Modified cholic acid conjugates

Abstract

The present disclosure provides compounds, compositions, and methods wherein the compounds comprise a modified cholic acid or cholic acid component and at least a second component wherein the second component is conjugated to the modified cholic acid or cholic acid, the compounds having the structure: # imgabs0 #, in some instances, as compared to a non-conjugated component alone, the conjugate compounds provide reduced cytotoxicity, greater efficacy, and more robust MOA (e.g., easier entry into cells or multiple modes of action occur). In some cases, the second component, such as biotin, is covalently linked to the modified cholic acid or cholic acid.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 406,667, filed on September 14, 2022, the content of which is incorporated herein by reference for all purposes. Background of the Invention

[0003] Broad - spectrum antimicrobials are compounds that treat a range of bacteria, including Gram - positive and Gram - negative bacteria, as well as a variety of infections. Antimicrobials that treat bacteria, viruses, and fungi are scarce.

[0004] Cholic acid is synthesized from cholesterol in the liver and is a primary bile acid. Cholic acid is hydroxylated at C3, C7, and C12 and promotes fat absorption and cholesterol excretion. The U.S. FDA has approved cholic acid formulated as Cholbam capsules for the treatment of children and adults with bile acid synthesis disorders due to single - enzyme defects.

[0005] WO 2018232502 teaches transfection reagents for delivering nucleic acids, which comprise lipids, e.g., cholic acid or deoxycholic acid conjugated to a polymer such as polyethyleneimine with a molecular weight up to 2.0 kilodaltons (kDa).

[0006] US20180050048 teaches conjugate compounds having a moiety derived from, for example, ursodeoxycholic acid and at least one moiety derived from, for example, berberine, or L - carnitine, or metformin, etc. This application relates to pharmaceutical compositions, methods for preparing these conjugates, and uses for treating various conditions, diabetes, diabetic complications, dyslipidemia, obesity, metabolic syndrome, and other indications.

[0007] US20050239204 teaches multifunctional molecular complexes for transferring nucleic acid compositions to target cells, which comprise: 1) a nucleic acid composition; 2) one or more cationic polyamine components bound to the nucleic acid composition, each of which contains 3 to 12 nitrogen atoms; 3) one or more endosome - membrane - disrupting promoting components, which include a) at least one lipophilic long - chain alkyl group, b) a fusion peptide, or c) cholic acid or cholestenyl or derivatives; and optionally present 4) one or more receptor - specific binding components, which are ligands of natural receptors of the target cells.

[0008] In view of the foregoing, there is a need for new broad - spectrum antimicrobials that are effective against bacteria, viruses, and fungi to explore and improve therapeutic effects for a range of purposes, such as improving the stability of drug aqueous solutions, triggering drug release for drug delivery, improving drug bioavailability and humoral circulation time, avoiding drug degradation failures, and improving efficacy. This disclosure also meets these needs and provides other advantages. Summary of the Invention

[0009] The present disclosure provides compounds, compositions, and methods, wherein the compounds comprise a first component of a bile acid or a modified bile acid (MCA) and a second component linked thereto, and the second component comprises biotin, desthiobiotin, or a biotin mimetic. Accordingly, the present invention includes bile acids linked to biotin, desthiobiotin, or a biotin mimetic, and similarly modified bile acids (MCAs) linked to biotin, desthiobiotin, or a biotin mimetic.

[0010] In certain cases, the conjugate compound provides reduced cytotoxicity in use relative to the use of the modified bile acid component alone. In certain cases, the conjugate compound has a more robust mode of action (MOA), e.g., multiple modes of action, which facilitate entry into cells. In certain cases, the second component is covalently bonded or linked to the modified bile acid. In certain aspects, the second component is biotin, or a biotin moiety. In certain cases, the conjugate compound provides comparable or higher efficacy in therapeutic treatment relative to treatment with unconjugated MCA.

[0011] Accordingly, in one embodiment, the present disclosure provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0012]

[0013] Wherein:

[0014] R 1 -R 4 、R 6 、R 7 、R 11 、R 12 、R 15 and R 16 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10)aminoalkylcarboxyl, substituted or unsubstituted (C1-C 10 )aminoalkylaminocarbonyl, substituted or unsubstituted (C1-C 10 )aminoalkylcarboxamido, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 )guanidinoalkoxy and (C1-C 10 )guanidinoalkylcarboxyl;

[0015] R 5 , R 8 , R 9 , R 10 , R 13 and R 14 Each is independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 )alkyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 )alkoxy-(C1-C 10 )alkyl, substituted or unsubstituted (C1-C 10 )aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 )aminoalkoxy, substituted or unsubstituted (C1-C 10 )aminoalkylcarboxyl, substituted or unsubstituted (C1-C 10 )aminoalkylaminocarbonyl, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 )guanidinoalkoxy and (C1-C 10 )guanidinoalkylcarboxyl;

[0016] L 1 is a linking group; and

[0017] B 1 is biotin, biotin, desthiobiotin, or a biotin mimetic.

[0018] In another embodiment, the present disclosure provides a method for treating a microbial infection or delaying the spread of a microbial infection, the method comprising:

[0019] The microorganism is contacted with an antimicrobial amount of a compound having Formula I or a pharmaceutically acceptable salt thereof:

[0020]

[0021] in:

[0022] R 1 -R 4 、R 6 、R 7 、R 11 、R 12 、R 15 and R 16 each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl formamido, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl;

[0023] R 5 、R 8 、R 9 、R 10 、R 13 and R 14 each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo group, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl;

[0024] L 1 is a linking group; and

[0025] B 1 is biotin or a biotin moiety, wherein the compound of formula I kills the microorganism or slows the spread of the microorganism. In some cases, the compounds of the present disclosure act as immunomodulators that can stimulate or inhibit the immune system.

[0026] These and other aspects, objects, and embodiments will become more apparent when reading the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shows the cytotoxicity of different CSA-44 concentrations (modified cholic acid) in human foreskin fibroblasts (hFF).

[0028] Figure 2 Shows the cytotoxicity of different concentrations of embodiments of the present disclosure in human foreskin fibroblasts (hFF).

[0029] Figure 3 Shows that embodiments of the present disclosure have an IC of 154 μM in Cal-u3 cells 50 .

[0030] Figure 4 Shows the protease inhibitory activity of embodiments of the present disclosure.

[0031] Figure 5A -B shows the antiviral activity of embodiments of the present disclosure. Figure 5A Shows the reduction of virus strain WA in the presence of the compounds of the present disclosure, and Figure 5B shows the reduction of virus strain BA.5 in the presence of the compounds of the present disclosure.

[0032] Figure 6 Shows the in vivo levels of virus in the lung tissues of the control group (PBS) and the group administered with embodiments of the present disclosure.

[0033] Figures 7A-H show imaging experiments of H&E staining. Figure 7A - B Showing lung tissue with normal morphology from uninfected mice; Figure 7C - D Showing uninfected tissue treated with an embodiment of the present disclosure, which shows normal lung morphology; Figure 7E - F Showing infected tissue treated with PBS, which exhibits structural changes in the lung; Figure 7G - H Showing infected tissue treated with an embodiment of the present disclosure, which shows normal lung morphology.

[0034] Figure 8A -B shows a comparison of cytokine levels between the control group (infected but untreated) and the infected group treated with an embodiment of the present disclosure ( Figure 8A ). Figure 8B Showing a comparison of cytokine levels between the uninfected and infected groups treated with an embodiment of the present disclosure. Detailed Description

[0035] I. Definition

[0036] As used herein, "active ester" includes carboxyl group derivatives (such as NHS ester, sulfo-NHS ester, PAM ester or halophenyl ester) that are more prone to displacement by nucleophilic addition and elimination than an ethyl ester group. Representative carbonyl substituents of the active ester include succinimidyloxy (-OC4H4NO2), sulfosuccinimidyloxy (-OC4H3NO2SO3H), -1-oxobenzotriazolyl (-OC6H4N3); 4-sulfo-2,3,5,6-tetrafluorophenyl; or an aryloxy group, which is optionally substituted one or more times by an electron-withdrawing substituent such as nitro, fluorine, chlorine, cyano, trifluoromethyl or a combination thereof (such as pentafluorophenyloxy or 2,3,5,6-tetrafluorophenyloxy). Preferred active esters include succinimidyloxy esters, sulfosuccinimidyloxy esters and 2,3,5,6-tetrafluorophenyloxy esters.

[0037] As used herein, "acyl" includes the alkanoyl, aroyl, heterocyclic acyl or heteroaroyl groups as defined herein. Representative acyl groups include acetyl, benzoyl, nicotinoyl, etc.

[0038] As used herein, "alkanoyl" includes an alkyl-C(O)- group, wherein the alkyl group is as defined herein. Representative alkanoyl groups include acetyl, ethanoyl, valeryl, hexanoyl, etc.

[0039] As used herein, "alkenyl" includes straight-chain or branched-chain aliphatic hydrocarbon groups having from 2 to about 15 carbon atoms and containing at least one carbon-carbon double or triple bond. Preferred alkenyl groups have from 2 to about 12 carbon atoms. More preferred alkenyl groups contain from 2 to about 6 carbon atoms. In one aspect, hydrocarbon groups containing a carbon-carbon double bond are preferred. In a second aspect, hydrocarbon groups containing a carbon-carbon triple bond (i.e., alkynyl) are preferred. As used herein, "lower alkenyl" includes alkenyl groups having from 2 to about 6 carbon atoms. Representative alkenyl groups include vinyl, allyl, n-butenyl, 2-butenyl, 3-methylbutenyl, n-pentenyl, heptenyl, octenyl, decenyl, propynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, and the like. The alkenyl groups can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the alkenyl group can be independently replaced in part by groups selected from the following: fluorine, hydroxy, alkoxy, amino, alkylamino, acylamino, mercapto (thio), and alkylthio.

[0040] As used herein, "alkenylene" includes straight-chain or branched-chain divalent hydrocarbon chains containing at least one carbon-carbon double or triple bond. Preferred alkenylene groups contain from 2 to about 12 carbons in the chain, and more preferred alkenylene groups contain from 2 to 6 carbons in the chain. In one aspect, hydrocarbon groups containing a carbon-carbon double bond are preferred. In a second aspect, hydrocarbon groups containing a carbon-carbon triple bond are preferred. Representative alkenylene groups include -CH=CH-, -CH2-CH=CH-, -C(CH3)=CH-, -CH2CH=CHCH2-, ethynylene, propynylene, n-butynylene, and the like.

[0041] As used herein, "alkoxy" includes an alkyl-O- group, where the alkyl group is as defined herein. Representative alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, heptyloxy, and the like. The alkoxy groups can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the alkoxy group can be independently replaced in part by groups selected from the following: fluorine, hydroxy, alkoxy, amino, alkylamino, acylamino, mercapto, and alkylthio.

[0042] As used herein, "alkoxyalkyl" includes an alkyl-O-alkylene- group, where alkyl and alkylene are as defined herein. Representative alkoxyalkyl groups include methoxyethyl, ethoxymethyl, n-butoxymethyl, and cyclopentylmethoxyethyl.

[0043] As used herein, "alkoxycarbonyl" includes an ester group; i.e., an alkyl-O-CO- group, where the alkyl group is as defined herein. Representative alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, and the like.

[0044] As used herein, "alkoxycarbonylalkyl" includes an alkyl-O-CO-alkylene-group, wherein the alkyl and alkylene are as defined herein. Representative alkoxycarbonylalkyl groups include methoxycarbonylmethyl, ethoxycarbonylmethyl, methoxycarbonylethyl, and the like.

[0045] As used herein, "alkyl" includes aliphatic hydrocarbon groups which may be straight-chain or branched-chain and have from about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups have 1 to about 12 carbon atoms in the chain. More preferred alkyl groups have 1 to 6 carbon atoms in the chain. As used herein, "branched-chain" includes: one or more lower alkyl groups such as methyl, ethyl or propyl groups attached to a straight-chain alkyl chain. As used herein, "lower alkyl" contains 1 to about 6 carbon atoms in the chain, preferably 5 or 6 carbon atoms, and may be straight-chain or branched-chain. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl and 3-pentyl. In some cases, a bond on the backbone such as without an attached substituent indicates a methyl group attached thereto.

[0046] Alkyl groups may be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the alkyl group may be independently replaced in part by groups selected from the following: halo such as chlorine, fluorine, bromine or iodine, hydroxy, alkoxy, amino, alkylamino, acylamino, mercapto and alkylthio. As used herein, "alkylene" contains a straight-chain or branched-chain divalent hydrocarbon chain having 1 to about 6 carbon atoms. Preferred alkylene groups are lower alkylene groups having 1 to about 4 carbon atoms. Representative alkylene groups include methylene, ethylene, and the like.

[0047] As used herein, "alkylamino" includes an alkyl-NRR'-group, wherein the alkyl group is as defined herein. Alkylamino groups are those in which the alkyl is a C1-C 10 alkyl group. Representative alkylamino groups include methylamino, ethylamino, isopropylamino, heptylamino, and the like. The amino group may be a substituted amino group. The NRR' group, wherein R and R' are independently selected from H and alkyl.

[0048] As used herein, "alkylcarboxy" and "alkylcarboxyl" include RC(O)O- groups (i.e., esters). Representative alkylcarboxy groups include methylcarboxy, ethylcarboxy, tert-butylcarboxy, and the like. In some cases, the alkyl group is a lower alkyl group containing from 1 to about 6 carbon atoms in the chain, preferably 5 or 6 carbon atoms, which may be straight-chain or branched-chain. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, and 3-pentyl. The alkyl group may be substituted as defined herein.

[0049] As used herein, "alkylformamido" includes an alkyl-C(O)-NR’R”- group, where the alkyl group is as defined herein. The group of alkyl-C(O)-NRR’ is one in which the alkyl group is C1-C 10 those of alkyl. Representative alkyl-C(O)-NH- groups include methyl-C(O)-amino, ethyl-C(O)-amino, isopropyl-C(O)-amino, heptyl-C(O)-amino, and the like. The NRR’ group, where R and R’ are independently selected from H and C1-C 10 alkyl. More preferably, at least one of R and R’ is H.

[0050] As used herein, "alkylheterocyclyl-alkylformamido" includes an alkyl-heterocyclyl-alkyl-C(O)-NRR’ group. The heterocyclyl group may be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms of the group (e.g., 1 to 4, 1 to 2, or 1) may be independently replaced by moieties selected from the following: fluorine, hydroxy, alkoxy, amino, alkylamino, acylamino, mercapto, and alkylthio. In some aspects, the substituted heterocyclyl group may include exocyclic or endocyclic olefins (e.g., cyclohex-2-en-1-yl). In some aspects, the heterocyclyl group is unsubstituted or non-optionally substituted. The heterocyclyl group may be aromatic or non-aromatic.

[0051] As used herein, "alkylthio" includes an alkyl-S- group, where the alkyl group is as defined herein. Preferred alkylthio groups are those in which the alkyl group is a lower alkyl. Representative alkylthio groups include methylthio, ethylthio, isopropylthio, heptylthio, and the like.

