administered to a patient in need thereof in such amounts and for such a time as is necessary or sufficient to achieve at least one desired result. As will be appreciated by one skilled in the art, the desired result may vary depending on the condition to be treated, the extent of an infection, the amount of ionic liquid(s) given, and the number of

administrations. Thus, for example, in certain embodiments, one or more of the aforementioned ionic liquids may be administered to a patient suffering from a bacterial

B3806550.2 - 51 - Atty. Docket No. BUX-008.25 infection in such amounts and for such a time that it kills or prevents the growth of the bacteria. In other embodiments, one or more of the aforementioned ionic liquids may be administered to the eye of a patient prior to (prophylactic), during, or after undergoing surgery (e.g., cataract surgery) in such amounts that it kills or prevents the growth of the bacteria. In yet other embodiments, one or more the aforementioned ionic liquids may be administered to the skin of a patient undergoing a surgical or cosmetic procedure in such amounts and for such a time that it kills or prevents the growth of the bacteria. In certain embodiments, the aforementioned ionic liquid used is multicationic (i.e. the cationic portion of the ionic liquid comprises multiple positive charges). In certain embodiments, the aforementioned ionic liquid used is multianionic (i.e. the anionic portion of the ionic liquid comprises multiple negative charges).

In certain embodiments, a treatment according to the present invention may consist of a single dose or a plurality of doses over a period of time. Administration may be one or multiple times daily, weekly (or at some other multiple day interval) or on an intermittent schedule. The exact amount of an inventive ionic liquid(s), or a pharmaceutical composition thereof, to be administered will vary from subject to subject and will depend on several factors (as outlined herein).

Any of the aforementioned ionic liquids, or pharmaceutical compositions thereof, may be administered using any route of administration effective for achieving the desired effect. Administration can be applied topically, locally or systemically. Methods of local administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, and intra-articular routes.

Depending on the route of administration, effective doses may be calculated according to the body weight, body surface area, or organ size of the subject to be treated. Optimization of the appropriate dosages can readily be made by one skilled in the art in light of pharmacokinetic data observed in human clinical trials. Alternatively or additionally, the dosage to be administered can be determined from studies using animal models for the particular type of condition to be treated, and/or from animal or human data obtained from agents which are known to exhibit similar pharmacological activities. The final dosage regimen will be determined by the attending surgeon or physician, considering various factors which modify the action of active agent, e.g., the agent's specific activity, the agent's specific half-life in vivo, the severity of the condition and the responsiveness of

B3806550.2 - 52 - Atty. Docket No. BUX-008.25 the patient, the age, condition, body weight, sex and diet of the patient, the severity of any present infection, time of administration, the use (or not) of other concomitant therapies, and other clinical factors.

In certain embodiments, compositions according to the present invention are prepared so that a dosage unit contains between about 0.01 mg and about 250 mg of the aforementioned ionic liquid. In certain embodiments, compositions according to the present invention are prepared so that a dosage unit contains between about 0.01 mg and about 0.1 mg of the aforementioned ionic liquid. In certain embodiments, compositions according to the present invention are prepared so that a dosage unit contains between about 0.1 mg and about 1 mg of the aforementioned ionic liquid. In certain embodiments, compositions according to the present invention are prepared so that a dosage unit contains between about 1 mg and about 100 mg of the aforementioned ionic liquid. In certain embodiments, compositions according to the present invention are prepared so that a dosage unit contains between about 100 mg and about 250 mg of the aforementioned ionic liquid.

Combination Therapies

In certain embodiments, methods of treatment of the present invention can be employed in combination with additional therapies (i.e., a treatment according to the present invention can be administered concurrently with, prior to, or subsequently to one or more desired therapeutics or medical procedures). The particular combination of therapies (therapeutics or procedures) to employ in such a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved.

Thus, for example, in methods where an ionic liquid(s) of the present invention is administered as an antibiotic to a patient suffering from a bacterial infection, the patient may further receive a non-steroidal or steroidal anti-inflammatory drug, antibiotics, antimicrobials, and/or may undergo other therapies.

Thus, for example, in methods where an ionic liquid(s) of the present invention may be administered in combination with another aqueous soluble polymer, e.g., PEG, PEO, PAA

B3806550.2 - 53 - Atty. Docket No. BUX-008.25

In many methods of the present invention, an ionic liquid(s) is administered as part of a surgical or clinical procedure. For example, a polymer used as an antibiotic or antifungal and can be administered during an ocular surgery, cosmetic, orthopedic, thoracic, cardiovascular surgery.

Pharmaceutical Compositions

As mentioned above, methods of treatment of the present invention include administration of one or more the aforementioned ionic liquid(s) per se or in the form of a pharmaceutical composition. A pharmaceutical composition will generally comprise an effective amount of at least one of the aforementioned ionic liquids and at least one pharmaceutically acceptable carrier or excipient.

Pharmaceutical compositions of the present invention may be formulated according to general pharmaceutical practice (see, for example, "Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology", J. Swarbrick, and J.C. Boylan (Eds.), Marcel Dekker, Inc: New York, 1988). The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered compounds. Formulation will preferably produce liquid or semi-liquid (e.g., gel) pharmaceutical compositions.

Pharmaceutical compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "unit dosage form," as used herein, refers to a physically discrete unit of the aforementioned ionic liquid(s) for the patient to be treated. Each unit contains a predetermined quantity of active material calculated to produce the desired effect. It will be understood, however, that the total dosage of the composition will be decided by the attending physician within the scope of sound medical judgment.

Formulation of pharmaceutical compositions of the present invention will mainly depend on the form of administration chosen. In certain embodiments, injectable formulations (e.g., solutions, dispersions, suspensions, emulsions) will be preferred, for example, for administration to a joint (e.g., knee), an intervertebral disc, the urinary system, or the vocal cord.

B3806550.2 - 54 - Atty. Docket No. BUX-008.25

Injectable formulations can also be used for certain reconstruction or cosmetic procedures. Other procedures may alternatively use gels, lotions, creams, ointments, plasters, bandages, sheets, foams, films, sponges, dressings, or bioadsorbable patches that can be applied to the area in need of treatment.

Physiologically acceptable carriers, vehicles, and/or excipients for use with pharmaceutical compositions of the present invention can be routinely selected for a particular use by those skilled in the art. These include, but are not limited to, solvents, buffering agents, inert diluents or fillers, suspending agents, dispersing or wetting agents, preservatives, stabilizers, chelating agents, emulsifying agents, anti-foaming agents, ointment bases, penetration enhancers, humectants, emollients, and skin protecting agents.

Examples of solvents include water, Ringer's solution, U.S.P., isotonic sodium chloride solution, alcohols, vegetable, marine and mineral oils, polyethylene glycols, propylene glycols, glycerol, and liquid polyalkylsiloxanes. Inert diluents or fillers may be sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate.

Examples of buffering agents include citric acid, acetic acid, lactic acid,

hydrogenophosphoric acid, and diethylamine. Suitable suspending agents include, for example, naturally-occurring gums (e.g., acacia, arabic, xanthan, and tragacanth gum), celluloses (e.g., carboxymethyl-, hydroxyethyl-, hydroxypropyl-, and

hydroxypropylmethylcellulose), alginates and chitosans. Examples of dispersing or wetting agents are naturally-occurring phosphatides (e.g., lecithin or soybean lecithin),

condensation products of ethylene oxide with fatty acids or with long chain aliphatic alcohols (e.g., polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate).

Preservatives may be added to a pharmaceutical composition of the present invention to prevent microbial contamination that can affect the stability of the formulation and cause infection in the patient. Suitable examples of preservatives include parabens (such as methyl-, ethyl-, propyl-, p-hydroxy-benzoate, butyl-, isobutyl- and isopropyl- paraben), potassium sorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDM hydantoin, iodopropylnyl butylcarbamate, benzalconium chloride, cetrimide, and benzylalcohol. Examples of chelating agents include sodium EDTA and citric acid.

B3806550.2 - 55 - Atty. Docket No. BUX-008.25

Examples of emulsifying agents are naturally-occurring gums, naturally-occurring phosphatides (e.g., soybean lecithin, sorbitan mono-oleate derivatives), sorbitan esters, monoglycerides, fatty alcohols, and fatty acid esters (e.g., triglycerides of fatty acids). Anti-foaming agents usually facilitate manufacture, they dissipate foam by destabilizing the air-liquid interface and allow liquid to drain away from air pockets. Examples of anti- foaming agents include simethicone, dimethicone, ethanol, and ether.

