Compounds for carbonyl sulfide/carbon disulfide/hydrogen sulfide release and methods of making and using the same
10725055 ยท 2020-07-28
Assignee
Inventors
Cpc classification
C12Y402/01001
CHEMISTRY; METALLURGY
C07C333/08
CHEMISTRY; METALLURGY
C07C333/24
CHEMISTRY; METALLURGY
G01N33/52
PHYSICS
C07C329/06
CHEMISTRY; METALLURGY
C07C329/00
CHEMISTRY; METALLURGY
C07C329/16
CHEMISTRY; METALLURGY
C07C275/70
CHEMISTRY; METALLURGY
International classification
G01N33/52
PHYSICS
C07C333/08
CHEMISTRY; METALLURGY
Abstract
Disclosed herein are embodiments of donor compounds that can be used to produce H.sub.2S from COS or CS.sub.2 released from the donor compounds. In some embodiments, the donor compounds can indirectly produce H.sub.2S after being exposed to a reactive component in a triggering event. In other embodiments, the donor compounds can indirectly regenerate H.sub.2S after reacting with an H.sub.2S analyte. The donor compounds disclosed herein can be used for analytical techniques, disease diagnostics, and/or therapeutic applications. Methods of making and using the donor compounds also are provided herein.
Claims
1. A donor compound represented by Formula 1: ##STR00075## wherein ring B is an aryl group or a heteroaryl group selected from pyridyl, pyrrole, or thiophene; each of W and Y independently is oxygen or sulfur; V is oxygen, sulfur, or nitrogen; wherein each of W, Y, and V are selected to provide a carbamodithioate, a carbonodithioate, a carbonothioate, a carbonotrithioate, a thiocarbamate, or a thiocarbonate; or wherein W is oxygen and Y and V are both sulfur or Y is oxygen and W and V are both sulfur; R.sup.1, if present, is hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl; R.sup.2 is heteroaliphatic, aryl, heteroaryl, a saccharide, a targeting group, a detectable moiety, a drug molecule, or combinations thereof; each R.sup.3 independently is aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, hydroxyl, thiol, amine, imine, azide, a boronate ester, a boronic acid, a disulfide, an ester, a heteroaliphatic ester group, nitro, a silyl ether, a phosphoric ester, a sulfuric ester, 3,5-dinitrobenzenesulfonic acid, or a combination thereof; and n is an integer selected from 1 to 5; provided that when ring B is an aryl or heteroaryl group selected from pyridyl, pyrrole, or thiophene, the aryl or heteroaryl group comprises at least one ortho- or para-positioned R.sup.3 group, relative to the ##STR00076## group, wherein the ortho-positioned R.sup.3 group is a thiol; an amine of formula NR.sup.bR.sup.c, wherein each of R.sup.b and R.sup.c independently is selected from hydrogen, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and any combination thereof; an imine; a boronate ester; a boronic acid; a disulfide; an ester; a heteroaliphatic ester group; a nitro group; a silyl ether; a phosphoric ester; a sulfuric ester; or 3,5-dinitrobenzenesulfonic acid; and/or wherein the para-positioned R.sup.3 group is a thiol; an amine, an imine, an azide, a boronate ester, a boronic acid, a disulfide, an ester, a heteroaliphatic ester group, a nitro group, a silyl ether, a phosphoric ester, a sulfuric ester, or 3,5-dinitrobenzenesulfonic acid; if R.sup.1 is hydrogen and R.sup.2 is aryl, then the aryl group is -phenyl-(R.sup.4).sub.m wherein m is an integer selected from 0 to 5 and R.sup.4 is alkoxy, thioether, amide, amine, hydroxyl, thiol, acyloxy, azide, aliphatic, aryl, aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, quaternary amine, bromo, iodo, alkyl halide, heterocyclyl, pyridinyl, or pyridinyl wherein the nitrogen atom is functionalized with an aliphatic or aryl group; if R.sup.1 is hydrogen and V is N, then R.sup.2 is heteroaliphatic, aryl, heteroaryl, a saccharide, a targeting group, a detectable moiety, a drug molecule, or a combination thereof; and the donor compound is not or is other than ##STR00077## ##STR00078##
2. The donor compound of claim 1, wherein ring B is a phenyl group a pyridyl group, a thiophene group, or a pyrrole group; R.sup.2 is phenyl or phenyl comprising one or more R.sup.4 groups, wherein R.sup.4 is alkoxy; thioether; amide; amine; hydroxyl; thiol; acyloxy; aliphatic; aryl; aldehyde; ketone; ester; carboxylic acid; acyl; acyl halide; cyano; sulfonate; nitro; quaternary amine; heterocyclyl; pyridinyl; pyridinyl comprising a nitrogen atom functionalized with an aliphatic or aryl group; alkyl halide; halogen; 3H-spiro[isobenzofuran-1,9-xanthen]-3-one; 4-methyl-2H-chromen-2-one; or 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione; the ortho-positioned R.sup.3 group, if present, is a methyl ester, a pinacol boronate ester, a nitro, OCH.sub.2OC(O)t-butyl, silyl ether, SS-pyridinyl, or SS-phenyl; the para-positioned R.sup.3 group, if present, is a methyl ester, a pinacol boronate ester, an azide, a nitro, OCH.sub.2OC(O)t-butyl, silyl ether, SS pyridinyl, or SS-phenyl; and any other R.sup.3 groups are independently selected from aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, hydroxyl, thiol, amine, imine, azide, a boronate ester, a boronic acid, a disulfide, an ester, a heteroaliphatic ester group, nitro, a silyl ether, a phosphoric ester, a sulfuric ester, 3,5-dinitrobenzenesulfonic acid, or a combination thereof.
3. The donor compound of claim 1, represented by any one or more of Formulas 2A-2I: ##STR00079##
4. The donor compound of claim 1, represented by any one of Formulas 3A-3I: ##STR00080## wherein ring A is aryl or heteroaryl; and each R.sup.4 independently is alkoxy; thioether; amide; amine; hydroxyl; thiol; acyloxy; aliphatic; aryl; aldehyde; ketone; ester; carboxylic acid; acyl; acyl halide; cyano; sulfonate; nitro; quaternary amine; heterocyclyl; pyridinyl; pyridinyl comprising a nitrogen atom functionalized with an aliphatic or aryl group; alkyl halide; halogen; or together with one or more other R.sup.4 groups and ring A forms a 4-methyl-2H-chromen-2-one group; a 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione group, or a 3H-spiro[isobenzofuran-1,9-xanthen]-3-one group; and m is an integer selected from 0 to 5.
5. The donor compound of claim 1, represented by having a structure satisfying any one of Formulas 4-7: ##STR00081## wherein V is oxygen, sulfur, or NR.sup.1 Z is CH or nitrogen; X is S or NR, wherein R is selected from hydrogen, aliphatic, aryl, heteroaliphatic, or heteroaryl; each R.sup.4 independently is alkoxy; thioether; amide; amine; hydroxyl; thiol; acyloxy; aliphatic; aryl; aldehyde; ketone; ester; carboxylic acid; acyl; acyl halide; cyano; sulfonate; nitro; quaternary amine; heterocyclyl; pyridinyl; pyridinyl comprising a nitrogen atom functionalized with an aliphatic or aryl group; alkyl halide; halogen; or together with one or more other R.sup.4 groups and ring A forms a 4-methyl-2H-chromen-2-one group; a 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione group, or a 3H-spiro[isobenzofuran-1,9-xanthen]-3-one group; and m is an integer selected from 0 to 5.
6. The donor compound of claim 1, represented by any one of Formulas 4A-4I, 5A-5I, 6A-6I, or 7A-7K: ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## wherein Z is CH or nitrogen; X is S or NR, wherein R is selected from hydrogen, aliphatic, aryl, heteroaliphatic, or heteroaryl; each R.sup.4 independently is alkoxy; thioether; amide; amine; hydroxyl; thiol; acyloxy; aliphatic; aryl; aldehyde; ketone; ester; carboxylic acid; acyl; acyl halide; cyano; sulfonate; nitro; quaternary amine; heterocyclyl; pyridinyl; pyridinyl comprising a nitrogen atom functionalized with an aliphatic or aryl group; alkyl halide; halogen; or together with one or more other R.sup.4 groups and ring A forms a 4-methyl-2H-chromen-2-one group; a 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione group, or a 3H-spiro[isobenzofuran-1,9-xanthen]-3-one group; and m is an integer selected from 0 to 5.
7. The donor compound of claim 6, wherein m is 0 or 1 and each R.sup.4, if present, independently is selected from alkyl, azide, fluoro, bromo, chloro, iodo, or morpholinyl.
8. The donor compound of claim 1, wherein the donor compound is selected from ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##
9. A method, comprising: administering to a sample a donor compound of claim 1, or a composition thereof; and exposing the donor compound to a reactive component to release COS, CS.sub.2, H.sub.2S, or any combination thereof.
10. The method of claim 9, wherein the reactive component is an oxidant, a reductant, an enzyme, a nucleophile, light, an acid, a base, or any combination thereof.
11. The method of claim 9, further comprising exposing the sample, released COS, CS.sub.2, or a combination thereof to carbonic anhydrase, nitrogenase, RuBisCO, CO dehydrogenase, COSase, CS.sub.2 hydrolase, water, or a combination thereof.
12. The method of claim 9, further comprising analyzing the sample to detect a reaction between the donor compound and the reactive component, measuring an amount of COS, CS.sub.2, or H.sub.2S produced, or any combination thereof.
13. The method of claim 12, wherein analyzing the sample comprises detecting a color change and/or fluorescence change produced by a reaction product of the reaction between the donor compound and the reactive component.
14. The method of claim 9, wherein the sample is a biological sample selected from a cell, tissue, and/or bodily fluid.
15. A method, comprising: administering to a sample a donor compound of claim 1; exposing the sample to one or more of H.sub.2S, reactive oxygen, sulfur, and nitrogen species, to form a composition comprising an amine-terminated compound, COS, CS.sub.2, a cyclic by-product represented by a formula selected from: ##STR00110## or any combination thereof, wherein each Y.sup.a independently is selected from oxygen, NH, or sulfur and each X.sup.a independently is selected from NH or sulfur; or any combination of the amine-terminated compound, the COS, CS.sub.2, and the cyclic by-product; and regenerating H.sub.2S by exposing the composition to H.sub.2O, carbonic anhydrase, nitrogenase, RuBisCO, CO dehydrogenase, COSase, CS.sub.2 hydrolase, or a combination thereof.
16. The method of claim 15, further comprising detecting a reaction between the donor compound and the reactive component, measuring an amount of COS, CS.sub.2, and/or H.sub.2S produced, or any combination thereof, wherein analyzing the sample comprises detecting a color change and/or fluorescence change produced by a reaction product of a reaction between the donor compound and one or more of the H.sub.2S, other reactive oxygen, sulfur, and/or nitrogen species.
17. A method, comprising: administering to a sample from a subject a donor compound of claim 1; exposing the sample to a reactive component; and detecting a reaction in the sample between the donor compound and a reactive component and/or measuring an amount of COS, CS.sub.2, and/or H.sub.2S produced by the reaction.
18. The method of claim 17, further comprising determining if a subject has a disease associated with H.sub.2S misregulation or a disease associated with carbonic anhydrase overexpression.
19. A kit, comprising: a donor compound of claim 1; and a filter, a multi-well plate, a test strip, a slide, a disc, a container, an enzyme, carbonic anhydrase, nitrogenase, RuBisCO, CO dehydrogenase, COSase, CS.sub.2 hydrolase, water, a solubilizing agent, or a combination thereof.
