Methods of enhancing levodopa therapeutic efficacy

Abstract

The present disclosure provides methods of enhancing levodopa therapeutic efficacy for the treatment of Parkinson's disease. The present methods can include administration of compounds having a triphenylphosphonium moiety. Advantageously, the present method can mitigate microbial metabolism of levodopa to dopamine in the gut, improve bioavailability of levodopa in brain, and increase dopamine levels in the brain.

Description

METHODS OF ENHANCING LEVODOPA THERAPEUTIC EFFICACY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Provisional Patent Application No. 63 / 596,151, filed November 3, 2023, the content of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] A major hallmark of Parkinson’s disease (PD) is the loss of dopamine-producing (dopaminergic) neurons in the striatum, resulting in motor problems. PD is a progressive, debilitating neurodegenerative disease that results from the loss of dopaminergic neurons in the substantia nigra. At present, there is no clinical treatment to halt the progression of PD. For over 50 years, levodopa (L-dopa) has been a drug of choice for treatment of PD. L-dopa is converted to dopamine in the central nervous system, and thus serves to replace the lost dopamine and normalize signaling in the brain.

[0003] L-dopa therapy does not reverse or slow degeneration of dopaminergic neurons in PD, but it significantly reduces motor symptoms and increases quality of life, particularly early in its therapeutic course. However, as PD progresses, L-dopa doses often need to be increased to manage the PD symptoms, resulting in L-dopa-induced dyskinesia (LID). LID, which occurs in more than 50% of L-dopa treated patients, is characterized by abnormal and excessive voluntary movements that are so severe that they interfere with gross and fine movements needed for daily life. LIDs present a therapeutic conundrum as their appearance necessitates reducing L-dopa doses, resulting in increased PD symptoms.

[0004] Thus, there remains a need for methods of enhancing L-dopa therapeutic efficacy in Parkinson’s disease. SUMMARY OF THE INVENTION

[0005] In one aspect, the present disclosure provides a method of modulating microbial metabolism of levodopa in gut of a subject in need thereof, the method comprises administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl;R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;Mito is ; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0006] In another aspect, the present disclosure provides a method of mitigating microbial degradation of levodopa in gut of a subject in need thereof, the method comprises administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;Mito is; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0007] In another aspect, the present disclosure provides a method of improving bioavailability of levodopa in brain of a subject in need thereof, the method comprises administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0008] In yet another aspect, the present disclosure provides a method of treating Parkinson’s disease in a subject in need thereof, the method comprises administering to the subject an effective amount of levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0009] In some embodiments, the uptake of levodopa in the brain of the subject is increased and / or formation of dopamine in the brain of the subject is increased.

[0010] In some embodiments, the microbial metabolism of levodopa to dopamine in the gut of the subject is decreased.

[0011] In some embodiments, the levodopa is administered to the subject in a combination with carbidopa.

[0012] The present disclosure also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, an effective amount of levodopa, and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0013] In some embodiments, the pharmaceutical composition further comprises carbidopa.

[0014] In another aspect, the present disclosure provides a compound of formula (III-a) or (IV-a), or a pharmaceutically acceptable salt thereof,w ere n one of R3and R4is Mito, the other is –(CH2CH2O)v–RA; v is 1-20; RAis H or C1-4alkyl;Mito is; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0015] In another aspect, the present disclosure provides a compound of formula (V-a) or (VI-a), or a pharmaceutically acceptable salt thereof,wherein each of R5and R6is –(CH2CH2O)w–RAor RB; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl.

[0016] In another aspect, the present disclosure provides a compound of formula (VII), or a pharmaceutically acceptable salt thereof, wherein R7is Mito; Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For a more complete understanding of the embodiments and the advantages thereof, reference is now made to the following description, in conjunction with the accompanying figures briefly described as follows:

[0018] FIGS. 1A-1D show the effects of Mito-ortho-HNK analogs on the bacterial proliferation of E. faecalis. The effects of HNK (FIG.1A), Mito-ortho-HNK (FIG. 1B), Mito-PEG4-HNK (FIG.1C), Dec-HNK (FIG.1D) on the proliferation of E. faecalis were monitored at OD600for 6 h. Data shown are the mean±SD, n=4.

[0019] FIGS. 2A-2H show effects of Mito-ortho-HNK on the bacterial L-dopa consumptions with or without carbidopa. E. faecalis was treated with the Mito-ortho-HNK as indicated in presence of 1 mM L-dopa alone (FIGS.2A-2D) or with additional 0.22 mM of carbidopa (FIGS. 2E-2H). The effects of Mito-ortho-HNK on the proliferation inpresence of L-dopa alone (FIG. 2A) or in presence of L-dopa and carbidopa combination (FIG. 2E) were monitored at OD600for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. High-performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG. 2B and FIG.2F). The effects of Mito-ortho-HNK on L-dopa consumption (FIG.2C and FIG. 2G) and dopamine formation (FIG. 2D and FIG. 2H) are shown in presence of 1 mM L-dopa alone (FIGS. 2A-2D) or with additional 0.22 mM of carbidopa (FIGS. 2E- 2H) respectively. Statistical significance was determined using a Student’s t-test. *, P<0.05 or **, P<0.01 vs control group at each collection time point. Data shown are the mean±SD, n=3.

[0020] FIGS. 3A-3D show effects of Mito-PEG5-ATO on the bacterial L-dopa consumptions. E. faecalis was treated with Mito-PEG5-ATO as indicated in presence of L- dopa (1 mM). The effects of Mito-PEG5-ATO on the proliferation (FIG. 3A) were monitored at OD600for 6 h and culture media were collected at indicated time points for L- dopa and dopamine measurements. High-performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG.3B). The effects of Mito- PEG5-ATO on L-dopa consumption and dopamine formation were shown in FIGS.3C and 3D, respectively. Statistical significance was determined using a Student’s t-test. *, P<0.05 or **, P<0.01 vs control group at each collection time point. Data shown are the mean±SD, n=3.

[0021] FIGS. 4A-4D show the effects of commonly used antibiotics and Mito-PEG- ATO analogs on the proliferation of gram-positive and gram-negative bacteria. The effects of commonly used antibiotics (chloramphenicol [Cam] and ampicillin [Amp]), Mito-PEG2- ATO, and Mito-PEG5-ATO on the proliferation were monitored at OD600 for 6 h in gram- positive E. faecalis (FIG.4A) and S. aureus (FIG.4B) and in gram-negative E. coli (FIG. 4C) and P. aeruginosa (FIG.4D). Data shown are the mean±SD, n=4.

[0022] FIGS.5A-5D show effects of Mito-ortho-HNK and commonly used antibiotics on the bacterial membrane potential. (FIGS.5A and 5B) E. faecalis was treated with Mito- ortho-HNK (FIG. 5A) or commonly used antibiotics (FIG. 5B, chloramphenicol [Cam] and ampicillin [Amp]) as indicated for 1 h. The effects on the membrane potential were measured by TMRM dye (50 nM), the fluorescence indicator, to determine the percentage change in TMRM fluorescence intensity between the control and treatments groups. The lower levels of TMRM fluorescence resulting from treatment reflect the depolarization of mitochondrial membrane potential. Statistical significance was determined using aStudent’s t-test. **, P<0.01 vs control group at each collection time point. Data shown are the mean±SD, n=4. (FIGS. 5C and 5D) E. faecalis was treated with Mito-ortho-HNK (FIG.5C) or commonly used antibiotics (FIG.5D) as indicated for 3 h, and cell death was monitored in real time by SYTOX Green staining. Data shown are the mean±SD for n=4.

[0023] FIGS. 6A-6F show effects of Mito-ortho-HNK and antibiotics on ATP. E. faecalis were treated with Mito-ortho-HNK (FIG. 6A) or commonly used antibiotics (chloramphenicol [Cam], FIG. 6B; ampicillin [Amp], FIG. 6C) as indicated. FIGS.6A- 6C show the effects of Mito-ortho-HNK or antibiotics on E. faecalis proliferation shown as absorbance at OD600. FIGS.6D-6F show the effects of Mito-ortho-HNK or antibiotics on intracellular ATP levels as total luciferin luminescence. Data shown are the mean±SD, n=4.

[0024] FIGS.7A-7D show effect of Mito-ortho-HNK on L-dopa / dopamine metabolism in vivo. Mice were orally treated with 150 mg / kg L-dopa-d3 with or without Mito-ortho- HNK for 2 h. The effects of Mito-ortho-HNK on dopamine-d3 formation and L-dopa-d3 consumption were measured. L-dopa-d3 and dopamine-d3 were separated and monitored by LC-MS-SIM. Standards are shown in (FIG. 7A). Effects of Mito-ortho-HNK on L- dopa-d3 consumption and dopamine-d3 formation in the mice gut samples were measured, and the representative LC-MS-SIM trace are shown in (FIG. 7B). The formation of dopamine-d3 in mice brain samples was measured as described in the Methods section. (FIG.7C) The amount of dopamine-d3 in the brain is based on the area under the curve of the LC-MS-SIM dopamine-d3 peak. Statistical significance was determined using a Student’s t-test. The values shown are the fold change in Mito-ortho-HNK-treated mice compared with controls. (**) Statistically significant increase in dopamine-d3 formation compared with control (fold change>15 and P<0.01). The representative LC-MS-SIM traces are shown in (FIG.7D).

[0025] FIGS. 8A-8B illustrate gut microbial metabolism. (FIG. 8A) shows the gut metabolism of L-dopa and m-tyramine, and (FIG. 8B) shows MTD inhibition of gut bacterial metabolism of L-dopa to dopamine and m-tyramine.

[0026] FIGS. 9A-9B illustrate chemical structures of drugs, MTDs and PEGylated MTDs, and L-dopa and analogs. Chemical structures of drugs, MTDs, and PEGylated MTDs are shown in (FIG.9A), and of L-dopa and analogs are shown in (FIG.9B).

[0027] FIGS.10A-10B show effects of Mito-LND analogs and Mito-MGN analogs on the bacterial proliferation of E. faecalis. The effects of LND (FIG. 10A) and Mito-LND(FIG.10B) on the proliferation of E. faecalis were monitored at OD 600 nm for 6 h. Data shown are the mean±SD, n=4.

[0028] FIG. 11 shows the effects of common antibiotics and Mito-PEG-ATO analogs on the bacterial ATP level. S. aureus cells were treated with common antibiotics ampicillin (Amp) and chloramphenicol (Cam), Mito-PEG2-ATO, and Mito-PEG5-ATO for 1 h. Intracellular ATP levels were measured using a luciferase-based assay. Data are represented as a percentage of control (non-treated) cells after normalization to total cell number (OD 600 nm). The relative cell ATP levels (control is taken as 100%) are plotted against concentration. Dashed lines represent the fitting curves used to determine the IC50values as indicated. Data shown are the mean±SD.

[0029] FIG. 12 shows the effects of Mito11-APO on the bacterial proliferation of E. faecalis. The effects of Mito11-APO on the proliferation of E. faecalis were monitored at OD600 for 6 h. Data shown are the mean±SD, n=4.

[0030] FIGS. 13A-13D show the effects of Mito11-APO on the bacterial L-dopa consumptions with carbidopa. E. faecalis was treated with the Mito11-APO as indicated in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa. The effects of Mito11-APO on the proliferation in presence of L-dopa and carbidopa combination (FIG. 13A) were monitored at OD600 for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. High-performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG.13B). The effects of Mito11-APO on L-dopa consumption (FIG.13C) and dopamine formation (FIG.13D) are shown in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD, n=4.

[0031] FIG. 14 shows the effects of Mito10-APO on the bacterial proliferation of E. faecalis. The effects of Mito10-APO on the proliferation of E. faecalis were monitored at OD600 for 6 h. Data shown are the mean±SD, n=4.

[0032] FIGS. 15A-15D show the effects of Mito10-APO on the bacterial L-dopa consumptions with carbidopa. E. faecalis was treated with the Mito10-APO as indicated in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa. The effects of Mito10-APO on the proliferation in presence of L-dopa and carbidopa combination (FIG. 15A) were monitored at OD600 for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. High-performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG.15B). The effectsof Mito10-APO on L-dopa consumption (FIG.15C) and dopamine formation (FIG.15D) are shown in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD, n=4.

[0033] FIG. 16 shows the effects of Mito2-APO on the bacterial proliferation of E. faecalis.

[0034] FIGS.17A-17B show the effects of MitoQ analogs on the bacterial proliferation of E. faecalis. The effects of MitoQ (FIG. 17A), DM-MitoQ (FIG. 17B) on the proliferation of E. faecalis were monitored at OD600 for 6 h. Data shown are the mean±SD, n=4.

[0035] FIGS.18A-18D show the effects of MitoQ and DM-MitoQ on the bacterial L- dopa consumptions with carbidopa. E. faecalis was treated with the MitoQ or DM-MitoQ as indicated in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa. The effects of MitoQ or DM-MitoQ on the proliferation in presence of L-dopa and carbidopa combination (FIG.18A) were monitored at OD600 for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. High-performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG. 18B). The effects of MitoQ or DM-MitoQ on L-dopa consumption (FIG. 18C) and dopamine formation (FIG. 18D) are shown in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD, n=3.

[0036] FIG. 19 shows the structures of the Mito-APO, Mito-PEG-APO, and Mito-Q analogs.

[0037] The drawings illustrate only example embodiments and are therefore not to be considered limiting of the scope of the embodiments described herein, as other embodiments are within the scope of the disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0038] Before the present materials and methods are described, it is 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 will be limited only by the appended claims.

[0039] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a compound” should be interpreted to mean “one or more compounds” unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.”

[0040] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it 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.

[0041] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0042] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March ‘s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0043] The term “alkyl” as used herein, means a straight or branched chain saturated hydrocarbon. The alkyl can be a С1-4alkyl. Representative examples of alkyl include, butare 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.

[0044] The term “alkylene,” as used herein, means a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)CH2-, and CH2CH(CH3)CH(CH3)CH2-.

[0045] The term “alkene” as used herein, means an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10- alkenyl, and C2-C6-alkenyl, respectively.

[0046] The term “alkenylene” as used herein, means a divalent group derived from a straight or branched alkene, which attaches to the parent molecule at two different carbon atoms.

[0047] The term “aryl,” as used herein, means a carbocyclic aromatic group (e.g., phenyl or a bicyclic aryl). The term “aryl” includes polycyclic ring systems having one or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls or cycloalkenyls. For example, a bicyclic aryl can be a phenyl fused to a cycloalkyl moiety. Examples of aryl include naphthyl, dihydronaphthalenyl, tetrahydronaphthalenes, indanyl, or indenyl. The aryl (e.g., phenyl and bicyclic aryls) is attached to the parent molecular moiety through any carbon atom contained within the aryl.

[0048] The term “arylene” as used herein, means a divalent group derived from an aryl as described herein, which attaches to the parent molecule at two different ring carbon atoms. Examples of arylene includes, but are not limited to, phenylene, which is a divalent group derived from benzene and attaches to the parent molecule at two different ring carbon atoms (e.g., at 1,2-, 1,3-, or 1,4-positions).

[0049] The term “cycloalkyl” as used herein, means a monovalent group derived from an all-carbon ring system containing zero heteroatoms as ring atoms, and zero double bonds. The all-carbon ring system can be a monocyclic, bicylic, or tricyclic ring system, and can be a fused ring system, a bridged ring system, or a spiro ring system, or combinations thereof. Examples of cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and . The cycloalkyl groups described herein can be appended to the parent molecular moiety through any substitutable carbon atom.

[0050] The term “cycloalkylene” as used herein, means a divalent group derived from an all-carbon ring system containing zero heteroatoms as ring atoms and zero double bonds, which attaches to the parent molecule at two different ring carbons atoms. The all-carbon ring system can be a monocyclic, bicylic, or tricyclic ring system, and can be a fused ring system, a bridged ring system, or a spiro ring system. Representative examples of cycloalkylene include, but are not limited to, those derived from C3-10rings, such as

[0051] The term “halogen” or “halo” means a chlorine, bromine, iodine, or fluorine atom.

