INHIBITORS OF NT5C2 AND USES THEREOF

FIELD OF INVENTION

[0001] The present invention relates to compounds and pharmaceutical compositions capable of treating and/or restoring or increasing sensitivity of NT5C2 mutant leukemia cells to chemotherapy, as well as preventing therapy resistance for leukemia patients.

BACKGROUND OF THE INVENTION

[0002] Although the outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically, up to 15% of patients relapse and most succumb to the disease, making ALL one of the leading causes of cancer-related death in children. The most frequent cause of resistance involves mutations in NT5C2, which encodes a 5’ nucleotidase. Mutant NT5C2 exhibits increased enzymatic activity with neomorphic properties (e.g., altered substrate specificity and drug transport), that confer resistance to thiopurines, the cornerstone of ALL therapy. Furthermore, detailed structural studies of NT5C2 mutants showed that they become independent of allosteric effects of ATP and thus become constitutively active. Patients harboring relapse-specific NT5C2 mutations relapse early and have the worst prognosis.

[0003] Current treatment options for children with ALL do not include NT5C2 targeting compounds. Many children who relapse have a mutation inNT5C2 that directly leads to resistance to the current standard of care.

[0004] Thus, there exists an urgent, unmet need for NT5C2 targeting pharmaceuticals for deployment in novel therapeutic strategies to treat ALL.

SUMMARY OF THE INVENTION

[0005] Various non-limiting aspects and embodiments of the invention are described below.

[0006] In one aspect, the present application is directed to a compound having the structure according to Formula (I) or Formula (la): wherein:

R ¹ is aryl or heteroaryl, optionally substituted by one or more R ^a ; wherein R ^a is halogen, alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN, NO ₂ , OR ^b , SR ^b , NHOR ^b , C(O)R ^b , C(O)NR ^b R ^b , C(O)OR ^b , OC(O)R ^b , OC(O)NR ^b R ^b , NHR ^b , NR ^b R ^b , NR ^b C(O)R ^b , NR ^b C(O)OR ^b , NR ^b C(O)NR ^b R ^b , C(=NR ^b )R ^b , C(=NR ^b )NR ^b R ^b , NR ^b C(=NR ^b )NR ^b R ^b , NR ^b C(=NOH)NR ^b R ^b , NR ^b C(=NCN)NR ^b R ^b , NR ^b S(O)R ^b , NR ^b S(O) ₂ R ^b , NR ^b S(O) ₂ NR ^b R ^b , S(O)R ^b , S(O)NR ^b R ^b S(O) ₂ R ^b , SF ₅ , P(O)R ^b R ^b , P(O)(OR ^b )(OR ^b ), B(OR ^b ) ₂ and S(O) ₂ NR ^b R ^b ; and wherein R ^b is independently selected from H, Ci-6 alkyl, C1.4 haloalkyl, C2-6 alkenyl, C2-6 alkynyl, Ce-io aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl, or 4-14 membered heterocycloalkyl;

R ² is an acidic moiety having a pK _a between about 3 and about 6; or a pharmaceutically acceptable salt thereof.

[0007] In another aspect of the compound of Formula (I) or Formula (la), each R ^a is independently halogen, alkyl, alkoxy, or CF ₃ .

[0008] In another aspect, the present application is directed to a compound having the structure according to Formula (II): wherein:

R ³ is an optionally substituted Ci-6 alkyl, C2-6 alkenyl, or heteroaryl;

R ⁴ and R ⁵ are each independently H or C1.3 alkyl;

R ⁶ is heteroaryl, optionally substituted by one or more R ^d ; wherein R ^d is halogen, alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CF ₃ , CN, NO ₂ , OR ^e , SR ^e , NHOR ^e , C(O)R ^e , C(O)NR ^e R ^e , C(O)OR ^e , OC(O)R ^e , OC(O)NR ^e R ^e , NHR ^e , NR ^e R ^e , NR ^e C(O)R ^e , NR ^e C(O)OR ^e , NR ^e C(O)NR ^e R ^e , C(=NR ^e )R ^e , C(=NR ^e )NR ^e R ^e , NR ^e C(=NR ^e )NR ^e R ^e , NR ^e C(=NOH)NR ^e R ^e , NR ^C C(=NCN)NR ^C R ^C , NR ^e S(O)R ^e , NR ^e S(O) ₂ R ^e , NR ^e S(O) ₂ NR ^e R ^e , S(O)R ^e , S(O)NR ^e R ^e S(O) ₂ R ^e , SF ₅ , P(O)R ^e R ^e , P(O)(OR ^e )(OR ^e ), B(OR ^e ) ₂ and S(O) ₂ NR ^e R ^e ; and wherein R ^e is independently selected from H, Ci-6 alkyl, CM haloalkyl, C2-6 alkenyl, C2-6 alkynyl, Ce-io aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl, or 4- 14 membered heterocycloalkyl, or a pharmaceutically acceptable salt thereof.

[0009] In another aspect of the compound of Formula (II), R ^d is halogen or alkyl.

[00010] In another aspect, the present application is directed to a pharmaceutical composition comprising any of the herein described compounds or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[00011] In another aspect, the present application is directed to a pharmaceutical dosage form comprising any of the herein-described compounds or pharmaceutical compositions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[00012] In another aspect, the present application is directed to a method of targeting a cell having NT5C2 mutation comprising contacting the cell with any of the herein-described compounds or pharmaceutical compositions or a pharmaceutically acceptable salt thereof, in combination with 6-mercaptopurine.

[00013] In another aspect, the present application is directed to a method of restoring sensitivity to chemotherapy in a subject in need thereof, comprising administering to the subject an effective amount of any of the herein-described compounds or pharmaceutical compositions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[00014] In another aspect, the present application is directed to a method of reducing dosing of a chemotherapeutic agent in a subject in need thereof, comprising administering to the subject an effective amount of any of the herein-described compounds or pharmaceutical compositions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[00015] In another aspect, the present application is directed to a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the herein-described compounds or pharmaceutical compositions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, in combination with 6- mercaptopurine.

[00016] These and other aspects of the present invention will become apparent to those skilled in the art after a reading of the following detailed description of the invention, including the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[00017] Figure 1 illustrates the results of a cell viability assay for the NT5C2 WT cell line upon treatment with compound 15 and 6-mercaptopurine.

[00018] Figure 2 illustrates the results of a cell viability assay for the NT5C2 R37Q mutant cell line upon treatment with compound 15 and 6-mercaptopurine.

[00019] Figure 3 illustrates the results of a cell viability assay for both the NT5C2 WT and R37Q cell lines upon treatment with compound 15 alone.

[00020] Figure 4 illustrates the results of a cell viability assay for the NT5C2 WT cell line upon treatment with compound 14 and 6-mercaptopurine.

[00021] Figure 5 illustrates the results of a cell viability assay for the NT5C2 R37Q cell line upon treatment with compound 14 and 6-mercaptopurine

[00022] Figure 6 illustrates the results of a cell viability assay for both the NT5C2 WT and R37Q cell lines upon treatment with compound 14 alone.

[00023] Figure 7 illustrates the results of a cell viability assay for the NT5C2 WT cell line upon treatment with compound 15 and doxorubicin.

[00024] Figure 8 illustrates the results of a cell viability assay for the NT5C2 R37Q cell line upon treatment with compound 15 and doxorubicin.

DETAILED DESCRIPTION

[00025] Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention is intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

[00026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [00027] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure. [00028] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[00029] A “subject” or “patient” or “individual” or “animal”, as used herein, refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[00030] As used herein the term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[00031] The phrase “pharmaceutically acceptable”, as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[00032] Ranges can be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.

[00033] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if the other such compounds, material, particles, or method steps have the same function as what is named.

[00034] Compounds of the present invention include those described generally herein, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001 , the entire contents of which are hereby incorporated by reference.

[00035] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon, bicyclic hydrocarbon, or tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-30 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[00036] The term “cycloaliphatic,” as used herein, refers to saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 14 members, wherein the aliphatic ring system is optionally substituted as defined above and described herein. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons. The terms “cycloaliphatic,” may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring. In some embodiments, a carbocyclic group is bicyclic. In some embodiments, a 'carbocyclic group is tricyclic. In some embodiments, a carbocyclic group is polycyclic. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3-C6 hydrocarbon, or a Cs-Cio bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule, or a C9-C16 tricyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.

[00037] As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C20 for straight chain, C2-C20 for branched chain), and alternatively, about 1-10 carbon atoms, or about 1 to 6 carbon atoms. In some embodiments, a cycloalkyl ring has from about 3-10 carbon atoms in their ring structure where such rings are monocyclic or bicyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyls).

[00038] As used herein, the term “alkenyl” refers to an alkyl group, as defined herein, having one or more double bonds.

[00039] As used herein, the term “alkynyl” refers to an alkyl group, as defined herein, having one or more triple bonds.

