Mitochondrially Targeted N-Acetylcysteine ​​and Analogs

Abstract

The present disclosure provides mitochondria-targeted N-acetylcysteine ​​compounds and pharmaceutical compositions thereof. The compounds may be useful for treating cancer and enhancing CAR-T cell therapy. The compounds may be isotopically labeled, which may be useful for sample labeling and analysis. The compounds target mitochondria in cancer cells and are expected to improve antitumor efficacy and reduce toxicity.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 63 / 413,461, filed October 5, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] [Statement Regarding Federal Research or Development] Not applicable. [Background technology]

[0003] N-acetylcysteine ​​(NAC) has been used as an antioxidant in tumor cells and preclinical mouse tumor xenografts and has been reported to improve adaptive immunotherapy in melanoma. However, NAC has poor bioavailability and is used at high concentrations.

[0004] There remains a need for compounds that are effective in inhibiting cancer cell proliferation and have improved properties, including increased potency and reduced cytotoxicity. Summary of the Invention

[0005] Disclosed herein are modified N-acetylcysteine ​​(NAC) compounds, pharmaceutical compositions containing the compounds, kits, and methods of their use.

[0006] In one aspect, the disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof: JPEG2025535717000002.jpg33163(I) where: R 1 is H, C1-C4 alkyl, or JPEG2025535717000003.jpg46164, R 2 is H or C1-C4 alkyl, W is NH, O, or S; L is C1-C 20Alkylene, C2-C 20 Alkenylene, L1-R A -L2, or an amino acid, L1 and L2 are each independently absent or C1-C 10 is alkylene, R A Ha-(CH2CH2O) q -, arylene, or cycloalkylene; q is 1-20, X is a counterion, each Y is independently CF, Me, Cl, OMe, C(O)CH, NO, N(Me), or OH; m in each occurrence is independently 0, 1, 2, 3, 4, or 5, with the proviso that the compound is not (R)-2-acetamido-3-mercapto-N-methylpropanamide or (R)-2-acetamido-3-mercapto-N-ethylpropanamide.

[0007] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, is isotopically labeled. For example, the compound is 13 It can be labeled with C.

[0008] In another aspect, the present disclosure provides a pharmaceutical composition. The pharmaceutical composition comprises a compound according to any one of the methods disclosed herein. The compound of formula (I) or (II) above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0009] In a further aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

[0010] In another aspect, the present disclosure provides a method for enhancing CAT-T cell therapy in a subject in need of treatment for cancer, the method comprising administering to the subject an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

[0011] In another aspect, the present disclosure provides a method of analyzing a sample, the method comprising: 13 C-labeled compound) or a pharmaceutically acceptable salt thereof, thereby producing a labeled sample, and analyzing the labeled sample.

[0012] In yet another aspect, the present disclosure provides a kit, which includes a pharmaceutical composition described herein and instructions. [Brief explanation of the drawings]

[0013] [Figure 1A] Figure 1A shows the structures of NAC and Mito10-NAC analogs. [Figure 1B-1] Figure 1B-1 shows the calculated partition coefficients and relative hydrophobic regions for NAC and Mito10-NAC analogs. [Figure 1B-2] Figure 1B-2 shows the calculated partition coefficients and relative hydrophobic regions for NAC and Mito10-NAC analogs. [Figure 2A] Figures 2A-2B show the effects of NAC and Mito10-NAC on the proliferation of cells derived from various cancers. Figure 2A shows the effects of NAC and Mito10-NAC on MiaPaCa-2 cell proliferation monitored using an IncuCyte live cell analysis system. The IncuCyte analyzer provides real-time updates on cell confluence based on segmentation of high-resolution phase-contrast images. Representative cell images were displayed as a brown segmentation mask at the time when control cells reached 90% confluence (vertical black line). [Figure 2B]Figures 2A-2B show the effects of NAC and Mito10-NAC on the proliferation of cells derived from various cancers. Figure 2B shows that the same growth monitoring method was used for all cell lines, as indicated. IC50 values ​​were measured when control cells reached approximately 90% confluence. Relative cell confluence (control = 100%) is plotted against concentration. Dashed lines represent the fitting curves used to determine IC50 values, as indicated. Data shown are mean ± SD, n = 4. [Figure 3A] Figure 3A shows a comparison of the cell growth inhibitory effects of mitochondrial-targeted drugs and their parent compounds in human pancreatic cancer (MiaPaCa-2) cells. The effects of mitochondrial-targeted drugs and their parent compounds on MiaPaCa-2 cell proliferation were monitored using an IncuCyte live cell analysis system. IC50 values ​​were measured when control cells reached approximately 90% confluency. Relative cell confluency (control defined as 100%) is plotted against the concentration. The dashed lines represent the fitting curves used to determine the IC50 values ​​shown in the figure. The differences shown in the figure were calculated based on the potency differences in IC50 values ​​between each mitochondrial-targeted drug and its parent compound. [Figure 3B-1] Figure 3B shows a comparison of IC50 values ​​between Mito-Met and metformin, Mito-LND and LND, Mito-HNK and HNK, and Mito-ATO and ATO. [Figure 3B-2] Figure 3B shows a comparison of IC50 values ​​between Mito-Met and metformin, Mito-LND and LND, Mito-HNK and HNK, and Mito-ATO and ATO. [Figure 4A-1]Figures 4A-4B show the effects of Mito10-NAC analogs on the proliferation of human pancreatic cancer (MiaPaCa-2) cells. Figure 4A shows the effects of Mito10-NAC-SMe, Mito10-PEG-NAC, NAC-SMe, and NAC amide on MiaPaCa-2 cell proliferation using an IncuCyte live-cell analysis system. The IncuCyte analyzer provides real-time updates on cell confluence based on segmentation of high-resolution phase-contrast images. When control cells reached 90% confluence (vertical solid black line), a representative cell image was displayed as a segmentation mask, shown in brown. [Figure 4A-2] Figures 4A-4B show the effects of Mito10-NAC analogs on the proliferation of human pancreatic cancer (MiaPaCa-2) cells. Figure 4A shows the effects of Mito10-NAC-SMe, Mito10-PEG-NAC, NAC-SMe, and NAC amide on MiaPaCa-2 cell proliferation using an IncuCyte live-cell analysis system. The IncuCyte analyzer provides real-time updates on cell confluence based on segmentation of high-resolution phase-contrast images. When control cells reached 90% confluence (vertical solid black line), a representative cell image was displayed as a segmentation mask, shown in brown. [Figure 4B] Figures 4A-4B show the effects of Mito10-NAC analogs on the proliferation of human pancreatic cancer (MiaPaCa-2) cells. Figure 4B shows the IC50 values ​​measured when control cells reached approximately 90% cell density. The relative cell density, defined as 100% for control cells, is plotted against the concentration. The dashed line indicates the fitting curve used to determine the IC50 values. Data shown are the mean ± SD, n = 4. [Figure 5A]Figures 5A and 5B show the effects of NAC and Mito10-NAC on intracellular ATP levels and cell death in human pancreatic cancer (MiaPaCa-2) cells. Figure 5A shows the effects of NAC and Mito10-NAC on intracellular ATP levels. MiaPaCa-2 cells were treated with NAC or Mito10-NAC for 24 hours, and the concentration-dependent inhibition of intracellular ATP levels was measured. [Figure 5B-1] Figures 5A-5B show the effects of NAC and Mito10-NAC on intracellular ATP levels and cell death in human pancreatic cancer (MiaPaCa-2) cells. Figure 5B shows a SYTOX Green assay monitoring the cytotoxicity of NAC and Mito10-NAC in MiaPaCa-2 cells. MiaPaCa-2 cells were treated with NAC and Mito10-NAC at the indicated concentrations (IC50 values ​​in Figures 6A-6B) for 24 and 48 hours. Cell death, which exhibited strong green fluorescence intensity, was monitored by SYTOX Green staining using an IncuCyte live cell analysis system. Corresponding representative fluorescence images are shown in the left (24 hour) and right (48 hour) panels. Data are shown as mean ± SD, n = 4. [Figure 5B-2] Figures 5A-5B show the effects of NAC and Mito10-NAC on intracellular ATP levels and cell death in human pancreatic cancer (MiaPaCa-2) cells. Figure 5B shows a SYTOX Green assay monitoring the cytotoxicity of NAC and Mito10-NAC in MiaPaCa-2 cells. MiaPaCa-2 cells were treated with NAC and Mito10-NAC at the indicated concentrations (IC50 values ​​in Figures 6A-6B) for 24 and 48 hours. Cell death, which exhibited strong green fluorescence intensity, was monitored by SYTOX Green staining using an IncuCyte live cell analysis system. Corresponding representative fluorescence images are shown in the left (24 hour) and right (48 hour) panels. Data are shown as mean ± SD, n = 4. [Figure 6A-1]Figures 6A-6B show the effects of NAC and Mito10-NAC on mitochondrial oxygen consumption by healthy cells or mitochondrial complex I in human pancreatic cancer (MiaPaCa-2) cells. Figure 6A shows the effects of NAC and Mito10-NAC on mitochondrial oxygen consumption by healthy cells. MiaPaCa-2 cells were treated with NAC or Mito10-NAC for 24 hours, and concentration-dependent inhibition of mitochondrial respiration (OCR) was measured in healthy MiaPaCa-2 cells. After eight baseline OCR measurements, responses to mitochondrial modulators (oligonucleotides, DNP, and rotenone / antimycin A, as described in Methods) were recorded. *p<0.05, **p<0.01 compared to the control group at the final baseline measurement. Data are shown as mean ± SD, n=4. [Figure 6A-2] Figures 6A-6B show the effects of NAC and Mito10-NAC on mitochondrial oxygen consumption by healthy cells or mitochondrial complex I in human pancreatic cancer (MiaPaCa-2) cells. Figure 6A shows the effects of NAC and Mito10-NAC on mitochondrial oxygen consumption by healthy cells. MiaPaCa-2 cells were treated with NAC or Mito10-NAC for 24 hours, and concentration-dependent inhibition of mitochondrial respiration (OCR) was measured in healthy MiaPaCa-2 cells. After eight baseline OCR measurements, responses to mitochondrial modulators (oligonucleotides, DNP, and rotenone / antimycin A, as described in Methods) were recorded. *p<0.05, **p<0.01 compared to the control group at the final baseline measurement. Data are shown as mean ± SD, n=4. [Figure 6B-1]Figures 6A-6B show the effects of NAC and Mito10-NAC on mitochondrial oxygen consumption by healthy cells or mitochondrial complex I in human pancreatic cancer (MiaPaCa-2) cells. Figure 6B shows the effects of NAC and Mito10-NAC on mitochondrial complex I oxygen consumption. The dose-dependent effects of NAC or Mito10-NAC were measured by direct injection of NAC or Mito10-NAC into permeabilized MiaPaCa-2 cells. Mitochondrial complex I activity was monitored using a Seahorse XF-96 extracellular flux analyzer. Rotenone (a complex I inhibitor) was then rapidly added. Mitochondrial complex I-dependent oxygen consumption was expressed and calculated as rotenone-inhibitable OCR. Mitochondrial basal OCR (A, bottom) or mitochondrial complex I-dependent OCR (B, bottom) was plotted against treatment concentration. The dashed line indicates the fitting curve used to determine IC50 values. Data shown are mean ± SD (n = 4). [Figure 6B-2] Figures 6A-6B show the effects of NAC and Mito10-NAC on mitochondrial oxygen consumption by healthy cells or mitochondrial complex I in human pancreatic cancer (MiaPaCa-2) cells. Figure 6B shows the effects of NAC and Mito10-NAC on mitochondrial complex I oxygen consumption. The dose-dependent effects of NAC or Mito10-NAC were measured by direct injection of NAC or Mito10-NAC into permeabilized MiaPaCa-2 cells. Mitochondrial complex I activity was monitored using a Seahorse XF-96 extracellular flux analyzer. Rotenone (a complex I inhibitor) was then rapidly added. Mitochondrial complex I-dependent oxygen consumption was expressed and calculated as rotenone-inhibitable OCR. Mitochondrial basal OCR (A, bottom) or mitochondrial complex I-dependent OCR (B, bottom) was plotted against treatment concentration. The dashed line indicates the fitting curve used to determine IC50 values. Data shown are mean ± SD (n = 4). [Figure 7A]Figures 7A-7F show the cell growth inhibitory effects of Mito10-NAC in combination with NAC or AZD3965 in human pancreatic cancer (MiaPaCa-2) cells. MiaPaCa-2 cells were treated with Mito10-NAC (as indicated) alone or in combination with NAC (Figures 7A and 7C) or AZD3965 (Figures 7B and 7D), and cell growth was continuously monitored. Data shown are mean ± SD (n = 5). Representative cell images are shown as segmentation masks, indicated in brown when control cells reached approximately 90% confluency (vertical dashed line). **p < 0.01 compared to control. ##p < 0.01 compared to single compounds. (Figures 7E and 7F) Cell confluency (when control cells reached approximately 90% confluency) is plotted against concentration to calculate synergistic effects. Panel B shows the combination index-percent effect plot. The percent effect parameter was used as a measure of drug efficiency, with a value of 0 indicating complete inhibition of cell confluency and a value of 1 indicating no effect on cell confluency. The Mito-NAC concentration ranges used to calculate confidence intervals were 2, 4, 6, 8, 10, 12.5, 15, 25, and 50 µM. Data shown are mean ± SD, n = 4. [Figure 7B]Figures 7A-7F show the cell growth inhibitory effects of Mito10-NAC in combination with NAC or AZD3965 in human pancreatic cancer (MiaPaCa-2) cells. MiaPaCa-2 cells were treated with Mito10-NAC (as indicated) alone or in combination with NAC (Figures 7A and 7C) or AZD3965 (Figures 7B and 7D), and cell growth was continuously monitored. Data shown are mean ± SD (n = 5). Representative cell images are shown as segmentation masks, indicated in brown when control cells reached approximately 90% confluency (vertical dashed line). **p < 0.01 compared to control. ##p < 0.01 compared to single compounds. (Figures 7E and 7F) Cell confluency (when control cells reached approximately 90% confluency) is plotted against concentration to calculate synergistic effects. Panel B shows the combination index-percent effect plot. The percent effect parameter was used as a measure of drug efficiency, with a value of 0 indicating complete inhibition of cell confluency and a value of 1 indicating no effect on cell confluency. The Mito-NAC concentration ranges used to calculate confidence intervals were 2, 4, 6, 8, 10, 12.5, 15, 25, and 50 µM. Data shown are mean ± SD, n = 4. [Figure 7C]Figures 7A-7F show the cell growth inhibitory effects of Mito10-NAC in combination with NAC or AZD3965 in human pancreatic cancer (MiaPaCa-2) cells. MiaPaCa-2 cells were treated with Mito10-NAC (as indicated) alone or in combination with NAC (Figures 7A and 7C) or AZD3965 (Figures 7B and 7D), and cell growth was continuously monitored. Data shown are mean ± SD (n = 5). Representative cell images are shown as segmentation masks, indicated in brown when control cells reached approximately 90% confluency (vertical dashed line). **p < 0.01 compared to control. ##p < 0.01 compared to single compounds. (Figures 7E and 7F) Cell confluency (when control cells reached approximately 90% confluency) is plotted against concentration to calculate synergistic effects. Panel B shows the combination index-percent effect plot. The percent effect parameter was used as a measure of drug efficiency, with a value of 0 indicating complete inhibition of cell confluency and a value of 1 indicating no effect on cell confluency. The Mito-NAC concentration ranges used to calculate confidence intervals were 2, 4, 6, 8, 10, 12.5, 15, 25, and 50 µM. Data shown are mean ± SD, n = 4. [Figure 7D]Figures 7A-7F show the cell growth inhibitory effects of Mito10-NAC in combination with NAC or AZD3965 in human pancreatic cancer (MiaPaCa-2) cells. MiaPaCa-2 cells were treated with Mito10-NAC (as indicated) alone or in combination with NAC (Figures 7A and 7C) or AZD3965 (Figures 7B and 7D), and cell growth was continuously monitored. Data shown are mean ± SD (n = 5). Representative cell images are shown as segmentation masks, indicated in brown when control cells reached approximately 90% confluency (vertical dashed line). **p < 0.01 compared to control. ##p < 0.01 compared to single compounds. (Figures 7E and 7F) Cell confluency (when control cells reached approximately 90% confluency) is plotted against concentration to calculate synergistic effects. Panel B shows the combination index-percent effect plot. The percent effect parameter was used as a measure of drug efficiency, with a value of 0 indicating complete inhibition of cell confluency and a value of 1 indicating no effect on cell confluency. The Mito-NAC concentration ranges used to calculate confidence intervals were 2, 4, 6, 8, 10, 12.5, 15, 25, and 50 µM. Data shown are mean ± SD, n = 4. [Figure 7E]Figures 7A-7F show the cell growth inhibitory effects of Mito10-NAC in combination with NAC or AZD3965 in human pancreatic cancer (MiaPaCa-2) cells. MiaPaCa-2 cells were treated with Mito10-NAC (as indicated) alone or in combination with NAC (Figures 7A and 7C) or AZD3965 (Figures 7B and 7D), and cell growth was continuously monitored. Data shown are mean ± SD (n = 5). Representative cell images are shown as segmentation masks, indicated in brown when control cells reached approximately 90% confluency (vertical dashed line). **p < 0.01 compared to control. ##p < 0.01 compared to single compounds. (Figures 7E and 7F) Cell confluency (when control cells reached approximately 90% confluency) is plotted against concentration to calculate synergistic effects. Panel B shows the combination index-percent effect plot. The percent effect parameter was used as a measure of drug efficiency, with a value of 0 indicating complete inhibition of cell confluency and a value of 1 indicating no effect on cell confluency. The Mito-NAC concentration ranges used to calculate confidence intervals were 2, 4, 6, 8, 10, 12.5, 15, 25, and 50 µM. Data shown are mean ± SD, n = 4. [Figure 7F]Figures 7A-7F show the cell growth inhibitory effects of Mito10-NAC in combination with NAC or AZD3965 in human pancreatic cancer (MiaPaCa-2) cells. MiaPaCa-2 cells were treated with Mito10-NAC (as indicated) alone or in combination with NAC (Figures 7A and 7C) or AZD3965 (Figures 7B and 7D), and cell growth was continuously monitored. Data shown are mean ± SD (n = 5). Representative cell images are shown as segmentation masks, indicated in brown when control cells reached approximately 90% confluency (vertical dashed line). **p < 0.01 compared to control. ##p < 0.01 compared to single compounds. (Figures 7E and 7F) Cell confluency (when control cells reached approximately 90% confluency) is plotted against concentration to calculate synergistic effects. Panel B shows the combination index-percent effect plot. The percent effect parameter was used as a measure of drug efficiency, with a value of 0 indicating complete inhibition of cell confluency and a value of 1 indicating no effect on cell confluency. The Mito-NAC concentration ranges used to calculate confidence intervals were 2, 4, 6, 8, 10, 12.5, 15, 25, and 50 µM. Data shown are mean ± SD, n = 4. [Figure 8A-1] Figures 8A-8B show the effects of NAC and Mito10-NAC on melanoma cancer (UACC-62) cell proliferation. Figure 8A shows the effects of NAC and Mito10-NAC on UACC-62 cell proliferation monitored using an IncuCyte live-cell analysis system. The IncuCyte analyzer provides real-time updates of cell confluency based on segmentation of high-resolution phase-contrast images. Representative cell images are displayed as brown segmentation masks at the time when control cells reached 90% confluency (vertical black line). [Figure 8A-2]Figures 8A-8B show the effects of NAC and Mito10-NAC on melanoma cancer (UACC-62) cell proliferation. Figure 8A shows the effects of NAC and Mito10-NAC on UACC-62 cell proliferation monitored using an IncuCyte live-cell analysis system. The IncuCyte analyzer provides real-time updates of cell confluency based on segmentation of high-resolution phase-contrast images. Representative cell images are displayed as brown segmentation masks at the time when control cells reached 90% confluency (vertical black line). [Figure 8B] Figures 8A-8B show the effects of NAC and Mito10-NAC on the proliferation of melanoma cancer (UACC-62) cells. Figure 8B shows IC50 values ​​measured when control cells reached approximately 90% confluency. Relative cell confluency (control cells defined as 100%) is plotted against concentration. Dashed lines represent fitting curves used to determine IC50 values, as indicated. Data shown are means ± SD. [Figure 9] Figure 9 shows the effect of Mito-NAC on the proliferation of pancreatic cancer (MiaPaCa-2) cells under different treatment schedules. The effect of Mito-NAC on MiaPaCa-2 cell proliferation was monitored using an IncuCyte live cell analysis system. MiaPaCa-2 cells were treated with Mito-NAC once at the start of the experiment or received new treatments every 48 hours as indicated. Representative cell images at the time when control cells reached 90% confluence (vertical black line) are shown as segmentation masks in brown. No significant differences were observed between the two treatment schedules at the same concentration. Data shown are mean ± SD, n = 4. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Detailed Description of the Invention] Before describing the materials and methods of the present invention, it is to be understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0015] As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, the term "a compound" should be interpreted as meaning "one or more compounds" unless the context clearly dictates otherwise. As used herein, the term "plurality" means "two or more."

