GOLD(III) COMPOUNDS AND CANCER CELL-SELECTIVE MODULATION OF MITOCHONDRIAL RESPIRATION AND METABOLISM
20230265108 · 2023-08-24
Inventors
Cpc classification
C07F1/00
CHEMISTRY; METALLURGY
C07F9/6596
CHEMISTRY; METALLURGY
International classification
Abstract
A compound of formula (I) is provided. Useful compounds and compositions, and methods of making and methods of using such compounds and compositions. The subject matter also relates to methods of treating cancer using compounds and compositions provided.
Claims
1-45. (canceled)
46. A compound having the formula (I) or a pharmaceutically acceptable salt thereof: ##STR00054## wherein, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; R is selected from the group consisting of H, aryl, alkyl, electron withdrawing group (EWG), or electron donating group (EDG); R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl, or, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is optionally substituted with Z; and Z is selected from the group consisting of haloaryl and alkoxyaryl.
47. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00055## wherein, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl, or, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is optionally substituted with Z; and Z is selected from the group consisting of haloaryl and alkoxyaryl.
48. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00056## wherein, R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl, or, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is optionally substituted with Z; and Z is selected from the group consisting of haloaryl and alkoxyaryl.
49. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00057## wherein, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; and Y is CH.sub.2 or NH.
50. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00058## wherein Y is CH.sub.2 or NH.
51. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00059## wherein, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; Y is CH or N; and Z is haloaryl or alkoxyaryl.
52. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00060## wherein Y is CH or N, and Z is haloaryl or alkoxyaryl.
53. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00061## wherein, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; R is selected from the group consisting of H, aryl, alkyl, electron withdrawing group (EWG), or electron donating group (EDG); R.sub.1 is selected the group consisting of alkyl and substituted alkyl; and n is 2, 4, 6, 8, 10, 12, or 16.
54. The compound of claim 46, having the following formula or pharmaceutically acceptable salt thereof: ##STR00062## wherein, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; R is selected from the group consisting of H, aryl, alkyl, electron withdrawing group (EWG), or electron donating group (EDG); and each n is independently selected from 2, 4, 6, 8, 10, 12, or 16.
55. The compound of claim 46, wherein X is: (a) selected from the group consisting of CH and C═O; (b) aryl, selected from the group consisting of phenyl (Ph), benzyl (Bz), thiophenyl, furyl, and pyridyl; (c) selected from the group consisting of ##STR00063## or (d) X is C═NO-alkynyl.
56. The compound of claim 46, wherein R is in ortho, para, or meta position to Au.
57. The compound of claim 46, wherein R is: (a) aryl, selected from the group consisting of Ph, Bz, thiophenyl, furyl, and pyridyl; (b) alkyl including 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms; (c) EWG, selected from the group consisting of —CF.sub.3, NO.sub.2, —F, —Br, —Cl, Aldehyde —COH, —COOH, and —CN; or (d) EDG, selected from the group consisting of —OCH.sub.3, —OH, CH.sub.3, —C═C, -alkynyl group, and —NH.sub.2.
58. The compound of claim 46, wherein R.sub.1 is: (a) alkyl including 1-33 carbon atoms; (b) ##STR00064## wherein n is 2, 4, 6, 8, 10, 12, or 16; or (c) methyl or ethyl.
59. The compound of claim 46, wherein R.sub.2 is: (a) alkyl including 1-32 carbon atoms; (b) ##STR00065## wherein n is 2, 4, 6, 8, 10, 12, or 16; or (c) methyl or ethyl.
60. The compound of claim 46, wherein R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that not substituted.
61. The compound of claim 46, wherein R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is substituted with Z.
62. A pharmaceutical composition comprising the compound of claim 46 and a pharmaceutically-acceptable carrier.
63. A method of killing a cancer cell, comprising: contacting a cancer cell with an effective amount of the compound of claim 46.
64. A method of increasing reactive oxygen species (ROS) in a cell, comprising: contacting a cell with an effective amount of the compound of claim 46.
65. The method of claim 64, wherein the cell is a cancer cell.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:
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DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0076] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
[0077] Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
[0078] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which need to be independently confirmed.
[0079] The presently-disclosed subject matter includes unique Au(III) compounds and compositions. The presently-disclosed subject matter further includes methods of increasing reactive oxygen species in a cell. The presently-disclosed subject matter further includes methods of killing a cancer cell. The syntheses of Au(III) compounds is disclosed herein, which incorporate [C{circumflex over ( )}N] to improve complex stability and to provide unique ligands for structure activity relationship (SAR). As disclosed herein, the compounds display high potency in cancer cells.
[0080] In some embodiments, the presently-disclosed subject matter relates to compounds, or pharmaceutically acceptable derivatives thereof, useful inhibitors for the treatment of cancer. It is also understood that the disclosed compounds can all be employed as corresponding pharmaceutical compositions.
[0081] In some embodiments, the presently disclosed subject matter includes a compound having the following formula (I):
##STR00019##
[0082] The compounds disclosed herein can include all salt forms, for example, salts of both basic groups, inter alia, amines, as well as salts of acidic groups, inter alia, carboxylic acids. The following are non-limiting examples of anions that can form salts with protonated basic groups: chloride, bromide, iodide, sulfate, bisulfate, carbonate, bicarbonate, phosphate, formate, acetate, propionate, butyrate, pyruvate, lactate, oxalate, malonate, maleate, succinate, tartrate, fumarate, citrate, and the like. The following are non-limiting examples of cations that can form salts of acidic groups: ammonium, sodium, lithium, potassium, calcium, magnesium, bismuth, lysine, and the like.
[0083] The analogs (compounds) of the present disclosure are arranged into several categories to assist the formulator in applying a rational synthetic strategy for the preparation of analogs which are not expressly exampled herein. The arrangement into categories does not imply increased or decreased efficacy for any of the compositions of matter described herein.
[0084] In some embodiments of the compounds of formula (I) as disclosed herein, X is selected from the group consisting of C—Y, N—Y, O—Y, or S—Y, where Y is a substituent such as H, ═OR.sub.3, ═NR.sub.3, ═NOR.sub.3, —R.sub.3, where R.sub.3 is alkyl, aryl, or alkynyl. In some embodiments, X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl. In some embodiments, X is selected from the group consisting of CH and C═O. In some embodiments, X is aryl, selected from the group consisting of phenyl (Ph), benzyl (Bz), thiophenyl, furyl, and pyridyl. In some embodiments, X is selected from the group consisting of
##STR00020##
In some embodiments, X is C═NO-alkynyl.
[0085] In some embodiments of the compounds of formula (I) as disclosed herein, R is selected from the group consisting of H, aryl, alkyl, electron withdrawing group (EWG), or electron donating group (EDG). In some embodiments, R is in ortho, para, or meta position to Au. In some embodiments, R is aryl, selected from the group consisting of Ph, Bz, thiophenyl, furyl, and pyridyl. In some embodiments, R is alkyl including 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In some embodiments, R is EWG, selected from the group consisting of —CF.sub.3, NO.sub.2, —F, —Br, —Cl, Aldehyde —COH, —COOH, and —CN. In some embodiments, R is EDG, selected from the group consisting of —OCH.sub.3, —OH, CH.sub.3, —C═C, -alkynyl group, and —NH.sub.2.
[0086] In some embodiments of the compounds of formula (I) as disclosed herein, R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl.
[0087] In some embodiments of the compounds of formula (I) as disclosed herein, R.sub.1 is alkyl including 1-33 carbon atoms. In some embodiments, R.sub.1 is
##STR00021##
wherein n is 2, 4, 6, 8, 10, 12, or 16. In some embodiments, R.sub.1 is selected from the group consisting of methyl and ethyl.
[0088] In some embodiments of the compounds of formula (I) as disclosed herein, R.sub.2 is alkyl including 1-32 carbon atoms. In some embodiments, R.sub.2 is
##STR00022##
wherein n is 2, 4, 6, 8, 10, 12, or 16. In some embodiments, R.sub.2 is selected from the group consisting of methyl and ethyl.
[0089] In some embodiments of the compounds of formula (I) as disclosed herein, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that not substituted. In some embodiments, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is substituted with Z.
[0090] In some embodiments of the compounds of formula (I) as disclosed herein, Z is selected from the group consisting of haloaryl and alkoxyaryl.
[0091] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00023##
wherein X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl, or R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is optionally substituted with Z; and Z is selected from the group consisting of haloaryl and alkoxyaryl.
[0092] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00024##
wherein R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl, or, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is optionally substituted with Z; and Z is selected from the group consisting of haloaryl and alkoxyaryl.
[0093] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00025##
wherein R.sub.1 and R.sub.2 are independently selected the group consisting of alkyl and substituted alkyl, or, R.sub.1 and R.sub.2, taken together with the N to which they are bound, form a piperidine or piperazine that is optionally substituted with Z; and Z is selected from the group consisting of haloaryl and alkoxyaryl.
[0094] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00026##
wherein X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; and Y is CH.sub.2 or NH.
[0095] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00027##
wherein Y is CH.sub.2 or NH.
[0096] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
wherein Y is CH.sub.2 or NH.
##STR00028##
[0097] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00029##
wherein X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; Y is CH or N; and Z is haloaryl or alkoxyaryl.
[0098] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00030##
wherein Y is CH or N, and Z is haloaryl or alkoxyaryl.
[0099] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00031##
wherein Y is CH or N, and Z is haloaryl or alkoxyaryl.
[0100] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00032##
wherein X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl R is selected from the group consisting of H, aryl, alkyl, electron withdrawing group (EWG), or electron donating group (EDG); R.sub.1 is selected the group consisting of alkyl and substituted alkyl; and n is 2, 4, 6, 8, 10, 12, or 16.
[0101] In some embodiments of the compounds of formula (I) as disclosed herein, the compound has the following formula:
##STR00033##
wherein X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl; R is selected from the group consisting of H, aryl, alkyl, electron withdrawing group (EWG), or electron donating group (EDG); and each n is independently selected from 2, 4, 6, 8, 10, 12, or 16.
[0102] Some embodiments of the presently-disclosed subject matter includes a compound having the following formula:
##STR00034##
wherein X is selected from the group consisting of CH, C═O, C═NO-alkynyl, NH, O, S, or aryl. In some embodiments of the compound, X is CH or C═O.
[0103] In some embodiments of the compounds of formula (I) as disclosed herein, the compound is selected from the following formulae:
##STR00035## ##STR00036## ##STR00037##
[0104] In some embodiments, the presently-disclosed subject matter is related to a pharmaceutical composition. Pharmaceutical compositions as disclosed herein comprise one or more compounds as disclosed herein and a pharmaceutically-acceptable carrier.
