METHODS AND COMPOSITIONS FOR TREATMENT OR PREVENTION OF SYMPTOMS OF CLASSIC GALACTOSEMIA

CROSS-REFERENCE TO RELATED APLICATIONS

[0001] This claims priority to U.S. Provisional Patent Application No. 62/520,749, filed on June 16, 2017 and U.S. Provisional Patent Application No. 62/584,662, filed on

November 10, 2017, the entire contents of each of which are fully incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to methods for treating and preventing symptoms related to galactosemia.

BACKGROUND

[0003] Galactosemia is an inherited metabolic enzyme disorder transmitted as an autosomal recessive trait. Galactosemia is characterized by the inability of the body to metabolize the simple sugar galactose, causing the accumulation of galactose 1 -phosphate (GIP) in the body, which, in turn, can cause damage to the liver, central nervous system (e.g., brain), kidneys, eyes, and other body systems. Individuals with galactosemia cannot tolerate any form of milk, human or animal, and must carefully watch their intake of dairy and any other galactose-containing foods. There are 3 forms of the disease: galactose- 1 -phosphate uridyltransferase deficiency or classic galactosemia, galactose kinase deficiency and galactose-6-phosphate epimerase deficiency.

[0004] Classic galactosemia is a potentially lethal form that results from deleterious mutations in the GALT gene, which encodes the enzyme galactose- 1 -phosphate

uridylyltransferase. GALT is the second enzyme in the Leloir pathway of galactose metabolism, which catalyzes the conversion of galactose-1 -phosphate (gal-IP) and UDP- glucose to UDP-galactose and glucose- 1 -phosphate (FIG. 1). Although newborn screening programs and a galactose-restricted diet resolve the potentially lethal acute toxicity syndrome in the neonatal period, they fail to avert the long-term complications that include growth restriction, intellectual deficit, ataxia, speech dyspraxia and primary ovarian insufficiency (POI) that are frequently seen in many patients. The molecular mechanisms of these acute and long-term complications are still poorly understood despite several hypotheses that have been presented in the literature. Therefore, there remains a need for new therapeutic approaches for these long-term complications of classic galactosemia. BRIEF SUMMARY

[0005] Disclosed herein are methods and compositions for treating and preventing symptoms related to galactosemia.

[0006] In one aspect, the disclosure provides a method of treating or preventing a symptom of galactosemia comprising administering to a subject with galactosemia, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof,

(I)

wherein

R ¹ is CC1 ₃ , CHCI2, or CBr ₃ ;

R ² is aryl, heteroaryl, or a C3- ₈ cycloalkyl, wherein R ² is optionally substituted with 1-4

substituents independently selected from halogen, cyano, Ci- ₄ alkyl, -OCi- 4alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), and -N(Ci-4alkyl) ₂ ;

L ¹ is -C(R ⁴ ) ₂ -C(R ⁴ - -CR ⁴ =CR ⁴ -, -≡-, or "¾ ^ ;

G ¹ is -C(0)NH-R ³ , , -C(0)G ² , or -C(S)G ² ;

R ³ is G ³ , -Ci _-3 alkylene-G ³ , Ci _-6 alkyl, or -C _2-6 alkylene-OCi _-4 alkyl;

R ⁴ , at each occurrence, is independently hydrogen or Ci-4alkyl;

G ² is a 4- to 8-membered monocyclic heterocyclyl containing 1 or 2 heteroatoms

independently selected from nitrogen, oxygen, and sulfur and optionally substituted with 1-4 substituents independently selected from halogen, Ci- ₄ alkyl, and -OCi- 4alkyl; and

G ³ is aryl, heteroaryl, or C3- ₈ cycloalkyl, wherein G ³ is optionally substituted with 1-4

substituents independently selected from halogen, cyano, Ci- ₄ alkyl, -OCi- 4alkyl, -C(0)OCi _-4 alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), -N(Ci _-4 alkyl) ₂ , -C(0)NH ₂ , - C(0)NH(Ci _-4 alkyl), and -C(0)N(Ci _-4 alkyl) ₂ .

[0007] This disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying figures. BRIEF DESCRIPTIONS OF THE DRAWINGS

[0008] FIG. 1 is a schematic of the proposed pathological role of Endoplasmic Reticulum (ER) stress in disease-relevant phenotypes in the GalT-deficient mouse model. The Leloir pathway of galactose metabolism is high-lighted in green. UDP-galactose can also be produced via the UDP-4' galactose epimerase (GALE) reaction (not shown). In the absence of GALT activity, accumulation of toxic galactose metabolites may lead to elevated level of ER stress, which in turns may cause reduced PI3K/Akt signaling. In susceptible tissues, such attenuated PI3K/Akt signaling could lead to tissue-specific disease phenotypes.

[0009] FIG. 2A, FIB. 2B, FIG. 2C and FIG. 2D show the relative protein abundance of key regulators of PI3K/Akt pathway in normal and GalT-deficient mice. Protein expression levels of pAkt (Ser473), pGsk3 , Hsp90 and BiP from ovaries (FIG. 2A) and cerebella (FIG. 2C) of normal (NN) and mutant mice (GG) (n = 3 for each group) were compared by Western Blot analysis. Quantified results for the abundance of proteins from ovaries (FIG. 2B) and cerebella (FIG. 2D) are shown in bar graphs.

[0010] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E and FIG. 3F show the expression of PI3K/Akt signaling proteins in GalT-deficient mice tissues of different age groups. Protein expression levels of pAkt473, /?a«Akt, BiP, Hsp90 and pGsk3 from ovaries and cerebella of three wild type and GalT-deficient mice were compared by western blotting. The quantified results of the abundance of the proteins were depicted on the graphs on right. FIG. 3A and FIG. 3B show results from ovaries isolated from 4-month-old Normal (NN) and GalT- deficient mice (GG). FIG. 3C and FIG. 3D show results from cerebella isolated from 4- month-old Normal (NN) and GalT- deficient mice (GG). FIG. 3E and FIG. 3F show results from ovaries isolated from 8-month-old Normal (NN) and GalT- deficient mice (GG).

[0011] FIG. 4A and FIG. 4B show the effects of salubrinal of PI3K/Akt signaling in GalT-deficient mouse fibroblasts. FIG. 4A shows the Western Blot analysis of expression levels of pAkt (Ser473), pGsk3 , and BiP from a Salubrinal -treated mutant fibroblast line at different durations. FIG. 4B is a bar graph quantifying the results for the abundance of the selected proteins from three different mutant fibroblast lines with Salubrinal treatment.

[0012] FIG. 5A, FIG. 5B. FIG. 5C and FIG. 5D show the role of Salubrinal in alleviating ER stress and upregulating PI3K/Akt signaling in GalT-deficient mice. Mice were treated with DMSO (n=3) and Salubrinal (5mg/kg) (n=4) respectively per os once daily for 14 days. The protein expression level of BiP, pAkt(Ser473), Hsp90 and pan Akt from ovaries (FIG. 5A) and cerebella (FIG. 5C) were detected with Western Blot analysis. Quantitatively analyzed data from ovaries (FIG. 5B) and cerebella (FIG. 5D) are shown in bar graphs.

[0013] FIG. 6A and FIG. 6B show histological studies of cerebella of untreated, vehicle- treated and Salubrinal-treated GalT-deficient mice. Mice were treated with DMSO (vehicle) (n=4) and Salubrinal (5mg/kg) (n=4) respectively per os once daily for 21 days. FIG. 6A shows the density and distribution of Purkinje cells in untreated-control, vehicle-treated control and Salubrinal- treated control. FIG. 6B is a bar graph showing the quantitation of the number of Purkinje cells from untreated-control, vehicle-treated control and Salubrinal- treated control.

[0014] FIG. 7 is a bar graph showing the effects of Salubrinal on the number of primordial follicles in GalT-deficient mice. Primordial follicles were counted in an ovary collected from animals that were either untreated, treated with Salubrinal, or treated with vehicle alone for 21 days (n=3 for Untreated animals, n=4 for vehicle-treated animals, and n=4 for Salubrinal-treated group). Letters denote statistically different mean primordial follicle number as calculated by ANOVA, /? < 0.05.