[0016] As used herein, the terms "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If there are uses that are not clear to persons of ordinary skill in the art given the context in which the term is used, "about" and "approximately" will mean up to plus or minus 10% of the particular term, and "substantially" and "significantly" will mean more than plus or minus 10% of the particular term.

[0017] As used herein, the terms "comprise" and "comprising" have the same meaning as the terms "comprise" and "comprising." The terms "comprise" and "comprising" should be interpreted as "open" transitional terms that allow for the inclusion of additional components in addition to those recited in the claims. The terms "consisting of" and "consisting of" should be interpreted as "closed" transitional terms that do not allow for the inclusion of additional components other than those recited in the claims. The term "consisting essentially of" is partially closed and should be interpreted as allowing for the inclusion of only additional components that do not fundamentally alter the nature of the claimed subject matter.

[0018] The definitions of specific functional groups and chemical terms are explained in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS edition, Handbook of Chemistry and Physics, 75th Ed., inside cover), and specific functional groups are generally defined as described therein. Furthermore, the general principles of organic chemistry, as well as specific functional groups and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987, the entire contents of which are incorporated herein by reference.

[0019] As used herein, the term "alkyl" means a straight or branched chain saturated hydrocarbon. Alkyl is C 1-4 It may be alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0020] The term "alkylene," as used herein, means a divalent group derived from a straight-chain or branched-chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, -CH-, -CHCH-, -CHCHCH-, -CHCH(CH)CH-, and CHCH(CH)CH(CH)CH-.

[0021] As used herein, the term "alkene" refers to an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond, e.g., a straight-chain or branched group having 2 to 12, 2 to 10, or 2 to 6 carbon atoms, and is defined herein as C2 to C6, respectively. 12 -Alkenyl, C2-C 10 -alkenyl, and C2-C6-alkenyl.

[0022] The term "alkenylene," as used herein, means a divalent group derived from a straight or branched chain alkene and attached to two different carbon atoms of the parent molecule.

[0023] As used herein, the term "aryl" refers to a carbocyclic aromatic group (e.g., phenyl or bicyclic aryl). The term "aryl" includes polycyclic ring systems having one or more carbon rings, where two or more carbons are common to two adjacent rings (these rings are "fused rings"), where at least one of the rings is aromatic and the other ring may be, for example, a cycloalkyl or cycloalkenyl. For example, a bicyclic aryl may be a phenyl fused to a cycloalkyl moiety. Examples of aryls include naphthyl, dihydronaphthalenyl, tetrahydronaphthalene, indanyl, or indenyl. Aryls (e.g., phenyl and bicyclic aryls) are attached to the parent molecular moiety through any carbon atom contained within the aryl.

[0024] The term "arylene," as used herein, refers to a divalent group derived from an aryl, as described herein, that is bonded to two different ring carbon atoms of a parent molecule. Examples of arylene include, but are not limited to, phenylene, a divalent group derived from benzene that is bonded to two different ring carbon atoms of a parent molecule (e.g., 1,2-positions, 1,3-positions, or 1,4-positions).