[0105] In some embodiments, the presently-disclosed subject matter is related to a method of killing a cancer cell, which involves contacting the cancer cell with an effective amount of one or more compounds or compositions as disclosed herein. In some embodiments, the effective amount is from about 10 nM to about 100 uM. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in a subject. In some aspects of the disclosed method, the subject has been diagnosed with a need for treatment of cancer. In some embodiments, the subject is a mammal.
[0106] In some embodiments, the presently-disclosed subject matter is related to a method of increasing reactive oxygen species (ROS) in a cell, which involves contacting the cancer cell with an effective amount of one or more compounds or compositions as disclosed herein. In some embodiments, the effective amount is from about 10 nM to about 100 uM. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a cultured cell. In some embodiments, the cell is in a subject. In some embodiments, the subject is a mammal.
[0107] In some embodiments, the presently-disclosed subject matter is related to use of one or more compounds or compositions as disclosed herein in a medicament for the treatment of a cancer.
[0108] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
[0109] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
[0110] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).
[0111] A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more —OCH.sub.2CH.sub.2O— units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more —CO(CH.sub.2).sub.8CO— moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.
[0112] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
[0113] In defining various terms, “A.sup.1,” “A.sup.2,” “A.sup.3,” and “A.sup.4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.
[0114] The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six carbon atoms.
[0115] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.
[0116] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.
[0117] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.
[0118] The term “polyalkylene group” as used herein is a group having two or more CH.sub.2 groups linked to one another. The polyalkylene group can be represented by a formula —(CH.sub.2).sub.a—, where “a” is an integer of from 2 to 500.
[0119] The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as —OA.sup.1 where A.sup.1 is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as —OA.sup.1-OA.sup.2 or —OA.sup.1-(OA.sup.2).sub.a-OA.sup.3, where “a” is an integer of from 1 to 200 and A.sup.1, A.sup.2, and A.sup.3 are alkyl and/or cycloalkyl groups.
[0120] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A.sup.1A.sup.2)C═C(A.sup.3A.sup.4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C═C. The alkenyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.
[0121] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.
[0122] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.
[0123] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.
[0124] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
[0125] The term “aldehyde” as used herein is represented by a formula —C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C═O.
[0126] The terms “amine” or “amino” as used herein are represented by a formula NA′A.sup.2A.sup.3, where A.sup.1, A.sup.2, and A.sup.3 can be, independently, hydrogen or optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0127] The term “carboxylic acid” as used herein is represented by a formula —C(O)OH.
[0128] The term “ester” as used herein is represented by a formula —OC(O)A.sup.1 or —C(O)OA.sup.1, where A.sup.1 can be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by a formula -(A.sup.1O(O)C-A.sup.2-C(O)O).sub.a— or -(A.sup.1O(O)C-A.sup.2-OC(O)).sub.a—, where A.sup.1 and A.sup.2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.
[0129] The term “ether” as used herein is represented by a formula A.sup.1OA.sup.2, where A.sup.1 and A.sup.2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by a formula -(A.sup.1O-A.sup.2O).sub.a—, where A.sup.1 and A.sup.2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.
[0130] The term “halide” as used herein refers to the halogens fluorine, chlorine, bromine, and iodine.
[0131] The term “heterocycle,” as used herein refers to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Heterocycle includes pyridinde, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, including 1,2,4-triazine and 1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like.
[0132] The term “hydroxyl” as used herein is represented by a formula —OH.
[0133] The term “ketone” as used herein is represented by a formula A.sup.1C(O)A.sup.2, where A.sup.1 and A.sup.2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0134] The term “azide” as used herein is represented by a formula —N.sub.3.
[0135] The term “nitro” as used herein is represented by a formula —NO.sub.2.
[0136] The term “nitrile” as used herein is represented by a formula —CN.
[0137] The term “silyl” as used herein is represented by a formula —SiA.sup.1A.sup.2A.sup.3, where A.sup.1, A.sup.2, and A.sup.3 can be, independently, hydrogen or an optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0138] The term “sulfo-oxo” as used herein is represented by a formulas —S(O)A.sup.1, —S(O).sub.2A.sup.1, —OS(O).sub.2A.sup.1, or —OS(O).sub.2OA.sup.1, where A.sup.1 can be hydrogen or an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S═O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by a formula —S(O).sub.2A.sup.1, where A.sup.1 can be hydrogen or an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by a formula A.sup.1S(O).sub.2A.sup.2, where A.sup.1 and A.sup.2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by a formula A.sup.1S(O)A.sup.2, where A.sup.1 and A.sup.2 can be, independently, an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.
[0139] The term “thiol” as used herein is represented by a formula —SH.
[0140] The term “organic residue” defines a carbon containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.
[0141] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compounds disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.
[0142] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis/trans (E/Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.
[0143] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers. Additionally, unless expressly described as “unsubstituted”, all substituents can be substituted or unsubstituted.
[0144] In some aspects, a structure of a compound can be represented by a formula:
##STR00038##
which is understood to be equivalent to a formula:
##STR00039##
wherein n is typically an integer. That is, R.sup.n is understood to represent five independent substituents, R.sup.n(a), R.sup.n(b), R.sup.n(c), R.sup.n(d), R.sup.n(e). By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance R.sup.n(a) is halogen, then R.sup.n(b) is not necessarily halogen in that instance. Likewise, when a group R is defined as four substituents, R is understood to represent four independent substituents, R.sup.a, R.sup.b, R.sup.c, and R.sup.d. Unless indicated to the contrary, the substituents are not limited to any particular order or arrangement.
[0145] The following abbreviations are used herein. DMSO: dimethylsulfoxide. MeOH: methanol. EtOH: ethanol. TFA: trifluoroacetic acid. MeCN: acetonitrile.
[0146] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the methods of the invention.
[0147] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.
[0148] As used herein, the term “subject” includes both human and animal subjects. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
[0149] As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
[0150] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
[0151] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, “diagnosed with cancer” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can favorably inhibit or kill cancer cells.
[0152] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
[0153] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.
[0154] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
[0155] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
[0156] The present application can “comprise” (open ended) or “consist essentially of” the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.
[0157] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
[0158] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
[0159] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.
[0160] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
[0161] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.
[0162] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.
EXAMPLES
[0163] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.
[0164] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
[0165] Several methods for preparing the compounds of this invention are illustrated in the following Examples. Starting materials and the requisite intermediates are in some cases commercially available, or can be prepared according to literature procedures or as illustrated herein.
Example 1. General Experimental Details for Synthesis
[0166] The [C{circumflex over ( )}N]-cyclometalated gold(III) compounds were synthesized from previously reported methods..sup.54 Treatment of [C{circumflex over ( )}N]—Au(III)Cl.sub.2 with dithiocarbamate ligands in methanol for 16 h at room temperature followed by treatment with an aqueous saturated solution of NH.sub.4PF.sub.6 gave the desired compounds in respectable yields (Scheme 1).
##STR00040##
[0167] All reactions were carried under ambient conditions in air unless otherwise noted. Solvents were of ACS grade (Pharmco-Aaper) and used as is. The starting Au(III) cyclometalated complexes; [2-benzylpyridine]Au(III)Cl.sub.2 and [2-benzoylpyridine]Au(III)Cl.sub.2 were prepared according to a modified procedure as previously reported..sup.1 Sodium dimethyl dithiocarbamate and sodium diethyl dithiocarbamate salts were purchased from Alfa Aesar. 1-(4-bromophenyl)piperazine and 1-(4-Methoxyphenyl)piperazine were purchased from Sigma Aldrich. Ammomium hexafluorophosphate was purchased from Matrix Scientific. Carbon disulfide was purchased from Millipore Sigma and distilled prior to use. Sodium hydroxide pellets were purchased from VWR. Deuterated solvents were purchased from Cambridge Isotope Laboratories (Andover, Mass.). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorofluorescin diacetate (DCF-DA), and JC-1 were purchased from Cayman Chemicals. NMR spectra were recorded on a Bruker Avance NEO 400 MHz spectrometer and samples calibrated for: .sup.1H NMR (CD.sub.3CN δ=1.94 ppm and DMSO-d.sub.6 δ=2.50 ppm), .sup.13C NMR (CD.sub.3CN δ=118.26 and 1.32 ppm and DMSO-d.sub.6 δ=49.00 ppm), and .sup.19F NMR externally referenced to CFCl.sub.3 δ=0.00). Electrospray ionization mass spectrometry (ESI-MS) was performed on an Agilent Technologies 1100 series liquid chromatography/MS instrument. High-resolution mass spectra (HRMS) were obtained by direct flow injection (injection volume=2 μL) using ElectroSpray Ionization (ESI) on a Waters Qtof API US instrument in the positive mode (CIC, Boston University). Typical conditions are as follows: capillary=3000 kV, cone=35 or 15, source temperature=120° C., and a desolvation temperature=350° C. In addition to spectroscopic characterization, the purity of all compounds was assessed by RP-HPLC using an Agilent Technologies 1100 series HPLC instrument and an Agilent Phase Eclipse Plus C18 column (4.6 mm×100 mm; 3.5 μm particle size). All compounds were found to be 97% pure.
Example 2
[0168] ##STR00041##
[0169] Sodium piperazine dithiocarbamate: Prepared from a modified procedure in the literature..sup.2-3 A round bottom was charged with a piperazine (850 mg, 10 mmol) and dissolved in 80 mL of ethanol. Sodium hydroxide pellets (400 mg, 10 mmol) were crushed and added portion wise. The mixture was sonicated (15 minutes) until all the NaOH was dissolved. The reaction was cooled to 0° C. using an ice-bath. Carbon disulfide (1.53 g, 20 mmol) was placed in an addition funnel and added dropwise over 10 minutes. The reaction was allowed to warm to room temperature naturally and stirred for 12 h. All solvent was removed in vacuo at 50° C. to leave a yellow/white solid. The solid was dissolved in 20 mL of acetone and 250 mL of ether was added to precipitate a white solid, which was vacuum filtered, washed with excess ether, and dried to afford the sodium dithiocarbamate salt. Yield 1.62 g, 88%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=4.27 (t, J=8 Hz, 4H), 1.52-1.58 (m, 2H), 1.38-1.44 (m, 4H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ=213.09, 50.71, 26.22, 24.86.
