WO2024059005A1

WO2024059005A1 - Treating pku with correctors of mammalian slc6a19 function

Info

Publication number: WO2024059005A1
Application number: PCT/US2023/032413
Authority: WO
Inventors: Ryan A. HOLLIBAUGH; Dean G. Brown; Giovanni MUNCIPINTO; John A. MALONA
Original assignee: Jnana Therapeutics Inc.
Priority date: 2022-09-14
Filing date: 2023-09-11
Publication date: 2024-03-21

Abstract

Disclosed are compounds, compositions, and methods useful for treating or preventing a disease or disorder associated with abnormal levels of amino acids by modulation of SLC6A19 transport.

Description

TREATING PKU WITH CORRECTORS OF MAMMALIAN SLC6A19 FUNCTION

RELATED APPLICATION

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/406,443, filed September 14, 2022.

BACKGROUND

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH), the enzyme responsible for metabolizing phenylalanine. PKU is an autosomal recessive metabolic disorder in which phenylalanine is not properly metabolized and results in abnormally high levels of plasma phenylalanine. People who have PKU have abnormally high blood levels of phenylalanine, which if untreated can lead to irreversible neurological damage resulting in a spectrum of complications such as intellectual disabilities, seizures, neurodevelopmental and behavioral disorders. PKU is difficult to treat because blood levels of phenylalanine are directly related to diet. Patients must adhere to a life-long and strict diet that impacts all aspects of patients’ lives. Current standard of care are enzyme co-factor and enzyme substitution therapy but these therapies are not effective in all patients, and carry potential risk for adverse events.

The enzyme responsible for metabolizing phenylalanine, and thus maintaining phenylalanine homeostasis is phenylalanine hydroxylase (PAH). Loss-of-function (LOF) mutations at PAH gene at chromosome 12q23.2 are known to cause most forms of PKU. These LOF mutations resulting in PKU can be diagnosed as classical PKU (the most severe form), and “mild PKU” or “hyperphe” a less severe form. In addition to PAH, mutations in other enyzmes that affect phenylalanine metabolism, such as dihydropteridine reductase (DHPR), the enzyme responsible for synthesis of co-factors required for PAH activity, may also result in elevated levels of phenylalanine. In addition to diet, blood amino acid levels, including levels of phenylalanine, are regulated by SLC6A19. SCL6A19 is located in the proximal tubule of the kidney and is responsible for reabsorption of amino acids back into the blood.

SUMMARY

One aspect of the invention provides compounds, compositions, and methods useful for treating or preventing a disease or disorder associated with abnormal levels of amino acids by modulation of SLC6A19 transport. Accordingly, provided herein is a compound having the structure of Formula (I):

wherein: n is 0 or 1;

Li is absent or selected from -NH-, -N(CH3)-, -O-, and -CH2-;

L2 is -alkyl-

L3 is -(5-membered heteroaryl)-;

Xi is -C(RI)(R₂)(R₃);

X2 is an optionally substituted aryl or heteroaryl;

X3 is selected from -H, alkyl, and haloalkyl;

Ri is selected from -H, halo, hydroxyl, amido, amino, alkylamino, and aminoalkyl; and

R2 and R3 are each independently selected from -H and alkyl; or R2 and R3 taken together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl or cycloheteroalkyl; provided the compound is not selected from

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to methods of treating or preventing a disease or disorder associated with a genetic defect in phenylalanine hydroxylase in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I). Another aspect of the invention relates to methods of treating or preventing phenylketonuria, hyperphenylalaninemia, tyrosinemia, nonketotic hyperglycinemia, isovaleric acidemia, methylmalonic acidemia, propionic acidemia, maple syrup urine disease, DNAJC12 deficiency, urea cycle disorders, or hyperammonemia in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

Another aspect of the invention relates to methods of modulating SLC6A19 transport in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features, objects, and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table summarizing isoleucine transport data for exemplary compounds of the invention. A = IC50 <500 nM; B = IC50 500 nM - 1500 nM; C = IC50 >1500 nM - 5000 nM; D = IC50 >5000 nM - 10000 nM; and E = IC50 >10000 nM.

DETAILED DESCRIPTION

Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

In order for the present invention to be more readily understood, certain terms and phrases are defined below and throughout the specification. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-i somers, R- and 5-enantiomers, diastereomers, (D)-isomers, (inisomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

“Geometric isomer" means isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring, or to a bridged bicyclic system. Atoms (other than H) on each side of a carbon- carbon double bond may be in an E (substituents are on opposite sides of the carbon- carbon double bond) or Z (substituents are oriented on the same side) configuration. "R," "S," "S*," "R*," "E," "Z," "cis," and "trans," indicate configurations relative to the core molecule. Certain of the disclosed compounds may exist in “atropisomeric” forms or as “atropisomers.” Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the isolation of the conformers. The compounds of the invention may be prepared as individual isomers by either isomer-specific synthesis or resolved from a mixture of isomers. Conventional resolution techniques include forming the salt of a free base of each isomer of an isomeric pair using an optically active acid (followed by fractional crystallization and regeneration of the free base), forming the salt of the acid form of each isomer of an isomeric pair using an optically active amine (followed by fractional crystallization and regeneration of the free acid), forming an ester or amide of each of the isomers of an isomeric pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and removal of the chiral auxiliary), or resolving an isomeric mixture of either a starting material or a final product using various well known chromatographic methods.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

Percent purity by mole fraction is the ratio of the moles of the enantiomer (or diastereomer) or over the moles of the enantiomer (or diastereomer) plus the moles of its optical isomer. When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least about 60%, about 70%, about 80%, about 90%, about 99% or about 99.9% by mole fraction pure relative to the other stereoisomers. When a single enantiomer is named or depicted by structure, the depicted or named enantiomer is at least about 60%, about 70%, about 80%, about 90%, about 99% or about 99.9% by mole fraction pure. When a single diastereomer is named or depicted by structure, the depicted or named diastereomer is at least about 60%, about 70%, about 80%, about 90%, about 99% or about 99.9% by mole fraction pure.

When a disclosed compound is named or depicted by structure without indicating the stereochemistry, and the compound has at least one chiral center, it is to be understood that the name or structure encompasses either enantiomer of the compound free from the corresponding optical isomer, a racemic mixture of the compound or mixtures enriched in one enantiomer relative to its corresponding optical isomer. When a disclosed compound is named or depicted by structure without indicating the stereochemistry and has two or more chiral centers, it is to be understood that the name or structure encompasses a diastereomer free of other diastereomers, a number of diastereomers free from other diastereomeric pairs, mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures of diastereomers in which one diastereomer is enriched relative to the other diastereomer(s) or mixtures of diastereomers in which one or more diastereomer is enriched relative to the other diastereomers. The invention embraces all of these forms.

Structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by the replacement of a hydrogen with deuterium or tritium, or of a carbon with a ¹³C- or ¹⁴C- enriched carbon are within the scope of this invention.

The term “prodrug” as used herein encompasses compounds that, under physiological conditions, are converted into therapeutically active agents. A common method for making a prodrug is to include selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal.