20. A donor compound represented by Formula 2A: ##STR00111## wherein ring B is an aryl group or a heteroaryl group; R.sup.1 is hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl; R.sup.2 is aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, a saccharide, a targeting group, a detectable moiety, a drug molecule, or combinations thereof; each R.sup.3 independently is aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, hydroxyl, thiol, amine, imine, azide, a boronate ester, a boronic acid, a disulfide, an ester, a phosphoric ester, a sulfuric ester, a heteroaliphatic ester group, nitro, a silyl ether, 3,5-dinitrobenzenesulfonic acid, or a combination thereof; and n is an integer selected from 1 to 5; provided that when ring B is an aryl or heteroaryl group, the aryl or heteroaryl group comprises at least one ortho- or para-positioned R.sup.3 group, relative to the ##STR00112## group, wherein the ortho-positioned R.sup.3 group is a hydroxyl, a thiol, an amine, a heteroaliphatic group, an imine, a boronate ester, a boronic acid, a disulfide, an ester, a heteroaliphatic ester group, a nitro group, a silyl ether, a phosphoric ester, a sulfuric ester, or 3,5-dinitrobenzenesulfonic acid; and/or wherein the para-positioned R.sup.3 group is a hydroxyl, a thiol, an amine, a heteroaliphatic group, an imine, an azide, a boronate ester, a boronic acid, a disulfide, an ester, a heteroaliphatic ester group, a nitro group, a silyl ether, a phosphoric ester, a sulfuric ester, or 3,5-dinitrobenzenesulfonic acid; if R.sup.1 is hydrogen and R.sup.2 is aryl, then the aryl group is -phenyl-(R.sup.4).sub.m wherein m is an integer selected from 0 to 5 and R.sup.4 is alkoxy, thioether, amide, amine, hydroxyl, thiol, acyloxy, azide, aliphatic, aryl, aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, quaternary amine, fluoro, bromo, iodo, alkyl halide, heterocyclyl, pyridinyl, or pyridinyl wherein the nitrogen atom is functionalized with an aliphatic or aryl group; if R.sup.1 is hydrogen then R.sup.2 is non-aromatic aliphatic; or heteroaliphatic, aryl, heteroaryl, a saccharide, a targeting group, a detectable moiety, a drug molecule, or a combination thereof; and the donor compound is not or is other than ##STR00113## ##STR00114##
21. A donor compound represented by any one or more of the following formulas: ##STR00115## wherein each Z independently is CH or nitrogen; each X independently is O, S, or NR, wherein R is selected from hydrogen, aliphatic, aryl, heteroaliphatic, or heteroaryl; each R.sup.3 independently is aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, hydroxyl, thiol, amine, azide, a boronate ester, a boronic acid, a disulfide, an ester, nitro, a phosphoric ester, a sulfuric ester, a silyl ether, 3,5-dinitrobenzenesulfonic acid, or a combination thereof; R.sup.4 is alkoxy, thioether, amide, amine, hydroxyl, thiol, acyloxy, aliphatic, aryl, aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, quaternary amine, fluoro, bromo, iodo, alkyl halide, pyridinyl, pyridinyl wherein the nitrogen atom is functionalized with an aliphatic or aryl group; and n is an integer selected from 1 to 3; and provided that the donor compound is not ##STR00116##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
I. Explanation of Terms
(26) The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, comprising means including and the singular forms a or an or the include plural references unless the context clearly dictates otherwise. The term or refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.
(27) Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.
(28) Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term about. Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word about is recited. Furthermore, not all alternatives recited herein are equivalents.
(29) To facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided. Certain functional group terms include an R.sup.a group that, though not part of the defined functional group, indicates how the functional group attaches to the donor compound to which it is bound. Also, a dashed bond (i.e., ---) as used in certain formulas described herein indicates an optional bond (that is, a bond that may or may not be present). A person of ordinary skill in the art would recognize that the definitions provided below and the donor compounds and formulas included herein are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. In formulas and donor compounds disclosed herein, a hydrogen atom is present and completes any formal valency requirements (but may not necessarily be illustrated) wherever a functional group or other atom is not illustrated. For example, a phenyl ring that is drawn as
(30) ##STR00001##
comprises a hydrogen atom attached to each carbon atom of the phenyl ring other than the a carbon, even though such hydrogen atoms are not illustrated. Any functional group disclosed herein and/or defined above can be substituted or unsubstituted, unless otherwise indicated herein.
(31) Acyloxy: R.sup.aOC(O)R.sup.b, wherein R.sup.a is the atom of the formulas disclosed herein to which the acyloxy group is attached and R.sup.b is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, or heteroaryl.
(32) Acyl Halide: R.sup.aC(O)X, wherein R.sup.a is the atom of the formulas disclosed herein to which the acyl halide group is attached and X is a halogen, such as Br, F, I, or Cl.
(33) Aldehyde: R.sup.aC(O)H, wherein R.sup.a is the atom of the formulas disclosed herein to which the aldehyde group is attached.
(34) Aliphatic: A hydrocarbon, or a radical thereof, having at least one carbon atom to 50 carbon atoms, such as one to 25 carbon atoms, or one to ten carbon atoms, and which includes alkanes (or alkyl), alkenes (or alkenyl), alkynes (or alkynyl), including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well.
(35) Alkenyl: An unsaturated monovalent hydrocarbon having at least two carbon atoms to 50 carbon atoms, such as two to 25 carbon atoms, or two to ten carbon atoms, and at least one carbon-carbon double bond, wherein the unsaturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent alkene. An alkenyl group can be branched, straight-chain, cyclic (e.g., cycloalkenyl), cis, or trans (e.g., E or Z).
(36) Alkyl: A saturated monovalent hydrocarbon having at least one carbon atom to 50 carbon atoms, such as one to 25 carbon atoms, or one to ten carbon atoms, wherein the saturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent compound (e.g., alkane). An alkyl group can be branched, straight-chain, or cyclic (e.g., cycloalkyl).
(37) Alkynyl: An unsaturated monovalent hydrocarbon having at least two carbon atoms to 50 carbon atoms, such as two to 25 carbon atoms, or two to ten carbon atoms and at least one carbon-carbon triple bond, wherein the unsaturated monovalent hydrocarbon can be derived from removing one hydrogen atom from one carbon atom of a parent alkyne. An alkynyl group can be branched, straight-chain, or cyclic (e.g., cycloalkynyl).
(38) Alkoxy: R.sup.aO-alkyl, R.sup.aO-alkenyl, or R.sup.aO-alkynyl, wherein R.sup.a is the atom of the formulas disclosed herein to which the alkoxy is attached with exemplary embodiments including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy.
(39) Amide: R.sup.aC(O)NR.sup.bR.sup.c wherein R.sup.a is the atom of the formulas disclosed herein to which the amide is attached, and each of R.sup.b and R.sup.c independently is selected from hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, or any combination thereof.
(40) Amine: R.sup.aNR.sup.bR.sup.c, wherein R.sup.a is the atom of the formulas disclosed herein to which the amine is attached, and each of R.sup.b and R.sup.c independently is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and any combination thereof.
(41) Amine-terminated Compound: A compound produced by a reaction between a reactive component and a donor compound contemplated by the present disclosure. Amine-terminated compounds comprise a terminal amine group that is obtained from the fragmentation or bond breaking of the carbon-nitrogen bond present in a WC(Y)V group wherein V is nitrogen.
(42) Aryl: An aromatic carbocyclic group comprising at least five carbon atoms to 15 carbon atoms, such as five to ten carbon atoms, having a single ring or multiple condensed rings, which condensed rings can or may not be aromatic provided that the point of attachment is through an atom of the aromatic carbocyclic group.
(43) Boronate Ester: A functional group comprising a boron atom covalently bound to two oxygen atoms that are in turn bound to one another through an aliphatic, aryl, heteroaliphatic, or heteroaryl group.
(44) Boronic acid: A functional group comprising a boron atom covalently bound to two hydroxyl groups.
(45) Carbamodithioate: A functional group satisfying a formula WC(Y)V of the donor compounds described herein, wherein W is sulfur, C is carbon, Y is sulfur, and V is nitrogen connected to an R.sup.1 group (i.e., S(CS)NR.sup.1).
(46) Carbonodithioate: A functional group satisfying a formula WC(Y)V of the donor compounds described herein, wherein W is sulfur, C is carbon, Y is sulfur, and V is oxygen (i.e., S(CS)O).
(47) Carbonothioate: A functional group satisfying a formula WC(Y)V of the donor compounds described herein, wherein W is oxygen, C is carbon, Y is sulfur, and V is oxygen (i.e., O(CS)O).
(48) Carbonotrithioate: A functional group satisfying a formula WC(Y)V of the donor compounds described herein, wherein W is sulfur, C is carbon, Y is sulfur, and V is sulfur (i.e., S(CS)S).
(49) Carboxyl: R.sup.aC(O)OR.sup.b, wherein R.sup.a is the atom of the formulas disclosed herein to which the carboxyl group is attached and wherein R.sup.b is alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, hydrogen, and any combination thereof.
(50) Cyclic By-Product: A compound having a cyclic core that is formed by a reaction between a reactive component and a donor compound contemplated by the present disclosure. In particular disclosed embodiments, the cyclic by-product has a structure meeting any one or more of the following formulas:
(51) ##STR00002##
wherein Y.sup.a and X.sup.a independently is selected from oxygen, NR (wherein R is selected from hydrogen, aliphatic, or aryl), or sulfur.
(52) Detectable signal: A color change that occurs when a donor compound disclosed herein comprising a detectable moiety (e.g., a fluorophore or a dye) reacts with a reactive component to release the detectable moiety.
(53) Disulfide: R.sup.aSSR.sup.b, wherein R.sup.a is the atom of the formulas disclosed herein to which the disulfide group is attached and wherein R.sup.b is selected from hydrogen, aliphatic, aryl, heteroaliphatic, or heteroaryl.
(54) Donor Compound: A compound having a structure satisfying any one or more of the formulas described herein, such as Formulas 1, 2A-2I, 3A-3I, 4-7, 4A-4I, 5A-5I, 6A-6I, or 7A-7K as disclosed herein. In some embodiments, a donor compound is also called a compound contemplated by the present disclosure, or donor compound contemplated by the present disclosure.
(55) Ester: R.sup.aC(O)OR.sup.b, wherein R.sup.a is the atom of the formulas disclosed herein to which the aldehyde group is attached and R.sup.b is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, or heteroaryl.
(56) Fluorophore: A functional group, or portion, of a donor compound disclosed herein that causes the donor compound or a part of the donor compound (e.g., an amine-terminated group) to fluoresce when exposed to an excitation source. Exemplary fluorophores are selected from, but not limited to, methylrhodol, 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, 4-methyl-2H-chromen-2-one, coumarin, naphthalimide, fluorescein, rhodamine, rhodol, Cy3, or Cy5.
(57) Haloaliphatic: An aliphatic group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo.
(58) Haloalkyl: An alkyl group wherein one or more hydrogen atoms, such as one to 10 hydrogen atoms, independently is replaced with a halogen atom, such as fluoro, bromo, chloro, or iodo. In an independent embodiment, haloalkyl can be a CX3 group, wherein each X independently can be selected from fluoro, bromo, chloro, or iodo.
(59) Heteroaliphatic: An aliphatic group comprising at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, selenium, phosphorous, and oxidized forms thereof within the group.
(60) Heteroalkyl/Heteroalkenyl/Heteroalkynyl: An alkyl, alkenyl, or alkynyl group (which can be branched, straight-chain, or cyclic) comprising at least one heteroatom to 20 heteroatoms, such as one to 15 heteroatoms, or one to 5 heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, selenium, phosphorous, and oxidized forms thereof within the group.
(61) Heteroaryl: An aryl group comprising at least one heteroatom to six heteroatoms, such as one to four heteroatoms, which can be selected from, but not limited to oxygen, nitrogen, sulfur, selenium, phosphorous, and oxidized forms thereof within the ring. Such heteroaryl groups can have a single ring or multiple condensed rings, wherein the condensed rings may or may not be aromatic and/or contain a heteroatom, provided that the point of attachment is through an atom of the aromatic heteroaryl group.
(62) Ketone: R.sup.aC(O)R.sup.b, wherein R.sup.a is the atom of the formulas disclosed herein to which the ketone is attached, and R.sup.b is selected from aliphatic, heteroaliphatic, aryl, heteroaryl, and any combination thereof.
(63) Reactive Component: A compound that reacts (e.g., via reduction, oxidation, nucleophilic attack, photolysis, or enzymatic cleavage) with the donor compounds contemplated by the present disclosure so as to initiate COS or CS.sub.2 release from the donor compound. Exemplary precursor compounds include, but are not limited to, donor compounds comprising a thiocarbamate, a carbamodithioate, a carbonothioate, a carbonodithioate, a carbonotrithioate, or a thiocarbonate. Exemplary reactive components include, but are not limited to oxidants (e.g., H.sub.2O.sub.2, benzoyl peroxide, and other peroxides, F.sub.2, Cl.sub.2, Br.sub.2, I.sub.2, or the like), reductants (e.g., formic acid, ascorbic acid, dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), oxalic acid, hydrazine, hydride-based reductants, hydrogen sulfide, and the like), enzymes (e.g., esterases, such as acetylesterases, thioesterases, phosphatases, sulfatases, and the like), acids (e.g., mineral acids, such as sulfuric acid, fluorosulfuric acid, phosphoric acid; sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, tosylic acid, and triflic acid; carboxylic acids, such as acetic acid, citric acid, formic acid, gluconic acid, ascorbic acid, lactic acid, oxalic acid, and tartaric acid; halogenated carboxylic acids, such as fluoroacetic acid and trifluoroacetic acid; or combinations thereof), bases (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, and amine-containing bases), nucleophiles (e.g., fluoride (F.sup.) sources, such as tetra-n-butylammonium fluoride (TBAF) or tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF), or hydrogen sulfide), light (e.g., UV light) or a combination thereof. In particular disclosed embodiments, the reactive component can also be hydrogen sulfide, or other reactive sulfur, oxygen, or nitrogen species (e.g., cysteine, homocysteine, thiosulfate, sulfite, sulfate, or nitrogen oxide).
(64) Saccharide: A sugar that can be selected from monosaccharides, disaccharides, oligosaccharides, and/or polysaccharides.
(65) Silyl Ether: A functional group comprising a silicon atom covalently bound to an alkoxy group.