Examples of gel bases or viscosity-increasing agents are liquid paraffin, polyethylene, fatty oils, colloidal silica or aluminum, glycerol, propylene glycol, carboxyvinyl polymers, magnesium-aluminum silicates, hydrophilic polymers (such as, for example, starch or cellulose derivatives), water-swellable hydrocolloids, carragenans, hyaluronates, and alginates. Ointment bases suitable for use in the pharmaceutical compositions of the present invention may be hydrophobic or hydrophilic; and specific examples include paraffin, lanolin, liquid polyalkylsiloxanes, cetanol, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids, polyethylene glycols, and condensation products between sorbitan esters of fatty acids, ethylene oxide (e.g., polyoxyethylene sorbitan monooleate), and polysorbates.

Examples of humectants are ethanol, isopropanol glycerin, propylene glycol, sorbitol, lactic acid, and urea. Suitable emollients include cholesterol and glycerol.

Examples of skin protectants include vitamin E, allatoin, glycerin, zinc oxide, vitamins, and sunscreen agents.

In certain embodiments, pharmaceutical compositions of the present invention may, alternatively or additionally, comprise other types of excipients including, thickening agents, bioadhesive polymers, and permeation enhancing agents.

Thickening agents are generally used to increase viscosity and improve bioadhesive properties of pharmaceutical compositions. Examples of thickening agents include, but are not limited to, celluloses, polyethylene glycol, polyethylene oxide, naturally occurring gums, gelatin, karaya, pectin, alginic acid, and povidone. In certain embodiments, a thickening agent is selected for its thioxotropic properties (i.e., has a viscosity that is decreased by shaking or stirring). The presence of such as an agent in a pharmaceutical composition allows the viscosity of the composition to be reduced at the time of

B3806550.2 - 56 - Atty. Docket No. BUX-008.25 administration to facilitate its application, e.g., to a skin area to be repaired, and to increase after application so that the composition remains at the site of administration.

Permeation enhancing agents are vehicles containing specific agents that affect the delivery of active components through the skin. Permeation enhancing agents are generally divided into two classes: solvents and surface active compounds (amphiphilic molecules). Examples of solvent permeation enhancing agents include alcohols (e.g., ethyl alcohol, isopropyl alcohol), dimethyl formamide, dimethyl sulfoxide, 1 dodecylazocyloheptan-2- one, N-decyl-methylsulfoxide, lactic acid, N,N-diethyl-m-toluamide, N-methyl pyrrolidone, nonane, oleic acid, petrolatum, polyethylene glycol, propylene glycol, salicylic acid, urea, terpenes, and trichloroethanol. The surfactant permeation enhancing agent in the present inventive pharmaceutical compositions may be nonionic, amphoteric, cationic, anionic, or zwitterionic. Suitable nonioinic surfactants include poly(oxyethylene)-poly(oxypropylene) block copolymers, commercially known as poloxamers; ethoxylated hydrogenated castor oils; polysorbates, such as Tween 20 or Tween 80. Amphoteric surfactants include quaternized imidazole derivatives, cationic surfactants include cetypyridinium chloride, cationic surfactants include "soap" (fatty acid), alkylsulfonic acid salts (the main component of synthetic detergent, such as linear alkyl benzene sulfonate (LAS)), fatty alcohol sulfate (the main component of shampoo or old neutral detergents), and zwitterionic surfactants include the betaines and sulfobetaines.

Injectable or topical formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, GAMA irradiation sterilization, E-Beam irradiation sterilization or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

For the topical application onto skin, a wound or a mucous membrane the composition comprising compounds of the present invention is preferably prepared in the form of an emulsion, a gel, an ointment, a foam, a band-aid, a cream of a mixed-phase and amphiphilic, respectively emulsion system (oil/water-water/oil-mixed-phase), a liposome or transferosome. These medicinal formulations are known in the prior art and the skilled practitioner can prepare compounds of the present invention without undue burden as a composition having one of those medicinal formulations. In an especially preferred

B3806550.2 - 57 - Atty. Docket No. BUX-008.25 embodiment, the composition is prepared in form of a cream, especially basis cream DAC (Deutsche Arzneimittel Codex) Basiscreme.

Further formulations, which can be topically applied are powders, pastes or solutions. Pastes often comprise as a base component lipophilic and hydrophilic additives with high solid content to provide consistency. The powders, in particular topically applied powders, can comprise for the increase the dispersity as well as the fluidity and the slideability as well as for the prevention of agglomerates, starches like wheat or rice starch, flame dispersion silicon dioxide and/or silica. These additives can also function as diluent.

In certain embodiments of the present invention the compositions of the present invention comprising an ionic liquid are prepared as an ointment, a gel, a band-aid, an emulsion, a lotion, a foam, a cream of mixed-phase or amphiphilic emulsion systems (oil- water/water-oil mixed phase), a liposome, a transferosome, a paste, or a powder.

Particular suitable adjuvants and carriers, respectively, for the preparation of topically applied compositions of the present invention are for example sodium alginate as gel-forming agent for the production of a suitable base or cellulose derivatives like, e.g. guar or xanthane gum, inorganic gel-forming agents like, e.g. aluminium hydroxide or betonite (so called thixotrope gel-forming agent), polyacrylic acid derivatives,

polyvinylpyrrolidone, microcrystalline cellulose or carboxymethyl cellulose. Furthermore biocompatible polyoxameres can be used which form a thermoreversible gel. Furthermore phospholipids or amphiphilic low or high molecular weight compounds can be considered. The gels can either be hydrogels based on water or hydrophobic organogels, for example, on the basis of mixtures of lower and higher molecular weight paraffin carbohydrates and Vaseline.

Skin soothing and/or anti-inflammation additives known to someone of skill in the art like, for example, synthetically produced substances and/or abstracts and/or substances from medicinal plants in particular bisobolol and panthenol can also be added to the composition. Furthermore coloring agents like, for example, yellow and/or red ferrous oxide and/or titanium dioxide for the adjustment of color and/or fragrances can be added to the composition.

In addition the compositions usable according to the present invention can comprise emulsifying agents. Suitable emulsifying agents are neutral, anionic or cationic tensides, for

B3806550.2 - 58 - Atty. Docket No. BUX-008.25 example alkali soaps, metal soaps, amine soaps, sulfurated and sulfonated compounds, invert soaps, long-chain fatty alcohols, partial fatty acid ester of sorbitans and

polyoxyethylene sorbitans, e.g. lanette-types, woolwax, lanoline or other synthetic products, which are suitable for the production of oil/water and/or water/oil emulsions. Hydrophilic organogels can be prepared, for example, on the basis of high molecular weight polyethylene glycols. This gel-type formulations are washable. Employed as lipids in the form of fatty and/or oily and/or waxy components for the preparation of ointments, crimes or emulsions are Vaseline, natural and/or synthetic waxes, fatty acids, fatty alcohols, fatty acid esters, e.g. as mono-, di- or triglycerides, paraffin oils or vegetable oils, hardened castor oils or coconut oils, lard, synthetic fats, e.g. on the basis of caprylic, caprinic, lauric and stearic acid or mixtures of triglycerides.

To adjust pH values it is possible to use osmotically effective acids and bases, e.g. hydrochloric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, further buffer-systems like, e.g. citrate, phosphate, Tris buffer, or

triethanolamine. Furthermore the stability can be improved by the addition of preservatives like, e.g. methyl or propylene benzoate (parabene) or sorbic acid.

Additional Bioactive Agents

In certain embodiments, the aforementioned ionic liquid(s) is(are) the only active ingredient(s) in an inventive pharmaceutical composition. In other embodiments, the pharmaceutical composition further comprises one or more bioactive agents. In certain embodiments, a bioactive agent may be associated with the ionic liquid. Alternatively or additionally, a bioactive agent may be added to the composition of polymer and does not form any associations with the polymer.

As will be appreciated by one skilled in the art, selection of one or more bioactive agents as component(s) of an inventive pharmaceutical composition will be based on the intended purpose of the pharmaceutical composition (e.g., use in viscosupplementation in the treatment of joints, use as viscoelastics in cataract surgery, use as tissue space fillers for cosmetic procedures, treatment of urinary incontinence or treatment of vocal cord problems, or use as anti-adhesives for wound care).

