ROS-activated co prodrugs for cancer treatment

Abstract

This application generally relates to carbon monoxide releasing compounds and compositions, and their use as carbon monoxide prodrugs. The compounds and composition can be used for treating cancer in a subject. Kits comprising the compounds and compositions described herein are also provided. Also described herein are methods of delivering carbon monoxide to a cell.

Description

ROS-ACTIVATED CO PRODRUGS FOR CANCER TREATMENTCROSS-REFERENCE TO RELATED APPLICATIONThis application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 593,452, filed on October 26, 2023, which is hereby incorporated by reference in its entirety for all purposes.BACKGROUNDCarbon monoxide (CO), generated by heme oxygenase-mediated heme degradation, is an endogenous signaling molecule with strong cytoprotective and anti-inflammatory effects. There have been two general types of CO donors: metal-carbonyl complexes and metal-free donors.SUMMARYDescribed herein are compounds comprising a cyclopentenone moiety and a reactive moiety, wherein exposure of the compounds to reactive oxygen species (ROS) under physiological conditions results in formation of the oxidation of a sulfide group to a sulfoxide group (a leaving group). Subsequent elimination of the sulfoxide leads to the generation of a norbonadien-7-one scaffold and the consequent release of carbon monoxide via a cheletropic reaction. Also described herein are methods of using these compounds. Optionally, the compounds can be used as carbon monoxide (CO) prodrugs.In some examples, the disclosure is directed to compounds of the following structures:and pharmaceutically acceptable salts thereof.Also described herein is a compound of Formula I:wherein R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether; R3is OR4or COOR5; R4is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; and R5is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; or a pharmaceutically acceptable salt thereof.Also described herein are compounds of Formula II:wherein R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether; and R6is H, a substituted alkyl, or an unsubstituted alkyl; and n is an integer from 0 to 20; or a pharmaceutically acceptable salt thereof.Also described herein is a compound of Formula Ila:wherein n is 3 or 4, or a pharmaceutically acceptable salt thereof.Also described herein is a compound of Formula lib :wherein n is 3 or 4, or a pharmaceutically acceptable salt thereof.Pharmaceutical compositions and methods of treating diseases with carbon monoxide prodrugs are also described herein.The details of one or more examples are set forth in the drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1, Panel A, is a plot showing CO release kinetics of compound 7a. FIG. 1, Panel B is a plot showing CO release kinetics of compound 7b.FIG. 2, Panel A, is a plot showing CO release kinetics of compound 14a. FIG. 2, Panel B is a plot showing CO release kinetics of compound 14b.FIG. 3, Panel A, is a plot showing a CO-myoglobin assay of compound 7a. FIG. 3, Panel B is a plot showing the CO release kinetics of compounds 7a and 14a. FIG. 3, Panel C is a plot showing CO release in response of compound 7a to different ROS species. FIG. 3, Panel D is a plot showing CO release in response of compound 14a to different ROS species.FIG. 4, Panel A, is a plot showing cell-imaging studies of CO release from compounds 7a and 14a in 4T1 cells. FIG. 4, Panel B is a plot showing red fluorescence intensity of the cell-imaging studies shown in FIG. 4, Panel A.FIG. 5, Panel A, is a plot showing the cytotoxicity studies of some of the compounds described herein in 4T1 cells. FIG. 5, Panel B is a plot showing the cytotoxicity studies of compounds described herein in Panc02 cells.FIG. 6, Panel A, shows microscopic photographs of 4T1 tumor sections after administration of compounds described herein. FIG. 6, Panel B is a plot showing the corresponding red fluorescence intensity of FIG. 6, Panel A.FIG. 7, Panel A, is a schematic illustration of an experimental schedule for in vivo antitumor therapy. FIG. 7, Panel B is a plot showing tumor growth profiles of mice bearing 4T1 subcutaneous tumors that were subjected to various treatments as described herein. FIG. 7, Panel C is a plot showing individual tumor growth curves of 4T1 tumor-bearing mice after various treatments described herein. FIG. 7, Panel D is a plot showing the body weight changes of each group of mice after undergoing some of the various treatments described herein. FIG. 7, Panel E is a plot showing the survival curves of each group of mice after undergoing some of the various treatments described herein.FIG. 8 shows a scheme of some of the compounds reacting with H2O2 (top) and HPLC traces of some of the compounds described herein in the presence and absence of H2O2 (bottom).FIG. 9 shows a scheme of some of the compounds reacting with H2O2 (top) and HPLC traces of some of the compounds described herein in the presence and absence of H2O2 (bottom).FIG. 10 shows a scheme of some of the compounds reacting with H2O2 (top) and HPLC traces of some of the compounds described herein in the presence and absence of H2O2 (bottom).FIG. 11, Panel A, shows HPLC studies of CO release reaction by a compound described herein. FIG. 11, Panel B shows CO release kinetics from a compound described herein.FIG. 12, Panel A, shows the anti-inflammatory effects of compounds described herein and their inactive products using the TNF-a level as an indicator. FIG. 12, Panel B shows cytotoxicity studies of compounds described herein and their inactive products using a MTT assay.FIG. 13, Panels A-D, show fluorescence microscopy images of CO in live cells that were treated with compounds described herein.FIG. 14, Panels A-D, show HPLC analysis of intracellular concentrations of compounds described herein.FIG. 15, Panels A-C, show in vivo COHb profiles in mice after oral administration of compounds described herein.DETAILED DESCRIPTIONI. GeneralDescribed herein are molecules that can release CO in vivo and in vitro in response to reactive oxygen species (ROS) . Methods for treating diseases and conditions using the carbon monoxide releasing compounds (also referred to as “CO prodrugs”) are also described. Metal-free, ROS-sensitive CO prodrugs are provided. In certain examples, the ROS-sensitive prodrugs are stable under physiological conditions unless exposed to ROS. The CO release after ROS-oxidation is also pH dependent with the ability to release CO at physiological pH (7.4). Release rate is diminished at lower pH. The tunable and predictable release rates exhibited by the compounds provides for lower gastrointestinal (GI) delivery in disease conditions where release in the upper GI tract is not needed or is counter-indicated. The oxidation-sensitive CO prodrugs can selectively deliver CO to cells with elevated ROS levels (e.g., cancer cells and inflammatory cells).II. DefinitionsAs used herein, the term “physiological conditions” refers to one or more of physiological temperature, pH, and tonicity. Body temperature is typically from about 33 °C to about 40 °C, preferably from about 35 °C to about 38 °C, such as about 37 °C. Physiological pH is typically from about 6.8 to 8, preferably 6.8 to about 7.5, such as about 7.0. However, the pH may be lower or higher at specific sites and / or due to a particular disease state. For example, the pH of gastric acid ranges from around 1.5 to around 3.5 in the human stomach lumen, and lower pH is often associated with diseased tissue such as tumor tissue.As used herein, the term “alkyl,” by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C1-2, C1-3, Ci-4, C1-5, C1-6, C1-7, C1-8, C1-9, Ci-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. For example, C1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted alkyl”groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.As used herein, the term “alkoxy,” by itself or as part of another substituent, refers to a group having the formula -OR, wherein R is alkyl as described above.As used herein, the term “thioether,” by itself or as part of another substituent, refers to a group having the formula -SR, wherein R is alkyl as described above.As used herein, the term “selenoether,” by itself or as part of another substituent, refers to a group having the formula -SeR, wherein R is alkyl as described above.As used herein, the term “cycloalkyl,” by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbomadiene. When cycloalkyl is a saturated monocyclic C3-8 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted cycloalkyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.As used herein, the term “alkylene” refers to an alkyl group, as defined above, linking at least two other groups (i.e., a divalent alkyl radical). The two moi eties linked to the alkylene group can be linked to the same carbon atom or different carbon atoms of the alkylene group.As used herein, the terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.As used herein, the term “haloalkyl,” by itself or as part of another substituent, refers to an alkyl group where some or all of the hydrogen atoms are replaced with halogen atoms. As for alkyl groups, haloalkyl groups can have any suitable number of carbon atoms, such as Ci-6. For example, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In some instances, the term “perfluoro” can be used to define a compound or radical where all the hydrogens are replaced with fluorine. For example, perfluoromethyl refers to 1,1,1 -trifluoromethyl .As used herein, the term “aryl,” by itself or as part of another substituent, refers to an aromatic ring system having any suitable number of carbon ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as Ce, C7, Cs, C9, C10, C11, C12, C13, C14, C15 or Ci6, as well as Ce-io, C6-12, or Ce-14. Aryl groups can be monocyclic, fused to form bicyclic (e.g., benzocyclohexyl) or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted aryl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.As used herein, the term “heteroaryl,” by itself or as part of another substituent, refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Heteroaryl groups can include any number of ring atoms, such as C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, or C3-12, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4; or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. For example, heteroaryl groups can be C5-8 heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C5-8 heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms; or C5-6 heteroaryl, wherein 1 to 4 carbon ring atoms are replaced with heteroatoms; or C5-6 heteroaryl, wherein 1 to 3 carbon ring atoms are replaced with heteroatoms. The heteroaryl group can include groups such as pyrrole, pyridine, imidazole,pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3, 5 -isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroaryl groups can also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted. Unless otherwise specified, “substituted heteroaryl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.The heteroaryl groups can be linked via any position on the ring. For example, pyrrole includes 1-, 2- and 3 -pyrrole, pyridine includes 2-, 3- and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazole includes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and 5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes 2-, 4-, 5- and 6- pyrimidine, pyridazine includes 3- and 4-pyridazine, 1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-, 5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiophene includes 2- and 3 -thiophene, furan includes 2- and 3 -furan, thiazole includes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and 5-isothiazole, oxazole includes 2-, 4- and 5- oxazole, isoxazole includes 3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindole includes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline, isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2- and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline, benzothiophene includes 2- and 3 -benzothiophene, and benzofuran includes 2- and 3 -benzofuran.Some heteroaryl groups include those having from 5 to 10 ring members and from 1 to 3 ring atoms including N, O or S, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include those having from 5 to 8 ring members and from 1 to 3 heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Some other heteroaryl groups include those having from 9 to 12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, benzofuran and bipyridine.Still other heteroaryl groups include those having from 5 to 6 ring members and from 1 to 2 ring atoms including N, O or S, such as pyrrole, pyridine, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.Some heteroaryl groups include from 5 to 10 ring members and only nitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, and cinnoline. Other heteroaryl groups include from 5 to 10 ring members and only oxygen heteroatoms, such as furan and benzofuran. Some other heteroaryl groups include from 5 to 10 ring members and only sulfur heteroatoms, such as thiophene and benzothiophene. Still other heteroaryl groups include from 5 to 10 ring members and at least two heteroatoms, such as imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline, quinazoline, phthalazine, and cinnoline.As used herein, the term “heterocyclyl,” by itself or as part of another substituent, refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms of N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Heterocyclyl groups can include any number of ring atoms, such as, C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, or C3-12, wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of carbon ring atoms can be replaced with heteroatoms in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The heterocyclyl group can include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. The heterocyclyl groups can also be fused to aromatic or non-aromatic ring systems to form members including, but not limited to, indoline. Heterocyclyl groups can be unsubstituted or substituted. Unless otherwise specified, “substituted heterocyclyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo, alkylamino, amido, acyl, nitro, cyano, and alkoxy.The heterocyclyl groups can be linked via any position on the ring. For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or 2- azetidine, pyrrolidine can be 1-, 2-or 3 -pyrrolidine, piperidine can be 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or 4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine, piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1- or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine, isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be 2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or 5- isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.When heterocyclyl includes 3 to 8 ring members and 1 to 3 heteroatoms, representative members include, but are not limited to, pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane and dithiane. Heterocyclyl can also form a ring having 5 to 6 ring members and 1 to 2 heteroatoms, with representative members including, but not limited to, pyrrolidine, piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, and morpholine.As used herein, the term “protecting group” (or “PG”) refers to a chemical moiety that renders a functional group (e.g., an amino group) unreactive, but is also removable so as to restore the amino group. Examples of protecting groups include, but are not limited to, ethers (e.g., methoxymethyl ether, -methoxybenzyl ether, and the like); silyl ethers (e.g, trimethyl silyl ether, te / 7-butyl di phenyl si lyl ether, and the like); benzyloxycarbonyl (Z or Cbz); 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); allyloxycarbonyl (Alloc); - toluene sulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2, 2, 4,6,7- pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy- 2,3,6-trimethylphenylsulfonyl (Mtr); acetamido; phthalimido; and the like. Other protecting groups are known to those of skill in the art including, for example, those described by Green and Wuts (Protective Groups in Organic Synthesis, 4thEd. 2007, Wiley-Interscience, New York).As used herein, the term “carbonyl,” by itself or as part of another substituent, refers to — C(O)~ , i.e., a carbon atom double-bonded to oxygen and bound to two other groups in the moiety having the carbonyl.As used herein, the term “amino” refers to a moiety -NR2, wherein each R group is H or alkyl. An amino moiety can be ionized to form the corresponding ammonium cation. “Dialkylamino” refers to an amino moiety wherein each R group is alkyl.As used herein, the term “sulfonyl” refers to a moiety -SO2R, wherein the R group is alkyl, haloalkyl, or aryl. A sulfonyl moiety can be ionized to form the corresponding cation. “Alkyl sulfonyl” refers to a sulfonyl moiety wherein the R group is alkyl.As used herein, the term “hydroxy” refers to the moiety -OH.As used herein, the term “cyano” refers to a carbon atom triple-bonded to a nitrogen atom (z.e., the moiety -ON).As used herein, the term “carboxy” refers to the moiety -C(O)OH. A carboxy moiety can be ionized to form the corresponding