US 20140357591A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0357591 A1 BAASOV et al. (43) Pub. Date: Dec. 4, 2014

(54) CONJUGATED ANTIMICROBIAL AGENTS Publication Classification (71) Applicant: Technion Research & Development (51) Int. Cl. Foundation Limited, Haifa (IL) A647/48 (2006.01) AOIN 43/60 (2006.01) (72) Inventors: Timor BAASOV. Haifa (IL); Varvara (52) U.S. Cl. Pokrovskaya, Nesher (IL); Valery CPC ...... A61K47/48092 (2013.01); A0IN 43/60 Belakhov, Haifa (IL); Mariana (2013.01) Hainrichson, Kiryat-Haim (IL) USPC ...... 514/39; 514/41 (21) Appl. No.: 14/461,494 (57) ABSTRACT Provided herein are antimicrobial conjugates of two antibi (22) Filed: Aug. 18, 2014 otic agents, exhibiting improved activity also against resistant bacteria, compared to each of the agents separately or their Related U.S. Application Data mixture, and having Substantially no resistance emerged (62) Division of application No. 13/260,590, filed on Sep. thereagainst, aS well as processes for preparation the same, compositions containing the same, and uses thereof in medi 27, 2011, now Pat. No. 8,809,286, filed as application cal treatments against pathogenic microorganisms. The dis No. PCT/IL2010/000257 on Mar. 25, 2010. closed antimicrobialents against conjugates paunog are composed9. of aminogly (60) Provisional application No. 61/164,951, filed on Mar. cosides and non-ribosomal active antibiotics. Some of the 31, 2009. antimicrobial conjugates are prepared via "click' chemistry. Patent Application Publication Dec. 4, 2014 US 2014/0357591 A1

nicked Open 1 2 3 4 5 6 7 8 Circular DNA^ - (topoisomers)relaxed DNA SuperColled DNA

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rinaCipro Cipro"NeOB NeoB .i FG. 2A US 2014/0357591 A1 Dec. 4, 2014

CONUGATED ANTIMICROBAL AGENTS ofexisting aminoglycoside drugs 1, 2. Earlier investigations in this direction have yielded several semi-synthetic drugs RELATED APPLICATIONS Such as amikacin, dibekacin, and arbekacin 1, 3]. However, 0001. This application is a division of U.S. patent appli new resistance to these drugs has emerged soon after their cation Ser. No. 13/260,590 filed on Sep. 27, 2011, which is a introduction to the clinic 4, 5. National Phase of PCT Patent Application No. PCT/IL2010/ 0008. One strategy that has been pursued in recent years to 000257 having International filing date of Mar. 25, 2010, overcome bacterial resistance to aminoglycoside drugs which claims the benefit of priority under 35 USC S119(e) of employs a combination of two different drugs in one mol U.S. Provisional Patent Application No. 61/164,951 filed on ecule 6. With this strategy, each drug moiety is designed to Mar. 31, 2009. The contents of the above applications are all bind independently to two different biological targets and incorporated herein by reference. synchronously accumulate at both target sites. Such dual action drugs, also referred to as hybrid drugs or conjugate FIELD AND BACKGROUND OF THE drugs, offer the possibility to overcome current resistance. In INVENTION addition, these conjugate drugs may reduce the appearance of new resistant strains 7. 0002 The present invention, in some embodiments 0009. Several applications of this approach have been thereof, relates to antimicrobial agents, and more particularly, reported 8-10. The dual action compounds, combining fluo but not exclusively, to non-resistance inducing antimicrobial roquinolone (enrofloxacin or norfloxacin) and cephalosporin conjugates which are effective also against resistant bacteria, (cefamandole) moieties with an amide linkage, were found and to uses thereof in treating infections. potent against Enterobacter species 9. Fluoroquinolone 0003. Early advancements in the field of antibiotics had anilinouracil conjugates linked via their secondary amino transformed medical care and dramatically reduced illness groups have also been synthesized 10. A series of oxazoli and death from infectious diseases. However, over the dinone-quinolone conjugate structures, which simulta decades, almost all the prominent infection-causing bacterial neously act on two different cellular functions, DNA replica strains have developed resistance to antibiotics. Among the tion and protein synthesis, have been reported 7, 11. Lead different classes of clinically important antibiotics that compounds of this series exhibited a balanced dual mode of largely suffered from the resistance problem during the last action and overcome the majority of known resistance few decades, is the aminoglycoside class of drugs. These mechanisms to quinolones and lineZolid in clinically relevant antibiotics have broad-spectrum of activity against both Gram-positive pathogens. Gram-negative and Gram-positive bacteria by selectively tar 0010 Investigations towards glycopeptide/beta-lactam geting bacterial protein synthesis machinery, and have been heterodimers were reported, employing Vancomycin and used for over fifty years. Such a prolonged clinical and vet cephalosporin synthons which were chemically linked to erinary use of currently available aminoglycosides has yield heterodimer antibiotics 12. resulted in effective selection of resistance, which severely (0011 U.S. Pat. No. 7,635,685 (see also 13) teaches limits their usefulness. modifications of aminoglycoside neomycin B (NeoB) by 0004. Due to the limitations associated with the use of linking a variety of Sugars at C5'-OH group via glycosidic classical antibiotics, extensive studies have been focused on linkage, which results with a class of pseudo-pentasaccha finding novel, efficient and non-resistance inducing antimi rides that exhibited similar or better antibacterial activities to crobial/antibacterial agents. that of the parent NeoB against selected bacterial strains. 0005. The most prevalent mechanism inclinical isolates of However, while the specificity constant values (kcat/Km) of resistant bacteria is the bacterial acquisition of aminoglyco these derivatives with the aminoglycoside resistance enzyme side-modifying enzymes, which modify the antibiotics by APH(3)-IIIa were in general lower than that of NeoB, the N-acetyltransferase (AAC), 0-phosphotransferase (APH), compounds exhibited inhibition values about 10-fold lower and 0-nucleotidylyltransferase (ANT) activities. Among than that of NeoB, suggesting that several different confor these enzymes families, aminoglycoside 3'-phosphotrans mations of the designed structures can bind the APHC3")-IIIa ferases (APHC3")s), of which seven isozymes are known, are productively and lead to the enzyme-catalyzed phosphoryl widely represented. These enzymes catalyZephosphorylation transfer process. at the 3'-OH to of both neomycin and kanamycin classes of 0012 Several other derivatives of non-sugar modifications aminoglycosides, rendering the resulting phosphorylated of NeoB at the C5"-position were reported to exhibit products inactive. enhanced antibacterial activity compared to the parent NeoB 0006 Although most of these enzymes are typically 14, however these derivatives also exhibited substrate pro monofunctional enzymes, the recent emergence of genes miscuity with respect to APHC3")-IIIa. encoding bifunctional aminoglycoside-modifying enzymes 0013 Additional background art includes a review of is another complication relevant to the clinical use of ami recent patent literature concerning heterodimers antibiotics noglycosides. Among these enzymes, the bifunctional AAC 15, WO 2003/044034 and U.S. Patent Application having (6')/APH(2") enzyme has been detected in Enterococcus, Sta phylococcus, and Streptococcus isolates, including the publication No. 2008300199. methicillin-resistant Staphylococcus aureus (MRSA), and has been the most extensively investigated, due to the large SUMMARY OF THE INVENTION number of clinically importantaminoglycosides that are sus 0014. The present invention, in some embodiments ceptible for modification with this enzyme. thereof, relates to antimicrobial agents, and more particularly, 0007 To tackle the problem of bacterial resistance caused but not exclusively, to non-resistance inducing antimicrobial by enzymatic modification, many analogs of aminoglyco conjugates effective against non-resistant and resistant bac sides have been synthesized by direct chemical modification teria. US 2014/0357591 A1 Dec. 4, 2014

0015 The present inventors have surprisingly uncovered Ciprobay, Ciproxin), balofloxacin (Baloxin), cinoxacin (Ci that conjugation of aminoglycosides with non-ribosomal nobac), clinafloxacin, danofloxacin (Advocin, Advocid), active antimicrobial agents results in a series of efficacious delafloxacin, difloxacin (Dicural, Vetequinon), enoxacin novel antimicrobial conjugate agents which are further char (Enroxil, Penetrex), enrofloxacin (Baytril), fleroxacin (Meg acterized by lack of emergence of resistance thereagainst. alone, Roquinol), flumequine (Flubactin), garenoxacin The present inventors have utilized the "click' chemistry for (Geninax), gatifloxacin (Tequin, Zymar), gemifloxacin (Fac preparing a class of such novel antimicrobial conjugates. tive), grepafloxacin (Raxar), ibafloxacin (Ibaflin), levofloxa 0016 Hence, according to embodiments of one aspect of cin (Cravit, Levaquin), lomefloxacin (Maxaquin), marbof the present, there is provided a conjugate having the general loxacin (Marbocyl. Zenequin), moxifloxacin (AVelox, formula I: Vigamox), nadifloxacin (Acuatim, Nadoxin, Nadixa), nalid Formula I ixic acid (NegGam, Wintomylon), norfloxacin (Lexinor, Noroxin, Quinabic, Janacin), ofloxacin (Floxin, Oxaldin, 0017 wherein: Tarivid), orbifloxacin (Orbax, Victas), oxolinic acid (Uroxin), 0018) A is a non-ribosomal-active antimicrobial agent paZufloxacin (Pasil, PaZucross), pefloxacin (Peflacine), pipe moiety; midic acid (Dolcol), piromidic acid (Panacid), prulifloxacin 0019 B is an aminoglycoside-based antimicrobial (Quisnon), roSoxacin (Eradacil), rufloxacin (Uroflox), Sara agent moiety; floxacin (Floxasol, Saraflox, Sarafin), sitafloxacin (Gracevit), 0020 X is a first spacer moiety, covalently bound to A, sparfloxacin (Zagam), temafloxacin (Omniflox), toSufloxacin or absent; (OZex, Tosacin) and trovafloxacin (Trovan). 0021 Y is a second spacer moiety, covalently bound to 0033 According to some embodiments of the invention, B, or absent; and the quinolone-based antimicrobial agent is ciprofloxacin. 0022 D is a linking moiety having the general formula 0034. According to some embodiments of the invention, II: the ciprofloxacin is covalently bound to X via the terminal nitrogen of the piperazine moiety thereof.

Formula II 0035. According to some embodiments of the invention, NN the aminoglycoside-based antimicrobial agent is selected / from the group consisting of neomycin B, neomycin C, strep Na tomycin, framycetin, paromomycin, ribostamycin, kanamy cin, amikacin, arbekacin, bekanamycin, dibekacin, tobramy cin, spectinomycin, hygromycin, paromomycin, gentamicin, R netilmicin, Sisomicin, isepamicin, Verdamicin and astromi cin. 0023 whereas each of the wiggled lines denote covalent 0036. According to some embodiments of the invention, bond to either A-X— or B Y , and R is selected the aminoglycoside-based antimicrobial agent is covalently from the group consisting of hydrogen, alkyl and alk bound to Y via the C5"-position thereof. enyl. 0037 According to some embodiments of the invention, 0024. According to some embodiments of the invention, the aminoglycoside-based antimicrobial agent is neomycin the non-ribosomal-active antibiotic agent is selected from the B group consisting of an anti-metabolite-based antimicrobial agent, a quinolone-based antimicrobial agent, a B-lactam 0038 According to some embodiments of the invention, based antimicrobial agent, a glycopeptide-based antimicro the aminoglycoside-based antimicrobial agent is covalently bial agent, a benzyl-2,4-diaminopyrimidine-based antimicro bound to Y via the C1-N-position thereof. bial agent, a Sulfonamide-based antimicrobial agent, a 0039. According to some embodiments of the invention, Sulfanilamide-based antimicrobial agent, a peptide-based the aminoglycoside-based antimicrobial agent is kanamycin antimicrobial agent, a pseudo-peptide-based antimicrobial A agent and a peptidomimetic-based antimicrobial agent. 0040. According to some embodiments of the invention, 0025. According to embodiments of another aspect of the each of X and Y, when present, is independently selected from present, there is provided a conjugate having the general the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, formula III: heteroalicyclic, aryl, heteroaryland a hydrocarbon chain hav ing 1-20 carbon atoms and ending or interrupted by at least A-X W Y B Formula III one heteroatom selected from the group consisting of O. S 0026 wherein: and N and/or containing from 0 to 19 unsaturated carbon 0027 A' is a quinolone-based antimicrobial agent moi carbon or carbon-heteroatom bonds. ety; 0041 According to some embodiments of the invention, 0028 B is an aminoglycoside-based antimicrobial each of X and Y, when present, is independently selected from agent moiety; the group consisting of —CH2—, —CH2—O— —(CH2) 0029 X is a first spacer moiety, covalently bound to A, , —(CH2). O— —(CH2). , —(CH2). O—, or absent; (CH) , —(CH) , —(CH) , —(CH(CH))— 0030 Y is a second spacer moiety, covalently bound to CH-, -CH=CH-CH=CH-, -C=C C=C , B, or absent; and —CHCH(OH)CH , —CH, O CH , —CH, O 0031 W is a linking moiety. CH-O-, -(CH2) O-(CH) , —(CH), O 0032. According to some embodiments of the invention, (CH2). O—, —CH2—mCH, CH2—, —CH2-pCH the quinolone-based antimicrobial agent is selected from the CH , —CH NHCO—, —CH NHCO , —CH group consisting of a fluoroquinolone, ciprofloxacin (Cipro, US 2014/0357591 A1 Dec. 4, 2014

0042. According to some embodiments of the invention, 0049 According to some embodiments of the invention, W is selected from the group consisting of a covalent bond, the conjugate presented herein is identified for use in the amide, carboxylate, cycloalkene, cyclohexene, heteroalicy treatment of a medical condition associated with a pathogenic clic, heteroaryl, triazine, triazole, disulfide, lactone, lactam, microorganism in a Subject. imine, aldimine, ketimine, hydraZone and semicarbazone. 0050. According to embodiments of another aspect of the present, there is provided a process of preparing the conjugate 0043. According to some embodiments of the invention, of any of claims 1-20, the process is effected by: W is having the general formula II: 0051 reacting a compound having the general formula IV: Formula II A-X—RG Formula IV 0052 wherein: 0.053 A is a non-ribosomal-active antimicrobial agent moiety; 0.054 X is a first spacer moiety, covalently bound to A, or absent; and 0055 RG is a first reactive group; 0044 whereas each of the wiggled lines denote covalent 0056 with a compound having the general formula V: bond to either A-X— or B Y , and R is selected RG-Y-B Formula V from the group consisting of hydrogen, alkyl and alk enyl. 0057 wherein: 0.058 B is an aminoglycoside-based antimicrobial 0045. According to some embodiments of the invention, agent moiety; R is hydrogen. 0059 Y is a second spacer moiety, covalently bound to 0046 According to some embodiments of the invention, A B, or absent; and is selected from the group consisting of neomycin B and 0060 RG is a second reactive group; kanamycin A. 0061 whereas one of RG or RG is alkynyl and the other is azide; 0047 According to some embodiments of the invention, B 0062 thereby forming the linking moiety D and obtain is ciprofloxacin. ing the conjugate. 0048. According to some embodiments of the invention, 0063. According to some embodiments of the invention, the conjugate presented herein is selected from the group the conjugation reaction is performed in the presence of a consisting of N-(4-(1-(2-(ciprofloxacin)ethyl)-1H-1,2,3-tria copper(I) catalyst. Zol-4-yl)-5'-phenyl) neomycin, N-(4-(1-(2-(ciprofloxacin) 0064. According to some embodiments of the invention, propyl)-1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin Ca the conjugation reaction is performed at room temperature. boxamide, N-(4-(1-(2-(ciprofloxacin)butyl)-1H-1,2,3- 0065 According to some embodiments of the invention, triazol-4-yl)-5'-phenyl)neomycin carboxamide, N-(4-(1-(2- the conjugation reaction is performed under mild microwave (ciprofloxacin)pentyl)-1H-1,2,3-triazol-4-yl)-5'-phenyl) irradiation. neomycin carboxamide, N-(4-(1-(2-(ciprofloxacin)hexyl)- 0066. According to some embodiments of the invention, 1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin carboxamide, the conjugation reaction is a "click” reaction. N-(4-(1-(2-hydroxy-3-(ciprofloxacin)propyl)-1H-1,2,3-tria 0067. According to embodiments of one aspect of the Zol-4-yl)-5'-phenyl)neomycin carboxamide, N-(4-(1-(2-(2- present, there is provided a process of preparing the conjugate (ciprofloxacin)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5'-phe of claim 3, the process is effected by: nyl)neomycin carboxamide, N-(4-(1-(4-((ciprofloxacin) 0068 reacting a compound having the general formula methyl)benzyl)-1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin VI: carboxamide, N-(4-(1-(3-((ciprofloxacin)methyl)benzyl)- 1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin carboxamide, A'-X-RG' Formula VI N-((1-(4-(ciprofloxacin)ethyl)-1H-1,2,3-triazol-4-yl)-5"- 0069 wherein: methyl)neomycin carboxamide, N-((1-(4-(ciprofloxacin) 0070 A' is a quinolone-based antimicrobial agent moi propyl)-1H-1,2,3-triazol-4-yl)-5'-methyl)neomycin Ca ety; boxamide, N-((1-(4-(ciprofloxacin)pentyl)-1H-1,2,3-triazol 0071 X is a first spacer moiety, covalently bound to A, 4-yl)-5'-methyl)neomycin carboxamide, N-((1-(2-hydroxy or absent; and 3-(ciprofloxacin)propyl)-1H-1,2,3-triazol-4-yl)-5'-methyl) 0.072 RG' is a first reactive group: neomycin carboxamide, N-((1-(2-(2-(ciprofloxacin)ethoxy) 0.073 with a compound having the general formula VII: ethyl)-1H-1,2,3-triazol-4-yl)-5'-methyl)neomycin carboxamide, N-((1-(3-((ciprofloxacin)methyl)benzyl)-1H RG-Y-B Formula VII 1,2,3-triazol-4-yl)-5'-methyl)neomycin carboxamide, 0074 wherein: N-((1-(4-((ciprofloxacin)methyl)benzyl)-1H-1,2,3-triazol 0075 B is an aminoglycoside-based antimicrobial 4-yl)-5'-methyl)neomycin carboxamide, 4-((5"-neomycin agent moiety; methoxy)methyl)-1-(2-(ciprofloxacin)ethyl)-1H-1,2,3-triaz 0.076 Y is a second spacer moiety, covalently bound to ole, 4-(1-N-kanamycin methyl)-1-(2-(ciprofloxacin)ethyl)- B, or absent; and 1H-1,2,3-triazole, 1-(4-(1-N-kanamycin methyl)-1H-1,2,3- 0.077 RG' is a second reactive group: triazol-1-yl)-3-(ciprofloxacin)propan-2-ol and 4-(1-N- 0078 thereby forming W from RG' and RG'. kanamycin methyl)-1-(4-((ciprofloxacin)methyl)benzyl)- 0079 According to some embodiments of the invention, 1H-1,2,3-triazole. one of RG' or RG' is selected from the group consisting of US 2014/0357591 A1 Dec. 4, 2014 alkynyl, amine, alkoxy, aryloxy, diene, Sulfhydryl, imino, monella typhi, Salmonella paratyphi, Salmonella typhi carboxylate, hydrazide, hydrazide and hydroxylamine, and murium, Salmonella virchow, Shigella spp., Yersinia entero the other is selected from the group consisting of azide, aziri colitica, Acinetobacter calcoaceticus, Flavobacterium spp., dine, epoxy, carbonyl, thiocarbonyl, aldehyde, chloride, bro Haemophilus influenzae, Pseudomonas aeruginosa, Campy mide, iodide, dienophile, mesylate, tresylate and tosylate. lobacter jejuni, Vibrio parahaemolyticus, Brucella spp., 0080 According to embodiments of one aspect of the Neisseria meningitidis, Neisseria gonorrhoea, Bacteroides present, there is provided a pharmaceutical composition fragilis, Fusobacterium spp., Mycobacterium tuberculosis which includes, as an active ingredient, the conjugate to pre and Mycobaterium Smegmatis. sented herein and a pharmaceutically acceptable carrier. I0088 According to some embodiments of the invention, 0081. According to some embodiments of the invention, the microorganism comprises at least one bacterial Strain the pharmaceutical composition presented herein is packaged which is resistant to at least one antibiotic agent. in a packaging material and identified in print, in or on the I0089. According to some embodiments of the invention, packaging material, for use in the treatment of a medical the bacterial strain is selected from the group consisting of condition associated with a pathogenic microorganism. 0090 (a) Gram-positive bacteria selected from the 0082. According to embodiments of another aspect of the group consisting of Strep. pyogenes (Group A), Strep. present invention, there is provided a use of the conjugate pneumoniae, Strep. GpB, Strep. viridans, Strep. GpD presented herein in the preparation of a medicament. (Enterococcus), Strep. GpC and GpG, Staph. aureus, 0083. According to some embodiments of the invention, Staph. epidermidis, Bacillus subtilis, Bacillus anthraxis, the medicament is for treating a medical condition associated Listeria monocytogenes, Anaerobic cocci, Clostridium with a pathogenic microorganism. spp., Clostridium difficile and Actinomyces spp.; and 0084. According to embodiments of one aspect of the 0.091 (b) Gram-negative bacteria selected from the present, there is provided a method of treating a medical group consisting of Escherichia coli, Enterobacter aero condition associated with a pathogenic microorganism in a genes, Kiebsiella pneumoniae, Proteus mirabilis, Pro subject, the method is effected by administering to the subject teus vulgaris, Morganella morganii, Providencia Stuar an effective amount of the conjugate presented herein. tii, Serratia marcescens, Citrobacter freundii, 0085. According to some embodiments of the invention, Salmonella typhi, Salmonella paratyphi, Salmonella the medical condition is selected from the group consisting of typhimurium, Salmonella virchow, Shigella spp., Yers actinomycosis, anthrax, aspergillosis, bacteremia, bacterial inia enterocolitica, Acinetobacter calcoaceticus, Fla skin diseases, bartonella infections, botulism, brucellosis, vobacterium spp., Haemophilus influenzae, Pseudomo burkholderia infections, campylobacter infections, candidi nas aueroginosa, Campylobacter jejuni, Vibrio asis, cat-scratch disease, chlamydia infections, cholera, parahaemolyticus, Brucella spp., Neisseria meningiti clostridium infections, coccidioidomycosis, cryptococcosis, dis, Neisseria gonorrhoea, Bacteroides fragilis, and dermatomycoses, dermatomycoses, diphtheria, ehrlichiosis, Fusobacterium spp., Acinetobacter baumanii, epidemic louse borne typhus, Escherichia coli infections, Pseudomonas aeruginosa; and fiisobacterium infections, gangrene, general infections, gen (c) Mycobacterium tuberculosis. eral mycoses, gram-negative bacterial infections, Gram-posi 0092 tive bacterial infections, histoplasmosis, impetigo, klebsiella 0093. Abbreviations used herein include, “AAC forami infections, legionellosis, leprosy, leptospirosis, listeria infec noglycoside N-acetyltransferase: “APH' for aminoglycoside tions, lyme disease, maduromycosis, melioidosis, mycobac O-phosphotransferase; “ANT for aminoglycoside O-nucle terium infections, mycoplasma infections, necrotizing fascii otidylyltransferase; “MRSA for methicillin-resistant Sta tis, nocardia infections, onychomycosis, ornithosis, phylococcus aureus; "NeoB' for neomycin B: “Cipro' for pneumococcal infections, pneumonia, pseudomonas infec ciprofloxacin; “TIPSC1' for triisopropylchlorosilane; tions, Q fever, rat-bite fever, relapsing fever, rheumatic fever, “PMB' for para-methoxybenzyl: “TBAF" for tetra-n-buty rickettsia infections, Rocky-mountain spotted fever, salmo lammonium fluoride; "CAN' for cerium ammonium nitrate; nella infections, Scarlet fever, Scrub typhus, sepsis, sexually “DCC” for N,N'-dicyclohexylcarbodiimide: “TEMPO for transmitted bacterial diseases, staphylococcal infections, 2.2.6,6-tetramethylpiperidine-1-oxyl; “BAIB for bis(ac streptococcal infections, Surgical site infection, tetanus, tick etoxy)iodobenzene: “HOBT for hydroxybenzotriazole; borne diseases, tuberculosis, tularemia, typhoid fever, urinary “MIC” for minimal inhibitory concentration: “Topol V for tract infection, vibrio infections, yaws, versinia infections, topoisomerase IV; and “IC50 for half maximal inhibitory Yersinia pestis plague, Zoonoses and Zygomycosis. concentration. I0086 According to some embodiments of the invention, 0094. As used herein the term “about refers to +10%. the microorganism includes at least one bacterial strain. (0095. The terms “comprises”, “comprising”, “includes”, 0087. According to some embodiments of the invention, “including”, “having and their conjugates mean “including the bacterial strain is selected from the group consisting of a but not limited to’. The term “consisting of means “includ Gram negative organism, a Gram positive organism or a ing and limited to’. mycobacteria Strain selected from the group consisting of 0096. The term “consisting essentially of means that the Strep. pyogenes (Group A), Strep. pneumoniae, Strep. GpB. composition, method or structure may include additional Strep. viridans, Strep. GpID (Enterococcus), Strep. GpC and ingredients, steps and/or parts, but only if the additional GpG, Staph. aureus, Staph. epidermidis, Bacillus subtilis, ingredients, steps and/or parts do not materially alter the basic Bacillus anthracis, Listeria monocytogenes, Anaerobic and novel characteristics of the claimed composition, method cocci, Clostridium spp., Actinomyces spp., Escherichia coli, Or Structure. Enterobacter aerogenes, Kiebsiella pneumoniae, Proteus 0097. As used herein, the singular form “a”, “an and mirabilis, Proteus vulgaris, Morganella morganii, Providen “the include plural references unless the context clearly cia Stuartii, Serratia marcescens, Citrobacter freundii, Sal dictates otherwise. For example, the term “a compound” or US 2014/0357591 A1 Dec. 4, 2014