The phrase “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ or portion of the body, to another organ or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, not injurious to the patient, and substantially non-pyrogenic. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. In certain embodiments, pharmaceutical compositions of the present invention are non-pyrogenic, i.e., do not induce significant temperature elevations when administered to a patient. The term “pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the compound(s). These salts can be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting a purified compound(s) in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts, and the like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the compounds useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts” in these instances refers to the relatively non-toxic inorganic and organic base addition salts of a compound(s). These salts can likewise be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting the purified compound(s) in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, di ethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).

The term “pharmaceutically acceptable cocrystals” refers to solid coformers that do not form formal ionic interactions with the small molecule.

A “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment, refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.

The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

The term “patient” or “subject” refers to a mammal in need of a particular treatment. In certain embodiments, a patient is a primate, canine, feline, or equine. In certain embodiments, a patient is a human.

An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyl defined below. A straight aliphatic chain is limited to unbranched carbon chain moieties. As used herein, the term “aliphatic group” refers to a straight chain, branched-chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, or an alkynyl group.

“Alkyl” refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having the number of carbon atoms specified, or up to 30 carbon atoms if no specification is made. For example, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and those moieties which are positional isomers of these moieties. Alkyl of 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer. Alkyl goups may be substituted or unsubstituted.

As used herein, the term “heteroalkyl” refers to an alkyl moiety as hereinbefore defined which contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms.

As used herein, the term “haloalkyl” refers to an alkyl group as hereinbefore defined substituted with at least one halogen.

As used herein, the term “hydroxyalkyl” refers to an alkyl group as hereinbefore defined substituted with at least one hydroxyl.

As used herein, the term “alkylene” refers to an alkyl group having the specified number of carbons, for example from 2 to 12 carbon atoms, that contains two points of attachment to the rest of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include methylene -(CH2)-, ethylene -(CH2CH2)-, n-propylene - (CH2CH2CH2)-, isopropylene -(CH2CH(CH3))-, and the like. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moiety, and may be optionally substituted with one or more substituents.

"Cycloalkyl" means mono- or bicyclic or bridged or spirocyclic, or polycyclic saturated carbocyclic rings, each having from 3 to 12 carbon atoms. Preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3-6 carbons in the ring structure. Cycloalkyl groups may be substituted or unsubstituted.

As used herein, the term “halocycloalkyl” refers to an cycloalkyl group as hereinbefore defined substituted with at least one halogen.

"Cycloheteroalkyl" refers to an cycloalkyl moiety as hereinbefore defined which contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms. Preferred cycloheteroalkyls have from 4-8 carbon atoms and heteroatoms in their ring structure, and more preferably have 4-6 carbons and heteroatoms in the ring structure. Cycloheteroalkyl groups may be substituted or unsubstituted.

Unless the number of carbons is otherwise specified, “lower alkyl,” as used herein, means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In certain embodiments, a substituent designated herein as alkyl is a lower alkyl.

“Alkenyl” refers to any cyclic or acyclic, branched or unbranched unsaturated carbon chain moiety having the number of carbon atoms specified, or up to 26 carbon atoms if no limitation on the number of carbon atoms is specified; and having one or more double bonds in the moiety. Alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, and tetracosenyl, in their various isomeric forms, where the unsaturated bond(s) can be located anywhere in the moiety and can have either the (Z) or the (E) configuration about the double bond(s).

“Alkynyl” refers to hydrocarbyl moieties of the scope of alkenyl, but having one or more triple bonds in the moiety.

The term “aryl” as used herein includes 3- to 12-membered substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon (i.e., carbocyclic aryl) or where one or more atoms are heteroatoms (i.e., heteroaryl). Preferably, aryl groups include 5- to 12-membered rings, more preferably 6- to 10-membered rings The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Carboycyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. Heteroaryl groups include substituted or unsubstituted aromatic 3- to 12-membered ring structures, more preferably 5- to 12- membered rings, more preferably 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl and heteroaryl can be monocyclic, bicyclic, or polycyclic.

The term “halo”, “halide”, or “halogen” as used herein means halogen and includes, for example, and without being limited thereto, fluoro, chloro, bromo, iodo and the like, in both radioactive and non-radioactive forms. In a preferred embodiment, halo is selected from the group consisting of fluoro, chloro and bromo.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to 12-membered ring structures, more preferably 5- to 12-membered rings, more preferably 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can be monocyclic, bicyclic, spirocyclic, or polycyclic. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, and the like.

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. In preferred embodiments, the substituents on substituted alkyls are selected from Ci-6 alkyl, C3-6 cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments, the substituents on substituted alkyls are selected from fluoro, carbonyl, cyano, or hydroxyl. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da). In some embodiments, a “small molecule” refers to an organic, inorganic, or organometallic compound typically having a molecular weight of less than about 1000. In some embodiments, a small molecule is an organic compound, with a size on the order of 1 nm. In some embodiments, small molecule drugs of the invention encompass oligopeptides and other biomolecules having a molecular weight of less than about 1000.

An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition depends on the composition selected. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions described herein can include a single treatment or a series of treatments.

The terms “decrease,” “reduce,” “reduced”, “reduction”, “decrease,” and “inhibit” are all used herein generally to mean a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, up to and including, for example, the complete absence of the given entity or parameter ascompared to the reference level, or any decrease between 10-99% as compared to the absence of a given treatment.

The terms “increased”, “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

As used herein, the term “modulate” includes up-regulation and down-regulation, e.g., enhancing or inhibiting a response.

A “radiopharmaceutical agent,” as defined herein, refers to a pharmaceutical agent which contains at least one radiation-emitting radioisotope. Radiopharmaceutical agents are routinely used in nuclear medicine for the diagnosis and/or therapy of various diseases. The radiolabelled pharmaceutical agent, for example, a radiolabelled antibody, contains a radioisotope (RI) which serves as the radiation source. As contemplated herein, the term “radioisotope” includes metallic and non-metallic radioisotopes. The radioisotope is chosen based on the medical application of the radiolabeled pharmaceutical agents. When the radioisotope is a metallic radioisotope, a chelator is typically employed to bind the metallic radioisotope to the rest of the molecule. When the radioisotope is a non-metallic radioisotope, the non-metallic radioisotope is typically linked directly, or via a linker, to the rest of the molecule.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Compounds o f the Invention

One aspect of the invention relates to a compound of Formula (I):

wherein: n is 0 or 1;

Li is absent or selected from -NH-, -N(CH3)-, -O-, and -CH2-;

L2 is -alkyl-

L3 is -(5-membered heteroaryl)-;

Xi is -C(RI)(R₂)(R₃); X2 is an optionally substituted aryl or heteroaryl;

X3 is selected from -H, alkyl, and haloalkyl;

or a pharmaceutically acceptable salt thereof.

In certain embodiments, R2 and R3 are each independently selected from -H and alkyl; or R2 and R3 taken together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl, which is not cyclopropyl, or cycloheteroalkyl.

In certain embodiments, at least one of Ri, R2, and R3 is not -H. In other embodiments, at least two of Ri, R2, and R3 are not -H. In other embodiments, each of Ri, R2, and R3 is not -H.

