COMPOUNDS USEFUL FOR TREATING NEURODEGENERATIVE DISORDERS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 61/532,048, filed September 7, 201 1, the disclosure of which is incorporated in its entirety herein by reference.

TECHNICAL FIELD OF INVENTION

[0002] The present invention relates to pharmaceutically active compounds useful for treating, or lessening the severity of, neurodegenerative disorders.

BACKGROUND OF THE INVENTION

[0003] The central role of the long form of amyloid beta-peptide, in particular Αβ(1- 42), in Alzheimer's disease has been established through a variety of histopathological, genetic and biochemical studies. See Selkoe, DJ, Physiol. Rev. 2001, 81 :741-766, Alzheimer's disease: genes, proteins, and therapy, and Younkin SG, J. Physiol. Paris. 1998, 92:289-92, The role of A beta 42 in Alzheimer's disease. Specifically, it has been found that deposition in the brain of Αβ(1-42) is an early and invariant feature of all forms of Alzheimer's disease. In fact, this occurs before a diagnosis of Alzheimer's disease is possible and before the deposition of the shorter primary form of A-beta, Αβ(1-40). See Parvathy S, et a , Arch. Neurol. 2001, 58:2025-32, Correlation between Abetax-40-, Abetax-42-, and Abetax-43 -containing amyloid plaques and cognitive decline. Further implication of Αβ(1- 42) in disease etiology comes from the observation that mutations in presenilin (gamma secretase) genes associated with early onset familial forms of Alzheimer's disease uniformly result in increased levels of Αβ(1-42). See Ishii K., et ah, Neurosci. Lett. 1997 ^' , 228: 17-20, Increased A beta 42(43)-plaque deposition in early-onset familial Alzheimer's disease brains with the deletion of exon 9 and the missense point mutation (H163R) in the PS-1 gene. Additional mutations in the amyloid precursor protein APP raise total Αβ and in some cases raise Αβ(1-42) alone. See Kosaka T, et ah, Neurology, 48:741-5, The beta APP717 Alzheimer mutation increases the percentage of plasma amyloid-beta protein ending at A beta42(43). Although the various APP mutations may influence the type, quantity, and location of Αβ deposited, it has been found that the predominant and initial species deposited in the brain parenchyma is long Αβ (Mann). See Mann DM, et al, Am. J. Pathol. 1996, 148: 1257-66, "Predominant deposition of amyloid-beta 42(43) in plaques in cases of Alzheimer's disease and hereditary cerebral hemorrhage associated with mutations in the amyloid precursor protein gene".

[0004] In early deposits of Αβ, when most deposited protein is in the form of amorphous or diffuse plaques, virtually all of the Αβ is of the long form. See Gravina SA, et al., J. Biol. Chem., 270:7013-6, Amyloid beta protein (A beta) in Alzheimer's disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at A beta 40 or A beta 42(43); Iwatsubo T, et al, Am. J. Pathol. 1996, 149: 1823-30, Full-length amyloid-beta (1-42(43)) and amino-terminally modified and truncated amyloid-beta 42(43) deposit in diffuse plaques; and Roher AE, et al, Proc. Natl. Acad. Sci. U S A. 1993, 90: 10836-40, beta-Amyloid-(l-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease. These initial deposits of Αβ(1- 42) then are able to seed the further deposition of both long and short forms of Αβ. See Tamaoka A, et al, Biochem. Biophys. Res. Commun. 1994, 205:834-42, Biochemical evidence for the long-tail form (A beta 1-42/43) of amyloid beta protein as a seed molecule in cerebral deposits of Alzheimer's disease.

[0005] In transgenic animals expressing Αβ, deposits were associated with elevated levels of Αβ(1-42), and the pattern of deposition is similar to that seen in human disease with Αβ(1-42) being deposited early followed by deposition of Αβ(1-40). See Rockenstein E, et al, J. Neurosci. Res. 2001, 66:573-82, Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(l-42); and Terai K, et al, Neuroscience 2001, 104:299-310, beta-Amyloid deposits in transgenic mice expressing human beta-amyloid precursor protein have the same characteristics as those in Alzheimer's disease. Similar patterns and timing of deposition are seen in Down's syndrome patients in which Αβ expression is elevated and deposition is accelerated. See Iwatsubo T., et al, Ann. Neurol. 1995, 37:294-9, Amyloid beta protein (A beta) deposition: A beta 42(43) precedes A beta 40 in Down syndrome.

[0006] Accordingly, selective lowering of Αβ(1-42) thus emerges as a disease-specific strategy for reducing the amyloid forming potential of all forms of Αβ, slowing or stopping the formation of new deposits of Αβ, inhibiting the formation of soluble toxic oligomers of Αβ, and thereby slowing or halting the progression of neurodegeneration.

SUMMARY OF THE INVENTION

[0007] As described herein, the present invention provides compounds useful for treating or lessening the severity of a neurodegenerative disorder. The present invention also provides methods of treating or lessening the severity of such disorders wherein said method comprises administering to a patient a compound of the present invention, or composition thereof. Said method is useful for treating or lessening the severity of, for example, Alzheimer's disease.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

1. General Description of Compounds of the Invention

[0008] According to one embodiment, the present invention provides a compound of formula I:

I

or a pharmaceutically acceptable salt thereof, wherein:

Ring A is selected from:

each m is independently 0, 1, 2, 3, or 4;

L is a covalent bond, or a straight or branched C1-5 saturated or unsaturated, straight or branched, divalent hydrocarbon chain;

each R ¹ is independently hydrogen, straight or branched Ci_6 alkyl, 3-6 membered cycloalkyl, or 3-6 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur, wherein each R ¹ is optionally and independently substituted with 1-4 R ³ groups, or:

R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form an optionally substituted 3-7 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached; or:

R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached; each R ² is independently R, deuterium, -OR, oxo, or:

two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused 3-7 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur; or:

two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-7 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur; or:

two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged saturated carbocyclic or a 4-7 membered bridged heterocyclic ring having 1 -2 heteroatoms independently selected from oxygen, nitrogen, or sulfur;

each R ³ is independently R, halogen, -C(0)N(R)2, -OR, C _1-3 alkyl optionally substituted with one or two -OH groups, or:

two R ³ groups on the same carbon atom are taken together to form an optionally substituted 3-6 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur; and

each R is independently hydrogen, C _1-4 aliphatic, or :

two R groups on the same nitrogen atom are taken together to form an optionally substituted 4-8 membered saturated or partially unsaturated ring. 2. Definitions

[0009] Compounds of this invention include those described generally above, and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5 ^th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0010] As described herein, compounds of the invention may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." In general, the term "substituted," whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.

[0011] The term "stable," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40 °C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

[0012] 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 "carbocycle" "cycloaliphatic" or "cycloalkyl"), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In yet other embodiments aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C8 hydrocarbon or bicyclic C ₈ -C ₁₂ 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 wherein any individual ring in said bicyclic ring system has 3-7 members. 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. In other embodiments, an aliphatic group may have two geminal hydrogen atoms replaced with oxo (a bivalent carbonyl oxygen atom =0), or a ring-forming substituent, such as -0-(straight or branched alkylene or alkylene)-0- to form an acetal or ketal. The term "alkylene," as used herein, refers to a bivalent straight or branched saturated or unsaturated hydrocarbon chain. In some embodiments, an alkylene group is saturated.

[0013] In certain embodiments, exemplary aliphatic groups include, but are not limited to, ethynyl, 2-propynyl, 1-propenyl, 2-butenyl, 1,3-butadienyl, 2-pentenyl, vinyl (ethenyl), allyl, isopropenyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, neo-pentyl, tert-pentyl, cyclopentyl, hexyl, isohexyl, sec-hexyl, cyclohexyl, 2-methylpentyl, tert-hexyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,3- dimethylbutyl, and 2,3-dimethyl but-2-yl.

[0014] The term "heterocycle," "heterocyclyl," "heterocycloaliphatic," or "heterocyclic" as used herein means non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members is an independently selected heteroatom. In some embodiments, the "heterocycle," "heterocyclyl," "heterocycloaliphatic," or "heterocyclic" group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members. [0015] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and, when specified, any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.

[0016] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), ΝΗ (as in pyrrolidinyl) or NR ⁺ (as in N-substituted pyrrolidinyl).

[0017] The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0018] As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0019] The term "aryl" used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein one or more ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". The term "aryl" also refers to heteroaryl ring systems as defined herein. In certain embodiments of the present invention, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term "aryl," as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0020] The term "heteroaryl," used alone or as part of a larger moiety as in "heteroaralkyl" or "heteroarylalkoxy," refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein one or more ring in the system is aromatic, one or more ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic". Heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.

[0021] The terms "heteroaryl" and "heteroar-," as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings. Examplary heteroaryl rings include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3- b]- 1 ,4-oxazin-3 (4H)-one.

[0022] As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0023] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH ₂ )o-4R°; -(CH ₂ )o-40R°; - 0(CH ₂ )o- ₄ R°, -0-(CH ₂ )o_ ₄ C(0)OR°; -(CH ₂ ) ₀ ^CH(OR°) ₂ ; -(CH ₂ ) ₀ _ ₄ SR ^o ; -(CH^Ph, which may be substituted with R°; which may be substituted with R°; - CH=CHPh, which may be substituted with R°; which may be substituted with R°; -N0 ₂ ; -CN; -N ₃ ; -(CH ₂ ) ₀ - ₄ N(R°) ₂ ; -(CH ₂ ) ₀ - ₄ N(R ^o )C(O)R°; - N(R°)C(S)R°; -(CH ₂ )o^N(R°)C(0)NR° ₂ ; -N(R°)C(S)NR° ₂ ; -(CH ₂ ) ₀ ^N(R ^o )C(O)OR°; - N(R°)N(R°)C(0)R°; -N(R°)N(R ⁰ )C(0)NR° ₂ ; -N(R°)N(R°)C(0)OR°; -(CH ₂ ) ₀ -4C(O)R°; - C(S)R°; -(CH ₂ )o^C(0)OR°; -(CH ₂ )C C(0)SR ⁰ ; -(CH ₂ )c C(0)OSiR ⁰ ₃ ; -(CH ₂ ) ₀ _ ₄ OC(O)R ^o ; -OC(0)(CH ₂ )o^SR°, SC(S)SR°; -(CH ₂ ) ₀ - ₄ SC(O)R°; -(CH ₂ ) ₀ -4C(O)NR° ₂ ; -C(S)NR° ₂ ; - C(S)SR°; -SC(S)SR°, -(CH ₂ ) ₀ - ₄ OC(O)NR° ₂ ; -C(0)N(OR°)R°; -C(0)C(0)R°; - C(0)CH ₂ C(0)R°; -C(NOR°)R°; -(CH^SSR ⁰ ; -(CH ₂ ) ₀ ^S(O) ₂ R°; -(CH ₂ ) ₀ ^S(O) ₂ OR°; - (CH ₂ )C OS(0) ₂ R ⁰ ; -S(0) ₂ NR° ₂ ; -(CH ₂ ) ₀ ^S(O)R°; -N(R°)S(0) ₂ NR° ₂ ; -N(R°)S(0) ₂ R°; - N(OR°)R°; -C( H)NR° ₂ ; -P(0) ₂ R°; -P(0)R° ₂ ; -OP(0)R° ₂ ; -OP(0)(OR°) ₂ ; SiR° ₃ ; -(d^ straight or branched alkylene)0-N(R°) ₂ ; or -(Ci_ ₄ straight or branched alkylene)C(0)0- N(R°) ₂ , wherein each R° may be substituted as defined below and is independently hydrogen, Ci_ ₆ aliphatic, -CH ₂ Ph, -0(CH ₂ )o_iPh, -CH ₂ -(5-6 membered heteroaryl ring), or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0^1 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3—12— membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0024] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH ₂ y ₂ R ^e , -(haloR*), -(CH ₂ y ₂ OH, -(CH ₂ y ₂ OR ^e , -(CH ₂ y ₂ CH(OR') ₂ ; -0(haloR ^e ), -CN, -N ₃ , -(CH ₂ y ₂ C(0)R ^e , -(CH ₂ y ₂ C(0)OH, -(CH ₂ y ₂ C(0)OR ^e , -(CH ₂ y ₂ SR ^e , -(CH ₂ y ₂ SH, -(CH ₂ y ₂ NH ₂ , -(CH ₂ y ₂ NHR ^e , -(CH ₂ y ₂ NR' ₂ , -N0 ₂ , -SiR' ₃ , -OSiR' ₃ , -C(0)SR ^e -(Ci- ₄ straight or branched alkylene)C(0)OR ^e , or -SSR* wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from Ci_ ₄ aliphatic, -CH ₂ Ph, -0(CH ₂ )o_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0025] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =0, =S, = NR ^* ₂ , =NNHC(0)R ^* , = NHC(0)OR ^* , =NNHS(0) ₂ R ^* , =NR ^* , =NOR ^* , -0(C(R ^* ₂ )) ₂ _ ₃ 0-, or -S(C(R ^* ₂ )) ₂ _ ₃ S- and =C(R ^* ) ₂ ,wherein each independent occurrence of R is selected from hydrogen, Ci_ ₆ aliphatic which may be ¾iih¾†T†n† ^p fl a¾ rl ^p fin ^p rl h ^p lnw nr an iin¾iih¾†T†n† ^p rl 5— 6— membered saturated, partially unsaturated, or aryl ring having 0M heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group include: -0(CR ^* 2)2- ₃ 0-, wherein each independent occurrence of R ^* is selected from hydrogen, Ci_6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0M heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0026] Suitable substituents on the aliphatic group of R include halogen, -R , -(haloR*), -OH, -OR", -O(haloR'), -CN, -C(0)OH, -C(0)OR ^e , -NH ₂ , -NHR", -NR' ₂ , or -