(66) Solubilizing Agent: An agent that increases the solubility of a donor compound disclosed herein in aqueous media. Solubilizing agents can be selected from, but are not limited to, sodium bicarbonate, glucose, polyalkylene ethers or glycols (e.g., polyethylene glycol, polypropylene glycol, and the like), surfactants (e.g., sorbitan esters), and other solubilizing agents known in the art.
(67) Sulfonyl/Sulfonate: A functional group having a formula R.sup.aSO.sub.2R.sup.b, wherein R.sup.a is the atom of the formulas disclosed herein to which the sulfonyl or sulfonate is attached, and R.sup.b is selected from hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, and any combination thereof.
(68) Targeting Group: A group that is capable of targeting a cell, an organelle, or the like and thereby directing the donor compound comprising the targeting group to the cell, organelle, or the like. In some embodiments, a targeting group can be a morpholine, a phosphonium or phosphine group, a quaternary amine, or the like. Other targeting components can include thiol or hydroxyl functional groups, which can be used to target surfaces or monolayers.
(69) Thiocarbamate: A functional group satisfying a formula WC(Y)V of the donor compounds described herein, wherein W is sulfur or oxygen, C is carbon, Y is sulfur, and V is nitrogen attached to an R.sup.1 group. In embodiments where W is sulfur, the functional group is an S-thiocarbamate having a structure S(CS)NR.sup.1. In embodiments where W is oxygen, the functional group is an O-thiocarbamate having a structure O(CS)NR.sup.1. As used herein, the term thiocarbamate includes S-thiocarbamate and O-thiocarbamate, unless expressly indicated otherwise.
(70) Thiocarbonate: A functional group satisfying a formula WC(Y)V of the donor compounds described herein, wherein W is sulfur, C is carbon, Y is oxygen, and V is oxygen (i.e., S(CO)O).
II. Introduction
(71) Conventional tools for analyte donation and reaction-based detection has provided significant insights into the roles of reactive sulfur, oxygen, and nitrogen species (RSONs) in biology; however, there are challenges associated with conventional methods used for both of these processes. For example, most donor constructs require consumption of other RSONs to release the caged analyte, thus shifting the RSON landscape as a consequence of caged analyte release. Donor constructs that can be triggered to release H.sub.2S by readily-modifiable triggers would provide a versatile platform for H.sub.2S donation in different contexts (
(72) To address these limitations, the present inventors have developed novel compounds and methods to access caged, triggerable COS/H.sub.2S donor motifs and CS.sub.2/H.sub.2S donor motifs that have direct applications in both caged analyte release and analyte-replacement probe development. In some embodiments, self-immolative H.sub.2S donors have been made to provide a new class of easily-modifiable H.sub.2S donors that can be triggered by external stimuli, and translate this caged H.sub.2S motif to address the issue of analyte consumption in reaction-based fluorescent probe design. Analyte replacement fluorescent probes, which upon reaction with H.sub.2S activate a fluorescence response and also release H.sub.2S, also are described herein and can provide a significant advance in addressing the problem of analyte homeostasis in reaction-based probe development (
(73) The donor compounds and methods described herein are not only useful research tools, but also have many potential therapeutic applications, such as providing a platform for protection in patients with high, eminent MI markers. The donor compounds described herein can be used as a modular donation platform in which the trigger can readily be modified to respond to different events, including biological (such as enzymatic), chemical (such as peroxy), and photolytic (such as UV irradiation) events. In some embodiments, the donor compounds described herein contain a caged molecule of H.sub.2S, which is released upon donor activation. In these systems, the donor compounds can release carbonyl sulfide (COS), which is rapidly converted to H.sub.2S by the ubiquitous enzyme carbonic anhydrase (CA), or by water. In yet additional embodiments, the donor compounds can release carbon disulfide (CS.sub.2), which can be converted to H.sub.2S using water or enzymes such as nitrogenase, RuBisCO, CO dehydrogenase, COSase, CS.sub.2 hydrolase, or a combination thereof. The inventive H.sub.2S-releasing compounds described herein are highly modular, allowing for both the triggering moieties and the rate of H.sub.2S release from the platform to be readily modified. There are a number of diseases, such as glaucoma, that are characterized by an over-expression of CA but which can also be treated by H.sub.2S addition. Such diseases are particularly attractive platforms for the disclosed COS/H.sub.2S donor motifs and the disclosed CS.sub.2/H.sub.2S donor motifs.
III. Compounds
(74) Disclosed herein are donor compounds that can be used to trigger production of H.sub.2S and/or to detect the presence of H.sub.2S while further regenerating H.sub.2S. Such donor compounds react with a reactive component to produce COS or CS.sub.2, which can be further converted to H.sub.2S. In some embodiments, the donor compounds described herein can have structures meeting Formula 1.
(75) ##STR00003##
(76) With reference to Formula 1, each of W and Y independently can be selected from oxygen or sulfur; V can be selected from oxygen, sulfur, or nitrogen (wherein if V is nitrogen, then the dashed bond of Formula 1 represents a bond between R.sup.1 and the nitrogen atom; or if V is oxygen or sulfur, then the dashed bond and R.sup.1 are not present); R.sup.3 can be a functional group selected to provide donor compounds responsive to oxidative stress, intracellular esterases, endogenous stimuli, light, or external biorthogonal stimuli; and n can be an integer selected from 0 to 5. Solely by way of example, R.sup.3 can be an ester group, a heteroaliphatic ester group (e.g., O(CH.sub.2).sub.rOCOR.sup.b, S(CH.sub.2).sub.rOCOR.sup.b, or NH(CH.sub.2).sub.rOCOR.sup.b, wherein R.sup.b is aliphatic and r is 1-4), phosphoric esters, sulfuric esters, azo groups, nitro groups, or thioesters that can be hydrolyzed by enzymes. In yet other embodiments, R.sup.3 can be a boronate ester or boronic acid that reacts with H.sub.2O.sub.2 to generate a phenol. In additional disclosed embodiments, R.sup.3 can be a biorthogonal functional group, such as a silyl ether that can be converted to a phenol upon exposure to a fluoride (F.sup.) source, or a silyl ester.
(77) In particular disclosed embodiments, ring B can be an aryl group or a heteroaryl group; R.sup.1 can be selected from hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl; R.sup.2 can be selected from aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, sugars, organelle targeting groups, fluorophores, drug molecules, or combinations thereof; each R.sup.3 independently can be selected from aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, hydroxyl, thiol, amine, azide, a boronate ester, a boronic acid, a disulfide, an ester, a heteroaliphatic ester group (e.g., O(CH.sub.2).sub.rOCOR.sup.b, S(CH.sub.2).sub.rOCOR.sup.b, or NH(CH.sub.2).sub.rOCOR.sup.b, wherein R.sup.b is aliphatic and r is 1-4), nitro, a silyl ether, 3,5-dinitrobenzenesulfonic acid, or a combination thereof; and n can be 1-5. In some embodiments, ring B is selected from furan, thiophene, phenyl, pyridyl, or pyrrole. In some embodiments, R.sup.1 is hydrogen. In some embodiments, R.sup.2 is -phenyl-(R.sup.4).sub.m, (wherein m can be 0-5 and R.sup.4 is as described below), or a fluorophore, such as methylrhodol, 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, 4-methyl-2H-chromen-2-one, or a combination thereof.
(78) In some embodiments, the donor compounds can have structures satisfying any one of Formulas 2A-2I.
(79) ##STR00004##
(80) With reference to Formulas 2A-2I, R.sup.3 can be as described above for Formula 1. For example, R.sup.3 can be a functional group selected to provide donor compounds responsive to oxidative stress, intracellular esterases, endogenous stimuli, or external biorthogonal stimuli. In some embodiments, R.sup.3 can be a group that can be hydrolyzed by esterases, such as an ester group, phosphoric esters, sulfuric esters, a heteroaliphatic ester group (e.g., O(CH.sub.2).sub.rOCOR.sup.b, S(CH.sub.2).sub.rOCOR.sup.b, or NH(CH.sub.2).sub.rOCOR.sup.b, wherein R.sup.b is aliphatic and r is 1-4), or thioesters. In yet other embodiments, R.sup.3 can be a boronate ester or boronic acid that reacts with H.sub.2O.sub.2 to generate a phenol. In additional disclosed embodiments, R.sup.3 can be a biorthogonal functional group, such as a silyl ether that can be converted to a phenol upon exposure to a fluoride (F.sup.) source. In yet additional embodiments, R.sup.3 can be a light triggerable group (e.g., nitro). In particular disclosed embodiments, ring B can be an aryl group or a heteroaryl group; R.sup.1 can be selected from hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl; R.sup.2 can be selected from aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, sugars, organelle targeting groups, fluorophores, drug molecules, or combinations thereof; each R.sup.3 independently can be selected from aliphatic, heteroaliphatic, aryl, heteroaryl, halogen, hydroxyl, thiol, amine, azide, a boronate ester, a boronic acid, a disulfide, an ester, OCH.sub.2OCOR.sup.b (wherein R.sup.b is aliphatic), nitro, a silyl ether, 3,5-dinitrobenzenesulfonic acid, or a combination thereof; and n can be 0-5. In some embodiments, ring B is selected from furan, thiophene, phenyl, pyridyl, or pyrrole. In some embodiments, R.sup.1 is hydrogen. In some embodiments, R.sup.2 is -phenyl-(R.sup.4)m (wherein m can be 0-5 and R.sup.4 is as described below), or a fluorophore, such as methylrhodol, 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, 4-methyl-2H-chromen-2-one, or a combination thereof.
(81) In some embodiments, the donor compounds can have structures satisfying Formula 3A-3I.
(82) ##STR00005##
With reference to Formulas 3A-3I, each of ring B, R.sup.1, R.sup.3, and n can be as recited above for Formulas 1 and 2A-2I; ring A can be aryl or heteroaryl; each R.sup.4 independently can be selected from aliphatic, aryl, heteroaliphatic, heteroaryl, or a combination thereof; and m can be 0 to 5. In particular disclosed embodiments, R.sup.4 can be selected to control the rate of COS or CS.sub.2 (and as such the rate of H.sub.2S) released from the donor compound. For example, selecting electron-withdrawing groups or electron-donating groups for R.sup.4 can result in the ability to control/tune the rate of COS or CS.sub.2 release. As such, R.sup.4 can be selected from electron-donating groups (e.g., alkoxy, thioether, amide, amine, hydroxyl, thiol, acyloxy, azide, aliphatic [e.g., alkyl, alkenyl, alkynyl)], aryl) or from electron-withdrawing groups (e.g., aldehyde, ketone, ester, carboxylic acid, acyl, acyl halide, cyano, sulfonate, nitro, quaternary amine, pyridinyl [or pyridinyl wherein the nitrogen atom is functionalized with an aliphatic or aryl group], alkyl halide, heterocyclyl, or halogen). In some embodiments, two or more R.sup.4 groups can be joined to form a multicyclic fused ring system with Ring A. Exemplary fused ring systems formed when two R.sup.4 groups join together include, but are not limited to methylrhodol, 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, 4-methyl-2H-chromen-2-one.
(83) In particular disclosed embodiments, the donor compounds can have structures satisfying any one or more of Formulas 4-7 and/or Formulas 4A-4I, 5A-51, 6A-6I, and 7A-7K.
(84) ##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
With reference to the formulas illustrated above, each R.sup.3, R.sup.4, n, and m independently can be as recited above for Formulas 1, 2A-2I, and 3A-3I; Z can be CH or nitrogen; and X can be O, S, or NR (wherein R is selected from hydrogen, aliphatic, or aryl). In particular disclosed embodiments, R.sup.3 can be selected from pinocol boronate, NC(H).sub.2, NC(alkyl).sub.2, OC(O)(CH.sub.2).sub.pCH.sub.3 (wherein p is 0-20), alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy), ester (e.g., OC(O)aliphatic, such as OC(O)methyl, OC(O)ethyl, OC(O)propyl, OC(O)i-propyl, OC(O)butyl, OC(O)t-butyl, OC(O)i-butyl, and the like), heteroaliphatic ester (e.g., O(CH.sub.2).sub.rOCOR.sup.b, S(CH.sub.2).sub.rOCOR.sup.b, or NH(CH.sub.2).sub.rOCOR.sup.b, wherein R.sup.b is aliphatic and r is 1-4), N.sub.3, F, Cl, I, Br, NO.sub.2, OTBS, OTMS, and SS-pyridyl; and R.sup.4 can be selected from F, Cl, Br, I, morpholinyl (wherein the morpholine group is attached via the nitrogen atom of the morpholine group), alkyl (e.g., methyl, ethyl, propyl, butyl, and the like), and hydrogen. In some embodiments, two or more R.sup.4 groups can be joined to form a multicyclic ring system with the phenyl group to which it is attached. Exemplary fused ring systems formed when two R.sup.4 groups join together include, but are not limited to methylrhodol, 2-(2-methoxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, 4-methyl-2H-chromen-2-one.
(85) Exemplary donor compound embodiments are provided below.