In certain embodiments, the compound having the structure:

In certain embodiments, Li is selected from -NH- and -N(CH3)-.

In certain embodiments, L2 is selected from -CH2-, -CH2CH2-, and -CH2CH2CH2-.

In certain embodiments, Ri is selected from -H, -F, -OH, -NH2, -CH2NH2, -N(H)(CH₃), -N(CH₃)₂, and -C(O)NH₂.

In certain embodiments, Ri is -H. In other embodiments, Ri is -NH2. In certain embodiments, R2 and R3 are each -H. In other embodiments, R2 and R3 are each -CH3.

In certain embodiments, R2 and R3 taken together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl.

In certain embodiments, R2 and R3 taken together with the carbon atom to which they are bonded form an unsubstituted cyclopropyl or cyclobutyl.

In certain embodiments, R2 and R3 taken together with the carbon atom to which they are attached form an optionally substituted cycloheteroalkyl.

In certain embodiments, R2 and R3 taken together with the carbon atom to which they are bonded form an unsubstituted azetidinyl, pyrrolidinyl, piperidinyl, or lactam.

In certain embodiments, R2 and R3 taken together with the carbon atom to which they are bonded form a substituted azetidinyl, pyrrolidinyl, piperidinyl, or lactam.

In certain embodiments, the azetidinyl, pyrrolidinyl, piperidinyl, or lactam is A -alkyl or /'/-acetyl substituted.

In certain embodiments, Xi is selected from

In certain embodiments, Xi is selected from

In certain embodiments, X2 is unsubstituted aryl.

In certain embodiments, the unsubstituted aryl is unsubstituted phenyl.

In certain embodiments, X2 is substituted aryl.

In certain embodiments, the substituted aryl is substituted phenyl.

In certain embodiments,

R4, R5, Rs, R7, and Rs are independently selected from -H, halogen, -CN, -CF3, -CHF2, -OCF3, -OCHF2, alkyl, alkenyl, alkynyl, and cycloalkyl; provided that at least one of R4, Rs, Rs, R7, and Rs is not -H.

In certain embodiments, R4, Rs, Rs, R7, and Rs are independently selected from -H, -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl; provided that at least one of R4, Rs, Rs, R7, and Rs is not -H.

In certain embodiments,

selected from -Cl, -Br, -F, -CN,

-CF3, -OCF3, -CH3, and cyclopropyl. Zy Rs

In certain embodiments, X2 is ; and R5 is selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

In certain embodiments,

selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

In certain embodiments,

are independently selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

In certain embodiments,

In certain embodiments,

In certain embodiments, X3 is -H, -CH3 or -CF3. In other embodiments, X3 is -CF3.

In certain embodiments, n is 1. In other embodiments, n is 2.

In certain embodiments, the compound having the structure:

In certain embodiments, the compound having the structure:

In certain embodiments, the compound having the structure:

In certain embodiments, the compound having the structure:

In certain embodiments, the compound having the structure:

In certain embodiments, the compound having the structure:

In some embodiments, the compound is selected from the following Table 1 :

Table 1.

In some embodiments, the compounds are atropisomers. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by the replacement of a hydrogen with deuterium or tritium, or of a carbon with a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. For example, in the case of variable R¹, the (Ci-C4)alkyl or the -O-(Ci-C4)alkyl can be suitably deuterated (e.g., -CD3, -OCD3).

Any compound of the invention can also be radiolabed for the preparation of a radiopharmaceutical agent.

Methods o f Treatment

One aspect of the invention provides compounds, compositions, and methods useful for treating or preventing a disease or disorder associated with abnormal levels of amino acids by modulation of SLC6A19 transport.

Another aspect of the invention relates to methods of treating or preventing a disease or disorder associated with a genetic defect in phenylalanine hydroxylase in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

In certain embodiments, the invention relates to methods of treating or preventing phenylketonuria in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

In certain embodiments, the invention relates to methods of treating or preventing hyperphenylalaninemia in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

In some embodiments, the compound reduces systemic phenylalanine levels in the subject.

In certain embodiments, the invention relates to methods of treating or preventing tyrosinemia (Type I, II, or III) in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

In certain embodiments, the compound reduces systemic glycine levels in the subject. In certain embodiments, the invention relates to methods of treating or preventing isovaleric acidemia, methylmalonic acidemia, propionic acidemia, maple syrup urine disease, DNAJC12 deficiency, urea cycle disorders, or hyperammonemia in a subject in need thereof comprising administering to the subject an effective amount of a compound of Formula (I).

In certain embodiments of any one of the disclosed methods, the compound modulates SLC6A19 in the subject.

In certain embodiments of any one of the disclosed methods, the compound inhibits SLC6A19 in the subject.

In certain embodiments of any one of the disclosed methods, the compound modulates SLC6A19 transport in the subject.

In certain embodiments of any one of the disclosed methods, the compound inhibits SLC6A19 transport in the subject.

In certain embodiments, the compound reduces systemic levels of an amino acid in the subject.

In certain embodiments of any one of the disclosed methods, wherein the subject is a mammal. In certain embodiments of any one of the disclosed methods, the mammal is a human.

In certain embodiments of any one of the disclosed methods, the compound of Formula (I) is defined as:

wherein: n is 0 or 1;

Li is absent or selected from -NH-, -N(CH3)-, -O-, and -CH2-;

L2 is -alkyl-

L3 is -(5-membered heteroaryl)-;

Xi is -C(RI)(R₂)(R₃);

X2 is an optionally substituted aryl or heteroaryl;

X3 is selected from -H, alkyl, and haloalkyl;

R2 and R3 are each independently selected from -H and alkyl; or R2 and R3 taken together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl or cycloheteroalkyl; or a pharmaceutically acceptable salt thereof.

In certain embodiments of any one of the disclosed methods, the compound is selected from the structure of any one of the compounds recited in Table 1.

In certain embodiments of any one of the disclosed methods, the compound is selected from:

wherein: n is 0 or 1;

Li is absent or selected from -NH-, -N(CH3)-, -O-, and -CH2-;

L2 is -alkyl-

L3 is -(5-membered heteroaryl)-;

Xi is -C(RI)(R₂)(R₃);

X2 is an optionally substituted aryl or heteroaryl;

X3 is selected from -H, alkyl, and haloalkyl;

or a pharmaceutically acceptable salt thereof.

Pharmaceutical Compositions, Routes of Administration, and Dosins

In certain embodiments, the invention is directed to a pharmaceutical composition, comprising a compound of the invention and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises a plurality of compounds of the invention and a pharmaceutically acceptable carrier.

In certain embodiments, a pharmaceutical composition of the invention further comprises at least one additional pharmaceutically active agent other than a compound of the invention.

Pharmaceutical compositions of the invention can be prepared by combining one or more compounds of the invention with a pharmaceutically acceptable carrier and, optionally, one or more additional pharmaceutically active agents.

As stated above, an “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the invention and/or other therapeutic agent without necessitating undue experimentation. A maximum dose may be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and “dosage” are used interchangeably herein.

