Neutral sphingomyelinase inhibitors

Abstract

Compounds and their use in treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression are disclosed.

Description

[0001] NEUTRAL SPHINGOMYELINASE INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 512,538, filed July 7, 2023, the contents of which are incorporated herein by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants AG059799 and MH075673 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND Neutral sphingomyelinase (nSMase) catalyzes the hydrolysis of sphingomyelin into ceramide and phosphocholine. As its name implies, the catalytic phosphodiesterase activity of nSMase is optimal at neutral pH. In mammals, four distinct genes, SMPD2, SMPD3, SMPD4, AND SMPD5, have been reported to encode nSMase1, nSMase 2, and nSMase3, Tomiuk et al., 1998; Hofmann et al., 2000; and Krut et al., 2006, and mitochondria- associated nSMase, Wu et al., 2010a, respectively. These isoforms of nSMase have different tissue distribution and subcellular localization, suggesting distinct physiological and pathogenic roles. Wu et al., 2010b. Among these isoforms, nSMase2 is prominently expressed in the brain and has gained increasing attention as a therapeutic target due to its role in the biogenesis of extracellular vesicles in various disease conditions including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and infectious diseases. Tallon et al., 2021a. SUMMARY In some aspects, the presently disclosed subject matter provides a compound of formula (I):

[0002] 1 41967.601_P17853-02 I); wherein: L can be present or absent and when present is a linker comprising a substituted or unsubstituted C1-C3 alkylene chain, wherein 1 to 3 methylene units of the alkylene chain can be optionally and independently replaced with -O-, -NRz-, -S-, -SO-, or -SO2-, wherein each Rzis independently selected from H, -(CH2)0-3ORy, -O(CH2)0-3C(O)ORy, or an optionally substituted C1-C6 alkyl group, and wherein each Ryis independently H or substituted or unsubstituted C1-C6alkyl; R1and R2are each independently selected from substituted or unsubstituted C1-C4alkyl and substituted or unsubstituted deuterated C1-C4 alkyl; R3and R4are each independently selected from H, C1-C4alkyl, and substituted or unsubstituted benzyl, provided that R3and R4are not H at the same time; or if L is absent, R3 and R4 can together form a 5- to 6-membered heterocylic ring, which can be fused to a 3- or 5-membered cycloalkyl, provided that neither R1 nor R2 form a cyclic ring with R3or R4; R5, R6, and R7 are each independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone; and pharmaceutically acceptable salts thereof. In certain aspects, the compound of formula (I) comprises a compound of formula (Ia): ; wherein: L comprises a alkylene chain, wherein 1 to 3 methylene units of the alkylene chain are optionally and independently replaced with -O-,

[0003] 2 41967.601_P17853-02 -NRz-, -S-, -SO-, or -SO2-, wherein each Rzis independently selected from hydrogen, -(CH2)0-3ORy, -O(CH2)0-3C(O)ORy, or an optionally substituted C1-C6 alkyl group; each Ryis independently H or substituted or unsubstituted C1-C6 alkyl; each R1, R2, R3, R4, R5, R6, R7is defined hereinabove and described in classes and subclasses herein, both singly and in combination; and pharmaceutically acceptable salts thereof. In some aspects, the presently disclosed subject matter provides a compound of formula (II): ; wherein: CyAis a substituted or unsubstituted 3- to 10-membered saturated or partially unsaturated monocyclic or bicyclic carbocyclyl or heterocyclyl having 0-6 heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein CyAis substituted with 0- 4 RAgroups; each RAis independently selected from oxo, halogen, -CN, -C(O)Ry, -C(O)2Ry-, -C(O)N(Ry)2, -NO2, -N(Ry)2, -N(R)C(O)Ry, -N(R)C(O)2Ry, -ORy, -OC(O)Ry, -OC(O)N(Ry)2, -SRy, -S(O)Ry, -S(O)2Ry, -S(O)N(Ry)2, -S(O)2N(Ry)2, or a substituted or unsubstituted group selected from C1-C6alkyl, phenyl, 3- to 7-membered saturated or partially unsaturated monocyclyl, or 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein each Ryis independently H or substituted or unsubstituted C1-C6alkyl; R1and R2are each independently selected from substituted or unsubstituted C1-C4alkyl and substituted or unsubstituted deuterated C1-C4 alkyl; R5, R6, and R7 are each independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone; and pharmaceutically acceptable salts thereof.

[0004] 3 41967.601_P17853-02 In certain aspects, R1and R2are each independently selected from -CH3, -CD3, and - CH2CF3. In certain aspects, R3 and R4 together form a 5- or 6-membered heterocyclic ring, which can be fused with a 3- or 5-membered cycloalkyl, each of which can be substituted or unsubstituted. In particular aspects, the 5- or 6-member heterocyclic ring is selected from substituted or unsubstituted pyrrolidinyl, piperidinyl, morpholinyl, octahydrocyclopenta[c]pyrrolyl, azabicyclo[3.1.0]hexanyl, and azabicyclo[3.1.1]heptanyl. In certain aspects, the 5- or 6-membered heterocyclic ring is substituted with one or more substituent groups selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone. In more certain aspects, the 5- or 6-membered heterocyclic ring is substituted with a substituent group selected from C1-C4 alkyl, hydroxyl, and phenyl. In particular aspects, the 5- or 6- membered heterocyclic ring is selected from pyrrolidinyl, methylpyrrolidinyl, phenylpyrrolidinyl, octahydrocyclopenta[c]pyrrolyl, piperidinyl, hydroxypiperidinyl, morpholinyl, azabicyclo[3.1.0]hexanyl, and azabicyclo[3.1.1]heptanyl. In certain aspects, one of R3or R4is H. In certain aspects, one of R3and R4is substituted or unsubstituted benzyl. In more certain aspects, one of R3 and R4 is H and the other is substituted or unsubstituted benzyl. In particular aspects, the benzyl is substituted with one or more substituent groups selected from alkoxyl, amine, and -CF3. In certain aspects, R5, R6, and R7are each independently H. In other aspects, the presently disclosed subject matter provides a method for treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression, the method comprising administering to a subject in need of treatment thereof an effective amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof. In certain aspects, the condition, disease, or disorder is associated with an elevated level of ceramide in the subject in need of treatment compared to a control subject not afflicted with the condition, disease, or disorder. In certain aspects, the administration of an effective amount of a compound of formula (I) or formula (II) to the subject decreases an nSMase2 activity or expression or decreases a level of ceramide in the subject.

[0005] 4 41967.601_P17853-02 In certain aspects, the condition, disease, or disorder is selected from the group consisting of a neurological, an oncological, an inflammatory, a cardiovascular disease, and an infectious condition, disease, or disorder. In particular aspects, the neurological condition, disease, or disorder is selected from Alzheimer's disease (AD), Parkinson's disease, HIV-associated neurocognitive disorder (HAND), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), a prion disease, Duchenne muscular dystrophy (DMD), and retinal degeneration. In other aspects, the presently disclosed subject matter provides a method for inhibiting neutral sphingomyelinase 2 (nSMase2), the method comprising administering to a subject, cell, or tissue an amount of a compound of formula (I) or formula (II) effective to inhibit nSMase2. In other aspects, the presently disclosed subject matter provides a method for inhibiting ceramide biosynthesis, the method comprising contacting a cell with an amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof. In other aspects, the presently disclosed subject matter provides use of a compound of formula (I) or formula (II) for preparing a medicament for treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression. Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawing as best described herein below. BRIEF DESCRIPTION OF THE DRAWING The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying drawing, which not necessarily drawn to scale, and wherein:

[0006] 5 41967.601_P17853-02 FIG. 1 shows plasma and brain concentrations of compound 11j and PDDC after oral administration in rats (10 mg / kg). DETAILED DESCRIPTION The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying drawing, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawing. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. A. Compounds of Formula (I) or Formula (II) In some embodiments, the presently disclosed subject matter provides a compound of formula (I): ; wherein: L can be present or absent and when present is a linker comprising a substituted or unsubstituted C1-C3 alkylene chain, wherein 1 to 3 methylene units of the alkylene chain can be optionally and independently replaced with -O-, -NRz-, -S-, -SO-, or -SO2-, wherein each Rzis independently selected from H, -(CH2)0-3ORy, -O(CH2)0-3C(O)ORy, or an optionally substituted C1-C6 alkyl group, and wherein each Ryis independently H or substituted or unsubstituted C1-C6alkyl;

[0007] 6 41967.601_P17853-02 R1and R2are each independently selected from substituted or unsubstituted C1-C4alkyl and substituted or unsubstituted deuterated C1-C4 alkyl; R3 and R4 are each independently selected from H, C1-C4 alkyl, and substituted or unsubstituted benzyl, provided that R3and R4are not H at the same time; or if L is absent, R3 and R4 can together form a 5- to 6-membered heterocylic ring, which can be fused to a 3- or 5-membered cycloalkyl, provided that neither R1 nor R2 form a cyclic ring with R3or R4; R5, R6, and R7 are each independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone; and pharmaceutically acceptable salts thereof. In certain embodiments, the compound of formula (I) comprises a compound of formula (Ia): ; wherein: L comprises a alkylene chain, wherein 1 to 3 methylene units of the alkylene chain are optionally and independently replaced with -O-, -NRz-, -S-, -SO-, or -SO2-, wherein each Rzis independently selected from hydrogen, -(CH2)0-3ORy, -O(CH2)0-3C(O)ORy, or an optionally substituted C1-C6alkyl group; each Ryis independently H or substituted or unsubstituted C1-C6 alkyl; each R1, R2, R3, R4, R5, R6, R7 is defined hereinabove and described in classes and subclasses herein, both singly and in combination; and pharmaceutically acceptable salts thereof. In some embodiments, the presently disclosed subject matter provides a compound of formula (II): ; 41967.601_P17853-02 wherein: CyAis a substituted or unsubstituted 3- to 10-membered saturated or partially unsaturated monocyclic or bicyclic carbocyclyl or heterocyclyl having 0-6 heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein CyAis substituted with 0- 4 RAgroups; each RAis independently selected from oxo, halogen, -CN, -C(O)Ry, -C(O)2Ry-, -C(O)N(Ry)2, -NO2, -N(Ry)2, -N(R)C(O)Ry, -N(R)C(O)2Ry, -ORy, -OC(O)Ry, -OC(O)N(Ry)2, -SRy, -S(O)Ry, -S(O)2Ry, -S(O)N(Ry)2, -S(O)2N(Ry)2, or a substituted or unsubstituted group selected from C1-C6 alkyl, phenyl, 3- to 7-membered saturated or partially unsaturated monocyclyl, or 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein each Ryis independently H or substituted or unsubstituted C1-C6 alkyl; R1 and R2 are each independently selected from substituted or unsubstituted C1-C4 alkyl and substituted or unsubstituted deuterated C1-C4alkyl; R5, R6, and R7 are each independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone; and pharmaceutically acceptable salts thereof. In certain embodiments, R1and R2are each independently selected from -CH3, -CD3, and -CH2CF3. In certain embodiments, R3 and R4 together form a 5- or 6-membered heterocyclic ring, which can be fused with a 3- or 5-membered cycloalkyl, each of which can be substituted or unsubstituted. In particular embodiments, the 5- or 6-member heterocyclic ring is selected from substituted or unsubstituted pyrrolidinyl, piperidinyl, morpholinyl, octahydrocyclopenta[c]pyrrolyl, azabicyclo[3.1.0]hexanyl, and azabicyclo[3.1.1]heptanyl. In certain embodiments, the 5- or 6-membered heterocyclic ring is substituted with one or more substituent groups selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone. In more certain embodiments, the 5- or 6-membered heterocyclic ring is substituted with a substituent group

[0008] 8 41967.601_P17853-02 selected from C1-C4alkyl, hydroxyl, and phenyl. In particular embodiments, the 5- or 6- membered heterocyclic ring is selected from pyrrolidinyl, methylpyrrolidinyl, phenylpyrrolidinyl, octahydrocyclopenta[c]pyrrolyl, piperidinyl, hydroxypiperidinyl, morpholinyl, azabicyclo[3.1.0]hexanyl, and azabicyclo[3.1.1]heptanyl. In certain embodiments, one of R3 or R4 is H. In certain embodiments, one of R3 and R4 is substituted or unsubstituted benzyl. In more certain embodiments, one of R3 and R4 is H and the other is substituted or unsubstituted benzyl. In particular embodiments, the benzyl is substituted with one or more substituent groups selected from alkoxyl, amine, and -CF3. In certain embodiments, R5, R6, and R7are each independently H. In particular embodiments, the compound of formula (I) or formula (II) is selected from: , , 02 s); In particular embodiments, the compound of formula (Ia) is: . In particular is: . 41967.601_P17853-02 In certain embodiments, R1and R2are not H. In certain embodiments, R1and R2are not -C(=O)R’. In some embodiments, R1 and R2 are not -C(=NR)R’. In particular embodiments, R1 and R2 together do not form a heterocyclic substituent group selected from: and . is: . embodiments, the compound of formula (I) does not have a structure selected from: . compounds of formula (I) or formula (II), and pharmaceutical compositions thereof, include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and include salts of active compounds that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. The parent form of the compound can differ from the various salt forms in certain physical properties, such as solubility, and the like. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion

[0009] 11 41967.601_P17853-02 exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Compounds containing both basic and acidic functionalities allow such compounds to be converted into either base or acid addition salts. Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, monohydrogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, galactonate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate, monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate, succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (+)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p-toluenesulfonate and trifluoroacetate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0010] 12 41967.601_P17853-02 The term “hydrocarbon” as used herein, refers to any chemical group comprising hydrogen and carbon. A hydrocarbon group may be substituted or unsubstituted. As would be known to one of ordinary skill in the art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. 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 or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 alipatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 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. 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. The term “alkane” refers to acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, and therefore consisting entirely of hydrogen atoms and saturated carbon atoms. The term “alkyl” refers to a univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom and having the chemical formula of -CnH2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R ≠ H), and R3C (R ≠ H) are primary, secondary and tertiary alkyl groups, respectively. An alkyl can be a straightchain (i.e., unbranched) or branched acyclic hydrocarbon

[0011] 13 41967.601_P17853-02 having the number of carbon atoms designated (i.e., C1-10means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons). In particular embodiments, the term “alkyl” refers to C1-20 inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. In other embodiments, the alkyl can be a C1-C4alkyl, including 1, 2, 3, and 4 carbons. In yet other embodiments, the alkyl can be a C1-C6 alkyl, including 1, 2, 3, 4, 5, and 6 carbons. In even yet other embodiments, the alkyl can be a C1-C8 alkyl, including 1, 2, 3, 4, 5, 6, 7, and 8 carbons. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers to straight-chain alkyls. In other embodiments, “alkyl” refers to branched alkyls. In certain other embodiments, “alkyl” refers to straight-chain and / or branched alkyls. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl. Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more substituents, which can be the same or different. Such substituent groups include, but are not limited to, alkyl, substituted alkyl, cycloalkyl, halogen, acyl, carboxyl, oxo, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto. The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain having from 1 to 20 carbon atoms or heteroatoms consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2- NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3,

[0012] 14 41967.601_P17853-02 -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)- CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The term “cycloalkane” refers to saturated monocyclic hydrocarbons (with or without side chains), e.g., cyclobutane. Unsaturated monocyclic hydrocarbons having one endocyclic double or one triple bond are called cycloalkenes and cycloalkynes, respectively. Those having more than one such multiple bond are cycloalkadienes, cycloalkatrienes, and the like. The inclusive terms for any cyclic hydrocarbons having any number of such multiple bonds are cyclic olefins or cyclic acetylenes. The term “cycloalkyl” refer to a univalent group derived from a cycloalkane by removal of a hydrogen atom from a ring carbon atom. Cycloalkyls can be a mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group also can be optionally substituted with a substituent group provided hereinabove for alkyl groups. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like. The term “cycloalkylalkyl” as used herein, refers to a cycloalkyl group, which is attached to the parent molecular moiety through an alkylene moiety, also as defined above, e.g., a C1-20alkylene moiety. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl. The terms “cycloheteroalkyl” and “heterocycloalkyl” (or more generally “heterocyclic”) are used interchangeably and refer to an unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), in which the nitrogen, sulfur, and phosphorus heteroatoms may be oxidized and the nitrogen heteroatom may be quaternized. The cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl,

[0013] 15 41967.601_P17853-02 pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like. The terms “cycloalkylene” and “heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively. As used herein the terms “bicycloalkyl” and “bicycloheteroalkyl” refer to two cycloalkyl or cycloheteroalkyl groups that are bound to one another. Non-limiting examples include bicyclohexane and bipiperidine. An “unsaturated hydrocarbon” has one or more double bonds or triple bonds. As used herein, the term “alkene” refers to an acyclic branched or unbranched hydrocarbons having one carbon–carbon double bond and the general formula CnH2n. Acyclic branched or unbranched hydrocarbons having more than one double bond are alkadienes, alkatrienes, and the like. More particularly, the term “alkenyl” as used herein refers to a monovalent group derived from a C2-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen molecule. Alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), 2-propenyl, butenyl, 1-methyl-2-buten-1- yl, pentenyl, 2-isopentenyl, hexenyl, octenyl, allenyl, butadienyl, crotyl (but-2-en-1-yl), 2- (butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), and the like, including higher homologs and isomers. The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3- cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl. The term “alkyne” as used herein refers to an acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula CnH2n-2, RC≡CR. Acyclic branched or unbranched hydrocarbons having more than one triple bond are known as alkadiynes, alkatriynes, and the like. The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C2-20hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not

[0014] 16 41967.601_P17853-02 limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and heptynyl groups, and the like. As used herein, the term “alkylene” refers to an alkanediyl group having the free valencies on adjacent carbon atoms, e.g. –CH(CH3)CH2– propylene (systematically called propane-1,2-diyl). More particularly, the term “alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (–CH2–); ethylene (– CH2–CH2–); propylene (–(CH2)3–); cyclohexylene (–C6H10–); –CH=CH–CH=CH–; – CH=CH–CH2–; -CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, - CH2CH2CH(CH2CH2CH3)CH2-, -(CH2)q-N(R)-(CH2)r–, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (–O–CH2– O–); and ethylenedioxyl (-O-(CH2)2–O–). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms also can occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and –R’OC(O)-.

[0015] 17 41967.601_P17853-02 The term “arene” refers to a monocyclic and polycyclic aromatic hydrocarbon. The term “aryl” refers to a group derived from arenes by removal of a hydrogen atom from a ring carbon atom. Groups similarly derived from heteroarenes are sometimes subsumed in this definition. An aryl group can include, for example, a single ring or multiple rings (such as from 2 to 3 rings), which are fused together or linked covalently. The term “heteroaryl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m(l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group. The term “heteroaryl” refers to the class of heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom from any ring atom. A “heteroaryl” group can include from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2- pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4- oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5- isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms “arylene” and “heteroarylene” refer to the divalent forms of aryl and heteroaryl, respectively. For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms “arylalkyl” and “heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like). However, the

[0016] 18 41967.601_P17853-02 term “haloaryl,” as used herein is meant to cover only aryls substituted with one or more halogens. Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g. “3 to 7 membered”), the term “member” refers to a carbon or heteroatom. Each of above terms defined hereinabove (e.g. , “alkyl,” “heteroalkyl,” “cycloalkyl, and “heterocycloalkyl”, “alkenyl”, “alkynyl,” “aryl,” “heteroaryl,” as well as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below. As used herein, the term “acyl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m(l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group. For example, in some embodiments, the term acyl includes an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as a 2-(furan-2- yl)acetyl)- and a 2-phenylacetyl group. Specific examples of acyl groups include acetyl and benzoyl. Acyl groups also are intended to include amides, -RC(=O)NR’, esters, -RC(=O)OR’, ketones, -RC(=O)R’, and aldehydes, -RC(=O)H. The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl–O–) or unsaturated (i.e., alkenyl–O– and alkynyl–O–) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, tert-butoxyl, and n- pentoxyl, neopentoxyl, n-hexoxyl, and the like. The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group. “Aryloxyl” refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to

[0017] 19 41967.601_P17853-02 phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl. “Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl. “Aralkyloxyl” refers to an aralkyl-O– group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl, i.e., C6H5-CH2-O-. An aralkyloxyl group can optionally be substituted. “Alkoxycarbonyl” refers to an alkyl-O-C(=O)– group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and tert- butyloxycarbonyl. “Aryloxycarbonyl” refers to an aryl-O-C(=O)– group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl. “Aralkoxycarbonyl” refers to an aralkyl-O-C(=O)– group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl. The term “acyloxyl” refers to an oxygen-centered radicals consisting of an acyl radical bonded to an oxygen atom, e.g., an acyl-O- group wherein acyl is as previously described. The term “amine” refers to a compound formally derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbyl groups, and having the general structures RNH2(primary amines), R2NH (secondary amines), R3N (tertiary amines). In some embodiments, the term amino refers to the –NH2 group. More generally, the amino group is -NR'R”, wherein R' and R” are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. The terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively. An “aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined,

[0018] 20 41967.601_P17853-02 attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure –NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure –NR’R”, wherein R’ and R” are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure –NR’R”R”’, wherein R’, R”, and R’” are each independently selected from the group consisting of alkyl groups. Additionally, R’, R”, and / or R’” taken together may optionally be –(CH2)k– where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino, trimethylamino, and propylamino. The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl–S–) or unsaturated (i.e., alkenyl–S– and alkynyl–S–) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like. “Acylamino” refers to an acyl-NH– group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH– group wherein aroyl is as previously described. The term “carbonyl” refers to a compound containing the carbonyl group, -C(=O)-. The term is commonly used in the restricted sense of aldehydes (R-C(=O)H) and ketones, although it actually includes carboxylic acids and derivatives. The term “carboxylic acid” refers to an oxoacids having the structure RC(=O)OH. The term is used as a suffix in systematic name formation to denote the –C(=O)OH group including its carbon atom. In some embodiments, the term “carboxyl” refers to the –COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety. “Carbamoyl” refers to an amide group of the formula –C(=O)NH2. “Alkylcarbamoyl” refers to a R’RN–C(=O)– group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and / or substituted alkyl as previously described. “Dialkylcarbamoyl” refers to a R’RN–C(=O)– group wherein each of R and R’ is independently alkyl and / or substituted alkyl as previously described. The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula - O-C(=O)-OR. The term “cyano” refers to the -C≡N group.

[0019] 21 41967.601_P17853-02 The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3- bromopropyl, and the like. The term “hydroxyl” refers to the –OH group. The term “hydroxyalkyl” refers to an alkyl group substituted with an –OH group. The term “mercapto” refers to the –SH group. The term “oxo compound” refers to a compounds containing an oxygen atom, =O, doubly bonded to carbon or another element. The term thus embraces aldehydes, carboxylic acids, ketones, sulfonic acids, amides and esters. Oxo used as an adjective (and thus separated by a space) modifying another class of compound, as in oxo carboxylic acids, indicates the presence of an oxo substituent at any position. To indicate a double-bonded oxygen that is part of a ketonic structure, the term keto is sometimes used as a prefix, but such use has been abandoned by IUPAC for naming specific compounds. A traditional use of keto is for indicating oxidation of CHOH to C=O in a parent compound that contains OH groups, such as carbohydrates, e.g. 3-ketoglucose. In some embodiments, the term “oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element. The term “nitro” refers to the –NO2group. The term “thio” refers to replacement of an oxygen by a sulfur, e.g., PhC(=S)NH2, thiobenzamide. The term “thiol” refers to a compounds having the structure RSH (R ≠ H), e.g., MeCH2SH ethanethiol. A thiol also is known by the term “mercaptan.” The term “thiohydroxyl” or “thiol,” as used herein, refers to a group of the formula – SH. The term “sulfate” refers to the –SO4group. The term “sulfide” refers to a compound having the structure RSR (R ≠ H) and also are referred to as “thioethers.” The term “sulfone” refers to a compound having the structure, RS(=O)2R (R ≠ H), e.g., C2H5S(=O)2CH3ethyl methyl sulfone.

[0020] 22 41967.601_P17853-02 The term “sulfoxide” refers to a compound having the structure R2S=O (R ≠ H), e.g., Ph2S=O diphenyl sulfoxide. The term “ureido” refers to a urea group of the formula –NH—CO—NH2. One of ordinary skill in the art would recognize that a structure represented generally by, for example, the formula: (R)nas used herein refers to a to a 3-carbon, a 4- carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and / or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to: and the like. A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure. The symbol ( ) denotes the point of attachment of a moiety to the remainder

[0021] 23 41967.601_P17853-02 of the molecule. When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond. Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist. Certain compounds of the present disclosure may possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as D- or L- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and / or isolate. The present disclosure is meant to include compounds in racemic, scalemic, and optically pure forms. Optically active (R)- and (S)-, or D- and L-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure. As used herein, the term “congener” refers to one of two or more substances related to each other by origin, structure, or function. The term “enantiomer” refers to one of a pair of molecular entities which are mirror images of each other and non-superposable. The term “stereoisomer” refers to an isomer that possess identical constitution, but which differ in the arrangement of their atoms in space. The term “racemate” refers to an equimolar mixture of a pair of enantiomers. It does not exhibit optical activity. The chemical name or formula of a racemate is distinguished

[0022] 24 41967.601_P17853-02 from those of the enantiomers by the prefix (±)- or rac- (or racem-) or by the symbols RS and SR. The term “diastereoisomerism” refers to stereoisomerism other than enantiomerism. Diastereoisomers (or diastereomers) are stereoisomers not related as mirror images. Diastereoisomers are characterized by differences in physical properties, and by some differences in chemical behavior towards achiral as well as chiral reagents. It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures with the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure. The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure. B. Methods of Treatment In other embodiments, the presently disclosed subject matter provides a method for treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression, the method comprising administering to a subject in need of treatment thereof an effective amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof. In certain embodiments, the condition, disease, or disorder is associated with an elevated level of ceramide in the subject in need of treatment compared to a control subject not afflicted with the condition, disease, or disorder. As used herein, the term “elevated,” as