[0015] FIG. 8 is a graph showing the effect of Salubrinal treatment on ovulation. Wild type and GalT-deficient mice at 8 weeks of age were treated with vehicle (VEH; DMSO) or Salubrinal (Sal) at 5 mg/kg per os daily for 21 days. Ovulated oocytes were collected from oviducts 16 hours following hCG injection and counted (n=6 in each group). An untreated group of mice (n=6) were also kept as controls.

[0016] FIG. 9A and FIG. 9B are images showing the effect of Salubrinal treatment on embryo development in wild type and GalT-deficient oocytes. Mice at 8 weeks of age were treated with salubrinal (SAL) at 5 mg/kg or vehicle (DMSO) daily for 21 days. Ovulated oocytes were collected from oviducts 16 h following hCG injection and fertilized in vitro (n=6 for each group). Fertilized oocytes were followed for 72 hours. FIG. 9A shows two-cell embryos developed from viable oocytes by day 2. FIG. 9B shows blastocysts developed from two-cell embryos by day 4.

[0017] FIG. 10A, FIG. 10B, FIG. IOC, and FIG. 10D are scatter plots showing the effects of Salubrinal treatment on litter size of GalT-deficient mice. FIG. 10A shows the size of a first litter, FIG. 10B shows the size of a second litter, FIG. IOC shows the size of a third litter, and FIG. 10D shows the combined data from the three litters. Each data point represents the litter size of one female mouse. Not all females gave birth or were pregnant, notably the DMSO control mice, at the time when the data were collected for the second litters. [0018] FIG. 11 is a scatter plot showing the effect of Salubrinal treatment on the time-to- pregnancy in GalT-deficient mice.

[0019] FIG. 12 is a bar chart showing the effect of Salubrinal on the body weight of pups born to GalT-deficient mothers that, during pregnancy, were untreated (leftmost bar for each age), treated with DMSO (middlemost bar for each age) and treated with Salubrinal

(rightmost bar for each age).

[0020] FIG. 13 is a scatter plot showing the effect of Salubrinal on the weight of the organs of pups born to GalT-deficient mothers that were untreated (circles) treated with DMSO (squares) and treated with Salubrinal (triangles) during pregnancy.

DETAILED DESCRIPTION

[0021] Before any embodiments of the methods and compositions of the present disclosure are explained in detail, it is to be understood that the inventions disclosed herein are not limited in application to the details set forth in the description or illustrated in the drawings. The inventions are capable of other embodiments and of being practiced or of being carried out in various ways.

[0022] The present disclosure provides methods and compositions for treatment or prevention of symptoms of galactosemia in a subject by administering compounds of formula (I), or pharmaceutically acceptable salts thereof. As disclosed herein, compounds of formula (I) effectively reversed the down-regulated PI3K/Akt signaling pathway in GalT-deficient cells and animals to levels close to those of their normal counterparts. Compounds of formula (I) significantly slowed down the loss of Purkinje cells in the cerebella, as well as the premature loss of primordial ovarian follicles in young mutant mice. Treatment with compounds of formula (I) dramatically increased the number of primordial follicles to almost twice that of untreated GalT-deficient animals. Treatment with compounds of formula (I) led to a significantly larger number of ovulated eggs in GalT-deficient animals, but had no impact upon egg production in normal animals. The ovulated eggs could be fertilized and developed into blastocysts at a rate similar to that of the normal animals. Treatment with compounds of formula (I) to GalT-deficient female mice not only led to significantly larger litters relative to the litters of the untreated animals but also decreased the time to pregnancy. 1. Definitions

[0001] As described herein, compounds of the present disclosure can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables in formula(I) encompass specific groups, such as, for example, aryl and heteroaryl. As one of ordinary skill in the art will recognize, combinations of substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

[0023] 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. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0024] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0025] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression "from about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean from 0.9-1.1. Other meanings of "about" may be apparent from the context, such as rounding off, so, for example "about 1" may also mean from 0.5 to 1.4.

[0026] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0027] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March 's Advanced Organic Chemistry, 5 ^th Edition, John Wiley & Sons, Inc., New York, 2001 ; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 ^rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0028] The term "alkyl" as used herein, means a straight or branched chain saturated hydrocarbon. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0029] The term "alkylene," as used herein, means a divalent group derived from a straight or branched chain saturated hydrocarbon. Representative examples of alkylene include, but are not limited to, -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH ₂ CH(CH ₃ )CH ₂ -, and CH ₂ CH(CH ₃ )CH(CH ₃ )CH ₂ -.

[0030] The term "aryl," as used herein, means phenyl or a bicyclic aryl. The bicyclic aryl is naphthyl, dihydronaphthalenyl, tetrahydronaphthalenyl, indanyl, or indenyl. The phenyl and bicyclic aryls are attached to the parent molecular moiety through any carbon atom contained within the phenyl or bicyclic aryl. [0031] The term "cycloalkyl" as used herein, means a monovalent group derived from an all-carbon ring system containing zero heteroatoms as ring atoms, and zero double bonds. The all-carbon ring system can be a monocyclic, bicylic, or tricyclic ring system, and can be a fused ring system, a bridged ring system, or a spiro ring system, or combinations thereof. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The cycloalkyl groups described herein can be appended to the parent molecular moiety through any substitutable carbon atom.

[0032] The term "cycloalkylene" as used herein, means a divalent group derived from an all-carbon ring system containing zero heteroatoms as ring atoms and zero double bonds, which attaches to the parent molecule at two different ring carbons atoms. The all-carbon ring system can be a monocyclic, bicylic, or tricyclic ring system, and can be a fused ring system, a bridged ring system, or a spiro ring system. Representative example those derived from C3-10 rings, such as

[0033] The term "halogen" means a chlorine, bromine, iodine, or fluorine atom.

[0034] The term "haloalkyl," as used herein, means an alkyl, as defined herein, in which one, two, three, four, five, six, or seven hydrogen atoms are replaced by halogen. For example, representative examples of haloalkyl include, but are not limited to, 2-fluoroethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l , 1 -dimethylethyl, and the like.

[0035] The term "heteroaryl," as used herein, means an aromatic heterocycle, i.e., an aromatic ring that contains at least one heteroatom selected from O, N, or S. A heteroaryl may contain from 5 to 12 ring atoms. A heteroaryl may be a 5- to 6-membered monocyclic heteroaryl or an 8- to 12-membered bicyclic heteroaryl. A 5-membered monocyclic heteroaryl ring contains two double bonds, and one, two, three, or four heteroatoms as ring atoms. Representative examples of 5-membered monocyclic heteroaryls include, but are not limited to, furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl (thiophenyl), and triazolyl. A 6-membered heteroaryl ring contains three double bonds, and one, two, three or four heteroatoms as ring atoms. Representative examples of 6-membered monocyclic heteroaryls include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl. The bicyclic heteroaryl is an 8- to 12-membered ring system having a monocyclic heteroaryl fused to an aromatic, saturated, or partially saturated carbocyclic ring, or fused to a second monocyclic heteroaryl ring. Representative examples of bicyclic heteroaryl include, but are not limited to, benzofuranyl, benzoxadiazolyl, 1,3-benzothiazolyl, benzimidazolyl, benzothienyl, indolyl, indazolyl, isoquinolinyl, naphthyridinyl, oxazolopyridine, quinolinyl, thienopyridinyl, and 5 ,6, 7 ,8-tetrahydroquinolinyl. The heteroaryl groups are connected to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the groups.

