ENANTIOMERS OF 5-((7-CHLOROISOQUINOLIN-l-YL)AMINO)-N-(6- METHOXY- 1 ,2,3 ,4-TETRAHYDRONAPHTHALEN-2- YL)PICOLINAMIDE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to European Patent Application No. EP21382846.0, filed on September 20, 2021, the entirety of which is incorporated by reference herein.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to enantiomers of isoquinoline compounds, processes for their preparation, and to the use thereof in the treatment and/or prevention of conditions associated with the alteration of the activity of beta galactosidase, specifically galactosidase beta-1 or GLB1, including GM1 gangliosidoses and Morquio syndrome, type B. In one aspect, the present disclosure is directed to enantiomers of 5- ((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy- 1,2,3, 4-tetrahydronaphthalen-2- yl)picolinamide, and pharmaceutically acceptable salts and solvates thereof, and their use. In one aspect, the present disclosure is directed to the S-enantiomer of 5-((7- chloroisoquinolin- 1 -yl)amino)-N-(6-m ethoxy- 1 ,2,3 ,4-tetrahydronaphthalen-2- yl)picolinamide, and pharmaceutically acceptable salts and solvates thereof, and their use.

BACKGROUND OF THE DISCLOSURE

[0003] WO 2018/122746 Al describes racemic mixtures of certain isoquinoline compounds, including 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy-l, 2,3,4- tetrahydro-naphthalen-2-yl)picolinamide, and their use. The preparation of the racemic mixture of 5-((7-chl oroisoquinolin-l-yl)amino)-N-(6-methoxy-l, 2,3,4- tetrahydronaphthalen-2-yl)picolinamide has been described in Example 40 of WO 2018/122746 Al having the following chemical structure: [0004] Compounds described in WO 2018/122746 Al are expected to be useful in treating GM1 gangliosidosis and Morquio B syndrome, both arising from betagalactosidase (GLB1) deficiency. GM1 gangliosidosis and Morquio B syndrome are very rare lysosomal storage diseases with an incidence of about 1 : 100,000-1 :200,000 live births worldwide (Caciotti A. et al.. Biochim Biophys Acta 1812(7):782-890 (July 2011)). Said conditions associated with GLB1 are known to be caused by a deficiency of the enzyme P-galactosidase due to mutations in the GLB1 gene.

[0005] P-Galactosidase cleaves P-galactose from different substrates, and deficiencies in its activity cause said substrates (i.e., gangliosides, and oligosaccharides carrying terminal P-linked galactose, such as ganglioside GM1 and glycosaminoglycans such as keratin sulfate) to accumulate in patients suffering from conditions associated with GLB 1 activity such as GM1 gangliosidosis and Morquio B syndrome.

[0006] Suzuki et al. (Cell. Mol. Life Sci. 65:351-353 (2008)) reported that the mutations of the GLB1 gene result in an unstable mutant P-galactosidase enzyme protein with normal or near-normal biological activity. The mutant enzyme protein seems to be unstable at neutral pH in the endoplasmic reticulum (ER)/Golgi apparatus, and rapidly degraded because of inappropriate molecular folding and this is the reason for its impaired activity. The authors also reported that the use of a competitive inhibitor binding to misfolded mutant protein as a molecular chaperone (i.e. a small molecule that interacts with a misfolded protein to achieve a recovery on its activity) resulted in the formation of a stable molecular complex at neutral pH. The protein-chaperone complex was safely transported to the lysosome, where it dissociated under the acidic conditions. In this way the mutant enzyme remained stabilized, and its catalytic function was enhanced.

[0007] Therefore, small molecules capable of binding allosterically or competitively to mutated P-galactosidase enzyme thereby stabilizing the enzyme against degradation (chaperones) constitute an important therapeutic target in conditions associated with the alteration of the activity of beta galactosidase, specially galactosidase beta-1 or GLB1.

[0008] There is a need for compounds with improved properties for treating or preventing conditions associated with the alteration of the activity of beta galactosidase, and specifically galactosidase beta-1 or GLB1, including GM1 gangliosidoses and Morquio syndrome, type B. BRIEF SUMMARY OF THE INVENTION

[0009] It has been surprisingly found that the S-enantiomer of 5-((7-chloroisoquinolin-l- yl)amino)-N-(6-methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)pic olinamide, or a pharmaceutically acceptable salt or solvate thereof, exhibits properties that are superior to the corresponding racemic mixture or the R-enantiomer.

[0010] The present disclosure is related to the discovery that compounds represented by Formula S-I or S-II, and pharmaceutically acceptable salts and solvates thereof, exhibit improved pharmacokinetic properties and better metabolic stability than the corresponding racemic compounds and corresponding R-isomers.

[0011] In one aspect, the present disclosure provides compounds represented by Formula S-I or S-II, as described herein, and pharmaceutically acceptable salts and solvates thereof, collectively referred to herein as “Compounds of the Disclosure” (each individually referred to hereinafter as a “Compound of the Disclosure”).

[0012] In some embodiments, Compounds of the Disclosure have an enantiomeric excess of no less than about 50%, no less than about 60%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 95%, or no less than about 99%.

[0013] In another aspect, the present disclosure provides a compound of Formula S-II substantially free from the corresponding R-enantiomer: or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the compound S-II is more than 99% free from the corresponding R-enantiomer.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, as defined herein and at least one pharmaceutically acceptable excipient.

[0015] In another aspect, the present disclosure provides a Compound of the Disclosure as defined herein, or pharmaceutically acceptable salts or solvates thereof, for use in the prevention or treatment of a condition associated with the alteration of the activity of GLB1.

[0016] In another aspect, the present disclosure provides use of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, as defined herein, in the preparation of a medicament for the prevention or treatment of a condition associated with the alteration of the activity of GLB1.

[0017] In another aspect, the present disclosure provides a method of treating or preventing a condition associated with the alteration of the activity of GLB 1 in a patient, comprising administering to the patient in need thereof an effective amount of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof.

[0018] In another aspect, the present disclosure provides a method of treating GM1 gangliosidosis or Morquio B syndrome in a patient, comprising administering to the patient in need thereof an effective amount of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof.

[0019] In another aspect, the present method of treating GM1 gangliosidosis or Morquio B syndrome in a patient further comprises administering to the patient an effective amount of an enzyme for enzyme replacement therapy. In one embodiment, the enzyme is P-galactosidase or an analog thereof.

[0020] In another aspect, the method further comprises administering to the patient a small molecule chaperone. In one embodiment, the small molecule chaperone binds competitively to an enzyme. In another embodiment, the small molecule chaperone is selected from the group consisting of iminoalditols, iminosugars, aminosugars, thiophenylglycosides, glycosidase, sulfatase, glycosyl transferase, phosphatase, and peptidase inhibitors.

[0021] In another aspect, the present disclosure provides a method of increasing P- galactosidase activity in a patient in need thereof, comprising administering to the patient an effective amount of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof.

[0022] Other aspects and advantages of the disclosure will be readily apparent from the following detailed description of the disclosure. The embodiments and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. [0023] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and are not restrictive of the disclosure as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0024] FIG. 1 is a bar graph representing the concentration of the racemic mixture, the (S)-enantiomer, or the (R)-enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6- methoxy-l,2,3,4-tetrahydro-naphthalen-2-yl)picolinamide in brain tissue and in plasma after a single i.v. administration of each compound at 10 mg/kg in male C57/BL6 mice.

[0025] FIGS. 2 A and 2B are line graphs representing the concentrations of the (S)- enantiomer and (R)-enantiomer in plasma of a C57/BL6 mouse after a single oral administration of 10 mg/kg of the racemic mixture, the (S)-enantiomer, or the (R)- enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy-l,2,3,4-tet rahydro- naphthalen-2-yl)picolinamide. FIG. 2A shows a concentration obtained for each enantiomer present in the racemic mixture. In FIG. 2B, the concentrations of the (S)- and (R)-enantiomers obtained for the racemic mixture are combined to show the concentration for the racemic mixture.

[0026] FIG. 3 is a line graph representing a percent of the area of the tested compound, the racemic mixture, the (S)-enantiomer, or the (R)-enantiomer of 5-((7- chloroisoquinolin- 1 -yl)amino)-N-(6-m ethoxy- 1 ,2,3 ,4-tetrahydro-naphthalen-2- yl)picolinamide, remaining in human liver microsomes at various incubation times with respect to the area of the tested compound at time 0 min.

[0027] FIG. 4 is a line graph representing a percent of the area of the tested compound, the racemic mixture, the (S)-enantiomer, or the (R)-enantiomer of 5-((7- chloroisoquinolin- 1 -yl)amino)-N-(6-m ethoxy- 1 ,2,3 ,4-tetrahydro-naphthalen-2- yl)picolinamide, remaining in mouse liver microsomes at various incubation times with respect to the area of the tested compound at time 0 min.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The headings provided herein are not limitations of the various aspects described herein, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Definitions

[0029] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. 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 technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0030] The articles "a," "an," and "the" 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.

[0031] The term "about", as used herein in connection with a measured quantity, refers to the normal variations in that measured quantity, as expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of measurement and precision of the measuring equipment. Typically, the term “about” includes the recited number ± 10%. Thus, “about 10” means 9 to 11.