(86) ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##
(87) In an independent embodiment, the donor compounds are not, or are other than any of the following:
(88) ##STR00037## ##STR00038## ##STR00039## ##STR00040## ##STR00041##
IV. Methods of Making and Using COS/H.SUB.2.S and CS.SUB.2./H.SUB.2.S Donor/Regenerating Compounds
(89) The donor compounds disclosed herein can be made using coupling reactions between hydroxyl-containing compounds (e.g., benzyl phenols, hydroxyl-containing furan, thiophene, or pyrrole compounds) and isothiocyanate-containing compounds (e.g., isothiocyanate-substituted phenyl compounds, isothiocyanate-substituted fluorophores or dyes, or isothiocyanate-substituted aliphatic compounds). Exemplary methods of making donor compounds disclosed herein are provided below in Schemes 1A and 1B. With reference to Schemes 1A and 1B, each of Y, W, V, R.sup.1, R.sup.2, R.sup.3, R.sup.4, and n can be as recited herein for the formulas described above.
(90) ##STR00042##
(91) ##STR00043##
(92) Another exemplary embodiment of a method for making the donor compounds disclosed herein is provided in Schemes 2A and 2B. As shown in Scheme 2B, a fluorophore can be coupled to a phenol starting material to provide the fluorophore-coupled thiocarbamate compound. The method illustrated in Scheme 2B can be used for other fluorophores and/or dyes comprising an amine functional group.
(93) ##STR00044##
(94) ##STR00045##
(95) The donor compounds disclosed herein can be used to detect the presence of H.sub.2S and/or produce H.sub.2S. In contrast to conventional H.sub.2S detection/release probes, the donor compounds disclosed herein are capable of releasing COS or CS.sub.2, which can then be converted to H.sub.2S. As such, the disclosed donor compounds can be used for controlled release of H.sub.2S (from COS or CS.sub.2 produced from the disclosed donor compounds), which can be triggered by external chemical stimuli. In particular disclosed embodiments, the donor compounds, which can act as COS or CS.sub.2 donors, can be used to target diseases in which carbonic anhydrase is overexpressed and/or as therapeutics for diseases associated with H.sub.2S misregulation, and therefore are useful in targeted therapy. In such embodiments, a therapeutically effective amount of the donor compound, or a sample comprising the donor compound, can be administered. The donor compounds also can be used as pro-drug conjugates. In yet additional embodiments, the donor compounds disclosed herein can be used to generate a detectable (e.g., fluorescent or colorimetric) signal upon COS or CS.sub.2 (or H.sub.2S) donation. As such, the rates and quantities of H.sub.2S can be measured by evaluating the detectable signal produced by the donor compounds after COS/H.sub.2S release or CS.sub.2/H.sub.2S release. The intensity of the detectable signal can be modified to thereby provide the ability to produce different functional donor concentration ranges with the same reaction platform. For example, the intensity can be modified by changing the brightness of the fluorophore or dye attached to the donor compound (e.g., by modifying the fluorophore/dye species and/or by changing the concentration of the donor compound comprising the fluorophore). In yet additional embodiments, the donor compounds disclosed herein can be used to replenish supplies of H.sub.2S as the donor compounds can be chemical tuned to release H.sub.2S upon reaction with H.sub.2S that activates the donor compound, thereby regenerating the H.sub.2S.
(96) In particular disclosed embodiments, methods contemplated by the present disclosure can include administering to a sample (or providing) one or more donor compounds having a structure satisfying any one of the formulas described herein and exposing the sample or the donor compound to a reactive compound to release COS, CS.sub.2, H.sub.2S, or a combination thereof. In some embodiments, exposing the sample or the donor compound to different pH environments can further facilitate COS/H.sub.2S or CS.sub.2/H.sub.2S release. In such embodiments, the pH of the sample can be controlled (such as by adding a reactive component selected from an acid or base) to effect COS or CS.sub.2 release from the COS/H.sub.2S donor compound or CS.sub.2/H.sub.2S donor compound used in the method. Exemplary reactive components include, but are not limited to an oxidant (e.g., H.sub.2O.sub.2 and other peroxides, F.sub.2, Cl.sub.2, Br.sub.2, I.sub.2, or the like), a reductant (e.g., formic acid, ascorbic acid, dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), oxalic acid, hydrazine, hydride-based reductants, and the like), an enzyme (e.g., esterases, such as acetylesterases, thioesterases, phosphatases, sulfatases, and the like), a fluoride (F.sup.) source, an acid (e.g., mineral acid, such as sulfuric acid, fluorosulfuric acid, phosphoric acid; sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, tosylic acid, and triflic acid; carboxylic acids, such as acetic acid, citric acid, formic acid, gluconic acid, ascorbic acid, lactic acid, oxalic acid, and tartaric acid; halogenated carboxylic acids, such as fluoroacetic acid and trifluoroacetic acid; or combinations thereof), a base (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, and amine-containing bases), a nucleophile, light, or a combination thereof.
(97) In particular disclosed embodiments, an ester group (or a heteroaliphatic ester group) can be used as the triggering group, providing slow-release COS/H.sub.2S donor compounds or CS.sub.2/H.sub.2S donor compounds upon ester cleavage by intracellular esterases that do not require consumption of cellular nucleophiles for activation (Scheme 3). By using intracellular esterases to promote cleavage, it is possible to impart cellular trappability, improve membrane permeability, and in the activation of caged pro-drugs and other biological payload. Additionally, activation by intracellular esterases eliminates the consumption of cellular nucleophiles for activation.
(98) ##STR00046##
(99) In additional embodiments, light can be used to initiate COS or CS.sub.2 and H.sub.2S formation from donor compounds disclosed herein. In such embodiments, the donor compounds can comprise functional group capable of being activated by exposure to light. For example, the donor compounds can comprise a nitro group, ketone group, or other photoactivatable groups. In particular disclosed embodiments, the donor compounds comprise at least one R.sup.3 group that is a nitro functional group and that is located at a position on Ring B that is ortho to Ring B's connection to a W group (as illustrated below).
(100) ##STR00047##
By exposing the donor compounds to light, the nitro group, ketone group, or other photoactivatable group will promote cleavage of Ring B to provide intermediate 400, which can then react to provide COS or CS.sub.2, as illustrated below in Scheme 4. The COS or CS.sub.2 can then be exposed to a reactive component to provide H.sub.2S as illustrated and discussed below. In particular disclosed embodiments, UV light can be used to trigger COS or CS.sub.2 release from the donor compounds described herein. For example, light having wavelengths ranging from 250 nm to 450 nm can be used, such as 300 nm to 400 nm, or 325 nm to 350 nm.
(101) ##STR00048##
(102) In some embodiments, the method can further comprise exposing the sample or the released COS or CS.sub.2 produced from the reaction between the reactive component and the donor compound to carbonic anhydrase, nitrogenase, RuBisCO, CO dehydrogenase, COSase, CS.sub.2 hydrolase, water, or a combination thereof. In certain embodiments, the sample can be exposed to the donor compound and the reactive component for a time period ranging from greater than 0 to at least 120 minutes, such as 30 seconds to 120 minutes, or 1 minute to 60 minutes, or 1 minute to 45 minutes, or 1 minute to 30 minutes, or 1 minute to 20 minutes. In particular disclosed embodiments, the sample can be contacted with the donor compound or reactive component at a certain pH, such as greater than 0 to at least 12, such as 3 to 12, or 6 to 9, or 7 to 7.8, or 7 to 7.4. The pH can be maintained using an appropriate buffer, such as a phosphate buffer (e.g., 3-{[tris(hydroxymethyl)methyl]amino} propanesulfonic acid, tris(hydroxymethyl)methylamine, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid, piperazine-N,N-bis(2-ethanesulfonic acid), or the like). In some embodiments, the sample can be further analyzed to determine the progress or completion of the reaction between the donor compound and the reactive component and/or to measure the amount of COS or CS.sub.2 or H.sub.2S produced by the reaction. Such embodiments can comprise detecting a color change or fluorescent signal produced by a reaction product formed from the reaction, such as an amine-terminated fluorophore or amine-terminated dye. Such embodiments also can further comprise exposing the sample to a reagent that facilitates analytical measurement of sulfide, such as monobromobimane (mBB) or methylene blue (MB).
(103) In some embodiments, the method of using the donor compounds described herein can comprise administering to a sample one or more donor compounds having a structure satisfying any of the formulas described herein, exposing the sample to H.sub.2S or another type of RSON species to form a composition comprising an amine-terminated by-product, COS or CS.sub.2, a cyclic by-product having a formula selected from:
(104) ##STR00049##
or any combination thereof, wherein each Y.sup.a and X.sup.a independently is selected from oxygen, NH, or sulfur; or any combination of the amine-terminated by-product, the COS or CS.sub.2, and the cyclic by-product. In some embodiments, the method can further comprise exposing the composition to H.sub.2O or carbonic anhydrase to regenerate H.sub.2S. In some embodiments, the donor compounds, when exposed to H.sub.2S (or another RSON species) or other reactive component, such as those described herein, can produce a mixture of by-products. The mixture of by-products, in some embodiments, can comprise one or more of an amine-terminated by-product, COS or CS.sub.2, or a cyclic by-product as described herein (or a combination of two or more cyclic by-products). In some embodiments, the composition comprises two or more of an amine-terminated by-product, COS or CS.sub.2, or a cyclic by-product as described herein (or a combination of two or more cyclic by-products).
(105) An exemplary embodiment of using the disclosed donor compounds to produce H.sub.2S using oxidative stress activation is shown below in Scheme 5.
(106) ##STR00050##
(107) In particular disclosed embodiments, the donor compounds disclosed herein can be converted to various by-products. In some embodiments, the donor compounds can be converted to a mixture of any of these by-products. Exemplary methods and mechanisms for the reaction process are illustrated below in Schemes 6A, 6B, 7A, 7B, 8, and 9. Schemes 6A, 6B, 7A, 7B, 8, and 9 also illustrate the types of by-products that can be obtained using the donor compounds and methods described herein. The presence of such by-products can be determined using, for example, an H.sub.2S electrode, NMR (.sup.1H, .sup.13O, or .sup.19F NMR), and/or fluorescent or colorimetric detection methods.
(108) ##STR00051##
(109) ##STR00052##
(110) ##STR00053##
(111) ##STR00054##
(112) ##STR00055## ##STR00056## ##STR00057##
(113) ##STR00058##
(114) Also disclosed herein are kits comprising the donor compounds described herein. In some embodiments, the kit comprises a donor compound having a structure satisfying any of the formulas provided herein and a filter, a multi-well plate, a test strip, a slide, a disc, a container, or combination thereof. In some embodiments, the kit can further comprise an enzyme, carbonic anhydrase, water, solubilizing agents, or a combination thereof.
V. Examples
(115) Materials and Methods.
(116) Reagents were purchased from Sigma-Aldrich or Tokyo Chemical Industry (TCI) and used as received. p-Azidobenzylalcohol, O-(4-azidobenzyl)-N-tolylthiocarbamate, methylrhodol, and COS gas were synthesized as previously reported. Spectroscopic grade, inhibitor-free THF was deoxygenated by sparging with argon followed by passage through a Pure Process Technologies solvent purification system to remove water and then stored over 4 molecular sieves in an inert atmosphere glove box. Deuterated solvents were purchased from Cambridge Isotope Laboratories and used as received. Silica gel (SiliaFlash F60, Silicycle, 230-400 mesh) was used for column chromatography. Preparatory chromatography was performed on Silicycle SiliaPlates (1 mm thickness). .sup.1H, .sup.13C{.sup.1H}, and .sup.19F NMR spectra were recorded on a Bruker 600 MHz instrument. Chemical shifts are reported in ppm relative to residual protic solvent resonances. H.sub.2S electrode data were acquired with a World Precision Instruments (WPI) ISO-H.sub.2S-2 sensor connected to a TBR4100 Free Radical Analyzer. Fluorescence spectra were obtained on a Quanta Master 40 spectrofluorometer (Photon Technology International) equipped with a Quantum Northwest TLC-50 temperature controller at 37.00.05 C. UV-visible spectra were acquired on a Cary 100 spectrometer equipped with a Quantum Northwest TLC-42 dual cuvette temperature controller at 37.000.05 C. All air-free manipulations were performed under an inert atmosphere using standard Schlenk techniques or an Innovative Atmospheres N2-filled glove box.
Spectroscopic Materials and Methods.
Phosphate buffered saline (PBS) tablets (1X, CalBioChem) and cetyl trimethylammonium bromide (CTAB) were used to make buffered solutions (PBS, 140 mM NaCl, 3 mM KCl, 10 mM phosphate, 1 mM CTAB, pH 7.4) in Millipore water. Buffer solutions were sparged with N.sub.2 to remove dissolved oxygen and stored in an N.sub.2-filled glovebox. Anhydrous sodium hydrosulfide (NaSH) was purchased from Strem Chemicals and handled under nitrogen. Aqueous stock solutions of NaSH were prepared in buffer in an N.sub.2-filled glovebox immediately prior to use. Stock solutions of MeRho-TCA were prepared in an N.sub.2-filled glovebox in DMSO and stored at 25 C. until immediately before use. Septum-sealed cuvettes from Starna Scientific were used to obtain spectroscopic measurements under anaerobic conditions.
