Derivatives of Xanthone Compounds

Abstract

The present invention relates to xanthone analogs. Such compounds may be used in the treatment of bacterial infections.

Claims

1.-36. (canceled)

37. A compound of Formula (II) or a pharmaceutically acceptable salt thereof: ##STR00115## wherein m is 1; Y is O; B is NR.sup.11R.sup.24; each of R.sup.3 and R.sup.10 is independently hydrogen or alkyl; R.sup.4 and R.sup.5 taken together with the carbon to which they are bonded form (CO); R.sup.6 and R.sup.7 taken together form a bond; R.sup.8 and R.sup.9 taken together form a bond; R.sup.11 for each occurrence is hydrogen; each of R.sup.12 and R.sup.13 independently for each occurrence is hydrogen or optionally substituted alkyl; R.sup.24 independently for each occurrence is ##STR00116## wherein n is 4, and each of R.sup.15 and R.sup.16 independently for each occurrence is hydrogen or (CO)NR.sup.12R.sup.13; and R.sup.23 independently for each occurrence is N(R.sup.13)(CNR.sup.12)NR.sup.12R.sup.13.

38. The compound of claim 37, wherein R.sup.24 independently for each occurrence is ##STR00117##

39. The compound of claim 38, wherein R.sup.24 independently for each occurrence is ##STR00118##

40. The compound of claim 39, wherein each occurrence of R.sup.13 in Formula (IIa-2) is independently optionally substituted alkyl.

41. The compound of claim 40, wherein R.sup.23 independently for each occurrence is N(H)(CNH)NH.sub.2.

42. The compound of claim 37, wherein R.sup.3 is hydrogen, and R.sup.10 is alkyl.

43. The compound of claim 37, wherein the compound is: ##STR00119## or a pharmaceutically acceptable salt thereof.

44. The compound of claim 37, wherein the compound is: ##STR00120## or a pharmaceutically acceptable salt thereof.

45. A process of preparing a compound of claim 37 or a pharmaceutically acceptable salt thereof, which comprises: reacting a compound having the formula ##STR00121## wherein m is 1; Y is O; each of R.sup.3 and R.sup.10 is independently hydrogen or alkyl; R.sup.4 and R.sup.5 taken together with the carbon to which they are bonded form (CO); R.sup.6 and R.sup.7 taken together form a bond; and R.sup.8 and R.sup.9 taken together form a bond; with a compound having the formula HN(R.sup.11)R.sup.24, wherein R.sup.11 in each occurrence is independently is hydrogen; and R.sup.24 is ##STR00122## wherein n is 4, and each of R.sup.15 and R.sup.16 independently for each occurrence is hydrogen or (CO)NR.sup.12R.sup.13; R.sup.23 independently for each occurrence is N(R.sup.13)(CNR.sup.12)NR.sup.12R.sup.13; and each of R.sup.12 and R.sup.13 independently for each occurrence is hydrogen or optionally substituted alkyl.

46. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 37, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

47. The pharmaceutical composition of claim 46, further comprising one or more additional therapeutic agents.

48. A method for treating a microbial infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound according to claim 37, or a pharmaceutically acceptable salt thereof.

49. The method of claim 48, wherein the microbial infection is a Gram negative bacterial infection or a Gram positive bacterial infection.

50. The method of claim 49, wherein the Gram positive bacteria is selected from the group consisting of Streptococcus spp., Staphylococcus spp., Bacillus spp., Carynebacterium spp., Clostridium spp., Listeria spp., and Enterococcus spp.

51. The method of claim 50, wherein the Gram positive bacteria is Staphylococcus aureus.

52. The method of claim 51, wherein the Staphylococcus aureus is Methicillin resistant Staphylococcus aureus.

53. The method of claim 48, the method further comprising administering one or more additional therapeutic agents.

54. A compound of Formula (II) or a pharmaceutically acceptable salt thereof: ##STR00123## wherein m is 1; Y is O; B is NR.sup.11R.sup.24; each of R.sup.3 and R.sup.10 is independently hydrogen or alkyl; R.sup.4 and R.sup.5 taken together with the carbon to which they are bonded form (CO); R.sup.6 and R.sup.7 taken together form a bond; R.sup.8 and R.sup.9 taken together form a bond; R.sup.11 in each occurrence is hydrogen; R.sup.24 is ##STR00124## wherein n is 0; each of R.sup.15 and R.sup.16 independently for each occurrence is hydrogen; and R.sup.23 independently for each occurrence is arginine or an arginine derivative.

55. The compound of claim 54, wherein R.sup.3 is hydrogen, and R.sup.10 is alkyl.

56. The compound of claim 54, wherein the arginine derivative independently for each occurrence is ##STR00125## wherein each of R.sup.12 and R.sup.13 independently for each occurrence is hydrogen or optionally substituted alkyl.

57. The compound of claim 54, wherein the compound is selected from the group consisting of: ##STR00126## ##STR00127## or a pharmaceutically acceptable salt thereof.

58. The compound of claim 54, wherein the compound is selected from the group consisting of: ##STR00128## ##STR00129## or a pharmaceutically acceptable salt thereof.

59. A process of preparing a compound of claim 54 or a pharmaceutically salt thereof, which comprises: reacting a compound having the formula ##STR00130## wherein m is 1; Y is O; each of R.sup.3 and R.sup.10 is independently hydrogen or alkyl; R.sup.4 and R.sup.5 taken together with the carbon to which they are bonded form (CO); R.sup.6 and R.sup.7 taken together form a bond; and R.sup.8 and R.sup.9 taken together form a bond with a compound of formula HNR.sup.11R.sup.24, wherein R.sup.11 for each occurrence is hydrogen; and R.sup.24 independently for each occurrence is R.sup.23, wherein R.sup.23 independently for each occurrence is arginine or an arginine derivative.

60. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 54, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

61. The pharmaceutical composition of claim 60, further comprising one or more additional therapeutic agents.

62. A method for treating a microbial infection in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound according to claim 54, or a pharmaceutically acceptable salt thereof.

