Modified Aminoglycoside Compounds And Uses Thereof In Disabling Bacterial Ribosome Patent Application (2025)

U.S. patent application number 17/325361 was filed with the patent office on 2021-09-09 for modified aminoglycoside compounds and uses thereof in disabling bacterial ribosome. This patent application is currently assigned to Technion Research & Development Foundation Limited. The applicant listed for this patent is Technion Research & Development Foundation Limited. Invention is credited to Timor BAASOV, Valery BELAKHOV, Alina KHONONOV, Michal SHAVIT KISHKOBER, Boris SMOLKIN.

MODIFIED AMINOGLYCOSIDE COMPOUNDS AND USES THEREOF IN DISABLINGBACTERIAL RIBOSOME

Abstract

Modified aminoglycoside compounds represented by Formula I asdefined and described in the specification are provided. Themodified aminoglycosides feature a diamine-containing functionalmoiety at one or more of positions 3', 4' and 6'. Uses of themodified aminoglycosides as antimicrobial (e.g., antibacterial)agents, and in treating medical conditions associated withmicroorganisms, are also provided.

Related U.S. Patent Documents

Claims

1. A compound represented by Formula I: ##STR00013## or apharmaceutically acceptable salt thereof, wherein: the dashed lineindicates a stereo-configuration of position 6' being an Rconfiguration or an S configuration; X.sub.1 is O or S; Rx1, Rx2,Ry1 and Rz are each independently selected from hydrogen, alkyl andcycloalkyl; Ry2-Ry9 and Rw1-Rw3 are each independently selectedfrom hydrogen, alkyl, and cycloalkyl; R.sub.1, R.sub.3 and R.sub.4are each independently NR.sub.23R.sub.24, OR.sub.20 or adiamine-containing moiety, wherein R.sub.20 is hydrogen, alkyl,cycloalkyl or the diamine-containing moiety, and each of R.sub.23and R.sub.24 is independently hydrogen, alkyl, cycloalkyl or acyl,provided that at least one of R.sub.1, R.sub.3 and R.sub.4 is orcomprises the diamine-containing moiety; R.sub.5 and R.sub.6 areeach independently selected from hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heteroalicyclic, aryl, heteroaryl and OR.sub.16,wherein Rib is independently selected from hydrogen, amonosaccharide moiety and an oligosaccharide moiety; andR.sub.7-R.sub.9 are each independently selected from the groupconsisting of hydrogen and acyl, wherein said diamine-containingmoiety comprises at least two amine-containing groups and at leastone linking group linking said at least two amine-containinggroups, and wherein said amine-containing groups and said at leastone linking group are arranged such that: (i) a difference in thepKa of at least two of said amine-containing groups is at least 1;and/or (ii) when the compound is in a physiological environment, atleast one of said amine-containing groups is protonated atphysiological pH while at least another of said amine-containinggroups is non-protonated; and/or (iii) when the compound interactswith a prokaryotic ribosomal RNA decoding site (A-site), the RNAundergoes a conformational change such that an O--P--O angle of atleast one phosphodiester bond is higher than 100.degree.; and/or(iv) when the compound interacts with a prokaryotic ribosomal RNAdecoding site (A-site), the functional moiety is capable ofadopting a configuration in which one of said amine-containinggroups is in close proximity and suitable orientation so as tointeract with a 2'-OH group of a ribose of a nucleotide in said RNAand another amine-containing moiety is in close proximity andsuitable orientation so as to interact with a phosphate group of anucleotide of an adjacent nucleotide.

2. The compound of claim 1, wherein said diamine-containingfunctional moiety is represented by the Formula:-(L1)n-N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b wherein: each of L1, L2,L3 and L4 is independently said linking group; each of N1, N2, N3and N4 is an amine-containing group; and each of a, b, n, m, k, andj is independently 0 or 1.

3. The compound of claim 1, wherein each of said amine-containinggroups is independently selected from amine, amide, guanyl,guanidyl, amide and hydrazine.

4. The compound of claim 1, wherein each of said linking groups isindependently a hydrocarbon group being of 1 to 6 carbon atoms inlength.

5. The compound of claim 4, wherein each of said linking groups isindependently an alkylene chain being of 1 to 6, or of 1 to 4, orof 2 or 3, carbon atoms in length.

6. The compound of claim 1, wherein said diamine-containingfunctional group is or comprises at least one of an ethylenediamine moiety, a methyl ethylenediamine moiety, adiethylenetriamine moiety, a N-(2-aminoethyl)pyrrolidone moiety,and a guanidine-ethyleneamine moiety.

7. The compound of claim 1, wherein R.sub.4 is or comprises saiddiamine-containing moiety.

8. The compound of claim 7, wherein R.sub.4 is OR.sub.20 andR.sub.20 is: -(L1)n-N1-(L2)m-N2 wherein: n and m are each 1; L1 andL2 are each independently an alkylene of 2 or 3 carbon atoms inlength; and N1 and N2 are each independently selected from amineand guanidyl.

9. The compound of claim 7, wherein R.sub.4 is:-N1-(L2)m-N2-(L3)k-(N3) wherein: m and k are 1; L2 and L3 are eachindependently an alkylene of 1, 2 or 3 carbon atoms in length; N1is amide; and each of N2 and N3 is independently an amine.

10. The compound of claim 1, wherein R.sub.1 is or comprises saiddiamine-containing moiety, and is:N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b wherein: m and k are each 1; jis 0 or 1; a is 1; b is 0 or 1; L2, L3 and L4, if present, are eachindependently an alkylene of 1, 2 or 3 carbon atoms in length; N1is amide; and each of N2, N3 and N4, if present, is independentlyan amine.

11. The compound of claim 10, wherein R.sub.4 isNR.sub.23R.sub.24.

12. The compound of claim 1, wherein at least one of R.sub.5 andR.sub.6 is OR.sub.16, and R.sub.16 is a monosacchride or anoligosaccharide.

13. The compound of claim 1, wherein each of Rx1, Rx2, Ry1 and Rzis hydrogen.

14. The compound of claim 1, wherein each of Ry2-Ry9 and Rw1-Rw3 ishydrogen.

15. The compound of claim 1, wherein each of R.sub.7 and R.sub.9 ishydrogen.

16. The compound of claim 1, selected from: ##STR00014####STR00015## ##STR00016## ##STR00017##

17. A pharmaceutical composition comprising a compound according toclaim 1.

18. A method of treating a medical condition associated with apathogenic microorganism in a subject in need thereof, the methodcomprising administering to the subject a compound according toclaim 1.

19. The method of claim 18, wherein said pathogenic microorganismis a bacterium.

20. The method of claim 18, wherein said pathogenic microorganismis an aminoglycoside-resistant microorganism.

Description

RELATED APPLICATIONS

[0001] This application is a Continuation of PCT Patent ApplicationNo. PCT/IL2019/051277 having International filing date of Nov. 22,2019, which claims the benefit of priority under 35 USC .sctn.119(e) of U.S. Provisional Patent Application No. 62/770,761, filedon Nov. 22, 2018. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

SEQUENCE LISTING STATEMENT

[0002] The ASCII file, entitled 86972SequenceListing.txt, createdon May 20, 2021, comprising 1,207 bytes, submitted concurrentlywith the filing of this application is incorporated herein byreference. The sequence listing submitted herewith is identical tothe sequence listing forming part of the internationalapplication.

FIELD AND BACKGROUND OF THE INVENTION

[0003] The present invention, in some embodiments thereof, relatesto aminoglycosides and, more particularly, but not exclusively, tonewly designed aminoglycoside compounds which are aimed atdisabling bacterial ribosome and are usable in treating medicalconditions associated with pathogenic microorganisms such aspathogenic bacteria, including drug-resistant bacterialstrains.

[0004] The ongoing emergence of multidrug-resistant pathogenicmicroorganism (pathogens) requires continuous intensive search fornovel antimicrobial agents (e.g., antibiotics). Unfortunately, onlytwo new classes of antibiotics, oxazolidinones and lipoproteinshave been introduced into clinical practice during the lastdecades. Furthermore, it is well documented that once a newantibiotic is introduced into the clinic, whether it is a novelchemical entity acting at a distinct bacterial target or asemisynthetic derivative that counters the resistance to its parentdrug, within only a short matter of time new resistance will emergeand create a serious public health problem. Some bacterial strainshave developed multidrug resistance that covers the majority ofcurrently available antibiotics. The significance of this healthproblem has re-energized the search for new antibacterial agentsand novel approaches.

[0005] One innovative approach is the development of catalyticantibiotics: the pharmacophore of an existing antibiotic ismodified to include a "catalytic warhead" that disables the targetin a catalytic manner. Unlike conventional antibiotics that act ontheir targets in either a reversible (non-covalent interaction) oran irreversible manner (covalent interaction), the antibioticsacting in a catalytic manner promote multiple turnovers of acatalytic cycle. The possible benefits include 1) activity at lowerdosages and consequently reduced side effects, 2) activity againstdrug-resistant bacteria, and 3) reduced potential for generatingnew resistance.

[0006] Catalytic drugs have been reported previously [Z. Yu, J. A.Cowan, Chem. Eur. J. 2017, 23, 14113-14127], and include, forexample, numerous peptide-cleaving agents based on small-moleculemetal complexes as artificial proteases [J. Suh, W. S. Chei, Curr.Opin. Chem. Biol. 2008, 12, 207-213], site-specific RNA-cleavingagents that combine a reactive moiety (phosphodiester cleavagedirected, nonmetallic warhead) with a recognition element(sequence-specific hybridization to target RNA) [T. Niittym-ki, H.Lonnberg, Org. Biomol. Chem. 2006, 4, 15-25], and nonmetallicsmall-organic molecules as artificial ribonucleases [T. Lcnnberg,K. M. Kero, Org. Biomol. Chem. 2012, 10, 569-574; R. Salvio, R.Cacciapaglia, L. Mandolini, F. Sansone, A. Casnati, RSC Adv. 2014,4, 34412-34416].

[0007] Aminoglycosides are highly potent, broad-spectrumantibiotics with many desirable properties for the treatment oflife-threatening infections.

[0008] It is accepted that the mechanism of action ofaminoglycoside antibiotics, such as paromomycin, involvesinteraction with the prokaryotic ribosome, and, more specifically,involves binding to the decoding A-site of the 16S ribosomal RNA,which leads to protein translation inhibition and interference withthe translational fidelity.

[0009] Several achievements in bacterial ribosome structuredetermination, along with crystal and NMR structures of bacterialA-site oligonucleotide models, have provided useful information forunderstanding the decoding mechanism in prokaryote cells andunderstanding how aminoglycosides exert their deleteriousmisreading of the genetic code. It has been shown thataminoglycosides exert their therapeutic (bactericidal) effect byselectively binding to the aminoacyl tRNA binding site (A-site) ofthe bacterial 16S rRNA, thereby interfering with translationalfidelity during protein synthesis [S. Magnet, J. S. Blanchard,Chem. Rev. 2005, 105, 477-498].

[0010] Previous reports on the ability of copper-aminoglycosidecomplexes to promote hydrolytic and oxidative cleavage of RNA haveprompted the potential use of these complexes as metallodrugs withpotent antibacterial activity. See, for example, A. Sreedhara, A.Patwardhan, J. A. Cowan, Chem. Commun. 1999, 2, 1147-1148; A.Patwardhan, J. A. Cowan, Dalton Trans. 2011, 40, 1795-1801; W.Szczepanik, A. Krezel, M. Brzezowska, E. Dworniczek, M.Jezowska-Bojczuk, Inorg. Chim. Acta 2008, 361, 2659-2666; and W.Szczepanik, J. Ciesiolka, J. Wrzesin'ski, J. Skala, M.Jezowska-Bojczuk, Dalton Trans. 2003, 1488-1494. However,antibacterial tests showed no significant enhancement in theactivity of the copper-aminoglycoside complex relative to that ofthe parent aminoglycoside [W. Szczepanik, E. Dworniczek, J.Ciesiolka, J. Wrzesin'ski, J. Skala, M. Jezowska-Bojczuk, J. Inorg.Biochem. 2003, 94, 355-364].

[0011] Some studies have shown that several simple oligoamines[Komiyama et al., J. Org. Chem. 1997, 62, 2155-2160; Yoshinari etal., Chem. Lett. 1990, 519-522], as well as basic polypeptides[Oivanen et al., Chem. Rev. 1998, 98, 961-990] have shown catalyticcleavage of RNA. It has also been shown that Neomycin B (NeoB),which has three times as many amines as 1,3-propanediamine,catalyzes hydrolysis of adenylyl(3'-5')-adenosine (ApA) 3-foldfaster than 1,3-propanediamine [Kirk et al., Chem. Commun. 1998,147-148]. NeoB consists of the meso-1,3-diaminocyclitol(2-deoxystreptamine, 2-DOS) ring for which the pK.sub.a values of5.74 and 8.04 were reported.

[0012] Yan et al. [Bioorg. Med. Chem. 2011, 19, 30-40] reported aseries of new derivatives of kanamycin B modified at the 4'-OHposition that showed antibacterial activity against both wild-typeand resistant bacteria. Therein, it is described that theside-chain-free amine is best tolerated by the ribosome; and thatthe A-site of the ribosome can accommodate bulky substituentslinked at the 4'-position.

[0013] Several studies have showed that a successful cleavage of anRNA phosphodiester bond requires substantial motion in theHO--C2'-C3'-O--P bonds of the ribose-3'-phosphate region to reachthe necessary low energy transition state wherein the C2'-OH groupis orientated for in-line nucleophilic attack on the scissile bond[T. Lcnnberg, K. M. Kero, Org. Biomol. Chem. 2012, 10, 569-574].Such flexibility is usually achieved by the enzyme-induced flippingof the base attached to the RNA scissile bond, as supported, forexample, by S. M. K. Takahashi, Acad. Press. New York 1982,435-468; X. J. Yang, T. Gerczei, L. Glover, C. C. Correll, Nat.Struct. Biol. 2001, 8, 968-973; and P. B. Rupert, A. R.Ferre-D'Amare, Nature 2001, 410, 780-786.

[0014] A mechanism for colicin E3 (ColE3), a natural enzymatictoxin produced in several E. coli strains, that selectively cleavesa phosphodiester bond between A1493 and G1494 of 16S rRNA, has beenproposed recently [C. L. Ng, K. Lang, N. A. G. Meenan, A. Sharma,A. C. Kelley, C. Kleanthous, V. Ramakrishnan, Nat. Struct. Mol.Biol. 2010, 17, 1241-1246]. This cleavage impairs the proteintranslation process and consequently leads to cell death. Theproposed mechanism of ColE3 also explains why this naturalribonuclease cleaves the specific position in the A site of rRNA,between A1493 and G1494. This region of the A site is veryimportant functionally (for correct proofreading) and is also oneof the most flexible and accessible regions in the whole ribosomebecause it needs to accommodate the incoming aminoacyl-tRNA.

[0015] WO 2017/118967 describes modified aminoglycosides featuringa core structure based on Rings I, II and optionally III ofparomomycin.

[0016] U.S. Pat. No. 7,635,586 discloses aminoglycosides derivedfrom Neomycin B, and their use as highly potent and effectiveantibiotics, while reducing or even blocking antibioticresistance.

[0017] Additional background art includes Nudelman, I., et al.,Bioorg Med Chem Lett, 2006. 16(24): p. 6310-5; Hobbie, S. N., etal., Nucleic Acids Res, 2007. 35(18): p. 6086-93; Kondo, J., etal., Chembiochem, 2007. 8(14): p. 1700-9; Rebibo-Sabbah, A., etal., Hum Genet, 2007. 122(3-4): p. 373-81; Azimov, R., et al., Am JPhysiol Renal Physiol, 2008. 295(3): p. F633-41; Hainrichson, M.,et al., Org Biomol Chem, 2008. 6(2): p. 227-39; Hobbie, S. N., etal., Proc Natl Acad Sci USA, 2008. 105(52): p. 20888-93; Hobbie, S.N., et al., Proc Natl Acad Sci USA, 2008. 105(9): p. 3244-9;Nudelman, I., et al., Adv. Synth. Catal., 2008. 350: p. 1682-1688;Nudelman, I., et al., J Med Chem, 2009. 52(9): p. 2836-45;Venkataraman, N., et al., PLoS Biol, 2009. 7(4): p. e95; Brendel,C., et al., J Mol Med (Berl), 2010. 89(4): p. 389-98; Goldmann, T.,et al., Invest Ophthalmol Vis Sci, 2010. 51(12): p. 6671-80; Malik,V., et al., Ther Adv Neurol Disord, 2010. 3(6): p. 379-89;Nudelman, I., et al., Bioorg Med Chem, 2010. 18(11): p. 3735-46;Warchol, M. E., Curr Opin Otolaryngol Head Neck Surg, 2010. 18(5):p. 454-8; Lopez-Novoa, J. M., et al., Kidney Int, 2011. 79(1): p.33-45; Rowe, S. M., et al., J Mol Med (Berl), 2011. 89(11): p.1149-61; Vecsler, M., et al., PLoS One, 2011. 6(6): p. e20733; U.S.Pat. Nos. 3,897,412, 4,024,332, 4,029,882, and 3,996,205; Greenberget al., J. Am. Chem. Soc., 1999, 121, 6527-6541; Kotra et al.,antimicrobial agents and chemotherapy, 2000, p. 3249-3256; Haddadet al., J. Am. Chem. Soc., 2002, 124, 3229-3237; Kandasamy, J. etal., J. Med. Chem. 2012, 55, pp. 10630-10643; Duscha, S. et al.,MBio, 2014, 5(5), p. e01827-14; Huth, M. E. et al., J Clin Invest.,2015, 125(2), pp. 583-92; Shulman, E. et al., J Biol Chem., 2014,289(4), pp. 2318-30 and FR Patent No. 2,427,341, JP Patent No.04046189.

[0018] Further background art includes T. Lcnnberg, K. M. Kero,Org. Biomol. Chem. 2012, 10, 569-574; C. L. Ng, K. Lang, N. A. G.Meenan, A. Sharma, Nat. Struct. Mol. Biol. 2010, 17, 1241-1246; M.Komiyama, K. Yoshinari, J. Org. Chem. 1997, 62, 2155-2160; K.Yoshinari, M. Komiyama, Chem. Lett. 1990, 19, 519-522; R.-B. B.Yan, M. Yuan, Y. F. Wu, X. F. You, X.-S. S. Ye, Bioorg. Med. Chem.2011, 19, 30-40; K. C. Nicolaou, V. A. Adsool, C. R. H. Hale, Org.Lett. 2010, 12, 1552-1555; N. S. Chindarkar, A. H. Franz, ARKIVOC(Gainesville, Fla., U.S.) 2008, 21; R. Pathak, D. Perez-Fernandez,R. Nandurdikar, S. K. Kalapala, E. C. Bottger, A. Vasella, Helv.Chim. Acta 2008, 91, 1533-1552; R. Pathak, E. C. C. Bcttger, A.Vasella, Helv. Chim. Acta 2005, 88, 2967-2985; E. D.Goddard-Borger, R. V. Stick, Org. Lett. 2007, 9, 3797-3800; B. a.Maguire, L. M. Wondrack, L. G. Contillo, Z. Xu, RNA 2008, 14,188-195; P. Pfister, S. Hobbie, Q. Vicens, E. C. Bcttger, E.Westhof, ChemBioChem 2003, 4, 1078-1088; Q. Vicens, E. Westhof,Chem. Biol. 2002, 9, 747-755; Y. Miao, V. A. Feher, J. A. McCammon,J. Chem. Theory Comput. 2015, 11, 3584-3595; Y. T. Pang, Y. Miao,Y. Wang, J. A. McCammon, J. Chem. Theory Comput. 2017, 13, 9-19; C.C. Correll, X. Yang, T. Gerczei, J. Beneken, M. J. Plantinga, J.Synchrotron Radiat. 2004, 11, 93-96; M. J. Belousoff, B. Graham, L.Spiccia, Y. Tor, Org. Biomol. Chem. 2009, 7, 30-33; R. J.Leatherbarrow, GraFit 5, Erithacus Software Ltd., Horley, U.K.,2001; S. Carr, D. Walker, R. James, C. Kleanthous, A. M. Hemmings,Structure 2000, 8, 949-960; and T. Baasov, B. Smolkin, A. Khononov,M. Shavit, V. Belakhov, ChemBioChem 2019, 20, 247-259. Theteachings of all of these documents are incorporated by referenceas if fully set forth herein.

SUMMARY OF THE INVENTION

[0019] The emergence of multidrug-resistant pathogens that areresistant to the majority of currently available antibiotics is asignificant clinical problem. The development of new antibacterialagents and novel approaches is therefore extremely important.

[0020] The present inventors have designed and practiced a seriesof new derivatives of the natural aminoglycoside antibiotics, whichwere shown to exhibit significant antibacterial activity againstwild-type bacteria and were especially potent against resistant andpathogenic strains, and which may be potentially used as a basisfor the design of catalytic antibiotics.

[0021] According to an aspect of some embodiments of the presentinvention there is provided a compound (a modified aminoglycoside)represented by Formula I:

##STR00001##

[0022] or a pharmaceutically acceptable salt thereof,

[0023] wherein:

[0024] the dashed line indicates a stereo-configuration of position6' being an R configuration or an S configuration;

[0025] X.sub.1 is O or S;

[0026] Rx1, Rx2, Ry1 and Rz are each independently selected fromhydrogen, alkyl and cycloalkyl;

[0027] Ry2-Ry9 and Rw1-Rw3 are each independently selected fromhydrogen, alkyl, and cycloalkyl;

[0028] R.sub.1, R.sub.3 and R.sub.4 are each independentlyNR.sub.23R.sub.24, OR.sub.20 or a diamine-containing moiety,wherein R.sub.20 is hydrogen, alkyl, cycloalkyl or thediamine-containing moiety, and each of R.sub.23 and R.sub.24 isindependently hydrogen, alkyl, cycloalkyl or acyl, provided that atleast one of R.sub.1, R.sub.3 and R.sub.4 is or comprises thediamine-containing moiety;

[0029] R.sub.5 and R.sub.6 are each independently selected fromhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic,aryl, heteroaryl and OR.sub.16, wherein R.sub.16 is independentlyselected from hydrogen, a monosaccharide moiety and anoligosaccharide moiety; and

[0030] R.sub.7-R.sub.9 are each independently selected from thegroup consisting of hydrogen and acyl,

[0031] wherein the diamine-containing moiety comprises at least twoamine-containing groups and at least one linking group linking theat least two amine-containing groups, and wherein theamine-containing groups and the at least one linking group arearranged such that:

[0032] (i) a difference in the pKa of at least two of theamine-containing groups is at least 1; and/or

[0033] (ii) when the compound is in a physiological environment, atleast one of the amine-containing groups is protonated atphysiological pH while at least another of the amine-containinggroups is non-protonated; and/or

[0034] (iii) when the compound interacts with a prokaryoticribosomal RNA decoding site (A-site), the RNA undergoes aconformational change such that an O--P--O angle of at least onephosphodiester bond is higher than 100.degree.; and/or

[0035] (iv) when the compound interacts with a prokaryoticribosomal RNA decoding site (A-site), the functional moiety iscapable of adopting a configuration in which one of theamine-containing groups is in close proximity and suitableorientation so as to interact with a 2'-OH group of a ribose of anucleotide in the RNA and another amine-containing moiety is inclose proximity and suitable orientation so as to interact with aphosphate group of a nucleotide of an adjacent nucleotide.

[0036] According to some of any of the embodiments describedherein, the diamine-containing functional moiety is represented bythe Formula:

-(L1)n-N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b

[0037] wherein: each of L1, L2, L3 and L4 is independently thelinking group; each of N1, N2, N3 and N4 is an amine-containinggroup; and each of a, b, n, m, k, and j is independently 0 or1.

[0038] According to some of any of the embodiments describedherein, each of the amine-containing groups is independentlyselected from amine, amide, guanyl, guanidyl, amide andhydrazine.

[0039] According to some of any of the embodiments describedherein, each of the linking groups (in Formula I and any of therespective embodiments and combinations thereof) is independently ahydrocarbon group being of 1 to 6 carbon atoms in length.

[0040] According to some of any of the embodiments describedherein, each of the linking groups (in Formula I and any of therespective embodiments and combinations thereof) is independentlyan alkylene chain being of 1 to 6, or of 1 to 4, or of 2 or 3,carbon atoms in length.

[0041] According to some of any of the embodiments describedherein, the diamine-containing functional group is or comprises atleast one of an ethylene diamine moiety, a methyl ethylenediaminemoiety, a diethylenetriamine moiety, a N-(2-aminoethyl)pyrrolidonemoiety, and a guanidine-ethyleneamine moiety.

[0042] According to some of any of the embodiments describedherein, R.sub.4 is or comprises the diamine-containing moiety.

[0043] According to some of any of the embodiments describedherein, R.sub.4 is OR.sub.20 and R.sub.20 is the diamine-containingmoiety.

[0044] According to some of any of the embodiments describedherein, R.sub.20 is

-(L1)n-N1-(L2)m-N2

[0045] wherein: n and m are each 1; L1 and L2 are eachindependently an alkylene of 2 or 3 carbon atoms in length; and N1and N2 are each independently selected from amine and guanidyl.

[0046] According to some of any of the embodiments describedherein, R.sub.4 is the diamine-containing moiety.

[0047] According to some of any of the embodiments describedherein, R.sub.4 is:

-N1-(L2)m-N2-(L3)k-(N3)

[0048] wherein: m and k are 1; L2 and L3 are each independently analkylene of 1, 2 or 3 carbon atoms in length; N1 is amide; and eachof N2 and N3 is independently an amine.

[0049] According to some of any of the embodiments describedherein, R.sub.1 is or comprises the diamine-containing moiety.

[0050] According to some of any of the embodiments describedherein, R.sub.1 is:

-N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b

[0051] wherein: m and k are each 1; j is 0 or 1; a is 1; b is 0 or1; L2, L3 and L4, if present, are each independently an alkylene of1, 2 or 3 carbon atoms in length; N1 is amide; and each of N2, N3and N4, if present, is independently an amine.

[0052] According to some of any of the embodiments describedherein, R.sub.4 is NR.sub.23R.sub.24.

[0053] According to some of any of the embodiments describedherein, at least one of R.sub.5 and R.sub.6 is OR.sub.16, andR.sub.16 is a monosaccharide or an oligosaccharide.

[0054] According to some of any of the embodiments describedherein, R.sub.5 is OR.sub.16 and R.sub.16 is an oligosaccharide(e.g., a di-saccharide).

[0055] According to some of any of the embodiments describedherein, R.sub.6 is OR.sub.16 and R.sub.16 is hydrogen.

[0056] According to some of any of the embodiments describedherein, the compound features a Neomycin B skeleton (e.g., as shownin Formula Ic, III or IV).

[0057] According to some of any of the embodiments describedherein, each of Rx1, Rx2, Ry1 and Rz is hydrogen.

[0058] According to some of any of the embodiments describedherein, each of Ry2-Ry9 and Rw1-Rw3 is hydrogen.

[0059] According to some of any of the embodiments describedherein, each of R.sub.7 and R.sub.9 is hydrogen.

[0060] According to some of any of the embodiments describedherein, the compound is one or more of Compounds 1-10, as describedherein.

[0061] According to some of any of the embodiments describedherein, the compound is Compound 8, as described herein.

[0062] According to an aspect of some embodiments of the presentinvention there is provided a pharmaceutical composition comprisinga compound (a modified aminoglycoside) as described herein in anyof the respective embodiments and any combination thereof.

[0063] According to an aspect of some embodiments of the presentinvention there is provided a compound (a modified aminoglycoside)as described herein in any of the respective embodiments and anycombination thereof or the pharmaceutical composition as describedherein, for use in the treatment a medical condition associatedwith a pathogenic microorganism.

[0064] According to an aspect of some embodiments of the presentinvention there is provided a method of treating a medicalcondition associated with a pathogenic microorganism, the methodcomprising administering to a subject in need thereof (e.g., asubjected afflicted with medical condition as described herein), acompound (a modified aminoglycoside) as described herein in any ofthe respective embodiments and any combination thereof or thepharmaceutical composition as described herein, thereby treatingthe medical condition in the subject.

[0065] According to some of any of the embodiments describedherein, the pathogenic microorganism is a bacterium.

[0066] According to some of any of the embodiments describedherein, the pathogenic microorganism is an aminoglycoside-resistantmicroorganism.

[0067] According to an aspect of some embodiments of the presentinvention there are provided processes of preparing the compoundsas described herein, which are essentially as described herein.

[0068] According to an aspect of some embodiments of the presentinvention there are provided compounds presented herein asintermediates in the above-mentioned processes, for example, theintermediate compounds presented in FIGS. 2A, 2B and 3.

[0069] Unless otherwise defined, all technical and/or scientificterms used herein have the same meaning as commonly understood byone of ordinary skill in the art to which the invention pertains.Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing ofembodiments of the invention, exemplary methods and/or materialsare described below. In case of conflict, the patent specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and are not intended tobe necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0070] The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Officeupon request and payment of the necessary fee.

[0071] Some embodiments of the invention are herein described, byway of example only, with reference to the accompanying drawings.With specific reference now to the drawings in detail, it isstressed that the particulars shown are by way of example and forpurposes of illustrative discussion of embodiments of theinvention. In this regard, the description taken with the drawingsmakes apparent to those skilled in the art how embodiments of theinvention may be practiced.

[0072] In the drawings:

[0073] FIG. 1 presents the chemical structures of exemplarycompounds according to some embodiments of the presentinvention.

[0074] FIG. 2A is a scheme showing an exemplary synthetic pathwayfor preparing Compound 1. a) AcCl, MeOH; b) TfN.sub.3, CuSO.sub.4(90%); c) PhCH(OMe).sub.2, CSA, DMF (83%); d) BnBr, NaH, DMF (88%);e) AcOH/H.sub.2O (90%); f) TsCl, py; g) NaN.sub.3, DMF (68%); h)allyl bromide, NaH, DMF (97%); i) K.sub.2OsO.sub.4, NMO,acetone/H.sub.2O (80%); j) PhI(OAc).sub.2, CH.sub.2Cl.sub.2; k)2-azidoethanamine; 1) NaBH(OAc).sub.3 (66%); m) PMe.sub.3, NaOH; n)Na/NH.sub.3, THF (65%).

[0075] FIG. 2B is a scheme showing an exemplary synthetic pathwayfor preparing Compounds 2-5. a) ImSO.sub.2N.sub.3.HCl, CuSO.sub.4(70%); b) PhCH(OMe).sub.2, CSA, DMF (88%); c) BnBr, NaH, DMF (60%);d) AcOH/H.sub.2O (61%); e) Trisyl chloride, py; f) NaN.sub.3, DMF(60%); g) allyl bromide, NaH, DMF (92%); h) K.sub.2OsO.sub.4, NMO,acetone/H.sub.2O (89%); i) PhI(OAc).sub.2, CH.sub.2Cl.sub.2; j)amines A, B, 1-(2-aminoethyl)pyrrolidine, C; k) NaBH(OAc).sub.3; l)trifluoroacetic acid, CH.sub.2Cl.sub.2; m) PMe.sub.3, NaOH; n)Na/NH.sub.3, THF.

[0076] FIG. 3 is a scheme showing an exemplary synthetic pathwayfor preparing Compounds 6-10. a) DMP, CH.sub.2Cl.sub.2 (86%); b)NaBH.sub.4, MeOH (82%); c) Tf.sub.2O, py/CH.sub.2Cl.sub.2; d)acetone/NH.sub.3 (41%); e) chloroacetyl chloride, NaHCO.sub.3, THF(98%); f) amines A, B, diethylenetriamine; g) PMe.sub.3, NaOH; h)Na/NH.sub.3, THF; CBz=benzyloxycarbonyl.

[0077] FIGS. 4A-B present a ball-and-stick representation ofCompound 2-induced cleavage site in the bacterial rRNA A-site.Modeling was performed by superimposition of Compound 2 with theNeoB structure in the crystal structure of NeoB bound to the rRNAoligonucleotide model (PDB ID: 2ET4), having SEQ ID NO: 3, by usingPyMOL (FIG. 4A) and a schematic illustration of a proposed actionof compound 2 on the hydrolysis of the phosphodiester bond betweenG1491 and A1492 (FIG. 4B).

[0078] FIG. 5 presents Table 1 showing comparative antibacterialactivity (MIC values) and inhibition of protein translation (IC50values) in the prokaryotic system of NeoB and exemplary Compounds1-10.

[0079] FIGS. 6A, 6B, 6C and 6D presents the data obtained incleavage experiments of E. coli ribosomes in the presence ofethylenediamine, colicin E3, NeoB, and Compound 3. FIG. 6A, Lane 1:E. coli ribosomes (control); lanes 2-5: ribosomes treated withincreasing concentrations of ethylenediamine. FIG. 6B, Lane 1:control; lanes 2-6: ribosomes treated with decreased concentrationsof colicin E3. FIG. 6C, Lane 1: control; lane 2: ribosomes treatedwith 7.3 mm colicin E3 (ColE3); lanes 3-6: ribosomes treated withincreasing concentrations of NeoB. FIG. 6D, Lane 1: control; lanes2-4: ribosomes treated with increasing concentrations of Compound3. rRNA fragments were analyzed on 6% acrylamide TBE/urea gel,stained with SYBR Gold and were analyzed by fluorescence.

[0080] FIGS. 7A-B present a two dimensional representation of thedouble-A site oligonucleotide model containing two identicalbinding sites of aminoglycosides, site I and site II, each havingSEQ NO. 2 (FIG. 7A), showing the attachment sites of thefluorescent tag at the 3'-end; and the sequence of RNA containing23 bases with the covalently attached fluorescent tag, Cy3 (aspurchased from Dharmacon Ltd.), having SEQ ID NO:3 (FIG. 7B). Thestructure of Cy3 and the cleavage sites of colicin E3 toxin(between A1493 and G1494) along with the proposed cleavage site ofthe designer aminoglycosides (between G1491 and A1492) areshown.

[0081] FIGS. 8A-B presents the data obtained in cleavageexperiments of the A-site oligonucleotide model rRNA (SEQ ID NO:1),incubated for 24 hours, pH 8, 37.degree. C. in the presence ofethylenediamine and Compound 6. FIG. 8A, Lane 1: RNA markers; lane2: blank lane; lane 3: not treated (control); lanes 4-7: rRNAoligonucleotide treated with increased concentrations ofethylenediamine. FIG. 8B, Lane 1: RNA markers; lanes 2-7: rRNAoligonucleotide treated with increased concentrations of compound6; lanes 8 and 9: rRNA oligonucleotide treated with 500 mm1,2-cyclohexanediamine (Cyclo) and ethylenediamine (N2N),respectively; lane 10: not treated (control). rRNA fragments wereanalyzed on 20% TBE/urea gel and were visualized by fluorescence.DS: double-stranded rRNA; SS: single-stranded rRNA.

[0082] FIG. 9 presents a simulated system used in the MDsimulations described I Example 5, which is composed of RNA (shownin red), aminoglycoside (green), water (blue surface), sodium(yellow), and chloride ions (purple).

[0083] FIG. 10 presents the normalized occurrence of the twoconformational states of Compound 2 warhead as a function of theintramolecular distance between the N6' ammonium of ring I and theN1 amine of the warhead. The representative structures arepresented. For clarity, only ring I of the aminoglycoside (green)and hydrogen atoms crucial for the interactions of the warhead areshown. Black dashed lines denote donor-acceptor short-rangeinteractions.

[0084] FIG. 11 presents the normalized occurrence of the threeconformational states of Compound 5 warhead as a function of twointramolecular distances N6'-N1 and N6'-N2. The representativestructures are presented. For clarity, only ring I of theaminoglycoside (green) and hydrogen atoms crucial for theinteractions of the warhead are shown. Black dashed lines denotedonor-acceptor short-range interactions

[0085] FIGS. 12A, 12B, 12C and 12D present the normalizedoccurrence of the two conformational states of Compound 10 warheadas a function of the dihedral angle N2-C3-C4-N3 (FIG. 12D). Therepresentative structures are presented (FIG. 12A and FIG. 12c).For clarity, only ring I of the aminoglycoside (green) and hydrogenatoms crucial for the interactions of the warhead are shown (FIG.12B). Black dashed lines denote donor-acceptor short-rangeinteractions.

[0086] FIG. 13 presents the normalized occurrence of the threebinding modes of Compound 8 warhead to A-site as a function of thetwo intermolecular distances N3-O2' and N4-(OP1, OP2). Therepresentative structures are presented. For clarity, only ring Iof the aminoglycoside (in green) and hydrogen atoms crucial forinteractions of the warhead are shown. Black dashed lines denotedonor-acceptor short-range interactions.

[0087] FIG. 14 presents the distributions of the O--P--O angle forNeoB (black) and Compounds 2 (red), 5 (yellow), 8 (green), and 10(blue) in GaMD simulations. Above is shown the O--P--O angle in therepresentative structures of NeoB (left) and compound 10 (right).The donor-acceptor short-range interactions important forstabilization of the O--P--O angle are marked by black dashedlines. For clarity, only ring I of the aminoglycosides (in green)and selected hydrogen atoms are shown.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0088] The present invention, in some embodiments thereof, relatesto aminoglycosides and, more particularly, but not exclusively, tonewly designed aminoglycoside compounds which are aimed atdisabling bacterial ribosome and are usable in treating diseasesand disorders associated with a pathogenic microorganism such aspathogenic bacteria, including drug-resistant bacterialstrains.

[0089] Before explaining at least one embodiment of the inventionin detail, it is to be understood that the invention is notnecessarily limited in its application to the details set forth inthe following description or exemplified by the Examples. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways.

[0090] The present inventors have designed and successfullysynthesized and practiced a series of new derivatives of thenatural aminoglycoside antibiotics.

[0091] The newly designed aminoglycoside derivatives (which arealso referred to herein as "modified aminoglycosides") weredesigned while considering structural and mechanistic data on thebacteriocin ColE3 and on aminoglycoside antibiotics.

[0092] The modified aminoglycosides were designed while aiming atmimicking the interactions of ColE3 with the prokaryotic rRNA(ribosomal RNA) decoding site.

[0093] The design principles included a choice of the "target"phosphodiester bond in the prokaryotic rRNA decoding site, thechemical structure of the functional moiety that is aimed atinteracting with the target position in the prokaryotic rRNA, whichis also referred to herein as the "warhead", and the attachmentsite of this functional moiety on the aminoglycoside scaffold. Asthe target bond, the phosphodiester bond between the rRNA basesG1491 and A1492 was selected as the potential cleavage site and the4', 3' and/or 6' positions (ring I) of the natural aminoglycosidewere selected as attachment site(s) for the functional moieties(warheads). As the functional moiety, a series of differentmoieties that feature two or more amines or amine-containing groups(also referred to herein as "diamine-containing functional moiety")was used.