[0032] As used herein, the term “optionally substituted” refers to a group that can be unsubstituted or substituted.

[0033] As used herein, the terms “halogen” or “halo” refer to -F, -Cl, -Br, or -I.

[0034] As used herein, the term “hydroxyl” or “hydroxy” refers to the group -OH.

[0035] As used herein, the term “alkyl” refers to a linear or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing no unsaturation, which is attached to the rest of the molecule by a single bond and, unless otherwise specified, an alkyl radical typically has from 1 to 4 carbon atoms, i.e., Ci-4 alkyl. Exemplary Ci-4 alkyl groups can be methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, i-butyl and sec-butyl. In another embodiment, the alkyl is C1-2 alkyl (methyl or ethyl). [0036] As used herein, the term “Ci-4 alkoxy” refers to oxygen substituted by one of the Ci-4 alkyl groups mentioned above (e.g., methoxy, ethoxy, propoxy, iso-propoxy, butoxy, tert-butoxy, iso-butoxy, and sec-butoxy), for example by one of the C1-2 alkyl groups.

[0037] As used herein, the term “cycloalkyl” embraces saturated carbocyclic radicals and, unless otherwise specified, a cycloalkyl radical typically has from 3 to 6 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. It is, for example, cyclopropyl, cyclopentyl and cyclohexyl. In another embodiment, the cycloalkyl group is C3-10 cycloalkyl.

[0038] As used herein, the terms “heterocyclyl” or “heterocyclic group” embrace typically a monocyclic or polycyclic, non-aromatic, saturated or unsaturated C2-10 carbocyclic ring, such as a 5- to 10-membered radical, in which one or more, for example 1, 2, 3 or 4 of the carbon atoms, for example, 1 or 2 of the carbon atoms are replaced by a heteroatom selected from N, O and S. In one embodiment, the heterocyclyl is a C3-7 heterocyclyl, i.e., a heterocycle having 3-7 carbon atoms and at least one heteroatom. In another embodiment, a heterocyclyl is a (5- to 10-membered)-Ci-9 heterocyclyl, i.e., a heterocycle having 5- to 10-members, of which 1-9 members are carbon. In another embodiment, the heteroatom is N. In another embodiment, the heteroatom is O.

[0039] In another embodiment, the heterocyclyl radicals are saturated. A heterocyclic radical may be a single ring or two or more fused rings wherein at least one ring contains a heteroatom. When a heterocyclyl radical carries one or more substituents, the substituents may be the same or different.

[0040] A said optionally substituted heterocyclyl is typically unsubstituted or substituted with 1, 2 or 3 substituents which may be the same or different. Examples of heterocyclic radicals include piperidyl, pyrrolidyl, pyrrolinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrazolinyl, pyrazolidinyl, quinuclidinyl, tetrazolyl, cromanyl, isocromanyl, imidazolidinyl, oxiranyl, azaridinyl, 4,5-dihydro-oxazolyl and 3-aza- tetrahydrofuranyl. The substituents are, for example, selected from halogen atoms, for example, fluorine or chlorine atoms, hydroxy groups, alkoxycarbonyl groups in which the alkyl moiety has from 1 to 4 carbon atoms, hydroxycarbonyl groups, carbamoyl groups, nitro groups, cyano groups, Ci-4 alkyl groups optionally substituted by one or more halogen atoms, Ci-4 alkoxy groups, optionally substituted by one or more halogen atoms and Ci-4 hydroxyalkyl groups. [0041] As used herein, the term “aryl” designates typically a Ce-io monocyclic or polycyclic aryl radical such as phenyl and naphthyl. In another embodiment, the aryl is phenyl. A said optionally substituted aryl radical is typically unsubstituted or substituted with 1, 2 or 3 substituents which may be the same or different. The substituents are, for example, selected from halogen atoms, for example, fluorine or chlorine atoms, hydroxy groups, alkoxycarbonyl groups in which the alkyl moiety has from 1 to 4 carbon atoms, hydroxycarbonyl groups, carbamoyl groups, nitro groups, cyano groups, Ci-4 alkyl groups optionally substituted by one or more halogen atoms, Ci-4 alkoxy groups, optionally substituted by one or more halogen atoms and Ci-4 hydroxyalkyl groups. When an aryl radical carries 2 or more substituents, the substituents may be the same or different. Unless otherwise specified, the substituents on an aryl group are typically themselves unsubstituted.

[0042] As used herein, the term “heteroaryl” designates typically a 5- to 10-membered ring system, comprising at least one heteroaromatic ring and containing at least one heteroatom selected from O, S and N, typically 1, 2, 3, or 4 heteroatoms.

[0043] A heteroaryl group may comprise a single ring or two or more fused rings wherein at least one ring contains a heteroatom. A said optionally substituted heteroaryl group is typically unsubstituted or substituted with 1, 2 or 3 substituents which may be the same or different. The substituents are, for example, selected from halogen atoms, for example, fluorine, chlorine or bromine atoms, alkoxycarbonyl groups in which the alkyl moiety has from 1 to 4 carbon atoms, carbamoyl groups, nitro groups, hydroxy groups, Ci-4 alkyl groups, optionally substituted by one or more halogen atoms and Ci-4 alkoxy groups, optionally substituted by one or more halogen atoms. When a heteroaryl radical carries 2 or more substituents, the substituents may be the same or different. Unless otherwise specified, the substituents on a heteroaryl radical are typically themselves unsubstituted.

[0044] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furyl, tetrazolyl, benzofuranyl, oxadiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, thiadiazolyl, thienyl, pyrrolyl, pyridinyl, benzothiazolyl, indolyl, indazolyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinolizinyl, cinnolinyl, triazolyl, indolizinyl, indolinyl, isoindolinyl, isoindolyl, imidazolidinyl, pteridinyl, thianthrenyl, pyrazolyl, 2H- pyrazolo[3,4-d]pyrimidinyl, lH-pyrazolo[3,4-d]pyrimidinyl, thieno[2,3-d]pyrimidinyl, and the various pyrrol opyridyl radicals.

[0045] In another embodiment, the heteroaryl is a (5- to 10-membered)-Ci-9 heteroaryl, i.e., a heteroaryl having 5- to 10-members, of which 1-9 members are carbon. In another embodiment, the heteroatom is N. In another embodiment, the heteroarom is O. In another embodiment, the heteroaryl is optionally substituted with 1, 2, or 3 groups independently selected from the group consisting of halogen, hydroxy, -CN, -ORb, -SRb, -N(Rb)2, -Ci-4alkyl optionally substituted with 1, 2, or 3 halogen atoms, optionally substituted Ce-io aryl, optionally substituted (5- to 10-membered)-Ci-9 heteroaryl, and (5- to 10-membered)-Ci-9 heterocyclyl; said cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, heterocyclyl, and alkylheterocyclyl is optionally fused to a further (second) ring.

[0046] The mention of optionally substituted heteroaryl radicals or rests within the present disclosure is intended to cover the N-oxides obtainable from these radicals when they comprise N-atoms.

[0047] As used herein, the term "stereoisomers" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).

[0048] The term "chiral center" or "asymmetric carbon atom" refers to a carbon atom to which four different groups are attached.

[0049] The term "epimer" refers to diastereomers that have opposite configuration at only one of two or more tetrahedral streogenic centers present in the respective molecular entities.

[0050] The term "stereogenic center" is an atom, bearing groups such that an interchanging of any two groups leads to a stereoisomer.

[0051] The terms "enantiomer" and "enantiomeric" refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light in the opposite direction.

[0052] The term "racemic" refers to a mixture of equal parts of enantiomers and which mixture is optically inactive. [0053] The term "absolute configuration" refers to the spatial arrangement of the atoms of a chiral molecular entity (or group) and its stereochemical description, e.g., R or S.

[0054] The term "resolution" refers to the separation or concentration or depletion of one of the two enantiomeric forms of a molecule.

[0055] The stereochemical terms and conventions used in the specification are meant to be consistent with those described in Pure & Appl. Chem 65:2193 (1996), unless otherwise indicated.

[0056] The term "enantiomeric excess" or "ee" refers to a measure for how much of one enantiomer is present compared to the other. For a mixture of R and S enantiomers, the percent enantiomeric excess is defined as | R - S | *100, where R and S are the respective mole or weight fractions of enantiomers in a mixture such that R + S = 1. With knowledge of the optical rotation of a chiral substance, the percent enantiomeric excess is defined as ([a]obs/[a]max)*100, where [a]obs is the optical rotation of the mixture of enantiomers and [oc]max is the optical rotation of the pure enantiomer. Accordingly, the value of ee will be a number from 0 to 100, 0 being racemic and 100 being pure, single enantiomer. Determination of enantiomeric excess is possible using a variety of analytical techniques, including NMR spectroscopy, chiral column chromatography or optical polarimetry.