In general, the amount of bioactive agent present in a pharmaceutical composition will be the ordinary dosage required to obtain the desired result through local

B3806550.2 - 59 - Atty. Docket No. BUX-008.25 administration. Such dosages are either known or readily determined by the skilled practitioner in the pharmaceutical and/or medical arts.

Examples of bioactive agents that can be present in a pharmaceutical composition of the present invention include, but are not limited to, analgesics, anesthetics, pain-relieving agents, antimicrobial agents, antibacterial agents, antiviral agents, antifungal agents, antibiotics, anti-inflammatory agents, antioxidants, antiseptic agents, antipruritic agents, immunostimulating agents, and dermatological agents. Specific examples of suitable bioactive agents are provided and discussed below.

Anti-infective agents for use in pharmaceutical compositions of the present invention are compounds, molecules or drugs which, when administered locally, have an anti-infective activity (i.e., they can decrease the risk of infection; prevent infection; or inhibit, suppress, combat or otherwise treat infection). Anti-infective agents include, but are not limited to, antiseptics, antimicrobial agents, antibiotics, antibacterial agents, antiviral agents, antifungal agents, anti-protozoan agents, and immunostimulating agents.

Antibiotics and other antimicrobial agents may be selected from the group consisting of bacitracin; the cephalosporins (such as cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine, cefaclor, cefamandole, cefonicid, ceforanide, cefoxitin, cefuroxime, cefoperazone, cefotaxime, cefotetan, ceftazidime, ceftizoxime, ceftriaxone, and meropenem); cycloserine; fosfomycin, the penicillins (such as amdinocillin, ampicillin, amoxicillin, azlocillin, bacamipicillin, benzathine penicillin G, carbenicillin, cloxacillin, cyclacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin); ristocetin; vancomycin; colistin; novobiocin; the polymyxins (such as colistin, colistimathate, and polymyxin B); the aminoglycosides (such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, spectinomycin, streptomycin, and tobramycin), the tetracyclines (such as demeclocycline, doxycycline, methacycline, minocycline, and oxytetracycline); carbapenems (such as imipenem);

monobactams (such as aztreonam); chloramphenicol; clindamycin; cycloheximide; fucidin; lincomycin; puromycin; rifampicin; other streptomycins; the macrolides (such as erythromycin and oleandomycin); the fluoroquinolones; actinomycin; ethambutol; 5- fluorocytosine; griseofulvin; rifamycins; the sulfonamides (such as sulfacytine,

sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfamethizole, and sulfapyridine); and trimethoprim.

B3806550.2 - 60 - Atty. Docket No. BUX-008.25

Other antibacterial agents include, but are not limited to, bismuth containing compounds (such as bismuth aluminate, bismuth subcitrate, bismuth subgalate, and bismuth subsalicylate); nitrofurans (such as nitrofurazone, nitrofurantoin, and furazolidone);

metronidazole; tinidazole; nimorazole; and benzoic acid.

Antiseptic agents may be selected from the group consisting of benzalkonium chloride, chlorhexidine, benzoyl peroxide, hydrogen peroxide, hexachlorophene, phenol, resorcinol, and cetylpyridinium chloride.

Definitions

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the

B3806550.2 - 61 - Atty. Docket No. BUX-008.25 inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.

This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as

"comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

B3806550.2 - 62 - Atty. Docket No. BUX-008.25

The term "ionic liquid" or "IL" as used herein means a salt or hydrate thereof with a melting point less than about 300 °C. In a preferred embodiment, the ionic liquid has a melting point of less than about 200 °C. In a preferred embodiment, the ionic liquid has a melting point of less than about 150 °C. In a preferred embodiment, the ionic liquid has a melting point of less than about 100 °C. In a preferred embodiment, the ionic liquid has a melting point of less than about 50 °C. In a preferred embodiment, the ionic liquid has a melting point of less than about room temperature. The ionic liquids of the present invention may comprise one or more compounds. Thus, the ionic liquid may be a pure compound or may be a mixture of compounds. Each compound comprises an anion or a mixture of anions; and a cation or a mixture of cations. Ionic liquids, when used herein, includes both ionic liquids as described above as well as "ionic viscoelastics".

As used herein, a "viscoelastic" material is a liquid (or solid) with both viscous and elastic properties. A viscoelastic liquid will deform and flow under the influence of an applied shear stress, but when the stress is removed the liquid will slowly recover from some of the deformation.

As used herein, the term "nucleophile" is recognized in the art, and means a chemical moiety having a reactive pair of electrons. Examples of nucleophiles include uncharged compounds such as water, amines, mercaptans and alcohols, and charged moieties such as alkoxides, thiolates, carbanions, and a variety of organic and inorganic anions. Illustrative anionic nucleophiles include simple anions such as hydroxide, azide, cyanide, thiocyanate, acetate, formate or chloroformate, and bisulfite. Organometallic reagents such as organocuprates, organozincs, organolithiums, Grignard reagents, enolates, acetylides, and the like may, under appropriate reaction conditions, be suitable

nucleophiles. Hydride may also be a suitable nucleophile when reduction of the substrate is desired. Further examples of nucleophiles can be found in Figure 1.

As used herein, the term "electrophile" is art-recognized and refers to chemical moieties which can accept a pair of electrons from a nucleophile as defined above.

Electrophiles useful in the method of the present invention include cyclic compounds such as epoxides, aziridines, episulfides, cyclic sulfates, carbonates, lactones, lactams and the like. Non-cyclic electrophiles include sulfates, sulfonates (e.g., tosylates), chlorides, bromides, iodides, and the like. Further examples of electrophiles can be found in Figure 1.

B3806550.2 - 63 - Atty. Docket No. BUX-008.25

The terms "electrophilic atom", "electrophilic center" and "reactive center" as used herein refer to the atom of the substrate which is attacked by, and forms a new bond to, the nucleophile. In most (but not all) cases, this will also be the atom from which the leaving group departs.

The term "diene" refers to hydrocarbons which contain two carbon-carbon double bonds. The term "dienophile" refers to a substituted alkene which can combine with a diene in a Diels-Alder reaction to form a six-membered ring.

A 1,3-dipolar cycloaddition is the reaction between a 1,3-dipole and a dipolarophile, most of which are substituted alkenes or alkynes, to form a five-membered ring. Examples of 1 ,3-dipolar cycloadditions include the azide-alkyne cycloadditions (azides and alkynes); diazo-alkene cycloadditions (diazo compounds and alkenes); nitrone-alkene (nitrones and alkenes). A "1 ,3-dipole" is a type of organic compound with a three-atom pi-electron system containing 4 electrons delocalized over three atoms. They are reactants in 1 ,3- dipolar cycloadditions (with dipolarophiles). Known 1,3-dipoles include, azides, ozone, nitro compounds, diazo compounds, azoxide compounds, carbonyl oxides, nitrile oxides, nitrous oxide, nitrones, azomethine imine, nitrilimines, carbonyl imines, azomethine ylide. nitrile ylide and carbonyl ylide.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl), branched-chain alkyl groups (e.g., i-propyl, i-butyl, t-butyl), cycloalkyl (alicyclic) groups (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. For example, Ci_6 alkyl means a straight or branched alkyl chain containing from 1 to 6 carbon atoms; examples of such group include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, 3 -methyl-butyl, hexyl and 2,3-dimethylbutyl and like. Similarly, the term Ci_ alkyl means a straight or branched

B3806550.2 - 64 - Atty. Docket No. BUX-008.25 alkyl chain containing from 1 to 4 carbon atoms. Likewise, C_4-10 cycloalkyls have from 4- 10 carbon atoms in their ring structure.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The term "alkylene," is art-recognized, and as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated. Examples of linear saturated Ci_i₀alkylene groups include, but are not limited to, -(CH₂)_n- where n is an integer from 1 to 10, for example, - CH₂- (methylene), -CH₂CH₂- (ethylene), -CH₂CH₂CH₂- (propylene), -CH₂CH₂CH₂CH₂- (butylene), -CH₂CH₂CH₂CH₂CH₂- (pentylene) and -CH₂CH₂CH₂CH₂CH₂CH₂- (hexylene). Examples of branched saturated Ci_i₀alkylene groups include, but are not limited to, -CH(CH₃)-, -CH(CH₃)CH₂-, -CH(CH₃)CH₂CH₂-, -CH(CH₃)CH₂CH₂CH₂-, - CH₂CH(CH₃)CH₂-, -CH₂CH(CH₃)CH₂CH₂-, -CH(CH₂CH₃)-, -CH(CH₂CH₃)CH₂-, and - CH₂CH(CH₂CH₃)CH₂-. Examples of linear partially unsaturated Ci_ioalkylene groups include, but are not limited to,-CH=CH- (vinylene), -CH=CH-CH₂-, -CH=CH-CH₂-CH₂-, - CH=CH-CH₂-CH₂-CH₂-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH₂-, -CH=CH-CH=CH- CH₂-CH₂-, -CH=CH-CH₂-CH=CH-, and -CH=CH-CH₂-CH₂-CH=CH-. Examples of branched partially unsaturated Ci_ioalkylene groups include, but are not limited to, -C(CH₃)=CH-, -C(CH₃)=CH-CH₂-, and -CH=CH-CH(CH₃)-. Examples of alicyclic saturated Ci_ioalkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex- 1 ,4-ylene). Examples of alicyclic partially unsaturated Ci_ioalkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), and cyclohexenylene (e.g., 2-cyclohexen- 1 ,4-ylene, 3- cyclohexen-l,2-ylene, and 2,5-cyclohexadien-l,4-ylene).