[0023] 25 41967.601_P17853-02 in “an elevated level of ceramide,” refers to a level of GCPII in a subject having or suspected of having a disease, disorder, or condition associated with an elevated level of ceramide compared to a level of ceramide in a normal subject, i.e., a subject who does not have or is not suspected of having a disease, disorder, or condition associated with an elevated level of ceramide, such as an increase of approximately 50%, 100%, 200%, 300%, 400%, 500%, or more. In certain embodiments, the administration of an effective amount of a compound of formula (I) or formula (II) to the subject decreases an nSMase2 activity or expression or decreases a level of ceramide in the subject. In certain embodiments, the condition, disease, or disorder is selected from the group consisting of a neurological, an oncological, an inflammatory, a cardiovascular disease, and an infectious condition, disease, or disorder. In particular embodiments, the neurological condition, disease, or disorder is selected from Alzheimer's disease (AD), Parkinson's disease, HIV-associated neurocognitive disorder (HAND), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), a prion disease, Duchenne muscular dystrophy (DMD), and retinal degeneration. In particular embodiments, the oncological condition, disease, or disorder is selected from breast cancer, cervical cancer, colon cancer, colorectal cancer (CRC), duodenal cancer, gastric cancer, lung cancer, multiple myeloma, oral cancer, pancreatic cancer, prostate cancer, and skin cancer. In other embodiments, the method further comprises treating the oncological condition, disease, or disorder in combination with immunotherapy to enhance systemic antitumor immunity. In certain embodiments, the inflammatory condition, disease, or disorder is selected from the group consisting of an inflammatory airway disease, including an allergic airway inflammation, an ischemia-reperfusion injury, including cerebral ischemia and hepatic ischemia-reperfusion injury, sepsis, atherosclerosis, myocardial infarction, and inflammatory bowel disease. In certain embodiments, the cardiovascular disease is a myocardial disease involving myocyte hypertrophy, fibroblast-derived cardiac hypertrophy, heart failure, heart hypertrophy, diastolic and / or systolic ventricular dysfunction and / or a cardiovascular disease

[0024] 26 41967.601_P17853-02 involving fibrosis, aortic stenosis, atrial fibrillation, genetic forms of cardiomyopathy, cardiac storage diseases and / or Fabry disease. In certain embodiments, the infectious condition, disease, or disorder comprises a viral infection selected from the group consisting of human immunodeficiency virus (HIV), Zika virus, rabies virus, Dengue virus, hepatitis C (HCV), hepatitis E (HEV), cytomegalovirus (HCMV), Newcastle disease virus (NDV), and Langat virus. In particular embodiments, the infectious disease is associated with a toxin produced from a bacterial infection. In more particular embodiments, the toxin produce from a bacterial infection is selected from Shiga toxin released by Escherichia coli and epsilon toxin released by Clostridum perfringens. In certain embodiments, the condition, disease, or disorder comprises an extracellular vesicle-mediated disease. In more certain embodiments, administration of the compound of formula (I) or formula (II) inhibits exosome biosynthesis. In other embodiments, the presently disclosed subject matter provides a method for inhibiting neutral sphingomyelinase 2 (nSMase2), the method comprising administering to a subject, cell, or tissue an amount of a compound of formula (I) or formula (II) effective to inhibit nSMase2. In some embodiments, performing the presently disclosed method results in inhibiting nSMase2 activity in a subject. As used herein, the term “inhibit” means to decrease or diminish the nSMase2 activity found in a subject, e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 985, 99%, or 100% of the nSMase2 activity compared to an untreated control subject or a subject without the disease or disorder. The term “inhibit” also may mean to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease, disorder, In other embodiments, the presently disclosed subject matter provides a method for inhibiting ceramide biosynthesis, the method comprising contacting a cell with an amount of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof. In other embodiments, the presently disclosed subject matter provides use of a compound of formula (I) or formula (II) for preparing a medicament for treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression.

[0025] 27 41967.601_P17853-02 As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition. The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject. In general, the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the makeup of the pharmaceutical composition, the target tissue, and the like. The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a compounds of formula (I) or formula

[0026] 28 41967.601_P17853-02 (II) and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state. Further, the compounds of formula (I) or formula (II) can be administered alone or in combination with adjuvants that enhance stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies. The timing of administration of a compounds of formula (I) or formula (II) and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a compound described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject. When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one

[0027] 29 41967.601_P17853-02 another. Where the compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents. When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times. In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually. Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by: Qa / QA + B / QB = Synergy Index (SI) wherein: QAis the concentration of a component A, acting alone, which produced an end point in relation to component A; Qa is the concentration of component A, in a mixture, which produced an end point; QBis the concentration of a component B, acting alone, which produced an end point in relation to component B; and Qb is the concentration of component B, in a mixture, which produced an end point. Generally, when the sum of Qa / QAand Qb / QBis greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than

[0028] 30 41967.601_P17853-02 one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition. Depending on the specific conditions being treated, such the compounds of formula (I) or formula (II), i.e., the “agent(s)”, may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20thed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery. For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0029] 31 41967.601_P17853-02 For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons. In particular embodiments, the compounds of formula (I) or formula (II) is administered intranasally in a form selected from the group consisting of a nasal spray, a nasal drop, a powder, a granule, a cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures. Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the compound(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound(s), and the preference and experience of the attending physician. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee

[0030] 32 41967.601_P17853-02 cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethyl-cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added. Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a subject” includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth. Throughout this specification and the claims, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes,

[0031] 33 41967.601_P17853-02 formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term “about,” when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions. Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range. EXAMPLES The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0032] 34 41967.601_P17853-02 EXAMPLE 1 Neutral Sphingomyelinase 2 Inhibitors Based on a Pyrazolo[1,5-a]pyrimidine Scaffold 1.1 General Materials and Methods All solvents were reagent grade or HPLC grade. All materials were obtained from commercial suppliers and used without further purification.1H NMR spectra were recorded at 500 MHz and13C NMR at 126 MHz on a Bruker AVANCE 500 NMR spectrometer. All reactions were run under inert conditions unless otherwise stated. Preparative HPLC purification was performed on an Agilent 1200 series HPLC system equipped with an Agilent G1315D DAD detector using a Phenomenex Luna 5 μm C18 column (21.2 mm × 250 mm, 5 μm). Analytical HPLC was performed on an Agilent 1200 series HPLC system equipped with an Agilent G1315D DAD detector (detection at 220 nm) and an Agilent 6120 quadrupole MS detector using an Agilent Eclipse Plus C18 column (2.1 mm × 50 mm, 3.5 μm) at a flow rate of 1.25 mL / min. The HPLC solvent system consisted of deionized water and acetonitrile, both containing 0.1% formic acid. The mobile phase in HPLC consisted of a gradient from 0% MeCN / 100% water to 15% MeCN / 85% water over 0.25 min followed by a gradient to 50% MeCN / 50% water over 1.75 min and then a gradient to 100% MeCN / 0% water over 1.00 min. All final biologically tested compounds were confirmed to be of ≥95% purity by the HPLC and NMR methods described above. 1.2 Chemical Synthesis Compounds 5–8 containing various substituents at the 3 position of the pyrazolo[1,5- a]pyrimidine scaffold were synthesized from the same precursor, pyrazolo[1,5- a]pyrimidine-3-amine 3 (Scheme 1). Compound 5 was formed by the reductive amination of compound 3 with paraformaldehyde in presence of NaBH3CN as the reducing agent. Similarly, reductive amination with in situ generated butane-1,4-dial in the presence of NaBH3CN was performed to prepare pyrrolidine derivative 6. Compound 7 was prepared by N-acetylation of amine 3 using acetic anhydride in presence of pyridine. Compound 8 was synthesized by sequential nucleophilic substitution of 4-nitrophenyl chloroformate by amine 3 and pyrrolidine.

[0033] 35 41967.601_P17853-02

[0034] Scheme 1: Synthesis of pyrazolo[1,5-a]pyrimidine derivatives 5–8. Reagents and conditions: (i) paraformaldehyde, 1M NaOH, NaBH3CN, rt, 25 %; (ii) 2,5- dimethoxytetrahydrofuran, 1M HCl, NaBH3CN, MeOH, rt, 32 %; (iii) Ac2O, pyridine, DCM, rt, 61 %; (iv) 4-nitrophenyl chloroformate, pyrrolidine, DIPEA, DCM, 0 ºC to rt, 15 %. Compounds 10a–j were synthesized by nucleophilic substitution of the chlorine atom of 5-chloropyrazolo[1,5-a]pyrimidin-3-amine 9 by dimethylamine (10a), pyrrolidine (10b), 2-methylpyrrolidine (10c), 3-methylpyrrolidine (10d), 3-phenylpyrrolidine (10e), 3- azabicyclo[3.1.0]hexane (10f), octahydrocyclopenta[c]pyrrole (10g), piperidine (10h), 3- azabicyclo[3.1.1]heptane (10i) and morpholine (10j) (Scheme 2). Derivatives 11a–j with the dimethylamine moiety were prepared by reductive amination of precursors 10a–j with paraformaldehyde using NaBH3CN as the reducing agent. Scheme 2: Synthesis of pyrazolo[1,5-a]pyrimidine derivatives 11a – j. Reagents and conditions: (i) MeCN; for 10a: 40 % Me2NH in water, 50 ºC, 16 h, 35 %; for 10b: pyrrolidine, 50 ºC, 2.5 h, 86 %; for 10c: 2-methylpyrrolidine hydrochloride, Et3N, 70 ºC, 20 h, 19 %; for 10d: 3-methylpyrrolidine hydrochloride, Et3N, 70 ºC, 4 h, 47 %; for 10e: 3-

[0035] 36 41967.601_P17853-02 phenylpyrrolidine hydrochloride, Et3N, 70 ºC, 20 h, 34 %; for 10f: 3- azabicyclo[3.1.0]hexane hydrochloride, Et3N, reflux, 24 h; 27 %; for 10g: octahydrocyclopenta[c]pyrrole, Et3N, reflux, 24 h, 49 %; for 10h: piperidine, 50 ºC, 16 h, 88 %; for 10i: 3-azabicyclo[3.1.1]heptane hydrochloride, Et3N, reflux, 4 h, 42 %; for 10j: morpholine, 50 ºC, 4 h, 51 %; (ii) paraformaldehyde, 1M NaOH, NaBH3CN, rt, 17 h, 27 – 83 %. Nucleophilic substitution of compound 9 by hydroxypiperidine generated inseparable mixtures of the desired product (compound 10k-m) and the corresponding hydroxypiperidine starting material (4-hydroxypiperidine for 10k, (R)-3-hydroxypiperidine for 10l and (S)-3-hydroxypiperidine for 10m). In an alternative strategy, nucleophilic addition of hydroxypiperidine to 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine (12) gave nitro compounds 13k-m (Scheme 3). Purification of 13k-m was uncomplicated. Compounds 10k–m were prepared by Pd-catalyzed hydrogenation of 13k–m. Finally, reductive amination of 10k–m with paraformaldehyde in presence of NaBH3CN yielded N,N- dimethylpyrazolo[1,5-a]pyrimidin-3-amine derivatives 11k–m. Scheme and conditions: (i) Et3N, MeCN, 50 ºC; for 13k: 4-hydroxypiperidine, 1.5 h, 57 %; for 13l: (R)- 3-hydroxypiperidine hydrochloride, 16 h, 67 %; for 13m: (S)-3-hydroxypiperidine hydrochloride, 1.5 h, 98 %; (ii) Pd / C, H2, MeOH, rt, 44 – 94 %; (iii) paraformaldehyde, 1M NaOH, NaBH3CN, rt, 17 h, 22 – 66 %.

[0036] 37 41967.601_P17853-02 Compounds 11n-u were synthesized by reductive amination of compound 9 with formaldehyde using NaBH3CN as the reducing agent followed by nucleophilic substitution of the chlorine atom of compound 14 with a primary amine. conditions: (i) formaldehyde (37 wt. % in H2O), NaBH3CN, MeOH, rt, 81%; (ii) DIEA, primary amine, isopropanol, 80 ºC, 10–78 %. 1.2.1 Synthesis of compounds 5-8. N,N-Dimethylpyrazolo[1,5-a]pyrimidin-3-amine (5) Compound 3 (200 mg, 1.49 mmol, 1.0 equiv.) and paraformaldehyde (99 mg, 3.28 mmol, 2.2 equiv.) were dissolved in 1M NaOH (8 mL) and stirred at rt for 1 h. The pH was adjusted to 3 with AcOH and NaBH3CN (412 mg, 6.56 mmol, 4.4 equiv.) was added in one portion. The reaction mixture was stirred at rt for 16 h. The solution was diluted with EtOAc (100 mL), washed with sat. NaHCO3(50 mL) and brine (50 mL), dried over MgSO4, and concentrated in vacuo. The residue was purified with preparative HPLC (grad. 0 – 100 % MeCN / H2O + 0.1 % FA over 50 min) to obtain the desired product 5 (60 mg, 25 %) as a red oil.1H NMR (500 MHz, CD3OD) δ 2.88 (s, 6H), 6.77 (dd, J = 7.2, 3.9 Hz, 1H), 7.79 (s, 1H), 8.26 (dd, J = 3.9, 1.6 Hz, 1H), 8.62 (dd, J = 7.2, 1.6 Hz, 1H);13C NMR (126 MHz, CD3OD) δ 44.17 (2C), 109.08, 127.56, 134.29, 136.04, 139.65, 147.49. LCMS: tR 0.25 min, m / z 163.1 [M+H]+. 3-(Pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine (6)