[00040] The term “heteroalkyl” is given its ordinary meaning in the art and refers to alkyl groups as described herein in which one or more carbon atoms is replaced with a heteroatom (e g., oxygen, nitrogen, sulfur, and the like). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[00041] The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic or bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term “aryl” may be used interchangeably with the term “aryl ring.” In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. [00042] The terms “heteroaryl” and “heteroar-,” used alone of as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to groups having 5 to 10 ring atoms (i.e., monocyclic or bicyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, such rings have 6, 10, or 147t electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quatemized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H — quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. A heteroaryl group may be monocyclic, bicyclic, tricyclic, tetracyclic, and/or otherwise polycyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[00043] As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen.

[00044] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic, tricyclic, tetracyclic, and/or otherwise polycyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[00045] As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[00046] The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring.

[00047] The term “unsaturated,” as used herein, means that a moiety has one or more units of unsaturation.

[00048] The term “halogen” means F, Cl, Br, or I; the term “halide” refers to a halogen radical or substituent, namely -F, -Cl, -Br, or -I.

[00049] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those 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, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[00050] 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.

[00051] Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[00052] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ⁿ C- or ¹³ C- or ¹⁴ C -enriched carbon are within the scope of this invention.

[00053] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.

[00054] Unless otherwise stated, all crystalline forms of the compounds of the invention and salts thereof are also within the scope of the invention. The compounds of the invention may be isolated in various amorphous and crystalline forms, including without limitation forms which are anhydrous, hydrated, non-solvated, or solvated. Example hydrates include hemihydrates, monohydrates, dihydrates, and the like. In some embodiments, the compounds of the invention are anhydrous and non-solvated. By "anhydrous" is meant that the crystalline form of the compound contains essentially no bound water in the crystal lattice structure, i.e., the compound does not form a crystalline hydrate.

[00055] As used herein, "crystalline form" is meant to refer to a certain lattice configuration of a crystalline substance. Different crystalline forms of the same substance typically have different crystalline lattices (e.g., unit cells) which are attributed to different physical properties that are characteristic of each of the crystalline forms. In some instances, different lattice configurations have different water or solvent content. The different crystalline lattices can be identified by solid state characterization methods such as by X-ray powder diffraction (PXRD). Other characterization methods such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), solid state NMR, and the like further help identify the crystalline form as well as help determine stability and solvent/water content.

[00056] Crystalline forms of a substance include both solvated (e.g., hydrated) and non- solvated (e.g., anhydrous) forms. A hydrated form is a crystalline form that includes water in the crystalline lattice. Hydrated forms can be stoichiometric hydrates, where the water is present in the lattice in a certain water/molecule ratio such as for hemihydrates, monohydrates, dihydrates, etc. Hydrated forms can also be non-stoichiometric, where the water content is variable and dependent on external conditions such as humidity.

[00057] In some embodiments, the compounds are substantially isolated. By "substantially isolated" is meant that a particular compound is at least partially isolated from impurities. For example, in some embodiments a compound of the invention comprises less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities. Impurities generally include anything that is not the substantially isolated compound including, for example, other crystalline forms and other substances.

THE COMPOUNDS

[00058] In one aspect, the present application is directed to a compound having the structure according to Formula (I) or Formula (la): wherein:

R ¹ is aryl or heteroaryl, optionally substituted by one or more R ^a ; wherein R ^a is halogen, alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CF ₃ , CN, NO ₂ , OR ^b , SR ^b , NH0R ^b , C(O)R ^b , C(O)NR ^b R ^b , C(O)OR ^b , OC(O)R ^b , OC(O)NR ^b R ^b , NHR ^b , NR ^b R ^b , NR ^b C(O)R ^b , NR ^b C(O)OR ^b , NR ^b C(O)NR ^b R ^b , C(=NR ^b )R ^b , C(=NR ^b )NR ^b R ^b , NR ^b C(=NR ^b )NR ^b R ^b , NR ^b C(=NOH)NR ^b R ^b , NR ^b C(=NCN)NR ^b R ^b , NR ^b S(O)R ^b , NR ^b S(O) ₂ R ^b , NR ^b S(O) ₂ NR ^b R ^b , S(O)R ^b , S(O)NR ^b R ^b S(O) ₂ R ^b , SF ₅ , P(O)R ^b R ^b , P(O)(OR ^b )(OR ^b ), B(OR ^b ) ₂ and S(O) ₂ NR ^b R ^b ; and wherein R ^b is independently selected from H, Ci-6 alkyl, CM haloalkyl, C ₂ . 6 alkenyl, C ₂ -6 alkynyl, Ce-io aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl, or 4-14 membered heterocycloalkyl;

R ² is an acidic moiety having a pK _a between about 3 and about 6; or a pharmaceutically acceptable salt thereof.

[00059] In one embodiment of the compound of Formula (I) or Formula (la), R ¹ is phenyl or a five membered heteroaryl comprising one or two heteroatoms selected from S, O, and N. In another embodiment, R ¹ is a five membered heteroaryl comprising one S or O atom.

[00060] In one embodiment of the compound of Formula (I) or Formula (la), R ^a is halogen, alkyl, alkoxy, or CF3.

[00061] In one embodiment of the compound of Formula (I) or Formula (la), R ^a is F or C1.3 alkyl. [00062] In one embodiment of the compound of Formula (I) or Formula (la), R ² is an acidic moiety having a pK _a between about 3.8 and about 5.2. In another embodiment, R ² is CO ₂ H, C(O)NHOH, wherein R ^c is phenyl or CF3.

[00063] In one embodiment of the compound of Formula (I) or Formula (la), the compound has the formula according to Formula (1-1) or Formula (la-I): wherein n is an integer from 0 to 5, or a pharmaceutically acceptable salt thereof.

[00064] In one embodiment of the compound of Formula (1-1) or Formula (la-1 ), R ^a is F. In another embodiment, yet another embodiment, n is 0 or 1. [00065] In one embodiment, of the compound of Formula (I) or Formula (la), the compound has the structure according to Formula (1-2) or (la-2): wherein n is an integer from

0 to 3, or a pharmaceutically acceptable salt thereof.

[00066] In one embodiment of the compound of Formula (1-2) or (la-2), R ^a is CH3. In another embodiment, wherein R ^c is phenyl or

CF3. In yet another embodiment, n is 0 or 1.

[00067] In one embodiment of the compound of Formula (I) or (la), the compound has the structure according to Formula (1-3) or Formula (la-3): wherein n is an integer from 0 to 3, or a pharmaceutically acceptable salt thereof.

[00068] In one embodiment of the compound of Formula (1-3) or Formula (la-3), R ² is CO2H or another embodiment, n is 0.

[00069] In one embodiment of the compound of Formula (I) or Formula (la), the compound has the structure according to Formula (1-4) or Formula (la-4): wherein n is an integer from 0 to 3, or a pharmaceutically acceptable salt thereof.

[00070] In one embodiment of the compound of Formula (1-4) or Formula (la-4), R ² is CO2H. In another embodiment, n is 0.

[00071] In one embodiment of the compound of Formula (I) or Formula (la), the compound has the structure selected from the group consisting of:

[00072] In another aspect, the present application is directed to a compound having the structure according to Formula (II):

R ¹ is an optionally substituted Ci-6 alkyl, C2-6 alkenyl, or heteroaryl;

R ⁴ and R ⁵ are each independently H or C1.3 alkyl;

R ⁶ is heteroaryl, optionally substituted by one or more R ^d ; wherein R ^d is halogen, alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, CN, NO ₂ , OR ^e , SR ^e , NH0R ^e , C(O)R ^e , C(O)NR ^e R ^e , C(O)OR ^e , OC(O)R ^e , OC(O)NR ^e R ^e , NHR ^e , NR ^e R ^e , NR ^e C(O)R ^e , NR ^e C(O)OR ^e , NR ^e C(O)NR ^e R ^e , C(=NR ^e )R ^e , C(=NR ^e )NR ^e R ^e , NR ^e C(=NR ^e )NR ^e R ^e , NR ^e C(=NOH)NR ^e R ^e , NR ^e C(=NCN)NR ^e R ^e , NR ^e S(O)R ^e , NR ^e S(O) ₂ R ^e , NR ^e S(O) ₂ NR ^e R ^e , S(O)R ^e , S(O)NR ^e R ^e S(O) ₂ R ^e , SF ₅ , P(O)R ^e R ^e , P(O)(OR ^e )(OR ^e ), B(OR ^e ) ₂ and S(O) ₂ NR ^e R ^e ; and wherein R ^e is independently selected from H, C 1-6 alkyl, C1-4 haloalkyl, C ₂ -6 alkenyl, C ₂ -6 alkynyl, Ce-io aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl, or 4- 14 membered heterocycloalkyl, or a pharmaceutically acceptable salt thereof.

[00073] In one embodiment of the compound of Formula (II), R ³ is Ci-6 alkyl, C ₂ -6 alkenyl, or heteroaryl, optionally substituted with one or more groups selected from C1.3 alkyl, CN, and OH. [00074] In one embodiment of the compound of Formula (II), R ^d is halogen or alkyl.

[00075] In one embodiment of the compound of Formula (II), the compound has the structure according to Formula (II- 1): wherein R ⁷ is H or C1.3 alkyl, wherein n is an integer from 0 to 5, or a pharmaceutically acceptable salt thereof.

