Methods and compositions for activation of tubulin tyrosination

Abstract

Disclosed are compounds, compositions, and methods for activating tubulin tyrosination. Compositions include a resveratrol, such as cis-resveratrol, or a compound of Formula I or Formula II. Compounds of Formula I and Formula II have the following structures where the variables, e.g., Y1, Y2, Y3, R1, R2, R3 the A-ring, and W are defined herein. Said compositions may be utilized in treating disorders that affects tubulin tyrosination such as aging and age-associated neurocognitive and metabolic disorders including ASDs, CVDs, and cancer.

Description

Attorney Docket No.: WKM0004PCT (1696) METHODS AND COMPOSITIONS FOR ACTIVATION OF TUBULIN TYROSINATION GOVERNMENT SUPPORT CLAUSE

[0001] This invention was made with government support under grant P20 GM109091 awarded by National Institutes of Health (NIH). The government has certain rights in the invention. CROSS REFERENCE TO RELATED APPLICATION

[0002] This application claims priority from U.S. Provisional Application No.63 / 572,547, filed April 1, 2024, which is hereby incorporated by reference in its entirety. BACKGROUND

[0003] Tyrosine (Tyr) and phenylalanine (Phe) are not only important for protein synthesis and cellular energy production, but also act as substrates for tubulin tyrosine ligase (TTL), an enzyme that adds tyrosine or phenylalanine to the C-terminal glutamic residue of α-tubulin (Tyr / Phe- tubulin). In contrast, the vasohibin / small vasohibin binding protein complexes (VASH1 / 2-SVBP) remove the C-terminal tyrosine / phenylalanine from the α -tubulin. TTL-dependent tubulin tyrosination levels are decreased during aging and age-associated neurocognitive and degenerative disorders. Also, serum tyrosine and phenylalanine levels are increased in said disorders. Interestingly, Alzheimer's disease (AD)-affected brain regions have been shown to have decreased tyrosyl-tRNA synthetase (TyrRS / YARS1) protein levels, and tyrosine / phenylalanine inhibits protein synthesis at the elongation step to deplete neuronal TyrRS. Therefore, strategies to activate tubulin tyrosination would help to decrease serum tyrosine / phenylalanine levels. Progressive age- associated neurocognitive diseases, such as AD, remain incurable. The amyloid precursor protein (APP) and its proteolytic amyloid beta (Aβ) peptides, the precursors to the amyloid plaques, deposited in the brains of AD patients, are genetically and biochemically linked to AD and other neurocognitive diseases. Aβ peptides are generated by the cleavage of amyloid precursor protein at the plasma membrane by the consecutive proteolytic actions of β-site APP-cleaving enzyme-1 (BACE- 1) and presenilin (PS1 / PS2), the catalytic subunit of γ- secretase. Major forms of Aβ consist of 40 or 42 amino acids (Aβ40 and Aβ42), which are produced at picomolar concentrations in healthy subjects and exhibit age-dependent increase in plasma Aβ42 levels. Since Aβ42 is more prone to aggregation, overproduction of Aβ42 or reduced brain Aβ42 clearance may trigger the development of AD. Consistently, AD brains accumulate Aβ, which eventually forms amyloid plaques and positively correlate with all-cause dementia. Previous studies found that higher CSF Aβ42 levels positively correlate with cognition and anti-inflammatory effects, emphasizing a critical role of Aβ not only in cognition and memory but also in inflammatory response in the CNS.Attorney Docket No.: WKM0004PCT (1696) Moreover, the development of Aβ plaques decreases Aβ42 / 40 ratio, suggesting that Aβ42 and Aβ40 may have distinct functions in memory formation and loss of Aβ42 function may contribute to the etiology of AD.

[0004] Consistent with observations that picomolar Aβ is a product of neuronal activity that stimulates synaptic plasticity and memory in mice, neuronal protein synthesis is also regulated in an activity-dependent manner and de novo protein synthesis is required for long-term memory formation. However, AD brains exhibit decreased protein synthesis along with depletion of TyrRS that activates tyrosine for protein synthesis in the AD-affected brain regions, suggesting that physiological Aβ may positively regulate neuronal protein synthesis.

[0005] As such, a need exists for compositions and methods to activate neuronal tubulin tyrosination, which may restore the function of crucial neuroprotective factor associated with cognitive performance and memory formation in AD brains and other neurocognitive disorders. In particular, a need exists for compositions and methods for activating tubulin tyrosination and treating aging and age-associated neurocognitive and metabolic disorders. SUMMARY

[0006] In general, the present disclosure is directed compositions and methods for activating tubulin tyrosination. Compositions disclosed herein may include a resveratrol derivative or its conjugates with amino acids derivatives or a pharmaceutically acceptable salt thereof. Said compositions may be utilized in treating neurodegradation that leads to constitutive aging and age- associated neurocognitive and metabolic disorders. The resveratrol derivatives or analogues include compounds of Formula I and Formula II and the pharmaceutically acceptable salts of compounds of Formula I and Formula II.

[0007] The disclosure includes a method of activating tubulin tyrosination in a cell, the method comprising contacting the cell with an effective amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof. The cell may either be in vitro or in vivo.

[0008] The disclosure also includes a method of treating a disorder modulated by tubulin tyrosination in a subject (such as a human patient), the method comprising administering to the subject a therapeutically effective amount a compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof.

[0009] Formula I isAttorney Docket No.: WKM0004PCT (1696) . The bond indicates the compound of Formula I can be in or a mixture of cis and trans conformations.

[0010] Within Formula I, the variables, e.g. Y1, Y2, and Y3have the following definitions.

[0011] Each of Y1, Y2, and Y3is independently selected from a H or a group .is independently chosen at each occurrence from the 20 native amino acid side chains where any hydroxyl group in the amino acid side chain is optionally acylated with a C2-C6acyl group and when R is a proline the proline pyrrolidine ring is formed by R and RAbeing joined by a -CH2CH2CH2- chain; and

[0013] RAand RBare independently chosen at each occurrence from H, C2-C6acyl, C1-C6alkyl, (C3-C6cycloalkyl)C0-C4alkyl, phenyl, and benzyl.

[0014] Formula II is .is a double or single bond;

[0016] The A ring ( ) is phenyl, C3-C6cycloalkyl, or a 4- to 6-membered heterocyclicgroup.

[0017] .

[0018] selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, (C3-C6cycloalkyl)C0-C2alkyl, C1-C2haloalkyl, and C1- C2haloalkoxy.Attorney Docket No.: WKM0004PCT (1696)

[0019] R2is absent or is 1 to 5 substituents independently selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, (C3-C6cycloalkyl)C0-C2alkyl, C1-C2haloalkyl, and C1- C2haloalkoxy.

[0020] R3is absent or is 1 to 4 substituents independently selected from halogen, C1-C2alkyl, C1-C2alkoxy, cyclopropyl, C1-C2haloalkyl, and C1-C2haloalkoxy.

[0021] Y is NH or O.

[0022] Z is H, hydroxyl, methyl, proline where the proline pyrrolidine ring is formed by Z and the nearest NH being joined by a -CH2CH2CH2- chain, or Z is a group -CH2X.

[0023] X is an amino acid side chain selected from H, -CH3, -CH2SH, -CH2CO2H, -CH2OH, -CH(CH3)OH, -CH2CH2CO2H, -CH2CH2CONH2, ,

[0024] Other features and aspects of the present disclosure are discussed in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, in which:

[0026] FIG.1 presents illustration of the role of tubulin tyrosination and detyrosination cycle in modulating cellular tyrosine and phenylalanine levels.

[0027] FIG.2 presents quantitative IF analysis of tyrosinated tubulin (Tyr-Tub) levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with tyrosine (500 mM) for 4 hr.

[0028] FIG.3 presents quantitative IF analysis of tyrosinated tubulin (Tyr-Tub) levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with phenylalanine (500 mM) for 4 hr.

[0029] FIG.4 presents quantitative IF analysis of tyrosinated tubulin (Tyr-Tub) (left) and TyrRS (right) levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub and anti-TyrRS antibodies after treatment with EpoY.Attorney Docket No.: WKM0004PCT (1696)

[0030] FIG.5A presents quantitative IF analysis of tyrosinated tubulin (Tyr-Tub) levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with thiorphan (100 nM) for 2 hr.

[0031] FIG.5B presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with verubecestat (50 nM) for 4 hr.

[0032] FIG.5C presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with Aβ40 and Aβ42 (50 pM) for 4 hr.

[0033] FIG.5D presents quantitative IF analysis of Tyr- Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with Aβ42 (50 pM) for 1 hr and 4 hr.

[0034] FIG.5E presents representative images of western blot (WB) of tubulin tyrosination and detyrosination levels determined in a quantitative WB analysis using anti-Tyr-Tub and anti-deTyr- Tub antibodies after treatment with increasing concentrations of Aβ40 and Aβ42 (10-50 pM) for 4 hr

[0035] FIG.6 presents quantitative IF analysis of Tyr- Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with Aβ40 (50 pM) for 1 hr and 4 hr.

[0036] FIG.7 presents quantitative IF analysis of Tyr-Tub and TyrRS levels in the rat cortical neurons (DIV 9 / 10) using anti-Tyr-Tub and anti-TyrRS antibodies after treatment with verubecestat (50 nM) for 4 hr.

[0037] FIG.8A presents quantitative IF analysis of TTL levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-TTL antibody after treatment with Aβ40 and Aβ42 (50 pM) for 4 hr.

[0038] FIG.8B presents quantitative IF analysis of TTL levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-TTL antibody after treatment with tyrosine (500 µM) for 4 hr.

[0039] FIG.8C presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with Aβ42 (50 pM) alone or in combination with tyrosine (500 µM) for 4 hr.