63. The method of claim 62, wherein the microbial infection is a Gram negative bacterial infection or a Gram positive bacterial infection.

64. The method of claim 63, wherein the Gram positive bacteria is selected from the group consisting of Streptococcus spp., Staphylococcus spp., Bacillus spp., Carynebacterium spp., Clostridium spp., Listeria spp., and Enterococcus spp.

65. The method of claim 64, wherein the Gram positive bacteria is Staphylococcus aureus.

66. The method of claim 65, wherein the Staphylococcus aureus is Methicillin resistant Staphylococcus aureus.

67. The method of claim 62, the method further comprising administering one or more additional therapeutic agents.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0182] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0183] FIG. 1 shows a kill curve for MRSA strain DM21455 exposed to compound AM-016 as described herein.

[0184] FIG. 2 shows an absorbance curve of varying concentrations of AM-016 in a saline solution.

[0185] FIG. 3 shows an absorbance curve of varying concentrations of AM-016 in water.

[0186] FIG. 4a-4b shows the results of an in vivo cytotoxicity assay on a rabbit eye.

[0187] FIG. 5 is shows the results of a comparative test for cytotoxicity between AM-016 and vancomycin in a rabbit corneal wound healing model.

[0188] FIG. 6 graphically illustrates the percentage wound closing in a rabbit corneal wound healing model over a 3 day period using vancomycin, AM-016 or saline solution.

[0189] FIG. 7 graphically illustrates a drug resistance curve for vancomycin and AM-016.

[0190] FIG. 8. is a plot MICs (g/mL) of AM016 against E. faecalis ATCC29212, S. aureus DM21455(MRSA), vancomycin intermediate-resistance S. aureus(VISA) and S. aureus ATCC 700699.

[0191] FIG. 9 shows the results of a depolarization study of AM-016 with Clinical Staphylococcus aureus DM4001(source: Eye).

[0192] FIG. 10 shows the results of a depolarization study of AM-005 with Clinical Staphylococcus aureus DM4001(source: Eye).

[0193] FIG. 11 shows the effect of membrane permeabilization induced by compounds described herein using SYTOX green assay.

[0194] FIG. 12 shows the results of observable SYTOX green fluorescence emission induced by compounds described herein against Gram-negative bacteria (E. coli ATCC8739).

[0195] FIG. 13 shows the effect of membrane permeabilization of compounds described herein against Gram-positive and Gram-negative bacteria.

[0196] FIG. 14 shows fluorescence microscopy images demonstrating that active compounds as described herein induce a significant amount of SYTOX green stained S. aureus (fluoresces in green).

[0197] FIG. 15 shows the results of an extracellular ATP leakage assay.

[0198] FIG. 16 shows the antimicrobial activity of AM-016, vancomycin and daptomycin against strain MRSA-21455.

[0199] FIG. 17 shows show the antimicrobial activity of AM-016, vancomycin and daptomycin against strain MRSA-09080R.

[0200] FIG. 18 shows show the antimicrobial activity of AM-016, vancomycin and daptomycin against strain MRSA-42412

[0201] FIG. 19 shows show the antimicrobial activity of AM-016, vancomycin and daptomycin against strain MRSA-21595.

[0202] FIG. 20 shows show the antimicrobial activity of AM-016, vancomycin and daptomycin against strain MRSA-700699.

EXAMPLES

[0203] Non-limiting examples of the invention, including the best mode, and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

EXAMPLES

Example 1Synthesis of Alpha Mangostin Derivatives

[0204] The hydroxyl groups of alpha-mangostin at C3 and C6 position were modified to mimic an antimicrobial peptide structure, which consists of a hydrophobic core and cationic side groups. The general modification strategy is described below.

##STR00079##

[0205] The above structure illustrates the general structure of the synthesized compounds described herein. Functional groups were introduced to the 3,6-position of alpha mangostin to mimic the antimicrobial peptides. n refers to the length of link space, from 1 to 20. The R moieties are selected from different functional group, such as halogen, aliphatic amines, aromatic amines, amino acid, guanidine and the like. The alpha-mangostin based synthetic antibiotics were classified into three types by the properties of the R functional groups, including cationic modification, neutral modification and anionic modification.

1.1 Cationic Modification of Alpha-Mangostin

Synthesis of Cationic Alpha-Mangostin Derivatives by Dibromo-Substituted Approach.

[0206] We first synthesized different length spacers of w-bromoalkyl substituted alpha mangostin, the length of the spacer was from 2-20. The intermediates were conjugated with three types of amines to obtain cationic modification of the alpha-magostin derivatives. These three types of amines including linear aliphatic amines such as ethanamine, propan-1-amine, dimethylamine, diethyl amine, dipropylamine, diisopropylamine, triethylamine, trimethylamine, tripropylamine, n-propylpentan-1-amine, butan-1-amine; cyclic aliphatic amines such as thiazolidine, isoxazolidine, oxazolidine, pyrrolidine, morpholine, piperazine, thiomorpholine, 1-methylimidazolidine; and aromatic amines such as 1,2,4-triazole, 1H-imidazole, pyrazole, pyridine, pyridazine, pyrimidine, 1H-1,2,3-triazole and the like (see Scheme 1-1)

##STR00080##

Scheme 1-1

[0207] The general synthesis route of cationic alpha-mangostin derivatives by dibromo-substituted approach.

[0208] Specific examples of this type of analogue are illustrated as below:

##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##

Synthesis of AM005

[0209] alpha-Mangostin (1.0 g, 2.44 mmol) was dissolved in 15 mL of acetone, then potassium carbonate (1.6 g, 12.20 mmol) and 1,4-dibromobutane (4.34 mL, 36.6 mmol) were added. The mixture was refluxed for 24 h. After the reaction was completed (TLC), the solvent was removed under reduced pressure. The oil residue was diluted with EtOAc and washed twice with saturated brine and once with water. The organic phase was dried over anhydrous Na.sub.2SO.sub.4 then purified via silica gel column chromatography (petroleum ether/EtOAc, 20/1, v/v), affording 1.27 g of product AM005 as a light yellow solid in 76.5% yield.

