WO2009058327A1 - Cyclodextrin derivatives as potentiators for antibiotics - Google Patents

Abstract

The invention provides a new class of antibiotics that are derivatives of cyclodextrin, which is a cyclic molecule comprising D-glucose units. In addition, the invention provides a method for potentiating the activity of antibiotic to inhibit the growth of a bacterium which is resistant to said antibiotic whereby cyclodextrin derivatives are administered with an antibiotic.

Description

CYCLODEXTRIN DERIVATIVES AS POTENTIATORS FOR

ANTIBIOTICS

(Attorney Docket No. PIN-006PC)

FIELD OF THE INVENTION

[001] The invention relates to cyclodextrin derivatives and their use as antibiotic and/or potentiators for antibiotics against pathogenic bacteria.

BACKGROUND OF THE INVENTION

[002] Numerous bacteria are known to cause diseases in humans. Among these bacteria are Enterococcus faecium, Eschericia coli, Pseudomonas aeruginosa, Bacillus atrophaeus, Staphylococcus aureus, Salmonella choleraesuis, Bacillus anthrasis, Pseudomonas aeruginosa, and many others. A disturbing recent trend has been the development of resistance to existing antibiotics in numerous pathogenic bacteria. [003] Conventional drugs consist of simple, fast-acting chemical compounds that are dispensed orally (as solid pills and liquids) or as injectables. Many of the pharmacological properties of these drugs can be improved through the use of drug delivery systems. These include soluble polymers, microparticles made of insoluble or biodegradable natural and synthetic polymers, microcapsules, lipoproteins, liposomes, micelles, etc. Drug delivery systems are designed to alter the pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity, biorecognition, and efficacy of their associated drugs, or to function as drug reservoirs (i.e., as sustained release systems), or both.

[004] In recent years, considerable efforts have been made to exploit cyclodextrins and their derivatives in different areas of drug delivery, particularly in protein and peptide delivery and gene delivery. Cyclodextrins are cyclic (α-l→4)-linked oligosaccharides of α-D- glucopyranose containing a relatively hydrophobic central cavity and a hydrophilic outer surface. Cyclodextrins are designated α, β and γ corresponding to 6, 7 or 8 glycopyranose units, with cavity diameters of 4.7-5.3, 6.0-6.5 and 7.5-8.3 A, respectively (Figure 1). Due to their unique ability to bind hydrophobic molecules within their cavity, cyclodextrins have been successfully applied in pharmaceutical formulations to enhance solubility, chemical stability and absorption of bioactive molecules. In addition, cyclodextrins are readily available at low cost, are not toxic, are nonimmunogenic, and exhibit an excellent pharmacokinetic profile.

[005] Bacterial resistance continues to develop and pose a significant threat both in hospitals and in the community. Although antibiotic therapy has considerably improved the clinical condition of patients and their quality of life, it often fails to lead to eradication of the pathogens. The decline of antibacterial research by many of the big pharmaceutical companies has led to a shortfall in new and better agents to fight the present threat of drug resistance. As a consequence, novel approaches to prevention and treatment are urgently required. Such approaches include the identification of novel agents active against previously unexploited targets or the discovery of novel entities to potentiate the activity of known antibiotics. The present invention involves the encapsulation of an antibiotic into a cyclodextrin derivative, thus permitting the facilitated transport of the antibiotic through bacterial cell membrane, and/or protection against degrading enzymes and efflux pumps. [006] Gram-negative bacterial infections are the cause of significant morbidity and mortality. Infections due to one such Gram negative organism, P. aeruginosa, are recognized by the medical community as particularly difficult to treat. For example chronic lung infection with Pseudomonas aeruginosa is responsible for the pulmonary deterioration and reduced life expectancy in patients with cystic fibrosis (CF). Once P. aeruginosa has taken residence in the CF lungs, it is rarely possible to eradicate it by antimicrobial chemotherapy. This problem created an urgent need for the development of new antibacterial agents that are directed towards novel targets. [007] Almost since the beginning of the antibiotic era, bacterial resistance has been major obstacle to successful treatment. Hardly any group of antibiotics has been introduced into clinical practice to which some bacterium has not developed resistance. The various antibiotic resistance mechanisms include alteration/modification of the target site, degradation of the antibiotic molecule and reduction of the intracellular antibiotic concentration as a result of reduced drug intake (low outer membrane permeability) or active drug export (efflux pumps). Since cyclodextrins are able to form inclusion complexes with a plethora of "guest" molecules, they can play a dual role in: i) increasing the stability of guest molecules, and ii) transporting drug molecules across membranes, due to their ability to recognize specific cellular receptors, such as lectins located on the bacterial cell surface. Moreover, recent studies showed that the inclusion of ampicillin in a cyclodextrin provided some protection against degradation by β-lactamase enzymes.

[008] Many antibiotics enter the Gram negative outer membrane through channel-forming proteins called porins. These are essential also for the uptake of hydrophilic molecules and nutrients across lipid bilayer membranes in Gram negative and some Gram positive bacteria. The porin superfamily contains a number of homotrimeric, transmembrane proteins that form water-filled pores across the outer cell membranes of gram-negative bacteria. A number of porins in their active form have been isolated and had their crystal structures determined. Among the best studied examples of substrate-specific porins is a maltoporin known as the LamB porin. It is responsible for the guided diffusion of maltose and maltodextrins into E. coli cells. Antibacterial resistance resulting from loss of a porin or from a mutation that lead to a narrowing of the porin channel have been reported.

[009] Recently the existence of a specific transport system for cyclodextrins in the bacterial cell wall has been reported (Cym transport system), initially in Klebsiella oxytoca. CymA encodes a "cycloporin", enabling cyclodextrins to penetrate the bacterial cell outer membrane. Cyclodextrins have also been shown to reversibly enter and modify the pore properties of CymA bacterial porin and fully block the ionic conductance of CymA pores.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 represents the structures of α, β and γ cyclodextrins.

[0011] Figure 2 represents partial ¹H NMR spectra of free doxycycline, free Compound No.

141, and 1 :1 Compound No. 141 -doxycycline complex in D₂O at 22 ºC.

[0012] Figure 3 represents a partial NOESY spectrum of Compound No. 141 :doxycycline

(1 :1) in D₂O at 22 ºC.

[0013] Figure 4 represents the HPLC profile of Compound No. 106.

[0014] Figure 5 represents the HPLC profile of a Compound No. 106 Iosmer.

[0015] Figure 6 represents the HPLC profile of a Compound No. 106 Isomer.

[0016] Figure 7 represents the HPLC profile of a Compound No. 138 Isomer.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] The invention relates to cyclodextrins as potentiators of known antibiotics against pathogenic bacteria, particularly those bacteria that have developed resistance to known antibiotics. The cyclodextrins are derivatives of α, β, or γ cyclodextrins. Particularly, the cyclodextrins of the present invention are β-cyclodextrins (β-CD), which are cyclic molecules comprising seven D-glucose units. Preferably, the β-CD derivatives are represented by General Formula 1.

wherein R₂ is independently H, OH, OAc, OMe, 0-lower alkyl, or 0(CH₂CH₂O)_n; R₃ is independently H, OH, OAc, OMe, O-lower alkyl, OSO₃Na, or NH₂; and R₆ is independently H, NH₂, S(CH₂)_mNH₂, N₃, 1, SH, lower-alkyl, S-alkylguanidyl, O-alkylguanidyl, S- alkylphthalimido, S-aralkylphthalimido, S-alkyl, S-aryl, S-arylalkyl, S-heterocyclic, S- heterocylic alkyl, S-aminoalkyl, O-aminoalkyl, aminoalkyl, 0-lower alkyl, aralkyl, aryl, heterocyclic ring(s), OSO₃Na or N which is mono, di or tri-substituted with alkyl, aralkyl, aryl, acyl, heterocyclic ring or heterocyclic alkyl, and any of which substituents can be further substituted with N, O or S which can be further substituted with H, alkyl, aralkyl, acyl, heterocyclic ring, heterocyclic alkyl or aryl, wherein for each of R₂, R₃ and R₆ any one or more of the carbon atoms may be optionally replaced by S, N or O, and wherein _n is from about 1 to about 15, preferably from about 1 to about 10, and wherein _m is from about 1 to about 15, preferably from about 1 to about 10. Preferably, R₆ is an ornthine, aminoalkyl, aryl amino, aminoamide, thioureido, S-aminoalkyl or an N₃ group. [0018] For puφoses of the invention, the term "lower alkyl" means an alkyl group from 1 to 7 carbon atoms. The terms "alkyl" and "aryl" include alkyl or aryl groups which may be substituted or unsubstituted. Preferred substitutions include, without limitation, substitution with nitrogen containing moieties, including amino groups, which may be mono or disubstituted, preferably with alkyl or aryl groups. Also, for purposes of the invention the term "alkyl" includes chains of 1-7 atoms with one or more nitrogen atoms and the remainder carbon atoms.