[0040] FIG.8D presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr- Tub antibody after treatment with Aβ40 (50 pM) alone or in combination with tyrosine (500 µM) for 4 hr.Attorney Docket No.: WKM0004PCT (1696)

[0041] FIG.9 presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of cortical neurons (DIV 9 / 10) using anti-Tyr-Tub antibody after treatment with Aβ42 (50 pM) alone or in combination with phenylalanine (500 µM) for 4 hr.

[0042] FIG.10 presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr- Tub antibody after treatment with Aβ40 (50 pM) alone or in combination with phenylalanine (500 µM) for 4 hr

[0043] FIG.11 presents quantitative IF analysis of Tyr-Tub levels in the soma and neurites of rat cortical neurons (DIV 9 / 10) using anti-Tyr- Tub antibody after treatment with ISRIB (50 nM) for 4 hr

[0044] FIG.12A presents quantitative IF analysis using anti-Tyr-Tub antibody after treatment with cis-RSV (50 µM) for 4 hr.

[0045] FIG.12B presents representative WB images of TTL levels. Primary cortical neurons (DIV 9 / 10) were treated with cis- and trans-RSV (5-50 µM) for 8 hr and the changes in the TTL levels were determined by WB using anti-TTL antibody.

[0046] FIG.12C presents representative WB images delta2-Tub and Tyr-Tub levels. Primary cortical neurons (DIV 9 / 10) were treated with cis- and trans-RSV (50 µM) for up to 16 hr and the changes in the levels of Tyr-Tub and delta2-Tub were determined by WB using their respective antibodies.

[0047] FIG.12D presents primary rat cortical neurons that were treated with paclitaxel (10 nM) for up to 4 hr and the changes in the TyrRS levels were determined by WB using anti- TyrRS antibody.

[0048] FIG.12E presents primary rat cortical neurons that were treated with parthenolide (10 µM) for up to 4 hr and the changes in the TyrRS levels were determined by WB using anti-TyrRS antibody.

[0049] FIG.13A presents IF analysis using anti-Tyr-Tub antibody after treatment with nMAb42 (50 nM) for 4 hr.

[0050] FIG.13B presents quantitative IF analysis of nucleotide incorporation in a DNA fiber after treatment with nMAb42 (50 nM) for 24 hr.

[0051] FIG.13C presents quantitative analysis of rat cortical neurons after treatment with either cis- and trans-RSV (50 mM) alone or in combination with nMAb42 (50 nM) for 24 hr. Neurite degeneration is quantified based on MAP2 staining.

[0052] FIG 14. The effect of compound II-8 (TTC-369) on TyrRS (tyrosyl-tRNA synthetase), PheRSβ (phenylalanyl-tRNA synthetase beta), and PheRSα (phenylalanyl-tRNA synthetase alpha) levels in primary rat coritical neurons. FIG.14A. Rat cortical neurons were treated with compound II-8 at concentrations of 0.1 nM 1.0 nM for 2 hours and then detecting TyrRS, PheRSβ,Attorney Docket No.: WKM0004PCT (1696) and PheRSα in the rat cortical neurons via Western blot using anti-TyrRS and anti-PheRS antibodies. FIG.14B. TyrRS and PheRSβ levels as a function of increasing compound II-8 concentration. TyrRS and PheRSβ levels were increased in rat cortical neurons treated with II-8 at all tested concentrations between 0.1 nM to 1.0 nM relative to TyrRS and PheRSβ levels in untreated neurons.

[0053] FIG.15. The effect of compound II-8 on PARylation in primary rat cortical neurons. FIG.15A. Rat cortical neurons were treated with II-8 at concentrations from 0.25 to 2.5 nM for 15 minutes and then quantified PARylation levels in untreated and II-8 treated neurons via western blot analysis using anti-PAR antibody. FIG.15B. II-8 treatment increased PARylation in rat cortical neurons relative to untreated rat cortical neurons at all tested concentrations. The maximum effect was at 1 nM.

[0054] FIG.16. II-8 protects against tyrosine, NMDA, and etoposide (ETO)-induced DNA damage accumulation and neurotoxicity in rat cortical neurons. FIG.16A. Rat cortical neurons were treated with 1mM tyrosine either alone or in combination with II-8 (50 nM) for 16 hr and the quantified the changes in γ-H2AX foci levels (a marker of DNA damage) using immunofluorescent (IF) analysis. The number of γ-H2AX foci decreased slightly relative to the untreated group in response to II-8 treatment alone. The number of γ-H2AX foci increased approximately 3-fold relative to untreated in response to Tyrosine treatment but was similar to the untreated group when neurons were treated with both tyrosine and II-8. FIG.16B. Quantification of number of γ-H2AX foci in II-8, Tyrosine only, and Tyrosine + II-8 treated groups.

[0055] FIG.17. FIG.17A. Rat cortical neurons treated with NMDA alone and II-8 alone and in combination at II-8 concentrations of 100 nM and 250 nM for 24 hours to assess the effect of II- 8 on NMDA damage. The MTT assay was used to quantify neuronal viability. FIG.17B. To evaluate the effect of II-8 on etoposide mediated damage, rat cortical neurons were treated with ETO alone and II-8 alone and in combination at II-8 concentrations of 0.1 to 1 nM for 24 hours, again using the MTT assay to quantify neuronal viability. Treatment with NMDA alone or etoposide alone decreased neuronal viability. Treatment with II-8 in combination with NMDA or etoposide restored viability in a concentration dependent manner.

[0056] FIG.18. II-8 protects against nanomolar amyloid beta 42 (Aβ42)-mediated neurite degeneration. Rat cortical neurons with either 50 nM Aβ42 alone (FIG.18A) or in combination with 50 nM II-8 (FIG.18B) for 24 hours. Quantification via the neurite degeneration index showed that treatment II-8 protects neurons from Aβ42 induced neurite degeneration.

[0057] FIG.19. II-8 mimics cis-RSV in protecting against nanomolar amyloid beta 42 (Aβ42)- mediated neurotoxicity. Rat cortical neurons were treated with 50 nM Aβ42 or in combination withAttorney Docket No.: WKM0004PCT (1696) cis-RSV or trans-RSV or II-8 (100 pM, supplemented every 3rdday) for 25 days and the neuronal viability was quantified using MTT assay.

[0058] FIG.20. II-8 protects against tyrosine-mediated neuronal DNA repair. Primary cortical neurons (DIV 9 / 10) were treated with II-8 (1 nM) either alone or in combination with tyrosine (1 mM) for 16 hr and the percentages of DNA in the comet tail were quantified

[0059] FIG.21. II-8 protects against tyrosine-mediated nucleotide incorporation in cortical neurons. Rat cortical neurons (DIV 9 / 10) were treated with CldU (100 µM) either alone or in combination with tyrosine (1 mM) or II-8 (1 nM) or their combination for 4 hr and nucleotide incorporation was determined using DNA fiber assay after staining with anti- CidU antibody using immunofluorescence (IF).

[0060] Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention. DETAILED DESCRIPTION TERMINOLOGY

[0103] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary.

[0104] Unless specifically stated, terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and / or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and / or” unless expressly stated otherwise.

[0105] Furthermore, although items, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. The presence of broadening words and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

[0106] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0107] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those withinAttorney Docket No.: WKM0004PCT (1696) experimental error (which can be determined by e.g. given data set, art accepted standard, and / or with e.g. a given confidence interval (e.g.90%, 95%, or more confidence interval from the mean), such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosure. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0109] As will be apparent to those of skill in the art reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0110] Where a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to anyAttorney Docket No.: WKM0004PCT (1696) specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0111] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0112] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub- ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0113] Formula I includes all subformulae and compounds that fall within the scope of Formula I and Formula II includes all subformulae and compounds that fall within the scope of Formula II.

[0114] In certain situations, the compounds of Formula I or Formula II may contain one or more asymmetric elements such as stereogenic centers, stereogenic axes and the like, e.g. asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.Attorney Docket No.: WKM0004PCT (1696) Unless stated otherwise or clearly indicated from the context, Formula I and Formula II include all stereoisomeric forms, including racemates, optically enriched, and optically pure forms. In addition, compounds with carbon-carbon double bonds may occur in Z- and E-forms, with all isomeric forms of the compounds being included in the present disclosure. In these situations, the single enantiomers, i.e., optically active forms can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.

[0115] Formula I and Formula II includes all isotopically enriched versions of compounds of Formula I, Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include11C,13C, and14C, isotopes of fluorine include19F, isotopes of nitrogen include15N, and isotopes of oxygen include17O. Isotopically enriched versions of compounds of Formula I and Formula II, include, e.g., compounds in which one or more C in a compound of Formula I or Formula II is13C enriched, in which one or more hydrogen position in a compound of Formula I or Formula II is deuterated or tritiated, in which one or more N in a compound of Formula I or Formula II is15N enriched.

[0116] Certain compounds are described herein using a general formula that includes variables, e.g., R1, R2, R3, Y1, Y2, Y3, RA, RB, R, W, X, Y, and Z. Unless otherwise specified, each variable within such a formula is defined independently of other variables. Thus, if a group is said to be substituted, e.g., with 0-2 R*, then said group may be substituted with up to two R* groups and R* at each occurrence is selected independently from the definition of R*.

[0117] Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent.

[0118] The term “substituted” means that any one or more hydrogen atoms bound to the designated atom or group is replaced with a selection from the indicated group, provided that the designated atom's normal valence is not exceeded. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation from a reaction mixture, and subsequent formulation into an effective therapeutic agent. Unless otherwise specified substituents are named into the core structure.Attorney Docket No.: WKM0004PCT (1696)

[0119] Substituents are named into the ring unless otherwise indicated. A dash ("-") or a double bond (“=”) that is not between two letters or symbols indicates the point of attachment for a substituent. For example, -CONH2is attached through the carbon atom.