“at least one compound may include a plurality of com rithmic plot of Topol V inhibition, measured for Cipro and pounds, including mixtures thereof. Compound 1 f while the percentages of the supercoiled DNA 0098. Throughout this application, various embodiments were calculated from the electrophoresis images by using of this invention may be presented in a range format. It should Image.J Launcher program (Rasband, W. Bethesda, Md., be understood that the description in range format is merely USA), and plotted as functions of drug concentration (each for convenience and brevity and should not be construed as an data point represents the average of 2-3 independent experi inflexible limitation on the scope of the invention. Accord mental results); and ingly, the description of a range should be considered to have 0105 FIGS. 2A-B present comparative data on the emer specifically disclosed all the possible Subranges as well as gence of resistance in E. coli (FIG. 2A) and B. subtilis (FIG. individual numerical values within that range. 2B) after 15 serial passages in the presence of Cipro, NeoB, 0099. Whenever a numerical range is indicated herein, it is Cipro-i-NeoB mixture (1:1 molar ratio) and an exemplary meant to include any cited numeral (fractional or integral) conjugate, according to Some embodiments of the present within the indicated range. The phrases “ranging/ranges invention, Compound 1i, wherein relative MIC is the normal between a first indicate number and a second indicate num ized ratio of MIC obtained for a give subculture to MIC ber and “ranging/ranges from a first indicate number “to a obtained upon first exposure. second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers DESCRIPTION OF EMBODIMENTS OF THE and all the fractional and integral numerals therebetween. INVENTION 0100. As used herein the term “method’ refers to manners, 0106 The present invention, in some embodiments means, techniques and procedures for accomplishing a given thereof, relates to antimicrobial agents, and more particularly, task including, but not limited to, those manners, means, but not exclusively, to non-resistance inducing antimicrobial techniques and procedures either known to, or readily devel conjugates which are effective also against resistant bacteria, oped from known manners, means, techniques and proce and to uses thereof in treating infections. dures by practitioners of the chemical, pharmacological, bio 0107 Before explaining at least one embodiment of the logical, biochemical and medical arts. invention in detail, it is to be understood that the invention is 0101 Unless otherwise defined, all technical and/or sci not necessarily limited in its application to the details set forth entific terms used herein have the same meaning as com in the following description or exemplified by the Examples. monly understood by one of ordinary skill in the art to which The invention is capable of other embodiments or of being the invention pertains. Although methods and materials simi practiced or carried out in various ways. lar or equivalent to those described herein can be used in the 0108. In search for a solution to the prevalent problem of practice or testing of embodiments of the invention, exem bacterial resistance caused by enzymatic modification, as plary methods and/or materials are described below. In case discussed hereinabove, the present inventors have devised of conflict, the patent specification, including definitions, will and Successfully prepared and practiced novel antimicrobial control. In addition, the materials, methods, and examples are conjugates that act by applying bactericidal pressure on two illustrative only and are not intended to be necessarily limit different essential systems of the microorganisms while being 1ng. present in the microorganism cell as one conjugated mol ecule. To this end, the present inventors have utilized the BRIEF DESCRIPTION OF THE DRAWINGS "click chemistry to conjugate aminoglycosides, which are 0102 Some embodiments of the invention are herein known to act on ribosomal-related mechanisms, to antimicro described, by way of example only, with reference to the bial agents which exert their antibiotic effect on non-riboso accompanying drawings and images. With specific reference mal mechanisms. now to the drawings and images in detail, it is stressed that the 0109 The present invention, according to some embodi particulars shown are by way of example and for purposes of ments thereof, describes the synthesis and biological evalua illustrative discussion of embodiments of the invention. In tion of antimicrobial conjugates, comprising antimicrobial this regard, the description taken with the drawings and agents such as, for example, members of the fluoroquinolone images makes apparent to those skilled in the art how embodi family (e.g., ciprofloxacin or Cipro) and the aminoglycoside ments of the invention may be practiced. family (e.g., neomycin B or NeoB). These conjugates com 0103) In the drawings: prise two pharmacophores which are active by different anti 0104 FIGS. 1A-D present comparative data for the inhi microbial mechanism; one side is anti-ribosomal, while the bition of DNA gyrase (FIGS. 1A-B) and Topol V (FIGS. other is non-ribosomal. 1C-D) with Cipro and exemplary Compound 1 f, wherein 0110. The present inventors have shown that by utilizing FIG. 1A is a photograph of a 1% agarose gel showing the the "click chemistry” described herein, combinatorial synthe inhibitory activity of Compound 1 fagainst DNA gyrase (lane sis from relatively simple building blocks can afford a wide 1, relaxed DNA: lane2, supercoiling reaction by DNA gyrase family of novel antimicrobial conjugates. Particularly, the without presence of inhibitor; lanes 3-8 are the same as lane 1 present inventors have devised and Successfully practiced but in the presence of 30, 60, 100, 150, 200, and 300 nM of novel methods for preparing azides and alkynes bearing anti Compound 1f); FIG.1B is a to semilogarithmic plot of invitro microbial agent moieties, such as fluoroquinolone and ami DNA gyrase Supercoiling reaction inhibition, measured for noglycosides, which can be efficiently utilized for forming Cipro and Compound 1f FIG. 1C is a photograph of a 1% this novel family of conjugates. agarose gel showing the inhibitory activity of Compound 1 f 0111. These conjugates, according to some embodiments against Topol V (lane 1, supercoiled DNA: lane 2, relaxation of the present invention, exhibit high potency against both reaction by Topol V without the presence of inhibitor; lanes Gram-negative and Gram-positive bacteria including MRSA 3-8 are the same as lane 1 but in the presence of 0.2,0.3, 0.5, and strains harboring either the monofunctional APH(3') 0.8, 1.2, and 10 uM of Compound 1f FIG. 1D is a semiloga enzyme or the bifunctional AAC(6')/APH(2") enzyme. The US 2014/0357591 A1 Dec. 4, 2014 conjugates, according to some embodiments of the present DNA and DNA gyrase or TopolV. The quinolone-gyrase invention, overcome currently resistant bacteria, and in addi DNA or quinolone-TopolV-DNA complex formation inhibits tion, reduce the appearance of new resistant strains there DNA replication and cell growth, and is responsible for the against. The conjugates, according to some embodiments of bactericidal action of quinolones 20. the present invention, also exhibit a dual mode of antimicro 0117. As demonstrated in the Examples section that fol bial activity by inhibiting both bacterial protein synthesis and lows and in the accompanyingTables and Figures, a variety of topoisomerase/gyrase enzyme activity. 20 exemplary conjugates of the aminoglycosides NeoB and 0112 An exemplary conjugate which comprises neomy KanA attached to Cipro where prepared by "click' chemistry, cin B and ciprofloxacin, according to Some embodiments of and tested for antimicrobial activity, rate of resistance emer the present invention, was significantly more potent than the gence, inhibition of prokaryotic protein translation and inhi parent compound neomycin B, and showed antimicrobial bition of DNA girase and topoisomerase IV enzymes. The activity against most prevalent types of resistance associated conjugates presented herein have been shown to possess with aminoglycosides. The exemplary conjugate Cipro highly effective antimicrobial trait while not evoking resis NeoB, according to some embodiments of the present inven tance thereto. tion, inhibited bacterial protein synthesis with the potencies 0118. According to optional embodiments of the inven similar to or better than that of neomycin B, and were up to tion, the two antimicrobial agents are linked together through 32-fold more potent inhibitors than ciprofloxacin for the fluo various linking and spacer moieties. roquinolone targets, DNA gyrase and toposiomerase IV, indi 0119 Hence, according to an aspect of some embodi cating a balanced dual mode of action. Significant delay of ments of the invention, there is provided a conjugate having resistance formation was observed in both E. coli and B. the general formula III: subtilis to the treatment with the exemplary Cipro-NeoB con A-X W Y B Formula III jugate, according to Some embodiments of the present inven tion, compared to that of each Cipro and NeoB used sepa 0120 wherein: rately or as a 1:1 mixture. 0121 A' is a quinolone-based antimicrobial agent moi 0113. While the mechanism of action of aminoglycosides ety; is not fully understood, it is accepted that among several 0.122 B is an aminoglycoside-based antimicrobial potential antibiotic mechanisms, their main mechanism of agent moiety; action involves direct interaction with one or more ribosomal 0123 X is a first spacer moiety, covalently bound to A, subunits by binding to the ribosomal RNA (rRNA), thereby or absent; affecting microbial protein synthesis at the -translation level 0.124 Y is a second spacer moiety, covalently bound to by inhibiting translocation step and total translation process. B, or absent; and Aminoglycosides also interfere with the fidelity of protein 0.125 W is a linking moiety. synthesis through affecting the proofreading process, causing I0126. As further demonstrated in the Examples section the misreading and increased rate of error in synthesis with pre follows, and is further discussed in detail hereinbelow, the mature termination 16. The present inventors have hypoth present inventors have successfully utilized the "click' chem esized that due the presence of positive charge which typi istry for preparing novel conjugates of aminoglycosides, and cally characterizes aminoglycosides, conjugates of to this effect, have prepared versatile derivatives of aminogly aminoglycosides could afford favorable binding to DNA and/ cosides that could participate in a "click” reaction with or DNA-protein interface, and therefore exhibit better inhibi complementary derivatives of non-ribosomal antimicrobial tion and improved antibacterial activity when conjugated agents. with antimicrobial agents, such as quinolones, which also I0127 Hence, according to an aspect of some embodi interact with nucleic acids. The well-established binding of ments of the invention, there is provided a conjugate having aminoglycosides to DNA 17, along with the inhibition of the general formula I: various nucleic acid metabolizing enzymes by aminoglyco A-X-D-Y B Formula I sides 18, Supported this hypothesis. 0.128 wherein: 0114. The present inventors hypothesized that conjugates 0129. A is a non-ribosomal-active antimicrobial agent of aminoglycosides and non-ribosomal antimicrobial agents moiety; would provide superior antimicrobial effects, particularly 0.130 B is an aminoglycoside-based antimicrobial when fighting resistant Strains of pathogenic microorgan agent moiety; isms. The concept was reduced to practice with quinolones 0131 X is a first spacer moiety, covalently bound to A, conjugated to aminoglycosides, wherein the conjugation was or absent; exemplified by use of "click' chemistry. 0.132. Y is a second spacer moiety, covalently bound to 0115 The present inventors have thus prepared and tested B, or absent; and a family of conjugates of neomycin B (NeoB, an aminogly 0.133 D is a linking moiety having the general formula coside) and the fluoroquinolone ciprofloxacin (Cipro, a qui II, as presented hereinbelow. nolone), prepared via “click' chemistry and linked via 1.2.3- I0134. In the context of the present embodiments, the terms triazole moiety (Cipro-NeoB or Compound 1). “antimicrobial agent”, “antibacterial agent”, “antibiotic 0116. The rational behind the selection of these two exem agent” and “bactericidal agent” are used interchangeably. plary antimicrobial agents to form the parts of a new antimi I0135. As used herein, the term "moiety” describes portion crobial conjugate stems from their different mechanism of of a molecule, and typically a major portion thereof. antimicrobial activity. Quinolones exert their antimicrobial 0.136 The phrase “non-ribosomal-active antimicrobial activity by targeting bacterial DNA gyrase and topoisomerase agent, as used herein, refers to an agent which has a mecha IV (TopolV) and inhibiting DNA replication process 19. nism of antimicrobial action which does not involve direct Particularly, quinolones bind to complexes that form between interaction with a ribosomal subunit. Alternatively, this US 2014/0357591 A1 Dec. 4, 2014

phrase refers to any antimicrobial agent having an antimicro tibuten, ceftiolene, ceftizoxime, oxacephem (flomoxeflata bial mechanism which is different from that of aminoglyco moxef), cefepime, cefoZopran, cefpirome, cefauinome, sides, as it is known in the art. Typically, the mechanism of ceftobiprole, ceftiofur, cefauinome, cefoVecin, aztreonam, antimicrobial action attributed to aminoglycosides is gener tigemonam, penam (Sulbactam, taZobactam) and clavam (cla ally referred to as anti-protein-biosynthesis activity. Vulanic acid). 0.137 Exemplary non-ribosomal-active antimicrobial 0141 Glycopeptides, which generally inhibit bacterial agents include, without limitation, anti-metabolite-based peptidoglycan synthesis, include, without limitation, Vanco antimicrobial agents, quinoline- and fluoroquinolone-based mycin (oritavancin, telavancin), teicoplanin (dalbavancin) antimicrobial agents (jointly referred to herein as quinolo and ramoplanin. nes), 3-lactam-based antimicrobial agents, glycopeptide 0.142 Benzyl-2,4-diaminopyrimidines, which generally based antimicrobial agents, benzyl-2,4-diaminopyrimidine inhibit bacterial dihydrofolate reductase, include, without based antimicrobial agents, Sulfonamide-based antimicrobial limitation, trimethoprim, brodimoprim, tetroXoprimandicla agents, sulfanilamide-based antimicrobial agents, peptide prim. based antimicrobial agents, pseudo-peptide-based antimicro 0.143 Topoisomerase inhibitors, which generally inhibit bial agents and peptidomimetic-based antimicrobial agents. bacterial DNA replication, include cinoxacin, flumequine, 0138 Quinolones, which generally interfere with bacte nalidixic acid, oxolinic acid, pipemidic acid, piromidic acid rial DNA replication, include, without limitation, ciprofloxa and roSoxacin. cin (Cipro, Ciprobay, Ciproxin), balofloxacin (Baloxin), 0144 RNA polymerase inhibitors, which generally inhibit cinoxacin (Cinobac), clinafloxacin, danofloxacin (Advocin, bacterial RNA synthesis, include rifampicin, rifabutin, rifap Advocid), delafloxacin, difloxacin (Dicuiral, Vetequinon), entine and rifaximin. enoxacin (Enroxil, Penetrex), enrofloxacin (Baytril), fleroxa 0145 Sulfonamides, which generally inhibit bacterial cin (Megalone, Roquinol), flumequine (Flubactin), DNA and RNA synthesis, include sulfaisodimidine, sulfame garenoxacin (Geninax), gatifloxacin (Tequin, Zymar), gemi thizole, sulfadimidine, sulfapyridine, sulfafurazole, sulfanil floxacin (Factive), grepafloxacin (Raxar), ibafloxacin (Ibaf amide (prontosil), Sulfathiazole, Sulfathiourea, Sulfamethox lin), levofloxacin (Cravit, Levacquin), lomefloxacin (Max azole, Sulfadiazine, Sulfamoxole, Sulfadimethoxine, aquin), marbofloxacin (Marbocyl, Zenequin), moxifloxacin Sulfalene, Sulfametomidine, Sulfametoxydiazine, Sul (AVelox, Vigamox), nadifloxacin (Acuatim, Nadoxin, famethoxypyridazine, Sulfaperin, Sulfamerazine, Sul Nadixa), nalidixic acid (NegGam, Wintomylon), norfloxacin faphenazole and SulfamaZone. (Lexinor, NoroXin, Quinabic, Janacin), ofloxacin (Floxin, Oxaldin, Tarivid), orbifloxacin (Orbax, Victas), oxolinic acid 0146 Sulfanilamides, which are generally regarded as (Uroxin), paZufloxacin (Pasil, PaZucross), pefloxacin (Pefla bacterial PABA-antimetabolite, include sulfanilamide (sul cine), pipemidic acid (Dolcol), piromidic acid (Panacid), pru facetamide, Sulfametrole), furosemide, Sulfadiazine and Sul lifloxacin (Quisnon), roSoxacin (Eradacil), rufloxacin (Urof famethoxazole. lox), Sarafloxacin (Floxasol, Saraflox, Sarafin), sitafloxacin 0.147. Other non-ribosomal (antimetabolite and/or not (Gracevit), sparfloxacin (Zagam), temafloxacin (Omniflox), against protein biosynthesis) active antibiotics include, with to sufloxacin (OZex, Tosacin) and trovafloxacin (Trovan). out limitation, D-cycloserine (inhibiting bacterial alanine 0.139. As presented in the Examples section that follows racemase and D-ala-D-ala ligase), fosfomycin (inhibiting below, ciprofloxacin (Cipro) was used as exemplary non bacterial UDP-N-acetylglucosamine enolpyruvyl trans ribosomal-active antimicrobial agent in the preparation of ferase), as well as metronidazole, tinidazole, ornidazole, two series of conjugates, according to some embodiments of nitrofurantoin and nifurtoinol (anaerobic bacteria's DNA the present invention. inhibitors). 0140 B-Lactams, which generally inactivate bacterial 0.148. The other antimicrobial agent member of the con transpeptidase enzymes, include, without limitation, penicil jugates, according to to embodiments of the present inven lins, aminopenicillins, amoxicillin, amplicillin (pivampicillin, tion, is an aminoglycoside-based antimicrobial agent, which hetacillin, bacampicillin, metampicillin, talampicillin), epi is selected from the group consisting of neomycin B and cillin, carboxypenicillins, carbenicillin (carindacillin), ticar neomycin C, Streptomycin, framycetin, paromomycin, ribos cillin, temocillin, ureidopenicillins, azlocillin, piperacillin, tamycin, kanamycin A, kanamycin B and kanamycin C, ami meZlocillin, mecillinam (pivmecillinam), Sulbenicillin, ben kacin, arbekacin, bekanamycin, dibekacin, tobramycin, spec Zylpenicillins, clometocillin, benzathine benzylpenicillin, tinomycin, hygromycin, paromomycin, gentamicin, procaine benzylpenicillin, azidocillin, penamecillin, phe netilmicin, Sisomicin, isepamicin, Verdamicin, astromicin noxymethylpenicillins, propicillin, benzathine phenoxym and any derivative thereof. ethylpenicillin, pheneticillin, cloxacillin (dicloxacillin, flu 0149. As presented in the Examples section that follows cloxacillin), oxacillin, meticillin, nafcillin, faropenem, below, neomycin Band kanamycin A were used as exemplary biapenem, doripenem, ertapenem, imipenem, meropenem, aminoglycosides in the preparation of two series of conju panipenem, cefazolin, cefacetrile, cefadroxil, cefalexin, cefa gates, according to some embodiments of the present inven loglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefa tion. trizine, cefazedone, cefazaflur, cefradine, cefroxadine, ceft 0150. According to some embodiments of the present eZole, cefaclor, cefamandole, cefiminox, cefonicid, invention, the conjugate comprises a linking moiety ceforanide, cefotiam, cefprozil, cefbuperaZone, cefuroxime, covalently attached to each of the antimicrobial agents in the cefuZonam, cephamycin (cefoxitin, cefotetan, cefimetazole), conjugate, either directly or indirectly, via a spacer moiety, as carbacephem (loracarbef), cefixime, ceftazidime, ceftriax this term is defined and exemplified hereinafter. one, cefcapene, cefdaloXime, cefdinir, cefditoren, cefetamet, 0151. According some embodiments of the present inven cefnenoXime, cefodizime, cefoperaZone, cefotaxime, cefpi tion, the antimicrobial agent moieties are attached to the mizole, cefpiramide, cefpodoxime, cefsulodin, cefteram, cef linking moiety via spacer moieties. US 2014/0357591 A1 Dec. 4, 2014

0152. As used herein, the phrase “spacer moiety’ 0159. As used herein, the term “alkyl describes an ali describes a chemical moiety that typically extends between phatic hydrocarbon including straight chain and branched two chemical moieties and is attached to each of the chemical chain groups. Preferably, the alkyl group has 1 to 20 carbon moieties via covalent bonds. The spacer moiety may be linear atoms, and more preferably 1-10 carbon atoms. Whenever a or cyclic, be branched or unbranched, rigid or flexible. numerical range; e.g., "1-10, is stated herein, it implies that 0153. The nature of the spacer moieties can be regarded as the group, in this case the alkyl group, may contain 1 carbon having an effect on two aspects, the synthetic aspect, namely atom, 2 carbonatoms, 3 carbonatoms, etc., up to and includ the influence of the spacer moieties on the process of prepar ing 10 carbon atoms. The alkyl can be substituted or unsub ing the conjugates presented herein, and the influence of the stituted. When substituted, the substituent can be, for spacer moieties on the biology activity of the conjugates in example, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an terms of antimicrobial activity, bioavailability and other aryl, a heteroaryl, a halide, a hydroxy, an alkoxy and a ADME-Tox considerations. hydroxyalkyl as these terms are defined hereinbelow. 0154 According to some embodiments of the present 0160 The term “alkenyl' describes an unsaturated alkyl, invention, the spacer moieties are selected Such that they as defined herein, having at least two carbon atoms and at allow and/or promote the conjugation reaction between the least one carbon-carbon double bond. The alkenyl may be two halves of the conjugates and reduce the probability for the substituted or unsubstituted by one or more substituents, as formation of side-products due to undesired reactions. Such described for alkyl hereinabove. traits can be selected for in terms of spacer's length, flexibil 0.161 The terms “alkynyl or “alkyne', as defined herein, ity, structure and specific chemical reactivity or lack thereof. is an unsaturated alkyl having at least two carbon atoms and Spacer moieties with fewer reactive groups will present a at least one carbon-carbon triple bond. The alkynyl may be simpler synthetic challenge, requiring less protection/depro substituted or unsubstituted by one or more substituents, as tection steps and affording higher chemical yields. For described hereinabove. example, to saturated and linear alkyls of 1-10, or 1-5 carbon 0162 The term “cycloalkyl describes an all-carbon atoms, having one reactive group at the end atom for conju monocyclic or fused ring (i.e., rings which share an adjacent gation with a corresponding reactive group, would afford pair of carbon atoms) group where one or more of the rings substantially higher yield and fewer side products. Similarly, does not have a completely conjugated pi-electron system. a spacer moiety based on one or two chained benzyl rings The cycloalkyl group may be substituted or unsubstituted as would also lead to an efficient conjugation reaction. described for alkyl hereinabove. O155 On the other hand, the spacer moiety is selected such (0163 The term "heteroalicyclic” describes a monocyclic that its attachment to the antimicrobial agent, be it A A' or B or fused ring group having in the ring(s) one or more atoms in formulae I and III, even at Suitable positions as discussed Such as nitrogen, oxygen and Sulfur. The rings may also have hereinbelow, does not obliterate or otherwise preclude the one or more double bonds. However, the rings do not have a antimicrobial activity of the antimicrobial agents Substan completely conjugated pi-electron system. The heteroalicy tially by virtue of its own chemical structure, namely due to its clic may be substituted or unsubstituted as described for alkyl steric effect, physical effect (solubility, charge etc.), or hereinabove. Representative examples are piperidine, pipera chemical reactivity. For example, highly hydrophobic spacer Zine, tetrahydrofurane, tetrahydropyrane, morpholino and the moieties would lower the bioavailability of the conjugate by like. lowering its aqueous solubility. Highly flexible and long 0164. The term “aryl' describes an all-carbon monocyclic spacer moieties may lower the binding constant of the con or fused-ring polycyclic (i.e., rings which share adjacent pairs jugates to their biological targets and may also present steric of carbon atoms) groups having a completely conjugated hindrance issues, adversely affecting binding. pi-electron system. The aryl group may be substituted or 0156. As can be seen in the comparative experimental unsubstituted. Substituted aryl may have one or more sub results presented hereinbelow, a simple spacer moiety, based stituents as described for alkyl hereinabove. on a saturated alkyl with or without a heteroatom along its 0.165. The term "heteroaryl describes a monocyclic or chain, or a spacer moiety based on an aromatic ring, both fused ring (i.e., rings which share an adjacent pair of atoms) having a length that corresponds to 2-7 carbon-carbon bonds group having in the ring(s) one or more atoms, such as, for and a relatively small number of degrees of rotational free example, nitrogen, oxygen and Sulfur and, in addition, having dom, would afford an effective antimicrobial conjugate, a completely conjugated pi-electron system. Examples, with according to some embodiments of the present invention, out limitation, of heteroaryl groups include pyrrole, furane, which is also reasonably simple to produce. thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, 0157. In the context of the present embodiments, the pyrimidine, quinoline, isoquinoline and purine. The het spacer moiety which is attached to A in formula I (or to A' in eroaryl group may be substituted or unsubstituted as formula III) is denoted X and referred to as the first spacer described for alkyl hereinabove. Representative examples of moiety, and the spacer moiety which is attached to B in heteroaryls include triazole, furane, imidazole, indole, iso formula I (and in formula III) is denoted Y and referred to as quinoline, oxazole, pyrazole, pyridine, pyrimidine, pyrrole, the second spacer moiety. quinoline, thiazole, thiophene, triazine, purine and the like. 0158. According to some embodiments of the present 0166 According to some embodiments of the present invention, each of X and Y can be independently alkyl, alk invention, the spacer moieties include, without limitation, enyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, heteroaryl CH s CH O s (CH2) s (CH2). s (CH) and/or a hydrocarbon chain having 1-20 carbon atoms and — —(CH2). O— —(CH) , —(CH) , —(CH) ending or interrupted by at least one heteroatom selected from —(CH(CH))—CH2—, —CH=CH-CH=CH-. the group consisting of O. S and N and/or containing from 0 - C=C C=C , —CHCH(OH)CH , —CH-O- to 19 unsaturated carbon-carbon or carbon-heteroatom CH2—, CH-O-CH2—O—, (CH2). O—(CH) bonds. 2 3 (CH2). O—(CH)—O—, CH mCH - US 2014/0357591 A1 Dec. 4, 2014