Example 3. Sodium 1-(4-bromophenyl)piperazine dithiocarbamate
[0170] ##STR00042##
[0171] 1-(4-bromophenyl)piperazine (241 mg, 1 mmol) and crushed NaOH pellets (40 mg, 1 mmol) were added in 25 mL of methanol and sonicated for 15 minutes to dissolve the NaOH pellets. The reaction was cooled to 0° C. Carbon disulfide (77 mg, 1 mmol) was placed in an addition funnel and added dropwise over 5 minutes. The reaction was allowed to warm up to room temperature naturally and stirred for 2 h. The solvent was removed in vacuo at 70° C. to leave a white solid. The solid was dissolved in acetone (5 mL) and ether (75 mL) was added to precipitate a white solid, which was vacuum filtered, washed with ether, and dried. Yield 200 mg, 59%. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.35 (d, J=4 Hz, 2H), 6.88 (d, J=4 Hz, 2H), 4.45 (t, J=8 Hz, 4H), 3.11 (t, J=8, 4H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ=214.73, 150.50, 131.86, 117.56, 48.88, 48.23.
Example 4. Sodium 1-(4-methoxyphenyl)piperazine dithiocarbamate
[0172] ##STR00043##
[0173] Prepared analogously to sodium 1-(4-bromophenyl)piperazine dithiocarbamate. 1-(4-methoxyphenyl)piperazine (192 mg, 1 mmol), NaOH (40 mg, 1 mmol), CS2 (77 mg, 1 mmol). Yield 215 mg, 74%. .sup.1H NMR (400 MHz, DMSO-d6) δ=6.82 (d, J=4 Hz, 2H), 6.61 (d, J=4 Hz, 2H), 4.45 (t, J=8 Hz, 4H), 3.68 (s, 3H), 2.86 (t, J=8 Hz, 4H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ=214.49, 153.45, 145.85, 118.03, 114.77, 55.69, 51.31, 50.30, 49.40, 46.12.
Example 5. General Procedure for the Preparation of Au(III) [C{circumflex over ( )}N] Dithiocarbamate Complexes 1a-e and 2a-e
[0174] Complexes were prepared following a slightly modified procedure from the literature..sup.4 For complexes 1a-1e, the corresponding Au(III) starting material (2-benzoylpyridine)Au(III)Cl.sub.2 (50 mg, 0.11 mmol) was suspended in 20 mL of MeOH in a 250 mL Erlenmeyer flask and stirred at room temperature. A separate solution of the corresponding sodium dithiocarbamate salt (0.13 mmol) was dissolved in 10 mL of MeOH and added dropwise (500 μL/minute). The mixture gradually turned yellow upon addition of the dithiocarbamate solution. The reaction was stirred at room temperature for 10-12 h. A saturated solution of NH.sub.4PF.sub.6 in DI H.sub.2O was made and added to the MeOH mixture. Excess DI H.sub.2O was added until a precipitate was observed. The solution was filtered and the solid washed with DI H.sub.2O (10 mL), excess Et.sub.2O and vacuum dried for 30 minutes to afford the Au(III) complexes 1a-1e. Complexes 2a-e were prepared analogously to 1a-1e but with the Au(III) starting material (2-benzylpyridine)Au(III)Cl.sub.2 (50 mg, 0.11 mmol).
Example 6.
[0175] ##STR00044##
[0176] Prepared as described in the general procedure. [(2-benzoylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and NaDMDTC (19 mg, 0.13 mmol). Yield 61 mg, 86%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=9.01 (d, J=4 Hz, 1H), 8.51 (d, J=4 Hz, 2H), 7.97 (quint., J=8 Hz, 2H), 7.58 (sext., J=12 Hz, 3H), 3.44 (d, J=8 Hz, 6H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=194.11, 190.33, 152.69, 147.60, 145.03, 142.14, 136.13, 131.75, 131.58, 131.36, 130.09, 129.09, 42.18, 41.07; 19F NMR (376 MHz, MeCN−d.sub.3) δ=−131.02, −135.41, −139.81, −144.20, −148.59, −157.37. HRMS (m/z) calcd. 499.0213, found 499.0212 [M-PF.sub.6]+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=6.20 minutes using the following method: Flow rate: 1 mL/min; X=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH) 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 7. 1b
[0177] ##STR00045##
[0178] Prepared as described in the general procedure. [(2-benzoylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and NaDEDTC (22 mg, 0.13 mmol). Yield 62 mg, 84%. .sup.1H NMR (400 MHz, MeCN-ds) (=9.03 (d, J=4 Hz, 1H), 8.54 (d, J=4 Hz, 2H), 8.02 (quint., J=8 Hz, 2H), 7.60 (sext., J=12 Hz, 3H), 3.88 (dq, J=20, 8 Hz, 4H), 1.39 (dt, J=24, 8 Hz, 6H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=193.47, 190.30, 152.60, 147.61, 145.02, 142.32, 136.12, 131.75, 131.56, 131.35, 130.09, 129.09, 49.02, 47.67, 47.47, 12.31, 12.07, 12.02; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−131.02, −135.41, −139.81, −144.20, −148.59, −157.37. HRMS (m/z) calcd. 527.0526, found 527.0516 [M-PF.sub.6].sup.+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=7.00 minutes using the following method: Flow rate: 1 mL/min; λ=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 8. 1c
[0179] ##STR00046##
[0180] Prepared as described in the general procedure. [(2-benzoylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and NaPipDTC (24 mg, 0.13 mmol). Yield 55 mg, 73%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.99 (d, J=4, 1 Hz H), 8.51 (d, J=4 Hz, 2H), 7.98 (quint., J=8 Hz, 2H), 7.58 (sext., J=12 Hz, 3H), 3.90 (dt, J=16, 4 Hz, 4H), 1.74-1.82 (m, 6H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=191.37, 190.34, 152.64, 147.60, 145.01, 142.37, 136.12, 131.76, 131.64, 131.37, 130.07, 129.09, 52.99, 51.16, 25.97, 24.02; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−130.85, −135.24, −139.63, −144.02, −148.41, −157.20. HRMS (m/z) calcd. 539.0526, found 539.0527 [M-PF.sub.6]+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=7.12 minutes using the following method: Flow rate: 1 mL/min; λ=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 9. 1d
[0181] ##STR00047##
[0182] Prepared as described in the general procedure. 2-benzoylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and Na(4-bromophenylpiperazine)DTC (44 mg, 0.13 mmol). Yield 30 mg, 32%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=9.04 (d, J=4 Hz, 1H), 8.54 (d, J=4 Hz, 2H), 8.01 (quint., J=8 Hz, 2H), 7.63 (sext., J=12 Hz, 3H), 7.44 (d, J=8 Hz, 2H), 6.95 (d, J=8 Hz, 2H), 4.12 (t, J=8 Hz, 4H), 3.48 (t, J=12 Hz, 4H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=194.06, 152.66, 149.61, 144.92, 136.11, 132.58, 131.67, 131.64, 131.43, 130.05, 129.03, 112.64, 50.78, 49.14, 48.07; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−130.66, −135.05, −139.44, −143.84, −148.23, −152.62. HRMS (m/z) calcd. 693.9897, found 693.9899 [M-PF.sub.6].sup.+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=9.26 minutes using the following method: Flow rate: 1 mL/min; λ=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 10. 1e
[0183] ##STR00048##
[0184] Prepared as described in the general procedure. [(2-benzoylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and Na(4-methoxyphenylpiperazine)DTC (39 mg, 0.13 mmol). Yield 42 mg, 48%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.87 (d, J=8 Hz, 1H), 8.31 (t, J=8 Hz, 2H), 7.82 (t, J=12 Hz, 1H), 7.61 (d, J=8 Hz, 1H), 7.50 (t, J=8 Hz, 1H), 7.43 (d, J=8 Hz, 4H), 7.26 (d, J=10 Hz, 1H), 6.97 (d, J=8 Hz, 2H), 6.88 (d, J=8 Hz, 2H), 4.01 (dt, J=20, 8 Hz, 4H), 3.74 (s, 3H), 3.28 (quint., J=8 Hz, 4H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=203.55, 155.21, 151.22, 144.80, 134.96, 132.54, 132.36, 128.49, 127.39, 119.54, 115.05, 55.64, 50.69, 50.08, 49.10; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−135.41, −139.80, −144.19, −148.58, −152.98, −157.36. HRMS (m/z) calcd. 646.0897, found 646.0896 [M-PF.sub.6]+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=7.14 minutes using the following method: Flow rate: 1 mL/min; =260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 11. 2a
[0185] ##STR00049##
[0186] Prepared as described in the general procedure. [(2-benzylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and NaDMDTC (19 mg, 0.13 mmol). Yield 64 mg, 92%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) 0=8.86 (d, J=8 Hz, 1H), 8.22 (t, J=8 Hz, 1H), 7.95 (d, J=8 Hz, 1H), 7.62 (t, J=8 Hz, 1H), 7.42 (t, J=8 Hz, 1H), 7.32 (t, J=8 Hz, 1H), 7.19 (t, J=8 Hz, 1H), 4.43 (s, 2H), 3.45 (d, J=4 Hz, 6H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) 0=195.79, 157.04, 152.22, 146.36, 144.17, 13357, 131.11, 129.61, 129.52, 129.23, 127.73, 126.21, 47.31, 42.12, 40.89; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−130.53, −134.92, −139.31, −143.70, −148.10, −152.48. HRMS (m/z) calcd. 485.0421, found 485.0418 [M-PF.sub.6].sup.+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=6.26 minutes using the following method: Flow rate: 1 mL/min; X=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 12. 2b
[0187] ##STR00050##
[0188] Prepared as described in the general procedure. [(2-benzylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and NaDEDTC (22 mg, 0.13 mmol). Yield 64 mg, 88%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.85 (d, J=8 Hz, 1H), 8.21 (t, J=8 Hz, 1H), 7.91 (d, J=8 Hz, 1H), 7.60 (t, J=8 Hz, 1H), 7.40 (t, J=8 Hz, 1H), 7.31 (t, J=8 Hz, 1H), 7.16 (t, J=8 Hz, 1H), 4.45 (s, 2H), 3.65 (td, J=24, 16, 8 Hz, 4H), 1.32-1.48 (m, 6H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=195.12, 157.03, 152.17, 146.48, 144.16, 133.56, 131.09, 129.60, 129.50, 129.22, 127.75, 126.23, 48.87, 47.37, 47.30, 12.32, 12.04; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−134.25, −138.64, −143.03, −147.42, −151.81, −160.60. HRMS (m/z) calcd. 513.0734, found 513.0740 [M-PF.sub.6]-1, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=7.34 minutes using the following method: Flow rate: 1 mL/min; X=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 13. 2c
[0189] ##STR00051##