[0057] The term “enantiomerically enriched” as used herein refers to Compounds of the Disclosure having an enantiomeric excess of the S-enantiomer compared to the R- enantiomer. In some embodiments, Compounds of the Disclosure are enantiomerically enriched by having the ee of about 5% or more. In another embodiment, the ee is about 10%. In another embodiment, the ee is about 20%. In another embodiment, the ee is about 30%. In another embodiment, the ee is about 40%. In another embodiment, the ee is about 50%. In another embodiment, the ee is about 60%. In another embodiment, the ee is about 70%. In another embodiment, the ee is about 80%. In another embodiment, the ee is about 85%. In another embodiment, the ee is about 90%. In another embodiment, the ee is about 91%. In another embodiment, the ee is about 92%. In another embodiment, the ee is about 93%. In another embodiment, the ee is about 94%. In another embodiment, the ee is about 95%. In another embodiment, the ee is about 96%. In another embodiment, the ee is about 97%. In another embodiment, the ee is about 98%. In another embodiment, the ee is about 99%. [0058] The term, "substantially free from the R-enantiomer" as used herein refers to a Compound of the Disclosure (such as, e.g., a compound of Formula S-II) that is at least about 85% or more, free from the corresponding R-enantiomer. In particular embodiments, the Compound of the Disclosure is at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or about 99.9% free from the corresponding R-enantiomer.

[0059] The term “pharmaceutically acceptable” refers to compositions and molecular entities that are physiologically tolerable and do not typically produce an allergic reaction or a similar unfavorable reaction, such as gastric disorders, dizziness and suchlike, when administered to a human or animal. For example, the term “pharmaceutically acceptable” means it is approved by a regulatory agency of a state or federal government or is included in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0060] The term "treatment" or "treating" refers to administering a therapy in an amount, manner or mode effective to improve a condition, symptom, or parameter associated with a condition or to prevent progression of a condition, to either a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and can be tailored to the patient.

[0061] By an "effective" amount or a "therapeutically effective amount" of a drug or pharmacologically active agent is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0062] The term "prevention" or "to prevent" refers to the reduction in the risk of acquiring or developing a given disease or disorder, or the reduction or inhibition of the recurrence or a disease or disorder.

[0063] The term “patient” as used herein refers to a human. In some embodiments, the patient is an adult. In some embodiments, the patient is a geriatric patient. In some embodiments, the patient is a child. In some embodiments, the patient is an infant. In some embodiments, the patient is a toddler. In some embodiments, the patient is a preadolescent. In some embodiments, the patient is an adolescent.

[0064] As used herein, the term "child" is a human being between the stages of birth and puberty.

[0065] The term "puberty" is the process of physical changes through which a child's body matures into an adult body capable of sexual reproduction. On average, girls begin puberty around ages 10-11 and end puberty around 15-17; boys begin around ages 11-12 and end around 16-17.

[0066] As used herein, the term "infant" is the synonym for "baby," the very young offspring of a human. The term "infant" is typically applied to young children under one year of age.

[0067] As used herein, the term "toddler" refers to a child of 12 to 36 months old.

[0068] As used herein, the term "preadolescent" refers to a person of 10-13 years old.

[0069] As used herein, the term "adolescent" refers to a person between ages 10 and 19.

[0070] The term “solvate” means any form of the active compound of the disclosure which has another molecule (for example a polar solvent such as water or ethanol, a cyclodextrin or a dendrimer) attached to it through noncovalent bonds. Methods of solvation are known within the art.

[0071] The disclosure also provides salts of the Compounds of the Disclosure. Nonlimiting examples are sulphates; hydrohalide salts; phosphates; lower alkane sulphonates; aryl sulphonates; salts of C1-20 aliphatic mono-, di- or tribasic acids which can contain one or more double bonds, an aryl nucleus or other functional groups such as hydroxy, amino, or keto; salts of aromatic acids in which the aromatic nuclei may or may not be substituted with groups such as hydroxyl, lower alkoxyl, amino, mono- or di- lower alkylamino sulphonamido. Also included within the scope of the disclosure are quaternary salts of the tertiary nitrogen atom with lower alkyl halides or sulphates, and oxygenated derivatives of the tertiary nitrogen atom, such as the N-oxides. In preparing dosage formulations, those skilled in the art will select the pharmaceutically acceptable salts.

[0072] Solvates and salts can be prepared by methods known in the state of the art. Note that the non-pharmaceutically acceptable solvates also fall within the scope of the disclosure because they can be useful in preparing pharmaceutically acceptable salts and solvates.

[0073] The Compounds of the Disclosure also seek to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a carbon enriched in ^U C, ¹³ C or ¹⁴ C or the replacement of a nitrogen by a ¹⁵ N enriched nitrogen are within the scope of this disclosure.

[0074] As used herein, the term "enzyme replacement therapy," or "ERT" refers to administering an exogenously-produced natural or recombinant enzyme or analog thereof to a patient in need thereof. In the case of a lysosomal storage disease, for example, the patient accumulates harmful levels of a substrate (i.e., material stored) in lysosomes due to a deficiency or defect in an enzyme responsible for metabolizing the substrate, or due to a deficiency in an enzymatic activator required for proper enzymatic function. Enzyme replacement therapy is provided to the patient to reduce the levels of (i.e., debulk) accumulated substrate in affected tissues. Enzyme replacement therapies for treating lysosomal storage diseases are known in the art. In accordance with a combination therapy of the disclosure, a lysosomal enzyme, e.g., P-galactosidase, can be used for enzyme replacement therapy to reduce the levels of corresponding substrate, e.g., GM1- ganglioside, glycoprotein, keratan sulfate, in a patient having GM1 gangliosidosis or Morquio B syndrome.

[0075] As used herein, an "effective amount" of an enzyme," when administered to a subject in a combination therapy of the disclosure, is an amount sufficient to improve the clinical course of a lysosomal storage disease, where clinical improvement is measured by any of the variety of defined parameters well known to the skilled artisan.

[0076] As used herein the term "small molecule chaperone" refers to a compound, other than a Compound of the Disclosure, that is capable of binding allosterically or competitively to a mutated enzyme, e.g., P-galactosidase, thereby stabilizing the enzyme against degradation. In some embodiments, the small molecule chaperone facilitates proper folding and transport of an enzyme to its site of action. Small molecule chaperones for the treatment of lysosomal storage diseases are known in the art. See, e.g., US 2016/0207933 Al and WO 2011/049737 Al. Compounds of the Disclosure

[0077] The inventors have unexpectedly discovered that S-enantiomers of a certain group of isoquinoline compounds exhibit properties that are superior compared to the corresponding racemic compounds. In particular, the S-enantiomer of 5-((7- chloroisoquinolin- 1 -yl)amino)-N-(6-m ethoxy- 1 ,2,3 ,4-tetrahydronaphthalen-2- yl)picolinamide (also described herein as 5-[(7-chloro-l-isoquinolyl)amino]-N-[(2S)-6- methoxytetralin-2-yl]pyridine-2-carboxamide) having the structure: has been found to, for example, exhibit surprisingly better pharmacokinetic properties, better brain penetration, and an improved metabolic stability than those exhibited by the racemic compound, or even the R-enantiomer. Compounds of the Disclosure thus potentially offer an advantage over treating patients in need thereof with the corresponding racemic mixtures.

[0078] In one embodiment, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I: and the pharmaceutically acceptable salts and solvates thereof, wherein

[0079] R ¹ and R ² are each independently selected from the group consisting of hydrogen, halogen, -CN, -ORb, and -Ci-4 alkyl, wherein said -Ci-4 alkyl group is optionally substituted by 1, 2 or 3 independently selected halogen atoms; [0080] R ³ is selected from the group consisting of halogen, -CN, -ORb, and -Ci-4 alkyl, wherein said -Ci-4 alkyl group is optionally substituted by 1, 2 or 3 independently selected halogen atoms;

[0081] each R ⁴ is independently selected from the group consisting of halogen, hydroxy, -CN, -ORb, -SRb, -N(Rb)2, -Ci-4alkyl optionally substituted with 1, 2, or 3 halogen atoms, optionally substituted -Ce-io aryl, optionally substituted -(5- to 10-membered)-Ci-9 heteroaryl, and -(5- to 10-membered)-Ci-9 heterocyclyl;

[0082] m is 0, 1, 2, or 3; and

[0083] each Rb is independently selected from the group consisting of hydrogen, -Ci-4 alkyl, -C3-10 cycloalkyl, and -(5- to 10-membered)-Ci-9 heterocyclyl, wherein said alkyl, cycloalkyl or heterocyclyl groups are optionally substituted by 1, 2 or 3 fluorine atoms.

[0084] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein R ³ is selected from the group consisting of halogen, -CN, and -ORb, wherein Rb is as defined for Formula S-I. In some embodiments, R ³ is selected from the group consisting of halogen, -CN, -OH, and -O(Ci-4 alkyl) optionally substituted with 1, 2, or 3 fluorine atoms. In some embodiments, R ³ is a halogen. In some embodiments, R ³ is -Cl, -Br, or -F. In some embodiments, R ³ selected from the group consisting of -Cl, -CN, and -OCH3. In some embodiments, R ³ is -Cl.

[0085] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein R ¹ and R ² are both H and R ³ is selected from the group consisting of halogen, -CN, and -ORb, wherein Rb is as defined for Formula S-I. In another embodiment, R ³ is selected from the group consisting of -Cl, -CN, and -OCH3.