[0019] Table 1 depicts several non-limiting examples of β-CD derivatives according to the invention. Table 2 depicts antibacterial and/or antibiotic potention properties of several of the β-CD derivatives disclosed herein.

20

Table 2

[0020] In another embodiment of the invention, the cyclodextrins according to the present invention are α or γ cyclodextrins, which have 6 or 8 D-glucose units, respectively. These cyclodextrins are represented by General Formulas 2 and 3:

wherein R₂, R₃, and R₆ are as defined in General Formula 1.

[0021] The invention additionally provides methods for potentiating the activity of antibiotics to inhibit the growth of a bacterium which are resistant to clinically used antibiotics, to treat or prevent an infection by these bacteria. The methods according to this aspect of the invention comprise contacting the bacterium with said antibiotic and one or more members of the compound of General Formulas 1, 2, or 3. Particularly, the cyclodextrin derivatives of the present invention are effective in potentiating the activity of vancomycin against

Vanocomycin-resistant Enterococci (VRE), drug resistant Klebsiella and methicillin against

Methicillin-resistant Staphylococcus aureus (MRSA).

[0022] For purposes of the invention, the term "resistant" or "resistance" to a bacterium or bacterial infection to an antibiotic includes a complete resistance to the antibiotic or a partial resistance which is defined herein as a circumstance in which the minimum inhibitory concentration (MIC) of an antibiotic toward the organism in question has increased or renders the antibiotic clinically inoperative due to lack of effectiveness or excessive side effects.

Examples of increased MIC include, but are not limited to, situations where the MIC has increased by 25%, 50%, or 100% and increments therein.

[0023] For purposes herein, potentiation may be defined as a circumstance in which a compound substantially lowers the MIC of an antibacterial agent toward one or more organisms. It includes the case in which it effectively restores the therapeutic utility of an antibacterial agent whose utility has been compromised by bacterial resistance.

[0024] In another embodiment, the invention provides pharmaceutical compositions. These compositions comprise one or more members of the compounds of the invention, a known antibiotic, and a physiologically acceptable carrier.

[0025] As used herein, the term "physiologically acceptable" refers to a material that does not interfere with the effectiveness of the compounds of the first or third aspects of the invention and is compatible with a biological system such as a cell, cell culture, tissue, or organism. In certain embodiments, the biological system is a living organism, such as a mammal. In certain embodiments, the mammal is a human.

[0026] As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990, ISBN: 0-912734-04-3.

[0027] In a further embodiment of this aspect, the invention provides methods for treating a bacterial infection. The method according to this embodiment of the invention comprises administering to a mammal with a bacterial infection one or more members of the compound of General Formulas 1, 2, or 3, in conjunction with a known antibiotic. [0028] In a further embodiment of this aspect, the invention provides methods for preventing a bacterial infection. The method according to this embodiment of the invention comprises administering to a mammal susceptible to a bacterial infection one or more members of the compound of General Formulas 1, 2, or 3 in conjunction with a known antibiotic. [0029] In one embodiment of the invention, the cyclodextrins may be used to potentiate the activity of known antibiotics, such as those approved by the FDA or other regulatory agencies for the treatment of bacterial infections. Antibiotics useful in the present invention include, but are not limited to, glycopeptides, aminoglycosides, beta-lactams, quinolones, sulfonamides, rifampins, monobactams, carbepenems, macrolides, lincosamines, fluoroquinolones, penicillins, oxazolidinones, and tetracyclines. Specific examples include, but are not limited to, vancomycin, ceftriaxone, methicillin, tobramycin, ciprofloxacin, penicillin V, chloramphenicol, norfloxacin, doxycycline, paromycin, kanamycin, neomycin, gentamycin, azythromycin (trade name Zythromax) and streptomycin. [0030] In the methods according to this aspect of the invention the bacteria is in a mammal. Preferably, the mammal is a human. [0031] In the methods according to this aspect of the invention, administration of the compound can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, aerosol, intraocular, intratracheal, intrarectal or vaginal. Administration of the therapeutic compositions can be carried out using known procedures at dosages and for periods of time effective to reduce symptoms or surrogate markers of the infection. A doctor can determine the appropriate dose to administer or therapeutic protocol useful for preventing or preventing a bacterial infection. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode. Preferred methods of administration include parenteral (IV, IM, SC), intranasal, ocular (eye) delivery, and oral administration (as tablet, solution, capsule, etc.).

EXAMPLES

[0032] The following examples are intended to further illustrate certain particularly preferred embodiments of the invention and are not intended to limit the scope of the invention. Indeed, the chemical syntheses described below are equally applicable to α and γ cyclodextrins, as well as the β-cyclodextrins of the following examples.

A. Synthesis of selected CD derivatives

[0033] Triphenylphosphine (54.1 g, 0.2 mol) was dissolved with stirring in 210 rtiL of anhydrous DMF. To this solution was added 51 g (0.2 mol) of iodine over a 15 min. period with evolution of heat. Dry β-cyclodextrin (10.41 g, 9.65 mmol) was added to the dark brown solution and the mixture stirred at 70 ºC for 20 h. The solution was then concentrated to about one-half the volume (~ 100 mL). 3 M NaOMe in MeOH (57 niL) was prepared by adding 3.78 g of sodium to cooled (0 °C) dry methanol (57 mL) under argon. This NaOMe solution was added dropwise to the reaction mixture at 0 ºC and the resulting mixture stirred for 30 min. The reaction mixture was poured into 750 mL of MeOH and stirring was continued for 20 min. The yellow precipitate formed was filtered over a filter paper and washed with three 75 mL portions of MeOH and two 50-mL portions of acetone. The solid was suspended in water (250 mL) and stirred for 20 min. then filtered, washed with two 50- mL portions of water, 50-mL portions of MeOH and three 100-mL portions of acetone. After drying under vacuum, per-6-iodo-6-deoxy-β-cyclodextrin was obtained as a yellow powder: yield 13.2 g (75%).

[0034] Alternatively, per-6-bromo-6-deoxy-β-CD may be synthesized according to the procedure set forth below.

[0035] Bromine (27.5 mL, 0.53 mol) was added under argon to a solution of triphenylphosphine (14O g, 0.53 mol) in anhydrous DMF (60 mL) with vigorous stirring. During the addition, the reaction mixture was not allowed to warm above 60 ºC. The resulting precipitate was further stirred at rt for 1 h. Freshly crystallized and oven dried β- CD (34.0 g, 30.0 mmol) in anhydrous DMF (400 mL) was added dropwise and the reaction mixture was stirred under argon at 75-80 ºC for 18 h. The solvent was removed under diminished pressure and the residue was treated at 0 ºC with a cooled 3 M solution of NaOMe in MeOH (500 mL). After 1 h stirring at rt, the product was precipitated in a large amount of cold water (~ 3.0 L) and filtered, followed by successive washing with MeOH (3 x 250 mL). The residue was dried, suspended in MeOH (1.0 L) and stirred for 1 h at 55 ºC. The product was again filtered while hot and washed with warm MeOH (5 x 100 mL) to remove any residual triphenylphosphine oxide. Per-6-bromo-6-deoxy-β-CD was dried under vacuum and was obtained as a colorless solid: yield 43.8 g (93 %); ¹H NMR (DMSO-d₆, 300 MHz) δ 6.05 (br s, 7H), 5.91 (br s, 7H), 4.97 (br d, 7H), 4.00 (d, 7H, J= 10.2 Hz), 3.81 (t, 7H, J= 8.7 Hz), 3.69- 3.59 (m, 14H), 3.32-3.39 (m, 14 H, hidden by HDO peak); ¹³C NMR (DMSO-d₆, 75 MHz) δ 102.02, 84.50, 72.17, 71.96, 70.85, 34.25.

[0036] To a solution of 1.99 g (1.04 mmol) of per-6-deoxy-6-iodo-β-cyclodextrin in 50 mL of dry DMF was added 1.0 g (15.4 mmol) of NaN₃. The resulting suspension was stirred at 60 ºC for 48 h. The suspension was then concentrated under diminished pressure to a few mL before a large excess (-150 mL) of H₂O was added. The mixture was stirred for 15 min. and the formed white precipitate was filtered. The precipitate was washed with two 25-mL portions of H₂O, three 25-mL portions of acetone and dried under vacuum to give per-6- azido-6-deoxy-β-cyclodextrin as an off-white solid: yield 972 mg (71%). ound No. 2)

C₄₂H₇₇N₇O₂₈

Mw: 1 128.09

To a solution of 10 g of per-6-azido-6-deoxy-β-cyclodextrin (7.63 mmol) in 200 mL of dry DMF was added 31.8 g of triphenylphosphine (121 mmol). The resulting solution was stirred at rt for 2 h. Concentrated aqueous NH₃ (~ 75 mL) was added dropwise to the solution upon which the mixture turned into an off-white suspension. Stirring was continued at rt for 20 h and the solvent evaporated under diminished pressure to approximately 40 mL. The product was precipitated by addition of 500 mL of EtOH and filtered. The residue was washed thoroughly with hot MeOH and dried under vacuum. Per-6-amino-6-deoxy-β-cyclodextrin was obtained as a white solid: yield 8.2 g (95%). To allow characterization by NMR, a small amount of per-6-amino-6-deoxy-β-CD was converted to its hydrochloride salt. ¹H NMR (D₂O) δ 3.24 (dd, I H, J= 7.2, 13.5 Hz), 3.43 (dd, IH, J= 3.2, 13.5 Hz), 3.57 (t, I H, J= 9.3 Hz), 3.65 (dd, I H, J= 3.3, 9.9 Hz), 3.97 (t, IH, J= 9.4 Hz), 4.18 (m, IH), 5.15 (d, IH, J = 3.3 Hz).

