WO2004093821A2 - Treatment of anthrax infection using inhibitors of lethal factor protease activity - Google Patents

Abstract

Compounds containing spaced N and/or O, by virtue of their ability to inhibit the protease activity of lethal factor from B. anthracis, are useful in the prevention and treatment of anthrax toxicity. Libraries of these compounds are also useful as substrates for screening methods to identify LF inhibitors.

Description

TREATMENT OF ANTHRAX INFECTION USING INHIBITORS OF LETHAL FACTOR PROTEASE ACTIVITY

Cross-Reference to Related Application

[0001] This application claims priority to U.S. provisional application Serial No. 60/464,923 filed 22 April 2003, which is incorporated herein by reference.

Statement of Rights to Inventions Made Under Federally Sponsored Research

[0002] This invention was supported in part by a grant from the National Institutes of Health. The U.S. Government has certain rights in this invention.

Technical Field

[0003] The invention relates to the field of amelioration of anthrax infection. More particularly, it relates to treatment of such infection using compounds such a polyamines, aminoglycoside derivatives, aminocyclitol derivatives, or aryl- or heteroaryl-containing compounds. More particularly, the derivatives of these compounds contain guanidinyl substituents.

Background Art

[0004] Bacillus anthracis is a spore-forming Gram positive bacterium that is the causative agent of anthrax infection. Generally, the spores can enter a subject by oral ingestion, through the skin, or by inhalation. The spores are phagocytized and travel to regional lymph nodes where they germinate and release three proteins (toxins) that are the causative agents ofthe symptoms ofthe infection. While antibiotics such as ciprofloxacin, penicillins, and tetracyclines may be effective in reducing the bacterial infection itself, once the proteins are released, reduction ofthe infection itself does not significantly arrest the course ofthe disease. In addition, in light ofthe attractiveness of anthrax as a biological weapon, modification of wildtype B. anthracis to provide antibiotic resistance is a distinct possibility.

[0005] Therefore, it is important to provide a means to treat anthrax which is independent of antibiotic resistance, and which will be effective even after the toxins have been released from an infection that has not been prevented or treated sufficiently promptly. [0006] The toxins released are in the form of three proteins: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA generates proteins that shuttle LF and EF into an intracellular endosomal compartment. When PA binds to a cell surface anthrax toxin receptor, a 20 kD fragment is cleaved, allowing the PA to heptamerize. The heptamer behaves as the shuttle. PA+LF is lethal toxin (LT) and PA+EF is edema toxin (ET). When LT and ET enter the endosomes, dissociation occurs and LF and EF are released into the cytoplasm to exert toxic effects. EF is the less toxic and is a calmodulin-dependent adenylate cyclase that synthesizes cAMP, leading to edema. LF is a protease that specifically cleaves mitogen-activated protein kinase kinase (MAPKK) in macrophages. Because MAPKK is a critical signaling molecule, removal by this protease results in cell death. The dead cells release cytokines and NO which cause septic shock and death ofthe subject.

[0007] Compounds that inhibit LF protease activity and thus are useful in the prevention or treatment of anthrax infections have been described in U.S. 6,436,933, the disclosure of which is incorporated herein by reference. The compounds described in this patent are l-hydroxy-hydropyrazin-2-ones. These compounds were assessed for their ability to inhibit anthrax lethal factor by assessing their ability to inhibit the cleavage of a GST-MEK1 fusion protein, detected by determining the levels of GST liberated. In addition, inhibiting lethal factor and treating anthrax using a group of compounds having a mandatory sulfonamide core is disclosed in WO03/101382. Further, after the provisional application's filing date ofthe present invention, Lee, L. V. et al., J. Am. Chem Soc. Internet Publication (2004) describes the inhibition constant of neomycin B with respect to lethal factor.

[0008] Recently, Cummings, R.T., et al, Proc. Natl. Acad. Sci. USA (2002) 99:6603-6606 developed a fluorescence resonance energy transfer (FRET) assay to determine the activity of lethal factor protease. In this high throughput assay, a consensus derived peptide was labeled at the N-terminus with a fluorophore and at the C-terminus with a quencher. Cleavage ofthe substrate peptide therefore results in enhanced fluorescence, and thus inhibitors can be assessed by their ability to reduce the level of fluorescence emitted in the assay.

[0009] The present invention resides in the surprising discovery that certain compounds, including amino glycoside-based antibiotics are able to inhibit the protease activity of LF. In general, aminoglycoside antibiotics have been used for treatment of Gram negative bacterial infection rather than Gram positive infections such as anthrax or botulism. A study by Bhatnagar, R., et al, Cell Signal (1999) 11:111-116 showed that neomycin, an aminoglycoside antibiotic, could protect cells pretreated with this compound against anthrax LT. This protection was attributed to the known ability of neomycin to inhibit phospholipase C. This inhibition was considered to control the initiation ofthe inositol-l,4,5-triphosphate signaling cascade initiated by anthrax LT in the test J774A.1 cells.

[0010] Studies have also been conducted ofthe effects of aminoglycosides on mammalian enzymes as described in Asch, H.L., et al, J. Infect. Dis. (1978) 138:257-259; Grimwood, K., et al, Anti-Microb. Agent & Chemo. (1989) 33:41-47; Olbricht, C.J., et al, Kidney Int. (1991) 39:639-646; Levy, F., et al, J. Immunol. (2002) 169:4161-4171; and Sekini, K., et al, Int. J. Cancer (2001) 94:485-491.

[0011] Perguanidinylation of particular aminogylcosides, rather than selective guanidinylation, using a sulfonyl-containing compound is described in U.S. Pat No. 6,525,182. Guanidinylation reagents are described in Feicheinger (Feichtinger, K. et al, J. Org. Chem. <53:8432-8439 (1998)). In addition, the synthesis of particular guanidino aminoglycoside derivatives for the exploration of general antiobiotic potential have been described in Streicher (Streicher, W. et al, Drugs Exptl. Clin. Res. D (8/9) 591-598 (1983)), namely 6-N-guanidino kanamycin A, 1-N-guanidino gentamicins, 2'-and 3-N-guanidino gentamicin CI, 2'-and 6'-N- guanidino gentamicin Cla; and 6'-N-guanidino gentamicin C2.

[0012] With respect to one aminoglycoside in particular, neamine, Ryu, D. H. et al, Bioorg. & Med. Chem. 73:901-903 (2003) discloses the synthesis of enantiomers thereof, the disclosure of which is incorporated herein by reference. ■

[0013] The use of analogs of another class of antibiotics, the tetracyclines, to inhibit mammalian matrix metalloproteases and mammalian serine proteases is described in U.S. patents 6,277,061 and 5,773,430, respectively.

[0014] None ofthe references cited herein are admitted to be prior art.

Disclosure ofthe Invention

[0015] The invention is directed to the prevention and treatment ofthe symptoms of anthrax toxicity by the administration of compounds such as aminoglycosides, aminocyclitols, polyamines, aryl- or heteroaryl-containing compounds or derivatives thereof that inhibit lethal factor protease activity and thus ameliorate lethal factor intoxication. Particularly useful are compounds that have a guanidinyl substituent. Because the compounds ofthe invention are able to effect this inhibition, they can be administered at any time during the course ofthe infection; their ability to inhibit this enzyme results in an amelioration ofthe toxic effects ofthe products of 5. anthracis, and is independent of whether activity as antibiotics is exhibited. Further, the invention provides a high throughput assay to identify aminoglycoside, aminocyclitol, polyamine, aryl- and heteroaryl-containing and fused ring antibiotic inhibitors of this activity.

[0016] A class of compounds useful in the methods and compositions ofthe invention can be defined as compounds having a Ki with respect to inhibition of lethal factor (LF) of <500 μM. More preferably, the compounds ofthe invention have a Ki that is <300 μM, <100 μM, or <20 μM, and even more preferably <5 μM, and most preferably <1 μM. A subset of these compounds also has desired specificity such that its inhibitory activity with respect to LF is at least 10-fold greater than with mammalian proteases which might be present in a vertebrate subject to be treated. Still another subset is highly specific for LF as opposed to other bacterial toxins such as botulism toxin by a factor of 10. The assays ofthe invention are designed to provide rapid information with regard to both to activity against anthrax toxicity and with regard to specificity. The Ki values with respect to LF inhibition for preferred compounds useful in methods and compositions ofthe invention are listed in Tables A and B. Table B further includes Ki values for other proteases.

[0017] In one embodiment ofthe invention, the compounds useful in the methods and compositions ofthe invention have specificity for at least one protease activity in addition to LF. Assays ofthe invention provide a determination with respect to a specificity for an such additional protease such as furin, botulinum A, trypsin, cathepsin B, MM-9 and cathepsin D. Preferably, the useful compounds ofthe invention are dual inhibitors of LF and botulinum A, or LF and furin.

[0018] In one aspect, the invention is directed to a method to prevent or treat anthrax infection and/or lethal factor intoxication which method comprises administering to a subject in need of such prevention or treatment an amount of a compound comprising specifically spaced, staggered O and/or N atoms that inhibits LF activity sufficient to effect such prevention or treatment.

[0019] In one aspect, the compounds useful in the invention are cation-based lethal factor inhibitors. Cation-based compounds includes compounds that have the ability to attract a proton and form a positively charged entity in solution. Such cation-based compounds are believed to be useful due to a structure activity relationship (S AR) in common with the most potent LF inhibitors.

[0020] One class of compounds useful in the invention is that of aminoglycosides which are hexoses in which one or more hydroxyl groups has been replaced by an amine, or more generally, are hexoses that contain at least one N-containing substituent. The hexoses may be further derivatized through either the amino group(s) or hydroxyl group(s) or both. In addition, the aminoglycosides may contain as substituents aminocyclitols or may be coupled to other sugars or other substituents.

[0021] Another class of compounds useful in the methods and compositions ofthe invention is that of aminocyclitols which are cyclohexane moieties which contain hydroxyl and amino substituents in varying numbers. The cyclohexanes may be further derivatized through either the amino group(s) or hydroxyl group(s) or both. The aminocyclitols may contain as substituents aminoglycosides or may be coupled to other aminocyclitols or other substituents. It is clear that some compounds in this class may overlap the class of previously described aminoglycosides. For example, neamine is an aminoglycoside linked to a aminocyclitol.

[0022] Throughout this application, the common name "neamine" will be used for the structure shown below.

[0023] Neamine is also known as neomycin A and ((17?)-4t,6t-diamino-2t,3c-dihydroxy- cyclohex-r-yl)-[2,6-diamino-2,6-dideoxy-a-D-glucopyranoside. Autonom (v.2.2) provides its name as 5-amino-2-aminomethyl-6-(4,6-diamino-2,3-dihydroxy-cyclohexyloxy)-tetrahydro- pyran-3,4-diol. Its CAS registry number is 3947-65-7 (free base).

[0024] Another class of compounds useful in the methods and compositions ofthe invention includes polyamines. Polyamines include diamino alkylenes that optionally contain interspersed nitrogens in the alkylene chain. Polyamines may be linear such as spermine, putrescine, spermidine, diethylenetriamine, N-(3-aminopropyl)-l,3-propanediamme, N-(2-aminopropyl)~ 1,3-propanediamine, and 1,8-diaminooctane. Polyamines also include dentrimers having tetraamino, octaamino, hexadecaamino or other plural amino alkylenes that optionally contain interspersed nitrogens in the branched alkylene chain, such as polypropyleneimine tetraamine dentrimer, polypropyleneimine octaamine dentrimer, or polypropyleneimine hexadecaamine dentrimer. Polyamines may also be ring structures containing interspersed nitrogens in a cycloalkylene ring, such as 1,4,8,11-tetraaza-cyclotetradecane and cyclen. Combinations of polyamines such as cyclic and linear polyamines are also contemplated such as N,N'-bis(3- aminopropyl)piρerazine.

[0025] Polyamines also include polyamino acids, such as polyglutamic acid, polylysine, polyarginine, polyornithine or amino acid copolymers. Specific examples include poly-L- arginine hydrochloride salt, Ac-RRRRRR-NH2, H-RRRRRR-NH2, H-rrrrrr-NH2, poly-L- ornithine hydrobromide salt, Ac-CRAT10 iRRRRRR-NH2₅ poly-L-lysine hydrobromide salt, Ac CRATKRRR-NH2, Ac CRATKMrrr-NH2, Ac CRRRR-NH2, H-RRRRRR-OH, Ac CRARRR-NH2, Ac CRATKMRRR-NH2, Ac-CRRR-NH2, Ac-RRRRRR-OH, Ac-lmtarc-NH2, Ac-CRATKRSR-NH2, Ac-CRATRRR-NH2, Ac-CRARKML-NH2, H-KKKKK-OH (pentalysine) acetate salt, Ac-CRRTKML-NH2, Ac-CRATKRL-NH2, H-KKKK-OH (tetralysine) acetate salt, poly (D-Glu-D-Lys) HBr, H-KKK-OH (trilysine), poly glu sodium salt, and osteoblast adhesive peptide (H-KRSR-OH).

[0026] The class of polyamines also includes large proteins such as lactoferrin, myeloperoxidase (MPO), and alpha 1-PDX.

[0027] Still another class of compounds useful in the methods and compositions ofthe invention includes aryl- and heteroaryl-containing compounds optionally fused to another ring, such as 1,10-phenanthroline, 7-chloro-kynurenic acid, CNQX disodium, alpha tocopherol, guanosine hydrate, l-benzyl-4-(5-nitro-pyridin-2-yl)-piperazine, (l-benzyl-piperidin-4-yl)-(3- nitro-pyridin-2-yl)-amine, and DL-phenylalanine hydroxamate. This class of compounds includes fused ring antibiotics such as tetracycline and echinomycin. Such compounds are characterized by fused ring systems substituted with N and O atoms, and optionally S atoms and which may contain a lactone.

[0028] The compounds that are directly able to inhibit lethal factor activity may be administered as such, as their salts where appropriate, or as prodrugs that release the inhibitory compounds either quickly or over the course of time. One class of such prodrugs includes derivatives that allow binding to serum albumins to protect the compounds from excretion. An example of such a prodrug form of gentamicin is described by Shechter, Y., et al, J. Med. Chem. (2002) 45:4264-4270. Any modified form ofthe inhibitors ofthe invention which will release the inhibitors in vivo may be used. [0029] In another aspect, the invention is directed to pharmaceutical compositions designed for treatment of anthrax infection which comprise, as an active ingredient, one or more compounds ofthe invention, such as aminoglycoside derivatives, aminocyclitol derivatives, polyamine derivatives, aryl- or heteroaryl-containing derivatives and/or fused ring antibiotics.

[0030] In still another aspect, the invention is directed to high throughput methods to identify compounds of these classes useful in treatment of anthrax infection wherein members of an appropriate library are assessed for their ability to inhibit the cleavage by anthrax lethal factor of a peptide substrate which has been provided a fluorescent moiety and a quenching moiety. The specificity of inhibition is further assessed by determining the ability ofthe compound to inhibit the protease activity of mammalian or other vertebrate proteases, such as furin, trypsin, cathepsin B, human Matrix Metalloprotease-9 (MMP-9) and/or cathepsin D and/or to inhibit the protease activity of Botulism neurotoxin subtype A (BoNT/A).

Modes of Carrying Out the Invention

[0031] The compounds useful in the invention are characterized by including, in their structures, staggered, spaced, electronegative atoms able to form H-bonds, i.e., N and/or O. At least two such N and/or O atoms are maintained in the structure at a distance that would be conferred by the presence of 2-11, preferably 2-5, intervening methylene groups. These atoms may be spaced farther in the molecule, so long as sufficient flexibility is provided that a distance typified by that permitted by 2-11, preferably 2-5, intervening methylene groups can be achieved.

[0032] The compounds ofthe invention thus share, in general, a structure ofthe form

X — Y — X (1) wherein each X is independently N or O with appropriate further substitution; Y simply links the two X groups and may itself contain further N and/or O atoms. The structure ofthe molecule is such that at least in some conformations, the distance between the two X groups shown is approximately that conferred by 2-11, or more preferably 2-5 methylene groups. Portions ofthe compound of formula (1) may be in the form of a ring which may include a bridge between the two X moieties as well as a bridge between a portion ofthe Y linker and the X moiety. As used throughout the application, "linker" refers to a bond, where appropriate and/or defined as such, or moiety, which is generally divalent, that connects one moiety to another. [0033] Another group of compounds that exhibits the property of LF inhibition is the group known as aminoglycosides. These compounds are hexoses wherein one or more ofthe hydroxyl groups is replaced by an amino group. Preferably, the hexoses are pyranoses. As hexoses have a multiplicity of chiral centers, the compounds ofthe invention may exist in stereoisomerically pure forms, or as mixtures of stereoisomers. Thus, in general, the compounds useful in the invention are ofthe formula

wherein each of Y^1', Y^2' and Y^Y⁶ is independently R, OR, SR or NR₂ wherein each R is independently H or a substituent that does not interfere with the inhibitory activity ofthe aminoglycoside, and wherein - represents an optional double bond. At least one of Y^ must be NR₂, and no more than 5 of Y¹ , Y² , and Y -Y⁶ can be H. In some embodiments, two of said Y substituents are NR₂, and in some embodiments, three Y substituents are NR₂.

