Polyphenolic drugs with short-chain fatty acids

Abstract

The present disclosure provides polyphenolic compounds with short chain fatty acids and the use thereof in treating diseases, such as Parkinson's disease and cancer.

Description

Attorney Docket: 650053.01246POLYPHENOLIC DRUGS WITH SHORT-CHAIN FATTY ACIDS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Patent Application No.63 / 709,772, filed October 21, 2024, the content of which is hereby incorporates by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 CA208648 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Emerging evidence suggests that therapeutic modulation of the gut microbiome may offer a new approach to treating Parkinson’s disease (PD) symptoms. Reports indicate that the gut microbiome and its metabolites, including short-chain fatty acids (SCFAs) such as butyrate and butyrate-producing bacteria, are decreased in patients with PD. Serum SCFAs, such as butyrate, play a crucial role in motor and non-motor symptoms, with decreased butyrate levels linked to increased depression in PD patients. Furthermore, sodium butyrate / butyric acid administration has been shown to be effective in a rodent model of PD, improving motor deficits and neuronal dopamine levels.

[0004] There remains a need for new ways of pharmacologically enhancing the gut microbiome and its metabolites to counteract deficiencies of butyrate-producing bacteria in PD subjects.SUMMARY

[0005] In one aspect, the present disclosure provides a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof.whereinone of R1and R2is C(O)RA, the other is C(O)RA, RB, or Mito;Attorney Docket: 650053.01246RAat each occurrence is independently Ci-2oalkyl, C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl,, amino acid, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein v is 0-20 and Rwis H or C(0)Ci-2oalkyl;RBis H, Ci-2oalkyl, C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl, amino acid, (CH2CH20)k-Ci-2oalkyl, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein k is 1-20;MitoLis Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;X is a counterion;Y at each occurrence is independently CFs, Me, Cl, OMe. C(O)CH3, NO2, N(Me)2, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0006] For example, the compound can have a structure of formula (I-a), (I-b), (I-c), (I-d), (I-e), (Il-a), (ILb), or (II-c)Attorney Docket: 650053.01246

[0007] In another aspect, the present disclosure provides a method of modulating microbial metabolism of levodopa in gut of a subject in need thereof. The method can comprise administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof

[0008] 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 can comprise administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof

[0009] In another aspect, the present disclosure provides a method of treating Parkinson’s disease in a subject in need thereof. The method can comprise administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0010] In some embodiments, uptake of levodopa in the brain of the subject is increased and / or formation of dopamine in the brain of the subject is increased. In some embodiments, microbialAttorney Docket: 650053.01246metabolism of levodopa to dopamine in the gut of the subject is reduced. In some embodiments, alpha-synuclein aggregation in the subject is decreased.

[0011] In another aspect, the present disclosure provides a method of reducing or inhibiting cancer growth in a subject in need thereof. The method can comprise administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0012] In another aspect, the present disclosure provides a method of inhibiting or reducing metastasis of a cancer in a subject in need thereof. The method can comprise administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof. The cancer can be, for example, melanoma, lung cancer, colon cancer, or pancreatic cancer.

[0013] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a compound as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0015] FIG. 1 shows the chemical structures of HNK-SCFA-esters.

[0016] FIG. 2 shows reagents and conditions for the preparation of HNK-SCFA-esters (i, TEA. CH2CI2, 24-45%).

[0017] FIG. 3 shows esterase-induced metabolism of HNK-SCFA-esters. (A) Schemes showing the hydrolysis of HNK-SCFA-esters and the bis-esters. (B) Kinetics of esterase-induced hydrolysis of HNK-SCFAs. (C) Kinetics of esterase-induced hydrolysis of HNK-SCFA-bis-esters.

[0018] FIG. 4 shows the effects of HNK analogs on the bacterial proliferation of E. faecalis. The effects of HNK- Ac (A), HNK-PAc (B), HNK-BAc (C), HNK-HAc (D), acetate (E), propionate (F), buty rate (G), and hexaonate (H) and on the proliferation of E. faecalis were monitored at OD 600 nm for 6 h. Data shown are the mean±SD, n=4.

[0019] FIG. 5A shows the uptake and hydrolysis of HNK SCFA analogs in E. faecalis cells. E. faecalis cells were treated with 50 pM of HNK, HNK-Ac, HNK-PAc HNK-BAc, or HNK-Attorney Docket: 650053.01246HAc for 1 h. The uptake and percent hydrolysis of these compounds in E. faecalis cells are shown for each analog.

[0020] FIG. 5B shows the uptake of HNK-SCFA esters.

[0021] FIG. 6 shows the effects of HNK-Ac, HNK-PAc, HNK-BAc and HNK-HAc on the bacterial L-dopa degradation in the presence of carbidopa. E. faecalis was treated with HNK- Ac (A), HNK-PAc (C), HNK-BAc (B), and HNK-HAc (D) as indicated, in the presence of 1 mM L-dopa and 0.22 mM of carbidopa. The effects of each analog (Ze f), on the proliferation were monitored at OD600 for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. HPLC traces of representative samples and standards are shown in the middle left panel. The effects on L-dopa consumption and dopamine formation are shown respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SD. n=4.

[0022] FIG. 7 shows the effects of HNK short chain fatty acid analogs on the bacterial membrane potential. E. faecalis was treated with HNK, HNK-Ac, HNK-PAc, HNK-BAc, and HNK-HAc (A) and acetate, propionate, butyrate (B) as indicated for 1 h. The effects on the membrane potential were measured by TMRM dye, 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. **, P<0.01 vs control group at each collection time point. Data shown are the mean±SD. n=4.

[0023] FIG. 8 shows the effects of HNK short chain fatty acid analogs on the ATP level. E. faecalis was treated with HNK-Ac (A), HNK-PAc (B), HNK-BAc (C), and HNK-HAc (D) as indicated for 0.5, 1, and 2 h.

[0024] FIG. 9 shows the chemical structures of butyrate derivatives.

[0025] FIG. 10 shows the enzymatic hydrolysis of HNK-Ac and HNK-BA-Ac.

[0026] FIG. 11 shows the effects of HNK analogs on the bacterial cytotoxicity. E. faecalis was treated with HNK analogs as indicated for 3 h, and cell death was monitored in real time by SYTOX Green staining. Data shown are the means ± SD for n=4.

[0027] FIG. 12 shows the effects of SCFAs on the ATP level. E. faecalis was treated with acetate (A), propionate (B), butyrate (C), and hexanoate (D) as indicated for 0.5 h, 1 h, and 2 h.

[0028] FIG. 13 shows the chemical structures of Mito-HNK-SCFA-esters.Attorney Docket: 650053.01246

[0029] FIG. 14 shows the esterase-induced metabolism of Mito-HNK-SCFA-esters (A) Resulting chemical structures of the enzymatic hydrolysis of Mito-HNK-SCFA-esters. (B) Enzymatic hydrolysis of Mito-HNK-SCFA-esters using esterase

[0030] FIG. 15 shows the effects of Mito-HNK analogs on the bacterial proliferation of E. faecalis. The effects of Mito-HNK-BAc (A), Mito-HNK-PAc (B), Mito-HNK-HAc (C), on the proliferation of A. faecalis were monitored at OD 600 nm for 6 h. Data shown are the mean±SD, n=4.

[0031] FIG. 16 shows the uptake and hydrolysis of Mito-HNK-BAc in E. faecalis cells. E. faecalis cells were treated with either 2 pM of Mito-HNK-BAc for 1 h. For both, total compound uptake and hydrolysis were measured by LC-MS.

[0032] FIG. 17 shows the effects of Mito-HNK-BAc and Mito-ortho-HNK on the bacterial L- dopa consumptions in the presence of carbidopa. E. faecalis was treated with Mito-HNK-BAc (left), or Mito-ort / io-HNK (right), as indicated, in the presence of 1 mM L-dopa and 0.22 mM of carbidopa. The effects of Mito-HNK-BAc (left), or Mito-o / 'tAo-HNK (right) on the proliferation (A) were monitored at OD600 for 6 h and culture media were collected at indicated time points for L-dopa and dopamine measurements. HPLC traces of representatives’ samples and standards are shown in panel (B). The effects on L-dopa consumption (C) and dopamine formation (D) are shown respectively. *, P<0.05 vs control group at each collection time point. Data shown are the mean±SEM, n=4.

[0033] FIG. 18 shows the effects of Mito-HNK short chain fatty acid analogs on the bacterial membrane potential. E. faecalis was treated with Mito-HNK (A), Mito-HNK-PAc (B), Mito- HNK-BAc (C) and Mito-HNK-HAc (D) as indicated for 1 h. The effects on the membrane potential were measured by TMRM dye, 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. **, P<0.01 vs control group at each collection time point. Data shown are the mean±SD, n=4.

[0034] FIG. 19 shows the esterase-induced metabolism of Mito-HNK-PAc and Mito-HNK- BAc.

[0035] FIG. 20 shows the antiproliferative effects of Mito-HNK and Mito-HNK-PAC in pancreatic cancer (MiaPaCa-2) cells. (A) The effects of Mito-HNK and Mito-HNK-PAc on the proliferation of MiaPaCa-2 cells were monitored in the IncuCyte Live-Cell Analysis system. The IncuCyte analyzer provides real-time updates on cell confluence based on segmentation of high- definition phase-contrast images. Representative cell images were shown asAttorney Docket: 650053.01246segmentation mask illustrated in brown when control cells reached 90% confluence (vertical solid black line). (B) The IC50 values for MiaPaCa-2 cells were determined at the point at which control cells reached -90% confluence (vertical solid black line). Relative cell confluence (control is taken as 100%) is plotted against concentration. Dashed lines represent the fitting curves used to determine the IC50 values as indicated. Data shown are the mean ± SD.

[0036] FIG.21 shows the efficacy of HNK-BAC, 4-Me-HNK-BAC (40MEHNK), and MAG-BAC in mice syngraft tumors. An LKR syngeneic mouse model was used and LKR tumor cells were subcutaneously injected into the flanks of SV 129 mice, which were treated by gavage with 20 mg / kg of the respective polyphenol-butyrate. Results show that these compounds effectively caused the regression of tumors. An LKR syngeneic mouse model was used to determine whether polyphenol-buty rates could enhance the efficacy of anti-PD-1 checkpoint blockade. LKR tumor cells were subcutaneously injected into the flanks of SV 129 mice, which were then treated with HNK-BAC (20 mg / kg body weight, oral gavage) anti-PD-1 antibody, BAC (100 mM), and HNK-BAC plus anti-PD-1 antibody).

[0037] FIG. 22 shows HNK-BAC improves the anti-tumor efficacy of anti-PD-1 antibody in the LKR model.

[0038] FIG. 23 shows the effects of HNK-PBA and Mito-HNK-PBA analogs on the bacterial proliferation of E. faecalis. The effects of HNK-PBA (A), HNK-R,S-PBA (B), Mito-HNK-PBA (C), and Mito-PEG-HNK-PBA (D) on the proliferation of E. faecalis were monitored at OD600 for 6 h. Data shown are the mean±SD, n=4.

[0039] FIG. 24 shows the chemical structures of some butyrate and hydroxybutyrate derivatives, according to an aspect of the disclosure.

[0040] FIG. 25 shows chemical structures of hypothetical hydroxy butyrate derivatives, according to an aspect of the disclosure.

[0041] FIG. 26 shows the cellular, mitochondria uptake and hydrolysis via esterases of HNK-BAc, Mito-HNK-BAc, HNK-BHB, Mito-HNK-BHB and derivatives.

[0042] FIG. 27 shows (A) Syntheses of HNK-BAc, 4Me-HNK-BAc and MGN-HNK-BAc. Reagents and conditions: i, butyryl chloride. TEA, CH2CI2, 40%; ii. K2CO3, Mel. DMF, 40°C, 24h, 35%.; iii, butyryl chloride, TEA, CH2CI2, 90%; iv, butyryl chloride, TEA, CH2CI2. 40%. (B) Esterase-induced cleavage of HNK-butyrate. Hydrolysis of HNK-butyrate esters to HNK and B Ac. (C) Kinetics of esterase-induced hydrolysis of HNK-BAc.

[0043] FIG. 28 shows (A) Control and LRRK2 G2019S iPSCs generate tyrosine hydroxylase (TH; red) positive dopamine neurons. Nuclei labeled with Hoechst (blue). (B) Neurite length was significantly shorter in LRRK2 G2019S mutant PD iPSC-derived dopaminergic neuronsAttorney Docket: 650053.01246compared to control at all time points tested, but did significantly lengthen by 9 weeks in culture. *p<0.01 compared to control; #p<0.01 comparing 9 weeks to 5 and 7 weeks by ANOVA. N=4 independent experiments from each of 3 independent control and 3 independent LRRK2 iPSC lines. Scale bar = 50pm.

[0044] FIG. 29 shows (A) All three LRRK2 G2019S iPSC-derived dopaminergic cultures displayed diminished (A) ATP-linked. (B) maximal, and (C) spare respiration compared to all three of the respective control cultures. *p<0.05 by one-way repeated measures ANOVA with Tukey’s post-hoc test. n=4 independent experiments.

[0045] FIG.30 shows (A, B) Both a-syn triplication neurons (SNCA 3x) and LRRK2 G2019S neurons show increase a-syn expression compared to control. (C-E) SNCA neurons do not have altered p62 or LC3-II expression, but LRRK2 G2019S neurons have significantly higher expression compared to control. LRRK2 kinase inhibition (LRRK2-IN-1) does not normalize p62 or LC3-II expression. N=3 *p<0.05

[0046] FIG. 31 shows (A) Synthesis of Mito-HNK and Mito-HNK-BAc. Reagents and conditions: i, 10-Bromodecyltriphenylphosphonium, DMF. K2CO3, 40°C, 36h, 25%; ii) butyryl chloride, TEA, CH2CI2, 90%. (B) Esterase-induced cleavage of Mito-HNK-butyrate. (C) Hydrolysis of Mito-HNK-but rate ester. Kinetics of esterase-induced hydrolysis of Mito-HNK-BAc. (D) Uptake of HNK-BAc. HPLC traces of standards and HNK-BAc in cells.DETAILED DESCRIPTION

[0047] 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.

[0048] 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.”

[0049] 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 upAttorney Docket: 650053.01246to plus or minus 10% of the particular term and “substantially’' and “significantly"’ will mean more than plus or minus 10% of the particular term.

[0050] 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

[0051] 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 Modem Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0052] The term “alky l” as used herein refers to a monovalent saturated straight or branched hydrocarbon, such as a straight or branched group of 1-20, 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C20 alkyl (or Cmoalkyl), C1-C12 alkyl (or Ci-12alkyl), C1-C10 alkyl (or Ci-ioalkyl), or Ci-Ce alkyl (or Ci-ealkyl), respectively.

[0053] The term “alkylene” refers to a divalent saturated straight or branched hydrocarbon group, such as a straight or branched group having 1-20, 1-12, 1-10, or 1-6 carbon atoms, referred to herein as a C1-C20 alkylene (or Ci-2oalkylene), C1-C12 alkylene (or Ci-i2alkylene), C1-C10 alkylene (or Ci-ioalkylene), or Ci-Ce alkylene (or Ci-ealkylene), respectively. An exemplary alkylene group is -CH2CH2-.

[0054] The term “alkenyl” refers to a monovalent straight or branched hydrocarbon group having one or more double bonds. An alkenyl group having up to 20 carbon atoms is referred to as a C2-C20 alkenyl (or C2-2oalkenyl). Likewise, for example, an alkenyl having up to 6 carbon atoms is referred to as a C2-C6 alkenyl (or C2-6alkenyl).Attorney Docket: 650053.01246

[0055] The term "alkenylene" refers to a divalent straight or branched hydrocarbon group having one or more double bonds. An alkenylenyl group having up to 20 carbon atoms is referred to as a C2-C20 alkenylene (or C2-2oalkenylene). Likewise, for example, an alkenylene having up to 6 carbon atoms is referred to as a C2-C6 alkenylene (or C2-6alkenylene).

[0056] The term "alkoxy" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, / e / 7-butoxy. pentyloxy, and hexyloxy.

[0057] The term “carboxy” or "carboxyl" as used herein refers to the group -COOH or its corresponding salts, e.g. -COONa, etc.

[0058] The term "and" is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, and the like. The term "aryl" includes polycyclic ring systems having two 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, cycloalkenyls, cycloalkynyls, and / or aryls.

[0059] The term “arylene” refers to a divalent carbocyclic aromatic group. Representative ary lene groups include -CsLL-, -CioHe-, and the like. The term "arylene" includes polycyclic ring systems having two 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, cycloalkenyls, cycloalkynyls, and / or aryls.

[0060] The term "phenyl" refers to a mono-substituted benzene ring and has a formula of -C6H5

[0061] The term "heteroaryl" is art-recognized and refers to a heterocyclic aromatic group. Representative heteroaryl groups include pyridinyl, quinolinyL furanyl, thionyl, and the like. The term "heteroaryl" includes polycyclic ring systems having two or more heterocyclic rings in which two or more carbon or heteroatom are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is a heterocyclic aromatic group and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. In certain embodiments, the heteroaryl group is a 6-10 membered ring structure. The term "pyridyl" refers to a group derived from pyridine by removal of a hydrogen atom from a ring carbon atom in pyridine. The pyridyl group has a formula -C5H4N.

[0062] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein,Attorney Docket: 650053.01246e.g., as "C4-8-cycloalkyl," derived from a cycloalkane. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0063] The term “cycloalkylene” refers to a divalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group having, for example, 3-12, 3-8, 4-8, or 4-6 carbons derived from a cycloalkane. An exemplary cycloalkylene group is -C3H4-. Unless specified otherwise, cycloalkylene groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato. phosphinato. sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkylene group is not substituted, i.e., it is unsubstituted.

[0064] The terms "heterocycloalkyl" and "heterocyclic group" are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3-to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using Cx-Cy or Cx-y nomenclature where x and y are integers specifying the number of ring atoms. For example, a C3-C7 (or C3-7) heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation "C3-C7" or “C3-7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. In one embodiment, the heterocyclyl is piperidinyl

[0065] The term "halogen" refers to halogen atoms F, Cl, Br, and I, or halogen substituents fluoro (-F). chloro (-Cl), bromo (-Br), and iodo- (-1).

[0066] The term “haloalkyl” is art-recognized and refers to an alkyl group, as defined above, having halogen atoms, as defined above, replacing one or more hydrogen atoms. Representative haloalkyl groups include trifluoromethyl, dibromoethyl, monochloropropyl, and the like.

[0067] The term "hydroxy" refers to a group of the form -OH.Attorney Docket: 650053.01246

[0068] The term “hydroxyalkyl” refers to an alkyl means an alkyl, as defined herein, in which a hydrogen atom is replaced by -OH. Representative examples of hydroxyalkyl include, but are not limited to those derived from Ci-6 alkyls, such as -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, and the like.

[0069] The term "nitro" refers to a group of the form -NO2.

[0070] The term "cyano" refers to a group of the form -CN.

[0071] Terms such as "alkyl," "cycloalkyl," "alkylene," "cycloalkylene." etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “Cwalkyl”, “Ci-C4alkyl”, “Cs-ecycloalkyl”, “C.i-Cecycloalkyl”, “Ci-4alkylene”, “Ci-C4alkylene”). 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, "Csalkyl" is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in "Ci-C4" or "Ci-4" the members of the group that follows may have any number of carbon atoms falling within the recited range. A "Ci-C4alkyl" or "Ci-4alkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0072] If a group is described as being "substituted", a non-hydrogen substituent group is in the place of hydrogen on a carbon or nitrogen of that group. Thus, for example, a substituted alkyl is an alkyl in which at least one non-hydrogen group is in the place of a hydrogen on the alkyl. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro group, and difluoroalkyl is alkyl substituted with two fluoro groups. It should be recognized that if there is more than one substitution on a substituent, each non-hydrogen group may be identical or different (unless otherwise stated). Substituent groups include, but are not limited to, halogen, =0, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0073] When a group is referred to as "unsubstituted" or not referred to as "substituted" or "optionally substituted", it means that the group does not have any substituents. If a group is described as being "optionally substituted", the group may be either (1) not substituted or (2) substituted. If a group is described as being optionally substituted with up to a particular number of non-hydrogen substituents, that group may be either (1) not substituted; or (2)Attorney Docket: 650053.01246substituted by up to that particular number of substituent groups or by up to the maximum number of substitutable positions on that group, whichever is less.

[0074] 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).