[0086] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein m is 0. In some embodiments, Compounds of the Disclosure are compounds of Formula S-I, and the salts and solvates thereof, wherein m is 1 or 2 and R ⁴ is independently selected from the group consisting of halogen, hydroxy, -CN, -ORb, -SRb, -N(Rb)2, -Ci-4 alkyl optionally substituted with 1, 2, or 3 halogen atoms, optionally substituted -Ce-io aryl, optionally substituted -(5- to 10-membered)-Ci-9 heteroaryl, and - (5- to 10-membered)-Ci-9 heterocyclyl. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, R ⁴ is independently selected from the group consisting of halogen, hydroxy, -CN, -ORb, -SRb, -N(Rb)2, and -Ci-4 alkyl optionally substituted with 1, 2, or 3 halogen atoms, wherein each Rb is independently selected from the group consisting of hydrogen and -Ci-4 alkyl optionally substituted by 1, 2 or 3 fluorine atoms. In some embodiments, R ⁴ is independently methyl, ethyl, methoxy, or ethoxy. In another embodiment, R ⁴ is methoxy.

[0087] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein m is 1 and R ⁴ is at the 5-position of the tetrahydronaphthalen-2- yl ring:

[0088] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein m is 1 and R ⁴ is at the 6-position of the tetrahydronaphthalen-2- yl ring:

[0089] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein m is 1 and R ⁴ is at the 7-position of the tetrahydronaphthalen-2- yl ring:

[0090] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein m is 1 and R ⁴ is at the 8-position of the tetrahydronaphthalen-2- yl ring:

[0091] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein R ¹ and R ² are both hydrogen and R ³ is selected from the group consisting of halogen, -CN, -ORb, and -Ci-4 alkyl, wherein said -Ci-4 alkyl group is optionally substituted by 1, 2, or 3 independently selected halogen atoms, and wherein Rb is as defined above for Formula S-I. In another embodiment, Rb is hydrogen or -Ci-4 alkyl optionally substituted by 1, 2, or 3 independently selected halogen atoms.

[0092] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein R ¹ is hydrogen and R ² and R ³ are each independently selected from the group consisting of halogen, -CN, -ORb, and -Ci-4 alkyl, wherein said -Ci-4 alkyl group is optionally substituted by 1, 2, or 3 independently selected halogen atoms, and wherein Rb is as defined above. In another embodiment, Rb is hydrogen or -Ci-4 alkyl optionally substituted with 1, 2, or 3 independently selected halogen atoms.

[0093] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein R ² is hydrogen and R ¹ and R ³ are each independently selected from the group consisting of halogen, -CN, -ORb, and -Ci-4 alkyl, wherein said -Ci-4 alkyl group is optionally substituted by 1, 2, or 3 independently selected halogen atoms, and wherein Rb is as defined above. In another embodiment, Rb is hydrogen or -Ci-4 alkyl optionally substituted with 1, 2, or 3 independently selected halogen atoms.

[0094] In some embodiments, Compounds of the Disclosure are enantiomerically enriched compounds of Formula S-I, and the pharmaceutically acceptable salts and solvates thereof, wherein R ¹ , R ² , and R ³ , when other than hydrogen, are each independently selected from the group consisting of chlorine, fluorine, -CN, unsubstituted -Ci-4 alkyl (such as methyl or ethyl), -Ci-4 alkyl substituted with 1, 2, or 3 fluorine atoms (such as fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 1,1-difluoroethyl, or 1,1,1 -trifluoroethyl), and -ORb, wherein Rb is hydrogen, unsubstituted -Ci-4 alkyl (such as methyl or ethyl), or -Ci-4 alkyl substituted with 1, 2, or 3 fluorine atoms (such as fluorom ethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 1,1-difluoroethyl, or 1,1,1- trifluoroethyl). In some embodiments, R ¹ , R ² , and R ³ , when other than hydrogen, are each independently selected from the group consisting of chlorine and -ORb, wherein Rb is hydrogen or unsubstituted -Ci-4 alkyl. In another embodiment, Rb is hydrogen or -Ci-4 alkyl.

[0095] In another embodiment, a Compound of the Disclosure is enantiomerically enriched compound of Formula S-II: and the pharmaceutically acceptable salts and solvates thereof.

[0096] In some embodiments, the Compound of the Disclosure is enantiomerically enriched compound of Formula S-II:

[0097] In some embodiments, the Compound of the Disclosure is a pharmaceutically acceptable salt of the enantiomerically enriched compound of Formula S-II. In some embodiments, the Compound of the Disclosure is the hydrochloride (HC1) salt of the enantiomerically enriched compound of Formula S-II, or a solvate thereof.

[0098] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess (ee) is about 5% or more. In another embodiment, the ee is about 10%. In another embodiment, the ee is about 20%. In another embodiment, the ee is about 30%. In another embodiment, the ee is about 40%. In another embodiment, the ee is about 50%. In another embodiment, the ee is about 60%. In another embodiment, the ee is about 70%. In another embodiment, the ee is about 80%. In another embodiment, the ee is about 85%. In another embodiment, the ee is about 90%. In another embodiment, the ee is about 91%. In another embodiment, the ee is about 92%. In another embodiment, the ee is about 93%. In another embodiment, the ee is about 94%. In another embodiment, the ee is about 95%. In another embodiment, the ee is about 96%. In another embodiment, the ee is about 97%. In another embodiment, the ee is about 98%. In another embodiment, the ee is about 99%.

[0099] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 50%.

[0100] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 60%.

[0101] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 70%.

[0102] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 80%.

[0103] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 90%.

[0104] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 95%.

[0105] In some embodiments, Compounds of the Disclosure are compounds of any one of Formulae S-I or S-II, and the pharmaceutically acceptable salts and solvates thereof, wherein the enantiomeric excess is no less than about 99%.

[0106] In some embodiments, the pharmaceutically acceptable salt of a compound of any one of Formulae S-I or S-II is a hydrochloride salt (a HCl-salt).

Synthesis of Compounds of the Disclosure

[0107] Compounds of the Disclosure can be synthesized, for example, by using methods described in WO 2018/122746 Al to obtain a racemic mixture followed by chiral column separation of the enantiomers. [0108] In addition, Compounds of the Disclosure can be prepared by first preparing an acyl chloride intermediate by, for example using methods described in WO 2018/122746 Al, and then reacting the intermediate with a suitable chiral amine. An exemplary reaction for synthetizing the intermediate (e) is presented in Scheme 1 and the reaction with a chiral amine to obtain compounds of Formula S-I is presented in Scheme 2 below.

Scheme 1 wherein R ¹ , R ² , and R ³ are as defined above for Formula S-I.

Scheme 2 wherein R ¹ , R ² , R ³ , R ⁴ , and m are as defined above for Formula S-I.

[0109] Compounds of Formula S-I can be further converted to a salt form, such as a HC1- salt, by methods known in the art. Use of Compounds of the Disclosure

[0110] Compounds of the Disclosure described herein exhibit improved properties over their racemic mixtures. For example, as described in Example 4, the S-enantiomer of 5- ((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy- 1,2,3, 4-tetrahydronaphthalen-2- yl)picolinamide (S-II) (also described herein as 5-[(7-chloro-l-isoquinolyl)amino]-N- [(2S)-6-methoxytetralin-2-yl]pyridine-2-carboxamide) is more brain penetrant than the racemic compound. As shown in FIG. 1, the brain tissue has about 40% higher concentration of S-enantiomer (S-II) than the racemic mixture or the R-enantiomer (R-II) after a single i.v. administration of each compound at 10 mg/kg in male C57/BL6 mice. In addition, FIGS. 2 A and 2B show that the S-enantiomer has a lower in vivo clearance than the racemic mixture of the R-enantiomer after a single oral administration of 10 mg/kg of the racemic mixture, the (S)-enantiomer, or the (R)-enantiomer to C57/BL6 mice. In view of the data presented in Example 5, Table 2, the racemic mixture exhibits about 58% higher clearance in this in vivo test than the S-enantiomer (S-II) and, thus, the racemic mixture is expected to have a lower systemic exposure in a human patient than the S-enantiomer. As shown in FIGS. 3 and 4, the S-enantiomer (S-II) is more metabolically stable in human and mouse liver microsomes, respectively, than the racemic mixture. Therefore, Compounds of the Disclosure are expected to provide a more effective therapy for patients in need of treatment or prevention of a condition associated with the alteration of the activity of GLB1, such as GM1 gangliosidosis or Morquio B syndrome, than the corresponding racemic mixtures.

[0111] Compounds of the Disclosure described herein can be used/administered to treat and/or prevent conditions associated with the alteration of the activity of B-galactosidase, specifically galactosidase B-l or GLB1, including GM1 gangliosidoses and Morquio syndrome, type B, in a patient suffering from said condition.

[0112] In one aspect, the present disclosure is directed to a method of treating or preventing a condition associated with the alteration of the activity of GLB 1 in a patient, comprising administering to the patient in need thereof an effective amount of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the Compound of the Disclosure is a compound of Formula S-I, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the Compound of the Disclosure is a compound of Formula S-II, or a pharmaceutically acceptable salt or solvate thereof, as described herein.