CH2—, —CH2-mCH CH-O-, -CH2-pCH agent of formula I, or the quinolone-based antimicrobial CH2—, —CH2-pCH CH-O , —CH2—NHCO—, agent of formula III is ciprofloxacin, it is attached to X via the CH NHCO , CH, O CH and terminal nitrogen of the piperazine moiety thereof. However, —CH=CH-CH NH-(CH) . other positions on the skeleton or substituents of ciprofloxa 0167. It is noted herein that in some embodiments, for the cinto which a spacer moiety, if present, is attached are also sake of clarity and simplicity of presentation, the spacer moi contemplated. ety Y may be defined as including the C5" carbon atom of the aminoglycoside-based antimicrobial agent moiety in cases 0172. Several SAR studies on aminoglycosides have dem such as when Y is an amide —NHCO—, and the carbonatom onstrated a high tolerance for structural modifications at dif of the amide group is the C5" atom. Similarly, the spacer ferent positions. For 4.5-disubstituted 2-deoxystrepamine moiety Y may be defined as not including the oxygenatom of family of aminoglycosides the positions that were identified the C5"-O-group of the aminoglycoside-based antimicro for such modifications were C5"-position 13, 14, C2"-po bial agent moiety. sition 23-26 and N1 position 25. For 4,6-disubstituted 0168 The positions at which the first and second spacer 2-deoxystrepamine family of aminoglycosides, however, moieties are attached to the antimicrobial agents are generally substantial tolerance for structural variations was observed at selected Such that the attachment or presence of a spacer position C1-NH-27, 28. moiety on the antimicrobial agent does not preclude the anti microbial activity of the antimicrobial agents substantially. 0173 As mentioned in the background section, the present The ability to be modified structurally without substantially inventors have previously disclosed that some structural posi losing antimicrobial activity is referred to herein and in the art tions on the skeleton of known aminoglycosides are more as “tolerance for structural modifications of any given anti Suitable for chemical modifications, meaning that appending microbial agent. Suitable positions depend on the type of chemical moieties at these positions does not abolish or pre antimicrobial agent, hence discussion and exemplary posi clude the antimicrobial activity thereof. tions are provided herein below. 0.174 Scheme 2 presents the optional positions at which 0169 SAR studies on fluoroquinolones have demon the second spacer moiety can be attached to a 4.5-disubsti strated the highest tolerance for structural modifications at the tuted-2-deoxistreptamine type aminoglycoside, which terminal nitrogen of the piperazine ring 7, 9-11, 21, 22. include neomycin B, ribostamycin and paromomycin. Exem 0170 Scheme 1 presents an optional position at which the plary Suitable attachment sites, marked by arrows in Scheme first spacer moiety can be attached to a fluoroquinolone type 2, include the C5'-position, the C2"-position and the antimicrobial agent, which includes, without limitation, C1-NH position. ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, lom efloxacin, to norfloxacin, orbifloxacin, Sarafloxacin and temafloxacin. An exemplary Suitable attachment site is the terminal nitrogen position at the piperazine moiety, marked Scheme 2 by an arrow in Scheme 1. 6

Scheme 1 R O

F COOH

R3 r2 OH NH2 N^ X a HN R HN ~2s2.1 JRibostamycin. OH OH R4

R X R R2 R3 R4 Ribostamycin NH, Ciprofloxacin CH cyclopropane H H H Enoxacin N Et H H H Neomycin B NH, Gatifloxacin COCH cyclopropane H H CH Paromomycin OH Grepafloxacin CH cyclopropane CH. H. CH Lomefloxacin CF Et H H CH Norfloxacin CH Et H H H Orbifloxacin CF cyclopropane F CH, CH 0.175 Scheme 3 presents the optional position at which the Sarafloxacin CH p-fluorobenzene H H H second spacer moiety can be attached to a 4.6-disubstituted Temafloxacin CH 2,4-difluorobenzene H H CH 2-deoxistreptamine type aminoglycoside, which include kanamycin A and B, tobramycin and dibekacin. An exem 0171 Hence, according to some embodiments of the plary suitable attachment site is the C1-NH position, present invention, when the non-ribosomal antimicrobial marked by an arrow in Scheme 3. US 2014/0357591 A1 Dec. 4, 2014 10

0181. The phrase “covalent bond, as used herein, refers to Scheme 3 one or more pairs of electrons which are shared between atoms in a form of chemical bonding. 0182. The term “amide' describes a NR' C(=O) R" or a C(=O)—NR'R' end groups or a NR' C(=O)— linking moiety, where each of R' and R" is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or het 2 NH2 eroaryl, as these terms are defined herein. 0183. The term “carboxylate” or “ester', as used herein, HO NH -- refers to a C(=O)—O R' end group, where R is as 6 defined herein, or a —C(=O)—O— linking moiety. Ol 0.184 The term “triazine' refers to a heterocyclic ring, OH analogous to the six-membered benzene ring but with three HO O carbons replaced by nitrogen atoms. The three isomers of OH triazine are distinguished from each other by the positions of HN their nitrogen atoms, and are referred to as 1,2,3-triazine, 1,2,4-triazine, and 1.3.5-triazine. Other aromatic nitrogen heterocycles include pyridines with 1 ring nitrogen atom, diazines with 2 nitrogenatoms in the ring and tetrazines with R R2 R 4 ring nitrogen atoms. Kanamycin A OH OH OH 0185. The term “triazole' refers to either one of a pair of Kanamycin B OH OH NH, isomeric chemical compounds with molecular formula Tobramycin OH H NH, CHN, having a five-membered ring of two carbon atoms Dibekacin H H NH, and three nitrogen atoms, namely 1,2,3-triazoles and 1,2,4- triazoles. 0176 Following similar guidelines and rational, other 0186 The term “disulfide' refers to a S S linking positions on similar or other aminoglycosides and non-ribo moiety. Somal active antimicrobial agents can be identified and used 0187. The term “imine', which is also referred to in the art to construct the conjugates, according to embodiments of the interchangeably as “Schiff-base', describes a —N=CR'— present invention. linking moiety, with R" as defined herein or hydrogen. As is 0177 According to some embodiments of the present well known in the art, Schiff bases are typically formed by invention, when the aminoglycoside-based antimicrobial reacting an aldehyde or a ketone and an amine-containing agent is neomycin B, it is covalently bound to Y via the moiety Such as amine, hydrazine, hydrazide and the like, as C5"-position thereof, and when the aminoglycoside-based these terms are defined herein. The term “aldimine” refers to antimicrobial agent is kanamycin A, it is covalently bound to a —CH=N imine which is derived from an aldehyde. The Y via the C1-N-position thereof. term "ketimine” refers to a —CR'—N— imine which is 0178. In the context of the present embodiments, the derived from a ketone. spacer moieties, when present, connect between the antimi 0188 The term “hydrazone” refers to a R'C—N- crobial agent moieties and the linking moiety. As used herein, NR" linking moiety, wherein R and R" are as defined the phrase “linking moiety” describes a chemical moiety that herein. links two other chemical moieties via one or more covalent 0189 The term “semicarbazone' refers to a linking moi bonds. In general, the linking moiety can be formed during a ety which forms in a condensation reaction between an alde chemical reaction, Such that by reacting two or more reactive hyde or ketone and semicarbazide. A semicarbazone linking groups, the linking moiety is formed as a new chemical entity moiety stemming from a ketone is a —R'C=NNR"C(=O) which can comprise a bond (between two atoms), or one or NR" , and a linking moiety stemming from an aldehyde is more bonded atoms. Alternatively, the linking moiety can be a CR'—NNR"C(=O)NR" , wherein R and R" are as an independent chemical moiety comprising two or more defined herein and R" or as defined for R'. reactive groups to which the reactive groups of other com 0190. As used herein, the term “lactone' refers to a cyclic pounds can be attached, either directly or indirectly, as is ester, namely the intra-condensation product of an alcohol detailed hereinunder. group —OH and a carboxylic acid group —COOH in the 0179 Herein throughout, the phrase “end group' same molecule. describes a group (a Substituent) that is attached to another 0191 As used herein, the term “lactam” refers to a cyclic moiety in the compound via one atom thereof, and the phrase amide, as this term is defined herein. A lactam with two “linking moiety’ describes a group that is attached to two carbonatoms beside the carbonyl and four ring atoms in total other moieties via two or more atoms therein. is referred to as a B-lactam, a lactam with three carbon atoms 0180 Exemplary linking moieties, according to some beside the carbonyl and five ring atoms in total is referred to embodiments of the present invention, include without limi as a Y-lactam, a lactam with four carbon atoms beside the tation, amide, lactone, lactam, carboxylate, cycloalkene, carbonyl and six ring atoms in total is referred to as a 6-lac cyclohexene, heteroalicyclic, heteroaryl, triazine, triazole, tam, and so on. disulfide, imine, aldimine, ketimine, hydraZone, semicarba 0.192 According to some embodiments of the present Zone and the likes. Other linking moieties are defined here invention, the linking moiety, denoted D in formula I and W inbelow. in formula III, is a triazole having the general formula II: US 2014/0357591 A1 Dec. 4, 2014 11

an extended time period at which the conjugate can exert its Formula II antimicrobial activity when the agents comprising the conju gate are conjugated, up to the time it is secreted or otherwise removed from the infected system or body. An exemplary biostable linking moiety is a triazole-based linking moiety. 0.197 According to some embodiments of the present invention, the linking moiety is a biocleavable linking moiety. Representative examples of biocleavable moieties include, without limitation, amides, carboxylates, carbamates, phos 0193 wherein each of the wiggled lines denote covalent phates, hydrazides, thiohydrazides, disulfides, epoxides, per bond to either A-X, A-X or B Y, and R is selected OXO and methyleneamines. Such moieties are typically Sub from the group consisting of hydrogen, alkyl, jected to enzymatic cleavages in a biological system, by cycloalkyl, and alkenyl, each of which can be branched, enzymes such as, for example, hydrolases, amidases, kinases, unbranched, Substituted or unsubstituted. According to peptidases, phospholipases, lipases, proteases, esterases, Some embodiments of the present invention, R is hydro epoxide hydrolases, nitrilases, glycosidases and the like. gen, alkyl, aryl orcycloalkyl, as these are defined herein. 0198 As used herein, the phrase “biocleavable moiety’ According to some embodiments, R is hydrogen. describes a chemical moiety, which undergoes cleavage in a 0194 A linking group having Formula II hereinabove can biological system Such as, for example, the digestive system be a result of a reaction between two reactive groups, alkynyl of an organism or a metabolic system in a living cell. and azide, which reaction is referred to herein and in the art as 0199 The rational behind having a biocleavable linking "click chemistry’ or "click reaction’, as discussed hereinbe moiety in a conjugate according to some embodiments of the low and demonstrated in the Examples section that follows. present invention, stems from the assumption that under a 0.195 Other linking groups can also be a result of a reac particular dosing regime, the conjugate administered in each tion between two reactive groups, as further detailed herein dose would exert its antimicrobial activity within a certain below. Alternatively, a desired linking group is first generated time-frame, and thereafter be degraded to Sub-components by and the antimicrobial agents and/or spacer moieties are the Subject's metabolic systems, thereby rendering it easier attached thereto. for the subject’s secretion system to be rid thereof. 0196. According to some embodiments of the present 0200. The conjugates presented herein, according to some invention, the linking moiety (e.g., D in Formula I or W in embodiments of the present invention, can be formed from Formula III) is stable at physiological conditions, namely the neomycin B or kanamycin A, and ciprofloxacin. Exemplary linking moiety of the conjugate does not disintegrate for the conjugates, according to some embodiments of the present duration of exposure to the physiological environment in the invention, have been prepared, as presented and demon subjects body. Such linking moiety is referred to herein a strated in the Examples section below for neomycin B (Table “biostable'. Biostable linking moieties offer the advantage of 1 below), and for kanamycin A (Table 2 below). TABLE 1.

OH N OH NH2

F HN OH

Conjugate Full chemical name X Y Compound 1a N-(4-(1-(2- —(CH2)2— —CH-NHCO— (ciprofloxacin)ethyl)- 1H-1,2,3-triazol-4-yl)- 5'-phenyl)neomycin Compound 1b N-(4-(1-(2- —(CH2)— —CH-NHCO— (ciprofloxacin)propyl)- 1H-1,2,3-triazol-4-yl)- 5'-phenyl)neomycin carboxamide Compound 1c N-(4-(1-(2- —(CH2)— —CH-NHCO— (ciprofloxacin)butyl)- 5'-phenyl)neomycin carboxamide

US 2014/0357591 A1 Dec. 4, 2014 14

TABLE 2-continued HN F O / \ N=N O OH NH2 OH N N-X-N 21 Yn OH NH HOOC \ \ / VAZ OH N O HO O OH HO NH2

Conjugate Full chemical name X Y Compound 11i 4-(1-N-kanamycin CH2 pCH-CH2 CH2 methyl)-1-(4- ((ciprofloxacin)methyl) benzyl)-1H-1,2,3- triazole

0202 According to some embodiments of the present 0218 B is an aminoglycoside-based antimicrobial invention, other conjugates can be prepared following the agent moiety; general procedures presented in the Examples section below 0219 Y is a second spacer moiety, covalently bound to and following the general processes for preparing the conju B, or absent; and gates, as follows. 0220 RG' is a second reactive group. 0203 Hence, according to another aspect of the present 0221) A reactive group (RG) in each of the antimicrobial invention, there is provided a process of preparing the conju agent derivatives that forms the conjugate presented herein, gates presented herein under formula I. The process is namely A-X—and B. Y—, serves for covalently binding the effected by reacting a compound having the general formula antimicrobial agents to one another, so as to form the conju IV: gate. The reactive groups, referred to herein as RG (attached A-X-RG Formula IV to antimicrobial agent derivative A-X—) and RG (attached to antimicrobial agent derivative B-Y ) may form a part of 0204 wherein: the antimicrobial agent in cases where X and/orY are absent, 0205 A is a non-ribosomal-active antimicrobial agent or be present substantially at the end of each of the spacer moiety; moieties X and/or Y which are attached to the antimicrobial 0206 X is a first spacer moiety, covalently bound to A, agents. or absent; and 0222. The phrase “reactive group', as used herein, refers 0207 RG is a first reactive group; to a chemical group that is capable of undergoing a chemical 0208 with a compound having the general formula V: reaction that typically leads to the formation a covalent bond. Chemical reactions that lead to a bond formation include, for RG-Y-B Formula V example, cycloaddition reactions (such as the Diels-Alder's 0209 B is an aminoglycoside-based antimicrobial reaction, the 1,3-dipolar cycloaddition Huisgen reaction, and agent moiety; the similar "click reaction'), condensations, nucleophilic and 0210 Y is a second spacer moiety, covalently bound to electrophilic addition reactions, nucleophilic and electro B, or absent; and philic Substitutions, addition and elimination reactions, alky 0211 RG is a second reactive group. lation reactions, rearrangement reactions and any other 0212. According to yet another aspect of the present known organic reactions that involve a reactive group. invention, there is provided a process of preparing the conju 0223 Representative examples of reactive groups include, gates presented herein under formula III, which is effected by without limitation, acyl halide, aldehyde, alkoxy, alkyne, amide, amine, aryloxy, azide, aziridine, azo, carbamate, car reacting a compound having the general formula VI: bonyl, carboxyl, carboxylate, cyano, diene, dienophile, A'-X-RG' Formula VI epoxy, guanidine, guanyl, halide, hydrazide, hydrazine, hydroxy, hydroxylamine, imino, isocyanate, nitro, phos 0213 wherein: phate, phosphonate, Sulfinyl, Sulfonamide, Sulfonate, thio 0214 A' is a quinolone-based antimicrobial agent moi alkoxy, thioaryloxy, thiocarbamate, thiocarbonyl, thiohy ety; droxy, thiourea and urea, as these terms are defined 0215 X is a first spacer moiety, covalently bound to A, hereinafter. or absent; and 0224. As used herein, the term “aldehyde' refers to an 0216 RG' is a first reactive group: —C(=O)—H group. 0217 with a compound having the general formula VII: 0225. The term “hydroxy' as used herein describes an RG'-Y B Formula VII —OH group. US 2014/0357591 A1 Dec. 4, 2014

0226. The terms “thio”, “sulfhydryl' or “thiohydroxy” as 0248. As used herein, the term “hydrazine' describes a used herein describe an —SH group. - NR NR"R" group, wherein R', R" and R" are each inde 0227. The term “disulfide' as used herein describes an pendently hydrogen, alkyl, cycloalkyl or aryl, as these terms —S-S-linking moiety. are defined herein. 0228. The term “alkoxy” as used herein describes an —O- 0249. The term “hydroxylamine', as used hereon, refers to alkyl, an —O-cycloalkyl, as defined hereinabove. The ether either a –NHOH group or a —ONH2. group —O— is also a possible linking moiety. 0250. The term "nitro describes an —NO group. 0229. The term “aryloxy” as used herein describes an (0251. The term “acyl halide” describes a —(C=O)R" —O-aryl group. group wherein R" is halide, as defined hereinabove. 0230. The term “thioalkoxy” as used herein describes an 0252) The term “phosphate” describes an - O P(=O), —S-alkyl group. The thioether group—S—is also a possible (OR") end or reactive group or a —O P(=O)(O)—linking linking moiety. moiety, as these phrases are defined hereinabove, with R" as 0231. The term “thioaryloxy” as used herein describes an defined herein. —S-aryl group. The thioarylether group —S-aryl- is also a possible linking moiety. (0253) The term “phosphonate” describes a - P(=O) (OR)(OR") end or reactive group or a P(=O)(OR)(O)– 0232. As used herein, the term “amine” refers to an linking moiety, as these phrases are defined hereinabove, with —NR'R" group where R' and R" are each hydrogen, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl (bonded through a ring R" and R" as defined herein. carbon) or heteroalicyclic (bonded through a ring carbon) as (0254 The term “sulfoxide' or “sulfinyl describes a defined hereinbelow. —S(=O)R’ end or reactive group or an —S(=O)—linking moiety, as these phrases are defined hereinabove, where R is 0233. The terms “halide' or “halo' refer to fluorine, chlo as defined hereinabove. rine, bromine or iodine. 0255. The term "sulfonamide' encompasses the term 0234. As used herein, the term “azide refers to a N. “S-sulfonamide' which describes a S(=O), NR'R" end (-N=N*—N) group or reactive group or a —S(=O), NR'—linking moiety, as 0235. The term “aziridine', as used herein, refers to a these phrases are defined hereinabove, with R" and R" as reactive group which is a three membered heterocycle with defined herein; and the term "N-sulfonamide' which one amine group and two methylene groups, having a describes an RS(=O), NR"— end or reactive group or a molecular formula of -CH-NH. —S(=O) NR— linking moiety, as these phrases are 0236. The term “diene', as used herein, refers to a defined hereinabove, where R' and R" are as defined herein. —CR'—CR" CR"—CR" group, wherein R as defined 0256 The term “sulfonate” describes a S(=O) R' hereinabove, and R", R" and R" are as defined for R'. end or reactive group or an —S(=O) - linking moiety, as 0237. The term “dienophile', as used herein, refers to a these phrases are defined hereinabove, where R' is as defined reactive group that reacts with a diene, typically in a Diels herein. Alder reaction mechanism, hence a dienophile is typically a double bond or an alkenyl. 0257 According to some embodiments of the present invention, the linking moiety is formed as a result of a bond 0238. The term “epoxy’, as used herein, refers to a reac forming reaction between two reactive groups, namely RG tive group which is a three membered heterocycle with one and RG2, which can be present in either sides of the conju oxygen and two methylene groups, having a molecular for gate's moieties before these are reacted together to form the mula of —CHO. conjugate. For example, a reaction between a diene reactive 0239. The term “azo’ or “diazo describes an N—NR' group and a dienophile reactive group, e.g. a Diels-Alder reactive group or an —N=N-linking moiety, as these reaction, would form a cycloalkene linking moiety, and in phrases are defined hereinabove, with R" as defined herein most cases a cyclohexene linking moiety. In another example, above. an amine reactive group would forman amide linking moiety 0240. The term “carbamate” refers to a NR'(C=O)CH when reacted with a carboxyl reactive group. In another (carbamic acid) end or reactive group, ora—NR'(C=O)O— example, a hydroxyl reactive group would form an ester link linking moiety, with R" as defined hereinabove. ing moiety when reacted with a carboxyl reactive group. In 0241. The term “thiocarbamate” refers to a NR'(C=S) another example, a sulfhydryl reactive group would form a OH end or reactive group, or a —NR'(C—S)O— linking disulfide ( S S ) linking moiety when reacted with moiety, with R" as defined hereinabove. another sulfhydryl reactive group under oxidation conditions, 0242. The term “carbonyl refers to a —(C=O)—group. or a thioether (thioalkoxy) linking moiety when reacted with a halo reactive group or another leaving-reactive group. In 0243 The term “thiocarbonyl refers to a —(C=S)– another example, an alkynyl reactive group would form a group. triazole linking moiety by "click reaction' when reacted with 0244 As used herein, the term “carboxyl refers to an an azide reactive group. 0258. The "click reaction', also known as "click chemis 0245. The term "cyano describes a C=N group. try’ is a name used to describe a Cu(I)-catalyzed stepwise 0246 The term "isocyanate” describes an —N=C=O variant of the Huisgen 1,3-dipolar cycloaddition of azides and group. alkynes to yield 1,2,3-triazole. This reaction is carried out 0247 The term “hydrazide', as used herein, refers to a under ambient conditions, or under mild microwave irradia —C(=O) NR NR"R" group wherein R', R" and R" are tion, and with exclusive regioselectivity for the 1,4-disubsti each independently hydrogen, alkyl, cycloalkyl or aryl, as tuted triazole product when mediated by catalytic amounts of these terms are defined herein. Cu(I) salts V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. US 2014/0357591 A1 Dec. 4, 2014

Sharpless, Angew. Chem. Int. Ed. 2002, 41,2596; H. C. Kolb, 0266. In some embodiments, the hydrazine- or hydrazide M. Finn, K. B. Sharpless, Angew Chem., Int. Ed. 2001, 40, containing antimicrobial agent derivative will have the struc 2004). ture A/B X/Y (C=O) NH-NH where (C=O) is 0259. As demonstrated in the Examples section that fol optionally a carbonyl group that can be present or absent. lows, the "click reaction' is particularly suitable to form the 0267. In some embodiments, the semicarbazide-contain conjugates presented herein since it is carried out under con ing antimicrobial agent derivative will have the structure ditions which are non-distructive to the antimicrobial agent A/B X/Y NH (C=O) NH NH. moieties, according to some embodiments of the present 0268. The particular chemistry which defines the linking invention, and it affords the conjugates at high chemical moiety is selected considering the same aspects used for yields using mild conditions in aqueous media. The selectiv selecting the spacer moieties, namely the effect it has on the ity of this reaction allows to perform the conjugation with synthesis and the effect it has on the biological activity of the minimized or nullified use of protecting groups, which use resulting conjugate, with emphasis on the former. The nature often results in multistep cumbersome synthetic processes. of the reactive groups with form the linking moiety should be 0260 Hence, when D and W of formulae I and III respec selected in Such a way so as to minimize the required protec tively are having the structure represented in formula II, tion and deprotection steps, aimed at preventing side-reac namely a 4-yl-1-yl-1,2,3-triazole, one of RG or RG of for tions at various undesired positions on both sides of the con mulae IV and V respectively, or RG' or RG' of formulae VI jugate. For example, an amide linking moiety is simple to and VII respectively, is alkynyl and the other is azide. When form between an amine and a carboxyl groups, however the the process used for preparing the conjugates is based on the synthetic process would require specific protection of Such "click chemistry’, the reaction is effected in the presence of a groups on the antimicrobial agent moieties, particularly copper catalyst. amines on the aminoglycoside moiety. 0261 Alternatively, RG' or RG' of formulae VI and VII 0269. As demonstrated in the Examples section that fol respectively are selected Such that other linking moieties can lows, the conjugates according to Some embodiments of the be formed from their inter-reaction. According to some present invention are highly effective in treating medical con embodiments of the present invention, a strong nucleophile ditions associated with a pathogenic microorganism in a Sub (including but not limited to, hydrazine, hydrazide, hydroxy ject. lamine (aminooxy, —O—NH), semicarbazide), present as a 0270. The conjugates presented herein are also highly terminal group in one of the antimicrobial agent derivatives, effective in treating medical conditions associated with can be reacted with an aldehyde or ketone group present in the pathogenic microorganisms which have already developed other antimicrobial agent derivative to form an imine, a resistance to any antibiotic agent. The conjugates presented hydrazone, an oxime or a semicarbazone, as applicable, herein are particularly effective in fighting pathogenic micro which in some cases can be further reduced by treatment with organisms since they suppress the emergence of resistance an appropriate reducing agent. In Such embodiments, one of thereto. RG' or RG" can be a nucleophile group (Nu), and the other 0271 The phrases “effective in treating medical condi can be a leaving group (L), and their inter-reaction would tions associated with pathogenic microorganisms”, “effective form a conjugate according to Scheme 4 below. in treating a subject diagnosed with a medical conditions AB XY Nu-L-XY-AB->AB XY Nu-XY associated with pathogenic microorganisms' and/or “identi AB Scheme 4 fied for use in the treatment of a medical condition associated 0262. As shown in Scheme 4, one of the antimicrobial with a pathogenic microorganism in a subject', as used agent derivatives has the formula A/B X/Y Nu and the herein, refer to characteristics of a Substance, such as the other antimicrobial agent derivative has the formula L-X/Y- conjugates according to Some embodiments of the present A/B, and the resulting linking moiety is formed essentially invention, that can effect death, killing, eradication, elimina from the nucleophile group. tion, reduction in number, reduction of growth rate, reduction 0263. Examples of nucleophile groups include, but are not of a load, and a change in population distribution of one or limited to, amine, alkoxy, aryloxy, Sulfhydryl, imino, car more species of pathogenic microorganisms, as well as effect boxylate, hydrazide, aminooxy groups that would react pri ing a reduction or prevention of the emergence of resistance marily via a SN2-type mechanism. Additional examples of of such microorganisms to the Substance. nucleophile groups include those functional groups that 0272. Herein throughout, the phrase “pathogenic micro would react primarily via a nucleophilic addition reaction. organism' is used to describe any microorganism which can Examples of leaving groups include chloride, bromide, cause a disease or disorder in a higher organism, Such as iodide, mesylate, tresylate, and tosylate and other groups mammals in general and a human in particular. The patho expected to undergo nucleophilic displacement as well as genic microorganism may belong to any family of organisms ketones, aldehydes, thioesters, olefins, alpha-beta unsatur Such as, but not limited to prokaryotic organisms, eubacte ated carbonyl groups, carbonates and other electrophilic rium, archaebacterium, eukaryotic organisms, yeast, fungi, groups expected to undergo addition by nucleophiles. algae, protozoan, and other parasites. 0264. Hence, in some embodiments of the present inven 0273. Non-limiting examples of pathogenic microorgan tion, one of the antimicrobial agent derivatives A-X or B Y ism include Plasmodium falciparum and related malaria includes a terminal carbonyl-containing reactive group (alde causing protozoan parasites, Acanthamoeba and other free hyde or ketone), and the other antimicrobial agent derivative living amoebae, Aeromonas hydrophila, Anisakis and related contains a terminal hydrazine, hydroxylamine, hydrazide or worms, and further include, but not limited to Acinetobacter semicarbazide reactive group. baumanii, Ascaris lumbricoides, Bacillus cereus, Brevundi 0265. In some embodiments, the hydroxylamine-terminal monas diminuta, Campylobacter jejuni, Clostridium botuli antimicrobial agent derivative will have the structure A/B- num, Clostridium perfingens, Cryptosporidium parvum, X/Y O NH. Cyclospora Cayetanensis, Diphyllobothrium, Entamoeba US 2014/0357591 A1 Dec. 4, 2014