Synthesis of AM012

[0210] To a solution of AM005(100 mg, 0.147 mmol) in acetone (4 mL), 1H-pyrazole (100 mg, 1.47 mmol) and potassium carbonate (101 mg, 7.35 mmol) were added. The mixture was refluxed for 48 h. After the end of the reaction, the solvent was removed under reduced pressure. The residue was diluted with 50 mL of ethyl acetate and washed three times with saturated brine, dried over anhydrous Na.sub.2SO.sub.4. After removal of solvent, the residual mixture was purified via silica gel column chromatography (EtOAc/MeOH/Et3N, 100/2/1, v/v), affording 73.9 mg of product AM012 as a light yellow solid in 76.8% yield.

Synthesis of AM016

[0211] To a solution of AM005(100 mg, 0.147 mmol) in DMSO (4 mL), diethylamine (4 mL) was added. The mixture was stirred at room temperature for 3 h. After the end of the reaction, the mixture was diluted with 50 mL ethyl acetate, then washed with aqueous NaHCO.sub.3 and saturated brine (each three times). The organic phase was dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The residual crude oil was purified via silica gel column chromatography (EtOAc/MeOH/Et3N, 100/2/1, v/v), affording 80.8 mg of pure product AM016 as a yellow oil in 82.7% yield.

1.1.2 Synthesis of Cationic Alpha-Mangostin Derivatives by a Dicarboxyl-Substituted Approach.

[0212] Cationic modified alpha-mangostin derivatives were synthesized using a dicarboxyl substituted alpha-mangostin derivative. In order to obtain cationic modification alpha-mangostin derivatives, the intermediate was conjugated with different types of amines or peptides, such as arginine, histidine, lysine, spermidine, N,N-dimethyldipropylenetriamine, diethylenetriamine, ethanamine, N,N-diethyldiethylenetriamine, triethylenetetramine, pentaethylenehexamine, ethylenediamine, propan-1-amine, dimethylamine, diethylamine, 1,3-di-boc-2-(2-hydroxyethyl)guanidine et al. Some of this AM series analogues, especially AM-052, showed excellent antimicrobial activity against Gram-positive and Gram-negative bacteria, low cytotoxicity with a lower hemolytic activity with HC.sub.50 of 238 g/mL.

##STR00090##

Scheme 1-2

[0213] General synthesis route of key intermediate compounds of dicarboxyl-alkyl-substituted mangostin and some target molecules of cationic modification of alpha-mangostin by mimicking of AMPs. (Arg=Arginine, Lys=Lysine, His=Histidine).

[0214] Specific examples of this type of analogue are illustrated as below:

##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##

Synthesis of AM050

[0215] A mixture of alpha-mangostin (0.5 g, 1.22 mmol), methyl bromoacetate (1.12 g, 7.3 mmol) and KOH (341.6 mg, 6.1 mmol) in ethanol (30 mL) was refluxed for 72 h. After cooling down, the mixture was diluted with ethyl acetate and washed with 3 times NaCl solution (350 ml). Organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to give crude product as oil. And then continue the next step reaction without further purification.

Synthesis of AM051

[0216] To a solution of AM50 in 20 ml THF, was added a solution of LiOH(87.84 mg, 3.66 mmol) in 10 ml water. After stirring at room temperature for 2 h, DCM was added and the layers were separated. The mixture was washed with 3 times DCM (320 ml). And then the aqueous layer was acidified with diluted hydrochloric acid. The mixture was diluted with butanol and washed with 3 times NaCl solution (350 ml). Organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate crude residue, which was purified by column chromatography (silica gel, PE/EtOAc/CH.sub.3COOH, 3:1:0.04) to give 345.6 mg yellow solid. The two step yield is 53.8%.

Synthesis of AM052

[0217] HOBt (64.1 mg, 0.475 mmol) was added to AM51(100 mg, 0.19 mmol) in anhydrous DMF (5 mL). At 10 C., DIC (59.9 mg, 0.475 mmol), H-Arg-OMe.Math.2HCl (124 mg, 0.475 mmol) were added, and stir at 10 C. for 1 h. After stirring at room temperature overnight, the mixture was diluted with ethyl acetate and washed with 3 times NaCl solution (350 ml). Organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate crude residue, which was purified by chromatography to give yellow solid.

1.1.3 Synthesis of Cationic Alpha-Mangostin Derivatives by Diepoxy Ethyl-Substituted Approach.

[0218] Cationic modified alpha-mangostin derivatives were also synthesized using diepoxy ethyl-substituted alpha mangostin derivatives. In order to obtain cationic modification alpha-mangostin derivatives, the intermediate was conjugated with different types of amines, such as ethanamine, propan-1-amine, dimethylamine, diethyl amine, dipropylamine, diisopropylamine, triethylamine, trimethylamine, tripropylamine, n-propylpentan-1-amine, butan-1-amine, thiazolidine, isoxazolidine, oxazolidine, pyrrolidine, morpholine, piperazine, thiomorpholine, 1-methylimidazolidine, 1,2,4-triazole, 1H-imidazole, pyrazole, pyridine, pyridazine, pyrimidine, 1H-1,2,3-triazole and the like. (see Scheme 1-3).

##STR00098##

Scheme 1-3

[0219] General synthesis route of key intermediate compounds of diepoxy substituted mangostin and target molecules of cationic modification of alpha-mangostin.

[0220] Specific examples of this type of analogue are illustrated as below:

##STR00099## ##STR00100## ##STR00101##

Synthesis of AM058

[0221] A mixture of alpha-mangostin (0.5 g, 1.22 mmol), 1-bromo-2,3-epoxypropane (2.507 g, 18.3 mmol) and KOH (341.6 mg, 6.1 mmol) in ethanol (30 mL) was refluxed for 24 h. After cooling down, the mixture was diluted with ethyl acetate and washed with 3 times NaCl solution (350 ml). Organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated to generate crude residue, which was purified by column chromatography (silica gel, PE/EtoAc/, 3:1) to give yellow solid (yield: 133.9 mg, 21%)

Synthesis of AM059

[0222] To a solution of AM058(100 mg, 0.191 mmol) in methanol (4 mL), diethylamine (2 mL) was added. The mixture was refluxed for 6 h. After the end of the reaction, the mixture was evaporated to remove the excess amine and solvent, and then diluted with 40 mL ethyl acetate, then washed with aqueous NaHCO.sub.3 and saturated brine (each three times). The organic phase was dried over anhydrous Na.sub.2SO.sub.4 and concentrated under vacuum. The residual crude oil was purified purified by column chromatography (silica gel, EtOAc/MeOH/Et.sub.3N, 100/2/0.5, v/v) to give yellow solid. (yield: 93.3 mg, 73.1%).