NO2, wherein each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci^ aliphatic, -CH ₂ Ph, -0(CH ₂ )o-iPh, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0M heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0027] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R ^† , -NR ^† ₂ , -C(0)R ^† , -C(0)OR ^† , -C(0)C(0)R ^† , -C(0)CH ₂ C(0)R ^† , - S(0) ₂ R ^† , -S(0) ₂ NR ^† ₂ , -C(S)NR ^† ₂ , -C(NH)NR ^† ₂ , or -N(R ^† )S(0) ₂ R ^† ; wherein each R ^† is independently hydrogen, Ci_6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having C heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R ^† , taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0M heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0028] Suitable substituents on the aliphatic group of R ^† are independently halogen, - R", -(haloR*), -OH, -OR", -O(haloR'), -CN, -C(0)OH, -C(0)OR ^e , -NH ₂ , -NHR*, -NR' ₂ , or -NO2, wherein each R' is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_4 aliphatic, -CH ₂ Ph, -0(CH ₂ )o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0M heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0029] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.

[0030] Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0031] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ⁿ C- or ¹³ C- or ¹⁴ C- enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

3. Description of Exemplary Compounds

[0032] As described generally above, the present invention provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and described in classes and subclasses above and herein.

[0033] In certain embodiments, the present invention provides a compound of formula I having the stereochemistry depicted in formula I-a, below:

I-a

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and described in classes and subclasses above and herein for compounds of formula I. s defined enerall above, Ring A is selected from

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0035] In certain embodiments, Ring A is selected from

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0036] In certain embodiments, Ring A is selected from

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0037] In certain

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0038] In certain embodiments, Ring A is selected from

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0039] In certain embodiments Rin A is selected from

wherein each of m, R ¹ and R ² is independently as defined above and described herein. [0040] In certain embodiments, Ring A is of the following formula:

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0041] In certain embodiments, Ring A is of the following formula:

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0042] In certain embodiments, Ring A is of the following formula

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0043] In certain embodiments, Ring A is of the following formula

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0044] In certain embodiments, Ring A is of the following formula

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0045] In certain embodiments, Ring A is of the following formula

wherein each of m, R ¹ and R ² is independently as defined above and described herein.

[0046] As defined generally above and herein, each m is independently 0, 1, 2, 3, or 4. In some embodiments, each m is independently 1-2. In some embodiments, each m is independently 1-3. In certain embodiments, each m is independently 2 or 3. In some embodiments, each m is independently 1-4. In some embodiments, each m is 0. In some embodiments, each m is 1.

[0047] As defined generally above and herein, each R ¹ is independently hydrogen, straight or branched Ci_6 alkyl, 3-6 membered cycloalkyl, or 3-6 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur, wherein each R ¹ is optionally and independently substituted with 1-4 R ³ groups, or:

R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form an optionally substituted 3-7 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached; or:

R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached; wherein each R ² and R ³ is independently as defined above and described herein.

[0048]

wherein each of m and R ² is independently as defined above and described herein.

[0049] In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl, 3-6 membered cycloalkyl, or 3-6 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur, wherein each R ¹ is optionally and independently substituted with 1-4 R ³ groups, or:

R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form an optionally substituted 3-7 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached; or:

R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached; wherein each R ² and R ³ is independently as defined above and described herein.

[0050] In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is optionally and independently substituted with 1-4 R ³ groups, wherein each R ³ is independently as defined above and described herein.

[0051] In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl. In certain embodiments, each R ¹ is independently straight or branched C1-5 alkyl. In certain embodiments, each R ¹ is independently straight or branched C1-4 alkyl. In certain embodiments, each R ¹ is independently straight or branched C1-3 alkyl. In certain embodiments, each R ¹ is independently straight or branched hexyl. In certain embodiments, each R ¹ is independently straight or branched pentyl. In certain embodiments, each R ¹ is independently straight or branched butyl. In certain embodiments, each R ¹ is independently straight or branched propyl.

[0052] In certain embodiments, each R ¹ is w-pentyl. In certain embodiments, each R ¹ is 1 -methylbutyl. In certain embodiments, each R ¹ is (R)-l -methylbutyl. In certain embodiments, each R ¹ is (S)-l -methylbutyl. In certain embodiments, each R ¹ is 2- methylbutyl. In certain embodiments, each R ¹ is (R)-2-methylbutyl. In certain embodiments, each R ¹ is (5 ^* )-2-methylbutyl. In certain embodiments, each R ¹ is 3 -methylbutyl. In certain embodiments, each R ¹ is 1 , 1 -dimethylpropyl. In certain embodiments, each R ¹ is 2,2- dimethylpropyl. In certain embodiments, each R ¹ is 1 -ethylpropyl. In certain embodiments, each R ¹ is neopentyl.

[0053] In certain embodiments, each R ¹ is independently w-butyl. In certain embodiments, each R ¹ is 1-methylpropyl. In certain embodiments, each R ¹ is (R)-l- methylpropyl. In certain embodiments, each R ¹ is (S)- 1-methylpropyl. In certain embodiments, each R ¹ is 2-methylpropyl. In certain embodiments, each R ¹ is tert-butyl.

[0054] In certain embodiments, each R ¹ is w-propyl. In certain embodiments, each R ¹ is isopropyl.

[0055] In certain embodiments, each R ¹ is ethyl.

[0056] In certain embodiments, each R ¹ is methyl. [0057] In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is substituted with 1-4 R ³ groups, wherein each R ³ is independently as defined above and described herein.

[0058] In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is substituted with 1, 2, 3, or 4 R ³ groups, wherein each R ³ is independently as defined above and described herein. In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is substituted with 1, 2, or 3 R ³ groups, wherein each R ³ is independently as defined above and described herein. In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_ 6 alkyl is substituted with 1 or 2 R ³ groups, wherein each R ³ is independently as defined above and described herein. In certain embodiments, each R ¹ is independently straight or branched Ci-6 alkyl wherein the Ci-6 alkyl is substituted with 4 R ³ groups, wherein each R ³ is independently as defined above and described herein. In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is substituted with 3 R ³ groups, wherein each R ³ is independently as defined above and described herein. In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is substituted with 2 R ³ groups, wherein each R ³ is independently as defined above and described herein. In certain embodiments, each R ¹ is independently straight or branched Ci_6 alkyl wherein the Ci_6 alkyl is substituted with one R ³ group, wherein each R ³ is independently as defined above and described herein.

[0059] As defined generally above and herein, each R ³ is independently halogen, - C(0)N(R) ₂ , -OH, -0(Ci_4 alkyl), Ci_ ₃ alkyl optionally substituted with one or two -OH groups, or:

two R ³ groups on the same carbon atom are taken together to form a 3-6 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur;

wherein each R is independently as defined above and described herein.

[0060] In certain embodiments, each R ³ is independently R wherein each R is independently as defined above and described herein.

[0061] In certain embodiments, each R ³ is hydrogen. [0062] In certain embodiments, each R ³ is independently halogen. In certain embodiments, each R ³ is-F. In certain embodiments, each R ³ is-Cl. In certain embodiments, each R ³ is-Br. In certain embodiments, each R ³ is-I.

[0063] In certain embodiments, each R ³ is independently -C(0)N(R)2 wherein each R is independently as defined above and described herein.

[0064] In certain embodiments, each R ³ is -C(0)NH ₂ .

[0065] In certain embodiments, each R ³ is independently -OR wherein each R is independently as defined above and described herein.

[0066] In certain embodiments, each R ³ is independently -OH.

[0067] In certain embodiments, each R ³ is independently C _1-3 alkyl optionally substituted with one or two -OR groups wherein each R is independently as defined above and described herein.

[0068] In certain embodiments, each R ³ is w-propoxy. In certain embodiments, each R ³ is isopropoxy.

[0069] In certain embodiments, each R ³ is ethoxy.

[0070] In certain embodiments, each R ³ is methoxy.

[0071] In certain embodiments, each R ³ is independently C _1-3 alkyl optionally substituted with one or two -OH groups.

[0072] In certain embodiments, each R ³ is independently C _1-3 alkyl. In certain embodiments, each R ³ is independently C _1-3 alkyl substituted with one -OH group. In certain embodiments, each R ³ is independently C _1-3 alkyl optionally substituted with two -OH groups.

[0073] In certain embodiments, each R ³ is independently C _1-3 alkyl optionally substituted with one or two -OH groups. In certain embodiments, each R ³ is independently Ci-2 alkyl optionally substituted with one or two -OH groups. In certain embodiments, each R ³ is independently C ₃ alkyl optionally substituted with one or two -OH groups. In certain embodiments, each R ³ is ethyl optionally substituted with one or two -OH groups. In certain embodiments, each R ³ is methyl optionally substituted with one or two -OH groups.

[0074] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-6 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. [0075] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-6 membered saturated carbocyclic ring. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-5 membered saturated carbocyclic ring. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-4 membered saturated carbocyclic ring.

[0076] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a cyclohexyl, cyclopentyl, cyclobutyl, or cyclopropyl ring.

[0077] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-6 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-5 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-4 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[0078] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 6 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 5 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 4 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[0079] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 heteroatom independently selected from oxygen, nitrogen, or sulfur. [0080] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 oxygen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-6 membered heterocyclic ring having 1 oxygen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-5 membered heterocyclic ring having 1 oxygen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-4 membered heterocyclic ring having 1 nitrogen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form oxepanyl, tetrahydro-2H-pyranyl, tetrahydrofuranyl, or oxetanyl.

[0081] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 nitrogen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-6 membered heterocyclic ring having 1 nitrogen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-5 membered heterocyclic ring having 1 nitrogen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-4 membered heterocyclic ring having 1 nitrogen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form azepanyl, piperidinyl, pyrrolidinyl, azetidinyl, or aziridinyl.

[0082] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-6 membered heterocyclic ring having 1 sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-5 membered heterocyclic ring having 1 sulfur.