In certain embodiments, intravenous administration of a compound may typically be from 0.1 mg/kg/day to 20 mg/kg/day. In one embodiment, intravenous administration of a compound may typically be from 0.1 mg/kg/day to 2 mg/kg/day. In one embodiment, intravenous administration of a compound may typically be from 0.5 mg/kg/day to 5 mg/kg/day. In one embodiment, intravenous administration of a compound may typically be from 1 mg/kg/day to 20 mg/kg/day. In one embodiment, intravenous administration of a compound may typically be from 1 mg/kg/day to 10 mg/kg/day.

Generally, daily oral doses of a compound will be, for human subjects, from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral doses in the range of 0.5 to 50 milligrams/kg, in one or more administrations per day, will yield therapeutic results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from one order to several orders of magnitude lower dose per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.

For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

The formulations of the invention can be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

For use in therapy, an effective amount of the compound can be administered to a subject by any mode that delivers the compound to the desired surface. Administering a pharmaceutical composition may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.

For intravenous and other parenteral routes of administration, a compound of the invention can be formulated as a lyophilized preparation, as a lyophilized preparation of liposome-intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a salt complex. Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.

For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts”, In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al., J Appl Biochem 4: 185-9 (1982). Other polymers that could be used are poly-1, 3-dioxolane and poly-1, 3, 6-tioxocane. For pharmaceutical usage, as indicated above, polyethylene glycol moieties are suitable.

For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the invention (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

The therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the compound of the invention (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, oc-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An anti -frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride. Potential non-ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound of the invention or derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For topical administration, the compound may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

For administration by inhalation, compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the compounds disclosed herein (or salts thereof). The compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63: 135-144 (1990) (leuprolide acetate); Braquet et al., J Car diovasc Pharmacol 13(suppl.

5): 143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med3.2Q6-2\2 (1989) (ocl- antitrypsin); Smith et al., 1989, J Clin Invest 84: 1145-1146 (a- 1 -proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated by reference). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 (incorporated by reference), issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for the dispensing of the compounds of the invention. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified compound of the invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise a compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active compound of the invention per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for inhibitor stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compound of the invention caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, di chlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing compound of the invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The compound of the invention (or derivative) should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers (pm), most preferably 0.5 to 5 pm, for most effective delivery to the deep lung.

Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, a compound may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249: 1527-33 (1990).

The compound of the invention and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt or cocrystal. When used in medicine the salts or cocrystals should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts or cocrystals may conveniently be used to prepare pharmaceutically acceptable salts or cocrystals thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical compositions of the invention contain an effective amount of a compound as described herein and optionally therapeutic agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The therapeutic agent(s), including specifically but not limited to a compound of the invention, may be provided in particles. Particles as used herein means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of the compound of the invention or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the compound of the invention in a solution or in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems. The term “controlled release” is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term “sustained release” (also referred to as “extended release”) is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term “delayed release” is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. “Long-term” release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the compositions and methods described herein are readily apparent from the description of the invention contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: SLC6A19 Isoleucine transport assay

Cell line generation and maintenance

The Flp-In™ T-REx™ 293 cell line was purchased from Thermo Fisher Scientific. The line was used to generate a stable cell line inducibly expressing human SLC6A19 with a C-terminal V5 tag and stably expressing human TMEM27 (also known as Collectrin) with a C-terminal myc-DDK tag. The stable cell line was generated by transfecting SLC6A19- and TMEM27-encoding plasmids using standard protocols, followed by antibiotic selection. Stable cells were maintained in DMEM/F12 supplemented with Glutamax, 10% fetal bovine serum, 100 U/mL penicillin, 100 ug/mL streptomycin, 200 ug/mL hygromycin, 10 ug/mL blasticidin and 300 ug/mL neomycin (Thermo Fisher).

Assay: Isoleucine transport assay in 96-well format Stable cell lines were seeded at a density of 35,000 cells per well in a poly-D-lysine coated 96-well cell culture-treated plate on day 0. On day 1 the expression of SLC6A19 was induced by dispensing tetracycline at a final concentration of 1 ug/mL using a Tecan D300e digital dispenser. On day 2 the transport assay was run. Media was removed from the plate using the GentleSpin setting of a Centrifugal Blue Washer (Blue Cat Bio) and cells were washed with 175 uL live cell imaging solution (Thermo Fisher) using the Blue Washer. Following washing, cells were treated with 70 uL of either DMSO, positive control or compound, diluted in Krebs buffer (140 mM NaCl, 4.7 mM KC1, 2.5 mM CaCL, 1.2 mM MgCh, 11 mM HEPES, 10 mM Glucose, pH 7.4) at room temperature. After 20-60 minutes 30 uL of a 3.3 mM solution of 13C6,15N-L-isoleucine (Cambridge Isotope Laboratories) was added. After 20 min incubation with the isoleucine substrate at room temperature cells were washed with 175 uL live cell imaging solution using the Blue Washer. Cells were then lysed in 150 uL of 15 uM D-Leucine-dlO (CDN Isotopes) in ultrapure water. Plates were put on a shaker at 700 rpm for a minimum of 40 minutes to facilitate lysis. Following lysis, a standard dilution curve of 13C6,15N-L-isoleucine was added to wells containing lysates of untreated cells. Plates were returned to the shaker for a minimum of 2 minutes to ensure proper mixing of the standard curve. Plates were then centrifuged for 5 min at 4,000 rpm to pellet cellular debris and precipitate. Supernatants were diluted 1: 10 in acetonitrile + 0.1% formic acid in polypropylene plates.

Assay: Isoleucine transport assay in 384-well format

On day 0, stable cell lines were seeded at a density of 20,000 cells per well in a poly- D-lysine coated 384-well cell culture-treated plate in media containing 1 ug/mL tetracycline using a Viaflo 384-well pipette. Transport assays were run the following day (day 1). Media was removed from the plate using the GentleSpin setting of a Centrifugal Blue Washer (Blue Cat Bio) and cells were washed with 80 uL live cell imaging solution (Thermo Fisher) using the Blue Washer. Following washing, cells were treated with 20 uL of either DMSO, positive control or compound, diluted in Krebs buffer (140 mM NaCl, 4.7 mM KC1, 2.5 mM CaCL, 1.2 mM MgCL, 11 mM HEPES, 10 mM Glucose, pH 7.4) using a TECAN liquid handler. After 20-60 minutes incubation at room temperature 8.6 uL of a 3.3 mM solution of 13C6,15N-L-isoleucine (Cambridge Isotope Laboratories) was added. After 20 min incubation with the isoleucine substrate at room temperature cells were washed with 80 uL live cell imaging solution using the Blue Washer. Cells were then lysed in 80 uL of 15 uM D- Leucine-dlO (CDN Isotopes) in ultrapure water. Plates were put on a shaker at 700 rpm for a minimum of 2 hours to facilitate lysis. Following lysis, a standard dilution curve of 13C6,15N-L-isoleucine was added to wells containing lysates of untreated cells. Plates were returned to the shaker for a minimum of 5 minutes to ensure proper mixing of the standard curve. Plates were then centrifuged for 10 min at 4,000 rpm to pellet cellular debris and precipitate. Supernatants were diluted 1 : 10 in acetonitrile + 0.1% formic acid in polypropylene plates.