[0036] The terms "heterocycle," "heterocyclic" or "heterocyclyl" refer generally to ring systems containing at least one heteroatom as a ring atom where the heteroatom is selected from oxygen, nitrogen, and sulfur. In some embodiments, a nitrogen or sulfur atom of the heterocycle is optionally substituted with oxo. Heterocycles may be a monocyclic heterocycle, a fused bicyclic heterocycle, or a spiro heterocycle. The monocyclic heterocycle is generally a 4, 5, 6, 7, or 8-membered non-aromatic ring containing at least one heteroatom selected from O, N, or S. The 4-membered ring contains one heteroatom and optionally one double bond. The 5-membered ring contains zero or one double bond and one, two or three heteroatoms. The 6, 7, or 8-membered ring contains zero, one, or two double bonds, and one, two, or three heteroatoms. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, diazepanyl, 1,3-dioxanyl, 1 ,4-dioxanyl, 1,3- dioxolanyl , 4,5-dihydroisoxazol-5-yl, 3,4-dihydropyranyl, 1,3-dithiolanyl, 1,3-dithianyl, homomorpholinyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl, thiopyranyl, and trithianyl. The fused bicyclic heterocycle is a 7-12-membered ring system having a monocyclic heterocycle fused to a phenyl, to a saturated or partially saturated carbocyclic ring, or to another monocyclic heterocyclic ring, or to a monocyclic heteroaryl ring. Representative examples of fused bicyclic heterocycle include, but are not limited to, l,3-benzodioxol-4-yl, 1,3-benzodithiolyl, 3-azabicyclo[3.1.0]hexanyl, hexahydro-lH- furo[3,4-c]pyrrolyl, 2,3-dihydro-l,4-benzodioxinyl, 2,3-dihydro-l -benzofuranyl, 2,3-dihydro- 1 -benzothienyl, 2,3-dihydro-lH-indolyl, 5,6,7,8-tetrahydroimidazo[l,2-a]pyrazinyl, and 1,2,3,4-tetrahydroquinolinyl. Spiro heterocycle means a 4-, 5-, 6-, 7-, or 8-membered monocyclic heterocycle ring wherein two of the substituents on the same carbon atom form a second ring having 3, 4, 5, 6, 7, or 8 members. Examples of a spiro heterocycle include, but are not limited to, l,4-dioxa-8-azaspiro[4.5]decanyl, 2-oxa-7-azaspiro[3.5]nonanyl, 2-oxa-6- azaspiro[3.3]heptanyl, and 8-azaspiro[4.5]decane. The monocyclic heterocycle groups may contain an alkylene bridge of 1, 2, or 3 carbon atoms, linking two nonadjacent atoms of the group. Examples of such a bridged heterocycle include, but are not limited to, 2,5- diazabicyclo[2.2.1]heptanyl, 2-azabicyclo[2.2.1]heptanyl, 2-azabicyclo[2.2.2]octanyl, and oxabicyclo[2.2.1]heptanyl. The monocyclic, fused bicyclic, and spiro heterocycle groups are connected to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the group.

[0037] The term "hydroxy" as used herein, means an -OH group.

[0038] The term hydroxyalkyl as used herein means an alkyl, as defined herein, in which a hydrogen atom is replaced by -OH. For example, representative examples of hydroxyalkyl include, but are not limited to those derived from CI -6 alky Is, such as -CH20H,

-CH2CH20H, -CH2CH2CH20H, and the like.

[0039] Terms such as "alkyl," "cycloalkyl," "alkylene," "cycloalkylene," etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., "Ci- ₄ alkyl," "Ci- ₄ alkylene"). These designations are used as generally understood by those skilled in the art. For example, the representation "C" followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, "C ₃ alkyl" is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in "C1-4," the members of the group that follows may have any number of carbon atoms falling within the recited range. A "Ci- ₄ alkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0040] If a group is described as being "substituted", a non-hydrogen substituent group is in the place of hydrogen radical on a carbon or nitrogen of that group. Thus, for example, a substituted alkyl is an alkyl in which at least one non-hydrogen radical is in the place of a hydrogen radical on the alkyl. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro radical, and difluoroalkyl is alkyl substituted with two fluoro radicals. It should be recognized that if there is more than one substitution on a substituent, each non-hydrogen radical may be identical or different (unless otherwise stated). Substituent groups include, but are not limited to, halogen, =0, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl,

fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0041] When a group is referred to as "unsubstituted" or not referred to as "substituted" or "optionally substituted", it means that the group does not have any substituents. If a group is described as being "optionally substituted", the group may be either (1) not substituted or (2) substituted. If a group is described as being optionally substituted with up to a particular number of non-hydrogen radicals, that group may be either (1) not substituted; or (2) substituted by up to that particular number of substituent groups or by up to the maximum number of substitutable positions on that group, whichever is less.

[0042] If substituents are described as being independently selected from a group, each substituent is selected independent of the other. Each substituent, therefore, may be identical to or different from the other substituent(s).

[0043] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single

stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Thus, included within the scope of the invention are tautomers of compounds of formula I. The structures also include zwitterionic forms of the compounds or salts of formula I where appropriate.

[0044] The term "ataxia" refers to a lack of voluntary coordination of muscle movements, loss of full control of bodily movements, or loss of balance. It may include gait abnormality. Ataxia can be limited to one side of the body, referred to as hemiataxia, or affect both sides of the body.

[0045] The terms "down-regulate" and "down-regulation" refer to the ability of a cell to decrease the quantity of a cellular component or decrease the cascade of signaling event either by decreases the quantity of a cellular component or reversing the effect necessary to mediate the signaling event. [0046] The terms "dyspraxic speech" or "verbal dyspraxia," as used herein, refer to a sensory motor disorder of articulation characterized by impaired capacity to plan the positioning of speech musculature and muscle movements for the production of speech sounds. While it is primarily an articulation disorder, there are a number of other related communication problems associated with dyspraxia, such as: problems of syntax (word order), language organization, and pragmatics (set of rules governing conversation).

[0047] The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the regenerative cells, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).

[0048] As used herein, "Gal-T," "GalT," and "GALT" are used interchangeably to mean the enzyme galactose- 1 -phosphate uridyltransferase.

[0049] As used herein, "growth restriction" refers to delayed growth during childhood and early adolescence or through the late teens. Final adult heights or sizes may be within the normal range or may be decreased over the mean parental height or size.

[0050] As used herein, "motor impairment" refers to the partial or total loss of function of a body part, usually a limb or limbs. This may result in muscle weakness, poor stamina, lack of muscle control, or total paralysis. It may also manifest as fine-motor tremors, problems with coordination, gait and balance, dysarthria, ataxia, dystonia or a combination thereof.

[0051] As used herein, the term "preventing" refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.

[0052] As used herein, "primordial follicles" refer to the most immature stage of an ovarian follicle. During the first five months of development, a finite number of primordial follicles form in the fetal ovary. These follicles consist of oocytes surrounded by a single layer of squamous follicular cells. These primordial follicles remain in the process of the first meiotic division until puberty when they enter the stages of follicular growth to form a mature oocyte able to be released for fertilization.

[0053] As used herein, "Purkinje cells" refer to a nerve cell or neuron found in the cortex of the cerebellum. Purkinje cells send inhibitory projections to the deep cerebellar nuclei, and constitute the sole output of all motor coordination in the cerebellar cortex.

[0054] As used herein, "treat," "treating" and the like means a slowing, stopping or reversing of progression of a disease or disorder when provided a composition described herein to an appropriate control subject. The term also means a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the cell proliferation. As such, "treating" means an application or administration of the compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or symptoms of the disease.

[0055] A "subject" or "patient" may be human or non-human and may include, for example, animal strains or species used as "model systems" for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g. , children). Moreover, patient may mean any living organism, preferably a mammal (e.g. , human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0056] As used herein, the terms "providing", "administering," "introducing," are used interchangeably herein and refer to the placement of the compositions of the disclosure into a subject by a method or route which results in at least partial localization of the composition to a desired site. The compositions can be administered by any appropriate route which results in delivery to a desired location in the subject.

2. Methods for Treating or Preventing Symptoms of Galactosemia

[0057] The present disclosure provides methods and compositions for treating or preventing a symptom of galactosemia. The methods comprise administering to a subject with galactosemia, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , L ¹ and G ¹ are as defined herein, a. Compounds

[0058] The methods described herein comprise administering to a subject with galactosemia, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof

(I)

wherein R ¹ , R ² , L ¹ and G ¹ are as defined herein.