[0083] In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 2 oxygen atoms. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 2 nitrogen atoms. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 2 sulfur atoms. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 oxygen and 1 nitrogen. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 oxygen and 1 sulfur. In certain embodiments, two R ³ groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having 1 sulfur and 1 nitrogen.

[0084] Exemplary heteroatom rings formed by two R ³ on the same carbon atom are depicted below:

[0085] As defined generally above and herein, each R is independently hydrogen or Ci_ 4 aliphatic, or :

two R groups on the same nitrogen atom are taken together to form a 4-8 membered saturated or partially unsaturated ring.

[0086] In certain embodiments, each R is independently hydrogen. [0087] In certain embodiments, each R is C _1-4 independently aliphatic. In certain embodiments, each R is independently straight or branched C _1-4 alkyl. In certain embodiments, each R is independently straight or branched C _1-3 alkyl. In certain embodiments, each R is independently straight or branched butyl. In certain embodiments, each R is independently straight or branched propyl. In certain embodiments, each R is ethyl. In certain embodiments, each R is methyl.

[0088] In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-8 membered saturated or partially unsaturated ring.

[0089] In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-8 membered saturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-7 membered saturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-6 membered saturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-5 membered saturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form azocanyl, azepanyl, piperidinyl, pyrrolidinyl, azetidinyl, or aziridinyl.

[0090] In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-8 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-7 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-6 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4-5 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 4 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 5 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 6 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form a 7 membered partially unsaturated ring. In certain embodiments, two R groups on the same nitrogen atom are taken together to form an 8 membered partially unsaturated ring.

[0091] Exemplary R ³ groups are depicted below:

[0092] In certain embodiments, each R ¹ is independently 3-6 membered cycloalkyl optionally and independently substituted with 1-4 R ³ groups wherein each R ³ is independently as defined above and described herein.

[0093] In certain embodiments, each R ¹ is independently 3-6 membered cycloalkyl independently substituted with 1-4 R ³ groups wherein each R ³ is independently as defined above and described herein.

[0094] In certain embodiments, each R ¹ is independently 3-6 membered cycloalkyl. In certain embodiments, each R ¹ is independently 3-5 membered cycloalkyl. In certain embodiments, each R ¹ is independently 3-4 membered cycloalkyl.

[0095] In certain embodiments, each R ¹ is independently cyclohexyl. In certain embodiments, each R ¹ is independently cyclopentyl. In certain embodiments, each R ¹ is independently cyclobutyl. In certain embodiments, each R ¹ is independently cyclopropyl.

[0096] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[0097] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 3-5 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 3-4 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 6 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 5 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 4 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 3 membered saturated heterocyclyl having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[0098] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 heteroatom independently selected from oxygen, nitrogen, or sulfur.

[0099] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 oxygen. In certain embodiments, each R ¹ is independently 3-5 membered saturated heterocyclyl having 1 oxygen. In certain embodiments, each R ¹ is independently 3-4 membered saturated heterocyclyl having 1 oxygen. In certain embodiments, each R ¹ is independently tetrahydro-2H-pyranyl, tetrahydrofuranyl, or oxetanyl.

[00100] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 nitrogen. In certain embodiments, each R ¹ is independently 3-5 membered saturated heterocyclyl having 1 nitrogen. In certain embodiments, each R ¹ is independently 3-4 membered saturated heterocyclyl having 1 nitrogen. In certain embodiments, each R ¹ is independently piperidinyl, pyrrolidinyl, azetidinyl, or aziridinyl.

[00101] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 sulfur. In certain embodiments, each R ¹ is independently 3-5 membered saturated heterocyclyl having 1 sulfur.

[00102] In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 2 oxygen atoms. In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 2 nitrogen atoms. In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 2 sulfur atoms. In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 oxygen and 1 sulfur. In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 oxygen and 1 nitrogen. . In certain embodiments, each R ¹ is independently 3-6 membered saturated heterocyclyl having 1 sulfur and 1 nitrogen.

[00103] Exemplary R ¹ groups are depicted below:

[00104] In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-7 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached.

[00105] In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-7 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-6 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-5 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-4 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 4 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 5 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 6 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 7 membered heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached.

[00106] In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-7 membered heterocyclic ring having 0 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-7 membered heterocyclic ring having 1 heteroatom independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon adjacent to R ¹ are taken together to form a 3-7 membered heterocyclic ring having 2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached.

[00107] In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-7 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-6 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-5 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 5 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 6 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-7 membered bridged heterocyclic ring having 0-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached.

[00108] In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-7 membered bridged heterocyclic ring having 0 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-7 membered bridged heterocyclic ring having 1 heteroatom independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached. In certain embodiments, R ¹ and an R ² group on a carbon non-adjacent to R ¹ are taken together with their intervening atoms to form a 4-7 membered bridged heterocyclic ring having 2 heteroatoms independently selected from oxygen, nitrogen, or sulfur in addition to the nitrogen atom where R ¹ is attached.

[00109] Exemplary Ring A groups, wherein R ¹ and R ² are taken together to form a ring, are depicted below:

[00110] As defined generally above and herein, each R ² is independently R, deuterium, - OR, oxo, or:

two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused 3-7 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur; or:

two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-7 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur; or:

two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged saturated carbocyclic or a 4-7 membered bridged heterocyclic ring having 1 -2 heteroatoms independently selected from oxygen, nitrogen, or sulfur;

[00111] In certain embodiments, each R ² is independently R wherein each R is independently as defined above and described herein.

[00112] In certain embodiments, each R ² is hydrogen.

[00113] In certain embodiments, each R ² is independently C _1-3 alkyl. In certain embodiments, each R ² is independently methyl. In certain embodiments, each R ² is independently ethyl. In certain embodiments, each R ² is independently w-propyl. In certain embodiments, each R ² is independently isopropyl.

[00114] In certain embodiments, each R ² is deuterium.

[00115] In certain embodiments, each R ³ is independently -OR wherein each R is independently as defined above and described herein. [00116] In certain embodiments, each R ³ is -OH.

[00117] In certain embodiments, each R ² is oxo.

[00118] In certain embodiments, two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused 3-7 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[00119] In certain embodiments, two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused 3-7 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused 3-6 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused 3-5 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on the same carbon are taken together to form an optionally substituted spiro- fused 3-4 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on the same carbon are taken together to form an optionally substituted spiro-fused cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl ring.

[00120] In certain embodiments, two R ² groups on the same carbon are taken together to form a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on the same carbon are taken together to form a 3-6 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on the same carbon are taken together to form a 3-5 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on the same carbon are taken together to form a 3-4 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[00121] In certain embodiments, two R ² groups on the same carbon atom are taken together to form a 3-7 membered heterocyclic ring having one heteroatom independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on the same carbon atom are taken together to form a 3-6 membered heterocyclic ring having one heteroatom independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on the same carbon atom are taken together to form a 3-5 membered heterocyclic ring having one heteroatom independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on the same carbon atom are taken together to form a 3-4 membered heterocyclic ring having one heteroatom independently selected from oxygen, nitrogen, or sulfur.

[00122] In certain embodiments, two R ² groups on the same carbon are taken together to form an aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or piperazinyl ring.

[00123] In certain embodiments, two R ² groups on the same carbon are taken together to form an oxiranyl, oxetanyl, tetrahydrofuranyl or tetrahydro-2H-pyranyl ring.

[00124] In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-7 membered saturated carbocyclic or a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[00125] In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-7 membered saturated carbocyclic ring. In certain embodiments , two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-6 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-5 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted 3-4 membered saturated carbocyclic ring. In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl ring.

[00126] In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form a 3-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments , two R ² groups on adjacent carbon atoms are taken together to form a 3-6 membered heterocyclic ring having 1- 2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments , two R ² groups on adjacent carbon atoms are taken together to form a 3-5 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments , two R ² groups on adjacent carbon atoms are taken together to form a 3-4 membered heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. [00127] In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl or piperazinyl ring.

[00128] In certain embodiments, two R ² groups on adjacent carbon atoms are taken together to form an oxiranyl, oxetanyl, tetrahydrofuranyl or tetrahydro-2H-pyranyl ring.

[00129] In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged saturated carbocyclic or a 4-7 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[00130] In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted 4-7 membered bridged saturated carbocyclic ring. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted 4-6 membered bridged saturated carbocyclic ring. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted 4-5 membered bridged saturated carbocyclic ring. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form an optionally substituted bridged cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl ring.

[00131] In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 4-7 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 4-6 membered bridged heterocyclic ring having 1 -2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 4-5 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 4 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 5 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 6 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together with their intervening atoms to form a 7 membered bridged heterocyclic ring having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur.

[00132] In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together to form a bridged azetidinyl, pyrrolidinyl, piperidinyl or piperazinyl ring.

[00133] In certain embodiments, two R ² groups on non-adjacent carbon atoms are taken together to form a bridged oxetanyl, tetrahydrofuranyl or tetrahydro-2H-pyranyl ring.

[00134] Exemplary Ring A groups are depicted below:

[00135] As defined generally above, L is a covalent bond, or a straight or branched C _1-5 saturated or unsaturated, straight or branched, divalent hydrocarbon chain. In certain embodiments, L is a covalent bond, or a straight or branched 0 _1-5 alkylene chain.

[00136] In certain embodiments, L is a covalent bond and Ring A is selected from: wherein each of m, R ¹ , and R ² is as defined above and described herein.

[00137] In certain embodiments, L is a covalent bond and the present invention provides a compound of formul

II

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00138] In certain embodiments, the present invention provides a compound of formula II having the stereochemistry depicted in formula Il-a, below:

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00139] In certain embodiments, L is a covalent bond, or a straight or branched C1-5 saturated or unsaturated, straight or branched, divalent hydrocarbon chain, and Ring A is selected from:

wherein each of m, R ¹ , and R ² is as defined above and described herein.

[00140] In certain embodiments, L is a straight or branched C1-5 saturated or unsaturated, straight or branched, divalent hydrocarbon chain. In some embodiments, L is a straight or branched C1-5 alkylene chain. In certain embodiments, L is a straight or branched C1-4 alkvlene chain. In certain embodiments, L is a straight or branched C1-3 alkylene chain. In certain embodiments, L is a straight or branched C _1-2 alkylene chain. In certain embodiments, L is a straight or branched pentylene. In certain embodiments, L is a straight or branched butylene. In certain embodiments, L is a straight or branched propylene. In certain embodiments, L is a straight or branched ethylene. In certain embodiments, L is methylene.

[00141] In certain embodiments, the present invention provides a compound of formula III:

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00142] In certain embodiments, the present invention provides a compound of formula III having the stereochemistry depicted in formula Ill-a, below:

Ill-a

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00143] In certain embodiments, the present invention provides a compound of formula IV:

IV

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00144] In certain embodiments, the present invention provides a compound of formula IV having the stereochemistry depicted in formula IV-a, below:

IV-a

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00145] In certain embodiments, the present invention provides a compound of formula IV having the stereochemistry depicted in formula IV-b, below:

IV-b

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00146] In certain embodiments, the present invention provides a compound of formula V:

V or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00147] In certain embodiments, the present invention provides a compound of formula V having the stereochemistry depicted in formula V-a, below:

V-a

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00148] In certain embodiments, the present invention provides a compound of formula VI:

VI

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00149] In certain embodiments, the present invention provides a compound of formula VI having the stereochemistry depicted in formula Vl-a, below:

or a pharmaceutically acceptable salt thereof, wherein each variable is defined above and in classes and subclasses described above and herein.

[00150] Exemplary compounds of formula I are set forth in Table 1, below. Table 1. Exemplary Compounds

1-9 1-10

1-13 1-14

4. General Methods of Providing the Present Compounds

[00151] The compounds of this invention may be prepared or isolated in general by synthetic and/or semi-synthetic methods known to those skilled in the art for analogous compounds and by methods described in detail in the Examples, herein. Methods and intermediates of the present invention are useful for preparing compounds as described in, e.g. United States patent application serial number 13/040, 166, filed March 3, 2011, in the name of Bronk et al, the entirety of which is incorporated herein by reference.