[0190] Prepared as described in the general procedure. [(2-benzylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and NaPipDTC (24 mg, 0.13 mmol). Yield 51 mg, 69%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.83 (d, J=8 Hz, 1H), 8.23 (t, J=8 Hz, 1H), 7.96 (d, J=8 Hz, 1H), 7.62 (t, J=8 Hz, 1H), 7.42 (t, J=8 Hz, 1H), 7.32 (t, J=8 Hz, 1H), 7.20 (t, J=8 Hz, 1H), 4.44 (s, 2H), 3.94 (dt, J=12, 4 Hz, 4H), 1.81-1.85 (m, 6H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=193.10, 157.05, 152.22, 146.55, 144.14, 133.59, 131.18, 129.60, 129.49, 129.22, 127.74, 126.22, 52.81, 50.80, 47.33, 26.01, 25.78, 24.03; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−128.20, −132.59, −136.98, −141.38, −145.77, −150.16. HRMS (m/z) calcd. 525.0734, found 525.0731 [M-PF.sub.6].sup.+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=7.48 minutes using the following method: Flow rate: 1 mL/min; =260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 14. 2d
[0191] ##STR00052##
[0192] Prepared as described in the general procedure. [(2-benzylpyridine)Au(III)Cl.sub.2] (50 mg, 0.11 mmol) and Na(4-bromophenylpiperazine)DTC (44 mg, 0.13 mmol). Yield 39 mg, 43%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.86 (d, J=8 Hz, 1H), 8.21 (t, J=8 Hz, 1H), 7.94 (d, J=8 Hz, 1H), 7.61 (t, J=8 Hz, 1H), 7.39-7.45 (m, 4H), 7.33 (t, J=8 Hz, 1H), 7.19 (t, J=8 Hz, 1H), 6.92 (q, J=12 Hz, 2H), 4.42 (s, 2H), 4.06-4.11 (m, 4H), 3.42-3.48 (m, 4H); .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=195.05, 156.34, 151.65, 149.05, 145.68, 143.57, 132.87, 131.97, 130.53, 129.04, 128.64, 127.14, 125.60, 118.20, 112.02, 50.07, 48.25, 47.54, 46.65; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−131.38, −135.77, −140.16, −144.55, −148.94, −157.73. HRMS (m/z) calcd. 680.0104, found 680.0104 [M-PF.sub.6].sup.+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=9.40 minutes using the following method: Flow rate: 1 mL/min; X=260 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 15. 2e
[0193] ##STR00053##
[0194] Prepared as described in the general procedure. [(2-benzylpyridine)Au(III)Cl.sub.2] (57 mg, 0.11 mmol) and Na(4-bromophenylpiperazine)DTC (39 mg, 0.13 mmol). Yield 49 mg, 43%. .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.86 (d, J=4 Hz, 1H), 8.21 (t, J=8 Hz, 1H), 7.93 (d, J=8 Hz, 1H), 7.61 (t, J=8 Hz, 1H), 7.40 (t, J=8 Hz, 2H), 7.30 (t, J=8 Hz, 1H), 7.18 (t, J=8 Hz, 1H), 6.98 (d, J=12 Hz, 2H), 6.89 (d, J=8 Hz, 2H), 4.42 (s, 2H), 4.06-4.12 (m, 4H), 3.75 (s, 3H), 3.283.33 (m, 4H); .sup.13C NMR (101 MHz, MeCN-d.sub.3)=194.74, 156.43, 154.69, 151.74, 145.79, 144.26, 143.64, 132.98, 130.63, 129.12, 129.01, 128.72, 127.21, 125.67, 119.01, 114.53, 55.13, 50.70, 49.61, 48.83, 46.72; 19F NMR (376 MHz, MeCN-d.sub.3) δ=−130.66, −135.05, −139.44, 143.84, −148.23, −152.62. HRMS (m/z) calcd. 632.1105, found 632.1100 [M-PF.sub.6].sup.+, Purity was demonstrated to be >97% by RP-HPLC: R.sub.f=8.48 minutes using the following method: Flow rate: 1 mL/min; 2\, =260 nm; Eluent A=H2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH).
Example 16. NMR and MS Characterization
[0195] The compounds (1a-e and 2a-e) where characterized by .sup.1H-NMR, .sup.13C-NMR, and .sup.19F-NMR and high-resolution mass spectrometry (HRMS). The purity of the compounds was verified by HPLC (>95%). In the instant invention, the structural architecture of Au(III) dithiocarbamate complexes were expanded to include cyclic and aromatic side chains in addition to cyclometalation, which provide stabilization by strong σ-donation to the gold center..sup.55-56 It has been demonstrated that this stabilization strategy has positive implications on the solution chemistry, biological and electrochemical behavior of complexes..sup.57 Two different [C{circumflex over ( )}N]Au(III)Cl.sub.2 complexes with electronic variations were used in the synthesis with the aim of establishing structural diversity. Complexes 1a-e possess a carbonyl at the methine bridge of the benzylpyridine framework, whereas 2a-e does not. Studies from the instant observations show that the difference impart unique reactivity and kinetics towards nucleophiles. Alternate metal-based drugs to platinum, the first-line of chemotherapy for several cancer types are desperately needed to overcome the toxicity and resistance associated with platinum drugs..sup.58-62 Gold compounds including auranofin have been used in preclinical and clinical trials with great promise..sup.63-67 A more systematic design and the elucidation of the mechanism of action of gold compounds will expand the utility of well-defined gold anticancer drug candidates with reduced susceptibility to resistance and toxic side effects. A small library of gold(III) compounds supported by different cyclometalated and dithiocarbamate ligands to obtain cationic complexes complemented by hexafluorophosphate ions was developed. Structural diversity was achieved by using two different cyclometalated gold(III) starting materials and five dithiocarbamate ligands of distinct structural and electronic variety. Consequently, the library enabled exploration of the biological activity of novel gold(III) dithiocarbamates in the context of cancer as described in this report.
Example 17. X-Ray Crystallography
[0196] Single crystals of compounds 1c, 2a, 2b, 2c, and 2e (
[0197] All crystals were mounted using polyisobutene oil on the end of a glass fibre, which had been mounted to a copper pin using an electrical solder. It was placed directly in the cold gas stream of a liquid nitrogen cryostat.sup.5-6 A Bruker D8 Venture diffractometer with graded multilayer focused MoKα X-rays (λ=0.71073 Å) was used to collect diffraction. Raw data were integrated, scaled, merged, and corrected for Lorentz-polarization effects using the APEX3 package..sup.7-9 Space group determination and structure solution and refinement were carried out with SHELXT and SHELXL respectively..sup.10-11 All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were placed at calculated positions and refined using a riding model with their isotropic displacement parameters (Uiso) set to either 1.2 Uiso or 1.5 Uiso of the atom to which they were attached. Ellipsoid plots were drawn using SHELXTL-XP..sup.12 The structures, deposited in the Cambridge Structural Database, were checked for missed symmetry, twinning, and overall quality with PLATON,.sup.13 an R-tensor,.sup.14 and finally validated using CheckCIF..sup.13
[0198] Compounds 2a, 2b, 2c, and 2e crystallize out in the solid state with one molecule per asymmetric unit while compound 1c contains two cation/anions pair. Each cyclometalated ligand, 2-benzylpyridine and 2-benzoylpyridine have a slightly puckered twist. The bridging methine carbon allows for the formation of a 6-membered ring containing the Au atom, leading to a less strained cyclometalated system. Each molecule is distorted around the Au—C and Au—N bonds resulting in half occupancy among the two atoms. Analyzing the bonding motif of the [C{circumflex over ( )}N] framework reveals a significant trans-effect on the binding dithiocarbamate ligand. The Au—S bond trans to the nitrogen of the [C{circumflex over ( )}N] ligand is significantly shorter (on average 0.156 Å) than the Au—S bond trans to the carbon (Table 1). This results in a slightly distorted square planar geometry around the Au(III) center in comparison to other Au(III) bis-dithiocarbamate complexes bearing symmetrical dithiocarbamate ligands which contain four Au—S bonds equidistant to one another (˜2.33 Å)..sup.47, 68
TABLE-US-00001 TABLE 1 Selected interatomic distances of complexes 1c, 2a, 2b, 2c, and 2e. Compound Bond (Å) 1c 2a 2b 2c 2e Au—C 2.042 (19) 2.043 (3) 2.061 (5) 2.042 (3) 2.045 (19) Au—N 2.062 (18) 2.071 (2) 2.065 (5) 2.065 (3) 2.069 (17) Au—S.sub.(trans to N) 2.283 (8) 2.280 (7) 2.278 (16) 2.276 (9) 2.298 (5) Au—S.sub.(trans to C) 2.376 (9) 2.404 (8) 2.392 (14) 2.499 (8) 2.379 (5)
Example 18. Uv-Vis Spectrometry of Complexes
[0199] The photophysical properties of all ten complexes were evaluated by scanning their absorption profile in DMSO (
[0200] The UV-vis profile was then obtained by scanning from 600 to 200 nm. The spectra were normalized to maximum absorbance and plotted in GraphPad Prism 6.
[0201] Each complex displayed similar absorption profiles with a high-energy transition at 260 nm and a corresponding lower energy transition at 295-305 nm. The high-energy transition at 260 nm can be attributed to intraligand transitions of the 2-benzylpyridine or 2-benzoylpyridine fragments in the [C{circumflex over ( )}N] ligands. The low-energy transition is attributed to ligand-to-metal-charge transfer from the donor dithiocarbamate ligand to the electrophilic Au(III) metal center..sup.39 The different dithiocarbamate ligands utilized had minimal effect on the measured absorbance.
Example 19. UV-Vis Stability in DMEM and PBS
[0202] The stability of the complexes were evaluated in two relevant biological media, PBS (phosphate-buffered saline) and DMEM (Dulbecco's modified eagle medium). DMEM, which contains several biological nucleophiles such as amino acids, are common sources of reductants.
[0203] All spectra were recorded on a Shimadzu UV-1280 model instrument. DMEM was used as purchased from Corning® (4.5 g/L glucose, L-glutamine and sodium pyruvate). PBS was used as received from Corning® (without calcium or magnesium). Each medium was warmed to room temperature prior to dilution of the complexes. All complexes were freshly prepared prior to use as a 1 mM stock in DMSO. The solutions were then diluted to 50 μM×4 mL with the respective biological medium. The amount of DMSO in each solution was 5%, therefore the instrument was blanked with either a 5% DMSO/PBS solution or 5% DMSO/DMEM solution prior to each scan. Time periods of t=0 h (after preparation of sample), 1 h, 6 h, 12 h, and 24 h were recorded. Prior to each time point, the instrument was blanked with corresponding 5% DMSO solution. The absorbance profile was scanned from 650 nm to 240 nm for each scan. For each complex, the data were normalized to the highest absorbance at t=0 h and plotted in GraphPad Prism 6. Each respective graph can be seen in the appended graphs.