[0034] The non-interfering substituents represented by R, in addition to H, may be optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, and the like. In one aspect, alkyl and acyl contain 1-20C, alkenyl contains 2-40C. In another aspect, the alkyl and aryl groups may contain 1-lOC, preferably 1-5C. In addition, alkenyl and alkynyl groups may contain 2- 10C, preferably 2-6C; aryl groups may contain 5-12 ring members and acyl groups contain 1-lOC, preferably 1-5C. In all ofthe foregoing substituents, one or more ofthe carbon atoms may be replaced with a heteroatom such as N, S or O. In another embodiment, Y¹ and Y¹ may together form an optionally substituted spiro union.

[0035] In general, it is preferred that the substituents do not include adjacent heteroatoms, and in particular, that S and N are not adjacent. The foregoing substituents may alternatively, or in addition, be further substituted for example by OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH and -C(NH )NH and the like. In these substituents, R is defined as set forth above.

[0036] Particularly preferred substituents include additional sugar moieties, including pyranoses, furanoses, and reduced forms thereof. Also preferred as substituents are aminocyclitols. Aminocyclitols are cyclohexane moieties which contain hydroxyl and amino substituents in varying numbers. In another preferred embodiment, the aminoglycosides contain one or more, preferably 1-6, more preferably 1 , 2, 3, 4, 5, or 6 guanidinyl substitutents. A guanidinyl substituent is -NHC(=NH)NH₂. A -C(=NH)-NH₂ group is referred to as an amidino or carbamimidoyl group, and may be attached to an N to form a guanidinyl group.

[0037] Preferred non-interfering substituents on an aminoglycoside include H, OH, NH₂, -NHC(NH)NH₂, -C(NH)NH₂, -CH₃, -CH₂CH₃, -CH(OH)CH₃, -CH₂NH_2; -CH₂NHCH₃, -CH(CH₃)NH₂, -CH(CH₃)NHCH₃,

[0038] An asterisk (*) is used in the depiction of some non-interfering substituents above to show the preferred attachment site, in most cases, to the aminoglycoside. Such an asterisk also has the same meaning with respect to the aminocyclitol and aryl- or hetero-aryl-containing compounds below.

[0039] Particularly useful among the aminoglycosides or derivatives thereof in the invention are those shown in Table B, such as neomycin B hexaguanyl hexatrifluoroacetate salt, tetraguanyl neamine TFA salt, neomycin B (free base), neomycin B tris-sulfate, sisomicin sulfate, netilmicin, gentamicin (mixture), streptomycin, G418, paromomycin, dibeckacin, bekanamycin (kanamycin B), amikacin, neamme (free base), dihydrostreptomycin sesquisulfate, lividomycin A, kanamycin A, apramycin, butirosin B, amphotericin B, tetracycline, ribostamicin, tobramycin, hygromycin B. Particularly preferred guanidinylated neamine comounds are shown in Examples 1-16. As noted above, by the criterion of activity with respect to LF, all of these compounds are satisfactory for this purpose.

[0040] In a preferred embodiment, the aminoglycoside has the formula A, B, C and D below which are respectively 2,6-diamino-2,6-dideoxy-D-glucose, neosamine B, garosamine and 2- deoxystreptamine. Thus, such a preferred aminoglycoside may be selected from the group consisting of

c or an enantiomer thereof wherein each R is independently H or is 1-3 first substituents selected from the group

consisting of A, B, C, and

_?

D or an enantiomer thereof. Each R on the first substituent is independently H, -C(NH₂)NH, a first linker to the aminoglycoside, or a second substituent selected from the group consisting of A, B, C, and D. Each R on the second substituent is H, -C(NH₂)NH, or a second linker to the first substituent. The first and second linkers are independently selected from a bond, a C1-C12 acyl group, a C2-C12 alkyl, a C3-C8 cyclic alkyl, aryl, or heteroaryl. When the acyl group is a carbonyl, it forms preferred connections between O and N or two N's, namely carbamate and urea connections, preferably between two rings. Substitutions as described above may result in two adjacent heteroatoms which are not preferred. As such, where a substitution results in -NH-NH -or -O-O-, it is replaced with -NH- or -O- respectively. Further, when a substitution results in -NH-O -or -O- NH-, either is replaced with either -NH- or -O-. These preferred aminoglycosides contain at least two rings, and preferably 1-3 R groups on the aminoglycoside and the first substituent is A, B, C, or D. Further, 0-4 R groups on the aminoglycoside, the first substituent and the second substituent may contain a guanidinyl group, and guanidinylated compounds are preferred. If an enantiomer is present, an enantiomer of A is the preferred enantiomer.

[0041] Deoxystreptamine (D), or more specifically 2-deoxystreptamine, is preferably included in combinations thereof as this aminocyclitol has structural similarities based on the most potent guanidinyl neamine derivatives with respect to LF inhibition, which include a dihydroxystreptamine moiety. The following compound illustrates the numbering scheme for 2- deoxystreptamine:

2-deoxystreptamine

[0042] This numbering is the same as the numbering as the 2-deoxystreptamine ring in neamine, described above. Deoxystreptamine substituted with 1-4 guanidinyl groups is most preferred.

[0043] Also contemplated is a library comprising these aminoglycosides. In a preferred embodiment, an anomeric carbon of A, B, or C, is linked to D through one ofthe O atoms of D. In another preferred embodiment, the resulting compound is neamine or garamine. A structure of garamine is shown below:

Even more preferably the compound contains 1-4 guanidinyl groups.

[0044] Above and throughout the application, an "anomeric carbon" refers to the position of a carbon in a cyclic stereoisomer of a carbohydrate with isomerism involving only the arrangement of atoms or groups at the aldehyde or ketone position. In the present compounds, it preferably refers to a carbon in the backbone of a cyclic compound such as an aminoglycoside or cyclic sugar that is adjacent an O in the backbone of such cyclic compound, wherem the C is also linked to an O in an O-containing substituent.

[0045] Also contemplated is a library of LF inhibitors which compounds comprise the A, B and C aminoglycosides as defined above. [0046] Compounds ofthe invention and/or useful in methods and compositions ofthe invention, which exhibit the property of LF inhibition, may also have an aminocyclitol core. In one aspect the compound comprises an aminocyclitol of formula (3),

wherein each of X^X⁷ is independently R, OR, or NR₂; and wherein at least 2 of X^!-X⁵ are OR, wherein R is independently H or a substituent that does not interfere with the inhibitory activity ofthe aminocyclitol.

[0047] Preferred R groups are similar to those on an aminoglycoside, such as, in addition to H, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-12 ring members and wherem one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O. As with aminoglycosides, it is preferred that substituents do not include adjacent heteroatoms. Optional substituents may be selected from OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH, or -C(NH₂)NH, wherein R is defined as above. Particularly preferred substituents include sugar moieties, including pyranoses, furanoses, and reduced forms thereof.

[0048] Particularly preferred non-interfering substituents on an aminocyclitol include OH, NH₂, -C(NH)NH₂, -NHC(NH)NH₂, -CH₃, -CH₂CH₃,

or

[0049] In one aspect, the aminocyclitol useful in methods and compositions ofthe invention has the formula

E F wherein X⁶ is H or -C(NH₂)NH and wherem X³ is -OR or -R where R is alkyl, aryl, heteroaryl, and alkylaryl substituted with 0-3 OH, 0-3 NH₂, and 1-3 -NHC(NH)NH₂ or - C(NH)NH₂ groups. Libraries based on this preferred group of LF inhibitors are also contemplated.

[0050] Further, in a preferred aspect, novel aminocyclitols have the formula E or F, wherein X⁶ is H or -C(NH )NH; wherein X³ is -OR, -NHR or -R where R is alkyl, aryl, heteroaryl, and alkylaryl substituted with 1-3 -NHC(NH)NH₂ or -C(NH)NH₂ groups; and wherein X³ is not -OR when the O is connected to an anomeric carbon. Libraries based on this preferred group of LF inhibitors is also contemplated.

[0051] Preferred aminocyclitol compounds, aminocyclitols useful in the methods and compositions, and the libraries are based on the analysis ofthe SAR from Table A, particularly with respect to a library of compounds that are structurally similar to the compounds in Example 8. In particular, it is preferred that the guanidinyl groups are 8 bonds or fewer from each other when measuring from the closest N of each ofthe guanidinyl groups. In some embodiments X is H, and in others, -C(NH₂)NH is preferred. [0052] In another aspect, a preferred aminocyclitol compound has the formula

or an enantiomer thereof, wherein each R is independently H, -C(NH )NH, or is 1-3 first substituents selected from the group consisting of

or an enantiomer thereof, wherein each R is independently H, -CH₃ or is 1-3 first substituents selected from the group consisting of A, B, and C. Each R on the first substituent is independently H, -C(NH )NH, a first linker to the aminocyclitol, or a second substituent selected from the group consisting of A, B, C, and D. Each R on the second substituent is H, -CH₃, - C(NH₂)NH, or a second linker to the first substituent, wherein the first and second linkers are independently selected from a bond, a C1-C12 acyl, a C2-C12 alkyl, a C3-C8 cyclic alkyl, aryl, or heteroaryl. When the acyl group is a carbonyl, it forms preferred connections between O and N or two N's, namely carbamate and urea connections, preferably between two rings. As described above with respect to aminoglycosides, adjacent heteroatoms are not preferred and thus a substitution that results in -NH-NH -or -O-O- is replaced with -NH- or -O- respectively. Similarly, when a substitution results in -NH-O -or -O-NH-, either is replaced with -NH- or -O-. As preferred aminocyclitols contain two or more rings, 1-3 R groups on the aminocyclitol and the first substituent are A, B, C, or D. Further, no more than 0-4 R groups on the aminocyclitol, the first substituent and the second substituents may contain a guanidinyl group. If an enantiomer is present, an enantiomer of A is the preferred enantiomer.

[0053] Deoxystreptamine (D) is preferably included in combinations with itself as this aminocyclitol has structural similarities based on the most potent guanidinyl neamine derivatives with respect to LF inhibition, which include a dihydroxystreptamine moiety. Deoxystreptamine substituted with 1-4 guanidinyl is most preferred. A library of aminocyclitol LF inhibitors is also comtemplated.

[0054] A novel compound is also contemplated having formula (2) or (3) described above or a salt or prodrug thereof, and having at least one guanidinyl group and at least one NR₂ group wherein NR₂ is not guanidinyl and wherein the compound is not streptomycin or derivatives thereof, 6-N-guanidino kanamycin A, 1-N-guanidino gentamicins, 2 '-and 3-N-guanidino gentamicin C_1; 2'-and 6'-N-guanidino gentamicin C_la; or 6'-N-guanidino gentamicin C₂. Preferably the compound has at least one and may have 1, 2, 3, 4, 5, or 6 NR₂ groups. With respect to the guanidinyl groups, 1-5 groups may be present, and also 1-4 and 1-3, as well as 1-2 guanidinyl groups may be present. Preferred compounds are a) a neamine containing 1-3 guanidinyl groups or enantiomers thereof; b) a neomycin B containing 1-5 guanidinyl groups or enantiomers thereof; or c) l,3,2',6'-tetraguanidino-l,3,2',6'-tetradeamino-5,6,3',4'-tetra-0- methylneamine hydrochloride or enantiomers thereof. Even more preferred compounds are described in Examples 1-16, namely, 1-guanidino-l-deaminoneamine, 3-guanidino-3- deaminoneamine, 2'-guanidino-2'-deaminoneamine, 6'-guanidino-6'-deaminoneamine, 1,3- diguanidino-1 ,3-dideaminoneamine, 1 ,2'-diguanidino-l ,2'-dideaminoneamine, 1 ,6'- diguanidino- 1,6' -dideaminoneamine, 3,2' -diguanidino-3 ,2 ' -dideaminoneamine, 3,6'- diguanidino-3,6'-dideaminoneamine, 2',6'-diguanidino-2',6'-dideaminoneamine, 1 ,3,2'- triguanidino- 1,3,2' -trideaminoneamine, 1,3,6' -triguanidino- 1,3,6' -trideaminoneamine, 1 ,2 ' ,6 ' - triguanidino- 1 ,2 ' ,6 ' -trideaminoneamine, 3 ,2 ' ,6' -triguanidino-3 ,2 ' ,6 '-trideaminoneamine, or l,3,2',6'-tetraguanidino-l,3,2',6'-tetradeaminoneamine, or the salt or prodrug thereof, wherein the compound is not l,3,2',6'-tetraguanidino-l,2,2',6'-tetradeaminoneamine trifluoroacetate.

[0055] As guanidinyl groups are important substituents to the compounds ofthe invention, methods are provided to selectively form a guanidinyl derivative of a compound. In one aspect, a method of selectively guanidinylating a compound of formula (2) or (3), wherem the compound of formula (2) contains an aminocyclitol group is provided. The compound, which may optionally be selectively protected, and which contains at least one group that is capable of forming a guanidinyl group with N,N'-di-tert-butoxycarbonyl-N"-triflylguanidine orN,N'-di- (benzyloxycarbonyl)-N' '-triflylguanidine, such as an amino group, is reacted to form an optionally protected guanidinyl derivative or salt thereof having at least one guanidinyl group. In one aspect, the reactant is Ν,Ν'-di-tβrt-butoxycarbonyl-Ν''-triflylguanidine. The guanidinyl derivative, if it was protected, may then be optionally deprotected. [0056] Preferably the guanidinyl derivative is a neamine derivative having 1-3 guanidinyl groups, such as 1-guanidino-l-deaminoneamine, 3-guanidino-3-deaminoneamine, 2'-guanidino- 2'-deaminoneamine, 6'-guanidino-6'-deaminoneamine, 1 ,3-diguanidino-l ,3-dideaminoneamine, 1,2' -diguanidino- 1 ,2 ' -dideaminoneamine, 1,6' -diguanidino- 1,6' -dideaminoneamine, 3,2'- diguanidino-3 ,2 ' -dideaminoneamine, 3,6' -diguanidino-3 ,6 ' -dideaminoneamine, 2 ' ,6 ' - diguanidino-2 ' ,6 ' -dideaminoneamine, 1 ,3 ,2 ' -triguanidino- 1 ,3 ,2 '-trideaminoneamine, 1,3,6'- triguanidino- 1,3,6' -trideaminoneamine, 1 ,2 ' ,6 ' -triguanidino- 1 ,2 ' ,6 ' -trideammoneamine, and 3, 2', 6 '-triguanidino-3, 2 ',6 '-trideaminoneamine, or a salt or prodrug thereof. The procedure for the synthesis of such compounds is found in Examples 1-16.

[0057] Optionally protected compounds used in selective guanidinylation methods may be selected from the group consisting of 3,6'-di-N-(tert-butoxycarbonyl)neamine, l,3,6'-tri-N-(tert- butoxycarbonyl)neamine, neamine, 6'-N-(tert-butoxycarbonyl)neamine, 3,6'-di-N-(tert- butoxycarbonyl)neamine, 6 ' - [NN '-di-tert-(butoxycarbonyl)guanidino] -6 ' -deaminoneamine, 1 ,6 ' -di-N-(tert-butoxycarbonyl)neamine, 3,6' -di-[NN'-di-(tert-butoxycarbonyl)guanidino]- 3 ,6 ' -dideaminoneamine, 1 ,2 ' ,6' -tri-[NN'-di-(tert-butoxycarbonyl)guanidino]- 1 ,2' ,6 ' - trideaminoneamine, l-N-(tert-butoxycarbonyl)-6'-[NN'-di-tert-(butoxycarbonyl)guanidino]-6'- deaminoneamine, and 1 -N-(tert-butoxycarbonyl)-6'-[NN'-di-tert-(butoxycarbonyl)guanidino]- 6 '-deaminoneamine, as discussed in Examples 1-16.

[0058] Azides also may be used as protecting groups to make derivatives ofthe compounds ofthe invention. Azides ofthe invention are intermediates that allow for the selective functionalization of amino and hydroxyl groups on neamine and other aminoglycoside- and aminocyclitol-containing compounds. The mono-, di-, or tri-azido derivatives are preferred, and the mono- or di- are more preferred. Tetra-azido neamine is not preferred for selective functionalization of neamme. Selective functionalization includes functionalizing one or more R groups on a compound ofthe invention, preferably on an aminocyclitol, where the azide group is used as a protecting group while an OH group, for example, is reacted to form OR, such as in Examples 21 and 29-43. Preferred R groups may include but are not limited to alkyl, aryl, or heteroaryl, preferably substituted with one or more OH, ΝO₂, or CN. Illustrative R groups include hydroxyethyl, cyanomethyl, nitrophenyl, cyanophenyl, dinitrophenyl, or nitropyridinyl. An azide group may be further derivatized to form one or more guanidinyl group. Using limiting amounts of a guandinylation agent, such as N,N'-di-tert-butoxycarbonyl-N"- triflylguanidine orN,N'-di-(benzyloxycarbonyl)-N"-triflylguanidine, the number of protecting groups that may be converted to a guandinyl group may be controlled. Azides may also be used as intermediates to generate triazole heterocyclic derivatives as in Example 23.