[0120] The term "amino acid" as used herein refers to native amino acids, non-native amino acids, and amino acid analogs. Native amino acids include, for instance, the 20 (L)-amino acids commonly utilized during protein biosynthesis. Non-native amino acids include the 20 (D)-amino acids not utilized for protein biosynthesis, selenocysteine, and other plant-derived non-proteogenic amino acids such as pyrrolysine. Non-native amino acids include, for instance, selenocysteine, homoserine, ornithine, canvanine, N-methyl-L-alanine, L-DOPA (3,4-dihydroxyphenylalanine), Dap, and Dap. Amino acid analogs may include modified forms of naturally or non-naturally occurring amino acids, for instance, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Pomaglumetad (LY-404,039) is an amino acid analog.

[0121] “Alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms. Thus, the term C1- C6alkyl includes alkyl groups having from 1 to about 6 carbon atoms. When C0-Cnalkyl is used herein in conjunction with another group, for example, (cycloalkyl)C0-C2alkyl, the indicated group, in this case cycloalkyl, is either directly bound by a single covalent bond (C0), or attached by an alkyl chain having the specified number of carbon atoms, in this case from 1 to about 2 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and sec- pentyl. C1-C4alkyl includes alkyl groups having 1, 2, 3, or 4 carbon atoms.

[0122] “Alkoxy” is an alkyl group as defined above with the indicated number of carbon atoms attached to the group it substitutes through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2- pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3- methylpentoxy. Similarly, an “Alkylthio” or a “thioalkyl” group is an alkyl group as defined above with the indicated number of carbon atoms covalently bound to the group it substitutes by a sulfur bridge (-S-).

[0123] “Acyl” is a group of the formula alkylC(=O)-, where alkyl has the definition given above. A C2-C6acyl group has an alkyl group of 1 to 5 carbon covalently bound to a keto (C=O) group which is in turn covalently bound to the group it substitutes.

[0124] “Halo” or “halogen” as used herein is fluoro, chloro, bromo, or iodo.

[0125] “Haloalkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups, having the specified number of carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms. Thus, the term C1- C6haloalkylAttorney Docket No.: WKM0004PCT (1696) includes haloalkyl groups having from 1 to about 6 carbon atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, chloromethyl, chloroethyl, and penta-fluoroethyl. C1-C2alkyl includes alkyl groups having 1or 2 carbon atoms, substituted with 1 or more halogen atoms, generally up to the maximum allowable number of halogen atoms.

[0126] “Haloalkoxy” is an haloalkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of haloalkoxy include, but are not limited to, chloromethoxy, chloroethoxy, bromo-n-propoxy, bromo-i-propoxy, iodo-n-butoxy, iodo-2-butoxy, or chloro-n-pentoxy.

[0127] A “heterocyclic group” is a monocyclic saturated, partially unsaturated, or aromatic ring containing from 1 to 4 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a bicyclic saturated, partially unsaturated, or aromatic heterocycle containing at least 1 heteroatom chosen from N, O, and S in one of the two rings of the two ring system and containing up to about 4 heteroatoms independently chosen from N, O, and S in each ring of the two ring system. The rings of a bicyclic heterocyclic group can be in fused, bridged, pendant, or spiro orientation. Usually, each ring of the heterocycle contains from 4-6 ring atoms but some other number of ring atoms may be specified. Unless otherwise indicated, the heterocycle may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. When indicated the heterocycles described herein may be substituted on carbon, sulfur, or nitrogen atom if the resulting compound is stable. It is preferred that the total number of heteroatoms in a heterocycle is not more than 4 and that the total number of S and O atoms in a heterocycle is not more than 2, more preferably not more than 1. Examples of heterocycles include, pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benz[b]thiophenyl, isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, dihydroisoindolyl, 5,6,7,8-tetrahydroisoquinoline, pyrazolyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl. In certain embodiments a heterocycle is chosen from pyridinyl, pyrimidinyl, furanyl, thienyl, and pyrrolyl.

[0128] Additional examples of heterocycles include, but are not limited to, phthalazinyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzoisoxolyl, dihydro-benzodioxinyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl, imidazopyridinyl, isothiazolyl, naphthyridinyl, cinnolinyl, carbazolyl, beta-carbolinyl, isochromanyl, chromanonyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl, phenothiazinyl, 5 pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyranyl,Attorney Docket No.: WKM0004PCT (1696) benzothiopyranyl, coumarinyl, isocoumarinyl, chromanyl, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide, pyrirnidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N oxide, isoquinolyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N- oxide, thiazolyl N-oxide, indolizinyl N oxide, indazolyl N-oxide, benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide, thiadiazolyl N-oxide, tetrazolyl N- oxide, benzothiopyranyl S-oxide, and benzothiopyranyl S,S-dioxide.

[0129] “Heteroaryl” is a stable monocyclic aromatic ring having the indicated number of ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered aromatic ring which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms. In some embodiments bicyclic heteroaryl groups are 9- to 10-membered heteroaryl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to 7-member aromatic ring is fused to a second aromatic or non-aromatic ring. When the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. It is particularly preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include, but are not limited to, oxazolyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzo[d]oxazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl, dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, and isoxazolyl.

[0130] “Heterocycloalkyl” is a stable monocyclic ring having the indicated number of ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered cyclic ring which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heterocycloalkyl groups typically have from 5 to 7 ring atoms. The stable monocyclic heterocycloalkyl may have 3-10 ring atoms which contains from 1 to 3, or in some embodiments from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. In some embodiments bicyclic heterocycloalkyl groups are 9- to 10-membered heterocycloalkyl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to 7-memberAttorney Docket No.: WKM0004PCT (1696) cyclic ring is fused to a second aromatic or non-aromatic ring. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. Examples of heterocycloalkyl groups include, but are not limited to, oxiranyl, oxetanyl, tetrahydrofuranyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, or piperazinyl.

[0131] The term "administering" includes all modes and routes of administration which allow a compound to perform its intended function. In general, the compounds disclosed herein may be administered to a subject according to known methods, including injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The compound may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The compound also may be administered as a prodrug, which is converted to its active form in vivo.

[0132] “Pharmaceutical compositions” are compositions comprising at least one active agent, such as a compound, salt, or hydrate of a named Formulae of the disclosure (Formula I or II) and at least one other excipient. “Excipients” are any materials of a pharmaceutical composition other than the active agent or agents. Excipients include carriers and diluents, which may be added to the pharmaceutical compositions. Pharmaceutical compositions meet the U.S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs.

[0133] “Pharmaceutically acceptable salt” includes derivatives of the disclosed compounds wherein the parent compound is modified by making non-toxic acid or base salts thereof, and further refers to pharmaceutically acceptable hydrates or solvates of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non- toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxylmaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n- COOH where n is 0-4, and the like. Lists of additional suitable salts may be found, e.g., in G. Steffen Paulekuhn, et al., Journal of Medicinal Chemistry 2007, 50, 6665 and Handbook ofAttorney Docket No.: WKM0004PCT (1696) Pharmaceutically Acceptable Salts: Properties, Selection and Use, P. Heinrich Stahl and Camille G. Wermuth Editors, Wiley-VCH, 2002.

[0134] A “patient” is a human or non-human animal in need of medical treatment. Medical treatment can include treatment of an existing condition, such as a disease or disorder or diagnostic treatment. In some embodiments the patient is a human patient.

[0135] “Treatment” or “treating” means providing an active compound to a patient in an amount sufficient to measurably reduce any existing condition or slow existing condition progression.

[0136] A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s T-test, where p < 0.05.

[0137] The term "therapeutically effective amount" of a compound of the disclosure, means an amount effective, when administered to a patient, to provide a therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the symptoms of a neurocognitive or neurodegenerative disorder, and including an amount sufficient to reduce or inhibit DNA repair or an amount effect to decrease neuronal tyrosine levels. Thus, a therapeutically effective amount of a compound is also an amount sufficient to significantly reduce the indicia of the disease or condition being treated. A significant reduction is any detectable negative change that is statistically significant in a standard parametric test of statistical significance, such as Student’s t-test, in which p < 0.05. CHEMICAL DESCRIPTION

[0138] Reference will now be made in detail to various embodiments of the presently disclosed subject matter, one or more examples of which are set forth below. Each embodiment is provided by way of explanation, not limitation, of the subject matter. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment. Thus, it is intended that the present disclosure cover such modifications and variations as come within the scope of the appended claims and their equivalents.

[0139] In general, the present disclosure is directed to compositions and methods for activating tubulin tyrosination. Compositions disclosed herein may be utilized in treating aging and age- associated neurocognitive and metabolic disorders. In particular, compositions disclosed herein are neuroprotective against tyrosine-mediated neurodegeneration. Also, advantageously, compositions disclosed herein are neuroprotective against tyrosine-mediated tyrosyl-tRNA synthetase (TyrRS) depletion.

[0140] Compositions disclosed herein may include a DNA repair inhibitor, such as resveratrolAttorney Docket No.: WKM0004PCT (1696) or a derivative thereof. For instance, the composition may include a resveratrol analogue of Formula I: , or a wherein the variables, e.g., Y1, Y2, and Y3carry the definitions

[0141] The bond, , indicates the compound of Formula I can be in either the cis or trans conformation or a mixture of cis and trans conformations. The cis and trans conformations are O Y1

[0142] In certain embodiments the compound of Formula I, or salt thereof, is in the cis conformation.

[0143] The variables, Y1, Y2, and Y3, can each independently have any value set forth in the claims or specification so long as a stable compound results. In certain embodiments the variables, Y1, Y2, and Y3, can have any of the following definitions.

[0144] In an embodiment Y1, Y2, and Y3are and RAand RBare both H and R isindependently chosen at each occurrence from acid side chains where any hydroxyl group in the amino acid side chain is optionally acylated with a C2-C6acyl group and when R is a proline the proline pyrrolidine ring is formed by R and RAbeing joined by a - CH2CH2CH2- chain. The dash (-) indicated the point of attachment of the group to one of the 3 oxygen atoms shown in Formula I.

