Antibiotic-resistant artificial nucleoside, composition and method

Abstract

Antibiotic-resistant artificial nucleosides may be used in combination with antibiotics. The artificial nucleosides may be selected from: (I), (II), (III),(IV) wherein R is as defined herein.

Description

ANTIBIOTIC-RESISTANT ARTIFICIAL NUCLEOSIDE, COMPOSITION AND METHOD

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 420,875 filed October 31, 2022, and titled “ANTIBIOTIC-RESISTANT ARTIFICIAL NUCLEOSIDE, COMPOSITION AND METHOD,” which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. R01AI165997-01AI awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND

[0003] Antibiotic resistant bacteria are an urgent, global health threat. In the United States alone, approximately 3 million antibiotic-resistant infections occur annually, and more than 35,000 individuals die from these infections. Multidrug-resistant bacteria produce β-lactamases that can inactivate virtually all β-lactam antibiotics. This represents a significant challenge that hinders the development of new β-lactams with improved clinical efficacy. An additional complication is that β-lactams produce reactive oxygen species that, by damaging DNA, promote mutagenesis to further drive drug resistance. More importantly, the genomic insult produced by reactive oxygen species induces infectious bacteria to survive treatment by forming bacterial persisters. These persisters are extremely dangerous as they can remain dormant for an extended period of time even in spite of treatment and then rapidly re-emerge once the treatment has ended.

[0004] β-lactam antibiotics typically kill bacteria by interfering with cell wall biosynthesis. However, these compounds can also produce multiple cell stress responses such as the production of reactive oxygen species (ROS). ROS induce the SOS response, a DNA damage-induced state in which bacteria have an increased capacity for mutagenesis. FIG.1 illustrates a model of this effect. During the SOS response, a fractionof the infecting bacterial population also survives despite continuous exposure to β- lactams. This population of drug-resistant bacteria are called persisters and can remain induced for SOS for an extended period of time. Under these conditions, persisters form long filaments due to SOS-mediated arrest of cell septation as illustrated in FIG.2. The SOS response is not only involved in persister formation, but mutations in persister cells accumulate due to failed DNA repair followed by subsequent replication by various DNA polymerases. The accumulation of mutations contributes to subsequent antimicrobial resistance as well as the return of persisters to a metabolically active growing state.

[0005] It would be desirable to develop new compositions, systems, and methods which reduce or eliminate the aforementioned problems. BRIEF DESCRIPTION

[0006] Disclosed, in some embodiments, is an anti-microbial composition containing an artificial nucleoside and an anti-microbial agent.

[0007] The anti-microbial agent may be a β-lactam antibiotic.

[0008] In some embodiments, the artificial nucleoside is of formula (I): wherein Het is athe group consisting of:wherein R1 is OH, monoph , diphosphate (H3(O3PO)2- or ((O3PO)2-)3-), triphosphate (H4(O3PO)3-, ((O3PO)3-)4-), or ORa, where Ra comprises at least one of a straight chain, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, fatty acid residue, or a mono, di, or triphosphate thereof;wherein R4, R5, R6, R7, R9, R10, R11, R12, R15, R16, and R18, each independently represent substituents selected from the group consisting of hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhydryl, C1- C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2- C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2- C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono- (C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1-C24 alkyl)- substituted carbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)- NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (- N═N+═N−), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1- C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2- C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2- aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof, and further wherein any two adjacent (ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six-membered rings, and fused five-membered and / or six- membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogen substituents and zero to 3 heteroatoms; and with the proviso that at least one of R4, R5, R6, R7, R9, R10, R11, R12, R15, and R18 is other than hydrogen and that where R9 is amino R10 is other than hydrogen; andwherein at least one of R2, R3, R8, R13, R14, R17, and R19 is independently selected from hydrogen and a click-reactive functional group that is directly or indirectly bound to the purine or indole ring of the compound.

[0009] Disclosed, in other embodiments, is a method for treating a bacterial infection including administering an artificial nucleoside to a patent.

[0010] In some embodiments, the artificial nucleoside is 5-nitro-indolyl-2'- deoxynucleoside (5-NIdR).

[0011] Disclosed, in further embodiments, is a treatment method including: administering an artificial nucleoside to a patient; and administering an antimicrobial agent to the patient.

