Antibacterial cell-penetrating peptides

Abstract

The present disclosure relates to novel antibacterial cell-penetrating peptides and derivatives, and methods to make and use the novel antibacterial cell-penetrating peptides and derivatives. The novel antibacterial cell-penetrating peptides of the present invention with shorter linker between a pyrrolidine ring and a guanidine group provide unexpectedly higher potency against a broader scope of bacterial.

Claims

1. A compound of Formula I: ##STR00016## or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, or derivative thereof, wherein: R.sup.1 is H, an optionally substituted straight, branched or cyclic alkyl or acyl, an optionally substituted aryl or aroyl, an optionally substituted heteroaryl or heteroaroyl, an amino acid moiety, a dye moiety, a fluorophore moiety, a pharmaceutical conjugating agent moiety, an antibiotic moiety, or ##STR00017## R.sup.2 and R.sup.3 are each independently H, a C.sub.1-C.sub.8 branched or unbranched alkyl chain, a C.sub.3-C.sub.8 cyclic alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; R.sup.4 is H, a C.sub.1-C.sub.8 branched or unbranched alkyl chain, or a C.sub.3-C.sub.8 cyclic alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; R.sup.5 is H, a dye moiety, a fluorophore moiety, a pharmaceutical conjugating agent moiety, or an antibiotic moiety; L is C.sub.1-C.sub.8 branched or unbranched alkyl chain, or a C.sub.3-C.sub.8 cyclic alkyl; X and Y are each independently a bond, or a C.sub.1-C.sub.3 linker, wherein one of the carbon of the C.sub.1-C.sub.3 linker is optionally replaced with a heteroatom selected from the group consisting of N, O, and S; Z is a bond, or a linker comprising an optionally substituted straight, branched or cyclic alkyl, an amide group, a carbonyl group, a heteroatom selected from the group consisting of N, O, and S, a disulfide bond (SS bond), or any combination thereof; and n is 2-8.

2. The compound of claim 1, wherein R.sup.1 and/or R.sup.5 each independently represents the moiety of an aminoglycoside antibiotics or any derivative thereof.

3. The compound of claim 1, wherein R.sup.1 and/or R.sup.5 each independently represents an antibiotic moiety, wherein the antibiotic moiety is of an antibiotics selected from the group consisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin, Linezolid, Erythromycin, Eperezolid, and any derivative thereof.

4. The compound of claim 1, wherein R.sup.2 and R.sup.3 are each independently C.sub.1-C.sub.4 branched or unbranched alkyl chain.

5. The compound of claim 1, wherein R.sup.2 and R.sup.3 are isobutyl group.

6. The compound of claim 1, wherein R.sup.4 is hydrogen.

7. The compound of claim 1, wherein L is (CH.sub.2).

8. The compound of claim 1, wherein R.sup.1 and/or R.sup.5 each independently represents an antibiotic moiety, wherein the antibiotic moiety is of an antibiotics selected from the group consisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin, Linezolid, Erythromycin, Eperezolid, and any derivative thereof; wherein R.sup.2 and R.sup.3 are isobutyl group; R.sup.4 is hydrogen; L is (CH.sub.2); X and Y are a bond; Z is a carbonyl group; and n is 4.

9. The compound of claim 1 wherein, R.sup.1 and/or R.sup.5 each independently represents an aminoglycoside antibiotics moiety, and the aminoglycoside antibiotics is selected from the group consisting of Gentamicin, Kanamycin, Tobramycin, Amikacin, Neomycin, Plazomicin, and any derivative thereof; R.sup.2 and R.sup.3 are isobutyl group; R.sup.4 is hydrogen; L is (CH.sub.2); X and Y are a bond; Z is a carbonyl group; and n is 4.

10. The compound of claim 1, wherein the compound is: ##STR00018## ##STR00019## or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, or derivative thereof.

11. A method for treating a subject in need thereof, having a bacterial biofilm infection with an effective amount of compound of claim 1, or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, or derivative thereof.

12. The method of claim 11, wherein the compound of claim 1 inhibits the formation of a biofilm.

13. The method of claim 11, wherein the compound of claim 1 inhibits the growth of an established biofilm.

14. The method of claim 11, wherein the compound of claim 1 is anti-inflammatory.

15. The method of claim 11, wherein the compound of claim 1 is: ##STR00020## ##STR00021## or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, or derivative thereof.

Description

DETAILED DESCRIPTION

(1) For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

(2) In the present disclosure the term about can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

(3) As used herein, the term salts and/or pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.