[0025] The term "cycloalkyl" as used herein refers to a monovalent group derived from an all-carbocyclic ring system containing zero heteroatoms as ring atoms and zero double bonds. The all-carbocyclic ring system can be a monocyclic, bicyclic, or tricyclic ring system, and can be a fused, bridged, or spirocyclic ring system, or combinations thereof. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. Examples include, but are not limited to, JPEG2025535717000004.jpg18163. The cycloalkyl groups described herein can be attached to the parent molecular moiety through any substitutable carbon atom.

[0026] The term "cycloalkylene" as used herein refers to a divalent group derived from an all-carbocyclic ring system containing zero heteroatoms as ring atoms and zero double bonds, and attached to two different ring carbon atoms of the parent molecule. The all-carbocyclic ring system may be a monocyclic, bicyclic, or tricyclic ring system, and may be a fused, bridged, or spiro ring system. Representative examples of cycloalkylene include: C, such as JPEG2025535717000005.jpg24164 3-10 Examples include, but are not limited to, those derived from rings.

[0027] The term "halogen" or "halo" means a chlorine, bromine, iodine, or fluorine atom.

[0028] Terms such as "alkyl," "cycloalkyl," "alkylene," "arylene," or "cycloalkylene" may be preceded by a symbol indicating the number of atoms present in the group in a particular instance (e.g., "C1-C4 alkyl," "C 1-4 Alkyl," "C 3-6 cycloalkyl," "C 1-4 These symbols are used as commonly understood by those skilled in the art. For example, a "C" followed by a subscript number indicates the number of carbon atoms present in the group that follows. Thus, a "C alkyl" is an alkyl group having three carbon atoms (i.e., n-propyl, isopropyl). "C-C" or "C 1-4 When a range is given, such as "C1-C4 alkyl," the subsequent group members can have any number of carbon atoms within the stated range. For example, "C1-C4 alkyl" or "C 1-4 "Alkyl" refers to an alkyl group having from 1 to 4 carbon atoms in any arrangement (ie, straight or branched).

[0029] When substituents are described as being independently selected from a group, each substituent is selected independently of the other substituents. Thus, each substituent may be the same or different from the other substituents. good.

[0030] Unless otherwise specified, structures depicted herein are intended to encompass all isomers of the structure (e.g., enantiomers, diastereomers, positional isomers, and geometric isomers (or conformational isomers)), such as the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention are within the scope of the invention. Compounds disclosed herein may exist as positional isomers or mixtures of positional isomers. Unless otherwise specified, all tautomers and positional isomers of the compounds of the invention are within the scope of the invention.

[0031] The term "a pharmaceutically acceptable salt thereof" refers to a salt prepared by combining a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), and (If) with an acid whose anion is generally considered suitable for human consumption, or a base whose cation is generally considered suitable for human consumption. Pharmaceutically acceptable salts are particularly useful as products of the methods of the present invention due to their greater water solubility relative to the parent compound. For pharmaceutical use, salts of the compounds of the present invention are non-toxic "pharmaceutically acceptable salts." Salts encompassed by the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the present invention, typically prepared by reacting the free base with a suitable organic or inorganic acid.

[0032] Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention include, where possible, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, boric acid, fluoroboric acid, phosphoric acid, metaphosphoric acid, nitric acid, carbonic acid, sulfonic acid, sulfuric acid, and the like, and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glycolic acid, isothionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, methanesulfonic acid, trifluoromethanesulfonic acid, succinic acid, toluenesulfonic acid, tartaric acid, and trifluoroacetic acid. Suitable organic acids generally include, for example, aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids. Specific examples of suitable organic acids include acetic acid, trifluoroacetic acid, formic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, digluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, stearic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonate (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, toluenesulfonic ... Examples of the carboxylic acids include sulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, p-hydroxybutyric acid, galactaric acid, galacturonic acid, adipic acid, alginic acid, butyric acid, camphoric acid, camphorsulfonic acid, cyclopentanepropionic acid, dodecylsulfuric acid, glycoheptanoic acid, glycerophosphoric acid, heptanoic acid, hexanoic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, palmoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, thiocyanic acid, and undecanoic acid.

[0033] Furthermore, when the compounds of the present invention contain an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts, i.e., sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and salts formed with suitable organic ligands, such as quaternary ammonium salts. In another embodiment, base salts are formed from bases which form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine, and zinc salts.

[0034] Organic salts can be prepared from secondary, tertiary, or quaternary amine salts such as tromethamine, diethylamine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, etc. Basic nitrogen-containing groups can be quaternized with reagents such as lower alkyl (C1-C6) halides (e.g., methyl, ethyl, propyl, butyl chlorides, bromides, iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, diamyl sulfate), long-chain halides (e.g., decyl, lauryl, myristyl, stearyl chlorides, bromides, iodides), aryl alkyl halides (e.g., benzyl bromide, phenethyl bromide), etc.

[0035] The term "isotopically labeled" refers to compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), and (If) in which one or more atoms have been replaced with an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. Examples of isotopes suitable for inclusion in compounds of the present disclosure include hydrogen ( 2 H, 3 H, etc.), carbon ( 11 C. 13 C. 14 C), chlorine ( 36 Cl), fluorine ( 18 F), iodine ( 123 I, 125 I, etc.), nitrogen (13 N, 15 N, etc.), oxygen ( 15 O. 17 O. 18 O, etc.), phosphorus ( 32 P, etc.), sulfur ( 35 Certain isotopically labeled compounds of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), and (If), for example, compounds containing a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. 3 H) and carbon-14 ( 14 Deuterium (C) is particularly useful for this purpose due to its ease of incorporation and ease of detection. 2 Substitution with heavier isotopes, such as 1H, may offer therapeutic advantages due to increased metabolic stability (e.g., longer in vivo half-life and reduced dosage), and may be preferable in some circumstances. 11 C. 18 F, 15 O. 13 Substitution with aryl groups (e.g., N) is useful for studying substrate receptor occupancy by positron emission topography (PET). Isotopically labeled compounds of formulae (I), (Ia), (Ib), (Ic), (Id), (Ie), and (If) can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the accompanying Examples, substituting the appropriate isotopically labeled reagent for the non-labeled reagent used in the prior art.

[0036] The site-specific substitution of an atom with the same atomic number but different from the predominant atomic mass or mass number in nature can be considered as a substituent of the compounds of the present disclosure. A sample of a compound having such an isotope as a substituent incorporates at least 50% of the isotope at the labeled position. The concentration of such an isotope, for example, deuterium, can be defined by the isotopic enrichment factor. As used herein, the term "isotopic enrichment factor" refers to the ratio between the isotopic abundance and the natural abundance of a specific isotope. For example, if a substituent in a compound of the invention is designated as deuterium, such compounds have an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes, including describing and disclosing chemicals, cell lines, vectors, animals, instruments, statistical analyses, and methodologies reported in the publications that may be used in connection with the present invention. All references cited herein should be construed as indicative of the state of the art. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0038] The effect of NAC is believed to be due to its antioxidant and redox signaling role in mitochondria.There is a need for new thiol-containing molecules that target mitochondria.In various embodiments, the present disclosure provides NAC analogs that target mitochondria and their uses.

[0039] [Compound]

[0040] In one aspect, the disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt thereof: JPEG2025535717000006.jpg32164In formula, R 1 is H, C1-C4 alkyl, or JPEG2025535717000007.jpg46164, R 2 is H or C1-C4 alkyl, W is NH, O, or S; L is C1-C 20 Alkylene, C2-C 20 Alkenylene, L1-R A -L2, or an amino acid, L1 and L2 are each independently absent or C1-C 10 is alkylene, R A Ha-(CH2CH2O) q -, arylene, or cycloalkylene; q is 1-20, X is a counterion, each Y is independently CF, Me, Cl, OMe, C(O)CH, NO, N(Me), or OH; m in each occurrence is independently 0, 1, 2, 3, 4, or 5, but the compound is not (R)-2-acetamido-3-mercapto-N-methylpropanamide or (R)-2-acetamido-3-mercapto-N-ethylpropanamide.

[0041] In some embodiments, X is F, Cl, Br, or I. In some embodiments, X is Br. In some embodiments, X is 2,2,2-trifluoroacetic acid or acetate.

[0042] In some embodiments, R 1 teeth JPEG2025535717000008.jpg46164. In some embodiments, R 1 is H or C1-C4 alkyl.

[0043] In some embodiments, R 2 is H. In some embodiments, W is NH or O. In some embodiments, R 2 is H and W is NH or O.

[0044] In some embodiments, L is C-C 20 In some embodiments, L is L-R A In some embodiments, L is L-R A -L2 and R A Ha-(CH2CH2O) q In some embodiments, L is L-R A -L2 and R A is arylene. In some embodiments, L is L-R A -L2 and R A is cycloalkylene.

[0045] In some embodiments, m is 0 or 1. In some embodiments, Y, at each occurrence, is independently CF, Me, Cl, or OMe. In some embodiments, m is 1 and Y, at each occurrence, is independently CF, Me, Cl, or OMe.

[0046] In some embodiments, R 2 is methyl. In some embodiments, R 1 teeth JPEG2025535717000009.jpg13164, R 2 is methyl. In some embodiments, R 1 teeth JPEG2025535717000010.jpg13164, R 2 is methyl, W is NH, and L is C1-C 20 In some embodiments, the compound is JPEG2025535717000011.jpg29164, where n is 1-20. In some embodiments, n is 6, 8, 10, 12, 14, or 16. In some embodiments, n is 10 and the compound is The file is JPEG2025535717000012.jpg31164.

[0047] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, has the structure of Formula (Ia): JPEG2025535717000013.jpg47164In the formula, n is 1 to 20.

[0048] In some embodiments, a compound of Formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, In some embodiments, m is 0 or 1 and X is Br. In some embodiments, m is 0 and X is Br. In some embodiments, m is 1, X is Br, and each Y is independently Me, OMe, Cl, or CF.

[0049] In some embodiments, the compound JPEG2025535717000014.jpg29164, and n is 1 to 20. For example, n is 6, 8, 10, 12, 14, or 16. In some such embodiments, n is 10 and the compound is In some such embodiments, n is 12 and the compound is In some such embodiments, n is 16 and the compound is JPEG2025535717000017.jpg30163.

[0050] In some embodiments, m is 1, X is Br, and Y is Me. In some embodiments, the compound JPEG2025535717000018.jpg45164, where n is a number between 1 and 20. For example, n is 6, 8, 10, 12, 14, or 16.

[0051] In some embodiments, m is 1, X is Br, and Y is OMe. In some embodiments, the compound JPEG2025535717000019.jpg45164, where n is a number between 1 and 20. For example, n is 6, 8, 10, 12, 14, or 16.

[0052] In some embodiments, m is 1, X is Br, Y is Cl, and the compound JPEG2025535717000020.jpg42164, where n is 1 to 20. For example, n is 6, 8, 10, 12, 14, or 16.

[0053] In some embodiments, m is 1, X is Br, and Y is CF. In some embodiments, the compound JPEG2025535717000021.jpg46164 structure, where n is 1 to 20. For example, n is 6, 8, 10, 12, 14, or 16.

[0054] In some embodiments, R 1 teeth JPEG2025535717000022.jpg13164, R 2 is H, W is NH, and L is L1-R A -L2. For example, L1 and L2 are methylene and R A is arylene or cycloalkylene. In some embodiments, the compound JPEG2025535717000023.jpg36164. For example, A is phenylene. As a non-limiting example, the compound is JPEG2025535717000024.jpg35164.

[0055] In some embodiments, the compound of Formula (I) has the structure of Formula (Ib): JPEG2025535717000025.jpg47164, where n is 1 to 10. For example, n is 3, 4, 5, or 6. In some embodiments, n is 4.

[0056] In some embodiments, R 1 teeth JPEG2025535717000026.jpg12164, R 2 is H, W is O, and L is L1-R A -L2. For example, L1 is absent, L2 is ethylene, and R A Ha-(CH2CH2O) q - and q is 1 to 20. In some embodiments, the compound JPEG2025535717000027.jpg31164, and q is 1 to 19. For example, q is 3, 4, 5, or 6. As a non-limiting example, the compound is JPEG2025535717000028.jpg34164.