[0075] A person of ordinary skill in the art would be able to choose the substituents that fulfill the valency rules. For example, in a non-solvated or non-salt form of a compound, nitrogen typically has three bonds attached to it and oxygen typically has two bonds attached to it.

[0076] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term "stereoisomers" when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereo isomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated "(±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise.

[0077] As used herein, "salt" refers to acid addition salts and basic addition salts. It may also refer to those salts that may be prepared in situ during the final isolation and purification of the present compounds. The term "pharmaceutically acceptable salt thereof’ means a salt prepared by combining a compound as described herein 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.

[0078] 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,Attorney Docket: 650053.01246benzoic, 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.

[0079] 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.

[0080] 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 quatemized with agents such as lower alkyl (Ci-Cs) 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.

[0081] 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 hereinAttorney Docket: 650053.01246may 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.

[0082] 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 specifically mentioned 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.

[0083] The present disclosure relates to polyphenolic compounds, such as honokiol (HNK) and magnolol (MGN), modified with short chain fatty acid (SCFA, such C2-10 carboxylic acids). These compound can further include a triphenylphosphonium cation (TPP+)-based mitochondria-targeting moiety. The disclosed compounds may be useful as novel therapeutic agents for treatment of Parkinson’s disease or cancer.

[0084] Compounds

[0085] In one aspect, the present disclosure provides a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof,whereinone of R1and R2is C(O)RA, the other is C(O)RA, RB, or Mito;Attorney Docket: 650053.01246RAat each occurrence is independently Ci-2oalkyl, C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl,, amino acid, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein v is 0-20 and Rwis H or C(0)Ci-2oalkyl;RBis H, Ci-2oalkyl, C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl, amino acid, (CH2CH20)k-Ci-2oalkyl, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein k is 1-20;MitoLis Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;X is a counterion;Y at each occurrence is independently CFs, Me, Cl, OMe. C(O)CH3, NO2, N(Me)2, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0086] In some embodiments, R1is -C(O)RA, and R2is -C(O)RA, RB, or Mito.

[0087] In some embodiments, R1is -C(O)RA, RB, or Mito, and R2is -C(O)RA

[0088] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has a structure of formula of (I-a), (I-b), (I-c), (I-d), or (I-e)Attorney Docket: 650053.01246

[0089] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has a structure of formula of (Il-a), (Il-b), or (II-c)Attorney Docket: 650053.01246

[0090] In some embodiments, RAat each occurrence is independently Ci-2oalkyl, Ci-ioalkyl, Ci-salkyl, or Ci-3alkyl. In some embodiments, each RAis independently CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, CH2CH2CH2CH2CH3, or CH(CH2CH2CH3)2. In some embodiments, each RAis independently CH3, CH2CH3, or CH2CH2CH3. In some embodiments, RAis CH2CH2CH3

[0091] In some embodiments. RAat each occurrence is independently Ci-2oalkylene-phenyl, Ci-ioalkylene-phenyl, Ci-salkylene-phenyl, or Ci-3alkylene-phenyl. In some embodiments, each RAis independently CH2C6H5, CH2CH2C6H5, CH2CH2CH2C6H5, orCH2CH2CH2CH2C6C5. In some embodiments, RAis CH2CH2CH2C6H5 (or

[0092] In some embodiments, RAat each occurrence is independentlysome embodiments, v is 0-10, for example, v is 0, 1, 2, or 3. In some embodiments, Rwis H. In some embodiments, Rwis C(0)Ci-ioalkyl, C(O)Ci-5alkyl, or C(O)Ci-3alkyl. In some embodiments, RwC(O)CH2CH2CH3. In some embodiments, each RAis independentlyembodiments, each RAis independently OH.

[0093] In some embodiments, X is halogen, trifluoroacetate, or acetate. For example, X can be Br or trifluoroacetate.

[0094] In some embodiments, L is Ci-2oalkylene. For example, L can be -(CH2)4-, -(CH2)6-, -(CH2)8-, -(CH2)IO-, or - (CH2)i2-. In some embodiments, L is -(CH2)10-

[0095] In some embodiments, L is LI-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. Li is absent or-CH2CH2-. In some embodiments, L2 is absent or -CH2CH2-. In some embodiments, L is -(CH2CH2O)3-CH2CH2- or -(CH2CH2O)4-CH2CH2-.

[0096] In some embodiments, m is 0.Attorney Docket: 650053.01246

[0097] In some embodiments. Mito is -L-PPh3+. In some embodiments, Mito is -(CH2)6-PPhs+, -(CH2)8-PPh3+, -(CH2)10-PPh3+. In some embodiments, Mito is -(CH2CH2O)3-CH2CH2-PPh3+or -(CH2CH2O)4-CH2CH2- PPh3+.

[0098] In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has a structure of formula (I-a) or (I-b), in which RAis Ci-salkyl (such as CH2CH2CH3) and L is C8-15alkylene (such as -(CH2)10-). For example, in the compound of formula (I-a) or (I-b), or a pharmaceutically acceptable salt thereof, RAcan be CH2CH2CH3 and Mito can be -(CH2)io-PPh3+.

[0099] In some embodiments, the present compounds, or a pharmaceutically acceptable salt thereof, is selected from the group consisting ofAttorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246

[0100] In some embodiments, the present compounds, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of, and

[0101] Pharmaceutical Compositions

[0102] Another aspect of the disclosure provides a pharmaceutical composition. The pharmaceutical composition comprises the compound as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0103] The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight. In some embodiments, the pharmaceutical composition may beAttorney Docket: 650053.01246administered to provide the compound at a daily dose of about 0.5 to about 500 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg / kg body weight. 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 pM, 0.005 pM, 0.01 pM, 0.5 pM, 0.1 pM. 1.0 pM, 10 pM, and 100 pM (e.g, 0.1 pM - 1.0 pM).

[0104] The present compound may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0105] The compounds may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants. and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcry stallinc cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as buty lparaben, alcohols such as ethyl or benzy l alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0106] Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.Attorney Docket: 650053.01246

[0107] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, com starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0108] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0109] Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0110] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0111] 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.

[0112] 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.

[0113] 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 compositionsAttorney Docket: 650053.01246adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable earner, especially an aqueous solvent.

[0114] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

[0115] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0116] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i. e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0117] Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0118] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0119] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0120] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may beAttorney Docket: 650053.01246presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil. oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0121] Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat Parkinsons' disease or cancers. 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 one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating Parkinsons’ disease or cancers.

[0122] Methods of Use

[0123] Therapeutic targeting of the gut microbiome is emerging as a new way to treat Parkinson’s disease (PD) symptoms. Emerging research suggests levodopa (L-dopa) is metabolized to dopamine in the gut of some PD patients. This greatly decreases the amount of L-dopa that reaches the brain and its conversion to dopamine in the brain. Thus, it is essential to inhibit the bacterial degradation of L-dopa in the gut. We have recently shown that polyphenols such as honokiol (HNK) and the mitochondria-targeted analog of honokiol (Mito-HNK) dose-dependently and reversibly inhibit the proliferation of Enterococcus faecalis and the metabolism of L-dopa to dopamine in the gut.

[0124] Short-chain fatty acids (SCFAs), i.e., acetate with two carbons; propionate with three carbons; butyrate with four carbons; valerate with five carbons; caproate with six carbons; and valproate with 8 carbons are released in the colonic microbiota from anaerobic fermentation of dietary fibers. The relative amounts of SCFA released in the gut are dependent on the type and amount of ingested fiber. Propionate is a major microbial fermentation-induced metabolite in the human gut that mediates neuroprotection in PD. Butyrate-generating bacteria have alsoAttorney Docket: 650053.01246been linked to neuroprotection in PD. The increase of gut butyrate formed from prebiotic butyrogenic fibers was viewed as a potential therapeutic approach.

[0125] The extracellular pH in the gut is very acidic, ranging between 1 and 4. In vitro experiments suggest that even with prolonged incubation of the Mito-HNK-SCFA-esters in formate buffer (pH 3) at 37°C there was no hydrolysis. The influence of chain length on the esterase-mediated hydrolysis of esters and their structure-function relationship are well established.

[0126] Reports indicate that patients with PD have decreased levels of SCFAs, such as butyrate, in their serum and feces, and butyrate-generating bacteria in their gut. Serum SCFAs alter motor and nonmotor symptoms in patients with PD. Low butyrate levels are linked to increased depression and alpha-synuclein aggregation in PD. In animal models of PD, oral administration of sodium butyrate or butyric acid improved motor function, and increased dopamine levels and histone acetylation. Therefore, pharmacologically enhancing butyrate levels in the gut microbiome may therapeutically counter the butyrate-producing bacteria deficiency in PD subjects.

[0127] In another 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 an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0128] 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 an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0129] In another aspect, the present disclosure provides a method of improving bioavailability of L-dopa in brain of a subject in need thereof, the method comprising administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0130] In another aspect, the present disclosure provides a method of treating Parkinson’s disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0131] In some embodiments, by the present methods, uptake of levodopa in the brain of the subject is increased, or formation of dopamine in the brain of the subject is increased, or both.Attorney Docket: 650053.01246

[0132] In some embodiments, by the present methods, microbial metabolism of levodopa to dopamine in the gut of the subject is reduced.

[0133] In some embodiments, by the present method, alpha-synuclein aggregation in the subject is decreased. Without being limited by any theory, it is hypothesized that the present compounds may block the buildup of toxic a-synuclein protein in brain cells from Parkinson’s patients. In some embodiments, by improving mitochondrial health and reducing inflammation, the dual-action compounds as described herein may lead to new treatments for Parkinson’s and related brain disorders.

[0134] Mitochondria-targeted drugs (MTDs) inhibiting oxidative phosphorylation (OXPHOS) mitigate tumor cell proliferation. Mitochondria-targeted OXPHOS inhibitors decreased myeloid-derived suppressor cells (MDSCs). regulatory T cells (Tregs), and activated cytotoxic T cells in the tumor microenvironment. In addition, short-chain fatty acids (SCFA, e.g., acetate with two carbons, propionate with three carbons, butyrate with four carbons, valerate with five carbons, and caproate with six carbons) released in the colonic microbiota inhibit tumorigenesis, activate T cells, and improve immunotherapy through epigenetic modification. Without being limited by any theory, it is hypothesized that a molecule that combines the OXPHOS-inhibiting drug moiety and a short chain fatty acid ester may amplify tumor inhibition through immune modulation. In particular, the esterase enzymes in the gut can cleave the short-chain fatty acid esters and generate in situ short-chain fatty acids and the parent drug.

[0135] In another aspect, the present disclosure provides a method of reducing or inhibiting cancer growth in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof..

[0136] In some embodiments, the method further comprises treating the patient with surgery, radiation therapy (RT), or chemotherapy (CT) prior to or concurrently with administering the pharmaceutical composition.

[0137] In another aspect, the present disclosure provides a method of inhibiting or reducing metastasis of a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound as described herein, or a pharmaceutically acceptable salt thereof.

[0138] As used herein, the term "tumor" or "cancer" refers to any abnormal proliferation of tissues, including solid and non-solid tumors. For instance, the composition and methods of the present disclosure can be utilized to treat cancers that manifest solid tumors such as breast cancer, colon cancer, gastric cancer, glioblastoma, head and neck cancer, kidney cancer, liverAttorney Docket: 650053.01246cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and the like. In some embodiments, the cancer is melanoma, lung cancer, colon cancer, or pancreatic cancer.

[0139] In some embodiments, the method comprises administrating a compound having a structure of formula (I-a), (I-b), (I-c), or (Il-a), or a pharmaceutically acceptable salt thereof.

[0140] As used herein, the term "subject" refers to mammals, non-mammals, and / or cells. "Mammals" means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory’ animals including rodents, such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. The term "subject" does not denote a particular age or sex. Preferably, the subject is a human, particularly a human having PD or cancer.

[0141] As used herein, the term "treat" or "treating" refers to the management and care of a subject for the purpose of combating the disease, condition, or disorder. Treating includes the administration of a compound or a pharmaceutical composition of the present disclosure to inhibit, ameliorate and / or improve the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.

[0142] As used herein, the term "administering" refers to any means for introducing the compounds and the pharmaceutical compositions as disclosed herein into the body, preferablyAttorney Docket: 650053.01246into the systemic circulation. Examples include but are not limited to oral, buccal, sublingual, pulmonary, transdermal. transmucosal, as well as subcutaneous, intraperitoneal, intravenous, and intramuscular injection.

[0143] The compounds and pharmaceutical compositions utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0144] In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg. 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg. 200 mg, 500 mg. 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as endpoints any of these disclosed doses (e.g., 2.5 mg - 200 mg).

[0145] In some embodiments, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000. 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000. 10000, 15000, or 20000 ng / kg body weight of the subject. Minimal and / or maximal dose levels of the compounds for achieving therapy in the disclosed methodsAttorney Docket: 650053.01246of treatment may include dose levels falling within ranges having as endpoints any of these disclosed dose levels (e.g.. 500 - 2000 ng / kg body weight of the subject).

[0146] Kits

[0147] Another aspect of the disclosure provides a kit comprising a pharmaceutical composition comprising the compounds as disclosed herein and instructional material.

[0148] 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.EXAMPLES

[0149] The development of new pharmacotherapies for PD requires the synthesis and development of new molecules. Phenolic lipid has previously been used as a prodrug of butyric acid showing antibacterial effects, and polyphenols conjugated to SCFAs have previously been developed. We propose a novel class of molecules that combine HNK and Mito-HNK with SCFA esters (e.g., HNK-SCFA-esters and Mito-HNK-SCFA-esters). These molecules can release HNK or Mito-HNK and SCFAs by the action of esterase enzymes. For instance, honokiol butyric acid (HNK-BAc) and mitochondria-targeted honokiol butyric acid (Mito-HNK-BAc) will release the parent drug. HNK and Mito-HNK, and butyrate (FIGS 3 and 13). In this study, we report the antimicrobial potential of mitochondria-targeted drug (MTD)-containing esterase-cleavable SCFAs. We demonstrate that HNK-SCFA-esters and Mito-HNK-SCFA-esters (FIGS 1 and 12) can effectively delay the growth and degradation of L-dopa and its metabolism to dopamine in a dose-dependent manner.

[0150] Example 1

[0151] Disclosed herein are conjugates of honokiol (HNK) with short-chain fatty acids (SCFAs) and the effects of the conjugates on gut bacterium Enterococcus faecalis.

[0152] Introduction

[0153] Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by motor dysfunction and non-motor symptoms. Honokiol (HNK), a naturally occurringAttorney Docket: 650053.01246polyphenol derived from the bark of the Magnolia genus, has demonstrated neuroprotective effects in several PD mouse models. To enhance its therapeutic potential, we investigated a novel strategy involving the synthesis of HNK conjugates with short-chain fatty acids (SCFAs), generating a series of HNK-SCFA-esters (FIGs. 1 and 2). These esters are designed to undergo hydrolysis by gut-derived esterases, thereby releasing both HNK and SCFAs in the gut.

[0154] Representative HNK-SCFA conjugates include honokiol acetic acid (HNK-Ac), propionic acid (HNK-PAc), butyric acid (HNK-BAc), and hexanoic acid (HNK-HAc), along with their Z> A-ester counterparts. The dual release of HNK and SCFAs offers the potential for synergistic neuroprotective effects. Although similar phenolic lipids have previously been explored as prodrugs of butyric acid for antibacterial applications, and polyphenol-SCFA conjugates have been described, their relevance to PD pathophysiology has not been established. Considering the unmet need for novel pharmacological approaches targeting both motor and non-motor PD symptoms, the development of HNK-SCFA esters represents a promising therapeutic strategy.

[0155] Recent advances in microbiome research have highlighted the gut-brain axis as a key modulator of PD progression. Patients with PD consistently exhibit decreased gut microbial diversity and diminished levels of SCFAs, particularly butyrate, due to decreased abundance of butyrate-producing bacteria. This depletion correlates with the severity of both motor and non-motor symptoms, including depression in PD. Moreover, sodium butyrate administration has been shown to restore striatal dopamine levels and improve motor performance in preclinical PD models. Strategies aimed at augmenting gut-derived butyrate, through dietary supplementation, microbial modulation, or drug delivery, are of growing therapeutic interest in PD.

[0156] An additional microbiome-related challenge in PD is the compromised bioavailability of oral levodopa (L-dopa). Gut bacteria, particularly Enterococcus faecalis, metabolize L-dopa to dopamine via tyrosine decarboxylase in the gut, thereby reducing L-dopa’s systemic absorption and delivery to the brain. Because dopamine produced in the gut cannot cross the blood-brain barrier, this microbial metabolism leads to decreased central dopaminergic activity. Importantly, deletion of the tyrosine decarboxylase gene in E. faecalis abolished L-dopa metabolism in the gut, underscoring its central role in this detrimental pathway.

[0157] Although carbidopa, a peripheral aromatic L-amino acid decarboxylase inhibitor, is coadministered with L-dopa to prevent extracerebral metabolism, it does not inhibit bacterial tyrosine decarboxylases. Consequently, gut microbial metabolism remains a barrier to therapeutic efficacy of L-dopa. In this study, we demonstrate that selected esterase-cleavableAttorney Docket: 650053.01246HNK-SCFA conjugates delay bacterial L-dopa metabolism and attenuate dopamine formation in a dose-dependent manner.

[0158] To our knowledge, this is the first study to describe polyphenol-based SCFA conjugates with esterase-labile and antimicrobial properties relevant to PD. These findings establish a mechanistic basis for a multifunctional therapy that integrates gut microbiome modulation, neuroprotection, and preservation of L-dopa bioavailability. This approach represents a significant step toward the development of gut-targeted pharmacological interventions for PD. This study investigates the antimicrobial properties of honokiol (HNK), a naturally occurring polyphenol, when conjugated with short-chain fatty acids (SCFAs) such as butyrate. The inventors examined effects of HNK-SCFA ester conjugates on Enterococcus faecalis, a gut bacterium that metabolizes levodopa, a drug used to manage Parkinson’s disease symptoms. Our findings indicate that HNK-SCFA-esters (e.g., HNK-acetate, HNK-propi onate, HNK-butyrate, and HNK- hexanoate) inhibit E. faecalis growth in a dose-dependent manner, followed by a temporary recovery period during which levodopa remains intact and unmetabolized. Notably, HNK-SCFAs exhibit enhanced cellular permeability and are hydrolyzed within bacterial cells, releasing HNK and SCFAs. These results suggest that HNK-SCFAs may reversibly modulate the gut metabolism of levodopa to dopamine, potentially enhancing its therapeutic efficacy in treating Parkinson's disease (PD).

[0159] Results

[0160] Syntheses of HNK-SCFAs

[0161] Representative HNK-SCFA compounds were synthesized, and their chemical structures are shown in FIG. 1. In some cases, HNK-SCFAs were obtained by reacting HNK with the appropriate acyl chloride in the presence of triethylamine in dichloromethane (FIG. 2). The structures and purities of the products were confirmed by NMR analyses (below).

[0162] Esterase-induced hydrolysis of HNK-SCFAs HNK-SCFA-esters (HNK-Ac, HNK-PAc, HNK-BAc, HNK-HAc, and corresponding bis-esters) were hydrolyzed by esterase enzymes. FIG. 3 illustrates the time-dependent formation of HNK and the corresponding SCFA. Notably, in vitro experiments indicated no hydrolysis of HNK-SCFAs in formate buffer (pH=3) at 37 C, even after prolonged incubation (data not shown). Results showed that HNK-Ac and HNK-Bis-Ac underwent esterase-induced hydrolysis very rapidly (FIG. 10). As the chain length increased, the rate of hydrolysis decreased, following the order HNK-PAc < HNK-BAc < HNK-HAc.

[0163] Inhibition of E. faecalis proliferation by HNK-SCFA-esters Interestingly, unlike HNK (Fig. 4A), HNK-SCFAs with the esterase-cleavable SCFAs e.g., HNK-BAc) induced aAttorney Docket: 650053.01246dose-dependent time delay in E. faecalis proliferation (FIG. 4, panels B, C, and D). After the time lag, bacterial growth resumed at a similar rate. In contrast, SCFAs alone inhibited E. faecalis proliferation without a time lag at millimolar levels (FIG. 4, panels E-H).