(4) As used herein, the term nitrogen-protecting group in the present disclosure may be any functional group that can make the amine nitrogen to be protected as any form of carbamate, benzyl amine, amide, thioamide, sulfonamide, urea, or thiourea. The nitrogen-protecting group may include but is not limited to benzoyl, benzyloxycarbonyl, t-butoxycarbonyl (Boc), benzene sulfonyl, toluene sulfonyl, benzyl, benzhydryl, trityl, acetyl, fluorenylmethyloxycarbonyl protecting group (Fmoc), or trifluoroacetyl.

(5) As used herein, a fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several bonds. A fluorophore for the present disclosure may be but is not limited to fluorescein, rhodamine, Oregon green, eosin, and Texas red, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, squaraines, SeTau, Square dyes, naphthalene, or derivatives thereof.

(6) As used herein, the term dye is a colored substance that has an affinity to the substrate to which it is being applied. A dye may be a fluorophore material.

(7) As used herein, the term antibiotics refers to any anti-bacterial antibiotics, which are antibiotics that do not have activity against viruses, fungi and other non-bacterial microbes. The anti-bacterial antibiotics include bactericidal antibiotics, which destroy bacteria, and bacteriostatic antibiotics which prevent bacteria from multiplying. The anti-bacterial antibiotics further include narrow-spectrum antibiotics which target particular types of bacteria, such as Gram negative or Gram-positive bacteria, and broad spectrum antibiotics which affect a wide range of bacteria. Likewise, the anti-bacterial antibiotics include antibiotics for ingestion as well as antibiotics for intravenous administration which are often used to treat serious infections such as deep-seeded systemic infections, and antibiotics for topical and inhalation administration. The anti-bacterial antibiotics comprise antibiotics within the following presently recognized classes: aminoglycoside antibiotics, Ansamycins, Beta-lactam antibiotics (including the carbacephem, carbapenems, cephalosporins (first, second, third and fourth generations), monobactams and penicillins, Glycopeptides, Macrolides, lincosamides, Polypeptides, Quinolones, Sulphonamides, Tetracyclines, Cyclic lipopeptides, Glycylcyclines, Oxazolidinones, diaminopyrimidines, Nitrofurans, Rifamycins, antibiotic peptides, amphenicols, nitroimidazoles, streptogramins and phosphomycins.

(8) As used herein, the particular term aminoglycoside antibiotics refers to a medicinal and bacteriologic category of traditional antibacterial therapeutic agents that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside (sugar). The term can also refer more generally to any organic molecule that contains amino sugar substructures. Aminoglycoside antibiotics may include but is not limited to Gentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, or any derivative thereof.

(9) As used herein, the term antibiotic moiety as referred in a chemical structure refers to the structure of an antibiotic agent that is chemically linked to the structure of another chemical or partial structure of another chemical, through a chemical functional groups such as an ester group, an amide group or an acyl amide group through the hydroxyl, amino or amide group of the antibiotic agent.

(10) As used herein, the term pharmaceutical conjugating agent moiety refers to the partial structure of any suitable pharmaceutical agent such as a FDA approved drug that can be chemically linked to the structure of another chemical or partial structure of another chemical, through a chemical functional groups.

(11) The term cleavable means that the compound of the present disclosure can be cleaved from a disulfide bond (SS).

(12) Comparing with previously disclosed antibacterial cell-penetrating peptides, the present disclosure provides novel antibacterial cell-penetrating peptides that have shorter linker between a pyrrolidine ring and a guanidine group. Such modified peptides with shorter linkers, such as Example 1, provided unexpectedly higher potency against a broader scope of bacterial (see Table 1 of the present disclosure).

(13) The present invention provides novel cleavable conjugated cell penetrating peptides. Accordingly, the present invention provides a compound of Formula I:

(14) ##STR00003##
or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, or derivative thereof, wherein:

(15) R.sup.1 is H, an optionally substituted straight, branched or cyclic alkyl or acyl, an optionally substituted aryl or aroyl, an optionally substituted heteroaryl or heteroaroyl, an amino acid moiety, a dye moiety, a fluorophore moiety, a pharmaceutical conjugating agent moiety, an antibiotic moiety, or

(16) ##STR00004##

(17) R.sup.2 and R.sup.3 are each independently H, a C.sub.1-C.sub.8 branched or unbranched alkyl chain, a C.sub.3-C.sub.8 cyclic alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl;

(18) R.sup.4 is H, a C.sub.1-C.sub.8 branched or unbranched alkyl chain, or a C.sub.3-C.sub.8 cyclic alkyl, an optionally substituted aryl, or an optionally substituted heteroaryl;

(19) R.sup.5 is H, a dye moiety, fluorophore moiety, a pharmaceutical conjugating agent moiety, or an antibiotic moiety;

(20) L is C.sub.1-C.sub.8 branched or unbranched alkyl chain, or a C.sub.3-C.sub.8 cyclic alkyl;

(21) X and Y are each independently a bond, or a C.sub.1-C.sub.3 linker, wherein one of the carbon of the C.sub.1-C.sub.3 linker is optionally replaced with a heteroatom selected from the group consisting of N, O, and S;

(22) Z is a bond, or a linker comprising an optionally substituted straight, branched or cyclic alkyl, an amide group, a carbonyl group, a heteroatom selected from the group consisting of N, O, and S, a disulfide bond (SS bond), or any combination thereof; and

(23) n is 2-8.