[0037] Per-6-deoxy-6-iodo-β-cyclodextrin (952 mg, 0.5 mmol) was dissolved in 10 mL of DMF, 297 mg (3.9 mmol) of thiourea was then added and the reaction mixture heated to 70 ºC. After 19 h, the DMF was removed under diminished pressure to give an orange oil, which was triturated with 25 mL of acetone and stirred at reflux for 10 min. The product was collected by filtration, washed with two 5-mL portions of acetone and dried under vacuum. Per-6-deoxy-6-S-thioureido-β-cyclodextrin hydroiodide was obtained as a colorless powder: yield 610 mg (50%). e

[0038] To a solution of 1.0 g (0.52 mmol) of per-6-iodo-β-cyclodextrin in 5 mL of dry pyridine was added 7.5 mL OfAc₂O and 6.5 mg (0.05 mmol) of 4,4-dimethylaminopyridine. The reaction mixture was stirred at 23 °C under argon for 48 h. The reaction was quenched by the addition of 15 mL of MeOH and the solvent was concentrated under diminished pressure. Co-evaporation with three 4-mL portions of MeOH and three 4-mL portions of toluene gave a brown residue, which was purified on a silica gel column (20 x 3 cm). Elution with a step gradient of 1 :1 to 1 :4 hexane-EtOAc gave heptakis (2,3-di-O-acetyl-6-deoxy-6- iodo)cyclomaltoheptaose as a colorless foam, which crystallized upon trituration with ether: yield 1.06 g (81%): mp 180-182 ºC (lit. 172-177 ºC); ¹H NMR (CDCl₃) δ 2.05 (s, 3H), 2.09 (s, 3H), 3.58-3.81 (m, 4H), 4.83 (dd, IH, J= 9.9, 3.9 Hz), 5.20 (d, IH, J= 3.6 Hz), and 5.33

(br t, 1 H, J = 8.4 Hz); mass spectrum (MALDI), m/z 2514.9 [M+Na] , theoretical 2514.8. mp = 180- 182 ºC; Literature 176-180 ºC.

[0039] To a solution of 0.2 mmol of heptakis (2,3-di-(O-acetyl-6-deoxy-6- iodo)cyclomaltoheptaose and 3.0 mmol of (n-phthalimidolkyl)isothiouronium hydrobromide in 20 mL of dry DMF was added 1.63 g (5.0 mmol) of Cs₂CO3. The reaction mixture was stirred at 23 ºC under argon for 48 h. The reaction mixture was poured onto 40 g ice and 200 mL of 0.5 N HCl was added. The aqueous layer was extracted with three 50-mL portions of dichloromethane. The combined organic phase was washed successively with 200 mL of 0.5 N HCl and 100 mL of brine, dried (MgSO₄), and concentrated under diminished pressure. The residue was purified on a silica gel column (21 x 3 cm); elution with EtOAc. [0040] n= 2, ethyl: colorless foam, yield 0.14O g (23%). An additional 165 mg of the product was obtained in a slightly impure form; ¹H NMR (CDCl₃) δ 2.01 (s, 3H), 2.05 (s,

3H), 2.64 (m, 2H), 3.03 (m, 2H), 3.63 (m, 2H), 3.87 (t, IH, J= 8.4 Hz), 4.15 (m, IH), 4.80

(m, IH), 5.10 (br s, IH), 5.25 (t, I H, J= 8.7 Hz), 7.62 (m, 2H) and 7.73 (m, 2H); mass spectrum (MALDl), m/z 3068.8 [M+Na]⁺, theoretical 3068.7.

[0041] n= 3, propyl: colorless foam, yield 0.372 g (59%); ¹H NMR (CDCl₃) δ 1.91 (m, 2H),

2.02 (s, 3H), 2.05 (s, 3H), 2.60 (m, 2H, J= 12.9, 5.9 Hz), 3.03 (m, 2H), 3.66 (t, 2H, J = 6.9

Hz), 3.84 (t, IH), 4.12 (m, IH), 4.80 (dd, IH, J= 9.7, 3.8 Hz), 5.06 (d, IH, J= 3.8 Hz), 5.23

(dd, IH, J= 9.6, 8.3 Hz), 7.58 (dd, 2H, J= 5.4, 3.1 Hz) and 7.70 (dd, 2H, J= 5.5, 3.0 Hz); mass spectrum (MALDI), m/z 3166.8 [M+Na]⁺, theoretical 3166.8.

[0042] n=4, butyl: colorless foam, yield 0.156 g (24%). An additional 132 mg was obtained in a slightly impure form; ¹H NMR (CDCl₃) δ 1.61 (m, 2H), 1.73 (m, 2H), 2.02 (s, 3H), 2.06

(s, 3H), 2.65 (m, 2H), 3.03 (m, 2H), 3.63 (m, 2H), 3.88 (m, IH), 4.15 (m, IH), 4.80 (dd, IH, J

= 9.8, 3.7 Hz), 5.12 (d, IH, J= 3.6 Hz), 5.26 (m, IH), 7.64 (m, 2H), and 7.74 (m, 2H); mass spectrum (MALDI), m/z 3267.3 [M+Na]⁺, theoretical 3267.5.

[0043] n=5, pentyl: colorless foam, yield 0.435 g (65%); ¹³C NMR (acetone-d6) δ 20.99,

21.05, 26.79, 28.97, 34.16, 34.42, 38.39, 71.55, 72.63, 79.68, 97.72, 123.70, 133.18, 134.85,

168.76, 170.1 1, and 171.00.

[0044] n=6, hexyl: colorless foam, yield 0.462 g (67%); ¹³C NMR (acetone-d₆) δ 20.96,

21.04, 27.32, 30.79, 34.27, 34.93, 38.46, 71.50, 72.81, 79.58, 97.55, 123.71, 133.21, 134.89,

168.79, 170.1 1, and 170.96; mass spectrum (MALDI) m/z 3,464.6 [M+Na]-, theoretical

3,463.9.

[0045] n=10, decyl: yellow foam, yield 0.530 g (69%); ¹³C NMR (acetone-d₆) δ 20.35,

20.44, 27.02, 29.38, 30.12, 33.88, 37.85, 70.95, 72.1 1, 79.03, 96.98, 123.05, 132.56, 134.27,

168.13, 169.48, and 170.35. Example 6. Per-6-(n-aminoalkylthio)-β-cyclodextrin.

Compound No. 3

[0046] A mixture of 100 mg (31.9 mmol) of heptakis [2,3-di-O-acetyl-6-deoxy-6-(2- phthalimidoethyl)-thio]cyclomaltoheptaose and 1.55 mL (31.9 mmol) of hydrazine monohydrate in 1.5 mL of 1 : 1 EtOH-H₂O was stirred at 60 ºC for 18 h. The solvent was concentrated under diminished pressure to give a solid that was suspended in 5 mL of 1 N HCl and stirred at 23 ºC for 8 h. The insoluble material was filtered and the filtrate was diluted with 25 mL acetone, causing the product to precipitate. The supernatant was removed by centrifugation and the product was washed with four 25-mL portions of acetone and dried in vacuo. Compound No. 3 was obtained as a colorless solid: yield 46 mg (89%); mp 180- 182 ºC (dec); ¹³C NMR (DMSO-d₆) δ 102.07, 84.49, 72.51, 72.18, 71.28, 32.70 and 29.60; mass spectrum (MALDI), mfz 1548.8 [M] , theoretical 1548.9. n= 3, Compound No. 4

