# Patent

## Title
PRMT5 inhibitors and uses thereof

## Abstract

Described herein are compounds of Formula (A), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds of the present invention are useful for inhibiting PRMT5 activity. Methods of using the compounds for treating PRMT5-mediated disorders are also described.

## Background
RELATED APPLICATIONS
The present application is a national stage filing under 35 U.S.C. §371 of International PCT application PCT/US2013/077221, filed Dec. 20, 2013 which claims priority under 35 U.S.C. §119(e) to U.S. provisional patent applications, U.S. Ser. No. 61/745,477, filed Dec. 21, 2012, and U.S. Ser. No. 61/785,025, filed Mar. 14, 2013, the entire contents of each of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
Epigenetic regulation of gene expression is an important biological determinant of protein production and cellular differentiation and plays a significant pathogenic role in a number of human diseases.
Epigenetic regulation involves heritable modification of genetic material without changing its nucleotide sequence. Typically, epigenetic regulation is mediated by selective and reversible modification (e.g., methylation) of DNA and proteins (e.g., histones) that control the conformational transition between transcriptionally active and inactive states of chromatin. These covalent modifications can be controlled by enzymes such as methyltransferases (e.g., PRMT5), many of which are associated with specific genetic alterations that can cause human disease.
Disease-associated chromatin-modifying enzymes (e.g., PRMT5) play a role in diseases such as proliferative disorders, metabolic disorders, and blood disorders. Thus, there is a need for the development of small molecules that are capable of inhibiting the activity of PRMT5.


DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Protein arginine methyltransferase 5 (PRMT5) catalyzes the addition of two methyl groups to the two w-guanidino nitrogen atoms of arginine, resulting in ω-NG, N′G symmetric dimethylation of arginine (sDMA) of the target protein. PRMT5 functions in the nucleus as well as in the cytoplasm, and its substrates include histones, spliceosomal proteins, transcription factors (See e.g., Sun et al., PNAS (2011) 108: 20538-20543). PRMT5 generally functions as part of a molecule weight protein complex. While the protein complexes of PRMT5 can have a variety of components, they generally include the protein MEP50 (methylosome protein 50). In addition, PRMT5 acts in conjunction with cofactor SAM (S-adenosyl methionine).
PRMT5 is an attractive target for modulation given its role in the regulation of diverse biological processes. It has now been found that compounds described herein, and pharmaceutically acceptable salts and compositions thereof, are effective as inhibitors of PRMT5.
Such compounds have the general Formula (A):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R, R5, R6, R7, R8, Rx, R12, R13, and n are as defined herein.

In some embodiments, the inhibitors of PRMT5 have the general Formula (I):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R, R5, R6, R7, R8, Rx, and n are as defined herein.

In some embodiments, pharmaceutical compositions are provided which comprise a compound described herein (e.g., a compound of Formula (A), e.g., Formula (I)), or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.
In certain embodiments, compounds described herein inhibit activity of PRMT5. In certain embodiments, methods of inhibiting PRMT5 are provided which comprise contacting PRMT5 with an effective amount of a compound of Formula (A), e.g., Formula (I), or a pharmaceutically acceptable salt thereof. The PRMT5 may be purified or crude, and may be present in a cell, tissue, or a subject. Thus, such methods encompass inhibition of PRMT5 activity both in vitro and in vivo. In certain embodiments, the PRMT5 is wild-type PRMT5. In certain embodiments, the PRMT5 is overexpressed. In certain embodiments, the PRMT5 is a mutant. In certain embodiments, the PRMT5 is in a cell. In certain embodiments, the PRMT5 is in an animal, e.g., a human. In some embodiments, the PRMT5 is in a subject that is susceptible to normal levels of PRMT5 activity due to one or more mutations associated with a PRMT5 substrate. In some embodiments, the PRMT5 is in a subject known or identified as having abnormal PRMT5 activity (e.g., overexpression). In some embodiments, a provided compound is selective for PRMT5 over other methyltransferases. In certain embodiments, a provided compound is at least about 10-fold selective, at least about 20-fold selective, at least about 30-fold selective, at least about 40-fold selective, at least about 50-fold selective, at least about 60-fold selective, at least about 70-fold selective, at least about 80-fold selective, at least about 90-fold selective, or at least about 100-fold selective relative to one or more other methyltransferases.
In certain embodiments, methods of altering gene expression in a cell are provided which comprise contacting a cell with an effective amount of a compound of Formula (A), e.g., Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the cell in culture in vitro. In certain embodiments, cell is in an animal, e.g., a human.
In certain embodiments, methods of altering transcription in a cell are provided which comprise contacting a cell with an effective amount of a compound of Formula (A), e.g., Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the cell in culture in vitro. In certain embodiments, the cell is in an animal, e.g., a human.
In some embodiments, methods of treating a PRMT5-mediated disorder are provided which comprise administering to a subject suffering from a PRMT5-mediated disorder an effective amount of a compound described herein (e.g., a compound of Formula (A), e.g., Formula (I)), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the PRMT5-mediated disorder is a proliferative disorder, a metabolic disorder, or a blood disorder. In certain embodiments, compounds described herein are useful for treating cancer. In certain embodiments, compounds described herein are useful for treating hematopoietic cancer, lung cancer, prostate cancer, melanoma, or pancreatic cancer. In certain embodiments, compounds described herein are useful for treating a hemoglobinopathy. In certain embodiments, compounds described herein are useful for treating sickle cell anemia. In certain embodiments, compounds described herein are useful for treating diabetes or obesity. In certain embodiments, a provided compound is useful in treating inflammatory and autoimmune disease.
Compounds described herein are also useful for the study of PRMT5 in biological and pathological phenomena, the study of intracellular signal transduction pathways mediated by PRMT5, and the comparative evaluation of new PRMT5 inhibitors.
This application refers to various issued patent, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference.
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
It is to be understood that the compounds of the present invention may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present invention, and the naming of any compound described herein does not exclude any tautomer form.




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, replacement of 19F with 18F, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.
The term “aliphatic,” as used herein, includes both saturated and unsaturated, nonaromatic, straight chain (i.e., unbranched), branched, acyclic, and cyclic (i.e., carbocyclic) hydrocarbons. In some embodiments, an aliphatic group is optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl moieties.
When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C1-6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
“Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. In certain embodiments, each instance of an alkyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents. In certain embodiments, the alkyl group is unsubstituted C1-10 alkyl (e.g., —CH3). In certain embodiments, the alkyl group is substituted C1-10 alkyl.
In some embodiments, an alkyl group is substituted with one or more halogens. “Perhaloalkyl” is a substituted alkyl group as defined herein wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the alkyl moiety has 1 to 8 carbon atoms (“C1-8 perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 6 carbon atoms (“C1-6 perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 4 carbon atoms (“C1-4 perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 3 carbon atoms (“C1-3 perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 2 carbon atoms (“C1-2 perhaloalkyl”). In some embodiments, all of the hydrogen atoms are replaced with fluoro. In some embodiments, all of the hydrogen atoms are replaced with chloro. Examples of perhaloalkyl groups include —CF3, —CF2CF3, —CF2CF2CF3, —CCl3, —CFCl2, —CF2Cl, and the like.
“Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. In certain embodiments, each instance of an alkenyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is unsubstituted C2-10 alkenyl. In certain embodiments, the alkenyl group is substituted C2-10 alkenyl.
“Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more double bonds (“C2-20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. In certain embodiments, each instance of an alkynyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C2-10 alkynyl.
“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or is a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) and can be saturated or can be partially unsaturated. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. In certain embodiments, each instance of a carbocyclyl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is unsubstituted C3-10 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3-10 carbocyclyl.
In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6 cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3 8 cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). In certain embodiments, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C3-10 cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C3-10 cycloalkyl.
“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In certain embodiments, heterocyclyl or heterocyclic refers to a radical of a 3-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. In certain embodiments, each instance of heterocyclyl is independently optionally substituted, e.g., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.
In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 it electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. In certain embodiments, each instance of an aryl group is independently optionally substituted, e.g., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-14 aryl. In certain embodiments, the aryl group is substituted C6-14 aryl.
“Heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 it electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-14 membered heteroaryl”). In certain embodiments, heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).
In some embodiments, a heteroaryl group is a 5-14 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, each instance of a heteroaryl group is independently optionally substituted, e.g., unsubstituted (“unsubstituted heteroaryl”) or substituted (“substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.
Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
“Fused” or “ortho-fused” are used interchangeably herein, and refer to two rings that have two atoms and one bond in common, e.g.,




“Bridged” refers to a ring system containing (1) a bridgehead atom or group of atoms which connect two or more non-adjacent positions of the same ring; or (2) a bridgehead atom or group of atoms which connect two or more positions of different rings of a ring system and does not thereby form an ortho-fused ring, e.g.,




“Spiro” or “Spiro-fused” refers to a group of atoms which connect to the same atom of a carbocyclic or heterocyclic ring system (geminal attachment), thereby forming a ring, e.g.,




Spiro-fusion at a bridgehead atom is also contemplated.