(24) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents the moiety of an aminoglycoside antibiotics or any derivative thereof.

(25) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents an aminoglycoside antibiotics moiety, wherein the aminoglycoside antibiotics is selected from the group consisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, and any derivative thereof.

(26) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents Vancomycin, Linezolid, Erythromycin, Eperezolid, or any derivative thereof.

(27) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents an antibiotics moiety of an antibiotics, and wherein the antibiotics is selected from the group consisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin, Linezolid, Erythromycin, Eperezolid, and any derivative thereof.

(28) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.2 and R.sup.3 are each independently C.sub.1-C.sub.4 branched or unbranched alkyl chain.

(29) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.2 and R.sup.3 are isobutyl group.

(30) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.4 is hydrogen.

(31) In one embodiment, the present invention provides a compound of Formula I, wherein L is C.sub.1-C.sub.4 branched or unbranched alkyl chain. In one aspect, L is a methylene group.

(32) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents an antibiotics moiety of an antibiotics, and wherein the antibiotics is selected from the group consisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin, Linezolid, Erythromycin, Eperezolid, and any derivative thereof; R.sup.2 and R.sup.3 are isobutyl group; R.sup.4 is hydrogen; and L is (CH.sub.2); X and Y are each independently a bond; and n is 4.

(33) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents an aminoglycoside antibiotics moiety of an antibiotics, and wherein the antibiotics is selected from the group consisting of Gentamicin, Kanamycin, Tobramycin, Amikacin, Neomycin, Plazomicin, and any derivative thereof; R.sup.2 and R.sup.3 are isobutyl group; R.sup.4 is hydrogen; L is (CH.sub.2); X and Y are each independently a bond; and n is 4.

(34) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.1 and/or R.sup.5 represents an aminoglycoside antibiotics moiety, and the aminoglycoside antibiotics is Kanamycin or any derivative thereof; R.sup.2 and R.sup.3 are isobutyl group; R.sup.4 is hydrogen; L is (CH.sub.2); X and Y are each independently a bond; and n is 4.

(35) In one embodiment, the present invention provides a compound of Formula I, wherein R.sup.5 is a fluorescein moiety, R.sup.2 and R.sup.3 are isobutyl group; R.sup.4 is hydrogen; L is (CH.sub.2); X and Y are a bond; Z is a carbonyl group; and n is 4 (Formula II):

(36) ##STR00005##

(37) In one embodiment, the present invention provides a compound selected from the group consisting of:

(38) ##STR00006## ##STR00007##
a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, derivative, and prodrug thereof.

(39) In one embodiment, the present invention provides a method for treating a patient, either mammal or animal, having a microbial infection with any compound of Formula (I), or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, or derivative thereof. In one aspect, the compound of Formula (I) is used for inhibiting the formation of a biofilm. In one aspect, the compound of Formula (I) is used for inhibiting the growth of an established biofilm. In one aspect, the compound of Formula (I) is used for anti-inflammatory purpose. In one aspect, the method comprises using a compound selected from the group consisting of:

(40) ##STR00008## ##STR00009##
a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, derivative, and prodrug thereof.

EXPERIMENTAL SECTION

Synthesis of Example 1

(41) ##STR00010##

Example 1

About 1:1 Mixture Originated from the about 1:1 Mixture of 5-(and 6-)Carboxyfluorescein

Step 1: Fmoc-GAP(Boc).SUB.2 .(2) Synthesis

(42) ##STR00011##

(43) To Fmoc-L-Pro(4-NHBoc)-OH(2S,4R) (1) (200 mg, 0.44 mmol) in a 25 mL round bottom flask with a stir bar was added a triflouroacetic acid (TFA) and dichloromethane (DCM) solution (1:1, 2 mL). The reaction was stirred for 3 h at room temperature. The TFA/DCM solution was removed in vacuo and dried under high vacuum for 12 h. The residue was solubilized in DCM (2 mL) with sonication and N,N-di-tert-butoxycarbonyl-1H-pyrazole-1 (150.9 mg, 0.49 mmol) was added to the reaction flask followed by triethylamine (TEA) (249 L, 1.77 mmol). The resulting solution was stirred for 12 h at room temperature. The reaction mixture was extracted with a saturated sodium bicarbonate solution (3). Followed by extraction with brine (2). The organic layer was dried over sodium sulfate and the organic solvent was removed in vacuo. The crude material was purified by flash chromatography on silica gel with an eluent composed of 93% DCM and 7% methanol (MeOH). The desired fractions were collected and the solvent was removed in vacuo to yield a white solid, Fmoc-GAP(Boc).sub.2 (2) in 60% yield. MW: 594, ESI.sup.+MS: 595 m/z (M+H.sup.+).