[0047] A mixture of 100 mg (31.4 mmol) of heptakis[2,3-di-O-acetyl-6-deoxy-6-(3- phthalimidopropyl)-thio]cyclomaltoheptaose and 1.54 mL (31.78 mmol) of hydrazine monohydrate in 1.5 mL of 1 : 1 EtOH-H₂O was stirred at 60 ºC for 16 h. The solvent was concentrated under diminished pressure to give a solid, which was suspended in 5 mL of 1 N HC! and stirred at 23 ºC for 4 h. The insoluble material was filtered and the filtrate was diluted with 25 mL acetone, causing the product to precipitate. The supernatant was removed by centrifugation and the product was washed with four 25-mL portions of acetone and dried in vacuo. Compound No. 4 was obtained as a colorless solid: yield 53 mg (85%); mp 161- 163 ºC (dec); ¹³C NMR (DMSOd₆) δ 26.85, 29.71, 33.03, 37.79, 71.41, 72.23, 72.48, 84.52 and 102.09; mass spectrum (MALDI), m/z 1668.8 [M+Na]⁺, theoretical 1668.6. n= 4, Compound No. 5

[0048] A mixture of 80 mg (25.3 mmol) of heptakis[2,3-di-Oacetyl-6-deoxy-6-(4- phthalimidobutyl)-thio]cyclomaltoheptaose and 1.22 mL (25.3 mmol) of hydrazine monohydrate in 1.2 mL of 1 : 1 EtOH-H₂O was stirred at 60 ºC for 24 h. The solvent was concentrated under diminished pressure to give a solid, which was suspended in 5 mL of 1 N HCl and stirred at 23 ºC for 4 h. The insoluble material was filtered and the filtrate was diluted with 25 mL of acetone, causing the product to precipitate. The supernatant was removed by centrifugation and the product was washed with four 25-mL portions of acetone and dried in vacuo. Compound No. 5 was obtained as a colorless solid: yield 40 mg (94%); mp 172-174 C (dec); ¹³C NMR (DMSO-d₆) δ 26.12, 32.15, 32.85, 38.40, 71.49, 72.22, 72.47, 84.43 and 102.05; mass spectrum (MALDI), m/z 1745.9 [M+Na]⁺, theoretical 1745.3. n=5, Compound No. 27

[0049] A suspension of 1.19 g (0.35 mmol) of heptakis[2,3-di-0-acetyl-6-deoxy-6-(5- phthalimidopentyl)-thio]cyclomaltoheptaose in 20 mL of 1 :1 H₂O-EtOH was treated with 20 mL of hydrazine monohydrate and heated at 70 ºC for 18 h. The cooled reaction mixture was concentrated under diminished pressure. The residue was triturated with 40 mL of 1 N HCl, and the mixture was stirred at 23 ºC for 2 h. The insoluble material was removed by centrifugation, and the supernatant was treated with acetone until the product precipitated. The product was collected, washed with three 20-mL portions of acetone, and dried under vacuum. Compound No. 27 was obtained as a colorless solid: yield 395 mg (53%); mp 192- 194°C (dec); ¹³C NMR (DMSO-d₆) δ 26.12, 27.53, 29.81, 33.49, 34.08, 72.26, 73.46, 85.51 and 102.99. n=6, Compound No. 28

[0050] A suspension of 1.0 g (0.29 mmol) of heptakis[2,3-di-O-acetyl-6-deoxy-6-(6- phthalimidohexyl)-thio]cyclomaltoheptaose in 15 mL of 1 : 1 H₂O-EtOH was treated with 15 mL of hydrazine monohydrate and heated at 70 ºC for 20 h. The reaction mixture was cooled to room temperature, and the solvent was concentrated under diminished pressure. The residue was triturated with 40 mL of 1 N HCl, and the mixture was stirred at 23 °C for 18 h. The insoluble material was removed by centrifugation, and the supernatant was treated with acetone (200 mL, or until the product precipitated). The product was collected, washed with three 25-mL portions of acetone, and dried under vacuum. Compound No. 28 was obtained as a colorless solid: yield 523 mg (82%); mp 216-218 ºC (dec); ¹³C NMR (DMSO-d₆) δ 25.67, 26.86, 27.81, 29.13, 32.56, 33.09, 71.46, 72.22, 72.49, 84.47 and 101.97; mass spectrum (ESI), m/z 1,942.1 [M⁺], theoretical 1,941.7. n= 10, Compound No. 136

[0051] A suspension of 760 mg (0.19 mmol) of heptakis[2,3-di-O-acetyl-6-deoxy-6-(10- phthalimidodecyl)thio]cyclomaltoheptaose in 1 1 mL of 1 :1 H₂O-EtOH was treated with 1 1 mL of hydrazine monohydrate and heated at 80 ºC for 15 h. The cooled reaction mixture was concentrated under diminished pressure. The residue was triturated with 15 mL of 1 N HCl, and the mixture was stirred at 23 ºC for 1 h. The insoluble material was removed by centrifugation, and the supernatant was treated with acetone (150 mL, or until the product precipitated). The product was collected, washed with three 20-mL portions of acetone, and dried under vacuum. Compound No. 136 was obtained as a yellow solid: yield 174 mg (34%); mp 148 to 150 ºC (dec); ¹³C NMR (DMSO-d₆) δ 26.73, 27.68, 29.74, 33.57, 73.40, 85.43, and 102.86.

Synthesis of Compound No. 106

[0052] The crude product is isolated as the hydrobromide salt as judged by the amount of material isolated, solubility in water (soluble), and a silver nitrate test (positive). [0053] The following positional isomers of Compound Nos. 106 and 138 may also be synthesized. HPLC spectra of these derivatives are shown in Figures 5-8.

[0054] To a mixture of 0.32 mmol of peracetylated 6-iodo-β-CD and 6.72 mmol of the thiouronium salt in 30 mL of anhydrous DMF was added 2.6 g (8.0 mmol) OfCs₂CO₃. The mixture was stirred at 23 ºC under argon for 64 h. The insoluble material was filtered and the solvent was concentrated under diminished pressure. The residue was partitioned between 50 mL of EtOAc and 50 mL of water. The organic layer was separated, dried (MgSO₄) and concentrated under diminished pressure. The crude product was dissolved in 24 mL of pyridine and 36 mL OfAc₂O, 20 mg of dimethylaminopyridine were added and the mixture stirred at 23 ºC for 48 h under argon. The reaction was quenched by slow addition of 35 mL of MeOH and the solvent was concentrated under diminished pressure. The residue was partitioned between 75 mL of water and 65 mL of EtOAc. The organic layer was dried (MgSO₄) and concentrated under diminished pressure. The crude product was purified on a silica gel column (4 x 20 cm), eluting with 1 :4 hexane-EtOAc then EtOAc. [0055] ortho isomer: colorless foam, yield 0.835 g (73%); ¹H NMR (CDCl₃) δ 1.97 (s, 3H), 2.05 (s, 3H), 2.06 (m, 2H), 4.00 (m, 2H), 4.18 (m, 2H), 4.79 (m, 3H), 5.06 (d, IH, J= 3.9 Hz), 5.26 (t, I H, J= 8.8 Hz), 6.90 (m, 2H), 7.12 (m, 2H) and 7.52 (m, 4H); ¹³C NMR (acetone-d6) δ 21.03, 21.10, 34.44, 36.27, 39.24, 71.63, 73.00, 79.51, 97.91, 123.84, 128.26, 128.43, 129.58, 131.62, 133.00, 134.92, 136.16, 137.15, 168.65, 170.18 and 170.96; mass spectrum (MALDI), m/z 3604.8 [M+H+Na]⁺, theoretical 3604.8.

[0056] nteta isomer: yellow foam, yield 0.584 g (51%); ¹³C NMR (acetone-d₆) δ 20.40,

20.49, 33.32, 37.51, 41.37, 71.03, 72.35, 79.27, 97.15, 123.30, 126.81, 128.56, 128.62,

128.98, 129.23, 132.43, 134.38, 134.50, 137.25, 139.69, 167.90, 169.51 and 170.37; mass spectrum (MALDI), m/z 3607.8 [M+4H+Na]⁺, theoretical 3608.2.

[0057] para isomer: colorless foam, yield 0.424 g (37%); ¹³C NMR (acetone-d₆) δ 20.34,

20.44, 33.51, 37.34, 41.12, 70.98, 72.07, 78.88, 96.96, 123.24, 128.43, 129.51, 132.42,

134.32, 135.70, 138.61, 167.88, 169.44 and 170.31 ; mass spectrum (MALDI), m/z 3603.8

[M+Na], theoretical 3604.5.

[0058] A suspension of 600 mg (0.17 mmol) of starting material in 10 mL of 1:1 H₂O-EtOH was treated with 10 mL of hydrazine monohydrate and heated at 70 ºC for 18 h. The cooled reaction mixture was concentrated under diminished pressure. The residue was triturated with 30 mL of IN HCl and the mixture was stirred at 23 ºC for 3 h. The insoluble material was removed by centrifugation and the supernatant was treated with acetone until the product precipitated. The product was collected, washed with three 20-mL portions of acetone and dried under vacuum. Compound No. 141 was obtained as a colorless solid: 306 mg (78%); mp 185-187 ºC (dec); ¹³C NMR (DMSO-d₆) δ 33.14, 35.02, 72.82, 73.05, 73.23, 85.18, 102.92, 128.13, 128.94, 130.10, 131.27, 133.14 and 137.46; mass spectrum (MALDI), m/z 2081.6 [M]⁺, theoretical 2082.0.