[0037] 38 41967.601_P17853-02 2,5-Dimethoxytetrahydrofuran (580 µL, 4.47 mmol, 3.0 equiv.) was stirred in 1M HCl (1.5 mL) at rt for 16 h to form butane-1,4-dial in situ. After adjusting the pH to 10 with sat. Na2CO3, the mixture was added dropwise to the solution of arylamine 3 (200 mg, 1.49 mmol, 1.0 equiv.) in MeOH (8 mL). NaBH3CN (281 mg, 4.47 mmol, 3.0 equiv.) was added in one portion and the reaction mixture was stirred at rt for 16 h. The solution was concentrated in vacuo. The residue was diluted with EtOAc (200 mL), washed with dist. H2O (20 mL) and brine (20 mL), dried over MgSO4and concentrated in vacuo. The residue was purified with preparative HPLC (grad. 0 – 100 % MeCN / H2O + 0.1 % FA over 50 min) to obtain the desired product 6 (90 mg, 32 %) as a red solid.1H NMR (500 MHz, CD3OD): δ 1.99 – 2.01 (m, 4H), 3.36 – 3.42 (m, 4H), 6.78 (dd, J = 7.2, 3.9 Hz, 1H), 7.75 (s, 1H), 8.24 (dd, J = 3.8, 1.7 Hz, 1H), 8.62 (dd, J = 7.2, 1.7 Hz, 1H);13C NMR (126 MHz, d4-MeOD): δ 25.29 (2C), 52.55 (2C), 108.95, 126.15, 132.97, 135.77, 138.83, 146.87. LCMS: tR 0.95 min, m / z 189.1 [M+H]+. N-(Pyrazolo[1,5-a]pyrimidin-3-yl)acetamide (7) Compound 3 (100 mg, 0.745 mmol, 1.0 equiv.) was dissolved in anhydrous DCM (3 mL) and pyridine (121 µL, 1.49 mmol, 2.0 equiv.) was added. After 10 min, acetic anhydride (106 µL, 1.11 mmol, 1.5 equiv.) was added and the reaction mixture was stirred at rt for 1 h under inert atmosphere. The mixture was concentrated in vacuo and the residue was purified with preparative HPLC (grad. 0 – 100 % MeCN / H2O + 0.1 % FA over 50 min) to obtain the desired product 7 (80 mg, 61 %) as a yellow powder.1H NMR (500 MHz, d6- DMSO): δ 2.09 (s, 3H), 6.99 (dd, J = 7.1, 3.9 Hz, 1H), 8.48 (dd, J = 3.9, 1.6 Hz, 1H), 8.53 (s, 1H), 9.00 (dd, J = 7.1, 1.6 Hz, 1H), 10.11 (s, 1H);13C NMR (126 MHz, d6-DMSO): δ 22.73, 108.32, 110.62, 135.55, 137.59, 138.31, 148.12, 167.98. LCMS: tR 1.01 min, m / z 177.1 [M+H]+. N-(Pyrazolo[1,5-a]pyrimidin-3-yl)pyrrolidine-1-carboxamide (8) Compound 3 (100 mg, 0.745 mmol, 1.0 equiv.) was dissolved in anhydrous DCM (3 mL) and the solution was cooled to 0 ºC. DIPEA (260 µL, 1.49 mmol, 2.0 equiv.) was added and, after 5 min a solution of 4-nitrophenyl chloroformate (165 mg, 0.820 mmol, 1.1 equiv.) in anhydrous DCM (1 mL) was added dropwise. The reaction mixture was stirred at 0 ºC for 30 min and at rt for 2 h under inert atmosphere. The solution was concentrated in vacuo. The residue was dissolved in pyrrolidine (2 mL) and the resulting solution was stirred at rt for 30

[0038] 39 41967.601_P17853-02 min. The mixture was concentrated in vacuo and the residue was purified with preparative HPLC (grad. 0 – 40 % MeCN / H2O + 0.1 % FA over 50 min) to obtain the desired product 8 (25 mg, 15 %) as a yellow powder.1H NMR (500 MHz, CDCl3): δ 1.96 – 1.99 (m, 4H), 3.50 – 3.53 (m, 4H), 6.53 (s, 1H), 6.70 (dd, J = 7.1, 3.9 Hz, 1H), 8.28 (dd, J = 4.0, 1.6 Hz, 1H), 8.53 (dd, J = 7.1, 1.6 Hz, 1H), 8.62 (s, 1H);13C NMR (126 MHz, CDCl3): δ 25.77 (2C), 46.04 (2C), 107.49, 112.24, 134.90, 137.51, 138.32, 146.79, 154.01. LCMS: tR 1.31 min, m / z 232.1 [M+H]+. 1.2.2 General procedure for synthesis of compounds 10a-j. Method A. 5-Chloropyrazolo[1,5-a]pyrimidin-3-amine (9) (100 mg, 0.593 mmol, 1.0 equiv.) was suspended in anhydrous MeCN (3 mL) and secondary amine (1.5 or 10 equiv.) was added. The mixture was stirred at 50 – 90 ºC for 2.5 – 24 h and then concentrated in vacuo. The work up (if any) and purification depended on each individual reaction. Method B. Secondary amine hydrochloride (1.1 or 1.5 equiv.) and triethylamine (2.2 – 4.5 equiv.) were dissolved in anhydrous MeCN (3 mL) and stirred at rt for 5 min. 5- Chloropyrazolo[1,5-a]pyrimidin-3-amine (9) (100 mg, 0.593 mmol, 1.0 equiv.) was added. The mixture was stirred at 50 – 90 ºC for 2.5 – 24 h and then concentrated in vacuo. The work up (if any) and purification depended on each individual reaction. N5,N5-Dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (10a) Method A. Dimethylamine (40% aqueous solution, 1 mL), 50 ºC, 16 h; chromatography on silica gel (DCM / MeOH, 10:1), then further purified with preparative HPLC (grad. 0 – 40 % of MeCN / water over 50 min), 37 mg, 35 %, brown solid.1H NMR (500 MHz, CD3OD): δ 3.12 (s, 6H), 4.87 (bs, 2H), 6.37 (d, J = 7.9 Hz, 1H), 7.57 (s, 1H), 8.16 (dd, J = 7.7, 1.2 Hz, 1H);13C NMR (126 MHz, CD3OD): δ 38.18 (2C), 97.73, 113.70, 135.62, 137.48, 140.33, 156.22. LCMS: tR 0.14 min, m / z 178.1 [M+H]+. 5-(Pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10b) Method A. Pyrrolidine (495 µL, 5.93 mmol, 10 equiv.), 50 ºC, 2.5 h; diluted with DCM (100 mL) and washed with 50% solution of NaCl in water (2 × 50 mL), the organic layer was dried over MgSO4and concentrated in vacuo, 104 mg, 86 %, brown solid.1H NMR (500 MHz, CDCl3): δ 1.98 – 2.07 (m, 4H), 2.93 (bs, 2H), 3.51 – 3.61 (m, 4H), 6.04 (d,

[0039] 40 41967.601_P17853-02 J = 7.6 Hz, 1H), 7.62 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 25.40 (2C), 46.83 (2C), 96.97, 112.53, 134.63, 137.37, 139.57, 152.42. LCMS: tR 1.14 min, m / z 204.1 [M+H]+. 5-(2-Methylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10c) Method B. 2-Methylpyrrolidine hydrochloride (108 mg, 0.889 mmol, 1.5 equiv.), triethylamine (372 µL, 2.67 mmol, 4.5 equiv.), 70 ºC, 20 h; diluted in DCM (150 mL) and washed with dist. water (50 mL), the water phase was extracted with DCM (100 mL), combined organic layers dried over MgSO4 and concentrated in vacuo, chromatography on silica gel (DCM / MeOH, 10:1), 25 mg, 19 %, yellow amorphous oil.1H NMR (500 MHz, CDCl3): δ 1.24 (d, J = 6.3 Hz, 3H), 1.68 – 1.79 (m, 1H), 1.88 – 2.01 (m, 1H), 2.02 – 2.17 (m, 2H), 2.86 (bs, 2H), 3.44 – 3.49 (m, 1H), 3.61 – 3.65 (m, 1H), 4.16 – 4.25 (m, 1H), 6.02 (d, J = 7.7 Hz, 1H), 7.60 (s, 1H), 8.08 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 19.73, 23.24, 32.96, 47.33, 53.49, 97.30, 112.68, 134.74, 137.35, 139.72, 152.16. LCMS: tR 1.33 min, m / z 218.1 [M+H]+. 5-(3-Methylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10d) Method B. 3-Methylpyrrolidine hydrochloride (108 mg, 0.890 mmol, 1.5 equiv.), triethylamine (372 µL, 2.67 mmol, 4.5 equiv.), 70 ºC, 4 h; diluted in DCM (150 mL) and washed with dist. water (50 mL), the water phase was extracted with DCM (100 mL), combined organic layers dried over MgSO4 and concentrated in vacuo, chromatography on silica gel (DCM / MeOH, 20:1), 60 mg, 47 %, yellow amorphous oil.1H NMR (500 MHz, CDCl3) δ 1.14 (d, J = 6.7 Hz, 3H), 1.61 – 1.68 (m, 1H), 2.12 – 2.18 (m, 1H), 2.36 – 2.44 (m, 1H), 2.90 (bs, 2H), 3.06 – 3.10 (m, 1H), 3.48 – 3.53 (m, 1H), 3.68 – 3.75 (m, 2H), 6.02 (d, J = 7.7 Hz, 1H), 7.62 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 17.94, 33.18, 33.29, 46.70, 53.95, 96.84, 112.49, 134.66, 137.45, 139.64, 152.42. LCMS: tR1.35 min, m / z 218.1 [M+H]+. 5-(3-Phenylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10e) Method B. 3-Phenylpyrrolidine hydrochloride (163 mg, 0.890 mmol, 1.5 equiv.), triethylamine (372 µL, 2.67 mmol, 4.5 equiv.), 70 ºC, 20 h; diluted with EtOAc (200 mL), washed with dist. water (50 mL), the organic layer was dried over MgSO4 and concentrated in vacuo, chromatography on silica gel (DCM / MeOH, 20:1) furnished low purity 10e that was then further purified with preparative HPLC (grad. 0 – 70 % of MeCN / water over 50

[0040] 41 41967.601_P17853-02 min), 57 mg, 34 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 2.13 – 2.21 (m, 1H), 2.42 – 2.48 (m, 1H), 2.89 (bs, 2H), 3.50 – 3.66 (m, 3H), 3.80 – 3.87 (m, 1H), 4.03 – 4.10 (m, 1H), 6.07 (d, J = 7.7 Hz, 1H), 7.27 – 7.37 (m, 5H), 7.64 (s, 1H), 8.14 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3) δ 33.14, 44.01, 46.97, 53.46, 96.90, 112.86, 127.07, 127.20 (2C), 128.84 (2C), 135.06, 137.68, 139.68, 141.57, 152.47. LCMS: tR 1.83 min, m / z 280.1 [M+H]+. 5-(3-Azabicyclo[3.1.0]hexan-3-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10f) Method B. 3-Azabicyclo[3.1.0]hexane hydrochloride (78 mg, 0.653 mmol, 1.1 equiv.), triethylamine (182 µL, 1.31 mmol, 2.2 equiv.), reflux, 24 h; preparative HPLC (grad. 0 – 70 % of MeCN / water over 50 min), then further purified by chromatography on silica gel (DCM / MeOH, 15:1), 34 mg, 27 %, yellow amorphous oil.1H NMR (500 MHz, CDCl3) δ 0.25 (q, J = 4.3 Hz, 1H), 0.75 – 0.79 (m, 1H), 1.63 – 1.67 (m, 2H), 2.84 (s, 2H), 3.46 – 3.58 (m, 2H), 3.69 – 3.84 (m, 2H), 5.97 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 8.08 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 10.54, 15.97 (2C), 49.55 (2C), 97.10, 112.83, 134.87, 137.54, 139.47, 153.27. LCMS: tR 1.25 min, m / z 216.1 [M+H]+. 5-(Hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10g) Method A. Octahydrocyclopenta[c]pyrrole (99 mg, 0.890 mmol, 1.5 equiv.), triethylamine (248 µL, 1.78 mmol, 3.0 equiv.), reflux, 24 h; chromatography on silica gel (DCM / MeOH, 15:1), 70 mg, 49 %, brown amorphous oil.1H NMR (500 MHz, CDCl3): δ 1.49 – 1.59 (m, 2H), 1.61 – 1.70 (m, 1H), 1.75 – 1.84 (m, 1H), 1.84 – 1.95 (m, 2H), 2.37 – 2.98 (m, 4H), 3.28 – 3.41 (m, 2H), 3.69 – 3.80 (m, 2H), 6.00 – 6.07 (m, 1H), 7.62 (s, 1H), 8.07 – 8.14 (m, 1H);13C NMR (126 MHz, CDCl3) δ 25.56, 32.34 (2C), 42.97 (2C), 53.38 (2C), 97.20, 112.70, 134.72, 137.52, 139.64, 152.69. LCMS: tR 1.52 min, m / z 244.1 [M+H]+. 5-(Piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10h) Method A. Piperidine (586 µL, 5.93 mmol, 10 equiv.), 50 ºC, 16 h; diluted with DCM (100 mL), washed with dist. H2O (2 × 50 mL), the organic layer dried over MgSO4and concentrated in vacuo, 113 mg, 88 %, brown solid.1H NMR (500 MHz, CDCl3): δ 1.61 – 1.73 (m, 6H), 2.94 (s, 2H), 3.63 – 3.66 (m, 4H), 6.25 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 1.7 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 24.50, 25.51 (2C), 45.89