[0164] Uptake and hydrolysis of HNK-SCFA conjugates in E. faecalis cells FIG. 5A illustrates the uptake and hydrolysis of HNK-Ac, HNK-PAc, HNK-BAc, and HNK-HAc by E. faecalis over 1 h. The concentrations of HNK-SCFA conjugates in E. faecalis cells increased with chain length (HNK-HAc = HNK-BAc > HNK-PAc > HNK-Ac). (FIG. 5B) However, bacterial esterase-induced hydrolysis of HNK-SCFAs decreased with increasing chain length. Approximately 80% of HNK-Ac, 50% of HNK-PAc, 16% of HNK-BAc, and 10% of HNK- HAc were hydrolyzed to HNK. SCFAs (e.g., acetate, propionate, and butyrate) released from the hydrolysis of HNK-SCFA conjugates were not detectable using LC-MS, suggesting the need for further investigation using other mass spectrometry approaches such as GC-MS analyses.

[0165] Effect of HNK-SCFAs on L-dopa metabolism in E. faecalis cells L-dopa is routinely used in combination with carbidopa in PD management. Therefore, we examined the effect of carbidopa (using the same L-dopa: carbidopa ratio as in clinical use) on L-dopa metabolism by E. faecalis treated with HNK-SCFAs. At clinically relevant ratios, carbidopa did not affect L- dopa metabolism, as it is a poor substrate for bacterial tyrosine decarboxylase. FIG. 6, panels A-D show that HNK-SCFAs dose-dependently decreased L-dopa degradation and dopamine formation by E. faecalis. Notably, both HNK-PAc and HNK-BAc inhibited dopamine formation more effectively than HNK-Ac. In contrast, HNK did not exhibit a similar pattern; with increasing doses, there was a total inhibition of E. faecalis proliferation (Cheng et al., B. (2024), Commun Biol 7, 668).

[0166] Effect of HNK-SCFA-esters on E. faecalis membrane potential FIG. 7. panel A demonstrates that HNK-PAc, HNK-BAc, and HNK-HAc increased the membrane potential of E. faecalis at higher concentrations, with HNK-BAc exhibiting the strongest effect among the HNK-SCFA conjugates. Conversely, acetate, propionate, and butyrate did not affect membrane potential across a range of concentrations (FIG. 7, panel B). These findings are consistent with the observed enhanced uptake of HNK-SCFA-esters in the E. faecalis system (FIG. 6). The paradoxical effects of SCFAs in bacterial versus mammalian cells are discussed further.

[0167] Effects of HNK-SCFAs on ATP levels in E. faecalis Intracellular ATP was measured in E. faecalis cells exposed to HNK-SCFAs and SCFAs. As shown, the effects of HNK-SCFAs on ATP formation varied (FIG. 8). Initially, HNK-SCFAs enhanced ATP production followedAttorney Docket: 650053.01246by a decrease. SCFAs alone (FIG. 11, panel B) increased ATP levels in E. faecalis cells at millimolar concentrations.

[0168] HNK-BAc induced a dose- and time-dependent increase in ATP in the E. faecalis system. In HNK-BAc treated E. faecalis cells, ATP levels were initially higher in the presence of HNK-SCFAs, likely due to decreased ATP utilization under suppressed proliferation. After a dose-dependent time lag, the proliferation rates of E. faecalis increased over time in the presence of HNK-SCFAs. Accordingly, ATP levels began to decrease due to utilization (FIG.8).

[0169] In the presence of SCFAs alone, ATP levels increased (FIG. 8, panel B). This increase is likely due to the utilization of SCFAs, such as acetate, in the E. faecalis system to generate more ATP. However, it is important to note that high millimolar concentrations of SCFAs were used in this experiment.

[0170] Antimicrobial effects of HNK-SCFAs Antimicrobial activity was assessed using a standard micro-dilution assay to determine minimal inhibitory concentration (MIC), the lowest concentration that completely inhibits bacterial growth during overnight incubation at 37°C. HNK-Ac had a MIC of 180 pM when tested against E. faecalis. None of the other HNK derivatives exhibited antimicrobial activity at the highest concentration tested (180 pM). This is consistent with results from proliferation assays indicating that these compounds inhibit bacterial growth for periods of up to 6 hours, after which proliferation recovers.

[0171] Cytotoxicity of HNK-SCFAs Cytotoxicity was monitored in real time by SYTOX Green staining. The SYTOX measurements showed that HNK-SCFAs were not cytotoxic at the concentration inhibiting >90% proliferation of E. faecalis. (FIG. 12) In contrast, the antibiotic ampicillin was cytotoxic at concentrations inhibiting E. faecalis proliferation.

[0172] Calculated values of the octanol / water partition coefficients. The hydrophobicity of HNK-SCFAs was assessed by calculating the log P partition coefficients. The calculated log P values for HNK, HNK-Ac, HNK-PAc, HNK-BAc, and HNK-HAc are 5.2, 5.1, 5.9, 6.3, and 7.2, respectively (Table 1). The log P values of HNK-ester analogs (mono and 6 / s-esters) were assessed using a QSAR (quantitative structure-activity relationship) analysis and rational drug design as a measure of molecular hydrophobicity (Table 1). This method also uses a consensus model built using the ChemAxon software (San Diego, CA).Attorney Docket: 650053.01246 Table 1. Calculated values of the octanol / water partition coefficientsAttorney Docket: 650053.01246Discussion

[0173] This study has significant scientific impact as it reveals a novel therapeutic potential to mitigate the gut metabolism of L-dopa to dopamine using nontoxic SCFA conjugates of HNK, a naturally occurring polyphenol. Both HNK and SCFAs have been shown to be nontoxic and neuroprotective. HNK is a commercially available nutritional supplement that is also widely used as a drug to induce sleep. SCFAs are key players in the interplay between diet, microbiota, and health. SCFAs, including acetate (two carbons), propionate (three carbons), and butyrate (four carbons), are produced through anaerobic fermentation of dietary’ fibers by the colonicAttorney Docket: 650053.01246microbiome. The relative amounts of SCFA released in the gut depend on the type and amount of ingested fiber.

[0174] Propionate, a major microbial fermentation-induced metabolite in the human gut, has been shown to be neuroprotective in PD. Additionally, butyrate-generating bacteria have been associated with neuroprotection in PD. Increasing gut butyrate through prebiotic butyrogenic fibers has been proposed as a potential therapy. The bioavailability of L-dopa in the brain decreases in patients with PD due to the increased metabolism of L-dopa to dopamine by gut bacteria, specifically E. faecalis. The abundance of E. faecalis in human gut microbiota samples strongly correlates with L-dopa metabolism, and patients with PD have varying levels of these bacteria. Thus, decreasing bacterial metabolism is a promising therapeutic approach to enhance the bioavailability of L-dopa in the brain. Previously, we showed that HNK, conjugated to a triphenylphosphonium moiety, mitigated the metabolism of L-dopa — alone or combined with carbidopa — to dopamine. Mito-ortho-HNK suppressed the growth of E. faecalis, decreased dopamine levels in the gut, and increased dopamine levels in the brain. Here, we show that mitigating the gut bacterial metabolism of L-dopa using a hybrid molecule consisting of a naturally occurring molecule and an endogenous gut metabolite could enhance the efficacy of L-dopa.

[0175] Results indicate that HNK-SCFAs enhanced the membrane potential of E. faecalis, resulting in hyperpolarization (Fig. 6A). Hyperpolarization results when the membrane potential becomes more negative, whereas depolarization occurs when the membrane potential becomes less negative or more positive. However, HNK and SCFAs alone did not affect the membrane potentials (Fig. 6B). The increase in membrane potential followed the order: HNK- BAc > HNK-PAc > HNK-HAc > HNK- Ac (Fig. 6A). This finding contradicts previous studies. Under physiological pH values, the membrane potential of anaerobic gut commensal bacteria remained unchanged but slightly decreased at lower pH levels. In a model of colonic SCFA absorption using basolateral membrane vesicles from rat distal colonic mucosa, butyrate uptake was significantly higher at acidic extravesicular pH=5.5 than at pH=7.5. Butyrate was reported to cause a reversible hyperpolarization in neurons due to increased intracellular calcium ions. Propionate induced hyperpolarization in gallbladder epithelial cells. Acetate- induced hyperpolarization was attributed differences in calcium permeability.

[0176] HNK-SCFAs, as a novel class of prodrugs, have the potential to release two neuroprotective molecules: HNK and SCFAs like butyrate. Both molecules have been shown to inhibit neuroinflammation and reverse neurodegeneration. HNK activates mitochondrialAttorney Docket: 650053.01246sirtuin-3 (Sirt-3), which induces antitumor and anti-inflammatory effects. Sirt-3 is implicated as a potential target for PD.

[0177] HNK promotes mitophagy and mitochondrial dynamics through a Sirt-3-dependent mechanism involving the AMPK-PGC- 1 alpha signaling pathway. Additionally, HNK activates the NAD+-consuming enzyme Sirt-3 to prevent neuron death and improve motor performance in a rat model of PD.

[0178] HNK also decreases alpha-synuclein mRNA levels, potentially decreasing alpha-synuclein aggregation and the onset of neurological disorders collectively known as “synucleinopathies” including PD. This study revealed a novel therapeutic target for modulating alpha-synuclein expression. Furthermore, propionate supplementation has been shown to reverse alpha-synuclein-induced neurodegeneration in Caenorhabditis elegans. Enhancing SCFAs, such as propionate, in the gut through pharmacotherapy was shown to be beneficial in protecting against alpha-synuclein-induced neurodegeneration. Therefore, HNK-SCFAs may provide a synergistic therapeutic effect in combating synucleinopathy. The therapeutic signaling of SCFA receptors has been proposed as a treatment for neuroinflammatory disorders.

[0179] Although butyrate has been used as a nutritional supplement, its pungent and unfavorable odor has limited its widespread applications (Fig. S7). To overcome this problem, modified forms of butyrate were synthesized as prodrugs that release butyric acid through enzymatic hydrolysis (structures shown in Fig. S7). Proper administration of these drugs could offer advantages over probiotics. One such drug is arginine butyrate, an ester formed by combining arginine and butyrate, which is hydrolyzed to release arginine and butyrate in vivo. Administering low- doses of arginine butyrate restored membrane integrity and improved neuromuscular abnormalities in dystrophic mouse models. These beneficial effects are attributed to HDAC inhibition by butyrate and the inhibitory effects of nitric oxide synthase / arginine on intracellular calcium activity. Another prodrug, tributyrin (propane-1, 2,3-triyl tributanoate), is a triglyceride derived from glycerol and three molecules of butyric acid. Present in butter and used in margarine, tributyrin serves as a postbiotic microbiome supplement. It is rapidly absorbed as a prodrug that is hydrolyzed by the lipase enzyme to butyric acid, inducing apoptosis and inhibiting prostate cancer cells, and modulating gene transcription through HDAC inhibition. Phenylalanine-butyramide, a novel butyrate derivative, has demonstrated protective effects against doxorubicin-induced cardiotoxicity. It is also considered as a postbiotic that improves gut health by releasing butyrate. However, the mechanism of butyrate formation remains unclear. Additionally, amino acid-conjugatedAttorney Docket: 650053.01246butyrate (e.g., serine-conjugated butyrate) has been used as a prodrug in autoimmune arthritis and neuroinflammation in preclinical mouse models. Butyryl-L-camitine, a butyrate ester of carnitine, is proposed as a prodrug for delivering carnitine and butyrate in the gut and preventing inflammation.

[0180] Research suggests that L-dopa responsiveness in patients with PD may be associated with the abundance of the tyrosine decarboxylase gene in the gut. Although this gene is found in bacteria like Lactobacillus brevis, in humans, this gene is primarily associated with E. faecalis. Individuals with PD reportedly have decreased levels of SCFAs in their gut microbiome. Additionally, bacteria that metabolize L-dopa in the small intestine have been detected in the feces of people with PD. The metabolism of L-dopa by gut microbes and amino acid carboxylases in peripheral tissues contributes to the reduced availability of L-dopa in the brain.

[0181] A combination of L-dopa and carbidopa is the preferred treatment for managing PD symptoms. While carbidopa does not prevent gut metabolism of L-dopa, it does inhibit peripheral metabolism of L-dopa by acting as a substrate inhibitor of peripheral amino carboxylases. Interestingly, HNK-SCFAs may enhance the efficacy of L-dopa / carbidopa therapy by directly inhibiting both gut bacteria metabolism and peripheral metabolism of L- dopa

[0060] , Thus, a potential clinical implication of this work is the development of adjunctive pharmacomicrobiome therapy targeting the gut-brain axis.

[0182] This study has some limitations. Notably, it did not demonstrate the enhanced therapeutic efficacy of L-dopa / carbidopa / HNK-SCFAs in a PD-relevant genetic model, such as the MitoPark mouse. Additionally, the study did not investigate the effects of HNK-SCFAs on L-dopa-induced dyskinesia in a mouse model.

[0183] 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. We propose that the benefits of treating human PD with L-dopa can be potentiated by the use of adjunctive treatments utilizing HNK-SCFAs that inhibit the breakdown of L-dopa to dopamine in the gastrointestinal tract, thereby enhancing L-dopa conversion to dopamine in the brain. The MitoPark transgenic mouse model is particularly relevant, as it replicates key PD features including gastrointestinal dysfunction associated with PD. In addition, it will be important to determine if the beneficial effects of HNK-SCFAs observed in the current study are maintained with repeated treatments over an extended period of time. Collaboratively, we have previously published the neuroprotective effects of mitochondria-targeted drugs in theAttorney Docket: 650053.01246MitoPark mouse. Studies using MitoPark mice are not feasible at this time; however, future collaborative research will investigate these aspects.

[0184] Methods

[0185] Syntheses of honokiol acetic acid (HNK-Ac), honokiol bis acetic acid (HNK-Bis-Ac). honokiol propionic acid (HNK-PAc), honokiol bis propionic acid (HNK-5 / .s-PAc), honokiol butyric acid (HNK-BAc), honokiol bis butyric acid (HNK-Bis-BAc), and honokiol hexanoic acid (HNK-HAc) are presented in the subsequent sections. Chemical structures for butyrate derivatives are shown in FIG. 9.HNK HNK-AcScheme 1. Synthesis of HNK-Ac. Reagents and conditions: i, acetyl chloride, TEA, CH2CI2, 25%.

[0186] Bacterial strain and culture conditions

[0187] E. faecalis (Cat# OG1RF) was obtained from the American Type Culture Collection (ATCC). E. faecalis was cultured and grown overnight in TSB (tryptic soy broth), diluted 1:200 into fresh TSB and then 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.

[0188] Synthesis and purification of NK-SCFA conjugates

[0189] All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin layer chromatography using silica gel Merck 60F254. Crude materials were purified by flash chromatography on Merck Silica gel 60 (0.040-0.063 mm). 1HNMR spectra were recorded at 400.13 MHz respectively using a Bruker DPX AVANCE 400 spectrometer equipped with a quattro nucleus probe.1H NMR and31P were taken in deuterated chloroform (CDCl3) using tetramethyl silane as internal reference respectively. Chemical shifts (5) are reported in ppm and J values in Hertz.

[0190] Measurement of bacterial cell proliferationAttorney Docket: 650053.01246

[0191] For all proliferation assays, cells were diluted to the final OD600 of 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 even’ 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.

[0192] Measurement of bacterial membrane potential

[0193] Membrane potential was measured using the fluorescence dye tetramethylrhodamine methyl ester (TMRM). Briefly, bacteria in the exponential growth phase (OD600 of 0.4) were treated with test compounds as previously 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., Cary, NC). Data were collected as the mean fluorescent intensity and were normalized to the total OD600 as the total bacteria number. The effects on membrane potential were compared for potential correlation with the MIC and minimum bactericidal concentration values.

[0194] Uptake and intracellular hydrolysis ofHNK-SCFAs

[0195] E. faecalis cells in the exponential growth phase (OD600=0.3-0.5) were diluted to the final OD600 of 0.1 at 20 mL volume, then treated with HNK or HNK-SCFAs as indicated for 1 h. Cell pellets were collected by centrifugation at 2,500 g × 5 min at 4°C and stored at -80°C before extraction was performed. The cell pellet was dissolved in dimethylsulfoxide (100 pL) and taken for high-performance liquid chromatography (HPLC) analysis.

[0196] HNK-SCFAs and HNK formed from hydrolysis were separated and monitored by HPLC using an Agilent 1200 apparatus equipped with ultraviolet-visible absorption. Typically, 4 pL of a sample was injected on a Phenomenex reverse phase column (Kinetex 2.6 p, 100 mm*4.6 mm). The absorption traces were collected at 254 nm. The compounds were separated by a linear increase in acetonitrile phase concentration from 10% to 100% over 14 min and until 17 min at 100% acetonitrile containing 0.1% (v / v) trifluoroacetic acid. The flow rate used was 1.3 mL / min.

[0197] Measurement of intracellular ATP

[0198] Intracellular ATP was quantified using a luciferase-based ATP determination kit, according to the manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, Cat# FLLAA). Following cell lysis, a luciferase / luciferin agent (Cat# FLAAM) was added to the cell lysates.Attorney Docket: 650053.01246After swirling, luminescence was measured using a luminometer, and results were normalized to total cell numbers, which was represented as absorbance at 600 nm (OD600nm).

[0199] Cytotoxicity measurements

[0200] Cytotoxicity was evaluated using the SYTOX Green (Invitrogen, Cat# S7020)

[0020] , The SYTOX method labels the nuclei of dead cells, yielding green fluorescence. Fluorescence (Ex: 485 nm. Em: 535 nm) from the dead cells in the 96-well plate were recorded every 5 min for 3 h using a plate reader (BMG Labtech, Inc.) equipped with an atmosphere controller set at 37°C.

[0201] E. faecalis cells in the exponential growth phase (OD600=0.4) were treated with HNK-SCFAs in a black, clear-bottom 96-well plate for 3 h, and dead cells were monitored in the presence of 200 nM SYTOX Green. Cell lysis reagent (B-PER complete bacterial protein extraction reagent, Thermo Scientific, Cat# 89821) was used as a positive control.

[0202] L-dopa metabolism and LC-MS analysis

[0203] E. faecalis cells in the exponential growth phase (OD600 of ~0.4) were diluted to a final OD600 of 0.1 and treated with L-dopa (1 mM) alone or in combination with carbidopa (0.22 mM) as previously described

[0020] . At the indicated time points ( 1-6 h), samples (1 ml of media) were collected by centrifugation at 2,500 g x 5 min at 4°C, and the supernatant was lyophilized. The dry residue consisting of L-dopa and metabolites was resuspended in ice-cold methanol (100 pL) and analyzed by LC-MS using an Agilent 1200 apparatus equipped with ultraviolet-visible absorption and a mass spectrometry detector (single quadrupole). Typically, 2 pL of a sample was injected on an Agilent Poroshell column (120 HILIC-Z, PEEK, 100 mm x 2.1 mm, 2.7 pm, 25°C), with absorbance monitored at 280 nm.

[0204] Esterase-mediated hydrolysis ofHNK-SCFAs

[0205] HNK-SCFA-esters (1 mM) solutions were prepared in 100 mM phosphate buffer (pH=7.4). Then, HNK-SCFA-esters (100 pM) were incubated with esterase (30 U / mL) (Sigma Aldrich, St. Louis, MO). A 4 pL mixture was injected into HPLC to monitor the cleavage of the esters with the release of HNK and the corresponding SCFAs.

[0206] HPLC analyses were performed using an Agilent 1200 system equipped with absorption detectors. The samples (5 pL) were injected into a reverse phase column (Phenomenex, Kinetex C18, 100 mm x 4.6 mm, 2.6 pm) equilibrated with 10% (v / v) MeCN, 90% (v / v) water containing 0.1% (v / v) trifluoroacetic acid.

[0207] HNK esters derivatives were eluted by increasing the content of MeCN (v / v) from 20% to 100% over 14 min and until 17 min at 100% acetonitrile at a flow rate of 1.3 mL / min. The absorption used to monitor the ester cleavages was 254 nm.Attorney Docket: 650053.01246

[0208] Statistical analysis

[0209] All data were expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM), as indicated. Comparisons between treatment and control groups were performed using an unpaired Student’s t-test analysis. A p-value of less than 0.05 was considered statistically significant. Sample sizes (n) are indicated in figure legends.