[0094] In one subset of newly designed compounds, modificationswere made to the natural aminoglycoside NeoB or the disaccharidecore neamine, while attaching different diamine-containing moietiesto the 4'-OH position (see, Compounds 1-5, FIG. 1). In anothersubset modifications were made to the natural aminoglycoside NeoBwhile attaching different diamine-containing moieties directly tothe 4' position (see, Compounds 6-8, FIG. 1). In another subsetmodifications were made to the natural aminoglycoside NeoB, whileattaching different diamine-containing moieties to the 6' position(see, Compounds 9 and 10, FIG. 1).

[0095] All these compounds were successfully synthesized, using thesynthetic pathways shown in FIGS. 2A, 2B and 3.

[0096] The suggested interaction of such an exemplary compound,Compound 2, with the A-site of a prokaryotic rRNA (e.g., having SEQID NO:3) is presented in FIGS. 4A-B, and provides insights on therequired configuration of a modified aminoglycoside for providingan interaction that would disable the ribosome.

[0097] The new derivatives showed significant antibacterialactivity against wild-type strains of both Gram-negative andGram-positive bacteria and display significantly improved activity(compared to NeoB) against highly aminoglycoside-resistant strainsand pathogenic strains, and exhibited inhibition of proteinsynthesis, as shown in Table 1, presented in FIG. 5.

[0098] The observed anti-bacterial activity indicates that themodifications on NeoB did not hinder bacterial cell permeability orthe binding affinity of the aminoglycoside scaffold to the targetsite.

[0099] Some compounds displayed hydrolytic RNase activity, as shownin FIGS. 6B, 6C, 6D, 7A, 7B, 8A, 8B.

[0100] FIGS. 9, 10, 11, 12A, 12B, 12C, 12D, 13, 14 presentfull-atom GaMD simulations on the crystal structure of NeoB boundto the oligonucleotide model of the A-site rRNA (according to PDBID: 2ET4; see, SEQ ID NO:3). These data provide additionalinformation on the conformational states of the diamine-containingfunctional moieties when interacted with the selected prokaryoticrRNA decoding site, and on the resulting change in the O--P--Oangle of the phosphodiester bond linking A1492 and G1491 of theselected model, which can lead to its activation towards cleavageand/or to disabling of the ribosome activity.

[0101] Embodiments of the present invention relate to newlydesigned aminoglycosides that are aimed at interacting with aprokaryotic rRNA decoding site (A-site) to thereby disable thebacterial ribosome, to processes of preparing such compounds and touses thereof in the treatment of medical conditions associated withpathogenic microorganisms.

[0102] Herein, the phrase "prokaryotic rRNA decoding site" refersto a typically conserved site of a prokaryotic ribosomal RNA whichis also known in the art as the A-site of the rRNA. In someembodiments, this phrase refers to the 16S unit of the ribosomalRNA that comprises SEQ ID NO:2 or 3. In some embodiments, aninteraction of the aminoglycoside compounds of the presentembodiments, and of aminoglycoside compounds in general (e.g.,anti-bacterial aminoglycosides), relates to the sequence of the 5nucleotides between G1491 and G1494, which is referred to herein asthe aminoglycosides binding site in prokaryotic ribosomal RNA.

[0103] Modified Aminoglycosides:

[0104] According to some embodiments of the present invention thereare provided newly designed compounds, which are also referred toherein as aminoglycoside derivatives or as modifiedaminoglycosides.

[0105] According to some embodiments of the present invention, thenewly designed aminoglycoside compounds feature a di-, tri-, ortetra-pseudosaccharide structure, and one or more di-functionalmoiety or moieties, e.g., diamine-containing functionalmoiety/moieties, attached to one or more positions of Ring I of theaminoglycoside. The functional moiety/moieties are such that wheninteracting with the prokaryotic rRNA A-site, a change in theO--P--O angle of at least one phosphodiester bond in theaminoglycoside binding site of a prokaryotic ribosomal RNA, whichis typically from G1491 to G1494, occurs. The one or morephosphodiester bonds in which a change in O--P--O angle occurs canbe any such bond that links any adjacent oligonucleotides in theabove mentioned rRNA A-site sequence, preferably in theabove-mentioned aminoglycoside binding site, and in someembodiments it is the bond between G1491 and A1492. In someembodiments the change in O--P--O angle results in an angle that ishigher than 100.degree., higher than 120.degree., higher than140.degree., or preferably higher than 150.degree., and even higher(e.g., the closest to 180.degree.).

[0106] In some embodiments, the O--P--O angle of thisphosphodiester bond is such that facilitates a nucleophilic attackof one of the amine-containing groups (a basic group), that isfurther activated by another amine-containing group (an acidicgroup), which may lead to cleavage of the phosphodiester bond.

[0107] In some embodiments, the di-functional moiety is such thatfeatures at least one acidic amine-containing group, as definedherein, and at least one basic amine-containing group, as definedherein, and at least one linking group that links these groups toone another. In some embodiments, an intramolecular distance and/orthe conformational variability between these acidic and basicgroups is such that when the compound interacts with a prokaryoticrRNA A-site, the basic amine-containing group is capable ofinteracting with the 2'-hydroxy group of the ribose of oneoligonucleotide in the aminoglycoside binding site, e.g., G1491,and the acidic amine-containing group is capable of interactingwith the phosphate group of an adjacent oligonucleotide in theaminoglycoside binding site, e.g., A1492 and may also be capable todonate proton to the 5'-oxygen of the phosphate linkage.

[0108] In some embodiments, the functional moiety is such thatfeatures at least one acidic amine-containing group, as definedherein, and at least one basic amine-containing group, as definedherein, and a linking group that links these groups to one another,and an intramolecular distance and/or the conformationalvariability between the basic and acidic groups is such that whenthe compound interacts with a prokaryotic rRNA A-site, the basicamine-containing group is capable of interacting with the2'-hydroxy group of the ribose of one nucleotide as describedherein (e.g., G1491), and the acidic amine-containing group iscapable of interacting with the phosphate group of an adjacentoligonucleotide (e.g., A1492), as described herein, and optionallyalso of A1493, and these interactions result in an O--P--O angle ofthe phosphodiester bond between these adjacent oligonucleotides(e.g., G1491 and A1492) which is higher than 100.degree., higherthan 120.degree., higher than 140.degree., or higher than150.degree., as described herein.

[0109] Herein throughout, an "amine-containing group" describes achemical group that comprises or consists of at least one --NR'--or --NR'R'' group, with R' and R'' is each independently hydrogen,alkyl, or cycloalkyl, or R' and R'' form together a heterocyclic(e.g., alicyclic) group, or as defined hereinafter.

[0110] An amine-containing group can be --NR'-- or --NR'R'' groupper se (e.g., --NH-- or --NH.sub.2), as defined herein, or aprotonated (ammonium) form thereof, that is, --N.sup.+R'R''-- or--N.sup.+R'R''R'''--, with R''' being as defined for R' and R''(e.g., --N.sup.+H.sub.2-- or --N.sup.+H.sub.3). Preferably R''' ishydrogen. Preferably, at least one of R', R'' and R''' ishydrogen.

[0111] An amine-containing group can alternatively be a chemicalgroup that comprises one or more --NR'-- or --NR'R'' group(s) asdefined herein, or a protonated or ammonium form thereof, asdefined herein, as part of a larger group that comprises additionalchemical groups. Examples of such groups include, withoutlimitation, amide, thioamide, guanyl, guanidyl, carbamate,thiocarbamate, hydrazine, hydrazide, thiohydrazide, urea, andthiourea. In some embodiments, such groups include amide, guanyl,guanidyl, and hydrazine.

[0112] A basic amine-containing group, as used herein, generallydescribes a nucleophilic amine-containing group, or a group which,at a physiological pH and/or environment, can function as a protonacceptor group, and which can be in a protonated at a physiologicalpH and/or a physiological environment. In some embodiments, a basicamine-containing group features pKa higher than 8, or higher than9.

[0113] An acidic amine-containing group, as used herein, generallydescribes an electrophilic amine-containing group, or a groupwhich, at a physiological pH and/or environment, can function as aproton donor. In some embodiments, an acidic amine-containing groupfeatures pKa that is lower from that of the basic amine-containinggroup by at least 1 pKa unit, for example, by 1, 1.2, 1.4, 1.5,1.6, 1.8, 2, and even more.

[0114] In some embodiments, an acidic amine-containing group is orcomprises a positively charged ammonium group that can act as aproton donor, e.g., is protonated at physiological pH, for example,a --N.sup.+R'R''-- or --N.sup.+R'R''R'''-- group in which at leastone of R', R'' and R''' (if present) is hydrogen.

[0115] In some embodiments, a diamine-containing moiety is suchthat comprises at least two amine-containing groups as describedherein and a linking group that links these groups, and theamine-containing groups and the linking group are such that in aphysiological environment (e.g., pH), one of the amine-containinggroup is protonated (and functions as an acidic group, or a protondonor, or an electrophile) and the other amine-containing group isnot protonated (and functions as a basic group or a proton acceptoror a nucleophile).

[0116] In some embodiments, a diamine-containing moiety is suchthat comprises at least two amine-containing groups as describedherein and a linking group that links these groups, and theamine-containing groups and the linking group are such that in aphysiological environment (e.g., pH), a difference in the pKa ofamine-containing groups is at least 1 pKa unit, as describedherein.

[0117] It is to be noted that when reference is made to aprotonated group, it is meant that an abundance of such aprotonated form at a physiological pH (pH of about 7) is more than5%, or more than 10%, or more than 20%, or more than 30%, or morethan 40%, or more than 50%.

[0118] According to some of any of the embodiments describedherein, the modified aminoglycosides can be collectivelyrepresented by Formula I:

##STR00002##

[0119] or a pharmaceutically acceptable salt thereof,

[0120] wherein:

[0121] the dashed line indicates a stereo-configuration of position6' being an R configuration or an S configuration;

[0122] X.sub.1 is O or S;

[0123] Rx1, Rx2, Ry1 and Rz are each independently selected fromhydrogen, alkyl and cycloalkyl;

[0124] Ry2-Ry9 and Rw1-Rw3 are each independently selected fromhydrogen, alkyl, and cycloalkyl;

[0125] R.sub.1, R.sub.3 and R.sub.4 are each independentlyNR.sub.23R.sub.24, OR.sub.20 or a diamine-containing moiety,wherein R.sub.20 is hydrogen, alkyl, cycloalkyl or thediamine-containing moiety, and each of R.sub.23 and R.sub.24 isindependently hydrogen, alkyl, cycloalkyl or acyl, provided that atleast one of R.sub.1, R.sub.3 and R.sub.4 is or comprises thediamine-containing moiety;

[0126] R.sub.5 and R.sub.6 are each independently selected fromhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic,aryl, heteroaryl and OR.sub.16, wherein R.sub.16 is independentlyselected from hydrogen, a monosaccharide moiety and anoligosaccharide moiety; and

[0127] R.sub.7-R.sub.9 are each independently selected from thegroup consisting of hydrogen and acyl,

[0128] wherein the diamine-containing moiety comprises at least twoamine-containing groups, as described herein in any of therespective embodiments, and at least one linking group, asdescribed herein in any of the respective embodiments, linking theat least two amine-containing groups.

[0129] According to some of any of the embodiments describedherein, the at least two amine-containing groups and the at leastone linking group are arranged such that:

[0130] (i) a difference in the pKa of at least two of theamine-containing groups is at least 1; and/or

[0131] (ii) when the compound is in a physiological environment, atleast one of the amine-containing groups is protonated atphysiological pH while at least another of the amine-containinggroups is non-protonated; and/or

[0132] (iii) when the compound interacts with a prokaryoticribosomal RNA decoding site (A-site), the RNA undergoes aconformational change such that an O--P--O angle of at least onephosphodiester bond is higher than 100.degree.; and/or

[0133] (iv) when the compound interacts with a prokaryoticribosomal RNA decoding site (A-site) (e.g., comprising SEQ ID NO:2or 3), the functional moiety is capable of adopting a configurationin which one of the amine-containing groups is in close proximityand suitable orientation so as to interact with a 2'-OH group of aribose of a nucleotide in the RNA and another amine-containingmoiety is in close proximity and suitable orientation so as tointeract with a phosphate group of a nucleotide of an adjacentnucleotide.

[0134] According to some of any of the embodiments describedherein, the at least two amine-containing groups and the at leastone linking group are arranged such that any two, any three or allof (i), (ii), (iii) and (iv) as described herein are fulfilled.

[0135] According to some of any of the embodiments describedherein, the at least two amine-containing groups and the at leastone linking group are arranged such that a difference in the pKa ofat least two of the amine-containing groups is at least 1, asdescribed herein in any of the respective embodiments.

[0136] It is to be noted that two identical amine-containing groupswithin the same can feature different pKa values, due tointramolecular electrostatic interactions between one anotherand/or between each of these groups with other groups in thecompound, and/or due to intermolecular electrostatic interactionswith a surrounding environment (e.g., physiological environmentand/or interaction with the rRNA as described herein).

[0137] According to some of any of the embodiments describedherein, the at least two amine-containing groups and the at leastone linking group are arranged such that when the compound is in aphysiological environment, at least one of the amine-containinggroups is protonated at physiological pH, as described herein inany of the respective embodiments, while at least another of theamine-containing groups is non-protonated, as described herein inany of the respective embodiments. The physiological environmentcan be a physiological pH and/or a presence of additionalphysiological salts and/or interaction with the rRNA as describedherein.

[0138] According to some of any of the embodiments describedherein, the at least two amine-containing groups and the at leastone linking group are arranged such that when the compoundinteracts with a prokaryotic ribosomal RNA decoding site (A-site)as described herein (e.g., comprising SEQ ID NO:2 or 3), the RNAundergoes a conformational change such that an O--P--O angle of atleast one phosphodiester bond within said site is higher than100.degree., as described herein in any of the respectiveembodiments. See, for example, FIG. 14.

[0139] According to some these embodiments, when the compoundinteracts with the prokaryotic rRNA A-site as described herein, thediamine-containing functional moiety adopts a configuration,presumably upon the interactions formed between theamine-containing groups and chemically compatible groups in the RNAA-site, e.g., as described herein, which leads to theabove-indicated conformational change of the RNA.

[0140] In some of these embodiments, the O--P--O is determined byMD simulations as described herein in the Examples section thatfollows, using structural conformations with respect to prokaryoticrRNA portion having SEQ ID NO:3 or 2.

[0141] According to some of these embodiments, the phosphodiesterbond is one or more of the phosphodiester bonds within thenucleotide sequence G1491-G1494 of the rRNA portion as describedherein, e.g., the rRNA comprising the oligonucleotide sequencehaving SEQ ID NO:3 or 2.

[0142] According to some of these embodiments, the phosphodiesterbond is between G1491 and A1492 of the rRNA as described herein,e.g., the rRNA comprising the oligonucleotide sequence having SEQID NO:3 or 2.

[0143] According to some of any of the embodiments describedherein, the at least two amine-containing groups and the at leastone linking group are arranged such that when the compoundinteracts with a prokaryotic ribosomal RNA decoding site (A-site)as described herein, the functional moiety is capable of adopting aconfiguration in which one of the amine-containing groups is inclose proximity and suitable orientation so as to interact with a2'-OH group of a ribose of a nucleotide in the RNA and anotheramine-containing moiety is in close proximity and suitableorientation so as to interact with a phosphate group of anucleotide of an adjacent nucleotide.

[0144] Herein throughout, by "proximity and orientation" it ismeant that an indicated group or moiety is sufficiently close andproperly oriented so as to strongly interact with a respectivegroup (chemically compatible group) in the indicated binding site(the prokaryotic rRNA A-site, preferably the aminoglycoside bindingsite therein).

[0145] By "interacting" or "interact", in the context of groups ormoieties in a compound as described herein and a respective moietyin the rRNA, it is meant a chemical interaction as a result of, forexample, non-covalent interactions such as, but not limited to,electrostatic interactions, Van der Waals interactions and/orhydrogen bonding.

[0146] In some embodiments, the indicated close proximity andorientation of the functional moiety result from the spatialarrangement of the amine-containing groups, when the compoundcontacts the respective rRNA binding site, and the partial chargeof each of these groups that allows interaction with the respectiveindicated groups in the rRNA binding site. The spatial arrangementcan depend, for example, on the conformational variability of thefunctional moiety.

[0147] In some of any of these embodiments, the proximity andorientation of the amine-containing groups is determined by meansof crystallographic models, in silico modeling and/or MDsimulations, as described herein in the Examples section thatfollows. In some of these embodiments, the proximity andorientation is determined using such models for structuralconformations of the compound with respect to prokaryotic rRNAoligonucleotide sequence portion having SEQ ID NO:3 or 2.

[0148] According to some of these embodiments, the phosphodiesterbond is one or more of the phosphodiester bonds within thenucleotide sequence G1491-G1494 of the rRNA, e.g., the rRNA portioncomprising the oligonucleotide sequence having SEQ ID NO:3 or2.

[0149] In some of any of these embodiments, in any of theabove-indicated models, the distance between the amine-containinggroup that interacts with the 2'-OH group of the indicated riboseis no more than 4 angstroms, or no more than 3.5 angstroms, or nomore than 3 angstroms, and can be, for example, from 1 to 4, orfrom 2 to 4, or from 2 to 3.5, or from 2.5 to 2.5, angstroms. Suchamine-containing group is a "basic" amine group, as describedherein, which is non-protonated in physiological environment (e.g.,physiological pH) and/or can act as a nucleophile and/or as aproton acceptor.

[0150] In some of any of these embodiments, in any of theabove-indicated models, the distance between the amine-containinggroup that interacts with the indicated phosphate group, and thephosphate group is no more than 5 angstroms, or no more than 4.5angstroms, or no more than 4 angstroms, and can be, for example,from 1 to 5, or from 2 to 5, or from 2 to 4, or from 3 to 4,angstroms. Such amine-containing group is an "acidic" amine group,as described herein, which is protonated in physiologicalenvironment (e.g., physiological pH), as defined herein, and/or canact as a proton donor and/or as an electrophile.

[0151] The amine-containing group that interacts with the indicatedphosphate group typically interacts with one or both of the oxygenatoms of the phosphate group.

[0152] According to some of these embodiments, the 2'-OH group ofthe ribose is of the G1491 nucleotide and the phosphate is of theadjacent A1492 nucleotide of the rRNA, e.g., the rRNA portioncomprising the oligonucleotide sequence having SEQ ID NO:3 or2.

[0153] In some of any of the embodiments described herein, theproximity and orientation of the amine-containing groups towardsthe 2'-OH group of the indicated ribose the indicated phosphategroup results in the conformational change of the rRNA as describedherein, e.g., in a O--P--O angle of the phosphodiester bond asdescribed herein in any of the respective embodiments.

[0154] According to some of any of the embodiments describedherein, the diamine-containing functional moiety can be representedby the following Formula:

-(L1)n-N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b

wherein:

[0155] each of L1, L2, L3 and L4 is independently a linking group,as described herein in any of the respective embodiments, wherebywhen two or more linking groups are present, they can be the sameor different;

[0156] each of N1, N2, N3 and N4 is an amine-containing group, asdescribed herein in any of the respective embodiments; and

[0157] each of a, b, n, m, k, and j is independently 0 or 1.

[0158] In some of any of the embodiments described herein, at leastone of the linking groups, or each of the linking groups, in casethere are two or more linking groups, is independently ahydrocarbon group being of 1 to 6 carbon atoms in length.

[0159] Preferably, the hydrocarbon is a linear, aliphatic andnon-branched hydrocarbon, and further preferably it isunsubstituted.

[0160] In some of any of the embodiments described herein, at leastone of the linking groups, or each of the linking groups, in casethere are two or more linking groups, is independently an alkylenechain being of 1 to 6, or of 1 to 4, or of 2 or 3, carbon atoms inlength. Preferably, the alkylene chain is non-branched andunsubstituted.

[0161] Exemplary diamine-containing functional groups include, butare not limited to, moieties that consist of or comprise at leastone of an ethylene diamine moiety, a methyl ethylenediamine moiety,a diethylenetriamine moiety, an N-(2-aminoethyl)pyrrolidone moiety,and a guanidine-ethyleneamine moiety.

[0162] In some of any of the embodiments described herein, thecompound comprises one diamine-containing functional moiety.

[0163] In some of any of the embodiments described herein, R.sub.4is or comprises the diamine-containing moiety, such that thismoiety is at the 4' position of the aminoglycoside.

[0164] In some of these embodiments, R.sub.4 is OR.sub.20 andR.sub.20 is a diamine-containing moiety as described herein in anyof the respective embodiments and any combination thereof.

[0165] In exemplary embodiments, R.sub.20 is

-(L1)n-N1-(L2)m-N2

[0166] wherein:

[0167] n and m are each 1;

[0168] L1 and L2 are each independently an alkylene of 1, 2 or 3,preferably 2 or 3, carbon atoms in length; and

[0169] N1 and N2 are each independently selected from amine andguanidyl.

[0170] Exemplary such compounds include Compounds 1-5 as shown inFIG. 1 and hereinafter.

[0171] According to some of any of the embodiments describedherein, R.sub.4 is a diamine-containing moiety as described hereinin any of the respective embodiments and any combinationthereof.

[0172] In exemplary embodiments, R.sub.4 is:

-N1-(L2)m-N2-(L3)k-(N3)

[0173] wherein:

[0174] m and k are each 1;

[0175] L2 and L3 are each independently an alkylene of 1, 2 or 3carbon atoms in length;

[0176] N1 is amide; and

[0177] each of N2 and N3 is independently an amine.

[0178] In some of these embodiments, L2 is an alkylene of 1 carbonatom in length (e.g., methylene). In some of any of theseembodiments, L3 is an alkylene of 2 carbon atoms in length (e.g.,ethylene).

[0179] Exemplary such compounds include Compounds 6 and 7 as shownin FIG. 1 and hereinafter.

[0180] In exemplary embodiments, R.sub.4 is:

-N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b

[0181] wherein:

[0182] m, k and j are each 1;

[0183] L2, L3 and L4 are each independently an alkylene of 1, 2 or3 carbon atoms in length;

[0184] N1 is amide; and

[0185] each of N2, N3 and N4 is independently an amine.

[0186] In some of these embodiments, L2 is an alkylene of 1 carbonatom in length (e.g., methylene). In some of any of theseembodiments, L3 and L4 are each an alkylene of 2 carbon atoms inlength (e.g., ethylene).

[0187] An exemplary such compound is Compound 8 as shown in FIG. 1and hereinafter.

[0188] In some of any of the embodiments described herein, R.sub.1is or comprises the diamine-containing moiety.

[0189] In exemplary embodiments, R.sub.1 is:

-N1-(L2)m-N2-(L3)k-(N3)a-(L4)j-(N4)b

[0190] wherein:

[0191] m and k are each 1;

[0192] j is 0 or 1;

[0193] a is 1;

[0194] b is 0 or 1;

[0195] L2, L3 and L4, if present, are each independently analkylene of 1, 2 or 3 carbon atoms in length;

[0196] N1 is amide; and

[0197] each of N2, N3 and N4, if present, is independently anamine.

[0198] In some of these embodiments, L2 is an alkylene of 1 carbonatom in length (e.g., methylene). In some of any of theseembodiments, L3 and L4, if present, are each an alkylene of 2carbon atoms in length (e.g., ethylene).

[0199] In some of these embodiments, R.sub.4 is NR.sub.23R.sub.24,as described herein, and in some of these embodiments each ofR.sub.23 and R.sub.24 is hydrogen.

[0200] Exemplary such compounds are Compounds 9 and 10 as shown inFIG. 1 and hereinafter.

[0201] According to some of any of the embodiments describedherein, X is O.

[0202] According to some of any of the embodiments describedherein, the compound is a disaccharide, such that none of R.sub.5and R.sub.6 is a monosaccharide or an oligosaccharide moiety. Insome of these embodiments, each of R.sub.5 and R.sub.6 isOR.sub.16, and in some embodiments R.sub.16 is hydrogen.

[0203] According to some of any of the embodiments describedherein, the compound is a tri-, tetra- or higher oligosaccharide,and at least one of R.sub.5 and R.sub.6 is OR.sub.16, whereinR.sub.16 is a monosaccharide or an oligosaccharide, as describedherein.

[0204] Such compounds can include 3, 4, or more saccharide units(moieties) linked to one another, and can adopt, for example, askeleton of any of the aminoglycosides known to exhibit anantimicrobial (e.g., antibacterial) activity.

[0205] These include, for example, amikacin, apramycin, arbekacin,butirosin, dibekacin, fortimycin, G-418, gentamycin, hygromycin,habekacin, dibekacin, netlmicin, istamycin, isepamycin, kanamycinB, lividomycin, neomycin B, paromomycin, ribostamycin, sisomycin,spectinomycin, streptomycin and tobramycin.

[0206] In some of these embodiments, the aminoglycoside skeleton issuch that Ring I of the aminoglycoside interacts with theprokaryotic rRNA aminoglycoside binding site similarly to NeomycinB (NeoB).

[0207] Whenever a skeleton of an aminoglycoside is referred to, itis meant that the type (monosaccharide or oligosaccharide) ofR.sub.16 and the position of the respective OR.sub.16 is inaccordance with the skeleton of the aminoglycoside. It is furthermeant that all the substituents of each position in theaminoglycoside, except the position bearing the diamine-containingfunctional group as described herein, are substantially the same asin the respective aminoglycoside.

[0208] In some of any of the embodiments described herein, R.sub.5is OR.sub.16 and R.sub.16 is an oligosaccharide, e.g., adi-saccharide.

[0209] In some of these embodiments, R.sub.6 is OR.sub.16 andR.sub.16 is hydrogen.

[0210] In exemplary embodiments, the compound features a Neomycin Bskeleton, as described herein.

[0211] In some of any of the embodiments described herein, each ofRx1, Rx2, Ry1 and Rz is hydrogen.

[0212] In some of any of the embodiments described herein, each ofRy2-Ry9 and Rw1-Rw3 is hydrogen.

[0213] In some of any of the embodiments described herein, each ofR.sub.7 and R.sub.9 is hydrogen.

[0214] In exemplary embodiments, the compound is selected fromCompounds 1-10 as presented in FIG. 1 and hereinbelow.

[0215] In exemplary embodiments, the compound is selected fromCompounds 2-10 as presented in FIG. 1 and hereinbelow.

[0216] In exemplary embodiments, the compound is selected fromCompounds 2, 5, 8 and 10 as presented in FIG. 1 andhereinbelow.

[0217] In exemplary embodiments, the compound is Compound 8 aspresented in FIG. 1 and hereinbelow.

[0218] The term "monosaccharide", as used herein and is well knownin the art, refers to a simple form of a sugar that consists of asingle saccharide molecule which cannot be further decomposed byhydrolysis. Most common examples of monosaccharides include glucose(dextrose), fructose, galactose, and ribose. Monosaccharides can beclassified according to the number of carbon atoms of thecarbohydrate, i.e., triose, having 3 carbon atoms such asglyceraldehyde and dihydroxyacetone; tetrose, having 4 carbon atomssuch as erythrose, threose and erythrulose; pentose, having 5carbon atoms such as arabinose, lyxose, ribose, xylose, ribuloseand xylulose; hexose, having 6 carbon atoms such as allose,altrose, galactose, glucose, gulose, idose, mannose, talose,fructose, psicose, sorbose and tagatose; heptose, having 7 carbonatoms such as mannoheptulose, sedoheptulose; octose, having 8carbon atoms such as 2-keto-3-deoxy-manno-octonate; nonose, having9 carbon atoms such as sialose; and decose, having 10 carbon atoms.Monosaccharides are the building blocks of oligosaccharides likesucrose (common sugar) and other polysaccharides (such as celluloseand starch).

[0219] The term "oligosaccharide" as used herein refers to acompound that comprises two or more monosaccharide units, as theseare defined herein, linked to one another via a glycosyl bond(--O--) or a thioglycosyl bond (--S--). Preferably, theoligosaccharide comprises 2-6 monosaccharides, more preferably theoligosaccharide comprises 2-4 monosaccharides and most preferablythe oligosaccharide is a disaccharide moiety, having twomonosaccharide units.

[0220] In some of any of the embodiments described herein, themonosaccharide is a pentose moiety, such as, for example,represented by Formula II. Alternatively, the monosaccharide moietyis hexose.

[0221] In some of any of the embodiments described herein, themonosaccharide moiety is a ribose, represented by Formula II:

##STR00003##

[0222] wherein:

[0223] the curved line denotes a position of attachment;

[0224] the dashed line indicates a stereo-configuration of position5'' being an R configuration or an S configuration;

[0225] X.sub.2 is OR.sub.13 or NR.sub.14R.sub.15;

[0226] each of R.sub.10-R.sub.13 is independently hydrogen, alkyl,cycloalkyl, acyl, a monosaccharide moiety or an oligosaccharidemoiety, as defined herein; and

[0227] each of R.sub.14 and R.sub.15 is independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, and acyl.

[0228] In some embodiments, X.sub.2 is OR.sub.13, and R.sub.13 ishydrogen.

[0229] In some embodiments, X.sub.2 is NR.sub.14R.sub.15.

[0230] In some of any of these embodiments, the compound isrepresented by Formula Ib:

##STR00004##

[0231] with the variables being as described herein for Formulae Iand II, including any combination thereof.

[0232] In some of any of the embodiments of Formula Ib, R.sub.11 isa monosaccharide moiety or an oligosaccharide moiety (e.g., adi-saccharide moiety), as described herein in any of the respectiveembodiments and any combination thereof, such that altogether, Ribis an oligosaccharide moiety.

[0233] In some of these embodiments, the compound is represented byFormula Ic:

##STR00005##

[0234] wherein R.sub.17-R.sub.19 and R.sub.21 being as definedherein for R.sub.10-R.sub.13, X.sub.3 being as defined herein forX.sub.2, and all other variables being as described herein forFormulae I and II, including any combination thereof.

[0235] According to some of any of the embodiments describedherein, the compound is represented by Formula III:

##STR00006##

[0236] or a pharmaceutically acceptable salt thereof, whereinR.sub.4 is or comprises a diamine-containing moiety, as definedherein in any of the respective embodiments and any combinationthereof.

[0237] In some of the embodiments of Formula III, R.sub.4 isOR.sub.20, and R.sub.20 is the diamine-containing functionalmoiety, as defined herein in any of the respective embodiments andany combination thereof.

[0238] Exemplary such compounds include Compounds 2-5, asfollows:

##STR00007## ##STR00008##

[0239] In some of the embodiments of Formula III, R.sub.4 is thediamine-containing functional moiety, as defined herein in any ofthe respective embodiments and any combination thereof.

[0240] Exemplary such compounds include Compounds 6-8, asfollows:

##STR00009##

[0241] According to some of any of the embodiments describedherein, the compound is represented by Formula IV:

##STR00010##

[0242] or a pharmaceutically acceptable salt thereof, whereinR.sub.1 is a diamine-containing functional moiety as defined hereinin any of the respective embodiments and any combinationthereof.

[0243] Exemplary such compounds include Compounds 9 and 10, asfollows:

##STR00011##

[0244] Some embodiments of the present invention relate toprocesses of preparing the modified aminoglycosides describedherein.

[0245] Generally, the compounds can be prepared using methodologiesknown in the art for preparing modified aminoglycosides, whichinvolve selectively protecting and deprotecting the amine groupsand hydroxy groups present within the aminoglycoside skeleton asdesired, and introducing the required substituted at the desiredposition.

[0246] In some embodiments, preparing a compound as describedherein involves selecting or generating a suitable aminoglycosidecompound to be modified, which feature 2, 3, or 4 saccharide units,each being substituted by amine and hydroxy substituents,protecting the amine groups, protecting the hydroxyl groups andthen selectively deprotecting the protected group at the positionto which a diamine-containing functional moiety should beintroduced.

[0247] The phrase "protected group", as used herein, refers to agroup that is substituted or modified so as to block itsfunctionality and protect it from reacting with other groups underthe reaction conditions. A protected group is re-generated byremoval of the substituent or by being re-modified.

[0248] When an "amino-protected group" or "hydroxyl-protectedgroup" are used, it is meant that a protecting group is attached orused to modify the respective group so as to generate the protectedgroup.

[0249] The phrase "protecting group", as used herein, refers to asubstituent or a modification that is commonly employed to block orprotect a particular functionality while reacting other functionalgroups on the compound. The protecting group is selected so as torelease the substituent or to be re-modified, to thereby generatethe desired unprotected group.

[0250] For example, an "amino-protecting group" or"amine-protecting group" is a substituent attached to an aminogroup, or a modification of an amino group, that blocks or protectsthe amino functionality in the compound, and prevents it fromparticipating in chemical reactions. The amino-protecting group isremoved by removal of the substituent or by a modification thatre-generates an amine group.

[0251] Suitable amino-protected groups include azide (azido),N-phthalimido, N-acetyl, N-trifluoroacetyl, N-t-butoxycarbonyl(BOC), N-benzyloxycarbonyl (CBz) andN-9-fluorenylmethylenoxycarbonyl (Fmoc).

[0252] A "hydroxy-protecting group" or "hydroxyl-protecting group"refers to a substituent or a modification of a hydroxyl group thatblocks or protects the hydroxyl functionality, and prevents it fromparticipating in chemical reactions. The hydroxy-protecting groupis removed by removal of the substituent or by a modification thatre-generates a hydroxy group.

[0253] Suitable hydroxy protected groups include isopropylideneketal and cyclohexanone dimethyl ketal (forming a 1,3-dioxane withtwo adjacent hydroxyl groups), 4-methoxy-1-methylbenzene (forming a1,3-dioxane with two adjacent hydroxyl groups), O-acetyl,O-chloroacetyl, O-benzoyl (OBn) and O-silyl.

[0254] For a general description of protecting groups and theiruse, see T. W. Greene, Protective Groups in Organic Synthesis, JohnWiley & Sons, New York, 1991.

[0255] It is noted herein that when applicable, a "protected group"refers to a moiety in which one reactive function on a compound isprotected or more than one function are protected at the same time,such as in the case of two adjacent functionalities, e.g., twohydroxyl groups that can be protected at once by a isopropylideneketal.

[0256] Exemplary synthetic pathways are described in Example 2 inthe Examples section that follows, and are presented in FIGS. 2A,2B and 3.

[0257] Some embodiments of the present invention relate tointermediates formed while preparing compounds as describedherein.

[0258] Exemplary such intermediates include Compounds 11-18, shownin FIG. 2A, Compounds 19-29 shown in FIG. 2B, and Compounds 30-36shown in FIG. 3.

[0259] According to some of any of the embodiments describedherein, any of the compounds prepared or provided according to thepresent embodiments can be in a form of a pharmaceuticallyacceptable salt thereof.

[0260] As used herein, the phrase "pharmaceutically acceptablesalt" refers to a charged species of the parent compound and itscounter-ion, which is typically used to modify the solubilitycharacteristics of the parent compound and/or to reduce anysignificant irritation to an organism by the parent compound,and/or to improve its stability, while not abrogating thebiological activity and properties of the administered compound. Apharmaceutically acceptable salt of a compound as described hereincan alternatively be formed during the synthesis of the compound,e.g., in the course of isolating the compound from a reactionmixture or re-crystallizing the compound.

[0261] In the context of some of the present embodiments, apharmaceutically acceptable salt of the compounds described hereinmay optionally be an acid addition salt comprising at least onebasic (e.g., an amine-containing group such as amine and/orguanidyl and/or guanyl) group of the compound which is in apositively charged form (e.g., wherein the basic group isprotonated), in combination with at least one counter-ion, derivedfrom the selected base, that forms a pharmaceutically acceptablesalt.

[0262] The acid addition salts of the compounds described hereinmay therefore be complexes formed between one or more basic groupsof the compound and one or more equivalents of an acid.

[0263] Depending on the stoichiometric proportions between thecharged group(s) in the compound and the counter-ion in the salt,the acid additions salts can be either mono-addition salts orpoly-addition salts.

[0264] The phrase "mono-addition salt", as used herein, refers to asalt in which the stoichiometric ratio between the counter-ion andcharged form of the compound is 1:1, such that the addition saltincludes one molar equivalent of the counter-ion per one molarequivalent of the compound.

[0265] The phrase "poly-addition salt", as used herein, refers to asalt in which the stoichiometric ratio between the counter-ion andthe charged form of the compound is greater than 1:1 and is, forexample, 2:1, 3:1, 4:1 and so on, such that the addition saltincludes two or more molar equivalents of the counter-ion per onemolar equivalent of the compound.

[0266] An example, without limitation, of a pharmaceuticallyacceptable salt would be an ammonium cation or guanidinium cationand an acid addition salt thereof.

[0267] The acid addition salts may include a variety of organic andinorganic acids, such as, but not limited to, hydrochloric acidwhich affords a hydrochloric acid addition salt, hydrobromic acidwhich affords a hydrobromic acid addition salt, acetic acid whichaffords an acetic acid addition salt, ascorbic acid which affordsan ascorbic acid addition salt, benzenesulfonic acid which affordsa besylate addition salt, camphorsulfonic acid which affords acamphorsulfonic acid addition salt, citric acid which affords acitric acid addition salt, maleic acid which affords a maleic acidaddition salt, malic acid which affords a malic acid addition salt,methanesulfonic acid which affords a methanesulfonic acid(mesylate) addition salt, naphthalenesulfonic acid which affords anaphthalenesulfonic acid addition salt, oxalic acid which affordsan oxalic acid addition salt, phosphoric acid which affords aphosphoric acid addition salt, toluenesulfonic acid which affords ap-toluenesulfonic acid addition salt, succinic acid which affords asuccinic acid addition salt, sulfuric acid which affords a sulfuricacid addition salt, tartaric acid which affords a tartaric acidaddition salt and trifluoroacetic acid which affords atrifluoroacetic acid addition salt. Each of these acid additionsalts can be either a mono-addition salt or a poly-addition salt,as these terms are defined herein.

[0268] The present embodiments further encompass any enantiomers,diastereomers, prodrugs, solvates, hydrates and/or pharmaceuticallyacceptable salts of the compounds described herein.

[0269] As used herein, the term "enantiomer" refers to astereoisomer of a compound that is superposable with respect to itscounterpart only by a complete inversion/reflection (mirror image)of each other. Enantiomers are said to have "handedness" since theyrefer to each other like the right and left hand. Enantiomers haveidentical chemical and physical properties except when present inan environment which by itself has handedness, such as all livingsystems. In the context of the present embodiments, a compound mayexhibit one or more chiral centers, each of which exhibiting an R-or an S-configuration and any combination, and compounds accordingto some embodiments of the present invention, can have any theirchiral centers exhibit an R- or an S-configuration.

[0270] The term "diastereomers", as used herein, refers tostereoisomers that are not enantiomers to one another.Diastereomerism occurs when two or more stereoisomers of a compoundhave different configurations at one or more, but not all of theequivalent (related) stereocenters and are not mirror images ofeach other. When two diastereoisomers differ from each other atonly one stereocenter they are epimers. Each stereo-center (chiralcenter) gives rise to two different configurations and thus to twodifferent stereoisomers. In the context of the present invention,embodiments of the present invention encompass compounds withmultiple chiral centers that occur in any combination ofstereo-configuration, namely any diastereomer.