The term "arylene," is art-recognized, and as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of an aromatic ring, as defined below for aryl (the corresponding monodentate moiety). The term "aralkylene," "heteroarylene," and

B3806550.₂ - 65 - Atty. Docket No. BUX-008.25 the like are likewise defined (i. e. the diradical of the corresponding monodentate moiety). The term "aralkylene" and the like encompass aryl groups substituted with two alkyl radicals, as well as bidentate moieties wherein one or both of the radicals are on the aryl ring. Arylene, aralkylene, heteroarylene are all subsets of alkylene.

The term "halo" designates -F, -CI, -Br or -I. The term "haloalkyl" refers to alkyl as defined above substituted with one are more halogen, where the halogen is a fluorine, chlorine, bromine or iodine atom. The term "perhaloalkyl" as used herein as a group or a part of a group refers to a straight or branched carbon chain containing the specified number of carbon atoms and the requisite number of halogens so that the resulting group is fully saturated. For example, Ci_6 perhaloalkyl means a straight or branched alkyl chain containing from 1 to 6 carbon atoms and from 3 to 13 halogens; examples of such group include trifluoromethyl, pentafluoroethyl, heptafluoropropyl, heptafluoroisopropyl and like. Similarly, the term Ci_4 perfluoroalkyl means a straight or branched alkyl chain containing from 1 to 4 carbon atoms and 3 to 9 fluorine atoms.

The term "aralkyl" is art-recognized and refers to an alkyl group substituted with an aryl group (as defined below). The term "heteroaralkyl" is art-recognized and refers to an alkyl group substituted with an heteroaryl group (as defined below).

The terms "alkenyl" and "alkynyl" refer to radicals of unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described herein, but which contain at least one double or triple carbon-carbon bond, respectively.

The term "carbonyl" is art-recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an -0-, -S- or -N(R ), and R represents a

pharmaceutically acceptable salt, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)_bR²⁰⁰, wherein b is 1-10 and R²⁰⁰ represents a group permitted by the rules of valence, such as hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl, and R¹⁰⁶ represents

B3806550.2 - 66 - Atty. Docket No. BUX-008.25 hydrogen, halo, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)_bR²°°, wherein b and R²⁰⁰ are defined above. Where X is an oxygen and R¹⁰⁵ or R¹⁰⁶ is not hydrogen, the formula represents an "ester" radical. Where X is an oxygen, and R¹⁰⁵ is as defined above, the moiety is referred to herein as a "carboxyl", and particularly when R¹⁰⁵ is a hydrogen, the formula represents a "carboxylic acid" radical. Where X is an oxygen, and R¹⁰⁶ is hydrogen, the formula represents a "formyl". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl". Where X is a sulfur and R¹⁰⁵ or R¹⁰⁶ is not hydrogen, the formula represents a "thioester" radical. Where X is a sulfur and R¹⁰⁵ is hydrogen, the formula represents a "thiocarboxylic acid" radical. Where X is a sulfur and R¹⁰⁶ is hydrogen, the formula represents a "thio formyl." On the other hand, where X is a bond, and R¹⁰⁵ is not hydrogen, the above formula represents a "ketone" radical. Where X is a bond, and R¹⁰⁶ is hydrogen, the above formula represents an "aldehyde" radical. Where X is a bond, and R¹⁰⁶ is halo, the above formula represents an "acyl halide" (e.g. -C(=0)C1 is an acyl chloride).

The term "thiocarbonyl" as used herein refers to carbonyls, as described above, wherein the C=0 is replaced with a C=S.

The term "oxime" is art-recognized and refers to a radical that can be represented by the general formula:

wherein R and R are as defined herein.

The term "amino" is art-recognized and as used herein refers to radicals of both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:

wherein R , R and R each independently represent hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -(CH2)bR²°°, wherein b and R²⁰⁰ are defined above.

B3806550.2 - 67 - Atty. Docket No. BUX-008.25

The term amino also includes "acylamino," which refers to a radical that represented by the general formula:

wherein R¹⁰¹ is as defined above, and R¹⁰⁴ represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)_bR²⁰°, wherein b and R²⁰⁰ are defined above.

The term "amido" as used herein, refers to a radical that can be represented by the general formula:

wherein R and R are as defined above. Preferred embodiments of the amide will not include those which are unstable.

The term "carbamoyl" as used herein, refers to a radical that can be represented by the general formula:

wherein R and R are defined above.

The term "imidyl" as used herein, refers to a radical that can be represented by the general formula:

B3806550.2 - 68 - Atty. Docket No. BUX-008.25

wherein R and R are defined above.

The term "phthalimidyl" as used herein, refers to a radical that can be represented by the general formula:

wherein R¹¹⁵ and R¹¹⁶ are hydrogen or taken together with the carbons to which they are bound represents alkylene, arylene, heteroarylene or cycloalkylene. When R¹¹⁵ and R¹¹⁶ are hydrogen, the moiety is referred to herein as "succinimidyl." When R¹¹⁵ and R¹¹⁶, taken together with the carbons to which they are bound, are -C=C-, the moiety is referred to herein as "maleimidyl." When R¹¹⁵ and R¹¹⁶, taken together with the carbons to which they are bound, are phenyl, the moiety is referred to herein as "isoindolinyl-l,3-dione."

The term "anhydridyl" as used herein, refers to a radical that can be represented by the general formula:

wherein R is defined above.

The term "hydroxyl" means -OH. The term "alkoxy", as used herein, refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an

B3806550.2 - 69 - Atty. Docket No. BUX-008.25 oxygen atom. Examples of "alkoxy" radicals as used herein include, but are not limited to, methoxy, ethoxy, propoxy, prop-2-oxy, butoxy, but-2-oxy, 2-methylprop-l-oxy and 2- methylprop-2-oxy. The terms "aryloxy", "heteroaryloxy", "aralkyloxy" and

"heteroaralkyloxy" are likewise defined.

The term "oxo" means =0.

The term "nitro" means -N0₂.

The term "cyano" means -C≡N. The term "isocyano" means "-N≡C".

The term "isocyanate" means "-N=C=0". The term thioisocyanate means "- N=C=S".

The term "azide" means "-N-N≡N". The term "acyl azide" means "-C(=0)N-

N≡N".

The term "hydrazine" as used herein, refers to a radical that can be represented by the general formula:

wherein R¹⁰¹, R¹⁰² and R¹⁰³ are as defined above.

The term "sulfhydryl" means -SH. The term "alkylthio", as used herein, refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an sulfur atom (i.e., an alkyl sulfenyl group). The terms "arylthio", "heteroarylthio",

"aralkylthio" and "heteroaralkylthio" are likewise defined.

The term "sulfoxido" as used herein, refers to a radical that can be represented by the general formula:

B3806550.2 - 70 - Atty. Docket No. BUX-008.25 wherein R¹¹² represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH2)bR²⁰⁰, wherein b and R²⁰⁰ are defined above.

The term "sulfonyl", as used herein, refers to a radical that can be represented by the general formula:

wherein R¹¹¹ represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)_bR wherein b and R²⁰⁰ are defined above.

The term "sulfonyloxy" is art-recognized and includes a radical that can be represented by the general formula:

in which R¹⁰⁹ represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)_bR² wherein b and R²⁰⁰ are defined above.