H.sub.2S Electrode Materials and Methods.
Phosphate buffered saline (PBS) tablets (1X, CalBioChem) either with or without cetyl trimethylammonium bromide (CTAB) were used to make buffered solutions (PBS, 140 mM NaCl, 3 mM KCl, 10 mM phosphate, 1 mM CTAB, pH 7.4) in Millipore water. Buffer solutions were sparged with N.sub.2 to remove dissolved oxygen and stored in an N.sub.2-filled glovebox. Carbonic anhydrase (CA) from bovine erythrocytes (3,500 W/A units/mg) was obtained from Sigma Aldrich and a 1% CA stock solution was prepared in deoxygenated buffer (50 mM PIPES, 100 mM KCl, pH 7.4) in a glovebox, and the absorbance at 280 nm (1 cm path length cuvette) at 37 C. was measured to be 3.825. The concentration of the stock solution was calculated to be 67 M using 1%=19 for CA. The stock solution was stored under nitrogen at 4 C. and warmed to room temperature immediately before use. Stock solutions of tris(2-carboxyethyl)phosphine (TCEP) and acetazolamide (AAA) were prepared under N.sub.2 with degassed buffer (PBS, pH 7.4) immediately prior to use. Thiocarbamate and carbamate stock solutions were prepared in an N.sub.2-filled glovebox in DMSO and stored at 25 C. until immediately before use.
General Procedure for H.sub.2S Electrode Examples.
Scintillation vials containing 20.00 mL of phosphate buffer (140 mM NaCl, 3 mM KCl, 10 mM phosphate, 1 mM CTAB, pH 7.4) were prepared in an N.sub.2-filled glovebox. A split-top septum cap was placed on the vial after probe insertion and the headspace was sparged with N.sub.2. The WPI electrode was then inserted into the vial and the measured current was allowed to equilibrate before starting. With moderate stirring, the CA stock solution (50 L, 67 M) was injected, followed by subsequent injections of acetazolamide (10-50 L of a 10 mM stock solution in PBS buffer), COS gas (10 L, 0.345 mol), TCA stock solution (10 mM in DMSO), or TCEP stock solution (10 mM in PBS buffer).
General Procedure for Fluorescence Measurements.
In an N.sub.2-filled glovebox, a septum-sealed cuvette was charged with 3.00 mL of buffer (140 mM NaCl, 3 mM KCl, 10 mM phosphate, 1 mM CTAB, pH 7.4). The cuvette was removed from the glovebox and MeRho-TCA (60 L of a 1 mM stock solution) was injected into the vial, after which a background spectrum was recorded. The desired analytes were then introduced, and the fluorescence spectrum was measured at designated time points.
General Procedure for Measurement of Total Sulfide in Mouse Blood.
Mice were maintained at the association for assessment and accreditation of laboratory animal care international-accredited Louisiana State University Health Science Center-Shreveport animal resource facility and maintained in accordance with the National Research Council's guide for care and use of laboratory animals. All animal studies were approved by the institutional animal care and use committee (protocol P-12-011) and conformed to the guide for the care and use of laboratory animals published by the National Institutes of Health. Mice were anesthetized by IP injection with 150 mg/kg ketamine and 10 mg/kg xylazine. Mouse whole blood was collected from the retroorbital capillary plexus from three C57BL/6J male mice and diluted 1:50 in PBS (phosphate buffered saline pH 7.4) or red blood cell lysing buffer (Sigma, St Louis). Samples were treated with either 3 or 4 (see
H.sub.2S Calibration Curve
To 1.5 mL-UV cuvettes were added 0.5 mL of MB cocktail (vide infra) and 0.5 mL PBS buffer (pH 7.4, 10 mM). The resultant solution was mixed thoroughly, followed by the addition of an NaSH stock solution (1 mM) to make the final H.sub.2S concentrations of 1, 3, 5, 10, 15, and 20 M. The MB solution was allowed to react with H.sub.2S for 1 hour before measuring the absorbance at 670 nm.
H.sub.2S Release from PhotoTCM in PBS
A PhotoTCM stock solution (50 L, 20 mM in DMSO) was added to 20 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a 25-mL Pyrex round bottom flask. One high power LED bulb (800 mW) was placed 1 cm away from the flask. After stirring the reaction solution at room temperature for 2 min, the LED bulb was turned on and 0.5 mL of the reaction aliquots were taken to UV cuvettes containing 0.5 mL of MB cocktail (0.1 mL zinc acetate (1% w/v), 0.2 mL FeCl.sub.3 (30 mM in 1.2 M HCl), and 0.2 mL N,N-dimethyl-p-phenylene diamine (20 mM in 7.2 M HCl)) periodically. The absorbance at 670 nm was then measured after 1 hour and was converted to H.sub.2S concentration by using a H.sub.2S calibration curve.
A PeroxyTCM or TCM stock solution (50 L, 20 mM in DMSO) was added to 20 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a scintillation vial. After stirring at room temperature for 5 min, the H.sub.2O.sub.2 stock solution (1.0 M in H.sub.2O) was added to reach the desired final H.sub.2O.sub.2 concentration, and the H.sub.2S release was monitored using an H.sub.2S electrode.
Effects of CA, UV and Acetazolamide (AAA) on H.sub.2S Release from PhotoTCM-1 and H.sub.2S Release from PhotoCM-1 and TCM-1 (
Bar #1:10 mL of PBS containing CA (25 g/mL) was irradiated by UV light in a 25-mL Pyrex round bottom flask for 10 min and 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #2: The PhotoTCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) in a 25-mL Pyrex round bottom flask and the solution was stirred for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #3: The PhotoTCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a 25-mL Pyrex round bottom flask and the solution was stirred for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #4: The PhotoTCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) in a 25-mL Pyrex round bottom flask and the solution was irradiated by UV light for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #5: The TCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a 25-mL Pyrex round bottom flask and the solution was irradiated by UV light for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #6: The PhotoCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a 25-mL Pyrex round bottom flask and the solution was irradiated by UV light for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #7: The PhotoTCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a 25-mL Pyrex round bottom flask and the solution was irradiated by UV light for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Bar #8: The PhotoTCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) and AAA (10 M) in a 25-mL Pyrex round bottom flask and the solution was irradiated by UV light for 10 min. Then 0.5 mL of the reaction aliquot was taken to a UV cuvette containing 0.5 mL of MB cocktail. The absorbance at 670 nm was then measured after 1 hour by the MB assay.
Effects of Amino Acids on H.sub.2S Release from PhotoTCM-1 (
A PhotoTCM-1 stock solution (25 L, 20 mM in DMSO) was added to 10 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a 25-mL Pyrex round bottom flask. After stirring at room temperature for 5 minutes, the amino acid stock solution was added to reach the final concentration of 500 M (5 mM for GSH). The resultant solution was irradiated under UV light for 10 min and the absorbance at 670 nm was then measured after 1 hour by the MB assay.
Initial Mechanistic Insights into H.sub.2S Release from PhotoTCM-1
PhotoTCM-1 (15 mg) was dissolved in 4 mL of DMSO, followed by the addition of 2 mL of PBS (pH 7.4). The resultant solution was irradiated under UV light at 365 nm for 2 hours. H.sub.2O (15 mL) was then added and the aqueous solution was extracted with ethyl acetate (15 mL3). The organic layers were combined, washed with brine (15 mL3), dried over MgSO4, and removed under vacuum. The solid residue was dissolved in DMSO and injected into the Mass Spectrometer for product analysis.
MTT assay.
The MTT assay was performed as reported. Briefly, BEAS 2B cells were incubated with vehicle (DMSO), 1 (0.3, 1, 3, 10 M) or 2 (0.3, 1, 3, 10 M) for 24 hours, then 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to the cells at a final concentration of 0.5 mg/mL and cells were cultured at 37 C. for 1 hour. The cells were washed with PBS and the formazan dye was dissolved in isopropyl alcohol. The amount of converted formazan dye was measured at 570 nm with background measurement at 690 nm on a Powerwave reader (Biotek).
LDH Assay.
(117) Lactate dehydrogenase (LDH) release was determined as a cytotoxicity assay, a measurement of necrotic cell death, as described previously. Briefly, 30 L of supernatant was saved before addition of MTT and mixed with 100 L of freshly prepared LDH assay reagent to reach final concentrations of 85 mM lactic acid, 1040 mM nicotinamide adenine dinucleotide (NAD+), 224 mM N-methylphenazonium methyl sulfate (PMS), 528 mM 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) and 200 mM Tris (pH 8.2). The changes in absorbance were read kinetically at 492 nm for 15 min (kinetic LDH assay) on a monochromator-based reader (Powerwave HT, Biotek) at 37 C. LDH activity values are shown as V.sub.max for the kinetic assays in mOD/min.
(118) Bioenergetic Analysis in Cultured Cells.
(119) The XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, Mass.) was used to measure bioenergetic function as described previously. Briefly, BEAS 2B cells were incubated with vehicle (DMSO), 1 (1, 3, 10 M) or 2 (1, 3, 10 M) for 24h. Oxygen consumption rate (OCR) after oligomycin (1.5 g/mL) was used to assess ATP production rate and OCR after FCCP (0.5 M) to assess maximal mitochondrial respiratory capacity. Antimycin A (2 g/mL) and rotenone (2 M) were used to inhibit the flux of electrons through complex III and I, to detect residual non-mitochondrial oxygen consumption rate, which is considered to be due to cytosolic oxidase enzymes.
H.sub.2O.sub.2-Mediated COS-to-H.sub.2S Conversion
H.sub.2O.sub.2 was injected to PBS (pH 7.4, 10 mM, 20 mL) containing PeroxyTCM-1 in the presence or absence of CA. H.sub.2S release was monitored by using an H.sub.2S electrode (
Preparation of RSONS Solutions
Hydrogen peroxide (H.sub.2O.sub.2): H.sub.2O.sub.2 was delivered from 30% aqueous solutions. The concentration of H.sub.2O.sub.2 was determined using the absorption at 240 nm (c=43.6 M.sup.1 cm.sup.1).
Superoxide (O.sub.2.sup.): O.sub.2.sup. was prepared by dissolving KO.sub.2 in H.sub.2O.
Hypochlorite (OCl.sup.): OCl.sup. was delivered from 5% aqueous solutions. The concentration of ClO.sup. was determined using the absorption at 292 nm (c=360 M.sup.1 cm.sup.1).
Hydroxy radical (.OH): .OH was generated by reaction of FeSO.sub.4 (5 equiv.) with H.sub.2O.sub.2 (1 equiv.). The .OH concentration was equal to H.sub.2O.sub.2 concentration.
Singlet oxygen (.sup.1O.sub.2):.sup.1O.sub.2 was generated by mixing NaClO (3 equiv.) and H.sub.2O.sub.2 (1 equiv.). The .sup.1O.sub.2 concentration was equal to H.sub.2O.sub.2 concentration.
Peroxynitrite (ONOO.sup.): A mixture of NaNO.sub.2 (0.6 M) and H.sub.2O.sub.2 (0.7 M) was acidified with HCl (0.6 M) and KOH (1.5 M) was added immediately to make the solution alkaline. Manganese dioxide (MnO.sub.2) was added and the resultant mixture was stirring vigorously at r.t. for 20 min to remove excess H.sub.2O.sub.2. The concentration of ONOO.sup. was determined using the absorption at 302 nm (=1670 M.sup.1 cm.sup.1).
tert-Butyl hydroperoxide (TBHP): TBHP was delivered from 70% aqueous solutions.
tert-Butoxy radical (.OtBu): .OtBu was generated by reacting FeSO4 (5 equiv.) with TBHP (1 equiv.). The .OtBu concentration was equal to TBHP concentration.
S-Nitrosoglutathione (GSNO): Glutathione (0.2 M in H.sub.2O) was mixed with HCl (0.2 M), followed by the addition of NaNO.sub.2 (0.2 M in H.sub.2O). The reaction solution was stirred in dark for 30 min. The concentration of GSNO was determined using the absorption at 335 nm (=922 M.sup.1 cm.sup.1).
Nitric oxide (NO): NO was delivered by using DEA-Nonoate as a NO donor.
Nitroxyl (HNO): HNO was delivered by using Angeli's salt as an HNO donor.
RSONS Effects on H.sub.2S Release from PeroxyTCM-1
A PeroxyTCM-1 stock solution (50 L, 20 mM in DMSO) was added to 20 mL of PBS (pH 7.4, 10 mM) containing CA (25 g/mL) in a scintillation vial. After stirring at room temperature for 5 minutes, the RSONS stock solution was added to reach the final concentration to 500 M (5 mM for GSH). The H.sub.2S release was monitored using an H.sub.2S electrode for 20 min. The experiments were repeated in triplicate, and the results are reported as meanSD (n=3).