[0057] In some embodiments, R 1 is H or C1-C4 alkyl, and R 2 is H and W is NH or O.

[0058] In some embodiments, R 1 is H or C1-C4 alkyl, and R 2 is H, W is NH or O, and the compound has the structure of formula (Ic), (Id), or (Ie). JPEG2025535717000029.jpg89164In the formula, t is 1 to 20, u is 1 to 10, and v is 1 to 10.

[0059] In some embodiments, in the compound of Formula (Ic), or a pharmaceutically acceptable salt thereof, R 1 is methyl. In some embodiments, the compound JPEG2025535717000030.jpg31164. For example, t is 5, 7, 9, 11, 13, or 15. As a non-limiting example, t is 9 and the compound is The file is JPEG2025535717000031.jpg31164.

[0060] In some embodiments, in the compound of Formula (Id), or a pharmaceutically acceptable salt thereof, R 1 is methyl. In some embodiments, the compound JPEG2025535717000032.jpg31164. For example, u can be 3, 4, 5, or 6.

[0061] In some embodiments, in the compound of Formula (Ie), or a pharmaceutically acceptable salt thereof, R 1 is methyl. In some embodiments, the compound JPEG2025535717000033.jpg34164. For example, v can be 3, 4, 5, or 6.

[0062] The compound of formula (I) JPEG2025535717000034.jpg145164 or a pharmaceutically acceptable salt thereof.

[0063] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is isotopically labeled.

[0064] In some embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is 13 For example, the carbon atom of the carbonyl group (C=O) of this compound is 13 It can be labeled with C.

[0065] In some embodiments, 13 The C-labeled compound of formula (I), or a pharmaceutically acceptable salt thereof, has the structure of formula (If): JPEG2025535717000035.jpg38164

[0066] In some embodiments, the compound of formula (If) JPEG2025535717000036.jpg36164, where n is a number between 1 and 20. For example, n is 6, 8, 10, 12, 14, or 16.

[0067] As described herein 13 C-labeled compounds were 13 For C atoms, it may have an isotopic enrichment factor of at least 5, at least 10, at least 20, at least 50, or at least 90.

[0068] [Pharmaceutical composition]

[0069] Another aspect of the present disclosure provides a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0070] The pharmaceutical composition may contain the compound in the range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may contain the compound in the range of about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may contain the compound in the range of about 1 to 100 mg. The pharmaceutical composition allows the compound to be administered in a daily dose of about 0.1 to about 1000 mg / kg of body weight. In some embodiments, the pharmaceutical composition allows the compound to be administered in a daily dose of about 0.5 to about 500 mg / kg of body weight. In some embodiments, the pharmaceutical composition allows the compound to be administered in a daily dose of about 50 to about 100 mg / kg of body weight. In some embodiments, the pharmaceutical composition is administered to a subject. After administration (e.g., about 1, 2, 3, 4, 5, or 6 hours after administration), the concentration of the compound at the site of action can be within a concentration range bounded by an endpoint selected from 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM (e.g., 0.1 μM to 1.0 μM).

[0071] The compound can be formulated as a pharmaceutical composition containing a carrier, for example, the carrier can be selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0072] The compounds can be formulated into pharmaceutical compositions containing one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers include lactose monohydrate, anhydrous lactose, and various starches. Binders include various celluloses, cross-linked polyvinylpyrrolidone, microcrystalline cellulose (e.g., Avicel™ PH101 and Avicel™ PH102), microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC). TM). Suitable lubricants that affect the flowability of the powder to be compressed include colloidal silicon dioxide (e.g., Aerosil™ 200), talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners include natural or artificial sweeteners such as sucrose, xylitol, sodium saccharin, sodium cyclamate, aspartame, and axulfam. Examples of flavorings include Magna Sweet™ (a trademark of MAFCO), bubble gum flavor, fruit flavors, and the like. Examples of preservatives include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl alcohol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0073] Suitable diluents include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dicalcium phosphate, sugars, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose (such as Avicel™ PH101 and Avicel™ PH102), lactose (such as lactose monohydrate, anhydrous lactose, Pharmatose™ DCL21), dicalcium phosphate (such as Emcompress™), mannitol, starch, sorbitol, sucrose, glucose, etc.

[0074] Suitable disintegrants include lightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, maize starch, modified starch, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof.

[0075] Examples of effervescent agents include effervescent compounds such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, alginic acid, and their anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent compound may be present.

[0076] Pharmaceutical compositions containing the compounds may be adapted for administration by any suitable route, for example, oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. Such formulations may be used in pharmaceutical They may be prepared by any method known in the art, for example by combining the active ingredient with the carrier(s) or excipient(s).

[0077] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets, powders or granules, solutions or suspensions in aqueous or non-aqueous liquids, edible foams or whips, or oil-in-water or water-in-oil liquid emulsions.

[0078] Pharmaceutical compositions suitable for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time, for example, the active ingredient may be delivered from the patch by iontophoresis.

[0079] Pharmaceutical compositions suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols, or oils, and can contain suitable conventional additives such as preservatives, solvents to aid drug penetration, and emollients in ointments and creams.

[0080] For application to the eye or other external tissues, such as the mouth and skin, the pharmaceutical composition is applied in some embodiments as a topical ointment or cream.When formulated as an ointment, the compound can be used with a paraffinic or water-miscible ointment base.Alternatively, the compound can be formulated into a cream using an oil-in-water cream base or a water-in-oil base.Pharmaceutical compositions suitable for topical administration to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent.

[0081] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouthwashes.

[0082] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or as enemas.

[0083] Pharmaceutical compositions suitable for nasal administration wherein the carrier is a solid include coarse powders having a particle size (for example in the range 20 to 500 microns) which are administered like snuff (i.e. by holding a container of the powder close to the nose and inhaling rapidly through the nasal passage). Where the carrier is a liquid, suitable formulations for administration as a nasal spray or solution include aqueous or oil solutions of the active ingredient.

[0084] Pharmaceutical compositions suitable for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.

[0085] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.

[0086] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the patient's blood, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials, and may be stored in a lyophilized (lyophilized) state, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0087] Tablets and capsules for oral administration may be in unit dose form and may contain binders (e.g., syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone). The tablets may contain conventional excipients such as starch, sorbitol, sorbitol, glycine ... Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats and oils; emulsifiers such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles such as almond oil (which may contain edible oils); oily esters such as glycerin, propylene glycol, or ethyl alcohol; preservatives such as methyl parahydroxybenzoate or propyl parahydroxybenzoate or sorbic acid; and, if desired, conventional flavors or colorants.

[0088] Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds can be administered with, optionally in combination with, additional therapeutic agents to treat cancer. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agents are administered before, simultaneously with, or after administration of the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical compositions are formulated to comprise a disclosed compound and further include one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cancer.

[0089] How to use

[0090] N-acetylcysteine ​​(NAC) was first approved by the U.S. Food and Drug Administration (FDA) in 1963 as a drug to treat excessive mucus production in respiratory diseases, including cystic fibrosis. It was subsequently used as a treatment for paracetamol (i.e., acetaminophen or Tylenol) poisoning. NAC has also been used as a direct scavenger of reactive oxygen species (especially hydrogen peroxide) and as an antioxidant in cancer biology and immuno-oncology. NAC is frequently used as an antioxidant in studies using tumor cells, immune cells, and preclinical mouse models. In both in vitro and in vivo studies, NAC is used at high concentrations due to its relatively low bioavailability. Reportedly, the effects of NAC are cancer cell-dependent and stage-specific. NAC is membrane-permeable and can cross the blood-brain barrier depending on the dose and method of administration. The effects of NAC are thought to be due to its thiol-modulating effects on cells.

[0091] The present disclosure demonstrates the efficacy of mitochondrial-targeted thiol compounds, such as the compounds of formula (I) described herein, on cancer cell proliferation. Numerous reports have demonstrated the efficacy of triphenylphosphonium (TPP) compounds linked via alkyl side chains in both in vitro and in vivo cancer studies. +It has been shown that binding of drugs to the TPP site can selectively target mitochondria in cancer cells over normal cells. Compared to control, non-transformed cells, the more negative mitochondrial membrane potential in cancer cells is a key factor in determining whether TPP is involved. + TPP is involved in improving the uptake and retention of positively charged drugs bound to it. + In mouse xenografts administered with a modifying drug (e.g., mitochondrial vitamin E), TPP was transferred to tumor tissue. + Enhanced accumulation of the modified drug was observed. + By adding an alkyl side chain containing the moiety, mitochondrial-targeted NAC (Mito 10 Mitochondrial ATP (NAC) was synthesized (Figure 1A). 10 -NAC has a free sulfhydryl group, It may exhibit antioxidant and redox-regulating effects similar to those of mitochondrial ATP. Age-related decline in mitochondrial function is thought to be due to a decline in intracellular amino acid homeostasis, particularly cysteine. Cysteine ​​is the most toxic to mitochondria, and increased non-vacuolar cysteine ​​levels inhibit mitochondrial respiration.

[0092] Furthermore, the present disclosure provides a method for detecting NAC, Mitochondrial cytotoxicity in multiple cancer cells. 10 The relative antiproliferative activities of Mitochondrial NAC and its methylated analogs were compared. 10 showed that -NAC is approximately 1,500-2,000 times more potent than NAC, and that its antiproliferative effect is enhanced by methylation of the free sulfhydryl groups (Mito 10 -MeNAC IC 50 is 1.9 μM, while Mito 10 (The mitochondrial targeting compounds described herein exert antiproliferative effects in cancer cells.)

[0093] Cancer treatment methods

[0094] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof. In one embodiment, one compound disclosed herein can be administered, while in alternative embodiments, multiple compounds disclosed herein can be administered.

[0095] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired result, including the prevention or treatment of a disease. The term "effective amount" includes a "therapeutically effective amount." The term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as reducing or inhibiting cell proliferation in the case of cancer. The therapeutically effective amount of a compound disclosed herein may vary depending on factors such as the subject's condition, age, sex, and weight, and the ability of the disclosed compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide an optimal therapeutic response. A therapeutically effective amount is also an amount in which the toxicity or adverse effects of a compound disclosed herein are reduced compared to known compounds and are outweighed by therapeutically beneficial effects.

[0096] As used herein, the term "tumor" or "cancer" refers to an abnormal growth of tissue, including solid and non-solid tumors. For example, the compositions and methods of the present disclosure can be used to treat cancers that present as solid tumors, such as pancreatic cancer, breast cancer, colon cancer, lung cancer, prostate cancer, thyroid cancer, ovarian cancer, and skin cancer. The compositions and methods of the present disclosure can also be used to treat non-solid tumor cancers, such as non-Hodgkin's lymphoma and leukemia.

[0097] As used herein, the term "subject" refers to a mammal, a non-mammal, and / or a cell. "Mammal" refers to any member of the class Mammalia, including, but not limited to, humans, non-human primates such as apes and monkeys, such as chimpanzees, and domestic animals, such as cows, horses, sheep, goats, and pigs. Animal subjects include domestic animals, such as rabbits, dogs, and cats, and laboratory animals, including rodents, such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term "subject" does not denote a particular age or sex. Preferably, the subject is a human, particularly a human with cancer.

[0098] As used herein, the term "treat" or "treating" refers to the management and care of a subject for the purpose of addressing a disease, condition, or disorder. Treatment may include preventing, alleviating, and / or ameliorating the onset of symptoms or complications, reducing symptoms or complications, or includes administering a compound of the present disclosure to eliminate a disease, condition, or disorder.

[0099] As used herein, the term "administering" refers to any means for introducing a compound disclosed herein into the body, preferably into the systemic circulation. Examples include, but are not limited to, oral, buccal, sublingual, pulmonary, transdermal, transmucosal, and subcutaneous, intraperitoneal, intravenous, and intramuscular injection.

[0100] The compounds utilized in the methods disclosed herein can be administered in conventional dosage forms prepared by combining the active ingredients with standard pharmaceutical carriers or diluents according to conventional methods well known in the art, which methods may include mixing, granulating, compressing or dissolving the ingredients, depending on the desired preparation.