[0059] In some embodiments, R ¹ is CCI3, CHCI2, or CBr3. In exemplary embodiments,

[0060] In some embodiments, R ² is aryl, heteroaryl, or a Cs-gcycloalkyl, wherein R ² is optionally substituted with 1-4 substituents independently selected from halogen, cyano, Ci_ ₄ alkyl, Ci _-4 haloalkyl, -OCi _-4 alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), and -N(Ci _-4 alkyl) ₂ . In further embodiments, R ² is phenyl, a 5- or 6-membered monocyclic heteroaryl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a C5- ₇ cycloalkyl, wherein R ² is optionally substituted with 1-4 substituents independently selected from halogen, cyano, Ci _-4 alkyl, Ci _-4 haloalkyl, -OCi _-4 alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), and - N(Ci- ₄ alkyl) ₂ . In still further embodiments, R ² is phenyl, thiophenyl, pyridinyl, or cyclohexyl, wherein R ² is optionally substituted with 1-2 substituents independently selected from halogen, CH ₃ , CF ₃ , -OCH ₃ , nitro, and -NH ₂ . In still further embodiments, R ² is phenyl, thiophen-2-yl, pyridin-3-yl, or cyclohexyl, wherein the phenyl is optionally substituted with 1-2 substituents independent selected from halogen,CH ₃ , CF ₃ , -OCH ₃ , nitro, and -NH ₂ . In still further embodiments, R ² is phenyl, thiophen-2-yl, pyridin-3-yl, or cyclohexyl,

[0061] In some embodiments, L ¹ is -C(R ⁴ ) ₂ -C(R ⁴ ) ₂ - -CR ⁴ =CR ⁴ -, -≡-, or ^ In further embodiments, L ¹ is -CH2-CH2- or -CH=CH-. In exemplary embodiments, L ¹ is

[0062] In some embodiments, G ¹ is -C(0)NH-R ³ , -C(S)NH-R ³ , ** ^N , -C(0)G ² , or -C(S)G . In still further embodiments, G is ^{" i} . In still further embodiments, G is -C(S)G ² . In exemplary embodiments, G ¹ is -C(S)NH-R ³ .

[0063] In some embodiments, R is G ³ , -Ci _-3 alkylene-G ³ , -Ci- ₆ alkyl, or -C ₂ - ₆ alkylene- OCi- ₄ alkyl. In further embodiments, R ³ is G ³ , -CH ₂ -G ³ , C _3-6 alkyl, or -C ₂ - ₄ alkylene-OCi-

V

[0064] In some embodiments, R ⁴ , at each occurrence, is independently hydrogen or Ci_ ₄ alkyl. [0065] In some embodiments, G ² is a 4- to 8-membered monocyclic heterocyclyl containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur and optionally substituted with 1-4 substituents independently selected from halogen, Ci- ₄ alkyl, Ci- ₄ haloalkyl, and -OCi- ₄ alkyl. In further embodiments, G ² is a 5- to 7-membered monocyclic heterocyclyl containing 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur and optionally substituted with 1-4 substituents independently selected from halogen, Ci- ₄ alkyl, and -OCi- ₄ alkyl. In still further embodiments, G ² is a morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, azepanyl, or homomorpholinyl. In yet still further embodiments,

[0066] In some embodiments, G ³ is aryl, heteroaryl, or Cs-gcycloalkyl, wherein G ³ is optionally substituted with 1-4 substituents independently selected from halogen, cyano, Ci_ ₄ alkyl, Ci _-4 haloalkyl, -OCi _-4 alkyl, -C(0)OCi _-4 alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), -N(Ci_ ₄ alkyl) ₂ , -C(0)NH ₂ , -C(0)NH(Ci _-4 alkyl), and -C(0)N(Ci _-4 alkyl)2. In further embodiments,

G ³ is phenyl, naphthyl, quinolinyl, a 5- or 6-membered monocyclic heteroaryl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a C5- ₇ cycloalkyl, wherein G ³ is optionally substituted with 1-4 substituents independently selected from halogen, cyano, -OCi- ₄ alkyl, -C(0)OCi- ₄ alkyl, nitro, -NH ₂ , - NH(Ci _-4 alkyl), -N(Ci _-4 alkyl) ₂ , -C(0)NH ₂ , -C(0)NH(Ci _-4 alkyl), and -C(0)N(Ci _-4 alkyl) ₂ . In still further embodiments, G ³ is phenyl, pyridinyl, quinolinyl, cyclohexyl, or cycloheptyl, wherein the phenyl is optionally substituted with 1-2 substituents independently selected from halogen, Ci- ₄ alkyl, -OCi- ₄ alkyl, and -C(0)OCi- ₄ alkyl.

ound of formula (I) is

[0068] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomer, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1 -forms; (+) and ( ) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal- forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[0069] Compounds may be in racemic form or as individual enantiomers or

diastereomers by either stereospecific synthesis or by resolution. The compounds may, for example, be resolved into their component enantiomers or diastereomers by standard techniques, such as the formation of stereoisomeric pairs by salt formation with an optically active base, followed by fractional crystallization and regeneration of the free acid. The compounds may also be resolved by formation of stereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. The enantiomers also may be obtained from kinetic resolution of the racemate of corresponding esters using lipase enzymes.

[0070] Exemplary tautomeric forms include, for example, the following tautomeric pairs: keto/enol and imine/enamine.

[0071] In another embodiment, the compounds include isotope-labeled forms. An isotope-labeled form of a compound is identical to the compound apart from the fact that one or more atoms of the compound have been replaced by an atom or atoms having an atomic mass or mass number which differs from the atomic mass or mass number of the atom which usually occurs in greater natural abundance. Examples of isotopes which are readily commercially available and which can be incorporated into a compound by well-known methods include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, for example ² H, H, ¹ C, ¹⁴ C, ¹⁵ N, ¹⁸ 0, ¹⁷ 0, ¹⁸ F and ⁶ C1. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples include using an appropriate isotopically-labeled reagent in place of non-isotopically -labeled reagent. b. Symptoms of Galactosemia

[0072] The methods described herein may be used for treating or preventing a symptom of galactosemia. These treatment methods may comprise administering to a subject with galactosemia, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0073] Even with early diagnosis and careful restriction of lactose and galactose from the diet, subjects with galactosemia remain at increased risk for long-term complications that include symptoms of speech and language deficiencies, fine and gross motor skill delays, specific learning or cognitive and behavioral disabilities and cataracts. Primary or premature ovarian insufficiency (POI) is also very common among girls and women with

galactosemia. Some patients experience many of these complications; others do not.

[0074] The methods disclosed herein may be used to treat or prevent one or more of the symptoms relating to galactosemia. In some embodiments, the symptoms may comprise infertility or reduced fertility, motor impairment, growth restriction, dyspraxic speech, cataract, intellectual deficit, or a combination thereof.

[0075] In some embodiments, the symptoms may comprise infertility or reduced fertility.

[0076] In certain embodiments, the infertility or reduced fertility may comprise primary ovarian insufficiency. Primary ovarian insufficiency can vary from reduced fertility, to early development of irregular menstrual cycles and infertility, to primary amenorrhea and absence of spontaneous puberty.

[0077] Primary ovarian insufficiency may be due to the formation of a smaller primordial follicle pool or more rapid loss of primordial follicles and there is evidence for both mechanisms in classic galactosemia. In some embodiments, the reduced fertility comprises decreased numbers of primordial follicles, loss of primordial follicles or a combination thereof.

[0078] In some embodiments, the therapeutically effective amount of a compound of formula (I) increases fertility. Increased fertility may be shown by an increased number of ovulated eggs. Increased fertility may result in an increase in pregnancy rates.

[0079] In some embodiments, the therapeutically effective amount of a compound of formula (I) increases primordial follicle numbers. Compounds described herein may increase the primordial follicle numbers by as much as 2 fold. In some embodiments, the

therapeutically effective amount of a compound of formula (I) reverses the loss of primordial follicles.

[0080] In some embodiments, the symptom of galactosemia is motor impairment. Loss of motor function may cause fine-motor tremors, problems with coordination, gait and balance, dysarthria, ataxia, dystonia or a combination thereof.