[0113] In another aspect, the present disclosure is directed to a method of treating GM1 gangliosidosis or Morquio B syndrome in a patient, comprising administering to the patient in need thereof an effective amount of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the Compound of the Disclosure is a compound of Formula S-I, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the Compound of the Disclosure is a compound of Formula S-II, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the method of treating GM1 gangliosidosis or Morquio B syndrome in a patient further comprises administering to the patient an effective amount of an enzyme for enzyme replacement therapy. In some embodiments, the enzyme is P-galactosidase or an analog thereof. In some embodiments, the method further comprises administering to the patient a small molecule chaperone. In some embodiments, the small molecule chaperone binds competitively to an enzyme. In some embodiments, the small molecule chaperone is selected from the group consisting of iminoalditols, iminosugars, aminosugars, thiophenylglycosides, glycosidase, sulfatase, glycosyl transferase, phosphatase, and peptidase inhibitors. In some embodiments, suitable small molecule chaperones are selected from the group consisting of 1-deoxygalactonojirimycin (DGJ), N- nonyldeoxynojirimycin (NN-DNJ), N-butyldeoxygalactonojirimycin (NB-DGJ), galactose, fluorous iminoalditol, and epi-isofagomine.

[0114] In another aspect, the present disclosure is directed to a method of increasing P- galactosidase activity in a patient in need thereof, comprising administering to the patient an effective amount of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the Compound of the Disclosure is a compound of Formula S-I, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the Compound of the Disclosure is a compound of Formula S-II, or a pharmaceutically acceptable salt or solvate thereof, as described herein.

[0115] In another aspect, the present disclosure is also directed to the use of a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for treating and/or preventing a condition associated with the alteration of the activity of 13-galactosidase, specifically galactosidase 13-1 or GLB1, including GM1 gangliosidoses and Morquio syndrome, type B, in a patient suffering from said condition. In some embodiments, the Compound of the Disclosure is a compound of Formula S-I, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the Compound of the Disclosure is a compound of Formula S-II, or a pharmaceutically acceptable salt or solvate thereof, as described herein.

[0116] In another aspect, the present disclosure is directed to a Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, for use in treating GM1 gangliosidosis or Morquio B syndrome in a patient. In some embodiments, the Compound of the Disclosure is a compound of Formula S-I, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the

Compound of the Disclosure is a compound of Formula S-II, or a pharmaceutically acceptable salt or solvate thereof, as described herein. In some embodiments, the

Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, is administered to the patient in combination with an effective amount of an enzyme for enzyme replacement therapy. In some embodiments, the enzyme is P-galactosidase or an analog thereof. In some embodiments, the Compound of the Disclosure, or a pharmaceutically acceptable salt or solvate thereof, is administered to the patient in combination with a small molecule chaperone. In some embodiments, the small molecule chaperone binds competitively to an enzyme. In some embodiments, the small molecule chaperone is selected from the group consisting of iminoalditols, iminosugars, aminosugars, thiophenylglycosides, glycosidase, sulfatase, glycosyl transferase, phosphatase, and peptidase inhibitors. In some embodiments, suitable small molecule chaperones are selected from the group consisting of 1 -deoxy galactonojirimycin (DGJ), N-nonyldeoxynojirimycin (NN-DNJ), N-butyldeoxygalactonojirimycin (NB-DGJ), galactose, fluorous iminoalditol, and epi-isofagomine.

Pharmaceutical compositions

[0117] The present disclosure is also directed to pharmaceutical compositions, comprising an effective amount of a Compound of the Disclosure and at least one pharmaceutically acceptable excipient. In some embodiments, the composition comprises an effective amount of a compound of Formula S-I or Formula S-II, or a pharmaceutically acceptable salt or solvate thereof, as described herein, and at least one pharmaceutically acceptable excipient.

[0118] Due to their activity, the Compounds of the Disclosure can be used in human medicine. As described above, the Compounds of the Disclosure are useful for treating or preventing a condition associated with the alteration of the activity of B-galactosidase. The Compounds of the Disclosure can be administered to any patient suffering said condition. The term “patient” as used herein refers to any human that may experience the beneficial effects of a Compound of the Disclosure.

[0119] When administered to a patient, a Compound of the Disclosure can be administered as a component of a composition that comprises a pharmaceutically acceptable excipient or carrier.

[0120] The Compound of the Disclosure can be administered in combination with at least one other therapeutic agent. In one embodiment, the therapeutic agent comprises an enzyme for enzyme replacement therapy. In another embodiment, the therapeutic agent comprises a small molecule chaperone. Administration of the Compound of the Disclosure with at least one other therapeutic agent can be sequential or concurrent. In one embodiment, the Compound of the Invention and the at least one other therapeutic agent are administered in separate dosage forms. In another embodiment, the Compound of the Invention and the at least one other therapeutic agent are administered concurrently in the same dosage form.

[0121] The term “excipient” refers to a vehicle, diluent, or adjuvant that is administered with the active ingredient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similar. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, for example, for injectable solutions, may be used as vehicles. Suitable pharmaceutical vehicles are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin, 21 ^st Edition, 2005; or “Handbook of Pharmaceutical Excipients,” Rowe C.R.; Paul J.S.; Marian E.Q., sixth Edition, incorporated herein by reference.

[0122] Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid compositions (solutions, suspensions, or emulsions) for oral, topical, or parenteral administration. [0123] In another embodiment, the pharmaceutical compositions are in an oral delivery form. Pharmaceutical forms suitable for oral administration can be tablets and capsules, and can contain conventional excipients known in the art, such as binders, for example syrup, gum Arabic, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, cornstarch, calcium phosphate, sorbitol, or glycine; lubricants for the preparation of tablets, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycolate, or microcrystalline cellulose; or pharmaceutically acceptable wetting agents, such as sodium lauryl sulphate.

[0124] Solid oral compositions can be prepared by conventional methods of blending, filling, or preparation of tablets. Repeated blending operations can be used to distribute the active ingredient in all the compositions that use large amounts of fillers. Such operations are conventional in the art. The tablets can be prepared, for example, by dry or wet granulation and optionally can be coated by well known methods in normal pharmaceutical practice, in particular using enteric coating.

[0125] Pharmaceutical compositions can also be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in the appropriate unit dosage form. Suitable excipients, such as fillers, buffering agents, or surfactants can be used.

[0126] The mentioned formulations can be prepared using standard methods, such as those described or referred to in the Spanish and U.S. Pharmacopoeias and similar reference texts.

[0127] In general, the effective amount of a Compound of the Disclosure to be administered depends on the relative efficacy of the compound chosen, the severity of the condition or disorder being treated, and the patient’s weight. The active compound can be administered one or more times a day, for example 1, 2, 3, or 4 times daily, with typical total daily doses in the range from about 0.01 mg/kg of body weight/day to about 1000 mg/kg of body weight/day. In another embodiment, the effective dosage amount of a Compound of the Disclosure is about 500 mg/kg of body weight/day or less. In another embodiment, the effective dosage amount of a Compound of the Disclosure is about 100 mg/kg of body weight/day or less.

[0128] In another embodiment, the effective dosage amount ranges from about 0.01 mg/kg of body weight/day to about 100 mg/kg of body weight/day of a Compound of the Disclosure; in another embodiment, from about 0.02 mg/kg of body weight/day to about 50 mg/kg of body weight/day of a Compound of the Disclosure; and in another embodiment, from about 0.025 mg/kg of body weight/day to about 20 mg/kg of body weight/day of a Compound of the Disclosure.

[0129] A composition of the disclosure can be prepared by a method comprising admixing a Compound of the Disclosure with a pharmaceutically acceptable excipient or carrier. Admixing can be accomplished using methods known for admixing a compound and a pharmaceutically acceptable excipient or carrier. In another embodiment, the Compound of the Disclosure is present in the composition in an effective amount.

[0130] The following examples are illustrative, but not limiting, of the compounds, compositions and methods of the present disclosure. Suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art in view of this disclosure are within the spirit and scope of the disclosure.

EXAMPLES

[0131] Hereinafter, the term “h” means hours, “min” means minutes, “UPLC” means ultra-performance liquid chromatography, “UPLC-MS” or “HPLC-MS” means Liquid chromatography-mass spectrometry, “UV-DAD” means diode array ultraviolet detector method, “DMSO-de” means deuterated dimethyl sulfoxide, “DCM” means Di chloromethane, “MeOH” mean methanol, “EtOH” mean ethanol, “EtOAc” means ethyl acetate, “TEA” means triethylamine, “Pd2(dba)3” means tris(dibenzylideneacetone)- dipalladium(O), and “XantPhos” means 4,5-Bis(diphenylphosphino)-9,9- dimethylxanthene.

[0132] Purity and enantiomeric excess analysis of the compounds were conducted as per one of the following methods.

[0133] All the reagents used are commercially available.

[0134] Method UPLC-MS: Aquity UPLC BEH C18 (2.1 mm x 100 mm, 1.7 pm); wavelength: 210 nm - 400 nm; run time: 8.0 min; Mobile phase A: 0.1% of formic acid in water and B: 1.0% formic acid in acetonitrile; Time and mobile phase-gradient (time in min/%B): 0.00/3, 0.50/3, 7.00/97, 7.50/97, 7.60/3, 8.00/3; MASS: Agilent 1260 system coupled with diode array detector, and Q-Tof MS detector (G6540B). [0135] Method Chiral HPLC: Chiralpak® IA (3.0 mm x 150 mm, 3 pm); wavelength: 190 nm - 400 nm; run time: 20.0 min; Mobile phase and gradient: isocratic elution of 0.1% formic acid in water: 0.1% formic acid in acetonitrile = 30:70, 0.5 mL min' ¹ .