“Partially unsaturated” refers to a group 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 aromatic groups (e.g., aryl or heteroaryl groups) as herein defined. Likewise, “saturated” refers to a group that does not contain a double or triple bond, i.e., contains all single bonds.
In some embodiments, aliphatic, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” aliphatic, “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, including any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
Exemplary carbon atom substituents include, but are not limited to, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —ON(Rbb)2, —N(Rbb)2, —N(Rbb)3+X−, —N(ORcc)Rbb, —SH, —SRaa, —SSRcc, —C(═O)Raa, —CO2H, —CHO, —C(ORcc)2, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —OC(═NRbb)Raa, —OC(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —OC(═NRbb)N(Rbb)2, —NRbbC(═NRbb)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, —SO2ORaa, —OSO2Raa, —S(═O)Raa, —OS(═O)Raa, —Si(Raa)3, —OSi(Raa)3—C(═S)N(Rbb)2, —C(═O)SRaa, —C(═S)SRaa, —SC(═S)SRaa, —SC(═O)SRaa, —OC(═O)SRaa, —SC(═O)ORaa, —SC(═O)Raa, —P(═O)2Raa, —OP(═O)2Raa, —P(═O)(Raa)2, —OP(═O)(Raa)2, —OP(═O)(ORcc)2, —P(═O)2N(Rbb)2, —OP(═O)2N(Rbb)2, —P(═O)(NRbb)2, —OP(═O)(NRbb)2, —NRbbP(═O)(ORcc)2, —NRbbP(═O)(NRbb)2, —P(Rcc)2, —P(Rcc)3, —OP(Rcc)2, —OP(Rcc)3, —B(Raa)2, —B(ORcc)2, —BRaa(ORcc), C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
or two geminal hydrogens on a carbon atom are replaced with the group ═O, ═S, ═NN(Rbb)2, ═NNRbbC(═O)Raa, ═NNRbbC(═O)ORaa, ═NNRbbS(═O)2Raa, ═NRbb, or ═NORcc;
each instance of Raa is, independently, selected from C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
each instance of Rbb is, independently, selected from hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2R, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2OR, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2, —P(═O)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rbb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;
each instance of Rcc is, independently, selected from hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

    
    
        each instance of Rdd is, independently, selected from halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —ON(Rff)2, —N(Rff)2, —N(Rff)3+X−, —N(ORee)Rff, —SH, —SRee, —SSRee, —C(═O)Ree, —CO2H, —CO2Ree, —OC(═O)Ree, —OCO2Ree, —C(═O)N(Rff)2, —OC(═O)N(Rff)2, —NRffC(═O)Ree, —NRffCO2Ree, —NRffC(═O)N(Rff)2, —C(═NRff)ORee, OC(═NRff)Ree, —OC(═NRff)ORee, —C(═NRff)N(Rff)2, —OC(═NRff)N(Rff)2, —NRffC(═NRff)N(Rff)2, —NRffSO2Ree, —SO2N(Rff)2, —SO2Ree, —SO2ORee, —OSO2Ree, —S(═O)Ree, —Si(Ree)3, —OSi(Ree)3, —C(═S)N(Rff)2, —C(═O)SRee, —C(═S)SRee, —SC(═S)SRee, —P(═O)2Ree, —P(═O)(Ree)2, —OP(═O)(Ree)2, —OP(═O)(ORee)2, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can be joined to form ═O or ═S;
    
    


each instance of Ree is, independently, selected from C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
each instance of Rff is, independently, selected from hydrogen, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, 3-10 membered heterocyclyl, C6-10 aryl and 5-10 membered heteroaryl, or two Rff groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and
each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OC1-6 alkyl, —ON(C1-6 alkyl)2, —N(C1-6 alkyl)2, —N(C1-6 alkyl)3+X−, —NH(C1-6 alkyl)2+X−, —NH2(C1-6 alkyl)+X−, —NH3+X−, —N(OC1-6 alkyl)(C1-6 alkyl), —N(OH)(C1-6 alkyl), —NH(OH), —SH, —SC1-6 alkyl, —SS(C1-6 alkyl), —C(═O)(C1-6 alkyl), —CO2H, —CO2(C1-6 alkyl), —OC(═O)(C1-6 alkyl), —OCO2(C1-6 alkyl), —C(═O)NH2, —C(═O)N(C1-6 alkyl)2, —OC(═O)NH(C1-6 alkyl), —NHC(═O)(C1-6 alkyl), —N(C1-6 alkyl)C(═O)(C1-6 alkyl), —NHCO2(C1-6 alkyl), —NHC(═O)N(C1-6 alkyl)2, —NHC(═O)NH(C1-6 alkyl), —NHC(═O)NH2, —C(═NH)O(C1-6 alkyl), —OC(═NH)(C1-6 alkyl), —OC(═NH)OC1-6 alkyl, —C(═NH)N(C1-6 alkyl)2, —C(═NH)NH(C1-6 alkyl), —C(═NH)NH2, —OC(═NH)N(C1-6 alkyl)2, —OC(NH)NH(C1-6 alkyl), —OC(NH)NH2, —NHC(NH)N(C1-6 alkyl)2, —NHC(═NH)NH2, —NHSO2(C1-6 alkyl), —SO2N(C1-6 alkyl)2, —SO2NH(C1-6 alkyl), —SO2NH2, —SO2C1-6 alkyl, —SO2OC1-6 alkyl, —OSO2C1-6 alkyl, —SOC1-6 alkyl, —Si(C1-6 alkyl)3, —OSi(C1-6 alkyl)3-C(═S)N(C1-6 alkyl)2, C(═S)NH(C1-6 alkyl), C(═S)NH2, —C(═O)S(C1-6 alkyl), —C(═S)SC1-6 alkyl, —SC(═S)SC1-6 alkyl, —P(═O)2(C1-6 alkyl), —P(═O)(C1-6 alkyl)2, —OP(═O)(C1-6 alkyl)2, —OP(═O)(OC1-6 alkyl)2, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents can be joined to form ═O or ═S; wherein X is a counterion.
A “counterion” or “anionic counterion” is a negatively charged group associated with a cationic quaternary amino group in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F−, Cl−, Br−, I−), NO3−, ClO4−, OH−, H2PO4−, HSO4−, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).
“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).
Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary nitrogen atom substitutents include, but are not limited to, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2, —P(═O)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rcc groups attached to a nitrogen atom are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.
In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to, —OH, —ORaa, —N(Rcc)2, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRcc)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc, and Rdd are as defined herein. Nitrogen 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, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
Amide nitrogen protecting groups (e.g., —C(═O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.
Carbamate nitrogen protecting groups (e.g., —C(═O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.
Sulfonamide nitrogen protecting groups (e.g., —S(═O)2Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups include, but are not limited to, —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Oxygen 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, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxyl)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4′′-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4′′-tris(levulinoyloxyphenyl)methyl, 4,4′,4′′-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4′′-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), t-butyl carbonate (BOC), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups include, but are not limited to, —Raa, —N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa, —C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3, —P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Sulfur 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, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.
As used herein, a “leaving group”, or “LG”, is a term understood in the art to referred to a molecular fragment that departs with a pair of electrons upon heterolytic bond cleavage, wherein the molecular fragment is an anion or neutral molecule. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Examples of suitable leaving groups include, but are not limited to, halides (such as chloride, bromide, or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, haloformates, —NO2, trialkylammonium, and aryliodonium salts. In some embodiments, the leaving group is a sulfonic acid ester. In some embodiments, the sulfonic acid ester comprises the formula —OSO2RLG1 wherein RLG1 is selected from the group consisting alkyl optionally, alkenyl optionally substituted, heteroalkyl optionally substituted, aryl optionally substituted, heteroaryl optionally substituted, arylalkyl optionally substituted, and heterarylalkyl optionally substituted. In some embodiments, RLG1 is substituted or unsubstituted C1-C6 alkyl. In some embodiments, R is methyl. In some embodiments, RLG1 is —CF3. In some embodiments, RLG1 is substituted or unsubstituted aryl. In some embodiments, RLG1 is substituted or unsubstituted phenyl. In some embodiments RLG1 is:




These and other exemplary substituents are described in more detail in the Detailed Description, Examples, and claims. The present disclosure is not intended to be limited in any manner by the above exemplary listing of substituents.
“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 other animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds describe herein 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+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, quaternary salts.
A “subject” to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other non-human animals, for example, non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys), rodents (e.g., rats and/or mice), reptiles, amphibians, and fish. In certain embodiments, the non-human animal is a mammal. The non-human animal may be a male or female at any stage of development. A non-human animal may be a transgenic animal.
“Condition,” “disease,” and “disorder” are used interchangeably herein.
“Treat,” “treating” and “treatment” encompasses an action that occurs while a subject is suffering from a condition which reduces the severity of the condition or retards or slows the progression of the condition (“therapeutic treatment”). “Treat,” “treating” and “treatment” also encompasses an action that occurs before a subject begins to suffer from the condition and which inhibits or reduces the severity of the condition (“prophylactic treatment”).
An “effective amount” of a compound refers to an amount sufficient to elicit the desired biological response, e.g., treat the condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment.
A “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.
A “prophylactically effective amount” of a compound is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
As used herein, the term “methyltransferase” represents transferase class enzymes that are able to transfer a methyl group from a donor molecule to an acceptor molecule, e.g., an amino acid residue of a protein or a nucleic base of a DNA molecule. Methytransferases typically use a reactive methyl group bound to sulfur in S-adenosyl methionine (SAM) as the methyl donor. In some embodiments, a methyltransferase described herein is a protein methyltransferase. In some embodiments, a methyltransferase described herein is a histone methyltransferase. Histone methyltransferases (HMT) are histone-modifying enzymes, (including histone-lysine N-methyltransferase and histone-arginine N-methyltransferase), that catalyze the transfer of one or more methyl groups to lysine and arginine residues of histone proteins. In certain embodiments, a methyltransferase described herein is a histone-arginine N-methyltransferase.
As generally described above, provided herein are compounds useful as PRMT5 inhibitors. In some embodiments, the present disclosure provides a compound of Formula (A):




or a pharmaceutically acceptable salt thereof,

wherein

 represents a single or double bond;
R12 is hydrogen, halogen, or optionally substituted C1-3alkyl;
R13 is hydrogen, halogen, optionally substituted C1-3alkyl, —NRA1RA2, or —OR1;
RA1 and RA2 are each independently hydrogen, optionally substituted C1-3 alkyl, a nitrogen protecting group, or RA1 and RA2 are taken together with the intervening nitrogen atom to form an optionally substituted 3-6 membered heterocyclic ring;
Ring A is an optionally substituted, 5- to 12-membered, monocyclic or bicyclic, heterocyclyl or heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur;
R1 is hydrogen, Rz, or —C(O)Rz, wherein Rz is optionally substituted C1-6 alkyl;
Y is O or S;
R5, R6, R7, and R8 are each independently hydrogen, halo, or optionally substituted aliphatic;

    
    
        each Rx is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR′, and —N(Raa)2;
    
    


R′ is hydrogen or optionally substituted aliphatic;

    
    
        each R′′ is independently hydrogen or optionally substituted aliphatic, or two R′′ are taken together with their intervening atoms to form a heterocyclic ring; and
    
    


n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits.
In some embodiments, the carbon attached to R12 has (S)-stereochemistry. In some embodiments, the carbon attached to R12 has (R)-stereochemistry. In some embodiments, the carbon attached to R13 has (S)-stereochemistry. In some embodiments, the carbon attached to R13 has (R)-stereochemistry. In some embodiments, R12 is hydrogen. In some embodiments, R13 is hydrogen. In some embodiments, both R12 and R13 are hydrogen. In some embodiments, R12 is optionally substituted C1-3 alkyl. In some embodiments, R13 is optionally substituted C1-3 alkyl. In some embodiments, both R12 and R13 are optionally substituted C1-3alkyl. In some embodiments, R is halogen e.g., fluoro, bromo, chloro, or iodo, provided that R is not —OR1. In some embodiments, R is halogen e.g., fluoro, bromo, chloro, or iodo. In some embodiments, both R12 and R13 are halogen e.g., fluoro, bromo, chloro, or iodo. In some embodiments, R12 is halogen e.g., fluoro, bromo, chloro, or iodo and R13 is optionally substituted C1-3alkyl. In some embodiments, R12 is optionally substituted C1-3alkyl and R13 is halogen e.g., fluoro, bromo, chloro, or iodo. In some embodiments, R13 is —OR1. In some embodiments, R12 is optionally substituted C1-3alkyl and R13 is —OR1. In some embodiments, R12 is hydrogen and R13 is —OR1. In some embodiments, R12 is hydrogen and R13 optionally substituted C1-3alkyl. In some embodiments, R12 is optionally substituted C1-3alkyl and R is hydrogen. In some embodiments, R is halogen e.g., fluoro, bromo, chloro, or iodo and R13 is hydrogen. In some embodiments, R12 is hydrogen and R13 is halogen e.g., fluoro, bromo, chloro, or iodo.
As generally defined above, R12 is hydrogen, halogen, or optionally substituted C1-3alkyl. In certain embodiments, R12 is hydrogen. In certain embodiments, R12 is optionally substituted C1-3alkyl, e.g., optionally substituted with halogen. In certain embodiments, R12 is optionally substituted C1 alkyl, e.g., methyl or trifluoromethyl. In certain embodiments, R12 is optionally substituted C2 alkyl, e.g., ethyl. In certain embodiments, R is optionally substituted C3 alkyl, e.g., propyl. In certain embodiments, R12 is fluoro, provided that R is not —OR1. In certain embodiments, R is chloro, provided that R3 is not —OR1. In certain embodiments, R12 is bromo, provided that R is not —OR1. In certain embodiments, R12 is iodo, provided that R13 is not —OR1.
As generally defined above, R13 is hydrogen, halogen, optionally substituted C1-3alkyl, —NRA1RA2 or —OR1. In certain embodiments, R13 is hydrogen. In certain embodiments, R13 is optionally substituted C1-3alkyl, e.g., optionally substituted with halogen. In certain embodiments, R13 is optionally substituted C1alkyl, e.g., methyl or trifluoromethyl. In certain embodiments, R13 is optionally substituted C2 alkyl, e.g., ethyl. In certain embodiments, R is optionally substituted C3 alkyl, e.g., propyl. In certain embodiments, R13 is fluoro. In certain embodiments, R is chloro. In certain embodiments, R is bromo. In certain embodiments, R13 is iodo.
For example, in some embodiments of Formula (A), wherein R13 is hydrogen, the present disclosure provides a compound of Formula (A-1):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R5, R6, R7, R8, Rx, R12, and n are as described herein.

In some embodiments of Formula (A), wherein R12 is hydrogen, the present disclosure provides a compound of Formula (A-2):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R5, R6, R7, R8, Rx, R13, and n are as described herein.

In some embodiments of Formula (A), wherein both R12 and R13 are hydrogen, the present disclosure provides a compound of Formula (A-3):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R5, R6, R7, R8, Rx, and n are as described herein.

In some embodiments of Formula (A), wherein R13 is —OR1, the present disclosure provides a compound of Formula (A-4):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R, R5, R6, R7, R8, Rx, R12, and n are as described herein.

In some embodiments of Formula (A), wherein R13 is —NRA1RA2, the present disclosure provides a compound of Formula (A-5):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R5, R6, R7, R, Rx, R2, RA1, and RA2, and n are as described herein.

In some embodiments, the present disclosure provides a compound of Formula (I):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R, R5, R6, R7, R8, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (I-a):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R1, R5, R6, R7, R8, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (I-b):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R, R5, R6, R7, R8, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (I-c):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R1, Rx, and n are as described herein.

In some embodiments of Formula (A), the present disclosure provides a compound of Formula (A-6):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, Rx, R12, R13, and n are as described herein.

In certain embodiments, a provided compound is of Formula (I′):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R1, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (I′-a):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R1, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (I′-b):




or a pharmaceutically acceptable salt thereof, wherein Ring A, Y, R1, Rx, and n are as described herein.