Step 2: P14-GAP Peptide Synthesis

(44) To a 10 mL peptide synthesis flask was added Rink-Amide ChemMatrix resin (50 mg, 0.24 mmol/g loading). The resin was washed with dimethylformamide (DMF) (2, about 4 mL each). In a 13100 mm culture tube amino acid, Fmoc-P.sub.L, (Compound 3, FIG. 1, JACS, 2005, 127, 11798-11803) (24.6 mg, 0.06 mmol) was activated with hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (22.8 mg, 0.24 mmol) and diisopropylethylamine (DIEA) (20.9 L, 0.12 mmol) in DMF (4 mL) with sonication for 10 min. The resulting solution was added to the peptide synthesis flask, and the flask was agitated for 4 h. The solution was drained from the synthesis flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Piperidine in DMF (20%, 4 mL) was added to the reaction flask, and the flask was agitated for 30 min. The piperidine was drained and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Next, amino acid Fmoc-GAP(Boc).sub.2 (Compound 2, FIG. 1) (35.7 mg, 0.06 mmol) was activated with HATU (22.8 mg, 0.24 mmol) and DIEA (20.9 L, 0.12 mmol) in DMF (4 mL) in a 13100 mm culture tube with sonication for 10 min. The resulting solution was added to the peptide synthesis flask, and the flask was agitated for 4 h. The solution was drained from the synthesis flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Piperidine in DMF (20%, 4 mL) was added to the reaction flask, and the flask was agitated for 30 min. The piperidine was drained and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). This procedure was repeated to until the desired peptide sequence was synthesized ending with P.sub.L as the last amino acid. Commercially available Fmoc-Glycine (Compound 4, FIG. 1) (21.39 mg, 0.072 mmol) was activated with HATU (27.4 mg, 0.072 mmol) and DIEA (25 L, 0.144 mmol) in DMF (4 mL) in a 13100 mm culture tube with sonication for 10 min. The resulting solution was added to the peptide synthesis flask, and the flask was agitated for 3 h. The solution was drained from the synthesis flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Piperidine in DMF (20%, 4 mL) was added to the reaction flask, and the flask was agitated for 30 min. The piperidine was drained and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Finally, commercially available NHS-Fluorescein (Compound 5, a 1:1 mixture of 5-(and 6-)carboxyfluorescein) (12.49 mg, 0.026 mmol) dissolved in DMF (4 mL) with added DIEA (8.3 L, 0.048 mmol) was added the reaction flask which was protected from light with foil. The flask was agitated for 24 h. The solvent was drained from the reaction flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM (2, about 4 mL each) followed by drying under high vacuum for 2 h.

(45) ##STR00012##

Step 3: Cleavage and Purification of P14-GAP Peptide

(46) A TFA cocktail composed of TFA, triisopropylsilyl (TIPS), and water (95:2.5:2.5, 10 mL) was added to the peptide synthesis flask. The flask was agitated for 2 h. The solution was drained into a 50 mL centrifuge tube, the resin was rinsed with DCM (5 mL) and the wash was added to the same centrifuge tube. The solvent was removed in vacuo, the crude peptide was precipitated using cold diethyl ether (25 mL) and the tube was placed in a 80 C. freezer overnight. The precipitate was collected by centrifugation at 3500 rpm and the supernatant was decanted. The resulting precipitate was dissolved in water and purified using reverse phase high performance liquid chromatography (RP-HPLC) using a C18 semi preparative column. The eluent was composed of solvent A (acetonitrile with 0.1% TFA) and solvent B (water with 0.1% TFA) at a gradient of 15%-65% eluent A, at a flow rate of 12 mL/min, and UV detection at 214 nm and 254 nm. Fractions of the desired peptide were combined and lyophilized to obtain Fl P14-GAP Example 1 as a yellow solid (about 1:1 mixture originated from the about 1:1 mixture of 5-(and 6-)carboxyfluorescein). Analysis of the purity of the peptide was achieved by analytical RP-HPLC using a C18 analytical column, a flow rate of 1.2 mL/min, and UV detection at 214 nm and 254 nm; a purity of >95% was obtained. Characterization was achieved using matrix assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry. MW: 2510, MALDI-TOF.sup.+MS: 2510 m/z.