[00152] In the Schemes below, where a particular protecting group, leaving group, or transformation condition is depicted, one of ordinary skill in the art will appreciate that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. Such groups and transformations are described in detail in March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, M. B. Smith and J. March, 5 ^th Edition, John Wiley & Sons, 2001, Comprehensive Organic Transformations, R. C. Larock, 2 ^nd Edition, John Wiley & Sons, 1999, and Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 ^rd edition, John Wiley & Sons, 1999, the entirety of each of which is hereby incorporated herein by reference.

[00153] As used herein, the phrase "oxygen protecting group" includes, for example, carbonyl protecting groups, hydroxyl protecting groups, etc. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 ^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2- trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta- (trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2- and 4-picolyl.

[00154] Amino protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 ^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable amino protecting groups include, but are not limited to, aralkylamines, carbamates, cyclic imides, allyl amines, amides, and the like. Examples of such groups include t- butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxocarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, and the like. In certain embodiments, the amino protecting group of the R ¹⁰ moiety is phthalimido. In still other embodiments, the amino protecting group of the R ¹⁰ moiety is a tert-butyloxycarbonyl (BOC) group. In certain embodiments, the amino protecting group is a sulphone (SO ₂ R).

[00155] Each of Ring A, R ¹ , R ² , R ³ and L in the below Schemes is as defined above and described in classes and subclasses above and herein.

Isolation of Material from Biomass [00156] Certain compounds used in methods of the present invention are isolated from black cohosh root, also known as cimicifuga racemosa or actaea racemosa. Commercial extracts, powders, and capsules of black cohosh root are available for treating a variety of menopausal and gynecological disorders. However, it has been surprisingly found that certain compounds present in black cohosh root are useful for modulating and/or inhibiting amyloid-beta peptide production. In particular, certain compounds have been isolated from black cohosh root and identified, wherein these compounds are useful as syntheteic precursors en route to compounds useful for modulating and/or inhibiting amyloid-beta peptide production, and in particular amyloid-beta peptide (1-42). These compounds may be isolated and utilized in a form substantially free of other compounds normally found in the root.

[00157] In some embodiments, methods of the present invention for use in preparing a compound of formula II use compounds found in extracts of black cohosh and related cimicifuga species, whether from roots and rhizome or aerial parts of these plants. One of ordinary skill in the art will recognize that synthetic precursors may be obtained from one or more cimicifuga species including, but not limited to, Cimicifuga racemosa, Cimicifuga dahurica, Cimicifuga foetida, Cimicifuga heracleifolia, Cimicifuga japonica, Cimicifuga acerina, Cimicifuga acerima, Cimicifuga simplex, and Cimicifuga elata, Cimicifuga calthaefolia, Cimicifuga frigida, Cimicifuga laciniata, Cimicifuga mairei, Cimicifuga rubifolia, Cimicifuga americana, Cimicifuga biternata, and Cimicifuga bifida or a variety thereof. This may be accomplished either by chemical or biological transformation of an isolated compound or an extract fraction or mixture of compounds. Chemical transformation may be accomplished by, but not limited to, manipulation of temperature, pH, and/or treatment with various solvents. Biological transformation may be accomplished by, but not limited to, treatment of an isolated compound or an extract fraction or mixture of compounds with plant tissue, plant tissue extracts, other microbiological organisms or an isolated enzyme from any organism.

[00158] In some embodiments, a precursor compound is extracted from a sample of biomass to provide a compound of formula A, as depicted in Scheme I below. Scheme I.

Biomass

[00159] The term "biomass," as used herein, refers to roots, rhizomes and/or aerial parts of the cimicifuga species of plant, as described above and herein.

[00160] In some embodiments, the process of obtaining a compound of formula A from biomass comprises a step of pre-treating the biomass. In some embodiments, the step of pretreating comprises a step of drying. In certain embodiments, the step of drying comprises use of one or more suitable methods for providing biomass of a desired level of dryness. For instance, in some embodiments the biomass is dried using vacuum. In some embodiments, the biomass is dried using heat. In some embodiments, the biomass is dried using a spray dryer or drum dryer. In some embodiments, the biomass is dried using two or more of the above methods.

[00161] In some embodiments, the step of pretreating comprises a step of grinding. In certain embodiments, the step of grinding comprises passing the sample of biomass through a chipper or grinding mill for an amount of time suitable to provide biomass of a desired particle size. In some embodiments, the biomass is dried prior to being ground to a suitable particle size.

[00162] In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.2 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.3 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.4 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.5 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.6 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.7 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.8 mm ³ to about 1.0 mm ³ . In some embodiments, a suitable particle size ranges from about 0.9 mm ³ to about 1.0 mm ³ . [00163] In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.9 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.8 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.7 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.6 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.5 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.4 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.3 mm ³ . In some embodiments, a suitable particle size ranges from about 0.1 mm ³ to about 0.2 mm ³ .

[00164] In some embodiments, biomass is dried and ground prior to being extracted. The term "extraction," as used herein, refers to the general process of obtaining a compound of formula A comprising a step of exposing biomass to one or more suitable solvents under suitable conditions for a suitable amount of time in order to extract a compound of formula A from the biomass. In some embodiments, extraction comprises agitating and heating a slurry comprised of biomass and one or more suitable solvents. In certain embodiments, the one or more suitable solvents comprise one or more alcohols, and optionally water. Suitable alcohols include, but are not limited to, methanol, ethanol, isopropanol, and the like. In certain embodiments, the alcohol is methanol. In certain embodiments, the alcohol is ethanol. In some embodiments, the slurry is heated to a temperature of about 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, and 70 °C. In some embodiments, an elevated temperature is a temperature of greater than about 70 °C. In certain embodiments, the slurry is heated to about 50 °C. In certain embodiments, the slurry is kept at ambient temperature.

[00165] In some embodiments, the biomass is exposed to one or more suitable solvents under suitable conditions for an amount of time ranging from about 0.1 h to about 48 h. In some embodiments, the amount of time ranges from about 0.1 h to about 36 h. In some embodiments, the amount of time ranges from about 0.1 h to about 24 h. In some embodiments, the amount of time ranges from about 0.5 h to about 24 h. In some embodiments, the amount of time ranges from about 1 h to about 24 h. In some embodiments, the amount of time ranges from about 2 h to about 24 h. In some embodiments, the amount of time ranges from about 2 h to about 22 h. In some embodiments, the amount of time ranges from about 2 h to about 20 h. In some embodiments, the amount of time ranges from about 2 h to about 4 h. In some embodiments, the amount of time ranges from about 20 h to about 24 h. In some embodiments, the amount of time is about 2 h. In some embodiments, the amount of time is about 22 h.

[00166] In some embodiments, once the slurry of biomass is heated and/or agitated for a suitable amount of time, the slurry is filtered through e.g., Celite, and concentrated down to the crude extract. In certain embodiments, the crude extract is further treated with an aqueous salt solution such as, e.g., 5% aqueous KCl, and cooled to a temperature of about 2°C to about 10 °C. Exemplary other salts for use in an aqueous salt solution include, but are not limited to, (NH ₄ )S0 ₄ , K ₂ S0 ₄ , NaCl, etc. In some embodiments, the aqueous salt solution has a concentration ranging from about 1% to about 50 %. In some embodiments, the aqueous salt solution has a concentration ranging from about 3% to about 30%. In some embodiments, the aqueous salt solution has a concentration ranging from about 5% to about 10%. In some embodiments, the aqueous salt solution has a concentration ranging from about 10% to about 20%. In some embodiments, the aqueous salt solution has a concentration ranging from about 20% to about 30%. In certain embodiments, the crude extract is cooled to a temperature of about 2 °C to about 6 °C. In certain embodiments, the crude extract is cooled to a temperature of about 4 °C. In some embodiments, the crude extract is cooled for about 1, 2, 3, 4, or 5 h. In certain embodiments, the crude extract is cooled for about 2 h. In some embodiments, the crude extract is cooled for more than about 5 h. In certain embodiments, the crude extract is cooled for about 5 h to about 10 h. In certain embodiments, the crude extract is cooled for about 10 h to about 15 h. In certain embodiments, the crude extract is cooled for about 15 h to about 20 h. In certain embodiments, the crude extract is cooled for about 20 h to about 25 h. In some embodiments, after the crude extract is cooled for an appropriate amount of time, the slurry is centrifuged and the resulting solids are collected and dried using any one or more methods known in the art.

[00167] In some embodiments, step S-l provides compound A in about 3-15% purity. General method for preparing compounds of formula I

[00168] In some embodiments, compound A serves as starting material in the synthesis of a compound of formula I, as illustrated in Scheme II, below. [00169] As depicted in step S-2 of Scheme II, a compound of formula A is converted by dehydration to provide carbonyl compound B, which, in step S-3 is oxidatively cleaved at the polyol moiety to afford dialdehyde C.

Scheme II

[00170] In some embodiments, the reductive amination of dialdehyde C in step S-4 provides D, wherein each variable is defined above and described in classes and subclasses above and herein, as illustrated in Scheme III, below. In step S-5, the carbonyl group of formula D generated in S-2 is reduced to the corresponding hydroxyl group to provide E, wherein each variable is defined above and described in classes and subclasses above and herein, followed by deprotection in step S-6 to provide formula I-a, wherein each variable is defined above and described in classes and subclasses above and herein.

Sc

[00171] In some embodiments, the reductive amination of dialdehyde C in step S-4 provides morpholine D-i, as illustrated in Scheme IV, below. In step S-5, the carbonyl group of D-i generated in S-2 is reduced to the corresponding hydroxyl group to provide alcohol E- i, which is then deprotected in step S-6 to afford free amine F. The reductive amination of amine F with appropriate aldehydes and ketones provides compounds of formula I-a, wherein each variable is defined above and described in classes and subclasses above and herein.

S

Description of synthetic steps

[00172] As depicted in step S-2 above, dehydration of A under suitable conditions provides carbonyl compound B. In some embodiments, the process is catalyzed by a Lewis acid.

[00173] As depicted in step S-3 above, the polyol of compound B is oxidatively cleaved upon exposure to a suitable oxidant to afford dialdehyde C. In some embodiments, a suitable oxidant is a hypervalent iodide. In certain embodiments, the oxidant is sodium periodate and the solvent is a mixture of an organic solvent and an aqueous solvent. In some embodiments, the organic solvent is an ethereal solvent such as a tetrahydrofuran or a dialkyl ether. In some embodiments, the solvent mixture comprises an ethereal solvent and water in a v/v ratio of 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5. In certain embodiments, the solvent mixture comprises THF and water in a v/v ratio of 3 : 1. In some embodiments, suitable conditions for cleaving the polyol include heating the reaction for a suitable amount of time until TLC analysis indicates that the reaction is complete. In some embodiments, the reaction is run at ambient temperature. In some embodiments, the reaction is heated to about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, or 70 °C. In certain embodiments, the reaction is heated to about 50 °C. In some embodiments the reaction is heated for about 10 hours to about 20 hours. In certain embodiments the reaction is heated for about 15-17 hours.

[00174] As depicted in step S-4 above, dialdehyde C undergoes reductive amination in the presence of a suitable amine salt to provide a compound of formula D (see Scheme III above) or D-i (see Scheme IV above). One of skill in the art will appreciate that the structure of the product of the reaction will be dictated by the structure of the amine salt reagent selected. For instance, in some embodiments, the amine salt is of the general formula shown below:

PG ¹ ^-^

wherein each variable is defined above and described in classes and subclasses above and herein. PG ¹ is any suitable protecting group, and the product of step S-4 is a compound of formula D. In some embodiments, the amine salt is of the general formula shown below:

PG ¹ — NH ₃ ⁺ C

and the product of step S-4 is a compound of formula D-i. In some embodiments, the PG ¹ is a Boc or benzyl protecting group and the compound is of the formula D-i. In some embodiments, the PG ¹ is a BOC protecting group. In some embodiments, the PG ¹ is a benzyl protecting group.