carboxylate anion.As used herein, the term “ester” refers to the moiety -R(O)OR’, wherein R and R’ are independently selected from C1-8 alkyl, C3-8 cycloalkyl, or Ce-io aryl as described above.As used herein, the term “acyl” refers to a moiety-C(O)R, wherein each R group is C1-8 alkyl, C3-8 cycloalkyl, or Ce-io aryl as defined herein.As used herein, the term “amido” refers to a moiety -NRC(O)R or -C(0)NR2, wherein each R group is H or alkyl.As used herein, the term “nitro” refers to the moiety -NO2.As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (i.e., O=).As used herein, the term “solubilizing moiety” refers to a moiety used to increase the solubility of compounds described herein in a solvent (e.g., water or an organic solvent). Examples of solubilizing moieties include, but are not limited to, sugars (monosaccharides, oligosaccharides, and polysaccharides); polyols (e.g., glycerol, propylene glycol, and the like), synthetic polymers (e.g., hydrophilic polymers such as oligo(ethylene glycol), poly(ethylene glycols) (PEGs), poly-trimethylene glycols, poly(N-isopropylacrylamides) (NIP AMs), polyvinylpyrrolidones, polyoxyethylene-polyoxypropylene block copolymers, and the like) and biopolymers (e.g., proteins, starch, cellulose, heparin, hyaluronic acid, and the like).As used herein, the term “monosaccharide” refers to a sugar having a fivemembered carbon backbone (i.e., a pentose) or a six-membered carbon backbone (i.e., a hexose). Examples of monosaccharides include, but are not limited to, glucose, ribose, fucose, xylose, arabinose, galactose, mannose, glucuronic acid, and iduronic acid. Monosaccharides also include pentoses and hexoses substituted with hydroxy groups, oxo groups, amino groups, acetylamino groups, and other functional groups.As used herein, the term “oligosaccharide” refers to a compound containing at least two monosaccharides covalently linked together. Oligosaccharides include disaccharides,trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, and the like. Covalent linkages for linking sugars generally consist of glycosidic linkages (i.e., C-O-C bonds) formed from the hydroxyl groups of adjacent sugars. Linkages can occur between the 1 -carbon (the anomeric carbon) and the 4-carbon of adjacent sugars (i.e., a 1-4 linkage), the 1-carbon and the 3-carbon of adjacent sugars (i.e., a 1-3 linkage), the 1-carbon and the 6-carbon of adjacent sugars (i.e., a 1-6 linkage), or the 1- carbon and the 2-carbon of adjacent sugars (i.e., a 1-2 linkage). Other linkages can be present in the oligosaccharide, depending on the particular sugar subunits present. Those of skill in the art will appreciate that a sugar can be linked within an oligosaccharide such that the glycosidic bond at the anomeric carbon is in the a- or P-configuration.As used herein, the term “polysaccharide” generally refers to a compound containing 10 or more sugars linked together as described for oligosaccharides.As used herein, the term “salt” refers to acid or base salts of the compounds described herein. Illustrative examples of pharmaceutically acceptable salts include mineral acid salts (salts of hydrochloric acid, hydrobromic acid, phosphoric acid, and the like), organic acid salts (salts of acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, and quaternary ammonium salts (salts of methyl iodide, ethyl iodide, and the like). It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington: The Science & Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins, Philadelphia, Pa., 2000, which is incorporated herein by reference.Pharmaceutically acceptable salts of the acidic compounds as described herein are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl- ammonium salts.Similarly acid addition salts, such as of mineral acids, organic carboxylic and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, are also possible provided a basic group, such as pyridyl, constitutes part of the structure.The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compounds described herein.Compounds described herein include all tautomers and stereoisomers thereof, either in admixture or in pure or substantially pure form. The compounds described herein can have asymmetric centers at the carbon atoms, and therefore the compounds described herein can exist in diastereomeric or enantiomeric forms or mixtures thereof. All conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic, diastereomeric and other mixtures of such isomers, as well as solvates, hydrates, isomorphs, polymorphs and tautomers are within the scope of the compounds described herein. Compounds described herein can be prepared using diastereomers, enantiomers or racemic mixtures as starting materials. Furthermore, diastereomer and enantiomer products can be separated by chromatography, fractional crystallization or other methods known to those of skill in the art.Certain compounds described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the compounds described herein. Certain compounds described herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by this application and are intended to be within the scope of the compounds described herein.Any compound or formula given herein, is intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine and iodine, such as2H,3H,nC,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,36C1,123I and125I, respectively. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients.The disclosure also includes “deuterated analogs” of compounds described herein in which from 1 to n hydrogens attached to a carbon atom is / are replaced by deuterium, in which n is the number of hydrogens in the molecule. Such compounds exhibit increased resistance to metabolism and are thus useful for increasing the half-life of any compound when administered to a mammal, particularly a human. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Metabolism,” Trends Pharmacol. Sci. 5( 12): 524-527(1984). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced by deuterium.Deuterium labelled or substituted therapeutic compounds of the disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and / or an improvement in therapeutic index. An18F,3H,nC labeled compound may be useful for PET or SPECT or other imaging studies. Isotopically labeled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. It is understood that deuterium in this context is regarded as a substituent in a compound described herein.The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of this disclosure any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specifically as “H” or “hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition.As used herein, the term “pharmaceutical composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product, which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.As used herein, the term “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to a subject. By “pharmaceutically acceptable,” it is meant that the excipient is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof. Pharmaceutical excipients may include, but are not limited to, binders, fillers, disintegrants, lubricants, glidants, coatings, sweeteners, flavors and colors.As used herein, the terms “treat”, “treating,” and “treatment” refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, e.g., the result of a physical examination.As used herein, the terms “effective amount” and “therapeutically effective amount” refer to a dose of a compound, such as a carbon monoxide releasing compound, that brings about a result, e.g., a therapeutic effect, for which the compound was administered, and includes the maximally tolerable dose. When “effective amount” is used to describe an in vivo method, the desired result can refer to a therapeutic effect. When “effective amount” is used to describe an ex vivo method the desired results can refer to a detectable level of carbon monoxide. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman ’s The Pharmacological Basis of Therapeutics, 11thEdition, 2006, Brunton, Ed., McGraw-Hill; and Remington: The Science and Practice of Pharmacy, 2 I '1Edition, 2005, Hendrickson, Ed., Lippincott, Williams & Wilkins).The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.9X to 1. IX. “About X” thus includes, for example, a value from 0.95X to 1.05X, or from 0.98X to 1.02X, or from 0.99X to 1.01X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.07X, 1.08X, 1.09X, and 1.10X. Accordingly, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”III. Carbon Monoxide Releasing (Donor) CompoundsThe carbon monoxide releasing compounds described herein include a cyclopentenone moiety (e.g., a bicyclo[2.2.1]hept-2-en-7-one) and a reactive moiety. Exposure of the reactive moiety to ROS under physiological conditions, upon administration to a subject, results in oxidation and then elimination of the reactive moiety and formation of an unstable dienone intermediate (e.g., a bicyclo[2.2.1]hepta-2,5-dien-7-one) which undergoes a pericyclic fragmentation process to release carbon monoxide.In some cases, the CO prodrugs described herein include compounds as shown below:In some cases, the CO prodrugs described herein are compounds according toFormula I:wherein R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether; R3is OR4or COOR5; R4is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; and R5is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; or pharmaceutically acceptable salts thereof. In some cases, R1and R2are sulfonyl. In some cases, R1and R2are SOPI12. In some cases, R3is OR4and R4is H. In some cases, R3is COOR5and R5is H.In some cases, the CO prodrugs described herein are compounds according to Formula II:wherein R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether; R6is H, a substituted alkyl, or an unsubstituted alkyl; and n is an integer from 0 to 20; or pharmaceutically acceptable salts thereof. In some cases, R1and R2are sulfonyl. In some cases, R1and R2are SOPI12. In some cases, R6is H or CH3. In some cases, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 0 to 10. In some cases, n is 0 to 5.In some cases, the CO prodrugs described herein are compounds according to Formula Ila:wherein n is 3 or 4, or pharmaceutically acceptable salts thereof.In some cases, the CO prodrugs described herein are compounds according to Formula lib :wherein n is 3 or 4, or pharmaceutically acceptable salts thereof.The compounds described herein may be prepared using the methods disclosed herein and routine modifications thereof, which will be apparent given the disclosure herein and methods well known in the art. Conventional and well-known synthetic methods may be used in addition to the teachings herein. The synthesis of typical compounds describedherein may be accomplished as described in the following examples. It will be appreciated that where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in Wuts, Greene’s Protective Groups in Organic Synthesis, 5thEd., Wiley & Sons, 2014, which is incorporated herein by reference in its entirety, including references cited therein.Furthermore, the compounds of this disclosure may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this disclosure, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, and the like.The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka- Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd’s Chemistry of Carbon Compounds, Volumes 1-5, and Suppiementals (Elsevier Science Publishers, 1989) organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March’s Advanced Organic Chemistry, (John Wiley, and Sons, 5thEdition, 2001), and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).The terms “solvent,” “inert organic solvent,” or “inert solvent” refer to a solvent inert under the conditions of the reaction being described in conjunction therewith (including,for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like). Unless specified to the contrary, the solvents used in the reactions of the present disclosure are inert organic solvents, and the reactions are carried out under an inert gas, preferably nitrogen.IV. Methods of Forming and Releasing Carbon MonoxideIn another aspect, described herein are methods for forming and / or releasing carbon monoxide. The methods include exposing a carbon monoxide releasing compound as described to physiological conditions, wherein the compound comprises a cyclopentenone moiety and a leaving group precursor. Changes in pH (e.g., a decrease in pH) and / or exposure to reactive oxygen species in the physiological milieu results in elimination of the reactive moiety, formation of a bicyclo[2.2.1]hepta-2,5-dien-7-one intermediate, and subsequent fragmentation to release carbon monoxide. In some examples, the reactive oxygen species is CIO', H2O2, superoxide, hydroxy radical, singlet oxygen, or ONOO'. In some examples, the carbon monoxide releasing compounds described herein function by a mechanism shown in Scheme 1 below.Scheme 1In some examples, the reactive moiety in the carbon monoxide releasing compound is a leaving group and a change in pH upon exposure to physiological conditions results in elimination of the leaving group. In some examples, increasing the pH from below 4 to around 7.2 (or higher) results in elimination of the leaving group. For example, the pH can be increased from below 7 to around 7.2 or higher, or from below 6 to around 7.2 or higher, or from below 5 to around 7.2 or higher, or from below 4 to around 7.2 or higher, or from below 3 to around 7.2 or higher.In certain examples, the reactive moiety in the carbon-monoxide releasing compound is a leaving group precursor. Exposure of the leaving group precursor to reactive oxygen species upon exposure to physiological conditions results in formation of a leaving group and elimination of the leaving group. In certain examples, the leaving group precursor is a thiol group, which is sensitive to reactive oxygen species (ROS) such as peroxides, superoxide, hydroxyl radicals, ONOO', and singlet oxygen in physiological environments. Elevated levels of ROS are frequently present in cells and tissues affected by injuries of various types including cancer, bacterial infection, and inflammation.Compounds of Formulas I, II, Ila, and lib undergo a similar mechanism as described in Scheme 1 for the release of carbon monoxide, where the leaving group undergoes P-elimination to form norbornadienone and carbon monoxide is released. In some cases, the leaving group is sulfonyl, thioether, alkoxy, selenoether, or (NR4)+. In some cases, the leaving group is SChPh.V. Pharmaceutical FormulationsIn a related aspect, described herein are pharmaceutical compositions containing a compound as described herein and a pharmaceutically acceptable carrier or excipient, including but not limited to, purified water, buffer, or other pharmaceutically acceptable solvent. The carbon monoxide releasing compounds can also be formulated in a liposome or micelle. The amount of compound to be administered can be readily determined based on the amount of carbon monoxide to be generated.The compounds can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations. The term “administration by injection” includes intravenous, intramuscular, subcutaneous and parenteral injections, as well as use of infusion techniques. One or more compounds can be present in association with one or more nontoxic pharmaceutically acceptable carriers and if desired other active ingredients.Parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. More details on non-aqueous liquid formulations are disclosed below.Solutions and dispersions of the carbon monoxide releasing compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptableexcipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Pol oxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-P-alanine, sodium N-lauryl-P-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.Sterile injectable solutions can be prepared by incorporating the compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from thoselisted above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.Compositions intended for oral use can be prepared according to any suitable method known to the art for the manufacture of pharmaceutical compositions. Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Such compositions can contain one or more agents selected from diluents, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide palatable preparations. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.Formulations may be prepared using a pharmaceutically acceptable carrier. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. Delayed release dosage formulations may be prepared as described in standard references. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.Optional pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage formso that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium carbonate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, sodium phosphate, sodium carbonate, dry starch, hydrolyzed starches, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.Disintegrating and granulating agents are used to facilitate dosage form disintegration or “breakup” after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross-linked polymers, such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp).Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).The tablets can be uncoated or they can be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. These compounds can also be prepared in solid, rapidly released form.Compositions for oral use can also be formulated as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.Aqueous suspensions contain the carbon monoxide releasing compounds in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally occurring phosphatide, for example, lecithin, or condensation products or an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compounds in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring and coloring agents, can also be present.The compounds can also be in the form of non-aqueous liquid formulations, e.g., oily suspensions which can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or peanut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.Pharmaceutical compositions described herein can also be in the form of oil-in- water emulsions. The oily phase can be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.The compounds can also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.VI. Methods of TreatmentThe compounds (also referred to as carbon monoxide releasing compounds) and methods of the present disclosure have applications in any therapeutic approach in which carbon monoxide requirements are addressed. The requirement may be due to deficiency of carbon monoxide in a subject. The compounds and methods can also be used to treat patients having normal levels of endogenous carbon monoxide but who would benefit from an increase in carbon monoxide. The disclosure therefore relates to methods for treating and preventing diseases that are mediated at least in part by endogenous carbon monoxide.Accordingly, another aspect of the disclosure affords a method of providing carbon monoxide to a subject in need thereof. In certain examples, the method includes administering a compound as described herein, or a pharmaceutical composition containing a composition described herein, to a subject under conditions sufficient to form carbon monoxide.In some examples, the carbon monoxide releasing compounds are used for treatment of cancers. Examples of different types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (e.g., hepatocarcinoma), renal cancer (c.g, renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, pleural cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer, choriocarcinoma, head and neck cancer, blood cancer, osteogenic sarcoma,fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt’s lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,, multiple myeloma, brain cancer, bone cancer, oral cancer, rectal cancer, retinal cancer, uterine cancer, and vaginal cancer. Optionally, the cancer can be a carcinoma, a sarcoma, an adenocarcinoma, a lymphoma, a leukemia, and a solid and lymphoid cancer.In some examples, the methods described herein include a method of treating breast cancer with a compound described herein. In some examples, the compound is 7a, 7b, 10a, 10b, 14a, or 14b. In some examples, the compound is a compound of formulas (I) or (II). In some examples, the compound is a compound of formulas (Ila) or (lib). In some examples, the methods described herein include a method of treating pancreatic cancer with a compound described herein. In some examples, the compound is 7a, 7b, 10a, 10b, 14a, or 14b. In some examples, the compound is a compound of formulas (I) or (II). In some examples, the compound is a compound of formulas (Ila) or (lib).The specific dose level selected for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the condition undergoing therapy.Carbon monoxide releasing compounds can be administered at any suitable dose in the methods described herein. In general, a carbon monoxide releasing compound is administered at a dose ranging from about 0.1 milligrams to about 500 milligrams per kilogram of a subject’s body weight (i.e., about 0.1-500 mg / kg). The dose of the carbon monoxide releasing compound can be, for example, about 0.1-500 mg / kg, or about 1-250 mg / kg, or about 25-125 mg / kg, or about 50-100 mg / kg. The dose of the carbon monoxide releasing compound can be, for example, about 10-20 mg / kg, or 5-25 mg / kg, or 1-50 mg / kg, or 0.1-100 mg / kg. The dose of the compound can be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg. The dose of the carbon monoxide releasing compound can be administered at a dose below about 1, below about 2, below about 3, below about 4, below about 5, below about 10, below about 15, below about 20, below about 25, below about 30, below about 35, below about 40, below about 45, below about 50, below about 55, below about 60, below about 65, below about 70, below about 75, below about 85, below about 90, below about 95, below about 100, below about 150, below about 200, below about 250, below about 300, below about 350, below about 400, below about 450, or below about 500 mg / kg.In some examples, the dose of the compound is sufficient to release carbon monoxide in an amount such that no more than about 20% of the hemoglobin in a blood sample obtained from the subject is present as carboxyhemoglobin (HbCO). In some examples, the dose of the compound is sufficient to release carbon monoxide in an amount such that no more than about 15% of the hemoglobin in a blood sample obtained from the subject is present as HbCO. In some examples, the dose of the compound is sufficient to release carbon monoxide in an amount such that the amount of HbCO in a blood sample taken from the subject ranges from about 5% to about 15% (e.g., from about 5-12%) based on the total amount of hemoglobin in the sample. Sampling and HbCO quantification can be conducted over periods of time ranging from minutes to hours, or longer, following compound administration, and HbCO concentrations can be determined using known quantitative methods and devices (as described, for example, in U.S. Pat. Nos. 6,397,093; 5,491,341; and 4,997,769). Dosage can be adjusted such that the peak HbCO concentration does not exceed a certain level (e.g., 15%).The dosages can be varied depending upon the needs of the patient, the particular formulation being administered, and other factors. The dose administered to a patient should be sufficient to result in a beneficial therapeutic response in the patient. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of the drug in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the typical practitioner. The total dosage can be divided and administered in portions over a period of time suitable to address the carbon monoxide requirement.Administration of a compound as described herein can be conducted for a period of time which will vary depending upon the nature of the particular carbon monoxide requirement, its severity and the overall condition of the patient. Administration can be conducted, for example, hourly, every 2 hours, three hours, four hours, six hours, eight hours, or twice daily including every 12 hours, or any intervening interval thereof. Administration can be conducted once daily, or once every 36 hours or 48 hours, or once every month or several months. Following treatment, a patient can be monitored for changes in his or her condition and for alleviation of the symptoms of the carbon monoxide requirement. The dosage of the carbon monoxide releasing compound can either be increased in the event the patient does not respond significantly to a particular dosage level, or the dose can be decreased if an alleviation of the symptoms of the carbon monoxide requirement is observed,or if the carbon monoxide requirement has been ablated, or if unacceptable side effects are seen with a particular dosage.A therapeutically effective amount of carbon monoxide releasing compound can be administered to the subject in a treatment regimen comprising intervals of at least 1 hour, or 6 hours, or 12 hours, or 24 hours, or 36 hours, or 48 hours between dosages. Administration can be conducted at intervals of at least 72, 96, 120, 168, 192, 216, or 240 hours, or the equivalent amount of days. The dosage regimen can consist of two or more different interval sets. For example, a first part of the dosage regimen can be administered to a subject multiple times daily, daily, every other day, or every third day. The dosing regimen can start with dosing the subject every other day, every third day, weekly, biweekly, or monthly. The first part of the dosing regimen can be administered, for example, for up to 30 days, such as 7, 14, 21, or 30 days. A subsequent second part of the dosing regimen with a different interval administration administered weekly, every 14 days, or monthly can optionally follow, continuing for 4 weeks up to two years or longer, such as 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104 weeks. Alternatively, if the carbon monoxide requirement decreases, the dosage may be maintained or kept at lower than maximum amount. If the requirement increases, the first dosage regimen can be resumed until an improvement is seen, and the second dosing regimen can be implemented again. This cycle can be repeated multiple times as necessary.VII. ExamplesExample 1. Synthesis and activity of oxidation-sensitive CO releasing compounds.Scheme 2= -n-Pentyl = -n-Pentyl = -n-Pentyl2a. R-] > 4-Me-phenylD1 =4-Me-phenyl 4a. R phenyl2b: R1H 73b. R14b:Scheme 2 shows the synthesis of ROS-sensitive CO prodrugs with an aldehyde as the electron withdrawing group (“EWG”). Reagents and conditions: a) CF3CH2OH, CH3CN, 0 °C to rt, 10 min, 89-95%; b) Diisobutylaluminium hydride (DIBAL), dry CH2CI2, -78 °C to room temperature (rt), 30 min, 96%; c) bromomethyl methyl ether (“MOMBr”), dry CH2CI2, N,N-Diisoprpoylethylamine (“DIPEA”), 0 °C to room temperature, 30 minutes, 80-90%; d) toluene, reflux, 24 hours; then HC1, methanol (“MeOH”), reflux, 1 hour, 30-50%; e) pyridinium chlorochromate (“PCC”), CH2CI2, reflux, 1 hour, 50-70%.General Procedure. All reagents and solvents were of reagent grade. Column chromatography was carried out using flash silica gel (Sorbent 200-300 mesh). Thin layer chromatograph (“TLC”) analysis was conducted on silica gel plates (Sorbent Silica G UV254). Nuclear magnetic resonance (“NMR”) spectra were recorded on 300, 400 or 600 MHz spectrometers. Chemical shifts (b values) and coupling constants (J values) are given in ppm and hertz, respectively, using the respective solvent (XH NMR,13C NMR) as the internal reference. PE: petroleum ether; EA: ethyl acetate; DCM: dichloromethane. The purities of the CO prodrugs along with their CO-spent products were determined to be >95% with HPLC using A CSTCHROM C18 column (5 pm, 4.6 x 250 mm) at 1 mL / min flow rate monitored by UV at 254 nm.General procedure for the synthesis of compounds 2a and 2b. To a solution of substituted thiol (1 equiv.) and EtsN (1 equiv.) in CH3CN / CF3CH2OH (10 mL, 4: 1) wasadded methyl propiolate (1.1 equiv.) dropwise at 0 °C. The resulting mixture was stirred at room temperature overnight, and then extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with water and brine successively, and dried over anhydrous Na2SO4. After filtration and concentration, the crude product was purified on a silica gel column (PE: EA = 80: 1) to obtain the title compounds.2a (colorless oil, yield: 89%): 'HNMR (300 MHz, CDCh) 5 7.07 (dd, J= 10.1, 3.9 Hz, 1H), 5.93 - 5.70 (m, 1H), 3.69 (dd, J= 3.5, 1.5 Hz, 3H), 2.72 (dd, J= 8.5, 4.5 Hz, 2H), 1.63 (s, 2H), 1.32 (d, J= 1.6 Hz, 4H), 0.86 (d, J= 3.8 Hz, 3H).13C NMR (150 MHz, CDCh) 5 167.1, 150.8, 112.5, 77.4, 77.2, 77.0, 51.2, 36.1, 30.6, 30.0, 22.3, 13.9. HRMS (El): calculated for C9H16O2S [M]+: m / z 188.0871; found: 188.0870.2b (colorless oil, yield: 95%): 'HNMR (400 MHz, CDCh) 5 7.39 - 7.36 (m, 2H), 7.24 (d, J= 10.1 Hz, 1H), 7.17 (d, J = 7.9 Hz, 2H), 5.89 (d, J = 10.1 Hz, 1H), 3.78 (s, 3H), 2.36 (s, 3H).General procedure for the synthesis of compounds 3a and 3b. To a solution of 2a or 2b (1 equiv.) in dry CH2CI2 (20 mL) was added DIBAL (2.5 equiv.) dropwise under N2 at -78 °C. The resulting reaction mixture was stirred for another 1 hour at -78 °C. After the completion of the reaction, the resulting mixture was quenched with saturated Rochelle salt solution (30 mL) for another 30 minutes at room temperature. The organic layer was separated, and the aqueous layer was extracted with CH2CI2 (2 x 20 mL). The combined organic layer was washed with brine, and dried over anhydrous Na2SO4. After filtration and concentration, the obtained residue was purified on a silica gel column (PE: EA = 10: 1 - 4: 1) to obtain the title compounds.3a (colorless oil, yield: 96%): 'HNMR (300 MHz, CDCh) 5 6.03 (d, J= 9.6 Hz, 1H), 5.68 (d, J= 6.4 Hz, 1H), 4.18 (d, J= 4.6 Hz, 2H), 2.61 (t, J= 7.2 Hz, 2H), 2.46 (s, 1H), 1.57 (s, 2H), 1.29 (s, 4H), 0.85 (s, 4H).13C NMR (150 MHz, CDCh) 5 127.9, 127.5, 77.4, 77.2, 77.0, 59.8, 34.3, 30.7, 30.0, 22.3, 14.0. HRMS (El): calculated for CsHieOS [M]+: m / z 160.0922; found: 160.0920.3b (colorless oil, yield: 96%, Z:E=1 :2): 'HNMR (300 MHz, CDCh) 5 7.27 (t, J= 8.7 Hz, 6H), 7.17 - 7.06 (m, 6H), 6.41 (d, J= 15.0 Hz, 2H), 6.31 (d, J= 9.4 Hz, 1H), 5.95 - 5.77 (m, 3H), 4.34 (d, J= 6.1 Hz, 2H), 4.15 (d, J= 5.6 Hz, 4H), 2.33 (s, 9H).General procedure for the synthesis of compounds 4a and 4b. To a solution of 3a or 3b (1 equiv.) and DIPEA (3 equiv.) in dry CH2CI2 (20 mL) was added MOMBr (4 equiv.) dropwise under N2 at room temperature. The resulting reaction mixture was stirred for another 1 hour at room temperature. Then the reaction solution was washed with water andbrine, and the organic layer was dried over anhydrous Na2SO4. After filtration and concentration, the obtained residue was purified on a silica gel column (PE: EA = 60: 1) to obtain the title compounds.4a (colorless oil, yield: 82%): 'HNMR (300 MHz, CDCh) 5 6.14 (d, J= 8.3 Hz, 1H), 5.68 (d, J= 2.3 Hz, 1H), 4.62 (d, J= 4.6 Hz, 2H), 4.14 (d, J= 6.2 Hz, 2H), 3.36 (d, J=4.6 Hz, 3H), 2.75 - 2.55 (m, 2H), 1.59 (s, 2H), 1.32 (s, 4H), 0.88 (d, J= 5.2 Hz, 3H).13C NMR (150 MHz, CDCh) 5 129.5, 124.5, 96.0, 77.4, 77.2, 77.0, 64.3, 55.4, 34.3, 30.8, 30.1,22.3, 14.0. HRMS (ESI): calculated for C10H20O2S [M+H]+: m / z 205.1262; found: 205.1266.4b (colorless oil, yield: 45%, Z:E=1 :2): 'HNMR (600 MHz, CDCh) 5 7.29 (d, J= 8.1 Hz, 4H), 7.26 (d, J= 8.2 Hz, 2H), 7.13 (d, J= 8.1 Hz, 4H), 7.12 (d, J= 8.4 Hz, 2H), 6.43 (d, J= 15.1 Hz, 2H), 6.38 (d, J= 9.5 Hz, 1H), 5.87 (dt, J= 9.5, 6.3 Hz, 1H), 5.77 (dt, J= 15.1, 6.2 Hz, 2H), 4.68 (s, 2H), 4.63 (s, 4H), 4.27 (d, J= 6.4 Hz, 2H), 4.08 (d, J= 6.2 Hz, 4H), 3.41 (s, 3H), 3.36 (s, 6H), 2.33 (s, 6H), 2.32 (s, 3H).13C NMR (150 MHz, CDCh) 5137.5, 137.0, 132.0, 131.1, 130.6, 130.0, 130.0, 130.0, 128.3, 128.2, 126.7, 126.5, 96.1, 95.6,67.4, 64.2, 55.5, 55.4, 21.2, 21.1. HRMS (ESI): calculated for C12H16O2S [M+H]+: m / z 225.0871; found: 225.0868.General procedure for the synthesis of compounds 6a and 6b. A mixture of compounds 4a or 4b (2 equiv.) and dienone 5 (1 equiv.) in toluene (2 mL) was heated to 120- 130 °C in a sealed tube for 24 - 48 hours. After the completion of the reaction, the resulting solution was concentrated under vacuum. The obtained residue was purified on a silica gel column (PE:EA = 60: 1) to give a yellowish oil, which was dissolved in MeOH (5 mL) containing 1 mL of HC1 aqueous solution (37%). The resulting mixture was heated under reflux for another 1 hour. The reaction mixture was then cooled to room temperature and was concentrated under vacuum. The obtained residue was redissolved in ethyl acetate (20 mL), washed with brine, and dried over anhydrous Na2SO4. After filtration and concentration, the crude product was purified on a silica gel column (PE: EA = 8: 1) to obtain the title compounds.6a (white solid, yield: 50%): 'HNMR (600 MHz, CDCh) 5 7.23 (dd, J= 7.8, 1.7 Hz, 2H), 7.20 - 7.13 (m, 6H), 6.99 (dd, J = 7.8, 1.6 Hz, 2H), 3.88 - 3.73 (m, 2H), 3.29 (d, J=9.6 Hz, 1H), 2.75 - 2.65 (m, 4H), 1.64 (ddd, J= 14.8, 7.9, 2.6 Hz, 2H), 1.40 - 1.36 (m, 2H),I.36 (s, 3H), 1.35 (s, 3H), 1.32 (dd, J= 11.3, 4.1 Hz, 2H), 1.23 (dd, J= 11.6, 4.6 Hz, 1H), 0.91 (t, J= 1A Hz, 3H).13C NMR (150 MHz, CDCh) 5 203.8, 142.8, 142.4, 134.9, 134.8,130.5, 129.8, 128.2, 127.8, 127.2, 62.0, 59.6, 56.1, 54.1, 51.2, 35.4, 31.12, 3.14, 22.4, 14.1,I I.6. HRMS (El): calculated for C27H32O2S [M]+: m / z 420.2123; found: 420.2130.6b (white solid, yield: 30%): 'HNMR (300 MHz, CDCh) 5 7.32 (t, J= 7.7 Hz, 4H), 7.23 (t, J= 8.9 Hz, 6H), 7.08 (d, J= 6.2 Hz, 4H), 3.78 (s, 2H), 3.60 (d, J= 5.6 Hz, 1H), 2.32 (s, 3H), 2.15 (d, J= 5.1 Hz, 1H), 1.61 (s, 1H), 1.25 (s, 3H), 1.21 (s, 3H).13C NMR (150 MHz, CDCh) 5204.9, 144.7, 142.6, 137.9, 134.9, 134.4, 132.9, 131.7, 130.5, 130.0, 129.5, 128.2, 127.8, 127.5, 127.1, 61.1, 58.8, 55.5, 54.7, 53.9, 21.2, 11.7, 9.3. HRMS (El): calculated for C29H28O2S [M]+: m / z 440.1810; found: 440.1812.General procedure for the synthesis of compounds 7a and 7b. To a solution of 6a or 6b (1 equiv.) in CH2CI2 (10 mL) was added PCC (4 equiv.). Then the mixture was heated to 60 °C under reflux for one hour. After the completion of the reaction, the resulting mixture was filtered through Celite. Then the filtrate was concentrated under vacuum. The obtained residue was purified on a silica gel column (PE: EA = 60: 1) to afford the title compounds.