13C6,15N-L-isoleucine analysis was performed using a RapidFire365-QTOF 6545 (Agilent). Quantitative sample analysis utilizes automated solid-phase extraction (HILIC H6 cartridge) prior to mass spec injection. Samples were loaded using 95% acetonitrile, 0.1% formic acid and eluted from the cartridge with 5% acetonitrile, 0.1% formic acid directly for ESI-MS (electrospray ionization) analysis. Quantification of the analytes were performed using Agilent Masshunter Quant software from the high-resolution full scan data.

Example 2: Synthesis of Exemplary Compounds

Procedure 1: Synthesis of (S}-l-(tert-butoxycarbonyl}-3-(trifluoromethyl}pyrrolidine-3- carboxylic acid

Step 1: To a mixture of 2-(trifluoromethyl)acrylic acid (15.0 g, 107.1 mmol) and A-benzyl-1- methoxy-A-((trimethylsilyl)methyl)methanamine (25.4 g, 107.1 mmol) in DCM (150 mL) was added TFA (1.22 g, 10.7 mmol) dropwise at 0° C. The resulting mixture was stirred at room temperature for 17 hrs. Then the mixture was diluted with petroleum ether (600 mL) and filtered after stirring at room temperature for 30 mins. Then the filter cake was washed with a solution of PE:EtOAc = 3: 1 (60 mL) and concentrated under vacuum to give Al (24.5 g) as white solid.

The enantiomers of Al could be separated by chiral SFC (Shimadzu E-UC SFC; CHIRALPAK IC, 5*25 cm, 5pm) to afford (R)-A1 (10.2 g, 34.85 % yield) and (S)-Al (12.1 g, 41.35 % yield) as white solid. LC/MS (ESI) m/z: 274 (M+H)⁺. Step 2:To a solution of (S)-Al (8.0 g, 29.28 mmol) in MeOH (150 mL) was added 20% of Pd/C (1.6 g, w/w). The resulting mixture was stirred at room temperature for 2 hrs under H2 atmosphere. Then the mixture was filtered and the filtrate was concentrated under vacuum to give (S)-3-(trifluoromethyl)pyrrolidine-3-carboxylic acid (5.1 g, 95.12% yield) as light-yellow oil which was used at the next step directly without further purification. LC/MS (ESI) m/z: 184 (M+H)⁺.

Step 3: To a mixture of (S)-3-(trifluoromethyl)pyrrolidine-3 -carboxylic acid (5.1 g, 27.85 mmol) and TEA (5.64 g, 55.70 mmol) in DCM (70 mL) was added BOC2O (6.69 g, 30.63 mmol) drop-wise at 0 °C. The resulting mixture was stirred at room temperature for 2 hrs. Then the mixture was concentrated under vacuum and the crude product was purified by column chromatography on silica gel (DCM: MeOH = 100: 0 to 12: 1) to give 5 (7.5 g, 95.08% yield) as colorless oil. LC/MS (ESI) m/z: 282 (M-H)'.

Procedure 2: Synthesis of Aryl Amidoximes

Aryl amidoximes referenced below could be prepared according to an appropriate modification of the following procedure from the appropriately substituted aryl nitrile.

Synthesis of 4-cyano-N-hydroxybenzimidamide (Bl}

To a mixture of terephthalonitrile (12.8 g, 99.90 mmol) and TEA (11.12 g, 109.88 mmol) in EtOH (250 mL) was added hydroxylamine hydrochloride (6.94 g, 99.90 mmol). The resulting mixture was stirred at 70 °C for 2 hrs. After the SM was consumed, the mixture was concentrated under vacuum and the crude product was purified by column chromatography on silica gel (DCM: MeOH = 100: 0 to 10: 1) to give Bl (11.20 g, 69.57% yield) as yellow solid. LC/MS (ESI) m/z: 162 (M+H)⁺.

Procedure 3: Synthesis of (S}-3-Aryl-5-(3-(trifluoromethyl}vyrrolidin-3-yl}-l,2,4- oxadiazoles 1,2,4-oxadiazoles of type C2 referenced below could be prepared according to an appropriate modification of the following procedure from substituted aryl amidoxines such as Bl and (S)- A2 or racemic A2.

Synthesis of 4-cyano-N-hydroxybenzimidamide (C2)

Step 1: To a solution of (S)-A2 (7.5 g, 26.48 mmol) in DMF (250 mL) was added CDI (6.44 g, 39.72 mmol). The mixture was stirred at 85 °C for 1 hr. Then Bl (5.50 g, 34.42 mmol) was added into the above mixture. The resulting mixture was stirred at 85 °C for another 10 hrs. After cooling, the mixture was diluted with water (100 mL) and washed with EtOAc (60 mL) twice. The combined organic layers were separated, washed with saturated NH4CI solution (80 mL) and brine (80 mL), dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (PE: EtOAc = 100: 0 to 8: 1) to give Cl (9.60 g, 88.78% yield) as colorless oil. LC/MS (ESI) m/z: 409 (M+H)⁺.

Step 2: To a solution of 4 N HCl/dioxane solution (120 mL) was added Cl (9.60 g, 23.51 mmol) in portions at 0 °C under N2 atmosphere. The resulting mixture was stirred at room temperature for 2 hrs. Then the mixture was concentrated under reduced pressure to give crude C2 (7.25 g, 89.51% yield) as light yellow oil which was used at the next step directly without further purification. LC/MS (ESI) m/z: 309 (M+H)⁺.

Examples 1 - 42: The compounds in the table below could be prepared from a substituted pyrrolidine such as C2 and the appropriate Boc-protected diamine such as DI according to a suitable modification of the following procedure.

Step 1: To a mixture of C2 (5.65 g, 28.22 mmol) and DIEA (9.12 g, 70.56 mmol) in DMF (80 mL) was added CDI (4.58 g, 28.22 mmol) at 0 °C. The mixture was stirred at room temperature for 30 mins. Then DI (7.25 g, 23.51 mmol) was added into the above mixture. The resulting mixture was stirred at 50 °C for 3 hrs. After cooling, the mixture was quenched with aq. HC1 (120 mL, 2N) and extracted with EtOAc (100 mL) twice. The combined organic layers were separated, washed with brine (120 mL), dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel (DCM: MeOH = 100: 0 to 18: 1) to give D2 (11.6 g, 92.27% yield) as off-white solid. LC/MS (ESI) m/z: 435 (M-100+H)⁺.

Step 2: To a solution of 4N HCl/dioxane (120 mL) at 0 °C under was added D2 (11.6 g, 21.7 mmol) under a N2 atmosphere. The resulting mixture was stirred at room temperature for 2 hrs. Then the mixture was concentrated under reduced pressure to give crude product (10.6 g) as light yellow oil. Then the crude product was diluted with MTBE (100 mL) and the resulting slurry was filtered after stirring at r.t. for 5 hrs. The filter cake was washed with MTBE (100 mL) and concentrated under vacuum to give D3 (7.10 g, 75.31% yield) as white solid.

Examples 43 - 66: The compounds in the table below could be prepared from a substituted pyrrolidine such as E2, itself prepared according to procedure 3, and the appropriate diamine such as El according to a suitable modification of the following procedure.