[0059] A suspension of 757 mg (0.21 mmol) of starting material in 12 mL of 1 :1 H₂O-EtOH was treated with 12 mL of hydrazine monohydrate and heated at 70 ºC for 18 h. The cooled reaction mixture was concentrated under diminished pressure. The residue was triturated with 40 mL of IN HCl and the mixture was stirred at 23 ºC for 21 h. The insoluble material was removed by centrifugation and the supernatant was treated with acetone until the product precipitated. The product was collected, washed with three 20-mL portions of acetone and dried under vacuum. Compound No. 116 was obtained as a colorless solid: yield 316 mg (64%); mp 190-192 ºC (dec); ¹³C NMR (DMSO-d₆) δ 33.82, 37.61, 42.90, 72.90, 73.22, 73.37, 85.40, 103.06, 128.15, 129.46, 129.79, 130.25, 134.98 and 139.90; mass spectrum (MALDI), m/z 2081.6 [M]⁺, theoretical 2082.0. Compound No. 115 (para)

[0060] A suspension of 144 mg (0.04 mmol) of starting material in 3 mL of 1 :1 H₂O-EtOH was treated with 3 mL of hydrazine monohydrate and heated at 65 ºC for 19 h. The cooled reaction mixture was concentrated under diminished pressure. The residue was triturated with 8 mL of IN HCl and the mixture was stirred at 23 ºC for 22 h. The insoluble material was removed by centrifugation and the supernatant treated with acetone until the product precipitated. The product was collected, washed with three 10-mL portions of acetone and dried under vacuum. Compound No. 1 15 was obtained as a colorless solid: 66 mg (70%); mp 190-192 ºC (dec); ¹³C NMR (DMSO-d₆) δ 33.77, 37.46, 42.60, 72.79, 73.07, 73.20, 85.37, 102.94, 129.74, 133.19, 139.68; mass spectrum (MALDI), m/z 2081.6 [M] ⁺, theoretical m/z 2082.4.

[0061] A mixture of 2.0 g (0.104 mmol) of per-6-iodo-β-cyclodextrin and 15.6 g (146 mmol) of p-toluidine in 50 rtiL of anhydrous DMF was stirred under argon at 70-75 ºC for 70 h. The insoluble material was filtered through a filter paper and the filtrate was concentrated under diminished pressure. The dark brown oil was dissolved in 100 mL of EtOH, stirred for 15 min and the precipitate collected by centrifugation. The residue was washed with three 50- mL portions of EtOH, four 50-mL portions of acetone and dried under vacuum. Per-6-(p- tolylamino)-β-cyclodextrin (Compound No. 105) was obtained as an off-white powder: yield 1.27 g (68%).

[0062] N^α-Boc-N^δ-benzyloxy-L-ornithine (0.587 g, 1.60 mmol) was dissolved in 10 mL of dry DMF. HOBt (0.249 g, 1.63 mmol) was added and the solution was cooled to 0 ºC in an ice bath. DCC (0.330 g, 1.60 mmol) was added and the reaction mixture was stirred at 0 ºC for 1 h. The reaction mixture was allowed to warm to room temperature and was stirred for an additional 1 h. To the reaction vessel was added 250 mg (0.22 mmol) of per-6-amino-β- cyclodextrin and 0.2 mL of N-methylmorpholine. The resulting mixture was stirred for an additional 20 h. The precipitated dicyclohexylurea was filtered and the filtrate was concentrated under diminished pressure at 60 ºC to yield a slightly colored oil. Saturated aqueous sodium bicarbonate was added, resulting in a white suspension which was stirred for 1 h and filtered. The precipitate was washed with water and dried under vacuum. To remove the N^δ protecting group, the solid was dissolved in MeOH and 200 mg (50% wet) of palladium hydroxide-on-carbon was added with stirring. A hydrogen atmosphere (balloon pressure) was applied to the reaction mixture which was stirred at room temperature for an additional 20 h. The suspension was filtered, and the filtrate was concentrated under diminished pressure. The residue was dissolved in 5 mL of water, diluted with 75 mL of acetone, and the resulting suspension was sonicated. The precipitate was filtered and dried under vacuum to yield Compound No. 41 as an off-white foam: yield 0.501 g (86%); ¹³C NMR (DMSO-d₆) δ 21.2, 24.4, 27.9, 30.6, 33.3, 40.9, 50.3, 72.2, 77.7, 101.2, 155.3 and 170.1 ; mass spectrum (MALDI), m/z 2651.72 [M+2H+Na]⁺, theoretical 2651.41.

[0063] Per-6-[(N^α-Boc-ornithinyl)amino]-β-cyclodextrin hydrochloride (0.300 g, 0.1 14 mmol) was dissolved in 3 mL of neat trifluoracetic acid and stirred at room temperature for 1 h. The reaction mixture was concentrated under diminished pressure to obtain a pale orange oil. Diethyl ether was added and the suspension was sonicated. The precipitate was filtered, dried under vacuum and re-dissolved in 5 mL of 1 N aqueous HCl. Acetone (65 mL) was added and the resulting suspension was again sonicated. The precipitate was filtered and dried under vacuum to yield Compound No. 42 as a tan foam: yield 0.18O g (65%); ¹³C NMR (DMSO-d₆) δ 22.3, 28.25, 38.0, 51.4, 72.0, 72.6, 102.3 and 169.4; mass spectrum (MALDI), m/z 1950.26 [M+Na]⁺, theoretical 1950.1 1. [0064] Additional examples of cyclodextrins prepared include:

B. Inclusion Complexes

Example 12. Inclusion complex of Compound No. 141 and doxycycline

[0065] Compound No. 141 and doxycycline were dissolved separately in water at room temperature (7.0 x 10^-5 mol). A 1 :1 (mol/mol) cyclodextrin inclusion complex solution (3.5 x 10^'5 mol) of Compound No. 141 with doxycycline was obtained by directly mixing each aqueous solution of the cyclodextrin and the antibiotic. The complex solution was immediately subjected to NMR study.

Characterization of the inclusion complex

[0066] The ¹H and ¹³C NMR spectra were recorded using tetramethylsilane (TMS) as an internal standard on a 500 MHz spectrometer at room temperature. Chemical shifts were expressed as ppm (δ).

[0067] Free Doxycycline: ¹H NMR (500 MHz, D₂O) δ 1.231 (3 H, br s), 2.200 (1 H, m),

2.725 (3 H, br s), 2.821 (3 H, br s), 3.498 (1 H, m), 3.518 (1 H, m), 4.175 (1 H, br s), 6.370 (1

H, br d, J= 7.0 Hz), 6.529 (1 H, br d, J= 6.0 Hz), 7.124 (1 H, br s); ¹³C NMR (125 MHz,

D₂O) δ 15.054, 16.715, 38.344, 40.542, 40.574, 40.593, 41.100, 42.328, 42,483, 42.524,

45.466, 57.329, 65.197, 68.042, 72.959, 95.281, 107.208, 1 14.896, 1 15.870, 1 16.001,

136.752, 146.833, 160.407, 169.346, 171.576, 186.211, 193.108, and 193.225.

[0068] Compound No. 141: ¹H NMR (500 MHz, D₂O) δ 2.671 (1 H, dd, J= 13.5, 8.5 Hz),

2.865 (1 H, d, J= 13.5 Hz), 3,477 (1 H, t, J= 8.5 Hz), 3.552 (1 H, dd, J= 8.5, 2.5 Hz), 3.675

~ 3.779 (4 H, m), 4.048 (1 H, d, J= 14.0 Hz), 4.095 (1 H, d, J= 14.0 Hz), 5.020 (1 H, d, J =

2.5 Hz), 7.001 (1 H, br d, J= 7.5 Hz), 7.069 (1 H, br t, J= 7.5 Hz), 7.245 (1 H, br t, J= 7.5

Hz), and 7.303 (1 H, d, J= 7.5 Hz); ¹³C NMR (125 MHz, D₂O) δ 32.272, 33.593, 39.637,

71.220, 71.661, 72.617, 83.513, 100.930, 128.399, 129.159, 130.030, 130.896, 131.1 15, and