histolytica, certain Strains of Escherichia coli, Eus prone to or capable of developing resistance to at least one trongylides, Giardia lamblia, Klebsiella pneumoniae, List antimicrobial strain. Non-limiting examples of Suchbacterial eria monocytogenes, Nanophyetus, Plesiomonas Shigel strains include: loides, Proteus mirabilis, Pseudomonas aeruginosa, 0278 (a) Gram-positive bacteria such as Strep. pyo Salmonella, Serratia Odorifera, Shigella, Staphylococcus genes (Group A), Strep. pneumoniae, Strep. GpB, Strep. aureus, Stenotrophomonas maltophilia, Streptococcus, Tri viridans, Strep. GpID-(Enterococcus), Strep. GpC and churis trichiura, Vibrio cholerae, Vibrio parahaemolyticus, GpG, Staph. aureus, Staph. epidermidis, Bacillus subti Vibrio vulnificus and other vibrios, Yersinia enterocolitica, lis, Bacillus anthraxis, Listeria monocytogenes, Anaero Yersinia pseudotuberculosis and Yersinia kristensenii. bic cocci, Clostridium spp., and Actinomyces spp.; and 0279 (b) Gram-negative bacteria such as Escherichia 0274. Other pathogens include Strep. pyogenes (Group A), coli, Enterobacter aerogenes, Kiebsiella pneumoniae, Strep. pneumoniae, Strep. GpB, Strep. viridans, Strep. GpD Proteus mirabilis, Proteus vulgaris, Morganella morga (Enterococcus), Strep. GpC and GpG, Staph. aureus, Staph. nii, Providencia Stuartii, Serratia marcescens, Citro epidermidis, Bacillus subtilis, Bacillus anthracis, Listeria bacter freundii, Salmonella typhi, Salmonella monocytogenes, Anaerobic cocci, Clostridium spp., Actino paratyphi, Salmonella typhi murium, Salmonella vir myces spp., Escherichia coli, Enterobacter aerogenes, Kieb chow, Shigella spp., Yersinia enterocolitica, Acineto siella pneumoniae, Proteus mirabilis, Proteus vulgaris, Mor bacter calcoaceticus, Flavobacterium spp., Haemophi ganella morganii, Providencia Stuartii, Serratia marcescens, lus influenzae, Pseudomonas aueroginosa, Citrobacter freundii, Salmonella typhi, Salmonella Campylobacter jejuni, Vibrio parahaemolyticus, Bru paratyphi, Salmonella typhi murium, Salmonella virchow, cella spp., Neisseria meningitidis, Neisseria gonor Shigella spp., Yersinia enterocolitica, Acinetobacter cal rhoea, Bacteroides fragilis, and Fusobacterium spp. coaceticus, Flavobacterium spp., Haemophilus influenzae, 0280 According to some embodiments of the present Pseudomonas aeruginosa, Campylobacter jejuni, Vibrio invention, the conjugates to presented herein can be effec parahaemolyticus, Brucella spp., Neisseria meningitidis, tively used against bacterial strains which have developed or Neisseria gonorrhoea, Bacteroides fragilis, Fusobacterium are prone to or capable of developing resistance to at least one spp., Mycobacterium tuberculosis (including MDR and XDR antimicrobial strain, such as, but not limited to, E. coli R477 strains from hospital origins isolated from patients) and 100, E. coli ATCC 25922, E. coli AG100E, E. coli AG100A, Mycobaterium Smegmatis. B. subtilis ATCC 6633, MRSA ATCC 43300 and E. coli 0275 Accordingly, a condition associated with a patho ATCC 35218, genic microorganism describes an infectious condition that 0281. Thus, according to one aspect of the present inven results from the presence of the microorganism in a subject. tion there is provided a method of treating a medical condition The infectious condition can be, for example, a bacterial associated with a pathogenic microorganism in a Subject. The infection, a fungal infection, a protozoal infection, and the method is effected by administering to that subject, a thera like. peutically effective amount of a conjugate as presented herein. 0276 Some higher forms of microorganisms are patho 0282. As used herein, the phrase “therapeutically effective genic per-se, and other harbor lower forms of pathogenic amount describes an amount of an active agent being admin bacteria, thus present a medical threat expressed in many istered, which will relieve to some extent one or more of the medical conditions, such as, without limitation, actinomyco symptoms of the condition being treated. In the context of the sis, anthrax, aspergillosis, to bacteremia, bacterial skin dis present embodiments, the phrase “therapeutically effective eases, bartonella infections, botulism, brucellosis, burkhold amount describes an amount of a conjugate being adminis eria infections, campylobacter infections, candidiasis, cat tered and/or re-administered, which will relieve to some scratch disease, chlamydia infections, cholera, clostridium extent one or more of the symptoms of the condition being infections, coccidioidomycosis, cryptococcosis, dermatomy treated by being at a level that is harmful to the target micro coses, dermatomycoses, diphtheria, ehrlichiosis, epidemic organism(s), and cause a disruption to the life-cycle of the louse borne typhus, Escherichia coli infections, fusobacte target microorganism(s), namely a bactericidal level or oth rium infections, gangrene, general infections, general erwise a level that inhibits the microorganism growth or mycoses, gram-negative bacterial infections, Gram-positive eradicates the microorganism. bacterial infections, histoplasmosis, impetigo, klebsiella 0283. The efficacy of any antimicrobial agent, including infections, legionellosis, leprosy, leptospirosis, listeria infec the conjugates presented herein, is oftentimes referred to in tions, lyme disease, maduromycosis, melioidosis, mycobac minimal inhibitory concentration units, or MIC units. AMIC terium infections, mycoplasma infections, necrotizing fascii is the lowest concentration of an antimicrobial agent, typi tis, nocardia infections, onychomycosis, ornithosis, cally measured in micro-molar (LM) or micrograms per mil pneumococcal infections, pneumonia, pseudomonas infec liliter (ug/ml) units, which can inhibit the growth of a micro tions, Q fever, rat-bite fever, relapsing fever, rheumatic fever, organism after a period of incubation, typically 24 hours. rickettsia infections, Rocky-mountain spotted fever, salmo MIC values are used as diagnostic criteria to evaluate resis nella infections, Scarlet fever, Scrub typhus, sepsis, sexually tance of microorganisms to an antimicrobial agent, and for transmitted bacterial diseases, staphylococcal infections, monitoring the activity of an antimicrobial agent in question. streptococcal infections, Surgical site infection, tetanus, tick MICs are determined by standard laboratory methods, as borne diseases, tuberculosis, tularemia, typhoid fever, urinary these are described and demonstrated in the Examples section tract infection, vibrio infections, yaws, versinia infections, that follows. Standard laboratory methods typically follow a Yersinia pestis plague, Zoonoses and Zygomycosis. standard guideline of a reference body such as the Clinical 0277. The conjugates presented herein can be effectively and Laboratory Standards Institute (CLSI), British Society used against bacterial Strains which have developed or are for Antimicrobial Chemotherapy (BSAC) or The European US 2014/0357591 A1 Dec. 4, 2014

Committee on Antimicrobial Susceptibility Testing (EU 0294 The pharmaceutical composition may be formu CAST). In clinical practice, the minimum inhibitory concen lated for administration in either one or more of routes trations are used to determine the amount of antibiotic agent depending on whether local or systemic treatment or admin that the subject receives as well as the type of antibiotic agent istration is of choice, and on the area to be treated. Adminis to be used. tration may be done orally, by inhalation, or parenterally, for 0284 As presented in the Examples section that follows, example by intravenous drip or intraperitoneal, Subcutane the conjugates described herein exhibit MIC values in the ous, intramuscular or intravenous injection, or topically (in range of 0.2-20 ug/ml. cluding ophtalmically, vaginally, rectally, intranasally). 0285 According to another aspect of embodiments of the 0295 Formulations for topical administration may present invention, each of the conjugates described herein is include but are not limited to lotions, ointments, gels, creams, identified for use in treating a subject diagnosed with a medi Suppositories, drops, liquids, sprays and powders. Conven cal condition associated with a pathogenic microorganism. tional pharmaceutical carriers, aqueous, powder or oily bases, 0286 According to another aspect of embodiments of the thickeners and the like may be necessary or desirable. present invention, there is provided a use of any of the con 0296 Compositions for oral administration include pow jugates described herein as a medicament. In some embodi ders or granules, Suspensions or Solutions in water or non ments, the medicament is for treating a subject diagnosed aqueous media, Sachets, pills, caplets, capsules or tablets. with a medical condition associated with a pathogenic micro Thickeners, diluents, flavorings, dispersing aids, emulsifiers organism. or binders may be desirable. 0287. In any of the methods and uses described herein, the 0297 Formulations for parenteral administration may conjugate can be administered as a part of a pharmaceutical include, but are not limited to, sterile solutions which may composition, which further comprises a pharmaceutical also contain buffers, diluents and other suitable additives. acceptable carrier, as detailed hereinbelow. The carrier is Slow release compositions are envisaged for treatment. selected suitable to the selected route of administration. 0298. The amount of a composition to be administered 0288 The conjugates presented herein can be adminis will, of course, be dependent on the subject being treated, the tered via any administration route, including, but not limited severity of the affliction, the manner of administration, the to, orally, by inhalation, or parenterally, for example, by intra judgment of the prescribing physician, etc. venous drip or intraperitoneal, Subcutaneous, intramuscular 0299 Pharmaceutical compositions for use in accordance or intravenous injection, or topically (including ophtalmi with embodiments of the invention thus may beformulated in cally, Vaginally, rectally, intranasally). conventional manner using one or more pharmaceutically 0289 Hence, according to another aspect of embodiments acceptable carriers comprising excipients and auxiliaries, of the invention, there is provided a pharmaceutical compo which facilitate processing of the conjugates presented herein sition which comprises, as active ingredients, one or more of into preparations which can be used pharmaceutically. Proper the conjugates presented herein and a pharmaceutically formulation is dependent upon the route of administration acceptable carrier. According to some embodiments, the chosen. Toxicity and therapeutic efficacy of the conjugates composition is packaged in a packaging material and identi presented herein can be determined by standard pharmaceu fied in print, in or on the packaging material, for use in the tical procedures in experimental animals, e.g., by determin treatment of a medical condition associated with a pathogenic ing the ECso, the ICso and the LDso (lethal dose causing death microorganism in a Subject. in 50% of the tested animals) for a subject combination of 0290. As used herein the phrase “pharmaceutical compo antimicrobial agent(s) and polymer(s). The data obtained sition' or the term “medicament” refer to a preparation of the from these activity assays and animal studies can be used in conjugates presented herein, with other chemical compo formulating a range of dosage for use in human. nents such as pharmaceutically acceptable and Suitable car 0300. The dosage may vary depending upon the dosage riers and excipients, and optionally with additional active form employed and the route of administration utilized. The agents, such as an antimicrobial agent. The purpose of a exact formulation, route of administration and dosage can be pharmaceutical composition is to facilitate administration of chosen by the individual physician in view of the patients the conjugate to a subject. condition. (See e.g., Finglet al., 1975, in “The Pharmacologi 0291 Hereinafter, the term “pharmaceutically acceptable cal Basis of Therapeutics’. Ch. 1 p. 1). In general, the dosage carrier” refers to a carrier or a diluent that does not cause is related to the efficacy of the active ingredient which, in the significant irritation to an organism and does not abrogate the context of embodiments of the invention, is related to its biological activity and properties of the administered conju minimal inhibitory concentration (MIC) and the particular gate. Examples, without limitations, of carriers are: propy pharmacokinetics and pharmacology thereof for absorption, lene glycol, Saline, emulsions and mixtures of organic Sol distribution, metabolism, excretion and toxicity (ADME vents with water, as well as Solid (e.g., powdered) and Tox) parameters. The amount of a composition to be admin gaseous carriers. istered will, of course, be dependent on the subject being 0292 Herein the term “excipient” refers to an inert sub treated, the severity of the affliction, the manner of adminis stance added to a pharmaceutical composition to further tration, the judgment of the prescribing physician, etc. facilitate administration of a conjugate. Examples, without 0301 Compositions of the present invention may, if limitation, of excipients include calcium carbonate, calcium desired, be presented in a pack or dispenser device, such as an phosphate, various Sugars and types of starch, cellulose FDA (the U.S. Food and Drug Administration) approved kit, derivatives, gelatin, vegetable oils and polyethylene glycols. which may contain one or more unit dosage forms containing 0293 Techniques for formulation and administration of the active ingredient. The pack may, for example, comprise drugs may be found in “Remington's Pharmaceutical Sci metal or plastic foil. Such as, but not limited to ablister pack ences’ Mack Publishing Co., Easton, Pa., latest edition, or a pressurized container (for inhalation). The pack or dis which is incorporated herein by reference. penser device may be accompanied by instructions for admin US 2014/0357591 A1 Dec. 4, 2014

istration. The pack or dispenser may also be accompanied by acids. Conventional nontoxic salts include those derived from a notice associated with the container in a form prescribed by inorganic acids Such as hydrochloric, hydrobromic, Sulfuric, a governmental agency regulating the manufacture, use or Sulfamic, phosphoric and nitric acid; and the salts prepared sale of pharmaceuticals, which notice is reflective of approval from organic acids such as acetic, propionic, Succinic, gly by the agency of the form of the compositions for human or colic, Stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, Veterinary administration. Such notice, for example, may be maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, of labeling approved by the U.S. Food and Drug Administra salicylic, Sulfanilic, 2-acetoxybenzoic, fumaric, toluene tion for prescription drugs or of an approved product to insert. Sulfonic, methanesulfonic, ethane disulfonic, oxalic and Compositions comprising the conjugates presented herein isethionic acids. The pharmaceutically acceptable salts can be formulated in a compatible pharmaceutical carrier may also synthesized from the parent compound, which contains a be prepared, placed in an appropriate container, and labeled basic or acidic moiety, by conventional chemical methods. for treatment of an indicated condition, as is detailed herein. Generally, such salts can be prepared by reacting the free acid 0302) The present embodiments further encompass any or base forms of these compounds with a stoichiometric enantiomers, prodrugs, Solvates, hydrates and/or pharmaceu amount of the appropriate base or acid in water or in an tically acceptable salts of the conjugates described herein and organic solvent, or in a mixture of the two: generally, non methods, compositions and uses utilizing enantiomers, pro aqueous media like ether, ethyl acetate, ethanol, isopropanol, drugs, Solvates, hydrates and/or pharmaceutically acceptable oracetonitrile are preferred. Lists of suitable salts are found in salts of the conjugates described herein. Remington's Pharmaceutical Sciences, 17th ed. (Mack Pub 0303 As used herein, the term “enantiomer' refers to a lishing Company, Easton, Pa., 19143, p. 1418). Stereoisomer of a conjugate that is Superposable with respect 0309 Representative examples of conjugate pharmaceu to its counterpart only by a complete inversion/reflection tically acceptable salts that can be efficiently used in the (mirror image) of each other. Enantiomers are said to have context of the present invention include, without limitation, “handedness' since they refer to each other like the right and conjugate hydrochloride and conjugate mesylate. left hand. Enantiomers have identical chemical and physical 0310. According to some embodiments of the present properties except when present in an environment which by invention, the compositions, uses and method of treatment, itself has handedness, such as all living systems. according to some embodiment of the present invention, may 0304. The term “prodrug” refers to an agent, which is include the co-administration of at least one additional thera converted into the active conjugate (the active parent drug) in peutically active agent, as this is defined and discussed herein. vivo. Prodrugs are typically useful for facilitating the admin 0311. The conjugates described herein can be beneficially istration of the parent drug. They may, for instance, be bio utilized per-se in the treatment of pathogenic microorganism available by oral administration whereas the parent drug is infections, as these are defined hereinbelow. As to demon not. A prodrug may also have improved solubility as com strated in the Example section that follows, such conjugates pared with the parent drug in pharmaceutical compositions. are by themselves capable of exerting antimicrobial activity. Prodrugs are also often used to achieve a Sustained release of The option to include an additional therapeutically active the active conjugate in vivo. An example, without limitation, agent may thus act synergistically or cooperatively as toxic of a prodrug would be a conjugate according to some embodi agents against various bacteria, fungi and other microorgan ments of the present invention, having one or more carboxylic isms. acid moieties, which is administered as an ester (the “pro 0312 Exemplary additional therapeutically active agents drug'). Such a prodrug is hydrolyzed in vivo, to thereby include, but are not limited to, an antibiotic agent, an anti provide the free conjugate (the parent drug). The selected inflammatory agent, an anti-pruritic agent, an anti-prolifera ester may affect both the solubility characteristics and the tive agent and the likes. hydrolysis rate of the prodrug. 0313 While most known antibiotics act by interfering 0305. The term “solvate” refers to a complex of variable selectively with the biosynthesis of one or more of the Stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), molecular constituents of the cell-membrane, proteins or which is formed by a solute (the conjugates described herein) nucleic acids, the conjugates presented herein also act by and a solvent, whereby the solvent does not interfere with the reducing or preventing the adverse effects of microbial drug biological activity of the solute. Suitable solvents include, for resistance, and therefore may also act to increase the sensi example, ethanol, acetic acid and the like. tivity of pathogenic microorganism to the antibiotic treat (0306 The term “hydrate” refers to a solvate, as defined ment, including resistant strains. hereinabove, where the solvent is water. 0314. In general, the additional therapeutically active 0307 The phrase “pharmaceutically acceptable salt agent is selected such that it exerts a beneficial effect in the refers to a charged species of the to parent conjugate and its treatment againstan infection, hence the additional therapeu counter ion, which is typically used to modify the solubility tically active agent may be an antimicrobial agent, as this is characteristics of the parent conjugate and/or to reduce any defined hereinabove. significant irritation to an organism by the parent conjugate, 0315. It is expected that during the life of a patent maturing while not abrogating the biological activity and properties of from this application many relevantantimicrobial conjugates the administered conjugate. will be developed and the scope of the phrase “antimicrobial 0308 Examples of pharmaceutically acceptable salts conjugates' is intended to include all Such new technologies include, but are not limited to, mineral or organic acid salts of a priori. basic residues such as amines, and alkali or organic salts of 0316. It is appreciated that certain features of the inven acidic residues such as carboxylic acids. The pharmaceuti tion, which are, for clarity, described in the context of separate cally acceptable salts include the conventional non-toxic salts embodiments, may also be provided in combination in a or the quaternary ammonium salts of the parent compound single embodiment. Conversely, various features of the formed, for example, from non-toxic inorganic or organic invention, which are, for brevity, described in the context of a US 2014/0357591 A1 Dec. 4, 2014 20 single embodiment, may also be provided separately or in any 0325 IR spectra (CHCl) were recorded on a Bruker vec suitable subcombination or as suitable in any other described tor 22 spectrophotometer, and only significant peaks were embodiment of the invention. Certain features described in identified. Microwave assisted reactions were carried out in the context of various embodiments are not to be considered domestic microwave oven Sauter SG251. Analytical HPLC essential features of those embodiments, unless the embodi was performed on Hitachi LC system equipped with auto ment is inoperative without those elements. sampler, by using Superspher(R) 100 RP-18 column and a 0317 Various embodiments and aspects of the present detection wavelength was 271 nm. invention as delineated hereinabove and as claimed in the 0326 All syntheses reactions were carried out under an claims section below find experimental support in the follow argon atmosphere with anhydrous solvents, unless otherwise ing examples. noted. 0327 Materials: EXAMPLES 0328 1-Bromo-2-chloroethane, 1-bromo-3-chloropro pane, 1-bromo-4-chlorobutane, 1-bromo-5-chloropentane, 0318 Reference is now made to the following examples, 1,6-dibromohexane, 1,3-dibromo-2-propanol, 2-bromoethyl which together with the above descriptions, illustrate some ether, C.C.'-dibromo-meta-xylene, and C.C.'-dibromo-para embodiments of the invention in a non limiting fashion. Xylene as well as 4-ethynylaniline and propargylamine were Materials and Methods obtained from Sigma-Aldrich Israel. 0329 Compound 7 was prepared as previously reported 0319 Methods: 29. 0320 "H NMR spectra (including DEPT, 2D-COSY. 2D 0330 Purity of the Compounds 1a-q was determined by TOCSY. 1D TOCSY. HMOC, HMBC) were recorded on a using HPLC analysis which indicated more than 95% purity Bruker AvanceTM 500 spectrometer, and chemical shifts of each products. reported (in ppm) are relative to internal MeSi (8-0.0) with 0331 All other chemicals, reagents and solvents were CDC1 as the solvent, and to HOD (8–4.63) with DO as the obtained from commercial sources such as Sigma-Aldrich, solvent. Fluka and Acros unless stated otherwise. 0321 °C NMR spectra were recorded on a Bruker 0332 PlasmidpETSACG1 carrying the APH(3')-IIIa gene AvanceTM 500 spectrometer at 125.8 MHz, and the chemical (Gene bank Accession No. VO1547) was generously provided shifts reported (in ppm) relative to the residual solvent signal by Prof. A. Berghuis, McGill University. Plasmid pSF815 for CDC1 (8–77.00), or to external sodium 2,2-dimethyl-2- carrying the AAC(6')-APH(2") gene was generously pro silapentane sulfonate (8-0.0) for DO as the solvent. vided by Prof. S. Mobashery, University of Notre Dame. 0322 Mass spectra (MS) analysis were obtained either on Plasmid plT9d carrying the APHC3')-Ia gene was obtained a Bruker Daltonix Apex 3 mass spectrometer under electron from New England Biolabs. spray ionization (ESI), or by a TSQ-70B mass spectrometer (Finnigan Mat). Example 1 0323 Reactions were monitored by TLC on silica gel 60 Fs (0.25 mm, Merck), and spots were visualized by charring Syntheses with a yellow solution containing (NH4)Mo.O4.HO (120 0333. The general synthesis of the Cipro-NeoB conjugates grams) and (NH4)2Ce(NO) (5 grams) in 10% HSO (800 (Compound 1), according to Some embodiments of the mL). present invention, from Compound 2 (the “Cipro' moiety) 0324 Flash column chromatography was performed on and Compound 3 (the "NeoB' moiety), is illustrated in silica gel 60 (70-230 mesh). Scheme 5 below.

Scheme 5

N 7 N

... CCCO 2 US 2014/0357591 A1 Dec. 4, 2014

-continued

Cipro moiety NeoB moiety

0334 X and Y represent various first and second spacer 0341 Preparation of Compound 2 General Procedure B: moieties respectively, which form the connecting moiety 0342. Several SAR studies on fluoroquinolones have dem together with the resultant 4-yl-1-yl-1H-1,2,3-triazole link onstrated a high tolerance for structural variations at the 7-po ing moiety. sition of the phenyl ring (marked with “7” in Scheme 5 and 0335) Nine exemplary azido-derivatives of Cipro (referred Scheme 6), including alkylations at the terminal nitrogen of to herein and presented below as Compounds 2a-i), and three the piperazine moiety 7, 9-11, 21, 22. exemplary alkyne-derivatives of NeoB (referred to herein and (0343 Hence, Compound 2 (the “Cipro' moiety), is pre presented below as Compounds 3a-c), were prepared and pared as illustrated in Scheme 6 below, wherein X represents coupled via “click reaction 30 to afford a library of 17 first spacer moiety and D represents a halo group. exemplary Cipro-NeoB conjugates, according to some embodiments of the present invention, which are also referred to to herein and presented below as Compounds 1a-q. The Scheme 6 first and second spacer moieties, namely X and Y, were selected to vary both the length and chemical nature of the O O linkage between the two pharmacophores, the Cipro moiety F and the NeoB moiety. HO 0336 Preparation of Compound 1—General Procedure A: 7 -- 0337 Exemplary conjugates, according to some embodi ments of the present invention, referred to herein in general as N -> Compound 1, were prepared as illustrated in Scheme 5 above. / \ Null 0338 A solution of Compound 2 (0.06 mmol), and Com pound 3 (0.05 mmol), (CHCN). CuPF (0.025 mmol) in a ciprofloxacin ("Cipro") solution of EtN in water (7%, 5 ml) is placed in a glass vial "Nv 1st - - (25 ml). The vial is closed with a stopper and heated in a X NS. domestic microwave oven for 40 seconds at maximum power. N Propagation of the reaction is monitored by TLC using a X mixture of 10:15:6:15 CHCl/MeOH/HO/MeNHas eluent v r YN, (for a sample containing 33% reaction solution in EtOH). After completion, the reaction mixture is purified on a short N 7 Nu column of Amberlite CG-50 (H"-form). The column is sequentially washed by MeOH, MeOH/MeNH. (33% solu tion in EtOH) 95:5, MeOH/MeNH. (33% solution in EtOH) 9:1 and MeOH/MeNH. (33% solution in EtOH)4:1. Frac O tions containing the product were combined, evaporated, re 2 dissolved in water and evaporated again to afford the free amine form of the product. 0339. The product is dissolved in water; the pH is adjusted 0344. The azido-containing first spacer moiety precursor to 3.2 with TFA (0.01 M), and lyophilized to afford the TFA compounds are synthesized from the corresponding dibromo salt of the final product, typically as a white foamy solid. or bromochloro compounds according to published proce 0340 Chemical yields of the obtained Compounds 1a-q dure 31. A mixture of ciprofloxacin (Cipro, 1 mmol) and an are given in Table 4 hereinbelow and their complete analytical azido-containing first spacer moiety precursor compound (5 data are provided hereinbelow. mmol) in acetonitrile (15 mL) is refluxed in the presence of US 2014/0357591 A1 Dec. 4, 2014 22 powdered NaHCO (1 mmol) for 12-24 hours. When a TLC -continued (MeOH/CHC1, 1:9) indicates completion of the reaction N (within 2-14 hours), the mixture is filtered, washed with excess MeOH/CHCl (1:1) and the combined filtrates are 1) CAN, CHCN/HO evaporated to dryness under reduced pressure. N (69%) 0345 The residue is purified by column chromatography Ns -e-2) PMe, NaOH (silica gel, MeOH/CHCl, 1:10) to yield the product, typi OPMB 0.1M, THF cally as a slightly yellow solid. (89%)

0346 Cipro was subjected to modifications at the terminal O OPMB nitrogen of the piperazine moiety with various azido-contain or 4-2 ing first spacer moiety precursor compounds to afford the OPMB exemplary Compounds 2a-I, which were prepared as Compound 6, Y = -CH2 described in General Procedure B by direct coupling of the commercial Cipro with the corresponding bromo/chloro azides under reflux and base conditions (NaHCO, CHCN). NH2

Chemical yields of these products are summarized in Table 3 hereinbelow. 0347 Preparation of Compound 3—General Procedure C: 0348. The synthesis of two types of alkyne derivatives of NeoB (Compounds 3) is illustrated in Scheme 7 below.