Step 1 : To a mixture of E2 (156 mg, 0.440 mmol) and DIEA (74 mg, 0.572 mmol) in DMF (1 mL) was added CDI (82 mg, 0.572 mmol) in portions at 0 °C. The reaction mixture was stirred at room temperature for 40 mins. Then El (88 mg, 0.88 mmol) was added into the above mixture and the resulting mixture was stirred at 50 °C for 3 hrs. Then the mixture was concentrated to dryness. The residue was purified by preparative reverse phase HPLC to give E3 as a colorless oil. LC/MS (ESI) m/z: 444 (M+H)⁺.

Examples 67 - 73: The compounds in the table below could be prepared from a substituted pyrrolidine such as C2 and the appropriate diamine such as Fl according to a suitable modification of the following procedure.

Step 1: To a mixture of C2 (40 mg, 0.19 mmol) and DIEA (34 mg, 0.259 mmol) in DMF (0.5 mL) was added CDI (33 mg, 0.234 mmol). The reaction mixture was stirred at room temperature for 15 mins. Then Fl (40 mg, 0.194 mmol) was added into the above mixture and the resulting mixture was stirred at 50 °C for 3 hrs. Then the mixture was concentrated to dryness. The residue was taken up in 4N HCl/Dioxane solution (Im) and stirred for 30m, then concentrated again to dryness to afford F2, which was used without further purification.

LC/MS (ESI) m/z: 439 (M+H)⁺.

Step 2: To a solution of F2 HC1 (43mg, 98 umol) in methanol (1.5mL) was added DIPEA(38 mg, 292 umol) and 37% formaldehyde solution (22 uL, 292 umol). Sodium triacetoxyborohydride (42 mg, 195 umol) was added and the solution was stirred at room temperature. Additional portions of sodium triacetoxyborohydride were added until LCMS indicated full consumption of the starting material. The resultant solution was directly purified by preparative reverse phase HPLC to give F3 as a colorless oil. LC/MS (ESI) m/z: 453 (M+H)⁺.

Examples 74 - 76: The compounds in the table below could be prepared from l-(tert- butoxycarbonyl)-4-(trifluoromethyl)piperidine-4-carboxylic acid, the appropriate diamine, and the appropriate aryl nitrile by methods analogous to those used to prepare the pyrrolidines above.

Procedure 3: Synthesis of (S}-3-Aryl-5-(3-(trifluoromethyl}vyrrolidin-3-yl}-l,2,4- oxadiazoles

1,2,4-oxadiazoles of type G5 referenced below could be prepared according to an appropriate modification of the following procedure from nitrile G1 and an appropriately substituted benzoate such as G3 according to the following procedure.

Step 1: To a solution of G1 (1.07 g, 4.21 mmol) in ethanol (10 mL) was added hydroxylamine hydrochloride (585mg, 8.42 mmol) and triethylamine (1.17 mL, 8.42 mmol). The solution was heated at 60C for two hours, then cooled to room temperature. The solution was diluted with water and ethyl acetate and the phases separated. The aqueous phase was extracted twice more with ethyl acetate, and then the combined organic phases were washed with water, satd. Aq. NaCl, dried over MgSO4 and concentrated to give G2 (1.3g, 4.53 mmol) as a white solid which was used without further purification. LC/MS (ESI) m/z: 288.0 (M+H)⁺.

Step 2: To a mixture of G2 (324mg, 1.13mmol) and G3 (176 mg, 1.13mol) in dioxane (3 mL) was added DCC (256 mg, 1.24 mmol) at room temperature. The solution was then heated at 90C overnight, then cooled to room temperature. The precipitate was removed by filtration and the filtrate concentrated to an oil, which was then purified by column chromatography (0-100% Ethyl Acetate/Heptane) to afford the desired oxadiazole G4 (125.4 mg) as a colorless residue. LC/MS (ESI) m/z: 399.1 (M+H)⁺.

Step 3: To a solution of G4 (125.4 mg, 0.294 mmol) in DCE (1 mL) was added 1-chloroethyl chloroformate (67 uL, 0.614 mmol) at room temperature. The solution was stirred at 50C for 75m, then methanol (ImL) was added and the solution was heated at 60C for one hour. The solution was then concentrated to afford G5 HC1, which was used without further purification. LC/MS (ESI) m/z: 309.1 (M+H)⁺.

Examples 77 - 79: The compounds in the table below could be prepared from a 1,2,4- oxadiazoles of type G5 and the appropriate diamine by suitable modification of the methods reported above.

Example 80: Example 80 was prepared according to the following method.

Step 1: To a mixture of Al (210 mg, 0.77 mmol) in DMF (5 mL) was added DIPEA (199 mg, 1.54 mmol) and 2-bromo-l-(4-bromophenyl)ethan-l-one (213 mg, 0.77 mmol). The resulting mixture was stirred at room temperature for 3 hrs. Then the mixture was diluted with water (20 mL) and extracted with EtOAc (15 mL) twice. The combined organic layers were washed with saturated NH4CI solution and brine, dried over anhydrous Na2SC>4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (eluted with PE: EtOAc= 100: 0 to 40: 1) to give Hl (350 mg, 97.01 % yield) as colorless oil. LC/MS (ESI) m/z: 470/472 (M+H)⁺.

Step 2: To a solution of Hl (350 mg, 0.75 mmol) in toluene (15 mL) were added acetamide (1.28 g, 21.75 mmol) and Boron trifluoride diethyl etherate (1.5 mL, 0.73 mmol). The resulting mixture was stirred at 150 °C for 6 hrs in a seal tube. The reaction was continued until TLC indicated full consumption of starting material (PE: EtOAc= 10: 1). Then the mixture was quenched with saturated NaHCOs solution (40 mL) at 0 °C and extracted with EtOAc (30 mL><2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (PE: EtOAc= 100: 0 to 100: 3) to give H2 (135 mg, 40.20 % yield) as colorless oil. LC/MS (ESI) m/z: 451/453 (M+H)⁺.

Step 3: To a mixture of H2 (135 mg, 0.30 mmol) and Zn(CN)2 (53 mg, 0.45 mmol) in DMF (7 mL) was added Pd(PPh3)4 (35 mg, 0.03 mmol) and the resulting mixture was stirred at 120 °C for 16 hrs under N2 atmosphere. Then the mixture was diluted with H2O (20 mL) and extracted with EtOAc (15 mL) twice. The combined organic layers were washed with saturated NH4CI solution and brine, dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (eluted with PE: EtOAc= 100: 1 to 4: 1) to give H3 (83 mg, 69.82 % yield) as colorless oil. LC/MS (ESI) m/z: 398 (M+H)⁺.

Step 4: To a solution of H3 (83 mg, 0.21 mmol) in DCM (5 mL) was added chloroethyl chloroformate (90 mg, 0.63 mmol), the resulting mixture was stirred at 50 °C for 20 hrs under N2 atmosphere. Then MeOH (3 mL) was added into the above mixture and the mixture was stirred at 75 °C for another 2 hrs. Then the mixture was concentrated to give crude H4 HC1 (64 mg, 99.71 % yield) as colorless oil. LC/MS (ESI) m/z: 308 (M+H)⁺.