[00175] In some embodiments, the amine salt of step S-4 is commercially available. In some embodiments, the amine salt of step S-4 is generated immediately prior to the reductive amination reaction taking place. For instance, in certain embodiments the amine salt is generated by dissolving an amine in a suitable solvent and adding said solvent to an aqueous solution of a desired acid (e.g., aqueous HC1) which, upon removal of the solvent mixture, affords the corresponding amine HC1 salt for use in the reductive amination. In certain embodiments, a suitable solvent for generation of the amine is an alcoholic solvent such as ethanol. In certain embodiments, a suitable solvent for generation of the amine is a mixture of two or more alcoholic solvents, such as ethanol, methanol, and isopropanol. In some embodiments, the mixture is stirred for an amount of time and the solvent is removed at ambient temperature to provide the desired amine salt. In some embodiments, the solvent is removed at elevated temperatures to provide the desired amine salt. Methods of making amine salts are known in the chemical arts and described herein in the Exemplification. [00176] In some embodiments, a reaction solvent for use in the reductive amination of step S-4 is a polar protic solvent. In certain embodiments, the polar protic solvent is an alcoholic solvent such as ethanol. In some embodiments, dialdehyde C is premixed with the amine salt in the presence of an acid. In certain embodiments, the acid is acetic acid and the mixture is stirred for about 10, 15, 20, 25, or 30 minutes prior to addition of the reducing agent. In some embodiments, the reducing agent is a borohydride reducing agent such as, e.g., NaBH(OAc)3 and is used in molar excess with respect to the amount of amine salt present. In some embodiments, the reaction is allowed to proceed for 1, 2, 3, 4, or 5 hours, or until TLC or HPLC-MS analysis indicates completion. In some embodiments, upon reaction completion the product is treated to remove residual acid (e.g., via a toluene azeotrope), dried under vacuum, and carried on without further purification.

[00177] As depicted in step S-5 above, the carbonyl moiety of D or D-i is reduced upon exposure to sodium borohydride to afford alcohol E or E-i, respectively. In certain embodiments, sodium borohydride is premixed in a suitable solvent until at least partially dissolved. Exemplary such solvents include polar protic solvents (e.g., ethanol). In some embodiments, D or D-i is dissolved separately in a polar aprotic solvent such as ethyl acetate and added to the sodium borohydride reaction mixture over a period of time. In certain embodiments, D or D-i is added over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In certain embodiments, the reaction is run at ambient temperature for an amount of time of about 5, 10, 15, 20, 25, 30, 35, 40 or 45 minutes. In some embodiments, the reaction is quenched with an acid (e.g., acetic acid) and the product is treated to remove residual acid (via e.g., a toluene azeotrope), dried under vacuum, and purified before being used in the next step.

[00178] In some embodiments, the deprotection step of S-6 occurs through catalytic hydrogenation. In some embodiments, D or D-i is dissolved in a polar solvent. In some embodiments, D or D-i is dissolved in a polar protic solvent and exposed to an acid under conditions suitable to remove the protecting groups. In certain embodiments, the polar protic solvent is an alcoholic solvent (e.g., methanol) and the acid is Bronsted acid such as aqueous HC1. In some embodiments, the reaction occurs at elevated temperatures of about 30, 40, 50, or 60 °C. In certain embodiments, the reaction occurs at 50 °C.

V. Uses, Formulation and Administration

Pharmaceutically acceptable compositions [00179] According to another aspect of the present invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise any of the compounds as described herein, and optionally comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

[00180] It will also be appreciated that certain of the compounds of present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable salt thereof.

[00181] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt or salt of an ester of a compound of this invention that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or a pharmaceutically active metabolite or residue thereof. As used herein, the term "pharmaceutically active metabolite or residue thereof means that a metabolite or residue thereof is also a pharmaceutically active compound in accordance with the present invention.

[00182] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et ah, describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (Ci_4 alkyl) ₄ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersable products may be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[00183] In some cases, compounds of the present invention may contain one or more acidic functional groups and, thus, may be capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. See, for example, Berge et ah, supra.

[00184] The compositions of the present invention may additionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[00185] The compositions provided by the present invention can be employed in combination therapies, meaning that the present compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutic agents or medical procedures. The particular combination of therapies (therapeutic agents or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutic agents and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a compound described herein may be administered concurrently with another therapeutic agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects).

[00186] For example, known agents useful for treating neurodegenerative disorders may be combined with the compositions of this invention to treat neurodegenerative disorders, such as Alzheimer's disease. Examples of such known agents useful for treating neurodegenerative disorders include, but are not limited to, treatments for Alzheimer's disease such as acetylcholinesterase inhibitors, including donepezil, Exelon ^® and others; memantine (and related compounds as NMDA inhibitors), treatments for Parkinson's disease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine, pergolide, trihexephendyl, and amantadine; agents for treating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex ^® and Rebif ^® ), Copaxone ^® , and mitoxantrone; riluzole, and antiparkinsonian agents. For a more comprehensive discussion of updated therapies useful for treating neurodegenerative disorders, see, a list of the FDA approved drugs at http://www.fda.gov, and The Merck Manual, Seventeenth Ed. 1999, the entire contents of which are hereby incorporated by reference.

[00187] Additional examples of such known agents useful for treating neurodegenerative disorders include, but are not limited to, beta-secretase inhibitors/modulators, gamma- secretase inhibitors/modulators, HMG-CoA reductase inhibitors, NSAID's including ibuprofen, vitamin E, anti-amyloid antibodies, including humanized monoclonal antibodies, inhibitors/modulators of tau phosphorylation (such as GSK3 or CDK inhibitors/modulators) and/or aggregation, CB-1 receptor antagonists or CB-1 receptor inverse agonists, antibiotics such as doxycycline and rifampin, N-methyl-D-aspartate (NMDA) receptor antagonists, such as mematine, cholinesterase inhibitors such as galantamine, rivastigmnine, donepezil and tacrine, growth hormone secretagogues such as ibutamoren, ibutamoren mesylate and capromorelin, histamine H3 antagonists, AMPA agonists, PDE -IV, -V, -VII, -VIII, and -IX inhibitors, GABAA inverse agonists, and neuronal nicotinic agonists and partial agonists, serotonin receptor antagonists.

[00188] In other embodiments, the compounds of the present invention are combined with other agents useful for treating neurodegenerative disorders, such as Alzheimer's disease, wherein such agents include beta-secretase inhibitors/modulators, gamma-secretase inhibitors/modulators, anti-amyloid antibodies, including humanized monoclonal antibodies aggregation inhibitors, metal chelators, antioxidants, and neuroprotectants and inhibitors/modulators of tau phosphorylation (such as GSK3 or CDK inhibitors/modulators) and/or aggregation.

[00189] In some embodiments, compounds of the present invention are combined with gamma secretase modulators. In some embodiments, compounds of the present invention are gamma secretase modulators combined with gamma secretase modulators. Exemplary such gamma secretase modulators include, inter alia, certain NSAIDs and their analogs (see WO01/78721 and US 2002/0128319 and Weggen et al, Nature, 414 (2001) 212-16; Morihara et al, J. Neurochem., 83 (2002), 1009-12; and Takahashi et al, J. Biol. Chem., 278 (2003), 18644-70).

[00190] As used herein, the term "combination," "combined," and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this invention. For example, a compound of the present invention may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising a provided compound, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[00191] Other examples of agents the compounds of this invention may also be combined with include, without limitation: treatments for asthma such as albuterol and Singulair ^® ; agents for treating schizophrenia such as zyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agents such as corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents such as cyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids, cyclophosphamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anti-convulsants, ion channel blockers, agents for treating cardiovascular disease such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, and statins; agents for treating liver disease such as corticosteroids, cholestyramine, interferons, and antiviral agents; agents for treating blood disorders such as corticosteroids, anti-leukemic agents, and growth factors; and agents for treating immunodeficiency disorders such as gamma globulin.

[00192] The amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. In certain embodiments, the amount of additional therapeutic agent in the present compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

[00193] In an alternate embodiment, the methods of this invention that utilize compositions that do not contain an additional therapeutic agent, comprise the additional step of separately administering to said patient an additional therapeutic agent. When these additional therapeutic agents are administered separately they may be administered to the patient prior to, sequentially with or following administration of the compositions of this invention.

[00194] The pharmaceutically acceptable compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, as an oral or nasal spray, or the like, depending on the severity of the disorder being treated. In certain embodiments, the compounds of the invention may be administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

[00195] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[00196] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[00197] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[00198] In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[00199] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[00200] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with one or more inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar— agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[00201] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

[00202] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with one or more inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[00203] Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[00204] In some embodiments, the present invention provides a composition containing a provided compound in an amount of about 1 weight percent to about 99 weight percent. In other embodiments, the composition contains a provided compound wherein the composition contains no more than about 10.0 area percent HPLC of other components of black cohosh root relative to the total area of the HPLC chromatogram. In other embodiments, the composition containing a provided compound contains no more than about 8.0 area percent HPLC of other components of black cohosh root relative to the total area of the HPLC chromatogram, and in still other embodiments, no more than about 3 area percent.

Uses of Compounds and Pharmaceutically Acceptable Compositions

[00205] Alzheimer's Disease (AD) is believed to result from the deposition of quantities of a peptide, amyloid-beta ("A-beta"), within the brain. This peptide is produced by enzymatic cleavage of amyloid protein precursor ("APP") protein. The C-terminus of A-beta is generated by an enzyme termed gamma-secretase. Cleavage occurs at more than one site on APP producing different length A-beta peptides, some of which are more prone to deposition, such as A-beta 42. It is believed that aberrant production A-beta 42 in the brain leads to AD.

A-beta, a 37-43 amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP), is the major component of amyloid plaques. APP is expressed and constitutively catabolized in most cells. APP has a short half-life and is metabolized rapidly down two pathways. In one pathway, cleavage by an enzyme known as alpha-secretase occurs while APP is still in the trans-Golgi secretory compartment. This cleavage by alpha- secretase occurs within the A-beta portion of APP, thus precluding the formation of A-beta.

[00206] In contrast to this non-amyloidogenic pathway involving alpha-secretase described above, proteolytic processing of APP by beta-secretase exposes the N-terminus of A-beta, which after gamma-secretase cleavage at the variable C-terminus, liberates A-beta. Peptides of 40 or 42 amino acids in length (A-beta 1-40 and A-beta 1-42, respectively) predominate among the C-termini generated by gamma-secretase, however, a recent report suggests 1-38 is a dominant species in cerebrospinal fluid. A-beta 1-42 is more prone to aggregation than A-beta 1-40, the major component of amyloid plaque, and its production is closely associated with the development of Alzheimer's disease. The bond cleaved by gamma-secretase appears to be situated within the transmembrane domain of APP. In the amyloidogenic pathway, APP is cleaved by beta-secretase to liberate sAPP-beta and CTF- beta, which CTF-beta is then cleaved by gamma-secretase to liberate the harmful A-beta peptide.

[00207] While abundant evidence suggests that extracellular accumulation and deposition of A-beta is a central event in the etiology of AD, recent studies have also proposed that increased intracellular accumulation of A-beta or amyloid containing C- terminal fragments may play a role in the pathophysiology of AD. For example, over- expression of APP harboring mutations which cause familial Alzheimer's disease (AD) results in the increased intracellular accumulation of CTF-beta in neuronal cultures and A- beta 42 in HEK 293 cells.