[0012] The artificial nucleoside and the antimicrobial agent may be administered to the patient simultaneously.

[0013] In some embodiments, the artificial nucleoside and the antimicrobial agent are administered in the same composition.

[0014] The antimicrobial agent may be administered orally and the artificial nucleoside may be administered via an injection.

[0015] Artificial nucleosides are also disclosed. The artificial nucleoside may be selected from:; wherein R is selected from ed from the group consisting of hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6- C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (- CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6- C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2- C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (- COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-N(C1- C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (- (CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5- C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH- (CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (- NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (- (SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5- C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof. Antimicrobial compositions containing the artificial nucleoside and an antimicrobial agent(e.g., a β-lactam antibiotic) are also disclosed. Further disclosed are treatment methods including administering the antimicrobial composition to a patient.

[0016] The artificial nucleoside may be of the formula: ; wherein each R is of O, S, C, and N.Antimicrobial compositions containing the artificial nucleoside and an antimicrobial agent (e.g., a β-lactam antibiotic) are also disclosed. Further disclosed are treatment methods including administering the antimicrobial composition to a patient.

[0017] These and other non-limiting characteristics are more particularly described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

[0019] FIG. 1 illustrates a model for increased mutagenesis and drug resistance caused by translesion DNA synthesis.

[0020] FIG.2 shows that resistance to β-lactam antibiotics through the formation of filamentous persisters occurs with hours of treatment.

[0021] FIG. 3A-F are checkerboard analysis charts showing synergistic cell-killing effects of combining 5-NIdR with aztreonam / avibactam in drug-resistant bacteria (FIG. 3A: IMP-1; FIG.3B: VIM-2; FIG.3C: SPM-1; FIG.3D: PA01 WT; FIG.3E: ATCC 27853; and FIG.3F: OXA-50).

[0022] FIG.4 illustrates that the reversion of filamentous persisters after removal of antibiotics is very rapid.

[0023] FIG.5 shows how the combination of 5-NIdR and β-lactams affects the viability of filamentous persisters.

[0024] FIG. 6A-D are SEM images demonstrating the combination of 5 NIdR with aztreonam as a strategy to impede persistence (FIG.6A: PA01 WT: Aztreonam; FIG.6B: PA01 ΔdinB: Aztreonam; FIG.6C: PA01 dinB overexpressor: Aztreonam; and FIG.6D: PA01 WT: Aztreonam + 5-NIdR). DETAILED DESCRIPTION

[0025] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent can be used in practice or testing of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and articles disclosed herein are illustrative only and not intended to be limiting.

[0027] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0028] As used in the specification and in the claims, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of” the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0029] Unless indicated to the contrary, the numerical values in the specification should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of the conventional measurement technique of the type used to determine the particular value.

[0030] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 to 10” is inclusive of the endpoints, 2 and 10, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0031] As used herein, approximating language may be applied to modify any quantitative representation that may vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1.

[0032] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0033] The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups, such as cyclopentyl, cyclohexyl, and the like. Generally, although again not necessarily, alkyl groups herein contain 1 to about 18 carbon atoms, preferably 1 to about 12 carbon atoms. The term “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms. Substituents identified as “C1-C6 alkyl” or “lower alkyl” can contain 1to 3 carbon atoms, and more particularly such substituents can contain 1 or 2 carbon atoms (i.e., methyl and ethyl). “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the terms “alkyl” and “lower alkyl” include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom- containing alkyl or lower alkyl, respectively.

[0034] The term “alkenyl” refers to a linear, branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms containing at least one double bond, such as ethenyl, n- propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally, although again not necessarily, alkenyl groups can contain 2 to about 18 carbon atoms, and more particularly 2 to 12 carbon atoms. The term “lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, and the specific term “cycloalkenyl” intends a cyclic alkenyl group, preferably having 5 to 8 carbon atoms. The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer to alkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkenyl” and “lower alkenyl” include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0035] The term “alkynyl” refers to a linear or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, and the like. Generally, although again not necessarily, alkynyl groups can contain 2 to about 18 carbon atoms, and more particularly can contain 2 to 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 6 carbon atoms. The term “substituted alkynyl” refers to alkynyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkynyl” and “heteroalkynyl” refer to alkynyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms “alkynyl” and “lower alkynyl” include linear, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0036] The term “alkoxy” refers to an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group may be represented as -O-alkyl where alkyl is as defined above. A “lower alkoxy” group intends an alkoxy group containing 1 to 6 carbon atoms, and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Preferred substituents identified as “C1-C6 alkoxy” or “lower alkoxy” herein contain 1 to 3 carbon atoms, and particularly preferred such substituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