Synthesis of Example 2 and 3

(47) ##STR00013##

Example 2

About 1:1 Mixture Originated from the about 1:1 Mixture of 5-(and 6-)Carboxyfluorescein

Scheme 2 Provides the Synthetic Route for Preparing Compound 12

(48) ##STR00014##

Synthesis of Compound 7

(49) A flamed dried 1000 mL rb flask under N.sub.2 (g) was charged with a magnetic stir bar, cis-N-boc-hydroxyproline, compound 6 (5.00 g, 21.6 mmol) and dry THF (430 mL). The flask was placed on an ice bath and to this stirring solution was added benzylbromide (3.35 mL, 28.1 mmol) and trimethylamine (3.91 mL, 28.1 mmol) dropwise. The reaction was allowed to warm up to room temperature and stirred for 24 hr. At this time, solvent was removed by rotary evaporation and the residue was dissolved in DCM (400 mL), washed with 10% HCl (200 mL), water (200 mL), sat. NaHCO.sub.3 (200 mL) and brine (200 mL). The organic layer was then dried (MgSO.sub.4, anhydrous), filtered and concentrated by rotary evaporation. The residue was dissolved in minimal DCM and purified by flash chromatography (silica gel, 5% MeOH/DCM) to yield Compound 7 as a colorless oil (5. 90 g, 18.3 mmol). R.sub.f=0.65 in 10% MeOH/DCM. .sup.1H NMR (CDCl3, 400 MHz): 7.35 (m, 5H), 5.36 (m, 1H), 5.32 (m, 1H), 5.56 (m, 2H), 3.15 (br, 1H), 2.30 (m, 1H), 2.07 (dt, J=16 Hz, 4 Hz, 1H), 1.39 (s, 9H).

Synthesis of Compound 8

(50) A flame dried 200 mL rb flask under N.sub.2(g) was charged with a magnetic stir bar, Compound 7 (4.25 g, 13.2 mmol) and dry DCM (50 mL). The flask was placed on an ice bath and to this stirring solution was added trimethylamine (3.70 mL, 26.4 mmol) followed by addition of tosyl chloride (3.77 g, 19.8 mmol) in portions over 30 min. (on smaller scales I usually add TsCl all at once). Reaction was allowed to warm up to room temperature and stirred for 24 hr. At this time the reaction was diluted with DCM (150 mL), washed with water (275 mL), dried (MgSO.sub.4, anhydrous), filtered and concentrated. The residue was dissolved in a minimal amount of DCM and purified by flash chromatography (35% EtOAc/Hexanes) to yield Compound 8 as a colorless oil (5.02 g, 10.6 mmol, 80% yield). R.sub.f=0.3 in 35% EtOAc/Hexanes. .sup.1H NMR (CDCl3, 400 MHz): 7.70 (d, J=12, 2H), 7.31 (m, 7H), 5.10 (m, 2H), 5.08 (m, 1H), 4.42 (t, J=6 Hz, 1H), 3.65 (d, J=6 Hz, d, 1H), 3.57 (td, J=14 Hz, 4 Hz, 1H), 2.44 (m, 1H), 2.42 (s, 3H), 2.35 (q, J=4 Hz, 1H), 1.35 (s, 9H).

Synthesis of Compound 9

(51) A flame dried 2-neck round-bottom flask was equipped with a reflux condenser and charged with a magenetic stir bar and Compound 8 (4.70 g, 9.99 mmol). The reaction flask was purged with N.sub.2 (g) and DMSO (67 mL) was added. To this stirring solution was added KCN (0.976 g, 15.0 mmol) and the reaction was heated to 60 C. for 4 hrs. After allowing to cool to room temperature, the reaction was diluted with EtOAc (150 mL) and washed with water (275 mL) and brine (75 mL). The organic layer was dried (MgSO.sub.4, anhydrous), filtered and concentrated. The residue was dissolved in minimal amount of DCM and purified by flash chromatography (20% EtOAc/Hexanes) to yield Compound 9 as a white solid (2.15 g, 6.49 mmol, 65% yield). R.sub.f=0.30 in 20% EtOAc/Hexanes. .sup.1H NMR (CDCl3, 400 MHz): 7.35 (m, 5 H), 5.17 (m, 2H), 4.47 (m, 1H), 3.89 (m, 1H), 3.65 (m, 1H), 3.21 (quint, J=8 Hz, 1H), 2.52 (m, 1H), 2.34 (m, 1H), 1.40 (s, 9H).