[0041] 42 41967.601_P17853-02 (2C), 96.04, 113.00, 134.86, 137.14, 138.85, 153.79. LCMS: tR1.25 min, m / z 218.1 [M+H]+. 5-(3-Azabicyclo[3.1.1]heptan-3-yl)pyrazolo[1,5-a]pyrimidin-3-amine (10i) Method B. 3-Azabicyclo[3.1.1]heptane hydrochloride (118 mg, 0.890 mmol, 1.5 equiv.), triethylamine (248 µL, 1.78 mmol, 3.0 equiv.), reflux, 4 h; chromatography on silica gel (DCM / MeOH, 15:1), 57 mg, 42 %, brown amorphous oil.1H NMR (500 MHz, CDCl3) δ 1.36 – 1.45 (m, 2H), 2.14 – 2.24 (m, 2H), 2.54 – 2.63 (m, 2H), 2.87 (bs, 2H), 3.61 – 3.86 (m, 4H), 6.12 – 6.18 (m, 1H), 7.58 – 7.63 (m, 1H), 8.10 – 8.16 (m, 1H);13C NMR (126 MHz, CDCl3): δ 31.76 (2C), 32.33 (2C), 51.53 (2C), 77.16, 112.77, 134.76, 137.39, 139.50, 154.29. LCMS: tR1.36 min, m / z 230.1 [M+H]+. 5-Morpholinopyrazolo[1,5-a]pyrimidin-3-amine (10j) Method A. Morpholine (519 µL, 5.93 mmol, 10 equiv.), 50 ºC, 4 h; chromatography on silica gel (DCM / MeOH, 20:1) furnished low purity 10j that was then further purified with preparative HPLC (grad. 0 – 50 % MeCN / water over 50 min), 66 mg, 51 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 2.93 (bs, 2H), 3.59 (t, J = 4.8 Hz, 4H), 3.77 (t, J = 4.2 Hz, 4H), 6.17 (dd, J = 7.8, 2.0 Hz, 1H), 7.61 (d, J = 2.0 Hz, 1H), 8.13 (dd, J = 8.0, 2.1 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 45.27 (2C), 66.61 (2C), 95.75, 113.83, 135.40, 137.37, 138.42, 154.06. LC / MS: tR 1.00 min, m / z 220.1 [M+H]+. 1.2.3 General procedure for synthesis of compounds 13k-m 5-Chloro-3-nitropyrazolo[1,5-a]pyrimidine (12) (100 mg, 0.504 mmol, 1.0 equiv.) and secondary amine (1.5 equiv.) were suspended in anhydrous MeCN and triethylamine (3 – 3.5 equiv.) was added. The reaction mixture was stirred at 50 ºC for 1.5 – 16 h under inert atmosphere. The mixture was concentrated in vacuo and the residue was purified with preparative HPLC. 1-(3-Nitropyrazolo[1,5-a]pyrimidin-5-yl)piperidin-4-ol (13k) 4-Hydroxypiperidine (76 mg, 0.755 mmol, 1.5 equiv.), MeCN (3 mL), triethylamine (211 µL, 1.51 mmol, 3.0 equiv.), 1.5 h; preparative HPLC (grad. 0 – 70 % MeCN / water over 50 min), 76 mg, 57 %, yellow solid.1H NMR (500 MHz, d6-DMSO): δ 1.37 – 1.47 (m, 2H), 1.81 – 1.89 (m, 2H), 3.41 – 3.65 (m, 2H), 3.78 – 3.85 (m, 1H), 3.90 – 4.50 (m, 2H), 4.84 (d, J = 3.9 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 8.57 – 8.59 (m, 1H), 8.75 (d, J = 7.9 Hz, 1H). LCMS: tR1.44 min, m / z 264.1 [M+H]+.

[0042] 43 41967.601_P17853-02 (R)-1-(3-Nitropyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ol (13l) (R)-3-Hydroxypiperidine hydrochloride (1.04 g, 7.55 mmol, 1.5 equiv.), MeCN (25 mL), triethylamine (2.46 mL, 17.6 mmol, 3.5 equiv.), 16 h; preparative HPLC (grad. 0 – 100 % MeCN / water over 50 min), 889 mg, 67 %, yellow solid.1H NMR (500 MHz, d6- DMSO): δ 1.37 – 1.61 (m, 2H), 1.75 – 1.96 (m, 2H), 3.05 – 3.28 (m, 1H), 3.35 – 3.51 (m, 1H), 3.56 – 3.69 (m, 1H), 3.73 – 3.89 (m, 1H), 3.93 – 4.15 (m, 1H), 4.55 – 4.79 (m, 1H), 6.97 (d, J = 7.9 Hz, 1H), 8.58 (s, 1H), 8.74 (d, J = 7.9 Hz, 1H). LCMS: tR1.51 min, m / z 264.1 [M+H]+. (S)-1-(3-Nitropyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ol (13m) (S)-3-Hydroxypiperidine hydrochloride (104 mg, 0.755 mmol, 1.5 equiv.), MeCN (3 mL), triethylamine (246 µL, 1.76 mmol, 3.5 equiv.), 1.5 h; preparative HPLC (grad. 0 – 30 % MeCN / water over 50 min), 130 mg, 98 %, yellow solid.1H NMR (500 MHz, d6- DMSO): δ 1.41 – 1.57 (m, 2H), 1.77 – 1.94 (m, 2H), 3.05 – 3.29 (m, 1H), 3.35 – 3.48 (m, 1H), 3.57 – 3.72 (m, 1H), 3.72 – 3.90 (m, 1H), 3.91 – 4.05 (m, 1H), 4.88 – 5.15 (m, 1H), 6.96 (d, J = 8.0 Hz, 1H), 8.58 (s, 1H), 8.73 (d, J = 7.9 Hz, 1H). LCMS: tR 1.50 min, m / z 264.1 [M+H]+. 1.2.4 General procedure for synthesis of compounds 10k-m Compound 13k-m (1.0 equiv.) and Pd / C (0.5 equiv.) were suspended in anhydrous MeOH and the suspension was stirred at rt for 5 – 16 h under H2 atmosphere. The suspension was filtered through filter paper and the filtrate was concentrated in vacuo. 1-(3-Aminopyrazolo[1,5-a]pyrimidin-5-yl)piperidin-4-ol (10k) Compound 13k (71 mg, 0.270 mmol, 1.0 equiv.), Pd / C (14 mg, 0.135 mmol, 0.5 equiv.), MeOH (4 mL), 5 h; 51 mg, 81 %, brown solid.1H NMR (500 MHz, d6-DMSO): δ 1.30 – 1.42 (m, 2H), 1.75 – 1.84 (m, 2H), 3.15 – 2H), 3.56– 3.69 (m, 2H), 3.69 – 3.77 (m, 1H), 4.02 – 4.11 (m, 2H), 4.70 – 4.76 (m, 1H), 6.52 (dd, J = 7.9, 1.8 Hz, 1H), 7.46 (d, J = 1.8 Hz, 1H), 8.36 (dd, J = 7.9, 1.8 Hz, 1H);13C NMR (126 MHz, d6-DMSO): δ 33.90 (2C), 42.24 (2C), 65.96, 96.08, 114.88, 135.36, 135.37, 136.47, 152.93. LCMS: tR0.13 min, m / z 234.1 [M+H]+. (R)-1-(3-Aminopyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ol (10l) Compound 13l (200 mg, 0.760 mmol, 1.0 equiv.), Pd / C (40 mg, 0.380 mmol, 0.5 equiv.), MeOH (10 mL), 16 h; 167 mg, 94 %, yellow solid.1H NMR (500 MHz, CDCl3): δ

[0043] 44 41967.601_P17853-02 1.49 – 1.60 (m, 1H), 1.60 – 1.70 (m, 1H), 1.81 – 1.97 (m, 2H), 3.00 (bs, 3H), 3.39 – 3.53 (m, 2H), 3.64 – 3.74 (m, 1H), 3.81 – 3.93 (m, 2H), 6.19 (dd, J = 8.0, 2.5 Hz, 1H), 7.61 (d, J = 2.6 Hz, 1H), 8.07 (dd, J = 7.9, 2.6 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 22.26, 32.78, 45.80, 51.77, 66.26, 96.44, 113.22, 135.25, 137.41, 138.75, 154.47. LCMS: tR0.14 min, m / z 234.1 [M+H]+. (S)-1-(3-Aminopyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ol (10m) Compound 13m (130 mg, 0.494 mmol, 1.0 equiv.), Pd / C (26 mg, 0.247 mmol, 0.5 equiv.), MeOH (10 mL), 5 h; chromatography on silica gel (DCM / MeOH, 10:1 to 5:1), 69 mg, 60 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 1.46 – 1.55 (m, 1H), 1.57 – 1.64 (m, 1H), 1.78 – 1.86 (m, 1H), 1.87 – 1.94 (m, 1H), 3.28 (bs, 3H), 3.33 – 3.41 (m, 2H), 3.65 – 3.71 (m, 1H), 3.77 – 3.82 (m, 1H), 3.86 – 3.91 (m, 1H), 6.13 (dd, J = 8.0, 2.1 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 8.03 (dd, J = 8.0, 2.1 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 22.28, 32.74, 45.59, 51.77, 66.04, 96.37, 113.07, 135.10, 137.25, 138.74, 154.32. LC / MS: tR 0.14 min, m / z 234.1 [M+H]+. 1.2.5 General procedure for synthesis of compounds 11a-m Compound 10a-m (1.0 equiv.) and paraformaldehyde (2.2 equiv.) were suspended in 1M NaOH (2.5 – 7 mL) and the mixture was stirred at rt for 1h. After adjusting the pH to 3 with AcOH, NaBH3CN (4.4 equiv.) was added in one portion. The reaction mixture was stirred at rt for 16 h. The mixture was diluted with EtOAc or DCM and washed with sat. NaHCO3. The organic layer was dried over MgSO4and concentrated in vacuo. The residue was purified on silica gel and / or purified with preparative HPLC. N3,N3,N5,N5-Tetramethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (11a) Compound 10a (35 mg, 0.198 mmol, 1.0 equiv.), paraformaldehyde (13 mg, 0.435 mmol, 2.2 equiv.), 1M NaOH (2.5 mL), NaBH3CN (55 mg, 0.869 mmol, 4.4 equiv.); EtOAc (75 mL), sat. NaHCO3 (50 mL), preparative HPLC (grad. 0 – 50 % of MeCN / water over 50 min), 11 mg, 27 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 2.86 (bs, 6H), 3.15 (s, 6H), 6.21 (dd, J = 7.8, 2.0 Hz, 1H), 7.58 (s, 1H), 8.13 (dd, J = 7.8, 2.0 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 38.08 (2C), 44.06 (2C), 96.09, 122.35, 134.69, 135.04, 138.96, 153.94. LCMS: tR 1.21 min, m / z 206.1 [M+H]+. N,N-Dimethyl-5-(pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (11b)

[0044] 45 41967.601_P17853-02 Compound 10b (90 mg, 0.443 mmol, 1.0 equiv.), paraformaldehyde (29 mg, 0.974 mmol, 2.2 equiv.), 1M NaOH (5 mL), NaBH3CN (122 mg, 1.95 mmol, 4.4 equiv.); EtOAc (80 mL), sat. NaHCO3 (50 mL), preparative HPLC (grad. 0 – 50 % of MeCN / water over 50 min), 49 mg, 48 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 1.94 – 2.00 (m, 4H), 2.82 (s, 6H), 3.42 – 3.59 (m, 4H), 6.02 (dd, J = 7.7, 1.8 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 8.08 (dd, J = 7.8, 1.8 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 25.47 (2C), 43.97 (2C), 46.86 (2C), 97.24, 122.08, 134.44, 134.76, 139.27, 151.86. LCMS: tR1.35 min, m / z 232.1 [M+H]+. N,N-Dimethyl-5-(2-methylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (11c) Compound 10c (19 mg, 0.088 mmol, 1.0 equiv.), paraformaldehyde (6 mg, 0.194 mmol, 2.2 equiv.), 1M NaOH (2.5 mL), NaBH3CN (24 mg, 0.387 mmol, 4.4 equiv.); DCM (100 mL), sat. NaHCO3 (2×50 mL), preparative HPLC (grad. 0 – 100 % of MeCN / water over 40 min), 9 mg, 43 %, yellow amorphous oil.1H NMR (500 MHz, CDCl3) δ 1.26 (d, J = 6.3 Hz, 3H), 1.70 – 1.76 (m, 1H), 1.95 – 2.02 (m, 1H), 2.03 – 2.13 (m, 2H), 2.86 (s, 6H), 3.45 – 3.50 (m, 1H), 3.62 – 3.66 (m, 1H), 4.18 – 4.34 (m, 1H), 6.06 (d, J = 7.7 Hz, 1H), 7.57 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 19.56, 23.20, 32.85, 44.00 (2C), 47.27, 53.42, 97.38, 112.15, 134.44, 134.74, 139.20, 151.41. LCMS: tR1.53 min, m / z 246.1 [M+H]+. N,N-Dimethyl-5-(3-methylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (11d) Compound 10d (50 mg, 0.230 mmol, 1.0 equiv.), paraformaldehyde (15 mg, 0.506 mmol, 2.2 equiv.), 1M NaOH (5 mL), NaBH3CN (63 mg, 1.01 mmol, 4.4 equiv.); DCM (100 mL), sat. NaHCO3 (2×50 mL), preparative HPLC (grad. 0 – 100 % of MeCN / water over 40 min), 38 mg, 68 %, yellow amorphous oil.1H NMR (500 MHz, CDCl3) δ 1.11 (d, J = 6.7 Hz, 3H), 1.57 – 1.65 (m, 1H), 2.08 – 2.14 (m, 1H), 2.33 – 2.40 (m, 1H), 2.83 (s, 6H), 3.02 – 3.09 (m, 1H), 3.45 – 3.51 (m, 1H), 3.60 – 3.80 (m, 2H), 6.02 (d, J = 7.7 Hz, 1H), 7.56 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 17.98, 33.33, 43.95 (2C), 46.67, 53.93, 58.37, 97.05, 122.06, 134.44, 134.74, 139.27, 151.79. LCMS: tR1.54 min, m / z 246.1 [M+H]+. N,N-Dimethyl-5-(3-phenylpyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (11e) Compound 10e (50 mg, 0.179 mmol, 1.0 equiv.), paraformaldehyde (12 mg, 0.394 mmol, 2.2 equiv.), 1M NaOH (5 mL), NaBH3CN (49 mg, 0.788 mmol, 4.4 equiv.); EtOAc