[0081] In some embodiments, motor impairment comprises one or more of tremors and ataxia. Ataxia is a total or partial inability to coordinate voluntary bodily movements, for example, during walking. Ataxia may include dysmetria. Dysmetria is improper estimation of distance during muscular activity. Dysmetria includes both hypo- and hypermetria. With hypermetria, voluntary muscular movement overreaches the intended goal; with hypometria, voluntary movement falls short of the intended goal. Hypermetria is more commonly recognized than hypometria. Tremors are rhythmic, involuntary muscular contractions characterized by oscillations, or back and forth shaking, of a part of the body. Tremors can affect various body parts such as the hands, head, facial structures, vocal cords, trunk, and legs. Most tremors, however, occur in the hands. There are many different types of tremors. Some types of tremors occur while the muscle is relaxed, however, most types of tremors occur during the voluntary movement of a muscle.

[0082] In some embodiments, the motor impairment comprises decreased numbers of Purkinje cells in the cerebellum, loss of Purkinje cells in the cerebellum or a combination thereof. Purkinje cells send inhibitory projections to the deep cerebellar nuclei, and constitute the sole output of all motor coordination in the cerebellar cortex.

[0083] In some embodiments, the therapeutically effective amount of a compound of formula (I) increases Purkinje cells in the cerebellum. Compounds described herein may increase the number of Purkinje cells by approximately 20%. Compounds described herein may increase the number of Purkinje cells by 15-20%, 10-20%, 20-25%, or 20-30%.

[0084] In some embodiments, the symptoms comprise growth restriction. Growth restriction may include delayed growth during childhood and early adolescence or through the late teens. Final adult heights may be within the normal range or may be decreased over the mean parental height.

[0085] In some embodiments, the symptoms comprise dyspraxic speech. Dyspraxia is not a developmental delay of speech but rather a sensory motor disorder of articulation characterized by impaired capacity to plan the positioning of speech musculature and muscle movements for the production of speech sounds. Other related communication problems associated with dyspraxia include: problems of syntax or word order, language organization and pragmatics.

[0086] In some embodiments, the symptoms comprise intellectual deficit. Intellectual deficit may include any learning or cognitive and behavioral disabilities or disorders that interfere with a person's ability to master a skill (such as reading, writing, arithmetic concepts, etc.). These disorders can manifest themselves in many ways such as, specific difficulties with spoken and written language, coordination, self-control, or attention. Some of the disabilities that have been observed in patients with galactosemia include: speech and language difficulties, fine and/or gross motor difficulties, and difficulty with math or reading in school.

[0087] In some embodiments, the symptoms comprise cataract. Cataracts may be the result of the accumulation of galactitol in the lens of the eye. The cataract may be in one or both of the eyes. The cataract may be associated with osmotic swelling of the lens cells or increased osmotic pressure within the lens. c. PBK/Akt Signaling Pathway

[0088] The PI3K-Akt pathway is an intracellular signal transduction pathway that has a critical role in diverse cellular functions including metabolism, proliferation, cell survival, growth and angiogenesis in response to extracellular signals. The pathway is mediated through serine and/or threonine phosphorylation of a range of downstream substrates. Akt is fully activated following phosphorylation of the serine residue at position 473 (Ser 473) and thereby can regulate a wide range of proteins or target substrates by phosphorylation.

Phosphorylation by Akt can be inhibitory or stimulatory, either suppressing or enhancing the activity of target proteins. Target protein of Akt include, but are not limited to, the forkhead family of transcription factors (FoxO), cAMP response element binding protein (CREB), mechanistic target of rapamycin (mTOR) and glycogen synthase kinase 3 (Gsk3).

[0089] Dysfunction of metabolic enzymes can affect the regulation of the PBK/Akt growth signaling. In some embodiments, the subject with galactosemia has down-regulated PBK/Akt signaling compared to a subject without galactosemia. The down-regulation of the PBK/Akt pathway may occur at both mRNA and protein abundance levels. For example, galactosemia may decrease the protein expression levels of pAkt(Ser 473), panA t, pGsk3 , and Hsp90 in the ovaries and/or the cerebellum. Galactosemia may decrease the protein expression levels of pAkt(Ser 473), panAkt, pGsk3 and Hsp90 by at least 30% or by as much as 95%. Galactosemia may cause a decrease in the level of phosphorylation of pAkt(Ser 473) and/or pGsk3 .

[0090] In some embodiments, the therapeutically effective amount of a compound of formula (I) is an amount that increases PBK/Akt signaling. The compounds described herein may increase the mRNA and/or the protein abundance levels of PBK/Akt signaling molecules. The compounds described herein may increase the mRNA and/or the protein abundance levels of PBK/Akt signaling molecules by between 50% and 75%. The compounds described herein may increase the phosphorylation of pAkt(Ser 473) and/or pGsk3 . The phosphorylation may increase by about 200%. The phosphorylation may increase by between 180 and 220%.

[0091] In some embodiments, the therapeutically effective amount of a compound of formula (I) is an amount that reverses down-regulated PI3K/Akt signaling. In some embodiments, the compounds described herein normalize the down-regulated PI3K/Akt signaling to approximately 70% of the levels seen in normal subjects.

[0092] Compounds of formula (I) have been shown to have activity through inhibition of glycine transporters. The GlyTl glycine transporter is expressed by astrocytes in the spinal cord, brainstem, and brain hemispheres. The GlyT2 uptake system is restricted to the spinal cord, brainstem and cerebellum, and is not found in the cortex and other regions of the brain hemispheres.

[0093] However, the glycine transporter family does not have a role in PI3K/Akt signaling and surprisingly, the compounds of formula (I) are acting independently of a glycine transporter inhibitory mechanism. In some embodiments, the therapeutically effective amount of a compound of formula (I) used to treat or prevent a symptom of galactosemia does not inhibit a glycine transporter. In some embodiments, the

therapeutically effective amount of a compound of formula (I) used to treat or prevent a symptom of galactosemia does not inhibit GlyTl or GlyT2. d. Administration

[0094] Compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may be administered to subjects by a variety of methods. In any of the uses or methods described herein, administration can be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof.

[0095] The amount of the compound of the present disclosure, or pharmaceutically acceptable salts thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature and/or symptoms of galactosemia and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compounds disclosed herein in amounts that exceed, or even far exceed, the dosage ranges described herein.

[0096] In some embodiments, the compounds, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions as disclosed herein may be administered by inhalation, oral administration, or intravenous administration. In general, however, a suitable dose will often be in the range of from about 0.01 mg/kg to about 100 mg/kg, such as from about 0.05 mg/kg to about 10 mg/kg. For example, a suitable dose may be in the range from about 0.10 mg/kg to about 7.5 mg/kg of body weight per day, such as about 0.10 mg/kg to about 0.50 mg/kg of body weight of the recipient per day, about 0.10 mg/kg to about 1.0 mg/kg of body weight of the recipient per day, about 0.15 mg/kg to about 5.0 mg/kg of body weight of the recipient per day, about 0.2 mg/kg to 4.0 mg/kg of body weight of the recipient per day. The compound may be administered in unit dosage form; for example, containing 1 to 100 mg, 10 to 100 mg or 5 to 50 mg of active ingredient per unit dosage form.

[0097] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub- doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0098] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies. For example, useful dosages of a compound of the present invention, or pharmaceutically acceptable salts thereof, can be determined by comparing their in vitro activity, and in vivo activity in animal models.

[0099] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vivo and/or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, FIPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[00100] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions.

Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the galactosemia symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[00101] Compounds, salts and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, dogs or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, route of administration and/or regime.