[0145] In an embodiment Y1is , and Y2and Y3are each hydrogen or methyl and RAand RBare both hydrogen.

[0146] In some embodiments R is independently chosen at each occurrence from the amino acid side chain of tyrosine optionally substituted at the tyrosine hydroxyl with Ac, a C2-C6acylAttorney Docket No.: WKM0004PCT (1696) group, phenylalanine, leucine, isoleucine, and valine.

[0147] In some embodiments R is independently chosen at each occurrence from the amino acid side chain of tyrosine optionally substituted at the tyrosine hydroxyl with Ac, a C2-C6acyl group, phenylalanine, leucine, isoleucine, and valine.

[0148] Or, R can be the side chain of phenylalanine.

[0149] Formula I includes the following subformulae:

[0150] This disclosure also includes the following subformulae of Formula I:Attorney Docket No.: WKM0004PCT (1696) O O f);I and the pharmaceutically acceptable salts thereof:a tyrosine-mediated DNA repair inhibitor, such compounds of Formula II and the pharmaceutically acceptable salt thereof.includes compounds of the following subformulae of Formula II and the pharmaceutically acceptable salts thereof. b)Attorney Docket No.: WKM0004PCT (1696) . X, Y, Z, and the A-ring, can have the definitions set forthdefinitions set forth in this specification. Any of the variable definitions can be combined so long as a stable compound results. (i) R1and R2are each absent or are 1 to 2 substituents independently selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, C1-C2haloalkyl, and C1-C2haloalkoxy. (ii) R3is absent. (iii) Y is NH. (iv) Y is O. (v) X is H, -CH2OH, or -CH(CH3)OH. (vi) The bond is a double bond. (vii) The bond is a single bond.

[0155] This disclosure includes the following exemplified compound of Formula II and the pharmaceutically acceptable salts thereof. );Attorney Docket No.: WKM0004PCT (1696) [01y: ; .stereochemistry: .R may be an amino acid side chain and in Formula II the variable X may be an amino acid chain. The amino acid side chain represented by variable R or X may be from an acidic amino acid. For example, the amino acid residue has a negative charge due to loss of hydrogen ion at physiological pH. Amino acids having an acidic side chain may include glutamic acid and aspartic acid.

[0159] In one embodiment, the amino acid side chain may be from an acidic amino acid. For example, the amino acid residue has a negative charge due to loss of hydrogen ion at physiological pH. Amino acids having an acidic side chain may include glutamic acid and aspartic acid.

[0160] The amino acid side chain may be from a basic amino acid residue. For example, the residue may have a positive charge due to association with hydrogen ions at physiological pH or within one or two pH units thereof. Amino acids having a basic side chain include arginine, lysine, and histidine.Attorney Docket No.: WKM0004PCT (1696)

[0161] The amino acid side chain may be from a hydrophobic amino acid residue. For example, the residue is not charged at physiological pH. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine, and tryptophan.

[0162] The amino acid side chain may be from a neutral / polar amino acid residue. For instance, the residue is not charged at physiological pH and the residue is not sufficiently repelled by aqueous solution so that it would seek inner positions in the conformation of a peptide or protein in which it is contained when the peptide is in an aqueous medium. Amino acids having a neutral / polar side chain include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0163] The compounds of Formula I and Formula II are resveratrol analogues. Resveratrol has two stereoisomers shown below.derivative can have opposing effects on tyrosine-mediated neurotoxic effects. For instance, phenylalanine, tyrosine, and trans- RSV and derivatives thereof can inhibit tubulin tyrosination. Unexpectedly, cis-RVS derivatives that increase neuronal TyrRS protein levels stimulate neuronal tubulin tyrosination to protect against tyrosine-mediated neurodegeneration. As such, cis-resveratrol and tyrosine or other amino acids that would act as tyrosine antagonists or their derivatives may serve as a neuroprotectant against tyrosine-induced neurodegeneration by activating tubulin tyrosination. Similarly, cis- resveratrol and tyrosine or other amino acids that would act as tyrosine antagonists or their derivatives may also serve as activator of tubulin tyrosination to protect against phenylalanine- induced inflammatory response.

[0165] Compositions disclosed herein may be cis-resveratrol analogues. Resveratrol analogues may be synthesized through a scheme in which D / L amino acids or their amino group derivatives connected to cis / trans-resveratrol through an ester bond such that they will be metabolized in vivo or in vitro in the cell to release / regenerate cis-resveratrol and D / L amino acids or their amino group derivatives from the administered parent compound.

[0166] In a common embodiment, the cis / trans-resveratrol analogues of Formula I may be one of the following design:Attorney Docket No.: WKM0004PCT (1696) METHODS

[0167] in a subject (e.g. a patient) comprising administering an effective amount of a compound or salt thereof of Formula I or II to the subject. The disclosure further includes a method of treating a disease or disorder associated with tubulin tyrosination in a subject comprising administering an effective amount of a compound or salt thereof of Formula I or II to the subject. A patient can be a human patient or non- human patient such as a companion animal or livestock animal. The disclosure includes use of a compound or salt thereof of Formula I or II for inhibiting tubulin tyrosination in a subject (e.g. a patient). The disclosure includes use of a compound or salt thereof of Formula I or II for treating a disease or disorder associated with tubulin tyrosination in a subject (e.g. a patient). The disclosure includes methods of manufacturing a medicament of Formula I or II, or salt thereof, for use in inhibiting tubulin tyrosination in a subject (e.g. a patient) or for use in treating a disease or disorder associated with tubulin tyrosination in a subject.

[0168] In one embodiment, compositions disclosed herein may be utilized in methods for activating tubulin tyrosination. Methods for activating tubulin tyrosination may include administration of the composition disclosed herein in vitro or ex vivo (e.g., by contacting the cell with such compounds) or, alternatively, in vivo (e.g., administering the compound to a subject). Activation of tubulin tyrosination may be measured by an increase in nuclear TyrRS levels in a cell. For instance, TyrRS levels in a cell may be increased relative to a control amount by about 10% or more, such as about 20% or more, such as about 30% or more, such as about 40% or more, such as about 50% or more, or such as about 60% or more.

[0169] When tubulin tyrosination is inactive, neuronal tyrosine levels may be elevated which can inhibit protein synthesis at the elongation step to deplete neuronal TyrRS. In one embodiment, activation of tubulin tyrosination may be measured by a decrease in neuronal tyrosine levels. As such, following administration of the composition disclosed herein, tyrosine levels in a cell may be reduced relative to a control amount of about 10% or more, such as about 20% or more, such as about 30% or more, such as about 40% or more, such as about 50% or more, or such as about 60%Attorney Docket No.: WKM0004PCT (1696) or more.

[0170] Introduction of a compound or composition (e.g. a compound or salt disclosed herein such as a compound of Formula I or Formula II, or a salt thereof) into a cell may trigger degradation of one or more neuroinflammatory factors. For instance, the neuroinflammatory factors may include, but are not limited to, poly-ADP-ribose polymerase 1 (PARP1), signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB), topoisomerase 1 (TOP1), or a combination thereof. As such, following administration of a composition disclosed herein, levels of neuroinflammatory factors in a cell may be reduced relative to a control amount by about 10% or more, such as about 20% or more, such as about 30% or more, such as about 40% or more, such as about 50% or more, or such as about 60% or more.

[0171] The present disclosure provides methods for treating and / or preventing a disorder in a subject via administering a compound of Formula I or Formula II to the subject. In one embodiment, the disorder may be mediated by increased tyrosine levels. In another embodiment, the disorder may be aging.

[0172] In one embodiment, the disorder may be an age-associated neurocognitive disorder. The age-associated neurocognitive disorder may include, but is not limited to, insomnia, hypersomnia, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, epilepsy and seizures, learning disabilities, neuromuscular disorders, Cockayne syndrome, cerebral palsy, dystonia, spinocerebellar ataxia with axonal neuropathy-1 (SCAN1), Angelman Syndrome, COVID-19-related neurocognitive problems, chemotherapy-associated neurocognitive problems including ‘chemo brain’, autism spectrum disorder (ASD), delirium, mild-cognitive impairment, traumatic brain injury, phenylketonuria, or tyrosinemia. In one embodiment, the age-associated neurocognitive disorder may be Alzheimer’s disease.

[0173] In another embodiment, the disorder may be an age-associated metabolic disorder. For instance, the age-associated metabolic disorder may include, but is not limited to, heart failure, cardiovascular disease, autoimmune-related disorders, myocardial ischemia reperfusion injury, hypertension, stroke, septic encephalopathy, diabetes, obesity, sepsis, Systemic Lupus Erythematosus, or inflammation.

[0174] Similarly, in other embodiments, the disorder is a cancerous growth of tissues or cells, such as breast cancer, ovarian cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, brain tumor, leukemia, bone cancer, and cachexia.

[0175] The therapeutically effective amount of the composition can vary based on factors such as the disorder stage, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the subject. Further, compounds disclosed herein can be administered toAttorney Docket No.: WKM0004PCT (1696) a subject at one time or over a series of treatments and may be administered to the subject at any time.

[0176] Interestingly, in accordance with the present disclosure, nanomolar concentration of compounds disclosed herein may exacerbate undesirable neurotoxic effects that leads to the depletion of neuronal TyrRS and increases levels of neuroinflammatory factors. As such, compounds disclosed herein in the trans configuration may be introduced to a cell at a concentration less than 25 nM. As such, compounds disclosed herein may be introduced to a cell at a concentration from about 1 micromolar (µM) to about 100 µM, such as from about 2 µM to about 95 µM, such as from about 10 µM to about 85 µM, such as from about 20 µM to about 75 µM, such as from about 35 µM to about 50 µM, or any range therebetween. As expected, the dosage will be dependent on the condition, size, and age of the subject.