[0204] Complexes 1a and 2a in both PBS and DMEM displayed the highest stability over 24 h (
Example 20. Reactivity with GSH(UV-Vis Spectrometry)
[0205] Stock solutions of the complexes were prepared by dissolving enough compound to achieve 1 mM×1 mL in DMSO. The stock solutions were diluted down to 50 μM by taking 50 μl of the stock and diluting to 1 mL with PBS. A separate stock solution of GSH was prepared as a 1 mM stock×5 mL. This stock solution was then diluted to 50 μM in PBS. All spectra were recorded on a Shimadzu UV-1280 model instrument. A blank was prepared by mixing 50 μL of DMSO and 1.95 mL of PBS. Time intervals of 5 minutes and 12 h were recorded. Prior to each recording the instrument was blanked. The equimolar solutions of the complex (50 μM) and GSH (50 μM) were mixed in a 1:1 ratio to achieve a final concentration of 25 μM the UV-vis spectra recorded at the indicated time intervals. For each reaction, the spectrum was normalized to the highest absorbance and plotted in GraphPad Prism 6. Each respective graph can be seen in the appended graphs.
Example 21. Reactivity with NAC(UV-Vis Spectrometry)
[0206] The experimental procedure was performed identically to that of the reaction involving GSH noted above. For each reaction, the spectrum was normalized to the highest absorbance and plotted in GraphPad Prism 6. Each respective graph can be seen in the appended graphs.
Example 22. Reactivity with GSH (HPLC)
[0207] All solvents used were of HPLC grade. All spectra were recorded using an Agilent Technologies 1100 series HPLC instrument and an Agilent Phase Eclipse Plus C18 column (4.6 mm×100 mm; 3.5 μm particle size). Compound 2a was prepared as a 5 mM stock in MeCN. GSH was prepared as a 5 mM stock in H.sub.2O. The stocks were diluted to a final concentration of 2.5 mM with the appropriate solvent and each respective HPLC spectrum recorded. For the reaction of 2a+GSH, 1 mL from each of the 5 mM stocks were mixed in a 1:1 ratio to achieve a 2 mL solution with a final concentration of 2.5 mM. The sample was then subjected to HPLC analysis. All spectra were recorded using the following method: Flow rate: 1 mL/min; λ=240 nm; Eluent A=H.sub.2O with 0.1% TFA; Eluent B=MeOH with 0.1% TFA; Solvent Gradient: 0-3 min (50:50 H.sub.2O:MeOH), 5 min (40:60 H.sub.2O:MeOH), 7 min (30:70 H.sub.2O:MeOH), 9 min (0:100 H.sub.2O:MeOH), 10 min (20:80 H.sub.2O:MeOH), 12 min until end of run (100:0 H.sub.2O:MeOH). Data were plotted using GraphPad Prism 6. Each respective graph can be seen in the main text (
Example 23. Reactivity with GSH (.SUP.1.H NMR Spectroscopy)
[0208] Stock solutions comprised of a 1 mL, 20 mM solution of 2a or L-GSH in DMSO-d.sub.6 was prepared. The solution of GSH was sonicated for 5 minutes to dissolve all of the reagent. 500 μL of each solution were mixed in a 1:1 ratio to produce a final concentration of 10 mM in DMSO-d.sub.6. The solution was then analyzed by .sup.1H NMR spectroscopy. The solution was further analyzed at different time intervals, t=1 h, 6 h, 12 h, and 24 h. Spectra were plotted using Mestrenova. (
Example 24. LC-MS Analysis of the 2a+GSH Adduct
[0209] After performing the reaction of 2a+GSH, a single new peak was observed. The same solution was then subjected to LC-MS analysis (2.5 mM) using an Agilent 1200 HPLC with a direct flow injection with a HPLC auto sampler without a column, λ=280 nm, (injection volume: 40 μL, flow rate: 0.2 mL/min). ESI positive mode was taken with a source temperature of 120° C., desolvation temperature of 300° C., Capillary Vat 3.5 kV while Cone was set at 35. Data was plotted and analyzed using Mestrenova.
Example 25. Cyclic Voltammetry
[0210] All electrochemical measurements were recorded with a scan rate of 0.1 V/s with a three-segment sweep and a sample interval of 0.001 V. The quiet time was set to 2 seconds and sensitivity and 1×10.sup.−5 A/V. All solutions were freshly prepared prior to use. All spectra were recorded using a CH instruments 650E potentiostat. The electrodes used were all 3 mm: glassy carbon working electrode (CHI104), Ag/AgCl reference electrode (CHI111), and a platinum wire counter electrode (CHI115). Compound 2a (8.8 mg/7 mL), NaDMDTC (2 mg/7 mL), and (2-benzylpyridine)AuCl.sub.2 (6.1 mg/7 mL) were prepared as a 2 mM solution in anhydrous DMSO using 0.1 M NBu.sub.4PF.sub.6 (271 mg/7 mL) as the electrolyte. The samples were purged with nitrogen for 15 minutes and recorded. GSH (4.3 mg/7 mL) was prepared as a 2 mM solution in deoxygenated DI H.sub.2O using 0.1 M anhydrous NaClO.sub.4 (85 mg/7 mL) as the electrolyte. Due to poor solubility of GSH in DMSO, the reaction between 2a and GSH was performed in a mix of solvents. Compound 2a (8.8 mg/7 mL) was dissolved in anhydrous DMSO while GSH (4.3 mg/7 mL) was dissolved in deoxygenated DI H.sub.2O. 3.5 mL of each solution were mixed in a 1:1 ration to achieve a final concentration of 1 mM×7 mL. To this was then added 0.1 M NBu.sub.4PF.sub.6 as the electrolyte (271 mg). All data were plotted in GraphPad Prism 6.
Example 26. Reactivity of Gold(III) Dithiocarbamates with Cysteine Thiols
[0211] After establishing the stability of complexes, 1a or 2a in biological buffer and DMEM solutions, the reactivity of 2a with cysteine thiols was investigated using L-glutathione (GSH) and L-N acetyl cysteine as a models (
TABLE-US-00002 TABLE 2 IC.sub.50 values for 1a-e and 2a-e across a panel of cell lines. Cells were seeded at a density of 2,000 cells/well and treated for 72 h. IC.sub.50 values are plotted as the mean ± s.e.m (n = 3). IC.sub.50 (μM), 72 h MDA-MB-175 MDA-MB-231 A2780 RPE-NEO 1a 0.926 ± 0.10 0.531 ± 0.103 0.521 ± 0.081 10.29 ± 2.61 1b 1.26 ± 0.307 0.935 ± 0.16 0.924 ± 0.187 25.4 ± 1.98 1c 1.85 ± 0.194 0.951 ± 0.135 1.19 ± 0.126 12.11 ± 1.59 1d 0.842 ± 0.671 1.14 ± 0.08 0.816 ± 0.123 15.13 ± 2.19 1e 1.12 ± 0.431 1.36 ± 0.04 1.13 ± 0.065 14.8 ± 2.15 2a 0.618 ± 0.080 0.773 ± 0.117 0.741 ± 0.086 17.1 ± 1.86 2b 0.831 ± 0.099 2.11 ± 0.100 0.820 ± 0.073 15.1 ± 1.86 2c 1.031 ± 0.091 1.04 ± 0.101 1.20 ± 0.33 21.1 ± 18.6 2d 0.937 ± 0.399 0.849 ± 0.067 2.962 ± 0.71 14.5 ± 2.45 2e 1.17 ± 0.487 1.31 ± 0.088 0.811 ± 0.892 19.2 ± 2.81
Example 27. Cell Culture
[0212] All cell lines were purchased from ATCC and routinely grown in a humidified incubator at 37° C. with 5-10% CO2. MDA-MB-231, MDA-MB-175, MRCS, RPE-NEO, H460, and K562 were grown in DMEM supplemented with 10% FBS, 1% amphotericin and 1% penicillin/streptomycin. A2780 cells were grown in RPMI supplemented with 10% FBS, 1% amphotericin, and 1% penicillin/streptomycin, and 4 mM glutamine. All supplements along with PBS and trypsin-EDTA were purchased from Corning Inc. and used as is.
Example 28. Cellular Uptake
[0213] Cellular uptake studies were performed to determine what percentage of the compounds were getting into the cell after treatment. High cellular uptake generally corresponds to high efficacy of the drug candidate in hand, since most drug targets are intracellular. For this study, MDA-MB-231 were treated with all 10 compounds and Auranofin at 5 μM for 24 h (
Example 29. Anticancer Activity In Vitro
[0214] All the gold(III) dithiocarbamate compounds display remarkable cell killing potential in a panel of breast, ovarian, lung and leukemia cancers. Preliminary studies focused on different cancer cell lines (MDA-MB-231, MDA-MB-175, and A2780) and a normal retinal epithelial cell line (RPE-NEO). These adherent cells were exposed to a serial-diluted concentration of all ten compounds for 72 h. The cells were subsequently treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solutions, dissolved with DMSO and the absorbance at 570 nm was measured with a plate-reader. An initial screen showed promising half-maximal inhibitory concentration (IC.sub.50) values for all complexes across the three cancer cell lines (Table 2).
[0215] Specifically, the complexes exhibited high toxicity (0.5-1.5 μM) towards the TNBC cell line MDA-MB-231 (
[0216] With the completion of preliminary cytotoxicity screening, the efficacy of compound 2a was evaluated in different cancer tissue type beyond breast cancer. Both H460 (human large cell lung carcinoma) and K562 (human chronic myelogenous leukemia) were utilized (
Example 30. Differential Gene Expression and Biological Pathway Analysis
[0217] The whole-cell effect of compound 2a was evaluated by analyzing differentially expressed genes (DEG) from RNA-seq. MDA-MB-231 cells were treated with 1 μM of 2a for 12 h followed by the isolation of high-purity RNA for Illumina Hi-seq. There were 3019 DEG found with 1596 upregulated and 1423 downregulated genes in response to 2a (
Example 31. Gold(III) Dithiocarbamate Disrupts TNBC Cell Metabolism
[0218] The RNA-seq showed transcriptional suppression of key genes involved in the catalytic conversion of long-chain fatty acids such as ACSL4. Further analysis of DEG revealed the modulation of several genes within the mitochondria respiratory chain or oxidative phosphorylation. Activated DEG included NDUFS7, [complex I], COX7A1 gene [complex IV], and ATP5O, ATP5I genes [complex V] whereas the inhibited DEG were MT-ND4L [complex I], SDHD gene [complex II], and ATP5C, ATP5F [complex V]. Moreover, an uncoupling protein related gene, UCP3 was found to be upregulated in response to 2a. These findings prompted functional biology experiments to further corroborate the effect of 2a or its mechanism of action in TNBC. The effects of 2a on mitochondrial membrane potential were examined, assayed by JC-1 dye..sup.85 Following an extended 2a treatment and JC-1 staining (
Example 32. Bioenergetics
[0219] With the previous biological data pointing towards metabolic changes, the effect 2a had on redox metabolism was explored. The ETC is a complicated biological system that is constantly changing in response to external stress in order to achieve redox homeostasis within the cell. With the DEG data alluding to involvement of ETC genes (
Example 33. Apoptosis Evaluation
[0220] To further assess the mechanistic pathway upon treatment with 2a, the apoptotic effect in MDA-MB-231 was analyzed.