[0059] As such, in one aspect a method of making a derivative of a compound of formula (2) or (3), wherein the compound of formula (2) contains an aminocyclitol group and wherein the compound contains 1-4 hydroxyl groups, comprises reacting an azide derivative of a compound of formula (2) or (3) with a reactant for derivatizing the hydroxyl group or groups thereof. The reactant forms an optionally substituted alkyl, alkenyl, aryl, or alkylaryl derivative, wherein an O, S, or N may replace a C in said derivative, wherein the optional substituents maybe OH, CN, -NHC(NH)NH₂, or -C(NH)NH₂.. Preferably the derivative is selected from the group consisting of 5,6,3 ',4'-tetra-O-methylneamine hydrochloride, l,3-diazido-l,3-dideamino-4-< -(2- hydroxyethyl)-5,6-di-0-benzyl-2-deoxystreptamine, 1 ,3-diazido-l ,3-dideamino-4-t - cyanomethyl-5,6-di-( -benzyl-2 -deoxystreptamine, 1 ,3 ,-diazido- 1 ,3 ,-dideamino-4-O-(2- nitrophenyl)-5,6-di-0-benzyl-2-deoxystreptamine, 1 ,3-diazido-l ,3-dideamino-4-<9-(2- cyanophenyl)-5,6-di-O-benzyl-2-deoxystreptamine, 1 ,3,-diazido-l ,3,-dideamino-4-O- (4-nitrophenyl)-5,6-di-O-benzyl-2-deoxystreptamine, 1 ,3,-diazido-l ,3, -dideamino-4-O- (2,4-dinitrophenyl)-5,6-di-0-benzyl-2-deoxystreptamine, 1 ,3,-diazido-l, 3, -dideamino-4-O- (2-nitropyridinyl)-5,6-di-( -benzyl-2-deoxystreptamine, 1 ,3,-diazido-l ,3, -dideamino-4-O- (4-nitropyridinyl)-5,6-di-c -benzyl-2-deoxystreptamme, 1 ,3,-diazido-l ,3, -dideamino-4-O- (3-nitropyridinyl)-5,6-di-O-benzyl-2-deoxystreptamine, and 1 ,3,-diazido-l ,3, -dideamino-4-O- (2-nitrophenyl)-6-0-benzyl-2-deoxystreptamine, or protected form, unprotected form, prodrug or salt thereof. These novel azide intermediates are described in Examples 22 and 29-43.

[0060] An amidine derivative (i.e., containing a -C(NH)NH₂ group) may be formed in derivatives containing cyano groups by reducing the azide groups to amines and converting the cyano group to an amidine by treatment with ammonia followed by hydrochloric acid in methanol as shown in Examples 30 and 32.

[0061] A guanidinyl group may be added to the azide or amidine derivative described above by reacting the derivative with a N,N'-di-tert-butoxycarbonyl-N"-triflylguanidine or N,N',N"- tri-(tert-butoxycarbonyl)-guanidine, after appropriate deprotection, to form a derivative containing at least one guanidyl group, as described in Examples 22 and 29-43. Preferred guanidinylated derivatives are selected from the group consisting of l,3,2',6'-tetraguanidino- 1 ,3 ,2 ' ,6 ' -tetradeamino-5,6,3 ' ,4' -tetra-O-methylneamine hydrochloride, 1 ,3 -diguanidino- 1,3- dideamino-4-( -(2-guanidinoethyl)-2-deoxystreptamine, 1 ,3 -diguanidino- 1 ,3-dideamino-4-O- (carboxamidine)-2-deoxystreptamine, 1 ,3-diguanidino-l ,3-dideamino-4-0-(2-guanidinophenyl)- 2-deoxystreptamine, 1 ,3-diguanidino-l ,3-dideamino-4-< -(2-benzamidine)-2-deoxystreptamine, 3-guanidino-3-deamino-4-0-(2-benzamidine)-2-deoxystreptamine, l,3-diguanidino-l,3- dideamino-4-0-(4-guanidinophenyl)-2-deoxystreptamine, 1 ,3-diguanidino-l ,3-dideamino-4-O- (2,4-diguanidinophenyl)-2-deoxystreptamine, 3 -guanidino-3 -deamino-4-0-(2,4- diguanidinophenyl)-2-deoxystreptamine, 3-guanidino-3-deamino-4- -(2-guanidino-4- aminophenyl)-2-deoxystreptamine, l,3-diguaιιidino-l,3-dideamino-4-0-(2-guanidino-4- aminophenyl)-2-deoxystreptamine, 1 ,3-diguanidino-l , 3-dideamino-4-0-(2-guanidinopyridin-3- yl)-2 -deoxystreptamine, 1 ,3-diguanidino-l ,3-dideamino-4-0-(4-guanidinopyridin-3-yl)-2- deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-0-(3-guanidinopyridin-4-yl)-2- deoxystreptamine, 1 ,3 -diguanidino- 1 ,3 -dideamino-4-0-(2-guanidinophenyl)-2,5- dideoxystreptamine, and 3 -guanidino-3 -deamino-4-O-(2-guanidinophenyl)-2-deoxystreptamine, or a prodrug or salt thereof. A more detailed procedure ofthe synthesis of such compounds is found in Examples 22 and 29-43.

[0062] A method of making an azide derivative of a compound of formula (2) or (3), wherein the compound of formula (2) contains an aminocyclitol group is also contemplated. Such method comprises reacting the compound which optionally may be selectively protected, and which contains at least one group that is capable of being derivatized to an azide, with an azide reactant such as trifiyl azide to form an azide derivative or salt thereof having at least one azide group. The azide derivative then optionally may be deprotected. The optionally, selectively protected compound useful in the inventive methods may be selected from the group consisting of l,3,6'-tri-N-(tert-butoxycarbonyl)neamine, 3,6'-di-N-(tert- butoxycarbonyl)neamine, l,6'-di-N-(tert-butoxycarbonyl)neamine, and 1,3-di-N- (butyloxycarbonyl)-3,6'-di-N-(benzyloxycarbonyl)neamine. Preferably a deprotected azide derivative or a prodrug or salt thereof is made in accordance with the method shown in Examples 17-20.

[0063] Novel azide derivatives of a compound of formula (2) that contain an aminocyclitol group or formula (3) have an amino group which may be derivatized to an azide group, which preferably is not tetra-azidoneamine. Preferably, an azide derivative ofthe invention has the following formula

₅ or a salt thereof. The preparation of these compounds is described in Examples 17-20. [0064] Another class of compounds useful in the methods and compositions ofthe invention includes the compounds generally known as polyamines and closely related polyamine compounds, such as polymerized amino acids, or proteins. As used herein, "polyamine" refers to any compound that contains a) at least one nitrogen atom and either one sulfur atom or one oxygen atom, or b) at least two nitrogen atoms, able to assume a conformation wherein the space between the nitrogen and either the sulfur or oxygen atom, or between the nitrogen nuclei is that conferred by 2-11, preferably 2-5 methylene groups. The polyamine may be a cyclic polyamine, preferably having from 6-14, preferably 12-14, members in the ring backbone. Preferably the polyamines are linear, branched and/or cyclic compounds, diamino alkylenes, dentrimers, polyamino acids, or proteins. In a further preferred embodiment, the polyamine compound contains one or more, preferably 1-20, guanidinyl groups. [0065] This class of polyamines is broadly defined as comprising a heteroalkyl backbone which contains one or more linear, branched and/or cyclic portions thereof and contains 4-120 members wherein from 1-40 members of said backbone are nitrogen atoms and the remainder are carbon atoms, or a salt or prodrug thereof. A terminal nitrogen atom in the backbone may be NH₂ or =N. In addition, each nitrogen atom may be spaced from any other riitrogen atom by at least one carbon atom as long as the space conferred between two nitrogen atoms, or a nitrogen and an oxygen atom, is that conferred by 2-11, preferably 2-5, methylene groups. One or more carbon atoms may be optionally substituted with 0-20 =O, 0-5 hydroxyl, and 0-5 alkylthioalkyl or alkylsulfanyl groups.

[0066] Typically, linear polyamines are alkylene diamines that optionally include in the alkylene chain one or more, preferably 0-20, more preferably 0-14, interspersed non-adjacent nitrogens. In one aspect, polyamines useful in methods and compositions ofthe invention are linear and contain 6-14 members. Such linear polyamines have 2-4 members ofthe backbone that are nifrogen atoms and the remainder are carbon atoms. In addition, each nitrogen atom is spaced from another nitrogen atomin the backbone by at least two carbon atoms. The linear polyamines also contain two members which are NH₂. Such polyamines are typified by spermine, putrescine, spermidine, diethylenetriamine, N-(3-aminopropyl)-l,3-propanediamine, N-(2-aminopropyl)-l,3-propanediamine, and 1,8-diaminooctane as shown in Table B.

[0067] Polyamines may also be cyclic in nature or contain a cylic portion thereof, hi one aspect of such, a monocyclic backbone containing 6-14 members, wherem 2-4 members are nitrogen atoms and 1-2 members ofthe monocyclic backbone optionally are substituted with a linear alkylamine. Typical cyclic polyamines are 1,4,8,11-tetra-cyclotetradecane and cyclen. A typical combination of polyamines such as cyclic and linear polyamines is N,N'-bis(3- aminopropyl)piperazine.

[0068] A subset of polyamines, the dentrimers or branched polyamines, is another embodiment ofthe invention. In one aspect, therefore, the polyamine is branched and contains 1-16 NH . Each N is spaced from another nifrogen atom in the backbone, if present, by at least two carbon atoms. In a preferred embodiment, the compound contains from 1-7 guanidinyl groups. In another aspect, a branched polyamine is substituted with 1-15 =O, 0-3 hydroxyl, and 0-2 alkylthioalkyl or alkylsulfanyl, preferably methylthioethyl or methylsulfanyl. Preferred dentrimers are tetraamines, octaamines, and hexadecaamines.

[0069] Another subset of polyamines is polymerized amino acids or polypeptides. Typically, the structure includes a peptide backbone containing integral amides ofthe peptide bond optionally having one or more, preferably 2-500, and more preferably 2-150 amino acid side chains. Typical compounds contain at least one amino acid side chain and usually from 4-16 amino acid side chains. The term "amino acid" includes any one ofthe twenty naturally occurring amino acids or the D form of any one ofthe naturally occurring amino acids. Preferred amino acids include arginine, alanine, asparagine, cysteine, glycine, glutamine, glutamic acid, isoleucine, leucine, lysine, methionine, serine, and threonine. In addition, the term "amino acid" also includes other non naturally occurring amino acids besides the D amino acids, which are functional equivalents ofthe naturally occurring amino acids. Non naturally occurring amino acids include, for example, norleucine ("Nle"), norvaline ("Nva"), L or D naphthalanine, ornithine ("Orn"), homoarginine (homoArg) and others well known in the peptide art, such as those described in M. Bodanzsky, "Principles of Peptide Synthesis," 1st and 2nd revised ed., Springer Verlag, New York, NY, 1984 and 1993, and Stewart and Young, "Solid Phase Peptide Synthesis," 2nd ed., Pierce Chemical Co., Rockford, IL, 1984, both of which are incorporated herein by reference.

[0070] Polypeptides may contain D or L amino acids or combinations thereof. In Table B, the D or L configuration of an amino acid is reflected in the name ofthe polyamine using a lower case letter, which signifies the D configuration, or an upper case letter, which signifies the L configuration. For instance, for the single letter amino acid designation "r", the lower case "r" means D-arginine, and upper case "R" means L-arginine. Amino acids and amino acid analogs can be purchased commercially (Sigma Chemical Co.; Advanced Chemtech) or synthesized using methods known in the art. In one aspect, a polypeptide may contain a D and/or L isomer of lysine, arginine, glutamine, or ornithine, or combinations thereof.

[0071] Polymers or copolymers ofthe naturally or non-naturally occurring amino acids are used in accordance with the invention. Preferred polymerized amino acids include, for example, polylysine, polyarginine, polyglutamic acid, and polyoniithine, and copolymers formed from the monomers thereof, such as copolymers of two or more, preferably 2-20 more preferably 2-10, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, ofthe naturally or non-naturally occurring amino acids. The polymerization of these monomers results in proximal nitrogen atoms sufficient for activity as required.

[0072] The term "amino acid side chain" refers to a branched chain of atoms attached to the alpha carbon of an amino acid. Preferably, the 20 naturally occurring amino acid side chains are preferred substituents on the peptide backbone, however non naturally occurring amino acid side chains are also contemplated. Preferred amino acid side chains are those that confer a positive charge on the polypeptide, such as lysine, arginine, histidine, or ornithine.

[0073] The "n" with respect to the amino acid repeating unit as illustrated in Table B preferably is in the range of n = 2 to 147, where n is the number of amino acid subunits in the polymer; for example, poly-L-arginine hydrochloride was purchased at a molecular weight range of between 5,000 to 15,000 (n = 25 to 77). More preferably, "n" is from n = 5 to n = 77, and the most preferred is n = 2 to n=10.

[0074] Proteins are also included in the subset of polyamino acids. A "protein" that may be used in the compositions and methods ofthe invention can include any protein that has a net positive charge at a physiological pH and thus are cationic proteins. Proteins must contain proximal nitrogen atoms sufficient for activity as required. Preferred proteins include lactoferrin, myeloperoxidase (MPO), and alpha 1-PDX.

[0075] Still another class of compounds useful in the invention includes the aryl- or heteroaryl-containing compounds, which include the fused ring antibiotics. Representatives of these compounds are also shown in Table B as tetracycline and echinomycin. Tetracycline and echinomycin contains a fused ring system retaining the property that electronegative H-bonding- capable atoms, i.e., O and N are maintained at suitable distances. Thus, O and N may be substituents external to the fused ring system or may be contained in one or more ofthe fused rings. At least one ofthe O and/or N must be external to the fused rings. These particular compounds meet the criterion of exhibiting a Ki with respect to LF of <500 μM; thus, they are useful in methods which involve simply inhibiting this enzyme. Further, tetracycline, while useful in methods to inhibit LF in general, may present problems with regard to administering it to subjects for treatment of anthrax toxicity in view of its ability to inhibit certain vertebrate proteases. In view ofthe favorable properties of tetracycline with respect to LF, however, other compounds of this class may be found which have the relevant specificity. It is a further aspect ofthe invention that fused ring antibiotic compounds such as spectinomycin, which have weak inhibitory activity with respect to LF, are guanidinylated such that the LF activity is increased.

[0076] In one aspect, the aryl or heteroaryl ring is optionally fused to 1-3 alkyl, heteroalkyl, alkenyl, or heteroalkenyl rings which contains 0-4 O, S or N; and the ring or fused ring is optionally substituted with 0-11 substituents that do not interfere with the inhibitory activity of the compound. Preferred non-interfering substituents, which may be optionally substituted, may be selected from halo, NO₂, =O, or optionally substituted R, NR₂, OR, NRCO, CONR₂, or NH(C=O)NHR, wherein R is H, Na, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-14 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O. Optional substituents may be selected from OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH or -C(NH₂)NH wherein R is defined as above. In particular the non-interfering substituent is preferably selected from CI, -COOH, CN, OH, CH, =O, NH₂, NO₂,

[0077] It is contemplated that all ofthe compounds ofthe invention, or compounds useful in methods and compositions ofthe invention, described above, may further contain one or more guanidinyl groups. A "guanidinyl" group is -NHC(NH₂)NH. "Guanyl","guanidino", "guanidyl", "guanidine", and "guanidinyl" are referred to herein, and in the literature, as having the same meaning. The structure below is guanidine (free guanidine R=R'=R"=H).

In addition to the N,N',N"-trisubstituted pattern shown, mono-, N,N-di-, N,N'-di, N,N,N'-tri, N,N,N',N'-tetra-, and N,N,N',N"-tefrasubstitution is possible.

[0078] Methods ofthe invention preferably include compounds having one or more guanidinyl groups, which generally have been found to have a greater inhibitory effect with respect to LF than their non-guanidinylated counterparts. Preferred compounds, therefore, also contain one or more guanidinyl groups. More preferred embodiments contain two or more guanidinyl groups. In particular, it is preferred that the guanidinyl groups are 15 bonds or fewer, more preferably 10 bonds or fewer, and most preferably 8 bonds or fewer from each other when measuring from the closest nitrogen atom in each guanidinyl group.

[0079] It is preferred that perguanidinylated neamine TFA and perguanidinylated compounds of neomycin B, kanamycin A, kanamycin B, tobramycin, paromomycin, and gentamicins and salts thereof are not included in the compounds ofthe invention but are included in the methods and compositions ofthe invention.