[00208] A-beta 42 is the 42 amino acid long form of A-beta that is believed to be more potent in forming amyloid plaques than the shorter forms of A-beta.. Moreover, evidence suggests that intra- and extracellular A-beta are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial AD mutations in APP ("Swedish" and "London") is an increased intracellular accumulation of A- beta 42.

[00209] Without wishing to be bound by theory, it is believed that of importance in this A-beta-producing pathway is the position of the gamma-secretase cleavage. If the gamma- secretase proteolytic cut is at residue or before 711-712, shorter A-beta. (A-beta 40 or shorter) is the result; if it is a proteolytic cut after residue 713, long A-beta (A-beta 42) is the result. Thus, the gamma secretase process is central to the production of A-beta peptide of 40 or 42 amino acids in length (A-beta 40 and A-beta 42, respectively). For a review that discusses APP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447-453; Selkoe, 1994, Ann. Rev. Cell Biol. 10:373-403. See also, Esch et al, 1994, Science 248: 1122.

[00210] Cleavage of APP can be detected in a number of convenient manners, including the detection of polypeptide or peptide fragments produced by proteolysis. Such fragments can be detected by any convenient means, such as by antibody binding. Another convenient method for detecting proteolytic cleavage is through the use of a chromogenic .beta, secretase substrate whereby cleavage of the substrate releases a chromogen, e.g., a colored or fluorescent, product. More detailed analyses can be performed including mass spectroscopy.

[00211] Much interest has focused on the possibility of inhibiting the development of amyloid plaques as a means of preventing or ameliorating the symptoms of Alzheimer's disease. To that end, a promising strategy is to inhibit the activity of beta- and/or gamma- secretase, the two enzymes that together are responsible for producing A-beta. This strategy is attractive because, if amyloid plaque formation as a result of A-beta deposition is a cause of Alzheimer's disease, then inhibiting the activity of one or both of the two secretases would intervene in the disease process at an early stage, before late-stage events such as inflammation or apoptosis occur.

[00212] Modulators of gamma-secretase may function in a variety of ways. They may block gamma-secretase completely, or they may alter the activity of the enzyme so that less A-beta 42 and more of the alternative, soluble, forms of A-beta., such as A-beta 37, 38 or 39 are produced. Such modulators may thereby retard or reverse the development of AD.

[00213] Compounds are known, such as indomethacin, ibuprofen and sulindac sulphide, which inhibit the production of A-beta 42 while increasing the production of A-beta 38 and leaving the production of A-beta 40 constant.

[00214] In some embodiments, compounds of the present invention are useful gamma- secretase modulators. In some embodiments, compounds of the present invention modulate the action of gamma-secretase such that amyloid-beta (1-42) peptide production in a patient is attenuated. In certain embodiments, compounds of the present invention modulate the action of gamma-secretase so as to selectively attentuate amyloid-beta (1-42) peptide production in a patient. In some embodiments, such selective attenuation occurs without significantly lowering production of the total pool of Abeta, or the specific shorter chain isoformamyloid-beta (1-40) peptide. In some embodiments, such selective attenuation results in secretion of amyloid beta which has less tendency to self-aggregate and form insoluble deposits, is more easily cleared from the brain, and/or is less neurotoxic. In some embodiments, the ability of compounds of the present invention to modulate gamma- secretase is beneficial in that there is a reduced risk of side effects with treatment resulting from, e.g., minimal disruption of other gamma-secretase controlled signaling pathways.

[00215] In some embodiments, compounds of the present invention are gamma-secretase modulators useful for treating a patient suffering from AD, cerebral amyloid angiopathy, HCHWA-D, multi-infarct dementia, dementia pugilistica or traumatic brain injury and/or Down syndrome.

[00216] In some embodiments, one or more compounds of the present invention are administered to a patient suffering from mild cognitive impairment or age-related cognitive decline or pre-symptomatic AD or prodromal or predementia AD (Dubois et al The Lancet Neurology 10 (2010) 70223-4 A favourable outcome of such treatment is prevention or delay of the onset of AD. Age-related cognitive decline and mild cognitive impairment (MCI) are conditions in which a memory deficit is present, but other diagnostic criteria for dementia are absent (Santacruz and Swagerty, American Family Physician, 63 (2001), 703-13). As used herein, "age-related cognitive decline" implies a decline of at least six months' duration in at least one of: memory and learning; attention and concentration; thinking; language; and visuospatial functioning and a score of more than one standard deviation below the norm on standardized neuropsychologic testing such as the MMSE.

[00217] In some embodiments, compounds of the present invention are useful for modulating and/or inhibiting amyloid-beta (1-42) peptide production in a patient. Accordingly, compounds of the present invention are useful for treating, or lessening the severity of, disorders associated with amyloid-beta (1-42) peptide production in a patient.

[00218] In some embodiments, the compounds of the present invention are useful for modulating and/or inhibiting amyloid-beta (1-40) peptide production in a patient. Accordingly, the compounds of the present invention are useful for treating, or lessening the severity of, disorders associated with amyloid-beta (1-40) peptide production in a patient. In some embodiments, compounds of the present invention do not modulate and/or inhibit amyloid-beta (1-40) peptide production in a patient. [00219] In some embodiments, the compounds of the present invention are useful for modulating and/or inhibiting amyloid-beta (1-38) peptide production in a patient. Accordingly, the compounds of the present invention are useful for treating, or lessening the severity of, disorders associated with amyloid-beta (1-38) peptide production in a patient.

[00220] In some embodiments, the compounds of the present invention are useful for reducing both amyloid-beta (1-42) and amyloid beta (1-38). In some embodiments, the compounds of the present invention are useful for reducing amyloid-beta (1-42) and raising amyloid beta (1-38).

[00221] The compounds, extracts, and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for treating or lessening the severity of a neurodegenerative disorder. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like.

[00222] In certain embodiments, the present invention provides a method for modulating and/or inhibiting amyloid-beta (1-42) peptide production in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition comprising said compound. In other embodiments, the present invention provides a method of selectively modulating and/or inhibiting amyloid-beta (1-42) peptide production in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In still other embodiments, the present invention provides a method of reducing amyloid-beta (1-42) peptide levels in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In other embodiments, the present invention provides a method for reducing amyloid-beta (1-42) peptide levels in a cell, comprising contacting said cell with a provided compound. Another embodiment provides a method for reducing amyloid-beta (1-42) in a cell without substantially reducing amyloid-beta (1-40) peptide levels in the cell, comprising contacting said cell with a provided compound. Yet another embodiment provides a method for reducing amyloid-beta (1-42) in a cell and increasing one or more of amyloid-beta (1-37) and amyloid-beta (1-39) in the cell, comprising contacting said cell with a provided compound. [00223] As used herein, the term "reducing" or "reduce" refers to the relative decrease in the amount of an amyloid-beta achieved by administering a provided compound as compared to the amount of that amyloid-beta in the absence of administering a provided compound. By way of example, a reduction of amyloid-beta (1-42) means that the amount of amyloid-beta (1-42) in the presence of a provided compound is lower than the amount of amyloid-beta (1- 42) in the absence of a provided compound.

[00224] In still other embodiments, the present invention provides a method for selectively reducing amyloid-beta (1-42) peptide levels in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In certain embodiments, the present invention provides a method for reducing amyloid-beta (1-42) peptide levels in a patient without substantially reducing amyloid-beta (1-40) peptide levels, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00225] In certain embodiments, the present invention provides a method for reducing amyloid-beta (1-42) peptide levels in a patient and increasing one or more of amyloid-beta (1-37) and amyloid-beta (1-39), wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00226] In certain embodiments, the present invention provides a method for reducing amyloid-beta (1-42) peptide levels in a patient and increasing amyloid-beta (1-38), wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In certain embodiments, the present invention provides a method for reducing amyloid-beta (1-42) peptide levels in a patient and decreasing amyloid-beta (1-38), wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00227] The term "increasing" or "increase," as used herein in reference to an amount of an amyloid-beta, refers to the relative rise in the amount of an amyloid-beta achieved by administering a provided compound (or contacting a cell with a provided compound) as compared to the amount of that amyloid-beta in the absence of administering a provided compound (or contacting a cell with a provided compound). By way of example, an increase of amyloid-beta (1-37) means that the amount of amyloid-beta (1-37) in the presence of a provided compound is higher than the amount of amyloid-beta (1-37) in the absence of a provided compound. For instance, the relative amounts of either of amyloid-beta (1-37) and amyloid-beta (1-39) can be increased either by an increased production of either of amyloid- beta (1-37) and amyloid-beta (1-39) or by a decreased production of longer amyloid-beta peptides, e.g., amyloid-beta (1-40) and/or amyloid-beta (1-42). In addition, it will be appreciated that the term "increasing" or "increase," as used herein in reference to an amount of an amyloid-beta, refers to the absolute rise in the amount of an amyloid-beta achieved by administering a provided compound. Thus, in certain embodiments, the present invention provides a method for increasing the absolute level of one or more of amyloid-beta (1-37) and amyloid-beta (1-39), wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In other embodiments, the present invention provides a method for increasing the level of one or more of amyloid-beta (1-37) and amyloid-beta (1-39), wherein the increase is relative to the amount of longer amyloid-beta peptides, e.g., amyloid-beta (1-40) and/or amyloid-beta (1- 42), or total amyloid-beta, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00228] One of ordinary skill in the art will appreciate that overall ratio of amyloid-beta peptides is significant where selective reduction of amyloid-beta (1-42) is especially advantageous. In certain embodiments, the present compounds reduce the overall ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide. Accordingly, another aspect of the present invention provides a method for reducing the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide in a patient, comprising administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In certain embodiments, the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide is reduced from a range of about 0.1 to about 0.4 to a range of about 0.05 to about 0.08.

[00229] In other embodiments, the present invention provides a method for reducing the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide in a cell, comprising contacting the cell with a provided compound. In certain embodiments, the ratio of amyloid- beta (1-42) peptide to amyloid-beta (1-40) peptide is reduced from a range of about 0.1 to about 0.4 to a range of about 0.05 to about 0.08.

[00230] According to one aspect, the present invention provides a method for treating or lessening the severity of a disorder associated with amyloid-beta (1-42) peptide, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. Such disorders include neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Down's syndrome.

[00231] Such disorders also include inclusion body myositis (deposition of A-beta in peripheral muscle, resulting in peripheral neuropathy), cerebral amyloid angiopathy (amyloid in the blood vessels in the brain), and mild cognitive impairment and pre-symptomatic, prodromal or predementia AD.

[00232] "High A-beta42" is a measurable condition that precedes symptomatic disease, especially in familial patients, based on plasma, CSF measurements, and/or genetic screening or brain imaging. This concept is analogous to the relationship between elevated cholesterol and heart disease. Thus, another aspect of the present invention provides a method for preventing a disorder associated with elevated amyloid-beta (1-42) peptide, wherein said method comprises administering to said patient a provided compound or a pharmaceutically acceptable composition thereof.

[00233] In other embodiments, the present invention provides a method for treating diseases where A-beta amyloidosis may be an underlying aspect or a co-existing and exacerbating factor, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00234] In still other embodiments, the present invention provides a method for treating a disorder in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof, and wherein said disorder is Lewy body dementia (associated with deposition of alpha-synuclein into Lewy bodies in cognitive neurons; a-synuclein is more commonly associated with deposits in motor neurons and the etiology of Parkinson's disease), Parkinson's disease, cataract (where a-beta is aggregating in the eye lens), age-related macular degeneration, Tauopathies (e.g. frontotemporal dementia), Huntington's disease, ALS/Lou Gerhig's disease, Type 2 diabetes (IAPP aggregates in pancreatic islets, is similar in size and sequence to A-beta and having type 2 diabetes increases risk of dementia), transthyretin amyloid disease (TTR, an example of this disease is in heart muscle contributing to cardiomyopathy), prion disease (including Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru), and CJD.