[0001] 7a (white solid, yield: 60%): 'H NMR (300 MHz, CDCh) 5 9.84 (d, J= 5.4 Hz, 1H), 7.25 - 7.16 (m, 8H), 7.01 - 6.94 (m, 2H), 3.47 (d, J= 9.6 Hz, 1H), 3.05 - 2.97 (m, 1H), 2.61 - 2.50 (m, 2H), 1.58 - 1.51 (m, 2H), 1.42 (s, 3H), 1.34 (s, 3H), 1.33 - 1.27 (m, 4H), 0.89 (t, = 6.7 Hz, 3H).13C NMR (150 MHz, CDCh) 5202.1, 200.8, 143.5, 141.1, 134.3, 130.4, 129.8, 128.4, 128.1, 127.6, 77.4, 77.2, 77.0, 58.8, 58.6, 57.1, 52.5, 33.9, 31.0, 29.3, 22.3, 14.0, 11.7, 10.9. HRMS (ESI): calculated for C27H30O2S [M+H]+: m / z 419.2045; found: 419.2043.7b (white solid, yield: 56%): 'HNMR (300 MHz, CDCh) 5 9.61 (s, 1H), 7.27 (s, 10H), 7.10 (s, 4H), 3.94 (s, 1H), 3.00 (s, 1H), 2.33 (s, 3H), 1.36 (s, 3H), 1.28 (s, 3H).13C NMR (150 MHz, CDCh) 5 202.2, 198.7, 144.6, 141.4, 137.9, 133.5, 133.5, 132.5, 130.1, 129.8, 129.2, 128.3, 128.3, 127.8, 127.6, 126.6, 126.5, 64.2, 57.4, 48.6, 21.3, 12.3, 10.0. HRMS (ESI): calculated for C10H20O2S [M+H]+: m / z 439.1654; found: 439.1656.Scheme 3oScheme 3 shows the synthesis of CO prodrugs with the EWG being ester and amide groups. Reagents and conditions: a) R2SH, CF3CH2OH, CH3CN, 0 °C to room temperature, 10 minutes, 85-95%; b) toluene, reflux, 48 hours, 30-50%; c) trifluoroacetic acid (“TFA”), CH2CI2, reflux, 1 hour, 70-90%; d) 1 -hydroxypyrrolidine-2, 5-dione, N,N'- Dicyclohexylcarbodiimide (“DCC”), 4-Dimethylaminopyridine (“DMAP”), CH2CI2, overnight; then (CH3)2NH HC1, 3 hours, 60-70%.General procedure for the synthesis of compounds 2c and 2d. To a solution of substituted thiol (1 equiv.) and EtsN (1 equiv.) in CH3CN / CF3CH2OH (10 mL, 4: 1) was added substituted propynyl ester (1.1 equiv.) dropwise at 0 °C. Then the ice bath was removed and the mixture was stirred at room temperature for ten minutes. After the completion of the reaction, the resulting mixture was concentrated under reduced pressure. The obtained residue was purified on a silica gel column (PE:EA = 80: 1) to afford the title compounds.2c (colorless oil, yield: 95%): 'H NMR (300 MHz, CDCI3) 5 6.97 (d, J= 10.2 Hz, 1H), 5.75 (d, J= 10.1 Hz, 1H), 2.73 (t, J= 7.4 Hz, 2H), 1.65 (dd, J= 14.5, 7.3 Hz, 2H), 1.49 (s, 9H), 1.42 - 1.28 (m, 4H), 0.89 (t, J= 6.8 Hz, 3H).13C NMR (150 MHz, CDCI3) 5 166.4, 148.9, 115.0, 80.5, 77.4, 77.2, 76.95, 36.2, 30.8, 30.1, 28.4, 22.4, 14.1. HRMS (ESI): calculated for C12H22O2S [M+H]+: m / z 231.1341; found: 231.1339.2d (colorless oil, yield: 98%): ‘HNMR (400 MHz, CDCI3) 5 7.39 - 7.35 (m, 2H), 7.16 (d, 7= 7.9 Hz, 2H), 7.14 (d, J= 10.1 Hz, 1H), 5.80 (d, J= 10.1 Hz, 1H), 2.35 (s, 3H), 1.53 (s, 9H).13C NMR (100 MHz, CDCI3) 5 166.3, 149.1, 138.4, 133.1, 131.4, 130.2, 115.1,80.9, 28.4, 21.3. HRMS (ESI): calculated for CuHisChS [M+Na]+: m / z 273.0925; found: 273.0921.General procedure for the synthesis of compounds 8a 8b, and 8c. A mixture of 2a or 2c-2d (2 equiv.) and dienone 5 (1 equiv.) in toluene (2 mL) was heated to 120-130 °C in a sealed tube for 48 hours. After the completion of the reaction, the resulting solution was concentrated under vacuum. The obtained residue was purified on a silica gel column (PE: EA = 60: 1) to obtain the title compounds.8a (white solid, yield: 40%): 'HNMR (400 MHz, CDCh) 5 7.25 (dd, J= 8.2, 2.7 Hz, 2H), 7.18 - 7.12 (m, 8H), 3.64 (s, 3H), 3.49 (d, J= 10.3 Hz, 1H), 3.41 (d, J= 10.3 Hz, 1H), 2.63 (d, J= 12.8, 6.4 Hz, 2H), 1.61 (d, J= 6.2 Hz, 2H), 1.38 (s, 3H), 1.36 (s, 3H), 1.35 - 1.32 (m, 2H), 1.26 (s, 2H), 0.91 (t, J= 7.0 Hz, 3H).13C NMR (150 MHz, CDCh) 5 200.8, 170.1, 143.3, 142.0, 135.5, 134.8, 130.6, 127.8, 127.6, 127.1, 77.4, 77.2, 77.0, 59.0, 56.7,55.9, 54.0, 51.5, 34.7, 31.1, 29.8, 22.4, 14.1, 12.3, 11.3. HRMS (El): calculated for C28H32O3S [M]+: m / z 448.2072; found: 448.2066.8b (white solid, yield: 37%): 'HNMR (600 MHz, CDCh) 57.28 - 7.26 (m, 2H),7.19 - 7.09 (m, 8H), 3.37 (d, J= 10.3 Hz, 1H), 3.34 (d, J= 10.2 Hz, 1H), 2.70 - 2.58 (m, 2H), 1.65 - 1.59 (m, 2H), 1.39 (s, 3H), 1.38 (s, 9H), 1.37 - 1.35 (m, 2H), 1.35 (s, 3H), 1.34 - 1.29 (m, 2H), 0.90 (t, J= 7.1 Hz, 3H).13C NMR (150 MHz, CDCh) 5201.2, 168.3, 142.8,141.9, 135.3, 134.9, 130.7, 130.6, 127.6, 127.4, 126.9, 126.8, 81.4, 59.0, 56.9, 55.5, 53.8, 34.8, 31.1, 29.6, 28.1, 22.3, 14.0, 12.2, 11.3. HRMS (El): calculated for C31H38O3S [M]+: m / z 490.2542; found: 490.2537.8c (white solid, yield: 16%): 'H NMR (300 MHz, CDCh) 5 7.32 (dd, J= 10.1, 7.8 Hz, 4H), 7.18 (dd, J= 14.7, 6.2 Hz, 8H), 7.08 (d, J= 7.9 Hz, 2H), 3.88 (d, J= 10.3 Hz, 1H), 3.46 (d, J= 10.3 Hz, 1H), 2.31 (s, 3H), 1.41 (s, 3H), 1.35 (s, 9H), 1.26 (s, 3H).13C NMR (150 MHz, CDCh) 5200.6, 168.4, 143.2, 141.7, 136.7, 135.1, 134.1, 130.8, 129.8, 127.8, 127.6, 127.1, 81.9, 77.4, 77.2, 77.0, 59.5, 57.5, 56.9, 55.2, 28.2, 21.1, 12.2, 11.7. HRMS (ESI): calculated for C33H34O3S [M+H]+: m / z 511.2307; found: 511.2305.General procedure for the synthesis of compounds 9a and 9b. To a solution of 8b or 8c (1 equiv.) in CH2CI2 (6 mL) was added trifluoroacetic acid (1 mL). Then the mixture was heated to 60 °C under reflux for one hour. After the completion of the reaction, the resulting mixture was concentrated under vacuum. The obtained residue was purified on a silica gel column (CH2Ch:MeOH = 100: 1) to obtain the title compounds.9a (white solid, yield: 99%): 'HNMR (400 MHz, CDCh) 5 7.25 - 7.21 (m, 2H),7.19 - 7.13 (m, 3H), 7.11 - 7.03 (m, 5H), 3.49 (d, J= 10.2 Hz, 1H), 3.43 (d, J= 10.2 Hz,1H), 2.64 (dd, J= 13.5, 7.3 Hz, 2H), 1.64 - 1.55 (m, 2H), 1.40 (s, 3H), 1.37 (s, 3H), 1.35 -I.28 (m, 4H), 0.88 (t, J= 7.1 Hz, 3H).13C NMR (100 MHz, CDCh) 5200.5, 174.8, 143.1,142.1, 135.2, 134.6, 130.6, 130.5, 127.8, 127.6, 127.3, 127.1, 58.9, 57.0, 55.3, 53.6, 34.7,31.1, 29.7, 22.4, 14.1, 12.2, 11.3. HRMS (ESI): calculated for C27H30O3S [M-H] m / z 433.1916; found: 433.1920.9b (white solid, yield: 93%): 'HNMR (600 MHz, CDCh) 5 7.35 (d, J= 8.0 Hz, 2H), 7.31 (dd, J= 6.5, 2.8 Hz, 2H), 7.22 - 7.17 (m, 3H), 7.10 (s, 5H), 7.00 (d, J= 8.0 Hz, 2H), 3.84 (d, J= 10.3 Hz, 1H), 3.54 (d, J= 10.3 Hz, 1H), 2.26 (s, 3H), 1.41 (s, 3H), 1.39 (s, 3H).13C NMR (150 MHz, CDCI3) 5 200.0, 175.0, 143.4, 141.9, 137.70, 135.1, 134.7, 132.46, 130.7, 130.6, 130.0, 127.9, 127.6, 127.3, 127.1, 66.0, 59.3, 58.2, 57.1, 55.3, 21.2, 15.4, 12.1,I I.5. HRMS (ESI): calculated for C29H26O3S [M-H]+: m / z 453.1524; found: 453.1529.General procedure for the synthesis of compounds 10a and 10b. To a solution of 9a and 9b (1 equiv.) in CH2CI2 (5 mL) was successively added DCC (1.2 equiv.), DMAP (0.1 equiv.) and 1 -hydroxypyrrolidine-2, 5-dione (1.2 equiv.). The resulting solution was stirred at room temperature overnight. After the completion of the reaction, the resulting mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified on a silica gel column (PE:EA = 8: 1) to obtain a white solid. The white solid was dissolved in CH2CI2 (5 mL), followed by the addition of triethylamine (4 equiv.) and dimethylamine hydrochloride (3 equiv.). The resulting mixture was stirred at room temperature for 4 hours. After the completion of the reaction, the resulting mixture was concentrated under vacuum. The residue was purified on a silica gel column (PE: EA = 8: 1) to obtain the title compounds.10a (white solid, yield: 77%): 'HNMR (400 MHz, CDCh) 5 7.20 (m, J= 11.1, 7.2,5.1, 2.3 Hz, 8H), 7.04 - 7.00 (m, 2H), 3.61 (d, J= 4.9 Hz, 1H), 3.15 (d, J= 4.9 Hz, 1H), 3.12 (s, 3H), 3.02 (d, J= 3.9 Hz, 3H), 2.66 - 2.53 (m, 2H), 1.56 (d, J = 7.4 Hz, 2H), 1.39 (s, 3H), 1.33 - 1.25 (m, 4H), 1.15 (s, 3H), 0.89 (t, J= 7.1 Hz, 3H).13C NMR (150 MHz, CDCh) 5 201.9, 172.2, 134.5, 130.4, 129.6, 128.3, 127.8, 127.6, 127.3, 77.4, 77.2, 77.0, 56.8, 55.6, 54.8, 38.3, 36.0, 33.7, 31.1, 29.9, 22.4, 14.1, 11.4, 9.5. HRMS (El): calculated for C29H35NO2S [M]+: m / z 461.2389; found: 461.2390.10b (white solid, yield: 87%): 'HNMR (400 MHz, CDCh) 5 7.27 - 7.18 (m, 12H), 7.08 - 7.03 (m, 4H), 4.25 (d, J= 4.6 Hz, 1H), 3.13 (d, J= 4.6 Hz, 1H), 2.88 (s, 3H), 2.75 (s, 3H), 2.31 (s, 3H), 1.36 (s, 3H), 1.13 (s, 3H).13C NMR (150 MHz, CDCh) 5201.1, 172.1, 143.6, 143.2, 137.4, 134.5, 131.7, 130.9, 130.5, 129.9, 129.5, 128.4, 127.9, 127.6, 127.4, 77.4, 77.2, 77.0, 58.1, 57.1, 56.8, 54.1, 37.8, 35.8, 21.2, 11.5, 9.5. HRMS (ESI): calculated for C31H31NO2S [M+H]+: m / z 482.2154; found: 482.2151.Scheme 4Scheme 4 shows the synthesis of CO prodrugs with a cyano group as the electron withdrawing group. Reagents and conditions: a) MeOH, 0 °C to room temperature, overnight, 65-80%; b) trifluoroacetic anhydride (“TFAA”), EtsN, CH2CI2, 0 °C to room temperature, 30 minutes, 60-80%; c) toluene, reflux, 48 hours, 40-50%.General procedure for the synthesis of compounds 12a and 12b. To a solution of compound 11 (1 equiv.) and EtsN (1 equiv.) in MeOH (5 mL) was added substituted thiol (1 equiv.). The mixture was stirred at room temperature overnight. After the completion of the reaction, the resulting mixture was concentrated under vacuum, and the obtained residue was purified on a silica gel column (PE:EA = 4: 1-2: 1) to yield the title compounds as a mixture of ZZE isomers.12a (white solid, yield: 72%, Z:E=4: 1): *H NMR (400 MHz, CDCh) 5 7.59 (d, J= 14.9 Hz, 0.25H), 6.90 (d, J= 10.1 Hz, 1H), 5.98 (s, 1H), 5.82 (d, J= 10.1 Hz, 1H), 5.80 (d, J = 14.9 Hz, 0.21H), 5.60 (s, 1H), 2.79 - 2.73 (m, 0.51H), 2.73 - 2.67 (m, 2H), 1.70 - 1.61 (m, 2.56H), 1.40 - 1.27 (m, 5H), 0.88 (td, J= 7.1, 4.1 Hz, 3.84H).13C NMR (100 MHz, CDCh) 5 168.5, 166.8, 147.2, 144.8, 115.6, 114.7, 36.4, 32.5, 31.0, 30.7, 30.1, 28.8, 22.3, 22.3, 14.0. HRMS (ESI): calculated for CsHisNOS [M+Na]+: m / z 196.0772; found: 196.0769.12b (white solid, yield: 69%, Z:E=1 : 1): 'HNMR (600 MHz, CDCh) 5 7.69 (d, J= 14.7 Hz, 0.5H), 7.39 - 7.34 (m, 3H), 7.20 (d, J= 7.9 Hz, 1H), 7.16 (d, J= 7.9 Hz, 2H), 7.09 (d, J= 9.9 Hz, 2H), 5.98 (s, 1H), 5.87 (d, J= 9.9 Hz, 2H), 5.65 (d, J= 14.7 Hz, 1H), 5.60 (s, 2H), 2.36 (s, 1.5H), 2.34 (s, 3H).13C NMR (150 MHz, CDCh) 5 168.2, 166.6, 147.9, 145.2, 139.4, 138.3, 133.6, 133.2, 131.3, 130.5, 130.1, 127.3, 116.7, 114.3, 21.4, 21.2. HRMS (ESI): calculated for C10H11NOS [M+H]+: m / z 194.0640; found: 194.0640.General procedure for the synthesis of compounds 13a and 13b. To a solution of 12a and 12b (1 equiv.) and EtsN (5 equiv.) in CH2CI2 (5 mL) was added TFAA (3 equiv.) dropwise at 0 °C. The resulting mixture was then stirred at room temperature for 30 minutes. After the completion of the reaction, the reaction mixture was concentrated under vacuum,and the residue was purified on a silica gel column (PE: EA = 60: 1-30: 1) to obtain the title compounds.13a (colorless oil, yield: 71%): 'HNMR (300 MHz, CDCh) 5 7.14 (d, J= 10.3 Hz, 1H), 5.26 (d, J= 10.4 Hz, 1H), 2.88 (t, J= 7.1 Hz, 2H), 1.67 (m, 2H), 1.43 - 1.25 (m, 4H), 0.91 (t, J= 6.4 Hz, 3H).13C NMR (100 MHz, CDCh) 5 153.3, 116.3, 92.6, 34.6, 30.5, 30.1,22.3, 14.0. HRMS (ESI): calculated for CSHBNS [M+H]+: m / z 156.0847; found: 156.0843.13b (white solid, yield: 88%): 'HNMR (300 MHz, CDCh) 5 7.34 (d, J= 7.3 Hz, 2H), 7.24 (d, J= 10.3 Hz, 1H), 7.19 (d, J= 7.5 Hz, 2H), 5.32 (d, J= 10.3 Hz, 1H), 2.36 (s, 3H).13C NMR (100 MHz, CDCh) 5 153.5, 139.5, 131.8, 130.5, 128.5, 115.8, 93.1, 21.3. HRMS (ESI): calculated for C10H9NS [M+H]+: m / z 176.0534; found: 176.0531.General procedure for the synthesis of compounds 14a and 14b. A mixture of 