[0221] Apoptosis is a common cell death pathway for chemotherapeutics which can be characterized by distinct morphological features and biochemical mechanisms..sup.88-89 Apoptosis occurs normally in healthy cells to maintain a healthy population of cells during aging or development of tissues. Apoptosis occurs normally in healthy cells to maintain a healthy population of cells during aging or development of tissues. Some transition metal-based drugs can trigger apoptosis due to inhibition of p53 dependant pathway (a tumour suppressor gene)..sup.90-91 Populations of apoptotic cells can be determined by containing cells with Annexin V and PI..sup.92-93 Cells undergoing apoptosis contain ample amount of phosphatidylserine (PS) which can be bound by Annexin V. The Annexin is then labelled with FITC, a green fluorogenic dye which can be visualized by fluorescence activated cell sorting (FACS). PI is used to stain damaged DNA to distinguish apoptotic from necrotic cells. Such staining gives four separate quadrants upon analysis; i) lower left, healthy cells which are negative for both markers, ii) lower right, pre-apoptotic cells which are positive for FITC but not for PI, iii) upper right, apoptotic cells which are positive for both markers, iv) upper left, necrotic cells which are positive for only PI.
Example 34. ROS Analysis
[0222] The status of intracellular ROS induced by 2a was next examined. For this experiment, 2′,7′-dichlorofluoresceindiacetate (DCF-DA),.sup.103 a fluorogenic dye with an excitation/emission wavelength of 495/525 nm was used. DCF-DA enters into the cell and is subsequently deacetylated by cellular esterases where it is then oxidized by ROS to produce a fluorescent compound that can detected with flow cytometry (FACS) using the FITC channel..sup.104
[0223] To quantify the amount of ROS produced, MDA-MB-231 cells were subjected to 2a at 10 and 20 μM to assess concentration dependence. Within 1 hour of treatment, a 1.3× increase in ROS in cells treated with 2a versus DMSO (
Example 35. Cell Cycle Analysis
[0224] The effect of 2a on cell cycle was studied by flow cytometry. There was a time-dependent increase in G0/G1 cell cycle population over 24-72 h period. Analysis of RNA-seq data revealed the down regulation of several cell cycle related genes including cyclin D1 and cyclin dependent kinases (CDK1, CDK4, CCND1) (
Example 36. Cell Viability of 2a+GSH Adduct in MDA-MB-231
[0225] Cells were grown to confluency and added trypsin to collect the cells. The cells were washed with 2 mL of PBS and suspended in 10 mL of DMEM. The cells were centrifuged at 2000 rpm for 5 minutes. The pellet was washed with 2 mL of PBS and suspended in 5 mL of DMEM. The cells were then plated in three separate 96-well clear bottom plate at a density of 2,000 cells/well. The cells were allowed to adhere overnight. The adduct was prepared by taking a 10 mM stock of 2a in DMSO and GSH in DMEM and mixing in a 1:1 ratio to achieve a 5 mM stock of the adduct. The adduct solution was diluted to 100 μM working concentration with DMEM. The adduct was then added at a 3× dilution starting at 50 μM for the highest concentration and the cells incubated for 72 h at 37° C. with 5-10% CO.sub.2. The medium was removed and a solution of MTT (100 μL, prepared by dissolving MTT at 5 mg/mL and diluting by 10× with DMEM) was added to each well and incubated for 4 h at 37° C. with 5-10% CO.sub.2. The dye was removed from each well and 100 μL of DMSO was added to induce cell lysis. The plates were read using a Genios plate reader (λ=570 nm). The experiment was performed in triplicate. Data are plotted as the mean±s.e.m. (n=3) (
Example 37. Cell Viability of 1a-1e and 2a-2e (Adherent Cell Lines)
[0226] The cell viability of all 10 complexes were performed in MDA-MB-231, MDA-MB-175, A2780, and RPE-NEO. The cell viability of compound 2a was also determined in H460. Cells were grown to confluency and trypsin was added to detach and harvest cells. The cells were washed with 2 mL of PBS and suspended in 10 mL of the appropriate media. The cells were centrifuged at 2000 rpm for 5 minutes and the pellet washed with 2 mL of PBS then suspended in 5 mL of the appropriate media. The cells were plated at a density of 2,000 cells/well in a 96-well clear bottom plate and allowed to adhere overnight at 37° C. with 5-10% CO.sub.2. The compounds were prepared as a stock in DMSO and used fresh. The compounds were added at seven concentrations with a 3× serial dilution starting at 50 μM for the highest concentration and incubated at 37° C. for 72 h with 5-10% CO.sub.2. The medium was removed and a solution of MTT (100 μL, prepared by dissolving MTT at 5 mg/mL and diluting by 10× with DMEM) was added to each well and incubated for 4 h at 37° C. with 5-10% CO.sub.2. The dye was removed from each well and 100 μL of DMSO was added to induce cell lysis. The plates were read using a Genios plate reader (λ=570 nm). The experiment was performed in triplicate and data are plotted as the mean±s.e.m. (n=3). Data for 2a can be found in
Example 38. Cell Viability of 1a-1e and 2a-2e (Suspended Cell Lines)
[0227] The cell viability of 2a was determined in K562. Cells were grown to confluency and centrifuged at 2000 rpm for 5 minutes to collect the cell pellet. The cells were washed with 5 mL of PBS, suspended in 5 mL of DMEM, and centrifuged again at 2000 rpm for 5 minutes to collect the pellet. The pellet was then washed with 2 mL of PBS and suspended in 5 mL of DMEM. The cells were plated at density of 2,000 cells/well in a 96-well white bottom plate. 2a was prepared as a stock solution in DMSO and used fresh. The compounds were added at seven concentrations with a 3× serial dilution starting at 50 μM for the highest concentration and incubated at 37° C. for 72 h with 510% CO.sub.2. The cells were removed from the incubator and allowed to rest at room temperature for 30 minutes. To each designated well was then added 20 μL of CellTiter-Glo solution and orbitally shaken for 5 minutes and the luminescence (1000 ms integration and 150 ms gain) acquired on a Genios plate reader. The experiment was performed in triplicate. Data are plotted as the mean±s.e.m. (n=3) as seen in the main text (Table 2 and
Example 39. Apoptosis Analysis
[0228] MDA-MB-231 cells were seeded at a density of 5×10.sup.5 cells/well in a 6 well clear bottom plate with a final media volume of 2 mL. The cells were allowed to adhere overnight at 37° C. A stock of 2a was prepared fresh in DMSO and added to the desired well at a concentration of 1 μM with a final volume of 2.5 mL and incubated for 4 h at 37° C. A stock of H.sub.2O.sub.2 was prepared in PBS and the cells treated at a final concentration of 2 mM for 1 hour as a positive control. When ready for analysis, the media were removed and the wells washed with 5 mL of PBS. The cells were trypsinized (1 mL), 5 mL of DMEM were added to each well, and total volume collected and centrifuged to pellet the cells. The cells were resuspended in 2 mL of fresh media, counted, and reconstituted to a concentration of 1×10.sup.5 cells/mL. The cells were centrifuged again, and the pellet suspended in 500 μL of Annexin binding buffer. To each sample was added 5 μl of Annexin V-FITC and 5 μl PI and incubated in the dark at room temperature for 5 minutes. The samples were then subjected to FACS analysis. Graphs are representative of three technical replicates. Percentages are plotted as the mean±s.e.m. (n=3) as seen in the main text (
Example 40. Immunoblotting
[0229] MDA-MB-231 cells were seeded at density of 5×10.sup.5 cells/well in a 6 well clear bottom plate with a final volume of 2 mL and allowed to adhere overnight at 37° C. Compound 2a was prepared as a stock in DMSO and added to the respective wells at the specified concentrations of 0.1, 1, and 10 μM and treated for 12 h. The cells were washed with PBS (3×3 mL) and were scraped into SDS-PAGE loading buffer (64 mM Tris-HCl (pH 6.8)/9.6% glycerol/2% SDS/5% β-mercaptoethanol/0.01% bromophenol blue) and incubated at 95° C. on a heat block for 10 min. The samples were cooled and stored at −20° C. until ready for use. Whole cell lysates were resolved by 4-20% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE; 100 V for 35 min) followed by electro transfer to a PVDF (350 mA for 1 h). Membranes were blocked using 3% (w/v) bovine serum albumin (BSA) in PBST (PBS/0.1% Tween 20) and incubated with specific primary antibodies (Cell Signaling Technology) overnight at 4° C. On the following day, after washing with PBST (3×5 mL), the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) in freshly prepared BSA blocking solution. Immuno complexes were detected with the ECL detection reagent (BioRad) and analyzed using a BioRad imager with a chemiluminescence filter. Blots can be found in the main text (
Example 41. Whole Cellular Uptake
[0230] MDA-MB-231 cells were seeded at a density of 1×10.sup.6 cells/mL in a 6 well clear bottom plate with a volume of 2.5 mL and allowed to adhere overnight at 37° C. Compounds were prepared as a stock in DMSO and added to each well at a final concentration of 5 μM and treated for 24 h. Auranofin was used as a comparative control and treated at a final concentration of 5 μM and treated for 24 hours as well. The cells were then collected by trypsinization and centrifuged at 2000 rpm for 5 minutes to form a pellet. The pellet was suspended in 1 mL of DMEM, transferred to a 1.5 mL Eppendorf tube, and centrifuged again at 2000 rpm for 5 minutes. The media were removed, washed with PBS twice, and the pellet stored at −20° C. until analysis. Prior to analysis, the pellets were suspended in 0.5 mL of concentrated HCl and agitated for 1 minute. The solution was transferred to a 15 mL Falcon tube and then 4.5 mL of DI H.sub.2O was added. The samples were then subjected to analysis with ICP-OES. Data is represented as the mean±s.e.m. (n=3) as seen in the main text (
Example 42. Differential Gene Expression Using RNA-Sequencing
[0231] MDA-MB-231 cells were seeded on petri dish (100 mm×15 mm) and allowed to grow to 85% confluency. The cells were then treated with 2a at a concentration of 1 μM for 12 h at 37° C. Cells were harvested and 1×10.sup.7 cells were collected. High quality RNA was isolated using RNA Qiagen kit following manufacturer's protocol and subsequently sent to Novogene® for RNA-sequencing and analysis. Prior to analysis samples were required to pass three tests before library construction: 1) nanodrop for RNA purity (OD.sub.260/OD.sub.280), 2) agarose gel electrophoresis for RNA integrity and potential contamination, and 3) Agilent 2100 check RNA integrity. Next, the NEB library was constructed from mRNA enrichment and fragmentation, followed by reverse transcription, second strand cDNA synthesis, end repair, addition of adaptor, and finally amplification with PCR. After library construction, qPCR was used to accurately quantify the library effective concentration (>2 nM), in order to ensure the library quality. Raw reads were removed via the following parameters: 1) remove reads containing adaptors, 2) remove reads containing N>10% (N represents bases that could not be determined), 3) the Qscore (Quality value) of over 50% bases of the read was ≤5. Novogene® then uses STAR to accomplish the mapping reads to the reference genome. Gene expression level is then estimated by the abundance of transcripts (count of sequencing) that mapped to genome or exon where read counts are proportional to gene expression level, gene length and sequencing depth. Samples are then subjected to analysis using Pearson's correlation coefficient and principal component analysis for statistical significance.