136.157. [0069] 1:1 Complex Compound No. 141 and doxycycline: ¹H NMR (500 MHz, D₂O) for Compound No. 141 : δ_H 2.698 (1 H, dd, J = 13.5, 8.0 Hz), 2.859 (1 H, d, J= 13.5 Hz), 3,469 (1 H, t, J= 8.5 Hz), 3.551 (1 H, m), 3.630 - 3.733 (4 H, m), 3.986 (1 H, d, J= 14.0 Hz), 4.068 (1 H, d, J= 14.0 Hz), 4.978 (1 H, br s), 6.880 (1 H, dd, J= 8.0, 1.5 Hz), 6.954 (1 H, dt, J= 8.0, 1.5 Hz), 7.194 (1 H, dt, J= 8.0, 1.5 Hz), and 7.270 (1 H, dd, J= 8.0, 1.5 Hz); ¹³C NMR (125 MHz, D₂O) δ_c 32.403, 33.815, 39.591, 71.580, 71.854, 72.674, 84.41, 101.76, 128.432, 129.1 17, 130.108, 130.766, 131.123, and 136.100. ¹H NMR (500 MHz, D₂O) doxycycline: δ_H 1.415 (3 H, d, J= 5.0 Hz), 2.475 (1 H, ddd, J= 8.5, 8.5, 2.0 Hz), 2.624 ~ 3.733 (overlapping with other signals), 4.220 (1 H, d, J= 2.0 Hz ), 6.624 (1 H, dd, J= 7.5,

2.0 Hz), 6.880 (overlapping with other signal), and 7.373 (1 H, dd, J= 7.5, 2.0 Hz ); ¹³C NMR (125 MHz, D₂O) δ_c 15.212, 16.718, 38.269, 41.069, 45.878, 57.333, 65.703, 68.092, 73.243, 96.141, 107.598, 115.426, 115.649, 1 15.785, 136.211, 148.123, 160.804, 169.797, 171.797, 186.428, 192.903, and 193.586.

Investigation of Compound No. 141: doxycycline inclusion complex in aqueous solution by NMR spectroscopy

[0070] NMR techniques including two-dimensional NOE spectroscopy are commonly used to study inclusion complexes of cyclodextrins. Compared to the ¹H NMR shifts of the free Compound No. 141 and free doxycycline, the chemical shifts of the protons of Compound No. 141 : doxycycline inclusion complex were shifted in the ¹H NMR spectrum. The largest chemical shifts occurred between those of the aromatic protons on the cyclodextrin and the antibiotic. For Compound No. 141, the δ_H values of four protons shifted upfield from δ_H

7.001 (1 H, br d, J= 7.5 Hz), 7.069 (1 H, br t, J= 7.5 Hz), 7.245 (1 H, br t, J= 7.5 Hz), and 7.303 (1 H, d, J= 7.5 Hz) to δ_H 6.880 (1 H, dd, J= 8.0, 1.5 Hz), 6.954 (1 H, dt, J= 8.0, 1.5 Hz), 7.194 (1 H, dt, J= 8.0, 1.5 Hz), and 7.270 (1 H, dd, J= 8.0, 1.5 Hz) with Δδ_free - complex values of 0.033 ~ 0.121 ppm, while the chemical shifts of three protons on doxycycline were downshifted from δ_H 6.370 (1 H, br d, J= 7.0 Hz), 6.529 (1 H, br d, J= 6.0 Hz), 7.124 (1 H, br s) to corresponding δ_H 6.624 (1 H, dd, J= 7.5, 2.0 Hz), 6.880 (overlapping with other signal), and 7.373 (1 H, dd, J= 7.5, 2.0 Hz ) with Δδ_Co_mpiex-fr_ee (δ differential ) values of 0.249 - 0.351 ppm (Figure 3). Furthermore, the ¹³C NMR spectrum of the inclusion complex showed some up/down-shifts for the δc values compared with those of their free forms. The assignments of protons and carbons of the inclusion complex were confirmed through COSY, HMQC, HMBC, and NOESY spectral analyses. [0071] The NOESY spectrum of the complex Compound No. 141 : doxycycline clearly showed cross peaks between the two aromatic protons at δ 6.880 (Ha), and 6.624 (Hb) on the cyclodextrin, and the antibiotic, respectively (Figure 4). This NMR study clearly indicates that an inclusion complex between the cyclodextin and the antibiotic was formed in aqueous solution; the aromatic ring of the antibiotic is believed to bind within the cyclodextrin core. Investigation of Compound No. 141: doxycycline inclusion complex in aqueous solution by NMR spectroscopy

[0072] The ability of Compound No. 141 to form a complex with vancomycin was also studied by NMR. Admixture of the cyclodextrin and antibiotic resulted in the immediate appearance of new/shifted resonances. The complex was monitored by NMR over a period of 24 hours; no change in the initially formed complex was observed. [0073] Additional NMR studies confirmed the presence of Compound No. 106:Vancomycin and Compound No. 138: Vancomycin complexes as well.

C. Antibacterial Activity Testing 1. Materials

[0074] The following materials were used in antibacterial testing; Mueller-Hinton broth, Brain Heart infusion broth, Mueller-Hinton agar, Brain Heart infusion agar, 96 well, NUNC micro testing plates, Rainin micro-pipetors (l μl-1000μl), Rainin multi-channel micro- pipetors (12 channel, lμl-200μl), petri dishes (100x15mm), Rifampicin antibiotic. [0075] The following bacterial strains were used; Enterococcus faecium ATCC 19434, Vancomycin resistant Enterococcus faecium (VRE) ATCC51575, Eschericia coli ATCC 25404, Pseudomonas aeruginosa ATCC 10145, Pseudomonas aeruginosa Sus20 (clinical isolate), Pseudomonas aeruginosa Sus25 (clinical isolate), Pseudomonas aeruginosa MR 10 (clinical isolate), Pseudomonas aeruginosa MR 20 (clinical isolate), Pseudomonas aeruginosa AGRlO (clinical isolate), Pseudomonas aeruginosa AGR 21 (clinical isolate), Bacillus atrophaeus ATCC 9372, Bacillus subtilis ATCC 6051, Staphylococcus aureus ATCC 12600, Methicillin resistant Staphylococcus aureus (MRSA) ATCC 700698, drug resistant Klebsiella ATCC 700603, and Salomonella choleraesuis.

2. Methods a. MIC determination

[0076] Bacteria form liquid stocks (10-20 μL) were placed on the corresponding agar plates and grown at 37 ºC during 16-18 h. One colony was picked from the agar plate and transferred to 3 mL of Mueller-Hinton or Brain Heart) infusion media (for Enterococcus strains) and permitted to grow for 3-4 h at 37 ºC in a thermostated shaker. To standardize the concentration of bacteria in the prepared cultures McFarland standard was used. All cultures were used at concentrations of about 10⁸ per mL. One hundred μL of each standardized culture was inoculated in 20 mL of corresponding media and assayed in the presence of eleven 2-fold dilutions of tested compounds. Stock solutions of tested compounds (10 mg/mL) were prepared in DMSO. The testing was dependent on the specific. To determine the MIC of tested compounds, two-fold dilutions were prepared with use of corresponding medium as following: all wells of 96 well microplate were filled with 50 μL of corresponding medium, except the second well of each row (they contain 96 μL). The first well of each row was used as a control of cell growth without compounds. 4μL of each tested compound were added into the second well of corresponding row (see scheme), mixed and 50 μL was transferred to the next well. The process was repeated until rows 2-12 had been serially diluted. Fifty μL was removed from last well of each row and 50 μL of corresponding bacteria culture (prepared previously) was added to each well of plate. The plates were incubated at 37 ºC in a thermostated shaker during 20-24 h and cell growth was detected visually. The MIC for each tested compound was determined as the dilution without visible cell growth. To standardize MIC rifampicin (5 mm/mL) was assayed every time as a positive control.

b. Potentiation study

[0077] To study the potentiation activity of cyclodextrins, two different procedures were used (Selection and Verification).