[0080] Without being bound by theory, the compounds useful in the methods and compositions ofthe invention that contain a guanidininyl group, such as per-guanidinylated neomycin, may have a different mechanism of action than neomycin B or other non- guanidinylated compounds. Per-guanidinylated neomycin is an uncompetitive inhibitor whereas neomycin B and other non-guanidinylated compounds are (mixed) competitive inhibitors. Uncompetitive inhibitors bind to the enzyme-substrate complex rather than just to the enzyme, and thus offer concomitant advantages related to LF inhibition.

[0081] Methods for selective guanidinylation and the resulting compounds also are contemplated. It is preferred that 6-N-guanidino kanamycin A, 1-N-guanidino gentamicins, 2'- and 3-N-guanidino gentamicin CI, 2 '-and 6'-N-guanidino gentamicin Cla; and 6'-N-guanidino gentamicin C2 as described in Streicher (supra) are not included in the compounds ofthe invention but are included in the methods and compositions ofthe invention.

[0082] "Gentamicins" refer to all the members ofthe class of gentamicins wherein, with reference to the structure of gentamicin (mixtures) in Table B, for example, R =R =H, R¹⁼R²=CH_3; and R¹⁼CH₃ and R²=H. "Gentamicin (mixtures)" refers to a mixture of gentamicin where R^R^H, R¹=R²=CH₃, and R^CHs andR²=H.

[0083] "Derivatives of streptomycin" include the reduction products of streptomycin such as dihydrostreptomycin.

[0084] As used herein, a "noninterfering substituent" is a substituent which leaves the ability ofthe compound ofthe invention to inhibit lethal factor (LF) qualitatively intact. Thus, the substituent may alter the degree of inhibition of LF. However, as long as the compound ofthe invention retains the ability to inhibit LF, the substituent will be classified as "noninterfering." Example 44 is a novel assay that can determine the ability of a compound to inhibit LF. The positions which are occupied by "noninterfering substituents" can be substituted by conventional organic or inorganic moieties as is understood in the art. It is irrelevant to the present invention to test the outer limits of such substitutions. The essential features ofthe compounds are those set forth with particularity herein.

[0085] As used herein, the term "alkyl," "alkenyl" and "alkynyl" include straight- and branched-chain and cyclic monovalent substituents. Examples include methyl, ethyl, isopropyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. Typically, the alkyl, alkenyl and alkynyl substituents contain 1-40C (alkyl) or 2-40C (alkenyl or alkynyl). In most embodiments, they contain 1-12C (alkyl) or 2-16C (alkenyl or alkynyl). "Alkyl," "alkenyl" and "alkynyl" substituents may contain one or more heteroatoms, preferably 0-14 O, S and/or N, more typically 1 or 2 heteroatoms or combinations thereof within the backbone residue and may be referred to as heteroalkyl, heteroalkenyl and heteroalkynyl. "Alkenyl" or "alkynyl" may also refer to compounds that contain alkyl portions in addition to alkenyl or alkynyl portions on the substituent. "Alkylene" refers to a divalent "alkyl" moiety, for example, methylene. [0086] As used herein, "acyl" may consist of a carbonyl group (a divalent carbon double bonded to an oxygen), where appropriate, and may also encompasses the definitions of alkyl, alkenyl, alkynyl and the related hetero-forms which are coupled to an additional residue through a carbonyl group, which may be monovalent.

[0087] An "aromatic" moiety or "aryl" refers to a monocyclic or fused bicyclic, tricyclic, or multicyclic moiety such as phenyl or naphthyl; "heteroaromatic" also refers to monocyclic or fused bicyclic, tricyclic, or multicyclic ring systems containing one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits inclusion of 5 membered rings as well as 6 membered rings. Thus, typical aromatic systems include phenyl, naphthyl, pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyl and the like. Any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition. Typically, the ring systems contain 5-14 ring member atoms.

[0088] Similarly, "alkylaryl" and "heteroalkylaryl" refer to aromatic and heteroaromatic systems which are coupled to another residue tlirough a carbon chain, including substituted or unsubstituted, saturated or unsaturated, and/or linear, branched or cyclic carbon chains of 5-40C, and typically 5-25C. These carbon chains may also include a carbonyl group, thus making them able to provide substituents as an acyl moiety.

[0089] A "spiro union" has its ordinary meaning and refers to a linkage between two rings consisting of a single atom common to both rings. Typical spiro unions contain cycloalkyl rings or fused cycloalkyl rings or combinations thereof that may contain one or more O, S or N heteroatoms or combinations thereof within the backbone ofthe residue.

[0090] The compounds ofthe invention which are useful in the compositions and methods ofthe invention may be present in the free base form or in the salt or prodrug form thereof. Any pharmaceutically acceptable salt may be used. See, for example, "Pharmaceutical Salts," Berge, et al., J. Pharm. Sci. (1977) 66:1 19, which is incorporated herein by reference. Typical salts which are pharmaceutically acceptable salts are hydrochloride, sulfate, hydrobromide, tarfrate, mesylate, maleate, citrate, phosphate, acetate, trifluoro acetate, pamoate, hydroiodide, nitrate, lactate, methylsulfate and fumarate. With respect to the polyamino acids, it is assumed that the number of "n" salt units, such as HCI, that may be present is in a 1 : 1 ratio with NH₃ formed or equivalent to the amino acid repeating units plus 1. [0091] Several ofthe compounds were tested for botulinum A inhibition with LF as a secondary (specificity) assay. From this data, it is contemplated that the compounds ofthe invention may be dual inhibitors, meaning they are inhibitors for either botulinum or anthrax toxicities. Particularly preferred dual inhibitors are peptides, such as Ac-CRATKMRRR-NH₂ and related structures.

[0092] Further, the compounds ofthe invention may further inhibit furin. Furin is a host cell protease that cleaves the anthrax protein protective antigen (PA) from an inactive to an active form. Polyamine derivatives, such as hexapeptides have been demonstrated to inhibit furin. Cameron, A. et al, J. Biol. Chem., 275:36741-9 (2000). It is therefore a further aspect ofthe invention to provide methods, compounds and compositions that block both the furin processing step as well as the LF step.

[0093] For use in treatment, the compounds ofthe invention are administered in standard protocols, for example by injection, such as intravenous administration. A typical dosage level per day is about 10 mg/kg; however, this is merely a starting point as a number of factors need to be considered in determining dosage. In general, any satisfactory route of administration may be employed. In addition to intravenous administration, which is preferred, intramuscular, intraperitoneal, or subcutaneous injection may be used. The invention compounds may also be delivered through transmucosal or transdermal routes, or may be administered by inclusion in a controlled release matrix. In addition, liposomal preparations or other particulate preparations which effect drug delivery may be employed.

[0094] Some compounds may be administered orally. A typical dose regimen would include, for example, 1-4 doses per day using tablets or capsules containing approximately 500 mg of active ingredient, with 1-4 capsules or tablets being administered per dose. Optimization of dosage regimen is routine and will depend on the nature ofthe active ingredient, the severity ofthe infection, the condition ofthe subject, and the judgment ofthe practitioner. Optionally, a compound may be administered in a combination of oral and parenteral regimens.

[0095] "Subject", as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model, such as a mouse, rat, or rabbit. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.

[0096] The medications administered may contain more than one active ingredient, and thus the aminoglycoside, aminocyclitol, aryl or heteroaryl, or polyamine active agents, or mixtures thereof, may also be supplemented with additional medicaments either to complement the effect ofthe protease inhibition activity, to behave as standard antibiotics, or to ameliorate any possible side effects.

[0097] As stated above, the specific means of administration and the dosage level will be dependent on the nature ofthe active ingredient, the nature ofthe subject to be treated, the severity ofthe infection and/or lethal factor intoxication, the severity ofthe risk of infection and/or lethal factor intoxication, and the judgment ofthe practitioner.

[0098] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) lethal factor toxicity or having lethal factor toxicity in a subject. As used herein, the term "treatment" is defined as the application or administration of a compound or composition to a patient, or application or administration of a compound or composition to an isolated tissue from a patient, who has an infection, a symptom of an infection or a predisposition toward an infection, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the infection, the symptoms of infection or the predisposition toward infection.

[0099] The compounds may be formulated into pharmaceutical compositions appropriate for the administration route. Thus, various pharmaceutical excipients may be added to the active ingredient(s) to facilitate effective treatment. Suitable formulations are well known in the art and are found, for example, in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0100] The compounds ofthe present invention in treating subjects for the toxic effects of anthrax infection may be used in combination with additional compounds ofthe invention and/or in combination with additional antibiotics or other pharmaceutical active ingredients for the amelioration of side effects or enhanced effectiveness.

[0101] For practice ofthe method ofthe invention to identify aminoglycoside, polyamine or fused ring antibiotic analogs that inhibit LF, typically combinatorial libraries will be useful (but not necessary). For example, combinatorial libraries of aminoglycosides in general are available commercially, or may be synthesized de novo. Libraries based on glucose analogs containing an amino group at position 2 and substituted with various substituents are available from Alchemia Pty Ltd, Brisbane, Australia, and are described at the web address alchemia.com. These libraries are based upon the amino sugar of gentamicin/kanamycin. Similarly, libraries of polyamines and of fused ring antibiotics may be synthesized using either individual or combinatorial approaches. It is, of course, within the scope ofthe invention to test libraries that contain one or more of compounds which are aminoglycosides, aminocyclitols, polyamines, aryl- or heteroaryl- containing compounds or derivatives thereof, or mixtures of all compound types.

[0102] The compounds ofthe invention are synthesized using art-known means or may be isolated from natural sources. The libraries of compounds or compounds to be tested serially can similarly be constructed using art-recognized approaches.

[0103] Although the compounds described above, generally, are useful in the prevention or treatment of anthrax toxicity symptoms and sequelae, screening the libraries of these compounds is clearly useful in order to optimize the selection ofthe most active compounds. Other additional assays may also be employed to assure specificity and lack of undesirable side effects. The probability of finding a successful candidate is greatly enhanced by prior knowledge ofthe types of compounds likely to yield successful results. Thus, by limiting the libraries searched for example to aminoglycosides and/or aminocyclitols and/or aryl or heteroaryl compounds and/or polyamines and/or fused ring antibiotics containing N and O, the effort involved in screening is greatly minimized.

[0104] For one embodiment ofthe assays ofthe invention, enhancement of fluorescence is measured upon cleavage of substrate in a fluorescence resonance energy transfer (FRET) assay. Such an assay for B. anthracis lethal factor protease is described by Cummings, R.T., et al, Proc. Natl. Acad. Sci. USA (2002) 99:6603-6606. In this assay, developed with the use of peptides within the MAP kinase kinase (MAPKK) family, a substrate peptide is dissolved in buffer and the appropriate protease added. In principle, the hydrolysis ofthe substrate is monitored by any acceptable means, such as solubilization ofthe peptide, detection of a fragment with labeled antibody, but preferably, as in the FRET assay, by following the level of fluorescence as a function of hydrolysis when the substrate has been labeled with a fluorophore and quencher. In this preferred embodiment, one portion ofthe substrate peptide is coupled to a fluorophore and a portion which will be liberated by cleavage is labeled with a quencher. For example, the fluorophore may be fluorescein, o-aminobenzoic acid, or a coumarin fluorophore, such as that used by Cummings, et al, i.e., 7-hydroxy-4-methyl-3-acetylcoumarinyl, and the like. The quencher may be, for example, 4-dimethylaminoazobenzene-4' carboxyl, 2,4-dinitrophenol, dansyl, or N-(4-[(7-nitro-2,l,3-benzoxadiazol-4-yl)amino]phenyl)-acetyl. Any suitable combination of fluorophore and quencher may be used. As the hydrolysis occurs, the fluorophore and quencher are physically separated and fluorescence is enhanced.

[0105] Although assays which can be adapted to high throughput formats such as the FRET assay described above may be advantageous, other art-known assays, such as those based on SDS-PAGE or HPLC could also be used. See, for example, Hammond, S.A., et al, Infect. Immun. (1998) 66:2374-2378; Vitale, G., et al, Biochem. J. (2000) 352:739-745; and Duesbery, N.S., et al, Science (1998) 280:734-737.

[0106] The following examples are offered to illustrate but not to limit the invention.

Examples

[0107] Several Examples in the application were obtained through selective guanidinylation of amino groups. These are reactions in which an amino or hydroxyl group is transformed into a guanidinyl group, usually by a two step procedure involving protecting groups on the guanidinylating reagent. These transformations are known as 'guanidinylation' or 'guanylation' reactions (Feichtinger, K. et al, J. Org. Chem. 63:8432-8439 (1998); Wu, Y. et al. J. Org. Chem. 67(21):7553-7556 (2002)). Methods for nonselective guanidinylation at all the nitrogen positions of some aminoglycosides have been described (Baker, T. J. et al, J. Org. Chem., 65(26):9054-9058 (2000); Luedtke, N. W. et al, J. Am. Chem Soc. 122:12035-12036 (2000); Hui, Y. et al, Tet. Lett. 43:9255-9257 (2002); U. S. Patent No. 6,252,182, issued February 25, 2003 to Goodman, M. et al).

Scheme 1. Generic guanidinylation reactions

wherein R is the remainder of a compound defined herein.

Methods.

LC/MS analysis. [0108] For all N-(tert-butoxycarbonyl) protected compounds: LC/MS analysis performed in ESI positive mode with an Agilent 1100 LC/MSD VL system equipped with Agilent 1100 HP PDA and Sedex 75 ELSD detectors. Column: Zorbax Eclipse SD-C18, 5 μm, 4.6 x 75 mm; Temperature set at 25 ^°C; Mobile Phase: (%A = 0.025% trifluoroacetic acid-water, %B = 0.025% trifluoroacetic acid-acetonitrile); 80% A - 20% B (start) with step gradient to 100% B in 12 min.; Flow rate: 1.0 mL/min.; ELSD gain set @ 3.

[0109] For all other compounds: LC/MS analysis performed in ESI positive mode with an Agilent 1100 LC/MSD VL system equipped with Agilent 1100 HP PDA and Sedex 75 ELSD detectors. Column: Zorbax Eclipse SD-C18, 5 μm, 4.6 x 250 mm; Temperature set at 25 C; Mobile Phase: (A = 0.2% pentafluoropropionic acid-water, B = methanol); 55% A - 45% B (start) with step gradient to 95% B over 15 min.; Flow rate: 1.0 mL/min.; ELSD gain set @ 3.

[0110] Abbreviations used herein include: mg (milligram), mL milliliter, mmol (millimole), g (gram), LC/MS (liquid chromatography / mass spectrometry), TLC (thin layer chromatography), and bn (benzyl).

General procedure A: Deprotection of N-(tert-butoxycarbonyl)-protected neamine derivatives

[0111] This procedure removes the tert-butyloxy protecting group from all protected amino and guanidino groups in a single step. A reaction mixture of an N-(tert-butoxycarbonyl)- protected neamine derivative in 1:1 trifluoroacetic acid-dichloromethane (15 to 80 mL per g) was stirred at room temperature for 3 hours. Volatiles were removed under reduced pressure and the resulting crude material triturated or precipitated with diethyl ether to give the trifluoroacetate salt.

General procedure B: Ion exchange preparation of hydrochloride salts from trifluoroacetate salts

[0112] Dowex 1x2-200 quaternary amine resin was preactivated with hydrochloric acid and washed well with water prior to use. Trifluoroacetate salts of neamine derivatives were dissolved in water or methanol-water (1:1) and stirred with the preactivated resin (10 to 25 g of resin per 1 g of salt) for 15 minutes. Resin was removed by filtration and washed with water. The solution was concentrated and the hydrochloride salt obtained by precipitation or frituration with diethyl ether. Example 1 1-guanidino-l -deaminoneamine hydrochloride

[0113] A reaction mixture of 3,6'-di-N-(tert-butoxycarbonyl)neamine (522 mg, 1 mmol), NN'-di-(tert-butoxycarbonyl)-N"-triflylguanidine (391 mg, 1 mmol), and triethylamine (0.14 mL, 1 mmol) in methanol (20 mL) was stirred at room temperature for 18 hours and then concentrated. The residue was purified by silica gel chromatography eluting with chloroform- methanol-concentrated aqueous ammonium hydroxide (120:15:0.8). Concentration of pure fractions gave 1 -[NN'-di-tert-(butoxycarbonyl)guanidino] - 1 -deamino-3 ,6 ' -di-N-(tert- butoxycarbonyl)neamine (mass spectrum (LC/MS) m/z 765 [M+H⁺]) and 3,6'-di-N-(tert- butoxycarbonyl)-2'-[NN'-di-tert-(butoxycarbonyl)guanidino]-2'-deaminoneamine (mass spectrum (LC/MS) m/z 765 [M+H⁺]).

[0114] Deprotection and ion exchange of l-[NN'-di-(tert-butoxycarbonyl) guanidino]-l- deamino-3,6'-di-N-(tert-butoxycarbonyl)neamine were performed according to general procedures A and B to provide 1-guanidino-l -deaminoneamine hydrochloride. Mass spectrum (LC/MS) m/z 365 [M+H⁺].