In some embodiments of Formula (A), the present disclosure provides a compound of Formula (A-7):




or a pharmaceutically acceptable salt thereof, wherein Y, Rx, and n are as described herein,

G is NR2, CR3R4, O or S;
R12 is hydrogen, halogen, or optionally substituted C1-3alkyl;
R13 is hydrogen, halogen, optionally substituted C1-3alkyl, —NRA1RA2, or —OR1;
RA1 and RA2 are independently hydrogen, optionally substituted C1-3 alkyl, a nitrogen protecting group, or RA1 and RA2 are taken together with the intervening nitrogen atom to form an optionally substituted 3-6 membered heterocyclic ring;
R1 is hydrogen, Rz, or —C(O)Rz, wherein Rz is optionally substituted C1-6 alkyl;
R2 is selected from the group consisting of optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —C(O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(═NRB)RA, —C(═NRB)N(RB)2, —C(═S)RA, —C(═S)N(RB)2, —S(═O)RA, —SO2RA, and —SO2N(RB)2;
R3 is selected from the group consisting of hydrogen, halo, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA, —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2;
each RA is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
each RB is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring;
R4 is selected from the group consisting of hydrogen, halo, and optionally substituted aliphatic;
each Ry is independently selected from the group consisting of halo, —CN, —NO2, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA, —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2, or two adjacent Ry groups may be taken together with their intervening atoms to form a saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur;
p is 0, 1, or 2; and
m is 0, 1, 2, 3, or 4.
In certain embodiments, a provided compound is of Formula (II):




or a pharmaceutically acceptable salt thereof, wherein R1, G, Y, Ry, m, p, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-a):




or a pharmaceutically acceptable salt thereof, wherein R1, G, Y, Ry, m, p, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (II-b):




or a pharmaceutically acceptable salt thereof, wherein R1, G, Y, Ry, m, p, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (A-8):




or a pharmaceutically acceptable salt thereof, wherein R2, Ry, m, Rx, R12, R13, and n are as described herein.

In certain embodiments, a provided compound is of Formula (III):




or a pharmaceutically acceptable salt thereof, wherein R2, Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (III-a):




or a pharmaceutically acceptable salt thereof, wherein R2, Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (III-b):




or a pharmaceutically acceptable salt thereof, wherein R2, Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (A-9):




or a pharmaceutically acceptable salt thereof, wherein R3, Ry, m, Rx, R12, R13, and n are as described herein.

In certain embodiments, a provided compound is of Formula (IV):




or a pharmaceutically acceptable salt thereof, wherein R3, Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (IV-a):




or a pharmaceutically acceptable salt thereof, wherein R3, Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (IV-b):




or a pharmaceutically acceptable salt thereof, wherein R3, Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (A-10):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, R12, R13, and n are as described herein.

In certain embodiments, a provided compound is of Formula (V):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (V-a):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (V-b):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (A-11):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, R12, R13 and n are as described herein.

In certain embodiments, a provided compound is of Formula (VI):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (VI-a):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, and n are as described herein.

In certain embodiments, a provided compound is of Formula (VI-b):




or a pharmaceutically acceptable salt thereof, wherein Ry, m, Rx, and n are as described herein.

In some embodiments,  represents a single bond. In some embodiments,  represents a double bond.
As defined generally above, Ring A is an optionally substituted, 5- to 12-membered, monocyclic or bicyclic, heterocyclyl or heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. One of ordinary skill in the art will understand that Ring A comprises an amide or thioamide. In certain embodiments, Ring A is an optionally substituted, 5- to 6-membered, monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, Ring A is an optionally substituted, 5- to 7-membered, monocyclic heterocyclyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, Ring A is an optionally substituted, 8- to 10-membered, bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, Ring A is an optionally substituted, 8- to 12-membered, bicyclic heterocyclyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, Ring A is an optionally substituted piperdinone. In certain embodiments, Ring A is an optionally substituted pyridinone. In certain embodiments, Ring A is an optionally substituted piperazinone. In certain embodiments, Ring A is an optionally substituted isoindolinone. In certain embodiments, Ring A is an optionally substituted 2H-benzo[b][1,4]oxazin-3(4H)-one. In some embodiments, Ring A is




wherein G, Y, Ry, m, and p are as described herein.

In certain embodiments, Y is O. In certain embodiments, Y is S.
As defined generally above, R1 is hydrogen, Rz, or —C(O)Rz, wherein Rz is optionally substituted C1-6 alkyl. In certain embodiments, R1 is hydrogen. In some embodiments, R1 is optionally substituted C1-6 alkyl. In certain embodiments, R1 is unsubstituted C1-6 alkyl. In certain embodiments, R1 is methyl, ethyl, or propyl. In some embodiments, R1 is —C(O)Rz, wherein Rz is optionally substituted C1-6 alkyl. In certain embodiments, R1 is —C(O)Rz, wherein Rz is unsubstituted C1-6 alkyl. In certain embodiments, R1 is acetyl.
As defined generally above, G is NR2, CR3R4, O or S. In certain embodiments, G is NR2. In certain embodiments, G is CR3R4. In certain embodiments, G is O. In certain embodiments, G is S.
As defined generally above, R2 is selected from the group consisting of optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —C(O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(═NRB)RA, —C(═NRB)N(RB)2, —C(═S)RA, —C(═S)N(RB)2, —S(═O)RA, —SO2RA, and —SO2N(RB)2. In some embodiments, R2 is optionally substituted aryl. In certain embodiments, R2 is optionally substituted phenyl. In certain embodiments, R2 is unsubstituted phenyl. In certain embodiments, R2 is halophenyl. In certain embodiments, R2 is fluorophenyl. In certain embodiments, R2 is chlorophenyl. In some embodiments, R2 is phenyl substituted with optionally substituted C1-6 alkyl. In some embodiments, R2 is phenyl substituted with optionally substituted C1-3 alkyl. In certain embodiments, R2 is phenyl substituted with methyl. In certain embodiments, R2 is phenyl substituted with —CH2OOH. In some embodiments, R2 is phenyl substituted with a heterocyclic ring. In certain embodiments, R2 is phenyl substituted with morpholinyl. In certain embodiments, R2 is phenyl substituted with tetrahydropyranyl. In some embodiments, R2 is optionally substituted heteroaryl. In certain embodiments, R2 is optionally substituted quinoline. In certain embodiments, R2 is unsubstituted quinoline. In certain embodiments, R2 is substituted quinoline. In certain embodiments, R2 is optionally substituted pyridine. In certain embodiments, R2 is pyridine substituted with a heterocyclic ring. In some embodiments, R2 is optionally substituted aliphatic. In certain embodiments, R2 is unsubstituted aliphatic. In certain embodiments, R2 is —CH2-aryl. In certain embodiments, R2 is benzyl. In certain embodiments, R2 is —CH2-heteroaryl. In certain embodiments, R2 is —CH2-pyridyl. In some embodiments, R2 is —C(═O)RA. In certain embodiments, R2 is —C(═O)RA, wherein RA is optionally substituted aliphatic. In certain embodiments, R2 is acetyl. In certain embodiments, R2 is —SO2RA. In certain embodiments, R2 is —SO2RA, wherein RA is optionally substituted aliphatic. In certain embodiments, R2 is —SO2CH3.
In certain embodiments, R2 is selected from the group consisting of:




As defined generally above, R3 is selected from the group consisting of hydrogen, halo, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA, —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2. In some embodiments, R3 is selected from the group consisting of hydrogen, halo, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —OC(O)RA, —NRBC(═O)RA, —NRBC(═O)N(RB)2, —SC(═O)RA, —C(═NRB)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(═O)RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2.
In certain embodiments, R3 is hydrogen. In some embodiments, R3 is not hydrogen. In some embodiments, R3 is halo. In certain embodiments, R3 is fluoro. In some embodiments, R3 is optionally substituted aliphatic. In certain embodiments, R3 is optionally substituted C1-6 aliphatic. In certain embodiments, R3 is optionally substituted C1-6 alkyl. In certain embodiments, R3 is substituted C1-6 alkyl. In certain embodiments, R3 is —CF3, —CHF2, or —CH2F. In certain embodiments, R3 is unsubstituted C1-6 alkyl. In certain embodiments, R3 is methyl, ethyl, or propyl. In some embodiments, R3 is —CN or —NO2. In some embodiments, R3 is optionally substituted carbocyclyl, optionally substituted phenyl, optionally substituted heterocyclyl, or optionally substituted heteroaryl. In some embodiments, R is —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —OC(O)RA, —NRBC(O)RA, —NRBC(O)N(RB)2, —SC(O)RA, —C(═NRB)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —SO2RA, —NRBSO2RA, or —SO2N(RB)2. In some embodiments, R3 is optionally substituted aryl. In certain embodiments, R3 is optionally substituted phenyl. In certain embodiments, R3 is unsubstituted phenyl. In certain embodiments, R3 is halophenyl. In certain embodiments, R3 is fluorophenyl. In certain embodiments, R3 is chlorophenyl. In some embodiments, R3 is phenyl substituted with optionally substituted C1-6 alkyl. In some embodiments, R3 is phenyl substituted with optionally substituted C1-3 alkyl. In certain embodiments, R3 is phenyl substituted with methyl. In certain embodiments, R3 is phenyl substituted with —CH2OH. In some embodiments, R3 is phenyl substituted with a heterocyclic ring. In certain embodiments, R3 is phenyl substituted with morpholinyl. In certain embodiments, R3 is phenyl substituted with tetrahydropyranyl. In some embodiments, R3 is optionally substituted heteroaryl. In certain embodiments, R3 is optionally substituted quinoline. In certain embodiments, R3 is unsubstituted quinoline. In certain embodiments, R3 is substituted quinoline. In certain embodiments, R3 is optionally substituted pyridine. In certain embodiments, R3 is pyridine substituted with a heterocyclic ring. In some embodiments, R3 is optionally substituted aliphatic. In certain embodiments, R3 is unsubstituted aliphatic. In certain embodiments, R3 is —CH2-aryl. In certain embodiments, R3 is benzyl. In certain embodiments, R3 is —CH2-heteroaryl. In certain embodiments, R3 is —CH2-pyridyl.
As defined generally above, R4 is selected from the group consisting of hydrogen, halo, and optionally substituted aliphatic. In certain embodiments, R4 is hydrogen. In some embodiments, R4 is not hydrogen. In some embodiments, R4 is halo. In certain embodiments, R4 is fluoro. In some embodiments, R4 is optionally substituted aliphatic. In certain embodiments, R4 is optionally substituted C1-6 aliphatic. In certain embodiments, R4 is optionally substituted C1-6 alkyl. In certain embodiments, R4 is substituted C1-6 alkyl. In certain embodiments, R4 is unsubstituted C1-6 alkyl. In certain embodiments, R4 is methyl, ethyl, or propyl.
As defined generally above, R5, R6, R7, and R8 are each independently hydrogen, halo, or optionally substituted aliphatic. In some embodiments, R5, R6, R7, and R8 are hydrogen. In some embodiments, R6, R7, and R8 are hydrogen, and R5 is optionally substituted aliphatic. In some embodiments, R6, R7, and R8 are hydrogen, and R5 is optionally substituted C1-6 aliphatic. In some embodiments, R6, R7, and R8 are hydrogen, and R5 is optionally substituted C1-3 aliphatic. In some embodiments, R6, R7, and R8 are hydrogen, and R5 is methyl. In some embodiments, R6, R7, and R5 are hydrogen, and R is optionally substituted aliphatic. In some embodiments, R6, R7, and R5 are hydrogen, and R8 is optionally substituted C1-6 aliphatic. In some embodiments, R6, R7, and R5 are hydrogen, and R8 is optionally substituted C1-3 aliphatic. In some embodiments, R6, R7, and R5 are hydrogen, and R8 is methyl. In some embodiments, R5 is hydrogen. In some embodiments, R5 is halo. In certain embodiments, R5 is fluoro. In some embodiments, R5 is optionally substituted C1-6 aliphatic. In some embodiments, R5 is optionally substituted C1-3 alkyl. In certain embodiments, R5 is methyl. In some embodiments, R6 is hydrogen. In some embodiments, R6 is halo. In certain embodiments, R6 is fluoro. In some embodiments, R6 is optionally substituted C1-6 aliphatic. In some embodiments, R6 is optionally substituted C1-3 alkyl. In certain embodiments, R6 is methyl. In some embodiments, R7 is hydrogen. In some embodiments, R7 is halo. In certain embodiments, R7 is fluoro. In some embodiments, R7 is optionally substituted C1-6 aliphatic. In some embodiments, R7 is optionally substituted C1-3 alkyl. In certain embodiments, R7 is methyl. In some embodiments, R8 is hydrogen. In some embodiments, R8 is halo. In certain embodiments, R8 is fluoro. In some embodiments, R8 is optionally substituted C1-6 aliphatic. In some embodiments, R8 is optionally substituted C1-3 alkyl. In certain embodiments, R8 is methyl.
As defined generally above, p is 0, 1, or 2. In certain embodiments, p is 0. In certain embodiments, p is 1. In certain embodiments, p is 2.
As defined generally above, each Ry is independently selected from the group consisting of halo, —CN, —NO2, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA, —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2, wherein RA and RB are described herein.
In some embodiments, at least one Ry is halo. In certain embodiments, at least one Ry is fluoro. In certain embodiments, at least one Ry is chloro. In some embodiments, at least one Ry is —CN. In some embodiments, at least one Ry is —ORA, wherein RA is optionally substituted aliphatic. In some embodiments, at least one Ry is —ORA, wherein RA is unsubstituted C1-6 alkyl. In certain embodiments, at least one Ry is methoxy, ethoxy, or propoxy. In certain embodiments, at least one Ry is methoxy. In some embodiments, at least one Ry is —ORA, wherein RA is substituted C1-6 alkyl. In certain embodiments, at least one Ry is —OCH2CH2N(CH3)2. In some embodiments, at least one Ry is —ORA, wherein RA is optionally substituted heterocyclyl. In some embodiments, at least one Ry is —ORA, wherein RA is an optionally substituted 4- to 7-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, at least one Ry is —ORA, wherein RA is oxetanyl, tetrahydrofuranyl, or tetrahydropyranyl. In some embodiments, at least one Ry is —N(RB)2. In some embodiments, at least one Ry is —N(RB)2, wherein each RB is independently hydrogen or C1-6 alkyl. In some embodiments, at least one Ry is —NHRB. In some embodiments, at least one Ry is —N(C1-6 alkyl)2, —NH(C1-6 alkyl), or —NH2. In certain embodiments, at least one Ry is —NH2. In certain embodiments, at least one Ry is —NHCH3. In certain embodiments, at least one Ry is —N(CH3)2. In some embodiments, at least one Ry is —N(RB)2 or —NHRB, wherein at least one RB is optionally substituted heterocyclyl. In some embodiments, at least one Ry is —N(RB)2 or —NHRB, wherein at least one RB is an optionally substituted 4- to 7-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, at least one Ry is —N(RB)2 or —NHRB, wherein at least one RB is oxetanyl, tetrahydropyranyl, or tetrahydrofuranyl. In some embodiments, at least one Ry is —N(RB)2 or —NHRB, wherein at least one RB is optionally substituted piperidinyl or optionally substituted piperazinyl.
In some embodiments, at least one Ry is optionally substituted aliphatic. In certain embodiments, at least one Ry is substituted aliphatic. In certain embodiments, at least one Ry is unsubstituted aliphatic. In some embodiments, at least one Ry is optionally substituted C1-6 alkyl. In certain embodiments, at least one Ry is unsubstituted C1-6 alkyl. In certain embodiments, at least one Ry is substituted C1-6 alkyl. In certain embodiments, at least one Ry is methyl, ethyl, or propyl. In certain embodiments, at least one Ry is methyl. In certain embodiments, at least one Ry is —CF3, CHF2, or CH2F. In certain embodiments, at least one Ry is C1-6 alkyl substituted with aryl, heteroaryl, or heterocyclyl. In certain embodiments, at least one Ry is benzyl. In certain embodiments, at least one Ry is —(C1-6 alkyl)-aryl. In certain embodiments, at least one Ry is —(C1-6 alkyl)-heteroaryl. In certain embodiments, at least one Ry is —(C1-6 alkyl)-heterocyclyl. In certain embodiments, at least one Ry is —CH2-aryl. In certain embodiments, at least one Ry is —CH2-heteroaryl. In certain embodiments, at least one Ry is —CH2-heterocyclyl.
In some embodiments, at least one Ry is —C(O)N(RB)2. In certain embodiments, at least one Ry is —C(O)NHRB. In certain embodiments, at least one Ry is —C(O)NH2. In certain embodiments, at least one Ry is —C(O)N(RB)2, wherein the RB groups are taken together with their intervening atoms to form an optionally substituted 5- to 6-membered heterocyclyl. In certain embodiments, at least one Ry is —C(O)N(RB)2, wherein the RB groups are taken together with their intervening atoms to form an optionally substituted morpholinyl.
In some embodiments, at least one Ry is —SO2N(RB)2. In certain embodiments, at least one Ry is —SO2NHRB. In certain embodiments, at least one Ry is —SO2NH2. In certain embodiments, at least one Ry is —SO2N(RB)2, wherein neither RB is hydrogen. In certain embodiments, at least one Ry is —SO2NH(C1-6 alkyl) or —SO2N(C1-6 alkyl)2. In certain embodiments, at least one Ry is —SO2N(CH3)2. In certain embodiments, at least one Ry is —SO2N(RB)2, wherein the RB groups are taken together with their intervening atoms to form an optionally substituted 5- to 6-membered heterocyclyl. In certain embodiments, at least one Ry is —SO2-morpholinyl. In certain embodiments, at least one Ry is —SO2-piperidinyl, —SO2-piperazinyl, or —SO2-piperidinyl.
In some embodiments, at least one Ry is —SO2RA. In some embodiments, at least one Ry is —SO2RA, wherein RA is optionally substituted aliphatic. In some embodiments, at least one Ry is —SO2(C1-6 alkyl). In some embodiments, at least one Ry is —SO2CH3. In some embodiments, at least one Ry is —C(O)RA. In some embodiments, at least one Ry is —C(O)RA, wherein RA is optionally substituted aliphatic. In some embodiments, at least one Ry is —C(O)(C1-6 alkyl). In some embodiments, at least one Ry is —C(O)CH3.
In some embodiments, at least one Ry is —N(RB)C(O)RA. In certain embodiments, at least one Ry is —NHC(O)RA. In certain embodiments, at least one Ry is —NHC(O)(C1-6 alkyl). In certain embodiments, at least one Ry is —NHC(O)CH3.
In some embodiments, at least one Ry is —N(RB)SO2RA. In some embodiments, at least one Ry is —NHSO2RA. In some embodiments, at least one Ry is —N(C1-6 alkyl)SO2RA. In certain embodiments, at least one Ry is —NHSO2(C1-6 alkyl) or —N(C1-6 alkyl)SO2(C1-6 alkyl). In certain embodiments, at least one Ry is —NHSO2CH3. In certain embodiments, at least one Ry is —N(CH3)SO2CH3.
In some embodiments, at least one Ry is optionally substituted heterocyclyl, optionally substituted carbocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, at least one Ry is an optionally substituted 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is an optionally substituted 5-membered heterocyclyl having one heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is optionally substituted pyrrolidinyl. In certain embodiments, at least one Ry is pyrroldinyl, hydroxypyrrolidinyl, or methylpyrrolidinyl. In certain embodiments, at least one Ry is an optionally substituted 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is an optionally substituted 6-membered heterocyclyl having one heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is optionally substituted piperidinyl. In certain embodiments, at least one Ry is an optionally substituted 6-membered heterocyclyl having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is optionally substituted piperdinyl, optionally substituted piperazinyl, or optionally substituted morpholinyl. In certain embodiments, at least one Ry is morpholinyl, tetrahydropyranyl, piperidinyl, methylpiperidinyl, piperazinyl, methylpiperazinyl, acetylpiperazinyl, methylsulfonylpiperazinyl, aziridinyl, or methylaziridinyl. In some embodiments, at least one Ry is an optionally substituted 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is an optionally substituted 5-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is an optionally substituted 5-membered heteroaryl having one heteroatom selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is an optionally substituted 5-membered heteroaryl having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, at least one Ry is an optionally substituted 6-membered heteroaryl having 1-3 nitrogens. In certain embodiments, at least one Ry is an optionally substituted pyrazolyl. In certain embodiments, at least one Ry is an optionally substituted imidazolyl. In certain embodiments, at least one Ry is an optionally substituted pyridyl. In certain embodiments, at least one Ry is an optionally substituted pyrimidyl. In certain embodiments, at least one Ry is pyrazolyl, methylpyrazolyl, imidazolyl, or methylimidazolyl.
In some embodiments, Ry is —ORA. In some embodiments, Ry is —ORA, wherein RA is optionally substituted heterocyclyl. In some embodiments, Ry is —ORA, wherein RA is optionally substituted heteroaryl. In some embodiments, Ry is —ORA, wherein RA is optionally substituted cycloalkyl. In some embodiments, Ry is —N(RB)2. In some embodiments, Ry is —NHRB. In some embodiments, Ry is —NHRB, wherein RB is optionally substituted heterocyclyl. In some embodiments, Ry is —NHRB, wherein RB is optionally substituted heteroaryl. In some embodiments, Ry is —NHRB, wherein RB is optionally substituted cycloalkyl. In some embodiments, Ry is —N(RB)2, wherein one RB is optionally substituted heterocyclyl, and the other RB s C1-4 alkyl. In some embodiments, Ry is —N(RB)2, wherein one RB is optionally substituted heteroaryl, and the other RB is C1-4 alkyl. In some embodiments, Ry is —N(RB)2, wherein one RB is optionally substituted cycloalkyl, and the other RB is C1-4 alkyl.
As defined generally above, each Rx is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR′, and —N(R′′)2. In certain embodiments, at least one Rx is halo. In certain embodiments, at least one Rx is fluoro. In certain embodiments, at least one Rx is —CN. In certain embodiments, at least one Rx is optionally substituted aliphatic. In certain embodiments, at least one Rx is optionally substituted C1-6 alkyl. In certain embodiments, at least one Rx is methyl. In certain embodiments, at least one Rx is —CF3. In certain embodiments, at least one Rx is —OR′ or —N(R′′)2. In certain embodiments, Rx is not —OR′ or —N(R′′)2. In certain embodiments, at least one Rx is —OCH3. In certain embodiments, Rx is not —OCH3.
As is generally understood from the above disclosure, the ring system:




is a fused bicyclic ring system, i.e., a phenyl ring fused to a nitrogen containing ring, wherein the point of attachment to the parent moiety is on the nitrogen, and wherein the fused bicyclic system is optionally substituted with (Rx)n, wherein n and Rx are as defined herein. As is generally understood, each of the atoms of the phenyl ring and the nitrogen-containing ring can be independently optionally substituted with Rx, as valency permits.

In certain embodiments, the fused bicyclic ring system is optionally substituted with one or more Rx, with the proviso that when the nitrogen-containing ring is substituted at one of the positions alpha to the nitrogen, Rx is not —C(═O)Rx1, wherein Rx1 is optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, or —SRA, wherein RA and RB are as generally defined herein. In certain embodiments, the nitrogen-containing ring does not comprise an Rx substituent. In certain embodiments, only atoms of the phenyl ring are optionally substituted with one or more Rx.
In certain embodiments, the nitrogen-containing ring is optionally substituted and the fused bicyclic ring system is of the formula:




wherein Rx is as defined above, and n is 0, 1, 2, 3, or 4.

Thus, one of ordinary skill in the art will appreciate that an Rx group can be attached anywhere on the tetrahydroisoquinoline or dihydroisoquinoline ring. In certain embodiments, an Rx group is attached to the benzene portion of the tetrahydroisoquinoline or dihydroisoquinoline ring. In certain embodiments, an Rx group is attached to the tetrahydropyridine or dihydropyridine portion of the tetrahydroisoquinoline or dihydroisoquinoline ring. In certain embodiments, Rx groups are attached to both the benzene portion and the tetrahydropyridine (or dihydropyridine) portion of the tetrahydroisoquinoline (or dihydroisoquinoline) ring. See, for example, the structures shown below:




In certain embodiments, a provided compound is of Formula (VII):




or a pharmaceutically acceptable salt thereof.