[00076] In one embodiment of the compound of Formula (II- 1 ), R ^d is Br. In another embodiment, yet another embodiment, R ⁴ and R ⁵ are each H.

[00077] In one embodiment of the compound of Formula (II-l ), R ⁷ is H or CH3. In another embodiment, n is 0 or 1.

[00078] In one embodiment of the compound of Formula (II), the compound a structure according to Formula (II-2):

wherein n is an integer from 0 to 5, or a pharmaceutically acceptable salt thereof.

[00079] In one embodiment of the compound of Formula (II-2), R ³ is CH3. In another embodiment, R ⁴ is CH3 and R ⁵ is H. In yet another embodiment, n is 0.

[00080] In one embodiment of the compound of Formula (II), the compound has a formula selected from:

[00081] The present invention also includes salts of the compounds described herein. As used herein, “salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of salts include, but are not limited to, mineral acid (such as HC1, HBr, H2SO4) or organic acid (such as acetic acid, benzoic acid, trifluoroacetic acid salts of basic residues such as amines; alkali (such as Li, Na, K, Mg, Ca) or organic (such as trialkylammonium) salts of acidic residues such as carboxylic acids; and the like. The salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile (ACN) are preferred.

[00082] The present application also includes pharmaceutically acceptable salts of the compounds described herein. The “pharmaceutically acceptable salts” include a subset of the “salts” described above which are conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00083] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Greene Protective Groups in Organic Synthesis, 4th Ed., John Wiley & Sons: New York, 2006.

PHARMACEUTICAL COMPOSITIONS

[00084] When employed as pharmaceuticals, the compounds of this invention are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

[00085] Generally, the compounds of this invention are administered in a therapeutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound -administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[00086] The pharmaceutical compositions of this invention can be administered by a variety of routes including oral, rectal, intraocular, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intradermal, directly into cerebrospinal fluid, intratracheal, and intranasal. Depending on the intended route of delivery, the compounds of this invention are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.

[00087] The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the active compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

[00088] Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00089] Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10%) by weight with the remainder being the injectable carrier and the like.

[00090] Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10%) by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as an ointment, the active ingredients will typically be combined with either a paraffinic or a water- miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base. Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention. [00091] The compounds of this invention can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.

[00092] The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[00093] The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[00094] Pharmaceutical compositions containing the compounds of the invention can be prepared in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[00095] In some embodiments, the pharmaceutical composition of the invention is in liquid form. Liquid forms include, by way of non-limiting example, emulsions, solutions, suspensions, syrups, slurries, dispersions, colloids and the like. In some embodiments, a pharmaceutical composition described herein is in liquid, semi-solid or solid (e.g., powder) form. In specific embodiments, a pharmaceutical composition described herein is in semi-solid form, e.g., a gel, a gel matrix, a cream, a paste, or the like. In some embodiments, semi-solid forms comprise a liquid vehicle. In some embodiments, the pharmaceutical composition of the invention is a solid dosage form, such a tablet, a granule, a sachet, or a powder. Also provided are pharmaceutical compositions comprising a compound of the invention or a pharmaceutically acceptable salt thereof in the form of a dissolving tablet, a dissolving wafer, a capsule, or a gel capsule. In certain embodiments, solid dosage forms described herein comprise a solid vehicle (e.g., as used in a tablet), and/or a gaseous vehicle (e.g., as used in DPI).

[00096] In some embodiments, a composition is in a unit dose formulation for oral, intranasal, or other administration to a patient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[00097] The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[00098] In some embodiments, the compounds or compositions described herein are administered intranasally. As used herein, “nasal delivery-enhancing agents” include agents which enhance the release or solubility (e.g., from a formulation delivery vehicle), diffusion rate, penetration capacity and timing, uptake, residence time, stability, effective half-life, peak or sustained concentration levels, clearance and other desired nasal delivery characteristics (e.g., as measured at the site of delivery, or at a selected target site of activity such as the brain) of the compounds or compositions of the invention. Enhancement of mucosal delivery can thus occur by any of a variety of mechanisms, for example by increasing the diffusion, transport, persistence or stability of the compounds or compositions of the invention, enzyme inhibition, increasing membrane fluidity, modulating the availability or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilizing mucosal membrane components (e.g., lipids), changing nonprotein and protein sulfhydryl levels in mucosal tissues, increasing water flux across the mucosal surface, modulating epithelial junctional physiology, reducing the viscosity of mucus overlying the mucosal epithelium, reducing mucociliary clearance rates, increasing nasal blood flow and other mechanisms. Suitable mucosal delivery enhancing agents will be clear to a person skilled in the art of pharmacology and are further described hereafter.

[00099] Compositions of the invention can be simple aqueous (e.g., saline) solutions. Alternatively, they can contain various additional ingredients which enhance stability and/or nasal delivery of the compounds of the invention. Such additional ingredients are well known in the art. Non-limiting examples of useful additional ingredients for enhancing nasal delivery include, e.g., (a) aggregation inhibitory agents (e.g., polyethylene glycol, dextran, diethylaminoethyl dextran, and carboxymethyl cellulose), (b) charge modifying agents, (c) pH control agents, (d) degradative enzyme inhibitors (e.g., amastatin and bestatin [see, e.g., O'Hagan et al., Pharm. Res. 1990, 7: 772- 776 and WO 05/120551]; (e) mucolytic or mucus clearing agents (e.g., n-acetyl-cysteine, propyl gallate and cysteine methionine dimers, chaotropes [see, e.g., WO 04/093917]), (f) ciliostatic agents; (g) membrane penetration enhancing agents, (h) modulatory agents of epithelial junction physiology, such as nitric oxide (NO) stimulators, chitosan, and chitosan derivatives; (i) vasodilator agents, (j) selective transport-enhancing agents, and (k) stabilizing delivery vehicles, carriers, supports or complex-forming agents. See, e.g., EP 037943, EP 094157, EP 173990, EP 214898, EP 215697, EP 327756, EP 490806, U.S. Pat. No. 4,476,116, U.S. Pat. No. 5,759,565, WO 04/093917 and WO 05/120551.

[000100] Non-limiting examples of membrane penetration-enhancing agents useful in the compositions of the invention include, e.g., (i) a surfactant (e.g., Tween 80, Poloxamer 188, polysorbates; see also EP 490806, U.S. Pat. No. 5,759,565, and W004/093917), (ii) a bile salt or bile salt derivative (e.g., unsaturated cyclic ureas and Transcutol), (iii) a phospholipid or fatty acid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) a nitric oxide donor compound (e.g., S-nitroso-N-acetyl-DL-penicillamine, NORI, NOR4, which are preferably co-administered with an NO scavenger such as carboxy-PITO or doclofenac sodium), (vii) a long- chain amphipathic molecule (e.g., deacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid) (viii) a small hydrophobic penetration enhancer, (ix) sodium salicylate or a salicylic acid derivative (e.g., acetyl salicylate, choline salicylate, salicylamide, etc.), (x) a glycerol ester of acetoacetic acid, (xi) a cyclodextrin or betacyclodextrin derivative, (xii) a medium-chain fatty acid including mono- and diglycerides (e.g., sodium caprate - extracts of coconut oil, Capmul), (xiii) a chelating agent (e.g., citric acid, salicylates), (xiv) an amino acid or salt thereof (e.g. monoaminocarboxlic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc.; hydroxyamino acids such as serine; acidic amino acids such as aspartic acid, glutamic acid, etc; and basic amino acids such as lysine etc., inclusive of their alkali metal or alkaline earth metal salts), (xv) an N- acetylamino acid or salt thereof, (xvi) an enzyme degradative to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis, (xix) cationic polymers, or any combination thereof. The membrane penetration-enhancing agent can be also selected from small hydrophilic molecules, including but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones. Additional membrane penetration enhancers include emulsifiers (e.g. sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid esters, N- alkylpyrrolidones, proline acyl esters, and the like; mixed micelles; glycerol esters of acetoacetic acid (e.g., glyceryl-l,3-diacetoacetate or l,2-isopropylideneglycerine-3-acetoacetate), and triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810); cyclodextrins and P-cyclodextrin derivatives (e.g., 2-hydroxypropyl-P-cyclodextrin and heptakis (2,6-di-O-methyl-P-cyclodextrin) which can be optionally conjugated with Peptide and further optionally formulated in an oleaginous base; and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, Nacetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline, Nacetylhydroxyproline, etc.) and their salts (alkali metal salts and alkaline earth metal salts), as well as other penetration-promoting agents that are physiologically compatible for intranasal delivery. See, e.g., WO 04/093917, WO 05/120551 and Davis and Ilium (Clin. Pharmacokinet 2003, 42: 1107-1128).

[000101] Non-limiting examples of useful absorption enhancers include, e.g., surfactants, glycosides, cyclodextrin and glycols. Non-limiting examples of useful bioadhesive agents include, e.g., carbopol, cellulose agents, starch, dextran, and chitosan.