[0210] Synthesis and characterization of compounds

[0211] HNK-Ac (3',5-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl acetate) HNK-Ac was prepared by reacting HNK (0.4 g, 1.5 mmol) in dichloromethane (CH2CI2) (10 mL) with acetyl chloride (107 pL, 1.5 mmol) in the presence of triethylamine (217 pL, 1.5 mmol). (Scheme 1) The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by diethyl ether (Et20). The organic layer was dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure. Purification by flash chromatography (AcOEt / pentane, 85 / 15) delivered the corresponding HNK-Ac (115 mg, 25% yield) as a mixture of both isomers. HRMS calculated for HNK-Ac C20H20O3 [MNa]+331.1305, found. 331.1306. 'H NMR (400.13 MHz, CDCl3) δ, 7.38-7.33 (2H, m), 7.18-7.14 (1H, m), 7.11-7.07 (1H, m), 7.06-7.04 (1H, m), 6.92 (1H, d, J= 8.3), 6.06-5.84 (2H, m), 5.16-5.02 (5H, m), 3.40-3.33 (4H, m), 2.35 (3H, s).13C NMR (75 MHz, CDCl3) δ, 169.4, 150.8, 148.6, 137.6, 135.5, 135.2, 132.9, 132.3, 131.1, 130.3, 129.3, 128.2, 127.2, 123.2, 116.6, 115.9, 115.6, 39.3, 34.7, 20.9.

[0212] HNK-Bis-Ac (3',5-diallyl-[l,l'-biphenyl]-2,4'-diyl diacetate) HNK-Bis-Ac was prepared by reacting HNK (0.15 g, 0.56 mmol) in dichloromethane (CH2CI2) (6 mL) with acetyl chloride (107 pL, 1.5 mmol) in the presence of tri ethylamine (217 pL, 1.5 mmol). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by diethyl ether (Et20). The organic layer was dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure. Purification by flash chromatography (AcOEt / pentane, 85 / 15) delivered the corresponding HNK-Bis-Ac (160 mg, 81 % yield). HRMS calculated for HNK-Bis-Ac C22H22O4 [MNa]+373.1410, found, 373.1405.1H NMR (400.13 MHz, CDCl3), δ 7.27-7.22 (2H, m), 7.20-7.14 (2H, m), 7.08-7.02 (2H, m), 6.01-5.82 (2H, m), 5.13-5.03 (4H, m), 3.40 (2H, d, J= 6.9), 3.30 (2H, d, J= 6.6), 2.30 (3H, s), 2.06 (3H, s).13C NMR (75 MHz, CDCl3) δ 169.5. 169.3, 148.3, 145.9, 138.2, 136.9, 135.7, 135.5, 133.7. 131.6, 130.9, 130.8. 128.6, 127.9, 122.7. 122.3, 116.4, 116.3. 39.6. 34.7. 20.9.20.8.Attorney Docket: 650053.01246Scheme 2. Synthesis of HNK-PAc. Reagents and conditions: i, Propionyl chloride, TEA, CH2CI2, 55%.

[0213] HNK-PAc (3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl propionate) and HNK-Bis-PAc (3',5-diallyl-[l,l'-biphenyl]-2,4'-diyl dipropionate) To a mixture of HNK (0.25 g, 0.93 mmol) in CH2CI2 (4 mL) was added propionyl chloride (82 pL, 0.93 mmol) in the presence of tri ethylamine (200 pL, 0.97 mmol) (Scheme 2). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4. and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding HNK-PAc (136 mg, 45% yield) and honokiol para propionic acid (HNK-para-PAc) (30 mg, 10%). HRMS calculated for HNK-PAc C21H22O3 [MH]+340.1907, found, 340.1909.1H NMR (400.13 MHz, CDCl3) δ. 7.37-7.33 (2H, m), 7.18-7.13 (1H, m), 7.11-7.03 (2H, m), 6.91 (1H, d, J= 8.3), 6.07-5.84 (2H, m), 5.02-5.04 (5H, m), 3.40-3.33 (4H, m), 2.65 (2H, q, J= 7.6), 1.31 (3H, t, J=7.6).13C NMR (75 MHz, CDCl3) δ 172.9, 150.8, 148.6, 137.7, 135.6, 135.0, 132.9, 132.3, 131.1, 130.2, 129.2. 128.1, 127.3. 123.1, 116.6. 115.9, 115.6. 39.4, 35.1, 34.7, 27.7, 9.4.

[0214] HNK-Bis-PAc (3',5-diallyl-[1,1'-biphenyl]-2,4'-diyl dipropionate) To a mixture of HNK (0.15 g, 0.56 mmol) in CH2CI2 (5 mL) was added propionyl chloride (82 pL, 0.93 mmol) in the presence of tri ethylamine (300 pL, 1.45 mmol). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding HNK- / izs-PAc (181 mg, 85% yield). HRMS calculated for HNK-bis-PAc C24H26O4 [MH]+396.2169, found, 396.2171. 'H NMR (400.13 MHz, CDCl3), δ 7.27-7.24 (2H, m), 7.21-7.16 (2H. m). 7.08-7.02 (2H. m), 6.04-5.84 (2H, m), 5.16-5.02 (4H, m), 3.43-3.28 (4H, m), 2.61 (2H, q, J= 7.6), 2.36 (2H, q,,7= 7.6), 1.29 (3H, t, (6H, m, J= 7.6), 1.07 (3H, t, (6H, m, J= 7.6).13C NMR (75 MHz, CDCl3) δ 172.9, 172.7, 148.4, 146.1, 138.0, 136.9,Attorney Docket: 650053.01246135.8, 135.5, 133.9, 131.6, 130.9, 130.8, 128.6, 127.9, 122.7, 122.2, 116.3, 116.2, 39.6, 34.6, 27.7, 27.6, 9.1. 8.9.HNK HNK-BAcScheme 3. Synthesis of HNK-BAc. Reagents and conditions: i, butyryl chloride, TEA, CH2CI2.58%.

[0215] HNK-BAc (3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl butyrate [HNK-orf / zo-BAc]) To a mixture of HNK (0.25 g, 0.33 mmol) in CH2CI2 (4 mL) was added butyryl chloride (137 pL. 1 mmol) in the presence of triethylamine (200 μL, 0.97 mmol) (Scheme 3). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding HNK-BAc (101 mg, 45% yield) honokiol para butyric acid (HNK-para-BAc) (30 mg, 13%) HRMS calculated for HNK-BAc C22H24O3 [M+NH4]+, 354.2064. found.354.2062. 'H NMR (400.13 MHz, CDCl3), δ 7.36-7.34 (2H, m), 7.15 (1H, d, J = 8.1), 7.01-7.04 (2H, m), 6.91 (1H, d, J= 8.1), 6.01-5.86 (2H, m), 5.22-5.02 (5H, m), 3.37 (4H, dd, J= 1, 6.6), 2.60 (2H, t, J= 7.3), 1.83 (2H. sext., J= 7.5, 14.8), 1.09 (3H, t, J= 7.3).13C NMR (75 MHz, CDCl3) δ 172.0, 172.7, 150.8, 148.6, 137.7, 135.5, 135.1, 132.9, 132.3, 131.1, 130.2, 129.2, 128.1, 127.3, 123.1, 116.6, 115.9, 115.6, 39.3, 36.2, 34.6, 18.2, 13.7.

[0216] HNK-Bis-BAc (3',5-diallyl-[1,1'-biphenyl]-2,4'-diyl dibutyrate) To a mixture of HNK (0.15 g, 0.56 mmol) in CH2CI2 (4 mL) was added butyryl chloride (200 pL, 1.5 mmol) in the presence of triethylamine (153 pL, 1.15 mmol). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding HNK-Bis-BAc (201 mg, 87 % yield), ESI-MS (electrospray ionization mass spectrometry) for HNK-Bis-BAc C26H30O4 [MH]+407.8. 'H NMR (400.13 MHz, CDCl3), δ 7.28-7.25 (2H, m), 7.21-7.15 (2H, m), 7.08-7.02 (2H, m), 6.04-5.84 (2H, m). 5.16-5.02 (4H, m), 3.43-3.28 (4H, m),Attorney Docket: 650053.012462.57 (2H, t, J= 7.3), 2.33 (2H, t, J= 7.3), 1.81 (2H, sext, J = 7.5, 14.9), 1.60 (2H, sext., J= 7.5, 14.9), 1.08 (3H. t, J = 7.3). 0.87 (3H, t, J = 7.3).13C NMR (75 MHz, CDCl3) δ 172.1, 171.9, 148.4, 146.1, 138.0, 136.9, 135.7, 135.5, 134.0, 131.6, 130.9, 128.6, 128.0, 122.8, 122.2, 116.3, 116.2, 39.6, 36.2, 36.0, 34.6, 18.5, 18.2, 13.7, 13.5.HNK HNK-HAcScheme 4. Synthesis of HNK-HAc. Reagents and conditions: i, hexanoyl chloride, TEA, CH2CI2, 24%.

[0217] HNK-HAc (3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl hexanoate [HNK-ortrio-HAc] and 3',5-diallyl-4'-hydroxy-[l,r-biphenyl]-2-yl hexanoate [HNK-pura-HAc]) HNK-HAc was prepared by reacting HNK (0.35 g, 1.3 mmol) in CH2CI2 (4 mL) with hexanoyl chloride (186 pL, 1.3 mmol) in the presence of triethylamine (200 pL, 0.97 mmol) (Scheme 4). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et2O. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding HNK-HAc (115 mg, 24% yield) as a mixture of both isomers. HRMS calculated for HNK-HAc C24H28O3 [MNa]+387.1931, found, 387.1931. 'H NMR (400.13 MHz, CDCl3), δ 7.37-7.33 (1H, m), 7.23-6.77 (5H, m), 6.10-5.86 (2H, m), 5.22-4.99 (5H, m), 3.47-3.31 (4H. m). 2.61&2.35 (2H. 2t, J = 7.6. 7.3), 1.80 & 1.56 (2H, 2sext. J= 7.6, 14.9).1.46-1.38 (2H, m), 1.32-1.18 (2H, m), 0.96& 0.88 (3H, 2t, J= 7.1, 6.9).13C NMR (75 MHz, CDCl3) δ 172.3, 172.2, 153.6, 150.7, 148.6, 146.1, 137.9, 137.6, 137.1, 136.2, 135.4, 134.9, 134.4, 132.9. 132.3, 131.1. 131.0, 130.8, 130.2, 129.2, 128.1, 127.3, 123.2, 122.7, 116.7, 116.6, 116.1, 115.9, 115.6, 39.7, 39.4, 35.1, 34.6, 34.3, 34.2, 31.3, 31.2, 24.6, 24.4, 22.3, 22.2, 13.9, 13.8.Attorney Docket: 650053.01246HNK HNK-PBAcScheme 5. Synthesis of HNK-PBAc. Reagents and conditions: i, 4-Phenylbutanoylchloride, TEA. CH2CI2, 24%.

[0218] HNK-PBAc (3,5'-diallyl-2'-hydroxy-[l.r-biphenyl]-4-yl 4-phenylbutanoate and 3',5-diallyl-4'-hydroxy-[l, T-biphenyl]-2-yl 4-phenylbutanoate) HNK-PBAc was prepared by reacting HNK (0.25 g, 0.94 mmol) in CH2CI2 (4 mL) with 4-phenylbutanoylchloride (154 pL, 0.94 mmol) in the presence of triethylamine (128 pL, 0.94 mmol) (Scheme 5). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SOr, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 90 / 10) delivered the corresponding HNK-PBAc (102 mg, 26% yield) as a mixture of both isomers. HRMS calculated for HNK-PBAc C28H28O3 [MNa]+435.1931, found, 435.1932. 'H NMR (400.13 MHz, CDCk), 5 7.37-7.30 (4H, m), 7.26-7.21 (3H, m), 7.17-7.03 (3H, m), 6.93-6.91 (1H, 2s), 6.03-5.86 (2H, m), 5.13-5.04 (5H, m), 3.36 (4H, t, J= 5.6), 2.78 (2H, t, J= 7.3), 2.63 (2H, t, J = 7.3), 2.13 (2H, quint., J = 15.1, 7.8).3C NMR (75 MHz, CDCh) 8 171.9, 150.8, 148.6, 141.1, 137.7. 135.4, 135.1. 132.9, 132.4. 131.1, 130.2. 129.3, 128.5, 128.1, 127.3, 126.2, 123.2, 116.7, 115.9, 39.4, 35.1, 44.6, 33.5, 26.4.HNK HNK APAeAttorney Docket: 650053.01246Scheme 6. Synthesis of HNK-VAPAc. Reagents and conditions: i, Valproylchloride, TEA, CH2CI2.

[0219] A valproyl derivative, HNK-VAPAc, can be synthesized as shown in Scheme 6, using an approach similar to the sy nthesis of HNK-PBAc.

[0220] Example 2

[0221] In this study, we investigated the antimicrobial potentials of mitochondria-targeted honokiol (Mito-HNK), conjugated to esterase-cleavable short-chain fatty acids (SCFAs) such as buty rate, propionate, or acetate. Specifically, the goal was to understand the effect of these compounds (e.g.. Mito-HNK-butyrate. on the growth of Enterococcus faecalis (E. faecalis). E. faecalis is known to metabolize levodopa, a drug frequently used to manage symptoms of Parkinson’s disease (PD). Our findings revealed that Mito-HNK-SCFAs inhibit the growth of E. faecalis in a dose-dependent manner. Interestingly, after an initial time lag, E. faecalis resumed its normal grow th pattern. However, Mito-HNK-SCFAs more potently delayed the growth of E. faecalis. Notably, levodopa was not metabolized to dopamine during the time lag. Additionally, Mito-HNK-SCFAs were hydrolyzed within bacterial cells to yield Mito-HNK and the corresponding SCFA. Mito-HNK-SCFAs are nearly 10-times more potent than the corresponding HNK-SCFAs with respect to inhibition of E. faecalis proliferation, L-dopa degradation, and dopamine formation. Based on these results, we propose that Mito-HNK- SCFAs could be utilized to mitigate gut metabolism of levodopa to dopamine, enhancing the therapeutic efficacy of levodopa in PD patients.

[0222] Therapeutic targeting of the gut microbiome is emerging as a new way to treat Parkinson’s disease (PD) symptoms. Reports indicate that the gut microbiome and its metabolites including SCFAs are diminished in patients with PD. Serum short-chain fatty acids (SCFAs) alter motor and nonmotor symptoms in patients with PD. Reports suggest that patients with PD have decreased fecal levels of butyrate and butyrate-generating bacteria. Deceased buty rate was associated with increased depression in patients with PD. Buty rate levels are also affected by alpha-sy nuclein aggregation. Sodium butyrate / butyric acid administration was shown to be effective in a rodent model of PD. Pharmacologically enhancing buty rate levels in the gut microbiome should therapeutically counter the butyrate-producing bacteria deficiency in PD subjects. In a rodent model of PD, buty rate administration decreased motor deficits, elevated dopamine levels, and increased histone acetylation. A recent report suggests that supplementation with propionate reversed alpha-synuclein-induced neurodegeneration in C. elegans.Attorney Docket: 650053.01246

[0223] Development of new pharmacotherapies to treat PD first requires syntheses and development of new molecules. Thus, a molecule that combines mitochondria-targeted honokiol (Mito-HNK) and an SCFA ester e.g., Mito-HNK-SCFA-esters) is of interest, as this molecule could generate Mito-HNK and SCFAs in the gut in the presence of the esterase enzymes. For example, mitochondria-targeted honokiol butyric acid (Mito-HNK-BAc) will release the parent drug. Mito-HNK, and butyric acid or butyrate (FIG. 13). Polyphenols conjugated to SCFAs have previously been developed as a prodrug showing antibacterial effects. Here, we present the first report of mitochondria-targeted drug (MTD)-containing esterase-cleavable SCFAs with antimicrobial potential. In this study, we show that several naturally occurring, triphenylphosphonium anion (TPP+)-modified molecules, FDA-approved drugs, and esterase-cleavable SCFA esters (FIG. 13) potently induce a dose-dependent delay in the proliferation and microbial degradation of levodopa (L-dopa) and metabolism to dopamine.

[0224] Materials and Methods 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).

[0225] The effects of MTDs on bacterial respiration were measured using a Seahorse XF96 extracellular flux analyzer as previously described. Oxygen consumption rates and extracellular acidification rates were measured for ~5-7 min (to establish baseline) prior to injection of the MTD or antibiotic and then followed for 4-8 h. Oxygen consumption rates were normalized to the number of viable cells, which were quantitated from a parallel plate using the LIVE / DEAD bacterial viability' assay. Results with MTDs were compared with those obtained with classical antibiotics.

[0226] Measurement of bacterial membrane potential The effects of MTDs on bacterial membrane potential using the fluorescent dye tetramethylrhodamine (TMRM) were determined. Briefly, bacteria in the exponential growth phase were washed and resuspended in HEPES buffer containing 100 rnM potassium chloride and 20 mM glucose; then, they were incubated yvith TMRM until a stable reduction in fluorescence yvas observed due to membrane uptake and quenching of dye fluorescence in response to an intact membrane potential. An aliquot of MTD at the desired concentration yvas added, and fluorescence yvas monitored at an excitation of 548 nm and emission of 574 nm using a QM-4 spectrofluorometer (Photon Technology International Inc.. Birmingham. NJ). To ensure that the observed effects ere not due to general membrane disruption, we measured membrane permeability using SYTOXAttorney Docket: 650053.01246Green. Membrane permeabilization with 0.1% Triton X-100 was used as a positive control. The effects on membrane potential were compared for potential correlation with the minimum inhibitory concentrations (MIC) and minimum bactericidal concentration values.

[0227] Intracellular ATP levels 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 the total protein level in each well.

[0228] LC-MS measurements L-Dopa and its metabolites were separated and monitored by LC-MS using an Agilent 1200 apparatus equipped with an ultraviolet-visible (UV-Vis) absorption and mass spectrometry (MS) detector (single quadrupole). Typically. 2 pL of a sample was injected on an Agilent Poroshell column (120 HILIC-Z, PEEK, 100 mm x 2.1 mm, 2.7 pm, 25 °C). The absorption traces were collected at 280 nm.

[0229] L-Dopa-d3 and dopamine-d3 were separated and monitored by liquid chromatographymass spectrometry-single ion monitoring LC-MS-SIM using an Agilent 1200 apparatus equipped with a UV-Vis absorption and MS detector (single quadrupole). Typically, 2 pL of a sample was injected on an Agilent Poroshell column (120 HILIC-Z, PEEK, 100 mm x 2.1 mm, 2.7 pm, 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.

[0230] In addition, mass spectrometry-single ion monitoring (MS-SIM) detection parameters were set up using electrospray ionization (ESI). SIM was defined as follows: L-Dopa-d3 [m / z = 201 (+)] and dopamine-d3 [m / z = 157 (+)].

[0231] Hydrolysis of MTD compounds using esterase. We evidenced that Mito-HNK-SCFA-esters can be hydrolyzed by esterases allowing the formation of Mito-HNK with the corresponding SCFA (FIG. 14). Mito-HNK-SCFA-esters (1 mM) mother solutions were prepared in 100 mM phosphate buffer (pH= 7.4). Then, Mito-HNK-SCFA-esters (100 mM) were incubated in the presence of esterase (30 U / mL). 5 mL was injected into HPLC to monitor the cleavage of the esters with the release of Mito-HNK and the corresponding small chain fatty acids. HPLC analyses were performed using an Agilent 1100 system equipped with absorption detectors. The samples (5 pl) were injected into a reverse phase column (Phenomenex, Kinetex C18, 100 mm x 4.6 mm, 2.6 pm) equilibrated with 10% (v / v) MeCN,Attorney Docket: 650053.0124690% (v / v) water containing 0.1% (v / v) trifluoroacetic acid. Mito-HNK esters derivatives were eluted by increasing the content of MeCN (v / v) from 20% to 100% over 14 min at a flow rate of 1.3 mL / min. The absorption used to monitor the ester cleavages was 240 nm.

[0232] Statistical analysis Comparisons between the control and treatment groups were made using an unpaired Student’s t-test analysis. 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.

[0233] Results

[0234] Mito-HNK-SCFA-esters inhibit E. faecalis proliferation Mito-HNK, Mito-HNK-BAc, and Mito-HNK-PAc are nearly 20 times more effective than unmodified HNK in inhibiting E. faecalis proliferation (FIG. 15, panels B-E). At low concentrations, Mito-HNK analogs delayed the proliferation of E. faecalis. At lower doses of Mito-HNK and analogs, after a 2-3 h time lag, E. faecalis proliferation continued at nearly the same rate as the control. FIG.16 shows the uptake and hydroly sis of Mito-HNK-B Ac by E. faecalis. As shown, the levels of Mito-HNK taken up by E. faecalis were significantly higher than in E. faecalis treated with Mito-HNK.