[0078] The selection of cyclodextrins that can potentiate the activity of known antibiotics and Pinnacle antibacterial compounds was carried out in accordance with the procedure described below. The procedure employed below is not limited to the antibiotics specifically described in the following examples, and may be applied to any known antibiotic. For this purpose, all synthesized cyclodextrins were diluted with DMSO until the concentration was 0.5 mg/mL and placed in the corresponding well of 96-well plates (lOOμL well) (Scheme 2). To carry out the selections, 2 μL from each well the of prepared master plates were transferred into the corresponding well of a work plate. Fifty μL of bacterial culture, prepared as described above, was placed into 20 μL of medium, containing MIC/4 concentration of corresponding antibacterial agents. 98 μL of prepared culture was placed into each well of the work plates to a final concentration of cyclodextrin of 10 μg/mL. The plates were incubated at 37 ºC in a thermostated shaker during 20-24 h and cell growth was detected visually. Cyclodextrins whose presence in the medium led to the absence of visual cell growth, were selected as possible potentiators of the antibacterial compounds, used for the assay. [0079] In the next stage, verification was carried out in two ways. First, the MIC of antibacterial agents was determined for different bacterial strains in the absence and in the presence of selected cyclodextrins (10-20 μg/mL). Second, the antimicrobial agents were mixed with cyclodextrins (normally 1 : 1 molar ratio) and MICs were estimated for antibacterial agent alone, cyclodextrins alone and mixture. The protocol for MIC determination described above was used in both assays.

c. Summary of Results

[0080] A library of- 150 individual cyclodextrin derivatives were synthesized and surveyed for their ability to potentiate known antibiotics against four bacterial strains. A number of derivatives which increased the activity of these antibiotics have been identified from this survey. In particular, Pseudomonas aeruginosa was more sensitive to doxycylin when this antibiotic was combined with cyclodextrins bearing a lysinamide group (Compound No. 29), an aminoalkylthio group (Compound Nos. 4, 5, 27and 28) or aminobenzylthio group (Compound Nos .115, 116and 141) at the primary position. The activity of doxycycline increased 16-fold compared to doxycycline alone. The commercially available cyclodextrin HP-β- cyclodextrin did not have any effect on the activity of doxycycline. The activity was completely lost when doxycycline was tested with 2,6-DMCD and 2,3,6-TMCD. These cyclodextrins are more water soluble and are often used to increase the water solubility of drugs. The activity of norfloxacin was also potentiated by the same cyclodextrins {See Table 1 for potentiation against P. aeruginosa) and increased 4-fold compared to norfloxacin alone. [0081] The potentiating effect observed with the cyclodextrin described herein appears not to be a result of the increased solubility of doxycycline. For example, the water insoluble cyclodextrins 74 and 75 also increased the activity of norfloxacin more than 4-fold. [0082] An aminoglycoside resistant strain of Pseudomonas aeruginosa was also more sensitive to aminoglycosides when these antibiotics were administered in combination with cyclodextrins (Table 2). Most notably, the arylamino cyclodextrins 115, 1 16 and 141 were the most effective in decreasing the MICs of all aminoglycosides tested. These last three cyclodextrins were active both in tests against susceptible and resistant strains of Pseudomonas aeruginosa. A different family of cyclodextrin derivatives have been selected in the screen to identify cyclodextrins capable of potentiating antibiotics toward MRSA (Table 3).

[0083] These water insoluble cyclodextrins bearing an arylamino group at the primary positions were very effective in potentiating the activity of methicillin (more than 130-fold). This result also confirms that the potentiating effect observed is not the result of increased solubility of the antibiotic. Moreover, liphophilic cyclodextrins 74 and 75 also have a positive effect on the action of methicillin. Similar results were also observed for tobramycin when it was complexed with the same cyclodextrin derivatives.

[0084] The resistance of Enteroccocus to vancomycin was also reduced when vancomycin was combined with Compound Nos. 16 and 105. These two water insoluble cyclodextrins increased the activity of vancomycin more than 130-fold, compared to vancomycin alone. Other cyclodextrins bearing aminoaryl (80, 100, 1 10), aminobenzyl (138, 106) or aminoalkyl groups (74, 75) potentiated the activities of four different antibiotics against VRE. [0085] Testing of additional cyclodextrins revealed that Compound No. 111 is quite effective at potentiating the action of methicillin and tobramycin toward MRSA; Compound No. 59 potentiated the action of methicillin and penicillin toward P. aeruginosa. 1. Potentiation activity of four known antibiotics by β-cyclodextrin derivatives against Pseudomonas aeruginosa (Table 3).

[0086] A panel of cyclodextrins was selected and screened for their ability to potentiate the antibacterial activity of four known antibiotics: one aminoglycoside (tobramycin), one cephalosporin (ceftriaxone), one quinolone (norflaxocin) and one tetracycline (doxycycline), against P. aeruginosa (Table 3).

[0087] In the absence of cyclodextrin (Entry 1), two of the antibiotics showed good activity

(MIC= 0.78-0.39 μg/mL) and the other two showed only moderate activity (MIC= 25-12.5 μg/mL) against P. aeruginosa. When the antibiotics were combined with the commercially available cyclodextrins hydroxypropyl-β- cyclodextrin, 2,6-DMCD or 2,3,6-TMCD (1:1 molar ratio), no reduction in the MICs was observed (Entries 2-4). In addition, the antibacterial activity of doxycyclin was completely lost with addition of the methylated cyclodextrins. When coadministered with Compound No. 29, norfloxacin, ceftriaxone and doxycyclin showed a 13-16 fold increase in activity, whereas tobramycin appeared to be equally active against P. aeruginosa (entry 3). In the case of Compound Nos. 42 and 17 (entries 4 and 8), a 5-fold and 16-fold increase in activity was observed in the case norfloxacin and ceftriaxone, respectively. Compound No. 36 (entry 5) also showed a 13-fold increase in activity for norfloxacin and 8 and 16-fold increases in the case of ceftriaxone and doxycyclin, respectively. The structures of Compound Nos. 17, 29and 42 are related to a cyclodextrin bearing a α-aminoamide functionality at position 6, while Compound No. 36 has a thioureido group.

[0088] Another set of cyclodextrins incorporating linear alkylamino groups at the primary position has been screened for their ability to potentiate the four antibiotics against P. aeruginosa. Cyclodextrins with an aminoalkylthio group chain with 3 to 6 carbons (Table 3, entries 19-23) have the ability to decrease the MIC of norfloxacin, ceftriaxone and doxycyclin 3, 8 and 16-fold, respectively. They did not show any potentiation for tobramycin. Surprisingly, cyclodextrins with longer alkyl chain (n= 6, 7 and 8) did not potentiate any of the antibiotics, while Compound No. 136 (n=9, entry 6) showed an increase in the activity for all antibiotics tested.

[0089] An α-cyclodextrin tested (122, entry 18) was also effective in potentiating the activity of all antibiotics, except tobramycin.

[0090] Similarly, cyclodextrins with a benzylamino group at the primary position (1 15, 1 16 and 141) showed an 8-fold and 16-fold increase in activity for ceftriaxone and doxycyclin, respectively (entries 15-17), while Compound No. 116 was effective in combination with norfloxacin (entry 16). Table 3 also shows that two cyclodextrins with lipophilic side chains (74 and 75) were selective in potentiating the activities of norfloxacin and tobramycin (entries 1 1 and 12).

2. Potentiation activity of six aminoglycosides by β-cyclodextrin derivatives against aminoglycoside resistant Pseudomonas aeruginosa (Table 4).

[0091] Table 4 summarizes the potentiating effect of a series of cyclodextrin derivatives on aminoglycosides against an aminoglycoside resistant strain of P. Aeruginosa. From a library of -150 cyclodextrins , nine derivatives were able to lower the MICs of aminoglycosides when mixed in a 1 :1 molar ratio. HP-β- cyclodextrin did not have any noticeable effect on the antibacterial activities of aminoglycosides, as was also true for the methylated cyclodextrins. A slight potentiating effect was detected for Compound Nos. 16, 74 and 75 and 106 while a 4-fold decrease in MIC₉₀ was noted for tobramycin. In addition, Compound Nos. 74 and 75 slightly potentiate the activities neomycin and gentamycin. A more pronounced potentiating affect was observed with cyclodextrin derivatives possessing an alkylamino and arylamino group on the primary position. For example, Compound Nos. 1 15, 1 16and 141 can increase the activity of the aminoglycosides up to 30-fold, as observed in the case of neomycin and streptomycin.

3. Potentiation activity of four known antibiotics by β-cyclodextrin derivatives against methicillin-resistant Staphylococcus aureus (MRSA) (Table 5).

[0092] Methicillin-resistant Staphylococcus aureus (MRSA) strains represent a worldwide threat because of their virulence and broad distribution in the hospital setting and community. See Michel, M., Gutmann, L. Lancet 1997, 349, 1901-1906. Moreover, the MRSA strains are often resistant not only to β-lactam antibiotics but also to fluoroquinolones, chloramphenicol, clindamycin, tetracycline and aminoglycosides. In addition, resistance to recently developed anti-staphylococcus agents, such as the oxazolidinone-type antibiotics has emerged. See Meka, V. G.; Gold, H. S. Clin. Infect. Dis. 2004, 39(7), 1010-1015. Therefore, the development of new classes of anti-MRSA compounds is of particular importance. [0093] From a library of -150 cyclodextrin derivatives, about 15 cyclodextrin derivatives have been selected as potentiators for four known antibiotics against MRSA. The cyclodextrins have been divided into two classes: cyclodextrins without any significant activity against MRSA and cyclodextrins with intrinsic activity against MRSA. Table 5. Potentiation activity of four known antibiotics by β-cyclodextrin derivatives against MRSA

[0094] When the four antibiotics were tested with the commercially available cyclodextrins: HP-β- cyclodextrin, 2,6-DMCD and 2,3,6-TMCD (1 :1 molar ratio), no effect on the activity of these antibiotics have been observed (entry 1, Table 3). Seven cyclodextrin derivatives (lacking antibacterial activity) were selected for their ability to reduce the MICs of the antibiotics tested (entries 3-9). When combined with Compound No. 16 (entry 3), the activities of ceftriaxone, methicillin, tobramycin and ciprofloxacin increased 16, 65, 32 and 8-fold, respectively. A similar pattern was also observed for Compound Nos. 105, 136 and 141, albeit with some differences in the extent of potentiation of individual antibiotics. Most notable is that the activity of ceftriaxone was potentiated >130 times by Compound No. 141 (entry 9).