[0271] The term "prodrug" refers to an agent, which is convertedinto the active compound (the active parent drug) in vivo. Prodrugsare typically useful for facilitating the administration of theparent drug. They may, for instance, be bioavailable by oraladministration whereas the parent drug is not. A prodrug may alsohave improved solubility as compared with the parent drug inpharmaceutical compositions. Prodrugs are also often used toachieve a sustained release of the active compound in vivo. Anexample, without limitation, of a prodrug would be a compound ofthe present invention, having one or more carboxylic acid moieties,which is administered as an ester (the "prodrug"). Such a prodrugis hydrolyzed in vivo, to thereby provide the free compound (theparent drug). The selected ester may affect both the solubilitycharacteristics and the hydrolysis rate of the prodrug.

[0272] The term "solvate" refers to a complex of variablestoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on),which is formed by a solute (the compound of the present invention)and a solvent, whereby the solvent does not interfere with thebiological activity of the solute. Suitable solvents include, forexample, ethanol, acetic acid and the like.

[0273] The term "hydrate" refers to a solvate, as definedhereinabove, where the solvent is water.

[0274] Therapeutic Uses:

[0275] The compounds according to some embodiments of the presentinvention are effective in treating medical conditions associatedwith a pathogenic microorganism in a subject.

[0276] The compounds presented herein can also be effective intreating medical conditions associated with pathogenicmicroorganisms which have already developed resistance to anyantibiotic agent.

[0277] The phrases "effective in treating medical conditionsassociated with pathogenic microorganisms", "effective in treatinga subject diagnosed with a medical conditions associated withpathogenic microorganisms" and/or "for use in the treatment of amedical condition associated with a pathogenic microorganism in asubject", as used herein interchangeably, refer to characteristicsof a substance, such as the compounds according to some embodimentsof the present invention, that can effect death, killing,eradication, elimination, reduction in number, reduction of growthrate, reduction of a load, and/or a change in populationdistribution of one or more species of pathogenic microorganisms,as well as effecting a reduction or prevention of the emergence ofresistance of such microorganisms to the substance.

[0278] Herein throughout, the phrase "pathogenic microorganism" isused to describe any microorganism which can cause a disease ordisorder in a higher organism, such as mammals in general and ahuman in particular. The pathogenic microorganism may belong to anyfamily of organisms such as, but not limited to prokaryoticorganisms, eubacterium, archaebacterium, eukaryotic organisms,yeast, fungi, algae, protozoan, and other parasites.

[0279] Non-limiting examples of pathogenic microorganism includePlasmodium falciparum and related malaria-causing protozoanparasites, Acanthamoeba and other free-living amoebae, Aeromonashydrophila, Anisakis and related worms, and further include, butnot limited to Acinetobacter baumanii, Ascaris lumbricoides,Bacillus cereus, Brevundimonas diminuta, Campylobacter jejuni,Clostridium botulinum, Clostridium perfringens, Cryptosporidiumparvum, Cyclospora cayetanensis, Diphyllobothrium, Entamoebahistolytica, certain strains of Escherichia coli, Eustrongylides,Giardia lamblia, Klebsiella pneumoniae, Listeria monocytogenes,Nanophyetus, Plesiomonas shigelloides, Proteus mirabilis,Pseudomonas aeruginosa, Salmonella, Serratia odorifera, Shigella,Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus,Trichuris trichiura, Vibrio cholerae, Vibrio parahaemolyticus,Vibrio vulnificus and other vibrios, Yersinia enterocolitica,Yersinia pseudotuberculosis and Yersinia kristensenii.

[0280] Other pathogens include Strep. pyogenes (Group A), Strep.pneumoniae, Strep. GpB, Strep. viridans, Strep. GpD (Enterococcus),Strep. GpC and GpG, Staph. aureus, Staph. epidermidis, Bacillussubtilis, Bacillus anthracis, Listeria monocytogenes, Anaerobiccocci, Clostridium spp., Actinomyces spp, Escherichia coli,Enterobacter aerogenes, Kiebsiella pneumoniae, Proteus mirabilis,Proteus vulgaris, Morganella morganii, Providencia stuartii,Serratia marcescens, Citrobacter freundii, Salmonella typhi,Salmonella paratyphi, Salmonella typhi murium, Salmonella virchow,Shigella spp., Yersinia enterocolitica, Acinetobactercalcoaceticus, Flavobacterium spp., Haemophilus influenzae,Pseudomonas aeruginosa, Campylobacter jejuni, Vibrioparahaemolyticus, Brucella spp., Neisseria meningitidis, Neisseriagonorrhoea, Bacteroides fragilis, Fusobacterium spp., Mycobacteriumtuberculosis (including MDR and XDR strains from hospital originsisolated from patients) and Mycobaterium smegmatis.

[0281] Accordingly, a condition associated with a pathogenicmicroorganism describes an infectious condition that results fromthe presence of the microorganism in a subject. The infectiouscondition can be, for example, a bacterial infection, a fungalinfection, a protozoal infection, and the like, collectivelyreferred to herein as "microbial infection".

[0282] Some higher forms of microorganisms are pathogenic per-se,and other harbor lower forms of pathogenic bacteria, thus present amedical threat expressed in many medical conditions, such as,without limitation, actinomycosis, anthrax, aspergillosis,bacteremia, bacterial skin diseases, bartonella infections,botulism, brucellosis, burkholderia infections, campylobacterinfections, candidiasis, cat-scratch disease, chlamydia infections,cholera, clostridium infections, coccidioidomycosis,cryptococcosis, dermatomycoses, dermatomycoses, diphtheria,ehrlichiosis, epidemic louse borne typhus, Escherichia coliinfections, fusobacterium infections, gangrene, general infections,general mycoses, gram-negative bacterial infections, Gram-positivebacterial infections, histoplasmosis, impetigo, klebsiellainfections, legionellosis, leprosy, leptospirosis, listeriainfections, lyme disease, maduromycosis, melioidosis, mycobacteriuminfections, mycoplasma infections, necrotizing fasciitis, nocardiainfections, onychomycosis, ornithosis, pneumococcal infections,pneumonia, pseudomonas infections, Q fever, rat-bite fever,relapsing fever, rheumatic fever, rickettsia infections,Rocky-mountain spotted fever, salmonella infections, scarlet fever,scrub typhus, sepsis, sexually transmitted bacterial diseases,staphylococcal infections, streptococcal infections, surgical siteinfection, tetanus, tick-borne diseases, tuberculosis, tularemia,typhoid fever, urinary tract infection, vibrio infections, yaws,yersinia infections, Yersinia pestis plague, zoonoses andzygomycosis.

[0283] The compounds presented herein can be effectively usedagainst bacterial strains which have developed or are prone to orcapable of developing resistance to at least one antimicrobialagents. Non-limiting examples of such bacterial strainsinclude:

[0284] (a) Gram-positive bacteria such as Strep. pyogenes (GroupA), Strep. pneumoniae, Strep. GpB, Strep. viridans, Strep. GpD(Enterococcus), Strep. GpC and GpG, Staph. aureus, Staph.epidermidis, Bacillus subtilis, Bacillus anthraxis, Listeriamonocytogenes, Anaerobic cocci, Clostridium spp., and Actinomycesspp; and

[0285] (b) Gram-negative bacteria such as Escherichia coli,Enterobacter aerogenes, Kiebsiella pneumoniae, Proteus mirabilis,Proteus vulgaris, Morganella morganii, Providencia stuartii,Serratia marcescens, Citrobacter freundii, Salmonella typhi,Salmonella paratyphi, Salmonella typhi murium, Salmonella virchow,Shigella spp., Yersinia enterocolitica, Acinetobactercalcoaceticus, Flavobacterium spp., Haemophilus influenzae,Pseudomonas aueroginosa, Campylobacter jejuni, Vibrioparahaemolyticus, Brucella spp., Neisseria meningitidis, Neisseriagonorrhoea, Bacteroides fragilis, and Fusobacterium spp.

[0286] According to some embodiments of the present invention, thecompounds presented herein can be effectively used againstbacterial strains which have developed or are prone to or capableof developing resistance to at least one antimicrobial agent.

[0287] According to some embodiments of the present invention, thecompounds presented herein can be effectively used againstbacterial strains which have developed or are prone to or capableof developing resistance to at least one antibacterial agent.

[0288] According to some embodiments of the present invention, thecompounds presented herein can be effectively used againstbacterial strains which have developed or are prone to or capableof developing resistance to an aminoglycoside antibacterialagent.

[0289] Exemplary such bacterial strains include but not limited to,Escherichia coli strains such as E. coli R477-100, E. coli ATCC25922, E. coli AG100B, E. coli ATCC 35218 and E. coli AG100A, B.subtilis strains (e.g., ATCC 6633), MRSA strains (e.g., ATCC43300), and Pseudomonas aueroginosa strains.

[0290] Thus, according to one aspect of the present invention thereis provided a method of treating a medical condition associatedwith a pathogenic microorganism in a subject. The method iseffected by administering to that subject, a therapeuticallyeffective amount of a compound as presented herein.

[0291] As used herein, the phrase "therapeutically effectiveamount" describes an amount of an active agent being administered,which will relieve to some extent one or more of the symptoms ofthe condition being treated. In the context of the presentembodiments, the phrase "therapeutically effective amount"describes an amount of a compound being administered and/orre-administered, which will relieve to some extent one or more ofthe symptoms of the condition being treated by being at a levelthat is harmful to the target microorganism(s), and cause adisruption to the life-cycle of the target microorganism(s), namelya bactericidal level or otherwise a level that inhibits themicroorganism growth or eradicates the microorganism.

[0292] The efficacy of any antimicrobial agent, including thecompounds presented herein, is oftentimes referred to in minimalinhibitory concentration units, or MIC units. A MIC is the lowestconcentration of an antimicrobial agent, typically measured inmicro-molar (.mu.M) or micrograms per milliliter (.mu.g/ml) units,which can inhibit the growth of a microorganism after a period ofincubation, typically 24 hours. MIC values are used as diagnosticcriteria to evaluate resistance of microorganisms to anantimicrobial agent, and for monitoring the activity of anantimicrobial agent in question. MICs are determined by standardlaboratory methods, as these are described and demonstrated in theExamples section that follows. Standard laboratory methodstypically follow a standard guideline of a reference body such asthe Clinical and Laboratory Standards Institute (CLSI), BritishSociety for Antimicrobial Chemotherapy (BSAC) or The EuropeanCommittee on Antimicrobial Susceptibility Testing (EUCAST). Inclinical practice, the minimum inhibitory concentrations are usedto determine the amount of antibiotic agent that the subjectreceives as well as the type of antibiotic agent to be used.

[0293] According to another aspect of embodiments of the presentinvention, each of the compounds described herein is for use intreating a medical condition associated with a pathogenicmicroorganism and/or in treating a subject diagnosed with a medicalcondition associated with a pathogenic microorganism.

[0294] According to another aspect of embodiments of the presentinvention, there is provided a use of any of the compoundsdescribed herein as a medicament or in the manufacture of amedicament. In some embodiments, the medicament is for treating amedical condition associated with a pathogenic microorganism and/ora subject diagnosed with a medical condition associated with apathogenic microorganism.

[0295] The compounds presented herein can be administered via anyadministration route, including, but not limited to, orally, byinhalation, or parenterally, for example, by intravenous drip orintraperitoneal, subcutaneous, intramuscular or intravenousinjection, or topically (including ophthalmically, vaginally,rectally, intranasally).

[0296] As used herein, the term "treating" includes abrogating,substantially inhibiting, slowing or reversing the progression of acondition, substantially ameliorating clinical or aestheticalsymptoms of a condition or substantially preventing the appearanceof clinical or aesthetical symptoms of a condition.

[0297] As used herein, the phrase "therapeutically effectiveamount" describes an amount of the polymer being administered whichwill relieve to some extent one or more of the symptoms of thecondition being treated.

[0298] Pharmaceutical Compositions:

[0299] In any of the methods and uses described herein, thecompounds described herein can be utilized either per se or form apart of a pharmaceutical composition, which further comprises apharmaceutically acceptable carrier, as defined herein.

[0300] According to an aspect of some embodiments of the presentinvention, there is provided a pharmaceutical composition whichcomprises, as an active ingredient, any of the novel compoundsdescribed herein and a pharmaceutically acceptable carrier.

[0301] As used herein a "pharmaceutical composition" refers to apreparation of the compounds presented herein, with other chemicalcomponents such as pharmaceutically acceptable and suitablecarriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

[0302] Hereinafter, the term "pharmaceutically acceptable carrier"refers to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biologicalactivity and properties of the administered compound. Examples,without limitations, of carriers are: propylene glycol, saline,emulsions and mixtures of organic solvents with water, as well assolid (e.g., powdered) and gaseous carriers.

[0303] Herein the term "excipient" refers to an inert substanceadded to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin,vegetable oils and polyethylene glycols.

[0304] Techniques for formulation and administration of drugs maybe found in "Remington's Pharmaceutical Sciences" Mack PublishingCo., Easton, Pa., latest edition, which is incorporated herein byreference.

[0305] Pharmaceutical compositions of the present invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

[0306] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in conventional mannerusing one or more pharmaceutically acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of thecompounds presented herein into preparations which, can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

[0307] According to some embodiments, the administration iseffected orally. For oral administration, the compounds presentedherein can be formulated readily by combining the compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds presented herein to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitableauxiliaries if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparationssuch as, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone(PVP). If desired, disintegrating agents may be added, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof such as sodium alginate.

[0308] Pharmaceutical compositions, which can be used orally,include push-fit capsules made of gelatin as well as soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules may contain the active ingredientsin admixture with filler such as lactose, binders such as starches,lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the compounds presented herein maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers may be added. All formulations for oral administrationshould be in dosages suitable for the chosen route ofadministration.

[0309] For injection, the compounds presented herein may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline buffer with or without organic solvents suchas propylene glycol, polyethylene glycol.

[0310] For transmucosal administration, penetrants are used in theformulation. Such penetrants are generally known in the art.

[0311] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or drageecoatings for identification or to characterize differentcombinations of active aminoglycoside compounds doses.

[0312] For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

[0313] For administration by inhalation, the compounds presentedherein are conveniently delivered in the form of an aerosol spraypresentation (which typically includes powdered, liquefied and/orgaseous carriers) from a pressurized pack or a nebulizer, with theuse of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbondioxide. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of thecompounds presented herein and a suitable powder base such as, butnot limited to, lactose or starch.

[0314] The compounds presented herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unitdosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. The compositions may besuspensions, solutions or emulsions in oily or aqueous vehicles,and may contain formulatory agents such as suspending, stabilizingand/or dispersing agents.

[0315] Pharmaceutical compositions for parenteral administrationinclude aqueous solutions of the compounds preparation inwater-soluble form. Additionally, suspensions of the compoundspresented herein may be prepared as appropriate oily injectionsuspensions and emulsions (e.g., water-in-oil, oil-in-water orwater-in-oil in oil emulsions). Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fattyacids esters such as ethyl oleate, triglycerides or liposomes.Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents, whichincrease the solubility of the compounds presented herein to allowfor the preparation of highly concentrated solutions.

[0316] Alternatively, the compounds presented herein may be inpowder form for constitution with a suitable vehicle, e.g.,sterile, pyrogen-free water, before use.

[0317] The compounds presented herein may also be formulated inrectal compositions such as suppositories or retention enemas,using, e.g., conventional suppository bases such as cocoa butter orother glycerides.

[0318] The pharmaceutical compositions herein described may alsocomprise suitable solid of gel phase carriers or excipients.Examples of such carriers or excipients include, but are notlimited to, calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin and polymers such aspolyethylene glycols.

[0319] Pharmaceutical compositions suitable for use in context ofthe present invention include compositions wherein the activeingredients are contained in an amount effective to achieve theintended purpose. More specifically, a therapeutically effectiveamount means an amount of compounds presented herein effective toprevent, alleviate or ameliorate symptoms of the disorder, orprolong the survival of the subject being treated.

[0320] Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein.

[0321] For any compounds presented herein used in the methods ofthe present embodiments, the therapeutically effective amount ordose can be estimated initially from activity assays in animals.For example, a dose can be formulated in animal models to achieve acirculating concentration range that includes the mutationsuppression levels as determined by activity assays (e.g., theconcentration of the test compounds which achieves a substantialread-through of the truncation mutation). Such information can beused to more accurately determine useful doses in humans.

[0322] Toxicity and therapeutic efficacy of the compounds presentedherein can be determined by standard pharmaceutical procedures inexperimental animals, e.g., by determining the EC.sub.50 (theconcentration of a compound where 50% of its maximal effect isobserved) and the LD.sub.50 (lethal dose causing death in 50% ofthe tested animals) for a subject compound. The data obtained fromthese activity assays and animal studies can be used in formulatinga range of dosage for use in human.

[0323] The dosage may vary depending upon the dosage form employedand the route of administration utilized. The exact formulation,route of administration and dosage can be chosen by the individualphysician in view of the patient's condition. (See e.g., Fingl etal., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

[0324] Dosage amount and interval may be adjusted individually toprovide plasma levels of the compounds presented herein which aresufficient to maintain the desired effects, termed the minimaleffective concentration (MEC). The MEC will vary for eachpreparation, but can be estimated from in vitro data; e.g., theconcentration of the compounds necessary to achieve 50-90%expression of the whole gene having a truncation mutation, i.e.read-through of the mutation codon. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determineplasma concentrations.

[0325] Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, whichmaintains plasma levels above the MEC for 10-90% of the time,preferable between 30-90% and most preferably 50-90%.

[0326] Depending on the severity and responsiveness of the chroniccondition to be treated, dosing can also be a single periodicadministration of a slow release composition described hereinabove,with course of periodic treatment lasting from several days toseveral weeks or until sufficient amelioration is effected duringthe periodic treatment or substantial diminution of the disorderstate is achieved for the periodic treatment.

[0327] The amount of a composition to be administered will, ofcourse, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc. Compositions of the present inventionmay, if desired, be presented in a pack or dispenser device, suchas an FDA (the U.S. Food and Drug Administration) approved kit,which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal orplastic foil, such as, but not limited to a blister pack or apressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. Thepack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, whichnotice is reflective of approval by the agency of the form of thecompositions for human or veterinary administration. Such notice,for example, may be of labeling approved by the U.S. Food and DrugAdministration for prescription drugs or of an approved productinsert. Compositions comprising a compound according to the presentembodiments, formulated in a compatible pharmaceutical carrier mayalso be prepared, placed in an appropriate container, and labeledfor treatment of an indicated condition or diagnosis, as isdetailed hereinabove.

[0328] Thus, in some embodiments, the pharmaceutical composition ispackaged in a packaging material and identified in print, in or onthe packaging material, for use in the treatment of a medicalcondition associated with a pathogenic microorganism, as definedherein.

[0329] In any of the composition, methods and uses describedherein, the compounds can be utilized in combination with otheragents useful in the treatment of the medical conditions describedherein.

[0330] It is expected that during the life of a patent maturingfrom this application additional relevant aminoglycoside skeletonswill be developed and the scope of the term modified aminoglycosideis intended to include all such new technologies a priori.

[0331] It is expected that during the life of a patent maturingfrom this application additional relevant pathogenic microorganismswill be developed and the scope of this phrase is intended toinclude all such new technologies a priori.

[0332] As used herein the term "about" refers to .+-.10% or.+-.5%.

[0333] The terms "comprises", "comprising", "includes","including", "having" and their conjugates mean "including but notlimited to".

[0334] The term "consisting of" means "including and limitedto".

[0335] The term "consisting essentially of" means that thecomposition, method or structure may include additionalingredients, steps and/or parts, but only if the additionalingredients, steps and/or parts do not materially alter the basicand novel characteristics of the claimed composition, method orstructure.

[0336] As used herein, the singular form "a", "an" and "the"include plural references unless the context clearly dictatesotherwise. For example, the term "a compound" or "at least onecompound" may include a plurality of compounds, including mixturesthereof.

[0337] Throughout this application, various embodiments of thisinvention may be presented in a range format. It should beunderstood that the description in range format is merely forconvenience and brevity and should not be construed as aninflexible limitation on the scope of the invention. Accordingly,the description of a range should be considered to havespecifically disclosed all the possible subranges as well asindividual numerical values within that range. For example,description of a range such as from 1 to 6 should be considered tohave specifically disclosed subranges such as from 1 to 3, from 1to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., aswell as individual numbers within that range, for example, 1, 2, 3,4, 5, and 6. This applies regardless of the breadth of therange.

[0338] Whenever a numerical range is indicated herein, it is meantto include any cited numeral (fractional or integral) within theindicated range. The phrases "ranging/ranges between" a firstindicate number and a second indicate number and "ranging/rangesfrom" a first indicate number "to" a second indicate number areused herein interchangeably and are meant to include the first andsecond indicated numbers and all the fractional and integralnumerals therebetween.

[0339] As used herein the term "method" refers to manners, means,techniques and procedures for accomplishing a given task including,but not limited to, those manners, means, techniques and procedureseither known to, or readily developed from known manners, means,techniques and procedures by practitioners of the chemical,pharmacological, biological, biochemical and medical arts.

[0340] As used herein, the term "treating" includes abrogating,substantially inhibiting, slowing or reversing the progression of acondition, substantially ameliorating clinical or aestheticalsymptoms of a condition or substantially preventing the appearanceof clinical or aesthetical symptoms of a condition.

[0341] When reference is made to particular sequence listings, suchreference is to be understood to also encompass sequences thatsubstantially correspond to its complementary sequence as includingminor sequence variations, resulting from, e.g., sequencing errors,cloning errors, or other alterations resulting in basesubstitution, base deletion or base addition, provided that thefrequency of such variations is less than 1 in 50 nucleotides,alternatively, less than 1 in 100 nucleotides, alternatively, lessthan 1 in 200 nucleotides, alternatively, less than 1 in 500nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively,less than 1 in 10,000 nucleotides.

[0342] It is understood that any Sequence Identification Number(SEQ ID NO) disclosed in the instant application can refer toeither a DNA sequence or a RNA sequence, depending on the contextwhere that SEQ ID NO is mentioned, even if that SEQ ID NO isexpressed only in a DNA sequence format or a RNA sequence format.Similarly, though some sequences are expressed in a RNA sequenceformat (e.g., reciting U for uracil), depending on the actual typeof molecule being described, it can refer to either the sequence ofa RNA molecule comprising a dsRNA, or the sequence of a DNAmolecule that corresponds to the RNA sequence shown. In any event,both DNA and RNA molecules having the sequences disclosed with anysubstitutes are envisioned.

[0343] Herein throughout, the phrase "linking moiety" or "linkinggroup" describes a group that connects two or more moieties orgroups in a compound. A linking moiety is typically derived from abi- or tri-functional compound, and can be regarded as a bi- ortri-radical moiety, which is connected to two or three othermoieties, via two or three atoms thereof, respectively.

[0344] Exemplary linking moieties include a hydrocarbon moiety orchain, optionally interrupted by one or more heteroatoms, asdefined herein, and/or any of the chemical groups listed below,when defined as linking groups.

[0345] When a chemical group is referred to herein as "end group"it is to be interpreted as a substituent, which is connected toanother group via one atom thereof.

[0346] Herein throughout, the term "hydrocarbon" collectivelydescribes a chemical group composed mainly of carbon and hydrogenatoms. A hydrocarbon can be comprised of alkyl, alkene, alkyne,aryl, and/or cycloalkyl, each can be substituted or unsubstituted,and can be interrupted by one or more heteroatoms. The number ofcarbon atoms can range from 2 to 20, and is preferably lower, e.g.,from 1 to 10, or from 1 to 6, or from 1 to 4. A hydrocarbon can bea linking group or an end group.

[0347] As used herein, the term "amine" describes both a --NR'R''group and a --NR'-- group, wherein R' and R'' are eachindependently hydrogen, alkyl, cycloalkyl, aryl, as these terms aredefined hereinbelow.

[0348] The amine group can therefore be a primary amine, where bothR' and R'' are hydrogen, a secondary amine, where R' is hydrogenand R'' is alkyl, cycloalkyl or aryl, or a tertiary amine, whereeach of R' and R'' is independently alkyl, cycloalkyl or aryl.

[0349] Alternatively, R' and R'' can each independently behydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide,phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, carbonyl,C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate,urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl,guanidine and hydrazine.

[0350] Further alternatively, R' and R'' form together aheteroalicyclic nitrogen-containing ring.

[0351] The amine group as described herein can be in a protonatedor an ammonium form, as described herein.

[0352] The term "alkyl" describes a saturated aliphatic hydrocarbonincluding straight chain and branched chain groups. Preferably, thealkyl group has 1 to 30, or 1 to 20 carbon atoms. Whenever anumerical range; e.g., "1-20", is stated herein, it implies thatthe group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbonatoms. The alkyl group may be substituted or unsubstituted.

[0353] The alkyl group can be an end group, as this phrase isdefined hereinabove, wherein it is attached to a single adjacentatom, or a linking group, as this phrase is defined hereinabove,which connects two or more moieties via at least two carbons in itschain. When the alkyl is a linking group, it is also referred toherein as "alkylene" or "alkylene chain".

[0354] Alkene and Alkyne, as used herein, are an alkyl, as definedherein, which contains one or more double bond or triple bond,respectively.

[0355] The term "cycloalkyl" describes an all-carbon monocyclicring or fused rings (i.e., rings which share an adjacent pair ofcarbon atoms) group where one or more of the rings does not have acompletely conjugated pi-electron system. Examples include, withoutlimitation, cyclohexane, adamantine, norbornyl, isobornyl, and thelike. The cycloalkyl group may be substituted or unsubstituted.

[0356] The cycloalkyl group can be an end group, as this phrase isdefined hereinabove, wherein it is attached to a single adjacentatom, or a linking group, as this phrase is defined hereinabove,connecting two or more moieties at two or more positionsthereof.

[0357] The term "heteroalicyclic" describes a monocyclic or fusedring group having in the ring(s) one or more atoms such asnitrogen, oxygen and sulfur. The rings may also have one or moredouble bonds. However, the rings do not have a completelyconjugated pi-electron system. Representative examples arepiperidine, piperazine, tetrahydrofurane, tetrahydropyrane,morpholino, oxalidine, and the like.

[0358] The heteroalicyclic may be substituted or unsubstituted. Theheteroalicyclic group can be an end group, as this phrase isdefined hereinabove, where it is attached to a single adjacentatom, or a linking group, as this phrase is defined hereinabove,connecting two or more moieties at two or more positionsthereof.

[0359] The term "aryl" describes an all-carbon monocyclic orfused-ring polycyclic (i.e., rings which share adjacent pairs ofcarbon atoms) groups having a completely conjugated pi-electronsystem. The aryl group may be substituted or unsubstituted. Thearyl group can be an end group, as this term is definedhereinabove, wherein it is attached to a single adjacent atom, or alinking group, as this term is defined hereinabove, connecting twoor more moieties at two or more positions thereof.

[0360] The term "heteroaryl" describes a monocyclic or fused ring(i.e., rings which share an adjacent pair of atoms) group having inthe ring(s) one or more atoms, such as, for example, nitrogen,oxygen and sulfur and, in addition, having a completely conjugatedpi-electron system. Examples, without limitation, of heteroarylgroups include pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinolineand purine. The heteroaryl group can be an end group, as thisphrase is defined hereinabove, where it is attached to a singleadjacent atom, or a linking group, as this phrase is definedhereinabove, connecting two or more moieties at two or morepositions thereof. Representative examples are pyridine, pyrrole,oxazole, indole, purine and the like.

[0361] A "guanidine" or "guanidine" or "guanidinyl" or "guanidyl"group refers to an --RaNC(.dbd.NRd)-NRbRc group, where each of Ra,Rb, Rc and Rd can each be as defined herein for R' and R''.

[0362] A "guanyl" or "guanine" group refers to an RaRbNC(.dbd.NRd)-group, where Ra, Rb and Rd are each as defined herein for R' andR''.

[0363] In some of any of the embodiments described herein, theguanidine group is --NH--C(.dbd.NH)--NH.sub.2.

[0364] In some of any of the embodiments described herein, theguanyl group is H.sub.2N--C(.dbd.NH)-- group.

[0365] Any one of the amine (including modified amine), guanidineand guanine groups described herein is presented as a free baseform thereof, but is meant to encompass an ionized form thereof atphysiological pH, and/or within a salt thereof, e.g., apharmaceutically acceptable salt thereof, as described herein.

[0366] Whenever an alkyl, cycloalkyl, aryl, alkaryl, heteroaryl,heteroalicyclic, acyl and any other moiety as described herein issubstituted, it includes one or more substituents, each canindependently be, but are not limited to, hydroxy, alkoxy,thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, alkaryl, alkenyl,alkynyl, sulfonate, sulfoxide, thiosulfate, sulfate, sulfite,thiosulfite, phosphonate, cyano, nitro, azo, sulfonamide, carbonyl,thiocarbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate,O-thiocarbamate, oxo, thiooxo, oxime, acyl, acyl halide, azo,azide, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide,guanyl, guanidyl, hydrazine and hydrazide, as these terms aredefined herein.

[0367] The term "halide" and "halo" describes fluorine, chlorine,bromine or iodine.

[0368] The term "haloalkyl" describes an alkyl group as definedabove, further substituted by one or more halide.

[0369] The term "sulfate" describes a --O--S(.dbd.O).sub.2--OR' endgroup, as this term is defined hereinabove, or an--O--S(.dbd.O).sub.2--O-- linking group, as these phrases aredefined hereinabove, where R' is as defined hereinabove.

[0370] The term "thiosulfate" describes a--O--S(.dbd.S)(.dbd.O)--OR' end group or a--O--S(.dbd.S)(.dbd.O)--O-- linking group, as these phrases aredefined hereinabove, where R' is as defined hereinabove.

[0371] The term "sulfite" describes an --O--S(.dbd.O)--O--R' endgroup or a --O--S(.dbd.O)--O-- group linking group, as thesephrases are defined hereinabove, where R' is as definedhereinabove.

[0372] The term "thiosulfite" describes a --O--S(.dbd.S)--O--R' endgroup or an --O--S(.dbd.S)--O-- group linking group, as thesephrases are defined hereinabove, where R' is as definedhereinabove.

[0373] The term "sulfinate" describes a --S(.dbd.O)--OR' end groupor an --S(.dbd.O)--O-- group linking group, as these phrases aredefined hereinabove, where R' is as defined hereinabove.

[0374] The term "sulfoxide" or "sulfinyl" describes a --S(.dbd.O)R'end group or an --S(.dbd.O)-- linking group, as these phrases aredefined hereinabove, where R' is as defined hereinabove.

[0375] The term "sulfonate" describes a --S(.dbd.O).sub.2--R' endgroup or an --S(.dbd.O).sub.2-- linking group, as these phrases aredefined hereinabove, where R' is as defined herein.

[0376] The term "S-sulfonamide" describes a--S(.dbd.O).sub.2--NR'R'' end group or a --S(.dbd.O).sub.2--NR'--linking group, as these phrases are defined hereinabove, with R'and R'' as defined herein.

[0377] The term "N-sulfonamide" describes anR'S(.dbd.O).sub.2--NR''-- end group or a --S(.dbd.O).sub.2--NR'--linking group, as these phrases are defined hereinabove, where R'and R'' are as defined herein.

[0378] The term "disulfide" refers to a --S--SR' end group or a--S--S-- linking group, as these phrases are defined hereinabove,where R' is as defined herein.

[0379] The term "phosphonate" describes a --P(.dbd.O)(OR')(OR'')end group or a --P(.dbd.O)(OR')(O)-- linking group, as thesephrases are defined hereinabove, with R' and R'' as definedherein.

[0380] The term "thiophosphonate" describes a--P(.dbd.S)(OR')(OR'') end group or a --P(.dbd.S)(OR')(O)-- linkinggroup, as these phrases are defined hereinabove, with R' and R'' asdefined herein.

[0381] The term "phosphinyl" describes a --PR'R'' end group or a--PR'-- linking group, as these phrases are defined hereinabove,with R' and R'' as defined hereinabove.

[0382] The term "phosphine oxide" describes a --P(.dbd.O)(R')(R'')end group or a --P(.dbd.O)(R')-- linking group, as these phrasesare defined hereinabove, with R' and R'' as defined herein.

[0383] The term "phosphine sulfide" describes a--P(.dbd.S)(R')(R'') end group or a --P(.dbd.S)(R')-- linkinggroup, as these phrases are defined hereinabove, with R' and R'' asdefined herein.

[0384] The term "phosphite" describes an --O--PR'(.dbd.O)(OR'') endgroup or an --O--PH(.dbd.O)(O)-- linking group, as these phrasesare defined hereinabove, with R' and R'' as defined herein.

[0385] The term "carbonyl" or "carbonate" as used herein, describesa --C(.dbd.O)--R' end group or a --C(.dbd.O)-- linking group, asthese phrases are defined hereinabove, with R' as definedherein.

[0386] The term "thiocarbonyl" as used herein, describes a--C(.dbd.S)--R' end group or a --C(.dbd.S)-- linking group, asthese phrases are defined hereinabove, with R' as definedherein.

[0387] The term "oxo" as used herein, describes a (.dbd.O) group,wherein an oxygen atom is linked by a double bond to the atom(e.g., carbon atom) at the indicated position.

[0388] The term "thiooxo" as used herein, describes a (.dbd.S)group, wherein a sulfur atom is linked by a double bond to the atom(e.g., carbon atom) at the indicated position.

[0389] The term "oxime" describes a .dbd.N--OH end group or a.dbd.N--O-- linking group, as these phrases are definedhereinabove.

[0390] The term "hydroxyl" describes a --OH group.

[0391] The term "alkoxy" describes both an --O-alkyl and an--O-cycloalkyl group, as defined herein.

[0392] The term "aryloxy" describes both an --O-aryl and an--O-heteroaryl group, as defined herein.

[0393] The term "thiohydroxy" describes a --SH group.

[0394] The term "thioalkoxy" describes both a --S-alkyl group, anda --S-cycloalkyl group, as defined herein.

[0395] The term "thioaryloxy" describes both a --S-aryl and a--S-heteroaryl group, as defined herein.

[0396] The "hydroxyalkyl" is also referred to herein as "alcohol",and describes an alkyl, as defined herein, substituted by a hydroxygroup.

[0397] The term "cyano" describes a --C.ident.N group.

[0398] The term "isocyanate" describes an --N.dbd.C.dbd.Ogroup.

[0399] The term "isothiocyanate" describes an --N.dbd.C.dbd.Sgroup.

[0400] The term "nitro" describes an --NO.sub.2 group.

[0401] The term "acyl halide" describes a --(C.dbd.O)R'''' groupwherein R'''' is halide, as defined hereinabove.

[0402] The term "azo" or "diazo" describes an --N.dbd.NR' end groupor an --N.dbd.N-- linking group, as these phrases are definedhereinabove, with R' as defined hereinabove.

[0403] The term "peroxo" describes an --O--OR' end group or an--O--O-- linking group, as these phrases are defined hereinabove,with R' as defined hereinabove.

[0404] The term "carboxylate" as used herein encompassesC-carboxylate and O-carboxylate.

[0405] The term "C-carboxylate" describes a --C(.dbd.O)--OR' endgroup or a --C(.dbd.O)--O-- linking group, as these phrases aredefined hereinabove, where R' is as defined herein.

[0406] The term "O-carboxylate" describes a --OC(.dbd.O)R' endgroup or a --OC(.dbd.O)-- linking group, as these phrases aredefined hereinabove, where R' is as defined herein.

[0407] A carboxylate can be linear or cyclic. When cyclic, R' andthe carbon atom are linked together to form a ring, inC-carboxylate, and this group is also referred to as lactone.Alternatively, R' and O are linked together to form a ring inO-carboxylate. Cyclic carboxylates can function as a linking group,for example, when an atom in the formed ring is linked to anothergroup.

[0408] The term "thiocarboxylate" as used herein encompassesC-thiocarboxylate and 0-thiocarboxylate.

[0409] The term "C-thiocarboxylate" describes a --C(.dbd.S)--OR'end group or a --C(.dbd.S)--O-- linking group, as these phrases aredefined hereinabove, where R' is as defined herein.

[0410] The term "O-thiocarboxylate" describes a --OC(.dbd.S)R' endgroup or a --OC(.dbd.S)-- linking group, as these phrases aredefined hereinabove, where R' is as defined herein.

[0411] A thiocarboxylate can be linear or cyclic. When cyclic, R'and the carbon atom are linked together to form a ring, inC-thiocarboxylate, and this group is also referred to asthiolactone. Alternatively, R' and O are linked together to form aring in O-thiocarboxylate. Cyclic thiocarboxylates can function asa linking group, for example, when an atom in the formed ring islinked to another group.

[0412] The term "carbamate" as used herein encompasses N-carbamateand O-carbamate.

[0413] The term "N-carbamate" describes an R''OC(.dbd.O)--NR'-- endgroup or a --OC(.dbd.O)--NR'-- linking group, as these phrases aredefined hereinabove, with R' and R'' as defined herein.

[0414] The term "O-carbamate" describes an --OC(.dbd.O)--NR'R'' endgroup or an --OC(.dbd.O)--NR'-- linking group, as these phrases aredefined hereinabove, with R' and R'' as defined herein.

[0415] A carbamate can be linear or cyclic. When cyclic, R' and thecarbon atom are linked together to form a ring, in O-carbamate.Alternatively, R' and O are linked together to form a ring inN-carbamate. Cyclic carbamates can function as a linking group, forexample, when an atom in the formed ring is linked to anothergroup.

[0416] The term "carbamate" as used herein encompasses N-carbamateand O-carbamate.

[0417] The term "thiocarbamate" as used herein encompassesN-thiocarbamate and 0-thiocarbamate.

[0418] The term "O-thiocarbamate" describes a --OC(.dbd.S)--NR'R''end group or a --OC(.dbd.S)--NR'-- linking group, as these phrasesare defined hereinabove, with R' and R'' as defined herein.

[0419] The term "N-thiocarbamate" describes an R''OC(.dbd.S)NR'--end group or a --OC(.dbd.S)NR'-linking group, as these phrases aredefined hereinabove, with R' and R'' as defined herein.

[0420] Thiocarbamates can be linear or cyclic, as described hereinfor carbamates.

[0421] The term "dithiocarbamate" as used herein encompassesS-dithiocarbamate and N-dithiocarbamate.

[0422] The term "S-dithiocarbamate" describes a--SC(.dbd.S)--NR'R'' end group or a --SC(.dbd.S)NR'-- linkinggroup, as these phrases are defined hereinabove, with R' and R'' asdefined herein.