[0052] As used herein, "alkylthioalkyl" includes an alkylthio-alkylene- group, where alkylthio and alkylene are defined herein. Representative alkylthioalkyl groups include methylthiomethyl, ethylthiopropyl, isopropylthioethyl, and the like.

[0053] As used herein, "amido" includes a group of the formula Y1Y2N-C(O)-, where Y1 and Y2 are independently hydrogen, alkyl, or alkenyl; or Y1 and Y2, together with the nitrogen to which they are attached, form a 4- to 7-membered nitrogen heterocyclic group (e.g., piperidyl). Representative amido groups include primary amido (H2N-C(O)-), methylamido, dimethylamido, diethylamido, etc. Preferably, "amido" is a -C(O)NRR' group, where R and R' are independently selected from H and alkyl. More preferably, at least one of R and R' is H.

[0054] As used herein, "amidoalkyl" includes an amido-alkylene- group, where amido and alkylene are defined herein. Representative amidoalkyl groups include amidomethyl, amidoethylene, dimethylamidomethyl, etc.

[0055] As used herein, "amino" includes a group of the formula Y1Y2N-, where Y1 and Y2 are independently hydrogen, acyl, or alkyl; or Y1 and Y2, together with the nitrogen to which they are attached, form a 4- to 7-membered nitrogen heterocyclic group (e.g., piperidyl). Optionally, when Y1 and Y2 are independently hydrogen or alkyl, an additional substituent may be added to the nitrogen to form a quaternary ammonium ion. Representative amino groups include primary amino (H2N-), methylamino, dimethylamino, diethylamino, etc. Preferably, "amino" is a -NRR' group, where R and R' are independently selected from H and alkyl. Preferably, at least one of R and R' is H. In some cases, the amino group may be a cationic amine salt, such as -NH3 + Cl - 。

[0056] As used herein, "aminoalkyl" includes an amino-alkylene- group, where amino and alkylene are defined herein. Representative aminoalkyl groups include aminomethyl, aminoethyl, dimethylaminomethyl, etc.

[0057] As used herein, "aroyl" includes an aryl-CO- group, where aryl is defined herein. Representative aroyl groups include benzoyl, naphthalene-1-carbonyl, and naphthalene-2-carbonyl.

[0058] As used herein, "aroylalkyl" includes an aryl-CO-alkyl group, where aryl is defined herein. Representative aroylalkyl groups include benzoylalkyl, naphthalene-1-carbonyl-alkyl, and naphthalene-2-carbonyl-alkyl.

[0059] As used herein, "aryl" includes an aromatic monocyclic or polycyclic ring system having 6 to about 14 carbon atoms, preferably 6 to about 10 carbon atoms. Representative aryl groups include phenyl and naphthyl.

[0060] As used herein, the term "arylalkyl" or "aralkyl" includes an alkyl group as defined herein, wherein at least one hydrogen substituent is replaced by an aryl group as defined herein. Examples include but are not limited to benzyl, 1-phenylethyl, 4-methylbenzyl, and 1,1-dimethyl-1-phenylmethyl. The arylalkyl or aralkyl group may be unsubstituted or optionally substituted according to its constituent groups. For example but not limited to, the aryl group of the arylalkyl group may be substituted, such as in 4-methylbenzyl, 2,4,6-trimethylbenzyl, 4-tert-butylbenzyl, 4-isopropylbenzyl, etc. In some aspects, the group is unsubstituted or non-optionally substituted, especially if it includes defined substituents such as hydroxyalkyl or alkylaminoalkoxy groups.

[0061] As used herein, "aromatic ring" includes 5- to 12-membered aromatic monocyclic or fused polycyclic moieties, which may include from 0 to 4 heteroatoms selected from oxygen, sulfur, selenium, and nitrogen. Exemplary aromatic rings include benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, benzothiazoline, benzothiophene, benzofuran, indole, benzindole, quinoline, etc. The aromatic ring group may be substituted at one or more positions with halo, alkyl, alkoxy, alkoxycarbonyl, haloalkyl, cyano, sulfonato, aminosulfonyl, aryl, sulfonyl, aminocarbonyl, carboxyl, acylamino, alkylsulfonyl, amino, and substituted or unsubstituted substituents.

[0062] As used herein, "B 1"Or a biotin moiety" includes biotin (CAS 58-85-5), desthiobiotin (CAS 533-48-2), and biotin mimetics. Generally, a biotin mimetic is a peptide that binds streptavidin, or avidin, or neutravidin. Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles, and amphibians. Avidin-binding protein contains four identical subunits with a combined mass of 67,000 - 68,000 daltons. Removal of glycosylation from avidin yields neutravidin with a mass of 60,000 daltons. In some cases, a biotin mimetic contains the His-Pro-Gln (HPQ) tripeptide or the HPQN sequence (see Devlin, J.J.; Panganiban, L.C.; Devlin, P.E. Random peptide libraries: A source of specific protein binding molecules. Science 1990, 249, 404 - 406 or Luo et al., 1998 J. Biotechnol. 65:225 and references cited therein). A biotin mimetic can be from about 3 to about 20 amino acids. In some cases, a biotin mimetic is a moiety that binds a receptor that similarly binds biotin. In some cases, a biotin mimetic is any compound capable of binding avidin or streptavidin.

[0063] As used herein, "carboxy" and "carboxyl" include the HOC(O)- group (i.e., carboxylic acid) or its salts.

[0064] As used herein, "carboxyalkyl" includes the HOC(O)-alkylene- group, where alkylene is defined herein. Representative carboxyalkyls include carboxymethyl (i.e., HOC(O)CH2-) and carboxyethyl (i.e., HOC(O)CH2CH2-).

[0065] As used herein, a modified cholic acid ("MCA") conjugate is a synthetically produced chemical compound that contains the backbone of cholic acid, chenodeoxycholic acid, deoxycholic acid, or lithocholic acid, having the A - D fused ring system of formula I, where R 1 to R 16 , L 1 and B 1 are defined herein, and which can contain various charged groups (e.g., amines and cationic groups) attached to the backbone. The MCA conjugate has an appended B 1 , which includes biotin, biotin, desthiobiotin, and biotin mimetics, covalently linked to the backbone through a linking group. The MCA conjugate has antimicrobial properties.

[0066] As used herein, "cycloalkyl" includes non-aromatic monocyclic or polycyclic ring systems having from about 3 to about 10 carbon atoms, preferably from about 5 to about 10 carbon atoms. More preferably, the cycloalkyl ring contains 5 or 6 ring atoms. The cycloalkyl group optionally contains at least one sp 2 hybridized carbon (e.g., a ring containing an intra- or extra-ring olefin). Representative monocyclic cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, etc. Representative polycyclic cycloalkyls include 1-decalin, norbornyl, adamantyl, etc.

[0067] As used herein, "cycloalkylene" includes divalent cycloalkyl having from about 4 to about 8 carbon atoms. Preferred cycloalkylene groups include 1,2-, 1,3- or 1,4-cis- or trans-cyclooctylene.

[0068] The terms "condition", "disease" and "disorder" are used interchangeably herein to refer to the condition of an individual. A condition is an impairment or disorder that affects the normal functioning of the body and is typically the result of a bacterial, fungal or viral infection in an individual. A disease is a pathological condition of an organ, body part or system caused by various factors such as infection, genetic defect (e.g., epidermolysis bullosa (EB) is the name given to a rare group of genetic skin disorders that cause the skin to become very fragile. Any trauma or friction to the skin can cause painful blisters) or environmental stress and is characterized by a recognizable group of symptoms. A condition or disease can refer to a biofilm-related condition or a condition caused by a planktonic bacterial phenotype, which is characterized by bacterial growth associated with the disease. In some cases, a disease or condition is a physiological response in which the immune system causes the release of pro-inflammatory molecules that damage the body. Other diseases and conditions include respiratory diseases, reproductive disorders and skin conditions.

[0069] As used herein, the term "effective amount" or "effective dose" includes an amount sufficient to achieve the desired result and will thus depend on the component and the desired result. Nevertheless, once the desired effect has been identified, determining an effective amount is within the skill of those in the art.

[0070] As used herein, "halo" or "halogen" includes fluorine, chlorine, bromine or iodine.

[0071] As used herein, "heteroatom" includes atoms other than carbon or hydrogen. Representative heteroatoms include O, S, and N. The nitrogen or sulfur atom of the heteroatom is optionally oxidized to the corresponding N-oxide, S-oxide (sulfoxide), or S-dioxide (sulfone). In a preferred aspect, the heteroatom has at least two bonds to an alkylene carbon atom (e.g., -C1-C9 alkylene-O-C1-C9 alkylene-). In some embodiments, the heteroatom is further substituted with an acyl, alkyl, aryl, cycloalkyl, heterocyclic, or heteroaryl group (e.g., -N(Me)-; -N(Ac)-).

[0072] As used herein, "heteroaroyl" includes a heteroaryl-C(O)- group, wherein the heteroaryl is as defined herein. Representative heteroaroyl groups include thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, picolinoyl, and the like.

[0073] As used herein, "heterocycloyl" includes a heterocyclic-C(O)- group, wherein the heterocyclic is as defined herein. Representative heterocycloyl groups include N-methyl prolinoyl, tetrahydrofuranoyl, and the like.

[0074] As used herein, "hydroxyalkyl" includes an alkyl group as defined herein that is substituted with one or more hydroxy groups. Preferred hydroxyalkyls contain lower alkyls. Representative hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

[0075] As used herein, "heterocyclic" includes an aromatic or non-aromatic monocyclic or polycyclic ring system having from about 3 to about 10 ring atoms, preferably from about 5 to about 10 ring atoms, wherein one or more atoms in the ring system are one or more elements other than carbon, such as nitrogen, oxygen, or sulfur. Preferred heterocyclic groups contain from about 5 to about 6 ring atoms. The heterocyclic group optionally contains at least one sp 2Hybrid atoms (e.g., a ring containing a carbonyl group, an internal olefin or an external olefin). The prefixes "aza-", "oxa-" or "thia-" before a heterocyclic group indicate the presence of at least one nitrogen, oxygen or sulfur atom, respectively, as a ring atom. The nitrogen atom or sulfur atom of the heterocyclic group is optionally oxidized to the corresponding N-oxide, S-oxide or S-dioxide. Representative non-aromatic monocyclic heterocyclic groups include piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydrothiopyranyl, etc. Representative aromatic heterocyclic groups include pyrazinyl, furyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidinyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzazaindolyl, 1,2,4-triazinyl, benzothiazolyl, etc.

[0076] The heterocyclic group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the group can be independently replaced in part by a group selected from the following: fluorine, hydroxy, alkoxy, amino, alkylamino, acylamino, mercapto, and alkylthio. In some aspects, the substituted heterocyclic group can include an external olefin or an internal olefin (e.g., cyclohex-2-en-1-yl). In some aspects, the heterocyclic group is unsubstituted or non-optionally substituted.

[0077] As used herein, the term "hydrophobic moiety" or "hydrophobic group" includes a moiety or functional group that repels water. Examples can include, but are not limited to: a non-polar alkyl moiety, such as an unsubstituted alkyl group having more than 5 carbons; a phenyl group; and an anthracenyl group.

[0078] As used herein, the term "hydrophilic moiety" or "hydrophilic group" includes a moiety or functional group that has a strong affinity for water. Examples can include, but are not limited to, a charged moiety, such as a cationic moiety or anionic moiety, or a polar uncharged moiety, such as an alkoxy group or an amine group.

[0079] As used herein, the term "pharmaceutically acceptable salt" is generally formed from an acid that forms a non-toxic acid anion, such as hydrochloride, hydrobromide, sulfate, phosphate or acid phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate or gluconate. Salt formation can be used to increase solubility and thus increase the dissolution rate of the drug. Hydrochloride, mesylate, hydrobromide, acetate and fumarate are the conventional counterions for basic chemical entities, while sodium, calcium and potassium are the counterions for weakly acidic drugs.

[0080] As used herein, the terms "treat", "treating" or "treatment" include administering or applying a composition (e.g., a composition described herein) in an amount, manner (e.g., dosing regimen) and mode (e.g., route of administration) effective to ameliorate a disorder or its symptoms, or to prevent, delay or slow the progression of a disorder or its symptoms. Such amelioration can include, but is not limited to, alleviating or improving one or more symptoms or conditions, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, preventing the spread or dissemination of the disease, delaying or slowing the progression of the disease, improving or alleviating the disease state, reducing disease recurrence, and remission, whether partial or complete and whether detectable or undetectable.

[0081] A "water-solubilizing group" is a group that imparts more hydrophilicity to the modified bile acid component. The water-solubilizing group can be an ethylene oxide oligomer. In certain aspects, the water-solubilizing group includes one or more alkylene oxide repeating units. For example, the water-solubilizing group can contain one or more ethylene glycol units -(OCH2CH2) n -. The PEG group can be of any length, however, it generally includes from 1 to 20 ethylene glycol repeating units where n is 1 to 20. Other PEG derivatives such as CH3(OCH2CH2) n CO or CH3(OCH2CH2) n can be used, where n = 1 to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or n is from 4 to 8; alternatively, HOOCCH2CH2CO-, HOOCCH2CH2- or CH3CO- can be used. Those skilled in the art know other groups that impart more water solubility to the molecule.

[0082] II. Embodiment

[0083] The present disclosure provides compounds, compositions, and methods, where the compounds comprise a modified bile acid or bile acid moiety and at least a second moiety, where the second moiety is conjugated to the modified bile acid or bile acid moiety. In certain cases, it has surprisingly been found that, relative to using the modified bile acid moiety alone, the conjugate compounds provide reduced cytotoxicity in use. In certain cases, it has surprisingly been found that, relative to using the modified bile acid moiety alone, the conjugate compounds provide a more robust MOA (e.g., easier entry into cells or multiple modes of action) and increased efficacy in use. In certain aspects, the molecule conjugated to the modified bile acid is a biotin moiety as defined herein, which is attached to a linker defined as L. As used herein, conjugation means covalent attachment of the bile acid or modified bile acid to the biotin moiety. B 1 Or the biotin moiety includes biotin, desthiobiotin, and biotin mimetics.

[0084] The compositions according to the present disclosure can be formulated in any therapeutically acceptable manner, as described in more detail below. In addition, relative to treatment with unconjugated MCA added, the compositions and methods according to the present disclosure provide comparable efficacy in therapeutic treatment while also providing reduced cytotoxicity.

[0085] In certain aspects, the present disclosure provides bile acid conjugates, the modified compounds having beneficial effects in vivo (e.g., antimicrobial activity and / or immunomodulatory activity), but having reduced cytotoxicity when the compound is exposed to infected cells or cells in need of protection from infection. In certain cases, it has been observed that when the conjugate compound is exposed to infected cells or cells in need of protection from infection (e.g., prophylaxis), the compound provides greater efficacy and a more robust MOA (e.g., easier entry into cells or multiple modes of action).