[0045] 46 41967.601_P17853-02 (100 mL), NaHCO3(20 mL), chromatography on silica gel (DCM / MeOH, 20:1), then further purification on preparative HPLC (grad. 0 – 100 % of MeCN / water + 0.1% FA over 40 min), 38 mg, 69 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 2.12 – 2.19 (m, 1H), 2.38 – 2.48 (m, 1H), 2.85 (s, 6H), 3.50 – 3.63 (m, 3H), 3.76 – 3.90 (m, 1H), 3.99 – 4.16 (m, 1H), 6.08 (d, J = 7.7 Hz, 1H), 7.22 – 7.40 (m, 5H), 7.60 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 33.09, 44.03 (2C), 44.03, 46.89, 53.39, 97.05, 122.32, 127.00, 127.18 (2C), 128.78 (2C), 134.64, 135.05, 139.22, 141.59, 151.75. LCMS: tR2.01 min, m / z 308.2 [M+H]+. 5-(3-Azabicyclo[3.1.0]hexan-3-yl)-N,N-dimethylpyrazolo[1,5-a]pyrimidin-3-amine (11f) Compound 10f (29 mg, 0.135 mmol, 1.0 equiv.), paraformaldehyde (9 mg, 0.297 mmol, 2.2 equiv.), 1M NaOH (4 mL), NaBH3CN (37 mg, 0.593 mmol, 4.4 equiv.); DCM (100 mL), sat. NaHCO3 (50 mL), preparative HPLC (grad. 0 – 100 % of MeCN / water + 0.1% FA over 40 min), 27 mg, 82 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 0.20 – 0.30 (m, 1H), 0.70 – 0.82 (m, 1H), 1.58 – 1.70 (m, 2H), 2.84 (s, 6H), 3.48 – 3.59 (m, 2H), 3.64 – 3.97 (m, 2H), 6.01 (d, J = 7.7 Hz, 1H), 7.57 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, CDCl3) δ 10.59, 16.00 (2C), 44.02 (2C), 49.54 (2C), 97.32, 134.58, 134.94, 152.61. LCMS: tR1.43 min, m / z 244.1 [M+H]+. 5-(Hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-N,N-dimethylpyrazolo[1,5-a]pyrimidin-3- amine (11g) Compound 10g (65 mg, 0.267 mmol, 1.0 equiv.), paraformaldehyde (18 mg, 0.588 mmol, 2.2 equiv.), 1M NaOH (6 mL), NaBH3CN (74 mg, 1.18 mmol, 4.4 equiv.); DCM (100 mL), sat. NaHCO3 (200 mL), preparative HPLC (grad. 0 – 100 % of MeCN / water + 0.1% FA over 50 min), 60 mg, 83 %, brown oil.1H NMR (500 MHz, DMSO-d6): δ 1.41 – 1.51 (m, 2H), 1.52 – 1.60 (m, 1H), 1.66 – 1.74 (m, 1H), 1.77 – 1.83 (m, 2H), 2.73 (bs, 6H), 2.92 – 3.59 (m, 4H), 3.62 – 3.78 (m, 2H), 6.27 (d, J = 7.7 Hz, 1H), 7.53 (s, 1H), 8.41 (d, J = 7.7 Hz, 1H);13C NMR (126 MHz, DMSO-d6): δ 25.13, 31.88 (2C), 42.22 (2C), 52.83 (2C), 55.74 (2C), 97.55, 135.21, 135.21, 137.56, 151.49. LCMS: tR1.76 min, m / z 272.2 [M+H]+. N,N-Dimethyl-5-(piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine (11h) Compound 10h (125 mg, 0.575 mmol, 1.0 equiv.), paraformaldehyde (38 mg, 1.27 mmol, 2.2 equiv.), 1M NaOH (7 mL), NaBH3CN (159 mg, 2.53 mmol, 4.4 equiv.); EtOAc (150 mL), sat. NaHCO3(80 mL), preparative HPLC (grad. 0 – 60 % MeCN / water over 50

[0046] 47 41967.601_P17853-02 min), 59 mg, 42 %, brown solid.1H NMR (500 MHz, CDCl3): δ 1.56 – 1.67 (m, 6H), 2.83 (s, 6H), 3.57 – 3.63 (m, 4H), 6.25 (d, J = 7.8 Hz, 1H), 7.55 (s, 1H), 8.09 (dd, J = 7.9, 1.7 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 24.69, 25.65 (2C), 44.05 (2C), 46.13 (2C), 96.46, 122.50, 134.55, 135.11, 138.69, 153.42. LCMS: tR1.44 min, m / z 246.2 [M+H]+. 5-(3-Azabicyclo[3.1.1]heptan-3-yl)-N,N-dimethylpyrazolo[1,5-a]pyrimidin-3-amine (11i) Compound 10i (48 mg, 0.209 mmol, 1.0 equiv.), paraformaldehyde (14 mg, 0.461 mmol, 2.2 equiv.), 1M NaOH (5 mL), NaBH3CN (58 mg, 0.920 mmol, 4.4 equiv.); DCM (100 mL), sat. NaHCO3 (100 mL), preparative HPLC (grad. 0 – 100 % of MeCN / water + 0.1% FA over 50 min), 27 mg, 82 %, yellow solid.1H NMR (500 MHz, CDCl3) δ 1.39 – 1.43 (m, 2H), 2.17 – 2.22 (m, 2H), 2.57 – 2.59 (m, 2H), 2.86 (s, 6H), 3.57 – 3.91 (m, 4H), 6.20 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 8.17 (d, J = 7.8 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 31.79 (2C), 32.31 (2C), 43.98 (2C), 51.47 (2C), 95.71, 121.60, 134.65, 134.85, 139.31, 153.74. LCMS: tR 1.61 min, m / z 258.1 [M+H]+. N,N-Dimethyl-5-morpholinopyrazolo[1,5-a]pyrimidin-3-amine (11j) Compound 10j (100 mg, 0.456 mmol, 1.0 equiv.), paraformaldehyde (30 mg, 1.00 mmol, 2.2 equiv.), 1M NaOH (5 mL), NaBH3CN (126 mg, 2.01 mmol, 4.4 equiv.); EtOAc (150 mL), sat. NaHCO3(50 mL), chromatography on silica gel (DCM / MeOH, 15:1), then further purified with preparative HPLC (grad. 0 – 50 % MeCN / water over 50 min), 31 mg, 27 %, light orange solid.1H NMR (500 MHz, CDCl3): δ 2.84 (s, 6H), 3.61 (t, J = 4.8 Hz, 4H), 3.78 (t, J = 4.2 Hz, 4H), 6.23 (d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 8.17 (d, J = 7.8 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 44.02 (2C), 45.32 (2C), 66.66 (2C), 96.05, 123.09, 134.83, 135.54, 138.21, 153.56. LCMS: tR 1.20 min, m / z 248.1 [M+H]+. 1-(3-(Dimethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-4-ol (11k) Compound 10k (44 mg, 0.189 mmol, 1.0 equiv.), paraformaldehyde (12 mg, 0.415 mmol, 2.2 equiv.), 1M NaOH (4 mL), NaBH3CN (52 mg, 0.830 mmol, 4.4 equiv.); EtOAc (100 mL), sat. NaHCO3 (100 mL), chromatography on silica gel (DCM / MeOH, 20:1), 11 mg, 22 %, yellow solid.1H NMR (500 MHz, d6-DMSO): δ 1.30 – 1.43 (m, 2H), 1.75 – 1.83 (m, 2H), 2.74 (s, 6H), 3.16 – 3.27 (m, 2H), 3.73 (dq, J = 8.8, 4.4 Hz, 1H), 4.00 – 4.08 (m, 2H), 4.73 (d, J = 4.2 Hz, 1H), 6.61 (d, J = 7.9 Hz, 1H), 7.55 (s, 1H), 8.43 (d, J = 7.8 Hz, 1H);13C NMR (126 MHz, d6-DMSO): δ 33.77 (2C), 42.34 (2C), 43.32 (2C), 65.88, 96.69, 121.95, 133.73, 135.67, 137.00, 152.81. LCMS: tR1.17 min, m / z 262.2 [M+H]+.

[0047] 48 41967.601_P17853-02 (R)-1-(3-(Dimethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ol (11l) Compound 10l (50 mg, 0.214 mmol, 1.0 equiv.), paraformaldehyde (14 mg, 0.472 mmol, 2.2 equiv.), 1M NaOH (2.5 mL), NaBH3CN (59 mg, 0.943 mmol, 4.4 equiv.); EtOAc (100 mL), sat. NaHCO3(2 × 40 mL), chromatography on silica gel (DCM / MeOH, 20:1 to 15:1), then further purified with preparative HPLC (grad. 0 – 50 % MeCN / water over 50 min), 37 mg, 66 %, yellow solid.1H NMR (500 MHz, CDCl3): δ 1.51 – 1.68 (m, 2H), 1.81 – 1.97 (m, 2H), 2.37 – 2.75 (m, 1H), 2.84 (s, 6H), 3.39 – 3.51 (m, 2H), 3.69 – 3.77 (m, 1H), 3.82 – 3.92 (m, 2H), 6.24 (d, J = 7.8 Hz, 1H), 7.58 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H);13C NMR (126 MHz, CDCl3): δ 22.27, 32.85, 44.13 (2C), 45.68, 51.93, 66.28, 96.69, 122.51, 134.68, 135.30, 138.46, 153.89. LCMS: tR1.20 min, m / z 262.2 [M+H]+. (S)-1-(3-(Dimethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ol (11m) Compound 10m (62 mg, 0.266 mmol, 1.0 equiv.), paraformaldehyde (18 mg, 0.585 mmol, 2.2 equiv.), 1M NaOH (5 mL), NaBH3CN (73 mg, 1.17 mmol, 4.4 equiv.); EtOAc (100 mL), sat. NaHCO3(150 mL), chromatography on silica gel (DCM / MeOH, 20:1), then further purified with preparative HPLC (grad. 0 – 50 % MeCN / water over 50 min), 25 mg, 36 %, yellow solid.1H NMR (500 MHz, d6-DMSO): δ 1.34 – 1.50 (m, 2H), 1.70 – 1.83 (m, 1H), 1.83 – 1.95 (m, 1H), 2.74 (bs, 6H), 2.86 – 3.00 (m, 1H), 3.10 – 3.21 (m, 1H), 3.49 – 3.56 (m, 1H), 3.86 – 4.00 (m, 1H), 4.07 – 4.21 (m, 1H), 4.88 – 4.89 (m, 1H), 6.57 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H);13C NMR (126 MHz, d6-DMSO): δ 22.42, 33.11, 43.37 (2C), 44.56, 51.74, 65.02, 96.74, 108.85, 135.50, 135.58, 137.02, 152.93. LCMS: tR1.22 min, m / z 262.2 [M+H]+. 5-Chloro-N,N-dimethylpyrazolo[1,5-a]pyrimidin-3-amine (14) 5-Chloropyrazolo[1,5-a]pyrimidin-3-amine (9) (1.00 g, 5.93 mmol, 1 equiv.) was dissolved in MeOH (20 mL) and formaldehyde solution (37 wt. % in H2O, 972 µL, 13.1 mmol, 2.1 equiv.) was added. The reaction mixture was stirred at rt for 30 min and then acidified with acetic acid to pH 3. NaBH3CN (559 mg, 8.89 mmol, 1.5 equiv.) was added in one portion and the resulting solution was stirred at rt for 16 h. The mixture was concen- trated in vacuo, diluted with EtOAc (150 mL) and washed with NaOH solution (1M in H2O, 150 mL), distilled water (100 mL) and brine (100 mL). The organic layer was dried over an- hydrous MgSO4and concentrated in vacuo. The residue was purified on silica gel (DCM / MeOH, 15:1). Product 14 (950 mg, 81 %) was isolated as orange solid.1H NMR