1.2 Synthesis of Anionic Modification of Alpha-Mangostin.

[0223] In addition to the cationic modification of alpha-mangostin, anionic modifications of alpha-mangostin were synthesized (see Scheme 1-4).

##STR00102##

Scheme 1-4

[0224] General synthesis of anionic alpha-mangostin analogues.

[0225] Specific examples of this type of analogue are illustrated as below:

##STR00103##

Synthesis of AM071 and AM072

[0226] Alpha-mangostin (201.6 mg, 0.491 mmol) was reacted with chloroacetic acid (826.8 mg, 8.75 mmol) and NaOH (0.576 mg) in DMSO (5 ml) with stirring at 75 C. for 72 h. The reaction mixture was cooled down and hydrochloric acid was added until no additional precipitate was formed. Solvent was removed, then the residual oil was washed 3 times with saturated NaCl solution. The organic extracts were combined and dried overnight with anhydrous Na.sub.2SO.sub.4. Solvent was removed under reduced pressure. The resulting residue was purified by column chromatography (PE/EtoAc/AA,70/40/1,V/V/V). Yield: AM071, 45.2%; AM072, 16.9%

1.3. Neutral Hydrophilic Modification of Alpha-Mangostin.

[0227] Neutral modified alpha-mangostin derivatives were synthesised. In contrast to cationic and anionic modified alpha mangostin, neutral functional group without charge carrier such as halogens, alkyl groups, sulfonamide or polyethylene glycols (PEG) were used to modify the alpha-mangostin structure (see Scheme 1-5)

##STR00104##

Scheme 1-5

[0228] General synthesis of AM series of PEG-mangostin conjugate

[0229] The specific examples of this type of analogue are illustrated below:

##STR00105##

Synthesis of AM-076

[0230] A mixture of alpha-mangostin (100 mg, 0.244 mmol), PEG-OTs (0.4542 g, 0.502 mmol) and K.sub.2CO.sub.3(0.0488 g, 0.353 mmol) in DMF (5 ml) was stirred at 100 C. After the end of the reaction, the mixture was diluted with ethyl acetate and washed 3 times with NaCl solution. The resulting residue was purified by silica gel column chromatography (EtoAc/MeOH, 5/3, V/V) to give compound AM-076 as a brownish-yellow liquid in yield of 57.1%.

Example 2 Biological Activity

2.1 Minimum Inhibition Concentrations (MIC) and Selectivity.

[0231] The following tables show the antimicrobial activity of some of the compounds described herein.

TABLE-US-00001 TABLE 2-1 Antibacterial Activity (MIC), in vitro toxicity (HC.sub.50) and Selectivity of -mangostin derivatives. pKa of conjugated MIC.sub.99 [g mL.sup.1] HC.sub.50 [g Selectivity [HC.sub.50/MIC.sub.99] Compound n R amine, RH MRSA.sup.b S. aureus.sup.c mL.sup.1].sup.a MRSA S. aureus AM-011 3 [00106] 10.98 0.39 0.78 16.27 41.72 20.86 AM-016 4 [00107] 10.98 0.39 0.39 19.6 50.26 50.26 AM-008 5 [00108] 10.98 0.78 1.56 14 17.95 8.97 AM-010 6 [00109] 10.98 1.56 1.56 26.5 16.99 16.99 AM-015 4 [00110] 11.27 1.56 1.56 25 16.03 16.03 AM-017 4 [00111] 11.22 1.56 0.78 19.25 12.34 24.68 AM-009 4 [00112] 8.36 >200 >200 >200 .sup.d AM-012 4 [00113] 2.52 >200 >200 >200 AM-002 4 [00114] 2.20 >200 >200 >200 AM-005 4 Br NA.sup.e >200 >200 >200 AM-000 0 H NA 1.56 1.56 9 5.77 5.77

Example 2Antimicrobial Activity of Alpha-Mangostin Derivative AM016

[0232] The anti-microbial activity of AM016(3,6-bis-[4-(diethylamino)butoxy)]-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one; or 3,6-O-bis[4-(diethylamino)butyl]--mangostin) was tested.

[0233] As can be seen in Table 1A, compound AM0016 demonstrates very good antimicrobial activity against Gram positive bacteria, specifically a series of MRSA strains. Table 1a and 1b show the minimum inhibition concentration (MIC) (g/ml)/((M) of compound AM-016 needed for antimicrobial activity.

[0234] AM-016 also demonstrates a broad antimicrobial activity against Gram negative bacteria. AM016 is also effective against

[0235] Gram negative bacteria (Table 1b).