[0086] In certain aspects, the modified bile acid conjugate comprises a modified bile acid and a second compound, where the second compound comprises or is selected from a group of compounds such that when the conjugate so formed is exposed to a desired target, a desired therapeutic result is obtained. In certain cases, relative to the degree of cytotoxicity that occurs when the modified bile acid compound in its unconjugated form is exposed to the same target, the cytotoxicity at the target site is reduced. In certain cases, when exposed to the same target, the MOA at the target site is more robust relative to the modified bile acid compound in its unconjugated form.

[0087] In one embodiment, the present disclosure provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0088]

[0089] Wherein:

[0090] R 1 -R4 , R 6 , R 7 , R 11 , R 12 , R 15 and R 16 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl formamido, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl;

[0091] R 5 , R 8 , R 9 , R 10 , R 13 and R 14 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C10 ) aminoalkylamino-carbonyl, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkylcarboxy;

[0092] L 1 is a linking group; and

[0093] B 1 is biotin, desthiobiotin or a biotin mimic.

[0094] In certain aspects, R 3 , R 7 and R 12 are each independently selected from: substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkylcarboxy, substituted or unsubstituted (C1-C 10 ) aminoalkylamino-carbonyl and substituted or unsubstituted (C1-C 10 ) aminoalkylformamido; and R 5 , R 9 and R 13 are each independently selected from: hydrogen, hydroxy and substituted or unsubstituted (C1-C 10 ) alkyl.

[0095] In certain aspects, R 3 , R 7 and R 12 are each a substituted or unsubstituted (C1-C 10 ) aminoalkylcarboxy, wherein the amino is optionally quaternized; and R 5 , R 9 and R 13 are each independently hydrogen or a substituted or unsubstituted (C1-C3) alkyl.

[0096] In certain aspects, L 1 is -L-Y-Z, wherein L is independently selected from: a bond, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkylcarboxy, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10)aminoalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo group, substituted or unsubstituted (C1-C 10 )aminoalkoxy, substituted or unsubstituted (C1-C 10 )aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 )alkylformamido, substituted or unsubstituted (C1-C 10 )alkylformamido (wherein the amino group is substituted), and substituted or unsubstituted (C1-C 10 )alkylaminocarbonyl;

[0097] Y is optionally present and is selected from: substituted or unsubstituted (C1-C 10 )alkyl heterocyclic group (C1-C 10 )alkylformamido, substituted or unsubstituted (C1-C 10 )alkyl heterocyclic group, substituted or unsubstituted (C1-C 10 )alkyl heterocyclic group (C1-C 10 )alkyl carboxyl and substituted or unsubstituted (C1-C 10 )alkyl heterocyclic group (C1-C 10 )alkylaminocarbonyl; and

[0098] Z is selected from: substituted or unsubstituted C1-C 30 alkylene, C1-C 30 alkenylene, wherein the alkylene or alkenylene is optionally inserted with at least one heteroatom, substituted or unsubstituted (C1-C 10 )alkylamino, substituted or unsubstituted (C1-C 10 )alkylcarbonyl, PEG 1-30 , and the PEG 1-30 is optionally terminated with a member selected from: a bond, -O-, -S-, -NH-, -NHC(O)- and -C(O)NH-.

[0099] In certain aspects, B 1 is biotin, desthiobiotin or a biotin mimetic.

[0100] In certain aspects, the present disclosure provides a cholic acid conjugate, wherein the cholic acid is conjugated to biotin, desthiobiotin or a biotin mimetic.

[0101] In another embodiment, the present disclosure provides a method for treating a microbial infection or delaying microbial transmission, the method comprising:

[0102] contacting the microbe with an antimicrobial amount of a compound of formula I or a pharmaceutically acceptable salt thereof:

[0103]

[0104] Wherein:

[0105] R 1 -R 4 、R 6 、R 7 、R 11 、R 12 、R 15 and R 16 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl formamido, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl;

[0106] R 5 、R 8 、R 9 、R 10 、R 13 and R 14 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10) Haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo group, substituted or unsubstituted (C1-C 10 ) Aminoalkoxy, substituted or unsubstituted (C1-C 10 ) Aminoalkylcarboxy, substituted or unsubstituted (C1-C 10 ) Aminoalkylaminocarbonyl, (C1-C 10 ) Azidoalkoxy, (C1-C 10 ) Cyanoalkoxy, (C1-C 10 ) Guanidinoalkoxy and (C1-C 10 ) Guanidinoalkylcarboxy;

[0107] L 1 is a linking group; and

[0108] B 1 is biotin, desthiobiotin or a biotin mimetic, wherein the compound of formula I kills the microorganism or slows the spread of the microorganism.

[0109] In certain aspects, R 3 、R 7 and R 12 are each independently selected from: substituted or unsubstituted (C1-C 10 ) Aminoalkoxy, substituted or unsubstituted (C1-C 10 ) Aminoalkylcarboxy, substituted or unsubstituted (C1-C 10 ) Aminoalkylaminocarbonyl and substituted or unsubstituted (C1-C 10 ) Aminoalkylformamido; and R 5 、R 9 and R 13 are each independently selected from: hydrogen, hydroxy and substituted or unsubstituted (C1-C 10 ) alkyl.

[0110] In certain aspects, R 3 、R 7 and R 12 are each a substituted or unsubstituted (C1-C 10 ) Aminoalkylcarboxy, wherein the amino is optionally a cationic amine salt; and R 5 、R 9 and R 13 are each independently hydrogen or substituted or unsubstituted (C1-C3) alkyl.

[0111] In certain aspects, L 1 is -L-Y-Z, where L is independently selected from: a bond, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkylcarboxy, (C1-C10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkylcarboxyl, substituted or unsubstituted (C1-C 10 ) alkylformamido, substituted or unsubstituted (C1-C 10 ) alkylformamido (wherein the amino is substituted), and substituted or unsubstituted (C1-C 10 ) alkylaminocarbonyl;

[0112] Y is optionally present and is selected from: substituted or unsubstituted (C1-C 10 ) alkylheterocyclyl(C1-C 10 ) alkylformamido, substituted or unsubstituted (C1-C 10 ) alkylheterocyclyl, substituted or unsubstituted (C1-C 10 ) alkylheterocyclyl(C1-C 10 ) alkylcarboxyl and substituted or unsubstituted (C1-C 10 ) alkylheterocyclyl(C1-C 10 ) alkylaminocarbonyl; and

[0113] Z is selected from: substituted or unsubstituted C1-C 30 alkylene, C1-C 30 alkenylene, wherein the alkylene or alkenylene is optionally inserted with at least one heteroatom, substituted or unsubstituted (C1-C 10 ) alkylamino, substituted or unsubstituted (C1-C 10 ) alkylcarbonyl, PEG 1-30 and the PEG 1-30 is optionally terminated with a member selected from: a bond, -O-, -S-, -NH-, -NHC(O)- and -C(O)NH-.

[0114] In certain aspects, the biotin moiety includes biotin, desthiobiotin or a biotin mimetic.

[0115] In certain aspects, the microorganism is selected from microorganisms, bacteria, viruses and fungi.

[0116] In certain aspects, the microorganism is a virus.

[0117] In some aspects, the virus is a coronavirus, such as SARS-CoV-2, its variants, and subvariants.

[0118] In some aspects, the virus is a respiratory syncytial virus (RSV).

[0119] In some aspects, the virus is an influenza A virus.

[0120] In some aspects, the present disclosure provides a modified bile acid conjugated to a second compound, which in some cases is a biotin moiety. As used herein, the biotin moiety can be biotin, desthiobiotin, or a biotin mimetic.

[0121] Bile acid, (6R)-6-[(1R,3aS,3bR,4R,5aS,7R,9aS,9bS,11S,11aR)-4,7,11-trihydroxy-9a,11a-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-yl]heptanoic acid, with a CAS number of 81-25-4 and a chemical structure as follows:

[0122]

[0123] Compounds related to or derived from bile acid are compounds known as cationic steroid antimicrobials such as CSA-44. Cationic steroid antimicrobials are also known as ceragenins. The chemical structure of CSA-44 is as follows:

[0124]

[0125] In some aspects, the modified bile acid conjugate of the present disclosure has the structure of Formula Ia as follows:

[0126]

[0127] In yet another aspect, the modified bile acid conjugate is formed from a modified bile acid and a second compound, where the second compound is a biotin moiety, and the present disclosure provides a method of exposing cells in an infected mammal (e.g., infected with bacteria, virus, fungi, or a combination thereof) to the modified bile acid conjugate, wherein the method of exposure results in a cytotoxicity of the cells exposed to the conjugate that is less than the degree of cytotoxicity that occurs when the cells are exposed to the unconjugated form. In this method of the invention, the use of the conjugate achieves at least a two-fold reduction in cytotoxicity compared to the use of unconjugated ceragenin. In another aspect, the reduction is at least about four-fold (1X, 2X, 3X, 4X, or more).

[0128] In addition, other scaffolds for modifying bile acids include chenodeoxycholic acid, (4R)-4-[(1R,3aS,3bR,4R,5aS,7R,9aS,9bS,11aR)-4,7-dihydroxy-9a,11a-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-yl]pentanoic acid, with CAS number 474-25-9 and having the following structure:

[0129]

[0130] Another scaffold for modifying bile acids includes deoxycholic acid, (4R)-4-[(1R,3aS,3bR,5aR,7R,9aS,9bS,11S,11aR)-7,11-dihydroxy-9a,11a-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-yl]pentanoic acid, with CAS number 83-44-3 and having the following structure:

[0131]

[0132] Other scaffolds for modifying bile acids include lithocholic acid, (4R)-4-[(1R,3aS,3bR,5aR,7R,9aS,9bS,11aR)-7-hydroxy-9a,11a-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-1-yl]pentanoic acid, with CAS number 434-13-9 and having the following structure:

[0133]

[0134] In another aspect, the present disclosure comprises, consists of, or consists essentially of: modified cholic acid (MCA) conjugate molecules having a therapeutic index greater than 3, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In another aspect, the present disclosure comprises, consists of, or consists essentially of: MCAs having a therapeutic index of at least 15, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26. As illustrated herein, the toxicity and therapeutic efficacy of such compounds and compositions can be determined by standard pharmacological procedures conducted in cell cultures or experimental animals, such as for determining CC 50 (cytotoxic concentration or dose lethal to >50% of the population) and EC 50 (concentration or dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index ("TI"), and it can be expressed as the ratio CC 50 / EC 50 .

[0135] In some aspects, the present disclosure provides modified bile acids conjugated to biotin or a biotin moiety, which conjugates are added to a formulation and applied to cells infected with bacteria, fungi, or viruses. The modified bile acid conjugates are present in an amount that provides favorable antimicrobial (e.g., antibacterial, antiviral, antifungal, or combinations thereof) results while resulting in a level of cytotoxicity that is less than the level of cytotoxicity when such infected cells are exposed to the unconjugated form.

[0136] In some aspects, the present disclosure provides a formulation comprising, consisting essentially of, or consisting of: administering a modified bile acid conjugated to biotin or a biotin moiety to a mammal, human, or animal having a viral infection, wherein the degree of infection is reduced and wherein the cytotoxicity that occurs in the mammal exposed to the conjugate is less than the degree of cytotoxicity that occurs when the unconjugated form is administered to the mammal to treat such infection. The individual is typically a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can advantageously be used as individuals representing, for example, animal models of various conditions. In addition, the methods described herein can be used to treat domestic animals and / or pets. The individual can be male or female.

[0137] In some aspects, the present disclosure provides a formulation comprising, consisting essentially of, or consisting of: administering a modified bile acid conjugated to biotin or a biotin moiety to a mammal, particularly a human, having a viral infection, wherein the degree of infection is reduced and wherein the cytokine response is favorable compared to untreated infected mammals.

[0138] In some aspects, the present disclosure provides methods for treating a disease or condition. In one aspect, the present disclosure provides treating (or preventing) a viral infection, such as SARS-CoV-2 and its variants or influenza A in an individual, such as a human patient, comprising the step of administering to the patient a formulation comprising a modified bile acid conjugated to biotin or a biotin moiety in an amount effective to treat (or prevent) the infection and wherein the degree of cytotoxicity produced by the administration is less than the degree of cytotoxicity produced by administering the unconjugated form to the patient.

[0139] In some aspects, the present disclosure provides a treatment for coronavirus infections such as those caused by SARS-CoV-2 and its variants, or influenza A in a mammalian individual such as a human patient, comprising the step of administering to the patient a formulation comprising a modified bile acid conjugated to biotin or a biotin moiety, in an amount effective to treat the infection, and wherein the degree of cytotoxicity produced by the treatment is less than the degree of cytotoxicity produced by administering to the patient the modified bile acid in its unconjugated form, and wherein the conjugated form comprises biotin.

[0140] In some cases, compared to unconjugated MCA, the present disclosure provides MCA conjugates having increased efficacy and a more robust mode of action (e.g., easier entry into cells or multiple modes of action). Additionally, the present disclosure provides MCA conjugates having a therapeutic index of: greater than 3, or at least 15, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or even greater.

[0141] In some cases, the biotin or biotin moiety includes biotin, desthiobiotin, or a biotin mimetic. The biotin mimetic can be a short peptide comprising 2 - 12 amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. The single-letter and three-letter designations of amino acids are as follows:

[0142] Single - letter code Amino acid Three - letter code G Glycine Gly A Alanine Ala L Leucine Leu M Methionine Met F Phenylalanine Phe W Tryptophan Trp K Lysine Lys Q Glutamine Gln E Glutamic acid Glu S Serine Ser P Proline Pro V Valine Val I Isoleucine Ile C Cysteine Cys Y Tyrosine Tyr H Histidine His R Arginine Arg N Asparagine Asn D Aspartic acid Asp T Threonine Thr

[0143] Compounds of the present disclosure

[0144] In some aspects, the present disclosure provides a modified bile acid conjugated to a second compound, wherein the second compound is a vitamin such as biotin. Table 1 provides specific compounds of modified bile acids conjugated to biotin:

[0145] Table 1

[0146]

[0147]

[0148] In some aspects, the present disclosure provides a modified bile acid conjugated to biotin or a biotin moiety, comprising a modified bile acid and a second compound, wherein the second compound is desthiobiotin. Table 2 provides specific compounds of modified bile acids conjugated to desthiobiotin:

[0149] Table 2

[0150]

[0151] In some aspects, the present disclosure provides modified bile acids conjugated to biotin or a biotin moiety, which comprise a modified bile acid and a second compound, wherein the second compound is a biotin mimetic. Table 3 provides specific compounds of modified bile acids conjugated to biotin mimetics:

[0152] Table 3

[0153]

[0154] III. Synthesis

[0155] The modified bile acid can be linked to biotin or a biotin moiety to form a compound of formula I as defined above. "Click" chemistry provides a possible way to link the modified bile acid to biotin or a biotin moiety. Click chemistry uses simple, robust reactions, such as the copper-catalyzed cycloaddition of azides and alkynes, to generate intermolecular linkages. For a review of click chemistry, see Kolb, H.C.; Finn, M.G.; Sharpless, K.B., Angew. Chem. 2001, 40, 2004.