Scheme 7 a) OH 1) TfN, CuSO4 Compound 3a, Y = -CH2 EtN (86%) NeoB -> 2) TIPSCI, Pyr (75%) b)

N

O HO 1) PMBC1, HO N NaOH, TEMPO, N DMF BAIB TIPSO O O N. (78%) CHCl2.H2O 5. -e- 0 2) TBAF, (76%) OH THF -> OH N (72%)

N O O OH N ~4-2 OAc OA OH C Compound 4 Compound 7 N

N

N 3 Yn Y

N Her21 Br 21 NH, DCC OPMB Na, DMF HOBT, CH2Cl2 ~4-2 (80%) (72-86%) N O O OPMB N ~4-2O OAc OPMB OAc Compound 5 Compound 8 US 2014/0357591 A1 Dec. 4, 2014 23

-continued 0349 For the preparation of the alkyne-NeoB derivative N Compound 3a, commercial NeoB is converted to the corre sponding per-azido derivative according to published proce dure 32, followed by selective protection of the primary N hydroxyl to afford the intermediate Compound 4. Protection O O 1) MeNH2, of all the secondary hydroxyls with p-methoxybenzyl (PMB) EtOH ether and selective removal of silyl ether with TBAF affords Aa -e- OAc 2) PMe, Compound 5. Treatment of Compound 5 with propargyl bro NaOHATHF mide under alkaline conditions (e.g., NaH) affords the (80-94%, 5"-alkyne derivative Compound 6. Deprotection steps for OAc 2 steps) removal of PMB ether protections with CAN, followed by OAc Staudinger reaction to convert all the azides to the corre Compound 9a, Y = - -CH2 sponding amines, afford the alkyne-NeoB derivative Com Compound 9b, Y = -pCH pound 3a. NH2 0350. Two other alkyne derivatives of NeoB, Compounds

3b-c, contain a terminal alkyne group connected to the NeoB moiety at 5'-position via an amide linkage. For the assembly of these derivatives, the readily available 5"-alcohol 729 is converted to the corresponding 5'-acid Compound 8 accord ing to a published procedure 33. The resulting acid is there after coupled with the commercially available alkyne-amines in the presence of DCC to afford the corresponding amide Compounds 9a-b. Finally, removal of all the ester protections (MeNH, MeOH) followed by Staudinger reaction yield the alkyne-NeoB derivative Compound 3b-c in high yields. 0351 Preparation of Exemplary Compounds: Compound 3b, Y = -CH2 0352 Reagents used and chemical yield obtained in the Compound 3c, Y = -pCH preparation of exemplary Compounds 2a-i are presented in Table 3 below. All of Compounds 2a-i were prepared follow ing General Procedure B presented hereinabove. TABLE 3

Compounds 2a-i O

HOOC F

7 N N ~ N- N Nx1 N

azido-containing

US 2014/0357591 A1 Dec. 4, 2014 26

0382 "H NMR (500 MHz, CDC1): 8=1.16-1.19 (m, 2H, Preparation of 1,3,2',6'2",6"-Hexaazido-5'-triso cyclopropane), 1.34-1.38 (m. 2H, cyclopropane), 2.67-2.69 propylsilyloxy-neomycin (Compound 4) (t, J–4.5 Hz, 4H, piperazine), 3.35-3.37 (t, J–4.5 Hz, 4H, piperazine), 3.51-3.55 (m. 1H, cyclopropane), 3.62 (s. 2H, 0390 NCH), 4.35 (s. 2H, CHN), 7.22-7.24 (m. 1H, aromatic), 7.30-7.37 (m, 4H, aromatic, C. H), 7.86-7.89 (d. J=13.0 Compound 4 HZ, 1H, Cs—H), 8.67 (s, 1H, C, H) ppm. 0383 °C NMR (125 MHz, CDC1): 8=10.0 (CH, of cyclopropane), 31.4 (CH of cyclopropane), 51.5, 54.5, 56.5 (CHN), 64.4 (NCH), 106.6, 109.7, 113.8, 114.0, 121.2, 129.0, 130.7, 130.9, 137.3, 140.3, 140.9, 1544, 156.4, 168.8, 178.7 ppm. 0384 MALDITOFMS calculated for CHFNO (IM+ HI"): m/e=477.2: measured m/e=477.5. 7-(4-(4-(AZidomethyl)benzyl)piperazin-1-yl)-1-cy clopropyl-6-fluoro-4-OXO-1,4-dihydroquinoline-3- carboxylic acid (Compound 2i) 0385 0391 Commercially available NeoB was converted to the corresponding perazido derivative according to the published procedure 32. Hexaazido-NeoB (5.10 grams, 6.62 mmol) was dissolved in pyridine (30 ml), added with 4-DMAP (cata Compound 2i. lytic amount) and stirred at room temperature. After 15 min O O utes, triisopropylsilylchloride (TIPSC1) (1.91 grams, 9.93 F mmol) was added, and TLC (EtOAc, 100%) indicated HO N completion after 3 hours. The mixture was diluted with Nta N ^ N? EtOAc and washed with brine, HSO (2%), saturated NaHCO, and brine. The organic layers were combined, dried A N over MgSO, evaporated and the residue was purified by flash chromatography (silica gel, EtOAc/Hexane) to yield the silyl ether Compound 4 as a white powder (4.60 grams, 75% yield). 0386 H NMR (500 MHz, CDC1): 8=1.17-1.20 (m, 2H, 0392 HNMR (500 MHz, CDC1): 8–0.85-0.98 (m, 15H, cyclopropane), 1.35-1.39 (m. 2H, cyclopropane), 2.68-2.69 TIPS); ring I: 8–3.14-3.28 (m,3H, H-2, H-4, H-6), 3.33-3.51 (t, J=5.0 Hz, 4H, piperazine), 3.35-3.37 (t, J=5 Hz, 4H, pip (m. 1H, H-6), 3.63-3.74 (m. 1H, H-3), 3.91-3.95 (m, 1H, erazine), 3.52-3.54 (m. 1H, cyclopropane), 3.61 (s. 2H, H-5), 5.65-5.66 (d. J–3.5 Hz, 1H, H-1); ring II: 8=1.19-1.24 NCH), 4.34 (s. 2H, CHN), 7.29-7.31 (d. J–7.5 Hz, 2H, (ddd, JJ J = 12.5 Hz, 1H, H-2ax), 2.01-2.06 (dt, J=4.0, aromatic), 7.33-7.34 (d. J–7.0 Hz, 1H, C, H), 7.38-7.39 (d. 12.5 Hz, 1H, H-2eq), 3.14-3.28 (m, 2H, H-1, H-3), 3.33-3.51 J–7.5 Hz, 2H, aromatic), 7.91-7.94 (d. J=13.0 Hz, 1H, (m,3H, H-4, H-5, H-6); ring III: 8–3.33-3.51 (m. 1H, H-5'), Cs—H), 8.70 (s, 1H, C, H) ppm. 3.63-3.74 (m, 1H, H-5), 4.03-4.05 (m, 2H, H-2, H-4), 4.22 4.23 (dd, J–4.0, 4.5 Hz, 1H, H-3), 5.10-5.11 (d. J=4.5 Hz, 1H, (0387 °C NMR (125MHz, CDC1): 8–8.1 (CH, of cyclo H-1); ring IV: 8–2.91-2.94 (dd, J=4.0, 10.5 Hz, 1H, H-6), propane), 35.2 (CH of cyclopropane), 49.7, 52.6 (CHN), 3.14-3.28 (m, 2H, H-4, H-6), 3.63-3.74 (m. 1H, H-2), 3.78 54.5, 62.4 (NCH), 104.7, 107.9, 112.1, 112.3, 119.5, 128.2, 3.80 (t, J–3.5 Hz, 1H, H-3), 3.81-3.83 (m, 1H, H-5), 4.96 (d. 129.5, 1344, 137.9, 139.0, 145.9, 147.3, 152.6, 154.6, 167.0, J=1.0 Hz, 1H, H-1) ppm. 177.0 ppm. 0393 °C NMR (125 MHz, CDC1): 8=11.5, 17.4, 31.6 0388 MALDITOFMS calculated for CHFNO (IM+ (C-2), 50.8 (C-6), 51.1 (C-6"), 59.2, 59.4, 60.4, 63.2, 63.3, HI): m/e=477.1; measured m/e=477.5. 68.4, 68.7, 70.5, 70.9, 71.1, 73.6, 74.3, 75.0, 75.3, 76.3, 83.1, 0389) Exemplary Compounds 3a-c were prepared follow 85.1, 96.3 (C-1"), 98.6 (C-1'), 107.2 (C-1") ppm. ing General Procedure C presented hereinabove, using two 0394 MALDI TOFMS calcd for C.H.N.O.SiNa synthetic routes as presented in Scheme 7 hereinabove. (IM+Na"): m/e=949.6; measured m/e=949.4.

US 2014/0357591 A1 Dec. 4, 2014 28

78.1, 79.5, 79.7, 81.6, 82.1, 83.8, 96.2 (C-1"), 98.2 (C-1'), H-3), 5.36 (s, 1H, H-1); ring IV 8,83.08-3.13 (dd, J=8.5, 13.5 107.1 (C-1"), 113.7, 113.8, 114.0, 129.4, 129.51, 129.8, 130. HZ, 1H, H-6), 3.27-3.51 (m. 2H, H-2, H-6'), 3.75 (m. 1H, 0, 159.1, 159.2, 159.5 ppm. H-4), 4.14-4.17 (m. 1H, H-3), 4.23-4.28 (m. 1H, H-5), 5.22 (s, (0405) MALDITOFMS calcd for C.H.N.O. Na (IM+ 1H, H-1) ppm. Na"): m/e=1551.8; measured m/e=1551.4. 0410 °C NMR (125 MHz, DO): 8–29.6 (C-2), 42.2 (C-6'), 42.5 (C-6"), 50.2, 51.6, 52.6, 55.3, 60.3, 68.9, 69.4, Preparation of 5"-(Prop-2-ynyloxy)-neomycin 69.6, 71.3, 72.1, 73.2, 74.1,749, 76.2,772, 78.9, 81.1 (CH of (Compound 3a) triple bond), 81.6, 87.1,96.4(C-1"),96.9 (C-1'), 112.5 (C-1") ppm. 0406 0411 MALDITOFMS calcd for CHNO (IM+H"): m/e=653.3; measured m/e=653.3. Compound 3a Preparation of 1,3,2,6'2",6"-Hexaazido-6,3',4',2", NH 3",4'-hexaacetoxy-neomycin-4"-carboxylic acid

(Compound 8) 0412 NH

O Compound 8 OH

HN

0407 Compound 6 (0.43 grams, 0.28 mmol) was dis solved in acetonitrile (5 ml) and after stirring at -4°C. for 10 minutes, cerium (IV) ammonium nitrate (CAN) (1.0 grams, OAc 1.82 mmol) in 0.5 ml of water was added. The reaction progress was monitored by TLC (EtOAc/Hexane, 4:5, and EtOAc, 100%). After 3 hours, the reaction mixture was OAc diluted with EtOAc (100 ml) and washed with brine. The combined organic layer was dried over MgSO4, evaporated to 0413 Compound 7 (0.43 gram, 0.42 mmol, prepared dryness and used for the next step without further purifica according to Fridman, 2003 #301) was dissolved in CHCl, t1On. (30 ml) and cooled to 5° C. Thereafter, water (2.5 ml), 0408. The crude product from the previous step was dis TEMPO (0.013 grams, 0.08 mmol) and BAIB (0.34 grams, solved in THF (7 ml), NaOH 0.1M (1.5 ml) and stirred at 60° 1.06 mmol) were added. The reaction mixture was stirred at C. for 10 minutes after which PMe (1 M solution in THF, 4.0 5°C. for 40 minutes and then allowed slowly to warm to room ml. 4.0 mmol) was added. The reaction progress was moni temperature. The reaction progress was monitored by TLC tored by TLC (CH.Cl/MeOH/HO/MeNH. (33% solution with two solvent systems (EtOAc/Hexane, 1:1, and MeOH/ in EtOH), 10:15:6:15), which indicated completion after 3.5 CHCl. 1:9), which indicated completion after 4.5 hours. The hours. The reaction mixture was purified by flash chromatog mixture was cooled to 0°C., diluted with EtOAc, quenched raphy on a short column of silica gel and the column was with Na2SOs, and washed with brine. The combined organic washed as follows: THF. EtOH, MeOH, and finally with layer was dried over MgSO evaporated and purified by flash MeNH. (33% solution in EtOH). The fractions containing the chromatography (silica gel, MeOH/CHCl) to yield Com product were combined and evaporated to dryness, re-dis pound 8 (320 mg, 76% yield). Solved in water and evaporated again to afford Compound 3a 104.14 'H NMR (500 MHz, CDC1): 8–2.04 (s.3H, OAc), as a free amine (162.5 mg. 89% for both steps). This product 2.06 (s.3H, OAc), 2.12 (s.3H, OAc), 2.13 (s.3H, OAc), 2.15 was then dissolved in water, the pH was adjusted to 7.5 with (s.3H, OAc), 2.16 (s.3H, OAc); ring I: 8–3.27-3.44 (m. 1H, 0.01 M HSO, and lyophilized to give the sulfate salt of H-2), 3.50-3.57 (m. 2H, H-6, H-6), 4.41-4.43 (m. 1H, H-5), Compound 3a (220 mg) as a white foamy solid. 5.00 (t, J=9.5 Hz, 1H, H-4), 5.43 (t, J=10.5 Hz, 1H, H-3), 6.12 (0409 HNMR (500 MHz, D.O, pH=3.0): 8–2.96 (m. 1H, (s, 1H, H-1); ring II: 8-1.63 (ddd, J. J. J.-12.5 Hz, 1H, CH of triple bond), 4.23-4.28 (m. 2H, CH of linker); ring I: H-2ax), 2.37 (dt, J–3.5, 12.5 Hz, 1H, H-2eq), 3.27-3.44 (m, 8–3.27-3.51 (m, 4H, H-2, H-4, H-6, H-6), 3.82-3.93 (m, 2H, 2H, H-1, H-3), 3.73 (t, J–9.0 Hz, 1H, H-5), 3.96 (t, J=9.0 Hz, H-3, H-5), 6.07-6.08 (d. J=4.0 Hz, 1H, H-1); ring II: 8=1.96 1H, H-4), 4.93-4.97 (m. 1H, H-6); ring III 8, 4.71 (m. 1H, 2.03 (ddd, JJ J 12.5 Hz, 1H, H-2ax), 2.37-2.41 (dt, J–4. H-3), 4.81 (t, J=5.0 Hz, 1H, H-2), 4.87 (d, J-3.0 Hz, 1H, H-4), 0, 12.5 Hz, 1H, H-2eq), 3.27-3.51 (m, 2H, H-1, H-3), 3.64 5.54 (d. J-50 Hz, 1H, H-1); ring IV: 8=3.27-3.44 (m, 3H, 3.69(m. 1H, H-5), 4.00-4.04 (t, J=9.5 Hz, 1H, H-6), 4.14-4.17 H-2, H-6, H-6), 4.05-4.07 (m. 1H, H-5), 4.71 (m, 1H, H-4), (t, J=9.5 Hz, 1H, H-4); ring III: 8=3.64-3.69 (m. 1H, H-5), 4.95 (s, 1H, H-1), 5.05 (t, J=2.5 Hz, 1H, H-3) ppm. 3.82-3.93 (m. 1H, H-5'), 4.23-4.28 (m. 1H, H-4), 4.40 (dd, 0415 °C NMR (125 MHz, CDC1): 8–20.4, 20.7, 20.8, J–2.0, 4.0 Hz, 1H, H-2), 4.47-449 (dd, J=4.0, 7.0 Hz, 1H, to 21.0, 31.2 (C-2), 50.5 (C-6"), 50.9 (C-6'), 57.2, 58.0, 59.0, US 2014/0357591 A1 Dec. 4, 2014 29

60.7, 65.7, 68.6, 69.1, 69.3, 69.8, 73.0, 74.6, 75.2, 76.0, 79.3, 75.8, 76.9, 77.7, 80.4 (CH of triple bond), 80.9, 83.1, 83.3, 81.8, 96.5 (C-1'), 100.3 (C-1"), 106.3 (C-1"), 168.6, 169.7, 99.0 (C-1"), 102.3 (C-1), 106.7 (C-1"), 170.1, 170.4, 1714, 169.8, 170.1, 170.2 ppm. 171.8, 171.9 ppm. 0416 MALDI TOFMS calcd for CHNOK (M+ 0421 MALDITOFMS calcd for CHNO Na (M+ KI): m/e=1075.3; measured m/e=1075.4. Na"): m/e=1096.3; measured m/e=1096.3. Preparation of 1,3,2,6'2",6"-Hexaazido-6,3',4'2", Preparation of 4"-(Prop-2-ynylcarbamoyl)-neomycin 3",4"-hexaacetoxy-4"-(prop-2-ynylcarbamoyl)-neo (Compound 3b) mycin (Compound 9a) 0422 0417 Compound 3b Compound 9a NH2

NH2

O OH

HN N O 0423 Compound 9a (2.4 grams, 2.27 mmol) was dis solved in 33% solution of MeNH in EtOH (40 ml) and the 0418 Compound 8 (2.68 grams, 2.59 mmol) was dis mixture was stirred at room temperature for 30 hours. The solved in CHCl (35 ml), then DCC (0.53 grams, 2.57 mmol) reagent and the solvent were removed by evaporation and the and HOBT (0.25 grams, 1.85 mmol) were added at 0°C. The residue was dissolved in THF (50 ml), NaOH 0.1M (3 ml) and reaction mixture was stirred at 0° C. for 1 hour, allowed stirred at 60° C. for 10 minutes after which PMe (1 M solu slowly to warm to room temperature, and then added with tion in THF, 21.9 mL. 21.9 mmol) was added. Propagation of propargyl amine (0.43 grams, 7.81 mmol). Progress of the the reaction was monitored by TLC CHC1/MeoH/HO/ reaction was monitored by TLC with two solvent systems MeNH. (33% solution in EtOH), 10:15:6:15), which indi (EtOAc/Hexane, 1:1 and MeOH/CHC1, 1:9), which indi cated completion after 3.5 hours. The reaction mixture was cated completion after 4 hours. The mixture was diluted with purified by flash chromatography on a short column of silica EtOAc and washed with brine. The combined organic layer gel and the column was washed as follows: THF. EtOH, was dried over MgSO, evaporated and purified by flash MeOH, and finally with MeNH. (33% solution in EtOH). The chromatography (silica gel, EtOAc/Hexane) to afford Com fractions containing the product were evaporated under pound 9a (2.0 grams, 72% yield). vacuum, re-dissolved in water and evaporated again to afford 0419 H NMR (500 MHz, CDC1): 8–2.07 (s.3H, OAc), Compound 3b as a free amine (1.39 grams, 92% yield). This 2.10 (s.3H, OAc), 2.13 (s, 6H, OAc), 2.17 (s.3H, OAc), 2.19 product was then dissolved in water, the pH was adjusted to (s.3H, OAc), 2.25-2.26 (t, J=2.5 Hz, 1H, CH of triple bond), 7.5 with 0.01 MHSO and lyophilized to give the sulfate salt 3.91-3.95 (m, 1H, CH of linker), 4.14-4.17 (m, 1H, CH of of Compound 3b (1.88 grams) as a white foamy solid. linker), 7.41-7.44 (t, J=6.0 Hz, 1H, NH); ring I: 8–3.23-3.26 0424 'H NMR (500 MHz, D.O, pH=3.17): 8–2.59-2.60 (dd, J=3.0, 10.0 Hz, 1H, H-2), 3.31-3.44 (m, 2H, H-6, H-6'), (t, J=2.5 Hz, 1H, CH of triple bond), 3.86-4.02 (m, 2H, CH, 4.46-4.49 (m, 1H, H-5), 4.99-5.09 (m, 1H, H-4), 5.50-5.54 of linker); ring I: 8–3.20-3.24 (dd, J=3.5, 13.5 Hz, 1H, H-6), (dd, J=9.0, 11.0 Hz, 1H, H-3), 5.97-5.98 (d. J=4.0 Hz, 1H, 3.25-3.37 (m, 3H, H-2, H-4, H-6'), 3.86-4.02 (m, 2H, H-3, H-1); ring II 8, 1.62-1.70 (ddd, JJ J = 12.5 Hz, 1H, H-5), 6.03-6.04 (d. J=4.0 Hz, 1H, H-1); ring II: 8=1.92-1.98 H-2ax), 2.39-2.43 (dt, J=4.0, 12.5 Hz, 1H, H-2eq), 3.31-3.44 (ddd, JJ J =12.5 Hz, 1H, H-2ax), 2.37-2.41 (dt, J=4.0, (m. 1H, H-3), 3.54-3.58 (m, 1H, H-1), 3.74-3.78 (t, J=9.0 Hz, 12.5 Hz, 1H, H-2eq), 3.25-3.37 (m. 1H, H-1), 3.45-3.50 (m, 1H, H-4), 3.99-402 (t, J=9.0Hz, 1H, H-5), 4.99-5.09 (m. 1H, 1H, H-3), 3.67-3.71 (m, 1H, H-5), 3.86-4.05 (m. 1H, H-6), H-6); ring III: 8–4.57-4.60 (t, J=6.0 Hz, 1H, H-2), 4.65-4.66 4.15-4.19 (t, J=9.5 Hz, 1H, H-4); ring III: 8–4.42-4.43 (dd. (dd, J–3.5, 6.0 Hz, 1H, H-3), 4.82 (d. J=4.0 Hz, 1H, H-4), J=2.0, 4.5 Hz, 1H, H-3), 4.47-4.48 (d. J=7.5 Hz, 1H, H-4), 5.59-5.61 (d. J=6.0 Hz, 1H, H-1); ring IV: 8=3.31-3.44 (m, 4.60-4.62 (dd, J–4.5, 7.5 Hz, 1H, H-2), 5.44 (s, 1H, H-1); ring 2H, H-2, H-6), 3.54-3.58 (m, 1H, H-6), 4.09-4.13 (m, 1H, IV: 8–3.13–3.17 (dd, J=8.0, 13.5 Hz, 1H, H-6), 3.38-3.41 (dd, H-5), 4.71-4.72 (t, J-2.0 Hz, 1H, H-4), 4.99-5.09 (m, 2H, J=3.0, 13.5 Hz, 1H, H-6'), 3.53 (m. 1H, H-2), 3.67-3.71 (m, H-1, H-3) ppm. 1H, H-4), 4.15-4.19 (m, 1H, H-3), 4.20-4.23 (m. 1H, H-5), 0420 °C NMR (125 MHz, CDC1): 8–22.1, 22.5, 22.7, 5.20 (s, 1H, H-1) ppm. 22.8, 30.6 (CH of linker), 33.2 (C-2), 52.4 (C-6'), 52.7 (C-6'), 0425 °C NMR (125 MHz, DO): 8–29.6 (C-2), 30.7, 59.2, 59.9, 60.9, 62.0, 67.4, 70.6, 71.0, 71.1, 71.2, 73.5, 74.8, 42.1 (C-6'), 42.2 (C-6'), 50.2, 51.8, 52.5, 55.4, 68.7, 69.3,

US 2014/0357591 A1 Dec. 4, 2014 35

55.1, 55.3, 61.7, 68.8, 69.2, 69.7, 71.2, 72.2, 72.5, 74.2, 74.8, 3.25-3.65 (m,2H, H-2, H-6'), 3.73 (m, 1H, H-4), 4.11-4.13 (t, 77.3, 79.5, 81.1, 86.5, 96.6 (C-1"), 97.5 (C-1), 108.4, 112.4 J=3.0 Hz, 1H, H-3), 4.18-4.20(m, 1H, H-5), 5.17 (s, 1H, H-1) (C-1"), 116.9, 119.2, 121.0, 126.1 (CH of triazole), 130.3, ppm. 130.5, 133.7, 138.7, 140.6, 145.7, 150.0, 1540, 170.9, 172.8, 0506 °C (NMR 125 MHz, DO): 8–9.2 (cyclopro 177.7 ppm. pane), 29.7 (C-2),37.9, 42.1, 42.2, 46.4, 48.2, 50.2, 51.4, 52.6, 53.7, 55.2, 56.9, 65.1, 69.1, 69.3, 69.6, 71.2, 71.5, 0503 MALDI TOFMS calculated for CHFNOK 71.9, 72.7, 74.1, 75.2, 76.6, 77.9, 81.9, 86.9, 96.7 (M+K"): m/e=1180.6; measured m/e=1180.4. (C-1"), 97.3 (C-1'), 107.4, 108.4, 112.3 (C-1"), 116.9, 119.2, 127.5 (CH of triazole), 140.7, 145.8, 150.0, 154. 0504 Compound 1q was prepared according to General 0, 156.0, 170.7, 177.6 ppm. Procedure A presented hereinabove, using Compounds 2a (0507 MALDI TOFMS calculated for CHFNOK and Compound 3a, each prepared and characterized as pre (IM+KI): m/e=1091.2: measured m/e=1091.5. Following sented hereinabove. the design rational presented hereinabove, the synthesis of a series of kanamycin A-ciprofloxacin conjugates has been car 0505 H NMR (500 MHz, D.O, pH=3.2): 8=1.07 (m, 2H, ried out as follows. The general approach for the assembly of cyclopropane), 1.30 (m. 2H, cyclopropane), 3.25-3.65 (m, Such structures is similar to that of to Cipro-NeoB conjugates, 8H, piperazine), 3.78-3.85 (m. 1H, cyclopropane; 2H, pipera namely a series of alkyne-derivative of kanamycin A, gener zine), 4.68 (m,2H, CH, of linker), 4.91 (t, J=7.0Hz, 2H, CH, ally referred to hereinas Compound 11, has been synthesized, of linker), 7.29-7.31 (m. 2H, C, H, Cs—H), 8.11 (s, 1H, and reacted by "click chemistry” with a series of exemplary triazole hydrogen), 8.47 (S. 1H, C-H); aminoglycoside azido-derivatives of ciprofloxacinto afford a series of conju hydrogens: ring I: 8–3.14-3.18 (m. 1H, H-2), 3.25-3.65 (m. gates, according to some embodiments of the present inven 3H, H-4, H-6, H-6'), 3.78-3.85 (m, 2H, H-3, H-5), 5.96 (d. tion, also referred to herein as Cipro-KanA. J=4.0 Hz, 1H, H-1); ring II: 8=1.83-1.90 (ddd, JJJ-13.0 0508 Preparation of Compound 11—General Proce HZ, 1H, H-2ax), 2.36-2.40 (dt, J=4.0, 13.0 Hz, 1H, H-2eq), dure D: 3.25-3.65 (m, 2H, H-1, H-3), 3.78-3.85 (m. 1H, H-5), 3.88 0509. The general synthesis of the Cipro-KanA conju 3.92 (t, J=10.0 Hz, 1H, H-6), 4.03-4.07 (t, J=10.0 Hz, 1H, gates (Compound 11), according to some embodiments of the H-4); ring III: 8=3.25-3.65 (m, 2H, H-5, H-6), 4.27-4.29 (m, present invention, from Compound 2 (the “Cipro' moiety) 2H, H-2, H-4), 4.36-4.38 (t, J=5.0Hz, 1H, H-3), 5.33-5.34 (d. and Compound 21 (the “KanA' moiety), is illustrated in J=1.0 Hz, 1H, H-1); ring IV: 8–3.14-3.18 (m. 1H, H-6), Scheme 8 below.