[0177] Compounds disclosed herein may be administered, as appropriate or indicated, in a single dose as a bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, multiple times per week, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks, or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

[0178] In an embodiment methods of treatment include administering a compound of Formula I or Formula II or salt thereof and the therapeutically effective amount of the compound of Formula I or Formula II is 0.1 mg to 2000 mg daily, 0.5 mg to 15 g administered weekly, or 20 mg to 500 g administered monthly. The amount of the compound of Formula I or Formula II administered daily can be 0.1 mg to 2000 mg daily, 0.1 mg to 1500 mg daily, comprising 0.1 to 1000 mg daily, 0.1 to 900 mg daily, 0.1 to 700 mg daily, 0.1 to 600 mg daily, 0.1 to 500 mg daily, 0.1 to 400 mg daily, 0.1 to 300 mg daily, 0.1 to 200 mg daily, 0.1 to 100 mg daily, 0.1 to 50 mg daily, 0.1 to 25 mg daily, 0.1 to 10 mg daily, 0.1 to 5 mg daily, 1.0 to 1000 mg daily, 1.0 to 900 mg daily, 1.0 to 700 mg daily, 1.0 to 600 mg daily, 1.0 to 500 mg daily, 1.0 to 400 mg daily, 1.0 to 300 mg daily, 1.0 to 200 mg daily, 1.0 to 100 mg daily, 1.0 to 50 mg daily, 1.0 to 25 mg daily, 1.0 to 10 mg daily, 1.0 to 5 mg daily, 10 to 1000 mg daily, 10 to 900 mg daily, 10 to 700 mg daily, 10 to 600 mg daily, 10 to 500 mg daily, 10 to 400 mg daily, 10 to 300 mg daily, 10 to 200 mg daily, 10 to 100 mg daily, 10 to 50 mg daily, 100 to 1000 mg daily, 100 to 900 mg daily, 100 to 700 mg daily, 100 to 600 mg daily, or 100 to 500 mg daily.

[0179] When the compound of Formula I or Formula II is administered daily, it can be administered 1, 2, 3, or 4 or more times daily. Once or twice daily administration is preferred.Attorney Docket No.: WKM0004PCT (1696)

[0180] Methods of treatment include administering a sufficient amount of a compound of Formula I or Formula II or salt thereof to provide a plasma or blood Cmax of 100 ng / ml, 90 ng / ml, 80 ng / ml, 70 ng / ml, 60 ng / ml, 50 ng / ml, 40 ng / ml, 30 ng / ml, 20 ng / ml, or 10 ng / ml. PHARMACEUTICAL COMPOSITIONS

[0181] In one embodiment, a pharmaceutical composition comprising a compound of Formula I or Formula II or salt thereof together with at least one pharmaceutically compatible excipient can be delivered to the targeted cells or tissue via a pharmaceutically acceptable delivery system.

[0182] In one embodiment, a composition disclosed herein and / or a pharmaceutically compatible carrier comprising a composition can be delivered to the targeted cells or tissue via a pharmaceutically acceptable delivery system. The term "administering" is intended to include modes and routes of administration which allow a compound to perform its intended function. In general, the compounds disclosed herein may be administered to a subject according to known methods, including injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or retained in conjunction with a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The compound may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The compound also may be administered as a prodrug, which is converted to its active form in vivo.

[0183] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose in saline, etc. Solid supports, liposomes, nanoparticles, microparticles, nanospheres or microspheres may also be used as carriers for administration of a compound disclosed herein. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Supplementary agents can also be incorporated into the compositions.

[0184] Pharmaceutical excipients include carriers, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. In some instances definitions of these classes of excipients overlap.

[0185] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose in saline, etc. Solid supports, liposomes, nanoparticles, microparticles, nanospheresAttorney Docket No.: WKM0004PCT (1696) or microspheres may also be used as carriers for administration of a compound disclosed herein. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. The use of such media and agents for pharmaceutically active substances is well-known in the art. Supplementary agents can also be incorporated into the compositions.

[0186] It can be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the application is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0187] Pharmaceutical compositions for parenteral, intradermal, or subcutaneous injection can include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[0188] A composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like that can enhance the effectiveness of the active ingredient. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. A composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

[0189] For intravenous administration, suitable carriers include, without limitation, physiological saline, bacteriostatic water, Cremophor EL™ (BASF™, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, an injectable composition should be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action ofAttorney Docket No.: WKM0004PCT (1696) microorganisms such as bacteria and fungi. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0190] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[0191] Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of an orally ingestible composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel®, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0192] When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin (e.g., peanut oil, mineral oil, soybean oil, or sesame oil), or synthetic oils may be added. A liquid form may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. When administered in liquid form, a composition can contain from about 0.5 to 90% by weight of the cis-resveratrol derivative.

[0193] For administration by inhalation, a compound, e.g., a cis-resveratrol derivative may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0194] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.

[0195] In certain embodiments, a pharmaceutical composition can be formulated for sustainedAttorney Docket No.: WKM0004PCT (1696) or controlled release of the compound (e.g., cis-resveratrol derivative). Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0196] The pharmaceutical composition can be formulated for oral administration. The oral dosage form can comprise from 0.1 to 99% by weight (wt%) of the compound or salt thereof of Formula I or Formula II. Some embodiments contain from 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50 %, 1 to 40%, 1 to 30%, 1 to 25%, 1 to 20%, 5 to 99%, 5 to 90%, 5 to 80%, 5 to 70%, 5 to 60%, 5 to 50 %, 5 to 40%, 5 to 30%, 5 to 25%, 5 to 20%, 10 to 99%, 10 to 90%, 10 to 80%, 10 to 70%, 10 to 60%, 10 to 50 %, 10 to 40%, 10 to 30%, 20 to 99%, 20 to 90%, 20 to 80%, 20 to 70%, 20 to 60%, 20 to 50 %, or 20 to 40% by weight (wt%) of the compound or salt thereof of Formula I or Formula II.

[0197] In an embodiment the dosage form comprises a compound or salt of Formula I or Formula II and is an immediate or extended release dosage from comprising 0.1 to 1000 mg, 0.1 to 900 mg, 0.1 to 700 mg, 0.1 to 600 mg, 0.1 to 500 mg, 0.1 to 400 mg, 0.1 to 300 mg, 0.1 to 200 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 25 mg, 0.1 to 10 mg, 0.1 to 5 mg, 1.0 to 1000 mg, 1.0 to 900 mg, 1.0 to 700 mg, 1.0 to 600 mg, 1.0 to 500 mg, 1.0 to 400 mg, 1.0 to 300 mg, 1.0 to 200 mg, 1.0 to 100 mg, 1.0 to 50 mg, 1.0 to 25 mg, 1.0 to 10 mg, 1.0 to 5 mg, 10 to 1000 mg, 10 to 900 mg, 10 to 700 mg, 10 to 600 mg, 10 to 500 mg, 10 to 400 mg, 10 to 300 mg, 10 to 200 mg, 10 to 100 mg, 10 to 50 mg, 100 to 1000 mg, 100 to 900 mg, 100 to 700 mg, 100 to 600 mg, or 100 to 500 mg of the compound of Formula I or Formula II or salt thereof per unit dosage form.

[0198] In an embodiment the dosage form comprises a compound of Formula I or Formula II and is an extended release dosage form formulated for once weekly to once monthly administration that provides a plasma Cmaxof 0.1 to 100 nM of the compound of Formula I to the patient. The form can be an extended release dosage form.

[0199] The present disclosure may be better understood with reference to the following examples.

[0200] The therapeutically effective amount of the composition can vary based on factors such as the disorder stage, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the subject. Further, compounds disclosed herein can be administered to a subject at one time or over a series of treatments and may be administered to the subject at anyAttorney Docket No.: WKM0004PCT (1696) time.

[0201] Interestingly, in accordance with the present disclosure, nanomolar concentration of compounds disclosed herein may exacerbate undesirable neurotoxic effects that leads to the depletion of neuronal TyrRS and increases levels of neuroinflammatory factors. As such, compounds disclosed herein may be introduced to a cell at a concentration less than about 25 nM. As such, compounds disclosed herein may be introduced to a cell at a concentration from about 1 micromolar (µM) to about 100 µM, such as from about 2 µM to about 95 µM, such as from about 10 µM to about 85 µM, such as from about 20 µM to about 75 µM, such as from about 35 µM to about 50 µM, or any range therebetween. As expected, the dosage will be dependent on the condition, size, and age of the subject.

[0202] Compounds disclosed herein may be administered, as appropriate or indicated, in a single dose as a bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, multiple times per week, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks, or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

[0203] It can be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the application is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0204] Pharmaceutical compositions for parenteral, intradermal, or subcutaneous injection can include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. A composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like that can enhance the effectiveness of the active ingredient. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use ofAttorney Docket No.: WKM0004PCT (1696) surfactants. A composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

[0205] For intravenous administration, suitable carriers include, without limitation, physiological saline, bacteriostatic water, Cremophor EL™ (BASF™, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, an injectable composition should be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0206] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[0207] Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of an orally ingestible composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel®, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0208] When administered orally in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin (e.g., peanut oil, mineral oil, soybean oil, or sesame oil), or synthetic oils may be added. A liquid form may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. When administered in liquid form, a composition can contain from about 0.5 to 90% by weight of the cis-resveratrol derivative.

[0209] For administration by inhalation, a compound, e.g., a cis-resveratrol derivative may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.Attorney Docket No.: WKM0004PCT (1696)

[0210] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.

[0211] In certain embodiments, a pharmaceutical composition can be formulated for sustained or controlled release of the compound (e.g., cis-resveratrol derivative). Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0212] It is to be understood that the in vivo methods have application for both human and veterinary use. The methods of the present invention contemplate single, as well as multiple, administration, given either simultaneously or over an extended period of time.