[00102] A therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, disclosed herein may be administered alone or in combination with a therapeutically effective amount of at least one additional therapeutic agents. In some embodiments, the compounds as disclosed herein are administered in combination with at least one additional therapeutic agents. In some embodiments, the at least one additional therapeutic is administered prior to or following administration of the compounds or pharmaceutical compositions as disclosed herein. 3. Examples

[00103] It will be readily apparent to those skilled in the art that other suitable

modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

Example 1

[00104] Chemicals and Antibodies . Tunicamycin and Salubrinal were purchased from Sigma Inc. (St. Louis, MO). Antibodies against selected components of the PI3K/Akt signaling pathway were included in the Phospho-Akt Pathway Antibody Sampler Kit (Cell Signaling Technology, Danvers, MA). Anti-mouse BiP/GRP78 was purchased from BD Bioscience (San Jose, CA). Anti-mouse Hsp90 was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX). Anti-mouse Gapdh was purchased from Sigma Inc. (St. Louis, MO).

[00105] Primary fibroblast and Culture Conditions. Primary skin fibroblasts were isolated from young adult female GalT-deficient mice and their age-matched normal littermates using the method described by Seluanov and coworkers (J Vis Exp. 44 (2010) 2033). After confirmation of cell type identity by a Board-Certified pathologist, the isolated fibroblast strains were propagated in regular Dulbecco's Modified Eagle Medium (DMEM)

supplemented with 15% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin at

37 C with 5% ambient CO ₂ . Collagenase and cell culture media were purchased from Invitrogen (Waltham, MA). When indicated, cells at 70% confluence were treated with 50μΜ Salubrinal dissolved in DMSO for 24 or 48 hours. To induce ER stress in normal fibroblasts, tunicamycin was administered at a concentration of 2^g/ml.

[00106] Animals and Experimental Groups. All animal studies were conducted in full compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the University of Utah Institutional Animal Care and Use Committee. GalT-deficient mice used in this study was constructed as previously described (Eur J Hum Genet. 22 (2014) 1172-1179), and were fed with normal chow at all times since weaning. All mice were confirmed by genotyping (molecular and biochemical) using previously published protocols. Animals were randomly assigned into the following experimental groups, each containing at least three mice.

[00107] (a) Untreated Control Group: Animals did not receive any treatment.

[00108] (b) Control Group: The animals have been administered with a vehicle solution consisting of dimethylsulfoxide (DMSO) diluted with soymilk (final concentration of DMSO was 0.6%) per os once daily at indicated durations.

[00109] (c) Salubrinal (Sal) Group: The animals have been administered with a mixture of solution consisting of Salubrinal (1, and 5mg/kg) and DMSO which is then diluted with soymilk (the final concentration of DMSO was 0.6%) per os once a day at indicated durations.

[00110] Assessment of PI3K/Akt signaling in Salubrinal-treated fibroblasts /tissues.

Fibroblast cells were lysed after the indicated incubation period and expression of PI3K/Akt signaling molecules was analyzed by Western Blot. To harvest tissues, the animals were anesthetized by isoflurane and decapitated. After removal of the brains, the cerebella were dissected on a chilled ice plate and homogenized in ice-cold hypotonic lysis buffer (1 mM EDTA, 10 mM Tris-HCl, pH 7.4), supplemented with protease inhibitor cocktail (1 mg/ml each of aprotinin, pepstatin, and leupeptin; 100 mg/ml phenylmethylsulfonyl fluoride and 2 mM sodium orthovanadate). Ovaries were extracted and fat removed under a dissection microscope, were immediately transferred into ice-cold phosphate buffer saline (PBS) and homogenized in ice cold hypotonic lysis buffer. Homogenized samples were centrifuged at 18,000 x g at 4°C for 20 min and supernatant collected. Protein concentrations were determined by a BCA protein assay kit {Thermo Scientific, Product # 23223) using a 96-well microplate reader.

[00111] Sample preparation for histological studies.

[00112] (1) Brain: The animal cranium was extracted and placed in ice-cold PBS followed by 10 % formalin for 72 hours. Several washes in 70% ethanol were done over a period of the next 48 hours to rehydrate the tissues. The whole brain with attached cerebellum was then isolated from the skull and cut in half along the mid sagittal plane prior to tissue processing, slide preparation, and hematoxylin and eosin (H&E) staining performed by the Research Histology Core Facility of the ARUP Laboratories (Salt Lake City, UT, LISA). H&E-stained sections were sent to a Board-certified pathologist at the University of Florida for the analysis of the granular and molecular layers in the cerebellum in a single blinded manner as published before. Slides were also used for Purkinje cell count of each section.

[00113] (2) Ovary: Tissue processing, slide preparation, and H&E staining of the ovaries harvested from the animals were performed at Division of Reproductive Sciences,

Department of Obstetrics and Gynecology, University of Colorado, with the assistance of Prof. Joshua Johnson, Ph.D. Blinded histomorphometric evaluation of ovarian follicle and corpora lutea content were performed. These methods are briefly summarized as follows. At the time of collection, ovaries from all group animals were assigned unique codes for blinding purposes. Ovaries were fixed in Dietrich's fixative overnight at 4°C, and were stored in 70% ethanol at 4°C until paraffin embedding. After dehydration and embedding, pole-to- pole serial sections were cut and placed on glass slides. Sections were rehydrated, and stained with Weigert's Iron Hematoxylin and Picric Acid-Methyl blue. Slides were then dehydrated and mounted using Permount for evaluation.

[00114] Blind histomorphometric evaluation of ovarian follicles and corpora lutea. Intact immature (primordial, primary, and small preantrai) follicles were counted in every 5 ^th section, and the total numbers were estimated by multiplying by 5. Intact antral follicles and corpora lutea were catalogued in every serial section, and adjacent sections were viewed to ensure these structures were only counted once. In addition to counting these standard follicle and corpus iuteum classes, sections were evaluated for any follicular abnormalities. After all ovaries were evaluated, specimens were de-coded and data were analyzed according to treatment groups. Data from untreated GalT gene-trapped mice, GalT-deficient mice treated with Salubrinal as above, and those treated with vehicle alone were compared and ANOVA analysis was performed to detect significant differences in any counted category- using R.

[00115] Follicular maturation studies. Five-week-old wild-type (WT) and GalT-deficient female mice (n=4 each) were housed and treated with Salubrinal per os at a 5 mg/kg body weight concentration once daily for 21 days as described above. The same number of age- matched WT and GalT-deficient females was treated with 0.1% DMSO as controls. For superovulation, Pregnant Mare Serum Gonadotropin (PMSG) (5 IU) was injected (i.p.), followed by Human Chorionic Gonadotropin (hCG) at the same dose 48 h later. Oocytes were isolated from the oviduct 15 h after hCG administration and counted. Superovulated cumulus-oocyte complexes (COCs) were retrieved and transferred to plates with Global Fertilization Media containing 2x10 sperm/ml. Eggs were transferred to culture media and evaluated 24 and 72 hours post-sperm addition.

[00116] Breeding Studies. Five week old female GalT-deficient mice were treated with Salubrinal at a 5 mg/kg of body weight concentration once daily for five weeks. Other GalT- deficient mice were either treated with 0.1% DMSO or were untreated, as controls. After the five week treatment period, the female mice were bred with male mice. The number of pups born to the female mice was counted, and the time-to-pregnancy for the mice was recorded. The breeding experiments were conducted three times (i.e., three separate litters). FIG. 10A shows the number of pups in the first litter, FIG. 10B shows the number of pups in the second litter, FIG. IOC shows the number of pups in the third litter, and FIG. 10D shows the combined data from all three litters. FIG. 11 shows the time-to-pregnancy for the mice in each litter. In addition, after the pups were born, the pups bom to the GalT-deficient mothers that were untreated, treated with DMSO and treated with Salubrinal were weighed each day for 20 days to assess the effect of Salubrinal on the weight of the pups (see FIG. 12). 21 days after birth, once the pups were weaned, the organs from the pups were collected and weighed to assess the effect of Salubrinal on the weights of the pups' organs (see FIG. 13).