[0156] With the aid of the so-called "click chemistry" described in Sharpless et al., Angew. Chem, Int. Ed. 40:2004 (2001), the binding (or linking) of two fragments is generally achieved to prepare larger molecules or structures. This term is used to describe a series of bimolecular reactions between two different reactants such as azides and acetylenes. The formation of 1,2,3-triazoles in the 1,3-dipolar cycloaddition of azides to triple bonds is known, but the reaction is slow under ambient conditions because of the high activation energy of the acetylene-azide cycloaddition.

[0157] The utility of the reaction of azides with alkynes was extended by the discovery of Cu(I) catalysis. In organic or aqueous solutions, at room temperature, the 1,3-cycloaddition of azides to terminal alkynes is facile in the presence of a catalytic amount of cuprous salts.

[0158] U.S. Patent 7,807,619 to Bertozzi et al. teaches modified cycloalkyne compounds and methods of using such compounds in modified conjugates. Bertozzi et al. teach cycloaddition reactions that can be carried out under physiological conditions. As disclosed herein, the modified cycloalkyne reacts with an azide moiety on a target molecule to produce a covalently modified conjugate.

[0159] The present disclosure provides compounds of formula I having click chemistry functional groups for labeling biotin or a biotin moiety. Thus, in one aspect, the present disclosure provides modified bile acids or biotin or a biotin moiety having selected from azido, alkynyl, polyethylene glycolated azido, and polyethylene glycolated alkynyl.

[0160] In other aspects, the present invention relates to two components that interact with each other to form stable covalent bioorthogonal bonds. Bioorthogonal reactions are reactions of materials with each other, where each material has limited or substantially no reactivity with functional groups found in vivo. These components are used in chemical and biological assays, such as chemical reagents, medical imaging, and therapy, and more specifically, in nucleic acid modification techniques. According to a specific embodiment of the present invention, the covalent bioorthogonal bond is obtained by the [3+2] cycloaddition of azide and alkyne.

[0161] In other aspects, one of two components that interact with each other to form stable covalent bioorthogonal bonds. The starting material contains an azide group or an alkynyl group on MCA and is used as a reactant in click chemistry reactions, and the other reactant is biotin or a biotin moiety containing an alkynyl group or an azide group.

[0162] An azide-reactive group such as an alkyne compound can react with at least one 1,3-dipole-functional compound such as an alkyne-reactive group (e.g., azido group) in a cyclization reaction to form a heterocyclic compound. In certain embodiments, the reaction can be carried out in the presence of an added catalyst (e.g., Cu(I)). In other embodiments, the reaction is carried out in the absence of such a catalyst. Exemplary 1,3-dipole-functional compounds include, but are not limited to: azide-functional compounds, nitrile oxide-functional compounds, nitrone-functional compounds, azoxy-functional compounds, and / or acyl diazo-functional compounds. In certain cases, azide-functional compounds are used. The following Route I is one method for obtaining the compound of formula I:

[0163]

[0164] Suitable moieties for click reactions include, for example, biotin or a biotin moiety.

[0165] In one aspect, the compounds of formula I have sufficient solubility in aqueous solutions, and once they are conjugated to a soluble ligand, the ligand retains its solubility. In certain cases, the bioconjugates also have good solubility in organic media (e.g., DMSO or DMF), which provides considerable versatility to the synthetic methods for labeling of the desired materials.

[0166] In another aspect, the compounds of formula I can be prepared as shown in the following Route II. As shown therein, an active ester of cholic acid 7 is prepared by dropwise addition of benzyl in the presence of camphorsulfonic acid to generate compound 8.

[0167] Compound 9 was prepared by adding compound 8 to DCC, 4-DMAP and 3-((tert-butoxycarbonyl)amino)propanoic acid. After completion, the crude mixture was dried over anhydrous Na2SO4, the solvent was removed under reduced pressure, and the mixture was loaded onto diatomaceous earth. The crude mixture was then purified by SiO2 chromatography to give compound 9. Compound 10 was prepared by Pd reduction of compound 9 with hydrogen to give compound 10. Compound 12 was prepared by reacting compound 10 with compound 11 (N-(2-aminoethyl)-5-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide). A modified cholic acid biotin conjugate was generated.

[0168]

[0169] Peptide - based MCA

[0170] The present disclosure provides biotin mimetics, such as peptide-based mimetics. The disclosed peptide mimics the binding function of biotin to certain short peptides (3 - 20 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids), particularly peptides that contain the HPQN motif within the sequence. Solid-phase peptide synthesis (SPPS) techniques have greatly simplified the in vitro synthesis of peptides, including post-synthesis labeling and linker attachment (2). There are many peptide manufacturers that can prepare custom peptides to order as a standard service.

[0171] In the SPPS method, the peptide is built one amino acid at a time, each added to the N-terminus of the growing peptide chain. After each amino acid is added, the protecting group on its amine is removed so that the next amino acid can be added. When the sequence is complete, the protecting group at the N-terminus is removed. This can be left as a free amine for attachment to another molecule (e.g., by the activated acid chemistry discussed previously), or a linker group can be added as a spacer and / or to provide a different type of ligation chemistry, such as azides or alkynes for click reactions. When all the desired units have been added, the completed peptide is cleaved from the solid support.

[0172] Thus, the HPQN tetrapeptide with a suitable spacer / linker group at the N-terminus can be readily purchased from suppliers specializing in peptide manufacture. For example, 6-aminohexanoic acid and 6-azidohexanoic acid can be used. Many other linker groups can be selected, including direct attachment of MCA to the N-terminus of the peptide.

[0173] The compounds of the present disclosure are stable at a pH range of about 6 to about 8 (e.g., a pH of about 6.5 to about 7.5), or at a pH value of about 6, about 7, or about 8 (e.g., a pH of about 7). Storage stability in buffer is monitored over a period of at least 7 days to determine the effect of solution pH and degradation. For example, Compound 12 is stable at a pH of about 7 with no degradation for at least 7 days. Advantageously, during viral infection, stability at a neutral or near-neutral pH of about 7 accelerates wound healing, reduces inflammation, and reduces the risk of cytokine storm. In some aspects, the compounds of Formula I are formulated in a preparation having a pH of about 6 to about 8.

[0174] IV. Application

[0175] The present disclosure provides antimicrobial agents or antimicrobial conjugates that kill microorganisms or slow the spread of microorganisms. Microorganisms include bacteria, viruses, and fungi, such as molds and mildews. Antimicrobial conjugates are not limited to pharmaceuticals, and they can be used topically or in non-therapeutic settings to control the growth of microorganisms (e.g., bacteria, viruses, and fungi) or biofilms, or in non-limiting applications such as anti-inflammatory, antipruritic (itching relief), soothing, urticaria, eczema, pityriasis, psoriasis, erythema, heel fissures, and dermatitis. Additionally, the compounds disclosed herein can be used in applications that kill or control microorganisms upon contact. Microorganisms can also affect a variety of biological, medical, and processing operations. Methods and treatments using antimicrobial conjugates, or combinations of antimicrobial conjugates with additional compounds, can include killing, dissipating, treating, reducing, or preventing or inhibiting microbial infection or formation or proliferation.

[0176] In some aspects, the formation of biofilms is inhibited. In other aspects, pre-formed biofilms are dissipated. In other aspects, substantially all cells containing biofilms are killed.

[0177] In some aspects, the antimicrobial conjugates are suitable for therapeutic and / or prophylactic use. The latter approach is highly desirable to reduce the likelihood of viral infection in high-risk individuals (e.g., the elderly (especially the elderly in nursing homes), individuals with co-morbidities, and healthcare workers), thus greatly reducing the burden on the healthcare system. Antiviral agents with high safety profiles can be used pre-exposure or immediately after contact with an infected individual (post-exposure / treatment). The proposed multiple modes of action (MOA) would allow for pre-viral-infection treatment and post-viral-infection treatment.

[0178] In some aspects, the antimicrobial conjugate, alone or in combination with a similar composition having a sufficient amount of one or more active components, can be provided to an individual as an antimicrobial agent. A sufficient amount of one or more antimicrobial conjugates can be sufficient to have antibacterial and antifungal activity. A sufficient amount of one or more antimicrobial conjugates can be sufficient to have bactericidal, virucidal, or fungicidal activity. A sufficient amount of one or more antimicrobial conjugates can be used for prophylactic use. A sufficient amount of one or more antimicrobial conjugates can be used for the treatment of one or more susceptible pathogens. A sufficient amount of one or more antimicrobial conjugates can be used for the treatment of one or more susceptible and antimicrobial-resistant pathogens. Any of the antimicrobial conjugates described can be provided, with or without additional agents, in one or more suitable, safe, and effective formulations, such as for topical use and / or for in vivo use. When used for in vivo use, the antimicrobial conjugate can be formulated for oral ingestion. When used for in vivo use, the antimicrobial conjugate composition can be formulated for injection. When used for in vivo use, the antimicrobial conjugate can be formulated for inhalation, such as by a nebulizer, nasal spray, or lung inhaler. When used for in vivo use, the antimicrobial conjugate can be formulated for lavage of organs such as the uterus, bladder, stomach, and intestine.

[0179] In some aspects, the modified cholic acid (MCA) conjugate, alone or in combination with a similar composition, can be used for the therapeutic or prophylactic treatment of viral infections, such as enveloped or non-enveloped viruses, or for reducing or preventing the transmission of lipid-enveloped viral infections, such as SARS-CoV-2 and its variants, RSV, and influenza A virus.

[0180] In some aspects, the modified cholic acid (MCA) conjugate, alone or in combination with a similar composition, treats bacterial, fungal, or viral respiratory infections and any secondary infections, such as bacterial and / or fungal lung infections in the upper / lower respiratory tract.

[0181] In some aspects, the modified cholic acid (MCA) conjugate, alone or in combination with a similar composition, modulates cytokines and chemokines to reduce cytokine storms, sepsis, and other immune system-mediated responses such as potential autoimmune disorders.

[0182] In some aspects, the modified cholic acid (MCA) conjugate, alone or in combination with a similar composition, can be used for the treatment of respiratory diseases caused by viruses, bacteria, fungi, and other pathogens and is not affected by antimicrobial resistance. Treatment is delivered by nebulizer, nasal spray, lung inhaler, etc. Other delivery systems include oral administration. These applications can be done prophylactically as well as therapeutically.

[0183] In some aspects, the compounds disclosed herein can be used to treat Herpes Zoster, also known as shingles, which is a viral infection that causes a painful rash on the skin. Herpes Zoster is caused by the varicella-zoster virus, which also causes chickenpox. After a person has had chickenpox, the virus lies dormant in certain nerves for several years, and its reactivation causes the Herpes Zoster infection. In some cases, treatment can be used for the viral infection by applying an ointment / cream, foam, and / or spray to the viral rash or in the form of, for example, papules or blisters.

[0184] In some aspects, the compounds disclosed herein can be used to treat skin blisters (herpetic rash) caused by Herpes Simplex Virus (HSV-1). Additionally, another herpes virus (HSV-2) can cause genital herpes and can also be passed on to infants during birth. Both forms of the virus enter the nerve cells of the body, where they survive. The virus tends to lie dormant or asleep in the cells until something activates it and causes an outbreak of symptoms. Both Herpes type 1 and Herpes type 2 can be treated with an ointment / cream, spray, foam, or gel.

[0185] In some aspects, the microorganism is selected from microbes, bacteria, viruses, and fungi.

[0186] In some aspects, the microorganism is a virus, such as a coronavirus (e.g., SARS-CoV-2), RSV, influenza A, Herpes simplex virus type I and Herpes simplex virus type II, and norovirus.

[0187] In some aspects, the step of contacting the microorganism is carried out directly or indirectly by intervening in the viral life cycle.

[0188] In some aspects, the methods herein provide a beneficial effect in vivo as immunomodulators for modulating the toxic immune response to a disease.

[0189] Combinations with other antimicrobials

[0190] In certain aspects, the present disclosure provides compounds, compositions, or methods, such as industrial compositions, therapeutic compositions, or pharmaceutical compositions, that comprise an antimicrobial conjugate in combination with one or more additional active compositions.

[0191] In some cases, the antimicrobial conjugate can be administered alone or in combination with a second agent (such as a biocide, antibiotic, or antimicrobial agent) to kill, dissipate, treat, reduce, prevent, or inhibit bacteria, viruses, or fungi. Antimicrobial agents such as antibiotics, antifungals, or antivirals can be co-administered with the antimicrobial conjugate either sequentially or simultaneously.

[0192] Additional antimicrobial agents (including antibiotics) can be any compound known to those of ordinary skill in the art that can inhibit the growth of or kill bacteria, fungi, viruses, and other forms of pathogens. Non-limiting examples of useful antibiotics include lincosamides (clindamycin); chloramphenicols; tetracyclines (e.g., tetracycline, chlortetracycline, demeclocycline, methacycline, doxycycline, minocycline); aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, amikacin (smikacin), kanamycin, streptomycin, neomycin); β-lactams (e.g., penicillins, cephalosporins, imipenem, aztreonam); glycopeptide antibiotics (e.g., vancomycin); polypeptide antibiotics (e.g., bacitracin); macrolides (erythromycins), amphotericins; sulfonamides (e.g., sulfanilamide, sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole, sulfacytine, sulfadoxine, sulfamylon, para-aminobenzoic acid, trimethoprim-sulfamethoxazole); methenamin; nitrofurantoin; phenazopyridine; trimethoprim; rifampin; metronidazole; cefazolin; lincomycin; kitasamycin; mupirocin; quinolones (e.g., nalidixic acid, cinoxacin, norfloxacin, ciprofloxacin, pefloxacin, ofloxacin, enoxacin, fleroxacin, levofloxacin); novobiocin; polymyxins; gramicidin; and anti-Pseudomonas aeruginosa drugs (e.g., carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, piperacillin) or any salt or variant thereof. Such antibiotics can be commercially obtained, for example, from Daiichi Sankyo, Inc. (Parsipanny, NJ), Merck (Whitehouse Station, NJ), Pfizer (New York, NY), Glaxo Smith Kline (Research Triangle Park, NC), Johnson & Johnson (New Brunswick, NJ), AstraZeneca (Wilmington, DE), Novartis (East Hanover, NJ), and Sanofi-Aventis (Bridgewater, NJ). The antibiotics used depend on the type of bacterial infection.

[0193] In certain cases, the compounds of the present disclosure can be combined with one or more of the following, such as antifungal agents, including allylamines, azoles, polyenes, pyrimidines, tetraenes, thiocarbamates, sulfonamides, glucan synthesis inhibitors, and benzoic acid compounds. Specific antifungal agents include amorolfine, butenafine, naftifine, terbinafine, ketoconazole, fluconazole, econazole, econazole nitrate, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole, terconazole, butoconazole, thiabendazole, voriconazole, sertaconazole, sulconazole, fenticonazole, posaconazole, bifonazole, flutrimazole, nystatin, pimaricin, amphotericin B, flucytosine, natamycin, tolnaftate, mafenide, dapsone, caspofungin, actofunicone, griseofulvin, potassium iodide, gentian violet, ciclopirox, ciclopirox olamine, haloprogin, undecylenate, silver sulfadiazine, undecylenic acid, undecylenicalkanolamide, and carbol-fuchsin.