13a and 13b (2 equiv.) and compound 5 (1 equiv.) in toluene (2 mL) was heated to 120 °C in a sealed tube for 24-48 hours. After the completion of the reaction, the resulting solution was concentrated under reduced pressure, and the residue was purified on a silica gel column (PE: EA = 60: 1-30: 1) to obtain the title compounds.14a (white solid, yield: 20%): 'HNMR (400 MHz, CDCh) 5 7.20 (d, J= 1.4 Hz, 10H), 3.55 (d, J= 9.2 Hz, 1H), 3.37 (d, J= 9.2 Hz, 1H), 2.67 (m, 2H), 1.71 - 1.61 (m, 2H), 1.43 (s, 3H), 1.41 (s, 3H), 1.39 - 1.29 (m, 4H), 0.90 (t, J = 7.1 Hz, 3H).13C NMR (100 MHz, CDCh) 5 199.8, 144.1, 142.3, 133.9, 133.3, 130.4, 130.1, 127.9, 127.7 117.7, 57.9, 56.9, 51.6, 43.7, 33.5, 31.1, 29.5, 22.4, 14.1, 11.7, 11.3. HRMS (El): calculated for C27H29NOS [M]+: m / z 415.1970; found: 415.1964.14b (white solid, yield: 45%): 'HNMR (400 MHz, CDCh) 5 7.41 (d, J= 8.1 Hz, 2H), 7.27 - 7.21 (m, 10H), 7.13 (d, J= 8.0 Hz, 2H), 3.70 (d, J= 9.2 Hz, 1H), 3.55 (d, J= 9.2 Hz, 1H), 2.33 (s, 3H), 1.46 (s, 3H), 1.42 (s, 3H).13C NMR (100 MHz, CDCh) 5 199.4, 144.0, 142.8, 138.5, 133.9, 133.3, 133.2, 130.4, 130.3, 130.3, 130.1, 117.7, 58.1, 57.2, 56.3, 43.7,21.3, 11.6, 11.5. HRMS (ESI): calculated for C29H25NOS [M+K]+: m / z 474.1294; found: 474.1294.Example 2. CO release kinetics studies of ROS-Sensitive ProdrugsScheme 5Scheme 5 shows the CO release kinetics of all CO prodrugs were determined in 40% CH3CN in phosphate-buffered saline (“PBS”) (pH = 7.4 and pH = 8.4) at 37 °C by monitoring the consumption of CO prodrugs and the formation of byproducts using HPLC. The final concentrations of all CO prodrugs were kept at 25 pM. The HPLC eluent was acetonitrile / H2O. A chromatographic column (CSTCHROM Cis 5 pm, 4.6 x 250 mm) was used. The flow rate was set as 1 mL / min. Detection wavelength: 254 nm. Injection sample volume: 50 pL. Each experiment was triplicated. The results are summarized in Table 1 below.Table 1 below shows the CO release half-lives of CO prodrugs in the presence / absence of H2O2 (300 pM).Scheme 6. The synthesis of the CO-spent byproducts. Reagents and conditions: a) toluene, reflux, 2 hours, 80-98%; b) PCC, CH2CI2, reflux, 1 hour, 65%; c) TFA, CH2CI2, reflux, 1 hour, 90%; d) 1 -hydroxypyrrolidine-2, 5-dione, DCC, DMAP, CH2CI2, overnight; then (CH3)2NH HC1, 3 hours, 70%; e) TFAA, EtsN, CH2CI2, 0 °C to room temperature, 30 minutes, 95%.To facilitate the kinetics studies and also to better deconvolute the antiproliferative activities of the CO prodrugs, the corresponding byproducts after CO release from these CO prodrugs were also synthesized. As depicted in Scheme 6, the Diels Alder (“DA”) reaction between the respectively substituted alkynes and 5 yielded 16a-d. 16d was further oxidized by PCC to afford 17 as the control byproduct of CO prodrugs 7a / b. The hydrolysis of 16b followed by amidation produced 19 as the control byproduct of CO prodrugs lOa / b. Dehydration of 16c gave rise to 20 as the control byproduct of CO prodrugs 14a and 14b.With CO prodrugs 7a / b, 8a, lOa / b and 14a / b in hand, CO release kinetics of the compounds were tested in the presence / absence of H2O2by monitoring the formation of their byproducts using high-performance liquid chromatography (“HPLC”). Figure 1 shows the CO release kinetics of prodrugs 7a (25 pM, A) and 7b (25 pM, B) with and without H2O2 (400 pM) in 40% of CH3CN / PBS (pH 7.4) at 37 °C. The consumption of prodrugs 7a / b and the formation of compound 17 were simultaneously monitored using HPLC. As designed, 7a displayed a significant difference in the CO release half-lives depending on the presence (ti / 2: 3.4 hours) or absence of H2O2 (ti / 2: 81.6 hours, Figure 1, Panel A), indicating CO release in response to H2O2. In addition, no build-up of the oxidized intermediate was observed, suggesting a spontaneous elimination reaction. On the other hand, as for its structural analog7b with a leaving group of 4-methylphenyl thiolate, no significant difference in the CO release half-lives was observed between the reaction with (ti / 2: 2.2 hours) and without H2O2 (ti / 2: 3 hours, Figure 1, Panel B). Not to be bound by theory, the result can be explained by the understanding in the art that the 4-methylphenyl thiolate is a much better leaving group than the pentyl thiolate. In addition, the electron delocalization, as well as the steric repulsion effects of the phenyl group make the 4-methylphenyl thioether less prone to oxidation as compared to the pentyl thioether. Therefore, the addition of H2O2 did not significantly accelerate the formation of 17 in that the oxidation of the thioether did not significantly outcompete the elimination reaction from compound 7b.For CO prodrugs 8a, 10a and 10b, no formation of the byproducts was observed either in the presence or absence of H2O2 after 24 hours of incubation at 37 °C. In the presence of H2O2, the buildup of the oxidized intermediates was observed in these cases, yet no further elimination occurred due to the weak electron-withdrawing capacity of the ester and amide group (Figure 8, Figure 9, and Figure 10).Figure 8 shows HPLC traces of 8a in the absence (left) and presence (right) of H2O2 at different incubation time points. The eluent was 85% CH3CN.Figure 9 shows HPLC traces of 10a in the absence (left) and presence (right) of H2O2 at different incubation time points. The eluent was as follows: 0-20 minutes: 80% CH3CN (for the sample without H2O2); 0-30 minutes: 55% CH3CN, 30-50 minutes: 90% CH3CN (for the sample with H2O2).Figure 10 shows The HPLC traces of 10b in the absence (left) / presence (right) of H2O2 at different incubation time points. The eluent was as follows: 0-20 minutes: 80% CH3CN (for the sample without H2O2); 0-30 minutes: 55% CH3CN, 30-50 minutes: 90% CH3CN (for the sample with H2O2).For CO prodrugs 14a / b with a cyano EWG, no formation of the byproduct was witnessed in the absence of H2O2 after 24 hours of incubation at 37 °C in 40% of CH3CN / PBS. However, in the presence of H2O2 (400 pM), both 14a and 14b can be efficiently oxidized with a half-life of around 2 hours, and the formed oxidized intermediates slowly underwent the expected elimination to afford the byproduct 20 with a half-life of around 12 hours (Figure 2, Panels A and 2B).Figure 2 shows the release kinetics of CO from prodrugs 14a (25 pM, A) and 14b (25 pM, B) in the presence of H2O2 (400 pM) in 40% of CH3CN / PBS (pH 7.4) at 37 °C. The consumption of prodrugs 14a and 14b and the formation of compound 20 were simultaneously monitored using HPLC.These results demonstrate that the elimination rate was highly dependent on both the electron-withdrawing group and the leaving group, and the ROS-induced CO release rates can be tuned by varying the electron-withdrawing groups, which mainly affected the acidity of the proton at 5 -position. For amide and ester, of which the alpha proton possessed a pKa value higher than 25, no elimination occurred under physiological conditions. In the case of aldehyde, the alpha proton was much more acidic with a pKa of around 17, and thus the elimination happened smoothly. The alpha proton of the cyano group possessed a pKa value of around 25, and the elimination occurred very sluggishly with a half-life of 12 hours. Therefore, for ROS-sensitive CO prodrug with an alkylthiolate as the leaving group, a pKa value of 25 of the alpha protons seems the cutoff value for the beta-elimination under physiological conditions, and any electron-withdrawing group with a less acidic alpha proton than cyano was expected to undergo no or very sluggish elimination. Meanwhile, the half-life of ROS-sensitive CO prodrugs can also be fine-tuned to a broad range by introducing other EWG, of which the alpha proton’s pKa values were lower than 25, such as ketone, nitro and sulfone.To further validate CO release, 7a was tested using a CO-myoglobin assay.Figure 3, Panel A, shows CO-myoglobin (Mb) assay validating H2O2-triggered CO release from 7a. Figure 3, Panel B, shows CO release kinetics from 7a (25 pM) and 14a (25 pM in the presence or absence of H2O2 (400 pM) in 40% CH3CN / PBS (pH 7.4) at 37 °C. Figure 3, Panels C and D show CO release profiles of 7a (25 pM) and 14a (25 pM) in response to different ROS (400 pM) or other endogenously found species (400 pM) in 40% CH3CN / PBS at 37 °C for a period of 2 hours.As summarized in Figure 3, Panel A, H2O2-induced CO release from 7a was confirmed by the featured changes in the UV-vis spectra. In addition, a commercially available CO detector was also used to monitor CO release from 7a and 14a in the presence or absence of H2O2. As shown in Figure 3, Panel B, steady CO release from both 7a and 14a was observed in the presence of H2O2, while no CO release was detected in the absence of H2O2. Meanwhile, consistent with the results obtained in the HPLC-based reaction kinetics studies, the CO release yield of 7a was much higher than 14a under the same conditions. Since the thioether can be readily oxidized by a series of ROS, 7a and 14a should be able to release CO in response to other ROS as well. Toward this end, 7a and 14a were assayed for their CO release profiles in response to different ROS and other endogenously found species. As summarized in Figure 3, Panels C and D, two other tumor-associated ROS, hypochloriteand ONOO', also triggered significant CO release from both 7a and 14a, while no meaningful CO release was triggered by the other ROS and endogenously found species. These attributes presented 7a and 14a as promising ROS-triggered CO prodrugs for tumor-targeted CO delivery.General procedure for the synthesis of compounds 16a, 16b, 16c, and 16d. A mixture of the substituted alkynes (1.1 equiv.) and compound 5 (1 equiv.) in toluene (3 mL) was heated to 120 °C in a sealed tube for 2 hours. After the completion of the reaction, the resulting solution was concentrated under reduced pressure, and the residue was purified on a silica gel column (PE: EA = 60: 1-2: 1) to obtain the title compounds, as shown in Scheme 6 above.16a (white solid, yield: 95%): 'HNMR (300 MHz, CDCh) 5 7.75 (s, 1H), 7.22 - 6.99 (m, 6H), 6.92 (m, 4H), 3.94 (s, 3H), 2.25 (s, 3H), 2.10 (s, 3H).16b (white solid, yield: 97%): 'HNMR (300 MHz, CDCh) 5 7.60 (s, 1H), 7.10 (m, 6H), 6.90 (m, 4H), 2.20 (s, 3H), 2.08 (s, 3H), 1.63 (s, 9H).13C NMR (150 MHz, CDCh) 5168.5, 144.4, 143.0, 140.6, 140.4, 133.5, 132.4, 130.2, 129.8, 129.7, 127.6, 126.3, 126.2,81.5, 28.4, 20.9, 18.7. HRMS (El): calculated for C25H26O2 [M]+: m / z 358.1933; found: 358.1931.16c (white solid, yield: 93%): 'H NMR (300 MHz, CDCh) 5 7.37 (s, 1H), 7.19 - 7.04 (m, 6H), 6.98 - 6.86 (m, 4H), 6.22 (s, 1H), 5.90 (s, 1H), 2.17 (s, 3H), 2.08 (s, 3H).13C NMR (100 MHz, CDCh) 5 173.1, 143.5, 143.0, 140.3, 140.2, 135.4, 133.8, 131.3, 130.0,129.7, 127.7, 127.6, 127.3, 126.3, 20.9, 18.1. HRMS (ESI): calculated for C21H19NO [M+H]+: m / z 302.1545; found: 302.1550.16d (white solid, yield: 80%): 'HNMR (600 MHz, CDCh) 5 7.34 (s, 1H), 7.12 (td, J= 7.4, 3.5 Hz, 4H), 7.09 - 7.04 (m, 2H), 6.95 - 6.91 (m, 4H), 4.79 (d, J= 5.5 Hz, 2H), 2.10 (s, 3H), 2.07 (s, 3H).13C NMR (150 MHz, CDCh) 5 142.4, 141.3, 140.9, 140.8, 137.8, 133.7,131.7, 130.3, 130.1, 