Example 2 3 -guanidino-3 -deaminoneamine hydrochloride

[0115] In a manner similar to Example 5 but utilizing limiting amounts of N,N'-di-(tert- butoxycarbonyl)-N"-triflylguanidine as a starting material followed by chromatography, 3- guanidino-3 -deaminoneamine hydrochloride is obtained.

Example 3 2 ' - guanidino-2 ' -deaminoneamine hydrochloride

[0116] A reaction mixture of l,3,6'-tri-N-(tert-butoxycarbonyl)neamine (Grapsas, I. et al. J. Org. Chem. 59(7):1918-1922 (1994)Roestamadji, J., et al, Bioorg. Med. Chem. Lett. 8(24):3483-3488 (1998) (300 mg, 0.48 mmol), NN'-di-(tert-butoxycarbonyl)-N' '- triflylguanidine (566 mg, 1.45 mmol), and triethylamine (0.202 mL, 1.45 mmol) in methanol (5 mL) was stirred at room temperature for 18 hours and then concentrated. The residue was purified by silica gel chromatography eluting with dichloromethane-methanol and recrystallized from methanol-water to give 170 mg of l,3,6'-tri-N-(tert-butoxycarbonyl)-2'-[NN'-di-tert- (butoxycarbonyl)guanidino]-2'-deaminoneamine. Mass spectrum (LC/MS) m/z 865 [M+H⁺].

[0117] Deprotection and ion exchange of 150 mg of this material were performed according to general procedures A and B to provide 45 mg of 2 '-guanidino-2 '-deaminoneamine hydrochloride as a white solid. Mass spectrum (LC/MS) m/z 365 [M+H⁺].

Alternative procedure. [0118] Deprotection of 3,6'-di-N-(tert-butoxycarbonyl)-2'-[NN'-di-tert- (butoxycarbonyl)guanidino]-2'-deaminoneamine (isolated as a product in Example 1) according to general procedure A followed by ion exchange ofthe trifluoroacetate salt according to general procedure B provided 2'-guanidino-2'-deaminoneamine hydrochloride as a white solid. Mass spectrum (LC/MS) m/z 365 [M+H⁺]. Mass spectrum (LC/MS) m/z 365 [M+H⁺]. Example 4 6 ' - guanidino-6 ' -deaminoneamine hydrochloride

[0119] A reaction mixture of neamine (Dutcher, J., J. Am. Chem. Soc. 74:3420-3422 (1952); Grapsas, I. et al. J. Org. Chem. 59(7):1918-1922 (1994)) (3000 mg, 9.3 mmol) andNN'-di- (tert-butoxycarbonyl)-N' '-triflylguanidine (3278 mg, 8.37 mmol) in methanol (180 mL) and water (135 mL) was stirred at room temperature for 18 hours. Methanol was removed under reduced pressure and the aqueous solution was lyophilized. The crude material was purified by silica gel chromatography eluting with dichloromethane-methanol-concentrated aqueous ammonium hydroxide (4:1:0.25) to give 2960 mg of 6'-[NN'-di-tert- (butoxycarbonyl)guanidino] -6 '-deaminoneamine. TLC R_f 0.29 silica, dichloromethane- methanol-concentrated aqueous ammonium hydroxide (4:1 :0.25). 1H ΝMR (500 MHz, methanol-d6) d 5.21 (d, IH), 3.82-3.86 (m, IH), 3.75 (dd, IH), 3.60 (dd, IH), 3.51 (dd, IH), 3.40 (dd, IH), 3.18 (dd, IH), 3.16 (dd, IH), 3.10 (dd, IH), 2.66-2.77 (m, 3H), 1.98-2.03 (m, IH), 1.43-1.53 (18H), 1.18-1.25 (m, IH). Mass spectrum (LC/MS) m/z 565 [M+H⁺].

[0120] Deprotection of 200 mg of this material according to general procedure A followed by ion exchange ofthe trifluoroacetate salt according to general procedure B provided 135 mg of 6 '-guanidino-6 '-deaminoneamine hydrochloride as a light yellow solid. 1H ΝMR (500 MHz, D₂O) d 5.87 (d, IH), 3.96-4.02 (m, 3H), 3.73 (dd, IH), 3.58-3.65 (m, 4H), 3.54 (m, IH), 3.48 (dd, IH), 3.36-3.42 (m, IH), 2.56 (m, IH), 1.96 (m, IH). Mass spectrum (LC/MS) m/z 365 [M+H⁺]. Example 5 1.3 -diguanidino-l,3-dideaminoneamine trifluoroacetate

[0121] A reaction mixture of 6'-N-(tert-butoxycarbonyl)neamine Grapsas, I. et al. J. Org. Chem. 59(7):1918-1922 (1994) (650 mg, 1.539 mmol) NN'-di-(tert-butoxycarbonyl)-N"- triflylguanidine (1204 mg, 3.0 mmol), and triethylamine (0.488 mL, 3.5 mmol) in methanol (20 mL) and water (3 mL) was stirred at room temperature for 18 hours and then concentrated. The residue was purified by silica gel chromatography eluting with chloroform-methanol, followed by frituration from water-methanol (8:1) to give 647 mg of l,3-di-[NN'-di-tert- (butoxycarbonyl)guanidino]-l,3-dideamino-6^''-N-(tert-butoxycarbonyl)neamine as a white solid. TLC R_f 0.54 silica, chloroform-methanol-concentrated aqueous ammonium hydroxide (10:1:0.1). Mass spectrum (LC/MS) m/z 908 [M+H ].

[0122] Deprotection of 300 mg of this material was performed according to general procedure A to provide 270 mg of l,3-diguanidino-l,3-dideaminoneamine trifluoroacetate as a white solid. Mass spectrum (LC/MS) m/z 407 [M+H⁺].

Example 6 1.2'-diguanidino-l .2 '-dideaminoneamine hydrochloride

[0123] A reaction mixture of 3,6'-di-N-(tert-butoxycarbonyl)neamine (Grapsas, I. et al. J. Org. Chem. 59(7): 1918-1922 (1994)) (261.3 mg, 0.5 mmol) NN'-di-(tert-butoxycarbonyl)-N' '- triflylguanidine (1174 mg, 3.0 mmol), and triethylamine (0.42 mL, 3.0 mmol) in methanol was stirred at room temperature for 18 hours and then concentrated. The residue was purified by silica gel chromatography eluting with ethanol-dichloromethane, followed by trituration from hexanes to give 455 mg of l,2'-di-[ NN'-di-(tert-butoxycarbonyl)guanidino]-l,2'-dideamino- 3,6'-N-(tert-butoxycarbonyl)neamine as a white solid. TLC R_f 0.26 silica, dichloromethane- methanol (96:4). Mass spectrum (LC/MS) m/z 1008 [M+H⁺].

[0124] Deprotection of 227 mg of this material according to general procedure A provided 200 mg of l,2'-di-N-guanymeamine trifluoroacetate salt. Ion exchange of 170 mg ofthe trifluoroacetate salt according to general procedure B provided 100 mg of l,2'-di-guanidino- 1,2 '-dideaminoneamine hydrochloride salt as a white solid. Mass spectrum (LC/MS) m/z 407 [M+H⁺].

[0125] Alternative procedure: this compound was also prepared using a similar procedure but with 3,6'-di-N-(benzyloxycarbonyl)neamine (mass spectrum (LC/MS) m/z 1076 [M+H⁺]) as a starting material.

Example 7 1.6' -diguanidino- 1.6 '-dideaminoneamine trifluoroacetate

[0126] Reaction mixture of 6'-[N,N'-di-tert-(butoxycarbonyl)guanidino]-6'- deaminoneamine (500 mg, 0.886 mmol), NN'-di-(tert-butoxycarbonyl)-N"-triflylguanidine (347 mg, 0.886 mmol), and triethylamine (0.0.37 mL, 2.658 mmol) in methanol (25 mL) was stirred at room temperature for 20 hours and then concentrated. The residue was purified by silica gel chromatography eluting with methanol-dichloromethane-concentrated aqueous ammonium hydroxide, and pure fractions were concentrated to give l,6'-di-[NN'-di-(tert- butoxycarbonyl)guanidino]-l,6'-dideaminoneamine (TLC R_f 0.16 silica, dichloromethane- methanol-concentrated aqueous ammonium hydroxide (9:1:0.1), mass spectrum (LC/MS) m/z 807 [M+H⁺]), 3,6'-di-[N,N'-di-(tert-butoxycarbonyl)guanidino]-3,6'-dideaminoneamine (TLC R_f 0.34, mass spectrum (LC/MS) m/z 807 [M+H⁺]), l,3,6'-tri-[NN'-di-(tert- butoxycarbonyl)guanidino]-l,3,6'-trideaminoneamine (TLC R_f 0.92, mass spectrum (LC/MS) m/z 1049 [M+H⁺]), and l,2',6'-tri- [NN'-di-(tert-butoxycarbonyl)guanidino]-l,2',6'- trideaminoneamine (TLC R_f 0.85, mass spectrum (LC/MS) m/z 1049 [M+H⁺]) as white solids.

[0127] Deprotection of l,6'-di~[N,N'-di-(tert-butoxycarbonyl)guanidino]-l,6'- dideaminoneamine (55 mg) was performed according to general procedure A to provide 1,6'- diguanidino- 1,6 '-dideaminoneamine trifluoroacetate salt as a pale yellow solid (25 mg). Mass spectrum (LC/MS) m/z 407 [M+H⁺].

Example 8 3.2 ' -di guanidino-3.2 ' -dideaminoneamine hydrochloride

[0128] A reaction mixture of l,6'-di-N-(tert-butoxycarbonyl)neamine (Roestamadji, J., et al, J. Am. Chem. Soc. 117:11060-11069 (1995)) (261.3 mg, 0.5 mmol), NN'-di-(tert- butoxycarbonyl)-N"-triflylguanidine (1174 mg, 3 mmol), and triethylamine (0.42 mL, 3 mmol) in methanol was stirred at room temperature for 18 hours. The reaction mixture was concentrated and purified by silica gel chromatography eluting with ethyl acetate- dichloromethane. Concentration and precipitation with water gave 350 mg of l,6'-di-N-(tert- butoxycarbonyl)-3 ,2 ' -di-[N,N'-di-(tert-butoxycarbonyl)guanidino] 3 ,2 ' -dideaminoneamine as a white powder. TLC R_f 0.25 silica, dichloromethane-ethanol (95:5). Mass spectrum (LC/MS) m/z 1008 [M+H⁺]. [0129] Deprotection of 250 mg of this material according to general procedure A provided 200 mg of 3, 2 '-diguanidino-3 ,2 '-dideaminoneamine trifluoroacetate salt as a white powder. Ion exchange of 160 mg ofthe trifluoroacetate salt according to general procedure B provided 80 mg of 3,2 '-diguanidino-3 ,2 '-dideaminoneamine hydrochloride as a white solid. Mass spectrum (LC/MS) m/z 407 [M+H⁺].

Example 9 3.6'-diguanidino-3.6'-dideaminoneamine trifluoroacetate

[0130] Deprotection of 3,6'-di-[NN'-di-(tert-butoxycarbonyl)guanidino]-3,6'- dideaminoneamine (isolated as a product in Example 7) (35 mg) was performed according to general procedure A to provide 3,6'-diguanidino-3,6'-dideaminoneamine trifluoroacetate salt as a white solid (15 mg). Mass spectrum (LC/MS) m/z 407 [M+H⁺].

Example 10 2'.6'-diguanidino-2'.6'-dideaminoneamine trifluoroacetate

[0131] A reaction mixture of 6'-[N,N'-di-tert-(butoxycarbonyl)guanidino]-6'- deaminoneamine (300 mg, 0.531 mmol) and zinc diacetate (467 mg, 2.125 mmol) in methanol (10 mL) was stirred for 2 hours at room temperature, and di-tert-butyl dicarbonate (128 mg, 0.584 mmol) was added. After stirring overnight, concentration and purification by silica gel chromatography eluting with chloroform-methanol-concentrated ammonium hydroxide (4:1:0.25) gave 140 mg of l-N-(tert-butoxycarbonyl)-6'-[NN'-di-tert- (butoxycarbonyl)guanidino] -6 '-deaminoneamine as a white solid. TLC R_f 0.45 silica, chloroform-methanol-concentrated ammonium hydroxide (4:1:0.3).

[0132] A reaction mixture of l-N-(tert-butoxycarbonyl)-6'-[NN'-di-tert- (butoxycarbonyl)guanidino] -6 '-deaminoneamine (100 mg, 0.177 mmol) and nickel diacetate (176 mg, 0.708 mmol) in methanol (5 mL) and water (0.5 mL) was stirred for 2 hours, after which NN'-di-(tert-butoxycarbonyl)-N"-triflylguanidine (104 mg, 0.266 mmol) was added. After 4 days, the reaction was concentrated and the residue purified by silica gel chromatography eluting with chloroform-methanol-concentrated aqueous ammonium hydroxide (9:1:0.15) to give l-N-(tert-butoxycarbonyl)-2',6'-di-[N,N'-di-tert-(butoxycarbonyl)guanidino]- 2 ',6 '-dideaminoneamine as a white solid. TLC R_f 0.13 silica, chloroform-methanol-concentrated ammonium hydroxide (9:1:0.1). Mass spectrum (LC/MS) m/z 807 [M+H⁺].

[0133] Deprotection according to general procedure A gave 2',6'-diguanidino-2',6'- dideaminoneamine trifluoroacetate salt. Mass spectrum (LC/MS) m/z 407 [M+H⁺].

Example 11 1.32 ' -triguanidino- 1.32 ' -trideaminoneamine hydrochloride

[0134] A reaction mixture of 6'-N-(tert-butoxycarbonyl)neamine Grapsas, I. et al. J. Org. Chem. 59(7):1918-1922 (1994) (150 mg, 0.355 mmol) NN'-di-(tert-butoxycarbonyl)-N"- triflylguanidine (1174 mg, 3 mmol), and triethylamine (0.45 mL, 3.2 mmol) in methanol (5 mL) was stirred at room temperature for 18 hours and then concentrated. The residue was purified by silica gel chromatography eluting with methanol-dichloromethane, followed by trituration from hexanes to give 360 mg of l,3,2'-tri-[NN'-di-tert-(butoxycarbonyl)guanidino]-l,3,2'- trideamino-ι5'-N-(tert-butoxycarbonyl)neamine. TLC R_f 0.27 silica, dichloromethane-methanol (97:3). Mass spectrum (LC/MS) m/z 1150 [M+H⁺].

[0135] Deprotection and ion exchange of 120 mg of this material were performed according to general procedures A and B to provide 40 mg of l,3,2'-triguanidino-l,3,2'- trideaminoneamine hydrochloride as a white solid. Mass spectrum (LC/MS) m/z 449 [M+H ].

Example 12 1.3.6'-triguanidino-1.3,6'-trideaminoneamine trifluoroacetate

[0136] Deprotection of l,3,6'-tri-[N,N'-di-(tert-butoxycarbonyl)guanidino]-l,3,6'- trideaminoneamine (isolated as a product in Example 7) (100 mg) was performed according to general procedure A to provide l,3,6'-triguanidino-l,3,6'-trideaminoneamine trifluoroacetate salt as a white solid (50 mg). Mass spectrum (LC/MS) m/z 449 [M+H⁺].

Example 13 1 ,2',6'-triguanidino- 2'.6'-trideaminoneamine trifluoroacetate

[0137] Deprotection of l,2',6'-tri-[NN'-di-(tert-butoxycarbonyl)guanidino]-l,2',6'- trideaminoneamine (isolated as a product in Example 7) (50 mg) was performed according to general procedure A to provide l,2',6'-triguanidino-l,2',6'-trideaminoneamine trifluoroacetate salt as a pale yellow powder (20 mg). Mass spectrum (LC/MS) m/z 449 [M+H⁺].

Example 14 32 ' .6 ' -tri guanidino-3.2 ' .6 ' -trideaminoneamine trifluoroacetate

[0138] A reaction mixture of l-N-(tert-butoxycarbonyl)-6'-[NN'-di-tert- (butoxycarbonyl)guanidino] -6 '-deaminoneamine (isolated as an intermediate in Example 10) (100 mg, 0.151 mmol), NN'-di-(tert-butoxycarbonyl)-N"-triflylguanidine (147 mg, 0.376 mmol), and triethylamine (0. mL, 3 mmol) in methanol was stirred at room temperature for 24 hours, after which an additional 147 mg of NN'-di-(tert-butoxycarbonyl)-N"-triflylguanidine in 10 mL methanol was added. After 69 hours, solvent was removed and the residue purified by silica gel chromatography in a manner similar to Example 11 to provide l-N-(tert- butoxycarbonyl)-3 ,2 ' ,6 ' -tri- [NN'-di-tert-(butoxycarbonyl)guanidino] -3 ,2 ' ,6 ' - trideaminoneamine.

[0139] Deprotection according to general procedure A provided 3,2',6'-triguanidino-3,2',6⁵ trideaminoneamine trifluoroacetate salt. Mass spectrum (LC/MS) m/z 449 [M+H].