Cell Culture and Cytotoxicity of PeroxyTCM-1, PeroxyCM-1, and TCM-1
HeLa cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C. under 5% CO.sub.2. Confluent HeLa cells were incubated in FBS-free DMEM containing vehicle (0.5% DMSO), PeroxyTCM-1, PeroxyCM-1, or TCM-1 (10-100 M) for 2 hours in a 96-well plate. After the indicated treatment, 100 L of FBS-free DMEM containing 10% CCK-8 solution was added to each well, and cells were incubated for 3 hours at 37 C. The absorbance at 450 nm was measured by using a microplate reader. The cell viability was measured and normalized to the vehicle group. The results are expressed as meanSEM (n=6).
Cell Imaging of Exogenous H.sub.2O.sub.2-Induced H.sub.2S Release from PeroxyTCM-1
HeLa cells were plated in poly-D-lysine coated plates (MatTek) containing 2 mL of DMEM and incubated at 37 C. under 5% CO.sub.2 for 24 h. The confluent cells were washed with PBS and then co-incubated with PeroxyTCM-1 (50 M), HSN.sub.2 (5 M) and NucBlue nuclear dye (2 drops) in FBS-free DMEM for 30 min. After removal of extracellular PeroxyTCM-1 and HSN.sub.2 by washing with PBS, cells were incubated in FBS-free DMEM in the presence or absence of H.sub.2O.sub.2 (25 or 50 M) for 30 min. Prior to imaging, cells were washed with PBS and bathed in 2 mL of PBS. Cell imaging was performed on a Leica DMi8 fluorescent microscope.
Cell Imaging of PMA-Induced Endogenous ROS Generation
RAW 264.7 cells were plated in poly-D-lysine coated plates (MatTek) containing 2 mL of DMEM and incubated at 37 C. under 5% CO.sub.2 for 24 h. The confluent cells were washed with PBS and then co-incubated with DCFDA (10 M) and NucBlue nuclear dye (2 drops) in FBS-free DMEM for 30 min. After removal of extracellular DCFDA by washing with PBS, cells were incubated in FBS-free DMEM in the presence or absence of PMA (500 nM) for 3 h. Prior to imaging, cells were washed with PBS and bathed in 2 mL of PBS. Cell imaging was performed on a Leica DMi8 fluorescent microscope.
Cell Imaging of Endogenous ROS-Induced H.sub.2S Release from PeroxyTCM-1
RAW 264.7 cells were plated in poly-D-lysine coated plates (MatTek) containing 2 mL of DMEM and incubated at 37 C. under 5% CO.sub.2 for 24 h. The confluent cells were washed with PBS and then co-incubated with PeroxyTCM-1 (50 M), HSN2 (5 M), and NucBlue nuclear dye (2 drops) in FBS-free DMEM for 30 min. After removal of extracellular PeroxyTCM-1 and HSN.sub.2 by washing with PBS, cells were incubated in FBS-free DMEM in the presence or absence of PMA (500 nM) for 3 h. Prior to imaging, cells were washed with PBS and bathed in 2 mL of PBS. Cell imaging was performed on a Leica DMi8 fluorescent microscope.
Cytotoxicity of H.sub.2O.sub.2 in HeLa Cells
Confluent HeLa cells were incubated in FBS-free DMEM containing H.sub.2O.sub.2 (50-400 M) for 1 h. Cells were then washed with PBS. Cell viability was evaluated by using CCK-8 assay, and the results are expressed as meanSEM (n=5).
Cytoprotection of PeroxyTCM-1, PeroxyCM-1, and TCM-1 against H.sub.2O.sub.2-Induced Oxidative Stress
Confluent HeLa cells were incubated in FBS-free DMEM containing H.sub.2O.sub.2 (100 M) in the absence or presence of PeroxyTCM-1, PeroxyCM-1, or TCM-1 (10-50 M) for 1 hour in a 96-well plate. Cells were then washed with PBS, and 100 L of FBS-free DMEM containing 10% CCK-8 solution was added to each well. Cells were incubated for 3 hours at 37 C. The absorbance at 450 nm was measured by using a microplate reader. The cell viability was evaluated, and the results are expressed as meanSEM (n=5).
Representative Syntheses
General Procedure for the Preparation of Thiocarbamates.
(120) Sodium hydride (60% in oil, 1.25 mmol) was added to a solution of the isothiocyanate (1 mmol) and the benzyl alcohol (1 mmol) in anhydrous THF (6-12 mL). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. After the solvent was removed under reduced pressure, CH.sub.2Cl2 was added, and the resulting solution was washed with water and brine. The organic layer was dried over sodium sulfate and filtered. The solvent was removed by rotary evaporation and the crude product was purified using either column chromatography (hexanes:EtOAc gradient) or silica gel preparatory thin layer chromatography (3:2 hexanes:EtOAc).
(121) ##STR00059##
O-(4-Azidobenzyl)-N-(4-fluorophenyl)thiocarbamate (1)
(122) Purification via preparatory TLC (3:2 hexanes/EtOAc) yielded the product as a pure white solid (107.9 mg, 35% yield). .sup.1H NMR (600 MHz, DMSO-d6) (ppm): 7.23-7.50 (m, 4H), 7.06-7.13 (m, 4H), 7.44-7.48 (br s, 2H). Two rotomers were observable by .sup.13C{.sup.1H} and .sup.19F NMR: .sup.13C{.sup.1H} NMR (150 MHz, DMSO) (ppm): 189.4 (187.9 minor), 161.3 (159.7 minor), 140.6 (d, J=15.4 Hz), 135.4 (134.3 minor), 133.7 (133.0 minor), 126.8 (125.5 minor), 120.1 (130.9 minor), 116.5 (m), 71.0 (72.7 minor). .sup.19F NMR (470 MHz, DMSO-d6) (ppm): 115.9 (116.4 minor). FTIR (ATR, cm-1): 3199, 3039, 2109, 1607, 1543, 1503, 1404, 1339, 1280, 1220, 1168, 1014, 857, 787, 658.
(123) ##STR00060##
O-(4-Azidobenzyl)-N-(4-fluorophenyl)carbamate (2)
(124) p-Azidobenzyl alcohol (57.5 mg, 0.386 mmol) and NEt3 (54 L, 0.39 mmol) were combined in dry THF (2 mL), and 4-fluorophenylisocyanate (44 L, 0.39 mmol) was added dropwise. The reaction mixture was stirred under nitrogen at room temperature and shielded from light for 24 hours. The solvent was evaporated under reduced pressure, and the crude product was purified by preparatory TLC (2:1 hexanes/EtOAc) to yield the pure product as a white solid (61.8 mg, 56%). .sup.1H NMR (600 MHz, DMSO-d6) (ppm): 9.79 (s, 1H), 7.48-7.44 (m, 4H), 7.10-7.15 (m, 4H), 5.12 (s, 2H). .sup.13C{.sup.1H} NMR (150 MHz, DMSO-d6) (ppm): 158.4, 156.9, 153.4, 139.2, 135.4, 133.5, 130.0, 119.2, 115.4 (d, J=22.2 Hz), 65.2. .sup.19F NMR (470 MHz, DMSO-d6) (ppm): 120.82. HRMS (m/z): [M+Na]+ calcd for [C.sub.14H.sub.11FN.sub.4O.sub.2Na].sup.+ 309.0764, found 309.0601.
(125) ##STR00061##
O-(4-Benzyl)-N-tolylthiocarbamate (3)
(126) Purification via column chromatography (hexanes/EtOAc gradient) yielded the product as a pure white solid (173.7 mg, 68%). .sup.1H NMR (600 MHz, DMSO-d6) (ppm): 11.11 (s, 1H), 7.57-7.44 (m, 2H), 7.43-7.32 (m, 4H), 7.23-7.06 (m, 3H), 5.49-5.58 (br s, 2H), 2.26 (s, 3H). .sup.13C{.sup.1H} NMR (150 MHz, DMSO-d6) (ppm): 187.4, 186.8, 136.0, 135.6, 135.1, 134.4, 134.0, 129.2, 128.8, 128.4, 128.3, 128.2, 128, 122.9, 121.2, 72.0, 70.3, 20.4. HRMS (m/z): [M+H]+ calcd for [C.sub.15H.sub.16NOS].sup.+ 258.0953, found 258.0948.
Methylrhodol-isothiocyanate (MeRho-NCS)
(127) Methylrhodol (200 mg, 0.577 mmol) and NEt3 (480 L, 3.46 mmol) were combined in dry DMF (2 mL) in oven-dried glassware. Thiocarbonyldiimidazole (TCDl, 211 mg, 1.16 mmol) was dissolved in dry DMF (6 mL) and added dropwise to the solution. The reaction mixture was stirred under nitrogen at room temperature for 24 hours then quenched with water. The organic layer was extracted with EtOAc, washed three times with aqueous LiCl (5%, 20 mL), dried over MgSO4, and the solvent was evaporated under reduced pressure. The crude product was purified using preparatory TLC on an oven-dried prep plate using dry hexanes:EtOAc (1:1) and isolated as a white solid (134 mg, 60%). .sup.1H NMR (600 MHz, CDCl.sub.3) (ppm): 8.03 (d, J=7.5 Hz, 1H), 7.68 (t, J=7.4 Hz, 1H), 7.63 (t, J=7.5 Hz, 1H), 7.15-7.12 (m, 2H), 6.89 (dd, J1=8.5, J2=2.1 Hz, 1H), 6.80-6.76 (m, 2H), 6.70 (d, J=8.8 Hz, 1H), 6.64 (dd, J1=8.9, J2=2.5 Hz, 1H), 3.84 (s, 3H). .sup.13C{.sup.1H} NMR (150 MHz, CDCl.sub.3) (ppm): 169.3, 161.7, 153.0, 152.1, 151.9, 137.8, 135.4, 133.4, 130.2, 129.5, 129.2, 126.5, 125.4, 123.9, 121.4, 118.5, 114.2, 112.4, 110.8, 101.1, 82.2, 55.8. FTIR (ATR, cm-1): 2922, 2852, 2017, 1761, 1607, 1563, 1495, 1416, 1324, 1247, 1099, 1079, 941. HRMS (m/z): [M+H]+ calcd for [C.sub.22H.sub.13NO.sub.4SH].sup.+ 388.0644, found 388.0471.
(128) ##STR00062##
O-(4-Azidobenzyl)-N-methylrhodolthiocarbamate (MeRho-TCA)
(129) In oven-dried glassware, MeRho-NCS (134 mg, 0.346 mmol) was dissolved in dry THF (5 mL). p-Azidobenzylalcohol (150 mg, 1.00 mmol) was dissolved in dry THF (5 mL) and added to the solution of MeRho-NCS. Sodium hydride (60% in mineral oil, 18 mg, 0.45 mmol) was added and the reaction mixture was stirred at room temperature under nitrogen and protected from light for 24 hours. The solvent was evaporated under reduced pressure and the crude product was dissolved in EtOAc. The organic layer was washed with water and brine and dried over sodium sulfate. The product was purified by preparatory TLC (5% MeOH in DCM) while protected from light, to yield the pure product as a pale yellow solid (65 mg, 35%). .sup.1H NMR (600 MHz, CDCl.sub.3) (ppm): 8.26 (br s, 1H), 8.02 (d, J=7.6 Hz, 1H), 7.67 (td, J1=7.5, J2=1.3 Hz, 1H), 7.62 (td, J1=7.5, J2=1.0 Hz, 1H), 7.43 (d, J=8.4 Hz, 2H), 7.14 (d, J=7.5 Hz, 1H), 7.05 (d, J=8.4 Hz, 2H), 6.78 (d, J=2.5 Hz, 1H), 6.73 (d, J=8.2 Hz, 1H), 6.69 (d, J=8.8 Hz, 1H), 6.62 (dd, J1=8.8, J2=2.5 Hz, 1H), 5.59 (br s, 2H), 3.84 (s, 3H). .sup.13C{.sup.1H} NMR (150 MHz, CDCl.sub.3) (ppm): 206.9, 169.3, 161.5, 152.9, 152.3, 151.7, 140.6, 135.1, 130.3, 129.9, 129.0, 128.8, 126.6, 125.1, 123.9, 119.3, 116.9, 115.9, 112.0, 110.9, 109.2, 100.9, 82.5, 55.6, 53.5, 30.9, 29.7, 29.3. HRMS (m/z): [M+H]+ calcd for [C.sub.29H.sub.20N.sub.4O.sub.5SH].sup.+ 537.1233, found 537.0827.
(130) General Procedure for the Synthesis of Photolabile Compounds.
(131) The o-nitrobenzyl alcohol species (1.0 equiv.) was combined with p-fluorophenyl isothiocyanate (1.0 equiv.) in anhydrous THF (15 mL) at 0 C., followed by the addition of NaH (60% in paraffin liquid, 1.25 equiv.). The resultant mixture was stirred at 0 C. for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at room temperature until the completion of the reaction indicated by TLC. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (315 mL). The organic layers were combined, dried over MgSO4, and evaporated under vacuum. The crude product was purified by column chromatography. NMR spectra were obtained at 60 C. because thiocarbamates show two sets of NMR resonances at room temperature due to slow rotation around the thiocarbamate functional group at room temperature.