Scheme 8 HN

O OH NH2 OH / Yn NH O OH -- OH -as O HO O OH HO NH2

HOOC F HN

O r NN NH2 OH N- N nx1'Nay-> 1 OHO OH O HO O OH HO NH2 N - N - - Cipro moiety KanA moiety US 2014/0357591 A1 Dec. 4, 2014 36

0510 Wherein X and Y represent various first and second -continued spacer moieties respectively, which form the connecting moi NHCbz ety together with the resultant 4-yl-1-yl-1H-1,2,3-triazole linking moiety. O 0511 Preparation of Compound 21a: HO NHCbz HO , NH2 HO Compound 21a O

OH HO O OH FCOCHN 41

OH 0514 Selective Cbz-protection of two amino groups at C6' and C3 positions of KanA was carried out in the presence of HO OH Zinc acetate. The use of Zn salt allowed suitable temporary HN protection of vicinal amino alcohol functions as Zinc(II) che lates. Thereafter, unbound amino group reacted with benzy loxycarbonyloxy succinimide to afford the desired protected 0512 Compound 21a, wherein Y is a methylene KanA derivative. Treatment of this compound with ethyl (—CH2—) group, was prepared from commercially available trifluoroacetate in DMSO afforded KanA derivative, Com Kanamycin A (Shanghai FWD Chemicals Limited), accord pound 41, selectively unprotected at the C1-NH position in ing to previously published procedure Yamasaki, T., et al., J. high yield. Antibiot (Tokyo), 1991, 44, 646. 0515 Compound 41 was reacted with propargyl bromide 0513 Briefly, the Kanamycin Aderivative, Compound 41, in the presence of KCO to give protected alkyne derivative which has only one amine group non-protected at C1-NH of KanA, Compound 31a (Scheme 10). Cleavage of trifluo position, was prepared according to Scheme 9 presented roacetate group in Compound 31a with methyl amine fol below. lowed by Cbz deprotection in the presence of HBr in acetic acid afforded desired Compound 21a.

Scheme 9 NH2 Scheme 10

6 NHCbz O HO HO NH2 O HO 3 HOo NHCbz O NH2 CbzOS HO HO Zn(OAc) O NH2 Br1N DMSO 1Aa- Šs O K2CO3 (50%) of DMF (70%) HO O OH FCOCHN Kanamycin A 41

NHCbz NHCbz

O H O A HO NHCbz o CbzHN HO O EtOCOCF HO (\ayHO -e-1. MeNH2, EtOH NH2 O 2. HBiHOAC HO DMSO O (75% for two (100%) HO O OH steps) OH OH HO O OH FCOCHN HN 31a US 2014/0357591 A1 Dec. 4, 2014 37

-continued 0520. The purified product from the above step (100 mg. NH2 0.113 mmol) was dissolved in 33% solution of MeNH in EtOH (10 ml) and the mixture was stirred at room tempera ture for 12 hours. The reagent and the solvent were removed HO O / HO HN by evaporation and the residue was dissolved in AcOH (2 ml) HO HN and stirred at 15° C. for 10 minutes after which 30% solution O HO of HBr in AcOH (0.5 ml) was added. Propagation of the O reaction was monitored by TLC (CH.Cl/MeOH/HO/ OH MeNH. (33% solution in EtOH) 10:15:6:15), which indi HO O cated completion after 1 hour. 1N NaOH solution was added OH to the reaction mixture until pH became neutral, then the HN mixture was purified on a short column of Amberlite CG-50 21a (Htform). The column was sequentially washed by MeOH, MeOH/MeNH. (33% solution in EtOH) 95:5, MeOH/ 0516 Compound 41 (100 mg 0.115 mmol) was dissolved MeNH. (33% solution in EtOH) 9:1 and MeOH/MeNH in dry DMF (5 ml). Potassium carbonate (19 mg, 0.140 (33% solution in EtOH)4:1. Fractions containing the product mmol) and propargyl bromide (11.3 ul, 0.126 mmol) were were combined, evaporated, re-dissolved in water and evapo added to the resulted solution at ambient temperature. The rated again to afford the free amine form of the product, reaction propagation was monitored by TLC (MeOH/DCM Compound 21a (50 mg, 75%). This product was then dis 1:5), which indicated completion after overnight stiffing. The solved in water, the pH was adjusted to 7.5 with 0.01 M mixture was transferred on silica column and purified by flash HSO and to lyophilized to give the sulfate salt of Compound chromatography (silica, MeOH/DCM) to yield the corre 21a as a white foamy solid. sponding 1-N-alkilated product: 3,6'-diCbz-3"-trifluoro 0521. HNMR (500 MHz, D.O, pH=3.17): 8=3.00 (t, 1H, acetyl-1-prop-2-yn-1-kanamycin A (72 mg, 70% yield). CH of triple bond), 3.94-3.98 (dd, J–2.5, 15.5 Hz, 1H, 0517 H NMR (500 MHz, CDOD/CDC1): 8–2.44-2.45 —CH triple bond), 4.07-4.11 (dd, J=2.5, 15.5 Hz, 1H, (t, J=2.5 Hz, 1H, CH of triple bond), 3.63-4.08 (m, 2H, —CH-triple bond); ring I: 8–3.03-3.08 (dd, J=9.0, 13.5 —CH triple bond), 4.96-5.10 (m, 4H, CH of Cbz), 7.23 HZ, 1H, H-6), 3.24-3.28 (t, J=9.5 Hz, 1H, H-4), 3.35-3.38 (dd, 7.35 (m. 10H, aromatic); ring I: 8–3.37-3.54 (m, 5H, H-2, J=3.5, 9.0 Hz, 1H, H-6), 3.60-3.76 (m, 1H, H-2), 3.81-3.93 H-3, H-4, H-6, H-6), 3.63-4.08 (m. 1H, H-5), 4.96-5.10 (m, (m. 2H, H-3, H-5), 5.55-5.56 (d. J=4.0 Hz, 1H, H-1); ring II: 1H, H-1); ring II: 8=1.29-1.36 (ddd, J=JJ-12.5 Hz, 1H, 8=1.94-2.01 (ddd, JJ J-12.5 Hz, 1H, H-2ax), 2.56-2.58 H-2ax), 2.23-2.27 (dt, J=4.0, 12.5 Hz, 1H, H-2eq), 2.96-2.98 (dt, J=4.0, 12.5 Hz, 1H, H-2eq), 3.40-3.45 (m. 1H, H-3), (m. 1H, H-1), 3.37-3.54 (m. 1H, H-5), 3.63-4.08 (m,3H, H-3, 3.60-3.76 (m, 1H, H-5), 3.81-3.93 (m, 3H, H-1, H-4, H-6): H-4, H-6); ring III: 8=3.22-3.26 (dd, J=4.0, 13.0 Hz, 1H, ring III: 8–3.40-3.45 (m. 1H, H-3), 3.55-3.58 (dd, J=4.0, 10.5 H-6), 3.31-3.34 (m. 1H, H-6), 3.37-3.54 (m, 2H, H-2, H-4), HZ, 1H, H-4), 3.60-3.76(m,2H, H-6, H-6), 3.81-3.93 (m,2H, 3.63-4.08 (m, 2H, H-3, H-5), 4.96-5.10 (m. 1H, H-1) ppm. H-2, H-5), 5.10-5.11 (d. J=3.0 Hz, 1H, H-1) ppm. 0518 °C NMR (125 MHz, CD,OD/CDC1): 8–31.2 0522 °C NMR (125 MHz, DO): 8–25.4 (C-2), 35.9 (C-2), 35.0 (C-6"), 40.9 (C-6'), 50.1, 55.7, 61.2 (triple bond), (-CH triple bond), 40.4 (C-6'), 47.7, 50.0, 54.9, 55.6, 66.7, 66.8, 67.7, 69.7, 70.4, 714, 72.5, 72.8, 73.4, 73.9, 75.2, 59.8 (C-6"), 65.2, 68.1, 68.6, 70.7, 71.8, 72.4, 73.0, 73.7, 84.0, 85.5, 100.41 (C-1), 101.3 (C-1"), 114.87, 117.2, 127.8, 76.9, 78.8, 83.0, 96.0 (C-1'), 100.4 (C-1") ppm. 127.9, 128.0, 128.1, 128.4, 136.1, 136.2, 156.5, 157.9, 158.9, 159.2 ppm. 0523 MALDITOFMS calcd for CHNO (M+H"): 0519 MALDI TOFMS calcd for C.H.F.N.O. Na m/e323.2 measured m/e 523.3. (M+Na"): m/e=909.3; measured m/e=909.3. 0524 Preparation of Compound 11a:

Compound 11a. HN

F O OH O NN NH2 OH OH N/ VN-(CH2):-Ne --- N O HOOC > OH OH US 2014/0357591 A1 Dec. 4, 2014

0525 Compound 11a was prepared according to General C. H. C. H), 7.53-7.55 (d. J=10.0 Hz, 2H, aromatic), 8.21 Procedure D. presented hereinabove, using Compounds 2a (a (S, 1H, triazole hydrogen), 8.58 (S. 1 H. C. H); aminoglyco ciprofloxacin azido derivatives, see details hereinabove) and side hydrogens: ring I: 8–3.11-3.15 (dd, J–7.0, 13.0 Hz, 1H, Compound 21a. H-6), 3.21-3.88 (m, 5H, H-2, H-3, H-4, H-5, H-6), 5.48-5.49 0526 H NMR (500 MHz, D.O, pH=3.0): 8=1.17 (m, 2H, (d. J=3.0 Hz, 1H, H-1); ring II: 8=1.85-1.93 (ddd, cyclopropane), 1.38 (d. J–7.0 Hz, 2H, cyclopropane), 3.62 JJJ-12.0 Hz, 1H, H-2ax), 2.71-2.75 (dt, J=4.0, 12.0 Hz, 3.85 (m, 8H, piperazine; 1H, cyclopropane; 2H, CH of 1H, H-2eq), 3.21-3.88 (m, 4H, H-3, H-4, H-5, H-6), 3.94-3.98 linker), 4.44-4.47 (d. J=15.0 Hz, 1H, CH of linker), 4.61-4. (td, J=2.0, 10.0 Hz, 1H, H-1); ring III: 8=3.21-3.88 (m, 6H, 63 (d. J=15.0 Hz, 1H, CH, of linker), 5.00 (t, J=2.0 Hz, 2H, H-2, H-3, H-4, H-5, H-6, H-6"), 5.42 (s, 1H, H-1) ppm. CHoflinker), 7.50 (m. 1H, C, H), 7.56-7.59 (d. J=10.0Hz, 0537 °C (NMR 151 MHz, DO): 8–7.4 (cyclopropane), 1H, C, H), 7.51-7.56, 8.30 (s, 1H, triazole hydrogen), 8.66 25.4 (C-2), 39.7, 40.2, 46.3, 50.9, 53.5, 54.9, 56.2, 59.8, 65.2, (S, 1H, C-H); aminoglycoside hydrogens: ring I: Ö–3.11 68.1, 68.6, 70.6, 72.1, 72.2, 72.8, 77.9, 83.5, 95.7 (C-1), 3.15 (dd, J=7.0, 13.0 Hz, 1H, H-6), 3.29-3.35 (t, J=9.5 Hz, 1H, 100.8 (C-1'), 105.7, 115.1, 117.4, 119.7, 126.6 (CH of triaz H-4), 3.37-3.40 (dd, J=3.5, 9.0 Hz, 1H, H-6), 3.62-3.85 (m, ole), 128.4, 131.9, 136.7, 137.7, 138.9, 169.0 ppm. 3H, H-2, H-3, H-5), 5.50-5.51 (d. J=3.0 Hz, 1H, H-1); ring II: 0538 MALDITOFMS calculated for CHFNO-Na 8=1.92-1.99 (ddd, JJ J-12.0 Hz, 1H, H-2ax), 2.71-2.75 (IM+Na"): m/e=1021.6; measured m/e=1021.4. (dt, J=4.0, 12.0 Hz, 1H, H-2eq), 3.44-3.48 (t, J–10.0 Hz, 1H, H-4), 3.62-3.85 (m, 3H, H-3, H-5, H-6), 3.87-3.90 (td, J=2.0, Example 2 10.0 Hz, 1H, H-1); ring III: 8–3.62-3.85 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6), 5.07 (s, 1H, H-1) ppm. Antibacterial Activity 0527 °C (NMR 151 MHz, DO): 8–7.6 (cyclopropane), 0539 Exemplary conjugates, according to some embodi 25.7 (C-2), 36.7, 39.8, 40.4, 44.7, 46.5, 47.7, 52.2, 55.0, 56.4, ments of the present invention, namely the Cipro-NeoB con 60.0, 65.4, 68.3, 68.8, 70.8, 72.3, 72.4, 73.0, 78.2, 83.7, 95.2 jugate Compounds 1a-q, were tested for in vitro antibacterial (C-1'), 100.9 (C-1), 106.1, 107.0, 111.2, 115.5, 117.4, 119.4, activity against a panel of Susceptible and resistant bacterial 127.1 (CH of triazole), 138.5, 139.2, 144.2, 148.7, 152.8, strains using non-conjugated Cipro and NeoB as controls 154.2, 169.3 ppm. (see, Table 4 below). Data for selected Gram-negative and 0528 MALDITOFMS calculated for CHFNO-Na Gram-positive strains are reported as minimal inhibitory con (M+Na"): m/e=945.8; measured m/e=945.4. centration (MIC) values. Resistant strains included Escheri 0529 Preparation of Compound 11 f: chia coli AG100A and AG100E3 (Gram-negative), and methi 0530 Compound 11f was prepared according to General cillin-resistant S. aureus (MRSA) (ATCC 43300, Gram Procedure D. presented hereinabove, using Compounds 2f positive). E. coli AG100A and AG100B are aminoglycosides and Compound 21a. resistant laboratory strains that harbor Kan transposon 0531 H NMR (500 MHz, D.O, pH=3.1): 8–1.16 (m,2H, Tn903 (35). MRSA (ATCC 43300) is one of the leading cyclopropane), 1.38-1.39 (d. J=5.0 Hz, 2H, cyclopropane), causes of bacterial infections and exerts high level of resis 3.28-3.90 (m. 2H, CH of linker; 8H, piperazine; 1H, cyclo tance to aminoglycosides 36. propane), 4.47-4.49 (d. J=15.0 Hz, 1H, CH of linker), 4.50 0540. The MIC values for a set of exemplary conjugates 4.54 (m. 1H, CH of linker), 4.60-4.63 (d. J=15.0 Hz, 1H, CH, according to some embodiments of the present invention, oflinker), 4.63-4.65 (m,2H, CH, of linker)7.46-7.49 (m,2H, Compounds 1a-q, were determined using the double-mi Cs H. C. H), 8.20 (s, 1H, triazole hydrogen), 8.61 (s, 1H, crodilution method according to the National Committee for C-H); aminoglycoside hydrogens: ring I: 6–3.11-3.15 (dd. Clinical Laboratory Standards (NC=CLS) with starting con J=7.0, 13.0 Hz, 1H, H-6), 3.28-3.90 (m, 5H, H-2, H-3, H-4, centration of 384 ug/ml and 1.5 ug/ml of the tested com H-5, H-6), 5.48-5.49 (d. J=3.0 Hz, 1H, H-1); ring II: 8=1.90 pounds. All the experiments were performed in duplicates 1.97 (ddd, J=J. J.-12.0 Hz, 1H, H-2ax), 2.73-2.77 (dt, J–4. and analogous results were obtained in two to four different 0, 12.0 Hz, 1H, H-2eq), 3.28-3.90 (m, 4H, H-3, H-4, H-5, experiments. H-6), 3.93-4.00 (td, J=2.0, 10.0 Hz, 1H, H-1); ring III: 8–3. 0541 Resistance studies were performed in parallel with 28-3.90 (m, 6H, H-2, H-3, H-4, H-5, H-6, H-6), 5.05 (s, 1H, E. coli ATCC 35218 and B. Subtilis ATCC 6633 Strains in the H-1) ppm. presence of Cipro, NeoB, Cipro:NeoB mixture (1:1 molar 0532 °C (NMR 151 MHz, DO): 8–7.4 (cyclopropane), ratio), and Compound li. After the initial MIC experiments, 25.5 (C-2), 36.1, 39.6, 40.2, 46.1, 47.5, 53.5, 54.9, 56.2, 58.4, MICs were determined once in two days, for 15 passages as 59.8, 63.9, 65.2, 68.1, 68.7, 70.6, 72.1, 72.2, 72.8, 78.0, 83.6, follows: for each compound tested, bacteria from the one half 95.7 (C-1'), 100.8 (C-1'), 105.7, 106.6, 112.7, 115.1, 117.4, MIC well were diluted 100-fold (50 ul of the bacterial growth 127.4 (CH of triazole), 137.8, 138.9, 1440, 162.7, 169.0 in the total of 5 ml LB medium) and were grown overnight at ppm. 37°C. The OD600 of the bacteria was diluted to yield 5x10 0533. MALDITOFMS calculated for CHFNO, Na cells/ml in LB (according to a calibration curve) and used (M+Na"): m/e=975.6; measured m/e=975.4. again for MIC determination in the Subsequent generation. In 0534 Preparation of Compound 11 i: parallel, MIC evolution during these subcultures was com 0535 Compound 11i was prepared according to General pared concomitantly with each new generation, using bacte Procedure D. presented hereinabove, using Compounds 2i ria harvested from control wells (wells cultured without anti and Compound 21a. microbial agent from the previous generation). The relative 0536 H NMR (500 MHz, D.O, pH=3.2): 8–1.12 (m,2H, MIC was calculated for each experiment from the ratio of cyclopropane), 1.35-1.36 (m. 2H, cyclopropane), 3.21-3.88 MIC obtained for a given subculture to that obtained for (m, 8H, piperazine; 1H, cyclopropane), 4.42-4.44 (m, 3H, first-time exposure. CH of linker), 4.58-4.61 (d. J=15.0 Hz, 1H, CH of linker), 0542) MIC levels obtained for exemplary conjugates 5.68 (m. 2H, CH of linker), 7.42-7.47 (m, 4H, aromatic, according to some embodiments of the present invention, US 2014/0357591 A1 Dec. 4, 2014 39

Compounds 1a-q, against selected bacterial strains, are pre Gram-positive MRSA with the activities of 8- to 128-fold sented in Table 4 below. The MIC values represent the results better than that of NeoB. The exemplary conjugate Com obtained in parallel experiments with two different starting pounds li and Compound 1 q retained similar activity to that concentrations of the tested compound (384 and 1.5 Lig/ml). of NeoB in B. subtilis and displayed the most prominent AG100E3 and AG100A are two kanamycin resistant Escheri activity against the MRSA. chia coli strains expressing APHC3")-I aminoglycoside resis 0544 SAR analysis among the 17 exemplary conjugates tance enzyme 35. presented in Table 4 to hereinabove revealed that the length of TABLE 4 la-q NH2

HO O HO NH2 N=N HN Y o, NH2 1.Ne5 O N1 OH OH NH2 N N O O OH HN HOOC F OH

O

MIC ml

E. coi E. coi B. Subiiis MRSA Com- Yield R477- ATCC E. coi E. coi ATCC ATCC pound X Y (%) 1OO 25922 AG1 OOB AG1 OOA 6633 433OO “Cipro' - O.O2 O.O2 O.OS

0543. As can be seen in Table 4, all of the exemplary the linear aliphatic chain at position X (Compounds 1a-e and conjugates exhibited significant antibacterial activity. This Compounds 1j-l) has less influence on antibacterial activity, activity was especially improved in comparison to that of as the variation in antibacterial potency against individual NeoB alone. The most prominent improvement was observed strains is very little. This is valid when comparing between againstall E. coli strains including aminoglycosides-resistant Compounds 1a-e or between Compounds 1j-1 of the same sets strains E. coli AG100A and E. coli AG100E3. On average, the with respect to the Y spacer, or between Compounds 1a-e and conjugates showed 2-8 and 2-16 times better activity than Compounds 1j-1 of different sets. Conjugates consisting of NeoB against E. coli R477-100 and E. coli 25922, respec the linear aliphatic chain incorporating with alcohol function tively, and this was much higher against the resistant E. coli ality (Compounds 1f and 1 m), show better activity than those AG100A and E. coli AG100E3 strains with Compound 1i and incorporating with ether functionality (Compounds 1g and Compound 1 q as the most active derivatives. Compound li 1n) at position X. Among the conjugates containing an aro was 128-folds more potent than NeoB against E. coli matic linker at position X (Compounds 1h-i and Compounds AG100E3 and E. coli AG100A; Compound 1d was 32-fold lo-p), Compound li that contains para-Substituted benzene better thanNeoB against E. coli AG100E and 253-folds better ring at both X and Y positions displayed the most prominent against E. coli AG100A. All the conjugates displayed signifi activity againstall bacterial strains tested. The conjugate that cantly better potency against the aminoglycosides-resistant displayed similar spectrum of activity to that of Compound 1i US 2014/0357591 A1 Dec. 4, 2014 40 was Compound 1 q that contains the shortest spacers at both X 0548. As can be seen in Table 5, the MIC values for NeoB and Y positions, suggesting that a limited number of degrees are by more than 64-, 16- and 8-fold higher for the resistant of freedom in the spacer have a positive effect on activity. The strains than the respective non-resistant strains. As expected, observation that the majority of conjugates are more active the activity of NeoB against the resistant strain harboring than NeoB against Gram-negative bacteria (E. coli) while bifunctional AAC(6')/APH(2") resistance is significantly retaining moderate activities against the Susceptible Gram lower than those harboring monofunctional APH(3)-Ia or positive bacteria (B. subtilis) prompted the investigation of APH(3)-IIIa resistance. All the tested conjugates were less the activity of conjugates against E. coli Strains harboring effective than Cipro but displayed significant to excellent particular aminoglycosides resistant plasmids. activities against resistant Strains with the potencies far 0545 For this purpose, exemplary conjugates, according greater than that of the parent NeoB. The conjugates also to some embodiments of the present invention, were tested displayed similar (E. coli XL 1 blue) or better (E. coli BL21) against five isogenic E. coli strains. E. coli (pSF815) and E. antibacterial efficiency to that of NeoB against susceptible coli (pET9d) are laboratory resistant strains derived by trans strains. formation of E. coli XL 1 blue (background strain) with the 0549. As can further be seen in Table 5, the MIC ratio for pSF815 and pET9d plasmids, respectively. The pSF815 each tested conjugate (calculated by dividing the MIC value encodes for the bifunctional AAC(6')/APH(2") resistance against resistant strain to the MIC value against Susceptible enzyme, which catalyses acetylation of the amino group at strain) was significantly lower to that calculated for the NeoB. 6'-NH and phosphorylation at the 2"-OH. The pET9d It is noted herein that this MIC ratio was 1 for the majority of encodes for the APHC3")-Ia resistance enzyme, which cataly cases: for all the conjugates against E. coli (pETSACG1); for ses phosphorylation at the 3'-OH of both neomycin and kana Compounds li, 1 p and 1o against E. coli (pET9d), and for mycin families of aminoglycosides. The last two isogenic Compound 1p against E. coli (pSF815). strains used were E. coli BL21 (background strain) and E. coli (pETSACG1). The latter was derived by transformation of E. 0550. The observed identical MICs of the conjugates coli BL21 with the plTSACG1 plasmid that encodes for the against different isogenic pairs of bacteria Suggest that the APH(3)-IIIa resistance enzyme. These three enzymes are reason for the observed sensitivity of the E. coli harboring among the most prevalent modes of resistance found in ami either AAC(6')/APH(2") (in case of Compound 1p), APH(3)- noglycosides resistance strains 36-39. Ia (in cases of Compounds li, 1 p and lo) or APHC3")-IIIa (in 0546. Since the aminoglycoside resistance of the engi all the tested conjugates) is the inferior activity of these neered strains, E. coli (pSF815), E. coli (pET9d) and E. coli enzymes toward particular conjugates, rather than reduced (pETSACG1) is mediated only due to the presence of the permeability of the conjugates. respective cloned resistance enzyme, comparison of the MIC 0551. To further substantiate this observation, the detailed values against each pair of the resistant and background kinetic analysis of the purified APHC3")-IIIa enzyme with strains eliminates other effects that could affect the activity of NeoB along with the exemplary conjugate Compound 1m the tested compound, like penetration or solubility. Without (that displayed the MIC ratio of 1 against pETSACG1) was being bound by any particular theory, it is suggested that a carried out according to the previously reported procedure poorer substrate for the resistance enzyme should have a low 13. ratio between the MIC values of the resistant and non-resis 0552. The measured K (uM), k, (s) and k/K. tant strains, as demonstrated in several earlier studies 13, 14. (M'xs') values were: 5.7+0.7, 2.4+0.1 and 42x10 for 40. NeoBand 86.8+8.9, 2.5+0.1 and 2.9x10' for Compound 1m, 0547 Table 5 presents antibacterial activities of exem respectively. The kinetic constants measured for NeoB are plary conjugate according to some embodiments of the similar to previously reported values 13, 41. The observed present invention, against E. coli XL 1 blue and E. coli BL21 data indicate that Compound 1 m is a poorer Substrate of (Background Strains) and their Engineered Variants. The APH(3)-IIIa thanNeoB. The observed decrease inspecificity MIC ratios were calculated by dividing the MIC value against for Compound 1m is caused primarily by its poor ability to resistant strain to that against the respective background saturate the enzyme, as judged from its elevated K (87 uM) strain. compared to that of the NeoB (K-6 uM). TABLE 5