[0037] The term “aryl” refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups can contain 5 to 20 carbon atoms, and particularly preferred aryl groups can contain 5 to 14 carbon atoms. Exemplary aryl groups contain one aromatic ring or two fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the terms “heteroatom- containing aryl” and “heteroaryl” refer to aryl substituents, in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0038] The term “aryloxy” as used herein refers to an aryl group bound through a single, terminal ether linkage, wherein “aryl” is as defined above. An “aryloxy” group may be represented as -O-aryl where aryl is as defined above. Preferred aryloxy groups contain 5 to 20 carbon atoms, and particularly preferred aryloxy groups contain 5 to 14 carbon atoms. Examples of aryloxy groups include, without limitation, phenoxy, o-halo- phenoxy, m-halo-phenoxy, p-halo-phenoxy, o-methoxy-phenoxy, m-methoxy-phenoxy, p-methoxy-phenoxy, 2,4-dimethoxy-phenoxy, 3,4,5-trimethoxy-phenoxy, and the like.

[0039] The term “alkaryl” refers to an aryl group with an alkyl substituent, and the term “aralkyl” refers to an alkyl group with an aryl substituent, wherein “aryl” and “alkyl” are as defined above. Exemplary aralkyl groups contain 6 to 24 carbon atoms, and particularly preferred aralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4- benzylcyclohexylmethyl, and the like. Alkaryl groups include, for example, p- methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7- cyclooctylnaphthyl, 3-ethyl-cyclopenta-1,4-diene, and the like.

[0040] The term “cyclic” refers to alicyclic or aromatic substituents that may or may not be substituted and / or heteroatom containing, and that may be monocyclic, bicyclic, or polycyclic.

[0041] The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.

[0042] The term “heteroatom-containing” as in a “heteroatom-containing alkyl group” (also termed a “heteroalkyl” group) or a “heteroatom-containing aryl group” (also termed a “heteroaryl” group) refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term “heteroalkyl” refers to an alkyl substituent that is heteroatom-containing, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, the terms “heteroaryl” and heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom- containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, etc.

[0043] “Hydrocarbyl” refers to univalent hydrocarbyl radicals containing 1 to about 30 carbon atoms, preferably 1 to about 24 carbon atoms, more preferably 1 to about 18 carbon atoms, most preferably about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated, and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, and the like. “Substituted hydrocarbyl” refers to hydrocarbyl substituted with one or more substituent groups, and the term “heteroatom-containing hydrocarbyl” refers to hydrocarbyl in which at least one carbon atom is replaced with a heteroatom. Unless otherwise indicated, the term “hydrocarbyl” is to be interpreted as including substituted and / or heteroatom-containing hydrocarbyl moieties.

[0044] By “substituted” as in “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2- C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (- CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (- (CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)- substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1-C4 alkyl)-substituted carbamoyl (- (CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (- (CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-ON+C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6- C20 arylamido (-NH-(CO)-aryl), imino (-CR═NH where R=hydrogen, C1-C24 alkyl, C5- C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O−), C1- C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1- C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O−)2), phosphinato (-P(O)(O-)), phospho (-PO2), and phosphino (- PH2); and the hydrocarbyl moieties C1-C24 alkyl (preferably C1-C18 alkyl, more preferably C1-C12 alkyl, most preferably C1-C6 alkyl), C2-C24 alkenyl (preferably C2-C18 alkenyl, more preferably C2-C12 alkenyl, most preferably C2-C6 alkenyl), C2-C24 alkynyl (preferably C2- C18 alkynyl, more preferably C2-C12 alkynyl, most preferably C2-C6 alkynyl), C5-C20 aryl (preferably C5-C14 aryl), C6-C24 alkaryl (preferably C6-C18 alkaryl), and C6-C24 aralkyl (preferably C6-C18 aralkyl).