Example 1

Synthesis of general intermediate 5: 5-[(7-chloro-l-isoquinolyl)amino]pyridine -2-

[0136] Step 1: Synthesis of methyl 5-[(7-chloro-l-isoquinolyl)amino]pyri dinercarb oxy late (3).

[0137] 1,7-Dichloroisoquinoline (1) (10 g, 49 mmol), methyl 5-aminopyridine-2- carboxylate (2) (7.68 g, 49 mmol), Xantphos (5.84 g, 9.8 mmol) and CS2CO3 (32 g, 98 mmol) were suspended in degassed 1,4-dioxane (250 mL) under argon atmosphere. To the suspension, Pd2(dba)3 (4.45 g, 7.34 mmol) was added. The reaction mixture was stirred for 2.5 h at 130 °C, cooled to room temperature, passed through a pad of Celite and washed with EtOAc (400 mL). Obtained mother liquor was washed with water (2 x 140 mL), sat. aq. sol. NaHCOs (2 x 140 mL) and brine (140 mL), dried over Na2SO4 (anhydrous), filtered and evaporated to dryness. The crude product was adsorbed on silica and dry loaded in pre-column and purified by flash chromatography (eluent cyclohexane/EtOAc, 0 - 90 % EtOAc) to give the desired compound 3 (9.17 g, yield: 60%, light yellow solid, purity by UV-DAD: 98%); MS (ES+, m/z 314.0 [M+H] ⁺ ; ^X H NMR (DMSO-de, 600 MHz): 8 9.78 (s, 1H), 9.12 (d, J=2.6 Hz, 1H), 8.74 (s, 1H), 8.63 (dd, J=8.6, 2.6 Hz, 1H), 8.15 (d, J=5.7 Hz, 1H), 8.07 (d, J=8.6 Hz, 1H), 7.97 (d, J=8.8 Hz, 1H), 7.81 (dd, J=8.6, 2.0 Hz, 1H), 7.41 (d, J=5.7 Hz, 1H), 3.87 (s, 3H) ppm. [0138] Step 2: Synthesis of 5-[(7-chl oro-1 -isoquinolyl) amino]pyridine-2-carboxylic acid (4).

[0139] To a solution of methyl 5-[(7-chloro-l-isoquinolyl)amino]pyridine-2-carboxylate

(3) (9.17 g, 28.6 mmol) in methanol (200 mL), a solution of LiOH (6.01 g, 0.14 mol) in water (150 mL) was added. After 18 h of stirring at room temperature, the reaction was completed. Methanol was evaporated under reduced pressure, and additional amount of water (150 mL) was added. The pH of the suspension was adjusted to 4.5 with 6M HC1. The resulting solid was filtered under vacuum, washed with water (100 mL), and dried in the vacuum oven at 60 °C for 24 h to afford the desired compound 4 (8.56 g, yield: 94%, yellow solid, purity by UV-DAD: 94%); MS (ES+, m/z . 300.0 [M+H] ⁺ ; ^X H NMR (DMSO-de, 500 MHz): 8 9.74 (s, 1H), 9.11 (d, J=2.4 Hz, 1H), 8.73 (s, 1H), 8.59 (dd, J=8.7, 2.6 Hz, 1H), 8.14 (d, J=5.5 Hz, 1H), 8.05 (d, J=8.9 Hz, 1H), 7.96 (d, J=8.9 Hz, 1H), 7.80 (dd, J=8.7, 2.0 Hz, 1H), 7.39 (d, J=5.5 Hz, 1H) ppm.

[0140] Step 3: Synthesis of 5-[(7-chloro-l-isoquinolyl)amino]pyridine-2-carbonyl chloride (5).

[0141] To a suspension of 5-[(7-chloro-l-isoquinolyl)amino]pyridine-2-carboxylic acid

(4) (8.56 g, 26.8 mmol) in dry acetonitrile (250 mL), dry DMF (0.4 pL) at 0°C was added. Oxalyl chloride (6.91 mL, 80.5 mmol) was added at 0°C and the reaction mixture was left to stir at 0°C for 2 h, and then at room temperature overnight. Aliquot of the reaction mixture was dissolved in dry MeOH and checked by UPLC-MS. The desired product 5 (methyl ester) was observed, purity by UV-DAD: 96%, MS (ES+, m/z . 314.0 [M+H]+. The starting material was completely consumed. Reaction mixture was evaporated till dryness, and co-evaporated with toluene (2 x 50 mL). The mixture was concentrated in vacuo to yield crude compound 5 (8.95 g, yield: 92%, brown solid, purity by UV (254 nm): 88%).

Example 2

Synthesis of S- and R-enantiomers of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy- l,2,3,4-tetrahydronaphthalen-2-yl)picolinamide as HCl-salts

[0142] The synthesis of the S- and R-enantiomers of 5-((7-chloroisoquinolin-l- yl)amino)-N-(6-methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)pic olinamide HC1 follow the same synthetic route. The first step is an amidation of the acyl chloride 5 with enantiomerically pure amines (HC1 salts), followed by the HC1 salt formation.

Example 2.1

Synthesis of the S-enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy- l,2,3,4-tetrahydronaphthalen-2-yl)picolinamide (S-II) HC1

[0143] Step a: Synthesis of 5-[(7-chloro-l-isoquinolyl)amino]-N-[(2S)-6- methoxytetralin-2-yl]pyridine-2-carboxamide (S-II)

[0144] To a suspension of 5-[(7-chloro-l-isoquinolyl)amino]pyridine-2-carbonyl chloride (5) (4.31 g, 12.9 mmol) in dry dichloromethane (150 mL) previously cooled to 0°C , (2S)- 6-methoxytetralin-2-amine hydrochloride (6) (2.5 g, 11.7 mmol) and TEA (8.15 mL, 58.5 mmol) at 0 °C were added. Stirring of the reaction mixture was continued at 0 °C for 2 h. Water (150 mL) was added to the reaction mixture. The organic layer was separated, and the aqueous layer was extracted with DCM (2 x 100 mL). Combined organic layers were dried over Na2SO4 and evaporated to dryness to yield the crude product which was purified by flash chromatography (eluent: DCM / 10% MeOH in DCM; elution of the product at 30% of 10% MeOH in DCM) to afford a brownish solid product which was triturated with EtOAc. The light yellow precipitate was filtered and dried under vacuum affording 2.13 g of the title compound (S-II) (yield 39 %, purity by UV-D D: 98.0%).

[0145] MS (ES+, m/zy. 459.0 [M+H] ⁺ .

[0146] ’H NMR (DMSO-de, 300 MHz): 8 9.68 (s, 1H), 9.07 (d, J=2.1 Hz, 1H), 8.71 (s, 1H), 8.56 (dd, J=2.5, 8.5 Hz, 1H), 8.47 (d, J=8.2 Hz, 1H), 8.10 (d, J=5.8 Hz, 1H), 8.03 (d, J=8.9 Hz, 1H), 7.94 (d, J=8.9 Hz, 1H), 7.78 (dd, J=1.9, 8.5 Hz, 1H), 7.35 (d, J=5.3 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 6.73 - 6.64 (m, 2H), 4.26 - 4.10 (m, 1H), 3.71 (s, 3H), 3.00 - 2.73 (m, 4H), 2.05 - 1.91 (m, 1H), 1.91 - 1.77 (m, 1H).

[0147] Step b: Synthesis of 5-[(7-chloro-l-isoquinolyl)amino]-N-[(2S)-6- methoxytetralin-2-yl]pyridine-2-carboxamide (S-II) hydrochloride

[0148] HC1 in EtOH (1.25 M, 14.9 mL, 18.6 mmol) was added dropwise to a suspension of 5 - [(7-chloro- 1 -i soquinolyl)amino] -N- [(2 S)-6-methoxytetralin-2-yl]pyridine-2- carboxamide (S-II) (2.13 g, 4.64 mmol) in EtOH (dried on sieves, 30 mL) at 0°C. Upon addition of HC1 in EtOH, compound S-II was completely dissolved with stirring and immediately precipitate occurred. After 10 min of stirring at 0°C, the reaction mixture was taken out from the ice bath and left to stir at room temperature for 3 h. After three hours, the reaction mixture was evaporated till dryness, diethyl ether (3 x 50 mL) was added and the reaction mixture was evaporated in vacuo to obtain the desired title compound S-II HC1 (2.22 g, yield: 95%, purity by UV-DAD: 98.0%).

[0149] MS (ES+, m/z 459.0 [M+H] ⁺ ).

[0150] ¹ H NMR (DMSO-de, 600 MHz) 5 = 11.11 (br s, 1H), 9.05 (s, 2H), 8.72 (d, J=8.4

Hz, 1H), 8.45 (d, J=7.8 Hz, 1H), 8.20 (d, J=8.4 Hz, 1H), 8.04 (d, J=9.0 Hz, 1H), 7.96 (dd, J=1.8, 9.0 Hz, 1H), 7.91 (d, J=6.6 Hz, 1H), 7.46 (d, J=6.6 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.72 - 6.67 (m, 2H), 4.25 - 4.16 (m, 1H), 3.71 (s, 3H), 2.98 - 2.90 (m, 1H), 2.89 - 2.80 (m, 3H), 2.03 - 1.95 (m, 1H), 1.94 - 1.83 (m, 1H) ppm.