[0101] In some embodiments of the disclosed methods of treatment, the subject is administered 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg , 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg of the compound are administered once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times weekly to treat a disease or disorder in a subject. In some embodiments, the subject is administered 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 64 mg, 66 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72.5 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82.5 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92.5 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102 mg, 103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg, 110 mg, 111 mg, 112 mg, 113 mg, 114 mg, 115 mg, 116 mg, 117 mg, 118 mg, 119 mg, 120 mg, 121 mg, 122 mg, 123 mg, 124 mg, 125 mg, 126 mg, 127 mg, 128 High doses of the compounds, such as 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, are administered once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times weekly to treat a disease or disorder in a subject. The minimum and / or maximum doses of the compounds may include doses within a dose range (e.g., 2.5 mg to 200 mg) that endpoint at any of these disclosed doses.

[0102] In some embodiments, the minimum dosage level of the compound to achieve treatment in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg of subject body weight. In some embodiments, the maximum dosage level of compound to achieve treatment in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg of subject body weight. The minimum and / or maximum dose levels of the compounds to achieve treatment in the disclosed therapeutic methods can include dose levels within a range having any of these disclosed dose levels as an endpoint (e.g., 500-2000 ng / kg of subject body weight).

[0103] In some embodiments, the cancer to be treated is pancreatic cancer, breast cancer, melanoma cancer, non-small cell lung cancer, or a combination thereof.

[0104] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent, hi some embodiments, the additional therapeutic agent is AZD3965.

[0105] In some embodiments, the compounds described herein can be combined with ionizing radiation to inhibit the formation of tumor cells.

[0106] In some embodiments, the compounds described herein can be combined with immunotherapy to inhibit the formation of tumor cells. Suitable immunotherapies include, but are not limited to, immune checkpoint inhibitor therapy, cell therapy, and other immunotherapies known in the art. Immune checkpoint inhibitor therapy may involve inhibiting immune checkpoint proteins produced by immune system cells, such as T cells, from binding to partner proteins on other cells, such as tumor cells. In cell therapy, live cells can be infused, transplanted, or implanted into patients to replace or repair damaged tissues and / or cells.

[0107] In some embodiments, the compounds disclosed herein can be combined with conventional treatment protocols to enhance the effectiveness of conventional cancer treatments.

[0108] How to enhance CAR-T cell therapy

[0109] In another aspect, the present disclosure provides a method of enhancing CAR-T cell therapy in a subject in need thereof, comprising administering to the subject an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof.

[0110] "CAR-T cells" refer to chimeric antigen receptor-expressing T cells. Those skilled in the art will understand that chimeric antigen receptors (CARs) are a type of antigen-targeting receptor that fuses an intracellular T cell signaling domain with an extracellular tumor-binding site (most commonly a single-chain variable fragment (scFv) derived from a monoclonal antibody). CARs can directly recognize cell surface antigens, independent of MHC-mediated presentation, allowing for the use of a single receptor construct specific for any antigen in any patient. "CAR-T cell therapy" can be a therapeutic approach for the treatment of, for example, cancer-related (e.g., B-cell and T-cell lymphoma) and immune-related malignancies. CAR-TT cells can include, for example, patient-derived memory CDS+ T cells engineered to express a recombinant T cell receptor specific for a known antigen present on the subject's tumor. While the present disclosure is generally described in the context of using CAR-T cell therapy to treat cancer, it should be understood that such therapy is also useful for the treatment of other indications.

[0111] In some embodiments, the subject is a cell or a human.

[0112] In some embodiments, the subject is a human and the method comprises administering to the subject an effective amount of a compound, or a pharmaceutically acceptable salt thereof, in combination with CAR-T cell therapy.

[0113] In some embodiments, the subject is a human and the method comprises pretreating CAR-T cells with a compound, or a pharmaceutically acceptable salt thereof, and administering to the subject an effective amount of the pretreated CAR-T cells.

[0114] How to analyze the sample

[0115] Another aspect of the present disclosure provides a method of analyzing a sample, the method comprising contacting the sample with an isotopically labeled compound described herein, or a pharmaceutically acceptable salt thereof, thereby producing a labeled sample, and analyzing the labeled sample.

[0116] In some embodiments, the sample comprises cells.

[0117] In some embodiments, analyzing the labeled sample in the present methods includes analyzing a profile or image of the labeled sample. As used herein, the term "profile" includes a nucleic acid profile or a proteomic profile. A nucleic acid profile or a proteomic profile is an analysis of characteristics including, but not limited to, gene organization, gene expression, and protein modifications. Nucleic acid profiles and proteomic profiles using cDNA analyze gene organization or gene expression (i.e., gene products, which are RNA and protein), respectively. An "image" of the sample can be obtained by any of a variety of sophisticated microscopy and imaging techniques known to those skilled in the art. Examples include, but are not limited to, bright-field microscopy, dark-field microscopy, phase-contrast microscopy, and differential interference contrast (DIC) microscopy. These techniques combine magnification and contrast mechanisms to produce images with cellular or subcellular image resolution.

[0118] In some embodiments, analyzing the labeled sample comprises analyzing a profile of the labeled sample.

[0119] In some embodiments, the profile comprises a proteome profile. The term "proteome profile" is used to refer to a representation of the expression pattern of multiple proteins in a biological sample (e.g., a biological fluid at a particular time point). A proteome profile can be represented, for example, as a mass spectrum, but also includes other representations based on the physicochemical or biochemical properties of proteins.

[0120] In some embodiments, the method includes analyzing the proteomic profile of the labeled sample using mass spectrometry.

[0121] In some embodiments, analyzing the labeled sample comprises analyzing an image of the labeled sample.

[0122] In some embodiments, the method includes generating an image of the labeled sample using magnetic resonance imaging (MRI).

[0123] kit

[0124] Another aspect of the present disclosure provides a kit comprising a pharmaceutical composition comprising a compound disclosed herein and instructions.

[0125] The term "instructions" refers to a publication, record, diagram, or other medium of expression used to communicate the usefulness of the pharmaceutical composition to humans for one of the purposes described herein. The instructions can also include, for example, the appropriate dosage of the pharmaceutical composition. The instructions for the kit can be, for example, affixed to a container containing the pharmaceutical composition disclosed herein or shipped with a container containing the pharmaceutical composition. Alternatively, the instructions and pharmaceutical composition can be packaged together with the recipient's package. The instructions may be shipped separately from the container, intended for use by a person in combination. [Example]

[0126] The following examples, of course, are offered by way of illustration only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and following examples and fall within the scope of the appended claims.

[0127] In the following examples, a mitochondria-targeted NAC (Mito) with a 10-carbon alkyl side chain attached to a triphenylphosphonium group, which is functionally similar to NAC, is described. 10 -NAC) was synthesized and investigated. 10 -NAC has a free sulfhydryl group and is more hydrophobic than NAC. 10 -NAC has an inhibitory effect on some cancer cells, including pancreatic cancer cells, approximately 2,000 times that of NAC. 10-NAC methylation also inhibited cancer cell proliferation. 10 NAC inhibited mitochondrial complex I-driven respiration, and in combination with a monocarboxylate transporter 1 inhibitor synergistically reduced the proliferation of pancreatic cancer cells. These results suggest that NAC and Mito 10 These results suggest that the antiproliferative effects of -NAC are unlikely to be related to their antioxidant mechanisms (i.e., scavenging of reactive oxygen species) or sulfhydryl-dependent redox-modulating effects.

[0128] Example 1. Synthesis of Mitochondrially Targeted N-Acetylcysteine ​​and Its Analogues

[0129] The general synthesis of Mito-NAC compounds is shown in Scheme 1. Scheme 1. Synthesis of Mito-PEG-NAC and Mito-NAC. Reagents and conditions: i. HOBt, DIC, (aminoalkyl)-triphenylphosphonium bromide hydrochloride, triethylamine, CHCl, room temperature, 12 hours; ii. EtSi-H, TFA, room temperature, 1 hour, 83%; iii. HOBt, DIC, PEG bromide, pyridine, CHCl, room temperature, 12 hours; iv. PPh, CHCN, reflux, 48 hours; v. EtSi-H, TFA, room temperature, 1 hour.

[0130] 13 A general method for synthesizing C-labeled Mito-NAC compounds is shown in Scheme 2. JPEG2025535717000038.jpg58164 Scheme 2. Synthesis of labeled 13C-Mito-NAC. Reagents and conditions: i, triethylamine, CHCl, room temperature, 12 hours; ii, HOBt, DIC, (10-aminodecyl)-triphenylphosphonium bromide hydrochloride, pyridine, CHCl, room temperature, 12 hours; ii, EtSi-H, TFA, room temperature, 1 hour.

[0131] Mito 10 Synthesis of -NAC

[0132] Mito10 -NAC was synthesized in two steps by activating the carboxylic acid with N,N'-diisopropylcarbodiimide (DIC) / hydroxybenzotriazole (HOBt), followed by the addition of (10-aminodecyl)-triphenylphosphonium bromide in the presence of triethylamine in dichloromethane (CHCl). 2,2,2-trifluoroacetic acid (TFA) and triethylsilane were used to deprotect the thiol, and Mito 10 -NAC. Mito 10 The synthesis of -NAC is shown in Scheme 3. JPEG2025535717000039.jpg28164 Scheme 3.Mito 10 Synthesis of -NAC. Reagents and conditions: i. HOBt, DIC, (10-aminodecyl)-triphenylphosphonium bromide hydrochloride, triethylamine, CHCl, room temperature, 12 hours, 71%; ii. EtSi-H, TFA, room temperature, 1 hour, 83%.

[0133] A stirred solution of N-acetyl-S-trityl-L-cysteine ​​(0.3 g, 0.74 mmol) in CHCl / N,N-dimethylformamide (DMF) (15 mL / 100 mL) was cooled to 0 °C and treated sequentially with HOBt (0.2 g, 1.48 mmol) and DIC (0.19 g, 1.50 mmol). After stirring at room temperature for 2 h, (10-aminodecyl)-triphenylphosphonium bromide hydrochloride (0.35 g, 0.65 mmol) and triethylamine (188 μL, 0.13 mmol) were added to the mixture. The reaction mixture was stirred overnight at room temperature. Next, CHCl and water (HO) (25 mL) were added to the mixture. The organic layer was dried over sodium sulfate (NaSO). The solvent was removed under reduced pressure. The crude product was poured into 100 mL of ether and centrifuged. The insoluble salts were collected and purified by flash chromatography (CH2Cl2 / ethanol [EtOH] 9 / 1) to give the corresponding trityl-Mito 10 Trityl-Mito NAC (0.41 g, 71% yield) was obtained. High performance liquid chromatography mass spectrometry (HPLC-MS) showed that the product was sufficiently pure to be used without further purification. 10-NAC C 52 H 58 N2O2PS + [M] + , 806.1 electrospray ionization mass spectrometry (ESI-MS).

[0134] Trityl-Mito 10 -NAC (0.25 g, 0.31 mmol) and triethylsilane (100 μL, 0.60 mmol) were dissolved in trifluoroacetic acid (1 mL) and stirred at room temperature for 1 h. The mixture was then directly purified by reverse-phase chromatography using a C18 column (HO / acetonitrile [CHCN], 9 / 1 to 0 / 10, with 0.1% TFA added) to obtain the corresponding Mitochondrial. 10 -NAC (0.15 g, 83% yield) was obtained.

[0135] Mito 10 - HRMS calculated value of NAC C 33 H 44 N2O2PS + [M] + 563.2856, actual value 563.2856. 31 P NMR (400.13MHz, CDCl3) δ 23.76. 1 H NMR (400.13MHz, CDCl3), δ 7.83-7.77(3H, m), 7.71-7.59(12H, m), 7.44-7.37(1H, m), 7.12-7.01(1H, m), 6.90-6.75(1H, m), 4.59-4.50(1H, m), 3.25-3.08(4H, m), 2.97-2.71(2H, m), 2.01(3H, s), 1.58-1.62(3H, m), 1.52-1.38(4H, m), 1.26-1.16(9H, m). 13 C NMR (75MHz, CDCl3) δ 171.2, 170.2, 135.4, 135.3, 133.4, 133.2, 130.7, 130.5, 118.4, 117.5, 55.2, 39.5, 30.2 , 30.1, 28.9, 28.7, 28.6, 28.5, 28.3, 26.7, 26.4, 22.9, 22.4 (d, J=4.4), 22.3 (d, J=51.3).