Example 15 1.3.2'.6'-tetraguanidino-1.3.2'.6'-tetradeaminoneamine hydrochloride

[0140] l,3,2',6'-tetraguanidino-l,3,2',6'-tefradeaminoneamine trifluoroacetate salt (Hui, Y. et al, Tet. Lett. 43:9255-9257 (2002)) (170 mg) was loaded onto a column of Bio-Rad AG1-X2 resin (10 g) which had been pretreated with hydrochloric acid and washed with water to pH 7. Elution with dioxane-water (1:1) and concentration gave the desired l,3,2',6'-tetraguanidino- l,3,2',6'-tetradeaminoneamine hydrochloride as a light yellow solid (99 mg). Mass spectrum (LC/MS) m/z 491 [M+H⁺].

Example 16 1.3.2 ' .6 ' -tetraguanidino- 1.3.2 ' .6 ' -tetradeaminoneamine

[0141] l,3,2',6'-tetraguanidino-l,3,2',6'-tetradeaminoneamine trifluoroacetate salt (Hui, Y. et al, Tet. Lett. 43:9255-9257 (2002)) (150 mg) was loaded onto a column of Amberjet 4400 (OH^") resin. Elution with dioxane- water (1:1) and concentration gave the desired 1,3 ,2 ',6'- tetraguanidino-l,3,2',6'-tetradeaminoneamine hydrochloride as a light yellow solid (42 mg). Mass spectrum (LC/MS) m/z 491 [M+H⁺].

Example 17 2 ' -azido-2 ' -deaminoneamine hydrochloride

[0142] To a reaction mixture of 1,3,6 '-tri-N-(tert-butoxycarbonyl)neamine (Grapsas, I. et al. J. Org. Chem. 59(7): 1918-1922 (1994); Roestamadji, J., et al, Bioorg. Med. Chem. Lett. 8(24):3483-3488 (1998)) (930 mg, 1.49 mmol), triflyl azide Alper, P. B. et al, Tet. Lett., 37(34):6029-6032 (1996) (5.92 mmol), and sodium carbonate (310 mg, 2.92 mmol) in dichloromethane- water-methanol (16:3:15, 34 mL) at 4°C was added copper sulfate (0.2 mL of a 2% solution in water). After warming to room temperature overnight, the mixture was concentrated, taken up in aqueous sodium hydroxide and extracted with ethyl acetate. The organic fraction was concentrated and purified by chromatography on silica gel eluting with chloroform-methanol (10:1) to give 2'-azido-2'-deamino-l,3,6'-tri-N-(tert- butoxycarbonyl)neamine (630 mg).

[0143] 2'-azido-2'-deamino-l,3,6'-tri-N-(tert-butoxycarbonyl)neamine (49 mg) was treated with hydrochloric acid in dioxane (4.0 molar, 5.0 mL) for 3 hours and the mixture concentrated to provide 2 '-azido-2 '-deaminoneamine hydrochloride (35 mg). Mass spectrum (LC/MS) m/z 349 [M+H⁺].

Example 18 12 '-diazido-1.2 '-dideaminoneamine hydrochloride

[0144] In a manner similar to Example 19 but utilizing 3,6'-di-N-(tert- butoxycarbonyl)neamine as a starting material, 1,2 '-diazido-1, 2 '-dideaminoneamine hydrochloride is prepared.

Example 19 3.2' -diazido-3.2 ' -dideaminoneamine hydrochloride

[0145] To a reaction mixture of l,6'-di-N-(tert-butoxycarbonyl)neamine (Roestamadji, J., et al, J. Am. Chem. Soc. 117:11060-11069 (1995)) (450 mg, 0.861 mmol), triflyl azide (Alper, P. B. et al, Tet. Lett., 37(34):6029-6032 (1996)) (5.92 mmol), and sodium carbonate (250 mg, 2.36 mmol) in dichloromethane-water-methanol (16:3:15, 34 mL) at 4 °C was added copper sulfate (0.2 mL of a 2% solution in water). After warming to room temperature overnight, the mixture was concentrated, taken up in aqueous sodium hydroxide and extracted with ethyl acetate. The organic fraction was concentrated and purified by chromatography on silica gel eluting with chloroform-methanol (10:1) to give 3,2'-diazido-3,2'-dideamino-l,6'-di-N-(tert- butoxycarbonyl)neamine (371 mg).

[0146] 3,2'-diazido-3,2'-dideamino-l,6'-di-N-(tert-butoxycarbonyl)neamine (65 mg) was treated with hydrochloric acid in dioxane (4.0 molar, 5.0 mL) for 3 hours and the mixture concentrated to provide 3, 2 '-diazido-3 ,2 '-dideaminoneamine hydrochloride (60 mg). Mass spectrum (LC/MS) m/z 375 [M+H^1"].

Example 20 3.6' -diazido-3 ,6 '-dideaminoneamine hydrochloride

[0147] In a manner similar to the examples above, but using l,3-di-N-(butyloxycarbonyl)- 3,6'-di-N-(benzyloxycarbonyl)neamine as an intermediate, 1,2 '-diazido-1, 2 '-dideaminoneamine hydrochloride is prepared. Example 21 5.6.3 '.4'-tetra-O-methylneamine hydrochloride

[0148] A reaction mixture of l,3,2',6'-tetraazido-l,3,2',6'-tetradeaminoneamine (Alper, P. B. et al, Tet. Lett., 37(34): 6029-6032 (1996)) (200 mg, 0.47 mmol) and sodium hydride (60 mg 80%) dispersion, washed with 2 x 5 L tetrahydrofuran) in NN-dimethylformamide (2.0 mL) was stirred for 30 minutes. Methyl iodide (1.0 mL, 16.0 mmol) was added and stirring continued overnight. Concentration and chromatography on silica gel eluting with chloroform- hexanes (4:1) provided l,3,2',6'-tetraazido-l,3,2',6'-tetradeamino-5,6,3',4'-tetra-0- methylneamine (80 mg). Mass spectrum (LC/MS) m/z 505 [M+Νa+H⁺].

[0149] A reaction mixture of l,3,2',6'-tetraazido-l,3,2',6'-tetradeamino-5,6,3',4'-tetra-0- ' methylneamine (60 mg, 0.124 mmol) and 20% Pd(OH)₂ on carbon (60 mg) in ethanol (20 mL) was treated with hydrogen for 16 hours. Filtration and concentration gave a residue which was purified by silica gel chromatography, eluting with chloroform-methanol-concentrated aqueous ammonium hydroxide (3:2:0.2). Treatment of concentrated fractions with ethanol and hydrochloric acid followed by concentration gave 5,6,3 ',4'-tetra-0-methylneamine hydrochloride (43 mg) as a white solid. Mass spectrum (LC/MS) m/z 379 [M+H⁺].

Table A. Anthrax lethal factor inhibition data for guanidinoneamine derivatives.

Example 22 1.3.2\6'-tefraguanidino-1.3.2'.6'-tefradeammo-5.6.3'.4'-tefra-O-methylneamine hydrochloride

[0150] In a manner similar to the preparation ofthe above examples, 1,3,2',6'- tetraguanidino-l,3,2',6'-teti'adeamino-5,6,3',4'-tetra-0-methylneamine hydrochloride is prepared via 1 ,3 ,2 ' ,6 ' -tetra-N-[N,N'-di-tert-(butoxycarbonyl)guanidino]- 1 ,3 ,2' ,6 '-tetradeamino- 5,6,3 ',4'-tetra-O-methylneamine (mass spectrum (LC/MS) m/z 674 [M/2+H⁺]).

[0151] Selective guanidinylation of 5,6,3',4'-tetra-O-methylneamine (Example 21) may be performed according to methods similar to those described in the Examples above to provide selective mono-, di-, and tri-N-guanidinyl derivatives of 5,6,3 ',4'-tetra-O-methylneamine. Product mass spectrum (LC/MS) m/z 547 [M+H⁺].

Example 23 2'-(4-phenyl-[1.2.3]triazol-l-yl)-2'-deaminoneamine hydrochloride

[0152] A reaction mixture of 2'-azido-2'-deamino-l,3,6'-tri-N-(tert- butoxycarbonyl)neamine (obtained as an intermediate in Example 17) (120 mg, 0.185 mmol) and phenylacetylene (0.1 mL, 0.91 mmol), NN-diisopropylethylamine (0.25 mL, 0.15 mmol) and copper iodide (6 mg, 0.031 mmol) in NN-dimethylformamide (1 mL) was stirred in the dark overnight at room temperature. Volatiles were removed by concentration and the residue purified by column chromatography on silica, eluting with chloroform-methanol (10:1) to provide 136 mg of 2'-(4-phenyl-[l,2,3]triazol-l-yl)-2'-deamino-l,3,6'-tri-N-(tert- butoxycarbonyl)nearnine. Mass spectrum (LC/MS) m/z 751 [M+H⁺].

[0153] This material was treated with hydrochloric acid in 1,4-dioxane (4 molar) for 3 hours. Evaporation gave 2'-(4-phenyl-[l,2,3]triazol-l-yl)-2'-deaminoneamine hydrochloride as a solid. Mass spectrum (LC/MS) m/z 451 [M+H⁺].

Example 24

[0154] Selective guanidinylation, protection, and derivatization ofthe above isomer of neamine (Ryu, D. H., et al, Bioorg. Med. Chem. Lett. 13(5):901-903 (2003)) is performed in a manner similar to the methods from the Examples above to provide mono-, di-, tri- and tetraguanidino derivatives.

Example 25

[0155] Selective guanidinylation, protection, and derivatization ofthe above isomer of neamine (Ryu, D. H., et al, Bioorg. Med. Chem. Lett. 13(5):901-903 (2003)) is performed in a manner similar to the methods from the Examples above to provide mono-, di-, tri- and tetraguanidino derivatives.

Example 26

[0156] Selective guanidinylation, protection, and derivatization ofthe above isomer of neamine (Ryu, D. H., et al, Bioorg. Med. Chem. Lett. 13(5):901-903 (2003)) is performed in a manner similar to the methods from the Examples above to provide mono-, di-, tri- and tetraguanidino derivatives.

Example 27

[0157] Selective guanidinylation, protection, and derivatization ofthe above isomer of neamine (Ryu, D. H., et al, Bioorg. Med. Chem. Lett. 13(5):901-903 (2003)) is performed in a manner similar to the methods from the Examples above to provide mono-, di-, tri- and tetraguanidino derivatives. Example 28

[0158] Selective guanidinylation, protection, and derivatization ofthe above isomer of neamine (Ryu, D. H., et al, Bioorg. Med. Chem. Lett. 13(5):901-903 (2003)) is performed in a manner similar to the methods from the Examples above to provide mono-, di-, tri- and tetraguanidino derivatives.

Example 29 1.3-diguanidino-1.3-dideamino-4-0-(2-guanidinoethyl)-2-deoxystreptamine

[0159] Reaction of l,3-diazido-l,3-dideammo-5,6-di-O-benzyl-2-deoxystreptamine with 2- bromoethanol in the presence of a base such as sodium hydride or potassium carbonate in NN- dimethylformamide gives l,3-diazido-l,3-dideamino-4-( -(2-hydroxyethyl)-5,6-di-O-benzyl-2- deoxystreptamine which, under treatment withNN',N"-tri-(tert-butoxycarbonyl)guanidine, triphenylphosphine and diethyl or diisopropyl diazodicarboxylate in dichoromethane or tetrahydrofuran gives 1 ,3 -diazido- 1 ,3 -dideamino-4-( -[2-(N,N'-di-tert- butoxycarbonyl)guanidinoethyl]-5,6-di-( -benzyl-2-deoxystreρtamine. Reduction is accomplished under hydrogenation conditions (palladium on carbon, hydrogen balloon pressure) to give 4-t -[2-(NN'-di-tert-butoxycarbonyl)guanidinoethyl]-2-deoxystreptamine. This compound is then reacted with excess NN'-di-(tert-butoxycarbonyl)-N' '-triflylguanidine and triethylamine in a solvent mixture of dioxane and water or methanol and water. Under general procedure A and B, 1,3 -diguanidino- 1, 3 -dideamino-4-( -(2-guanidinoethyl)-2-deoxystreptamine hydrochloride is isolated. By limiting the amount of NN'-di-(tert-butoxycarbonyl)-N"- triflylguanidine used, diguanidinylated products can also be obtained.

Example 30 1.3-diguanidino-1.3-dideamino-4-0-rcarboxamidine -2 -deoxystreptamine

[0160] Reaction of l,3-diazido-l,3-dideamino-5,6-di-0-benzyl-2-deoxystreptamine with bromoacetonitrile in the presence of base such as sodium hydride or potassium carbonate in NN-dimethylformamide gives l,3-diazido-l,3-dideamino-4-O-cyanomethyl-5,6-di-O-benzyl-2- deoxystreptamine. This compound is reduced under hydrogenation conditions (palladium on carbon, hydrogen balloon pressure) to give 4-O-cyanomethyl-2-deoxystreptamine. The cyano group is converted into an amidine by treatment with ammonia followed by dry hydrochloric acid in methanol. A protocol similar to that described in Example 29 is followed to provide 1,3- diguanidino- 1 ,3 -dideamino-4-0-(carboxamidine)-2-deoxystreptamine hydrochloride.

Example 31 1.3-diguanidino-1.3-dideamino-4-0-(2-guanidinophenyl)-2-deoxystreptamine

[0161] Reaction of l,3-diazido-l,3-dideamino-5,6-di-0-benzyl-2-deoxystreptamine with 2- bromo- or 2-fluoro- or 2-chloronitrobenzene in the presence of base such as potassium carbonate in NN-dimethylformamide gives l,3,-diazido-l,3,-dideamino-4-O-(2-nitrophenyl)-5,6-di-0- benzyl-2-deoxystreptamine. Under hydrogenation conditions as described in Example 29, this compound is reduced to 4-c -(2-aminophenyl)-2-deoxystreptamine. Under guanidinylation and deprotection conditions similar to those described in Example 29, l,3-diguanidino-l,3- dideamino-4-O-(2-guanidinophenyl)-2-deoxystreptamine hydrochloride is obtained.

Example 32 L3-diguanidino-1.3-dideamino-4-0-f2-benzamidineV2-deoxystreptamine

[0162] Reaction of l,3-diazido-l,3-dideamino-5,6-di-0-benzyl-2-deoxystreptamine with 2- bromo or 2-fluoro- or 2-chlorocyanobenzene in the presence of base such as potassium carbonate in dimethylformamide gives l,3-diazido-l,3-dideamino-4-<9-(2-cyanophenyι)-5,6-di- O-benzyl-2-deoxystreptamine. Under hydrogenation conditions as described in Example 29, 4- O-(2-cyanophenyl)-2-deoxystreptamine is obtained, which is converted to an amidine as described in Example 30. Under guanidinylation and deprotection conditions similar to those described in Example 29, l,3-diguanidino-l,3-dideamino-4-c -(2-benzamidine)-2- deoxystreptamine hydrochloride is obtained.

Example 33 3-guanidino-3-deamino-4-O-(2-benzamidine)-2-deoxystreptamine

[0163] As described in Example 32, under the conditions of a limiting amount of NN'-di- (tert-butoxycarbonyl)-N"-triflylguanidine followed by chromatography, 3 -guanidino-3 - deamino-4-O-(2-benzamidine)-2-deoxystreptamine hydrochloride is obtained. Example 34 1.3 -diguanidino- 1.3-dideamino-4-O-f4-guamdinophenyl)-2 -deoxystreptamine

[0164] As described in Example 31 but with 4-bromo-, 4-fluoro- or 4-chloronitrobenzene used as starting material, 1,3 -diguanidino- 1,3 -dideamino-4-c -(4-guanidinophenyl)-2- deoxystreptamine hydrochloride is obtained.

Example 35 1.3-diguanidino-1.3-dideamino-4-t -(2.4-diguanidinopheιιyl)-2-deoxystreptamine

[0165] As described in Example 31 but with 1 -bromo-, 1 -fluoro- or l-chloro-2,4- dinitrobenzene used as starting material, l,3-diguanidino-l,3-dideamino-4-0-(2,4- diguanidinophenyl)-2-deoxystreptamine hydrochloride is obtained. Example 36 3-guanidino-3-deamino-4-( -(2,4-diguanidinophenyl -2-deoxystreptamine

[0166] As described in Example 35 under the conditions of a limiting amount of NN'-di- (tert-butoxycarbonyl)-N"-triflylguanidine followed by chromatography, 3 -guanidino-3 - deamino-4-0-(2,4-diguanidinophenyl)-2-deoxystreptamme hydrochloride is obtained.

Example 37 3 - guanidino-3 -deamino-4- Q-(2- guanidino-4-aminophenyl)-2-deoxystreptamine

[0167] As described in Example 36, 3-guanidino-3-deamino-4-O-(2-guanidino-4- aminophenyl)-2-deoxystreptamine hydrochloride is obtained.