The term "oxysulfonyl" is art-recognized and includes a radical that can be represented by the general formula:

in which R¹⁰⁷ is an electron pair, hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)_bR²⁰⁰, wherein b and R²⁰⁰ are defined above.

The term "sulfonylamino" is art-recognized and includes a radical that can be represented by the general formula:

B3806550.2 - 71 - Atty. Docket No. BUX-008.25

in which R¹⁰⁸ and R¹⁰⁹ independently represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)bR²⁰⁰, wherein b and R²⁰⁰ are defined above.

The term "sulfamoyl" is art-recognized and includes a radical that can be represented by the general formula:

wherein R¹¹⁰ independently for each occurrence represents hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl or -(CH₂)bR²⁰⁰, wherein b and R²⁰⁰ are defined above.

The term "carbocyclyl" is art-recognized and refers to univalent radical formed by removing a hydrogen atom from an benzene, napthalene, antracene or cycloalkane. Each of the rings of the carbocyclyl may be substituted with any of the radicals described herein.

The term "polycyclyl" is art-recognized and refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle may be substituted with any of the radicals described herein.

The term "aryl" refer to 5 to 10-membered mono-, bi- or tri-cyclic radicals (i.e., a univalent radical formed by removing a hydrogen atom from a benzene, napthalene or antracene). The aryl radical can be substituted at one or more ring positions with any of the radicals described herein.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur, phosphorus and selenium.

B3806550.2 - 72 - Atty. Docket No. BUX-008.25

The term "heteroaryl" refer to 5 to 10-membered mono-, bi- or tri-cyclic radicals which contain carbon atoms, hydrogen atoms, and one to four heteroatoms (i.e., a univalent radical formed by removing a hydrogen atom from a heteroaromatic compound). As is well known to those skilled in the art, heteroaryl rings have less aromatic character than their all- carbon counter parts. Thus, for the purposes of the invention, a heteroaryl group need only have some degree of aromatic character. Illustrative examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (l,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, isoxazolyl, and oxazolyl. The heteroaryl radical can be substituted at one or more ring positions with any of the radicals described herein.

The term "heterocyclyl" refers to 3 to 10-membered radical ring structures which contain one to four heteroatoms (i.e., univalent radicals formed by removing a hydrogen atom from a ring of a heterocyclic compound). Heterocyclic compounds include thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with any of the radicals described herein. "Non-aromatic heterocycyl" refers to

heterocyclyl radicals which are not heteroaryl radicals.

The term "silyl" is used herein includes hydrocarbyl derivatives of the silyl (H₃Si-) group (i.e., (hydrocarbyl^Si-), wherein a hydrocarbyl groups are univalent groups formed by removing a hydrogen atom from a hydrocarbon, e.g. ethyl, phenyl. The hydrocarbyl groups can be combinations of differing groups which can be varied in order to provide a number of silyl groups, such as trimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS), tert- butyldimethylsilyl (TBS/TBDMS), triisopropylsilyl (TIPS), and [2- (trimethylsilyl)ethoxy]methyl (SEM).

As used herein, the term "substituted" is contemplated to include all permissible number and types of substituents of organic compounds (e.g., monosubstituted,

B3806550.2 - 73 - Atty. Docket No. BUX-008.25 disubstituted, trisubstituted, tetrasubstituted, and the like). In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described hereinabove (such as alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, oxo, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, and heterocyclyl). The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and

nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

"Small molecule" is an art-recognized term. In certain embodiments, this term refers to a molecule which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu.

The term "macromolecule" as used herein refers to the four conventional biopolymers (nucleic acids, proteins, carbohydrates, and lipids), as well as non-polymeric molecules with large molecular mass such as macrocycles. Macromolecules are synthesized through the process of polymerization, during which monomers are assembled into macromolecules. An example of a macromolecule is an enzyme.

B3806550.2 - 74 - Atty. Docket No. BUX-008.25

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and ^-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or

chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

B3806550.2 - 75 - Atty. Docket No. BUX-008.25

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, "Handbook of Chemistry and Physics", 67th Ed., 1986-87, inside cover.

The term "microorganism" as used herein refers to disease causing or contributing bacteria (including Gram-negative and Gram-positive organisms, such as Staphylococci sps. {e.g. Staphylococcus aureus, Staphylococcus epidermis), Enterococcus sp. (E.

faecalis), Pseudomonas sp. (P. aeruginosa), Escherichia sp. (E. coli), Proteus sp. (P. mirabilis), fungi (including Candida albicans and Fusarium), viruses, protists, parasites, or other infective agents capable of causing disease in humans or non-human animals. It follows that antibiotics and antifungals are examples of antimicrobial agents.

The term 'Gram-positive bacteria' is an art recognized term for bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure. Representative Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium,

Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,

Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, and Streptococcus sanguis.

B3806550.2 - 76 - Atty. Docket No. BUX-008.25

The term "Gram-negative bacteria" is an art recognized term for bacteria characterized by the presence of a double membrane surrounding each bacterial cell.

Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pestis.

"Medical device" refers to a non-naturally occurring object that may be, is or has been inserted or implanted in a subject or applied to a surface of a subject. Medical devices can be made of a variety of biocompatible materials, including: metals, ceramics, polymers, gels and fluids not normally found within the human body. Examples of polymers useful in fabricating medical devices include such polymers as silicones, rubbers, latex, plastics, polyanhydrides, polyesters, polyorthoesters, polyamides, polyacrylonitrile, polyurethanes, polyethylene, polytetrafluoroethylene, polyethylenetetraphthalate and polyphazenes.

Medical devices can also be fabricated using certain naturally-occurring materials or treated naturally-occurring materials. As an example, a heart valve can be fabricated by combining a treated porcine heart valve with an affixation apparatus using artificial materials. Medical devices can include any combination of artificial materials, combinations selected because of the particular characteristics of the components. For example, a hip implant can include a combination of a metallic shaft to bear the weight, a ceramic artificial joint and a polymeric glue to affix the structure to the surrounding bone. An implantable device is one intended to be completely imbedded in the body without any structure left outside the body

B3806550.2 - 77 - Atty. Docket No. BUX-008.25

(e.g. heart valve). An insertable device is one that is partially imbedded in the body but has a part intended to be external (e.g. a catheter or a drain). Medical devices can be intended for short-term or long-term residence where they are positioned. A hip implant is intended for several decades of use, for example. By contrast, a tissue expander may only be needed for a few months, and is removed thereafter. Insertable devices tend to remain in place for shorter times than implantable devices, in part because they come into more contact with microorganisms that can colonize them.

An "implant" is any object intended for placement in a human body that is not a living tissue. Implants include naturally derived objects that have been processed so that their living tissues have been devitalized. As an example, bone grafts can be processed so that their living cells are removed, but so that their shape is retained to serve as a template for ingrowth of bone from a host. As another example, naturally occurring coral can be processed to yield hydroxyapatite preparations that can be applied to the body for certain orthopedic and dental therapies. An implant can also be an article comprising artificial components. The term "implant" can be applied to the entire spectrum of medical devices intended for placement in a human body.

While several embodiments of the present invention are described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles,

B3806550.2 - 78 - Atty. Docket No. BUX-008.25 materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 ~ Synthesis of a Dicationic Phosphoniuni Ionic Liquid

Trihexylphosphine (19.14 g, 94.7 mmol) and 1,10-dichlorodecane (10 g, 47.4 mmol) were mixed together and heated to 140 °C for 24 hours. Next, the mixture was placed under vacuum at 140 °C to remove any volatile components. A clear colorless liquid was obtained in 99% yield. ¾ NMR (CDC1₃): δ 0.89 (m, 18, C¾); 1.30-1.72 (br, 64, CH₂- CH₂); 2.38-2.52 (br, 16, CH₂-P). ¹ C NMR (CDC1₃): δ 14.52-14.60 (CH₃); 19.48-22.99 (CH₂); 30.98-31.89 (CH₂-P). ¹P NMR (CDC1₃): δ 33.92 (P⁺). ES MS: 747.68 m/z [MCI]^" (theory: 784.12 m/z [M]⁺). Elemental analysis: (theory: C, 70.46; H, 12.60) found C, 70.34; H, 12.40.

Following the above procedure, we have prepared a series of dicationic

phosphonium molecules (shown below). All of the compounds were characterized by ¹H, ¹³C, ³¹P NMR and were shown to be pure by elemental analysis.