Synthesis of Compound 10

(52) Compound 9 (1.90 g, 5.75 mmol) was dissolved in TFA:DCM (1:1, v/v, 65 mL) and stirred for 30 min. Solvent was removed by rotary evaporation and the residue was dissolved in 1:1 dioxane: sat. NaHCO.sub.3 (130 mL total). FmocOSu (2.52 g, 7.48 mmol) was added and the reaction was stirred overnight. The reaction solution was extracted with EtOAc (3200 mL) and the combined organic layers were dried (MgSO.sub.4, anhydrous), filtered and concentrated. The residue was dissolved in minimal DCM and purified by flash chromatography (35% EtOAc/Hexanes) to yield Compound 10 as a colorless oil (2.35 g, 5.12 mmol, 90% yield). R.sub.f=0.30 in 35% EtOAc/hexanes. .sup.1H NMR (CDCl3, 400 MHz): 7.78 (m, 2H), 7.53 (m, 2H), 7.40 (m, 2H), 7.32 (m, 7H), 7.13 (dd, J=24 Hz, 12 Hz), 5.15 (m, 2H), 4.44 (m, 3H), 4.14 (t, J=6 Hz, 1H), 3.94 (m, 1H), 3.71 (m, 1H), 3.25 (m, 1H), 2.52 (m, 1H), 2.38 (m, 1H).

Synthesis of Compound 11

(53) A flame dried 500 mL round-bottom flask under N.sub.2(g) was charged with a magnetic stir bar, Compound 10 (2.15 g, 475 mmol), and dry MeOH (120 mL). To this stirring solution was added CoCl.sub.2.6H.sub.2O (2.29 g, 9.62 mmol). The flask was placed on an ice bath and NaBH.sub.4 (1.80 g, 47.5 mmol) was added in portions. The reaction was stirred at 0 C. for 30 min. At this time 1H-Pyrazole-N,N-di-boc-carboxamidine (5.90 g, 19.0 mmol) was added in portions and the reaction was allowed to warm up to room temperature and stirred overnight. After 12 hr the solution was filtered through a celite pad and the celite pad was rinsed with MeOH. The filtrate was diluted with an equal amount of water and MeOH was removed by rotary evaporation. The aqueous layer was extracted with EtOAc (3100 mL), dried (MgSO.sub.4, anhydrous), filtered and concentrated by rotary evaporation. The residue was dissolved in minimal DCM and purified by flash chromatography (20% EtOAc/Hexanes) to yield Compound 11 as a colorless oil (2.04 g, 2.92 mmol, 61% yield). R.sub.f0.41 in 40% EtOAc/Hexanes. .sup.1H NMR (CDCl3, 400 MHz): 11.49 (s, 1H), 8.45 (m, 1H), 7.76 (m, 2H), 7.56 (m, 2H), 7.38 (m, 2H), 7.30 (m, 7H), 5.14 (m, 2H), 4.42 (m, 3H), 4.13 (t, J=8 Hz, 1H), 3.84 (dt, J=9 Hz, 8 Hz, 1H), 3.48 (m, 1H), 3.25 (m, 1H), 2.64 (m, 1H), 2.15 (m, 1H), 2.04 (m, 1H), 1.50 (s, 18H). ESI-MS [MH].sup.+ expected: 699.3388, observed 699.3384.

Synthesis of Compound 12

(54) Compound 11 (2.01 g, 2.88 mmol) was dissolved in 1:1 EtOAc/Ethanol (30 mL) and to this stirring solution was added 10% Pd/C (0.300 g, 10 mol %). The solution was purged with N.sub.2 (g) placing under H.sub.2 (g). After 4 hr, the solution was filtered through a celite pad and the celite pad was rinsed with MeOH. The filtrate was concentrated and the residue was purified by flash chromatography (3% MeOH/DCM) to yield Compound 12 as a white solid (1.44 g, 2.37 mmol, 82% yield). R.sub.f=0.47 in 10% MeOH/DCM. .sup.1H NMR (CDCl3, 400 MHz): 11.51 (br, 1H), 8.47 (m, 1H), 7.72 (d, J=8 Hz, d, 2H), 7.56 (m, 2H), 7.38 (m, 2H), 7.30 (m, 2H), 4.39 (m, 3H), 4.19 (t, J=8 Hz, 1H), 3.75 (m, 1 Hz), 3.48 (m, 2H), 3.18 (quint, J=8 Hz), 2.63 (m, 1H), 2.28 (m, 1H), 2.00 (m, 1H), 1.49 (br, 18H) ESI-MS [MH].sup.+ expected: 609.2919, observed: 609.2923.