(132) ##STR00063##
PhotoTCM-1 was prepared from o-nitrobenzyl alcohol and p-fluorophenyl isothiocyanate using the general synthetic procedure described above (520 mg, 85% yield). .sup.1H NMR (500 MHz, DMSO-d.sub.6 at 60 C.) (ppm): 11.20 (s, 1H), 8.13 (d, J=5.0 Hz, 1H), 7.64 (m, 5H), 7.18 (t, J=10.0 Hz, 2H), 5.89 (s, 2H). .sup.19F NMR (470 MHz, DMSO-ds at 60 C.) (ppm): 116.9. .sup.13C{.sup.1H} NMR (150 MHz, DMSO-d.sub.6 at 60 C.) (ppm): 187.8, 160.7, 159.1, 148.0, 134.5, 132.0, 130.0, 129.8, 125.4, 125.2, 115.8, 67.5.1R (cm.sup.1): 3170, 3007, 1521, 1501, 1332, 1207, 1185, 1154, 1057, 1046, 791, 727, 561. HRMS m/z [M+H].sup.+ calcd. for [C.sub.14H.sub.12FN.sub.2O.sub.3S]+307.0553; found 307.0558. M. P. ( C.): 120-122.
(133) ##STR00064##
PhotoTCM-2 was prepared from 4,5-dimethoxy-2-nitrobenzyl alcohol and p-fluorophenyl isothiocyanate using the general synthetic procedure described above (626 mg, 86% yield). .sup.1H NMR (500 MHz, DMSO-d.sub.6 at 60 C.) (ppm): 11.6 (s, 1H), 7.72 (s, 1H), 7.56 (m, 2H), 7.18 (t, J=10.0 Hz, 3H), 5.83 (s, 2H), 3.90 (s, 6H). .sup.19F NMR (470 MHz, DMSO-d.sub.6 at 60 C.) (ppm): 116.9. .sup.13C{.sup.1H} NMR (150 MHz, DMSO-ds at 60 C.) (ppm): 188.0, 160.7, 159.1, 153.8, 148.7, 140.4, 134.9, 125.6, 115.9, 112.3, 109.0, 69.4, 68.0, 56.7. IR (cm.sup.1): 3170, 3007, 1521, 1502, 1406, 1333, 1235, 1208, 1185, 1154, 1056, 1046, 791, 778, 726, 703, 681. HRMS m/z [M+H].sup.+ calcd. for [C.sub.16H.sub.16FN.sub.2O.sub.5S]+367.0764; found 367.0755. M. P. ( C.): 156-158.
(134) ##STR00065##
PhotoCM-1
(135) o-Nitrobenzyl alcohol (153 mg, 1.0 mmol, 1.0 equiv.) was combined with p-fluorophenyl isocyanate (137 mg, 1.0 mmol, 1.0 equiv.) in anhydrous toluene (30 mL) at room temperature, followed by the addition of triethylamine (101 mg, 1.0 mmol, 1.0 equiv.). The resultant mixture was stirred at 110 C. for 20 h, after which the oil bath was removed, and the reaction solution was cooled to room temperature. The reaction mixture was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (315 mL). The organic layers were combined, dried over MgSO.sub.4, and evaporated under vacuum. The crude product was purified by using column chromatography to afford pure PhotoCM-1 as white solid (220 mg, 76%). .sup.1H NMR (500 MHz, CDCl.sub.3) (ppm): 9.95 (s, 1H), 8.14 (d, J=10.0 Hz, 1H), 7.83 (t, J=5.0 Hz, 1H), 7.75 (t, J=10.0 Hz, 1H), 7.64 (t, J=5.0 Hz, 1H), 7.49 (m, 2H), 7.14 (t, J=10.0 Hz, 2H), 5.50 (s, 2H). .sup.19F NMR (470 MHz, CDCl.sub.3) (ppm): 120.6. .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 159.2, 157.3, 153.5, 147.8, 135.7, 134.6, 132.7, 129.8, 125.3, 120.4, 115.9, 63.0. IR (cm.sup.1): 3170, 3007, 1521, 1502, 1406, 1333, 1235, 1208, 1185, 1154, 1056, 846, 791, 778, 726, 703, 681, 561. HRMS m/z [M+H].sup.+ calcd. for [C.sub.14H.sub.12FN.sub.2O.sub.4].sup.+ 291.0781; found 291.0786. M. P. ( C.): 146-148.
(136) ##STR00066##
O-(4-pivaloylbenzyl)-N-(p-tolyl)thiocarbamate (5)
(137) 4-Pivaloylbenzyl alcohol (131 mg, 0.668 mmol) and p-tolylisothiocyanate (107 mg, 0.717 mmol) were combined in anhydrous THF (3 mL) and DBU (125 L, 0.835 mmol) was added dropwise. The reaction mixture was stirred under nitrogen at room temperature for 4 hours. The solvent was removed under reduced pressure and the crude mixture was purified using column chromatography (hexanes:EtOAc gradient) to yield the pure product as a white solid (123 mg, 52%). Note: The resonances of thiocarbamates are doubled as a result of slow rotation and the peaks associated with these rotamers are denoted with major and minor when delineation is possible. .sup.1H NMR (600 MHz, DMSO-d.sub.6) (ppm): 11.13 (s, 1H), 7.54-7.42 (m, 3H), 7.21-7.07 (m, 5H), 5.57 (minor); 5.48 (major) (s, 2H), 2.28 (major); 2.23 (minor) (s, 3H), 1.30 (s, 9H). .sup.13C{.sup.1H} NMR (150 MHz, DMSO-d.sub.6) (ppm): 187.8, 187.2, 176.9, 151.1, 136.5, 135.5, 134.9, 134.6, 134.0, 133.6, 130.2, 129.9, 129.7, 129.4, 123.4, 122.5, 122.3, 71.9, 70.3, 39.0, 27.2, 20.9. FTIR (ATR, cm.sup.1): 3207, 2972, 1750, 1704, 1541, 1423, 1107, 1043, 810. HRMS (m/z): [M+N].sup.+ calcd for [C.sub.20H.sub.23NO.sub.3SNa].sup.+ 380.1296, found 380.1299.
(138) ##STR00067##
O-(4-pivaloylbenzyl)-N-(p-tolyl)carbamate (6)
(139) 4-Pivaloyl benzyl alcohol (100 mg, 0.480 mmol) was dissolved in anhydrous THF (4 mL) and DBU (89.6 L, 0.600 mmol) was added. p-Tolylisocyanate (64.3 mg, 0.480 mmol) was added dropwise in anhydrous THF (1 mL). The reaction mixture was stirred under nitrogen at room temperature for 18 hours. The solvent was removed under reduced pressure and the crude mixture was purified using column chromatography (hexanes:EtOAc gradient) to yield the pure product as a clear oil (119 mg, 73%). .sup.1H NMR (600 MHz, DMSO-d.sub.6) (ppm): 9.65 (s, 1H), 7.47 (d, J=8.2 Hz, 2H), 7.32 (d, J=8.8 Hz, 2H), 7.07 (d, J=8.1 Hz, 2H), 6.85 (d, J=8.7 Hz, 2H), 5.05 (s, 2H), 2.23 (s, 3H), 1.30 (s, 9H). .sup.13C{.sup.1H} NMR (150 MHz, DMSO-d.sub.6) (ppm): 176.9, 153.8, 150.9, 137.1, 134.8, 131.9, 129.9, 129.6, 122.2, 118.6, 65.5, 39.0, 27.3, 20.8. FTIR (ATR, cm.sup.1): 3364, 2974, 1725, 1595, 1524, 1508, 1192, 1111, 1015, 812, 785, 766. HRMS (m/z): [M+Na].sup.+ calcd for [C.sub.20H.sub.23NO.sub.4Na].sup.+ 364.1525, found 364.1525.
General Procedure for the Synthesis of Boronate Ester Compounds.
The benzyl alcohol species (1.0 equiv.) was combined with substituted phenyl isothiocyanate (1.0 equiv.) in anhydrous THF (15 mL) at 0 C., followed by the addition of NaH (60% in paraffin liquid, 1.25 equiv.). The resultant mixture was stirred at 0 C. for 20 min, after which the ice bath was removed, and the reaction mixture was stirred at r.t. until the completion of the reaction indicated by TLC. The reaction was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (315 mL). The organic layers were combined, dried over MgSO.sub.4, and evaporated under vacuum. The crude product was purified by column chromatography. Certain compounds show two sets of NMR resonances at room temperature due to slow rotation around thiocarbamate functional groups.
(140) ##STR00068##
PeroxyTCM-1 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester and 4-fluorophenyl isothiocyanate using the general synthetic procedure described above (245 mg, 32% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) (ppm): 8.59 (s, 1H), 7.84 (s, 2H), 7.55 (s, 1H), 7.38 (br, 2H), 7.20 (br, 1H), 7.02 (br, 2H), 5.65 (br, 2H), 1.38 (s, 12H). .sup.19F NMR (470 MHz, CDCl.sub.3) (ppm): 115.3, 115.9. .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 188.5, 137.8, 135.1, 132.8, 127.3, 125.9, 124.0, 116.0, 115.8, 84.0, 74.0, 24.9. IR (cm.sup.1): 3196, 2978, 1736, 1612, 1541, 1507, 1451, 1391, 1357, 1210, 1194, 1140, 1087, 1059, 1020, 964, 859, 817, 724, 652. HRMS m/z [M+Na].sup.+ calcd. for [C.sub.20H.sub.23BFNNaO.sub.3S].sup.+ 410.1377; found 410.1367.
(141) ##STR00069##
PeroxyTCM-2 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester and 4-tolyl isothiocyanate using the general synthetic procedure described above (251 mg, 34% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) (ppm): 8.53 (s, 1H), 7.84 (s, 2H), 7.41 (s, 3H), 7.12 (m, 3H), 5.66 (s, 2H), 2.34, (s, 3H), 1.38 (s, 12H). .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 188.4, 135.0, 129.6, 127.3, 127.2, 121.8, 83.9, 73.9, 24.9, 20.9. IR (cm.sup.1): 3279, 2973, 2924, 1613, 1596, 1530, 1515, 1442, 1339, 1318, 1274, 1212, 1168, 1141, 1085, 1042, 1021, 962, 856, 818, 787, 728, 652. HRMS m/z [M+N].sup.+ calcd. for [C.sub.21H.sub.26BNNaO.sub.3S].sup.+ 406.1628; found 406.1632.
(142) ##STR00070##
PeroxyTCM-3 was prepared from 4-(hydroxymethyl)phenylboronic acid pinacol ester and phenyl isothiocyanate using the general synthetic procedure described above (157 mg, 21% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) (ppm): 8.56 (s, 1H), 7.85 (s, 2H), 7.30 (m, 7H), 5.67 (s, 2H), 1.38 (s, 12H). .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 188.4, 138.0, 136.8, 135.1, 129.1, 127.3, 125.6, 123.3, 121.7, 83.9, 74.0, 24.9.1R (cm.sup.1): 3177, 2981, 1616, 1593, 1535, 1491, 1449, 1401, 1367, 1332, 1223, 1176, 1144, 1094, 1027, 1018, 860, 842, 799, 747, 727. HRMS m/z [M+Na].sup.+ calcd. for [C.sub.20H.sub.24BNNaO.sub.3S]+392.1472; found 392.1477.
(143) ##STR00071##
TCM-1 was prepared from benzyl alcohol and 4-fluorophenyl isothiocyanate using the general synthetic procedure described above (205 mg, 79% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) (ppm): 8.63 (s, 1H), 7.94 (s, 1H), 7.40 (br, 5H), 7.22 (s, 1H), 7.03 (br, 2H), 5.62 (br, 2H). .sup.19F NMR (470 MHz, CDCl.sub.3) (ppm): 115.2, 116.0. .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 188.5, 134.9, 132.9, 128.7, 128.6, 128.2, 125.8, 123.9, 115.9, 74.1. IR (cm-1): 3204, 3039, 1610, 1550, 1497, 1450, 1401, 1344, 1301, 1289, 1233, 1208, 1192, 1049, 840, 815, 730, 693. HRMS m/z [M+Na].sup.+ calcd. for [C.sub.14H.sub.12FNNaOS].sup.+ 284.1521: found 284.0529.
(144) ##STR00072##
TCM-2 was prepared from benzyl alcohol and 4-tolyl isothiocyanate using the general synthetic procedure mentioned above (365 mg, 71% yield). .sup.1H NMR (500 MHz, CDCl.sub.3) (ppm): 8.43 (s, 1H), 7.94 (s, 1H), 7.41 (br, 5H), 7.13 (br, 3H), 5.62 (br, 2H), 2.34 (s, 3H). .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 188.4, 135.3, 134.4, 129.6, 128.6, 128.5, 128.2, 123.8, 121.7, 74.0, 21.0. IR (cm-1): 3197, 3028, 1593, 1545, 1498, 1451, 1401, 1346, 1309, 1288, 1207, 1186, 1081, 1055, 804, 692, 646. HRMS m/z [M+Na].sup.+ calcd. for [C.sub.15H.sub.15NNaOS].sup.+ 280.0772; found 280.0771.