Example 38 1.3-diguanidino-1.3-dideamino-4-O-(2-guanidino-4-aminophenylV2-deoxystreptamine

[0168] As described in Example 36, l,3-diguanidino-l,3-dideamino-4-0-(2-guanidino-4- aminophenyl)-2-deoxystreptamine hydrochloride is obtained.

Example 39 l,3-diguanidino-1.3-dideamino-4-O-(2-guanidinopyridin-3-ylV2-deoxystreptamine

[0169] As described in Example 31 but with 3-chloro-2-nitropyridine as a starting material, l,3-diguanidino-l,3-dideamino-4-0-(2-guanidinopyridin-3-yl)-2-deoxystreptamine hydrochloride is obtained.

Example 40 1.3 -diguanidino- 1.3 -dideamino-4-O-f 4-guanidinopyridin-3 -yl)-2-deoxystreptamine

[0170] As described in Example 39 but with 3-chloro-4-nitroρyridine as a starting material, l,3-diguanidino-l,3-dide nino-4-O-(4-gUcim^inopyridin-3-yl)-2-deoxysfreρtamine hydrochloride is obtained.

Example 41 1.3-diaιanidino-1.3-dideamino-4-0-f3-guanidinopyridin-4-ylV2-deoxystreptamine

[0171] As described in Example 39 but with 4-chloro-3-nitropyridine as a starting material, l,3-diguanidino-l,3-dideamino-4-( -(3-guanidinopyridin-4-yl)-2-deoxystreptamine hydrochloride is obtained.

Example 42 1.3-diguaιιidino-1.3-dideamino-4-0-(2-guanidinophenyl)-2,5-dideoxystreptamine

[0172] As described in Example 31 but with 1,3 -diazido-1, 3 -deamino-2,5- dideoxysfreptamine used as the starting material (Haviv, F. et al, Can. J. Chem. 56(20):2677- 2680 (1978)), l,3-diguanidino-l,3-dideamino-4-0-(2-guanidinophenyl)-2,5-dideoxystreptamine hydrochloride is obtained. Example 43 3-guanidino-3-deamino-4-O-(2-guanidinophenyl)-2 -deoxystreptamine

[0173] As described in Example 42 but under the conditions of a limiting amount of N,N'~ di-(tert-butoxycarbonyl)-N"-triflylguanidine followed by chromatography, 3-guanidino-3- deamino-4-c -(2-guanidinophenyl)-2-deoxystreptamine hydrochloride is obtained.

Example 44 LF Inhibition Assay

[0174] Compounds were tested to determine if they inhibit anthrax lethal factor-mediated cleavage of a synthetic substrate corresponding to the cleavage region of MAPKK (mitogen- activated protein kinase kinase). Lethal factor (20 nM final concentration) and MAPKK substrate (MAPKKide® 12.5 μM, final concentration) were purchased from List Biological Laboratories, Campbell, CA. The assay final volume was 50 μl (Fisher #3694 96-well half area plates) consisting of 5 μl inhibitor/test sample/buffer, 25 μl buffer (20 mM Hepes, pH 7.4), 10 μl enzyme and 10 μl substrate. Test sample, buffer and enzyme were incubated briefly at room temperature. Upon addition of substrate, the reaction was linear for 15 min at room temperature. Fluorescence intensity was determined in the kinetic mode (Ex: 320 nm, Em: 420 nm; 6-minute read time; Molecular Devices Gemini fluorescence plate reader) and data (IC50/Ki) was reduced by SoftMax Pro (Molecular Devices, Sunnyvale, CA).

[0175] Assays for inhibition of other proteases were carried out in 50 μl reaction volumes (in 96-well plates) using similar fluoresecence- or absorbance-based assays. For botulinum neurotoxin/A the buffer was 30mM Hepes pH 7.3, 5mM DTT, 0.25mM ZnCL2, 1 mg/mL BSA. Botulinum neurotoxin/A was purchased from the University of Wisconsin. Substrate (SΝAPtide) was purchased from List Laboratories (Campbell, CA, Cat # 5203A). Human cathepsin B (a representative cysteine protease) was carried out in 0.1 M Νa Phosphate pH 6.15, 1 mM EDTA, 100 mM ΝaCl, 1 mM DTT, .01% Tween 20, .01% Νa Azide) using enzyme from Enzyme Systems Products (Cat # CATB 1; final concentration = lOnM) and substrate ZRRAMC ( Calbiochem Cat # 219392). Cathepsin D (a representative aspartyl protease) was carried out in 50 mM Na Acetate pH 4.0, 50 mM NaCI using human spleen enzyme from Calbiochem (Cat #219394) and the substrate Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu- Lys(Dnp)D-Arg-NH2( Calbiochem Cat #219360). The trypsin assay (a representative serine protease) was conducted in a buffer composed of 50 mM Tris pH 8.2, 150 mM NaCI, 0.05% Tween 20. Bovine pancrease trypsin was purchased from Calzyme and substrate was purchased from Sigma (TosylGPKpNA-Cat # T6140). Matrix metalloprotease-9 was the representative human matrix metalloprotease assay. The assay conditions were Tris pH 7.6, 200 mM NaCI, 5mM CaC12, 20 uM ZnCL2, 0.05% BRIJ 35. The enzyme was purchased from Oncogene (Cat# PF024) and the substrate was purchased from Calbiochem (Cat# 444221).

[0176] The Ki with respect to inhibition LF, furin, botulinum A, trypsin, cathepsin B, MM-9 and cathepsin D is shown for compounds in the following Table B. An entry of zero in this chart indicates that the compound was tested and showed a 50% inhibitory concenfration of greater than 300 micromolar. Although individual stereoisomers are shown in Table B, in some cases mixtures of stereoisomers or a different stereoisomer were possibly present in the assays performed.

Table B. Structures and Ki Values

-4

-4

00

©

Claims

Claims

1. A method to prevent or treat anthrax lethal factor toxicity in a subject, which method comprises administering to a subject in need of such prevention or treatment an amount of a compound of formula (1) sufficient to effect said prevention or treatment, wherem said compound is ofthe formula:

X — Y — X (1) wherein each X is independently N or O, each N or O further coupled to substituents to satisfy its valence requirements; and

Y is a linker which permits at least one conformation which spaces the two X atoms at a distance that would be conferred by an alkylene chain consisting from 2-11 methylene units; wherein the compound of formula (1) may be administered as such, as a salt, and/or as a prodrug.

2. The method of claim 1 , wherein the compound of formula (1) is an aminoglycoside.

3. The method of claim 2, wherein said aminoglycoside is of the formula

wherein each of Y^1', Y^2' and Y^J-Y⁶ is independently R, OR, SR or NR₂ wherein each R is independently H or a substituent that does not interfere with the inhibitory activity ofthe aminoglycoside; wherem at least one of Y^!-Y⁶ must be NR₂, and no more than 5 of Y^1', Y^2', and Y*-Y⁶ can be H; and wherein ^: represents an optional double bond.

4. The method of claim 3, wherein said non-interfering substituents represented by R are independently, in addition to H, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-12 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; and wherein Y¹ and Y¹ may together form an optionally substituted spiro union.

5. The method of claim 4, wherein said optional substituents are selected from the group consisting of OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH and -C(NH₂)NH wherein R is defined as in claim 4.

6. The method of claim 5, wherein at least one R comprises an additional sugar moiety or an aminocyclitol, each optionally containing one or more guanidinyl groups.

7. The method as in claim 5, wherein optionally substituted R is selected from the group consisting of H, OH, NH₂, -NHC(NH)NH₂, -C(NH)NH₂, -CH₃, -CH₂CH₃, -CH(OH)CH₃, -CH₂NH_2> -CH₂NHCH₃, -CH(CH₃)NH₂, -CH(CH₃)NHCH₃,

8. The method defined in claim 3 wherein the aminoglycoside is selected from the group consisting of

A B C or an enantiomer thereof wherein each R is independently H or is 1-3 first substituents

selected from the group containing A, B, C, and

_}

D wherein each R on the first substituent is independently H, -C(NH₂)NH, a first linker to the aminoglycoside, or a second substituent selected from the group consisting of A, B, C, and D; wherein each R on the second substituent is H, -C(NH₂)NH, or a second linker to the first substituent; wherein the first and second linkers are independently selected from a bond, an acyl group, a C2-C12 alkyl, a C3-C8 cyclic alkyl, aryl, or heteroaryl; wherein a substitution that results in -NH-NH -or -O-O- is replaced with -NH- or -O- respectively; wherein a substitution that results in -NH-O -or -O-NH- is replaced with -NH- or -O-; wherein 1-3 R groups on the aminoglycoside and the first substituent is A, B, C, or D; and wherein 0-4 R groups on the aminoglycoside, the first substituent and the second substituent may contain a guanidinyl group.

9. The method defined in claim 8, wherein the anomeric carbon of A, B, or C, is linked to D through one ofthe O atoms of D.

10. The method defined in claim 9, wherein the compound is neamine or garamine; and wherein the compound contains 1-4 guanidinyl groups.

11. A library of aminoglycoside compounds as defined in claim 8.

12. The method defined in claim 1 wherem the compound comprises an aminocyclitol of formula (3),

1 1 1 wherein each of X -X is independently R, OR, or NR and wherein at least 2 of X -X are OR, wherein R is independently H or a substituent that does not interfere with the inhibitory activity ofthe aminocyclitol.

I ll

13. The method defined in claim 12, wherein each R is independently, in addition to H, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-12 ring members and wherem one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; and wherein optional substituents are selected from OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO R, =N, -NHC(NH₂)NH, or -C(NH₂)NH, wherein R is defined as above.

14. The method as defined in claim 13, wherein optionally substituted R is selected from the group consisting of H, OH, NH₂, -C(NH)NH₂, -NHC(NH)NH₂, -CH₃, -CH₂CH₃,

wherein R¹ and R²

are independently H or CH₃,

and

15. The method defined in claim 12 wherein the aminocyclitol has the formula

E F wherein X⁶ is H or -C(NH₂)NH; and wherein X³ is -OR or -R where R is alkyl, aryl, heteroaryl, and alkylaryl substituted with 0-3 OH, 0-3 NH₂, and 1-3 -NHC(NH)NH₂ or -C(NH)NH groups.

16. An aminocyclitol having the formula,

E F wherein X⁶ is H or -C(NH₂)NH; wherein X³ is -OR, -NHR or -R where R is alkyl, aryl, heteroaryl, and alkylaryl substituted with 1-3 -NHC(NH)NH₂ or -C(NH)NH₂ groups; and wherein X³ is not -OR when the O is connected to an anomeric carbon.

17. A library of aminocyclitol compounds defined in claim 16.

18. The method defined in claim 12, wherein the aminocyclitol is

or an enantiomer thereof, wherein each R is independently H, -C(NH₂)NH, or is 1-3 first substituents selected from the group consisting of

A B C D or an enantiomer thereof, wherein each R is independently H, -CH₃ or is 1-3 first substituents selected from the group consisting of A, B, and C; wherein each R on the first substituent is independently H, -C(NH₂)NH, a first linker to the aminocyclitol, or a second substituent selected from the group consisting of A, B, C, and D; wherein each R on the second substituent is H, -CH₃, -C(NH₂)NH, or a second linker to the first substituent, wherein the first and second linkers are independently selected from a bond, a C1-C12 acyl, a C2-C12 alkyl, a C3-C8 cyclic alkyl, aryl, or heteroaryl; wherein a substitution that results in -NH-NH -or -O-O- is replaced with -NH- or -O- respectively; wherein a substitution that results in -NH-O -or -O-NH- is replaced with -NH- or -O-; wherem 1-3 R groups on the aminocyclitol and the first substituent are A, B, C, or D; and wherein no more than 0-4 R groups on the aminocyclitol, the first substituent and the second substituents may contain a guanidinyl group.

19. A library comprising the compounds defined in claim 18.

20. The method of claim 1, wherein the compound of formula (1) is a polyamine, a polypeptide, or a protein, or a derivative thereof.

21. The method defined in claim 20 wherein the compound comprises a heteroalkyl backbone which contains one or more linear, branched and/or cyclic portions thereof and contains 4-120 members wherein from 1-40 members of said backbone are nitrogen atoms and the remainder are carbon atoms; wherein a terminal nitrogen in the backbone is NH or =N; wherein each nitrogen atom is spaced from any other nitrogen atom by at least one carbon atom; and wherein one or more carbon atoms is optionally substituted with 0-20 =O, 0-5 hydroxyl, and 0-5 alkylthioalkyl or alkylsulfanyl groups; or a salt or prodrug thereof.

22. The method defined in claim 21 , wherein the compound contains at least one amino acid side chain.

23. The method defined in claim 21, wherein the compound is a polypeptide which contains a D and/or L amino acid isomer.

24. The method defined in claim 21, wherein the polypeptide contains a D and/or L isomer of lysine, arginine, glutamine, or ornithine, or combinations thereof.

25. The method defined in claim 21 , wherein the compound is linear and contains 6-14 members in the backbone; wherein 2-4 members ofthe backbone are nitrogen atoms and the remainder are carbon atoms; wherein each nitrogen atom is spaced from another nitrogen atom in the backbone by at least two carbon atoms; and wherem two of said members are NH₂.

26. The method defined in claim 21 wherein the compound comprises a monocyclic backbone containing 6-14 members in the backbone; wherein 2-4 members are nitrogen atoms; and wherein 1-2 members ofthe monocyclic backbone optionally are substituted with a linear alkylamine.

27. The method defined in claim 21 wherein the compound is branched and contains 1-16 NH₂; and wherein each nitrogen atom is spaced from another nitrogen atom in the backbone, if present, by at least two carbon atoms.

28. The method defined in claim 21, wherem the compound contains from 1-7 guanidinyl groups.

29. The method defined in claim 28, wherein the compound is substituted with 1-15 =O, 0-3 hydroxyl, and 0-2 alkylthioalkyl or alkylsulfanyl.

30. The method defined in claim 29, wherein the alkylthiolalkyl group is methylthioethyl and the alkylsulfanyl group is methylsulfanyl.

31. The method defined in claim 20 wherein the compound is a protein.

32. The method defined in claim 31 selected from the group consisting of myeloperoxidase, lactoferrm, and alpha 1 PDX.

33. The method of claim 1, wherein the compound of formula (1) is a fused ring antibiotic containing N and/or O or a derivative thereof.

34. The method defined in claim 1 wherein the compound contains at least one aryl or heteroaryl ring optionally fused to 1-3 alkyl, heteroalkyl, alkenyl, or heteroalkenyl rings which contains 0-4 O, S or N; and wherein the ring or fused ring is optionally substituted with 0-11 substituents that do not interfere with the inhibitory activity ofthe compound.

35. The method defined in claim 34 wherein the non-interfering substituent, which may be optionally substituted, is selected from halo, NO₂, =O, or optionally substituted R, NR₂, OR, NRCO, CONR₂, or NH(C=O)NHR, wherein R is H, Na, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-14 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; and wherein the optional substituents are OR, SR, NR₂, =0, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH or -C(NH₂)NH wherein R is defined as above.

36. The method defined in claim 35 wherein the non-interfering substituent is selected from CI, -COOH, CN, OH, CH, =O,

NH₂, NO₂,

37. A compound of formula (2) or (3) or a salt or prodrug thereof,

-y5 -γ6

wherein each of Y^1', Y^2' and Y^Y⁶ is independently R, OR, SR or NR₂; wherein at least one of Y*-Y⁴ and Y⁶ must be NR₂, and no more than 5 of YX Y² , and Y^ can be Hj and wherein represents an optional double bond;

wherein each of X -X is independently R, OR, or NR₂ and wherein at least 2 of X -X are OR; wherein each R independently is H, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-12 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; wherein Y¹ and Y² may together form an optionally substituted spiro union; and wherein said optional substituents are selected from the group consisting of OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH and -C(NH₂)NH wherein R is defined above; and wherein the compound is not streptomycin or derivatives thereof, 6-N-guanidino kanamycin A, 1-N-guanidino gentamicins, 2 '-and 3-N-guanidino gentamicin , 2 '-and 6'-N- guanidino gentamicin C_la; or 6'-N-guanidino gentamicin C₂.

38. The compound defined in claim 37 which is a) a neamine containing 1-3 guanidinyl groups or enantiomers thereof; b) a neomycin B containing 1-5 guanidinyl groups or enantiomers thereof; or c) l,3,2',6'-tetraguanidino-l,3,2',6'-tetradeamino-5,6,3',4'-tetra-c - methylneamine hydrochloride or enantiomers thereof.