128.6, 127.5, 126.1, 126.0, 64.2, 20.9, 16.4. HRMS (El): calculated for C21H20O [M]+: m / z 288.1514; found: 288.1513.The synthesis of compound 17. To a solution of 16d (230 mg, 0.8 mmol, 1 equiv.) in CH2CI2 (5 mL) was added PCC (688 mg, 3.2 mmol, 4 equiv.). Then the mixture was heated to 60 °C under reflux for one hour. After the completion of the reaction, the resulting mixture was filtered through Celite. Then the filtrate was concentrated under vacuum, and the residue was purified on a silica gel column (PE: EA = 30: 1) to obtain the title compound, (white solid, 148 mg yield: 65%): 'HNMR (300 MHz, CDCh) 5 10.40 (s, 1H), 7.77 (s, 1H), 7.21 - 7.03 (m, 6H), 7.00 - 6.85 (m, 4H), 2.38 (s, 3H), 2.14 (s, 3H).The synthesis of compound 18. To a solution of 16b (80 mg, 0.22 mmol, 1 equiv.) in CH2CI2 (2 mL) was added trifluoroacetic acid (1 mL). Then the mixture was heated to 60 °C under reflux for two hours. After the completion of the reaction, the resulting mixture was concentrated under reduced pressure, and the residue was purified on a flash silica gel column (DCM: MeOH = 60: 1) to obtain compound 18 as a white solid (63 mg, yield: 95%):XH NMR (300 MHz, CDCh) 5 7.97 (s, 1H), 7.14 (m, 6H), 6.93 (m, 4H), 2.35 (s, 3H), 2.13 (s, 3H).13C NMR (150 MHz, DMSO-de) 5 169.5, 144.0, 142.5, 140.1, 139.8, 133.2, 132.8, 130.9, 129.8, 129.3, 127.6, 126.3, 126.2, 20.4, 18.3. HRMS (ESI): calculated for C21H18O2 [M-H] ’: m / z 301.1307; found: 301.1310.The synthesis of compound 19. To a solution of 18 (100 mg, 0.33 mmol, 1 equiv.) in CH2CI2 (2 mL) was added DCC (1.2 equiv.), DMAP (4 mg, 0.03 mmol, 0.1 equiv.) and 1- hydroxypyrrolidine-2, 5-dione (46 mg, 0.4 mmol, 1.2 equiv.). The resulting mixture was stirred at room temperature overnight. After the completion of the reaction, the resulting solution was filtered and the filtrate was concentrated under vacuum. The residue was purified on a silica gel column (PE: EA = 4: 1) to obtain a white solid. The white solid was dissolved in CH2CI2 (5 mL), followed by the addition of triethylamine (“TEA”) (184 pL, 1.32 mmol, 4 equiv.) and dimethylamine hydrochloride (81 mg, 0.99 mmol, 3 equiv.). The resulting mixture was stirred at room temperature for 4 hours. After the completion of the reaction, the resulting mixture was concentrated under vacuum, and the residue was purified on a silica gel column (PE: EA = 3: 1) to obtain compound 19 as a white solid (97 mg, yield: 89%).XH NMR (400 MHz, CDCh) 5 7.14 (s, 1H), 7.08 (m, 6H), 6.96 - 6.85 (m, 4H), 3.17 (s, 3H), 2.98 (s, 3H), 2.06 (s, 3H), 1.97 (s, 3H).13C NMR (150 MHz, CDCh) 5 172.1, 142.4, 142.0, 140.5, 140.4, 136.3, 134.1, 130.1, 129.8, 129.3, 127.5, 126.2, 126.2, 38.7, 34.7, 20.9, 17.5. HRMS (ESI): calculated for C23H23NO [M+H]+: m / z 330.1858; found: 330.1859.The synthesis of compound 20. To a solution of 16c (80 mg, 0.27 mmol, 1 equiv.) and EtsN (184 pL, 1.33 mmol, 5 equiv.) in CH2CI2 (2 mL) was added TFAA (0.1 mL) dropwise at 0 °C. Then the mixture was stirred at room temperature for 30 minutes. After the completion of the reaction, the resulting mixture was concentrated under vacuum, and the residue was purified on a silica gel column (PE: EA = 60: 1-30: 1) to obtain compound 20 as a white solid (70 mg, yield: 93%). 'HNMR (300 MHz, CDCh) 5 7.55 (s, 1H), 7.19 - 7.07 (m, 6H), 6.90 (m, 4H), 2.27 (s, 3H), 2.09 (s, 3H).13C NMR (100 MHz, CDCh) 5 146.4, 142.8, 139.3, 139.1, 137.7, 135.0, 132.7, 129.8, 129.3, 127.9, 127.8, 126.8, 126.8, 118.9, 112.2, 20.8, 19.5. HRMS (El): calculated for C21H17N [M]+: m / z 283.1361; found: 283.1355.CO release kinetics studies. The CO release kinetics of CO prodrugs including compounds 7a, 7b, 8a, 8c, 10a, 10b, 14a and 14b in the absence or presence H2O2 were determined in 40% of CH3CN in PBS (pH = 7.4) at 37 °C by monitoring the consumption of CO prodrugs and the formation of byproducts using HPLC. The final concentrations of all CO prodrugs and H2O2 were kept at 25 pM and 400 pM, respectively. The HPLC eluent was acetonitrile / H2O. A chromatographic column (CSTCHROM Cis 5 pm, 4.6 x 250 mm) was used. The flow rate was set to 1 mL / min. The detection wavelength was 254 nm. The injection sample volume was 50 pL. Each experiment was triplicated.CO-myoglobin assay. To further verify the release of CO, compound 7a was selected for CO-myoglobin assay. Specifically, a solution of myoglobin (0.5 mg / mL) in PBS (10 mL, pH = 7.4) was put in a transparent vial (20 mL), and a solution of 7a (21 mg) in 50% CH3CN / PBS (100 mL) was placed in a conical flask (150 mL). These two solutions were ultrasonically degassed with nitrogen bubbling for at least 30 minutes. Sodium dithionite (1 mL, 22 mg / mL) was added to the degassed solution of myoglobin. The obtained two solutions were connected by a cannula under a flow of nitrogen. H2O2 (100 pL, w% = 30%) was added to the solution of 7a, and these two solutions were stirred in an oil bath at 37 °C for 12 hours. After that, the myoglobin solution was cooled with an ice bath at 0 °C for 6 hours to increase the solubility of CO. The solution of 7a without the addition of H2O2 was used as the control. Then, the ultraviolet-visible spectroscopy of the resulting myoglobin solution was recorded.Quantification of CO release with a commercially available CO detector. The CO release profiles from 7a and 14a with H2O2 or without H2O2 were determined in 40% CH3CN in PBS (pH = 7.4) at 37 °C by a CO meter. The final concentrations of all CO prodrugs and H2O2 were kept at 25 pM and 400 pM, respectively, and each experiment was triplicated independently.CO release profiles of 7a and 14a in response to various endogenous stimuli. CO release profiles of 7a and 14a in response to other ROS as well as endogenously found small molecules were tested with a CO meter. Specifically, to a solution of 7a / 14a (25 pM) in 40% CH3CN in PBS (pH = 7.4) at 37 °C was added the tested species (NaClO, H2O2, HO», TBHP, LBuO»,XO2, ONOO', *02’, GSH, Cys with a final concentration of 400 pM), and CO release was continuously recorded with a CO meter for a period time of 2 hours. Example 3. Biology StudiesSince compounds 7a and 14a share the same release mechanism and possess very similar structures and byproducts but different release rates, they were then selected forsubsequent biology studies to probe the effects of CO release rate on antitumor effects. Initially, 7a and 14a were tested to see if they could deliver CO intracellularly in response to endogenous ROS. 8a was employed as a negative control because it shares high structural similarity with 7a and 14a, while displaying no CO release in response to ROS. Toward this end, a previously reported specific fluorescent CO probe 1-Ac was synthesized and employed to image CO release in murine mammary carcinoma 4T1 cells.Figure 4, Panel A shows cell-imaging studies of CO release from 7a (20 pM) and 14a (20 pM) in 4T1 cells tracked by 1-Ac (4 pM) (Scale bar = 20 pm). Figure 4, Panel B shows the quantification of the corresponding red fluorescence intensity in Figure 4, Panel A. Statistical significance was calculated via student’s t-test, ***p < 0.001.As shown in Figure 4, cells treated with probe only or the probe in combination with the negative control 8a did not exhibit meaningful red fluorescence. However, cells treated with probe plus 7a or 14a displayed significantly enhanced red fluorescence, indicating intracellular CO release from 7a and 14a in these cells. In addition, 7a delivered more CO than 14a did in 4T1 cells in the same time frame, as indicated by the much more- enhanced red fluorescence intensity (Figure 4, Panel B). Such results are consistent with the ROS response rate in the cell-free assay (Figure 3).Figure 5 shows cytotoxicity studies of CO prodrugs in (A) 4T1 and (B) Panc02 cells. Each experiment was triplicated independently.Having shown endogenous ROS-triggered CO release from 7a and 14a, their antiproliferative activities in cancer cells were tested. To facilitate the deconvolution of the results, 8a along with the CO-spent byproducts 17 and 20 were employed as the control. As summarized in Figure 5, compound 7a displayed very pronounced antiproliferative activities against 4T1 (ICso: 29.44 pM) and Panc02 (ICso: 72.47 pM) cancer cells, while its CO-spent byproduct 17 and its structural analog 8a with no CO release only exhibited minimal or no cytotoxicity. Thus, the observed antiproliferative activities are mainly attributed to the CO release from 7a rather than its byproducts. In addition, 14a with a slower ROS response rate also displayed some inhibitory activities against 4T1 cells (ICso > 100 pM), and its CO-spent byproduct 20 showed no obvious effects at concentrations up to 100 pM.While in the Panc02 cells, both 14a and 20 displayed no apparent antiproliferative activities at the concentrations tested. These results indicated a less potent antiproliferative activity of 14a as compared to 7a, implying that CO prodrug with a faster release rate is beneficial for antiproliferative activities.To determine whether these CO prodrugs could release CO at the tumor site in vivo, a 4T1 subcutaneous tumor model was also evaluated. The 4T1 tumor model is a validated, accepted, model for human breast cancer. For this purpose, CO prodrugs 7a, 8a and 14a were intravenously (“I.V.”) injected at a dose of 10 mg / kg to 4T1 tumor-bearing mice, and CO fluorescent probe 1-Ac (4 pM, 50 pL) was injected intratumorally at 6 hours post-injection. After another 2 hours, the mice were sacrificed, and the tumors were harvested.Figure 6, Panel A shows microscopic photographs of 4T1 tumor sections showing CO release (red) after intravenous administration of 7a, 8a and 14a (Dosage: 10 mg / kg, Scale bar = 20 pm), n = 3. Figure 6, Panel B shows the quantification of corresponding red fluorescence intensity in Figure 6, Panel A. Statistical significance was calculated via student’s t-test, **p < 0.01.As shown in Figure 6, the stained tumor sections confirmed CO release from 7a and 14a at the tumor site, while the control prodrug 8a did not exhibit any CO release. Consistent with the reaction kinetics with ROS and the cell culture-based results, 7a displayed much more enhanced red fluorescence as compared to 14a, indicating more efficient CO release in vivo from 7a.To probe whether CO release rates also matter for in vivo antitumor efficacy, the tumor suppression effects of CO prodrugs 7a and 14a (10 mg / kg) in vivo in a 4T1 subcutaneous mouse tumor model was tested. Compound 8a along with the byproducts 17 and 20 were used as negative controls.Figure 7, Panel A shows a schematic illustration of the experimental schedule for in vivo antitumor therapy. Figure 7, Panel B shows tumor growth profiles of the mice bearing 4T1 subcutaneous tumors with various treatments at the dose of 10 mg / kg. Figure 7, Panel C shows individual tumor growth curves of 4T1 tumor-bearing mice after various treatments. Figure 7, Panel D shows body weight changes of each group after various treatments. Figure 7, Panel E shows survival curves of each group after various treatments. Data were reported as mean ± standard deviation (s.d.) (n=5). Statistical