[0235] Mito-HNK-SCFA-esters inhibit E. faecalis-dependent L-dopa degradation and dopamine formation. FIG. 17 shows the dose-dependent decrease in L-dopa degradation by E. faecalis in the presence of Mito-HNK-B Ac. Mito-HNK-BAc was slightly more effective than Mito-HNK in inhibiting L-dopa degradation and dopamine formation (FIG. 17).

[0236] Effects of Mito-HNK-SCFA-esters on E. faecalis membrane potential FIG. 18 show that both HNK-BAc and HNK-PAc dose-dependently enhanced the E. faecalis membrane potential, with HNK-BAc being more potent than HNK-PAc. In contrast, neither butyrate nor propionate had an effect over a range of concentrations. This is consistent with the results showing enhanced uptake and hydrolysis of HNK-SCFA-esters (FIG. 17).

[0237] Discussion

[0238] SCFAs, i.e., acetate with tw o carbons; propionate with three carbons; butyrate with four carbons; valerate with five carbons; and caproate with six carbons are released in the colonic microbiota from anaerobic fermentation of dietary fibers. The relative amounts of SCFA released in the gut are dependent on the type and amount of ingested fiber. Propionate is a major microbial fermentation-induced metabolite in the human gut that mediates neuroprotection in PD. Butyrate-generating bacteria have also been linked to neuroprotection in PD. The increase of gut butyrate formed from prebiotic butyrogenic fibers was viewed as a potential therapeutic approach.Attorney Docket: 650053.01246

[0239] The extracellular pH in the gut is very' acidic, ranging between 1 and 4. In vitro experiments suggest that even with prolonged incubation of Mito-HNK-SCFA in formate buffer (pH 3) at 37 °C there was no hydrolysis (not shown). The influence of chain length on the esterase-mediated hydrolysis of esters and their structure-function relationship are well established. Enhancing SCFAs such as propionate in the gut through pharmacotherapy was shown to be beneficial in protecting against alpha-synuclein-induced neurodegeneration.

[0240] MTD-SCFA ester / L-dopa / carbidopa therapy may enhance MTD and SCFA levels in the gut which may augment neuroprotection.

[0241] Synthesis Synthesis of mitochondria-targeted honokiol propionic acid (Mito-HNK-PAc), mitochondria-targeted honokiol butyric acid (Mito-HNK-BAc), mitochondria-targeted valeric acid (Mito-HNK-VAc), mitochondria-targeted honokiol hexanoic acid (Mito-HNK-HAc), and the PEGylated mitochondria-targeted analog of honokiol propionic acid (Mito-PEGs-HNK-PAc) are presented in the following sections. All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin layer chromatography (TLC) using silica gel Merck60F254. Crude materials were purified by flash chromatography on Merck Silica gel 60 (0.040-0.063 mm). 'H NMR spectra were recorded at 400.13 MHz respectively using a Bruker DPX AVANCE 400 spectrometer equipped with a quattro nucleus probe. ’H NMR and31P were taken in deuterated chloroform (CDCh) using CDCh and tetramethyl silane (TMS) as internal reference respectively. Chemical shifts (d) are reported in ppm and J values in Hertz.Mito-HNK-BAc (n= 2)Scheme 7. Synthesis of Mito-HNK-PAc and Mito-HNK-BAc. Reagents and conditions: i, propionyl chloride (n=l) or butyryl chloride (n=2), TEA, CH2CI2, 55-64%.

[0242] Mito-HNK-PAc ([10-[3,5’-diallyl-2’-(propionyloxy)-(l,l ’-biphenyl)-4-yl]-oxy]-decyltriphenylphosphonium bromide) Mito-HNK-PAc was prepared by reacting mitochondria-targeted honokiol (Mito-HNK) with propionyl chloride in the presence ofAttorney Docket: 650053.01246triethylamine in CH2CI2. (Scheme 7). To a mixture of Mito-HNK (0.25 g, 0.33 mmol) in CH2CI2 (4 mL) was added propionyl chloride (58 pL, 0.67 mmol) in the presence of triethylamine (200 pl, 0.97 mmol). The mixture was stirred at room temperature for 2 h. The product was precipitated into Et20. Purification by flash chromatography (CHzCh / EtOH, from 100% to 85 / 15) delivered the corresponding Mito-HNK-PAc (171 mg, 64% yield). HRMS calculated for Mito-HNK-PAc C49H56O3P+[M]+723.3962, found, 723.3962.31P NMR (400.13 MHz, CDCh) 824.23. 'H NMR (400.13 MHz, CDCh), 8 7.84-7.63 (15H, m), 7.19-7.07 (4H, m), 6.98 (1H, d, J= 8.3), 6.82 (1H, d, J= 8.1), 6.01-5.87 (2H, m), 5.11-5.05 (4H, m), 3.93 (2H, t, J= 6.4), 3.73-3.61 (2H, m), 3.36 (4H, t, J= 7.3), 2.34 (2H, q, J= 7.6, 15.1), 1.75 (2H, sext., J = 6.6, 13.5), 1.60-1.56, 3H, m), 1.46-1.35 (2H, m), 1.31-1.17 (9H, m), 1.04 (3H. t. J= 7.6).13C NMR (75 MHz, CDCh) 8 172.8,156.1, 146.0, 137.7, 137.0, 136.8, 134.9, 134.8, 134.3, 133.5, 133.4, 130.7, 130.4, 130.3, 130.1, 129.4, 128.2, 127.8, 127.6, 122.5, 118.7, 117.8, 115.9, 115.2, 110.9, 67.9, 39.5, 34.4, 30.4, 30.2, 29.3, 29.2, 29.1, 29.0, 27.5, 25.6, 22.5 (d, J= 49.9), 22.6 (d, J= 5.1), 8.8.

[0243] Mito-HNK-BAc ([10-[3,5'-diallyl-2'-(butyryloxy)-(l, T-biphenyl)-4-yl]-oxy]-decyltriphenylphosphonium bromide) Mito-EINK-BAc was prepared by reacting Mito-HNK with butyryl chloride in the presence of triethylamine in CH2CI2. (Scheme 7). To a mixture of Mito-HNK (0.25 g. 0.33 mmol) in CH2CI2 (4 mL) was added butyryl chloride (137 pL, 1 mmol) in the presence of triethylamine (200 pl, 0.97 mmol). The mixture was stirred at room temperature for 2 h. The product was precipitated into Et20. Purification by flash chromatography (CH2Ch / EtOH, from 100% to 85 / 15) delivered the corresponding Mito-HNK-BAc (150 mg, 55% yield). HRMS calculated for Mito-HNK-BAc C50H58O3P+[M]+737.4118, found, 737.4122.31P NMR (400.13 MHz, CDCh) 8 24.35. 'H NMR (400.13 MHz, CDCh), 87.95-7.77 (15H, m), 7.37-7.29 (2H, m), 7.26-7.20 (2H, m), 7.09 (1H, d, J = 8.1), 6.93 (1H, d, J = 8.1), 6.13-5.98 (2H, m), 5.22-5.06 (4H, m), 4.05 (2H, t, J= 6.4), 3.88-3.78 (2H, m), 3.48 (4H, t, J = 1.6), 2.41 (2H, t, J = 1.6). 1.90-1.82 (2H, m). 1.77-1.61 (6H, m), 1.56-1.49 (2H, m), 1.43-1.26 (8H, m), 0.94 (3H, t, J= 1.6).13C NMR (75 MHz, CDCh) 8 172.1,156.2, 146.0, 137.8, 137.1, 136.9, 134.9, 134.8, 134.5, 133.7, 133.5, 130.1, 130.5, 130.3, 130.2, 129.5, 128.3, 127.9, 127.7, 122.6, 118.8, 117.9, 116.0, 115.3, 110.9, 68.0, 39.6, 36.0. 34.5. 30.4, 30.3, 29.6, 29.4. 29.23, 29.21. 29.15, 29.10. 26.0. 22.9. 22.7 (d, J= 49.9), 22.6 (d, J= 4.4), 18.1, 13.4.Attorney Docket: 650053.01246Mito-HNK-VAcScheme 8. Synthesis of Mito-HNK-VAc. Reagents and conditions: i, valeric chloride, TEA, CH2CI2, 49%.

[0244] Mito-HNK-VAc ([10-[3,5'-diallyl-2'-( pentanoyloxy)-(l, T-biphenyl)-4-yl]-oxy]-decyltriphenylphosphonium bromide) Mitochondria-targeted honokiol valeric acid (Mito-HNK-VAc) was prepared by reacting Mito-HNK (0.2 g, 0.26 mmol) in CH2CI2 (4 mL) with pentanoyl chloride (80 pL, 0.66 mmol) in the presence of tri ethylamine (72 pl, 0.56 mmol). The mixture was stirred at room temperature for 2 h. (Scheme 8) The product was precipitated into Et20. Purification by flash chromatography (CH2Ch / EtOH, from 100% to 85 / 15) delivered the corresponding Mito-HNK-VAc (107 mg, 49% yield). The synthesis of Mito-HNK-VAc is presented in Scheme 8. HRMS calculated for Mito-HNK-VAc C51H60O3P+[M]+751.4275, found, 751.4274.31P NMR (400.13 MHz, CDCh) 524.55.1HNMR(400.13 MHz, CDCI3), 5 7.90-7.66 (15H, m), 7.44-7.37 (1H, m), 7.20-7.16 (1H, m), 7.15-7.07 (2H, m), 7.03&7.00 (1H, 2d, J= 8.3, 8.2), 6.89&6.84 (1H, 2d, J= 8.3, 8.2), 6.05-5.84 (2H,m), 5.15-4.97 (4H, m), 3.96&3.90 (2H, 2t. J = 6.5, 6.5), 3.87-3.76 (2H, m), 3.44-3.28 (4H, m). 2.55&2.34 (2H, 2t,.7= 7.4, 7.4), 1.83-1.71 (2H, m), 1.69-1.59 (6H, m), 1.55-1.42 (3H, m), 1.28-1.16 (9H, m), 0.98&0.85 (3H, 2t, J= 7.3, 7.3).13C NMR (75 MHz, CDCh) 8 172.3, 172.2, 156.2, 154.4, 147.8, 146.1, 137.8, 137.7, 137.1, 136.9, 136.4, 136.0, 134.9, 134.8, 133.8, 133.7, 132.2, 131.6, 130.9, 130.5. 130.4, 128.4. 127.8, 122.7. 121.6, 118.9. 118.1, 116.1. 116.0, 115.5. 115.4, 113.9.112.7, 110.9, 68.6, 68.0, 39.6, 39.4, 34.7, 34.5, 34.1, 33.9, 30.5, 30.4, 30.3, 30.2, 29.4, 29.3, 29.2, 29.1, 29.0, 27.0, 26.7, 26.1, 25.9, 22.9, 22.7, 22.6, 22.4 (d, J= 53.5), 13.7, 13.6.Attorney Docket: 650053.01246Mito-HNK-HAcScheme 9. Synthesis of Mito-HNK-HAc. Reagents and conditions: i, Hexanoyl chloride, TEA, CH2CI2, 57%.

[0245] Mito-HNK-HAc (10-[3,5'-diallyl-2’-(hexanoyloxy)-(l,l'-biphenyl)-4-yl]-oxy-decyltriphenylphosphonium bromide) Mitochondria-targeted honokiol hexanoic acid (Mito-HNK-HAc) was prepared by reacting Mito-HNK (0.2 g, 0.26 mmol) in CH2CI2 (4 mL) with hexanoyl chloride (74 pL, 0.52 mmol) in the presence of triethylamine (72 pl, 0.56 mmol). The mixture was stirred at room temperature for 2 h. The product was precipitated into Et20. Purification by flash chromatography (CH2C12 / EtOH, from 100% to 85 / 15) delivered the corresponding Mito-HNK-HAc (130 mg, 57% yield). The synthesis of Mito-EINK-HAc is presented in Scheme 9. HRMS calculated for Mito-HNK-HAc C52H62O3P+[M]+765.4431, found, 765.4432.31P NMR (400.13 MHz, CDCh) 5 24.57. 'H NMR (400.13 MHz, CDCh), 57.91-7.66 (15H, m), 7.44-7.38 (1H, m), 7.35-7.07 (3H, m), 7.04&7.01 (1H, 2d, J= 8.3, 8.2), 6.89&6.84 (1H, 2d, J = 8.4, 8.3), 6.05-5.85 (2H, m), 5.17-4.97 (4H, m), 4.02-3.68 (4H, m), 3.44-3.27 (4H, m), 2.57&2.34 (2H, 2t, J = 7.4, 7.4), 1.84-1.73 (2H, m), 1.71-1.65 (2H, m), 1.60-1.35 (6H, m). 1.36-1.14 (12H, m), 0.94&0.85 (3H, 2t, J= 7.1. 7.1).13C NMR (75 MHz, CDCh) 5 172.3, 172.2, 156.2, 154.4, 147.8, 146.1, 137.8, 137.6, 137.1, 137.0, 136.9, 136.3, 135.9, 134.9, 134.8, 133.7, 133.6, 132.1, 131.6, 130.9, 130.8, 130.4, 130.3, 128.4, 128.3, 127.8, 127.7, 122.6. 121.6, 118.8, 118.0, 116.1, 116.0, 115.5, 115.3, 112.6, 110.9, 68.5, 67.9, 39.6, 39.3, 34.6, 34.4, 34.3, 34.2, 31.2, 31.1, 29.4, 29.3. 29.2. 29.1. 26.1. 25.9, 24.6, 24.3, 22.7 (d. J = 49.9), 22.6, 22.2, 13.9, 13.7.Attorney Docket: 650053.01246o--cScheme 10. Synthesis of Mito-HNK-VAPAc. Reagents and conditions: i, Valproylchloride, TEA, CH2CI2.

[0246] A valproyl derivative, Mito-HNK-VAPAc, could be synthesized as shown in Scheme 10, using an approach similar to the synthesis of Mito-HNK-HAc.Scheme 11. Synthesis of Mito-PEGs-HNK-PAc. Reagents and conditions: i, 1,14-dibromo-3,6,9, 12-tetraoxatetradecane, K2CO3, DMF, 40°C, 36h, 31%; ii, triphenylphosphine. CH3CN, reflux, 18h, 45%; iii, propionyl chloride, TEA, CH2CI2, 55%.

[0247] Mito-PEGs-HNK-PAc (14-((3',5-diallyl-4'-(propionyloxy)-[l,l'-biphenyl]-2-yl)oxy)-3,6,9, 12-tetraoxatetradecyl)triphenylphosphonium and (14-((3,5'-diallyl-2'-(propionyloxy)-[l, T-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)triphenylphosphonium Mito-PEGs-HNK was prepared in two steps, by reacting the appropriate PEGylated dibromoalkane withAttorney Docket: 650053.01246honokiol in the presence of potassium carbonate in DMF. The addition of triphenylphosphine on the bromopegylated honokiol (HNK-PEGs-Br) led to Mito-PEG-HNK. Then, Mito-HNK-PEGs-PAc was prepared by reacting propionyl chloride in the presence of triethylamine in CH2CI2 (Scheme 11).

[0248] To a mixture of honokiol (0.6 g, 2.25 mmol), anhydrous potassium carbonate (0.3 g, 2.4 mmol) in DMF (4 mL) was added l,14-dibromo-3,6,9,12-tetraoxatetradecane (0.8 g, 2.2 mmol). The mixture was stirred at 45°C for 24 h. The residue was taken up into water and extracted with Et20. The organic layer was dried over Na2SC>4, and the solvent was removed under reduced pressure. Purification by flash chromatography (pentane / Et2O, from 1 / 0 to 1 / 1) delivered the corresponding PEGs-HNK (0.38 g, 31% yield). PEGs-HNK was directly used for the next step. A mixture of PEGs-HNK (0.38 g, 0.69 mmol) and triphenylphosphine (0.18 g, 1.11 mmol) in acetonitrile (2 mL) was stirred at reflux for 18 h. 100 mL of Et20 was poured into the mixture. The precipitate was purified by flash chromatography (CFLCh / EtOH 9 / 1) and led to the corresponding Mito-PEGs-HNK (254 mg, 45% yield). FIRMS calculated for Mito-PEGs-HNK C46H52O6P+[M]+731.3496, found, 731.3500.31P NMR (400.13 MHz, CDCh) 5 25.45, 25.24. 'H NMR (400.13 MHz, CDCh) 8 7.81-7.66 (9H, m), 7.64-7.55 (6H, m), 7.28-7.21 (2H, m), 7.18 (1H, 2d, J= 8.2), 7.10-6.96 (2H, m), 6.85-6.80 (1H, m), 6.04-5.90 (2H, m), 5.10-4.95 (4H, m), 4.13-3.70 (10H, m), 3.65-3.45 (4H, m), 3.40-3.30 (6H, m), 3.29-3.11 (4H, m).13C NMR (75 MHz, CDCh) 8 155.5, 154.2, 151.9, 138.0, 137.7, 137.3, 136.8, 135.4, 134.6, 134.4, 133.94, 133.91, 133.84, 133.81, 132.6, 131.2, 131.0, 130.7, 130.6, 130.4, 130.0, 129.95, 129.91, 129.8, 128.2, 128.1, 127.5, 127.4, 119.2, 119.1, 118.4, 118.3, 116.4, 115.5, 115.4, 115.1. 113.0, 111.4, 70.9. 70.7. 70.5, 70.3, 70.2, 70.1, 69.8, 69.7, 68.6, 67.9,63.8, 63.7, 53.4, 39.4, 25.5, 24.9, 25.2 (d, J= 58.2).

[0249] To a mixture of Mito-PEGs-HNK (0.17 g, 0.21 mmol) in CH2CI2 (4 mL) was added propionyl chloride (30 pL, 0.34 mmol) in the presence of tri ethylamine (100 pl, 0.5 mmol). The mixture was stirred at room temperature for 2 h. The product was precipitated into Et20. Purification by flash chromatography (CH2Ch / EtOH, from 100% to 85 / 15) delivered the corresponding Mito-PEGs-HNK-PAc (0.1 g, 55% yield). HRMS calculated for Mito-PEGs-HNK-PAc C49H56O7P+[M]+787.3758, found, 787.3761.31P NMR (400.13 MHz, CDCh) 8 25.62, 25.60. 'H NMR (400.13 MHz, CDCh) 8 7.89-7.79 (6H, m), 7.77-7.60 (9H, m), 7.44-7.39 (1H, m), 7.22-7.09 (3H, m), 7.08-6.99 (1H, m), 6.89-6.80 (1H, m), 6.04-5.89 (2H, m), 5.16-4.98 (4H, m), 4.26-3.83 (7H, m), 3.75-3.45 (7H, m), 3.44-23 (10H, m), 2.61&2.36 (2H, 2q, J= 7.6, 15,4), 1.28&1.07 (3H, 2t. J= 7.6).13C NMR (75 MHz, CDCh) 8 172.9, 155.8,Attorney Docket: 650053.01246154.1, 146.0, 138.0, 137.5, 137.0, 136.8, 136.2, 136.0, 134.5, 134.1, 134.0, 131.5, 131.0, 130.8, 130.4, 130.0. 129.9, 128.6, 128.5, 128.4; 128.0, 127.8, 122.7, 121.7, 119.5, 118.6, 116.1, 115.7, 115.5, 113.1, 111.3, 70.9, 70.8, 70.6, 70.5, 70.4, 70.3, 70.2, 69.9, 69.87, 69.81, 69.6, 68.4, 67.9, 64.1, 64.0, 45.9, 39.6, 39.3, 34.7, 34.5, 27.7, 27.6, 25.4 (d, J= 52.1), 8.6, 8.9.

[0250] Schemes 12-13 show the representative synthesis of Mito-HNK-SCFAs (Scheme 12) And Mito-PEG-HNK-SCFAs (Scheme 13).Mito-HNK-PAcScheme 12. Synthesis of Mito-HNK-PAc. Reagents and conditions: i, TEA, CH2CI2, 2 h, ii, TEA, CH2CI2.Scheme 13. Synthesis of Mito-PEG-HNK-PAc. Reagents and conditions: i. TEA, CH2CI2, 2 h.

[0251] Analysis of L-DOPA and Dopamine using LC-MS or LC-MS-SIM The freeze-dried samples were prepared as follows: To the tubes containing the cells was added 200 pLAttorney Docket: 650053.01246of ammonium formate buffer (10 mM). Then, the tubes were shaken vigorously (vortex) for 10 s and centrifugated for 7 min x 20.000 g at room temperature. The supernatants (100 pL) were transferred into HPLC vials with conical inserts and analyzed by LC-MS, described as follows: 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. The gradient is shown in Table 2.Table 2. Gradient for analysis of L-DOPA and Dopamine using LC-MS

[0252] Example 3

[0253] Disclosed herein are MGN derivatives including, but not limited to, MitopEG5-MGN-BAc as shown below.