[000102] In various embodiments of the invention, a compound of the invention is combined with one or more of the nasal delivery-enhancing agents recited above. These nasal delivery-enhancing agents may be admixed, alone or together, with the nasal carrier and with the compound of the invention, or otherwise combined therewith in a pharmaceutically acceptable formulation or delivery vehicle. For nasal delivery-enhancing agents to be of value within the invention, it is generally desired that any significant changes in permeability of the mucosa be reversible within a time frame appropriate to the desired duration of drug delivery.

[000103] Furthermore, there should be no substantial, cumulative toxicity, nor any permanent deleterious changes induced in the barrier properties of the nasal mucosa with long term use.

[000104] In addition to the compound of the invention, the nasal carrier and, optionally, one or more further additives and/or agents, the composition of the invention may further comprise one or more additional therapeutic ingredients (or active substances). These therapeutic ingredients can be any compound that elicits a desired activity or therapeutic or biological response in the subject.

[000105] The proportion of each further component in the nasal composition of the invention may vary depending on the components used. For example, but without being limiting, the amount of nasal carrier may be in the range of from 0.1 to 99.9% by weight of the total weight or volume of the composition. When present, the amount surfactant may be in the range from about 0.01 to about 10% or higher and preferably about 0.05 to about 1.0% by weight of the total volume or weight of the composition, the amount depending on the specific surfactant used. The amount is generally kept as low as possible since above a certain level no further enhancement of absorption can be achieved and also too high of a surfactant level may cause irritation of the nasal mucosa. The amount of delivery enhancing agents may be at least 0.1%, suitably in the range from about 0.5 to 10% of the total weight of the composition. Where the composition is liquid, the enhancing agent may suitably be present in an amount of from 0.1 to 5% w/v of the total composition. Preserving agents may be present in an amount of from about 0.002 to 0.02% by weight of the total weight or volume of the composition.

[000106] The useful delivery volume of the pharmaceutical compositions of the invention is limited by the size of the nasal cavity. Suitable delivery volumes will be clear to a person skilled in the art of pharmacology. Preferably, the total composition quantity administered at each nasal application comprises from about 0.02 to 0.5 ml, preferably about 0.07 to 0.3 ml, typically about 0.09-0.1 ml.

[000107] The liquid compositions of the invention may be prepared by bringing into intimate admixture a compound the invention in the liquid carrier optionally together with the further ingredients, additives and/or agents. The solid nasal composition of the invention may be prepared in conventional manner. A compound of the invention may be admixed with the carrier particles, e.g. a polymer base or cellulose product in conventional manner, optionally with further ingredients, additives and/or agents as indicated above e.g. a mucosal delivery enhancing agent or surfactant such as disclosed. A compound of the invention may be in solution e.g. an aqueous or alcoholic solution when being mixed with the carrier particles and the solvent evaporated, e.g. under freeze-drying or spray drying. Such drying may be effected under the conventional conditions. Alternatively, the mixture may be compacted or granulated and then be pulverized and/or sieved. If desired the particles may be coated. In one embodiment of the invention, the nasal composition is prepared by lyophilisation. A homogeneous solution, preferably aqueous, containing a compound of the invention and optionally containing further ingredients, additives and/or agents as discussed above, is prepared and then submitted to lyophilisation in analogy with known lyophilisation procedures, and to subsequent drying. The resulting powder may then be dissolved in a liquid excipient or nasal carrier before administration, e.g. to reconstitute nasal drops, gel or spray. Alternatively, it may be administered as such in the form of lyophilized powder or it may be mixed with further ingredients, additives and/or agents as discussed above. For example, a lyophilized powder comprising a compound of the invention but free of any nasal carrier may be prepared and then admixed with the desired nasal carrier or mixture of nasal carriers.

[000108] The present invention encompasses any delivery device that is suitable for nasal administration of the compositions of the invention. Preferably, such means administers a metered dosage of the composition. The composition of the present invention may be packed in any appropriate form or container as long as a means is provided to deliver the composition to the nasal mucosa. Non-limiting examples of useful intranasal delivery devices include, e.g., instillation catheters, droppers, unit-dose containers, squeeze bottles pump sprays, airless and preservative- fee sprays, compressed air nebulizers, metered-dose inhalers, insufflators and pressurized metered dose inhalers.

[000109] For administration of a liquid in drop form, compositions of the invention can be placed in a container provided with a conventional dropper/closure device, e.g. comprising a pipette or the like, preferably delivering a substantially fixed volume of composition/drop.

[000110] For administration of an aqueous solution as a nasal spray, the aqueous solution may be dispensed in spray form by a variety of methods known to those skilled in the art. For example, such compositions will be put up in an appropriate atomising device, e.g. in a pump-atomiser, or the like. The atomising device will be provided with appropriate means, such as a spray adaptor for delivery of the aqueous spray to the naris. Preferably it will be provided with means ensuring delivery of a substantially fixed volume of composition/actuation (i.e. per spray -unit). Examples of nasal sprays include nasal actuators produced by Ing. Erich Pfeiffer GmbH, Radolfzell, Germany (see U.S. Pat. No. 4,511,069, U.S. Pat. No. 4,778,810, U.S. Pat. No. 5,203,840, U.S. Pat. No. 5,860,567, U.S. Pat. No. 5,893,484, U.S. Pat. No. 6,227,415, and U.S. Pat. No. 6,364,166. Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers.

[000111] Alternatively, the spray may be bottled under pressure in an aerosol device. The propellant may be a gas or a liquid (e.g. a fluorinated and/or chlorinated hydrocarbon). The spray composition may be suspended or dissolved in a liquid propellant. Stabilizing and/or suspending agents and/or co-solvents may be present.

[000112] A dry powder may be readily dispersed in an inhalation device as described in U.S. Pat. No. 6,514,496 and Garcia-Arieta et al., Biol. Pharm. Bull. 2001; 24: 1411-1416.

[000113] If desired a powder or liquid may be filled into a soft or hard capsule or in a single dose device adapted for nasal administration. The powder may be sieved before filled into the capsules such as gelatine capsules. The delivery device may have means to break open the capsule. The powdery nasal composition can be directly used as a powder for a unit dosage form. The contents of the capsule or single dose device may be administered using e.g. an insufflator. Preferably it will be provided with means ensuring dosing of a substantially fixed amount of composition. [0001 14] In another embodiment, the composition of the invention can be provided as a nasal insert having the compound of the invention dispersed therein. The insert may be retained in the naris, but flushed by the nasal mucus, and may be designed to release the compound of the invention at the same place in the naris. Suitable nasal insert types include nasal plugs, tampons and the like. Further examples of nasal inserts, their characteristics and preparation are described in EP 490806.

[000115] In one aspect, a composition or unit dosage form according to the invention is formulated for sublingual administration, wherein the unit dosage form is a film including one or more disintegrants (e.g., materials that favor disintegration or fast dissolution by virtue of their solubility in water, such as hydrolyzed starches, sugars, and glycerin, which may play a dual role as a plasticizer and disintegrant) and a plasticizing agent, the film having a first portion including apomorphine hydrochloride, and a second portion including pH neutralizing agent, wherein the unit dosage form includes from 0.5 to 5 mg, from 4 to 10 mg, or from 8 to 20 mg of apomorphine hydrochloride and the pH neutralizing agent is present in an amount sufficient to produce a solution having a pH of between 3.0 and 6.0, preferably between 4.5 and 6.5, (e.g., a pH of between 2.5 and 4.5, 3.0 and 6.0, 3.5 and 6.5, 4.5 and 6.5, or 5.0 and 6.0) when the unit dosage form is placed in unbuffered water at pH 7 (e.g., the pH observed within 5 minutes of placing the unit dosage form in 1, 5, or 10 mL of unbuffered water). The film can include from 1 to 50% (w/w) (e.g., l±0.75%, 2±1.5%, 3±0.5%, 5±2%, 7.5±2.5%, 10±2%, 14±3%, 18±4%, 22±5%, 25±5%, 30±5%, 35±5%, 40±5%, 45±5%, or 50±5% (w/w)) of the one or more disintegrants. In certain embodiments, the unit dosage form further includes a high molecular weight polymer having a weight average molecular weight of greater than 60 KDa selected from hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, and methyl cellulose. In other embodiments, the unit dosage form further includes a low molecular weight polymer having a weight average molecular weight of from 5 KDa to 50 KDa selected from hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, and methyl cellulose. The pH neutralizing agent can be an organic base (e.g., pyridoxine, meglumine, or any organic base described herein) or an inorganic base (e.g., magnesium hydroxide, sodium bicarbonate, or an inorganic base described herein). In particular embodiments, the unit dosage form includes 35±5% (w/w) disintegrant, from 0.5 to 5 mg, from 4 to 10 mg, or from 8 to 20 mg of apomorphine hydrochloride and pyridoxine present in an amount sufficient to produce a solution having a pH of between 4.5 and 6.5 when the unit dosage form is placed in unbuffered water at pH 7. Suitable film for oral administration of the compositions according to the invention is disclosed in, e.g., U.S. Pat. No. 8,846,074.