[0052] Terms such as “alkyl,” “cycloalkyl,” “alkylene,” “arylene,” or “cycloalkylene,” etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “С1-С4alkyl,” “C1-4alkyl,” “C3-6cycloalkyl,” “C1-4alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation “C” followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, “C3alkyl” is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in “C1-C4” or “C1-4,” the members of the group that follows may have any number of carbon atoms falling within the recited range. A “C1-C4alkyl” or “C1-4alkyl,” for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0053] If substituents are described as being independently selected from a group, each substituent is selected independent of the other. Each substituent, therefore, may be identical to or different from the other substituent(s).

[0054] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, regioisomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for eachasymmetric center, (Z) and I double bond isomers, and (Z) and I conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. The compound disclosed herein may exist as a regioisomer or a mixture of regioisomers. Unless otherwise stated, all tautomeric and regioisomeric forms of the compounds of the invention are within the scope of the invention.

[0055] The term “pharmaceutically acceptable salt thereof” means a salt prepared by combining a compound of formula (III), (IV), (V), or (VI) with an acid whose anion, 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 because of their greater aqueous solubility relative to the parent compound. For use in medicine, the salts of the compounds of this invention are non-toxic “pharmaceutically acceptable salts”. Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.

[0056] Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention when possible include those derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids. Specific examples of suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate (pamoate), methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, P-hydroxybutyrate, galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate, hexanoate, nicotinate, 2- naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, and undecanoate.

[0057] Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, i.e., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., 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.

[0058] Organic salts may be made from secondary, tertiary or quaternary amine salts, such as tromethamine, diethylamine, N, N’-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl (C1-C6) halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (i.e., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0059] The term “isotopically labelled” refers to compounds of formula (III), (IV), (V), or (VI) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Examples of isotopes suitable for inclusion in the compounds of the disclosure include isotopes of hydrogen, such as2H and3H, carbon, such as11C,13C and14C, chlorine, such as36Cl, fluorine, such as18F, iodine, such as123I and125I, nitrogen, such as13N and15N, oxygen, such as15O,17O and18O, phosphorus, such as32P, and sulfur, such as35S. Certain isotopically labelled compounds of formula (III), (IV), (V), or (VI), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon-14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such as11C,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds of formula (III), (IV), (V), or (VI) may generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using anappropriate isotopically labeled reagents in place of the non-labeled reagent previously employed.

[0060] Site specific substitution of atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number that predominates in nature can be regarded as a substituent of a compound of the present disclosure. A sample of a compound having such an isotope as a substituent has at least 50% isotope incorporation at the labelled position(s). The concentration of such isotopes, e.g., deuterium, may be defined by the isotopic enrichment factor. The term “isotopic enrichment factor” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. For example, if a substituent in a compound of this invention is denoted deuterium, such compound has 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).

[0061] As used herein, the term “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired result, including a desired therapeutic result, such as modulation of microbial metabolism of levodopa, mitigation of microbial degradation of levodopa, improvement of bioavailability of levodopa in the brain, and treatment of Parkinson’s disease. An effective amount of the compounds as disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the disclosed compounds to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compounds as disclosed herein are reduced as compared with known compounds and are outweighed by the therapeutically beneficial effects.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meanings 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 specificallymentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0063] Parkinson’s disease can be managed using levodopa; however, as Parkinson’s disease progresses, patients require increased doses of levodopa, which can cause undesirable side effects. Additionally, the oral bioavailability of levodopa decreases in Parkinson’s disease patients due to the increased metabolism of levodopa to dopamine by gut bacteria, Enterococcus faecalis, resulting in decreased neuronal uptake and dopamine formation. Parkinson’s disease patients have varying levels of these bacteria. Thus, decreasing bacterial metabolism can be a promising therapeutic approach to enhance the bioavailability of levodopa in the brain. However, there remains an unmet need for agents that effectively modulate bacterial metabolism of levodopa and improve bioavailability of the levodopa in the brain.

[0064] The present disclosure relates to methods of enhancing levodopa therapeutic efficacy in Parkinson’s disease. In various embodiments, the present method may involve inhibiting gut microbial metabolism of levodopa. In particular embodiments, the present disclosure demonstrates that Mito-ortho-HNK, formed by modification of a naturally occurring molecule, honokiol, conjugated to a triphenylphosphonium moiety, mitigates the metabolism of levodopa—alone or combined with carbidopa—to dopamine. Mito-ortho- HNK suppresses the growth of E. faecalis, decreases dopamine levels in the gut, and increases dopamine levels in the brain. Mitigating the gut bacterial metabolism of levodopa as shown here could enhance its efficacy.

[0065] For decades, levodopa (L-dopa) has been used to manage symptoms caused by the depletion of endogenous dopamine in the brains of patients with Parkinson’s disease (PD). A characteristic hallmark of PD is the loss of dopamine-producing neurons in the striatum. Orally administered L-dopa crosses the blood–brain barrier and is metabolized to dopamine in the brain by the enzyme aromatic L-amino acid decarboxylase. As PD progresses, patients require increased doses of L-dopa, the side effects of which are dyskinesia and drug toxicity. Unfortunately, as PD progresses, more dopaminergic neurons are lost, which—together with likely development of tolerance to dopamine effects at its receptors—results in an increased need for L-dopa to maintain therapeutic efficacy. As L-dopa doses are increased, however, the potential for the development of L-dopa induced dyskinesia is enhanced. L-dopa induced dyskinesia is extremely debilitating and often requires withdrawal of L-dopa therapy.

[0066] Nearly five decades ago, it was reported that L-dopa can be metabolized by the gut microbiota. Therapeutic implications of extracerebral metabolism of L-dopa in treatment of PD were also discussed. Only recently, the specific genes and enzymes of the microbial metabolism of L-dopa were identified. The decrease in the oral bioavailability of L-dopa in patients with PD was linked to its increased metabolism to dopamine by gut bacteria, resulting in decreased uptake of L-dopa and dopamine formation in the brain. Dopamine produced by decarboxylation of L-dopa in the gut does not cross the blood– brain barrier. In particular, the presence of commensal Enterococcus faecalis in the gut microbiota of PD patients correlates with increased metabolism of L-dopa in gut microbiota and decreased striatal dopamine levels. Thus, decreasing the microbial metabolism of L- dopa may provide an approach to enhancing its bioavailability and, as a result, its conversion to dopamine in the brain.

[0067] Using genome-mining techniques, E. faecalis was identified as the microbial species responsible for L-dopa metabolism. Investigators discovered that L-dopa is metabolized to dopamine by E. faecalis-derived tyrosine decarboxylases. Ex vivo human fecal suspensions from PD patients and healthy individuals were used to show that E. faecalis in gut microbiota is responsible for L-dopa metabolism. Thus, L-dopa metabolism by gut commensal bacteria results in reduced therapeutic efficacy of L-dopa as well as increased m-tyramine generation from dopamine, which can have serious adverse effects. Alpha-fluoromethyl amino acids, known inhibitors of tyrosine decarboxylases, and the L- tyrosine analog (S)-alpha-fluoromethyltyrosine, both inhibit L-dopa decarboxylation in E. faecalis. The role of the microbiota–gut–brain axis in regulating dopaminergic signaling is gaining increased attention.

[0068] Bacterial tyrosine decarboxylases is present in all but three of 655 E. faecalis genomes surveyed. Typically, carbidopa, an inhibitor of extracerebral aromatic L-amino acid decarboxylase, prevents its peripheral metabolism to dopamine. However, carbidopa is not an effective inhibitor of the enzyme tyrosine decarboxylases; it is 200-fold less active toward E. faecalis tyrosine decarboxylases relative to human dopa decarboxylase and is unable to prevent gut bacterial L-dopa metabolism.

[0069] Antimicrobial therapy has been suggested as a nontoxic, highly effective approach to mitigating the gut metabolism of L-dopa to dopamine and enhancing the uptakeand metabolism of L-dopa into the brain in murine models of PD. Cationic compounds can interact with bacterial membranes and inhibit bacterial growth, including triphenylphosphonium cation (TPP+)-based mitochondria-targeted compounds that have been shown to be effective antimicrobial agents. Compounds that decrease the electrochemical gradient of protons across the bacterial cytoplasmic membrane (also known as proton motive force) were reported to exert antibacterial effects. Compounds containing the hydrophobic cations such as TPP+with a delocalized positive charge exert protonophore-like activity in mitochondrial membranes. Although several TPP+-based mitochondria-targeted drugs (MTDs) exert antimicrobial effects, the present disclosure investigates the effect of mitochondria-targeted honokiol (Mito-ortho-HNK) and analogs on bacterial metabolism of L-dopa because these compounds have previously been used in preclinical animal models without significant toxicity.

[0070] In one aspect, the present disclosure provides a method of modulating microbial metabolism of levodopa in gut of a subject in need thereof, the method comprising administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein.

[0071] In another aspect, the present disclosure provides a method of mitigating microbial degradation of levodopa in gut of a subject in need thereof, the method comprising administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein.

[0072] In another aspect, the present disclosure provides a method of improving bioavailability of levodopa in brain of a subject in need thereof, the method comprising administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein.

[0073] In another aspect, the present disclosure provides a method of treating Parkinson’s disease in a subject in need thereof, the methods comprise administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein.

[0074] The foregoing methods include administration of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,w ere n one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl;R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20; Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0075] In some embodiments, the uptake of levodopa in the brain of the subject is increased and / or formation of dopamine in the brain of the subject is increased. For example, the present methods may include an increase in the uptake of levodopa in the brain of the subject, such as an increase of levodopa uptake of about 5%, about 10%, about 20%, about 50%, about 100%, about 200%, or about 500%. For example, the present methods may include an increase of the formation of dopamine in the brain of the subject, such as an increase of dopamine formation of about 5%, about 10%, about 20%, about 50%, about 100%, about 200%, or about 500%.

[0076] In some embodiments, the microbial metabolism of levodopa to dopamine in the gut of the subject is decreased. For example, the present methods may include a decreaseof levodopa metabolism of about 5%, about 10%, about 20%, about 50%, or about 80% in the gut of the subject.

[0077] In some embodiments, the compound used for the present methods is a compound of formula (III) or (IV), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has formula (III) or (IV), in which one of R3and R4in formula (III) or (IV) is Mito and the other is H. In some embodiments, the compound has formula (III), in which R3is Mito and R4is H. In some embodiments, the compound has formula (III), in which R3is H and R4is Mito. In some embodiments, the compound has formula (IV), in which R3is Mito and R4is H.

[0078] In some embodiments, the compound used for the present methods is a compound of formula (V) or (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has formula (V) or (VI), in which each of R5and R6in formula (V) and (VI), respectively, is Mito.

[0079] In some embodiments, the compound used for the present methods is a compound of formula (VII) or (VIII), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (VII), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (VIII), or a pharmaceutically acceptable salt thereof, in which LAis C(O), LBis O, f is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and R8is H.

[0080] In some embodiments, the compound used for the present methods is a compound of formula (IX) or (X), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has formula (IX). In some embodiments, the compound has formula (X). In some embodiments, the compound has formula (IX) or (X), in which u is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0081] In some embodiments, X in the compounds as described herein is halogen, trifluoroacetate, or acetate. For example, X can be Br–or trifluoroacetate.

[0082] In some embodiments, L in the compounds as described herein is C1-20alkylene. For example, L can be –(CH2)4–, –(CH2)6–, –(CH2)8–, –(CH2)10–, or –(CH2)12–. In some embodiments, L is –(CH2)10–.

[0083] In other embodiments, L in the compounds as described herein is L1-RC-L2, and RCis –(CH2CH2O)q–. In some embodiment, RCis –(CH2CH2O)q–, in which q is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, L is –(CH2CH2O)q–CH2CH2–, in which q is 1, 2, 3, 4, or 5. In some embodiments, L is –(CH2CH2O)3–CH2CH2– or –(CH2CH2O)4–CH2CH2–.

[0084] Examples of compounds as described herein include those disclosed in WO 2016 / 201188 A1, WO 2019 / 136154 A1, WO 2021 / 081500 A1, and WO 2010 / 126719 A1, which are incorporated herein by reference in their entireties.

[0085] In some embodiments, the compound is selected from the group consisting ofor a pharmaceutically acceptable salt thereof.

[0086] The present methods may include simultaneous, separate, or sequential administration of levodopa and a compound as described herein. For example, levodopa and a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof can be administrated to a subject in need thereof simultaneously, separately, or sequentially. The present methods may include administration of levodopa and a compound as described herein in a combination with an additional active agent. The additional active agent can include, for example, known medications or therapies used in the management and treatment of Parkinson disease. The addition active agent may modulate the gut and / or peripheral metabolism of levodopa. As a nonlimiting example, carbidopa is currently used with L-dopa in the treatment of PD symptoms. Carbidopa inhibits the peripheral metabolism of L-dopa by acting as a substrate inhibitor of peripheral amino carboxylases. Thus, a combination of the present compounds and carbidopa may inhibit both the microbial metabolism and the peripheral metabolism of L-dopa. In some embodiments, the present methods include administrating levodopa, the compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, to the subject in a combination with carbidopa.

[0087] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, an effective amount oflevodopa, and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,w ere n one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or ORAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20; Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0088] The pharmaceutical compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions may contain a therapeutically effective amount of levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein, which is related to the dose of the compound to be administered.

[0089] The compositions may contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used. The activity of the compounds employed in the compositions and methods disclosed herein are notbelieved to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy. In some embodiments, the pharmaceutical composition includes a compound as described herein in a range from about 0.1 to about 2000 mg, such as from about 0.5 to 500 mg or from about 1 to about 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.01 mg / kg to about 1000 mg / kg body weight, such as about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 1000 mg / kg, about 0.1 to about 500 mg / kg, about 0.1 to about 100 mg / kg, or about 50 to about 100 mg / kg body weight.

[0090] In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end- points 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 – 1.0 µM).

[0091] The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. Suitable pharmaceutically acceptable carriers include, but are not limited to, for example, suitable diluents, vehicles, excipients, preservatives, solubilizers, emulsifiers, liposomes, or nanoparticles, among others. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solutions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include isotonic solutions, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0092] The formulation should be selected according to the mode of administration. The compositions may include a pharmaceutical carrier, excipient, or diluent, which are nontoxic to the subject being exposed thereto at the dosages and concentrations employed. Examples of pharmaceutical carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN brand surfactant, polyethylene glycol (PEG), and PLURONICSTM surfactant.

[0093] Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.

[0094] Suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both oral and intravenous administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as well as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, topical, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and / or intrathecal administration but not suitable for intracerebral administration.

[0095] The inert ingredients and manner of formulation of the pharmaceutical compositions may be selected from conventional technologies. The usual methods of formulation used in pharmaceutical science may be used here. Suitable types of compositions include, but are not limited to, tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdermal patches, and suspensions.

[0096] Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders, but any suitable capsule formulation can be used.

[0097] Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants, anddisintegrators (in addition to the compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.

[0098] Tablets can be coated with sugar, e.g., as a flavor enhancer and sealant. The compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasting substances, such as mannitol, in the formulation. Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty that some patients experience in swallowing solid objects.

[0099] A lubricant can be used in the tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.

[0100] Tablets can also contain disintegrators. Disintegrators are substances that swell when wetted to break up the tablet and release the compound. They include starches, clays, celluloses, algins, and gums. As further illustration, corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation- exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.

[0101] The composition can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments and soluble in basic environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0102] Transdermal patches can also be used to deliver the compounds. Transdermal patches can include a resinous composition in which the compound will dissolve or partially dissolve; and a film which protects the composition, and which holds the resinous composition in contact with the skin. Other, more complicated patch compositions can also be used, such as those having a membrane pierced with a plurality of pores through which the compound is pumped by osmotic action.