[0213] The present disclosure may be better understood with reference to the following examples. EXAMPLES ABBREVIATIONS

[0214] AcOH Acetyl alcohol DCE 1,2-Dichloroethane DIBAL Diisobutylaluminum hydride Et3N Triethyl amine EtOH Ethanol IF Immunofluorescence Na(OAc)3BH Sodium triacetoxyhydroborate OTBS O-(tert-Butyldimethylsilyl)hydroxylamine rt or RT Room temperature TBAF Tetra-n-butylammonium fluoride THF TetrahydrofuranAttorney Docket No.: WKM0004PCT (1696) MATERIALS AND METHODS Primary Neuronal Culture

[0215] Primary cortical neurons were harvested from 18-day-old Sprague Dawley rat pups using Hibernate E (BrainBits) and dissociated with the Neural Tissue Dissociation kit (Miltenyi Biotec). Briefly, the cortices were minced cortices and incubated in a pre-heated enzyme mix at 37 °C for 15 minutes. The tissues were then strained using a 40 µm cell strainer, washed, and centrifuged. The neurons were then cultured on tissue culture plates coated with 50 µg / ml poly-D- Lysine (Sigma Aldrich). The culture medium consisted of NBActive-1 medium (BrainBits) supplemented with 100 U / ml of Penicillin-Streptomycin (Life Technologies), 2 mM L-Glutamine (Life Technologies), and 1X N21 supplement (R&D Systems) was used as culture medium. Control (non-targeting), TyrRS, and PARP1 siRNAs were obtained from Invitrogen (# AM4635, s443, and s130207, respectively).5 DIV neurons were transfected with 75 nM control or TyrRS siRNA using Dharmafect 3 Transfection Reagent. A second transfection was done two days later using 75 nM of TyrRS siRNA, followed by cell collection or assays after another 48 hr. For PARP1 siRNA, 7 DIV neurons were transfected with either 75 nM control or PARP1 siRNA. Western Blotting

[0216] Cultured primary rat cortical neurons (DIV 9 / 10) were prepared for analysis by washing with cold 1× PBS and lysing in cell lysis buffer. The lysates were then centrifuged at 15,000g for 15 min at 4 °C to separate the chromatin-bound and soluble fractions and equal amounts of protein were loaded onto a 4% to 12% gradient gel (NuPAGE-Invitrogen) for electrophoresis. The protein was transferred to a 0.2 µm NC membrane and the membrane was blocked with 5% non-fat milk in TBST . Primary antibodies were applied to the membrane and incubated overnight at 4 °C, followed by incubation with secondary antibodies for 1 hour at room temperature. The immobilon ECL Ultra Western HRP Substrate was used to detect the proteins, and the luminescent image analyzer (ChemiDoc Imaging System, Bio-Rad) was used for quantification. The western blots were quantified using ImageJ software (Version 1.53t).Attorney Docket No.: WKM0004PCT (1696) List of antibodies used for western blotting Antibody Company Catalog No. Dilution Cy

[0217] The cells were harvested with a cell scraper using chilled PBS and counted. The comet assay (Trevigen Inc, Gaithersburg, MD) was performed according to manufacturer's protocol using alkaline conditions). Briefly, after electrophoresis, the slides were washed twice in deionized water for 5 min and immersed in 70% ethanol for 5 min. Subsequently, the slides were dried at 37 °C for 30 min. DNA staining was done using SYBR Gold dye (Fisher Scientific, 1:10000 in Tris–EDTA buffer, pH 7.5) for 20 min in the dark at room temperature and then imaged using an epifluorescent microscope at 10X magnification. The images were quantified and scored for comet parameters such as tail length using the Tritek CometScore™ Freeware v1.5 image analysis software. Immunofluorescence (IF)

[0218] Cultured cortical neurons at DIV 9-10 were fixed in 4% formaldehyde for 15 minutes, permeabilized, and blocked with 5% BSA (PBS) and 0.1% Tween20 for 30 minutes at room temperature. Primary antibodies were added and incubated overnight at 4 °C, followed by secondary antibody incubation for 1 hour at room temperature. Alexa Fluor 647 (anti-chicken), Alexa Fluor 555 (anti-mouse), and Alexa Fluor 488 (anti- rabbit) from Invitrogen were used as secondary antibodies at a dilution of 1:1000. Coverslips were mounted with DAPI-supplemented mounting medium, Prolong Gold Antifade (Invitrogen), and imaged with a Leica DMI6000 epifluorescent microscope using an oil immersion 63x / NA 1.4 objective. The quantification of total protein levels in neurons was performed using ImageJ (Version 1.53c), with imaging parameters matched for exposure, gain, and offset.Attorney Docket No.: WKM0004PCT (1696) List of antibodies used for IF n

[0219] All drugs / inhibitors stock solutions (1000x) were prepared in DMSO or ethanol and diluted in culture media to their final concentration. The various compounds used for treatments and their stock concentrations are listed below: Compound Catalog Stock Final Solvent Concentration ConcentrationStatistical Analysis

[0220] The data was analyzed for statistical significance and differences between groups were determined. Depending on the number of groups being compared, either one-way ANOVA withAttorney Docket No.: WKM0004PCT (1696) multiple comparisons without correction or two-way ANOVA with multiple comparisons without correction were used. When comparing two groups, either paired or unpaired t-tests were used. The data analysis was performed using GraphPad statistical analysis software. EXAMPLE1. SYNTHESIS OFETHYL(E)-4-((2-(3,5-DIMETHOXYPHENYL)-3- PHENYLALLYL)AMINO)BENZOATE CN CNEXAMPLE 2. SYNTHESIS SCHEME OF ETHYL (E)-4-((2-(3,5-DIMETHOXYPHENYL)-3-PHENYLALLYL)AMINO)BENZOATE(II-2)MeO OMe MeO OMeNa(OAc)3BH, AcOH,benzoate (II-2), we prepared 3-(3,5-dimethoxyphenyl)-2-phenylpropan-1-amine (5) from nitrile 3 via the double reduction in addition to nitril group in the presence of CoCl2-NaBH4, the subsequent reductive amination of amine 5 with ethyl 4-formylbenzoate afforded the II-2 as shown in the Scheme above.Attorney Docket No.: WKM0004PCT (1696) EXAMPLE 3. SYNTHESIS OF SYNTHESIS OF ETHYL (Z)-4-((2,3-DIPHENYLALLYL)AMINO) BENZOATE (II- 3) ringslowered the cell survival activity of II-1. Therefore, in order to increase the potency of II-1, we modified it to defunctionalize the methoxy substituent groups on the aromatic ring of the stilbene segment. In addition, we also modified the amino-benzoate functional moiety of II-1 to benzamide derivatives with the attachment of serine and threonine amino acid side chain linkers, as these pharmaceutically important pharmacophores would have a potential impact on the broadening of multi-synthetase activity at the given instance of disease target. Therefore, we synthesized the modified stilbene core without methoxy groups on both the aromatic rings by following a three-step reaction sequence, starting from simple and commercially available starting materials, benzaldehyde and 2-phenyl-acetonitrile.

[0223] The first step in the sequence involving condensation of 2-phenyl-acetonitrile (6) and benzaldehyde (7) in presence of 40% aqueous ethanolic KOH provided the 2,3- diphenylacrylonitrile (8). However, unlike routine synthesis, a significant formation of 2- phenylacetic acid from the reactant 2-phenylacetonitrile under strong basic conditions limited the reaction outcome to moderate yield. Nevertheless, the crucial reduction of CN-group to the corresponding 2,3-diphenylacrylaldehyde 9 was achieved by using a mild reducing agent, diisobutylaluminium hydride (DIBAL-H) reaction conditions. Due to the slow reactivity of CN- group at lower reaction temperature, and an unwanted double bond reduction, CN group reduction of 8 to the corresponding amine, and further reduction of formed aldehyde 9 to the corresponding alcohol at higher temperatures had greatly hindered the scale-up reaction beyond 0.1 to 0.15 gram. Therefore, we conducted a series of small-scale reactions to obtain the required quantity in order to proceed further steps.

[0224] The subsequent reductive amination of the obtained 2,3-diphenylacrylaldehyde 9 with ethyl-4-aminobenzoate under sodium triacetoxyhydroborate (NaBH(OAc)3conditions afforded the pure stilbene-aminobenzoate conjugate II-3 in 25-30% yield over three steps.Attorney Docket No.: WKM0004PCT (1696) EXAMPLE 4. SYNTHESIS OF (E)-N1-(2,3-DIPHENYLALLYL)BENZENE-1,4-DIAMINE (II-5) AND (E)-4- ((2,3-DIPHENYLALLYL)AMINO)-N-HYDROXYBENZAMIDE(II-6)in isopropanol, refluxing at room temperature. Unfortunately, under these reactions condition the desired product could not be obtained. However, the conversion of ester to its acid chloride 10 followed by the treatment of crude reaction mixture with NH4OH in isopropanol resulted in the desired benzamide derivative II-5 in good yield. Similarly, the treatment of II-3 with hydroxylamine hydrochloride in presence of 1N KOH in methanol furnished the hydroxamic acid derivative II-6 in good yield as shown in the above scheme. EXAMPLE 5. SYNTHESIS SCHEME OF II-7 AND II-8: SYNTHESIS OF 4-((2,3-DIPHENYLALLYL)AMINO)- N-(2-HYDROXYETHYL)BENZAMIDE (II-7) AND 4-((2,3-DIPHENYLALLYL)AMINO)-N-(2- HYDROXYPROPYL)BENZAMIDE (II-8)

[0226] To connect a serine amino acide side chain to II-3, we initially synthesized theAttorney Docket No.: WKM0004PCT (1696) ethanolamine connected benzamide which upon reductive amination should provide the desired product. Nevertheless, our efforts with the treatment of 9 with the benzamide 11 was unsuccessful under reductive amination reaction conditions. Therefore, we have choosen the acid-amine coupling approach by converting the estre to acid chloride, and then treating the acid chloride with 2-methoxyethylamine under basic conditions to provide the desired II-7 in good yield over four steps.