Step 5: To a solution of DI HC1 (55 mg, 0.27 mmol) in DMF (4 mL) was added DIEA (76 mg, 0.59 mmol) and CDI (44 mg, 0.27 mmol), the mixture was stirred at 0 °C for 30 mins. Then H4 HC1 (64 mg, 0.21 mmol) was added into the above mixture, the resulting mixture was stirred at 50 °C for another 2 hrs. Then the mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL) twice. The combined organic layers were washed with saturated NH4CI solution and brine, dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (DCM: MeOH= 100: 0 to 100: 3) to give Boc-F5 (70 mg, 63.02 % yield) as colorless oil. LC/MS (ESI) m/z: 534 (M+H)⁺. A round-bottom flask was charged with Boc-H5 (70 mg, 0.13 mmol) and HCl/dioxane (4 mol/L, 4 mL) at 0 °C. The resulting mixture was stirred at room temperature for 1 hr. LCMS indicated the complete consumption of the starting material. Then the mixture was concentrated to dryness under reduced pressure. The residue was purified via prep-HPLC to give H5 (39.0 mg, 68.55 % yield) as white solid.

Example 81: Example 81 was prepared according to the following method.

Step 1: To a mixture of (rac)-A2 (5.18 g, 18.29 mmol) and DIEA (7.09 g, 54.86 mmol) in MeCN (100 mL) was added CDI (4.45 g, 27.43 mmol) at 0 °C. The mixture was stirred at 90 °C for 1 hr. Then N,O-Dimethylhydroxylamine hydrochloride (2.32 g, 23.77 mmol) was added into the above mixture. The resulting mixture was stirred at 90 °C for another 3 hrs. After cooling, the mixture was quenched with aq. HCI solution (100 mL, 2 N) and extracted with dichloromethane (100 mL) twice. The combined organic layers were washed brine (150 mL), dried over anhydrous Na2SC>4 and concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel (eluted with PE: EtOAc = 100:0 to 4: 1) to give II (5.09 g, 85.29 % yield) as white solid. LC/MS (ESI) m/z: 271 (M- 56+H)⁺.

Step 2: To a solution of II (2.0 g, 6.13 mmol) in dry DCM (50 mL) was added a solution of DIBAL-H in THF (18.4 mL, 1 M) dropwise at -78 °C under N2 atmosphere. The resulting mixture was stirred at -78 °C for 1 hr. Then the mixture was quenched with saturated potassium sodium tartrate tetrahydrate solution (50 mL) in portions at 0 °C and stirred at room temperature for 1 hr. Then the mixture was extracted with DCM (60 mL) twice. The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SC>4 and concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (eluted with PE: EtOAc = 100:0 to 20: 1) to give 12 (1.50 g, 91.58% yield) as white solid. LC/MS (ESI) m/z: 212 (M-56+H)⁺.

Step 3: To a solution of 12 (1.50 g, 5.61 mmol) in MeOH (50 mL) was added K2CO3 (2.33 g, 16.84 mmol) and (l-diazo-2-oxo-propyl)-phosphonic acid dimethyl ester (1.62 g, 8.42 mmol). The resulting mixture was stirred at room temperature for 2 hrs. Then the mixture was diluted with water (80 mL) and extracted with MTBE (80 mL) twice. The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4 and concentrated to dryness. The residue was purified by column chromatography on silica gel (eluted with PE: EtOAc = 100:0 to 40: 1) to give 13 (1.25 g, 84.60% yield) as white solid.

Step 4: To a mixture of 4-Azidobenzonitrile (70 mg, 0.48 mmol) and 13 (140 mg, 0.53 mmol) in DMSO (6 mL) was added Q1SO4 (8 mg, 0.05 mmol) and Sodium ascorbate (77 mg, 0.39 mmol) at room temperature. The resulting mixture was stirred at 60 °C for 3 hrs under N2 atmosphere. Then the mixture was diluted with water (30 mL) and extracted twice with DCM (15 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by column chromatography on silica gel (eluted with PE: EtOAc = 100:0 to 4: 1) to give Boc-I4 (94 mg, 47.51 % yield) as yellow oil. LC/MS (ESI) m/z: 408 (M+H)⁺. To the vessel containing Boc- 14 (94 mg, 0.23 mmol) was added 4 N HCl/dioxane solution (5 mL) at 0 °C under N2 atmosphere. The resulting mixture was stirred at room temperature for 2 hrs. Then the mixture was concentrated under reduced pressure to give crude 14 HC1 (75 mg, 95.07% yield) as light yellow oil which was used at the next step directly without further purification. LC/MS (ESI) m/z: 308 (M+H)⁺. Step 5: To a mixture of DI HCI (59 mg, 0.29 mmol) and DIPEA (95 mg, 0.73 mmol) in DMF (8 mL) was added CDI (47 mg, 0.29 mmol) at 0 °C. The mixture was stirred at room temperature for 1 hr. Then 14 HCI (75 mg, 0.23 mmol) was added into the above mixture and the resulting mixture was stirred at 40 °C for another 3 hrs. After cooling, the mixture was quenched with saturated NaHCOs aqueous (20 mL) and extracted with EtOAc (15 mL) twice. The combined organic layers were washed with saturated NH4CI solution (30 mL) and brine (30 mL), dried over anhydrous Na2SO4 and concentrated to dryness. The crude product was purified by column chromatography on silica gel (DCM: MeOH = 100: 0 to 15: 1) to give Boc-I5 (55 mg, 42.23% yield) as off-white solid. LC/MS (ESI) m/z: 434 (M-100+H)⁺. Boc- 15 (55 mg, 0.10 mmol) was added into 4 N HCl/dioxane solution (5 mL) in portions at 0 °C under N2 atmosphere. The resulting mixture was stirred at room temperature for 2 hrs. Then the mixture was concentrated under reduced pressure to dryness. The residue was purified via prep-HPLC to give 15 (18 mg, 40.29% yield) as colorless oil.

Example 82: Example 82 was prepared according to the following method.

To a solution of JI (15 mg, 53 umol) and 5-((tert-butoxycarbonyl)amino)pentanoic acid (15 mg, 69 umol) in DMF (0.5 mL) was added DIPEA (28.5 uL, 106 umol) and HATU (26.3 mg, 69 umol). The solution was stirred at room temperature, then concentrated to a residue, which was taken up in 4N HCl/Dioxane (1 mL). The solution was held at room temperature, then concentrated to a residue. The product was taken up in methanol then purified by preparative reverse phase HPLC to afford J2.

Example 83: Example 83 was prepared according to the following method.

Step 1: To a solution of (rac)-A2 (108 mg, 381 umol) in THF (2 mL) was added 4- Chlorobenzoic acid hydrazide (65.1 mg, 381 umol), Triethylamine (159 uL, 1.14mmol), and T3P (587 mg, 953 umol). The solution was heated at 75C for 3h, then at room temperature overnight. The solution was poured into water and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed twice with satd. Aqueous sodium bicarbonate, then washed once with brine, dried over MgSO4, filtered and concentrated to a residue which was purified by column chromatography to afford Hl as a white solid (117mg, 270 umol) LC/MS (ESI) m/z: 335.9 (M-100+H)⁺.