[0103] Pharmaceutical compositions adapted 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.

[0104] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0105] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

[0106] The composition can be prepared with materials (e.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans or non-human subjects. In some embodiments, the composition is suitable for use in humans. In some embodiments, the composition is prepared with materials having purity and / or other properties that render it suitable for administration to non-human subjects, but not suitable for administration to humans.

[0107] Each dosage unit may contain the dose of a given compound, for example, a daily dose, or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.

[0108] The composition may include a single compound or a combination of compounds as described herein for administration. For example, two or more of thecompounds described herein may be included in the composition. In addition, the composition may include solvate forms of the compounds or salts, esters, and / or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like.

[0109] The disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents. The additional therapeutic agent may include, for example, one or more known agent for treating Parkinson’s disease. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical compositions are formulated to comprise the disclosed compounds and further to comprise the one or more additional therapeutic agents.

[0110] In some embodiments, the compound used for the present pharmaceutical compositions is a compound of formula (III) or (IV), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has formula (III) or (IV), in which one of R3and R4in formula (III) or (IV) is Mito and the other is H. In some embodiments, the compound has formula (III), in which R3is Mito and R4is H. In some embodiments, the compound has formula (III), in which R3is H and R4is Mito. In some embodiments, the compound has formula (IV), in which R3is Mito and R4is H.

[0111] In some embodiments, the compound used for the present pharmaceutical compositions is a compound of formula (V) or (VI), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has formula (V) or (VI), in which each of R5and R6in formula (V) and (VI), respectively, is Mito.

[0112] In some embodiments, the compound used for the present pharmaceutical compositions is a compound of formula (VII) or (VIII), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (VII), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is a compound of formula (VIII), or a pharmaceutically acceptable salt thereof, in which LAis C(O), LBis O, f is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 and R8is H.

[0113]

[0113] In some embodiments, the compound used for the present pharmaceutical compositions is a compound of formula (IX) or (X), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has formula (IX). In someembodiments, the compound has formula (X). In some embodiments, the compound has formula (IX) or (X), in which u is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0114] In some embodiments, the pharmaceutical composition includes a compound as described herein, in which X is halogen, trifluoroacetate, or acetate.

[0115] In some embodiments, the pharmaceutical composition includes a compound as described herein, in which L is C1-20alkylene. In other embodiment, the pharmaceutical composition includes a compound as described herein, in which L is L1-RC-L2, and RCis – (CH2CH2O)q–.

[0116] In some embodiments, the pharmaceutical composition includes a compound as described herein, which is selected from the group consisting ofor a pharmaceutically acceptable salt thereof.

[0117] In some embodiments, the pharmaceutical composition further comprises an additional active agent, such as carbidopa.

[0118] In another aspect, the present disclosure provides a compound of formula (III-a) or (IV-a), or a pharmaceutically acceptable salt thereof,w ere n one of R3and R4is Mito, the other is –(CH2CH2O)v–RA; v is 1-20; RAis H or C1-4alkyl;Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0119] In some embodiments, the compound has formula (III-a), in which R3is Mito and R4is–(CH2CH2O)v–RA. In some embodiments, the compound has formula (III-a), in which R3is –(CH2CH2O)v–RAand R4is Mito. In some embodiments, the compound has formula (IV-a), in which R3is Mito and R4is–(CH2CH2O)v–RA. In some embodiments, the compound has formula (III-a) or (IV-a), in which v is 1, 2, 3, 4, or 5. In some embodiments, the compound has formula (III-a) or (IV-a), in which v is 1, 2, 3, 4, or 5 and RAis methyl.

[0120] Examples of compounds of formula (III-a) and (IV-a) include: oa p a aceu ca y accep a e sa e eo .

[0121] In another aspect, the present disclosure provides a compound of formula (V-a) or (VI-a), or a pharmaceutically acceptable salt thereof,wherein each of R5and R6is –(CH2CH2O)w–RAor RB; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl.

[0122] In some embodiments, the compound has formula (V-a) or (VI-a) , in which each of R5and R6is –(CH2CH2O)w–RAand w is 1, 2, 3, 4, or 5. In some embodiments, the compound has formula (V-a), in which R5is –(CH2CH2O)w–RA, w is 1, 2, 3, 4, or 5, and RAis methyl. In some embodiments, the compound has formula (V-a), in which R5is RB. For example, RBis C5, C6, C7, C8, C9, C10, or C11 alkyl.

[0123] In some embodiments, the compound has formula (VI-a), in which Z is NH, R6is –(CH2CH2O)w–RA, and w is 1, 2, 3, 4, or 5. In some embodiments, the compound has formula (VI-a), in which Z is O, R6is –(CH2CH2O)w–RA, and w is 1, 2, 3, 4, or 5. In some embodiments, RAis methyl.

[0124] Examples of compounds of formula (V-a) and (VI-a) include: Clor a pharmaceutically acceptable salt thereof.

[0125] In another aspect, the present disclosure provides a compound of formula (VII), or a pharmaceutically acceptable salt thereof,wherein R7is Mito; Mito isL is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0126] In some embodiments, the compound has formula (VII), in which L is L1-RC-L2, and RCis –(CH2CH2O)q–. In some embodiment, RCis –(CH2CH2O)q–, in which q is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, L is –(CH2CH2O)q–CH2CH2–, in which q is 1, 2,3, 4, or 5. In some embodiments, L is –(CH2CH2O)3–CH2CH2– or –(CH2CH2O)4– CH2CH2–.

[0127] In some embodiments, the compound has formula (VII), in which L is C1-20alkylene, such as –(CH2)4–, –(CH2)6–, –(CH2)8–, –(CH2)10–, or –(CH2)12–. In some embodiments, L is –(CH2)10–.

[0128] In some embodiments, the compound has formula (VII), in which X is halogen, trifluoroacetate, or acetate. For example, X can be Br–or trifluoroacetate.

[0129] Examples of compounds of formula (VII) include:or a pharmaceutically acceptable salt thereof.

[0130] Another aspect of the disclosure provides a kit comprising an effective amount of levodopa, and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein and instructional material.

[0131] The term "instructional material" refers to a publication, a recording, a diagram, or any other medium of expression which is used to communicate the usefulness of the present pharmaceutical composition for one of the purposes set forth herein in a human. The instructional material can also, for example, describe an appropriate dose of the present pharmaceutical composition. The instructional material of the present kit can, for example, be affixed to a container which contains a pharmaceutical composition as disclosed herein or be shipped together with a container which contains the pharmaceutical composition. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the pharmaceutical composition be used cooperatively by the recipient.

[0132] The present kit may include instruction for simultaneous, separate, or sequential administration of levodopa and a compound as described herein. For example, levodopa and a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, as described herein, can be administrated to a subject in need thereof simultaneously, separately, or sequentially.EXAMPLES

[0133] The following examples are shown to further illustrate the disclosed invention and are not intended to limit the disclosed invention, as recited in the claims.

[0134] Example 1

[0135] Results from this study show that Mito-ortho-HNK suppressed the growth of E. faecalis in a time- and dose-dependent manner. Mito-HNK decreased levodopa degradation and dopamine formation by E. faecalis. Additionally, it was shown that administration of Mito-ortho-HNK along with deuterated L-dopa to mice decreased deuterated dopamine formation in the gut and enhanced the uptake of deuterated L-dopa and formation of deuterated dopamine in the brain. These findings suggest a potential therapeutic pathway for enhancing the efficacy of L-dopa and L-dopa / carbidopa therapy in PD.

[0136] Mito-ortho-HNK and Mito-PEG4-HNK and analogs inhibit E. faecalis proliferation

[0137] Mito-ortho-HNK and PEGylated mitochondria-targeted honokiol (Mito-PEG4- HNK) are nearly 20 times more effective than unmodified honokiol (HNK) in inhibiting E. faecalis proliferation (FIGS.1A and 1C). At low concentrations, Mito-ortho-HNK analogs delayed the proliferation of E. faecalis. At lower doses of Mito-ortho-HNK and analogs, after a 2–3 h time lag, E. faecalis proliferation continued at nearly the same rate as the control. (FIGS.1B and 1D).

[0138] Mito-ortho-HNK and analogs inhibit L-dopa metabolism to dopamine by E. faecalis

[0139] Next, similar experiments was performed in the presence of L-dopa. As shown in FIG. 2A, Mito-ortho-HNK dose-dependently inhibited E. faecalis proliferation in the presence of L-dopa (1 mM). Samples collected at different time points were analyzed by high-performance liquid chromatography (FIG.2B). At concentrations of 3 µM and 4 µM of Mito-ortho-HNK, L-dopa degradation was inhibited together with reduced dopamine formation by E. faecalis (FIGS.2C and 2D). Samples collected at the 4 h time point showed that treatment with 3 µM of Mito-ortho-HNK caused 71% and 68% inhibition in L-dopa consumption and dopamine formation, respectively, compared with the control group in same period.

[0140] L-dopa is routinely used in combination with carbidopa in PD management. Further studies were conducted to investigate the effect of carbidopa (using the same ratio of L-dopa:carbidopa as used in clinic) on L-dopa metabolism by E. faecalis and the effect of Mito-ortho-HNK. As shown in FIGS. 2E-2H, Mito-ortho-HNK dose-dependentlyinduced a time lag in E. faecalis proliferation in the presence of L-dopa (1 mM) and carbidopa (0.2 mM). Samples collected at different points were analyzed by high- performance liquid chromatography (FIGS.2E-2H). At the concentration used, carbidopa did not affect the L-dopa metabolism as it is a poor substrate for bacterial tyrosine decarboxylase.

[0141] Mito-PEG5-ATO inhibits L-dopa metabolism to dopamine by E. faecalis

[0142] As shown in FIG. 3A, PEGylated mitochondria-targeted atovaquone (Mito- PEG5-ATO) dose-dependently inhibited E. faecalis proliferation in presence of L-dopa (1 mM). Samples isolated at various time points were analyzed by high-performance liquid chromatography (FIG. 3B). Mito-PEG5-ATO dose dependently inhibited L-dopa consumption (FIG.3C) by E. faecalis and the formation of dopamine (FIG.3C). Samples collected at the 2.5 h time point showed that treatment with 2.5 µM Mito-PEG5-ATO caused a 50% inhibition in L-dopa consumption and dopamine formation (FIGS. 3C and 3D).

[0143] Antimicrobial effects of MTDs on gut bacteria

[0144] To determine the impact of MTDs and related controls on bacterial growth, the minimal inhibitory concentration values were measured by micro broth-dilution assay. Minimal inhibitory concentration is the lowest concentration of an antibiotic that fully inhibits the growth of a bacterial strain. As shown in Table 1, all of the compounds were antimicrobial against a panel of representative gram-positive bacteria, with minimal inhibitory concentration values in the 0.5–4 µM range. Antimicrobial activity against two representative gram-negative bacteria, Escherichia coli (BL21) and Pseudomonas aeruginosa (PA01), was relatively weaker, with minimal inhibitory concentration values from 16 µM to >64 µM (Table 1). This may be due to several factors including reduced uptake across the more complex gram-negative outer membrane and the presence of a multidrug resistance pump. Table 1. Effect of mitochondria-targeted analogs on minimal inhibitory concentrations in gram negative and positive bacteria Minimal inhibitory concentrations (μM)

[0145] Comparison of antimicrobial effects of commonly used antibiotics and MTDs

[0146] Preliminary results also indicated that commonly used antibiotics chloramphenicol (1.25–20 µM) and ampicillin (2.5–20 µM) inhibited E. faecalis proliferation throughout the 6 h monitoring period (FIGS.4A-4D). However, Mito-ortho- HNK and analogs exhibited a time lag depending on the concentration. At 2.5 µM concentration, E. faecalis proliferation was maximally inhibited for 2 h, and then recovered to reach a similar level of confluence as the control (FIGS.2A-2H).

[0147] The effects of commonly used antibiotics and Mito-PEG-ATO analogs on the proliferation of E. faecalis are shown in FIGS.4A-4D. Results show that chloramphenicol and ampicillin dose-dependently inhibited E. faecalis proliferation. However, Mito-PEG- ATO and analogs exhibited a time lag depending on the concentration. At 2.5 µM concentration, E. faecalis proliferation was maximally inhibited for 2 h, and then it started to proliferate and reached a similar level of confluence as control.

[0148] This raises the possibility that L-dopa metabolism by E. faecalis can be inhibited in the presence of MTDs, including Mito-ortho-HNK and analogs or Mito-PEG-ATO analogs, and that the extent of inhibition is dependent on the timing of administration. If both L-dopa and Mito-ortho-HNK analogs were added at the same time, it is suggested thatL-dopa metabolism to dopamine would be inhibited during the initial time lag and would resume with time.

[0149] Effect of MTDs on membrane potential

[0150] The uptake of TPP+-modified compounds into bacteria is driven by the large, negative inside potential across the bacterial envelope. Membrane depolarization was observed for the MTD SkQ1 and proposed as its mechanism of action. FIG.5A shows that Mito-ortho-HNK dose-dependently decreased tetramethylrhodamine (TMRM) fluorescence due to depolarization of bacterial membrane potential. Treatment with a 4 µM concentration of Mito-ortho-HNK decreased the membrane potential by 15%. In contrast with MTDs, treatment with commonly used antibiotics chloramphenicol and ampicillin had no effect on TMRM fluorescence (FIG.5B).

[0151] Effect of Mito-ortho-HNK and commonly used antibiotics on cytotoxicity

[0152] Cytotoxicity was monitored in real time by SYTOX Green staining. The SYTOX measurements showed that Mito-ortho-HNK was not cytotoxic at the concentration inhibiting >90% proliferation of E. faecalis (FIG.5C). In contrast, the antibiotic ampicillin but not chloramphenicol was cytotoxic at concentrations inhibiting E. faecalis proliferation (FIG.5D). The Discussion section explores the mechanistic differences.

[0153] Effect of Mito-ortho-HNK and commonly used antibiotics on ATP

[0154] Intracellular ATP was measured in E. faecalis cells in the presence of Mito- ortho-HNK and the commonly used antibiotics chloramphenicol and ampicillin. As shown in FIGS. 6A-6F, the effects of Mito-ortho-HNK and antibiotics on ATP formation were completely different. Whereas Mito-ortho-HNK induced a dose- and time-dependent increase in ATP, ampicillin caused a dose- and time-dependent decrease in ATP compared with the control in an E. faecalis system. As shown in the proliferation data (FIG.6A), in Mito-ortho-HNK-treated E. faecalis, ATP levels were higher, probably due to decreased utilization of ATP under suppressed proliferation. After a dose-dependent time lag, the rates of proliferation of E. faecalis increased with time. Concomitantly, ATP levels started to decrease due to increased utilization of ATP (FIGS.6D-6F). In contrast with Mito-ortho- HNK, commonly used antibiotics chloramphenicol and ampicillin did not increase ATP levels in E. faecalis compared with the control. At higher concentrations, ATP levels decreased below the control levels, despite a decreased rate of proliferation.

[0155] Effect of MTD on in vivo metabolism L-dopa

[0156] The basal level of dopamine in the mouse brain is high. In order to monitor the impact of MTD treatment on L-dopa uptake, we used levodopa-d3 (L-dopa-d3), a stableform of L-dopa with a deuterium substituted aromatic ring that crosses the blood–brain barrier (FIGS.7A and 9B). The labeling with deuterium in L-dopa-d3 allowed us to track the distribution and metabolism of L-dopa-d3 to dopamine-d3 in the brain and gut (FIGS. 7A-7D).