[000116] In some embodiments, a composition or unit dosage form described herein is administered as an emulsion, a solution, a suspension, a syrup, a slurry, a dispersion, a colloid, a dissolving tablet, a dissolving wafer, a capsule, a gel capsule, a semi-solid, a solid forma gel, a gel matrix, a cream, a paste, a tablet, a granule, a sachet, a powder, or the like. In certain aspects, about 0.000001 mg to about 2000 mg, about 0.00001 mg to about 1000 mg, or about 0.0001 mg to about 750 mg, about 0.001 mg to about 500 mg, about 0.01 mg to about 250 mg, about 0.1 mg to about 100 mg, about 0.5 mg to about 75 mg, about 1 mg to about 50 mg, about 2 mg to about 40 mg, about 5 mg to about 20 mg, or about 7.5 mg to about 15 mg of compound of formula (I) per day or per dose is administered to an individual.

[000117] In some embodiments, the compound of the invention is present in a composition or a unit dose of a composition described herein in an amount of from about 0.01 mg to about 10 mg (e.g., about 0.1-10 mg, about 0.25-5 mg, about 0.25-2.5 mg, about 1-2 mg or about 2-3 mg, about 0.5 mg to about 2 mg, about 1 to about 2 mg, about 1 mg, or about 2 mg). In some embodiments, the amount of corticosteroid administered daily or in a unit dose is between about 0.5 mg and about 3 mg, between about 0.5 mg and about 4 mg, or between about 0.35 mg and about 4 mg. In other embodiments, the amount of the compound present in a unit dose or administered daily is between about 1 and about 3 mg, or between about 1 and about 2 mg, or between about 2 and about 3 mg. [000118] In certain aspects, about 0.05 mg to about 50 mg, about 0.25 mg to about 20 mg, about 0.25 mg to about 15 mg, about 0.25 mg to about 10 mg, or about 0.25 mg to about 5 mg (e.g., about 0. 1 to about 5 mg, about 0.25 to about 2.5 mg, about 0.3 mg to about 2 mg, about 0.5 mg to about 1 mg, about 0.7 mg to about 1.5 mg, about 0.375 mg, about 0.75 mg, about 1 mg, about 1.25 mg, about 1.5 mg or about 2 mg) of the compound per day or per dose is administered to a patient. [000119] In some embodiments, the compound is present in a unit dose in an amount of between about 5 mg and about 500 mg. In some embodiments, the amount of the compound administered daily or in a unit dose is between about 5 mg and about 300 mg. In other embodiments, the amount of the compound present in a unit dose or administered daily is between about 5 and about 250 mg, or between about 5 and about 200 mg, between about 5 mg and about 150 mg, between about 5 mg and about 100 mg, or between about 5 and about 50 mg.

[000120] In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

[000121] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of the compound of Formula I. When referring to these pre-formulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid pre-formulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.000001 to about 2000 mg of the active ingredient of the present application.

[000122] The tablets or pills containing the compound of Formula I can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[000123] The liquid forms in which the compounds and compositions of the present application can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[000124] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.

[000125] The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

[000126] The therapeutic dosage of the compounds of the invention can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compounds of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[000127] The present application also includes pharmaceutical kits useful, for example, in the treatment or prevention of diseases which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of the compounds of the invention. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

[000128] Delivery devices are important not only for delivering the compounds of the invention, but also for providing an appropriate environment for storage. This would include protection from microbial contamination and chemical degradation. The device and formulation should be compatible so as to avoid potential leaching or adsorption. The delivery device (or its packaging) can be optionally provided with a label and/or with instructions for use indicating that the composition should be used intranasally.

METHODS OF USE

[000129] In light of that which is understood in the art and described herein regarding the prominent role of NT5C2 mutations in diseases/conditions characterized by resistance to chemotherapy, methods are presented herein for treating such diseases/conditions, including but not limited to acute lymphoblastic leukemia, and other diseases related to mutant NT5C2 activity, including other cancers and chemotherapeutic resistance, which methods comprise administering to a subject in need thereof a compound described herein in a therapeutically effective amount. In a particular embodiment, at least one compound described herein is utilized, either alone or in combination with one or more known therapeutic agents. In a further particular embodiment, the present invention provides a method for treating NT5C2 mediated human diseases, wherein treatment alleviates one or more symptoms resulting from that disorder, the method comprising administration to a human in need thereof a therapeutically effective amount of a compound described herein.

[000130] Further to the above, the present compounds are inhibitors of NT5C2 and are used as therapeutic agents for the treatment of conditions in mammals that are causally related or attributable to NT5C2 activity. Accordingly, the compounds and pharmaceutical compositions of this invention find use as therapeutics for preventing and/or treating a variety of conditions related to, for example, acute lymphoblastic leukemia in mammals, including humans.

[000131] In a method of treatment aspect, this invention provides a method of treating a mammal susceptible to or afflicted with a condition associated with chemotherapeutic resistance, such as cancer, leukemia, or acute lymphoblastic leukemia, which method comprises administering an effective amount of one or more of the pharmaceutical compositions just described.

[000132] As a further aspect of the invention there is provided the present compounds for use as a pharmaceutical especially in the treatment or prevention of the aforementioned conditions and diseases. Also provided herein is the use of the present compounds in the manufacture of a medicament for the treatment or prevention of one of the aforementioned conditions and diseases. [000133] Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses. Modes of administration suitable for mucosal sites are also envisioned herein and include without limitation: intra-anal swabs, enemas, intranasal sprays, and aerosolized or vaporized compounds and/or compositions for delivery to the lung mucosa. One of skill in the art would choose an appropriate delivery mode/s based on a variety of parameters, including the organ or tissue site in a patient with a disease or condition that is most severely affected by the disease or condition.

[000134] When used to prevent or ameliorate onset or relapse of a condition such as acute lymphoblastic leukemia, the compounds of this invention will be administered to a patient at risk for developing the condition or disorder, typically on the advice and under the supervision of a physician, in the dosage forms described above. Patients at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.

[000135] The compounds of this invention can be administered as the sole active agent or they can be administered in combination with other agents, including other compounds that demonstrate the same or a similar therapeutic activity and are determined to safe and efficacious for such combined administration. In one non-limiting embodiment, the compounds of the present invention are administered in combination with 6-mercaptopurine.

EXAMPLES

[000136] The following examples illustrate specific aspects of the instant description. The examples should not be construed as limiting, as the examples merely provide specific understanding and practice of the embodiments and their various aspects.

General synthetic procedures

[000137] The compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[000138] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein. [000139] The compounds of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.

EXAMPLE 1 : Synthesis of analogs 1 and 2, 3, 4, 5, 6 according to the invention [000140] Compound 1 according to the present disclosure was prepared as shown in Scheme 1 from commercially available starting materials.

Scheme 1. Synthesis of 1.

Example 1A: Preparation of compound 1-2

[000141] To a solution of thiophene-2-carbaldehyde (1 g, 8.92 mmol) and NH2OH HCI (620 mg, 8.92 mmol) in EtOH (10 mL) was added NaOAc (1.46 g, 17.8 mmol). The reaction mixture was heated to 60 °C for 1 h then cooled to room temperature. The reaction solution was diluted with water (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic phases were washed with saturated brine (30 mL), dried over NazSCU, filtered and concentrated under vacuum to give crude thiophene-2-carbaldehyde oxime 1-2 (1.1 g) as a yellow oil which was used directly in the next step.

Example IB: Preparation of compound 1-3

[000142] To a solution of methyl prop-2-ynoate (2.17 g, 25.9 mmol) and thiophene-2- carbaldehyde oxime (1.1 g, 8.65 mmol) in MeCN (10 mL) and water (5 mL) at 0 °C was added PhI(OAc)2 (3.40 g, 10.6 mmol). The reaction was stirred at 0 °C for 10 min. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic phases were washed with saturated brine (30 mL), dried over NazSCL, fdtered and concentrated under vacuum to give a residue which was purified by flash silica gel chromatography using a gradient of EtOAc/petroleum ether from 1/100 to 1/10. Methyl 3-(2-thienyl)isoxazole-5- carboxylate 1-3 (86 mg) was obtained as a white solid. M + H ⁺ = 210.0 (LCMS); ^J H NMR (400 MHz, DMSO-t/e) 5 7.90 (s, 1H), 7.86 (dd, J= 1.2, 3.6 Hz, 1H), 7.80 (d, J= 5.2 Hz, 1H), 7.24 (dd, J= 3.6, 4.8 Hz, 1H), 3.93 (s, 3H).

Example 1C: Preparation of compound 1

[000143] To a solution of methyl 3-(2-thienyl)isoxazole-5-carboxylate (86 mg, 0.411 mmol) in MeOH (2 mL) was added a solution of LiOH H2O (60 mg, 2.09 mmol) in water (0.2 mL). The reaction was stirred at 20 °C for 10 min. The reaction mixture was diluted with water (20 mL), acidified with IN aqueous HC1 to pH 2-3 and extracted with EtOAc (20 mL x 3). The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (Phenomenex Synergi C18 column (150 x 25 mm, 10 pm); flow rate: 25 mL/min; gradient: 26% - 56% B over 10 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized. 3-(2-Thienyl)isoxazole-5-carboxylic acid 1 (16 mg) was obtained as a white solid. M + H ⁺ = 196.0 (LCMS); ^L H NMR (400 MHz, DMSO-t/ ₆ ) 8 7.83 (s, 1H), 7.78 (d, J = 52 Hz, 1H), 7.69 (s, 1H), 7.23 (s, 1H).