Synthesis of Example 2

(55) Peptide Synthesis: To a 10 mL peptide synthesis flask was added 50 mg of Rink-Amide ChemMatrix resin (0.24 mmol/g loading). The resin was washed with dimethylformamide (DMF) (2, about 4 mL each). In a 13100 mm culture tube amino acid, Compound 12 (24.6 mg, 0.06 mmol) was activated with hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (22.8 mg, 0.06 mmol) and diisopropylethylamine (DIEA) (20.9 L, 0.12 mmol) in DMF (4 mL) with sonication for 10 min. The resulting solution was added to the peptide synthesis flask, and the flask was agitated for 4 h. The solution was drained from the synthesis flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Piperidine in DMF (20%, 4 mL) was added to the reaction flask, and the flask was agitated for 30 min. The piperidine was drained and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Next, Fmoc-GAP(Boc).sub.2 (35.7 mg, 0.06 mmol) was activated with HATU (22.8 mg, 0.06 mmol) and DIEA (20.9 L, 0.12 mmol) in DMF (4 mL) in a 13100 mm culture tube with sonication for 10 min. The resulting solution was added to the peptide synthesis flask, and the flask was agitated for 4 h. The solution was drained from the synthesis flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Piperidine in DMF (20%, 4 mL) was added to the reaction flask, and the flask was agitated for 30 min. The piperidine was drained and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). This procedure was repeated to until the desired peptide sequence was synthesized. Commercially available Fmoc-glycine (21.39 mg, 0.072 mmol) was activated with HATU (27.4 mg, 0.072 mmol) and DIEA (25 L, 0.144 mmol) in DMF (4 mL) in a 13100 mm culture tube with sonication for 10 min. The resulting solution was added to the peptide synthesis flask, and the flask was agitated for 3 h. The solution was drained from the synthesis flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Piperidine in DMF (20%, 4 mL) was added to the reaction flask, and the flask was agitated for 30 min. The piperidine was drained and the resin was washed sequentially with DMF, DCM, MeOH, DCM and DMF (2, about 4 mL each). Completion of deprotection and coupling reactions were confirmed for primary and secondary amines using the Kaiser and Chloranil test, respectively. Finally, when appropriate, commercially available NHS-fluorescein (12.49 mg, 0.026 mmol) dissolved in DMF (4 mL) with added DIEA (8.3 L, 0.048 mmol) was added to the reaction flask which was protected from light with foil. The flask was agitated for 24 h. The solvent was drained from the reaction flask and the resin was washed sequentially with DMF, DCM, MeOH, DCM (2, about 4 mL each) followed by drying under high vacuum for 2 h.

(56) Cleavage and Purification of Peptides: About 5 mL of a TFA cocktail composed of 95% TFA, 2.5% triisopropylsilane (TIPS), and 2.5% water was added to the peptide synthesis flask. The flask was agitated for 2 h. The solution was drained into a 50 mL centrifuge tube, the resin was rinsed with 5 mL of TFA cocktail followed by 5 mL of DCM. The washes were drained into centrifuge tube containing the cleaved peptide. The solvent was removed in vacuo, the crude peptide was precipitated using cold diethyl ether (25 mL) and the tube was placed in a 80 C. freezer overnight. The precipitate was collected by centrifugation at 3500 rpm and the supernatant was decanted. The resulting precipitate was dissolved in deionized water (10 mg/mL) and purified using reverse phase high performance liquid chromatography (RP-HPLC) using a C18 semi preparative column. The eluent was composed of solvent A (acetonitrile with 0.1% TFA) and solvent B (water with 0.1% TFA) at a gradient of 15-70% eluent A, flow rate of 12 mL/min, and UV detection at 214 nm and 254 nm. Fractions of the desired peptide were combined, the solvent was removed in vacuo, and lyophilized to obtain the peptides. Analysis of the purity was achieved by analytical RP-HPLC using a C18 analytical column, a flow rate of 1.2 mL/min, and UV detection at 214 nm and 254 nm; a purity of >95% was obtained with retention times of 20.1 (Fl-P14GAPC1) min. The peptide product was characterized using matrix assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry. Mass expected (Fl-P14GAPC1): 2623, MALDI-ToF Obtained Mass: 2624 m/z.

Synthesis of Example 3

(57) ##STR00015##

Example 3

(58) Example 3 was prepared by the method substantially the same for making Example 1. In the peptide synthesis step, no fluorophore was added, instead the resin was treated with acetic anhydride (0.3 mL) and DIEA (0.17 mL) in DMF for 2 hours. In the cleavage and purification of peptide step, the eluent was composed of solvent A (acetonitrile with 0.1% TFA) and solvent B (water with 0.1% TFA) at a gradient of 15-65% eluent A, with retention time of 19.7 minutes. Mass expected (Ac-P13GAP): 2137, MALDI-ToF Obtained Mass: 2138 m/z.