MIC (ig/ml)

XL1 bluef XL1 bluef BL21. Compound XL1 pSF815 pET9d pETSACG1 Expressed blue AAC(6')- MIC APH(3)- MIC BL21 APH(3)- MIC enzyme – APH(2") ratio Ia ratio IIIa ratio

Cipro O.10 O.38 3.8 O.10 1 384 >64 96 16 6 48 8 1i 3 24 8 3 1 0.4 0.4 1 1m 6 48 8 12 2 O.2 O.2 1 1o 6 24 4 1 0.4 0.4 1 1p 6 6 1 1 0.4 0.4 1 1q. 3 12 4 12 4 O.2 O.2 1 US 2014/0357591 A1 Dec. 4, 2014 41

0553. The observed kinetic data with Compound 1 m is 0558 As can be seen in Table 6, the Cipro-Kana conju consistent with the antibacterial data presented in Table 5. In gates, according to some embodiments of the present inven fact, comparison of its K value (87 uM) with the MIC of 5.5 tion, exhibit notable improved antimicrobial activity over that uM (0.2 Lug/ml) against E. coli (pETSACG1), Suggests that of kanamycin A. the bacteria are killed at far lower concentration before the enzyme’s full activity is reached. Example 3 0554. As can further be seen in Table 5, the MIC ratio for Cipro in case of the isogenic E. coli XL 1 blue (pSF815)/E. coli XL 1 blue strains was 3.8, indicating a modification of this Biochemical Studies important clinical antibiotic by the aminoglycoside resistant AAC(6')/APH(2") enzyme. Since two other strains harboring 0559 To investigate the possibility of a dual mode of APH(3') activity displayed the MIC ratios of 1, it is suggested action of the conjugates according to some embodiments of that Cipro may undergo N-acetylation at the terminal nitro the present invention, the exemplary conjugates Compounds gen of its to piperazine moiety by AAC(6')/APH(2"). This lf, 1i, and 1q were tested for both the inhibition of protein Suggestion is Supported by recent reports demonstrating that synthesis in an in vitro transcription/translation assay, and the Some common aminoglycoside acetyltransferases (AACs), inhibition of the enzymes that are targeted by the quinolones, including AAC(6')s, are capable of performing N-acetylation DNA gyrase and Topol V (see, Table 7 and FIG. 1). of fluoroquinolones having a free amino group at 7-position 0560 Purification and kinetic analysis of the APH(3')-IIIa 42, 43. enzyme were performed according to previously described 0555. In addition, a close inspection of the data in Table 4 procedure 13. reveals that Cipro displayed particularly reduced activity against S. aureus (MRSA); MIC of 0.2 in comparison to MIC 0561 Protein translation inhibition by the different com values of 0.05 to less than 0.005 against other tested strains. pounds was quantified in a coupled transcription/translation Since the bifunctional AAC(6')/APH(2") enzyme is the most assay by using E. coli S30 extracts for circular DNA with the frequently encountered aminoglycoside-modifying enzyme pBEST/uc plasmid (Promega), according to the manufactur in staphylococci, including MRSA 44, the observed er's protocol. reduced activity of Cipro against S. aureus (MRSA) may be 0562 Translation reactions (25ul) that contained variable explained due to the modification of Cipro by AAC(6')-APH concentrations of the tested compound were incubated at 37° (2"). C. for 60 minutes, cooled on ice for 5 minutes, and diluted 0556. To further investigate these observations, purifica with a dilution reagent (tris-phosphate buffer (25 mM, pH tion of the bifunctional AAC(6')/APH(2") and the detailed 7.8), DTT (2 mM), 1,2-diaminocyclohexanetetraacetate (2 kinetic analysis of Cipro with the homogeneous enzyme, mM), glycerol (10%), Triton X-100 (1%) and BSA (1 along with the structural characterization of the enzymatic mg/ml)) into 96-well plates. The luminescence was measured product are performed. immediately after the addition of the Luciferase Assay 0557. Results of the antibacterial activity assays obtained Reagent (50 ul; Promega), and the light emission was for the exemplary Cipro-KanA conjugates, Compounds 11a, recorded with a Victor3TM Plate Reader (Perkin-Elmer). The 11f and 11i, against variety of bacterial strains, including concentration of half-maximal inhibition (ICs) was obtained resistant strains, are presented in Table 6 below. from fitting concentration-response curves to the data of at TABLE 6

O HO F

HO HN2 NN O HO H \ A V O N S. N-X-N N HO V / O / COOH OH N HO O OH HN

MIC ml

E. Coi E. coi E. coiXL1 Com- R477- E. coi E. coi E. coi Bacilius XL1 blue/pET9d pound X Y 1OO 25922 AG100B AG100A Subiiis blue aph(3)-Ia

Cipro O.O23 O.O23 O.O6 <0.003 O.O23 KanA 24 12 >384 384 1.5 3 >384 11a —(CH2)2— —CH2— 6 3 12 0.75 1.5 6 6 11f —CH-CH(OH)CH2— —CH2— 3 1.5 6 0.75 1.5 3 3 11i —CH2—pCH-CH2— —CH2— 6 6 12 0.75 3 3 3 US 2014/0357591 A1 Dec. 4, 2014 42 least two independent experiments by using Grafit 5 software tem. The ICso was defined as the drug concentration that (Leatherbarrow, R. J. Erithacus Software Ltd: Horley, U.K., reduced the enzymatic activity observed with drug-free con 2001). trols by 50%. 0563 DNA supercoiling activity was assayed with relaxed pBR322 DNA as a substrate (TopoCEN, Inc) according to the TABLE 7 manufacturer's protocol. The standard reaction mixture (20 Protein ul) contained 35 mM Tris-Cl pH 7.5, 24 mM KC1, 4 mM DNA gyrase TopolV synthesis MgCi 2 to mM dithiothreitol, 1.8 mM spermidine, 1 mM Compound ICso (IM) ICso (IM) ICso (IM) ATP, 6.5% glycerol, 0.1 mg/ml BSA, 12.5 lug/ul relaxed Cipro 1.3 0.1 10.8 O.3 inactive pBR322, and DNA gyrase protein. The reaction mixture was NeoB inactive inactive 10.5 + 0.1 incubated at 37° C. for 1 hour, and then was terminated by 1f O.O73 O.OOS O-580.04 2.20.6 addition of a loading dye and chloroform/isoamyl alcohol 1i O.O85 O.OO3 O55, O.O6 16.7 - 4.4 (24:1) mixture. After brief agitation the blue aqua phase was 1q. O.041 - 0.009 7.90 O.25 1814.9 analyzed by electrophoresis in 1% agarose. One unit of Super coiling activity was defined as the amount of DNA gyrase 0567 As can be seen in FIG. 1 and Table 7, the conjugates required to Supercoil 0.5ug of plasmid in 1 hour. The ICso was inhibited bacterial protein synthesis with the potencies simi defined as the drug concentration that reduced the enzymatic lar to or better than that of NeoB, confirming their strong activity observed with drug-free controls by 50%. aminoglycoside character and the observed antibacterial 0564) DNA relaxation activity was assayed with super activity. coiled p3R322 DNA as a substrate (Inspiralis Ltd) according 0568 Based on the observed reduced antibacterial activity to the manufacturers protocol. The standard reaction mixture of all the conjugates in comparison to that of Cipro (Tables 2 (20 ul) contained 40 mM HEPES-KOH pH 7.6, 100 mM and 3), it is expected that the conjugates, according to some potassium glutamate, 10 mM Mg(OAc), 10 mM dithiothrei embodiments of the present invention, should be weaker tol, 1 mM ATP 50 g/ml albumin, 5 ng/ul relaxed pBR322, DNA gyrase and Topol V inhibitors than Cipro. However, the and TopolV protein. The reaction mixture was incubated at exemplary conjugates Compound 1 f, li, and 1 q displayed far 37°C. for 30 minutes, and then was terminated by addition of greater activities than Cipro in both the DNA gyrase and 2 Jul 0.5 M EDTA, 3.5ul loading dye and 20 ul chloroform/ Topol V assays, indicating the dual mode of action of these isoamyl alcohol (24:1) mixture. After brief agitation the blue molecules. The measured ICso values for Compound 1 f, li. aqua phase was analyzed by electrophoresis in 1% agarose. and 1d were 18-, 15- and 32-fold superior than that of Cipro One unit of relaxation activity was defined as the amount of in DNA gyrase assay, and 19- 20- and 1.4-fold superior than TopolV required to relax 0.1 lug of plasmid in 30 minutes. The Cipro in Topol Vassay. It is of note herein that the ICs values ICso was defined as the drug concentration that reduced the determined for Cipro for the inhibition of DNA gyrase and enzymatic activity observed with drug-free controls by 50%. Topol V are very similar to those previously reported 11, 45. 0565 FIGS. 1A-D present comparative data for the inhi 0569. These data clearly confirm the design concept of the bition of DNA gyrase (FIGS. 1A-B) and Topol V (FIGS. Cipro-NeoB conjugates, and of the conjugates presented 1C-D) with Cipro and exemplary Compound 1 f, wherein herein in general, and their desired dual mode of action. The FIG. 1A is a photograph of a 1% agarose gel showing the observed difference between antibacterial performance and inhibitory activity of Compound 1 fagainst DNA gyrase (lane targets inhibition can be explained by the reduced cell pen 1, relaxed DNA: lane2, supercoiling reaction by DNA gyrase etration of the conjugates in comparison to Cipro. Both the without presence of inhibitor; lanes 3-8 are the same as lane 1 higher molecular weight and the higher overall charge of the but in the presence of 30, 60, 100, 150, 200, and 300 nM of conjugates than those of Cipro might contribute to their Compound 1f); FIG. 1B is a semilogarithmic plot of in vitro reduced cell penetration. The observed superior activity of the DNA gyrase Supercoiling reaction inhibition, measured for selected conjugates (like, for example, that of Compounds li Cipro and Compound 1f FIG. 1C is a photograph of a 1% and 1q) to that of the parent NeoB against a variety of Gram agarose gel showing the inhibitory activity of Compound 1 f negative and Gram-positive bacteria including resistant strains along with their established dual mode of action Sug against Topol V (lane 1, supercoiled DNA: lane 2, relaxation gest a role for these conjugates in treating infections by resis reaction by Topol V without the presence of inhibitor; lanes tant bacterial strains and in cases where bacterial resistance 3-8 are the same as lane 1 but in the presence of 0.2,0.3, 0.5, may develop. 0.8, 1.2, and 10 uM of Compound 1f FIG. 1D is a semiloga rithmic plot of Topol V inhibition, measured for Cipro and Compound 1 f; while the percentages of the supercoiled DNA Example 4 were calculated from the electrophoresis images by using Image.J Launcher program (Rasband, W. Bethesda, Md., Emergence of Resistance USA), and plotted as functions of drug concentration (each 0570. One advantage of the conjugates, according to some data point represents the average of 2-3 independent experi embodiments of the present invention, is their potential to mental results). slow the emergence of resistance, as defined in earlier studies 0566 Table 7 present that activity assay data measured for 6, 10. The underline hypothesis is that treatments that exemplary conjugates according to some embodiments of the inhibit multiple targets might delay and decrease the patho present invention, used as inhibitors for DNA expression of gen’s ability to accumulate simultaneous mutations that gyrase, Topol V, and bacterial protein synthesis. Super-coil affect the multiple targets 46. ing assay were conducted with E. coli DNA gyrase. Relax 0571 To assess the potential of emergence of antibacterial ation assays were conducted with E. coli Topol V. In vitro resistance to Cipro-NeoB conjugates, the present inventors transcription/translation assay with E. coli S30 extract sys have used a procedure of selective pressure in which both E. US 2014/0357591 A1 Dec. 4, 2014

coli ATCC 35218 and B. subtilis ATCC 6633 were exposed to side (NeoB) with potent antibacterial activity and dual mode subinhibitory (/2 MIC) concentrations of Cipro, NeoB, of action, has been designed, prepared and tested. It has been Cipro:NeoB mixture (1:1 molar ratio), and exemplary conju shown that the nature of the spacers between the fluoroqui gate Compound li during 15 Successive Subcultures (see, nolone and the aminoglycoside moieties may influence the FIG. 2). antibacterial activity. 0572. It is noted herein that the MIC values of the Cipro: 0578. The conjugates were significantly more potent than NeoB mixture (1:1 molar ratio) against the bacterial strains the parent NeoB, especially against Gram-negative bacteria (see, Table 4 hereinabove) were very similar to that of Cipro and Gram-positive MRSA, and overcome most prevalent (on the weight basis of the composition of Cipro into the types of resistance associated with aminoglycosides. mixture, data not shown). Therefore, these experiments were 0579. At the isolated target level, the conjugates inhibited designed to include, in addition to Cipro and NeoB, also the bacterial protein synthesis with the potencies similar to or mixture Cipro--NeoB. better than that of NeoB, and were up to 32-fold more potent 0573 FIGS. 2A-B and Table 8 hereinafter present com inhibitors than Cipro for the fluoroquinolone targets, DNA parative data on the emergence of resistance in E. coli (FIG. gyrase and Topol V. 2A) and B. subtilis (FIG. 2B) after 15 serial to passages in the 0580. The conjugates presented herein demonstrated a presence of Cipro, NeoB, Cipro-i-NeoB mixture (1:1 molar significant delay of resistance formation in both Gram-nega ratio) and an exemplary conjugate, according to some tive (E. coli) and Gram-positive (B. subtilis) bacteria to the embodiments of the present invention, Compound li, treatment with Cipro-NeoB conjugate in comparison to that wherein relative MIC is the normalized ratio of MIC obtained of each drug (Cipro and NeoB) separately or their 1:1 mix for a given subculture to MIC obtained upon first exposure. ture. 0574 Table 8 presents a summary of the data examining 0581 Although the invention has been described in con the potential of emergence of resistance of Gram-negative (E. junction with specific embodiments thereof, it is evident that coli ATCC 35218) and Gram-positive (B. subtilis ATCC many alternatives, modifications and variations will be appar 6633) bacteria against Cipro, NeoB, a mixture of Cipro ent to those skilled in the art. Accordingly, it is intended to NeoB (1:1 molar ratio) and the exemplary conjugate accord embrace all Such alternatives, modifications and variations ing to some embodiments of the present invention, Com that fall within the spirit and broad scope of the appended pound li. claims. 0582 All publications, patents and patent applications TABLE 8 mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same MIC (Lig/mL extent as if each individual publication, patent or patent appli E.coli ATCC 35218 B.Subilis ATCC 6633 cation was specifically and individually indicated to be incor 1st 15th 1st 15th porated herein by reference. In addition, citation or identifi Compound passage passage Ratio passage passage Ratio cation of any reference in this application shall not be construed as an admission that Such reference is available as Cipro O.O1 0.75 75 O.O2 0.75 37.5 prior art to the present invention. To the extent that section Cipro + O.O1 O.2 2O O.OS O.38 7.6 NeoB headings are used, they should not be construed as necessarily NeoB 12 48 4 0.75 6 8 limiting. 1i 3 3 1 3 3 1 REFERENCES CITED BY NUMERALS

0575. As can be seen in FIG. 2 and Table 8, the relative Other References are Cited in the Text MIC values of Cipro, NeoB and Cipro--NeoB mixture increased by 75-, 4- and 20-fold against E. coli and by 37.5- 0583. 1. Kondo, S. and K. Hotta, Semisynthetic aminogly 8- and 7.6-fold against B. subtilis, while that of the exemplary coside antibiotics. Development and enzymatic modifica conjugate, Compound li, remained unchanged against both tions. J. Infect. Chemother, 1999. 5(1): p. 1-9. E. coli and B. subtilis. Similar emergence of resistance under 0584 2. Ye, X.-S. and L.-H. Zhang, Aminoglycoside same experimental conditions, have been reported for Cipro mimetics as Small-molecule drugs targeting RNA. Curr. 47 and aminoglycosides 48. Med. Chem., 2002(9): p. 929-939. 0576. The ability of the conjugates presented herein to 0585 3. Umezawa, H. and I. R. Hooper, eds. Aminogly delay the emergence of resistance development was not dem coside Antibiotics. 1982, Springer-Verlag New York, onstrated for the previously reported antimicrobial conju Heidelberg. gates 6-11. As such, the observed delay of resistance devel 0586 4. Magnet, S. and J. S. Blanchard, Molecular opment towards the conjugate presented herein, compared to insights into aminoglycoside action and resistance. Chem that of either Cipro, NeoB and the mixture Cipro-i-NeoB in Rev. 2005. 105(2): p. 477-98. both the Gram-negative (E. coli) and Gram-positive (B. sub 0587 5. Vakulenko, S. B. and S. Mobashery, Versatility of tilis) bacteria, demonstrate their effectiveness. aminoglycosides and prospects for their future. Clin. Example 5 Microbiol. Rev., 2003. 16(3): p. 430-50. 0588 6. Bremner, J. B., J. I. Ambrus, and S. Samosorn, Dual action-based approaches to antibacterial agents. Conclusions Curr Med Chem, 2007. 14(13): p. 1459-77. 0577. A series of exemplary conjugates, according to 0589 7. Hubschwerlen, C., et al., Design, synthesis and Some embodiments of the present invention, containing a biological evaluation of oxazolidinone-quinolone hybrids. covalently linked fluoroquinolone (Cipro) and aminoglyco Bioorg Med Chem, 2003. 11(10): p. 2313-9. US 2014/0357591 A1 Dec. 4, 2014 44

0590 8. Grapsas, I., S.A. Lerner, and S. Mobashery, Con cific binding mode to an a-minor motifenhances in vitro joint molecules of cephalosporins and aminoglycosides. antibacterial activity. ChemMedChem, 2007. 2(11): p. Arch Pharm (Weinheim), 2001. 334 (8-9): p. 295-301. 1631-8. 0591 9. Johnson, D. M. and R. N. Jones, CQ-397 and 0609. 26. Linsell, M. G., Adam Aaron; Aggen, James: CQ-414: antimicrobial activity and spectrum of two fluo Moser, Heinz; Hanessian, Stephen; Pachamuthu, Kan roquinolone—cephalosporin, dual-action compounds dasamy; Klegraf, Ellen, Preparation of 1,4,5-substituted with carboxamido bonds. Clin Microbiol Infect, 1997. amino-glycoside analogs as antibacterial agents, P.W.A. 3(3): p. 335-344. C.A. 2008: 1247020, Editor. 2008. 0592) 10. Zhi, C., et al., Hybrid antibacterials. DNA poly 0610) 27. Haddad, J., et al., Design of novel antibiotics that merase-topoisomerase inhibitors. J Med Chem, 2006. bind to the ribosomal acyltransfer site. 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A stepwise huisgen sides: biochemical studies and antibacterial activity of cycloaddition process. Copper(I)-catalyzed regioselective neomycin B derivatives. Bioorg Med Chem, 2005. 13(20): "ligation' of azides and terminal alkynes. Angew Chem p. 5797-807. Int Ed Engl, 2002. 41 (14): p. 2596-9. 0596) 14. Zhang, J., et al., Surprising Alteration of Anti 0614 31.Yao, L., B.T. Smith, and J. Aube, Basepromoted bacterial Activity of 5"-Modified Neomycin against Resis reactions of bridged ketones and 1.3-and 1.4-haloalkyl tant Bacteria. J Med Chem, 2008. azides: competitive alkylation vs azidation reactions of 0597 15. Long, D. D. and D. G. Marquess, Novel het Ketone enolates. J Org Chem, 2004. 69(5): p. 1720-2. erodimer antibiotics: a review of recent patent literature. 0615. 32. Alper, P. B. H. M.: Sears, P.; and Wong, C.-H., Future Medicinal Chemistry, 2009. 1(6): p. 1037-1050 Probing the Specificity of 0598 16. Shakil, S., et al., Aminoglycosides versus bacte 0616) Aminoglycoside-Ribosomal RNA Interactions with ria—a description of the action, resistance mechanism, Designed Synthetic Analogs. JACS 1998. 120: p. 1965-1978. and nosocomial battleground. J Biomed Sci., 2008. 15(1): 0.617 33. van den Bos, L. J., et al. Thioglycuronides: p. 5-14. synthesis and application in the assembly of acidic oli 0599. 17. Zimmer, C., H. Triebel, and H. Thrum, Interac gosaccharides. Org Lett, 2004. 6(13): p. 216.5-8. tion of streptothricin and related antibiotics with nucleic 0618. 34. Thomas, J. R. X. Liu, and P. J. Hergenrother, acids. Biochim Biophys Acta, 1967. 145(3): p. 742-51. Size-specific ligands for RNA hairpin loops. JAm Chem 0600 18. Ren, Y. G., et al., Inhibition of Klenow DNA Soc, 2005. 127(36): p. 12434-5. polymerase and poly(A)-specific ribonuclease by ami 0619. 35. Okusu, H., D. Ma, and H. Nikaido, Acra Beflux noglycosides. Rina, 2002.8(11): p. 1393-400. pump plays a major role in the antibiotic resistance phe 0601 19. Drlica, K. and X. Zhao, DNA gyrase, topoi notype of Escherichia coli multiple-antibiotic-resistance Somerase IV and the 4-quinolones. Microbiol Mol Biol (Mar) mutants. J Bacteriol, 1996. 178(1): p. 306-8. Rev. 1997. 61(3): p. 377-92. 0620 36. Ida, T., et al., Identification of aminoglycoside 0602. 20. Hawkey, P.M., Mechanisms of quinolone action modifiving enzymes by susceptibility testing epidemiology and microbial response. J of methicillin-resistant Staphylococcus aureus in Japan. J 0603 Antimicrob Chemother, 2003.51 Suppl 1: p. 29-35. Clin Microbiol, 2001. 39(9): p. 3115-21. 0604, 21. Kerns, R. J., et al., Structural features of piper 0621 37. Wright, G. D., A. M. Berghuis, and S. Mobash azinyl-linked ciprofloxacin dimers required for activity ery, Aminoglycoside antibiotics. Structures, functions, and against drug-resistant Strains of Staphylococcus aureus. resistance. Adv Exp Med Biol, 1998. 456: p. 27-69. Bioorg Med Chem Lett, 2003. 13(13): p. 2109-12. 0622 38. Chandrakanth, R. K. S. Raju, and S. A. Patil, 0605) 22. Sriram, D., et al., Synthesis and antimycobacte Aminoglycoside-resistance mechanisms in multidrug-re rial evaluation of various 7-substituted ciprofloxacin sistant Staphylococcus aureus clinical isolates. Curr derivatives. Bioorg Med Chem, 2005. 13(20): p. 5774-8. Microbiol, 2008. 56(6): p. 558-62. 0606. 23. Hanessian, S., et al., Structure-based design, 0623 39. Schmitz, F. J., et al., The prevalence of ami synthesis, and A-site rRNA cocrystal complexes of func noglycoside resistance and corresponding resistance tionally novel aminoglycoside antibiotics. C2" ether ana genes in clinical isolates of staphylococci from 19 Euro logues of paromomycin. J Med Chem, 2007. 50(10): p. pean hospitals. J Antimicrob Chemother, 1999. 43(2): p. 2352-69. 253-9. 0607 24. Francois, B., et al., Antibacterial aminoglyco 0624 40. Hainrichson, M., et al., Overexpression and ini sides with a modified mode of binding to the ribosomal tial characterization of the chromosomal aminoglycoside RNA decoding site. Angew Chem Int Ed Engl, 2004. 3'-O-phosphotransferase APH(3)-IIb from Pseudomonas 43(48): p. 6735-8. aeruginosa. Antimicrob Agents Chemother, 2007. 51(2): 0608 25. Kondo, J., et al., Crystal structure of the bacte p. 774-6. rial ribosomal decoding site complexed with a synthetic 0625 41. McKay, G. A., P. R. Thompson, and G. D. doubly fitnctionalized paronomycin derivative. a new spe Wright, Broad spectrum aminoglycoside phosphotrans US 2014/0357591 A1 Dec. 4, 2014