[0254] Schemes 14-15 show representative syntheses of the Mito-MGN-SCFAs (Scheme 14) and Mito-PEG-MGN-SFCAs (Scheme 15).Scheme 14. Synthesis of Mito-MGN-SCFA. Reagents and conditions: i, TEA, CH2CI2, 2 h, ii, TEA, CH2CI2.Attorney Docket: 650053.01246Scheme 15. Synthesis of Mito-PEG-MGN-SFCA. Reagents and conditions: i, TEA, CH2CI2, 2 h.

[0255] Synthesis of mitochondria-targeted MGN-PAc (Mito-MGN-PAc) is shown in Scheme 16. Mito-MGN-PAc was prepared in two steps by reacting propionyl chloride with honokiol in the presence of triethylamine in CH2CI2 followed by the reaction with (10-bromodecyl)-triphenylphosphonium bromide in the presence of potassium carbonate in DMF.MGN-PAc Mito-MGN-PAc Scheme 16. Synthesis of Mito-MGN-PAc. Reagents and conditions: i, (10-Bromodecyl)-triphenylphosphonium bromide, K2CO3. DMF, 12 h; ii, Propionyl chloride. TEA, CH2CI2.

[0256] Example 4

[0257] Mitochondria-targeted drugs (MTDs) inhibiting oxidative phosphorylation (OXPHOS) mitigate tumor cell proliferation. Mitochondria-targeted OXPHOS inhibitors decreased myeloid-derived suppressor cells (MDSCs). regulatory T cells (Tregs), and activated cytotoxic T cells in the tumor microenvironment.

[0258] Short-chain fatty acids (acetate with two carbons, propionate with three carbons, buty rate with four carbons, valerate with five carbons, and caproate with six carbons) released in the colonic microbiota from anaerobic fermentation of dietary fibers inhibit tumorigenesis, activate T cells, and improve immunotherapy through epigenetic modification. The relative amounts of SCFA released in the gut are dependent on the type and amount of ingested fiber. Propionate is a major microbial fermentation-induced metabolite in the human gut.

[0259] It is hypothesized that a molecule that combines the OXPHOS-inhibiting drug moiety and a short chain fatty acid ester will amplify tumor inhibition through immune modulation.Attorney Docket: 650053.01246The rationale for the hypothesis is that the esterase enzymes in the gut can cleave the shortchain fatty acid esters and generate in situ short-chain fatty acids and the parent drug. For example, the mitochondria-targeted decyl (Mito-decyl) propionic ester will release the Mito-decyl compound and propionic acid, and mitochondria-targeted honokiol (Mito-HNK) propionic ester derivatives will release the parent drug, Mito-HNK and propionic acid (PAc), or butyric acid / bul rale (BAc) (FIG. 19).

[0260] Antitumor activity of MTDs: Activation of immune cells and epigenetic reprogramming It has been shown that mitochondria-targeted atovaquone (Mito-ATO) targets both granulocytic-myeloid-derived suppressor cells (G-MDSCs) and Tregs in the tumor microenvironment, resulting in significant decreases of both G-MDSCs and Tregs as determined by flow cytometry analysis. Intratumoral injection of Mito-ATO into primary tumors in a spontaneous tumor model triggered potent T cell immune responses locally and in distant tumor sites. Single-cell RNA sequencing revealed that Mito-ATO inhibits the expression of genes for OXPHOS and glycolysis in G-MDSCs and Tregs, and facilitates the infiltration of CD4+ T cells in the tumor immune microenvironment. This study shows for the first time the immune modulatory and tumor preventing effects of MTDs.

[0261] MTDs induce activation of AMP -activated protein kinase (AMPK) in several cancer cells. This is also responsible for activation of specific histone acety ltransferases (HAT) and for stimulation of histone acetylation. MTDs could indirectly act as histone deacetylase (HD AC) inhibitors.

[0262] Antitumor activity of SCFAs: Activation of immune cells through epigenetic reprogramming. Short chain fatty7acids (SCFAs) are generated in the gut microbiota from all carbohydrates and fuel the activities of gut friendly bacteria. Short chain fatty' acids (acetate [C-2], butyrate [C-4], propionate [C-3], valerate [C-5], and caproate [C-6]) are released in the colonic microbiota from anaerobic fermentation of dietary fibers. The relative amounts of SCFA released in the gut are dependent on the type and amount of ingested fiber. Previous studies have shown the enhancement of propionate levels in the gut via colonic delivery of prodrugs, such as insulin propionate ester, able to release propionate. Propionate is a major microbial fermentation-induced metabolite in the human gut.

[0263] SCFA (e.g., butyrate) inhibits human colon cancer cell (HT-29) proliferation. Butyrate is reported to exert cell cycle arrest in cancer cells through the induction of histone H4 hyperacetylation and the p21 gene. Although propionate and valerate caused growth arrest of colon cancer cells, histone hyperacetylation occurred to a smaller degree as compared with butyrate. Hyperacetylation of histones induced changes in chromatin packing due to disruptionAttorney Docket: 650053.01246in ionic interactions with the adjacent DNA backbone. The effects were negated by HD AC inhibitors.

[0264] Decrease in SCFAs mitigates the anti-inflammatory responses in diabetes. There is an emerging role of microbiota-derived SCFAs in cancer prevention. The ability of SCFAs to inhibit colon cancer and enhance immunotherapy was linked to mitochondrial function. For example, SCFAs (e.g., butyrate and pentanoate) enhance the antitumor activity of cytotoxic T lymphocytes and chimeric antigen receptor T cells (CAR-T) cells through metabolic and epigenetic reprogramming. In vitro treatment of immune cells (T cells) with SCFAs increases the function of mTOR (mammalian target of rapamycin) and inhibits class 1 histone deacety lase activity'. This reprogramming leads to increased production of effector molecules (CD25. interferon gamma, and tumor necrosis factor alpha) and enhances the antitumor activity of antigen-specific cytotoxic T lymphocytes and CAR-T cells. SCFAs enhance cellular cancer immunotherapy.

[0265] Antiproliferative effect of Mito-HNK-PAc FIG. 20 shows the relative antiproliferative effects of Mito-HNK and Mito-HNK-PAc in pancreatic cancer cells. Results show that the potency of Mito-HNK-PAc is nearly the same or slightly better than that of Mito-HNK. In summary, a mitochondria-targeted prodrug containing an SCFA ester generates a parent drug that inhibits OXPHOS in tumors and induces metabolic reprogramming in the tumor immune microenvironment (immune modulatory effects), and an SCFA propionate and other SCFAs with tumor inhibitory and immunomodulatory effects. The net effect of this dual combination is synergistic inhibition of tumor growth.

[0266] Antiproliferative and potential neuroprotective effects of Mito-HNK-SCFA Reports indicate that the gut microbiome and its metabolites including the short chain fatty acids are diminished in PD patients. Serum short chain fatty acids alter motor and non-motor symptoms in PD patients. Reports suggest that PD patients have decreased fecal levels of butyrate and butyrate-generating bacteria. Deceased butyrate was associated with increased depression in PD patients. Sodium butyrate / butyric acid administration was shown to be effective in PD patients. Therapeutic targeting of gut microbiome is emerging as a new way to treat PD symptoms.

[0267] It is hypothesized that a molecule that combines Mito-HNK and an SCFA ester (Mito-HNK-SCF-esters) in the presence of the esterase enzymes could generate in the gut Mito-HNK and short-chain fatty' acids. For example, the mitochondria-targeted honokiol (Mito-HNK) butyrate ester derivatives will release the parent drug. Mito-HNK and butyric acid or butyrate (BAc) (FIGs 14 and 19). Although prodrugs or amino acids conjugated to SCFAs haveAttorney Docket: 650053.01246previously been developed, this is the first report of a mitochondria-targeted drug containing esterase cleavable SCFAs with antimicrobial and neuroprotective potential.

[0268] Esterase-induced release of PB from HNK-PBAc and Mito-HNK-PBAc and analogs Phenylbutyrate (PB) is an FDA-approved drug for amyotrophic lateral sclerosis. PB also has been shown to inhibit progression of PD in cell culture and animal models. PB is administered as a suspension in glycerol or as a glycerol conjugate. PB is essentially an aromatic short-chain fatty acid. The inventors surmise that PB conjugated to HNK and Mito-HNK could be cleaved by esterase enzymes in the gut, releasing HNK or Mito-HNK and PB (Scheme 17). In addition, Mito-HNK-PBAc by itself could slow the grow th of E. faecalis and decrease L-dopa degradation and dopamine formation in the gut. The beneficial effects of PB in PD patients is well established. The in situ release of PB in the gut by esterases could have the dual benefit of the antimicrobial effect in the gut and the neuroprotective effect. HNK-PBAc and Mito-HNK-PBAc can be synthesized via the procedure disclosed in Examples 1-2.Scheme 17. Esterase-induced cleavage of Mito-HNK-PBAc to Mito-HNK and PB.

[0269] Efficacy of HNK-BAC, 4Me-HNK-BAC and MAG-BAC in mice syngraft tumors

[0270] To test the tumor inhibition ability of HNK-BAC, we established lung tumors via tail vein injection of LKR13 lung cancer cells. Three days after tumor inoculation, HNK-BAC was treated with lx HNK-BAC (37.5 pmol / kg.bw) or 5x (187.5 pmol / kg.bw) by oral gavage. IxHNK-BAC resulted in 55% inhibition of tumor growth, whereas 5xHNK-BAC decreased tumor growth by 75% (FIG. 21, panel A). We also tested the tumor inhibition ability of magnolol derivative MAG-BAC and honokiol derivative 4Me-HNK-BAC in the KPB25L breast cancer syngraft model. MAG-BAC or 4Me-HNK-BAC were treated with 4mM for four injections, the inhibition rate of MAG-BAC is 79%, and the inhibition rate of 4Me-HNK-BAC is 12% (FIG. 21, panel B).Attorney Docket: 650053.01246

[0271] Combination of HNK-BAC with anti-PD-1 antibody in syngraft lung models

[0272] An LKR lung cancer syngraft mouse model was used to determine whether HNK-BAC could enhance the efficacy of the anti-PD-1 checkpoint blockade. Subcutaneous injections of LKR tumor cells were done on the flanks of SV 129 mice, and mice were treated with BAC, anti-PD-1 antibody, or HNK-BAC with anti-PD-1. Notably, a significantly greater anti-tumor effect was observed with the combination of HNK-BAC and anti-PDl antibody than with anti-PD1 itself. As shown in FIG. 22, HNK-BAC improves the anti-tumor efficacy of anti-PD-1 antibody in the LKR model.

[0273] Example 5

[0274] Overview

[0275] Phenylbutyrate (PBA) is an aromatic short-chain fatty acid derivative of butyrate. PBA is a reversible inhibitor of histone decarboxylases (HDACs), which is attributed to its ability to modify epigenetic mechanisms, modify gene expression, decrease cellular proliferation, increase cell differentiation, and induce apoptosis. Sodium phenylbutyrate (i.e., the sodium salt of PBA) has numerous clinical applications. It has been approved by the US Food & Drug Administration (FDA) to treat urea cycle disorders by removing ammonia in the blood, and more recently as a drug in combination with Taurursodiol to slow the progression of motor neuron disease (also amyotrophic lateral sclerosis [ALS]).

[0276] PBA has been shown to mitigate motor impairment and dopaminergic neuronal death in Parkinson’s disease (PD). PBA upregulates the DJ-1 protein and protects neurons in preclinical models of PD. Alpha-synuclein undergoes aggregation and forms abnormal protein deposits in dopaminergic neurons, resulting in neuronal cell death in PD. The FDA-approved drug phenylbutyrate-triglyceride is in a Phase I clinical trial studying its ability to remove alpha-synuclein from the brains of patients with PD into the bloodstream. PBA was shown to mitigate cognitive deficit and decrease tau pathology in an Alzheimer’s disease mouse model.

[0277] PBA has a low bioavailability and is administered in very high doses in patients. Often PBA is administered as an esterase-cleavable conjugate (e.g., PBA-triglyceride). PBA-triglyceride contains three molecules of PBA attached to the hydroxyl groups of glycerol. In addition, modified forms of PBA have been developed because of its unfavorable odor. This odor problem can be overcome with modified PBA as prodrugs that release PBA through enzymatic hydrolysis. A strategy' is used here in which honokiol, a naturally occurring neuroprotective polyphenol, is combined with PBA to form HNK-PBA-esters and other more hydrophilic analogs, HNK-PEG-PBA. These esters release HNK and PBA in the presence of esterase enzymes that are typically present in the gut. The inventors also combinedAttorney Docket: 650053.01246mitochondria-targeted honokiol (Mito-HNK) and its pegylated analog (Mito-PEG-HNK) with PBA to form Mito-HNK-PBA and Mito-PEG-HNK-PBA. It is hypothesized that these newly synthesized prodrug conjugates may exert a synergistic anti-Parkinsonian effect. Polyphenols conjugated to PBA were not previously developed.

[0278] The oral bioavailability of levodopa in patients with PD is decreased due to enhanced metabolism to dopamine by gut bacteria, Enterococcus faecalis. present in the gut microbiome of patients with PD. PBA has antibacterial activity as do HNK and Mito-HNK analogs.

[0279] In this disclosure, synthesis and verification of the structures of prodrug conjugates of HNK-PBA, Mito-HNK-PBA, and their pegylated analogs. The inventors also tested their effects on gut bacteria, Enterococcus faecalis. Mito-PEG-HNK-PBA reversibly inhibits the proliferation of this gut bacteria, suggesting a promising therapeutic application of this class of modified forms of PBA in Parkinson’s research.

[0280] Synthesis of HNK-PB Analogs

[0281] HNK-PBA and HNK-BZs-PBA were prepared by reacting HNK with 4-phenylbutanoyl chloride in the presence of triethylamine in dichloromethane (CH2CI2) (Scheme 18).conditions: i, 4-phenylbutanoyl chloride, TEA, CH2CI2, 24%. ii, 4-phenylbutanoyl chloride, TEA, CH2CI2, 91%. iii, Br-PEG5-OMe, K2CO3, DMF, 32%. iv, 4-phenylbutanoyl chloride, TEA. CH2CI2, 70%.

[0282] Synthesis of Mito-HNK-PBA and Mito-PEG-HNK-PBA

[0283] Mito-HNK-PBA was prepared by reacting Mito-HNK1with 4-phenylbutanoyl chloride in the presence of tri ethylamine in CH2CI2 (Scheme 19). In addition, the peg lated mitochondria-targeted analog was prepared in three steps, by reacting the appropriate pegylated dibromoalcane with honokiol in the presence of potassium carbonate in DMF. Addition ofAttorney Docket: 650053.01246triphenylphosphine on the bromopegy dated honokiol (HNK-PEGs-Br) led to the Mito-PEG- HNK. Then, addition of 4-phenylbutanoyl chloride in the presence of triethylamine in CH2CI2 afforded the Mito-PEG-HNK-PBA (Scheme 20).Scheme 19. Synthesis of Mito-HNK-PBA. Reagents and conditions: i, 4-phenylbutanoyl chlonde, TEA, CH2CI2, 71%.Scheme 20. Synthesis of Mito-PEG-HNK-PBA. Reagents and conditions: i, 1,14-dibromo-3,6,9, 12-tetraoxatetradecane, K2CO3, DMF, 40°C, 12 h. 27%; ii, triphenylphosphine. CH3CN, reflux, 18 h, 32%; iii, 4-phenylbutanoyl chloride, TEA, CH2CI2, 83%.

[0284] Materials and Methods

[0285] General. All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin-layer chromatography (TLC) using silica gel Merck60F254. Crude materials were purified by flash chromatography on Merck Silica gel 60 (0.040-0.063 mm).31P nuclear magnetic resonance (NMR). 'H NMR and13C NMR spectra were recorded at 400.13 MHz spectrometers and 75.54 MHz, respectively. 'H NMR spectra were recorded using a Bruker DPX AVANCE 400 spectrometer (Marseille, PACA, France) equipped with a quattro nucleus probe. Chemical shifts (5) are reported in ppm and J values in Hertz.Attorney Docket: 650053.01246

[0286] HNK-PBA. HNK-PBA was prepared by reacting HNK (0.25 g, 0.94 mmol) in CH2CI2 (4 mL) with 4-phenylbutanoylchloride (154 pL, 0.94 mmol) in the presence of tri ethylamine (128 pL, 0.94 mmol) (Scheme 5). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by diethyl ether (Et20). The organic layer was dried over sodium sulfate (JSteSCU), and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 90 / 10) delivered the corresponding HNK-PBA (102 mg, 26% yield) as a mixture of both isomers. High-resolution mass spectrometry (HRMS) calculated for HNK-PBA C28H28O3 [MNa]+435.1931, found, 435.1932. 'H NMR (400.13 MHz, CDCh), 57.37-7.30 (4H, m), 7.26-7.21 (3H, m), 7.17-7.03 (3H, m), 6.93-6.91 (1H, 2s), 6.03-5.86 (2H, m), 5.13-5.04 (5H, m). 3.36 (4H, t, J= 5.6), 2.78 (2H, t, J = 7.3), 2.63 (2H, t, J = 7.3), 2.13 (2H, quint., J = 15.1, 7.8).13C NMR (75 MHz, CDCh) 5 171.9, 150.8, 148.6, 141.1, 137.7, 135.4, 135.1, 132.9, 132.4, 131.1, 130.2, 129.3, 128.5, 128.1, 127.3, 126.2, 123.2, 116.7, 115.9, 39.4, 35.1, 44.6, 33.5, 26.4.

[0287] HNK-Bis-PBA. HNK-BN-PB A was prepared by reacting HNK (0.25 g, 0.94 mmol) in CH2CI2 (5 mL) with 4-phenylbutanoylchloride (301 pL, 1.9 mmol) in the presence of tri ethylamine (256 pL, 1.9 mmol) (Scheme 18). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over NazSCL, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 95 / 05) delivered the corresponding HNK- / L.s-PBA (481 mg. 91% yield) as a mixture of both isomers. HRMS calculated for HNK-R,S-PBA C38H38O4 [MNa]+581.2662, found, 581.2657. T1 NMR (400.13 MHz, CDCh), 57.36-7.30 (4H, m), 7.26-7.11 (10H, m), 7.06-.99 (2H, m), 6.05-5.81 (2H, m), 5.18-4.98 (4H, m), 3.42 (2H, d, J= 6.6), 3.27 (2H, d, J= 6.4), 2.77 (2H, t, J= 7.6), 2.64-2.52 (4H, m), 2.37 (2H, t, J= 7.6), 2.11 (2H, quint., J= 15.1. 7.8), 1.87 (2H. quint., J= 15.1, 7.8).13C NMR (75 MHz, CDCh) 5 171.9, 171.7, 148.3, 146.0, 141.2, 141.1, 138.1, 136.9, 135.7, 135.5, 133.9, 131.6, 130.9, 128.6, 128.5,128.49, 128.46, 128.0, 126.1, 125.9, 122.7, 122.2, 116.4, 116.2, 39.6, 35.1, 34.9, 34.5, 33.5, 33.4, 26.4, 26.2.

[0288] Mito-HNK-PBA. Mito-HNK was prepared according to the procedure as previously described. To a mixture of Mito-HNK (0.33 g, 0.44 mmol), in CH2CI2 (10 mL) was added 4-phenyl butanoyl chloride (95 pL, 0.57 mmol) in the presence of triethylamine (82 pl, 0.57 mmol). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by CH2CI2. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatographyAttorney Docket: 650053.01246(CH2C12 / ethanol [EtOH], 9 / 1) delivered the corresponding Mito-HNK-PBA (0.28 g, 71% yield). HRMS calculated for Mito-HNK-PBA: C56H620sP+[M]+813.4431, found, 813.4434.