[0194] Additional known biocides include biguanides, chlorhexidine, triclosan, chlorine dioxide, etc. Although combinations of these biocides and MCAs can be envisioned, in other respects, the MCA conjugates can replace one or more of the foregoing.

[0195] Useful examples of antimicrobials include, but are not limited to, pyrithiones, especially zinc complexes (ZPT); dimethyldimethylol hydantoin methylchloroisothiazolinone / methylisothiazolinone (Kathon ); sodium sulfite; sodium bisulfite; imidazolidinyl urea (Germall ), diazolidinyl urea (Germall ); benzyl alcohol; 2-bromo-2-nitropropane-1,3-diol formalin (formaldehyde); iodopropenyl butylcarbamate (Polyphase PI ); chloroacetamide; methylamine; methyl dibromoglutaronitrile (1,2-dibromo-2,4-dicyanobutane or ); glutaraldehyde; 5-bromo-5-nitro-1,3-dioxane phenethyl alcohol; o-phenylphenol / sodium o-phenylphenate; sodium hydroxymethylglycinate (Suttocide ); polymethoxy bicyclic oxazolidine (Nuosept ); dimethoxane; thimerosal; dichlorobenzyl alcohol; captan; chlorphenesin; dichlorophen; chlorbutanol; glyceryl laurate; halogenated diphenyl ether; 2,4,4'-trichloro-2'-hydroxydiphenyl ether( or TCS); 2,2'-dihydroxy-5,5'-dibromodiphenyl ether; phenolic compounds; phenol; 2-methylphenol; 3-methylphenol; 4-methylphenol; 4-ethylphenol; 2,4-dimethylphenol; 2,5-dimethylphenol; 3,4-dimethylphenol; 2,6-dimethylphenol; 4-n-propylphenol; 4-n-butylphenol; 4-n-pentylphenol; 4-tert-pentylphenol; 4-n-hexylphenol; 4-n-heptylphenol; monoalkyl and polyalkyl halophenols and aromatic halophenols; p-chlorophenol; methyl p-chlorophenol; ethyl p-chlorophenol; n-propyl p-chlorophenol; n-butyl p-chlorophenol; n-pentyl p-chlorophenol; sec-pentyl p-chlorophenol; cyclohexyl p-chlorophenol; n-heptyl p-chlorophenol; n-octyl p-chlorophenol; o-chlorophenol; methyl o-chlorophenol; ethyl o-chlorophenol; n-propyl o-chlorophenol; n-butyl o-chlorophenol; n-pentyl o-chlorophenol; tert-pentyl o-chlorophenol; n-hexyl o-chlorophenol; n-heptyl o-chlorophenol; o-benzyl p-chlorophenol; o-benzyl m-methyl p-chlorophenol; o-benzyl-m,m-dimethyl p-chlorophenol; o-phenethyl p-chlorophenol; o-phenethyl m-methyl p-chlorophenol; 3-methyl p-chlorophenol; 3,5-dimethyl p-chlorophenol; 6-ethyl-3-methyl p-chlorophenol; 6-n-propyl-3-methyl p-chlorophenol; 6-isopropyl-3-methyl p-chlorophenol; 2-ethyl-3,5-dimethyl p-chlorophenol; 6-sec-butyl-3-methyl p-chlorophenol; 2-isopropyl-3,5-dimethyl p-chlorophenol; 6-diethylmethyl-3-methyl p-chlorophenol; 6-isopropyl-2-ethyl-3-methyl p-chlorophenol; 2-sec-pentyl-3,5-dimethyl p-chlorophenol; 2-diethylmethyl-3,5-dimethyl p-chlorophenol; 6-sec-octyl-3-methyl p-chlorophenol; p-chloro-m-cresol; p-bromophenol; methyl p-bromophenol; ethyl p-bromophenol; n-propyl p-bromophenol; n-butyl p-bromophenol; n-pentyl p-bromophenol; sec-pentyl p-bromophenol; n-hexyl p-bromophenol; cyclohexyl p-bromophenol; o-bromophenol; tert-pentyl o-bromophenol; n-hexyl o-bromophenol; n-propyl-m,m-dimethyl o-bromophenol; 2-phenylphenol; 4-chloro-2-methylphenol; 4-chloro-3-methylphenol; 4-chloro-3,5-dimethylphenol; 2,4-dichloro-3,5-dimethylphenol; 3,4,5,6-tetrabromo-2-methylphenol; 5-methyl-2-pentylphenol; 4-isopropyl-3-methylphenol; p-chloro-m-xylenol (PCMX); chlorothymol; phenoxyethanol; phenoxyisopropanol; 5-chloro-2-hydroxy diphenylmethane; resorcinol and its derivatives; resorcinol; methyl resorcinol; ethyl resorcinol; n-propyl resorcinol; n-butyl resorcinol; n-pentyl resorcinol; n-hexyl resorcinol; n-heptyl resorcinol; n-octyl resorcinol; n-nonyl resorcinol; phenyl resorcinol; benzyl resorcinol; phenethyl resorcinol; phenylpropyl resorcinol; p-chlorobenzyl resorcinol; 5-chloro-2,4-dihydroxy diphenylmethane; 4'-chloro-2,4-dihydroxy diphenylmethane;5-bromo-2,4-dihydroxydiphenylmethane; 4'-bromo-2,4-dihydroxydiphenylmethane; bisphenol compounds; 2,2'-methylenebis(4-chlorophenol); 2,2'-methylenebis(3,4,6-trichlorophenol); 2,2'-methylenebis(4-chloro-6-bromophenol); bis(2-hydroxy-3,5-dichlorophenyl) sulfide; bis(2-hydroxy-5-chlorobenzyl) sulfide; benzoates (parabens); methyl paraben; propyl paraben; butyl paraben; ethyl paraben; isopropyl paraben; isobutyl paraben; benzyl paraben; sodium methyl paraben; sodium propyl paraben; halogenated diphenyl ureas; 3,4,4'-trichlorocarbanilide (e.g., or TCC); 3-trifluoromethyl-4,4'-dichlorocarbanilide; 3,3',4-trichlorocarbanilide; chlorhexidine and its bigluconates; diacetates and dihydrochlorides; undecylenic acid; thiabendazole; hexetidine; poly(hexamethylene biguanide) hydrochloride

[0196] In some cases, the compounds of the present disclosure are combined with antiviral agents. Antiviral agents act as either virus targets or host factors. Virus-targeting antiviral agents act during the viral life cycle by direct or indirect means. Host-targeting antiviral agents include agents that target host proteins involved in the viral life cycle. Direct virus-targeting antiviral agents include attachment inhibitors, entry inhibitors, fusion inhibitors, uncoating inhibitors, protease inhibitors, polymerase inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and integrase inhibitors. Additional antiviral agents can be any compound known to those of ordinary skill in the art that can directly kill the virus, prevent its entry into cells, or delay or stop its replication within cells. In some cases, the antiviral agent is a nucleoside analogue, b) a non-nucleoside polymerase inhibitor, or a protease inhibitor. In some cases, host-targeting antiviral agents include cyclophilin inhibitors (alipovir), HIV-1 co-receptor antagonists (aplaviroc, vicriviroc, etc.). Indirect virus-targeting antiviral agents include those interaction blockers during the viral life cycle. In some cases, indirect virus-targeting antiviral agents include RTC blockers (BMS790052) and RNP blockers (Nucleozin). Useful non-limiting examples of antiviral agents include acyclovir, remdesivir, oseltamivir, maraviroc, ribavirin, tramantadine, nitazoxanide, lopinavir, ensitrelvir, and adefovir.

[0197] In certain instances, the compounds of the present disclosure can be combined with antiviral agents such as protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase inhibitors, entry inhibitors, maturation inhibitors, and their pharmaceutically acceptable salts and precursors. Specific examples include acyclovir, docosanol, edoxuridine, famciclovir, foscarnet, idoxuridine, penciclovir, trifluridine, tromantidine, valacyclovir, and vidarabine (all of which treat infections caused by one or more herpesviruses); adefovir, boceprevir, entecavir, ribavirin, and taribavirin (all of which treat infections caused by one or more hepatitis viruses); amantadine, arbidol, oseltamivir, peramivir, rimantadine, and zanamivir (all of which treat infections caused by one or more influenza viruses). Other antiviral drugs include amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (all of which are protease inhibitors); abacavir (ABC), amdoxovir, apricitabine (ATC), didanosine (ddI), elvucitabine, emtricitabine (FTC), entecavir (INN), lamivudine (3TC), racivir, stampidine, stavudine (d4T), zalcitabine (ddC), and zidovudine (AZT) (all of which are NRTIs); adefovir (also known as bis-POM PMPA) and tenofovir (both of which are NtRTIs); delavirdine, efavirenz, etravirine, lersivirine, loviride, nevirapine, and rilpivirine (all of which are NNRTIs); elvitegravir, globoidnan A, GSK-572, MK-2048, and raltegravir (all of which are integrase inhibitors); enfuvirtide, ibalizumab, maraviroc, and vicriviroc (all of which are fusion / entry inhibitors); bevirimat and vivecon (both of which are maturation inhibitors); and their pharmaceutically acceptable salts and precursors, and mixtures thereof.The compounds disclosed herein exhibit protease inhibitory activity and, accordingly, the present disclosure provides combination therapies utilizing compounds of Formula I and RNA-dependent RNA polymerase (RdRp) inhibitors such as Molnupiravir.

[0198] V. Formulation

[0199] The modified bile acid conjugate antimicrobials provided herein can be incorporated into pharmaceutical compositions. The antimicrobial conjugate, or a combination of the antimicrobial conjugate with additional compounds, can be incorporated into the pharmaceutical composition as a pharmaceutically acceptable salt or derivative. Some pharmaceutically acceptable derivatives of the antimicrobial conjugates of the present invention can include chemical groups that increase water solubility. As used herein, "pharmaceutically acceptable carrier" means a carrier that can be administered to an individual together with the antimicrobial conjugates described herein, or a combination of the antimicrobial conjugate and additional compounds, and that does not destroy its pharmacological activity. Pharmaceutically acceptable carriers include, for example, solvents, binders, dispersion media, coatings, preservatives, coloring agents, isotonic agents, absorption delaying agents, and the like that are compatible with drug administration. Supplementary active compounds can also be incorporated into the compositions.

[0200] Non-limiting examples of pharmaceutically acceptable carriers that can be used include poly(ethylene-co-vinyl acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyl acetate-co-vinyl alcohol), crosslinked poly(ethylene-co-vinyl acetate), crosslinked partially hydrolyzed poly(ethylene-co-vinyl acetate), crosslinked poly(ethylene-co-vinyl acetate-co-vinyl alcohol), poly-D,L-lactic acid, poly-L-lactic acid, polyglycolic acid, PGA, copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone, polypentrolactone, poly(anhydrides), copolymers of polycaprolactone and polyethylene glycol, copolymers of polylactic acid and polyethylene glycol, polyethylene glycol; and combinations and blends thereof.

[0201] Other carriers include, for example, aqueous gelatin, aqueous protein, polymeric carriers, cross-linking agents, or combinations thereof. In other cases, the carrier is a matrix. In other cases, the carrier includes water, pharmaceutically acceptable buffer salts, pharmaceutically acceptable buffer solutions, pharmaceutically acceptable antioxidants, ascorbic acid, one or more pharmaceutically acceptable polypeptides of low molecular weight, peptides comprising from about 2 to about 10 amino acid residues, one or more pharmaceutically acceptable proteins, one or more pharmaceutically acceptable amino acids, essential amino acids, one or more pharmaceutically acceptable carbohydrates, one or more pharmaceutically acceptable carbohydrate-derived materials, non-reducing sugars, glucose, sucrose, sorbitol, trehalose, mannitol, maltodextrin, dextrin, cyclodextrin, pharmaceutically acceptable chelating agents, EDTA, chelating agents for divalent metal ions, chelating agents for trivalent metal ions, glutathione, pharmaceutically acceptable non-specific serum albumin, or combinations thereof.

[0202] In other embodiments, the composition may further comprise a pharmaceutically acceptable carrier. In other embodiments, the effective amount is an amount effective to treat or prevent a disease or disorder. In some embodiments, the effective amount includes an amount effective to treat or prevent a biofilm.

[0203] In some embodiments, the compositions discussed herein further comprise an agent suitable for administration to a surface. In other embodiments, the composition is formulated as a wash solution, dressing, wound gel, or synthetic tissue. In additional embodiments, the composition is formulated as a tablet, pill, lozenge, capsule, liquid spray, aerosol spray, solution, suspension, gel, paste, lotion, ointment, cream, or foam. In some embodiments, the composition is formulated for parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, vaginal, or rectal administration.

[0204] In some embodiments, the antimicrobial conjugate, or a combination of the antimicrobial conjugate with at least one other composition, is formulated as a sustained-release formulation.

[0205] In some embodiments, the antimicrobial conjugate, or a combination of the antimicrobial conjugate with at least one other composition, is intended for medical applications, such as for active release or passive antimicrobial coatings for: irrigation solutions for open wounds, toothpaste additives, hand sanitizers, systemic prophylactic antimicrobials, catheter lock solutions, ophthalmic solutions for rinsing and contact lens cleaners, prophylactic dental inserts, high-level disinfectants, gastrointestinal (GI) tract oral medications for treating infections (such as those caused by Shigella, Cryptosporidium, Vibrio cholerae, or Clostridium difficile), topical ointments for treating skin complications (including infections, oral ulcers, psoriasis, herpes, chronic wounds, diaper rash, onychomycosis (athlete's foot), tinea unguium (toenail fungus), ulcers, or acne, etc.).

[0206] In some embodiments, the compounds can be used in skin care formulations, beauty personal care formulations, intimate hygiene formulations, and foot care formulations.

[0207] As is known to those of ordinary skill in the art, a pharmaceutical composition containing an antimicrobial conjugate, or a combination of an antimicrobial conjugate with additional compounds, can be formulated to be compatible with its intended route of administration. Non-limiting examples of routes of administration include parenteral such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, vaginal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration can include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0208] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition can be sterile and can be fluid to the extent that it is easy to inject. It should be stable under the conditions of preparation and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium that contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants, appropriate fluidity can be maintained. The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. It may be desirable to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including substances that delay absorption in the composition, such as aluminum monostearate and gelatin (see, for example, Remington: The Science and Practice of Pharmacy, 21 st nd edition, Lippincott Williams & Wilkins, Gennaro, ed. (2006)).