[00235] In some emnbodiments, the present invention provides a method for treating a disorder in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof, and wherein said disorder is mild cognitive impairment, pre-symptomatic AD, prodromal or predementia AD, Trisomy 21 (Down Syndrome), cerebral amyloid angiopathy, degenerative dementia, Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D), Creutzfeld- Jakob disease, prion disorders, amyotrophic lateral sclerosis, progressive supranuclear palsy, head trauma, stroke, Down syndrome, pancreatitis, inclusion body myositis, other peripheral amyloidoses, diabetes and atherosclerosis, cerebral amyloid angiopathy, HCHWA-D, multi- infarct dementia, and/or dementia pugilistica, or traumatic brain injury.

[00236] In other embodiments, the present invention provides a method for treating or lessening the severity of Alzheimer's disease in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00237] Without wishing to be bound by any particular theory, it is believed that the present compounds are modulators of gamma-secretase which selectively reduce levels of amyloid-beta (1-42). Accordingly, another embodiment of the present invention provides a method of modulating gamma-secretase in a patient, comprising administering to said patient a provided compound, or pharmaceutically acceptable composition thereof. In certain embodiments, the present compounds are inhibitors of gamma-secretase. Said method is useful for treating or lessening the severity of any disorder associated with gamma-secretase. Such disorders include, without limitation, neurodegenerative disorders, e.g. Alzheimer's disease. In some embodiments, such disorders include cerebral amyloid angiopathy, HCHWA-D, multi-infarct dementia, dementia pugilistica, traumatic brain injury and/or Down syndrome.

[00238] The Notch/Delta signaling pathway is highly conserved across species and is widely used during both vertebrate and invertebrate development to regulate cell fate in the developing embryo. See Gaiano and Fishell, "The Role of Notch in Promoting Glial and Neural Stem Cell Fates" Annu. Rev. Neurosci. 2002, 25:471-90. Notch interacts with the gamma-secretase complex and has interactions with a variety of other proteins and signaling pathways. Notchl competes with the amyloid precursor protein for gamma-secretase and activation of the Notch signaling pathway down-regulates PS-1 gene expression. See Lleo et al, "Notchl Competes with the Amyloid Precursor Protein for γ-Secretase and Down- regulates Presenilin-1 Gene Expression" Journal of Biological Chemistry 2003, 48:47370- 47375. Notch receptors are processed by gamma-secretase acting in synergy with T cell receptor signaling and thereby sustain peripheral T cell activation. Notch 1 can directly regulate Tbx21 through complexes formed on the Tbx21 promoter. See Minter et al, "Inhibitors of γ-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21," Nature Immunology 2005, 7:680-688. In vitro, gamma- secretase inhibitors extinguished expression of Notch, interferon-gamma and Tbx21 in ΤΉ1- polarized CD4+ cells. In vivo, administration of gamma-secretase inhibitors substantially impeded TH1 -mediated disease progression in the mouse experimental autoimmune encephalomyelitis model of multiple sclerosis suggesting the possibility of using such compounds to treat TH1 -mediated autoimmunity See Id. Inhibition of gamma-secretase can alter lymphopoiesis and intestinal cell differentiation (Wong et al, "Chronic Treatment with the γ-Secretase Inhibitor LY-41 1,575 Inhibits β-Amyloid Peptide Production and Alters Lymphopoiesis and Intestinal Cell Differentiation" Journal of Biological Chemistry 2004, 26: 12876-12882), including the induction of goblet cell metaplasia. See Milano et al, "Modulation of Notch Processing by g-Secretase Inhibitors Causes Intestinal Goblet Cell Metaplasia and Induction of Genes Known to Specify Gut Secretory Lineage Differentiation" Toxicological Sciences 2004, 82:341-358.

[00239] Strategies that can alter amyloid precursor protein ("APP") processing and reduce the production of pathogenic forms of amyloid-beta without affecting the release of Notch intracellular domain (NICD) following the processing of Notch are highly desirable are highly desirable. See Wanngren, J., et al, Second generation gamma secretase modulators exhibit different modulation of Notch beta and amyloid beta production, J. Biol. Chem. 2012, article in press; Okochi, M., et al, Secretion of the Notch- 1 amyloid beta-like peptide during Notch signaling, J. Biol. Chem. 2006, 281, 7890-7898. Moreover, as described above, the inhibition of gamma-secretase has been shown in vitro and in vivo to inhibit the polarization of Th cells and is therefore useful for treating disorders associated with Thl cells. Thl cells are involved in the pathogenesis of a variety of organ-specific autoimmune disorders, Crohn's disease, Helicobacter pylori-induced peptic ulcer, acute kidney allograft rejection, and unexplained recurrent abortions, to name a few.

[00240] According to one embodiment, the invention relates to a method of inhibiting the formation of Thl cells in a patient comprising the step of administering to said patient a compound of the present invention, or a composition comprising said compound. In certain embodiments, the present invention provides a method for treating one or more autoimmune disorders, including irritable bowel disorder, Crohn's disease, rheumatoid arthritis, psoriasis, Helicobacter pylori- induced peptic ulcer, acute kidney allograft rejection, multiple sclerosis, or systemic lupus erythematosus, wherein said method comprises administering to said patient a provided compound, prepared according to methods of the present invention, or a pharmaceutically acceptable composition comprising said compound.

[00241] In certain embodiments, the present invention provides a method for modulating and/or inhibiting amyloid-beta peptide production, without affecting the release of Notch intracellular domain (NICD) following the processing of Notch are highly desirable, in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition comprising said compound.

[00242] In certain embodiments, the present invention provides a method for inhibiting amyloid-beta (1-42) peptide production, without affecting the release of Notch intracellular domain (NICD) following the processing of Notch are highly desirable, in a patient, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition comprising said compound.

[00243] In certain embodiments, the present invention provides a method for reducing amyloid-beta (1-42) peptide levels in a patient and increasing one or more of amyloid-beta (1-37) and amyloid-beta (1-39), without affecting the release of Notch intracellular domain (NICD) following the processing of Notch are highly desirable, wherein said method comprises administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof.

[00244] Accordingly, another aspect of the present invention provides a method for reducing the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide in a patient, without affecting Notch processing, comprising administering to said patient a provided compound, or a pharmaceutically acceptable composition thereof. In certain embodiments, the ratio of amyloid-beta (1-42) peptide to amyloid-beta (1-40) peptide is reduced from a range of about 0.1 to about 0.4 to a range of about 0.05 to about 0.08.

[00245] The compounds of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts. The term "patient," as used herein, means an animal, preferably a mammal, and most preferably a human.

[00246] Various functions and advantages of these and other embodiments of the present invention will be more fully understood from the examples described below. The following examples are intended to illustrate the benefits of the present invention, but do not exemplify the full scope of the invention.

Exemplification

[00247] The following experimental describe preparation of exemplary compounds of the present invention. Melting points are uncorrected. ^X H and ¹³ C NMR spectra were measured at 400 and 100 MHz respectively in CDCI ₃ , CD ₃ OD, or pyridine-d5. Chemical shifts are downfield from trimethylsilane (TMS) as internal standards, and J values are in hertz. Mass spectra were obtained on AP 1-2000, or Hewlett Parkard series 1100 MSD with ESI technique. All solvents used were reagent grade. Gamma-oryzanol was purchased from ChemPacific Corporation (Baltimore, MD, USA). The black cohosh extract was obtained as a custom order from Hauser Pharmaceuticals. This extract is substantially equivalent to the USP preparation of black cohosh extract, in which about 50% aqueous ethanol is used to extract powdered root and then concentrated to near dryness. Other abbreviations include: AC ₂ O (acetic anhydride), DMAP (dimethylaminopyridine), PhI(OAc) ₂ (iodosobenzene diacetate), PDC (pyridinium dichromate), TFAA (trifluoroacetic acid), DMDO (dimethyldioxirane), DIPEA (Ν,Ν-Diisopropylethylamine), RB (round-bottom), TLC (thin layer chromatography), MeOH (methanol), MeOD (methanol d-4), /-PrOH (isopropanol), TBDMS (tert-butyldimethylsilyl-), TBS (tert-butyldimethylsilyl-), DHEA (dehydroepiandrosterone), TBHP (tert-butylhydroperoxide), DMSO (dimethylsulfoxide), KOt-Bu (potassium tert- butoxide), MS (mass spectrometry), Mom-Cl (Chloromethyl methyl ether), EtOAc (ethyl acetate), M.P. (melting point), EtPPhsI (ethyltriphenylphosphonium iodide), Ets (triethyl amine), mCPBA (met[alpha]-chloroperbenzoic acid), BF ₃ 0Et ₂ (trifluoroborane etherate), EtOH (ethanol), HPLC (high performance liquid chromatography), LCMS (liquid chromatography mass spectrometry), NMR (nuclear magnetic resonance).

[00248] General procedures: Reagents were acquired commercially and used without further purification except where noted. LC/MS spectra were acquired using an Agilent MSD with electrospray ionization and Agilent 1 100 series LC with a Zorbax C-18 column (2.1 x 30 mm, 3.5 micron particle size). Standard LC conditions utilized CH ₃ CN with 0.1% formic acid as the organic phase and water containing 0.1% formic acid as the aqueous phase, and were run as follows: Flow rate 1.000 mL/min; 0-1.80 minutes 2-98% organic-aqueous; 1.80-3.75 minutes 98% organic-aqueous, 3.75-3.76 minutes 98-2% organic-aqueous; 3.76- 4.25 minutes 2% organic-aqueous. LC/MS samples included here are of reaction mixtures pre-workup unless otherwise noted. Automatic integration over the entire non-background signal is included here, and selected key masses for individual regions have been added manually. NMR spectra were acquired using a Varian 400 MHz instrument and are acquired in CDC1 ₃ .

[00249] Example 1

Step SI:

Biomass

A

[00250] The black cohosh biomass was first dried and ground to a suitable particle size usually ranging from about 0.1 to about 1.0 mm ³ . This was accomplished by passage through a chipper or a grinding mill. The ground biomass (1.88 kg) was extracted with tech grade methanol (9.4 L) at 50°C for 2 hours. Other polar solvent such as an alcohol, preferably 95% ethanol could also be used. The extraction could also be done at RT for 22 hours. The extract solution was filtered through Celite using a basket centrifuge. The filter cake was rinsed with tech grade methanol and the filtrates were combined. The clear, homogeneous, dilute methanol extract was concentrated under vacuum with a max. temperature 33 °C reached, which provided 1.3 L of concentrated solution with suspended solids visible. The concentrated extract was added slowly to 5% KCl solution in water (5.2 L) and the resulting mixture was cooled to 4 °C and held for 2 hours. Other salts can also be used, including but not limited to, ( H ₄ ) ₂ S04, K ₂ S0 ₄ , NaCl, etc. The concentration of salt in water ranges from 3% to 30%. The holding time ranges from 2 to 24 hours. The precipitate containing compound A was formed, whichwas collected using a centrifuge and rinsed with water. An aqueous salt solution can also be used to rinse the solid, including but not limited to, 0 - 30 % (NH ₄ ) ₂ S0 ₄ , K ₂ S0 ₄ , KCl, NaCl, etc. Sometimes Celite is added as filter aid to facilitate the filtration. The collected solid was transferred to a dryer which provided 71 g of dry solid. Dryers include spray dryer, drum dryer, etc.

[00251] The above solid (71 g) was taken up in 210 mL of CH ₂ C1 ₂ and the obtained slurry was stirred at RT for 1 h, followed by addition of 268 mL of 10% NaCl. The organic phase was collected and the aqueous layer was extracted again with 70 mL of CH ₂ C1 ₂ . The combined organic phasewas evaporated to dryness, which afforded 56.7 g of solid, which contains 13% of A by HPLC-ELSD analysis.