[0095] Among the cyclodextrin derivatives having intrinsic antibacterial activity (entries 10- 18), the complexes of Compound Nos. 110, 137, 80, 138, 106and 100 with either methicillin or tobramycin showed >130 times higher activity than the antibiotics alone. Also, MRSA was more sensitive to all antibiotics when Compound No. 74 was added to the medium. These results show clearly that MRSA appears to be more sensitive when its viability was challenged by antibiotics admixed with the synthetic cyclodextrins described herein.

4. Potentiation activity of four known antibiotics by β-cyclodextrin derivatives against Vancomycin-Resistant Enterococcus (VRE). (Table 6).

[0096] Enterococci are normal colonizers of the human gastrointestinal tract and are organisms with relatively low virulence. However, the emergence of Enterococci with high level resistance to vancomycin (VRE) in the US and some other parts of the world during the 1990s has severely constrained therapeutic options for the management of serious infection, since Enterococci already possess intrinsic and acquired resistance to most other antimicrobials. Michel, M., Gutmann, L. Lancet 1997, 349, 1901-1906. Bonten, M. J. M., Willems, R., Weinstein, R.A. Lancet Infect. Dis. 2001, 1(5), 314-325. Thus, novel strategies to enhance the potency of glycopeptides, and other antibacterials, against VRE by chemical modifications or other means are of great interest.

Table 6. Potentiation activity of four known antibiotics by β-cyclodextrin derivatives against VRE

[0097] In the case of VRE, none of the four clinically used antibiotics showed any significant activity when tested alone (entry 1, Table 6) or in combination with HP-β- cyclodextrin, 2,6- DMCD or 2,3,6-TMCD (entry 2). The only exception was observed with methicillin with a 4-fold increase in activity, when this antibiotic was tested in combination with HP-β- cyclodextrin and 2,3,6-TMCD. However, the anti-VRE activity of vancomycin increased >130 fold when Compound No. 16 and 105 were added to the medium (entries 4 and 10). A similar effect was also observed for methicillin and ceftriaxone, when combined with Compound No. 80 (entry 14). The lipophilic cyclodextrins 74 and 75 (entries 8 and 9) were effective in decreasing the MICs of all 4 antibiotics tested, at least 16 fold. The same effect was noted with Compound Nos. 138, 106 and 110 (entries 15, 16 and 18). 5. Potentiation activity of antibiotics by β-cyclodextrin derivatives against Klebsiella. [0098] Initial studies with Klebsiella were carried out using a highly resistant strain (700603) available from ATCC. Klebsiella infections are presently treated clinically with either a monobactam (Aztreonam) or penicillin (Piperacillin) derivative. Five cyclodextrins (2, 106, 115, 1 16 and 141) were found to potentiate the action of these clinical agents v. Klebsiella. Additionally, Compound Nos. 134 and 135 exhibited fairly strong intrinsic activity toward Klebsiella.

[0099] Compound Nos. 106 and 138 have also been tested for acute toxicity in rats at 20 mg/kg; no adverse effect was observed (n=3 for 106 and 138). The acute toxicity of a combination of Compound No. 106 and equimolar vancomycin at 20 mg/kg was tested and no adverse effect was observed. (n=3). Table 7 shows the potentiation of positional isomers of Compound Nos. 106 and 138 with Vancomycin..

[00100] To characterize the activity of Compound No. 141 (NF), this cyclodextrin and its 1 :1 mixtures with 4 antibiotics have been tested against two resistant strains (MRSA and AGR-21 P. aeruginosa) (Table 8).

[00101] Certain cyclodextrins (Figure 9), were selected against MRSA ATCC 700698 as possible potentiator of antibacterial activity of methicillin and tobramycin, have been tested alone and in combination with these antibiotic for MIC determination (Table 9).

[00102] Certain cyclodextrins (Figure 10), were selected against P. aeruginosa ATCC

10145 as possible potentiator of ceftriaxone activity, and have been tested in combination with two β-lactam antibiotics (methicillin and penicillin V) (Table 10).

[00103] Antibacterial activity of 1 1 antibiotics have been tested Klebsiella pneumonia

ATCC700603 (Table 1 1).

[00104] The ability of certain cyclodextrins to potentiate the activity of piperacillin and aztreonam, has also been determined. Concentration of CDs - 10 μg/mL and concentration of both antibiotics - 50 μg/mL have been used in selection assays. Four CDs have been selected as possible potentiators of activity of both antibiotics and Compound No. 2 - only aztreonam (Figure 1 1). Compound Nos. 134 and 135 have shown the antibacterial activity at the tested concentration (10 μg/mL).

[00105] Activity of selected cyclodextrins themselves and in combination with four antibiotics (methicillin; piperacillin; aztreonam and penicillin V) against K. pneumonia has been determined. (Table 12).

[00106] CDs, selected as antibacterials in concentration less then 10 μg/mL

(Compound Nos. 134 and 135 have also been assayed (Table 13).

[00107] To check the time of complex formation, the MIC of mixtures of Compound

No. 141 after 2h incubation at room temperature and prepared immediately before testing have been compared (Table 14).

[00108] It is clear from the above results that the cyclodextrins effectively potentiate the effect of antibiotics against resistant bacterial strains. This result is entirely unexpected as demonstrated by the above results, specifically the high MIC of the cyclodextrins and antibiotics alone as compared to when the two are combined.

[00109] It is also clear that several bacterial strains are more sensitive to antimicrobial treatment when their viability was challenged by antibiotic molecules combined with cyclodextrin derivatives. Because new classes of antibiotics have been slow to be identified, this synergism between antibiotics and cyclodextrins constitutes a novel approach for combating bacterial resistance by increasing the effectiveness of currently available drugs. The foregoing results clearly demonstrate that the predominant factor influencing the potentiating activity is the nature of the substitution on the cyclodextrin core.

Claims

What is claimed is:

1. A method for potentiating the activity of an antibiotic to inhibit the growth of a bacterium which is resistant to said antibiotic, comprising contacting the bacterium with a composition comprising said antibiotic and a compound having the formula

wherein R₂ is independently H, OH, OAc, 0-lower alkyl, or 0(CH₂CH₂O)_n; R₃ is independently H, OH, OAc, O-lower alkyl, OSO₃Na, or NH₂; and R₆ is independently NH₂, S(CH₂)_mNH₂, N₃, S-alkylguanidyl, O-alkylguanidyl, S-alkylphthalimido, S- aralkylphthalimido, S-alkyl, S-aryl, S-arylalkyl, S-heterocyclic, S-heterocyclic alkyl, S-aminoalkyl, O-aminoalkyl, aminoalkyl, O-lower alkyl, aralkyl, aryl, heterocyclic ring(s), OSO₃Na, or N which is mono, di or tri-substituted with alkyl, aralkyl, aryl, acyl, heterocyclic ring or heterocyclic alkyl, and any of which substituents can be further substituted with N, O or S which can be further substituted with H, alkyl, aralkyl, acyl, heterocyclic ring, heterocyclic alkyl or aryl, wherein for each of R₂, R₃ and R₆ any one or more of the carbon atoms may be optionally replaced by S, N or O, and wherein _n is from about 1 to about 15, and wherein _m is from about 1 to about 15, wherein the bacterium is contacted with the compound and the antibiotic separately or simultaneously.

2. The method according to claim 1, wherein _n is from about 1 to about 10, and wherein m is from about 1 to about 10.

3. The method according to claim 1, wherein indenpendently for each of R₂ or R₃ the O- lower-alkyl is OMe.

4. The method of claim 1, wherein R₆ is N₃, and R₂ and R₃ are both hydrogen.

5. The method according to claim 1, wherein the bacterium is in a mammal.

6. The method according to claim 4, wherein the mammal is a human.

7. The method of claim 1, wherein the antibiotic is a glycopeptide, an aminoglycoside, a beta-lactam, a quinolone, a sulfonamide, a rifampin, a monobactam, a carbepenem, a macrolide, a lincosamine, a fluoroquinolone, an oxazolidinone, a tetracycline or mixture thereof.

8. The method of claim 1, wherein R₆ is N₃.

9. The method of claim 1, wherein the antibiotic is vancomycin, ceftriaxone, methicillin, tobramycin, ciprofloxacin, penicillin V, chloramphenicol, norfloxacin, doxycycline, paromycin, kanamycin, neomycin, gentamycin, azythromycin, streptomycin or mixtures thereof.