[0151] The enantiomeric excess determined by chiral HPLC-MS was more than 99 %.

Example 2.2

Synthesis of R-enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy-l, 2,3,4- tetrahydronaphthalen-2-yl)picolinamide (R-II) HC1

[0152] Step a: Synthesis of 5-[(7-chloro-l-isoquinolyl)amino]-N-[(2R)-6- methoxytetralin-2-yl]pyridine-2-carboxamide (R-II)

[0153] To a suspension of 5-[(7-chloro-l-isoquinolyl)amino]pyridine-2-carbonyl chloride (5) (3.5 g, 9.69 mmol) in dry di chloromethane (150 mL) previously cooled to 0°C , (2R)- 6-methoxytetralin-2-amine hydrochloride (6) (1.8 g, 8.42 mmol) and TEA (5.87 mL, 42.1 mmol) at 0 °C were added. Stirring of the reaction mixture was continued at room temperature for 18 h. Water (75 mL) was added to the reaction mixture. The organic layer was separated, and the aqueous layer was extracted with DCM (2 * 100 mL). Combined organic layers were dried over Na2SO4 and evaporated to dryness to yield crude title compound which was purified by flash chromatography (eluent: DCM / 10% MeOH in DCM; elution of the product at 30% of 10% MeOH in DCM) to afford a brownish solid product which was triturated with EtOAc. The light yellow precipitate was filtered and dried under vacuum affording 2.30 g of the tile compound (R-II) (yield 59 %, purity by UV-DAD: 99.5%).

[0154] MS (ES+, m/zy. 459.7 [M+H] ⁺ .

[0155] ’H NMR (DMSO-de, 500 MHz): 8 9.69 (s, 1H), 9.09 (d, J=2.4 Hz, 1H), 8.72 (s, 1H), 8.56 (dd, J=2.4, 8.9 Hz, 1H), 8.48 (d, J=8.2 Hz, 1H), 8.11 (d, J=5.8 Hz, 1H), 8.04 (d, J=8.5 Hz, 1H), 7.95 (d, J=8.9 Hz, 1H), 7.80 (dd, J=1.7, 8.7 Hz, 1H), 7.36 (d, J=5.8 Hz, 1H), 7.00 (d, J=8.2 Hz, 1H), 6.73 - 6.67 (m, 2H), 4.24 - 4.15 (m, 1H), 3.71 (s, 3H), 2.98 - 2.89 (m, 1H), 2.88 - 2.78 (m, 3H), 2.03 - 1.96 (m, 1H), 1.92 - 1.82 (m, 1H).

[0156] Step b: Synthesis of 5-[(7-chloro-l-isoquinolyl)amino]-N-[(2R)-6- methoxytetralin-2-yl]pyridine-2-carboxamide (R-II) hydrochloride

[0157] HC1 in EtOH (1.25 M, 20 mL, 25 mmol) was added dropwise to a suspension of 5-[(7-chloro-l-isoquinolyl)amino]-N-[(2R)-6-methoxytetralin- 2-yl]pyridine-2- carboxamide (R-II) (2.87 g, 6.25 mmol) in EtOH (dried on sieves, 40 mL) at 0°C. Upon addition of HC1 in EtOH, compound R-II was completely dissolved with stirring and immediately precipitate occurred. After 30 min of stirring at 0°C, the reaction mixture was taken out from the ice bath and left to stir at room temperature for 3 h. After three hours, the reaction mixture was evaporated till dryness, diethyl ether (3 x 75 mL) was added and the reaction mixture was evaporated in vacuo to obtain desired product (R-II) HC1 (2.73 g, yield: 86%, purity by UV-DAD: 98.0%).

[0158] MS (ES+, m/zy. 459.5 [M+H] ⁺ ).

[0159] ’H NMR (DMSO-de, 500 MHz) 8 = 10.74 (br s, 1H), 9.07 - 9.02 (m, 1H), 8.94 (s, 1H), 8.66 (d, J=8.2 Hz, 1H), 8.46 (d, J=7.9 Hz, 1H), 8.16 (d, J=8.5 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.97 - 7.90 (m, 2H), 7.44 (d, J=6.4 Hz, 1H), 7.00 (d, J=8.2 Hz, 1H), 6.74 - 6.66 (m, 2H), 4.26 - 4.16 (m, 1H), 3.71 (s, 3H), 2.98 - 2.90 (m, 1H), 2.89 - 2.78 (m, 3H), 2.03 - 1.95 (m, 1H), 1.94 - 1.83 (m, 1H) ppm.

[0160] The enantiomeric excess determined by chiral HPLC-MS was 97.5 %.

Example 3

Enhancement of P-galactosidase Activity Measured in GM1 Fibroblasts

[0161] The capacity of a certain Compound of the Disclosure, 5-[(7-chloro-l- isoquinolyl)amino]-N-[(2S)-6-methoxytetralin-2-yl]pyridine-2 -carboxamide (S-II), to enhance mutated beta-galactosidase activity levels in GM1 fibroblasts, as well as the R- enantiomer (R-II) and the racemic mixture, were assayed as follows.

Materials

[0162] Fibroblasts homozygous for a GM1 gangliosidosis missense mutation (GM11473) (canine fibroblasts homozygous for the GM1 gangliosidosis missense mutation p.R60H equivalent to human p.R59H mutation) were purchased from Coriell Institute for Medical Research (Camden, NJ, USA).

Cell Culture and Compound Treatment

[0163] Fibroblasts were seeded at 4x104 cells per well in 12-well cell culture plates in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% of fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S) (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37°C, 5% CO2 overnight for cell attachment. Subsequently, cells were incubated in the absence or presence of the compounds at the desired concentration for 4 days. After incubation, cells were washed twice with phosphate-buffered saline (“PBS”) and detached using Trypsin-EDTA solution (Sigma Aldrich, St. Louis, MO, USA) to prepare cell pellets. The pellets were stored at -80°C until activity assays were performed. Enzyme Activity Assay

[0164] P-Galactosidase activity in cell lysates was measured by using 4- Methylumbelliferyl-P-D-galactopyranoside substrate (Sigma Aldrich, St.Louis, MO, USA). Briefly, lysates were resuspended in 200 pL of 0.9% NaCl containing 0.01% triton X-100 lysis buffer to promote membrane disruption. The cell suspension was sonicated and centrifuged to remove insoluble materials. Then, lysates were mixed with 4-MU-P-D- galactopyranoside in 50 mM citrate buffer (pH=4) for 60 min at 37 °C. The reaction was terminated by adding 100 mM glycine-NaOH buffer (pH=10.7). The liberated 4-MU was measured on a GloMax® Discover plate reader (Promega™, Madison, WI, USA) with excitation at 340 nm and emission at 460 nm. Protein quantification was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Measurements were interpolated in a 4-MU standard curve and normalized by protein quantity. Enzyme activities were expressed in treated cells as X-fold increase in comparison with non-treated cells (X=l represents no enhancement).

[0165] The capacity of the Compound of the Disclosure to produce an increase in P- galactosidase enzyme activity in canine GM1 fibroblasts bearing p.R60H canine GLB1 mutation at concentrations between 6 and 50 pM is denoted as follows:

[0166] increase in comparison with non-treated of >2.5 fold is shown as A;

[0167] increase in comparison with non-treated of >1.7-2.5 fold is shown as B;

[0168] increase in comparison with non-treated of 1.2-1.7 fold is shown as C;

[0169] D means that no increase compared with non-treated cells was detected in this method; and

[0170] ND means “not determined.”

[0171] The results are presented in Table 1.

Table 1

Example 4

The S-enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy-l,2,3,4- tetrahydronaphthalen-2-yl)picolinamide exhibits an improved brain penetration

[0172] The brain penetration of the racemic mixture of 5-((7-chloroisoquinolin-l- yl)amino)-N-(6-methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)pic olinamide, the S- enantiomer (S-II), and the R-enantiomer (R-II) after i.v. injection were tested as follows.

Methods:

[0173] Nine male C57BL/6 mice were administered intravenously with a solution formulation of the tested compounds at 10 mg/kg dose. Blood samples (approximately 60 pL) were collected under light isoflurane anesthesia from a set of three mice at 0.25, 2 and 8 hr. Plasma was harvested by centrifugation of blood and stored at -70 ± 10 °C until analysis. Immediately after blood and brain samples were collected from each mouse at respective time points. Tissue samples were homogenized using ice-cold phosphate buffer saline (pH 7.4) and homogenates were stored below -70 °C until analysis. Total homogenate volume was three times the brain weight.

[0174] The plasma and brain concentration-time data were used for the pharmacokinetic analysis. Plasma, and brain samples were quantified by fit-for-purpose LC-MS/MS method (LLOQ: 1.00 ng/mL for plasma and 2.00 ng/mL for brain):

[0175] Dose: i.v.: 10 mg/kg;

[0176] Dose volume: i.v.: 5 mL/kg;

[0177] Feeding regimen: Food and water ad libitum,' and

[0178] Formulation used: i.v.: 5% v/v NMP (N-methyl-2-pyrrolidone), 10% 5% v/v Solutol® HS-15, 30% v/v PEG-400 and 60% v/v normal saline. Formulation Preparation'.