[0236] AM-016 eliminates 99.9% of MRSA in a sample within 20 minutes of application (FIG. 1)

TABLE-US-00002 TABLE 1a AM-016 AM-016 MRSA strain (g/ml) (M) MRSA DM21455 0.39 0.59 Source: Eye Clinical 1.56 2.35 Staphylococcus aureus DM4001 Source: Eye MRSA DM09808R 1.56 2.35 Source: Eye Clinical 0.78 1.17 Staphylococcus aureus DM4400R Source: Cornea Clinical 0.78 1.17 Staphylococcus aureus DM4583R Source: Eye MRSA DB57964/04 0.78 1.17 Clinical 1.56 2.35 Staphylococcus aureus DM4299 MRSA DM21595 0.78 1.17 Source: Wound MRSA DR42412 1.56 2.35 Source: Sputum MRSA DR68004 1.56 2.35 Source: Blood MRSA QC Strain 3.125 4.70 ATCC BAA1026 Staphylococcus 0.39 0.59 aureus ATCC29213 Staphylococcus 1.56 2.35 aureus ATCC 6538 Straphylococcus 0.78 1.17 aureus ATCC 6538P Straphylococcus 1.56 2.35 aureus ATCC 29737 Straphylococcus 3.125 4.70 aureus ATCC 25923

TABLE-US-00003 TABLE 1b AM-016 AM-016 Bacteria strain (g/ml) (M) Streptococcus 0.195 0.295 faecium ATCC 10541 Streptococcus 0.78 1.17 epidermidis ATCC 12228 Bacillus cereus 3.125 4.70 ATCC11778 Pseudomonas 50 75.20 aeruginosa ATCC27853 Klebsiella 40 60.16 pneumoniae ATCC10031 Enterococcus 0.78 1.17 faecalis ATCC 29212
Table 1a and 1b: Minimum inhibition concentration (MIC) (g/ml)/(M) of compound AM-016 for eliminating Gram positive and Gram negative bacteria.

Example 2Antimicrobial Activity of AM-052 Against Against MRSA and Gram-Negative Bacteria

[0237] AM-052 shows excellent antimicrobial activity against MRSA and Gram-negative bacteria, with low hemolytic activity with HC50 of 238 (g/mL) (See Table 3).

TABLE-US-00004 TABLE 2 Summary of MIC99 (g/mL), HC50 (g/mL) and Selectivity Index of AM-052 Strain of Selectivity bacteria MIC99 HC50 Index Methicillin- 3.125 238 76 resistant Staphylococcus aureus DM09808R Methicillin- 1.56 238 153 resistant Staphylococcus aureus DM21455 Source: Eye Staphylococcus 6.25 238 38 aureus ATCC 29213 Bacillus cereus 3.125 238 76 ATCC 11778 Pseudomonas 12.5 238 19 aeruginosa ATCC27853 Klebsiella 6.25 238 38 pneumoniae ATCC10031

Example 3Basic Physiochemical Properties of AM016 and AM052

3.1 Solubility

[0238] Table 3-1 shows the solubility of compounds AM016 and AM052 in different media.

TABLE-US-00005 TABLE 3-1 Solubility (ug/ml) Solvents AM016 AM052 Water 72.1 3.6 >2 mg/ml Saline 133.3 11.5 >1 mg/ml DMSO >7 mg/ml >2 mg/ml PBS >3 mg/ml not soluble

Table 3-1: Solubility of AM016 and AM052 in Different Media

[0239] The determination of solubility was made as follows: AM016 with known weight was added with 1 mL saline, pure water or phosphate buffer saline (PBS) 20 mM at pH7. The solvent was added stepwise until the compound was dissolved completely. Then, the stock solution was diluted to series of diluents and the absorbance of each diluent was determined.

[0240] In PBS buffer AM-016 has excellent solubility. The solubility in PBS is 3 mg/ml.

[0241] In saline solution FIG. 2 shows that the solubility of AM016 is 130 g/mL.

[0242] In pure water solubility is 70 g/ml, see FIG. 3.

3.2 pH Value of AM016 and AM052 in Different Media

[0243]

TABLE-US-00006 TABLE 3-2 pH value of AM016 in different media Concentration Concentration of compound pH of of compound pH of Solvents (g/ml) AM0016 (g/ml) AM052 Water 50 6.44 0.27 50 6.52 0.29 70 6.72 0.23 100 7.66 0.33 Saline 50 6.84 0.21 50 5.33 0.05 100 6.78 0.05 100 5.33 0.01 PBS 50 6.96 0.01 50 100 6.97 0.01 100
The pH values were obtained using an electronic pH meter.

Example 4Minimum Inhibition Concentration (MIC) (g/Ml)/(M) of Compounds AM002, AM005, AM008, AM009, AM010

[0244] AM002, AM005, AM008, AM009, AM010 were tested against Gram positive bacteria including several strains of Staphylococcus aureus. The results are shown in Table 2. It can be seen that compounds AM008, AM009 and AM010 display very good antimicrobial activity against certain strains of Staphylococcus aureus. AM-005, which is a neutral hydrophobic modification of alpha-mangostin by incorporation of dibromo alkyl substitutions, did not show activity against Gram-positive bacteria at 50 ug/ml (see Table 4).

TABLE-US-00007 TABLE 4 Minimum inhibition concentration (MIC) (g/ml)/(M)of compounds AM002, AM005, AM008, AM009, AM010 MRSA MRSA MRSA Clinical DM09808R DM21455 DM21595 Staphylococcus Bacilus Staphylococcus Source: Source: Source: aureus Cereus aureus S/N Eye Eye Eye DM4001 ATCC11778 ATCC29213 AM- >50 ND ND ND >50 >50 002 AM- >50 >50 ND ND ND >50 005 AM- 0.78 1.56 008 AM- >25 >25 009 AM- 1.56 1.56 010 Staphylococcus Klebsiella Pseudomonas Escherichia aureus pneumoniae aeruginosa coli S/N ATCC6538 ATCC10031 ATCC27853 ATCC8739 AM- ND >100 30 >100 002 AM- ND ND >50 ND 005

Example 5Biological Activity of Compounds AM071 and AM072

[0245] Table 5 shows the biological activity of compounds AM071 and AM072 against certain strains of Gram positive bacteria.

TABLE-US-00008 TABLE 5 MIC data of AM-071 and AM-072 for Gram positive bacteria AM-071 AM-071 AM-072 AM-072 Bacteria (g/ml) (mM) (g/ml) (mM) MRSA DM21455 25 53.36 6.25 13.34 Source: Eye Bacillus cereus 6.25 13.34 6.25 13.34 ATCC11778 Clinical 25 53.36 6.25 13.34 Staphylococcus aureus DM4001 Source: Eye Staphylococcus aureus 12.5 26.68 6.25 13.34 ATCC29213 As can be seen from Table 5 above, compounds AM071 and AM072 do not demonstrate very good antimicrobial activity against Staphylococcus aureus or Bacillus cereus

Example 6In Vivo Cytotoxicity Test of AM-016

[0246] AM-016 does not show cytotoxicity in the rabbit eye after 3 days of application of drug (FIGS. 5a and b).