[0157] First, the effect of Mito-ortho-HNK on L-dopa metabolism to dopamine in the gut was also investigated. Results show that Mito-ortho-HNK inhibits the conversion of L- dopa-d3 to dopamine-d3 in the gut (FIG.7B). Mice treated with L-dopa-d3 (150 mg / kg) or L-dopa-d3 (150 mg / kg) + Mito-ortho-HNK (10 mg / kg) by oral gavage show L-dopa metabolism and dopamine-d3 formation by gut microbiome from gut-homogenized samples (FIGS.7A and 7B). Mito-ortho-HNK inhibited dopamine-d3 formation (FIG.7B) and L-dopa-d3 consumption (FIG. 7B) as compared with the control mouse in gut homogenized samples. In the initial proof-of-concept study in regular, wild type mice treated with L-dopa-d3 or L-dopa-d3 + Mito-ortho-HNK, a higher amount of dopamine-d3 was detected in brain samples in mice treated with L-dopa-d3 + Mito-ortho-HNK compared with mice treated just with L-dopa-d3 (FIG.7C). This result indicates that Mito-ortho-HNK can inhibit L-dopa-d3 consumption by gut microbiome, thus increasing the amount of L- dopa-d3 reaching the mouse brain and, consequentially, increasing the formation of dopamine-d3 in the mouse brain.

[0158] In addition, as shown in FIGS. 1A-1D and FIGS. 10A-10B, the antibiotics ampicillin and chloramphenicol inhibit both gram-negative and gram-positive bacteria, and Mito-PEG-HNK, Mito-PEG-ATO, and Mito-LND (also referred to as Mito-LON) show selectivity. The intracellular ATP was measured in S. aureus cells in the presence of Mito- PEG-ATO analogs and the common antibiotics, ampicillin, and chloramphenicol. As shown in FIG.11, Mito-PEG5-ATO was nearly 50 times more potent than ampicillin and nearly 500 times more potent than chloramphenicol in inhibiting intracellular ATP levels and bioenergetics.

[0159] Discussion

[0160] Based on the results obtained from in vitro and in vivo experiments, it is hypothesized that the benefits of L-dopa treatment of PD in humans can be potentiated by the use of adjunctive treatments that inhibit L-dopa breakdown in the gastrointestinal tract. The scientific impact is the strong potential of MTDs as nontoxic, and highly effective agents in mitigating gut metabolism of L-dopa to dopamine and enhancing the uptake and metabolism of L-dopa into the brain in murine models of PD.

[0161] Studies have reported both beneficial and deleterious effects of antibiotics in the treatment of neurological disorders, including in PD. Tetracycline antibiotics such as doxycycline and minocycline were reported to be effective in mitigating the progression of neuronal dysfunction in mice models of PD. Neuroprotective mechanisms of antibiotics are distinctly different from their antibacterial mechanisms. Conventional antibiotics inhibit the growth of bacteria through inhibition of bacterial proteins, cell membranes, cell walls, or nucleic acid syntheses. In contrast, mitochondria-targeted agents exert cytostatic and not cytotoxic effects in E. faecalis. Studies report the identification of drug-resistant phenotypes and genes resistant to antibiotics in E. faecalis. E. faecalis isolates displayed a high level of resistance to several traditional antibiotics including to tetracycline antibiotics.

[0162] Mitochondria-targeted drugs may have some advantages over the traditional antimicrobials. Mitochondria-targeted drugs are more potent than the conventional antibiotics in inhibiting the proliferation of E. faecalis (Table 1, FIGS. 4A-4D). Other commensal species expected to be present in the gut may be further examined. Nonetheless, the results here suggest that the dose-dependence of this drug will likely differ for different species and different locations within the gut. However, the present findings indicate that the impact of mitochondria-targeted drugs (e.g., Mito-ortho-HNK and Mito- PEG5-ATO) is temporary and may provide a window of opportunity to enhance the therapeutic efficacy of L-dopa. Mito-ortho-HNK at 4 micromolar levels caused a substantial decrease in membrane potential in E. faecalis (FIG.5A). At this concentration, the proliferation of E. faecalis was inhibited (>90%) for 2–3 h (FIG. 5B). The SYTOX assay indicated that Mito-ortho-HNK was not cytotoxic at this concentration (FIG. 5B). Previously, it has been reported that bacterial cell division is dependent on membrane potential. The antibacterial effect of Mito-ortho-HNK is mechanistically different from those of chloramphenicol and ampicillin. Both chloramphenicol and ampicillin inhibited E. faecalis proliferation but did not affect its membrane potential (FIG. 5A). However, ampicillin inhibits bacterial cell wall synthesis, making the bacterial envelope more fragile, and did not affect membrane permeability (FIGS. 5A-5D). Chloramphenicol inhibits bacterial protein synthesis and did not affect the membrane permeability. Consistent with these findings, ampicillin enhanced the uptake of SYTOX Green dye and chloramphenicol had little or no effect on SYTOX Green uptake (FIG. 5B). At these concentrations, the proliferation of E. faecalis was inhibited (FIG.5A).

[0163] The antimicrobial efficacy of MTDs varies depending on the bacterial strain (Table 1). Most gram-positive bacteria were sensitive to MTDs, with MICs in the lowmicromolar to submicromolar range. However, MTDs exhibited less antibacterial efficacy toward E. coli and P. aeruginosa (Table 1, FIGS.4A-4D). Previous studies suggest that the presence of the highly effective multidrug resistance pump in E. coli is the reason why SkQ1, a decyl(triphenyl)phosphonium cation, conjugated to a quinone moiety was much less sensitive in E. coli (Table 1, FIGS. 4A-4D). E. coli mutants lacking this resistance pump showed increased sensitivity to MTDs. The multidrug resistance pump expelled the TPP+-containing SkQ1.

[0164] The lack of in vivo toxicity of Mito-HNK was previously reported in Pan, J. et al. (Mitochondria-targeted honokiol confers a striking inhibitory effect on lung cancer via inhibiting complex I activity. iScience 3, 192–207 (2018)). The potential toxicity and neurological changes induced by Mito-HNK were assessed in an eight-week toxicology study in A / J mice. Mice were treated with vehicle control and with various doses of Mito- HNK (7.5, 37.5, and 75 µmol / kg, which represent 2×, 10×, and 20× the therapeutically effective dose of 3.75 µmol / kg in the lung cancer mouse model, respectively), given via oral gavage five days per week for eight weeks. After eight weeks of treatment, no meaningful differences were observed between control mice and those treated with any dose of Mito-HNK, including the highest dose (75 µmol / kg). In addition, no histopathological changes were seen in either neural (frontal cortex and cerebellum) or skeletal muscles. Mito-HNK did not affect the motor function monitored in a rotarod assay.18Overall, Mito-HNK did not show any indications of toxicity at a dose that is 20- fold higher than the maximally tolerated dose.

[0165] PEGylation decreases the hydrophobicity of MTDs, and nearly all MTDs and Mito-PEG analogs have similar alkyl side-chain lengths but substantially different hydrophobicities (log P [octanol partition coefficients]). The calculated log P values for atovaquone, HNK, lonidamine, Mito10-ATO, Mito-ortho-HNK, Mito-lonidamine, Mito- PEG5-ATO, Mito-PEG4-HNK, and Mito-PEG4-APO are 5.1, 5.2, 4.5, 12.8, 13.0, 9.3, 9.2, 9.5, and 5.4, respectively. This approach enables further development to vary drug hydrophobicity and enhance the antimicrobial effects of MTDs.

[0166] Previous studies using another form of deuterated L-dopa, SD-1077 (where the alkyl side chain protons are deuterated), indicate a slower metabolism of the corresponding deuterated dopamine (due to the kinetic isotope effect), leading to enhanced stability and behavioral potency in an animal model of PD. The deuterated L-dopa used in this study has aromatic ring protons that are deuterated and, therefore, do not have the kinetic isotope effect. The endogenous levels of dopamine in the mouse brain are relatively high, and theincreased dopamine levels in the brain that result from use of the nondeuterated conventional form of L-dopa might make optimization of the method more difficult. The use of appropriately labeled L-dopa with the carbon-13-labeled isotope should help overcome the detection limitations (of dopamine) with the possibility of method optimization.

[0167] A genetically engineered mouse model, the MitoPark mouse, recapitulates many of the phenotypic features (mitochondrial dysfunction, microglial activation, dopaminergic degeneration, dopamine deficiency, and progressive neuronal deficits and protein occlusion) of PD. It is hypothesized that the benefits of treating human PD with L-dopa can be potentiated by the use of adjunctive treatments utilizing MTDs that inhibit the breakdown of L-dopa to dopamine in the gastrointestinal tract, thereby enhancing L-dopa conversion to dopamine in the brain (FIGS.8A-8B). Studies focusing on mouse models of PD, including MitoPark and LRRK2 transgenic mice, can be performed to provide additional insights relevant to gut microbial metabolism of L-dopa and its inhibition by Mito-ortho-HNK. Furthermore, MTDs (e.g., Mito-apocynin and Mito-quinone) prevent hyposmia and loss of motor function in LRRK2 PD mice, and they inhibit MPTP (1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neurotoxicity in a PD mouse model. A combination of L-dopa and carbidopa is the treatment of choice for managing PD symptoms. Carbidopa does not prevent gut metabolism of L-dopa but does inhibit the peripheral metabolism of L-dopa by acting as a substrate inhibitor of peripheral amino carboxylases. MTDs could enhance the efficacy of L-dopa / carbidopa therapy by directly reversing the gut bacteria metabolism of L-dopa. Thus, it is conceivable that a combination of MTD / L-dopa / carbidopa therapy may inhibit both the microbial metabolism and the peripheral metabolism of L-dopa.

[0168] Methods

[0169] Syntheses of MTDs

[0170] MTDs (e.g., Mito-ortho-HNK) were prepared by conjugating the mitochondria- targeting TPP+moiety to the corresponding parent molecule via alkyl linkers of different natures and lengths (FIG. 9A). Based on the data published by us and other laboratories, fine-tuning of the linker length and hydrophobicity is important for optimization of the uptake and biological activity of the TPP+-based MTDs.

[0171] To a mixture of HNK (1.3 g, 4.9 mmol), anhydrous potassium carbonate (0.69 g, 4.9 mmol) in DMF (40 mL) was added 10-bromodecyltriphenylphosphonium bromide (2.8 g, 4.9 mmol). The mixture was stirred at 40°C for 24 h. The solvent was removedunder vacuum, and the residue was taken up into water and extracted with dichloromethane. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. Purification by flash chromatography (diethyl ether, dichloromethane, and dichloromethane / ethanol, 9 / 1) delivered the corresponding mitochondria-targeted ortho-honokiol (Mito-ortho-HNK) (0.3 g, 8.1 % yield).

[0172] Bacterial proliferation assay

[0173] All in vitro experiments were conducted using validated bacterial strains acquired from the American Type Culture Collection for E. faecalis (Cat# OG1RF) and from National Collection of Type Cultures for Eggerthella lenta (Cat# NCTC 11813). E. faecalis was cultured in tryptic soy broth. E. faecalis cells were diluted 1:100 into fresh tryptic soy broth medium. Then, they were grown at 37°C in flasks on a rotating shaker at 250 rpm to reach the exponential growth phase (optical density at 600 nm [OD600] of 0.2– 0.5) before use in the in vitro assays. For all proliferation assays, cells were diluted to the final OD600of 0.1 with indicated treatments in a 96-well plate. Cell proliferation, which was represented as absorbance at 600 nm, was acquired in real time every 3 min for 6 h using a plate reader (BMG Labtech, Inc., Ortenberg, Germany) equipped with an atmosphere controller set at 37°C, 100% air.

[0174] Bacterial metabolism of L-dopa

[0175] E. faecalis cells in the exponential growth phase (OD600of ~0.4) were diluted to the final OD600 at 0.1. Then, they were treated in presence of L-dopa (1 mM) alone or in combination with carbidopa (0.22 mM) in the same manner as described for Mito-ortho- HNK. At the indicated time points (1–6 h), samples (1 ml of media) were collected by centrifugation at 2500 g × 5 min at 4°C and stored at −80°C before performing lyophilization. The dry residue was dissolved in ice-cold methanol (100 µl) and taken for liquid chromatography–mass spectrometry (LC-MS) analysis.

[0176] LC-MS experiments

[0177] L-Dopa and its metabolites were separated and monitored by LC-MS using an Agilent 1200 apparatus equipped with an ultraviolet-visible absorption and mass spectrometry detector (single quadrupole). Typically, 2 µL of a sample was injected on an Agilent Poroshell column (120 HILIC-Z, PEEK, 100 mm × 2.1 mm, 2.7 µm, 25°C). The absorption traces were collected at 280 nm.

[0178] L-Dopa-d3 and dopamine-d3 were separated and monitored by liquid chromatography–mass spectrometry–single ion monitoring (LC-MS-SIM) using an Agilent 1200 apparatus equipped with an ultraviolet-visible absorption and MS detector(single quadrupole). Typically, 2 µL of a sample was injected on an Agilent Poroshell column (120 HILIC-Z, PEEK, 100 mm × 2.1 mm, 2.7 µm, 25°C) equilibrated with 100% ammonium formate (10 mM, pH 3.0 containing acetonitrile / water, 9 / 1,). The compounds were separated by a linear increase in ammonium formate (10 mM, pH 3.0 containing acetonitrile / water, v / v) phase concentration from 0% to 80% over 14 min using a flow rate of 0.5 mL / min. The absorption traces were collected at 280 nm.

[0179] In addition, mass spectrometry–single ion monitoring (MS-SIM) detection parameters were set up using electrospray ionization. SIM was defined as follows: L-Dopa- d3 [m / z = 201 (+)] and dopamine-d3 [m / z = 157 (+)].

[0180] Bacterial membrane potential and cytotoxicity assay

[0181] The effects of MTDs on bacterial membrane potential were determined using the fluorescent dye TMRM. Briefly, bacteria in the exponential growth phase (OD600of 0.4) were treated as indicated for the MTDs or commonly used antibiotics in a black, clear- bottom 96-well plate; then, an aliquot of TMRM was added at a final concentration of 50 nM for 20 min. After incubation with TMRM, the plate was centrifuged twice at 2500 g for 5 min and washed with phosphate buffered saline. Fluorescence was monitored at an excitation of 544 nm and emission of 590 nm using a plate reader (BMG Labtech, Inc.). Data were collected as the mean fluorescent intensity. Results were normalized to the total OD600as the total bacteria number.

[0182] For kinetic monitoring of the cytotoxicity assay, E. faecalis cells in the exponential growth phase (OD600=0.4) were treated as indicated for the MTDs or commonly used antibiotics in a black, clear-bottom 96-well plate for 3 h, and dead cells were monitored in the presence of 200 nM SYTOX Green (Invitrogen, Waltham, MA). The SYTOX method labels the nuclei of dead cells, yielding green fluorescence. Fluorescence intensities from the dead cells in the 96-well plate were acquired in real time every 5 min for 3 h using a plate reader (BMG Labtech, Inc.) equipped with an atmosphere controller set at 37°C, 100% air using a fluorescence detection with 485 nm excitation and 535 nm emission. Data are represented as mean fluorescent intensity.

[0183] Intracellular ATP levels

[0184] A luciferase-based assay was used to measure intracellular adenosine triphosphate (ATP) levels according to the manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, Cat# FLLAA). Briefly, a mixture containing luciferase and luciferin (Cat# FLAAM) was added to cell lysates. After swirling, the light released was measured in a luminometer. The results were normalized to OD600 level in each well.