[0136] Mito 10 Synthesis of -NAC-SMe

[0137] Mitochondrial Targeted N-Acetylmethylated Cysteine ​​(Mito 10 -NAC-SMe) is Mito 10 It was prepared using the same reaction conditions as in the synthesis of NAC. 10 The synthesis of -NAC-SMe is shown in Scheme 4. JPEG2025535717000040.jpg30164 Scheme 4. Synthesis of Mito10-NAC-SMe. Reagents and conditions: i, HOBt, DIC, (10-aminodecyl)-triphenylphosphonium bromide hydrochloride, triethylamine, CHC l2 , room temperature, 12 hours, 43%.

[0138] A stirred solution of N-acetyl-S-methyl-L-cysteine ​​(0.15 g, 0.75 mmol) in CHCl / DMF (15 mL / 100 mL) was cooled to 0 °C and treated sequentially with HOBt (0.13 g, 0.96 mmol) and DIC (152 μL, 0.96 mmol). After stirring at room temperature for 2 h, the mixture was added with (10-aminodecyl)-triphenylphosphonium bromide hydrochloride (0.35 g, 0.65 mmol) and triethylamine (188 μL, 0.13 mmol). The reaction mixture was stirred overnight at room temperature. CHCl and H2O (25 mL) were then added to the mixture. The organic layer was dried over Na2SO4. The solvent was removed under reduced pressure. The crude product was poured into 100 mL of ether and centrifuged. The insoluble salts were collected and purified by reversed-phase chromatography using a C18 column (H2O / CH3CN 9 / 1 to 0 / 10, with 0.1% TFA) to give the corresponding Mito 10 -NAC-SMe (0.18 g, 43% yield) was obtained.

[0139] Mito 10 - HRMS calculated value of NAC-SMe C 34 H 46 N2O2PS + [M] + 577.3012, actual value 577.3015. 31P NMR (400.13MHz, CDCl3) δ 23.90. 1 H NMR (400.13MHz, CDCl3), δ 7.86-7.80(3H, m), 7.75-7.64(12H, m), 7.19-7.06(1H, m), 7.05-6.84(1H, m), 4.58-4.48(1H, m), 3.3 9-3.16 (4H, m), 2.89-2.82 (2H, m), 2.13 (3H, s), 2.04 (3H, s), 1.68-1.42 (6H, m), 1.32-1.19 (10H, m). 13 C NMR (75MHz, CDCl3) δ 170.8, 170.7, 135.3, 135.2, 133.3, 133.2, 130.6, 130.5, 118.3, 117.5, 52.8, 39.5, 36.4, 30.2, 30.1, 26.5, 22.9, 22.4(d, J=51.4), 22.3(d, J=4.4), 15.7.

[0140] Synthesis of Mito-PEG4-NAC

[0141] Mitochondria-targeted PEGylated N-acetylcysteine ​​(Mito-PEG4-NAC) was synthesized in three steps. Activation of the carboxylic acid with DIC / HOBt, followed by addition of the corresponding brominated PEG derivative in CHCl in the presence of pyridine, gave the PEGylated bromide derivative. Nucleophilic substitution of the bromide with triphenylphosphine gave the mitochondria-targeted intermediate. Deprotection of the thiol with TFA and triethylsilane gave Mito-PEG4-NAC. The synthesis of Mito-PEG4-NAC is shown in Scheme 5. Scheme 5. Synthesis of Mito-PEG4-NAC. Reagents and conditions: i) HOBt, DIC, (2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]ethanol, pyridine, CHCl, room temperature, 12 h, 58%; ii) PPh, CHCN, reflux, 48 h, 46%; iii) EtSi-H, TFA, room temperature, 1 h, 95%.

[0142] A stirred solution of N-acetyl-S-trityl-L-cysteine ​​(0.5 g, 1.2 mmol) in CHCl (10 mL) was cooled to 0°C and treated sequentially with HOBt (0.3 g, 2.4 mmol) and DIC (390 μL, 2.4 mmol). After stirring at room temperature for 2 hours, (2-[2-[2-(2-bromoethoxy)ethoxy]ethoxy]ethanol (0.28 g, 1.1 mmol) and pyridine (97 μL, 1.2 mmol) were added to the mixture. The reaction mixture was stirred at room temperature overnight. Then, CHCl and HO (25 mL) were added to the mixture. The organic phase was dried over NaSO. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography (CHCl / EtOH 9 / 1) to give the corresponding trityl-PEG-NAC (0.46 g, 58% yield). HPLC-MS showed that the product was sufficiently pure and could be used without further purification. ESI-MS C of trityl-PEG-NAC 32 H 38 BrNO6S [MH] + , 645.0. The insoluble salt was purified by flash chromatography (CHCl / EtOH) to give the corresponding trityl-Mito-PEG-NAC (0.3 g, 46% yield). HPLC-MS results indicated that the product was sufficiently pure to be used without further purification. ESI-MS C of trityl-Mito-PEG-NAC 50 H 53 NO6PS + [MH] + , 826.4.

[0143] A mixture of trityl-Mito-PEG-NAC (0.2 g, 0.2 mmol), triethylsilane (a few drops), trifluoroacetic acid (1 mL), and CHCl (1 mL) was stirred at room temperature for 1 h. The mixture was then directly purified by reverse-phase chromatography using a C18 column (HO / CHCN = 9 / 1 to 0 / 10, 0.1% TFA added) to obtain the corresponding Mito-PEG-NAC (0.14 g, 96% yield).

[0144] HRMS calculated C value of Mito-PEG4-NAC 31 H39 NO6PS + [M] + 584.2230, actual value 584.2232. 31 P NMR (400.13MHz, CDCl3)δ 25.09. 1 H NMR (400.13MHz, CDCl3), δ 7.83-7.63(15H, m), 6.88-6.77(1H, m), 6.71-6.62(1H, m), 4.87-4.78(1H, m), 4.44-4.19(2H, m), 3. 91-3.65(6H, m), 3.58-3.52(2H, m), 3.45-3.40(2H, m), 3.33(4H, s), 3.08-2.89(2H, m), 2.05(3H, s). 13 C NMR (75MHz, CDCl3) δ 171.2, 170.1, 134.83, 134.81, 133.9, 133.8, 130.2, 130.0, 119.2, 118.3, 70.5 , 70.3, 70.2, 70.0, 68.8, 64.5, 63.6, 63.5, 53.9, 26.7, 24.7 (d, J=53.5), 22.9.

[0145] Example 2. Synthesis of N-acetylcysteine ​​amide (NACA)

[0146] A general method for the synthesis of NACA is shown in Scheme 6. Scheme 6. Synthesis of Amide and Ester Derivatives of NAC. Reagents and Conditions: i. HOBt, DIC, aminoalkyl, triethylamine, CHCl, room temperature, 12 hours; ii. EtSi-H, TFA, room temperature, 1 hour; iii. HOBt, DIC, PEG-OH, pyridine, CHCl, room temperature, 12 hours; iv. EtSi-H, TFA, room temperature, 1 hour; v. HOBt, DIC, PEG-NH, triethylamine, CHCl, room temperature, 12 hours; vi. EtSi-H, TFA, room temperature, 1 hour.

[0147] Example 3. Antiproliferative effects of mitochondria-targeted N-acetylcysteine ​​and analogs in cancer cells

[0148] NAC and Mitochondrial Activity in Multiple Cancer Cells 10 The relative antiproliferative potencies of Mitochondrial NAC, Mitochondrial NAC, and their methylated analogs were compared. 10 -NAC was approximately 1,500-2,000 times more potent than NAC, and methylation of the free sulfhydryl groups enhanced its antiproliferative activity (Mito 10 -MeNAC half inhibitory concentration (IC 50 ) is 1.9 μM, while Mito 10 (The α-NAC concentration was 9.6 μM.) This suggests that the antiproliferative effect is not related to antioxidant or radical scavenging activity.

[0149] Mito 10 -Antiproliferative effects of NAC

[0150] Mitochondrial activity against the proliferation of multiple cancer cell lines derived from pancreatic, breast, and lung cancers (MiaPaCa-2, MDA-MB-231, MCF-7, A549) and a non-malignant breast cancer cell line (MCF-10A) as a control. 10 We investigated the effects of Mitochondrial NAC and NAC on the proliferation of MiaPaCa-2, MDA-MB-231, MCF-7, A549, and MCF-10A cells. 10 The effects of NAC and Mitochondrial Cell Growth Regulator (MGR) on these cells were examined. Figures 2A and 2B show the effects of NAC and Mitochondrial Cell Growth Regulator (MGR) on these cells. 10 The effect of NAC on mitochondria-targeted triphenylphosphonium (TPP) + ) and previously reported drugs inhibit cancer cell proliferation by 100–500 fold compared to their non-targeted parent drugs (Figure 3B). 10 TPP inhibited cancer cell proliferation by 1,500 to 2,400 times more than NAC (Figures 2A-2B, Figure 3A). This was a completely unexpected finding. + Such a large difference in efficacy of a drug-containing compound on cancer cells, as demonstrated with the Mito-NAC compound, is unprecedented. 10One of the reasons for this marked enhancement of the antiproliferative effect of -NAC is the combination of non-targeted analogs and TPP. + This may be related to the difference in the relative hydrophobicity of the binding analogues. Hydrophobicity calculations show that NAC is very hydrophilic, while Mito 10 -NAC has been shown to be relatively hydrophobic (NAC and Mito 10 -NAC log P values ​​are −0.7 and 6.4, respectively) (Fig. 1B).

[0151] To confirm that Mito-NAC degradation over time does not affect cell proliferation results, we compared the results of cell proliferation experiments in which Mito-NAC was administered as a bolus dose versus freshly administered doses every 48 hours. No significant differences in cell proliferation profiles were observed, indicating that Mito-NAC was relatively stable over the experimental period (Figure 9).

[0152] The results also suggest that the NAC-induced antiproliferative effects observed at very high concentrations in cells may be due to "off-target" effects rather than mitochondrial effects. The results of oxygen consumption experiments in MiaPaCa-2 cells also support this conclusion (Figures 6A-6B). Furthermore, MCF-10A cells, used as a non-malignant cell line control for breast cancer, may not be as effective or appropriate as a non-malignant cell line control for other types of cancer.

[0153] The effects of methyl-substituted NAC and Mito10-NAC on the proliferation of human pancreatic cancer (MiaPaCa-2) cells were investigated (Figures 4A-4B). The results suggest that methylation of sulfhydryl groups indeed enhanced the antiproliferative effect. This suggests that antioxidant mechanisms related to the scavenging of reactive oxygen species (superoxide and hydrogen peroxide) or reactive nitrogen species (peroxynitrite) are involved in the antiproliferative effects of Mito10-NAC. 10 These results suggest that NAC (or its methylated analogues) may not be involved in the antiproliferative effects it induces in cancer cells.

[0154] The effects of other NAC analogs (Mito-PEG4-NAC and NAC amide) on cell proliferation were also tested. Results showed that Mito-PEG4-NAC and NAC amide had IC values ​​of 0.01 to 0.01, indicating that they inhibit cell proliferation. 50 The values ​​were shown to be 36.7 μM and 4300 μM, respectively (FIGS. 4A-4B).

[0155] In MiaPaCa-2 cells, NAC and Mito 10 In the presence of NAC, we monitored both ATP (Figure 5A) and cytotoxicity as revealed by the SYTOX Green assay (Figure 5B). At much higher concentrations (>50 mM), NAC inhibited ATP levels, but significant cell death was observed under these conditions. At concentrations that inhibited human pancreatic cancer cell proliferation (10–20 μM), Mitochondrial activity was also observed. 10 -NAC had no effect on ATP concentrations or cell death (Figures 5A and 5B).

[0156] Melanoma (UACC-62) cells showed a completely different behavior from other cancer cells. 10 -NAC was more resistant, but Mito 10 -NAC was still much more potent than NAC. Another confounding factor was that NAC exerted a dose-dependent biphasic effect (Figures 8A-8B). Due to experimental constraints of the assay, it was not feasible to perform cell proliferation or Seahorse assays and ATP measurements at the same time points (Figures 4A, 4B, 5A, 5B, 6A, 6B).