As defined generally above, n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2.
As generally defined above, RA1 and RA2 are independently hydrogen, substituted or unsubstituted C1-3 alkyl, substituted or unsubstituted acyl, or a nitrogen protecting group. In some embodiments, RA1 is hydrogen. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is unsubstituted C1-3 alkyl. In some embodiments, RA1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, RA1 is substituted C1-3 alkyl. In some embodiments, RA1 is —CF3, —CHF2, —CH2F, or —CH(CF3)CH3. In some embodiments, RA1 is substituted or unsubstituted acyl. In some embodiments, RA1 is acetyl. In some embodiments, RA1 is a nitrogen protecting group. In some embodiments, RA1 is CH3SO2—. In some embodiments, RA2 is hydrogen. In some embodiments, RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA2 is unsubstituted C1-3 alkyl. In some embodiments, RA2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, RA2 is substituted C1-3 alkyl. In some embodiments, RA2 is —CF3, —CHF2, —CH2F, or —CH(CF3)CH3. In some embodiments, RA2 is substituted or unsubstituted acyl. In some embodiments, RA2 is acetyl. In some embodiments, RA2 is a nitrogen protecting group. In some embodiments, RA2 is CH3SO2—. In some embodiments, RA1 is hydrogen, and RA2 is hydrogen. In some embodiments, RA1 is hydrogen, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is hydrogen, and RA2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, RA1 is hydrogen, and RA2 is —CF3, —CHF2, —CH2F, or —CH(CF3)CH3. In some embodiments, RA1 is hydrogen, and RA2 is substituted or unsubstituted acyl. In some embodiments, RA1 is hydrogen, and RA2 is acetyl. In some embodiments, RA1 is hydrogen, and RA2 is a nitrogen protecting group. In some embodiments, RA1 is hydrogen and RA2 is CH3SO2—. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is methyl. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is ethyl. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is n-propyl. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is isopropyl. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is substituted or unsubstituted acyl. In some embodiments, RA1 is substituted or unsubstituted C1-3 alkyl, and RA2 is a nitrogen protecting group. In some embodiments, RA1 is methyl, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is methyl, and RA2 is methyl. In some embodiments, RA1 is methyl, and RA2 is ethyl. In some embodiments, RA1 is methyl, and RA2 is n-propyl. In some embodiments, RA1 is methyl, and RA2 is isopropyl. In some embodiments, RA1 is methyl, and RA2 is substituted or unsubstituted acyl. In some embodiments, RA1 is methyl, and RA2 is a nitrogen protecting group. In some embodiments, RA1 is ethyl, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is ethyl, and RA2 is methyl. In some embodiments, RA1 is ethyl, and RA2 is ethyl. In some embodiments, RA1 is ethyl, and RA2 is n-propyl. In some embodiments, RA1 is ethyl, and RA2 is isopropyl. In some embodiments, RA1 is ethyl, and RA2 is substituted or unsubstituted acyl. In some embodiments, RA1 is ethyl, and RA2 is a nitrogen protecting group. In some embodiments, RA1 is n-propyl, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is n-propyl, and RA2 is methyl. In some embodiments, RA1 is n-propyl, and RA2 is ethyl. In some embodiments, RA1 is n-propyl, and RA2 is n-propyl. In some embodiments, RA1 is n-propyl and RA2 is isopropyl. In some embodiments, RA1 is n-propyl, and RA2 is substituted or unsubstituted acyl. In some embodiments, RA1 is n-propyl and RA2 is a nitrogen protecting group. In some embodiments, RA1 is isopropyl and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is isopropyl and RA2 is methyl. In some embodiments, RA1 is isopropyl and RA2 is ethyl. In some embodiments, RA1 is isopropyl, and RA2 is n-propyl. In some embodiments, RA1 is isopropyl, and RA2 is isopropyl. In some embodiments, RA1 is isopropyl, and RA2 is substituted or unsubstituted acyl. In some embodiments, RA1 is isopropyl, and RA2 is a nitrogen protecting group. In some embodiments, RA1 is substituted or unsubstituted acyl, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is a nitrogen protecting group, and RA2 is substituted or unsubstituted C1-3 alkyl. In some embodiments, RA1 is a nitrogen protecting group and RA2 is methyl. In some embodiments, RA1 is a nitrogen protecting group, and RA2 is ethyl. In some embodiments, RA1 is a nitrogen protecting group, and RA2 is n-propyl. In some embodiments, RA1 is a nitrogen protecting group, and RA2 is isopropyl. In some embodiments, RA1 is a nitrogen protecting group, and RA2 is a nitrogen protecting group.
As generally defined above, RA1 and RA2 can be taken together with the intervening nitrogen atom to form a substituted or unsubstituted 3-6 membered heterocyclic ring. In certain embodiments, RA1 and RA2 can be taken together with the intervening nitrogen atom to form a substituted or unsubstituted azetidine. In certain embodiments, RA1 and RA2 can be taken together with the intervening nitrogen atom to form a substituted or unsubstituted pyrrolidine. In certain embodiments, RA1 and RA2 can be taken together with the intervening nitrogen atom to form a substituted or unsubstituted piperidine. In certain embodiments, RA1 and RA2 can be taken together with the intervening nitrogen atom to form a substituted or unsubstituted piperazine. In certain embodiments, RA1 and RA2 can be taken together with the intervening nitrogen atom to form a substituted or unsubstituted morpholine.
In some embodiments, e.g. for Formula (A), Formula (I), or any subgenera thereof, the provided compound is of a free base form. In some embodiments, e.g. for Formula (A), Formula (I), or any subgenera thereof, the provided compound is in the form of a pharmaceutically acceptable salt as generally defined herein. In some embodiments, the provided compound is a hydrochloride salt thereof. In some embodiments, the provided compound is a tartrate salt thereof. In some embodiments, the provided compound is a monotartrate salt thereof. In some embodiments, the provided compound is a bitartrate salt thereof.
In certain embodiments, a provided compound is a compound listed in Table 1A, or a pharmaceutically acceptable salt thereof.







TABLE 1A







Exemplary Compounds










Cmpd


LCMS m/z


No
Structure
Exact Mass
(M + H)





 1




322.1681
323.1


 


 2




284.1525
285.1


 


 3




379.226 
380.2


 


 4




416.2212
417.2


 


 5




399.1714
400.1


 


 6




383.2009
384.2


 


 7




399.1714
400.1


 


 8




451.2583
452.3


 


 9




399.1714
400.1


 


10




379.226 
380.2


 


11




383.2009
384.2


 


12




450.2631
451.2


 


13




360.1838
361.2


 


14




360.1838
361.2


 


15




379.226 
380.2


 


16




364.2151
365.2


 


17




364.2151
365.2


 


18




375.1947
376.2


 


19




375.1947
376.2


 


20




395.2209
396.2


 


21




331.1896
332.1


 


22




367.1566
368.1


 


23




373.2365
374.2


 


24




379.226 
380.1


 


25




380.2212
381.2


 


26




380.2212
381.1


 


27




303.1947
304.1


 


28




290.163 
291.1


 


29




408.2525
409.3


 


30




408.2525
407.2


 


31




338.163 
339.2


 


32




401.2315
402.2


 


33




411.2158
412.1


 


34




412.2111
413.1









In certain embodiments, a provided compound is a compound listed in Table 1B, or a pharmaceutically acceptable salt thereof.







TABLE 1B







Exemplary Compounds










Cmpd


LCMS m/z


No
Structure
Exact Mass
(M + H)





35




449.2791
—


 


36




463.2947
—


 


37




477.3104
—


 


38




491.2896
—


 


39




527.2566
—


 


40




502.2556
—


 


41




545.2978
—


 


42




517.3417
—


 


43




518.3369
—


 


44




519.3209
—


 


45




489.3104
—


 


46




503.3260
—









In certain embodiments, a provided compound inhibits PRMT5. In certain embodiments, a provided compound inhibits wild-type PRMT5. In certain embodiments, a provided compound inhibits a mutant PRMT5. In certain embodiments, a provided compound inhibits PRMT5, e.g., as measured in an assay described herein. In certain embodiments, the PRMT5 is from a human. In certain embodiments, a provided compound inhibits PRMT5 at an IC50 less than or equal to 10 μM. In certain embodiments, a provided compound inhibits PRMT5 at an IC50 less than or equal to 1 μM. In certain embodiments, a provided compound inhibits PRMT5 at an IC50 less than or equal to 0.1 μM. In certain embodiments, a provided compound inhibits PRMT5 in a cell at an EC50 less than or equal to 10 μM. In certain embodiments, a provided compound inhibits PRMT5 in a cell at an EC50 less than or equal to 1 μM. In certain embodiments, a provided compound inhibits PRMT5 in a cell at an EC50 less than or equal to 0.1 μM. In certain embodiments, a provided compound inhibits cell proliferation at an EC50 less than or equal to 10 μM. In certain embodiments, a provided compound inhibits cell proliferation at an EC50 less than or equal to 1 μM. In certain embodiments, a provided compound inhibits cell proliferation at an EC50 less than or equal to 0.1 μM. In some embodiments, a provided compound is selective for PRMT5 over other methyltransferases. In certain embodiments, a provided compound is at least about 10-fold selective, at least about 20-fold selective, at least about 30-fold selective, at least about 40-fold selective, at least about 50-fold selective, at least about 60-fold selective, at least about 70-fold selective, at least about 80-fold selective, at least about 90-fold selective, or at least about 100-fold selective for PRMT5 relative to one or more other methyltransferases.
It will be understood by one of ordinary skill in the art that the PRMT5 can be wild-type PRMT5, or any mutant or variant of PRMT5.
In some embodiments embodiment, the mutant or variant of PRMT5 contains one or more mutations (e.g., conservative substitutions). In some embodiments, provided herein is a PRMT5 point mutant. In some embodiments, the PRMT point mutant has an amino acid sequence that a degree of homology to the amino acid sequence of SEQ ID NO: 1 of at least about 80%, e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 97%. Further provided is a protein that has a degree of homology to the amino acid sequence of SEQ ID NO: 2 of at least about 80%, e.g., at least about 85%, at least about 90%, at least about 95%, or at least about 97%.
In certain embodiments, the PRMT5 is isoform A (GenBank accession no. NP006100) (SEQ ID NO.:1):