B3806550.₂ - 79 - Atty. Docket No. BUX-008.25

Example 2 ~ Synthesis of a Monocationic Phosphonium Ionic Liquid

Trihexylphosphine (8.3 g, 29 mmol) and 1 -chlorodecane (5.22 g, 29.6 mmol) were mixed together and heated to 140 °C for 24 hours. Next, the mixture was placed under vacuum at 140 °C to remove any volatile components. A clear colorless liquid was obtained in 99% yield. 'H NMR (CDC1₃): δ 0.78 and 0.797 (t, 12, CH₃); 1.12-1.26 (br, 24, G¾); 1.35-1.50 (br, 16, CH₂-CH₂-P); 2.30-2.38 (br, 8, C¾-P). ¹³C NMR (CDC1₃): δ 13.71 and 13.87 (CH₃); 19.27 and 18.80 (CH₂-CH₃); 21-22 (CH₂); 28.75-31.60 (CH₂-P). ³¹P NMR (CDC1₃): δ 35.46 (P⁺). ES MS: 427.44 m/z [MCI]^" (theory: 463.20 m/z [M]⁺).

Elemental analysis: (theory: C, 72.60; H, 13.20) found C, 72.53; H, 13.18.

Example 3— Anion Exchanges

A. (CH₃CH₂CH₂CH₂CH₂CH₂)₃P⁺-C₁₀H₂o-P⁺(CH₃CH₂CH₂CH₂CH₂CH₂)3,2Cl (lg, 1.6 mmol) and H0₂C-CioH₂o-C0₂H (373 mg, 1.6 mmol) were mixed together at 80 °C under vacuum for 2h to lead to a clear colorless viscous oil.

B. (CH₃CH₂CH₂CH₂CH₂CH₂)₃P⁺-CioH₂₀-P⁺(CH₂CH₂CH₂CH₂CH₂CH₃)₃,2Cr (lg, 1.6 mmol) and EDTA (237 mg, 0.8 mmol) were mixed together at 80 °C under vacuum for

2h to lead to a clear colorless viscous oil.

C. (CH₃CH₂CH₂CH₂CH₂CH₂)₃P⁺-C ₁₀H₂₀-P⁺(CH₂CH₂CH₂CH₂CH₂CH₃)₃,2Cr ( 1 g, 1.3 mmol) and Polyacrylic Acid, 240,000 Mw at 25% in water (734 mg, 0.76 μηιοΐ) were mixed together at 100 °C under vacuum for 2h to remove water and lead to a clear colorless viscoelastic. The material formed from mixing (CH₃CH₂CH₂CH₂CH₂CH₂)₃P -CioH₂o- P⁺(CH₂CH₂CH₂CH₂CH₂CH₃)₃,2Cr with polyacrylic acid (240,000 Mw at 25% in water) can be pulled to form a fiber-like material.

D. (CH₃CH₂CH₂CH₂CH₂CH₂)₃P⁺-C₁₀H₂o-P⁺(CH₂CH₂CH₂CH₂CH₂CH₃)₃,2Cr (lg, 1.3 mmol) and H0₂C-C₈Fi₆-C0₂H (625 mg, 1.3 mmol) were mixed together at 80 °C under vacuum for 2h to lead to a waxy material.

E. (CH₃CH₂CH₂CH₂CH₂CH₂)₃P⁺-(CH₂)₃-NHCO-(CH₂)io-CONH-(CH₂)₃- P⁺(CH₂CH₂CH₂CH₂CH₂CH₃)₃,2Cr (lg, lmmol) and EDTA (153 mg, 0.5 mmol) were mixed together at 80 °C under vacuum for 2h to lead to a solid material.

F. (CH₃CH₂CH₂CH₂CH2CH2)₃P⁺-(CH₂)₉CH₃,Cr (lg, 2.15mmol) and EDTA (315 mg, 1.08 mmol) were mixed together at 80 °C under vacuum for 2h to lead to a liquid. The

B3806550.₂ - 80 - Atty. Docket No. BUX-008.25 low viscous liquid is significantly different that the ionic liquids prepared with the di- phosphonium as described above.

G. (CH₃CH₂CH₂CH₂CH₂CH₂)₃P⁺-C ioH₂₀-P⁺-(CH₂CH₂CH₂CH₂CH₂CH₃)3,2Cr (1 g, 1.3mmol) and tetraethyl ester of EDTA (257 mg, 0.65 mmol) were mixed together at 80 °C under vacuum for 2h to lead to a liquid. Again a low viscous liquid is formed due to the inability to form a network with the ester analog of EDTA and is significantly different that with EDTA.

Example 4 ~ Synthesis of Tri-n-butyl(carboxypropyl)phosphoniuni Chloride

In a glove box, 1 eq. of tributylphosphine (1.57 g, 7.76 mmol) was mixed with 1 eq. of 4-chlorobutyric acid (952 mg, 7.76 mmol) in a microwave tube. The tube was microwave irradiated for 15 min. at 80 °C, power 250W. Tri-n- butyl(carboxypropyl)phosphonium chloride was obtained in a 99% yield. ^LH NMR

(CDC1₃): δ 0.95 (t, 9H), 1.53 (m, 12H), 1.90 (m, 4H), 2.29-2.58 (m, 6H), 2.69 (t, 2H). ¹³C (CDC1₃):5 14.0?CH₃), 17.92-27.97 (CH₂), 35.09 (CH₂-CO), 174.65 (COOH). ¹P (CDC1₃): 5 21.40.

Example 5— Tri-n-butyl[carboxy( V-hydroxysuccinimide)propyl]phosphonium Chloride

At 0 °C, tri-n-butyl(carboxypropyl)phosphonium chloride (1.0 eq) was dissolved in dry DCM then 1.1 eq NHS (525 mg, 4.56 mmol), 1.1 eq DCC (939 mg, 4.56 mmol) and a catalytic amount of HOBT were successively added. The mixture was allowed to stir at room temperature for 10 hrs. DCU was filtered and the filtrate was added drop-wise to a cold ether solution. The oily residue obtained was collected, redissolved in DCM and the previous operation was repeated three times to afford tri-n-butyl[carboxy(N- hydroxysuccinimide)propyl]phosphonium chloride (1.50 g, 86%). 1H MR (CDC1₃): δ 0.95 (t, 9H), 1.53 (m, 12H), 2.11 (m, 2H), 2.39 (m, 6H), 2.66 (m, 4H), 2.87 (s, 2H), 2.94 (t, 2H). ¹ C (CDCb): δ 14.07 (CH3), 17.92-27.97 (CH2), 35.09 (CH2-CO), 174.65 (COOH). 1P (CDC1₃): δ 21.40. HRMS: 386.243 m/z (M) (theory: 386.246 m/z (M⁺)). Elemental analysis: (theory: C, 62.15; H, 9.65; N, 3.62; O, 16.56; P, 8.01) found C, 62.34; H, 9.15; N, 3.58; O, 16.99.

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Example 6— Procedure for the Synthesis of Ionic Liquids using Ionic Liquid Reagents

The nucleophile (e.g. an amine or thiol) is dissolved in dry DCM, 1 eq of TEA and 1.2 eq of the ionic liquid reagent are successively added. The mixture is allowed to stir for 12 hrs then the yields are determined by ^!H NMR (CDC1₃).

As shown in Figure 2, the primary alkyl and benzyl amines reacted with tri-n- butyl[carboxy(N-hydroxysuccinimide)propyl]phosphonium chloride in good to high yield (greater than about 80%). As expected, a lowering of the amine p a either through para substitution of benzyl amine with fluorine or the use of aniline instead of benzyl amine reduced the coupling yield. The reaction was not limited to amines, an alkyl thiol reacted readily with the ionic liquid reagent in high yield to afford the thioester linkage and the corresponding ionic liquid.

Example 7— Preparation of Trihexylphosphonium Alkyne Salts

A. Trihexylphosphine (30 mmol) and 5-chloro-l-pentyne (30 mmol) were mixed together and heated to 100 °C for one week under nitrogen. Next, the mixture was placed under vacuum at 140 °C to remove any volatile components. The solution was rinsed with hexane. A clear colorless liquid was obtained in greater than about 90% yield and the trihexylphosphonium alkyne salt was characterized by NMR.

B. Under nitrogen, 30 mmol of tributylphosphine was mixed with 30 mmol of 4- chloro-l-butyne. The mixture was stirred for 5 days at 90 °C to give an oily residue. The product latter was rinsed with hexane, then DCM was added and the resulting solution was added dropwise to cold Et₂0. The resulting precipitate was rapidly filtered to afford the final tributylphosphonium alkyne salt in greater than about 80% yield and was

characterized by NMR.