[0423] The term "N-dithiocarbamate" describes an R''SC(.dbd.S)NR'--end group or a --SC(.dbd.S)NR'-linking group, as these phrases aredefined hereinabove, with R' and R'' as defined herein.

[0424] The term "urea", which is also referred to herein as"ureido", describes a --NR'C(.dbd.O)--NR''R''' end group or a--NR'C(.dbd.O)--NR''-- linking group, as these phrases are definedhereinabove, where R' and R'' are as defined herein and R''' is asdefined herein for R' and R''.

[0425] The term "thiourea", which is also referred to herein as"thioureido", describes a --NR'--C(.dbd.S)--NR''R''' end group or a--NR'--C(.dbd.S)--NR''-- linking group, with R', R'' and R''' asdefined herein.

[0426] The term "amide" as used herein encompasses C-amide andN-amide.

[0427] The term "C-amide" describes a --C(.dbd.O)--NR'R'' end groupor a --C(.dbd.O)--NR'-- linking group, as these phrases are definedhereinabove, where R' and R'' are as defined herein.

[0428] The term "N-amide" describes a R'C(.dbd.O)--NR''-- end groupor a R'C(.dbd.O)--N-- linking group, as these phrases are definedhereinabove, where R' and R'' are as defined herein.

[0429] An amide can be linear or cyclic. When cyclic, R' and thecarbon atom are linked together to form a ring, in C-amide, andthis group is also referred to as lactam. Cyclic amides canfunction as a linking group, for example, when an atom in theformed ring is linked to another group.

[0430] The term "guanyl" also describes a R'R''NC(.dbd.N)-- endgroup or a --R'NC(.dbd.N)-- linking group, as these phrases aredefined hereinabove, where R' and R'' are as defined herein.

[0431] The term "guanidine" also describes a--R'NC(.dbd.N)--NR''R''' end group or a --R'NC(.dbd.N)--NR''--linking group, as these phrases are defined hereinabove, where R',R'' and R''' are as defined herein.

[0432] The term "hydrazine" describes a --NR'--NR''R''' end groupor a --NR'--NR''-- linking group, as these phrases are definedhereinabove, with R', R'', and R''' as defined herein.

[0433] As used herein, the term "hydrazide" describes a--C(.dbd.O)--NR'--NR''R''' end group or a --C(.dbd.O)--NR'--NR''--linking group, as these phrases are defined hereinabove, where R',R'' and R''' are as defined herein.

[0434] As used herein, the term "thiohydrazide" describes a--C(.dbd.S)--NR'--NR''R''' end group or a --C(.dbd.S)--NR'--NR''--linking group, as these phrases are defined hereinabove, where R',R'' and R''' are as defined herein.

[0435] Herein throughout, the term "acyl" describes a--C(.dbd.O)--R group, wherein R is as described herein.

[0436] Herein throughout, the term "acyl" describes a--C(.dbd.O)--R group, with R being a substituted or unsubstitutedalkyl, cycloalkyl, aryl, alkaryl, a hydrocarbon chain, orhydrogen.

[0437] In exemplary embodiments, the acyl is such that R is analkyl or alkaryl or aryl, each of which being optionallysubstituted by one or more amine substituents.

[0438] In some embodiments, R is a substituted alkyl, and in someembodiments, R is substituted by hydroxy at the .alpha. positionwith respect to the carbonyl group, such that the acyl is.alpha.-hydroxy-acyl.

[0439] In some embodiments, the .alpha.-hydroxy-acyl is furthersubstituted by one or more amine groups, and is anamino-substituted .alpha.-hydroxy-acyl.

[0440] In some of the embodiments of an acyl group as describedherein, the amine substituents can be, for example, at one or moreof positions .beta., .gamma., .delta., and/or .omega. of the moietyR, with respect to the acyl.

[0441] Exemplary amino-substituted .alpha.-hydroxy-acyls include,without limitation, the moiety (S)-4-amino-2-hydroxybutyryl, whichis also referred to herein as AHB. According to some embodiments ofthe present invention, an alternative to the AHB moiety can be the.alpha.-hydroxy-.beta.-aminopropionyl (AHP) moiety. Additionalexemplary amino-substituted .alpha.-hydroxy-acyls include, but arenot limited to, L-(-)-.gamma.-amino-.alpha.-hydroxybutyryl,L(-)-.delta.-amino-.alpha.-hydroxyvaleryl,L-(-)-.beta.-benzyloxycarbonylamino-.alpha.-hydroxypropionyl, aL-(-)-.delta.-benzyloxycarbonylamino-.alpha.-hydroxyvaleryl.

[0442] It is noted herein that according to some embodiments of thepresent invention, other moieties which involve a combination ofcarbonyl(s), hydroxyl(s) and amino group(s) along a lower alkylexhibiting any stereochemistry, are contemplated as optionalsubstituents in place of AHB and/or AHP, including, for example,2-amino-3-hydroxybutanoyl, 3-amino-2-hydroxypentanoyl,5-amino-3-hydroxyhexanoyl and the likes.

[0443] It is appreciated that certain features of the invention,which are, for clarity, described in the context of separateembodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the invention, whichare, for brevity, described in the context of a single embodiment,may also be provided separately or in any suitable subcombinationor as suitable in any other described embodiment of the invention.Certain features described in the context of various embodimentsare not to be considered essential features of those embodiments,unless the embodiment is inoperative without those elements.

[0444] Various embodiments and aspects of the present invention asdelineated hereinabove and as claimed in the claims section belowfind experimental support in the following examples.

EXAMPLES

[0445] Reference is now made to the following examples, whichtogether with the above descriptions illustrate some embodiments ofthe invention in a non limiting fashion.

Materials and Experimental Methods

[0446] General Techniques:

[0447] NMR spectra (including .sup.1H, .sup.13C, DEPT, 2D COSY, 1DTOCSY, HMQC, HMBC) were recorded with a Bruker Avance 500spectrometer, and chemical shifts are reported relative to internalMe.sub.4Si (d=0.0 ppm) with CDCl.sub.3 as the solvent or to MeOD(d=3.35 ppm) as the solvent.

[0448] .sup.13C NMR spectra were recorded with a Bruker Avance 500spectrometer at 125.8 MHz, and the chemical shifts are reportedrelative to the solvent signal for CDCl.sub.3 (d=77.00 ppm) or tothe solvent signal for MeOD (d=49.0 ppm).

[0449] Mass spectra analyses were obtained with a Bruker DaltonixApex 3 mass spectrometer under electrospray ionization (ESI) or aTSQ-70B mass spectrometer (Finnigan Mat).

[0450] Reactions were monitored by TLC on Silica Gel 60 F254 (0.25mm, Merck), and spots were visualized by charring with a yellowsolution containing (NH.sub.4)Mo.sub.7O.sub.24.4H.sub.2O (120grams) and (NH.sub.4).sub.2Ce(NO.sub.3).sub.6 (5 grams) in 10%H.sub.2SO.sub.4 (800 mL).

[0451] Flash column chromatography was performed on Silica Gel 60(70-230 mesh).

[0452] All reactions were performed under an argon atmosphere withanhydrous solvents, unless otherwise indicated.

[0453] Neomycin B and paromomycin as analytical samples forcomparative biochemical assays were purchased from Sigma.

[0454] For chemical syntheses, large-scale paromomycin (used as astarting material) was purchased from Apollo Scientific (Stockport,UK).

[0455] All other chemicals and biochemicals, unless otherwiseindicated, were obtained from commercial vendors.

[0456] In all biological tests, all the tested aminoglycosides werein their sulfate salt forms, except Compound 5, which was used asits trifluoroacetate salt.

[0457] Biochemical Assays:

[0458] Prokaryotic in vitro translation inhibition by the differentstandard and synthetic aminoglycosides was quantified in coupledtranscription/translation assays by use of E. coli S30 extract forcircular DNA with the pBESTluc plasmid (Promega), according to themanufacturer's protocol.

[0459] Translation reactions (25 mL) containing variableconcentrations of the tested aminoglycoside were incubated at37.degree. C. for 60 minutes, cooled on ice for 5 minutes, anddiluted with a dilution reagent [Tris phosphate buffer (25 mm, pH7.8), dithiothreitol (DTT, 2 mm),1,2-diaminocyclohexanetetraacetate (2 mm), glycerol (10%), tritonX.sub.100 (1%), and bovine serum albumin (BSA, 1 mg/mL] into96-well plates.

[0460] The luminescence was measured immediately after the additionof Luciferase Assay Reagent (Promega) (50 mL), and light emissionwas recorded with a Victor3 Plate Reader (PerkinElmer).

[0461] The concentration of half-maximal inhibition (IC.sup.50) wasobtained from fitting concentration-response curves to the data ofat least three independent experiments by using Grafit 5software.

[0462] Comparative antibacterial activities were determined bymeasuring the MIC values by using the double-microdilution methodaccording to the National Committee for Clinical LaboratoryStandards (NCCLS).

[0463] All the experiments were performed in triplicate, andanalogous results were obtained in three different experiments.

[0464] For the rRNA cleavage experiments, the ribosomes wereisolated from E. coli cells (R477-100) by following the reportedprotocol.

[0465] Ribosomes were pelleted from pooled fractions (35 K for 15hours at 4.degree. C.) and were re-suspended in buffer for snapfreezing in liquid nitrogen and storage at -80.degree. C. The resinwas rinsed with water after use and was stored in 20% ethanol at48.degree. C. The catalytic domain of ColE3 was purified from itsimmunity protein as previously described [44]. Briefly, afterelution from the Ni-affinity column with 6M Gn.HCl, the ColE3 RNasebecame unfolded. It refolded upon dialysis in 50 mM potassiumphosphate or 20 mM Tris pH 7.5 buffer.

[0466] All parts of the purification procedure could be performedat room temperature, and the product was analyzed on 16%SDS-PAGE.

[0467] The cleavage experiments of rRNA with E. coli ribosomes wereperformed by incubation of freshly isolated ribosomes for 24 hours(5 minutes in the case of ColE3; 37.degree. C., pH 7.0) in thepresence of ethylenediamine, NeoB, Compound 3, or ColE3.

[0468] After incubation, RNA was phenol/chloroform extracted fromsamples and was electrophoresed on a 6% acrylamide TBE/urea gel for100 minutes at 180 V, stained with SYBR Gold, and analyzed byfluorescence.

[0469] A short RNA oligomer that represented the bacterial A-sitesequence labeled with a fluorescent tag (23 bases, for sequence,see FIGS. 7A and 7B and SEQ ID NOs: 2 and 3) was also used for rRNAcleavage experiments. This RNA sequence was purchased fromDharmacon and was used without further purification.

[0470] The cleavage experiments were performed by using gelelectrophoresis; the rRNA fragments were analyzed on 20% TBE/ureagel and were visualized by fluorescence.

[0471] Molecular Dynamics Simulations:

[0472] MD simulations were performed on the model of the A-sitecontaining two symmetric aminoglycoside binding sites by using thecrystal structure of the A-site with neomycin B bound (PDB ID:2ET4) [B. Fran.ANG.ois, R. J. M. Russell, J. B. Murray, F.Aboul-ela, B. Masquida, Q. Vicens, E. Westhof, Nucleic Acids Res.2005, 33, 5677-5690]. The oligonucleotide sequence used in thismodel is presented herein as SEQ ID NO: 3.

[0473] The MD simulation protocol consisted of energy minimization,thermalization, equilibration, and production phases. In the firsttwo phases, harmonic constraints with a force constant of 10kcal.andgate.mol.sup.-1.ANG..sup.-2 were imposed on heavy atoms ofthe solute.

[0474] First, all systems were energy minimized with the aboverestraints undergoing 5000 steps of steepest descent followed by4000 steps of conjugate gradient minimization by using sander(Amber 12).

[0475] The next phases were performed with NAMD [J. C. Phillips, R.Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot, R.D. Skeel, L. Kal8, K. Schulten, J. Comput. Chem. 2005, 26,1781-1802].

[0476] Second, during thermalization (in the NVT ensemble), eachsystem was heated from 10 to 310 K, increasing the temperature by10K every 100 ps. Then, 2 ns simulations at 310 K were performed.Third, equilibration was performed in the NpT ensemble with aconstant pressure of 1 Atm controlled by using the Langevin Pistonmethod and at constant temperature of 310 K regulated by Langevindynamics with a damping factor of 1 ps.sup.-1.

[0477] During 5 ns equilibration, the restraints were exponentiallydecreased in 50 time windows (scaled from 1 to 0.0065). Further,the 120 ns production runs were performed without anyrestraints.

[0478] Periodic boundary conditions and the Particle Mesh Ewaldmethod with a grid spacing of 1 .ANG. were used. The SHAKEalgorithm and an integration time step of 2 fs were applied.

[0479] For nonbonded interactions, a short-range cutoff of 12 .ANG.was used.

[0480] In order to calculate the GaMD acceleration parameters, theoriginal simulation experiments were followed by the GaMDsimulation [38, 39] experiments with an additional 2 ns of MDsimulation.

[0481] After adding the boost potential, the simulation wascontinued for 30 ns to equilibrate the system. Subsequently, tenindependent GaMD production runs were conducted for 100 ns each,starting with randomized initial atomic velocities.

[0482] The GaMD simulations were performed in the dual-boost mode,in which the boost potential was applied to the dihedral and totalpotential energy terms.

[0483] The threshold energy was set to the lower bound, that is,E=Vmax. The upper limit of the boost potential standard deviation,.sigma..sub.0, was set to 10 kcalmol.sup.-1 for the dihedral andtotal potential energetic terms.

Example 1

Rationale of the Design of Modified Aminoglycosides

[0484] The following aspects were first considered while designingthe novel aminoglycoside derivatives of the present embodiments:The choice of the phosphodiester bond in the A-site that should bethe most susceptible to cleavage; the potential "warhead" structurethat may exhibit a catalytic cleavage; and the attachment site of a"warhead" on the aminoglycoside structure.

[0485] It has been shown that successful cleavage of an RNAphosphodiester bond requires substantial motion in theHO--C2'-C3'-O--P bonds of the ribose-3'-phosphate region to reachthe necessary low-energy transition state where the C2'-OH group isorientated for in-line nucleophilic attack on the scissile bond [T.Lcnnberg, K. M. Kero, Org. Biomol. Chem. 2012, 10, 569-574].

[0486] Such flexibility is usually achieved by enzyme-inducedflipping of the base attached to the RNA scissile bond. Themechanisms suggested for RNase T1, RNase a-sarcin, and severalribozymes, mentioned in the Background section, are examples thatsupport this notion.

[0487] The proposed mechanism for colicinE3 (ColE3), a naturalenzymatic toxin produced in several Escherichia coli strains thatselectively cleaves a phosphodiester bond between A1493 and G1494of 16S rRNA is also of relevance [C. L. Ng, K. Lang, N. A. G.Meenan, A. Sharma, A. C. Kelley, C. Kleanthous, V. Ramakrishnan,Nat. Struct. Mol. Biol. 2010, 17, 1241-1246]. This cleavage impairsthe protein-translation process and, consequently, leads to celldeath.

[0488] The proposed mechanism of ColE3 explains why this naturalribonuclease cleaves a specific position in the A-site of rRNA,between A1493 and G1494. This region of the A-site is veryimportant functionally (for correct proofreading) and is also oneof the most flexible and accessible regions in the whole ribosome,because it needs to accommodate the incoming aminoacyl-tRNA.

[0489] The present inventors have assumed that the targetphosphodiester bond should be within the region of rRNA that uponbinding of an aminoglycoside undergoes the most extensiveconformational change. This region is virtually the same as that ofColE3 binding: G1491-A1492-A1493-G1494.

[0490] Given that the binding of most aminoglycosides inducesextensive flipping of the A1492 and A1493 base residues from thebulged-in (ligand-unbound ribosome) to the bulged-out conformation[B. Fran.ANG.ois, R. J. M. Russell, J. B. Murray, F. Aboul-ela, B.Masquida, Q. Vicens, E. Westhof, Nucleic Acids Res. 2005, 33,5677-5690], similar to that of ColE3 binding [C. L. Ng, K. Lang, N.A. G. Meenan, A. Sharma, Nat. Struct. Mol. Biol. 2010, 17,1241-1246], the present inventors have assumed that the best threephosphodiester bond candidates within the A site are betweenG1491-A1492, A1492-A1493, and A1493-G1494.

[0491] As indicated hereinabove, previous studies with simplediamines demonstrated their ability to accelerate cleavage ofadenylyl(3'-5')-adenosine (ApA) from one to three orders ofmagnitude more efficiently than the corresponding monoamines.

[0492] Ethylenediamine, methyl ethylenediamine, diethylenetriamine,N-(2-aminoethyl)pyrrolidine, and guanidine-ethyleneamine wereselected as potential "catalytic warheads" for preparing newlydesigned NeoB derivatives exemplified herein as Compounds 1-10(see, FIG. 1).

[0493] The 4'-hydroxy group (ring I) of NeoB (see, FIG. 1) wasfirst selected as the attachment site.

[0494] As shown in FIGS. 4A-B, preliminary molecular modelingstudies of the proposed warheads linked at the 4'-position showedthat the phosphodiester bond between G1491 and A1492 was theclosest one and that its cleavage may be feasible through acid-basecatalysis: the terminal amino group in its ammonium form canactivate the phosphate between G1491 and A1492 as a general acid(3.9 a distance), and the next-nearest amine can activate the2'-hydroxy group of G1491 as a general base (2.6 a distance).

[0495] Thus, G1491-A1492 was selected as the cleavage site, asschematically illustrated in FIG. 4B.

Example 2

Chemical Syntheses of Newly Designed Aminoglycoside Derivatives ofNeoB

[0496] Synthesis of 4'-O-linked Compounds (Compounds 1-5):

[0497] To selectively modify NeoB at the desired 4'-position, asynthetic pathway for its simplest fragment, that is, neamine,which consists of rings I and II of NeoB, was designed, andCompound 1 was prepared accordingly, as illustrated in FIG. 2A.

[0498] The synthesis started from commercial paromomycin sulfate;it was treated with anhydrous HCl [acetyl chloride (AcCl) in MeOH]at reflux, which resulted in highly regioselective hydrolysisbetween rings II and III to give paromamine as its hydrochloridesalt. The obtained salt was converted into the freebase form bypassing it through a column of Dowex 50W (H+ form). Paromamine inits free-base form was then converted into corresponding perazidoderivative 11 by a diazo-transfer reaction in the presence oftrifluoromethanesulfonyl azide (TfN.sub.3), CuSO.sub.4.5H.sub.2O,and Et.sub.3N.

[0499] Treatment of 11 with benzaldehyde dimethylacetal in dry DMFin the presence of camphorsulfonic acid (CSA) afforded thecorresponding benzylidene acetal 12, which was then O-benzylatedwith benzyl bromide (BnBr) in the presence of NaH in DMF to yieldtribenzyl ether 13. Removal of the benzylidene group (acetic acid,60.degree. C.) gave corresponding diol 14, which was thenselectively tosylated at the 6'-hydroxy group by using4-toluenesulfonyl chloride (TsCl) in pyridine (py); this wasfollowed by nucleophilic substitution with sodium azide to yieldcompound 15. Allylation of the 4'-hydroxy group with allyl bromidein the presence of NaH in DMF gave 4'-allyl derivative 16. Attemptsto convert 16 into the corresponding aldehyde by ozonolysisresulted in a mixture of products owing to partial oxidation of thebenzyl groups. To solve this problem, the double bond in 16 wasfirst converted into corresponding diol 17 by using a previouslydescribed procedure [24]. Oxidative cleavage of diol 17[PhI(OAc).sub.2, CH.sub.2Cl.sub.2] was followed by in situreductive amination with 2-azidoethanamine to yield corresponding4'-azido amine 18 in 66% yield. Finally, after several unsuccessfulattempts to remove the benzyl and azide protections in 18, asequential operation involving Staudinger and Birch reactions wasdetermined as the best protocol. Thus, the Staudinger reaction(PMe.sub.3, NaOH) followed by Birch reduction (Na/NH.sub.3, THF)gave target compound 1 in 65% yield.

[0500] The 4'-O-substituted derivatives of NeoB, Compounds 2-5(See, FIG. 1), were synthesized using the same strategy as thatdescribed for the synthesis of Compound 1 with some modifications,as illustrated in FIG. 2B.

[0501] The following modifications were applied:

[0502] Unlike the azidation of paromamine with TfN.sub.3 to yieldcorresponding perazido derivative 11 (FIG. 3), the same reaction onparomomycin gave a very low yield of desired perazido derivative19. In an attempt to improve the yield of the desired perazidoproduct, instead of TfN.sub.3, imidazole-1-sulfonyl azidehydrochloride (ImSO.sub.2N.sub.3.HCl) was used, and the tosylchloride was replaced with the more bulky triisopropylsulfonylchloride (trisyl chloride), which was more selective for protectionof the 6'-hydroxy group (conversion of Compound 22 into 23) andgave 60% yield over two steps (trisylation and azidation).

[0503] Common intermediate diol 25 was separately subjected to insitu oxidation and reductive amination steps with four differentamine linkers, compounds A, B, 1-(2-aminoethyl)pyrrolidine, and C(shown below), to afford the corresponding protected4'-O-derivatives of NeoB, compounds 26-29 (See, FIG. 2B).

##STR00012##

[0504] The Staudinger reaction (PMe.sub.3, NaOH) followed by theBirch reduction (Na/NH.sub.3, THF) gave Compounds 2-5 insatisfactory yields.

[0505] The structures of Compounds 1-5 were all confirmed bycombining various 1D and 2D NMR spectroscopy techniques, including2D .sup.1H-.sup.13C HMQC and HMBC, 2D COSY, and 1D selective TOCSYexperiments, along with mass spectrometry analysis.

[0506] The following describes the detailed syntheses of Compounds1-5 and the intermediates thereof.

Preparation of Compound4,6'-O-benzylidene-1,2',3-triazido-paromamine (12)

[0507] Compound 11 (1 gram, 2.49 mmol) was dissolved in dry DMF (20mL) and added with benzaldehyde dimethyl acetal (0.87 mL, 5.79mmol) and a catalytic amount of CSA. The reaction was stirred at60.degree. C. and the reaction progress was monitored by TLC (EtOAc60%, Hexane 40%), which indicated the completion of the reactionafter 2 hours. The reaction mixture was diluted with EtOAc andextracted with saturated aqueous solutions of NaHCO.sub.3 and NaCl.The combined organic layer was dried over MgSO.sub.4, filtered andconcentrated under reduced pressure. The crude product was purifiedby flash chromatography (EtOAc/hexane 1:1) to afford 12 (1.0 gram,83% yield).

[0508] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=6.01 (d, 1H,J=3.6 Hz, H-1), 4.77 (dd, 1H, J=10.2, 5.1 Hz, H-6), 4.67-4.61 (m,2H, H-3, H-6'), 4.24 (t, 1H, J=10.3 Hz, H-5), 4.02 (dd, 1H, J=9.5,9.3 Hz, H-4), 3.83 (dd, 1H, J=10.2, 4.2 Hz, H-2)); `Ring II`:.delta.H=4.02 (t, 1H, J=9.4 Hz, H-5), 3.91-3.76 (m, 4H, H-1, H-3,H-4, H-6), 2.77 (dt, 1H, J=12.7, 3.9 Hz, H-2eq), 1.93 (ddd, 1H,J=12.3, 10.6, 7.0 Hz, H-2ax); the additional peaks in the spectrumwere identified as follow: 8.02-7.97 (m, 2H, Ar), 7.84 (dd, 2H,J=5.1, 1.9 Hz, Ar), 6.05 (s, 1H, phCH).

[0509] .sup.13C NMR (125 MHz, MeOD): .delta.=C 137.58 (Ar), 129.54(Ar), 128.56 (Ar), 126.70 (Ar), 102.41 (phCH), 99.62 (C-1'), 82.16,81.33, 76.70, 76.50, 69.19 (C-5'), 69.06 (C-6'), 64.39, 63.59,60.53, 59.66, 32.49 (C-2).

[0510] MALDI TOFMS calcd for C.sub.19H.sub.24N.sub.9O.sub.7([M+H]+) m/e 490.4; measured m/e 490.0).

Preparation of4,6'-O-benzylidene-3',5,6-tri-O-benzyl-1,2',3-triazido-paromamine(13)

[0511] To a stirred solution of compound 12 (1 gram, 2.04 mmol) inanhydrous DMF (20 mL), TBAI (1 gram, 2.70 mmol), HMPA (5 mL) andBnBr (1.45 mL, 12.19 mmol) were added. After stirring for 20minutes, the mixture was cooled to -15.degree. C. and NaH (0.5gram, 12.5 mmol, 60% in oil) was added in portions. After beingstirred for 30 min at -15.degree. C., the mixture was allowed towarm to room temperature. The reaction progress was monitored byTLC (EtOAc 50%, Hexane 50%), which indicated completion after 1hour. The reaction mixture was diluted with EtOAc and washed withwater, 1M HCl, saturated aqueous NaHCO.sub.3 and brine. Thecombined organic layer was dried over anhydrous MgSO.sub.4,filtered and evaporated to dryness. The residue was purified byflash chromatography (EtOAc/hexane 1:5) to afford 13 (1.36 gram,88% yield).

[0512] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=5.50(d, 1H, J=4.0 Hz, H-1), 4.27 (dd, 1H, J=10.1, 5.0 Hz, H-6), 4.21(td, 1H, J=9.8, 4.9 Hz, H-5), 4.06 (t, 1H, J=9.5 Hz, H-3),3.71-3.62 (m, 2H, H-4, H-6), 3.29 (dd, 1H, J=10.0, 4.1 Hz, H-2);`Ring II`: .delta.H=3.59-3.53 (m, 2H, H-4, H-5), 3.45 (ddd, 1H,J=12.4, 9.8, 4.3 Hz, H-1), 3.40-3.32 (m, 2H, H-3, H-6), 2.28 (dt,1H, J=13.3, 4.5 Hz, H-2eq), 1.44 (ddd, 1H, J=12.8 Hz, H-2ax); theadditional peaks in the spectrum were identified as follow: 7.45(dd, 2H, J=7.5, 1.7 Hz, Ph), 7.37-7.19 (m, 18H, Ph), 5.52 (s, 1H,Bn), 4.97 (d, 1H, J=10.7 Hz, Bn), 4.93 (d, 1H, J=11.0 Hz, Bn), 4.88(d, 1H, J=10.7 Hz, Bn), 4.84 (d, 1H, J=10.5 Hz, Bn), 4.78 (d, 1H,J=10.5 Hz, Bn), 4.75 (d, 1H, J=11.0 Hz, Bn).

[0513] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.=C 138.11 (Ar),137.94 (Ar), 137.42 (Ar), 129.17 (Ar), 128.65 (Ar), 128.63 (Ar),128.55 (Ar), 128.42 (Ar), 128.38 (Ar), 128.28 (Ar), 128.19 (Ar),128.01 (Ar), 127.79 (Ar), 127.19 (Ar), 126.21 (Ar), 101.56 (Bn-CH),98.48 (C-1'), 84.82 (s), 84.46 (s), 82.72 (s), 78.05 (s), 76.39(C-3'), 76.07 (Bn), 75.41 (Bn), 75.18 (Bn), 69.00 (C-6'), 63.27(C-5'), 62.88 (C-2'), 60.37 (C-1), 59.36, 32.46 (C-2).

[0514] MALDI TOFMS calcd for C.sub.40H.sub.43N.sub.9O.sub.7([M+H]+) m/e 760.32; measured m/e 760.09).

Preparation of 3,5,6-tri-O-benzyl-1,2,3-triazido-paromamine(14)

[0515] Compound 13 (1.46 gram, 1.93 mmol) was dissolved in amixture of acetic acid (10 mL) and water (2 mL). The reactionmixture was left to stirred at 50.degree. C. overnight. Thecompletion of the reaction was indicated by TLC (EtOAc 50%, Hexane50%). The mixture was diluted with EtOAc and washed with saturatedaqueous NaHCO.sub.3 and brine. The combined organic layer was driedover anhydrous MgSO.sub.4, filtered and evaporated to dryness. Theresidue was purified by flash chromatography (EtOAc/hexane 1:5) toafford 14 (1.2 gram, 90% yield).

[0516] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=5.58(d, 1H, J=3.8 Hz, H-1), 3.99 (dt, 1H, J=9.9, 3.5 Hz, H-5), 3.84(dd, 1H, J=10.0, 9.0 Hz, H-3), 3.79-3.76 (m, 2H, H-6, H-6'), 3.62(ddd, 1H, J=9.6, 6.9, 3.7 Hz, H-4), 3.18 (dd, 1H, J=10.3, 3.8 Hz,H-2), 2.33 (d, 1H, J=3.6 Hz, OH-4), 1.81 (t, 1H, J=6.2 Hz, OH-6);`Ring II`: .delta.H=3.59-3.54 (m, 2H, H-4, H-5), 3.46 (ddd, 1H,J=12.4, 9.8, 4.5 Hz, H-1), 3.41-3.34 (m, 2H, H-3, H-6), 2.26 (dt,1H, J=13.3, 4.6 Hz, H-2eq), 1.41 (ddd, 1H, J=12.7 Hz, H-2ax); theadditional peaks in the spectrum were identified as follow:7.35-7.22 (m, 15H, Ph), 4.99 (d, 1H, J=10.8 Hz, Bn), 4.90 (d, 1H,J=11.2 Hz, Bn), 4.86 (d, 1H, J=10.8 Hz, Bn), 4.83 (d, 1H, J=10.5Hz, Bn), 4.78 (d, 1H, J=10.4 Hz, Bn), 4.73 (d, 1H, J=11.2 Hz,Bn).

[0517] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.=C 138.05 (Ar),137.99 (Ar), 137.41 (Ar), 128.86 (Ar), 128.67 (Ar), 128.65 (Ar),128.32 (Ar), 128.31 (Ar), 128.23 (Ar), 128.21 (Ar), 127.85 (Ar),127.19 (Ar), 97.80 (C-1'), 84.77 (C-6), 84.62 (C-5), 80.12 (C-3'),77.44 (C-4), 76.11 (Bn), 75.44 (Bn), 75.29 (Bn), 71.99 (C-5'),70.88 (C-4'), 62.97 (C-2'), 62.15 (C-6'), 60.42 (C-1), 59.79 (C-3),32.64 (C-2).

[0518] MALDI TOFMS calcd for C.sub.33H.sub.37N.sub.9O.sub.7Na([M+Na]+) m/e 694.27; measured m/e 694.03).

Preparation of 3,5,6-tri-O-benzyl-1,2,3,6'-tetraazido-paromamine(15)

[0519] Compound 14 (0.18 gram, 0.26 mmol) was treated withp-toluene sulfonyl chloride (2.37 grams, 12.4 mmol) in the presenceof pyridine (20 mL) and 4-DMAP (1 gram, 0.81 mmol) and heated to60.degree. C. The reaction progress was monitored by TLC (EtOAc30%, Hexane 70%), which indicated completion after 8 hours. Thenthe reaction mixture was diluted with EtOAc and washed with water,1M HCl, saturated aqueous NaHCO.sub.3 and brine. The combinedorganic layer was dried over anhydrous MgSO.sub.4, filtered andevaporated to dryness. The tosylate intermediate was then mixedwith sodium azide (2.37 grams, 12.4 mmol) and DMF (10 mL). Afterstirring at 60.degree. C. for 18 hours, the reaction mixture wasdiluted 50 with EtOAc washed with water, 1M HCl, saturated aqueousNaHCO.sub.3 and brine. The combined organic phase was dried overMgSO.sub.4 and concentrated under vacuum. The crude was purified bycolumn chromatography on silica gel (EtOAc/hexane 1:3) to afford 15(0.12 gram, 68%).

[0520] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=5.59(d, 1H, J=3.8 Hz, H-1), 4.16 (ddd, 1H, J=9.9, 4.6, 2.8 Hz, H-5),3.80 (dd, 1H, J=10.1, 9.0 Hz, H-3), 3.53-3.47 (m, 2H, H-4, H-6),3.43 (dd, 1H J=13.3, 4.9 Hz, H-6'), 3.22 (dd, 1H, J=10.3, 3.9 Hz,H-2), 2.08 (d, 1H, J=3.5 Hz, OH-4). `Ring II`: .delta.H=3.64-3.54(m, 2H, H-4, H-6), 3.50-3.42 (m, 1H, H-3), 3.43-3.35 (m, 2H, H-1,H-5), 2.28 (dt, 1H, J 13.2, 4.4, H-2eq), 1.45 (ddd, 1H, J 12.6,H-2ax); the additional peaks in the spectrum were identified asfollow: 7.41-7.17 (m, 15H, Ar), 5.00 (d, 1H, J=0.9 Hz, Bn), 4.94(d, 1H, J=11.3 Hz, Bn), 4.87 (d, 1H, J=10.9 Hz, Bn), 4.84 (d, 1H,J=10.4 Hz, Bn), 4.78 (d, 1H, J=10.4 Hz, Bn), 4.68 (d, 1H, J=11.3Hz, Bn).

[0521] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=137.99 (Ar),137.89 (Ar), 137.41 (Ar), 128.95 (Ar), 128.66 (Ar), 128.64 (Ar),128.46 (Ar), 128.33 (Ar), 128.24 (Ar), 128.20 (Ar), 127.81 (Ar),127.09 (Ar), 97.80 (C-1'), 84.78, 84.54, 80.19 (C-3'), 77.53, 76.12(Bn), 75.38 (Bn), 75.31 (Bn), 71.33 (C-5'), 71.09, 62.93 (C-2'),60.44, 59.66, 51.36 (C-6'), 32.55 (C-2).

[0522] MALDI TOFMS calcd for C.sub.33H.sub.36N.sub.12O.sub.6Na([M+Na]+) m/e 719.2; measured m/e 719.05).

Preparation of4'-O-Allyl-3',5,6-tri-O-benzyl-1,2',3,6'-tetraazido-paromamine(16)

[0523] Compound 15 (124 mg, 0.168 mmol) was dissolved in 10 mL ofDMF and cooled to -10.degree. C. The reaction was treated withsodium hydride (47 mg, 1.963 mmol, 60% in oil) followed by allylbromide (0.1 mL, 1.15 mmol). The reaction progress was monitored byTLC (EtOAc 20%, Hexane 80%), which indicated completion after 1hour. After completion the reaction mixture was diluted with EtOAcand washed with water, 1M HCl, saturated aqueous NaHCO.sub.3 andbrine. The combined organic layer was dried over anhydrousMgSO.sub.4, filtered and evaporated to dryness. The residue waspurified by flash chromatography (EtOAc/hexane 1:10) to afford 16(127 mg, 97% yield).

[0524] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=5.54(d, 1H, J=3.6 Hz, H-1), 4.21 (ddd, 1H, J=9.9, 4.0, 2.6 Hz, H-5),3.91 (dd, 1H, J=10.1, 9.1 Hz, H-3), 3.52 (dd, 1H, J=13.4, 2.3 Hz,H-6), 3.40 (dd, 1H, J=13.3, 4.5 Hz, H-6'), 3.36 (dd, 1H, J=9.9, 9.0Hz, H-4), 3.23 (dd, 1H, J=10.5, 4.3 Hz, H-2); `Ring II`: .delta.H3.60-3.53 (m, 2H, H-4, H-6), 3.47-3.41 (m, 1H, H-5), 3.40-3.34 (m,2H, H-1, H-3), 2.26 (dt, J=13.3, 4.4 Hz, H-2aq), 1.45 (ddd, J=12.6Hz, H-2ax); the additional peaks in the spectrum were identified asfollow: 7.37-7.22 (m, 15H, Ar), 5.85 (ddd, 1H, J=22.6, 10.7, 5.6Hz, Allyl), 5.23 (dd, 1H, J=17.2, 1.4 Hz, Allyl), 5.14 (dd, 1H,J=10.5, 1.2 Hz, Allyl), 4.98 (d, 1H J=10.9 Hz, Bn), 4.90 (d, 1H,J=10.8 Hz, Bn), 4.85-4.81 (m, 3H, Bn), 4.78 (d, 1H, J=10.4 Hz, Bn),4.27 (dd, 1H, J=12.5, 5.5 Hz, Allyl), 4.08 (dd, 1H, J=12.5, 5.7 Hz,Allyl).

[0525] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.00 (Ar),137.78 (Ar), 137.37 (Ar), 134.29 (Allyl), 128.56 (Ar), 128.53 (Ar),128.25 (Ar), 128.15 (Ar), 128.10 (Ar), 127.99 (Ar), 127.69 (Ar),127.00 (Ar), 117.41 (Allyl), 97.63 (C-1'), 84.69 (s), 84.41 (s),79.92 (C-3'), 78.55 (s), 77.65 (s), 76.01 (Bn), 75.52 (Bn), 75.31(Bn), 73.95 (Allyl), 71.08 (C-5'), 63.16 (C-2'), 60.31 (C-5), 59.42(s), 51.08 (C-6'), 32.39 (C-2).

[0526] MALDI TOFMS calcd for C36H.sub.41N1206 ([M+H]+) m/e 737.33;measured m/e 737.12).

Preparation of4'-O-(2,3-dihydroxypropyl)-3,5,6-tri-O-benzyl-1,2',3,6'-tetraazido-paroma-mine (17)

[0527] To a solution of compound 16 (300 mg, 0.407 mmol) in amixture of acetone:water (10:1) were added 4-methylmorpholineN-oxide (2 equiv, 0.814 mmol), and osmium tetroxide (0.02 equiv, 5mg, 0.008 mmol). When the starting material had been consumed asmonitored by TLC (EtOAc 20%, hexane 80%) the mixture was dilutedwith EtOAc and quenched with saturated aqueous sodium thiosulfateand brine. The combined organic phases were dried over anhydrousMgSO.sub.4, filtered and evaporated to dryness. The residue waspurified by column chromatography (EtOAc/hexane 55:45) to afford 17(245 mg, 80% yield) as a mixture of two diastereorners.

[0528] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=5.59(s, 1H, H-1), 4.21 (t, 1H J=10.9 Hz, H-5), 3.97-3.90 (m, 1H, H-3),3.58 (d, 1H, J=12.4 Hz, H-6), 3.47 (d, 1H, J=13.1 Hz, H-6'), 3.41(dd, 1H, J=16.7, 9.3 Hz, H-4), 3.27 (dd, 1H, J=10.9, 6.2 Hz, H-2);`Ring II`: .delta.H=3.66-3.56 (m, 2H, H-4, H-5), 3.56-3.46 (m, 1H,H-1), 3.46-3.35 (m, 2H, H-3, H-6), 2.39-2.28 (m, 1H, H-2eq), 1.49(ddd, 1H, J=12.6 Hz, H-2ax); the additional peaks in the spectrumwere identified as follow: 7.45-7.19 (m, 15H, Ar), 5.03 (d, 1H,J=11.0 Hz, PHJH), 4.93-4.85 (m, 3H, PHJH), 4.82 (d, 2H, J=10.6 Hz,PHJH), 3.85 (d, J=9.7 Hz), 3.78 (d, J=4.9 Hz), 3.69 (t, J=6.6 Hz),3.64-3.56 (m), 3.52 (d, J=7.1 Hz), 2.89-2.78 (m), 2.63-2.56 (m),1.88 (d, J=26.9 Hz). .sup.13C NMR (125 MHz, CDCl.sub.3):.delta.C=137.75 (Ar), 137.33 (Ar), 137.14 (Ar), 128.47 (Ar), 128.46(Ar), 128.43 (Ar), 128.41 (Ar), 128.09 (Ar), 128.06 (Ar), 127.97(Ar), 127.57 (Ar), 126.78 (Ar), 97.46 (C-1'), 84.54, 84.21, 79.77(C-3'), 79.20, 77.52, 77.46, 75.88, 75.52, 75.35, 75.15, 74.54,74.30, 71.07 (C-5'), 63.39 (CH2), 63.24 (C-2'), 60.16, 59.34(C-4'), 50.87 (C-6'), 32.28 (C-2).