[0289] Mito-HNK-PBA.31P (400.13 MHz, CDCh) 5 24.48. 'H NMR (400.13 MHz, CDCh) 57.90-7.63 (15H, m), 7.45-7.25 (4H, m), 7.25-6.80 (7H, m), 6.05-5.82 (2H, m), 5.14-4.91 (4H, m), 3.96-3.85 (2H, m), 3.85-3.74 (2H, m), 3.45-3.25 (4H, m), 2.76&2.52 (2H, 2t, J=7.3), 2.60&2.37 (2H, 2t. J=7.6), 2.09&1.86 (2H, 2quint., J=7.6, 15.1), 1.75-1.55 (6H, m), 1.37-1.13 (10H, m).13C NMR (75 MHz, CDCh) 5 171.8, 154.4, 147.7, 141.2, 137.7, 136.5, 135.6, 134.9.134.8, 133.7, 133.6, 132.1, 131.6, 130.9, 130.7, 130.4, 130.3, 129.8, 128.48, 128.43, 128.3, 128.2, 127.9, 127.7, 126.1, 125.9, 122.6, 121.6, 118.9, 118.0, 116.1, 115.5, 112.6, 110.9, 68.5, 67.9, 39.6, 39.3, 35.0, 34.8, 34.6, 34.4, 33.5, 30.4, 30.2, 29.3, 29.1, 29.0, 26.5, 26.2, 25.9, 22.7 (d,.7= 49.8), 22.6 (d, J= 4.4).

[0290] Mito-PEG-HNK-PBA. To a mixture of honokiol (0.6 g, 2.4 mmol), anhydrous potassium carbonate (0.3 g, 2.4 mmol) in DMF (5 rnL) was added 1,1 l-dibromo-3,6,9-trioxaundecane (0.82 g, 2.0 mmol). The mixture was stirred at 40 °C for 12h. The residue was taken up into water and extracted with Et20. The organic layer was dried over Na2SOr, and the solvent was removed under reduced pressure. Purification by flash chromatography (pentane / Et2O, 1 / 1) delivered the corresponding PEG-HNK (0.328 g, 27% yield). PEG-HNK was directly used for the next step.

[0291] 'H NMR (400.13 MHz, CDCh), 6 'H NMR (400.13 MHz, CDCh) 57.45-7.35 (1H, m), 7.26-7.21 (1H. m). 7.15-76.64 (4H, m), 6.13-5.92 (2H, m), 5.21-5.02 (5H, m), 4.22-4.15 (1H, m), 4.09-4.04 (1H, m), 3.94-3.72 (6H, m), 3.72-3.53 (10H, m), 3.49-3.43 (4H, m), 3.39-3.33 (2H, m).

[0292] A mixture of PEG-HNK (0.33 g, 0.6 mmol) and triphenylphosphine (0.24 g, 0.91 mmol) in acetonitrile (2 mL) was stirred at reflux for 48 hours. The mixture was poured in 100 mL of ether. The precipitate was purified by flash chromatography (CH2Ch / EtOH, 9 / 1) and led to the corresponding Mito-PEG-HNK (150 mg, 32% yield).

[0293] 'H NMR (400.13 MHz, CDCh), 5 'H NMR (400.13 MHz, CDCh) 5 7.78-7.68 (10H, m), 7.62-7.54 (5H, m), 7.27-6.76 (6H, m), 6.06-5.98 (2H, m). 5.12-4.91 (4H, m), 4.15-3.98 (2H, m), 3.97-3.43 (12H, m), 3.42-3.11 (10H. m).

[0294] To a mixture of Mito-PEG-HNK (0.15 g, 0.18 mmol), in CH2CI2 (10 mL) was added 4-phenylbutanoyl chloride (95 pL, 0.57 mmol) in the presence of triethylamine (41 □!, 0.28 mmol). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by CH2CI2. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatographyAttorney Docket: 650053.01246(CH2CI2 / EtOH, 9 / 1) delivered the corresponding Mito-PEG-HNK-PBA (0.15 g, 83% yield). HRMS calculated for Mito-PEG-HNK-PBA: C56H62O? P+[M]+877.4228. found, 877.4222.

[0295] 31P NMR (400.13 MHz, CDCh) 5 25.70 'H NMR (400.13 MHz, CDCh), 5 'H NMR (400.13 MHz, CDCh) 5 7.91-7.82 (6H, m), 7.76-7.61 (3H, m), 7.67-7.59 (6H, m), 7.43-7.28 (1H, m), 7.26-7.07 (8H, m), 7.01, 7.00 (1H, 2d, J= 8.9, 8.1), 6.81, 6.87 (1H, 2d, J= 9.1, 8.3), 6.04-5.85 (2H, m). 5.16-4.95 (4H, m), 4.33-4.22 (2H, m), 4.12-4.03 (2H, m), 4.02-3.91 (2H, m), 3.90-3.79 (2H, m), 3.73-3.69 (2H, m), 3.67-3.59 (2H. m). 3.56-3.52 (2H, m), 3.43-3.24 (10H, m), 2.76, 2.53 (2H, 2t, J= 7.4, 7.3), 2.60, 2.37 (2H, 2t, J= 7.4, 7.3), 2.10, 1.86 (2H, 2q, J = 1.9, 15.4).13C NMR (75 MHz, CDCh) 5 171.9, 155.9, 146.1, 141.3, 138.0, 137.1, 136.8, 134.5, 134.4, 134.1, 134.0, 130.9, 130.4, 130.0, 129.9, 128.4, 128.3, 128.1, 127.8, 125.9, 122.7, 119.6, 118.8. 116.1, 115.6. 70.9. 70.6, 70.5, 70.3, 70.2, 70.8, 70.4, 69.9, 69.8. 69.6. 64.2. 64.1, 45.8, 39.6, 34.9, 34.4, 33.5, 26.2, 25.2 (d, J = 52.8).

[0296] Calculated values of the octanol / water partition coefficients. The hydrophobicity of HNK-SCFAs was assessed by calculating the log P partition coefficients. The calculated log P values for HNK, Mito-HNK-PBA, HNK-PBA, Mitoio-HNK, Mitoio-HNK-Pac, Mito-PEG-HNK-PBA, HNK-PEG-PBA, Mito-PEG-HNK are 5.2, 15.7, 7.8, 13.0, 10.5, 12.1, 7.8, and 9.5. respectively (Table 3). The log P values of HNK-ester analogs (mono and to-esters) were assessed using a QSAR (quantitative structure-activity relationship) analysis and rational drug design as a measure of molecular hydrophobicity (Table 1). This method also uses a consensus model built using the ChemAxon software (San Diego, CA).

[0297] Effects of analogs on bacterial proliferation

[0298] The effects of HNK-PBA and Mito-HNK-PBA analogs on the bacterial proliferation of E. faecalis are shown in FIG. 23. Unlike HNK-PBA (Fig. 23 A) and HNK-Bis-PBA (Fig. 23B), Mito-HNK-PBA (Fig 23C) and Mito-PEG-HNK-PBA (Fig 23D) induced a dose-dependent delay in E. faecalis proliferation. After the time lag, bacterial growth resumed at a normal rate (Fig 23D).Attorney Docket: 650053.01246Table 3. Calculated values of the octanol / water partition coefficient.Attorney Docket: 650053.01246

[0299] Example 6Disclosed herein are derivatives of HNK, 4-OMe-HNK and MGN including buty rate and plhydroxy butyrate derivatives of HNK. Example structures are shown in FIGS. 24-25. FIG. 26 shows a schematic of cellular, mitochondria uptake and hydrolysis via esterases of HNK-BAc, Mito-HNK-BAc. HNK-BHB, Mito-HNK-BHB and derivatives thereof.

[0300] Syntheses of Magnolia bark derivatives

[0301] Magnolia bark derivatives were obtained by reacting HNK, MGN or 4-0-Me-HNK with the butyryl chloride in the presence of triethy lamine in dichloromethane (CH2CI2) (Scheme 21). Structures and the purity of products were confirmed by NMR analyses.Attorney Docket: 650053.01246Scheme 21. Synthesis of magnolia bark derivatives, i, buty ry l chloride, TEA, CH2CI2, 42%. ii, Mel. K2CO3, DMF, rt, 39%. iii. butyryl chloride, TEA. CH2CI2, 78%. iv, butyryl chloride, TEA, CH2CI2, 52%; v, EDC, HOBt, DIPEA, 2-hydroxybutyric acid, 27%.

[0302] HNK-BAc (3,5'-diallyl-2'-hydroxy-[l,l'-biphenyl]-4-yl buty rate and 3',5-diallyl-4'-hydroxy-[l,l'-biphenyl]-2-yl butyrate )

[0303] Synthesis: To a mixture of HNK (0.25 g, 0.93 mmol) in CH2CI2 (4 mL) was added butyryl chloride (137 pL, 1 mmol) in the presence of triethylamine (200 μL, 0.97 mmol). The mixture was stirred at room temperature for 2 h. (Scheme 22) Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding HNK-BAc (131 mg, 42% yield) as a mixture of both isomers. HRMS calculated for HNK-BAc C22H24O3 [M+NH4]+, 354.2064, found, 354.2062.

[0304] NMR Results: 'H NMR (400.13 MHz, CDC13).5 7.36-7.34 (2H, m), 7.15 (1H, d, J = 8.1), 7.01-7.04 (2H, m), 6.91 (1H. d. J = 8.1), 6.01-5.86 (2H, m), 5.22-5.02 (5H, m), 3.37 (4H. dd, J = 1, 6.6), 2.60 (2H, t, J = 7.3), 1.83 (2H, sext., J = 7.5, 14.8), 1.09 (3H, t, J = 7.3).13C NMR (75 MHz, CDCI3) 5 172.0, 172.7, 150.8, 148.6, 137.7, 135.5, 135.1, 132.9, 132.3, 131.1, 130.2, 129.2, 128.1, 127.3, 123.1, 116.6, 115.9, 115.6, 39.3, 36.2, 34.6. 18.2, 13.7.

[0305] MGN-BAc (5,5'-diallyl-2,-hydroxy-[l,r-biphenyl]-2-yl butyrate

[0306] Synthesis: To a mixture of MGN (0.5 g, 1.9 mmol) in CH2CI2 (4 mL) was added butyryl chloride (200 pL, 1.9 mmol) in the presence of triethylamine (256 pl, 1.9 mmol). (Scheme 21) The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding MGN-BAc (331 mg, 52% yield).

[0307] HRMS calculated for MGN-BAc C22H24O3 [M+Na]+, 359.1618, found, 359.1616.

[0308] NMR Results: *HNMR (400.13 MHz, CDCh),57.30-7.24 (1H, m), 7.21-7.18 (1H, m), 7.12-7.06 (2H. m), 6.95-6.89 (2H. m). 6.05-5.89 (2H, m). 5.18-4.96 (5H, m). 3.44 (2H. dd. J = 6.6), 3.33 (2H, dd, J = 6.6), 2.3 (2H, t, J = 7.3), 1.49 (2H, sext, J = 7.4, 14.8), 0.78 (3H, t, J = 7.4).13C NMR (75 MHz, CDCk) 5 172.4, 151.3, 146.9, 138.6, 137.7, 136.7, 131.9, 131.8, 130.5, 130.1, 129.6, 124.0, 122.9, 116.4, 116.3, 115.5, 39.5, 39.3, 35.8, 18.2, 13.3.

[0309] HNK-BHB (3',5-diallyl-4'-hydroxy-[l,l'-biphenyl]-2-yl 3-hydroxybutanoateAttorney Docket: 650053.01246

[0310] Synthesis: To a mixture of P-hydroxybutyric acid (0.4 g, 3.8 mmol) and HOBt (1g, 7.4 mmol) was added dropwise at 0°C DIC (1.2 mL, 7.7 mmol). (Scheme 21) The mixture was stirred for 2 hours. Then, HNK (1 g, 3.7 mmol) in CH2CI2 (5 mL) following by pyridine (300 pL. 3.8 mmol). The mixture was stirred overnight at room temperature. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (pentane / AcOEt, 6 / 4) delivered the corresponding BHB-BAc (361 mg, 27 % yield).

[0311] HRMS calculated for HNK-BHB, C22H24O4 [M+Na]+, 375.1567, found, 375.1562.

[0312] NMR Results:1HNMR(400.13 MHz, CDCH), 67.40-6.85 (6H, m), 6.11-5.86 (2H, m), 5.22-5.03 (4H, m), 4.44-4.31 & 4.12-4.06 (1H, 2m), 3.46-3.42 (4H, m), 2.85-2.70 & 2.58-2.44 (2H, 2m), 1.35 & 1.17 (3H, 2d, J = 6.3).13C NMR (75 MHz, CDCh) 5 171.3, 150.7, 148.2, 137.6, 136.9, 136.2, 135.5, 132.8, 132.4, 131.3, 130.9, 130.9, 130.2, 129.3, 128.2, 127.2, 123.0, 122.5, 116.7, 116.5, 116.2, 115.9, 115.8, 64.3, 64.0, 42.9, 42.8, 39.6, 39.3, 35.0, 34.7, 22.6, 22.2.

[0313] 4Me-HNK. 3'.5-Diallyl-4'-methoxy-[l,r-biphenyl]-2-ol

[0314] Synthesis: To a mixture of HNK (0.9 g, 3.3 mmol) and methyl iodide (221 pL, 3.5 mmol) in DMF (4 mL) was added K2CO3 (0.93 g, 6.7 mmol). The mixture was stirred at 35 °C for 6 h. Then, water was added to the mixture and the product was extracted by ethyl acetate. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding 4Me-HNK (0.37 g, 39% yield).

[0315] NMR Results: 'H NMR (400.13 MHz, CDCh), 5 7.30 (1H, dd, J = 8.4, 2.2), 7.25 (1H, d, J = 2.1), 7.09-7.03 (2H, m), 6.97 (1H, d, J = 8.4), 6.92 (1H, d, J = 8.1), 6.09-5.93 (2H, m), 5.16-5.02 (5H, m), 3.89 (3H. s), 3.45 (4H, d. J = 6.7), 3.37 (2H, d, J = 6.6);13C NMR (75 MHz, CDCh) 8 157.0, 150.8, 137.8, 136.5, 132.1, 132.2, 130.5, 130.2, 129.8, 129.0, 128.7, 127.9, 127.8, 115.8, 115.5, 110.9, 55.5, 39.4, 34.3.

[0316] 4Me-HNK-BAc. 3 5 -Dially l-4'-methoxy-[ 1,1 '-biphenyl] -2-yl buty rate

[0317] Synthesis: To a mixture of 4Me-HNK (0.38 g, 1.4 mmol) in CH2CI2 (4 mL) was added butyryl chloride (170 pL, 1.7 mmol) in the presence of tri ethylamine (360 pl, 2.6 mmol). The mixture was stirred at room temperature for 2 h. Then, water was added to the mixture and the product was extracted by Et20. The organic layer was dried over Na2SO4, and the solvent was removed under reduced pressure. Purification by flash chromatography (Et2O / pentane, 85 / 15) delivered the corresponding 4Me-HNK-BAc (370 mg, 78% yield). HRMS calculated for 4Me-HNK-BAc C23H26O3 [M+Na]+, 373.1774, found, 373.1775.Attorney Docket: 650053.01246

[0318] NMR Results:7.19 (2H, m), 7.15 (1H, dd, J = 8.1, 1.9), 7.02 (1H, d, J = 8.1), 6.89 (1H, d, J = 8.3), 6.07-5.92 (2H, m), 5.17-5.02 (5H, m), 3.87 (3H, s), 3.42 (4H, dd, J = 6.4, 1.0), 2.34 (2H, t, J = 7.4), 1.65-1.50 (2H, q, J = 7.4), 0.87 (3H, t, J = 7.4). 13C NMR (75 MHz, CDCk) 5 172.1, 156.7, 146.1, 137.9, 137.1, 136.8, 134.5, 130.9, 130.4, 129.9, 128.2, 128.0, 127.8, 122.7, 116.1, 110.2, 115.4, 55.5, 39.7, 36.1, 34.3, 18.2, 13.5.

[0319] Example 7

[0320] Disclosed herein are polyphenol -butyrate conjugates to effectively decrease or mitigate a-syn aggregation and toxicity, enhance autophagic clearance, and restore mitochondrial function in human PD dopaminergic neurons.

[0321] Parkinson’s disease (PD) affects more than 10 million people worldwide and currently has no cure. This project will test anew class of small molecules that combine a natural plant compound (honokiol) with a gut-derived nutrient (butyrate) to block the buildup of toxic a-synuclein protein in brain cells from Parkinson’s patients. By improving mitochondrial health and reducing inflammation, these dual-action compounds could lead to new treatments for Parkinson’s and related brain disorders.

[0322] Summary

[0323] Polyphenols such as honokiol (HNK) and 4-O-methylhonokiol (4-Me-HNK) decrease a-syn toxicity and exert neuroprotective effects, while the gut microbiota short-chain fatty acid metabolite butyrate (BAc) enhances mitochondrial function and suppresses inflammation through histone deacetylase inhibition. However, both have poor cell permeability. To overcome this limitation, we synthesized novel polyphenol-butyrate conjugates (HNK-BAc, MGN-BAc, 4-Me-HNK-BAc) that release active agents intracellularly following esterase cleavage. Because the mitochondrion is the central hub of a-syn toxicity, we developed the corresponding mitochondria-targeted polyphenol-butyrate derivatives (Mito-HNK-BAc, Mito-MGN-BAc) by incorporating a triphenylphosphonium moiety7for mitochondrial enrichment.

[0324] Aim 1 will test the ability of polyphenol-BAc conjugates to decrease a-syn aggregation and toxicity in patient-derived induced pluripotent stem cell (iPSC)-derived dopaminergic neurons carrying mutations in a-syn, Parkin, and LRRK2. Outcomes include a-syn aggregation, autophagy, proteasome activity7, mitochondrial function, oxidative stress, and cell survival.

[0325] Aim 2 will evaluate whether mitochondria-targeted conjugates provide superior neuroprotection compared with non-targeted analogs by quantifying mitochondrial uptake, esterase-mediated release, and effects on a-syn-associated phenotypes.Attorney Docket: 650053.01246

[0326] The innovative aspect of the proposed studies is developing a new class of dual-action, mitochondria-targeted neuroprotectants that simultaneously deliver polyphenol and butyrate in cells.

[0327] Specific Aims

[0328] Parkinson’s disease (PD) is a progressive neurodegenerative disorder affecting over 10 million people worldwide. A key hallmark of PD is the misfolding and aggregation of alpha-synuclein (a-syn), a presynaptic protein whose toxic forms dnve many of the pathological features of PD including mitochondrial dysfunction, oxidative stress, neuroinflammation, and dopaminergic neuron death. The current treatments for PD aim to restore dopamine levels (levodopa) or modulate the neural circuitry (deep brain stimulation), but neither impact an underlying neuronal pathology. Therefore, there remains an urgent need for therapies that directly target a-syn toxicity.

[0329] Honokiol (HNK), a polyphenol from magnolia bark, has shown neuroprotective effects in PD models, and our newly synthesized mitochondria-targeted version (Mito-HNK) enhances levodopa efficacy in vivo. Additionally, emerging evidence shows that PD patients have reduced levels of short-chain fatty acids (SCFAs), especially but rate (BAc) in their gut microbiota. Butyrate has neuroprotective properties, including histone deacetylase inhibition, mitochondrial support, and anti-inflammatory effects. Thus, reduced SCFAs implicates the gut-brain axis in PD pathogenesis. Unfortunately, butyrate itself has poor cellular uptake and bioavailability.

[0330] We hypothesize that conjugating butyrate to FINK and related analogs — magnolol (MGN) and 4-O-methylhonokiol (4-Me-HNK) — will improve delivery and, via esterase cleavage, release both active agents. Mitochondria-targeted versions (e.g., Mito-HNK-BAc) are designed to enrich delivery within mitochondria. Therefore, we have synthesized esterase-cleavable conjugates (HNK-BAc, MGN-BAc, 4-Me-HNK-BAc) and mitochondria-targeted analogs (Mito-HNK-BAc, Mito-MGN-BAc) that improved neuronal function and decreased oxidative stress in PD-relevant models.

[0331] Central Hypothesis: Polyphenol-butyrate conjugates will more effectively decrease a-syn aggregation and toxicity, enhance autophagic clearance, and restore mitochondrial function than in the unconjugated form in human PD dopaminergic neurons.

[0332] Aim 1: Test the ability of HNK-BAc, MGN-BAc, and Me-HNK-BAc to decrease a-syn aggregation and toxicity in PD iPSC-derived dopaminergic neurons.

[0333] Using patient-derived induced pluripotent stem cell (iPSC)-derived dopaminergic neurons from healthy control and PD patients expressing mutations in a-syn, Parkin, andAttorney Docket: 650053.01246LRRK2, we will compare the effects of poly phenol -butyrate conjugates to unconjugated polyphenols and butyrate. a-Syn aggregation will be assessed via thioflavin T fluorescence, a-syn-specific ELISA, western blot, and immunocytochemistry. We will measure autophagy via LC3-II and p62 western blot and proteasome activity via fluorescence substrate assays. Cytotoxicity7and mitochondrial function will be measured using caspase-3 activity, ATP levels, LDH release, mitochondrial membrane potential, reactive oxygen species, and Seahorse XF technology.