[0185] In vivo detection of dopamine-d3

[0186] All animal protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. Mice (C57BL / 6J, 6-8 weeks old, from Jackson Laboratories) were divided into two groups and then orally gavaged with either 250 mg / kg L-dopa-d3 only (control) or 250 mg / kg L-dopa-d3 + 20mg / kg Mito-ortho-HNK for 2 h. The mice were sacrificed, and the brain tissue and gut tissue (including stomachs and intestines) were harvested, snap frozen in liquid nitrogen, and then stored at −80°C before extraction. The protocol for extracting L-dopa and dopamine is the same as described previously. Once tissue weight was obtained, it was transferred to a homogenization tube containing 1 ml of ice-cold methanol. The tissue homogenization and extraction were performed using an Omni Bead Ruptor 24 homogenizer (Omni International, Kennesaw, GA). The homogenate was centrifuged at 16,000×g for 10 min 4°C; then, the supernatant was collected and transferred to a new 1.5 ml microcentrifuge tube for lyophilization. All samples were stored at −80°C until LC-MS analysis. L-dopa- d3 and dopamine-d3 were separated and monitored by LC-MS-SIM. The absorption traces were collected at 280 nm, and MS-SIM detection parameters were set up using electrospray ionization. SIM was defined as follows: L-dopa-d3 [m / z = 201 (+)] and dopamine-d3 [m / z = 157 (+)].

[0187] Statistics and reproducibility

[0188] Comparisons between the control and treatment groups were made using an unpaired Student’s t test analysis. Sample size and replicates are stated in figure legends, respectively. P-values of less than 0.05 were determined as statistically significant. Values denote mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). The number of replicates per treatment group are shown as n.

[0189] Synthesis of Mito-ortho-HNK and Mito-PEG4-HNK

[0190] Synthesis of mitochondria-targeted ortho-honokiol (Mito-ortho-HNK, Scheme1) and the PEGylated mitochondria-targeted analog of honokiol (Mito-PEG4-HNK, Scheme 2) are presented in the following sections. Mito-ATO, Mito-PEG2-ATO, and Mito- PEG5-ATO were prepared as previously described. Their nuclear magnetic resonance (NMR) data are presented below.

[0191] All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin layer chromatography using silica gel Merck60F254. Crude materials were purified by flash chromatography on Merck Silica gel 60 (0.040–0.063 mm).1H NMR spectra were recorded at 400.13 MHz respectively using aBruker DPX AVANCE 400 spectrometer equipped with a quattro nucleus probe.1H NMR and31P were taken in deuterated chloroform (CDCl3) using CDCl3and tetramethylsilane as internal reference respectively. Chemical shifts are reported in ppm and J values in Hertz.

[0192] Synthesis of Mito-ortho-HNK, Mito-PEG4-HNK and Mito-PEG4-HNKMe is presented in Schemes 1 and 2.

[0193] Preparation of Mito-ortho-HNK ([10-[3,5’-diallyl-2’-hydroxy-(1,1’- biphenyl)-4-yl]-oxy]-decyltriphenylphosphonium bromide)

[0194] Mito-ortho-HNK was prepared by reacting 10- bromodecyltriphenylphosphonium bromide (Mito-Br) with honokiol (HNK) in the presence of potassium carbonate in dimethylformamide (DMF) (Scheme 1).Scheme 1. Synthesis of Mito-ortho-HNK. Reagents and conditions: i, triphenylphosphine, neat, 90°C, 47%; ii, Mito-Br, K2CO3, DMF, 40°C, 38 h., 14%.

[0195] 10-Bromodecyltriphenylphosphonium bromide. The procedure was adapted from Pan et al.

[0040] . A mixture of triphenylphosphonium (1 g, 3.8 mmol) and dibromide (5.7 g, 19 mmol) was heated at 90°C for 6 h. After cooling, the crude product was washed by ether (Et2O) and purified by flash chromatography (pentane, Et2O, and dichloromethane [CH2Cl2] / ethanol (EtOH) 9:1) to afford the corresponding phosphonium salt as a white solid (1 g, 47% yield).31P (400.13 MHz, CDCl3) δ 24.32.1H NMR (400.13 MHz, CDCl3) δ 7.85-7.65 (15H, m), 3.73-3.66 (2H, m), 3.40-3.34 (2H, m), 1.80-1.75 (4H, m), 1.31-1.20 (12H, m).

[0196] Mito-ortho-HNK. To a mixture of HNK (1.3 g, 4.9 mmol), anhydrous potassium carbonate (0.69 g, 4.9 mmol) in DMF (40 mL) was added 10-bromodecyltriphenylphosphonium bromide (2.8 g, 4.9 mmol). The mixture was stirred at 40°C for 24 h. The solvent was removed under vacuum, and the residue was taken up into water and extracted with CH2Cl2. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O, CH2Cl2, and CH2Cl2 / EtOH) delivered the corresponding Mito-ortho-HNK (0.51 g, 14% yield).31P (400.13 MHz, CDCl3) δ 24.58.1H NMR (400.13 MHz, CDCl3) δ 7.86-7.65 (15H, m), 7.35-7.21 (2H, m), 7.04-6.89 (4H, m), 6.04-5.92 (2H, m), 5.57 (1H, s), 5.13-4.98 (4H, m), 3.99 (2H, t, J = 6.4), 3.87-3.75 (2H, m), 3.45-3.28 (4H, m), 1.84-1.74 (2H, m), 1.62-1.56 (3H, m), 1.51-1.12 (11H, m).13C NMR (75 MHz, CDCl3) δ 156.3, 151.1, 137.9, 136.7, 134.9, 134.8, 133.7, 133.6, 131.8, 130.4, 130.3, 130.1, 129.6, 129.0, 128.6, 127.9, 127.8, 118.8, 118.2, 115.7, 115.6, 115.4, 111.7, 68.0, 39.4, 34.5, 30.4, 30.3, 29.3, 29.2, 29.19, 29.14, 29.08, 29.04, 25.9, 22.7, (d, J = 49.2), 22.6, (d, J = 4.2). HRMS calculated for Mito-ortho-HNK C46H52O2P [MH]+ 667.3699, found, 667.3699. Two-dimensional NMR was performed on a 600 MHz NMR equipped with TCI (triple resonance inverse) probe in manual mode. ¹H-¹H COSY (correlation spectroscopy), HSQC (heteronuclear single quantum correlation), HMBC (heteronuclear multiple bond correlation), and NOESY (nuclear overhauser effect spectroscopy) were used.

[0197] Preparation of Mito-PEG4-HNK (2-(2-(2-(2-((3’,5-diallyl-4’-hydroxy-[1,1’- biphenyl]-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)triphenylphosphonium bromide and (2- (2-(2-(2-((3,5’-diallyl-2’-hydroxy-[1,1’-biphenyl]-4 yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)triphenylphosphonium bromide and Mito-HNK- PEGOMe (10-((4’-((2,5,8,11-tetraoxatridecan-13-yl)oxy)-3’,5-diallyl-[1,1’-biphenyl]-2- yl)oxy)decyl)triphenylphosphonium bromide and (10-((2’-((2,5,8,11-tetraoxatridecan-13- yl)oxy)-3,5’-diallyl-[1,1’-biphenyl]-4-yl)oxy)decyl)triphenylphosphonium bromide.

[0198] Mito-PEG4-HNK was prepared in two steps, by reacting the appropriate PEGylated dibromoalcane with honokiol in the presence of potassium carbonate in DMF. The addition of triphenylphosphine on the bromopegylated honokiol (HNK-PEG4-Br) led to Mito-PEG4-HNK. In addition, the Mito-HNK was pegylated by using similar procedure in the presence of bromo-2,5,8,11-tetraoxatridecane and led to Mito-HNK-PEGOMe (Scheme 2).Scheme 2. Synthesis of Mito-PEG4-HNK and Mito-HNK-PEGOMe. Reagents and conditions: i, 1,11-Dibromo-3,6,9-trioxaundecane, K2CO3, DMF, 40°C, 36h, 62%; ii, triphenylphosphine, CH3CN, reflux, 18h, 31%; iii, 10-Bromodecyltriphenylphosphonium bromide, K2CO3, DMF, 60°C, 31%; iv, bromo-2,5,8,11-tetraoxatridecane, K2CO3, DMF, 40°C, 48h, 61%.

[0199] To a mixture of honokiol (0.3 g, 1.2 mmol), anhydrous potassium carbonate (0.3 g, 2.4 mmol) in DMF (4 mL) was added 1,11-Dibromo-3,6,9-trioxaundecane (0.36 g, 1.1 mmol). The mixture was stirred at 40 °C for 36h. The residue was taken up into water and extracted with Et2O. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. Purification by flash chromatography (Pentane / Et2O, 1 / 1) delivered the corresponding PEG-HNK (0.35 g, 62% yield). PEG-HNK was directly used for the next step. A mixture of PEG-HNK (0.35 g, 0.69 mmol) and triphenylphosphine (0.24 g, 0.91 mmol) in acetonitrile (2 mL) was stirred at reflux for 18 hours. The mixture was poured in 100 mL of ether. The precipitate was purified by flash chromatography (CH2Cl2 / EtOH 9 / 1) and led to the corresponding Mito-PEG4-HNK (165 mg, 31% yield). HRMS calculated for Mito-PEG4-HNK C44H48O5P+[M]+687.3234, found, 687.3238.31P NMR (400.13 MHz, CDCl3) δ 25.39, 25.16.1H NMR (400.13 MHz, CDCl3), δ1H NMR(400.13 MHz, CDCl3) δ 7.79-7.67 (9H, m), 7.64-7.56 (6H, m), 7.32 (1H, ddd, J = 2.2, 4.4, 8.0), 7.24 (1H, 2d, J = 2.2, 2.2), 7.17 (1H, 2d, J = 8.0), 7.12-6.94 (2H, m), 6.89-6.80 (1H, m), 6.06-5.89 (2H, m), 5.11-4.95 (4H, m), 4.20-4.01 (2H, m), 3.99-3.80 (5H, m), 3.80-3.61 (3H, m), 3.50-3.40 (2H, m), 3.39-3.28 (5H, m), 3.27-3.15 (4H, m).13C NMR (75 MHz, CDCl3) δ 155.6, 154.3, 154.0, 151.8, 138.0, 137.8, 137.4, 136.8, 134.6, 134.57, 134.48, 134.4, 134.0, 133.9, 133.8, 132.5, 131.3, 130.9, 130.8, 130.7, 130.1, 130.0, 129.9, 129.8, 129.3, 128.9, 128.4, 128.3, 128.2, 127.5, 127.4, 125.4, 119.3, 119.2, 118.5, 118.4, 116.4, 115.6, 115.2, 115.1, 112.6, 111.6, 71.0, 70.8, 70.5, 70.2, 70.18, 70.10, 69.99, 69.93, 69.66, 69.63, 68.6, 68.0, 63.9, 63.8, 63.7, 63.6, 39.4, 34.5, 25.3 (d, J = 52.1), 25.2 (d, J = 52.8).

[0200] To a mixture of Mito-HNK (0.19 g, 0.25 mmol), anhydrous potassium carbonate (0.05 g, 0.36 mmol) in DMF (4 mL) was added the bromo-2,5,8,11-tetraoxatridecane (0.13 g, 0.36 mmol). The mixture was stirred at 40 °C for 48h. The mixture was taken up into water and extracted with CH2Cl2. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. The crude product was washed with ether before purification by flash chromatography (CH2Cl2 / EtOH, 9 / 1). Mito-HNK-PEGOMe is obtained as a white solid (145 mg, 61 % yield). HRMS calculated for Mito-HNK-PEGOMe : C55H70O6P+[M]+857.4905, found, 857.4899. Mito-HNK-PEGOMe.31P (400.13 MHz, CDCl3) δ^24.34.1H NMR (400.13 MHz, CDCl3) δ 7.85-7.74 (9H, m), 7.71-7.64 (6H, m), 7.35-7.28 (2H, m),7.09(1H,d,J = 2.2), 7.03 (1H,dd,J = 8.3, 2.4), 6.84 (1H, dd, J = 8.3, 4.4), 6.05-5.89 (2H, m), 5.10-4.95 (4H, m), 4.15-4.13 (2H, m), 3.89-3.84 (4H, m), 3.75- 3.59 (12H, m), 3.54-3.50 (2H, m), 3.41-3.36 (2H, m), 3.34 (3H, s), 3.34-3.30 (2H, m), 1.69- 1.64 (6H, m), 1.36-1.27 (2H, m), 1.26-1.11 (9H, m).13C NMR (75 MHz, CDCl3) δ 155.3, 154.3, 137.7, 137.0, 134.9, 134.8, 133.6, 133.5, 132.0, 131.09, 131.03, 130.7, 130.5, 130.38, 130.34, 128.1, 127.9, 127.7, 118.7, 117.9, 115.3, 115.2, 112.6, 111.0, 71.8, 77.0, 70.6, 70.51, 70.49, 70.39, 69.7, 68.5, 67.8, 58.9, 39.3, 34.4, 30.4, 30.2, 29.3, 29.09, 29.07, 29.01, 25.9, 22.7 (d, J = 49.2), 22.6.

[0201] Synthesis of Mito-PEG4-MGN is presented in Scheme 3.

[0202] Preparation of Mito-PEG4-MGN (2-(2-(2-(2-((5,5’-diallyl-2’-hydroxy-[1,1’- biphenyl]-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)triphenylphosphonium.

[0203] The pegylated mitochondria-targeted analog of magnolol (Mito-PEG4-MGN) was prepared in two steps, by reacting the appropriate pegylated dibromoalcane with magnolol in the presence of potassium carbonate in DMF. Addition of triphenylphosphine on the bromopegylated manolol (MGN-PEG4-Br) led to the Mito-PEG-MGN.   Scheme 3. Synthesis of Mito-PEG4-MGN. Reagents and conditions: I, 1,11-Dibromo- 3,6,9-trioxaundecane, K2CO3, DMF, rt., 24h, 40 %; ii, triphenylphosphine, CH3CN, reflux, 48h, 36 %.

[0204] To a mixture of magnolol (0.65 g, 2.4 mmol), anhydrous potassium carbonate (0.34 g, 2.5 mmol) in DMF (4 mL) was added 1,11-Dibromo-3,6,9-trioxaundecane (0.8 g, 2.5 mmol). The mixture was stirred at room temperature for 24h. The residue was taken up into water and extracted with Et2O. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Pentane / Et2O, 1 / 1) delivered the corresponding PEG-MGN (490 mg, 40% yield). PEG- MGN was directly used for the next step. δ1H NMR (400.13 MHz, CDCl3) δ 7.21-7.13 (2H, m)^^7.12-7.05 (2H, m), ^6.98-6.92 (2H, m), 6.06-5.92 (2H, m), 5.14-5.04 (4H, m), 4.23-4.17 (2H, m), 3.82-3.76 (4H, m), 3.68-3.61 (8H, m), 3.45 (2H, t, J = 3.4), 3.42-3.35 (4H, m).

[0205] A mixture of PEG-MGN (0.47 g, 0.93 mmol) and triphenylphosphine (0.29 g, 1.1 mmol) in acetonitrile (4 mL) was stirred at reflux for 48 hours. The mixture was poured in 100 mL of ether. The precipitate was purified by flash chromatography (CH2Cl2 / EtOH 9 / 1) and led to the corresponding Mito-PEG4-MGN (260 mg, 36 % yield). HRMS calculated for Mito-PEG-MGN C44H48O5P+[M]+687.3234, found, 687.3232.31P NMR (400.13 MHz, CDCl3) δ 25.53.1H NMR (400.13 MHz, CDCl3), δ1H NMR (400.13 MHz, CDCl3) δ 7.87-7.79 (6H, m), 7.75-7.55 (9H, m), 7.19-7.13 (2H, m), 7.10-7.03 (2H, m), ^6.98-6.89 (2H, m), 6.75 (1H, s), 6.11-5.90 (2H, m), 5.13-5.11 (1H, m), 5.10-5.02 (3H, m), 4.23-4.12 (4H, m), 3.97 (2H, dt, J = 22.4, 5.6), 3.79-3.72 (2H, m), 3.45-3.59 (2H, m), 3.43-3.24 (10H, m).13C NMR (75 MHz, CDCl3) δ 153.2, 152.4, 137.9, 137.4, 134.53, 134.50, 134.1, 134.0, 133.7, 132.6, 132.2, 131.4, 130.0, 129.9, 128.9, 128.8, 119.5, 118.6, 118.1, 116.5, 115.8, 115.4, 112.7, 70.7, 70.2, 69.9, 69.1, 64.1, 64.0, 39.4, 39.3, 25.4 (d, J = 52.1).