Example 43. Mitochondrial Membrane Potential (JC-1)
[0232] MDA-MB-231 cells were plated at a density of 5×10.sup.5 cells/plate using a glass bottom petri dish fitted with a #1.5 cover slip with a final volume of 1.5 mL and allowed to adhere overnight at 37° C. Compound 2a was prepared as a stock in DMSO and added at a final concentration of 10 μM. The cells were treated for 6 h at this concentration. CCCP was prepared as a stock in DMSO and added at a final concentration of 100 μM and the cells treated for 1 h. This was used as a positive control. After the indicated treatment time, a working solution of the JC-1 dye (Cayman Chemicals) was prepared by adding 100 μL of dye into 900 μL of DMEM. Note: the working solution of JC-1 should always be prepared fresh and not stored for long-term use. Then, 100 μL/mL of DMEM were added to the cells and incubated at 37° C. for 20 minutes. Prior to imaging, the media was removed and replaced with room temperature PBS (2 mL). The cells were then visualized using confocal microscopy on a Nikon MR Inverted Confocal Microscope. J-aggregates were imaged with (excitation/emission: 510/590 nm) and J-monomers with (excitation/emission: 488/525 nm). Each image is representative of three technical replicates as seen in the main text (
Example 44. ROS Analysis (DFC-DA)
[0233] MDA-MB-231 cells were seeded at a density of 5×10.sup.5 cells/well in a 6-well clear bottom plate with a final volume of 2 mL and allowed to adhere overnight at 37° C. Compound 2a was prepared as a stock in DMSO and added to the desired wells at a final concentration of both 5 and 10 μM and treated for 1 h. For the positive control, H.sub.2O.sub.2 was used and diluted with PBS and added at a final concentration of 1 mM with a treatment time of 1 h. For the wells that were pre-treated with NAC, a stock solution of NAC in DMEM was added at a concentration of 10 mM for 2 h prior to the addition of 2a. Following treatment, the media were removed, cells were washed with 5 mL of PBS, and collected via trypsinization by centrifuging at 2000 rpm for 5 minutes. The pellet was then suspended in 500 μL of a 10 μM DCF-DA solution in PBS and incubated for 30 minutes at 37° C. in the dark. The cells were then centrifuged again at 2000 rpm for 5 minutes, the pellet washed with PBS (3×1 mL) and suspended in 500 μL of PBS. The cells were then analyzed using FACS with the FITC channel (excitation, 2\, =488 nm). Each data set is representative of three technical replicates as seen in the main text (
Example 45. Mitochondrial Metabolism Analysis with Seahorse XF96 Analysis
[0234] The initial step of Seahorse XF96 analysis included optimization of the cell density. In this stage MDA-MB-231 cells were seeded at a range of densities from 2000 cells/well to 100,000 cells/well, followed by optimization of the FCCP injection concentration used (0.6 μM of 1.2 μM). The optimum conditions were determined to be 30,000 cells/well and an FCCP injection concentration of 0.6 μM. All Seahorse XF96 experiments with MDA-MB-231 were performed under these conditions. The cells were seeded the night prior to the experiment with a final volume of 100 μL and incubated overnight at 37° C. Compound 2a was prepared as a stock in DMSO and diluted to a working concentration of 200 μM with Seahorse XF96 assay buffer and then subsequently serial diluted by 3× to achieve multiple concentrations. The assay was performed using a pneumatic injection method of 2a, with the final injection concentrations of 0.1, 1, 3, and 11 μM. This was followed by injection of oligomycin (1.5 μM), FCCP (0.6 μM) and rotenone/antimycin A (0.5 μM). The metabolic parameters are calculated as seen in the supplementary information of the following papers..sup.15-16 The Seahorse XF96 analysis for MRCS was performed in an identical manner to MDA-MB-231 except that MRCS cells were seeded at a density of 50,000 cells/well and an FCCP injection concentration of 0.6 μM was used.
Example 46. Cell Cycle Analysis
[0235] MDA-MB-231 cells were seeded at a density of 2×10.sup.5 cells/well in a 6 well clear bottom plate with a final media volume of 2 mL and allowed to adhere overnight 37° C. Compound 2a was prepared fresh as a stock in DMSO and added at a final concentration of 0.1 μM with a final volume of 2.5 mL. Cells were treated with 2a for time periods of 24 h, 48 h, and 72 h. After the desired treatment period, the medium was removed and added to a 15 mL Falcon tube. The wells were washed with 5 mL of PBS and added to the Falcon tube. The cells were trypsinized (1 mL) and added 5 mL of fresh DMEM. All media were combined, and the tube centrifuged at 2000 rpm for 5 minutes to collect the pellet. The media were decanted, and the pellet suspended in 1 mL of PBS, which was then transferred to a 1 mL Eppendorf tube, centrifuged at 2000 rpm for 5 minutes and suspended in 70% EtOH/PBS solution. These solutions were stored at 4° C. until ready for analysis. Once all treatments had been collected, the cells were collected by centrifuging at 2000 rpm for 5 minutes. The cells were washed twice with PBS (1 mL) and suspended in a 504 of RNase solution (100 mg/mL) and 2004 of a 50 mg/mL PI solution. The solutions were then filtered through a 5 mL polystyrene round-bottom tube fit with a cell-strainer cap. The samples were then analyzed with FACS. Data are representative of three technical replicates with percentages plotted as the mean±s.e.m. (n=3) as seen in the main text (
Example 47. Mitochondrial Dysfunction and Metabolic Stress Via Loss of OCR
[0236] A metabolic stress test was performed on mitochondria using the Seahorse XF96 and measured the subsequent changes of OCR after injection AuDTC (
[0237] A loss of total OCR was seen across these panel of TNBCs upon treatment with AuDTC in a concentration dependent manner (
[0238] Taken together the data illustrates that the gold agent rapidly perturbs mitochondrial metabolism regardless of tumor type and it is selective for cancer compared to normal cells. Further, the data represent an innate capability of the gold agent to rapidly induce mitochondrial stress through perturbation of OXPHOS.
Example 48. Complex I Mediated Metabolic Stress
[0239] The underlying mechanism of mitochondrial respiration inhibition was evaluated by assessing the effect of 2a on specific mitochondrial complex activity. Mitochondrial metabolism is driven by the transfer of electrons through the ETC. The complex system is made of >100 proteins which form the OXPHOS machinery. The ETC is comprised of 5 complexes; I-IV and complex V which is a subunit known as F.sub.0F.sub.1 ATP synthase which drives respiration through a proton gradient converting ADP to ATP. Several studies have identified potent small molecules for inhibition of these various complexes, therefore any possible specific interaction between the gold agent and complex I were explored.
[0240] The effect of the gold agent on mitochondrial complex I-driven respiration was evaluated using the plasma membrane permeabilizer (XF PMP) assay (
[0241] To measure complex I driven OCR, MDA-MB-468 cells were subjected to pneumatic injection of 2a after the cells had been incubated with MAS buffer containing excess levels of pyruvate (10 mM) added with 1 mM malate to feed the TCA cycle and thus NADH linked respiration. In a concentration-dependent manner, a loss in OCR and respiratory function was observed at 33 μM (
Example 49. Perturbation of Mitochondrial Membrane Potential
[0242] Following the observations that compound 2a decreases cellular respiration, the compound was further evaluated to determine how its exposure to cells affects mitochondrial membrane potential (MMP). Healthy mitochondria are heavily reliant on maintaining a homeostatic balance within the organelle. The REDOX transfer of electrons through ETC protein complexes I-IV in the inner mitochondria membrane provides the energy to drive protons against their concentration gradient across the inner mitochondrial membrane..sup.405-407 Therefore, loss in MMP would subsequently lead to loss in bioenergetic health, which would result in loss of ETC activity.
[0243] The MMP depolarizing effect on the panel of TBNCs and healthy models was examined with the use of flow assisted cell sorting (FACS) (
Example 50. Glycolytic Effect and Metabolic Reprogramming
[0244] Drawing back to the dependency of cancer cells on both aerobic glycolysis and cellular metabolism, fundamental changes in cellular response upon exposure to the gold agent were further studied. Based on the claim that these agents induce loss of mitochondrial respiration and subsequently loss of bioenergetics, the effect of 2a on glycolysis was analyzed, that is, both direct effects and induced effects after exposure.