39. The compound defined in claim 37 which is selected from the group consisting of 1-guanidino-l -deaminoneamine, 3 -guanidino-3 -deaminoneamine, 2'-guanidino-2'- deaminoneamine, 6'-guanidino-6'-deaminoneamine, 1 ,3-diguanidino-l ,3 -dideaminoneamine, 1,2' -diguanidino- 1 ,2 ' -dideaminoneamine, 1,6' -diguanidino- 1,6' -dideaminoneamine, 3,2'- diguanidino-3 ,2 ' -dideaminoneamine, 3 ,6 '-diguanidino-3 ,6 ' -dideaminoneamine, 2 ' ,6 ' - diguanidino-2 ' ,6 ' -dideaminoneamine, 1 ,3 ,2 ' -triguanidino- 1,3,2' -trideaminoneamine, 1,3,6'- triguanidino- 1,3,6' -trideaminoneamine, 1 ,2 ' ,6 ' -triguanidino- 1 ,2 ' , 6 ' -trideaminoneamine, 3,2',6'-triguanidino-3,2',6'-trideammoneamine, and l,3,2',6'-tetraguanidino-l,3,2',6'- tetradeaminoneamine, or the salt or prodrug thereof, wherein the compound is not 1,3,2', 6'- tetraguanidino- 1 ,2,2' ,6 ' -tetradeaminoneamine trifluoroacetate.

40. A method of selectively forming a guanidinyl derivative of a compound of formula (2) or (3), wherein the compound of formula (2) contains an aminocyclitol group comprising:

wherein each of Y^1', Y^2' and Y^!-Y⁶ is independently R, OR, SR or NR₂; wherein at least one of Y^!-Y⁴ and Y⁶ must be NR₂, and no more than 5 of Y Y² , and Y^ can be Hj and wherein -^"■"— represents an optional double bond;

wherein each of X -X is independently R, OR, or NR₂ and wherein at least 2 of X -X are OR; wherein each R independently is H, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-12 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; wherein Y and Y may together form an optionally substituted spiro union; and wherein said optional substituents are selected from the group consisting of OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH and -C(NH₂)NH wherein R is defined above; reacting said compound, which optionally may be selectively protected, and which contains at least one group that is capable of forming a guanidinyl group, with N,N'-di-tert- butoxycarbonyl-N"-triflylguanidine or NN'-di-(benzyloxycarbonyl)-N' '-triflylguanidine to form an optionally protected guanidinyl derivative or salt thereof having at least one guanidinyl group; and optionally deprotecting the guanidinyl derivative.

41. The method as in claim 40, wherein the group that is capable of forming a guanidinyl group is an amino group; wherein the guanidinyl derivative is protected; wherein the method further comprises deprotecting the protected guanidinyl derivative; and wherem the guanidinyl derivative is a neamine derivative having 1-3 guanidinyl groups or a salt thereof.

42. The method as in claim 41 wherein the optionally protected compound is selected from the group consisting of 3,6'-di-N-(tert-butoxycarbonyl)neamine, l,3,6'-tri-N-(tert-butoxycarbonyl)neamine, neamine, 6'-N-(tert-butoxycarbonyl)neamine, 3,6'-di-N-(tert-butoxycarbonyl)neamine, 6'-[NN'-di-tert- (butoxycarbonyl)guanidino] -6 ' -deaminoneamine, 1,6' -di-N-(tβrt-butoxycarbonyl)neamine, Sje'-di-tNN'-di-^ert-butoxycarbony^guanidinoJ-S^'-dideammoneamine, l,2',6'-tri-[N,N'-di- (tert-butoxycarbonyl)guanidino] - 1 ,2 ' ,6 ' -trideaminoneamine, 1 -N-(tert-butoxycarbonyl)-6 ' - [NN'-di-tert-(butoxycarbonyl)guanidino]-6 '-deaminoneamine, and 1 -N-(tert-butoxycarbonyl)- 6 ' -[N,N'-di-tert-(butoxycarbonyl)guanidino] -6 ' -deaminoneamine; wherein the reactant is Ν,Ν'-di-tert-butoxycarbonyl-Ν"-triflylguanidine wherein the neamine derivative is selected from the group consisting of 1-guanidino- 1 -deaminoneamine, 3 -guanidino-3 -deaminoneamine, 2 ' -guanidino-2 ' -deaminoneamine, 6 ' -guanidino-6 ' -deaminoneamine, 1 ,3 -diguanidino- 1 ,3 -dideaminoneamine, 1,2' -diguanidino- 1 ,2 '-dideaminoneamine, 1,6' -diguanidino- 1,6' -dideaminoneamine, 3,2' -diguanidino-3 ,2 ' - dideaminoneamine, 3,6'-diguanidino-3,6'-dideaminoneamine, 2',6'-diguanidino- 2',6'-dideaminoneamine, l,3,2'-triguanidino-l,3,2'-trideaminoneamme, l,3,6'-triguanidino- l,3,6'-trideaminoneamine, l,2',6'-triguanidino-l,2',6'-trideaminoneamine, and 3,2',6'- triguanidino-3,2',6'-trideaminoneamine, or a salt thereof.

43. A method of making a derivative of a compound of formula (2) or (3),

wherein each of Y^1', Y^2' and Y^Y⁶ is independently R, OR, SR or NR₂; wherein at least one of Y^J-Y⁴ and Y⁶ must be NR₂, and no more than 5 of Y¹ , Y² , and Y^YXan be Hj and wherem - represents an optional double bond;

1 7 1 ^ wherein each of X -X is independently R, OR, or NR₂ and wherein at least 2 of X -X are OR; wherein each R independently is H, optionally substituted alkyl, alkenyl, aryl, or alkylaryl, wherem said alkyl contains 1-lOC, alkenyl contains 2-10C, aryl contains 5-12 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; wherein the compound of formula (2) contains an aminocyclitol group; wherein the compound contains 1-4 hydroxyl groups; comprising: reacting an azide derivative of a compound of formula (2) or (3) with a reactant for derivatizing the hydroxyl group or groups thereof; wherein the reactant forms an optionally substituted alkyl, alkenyl, aryl, or alkylaryl derivative, wherein an O, S, or N may replace a C in said derivative; and optionally deprotecting the derivative.

44. The method defined in claim 43, wherein the optional substituents are selected from the group consisting of OH, CN, -NHC(NH)NH₂, and -C(NH)NH₂.

45. The method defined in claim 43 wherein the derivative is selected from the group consisting of 5,6,3 ',4'-tetra-0- methylneamine hydrochloride, 1 ,3-diazido-l ,3-dideamino-4-0-(2-hydroxyethyl)-5,6-di-O- benzyl-2-deoxystreptamine, 1 ,3-diazido-l ,3-dideamino-4-0-cyanomethyl-5,6-di-0-benzyl-2- deoxystreptamine, 1 ,3,-diazido-l ,3,-dideamino-4-O-(2-nitrophenyl)-5,6-di-0-benzyl-2- deoxystreptamine, l,3-diazido-l,3-dideamino-4-c -(2-cyanophenyl)-5,6-di-O-benzyl-2- deoxystreptamine, l,3,-diazido-l,3,-dideamino-4-O-(4-nitrophenyl)-5,6-di-0-benzyl-2- deoxystreptamine, l,3,-diazido-l,3,-dideamino-4-O-(2,4-dinitrophenyl)-5,6-di-0-benzyl-2- deoxystreptamine, 1 ,3,-diazido-l ,3,-dideamino-4-O-(2-nitropyridinyl)-5,6-di-0-benzyl-2- deoxystreptamine, 1 ,3,-diazido-l ,3,-dideamino-4-O-(4-nitropyridinyl)-5,6-di-0-benzyl-2- deoxystreptamine, 1 ,3,-diazido-l ,3,-dideamino-4-O-(3-nitropyridinyl)-5,6-di-O-benzyl-2- deoxystreptamine, and 1 ,3 ,-diazido- 1 ,3 ,-dideamino-4-O-(2-nitroρhenyl)-6-( -benzyl-2- deoxystreptamine, or protected form, unprotected form, prodrug or salt thereof.

46. The method defined in claim 45 further comprising reacting the derivative with a N,N'-di-te7^Λt-butoxycarbonyl-N"-trifiylguanidine or N,N',N"-tri-(tβrt-butoxycarbonyl)-guanidine to form a derivative containing at least one guanidyl group.

47. The method defined in claim 46 wherein the derivative is selected from the group consisting of l,3,2',6'-tefraguanidino-l,3,2',6'-tetradeamino-5,6,3',4'-tetra-( - methylneamine hydrochloride, 1 ,3-diguanidino- 1 ,3-dideamino-4-O-(2-guanidinoethyl)-2- deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-0-(carboxamidine)-2-deoxystreptamine, 1 ,3-diguanidino- 1 ,3 -dideamino-4-O-(2-guanidinophenyl)-2-deoxystreptamine, 1 ,3- diguanidino-l,3-dideamino-4-( -(2-benzamidine)-2-deoxystreptamine, 3-guanidino-3- deamino-4-O-(2-benzamidine)-2-deoxystreptamme, l,3-diguanidino-l,3-dideamino-4-O-(4- guanidinophenyl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-0-(2,4- diguanidinophenyl)-2-deoxystreptamine, 3-guanidino-3-deamino-4-c -(2,4- diguanidinophenyl)-2-deoxystreptamine, 3-guanidino-3-deamino-4-0-(2-guanidino-4- aminophenyl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-c -(2-guanidino-4- aminophenyl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-0-(2-guanidinopyridin- 3-yl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-( -(4-guanidinopyridin-3-yl)-2- deoxystreptamine, 1 ,3-diguanidino- 1 ,3 -dideamino-4-0-(3 -guanidinopyridin-4-yl)-2- deoxystreptamine, 1 ,3-diguanidino- 1 ,3 -dideamino-4-{ -(2-guanidinophenyl)-2,5- dideoxystreptamine, and 3-guanidino-3-deamino-4-O-(2-guanidinophenyl)-2- deoxystreptamine, or a prodrug or salt thereof.

48. A compound selected from the groups consisting of l,3,2',6'-tetraguanidino- 1 ,3 ,2 ' ,6 '-tetradeamino-5,6,3 ',4 '-tetra-c -methylneamine hydrochloride, 1 ,3 -diguanidino- 1 ,3 - dideamino-4- -(2-guanidinoethyl)-2-deoxystreptamine, 1 ,3-diguanidino-l ,3-dideamino-4-0- (carboxamidine)-2-deoxystreptamine, 1 ,3-diguanidino-l , 3-dideamino-4-O-(2- guanidinophenyl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideammo-4-c -(2-benzamidine)- 2-deoxystreptamine, 3-guamdino-3-deammo-4-0-(2-benzamidine)-2-deoxystreptamine, 1,3- diguanidino-1, 3 -dideamino-4-0-(4-guanidinophenyl)-2 -deoxystreptamine, 1,3-diguanidino- l,3-dideamino-4-0-(2,4-diguanidinophenyl)-2-deoxystreptamine, 3-guanidino-3-deamino-4-0- (2,4-diguanidinophenyl)-2-deoxystreptamine, 3 -guanidino-3 -deamino-4-0-(2-guanidino-4- aminophenyl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideamino-4- -(2-guanidino-4- aminophenyl)-2-deoxystreptamine, l,3-diguanidino-l,3-dideamino-4-0-(2-guanidinopyridm- 3-yl)-2-deoxystreptamine, 1 ,3-diguanidino- 1 ,3-dideamino-4-0-(4-guanidinopyridin-3-yl)-2- deoxystreptamine, 1 ,3-diguanidino-l ,3 -dideamino-4-0-(3 -guanidmopyridin-4-yl)-2- deoxystreptamine, 1 ,3-diguanidino- 1 ,3-dideamino-4-0-(2-guamdinophenyl)-2,5- dideoxystreptamine, and 3-guanidino-3-deamino-4-O-(2-guanidinophenyl)-2- deoxystreptamine, or a prodrug or salt thereof.

49. A method of making an azide derivative of a compound of formula (2) or (3), wherein the compound of formula (2) contains an aminocyclitol group

wherein each

SR or NR₂; wherein at least one of Y*-Y⁴ and Y⁶ must be NR₂, and no more than 5 of Y¹ , Y² , and

Y^ can be Hj and wherein represents an optional double bond;

wherein each of X -X⁷ is independently R, OR, or NR₂ and wherein at least 2 of X -X⁵ are OR; wherein each R independently is H, optionally substituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, acyl or acylaryl, wherein said alkyl and acyl contain 1-20C, alkenyl contains 2-40C, aryl contains 5-12 ring members and wherein one or more ofthe carbon atoms may be replaced with a heteroatom selected from N, S and O; wherein Y and Y may together form an optionally substituted spiro union; and wherein said optional substituents are selected from the group consisting of OR, SR, NR₂, =O, CN, CF₃, halo, COR, OOCR, NRCOR, NROR, COOR, CONR₂, SO₂NR₂, NRSO₂R, =N, -NHC(NH₂)NH and -C(NH₂)NH wherein R is defined above; and comprising: reacting said compound which optionally may be selectively protected, and which contains at least one group that is capable of being derivatized to an azide, with an azide reactant to form an azide derivative or salt thereof having at least one azide group; and optionally deprotecting the azide derivative.

50. The method as in claim 49 wherein the compound is selected from the group consisting of 1,3,6 '-tri-N-(tert- butoxycarbonyl)neamme, 3,6'-di-N-(tert-butoxycarbonyl)neamine, l,6'-di-N-(tert- butoxycarbonyl)neamine, and l,3-di-N-(butyloxycarbonyl)-3,6'-di-N- (benzyloxycarbonyl)neamine; wherein the azide reactant is triflyl azide; and wherein a deprotected azide derivative is selected from the group consisting of 2'- azido-2' -deaminoneamine, 1 ,2'-diazido-l ,2 '-dideaminoneamine, 3,2 '-diazido-3 ,2'- dideammoneamine, and 3, 6 '-diazido-3, 6 '-dideaminoneamine; or a prodrug or salt thereof.

51. An azide derivative of a compound of formula (2) as set forth in claim 3, wherein the compound contains an aminocyclitol group and wherein an amino group is derivatized to an azide group.

52. An azide derivative of claim 51 wherein the compound is not tetra- azidoneamine.

53. The azide derivative of claim 52 having the following formula

or a salt thereof.

54. The method as in claim 1, wherein the compound further inhibits an activity of furin or botulinum A.

55. A pharmaceutical composition for the prevention or treatment of anthrax lethal factor toxicity which composition comprises a compound of formula (1) as set forth in claim 1 along with a pharmaceutically acceptable excipient.

56. A pharmaceutical composition for the prevention or treatment of anthrax lethal factor toxicity which composition comprises the aminoglycoside of formula (2) as set forth in claim 3 along with a pharmaceutically acceptable excipient.

57. A method to identify a compound useful to prevent or treat anthrax lethal factor toxicity which method comprises assessing the ability of a tested compound to inhibit the protease activity of B. anthracis lethal factor (LF) whereby a compound which inhibits said protease activity is identified as a compound that is useful in said prevention or treatment; wherein said compound tested is a compound of formula (1) as set forth in claim 1.

58. The method of claim 57, wherein said assessing is conducted as a screen and a multiplicity of compounds of formula (1) is thus assessed.

59. The method of claim 57 which comprises the steps of

(a) providing a substrate for B. anthracis lethal factor (LF) wherein said substrate is coupled to a fluorophore and a quencher for said fluorophore and wherein the fluorophore and quencher are disposed on said substrate such that cleavage of said substrate results in a portion to which the fluorophore is coupled and a separate portion to which the quencher is coupled; and

(b) contacting said substrate with LF in the presence of a candidate compound and in the absence of a candidate compound;

(c) measuring the fluorescence generated in the presence as compared to the absence of said candidate compound; whereby a candidate compound whose presence results in a diminution of fluorescence as compared to its absence is identified as a compound useful in preventing or treating anthrax toxicity.

60. The method of claim 59, wherein a multiplicity of candidate compounds is tested.

61. The method of claim 60, wherein said multiplicity consists essentially of aminoglycosides and/or aminocyclitols and/or polyamines and/or polyamino acids and/or proteins and/or aryl- or heteroaryl-contianing compounds and/or fused ring antibiotics containing N and/or O.

62. The method of claim 1 , wherein said compound of formula (1) is not neomycin, is not a l-hydroxyhydroρyrazin-2-one, and is not a hydroxamic acid sulfonamide.

63. The composition of claim 6, wherein said compound is not neomycin, is not a l-hydroxyhydropyrazin-2-one, and is not a hydroxamic acid sulfonamide.

64. The method of claim 57, wherein said compound of formula (1) is not neomycin, is not a l-hydroxyhydropyrazin-2-one, and is not a hydroxamic acid sulfonamide.

65. A method to inhibit B. anthracis lethal factor (LF) which method comprises contacting said lethal factor with a compound of formula (1) or a salt thereof in an amount sufficient to effect said inhibition, wherein said compound is ofthe formula:

X — Y — X (1) wherein each X is independently N or O, each N or O further coupled to substituents to satisfy its valence requirements,

Y is a linker which permits at least one conformation which spaces the two X atoms at a distance that would be conferred by an alkylene chain consisting of 2-11 methylene units.

66. The method of claim 65, wherein said compound of formula (1) is not a 1 -hydroxyhydropyrazin-2-one.

67. The compound having the formula 2'-(4-phenyl-[l,2,3]triazol-l-yl)-2'- deaminoneamine hydrochloride.