[0209] Sterile injectable solutions can be prepared as follows: The antimicrobial conjugate, or a combination of the antimicrobial conjugate with additional compounds, is added in the required amounts to a suitable solvent, along with one or a combination of the above-listed ingredients as needed, and then filtered sterilized. Generally, dispersions are prepared by adding the active compound to a sterile carrier that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods include, but are not limited to, vacuum drying and freeze drying, which yield a powder of the active ingredient and any additional required ingredients from its previously sterile filtered solution.

[0210] Oral compositions can contain an inert diluent or an edible carrier or binder. For the purpose of oral therapeutic administration, the antimicrobial conjugate, or a combination of antimicrobial conjugates, or a combination of an antimicrobial conjugate and another compound can be admixed with excipients and used in the form of tablets, chewing gums, lollipops, pills, lozenges, chewable tablets or capsules (e.g., gelatin capsules). Also included are delivery mechanisms to the stomach and gastrointestinal tract. Pharmaceutically compatible binders or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, chewable tablets, etc. can contain any of the following ingredients or compounds having similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate or orange flavoring.

[0211] For administration by inhalation, the antimicrobial conjugate, or a combination of an antimicrobial conjugate and another compound can be delivered by nebulizer, nasal spray, pulmonary inhaler, or in the form of an aerosol spray (which is from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide)), nasal spray, pulmonary inhaler, or a hospital or outpatient nebulizer.

[0212] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the permeation barrier is used in the formulation. Such penetrants are generally known in the art and include, but are not limited to, for example, transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal spray, pulmonary inhaler, nebulizer or suppository. For transdermal administration, the active compound and composition are formulated into a pharmaceutically acceptable formulation embodiment, such as an ointment, salve, gel, skin cleanser / detergent or cream, as is generally known in the art.

[0213] For the treatment of acute or chronic wounds, the antimicrobial conjugate, or a combination of an antimicrobial conjugate and another compound can be formulated as a dressing, wash solution, gel, salve, foam ointment or synthetic tissue, etc.

[0214] A pharmaceutical composition containing the antimicrobial conjugate, or a combination of an antimicrobial conjugate and another compound can also be prepared in the form of a suppository (e.g., having a conventional suppository base such as cocoa butter and other glycerides) or a retention enema for rectal delivery.

[0215] Some pharmaceutical compositions containing antimicrobial conjugates, or combinations of antimicrobial conjugates with additional compounds, can be prepared with carriers (which protect the antimicrobial conjugate, or the combination of the antimicrobial conjugate with the additional compound, against conjugate degradation and rapid elimination from the body), such as controlled-release formulations, including, for example, implants and microencapsulated delivery systems as described in Tan et al., Pharm. Res. 24:2297-2308 (2007).

[0216] Alternatively, biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available. Liposome suspensions (including liposomes targeted to specific cells, which have monoclonal antibodies to cell surface antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0217] The toxicity and therapeutic efficacy of such compounds and compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining the CC 50 (cytotoxic concentration or dose lethal to >50% of the population) and the EC 50 (concentration or dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index ("TI"), and it can be expressed as the ratio CC 50 / EC 50 . Although compounds and compositions showing toxic side effects can be used, care should be taken to design a delivery system that targets the active ingredient to the affected tissue site so as to minimize potential damage to normal cells and thus reduce side effects.

[0218] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for humans. The dosage of such compounds and compositions will generally lie within a range that includes the ED 50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration employed. For any compound or composition used in the methods described herein, the therapeutically effective dose can initially be estimated from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50(i.e., the concentration of the test compound or composition that achieves half-maximal inhibition of the symptom). Such information can be used to more accurately determine the useful dose in humans. Plasma levels can be measured, for example, by high performance liquid chromatography. Information on the preparation and testing of such compositions is known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 21 st ed., Lippincott Williams & Wilkins, Gennaro, ed. (2006).

[0219] Physicians will appreciate that certain factors may affect the dose required to effectively treat an individual, including but not limited to the severity of the disease or disorder, previous treatment, the general health or age of the individual, and the presence of other diseases. In addition, treating an individual with a therapeutically effective amount of an antimicrobial conjugate, or a combination of an antimicrobial conjugate and another compound, can include a single treatment or a series of treatments.

[0220] The compound or pharmaceutical composition can be included in a container, package, or dispenser together with instructions for administration. One of ordinary skill in the art will appreciate that the compounds or pharmaceutical compositions described herein can be formulated as single-dose vials, or single-dose or multi-dose packages, for nasal sprays, nebulizers, pulmonary inhalers, or other dispensing devices.

[0221] An antimicrobial conjugate, or a combination of an antimicrobial conjugate and another compound, can be suitable as an anti-biofilm active in personal care formulations, such as shampoos, bath additives, hair care formulations, liquid and solid soaps (based on synthetic surfactants and salts of saturated or unsaturated fatty acids), lotions and creams, deodorants, other aqueous or alcoholic solutions, such as skin cleansing solutions, wet cleansing cloths, oils, or powders.

[0222] VI. Example

[0223] Example 1 is a description of the synthesis of the cholanic acid biotin conjugate (Compound 12).

[0224] Cholanic acid was added to a round bottom flask containing a stir bar and solvent. Then, while stirring at room temperature, camphorsulfonic acid was added, followed by dropwise addition of benzyl alcohol. The reaction was continued with stirring at room temperature and monitored by TLC. After completion, the crude mixture was dried over anhydrous Na2SO4, the solvent was removed under reduced pressure, and the mixture was loaded onto silica gel. The crude mixture was then purified by SiO2 chromatography to give Compound 8.

[0225] Compound 9: Add DCM to a round-bottom flask containing a stir bar and cool to 0 °C in an ice bath. Add DCC, 4-DMAP, and 3-((tert-butoxycarbonyl)amino)propanoic acid and stir for 5 minutes while maintaining the temperature. Dissolve Compound 8 in the minimum amount of DCM and add to the reaction mixture with stirring. Allow the reaction to warm to room temperature and stir for an additional 3 h, monitoring by TLC. Upon completion, dry the crude mixture over anhydrous Na2SO4, remove the solvent under reduced pressure, and load onto silica gel. Then purify the crude mixture using SiO2 chromatography to afford Compound 9.

[0226] Compound 10: Flame-dry a two-neck round-bottom flask containing a stir bar and equipped with a septum, purge with N2 and backfill 3x. Then add EtOH, followed by Compound 9 and purge again, backfill with N2. Add Pd and allow the reaction to stir at room temperature under a positive pressure of H2 for 3 h. Upon completion, filter the reaction mixture through silica gel and wash with EtOAc 3x. Dry the combined crude mixture over anhydrous Na2SO4, remove the solvent under reduced pressure, and load onto silica gel. Purify the crude mixture using SiO2 chromatography to afford Compound 10.

[0227] Compound 12: Add dry THF, Compound 10, and N-(2-aminoethyl)-5-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (Compound 11) to a round-bottom flask containing a stir bar. Stir the reaction mixture and monitor by TLC. Then add a 1,4-dioxane solution of 4M HCl and continue to stir the reaction mixture. Upon completion, dry the crude mixture over anhydrous Na2SO4, remove the solvent under reduced pressure, and load onto silica gel. Purify the crude mixture using SiO2 chromatography to afford the cholic acid biotin conjugate Compound 12 as a white solid. 1 1H NMR (CD3OD, 500 MHz); HRMS ESI+ [M+H] + m / z 890.

[0228] Record 1 1H NMR and 13 13C NMR spectra and reference to TMS, residual CHD2OD ( 1 1H) or CD3OD ( 13 13C). Obtain mass spectrometry data. Obtain compound purity by ultra-high performance liquid chromatography. Reagents and solvents were purchased commercially and used as received.

[0229] Example 2Is the expected preparation of tris(3-(chloro-l5-ammonio)propionate)(3R,7R,9S,10S,12S,13R,14S,17S)-17-(4-(((1-(1-((3aS,4S,6aR)-3a,6a-dimethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-43,43-dimethyl-3,41-dioxo-7,10,13,16,19,22,25,28,31,34,37-undecaoxa-4,40-diaza-tetratetracontane-44-yl)-1H-1,2,3-triazol-4-yl)methyl)amino)-4-oxobutyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (6a). In Route 1, propargylamine can be used as the starting reagent to produce compound 6a.

[0230]

[0231] Example 3 Is the expected preparation of tris(3-aminopropionate)(3R,7R,10S,12S,13R)-10,13-dimethyl-17-((2R)-5-oxo-5-((6-(5-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexyl)amino)pent-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12a). Under the same conditions, compound 11 from Route 2 can be replaced with compound 11a, creating a longer alkyl chain linkage between biotin and the remaining amine to generate compound 12a.

[0232]

[0233] Example 3A Is the expected preparation of tris(3-aminopropionate)(3R,7R,10S,12S,13R)-10,13-dimethyl-17-((2R)-5-(octyl(2-(5-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)amino)-5-oxopent-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12a’). Under the same conditions, compound 11 from Route 2 can be replaced with compound 11a’, creating a longer alkyl chain linkage between biotin and the remaining amine to generate compound 12a’.

[0234]

[0235] Example 4is the expected preparation of tris(3-aminopropionic acid) (3R,7R,10S,12S,13R)-17-((2R)-5,16-dioxo-20-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9,12-dioxa-6,15-diazaicosan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12b). Under the same conditions, compound 11 from Route 2 can be replaced with compound 11b to create a longer alkyl chain linkage between biotin and the remaining amine to generate compound 12b.

[0236]

[0237] Example 5 is the expected preparation of tris(3-aminopropionic acid) (3R,7R,10S,12S,13R)-17-((27R)-5,24-dioxo-1-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9,12,16,20-tetraoxa-6,23-diazaoctacos-27-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12c). Under the same conditions, compound 11 from Route 2 can be replaced with compound 11c to create a longer chain linkage between biotin and the remaining amine to generate compound 12c.

[0238]

[0239] Example 6 is the expected preparation of tris(3-aminopropionic acid) (3R,7R,10S,12S,13R)-17-((2R)-5,34-dioxo-38-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9,12,15,18,21,24,27,30-octaoxa-6,33-diazatriacontan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12d). Under the same conditions, compound 11 from Route 2 can be replaced with compound 11d to create a longer chain linkage between biotin and the remaining amine to generate compound 12d.

[0240]

[0241] Example 7is the expected preparation of tris(3-aminopropionic acid)(3R,7R,10S,12S,13R)-17-((2R)-5,79-dioxo-83-((4R)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tricosaoxa-6,78-diazaoctatriacontan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12e). Under the same conditions, compound 11 from Route 2 can be replaced with compound 11e, creating a longer chain linkage between the biotin and the remaining amine to generate compound 12e.

[0242]

[0243] Example 8 is the expected preparation of tris(3-(chloro-l5-ammonio)propionic acid)(3R,7R,9S,10S,12S,13R,14S,17S)-17-(4-(((1-(2-(2-(2-(5-((3aS,4S,6aR)-3a,6a-dimethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)(octyl)amino)-4-oxobutyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (6b). Under the same reaction conditions, compound 4 from Route 1 can be replaced with the alternative azide-capped biotin 4b to generate compound 6b.

[0244]

[0245] Example 9 is the expected preparation of tris(3-(chloro-l5-ammonio)propionic acid)(3R,7R,9S,10S,12S,13R,14S,17S)-17-(4-(((1-(2-(2-(2-(5-((3aS,4S,6aR)-3a,6a-dimethyl-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-4-oxobutyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (6c). Under the same reaction conditions, compound 4 from Route 1 can be replaced with the alternative azide-capped biotin 4b, while replacing compound 1 with 1a to generate 3a, to generate compound 6c.

[0246]

[0247] Desthiobiotin MCA

[0248] Example 10 Is the expected preparation of tris(3-(chloro-l5-amino)propionic acid)(3R,7R,9S,10S,12S,13R,14S,17S)-10,13-dimethyl-17-(4-(octyl((1-(2-(2-(2-(6-((R)-4,5,5-trimethyl-2-oxoimidazolidin-4-yl)hexanamido)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-4-oxobutyl)hexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (6d). Compound 4 from Route 1 can be replaced with desthiobiotin derivative 4d. Desthiobiotin reacts with aminoazide to form the MCA compound 6d.

[0249]

[0250] Example 11 Is the expected preparation of tris(3-aminopropionic acid)(3R,7R,10S,12S,13R)-10,13-dimethyl-17-((2R)-21-((4R)-5-methyl-2-oxoimidazolidin-4-yl)-5,16-dioxo-9,12-dioxa-6,15-diazahenicosan-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (12e). Route 1 can be used to generate compound 12e by reacting desthiobiotin with diamine to form compound 11e and reacting it with compound 10 from Route 1 to form compound 12e.

[0251]

[0252] Example 12 Is the expected preparation of tris(3-(chloro-l,5-amino)propionic acid)(3R,7R,9S,10S,12S,13R,14S,17S)-10,13-dimethyl-17-(4-oxo-4-(((1-(2-(2-(2-(6-((R)-4,5,5-trimethyl-2-oxoimidazolidin-4-yl)hexanamido)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)butyl)hexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-tricarboxylate (6e). According to Route 1, biotin can be replaced with desthiobiotin derivative 4b. Desthiobiotin reacts with aminoazide to form the MCA compound 6e.

[0253]

[0254] Peptide-based MCA

[0255] In some embodiments, short sequence peptides (6 - 20 amino acids) containing the His - Pro - Gln - Asn (HPQN) motif within the sequence can be used to generate peptide-based MCA. In some embodiments, the N-terminus of the peptide can be retained as a free amine to be linked to additional molecules, such as a linker group, via the activation acid chemistry discussed previously.

[0256] Example 13 Is the expected preparation of compound 6f. Compound 4 from route 1 can be replaced with a linker - HPQN derivative 4f. The linker - HPQN can be prepared by the direct linking reaction of the N-terminus of peptide 4f with MCA.

[0257]

[0258] Example 14 Is the expected preparation of compound 12f: Compound 11 from route 2 can be replaced with a linker - HPQN derivative. The linker - HPQN can be prepared by directly linking to the N-terminus of the peptide via 6 - aminocaproic acid and reacting with MCA to form compound 12f.

[0259]

[0260] Example 15 Is the expected preparation of compound 6g: Compound 4 in route 1 can be replaced with a linker - HPQN derivative 4g, while compound 1 is replaced with compound 1a to remove the octyl group. The linker - HPQN 4g can be prepared by directly linking to the N-terminus of the peptide via azidohexanoic acid and reacting with MCA to form compound 6g.

[0261]

[0262] Example 16 Is the expected preparation of compound 6h: Compound 4 from route 1 can be replaced with a linker - decapeptide derivative 4h having the sequence Ser - Ala - Trp - Arg - His - Pro - Gln - Phe - Gly - Gly (SAWRHPQGG). The linker - SAWRHPQGG can react with MCA to form compound 6h.

[0263]

[0264] Example 17The expected preparation of compound 12g: Compound 11 from Route 2 can be replaced with, for example, a linker-decapeptide derivative having the sequence Ser-Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SAWRHPQGG). Linker-HPQN can react with MCA to form compound 12g.