ferase type III from Enterococcus. Overexpression, purifi paratyphi, Salmonella typhi murium, Salmonella virchow, cation, and substrate specificity. Biochemistry, 1994. Shigella spp., Yersinia enterocolitica, Acinetobacter cal 33(22): p. 6936-44. coaceticus, Flavobacterium spp., Haemophilus influenzae, 0626 42. Robicsek, A., et al., Fluoroquinolone-modifiving Pseudomonas aeruginosa, Campylobacter jejuni, Vibrio enzyme: a new adaptation of a common aminoglycoside parahaemolyticus, Brucella spp., Neisseria meningitidis, acetyltransferase. Nat Med, 2006. 12(1): p. 83-8. Neisseria gonorrhoea, Bacteroides fragilis, Fusobacterium 0627 43. Vetting, M.W., et al., Mechanistic and structural spp., Mycobacterium tuberculosis and Mycobaterium smeg analysis of aminoglycoside N-acetyltransferase AAC(6')- natis. Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib 4. The method of claim 1, wherein said microorganism cr variant. Biochemistry, 2008. 47(37): p. 9825-35. comprises at least one bacterial strain which is resistant to at 0628 44. Ardic, N., et al., Investigation of aminoglycoside least one antibiotic agent. modifiving enzyme genes in methicillin-resistant staphyllo 5. The method of claim 4, wherein said bacterial strain is cocci. Microbiol Res, 2006. 161(1): p. 49-54. selected from the group consisting of: 0629 45. Barnard, F. M. and A. Maxwell, Interaction (a) Gram-positive bacteria selected from the group consist between DNA gyrase and quinolones: effects of alanine ing of Strep. pyogenes (Group A), Strep. pneumoniae, mutations at Gyra subunit residues Ser(83) and Asp(87). Strep. GpB, Strep. viridans, Strep. GpD-(Enterococcus), Antimicrob Agents Chemother, 2001.45(7): p. 1994-2000. Strep. GpC and GpG, Staph. aureus, Staph. epidermidis, 0630 46. Walsh, C., Where will new antibiotics come Bacillus subtilis, Bacillus anthraxis, Listeria monocyto from? Nat Rev Microbiol, 2003. 1(1): p. 65-70. genes, Anaerobic cocci, Clostridium spp., Clostridium 0631 47. Radzishevsky, I. S., et al., Improved antimicro difficile and Actinomyces spp.; bial peptides based on acyl-lysine Oligomers. Nat Biotech (b) Gram-negative bacteria selected from the group con nol, 2007. 25(6): p. 657-9. sisting of Escherichia coli, Enterobacter aerogenes, 0632) 48. Mor, A., Peptide-based antibiotics: A potential Kiebsiella pneumoniae, Proteus mirabilis, Proteus vul answer to raging antimicrobial resistance. Drug Develop garis, Morganella morganii, Providencia Stuartii, Ser ment Research, 2000. 50: p. 440-447. ratia marcescens, Citrobacter freundii, Salmonella What is claimed is: typhi, Salmonella paratyphi, Salmonella typhimurium, 1. A method of treating a medical condition associated with Salmonella virchow, Shigella spp., Yersinia entero a pathogenic microorganism in a subject, the method com colitica, Acinetobacter Calcoaceticus, Flavobacterium prising administering to the subject an effective amount of a spp., Haemophilus influenzae, Pseudomonas auerogi conjugate having the general formula I: nosa, Campylobacter jejuni, Vibrio parahaemolyticus, A-X-D-Y B Formula I Brucella spp., Neisseria meningitidis, Neisseria gonor rhoea, Bacteroides fragilis, and Fusobacterium spp., wherein: Acinetobacter baumani, Pseudomonas aeruginosa; A is a non-ribosomal-active antimicrobial agent moiety; and B is an aminoglycoside-based antimicrobial agent moiety; (c) Mycobacterium tuberculosis. X is a first spacer moiety, covalently bound to A, or absent; 6. The method of claim 4, wherein said bacterial strain is Y is a second spacer moiety, covalently bound to B, or selected from the group consisting of S. aureus ATCC 43300 absent; and (MRSA), B. subtilis ATCC 6633, E. coli AG100E3, E. coli D is a linking moiety having the general formula II: AG100A. E. coli R477-100, E. coli ATCC 25922 and E. coli ATCC 35218. 7. The method of claim 1, wherein said non-ribosomal Formula II NN active antibiotic agentis selected from the group consisting of / an anti-metabolite-based antimicrobial agent, a quinolone based antimicrobial agent, a B-lactam-based antimicrobial agent, a glycopeptide-based antimicrobial agent, a benzyl-2, 4-diaminopyrimidine-based antimicrobial agent, a Sulfona R mide-based antimicrobial agent, a Sulfanilamide-based anti microbial agent, a peptide-based antimicrobial agent, a whereas each of the wiggled lines denote covalent bond to pseudo-peptide-based antimicrobial agent and a peptidomi either A-X— or B Y , and R is selected from the metic-based antimicrobial agent. group consisting of hydrogen, alkyl, cycloalkyl and alk 8. The method of claim 1, wherein said non-ribosomal enyl. active antibiotic agent is a quinolone-based antimicrobial 2. The method of claim 1, wherein said microorganism agent. comprises at least one bacterial strain. 9. The method of claim 8, wherein said quinolone-based 3. The method of claim 2, wherein said bacterial strain is antimicrobial agent is selected from the group consisting of a selected from the group consisting of Strep. pyogenes (Group fluoroquinolone, ciprofloxacin (Cipro, Ciprobay, CiproXin), A), Strep. pneumoniae, Strep. GpB, Strep. viridans, Strep. balofloxacin (Baloxin), cinoxacin (Cinobac), clinafloxacin, GpD (Enterococcus), Strep. GpC and GpG, Staph. aureus, danofloxacin (Advocin, Advocid), delafloxacin, difloxacin Staph. epidermidis, Bacillus subtilis, Bacillus anthracis, List (Dicural, Vetequinon), enoxacin (Enroxil, Penetrex), enrof eria monocytogenes, Anaerobic cocci, Clostridium spp., Acti loxacin (Baytril), fleroxacin (Megalone, Roquinol), flume nomyces spp., Escherichia coli, Enterobacter aerogenes, Kie quine (Flubactin), garenoxacin (Geninax), gatifloxacin (Te bsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, quin, Zymar), gemifloxacin (Factive), grepafloxacin (Raxar), Morganella morganii, Providencia Stuartii, Serratia marce ibafloxacin (Ibaflin), levofloxacin (Cravit, Levaquin), lom scens, Citrobacter freundii, Salmonella typhi, Salmonella efloxacin (Maxaquin), marbofloxacin (Marbocyl. Zenequin), US 2014/0357591 A1 Dec. 4, 2014 46 moxifloxacin (AVelox, Vigamox), nadifloxacin (Acuatim, bsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Nadoxin, Nadixa), nalidixic acid (NegGam, Wintomylon), Morganella morganii, Providencia Stuartii, Serratia marce norfloxacin (Lexinor, NoroXin, Quinabic, Janacin), ofloxacin scens, Citrobacter freundii, Salmonella typhi, Salmonella (Floxin, Oxaldin, Tarivid), orbifloxacin (Orbax, Victas), oxo paratyphi, Salmonella typhi murium, Salmonella virchow, linic acid (Uroxin), paZufloxacin (Pasil, PaZucross), pefloxa Shigella spp., Yersinia enterocolitica, Acinetobacter cal cin (Peflacine), pipemidic acid (Dolcol), piromidic acid coaceticus, Flavobacterium spp., Haemophilus influenzae, (Panacid), prulifloxacin (Quisnon), roSoxacin (Eradacil), Pseudomonas aeruginosa, Campylobacter jejuni, Vibrio rufloxacin (Uroflox), Sarafloxacin (Floxasol, Saraflox, Sara parahaemolyticus, Brucella spp., Neisseria meningitidis, fin), sitafloxacin (Gracevit), Sparfloxacin (Zagam), tema Neisseria gonorrhoea, Bacteroides fragilis, Fusobacterium floxacin (Omniflox), tosufloxacin (OZex, Tosacin) and trova spp., Mycobacterium tuberculosis and Mycobaterium smeg floxacin (Trovan). natis. 10. The method of claim 9, wherein said quinolone-based 18. The method of claim 15, wherein said microorganism antimicrobial agent is is ciprofloxacin. comprises at least one bacterial strain which is resistant to at 11. The method of claim 1, wherein said aminoglycoside least one antibiotic agent. based antimicrobial agent is selected from the group consist ing of neomycin B. neomycin C, streptomycin, framycetin, 19. The method of claim 18, wherein said bacterial strain is paromomycin, ribostamycin, kanamycin, amikacin, arbeka selected from the group consisting of: cin, bekanamycin, dibekacin, tobramycin, spectinomycin, (a) Gram-positive bacteria selected from the group consist hygromycin, paromomycin, gentamicin, netilmicin, Sisomi ing of Strep. pyogenes (Group A), Strep. pneumoniae, cin, isepamicin, Verdamicin and astromicin. Strep. GpB, Strep. viridans, Strep. GpD-(Enterococcus), 12. The method of claim 11, wherein said aminoglycoside Strep. GpC and GpG, Staph. aureus, Staph. epidermidis, based antimicrobial agent is selected from the group consist Bacillus subtilis, Bacillus anthraxis, Listeria monocyto ing of neomycin B and kanamycin A. genes, Anaerobic cocci, Clostridium spp., Clostridium 13. The method of claim 1, wherein each of X and Y, when difficile and Actinomyces spp.; present, is independently selected from the group consisting (b) Gram-negative bacteria selected from the group con of alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, sisting of Escherichia coli, Enterobacter aerogenes, heteroaryland a hydrocarbon chain having 1-20 carbonatoms Kiebsiella pneumoniae, Proteus mirabilis, Proteus vul and ending or interrupted by at least one heteroatom selected garis, Morganella morganii, Providencia Stuartii, Ser from the group consisting of O, S and N and/or containing ratia marcescens, Citrobacter freundii, Salmonella from 0 to 19 unsaturated carbon-carbon or carbon-heteroa typhi, Salmonella paratyphi, Salmonella typhimurium, tom bonds. Salmonella virchow, Shigella spp., Yersinia entero 14. The method of claim 1, wherein each of X and Y, when colitica, Acinetobacter Calcoaceticus, Flavobacterium present, is independently selected from the group consisting spp., Haemophilus influenzae, Pseudomonas auerogi nosa, Campylobacter jejuni, Vibrio parahaemolyticus, Brucella spp., Neisseria meningitidis, Neisseria gonor rhoea, Bacteroides fragilis, and Fusobacterium spp., Acinetobacter baumani, Pseudomonas aeruginosa; and (c) Mycobacterium tuberculosis. 20. The method of claim 18, wherein said bacterial strain is —CH=CH-CH NH (CH)—. selected from the group consisting of S. aureus ATCC 43300 15. A method of treating a medical condition associated (MRSA), B. subtilis ATCC 6633, E. coli AG100E3, E. coli with a pathogenic microorganism in a subject, the method AG100A. E. coli R477-100, E. coli ATCC 25922 and E. coli comprising, administering to said Subject an effective amount ATCC 35218. of a conjugate having the general formula III: 21. The method of claim 15, wherein said quinolone-based A-X W Y B Formula III antimicrobial agent is selected from the group consisting of a fluoroquinolone, ciprofloxacin (Cipro, Ciprobay, CiproXin), wherein: balofloxacin (Baloxin), cinoxacin (Cinobac), clinafloxacin, A' is a quinolone-based antimicrobial agent moiety; danofloxacin (Advocin, Advocid), delafloxacin, difloxacin B is an aminoglycoside-based antimicrobial agent moiety; (Dicural, Vetequinon), enoxacin (Enroxil, Penetrex), enrof X is a first spacer moiety, covalently bound to A, or absent; loxacin (Baytril), fleroxacin (Megalone, Roquinol), flume Y is a second spacer moiety, covalently bound to B, or quine (Flubactin), garenoxacin (Geninax), gatifloxacin (Te absent; and quin, Zymar), gemifloxacin (Factive), grepafloxacin (Raxar), W is a linking moiety. ibafloxacin (Ibaflin), levofloxacin (Cravit, Levaquin), lom 16. The method of claim 15, wherein said microorganism efloxacin (Maxaquin), marbofloxacin (Marbocyl. Zenequin), comprises at least one bacterial strain. moxifloxacin (AVelox, Vigamox), nadifloxacin (Acuatim, 17. The method of claim 16, wherein said bacterial strain is Nadoxin, Nadixa), nalidixic acid (NegGam, Wintomylon), selected from the group consisting of Strep. pyogenes (Group norfloxacin (Lexinor, NoroXin, Quinabic, Janacin), ofloxacin A), Strep. pneumoniae, Strep. GpB, Strep. viridans, Strep. (Floxin, Oxaldin, Tarivid), orbifloxacin (Orbax, Victas), oxo GpD (Enterococcus), Strep. GpC and GpG, Staph. aureus, linic acid (Uroxin), paZufloxacin (Pasil, PaZucross), pefloxa Staph. epidermidis, Bacillus subtilis, Bacillus anthracis, List cin (Peflacine), pipemidic acid (Dolcol), piromidic acid eria monocytogenes, Anaerobic cocci, Clostridium spp., Acti (Panacid), prulifloxacin (Quisnon), roSoxacin (Eradacil), nomyces spp., Escherichia coli, Enterobacter aerogenes, Kie rufloxacin (Uroflox), Sarafloxacin (Floxasol, Saraflox, Sara US 2014/0357591 A1 Dec. 4, 2014 47 fin), sitafloxacin (Gracevit), Sparfloxacin (Zagam), tema 33. The method of claim 32, wherein R is hydrogen. floxacin (Omniflox), tosufloxacin (OZex, Tosacin) and trova 34. The method of claim 15, being selected from the group floxacin (Trovan). consisting of N-(4-(1-(2-(ciprofloxacin)ethyl)-1H-1,2,3-tria 22. The method of claim 21, wherein said quinolone-based Zol-4-yl)-5'-phenyl) neomycin, N-(4-(1-(2-(ciprofloxacin) antimicrobial agent is ciprofloxacin. propyl)-1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin Ca 23. The method of claim 22, wherein said ciprofloxacin is boxamide, N-(4-(1-(2-(ciprofloxacin)butyl)-1H-1,2,3- covalently bound to X via the terminal nitrogen of the pip triazol-4-yl)-5'-phenyl)neomycin carboxamide, N-(4-(1-(2- erazine moiety thereof. (ciprofloxacin)pentyl)-1H-1,2,3-triazol-4-yl)-5'-phenyl) 24. The method of claim 15, wherein said aminoglycoside neomycin carboxamide, N-(4-(1-(2-(ciprofloxacin)hexyl)- based antimicrobial agent is selected from the group consist 1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin carboxamide, ing of neomycin B. neomycin C, streptomycin, framycetin, N-(4-(1-(2-hydroxy-3-(ciprofloxacin)propyl)-1H-1,2,3-tria paromomycin, ribostamycin, kanamycin, amikacin, arbeka Zol-4-yl)-5'-phenyl)neomycin carboxamide, N-(4-(1-(2-(2- cin, bekanamycin, dibekacin, tobramycin, spectinomycin, (ciprofloxacin)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5'-phe hygromycin, paromomycin, gentamicin, netilmicin, Sisomi nyl)neomycin carboxamide, N-(4-(1-(4-((ciprofloxacin) cin, isepamicin, Verdamicin and astromicin. methyl)benzyl)-1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin 25. The method of claim 24, wherein said aminoglycoside carboxamide, N-(4-(1-(3-((ciprofloxacin)methyl)benzyl)- based antimicrobial agent is covalently bound to Y via the 1H-1,2,3-triazol-4-yl)-5'-phenyl)neomycin carboxamide, C5"-position thereof. N-((1-(4-(ciprofloxacin)ethyl)-1H-1,2,3-triazol-4-yl)-5"- 26. The method of claim 25, wherein said aminoglycoside methyl)neomycin carboxamide, N-((1-(4-(ciprofloxacin) based antimicrobial agent is neomycin B. propyl)-1H-1,2,3-triazol-4-yl)-5'-methyl)neomycin Ca 27. The method of claim 24, wherein said aminoglycoside boxamide, N-((1-(4-(ciprofloxacin)pentyl)-1H-1,2,3-triazol based antimicrobial agent is covalently bound to Y via the 4-yl)-5'-methyl)neomycin carboxamide, N-((1-(2-hydroxy C1-N-position thereof. 3-(ciprofloxacin)propyl)-1H-1,2,3-triazol-4-yl)-5'-methyl) 28. The method of claim 25, wherein said aminoglycoside neomycin carboxamide, N-((1-(2-(2-(ciprofloxacin)ethoxy) based antimicrobial agent is kanamycin A. ethyl)-1H-1,2,3-triazol-4-yl)-5'-methyl)neomycin 29. The method of claim 15, wherein each of XandY, when carboxamide, N-((1-(3-((ciprofloxacin)methyl)benzyl)-1H present, is independently selected from the group consisting 1,2,3-triazol-4-yl)-5'-methyl)neomycin carboxamide, of alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl, N-((1-(4-((ciprofloxacin)methyl)benzyl)-1H-1,2,3-triazol heteroaryland a hydrocarbon chain having 1-20 carbonatoms 4-yl)-5'-methyl)neomycin carboxamide, 4-((5"-neomycin and ending or interrupted by at least one heteroatom selected methoxy)methyl)-1-(2-(ciprofloxacin)ethyl)-1H-1,2,3-triaz from the group consisting of O. S and N and/or containing ole, 4-(1-N-kanamycin methyl)-1-(2-(ciprofloxacin)ethyl)- from 0 to 19 unsaturated carbon-carbon or carbon-heteroa 1H-1,2,3-triazole, 1-(4-(1-N-kanamycin methyl)-1H-1,2,3- tom bonds. triazol-1-yl)-3-(ciprofloxacin)propan-2-ol and 4-(1-N- 30. The method of claim 29, wherein each of XandY, when kanamycin methyl)-1-(4-((ciprofloxacin)methyl)benzyl)- present, is independently selected from the group consisting 1H-1,2,3-triazole. 35. A method of inhibiting the growth of at least one micro organism comprising contacting the microorganism with a conjugate having the general formula I: A-X-D-Y B Formula I wherein: A is a non-ribosomal-active antimicrobial agent moiety; B is an aminoglycoside-based antimicrobial agent moiety; —CH=CH-CH NH (CH)—. X is a first spacer moiety, covalently bound to A, or absent; 31. The method of claim 15, wherein Wis selected from the Y is a second spacer moiety, covalently bound to B, or group consisting of a covalent bond, amide, carboxylate, absent; and cycloalkene, cyclohexene, heteroalicyclic, heteroaryl, triaz D is a linking moiety having the general formula II: ine, triazole, disulfide, lactone, lactam, imine, aldimine, ketimine, hydraZone and semicarbazone. Formula II 32. The method of claim 15, wherein W comprises the NN general formula II: /

Formula II NN R / whereas each of the wiggled lines denote covalent bond to either A-X— or B Y , and R is selected from the R group consisting of hydrogen, alkyl, cycloalkyl and alk enyl. whereas each of the wiggled lines denote covalent bond to 36. The method of claim 35, wherein said microorganism either A-X— or B Y , and R is selected from the comprises at least one bacterial strain. group consisting of hydrogen, alkyl, cycloalkyl and alk 37. The method of claim 36, wherein said bacterial strain is enyl. selected from the group consisting of Strep. pyogenes (Group US 2014/0357591 A1 Dec. 4, 2014 48

A), Strep. pneumoniae, Strep. GpB, Strep. viridans, Strep. Y is a second spacer moiety, covalently bound to B, or GpD (Enterococcus), Strep. GpC and GpG, Staph. aureus, absent; and Staph. epidermidis, Bacillus subtilis, Bacillus anthracis, List W is a linking moiety. eria monocytogenes, Anaerobic cocci, Clostridium spp., Acti 42. The method of claim 41, wherein said microorganism nomyces spp., Escherichia coli, Enterobacter aerogenes, Kie comprises at least one bacterial strain. bsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, 43. The method of claim 42, wherein said bacterial strain is Morganella morganii, Providencia Stuartii, Serratia marce selected from the group consisting of Strep. pyogenes (Group scens, Citrobacter freundii, Salmonella typhi, Salmonella A), Strep. pneumoniae, Strep. GpB, Strep. viridans, Strep. paratyphi, Salmonella typhi murium, Salmonella virchow, GpD (Enterococcus), Strep. GpC and GpG, Staph. aureus, Shigella spp., Yersinia enterocolitica, Acinetobacter cal Staph. epidermidis, Bacillus subtilis, Bacillus anthracis, List coaceticus, Flavobacterium spp., Haemophilus influenzae, eria monocytogenes, Anaerobic cocci, Clostridium spp., Acti Pseudomonas aeruginosa, Campylobacter jejuni, Vibrio nomyces spp., Escherichia coli, Enterobacter aerogenes, Kie parahaemolyticus, Brucella spp., Neisseria meningitidis, bsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Neisseria gonorrhoea, Bacteroides fragilis, Fusobacterium Morganella morganii, Providencia Stuartii, Serratia marce spp., Mycobacterium tuberculosis and Mycobaterium smeg scens, Citrobacter freundii, Salmonella typhi, Salmonella natis. paratyphi, Salmonella typhi murium, Salmonella virchow, 38. The method of claim 35, wherein said microorganism Shigella spp., Yersinia enterocolitica, Acinetobacter cal comprises at least one bacterial strain which is resistant to at coaceticus, Flavobacterium spp., Haemophilus influenzae, least one antibiotic agent. Pseudomonas aeruginosa, Campylobacter jejuni, Vibrio 39. The method of claim38, wherein said bacterial strain is parahaemolyticus, Brucella spp., Neisseria meningitidis, selected from the group consisting of: Neisseria gonorrhoea, Bacteroides fragilis, Fusobacterium (a) Gram-positive bacteria selected from the group consist spp., Mycobacterium tuberculosis and Mycobaterium smeg ing of Strep. pyogenes (Group A), Strep. pneumoniae, natis. Strep. GpB, Strep. viridans, Strep. GpD-(Enterococcus), 44. The method of claim 41, wherein said microorganism Strep. GpC and GpG, Staph. aureus, Staph. epidermidis, comprises at least one bacterial strain which is resistant to at Bacillus subtilis, Bacillus anthraxis, Listeria monocyto least one antibiotic agent. genes, Anaerobic cocci, Clostridium spp., Clostridium 45. The method of claim 44, wherein said bacterial strain is difficile and Actinomyces spp.; selected from the group consisting of: (b) Gram-negative bacteria selected from the group con (a) Gram-positive bacteria selected from the group consist sisting of Escherichia coli, Enterobacter aerogenes, ing of Strep. pyogenes (Group A), Strep. pneumoniae, Kiebsiella pneumoniae, Proteus mirabilis, Proteus vul Strep. GpB, Strep. viridans, Strep. GpD-(Enterococcus), garis, Morganella morganii, Providencia Stuartii, Ser Strep. GpC and GpG, Staph. aureus, Staph. epidermidis, ratia marcescens, Citrobacter freundii, Salmonella Bacillus subtilis, Bacillus anthraxis, Listeria monocyto typhi, Salmonella paratyphi, Salmonella typhimurium, genes, Anaerobic cocci, Clostridium spp., Clostridium Salmonella virchow, Shigella spp., Yersinia entero difficile and Actinomyces spp.; colitica, Acinetobacter Calcoaceticus, Flavobacterium (b) Gram-negative bacteria selected from the group con spp., Haemophilus influenzae, Pseudomonas auerogi sisting of Escherichia coli, Enterobacter aerogenes, nosa, Campylobacter jejuni, Vibrio parahaemolyticus, Kiebsiella pneumoniae, Proteus mirabilis, Proteus vul Brucella spp., Neisseria meningitidis, Neisseria gonor garis, Morganella morganii, Providencia Stuartii, Ser rhoea, Bacteroides fragilis, and Fusobacterium spp., ratia marcescens, Citrobacter freundii, Salmonella Acinetobacter baumani, Pseudomonas aeruginosa; typhi, Salmonella paratyphi, Salmonella typhimurium, and Salmonella virchow, Shigella spp., Yersinia entero (c) Mycobacterium tuberculosis. colitica, Acinetobacter Calcoaceticus, Flavobacterium 40. The method of claim38, wherein said bacterial strain is spp., Haemophilus influenzae, Pseudomonas auerogi selected from the group consisting of S. aureus ATCC 43300 nosa, Campylobacter jejuni, Vibrio parahaemolyticus, (MRSA), B. subtilis ATCC 6633, E. coli AG100E3, E. coli Brucella spp., Neisseria meningitidis, Neisseria gonor AG100A. E. coli R477-100, E. coli ATCC 25922 and E. coli rhoea, Bacteroides fragilis, and Fusobacterium spp., ATCC 35218. Acinetobacter baumani, Pseudomonas aeruginosa; 41. A method of inhibiting the growth of at least one micro and organism comprising contacting the microorganism with a (c) Mycobacterium tuberculosis. conjugate having the general formula formula III: 46. The method of claim 44, wherein said bacterial strain is A-X W Y B Formula III selected from the group consisting of S. aureus ATCC 43300 wherein: (MRSA), B. subtilis ATCC 6633, E. coli AG100E3, E. coli A' is a quinolone-based antimicrobial agent moiety; AG100A. E. coli R477-100, E. coli ATCC 25922 and E. coli B is an aminoglycoside-based antimicrobial agent moiety; ATCC 35218. X is a first spacer moiety, covalently bound to A, or absent;