[0529] MALDI TOFMS calcd for C.sub.36H.sub.42N.sub.12NaO.sub.8([M+Na]+) m/e 793.31; measured m/e 793.51).

Preparation of4'-O-(2-aminoethylazido)ethyl-3.sup.1,5,6-tri-O-benzyl-1,2',3,6'-tetraazi-do-paromamine (18)

[0530] To a solution of diol 17 (400 mg, 0.519 mmol) in anhydrousDCM (30 mL) was added PhI(OAc).sub.2 (1.2 equiv, 200 mg, 0.621mmol) at room temperature under Argon. After stirring for 2 hours2-azidoethanamine (2.6 equiv, 0.15 ml, 1.35 mmol) was added. Thereaction mixture was stirred for 30 min before sodiumtriacetoxyborohydride (2.8 equiv, 0.84 mmol, 1.453 mmol) was addedat room temperature. The reaction progress was monitored by TLC(EtOAc 50%, Hexane 50%), which indicated completion after 3 hours.After completion the reaction, the mixture was diluted with EtOAcand washed with saturated aqueous sodium bicarbonate and brine. Thecombined organic phases were dried over anhydrous MgSO.sub.4,filtered and evaporated to dryness. The residue was purified bycolumn chromatography (EtOAc/hexane 1:1) to afford 18 (280 mg, 66%yield).

[0531] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: 8H=5.57 (d, 1H,J=2.9 Hz, H-1), 4.23 (br d, 1H, J=9.9 Hz, H-5), 3.92 (t, 1H, J=9.5Hz, H-3), 3.58 (d, 1H, J=12.9 Hz, H-6), 3.49 (dd, 1H, J=13.3, 3.7Hz, H-6'), 3.38 (dd, 1H, J=9.7, 9.2 Hz, H-4), 3.28 (dd, 1H, J=10.6,5.2 Hz, H-2); `Ring II`: .delta.H=3.64-3.57 (m, 2H, H-4, H-5),3.53-3.47 (m, 1H, H-3), 3.46-3.40 (m, 2H, H-1, H-6) 2.33 (dt, 1H,J=13.2, 4.5 Hz, H-2eq), 1.50 (ddd, 1H, J=12.6 Hz, H-2ax); theadditional peaks in the spectrum were identified as follow:7.46-7.23 (m, 15H, Ar), 5.02 (d, 1H, J=10.9 Hz, Bn), 4.93 (d, 1H,J=10.9 Hz, Bn), 4.91-4.85 (m, 3H, J=8.1 Hz, Bn), 4.82 (d, 1H,J=10.5 Hz, Bn), 3.93-3.87 (m, 1H, CH.sub.2), 3.72-3.67 (m, 1H,CH.sub.2), 3.40-3.36 (m, 2H, CH.sub.2), 2.78-2.75 (m, 4H,CH.sub.2).

[0532] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.04 (Ar),137.84 (Ar), 137.40 (Ar), 128.63 (Ar), 128.62 (Ar), 128.31 (Ar),128.17 (Ar), 128.05 (Ar), 127.76 (Ar), 127.03 (Ar), 97.70 (C-1'),84.76, 84.45, 79.95 (C-3'), 79.19, 77.72, 76.08, 75.47, 75.37,72.36 (CH.sub.2), 71.10 (C-5'), 63.30 (C-2'), 60.37, 59.48, 51.30,51.15, 49.37 CH.sub.2), 48.45 (CH.sub.2), 32.47 (C-2).

[0533] MALDI TOFMS calcd for C.sub.37H.sub.45N.sub.16O.sub.6([M+H]+) m/e 808.36; measured m/e 809.10).

Preparation of 4'-O-(2-aminoethylamino)ethyl-paromamine (Compound1; FIGS. 1 and 2A)

[0534] Compound 18 (2.2 grams, 3.242 mmol) was dissolved in amixture of THF (10 mL) and aqueous NaOH (0.1M, 5 mL). This mixturewas stirred at room temperature for 10 minutes, after whichPMe.sub.3 (1M solution in THF, 38.91 mL, 38.91 mmol) was added.Propagation of the reaction was monitored by TLC[CH.sub.2Cl.sub.2/MeOH/H.sub.2O/MeNH.sub.2 (33% solution in EtOH),10:15:6:15], which indicated completion after 3 hour. The reactionmixture was purified by flash chromatography on a short column ofsilica gel. The column was washed with the following solvents:hexane (200 mL), THF (200 mL), CH.sub.2Cl.sub.2 (200 mL), EtOAc(200 mL), MeOH (400 mL). The product was eluted with the mixture of20% MeNH.sub.2 solution (33% solution in EtOH) in 80% MeOH.

[0535] Fractions containing the product were combined andevaporated under vacuum. THF (10 mL) was added via syringe to a drythree neck flask equipped with a Dewar condenser. Then ammonia(about 20 mL) was condensed into the reaction vessel. Small piecesof Na (300 mg, 13 mmol) were then allowed to dissolve in theammonia for 15 minutes. Then a solution of the aminoglycoside (fromthe above step) in a mixture of EtOH and THF (500 .mu.L each) wasadded in one portion and washed down with THF. The reaction wasstirred until the blue color was discharged. Then an aqueoussolution of ammonium formate (1 gram, 15.7 mmol) was added, and theammonia was allowed to evaporate. The remaining solvent was removedin Vacuum, and the residue was loaded onto a short column of silicagel. The column was washed with the following solvents: hexane (200mL), THF (200 mL), CH.sub.2Cl.sub.2 (200 mL), EtOAc (200 mL), MeOH(400 mL). The product was eluted with the mixture of 20% MeNH.sub.2solution (33% solution in EtOH) in 80% MeOH. Fractions containingthe product were combined and evaporated under vacuum. The productwas then dissolved in small volume of water and loaded on a columnof Amberlite CG-50 cation exchange resin (0.5 cm.times.10 cm) inits NH.sub.4+ form, washed with methanol (200 mL) and eluted with alinear gradient of 0% to 10% NH.sub.4OH solution. The productcontaining fractions were combined and evaporated under vacuum toafford 1 (727 mg, 65%).

[0536] The product was converted to its sulfate salt form asfollow: the free base was dissolved in water, the pH was adjustedto about 7 with H.sub.2SO.sub.4 (0.1 N) and lyophilized.

[0537] .sup.1H NMR (500 MHz, D.sub.2O): `Ring I`: .delta.H=6.05 (d,1H, J=3.8 Hz, H-1), 4.36 (dd, 1H, J=10.9, 8.8 Hz, H-3), 4.20 (dd,1H, J=9.5, 6.4 Hz, H-5), 3.55 (dd, 1H, J=13.2, 2.7 Hz, H-6), 3.50(dd, 1H, J=10.9, 4.7 Hz, H-2), 3.43 (dd, 1H, J=9.2, 9.2 Hz, H-4),3.27 (dd, 1H, J=13.3, 9.0 Hz, H-6'); `Ring II`: .delta.H=4.08 (dd,1H, J=10.1, 9.4 Hz, H-4), 3.75 (dd, 1H, J=9.2, 9.2 Hz, H-5), 3.65(dd, 1H, J=10.4, 9.3 Hz, H-6), 3.61-3.53 (m, 1H, H-3), 3.43-3.31(m, 1H, H-1), 2.52 (dt, 1H, J=12.4, 4.1 Hz, H-2eq), 2.04 (ddd, 1H,J=12.6 Hz, H-2ax); The additional peaks in the spectrum wereidentified as follow: 4.25-4.17 (m, 1H, CH.sub.2), 4.01 (dt, 1H,J=9.5, 4.5 Hz, CH.sub.2), 3.51-3.48 (m, 4H, CH.sub.2), 3.39 (t, 2H,J=4.9 Hz, CH.sub.2).

[0538] .sup.13C NMR (125 MHz, MeOD): .delta.C=94.66 (C-1'), 79.09,76.22 (C-4), 75.20 (C-5), 72.44 (C-6), 68.42 (C-3'), 68.18 (C-5'),67.16 (CH.sub.2), 53.39, 49.72, 48.46, 48.07 (CH.sub.2), 44.58(CH.sub.2), 40.49 (C-6'), 35.64 (CH.sub.2), 28.09 (C-2).

[0539] TOFMS calcd for C.sub.16H.sub.37N.sub.6O.sub.6 ([M+H]+) m/e409.28; measured m/e 409.09).

Preparation of 1,2',2''',3,6'''-pentaazido-paromomycin (19)

[0540] Commercially available paromomycin sulfate was neutralizedby passing through Dowex 50W resin column (H.sup.+ form). Then, thefree base (45.5 grams, 73.98 mmol) was dissolved in a mixture ofMeOH (1 L) and 1420 (1.00 mL) and stirred. To the fully dissolvedmixture, CuSO.sub.4 (1.28 gr, 8 mmol), K.sub.2CO.sub.3 (114.6grams, 830 mmol) and imidazole sulfonyl azide hydrochloride (86.9grams, 1.2 eq. per amine, 416 mmol) were added. The color of themixture changed from blue to dark green during the reaction. Thereaction progress was monitored by TLC(CH.sub.2Cl.sub.2/MeOH/H.sub.2O/MeNH.sub.2, 10:15:6:15) whichindicated completion after 18 hours. The reaction mixture wasevaporated, dissolved in MeOH (300 mL) and EtOAc (100 mL) andfiltered. Then, the solvent was evaporated and the crude wasdissolved in a minimum volume of H.sub.2O, the pH was adjusted to 3with HCl (3M) and then extracted with EtOAc. The combined organiclayers were dried over MgSO.sub.4 and concentrated under reducedpressure to yield compound 19 (35 grams, 70%) as a white solid.

[0541] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=5.76 (d, 1H,J=3.1 Hz, H-1), 3.92-3.86 (m, 2H, H-3, H-5), 3.78 (dd, 1H, J=11.9,1.6 Hz, H-6), 3.69 (dd, 1H, J=12.0, 4.3 Hz, H-6'), 3.35 (dd, 1H,J=10.1, 8.7 Hz, H-4), 3.03 (dd, 1H, J=10.7, 4.9 Hz, H-2); `RingII`: .delta.H=3.72-3.62 (m, 2H, H-4, H-5), 3.49-3.41 (m, 2H, H-3,H-6), 3.41-3.34 (m, 1H, H-1), 2.13 (dt, 1H, J=12.0, 4.0 Hz, H-2eq),1.33 (ddd, 1H, J=13.5 Hz, H-2ax); `Ring III`: .delta.H=5.35 (d, 1H,J=1.6 Hz, H-1), 4.40 (dd, 1H, J=7.4, 3.9 Hz, H-3), 4.26 (dd, 1H,J=4.8, 1.3 Hz, H-2), 4.09 (ddd, 1H, J=4.5, 3.3, 1.7 Hz, H-4), 3.78(dd, 1H, J=12.1, 2.4 Hz, H-5), 3.65 (dd, 1H, J=11.0, 3.9 Hz, H-5');`Ring IV`: .delta.H 5.08 (d, 1H, J=1.51 Hz, H-1), 3.98-3.96 (m, 1H,H-5), 3.90-3.88 (m, 1H, H-3), 3.63 (dd, 1H, J=4.8, 0.5 Hz, H-3),3.60 (m, 1H, H-6) 3.40 (d, 1H, J=2.3 Hz, H-4), 3.35 (m, 1H,H-6').

[0542] .sup.13C NMR (125 MHz, MeOD): .delta.C=108.95 (C-1''), 99.79(C-1'''), 98.03 (C-1'), 85.30 (C-5), 83.50 (C-4w), 77.11 (C-3''),77.01 (C-6), 76.31 (C-4), 75.56 (C-5'''), 75.21 (C-2''), 74.14(C-5'), 72.22 (C-3'), 71.93 (C-4'), 71.17 (C-3'''), 69.60 (C-4''),64.66 (C-2'), 63.28 (C-5''), 62.48 (C-6'), 61.85 (C-2'''), 61.83(C-1), 61.53 (C-3), 52.44 (C-6'''), 33.06 (C-2).

[0543] TOFMS calcd for C.sub.23H.sub.35N.sub.15O.sub.14Na ([M+Na]+)m/e 768.61; measured m/e 768.88).

Preparation of4',6'-O-benzylidene-1,2',2''',3,6'''-pentaazido-paromamine (20)

[0544] The titled compound was prepared as was described for thepreparation of compound 12 with the following quantities: compound19 (0.6 gram, 0.805 mmol), DMF (10 mL), benzaldehyde dimethylacetal (0.25 mL, 1.7 mmol), catalytic amount of camphorsulfonicacid to yield 20 (0.57 gram, 88%).

[0545] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=5.87 (d, 1H,J=3.6 Hz, H-1), 4.26 (dd, 1H, J=10.0, 5.0 Hz, H-6), 4.22-4.13 (m,2H, H-3, H-6'), 3.80 (t, 1H, J=10.1 Hz, H-5), 3.59 (dd, 1H, J=9.6,9.2 Hz, H-4), 3.29 (dd, 1H, J=10.3, 4.3 Hz, H-2); `Ring II`:.delta.H 3.77-3.67 (m, 2H, H-4, H-5), 3.59-3.52 (m, 1H, H-3),3.51-3.43 (m, 2H, H-1, H-6), 2.26 (dt, 1H, J=8.1, 4.0 Hz, H-2eq),1.45 (ddd, 1H, J=12.7 Hz, H-2ax); `Ring III`: .delta.H=5.43 (d, 1H,J=1.9 Hz, H-1), 4.47 (dd, 1H, J=7.0, 4.2 Hz, H-3), 4.36 (dd, 1H,J=4.8, 1.6 Hz, H-2), 4.19 (dt, 1H, J=4.9, 2.5 Hz, H-4), 3.88 (dd,1H, J=12.0, 2.5 Hz, H-5), 3.73 (dd, 1H, J=12.0, 5.4 Hz, H-5');`Ring IV`: .delta.H=5.18 (d, 1H, J=2.4 Hz, H-1), 4.08-4.04 (m, 1H,H-5), 3.98 (t, 1H, J=3.3 Hz, H-3), 3.72 (t, 1H, J=2.5 Hz, H-2),3.71 (dd, 1H, J=14.1, 8.4 Hz, H-6), 3.48 (t, 1H, J=2.5 Hz, H-4),3.43 (dd, 1H, J=12.2, 4.2 Hz, H-6'); The additional peaks in thespectrum were identified as follow: 7.55-7.52 (m, 2H, Ar),7.40-7.36 (m, 3H, Ar), 5.63 (s, 1H, PhCH).

[0546] .sup.13C NMR (125 MHz, MeOD): .delta.C=139.09 (Ar), 129.97(Ar), 129.06 (Ar), 127.55 (Ar), 109.66 (C-1''), 103.09 (PhCH),99.75 (C-1'''), 99.12 (C-1'), 85.19 (C-5), 83.42 (C-4''), 82.92(C-4'), 77.72 (C-6), 77.24 (C-4), 77.21 (C-3''), 75.60 (C-5'''),75.10 (C-2''), 71.11 (C-3'''), 69.79 (C-6'), 69.58 (C-3'), 69.53(C-4'''), 65.14 (C-2'), 64.54 (C-5'), 63.63 (C-5''), 61.83(C-2'''), 61.77 (C-1), 61.24 (C-3), 52.45 (C-6'''), 32.96(C-2).

[0547] TOFMS calcd for C.sub.30H.sub.39N.sub.15O.sub.14Na ([M+Na]+)m/e 856.27; measured m/e 856.39).

Preparation of4',6'-O-benzylidene-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6'''--pentaazido-paromomycin (21)

[0548] The titled compound was prepared as was described for thepreparation of compound 13 with the following quantities: compound20 (1 gram, 1.2 mmol), DMF (10 mL), TBAI (0.5 gram, 1.35 mmol),HMPA (3 mL, 17.2 mmol), BnBr (1.7 mL, 14.3 mmol, 2 eq. perhydroxyl), NaH (0.6 gram, 60% in oil, 25 mmol, 2 eq. per hydroxyl)to yield 21 (1.1 grams, 60%).

[0549] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.18(d, 1H, J=3.3 Hz, H-1), 4.33 (dd, 1H, J=10.2, 4.8 Hz, H-6),4.13-4.05 (m, 2H, H-3, H-5), 3.66 (dd, 1H, J=10.2 Hz, H-6'), 3.44(dd, 1H, J=9.9, 8.9 Hz, H-4), 3.08 (dd, 1H, J=10.1, 4.9 Hz, H-2);`Ring II`: .delta.H 3.95 (dd, 1H, J=11.1, 6.6 Hz, H-5), 3.67 (dd,1H, J=11.1, 6.2 Hz, H-4), 3.49-3.40 (m, 2H, H-1, H-3), 3.30 (dd,1H, J=9.5 Hz, H-6), 2.25 (dt, 1H, J=12.6, 4.3 Hz, H-2eq), 1.41(ddd, 1H, J=12.9 Hz, H-2ax); `Ring III`: .delta.H=5.67 (d, 1H,J=4.9 Hz, H-1), 4.32-4.28 (m, 2H, H-3, H-4), 3.96 (dd, 1H, J=9.3,3.9 Hz, H-2), 3.83 (dd, 1H, J=10.8, 2.2 Hz, H-5), 3.58 (dd, 1H,J=10.8, 3.0 Hz, H-5'); `Ring IV`: .delta.H=4.88 (d, 1H, J=1.5 Hz,H-1), 3.79-3.76 (m, 1H, H-5), 3.69-3.63 (m, 2H, H-3, H-6), 3.35(dd, 1H, J=2.7, 1.2 Hz, H-2), 3.13 (dd, 1H, J=3.7, 1.3 Hz, H-4),2.91-2.87 (m, 1H, H-6'); The additional peaks in the spectrum wereidentified as follow: 7.51-7.13 (m, 35H, Ar), 5.51 (s, 1H, PhCH),4.96 (d, 1H, J=10.7, PhCH.sub.2), 4.91 (d, 1H, J=11.3, PhCH.sub.2),4.78 (d, 1H, J=11.3, PhCH.sub.2), 4.73 (d, 1H, J=10.7, PhCH.sub.2),4.63 (d, 1H, J=12.0, PhCH.sub.2), 4.60 (d, 2H, J=11.2, PhCH.sub.2),4.54 (d, 1H, J=12.8, PhCH.sub.2), 4.47 (d, 1H, J=11.8, PhCH.sub.2),4.45 (d, 1H, J=11.8, PhCH.sub.2), 4.42 (d, 1H, J=12.0, PhCH.sub.2),4.27 (d, 1H, J=12.0, PhCH.sub.2).

[0550] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.36 (Ar),138.10 (Ar), 137.90 (Ar), 137.60 (Ar), 137.43 (Ar), 137.04 (Ar),136.96 (Ar), 128.69-126.11 (Ar), 106.25 (C1''), 101.39 (PhCH),98.60 (C-1'''), 96.50 (C-1'), 84.14 (C-6), 82.42 (C-2''), 82.13(C-4'), 82.11 (C-4''), 81.85 (C-5), 75.98 (C-3'), 75.51 (C-3''),75.42 (C-4), 75.03, 74.90, 74.39 (C-5'''), 73.26 (PhCH.sub.2),73.20 (PhCH.sub.2), 72.96 (C-3'''), 72.42 (PhCH.sub.2), 71.77(PhCH.sub.2), 71.60 (C-4'''), 70.37 (C-5''), 69.0 (C-6'), 62.97(C-5'), 62.87 (C-2'), 60.34 (C-1), 59.87 (C-3), 57.31 (C-2'''),51.14 (C-6'''), 32.39 (C-2).

[0551] TOFMS calcd for C.sub.72H.sub.75N.sub.15O.sub.14Na ([M+Na]+)m/e 1396.55; measured m/e 1396.55).

Preparation of2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6'''-pentaazido-paromomy-cin (22)

[0552] The titled compound was prepared as was described for thepreparation of compound 14 with the following quantities: compound21 (36 grams, 26.20 mmol), Acetic acid (240 mL), Water (50 mL) toyield 22 (20.5 grams, 61%).

[0553] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.27(d, 1H, J=3.1 Hz, H-1), 4.00-3.93 (m, 2H, H-3, H-5), 3.87 (ddd, 1H,J=10.8, 2.7, 1.5 Hz, H-6), 3.83-3.77 (m, 1H, H-6'), 3.51-3.45 (m,1H, H-4), 2.95 (dd, 1H, J=10.4, 5.0 Hz, H-2), 2.29-2.27, (m, 1H,OH-6), 1.72-1.67 (m, 1H, OH-4); `Ring II`: .delta.H=4.06 (dd, 1H,J=8.8 Hz, H-5), 3.72 (dd, 1H, J=9.2 Hz, H-4), 3.57-3.49 (m, 2H,H-1, H-3), 3.39 (dd, 1H, J=9.4 Hz, H-6), 2.32 (dt, 1H, J=13.3, 4.5Hz, H-2eq), 1.48 (q, 1H, J=12.7 Hz, H-2ax); `Ring III`:.delta.H=5.79 (d, 1H, J=5.1 Hz, H-1), 4.42-4.40 (m, 2H, H-3, H-4),4.08 (dd, 1H, J=6.4, 5.1 Hz, H-2), 3.93 (dd, 1H, J=9.9, 0.9 Hz,H-5), 3.68 (dd, 1H, J=9.4, 1.0 Hz, H-5'); `Ring IV`: .delta.H 5.00(d, 1H, J=2.5 Hz, H-1), 3.90-3.84 (m, 2H, H-3, H-5), 3.76 (dd, 1H,J=12.8, 8.1 Hz, H-6), 3.46-3.44 (m, 1H, H-2), 3.22-3.21 (m, 1H,H-4), 2.99-2.94 (m, 1H, H-6'); the additional peaks in the spectrumwere identified as follow: 7.47-7.23 (m, 30H, Ar), 5.08 (d, 1H,J=10.6 Hz, PhCH.sub.2), 5.01 (d, 1H, J=14.7 Hz, PhCH.sub.2), 4.81(d, 1H, J=10.6 Hz, PhCH.sub.2), 4.76 (d, 1H, J=11.4 Hz,PhCH.sub.2), 4.71 (d, 1H, J=12.1 Hz, PhCH.sub.2), 4.68 (d, 1H,J=11.8 Hz, PhCH.sub.2), 4.58-4.55 (m, 2H, PhCH.sub.2), 4.50 (d, 1H,J=12.0 Hz, PhCH.sub.2), 4.42-4.39 (m, 2H, PhCH.sub.2), 4.34 (d, 1H,J=12.1 Hz, PhCH.sub.2).

[0554] .sup.13C NMR (151 MHz, CDCl.sub.3): .delta.C=138.39 (Ar),138.19 (Ar), 138.02 (Ar), 137.62 (Ar), 137.08 (Ar), 137.02 (Ar),128.80 (Ar), 128.79 (Ar), 128.63 (Ar), 128.54 (Ar), 128.46 (Ar),128.41 (Ar), 128.38 (Ar), 128.33 (Ar), 128.25 (Ar), 128.21 (Ar),127.95 (Ar), 127.93 (Ar), 127.90 (Ar), 127.84 (Ar), 127.68 (Ar),127.61 (Ar), 127.23 (Ar), 106.23 (C-1''), 98.78 (C-1'''), 96.03(C-1'), 84.41 (C-6), 82.67 (C-2''), 82.29 (C-4''), 82.11 (C-5),79.77 (C-3'), 75.61 (C-3''), 75.19 (PhCH.sub.2), 75.07(PhCH.sub.2), 74.94 (C-4), 74.56 (C-5'''), 73.34 (PhCH.sub.2),73.28 (PhCH.sub.2), 72.92 (C-3'''), 72.48 (PhCH.sub.2), 71.70(C-5'), 71.55 (C-4'''), 70.50 (C-4'), 70.39 (C-5''), 62.74 (C-6'),62.19 (C-2'), 60.47 (C-1), 60.37 (C-3), 57.35 (C-2'''), 51.26(C-6'''), 32.66 (C-2).

[0555] TOFMS calcd for C.sub.65H.sub.71N.sub.15O.sub.14Na ([M+Na]+)m/e 1308.52; measured m/e 1308.00).

Preparation of2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6',6'''-hexaazido-neomyc-in (23)

[0556] The titled compound was prepared as was described for thepreparation of compound 15 with the following quantities: compound22 (26.3 grams, 20.45 mmol), pyr (130 mL), Trisyl chloride (insteadof tosyl chloride, 31.5 grams, 104.00 mmol), NaN.sub.3 (9.45 grams,145.38 mmol), DMF (100 mL), HMPA (30 mL) to yield 23 (16 grams,60%). 1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.21 (d, 1H,J=2.4 Hz, H-1), 4.04 (dd, 1H, J=9.4, 3.9 Hz, H-5), 3.84 (t, 1H,J=9.6 Hz, H-3), 3.49 (dd, 1H, J=13.6, 1.8 Hz, H-6), 3.33 (dd, 1H,J=13.2, 6.5 Hz, H-6'), 3.26-3.20 (m, 1H, H-4), 2.90 (dd, 1H,J=10.4, 5.3 Hz, H-2); `Ring II`: .delta.H=3.97 (t, 1H, J=9.0 Hz,H-5), 3.69 (t, 1H, J=9.3 Hz, H-4), 3.55-3.39 (m, 2H, H-1, H-3),3.31 (t, 1H, J=9.0 Hz, H-6), 2.25 (dt, 1H, J=13.2, 4.5 Hz, H-2eq),1.43 (ddd, 1H, J=12.7 Hz, H-2ax); `Ring III`: .delta.H=5.69 (d, 1H,J=5.2 Hz, H-1), 4.30-4.25 (m, 2H, H-3, H-4), 3.99-3.91 (m, 1H,H-2), 3.84-3.80 (m, 1H, H-5), 3.57 (dd, 1H, J=10.4, 2.8 Hz, H-5');`Ring IV`: .delta.H=4.93 (d, 1H, J=2.5 Hz, H-1), 3.83-3.71 (m, 2H,H-3, H-5), 3.70-3.62 (m, 1H, J=12.8, 8.1 Hz, H-6), 3.36 (s, 1H,H-2), 3.14-3.12 (m, 1H, H-4), 2.89-2.86 (m, 1H, H-6'); theadditional peaks in the spectrum were identified as follow:7.42-7.12 (m, 30H, Ar), 4.72 (d, 1H, J=10.6 Hz, PhCH.sub.2), 4.63(d, 1H, J=6.1 Hz, PhCH.sub.2), 4.61 (d, 2H, J=5.7 Hz, PhCH.sub.2),4.52 (d, 1H, J=11.9 Hz, PhCH.sub.2), 4.47 (dd, 2H, J=11.8, 5.2 Hz,PhCH.sub.2), 4.42 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.32 (d, 1H,J=12.0 Hz, PhCH.sub.2), 4.30-4.23 (m, 3H, PhCH.sub.2).

[0557] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.21 (Ar),137.88 (Ar), 137.59 (Ar), 136.96 (Ar), 136.90 (Ar), 128.77 (Ar),128.68 (Ar), 128.50 (Ar), 128.44 (Ar), 128.38 (Ar), 128.33 (Ar),128.25 (Ar), 128.19 (Ar), 128.16 (Ar), 127.82 (Ar), 127.78 (Ar),127.75 (Ar), 127.46 (Ar), 127.27 (Ar), 106.00 (C-1''), 98.59(C-1'''), 95.75 (C-1'), 84.35 (C-6), 82.56 (C-2''), 82.07 (C-4''),81.70 (C-5), 79.74 (C-3'), 75.49 (C-3''), 75.09 (PhCH.sub.2), 74.89(PhCH.sub.2), 74.42 (C-4), 73.34 (C-5'''), 73.21 (PhCH.sub.2),72.70 (PhCH.sub.2), 72.31 (C-3'''), 71.62 (PhCH.sub.2), 71.32(C-5'), 71.19 (C-4'''), 70.93 (C-4'), 70.19 (C-5''), 62.53(C-2'''), 60.34 (C-2'), 60.13 (C-1), 57.18 (C-3), 51.55 (C-6'),51.06 (C-6'''), 32.56 (C-2).

[0558] TOFMS calcd for C.sub.65H.sub.70N.sub.18O.sub.13Na ([M+Na]+)m/e 1333.54; measured m/e 1333.53).

Preparation of4'-O-Allyl-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6',6'''-hexaa-zido-neomycin (24)

[0559] The titled compound was prepared as was described for thepreparation of compound 16 with the following quantities: compound23 (11 grams, 8.393 mmol), DMF (100 mL), NaH (60% in oil, 1 gram,25.180 mmol), Allyl bromide (1.45 mL, 16.786 mL), TBAI (10 grams,27.073 mmol) to yield 24 (10.5 grams, 92.6%).

[0560] .sup.1H NMR (600 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.14(d, 1H, J=3.1 Hz, H-1), 4.12-4.06 (m, 1H, H-5), 3.95 (dd, 1H,J=10.0, 9.6 Hz, H-3), 3.50 (d, 1H, J=12.8 Hz, H-6), 3.33 (dd, 1H,J=13.3, 6.0 Hz, H-6), 3.13 (dd, 1H, J=9.9, 9.2 Hz, H-4), 2.97 (dd,1H, J=10.6, 5.0 Hz, H-2)); `Ring II`: .delta.H=3.94 (dd, 1H, J=8.4Hz, H-5), 3.66 (dd, 1H, J=9.2 Hz, H-4), 3.49-3.38 (m, 2H, H-1,H-3), 3.31 (dd, 1H, J=9.5, 8.6 Hz, H-6), 2.25 (dt, 1H, J=9.0, 5.0Hz, H-2eq), 1.43 (ddd, 1H, J=12.8 Hz, H-2ax); `Ring III`:.delta.H=5.66 (d, 1H, J=5.0 Hz, H-1), 4.28 (dt, 1H, J=3.0, 2.6 Hz,H-4), 4.25 (dd, 1H, J=4.1, 3.4 Hz, H-3), 3.95 (dd, 1H, J=6.0, 5.4Hz, H-2), 3.79 (dd, 1H, J=9.8, 1.4 Hz, H-5), 3.56 (dt, 1H, J=6.3,2.7 Hz, H-5'); `Ring IV`: .delta.H 4.90 (d, 1H, J=1.7 Hz, H-1),3.79-3.74 (m, 2H, H-3, H-5), 3.63 (dd, 1H, J=12.9, 9.0 Hz, H-6),3.35 (dd, 1H, J=7.1, 2.0 Hz, H-2), 3.12 (dd, 1H, J=2.6, 1.3 Hz,H-4), 2.92-2.84 (m, 1H, H-6'); The additional peaks in the spectrumwere identified as follow: 7.46-7.07 (m, 30H, Ar), 5.85 (m, 1H,Allyl), 5.24 (d, 1H, J=17.2 Hz, Allyl), 5.16 (d, 1H, J=10.6 Hz,Allyl), 4.95 (d, 1H, J=10.7 Hz, PhCH.sub.2), 4.81 (d, 1H, J=10.8Hz, PhCH.sub.2), 4.78 (d, 1H, J=10.9 Hz, PhCH.sub.2), 4.71 (d, 1H,J=10.6 Hz, PhCH.sub.2), 4.62 (d, 1H, J=12.2 Hz, PhCH.sub.2), 4.59(d, 1H, J=12.1 Hz, PhCH.sub.2), 4.53 (d, 1H, J=12.0 Hz,PhCH.sub.2), 4.45 (d, 2H, J=11.5 Hz, PhCH.sub.2), 4.42 (d, 1H,J=12.2 Hz, PhCH.sub.2), 4.32 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.27(m, 2H, PhCH.sub.2, Allyl), 4.05 (dd, 1H, J=12.3, 5.7 Hz,Allyl).

[0561] .sup.13C NMR (151 MHz, CDCl.sub.3): .delta.C=138.40 (Ar),138.06 (Ar), 138.01 (Ar), 137.77 (Ar), 137.13 (Ar), 137.06 (Ar),134.49 (Allyl), 128.79 (Ar), 128.61 (Ar), 128.53 (Ar), 128.50 (Ar),128.49 (Ar), 128.44 (Ar), 128.38 (Ar), 128.29 (Ar), 128.22 (Ar),127.94 (Ar), 127.90 (Ar), 127.86 (Ar), 127.58 (Ar), 127.55 (Ar),117.27 (Allyl), 106.27 (C-1''), 98.70 (C-1'''), 95.63 (C-1'), 84.34(C-6), 82.58 (C-2''), 82.16 (C-4''), 81.80 (C-5), 79.83 (C-3'),78.66, 75.65 (C-3''), 75.41 (PhCH.sub.2), 75.19 (PhCH.sub.2), 74.46(C-4), 73.82 (Allyl), 73.39 (C-5'''), 73.42 (PhCH.sub.2), 72.98(PhCH.sub.2), 72.48 (C-3'''), 71.93 (PhCH.sub.2), 71.82 (C-5'),71.58 (C-4'''), 71.07 (C-4'), 70.30 (C-5''), 63.13 (C-2'''), 60.48(C-2'), 60.09 (C-1), 57.37 (C-3), 51.49 (C-6'), 51.17 (C-6'''),32.60 (C-2).

[0562] TOFMS calcd for C.sub.68H.sub.74N.sub.18O.sub.13Na ([M+Na]+)m/e 1373.56; measured m/e 1373.43).

Preparation of4'-O-(2,3-dihydroxypropyl)-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2'''-,3,6',6'''-hexaazido-neomycin (25)

[0563] The titled compound was prepared as was described for thepreparation of compound 17 with the following quantities: compound24 (0.5 gram, 0.370 mmol), (3.4 mL), water (0.3 mL),4-methylmorpholine N-oxide (0.2 mL, 2 eq., 50% wt. in H.sub.2O),osmium tetroxide (2 mg, 0.008 mmol, 0.02 eq.) to yield 25 (0.45gram, 89%).

[0564] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.17(d, 1H, J=3.3 Hz, H-1), 4.07-4.02 (m, 1H, H-5), 3.93 (dd, 1H,J=12.3, 7.1 Hz, H-3), 3.52 (dd, 1H, J=12.9, 1.3 Hz, H-6), 3.36 (dd,1H, J=12.9, 5.4 Hz, H-6'), 3.16 (dd, 1H, J=18.0, 9.1 Hz, H-4), 2.91(dd, 1H, J=12.0, 6.0 Hz, H-2); `Ring II`: .delta.H=3.95 (dd, 1H,J=9.8, 8.8 Hz, H-5), 3.65 (dd, 1H, J=9.2 Hz, H-4), 3.50-3.41 (m,2H, H-1, H-3), 3.31 (dd, 1H, J=10.4, 9.2 Hz, H-6), 2.25 (dt, 1H,J=13.0, 4.4 Hz, H-2eq), 1.42 (ddd, 1H, J=12.9 Hz, H-2ax); `RingIII`: .delta.H=5.66 (d, 1H, J=4.7 Hz, H-1), 4.29-4.25 (m, 2H, H-3,H-4), 3.95 (dd, 1H, J=5.5 Hz, H-2), 3.80 (dd, 1H, J=10.0, 1.4 Hz,H-5), 3.56 (dd, 1H, J=10.2, 2.6 Hz, H-5'); `Ring IV`: .delta.H=4.91(d, 1H, J=2.0 Hz, H-1), 3.82-3.74 (m, 2H, H-3, H-5), 3.65 (dd, 1H,J=12.3, 8.7 Hz, H-6), 3.36 (dd, 1H, J=2.7, 1.2 Hz, H-2), 3.13 (dd,1H, J=2.5, 1.8 Hz, H-4), 2.92 (dd, 1H, J=9.9, 2.9 Hz, H-6'); theadditional peaks in the spectrum were identified as follow:7.45-7.10 (m, 30H, Ar), 4.95 (d, 1H, J=10.5 Hz, PhCH.sub.2), 4.87(d, 1H, J=10.7 Hz, PhCH.sub.2), 4.73 (d, 1H, J=6.7 Hz, PhCH.sub.2),4.71 (d, 1H, J=6.8 Hz, PhCH.sub.2), 4.62 (d, 1H, J=12.0 Hz,PhCH.sub.2), 4.59 (d, 1H, J=11.8 Hz, PhCH.sub.2), 4.52 (dd, 1H,J=11.9, 3.1 Hz, PhCH.sub.2), 4.48-4.40 (m, 3H, PhCH.sub.2), 4.32(d, 1H, J=12.0 Hz, PhCH.sub.2), 4.25 (d, 1H, J=12.1 Hz,PhCH.sub.2), 3.76-3.62 (m, 3H, Diol), 3.62-3.55 (m, 1H, Diol),3.52-3.46 (m, 1H, Diol), 3.01 (d, 1H, J=2.8 Hz, OH), 1.95-1.91 (m,1H, OH).

[0565] .sup.13C NMR (126 MHz, CDCl.sub.3): .delta.C=138.39 (Ar),138.37 (Ar), 137.99 (Ar), 137.79 (Ar), 137.75 (Ar), 137.53 (Ar),137.14 (Ar), 137.07 (Ar), 128.83 (Ar), 128.65 (Ar), 128.57 (Ar),128.49 (Ar), 128.43 (Ar), 128.35 (Ar), 128.24 (Ar), 127.98 (Ar),127.91 (Ar), 127.73 (Ar), 127.70 (Ar), 127.68 (Ar), 127.65 (Ar),127.61 (Ar), 106.34 (C-1''), 98.75 (C-1'''), 95.79 (C-1'), 84.38(C-6), 82.62 (C-5), 82.21 (C-2''), 81.88 (C-4''), 79.77, 79.65(C-4'), 79.53 (C-3'), 75.67 (Diol), 75.59 (C-4), 75.35 (Diol),75.26 (C-3''), 74.87, 74.54, 74.36 (C-5'''), 73.45, 73.00 (C-3'''),72.53, 71.86, 71.61, 71.36 (C-4'''), 71.21, 71.06 (C-5'), 70.34(C-5''), 63.60 (Diol), 63.25 (C-2'), 60.49 (C-1), 60.19 (C-3),57.40 (C-2'''), 51.39 (C-6'''), 51.24 (C-6'), 32.64 (C-2).