[0157] NAC and Mito 10 Effect of -NAC on mitochondrial complex I-induced oxygen consumption

[0158] Mitochondrial respiration (oxidative phosphorylation [OXPHOS]) and Complex I-induced oxygen consumption were assessed using the Seahorse method. MiaPaCa-2 cells were treated with various concentrations of NAC and Mito 10-NAC treatment and measurement of total oxygen consumption rate (OCR) (Figure 6A). A common bioenergetic indicator of mitochondrial stress was monitored. As shown in the figure, Mito 10 -NAC inhibited 50% of basal OCR at a concentration of 20 μM, whereas NAC required much higher IC concentrations (>100 mM) to inhibit basal OCR. 50 A value was required.

[0159] Treatment with NAC for 24 hours induced significant cell death (Fig. 5B). 10 By injecting NAC into permeabilized cells in real time, we tested whether it could directly inhibit mitochondrial complex I-dependent OCR (Figure 6B). Under these conditions, NAC inhibited complex I-induced oxygen consumption by only about 30% up to a concentration of 100 mM, and direct cytotoxicity was observed at such concentrations (Figure 5B). In contrast, Mito 10 -NAC directly inhibits complex I-induced oxygen consumption 50 The value is 37 μM. 10 NAC showed minimal cytotoxicity (Figure 5B). Thiol antioxidants (NAC and glutathione [GSH] esters) caused transient mitochondrial oxidation and inhibition of mitochondrial respiratory complex III in some cancer cells, including glioblastoma. However, our results using real-time monitoring of mitochondrial complex I-induced oxygen consumption suggest that NAC, even at high concentrations, does not affect mitochondrial respiration in MiaPaCa-2 cells.

[0160] NAC / Mito on pancreatic cancer cell growth 10 -NAC and Mito10-NAC / MCT-1 inhibitor combination effects

[0161] Monocarboxylate transporters have been used as therapeutic targets in cancer cells. It has been shown that simultaneous inhibition of monocarboxylate transporter 1 (MCT-1) and mitochondrial OXPHOS synergistically inhibits the proliferation of several cancer cells. More recently, these findings were confirmed in B-cell lymphoma xenografts using AZD3965 and another OXPHOS inhibitor. NAC was reported to inhibit the expression of monocarboxylate transporter 4 (MCT-4) in cancer cell lines. NAC reduced the stromal expression of MCT-4, which is used as a biomarker for breast cancer. Mito 10 -NAC may exhibit synergistic effects with NAC. 10 The synergistic effects of NAC and AZD3965 were compared. AZD3965 is an MCT-1 inhibitor currently undergoing Phase I / II clinical trials for cancer treatment. MiaPaCa-2 cells were treated with Mitochondrial receptor agonists (MTRs). 10 Cell proliferation was continuously monitored during treatment with either NAC and AZD3965 or NAC alone or in combination. Figures 7A-7F show the effect of the combination index on Mitochondrial proliferation. 10 -NAC has synergistic effects with the MCT-1 inhibitor (AZD3965), but not with NAC.

[0162] Relative inhibitory efficacy of mitochondrial-targeted drugs

[0163] Increasing the aliphatic chain length of the TPP+-linked molecule significantly enhanced its antiproliferative activity in tumor cells. As shown in Figure 3A, the fold difference between the parent compound and the TPP+-modified compound (with a 10-carbon linker side chain) depends on the parent compound, particularly its hydrophobicity. Figure 3A shows the dose-response characteristics of NAC and TPP+-modified analogs in MiaPaCa-2 cells. The difference between NAC and Mito10-NAC is 1,600-fold. While many factors contribute to the fold difference between the TPP+-modified drug and the unmodified drug, the hydrophobicity of the parent drug is a major factor. When the parent compound is highly hydrophilic (NAC), the mitochondrial membrane potential of tumor cells is more negative than that of normal cells, so TPP+ modification is likely to have a greater antiproliferative effect and mitochondrial respiration inhibition in tumor cells. TPP+ modification of mitochondrial-targeted drugs + It has been shown that incorporation of β-glucan into mitochondria is essential for its accumulation and antiproliferative effects in cancer cells.

[0164] Lack of radical scavenging mechanism

[0165] The paradoxical effects of reactive oxygen species (e.g., superoxide and hydrogen peroxide) have been previously reported in cancer cells. Superoxide and hydrogen peroxide have been reported to promote tumorigenesis and tumor progression at low concentrations, but induce cytotoxicity in tumor cells and inhibit metastasis at high concentrations. This suggests that inhibition of reactive oxygen species has different effects on tumorigenesis, tumor progression, and metastasis. Redox regulators (NAC) and lipid peroxidation inhibitors (vitamin E), chain-breaking antioxidants, enhanced lung cancer metastasis in mice. However, methylated Mito 10 -Based on the results obtained with NAC, Mito 10We conclude that reactive oxygen species or redox-modulating activity of -NAC is unlikely to play a significant role in its antiproliferative mechanism. The cell growth inhibition IC50 values ​​of Mito-NAC-SMe lacking -SH groups (Figures 7A–7F) are comparable to or slightly lower than those of Mito-NAC with -SH groups. The antiproliferative activity of Mito-NAC and Mito-NAC-SMe is unaffected regardless of the presence or absence of the redox-sensitive -SH group. Other studies have shown that blunting the activity of the nitroxide moiety in Mito-CP (i.e., removing the superoxide dismutase mimicking mechanism) does not affect its antiproliferative effect. Furthermore, reactive oxygen species generation is a key factor in the regulation of TPP in cancer cells. + It has also been shown that mitochondrial targeting is not responsible for the antiproliferative effects of drugs based on mitochondrial targeting.

[0166] Immunomodulatory effects of NAC and its antitumor immune function

[0167] Recently, NAC has found new applications in immunotherapy. Chimeric antigen receptor (CAR) T cells are genetically modified T cells that recognize and destroy proteins on cancer cells. In CAR T cell therapy, a patient's own T cells are reprogrammed to recognize and attack specific proteins in cancer cells, and then reinfused back into the patient. This ability is often impaired when oxidant-induced modifications in CAR T cells are enhanced. NAC has been shown to improve the efficacy of adoptive T cell immunotherapy in the treatment of melanoma. In a recent study, NAC-treated T cells cultured and then infused into a preclinical melanoma model as immunotherapy demonstrated improved outcomes. NAC-treated T cells were 33-fold more effective than T cells cultured without NAC. NAC improves the antitumor function of exhausted T cells, thereby improving the outcome of adoptive cell transfer (ACT) therapy. NAC enhances the antitumor function of T cells by activating PI3K / Akt, inhibiting Foxo1, and inhibiting reactive oxygen species. The opposing effects of NAC on T cells were concentration-dependent. At low concentrations, NAC exhibited immunostimulatory effects, whereas at higher concentrations, NAC exhibited inhibitory effects. Phase I clinical trials of NAC aimed at optimizing the tumor metabolic microenvironment are ongoing. Although NAC has low bioavailability, it is membrane permeable and has been shown to cross the blood-brain barrier in humans and rodents.

[0168] Mito 10 -NAC is highly effective in tumor cells, so Mito 10 The potential for enhancing CAR-T cell therapy with NAC may be explored. Published reports and ongoing clinical trials using NAC support this possibility. Mitochondrial-targeted drugs, such as Mito-ATO, reprogram the tumor microenvironment, inhibit tumor-suppressing immune cells, and activate T cells. 10Mito-ATO may reverse immune suppression by regulatory T cells by stimulating effector T cell function. Mito-ATO induced potent T cell immune responses at local and distant tumor sites and reduced myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment. Mito-ATO also increased tumor-infiltrating CD4+ T cells. Mito-ATO improved the efficacy of PD-1 blockade immunotherapy.

[0169] Synergistic antitumor effects of OXPHOS inhibitors and MCT-1 / 4 inhibitors

[0170] Mito 10 The antiproliferative effect of NAC was enhanced in the presence of AZD3965, an MCT-1 transporter inhibitor. The extent of the combination effect is consistent with our previously published heat map display of other mitochondrial-targeted drugs. AZD3965 has been reported to enhance intracellular acidosis through an increase in intracellular lactate and a decrease in extracellular lactate. Relatively high concentrations of AZD3965 were used to inhibit cancer cells. At these concentrations, AZD3965 exerts adverse side effects. Combination therapy with mitochondrial-targeted drugs is a promising option because it allows for a significant reduction in the concentration of AZD3965 used in cancer treatment. Other mitochondrial-targeted drugs (metformin and phenformin) have been used in brain tumor research as combination therapy with AZD3965. However, their effective concentrations vary significantly. In this study, we investigated the effects of mitochondrial-targeted drugs in combination with AZD3965 to achieve synergistic growth inhibition. 10 -NAC was used in combination with AZD3965 at low micromolar concentrations.

[0171] Hyperpolarized [1-13C]NAC probes in hyperpolarized 13C-MRI demonstrated that NAC was distributed throughout the body, including the brain. This finding is consistent with previous studies using the isotope-substituted NAC56. In both tumor cell and mouse xenograft studies, [13C]NAC was shown to form [13C]NAC-GSH dimers and other homodimers. Furthermore, [13C]GSH was not detected, suggesting that NAC may induce GSH synthesis by an indirect mechanism. Furthermore, the lack of [13C]GSH detection may be due to a shortened relaxation time. GSH induction by NAC is cell-dependent, and the mechanism by which GSH is formed remains to be elucidated.

[0172] The cysteine ​​residue Cys90 in the ND3 subunit of mitochondrial complex I has been reported to play an important role in mitochondrial function. Glutathionylation or nitrosation of this critical cysteine ​​residue regulates redox signaling. 10 -NAC may inhibit complex I by thiolating the mitochondrial cysteine ​​proteome. 10 Results using methylated analogs of -NAC demonstrated that disruption of the mitochondrial cysteine ​​proteome significantly increased the risk of mitochondrial damage. 10 This suggests that NAC may not be the cause of the inhibition of mitochondrial respiration. 10 The exact mitochondrial target of -NAC and other analogs has not yet been identified.

[0173] The antiproliferative effects of Mito-NAC may be due to cell cycle arrest. Previous studies have reported that mitochondrial-targeting drugs (e.g., Mito-magnolol) reduce AKT and Foxo1 phosphorylation and induce cell cycle arrest in the G1 phase of the cell cycle. Future studies investigating the effects of NAC, Mito-NAC, and their methylated analogs on AKT signaling and cell cycle arrest in cancer cells will further our understanding of these redox-sensitive thiols.

[0174] All experiments were performed in different cancer cell lines. Based on previous studies applied to in vivo mouse xenograft models, Mito 10 Mito-NAC and analogs may show similar efficacy in mouse xenografts. Results indicate that Mito-NAC is relatively stable over time in cell proliferation experiments. However, comprehensive analytical studies are needed to monitor the oxidative degradation of the compound over the course of the experiment.

[0175] NACA TPP + Conjugated analog

[0176] N-acetylcysteine ​​amide (NACA), a more lipophilic, membrane-permeable, and blood-brain barrier-permeable analog of the antioxidant thiol NAC, has been shown to be more potent than NAC in reversing paracetamol toxicity and oxidative stress-induced disease (see Example 2). NACA has been shown to enhance the therapeutic efficacy of neural stem cell-based anti-glioma oncolytic virus therapy. Multiple analogs of NACA and Mito-NACA can be synthesized. These compounds may enhance antitumor immune function.

[0177] Although the present invention has been described in considerable detail with reference to specific embodiments, those skilled in the art will recognize that the present invention may be applied to embodiments other than those presented for purposes of illustration. These embodiments are presented for purposes of illustration, not limitation. Accordingly, the scope of the appended claims should not be limited to the description of the embodiments contained herein.

[0178] method

[0179] Mito 10 The methods used for the synthesis of -NAC and its analogs, cellular experiments, and statistical analysis follow standard scientific methods that are routinely used and have been previously described.