Example 2

[00117] Down-regulation of the PI3K/Akt growth signaling pathway in GalT-deficient mice. To assess the relevance of reduced pro-survival PI3K/Akt signaling in the skin fibroblasts isolated from GalT-deficient mice in other disease-related phenotypes in the mutant animals, such as subfertility and motor impairment, the expression levels of the key components of the signaling pathway in different tissues were compared in normal and GalT- deficient mice of different ages (2, 4, and 7 months). As shown in FIG. 2B and FIG. 2C, PI3K/Akt is down-regulated in ovaries and cerebella harvested from 2-month-old mutant animals compared to age-matched normal (wild type) mice. The levels of pAkt (Ser473), pGsk3 , and Hsp90 in the isolated GalT-deficient ovaries were significantly (p < 0.05) reduced by 62.5%, 93.5% and 90%, respectively (FIG. 2A and FIG. 2B). Similarly, pAkt (Ser473), pGsk3 and Hsp90 in the GalT-deficient cerebella were reduced by 62%, 50%, and 30%, respectively (FIG. 2C and FIG. 2D). There was a 115%, and 148% higher ER stress (via the quantification of the ER stress response protein, BiP), respectively in ovaries and cerebella of mutant mice compared to those of normal mice. Comparable results were found in older mutant mice (FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E and FIG. 3F). Example 3

[00118] Reversal of attenuated PI3K/Akt signaling pathway by ER stress reducers on GalT -deficient mouse fibroblasts. To examine the role of ER stress plays in the down- regulation of the PI3K/Akt signaling pathway in GalT-deficient mouse fibroblasts and tissues, Salubrinal, an ER stress reducer, was used to test if attenuated PI3K/Akt signaling in GalT-deficient mouse fibroblasts could be reversed. Salubrinal is a specific inhibitor of eIF2a phosphatase enzymes and is primarily used experimentally to study stress responses in eukaryotic cells associated with the action of eIF2a.

[00119] In this study, mutant mouse fibroblasts were treated with Salubrinal for 24 and 48 hours, respectively at 50 μΜ, a dosage that is well-documented to protect against cultured cells from ER stress. Salubrinal treatment corresponded to increased phosphorylation of Akt and its downstream target in the mutant fibroblasts after 24 hours. Phosphorylation was increased further after 48 hours with 187% and 206%, respectively for pAkt (Ser473) and pGsk3p (FIG. 4A and FIG. 4B). A concomitant 73% reduction in BiP in the Salubrinal-treated mutant cells was also detected (FIG. 4A and FIG. 4B), reflecting a reduction in ER stress. Overall, these changes can be interpreted as normalizing the down-regulated PI3K/Akt signaling in GalT-deficient cells to approximately 70% of the levels seen in normal fibroblasts (data not shown). As a positive control, it was confirmed that the concentration of Salubrinal used reduced the ER stress in tunicamycin-treated normal cells (data not shown).

[00120] In addition to Salubrinal, the effectiveness of the chemical chaperone, and established ER stress reducer, 4-phenylbutyric acid (4-PBA) to reverse the attenuated PI3K/Akt pathway in the GALT-deficient fibroblasts was also tested. No positive effects were observed over a wide range of concentrations (data not shown). This result agrees well yeast model studies where 4-PBA did not confer any protective role in their growth assays.

Example 4

[00121] Reversal of attenuated PI3K/Akt signaling pathway by ER stress reducers on GalT -deficient mice. To determine the role played by excess ER stress in the down- regulation of the PI3K/Akt pathway in GalT-deficient mouse tissues in vivo, 5 mg/kg Salubrinal was administered, both daily and orally (per os), to seven GalT-deficient mice (n=3 for the vehicle-treated group and n=4 for the Salubrinal-treated group) for 14 days. After treatment, mice from all groups, including the untreated group, were euthanized and organs such as ovaries and cerebella were harvested. Consistent with the finding using cultured fibroblasts, Western Blot analysis results indicated that Salubrinal effectively reduced detectable ER stress in ovaries (FIG. 5A and FIG. 5B) and cerebella of the mutant mice (FIG. 5C and FIG. 5D). Salubrinal also increased the level of the pAkt (Ser473) by 56% and 75%, in the cerebella and ovaries, respectively. Levels of BiP were reduced to 57% and 67%, respectively in cerebella and ovaries.

[00122] The Salubrinal-mediated reversal of aberrant PI3K/Akt signaling in the cerebella of mutant mice was examined to see if it resulted in any improvement at the histological level. Gross histological evaluation revealed what appeared to be more intact Purkinje cells in the Salubrinal-treated mice (FIG. 6A) than in untreated or vehicle-treated control cerebella. Accordingly, a histomorphometric determination of the number of Purkinje cells per unit of cerebellar length showed that, Salubrinal treatment corresponded to an approximate 20% increase in the number of Purkinje cells compared to the two controls groups (FIG. 6B).

Example 5

[00123] Reversal of aberrant P13K/AM signaling is associated with improved disease phenotypes. Growth restriction and subfertility phenotypes were previously reported in the GalT-deficient mice. Recently, characterization of the mutant mice revealed motor impairment. In prior histomorphometric of the mutant female reproductive phenotype, the estimated overall ovarian follicle number and litter size were decreased in 6-month-old GalT- deficient females. To evaluate potential improvement(s) in ovarian parameters due to Salubrinal treatment, analysis of different follicle classes and corpora luteam number were performed. No significant differences in intact "healthy" primary, secondary, small preantral, or antral follicle classes, or, corpora lutea were detected between treatment groups (data not shown). Additionally, no significant differences were found in the numbers of overtly atretic ovarian follicles or zona pellucida remnants. However, treatment of GalT-deficient females with Salubrinal corresponded to a near-doubling of primordial follicles compared to untreated or vehicle-treated control animals (FIG. 7). Rescue of primordial follicles by treatment with an Akt agonist is consistent with the established role of PI3K/Akt/mTOR signaling in supporting the survival of immature ovarian follicles. Moreover, the mutant mice have approximately one-quarter the primordial follicles as slightly older wild-type mice when vehicle-treated, and this is increased to one-half the number seen in wild-type mice. Example 6

[00124] Salubrinal enhances follicular maturation in a mouse model of GalT -deficiency. Salubrinal treatment did not alter the number of ovulated eggs in WT animals after hormonal stimulation [untreated vs treated (31.5 ± 3.0 vs 32 ± 5.9)] (FIG. 8). Salubrinal treatment significantly increased the number of ovulated eggs in GalT-deficient animals [untreated vs treated (10 ± 3.0 vs 31.5 ± 6.1, /? < 0.05)] (FIG. 8). The mean number of ovulated eggs retrieved from vehicle-treated GalT-deficient animals (10 ± 3.0) was significantly lower than the number retrieved from vehicle-treated WT controls (31.5 ± 3.0, p < 0.05) (FIG. 8).

[00125] Ovulated oocyte complexes that had been collected from oviducts of

treated/untreated mice were fertilized in vitro, and oocyte viability and embryo development were examined. Fertilized eggs developed from two cells stage (FIG. 9A) to blastocyst (FIG. 9B) at a similar rate in both treated and untreated wild-type and GalT-deficient mice. The fraction of fertilized eggs (2-cell embryos) (-20%) was comparable among all groups (i.e., WT vs GalT-deficient; treated vs untreated) (FIG. 9A). The fraction of fertilized eggs that developed to blastocyst stage (-20%) was also comparable among all groups (FIG. 9B).

[00126] Results from the ovarian stimulation experiments extended the understanding of the GalT-deficient female mouse subfertility phenotype. GalT-deficient females produced fewer eggs than WT controls stimulated by a "superovulation" protocol. Treatment with Salubrinal led to a significantly larger number of ovulated eggs in the treated mutants, but had no impact upon egg production in WT mice (FIG. 8). The fact that the ovulated eggs from mutant animals could be fertilized and developed into blastocysts at a rate similar to that of the WT animals (FIG. 9A and FIG. 9B) further suggested that the major cause for the subfertility phenotype in the galactosemic model is the accelerated loss of the primordial follicle pool, which can be ameliorated by oral Salubrinal treatment.