[0566] TOFMS calcd for C.sub.68H.sub.76N.sub.18O.sub.15Na ([M+Na]+)m/e 1407.57; measured m/e 1407.51).

Preparation of4'-O-(2-aminoethylazido)ethyl-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2-''',3,6',6'''-hexaazido-neomycin (26)

[0567] The titled compound was prepared as was described for thepreparation of compound 18 with the following quantities: compound25 (700 mg, 0.505 mmol), DCM (30 mL), PhI(OAc).sub.2 (195 mg, 0.605mmol, 1.2 eq.), 2-azidoethanamine (0.11 ml, 1.3 mmol, 2.6 eq),triacetoxyborohydride (300 mg, 1.42 mmol, 2.8 eq.) to yield 26 (450mg, 62%).

[0568] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.14(d, 1H, J=3.9 Hz, H-1), 4.10-4.03 (m, 1H, H-5), 3.92 (dd, 1H,J=10.3, 9.4 Hz, H-3), 3.52 (dd, 1H, J=12.8, 1.1 Hz, H-6), 3.37 (dd,1H, J=13.2, 2.0 Hz, H-6'), 3.10 (dd, 1H, J=10.0, 9.2 Hz, H-4), 2.96(dd, 1H, J=11.0, 2.7 Hz, H-2); `Ring II`: .delta.H=3.92 (dd, 1H,J=9.0 Hz, H-5), 3.63 (dd, 1H, J=9.2 Hz, H-4), 3.48-3.37 (m, 2H,H-1, H-3), 3.28 (dd, 1H, J=8.9 Hz, H-6), 2.26 (dt, 1H, J=13.2, 4.5Hz, H-2eq), 1.41 (ddd, 1H, J=13.0 Hz, H-2ax); `Ring III`:.delta.H=5.66 (d, 1H, J=5.0 Hz, H-1), 4.33-4.23 (m, 2H, H-3, H-4),3.95 (t, 1H, J=5.9 Hz, H-2), 3.79 (dd, 1H, J=10.4, 1.7 Hz, H-5),3.56 (dd, 1H, J=10.5, 2.5 Hz, H-5'); `Ring IV`: .delta.H=4.90 (d,1H, J=1.2 Hz, H-1), 3.79-3.75 (m, 2H, H-3, H-5), 3.64 (dd, 1H,J=12.6, 9.1 Hz, H-6), 3.35 (d, 1H, J=4.6 Hz, H-2), 3.12 (s, 1H,H-4), 2.89 (dd, 1H, J=13.0, 4.0 Hz, H-6'); the additional peaks inthe spectrum were identified as follow: .delta. 7.42-7.14 (m, 30H,Ar), 4.95 (d, 1H, J=10.6 Hz, PhCH.sub.2), 4.83 (d, 1H, J=10.9 Hz,PhCH.sub.2), 4.78 (d, 1H, J=10.8 Hz, PhCH.sub.2), 4.72 (d, 1H,J=10.7 Hz, PhCH.sub.2), 4.62 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.59(d, 1H, J=11.8 Hz, PhCH.sub.2), 4.53 (d, 1H, J=11.9 Hz,PhCH.sub.2), 4.45 (d, 2H, J=11.8 Hz, PhCH.sub.2), 4.42 (d, 1H,J=11.9 Hz, PhCH.sub.2), 4.32 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.25(d, 1H, J=12.2 Hz, PhCH.sub.2), 3.88-3.83 (m, 1H, CH.sub.2),3.63-3.59 (m, 1H, CH.sub.2), 3.35 (t, 2H, J=5.6 Hz, CH.sub.2),2.78-2.69 (m, 4H, CH.sub.2).

[0569] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.44 (Ar),138.13 (Ar), 138.04 (Ar), 137.81 (Ar), 137.17 (Ar), 137.10 (Ar),131.03 (Ar), 128.95 (Ar), 128.83 (Ar), 128.65 (Ar), 128.57 (Ar),128.54 (Ar), 128.49 (Ar), 128.43 (Ar), 128.33 (Ar), 128.11 (Ar),127.98 (Ar), 127.94 (Ar), 127.91 (Ar), 127.64 (Ar), 127.60 (Ar),106.31 (C-1''), 98.75 (C-1'''), 95.76 (C-1'), 84.36 (C-6), 82.61(C-5), 82.21 (C-2''), 81.85 (C-4''), 79.79 (C-3'), 79.36 (C-4'),75.69, 75.29, 75.27 (C-3''), 75.23, 74.51 (C-5'''), 73.43(CH.sub.2), 73.03 (C-3'''), 72.53 (CH.sub.2), 71.87, 71.63(C-4'''), 71.14 (C-5'), 70.36 (C-5''), 68.31, 63.22 (C-2'), 60.52(C-1), 60.14 (C-3), 57.42 (C-2'''), 51.51 (C-6'''), 51.22 (C-6'),49.52 (CH.sub.2), 48.66 (CH.sub.2), 38.88, 32.63 (C-2), 30.51,29.85, 23.89, 23.13.

[0570] TOFMS calcd for C.sub.69H.sub.80N.sub.20O.sub.13K ([M+K]+)m/e 1461.58; measured m/e 1461.51).

Preparation of 4'-O-(2-aminoethylamino)ethyl-neomycin (Compound 2;FIGS. 1 and 2)

[0571] The titled compound was prepared as was described for thepreparation of compound 1 with the following quantities: Staudingerreaction: compound 26 (400 mg, 0.281 mmol), THF (10 mL), aqueousNaOH (0.1M, 5 mL), PMe.sub.3 (1M solution in THF, 6.7 mL, 6.7mmol). Birch reduction: THF (10 mL), ammonia (about 20 mL), Na (150mg, 7 mmol), ammonium formate (1 gran, 15.7 mmol). The analyticallypure product was obtained by passing the above product through ashort column of Amberlite CG50 (NH4.sup.+ form). The column wasfirst washed with MeOH and H.sub.2O, then the product was elutedwith a mixture of H.sub.2O/NH.sub.4OH (95:5) to afford compound 2(108 mg, 55% for two steps). For the storage and biological tests,compound 2 was converted to its sulfate salt form: the free basewas dissolved in water, the pH was adjusted around 7.0 withH.sub.2SO.sub.4 (0.1 N) and lyophilized.

[0572] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=5.47 (d, 1H,J=3.7 Hz, H-1), 3.74-3.69 (m, 1H, H-5), 3.67 (dd, 1H, J=10.5, 9.1Hz, H-3), 3.09 (dd, 1H, J=11.4, 1.3 Hz, H-6), 3.04 (dd, 1H, J=12.7,1.8 Hz, H-6'), 2.75 (dd, 1H, J=9.7, 9.2 Hz, H-4), 2.71 (dd, 1H,J=10.5, 2.9 Hz, H-2); `Ring II`: .delta.H=3.52 (dd, 1H, J=8.7 Hz,H-5), 3.45 (dd, 1H, J=8.5 Hz, H-4), 3.18-2.96 (m, 2H, H-1, H-3),2.75 (dd, 1H, J=10.9, 8.4 Hz, H-6), 2.01-1.95 (m, 1H, H-2eq), 1.23(ddd, 1H, J=10.9, 2.2 Hz, H-2ax); `Ring III`: .delta.H=5.32 (d, 1H,J=3.2 Hz, H-1), 4.41 (dd, 1H, J=5.7, 4.8 Hz, H-3), 4.17 (dd, 1H,J=5.7, 2.3 Hz, H-2), 4.02 (dd, 1H, J=8.6, 3.4 Hz, H-4), 3.84 (dd,1H, J=10.9, 2.6 Hz, H-5), 3.68 (dd, 1H, J=11.2, 3.4 Hz, H-5');`Ring IV`: .delta.H=4.90 (d, 1H, J=1.1 Hz, H-1), 3.92-3.86 (m, 2H,H-3, H-5), 3.44 (dd, 1H, J=5.1, 3.6 Hz, H-4), 3.36 (dd, 1H, J=10.9,8.7 Hz, H-6), 3.01 (dd, 1H, J=3.5, 1.1 Hz, H-2), 2.89 (dd, 1H,J=12.7, 4.1 Hz, H-6'); The additional peaks in the spectrum wereidentified as follow: 3.62-3.58 (m, 2H, CH.sub.2), 2.98-2.95 (m,2H, CH.sub.2), 2.78-2.75 (m, 1H, CH.sub.2), 2.71-2.65 (m, 3H,CH.sub.2).

[0573] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=108.56 (C-1''),101.68 (C-1'''), 99.86 (C-1'), 86.01 (C-6), 83.89 (C-5), 83.41(C-2''), 82.12 (C-4''), 79.16 (C-3'), 77.34 (C-4'), 75.65 (C-3''),75.29, 74.41 (C-5'''), 73.70 (CH.sub.2), 72.36 (C-3'''), 72.1(CH.sub.2), 71.08 (C-5'), 70.81 (C-4'''), 61.93 (C-5''), 57.55(C-2'), 54.73 (C-1), 52.37 (C-3), 51.29 (C-2'''), 50.52 (C-6'''),43.57 (C-6'), 43.29 (CH.sub.2), 42.81 (CH.sub.2), 37.31, 35.78(C-2), 28.59.

[0574] TOFMS calcd for C.sub.27H.sub.56N.sub.8O.sub.13Na ([M+Na]+)m/e 723.40; measured m/e 723.63).

Preparation of4'-O-(2-((benzyloxycarbonyl)(methyl)amino)ethylamino)ethyl-2'',3',3''',4'-'',5'',6-hexa-O-benzyl-1,2',2''',3,6',6'''-hexaazido-neomycin(27)

[0575] The titled compound was prepared as was described for thepreparation of compound 18 with the following quantities: compound25 (1 gram, 0.721 mmol), DCM (50 mL), PhI(OAc).sub.2 (280 mg, 0.870mmol, 1.2 eq.), N-Cbz-N-Methylethylenediamine (390 mg, 1.88 mmol,2.6 equiv), sodium triacetoxyborohydride (430 mg, 2.03 mmol, 2.8equiv). The residue was purified by column chromatography(EtOAc/hexane, 55:45) to afford compound 27 (590 mg, 58%).

[0576] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=5.48(d, 1H, J=3.7 Hz, H-1), 3.84 (dd, 1H, J=8.7, 3.6 Hz, H-5), 3.61(dd, 1H, J=10.8, 6.7 Hz, H-3), 3.24 (dd, 1H, J=6.5, 2.3 Hz, H-4),2.94 (dd, 1H, J=10.9, 2.7 Hz, H-6), 2.75 (dd, 1H, J=11.5, 2.9 Hz,H-6'), 2.43 (dd, 1H, J=11.2, 3.9 Hz, H-2); `Ring II`: .delta.H=3.94(dd, 1H, J=6.8, 2.2 Hz, H-5), 3.68 (dd, 1H, J=10.7, 7.2 Hz, H-4),3.35 (dd, 1H, J=11.2, 6.7 Hz, H-6), 2.98-2.84 (m, 2H, H-1, H-3),1.91 (dt, 1H, J=10.8, 1.6 Hz, H-2eq), 1.34 (ddd, 1H, J=11.9, 1.9Hz, H-2ax); `Ring III`: .delta.H=5.69 (d, 1H, J=3.1 Hz, H-1), 4.01(dd, 1H, J=5.7, 4.9 Hz, H-3), 3.92 (dd, 1H, J=5.2, 2.3 Hz, H-2),3.78 (dd, 1H, J=8.7, 4.1 Hz, H-4), 3.65 (dd, 1H, J=12.4, 3.7 Hz,H-5), 3.51 (dd, 1H, J=12.2, 3.5 Hz, H-5'); `Ring IV`: .delta.H=5.23(d, 1H, J=1.3 Hz, H-1), 3.86-3.79 (m, 2H, H-3, H-5), 3.58 (dd, 1H,J=5.2, 3.7 Hz, H-4), 3.49 (dd, 1H, J=12.3, 8.7 Hz, H-6), 3.16 (dd,1H, J=3.1, 1.5 Hz, H-2), 2.98 (dd, 1H, J=12.4, 8.2 Hz, H-6'); Theadditional peaks in the spectrum were identified as follow:7.54-7.28 (m, 35H, Ar), 4.93 (m, 2H, CH.sub.2 of Cbz), 4.87 (d, 1H,J=10.8 Hz, PhCH.sub.2), 4.81 (d, 1H, J=10.9 Hz, PhCH.sub.2), 4.76(d, 1H, J=10.8 Hz, P PhCH.sub.2), 4.69 (d, 1H, J=10.7 Hz,PhCH.sub.2), 4.62 (d, 1H, J=11.8 Hz, PhCH.sub.2), 4.53 (d, 1H,J=11.9 Hz, PhCH.sub.2), 4.47 (d, 1H, J=11.9 Hz, PhCH.sub.2), 4.34(d, 2H, J=11.8 Hz, PhCH.sub.2), 4.30 (d, 1H, J=11.7 Hz,PhCH.sub.2), 4.27 (d, 1H, J=12.1 Hz, Ph CH.sub.2), 4.22 (d, 1H,J=12.0 Hz, PhCH.sub.2), 3.36 (m, 2H, CH.sub.2), 3.17 (m, 3H,CH.sub.3), 3.08 (m, 2H, CH.sub.2), 2.82-2.76 (m, 4H, CH.sub.2).

[0577] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=139.48 (Ar),138.53 (Ar), 138.14 (Ar), 137.61 (Ar), 137.36 (Ar), 137.08 (Ar),134.09 (Ar), 129.96 (Ar), 128.83 (Ar), 128.54 (Ar), 128.37 (Ar),128.24 (Ar), 128.19 (Ar), 128.03 (Ar), 127.33 (Ar), 127.11 (Ar),125.98 (Ar), 125.74 (Ar), 125.51 (Ar), 124.64 (Ar), 124.58 (Ar),108.79 (C-1''), 102.45 (C-1'''), 99.76 (C-1'), 85.26 (C-6), 82.94(C-5), 82.67 (C-2''), 81.97 (C-4''), 79.34 (C-3'), 78.75 (C-4'),76.18 (CH.sub.2), 75.43 (C-3''), 74.84 (C-5'''), 73.58 (CH.sub.2),72.67 (C-3'''), 71.89 (C-5'), 71.26 (C-4'''), 67.85 (C-5''), 66.37(CH.sub.2 of Cbz), 62.74 (C-2'), 57.56 (C-1), 56.84 (C-3), 53.51(C-T''), 51.26 (C-6'''), 49.78 (CH.sub.2), 48.59 (C-6'), 46.54(CH.sub.2), 37.34 (CH.sub.3), 34.68 (C-2), 30.21.

[0578] TOFMS calcd for C.sub.78H.sub.88N.sub.20O.sub.15Na ([M+Na]+)m/e 1567.68; measured m/e 1567.75).

Preparation of 4'-O-(2-(methylamino)ethylamino)ethyl-neomycin(Compound 3; FIGS. 1 and 2B)

[0579] The titled compound was prepared as was described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: compound 27 (350 mg, 0.226 mmol), THF (10 mL), NaOH(0.1M, 5 mL), PMe.sub.3 (1M solution in THF, 6.5 mL, 62.77 mmol) toyield the compound as free amine form. Birch reduction: THF (10mL), ammonia (about 20 mL), small pieces of Na (120 mg, 5.6 mmol),ammonium formate (1 gram, 15.7 mmol). The analytically pure productwas obtained by passing the above product through a short column ofAmberlite CG50 (NH.sub.4+ form). The column was first washed withMeOH and H.sub.2O, then the product was eluted with a mixture ofH.sub.2O/NH.sub.4OH (93:7) to afford Compound 3 (100 mg, 62% fortwo steps).

[0580] For the storage and biological tests, compound was convertedto its sulfate salt form: the free base was dissolved in water, thepH was adjusted around 7.0 with H.sub.2SO.sub.4 (0.1 N) andlyophilized.

[0581] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=5.40 (d, 1H,J=3.4 Hz, H-1), 3.72 (dd, 1H, J=9.2, 3.8 Hz, H-5), 3.66 (dd, 1H,J=11.2, 7.1 Hz, H-3), 3.05 (dd, 1H, J=7.9, 2.1 Hz, H-4), 3.01 (dd,1H, J=12.4, 2.7 Hz, H-6), 2.79 (dd, 1H, J=12.5, 6.9 Hz, H-6'), 2.67(dd, 1H, J=10.4, 3.8 Hz, H-2); `Ring II`: .delta.H=3.52 (dd, 1H,J=7.5, 1.6 Hz, H-5), 3.41 (dd, 1H, J=10.2, 8.1 Hz, H-4), 3.20 (dd,1H, J=11.3, 7.7 Hz, H-6), 2.78-2.54 (m, 2H, H-1, H-3), 1.99-1.93(m, 1H, H-2eq), 1.20 (ddd, 1H, J=11.9, 1.9 Hz, H-2ax); `Ring III`:.delta.H=5.29 (d, 1H, J=2.7 Hz, H-1), 4.37 (dd, 1H, J=6.3, 5.1 Hz,H-3), 4.14 (dd, 1H, J=5.2, 2.1 Hz, H-2), 4.05 (dd, 1H, J=9.0, 3.9Hz, H-4), 3.79 (dd, 1H, J=12.2, 2.2 Hz, H-5), 3.71 (dd, 1H, J=12.2,3.5 Hz, H-5'); `Ring IV`: .delta.H=4.92 (d, 1H, J=1.0 Hz, H-1),3.96-3.90 (m, 2H, H-3, H-5), 3.48 (dd, 1H, J=4.8, 3.3 Hz, H-4),3.32 (dd, 1H, J=11.9, 8.9 Hz, H-6), 2.96 (dd, 1H, J=3.2, 1.3 Hz,H-2), 2.85 (dd, 1H, J=12.4, 8.2 Hz, H-6'); The additional peaks inthe spectrum were identified as follow: 4.92 (m, 2H, CH.sub.2 ofCbz), 3.61-3.57 (m, 2H, CH.sub.2), 3.23 (m, 1H, CH.sub.2),2.95-2.90 (m, 3H, CH.sub.3), 2.73-2.71 (m, 1H, CH.sub.2), 2.69-2.60(m, 4H, CH.sub.2). .sup.13C NMR (125 MHz, MeOD): .delta.C=109.70(C-1''), 100.67 (C-1'''), 100.28 (C-1'), 85.97 (C-6), 83.76 (C-5),83.27 (C-2''), 82.23 (C-4''), 78.95 (C-3'), 77.21 (C-4'), 75.95,75.56 (C-3''), 74.39 (C-5'''), 73.73 (CH.sub.2), 72.33 (C-3'''),72.04 (CH.sub.2), 71.05 (C-5'), 70.73 (C-4'''), 65.47 (CH.sub.2 ofCbz), 61.89 (C-5''), 57.59 (C-2'), 54.69 (C-1), 52.32 (C-3), 51.33(C-2'''), 50.48 (C-6'''), 43.61 (C-6'), 43.26 (CH.sub.2), 42.76(CH.sub.2), 37.27 (CH.sub.3), 35.80 (C-2), 28.64.

[0582] TOFMS calcd for C.sub.28H.sub.58N.sub.8O.sub.13Na ([M+Na]+)m/e 737.40; measured m/e 737.54).

Preparation of4-O-(2-(pyrrolidin-1-yl)ethylamino)ethyl-2'',3',3''',4''',5'',6-hexa-O-be-nzyl-1,2',2''',3,6',6'''-hexaazidoneomycin (28)

[0583] The titled compound was prepared as was described for thepreparation of compound 18 with the following quantities: compound25 (800 mg, 0.580 mmol), DCM (50 mL), PhI(OAc).sub.2 (224 mg, 0.7mmol, 1.2 eq.), N-(2-aminoethyl)pyrrolidine (0.2 mL, 1.5 mmol, 2.6equiv), sodium triacetoxyborohydride (344 mg, 1.62 mmol, 2.8equiv). The residue was purified by column chromatography(EtOAc/hexane, 60:40) to afford compound 28 (470 mg, 56%).

[0584] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.17(d, 1H, J=3.9 Hz, H-1), 4.12 (dt, 1H, J=6.8, 4.0 Hz, H-5), 3.96(dd, 1H, J=10.1, 8.9 Hz, H-3), 3.58 (dd, 1H, J=12.9, 1.7 Hz, H-6),3.42 (dd, 1H, J=12.2, 5.3 Hz, H-6'), 3.11 (dd, 1H, J=10.0, 8.8 Hz,H-4), 2.99 (dd, 1H, J=10.8, 3.1 Hz, H-2); `Ring II`: .delta.H=3.97(dd, 1H, J=9.9, 8.1 Hz, H-5), 3.69 (dd, 1H, J=9.7, 8.8 Hz, H-4),3.52-3.43 (m, 2H, H-1, H-3), 3.34 (dd, 1H, J=10.1, 8.2 Hz, H-6),2.31-2.25 (m, 1H, H-2eq), 1.46 (dt, 1H, J=12.3, 9.9 Hz, H-2ax);`Ring III`: .delta.H=5.69 (d, 1H, J=5.2 Hz, H-1), 4.35-4.25 (m, 2H,H-3, H-4), 3.99 (dd, 1H, J=5.7, 5.2 Hz, H-2), 3.83 (dd, 1H, J=10.2,1.8 Hz, H-5), 3.60 (dd, 1H, J=3.19, 10.52 Hz, H-5'); `Ring IV`:.delta.H=4.94 (d, 1H, J=1.7 Hz, H-1), 3.84-3.77 (m, 2H, H-3, H-5),3.67 (dd, 1H, J=12.8, 9.1 Hz, H-6), 3.39 (dd, 1H, J=4.8, 1.8 Hz,H-2), 3.16 (dd, 1H, J=2.1, 1.6 Hz, H-4), 2.89 (dd, 1H, J=9.2, 4.9Hz, H-6'). The additional peaks in the spectrum were identified asfollow: 3.89-3.86 (m, 1H, CH.sub.2), 3.65-3.63 (m, 1H, CH.sub.2),3.30 (m, 2H, CH.sub.2), 2.73-2.58 (m, 2H, CH.sub.2) 2.54 (m, 4H,CH.sub.2, Ring), 1.78 (m, 2H, Ring).

[0585] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.31 (Ar),138.08 (Ar), 137.92 (Ar), 137.68 (Ar), 137.04 (Ar), 136.96 (Ar),128.69 (Ar), 128.51 (Ar), 128.43 (Ar), 128.40 (Ar), 128.35 (Ar),128.29 (Ar), 128.20 (Ar), 127.96 (Ar), 127.85 (Ar), 127.81 (Ar),127.81 (Ar), 127.77 (Ar), 127.76 (Ar), 127.50 (Ar), 127.45 (Ar),106.17 (C-1''), 98.60 (C-1'''), 95.64 (C-1'), 84.22 (C-4''), 82.46(C-4'), 82.06 (C-4'''), 81.70 (C-2''), 79.62 (C-5), 79.55 (C-6),75.56 (C-3''), 75.14 (C-3'), 75.12, 75.07 (C-4), 74.38 (C-5'''),73.29 (C-3'''), 72.88 (C-5'), 72.39 (PhCH.sub.2), 71.73(PhCH.sub.2), 71.49 (PhCH.sub.2), 71.02 (PhCH.sub.2), 70.24(C-5''), 66.15 (Linker), 63.09 (C-2'), 60.40 (C-1), 59.93 (C-3),57.29 (C-2'''), 55.88 (Ring), 54.56 (Ring), 54.23 (Linker), 51.39(C-6'), 51.08 (C-6'''), 49.7 (Linker), 32.49 (C-2), 23.60 (Ring),23.44 (Ring).

[0586] TOFMS calcd for C.sub.73H.sub.86N.sub.20O.sub.13K ([M+K]+)m/e 1489.63; measured m/e 1489.86).

Preparation of 4-O-(2-(pyrrolidin-1-yl)ethylamino)ethyl-neomycin(Compound 4; FIGS. 1 and 2B)

[0587] The titled compound was prepared as described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: compound 28 (300 mg, 0.207 mmol), THF (10 mL), NaOH(0.1M, 5 mL), PMe.sub.3 (1M solution in THF, 6.1 mL, 58.9 mmol) toyield the compound as free amine form. Birch reduction: THF (10mL), ammonia (about 20 mL), small pieces of Na (170 mg, 7.9 mmol),ammonium formate (1 gram, 15.7 mmol). The analytically pure productwas obtained by passing the above product through a short column ofAmberlite CG50 (NH.sub.4.sup.+ form). The column was first washedwith MeOH and H.sub.2O, then the product was eluted with a mixtureof H.sub.2O/NH.sub.4OH (96:4) to afford Compound 4 (92 mg, 59% fortwo steps).

[0588] For the storage and biological tests, compound was convertedto its sulfate salt form: the free base was dissolved in water, thepH was adjusted around 7.0 with H.sub.2SO.sub.4 (0.1 N) andlyophilized.

[0589] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=5.44 (d, 1H,J=2.6 Hz, H-1), 3.77 (dd, 1H, J=9.8, 3.2 Hz, H-5), 3.69 (dd, 1H,J=10.5, 5.9 Hz, H-3), 3.08 (dd, 1H, J=12.7, 5.8 Hz, H-6), 3.03 (dd,1H, J=12.1, 2.6 Hz, H-6'), 2.84 (dd, 1H, J=13.5, 6.6 Hz, H-4), 2.72(dd, 1H, J=10.8, 5.0 Hz, H-2); `Ring II`: .delta.H 3.56 (dd, 1H,J=9.2, 6.6 Hz, H-5), 3.20 (dd, 1H, J=10.5, 7.7 Hz, H-4), 2.86-2.77(m, 2H, H-1, H-3), 2.66 (dd, 1H, J=9.7, 7.4 Hz, H-6), 2.05-1.91 (m,1H, H-2eq), 1.22 (ddd, 1H, J=11.3, 5.0 Hz, H-2ax); `Ring III`:.delta.H 5.32 (d, 1H, J=1.7 Hz, H-1), 4.39 (dd, 1H, J=6.1, 5.5 Hz,H-3), 4.16 (dd, 1H, J=5.1, 2.1 Hz, H-2), 4.05 (dd, 1H, J=6.8, 1.2Hz, H-4), 3.81 (dd, 1H, J=12.3, 1.9 Hz, H-5), 3.73 (dd, 1H, J=11.2,5.9 Hz, H-5'); `Ring IV`: .delta.H 4.95 (d, 1H, J=1.2 Hz, H-1),3.95 (dd, 1H, J=3.8, 1.4 Hz, H-3), 3.90 (m, 1H, H-5), 3.54 (dd, 1H,J=12.2, 8.6 Hz, H-6), 3.51 (dd, 1H, J=2.1, 1.1 Hz, H-4), 2.99 (dd,1H, J=4.0, 2.3 Hz, H-2), 2.72 (dd, 1H, J=9.0, 4.2 Hz, H-6'); theadditional peaks in the spectrum were identified as follow:3.92-3.90 (m, 1H, CH.sub.2), 3.51-3.49 (m, 1H, CH.sub.2), 3.23-3.19(m, 2H, CH.sub.2), 2.85-2.77 (m, 2H, CH.sub.2), 2.69-2.61 (m, 4H,CH.sub.2, Ring), 1.87-1.80 (m, 2H, Ring).

[0590] .sup.13C NMR (125 MHz, MeOD): .delta.C=108.28 (C-1''), 99.17(C-1'''), 98.69 (C-1'), 84.51 (C-4''), 82.18 (C-4'), 81.77 (C-5),80.93 (C-6), 77.50 (C-2''), 75.66 (C-3''), 74.26 (C-4'''), 74.12(C-3'''), 73.53 (C-5'), 72.92 (C-3'), 72.01 (C-4), 70.54 (C-5'''),69.88 (C-5''), 69.28 (CH.sub.2), 60.27 (C-2'), 56.07 (C-2'''),55.65 (Ring), 53.47 (CH.sub.2), 53.21 (C-1), 51.11 (C-3), 50.87(CH.sub.2), 42.09 (C-6'), 41.76 (C-6'''), 35.78 (C-2), 23.60(Ring), 22.75 (Ring).

[0591] TOFMS calcd for C.sub.31H.sub.62N.sub.8O.sub.13K ([M+K]+)m/e 793.41; measured m/e 793.38).

Preparation of4-O-(2-guanidinoethylamino)ethyl-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2-',2''',3,6',6'''-hexaazido-neomycin (29)

[0592] The titled compound was prepared as was described for thepreparation of compound 18 with the following changes andquantities: compound 25 (1 gram, 0.722 mmol), DCM (20 mL),PhI(OAc).sub.2 (1.2 equiv, 279 mg, 0.866 mmol),2-(2-Aminoethyl)-1,3-di-Boc-guanidine (3 equiv, 655 mg, 2.166mmol), sodium triacetoxyborohydride (3 equiv, 460 mg, 2.166 mmol).After purification the amine was then dissolved in a solution ofTFA in DCM (10 ml, 0.1M) and left to stir in room temperature.Reaction progress was monitored by TLC (EtOAc 90%, MeOH 10%), whichindicated completion after 48 hours. After completion the solventwas evaporated to dryness and the crude was purified by columnchromatography (EtOAc/MeOH 8:2) to yield 29 (385 mg, 37%).

[0593] .sup.1H NMR (500 MHz, MeOD): `ring I`: .delta.H=6.14 (d, 1H,J=4.07 Hz, H-1), 4.09-4.04 (m, 1H, H-5), 3.87 (dd, 1H, J=10.01,8.98 Hz, H-3), 3.53 (dd, 1H, J=14.13, 1.68 Hz, H-6), 3.39 (dd, 1H,J=13.43, 4.43 Hz, H-6'), 3.09 (dd, 1H, J=9.91, 8.67 Hz, H-4), 2.92(dd, 1H, J=10.84, 3.53 Hz, H-2); `ring II`: .delta.H=3.75 (dd, 1H,J=8.85, 8.85 Hz, H-5), 3.61 (dd, 1H, J=9.39, 9.39 Hz, H-4),3.52-3.42 (m, 2H, H-1, H-3), 3.25 (dd, 1H, J=9.59, 9.59 Hz, H-6),2.12 (dt, 1H, J=13.97, 5.07, 5.07 Hz, H-2eq), 1.32 (ddd, 1H,J=12.93, 12.26, 12.26 Hz, H-2ax); `ring III`: .delta.H=5.54 (d, 1H,J=4.39 Hz, H-1), 4.18 (dd, 1H, J=8.46, 4.23 Hz, H-3), 4.17-4.11 (m,1H, H-4), 3.94 (dd, 1H, J=4.88, 4.88 Hz, H-2), 3.74 (dd, 1H,J=10.63, 2.27 Hz, H-5), 3.50 (dd, 1H, J=10.86, 4.50 Hz, H-5');`ring IV`: .delta.H=4.81 (d, 1H, J=1.83 Hz, H-1), 3.81-3.74 (m, 1H,H-3), 3.80-3.78 (m, 1H, H-5), 3.53-3.46 (m, 1H, H-6), 3.28-3.26 (m,1H, H-4), 3.25-3.23 (m, 1H, H-2), 3.02-2.96 (m, 1H, H-6'); theadditional peaks in the spectrum were identified as follow:.delta.H 7.39-7.09 (m, 30H, Ar), 4.84-4.80 (m, 1H, BnCH.sub.2),4.75 (d, 1H, J=11.36 Hz, BnCH.sub.2), 4.73-4.64 (m, 2H,BnCH.sub.2), 4.54 (d, J=11.67 Hz, 1H), 4.49-4.38 (m, 5H,BnCH.sub.2), 4.31 (d, 2H, J=11.76 Hz, BnCH.sub.2), 4.03-3.99 (m,1H, CH.sub.2), 3.94 (dd, 1H, J=4.68, 4.68 Hz, CH.sub.2), 3.77-3.72(m, 1H, CH.sub.2), 3.52-3.46 (m, 1H, CH.sub.2), 3.43-3.35 (m, 2H,CH.sub.2), 3.06-2.96 (m, 2H, CH.sub.2).

[0594] .sup.13C NMR (126 MHz, CDCl.sub.3): .delta.C 157.50(Guanidine), 138.21 (Ar), 138.04 (Ar), 137.97 (Ar), 137.65 (Ar),137.46 (Ar), 137.38 (Ar), 128.20 (Ar), 128.09 (Ar), 128.04 (Ar),128.00 (Ar), 127.92 (Ar), 127.78 (Ar), 127.56 (Ar), 127.47 (Ar),127.26 (Ar), 106.95 (C-1''), 98.45 (C-1'''), 95.53 (C-1'), 83.95(C-6), 82.06 (C-4), 82.02 (C-5''), 81.47 (C-2''), 79.36 (C-3'),79.14 (C-4'), 75.80 (C-3''), 75.37 (C-4), 74.67 (PhCH.sub.2), 74.30(C-5'''), 74.18 (PhCH.sub.2), 73.15, 72.91 (PhCH.sub.2), 72.78(PhCH.sub.2), 72.15 (PhCH.sub.2), 71.97 (C-2'''), 71.57(PhCH.sub.2), 70.43 (C-5'), 70.27 (C-3''), 63.01 (C-2'), 60.33(C-1, C-3), 60.09 (CH.sub.2), 59.92, 57.08 (C-4'''), 50.90 (C-6',C-6'''), 46.37 (CH.sub.2), 37.88 (CH.sub.2), 31.77 (C-2).

[0595] MALDI TOFMS calcd for C.sub.70H.sub.81N.sub.22O.sub.22([M+H]+) m/e 1438.64; measured m/e 1439.00).

Preparation of 4'-O-(2-guanidinoethylamino)ethyl-neomycin (Compound5; FIGS. 1 and 2B)

[0596] Compound 29 (385 mg, 0.267 mmol) was dissolved in a mixtureof THF (10 mL) and aqueous NaOH (0.1M, 10 mL). This mixture wasstirred at room temperature for 10 minutes, after which PMe.sub.3(1M solution in THF, 9.63 mL, 38.91 mmol) was added. Propagation ofthe reaction was monitored by TLC[CH.sub.2Cl.sub.2/MeOH/H.sub.2O/MeNH.sub.2 (33% solution in EtOH),10:15:6:15], which indicated completion after 3 hours. The reactionmixture was purified by flash chromatography on a short column ofsilica gel. The column was washed with the following solvents:hexane (200 mL), THF (200 mL), CH.sub.2Cl.sub.2 (200 mL), EtOAc(200 mL), MeOH (400 mL). The product was eluted with the mixture of20% AcOH in 80% MeOH. Fractions containing the product werecombined and evaporated under vacuum. THF (10 mL) was added viasyringe to a dry three neck flask equipped with a Dewar condenser.Then ammonia (about 20 mL) was condensed into the reaction vessel.Small pieces of Na (300 mg, 13 mmol) were then allowed to dissolvein the ammonia for 15 min. Then a solution of the aminoglycoside ina mixture of EtOH and THF (500 .mu.L each) was added in one portionand washed down with THF. The reaction was stirred until the bluecolor was discharged. Then an aqueous solution of ammonium formate(1 gram, 15.7 mmol) was added, and the ammonia was allowed toevaporate. The remaining solvent was removed in Vacuum, and theresidue was loaded onto a short column of silica gel. The columnwas washed with the following solvents: hexane (200 mL), THF (200mL), CH.sub.2Cl.sub.2 (200 mL), EtOAc (200 mL), MeOH (400 mL). Theproduct was eluted with the mixture of 2% TFA in 98% MeOH. Thecontaining fractions were evaporated under vacuum to affordCompound 5 as a TFA salt (56 mg, 27%).

[0597] .sup.1H NMR (500 MHz, MeOD): `ring I`: .delta.H=5.98 (d, 1H,J=4.13 Hz, H-1), 4.18 (dd, 1H, J=11.87, 2.09 Hz, H-3), 4.05 (ddd,1H, J=9.17, 6.96, 4.13 Hz, H-5), 3.43-3.37 (m, 2H, H-2, H-4),3.27-3.23 (m, 1H, H-6), 3.14 (dd, 1H, J=13.20, 8.06 Hz, H-6');`ring II`: .delta.H=4.10-4.05 (m, 1H, H-4), 3.83 (dd, 1H, J=9.10,9.10 Hz, H-5), 3.53 (dd, 1H, J=9.79, 9.70 Hz, H-6), 3.44-3.30 (m,1H, H-3), 3.21-3.07 (m, 1H, H-1), 2.47-2.41 (m, 1H, H-2eq), 2.04(ddd, 1H, J=12.37, 12.37, 12.37 Hz, H-2ax); `ring III`:.delta.H=5.40 (d, 1H, J=2.39 Hz, H-1), 4.48 (dd, 1H, J=6.63, 4.35Hz, H-3), 4.34 (dd, 1H, J=5.20, 1.50 Hz, H-2), 4.18 (ddd, 1H,J=10.00, 3.97, 1.82 Hz, H-4), 3.87 (dd, 1H, J=5.54, 1.50 Hz, H-5),3.70 (dd, 1H, J=12.28, 4.17 Hz, H-5'); `ring IV`: .delta.H=5.26 (d,1H, J=1.73 Hz, H-1), 4.26 (ddd, 1H, J=7.85, 3.83, 1.45 Hz, H-5),4.11 (dd, 1H, J=3.17, 3.17 Hz, H-3), 3.66-3.65 (m, 1H, H-4),3.41-3.39 (m, 1H, H-2), 3.35 (dd, 1H, J=13.39, 7.41 Hz, H-6), 3.23(dd, 1H, J=13.38, 3.73 Hz, H-6'); the additional peaks in thespectrum were identified as follow: 4.19-4.17 (m, 1H, CH.sub.2),3.97-3.91 (m, 1H, CH.sub.2), 3.64-3.62 (m, 4H, CH.sub.2), 3.28-3.25(m, 2H, CH.sub.2).

[0598] .sup.13C NMR (126 MHz, CDCl.sub.3): .delta.C=161.28 (q,TFA-CO), 157.45 (GUA), 116.42 (q, TFA-CH.sub.3), 110.37 (C-1''),95.37 (C-1', C1'''), 85.06 (C-5), 81.90 (C-4'', C-3'), 79.69 (C-1),75.30 (C-3''), 75.17 (C-5'), 74.01 (C-2''), 72.71 (C-6), 70.52(C-5'''), 67.70 (C-3''', C-4'''), 67.66 (C-4), 67.13 (CH.sub.2),59.61 (C-5''), 53.87 (C-4'), 51.42 (C-2', C-2''), 49.91 (C-3),48.67, 47.73 (CH.sub.2), 46.04 (CH.sub.2), 40.06 (C-6', C-6'''),37.25 (CH.sub.2), 28.10 (C-2).

[0599] MALDI TOFMS calcd for C.sub.28H.sub.59N.sub.10O.sub.13([M+H]+) m/e 743.43; measured m/e 743.40).