(145) ##STR00073##
PeroxyCM-1
(146) 4-(Hydroxymethyl)phenylboronic acid pinacol ester (655 mg, 2.80 mmol, 1.4 equiv.) was combined with 4-fluorophenyl isocyanate (274 mg, 2.00 mmol, 1.0 equiv.) in anhydrous toluene (30 mL) at room temperature, followed by the addition of triethylamine (202 mg, 2.00 mmol, 1.0 equiv.). The resultant mixture was stirred at 110 C. for 16 h, after which the oil bath was removed, and the reaction solution was cooled to room temperature. The reaction mixture was quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (315 mL). The organic layers were combined, dried over MgSO.sub.4, and evaporated under vacuum. The crude product was purified by using column chromatography to afford pure PeroxyCM-1 as white solid (571 mg, 77%). .sup.1H NMR (300 MHz, CDCl.sub.3) (ppm): 7.84 (d, J=7.8 Hz, 2H), 7.35 (m, 4H), 7.01 (t, J=8.4 Hz, 2H), 6.79 (s, 1H), 5.22 (s, 2H), 1.37 (s, 12H). .sup.19F NMR (470 MHz, CDCl.sub.3) (ppm): 119.4. .sup.13C{.sup.1H} NMR (125 MHz, CDCl.sub.3) (ppm): 160.0, 158.1, 153.5, 139.0, 135.1, 133.7, 127.3, 120.5, 115.8, 83.9, 67.0, 24.9. IR (cm.sup.1): 3326, 2971, 1734, 1614, 1538, 1511, 1407, 1373, 1308, 1278, 1204, 1139, 1084, 1037, 855, 833, 784, 740, 656. HRMS m/z [M+Na].sup.+ calcd. for [C.sub.20H.sub.23BFNNaO.sub.4].sup.+ 394.1606; found 394.1602.
Example 1
(147) In some embodiments, model thiocarbamates were made to confirm that the proposed decomposition cascade to release COS occurs efficiently and to demonstrate the biological compatibility of this new H.sub.2S donor motif. In some embodiments, an azide in the para position of the benzylthiocarbamate was used to function as the H.sub.2S-responsive trigger for self-immolation and COS release. To facilitate NMR identification of the products, azidobenzylthiocarbamate (donor compound 1 of
(148) To confirm that the COS released from self-immolative thiocarbamates can serve as a potential source of H.sub.2S donation, it was established that independently prepared COS could be efficiently hydrolyzed to H.sub.2S by CA. Upon addition of COS gas to deoxygenated aqueous buffer (PBS, 1 mM CTAB, pH 7.4) containing CA from bovine erythrocytes, rapid H.sub.25 production was observed using an H.sub.2S-responsive electrode. In the absence of CA, negligible current was observed from COS alone, which is consistent with slow and pH-dependent, nonenzymatic hydrolysis in water (
Example 2
(149) In some embodiments, H.sub.2S release from model thiocarbamates in whole mouse blood was evaluated to expand on the efficacy of H.sub.2S release from thiocarbamates in more biologically-relevant contexts. Although murine systems provide a convenient model, mice have among the lowest CA levels in mammals, with murine blood only containing about 15% of the CA present in human blood, and thus represent a challenging target for sulfide release mediated by CA. To quantify total sulfide levels, the monobromobimane (mBB) method was used, which allows for the analytical measurement of different sulfide pools and is compatible with many types of biological samples. Measurement of the total sulfide, which includes free sulfide as well as bound sulfane-sulfur, revealed background levels of 8 170 M, which are higher than total sulfide levels commonly observed and reported in plasma, but are consistent with the high sulfane-sulfur content in red blood cells. Thiocarbamate 3 (
Example 3
(150) In additional embodiments, incorporating a fluorophore into the scaffold can be conducted to access an H.sub.2S-responsive fluorescent probe that releases H.sub.2S upon H.sub.2S detection. In some embodiments, methylrhodol (MeRho) was used as the fluorophore due to its high quantum yield and single fluorogenic amine, which could be readily converted into the desired thiocarbamate. Since the azide-functionalized scaffold would be triggered by H.sub.2S to release both MeRho and COS, this would function as a fluorescent H.sub.2S probe that would replenish sulfide through the release of COS. To access the desired scaffold, MeRho was treated with thiocarbonyldiimidazole (TODI) and NEt.sub.3 in DMF to afford methylrhodol isothiocyanate (MeRho-NCS) in 60% yield. Subsequent treatment with 4-azidobenzyl alcohol and NaH afforded the methylrhodol thiocarbamate azide (MeRho-TCA) in 35% yield. One benefit of this simple synthetic route is that almost any fluorophore containing a fluorogenic nitrogen can readily be functionalized with the benzylazide thiocarbamate group, thus providing access to a diverse library of fluorophores. With a sulfide-replenishing H.sub.2S probe in hand, the fluorescence response upon addition of sulfide was evaluated. Treatment of MeRho-TCA with 50 equivalents of NaSH in aqueous buffer (PBS, 1 mM CTAB, pH 7.4) resulted in a 65-fold fluorescence turn-on over 90 minutes (
Example 4
(151) In this example, the H.sub.2S releasing profiles of light-triggerable compounds were explored. A methylene blue (MB) assay was used to monitor H.sub.2S release from representative photo-triggerable compounds (e.g., PhotoTCM-1 (50 M) and PhotoTCM-2 (50 M)) upon irradiation with an LED UV light (365 nm) in PBS buffer (pH 7.4, 10 mM) containing CA (25 g/mL). A time-dependent H.sub.2S release was observed from both PhotoTCM-1 and PhotoTCM-2, with a peaking time for H.sub.2S release of 10-minutes (
(152) To confirm that the observed COS/H.sub.2S release was due to photocleavage of the ONB protecting group (ortho-nitrobenzyl), a series of control embodiments were performed. As shown in
(153) The cleavage mechanism of the photolabile groups is compatible with a thiocarbamate linking group and other WC(Y)V groups illustrated in the formulas described herein, resulting in generation of intermediates that decompose to produce COS and the by-products disclosed herein (e.g., 4-fluoroaniline). Consistent with this reaction mechanism, mass spectrometric analysis of the reaction mixture after UV irradiation clearly shows formation of 4-fluoroaniline (m/z: 106.1) and 2-nitrosobenzylaldehyde (m/z: 136.0). Additionally, this COS/H.sub.2S release does not proceed through an electrophilic quinone methide intermediate often required in self-immolative cascade reactions, thus increasing the biological compatibility of this bioorthogonal delivery approach.
(154) In summary, this example demonstrates that photolysis of photolabile groups is compatible with the WC(Y)V groups of the formulas described herein and thus provides a broad platform for reactivity. The time-dependent H.sub.2S release after UV-activation suggests that the donor compounds disclosed herein can be used as new efficacious photo-labile COS/H.sub.2S donors.
Example 5
(155) In this example, porcine liver esterase (PLE) is used to initiate a self-immolative decomposition reaction. After stirring the donor (14 mM) with PLE (28 U/mL) in PBS (pH 7.4) with 10% DMSO for 48 hours, the organic layer was extracted and analyzed by NMR spectroscopy and mass spectrometry (
(156) ##STR00074##
Example 6
(157) With esterase-triggered donors in hand, it was first confirmed that 5 was stable in aqueous solution in the absence of esterase. Next, it was verified that addition of 5 to pH 7.4 PBS buffer containing physiologically relevant levels of CA (25 g/mL) did not result in H.sub.2S generation when monitored using an H.sub.2S-selective electrode, confirming that the esterase does not cleave thiocarbamates directly (
Example 7
(158) Next, the cytotoxicity of 5-7 on BEAS 2B cells was evaluated by measuring the reduction of a tetrazolium compound (MTT) to formazan by metabolically active cells, and by measuring the release of lactate dehydrogenase (LDH) due to permeability of the plasma membrane, which is a sign of necrotic cell death. BEAS 2B human lung epithelial cells exhibit low expression of all three canonical H.sub.2S-producing enzymes, CBS, CSE, and 3-MST. To provide suitable comparisons with commonly-used synthetic donors, comparable cytotoxicity data for known H.sub.2S donors GYY4137, which does not localize in the mitochondria, was first obtained and also with mitochondrially-targeted AP39 (
(159) To investigate the underpinnings of the increased cytotoxicity of 5, and because H.sub.2S is a well-known inhibitor of mitochondrial cytochrome c oxidase, the mitochondrial respiration of BEAS 2B cells treated with 5-7 was evaluated using an Extracellular Flux Analyzer. This assay was selected because H.sub.2S is well known to impact mitochondrial respiration, even if the H.sub.2S is not delivered directly to the mitochondria, and because COS has been suggested to inhibit the mitochondrial respiratory chain. Incubation of BEAS 2B cells with increasing concentrations of 5 for 24 hours negatively affected all major bioenergetics parameters in this example, namely oxygen consumption linked with basal respiration, maximal respiration, and ATP synthesis (
(160) These examples illustrate that the easy-to-access, esterase-triggered COS/H.sub.2S donor compounds disclosed herein can be rapidly activated by esterases to generate H.sub.2S in vitro using isolated PLE.
Example 8
(161) In this example, donor compounds comprising boronate groups were evaluated to determine their ability to function as ROS-triggered H.sub.2S donors. Three thiocarbamate donors (peroxythiocarbamate: PeroxyTCM-1, PeroxyTCM-2, and PeroxyTCM-3) and two carbamate control compounds (thiocarbamates: TCM-1 and TCM-2) were prepared. The PeroxyTCM compounds are stable in aqueous buffer (pH 5-9) and are not hydrolyzed by esterases. The parent carbamate (peroxycarbamate-1, PeroxyCM-1) also was used and can also be activated by ROS, but releases CO.sub.2/H.sub.2O instead of COS/H.sub.2S. Access to these simple control compounds provides useful tools to determine whether observed biological activities of the donors are H.sub.2S-related or merely a product of the organic scaffold and/or byproducts.
(162) To evaluate the H.sub.2S release from the donor constructs in the presence of ROS, an H.sub.2S-selective electrode was used to monitor H.sub.2S release from PeroxyTCM-1 (50 M) upon treatment with H.sub.2O.sub.2 (50-1000 M) in PBS buffer (pH 7.4, 10 mM) containing CA (25 g mL.sup.1). H.sub.2O.sub.2-dependent H.sub.2S release from PeroxyTCM-1 was observed with corresponding second-order rate constant of 1.44 M.sup.1 s.sup.1 (
(163) The role of CA in converting COS into H.sub.2S was evaluated by incubating PeroxyTCM-1 with H.sub.2O.sub.2 (10 equiv) in the absence of CA. Although COS can be hydrolyzed to H.sub.2S under both acidic and basic conditions, this hydrolysis is much slower at physiological pH. Unexpectedly, a positive H.sub.2S release response was observed, indicating that COS could react directly with H.sub.2O.sub.2 to generate H.sub.2S in a CA-independent pathway (
(164) Specific reactive sulfur, oxygen, and nitrogen species (RSONS) were also evaluated to determine which RSONS could result in donor activation by measuring H.sub.2S release from PeroxyTCM-1 after incubation with different RSONS (
(165) Before investigating different potential biological applications of the PeroxyTCM compounds, the cytotoxicity of PeroxyTCM-1, PeroxyCM-1 and TCM1 (10-100 M) in HeLa cells was evaluated. No significant decrease in cell viability was observed after a 2 hour incubation, indicating that none of the three compounds exhibited appreciable cytotoxicity at the tested concentrations (
(166) The response of PeroxyTCM-1 to endogenous ROS generation also was evaluated. RAW 264.7 cells were incubated with phorbol 12-myristate 13-acetate (PMA), which is a well-established method to induce ROS and H.sub.2O.sub.2 production in macrophages. ROS generation was confirmed using 2,7-dichlorofluorescin diacetate (DCFDA;
(167) Also investigated was whether the developed ROS-activated donors could provide protection against ROS-related oxidative stress in simple cell culture models. ROS can play deleterious roles in various physiological and pathological systems ranging from aging to cardiovascular damage. In many cases, H.sub.2S administration can provide partial protection or rescue from these different disease states. For example, ROS generated during mitochondrial dysfunction are responsible for a wide range of damage in the cardiovascular system, including MI/R injury, and that exogenous H.sub.2S significantly preserved cardiac activity through a ROS scavenging pathways. On the basis of this H.sub.2S /ROS relationship, embodiments of donor compounds disclosed herein were evaluated to determine if the donor compounds would exhibit similar cytoprotective effects toward ROS-induced damage due to H.sub.2S release.
(168) To simulate increased cellular oxidative stress, HeLa cells were incubated with H.sub.2O.sub.2 (50-400 M) for 1 hour and observed a dose-dependent reduction of cell viability (
(169) In view of the many possible embodiments to which the principles of the present disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting. Rather, the scope of the present disclosure is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.