Characterization of the ConjugatesAnalytical HPLC

(59) Purity was determined by analytical RP-HPLC using a C18 reverse phase analytical column (5 m, 4 mm250 mm; Phenomenex Luna) at a flow rate of 1.2 mL/min. UV detection was at 214 nm. A 30 min gradient of 15-55%, 25-55% and 30-70% solvent A (A: acetonitrile and 0.05% trifluoroacetic acid (TFA) gradient of 15-55%, 25-55% and 30-70% solvent A (A: acetonitrile and 0.05% trifluoroacetic acid (TFA); B: water and 0.05% TFA) was used.

Characterization of the ConjugatesMass Spectrometry

(60) Peptides were further characterized using matrix associated laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Voyager DE, Applied Biosystems).

Method for In Vitro Antimicrobial Activity Assessment

Activities Against Pathogenic Bacteria

(61) The minimum inhibitory concentration (MIC) of the peptides (in uM/ml) and control antibiotics (vancomycin and gentamicin) in g/mL, were determined using the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) guidelines. See CLSI. 2007. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A7. CLSI, Wayne, Pa.

(62) TABLE-US-00001 TABLE 1 Comparision of MIC values [M] for the antibacterial activity MIC (uM)/MBC P14- P14-GAP Vanco- Genta- Strain ID LRR (Example 1) mycin micin Activity against ESKAPE pathogens E. faecium 700221 2 1 >32 >32 S. aureus NRS 384 16 2 0.5 0.5 K. pneumoniae 1706 >32 8 >32 2 K. pneumonia NR 41920 32 4 >32 1 K. pneumonia NR 41928 >32 1 >32 1 A. baumannii 1605 1 0.5 8 >32 P. aeruginosa 50573 16 2 >32 0.25 E. cloacae 1134 32 2 >32 0.5 Activity against more Staphylococcus spp. S. aureus NRS 383 32 2 4 >32 S. aureus NRS 382 32 2 1 0.5 S. aureus RN 4220 32 2 0.5 0.5 S. aureus ATCC 6538 16 1 0.5 0.125 S. epidermidis NRS 101 2 1 2 32 VRSA 5 16 2 4 0.5 VRSA 10 8 2 >32 0.5 Activity against more Pseudomonas aeruginosa P. aeruginosa 48982 32 4 >32 >32 P. aeruginosa 31040 32 2 >32 16 P. aeruginosa 31041 32 2 >32 32 Activity against more Acinetobacter baumannii A. baumannii 1747 1 0.5 >32 0.25 A baumannii 19606 2 1 32 16 Activity against enteric pathogens L. monocytogenes 191112 8 2 1 0.5 S. flexneri 1a 4 1 >32 0.5 S. enteritidis 4 1 >32 0.25 E. coli 21922 4 1 >32 2 S. typhimurium LT2 16 1 >32 0.5

(63) Antimicrobial activities of Example 1 (P14-GAP) and other anti-bacterial peptides, antibiotics were evaluated/compared with a series of Gram positive and negative bacteria, including a range of intracellularpathogens (Table 1). Comparing with previously disclosed antibacterial cell-penetrating peptides, the present disclosure provides novel antibacterial cell-penetrating peptides that have shorter linker between a pyrrolidine ring and a guanidine group. Such modified peptides with shorter linkers, such as Example 1, provided unexpectedly higher potency against a broader scope of bacterial. Example 1 is the compound that showed highest potency for every single bacterial. Because the activity of Example 1 came from the peptide moiety alone, the incorporation of other active moieties may provide further benefits.

Time-Kill Curves Against S. aureus MRSA US300 for Example 3

(64) Exponential-phase S. aureus MRSA US300 was diluted in CAMHB media and challenged with the indicated test agents at concentrations representing 2 MIC (a), or 4 MIC (b). Untreated control received phosphate buffered saline (PBS). Viability was enumerated at the indicated time points by serial dilution plating. Each point represents the mean Log.sub.10 CFU/ml +SD of triplicate determinations.

(65) Time to kill curves were obtained with Example 3 and the known antibiotics linezolid and vancomycin against S. aureus MRSA US300. A greater than 5 log reduction in bacteria levels was observed with Example 3 in 2 hours at both 2- and 4-times its MIC value, whereas vancomycin took 24 hours for a similar reduction and linezolid obtained only about a log reduction in 24 hours.

(66) Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.