significance was calculated via student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001.As illustrated in Figure 7, Panel A, the 4T1 cells were planted subcutaneously in mice, and the mice received treatment every 3 days when the average tumor volume reached -100 mm3. The tumor volumes of all the mice received 3 doses were continuously measured for 24 days, and the survival rates of each group were also monitored. As summarized in Figure 7, Panels B and C, the vehicle control group showed a rapid tumor growth with morethan 25.5 folds increase in the tumor volume, and both 7a and 14a exhibited very pronounced tumor suppression effects with only 9.4- and 16.5-fold increase in tumor volume, respectively. On the contrary, their CO-spent compounds 17 and 20 as well as the negative control 8a did not show similar anti-tumor effects, indicating that the observed tumor suppression effects were mainly ascribed to the CO release from 7a and 14a. Importantly, consistent with the in vitro antiproliferative results, 7a with a faster ROS response rate displayed more potent antitumor effects than 14a. In addition, 7a with the superior anti-tumor effects among all the compounds significantly improved the survival rate of the mice as compared to the control groups (from 27 day to 42 day, Figure 7, Panel E) and exhibited negligible body weight loss (Figure 7, Panel D).Experimental Methods for Biology Studies.General: Two cell lines (4T1 and Panc02) were used for in vitro studies (obtained from American Type Culture Collection). Cells were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium, Solarbio) containing 10% fetal bovine serum (Bio-Channel) and 1% penicillin-streptomycin (Solarbio) at 37 °C with 5% CO2, and the cells were passaged every other day. All animal experimental protocols were approved by the Animal Care and Use Committee of Soochow University and complied with all relevant ethical regulations (Protocol NO: 202210A655, date: Oct. 1st2022). All animals were housed in the laboratory animal center of Soochow University. Female BALB / c mice (6-8 weeks) purchased from Changzhou Cavens Technology Co. Ltd. were used in all studies. All the tested compounds were dissolved in 100% DMSO to yield the respective stock solution.Cytotoxicity studies. The cells were respectively inoculated in the 96-well plates at a density of 5000 / well, and were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with 5% CO2 for 24 hours. Then cells were treated with either 2%o DMSO or compounds 7a, 14a, 8a, 17, and 20 at concentrations of 0, 6.25, 12.5, 25, 50 and 100 pM for 24 hours. Then, 10 pL of Cell Counting Kit-8 (purchased from APExBIO) solution was added to each well and incubated for another 1-3 hours at 37 °C. The absorbance at 450 nm was measured by using a full-wavelength enzyme marker. The cell viability of the vehicle control was set as 100%, and other experimental groups were calculated accordingly.Cell-imaging studies. 4T1 cells were seeded in a 24-well plate with a density of 2.0* 105 / well. After incubating in the incubator for 24 hours, the fluorescent CO probe 1-Ac staining solution was added to the wells at a final concentration of 4 pM containing l%o DMSO. After 30 minutes of incubation in the dark, the cells were washed twice with PBS.Then, cells were treated with the tested compounds at a final concentration of 20 pM for 6 hours at 37 °C with 5% CO2. After that, the Hoechst dye was used to stain the cell nucleus and 4% paraformaldehyde (“PF A”) was used to fix the cells in the dark. The fluorescence images of the cells were acquired using a confocal laser scanning microscope (LSM-710, Zeiss).In vivo animal studies. All animal experiment protocols were approved by the Animal Care and Use Committee of Soochow University and complied with all relevant ethical regulations. 4T1 cells cancer cells (5 x 106) in 50 pL PBS were injected into the lateral right hindlimb of Balb / c healthy mice to establish the subcutaneous tumor model. The tumor-bearing mice were euthanized and sacrificed once the volume of the tumor reached 1500 mm3.Imaging of in vivo CO release. When the tumor volume was about 120 mm3, the mice were randomly divided into four groups (PBS, 7a, 14a and 8a). The tested compounds were dissolved with 5% DMSO containing 3% Tween-80 solution, and then the tested compounds were intravenously injected (200 pL) at a dose of 10 mg / kg. After 6 hours, 50 pL of CO probe 1-AC (4 pM) was injected intratum orally. After 1 hours, the tumors from each group were dissected and sectioned into 5 pm slices. The Hoechst dye was used to stain the cell nucleus. The fluorescence images of the tumor slices were acquired using a confocal laser scanning microscope (LSM-710, Zeiss).In vivo anticancer efficacy studies. When the tumor volume was about 100 mm3, the tumor-bearing mice were randomly divided into six groups (PBS, 7a, 14a, 8a, 17 and 20) with five mice in each group. The tested compounds were dissolved with 5% DMSO containing 3% Tween-80 solution, and then were intravenously injected (10 mg / kg, 200 pL) at Day 0, 3 and 5. During the 24-day observation period, the tumor size was measured with a digital caliper, and the tumor volume was calculated according to the following formula: volume = width2x length / 2. The tumor-bearing mice were euthanized and sacrificed once the volume of the tumor reached 1500 mm3, and the survival rates were plotted against the days. Example 4. Synthesis and Activity of Adamantane-fused Norbornen-7-ones.Synthesis. As shown in Scheme 7, CO-22X were each synthesized in 5-7 steps. Specifically, compounds 202a-b were obtained by ring expansion of compounds 201a-b in 68-74% yield by following a method described in Eguchi et al., J. Org. Chem. 1987;52(4):496-500, which is incorporated herein by reference. Compounds 202a-b were then oxidized to form diketone 203a-b in 51-63% yield. Condensation of compound 203a-b with 3 -pentanone in ethanol at room temperature afforded compounds 204a-b in 63-77%yield. Dehydration of compound 204a-b afforded compounds 205a-b. Diels-alder reactions of compounds 205a-b with cis- l,2-bis(phenylsulfonyl)ethylene afforded CO-220 and CO- 221 in 35-54% yield. The availability of a carboxyl group at the R2 position allows for the introduction of additional solubilizing groups. For this, a PEG group of variable length was introduced to give CO-222, -223, -224, and -225.Scheme 7Scheme 7 shows the synthetic route to CO-22X.Solubility Studies. With all the compounds on hand, the first property that was assessed was water solubility. The introduction of a PEG substantially improved the water solubility, where solubility of the compounds increased with increasing PEG length, as shown in Table 2. With CO-225, the water solubility was determined as 1.16 mM, and can be advantageously used in various biology experiments.Table 2. Solubility of CO prodrugs in waterCO Release Properties. CO release was examined for CO-22X using three methods: HPLC / LC-MS for product identity, GC for CO detection, and UV for reaction kinetics (Figure 11). For CO-220, Figure 11, Panel A shows the HPLC chromatogram profiles of CO release in PBS at 37 °C with almost full conversion to CP-220 (the side product after CO release) in about 2 hours. GC studies showed stoichiometric CO release from CO-220 (Figure 11, Panel B) with a ti / 2 of ~36 minutes. Similar half-lives were observed for other CO-22X under physiological pH. CO release from CO-220 was also observed to correlate to the compound’s pH and was slower at pH 4, as expected (Figure 11, Panel C).CO release kinetics were examined at different pH. For this experiment, UV was used to monitor reaction progress. This is because CP-220 has a UV absorbance peak at around 249 nm and CO-220 has no such absorbance. Thus, CO-release rate was determined by monitoring the increase of UV absorbance at 249 nm. As shown in Figure 11, Panel C, CO release at pH 7.4 in PBS is fast with a ti / 2 of ~36 min. Similar half-lives of other CO-22X were observed under physiological pH. CO release from CO-220 was also dependent on the pH of the buffer solution and was slower at pH 4, as expected (Figure 11, Panel C). Such results also indicate the minimal effect of the substituent at the R2 position on the release kinetics.Anti-inflammation Studies. Anti-inflammatory effects of these prodrugs were examined by assessing LPS-stimulated TNF-a secretion in RAW264.7 cells. CO-225 was chosen as a representative because it has the best water solubility. CO-220 and CO-221 were selected for comparisons because they have different solubilizing groups, such as a hydroxyl group or a carboxyl group, other than a PEG group. As shown in Figure 12, Panel A, CO-220 exhibited a pronounced dose-dependent inhibition of LPS-induced TNF-a secretion, indicating its anti-inflammatory effects. CO-221 showed modest inhibition while the effect of CO-225 on LPS-induced TNF-a secretion seems to be non-significant. Figure 12, Panel B shows the lack of cytotoxicity of these compounds.Without being limited by theory, the results indicate different potency for these analogs for reasons beyond CO release stoichiometry. Because of its easy escape from solution, the proximity of CO release to the site of action affects potency. Intracellular CO release was studied by conducting cellular CO imaging experiments (Figure 13) and determining the relative intracellular concentration of the CP product (Figure 14).Imaging Studies. Given the pronounced potency differences, these prodrugs were examined for CO release in a biological milieu. HeLa cells treated with CO-220 and CODP- 202 showed strong blue fluorescence (Figure 13). In contrast, treatment with CO-221 or CO- 225 plus CODP-202 led to attenuated blue fluorescence. Without being limited by theory, such results serve as indications of the relative amounts of CO released intracellularly.Intracellular Prodrug Concentration Determination. Based on the results above, and without being limited by theory, it seems that the propensity for various prodrugs to deliver CO intracellularly depends on their cellular penetration ability. For this, experiments were conducted to determine the intracellular concentrations of CP-22X, as surrogates of the concentration of CO-22X. Therefore, HeLa cells were co-incubated with 50 pM CO-22X for 1 hour. Afterward, the cell pellets were lysed using acetonitrile. The cell lysate was analyzed using HPLC. As shown in Figure 14, the intracellular concentration of CP-220 is greater than that of CP-221 or CP-225.Pharmacokinetic Studies. Pharmacokinetic studies were conducted on these CO prodrugs again using CO-220, CO-221, and CO-225. As shown in Figure 15, CO-220, CO- 221, and CO-225 elevated systemic CO levels using COHb as an indicator. Moreover, the peak COHb levels of CO-220, CO-221, and CO-225 were about 4%, 3%, and 4.5%, respectively. The plasma levels of the CO prodrugs or the CP metabolites (side products after CO release) were all below the detection limit (< 0.5 pM).Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A compound of the following formula:or a pharmaceutically acceptable salt thereof.