[00252] HPLC analysis conditions:

Column: Phenomenex Luna C18(2), 3 μιη, 4.6 mm x 150 mm;

Flow rate: 1.0 mL/min;

Detector: ELSD, Temp.: 55 °C, Gain 1 1;

Gradient:

Rt of Al (xyloside) = 7.9 min

Rt of A2 (arabinoside) = 7.2 min

[00253] Method S2-A: To a solution of the solid obtained from S-l (20.3 g, 13% A) in CH ₂ CI ₂ (162 mL) was added ZrCl ₄ (1.32 g) at 20 °C in three portions over 1 h. The mixture was stirred at 20 °C for additional 35 min and Celite (7.1 g) was added, followed by addition of Et ₃ (5 mL) within 5-15 min. The solids were filtered off and washed with CH ₂ CI ₂ (100 mL). The filtrates were combined and washed with half saturated aHC0 ₃ (100 mL). The aqueous layer was back extracted with CH2CI2 (25 mL). All the organic layers were combined and evaporated to dryness, which afforded crude product B (19.16 g). Purification of the crude on Si0 ₂ (100 g) with 0-7% MeOH/CH ₂ Cl ₂ provided B (4.07 g) in 58% purity based on HPLC-ELSD analysis. Precipitation of the solid in EtOH/water (41 mL/49 mL) at 5 °C followed by filtration and drying provided an upgraded compound B (2.4 g) in 83.3% purity by HPLC-ELSD analysis. HPLC-ELSD conditions: see above in S-l. Rt of Bl (xyloside) = 7.2 min. Rt of B2 (arabinoside) = 6.7 min.

[00254] Method S2-B: Alternatively, the solid obtained from S-l (32 g, 13% A) was dissolved in DMSO (70 mL), filtered through Celite and purified by reverse phase chromatography with C-18 column (40-63 μιη, 18.2 cm x 45 cm) using 60 - 70% MeOH/water as eluents. The fractions were analyzed using the analytical HPLC conditions described above. The selected fractions were combined and concentrated to about half of the original volume (1.1 L). NaCl (143 g) was added and the resulting mixture was extracted with CH2CI2 (2 x 340 mL). The combined organic phase was concentrated to dryness. Further drying in vacuo provides 4.0 g of solid A in 62.3 % purity by HPLC-ELSD analysis. To a solution of the above solid (62.3% A, 4.0 g) in CH ₂ C1 ₂ (80 mL) was added ZrCl ₄ (200 mg) at 20 °C. The mixture was stirred at 20 °C for 75 min and Celite (4.0 g) was added followed by addition of Ets (0.83 mL) within 5-15 min. The solids were filtered off and washed with CH ₂ CI ₂ (51 mL). The filtrates were combined and most solvent is removed by distillation at 30-40°C. The residue was azeotroped with EtOH to remove the rest of CH ₂ CI ₂ . Precipitation of the residue in EtOH/H20 (9/1 1) followed by filtration and drying provided compound B (1.2 g) in 96% purity by HPLC-ELSD analysis. HPLC-ELSD conditions: see above in S-l. Rt of Bl (xyloside) = 7.2 min. Rt of B2 (arabinoside) = 6.7 min.

Step S3:

[00255] In a 1-L round-bottomed flask, Compound B (50 g, 75.4 mmol) was dissolved in THF (600 mL) and H ₂ 0 (200 mL), treated with NaI0 ₄ (64.4 g, 301.7 mmol), and the resulting mixture was heated to 50 °C and stirred vigorously (>1000 rpm) for 17 h. The reaction was followed by LC/MS until no mono-oxidative cleavage product (m/z ([M+H] ⁺ ) = 661] was observed, thenwas cooled to RT and THF was removed in vacuo. The residue was diluted with CH ₂ C1 ₂ (300 mL) and H ₂ 0 (300 mL) and stirred at RT for 30 min. The mixture was then partitioned between CH ₂ C1 ₂ (800 mL) and H ₂ 0 (800 mL). A solution of aq. HC1 (1.0 M, 300 mL) was added and the layers were separated. The aqueous layer was extracted with CH ₂ C1 ₂ (1L, 2 x 500 mL) and the combined organic layers were washed with 10% NaOAc (300 mL), dried over Na ₂ S0 ₄ , filtered, and concentrated in vacuo. The dialdehyde compound Cwas obtained as a crude yellow solid (51.5 g) and was carried on to the next step without further purification, assuming quantitative yield.

Step S4:

[00256] The crude dialdehyde C was dissolved in 9: 1 EtOFLAcOH (200 mL) and the solution was treated with benzylamine hydrochloride salt (4.56 g, 31.6 mmol, 1.05 eq.), followed by NaBH(OAc)3 (19.24 g, 90.78 mmol, 3 eq.). The solution was stirred at RT and the reaction progress was monitored via LC/MS. After 1 h, the reaction was complete according to LC/MS (major product peak m/z ([M + H] ⁺ ) = 706, with some minor impurities at m/z ([M + H] ⁺ ) = 706 and m/z ([M + H] ⁺ ) = 706), was poured into 800 mL CH ₂ Cl ₂ /800 mL H20, and the layers were separated. The aqueous layer was extracted with CH ₂ C1 ₂ (4 x 400 niL), and the combined organic layers were dried over a ₂ S04, filtered, and concentrated under reduced pressure. To the residue was added toluene (100 mL), and the mixture was then re-concentrated to help azeotrope off any residual AcOH. This step was then repeated. The crude product D-i-1, a reddish-brown solid, was taken on without further purification, assuming quantitative yield. In some embodiments, it was preferred to concentrate from toluene multiple times to get the material very dry and free of AcOH to afford a reddish foam, not a reddish syrup.

Step S5:

[00257] A slurry of NaBH ₄ (1.256 g, 33.18 mmol, 1.1 eq.) in EtOH (20 mL) was stirred for 10 min, then a solution of crude D-i-1 in EtOAc (200 mL)was added, and the reaction was stirred at RT and monitored by LC/MS. After 10 min, LC/MS shows complete reaction (no starting material peak at m/z ([M + H] ⁺ ) = 706; complete conversion to product at m/z ([M + Hf) = 708). NaBH ₄ was quenched by adding AcOH (5.6 mL, 3.3 eq.) very slowly and dropwise at first as vigorous bubbling occured. After stirring for 5 min, the reaction mixture was poured into 800 mL CH2CI2/8OO mL H20, shaken, and the layers separated. The aqueous layer was extracted with CH2CI2 (2 x 400 mL), the combined organic layers were dried (Na ₂ S0 ₄ ), filtered, and concentrated. The crude product was dissolved in CH2CI2 and loaded onto a Biotage flash column (rough column loading was 1 g compound per 10 g silica gel) and gradient elutionwas used to purify (elution conditions: 1 column volume (CV) 25% EtO Ac/Hex, 8 CV gradient 25% to 100% EtO Ac/Hex, 2 CV 100% EtOAc). The purified product E-i-1 was collected as a pale yellow solid in 22-30% yield over the three steps from compound B, and the product usually contained about 5-10% impurity. The impurity did not affect the forward chemistry.

Step S6:

[00258] A solution of compound E-i-1 (4.258 g, 6.08 mmol) in EtOH (102 mL) and AcOH (2.3 mL) was degassed by bubbling N ₂ through (~2 L), then 20% Pd(OH) ₂ /C (0.560 g) was added and the reaction mixture was again degassed with N ₂ (~2 L). The N ₂ balloon was changed for a H ₂ balloon and H ₂ was continually bubbled through the reaction, changing balloon as required, for 5.5 h, then the reaction was stirred under an H ₂ atmosphere overnight. Additional 20% Pd(OH) ₂ /C (0.190 g) was added and H ₂ was bubbled through the reaction mixture for an additional 5 h. The reaction mixture was filtered through a plug of celite, the celite rinsed with MeOH, and the filtrate concentrated under reduced pressure. The residue was dissolved in CH ₂ C1 ₂ (300 mL) and washed twice with saturated aqueous NaHCC (100 mL each), dried over Na ₂ S0 ₄ , filtered and concentrated. The crude product was dissolved in CH ₂ C1 ₂ and loaded onto a Biotage 100 g Si0 ₂ flash column and gradient elution is used to purify (elution conditions: 10 CV gradient 0% to 10% MeOH/CH ₂ Cl ₂ , then 20 CV of 10% MeOH/CH ₂ Cl ₂ ). The desired product F was collected as 2.5397 g of a white solid (70% yield).

Step 57:

[00259] General Procedure for Reductive Amination. A 10-mL flask was charged with a solution of morpholine F (100 μιηοΐ, 1.0 equiv) and aldehyde or ketone (140 μιηοΐ, 1.4 equiv) in EtOH (0.9 mL), AcOH (0.1 mL) and DCM (0.1 mL). The reaction was stirred at room temperature while NaBH(OAc)3 (120 μιηοΐ, 1.2 equiv) is added. The reaction was stirred at room temperature until LC/MS indicates complete consumption of F, and was then applied to a CI 8 reverse phase chromatography column and eluted with MeCN-H ₂ 0 containing 0.1% formic acid.

Biological Assays: Αβ-42, Αβ-40, and Αβ-38 [00260] Assays are conducted to determine the ability of a Compound of Formula I to modulate Αβ-40, Αβ-40, and Αβ-38.

Procedure:

[00261] uELISA PLATES:

Human (6E10) Ab 3-PLEX ELISA kits are purchased from Meso Scale Discovery Labs, 9328 Gaither Road, Gaithersburg, MD 20877 (Catalog Number K15148E-3). Plates with capture antibodies are blocked for 1-2 hours at room temperature with 150 μϊ _^ of the manufactures blocking reagent.

[00262] CONDITIONED MEDIA:

- Culture 2B7 cells in 96 well plate with 250 of media per well until confluent;

- Prepare serial dilutions of compounds in DMSO at 100X the final desired concentration;

- Wash wells with 2B7 cells IX with 250 of media;

- Dilute DMSO stocks 1 : 100 into media:

- Add 250 of media containing compounds (1 % DMSO) to wells with 2B7 cells for 5 hours at 37 °C.

[00263] ELISA SAMPLE PREP:

-Dilute conditioned media: 1 part media with 1% DMSO and 1 part blocking buffer;

-150μΕ of the 250 μΐ _^ of conditioned media are used.

[00264] STANDARD CUR VE SAMPLE PREP: Prepare per manufacturer's protocol (see above).

-Seven point standard curve samples are prepared that contained Αβ-42, Αβ-40, and Αβ-38.

The highest concentration ΐΑβ-42 and Αβ-38 is 3,000 pg/mL and the highest concentration of Αβ-40 is 10,000 pg/mL. Subsequent serial dilutions are 1 :3 and the final composition of each sample is 1 part blocking buffer and 1 part cell medium containing 1% DMSO.

[00265] O VERNIGHT SAMPLE INCUBA TION:

-Blocked plates are washed 5X with MSD wash buffer with a plate washer;

-25 μΐ _^ of detection antibody and blocker G reagent in MSD blocking solution is added;

-25 μϊ _^ of samples (1 part conditioned media containing 1% DMSO and 1 part MSD blocking buffer) are then added;

-Plates are incubated overnight at 4 degrees C or 2 hours at room temp

[00266] FINAL WASH AND READOUT:

- Wash wells 5X with MSD wash buffer; - Add 150 μΐ, 2X MSD read buffer;

- Read with MSD imager.

[00267] BUFFERS: All reagents are in kit.

[00268] DATA ANALYSIS:

Αβ peptide levels for each peptide are calculated from the standard curve using the MSD software provided with the MSD 2400 Imager. Percent vehicle values for each compound dosage are then calculated and fit to a 4 parameter curve generating IC50 values.

[00269] CELL VIABILITY:

To the remaining 100 μΐ., of conditioned media in the tissue culture plate is added 100 μΐ., of CellTiter-Glo reagent from Promega. The plate is placed on an orbital rotator operating at 500 rpms for 2 minutes. The plate is left static for 10 minutes and then 150 \L of the lysates are transferred to a white plate and read in a luminometer.