[0179] Strength of formulation used for intravenous dose was 2 mg/mL: the compound (2.92 mg); NMP (0.066 mL); Solutol® HS-15 (5%) (0.066 mL); PEG-400 (30%) (0.395 mL); and normal saline (60%) (0.789 mL).

[0180] I .V.: Accurately weighed 2.92 mg of a tested compound was added in a labeled bottle for i.v. dosing. Individual excipient volumes were calculated to prepare a solution formulation at strength of 2 mg/mL. The volume of NMP was added and vortexed. The volumes of Solutol® HS-15, PEG-400 and normal saline were added followed by vertexing after each addition. The final formulation was vortexed for 2 minutes to obtain a clear solution.

Analysis method:

[0181] LC conditions: Thermo Scientific™ Accucore™, C18 (2.7 p, 50 mm X 2.1 mm) run time: 1.6 min; Injection volume: 1 pL; Flow rate: 0.8 mL/min; Mobile phase A: 0.1% formic acid in acetonitrile and B: 10 mM ammonium formate; and Time and mobile phase-gradient (time in min/%B): 0.00/95, 0.30/95, 0.50/5, 1.20/5, 1.40/95, 1.60/95.

[0182] MS/MS Source parameters: T 550°C, Gas 1 40, Gas 2 60, CUR 30, IS 5500, and CAD 8.

Extraction Procedure:

[0183] The extraction procedure for plasma/brain samples and the spiked plasma/brain calibration standards were identical:

[0184] A 25 pL (5 pL for CSF) of study sample plasma/brain (dilution was applied for few samples) or spiked plasma/brain calibration standard was added to individual prelabeled micro-centrifuge tubes followed by 100 pL of internal standard prepared in acetonitrile (glipizide, 100 ng/mL) was added except for blank, where 100 pL of acetonitrile was added. Samples were vortexed for 5 minutes. Samples were centrifuged for 10 minutes at a speed of 4000 rpm at 4 °C. Following centrifugation, 100 pL of clear supernatant was transferred in 96 well plates and analyzed using LC-MS/MS.

Results:

[0185] The results show that the S-enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N- (6-methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)picolinamide (S-II) exhibits an improved brain penetration when compared to that of the R-enantiomer (R-II) or the racemic mixture of 5-((7-chloroisoquinolin-l-yl)amino)-N-(6-methoxy- 1,2,3, 4-tetrahydro- naphthalen-2-yl)picolinamide.

[0186] FIG. 1 shows that the S-enantiomer (S-II) is present in brain at higher levels at 0.25 h than the R-enantiomer (R-II) or the racemate when administered intravenously (10 mg/kg) to the male mouse C57/BL6. Accordingly, the S-enantiomer shows to be more brain penetrant.

Example 5

Pharmacokinetic Assay after oral administration in mice

Administration and collection of samples

[0187] A total of twenty-seven mice (n=9/group) were divided into three groups and administered through oral route with a solution formulation described in Example 4 of each of the racemic mixture of compound 5-((7-chloroisoquinolin-l-yl)amino)-N-(6- methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)picolinamide, the S-enantiomer of the compound (S-II), and the R-enantiomer of the compound (R-II) at a 10 mg/kg dose.

[0188] Blood samples (approximately 60 uL) were collected under light isoflurane anesthesia from retro orbital plexus from a set of three mice at pre-dose and at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hr after administration, in micro-centrifuge tubes containing K2 EDTA anticoagulant. Immediately after blood collection, plasma was harvested by centrifugation at 4000 rpm, 10 min at 40 °C and samples were stored at -70±10°C until shipment.

[0189] Immediately after collection of blood, brain samples were collected from mice at 1, 4 and 24 hr after administration time points. Brain samples were homogenized using ice-cold phosphate buffer saline (pH 7.4) and homogenates were stored below -70 °C until analysis. The total homogenate volume was three times the brain weight. All samples were stored at -70±10°C.

Bio-analysis

[0190] QC plasma samples were prepared in mouse blank plasma adding 5 pl of each stock solution to 45 pl plasma and precipitating the protein by addition of 150 pl of cold acetonitrile (ACN) containing verapamil 200 ng/mL as an internal standard (IS). Samples were kept under mixing for 10 min and centrifuged for 15 min at 3000 g at 5 °C. Supernatants were injected into LC-MS/MS. Samples from kinetic study were analogously prepared from 50 pl of plasma.

[0191] Brain samples were homogenates in ammonium formate 20 mM 1 g/10 mL and samples were prepared as previously described for plasma.

[0192] Calibration range was 5-5000 ng/mL for both matrices. [0193] Samples were analyzed with a chiral method for the separation of the enantiomer.

Results

[0194] The results show that the S-enantiomer (S-II) has a lower in vivo clearance than the R-enantiomer (R-II) or the racemic mixture when administered orally (10 mg/kg) to mouse male C57/BL6. See FIGS. 2A and 2B. The results presented in FIG. 2A were obtained using a chiral HPLC. In FIG. 2B, the data for the two enantiomers of the racemic mixture presented in FIG. 2A are added together to provide one curve for the racemic mixture.

[0195] The data obtained in the pharmacokinetic study is presented in a table format below in Table 2.

Table 2 [0196] The S-enantiomer (S-II) has the lowest clearance and the R-enantiomer (R-II) has the highest clearance: S-enantiomer (3064.5) > racemic mixture (S + R)(1272.4) > R- enantiomer (1064.7).

Example 6

Metabolic stability in human and mouse liver microsomes

[0197] The metabolic stability of the racemic compound 5-((7-chloroisoquinolin-l- yl)amino)-N-(6-methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)pic olinamide and the enantiomers S-II and R-II, was tested evaluating possible enantiomeric conversion after incubation with mouse and human liver microsomes.

Assay protocol

[0198] The test compounds in duplicate were dissolved in DMSO to obtain 1 mg/mL solutions and pre-incubated, at the final concentration of 1 pg/mL (2 pM), for 10 min at 37 °C in potassium phosphate buffer 50 mM, pH 7.4, 3 mM MgCh, with mouse and human liver microsomes (Sigma) at the final concentration of 0.5 mg/mL.

[0199] After the pre-incubation period, reactions were started by adding the cofactors mixture (NADP, Glc6P, Glc6P-DH in 2% sodium bicarbonate).

[0200] Samples (25 pL) were taken at time 0, 15, 30, 60 and 120 min and added to 150 pL of acetonitrile with ketoprofen 40 ng/mL as Internal Standard (IS) to stop the reaction.

[0201] After centrifugation the supernatants were analyzed by LC-MS/MS (Acquity UPLC™ (Waters™) coupled with a API 3200 Triple Quadrupole AB Sciex™).

[0202] A control sample without cofactors was added in order to check the stability of test compounds in the matrix after 120 min.

[0203] 7 -Ethoxy coumarin (7-EC) was added as a reference standard.

[0204] The percent of the area of test compound remaining at the various incubation times were calculated in respect to the area of compound at time 0 min.

Description of LC/MS-MS method

[0205] Samples were analyzed on UPLC Acquity Premiere™ (Waters™) coupled with an API 3200 Triple Quadrupole AB Sciex™.

[0206] Mobile phase A: H2O with 0.1% formic acid; Mobile phase B: ACN 0.1% formic acid; Injection volume: 20 pL; Column: Chiralcel® OJ-RH or Chiralpak® AD (150 x 2,1 mm, 5 pm); Temperature: 40 °C; Flow 0.4 ml/min 60% Mobile phase A; 40% Mobile phase B in 40 min.

[0207] MS/MS Source parameters for PK study: T 450 °C, Gas 1 45, Gas 2 55, CUR 25, IS 4500, CAD 4.

[0208] MS/MS Source parameters for liver microsome study: T 450 °C, Gas 1 45, Gas 2 55, CUR 30, IS 5500, CAD 4.

Results

[0209] The results show that the S-enantiomer of 5-((7-chloroisoquinolin-l-yl)amino)-N- (6-methoxy-l,2,3,4-tetrahydronaphthalen-2-yl)picolinamide (S-II) is more metabolically stable than the racemic mixture or the R-enantiomer (R-II).

[0210] FIG. 3 exhibits the percent of the area of each of the test compounds remaining at the various incubation times in human liver microsomes with respect to the area of the test compound at time 0 min. As can be seen in FIG. 3, the S-enantiomer (S-II) is more metabolically stable in human liver microsomes than the R-enantiomer (R-II) or the racemic mixture.

[0211] FIG. 4 exhibits the percent of the area of each of the test compounds remaining at the various incubation times in mouse liver microsomes with respect to the area of the test compound at time 0 min. As also can be seen in FIG. 4, the S-enantiomer (S-II) is more metabolically stable in mouse liver microsomes than the R-enantiomer (R-II) or the racemic mixture.

[0212] All publications cited in this specification are incorporated herein by reference. While the disclosure has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the disclosure. Such modifications are intended to fall within the scope of the appended claims.