2. A compound of the following formula:or a pharmaceutically acceptable salt thereof.

3. A compound of the following formula:or a pharmaceutically acceptable salt thereof.

4. A compound of the following formula:or a pharmaceutically acceptable salt thereof.

5. A compound of the following formula:or a pharmaceutically acceptable salt thereof.

6. A composition comprising a compound of any one of claims 1-5 and a pharmaceutically acceptable carrier.

7. A kit comprising the compound of any one of claims 1-5.

8. A kit comprising the composition of claim 6.

9. A method for treating cancer in a subject, comprising: administering to the subject an effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein the cancer is breast cancer.

11. The method of claim 9, wherein the cancer is pancreatic cancer.

12. The method of claims 9, 10, or 11, further comprising administering a second compound to the subject.

13. The method of claim 12, wherein the second compound comprises a chemotherapeutic agent.

14. A method of delivering carbon monoxide to a cell, comprising: contacting a cell with an effective amount of a compound selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

15. The method of claim 14, wherein the contacting is performed in vitro or in vivo.

16. A compound of Formula I:R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether;R3is OR4or COOR5;R4is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; andR5is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; or a pharmaceutically acceptable salt thereof.

17. The compound of claim 16, wherein R3is OR4and R4is H.

18. The compound of claim 16, wherein R3is COOR5and R5is H.

19. The compound of any one of claims 16-18, wherein R1and R2are sulfonyl.

20. The compound of claim 19, wherein the sulfonyl is SChPh.

21. A compound of Formula II:(II), wherein:R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether; andR6is H, a substituted alkyl, or an unsubstituted alkyl; and n is an integer from 0 to 20; or a pharmaceutically acceptable salt thereof.

22. The compound of claim 21, wherein R6is H or CH3 and R1and R2are sulfonyl.

23. A compound of Formula Ila:wherein n is 3 or 4, or a pharmaceutically acceptable salt thereof.

24. A compound of Formula lib:wherein n is 3 or 4, or a pharmaceutically acceptable salt thereof.

25. A composition comprising a compound of any one of claims 16-24 and a pharmaceutically acceptable carrier.

26. A kit comprising the compound of any one of claims 16-24.

27. A kit comprising the composition of claim 25.

28. A method for treating cancer in a subject, comprising: administering to the subject an effective amount of a compound of Formula I:(I), wherein:R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether;R3is OR4or COOR5;R4is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; andR5is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; or a pharmaceutically acceptable salt thereof.

29. A method for treating cancer in a subject, comprising: administering to the subject an effective amount of a compound of Formula II:(II), wherein:R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether; andR6is H, a substituted alkyl, or an unsubstituted alkyl; and n is an integer from 0 to 10; or a pharmaceutically acceptable salt thereof.

30. The method of claims 28 or 29, wherein the cancer is breast cancer.

31. The method of claims 28 or 29, wherein the cancer is pancreatic cancer.

32. The method of claims 28-31, further comprising administering a second compound to the subject.

33. The method of claim 32, wherein the second compound comprises a chemotherapeutic agent.

34. A method of delivering carbon monoxide to a cell, comprising: contacting a cell with an effective amount of a compound of Formula I:(I), wherein:R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether;R3is OR4or COOR5;R4is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; and R5is H, a substituted or unsubstituted alkyl, or a solubilizing moiety; or a pharmaceutically acceptable salt thereof.

35. A method of delivering carbon monoxide to a cell, comprising: contacting a cell with an effective amount of a compound of Formula II:(II), wherein:R1and R2are each independently selected from the group consisting of sulfonyl, carbonyl, amido, ester, cyano, haloalkyl, alkoxy, and thioether, wherein one of R1or R2is sulfonyl, carbonyl, amido, ester, cyano, or haloalkyl, and wherein the other of R1or R2is sulfonyl, alkoxy, or thioether;R6is H, a substituted alkyl, or an unsubstituted alkyl; and n is an integer from 0 to 10; or a pharmaceutically acceptable salt thereof.

36. The method of claims 34 or 35, wherein the contacting is performed in vitro or in vivo.

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