Synthesis of 4'- and 6'-Amide-Linked Compounds (Compounds 6-10)

[0600] For the synthesis of the 4'-amide derivatives, alcohol 23(FIG. 2B) was first oxidized with Dess-Martin periodinane (DMP) toform corresponding 4'-ketone 30, which was then reduced with sodiumborohydride to afford compound 31 with an axial hydroxy group atthe 4'-position. Compound 31 was treated with triflic anhydride(Tf.sub.2O, pyridine, CH.sub.2Cl.sub.2) to form the corresponding4'-triflate, which was then treated with ammonia in acetone toyield 32 with an equatorial amine group at the 4'-position.

[0601] Next, 32 was treated with chloroacetyl chloride to give4'-chloride 33, which was then separately treated with threedifferent amines, compounds A and B (shown above) anddiethylenetriamine, to afford the corresponding 4'-amidederivatives of NeoB in their protected forms (compounds 34, 35, and36, respectively).

[0602] These products were then deprotected by using the two-stepprocedure described above (Staudinger and Birch) to afford thecorresponding 4'-amide derivatives of NeoB, Compounds 6, 7, and 8,in yields of 64, 68, and 20%, respectively (See, FIG. 5). Duringthe last deprotection step (the Birch reduction), it was uncoveredthat if this step was performed in the presence of an excess amountof sodium, transamidation rearrangement of the warhead, from the4'-position to the 6'-position, took place. The structure of therearrangement product (6'-amide) was confirmed by its isolation andsubsequent spectral assignment by using a combination of various 1Dand 2D NMR spectroscopy techniques. This rearrangement probablyoccurred as a result of the strong basic conditions generated afterquenching of the reaction, which resulted in the formation ofsodium hydroxide. This transformation was exploited by performingthe Birch reaction step with an excess amount of sodium andcorresponding 6'-amide-linked Compounds 9 and 10 were prepared inyields of 74 and 36%, respectively.

[0603] The following describes the detailed syntheses of Compounds6-10 and the intermediates thereof.

Preparation of4'-oxo-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6',6'''-hexaazido--neomycin (30)

[0604] Under argon, a solution of compound 23 (5 grams, 3.81 mmol)in CH.sub.2Cl.sub.2 (50 mL) was treated with Dess-Martinperiodinane (3.2 grams, 7.6 mmol, 2 eq.) and stirred for 4 hours atroom temperature. The reaction progress was monitored by TLC(Hexane/EtOAc, 7:3). After completion of the reaction, the mixturewas diluted with EtOAc and quenched with saturated aqueous sodiumthiosulfate, sodium bicarbonate and brine. The combined organicphases were dried over anhydrous MgSO.sub.4, filtered andevaporated to dryness. The residue was purified by columnchromatography (Hexane/EtOAc 75:25) to yield compound 30 (4.3grams, 86%).

[0605] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.34(d, 1H, J=2.1 Hz, H-1), 4.72 (dd, 1H, J=4.2, 1.0 Hz, H-5), 4.39 (d,1H, J=10.3 Hz, H-3), 3.46-3.36 (m, 3H, H-2, H-6, H-6'); `Ring II`:.delta.H=3.97 (dd, 1H, J=9.2, 8.7 Hz, H-5), 3.69 (dd, 1H, J=9.3,8.8 Hz, H-4), 3.55-3.39 (m, 2H, H-1, H-3), 3.32 (dd, 1H, J=8.9 Hz,H-6), 2.26 (dt, 1H, J=8.4, 4.0 Hz, H-2eq), 1.45 (ddd, 1H, J=12.9Hz, H-2ax); `Ring III`: .delta.H=5.68 (d, 1H, J=5.7 Hz, H-1),4.32-4.22 (m, 2H, H-3, H-4), 3.95 (t, 1H, J=5.9 Hz, H-2), 3.78 (dd,1H, J=9.7, 1.3 Hz, H-5), 3.55 (dd, 1H, J=10.4, 2.7 Hz, H-5'); `RingIV`: .delta.H 4.91 (d, 1H, J=2.5 Hz, H-1), 3.80-3.73 (m, 2H, H-3,H-5), 3.64 (dd, 1H, J=13.1, 9.2 Hz, H-6), 3.35 (dd, 1H, J=1.7, 1.1Hz, H-2), 3.11 (dd, 1H, J=3.2, 1.2 Hz, H-4), 2.86 (dd, 1H, J=6.6,4.5 Hz, H-6'); The additional peaks in the spectrum were identifiedas follow: 7.41-7.14 (m, 30H, Ar), 4.97 (d, 1H, J=10.5 Hz,PhCH.sub.2), 4.89 (d, 1H, J=13.2 Hz, PhCH.sub.2), 4.71 (d, 1H,J=10.4 Hz, PhCH.sub.2), 4.63 (d, 1H, J=12.1 Hz, PhCH.sub.2), 4.57(d, 2H, J=11.3 Hz, PhCH.sub.2), 4.47 (d, 1H, J=13.0 Hz,PhCH.sub.2), 4.39 (d, 1H, J=10.7 Hz, PhCH.sub.2), 4.32 (d, 1H,J=12.0 Hz, PhCH.sub.2), 4.26 (d, 2H, J=7.5 Hz, PhCH.sub.2), 4.24(d, 1H, J=7.8 Hz, PhCH.sub.2).

[0606] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=201.60 (C-4'),137.96 (Ar), 137.78 (Ar), 137.52 (Ar), 136.95 (Ar), 136.89 (Ar),136.75 (Ar), 128.65 (Ar), 128.47 (Ar), 128.45 (Ar), 128.39 (Ar),128.34 (Ar), 128.33 (Ar), 128.24 (Ar), 128.17 (Ar), 128.11 (Ar),127.79 (Ar), 127.77 (Ar), 127.75 (Ar), 127.60 (Ar), 127.49 (Ar),127.38 (Ar), 106.04 (C-1''), 98.66 (C-1'''), 95.40 (C-1'), 84.23(C-6), 82.55 (C-2''), 82.16 (C-4''), 81.70 (C-5), 78.90 (C-3'),75.82 (C-3''), 75.49 (PhCH.sub.2), 75.12 (PhCH.sub.2), 74.36 (C-4),73.90 (C-5'''), 73.48 (PhCH.sub.2), 73.38 (PhCH.sub.2), 73.29(C-3'''), 72.81 (PhCH.sub.2), 72.34 (C-5'), 71.67 (C-4'''), 69.95(C-5''), 63.62 (C-2'''), 60.22 (C-2'), 59.80 (C-1), 57.20 (C-3),51.05 (C-6'), 49.54 (C-6'''), 32.26 (C-2).

[0607] TOFMS calcd for C.sub.65H.sub.68N.sub.18O.sub.13Na ([M+Na]+)m/e 1331.51; measured m/e 1331.59).

Preparation of4'-hydroxy-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6',6'''-hexaa-zido-neomycin (31)

[0608] Under argon, a solution of compound 30 (4.3 grams, 3.3 mmol)in MeOH (50 mL) was cooled to -10.degree. C. Then, the mixture wastreated with NaBH.sub.4 (0.25 gram, 6.6 mmol, 2 eq.) and left tostir for 30 minutes then the mixture was allowed to warm to roomtemperature and was stirred for another 2 hours. The reactionpropagation was monitored by TLC (Hexane/EtOAc, 7:3). Aftercompletion, the mixture was diluted with EtOAc and washed with 1MHCl, saturated aqueous NaHCO.sub.3 and brine. The combined organicphases were dried over anhydrous MgSO.sub.4, filtered andevaporated to dryness. The residue was purified by columnchromatography (Hexane/EtOAc, 75:25) to obtain compound 31 (3.56grams, 82%).

[0609] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.23(d, 1H, J=3.1 Hz, H-1), 4.18 (dd, 1H, J=8.9, 5.9 Hz, H-5), 4.01(dd, 1H, J=10.9, 1.8 Hz, H-3), 3.96 (t, 1H, J=3.7 Hz, H-4), 3.62(dd, 1H, J=13.0, 3.5 Hz, H-6), 3.42 (dd, 1H, J=9.9, 4.5 Hz, H-2),3.30 (dd, 1H, J=12.2, 3.8 Hz, H-6'); `Ring II`: .delta.H=3.94 (dd,1H, J=9.2 Hz, H-5), 3.69 (dd, 1H, J=9.0, 1.1 Hz, H-4), 3.54-3.38(m, 2H, H-1, H-3), 3.30 (dd, 1H, J=9.0 Hz, H-6), 2.23 (dt, 1H,J=8.1, 4.8 Hz, H-2eq), 1.43 (ddd, 1H, J=12.4, 1.1 Hz, H-2ax); `RingIII`: .delta.H=5.66 (d, 1H, J=5.1 Hz, H-1), 4.28 (dd, 1H, J=3.9,1.8 Hz, H-4), 4.24 (dd, 1H, J=4.4, 3.0 Hz, H-3), 3.95 (dd, 1H,J=9.8, 3.7 Hz, H-2), 3.77 (dd, J=9.9, 1.3 Hz, H-5), 3.56 (dd,J=10.3, 2.8 Hz, H-5'); `Ring IV`: .delta.H=4.88 (d, 1H, J=1.4 Hz,H-1), 3.80-3.71 (m, 2H, H-3, H-5), 3.62 (dd, 1H, J=12.9, 8.3 Hz,H-6), 3.34 (dd, 1H, J=4.3, 1.5 Hz, H-2), 3.12 (dd, 1H, J=1.9, 1.3Hz, H-4), 2.89 (dd, 1H, J=11.9, 4.4 Hz, H-6'); the additional peaksin the spectrum were identified as follow: .delta. 7.38-7.14 (m,30H, Ar), 4.95 (d, 1H, J=10.6 Hz, PhCH.sub.2), 4.72 (d, 1H, J=11.5Hz, PhCH.sub.2), 4.68 (d, 1H, J=5.8 Hz, PhCH.sub.2), 4.64 (d, 1H,J=8.3 Hz, PhCH.sub.2), 4.62 (d, 1H, J=5.3 Hz, PhCH.sub.2), 4.60 (d,1H, J=8.2 Hz, PhCH.sub.2), 4.55 (d, 1H, J=11.8 Hz, PhCH.sub.2),4.48 (d, 1H, J=4.9 Hz, PhCH.sub.2), 4.45 (d, 1H, J=12.0 Hz,PhCH.sub.2), 4.42 (d, 1H, J=12.1 Hz, PhCH.sub.2), 4.32 (d, 1H,J=12.0 Hz, PhCH.sub.2), 4.26 (d, 1H, J=12.1 Hz, PhCH.sub.2).

[0610] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.27 (Ar),137.95 (Ar), 137.68 (Ar), 137.16 (Ar), 137.08 (Ar), 137.00 (Ar),128.79 (Ar), 128.70 (Ar), 128.52 (Ar), 128.44 (Ar), 128.36 (Ar),128.35 (Ar), 128.31 (Ar), 128.28 (Ar), 128.17 (Ar), 127.86 (Ar),127.80 (Ar), 127.77 (Ar), 127.57 (Ar), 127.49 (Ar), 127.35 (Ar),106.12 (C-1''), 98.66 (C-1'''), 95.82 (C-1'), 84.28 (C-6), 82.51(C-2''), 82.14 (C-5), 81.65 (C-3'), 75.66, 75.55 (C-3''), 75.07(PhCH.sub.2), 75.00 (C-4), 74.31 (C-5'''), 73.35 (PhCH.sub.2),72.97 (C-3'''), 72.41 (PhCH.sub.2), 72.25 (PhCH.sub.2), 71.77(PhCH.sub.2), 71.54 (C-5'), 70.25 (C-5''), 69.34 (C-4'''), 66.85(C-2'''), 60.40 (C-2'), 60.14 (C-1), 58.54 (C-3), 57.32 (C-4'),51.40 (C-6'), 51.05 (C-6'''), 32.49 (C-2).

[0611] TOFMS calcd for C.sub.65H.sub.70N.sub.18O.sub.13K ([M+K]+)m/e 1350.46; measured m/e 1350.78).

Preparation of4'-amino-4'-deoxy-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2',2''',3,6',6''-'-hexaazido-neomycin (32)

[0612] Compound 31 (3.56 grams, 2.71 mmol) was dissolved in amixture of pyridine (50 mL) and CH.sub.2Cl.sub.2 (20 mL). Thesolution was cooled to -10.degree. C., treated dropwise withTf.sub.2O (1.37 mL, 8.15 mmol, 3 eq.) and stirred for 3 hours. Thereaction propagation was monitored by TLC (Hexane/EtOAc, 7:3).After completion, the mixture was diluted with EtOAc and washedwith 1M HCl, saturated aqueous NaHCO.sub.3 and brine. The combinedorganic phases were dried over anhydrous MgSO.sub.4, filtered andevaporated to dryness. In the hydrogenation reactor, the crudecompound was dissolved in fresh distilled acetone (20 mL) andcooled to -78.degree. C. Then Ammonia (30 mL) was condensed in tothe reaction vessel, the mixture was allowed to warm to roomtemperature and was stirred for 48 hours. The solvents wereevaporated to dryness and the residue was purified by columnchromatography (Hexane/EtOAc, 6:4) to obtain compound 32 (1.46gram, 41%).

[0613] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.22(d, 1H, J=2.2 Hz, H-1), 3.82 (dd, 1H, J=7.6, 5.3 Hz, H-5), 3.62(dd, 1H, J=9.6 Hz, H-3), 3.50 (dd, 1H, J=12.2, 1.1 Hz, H-6), 3.31(dd, 1H, J=13.2, 5.8 Hz, H-6'), 2.89 (dd, 1H, J=10.5, 5.7 Hz, H-2),2.41 (dd, 1H, J=11.0, 5.4 Hz, H-4); `Ring II`: .delta.H=3.89 (dd,1H, J=9.0 Hz, H-5), 3.61 (dd, J=9.8, 8.1 Hz, H-4), 3.47-3.29 (m,2H, H-1, H-3), 3.21 (dd, J=9.7, 8.7 Hz, H-6), 2.13 (dt, 1H, J=7.8,4.8 Hz, H-2eq), 1.32 (ddd, 1H, J=12.2, 1.8 Hz, H-2ax); `Ring III`:.delta.H=5.66 (d, 1H, J=4.7 Hz, H-1), 4.39-4.15 (m, 2H, H-3, H-4),3.93 (dd, 1H, J=6.0 Hz, H-2), 3.78 (dd, 1H, J=10.4, 1.5 Hz, H-5),3.54 (dd, 1H, J=9.8, 1.8 Hz, H-5'); `Ring IV`: .delta.H=4.89 (d,1H, J=2.3 Hz, H-1), 3.77-3.69 (m, 2H, H-3, H-5), 3.61 (dd, 1H,J=12.7, 8.4 Hz, H-6), 3.31 (dd, 1H, J=3.0, 2.3 Hz, H-2), 3.08 (dd,1H, J=1.9, 1.1 Hz, H-4), 2.84 (dd, 1H, J=13.0, 3.3 Hz, H-6'). Theadditional peaks in the spectrum were identified as follow:7.32-7.12 (m, 30H, Ar), 4.93 (d, 1H, J=10.5 Hz, PhCH.sub.2), 4.90(d, 1H, J=8.7 Hz, PhCH.sub.2), 4.68 (d, 1H, J=10.4 Hz, PhCH.sub.2),4.59 (d, 1H, J=12.3 Hz, PhCH.sub.2), 4.55 (d, 1H, J=11.4 Hz,PhCH.sub.2), 4.49 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.44 (d, 1H,J=9.2 Hz, PhCH.sub.2), 4.42 (d, 1H, J=11.7 Hz, PhCH.sub.2), 4.38(d, 1H, J=12.0 Hz, PhCH.sub.2), 4.28 (d, 1H, J=12.2 Hz,PhCH.sub.2), 4.24 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.22 (d, 1H,J=12.4 Hz, PhCH.sub.2).

[0614] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=138.24 (Ar),137.89 (Ar), 137.85 (Ar), 137.63 (Ar), 136.99 (Ar), 136.93 (Ar),128.65 (Ar), 128.58 (Ar), 128.48 (Ar), 128.40 (Ar), 128.35 (Ar),128.31 (Ar), 128.24 (Ar), 128.22 (Ar), 128.16 (Ar), 128.06 (Ar),127.80 (Ar), 127.78 (Ar), 127.74 (Ar), 127.44 (Ar), 127.29 (Ar),106.02 (C-1''), 98.60 (C-1'''), 95.98 (C-1'), 84.33 (C-6), 82.55(C-2''), 82.09 (C-4''), 81.67 (C-5), 80.51 (C-3'), 75.54 (C-3''),75.06 (PhCH.sub.2), 75.00 (PhCH.sub.2), 74.88 (PhCH.sub.2), 74.36(C-4), 73.33 (C-5'''), 73.22 (C-3'''), 72.86 (PhCH.sub.2), 72.33(C-5'), 71.68 (PhCH.sub.2), 71.43 (C-4'''), 70.19 (C-5''), 63.32(C-2'''), 60.36 (C-2'), 60.24 (C-1), 57.21 (C-3), 54.36 (C-4'),52.14 (C-6'), 51.18 (C-6'''), 32.55 (C-2).

[0615] TOFMS calcd for C.sub.65H.sub.71N.sub.19O.sub.12 ([M]+) m/e1309.55; measured m/e 1309.80).

Preparation of4'-(2-chloroacetamido)-4'-deoxy-2'',3',3''',4''',5'',6-hexa-O-benzyl-1,2'-,2''',3,6',6'''-hexaazido-neomycin (33)

[0616] To a solution of amine 32 (1 gram, 0.76 mmol) in anhydrousTHF (10 mL), NaHCO.sub.3 (0.25 gram, 3 mmol) was added and themixture was stirred at room temperature. Then, the solution wastreated with chloroacetyl chloride (0.25 mL, 3.14 mmol, 4 eq.) andthe reaction was stirred for 1 hour. The reaction propagation wasmonitored by TLC (Hexane/EtOAc, 6:4). After completion, the mixturewas diluted with EtOAc and washed with 1M HCl and brine. Thecombined organic phases were dried over anhydrous MgSO.sub.4,filtered and evaporated to dryness. The crude product was purifiedby flash chromatography (Hexane/EtOAc, 75:25) to afford compound 33(1.1 gram, 98%).

[0617] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.24(d, 1H, J=3.8 Hz, H-1), 3.99 (dd, 1H, J=8.7, 6.5 Hz, H-5), 3.84(dd, 1H, J=9.2 Hz, H-3), 3.74 (dd, 1H, J=12.3, 1.6 Hz, H-4), 3.25(dd, 1H, J=13.5, 6.9 Hz, H-6), 3.16 (dd, 1H, J=13.1, 2.5 Hz, H-6'),3.03 (dd, 1H, J=10.0, 2.9 Hz, H-2); `Ring II`: .delta.H=3.94 (dd,1H, J=8.8 Hz, H-5), 3.67 (dd, 1H, J=9.2 Hz, H-4), 3.57-3.35 (m, 2H,H-1, H-3), 3.27 (dd, 1H, J=9.5 Hz, H-6), 2.19 (dt, 1H, J=13.2, 4.5Hz, H-2eq), 1.37 (ddd, 1H, J=12.8, 4.0 Hz, H-2ax); `Ring III`:.delta.H=5.65 (d, 1H, J=5.4 Hz, H-1), 4.25-4.22 (m, 2H, H-3, H-4),3.91 (dd, 1H, J=6.4, 5.3 Hz, H-2), 3.78 (dd, 1H, J=10.1, 1.4 Hz,H-5), 3.54 (dd, 1H, J=9.8, 1.4 Hz, H-5'); `Ring IV`: .delta.H=4.91(d, 1H, J=1.5 Hz, H-1), 3.81-3.74 (m, 2H, H-3, H-5), 3.63 (dd, 1H,J=12.8, 9.1 Hz, H-6), 3.32 (dd, 1H, J=4.7, 1.9 Hz, H-2), 3.09 (dd,1H, J=3.0, 1.5 Hz, H-4), 2.85 (dd, 1H, J=14.1, 2.8 Hz, H-6'); theadditional peaks in the spectrum were identified as follow:7.33-7.13 (m, 30H, Ar), 6.35 (d, 1H, J=9.2 Hz, Amide), 4.92 (d, 1H,J=10.4 Hz, PhCH.sub.2), 4.73 (d, 1H, J=11.4 Hz, PhCH.sub.2), 4.67(d, 1H, J=10.6 Hz, PhCH.sub.2), 4.59 (d, 1H, J=9.0 Hz, PhCH.sub.2),4.57 (d, 1H, J=8.7 Hz, PhCH.sub.2), 4.47 (d, 1H, J=11.8 Hz,PhCH.sub.2), 4.43 (d, 1H, J=11.7 Hz, PhCH.sub.2), 4.43 (d, 1H,J=11.8 Hz, PhCH.sub.2), 4.38 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.28(d, 1H, J=12.1 Hz, PhCH.sub.2), 4.24 (d, 1H, J=8.9 Hz, PhCH.sub.2),4.22 (d, 1H, J=12.0 Hz, PhCH.sub.2).

[0618] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=166.36 (Amide),138.39 (Ar), 138.15 (Ar), 137.92 (Ar), 137.83 (Ar), 137.30 (Ar),137.24 (Ar), 128.98 (Ar), 128.82 (Ar), 128.81 (Ar), 128.72 (Ar),128.65 (Ar), 128.63 (Ar), 128.58 (Ar), 128.49 (Ar), 128.31 (Ar),128.30 (Ar), 128.12 (Ar), 128.08 (Ar), 128.07 (Ar), 128.00 (Ar),127.78 (Ar), 106.32 (C-1''), 98.96 (C-1'''), 95.81 (C-1'), 84.65(C-6), 82.92 (C-2''), 82.43 (C-4''), 81.88 (C-5), 76.80 (C-3'),76.06 (C-3'''), 75.80 (C-3''), 75.44 (PhCH.sub.2), 74.73 (C-4),74.52 (PhCH.sub.2), 73.69 (PhCH.sub.2), 73.14 (C-5'''), 72.66(PhCH.sub.2), 72.00 (PhCH.sub.2), 71.74 (C-5'), 71.47 (C-4'''),70.90 (PhCH.sub.2), 70.41 (C-5''), 70.39 (C-4'), 62.75 (C-2'),60.62 (C-2'''), 60.52 (C-1), 57.52 (C-3), 52.33 (C-6'), 51.41(C-6'''), 32.83 (C-2).

[0619] TOFMS calcd for C.sub.66H.sub.70N.sub.19ClO.sub.13K ([M+K]+)m/e 1410.47; measured m/e 1410.67).

Preparation of4'-(2-(aminoethylazido)acetamido)-4'-deoxy-2'',3',3''',4''',5'',6-hexa-O--benzyl-1,2',2''',3,6',6'''-hexaazidoneomycin (34)

[0620] To a solution of compound 33 (0.5 gram, 0.36 mmol) inanhydrous DMF (10 mL), N,N-diisopropylethylamine (0.25 mL, 1.43mmol, 4 eq.) was added and the reaction was stirred at roomtemperature. Then, the solution was treated with azidoethyleneamine(0.124 gram, 1.44 mmol, 4 eq.), heated to 60.degree. C. and leftovernight. The reaction propagation was monitored by TLC(Hexane/EtOAc, 1:1). After completion, the mixture was diluted withEtOAc and washed with 1M HCl, saturated aqueous NaHCO.sub.3 andbrine. The combined organic phases were dried over anhydrousMgSO.sub.4, filtered and evaporated to dryness. The crude productwas purified by flash chromatography (Hexane/EtOAc, 6:4) to affordcompound 34 (0.375 gram, 72%).

[0621] .sup.1H NMR (500 MHz, CDCl.sub.3): `ring I`: .delta.H=6.22(d, 1H, J=3.83 Hz, H-1), 3.95 (dd, 1H, J=8.68, 8.68 Hz, H-5), 3.83(dd, 1H, J=9.95, 9.56 Hz, H-3), 3.72 (ddd, 1H, J=9.93, 9.93, 9.84Hz, H-4), 3.23 (dd, 1H, J=13.52, 7.02 Hz, H-6), 3.11 (d, 1H,J=12.53 Hz, H-6'), 2.97 (dd, 1H, J=9.53, 3.17 Hz, H-2); `ring II`:.delta.H=3.91 (dd, 1H, J=9.07, 9.07 Hz, H-5), 3.64 (dd, 1H, J=9.47,9.47 Hz, H-4), 3.43 (td, 1H, J=9.70, 9.67, 5.17 Hz, H-3), 3.36 (td,1H, J=9.77, 9.71, 5.13 Hz, H-1), 3.23 (dd, 1H, J=9.46, 9.46 Hz,H-6), 2.15 (dt, 1H, J=12.89, 4.52, 4.52 Hz, H-2eq), 1.34 (ddd, 1H,J=12.75, 12.75, 12.75 Hz, H-2ax); `ring III`: .delta.H=5.62 (d, 1H,J=6.03 Hz, H-1), 4.21-4.18 (m, 2H, H-3, H-4), 3.90-3.87 (m, 1H,H-2), 3.74 (dd, 1H, J=8.08, 2.95 Hz, H-5), 3.49 (dd, 1H, J=10.48,2.93 Hz, H-5'); `ring IV`: .delta.H=4.87 (d, 1H, J=2.26 Hz, H-1),3.72 (ddd, 1H, J=4.06, 4.06, 4.06 Hz, H-5), 3.68 (dd, 1H, J=2.82,2.82 Hz, H-3), 3.58 (dd, 1H, J=13.04, 8.51 Hz, H-6), 3.28 (dd, 1H,J=2.22, 2.22 Hz, H-2), 3.05 (d, 1H, J=2.66 Hz, H-4), 2.79 (dd, 1H,J=13.04, 3.83 Hz, H-6'); the additional peaks in the spectrum wereidentified as follow: 7.31-7.06 (m, 30H, Ar), 7.08 (d, 1H, J=9.22Hz, Amide) 4.89 (d, 1H, J=10.50 Hz, PhCH.sub.2), 4.72 (d, 1H,J=11.76 Hz, PhCH.sub.2), 4.63 (d, 1H, J=10.55 Hz, PhCH.sub.2), 4.54(d, 2H, J=12.45 Hz, PhCH.sub.2), 4.45-4.37 (m, 4H, PhCH.sub.2),4.34 (d, 1H, J=11.97 Hz, PhCH.sub.2), 4.24 (d, 1H, J=11.96 Hz,PhCH.sub.2), 4.17 (d, 2H, J=12.24 Hz, PhCH.sub.2), 3.15 (t, 2H,J=5.45, 5.45 Hz, CH.sub.2), 3.01 (d, 2H, J=8.18 Hz, CH.sub.2),2.55-2.44 (m, 2H, CH.sub.2).

[0622] .sup.13C NMR (126 MHz, CDCl.sub.3): .delta.C=171.40 (Amide),138.02 (Ar), 138.01 (Ar), 137.80 (Ar), 137.54 (Ar), 136.92 (Ar),136.86 (Ar), 128.59 (Ar), 128.42 (Ar), 128.33 (Ar), 128.30 (Ar),128.26 (Ar), 128.24 (Ar), 128.18 (Ar), 128.10 (Ar), 127.74 (Ar),127.71 (Ar), 127.68 (Ar), 127.61 (Ar), 127.58 (Ar), 127.39 (Ar),127.33 (Ar), 105.94 (C-1''), 98.58 (C-1'''), 95.46 (C-1'), 84.30(C-6), 82.53 (C-5), 82.08 (C-2''), 81.55 (C-4''), 77.01 (C-3'),75.51 (C-4), 75.29 (C-3''), 75.04 (PhCH.sub.2), 74.33 (C-5'''),73.44 (PhCH.sub.2), 73.31 (PhCH.sub.2), 73.27 (PhCH.sub.2), 72.76(C-3'''), 72.27 (PhCH.sub.2), 71.62 (PhCH.sub.2), 71.50 (C-5'),71.36 (C-4'''), 70.05 (C-5''), 62.41 (C-2'), 60.27 (C-1), 60.16(C-3), 57.15 (C-2'''), 52.19 (C-6'), 51.79 (CH.sub.2), 51.07(CH.sub.2), 51.05 (C-4'), 51.01 (C-6'''), 48.59 (CH.sub.2), 32.46(C-2).

[0623] MALDI TOFMS calcd for C.sub.69H.sub.78N.sub.23O.sub.13([M+H]+) m/e 1436.61; measured m/e 1436.67).

Preparation of 4'-(2-(aminoethylamino)acetamido)-4'-deoxy-neomycin(Compound 6; FIGS. 1 and 3)

[0624] The titled compound was prepared as was described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: compound 34 (0.417 gram, 0.29 mmol), THF (5 mL),PMe.sub.3 (3 equiv, 6.1 mL, 6.1 mmol), aqueous NaOH (5 mL, 0.1 M).Birch reduction: THF (10 mL), ammonia (about 20 mL), small piecesof Na (80 mg, 3.48 mmol), ammonium formate (1 gram, 15.7 mmol). Theanalytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH.sub.4.sup.+ form). Thecolumn was first washed with MeOH and H.sub.2O, then the productwas eluted with a mixture of H.sub.2O/NH.sub.4OH (95:5) to affordCompound 6 (92 mg, 45% for two steps). For the storage andbiological tests, Compound 6 was converted to its sulfate saltform: the free base was dissolved in water, the pH was adjustedaround 7.0 with H.sub.2SO.sub.4 (0.1 N) and lyophilized.

[0625] .sup.1H NMR (500 MHz, CDCl.sub.3): `ring I`: .delta.H=5.50(d, 1H, J=3.60 Hz, H-1), 3.84 (m, 1H, J=11.04 Hz, H-5), 3.78 (dd,J=9.85, 9.85 Hz, H-3), 3.69 (dd, J=10.37, 9.84 Hz, H-4), 2.85-2.66(m, 3H, H-2, H-6, H-6'); `ring II`: .delta.H=3.57-3.51 (m, 1H,H-5), 3.47-3.39 (m, 1H, H-4), 3.22 (dd, 1H, J=9.61, 9.61 Hz, H-6),2.82-2.75 (m, 1H, H-3), 2.67-2.60 (m, 1H, H-1), 1.99 (dt, 1H,J=12.78, 3.97, 3.97 Hz, H-2eq), 1.22 (ddd, 1H, J=12.43, 12.43,12.43 Hz, H-2ax); `ring III`: .delta.H 5=0.33 (d, 1H, J=3.27 Hz,H-1), 4.40-4.37 (m, H-3), 4.17 (dd, 1H, J=5.48, 2.46 Hz, H-2),4.09-4.06 (m, 1H, H-4), 3.82 (dd, 1H, J=11.95, 2.35 Hz, H-5), 3.73(dd, 1H, J=12.33, 3.90 Hz, H-5'); `ring IV`: .delta.H=4.95 (d, 1H,J=1.90 Hz, H-1), 3.95 (dd, 1H, J=3.11, 3.11 Hz, H-3), 3.90-3.85 (m,1H, H-5), 3.50 (d, 1H, J=3.44 Hz, H-4), 3.06 (dd, 1H, J=13.25, 8.98Hz, H-6), 2.98 (d, 1H, J=2.86 Hz, H-2), 2.86 (dd, 1H, J=13.34, 3.80Hz, H-6'); the additional peaks in the spectrum were identified asfollow: 3.37-3.30 (m, 2H, CH.sub.2), 3.26 (d, 2H, J=16.29 Hz,CH.sub.2), 2.78 (t, 2H, J=6.21, 6.21 Hz, CH.sub.2).

[0626] .sup.13C NMR (126 MHz, CDCl.sub.3): .delta.C=175.19 (Amide),109.73 (C-1''), 100.77 (C-1', C-1''), 86.15 (C-5), 83.90 (C-4),83.38 (C-4''), 79.08 (C-6), 77.36 (C-3''), 76.27 (C-5'''), 75.62(C-2''), 73.23 (C-5'), 72.11 (C-3'''), 71.59 (C-3'), 70.80(C-4'''), 62.11 (C-5''), 58.30 (C-2'), 54.74 (C-2'''), 540.4(C-4'), 53.04 (CH.sub.2), 52.46 (C-1), 52.33 (C-3), 51.99 (C-6'),43.36 (C-6'''), 41.69 (CH.sub.2), 37.48 (C-2).

[0627] MALDI TOFMS calcd for C.sub.27H.sub.57N.sub.9O.sub.14([M+H2O]+) m/e 731.40; measured m/e 731.84).

Preparation of4'-amino-6'-(2-(aminoethylamino)acetamido)-4'-deoxy-neomycin(Compound 9; FIGS. 1 and 3)

[0628] The titled compound was prepared as was described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: compound 34 (0.1 gram, 0.07 mmol), THF (5 mL), PMe.sub.3(3 equiv, 1.46 mL, 1.46 mmol), aqueous NaOH (5 mL, 0.1 M). Birchreduction: THF (10 mL), ammonia (about 20 mL), small pieces of Na(300 mg, 13 mmol), ammonium formate (1 gram, 15.7 mmol). Theanalytically pure product was obtained by passing the above productthrough a short column of Amberlite CG50 (NH.sub.4+ form). Thecolumn was first washed with MeOH and H.sub.2O, then the productwas eluted with a mixture of H.sub.2O/NH.sub.4OH (95:5) to affordCompound 9 (37 mg, 74% for two steps). For the storage andbiological tests, the compound was converted to its sulfate saltform: the free base was dissolved in water, the pH was adjustedaround 7.0 with H.sub.2SO.sub.4 (0.1 N) and lyophilized.

[0629] .sup.1H NMR (500 MHz, MeOD): `ring I`: .delta.H=5.50 (d, 1H,J=3.54 Hz, H-1), 3.72-3.67 (m, 1H, H-5), 3.43 (dd, 1H, J=9.77, 9.77Hz, H-3), 3.01 (dd, 1H, J=13.48, 2.72 Hz, H-6), 2.77 (dd, 1H,J=13.67, 6.80 Hz, H-6'), 2.68 (dd, 1H, J=10.21, 3.73 Hz, H-2), 2.49(dd, 1H, J=9.75, 9.75 Hz, H-4); `ring II`: .delta.H=3.57-3.49 (m,1H, H-5), 3.43 (dd, 1H, J=9.51, 9.51 Hz, H-4), 3.34 (dd, 1H,J=9.02, 9.02 Hz, H-6), 2.82-2.74 (m, 1H, H-3), 2.67-2.59 (m, 1H,H-1), 2.01-1.93 (m, 1H, H-2eq), 1.25-1.17 (m, 1H, H-2ax); `ringIII`: .delta.H=5.28 (d, 1H, J=3.47 Hz, H-1), 4.38-4.35 (m, 1H,H-3), 4.17-4.14 (m, 1H, H-2), 4.06-4.03 (m, 1H, H-4), 3.82-3.77 (m,1H, H-5), 3.73-3.68 (m, 1H, H-5'); `ring IV`: .delta.H=5.29 (d, 1H,J=3.68 Hz, H-1), 3.66-3.57 (m, 1H, H-5), 3.50 (dd, 1H, J=10.02,10.02 Hz, H-6), 3.12 (dd, 1H, J=12.77, 4.73 Hz, H-3), 2.73 (dd, 1H,J=9.88, 3.69 Hz, H-4), 2.52 (dd, 1H, J=11.59, 11.59 Hz, H-2),2.39-2.34 (m, 1H, H-6'); the additional peaks in the spectrum wereidentified as follow: .delta.H 3.91-3.86 (m, 1H, CH.sub.2),3.47-3.40 (m, 1H, CH.sub.2), 3.30 (s, 2H, CH.sub.2), 2.76-2.73 (m,2H, CH.sub.2).

[0630] .sup.13C NMR (126 MHz, MeOD): .delta.C=174.80 (Amide),110.05 (C-1''), 100.65 (C-1'''), 100.61 (C-1'), 85.91 (C-5), 85.50(C-6), 83.31 (C-3'), 83.16 (C-4, C-4''), 77.13 (C-3''), 75.74(C-5'''), 75.54 (C-2''), 72.02 (C-3'''), 70.69 (C-4'''), 69.08(C-5'), 62.07 (C-5''), 61.87 (CH.sub.2), 57.67 (C-2'), 56.13(C-4'), 54.67 (C-2'''), 52.31 (C-1, C-3), 48.83 (CH.sub.2), 43.68(C-6'), 43.23 (CH.sub.2), 43.20 (C-6'''), 37.38 (C-2).

[0631] MALDI TOFMS calcd for C.sub.27H.sub.57N.sub.9O.sub.14([M+H2O]+) m/e 731.40; measured m/e 731.84).

Preparation of4'-[2-(2-((benzyloxycarbonyl)(methyl)amino)ethylamino)acetamido]-4'-deoxy--2'',3',3''',4''',5'',6-hexa-Obenzyl-1,2',2''',3,6',6'''-hexaazido-neomyci-n (35)

[0632] The titled compound was prepared as was described for thepreparation of compound 27 with the following quantities: compound33 (0.5 gram, 0.36 mmol), DMF (10 mL), N,N-diisopropylethylamine(0.25 mL, 1.43 mmol, 4 eq.), N-Cbz-N-Methylethylenediamine (0.3gram, 1.44 mmol, 4 eq.) The crude product was purified by flashchromatography (Hexane/EtOAc, 6:4) to afford compound 35 (0.365gram, 65%).