[0227] A similar approach with 2-methoxypropan-1-amine was ineffective to provide the respective amide derivative II-8. However, the TBS-protected 1-aminopropan-2-ol (12) under acid- amine coupling reaction conditions furnished the desired threonyl side chain linker of the benzamide II-8 as depicted in the Scheme. Interestingly, the II-8 (EC50 = ≤ 25 nM) is more potent than the II-3 (EC50= ≥ 25 µM) as shown in MTT assay.

[0228] 1H NMR for compound II-8 (400 MHz, Chloroform-d) δ 7.61-7.52 (m, 2H), 7.30-7.19 (m, 3H), 7.14-7.06 (m, 2H), 7.01 (dd, J = 4.9, 1.9 Hz, 3H), 6.85 (dd, J = 6.8, 3.0 Hz, 2H), 6.55-6.51 (m, 3H), 4.30 (t, J = 6.0 Hz, 1H), 4.05 (d, J = 4.5 Hz, 2H), 3.90 (ddq, J = 12.5, 6.2, 2.9 Hz, 1H), 3.50 (ddd, J = 14.0, 6.4, 2.9 Hz, 1H), 3.27 (s, 1H), 3.19 (ddd, J = 14.0, 7.6, 5.2 Hz, 1H), 1.12 (d, J = 6.3 Hz, 3H).13C NMR (101 MHz, Chloroform-d) δ 168.59, 150.72, 139.09, 138.38, 136.31, 129.18, 128.91, 128.80, 128.56, 127.98, 127.64, 127.50, 126.87, 122.42, 112.17, 67.82, 51.60, 47.58, 20.99. HRMS (ESI) calcd for C25H27N2O2, 387.2073; found, 387.2069. EXAMPLE 6. TYROSINE AND PHENYLALANINE DECREASE NEURITE TUBULIN TYROSINATION

[0229] Tyrosine (Tyr) and phenylalanine (Phe) are not only important for protein synthesis and cellular energy production, but also act as substrates for tubulin tyrosine ligase (TTL) that adds tyrosine or phenylalanine to the C-terminal glutamic residue of α-tubulin (Tyr / Phe-tubulin). In contrast, the vasohibin-small vasohibin binding protein complexes (VASH1 / 2-SVBP) remove the C-terminal tyrosine / phenylalanine from the α -tubulin, as illustrated in the FIG.1. However, removing the penultimate glutamate residue generates delta2-tubulin and abrogates the Tyr / Phe addition. Brain tubulin tyrosination increases rapidly after single-shock contextual fear conditioning (30-60 min) and gradually decreases to its lowest level at 8 hr after the single-shock in the mouse brain. Mouse lacking TTL in the neocortex and hippocampus exhibits increased anxiety and impaired spatial learning, indicating its contribution to normal cognition and memory formation. In contrast, TTL is depleted in AD brains along with the accumulation of detyrosinated tubulin (deTyr- tubulin). Consistently, TTL levels positively correlate with cognitive performance, TyrRS levels, and negatively with AD progression. Since tyrosine and phenylalanine are increased in the brain of AD patients, we hypothesized that treatment with tyrosine and phenylalanine would negatively regulate tubulin tyrosination. We treated rat cortical neurons with either tyrosine (FIG.2) or phenylalanine (FIG.3) and immunoassayed for the levels of tubulin tyrosination. We found thatAttorney Docket No.: WKM0004PCT (1696) both tyrosine and phenylalanine decreased neurite tubulin tyrosination levels (FIG.2 and 3). However, unlike tyrosine, treatment with phenylalanine decreased tubulin tyrosination in the soma as well (FIG.3). Since tyrosine and phenylalanine deplete neuronal TyrRS protein levels, we wondered if inhibition of tubulin detyrosination using EpoY- a specific inhibitor of VASH1 / 2- SVBP, would affect neuronal TyrRS levels. We treated rat cortical neurons with EpoY and found that EpoY increased neuronal TyrRS levels along with increased tubulin tyrosination (FIG.4). Taken together, these data indicate that activation of tubulin tyrosination is a positive regulator of neuronal TyrRS protein levels that would act by decreasing cellular tyrosine and phenylalanine levels. EXAMPLE7. PICOMOLARAΒ40ANDAΒ42DISTINCTLY REGULATE NEURONAL TUBULIN TYROSINATIONIN CORTICAL NEURONS

[0230] Based on these observations and amyloid beta peptides are essential for cognition and memory formation, without wishing to be bound by theory, it was hypothesized that Aβ40 and Aβ42 would modulate tubulin tyrosination levels to regulate tyrosine and TyrRS levels in the neurons. To test this hypothesis, rat cortical neurons (DIV 9 / 10) were treated with thiorphan, BACEi, Aβ40, and Aβ42 and quantified Tyr-tubulin levels. While thiorphan increased Tyr-tubulin levels (FIG.5A), BACEi decreased it (FIG.5B). Furthermore, Aβ42 increased Tyr-tubulin levels (FIG.5C and D) with concomitant depletion of deTyr-tubulin in cortical cultures (FIG.5E). In contrast, Aβ40 decreased Tyr-tubulin levels only in the soma while increasing Tyr-Tub levels in the neurites (FIG.5C and FIG.6). Intriguingly, western blot (WB) analysis using whole cell lysate (WCL) showed that picomolar Ab40 and Ab42 have opposing effects on neuronal tubulin tyrosination and detyrosination levels and Ab40 is a stimulator of neuronal deTyr-tubulin levels in cortical cultures (FIG.5E). Intriguingly, treatment with verubecestat (BACE-1 inhibitor) decreased both tubulin tyrosination and TyrRS levels (FIG.7), suggesting that one of the major functions of cellular amyloid beta peptides include activation of tubulin tyrosination to regulate cellular tyrosine and phenylalanine levels. Since both Ab40 and Ab42 increased neurite tubulin tyrosination levels (FIG.5C, D and FIG.6) and in contrast, tyrosine and phenylalanine decreased neurite tubulin tyrosination levels (FIG.1 and 2), we wondered if they would reciprocally modulate the localization of TTL to the neurites. Interestingly, both Aβ40 and Aβ42 increased TTL levels in the neurites without affecting the soma (FIG.8A) whereas treatment with tyrosine decreased neurite TTL levels while accumulating its levels in the soma (FIG.8B), providing a potential molecular basis for Aβ-mediated neurite tubulin tyrosination in the neurites and tyrosine / phenylalanine- mediated depletion in the neurites. To test if Aβ could rescue tyrosine-mediated depletion of Tyr- tubulin, the rat cortical neurons (DIV 9 / 10) were co-treated with Aβ40 and Aβ42 and found that only Aβ40 rescues tyrosine mediated depletion of Tyr-tubulin in the neurites (FIG.8C and D).Attorney Docket No.: WKM0004PCT (1696) However, both Ab40 and Ab42 rescued neurite tyrosination levels in presence of phenylalanine (FIG.9 and FIG.10). These observations suggest that amyloid beta peptides-mediated regulation of tubulin tyrosination is negatively regulated by increased levels of tyrosine and phenylalanine.

[0231] Further, the relationship between neuronal TyrRS and Tyr-tubulin levels and protein synthesis activation were explored. We previously showed that inhibitor of integrated stress response (ISRIB) increases neuronal TyrRS levels. Similarly, treating rat cortical neurons (DIV 9 / 10) with ISRIB, it was found that ISRIB increased neuronal tubulin tyrosination levels (FIG.11), providing a potential novel molecular basis for ISRIB-mediated upregulation of neuronal TyrRS protein levels by decreasing cellular tyrosine / phenylalanine levels. Recently, it was observed that cis- and trans-resveratrol (cis- and trans-RSV) have opposite effects on neuronal TyrRS levels and protects against tyrosine-mediated neurotoxic effects. However, the mechanisms of cis- and trans- RSV-mediated regulation of neuronal TyrRS and tyrosine toxicity are not well understood. Since we recently showed that cis-RSV increases the nuclear and neurite levels of neuronal TyrRS, it was determined whether cis-RSV would affect Tyr-tubulin levels. Studies herein found that cis-RSV increased Tyr-tubulin levels both in the neurites and soma (FIG.12A), providing a unique ability of cis-RSV to protect against both phenylalanine and tyrosine-mediated downregulation of tubulin tyrosination. Furthermore, cis- and trans-RSV exhibited opposite effects on TTL, tubulin tyrosination, and delta2 tubulin levels (FIGs.12B and C).

[0232] To determine if changes in tubulin tyrosine would be sufficient to modulate neuronal TyrRS levels, cortical neurons were treated with paclitaxel that stabilizes microtubules through detyrosination and parthenolide that inhibits tubulin detyrosination. While treatment with paclitaxel depleted neuronal TyrRS (FIG.12D), treatment with parthenolide increased neuronal TyrRS (FIG. 12E), suggesting that tubulin tyrosination / detyrosination cycle is a major modulator of neuronal TyrRS. Together, these results suggest a hitherto unknown mechanism through which cis-RSV and Aβ may exploit the neuronal Tyr / deTyr-tubulin cycle to regulate neuronal TyrRS as well as tyrosine / phenylalanine levels and associated signaling events and regulatory mechanisms.