Example ID: Preparation of analogous compounds 2, 3, 4. 5, and 6

[000144] Table 1, below, depicts exemplary compounds synthesized according to an analogous procedure to the one described for 1.

Table 1. Compounds synthesized analogously to 1.

EXAMPLE 2: Synthesis of analog 7 according to the invention

[000145] Compound 7 according to the present disclosure was prepared as shown in Scheme 2 from commercially available starting materials.

Scheme 2. Synthesis of 7.

Example 2 A: Preparation of compound 7-2

[000146] To a mixture of ethyl 2-nitroacetate (0.6 g, 4.51 mmol) and ethynylbenzene (0.3 g, 2.94 mmol) in H2O (6 mL) was added TEMPO (0.05 g, 0.318 mmol). The reaction was stirred under an O2 atmosphere (balloon) at 60 °C for 6 h. The reaction solution was cooled to room temperature, diluted with water (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic phases were washed with saturated brine (30 mL), dried over NazSCO, filtered and concentrated under vacuum to give the crude product. The crude product was purified by silica gel chromatography using a gradient of EtOAc/petr oleum ether from 1/50 to 1/5. Ethyl 5-phenylisoxazole-3- carboxylate 7-2 (70 mg) was obtained as a white solid. M + H ⁺ = 218.1 (LCMS); ^J H NMR (400 MHz, DMSO-i/«) 8 7.74 - 7.72 (m, 2H), 7.41 - 7.39 (m, 3H), 6.84 (s, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.36 (t, J= 7.2 Hz, 3H).

Example 2B: Preparation of compound 7

[000147] To a solution of ethyl 5-phenylisoxazole-3-carboxylate (70 mg, 0.322 mmol) in MeOH (3 mL) was added LiOH (53 mg in 0.5 mL water, 1.25 mmol). The reaction solution was stirred at 20 °C for 10 min. The reaction solution was diluted with water (20 mL), acidified with IN aqueous HC1 to pH 2-3. The reaction mixture extracted with EtOAc (20 mL x 3) and the organic phases were washed with saturated brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (Phenomenex Synergi C18 column (150 x 25 mm, 10 pm); flow rate: 25 mL/min; gradient: 36% - 66% B over 10 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized. 5- Phenylisoxazole-3-carboxylic acid 7 (35 mg) was obtained as a white solid. M + H ⁺ = 190.1 (LCMS); 'H NMR (400 MHz, DMSO-tL) 8 14.09 (s, 1H), 7.96 - 7.94 (m, 2H), 7.57 - 7.55 (m, 3H), 7.41 (s, 1H).

EXAMPLE 3: Synthesis of analogs 8 and 9 according to the invention [000148] Compound 8 according to the present disclosure was prepared as shown in Scheme 3 from commercially available starting materials.

Scheme 3. Synthesis of 8.

Example 3 A: Preparation of compound 8-2

[000149] To a solution of thiophene-2-carbaldehyde (5 g, 44.6 mmol) in EtOH (50 mL) was added NaOAc (7.32 g, 89.2 mmol) and NH2OH HCI (3.10 g, 44.6 mmol). The mixture was stirred at 60 °C for 15 h. The reaction solution was cooled to room temperature, diluted with water (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic phases were washed with saturated brine (150 mL), dried over Na2SC>4, filtered and concentrated under vacuum to give the crude product. Thiophene-2-carbaldehyde oxime 8-2 (5.5 g, crude product) was obtained as yellow oil and used directly in the next step.

Example 3B: Preparation of compound 8-3

[000150] To a solution of thiophene-2-carbaldehyde oxime (5.10 g, 40.1 mmol) and methyl prop- 2-ynoate (8.43 g, 100 mmol) in H2O (100 mL) was added KC1 (2.99 g, 40.11 mmol) and Oxone® (16.0 g, 26. 1 mmol). The mixture was stirred at 20 °C for 1 h. The reaction solution was extracted with EtOAc (100 mL x 3), and the combined organic phases were washed with saturated brine (150 mL), dried overNa2SO4, filtered and concentrated under vacuum to give a residue which was purified by preparative HPLC (Waters Xbridge Cl 8 column (150 x 25 mm, 5 pm); flow rate: 20 mL/min; gradient: 19% - 49% B over 10 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized. Methyl 3-(2-thienyl)isoxazole-5-carboxylate 8-3 (1.3 g) was obtained as a white solid. M + H ⁺ = 210.0 (LCMS); 'H NMR (400 MHz, DMSO-^e) 8 7.90 (s, 1H), 7.86 (dd, J= 1.2, 3.6 Hz, 1H), 7.80 (d, J= 5.2 Hz, 1H), 7.24 (dd, J= 3.6, 4.8 Hz, 1H), 3.93 (s, 3H).

Example 3C: Preparation of compound 8-4

[000151] To a solution of methyl 3-(2-thienyl)isoxazole-5-carboxylate (1.2 g, 5.74 mmol) in MeOH (10 mL) was added LiOH H2O (1.18 g in 2 mL water, 28.7 mmol). The reaction was stirred at 20 °C for 15 h. The reaction solution was diluted with water (20 mL), acidified with IN aqueous HC1 to pH 2-3. The reaction mixture extracted with EtOAc (50 mL x 3). The combined organic phases were washed with saturated brine, dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. 3-(2-Thienyl)isoxazole-5-carboxylic acid 8-4 (0.86 g, crude product) was obtained as a white solid and used directly for the next step without further purification. M + H ⁺ = 196.0 (LCMS).

Example 3D: Preparation of compound 8-5

[000152] To a solution of 3-(2-thienyl)isoxazole-5-carboxylic acid (500 mg, 2.56 mmol) in DMF (5 mL) was added NH ₄ C1 (1.37 g, 25.62 mmol), DIPEA (993.18 mg, 7.68 mmol) and HATU (1.56 g, 4.10 mmol). The mixture was stirred at 20 °C for 2 h. The reaction mixture was poured into H2O (100 mL). The resulting solid was filtered and washed with water (50 mL), dried under vacuum and triturated with EtOAc (5 mL). 3-(2-Thienyl)isoxazole-5-carboxamide 8-5 (300 mg) was obtained as a yellow solid and used directly for the next step without further purification. M + H ⁺ = 195.0 (LCMS).

Example 3E: Preparation of compound 8-6

[000153] To a solution of 3-(2-thienyl)isoxazole-5-carboxamide (300 mg, 1.65 mmol) in DMF (1 mL) was added 2,4,6-trichloro-l,3,5-triazine (426 mg, 2.31 mmol). The mixture was stirred at 20 °C for 2 h. The reaction was quenched with H2O (30 mL) and the mixture was extracted with EtOAc (30 mL x 3). The combined organic phases were concentrated in vacuo to give a residue which was purified by flash silica gel chromatography using a gradient of EtOAc/petroleum ether from 1/100 to 1/2. 3-(2-Thienyl)isoxazole-5-carbonitrile 8-6 (200 mg) was obtained as a yellow solid. Example 3F: Preparation of compound 8

[000154] To a solution of 3-(2-thienyl)isoxazole-5-carbonitrile (200 mg, 0.851 mmol) in NMP (5 mL) was added pyridine hydrochloride (197 mg, 1.70 mmol) and NaNs (1.17 g, 18.0 mmol). The mixture was stirred at 120 °C for 2 h. The reaction solution was cooled to room temperature, diluted with water (20 mL), acidified with IN aqueous HC1 to pH 2-3 carefully, then extracted with EtOAc (20 mL x 3). The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered and concentrated (<30 °C) under reduced pressure carefully to give a residue. The residue was purified by preparative HPLC (Phenomenex Synergi Polar-RP column (100 x 25 mm, 4 pm); flow rate: 20 mL/min; gradient: 33% - 53% B over 7 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized to afford 5-(2Z/-tetrazol-5-yl)-3-(2- thienyl)isoxazole 8 (100 mg) as a pale solid. M + H ⁺ = 219.9 (LCMS). 'HNMR (400 MHz, DMSO- d ₆ ) 5 7.91 (dd, J= 0.8, 3.6 Hz, 1H), 7.87 (s, 1 H), 7.82 - 7.81 (m, 1H), 7.27 (dd, J= 3.6, 5.2 Hz, 1H).

Example 3G: Preparation of analogous compound 9

[000155] Table 2, below, depicts exemplary compounds synthesized according to an analogous procedure to the one described for 8.

Table 2. Compounds synthesized analogously to 8.

EXAMPLE 4: Synthesis of analog 10 according to the invention

[000156] Compound 10 according to the present disclosure was prepared as shown in Scheme 4 from commercially available starting materials.

Scheme 4. Synthesis of 10.