[0265]

[0266] Example 18 The expected preparation of compound 6i: Compound 4 in Route 1 can be replaced with, for example, a linker-decapeptide derivative 4h having the sequence Ser-Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SAWRHPQGG), while replacing compound 1 with compound 1a to remove the octyl group, to generate 3a. Linker-SAWRHPQGG will react by directly connecting to MCA at the N-terminus of peptide 4g to form compound 6i.

[0267]

[0268] Example 19 Illustrate the biological activities of compounds 6 and 12.

[0269] Table 4 compares the structures of compounds 6 and 12. Table 5 lists their biological activities.

[0270] Table 4

[0271]

[0272] Table 5

[0273] Molecule <![CDATA[CC 50 (mg / mL)]]> <![CDATA[EC 50 (mol / L)]]> TI Compound 12, Route II 65 3 21.6

[0274] Above, CC 50 is the 50% cytotoxic concentration; EC 50 is the 50% effective / inhibitory concentration, and TI is the therapeutic index / ratio. A higher TI value is better as the ratio quantifies the safety of the active agent.

[0275] It is believed that virus inactivation occurs by disrupting viral synthesis within the cell by preventing the migration of proteins from the cell's organelles (i.e., Golgi apparatus, endoplasmic reticulum) and thus depriving the virus of the proteins required for synthesis. Inactivation can occur through protonated amino groups (NH3 +)It occurs by attacking the negatively charged RNA or DNA of the virus. For example, the virus can be a non-enveloped virus such as Coxsackievirus CVRS3. As an example of a lipid-enveloped virus such as human coronavirus, NH3+ can attack viral capsid assembly, and thus the virus will be defective and unable to leave the cell. It is believed that virus inactivation can be produced by inhibiting the production of viral proteases (e.g., Mpro and PLpro), which induce cleavage and capsid proteins. This may be the case for inactivating both enveloped and non-enveloped viruses.

[0276] It is believed that conjugation with biotin results in reduced cytotoxicity because the latter has its own transport system across the cell membrane (sodium-dependent multivitamin transporter or SMVT), thus bypassing damage to the membrane. There are several existing methods for molecules to pass through without damaging the cell membrane. For example, CSA-44 can enter the cell by diffusing through membrane channels or by disrupting the phospholipid layer. In the latter case, this mode of entry may cause the cell to leak internal organelles and ultimately be destroyed. The conjugation process can affect functional amine or / and alkyl groups by steric hindrance, and thus can reduce the chance of contact with the phospholipid layer.

[0277] Example 20 Describe the cytotoxicity assay of CSA-44 (control) in human primary cell cultures.

[0278] (1) In each well of two 12-well tissue culture plates, seed HFF (human foreskin fibroblasts) cells (2x10 5 ) with 2 ml of DMEM medium supplemented with 10% FBS. (2) Culture the cells for 3 days. Add CSA-44 to test cytotoxicity. Test eleven concentrations: 40 μg / ml, 30 μg / ml, 20 μg / ml, 17.5 μg / ml, 15 μg / ml, 12.5 μg / ml, 10 μg / ml, 7.5 μg / ml, 5 μg / ml, 2.5 μg / ml, and 1 μg / ml. The sample with a concentration of 0 μg / ml is used as a control. Test each concentration in duplicate with two wells. (3) Culture the cells with CSA-44 for 48 hours. Use trypan blue staining to determine the number of live and dead cells on a hemocytometer. Calculate the percentage of live and dead cells in each sample. The results show that the cytotoxic concentration CC 50 (the concentration of the active substance that reduces cell viability by 50%) value of CSA-44 is approximately 15 μg / ml, as Figure 1 shown.

[0279] Example 21 Describe the therapeutic activity of CSA-44 (control) against human coronavirus 229E.

[0280] (1) In each well of four 24-well tissue culture plates, inoculate HFF cells (5x10 4 ) with 1 ml of DMEM medium supplemented with 10% FBS. (2) Culture the cells for 3 days until the HFF cells in each well reach 90% confluence (change the medium 1 day before infection). Add 10 μl of a 10-fold diluted original solution of the coronavirus (about 1x10 5 plaque-forming units, PFU) to each well. Allow infection to occur overnight. (3) Change the medium 24 hours after infection. Add CSA-44 to test its antiviral effect. Ten different concentrations of the drug were tested. They were 15 μg / ml, 10 μg / ml, 9 μg / ml, 8 μg / ml, 7 μg / ml, 6 μg / ml, 5 μg / ml, 4 μg / ml, 3 μg / ml, and 2 μg / ml, with 0 μg / ml as the control. Each concentration had 12 wells for testing. The cells were cultured for two weeks, and cell viability was quantified. CSA-44 effectively inhibited virus replication and growth. The EC 50 value (where EC is the concentration of the active substance that gives 50% of the maximum response) of CSA-44 was determined to be 4 μg / ml.

[0281] Example 22 Demonstrate the therapeutic activity of CSA-44 (control) against human coronavirus.

[0282] (1) In each well of four 24-well tissue culture plates, inoculate HFF cells (5x10 4 ) with 1 ml of DMEM medium supplemented with 10% FBS. (2) Culture the cells for 3 days until the HFF cells in each well reach 90% confluence (change the medium 1 day before infection). Add 10 μl of a 10-fold diluted original solution of the coronavirus (about 1x10 5 PFU) to each well. Allow infection to occur overnight. (3) Change the medium 24 hours after infection. Add different concentrations of CSA-44 to test its antiviral effect. Each concentration had 12 wells for testing. The cells were cultured in the presence of CSA-44 for 3 weeks, and cell viability was quantified. The results showed that CSA-44 effectively inhibited virus replication and growth. The EC 50 value of CSA-44 was estimated to be 4 μg / ml.

[0283]

[0284] Example 23 Demonstrate the determination of cell proliferation of the embodiments of the present disclosure.

[0285] The effect of Compound 12 on cell proliferation was determined using Calu-3 cells (ATCC# HTB-55), which are a transformed cell type commonly used to test cytotoxicity and antiviral activity.

[0286] The cells were cultured in MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin (Pen-strep). The cells were plated at 10,000 cells / well in 50 μl of growth medium in a white clear-bottom 96-well microplate. The next day, 2x compound dilutions were prepared in growth medium. 50 μl of the compound dilutions were added to the cells to achieve the desired final concentrations. 100 μl of growth medium was added to the cell-free control wells for measuring background luminescence. The cells were cultured at 37 °C and 5% CO2 for 3 days. At the end of the culture, the CellTiter Glo assay was performed as per the instructions: 100 μl of the reagent was added to each well and luminescence was read using a luminometer (BioTek Synergy TM 2 microplate reader).

[0287] The results showed that the CC 50 of Compound 12 was >200 μM. The CC 50 is the concentration of the test compound required to reduce cell viability by 50%.

[0288] Example 24 Description of the cytotoxicity assay of the CMA conjugate (Compound 12) in human primary cell cultures.

[0289] (1) In each well of two 12-well tissue culture plates, HFF cells (2x10 5 ) were seeded with 2 ml of DMEM medium supplemented with 10% FBS. (2) The cells were cultured for 3 days. The CMA conjugate was added to test cytotoxicity. 11 concentrations were tested. They were 100 μg / ml, 80 μg / ml, 60 μg / ml, 50 μg / ml, 40 μg / ml, 30 μg / ml, 25 μg / ml, 20 μg / ml, 15 μg / ml, 10 μg / ml, and 5 μg / ml. The sample with a concentration of 0 μg / ml was used as a control. Each concentration was tested in duplicate in two wells. (3) The cells were cultured with the CMA conjugate for 48 hours. Trypan blue staining was used to determine the number of live and dead cells on a hemocytometer. The percentage of live and dead cells in each sample was calculated. The results showed that the cytotoxic concentration CC 50 (the concentration of the active substance that reduces cell viability by 50%) of the CMA conjugate (Compound 12) was approximately 65 μg / ml, as Figure 2 shown.

[0290] Example 25Describe the cytotoxicity assay of the CMA conjugate (Compound 12).

[0291] One day before the test, Calu-3 cells were seeded in 96-well plates. At 80%-90% confluence, the growth medium was removed and replaced with medium containing a 50 μg / mL dilution of Compound 12. All treatments were added in quadruplicate or quintuplicate wells. The plates were incubated for 24 hours, then washed and replaced with fresh, untreated growth medium. MTS was added to each well according to the manufacturer's protocol (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) and incubated for 3 - 4 h.

[0292] After incubation, absorbance was measured at 490 nm on a SpectraMax M5 (Molecular Devices, LLC, San Jose, CA, USA) plate reader using SoftMax Pro 6.2.1 software. Figure 3 Show that Compound 12 has an IC of 154 μM 50 (i.e., CC 50 ).

[0293] Example 26 Describe the therapeutic and prophylactic activities of the CMA conjugate (Compound 12) against human coronavirus 229E.

[0294] A. Therapeutic activity of the CMA conjugate (Compound 12)

[0295] (1) In each well of four 24-well tissue culture plates, HFF cells (5 x 10 4 ) were seeded with 1 ml of DMEM medium supplemented with 10% FBS. (2) The cells were cultured for 3 days until the HFF cells in each well reached 90% confluence (the medium was changed 1 day before infection). 10 μl of a 10-fold diluted original solution of the coronavirus (approx. 1 x 10 5 PFU) was added to each well. Infection was allowed to proceed overnight. (3) The medium was changed 24 hours after infection. The CMA conjugate was added to test its antiviral effect. 10 different concentrations of the drug were tested. They were 15 μg / ml, 10 μg / ml, 9 μg / ml, 8 μg / ml, 7 μg / ml, 6 μg / ml, 5 μg / ml, 4 μg / ml, 3 μg / ml, and 2 μg / ml, with 0 μg / ml as the control. Each concentration had 12 wells for testing. The cells were cultured for two weeks, and cell viability was quantified. The CMA conjugate effectively inhibited virus replication and growth. Determine the EC of the CMA conjugate 50The value (where EC is the concentration of the active substance that gives 50% of the maximum response) is 3 μg / ml.

[0296] Preventive activity of the B.CMA conjugate (Compound 12)

[0297] A series of experiments were conducted to study whether cells could uptake Compound 12. In this study, human foreskin fibroblasts were used as a human primary cell culture model. The cells were cultured with 5 μg / ml of Compound 12 at different time points, then fixed and stained with avidin-conjugated fluorophore, and visualized with a fluorescence microscope. Within 15 minutes, it was found that the cells cultured with Compound 12 contained fluorescence. After 3 hours of culture, the fluorescence signal was still strong. These results indicate that the uptake is rapid and efficient, and Compound 12 is stable in the cells. In addition, Compound 12 seems to be mainly located in the cytoplasm, such as in the cellular membranous system (e.g., ER, Golgi apparatus, trans-Golgi network, etc.). Similar results were also found in cells infected with human coronavirus 229E. These results suggest that Compound 12 can be internalized into the cytoplasm of human cells, including the membranous system.

[0298] The effect of Compound 12 was studied in cellular structures and organelles such as the Golgi apparatus, mitochondria, and endoplasmic reticulum (ER). Treatment with Compound 12 at a concentration below the CC 50 value did not affect the overall structure and organization of these cellular structures and organelles. In summary, this study shows that (a) Compound 12 enters human cells, (b) Compound 12 seems to be mainly distributed and localized in the cytoplasm of human cells, including the membranous system, such as the Golgi apparatus and trans-Golgi network, and (c) Compound 12 does not seem to have a significant or substantial effect on the overall structure and organization of many intracellular structures and organelles (such as the Golgi apparatus and ER).

[0299] Example 27 Illustrate the mode of action (MOA) of the embodiments of the present disclosure.

[0300] Compound 12 has been measured to produce a high level of antiviral activity against 229E HCV after viral infection of cells, indicating that the compounds of the present disclosure are intracellular and can reduce the viral titer by targeting the released viral genetic material rather than the lipid viral envelope (which is lost upon entry into the cell).

[0301] In the "disinfectant" experiment, a stock solution of Compound 12 was produced by dissolving the compound in a 10% DMSO solution. Different amounts of Compound 12 from the stock solution were combined with 10 4Mixing of coronavirus 229E with PFU. After incubation at room temperature for 15 minutes, the mixture of the virus and compound 12 was added to the culture of primary human foreskin fibroblasts (HFF). At 24 hours post-infection, the medium was changed and the infected HFF were further cultured in the absence of compound 12. The levels of virus infection and inhibition produced were measured. The results showed that compound 12 had an EC 50 (μg / ml).

[0302] Multiple modes of action were confirmed by (1) limited envelope disruption in the assay above and (2) inhibition of 3CL protease (below).

[0303] The compound was dissolved in DMSO. A series of dilutions were prepared in 5% DMSO in 3CL protease assay buffer. 10 μl of the dilution was added to a 50 μl reaction such that the final concentration of DMSO was 1% in all reactions. The compound was pre-incubated in duplicate at room temperature for 30 minutes in a mixture containing 3CLPro assay buffer, 3CL enzyme, and the test compound. After 30 minutes, the enzymatic reaction was initiated by adding the 3CL protease substrate. The enzymatic reaction was carried out at room temperature for 4 hours. Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at an excitation of 360 nm and an emission of 460 nm.

[0304] 3CL protease (SARS-CoV-2) inhibitor assay #110922: IC 50 > 10 μM; 24% inhibition at 10 μM

[0305]

[0306] Figure 4 The protease activity of compound 12 of the present disclosure is shown. Protease inhibitors (PIs) are antiviral drugs that act by interfering with enzymes that cut proteins. Simply put, the PIs of the present disclosure prevent the virus from replicating itself. The calculated IC 50 is > 10 μM and there is 24% inhibition at 10 μM.

[0307] Example 28 Illustrates the activation of TNF-α by the compounds of the present disclosure.

[0308] It was confirmed that compound 12 stimulated the production of TNF-α in HEK293 cells. For example, 5 μg / ml of compound 12 induced cells to produce 7123 pg / ml of TNF-α, while untreated was -58.8 pg / ml.

[0309] The virus enters cells through angiotensin-converting enzyme-2 (ACE-2) and is (essentially) sensed by Toll-like receptor 7 (TLR7) present in endosomes. TLR7 activation leads to the production of interferon-α, TNF-α and the secretion of interleukin (IL)-12 and IL-6. This results in the formation of CD8+ specific cytotoxic T cells and, through CD4 + helper T cells lead to the formation of antigen-specific B cells and antibody production. This adaptive immune response controls viral infection and determines clinical recovery.

[0310] Example 29 Illustrate the antiviral activity of the compounds of the present disclosure.

[0311] Calu3 cells were seeded into 24-well plates until approximately 80% confluence. The cell culture medium was replaced with medium supplemented with compound 12 at 100 μM or an equivalent volume of vehicle solution (0.01% lactic acid) for a 1-hour incubation period. After incubation, the cells were inoculated with variant WA or strain BA.5 of the SARS-COV-2 virus at an MOI of 0.1 for a 1-hour adsorption period. After adsorption, the cells were washed with PBS to remove the virus, and then medium supplemented with compound 12 at 100 μM or an equivalent volume of vehicle solution medium was administered. The virus supernatant was collected 24 hours post-infection (hours post-infection (hpi)), and the medium was replenished with medium supplemented with compound 12 at 100 μM or an equivalent volume of vehicle solution. The collection and drug replenishment were repeated until 72 hpi.