[0045] In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.

[0046] When the term “substituted” appears prior to a list of possible substituted groups, it is intended that the term apply to every member of that group. For example, the phrase “substituted alkyl, alkenyl, and aryl” is to be interpreted as “substituted alkyl, substituted alkenyl, and substituted aryl.” Analogously, when the term “heteroatom- containing” appears prior to a list of possible heteroatom-containing groups, it is intended that the term apply to every member of that group. For example, the phrase “heteroatom- containing alkyl, alkenyl, and aryl” is to be interpreted as “heteroatom-containing alkyl, heteroatom-containing alkenyl, and heteroatom-containing aryl.”

[0047] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not. For example, the phrase “optionally substituted” means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a non-hydrogen substituent is not present.

[0048] When referring to a “compound” or “agent” herein, the term “compound” or “agent” is meant to encompass not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active analogs, including, but not limited to, salts, esters, amides, prodrugs, conjugates, active metabolites, and other such derivatives, analogs, and related compounds.

[0049] By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met therequired standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration. “Pharmacologically active” (or simply “active”) as in a “pharmacologically active” derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0050] As used herein, the term “pharmaceutically acceptable salts” or complexes refers to salts or complexes of the nucleosides that retain the desired biological activity of the parent compound and exhibit minimal, if any, undesired toxicological effects. Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, and polygalacturonic acid; (b) base addition salts formed with cations such as sodium, potassium, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with an organic cation formed from N,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c) combinations of (a) and (b); e.g., a zinc tannate salt or the like.

[0051] The term “prodrug”, as used herein, refers to a compound that is converted into the deoxynucleoside triphosphate on administration in vivo. Nonlimiting examples are pharmaceutically acceptable salts (alternatively referred to as “physiologically acceptable salts).

[0052] The phrases “parenteral administration” and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0053] By click-reactive functional groups it is meant that the functional group has click reactivity and / or can undergo a click reaction with a complementary click reactivefunctional group. A description of such groups is provided in U.S. Pat. No.9,988,680 to Berdis et al., which issued on June 5, 2018 and is incorporated by reference herein in its entirety.

[0054] The present disclosure relates to artificial nucleosides that combat antibiotic- resistance. Compositions containing the nucleosides and treatment methods utilizing the nucleosides are also disclosed. The artificial nucleosides may exhibit synergistic effects with antimicrobial agents, particularly antimicrobial agents that generate reactive oxygen species such as β-lactams.

[0055] The artificial nucleosides may be of formula (I):

[0056] where Het azaindene analog selected from the groupconsisting of:

[0057] where R1 is O or (O3PO-)2-), diphosphate (H3(O3PO)2- or ((O3PO)2-)3-), triphosphate (H4(O3PO)3-, ((O3PO)3-)4-), or ORa, where Ra comprises at least one of a straight chain, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, fatty acid residue, or a mono, di, or triphosphate thereof;

[0058] where R4, R5, R6, R7, R9, R10, R11, R12, R15, R16, and R18, each independently represent substituents selected from the group consisting of hydrogen, C1-C24 alkyl, C2- C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (- O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6- C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1- C24 alkyl)-substituted carbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2- C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.),alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2- aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof, and further wherein any two adjacent (ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six-membered rings, and fused five-membered and / or six- membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogen substituents and zero to 3 heteroatoms; and with the proviso that at least one of R4, R5, R6, R7, R9, R10, R11, R12, R15, and R18 is other than hydrogen and that where R9 is amino R10 is other than hydrogen; and

[0059] wherein at least one of R2, R3, R8, R13, R14, R17, and R19 are independently selected from hydrogen and a click-reactive functional group that is directly or indirectly bound to the purine or indole ring of the compound. In particular embodiments, at least one of R2, R3, R8, R13, R14, R17, and R19 is other than hydrogen.

[0060] The artificial nucleoside may be administered as a stabilized nucleotide prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the nucleoside. A number of nucleotide prodrug ligands are known. In general, alkylation, acylation or other lipophilic modification of the mono, di or triphosphate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that can replace one or more hydrogens on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. Bischofberger, Antiviral Research, 27 (1995) 1-17 which is incorporated by reference herein in its entirety. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect.