Example 4 A: Preparation of compound 10-2

[000157] To a solution of ethyl 3-phenylisoxazole-5-carboxylate (900 mg, 4.14 mmol) in MeOH (12 mL) was added LiOH FLO (781 mg in 2 mL water, 6.26 mmol). The reaction solution was stirred at 20 °C for 0.5 h. The reaction solution was diluted with water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic phases were washed with saturated brine (30 mL), dried over NazSCL, filtered and concentrated under vacuum to give a residue. The residue was purified by preparative HPLC (Phenomenex Synergi Cl 8 column (150 x 25 mm, 10 pm); flow rate: 25 mL/min; gradient: 31% - 61% B over 10 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized. 3-Phenylisoxazole-5-carboxylic acid 10-2 (700 mg) was obtained as a white solid. M + H ⁺ = 190.0 (LCMS).

Example 4B: Preparation of compound 10-3

[000158] To a solution of 3-phenylisoxazole-5-carboxylic acid (700 mg, 3.69 mmol) and NLLCl (700 mg, 13.1 mmol) in DMF (15 mL) was added DIEA (1654 mg, 14 mmol) and HATU (1.91 g, 5.02 mmol). The reaction solution was stirred at 20 °C for 2 h. The reaction solution was poured into water (100 mL) and the precipitate was collected by filtration. The filter cake was washed with water (50 mL) and dried over air to afford a crude product. 3-Phenylisoxazole-5-carboxamide 10-3 (400 mg, crude product) was obtained as a white solid which was used directly in the next step without further purification. M + H ⁺ = 189.1 (LCMS).

Example 4C: Preparation of compound 10-4 [000159] To a solution of 3-phenylisoxazole-5-carboxamide (180 mg, 0.957 mmol) in THF (5 mL) at 0 °C was added pyridine (196 mg, 2.48 mmol) and TFAA (302 mg, 1.44 mmol), and the reaction was stirred at 0 °C for 2 h. The reaction solution was diluted with water (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography using a gradient of EtOAc/petroleum ether from 1/100 to 1/5. 3-Phenylisoxazole-5-carbonitrile 10-4 (120 mg) was obtained as a white solid. M + H ⁺ = 171.0 (LCMS).

Example 4D: Preparation of compound 10

[000160] To a solution of 3-phenylisoxazole-5-carbonitrile (120 mg, 0.705 mmol) in NMP (5 mL) was added NaN3 (230 mg, 3.54 mmol) and pyridine hydrochloride (163 mg, 1.41 mmol). The reaction was heated to 130 °C for 1 h. The reaction solution was cooled to room temperature, diluted with water (20 mL), acidified with IN aqueous HC1 to pH 2-3 carefully, then extracted with EtOAc (20 mL x 3). The combined organic phases were washed with saturated brine, dried over Na2SO4, filtered and concentrated (<30 °C) under reduced pressure carefully to give a residue which was purified by preparative HPLC (Phenomenex Luna C18 column (75 x 30 mm, 3 pm); flow rate: 15 mL/min; gradient: 37% - 57% B over 7 min; mobile phase A: 0.04% aqueous formic acid, mobile phase B: acetonitrile) and lyophilized to give 3-phenyl-5-(2Z/-tetrazol-5- yl)isoxazole 10 (24 mg) as a white solid. M + H+ = 214.0 (LCMS). 1H NMR (400 MHz, DMSO- d6) 8 8.03 - 8.01 (m, 2H), 7.71 (s, 1H), 7.60 - 7.58 (m, 3H).

EXAMPLE 5: Synthesis of analog 11 according to the invention

[000161] Compound 11 according to the present disclosure was prepared as shown in Scheme 5 from commercially available starting materials.

Scheme 5. Synthesis of 11.

Example 5 A: Preparation of compound 11-2

[000162] To a solution of 3 -(2 -thienyl)isoxazole-5 -carboxylic acid (80 mg, 0.410 mmol) and O- (tetrahydropyran-2-yl)hydroxylamine (58 mg, 0.492 mmol) in DMF (1 mL) was added DIPEA (159 mg, 1.23 mmol) and HATU (234 mg, 0.615 mmol). The mixture was stirred at 20 °C for 1 h. The reaction was quenched with H2O (20 mL). The resulting mixture was extracted with EtOAc (30 mL x 3). The combined organic layers were dried over Na2SC>4, filtered, and concentrated under vacuum to give a residue. A-tetrahydropyran-2-yl oxy-3 -(2-thienyl)i soxazole-5- carboxamide 11-2 (100 mg, crude product) was obtained as a yellow oil, which was used directly in the next step without further purification. M + H ⁺ = 295. 1 (LCMS).

Example 5B: Preparation of compound 11

[000163] To a solution of A-tetrahydropyran-2-yloxy-3-(2-thienyl)isoxazole-5-carboxami de (90 mg, 0.306 mmol) in MeOH (1 mL) was added PTSA (105 mg, 0.612 mmol). The reaction mixture was stirred at 20 °C for 1 h. The solvent was removed in reduced pressure to give a residue. The residue was purified by preparative HPLC (Phenomenex Luna C18 column (75 x 30 mm, 3 pm); flow rate: 15 mL/min; gradient: 14% - 44% B over 7 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized to give 3-(2-thienyl)isoxazole-5-carbohydroxamic acid 11 (15 mg) as a white solid. M + H ⁺ = 211.4 (LCMS); ’H NMR (400 MHz, DMSO-t^) 3 11.97 - 11.57 (m, 1H), 9.71 - 9.41 (m, 1H), 7.82 - 7.76 (m, 2H), 7.57 - 7.50 (m, 1H), 7.24 (t, J= 4.4 Hz, 1H).

EXAMPLE 6: Synthesis of analogs 12 and 13 according to the invention

[000164] Compound 12 according to the present disclosure was prepared as shown in Scheme 6 from commercially available starting materials.

Scheme 6. Synthesis of 12.

Example 6A: Preparation of compound 12

[000165] To a solution of 3-(2-thienyl)isoxazole-5-carboxylic acid (80 mg, 0.410 mmol) and trifluoromethanesulfonamide (74 mg, 0.492 mmol) in DMF (1 mL) was added DIPEA (159 mg, 1.23 mmol) and HATU (235 mg, 0.615 mmol). The mixture was stirred at 20 °C for 1 hr. The reaction was quenched with H2O (10 mL). The resulting mixture was extracted with EtOAc (30 mL x 3). The combined organic layers were dried over NazSC , filtered, and concentrated under vacuum to give a residue. The residue was purified by preparative HPLC (Phenomenex Luna C18 column (75 x 30 mm, 3 pm); flow rate: 15 mL/min; gradient: 46% - 76% B over 7 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized. 3-(2-Thienyl)-A- (trifluoromethylsulfonyl)isoxazole-5-carboxamide 12 (10 mg, 7.48% yield) was obtained as a white solid. M - H ⁺ = 324.8 (LCMS); ^L H NMR (400 MHz, CDCh) 5 7.84 - 7.81 (m, 1H), 7.75 - 7.70 (m, 1H), 7.40 - 7.33 (m, 1H), 7.20 (dd, J= 4.0, 5.2 Hz, 1H).

Example 6B: Preparation of analogous compound 13

[000166] Table 3, below, depicts exemplary compounds synthesized according to an analogous procedure to the one described for 12.

Table 3. Compounds synthesized analogously to 12. EXAMPLE 7: Synthesis of analogs 14 and 15 according to the invention

[000167] Compound 14 according to the present disclosure was prepared as shown in Scheme 7 from commercially available starting materials.

Scheme 7. Synthesis of 14.

Example 7 A: Preparation of compound 14-2

[000168] To a solution of Ze/7-butyl A-(4-aminophenyl)carbamate (5 g, 24.0 mmol) and 5- methylisoxazole-4-carboxylic acid (4.58 g, 36.0 mmol) in DCM (100 m ) was added 50% T3P in DMF (15.28 g, 48.02 mmol) and DIPEA (9.31 g, 72.0 mmol). The mixture was stirred at 20 °C for 1 h. The reaction was quenched with H2O (50 ml). The resulting mixture was extracted with DCM (100 ml x 3) The combined organic layers were washed with saturated brine (150 ml), dried over anhydrous NazSO-i, filtered and concentrated under vacuum to afford a crude product, tert- Butyl A-[4-[(5-methylisoxazole-4-carbonyl)amino]phenyl]carbamate 14-2 (7.5 g, crude product) was obtained as a gray solid and used directly in the next step. M + H ⁺ = 318.1 (LCMS); ’H NMR (400 MHz, DMSO-tL) 8 9.95 (s, 1H), 9.31 (s, 1H), 9.06 (s, 1H), 7.54 (d, J= 92, 2H), 7.41 (d, J= 9.2, 2H), 2.67 (s, 3H), 1.47 (s, 9H).