[0245] The glycolytic rate assay was first performed in MDA-MB-231, a classic TNBC cell line which has a higher dependency on glycolysis than OXPHOS for tumorigenesis (
[0246] This injection is then followed by 2-deoxyglucose (2-DG) to block glycolytic acidification and confirm pathway specificity. This decrease then provides confirmation that the PER produced prior to the injection is due to glycolysis. Upon injection with the gold compound, no change was observed in the basal rate of PER (
[0247] The mitoOCR/glycoPER rate was compared (
[0248] To characterize cellular energy demands upon exposure to the gold agent AuDTC, the Seahorse XF analyzer was used to investigate ATP production rates linked to both glycolysis and mitochondrial metabolism in MDA-MB-468 (
[0249] Quantifying these real-time ATP rate results shows a >10-fold induced rate of glycoATP coupled with loss of mitoATP, illustrating a metabolic shift towards glycolysis; a result which is indicative of a faulty ETC (
[0250] Taken together, the glycolytic rate assay and ATP rate assay confirm in two independent manners that exposure to the gold agent induces a metabolic shift from mitochondrial metabolism to a glycolytic pathway (
Example 51. Induced Oxidative Stress and Damage
[0251] Reactive oxygen species are an integral part in maintaining cell homeostasis and are involved in a variety of cell signaling pathways. Innately, cells do require minimal amounts of ROS for proper function, however; increased amounts of ROS cause deleterious effects on cell proliferation and can ultimately lead to programmed cell death. With preceding data pointing to a loss of mitochondrial membrane potential and loss of cellular respiration, the effect of compound 2a was analyzed in the context of ROS production. First, the amount of ROS was analyzed by using DCF-DA, a fluorogenic dye, which is cleaved by intracellular esterases and subsequently oxidized by ROS species into a fluorescent molecule (
[0252] The treatment times adopted were to mimic the pneumatic injection method used when analyzing metabolic stress. However, the production of mtROS was evaluated in a time-dependent fashion, showing that at even a lower concentration of 1 μM there was initial increase in mtROS, followed by a steady increase with prolonged exposure (up to 24 hours) (
[0253] Although ROS is required for certain cell signaling pathways, the rapid increase will have harmful effects on mitochondrial function and overall cell health. These ROS species can directly interact with cellular components and induce deleterious effects including DNA damage, lipid peroxidation, and protein modifications. To analyze these downstream effects, the impact on γH2AX activation was assessed, which results from phosphorylation of the Ser-139 residue of the histone variant H2AX, in response to DNA damage. Given that these gold complexes are known to not directly interact with DNA and arrest cell cycle in the G1 phase, an increase of γH2AX was attributed to an increase in cellular ROS levels. With the use of immunocytochemistry, a persistent and significant increase in γH2AX levels was observed at both 12 hour and 24 hour time points upon exposure to the gold agent (5 μM) (
[0254] Images were taken using a 60× oil objective to visualize individual γH2AX foci within the nuclei. The cells were counterstained with Hoechst stain to show the overlay of γH2AX foci within the stained DNA. With the use of the Nikon-Elements software, a random section of nuclei (>10) were chosen for analysis and quantified. Prolonged exposure to the gold agent illustrated a significant increase between the 12-hour (18.5±7.5 foci/nucleus) and 24-hour (33.2±9.2 foci/nucleus) time points compared to the control (4.4±2.4 foci/nucleus) (
[0255] Next, oxidative stress was analyzed at the protein level by analyzing the loss of mTOR, a key regulatory protein in maintaining cell homeostasis and proliferation. Researchers have reported that loss of mTORC1 can be attributed to high levels of ROS and that ROS activates cytoplasmic ATM and AMPK, which both inhibit mTORC1. Therefore, it is worth investigating if exposure to the gold agent results in loss of this key regulatory protein. Using both confocal microscopy and immunoblotting, exposure to the gold agent was shown to result in a loss of mTOR. Confocal microscopy visually illustrated that MDA-MB-468 treated with the gold agent for 12 hours at 1 μM resulted in loss of mTOR (loss in red fluorescence) expression (
Example 52. Mitochondrial Specific Cytochrome C Release
[0256] Cytochrome c (Cyt c) is a prominent protein involved in cell regulation including the apoptotic pathway. Located in the mitochondrial intermembrane space, its classical function is to transfer electrons from the cytochrome c complex to cytochrome c oxidase which is located on the surface of the inner mitochondrial membrane. In recent years, researchers have found that cytochrome c can be released from the mitochondria upon exposure to apoptotic stimuli: depolarization of the MMP, ROS, and subsequent oxidative damage. Once released, cytochrome c can signal for caspase dependent apoptosis to promote cell death. Work by Korsmeyer gave insights into mechanistic implications of cytochrome c release which illustrate a BCL-2 regulated release. Further mechanistic insights show that opening of the mitochondrial permeability transition pore results in swelling of the mitochondrial matrix, rupture of the outer membrane, which then results in the release of the intermembrane components.
[0257] It is also reasonable to suggest that loss in ETC function and stability would result in release of this protein as well. Given the findings, the release of mitochondrial specific cytochrome c was investigated in a time dependent manner. MDA-MB-468 cells were permeabilized with digitonin, a detergent which permeabilizes the plasma membrane, keeping healthy mitochondria intact. By doing so, cytosolic cytochrome c is released and therefore only mitochondrial related cytochrome c will be measured with immunohistochemistry. When MDA-MB-468 cells are exposed to 2a at 1 μM, a 9% release of mitochondrial cytochrome c was observed within 4 hours (
Example 53. MitoTEMPO Rescues Oxidative and Bioenergetic Stress
[0258] Characterization of chemotherapeutic effects in TNBCs points to widespread bioenergetics stress and oxidative damage. The effect on pre-treating these cells with MitoTEMPO, a mitochondrial targeting radical scavenger, as an antioxidant was analyzed next to see if these deleterious effects could be alleviated upon treatment with the gold agent 2a. Given the increased levels of mtROS and loss of ETC efficiency, MitoTEMPO was selected to alleviate mitochondrial based oxidative stress. Generally, TNBCs were pretreated with MitoTEMPO for 2 hours followed by treatment with the gold agent to see if oxidative stress could be mitigated.
[0259] First, the effect of pre-treatment on mtROS production was examined using flow cytometry. Since MitoTEMPO is a specific scavenger for superoxide, the potential to alleviate mitochondrial oxidative damage was investigated by scavenging mtROS once produced. Pretreatment with MitoTEMPO resulted in 1.8-fold decrease in mtROS versus the non-pretreated group (
[0260] Next, the effect of MMP was examined when pretreated with MitoTEMPO (1 μM, 2 hours) (
[0261] The extent to which apoptosis was rescued when pre-treated with MitoTEMPO was analyzed (
[0262] Finally, the effect pretreatment with MitoTEMPO had on total bioenergetic stress was analyzed. To do this, plated MDA-MB-468 cells were subjected with 2 hours of with MitoTEMPO pretreatment (1 μM, 2 hours). ADTC was pneumatically injected at higher concentrations to see if there was a pronounced effect in rescuing bioenergetic stress. Overall, MitoTEMPO was able to alleviate significant loss in OCR in comparison to the non-pretreated group (
Example 54. Synthesis of dichloro(2-benzoylpyridine) gold(III)-alkyne ([C{circumflex over ( )}N]-alkyne)
[0263] [2-benzoylpyridine]Au(III)Cl.sub.2 (100 mg, 0.22 mmol) and o-2-propynylhydroxylamine hydrochloride (40 mg, 0.42 mmol) were suspended in DCM/MeOH (1:1) at a total volume of 6 mL and stirred at room temperature for 48 hours. The resulting mixture was transferred into a centrifuge tube and centrifuged briefly to separate the precipitate from the filtrate. The filtrate was removed by decanting and the precipitate was washed by resuspending it in equal volume of DCM/MeOH (1:1), centrifuged, and the filtrate decanted. The wash step was carefully done three times after which the precipitate was finally dissolved in DCM and dried under vacuum (40° C.) to afford an off-white, solid. Yield (46%, 52 mg). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=9.34 (d, J=6.3 Hz, 1H), 8.47 (td, J=7.8, 1.5 Hz, 1H), 8.32-8.27 (m, 1H), 7.97-7.89 (m, 1H), 7.54 (d, J=9.0 Hz, 1H), 7.41-7.27 (m, 3H), 5.02 (d, J=2.4 Hz, 2H), 3.67 (t, J=2.4 Hz, 1H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ=153.25, 143.68, 137.88, 133.77, 130.45, 129.16, 128.91, 128.23, 79.59, 79.37, 63.72.
Example 55. Synthesis of 4-alkyne
[0264] A suspension of [C{circumflex over ( )}N]-alkyne (10 mg, 0.02 mmol) in MeOH (5 mL) was added dropwise a solution of sodium dimethyldithiocarbamate (4.1 mg, 0.024 mmol) in 2 mL of MeOH. The suspension slowly turned to a yellow, clear solution and was allowed to mix for 12 hours at room temperature. A saturated solution of NaPF.sub.6 in DI H.sub.2O was prepared and added to the solution to precipitate a beige powder, which was vacuum filtered, washed with excess water and ether, and left to dry in air. Yield (79%, 10 mg). .sup.1H NMR (400 MHz, MeCN-d.sub.3) δ=8.87 (d, J=4 Hz, 1H), 8.32 (t, J=8 Hz, 2H), 7.69 (q, J=10 Hz, 1H), 7.59 (d, J=8 Hz, 1H), 7.41 (q, J=9.0 Hz, 2H), 7.27 (d, J=8 Hz, 1H), 4.78 (s, 2H), 3.36 (d, J=4 Hz, 6H), 2.58 (t, J=10 Hz, 1H). .sup.13C NMR (101 MHz, MeCN-d.sub.3) δ=153.25, 143.68, 137.88, 133.77, 130.45, 129.16, 128.91, 128.23, 79.59, 79.37, 63.72. .sup.19F NMR (376 MHz, MeCN-d.sub.3) δ=−71.8, −73.2. .sup.31P NMR (162 MHz, MeCN-d.sub.3) δ=−130.85, −135.24, −139.63, −144.02, −148.41, −157.20.
Example 56. In Vitro Click Chemistry and Colocalization
[0265] To further elucidate the targeting modality of 2a, a synthetic probe was developed with a free alkyne attached to be used as a clickable probe. The 2-benzoylpyridine framework of the cyclometalated gold was chemically transformed by reacting with o-2-propynylhydroxylamine hydrochloride to form an alkoxy imine with an alkyne handle. Replacement of the labile chloride ligands with the sodium salt of dimethyldithiocarbamate provided an alkyne modified version of 2a (4-alkyne) (
[0266] With this new tool in hand, in vitro click chemistry was performed with an azide fluorophore and counterstain with MitoTracker Red CM-H.sub.2XRos (MTR) to see the correlation between the two dyes. For this study, Alexa488-azide was used as the click counterpart and MitoTracker Red CM-H.sub.2XRos for mitochondrial tracking along with Hoechst as a nuclear counterstain (
[0267] Colocalization studies revealed that a large amount of 4-alkyne was colocalized with the red fluorescence from the MTR dye (
[0268] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:
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[0397] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.