[0061] In one aspect, the artificial nucleoside can be provided as a 5′-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside, at the 5′-OH position of the nucleoside or lipophilic preparations, include U.S. Pat. No.5,149,794; U.S. Pat. No.5,194,654, U.S. Pat. No. 5,223,263; U.S. Pat. No. 5,256,641; U.S. Pat. No. 5,411,947; U.S. Pat. No.5,463,092; U.S. Pat. No.5,543,389; U.S. Pat. No.5,543,390; U.S. Pat. No. 5,543,391; and U.S. Pat. No.5,554,728, all of which are incorporated herein by reference.

[0062] The artificial nucleoside may be administered orally, parenterally, rectally, vaginally, buccally, sublingually, nasally, by inhalation, topically, transdermally, or via an implanted reservoir in dosage forms containing conventional non-toxic pharmaceutically acceptable carriers and excipients. The term “parenteral” as used herein is intended to include subcutaneous, intravenous, and intramuscular injection. The amount of the indolyl deoxyriboside analogs of formula administered can, of course, be a therapeutically effective amount and can be dependent on the particular active agent, the condition or disorder being treated, the severity of the condition or disorder, the subject's weight, the mode of administration and other pertinent factors known to the prescribing physician. Generally, however, dosage can be in the range of approximately 0.001 μg / mL / day to 100 μg / mL / day, more preferably in the range of about 0.1 μg / mL / day to 10 μg / ml / day.

[0063] Depending on the intended mode of administration, the pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, caplets, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature.

[0064] For those compounds that are orally active, oral dosage forms are generally preferred, and include tablets, capsules, caplets, solutions, suspensions and syrups, and may also comprise a plurality of granules, beads, powders or pellets that may or may not be encapsulated. Preferred oral dosage forms are tablets and capsules.

[0065] Tablets may be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred. In addition to the active agent, tablets can generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, coloring agents, and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact. Suitable binder materials include, but arenot limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose, and lactose), polyethylene glycol, waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, microcrystalline cellulose, ethyl cellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricants are used to facilitate tablet manufacture, promoting powder flow and preventing particle capping (i.e., particle breakage) when pressure is relieved. Useful lubricants are magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums, or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, and microcrystalline cellulose, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, and sorbitol. Stabilizers, as well known in the art, are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions.

[0066] Capsules are also preferred oral dosage forms, in which case the active agent- containing composition may be encapsulated in the form of a liquid or solid (including particulates such as granules, beads, powders or pellets). Suitable capsules may be either hard or soft, and are generally made of gelatin, starch, or a cellulosic material, with gelatin capsules preferred. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like.

[0067] Oral dosage forms, whether tablets, capsules, caplets, or particulates, may, if desired, be formulated so as to provide for gradual, sustained release of the active agent over an extended time period. Generally, as will be appreciated by those of ordinary skill in the art, sustained release dosage forms are formulated by dispersing the active agent within a matrix of a gradually hydrolyzable material such as an insoluble plastic (e.g., polyvinyl chloride or polyethylene), or a hydrophilic polymer, or by coating a solid, drug- containing dosage form with such a material. Hydrophilic polymers useful for providing a sustained release coating or matrix include, by way of example: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate; and vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, and ethylene-vinyl acetate copolymer.

[0068] Preparations for parenteral administration include sterile nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Parenteral formulations may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. The formulations are rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat. They can also be manufactured using a sterile injectable medium.

[0069] The compound may also be administered through the skin or mucosal tissue using conventional transdermal drug delivery systems, wherein the active agent is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Alternatively, the drug- containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. Transdermal drug delivery systems may in addition contain a skin permeation enhancer.

[0070] Although the present compounds can generally be administered orally, parenterally, or transdermally, other modes of administration are suitable as well. For example, administration may be rectal or vaginal, preferably using a suppository that contains, in addition to the active agent, excipients such cocoa butter or a suppository wax. Formulations for nasal or sublingual administration are also prepared with standardexcipients well known in the art. The pharmaceutical compositions may also be formulated for inhalation, e.g., as a solution in saline, as a dry powder, or as an aerosol.