[0180] Cancer cell lines. The following routinely authenticated cell lines were obtained from the American Tissue Culture Collection (Manassas, VA): MiaPaCa-2 (catalog no. CRL-1420, human pancreatic cancer cells), MDA-MB-231 (catalog no. HTB-26, human breast cancer cells), and MCF-7 (catalog no. HTB-22, human breast cancer cells). The UACC-62 melanoma cell line was purchased and routinely authenticated from AddexBio (San Diego, CA, catalog no. C0020003). All cell lines were cultured at 37°C in 5% carbon dioxide. MiaPaCa-2 and MDA-MB-231 cells were cultured in DMEM medium (Thermo Fisher Scientific, catalog no. 11965) supplemented with 10% fetal bovine serum. MCF-7 cells were cultured in MEM-α medium (Thermo Fisher Scientific, catalog no. 12571) supplemented with 10% fetal bovine serum. UACC-62 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, catalog no. 11875) supplemented with 10% fetal bovine serum. All cells were stored in liquid nitrogen and used within 20 passages after thawing.

[0181] Cell proliferation measurements. Cell proliferation was monitored using the IncuCyte Live-Cell Analysis System. This imaging system is non-invasive and allows for continuous monitoring of cell confluency over several days. Cells were seeded in triplicate at 1,000 cells / well in 96-well plates and allowed to adhere overnight. Cells were then treated with the tested compounds at the indicated concentrations, and cell confluency was recorded over several days using the IncuCyte Live-Cell Analysis System.

[0182] Intracellular ATP levels. Cells were seeded overnight at 1,000 cells / well in a 96-well plate and then exposed to NAC analogs for 24 hours. Intracellular ATP levels were measured using a luciferase-based assay according to the manufacturer's instructions (catalog number FLAA, Sigma-Aldrich, St. Louis, MO). Briefly, an ATP assay mix solution consisting of luciferase and luciferin (catalog number FLAAM) was added to the cell lysate. After mixing, the amount of light produced was immediately recorded using a luminometer. Results were normalized to the total protein level measured in each well using the Bradford method (Bio-Rad Laboratories, Hercules, CA).

[0183] Mitochondrial respiration measurements. Mitochondrial oxygen consumption was measured using a Seahorse XF-96 extracellular flux analyzer (Agilent, North Billerica, MA). Bioenergetic function assays were used to assess cellular mitochondrial function in response to drug treatment. Cells were treated with either NAC or Mito 10 After 24 hours of treatment with -NAC, eight baseline OCR measurements were performed before injection of oligomycin (1 mg / mL) to inhibit ATP synthase, dinitrophenol (50 μM) to dissociate mitochondria and maximize OCR, and inhibitors of complexes I and III (1 μM rotenone and antimycin) to inhibit mitochondrial respiration. These measurements were used to determine indicators of mitochondrial function.

[0184] For mitochondrial complex I activity measurements, mitochondrial complex I-induced OCR measurements were performed using permeabilized cells in the presence of pyruvate / malate, a complex I substrate, and malonate (10 mM), a complex II inhibitor. 50 The values ​​were measured as previously reported.

[0185] Statistical analysis. Comparisons between control and treatment groups were performed using the unpaired Student's t-test. A p-value of less than 0.05 was considered statistically significant. All values ​​represent the mean ± standard deviation. The number of replicates per treatment group is indicated by n. IC 50 Values ​​and fitting curves were calculated using OriginPro 2016 (OriginLab Corporation, Northampton, MA).

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[0187] For completeness, various aspects of the invention are set forth in the following numbered clauses.

[0188] Clause 1. A compound of formula (I), or a pharmaceutically acceptable salt thereof. JPEG2025535717000043.jpg33168In formula, R 1 is H, C1-C4 alkyl, or JPEG2025535717000044.jpg46169, R 2 is H or C1-C4 alkyl, W is NH, O, or S; L is C1-C 20 Alkylene, C2-C 20 Alkenylene, L1-R A -L2, or an amino acid, L1 and L2 are each independently absent or C1-C 10 is alkylene, R A Ha-(CH2CH2O) q -, arylene, or cycloalkylene; q is 1-20, X is a counterion, Y is, independently at each occurrence, CF, Me, Cl, OMe, C(O)CH, NO, N(Me), or OH; m is independently 0, 1, 2, 3, 4, or 5; However, the compound is not (R)-2-acetamido-3-mercapto-N-methylpropanamide or (R)-2-acetamido-3-mercapto-N-ethylpropanamide.

[0189] Clause 2. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, wherein R 1 but JPEG2025535717000045.jpg45162.

[0190] Clause 3. A compound according to clause 2, or a pharmaceutically acceptable salt thereof, wherein R2 is H and W is NH or O.

[0191] Clause 4. A compound according to Clause 3, having the structure of formula (Ia), or a pharmaceutically acceptable salt thereof. JPEG2025535717000046.jpg47162In the formula, n is 1 to 20.

[0192] Clause 5. A compound according to Clause 3, having the structure of formula (Ib), or a pharmaceutically acceptable salt thereof. JPEG2025535717000047.jpg48162In the formula, n is 1 to 10.

[0193] Clause 6. A compound according to any one of clauses 1 to 5, or a pharmaceutically acceptable salt thereof, wherein m is 0 or 1.

[0194] Clause 7. A compound according to clause 1, or a pharmaceutically acceptable salt thereof, R1 is H or C1-C4 alkyl; R2 is H, and W is NH or O.

[0195] Clause 8. A compound according to Clause 7, having the structure of formula (Ic), (Id), or (Ie), or a pharmaceutically acceptable salt thereof: JPEG2025535717000048.jpg59162In formula, t is 1 to 20, u is between 1 and 10, and v is between 1 and 10.

[0196] Clause 9. A compound according to clause 1, JPEG2025535717000049.jpg134162, or a pharmaceutically acceptable salt thereof.

[0197] Clause 10. An isotopically labeled compound according to clause 1, or a pharmaceutically acceptable salt thereof.

[0198] Article 11. 13 11. A C-labeled compound according to clause 10, or a pharmaceutically acceptable salt thereof.

[0199] Clause 12. A compound according to Clause 11, having the structure of formula (If), or a pharmaceutically acceptable salt thereof. JPEG2025535717000050.jpg33162

[0200] Article 13. Compounds JPEG2025535717000051.jpg32161, 13. The compound according to clause 12, wherein n is 1 to 20.

[0201] Clause 14. A pharmaceutical composition comprising a compound according to any one of clauses 1 to 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0202] Clause 15. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to any one of clauses 1 to 13, or a pharmaceutically acceptable salt thereof.

[0203] Clause 16. The method of clause 15, wherein the cancer is pancreatic cancer, breast cancer, melanoma cancer, non-small cell lung cancer, or a combination thereof.

[0204] Clause 17. The method of any one of clauses 15 to 16, further comprising administering to the subject an additional therapeutic agent.

[0205] Clause 18. The method of clause 17, wherein the additional therapeutic agent is AZD3965.

[0206] Clause 19. A method of enhancing CAR-T cell therapy in a subject in need thereof, comprising administering to the subject an effective amount of a compound according to any one of clauses 1 to 13, or a pharmaceutically acceptable salt thereof.

[0207] Clause 20. The method of clause 19, wherein the subject is a cell or a human.

[0208] Clause 21. The method of clause 20, wherein the subject is a human, comprising administering to the subject an effective amount of a compound, or a pharmaceutically acceptable salt thereof, in combination with CAR-T cell therapy.

[0209] Clause 22. The method of clause 20, wherein the subject is a human, comprising pretreating CAR-T cells with a compound, or a pharmaceutically acceptable salt thereof, and administering to the subject an effective amount of the pretreated CAR-T cells.

[0210] Clause 23. A method of analyzing a sample, comprising contacting said sample with an isotopically labeled compound according to any one of clauses 10 to 13, or a pharmaceutically acceptable salt thereof, thereby producing a labeled sample, and analyzing said labeled sample.

[0211] Clause 24. The method of clause 23, wherein the sample comprises cells.

[0212] Clause 25. The method of any one of clauses 23 to 24, wherein the step of analyzing the labeled sample comprises analyzing a profile or image of the labeled sample.

[0213] Clause 26. The method of clause 25, wherein analyzing the labeled sample comprises analyzing a profile of the labeled sample.

[0214] Clause 27. The method of clause 26, wherein said profile comprises a proteomic profile.

[0215] Clause 28. The method of clause 27, comprising analyzing the proteomic profile of the labeled sample using mass spectrometry.

[0216] Clause 29. The method of clause 25, wherein analyzing the labeled sample comprises analyzing an image of the labeled sample.

[0217] Clause 30. The method of clause 23, comprising generating an image of the labeled sample using magnetic resonance imaging (MRI).

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof. During the ceremony, R １ is H, C １ -C ４ alkyl, or and R ２ is H or C １ -C ４ is alkyl, W is NH, O, or S; L is C １ -C ２０ Alkylene, C ２ -C ２０ Alkenylene, L １ -R Ａ -L ２ or an amino acid, L １ and L ２ are each independently absent or C １ -C １０ is alkylene, R Ａ Ha-(CH ２ CH ２ O) ｑ -, arylene, or cycloalkylene; q is 1-20; X is a counter ion, Each Y is independently CF ３ , Me, Cl, OMe, C(O)CH ３ , NO ２ , N(Me) ２ or OH, m in each occurrence is independently 0, 1, 2, 3, 4, or 5; However, the compound is not (R)-2-acetamido-3-mercapto-N-methylpropanamide or (R)-2-acetamido-3-mercapto-N-ethylpropanamide.

2. The R １ but 2. The compound of claim 1, wherein:

3. The R ２ is H and said W is NH or O, or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, having the structure of formula (Ia), or a pharmaceutically acceptable salt thereof: In the formula, n is 1 to 20.

5. The compound according to claim 3, having the structure of formula (Ib), or a pharmaceutically acceptable salt thereof: In the formula, n is 1 to 10.

6. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 0 or 1.

7. R １ is H or C1-C4 alkyl; R ２ is H, 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein W is NH or O.

8. 8. The compound of claim 7, having the structure of formula (Ic), (Id), or (Ie), or a pharmaceutically acceptable salt thereof: During the ceremony, t is 1-20, u is 1-10, v is 1-10.

9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

10. 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is isotopically labeled.

11. The compound １３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, which is C-labeled.

12. 12. The compound of claim 11, wherein the compound has the structure of formula (If):

13. The compound 13. The compound of claim 12, wherein n is 1 to 20.

14. 10. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

15. 10. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

16. 16. The method of claim 15, wherein the cancer is pancreatic cancer, breast cancer, melanoma cancer, non-small cell lung cancer, or a combination thereof.

17. 16. The method of claim 15, further comprising administering to the subject an additional therapeutic agent.

18. 18. The method of claim 17, wherein the additional therapeutic agent is AZD3965.

19. 1. A method for enhancing CAR-T cell therapy in a subject in need thereof, comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

20. 20. The method of claim 19, wherein the subject is a cell or a human.

21. 21. The method of claim 20, wherein the subject is a human, and the method comprises administering to the subject an effective amount of the compound, or a pharmaceutically acceptable salt thereof, in combination with CAR-T cell therapy.

22. The method of claim 20, wherein the subject is a human, comprising the steps of pretreating CAR-T cells with the compound, or a pharmaceutically acceptable salt thereof, and administering an effective amount of the pretreated CAR-T cells to the subject.

23. 1. A method of analyzing a sample, comprising: contacting the sample with the isotopically labeled compound of claim 1, or a pharmaceutically acceptable salt thereof, thereby producing a labeled sample; and analyzing the labeled sample.

24. 24. The method of claim 23, wherein the sample comprises cells.

25. 24. The method of claim 23, wherein analyzing the labeled sample comprises analyzing a profile or image of the labeled sample.

26. 26. The method of claim 25, wherein analyzing the labeled sample comprises analyzing the profile of the labeled sample.

27. 27. The method of claim 26, wherein the profile comprises a proteomic profile.

28. 28. The method of claim 27, comprising analyzing the proteomic profile of the labeled sample using mass spectrometry.

29. 26. The method of claim 25, wherein analyzing the labeled sample comprises analyzing the image of the labeled sample.

30. 24. The method of claim 23, comprising generating the image of the labeled sample using magnetic resonance imaging (MRI).

Images