[0206] Synthesis of Mito-(PEG)n-ATO and PEG-ATO is presented in Scheme 4.Scheme 4. Synthesis of Miton-ATO, Mito-ATO-PEGn and ATO-PEG. Reagents and conditions: I, K2CO3, DMF, 70°C, 7h-12h, 35-75%. ii, Br-PEGn-Br, K2CO3, DMF, 60°C, 8h, 35-81%; iii, triphenylphosphine, CH3CN, reflux, 18h, 14%-67%; iv, Br-PEG4-Br, K2CO3, DMF, 60°C, 33%; iv, Br-(CH2)n-CH3, DMF, 60°C, 8h, 81-83%.

[0207] Preparation of Mito4-ATO. To a mixture of atovaquone (0.3 g, 0.82 mmol) and potassium carbonate (0.15 g, 0.82 mmol) in DMF (3 mL) was added (4-bromobutyl)- triphenylphosphonium bromide, (0.39 g, 0.81 mmol). The mixture was stirred at 70°C for 9 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Then, Et2O was added to the mixture to precipitate the compound. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito4-ATO (0.47 g, 75% yield). HRMS calculated for Mito4-ATO C44H41ClO3P+[M]+683.2476, found, 683.2479.31P NMR (400.13 MHz, CDCl3) δ24.66.1H NMR (400.13 MHz, CDCl3,δ 8.06-8.03 (1H, m), 7.95-7.93 (1H, m), 7.92-7.85 (6H, m), 7.79-7.72 (3H, m), 7.71-7.63 (8H, m), 7.25-7.21 (2H, m), 7.15-7.12 (2H, m), 4.26 (2H, t, J = 5.6), 4.11- 4.02 (2H, m), 3.13-3.03 (1H, m), 2.58-2.48 (1H, m), 2.37-2.28 (2H, m), 2.10-1.98 (4H, m), 1.92-1.88 (2H, m), 1.65 (2H, dd, J = 12.7, 2.7), 1.54-1.39 (2H, m).13C NMR (75 MHz, CDCl3) δ 185.4, 181.7, 157.7, 145.7, 140.0, 134.9, 134.8, 133.8, 133.77, 133.68, 133.2, 132.3, 131.4, 131.3, 130.5, 130.3, 128.3, 128.2, 118.8, 117.9, 72.4, 43.1, 35.5, 34.3, 30.12 (d, 17.6), 30.1, 30.0, 22.2 (d, J = 50.6), 19.1 (d, J = 3.7).

[0208] Preparation of Mito10-ATO (Mito-ATO) To a mixture of atovaquone (0.4 g, 1.1 mmol) and potassium carbonate (0.17 g, 1.2 mmol) in DMF (5 mL) was added (10- bromodecyl)-triphenylphosphonium bromide, (0.61 g, 1.1 mmol). The mixture was stirredat 60°C overnight. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Then, Et2O was added to the mixture to precipitate the compound. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito-ATO (0.48 g, 52% yield). HRMS calculated for Mito-ATO C50H53ClO3P+[M]+767.3415, found, 767.3420.31P NMR (400.13 MHz, CDCl3) δ^ ^^^24.48.1H NMR (400.13 MHz, CDCl3),δ 8.08-7.98 (2H, m), 7.90-7.82 (6H, m), 7.81- 7.75 (3H, m), 7.73-7.64 (8H, m), 7.25-7.23 (2H, m), 7.18-7.16 (2H, m), 4.30 (2H, t, J = 6.6), 3.88-3.77 (2H, m), 3.25-3.15 (1H, m), 2.66-2.55 (1H, m), 2.24-2.11 (2H, m), 2.01- 1.92 (2H, m), 1.86-1.77 (2H, m), 1.76-1.68 (4H, m), 1.60-1.40 (6H, m), 1.38-1.25 (8H, m).13C NMR (75 MHz, CDCl3) δ^185.5, 181.9, 158.0, 146.0, 138.6, 134.9, 134.8, 133.8, 133.72, 133.67, 133.1, 132.4, 131.5, 131.4, 130.5, 130.4, 128.4, 128.2, 126.3, 125.9, 118.9, 118.1, 74.0, 43.3, 35.4, 34.5, 30.4 (d, J = 14.0) , 29.9, 29.5, 29.3, 29.2, 25.9, 22.8 (d, J = 49.1), 22.7 (d, J = 4.4).

[0209] Preparation of Mito12-ATO To a mixture of atovaquone (0.45 g, 1.2 mmol) and potassium carbonate (0.17 g, 1.2 mmol) in DMF (3 mL) was added (12-bromododecyl)- triphenylphosphonium bromide, (0.6 g, 1.0 mmol). The mixture was stirred at 70°C for 7 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Then, Et2O was added to the mixture to precipitate the compound. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito12-ATO (0.31 g, 35% yield). HRMS calculated for Mito12-ATO C52H57ClO3P+[M]+795.3728, found, 795.3729.31P NMR (400.13 MHz, CDCl3) δ 24.44.1H NMR (400.13 MHz, CDCl3), δ 8.07-7.97 (2H, m), 7.87-7.74 (9H, m), 7.72-7.63 (8H, m), 7.24-7.20 (2H, m), 7.18-7.13 (2H, m), 4.30 (2H, t, J = 6.6), 3.82-3.72 (2H, m), 3.25-3.13 (1H, m), 2.65- 2.55 (1H, m), 2.23-2.09 (2H, m), 1.99-1.92 (2H, m), 1.84-1.67 (7H, m), 1.63-1.42 (8H, m), 1.37-1.19 (9H, m).13C NMR (75 MHz, CDCl3) δ 185.5, 181.9, 158.1, 146.0, 138.6, 134.92, 134.89, 133.7, 133.6, 133.1, 132.4, 131.5, 131.4, 130.5, 130.4, 128.4, 128.2, 126.3, 125.9, 118.9, 118.1, 74.1, 43.3, 35.4, 34.5, 30.5, 30.4, 30.3 (d, J = 16.1), 29.9, 29.6, 29.5, 29.3, 29.24, 29.21, 25.9, 22.8 (d, J = 49.2), 22.4 (d, J = 4.4).

[0210] Preparation of Mito16-ATO To a mixture of atovaquone (0.13 g, 0.37 mmol) and potassium carbonate (0.05 g, 0.37 mmol) in DMF (2 mL) was added (12- bromohexadecyl)-triphenylphosphonium bromide, (0.2 g, 0.34 mmol). The mixture wasstirred at 70°C for 7 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Then, Et2O was added to the mixture to precipitate the compound. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito16-ATO (0.16 g, 54% yield). HRMS calculated for Mito16-ATO C56H65ClO3P+[M]+851.4354, found, 851.4360.31P NMR (400.13 MHz, CDCl3) δ 23.39.1H NMR (400.13 MHz, CDCl3),δ^8.00-7.93 (2H, m), 7.81-7.59 (17H, m), 7.21- 7.16 (2H, m), 7.11-7.09 (2H, m), 4.26 (2H, t, J = 6.7), 3.75-3.63 (2H, m), 3.20-3.08 (1H, m), 2.60-2.48 (1H, m), 2.17-2.03 (2H, m), 1.94-1.85 (2H, m), 1.80-1.73 (2H, m), 1.70-1.63 (2H, m), 1.68-1.38 (8H, m), 1.33-1.06 (20H, m).13C NMR (75 MHz, CDCl3) δ 185.54, 181.8, 158.0, 146.0, 138.5, 134.94, 134.91, 133.7, 133.6, 133.0, 132.3, 131.3, 131.4, 130.5, 130.4, 128.3, 128.1, 126.2, 125.9, 118.8, 118.0, 74.0, 43.3, 35.5, 34.5, 30.5, 30.4, 30.3, 29.9, 29.6, 29.59, 29.56, 29.52, 29.4, 29.3, 29.2, 29.1, 25.9, 22.8 (d, J = 49.9), 22.6 (d, J = 4.4).

[0211] Preparation of ATO-C4. To a mixture of atovaquone (0.4 g, 1.1 mmol) and potassium carbonate (0.15 g, 1.1 mmol) in DMF (3 mL) was added bromobutane, (0.15 g, 1.1 mmol). The mixture was stirred at 70°C for 7 hours. Then, Et2O was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Purification by flash chromatography (pentane / EtOAc 98 / 02) delivered the corresponding ATO-C4(0.38 g, 83% yield). HRMS calculated for ATO-C4C26H27ClO3[MH]+423.1721, found, 423.1718.1H NMR (400.13 MHz, CDCl3), δ^^^^8.01-7.90 (2H, m), 7.63-7.55 (2H, m), 7.19- 7.16 (2H, m), 7.11-7.08 (2H, m), 4.30 (2H, t, J = 6.7), 3.17-3.10 (1H, m), 2.57-2.49 (1H, m), 2.16-2.05 (2H, m), 1.90 (2H, dd, J = 13.5, 2.82), 1.79-1.71 (2H, m), 1.65 (2H, dd, J = 13.3, 3.2), 1.51-1.41 (4H, m), 0.94 (3H, t, J = 7.3).13C NMR (75 MHz, CDCl3) δ 185.4, 181.8, 158.0, 145.9, 138.6, 133.7, 133.0, 132.4, 131.7, 128.4, 128.1, 126.3, 125.9, 73.7, 43.3, 35.4, 34.5, 32.4, 29.9, 29.9, 19.1, 13.8.

[0212] Preparation of ATO-C10. To a mixture of atovaquone (0.4 g, 1.1 mmol) and potassium carbonate (0.15 g, 1.1 mmol) in DMF (3 mL) was added bromodecane, (0.24 g, 1.1 mmol). The mixture was stirred at 70°C for 7 hours. Then, Et2O was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Purification by flash chromatography (hexane / EtOAc 95 / 05) delivered the corresponding ATO-C10(0.45 g,81% yield). HRMS calculated for ATO-C10C32H39ClO3[MH]+507.2660, found, 507.2659.1H NMR (400.13 MHz, CDCl3),δ 8.00-7.94 (2H, m), 7.65-7.58 (2H, m), 7.21- 7.18 (2H, m), 7.12-7.09 (2H, m), 4.26 (2H, t, J = 6.6), 3.19-3.11 (1H, m), 2.59-2.51 (1H, m), 2.17-2.07 (2H, m), 1.90 (2H, dd, J = 13.6, 2.8), 1.80-1.73 (2H, m), 1.66 (2H, dd, J = 13.3, 3.2), 1.54-1.40 (4H, m), 1.36-1.16 (12H, m), 0.80 (3H, t, J = 6.7).13C NMR (75 MHz, CDCl3) δ 185.5, 181.9, 158.1, 146.0, 138.6, 133.7, 133.0, 132.4, 131.5, 128.4, 128.2, 126.3, 125.9, 73.7, 43.4, 35.4, 34.5, 31.9, 30.4, 29.9, 29.7, 29.6, 29.4, 29.3, 26.0, 22.7, 14.1.

[0213] Preparation of Mito-ATO-PEG2To a mixture of atovaquone (0.5 g, 1.36 mmol) and potassium carbonate (0.26 g, 1.88 mmol) in DMF (5 mL) was added 1-bromo- 2-[2-(2-bromoethoxy)ethoxy]ethane, (0.63 g, 2.7 mmol). The mixture was stirred at 60°C for 13 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was dried over Na2SO4. The solvent was removed under reduced pressure. Then, Purification by flash chromatography (Pentane / Et2O from 100% to 70 / 30) delivered the corresponding ATO-PEG2-Br (0.25 g, 35% yield). High-resolution mass spectrometry, (HRMS) calculated for ATO-PEG2-Br C26H26O4BrCl [M+H]+519.0756, found, 519.0758.1H NMR (400.13 MHz, CDCl3),δ 8.08-7.99 (2H, m), 7.72-7.63 (2H, m), 7.27-7.22 (2H, m), 7.20-7.13 (2H, m), 4.56-4.50 (2H, m), 3.90-3.85 (2H, m), 3.84 (2H, t, J = 6.1), 3.42 (2H, t, J = 6.1), 3.28-3.18 (1H, m), 2.68-2.57 (1H, m), 2.27-2.174 (2H, m), 2.00-1.91 (2H, m), 1.78-1.68 (2H, m), 1.61-1.47 (2H, m).

[0214] A mixture of ATO-PEG2-Br (0.2 g, 0.36 mmol) and triphenylphosphine (93 mg, 0.36 mmol) in acetonitrile was stirred at reflux for 18 hours. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito-ATO-PEG2(0.2 g, 67% yield). HRMS calculated for Mito-ATO-PEG2C44H41O4PCl+[M]+699.2426, found, 699.2411.31P NMR (400.13 MHz, CDCl3) δ 25.15.1H NMR (400.13 MHz, CDCl3), δ 8.10-8.03 (1H, m), δ 8.01-7.94 (1H, m), 7.89-7.77 (6H, m), 7.77-7.64 (6H, m), 7.64-7.58 (5H, m), 7.28-7.22 (2H, m), 7.18-7.11 (2H, m), 4.28-4.19 (2H, m), 4.19-4.00 (4H, m), 3.69-3.57 (2H, m), 3.16-3.03 (1H, m), 2.63-2.48 (1H, m), 2.15-1.97 (2H, m), 1.91- 1.80 (2H, m), 1.71-1.57 (2H, m), 1.57-1.40 (2H, m).13C NMR (75 MHz, CDCl3)^δ 185.2, 181.3, 157.4, 145.6, 139.1, 134.7, 134.6, 134.0, 133.9, 133.8, 133.2, 132.2, 131.5, 131.2, 130.1, 130.0, 128.4, 128.1, 126.4, 125.9, 119.2, 118.3, 71.7, 70.3, 64.3, 64.2, 43.2, 35.4, 34.4, 29.9, 25.5 (d, J = 52.1).

[0215] Preparation of Mito-ATO-PEG4To a mixture of atovaquone (0.5 g, 1.36 mmol) and potassium carbonate (0.26 g, 1.88 mmol) in DMF (5 mL) was added 1,11-Dibromo-3,6,9-trioxaundecane, (1 g, 3.12 mmol). The mixture was stirred at 60°C for 8 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was dried over Na2SO4. The solvent was removed under reduced pressure. Then, Purification by flash chromatography (Pentane / Et2O from 100% to 70 / 30) delivered the corresponding ATO-PEG4-Br (0.67 g, 81% yield). HRMS calculated for ATO-PEG4-Br C30H34O6BrCl [M+NH4]+624.1547, found, 624.1540.1NMR (400.13 MHz, CDCl3),δ 8.08-8.03 (1H, m), 8.02-8.00 (1H, m), 7.73-7.63 (2H, m), 7.27-7.23 (2H, m), 7.20-7.15 (2H, m), 4.58-4.52 (2H, m), 3.88-3.83 (2H, m), 3.78-3.72 (2H, m), 3.70-3.65 (2H, m), 3.63- 3.54 (6H, m), 3.45-3.39 (2H, m), 3.28-3.18 (1H, m), 2.68-2.57 (1H, m), 2.28-2.14 (2H, m), 2.00-1.92 (2H, m), 1.78-1.68 (2H, m), 1.61-1.47 (2H, m).13C NMR (75 MHz, CDCl3) δ 185.3, 181.8, 157.7, 145.9, 138.3, 133.7, 133.0, 132.3, 131.4, 128.3, 128.1, 126.2, 125.8, 72.3, 71.1, 70.7, 70.6, 70.5, 70.4, 43.2, 35.3, 34.4, 30.2, 29.8.