[0633] .sup.1H NMR (500 MHz, CDCl.sub.3): `Ring I`: .delta.H=6.27(d, 1H, J=3.7 Hz, H-1), 4.08-3.97 (m, 2H, H-5, H-3), 3.85 (dd,J=12.2, 9.9 Hz, H-4), 3.33 (dd, 1H, J=13.7, 7.2 Hz, H-6), 3.23 (dd,1H, J=13.1, 2.2 Hz, H-6'), 3.03 (dd, 1H, J=10.8, 2.4 Hz, H-2);`Ring II`: .delta.H=3.94 (dd, 1H, J=8.5 Hz, H-5), 3.69 (dd, 1H,J=8.8 Hz, H-4), 3.48-3.32 (m, 2H, H-1, H-3), 3.28 (dd, 1H, J=7.3Hz, H-6), 2.09 (dt, 1H, J=12.8, 4.6 Hz, H-2eq), 1.35 (ddd, 1H,J=12.3, 5.3 Hz, H-2ax); `Ring III`: .delta.H=5.68 (d, 1H, J=5.2 Hz,H-1), 4.31-4.26 (m, 2H, H-3, H-4), 3.95 (dd, 1H, J=6.1, 5.1 Hz,H-2), 3.82 (dd, 1H, J=10.1, 1.4 Hz, H-5), 3.56 (dd, 1H, J=10.4, 2.5Hz, H-5'); `Ring IV`: .delta.H=4.95 (d, 1H, J=1.5 Hz, H-1),3.83-3.74 (m, 2H, H-3, H-5), 3.67 (dd, 1H, J=12.7, 9.1 Hz, H-6),3.36 (dd, 1H, J=5.6, 1.8 Hz, H-2), 3.13 (dd, 1H, J=2.3, 1.6 Hz,H-4), 2.85 (dd, 1H, J=14.9, 2.9 Hz, H-6'); the additional peaks inthe spectrum were identified as follow: 7.45-7.12 (m, 35H, Ar), 7.2(d, 1H, J=8.79 Hz, Amide), 5.11 (d, 1H, J=12.4 Hz, PhCH.sub.2),5.09 (m, 2H, CH.sub.2 of Cbz), 4.95 (d, 1H, J=16.0 Hz, PhCH.sub.2),4.72 (d, 1H, J=10.6 Hz, PhCH.sub.2), 4.62 (d, 1H, J=12.5 Hz,PhCH.sub.2), 4.57 (d, 1H, J=11.2 Hz, PhCH.sub.2), 4.49 (d, 1H,J=12.6 Hz, PhCH.sub.2), 4.46 (m, 2H, PhCH.sub.2), 4.42 (d, 1H,J=12.0 Hz, PhCH.sub.2), 4.32 (d, 1H, J=12.0 Hz, PhCH.sub.2), 4.29(d, 1H, J=14.2 Hz, PhCH.sub.2), 4.25 (d, 1H, J=12.1 Hz,PhCH.sub.2), 3.37-3.35 (m, 2H, O.dbd.CCH.sub.2NHR), 3.24-3.21 (m,1H, CH.sub.2), 3.13 (m, 1H, CH.sub.2), 2.96 (m, 1H, CH.sub.2), 2.87(m, 1H, CH.sub.2), 2.17 (m, 3H, CH.sub.3).

[0634] .sup.13C NMR (125 MHz, CDCl.sub.3): .delta.C=170.42 (Amide),156.14 (Carbamate), 137.51 (Ar), 135.11 (Ar), 134.57 (Ar), 133.45(Ar), 132.45 (Ar), 132.13 (Ar), 131.95 (Ar), 131.54 (Ar), 130.97(Ar), 128.93 (Ar), 128.65 (Ar), 127.87 (Ar), 127.66 (Ar), 127.41(Ar), 127.13 (Ar), 126.49 (Ar), 125.83 (Ar), 111.85 (C-1''), 107.30(C-1'''), 106.73 (C-1'), 87.52 (C-4''), 86.63 (C-4'''), 83.21(C-2''), 77.40 (C-3''), 76.82 (C-3'''), 75.23 (C-6), 75.06 (C-3'),74.27 (C-5), 73.56 (C-5'), 73.35 (PhCH.sub.2), 73.04 (PhCH.sub.2),72.95 (C-5'''), 72.71 (PhCH.sub.2), 72.53 (C-4), 71.68(PhCH.sub.2), 70.82 (PhCH.sub.2), 68.51 (C-5''), 67.12 (CH.sub.2 ofCbz), 61.81 (C-2'''), 56.93 (C-4'), 56.12 (C-2'), 54.26 (CH.sub.2),53.50 (RCH.sub.2C.dbd.ONHR), 52.78 (C-3), 51.16 (C-1), 47.64(C-6'''), 45.23 (CH.sub.2), 44.87 (C-6'), 37.08 (CH.sub.3NHR),36.25 (C-2).

[0635] TOFMS calcd for C.sub.78H.sub.87N.sub.21O.sub.15Na ([M+Na]+)m/e 1580.66; measured m/e 1580.71).

Preparation of4'-[2-(2-((benzyloxycarbonyl)(methyl)amino)ethylamino)acetamido]-4'-deoxy--neomycin (Compound 7; FIGS. 1 and 3)

[0636] The titled compound was prepared as described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: compound 35 (365 mg, 0.234 mmol), THF (10 mL), NaOH(0.1M, 5 mL), PMe.sub.3 (1M solution in THF, 5.9 mL, 57 mmol) toyield the compound as free amine form. Birch reduction: THF (10mL), ammonia (about 20 mL), small pieces of Na (65 mg, 2.8 mmol),ammonium formate (1 gram, 15.7 mmol). The analytically pure productwas obtained by passing the above product through a short column ofAmberlite CG50 (NH.sub.4.sup.+ form). The column was first washedwith MeOH and H.sub.2O, then the product was eluted with a mixtureof H.sub.2O/NH.sub.4OH (91:9) to afford Compound 7 (95 mg, 68% fortwo steps). For the storage and biological tests, compound wasconverted to its sulfate salt form: the free base was dissolved inwater, the pH was adjusted around 7.0 with H.sub.2SO.sub.4 (0.1 N)and lyophilized.

[0637] .sup.1H NMR (500 MHz, MeOD): `Ring I`: .delta.H=5.74 (d, 1H,J=3.3 Hz, H-1), 4.10 (dd, 1H, J=9.6, 5.2 Hz, H-4), 3.83-3.71 (m,2H, H-5, H-3), 3.00-2.92 (m, 2H, H-6, H-6'), 2.84 (dd, 1H, J=12.2,2.1 Hz, H-2); `Ring II`: .delta.H=3.56 (dd, 1H, J=11.5, 7.4 Hz,H-5), 3.46 (dd, 1H, J=10.5, 8.9 Hz, H-4), 3.23 (dd, 1H, J=10.3, 9.1Hz, H-6), 2.81 (ddd, 1H, J=12.7, 9.1 Hz, H-1), 2.65 (ddd, 1H,J=12.7, 9.8 Hz, H-3), 2.00 (dt, 1H, J=9.1, 4.1 Hz, H-2eq), 1.24(ddd, 1H, J=12.3, 1.2 Hz, H-2ax); `Ring III`: .delta.H=5.41 (d, 1H,J=2.4 Hz, H-1), 4.40 (dd, 1H, J=6.5, 4.9 Hz, H-3), 4.18 (dd, 1H,J=5.1, 2.4 Hz, H-2), 4.09 (dd, 1H, J=6.0, 1.3 Hz, H-4), 3.91 (dd,1H, J=11.1, 1.5 Hz, H-5), 3.78 (dd, 1H, J=11.5, 3.3 Hz, H-5');`Ring IV`: .delta.H=4.97 (d, 1H, J=1.6 Hz, H-1), 3.97 (dd, 1H,J=4.0, 2.4 Hz, H-3), 3.53-3.51 (m, 2H, H-4, H-5), 3.00 (dd, 1H,J=3.6, 1.6 Hz, H-2), 2.82 (dd, 1H, 9.3 Hz, H-6), 2.68 (dd, 1H,J=2.9 Hz, H-6'); the additional peaks in the spectrum wereidentified as follow: 3.37-3.35 (m, 1H, RCH.sub.2C.dbd.ONHR), (m,2H, RHN(CH.sub.2).sub.2NHR), 2.62 (m, 3H, CH.sub.3NHR).

[0638] .sup.13C NMR (125 MHz, MeOD): .delta.C=174.58 (Amide),109.87 (C-1''), 100.70 (C-1'''), 100.61 (C-1'), 86.15 (C-5), 83.36(C-4''), 79.11 (C-6), 77.26 (C-3''), 76.12 (C-2''), 75.60 (C-3'),74.82 (C-3'''), 74.43 (C-4), 73.10 (C-5'''), 72.07 (C-5'), 71.02(C-4'''), 63.79 (C-2'''), 62.90 (RCH.sub.2C.dbd.ONHR), 62.03(C-5''), 59.42 (CH.sub.2), 58.87 (CH.sub.2), 58.18 (C-4'), 57.77(C-3), 56.07 (C-2'), 54.23 (C-1), 52.38, 51.62, 46.05, 45.10, 43.31(C-6'''), 42.74 (C-6'), 41.71, 39.20 (CH.sub.3NHR), 37.46(C-2).

[0639] TOFMS calcd for C.sub.28H.sub.57N.sub.9O.sub.13K ([M+K]+)m/e 766.41; measured m/e 766.67).

Preparation of4'-[2-(2-(2-aminoethylamino)ethylamino)acetamido]-4'-deoxy-2'',3',3''',4'-'',5'',6-hexa-O-benzyl-1,2',2''',3,6',6'''-hexaazido-neomycin(36)

[0640] Compound 33 (0.5 gram, 0.36 mmol) was dissolved in DMF (40mL) followed by the addition of diethylenetriamine (0.4 mL, 0.37mmol) and the reaction was left to stir at room temperature. Thereaction progress was monitored by TLC [EtOAc/MeOH/MeNH.sub.2 (33%solution in EtOH), 10:10:1] which indicated completion after 12hours. After completion of the reaction the solvent was evaporatedand the crude product was purified by flash chromatography(CHCl.sub.3/MeOH, 80:20) to afford compound 36 (0.62 gram,quantitative).

[0641] .sup.1H NMR (500 MHz, CDCl.sub.3): `ring I`: .delta.H=6.25(d, 1H, J=4.18 Hz, H-1), 4.33-4.27 (m, 1H, H-5), 4.00 (dd, 1H,J=9.88, 9.88 Hz, H-3), 3.91 (dd, 1H, J=10.73, 10.05 Hz, H-4),3.38-3.31 (m, 2H, H-6, H-6'), 3.15 (dd, 1H, J=10.27, 3.00 Hz, H-2);`ring II`: .delta.H=3.87 (dd, 1H, J=8.99, 8.99 Hz, H-5), 3.73 (dd,1H, J=10.16, 9.35 Hz, H-4), 3.65-3.55 (m, 2H, H-1, H-3), 3.35 (dd,1H, J=9.74, 8.93 Hz, -6H), 2.29 (dt, 1H, J=12.42, 4.37, 4.37 Hz,H-2eq), 1.45 (ddd, 1H, J=12.51, 12.49, 12.49 Hz, H-2ax); `ringIII`: .delta.H=5.65 (d, 1H, J=4.48 Hz, H-1), 4.30 (dd, 1H, J=4.15,4.15 Hz, H-3), 4.25-4.23 (m, 1H, H-4), 4.05 (dd, 1H, J=4.91, 4.91Hz, H-2), 3.84 (dd, 1H, J=10.69, 2.47 Hz, H-5), 3.61 (dd, 1H,J=10.54, 3.99 Hz, H-5'); `ring IV`: .delta.H=4.92 (d, 1H, J=1.99Hz, H-1), 3.92-3.86 (m, 2H, H-3, H-5), 3.60 (dd, 1H, J=12.35, 9.50Hz, H-6), 3.39-3.33 (m, 2H, H-2, H-4), 3.17-3.08 (m, 1H, H-6'); theadditional peaks in the spectrum were identified as follow:7.43-7.21 (m, 30H, Ar), 4.94 (d, 1H, J=10.93 Hz, BnCH.sub.2), 4.78(d, 1H, J=10.89 Hz, BnCH.sub.2), 4.74 (d, 1H, J=11.33 Hz,BnCH.sub.2), 4.64 (d, 1H, J=11.65 Hz, BnCH.sub.2), 4.60 (d, 1H,J=11.25 Hz, BnCH.sub.2), 4.57-4.50 (m, 5H, BnCH.sub.2), 4.44 (d,1H, J=12.12 Hz, BnCH.sub.2), 4.41 (d, 1H, J=11.61 Hz, BnCH.sub.2),3.21 (s, 2H, BnCH.sub.2), 2.86-2.73 (m, 4H, CH.sub.2), 2.70-2.60(m, CH.sub.2).

[0642] .sup.13C NMR (126 MHz, CDCl.sub.3): .delta.C=174.44 (Amide),139.63 (Ar), 139.52 (Ar), 139.16 (Ar), 138.95 (Ar), 138.87 (Ar),129.66 (Ar), 129.59 (Ar), 129.49 (Ar), 129.46 (Ar), 129.38 (Ar),129.37 (Ar), 129.22 (Ar), 129.02 (Ar), 128.92 (Ar), 128.78 (Ar),128.75 (Ar), 128.73 (Ar), 128.59 (Ar), 108.31 (C-1''), 99.95(C-1'''), 97.22 (C-1'), 85.44 (C-6), 83.53 (C-4''), 83.39 (C-5),83.00 (C-2''), 78.91, 78.23 (C-3'), 77.60, 77.29 (C-3''), 77.12(C-4), 76.08 (BnCH.sub.2), 75.72, 75.60, 75.14, 74.70, 74.62(BnCH.sub.2), 74.45 (BnCH.sub.2), 74.26 (BnCH.sub.2), 73.62(BnCH.sub.2), 73.49, 73.07 (BnCH.sub.2), 72.18, 72.12 (C-5'), 71.65(C-5''), 64.23 (C-2'), 62.88, 61.78, 61.14 (C-1, C-3), 58.59(C-2'''), 57.43, 53.00 (C-6'), 52.94 (C-4'), 52.76 (CH.sub.2),52.43, 52.37 (C-6'''), 49.68 (CH.sub.2), 49.62 (CH.sub.2), 41.46(CH.sub.2), 33.11 (C-2).

[0643] MALDI TOFMS calcd for C.sub.71H.sub.84N.sub.22O.sub.13([M+Na]+) m/e 1475.65; measured m/e 1475.23).

Preparation of 4'-amino-6'-[2-(2-(2-aminoethylamino)ethylamino)acetamido]-4'-deoxy-neomycin (Compound 10; FIGS. 1 and 3)

[0644] The titled compound was prepared as described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: Compound 36 (580 mg, 0.4 mmol), THF (10 mL), NaOH (0.1M,5 mL), PMe.sub.3 (1M solution in THF, 14.5 mL, 14.5 mmol) to yieldthe compound as free amine form. Birch reduction: THF (10 mL),ammonia (about 20 mL), small pieces of Na (500 mg, 21.7 mmol),ammonium formate (1 gram, 15.7 mmol). The analytically pure productwas obtained by passing the above product through a short column ofAmberlite CG50 (NH.sub.4+ form). The column was first washed withMeOH and H.sub.2O, then the product was eluted with a mixture ofH.sub.2O/NH.sub.4OH (91:9) to afford Compound 10 (109 mg, 36% fortwo steps). For the storage and biological tests, the compound wasconverted to its sulfate salt form: the free base was dissolved inwater, the pH was adjusted around 7.0 with H.sub.2SO.sub.4 (0.1 N)and lyophilized.

[0645] .sup.1H NMR (500 MHz, D.sub.2O): `ring I`: .delta.H=5.28 (d,1H, J=3.39 Hz, H-1), 3.77 (ddd, 1H, J=12.61, 5.62, 2.95 Hz, H-5),3.56 (dd, 1H, J=14.28, 4.50 Hz, H-6), 3.42 (dd, 1H, J=14.62, 3.09Hz, H-6'), 3.40 (dd, 1H, J=9.80, 9.80 Hz, H-3), 2.66 (dd, 1H,J=10.17, 3.71 Hz, H-2), 2.47 (dd, 1H, J=9.59, 9.59 Hz, H-4); `ringII`: .delta.H=3.64-3.55 (m, 1H, H-5), 3.43-3.32 (m, 1H, H-4),3.26-3.17 (m, 1H, H-6), 2.88-2.75 (m, 1H, H-3), 2.71-2.64 (m, 1H,H-1), 1.90 (dt, 1H, J=9.05, 3.92, 3.92 Hz, H-2eq), 1.22-1.07 (m,1H, H-2ax); `ring III`: .delta.H=5.43 (d, 1H, J=5.42 Hz, H-1),4.55-4.49 (m, 1H, H-3), 4.40-4.33 (m, 1H, H-2), 4.25-4.20 (m, 1H,H-4), 3.98-3.94 (m, 1H, H-5), 3.83-3.79 (m, 1H, H-5); `ring IV`:.delta.H=5.06 (d, 1H, J=2.40 Hz, H-1), 4.11 (dd, J=3.83, 3.83 Hz,H-3), 4.10-3.99 (m, 1H, H-5), 3.74 (dd, 1H, J=2.10, 2.10 Hz, H-4),3.19-3.09 (m, H, H-6), 3.12 (dd, 1H, J=4.01, 1.99 Hz, H-2),3.07-2.98 (m, 1H, H-6'); the additional peaks in the spectrum wereidentified as follow: 3.44 (s, 2H, CH.sub.2), 3.05-2.79 (m, 8H,CH.sub.2).

[0646] .sup.13C NMR (126 MHz, D20): .delta.C=174.39 (Amide), 108.57(C-1''), 99.49 (C-1'), 99.40 (C-1'''), 84.08 (C-5), 83.61 (C-4),81.69 (C-4''), 77.42 (C-6), 76.10 (C-3''), 75.17 (C-5'''), 73.47(C-2''), 73.08 (C-3'), 72.44 (C-5'), 70.71 (C-3'''), 68.62(C-4'''), 61.42 (C-5''), 55.86 (C-2'), 53.94 (C-4'), 52.76(C-2'''), 51.06 (CH.sub.2), 50.42 (C-1, C-3), 48.23 (CH.sub.2),47.62 (CH.sub.2), 47.26 (CH.sub.2), 41.17 (C-6'''), 39.99 (C-6'),39.06 (CH.sub.2), 35.63 (C-2).

[0647] MALDI TOFMS calcd for C.sub.29H.sub.60N.sub.10O.sub.13([M]+) m/e 756.43; measured m/e 756.40).

Preparation of4'-[2-(2-(2-aminoethylamino)ethylamino)acetamido]-4'-deoxy-neomycin(Compound 8; FIGS. 1 and 3)

[0648] The titled compound was prepared as described for thepreparation of Compound 1 with the following quantities: Staudingerreaction: Compound 36 (580 mg, 0.4 mmol), THF (10 mL), NaOH (0.1M,5 mL), PMe.sub.3 (1M solution in THF, 14.5 mL, 14.5 mmol) to yieldthe compound as free amine form. Birch reduction: THF (10 mL),ammonia (about 20 mL), small pieces of Na (55 mg, 2.31 mmol),ammonium formate (1 gram, 15.7 mmol). The analytically pure productwas obtained by passing the above product through a short column ofAmberlite CG50 (NH.sub.4+ form). The column was first washed withMeOH and H.sub.2O, then the product was eluted with a mixture ofH.sub.2O/NH.sub.4OH (91:9) to afford Compound 8 (60.2 mg, 20% fortwo steps). For the storage and biological tests, compound wasconverted to its sulfate salt form: the free base was dissolved inwater, the pH was adjusted around 7.0 with H.sub.2SO.sub.4 (0.1 N)and lyophilized.

[0649] .sup.1H NMR (500 MHz, MeOD): `ring I`: .delta.H=5.52 (d, 1H,J=3.52 Hz, H-1), 3.89-3.84 (m, 1H, H-5), 3.80 (dd, 1H J=10.12,10.12 Hz, H-3), 3.71 (dd, 1H, J=10.12, 9.16 Hz, H-4), 2.82 (ddd,1H, J=13.68, 2.48, 1.04 Hz, H-6), 2.77-2.69 (m, 2H, H-2, H-6');`ring II`: .delta.H=3.56 (dd, 1H, J=8.50, 4.82 Hz, H-5), 3.46 (dd,1H, J=8.89, 8.89 Hz, H-4), 3.24 (dd, 1H, J=11.10, 7.66 Hz, H-6),2.85-2.78 (m, 1H, H-3), 2.71-2.62 (m, 1H, H-1), 2.01 (dt, 1H,J=12.84, 3.76, 3.76 Hz, H-2eq), 1.24 (ddd, 1H, J=12.32, 12.32,12.32 Hz, H-2ax); `ring III`: .delta.H=5.37 (d, 1H, J=3.28 Hz,H-1), 4.43-4.39 (m, 1H, H-3), 4.23-4.18 (m, 1H, H-2), 4.12-4.09 (m,1H, H-4), 3.86 3.81 (m, 1H, H-5), 3.75 (dd, 1H, J=7.61, 4.44 Hz,H-5'); `ring IV`: .delta.H=4.97 (d, 1H, J=2.10 Hz, H-1), 3.98 (dd,1H, J=3.34, 3.34 Hz, H-3), 3.93-3.89 (m, 1H, H-5), 3.53 (bs, 1H,H-4), 3.08 (dd, 1H, J=12.89, 8.13 Hz, H-6), 3.01 (bs, 1H, H-2),2.89 (dd, 1H, J=13.67, 4.21 Hz, H-6'); the additional peaks in thespectrum were identified as follow: 3.43-3.23 (m, 2H, CH.sub.2),2.91-2.79 (m, 2H, CH.sub.2), 2.85-2.86 (m, 6H, CH.sub.2).

[0650] .sup.13C NMR (126 MHz, MeOD): .delta.C=175.18 (Amide),109.80 (C-1''), 100.73 (C-1', C-1'''), 86.06 (C-5), 83.95 (C-4),83.37 (C-2''), 79.01 (C-6), 77.42 (C-3''), 76.28 (C-5'''), 75.54(C-4''), 73.18 (C-5'), 72.11 (C-3''', C-3'), 70.77 (C-4'''), 62.20(C-5''), 58.26 (C-2'), 54.74 (C-2'''), 54.06 (C-4'), 52.44 (C-1),53.16 (CH.sub.2), 52.35 (C-3), 51.72 (CH.sub.2), 49.69 (CH.sub.2),43.52 (C-6'), 43.34 (C-6'''), 41.49 (CH.sub.2), 37.48 (C-2).

[0651] MALDI TOFMS calcd for C.sub.29H.sub.60N.sub.10O.sub.13([M]+) m/e 756.43; measured m/e 756.74).

Example 3

Antibacterial Activity and Protein Translation Inhibition

[0652] The minimal inhibitory concentration (MIC) values of thenewly designed exemplary Compounds 1-10 were determined againstwild-type (WT) Gram-negative and Gram-positive bacteria.

[0653] The bacterial strains that were included in these tests wereas follows:

[0654] Two wild-type (WT) E. coli strains (R477-100 and 25922) asrepresentatives of Gram-negative bacteria with unknown resistanceto aminoglycosides [V. Pokrovskaya, V. Belakhov, M. Hainrichson, S.Yaron, T. Baasov, J. Med. Chem. 2009, 52, 2243-2254] and two WTStaphylococcus epidermidis and Bacillus subtilis strains asrepresentatives of Gram-positive bacteria (the clinically usedaminoglycosides have significant antibacterial activity againstthese strains) [J. Kondo, M. Hainrichson, I. Nudelman, D.Shallom-Shezifi, C. M. C. M. Barbieri, D. S. D. S. Pilch, E.Westhof, T. Baasov, ChemBioChem 2007, 8, 1700-1709].

[0655] The resistant strains included MRSA, a Gram-positivebacterium, the treatment of which represents a great challenge inthe clinic; MRSA 252, which is known for its high resistance toaminoglycosides [M. T. G. Holden, E. J. Feil, J. A. Lindsay, S. J.Peacock, N. P. J. Day, M. C. Enright, T. J. Foster, C. E. Moore, L.Hurst, R. Atkin, et al., Proc. Natl. Acad. Sci. USA 2004, 101,9786-9791]; and MRSA CI 15877, which is resistant to naturalaminoglycosides [G. Kaneti, H. Sarig, I. Marjieh, Z. Fadia, A. Mor,FASEB J. 2013, 27, 4834-4843].

[0656] Other pathogens that were tested included several strains ofP. aeruginosa that have an inherent resistance to aminoglycosides[J. I. Sekiguchi, T. Asagi, T. Miyoshi-Akiyama, T. Fujino, I.Kobayashi, K. Morita, Y. Kikuchi, T. Kuratsuji, T. Kirikae,Antimicrob. Agents Chemother. 2005, 49, 3734-3742; M. Hainrichson,O. Yaniv, M. Cherniaysky, I. Nudelman, D. Shallom-Shezifi, S.Yaron, T. Baasov, Antimicrob. Agents Chemother. 2007, 51,774-776].

[0657] FIG. 5 presents a table showing the comparative MIC valuesof NeoB and Compounds 1-10 against the tested Gram-negative andGram-positive, pathogenic and resistant, strains.

[0658] The comparative data presented in FIG. 5 (Table 1) show thatall the new derivatives of NeoB, Compounds 2-10, exhibitsignificant antibacterial activity against both the WT andaminoglycoside-resistant strains, including Gram-negative andGram-positive bacteria.

[0659] The activities against the WT Gram-positive bacteria werediverse across the different strains tested. The activity of mostof the compounds against S. epidermidis is similar to or betterthan that of NeoB.

[0660] All new derivatives (Compounds 2-10) show significantlyimproved activity against the Gram-negative strains of pathogenicP. aeruginosa in comparison with NeoB.

[0661] P. aeruginosa is a nosocomial human pathogen known to beinherently resistant to aminoglycosides owing to the presence ofthe chromosomally encoded APH(3')-IIb enzyme. This enzyme catalyzesthe transfer of the ATP g-phosphoryl group to the 3'-hydroxy groupof many aminoglycosides, rendering them inactive as antibiotics[34] The observed improved activity of the new derivatives relativeto that of NeoB against the tested strains of P. aeruginosa can beexplained by the steric hindrance of the cationic warhead, whichintroduces unfavorable interactions with the APH(3')-IIb enzymeactive site.

[0662] An improvement in antibacterial performance of the Compounds2-10 versus that of NeoB was also observed against theGram-positive pathogenic MRSA strains. For example, Compounds 2 and8 exhibited MIC values that were 64 times lower than that of NeoB.The 4'-ether (Compounds 1-5) and 4'-amide (Compounds 6-8) compoundsexhibited substantially the same activity.

[0663] The antibacterial activity of Compound 1, a neamine-basedderivative, is substantially lower than that of the other compoundstested, indicating that its binding affinity to the A-site is muchlower.

[0664] Compounds 1-10 all showed antibacterial activity against theWT Geobacillus T1, also at 60.degree. C. Against the Geobacillus T1harboring the resistance to kanamycin, most of the new compoundsmaintained their high antibacterial activity, whereas NeoB almostlost its activity.

[0665] As can be seen, the introduced modifications to the NeoBstructure did not hinder the binding to the A-site and most of thederivatives retained significant antibacterial activity. Moreover,the new compounds overcame the existing resistance of P. aeruginosaand MRSA pathogens to aminoglycosides.

[0666] The protein translation inhibition was next tested bydetermining half-maximum inhibition levels (IC.sub.50 values, Table1). While most of the new compounds showed activity of the sameorder of magnitude as NeoB, the inhibitory potency of Compounds 6,9, and 10 with a 4'-nitrogen atom was two-fold higher than that ofNeoB (IC.sub.50 values of 0.006, 0.005, 0.006, and 0.01 forCompounds 6, 9, 10, and for NeoB, respectively).

[0667] Without being bound by any particular theory, it is assumedthat this may result from the additional interactions of the4'-amide (Compound 6) and 4'-amine (Compounds 9 and 10) groups ofthese compounds with the ribosomal A-site.

Example 4

RNase Activity Tests

[0668] The potential RNase activity of Compounds 1-10 was testedusing gel electrophoresis experiments, as previously reported forColE3 [C. L. Ng, K. Lang, N. A. G. Meenan, A. Sharma, Nat. Struct.Mol. Biol. 2010, 17, 1241-1246]. Experiments were first performedon full-size ribosomes isolated from E. coli, as previouslyreported [B. a. Maguire, L. M. Wondrack, L. G. Contillo, Z. Xu, RNA2008, 14, 188-195].

[0669] As a positive control, the RNase domain of the natural toxinColE3 was used (see, FIG. 6B). As shown in FIG. 6B, with ColE3,cleavage of approximately 40 bases from the 16S rRNA fragment(about 1540 nucleic bases) was observed, in a dose-dependentmanner.

[0670] As shown in FIGS. 6C and 6D, for the 5S and tRNA fragments,NeoB and Compound 3 did not show any signs of the cleaved productat concentrations up to 400 mm. At higher concentrations,solubility issues prevented detecting RNA cleavage.

[0671] As shown in FIG. 6A, within the same concentration range (upto 400 mm), ethylenediamine (a negative control) did not cleave thefull ribosome, suggesting that it is unable to bind to rRNAeffectively.

[0672] An A-site oligonucleotide model was next tested. Anoligonucleotide model similar to that used by Westhof andco-workers [P. Pfister, S. Hobbie, Q. Vicens, E. C. Bcttger, E.Westhof, ChemBioChem 2003, 4, 1078-1088; Q. Vicens, E. Westhof,Chem. Biol. 2002, 9, 747-755] for crystallographic studies wasselected. To improve RNA detection, a fluorescent Cy3 tag was addedat the 3' end (and not at the 5' end) to ensure that there was asignificant difference between the size of the full-length RNA andthe cleaved RNA, as shown in FIGS. 7A-B.

[0673] The cleavage experiments indicated that with ethylenediamine(N-2-N) non-specific cleavage was observed at high concentrations,100 and 200 mm of N-2-N, as shown in FIG. 8A.

[0674] In the presence of Compound 6, some RNA cleavage wasdetected at substantially lower concentrations, 10 mm, as shown inFIG. 8B. Double-stranded RNA (DS band in FIG. 8B) was alsoobserved, suggesting that the aminoglycoside binding stabilizeddouble-stranded RNA even though the gel was under denaturingconditions. Only nonspecific cleavage bands were observed at theconcentrations tested, and these fragments were longer than thoseexpected for specific and selective cleavage (<8 bases).

Example 5

MD Simulations

[0675] Conformational Dynamics of the Warheads and the Possibilityof RNA Cleavage:

[0676] Full-atom molecular dynamics (MD) followed by Gaussianaccelerated MD (GaMD) [Y. Miao, V. A. Feher, J. A. McCammon, J.Chem. Theory Comput. 2015, 11, 3584-3595; Y. T. Pang, Y. Miao, Y.Wang, J. A. McCammon, J. Chem. Theory Comput. 2017, 13, 9-19] wasperformed. The crystal structure of NeoB bound to theoligonucleotide model of the A-site rRNA (PDB ID:2ET4) [supra] wasused as a template for building the systems used in thesimulations.

[0677] The model of the A site contains two symmetricaminoglycoside binding sites using the crystal structure of the Asite with bound neomycin B (PDB code: 2ET4). The Compounds 2, 5, 8,and 10 were built with leap (Ambertools 17) based on the geometryof NeoB in the crystal structure (PDB ID: 2ET4). The initialstructures of the warheads were entirely linear not to favor anyconformation. All terminal amine groups in aminoglycosides wereprotonated. The aminoglycoside geometries were optimized at theHF/6-31G(d)/B3LYP/6-31G(d) level of theory and docked to the A siteby alignment to the neomycin moiety. The systems were then solvatedby adding 15 .ANG. layer of water molecules. Total molecular chargeof RNA was -40e and the charge of Compounds 2, 5, 8, and 10 was +7eeach. The negative charge of the system was neutralized with sodiumions and the ionic strength of 0.1 M NaCl was added. The atomiccharges of aminoglycosides were obtained using the RESP procedure2with Gaussian 09 and antechamber (Ambertools17) [D. A. Case, I. Y.Ben-Shalom, S. R. Brozell, D. S. Cerutti, T. E. Cheatham, III, V.W. D. Cruzeiro, T. A. Darden, R. E. Duke, D. Ghoreishi, M. K.Gilson, H. Gohlke, A. W. Goetz, D. Greene, R Harris, N. Homeyer, S.Izadi, A. Kovalenko, T. Kurtzman, T. S. Lee, S. LeGra, D. M. Y. andP. A. K. AMBER 2018. Univ. California, San Fr. 2018]. Their bondedand non-bonded parameters were assigned with GAFF2 usingantechamber and parmchk2 programs (Ambertools17). For RNA, theparameters of ff99OL3 were applied. TIP3P-FB model was used forexplicit water molecules [Wang, L. P.; Martinez, T. J.; Pande, V.S. Building Force Fields: An Automatic, Systematic, andReproducible Approach. J. Phys. Chem. Lett. 2014, 5 (11),1885-1891]. The simulated system is shown in FIG. 9. Compounds 2,5, 8, and 10, as representative examples, were simulated, and NeoBwas used as a control. The total MD and GaMD simulation time wasabout 5.5 ms. For compounds 2 and 5, two and three differentconformations of the warheads, respectively, were found, as shownin FIGS. 10 and 11. For both compounds, the dominant conformationof the warhead (82.7% of the population in 2 and 76.4% of thepopulation in 5) is characterized by a common intramolecularhydrogen bond between the N1 amine of the warhead and the N6'ammonium of the aminoglycoside ring I. These intramolecularhydrogen bonds may prevent the N1 amine of the warhead from actingas the general base to activate the 2'-OH group of the G1491 riboseas a nucleophile (see FIGS. 4A-B for the proposed mechanism).

[0678] As shown in FIGS. 12A-D and 13, the warhead of Compounds 8and 10, which represent the 4'- and 6'-amide derivatives of NeoB,is longer and does not form intramolecular interactions with therest of the molecule.

[0679] For Compound 10, as shown in FIGS. 12A-D, the largestconformational variability of the warhead was observed and isassociated with its rotation around the N2-C3-C4-N3 dihedral angle(FIG. 12B).

[0680] This coordinate was used in the clustering analysis and twomajor conformations of the warhead (72.2 (FIG. 12C) and 27.8% ofthe population (FIG. 12A)) were found (FIG. 12D). The short rangecontacts of the N4' ammonium with the phosphates of A1492 and A1493conformationally restrict the position of ring I in the A-site andalso the A1492 and A1493 backbone atoms.

[0681] For compound 8, three principal modes of binding of thewarhead to the rRNA were observed (see, FIG. 13). In the mostabundant binding mode (58.6% of the population), two short-rangeinteractions between the 2'-hydroxy group of the ribose (G1491) andthe N3 amine group of Compound 8 (proposed general base) andbetween the A1492 phosphate and the N4 ammonium of Compound 8(general acid) were observed. This conformational state of Compound8 is consistent with the suggested mechanism of A-site rRNAcleavage between G1491 and A1492, shown in FIG. 4B. In the secondmost-abundant binding mode of Compound 8 (21.6% of the population),the N2 amine group forms a hydrogen bond with the 2'-hydroxy groupof the ribose of G1491, which actually serves as the general base.The concomitant stabilization of the transition state through theinteraction of the warhead amines with the phosphate of A1492 islacking.

[0682] Compound 8 thus exhibits the interactions required for AsiterRNA cleavage between G1491 and A1492. The N3 amine of the warheadactivates the 2'-OH group of the G1491 ribose for nucleophilicattack, and the N4 ammonium of the warhead favorably binds to theOP2 and 03' atoms of the A1492 phosphate, which facilitatesnucleophilic attack.

[0683] In general, the efficiency of rRNA hydrolysis is highlydependent on the ability to induce the correct positioning of thenucleophile for in-line attack on the scissile bond. Enzymes, beinglarge, can mechanically achieve this step "easily" by distortingthe substrate to reach the conformation necessary for efficientcatalysis. For example, ColE3 [supra] and a-sarcin [C. C. Correll,X. Yang, T. Gerczei, J. Beneken, M. J. Plantinga, J. SynchrotronRadiat. 2004, 11, 93-96], the two bacterial toxins that cleave asingle phosphodiester bond of rRNA (in the small and largeribosomal subunits, respectively), both use RNA base flipping todock the substrate into the active site in such a manner so as tofacilitate crucial in-line attack.

[0684] In order to assess whether the aminoglycoside-warheadcombination can induce a similar conformational change in the rRNAA-site, the angle created between the 2'-OH (the G1491 ribose, thenucleophile), the phosphorus of the phosphate between G1491 andA1492, and the 5'-O (the leaving group), O--P--O angle, wasmeasured.

[0685] In the crystal structure of the Westhof model used for thesimulations, the O--P--O angle does not exceed 908. Thedistributions of the O--P--O angle, as obtained from GaMDsimulations of NeoB and Compounds 2, 5, 8, and 10 are shown in FIG.14. The smallest values of this angle are found for the NeoBcomplex, distributed in the range of 45 to 1058. The interactionsformed between O3' of NeoB (ring I) and OP2 of the A1492 phosphateand between O4' of NeoB (ring I) and OP2 of the A1493 phosphateseem to be the most important for orientation of the O--P--O angle.The modifications introduced into NeoB ring I to make Compounds 8and 10 clearly lead to an increase in this angle for bothderivatives, reaching as high as 170.degree. for Compound 10.

[0686] For Compound 8, this requirement is fulfilled thanks to thepersistent hydrogen bond formed by the 3'-hydroxy group of ring Iand concomitant stabilization of the A1492 phosphate by the N4ammonium group of the warhead. For Compound 10, the crucialshortrange interaction with the OP1 atom of the A1492 phosphate ismade by the N4' ammonium in ring I. Thus, stabilization of theO--P--O angle in the nearly in-line orientation is remarkable.

Example 6

Cytotoxicity and Eukaryotic Translation Inhibition

[0687] The cytotoxicity of the new compounds is determined in twokidney-derived cells, COS-7 and HEK-293 as previously reported [I.Nudelman, D. Glikin, B. Smolkin, M. Hainrichson, V. Belakhov, T.Baasov, Bioorganic Med. Chem. 2010, 18, 3735-3746; J. KandasamyAtia-Glikin, D., Belakhov, V. and Baasov, T., Med Chem Comm 2011,2, 165-171; A. Rebibo-Sabbah, I. Nudelman, Z. M. Ahmed, T. Baasov,T. Ben-Yosef, Hum. Genet. 2007, 122, 373-381].

[0688] The compounds are further tested for their selectivitytowards bacterial ribosomes versus eukaryotic cytoplasmic andmitochondrial ribosomes. For this purpose, the inhibition oftranslation in eukaryotic and mitochondrial systems is performed,as previously reported [Nudelman, D.

[0689] Glikin, B. Smolkin, M. Hainrichson, V. Belakhov, T. Baasov,Bioorganic Med. Chem. 2010, 18, 3735-3746; I. Nudelman, A.Rebibo-Sabbah, M. Cherniavsky, V. Belakhov, M. Hainrichson, F.Chen, J. Schacht, D. S. Pilch, T. Ben-Yosef, T. Baasov, J. Med.Chem. 2009, 52, 2836-2845; J. Kandasamy, D. Atia-Glikin, E.Shulman, K. Shapira, M. Shavit, V. Belakhov, T. Baasov, J. Med.Chem. 2012, 55, 10630-10643].

[0690] Although the invention has been described in conjunctionwith specific embodiments thereof, it is evident that manyalternatives, modifications and variations will be apparent tothose skilled in the art. Accordingly, it is intended to embraceall such alternatives, modifications and variations that fallwithin the spirit and broad scope of the appended claims.

[0691] It is the intent of the applicant(s) that all publications,patents and patent applications referred to in this specificationare to be incorporated in their entirety by reference into thespecification, as if each individual publication, patent or patentapplication was specifically and individually noted when referencedthat it is to be incorporated herein by reference. In addition,citation or identification of any reference in this applicationshall not be construed as an admission that such reference isavailable as prior art to the present invention. To the extent thatsection headings are used, they should not be construed asnecessarily limiting. In addition, any priority document(s) of thisapplication is/are hereby incorporated herein by reference inits/their entirety.

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