[0334] Rationale: With a-syn accumulation and mitochondrial malfunction both being critical aspects of PD pathology7, this has led to growing interest in small molecules that target a-syn aggregation and its associated neuronal toxicity. To date, HNK is the only compound among this class shown to decrease a-syn mRNA expression in neuronal cell-based models. The effects of magnolol (MGN) and 4-methylhonokiol (4-Me-HNK) on a-syn expression remains unknown. Butyrate, a microbial short-chain fatty7acid, has demonstrated context-dependent effects on a-syn aggregation. Therefore, here we will leverage our substantial expertise in small molecule development to evaluate a novel class of “hybrid” molecules that couple neuroprotective polyphenols with SCFAs like butyrate to combat a-syn associated pathology in human PD iPSC-derived dopaminergic neurons.

[0335] Preliminary Data

[0336] Synthesis of polyphenol-butyrate conjugates: Polyphenol -butyrate conjugates were synthesized by reacting HNK, MGN. or Me-HNK with butyryl chloride in the presence of tri ethylamine in di chloromethane (FIG. 27, panel A). The structures and purity of the resulting products were confirmed by NMR spectroscopy, liquid chromatography -mass spectrometry (LC-MS), and high-resolution mass spectrometry (HRMS). Both isomers of HNK-BAc were hydrolyzed by esterases, releasing the parent polyphenol (HNK) and corresponding short-chain fatty acid (BAc), respectively (FIG. 27, panel B and panel C).

[0337] Cellular phenotypes in PD iPSC-derived dopaminergic neurons: In our previous work, we have found that dopaminergic neurons derived from LRRK2 G2019S PD iPSCs show phenotypic neurite shortening by 5 weeks in culture compared to healthy control dopaminergic neurons (FIG. 28). Additionally, we have further shown that LRRK2 G2019S dopaminergic neurons have altered mitochondrial function and trafficking (FIG. 29 and data not shown). These data demonstrate that we can detect PD-relevant phenotypes in PD iPSC-derived dopaminergic neurons in vitro. Importantly, we have found that neurons derived from PD iPSCs expressing the a-syn triplication mutation and the LRRK2 G2019S mutation show increased a-syn expression (FIG. 30, panels A and B) and an increase in p62 and LC3-IIAttorney Docket: 650053.01246expression compared to healthy control dopaminergic neurons (FIG. 30, panels C-E). Interestingly, kinase inhibition (LRRK2-IN-1) treatment did not normalize p62 or LC2-II expression suggesting that other therapeutic targets may be needed to restore PD neuron function.

[0338] Experimental Design

[0339] Cell model and treatments: We will differentiate dopaminergic neurons from iPSCs generated from PD patients expressing mutations in LRRK2, parkin, and a-syn. We will follow standard differentiation procedures as we have done previously (FIG. 28). Briefly, iPSCs will be plated in appropriately sized culture dishes (e.g. 6 or 24 well plates, Seahorse plates) and patterned toward midbrain floor plate precursors via sonic hedgehog and WNT activation followed by dopaminergic neuron patterning and maturation. Based on our previous studies, we detect phenotypic alterations in PD iPSC-derived dopaminergic neurons by 5 weeks in vitro (FIG. 28). Therefore, we will treat dopaminergic neurons with each polyphenol-bulyrate conjugate (HNK-BAc, MGN-BAc, Me-HNK-Bac, 10-1000 nM), the corresponding unconjugated polyphenols (HNK, MGN, and 4-Me-HNK), or butyrate alone at ~28 days of differentiation. DMSO will be used as the diluent control. At various timepoints post-treatment, the following endpoints will be measured:

[0340] 1) Cytotoxicity7assessment: We will first assess compound toxicity' at 1-7 days posttreatment by measuring cleaved caspase-3 expression and activity as we have done previously, TUNEL expression as done previously, cellular ATP levels, and lactate dehydrogenase (LDH) release. We will also use TH immunofluorescence to measure neurite length and integrity as an indicator of overall neuronal health (FIG. 28). Based on these results, we will select the most appropriate dose for each compound for the remaining phenotypic assessments.

[0341] 2) Alpha-synuclein aggregation: We will assess a-syn aggregation using thioflavin T fluorescence (for amyloid fibrils), a-syn aggregation-specific ELISA using cell lysates as well as conditioned medium, western blotting to distinguish soluble and insoluble a-syn fractions, and immunocytochemistry to visualize aggregated inclusions based on our previous work.

[0342] 3) Mitochondrial bioenergetics: We will measure mitochondrial oxygen consumption rate (OCR) as an indicator of oxidative phosphorylation (OXPHOS) and extracellular acidification rate (ECAR) as a surrogate for glycolysis using the Seahorse XF96 Analyzer as we have done previously (Fig. 6). ATP production will be quantified using the luciferase-based ATP assay (Sigma) following the manufacturer's instructions. Mitochondrial membrane potential will be assessed by rhodamine- 123 accumulation via flow cytometry and immunofluorescence.Attorney Docket: 650053.01246

[0343] 4) Mitochondrial ROS: Mitochondrial ROS production will be assessed using immunofluorescence with specific fluorescent probes:80 1) Superoxide will be detected via hydroethidine oxidation to 2-hydroxyethidium, the diagnostic marker product, 81 and 2) hydrogen peroxide and peroxynitrite will be measured using boronate-based fluorescent probes.82

[0344] 5) Proteosome function and autophagy: To assess proteosome activity, we will measure the trypsin-like activity of the 26S proteosome using benzyloxycarbonyl-Leu-Leu-Lys-7-amido-4-methylcoumarin as a Anorogenic substrate. Proteolytic activity will be quantified by detecting release of 7-amido-4-methylcoumarin (excitation: 380 nm; emission: 460 nm). We will measure autophagy via LC3-II and p62 western blot as we have done previously (FIG. 30). Expression will be normalized to GAPDH or total protein as a loading control.

[0345] Expected Results and Alternate Strategies

[0346] We have extensive experience growing and differentiating iPSCs, many of the iPSC lines are in hand, and we have already developed the novel compounds. Therefore, we do not anticipate technical issues or experimental delays. Treatment concentrations and durations are based on our previous work with dopaminergic cell lines, so the treatment paradigms may need to be optimized for iPSC-derived dopaminergic neurons. Nevertheless, we expect to observe increased a-syn aggregation, mitochondrial malfunction, and cytotoxicity7in PD iPSC-derived dopaminergic neurons compared to healthy control dopaminergic neurons. Moreover, we anticipate that the SCFA-polyphenol conjugates will suppress a-syn aggregation and associated toxicity more effectively than their unconjugated counterparts. However, because we are using PD iPSCs that harbor different mutations, it is possible that the compounds may have different effects dependent upon the mutation background, which would lead to future mechanistic investigation.

[0347] Aim 2: Determine if Mito-HNK-BAc and Mito-MGN-BAc offer enhanced protection compared with non-targeted conjugates.

[0348] Buty rate conjugated compounds have previously been developed, but mitochondria-targeted prodrugs releasing buty rate have not been reported nor tested in human PD iPSC-derived dopaminergic neurons. We will synthesize mitochondria-targeted conjugates by linking a TPP+ moiety to HNK-BAc and MGN-BAc and measuring the intracellular release of Mito-HNK and Mito-MGN by mass spectrometry7. Using the cellular model outlined in Aim 1, we will then assess a-syn aggregation, mitochondrial function, autophagy, and cell viability7. We anticipate that mitochondria targeted compounds will be more effective at mitigating a-syn associated phenotypes than the non-mitochondria targeted compounds.Attorney Docket: 650053.01246

[0349] Rationale: Although prodrugs or amino acids conjugated to SCFAs have previously been developed, mitochondria-targeted drugs containing esterase cleavable SCFAs with neuroprotective potential have not been reported nor tested in human PD iPSC-derived dopaminergic neurons. We previously reported that mitochondria-targeted compounds improved neuronal function in Mito-Park and LRRK2-R1441G transgenic mice models. We will synthesize mitochondria-targeted conjugates that are designed to accumulate within mitochondria by linking a TPP cation to HNK-BAc and MGN-BAc (using our ortho-Mito-HNK scaffold) to test the impact on a-syn accumulation and related PD pathology in human dopaminergic neurons. Ongoing research in our laboratory suggests that Mito-HNK-BAc is taken up into bacterial cells and hydrolyzed intracellularly more effectively than HNK-BAc (not shown). Therefore, we will test the hypothesis that Mito-polyphenol-butyrate conjugates will exhibit superior efficacy in suppressing a-syn aggregation and toxicity compared with their non-targeted analogs.

[0350] Preliminary Data

[0351] Synthesis and esterase-induced hydrolysis of mitochondria-targeted HNK-BAc: Mito-HNK-BAc was obtained by reacting Mito-HNK with the butyryl chloride in the presence of tri ethylamine in dichloromethane (CH2CI2) (FIG. 31, panel A). Structures and the purity of products were confirmed by NMR, LC-MS and HRMS analyses. Additionally, we found that Mito-HNK-BAc can be hydrolyzed by esterases allowing the formation of Mito-HNK with the corresponding butyric acid (FIG. 31. panels B, C). Importantly, our preliminary data show that HNK-BAc undergoes complete hydrolysis in glioma cells after 1 h of treatment (FIG. 31, panel D).

[0352] Uptake and hydrolysis of HNK-BAc in U87-MG cells: U87-MG cells (3><106 / 10 cm dish) were treated with HNK or HNK-BAc (50 pM, 1 h), washed, pelleted, and snap-frozen. Cell extracts were prepared in DMSO and analyzed by HPLC (Agilent 1200, UV 254 nm). HNK-BAc and hydrolyzed HNK were separated on a Kinetex reverse-phase column using a 10-100% acetonitrile gradient with 0.1% TFA at 1.3 mL / min.

[0353] Experimental Design

[0354] Following differentiation of a-syn. Parkin, LRRK2 G2019S, and healthy control iPSCs into dopaminergic neurons, PD and control neurons will be treated with 10-1000 nM Mito-HNK-BAc or the parent compounds (HNK, but rate, and unconjugated controls) for 1-7 days to test toxicity. We will then select the most appropriate dose for the phenotypic analysis.

[0355] Next, to ensure direct mitochondrial targeting of the Mito-HNK-BAc compounds, we will assess:Attorney Docket: 650053.01246

[0356] 1) Intracellular release kinetics: The intracellular release of honokiol from Mito-HNK-BAc via esterase-mediated hydrolysis will be quantified using LC-MS / MS.

[0357] 2) HD AC measurements: Human iPSC-derived dopaminergic neurons will be treated with polyphenols (HNK, MGN, Mito-HNK, Mito-MGN) and their butyrate conjugates for 6-24 h. The experimental conditions will be similar to those used to determine a-syn aggregation. Nuclear extracts will be prepared and HD AC activity quantified using a fluorometric HDAC activity assay kit using an acetylated lysine substrate linked to a fluorophore, which upon deacetylation induces a fluorescent signal. Parallel treatments with butyrate and polyphenols will serve as positive controls. Cell-permeable pro-butyrates (e.g., serine-butyrate) will serve as an alternate positive control. Changes in HDAC activity after normalization to protein content will be correlated with histone H3 / H4 acetylation (by Western blot). Results from these experiments will provide a mechanistic link between polyphenol and butyrate treatment, HDAC inhibition, and a-syn toxicity'.

[0358] Finally, cells will be analyzed as outlined above for Aim 1 to assess a-syn aggregation, mitochondrial bioenergetics and ROS production, proteosome function, and autophagy. We will compare the results from Mito-HNK-BAc treatment to those collected in Aim 1 to determine whether targeting these compounds to the mitochondria is more effective than treatment with untargeted compounds.

[0359] Expected Results and Alternate Strategies

[0360] We anticipate that Mito-polyphenol-BAc conjugates will more effectively suppress a-syn aggregation and associated toxicity than non-targeted conjugates. Preliminary findings indicate that Mito-HNK-BAc is fully hydrolyzed intracellularly to generate Mito-HNK, while the released butyrate was not detectable by LC-MS, likely due to rapid metabolism;31 to overcome this, we will employ GC-MS and isotopically labeled butyrate to determine its metabolic fate.87,88 If we find that none of the Mito-HNK-BAc compounds reduces PD-associated phenotypes in iPSC-derived dopaminergic neurons, we will pivot to test alternative polyphenol-SCFA conjugates (e.g., propionate), given evidence that microbiome-derived metabolites protect against a-syn-induced neurodegeneration.

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Proc Natl Acad Sci U S A. 2015;112(27):8173-80. doi: 10.1073 / pnas.1509743112. PubMed PMID: 26100898; PMCID: PMC4500273.M. Huang, D. Xiong, J. Pan, Q. Zhang, Y. Wang, C. R. Myers, B. D. Johnson, M. Hardy, B. Kalyanaraman. M. You, Prevention of Tumor Growth and Dissemination by In Situ Vaccination with Mitochondria-Targeted Atovaquone, Advanced Science n / a(n / a) (2022) 2101267.M. Li, B. van Esch, G. T. M. Wagenaar, J. Garssen, G. Folkerts, P. A. J. Henricks, Pro- and antiinflammatory’ effects of short chain fatty acids on immune and endothelial cells, Eur J Pharmacol 831 (2018) 52-59M. D. Carretta, J. Quiroga, R. Lopez. M. A. Hidalgo. R. A. Burgos. Participation of Short-Chain Fatty Acids and Their Receptors in Gut Inflammation and Colon Cancer, Front Physiol 12 (2021) 662739.Maier, S., Reich, E., Martin, R., Bachem, M., Altug, V, Hautmann, R. E., and Gschwend, J. E. 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Mirzaei, A. Afaghi, S. Babakhani, M. R. Sohrabi, S. R. Hosseini-Fard, K. Babolhavaeji, S. Khani Ali Akbari, R. Yousefimashouf, S. Karampoor, Role of microbiota-derived short-chain fatty acids in cancer development and prevention, Biomed Pharmacother 139 (2021) 111619. Reynolds, D. A., Rajendran, V. M., and Binder, H. J. (1993) Bicarbonate-stimulated [14C] butyrate uptake in basolateral membrane vesicles of rat distal colon. Gastroenterology 105, 725-732Attorney Docket: 650053.01246Rocchi, P., Tonelli, R., Camerin, C., Purgato, S., Fronza, R., Bianucci, F., Guerra, F., Pession, A., and Ferreri, A. M. (2005) p21Wafl / Cipl is a common target induced by short-chain fatty acid HD AC inhibitors (valproic acid, tributyrin and sodium butyrate) in neuroblastoma cells. Oncol. Rep. 13, 1139-1144Russo, M., Guida, F., Paparo, L., Trinchese, G., Aitoro, R., Avagliano, C., Fiordelisi, A., Napolitano, F., Mercurio, V.. Sala, V., Li, M., Sorriento, D.. 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[0362] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0363] Clause 1. A compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof,whereinAttorney Docket: 650053.01246one of R1and R2is C(O)RA, the other is C(O)RA, RB, or Mito;RAat each occurrence is independently Ci-2o phenyl, cycloalkyl, Ci-2oalkylene-phenyl. Ci-2oalkylene-cycloalkyl,, amino acid, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein v is 0-20 and Rwis H or C(0)Ci-2oalkyl;RBis H, Ci-2oalkyl. C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl. amino acid, (CH2CH20)k-Ci-2oalkyl, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein k is 1-20;MitoLis Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene;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. or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

[0364] Clause 2. The compound of clause 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (I), whereinR1is -C(O)RA. andR2is -C(O)RA, RB, or Mito.

[0365] Clause 3. The compound of clause 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (I), whereinR1is -C(O)RA, RB. or Mito, andR2is -C(O)RA.Attorney Docket: 650053.01246

[0366] Clause 4. The compound of clause 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (I-a), (I-b), (I-c), (I-d), or (I-e)

[0367] Clause 5. The compound of clause 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (Il-a), (II-b), or (II-c)Attorney Docket: 650053.01246

[0368] Clause 6. The compound of clause 4, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (I-a) or (I-b), RAis Ci-salkyl, and L is C8-15alkylene.

[0369] Clause 7. The compound of clause 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting ofAttorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246

[0370] Clause 8. The compound of clause 6, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting ofAttorney Docket: 650053.01246, and

[0371] Clause 9. A method of modulating microbial metabolism of levodopa in gut of a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof.

[0372] Clause 10. A method of mitigating microbial degradation of levodopa in gut of a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof.

[0373] Clause 11. A method of improving bioavailability of L-dopa in brain of a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof.

[0374] Clause 12. A method of treating Parkinson's disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof.

[0375] Clause 13. The method of any one of clauses 9-12, 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.

[0376] Clause 14. The method of any one of clauses 9-13, whereby microbial metabolism of levodopa to dopamine in the gut of the subj ect is reduced.

[0377] Clause 15. The method of any one of clauses 9-14, whereby alpha-synuclein aggregation in the subject is decreased.Attorney Docket: 650053.01246

[0378] Clause 16. A method of reducing or inhibiting cancer growth in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof.

[0379] Clause 17. The method of clause 16, wherein the method further comprises treating the patient with surgery, radiation therapy (RT), or chemotherapy (CT) prior to or concurrently with administering the pharmaceutical composition.

[0380] Clause 18. A method of inhibiting or reducing metastasis of a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof.

[0381] Clause 19. The method of any one of clauses 16-18, wherein the cancer is melanoma, lung cancer, colon cancer, or pancreatic cancer.

[0382] Clause 20. The method of any one of clauses 9-19, wherein the compound has a structure of formula (I-a), (I-b), (I-c), or (Il-a), or a pharmaceutically acceptable salt thereof.

[0383] Clause 21. A pharmaceutical composition comprising the compound of any one of clauses 1-8, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0384] 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.

Claims

Attorney Docket: 650053.01246CLAIMSWe claim:

1. A compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof,whereinone of R1and R2is C(O)RA. the other is C(O)RA, RD, or Mito;RAat each occurrence is independently Ci-2oalkyl, C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl,, amino acid, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein v is 0-20 and Rwis H or C(0)Ci-2oalkyl;RBis H, Ci-2oalkyl, C2-2oalkenyl, phenyl, cycloalkyl, Ci-2oalkylene-phenyl, Ci-2oalkylene-cycloalkyl, amino acid, (CH2CH20)k-Ci-2oalkyl, or PEG, wherein the phenyl and cycloalkyl are optionally substituted, wherein k is 1 -20;Lis Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;Attorney Docket: 650053.01246q is 1-20:X is a counterion;Y at each occurrence is independently CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, or OH; m at each occurrence is independently 0, 1, 2, 3, 4, or 5.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (I), whereinR1is -C(O)RA. andR2is -C(O)RA, RB, or Mito.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (I), whereinR1is -C(O)RA, RB, or Mito, andR2is -C(O)RA.

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (1-a), (1-b), (1-c), (I-d), or (1-e)Attorney Docket: 650053.012465. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having a structure of formula (II-a), (Il-b), or (II-c)6. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (I-a) or (I-b), RAis Ci-salkyl, and L is C8-15alkylene.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting ofAttorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.01246Attorney Docket: 650053.012468. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting ofAttorney Docket: 650053.01246, and9. A method of modulating microbial metabolism of levodopa in gut of a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

10. A method of mitigating microbial degradation of levodopa in gut of a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

11. A method of improving bioavailability' of L-dopa in brain of a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

12. A method of treating Parkinson’s disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

13. The method of claim 9, 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.

14. The method of claim 9, whereby microbial metabolism of levodopa to dopamine in the gut of the subject is reduced.

15. The method of claim 9, whereby alpha-synuclein aggregation in the subject is decreased.

16. A method of reducing or inhibiting cancer grow th in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.Attorney Docket: 650053.0124617. The method of claim 16, wherein the method further comprises treating the patient with surgery, radiation therapy (RT). or chemotherapy (CT) prior to or concurrently with administering the pharmaceutical composition.

18. A method of inhibiting or reducing metastasis of a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

19. The method of claim 16, wherein the cancer is melanoma, lung cancer, colon cancer, or pancreatic cancer.

20. The method of claim 9, wherein the compound has a structure of formula (1-a), (1-b), (I-c), or (Il-a), or a pharmaceutically acceptable salt thereof.

21. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

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