Step 2: To a solution of Hl (118 mg, 268 umol) in MeCN (ImL) was added DIPEA (94 uL, 547 umol) and pTsCl (202 mg, 536 umol). The solution was stirred at room temperature for three hours, then diluted with water and ethyl acetate. The phases were separated and the organic phase was washed with satd. aq. NaHCO3, dried over MgSO4, filtered, and concentrated. The product was purified by column chromatography to afford H2 (85 mg, 203 umol) as a colorless oil. LC/MS (ESI) m/z: 362.2 (M-57+H)⁺.

Step 3: HCl/Dioxane (1 mL) was added to vial containing H2 (85 mg, 203 umol). The solution was maintained at room temperature for 30m, then concentrated to a afford H3 as a colorless residue, which was used without further purification.

Step 4a: To a suspension of CDI (2.11g, 14.68mmol) in DCM (lOmL) was added El (974 mg, 9.72 mmol) at 0C. The solution was stirred at room temperature for fifteen minutes, then concentrated to a residue and purified by column chromatography (0-100% DCM/Methanol). The product H4 (1.74g) was recovered as a thick oil.

Step 4b: To a solution of H3 (40mg, 113 umol) and DIPEA (40 uL, 225 umol) in DMF (0.5 mL) was added H4 (44mg, 225 umol). The solution was heated at 50C for 2h, then cooled to room temperature. The reaction mixture was directly purified by reverse phase preparative HPLC to afford H5 as a colorless oil.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

A compound of Formula (I):

wherein: n is 0 or 1;

Li is absent or selected from -NH-, -N(CH3)-, -O-, and -CH2-;

L2 is -alkyl-

L3 is -(5-membered heteroaryl)-;

Xi is -C(RI)(R₂)(R₃);

X2 is an optionally substituted aryl or heteroaryl;

X₃ is selected from -H, alkyl, and haloalkyl;

Ri is selected from -H, halo, hydroxyl, amido, amino, alkylamino, and aminoalkyl;

R2 and R₃ are each independently selected from -H and alkyl; or R2 and R₃ taken together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl or cycloheteroalkyl; provided the compound is not selected from

or a pharmaceutically acceptable salt thereof.

The compound of claim 1 having the structure:

3. The compound of claim 1 or 2, wherein Li is selected from -NH- and -N(CH₃)-.

4. The compound of any one of claims 1-3, wherein L2 is selected from -CH2-

-CH2CH2-, and -CH2CH2CH2-.

5. The compound of any one of claims 1-4, wherein Ri is selected from -H, -F, -OH, -NH₂, -CH2NH2, -N(H)(CH₃), -N(CH₃)₂, and -C(O)NH₂.

6. The compound of claim 5, wherein Ri is -H.

7. The compound of claim 5, wherein Ri is -NH2.

8. The compound of any one of claims 1-7, wherein R2 and R₃ are each -H.

9. The compound of any one of claims 1-7, wherein R2 and R₃ are each -CH₃.

10. The compound of any one of claims 1-7, wherein R2 and R₃ taken together with the carbon atom to which they are bonded form an optionally substituted cycloalkyl.

11. The compound of claim 10, wherein R2 and R₃ taken together with the carbon atom to which they are bonded form an unsubstituted cyclopropyl or cyclobutyl.

12. The compound of any one of claims 1-7, wherein R2 and R₃ taken together with the carbon atom to which they are attached form an optionally substituted cycloheteroalkyl.

13. The compound of claim 12, wherein R2 and R₃ taken together with the carbon atom to which they are bonded form an unsubstituted azetidinyl, pyrrolidinyl, piperidinyl, or lactam.

14. The compound of claim 12, wherein R2 and R3 taken together with the carbon atom to which they are bonded form a substituted azetidinyl, pyrrolidinyl, piperidinyl, or lactam.

15. The compound of claim 14, wherein the azetidinyl, pyrrolidinyl, piperidinyl, or lactam is A -alkyl or /'/-acetyl substituted.

17. The compound of any one of claims 1-16, wherein L3 is triazolyl, oxazolyl or oxadi azolyl.

18. The compound of any one of claims 1-17, wherein -L3-X2 is selected from

19. The compound of any one of claims 1-17, wherein -L3-X2 is

20. The compound of any one of claims 1-19, wherein X2 is unsubstituted aryl.

21. The compound of claim 20, wherein the unsubstituted aryl is unsubstituted phenyl.

22. The compound of any one of claims 1-19, wherein X2 is substituted aryl.

23. The compound of claim 22, wherein the substituted aryl is substituted phenyl.

The compound of claim 23, wherein

R4, Rs, Rs, R7, and Rs are independently selected from -H, halogen, -CN, -CF3, -CHF2, -OCF3, -OCHF2, alkyl, alkenyl, alkynyl, and cycloalkyl; provided that at least one of R4, Rs, Rs, R7, and Rs is not -H.

25. The compound of claim 24, wherein R4, R5, Rs, R7, and Rs are independently selected from -H, -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl; provided that at least one of R4, Rs, Rs, R7, and Rs is not -H.

The compound of claim 25, wherein

R4 is selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

The compound of claim 25, wherein X2 is

; and

Rs is selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, CH3, and cyclopropyl.

The compound of claim 25, wherein

Rs is selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

The compound of claim 23, wherein

Rs and Rs are independently selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

30. The compound of claim 23, wherein

R₄ and Re are independently selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

31. The compound of claim 23, wherein

Rs, Rs, and R7 are independently selected from -Cl, -Br, -F, -CN, -CF3, -OCF3, -CH3, and cyclopropyl.

The compound of claim 36, having the structure:

The compound of claim 36, having the structure:

The compound of claim 36, having the structure:

A compound or a pharmaceutically acceptable salt thereof having the structure of any one of the compounds recited in Table 1. A pharmaceutical composition, comprising a compound of any one of claims 1-43; and a pharmaceutically acceptable excipient. A method of treating or preventing a disease or disorder associated with a genetic defect in phenylalanine hydroxylase, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. A method of treating or preventing phenylketonuria, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. A method of treating or preventing hyperphenylalaninemia, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. The method of any one of claims 45-47, wherein the compound reduces systemic phenylalanine levels in the subject. A method of treating or preventing tyrosinemia (Type I, II, or III), comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. The method of claim 49, wherein the compound reduces systemic tyrosine levels in the subject. A method of treating or preventing nonketotic hyperglycinemia, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. The method of claim 51, wherein the compound reduces systemic glycine levels in the subject. A method of treating or preventing isovaleric acidemia, methylmalonic acidemia, propionic acidemia, maple syrup urine disease, DNAJC12 deficiency, urea cycle disorders, or hyperammonemia, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. A method of treating or preventing diabetes, chronic kidney disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, metabolic syndrome, obesity related disorders, or neurodevelopmental and autism-spectrum disorders, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-43. The method of any one of claims 45-54, wherein the compound inhibits SLC6A19 in the subject.