[0048] 49 41967.601_P17853-02 (500 MHz, CDCl3) δ 2.95 (s, 6H), 6.59 (d, J = 7.3 Hz, 1H), 7.74 (s, 1H), 8.34 (d, J = 7.3 Hz, 1H). LCMS: tR 0.31 min, m / z 197.1 [M+H]+. General procedure for synthesis of compound 11n-v. 5-Chloro-N,N-dimethylpyrazolo[1,5-a]pyrimidin-3-amine (14) (50 mg, 0.254 mmol, 1 equiv.) was dissolved in anhydrous isopropanol (0.4 mL) and DIEA (88 µL, 0.508 mmol, 2 equiv.) was added, followed by corresponding primary amine (0.508 mmol, 2 equiv.). The reaction mixture was stirred for 16 h at 80 ºC under inert atmosphere. The red solution was concentrated in vacuo. The residue was diluted with EtOAc (50 mL) and washed with dis- tilled water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The further purification dependeds on each individual reaction. N5-benzyl-N3,N3-dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (11n) Chromatography on silica gel: DCM / MeOH, 20:1. Yellow solid (46 mg, 68 %).1H NMR (500 MHz, CDCl3) δ1H NMR (500 MHz, Chloroform-d) δ 2.77 (s, 6H), 4.59 (d, J = 5.6 Hz, 2H), 5.05 – 5.15 (m, 1H), 5.86 (d, J = 7.6 Hz, 1H), 7.11 – 7.37 (m, 5H), 7.52 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H). LCMS: tR 2.11 min, m / z 268.2 [M+H]+. N3,N3-Dimethyl-N5-(4-(trifluoromethyl)benzyl)pyrazolo[1,5-a]pyrimidine-3,5-diamine (11o) Chromatography on silica gel: DCM / MeOH, 15:1. Yellow solid (34 mg, 40 %).1H NMR (500 MHz, CDCl3) δ 2.79 (s, 6H), 4.73 (d, J = 5.6 Hz, 2H), 5.07 – 5.15 (m, 1H), 5.96 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.4 Hz, 3H), 8.04 – 8.18 (m, 1H).19F NMR (471 MHz, Chloroform-d) δ -62.37. LCMS: tR1.99 min, m / z 336.2 [M+H]+. N5-(4-(Dimethylamino)benzyl)-N3,N3-dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (11p) Chromatography on silica gel: DCM / MeOH, 20:1. Brown amorphous compound (40 mg, 51 %).1H NMR (500 MHz, CDCl3) δ 2.87 (s, 6H), 2.94 (s, 6H), 4.54 (d, J = 5.2 Hz, 2H), 4.80 – 4.84 (m, 1H), 5.90 (d, J = 7.6 Hz, 1H), 6.68 – 6.74 (m, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.60 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H). LCMS: tR 1.31 min, m / z 311.2 [M+H]+. N5-(3,4-Dimethoxybenzyl)-N3,N3-dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (11q) Chromatography on silica gel: DCM / MeOH, 15:1. Yellow solid (97 mg, 58 %).1H NMR (500 MHz, CDCl3) δ 2.86 (s, 6H), 3.87 (d, J = 4.1 Hz, 6H), 4.59 (d, J = 5.3 Hz, 2H), 4.88 – 4.94 (m, 1H), 5.94 (d, J = 7.6 Hz, 1H), 6.82 – 6.83 (m, 1H), 6.90 – 6.93 (m, 2H), 7.61 (s, 1H), 8.10 (d, J = 7.5 Hz, 1H). LCMS: tR1.60 min, m / z 328.2 [M+H]+. N3,N3-Dimethyl-N5-(3,4,5-trimethoxybenzyl)pyrazolo[1,5-a]pyrimidine-3,5-diamine (11r)

[0049] 50 41967.601_P17853-02 Chromatography on silica gel: DCM / MeOH, 15:1. Brown amorphous compound (71 mg, 78 %).1H NMR (500 MHz, CDCl3) δ 2.86 (s, 6H), 3.83 (s, 3H), 3.84 (s, 6H), 4.59 (d, J = 5.4 Hz, 2H), 4.92 – 4.98 (m, 1H), 5.90 – 5.98 (m, 1H), 6.60 (s, 2H), 7.61 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H). LCMS: tR1.69 min, m / z 358.2 [M+H]+. N3,N3-Dimethyl-N5-(2-morpholinoethyl)pyrazolo[1,5-a]pyrimidine-3,5-diamine (11s) Chromatography on silica gel: DCM / MeOH, 5:1. Yellow amorphous compound (46 mg, 63 %).1H NMR (500 MHz, CDCl3) δ 2.48 – 2.51 (m, 4H), 2.62 (t, J = 5.9 Hz, 2H), 2.85 (s, 6H), 3.55 (q, J = 5.4 Hz, 2H), 3.71 – 3.76 (m, 4H), 5.29 – 5.31 (m, 1H), 5.97 (d, J = 7.6 Hz, 1H), 7.59 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H). LCMS: tR 0.14 min, m / z 291.2 [M+H]+. N5-Cyclopentyl-N3,N3-dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (11t) Chromatography on silica gel: DCM / MeOH, 15:1. Yellow amorphous compound (43 mg, 70 %).1H NMR (500 MHz, CDCl3) δ 1.47 (dq, J = 13.3, 6.5 Hz, 2H), 1.56 – 1.76 (m, 4H), 2.09 (dq, J = 12.8, 6.8 Hz, 2H), 2.84 (s, 6H), 4.29 (q, J = 6.3 Hz, 1H), 4.78 (d, J = 4.9 Hz, 1H), 5.91 (d, J = 7.6 Hz, 1H), 7.57 (s, 1H), 8.03 – 8.07 (m, 1H). LCMS: tR1.67 min, m / z 246.2 [M+H]+. 5-(((3-(Dimethylamino)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)-2-methoxyphenol (11u) Chromatography on silica gel: DCM / MeOH, 15:1. The isolated mixture of product 11t and starting material was further purified on preparative HPLC (grad. 15 – 60 % MeCN / water + 0.1 % FA over 30 min). Yellow solid (20 mg, 25 %).1H NMR (500 MHz, CDCl3) δ 2.86 (s, 6H), 3.85 (s, 3H), 4.48 – 4.60 (m, 2H), 5.08 – 5.22 (m, 2H), 5.91 (d, J = 7.5 Hz, 1H), 6.72 – 6.86 (m, 2H), 6.93 (s, 1H), 7.61 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H). LCMS: tR 1.52 min, m / z 314.2 [M+H]+. N5-(3,5-bis(trifluoromethyl)benzyl)-N3,N3-dimethylpyrazolo[1,5-a]pyrimidine-3,5-diamine (11v) Chromatography on silica gel: DCM / MeOH, 15:1. The isolated mixture of product 11u and starting material was further purified on preparative HPLC (grad. 20 – 70 % MeCN / water + 0.1 % FA over 50 min). Yellow solid (10 mg, 10 %).1H NMR (500 MHz, CDCl3) δ 2.76 (s, 6H), 4.79 (d, J = 5.6 Hz, 2H), 5.15 – 5.23 (m, 1H), 5.95 – 6.08 (m, 1H), 7.57 - 7.62 (m, 1H), 7.77 (s, 1H), 7.85 (s, 2H), 8.15 (d, J = 7.6 Hz, 1H).19F NMR (471 MHz, CDCl3) δ -62.70. LCMS: tR2.30 min, m / z 404.2 [M+H]+.

[0050] 51 41967.601_P17853-02 1.3 nSMase2 inhibitory potency The nSMase2 inhibitory potency was measured using a fluorescence-based coupled assay involving human nSMase2, alkaline phosphatase (AP), choline oxidase, and horseradish peroxidase as previously reported. Figuera-Losada et al., 2015. Briefly, human nSMase2 recombinant enzyme was used to catalyze the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Phosphorylcholine undergoes dephosphorylation by AP to produce choline which is subsequently oxidized by choline oxidase. H2O2generated in this oxidative step is detected by horseradish peroxidase-catalyzed conversion of Amplex Red® into resorufin (ex / em 530 / 590 nm). In order to assure that potent inhibitors identified in this coupled assay directly inhibited nSMase2, they were subsequently assessed in a counter assay using AP, choline oxidase, and horseradish peroxidase (but devoid of nSMase2 and sphingomyelin) in the presence of phosphorylcholine as previously described. Figuera- Losada et al., 2015. The results are summarized in Table 1. For compounds with an IC50value of 10 μM or below, we measured IC50 values in a counter assay in which test compounds were incubated with AP, choline oxidase, and horseradish peroxidase in the presence of phosphorylcholine. Compounds with ≤10-fold separation between nSMase2 and counter assays are marked (**) in Table 1 as these compounds likely interfere with AP, choline oxidase, and / or horseradish peroxidase in addition to (or instead of) nSMase2. Phenyl (R)- (1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)-pyrrolidin-3- yl)carbamate (PDDC) was used as a reference control compound. Rojas et al., 2019. Table 1. Inhibition of nSMase2

[0051] 52 41967.601_P17853-02 Table 1. Inhibition of nSMase2 Cmpd IC50(μM)*

[0052] 53 41967.601_P17853-02 Table 1. Inhibition of nSMase2 Cmpd IC50(μM)*

[0053] 54 41967.601_P17853-02 Table 1. Inhibition of nSMase2 Cmpd IC50(μM)* *Values are mean ± SD n of IC50values in nSMase2 and counter assays is ≤10-fold. 1.4 Evaluation of metabolic stability in liver microsomes The metabolic stability was evaluated using mouse, human and rat liver microsomes as previously described with minor modifications. Giancola et al., 2020. Briefly, reactions were carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl2, 0.4 U / mL glucose-6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions, in triplicate, were initiated by addition of the respective liver microsomes to the incubation mixture (compound final concentration was 5 μM; 0.5 mg / mL microsomes). After 60 min of incubation, aliquots of the mixture were removed and the reaction quenched by addition of three times the volume of ice-cold acetonitrile spiked with the internal standard. Compound

[0054] 55 41967.601_P17853-02 disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC / MS) method on a Dionex ultra-high-performance LC system coupled with a Q-Exactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham MA). Quantification of analytes and identification of the metabolites were performed in the full-scan mode (from m / z 50 to 1600) by comparing t = 0 samples and 60 min samples. Selected potent compounds were tested in mouse, rat, and human liver microsomes for metabolic stability (Table 2). Table 2. Metabolic stability of selected nSMase 2 inhibitors in 1.5 Evaluation of oral pharmacokinetics in rat All animal studies were performed as per protocols approved by the Institutional An- imal Care and Use Committee, at Johns Hopkins University. Male Wistar rats (6–8 weeks; weighing 200–250 g) obtained from Harlan®Laboratories (Indianapolis, IN) were main- tained in a controlled environment and allowed food and water available ad libitum. Dosing solution was prepared fresh prior to dosing (5% DMSO, 10% tween and 90% phosphate buffered saline). Compound 11j and PDDC were administered to rats as a solution via the oral gavage route at a dose of 10 mg / kg. At 30 min and 2h after dosing, blood samples were

[0055] 56 41967.601_P17853-02 collected in heparinized microtubes by cardiac puncture immediately before sacrifice. Brains were dissected after exsanguination and immediately flash frozen (−80° C). Plasma was pre- pared by centrifugation immediately after collection of blood samples. PDDC and 11j were assayed in plasma and tissues by the developed LC / MS / MS method described below. Quan- tification was performed using high-performance liquid chromatography with tandem mass spectrometry (LC-MS / MS). PDDC was quantified as previously published. Rojas et al., 2019. For 11j quantification, five microliters acetonitrile (100% v / v) containing internal standard (losartan, 0.5 μM) was added per mg tissue or microliter plasma and used to perform protein precipitation. The tissue samples were mechanically homogenized. Known concentrations of compound 11j were spiked into naive mouse plasma or brain homogenate to prepare calibration standards. Samples were vortex mixed and centrifuged at 10000g for 5 min at 4 °C. The supernatant was transferred to a 96 well plate and analyzed via LC– MS / MS. Chromatographic analysis was performed using a liquid chromatography system coupled to an Altis triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Separation of analyte was achieved at 35C using Agilent Eclipse Plus column (100 mm × 2.1 mm i.d.) packed with a 1.8 μm C18 stationary phase. The mobile phase consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in water with gradient elution. The [M+H]+ion transitions for 11j were m / z 248.15 → 165.05, 191.05, and for losartan (IS) they were m / z 423.15 → 207.13, 377.13. The results are shown in FIG. 1. 1.6 Aqueous solubility assessment The aqueous solubility of PDDC and compound 11j was determined by the shake flask method. Zhou et al., 2007. Test compounds were dissolved in PBS buffer (pH = 7.4) at 5 mg / mL concentration and incubated for 24 h at 37 °C on a thermomixer. Samples were subjected to constant mixing at a shaking speed of 1000 rpm to ensure saturation. After 24 hours the solution was filtered (0.45 μm PTFE) and the filtrate was diluted appropriately and solubility in the filtrate samples was determined against a calibration curve ranging 5-10000 nM. Samples were analyzed on a Dionex ultra-high-performance LC system coupled with Q Exactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham MA). The separation of analytes was achieved using the Agilent Eclipse Plus column (100 × 2.1

[0056] 57 41967.601_P17853-02 mm i.d.; maintained at 35 ºC) packed with a 1.8 µm C18 stationary phase. The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Pumps were operated at a flow rate of 0.3 mL / min for 8 min using gradient elution. The mass spectrometer controlled by Xcalibur software 4.0.27.13 (Thermo Scientific) was operated with a heated electrospray ionization (HESI) ion source in positive ionization mode. The [M + H]+ ion transitions of PDDC, compound 11j, and losartan (internal standard) were monitored in Full MS scan mode. Data were acquired and quantified with Xcalibur 2.2. The aqueous solubility of PDDC and compound 11j was 0.45 μg / mL and 1690 μg / mL, respectively. REFERENCES All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art. S. Tomiuk, K. Hofmann, M. Nix, M. Zumbansen, W. Stoffel, Cloned mammalian neutral sphingomyelinase: functions in sphingolipid signaling?, Proc Natl Acad Sci U S A, 95 (1998) 3638-3643. K. Hofmann, S. Tomiuk, G. Wolff, W. Stoffel, Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase, Proc Natl Acad Sci U S A, 97 (2000) 5895-5900. O. Krut, K. Wiegmann, H. Kashkar, B. Yazdanpanah, M. Kronke, Novel tumor necrosis factor-responsive mammalian neutral sphingomyelinase-3 is a C-tail-anchored protein, J Biol Chem, 281 (2006) 13784-13793.