[0071] The artificial nucleosides of the present disclosure can be combined with anti- microbial agents (e.g., β-lactams antibiotics) synergistically to generate an increased cytotoxic effect against antibiotic resistant bacteria and its adaptation through mutagenesis. This combination also affects the viability of bacterial persisters, thus providing a novel approach to combat drug resistance. Collectively, this unique drug combination provides a “first-in-class” approach to combat drug resistance in highly infectious microbial species, specifically targeting bacterial persistence. It should be understood that the artificial nucleoside(s) may be administered simultaneously or non- simultaneously with the antimicrobial agent(s). In simultaneous administration, the artificial nucleoside(s) and antimicrobial agent(s) may be administered in the same or different compositions. As a first non-limiting example, the antimicrobial agent (e.g., β- lactam antibiotic) is administered orally and the artificial nucleoside is injected. As a second non-limiting example, the artificial nucleoside and the antimicrobial agent are administered in the same pill, IV, or ointment.

[0072] The artificial nucleoside increases the potency and efficacy of β-lactam antibiotics. The artificial nucleoside is efficacious against β-lactam resistant bacteria. The artificial nucleoside adversely affects viability of microbial persisters.

[0073] The artificial nucleoside works in combination with existing antibiotics. The artificial nucleoside selectively targets the activity of a specific microbial DNA polymerase. The combination can prevent drug-resistance by interfering with mutagenic DNA replication.

[0074] One non-limiting example of an artificial nucleoside which may be used in the compositions and methods of the present disclosure is 5-nitro-indolyl-2'-deoxynucleoside (5-NIdR).

[0075] Additionally non-limiting examples of artificial nucleosides are illustrated below in formula (II)-(V).II); V);

[0076] wherein R is selected from the group consisting of selected from the group consisting of hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6- C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2- C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6- C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6- C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2- C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (- COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-N(C1- C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato(-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (- (CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5- C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH- (CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (- NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (- (SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5- C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof; and

[0077] whereinO, S, C, N, and any combination thereof (e.g., C-N, C-O, etc.). From left to right in Formula (V), -R-R- may be -O-S-; -O-C-; -O-N-; -S-O-; -S-C-; -S-N-; -C-O-; -C-S-; -C-N-; -N-O-; -N-S-; and / or -N-C-.

[0078] Non-limiting examples of antimicrobial agents include β-lactams, quinolone antibiotics, and other antibiotics that produce reactive oxygen species. The quinolone antibiotics may be fluoroquinolones such as ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, and ofloxacin.

[0079] The following examples are provided to illustrate the devices and methods of the present disclosure. The examples are merely illustrative and are not intended to limit the disclosure to the materials, conditions, or process parameters set forth therein. EXAMPLES

[0080] Experiments were conducted demonstrating that 5-NIdR can inhibit Pseudomonas aeruginosa proliferation and viability when used in combination with β-lactam antibiotics. FIG.3A-F show excellent inhibitory activity of 5-NIdR in checkerboard experiments in which variable concentrations of 5-NIdR are combined with variable concentrations of aztreonam / avibactam against clinical metallo-β-lactamase-producing carbapenem-resistant Pseudomonas aeruginosa strains (VIM-2 (FIG.3B), SPM-1 (FIG. 3C), IMP-1 (FIG.3A) , OXA-50 (FIG.3F)) which are resistant to nearly all anti-microbial agents, and wild type strains (ATCC 27853 (FIG.3E) and PA01 (FIG.3F)). In addition, these strains are resistant to nearly all commercially available β-lactams with only aztreonam / avibactam exhibiting minimal inhibitory concentrations (MICs) >8 µg / ml. 5- NIdR alone was not active, displaying individual MICs >512 µg / ml against these strains. However, when combined with β-lactams, 5-NIdR displays high potency, requiring concentrations as low as 0.5 µg / ml to achieve a >10-fold reduction in the MICs for aztreonam / avibactam against all Pseudomonas aeruginosa strains listed above.