[0247] Initial testing was carried out on a rabbit eye, using five applications per day at a concentration of 400 g/mL (513-1026MIC). No observable toxic effect was observed on the mouse cornea after AM016 was applied topically. FIG. 5a shows the rabbit eye before application of AM016 and FIG. 5b shows the rabbit eye after topical application of AM016.

Example 7Effect of AM016 on Wound Healing of Rabbit Cornea

[0248] FIG. 5 shows the results of a comparative test for cytotoxicity between AM-016 and vancomycin in a rabbit corneal wound healing model. The particular model used was a 5 mm diameter corneal epithelial abrasion model. Wound healing was monitored by a slit lamp using flourescein disclosure dye so that the size of the wound could be measured. The compounds were applied topically three times per day.

[0249] As can be seen AM-016 does not exhibit any cytotoxicity to the rabbit eye after 3 days of application of drug (see FIG. 5) compared with a normal control.

[0250] FIG. 6 graphically illustrates the percentage wound closing over the same 3 days using vancomycin, AM-016 or saline solution. As can be seen, the application of AM-016 does not delay wound healing indicating that AM-016 does not display any cytotoxic effects.

Example 8Drug Resistance Test of AM-016 with a MRSA Strain from an Eye

[0251] FIG. 7 shows that MRSA DM21455 has difficulty in developing drug resistance towards AM-016 during 20 serial passages of bacteria grown in media with a constant concentration of AM-016. In contrast, MRSA DM21455 develops drug resistance more quickly against vancomycin when compared to AM-016.

[0252] FIG. 8 shows the results of a multipassage resistance selection study to investigate the propensity of S. aureus and Enterococcus faecalis to develop endogeneous mutational resistance against AM016 during prolonged passages at the sub-inhibitory concentrations.

[0253] Resistance is defined as >4-fold increase in the original MIC developed in the multipassage study. FIG. 8 shows that there was no evidence of resistance or cross-resistance against AM016 in any of the 4 strains tested.

Example 9Action Mechanism Study of AM-016

[0254] Membrane potential-sensitive dye 3,3-dipropylthiadicarbocyanine iodide (DiSC3) was used to determine the cytoplasmic membrane depolarization activity of AM-016.

[0255] An overnight culture of clinical Staphylococcus aureus DM4001(source: Eye) was allowed to grow in Muller-Hinton broth (MHB) to OD620=0.3-0.4. Then, the bacteria was collected by centrifugation at 2800 r.p.m at 37 C. for 30 minutes and washed with 5 mM HEPES buffer at pH7. Then, the bacteria were re-suspended in the same buffer with with 0.2 mM EDTA to obtain an OD620 value of 0.9-0.1. The cell suspension was then incubated with 0.1M KCl and 0.4 M DiSC3 at 37 C. for 30 minutes. Dye uptake was monitored by fluorospectrometry (Photon Technology International (PTI) Photomultiplier Detection Systems, model 814) in a stirred cuvette until the fluorescence signal was stable. The desired concentration of AM-016 was added and the fluorescence was monitored at an excitation wavelength of 622 nm and at an emission wavelength of 670 nm. A blank with dye in the absence of bacteria with same concentration of AM-016 was used as a control.

[0256] FIG. 9 shows that when 12.5 ug/mL of AM-016 was added to the S. aureus culture, fluorescence increases. This signifies that AM-016 causes membrane depolarization and therefore DISC.sub.3 was released into medium and results in an increase in fluorescence. Thus, AM-016 is a potential membrane-target antimicrobial agent which is able to disrupt or dissipate the membrane potential of Gram positive bacteria. In contrast, interestingly, its precursor dibromo-substituted AM-005 (without tertiary amine as terminal groups) does not depolarize the membrane (FIG. 10).

[0257] In addition, time killing (see FIG. 1) and drug resistance (FIGS. 7 and 8) also support the suggested depolarization mechanism of AM-016.

[0258] FIG. 1 shows that more than 99.9% MRSA was killed within 20 minutes. For membrane targeted antimicrobial agents, the elimination time is very fast as the uptake of antimicrobial agent by the bacteria is not needed. The bacteria will be killed upon contact with antimicrobial agent.

[0259] FIG. 7 shows that MRSA has difficulty in developing drug resistance towards AM-016 during 20 serial passages. To develop drug resistance against a membrane target antimicrobial agent, bacteria have to change the composition of the cell wall or membrane to prevent interaction of the membrane with the antimicrobial agent. However, for substantial changes in the membrane composition to occur, the bacteria must undergo multiple rounds of mutation which may ultimately be incompatible with bacterial survival [1]. Vancomycin is an antimicrobial used to treat MRSA infection. The antimicrobial action mechanism of vancomycin involves inhibition of peptidoglycan polymerization. Drug resistance in Staphylococcus aureas is more pronounced for vancomycin when compared with AM-016. Taken together these results strongly suggest that AM-016 targets the membrane of bacteria and other microbes.

[0260] The depolarization study strongly suggests that AM-016 depolarizes a clinical S. aureus DM4001 bacteria membrane. In contrast the precursor dibromo-alkyl substituted AM-005 (without tertiary amine as terminal groups) does not depolarize the membrane. This result supports the MIC screening, the time killing and drug resistance results presented above.

SYTOX Green Assay

[0261] SYTOX green is a membrane impermeable dye. Interaction of SYTOX green with a nucleic acid enhances the fluorescence emission of SYTOX green significantly. S. aureus DM4001 was harvested at early exponential phase and suspended in 40 mM PBS until OD.sub.620 of 0.09 was obtained. Then, the bacteria suspension was incubated with 3 M of SYTOX Green in dark. The mixture was monitored in a stirring cuvette at an excitation wavelength of 504 nm and at an emission wavelength of 523 nm until the fluorescence was stable. Then, 10 M of AM compounds were added and the changed of fluorescence emissions were monitored. FIG. 4-2A shows that antimicrobial active compounds (AM-008, AM-010, AM-010, AM-011, AM-015, AM-016, AM-017) could induce membrane permeabilization and enhance the SYTOX green fluorescence emission. In contrast, inactive compounds including AM-002, AM-005, AM-009, AM-012, AM-044 and AM-045 were not able to trigger the membrane permeabilization (see FIG. 11). AM-043 had weaker membrane permeabilization effect.