[0216] A mixture of ATO-PEG4-Br (0.5 g, 0.82 mmol) and triphenylphosphine (0.24 g, 0.91 mmol) in acetonitrile was stirred at reflux for 18 hours. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito-ATO-PEG4(0.4 g, 56% yield). HRMS calculated for Mito-ATO-PEG4C48H49BrClO6P+[M]+787.2950, found, 787.2942.31P NMR (400.13 MHz, CDCl3) δ 25.59.1H NMR (400.13 MHz, CDCl3), δ8.10-8.09 (1H, m), 8.02-8.00 (1H, m), 7.91-7.82 (6H, m), 7.78-7.69 (5H, m), 7.69-7.62 (6H, m), 7.30-7.29 (1H, m), 7.27-7.26 (1H, m), 7.20-7.15 (2H, m), 4.56-4.50 (2H, m), 4.29-4.19 (2H, m), 4.01-3.89 (m, 2H), 3.87-3.80 (2H, m), 3.63-3.55 (2H, m), 3.42- 3.26 (2H, m), 3.34-3.28 (2H, m), 3.28-3.20 (3H, m), 2.69-2.59 (1H, m), 2.29-2.26 (2H, m), 2.03-1.92 (2H, m), 1.79-1.70 (2H, m), 1.62-1.40 (2H, m).13C NMR (75 MHz, CDCl3) δ 185.3, 181.7, 157.7, 145.9, 138.5, 134.6, 134.5, 134.1, 133.9, 133.8, 133.1, 132.3, 131.5, 131.3, 130.0, 129.9, 128.4, 128.1, 126.3, 125.8, 119.4, 118.5, 72.3, 70.6, 70.5, 70.2, 70.1, 69.9, 64.0, 63.9, 43.2, 35.3, 34.5, 29.8, 25.3 (d, J = 52.1).

[0217] Preparation of Mito-ATO-PEG5To a mixture of atovaquone (0.5 g, 1.36 mmol) and potassium carbonate (0.26 g, 1.88 mmol) in DMF (5 mL) was added 1,14- dibromo-3,6,9,12-tetraoxatetradecane (1 g, 2.73 mmol). The mixture was stirred at 60°C for 12 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was dried over Na2SO4. The solvent was removed under reduced pressure. Then, Purification by flash chromatography (Pentane / Et2O from 100% to 70 / 30) delivered the corresponding ATO-PEG5-Br (0.48 g, 54% yield). HRMS calculated for ATO-PEG5-Br C32H38O7BrCl [M+NH4]+668.1810, found, 668.1804.1H NMR (400.13 MHz,CDCl3),δ 8.05-8.00 (2H, m), 7.72-7.62 (2H, m), 7.27-7.22 (2H, m), 7.20-7.13 (2H, m), 4.56-4.50 (2H, m), 3.86-3.80 (2H, m), 3.76 (2H, t, J = 6.3), 3.69-3.52 (12H, m), 3.43 (2H, t, J = 6.2), 3.27-3.17 (1H, m), 2.66-2.56 (1H, m), 2.27-2.17 (2H, m), 2.00-1.88 (2H, m), 1.76-1.66 (2H, m), 1.60-1.46 (2H, m).13C NMR (75 MHz, CDCl3) δ 185.3, 181.7, 157.6, 145.9, 138.4, 134.53, 134.50, 134.0, 133.9, 133.8, 130.0, 129.0, 128.4, 128.1, 126.3, 125.8, 119.4, 118.5, 72.3, 70.6, 70.5, 70.4, 70.19, 70.18, 69.8, 64.0, 63.9, 58.2, 43.2, 35.3, 34.5, 29.8.

[0218] A mixture of ATO-PEG5-Br (0.48 g, 0.74 mmol) and triphenylphosphine (0.22 g, 0.84 mmol) in acetonitrile was stirred at reflux for 24 hours. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito-ATO-PEG5(0.35 g, 52% yield). HRMS calculated for Mito-ATO-PEG5C50H53O7PCl+[M]+831.3212, found, 831.3215.31P NMR (400.13 MHz, CDCl3) δ25.49.1H NMR (400.13 MHz, CDCl3),δ 8.06-8.02 (1H, m), 7.99-7.96 (1H, m), 7.87-7.77 (6H, m), 7.76-7.65 (5H, m), 7.67-7.58 (6H, m), 7.25-7.19 (2H, m), 7.18-7.12 (2H, m), 4.54-4.45 (2H, m), 4.19-4.07 (2H, m), 3.95-3.84 (m, 2H), 3.84-3.79 (2H, m), 3.67-3.61 (2H, m), 3.57-3.50 (2H, m), 3.47- 3.40 (2H, m), 3.37-3.26 (6H, m), 3.22-3.07 (1H, m), 2.26-2.12 (2H, m), 1.99-1.90 (2H, m), 1.76-1.65 (2H, m), 1.60-1.44 (2H, m).13C NMR (75 MHz, CDCl3) δ 185.3, 181.7, 157.6, 145.9, 138.4, 134.53, 134.50, 134.0, 133.9, 133.8, 133.1, 132.3, 131.4, 131.3, 130.0, 129.9, 128.4, 128.1, 126.3, 125.8, 119.4, 118.5, 72.3, 70.6, 70.5, 70.4, 70.19, 70.18, 69.8, 64.0, 63.9, 43.2, 35.3, 34.5, 29.8, 25.3 (d, J = 52.1).

[0219] Preparation of Mito-ATO-PEG9Triphenylphosphine (6.3 g, 12 mmol) was dissolved in acetonitrile (35 mL) under argon. Bromine (3.8 g, 24 mmol) was added drop by drop at 0°C. Then, 3, 6, 9, 12, 15, 18, 21, 24-octaoxahexacosane-1,26-diol (5.0 g, 12 mmol) was dissolved in acetonitrile (6 mL) and added dropwise. Reaction was stirred during 48 h at room temperature. The white residue was eliminated by filtration and the solvent was evaporated. The resulting orange residue was extracted several times with pentane. After evaporation of the combined extracts, the 1,26-dibromo-3, 6, 9, 12, 15, 18, 21, 24-octaoxahexacosane (Br-PEG8-Br) was obtained (4 g, 61%).1H NMR (400.13 MHz, CDCl3), δ 3.83 (4H, t, J = 6.4 Hz), 3.72-3.65 (28H, m), 3.49 (4H, t, J = 6.4 Hz).

[0220] To a mixture of atovaquone (0.4 g, 1.1 mmol) and potassium carbonate (0.26 g, 1.9 mmol) in DMF (5 mL) was added Br-PEG8-Br (1.2 g, 2.2 mmol) in 3mL of DMF. The mixture was stirred at 60°C for 16 hours. Then, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was dried over Na2SO4. The solvent was removed underreduced pressure. Then, Purification by flash chromatography (CH2Cl2 / Et2O, 50 / 50) delivered the corresponding ATO-PEG9-Br (0.80 g, 88% yield). The product was used without further purifications.

[0221] A mixture of ATO-PEG9-Br (0.8 g, 0.96 mmol) and triphenylphosphine (0.27 g, 1 mmol) in acetonitrile was stirred at reflux for 15 hours. Ether was added to precipitate the crude product. was Purification by flash chromatography (CH2Cl2 / EtOH 95 / 05 to 90 / 10) delivered the corresponding Mito-ATO-PEG9(0.14 g, 14% yield). HRMS calculated for Mito-ATO-PEG9C58H69O11PCl+[M]+1007.4261, found, 1007.4261.31P NMR (400.13 MHz, CDCl3) δ^25.65.1H NMR (400.13 MHz, CDCl3), δ 8.10-7.99 (2H, m), 7.91-7.83 (6H, m), 7.79-7.73 (3H, m), 7.72-7.62 (8H, m), 7.30-7.28 (1H, m), 7.27-7.26 (1H, m), 7.21-7.15 (2H, m), 4.58-4.53 (2H, m), 4.27-4.19 (2H, m), 3.98 (1H, t, J = 5.6), 3.92 (1H, t, J = 5.6), 3.88-3.83 (2H, m), 3.70-3.66 (2H, m), 3.63-3.55 (18H, m), 3.54-3.50 (2H, m), 3.40-3.37 (2H, m), 3.36-3.32 (2H, m), 3.31-3.26 (2H, m), 3.25-3.19 (1H, m), 2.69- 2.58 (1H, m), 2.29-2.15 (2H, m), 2.01-1.93 (2H, m), 1.79-1.70 (2H, m), 1.58-1.49 (2H, m).13C NMR (75 MHz, CDCl3) δ 185.5, 181.8, 157.7, 146.0, 138.4, 135.0, 134.6, 134.2, 133.9, 133.8, 133.1, 132.8, 131.5, 131.4, 130.1, 130.0, 128.4, 128.2, 127.0, 126.3, 126.0, 125.9, 119.4, 118.7, 72.4, 70.7, 70.5, 70.3, 69.9, 64.2, 64.1, 43.3, 43.2, 35.3, 34.52, 34.48, 34.3, 29.8, 29.2, 25.5 (d, J = 52.4).

[0222] Preparation of ATO-PEG To a mixture of atovaquone (0.3 g, 0.82 mmol) and potassium carbonate (0.11 g, 0.81 mmol) in DMF (3 mL) was added bromo-2,5,8,11- tetraoxatridecane, (0.22 g, 0.82 mmol). The mixture was stirred at 60°C for 12 hours. Then, Et2O was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Purification by flash chromatography (pentane / Et2O 80 / 20 to 100% Et2O) delivered the corresponding ATO-PEG (0.15 g, 33% yield). HRMS calculated for ATO-PEG C31H37O7Cl [M+NH4]+574.2566, found, 574.2566.1H NMR (400.13 MHz, CDCl3),δ 8.08-7.97 (2H, m), 7.72-7.62 (2H, m), 7.27-7.23 (2H, m), 7.20-7.15 (2H, m), 4.56-4.51 (2H, m), 3.87-3.82 (2H, m), 3.69-3.64 (2H, m), 3.61-3.56 (8H, m), 3.52-3.47 (2H, m), 3.34 (3H, s), 3.28-3.17 (1H, m), 2.66-2.56 (1H, m), 2.28-2.15 (2H, m), 2.00-1.90 (2H, m), 1.77- 1.68 (2H, m), 1.61-1.47 (2H, m).

[0223] Preparation of Mito-PEG4-LON The pegylated mitochondria-targeted analog of lonidamine (Mito-PEG-LON) was prepared in three steps, by reacting the freshly prepared acyl chloride of lonidamine with 2-{2-[2-(2-bromoethoxy)ethoxy]ethoxy}ethanol(Br-PEG4-OH) in the presence of TEA in CH2Cl2. Addition of triphenylphosphine on the bromopegylated lonidamine (PEG-LON-Br) led to Mito-PEG-LON in 40% yield. In addition, the pegylated lonidamine (PEG-LON) was prepared by using similar procedure in the presence of 2, 5, 8, 11-tetraoxatridecan-13-amine (MeO-PEG4-NH2) in 46% yield.Scheme 5. Synthesis of Mito-PEG-LON and PEG-LON. Reagents and conditions: I, (COCl)2, CH2Cl2, DMF, reflux, 2h., qt. Br-PEG4-OH, TEA, CH2Cl251%; iii, triphenylphosphine, CH3CN, reflux, 18h, 40%. iv, (COCl)2, CH2Cl2, DMF, reflux, 2h., qt. MeO-PEG4-NH2, TEA, CH2Cl2, 46%.

[0224] Preparation of Mito-PEG-LON. A solution of 1-(2, 4-dichlorobenzyl)-1H- indazole-3-carboxylic acid (lonidamine), (0.34 g, 1 mmol), oxalyl chloride (2 mL), and a catalytic amount of DMF (0.1 mL) in CH2Cl2(3 mL) was heated under reflux for 2 h. Unreacted oxalyl chloride and solvent were removed under reduced pressure to yield 1- (2,4-dichlorobenzyl)-1H-indazole-3-carbonyl chloride as a yellow solid. 2-{2-[2-(2- bromoethoxy)ethoxy]ethoxy}ethanol (Br-PEG4-OH) (0.27 g, 1 mmol) and triethylamine (0.284 mL, 2 mmol) were added to a solution of acyl chloride in CH2Cl2(10 mL), and the reaction mixture was stirred for 12 h at room temperature and then washed with water (30 mL). The organic layer was dried over Na2SO4and the solvent distilled under reduced pressure. Purification of the crude product by flash chromatography on a silica gel (CH2Cl2 / EtOH 95:05) afforded PEG-LON-Br (0.3 g, 51%).

[0225] A mixture of PEG-LON-Br (0.3 g, 0.54 mmol) and triphenylphosphine (0.15 g, 0.57 mmol) in 2 mL of acetonitrile was stirred at reflux for 18 hours. Then, Et2O was added to precipitate the product overnight. Purification by flash chromatography (CH2Cl2 / EtOHfrom 100% to 80 / 20) delivered the corresponding Mito-PEG-LON (0.16 g, 40% yield). HRMS calculated for Mito-PEG-LON C41H40Cl2N2O5P+[M]+741,2046 found, 741,2047.31P NMR (400.13 MHz, CDCl3) d 25.56.1H NMR (400.13 MHz, CDCl3) δ 8.22 (1H, dt, J = 8.2, 0.8), 7.89-7.81 (6H, m), 7.76-7.70 (3H, m), 7.69-7.61 (6H, m), 7.48-7.39 (3H, m), 7.36-7.31 (1H, m), 7.07 (1H, dd, J = 8.4, 2.2), 6.72 (1H, d, J = 8.3), 5.78 (2H, s), 4.59 (2H, t, J = 4.9), 4.18 (2H, m), 3.99-3.85 (4H, m), 3.64-3.59 (2H, m), 3.44-3.39 (2H, m), 3.35- 3.28 (4H, m).13C NMR (75 MHz, CDCl3) δ 162.3, 140.8, 134.5, 134.4, 134.1, 134.0, 133.1, 132.0, 130.0, 129.9, 129.5, 129.4, 127.6, 127.5, 123.8, 123.6, 122.4, 119.6, 118.4, 109.8, 70.6, 70.3, 70.2, 69.9, 69.1, 64.1, 63.9, 63.7, 50.4, 25.4 (d, J = 52.3).

[0226] Synthesis of PEG-LON. A solution of lonidamine, (0.19 g, 0.6 mmol), oxalyl chloride (2 mL), and a catalytic amount of DMF (0.1 mL) in CH2Cl2(3 mL) was heated under reflux for 2 h. Unreacted oxalyl chloride and solvent were removed under reduced pressure to yield 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbonyl chloride as a yellow solid. MeO-PEG4-NH2(0.13 g, 1 mmol) and triethylamine (284 μL, 2 mmol) were added to a solution of acyl chloride in CH2Cl2(10 mL), and the reaction mixture was stirred for 12 h at room temperature and then washed with water (30 mL). The organic layer was dried over Na2SO4and the solvent distilled under reduced pressure. Purification of the crude product by flash chromatography on a silica gel (CH2Cl2 / EtOH, 95:05) afforded LON-PEG (0.14 g, 46%). HRMS calculated for LON-PEG C24H29Cl2N3O5[M+H]+510.1557, found, 510.1563.1H NMR (400.13 MHz, CDCl3) δ 8.42 (1H, dt, J = 8.2, 1.1), 7.46-7.27 (5H, m), 7.12 (1H, dd, J = 8.3, 2.0), 6.64 (1H, d, J = 8.4), 5.68 (2H, s), 3.72-3.58 (14H, m), 3.55- 3.50 (2H, m), 3.36 (3H, s).

[0227] Synthesis of Mito-APO and Mito-PEG-APO is presented in Scheme 6.

[0228] Preparation of Mito10-APO (10-(4-acetyl-2- methoxyphenoxy)decyl)triphenylphosphonium bromide and Mito-PEG-APO (2-(2-(2-(2- (4-acetyl-2-methoxyphenoxy)ethoxy)ethoxy)ethoxy)ethyl)triphenylphosphonium bromide.