Example 8 ~ Huisgen Cycloaddition Using a Copper(I)-Catalyzed coupling of Azides and Terminal Alkynes

Phosphonium (trihexyl) pentyne (3 mmol) and (S)-3-azidopropane-l,2-diol (3 mmol) were suspended in a 1 : 1 mixture of water and tert-butyl alcohol (12 mL). Sodium ascorbate (0.3 mmol, 300 μί of freshly prepared solution in water) was added, followed by copper(I) sulfate pentahydrate (0.03 mmol, in 100 μΐ, of water). The heterogeneous mixture was stirred vigorously overnight, at which point it cleared and TLC analysis indicated

B3806550.2 - 82 - Atty. Docket No. BUX-008.25 complete consumption of the reactants. The reaction mixture was diluted with water (50 mL), cooled in ice, and the product was obtained as an oil.

Example 9— In vitro antibacterial activity

The minimal bacteriocidal concentration (MBC) activity of the diphosphonium dichlorides versus two model organisms, Staphylococcus aureus MZ100 and Escherichia coli W3110, both of which are well-characterized laboratory strains, as well as clinical isolates, was assessed. For all organisms, overnight TSB-grown cultures were diluted 1/100 into fresh TSB to a bacterial population of ~10⁷ colony forming units (CFU)/ml and exposed to the diphosphonium dichlorides at concentrations over a wide range (0.00032 - 5 mg/mL; 5 -fold serial dilutions) for ~16 hrs at 37 °C without shaking. After incubation, 2-3 \\L from each well was transferred to a TSA (trypticase soy agar) plate using a multi-prong device, and the plates were incubated overnight at 37 °C. The lowest concentration of antibiotic that resulted in complete loss of bacterial viability was assigned the MBC. The MBC determined for each compound is shown in Table 1 (below). The most effective compounds, abbreviated Di-Hex CIO and Di-Hex triazole, are highlighted in green. The clinically approved antibiotic Ciprofloxacin was used as a control and to serve as a reference. The diphosphonium dichlorides showed robust activity against both the Gram- positive and Gram-negative test organisms. In all cases, the minimal inhibitory concentration (MIC) of the diphosphonium dichloride was similar or identical to the MBC (data not shown). These data clearly establish that diphosphonium dichlorides have antimicrobial activity, and the antimicrobial activity varies with the structure and chemical properties of the compounds.

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Table 1. Activity versus lab strains.

aMBC=minimal bacteriocidal concentration, measured in TSB medium after ~16 hrs of exposure to antibiotics. Starting inoculum ~1 x 10⁷ CFU/imL

Example 10 ~ In vitro antibacterial activity against clinical strains

Table 2. Testing Di-Hex C10 for activity versus clinical isolates.

One isolate each of K. pneumonia and K. oxytoca.

bMBC calculated as for Table 1 .

°Methicillin resistant Staphylococcus aureus USA300 strains

Di-Hex CI O was also tested for its antimicrobial activity versus a wide range of clinical isolates (Table 2, above). Overall, the data showed that Di-Hex CIO had similar levels of antimicrobial activity versus the clinical isolates of S. aureus (n = 6) and S.

epidermidis (n = 2), and, significantly, versus four different community-acquired US A300 MRS A strains.

Additionally, Di-Hex C IO exhibited activity versus two different clinical isolates of the Gram-negative organisms Klebsiella that was similar to its activity versus Gram- positive organisms. Di-Hex C I O also demonstrated weak activity versus P. aeruginosa PA14, a well-known virulent laboratory strain.

Example 11— Kinetics of killing by the ILs.

The kinetics of killing by Di-Hex C IO was also assessed. These studies demonstrated how rapidly this agent kills the microbe and provides some insight into the

B3806550.2 - 84 - Atty. Docket No. BUX-008.25 mechanism by which these antimicrobial agents kill. Complete elimination of viable S. aureus MZ100 by 10 min (for technical reasons, this is the shortest exposure time tested) and E. coli W3110 was observed within 2 hrs when in the presence of Di-Hex CIO at a concentration of 40 μg/mL. These data suggest that for Gram-positive organisms, death is quite rapid, indicating perhaps that this antimicrobial agents target a pathway essential for cell viability, e.g., the plasma membrane.

Example 12— In vitro antifungal activity

Di-Hex CIO was tested for activity versus Candida albicans SC5314, a laboratory strain. Significantly, Di-Hex CIO showed robust activity versus this strain with an MBC = 12 μΜ.

Example 13— Cytotoxicity

A key property of any antimicrobial agent is that it should have minimal cytotoxic activity in the concentration range for which it displays antimicrobial activity. The cytotoxicity of the diphosphonium dichlorides was tested versus epithelial cells. In studies using a cell line of lung epithelial origin, no apparent cytoxicity (i.e., equivalent to vehicle control) was observed after exposure to 0.6 mM of Di-Hex CIO after 24 hrs of incubation (not shown).

Example 14— Surface Modification

Next the IL were attached to a surface using one of two methods. In the first, electrostatic interactions were used to assemble the IL on a Ti or Stainless Steel Surface. Both of these metals have an overall negative charge on their surface and thus the multicationic IL will adhere to the surface. In the second approach, the IL are modified to contain one or more thiol linkages and the thiol linkage is used to attached the IL to a gold surface.

Example 15 ~ Ionic liquid Dex CIO has low micromolar activity versus clinical bacterial strains, include antibiotic-resistant isolates

To determine the range of microbes against which Di-Hex CIO might be active, we determined the MBC for this compound versus clinical isolates of S. aureus (3 isolates), methicillin-resistant S. aureus (MRSA, 4 isolates of the USA300 strain), S. epidermidis (3 isolates), as well as the recent clinical isolate of Pseudomonas aeruginosa which is now

B3806550.2 - 85 - Atty. Docket No. BUX-008.25 widely used in the laboratory. Finally, we tested this compound for its activity versus a laboratory strain of a model fungal pathogen, Candida albicans.

As summarized in Table 3, Di-Hex CIO showed broad activity versus these clinical isolates, as well as activity versus P. aeruginosa. Of note, this compound has a MBC for C. albicans on par with that observed for Staphylococcus spp. Taken together, these data indicate that Di-Hex CIO has broad-spectrum activity versus bacteria, and may also be effective versus fungi.

Table 3. Testing Di-Hex C10 for activity versus clinical isolates.

Organism Tested # isolates MBC^a (mg/mL)

S. aureus clinical isolates 5 0.0016-0.04

S. epidermidis clinical isolates 3 0.0016-0.008

MRS A (USA300) 4 0.04

P. aeruginosa PA 14 1 0.2

Candida albicans 1 0.008

aMBC calculated as for Table 3

Example 16— Ionic liquid Dex CIO shows rapid bacterial killing

A desired feature of an antimicrobial agent is the ability to rapidly kill bacteria. Thus, we assayed the kinetics of killing for Dex CIO against S. aureus and E. coli at t = 0 (before addition of the antibiotic), then at 15 min, 30 min and 1 hr, 2 hr, 4 hr, 8 hr and 24 hr after addition of the compound. We find that Di-Hex CIO can kill S. aureus with an exposure time of less than 10 min at a concentration of, while 30 minutes is required to kill E. coli. Thus, for both Gram-positive and Gram-negative organisms, this IL can rapid result in bacterial cell death.

Example 17— Ionic liquid Dex CIO shows no obvious cytotoxicity in mouse corneas

As a first step to assess the efficacy of IL in vivo, we tested whether application to the eye resulted in any gross cytotoxicity. We applied the IL to the eye using a mouse model of corneal keratitis, in the absence of added bacteria, and assessed cytotoxicity by scoring opacity and epithelial damage, as well as histopathology of the treated organ.

B3806550.2 - 86 - Atty. Docket No. BUX-008.25

After scratching the mouse cornea, 9.6 μΜ Dex C-10 in PBS or PBS alone were applied, and the animals scored for corneal opacity and corneal epithelial damage at 24 hours. No corneal opacity or epithelial damage was observed for the Dex C-10-treated animals, and an identical finding was observed for the PBS-treated control (Figure 6A and 6B). Histological studies also revealed no difference between Dex C-10-treated and control PBS-treated animals in this analysis (Figure 6C). Taken together, these data show that a single treatment with low micromolar Dex C-10 does not negatively impact a wounded cornea.