[0233] Since higher concentrations of Ab42 is neurotoxic, we wondered if treatment with higher nanomolar (nM) concentrations would affect tubulin tyrosination levels. Therefore, we treated rat cortical neurons with nMAb42 (50 nM) and found that nMAb42 decreased tubulin tyrosination only in the soma (FIG.13A) whereas tubulin tyrosination in the neurites were increased (FIG.13A). Since cis-RSV increased tubulin tyrosination levels in the soma (FIG.12A), and we previously published that cis-RSV decrease nucleotide incorporation in cortical neurons, we wondered if treatment with nMAb42 would affect nucleotide incorporation in rat cortical neurons. We found that unlike pMAb42 that inhibits nucleotide incorporation (data not shown) and increase tubulin tyrosination in the soma (FIG.5C and D), nMAb42 did not prevent the nucleotideAttorney Docket No.: WKM0004PCT (1696) incorporation (FIG.13B). These observations suggest that pMAb42 and nMAb42 have opposing effects on soma tubulin tyrosination levels and nucleotide incorporation and agents that increase tubulin tyrosination in the soma and decrease nucleotide incorporation would protect against nMAb42-mediated neurotoxicity. Therefore, we treated rat cortical neurons (DIV9 / 10) with either cis-and trans-RSV (50 mM) or nMAb42 (50 nM) or their combination and found that only cis-RSV protected against nMAb42-mediated neurite degeneration and in contrast, trans-RSV by itself induced neurite degeneration and exacerbated nMAb42-induced neurite degeneration. These data for the first time demonstrate that cis- and trans-RSV have opposite effects on neuronal tubulin tyrosination and TTL levels and cis-RSV is a unique molecule that can protect against tyrosine / phenylalanine-mediated neurotoxic effects by activating tubulin tyrosination.

[0234] These and other modifications and variations to the present invention can be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole, or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims.

Claims

Attorney Docket No.: WKM0004PCT (1696) CLAIMS WHAT IS CLAIMED:

1. A method of activating tubulin tyrosination in a cell, the method comprising contacting the cell with an effective amount of a compound of Formula I: , salt thereof, wherein:of Formula I can be in either the cis or trans conformation or a mixture of cis and trans conformations; each of Y1, Y2, and Y3is independently selected from a H or a groupchosen at each occurrence from the 20 native amino acid side chains where any hydroxyl group in the amino acid side chain is optionally acylated with a C2-C6acyl group and when R is a proline the proline pyrrolidine ring is formed by R and RAbeing joined by a -CH2CH2CH2- chain; and RAand RBare independently chosen at each occurrence from H, C2-C6acyl, C1-C6alkyl, (C3- C6cycloalkyl)C0-C4alkyl, phenyl, and benzyl; or an effective amount of Formula II:salt thereof, wherein: the bond is a double or single bond; the A ring ( ) is phenyl, C3-C6cycloalkyl, or a 4- to 6-membered heterocyclic group;;Attorney Docket No.: WKM0004PCT (1696) R1is absent or is 1 to 5 substituents independently selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, (C3-C6cycloalkyl)C0-C2alkyl, C1-C2haloalkyl, and C1- C2haloalkoxy; R2is absent or is 1 to 5 substituents independently selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, (C3-C6cycloalkyl)C0-C2alkyl, C1-C2haloalkyl, and C1- C2haloalkoxy; R3is absent or is 1 to 4 substituents independently selected from halogen, C1-C2alkyl, C1- C2alkoxy, cyclopropyl, C1-C2haloalkyl, and C1-C2haloalkoxy; Y is NH or O; Z is H, hydroxyl, methyl, proline where the proline pyrrolidine ring is formed by Z and the nearest NH being joined by a -CH2CH2CH2- chain, or Z is a group -CH2X; X is an amino acid side chain selected from H, -CH3, -CH2SH, -CH2CO2H, -CH2OH, -CH(CH3)OH, -CH2CH2CO2H, -CH2CH2CONH2, ,2. A method of treating a disorder modulated by tubulin tyrosination in a subject, the method comprising administering to the subject a therapeutically effective amount a compound of Formula I: Y2O ,salt thereof, wherein: the bond indicates the compound of Formula I can be in either the cis or trans conformation or a mixture of cis and trans conformations; each of Y1, Y2, and Y3is independently selected from a H or a group RBAttorney Docket No.: WKM0004PCT (1696) R is independently chosen at each occurrence from the 20 native amino acid side chains where any hydroxyl group in the amino acid side chain is optionally acylated with a C2-C6acyl group and when R is a proline the proline pyrrolidine ring is formed by R and RAbeing joined by a -CH2CH2CH2- chain; and RAand RBare independently chosen at each occurrence from H, C2-C6acyl, C1-C6alkyl, (C3- C6cycloalkyl)C0-C4alkyl, phenyl, and benzyl; or an effective amount of Formula II: salt thereof, wherein:is a double or single bond; the A ring ( ) is phenyl, C3-C6cycloalkyl, or a 4- to 6-membered heterocyclic group;;selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, (C3-C6cycloalkyl)C0-C2alkyl, C1-C2haloalkyl, and C1- C2haloalkoxy; R2is absent or is 1 to 5 substituents independently selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1-C4alkoxy, (C3-C6cycloalkyl)C0-C2alkyl, C1-C2haloalkyl, and C1- C2haloalkoxy; R3is absent or is 1 to 4 substituents independently selected from halogen, C1-C2alkyl, C1- C2alkoxy, cyclopropyl, C1-C2haloalkyl, and C1-C2haloalkoxy; Y is NH or O; Z is H, hydroxyl, methyl, proline where the proline pyrrolidine ring is formed by Z and the nearest NH being joined by a -CH2CH2CH2- chain, or Z is a group -CH2X; X is an amino acid side chain selected from H, -CH3, -CH2SH, -CH2CO2H, -CH2OH, -CH(CH3)OH, -CH2CH2CO2H, -CH2CH2CONH2,Attorney Docket No.: WKM0004PCT (1696) ,3. The method of claim 1 or 2, wherein the compound is a compound of Formula I or a pharmaceutically acceptable salt thereof. RBRAN - 4. The method of claim 3, wherein Y1, Y2, and Y3areOand RAand RBare both H.RBRAN - 5. The method of claim 4, wherein Y1 O, and Y2and Y3are each hydrogen or methyl and RAand RBare both hydrogen.

6. The method of any one of claims 3 to 5, wherein each R is independently chosen at each occurrence from an C2-C6acyl group, and the side chain any of the following amino acids: tyrosine, tyrosine optionally substituted at the tyrosine hydroxyl with Ac, phenylalanine, leucine, isoleucine, and valine.

7. The method of any one of claims 3 to 5, wherein at least one R is the side chain of tyrosine.

8. The method of any one of claims 3 to 5, wherein at least one R is the side chain of phenylalanine.

9. The method of any one of claims 1 or 3 to 8, wherein the compound of Formula I is in the cis conformation.Attorney Docket No.: WKM0004PCT (1696) 10. The method of claim 1 or 2, wherein the compound is selected from: ; ;11. The method of claim 1 or 2, wherein the compound is selected from: OAttorney Docket No.: WKM0004PCT (1696) 12. The method of claim 1 or 2, wherein the compound is selected from: of the foregoing.

13. The method of claim 1 or 2, wherein the compound is a compound of Formula II or a pharmaceutically acceptable salt thereof.

14. The method of claim 13, wherein the compound is a compound of Formula (II-a)thereof.

15. The method of claim 13, wherein the compound is a compound of Formula (II-b)Attorney Docket No.: WKM0004PCT (1696) 16. The method of claim 13, wherein the compound is a compound of Formula (II-c)17. The method of any one of claims 13 to 16, where R1and R2are each absent or are 1 to 2 substituents independently selected from halogen, hydroxyl, amino, cyano, C1-C4alkyl, C1- C4alkoxy, C1-C2haloalkyl, and C1-C2haloalkoxy.

18. The method of any one of claims 13 to 17 wherein R3is absent.

19. The method of any of claims to 13 to 14 or 17 to 18 wherein Y is NH.

20. The method of any of claims 13 to 14 or 17 to 18 wherein Y is O.

21. The method of any of claims 13 to 20 wherein X is H, -CH2OH, or -CH(CH3)OH.

22. The method of any of claims 13 to 21 wherein the bond is a double bond.

23. The method of any of claims 13 to 22 wherein the bond is a single bond.

24. The method of claim 13, wherein the compound is a compound of Formula II selected fromAttorney Docket No.: WKM0004PCT (1696) and25. The method of claim 1 or 2, wherein the effective amount of the compound of Formula I or Formula II, or salt thereof, is an amount sufficient to increase the level TyrRS in the cell.

26. The method of claim 25, wherein the level of TyrRS in the cell is increased relative to a control amount by at least about 10%.

27. The method of claim 1 or 2, wherein the cell is a neuronal cell and the effective amount of the compound of Formula I or Formula II, or salt thereof, is an amount sufficient to decrease a level of tyrosine in the neuronal cell.

28. The method of claim 27, wherein the level tyrosine in the neuronal cell is decreased relative to a control amount by at least about 10%.

29. The method of claim 1 or 2, wherein the tubulin tyrosination comprises tubulin tyrosine ligase (TTL)-dependent tubulin tyrosination.

30. The method of claim 1 or 2, wherein the effective amount of the compound of Formula I or Formula II is a concentration of from about 1 µM to about 100 µM.Attorney Docket No.: WKM0004PCT (1696) 31. The method of claim 30, wherein the cell is a cultured cell that expresses poly-ADP- ribose polymerase 1 (PARP1).

32. The method of claim 2, wherein the disorder is a neurocognitive disorder.

33. The method of claim 32, where in the neurocognitive disorder is Alzheimer's disease, Parkinson's disease, autism spectrum disorder, delirium, mild-cognitive impairment, traumatic brain injury, phenylketonuria, or tyrosinemia.

34. The method of claim 2, wherein the disorder is a metabolic disorder.

35. The method of claim 34, wherein the metabolic disorder is selected from heart failure, cardiovascular disease, auto immune-related disorders, myocardial ischemia reperfusion injury, hypertension, stroke, diabetes, or obesity.

36. The method of claim 2, wherein the subject is a human.

37. The method of claim 2, wherein the amino acid side chain in the definition of R in Formula I or the definition of X in Formula II is the side chain of an amino acid selected from a group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

38. The method of claim 28, wherein R in Formula I or X in Formula II comprises a D- tyrosine side chain.

39. The method of claim 2, wherein a daily dose of about 0.1 mg to about 1000 mg of the compound of Formula I or the compound of Formula II is administered to the subject.

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