[0081] Scanning Electron Microscopy (SEM) analysis was used to evaluate if combining 5-NIdR with antibiotics affected the viability of bacterial persisters. As previously illustrated in FIG. 2, Pseudomonas aeruginosa treated with aztreonam / avibactam show a subpopulation of pronounced long filamentous, non- replicating persisters that emerge as early as 2 hours post-treatment. These filaments are >30 times the length of the original rod population and become established by 6 hours post-treatment. These persisters were unculturable from 72 to 168 hours on agar plates. However, when antibiotic treatment was discontinued after 168 hours (7 days), the long filamentous persister cells recovered within 2 days (216 hours) (FIG.4). Furthermore, the response of long filaments following drug removal in reversion experiments (FIG. 4) showed that budding of the long filaments started within 1 hour and complete reversion to the rod population occurred within 11 hours (FIG.4). These findings suggest that the formation of filamentous persisters is a survival mechanism against treatment with β- lactam and β-lactamase combinations.

[0082] FIG.5 shows the results of experiments demonstrating that combining 5-NIdR with aztreonam / avibactam influences the viability of bacterial persisters. The data show that a subpopulation of long filamentous persisters emerge after 24 hours post-treatment (panel c). The addition of 5-NIdR does not appear to influence formation of persisters induced by antibiotic treatment (panel d). However, close inspection of the fluorescence(panel d) and SEM data (panel e) shows the formation of “ghost” apoptotic saccules which represent non-viable persisters that do not undergo reversion to generate highly infectious rod populations. Taken together, these data demonstrate that the artificial nucleoside, 5-NIdR, combats antibiotic drug resistance by two (2) independent mechanisms. First, the artificial nucleoside inhibits TLS activity in response to ROS generated by β-lactams, and this serves to enhance the cytotoxicity of this class of antibiotics. Secondly, 5-NIdR adversely affects the viability of filamentous persisters that arise due to the SOS response to ROS induces by β-lactams. These two features represent a novel approach to attack drug-resistant bacteria.

[0083] FIGS. 6A-D demonstrate the combination of 5-NIdR with aztreonam as a strategy to impede persistence (FIG.6D). Against wild type PA01 P. aeruginosa, SEMs showed that aztreonam monotherapy resulted in filamentous persisters (FIG. 6A). However, there was an increased proclivity of 5-NIdR combinations to result in pronounced blebbing in the outer membrane at the ends of the long filaments: blebbing has been shown to be an intermediate step toward cell apoptosis in E. coli. The isogenic PA01 ∆dinB mutant exposed to aztreonam alone also exhibited blebbing (FIG. 6B), consistent with a role for Pol IV in tolerating DNA damage that accumulates in persisters, similar to how E. coli Pol V acts in persisters, as compared with the isogenic wild type and the isogenic dinB overexpressor strain (FIG.6C).

[0084] It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

CLAIMS:

1. An antimicrobial composition comprising: an artificial nucleoside; and an antimicrobial agent.

2. The antimicrobial composition of claim 1, wherein the antimicrobial agent is a β-lactam antibiotic.

3. The antimicrobial composition of any one of claims 1 and 2, wherein the artificial nucleoside is 5-nitro-indolyl-2'-deoxynucleoside (5-NIdR).

4. The antimicrobial composition of any one of claims 1 and 2, wherein the artificial nucleoside is of formula (I): wherein Het is afrom the group consisting of:wherein R1 is OH, monophosphate (H2O3PO- or (O3PO-)2-), diphosphate (H3(O3PO)2- or ((O3PO)2-)3-), triphosphate (H4(O3PO)3-, ((O3PO)3-)4-), or ORa, where Ra comprises at least one of a straight chain, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, fatty acid residue, or a mono, di, or triphosphate thereof;wherein R4, R5, R6, R7, R9, R10, R11, R12, R15, R16, and R18, each independently represent substituents selected from the group consisting of hydrogen, C1-C24 alkyl, C2- C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (- O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6- C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1- C24 alkyl)-substituted carbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2- C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2- aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof, and further wherein any two adjacent (ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six-membered rings, and fused five-membered and / or six- membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogen substituents and zero to 3 heteroatoms; and with the proviso that at least one of R4, R5, R6, R7, R9, R10, R11, R12, R15, and R18 is other than hydrogen and that where R9 is amino R10 is other than hydrogen; andwherein at least one of R2, R3, R8, R13, R14, R17, and R19 is independently selected from hydrogen and a click-reactive functional group that is directly or indirectly bound to the purine or indole ring of the compound.