[0229] The pegylated mitochondria-targeted analog of apocynine (Mito-PEG-APO) was prepared in two steps, by reacting the 1,11-Dibromo-3,6,9-trioxaundecane (Br-PEG- Br) with apocynine in the presence of potassium carbonate in DMF. Addition of triphenylphosphine on the bromopegylated apocynine (APO-PEG-Br) led to the Mito- PEG-APO. In addition, the non-pegylated mitochondria targeted apocynine (Mito10-APO)was prepared by using similar procedure in the presence of (10-bromodecyl)- triphenylphosphonium bromide (Mito-Br).Scheme 6. Synthesis of Mito-PEG-APO and Mito10-APO. Reagents and conditions: i, Br-PEGn-Br, K2CO3, DMF, 45°C, 18h, 47%; ii, triphenylphosphine, CH3CN, reflux, 18h, 59%; iii, Mito-Br, K2CO3, DMF, 80°C, 18h, 43%.

[0230] Synthesis of Mito-PEG-APO.

[0231] To a mixture of apocynine (0.26 g, 1.56 mmol) and potassium carbonate (0.43 g, 3.12 mmol) in DMF (4 mL) was added 1,11-Dibromo-3,6,9-trioxaundecane, (0.5 g, 1.56 mmol). The mixture was stirred at 45°C for 18 hours. Then, Et2O was added to the mixture as well as H2O (25 mL). The organic layer was dried over Na2SO4. The solvent was removed under reduced pressure. Then, Purification by flash chromatography (Pentane / Et2O from 100% to 10 / 90) delivered the corresponding APO-PEG-Br (0.3 g, 47% yield). The product was used without further purifications.

[0232] A mixture of APO-PEG-Br (0.26 g, 0.64 mmol) and triphenylphosphine (0.21 g, 0.80 mmol) in 2 mL of acetonitrile was stirred at reflux for 18 hours. Then, Et2O was added to precipitate the product overnight. Purification by flash chromatography (CH2Cl2 / EtOH from 100% to 80 / 20) delivered the corresponding Mito-PEG-APO (0.25 g, 59% yield). HRMS calculated for Mito-PEG-APO C35H40O6P+[M]+587.2557, found, 587.2561.31P NMR (400.13 MHz, CDCl3) δ 25.55.1H NMR (400.13 MHz, CDCl3), δ 7.89-7.78 (6H, m), 7.77-7.70 (3H, m), 7.69-7.59 (6H, m), 7.55 (1H, dd, J = 8.2, 2.0), 7.51 (1H, d, J = 2.0), 6.87 (1H, d, J = 8.3), 4.24-4.14 (4H, m), 3.98 (t, 1H, J = 5.5), 3.94-3.83 (6H, m), 3.65-3.59 (2H, m), 3.44-3.38 (m, 2H), 3.37-3.26 (4H, m), 2.56 (3H, s).13C NMR (75 MHz, CDCl3) δ 196.8, 152.5, 149.2, 134.5, 134.4, 134.1, 134.0, 130.7, 130.0, 129.8,123.1, 119.6, 118.4, 111.6, 110.5, 70.8, 70.3, 70.2, 69.9, 64.4, 68.4, 64.1, 64.0, 55.9, 26.2, 25.4 (d, J = 52.8).

[0233] Synthesis of Mito10-APO.

[0234] To a mixture of apocynine (0.16 g, 0.96 mmol) and potassium carbonate (0.26 g, 1.88 mmol) in DMF (4 mL) was added (10-bromodecyl)-triphenylphosphonium bromide, (0.5 g, 0.90 mmol). The mixture was stirred at 80°C for 18 hours. Then, after precipitation with Et2O, CH2Cl2was added to the mixture as well as H2O (20 mL). The organic layer was dried over Na2SO4. The solvent was removed under reduced pressure. Then, Purification by flash chromatography (CH2Cl2 / EtOH from 100% to 80 / 20) delivered the corresponding Mito10-APO (0.25 g, 43% yield). HRMS calculated for Mito10-APO C37H44O3P+[M]+567.3023, found, 567.3021.31P NMR (400.13 MHz, CDCl3) δ 24.47.1H NMR (400.13 MHz, CDCl3), δ 7.91-7.75 (9H, m), 7.74-7.65 (6H, m), 7.55 (1H, dd, J = 8.3, 2.2), 7.51 (1H, d, J = 2.2), 6.87 (1H, d, J = 8.3), 4.06 (t, 2H, J = 6.8), 3.90 (3H, s), 3.88- 3.74 (2H, m), 2.55 (3H, s), 1.89-1.76 (4H, m), 1.67-1.56 (4H, m), 1.47-1.33 (2H, m), 1.32- 120 (6H, m).13C NMR (75 MHz, CDCl3) δ 196.9, 152.9, 149.2, 134.9, 134.8, 133.7, 133.6, 130.5, 130.3, 130.1, 123.3, 119.0, 117.8, 111.1, 110.4, 69.0, 56.0, 30.4, 30.2, 29.3, 29.2, 29.0, 28.9, 26.2, 25.8, 22.7, (d, J = 4.4), 22.8 (d, J = 49.5).

[0235] Analysis of L-DOPA and Dopamine using LC-MS or LC-MS-SIM

[0236] The freeze-dried samples were prepared as follows: To the tubes containing the cells was added 200 μL of ammonium formate buffer (10 mM). Then, the tubes were shaken vigorously (vortex) for 10 s and centrifugated for 7 min × 20,000 g at room temperature. The supernatants (100 μL) were transferred into HPLC vials with conical inserts and analyzed by LC-MS, described as follows. Mobile phase: Mobile phase A, 10 mM ammonium formate, pH 3.0, 90% MeCN, 10% water; Mobile phase B, 10 mM ammonium formate, pH 3.0, 50% MeCN, 50% water. Gradient Time (min) Mobile phase A (%) Mobile phase B (%)

[0237] Calculated log P values for MTD Analogs

[0238] The calculated log P values of the MTD analogs were assessed using a QSAR (quantitative structure–activity relationship) analysis and rational drug design as a measure of molecular hydrophobicity (Table 2). This method also uses a consensus model built using the ChemAxon software (San Diego, CA). Table 2 Calculated values of the octanol / water partition coefficients

[0239] Example 2

[0240] In this Example, the effects of Mito-APO analogs and MitoQ analogs on the bacterial proliferation and L-dopa consumptions of E. faecalis were studied. FIG. 19 shows the structure of the Mito-APO analogs and MitoQ analogs.

[0241] FIGS. 12, 14 and 16 show the effects of Mito-APO analogs on the bacterial proliferation of E. faecalis. The effects of Mito11-APO (FIG.12), Mito10-APO (FIG.14), and Mito2-APO (FIG.16) on the proliferation of E. faecalis were monitored at OD600 for 6 h. Data shown are the mean±SD, n=4.

[0242] FIGS. 13A-13D show effects of Mito11-APO on the bacterial L-dopa consumptions with or without carbidopa. E. faecalis was treated with the Mito11-APO as indicated in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa. The effects of Mito11-APO on the proliferation in presence of L-dopa and carbidopa combination (FIG.13A) were monitored at OD600 for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. High-performance liquidchromatography traces of representatives’ samples and standards are shown in panel (FIG. 13B). The effects of Mito11-APO on L-dopa consumption (FIG. 13C) and dopamine formation (FIG.13D) are shown in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD, n=4.

[0243] FIGS. 15A-15D show the effects of Mito10-APO on the bacterial L-dopa consumptions with or without carbidopa. E. faecalis was treated with the Mito10-APO as indicated in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa. The effects of Mito10-APO on the proliferation in presence of L-dopa and carbidopa combination (FIG.15A) were monitored at OD600for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. High-performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG. 15B). The effects of Mito10-APO on L-dopa consumption (FIG. 15C) and dopamine formation (FIG.15D) are shown in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD, n=4.

[0244] Both Mito-APO-C10 and Mito-APO-C11 inhibit E. facecalis proliferation, suggesting that attaching the TPP+moiety to either the carboxylic group (-COOH) or the hydroxyl group (-OH) elicited a similar effect. It is noteworthy that the alkyl side chain length affects the observed effect on E. faecalis proliferation. While the long-chain Mito- APO-C10 potently inhibited E. faecalis proliferation, the short chain analog, Mito-APO- C2, had no effect on E. faecalis proliferation at the same concentration. One plausible reason is that Mito-APO-C10 is more hydrophobic than Mito-APO-C2 and Mito-APO-C10 is taken up more than Mito-APO-C2 by the bacteria.

[0245] The effects of MitoQ analogs on the bacterial proliferation and L-dopa consumptions of E.faecalis were also studied. FIGS.17A-17B show the effects of MitoQ analogs on the bacterial proliferation of E. faecalis. The effects of MitoQ (FIG.17A), DM- MitoQ (FIG.17B) on the proliferation of E. faecalis were monitored at OD600 for 6 h. Data shown are the mean±SD, n=4.

[0246] FIGS. 18A-18D show the effects of MitoQ analogs on the bacterial L-dopa consumptions with or without carbidopa. E. faecalis was treated with the MitoQ or DM- MitoQ as indicated in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa. The effects of MitoQ or DM-MitoQ on the proliferation in presence of L-dopa and carbidopa combination (FIG.18A) were monitored at OD600 for 6 h and culture mediawere collected at indicated time points for L-dopa and dopamine measurements. High- performance liquid chromatography traces of representatives’ samples and standards are shown in panel (FIG.18B). The effects of MitoQ or DM-MitoQ on L-dopa consumption (FIG.18C) and dopamine formation (FIG.18D) are shown in presence of 1 mM L-dopa alone with additional 0.22 mM of carbidopa respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD, n=4.

[0247] Methylated derivative DM-MitoQ, which is redox-inactive, had an effect (as the redox-active Mito-Q) in inhibiting E. faecalis proliferation. This suggests that the antioxidant mechanism of action is not responsible for their antiproliferative effects.

[0248] Preparation of DM-MitoQ.

[0249] DM-MitoQ was prepared in a two-step synthesis (Scheme 7). After the reduction of MitoQ using sodium borohydride, reduced MitoQ was dimethylated by methyl iodide in the presence of potassium carbonate in DMF, leading to DM-MitoQ. Scheme 7. Synthesis of DM-MitoQ. Reagents and conditions: i, NaBH4, MeOH, 30 min.; ii, MeI, K2CO3, DMF, 40°C, 12h, 30%.

[0250] To a mixture of MitoQ (0.11 g, 0.16 mmol), sodium borohydride (0.5 g, 13 mmol) in MeOH was added at 0°C under inert atmosphere. Hydrochloric acid (2 M) was added to the reaction mixture and the crude product was extracted by dichloromethane (CH2Cl2). The organic layer was dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure.

[0251] To a mixture of reduced MitoQ (0.11, 0.16 mmol), anhydrous potassium carbonate (0.5 g, 3.6 mmol) in dimethylformamide (DMF) (4 mL) was added methyl iodide (3 mL). The mixture was stirred at 40°C for 12 h. The solvent was removed under vacuum and the residue was washed with diethyl ether (Et2O). Purification by preparative high- performance liquid chromatography (HPLC) delivered the corresponding DM-MitoQ (30 mg, 25% yield). Electrospray ionization mass spectrometry calculated for DM-MitoQ C39H50O4P [M]+613.3. HRMS calculated for DM-MitoQ C39H50O4P [M]+613.3441,found, 613.3446.31P (400.13 MHz, CDCl3) δ^23.80^^1H NMR (400.13 MHz, CDCl3) δ7.90-7.65 (15H, m), 3.90 (3H, s),3.89 (3H, s), 3.81 (3H, s), 3.78 (3H, s), 3.30-3.20 (2H, m), 2.57-2.49 (2H, m), 2.15 (3H, s), 1.68-1.16 (16H, m).

[0252] Although the invention has been described in considerable detail with reference to certain embodiments, one skilled in the art will appreciate that the present invention can be used in alternative embodiments to those described, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein.

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Claims

CLAIMS What is claimed is:

1. A method of modulating microbial metabolism of levodopa in gut of a subject in need thereof, the method comprising administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA;each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20; Mito is; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

2. A method of mitigating microbial degradation of levodopa in gut of a subject in need thereof, the method comprising administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl;R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20; Mito is L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

3. A method of improving bioavailability of levodopa in brain of a subject in need thereof, the method comprising administering to the subject levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;  Mito is; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

4. A method of treating Parkinson’s disease in a subject in need thereof, the method comprising administering to the subject an effective amount of levodopa and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,w e e one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20;Mito is L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

5. The method of any one of claims 1-4, whereby uptake of levodopa in the brain of the subject is increased and / or formation of dopamine in the brain of the subject is increased.

6. The method of any one of claims 1-5, whereby microbial metabolism of levodopa to dopamine in the gut of the subject is decreased.

7. The method of any one of claims 1-6, wherein the compound is a compound of formula (III) or (IV), or a pharmaceutically acceptable salt thereof.

8. The method of claim 7, wherein one of R3and R4in formula (III) or (IV) is Mito and the other is H.

9. The method of any one of claims 1-6, wherein the compound is a compound of formula (V) or (VI), or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein each of R5and R6in formula (V) and (VI), respectively, is Mito.

11. The method of any one of claims 1-6, wherein the compound is a compound of formula (VII) or (VIII), or a pharmaceutically acceptable salt thereof.

12. The method of any one of claims 1-6, wherein the compound is a compound of formula (VII), or a pharmaceutically acceptable salt thereof.

13. The method of any one of claims 1-6, wherein the compound is a compound of formula (IX) or (X), or a pharmaceutically acceptable salt thereof.

14. The method of any one of claims 1-13, wherein X is halogen, trifluoroacetate, or acetate.

15. The method of any one of claims 1-14, wherein L in Mito is C1-20alkylene.

16. The method of any one of claims 1-14, wherein L in Mito is L1-RC-L2, and RCis – (CH2CH2O)q–.

17. The method of any one of claims 1-16, wherein the compound is selected from the group consisting of, ,, ,, or a pharmaceutically acceptable salt thereof.

18. The method any one of claims 1-17, wherein levodopa and the compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof, are administered to the subject in a combination with carbidopa.

19. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, an effective amount of levodopa, and an effective amount of a compound of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), or (X), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is–(CH2CH2O)v–RA; each of R5and R6is Mito, –(CH2CH2O)w–RA, or RB; v is 0-20; w is 1-20;Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl; R7is Mito; R8is H, COCH3, or CO(CH2)kCH3; LAis C(O), (CH=CH), or CH2; LBis O, NH, or C(O)O; f is 2-16; k is 1-16; u is 1-20; Mito is; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

20. The pharmaceutical composition of claim 19, further comprising carbidopa.

21. A compound of formula (III-a) or (IV-a), or a pharmaceutically acceptable salt thereof,wherein one of R3and R4is Mito, the other is –(CH2CH2O)v–RA; v is 1-20; RAis H or C1-4alkyl; Mito is; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene; RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

22. The compound of claim 21, wherein v is 1, 2, 3, 4, or 5.

23. A compound of formula (V-a) or (VI-a), or a pharmaceutically acceptable salt thereof,wherein each of R5and R6is –(CH2CH2O)w–RAor RB; w is 1-20; Z is NH or O RAis H or C1-4alkyl; RBis C1-20alkyl.

24. The compound of claim 23, wherein each of R5and R6is –(CH2CH2O)w–RAand w is 1, 2, 3, 4, or 5.

25. A compound of formula (VII), or a pharmaceutically acceptable salt thereof,wherein R7is Mito;Mito is ; L is C1-20alkylene, C2-20alkenylene, L1-RC-L2, or amino acid; L1and L2are each independently absent or C1-C10alkylene;RCis –(CH2CH2O)q–, arylene, or cycloalkylene; q is 1-20; X is a counterion; Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

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