Example 18— Ionic liquid Dex CIO protects against Pseudomonas aeruginosa infection in a mouse model of keratitis

We next assessed the efficacy of Dex C-10 against P. aeruginosa in the same mouse keratitis model.

Mice were anesthetized and positioned under an operating microscope. Three scratches were made in the corneal epithelium with a 25 gauge needle with the bevel positioned up. For the infection studies, 5 of the bacterial suspension of overnight- grown bacteria was resuspended at the desired bacterial concentration (~10⁵ CFU/5 μΤ) and was used as inoculum. To confirm the inoculum size, viable bacteria were determined from a small aliquot of the suspension by plating. For treatment conditions, IL, also in 5 μΐ_^ of PBS, was delivered to the infected eye at t=0 (the same time as infection). Infections were scored at days 1, 2, and 3 for opacity and epithelial damage. For epithelial damage, a score of 0 or 1 is assigned (0, intact corneal epithelium; 1, non-intact corneal epithelium). For opacity, scores ranging from 0-2 are assigned (0, no opacity; 1, partial opacity for 50% of the lesion; 2, partial opacity for 100% of the lesion). At the completion of the experiment, the animals were sacrificed, and treated eyes were either: i) enucleated for pathologic examination to test for cytotoxicity via histopathological study or ii) homogenized to determine bacterial burden by viable plating.

In this experiment, after wounding the eye, ~lxl0⁵ CFU of P. aeruginosa in 5 μΐ. PBS, was applied to the eye, followed immediately by application of 5 xL Dex C-10 at a final concentration of 9.6 μΜ in PBS, or the PBS vehicle control. Twenty-four animals later, the eyes were inspected visually, then enucleated and used for histopathological studies or to assess bacterial burden.

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As shown in Figure 7A, while 6/6 mice treated with the PBS control developed an infection by 24 hrs, only 2/6 mice treated with Dex C-10 developed an infection.

Representative infected and uninfected eyes are shown in Figure 7B. Quantitative analysis of epithelial cell damage and opacity revealed a statistically significant decrease in both these parameters (Figure 7C). Taken together, these data show that the IL Dex C-10 was capable of preventing infection by P. aeruginosa using a mouse model of keratitis without causing any detectable cytotoxicity.

Example 19— Efficacy of IL in an IP model of infection

The efficacy of Dex C-10 was tested in an intraperitoneal (IP) mouse model of infection. In the first experiment, S. aureus (~1 x 10⁶ CFU) was injected into mouse peritoneum. After ten minutes, the mice were injected either with Dex C-10 (9.6 micromolar) or the ethanol vehicle control. After thirty minutes later, the peritoneum was lavaged with 2 mL of saline, and bacterial viable counts determined. Using 2 mice per group, the results indicated that Dex C-10 completely eliminated the bacteria compared to ethanol (EtOH) vehicle control as shown in Figure 8.

A similar experiment was performed with P. aeruginosa. As shown in Figure 9, treatment with Dex C-10 markedly reduced the bacterial population in this IP infection model compared to vehicle control.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

B3806550.2 - 88 -

Claims

Atty. Docket No. BUX-008.25

An ionic liquid comprising an anionic component and a cationic component, wherein:

said anionic component is Y or X-[Y]_m;

m is 1-100 inclusive;

Y is selected, independently for each occurrence, from the group consisting of carboxylic acids, sulfonic acids, tetrafluoroborates, hexafluorophosphates, bis-trifluoromethane-sulfonimides, borates, phosphates, nitrates, sulfates, inflates, antimonates, phosphoniums, carboranes, poly-oxo metallates, and metalloboranes; said cationic component is W-[Z]_n;

n is 1-100 inclusive;

W is absent, or selected from the group consisting of linear or branched alkanes, linear or branched alkenes, linear or branched alkynes, monocyclic or polycyclic aromatics, monocyclic or polycyclic heteroaromatics, linear or branched polysilanes, linear or branched polyethylene glycols, linear or branched poly(propylene glycol), linear or branched polyalkylene oxides, linear or branched polysiloxanes, linear or branched polyacrylates, linear or branched polyacetals, linear or branched acrylics, linear or branched cellulosics, linear or branched polyethers, linear or branched halogenated polyethers, linear or branched halocarbons, linear or branched polyamides, linear or branched polycarbonates,

B3806550.2 - 89 - Atty. Docket No. BUX-008.25 linear or branched polyethylenes, linear or branched polypropylenes, linear or branched polystyrenes, and linear or branched polyurethanes, or a combination thereof;

Z is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-1,3- dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, R-N-R sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl, R ,

R , R , R , R , R , R , and R ;

R.

I ©

R-N-R

provided that at least one Z is selected from the group consisting of R

B3806550.2 - 90 - Atty. Docket No. BUX-008.25

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R -R ; any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7-membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

R^B is -C(=0)-, -C(=0)N(H>. -C(=0)0-, -C(=0)S-, arylene, heteroarylene or heterocyclylene;

R is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime,

B3806550.2 - 91 - Atty. Docket No. BUX-008.25 amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl or a macromolecule;

provided that exactly one R is a bond to W; and at least one R is alkylene-R^B-R^c.

2. An ionic liquid comprising an anionic component and a cationic component, wherein:

said anionic component is Y;

Y is selected, independently for each occurrence, from the group consisting of carboxylic acids, sulfonic acids, tetrafluoroborates, hexafluorophosphates, bis-trifluoromethane-sulfonimides, borates, phosphates, nitrates, sulfates, triflates, antimonates, phosphoniums, carboranes, poly-oxo metallates, and metalloboranes; said cationic component is Z;

Z is selected, independently for each occurrence, from the group consisting of

B3806550.2 - 92 - Atty. Docket No. BUX-008.25

R is a bond to W or is selected, independently for each occurrence, from the group consisting of hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl, silyl and alkylene-R^B-R^c; any two adjacent R, taken together with the atoms to which they are directly bound, may form a 5-membered, 6-membered, or 7-membered, saturated or unsaturated, carbocyclic or heterocyclic, ring; and

R^B is oxo, arylene, heteroarylene or heterocyclylene;

R is hydrogen, alkyl, halo, haloalkyl, perhaloalkyl, aralkyl, alkenyl, alkynyl, carbonyl, ester, carboxyl, carboxylic acid, formy, thiocarbonyl, thioester, thiocarboxylic acid, thioformyl, ketone, aldehyde, acyl halide, thiocarbonyl, oxime, amino, acylamino, amido, carbamoyl, imidyl, phthalimidyl, succinimidyl, maleimidyl, isoindolinyl-l,3-dione, anhydridyl, hydroxyl, alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, nitro, cyano, isocyano, isocyanate, thioisocyanate, azide, acyl azide, hydrazine, sulfhydryl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, sulfoxido, sulfonyl, sulfonyloxy, oxysulfonyl, sulfonylamino, sulfamoyl, carbocyclyl, polycyclyl, aryl, heteroaryl, heterocyclyl or silyl;)

provided that at least one R is alkylene-R^B-R^c.

3. An ionic liquid comprising an anionic component and a cationic component, wherein:

B3806550.2 - 93 - Atty. Docket No. BUX-008.25 said anionic component is Y;

R is alkyl;

R² is alkyl;

R³ is alkyl;

R⁴ is alkylene-R⁵;

4. A pharmaceutical composition comprising an ionic liquid of any one of claims 1-3; and a pharmaceutically acceptable excipient.

B3806550.2 - 94■ Atty. Docket No. BUX-008.25

5. A method of treating or preventing a microbial infection comprising the step of administering an effective amount of an ionic liquid of any one of claims 1-3, or a pharmaceutical composition of claim 4, to a patient in need thereof.

6. The method of claim 5, wherein the effective amount of an ionic liquid of any one of claims 1-3, or a pharmaceutical composition of claim 4, is administered topically.

7. The method of claim 5, wherein the effective amount of an ionic liquid of any one of claims 1-3, or a pharmaceutical composition of claim 4, is administered orally.

8. The method of claim 5, wherein said microbial infection is a bacterial infection.

9. The method of claim 5, wherein said microbial infection is a fungal infection.

10. The method of claim 5, wherein said microbial infection is drug resistant.

11. A medical device coated with an ionic liquid of any one of claims 1-3.

12. The medical device of claim 11 , wherein the device is an implant.

B3806550.2 - 95 -