5. A treatment method comprising: administering an artificial nucleoside to a patient; and administering an antimicrobial agent to the patient.

6. The treatment method of claim 5, wherein the artificial nucleoside and the antimicrobial agent are administered to the patient simultaneously.

7. The treatment method of any one of claims 5 and 6, wherein the artificial nucleoside and the antimicrobial agent are administered in the same composition.

8. The treatment method of any one of claims 5-7, wherein the antimicrobial agent is a β-lactam antibiotic.

9. The treatment method of any one of claims 5-8, wherein the artificial nucleoside is 5-nitro-indolyl-2'-deoxynucleoside (5-NIdR).

10. The treatment method of any one of claims 5-8, wherein the artificial nucleoside is of formula (I): wherein Het is afrom the group consisting of:wherein R1 is O3PO-)2-), diphosphate (H3(O3PO)2- or ((O3PO)2-)3-), triphosphate (H4(O3PO)3-, ((O3PO)3-)4-), or ORa, where Ra comprises at least one of a straight chain, branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, arylsulfonyl, aralkylsulfonyl, amino acid residue, fatty acid residue, or a mono, di, or triphosphate thereof; wherein R4, R5, R6, R7, R9, R10, R11, R12, R15, and R18, each independently represent substituents selected from the group consisting of hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (- O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6- C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1- C24 alkyl)-substituted carbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2- C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2- aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof, and further wherein any two adjacent (ortho) substituents may be linked to form a cyclic structure selected from five-membered rings, six-membered rings, and fused five-membered and / or six- membered rings, wherein the cyclic structure is aromatic, alicyclic, heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogen substituents and zero to 3 heteroatoms; and with the proviso that at least one of R4, R5, R6, R7, R9, R10, R11, R12, R15, and R18 is other than hydrogen and that where R9 is amino R10 is other than hydrogen; and wherein at least one of R2, R3, R8, R13, R14, R17, and R19 is independently selected from hydrogen and a click-reactive functional group that is directly or indirectly bound to the purine or indole ring of the compound.

11. The treatment method of any one of claims 5-8, wherein the artificial nucleoside is selected from the group consisting of: whereinof hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6- C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (- CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6- C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2- C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (- COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-N(C1- C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (- (CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5- C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (- NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (- (SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5- C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof.

12. The method of any one of claims 5-8, wherein the artificial nucleoside is: ;wherein each R is group of O, S, C, and N.

13. The method of any one of claim 5-12, wherein the antimicrobial agent is administered orally and the artificial nucleoside is administered via an injection.

14. An artificial nucleoside selected from the group consisting of:nd wherein nsisting of hydrogen, C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl, C3-C20 aryl, C6-C24 alkaryl, C6- C24 aralkyl, halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (- CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6- C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2- C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (- COOH), carboxylato (-COO−), carbamoyl (-(CO)-NH2), mono-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(C1-C24 alkyl)-substituted carbamoyl (-(CO)-N(C1- C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano(-CN), isocyano (-N+C−), cyanato (-O-CN), isocyanato (-O-N+═C−), isothiocyanato (-S-CN), azido (-N═N+═N−), formyl (-(CO)-H), thioformyl (- (CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5- C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH- (CO)-aryl), imino (-CR═NH where R is hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), alkylimino (-CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (- NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl;also termed “alkylthio”), arylsulfanyl (-S-aryl; also termed “arylthio”), C1-C24 alkylsulfinyl (- (SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5- C20 arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O-)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), and combinations thereof.

15. An antimicrobial composition comprising: the artificial nucleoside of claim 14; and an antimicrobial agent.

16. The antimicrobial composition of claim 15, wherein the antimicrobial agent is a β-lactam antibiotic.

17. A treatment method comprising: administering the antimicrobial composition of any one of claims 15 and 16 to a patient.

18. An artificial nucleoside of the formula: ;wherein each R is independently selected from the group consisting of O, S, C, and N.

19. An antimicrobial composition comprising: the artificial nucleoside of claim 18; and an antimicrobial agent.

20. The antimicrobial composition of claim 19, wherein the antimicrobial agent is a β-lactam antibiotic.

21. A treatment method comprising: administering the antimicrobial composition of any one of claims 19 and 20tient.

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