[0262] AM-008, AM-010, AM-011, AM-016, AM-015 and AM017 were found to be active against Gram-positive bacteria but inactive against Gram-negative bacteria. To further confirm that membrane permeabilization is important to kill the bacteria, the SYTOX green assay was tested using E. coli ATCC8739. FIG. 12 shows that addition of all AM-series compounds tested did not induce membrane permeabilization of E. coli as there was no observable increased of SYTOX green fluorescence emission. FIG. 13 shows the membrane permeabilization effects of selected compounds (active against Gram-positive bacteria) on Gram-positive and Gram-negative bacteria. The data clearly suggest that membrane targeting and inner membrane permeabilization are crucial for the compounds described herein to kill the bacteria.

[0263] In addition to the fluorescence spectroscopy, we further confirm the inner membrane targeting properties of active AM-series compounds using fluorescence microscopy (FIG. 14). Large numbers of stained bacteria were observed in the culture incubated with active AM-series compounds (AM-008, AM-010, AM-011, AM-015, AM-016, AM-017) at 10 M, clearly indicating that these AM-series compromised the living bacterial cytoplasmic membrane. In contrast, no significant staining was not observed in the culture treated with inactive compounds (AM-002, AM-005, AM-009, AM-012, AM-044, AM-045).

Calcein Leakage Study

[0264] To further investigate antimicrobial action of the active compounds via inner membrane targeting, a calcein leakage study was performed. In brief, liposomes which mimic bacterial membrane (DOPE: DOPG=3:1) and red blood cell (DOPC: DOPS=3:1) were synthesized. The lipids were dissolved in methanol/chloroform (1:2, by volume). The solvent was then dried gently using a constant stream of nitrogen gas. Then, the lipid film was placed under vacuum for at least two hours. The dried lipid film was then hydrated with calcein solution (80 mM calcein, 50 mM HEPES, 100 mM NaCl, 0.3 mM EDTA, pH 7.4). The hydrated vesicles were freeze-thaw (frozen in liquid nitrogen and warmed in water bath) for at least 7 cycles. Extrusion method using mini-extruder (Avanti Polar Lipid Inc.) was used to produce homogeneous large unilamellar vesicles (LUVs). The extrusion was done for at least 10 cycles using a polycarbonate membrane (Whatman, pore size 100 nm) to obtain LUVs with diameter of 100 nm. To separate the calcein encapsulated vesicles from free calcein, gel filtration column Sephadex G-50 was used. The concentrations of eluted liposomes were determined using total phosphorus determination assay. Leakage of calcein from the liposome induced by AM-series compounds could be monitored using fluorescence spectroscopy (Photon Technology International Model 814) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. An aliquot of the LUV suspension and lipid to AM-series compounds ratio of 2, 4 and 8 were mixed in a stirred cuvette and the fluorescence emission intensity was monitored. 0.1% Triton X-100 was use to determine the intensity at 100% leakage. Percentage of leakage (% L) was calculated with % L=[(I.sub.tI.sub.0)/(I.sub.I.sub.0)]*100], where I.sub.0 and I.sub.t are intensity before and after addition of -mangostin respectively and I.sub. is intensity after addition of 0.1% triton X-100. Table 4-3A shows the % calcein leakage from the liposome (DOPE:DOPG=3:1) induced by AM-series compounds. Inactive xanthones did not induce observable leakage up to compound to lipid ratio of 1:2. In addition, active AM-series compounds are also able to induce leakage of calcein from DOPC:DOPS=3:1 liposomes, which corroborate with the HC50 values for the compounds (Table 4-3B). Those compounds with high low HC50 values have stronger leakage %. Compounds with high HC50 values show negligible calcein leakage (<10%). To further investigate the inner membrane targeting action of AM-series compounds, E. coli lipid extract was used to construct the liposome. Although all AM-series compounds are inactive against E. coli, surprisingly, AM-010, AM-011, AM-008, AM-015, AM-016, AM017 were able to induce leakage of calcein from the E. coli lipid extract (Table 4-3C). Therefore, the outer layer of Gram-negative bacteria (LPS) may play important role to impede the AM-series compounds to reach inner membrane. Calcein leakage data strongly support the active compounds are inner membrane targeting.

TABLE-US-00009 TABLE 6 Percentage leakage of calcein from DOPE:DOPG = 3:1 liposomes induced by compounds as described herein. % Leakage AM-series compounds: Lipid ratio Liposome (DOPE:DOPG = 3:1) 1:2 1:4 1:8 AM-011 52.35 60.39 AM-016 54.43 37.00 21.00 AM-008 61.50 55.00 36.46 AM-010 67.00 54.34 46.57 AM-015 AM-017 AM-002, AM-012, AM-009, <10 AM-005

TABLE-US-00010 TABLE 7 Percentage leakage of calcein from DOPC:DOPS = 3:1 liposomes induced by compounds described herein. % Leakage AM-series compounds: Lipid ratio Liposome (DOPE:DOPG = 3:1) 1:2 1:4 1:8 AM-011 75.19 58.19 34.10 AM-016 88.43 72.76 37.66 AM-008 90.87 61.34 33.54 AM-010 91.00 62.05 37.31 AM-015 AM-017 AM-002, AM-012, AM-009, <10 AM-005

TABLE-US-00011 TABLE 8 Percentage leakage of calcein from E. coli lipid extract induced by compounds described herein. % Leakage AM-series compounds: Lipid ratio Liposome (E. coli extract) 1:2 1:4 1:8 AM-011 69.52 14.72 5.05 AM-016 57.82 58.43 31.70 AM-008 79.97 50.49 24.06 AM-010 73.85 48.11 28.51 AM-015 AM-017 AM-002, AM-012, AM-009, <10 AM-005