Example 7B: Preparation of compound 14-3

[000169] To a solution of /cvZ-butyl .V-[4-[(5-methylisoxazole-4- carbonyl)amino]phenyl]carbamate (7.5 g, 23.6 mmol) in DCM (50 ml) was added TFA (72.2 g, 633 mmol). The mixture was stirred at 20 °C for 1 h. The solvent was removed under reduced pressure to give a residue. The residue was basified with aqueous NaHCCh solution to pH 8. The resulting mixture was extracted with DCM (100 mL x 3). The combined organic layers were washed with saturated brine, dried over NazSCL, fdtered and concentrated under reduced pressure to give the crude product. A-(4-aminophenyl)-5-methyl-isoxazole-4-carboxamide 14-3 (4.5 g) was obtained as a yellow solid which was used directly in the next step. M + H ⁺ = 218.1 (LCMS); *H NMR (400 MHz, DMSO-t/e) 8 9.67 (s, 1H), 9.01 (s, 1H), 7.27 (d, J = 8.8, 2H), 6.56 - 6.52 (m, 2H), 4.96 (s, 2H), 2.65 (s, 3H).

Example 7C: Preparation of compound 14-4

[000170] To a solution of V-(4-aminophenyl)-5-methyl-isoxazole-4-carboxamide (400 mg, 1.84 mmol), 6-bromo-177-indole-2-carboxylic acid (500 mg, 2.08 mmol) and DMAP (30 mg, 0.246 mmol) in pyridine (20 mL) was added EDCI (600 mg, 3.13 mmol). The mixture was stirred at 25 °C for 15 h. The mixture was poured into water (200 mL). The resulting precipitate was collected by filtration and washed with water (100 mL). The solid was dried over air to give a crude product. A-[4-[(6-Bromo- l //-indole-2-carbonyl)amino]phenyl]-5-methyl-isoxazole-4- carboxamide 14-4 (500 mg, crude product) was obtained as a yellow solid. M + H ⁺ = 438.9, 440.9 (LCMS).

Example 7D: Preparation of compound 14

[000171] To a solution of A-[4-[(6-bromo-l//-indole-2-carbonyl)amino]phenyl]-5-methyl- isoxazole-4-carboxamide (300 mg, 0.683 mmol) in DMF (5 mL) was added TEA (350 mg, 3.46 mmol) . The mixture was stirred at 80 °C for 3 h. The mixture was cooled to room temperature, poured into water (40 mL) and extracted with EtOAc (100 mL x 3). The combined organic phases were washed with saturated brine. The resulting precipitate was collected by filtration and washed with water (100 mL). The collected solid was triturated from MeOH (20 mL) and dried under reduced pressure to afford 6-bromo-7V-[4-[[(£')-2-cyano-3 -hydroxy -but-2-enoyl]amino]phenyl]- 177-indole-2-carboxamide 14 (36 mg) as an off-white solid. M + H ⁺ = 438.9, 440.9 (LCMS). 'H NMR (400 MHz, DMSO-t/e) 6 12.03 (s, 1H), 11.86 (s, 1H), 10.16 (s, 1H), 7.66-7.60 (m, 4H), 7.48 (d, J= 8.8, 2H), 7.40 (s, 1H), 7.20(d, J= 8.4, 1H), 2.01 (s, 3H).

Example 7E: Preparation of compound 15 [000172] Table 4, below, depicts exemplary compounds synthesized according to an analogous procedure to the one described for 14.

Table 4. Compounds synthesized analogously to 14.

EXAMPLE 8: Synthesis of analog 16 according to the invention

[000173] Compound 16 according to the present disclosure was prepared as shown in Scheme 8 from commercially available starting materials.

16-3 16

Scheme 8. Synthesis of 16.

Example 8A: Preparation of compound 16-2

[000174] To a solution of A'-methyM-nitro-aniline (500 mg, 3.29 mmol) in THF (10 mL) at - 78 °C was added lithium bis(trimethylsilyl)amide (IM solution in THF, 4 mL). After being stirred for 30 min, acetic anhydride (500 mg, 4.90 mmol) was added dropwise. After being stirred for an additional 30 min at -78 °C, the reaction mixture was warmed to 0 °C and stirred for 1 h. The reaction mixture was poured into saturated aqueous NH4CI (30 mL) and extracted with EtOAc (30 mL x 3). The combined organic layers were dried over Na2SO4, fdtered, and concentrated under vacuum to give a residue which was purified by flash silica gel chromatography using a gradient of EtOAc/petroleum ether from 1/10 to 1/1. /V-Methyl-/V-(4-nitrophenyl)acetamide 16-2 (500 mg) was obtained as a yellow solid. M + H ⁺ = 195.2 (LCMS); NMR (400 MHz, DMSO-rL) 5 8.38 (d, J= 9.6 Hz, 2H), 7.63 (d, J= 8.8 Hz, 2H), 3.27 (s, 3H), 1.99 (s, 3H).

Example 8B: Preparation of compound 16-3

[000175] To a solution of /V-methyl-/V-(4-nitrophenyl)acetamide (500 mg, 2.57 mmol) in MeOH (10 mL) was added wet 10% Pd/C (200 mg, 0.189 mmol) under a nitrogen atmosphere. The reaction bottle was degassed and purged with H2 (balloon). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through a pad of celite. The filter cake was washed with MeOH (10 mL) and the filtrate was concentrated under reduced pressure. A-(4- Aminophenyl)-/V-methyl-acetamide 16-3 (400 mg, crude product) was obtained as a yellow oil which was used directly in the next step without purification.

Example 8C: Preparation of compound 16

[000176] To a solution of /V-(4-aminophenyl)-/V-methyl-acetamide (100 mg, 0.609 mmol) and benzo[Z>]thiophene-2-carboxylic acid (110 mg, 0.628 mmol) in DCM (5 mL) at 0 °C was added DIEA (160 mg, 1.24 mmol) and T3P (50% in ethyl acetate, 800 mg, 1.26 mmol). The reaction was stirred at 0 °C for 1 h. The mixture was poured into water (30 mL) and extracted with DCM (30 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to give a residue which was purified by flash silica gel chromatography using a gradient of EtOAc/petroleum ether from 1/5 to 2/1 to give a product. This product was further purified by preparative HPLC (Waters Xbridge C18 column (150 x 25 mm, 5 pm); flow rate: 20 mL/min; gradient: 33% - 62% B over 9 min; mobile phase A: 0.04% aqueous TFA, mobile phase B: acetonitrile) and lyophilized. W(4-(7V-Methylacetamido)phenyl)benzo[Z>]thiophene-2- carboxamide 16 (60 mg) was obtained as a white solid. M + H ⁺ = 325.0 (LCMS); 'H NMR (400 MHz, DMSO-t/Q 8 10.64 (s, 1H), 8.37 (s, 1H), 8.08 - 8.02 (m, 2H), 7.84 (d, J= 8.0 Hz, 2H), 7.51 - 7.48 (m, 2H), 7.34 (d, J= 8.0 Hz, 2H), 3.14 (s, 3H), 1.78 (s, 3H).

EXAMPLE 9: Improved sensitivity to chemotherapy treatment in cells overexpressing NT5C2 wildtype or R367Q mutant.

[000177] Cell Culture and Drug Treatment. Reh NT5C2 wildtype (WT) and R367Q (RQ) mutant cells previously generated were grown in RPMI1640 medium supplemented with 10% FBS, 1% Pen/Strep under 5% CO2 at 37 °C. 6-mercaptopurine (6MP) and doxorubicin (Dox) were serially diluted in RPMI before use at indicated concentrations. Compounds 15 and 14 were dissolved in DMSO at a concentration of 10 mM. The compounds were then diluted to indicated concentrations with the final amount of 3% DMSO in RPMI.

[000178] Cell Viability Assays. Cells were plated at 80,000 cells per well in a 96-well plate. Chemotherapy was added at indicated concentrations followed by addition of the compounds at a 10-fold dilution to bring the final DMSO amount to 0.3% for all wells. Controls included cells treated with just vehicle (0.3% DMSO), cells with chemotherapy alone, and cells with compound alone. Following 72 hours in culture, cell viability was measured using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega®). Cell viability was calculated by normalizing each cell line to its vehicle-treated control. Experiments were plated in triplicated and were repeated at least two times.

Results

[000179] The IC50 to 6MP for the WT cell line is l ie' ⁶ M compared to 2.3e' ⁶ M for the RQ cell line, validating the previously demonstrated increased resistance with this NT5C2 mutation. Both compounds 15 and 14 led to increased sensitivity to 6-MP treatment in both the WT and RQ cell lines in a dose-dependent manner (Figs. 1-6). However, the impact was more dramatic in the mutant. At the two highest doses (15 and 30 pM) of 15 the 6MP IC50 of WT significantly decreased to 5.9e' ⁷ M and 1 4e' ⁷ M and for RQ it significantly decreased to 8.8e' ⁷ M and 6.4e' ⁷ M, respectively (Fig. 2). In contrast, the compounds had minimal impact on the sensitivity to Doxorubicin, another drug used in ALL therapy (Figs. 7-8). The Dox IC50 of the WT was 1.8e' ⁹ M compared to 1.3e' ⁹ M with the highest dose of 15 and 2.2e' ⁹ M compared to 1.4e" ⁹ M for the RQ line. The inhibitor had no impact on cell viability when used alone. * * *

[000180] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present invention, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present invention. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variances which fall within the scope of the appended claims.

[000181] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.