[0057] 58 41967.601_P17853-02 B.X. Wu, V. Rajagopalan, P.L. Roddy, C.J. Clarke, Y.A. Hannun, Identification and characterization of murine mitochondria-associated neutral sphingomyelinase (MA- nSMase), the mammalian sphingomyelin phosphodiesterase 5, J Biol Chem, 285 (2010a) 17993-18002. B.X. Wu, C.J. Clarke, Y.A. Hannun, Mammalian neutral sphingomyelinases: regulation and roles in cell signaling responses, Neuromolecular Med, 12 (2010b) 320-330. C. Tallon, K.R. Hollinger, A. Pal, B.J. Bell, R. Rais, T. Tsukamoto, K.W. Witwer, N.J. Haughey, B.S. Slusher, Nipping disease in the bud: nSMase2 inhibitors as therapeutics in extracellular vesicle-mediated diseases, Drug Discov Today, 26 (2021a) 1656-1668. J. Skacel, B.S. Slusher, T. Tsukamoto, Small Molecule Inhibitors Targeting Biosynthesis of Ceramide, the Central Hub of the Sphingolipid Network, J Med Chem, 64 (2021) 279-297. C. Rojas, E. Barnaeva, A.G. Thomas, X. Hu, N. Southall, J. Marugan, A.D. Chaudhuri, S.W. Yoo, N. Hin, O. Stepanek, Y. Wu, S.C. Zimmermann, A.G. Gadiano, T. Tsukamoto, R. Rais, N. Haughey, M. Ferrer, B.S. Slusher, DPTIP, a newly identified potent brain penetrant neutral sphingomyelinase 2 inhibitor, regulates astrocyte-peripheral immune communication following brain inflammation, Sci Rep, 8 (2018) 17715. O. Stepanek, N. Hin, A.G. Thomas, R.P. Dash, J. Alt, R. Rais, C. Rojas, B.S. Slusher, T. Tsukamoto, Neutral sphingomyelinase 2 inhibitors based on the 4-(1H-imidazol- 2-yl)-2,6-dialkoxyphenol scaffold, Eur J Med Chem, 170 (2019) 276-289. C. Rojas, M. Sala, A.G. Thomas, A. Datta Chaudhuri, S.W. Yoo, Z. Li, R.P. Dash, R. Rais, N.J. Haughey, R. Nencka, B. Slusher, A novel and potent brain penetrant inhibitor of extracellular vesicle release, Br J Pharmacol, 176 (2019) 3857-3870. M. Sala, K.R. Hollinger, A.G. Thomas, R.P. Dash, C. Tallon, V. Veeravalli, L. Lovell, M. Kogler, H. Hrebabecky, E. Prochazkova, O. Nesuta, A. Donoghue, J. Lam, R. Rais, C. Rojas, B.S. Slusher, R. Nencka, Novel Human Neutral Sphingomyelinase 2 Inhibitors as Potential Therapeutics for Alzheimer's Disease, J Med Chem, 63 (2020) 6028- 6056. T. Bilousova, B.J. Simmons, R.R. Knapp, C.J. Elias, J. Campagna, M. Melnik, S. Chandra, S. Focht, C. Zhu, K. Vadivel, B. Jagodzinska, W. Cohn, P. Spilman, K.H. Gylys,

[0058] 59 41967.601_P17853-02 N.K. Garg, V. John, Dual Neutral Sphingomyelinase-2 / Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease, ACS Chem. Biol., 15 (2020) 1671-1684. C. Tallon, S. Picciolini, S.W. Yoo, A.G. Thomas, A. Pal, J. Alt, C. Carlomagno, A. Gualerzi, R. Rais, N.J. Haughey, M. Bedoni, B.S. Slusher, Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury, Biochem Pharmacol, 194 (2021b) 114796. X. Zhu, K.R. Hollinger, Y. Huang, A. Borjabad, B.H. Kim, T. Arab, A.G. Thomas, M. Moniruzzaman, L. Lovell, A. Turchinovich, K.W. Witwer, D.J. Volsky, N.J. Haughey, B.S. Slusher, Neutral sphingomyelinase 2 inhibition attenuates extracellular vesicle release and improves neurobehavioral deficits in murine HIV, Neurobiol Dis, 169 (2022) 105734. M. Figuera-Losada, M. Stathis, J.M. Dorskind, A.G. Thomas, V.V. Bandaru, S.W. Yoo, N.J. Westwood, G.W. Rogers, J.C. McArthur, N.J. Haughey, B.S. Slusher, C. Rojas, Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties, PLoS One, 10 (2015) e0124481. J.B. Giancola, A. Bonifazi, J. Cao, T. Ku, A.J. Haraczy, J. Lam, R. Rais, M.A. Coggiano, G. Tanda, A.H. Newman, Structure-activity relationships for a series of (Bis(4- fluorophenyl)methyl)sulfinylethyl-aminopiperidines and -piperidine amines at the dopamine transporter: Bioisosteric replacement of the piperazine improves metabolic stability, Eur J Med Chem, 208 (2020) 112674. L. Zhou, L. Yang, S. Tilton, J. Wang, Development of a high throughput equilibrium solubility assay using miniaturized shake-flask method in early drug discovery, J Pharm Sci, 96 (2007) 3052-3071. Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claim

[0059] 60 41967.601_P17853-02

Claims

THAT WHICH IS CLAIMED:

1. A compound of formula (I): ; wherein:L can be present or absent and when present is a linker comprising a substituted or unsubstituted C1-C3 alkylene chain, wherein 1 to 3 methylene units of the alkylene chain can be optionally and independently replaced with -O-, -NRz-, -S-, -SO-, or -SO2-, wherein each Rzis independently selected from H, -(CH2)0-3ORy, -O(CH2)0-3C(O)ORy, or an optionally substituted C1-C6 alkyl group, and wherein each Ryis independently H or substituted or unsubstituted C1-C6alkyl; R1and R2are each independently selected from substituted or unsubstituted C1-C4alkyl and substituted or unsubstituted deuterated C1-C4 alkyl; R3 and R4 are each independently selected from H, C1-C4 alkyl, and substituted or unsubstituted benzyl, provided that R3and R4are not H at the same time; or if L is absent, R3 and R4 can together form a 5- to 6-membered heterocylic ring, which can be fused to a 3- or 5-membered cycloalkyl, provided that neither R1 nor R2 form a cyclic ring with R3or R4; R5, R6, and R7 are each independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone; and pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein the compound of formula (I) comprises a compound of formula (Ia):61 41967.601_P17853-02a); wherein: L comprises a substituted or unsubstituted C1-C3 alkylene chain, wherein 1 to 3 methylene units of the alkylene chain are optionally and independently replaced with -O-, -NRz-, -S-, -SO-, or -SO2-, wherein each Rzis independently selected from hydrogen, -(CH2)0-3ORy, -O(CH2)0-3C(O)ORy, or an optionally substituted C1-C6 alkyl group; each Ryis independently H or substituted or unsubstituted C1-C6 alkyl; and pharmaceutically acceptable salts thereof.

3. A compound of formula (II): ; wherein:CyAis a substituted or unsubstituted 3- to 10-membered saturated or partially unsaturated monocyclic or bicyclic carbocyclyl or heterocyclyl having 0-6 heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein CyAis substituted with 0-4 RAgroups; each RAis independently selected from oxo, halogen, -CN, -C(O)Ry, -C(O)2Ry-, -C(O)N(Ry)2, -NO2, -N(Ry)2, -N(R)C(O)Ry, -N(R)C(O)2Ry, -ORy, -OC(O)Ry, -OC(O)N(Ry)2, -SRy, -S(O)Ry, -S(O)2Ry, -S(O)N(Ry)2, -S(O)2N(Ry)2, or a substituted or unsubstituted group selected from C1-C6alkyl, phenyl, 3- to 7-membered saturated or partially unsaturated monocyclyl, or 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1-2 heteroatoms selected from oxygen, nitrogen, and sulfur, wherein each Ryis independently H or substituted or unsubstituted C1-C6alkyl; R1and R2are each independently selected from substituted or unsubstituted C1-C4alkyl and substituted or unsubstituted deuterated C1-C4 alkyl;62 41967.601_P17853-02R5, R6, and R7are each independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone; and pharmaceutically acceptable salts thereof.

4. The compound of any one of claim 1 to claim 3, wherein R1 and R2 are each independently selected from -CH3, -CD3, and -CH2CF3.

5. The compound of claim 1 or claim 2, wherein R3 and R4 together form a 5- or 6- membered heterocyclic ring, which can be fused with a 3- or 5-membered cycloalkyl, each of which can be substituted or unsubstituted.

6. The compound of claim 5, wherein the 5- or 6-member heterocyclic ring is selected from substituted or unsubstituted pyrrolidinyl, piperidinyl, morpholinyl, octahydrocyclopenta[c]pyrrolyl, azabicyclo[3.1.0]hexanyl, and azabicyclo[3.1.1]heptanyl.

7. The compound of claim 6, wherein the 5- or 6-membered heterocyclic ring is substituted with one or more substituent groups selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl, each of which can be substituted or unsubstituted, alkoxyl, amine, -CF3, carbamoyl, carbonyl, cyano, halogen, hydroxyl, nitro, thiol, and sulfone.

8. The compound of claim 7, wherein the 5- or 6-membered heterocyclic ring is substituted with a substituent group selected from C1-C4 alkyl, hydroxyl, and phenyl.

9. The compound of any one of claim 5 to claim 8, wherein the 5- or 6-membered heterocyclic ring is selected from pyrrolidinyl, methylpyrrolidinyl, phenylpyrrolidinyl, octahydrocyclopenta[c]pyrrolyl, piperidinyl, hydroxypiperidinyl, morpholinyl, azabicyclo[3.1.0]hexanyl, and azabicyclo[3.1.1]heptanyl.

10. The compound of claim 1 or claim 2, wherein one of R3or R4is H.63 41967.601_P17853-0211. The compound of claim 10, wherein one of R3and R4is substituted or unsubstituted benzyl.

12. The compound of claim 11, wherein the benzyl is substituted with one or more substituent groups selected from alkoxyl, amine, and -CF3.

13. The compound of any one of claim 1 to claim 3, wherein R5, R6, and R7are each independently H.

14. The compound of any one of claim 1 to claim 3, wherein the compound of formula (I) or formula (II) is selected from: ,02nd 15. A method for treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression, the method comprising administering to a subject in need of treatment thereof an effective amount of a compound of any one of claim 1 to claim 14.

16. The method of claim 15, wherein the condition, disease, or disorder is associated with an elevated level of ceramide in the subject in need of treatment compared to a control subject not afflicted with the condition, disease, or disorder.65 41967.601_P17853-0217. The method of claim 15 or claim 16, wherein the administration of an effective amount of a compound of formula (I) or formula (II) to the subject decreases an nSMase2 activity or expression or decreases a level of ceramide in the subject.

18. The method of any one of claim 15 to claim 17, wherein the condition, disease, or disorder is selected from the group consisting of a neurological, an oncological, an inflammatory, a cardiovascular disease, and an infectious condition, disease, or disorder.

19. The method of claim 18, wherein the neurological condition, disease, or disorder is selected from Alzheimer's disease (AD), Parkinson's disease, HIV-associated neurocognitive disorder (HAND), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), a prion disease, Duchenne muscular dystrophy (DMD), and retinal degeneration.

20. The method of claim 18, wherein the oncological condition, disease, or disorder is selected from breast cancer, cervical cancer, colon cancer, colorectal cancer (CRC), duodenal cancer, gastric cancer, lung cancer, multiple myeloma, oral cancer, pancreatic cancer, prostate cancer, and skin cancer.

21. The method of claim 18, further comprising treating the oncological condition, disease, or disorder with immunotherapy to enhance systemic antitumor immunity.

22. The method of claim 18, wherein the inflammatory condition, disease, or disorder is selected from the group consisting of an inflammatory airway disease, including an allergic airway inflammation, an ischemia-reperfusion injury, including cerebral ischemia and hepatic ischemia-reperfusion injury, sepsis, atherosclerosis, myocardial infarction, and inflammatory bowel disease.

23. The method of claim 18, wherein the cardiovascular disease is a myocardial disease involving myocyte hypertrophy, fibroblast-derived cardiac hypertrophy, heart failure, heart hypertrophy, diastolic and / or systolic ventricular dysfunction and / or a cardiovascular66 41967.601_P17853-02disease involving fibrosis, aortic stenosis, atrial fibrillation, genetic forms of cardiomyopathy, cardiac storage diseases and / or Fabry disease.

24. The method of claim 18, wherein the infectious condition, disease, or disorder comprises a viral infection selected from the group consisting of human immunodeficiency virus (HIV), Zika virus, rabies virus, Dengue virus, hepatitis C (HCV), hepatitis E (HEV), cytomegalovirus (HCMV), Newcastle disease virus (NDV), and Langat virus.

25. The method of claim 18, wherein the infectious disease is associated with a toxin produced from a bacterial infection.

26. The method of claim 25, wherein the toxin produce from a bacterial infection is selected from Shiga toxin released by Escherichia coli and epsilon toxin released by Clostridum perfringens.

27. The method of claim 15, wherein the condition, disease, or disorder comprises an extracellular vesicle-mediated disease.

28. The method of claim 27, wherein administration of the compound of formula (I) or formula (II) inhibits exosome biosynthesis.

29. A method for inhibiting neutral sphingomyelinase 2 (nSMase2), the method comprising administering to a subject, cell, or tissue an amount of a compound of any of claim 1 to claim 14 effective to inhibit nSMase2.

30. A method for inhibiting ceramide biosynthesis, the method comprising contacting a cell with an amount of a compound of any one of claim 1 to claim 14 or a pharmaceutically acceptable salt thereof.67 41967.601_P17853-0231. Use of a compound of any one of claim 1 to claim 14 for preparing a medicament for treating a condition, disease, or disorder associated with an increased neutral sphingomyelinase 2 (nSMase2) activity or expression.

32. A pharmaceutical composition comprising a compound of any one of claims 1-14.68 41967.601_P17853-02

Images