[0312] Figure 5A Shows the reduction of virus strain WA in the presence of compound 12. Figure 5B Shows the reduction of virus strain BA.5 in the presence of compound 12.

[0313] Example 30 Illustrate the in vivo antiviral activity of the compounds of the present disclosure.

[0314] Figure 6 Shows a reduction in the in vivo viral load in the lung tissue of mice administered compound 12 compared to control mice not administered the compounds of the present disclosure. In the group administered compound 12, a significant (30 - 200-fold) reduction in virus replication and production was recorded at 2 and 3 days post-treatment. The results indicate that compound 12 inhibits HCV infection and growth in mice. The rebound seen at 6 days strongly suggests that a second dose (treatment) is required 2 - 3 days post-infection, or a single 1.0 mg / ml dose treatment would be effective.

[0315] Example 31 Illustrate the histopathological studies of infected and non-infected mice.

[0316] This example uses the following groups:

[0317] Group A : No infection, treated with PBS: Ten (10) 6-week-old female BALB / c mice were used for this group. First, the mice were anesthetized with isoflurane. Each mouse was treated intranasally with one dose of PBS (75 μl). The mice were sacrificed at 72 hours and 7 days after treatment. The lungs of these mice were collected for immunohistochemistry and cytokine analysis.

[0318] Group B : No infection, treated with Compound 12: Twenty (20) 6-week-old female BALB / c mice were used for this group. First, the mice were anesthetized with isoflurane. Each mouse was treated intranasally with one dose of 0.5 mg of Compound 12 diluted in PBS (75 μl). The mice were sacrificed at 24 hours, 48 hours, 72 hours, and 7 days after treatment. The lungs of these mice were collected for immunohistochemistry and cytokine analysis.

[0319] Group C : Infected with 229E, treated with PBS: Thirty-five (35) 6-week-old female BALB / c mice were used for this group. First, the mice were anesthetized with isoflurane. Each mouse was infected intranasally with 1×10 5 plaque-forming units (pfu) of HCoV-229E, and then treated with PBS (75 μl) 24 hours later. The mice were sacrificed at 24 hours after infection and at 24 hours, 48 hours, 72 hours, 6 days, and 7 days after treatment. The lungs of these mice were collected for viral load, immunohistochemistry, and cytokine analysis.

[0320] Group D : Infected with 229E, treated with Compound 12: Forty-five (45) 6-week-old female BALB / c mice were used for this group. First, the mice were anesthetized with isoflurane. Each mouse was infected intranasally with 1×10 5 pfu of HCoV-229E, and then treated with 0.5 mg of Compound 12 diluted in PBS (75 μl) for 24 hours. Mice from this group were sacrificed at 24 hours, 48 hours, 72 hours, 6 days, and 7 days after treatment. The lungs of these mice were collected for viral load, immunohistochemistry, and cytokine analysis.

[0321] H&E staining showed that lung tissues from non-infected mice showed normal morphology (Group A ( Figure 7A - B )); Group B ( Figure 7C - D ) 3 days after treatment). For tissues from Group D (infected mice treated with Compound 12), normal lung morphology was observed ( Figure 7G - H ), while in Group C (infected mice treated with PBS), the mice showed structural changes in the lungs ( Figure 7E - F ), which were consistent with HCV infection.

[0322] The staining and imaging results showed the successful delivery of Compound 12 in lung tissue, which was detected 1 - 2 days after treatment but not detected 3 days after treatment. At 3 days after treatment, administration of Compound 12 did not cause significant adverse effects on the morphology of the lung. These results indicate that Compound 12 does not exhibit significant toxicity in vivo at the dose used (0.5 mg / mouse). The results also showed subtle changes in the lung structure, associated with early signs of viral infection in Group C mice and relatively normal lung morphology in Group D mice. These results imply the antiviral activity and beneficial effects of the treatment.

[0323] Example 32 Illustrate that the embodiments of the present disclosure modulate cytokines as immunomodulators.

[0324] Figure 8A The comparison between Group C (control) and Group D (treated with Compound 12) is shown. The following cytokines were significantly reduced with treatment: IL-3, IL-17, CXCL11, CCL3, CCL4, and TNFα. The compounds of the present disclosure modulate cytokines and chemokines to reduce cytokine storms, sepsis, and other immune system-mediated responses, such as autoimmune disorders.

[0325] Figure 8B The comparison between Group B (no infection, treated with Compound 12) and Group D (HCoV-229 infected, treated with Compound 12) is shown, in which significant changes occurred in the relative levels of CCL11, IL-5, IL-3, GM-CSF, CXCL11, IL-10, CCL4, CCL1 / TCA, G-CSF, CCL17, IL-4, IL-17, BLC, TIMP-1, IL-7, CCL5, TNF-α, CXCL2.

[0326] Example 33 Illustrate the fungicidal and microbicidal activities of the embodiments of the present disclosure.

[0327] The following table shows that Compound 12 is active against yeasts and bacteria.

[0328]

[0329] Example 34 Illustrate the pH stability of the embodiments of the present disclosure.

[0330] The pH of an aqueous solution is a factor to consider for a drug formulated in an aqueous liquid. Compound 12 is stable at pH 7.0 (up to 7 days), which makes it easy to administer to the body. Using pH 7.0, molecular stability studies conducted using NMR analysis showed that it was stable for up to 7 days.

[0331] The pH of an aqueous solution is a factor considered for drugs prepared in the form of an aqueous liquid. The potential impact of pH on solubility is a factor in the stability of the drug to be administered. pH is important for the living system. The stability at pH 7 allows it to be formulated in a wide variety of components.

[0332] This property of stability at neutral pH is the factor behind its non-toxicity to human cells. Compound 12 has a CC 50 > 200 μM against transformed cells, and such cells tend to be more sensitive to many agents. In wound healing and in vivo experiments, it has been shown that a long-term strongly acidic wound environment prevents wound closure and re-epithelialization, while a long-term alkaline environment has no negative impact on wound closure or re-epithelialization. In addition, both in vitro and in vivo studies have shown that long-term acidic conditions significantly increase the expression of IL-1α in fibroblast cultures and wound fluid, while long-term alkaline conditions do not result in an elevated amount of IL-1α. The latter is interleukin 1α (IL-1α), a potent inflammatory cytokine that activates the inflammatory process, and its dysregulated signaling leads to destructive diseases manifested as severe acute or chronic inflammation. Therefore, wound treatment at neutral pH is highly preferred.

[0333] All references throughout this application, such as patent documents, including published or granted patents or equivalents and patent application publications, as well as non-patent literature materials or other original materials, are hereby incorporated by reference in their entirety as if individually incorporated by reference.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: Wherein: R 1 -R 4 、R 6 、R 7 、R 11 、R 12 、R 15 and R 16 each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl formamido, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl; R 5 、R 8 、R 9 、R 10 、R 13 and R 14 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl; L 1 is a linking group; and B 1 is biotin, desthiobiotin or a biotin mimetic.

2. The compound according to claim 1, wherein R 3 , R 7 and R 12 are each independently selected from: substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkylcarboxy, substituted or unsubstituted (C1-C 10 ) aminoalkylaminocarbonyl and substituted or unsubstituted (C1-C 10 ) aminoalkylformamido; and R 5 , R 9 and R 13 are independently selected from hydrogen, hydroxyl and substituted or unsubstituted (C1-C 10 )alkyl.

3. The compound according to any one of claims 1-2, wherein R 3 , R 7 and R 12 are each a substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, wherein the amino group is optionally a cationic amine salt; and R 5 、R 9 and R 13 each independently represents hydrogen or a substituted or unsubstituted (C1-C3) alkyl group.

4. The compound according to any one of claims 1-3, wherein L 1 is -L-Y-Z, where L is independently selected from: a bond, a substituted or unsubstituted (C1-C 10 ) alkyl, a substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, a (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, a (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, a substituted or unsubstituted (C1-C 10 ) aminoalkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, an oxo group, a substituted or unsubstituted (C1-C 10 ) aminoalkoxy, a substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, a substituted or unsubstituted (C1-C 10 ) alkyl formamido, a substituted or unsubstituted (C1-C 10 ) alkyl formamido (wherein the amino is substituted), and a substituted or unsubstituted (C1-C 10 ) alkylaminocarbonyl; Y is optionally present and is selected from: substituted or unsubstituted (C1-C 10 ) alkyl heterocyclic group (C1-C 10 ) alkyl formylamino, substituted or unsubstituted (C1-C 10 ) alkyl heterocyclic group, substituted or unsubstituted (C1-C 10 ) alkyl heterocyclic group (C1-C 10 ) alkyl carboxyl and substituted or unsubstituted (C1-C 10 ) alkyl heterocyclic group (C1-C 10 ) alkyl aminocarbonyl; and Z is selected from: substituted or unsubstituted C1-C 30 alkylene, C1-C 30 alkenylene, wherein the alkylene or alkenylene is optionally inserted with at least one heteroatom, substituted or unsubstituted (C1-C 10 ) alkylamino, substituted or unsubstituted (C1-C 10 ) alkylcarbonyl, PEG 1-30 , the PEG 1-30 is optionally terminated with a member selected from: a bond, -O-, -S-, -NH-, -NHC(O)-, and -C(O)NH-.

5. The compound according to any one of claims 1-4, wherein B 1 is biotin.

6. The compound according to any one of claims 1 - 5, wherein the compound is selected from:

7. The compound according to any one of claims 1-4, wherein B 1 is desthiobiotin.

8. The compound according to claim 7, wherein the compound is selected from:

9. The compound according to any one of claims 1-4, wherein B 1 is a biotin mimetic.

10. The compound according to claim 9, wherein the biotin mimetic compound is selected from:

11. The compound according to any one of claims 1 - 10, wherein the compound has a therapeutic or prophylactic index greater than 3 or at least 15.

12. A method for treating a microbial infection or delaying microbial spread in an individual, the method comprising: Contacting the microorganism with an antimicrobial amount of a compound of formula I or a pharmaceutically acceptable salt thereof: Wherein: R 1 -R 4 、R 6 、R 7 、R 11 、R 12 、R 15 and R 16 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl formamido, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl; R 5 、R 8 、R 9 、R 10 、R 13 and R 14 are each independently selected from: hydrogen, hydroxy, substituted or unsubstituted (C1-C 10 ) alkyl, (C1-C 10 ) hydroxyalkyl, (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl, substituted or unsubstituted aryl, (C1-C 10 ) haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, oxo, substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, (C1-C 10 ) azidoalkoxy, (C1-C 10 ) cyanoalkoxy, (C1-C 10 ) guanidinoalkoxy and (C1-C 10 ) guanidinoalkyl carboxyl; L 1 is a linking group; and B 1 is biotin, desthiobiotin or a biotin mimetic, wherein the compound of formula I kills the microorganism or slows the spread of the microorganism.

13. The method according to claim 12, wherein R 3 , R 7 and R 12 are each independently selected from: substituted or unsubstituted (C1-C 10 ) aminoalkoxy, substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, substituted or unsubstituted (C1-C 10 ) aminoalkyl aminocarbonyl, and substituted or unsubstituted (C1-C 10 ) aminoalkyl formamido; and R 5 , R 9 and R 13 are independently selected from hydrogen, hydroxyl and substituted or unsubstituted (C1-C 10 )alkyl.

14. The method according to any one of claims 12-13, wherein R 3 , R 7 and R 12 are each a substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, wherein the amino group is optionally a cationic amine salt; and R 5 、R 9 and R 13 are each independently hydrogen or a substituted or unsubstituted (C1-C3) alkyl group.

15. The method according to any one of claims 12 - 14, wherein L 1 is -L-Y-Z, where L is independently selected from: a bond, a substituted or unsubstituted (C1-C 10 ) alkyl, a substituted or unsubstituted (C1-C 10 ) alkyl carboxyl, a (C1-C 10 ) alkoxy-(C1-C 10 ) alkyl, a (C1-C 10 ) alkylamino-(C1-C 10 ) alkyl, a substituted or unsubstituted (C1-C 10 ) aminoalkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, an oxo group, a substituted or unsubstituted (C1-C 10 ) aminoalkoxy, a substituted or unsubstituted (C1-C 10 ) aminoalkyl carboxyl, a substituted or unsubstituted (C1-C 10 ) alkylformamido, a substituted or unsubstituted (C1-C 10 ) alkylformamido (wherein the amino group is substituted), and a substituted or unsubstituted (C1-C 10 ) alkylaminocarbonyl; Y is optionally present and is selected from: substituted or unsubstituted (C1-C 10 ) alkyl heterocyclyl (C1-C 10 ) alkylformamido, substituted or unsubstituted (C1-C 10 ) alkyl heterocyclyl, substituted or unsubstituted (C1-C 10 ) alkyl heterocyclyl (C1-C 10 ) alkyl carboxyl and substituted or unsubstituted (C1-C 10 ) alkyl heterocyclyl (C1-C 10 ) alkylaminocarbonyl; and Z is selected from: substituted or unsubstituted C1-C 30 alkylene, C1-C 30 alkenylene, wherein said alkylene or alkenylene is optionally inserted with at least one heteroatom, substituted or unsubstituted (C1-C 10 ) alkylamino, substituted or unsubstituted (C1-C 10 ) alkylcarbonyl, PEG 1-30 , said PEG 1-30 is optionally terminated with a member selected from: a bond, -O-, -S-, -NH-, -NHC(O)-, and -C(O)NH-.

16. The method according to any one of claims 12 - 15, wherein B 1 is biotin.

17. The method according to any one of claims 12 - 16, wherein the compound is selected from:

18. The method according to any one of claims 12-15, wherein B 1 is desthiobiotin.

19. The method according to claim 18, wherein the compound is selected from:

20. The method according to any one of claims 12 - 15, wherein B 1 is a biotin mimic.

21. The method according to claim 20, wherein the compound is selected from:

22. The method according to any one of claims 12 - 20, wherein the microorganism is selected from microorganisms, bacteria, viruses, and fungi.

23. The method according to claim 22, wherein the microorganism is a virus.

24. The method according to claim 23, wherein the virus is selected from coronavirus, RSV, influenza A, herpes simplex virus type I, herpes simplex virus type II, and norovirus.

25. The method according to claim 24, wherein the coronavirus is SARS-CoV-2.

26. The method according to any one of claims 12 - 25, wherein the step of contacting the microorganism is carried out directly or indirectly by intervening in the viral life cycle.

27. The method according to any one of claims 12 - 26, wherein the method provides a beneficial effect in vivo as an immunomodulator for modulating a toxic immune response to a disease.

28. The method according to any one of claims 12 - 27, wherein the compound of formula I is formulated in a preparation having a pH of about 6 to about 8.

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