Example 7

[00127] Salubrinal increases litter sizes in a mouse model of GalT -deficiency. To expand the histological and cellular results to a function level, litter sizes in GalT-deficient mice were monitored after Salubrinal treatment or treatment with a DMSO vehicle control in comparison to untreated animals. Young female GalT-deficient mice were treated with Salubrinal for 5 weeks as described above. Prior to breeding studies the treatment ceased. Salubrinal treatment of GalT-deficient female mice led to significantly larger litters relative to the litters of the untreated or DMSO vehicle control (See FIG. 10A, FIG. 10B, FIG. IOC and FIG. 10D). Example 8

[00128] GalT-deficient mice took longer to get pregnant than GalT-deficient mice treated with Salubrinal. In addition to the litter size of GalT-deficient mice, the time-to pregnancy was also analyzed. As shown in FIG. 11, GalT-deficient mothers treated with Salubrinal were, on average, able to get pregnant substantially sooner than untreated GalT-deficient mothers.

Example 9

[00129] Salubrinal had no adverse effects on body and organ weights of pups. As discussed above, after the pups were bom, those bom to the GalT-deficient mothers that were untreated, treated with DMSO and treated with Salubrinal were weighed each day for 20 days to assess the effect of Salubrinal on the weight of the pups. As shown in FIG 12, there was no discemible difference in weight, demonstrating that Salubrinal did not have an adverse effect on the body weight of the pups. 21 days after birth, once the pups were weaned, the organs from the pups were collected and weighed to assess the effect of Salubrinal on the weights of the pups' organs. As shown in FIG. 13, there was no discemible difference in the weight of the organs, demonstrating that Salubrinal did not have an adverse effect on the weight of the organs.

[00130] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[00131] Clause 1. A method of treating or preventing a symptom of galactosemia comprising administering to a subject with galactosemia, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof,

(I)

wherein

R ¹ is CC1 ₃ , CHCI2, or CBr ₃ ;

R ² is aryl, heteroaryl, or a C3- ₈ cycloalkyl, wherein R ² is optionally substituted with 1-4

substituents independently selected from halogen, cyano, Ci- ₄ alkyl, -OCi- 4alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), and -N(Ci. ₄ alkyl) ₂ ;

R ³ is G ³ , -Ci _-3 alkylene-G ³ , Ci _-6 alkyl, or -C2- ₆ alkylene-OCi _-4 alkyl;

R ⁴ , at each occurrence, is independently hydrogen or Ci- ₄ alkyl;

G ² is a 4- to 8-membered monocyclic heterocyclyl containing 1 or 2 heteroatoms

independently selected from nitrogen, oxygen, and sulfur and optionally substituted with 1-4 substituents independently selected from halogen, Ci- ₄ alkyl, and -OCi- 4alkyl; and

G ³ is aryl, heteroaryl, or C3- ₈ cycloalkyl, wherein G ³ is optionally substituted with 1-4

substituents independently selected from halogen, cyano, Ci- ₄ alkyl, -OCi- 4alkyl, -C(0)OCi _-4 alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), -N(Ci _-4 alkyl) ₂ , -C(0)NH ₂ , - C(0)NH(Ci- ₄ alkyl), and -C(0)N(Ci-4alkyl) ₂ .

[00132] Clause 2. The method of clause 1, wherein:

R ² is phenyl, a 5- or 6-membered monocyclic heteroaryl containing 1-3 heteroatoms

independently selected from nitrogen, oxygen, and sulfur, or a C5- ₇ cycloalkyl, wherein R ² is optionally substituted with 1-4 substituents independently selected from halogen, cyano, Ci _-4 alkyl, Ci _-4 haloalkyl, -OCi _-4 alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), and -N(Ci- 4alkyl) ₂ ;

L ¹ is -CH ₂ -CH ₂ - or -CH=CH-;

R ³ is G ³ , -CH ₂ -G ³ , C ₃ - ₆ alkyl, or

G ² is a 5- to 7-membered monocyclic heterocyclyl containing 1 or 2 heteroatoms

independently selected from nitrogen, oxygen, and sulfur and optionally substituted with 1-4 substituents independently selected from halogen, Ci- ₄ alkyl, and -OCi- 4alkyl; and

G ³ is phenyl, naphthyl, quinolinyl, a 5- or 6-membered monocyclic heteroaryl containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a C5-

₇ cycloalkyl, wherein G ³ is optionally substituted with 1-4 substituents independently selected from halogen, cyano, Ci- ₄ alkyl, -OCi- ₄ alkyl, -C(0)OCi- ₄ alkyl, nitro, -NH ₂ , -NH(Ci _-4 alkyl), -N(Ci _-4 alkyl) ₂ , -C(0)NH ₂ , -C(0)NH(Ci _-4 alkyl), and - C(0)N(Ci _-4 alkyl) ₂ . [00133] Clause 3. The method of clause 2, wherein:

R ² is phenyl, thiophenyl, pyridinyl, or cyclohexyl, wherein R ² is optionally substituted with 1-2 substituents independently selected from halogen, CH ₃ , CF ₃ , -OCH ₃ , nitro, and - NH ₂ ;

G ² is a morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, azepanyl, or homomorpholinyl; and

G ³ is phenyl, pyridinyl, quinolinyl, cyclohexyl, or cycloheptyl, wherein the phenyl is

optionally substituted with 1-2 substituents independently selected from halogen, Ci- 4alkyl, -OCi _-4 alkyl, and -C(0)OCi _-4 alkyl.

[00134] Clause 4. The method of any of clauses 1-3, wherein R ¹ is CC1 ₃ .

[00135] Clause 5. The method of any of clauses 1-4, wherein R ² is phenyl, thiophen-2-yl, pyridin-3-yl, or cyclohexyl, wherein the phenyl is optionally substituted with 1-2 substituents independent selected from halogen,CH ₃ , CF ₃ , -OCH ₃ , nitro, and -NH ₂ . phenyl is

[00137] Clause 7. The method of any of clauses 1-6, wherein L is ^"

[00138] Clause 8. The method of any of clauses 1 -7, wherein G ¹ is -C(S)NH-R ³

[00139] Clause 9. The method of any of clauses 1-7, wherein G 1 i ₁ s _Q

[00141] Clause 11. The method of any of clauses 1 -7, wherein G ¹ is -C(S)G ² N

[00142] Clause 12. The method of clause 11, wherein G 2 i:s- X or

[00143] Clause 13. The method of clause 1, wherein the compound of formula (I) is

[00144] Clause 14. The method of any of clauses 1-13, wherein the symptom of galactosemia is infertility or reduced fertility, motor impairment, growth restriction, dyspraxic speech, cataract, intellectual deficit, or a combination thereof.

[00145] Clause 15. The method of clause 14, wherein the infertility or reduced fertility comprises primary ovarian insufficiency.

[00146] Clause 16. The method of clause 14, wherein the infertility or reduced fertility comprises decreased numbers of primordial follicles, loss of primordial follicles or a combination thereof. [00147] Clause 17. The method of clause 14, wherein the motor impairment comprises one or more of tremors and ataxia.

[00148] Clause 18. The method of clause 14, wherein the motor impairment comprises decreased numbers of Purkinje cells in the cerebellum, loss of Purkinje cells in the cerebellum or a combination thereof.

[00149] Clause 19. The method of any of clauses 1-18, wherein the subject with galactosemia has down-regulated PI3K/Akt signaling compared to a subject without galactosemia.

[00150] Clause 20. The method of any of clauses 1-19, wherein the therapeutically effective amount is an amount that increases PI3K/Akt signaling.

[00151] Clause 21. The method of any of clauses 1-20, wherein the therapeutically effective amount is an amount that reverses down-regulated PI3K/Akt signaling.

[00152] Clause 22. The method of any of clauses 1-21, wherein the therapeutically effective amount is an amount that increases fertility.

[00153] Clause 23. The method of any of clauses 1-22, wherein the therapeutically effective amount is an amount that increases primordial follicle numbers or reverses the loss of primordial follicles.

[00154] Clause 24. The method of any of clauses 1-23, wherein the therapeutically effective amount is an amount that increases Purkinje cells in the cerebellum.

[00155] Clause 25. The method of any of clauses 1-24, wherein the therapeutically effective amount does not inhibit a glycine transporter.

[00156] Clause 26. The method of clause 25, wherein the glycine transporter is one or both of GlyTl or GlyT2.