Extracellular ATP Leakage Assay

[0265] ATP leakage assay is also an important assay to study the inner membrane targeting property of a compound. ATP is released from a membrane compromised cells and the amount of ATP released can be detected. In contrast, if a compound does not induce membrane disruption, minimum ATP released will be detected. In this study, a bioluminescence assay was used using recombinant firefly luciferase and its substrate D-luciferin. ATP is required for luciferase to interact with luciferin to produce light. First, bacteria suspension (OD=0.4) was incubated with AM-series compounds with desired concentration at 37 C. for 10 minutes. Then, the suspension was centrifugated at 3000 r.p.m. for 5 minutes. Standard reaction solution containing 0.5 mM D-luciferin, 1.25 g/mL firefly luciferase, 25 mM Tricine buffer at pH 7.8, 5 mM MgSO.sub.4, 100 M EDTA and 1 mM DTT was prepared and added into 96 well plates. 10 uL of supernatant of the culture solution was added and the ATP released was determined using Luminometer (TECAN Infinite M200Pro). 8 M of nisin was used to trigger complete ATP leakage. FIG. 4-4 shows the ATP leakage induced by the AM-series compounds. Compounds active against Gram-positive bacteria could induce strong ATP leakage. In contrast, inactive compounds were not able to induce ATP leakage. The result further shows that active AM-series compounds are inner membrane targeting (see FIG. 15).

[0266] The results of the antimicrobial action studies clearly show that AM-series compounds are inner membrane targeting. The mechanism is well correlated with the antimicrobial properties of active AM-series compounds such as AM016. FIG. 1 shows that more than 99.9% MRSA was killed within 20 minutes. For membrane target antimicrobial, the time killing is very fast as the uptake of antimicrobial by the bacteria is not needed. The bacteria will be killed upon it contacts with membrane target antimicrobial.

[0267] In addition, FIGS. 6 and 7 show that MRSA is difficult to develop drug resistance against AM-016 during 20 serial passages. To develop drug resistance against membrane target antimicrobial, bacteria have to change the compositions of cell wall or membrane to prevent the interaction with membrane target antimicrobial. However, tremendously changed of membrane composition requires multiple mutation and such alternation might be incompatible with cellular survival.

Example 10Antimicrobial Activity of AM-016 Compared with Vancomycin and Daptomycin Against Various Strains of Staphylococcus aureus

[0268] The antimicrobial activity of AM-016 was compared with vancomycin and daptomycin against various strains of Staphylococcus aureus using the Mueller Hinton broth dilution method.

[0269] FIGS. 16 to 20 show the antimicrobial activity of AM-016, vancomycin and daptomycin against various strains of Staphylococcus aureus. As can be seen AM-016 exhibits antimicrobial activity (as determined by the minimum inhibitory concentration in g/ml) against strains MRSA-21455(see FIG. 16), MRSA-09080R (see FIG. 17), MRSA-42412(see FIG. 18), MRSA-21595(see FIG. 19) and MRSA-700699(see FIG. 20). AM-016 shows superior antimicrobial activity against strains MRSA-09080R, MRSA-21595 and MRSA-42412 when compared to vancomycin. However, AM-016 shows superior antimicrobial activity against MRSA-700699 when compared to both daptomycin and vancomycin, with a minimum inhibitory concentration of 0.048 g/ml.

[0270] The minimum inhibitory concentrations (MIC) are as follows:

TABLE-US-00012 MIC values (g/ml) Daptomycin Vancomycin AM-016 MRSA-21455 (Eye) 0.78 0.78 1.56 MRSA-09808R (Eye) 0.195 0.395 3.125 MRSA-42412 (Sputum) 0.195 0.78 0.395 MRSA-700699 0.78 6.25 0.095 MRSA-21595 (Wound) 0.395 0.78 0.395

[0271] The minimum inhibitory concentrations for AM-016 and vancomycin against certain other strains of Gram positive bacteria are also presented below

TABLE-US-00013 Strains AM-016 Vancomycin Enterococcus faecalis 1.56 50 ATCC 51299 VRE Enterococcus faecalis 11: 0.098 Above 100 DS6527 (VRE 208/11) Enterococcus faecalis 11: 0.78 1.56 DU9345 VRE Staphylococcus aureus 10: 0.78 1.56 DB6506 (MRSA04/10) (VISA) Staphylococcus aureus 0.39 6.25 ATCC 700699 (VISA)
As can be seen, AM-016 demonstrates superior antimicrobial properties compared to vancomycin.

Example 11Antimicrobial Activity of Alpha-Mangostin Derivative Compounds Against Mycobacterium tuberculosis

[0272] Table 10 shows the antimicrobial activity of some alpha mangostin derivatives described herein against Mycobacterium tuberculosis.

TABLE-US-00014 TABLE 10 Antimicrobial activity against Mycobacterium tuberculosis, hemolytic activity and selectivity index of some typical AM-series compounds. MIC100 Hemolytic nmol/ml concentration, Selectivity Code number Mw (g/ml) HC50 (g/ml) index AM000 410.46 NI 6.5 (alpha- mangostin) AM002 656.77 NI AM005 680.46 NI AM008 692.97 4 (2.77) 14 5.05 AM009 692.88 8 (5.54) >1000 >180 AM012 654.79 NI AM-013 396.43 125 (49.55) 25 0.50 (Gamma- mangostin) AM015 660.88 4 (2.64) 25 9.46 AM016 664.91 4 (2.66) 19.6 7.37 AM071 526.53 250 37.5 0.28 AM072 556.64 250 ND Ethambutol 204.31 20 Note: NI: no activity at 250 nmol/mL

[0273] As can be seen from the results presented in Table 10 AM-009 appears to have the best selectivity against Mycobacterium tuberculosis.

REFERENCES

[0274] [1] Ooi, et al, XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity, Journal of Antimicrobial Chemotherapy (2009)64, 735-740.

APPLICATIONS

[0275] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.