MAAMAVGGAG GSRVSSGRDL NCVPEIADTL GAVAKQGFDF


 



LCMPVFHPRF KREFIQEPAK NRPGPQTRSD LLLSGRDWNT


 



LIVGKLSPWI RPDSKVEKIR RNSEAAMLQE LNFGAYLGLP


 



AFLLPLNQED NTNLARVLTN HIHTGHHSSM FWMRVPLVAP


 



EDLRDDIIEN APTTHTEEYS GEEKTWMWWH NFRTLCDYSK


 



RIAVALEIGA DLPSNHVIDR WLGEPIKAAI LPTSIFLTNK


 



KGFPVLSKMH QRLIFRLLKL EVQFIITGTN HHSEKEFCSY


 



LQYLEYLSQN RPPPNAYELF AKGYEDYLQS PLQPLMDNLE


 



SQTYEVFEKD PIKYSQYQQA IYKCLLDRVP EEEKDTNVQV


 



LMVLGAGRGP LVNASLRAAK QADRRIKLYA VEKNPNAVVT


 



LENWQFEEWG SQVTVVSSDM REWVAPEKAD IIVSELLGSF


 



ADNELSPECL DGAQHFLKDD GVSIPGEYTS FLAPISSSKL


 



YNEVRACREK DRDPEAQFEM PYVVRLHNFH QLSAPQPCFT


 



FSHPNRDPMI DNNRYCTLEF PVEVNTVLHG FAGYFETVLY


 



QDITLSIRPE THSPGMFSWF PILFPIKQPI TVREGQTICV


 



RFWRCSNSKK VWYEWAVTAP VCSAIHNPTG RSYTIGL






In certain embodiments, the PRMT5 is isoform B (GenBank accession no. NP001034708) (SEQ ID NO.:2)









MRGPNSGTEK GRLVIPEKQG FDFLCMPVFH PRFKREFIQE


 



PAKNRPGPQT RSDLLLSGRD WNTLIVGKLS PWIRPDSKVE


 



KIRRNSEAAM LQELNFGAYL GLPAFLLPLN QEDNTNLARV


 



LTNHIHTGHH SSMFWMRVPL VAPEDLRDDI IENAPTTHTE


 



EYSGEEKTWM WWHNFRTLCD YSKRIAVALE IGADLPSNHV


 



IDRWLGEPIK AAILPTSIFL TNKKGFPVLS KMHQRLIFRL


 



LKLEVQFIIT GTNHHSEKEF CSYLQYLEYL SQNRPPPNAY


 



ELFAKGYEDY LQSPLQPLMD NLESQTYEVF EKDPIKYSQY


 



QQAIYKCLLD RVPEEEKDTN VQVLMVLGAG RGPLVNASLR


 



AAKQADRRIK LYAVEKNPNA VVTLENWQFE EWGSQVTVVS


 



SDMREWVAPE KADIIVSELL GSFADNELSP ECLDGAQHFL


 



KDDGVSIPGE YTSFLAPISS SKLYNEVRAC REKDRDPEAQ


 



FEMPYVVRLH NFHQLSAPQP CFTFSHPNRD PMIDNNRYCT


 



LEFPVEVNTV LHGFAGYFET VLYQDITLSI RPETHSPGMF


 



SWFPILFPIK QPITVREGQT ICVRFWRCSN SKKVWYEWAV


 



TAPVCSAIHN PTGRSYTIGL






In certain embodiments, the PRMT5 is transcript variant 1 (GenBank accession no. NM_006109).
The present disclosure provides pharmaceutical compositions comprising a compound described herein, e.g., a compound of Formula (A), e.g., Formula (I), or a pharmaceutically acceptable salt thereof, as described herein, and optionally a pharmaceutically acceptable excipient. It will be understood by one of ordinary skill in the art that the compounds described herein, or salts thereof, may be present in various forms, such as amorphous, hydrates, solvates, or polymorphs. In certain embodiments, a provided composition comprises two or more compounds described herein. In certain embodiments, a compound described herein, or a pharmaceutically acceptable salt thereof, is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is an amount effective for inhibiting PRMT5. In certain embodiments, the effective amount is an amount effective for treating a PRMT5-mediated disorder. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective to prevent a PRMT5-mediated disorder.
In certain embodiments, the provided pharmaceutical compositions comprise a compound described herein, e.g., a compound of Formula (A), e.g., Formula (I), or any subgenera thereof, and optionally a pharmaceutically acceptable excipient, wherein the compound is of a free base form. In certain embodiments, the provided pharmaceutical compositions comprise a compound described herein, e.g., a compound of Formula (A), e.g., Formula (I), or any subgenera thereof, and optionally a pharmaceutically acceptable excipient, wherein the compound is in the form of a pharmaceutically acceptable salt as generally defined herein. In certain embodiments, the provided pharmaceutical compositions comprise a hydrochloride salt of a compound described herein and optionally a pharmaceutically acceptable excipient. In certain embodiments, the provided pharmaceutical compositions comprise a tartrate salt of a compound described herein and optionally a pharmaceutically acceptable excipient. In certain embodiments, the provided pharmaceutical compositions comprise a monotartrate salt of a compound described herein and optionally a pharmaceutically acceptable excipient. In certain embodiments, the provided pharmaceutical compositions comprise a bitartrate salt of a compound described herein and optionally a pharmaceutically acceptable excipient. In certain embodiments, the provided pharmaceutical compositions comprise a monotartrate salt and a bitartrate salt of a compound described herein and optionally a pharmaceutically acceptable excipient. In certain embodiments, the provided pharmaceutical compositions comprise a compound described herein in a form of free base, and a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.
Pharmaceutically acceptable excipients include any and all solvents, diluents, or other liquid vehicles, dispersions, 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. General considerations in formulation and/or manufacture of pharmaceutical compositions agents can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).
Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing a compound described herein (the “active ingredient”) into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60], sorbitan tristearate (Span 65), glyceryl monooleate, sorbitan monooleate (Span 80)), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CremophorTM), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.
Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.
Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.
Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise 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 (e.g., 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 include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the compounds described herein are mixed with solubilizing agents such as CremophorTM, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can 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 can 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.
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.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the compounds described herein 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 ingredient.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one 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, 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 comprise buffering agents.
Solid compositions of a similar type can 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 comprise opacifying agents and can 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 which can be used include polymeric substances and waxes. Solid compositions of a similar type can 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 active ingredient can 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 ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may 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 comprise buffering agents. They may optionally comprise opacifying agents and can 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 which can be used include polymeric substances and waxes.
Dosage forms for topical and/or transdermal administration of a provided compound may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and/or any desired preservatives and/or buffers as can be required. Additionally, the present disclosure encompasses the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration.
Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
A provided pharmaceutical composition can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A provided pharmaceutical composition can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
A provided pharmaceutical composition can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this disclosure.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
Compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of provided compositions will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease, disorder, or condition being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.
In certain embodiments, a compound described herein may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or 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.
In some embodiments, a compound described herein is administered one or more times per day, for multiple days. In some embodiments, the dosing regimen is continued for days, weeks, months, or years.
It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
It will be also appreciated that a compound or composition, as described herein, can be administered in combination with one or more additional therapeutically active agents. In certain embodiments, a compound or composition provided herein is administered in combination with one or more additional therapeutically active agents that improve its bioavailability, reduce and/or modify its metabolism, inhibit its excretion, and/or modify its distribution within the body. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.
The compound or composition can be administered concurrently with, prior to, or subsequent to, one or more additional therapeutically active agents. In certain embodiments, the additional therapeutically active agent is a compound of Formula (A), e.g., Formula (I). In certain embodiments, the additional therapeutically active agent is not a compound of Formula (A), e.g., Formula (I). In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of a provided compound with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved. In general, it is expected that additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
Exemplary additional therapeutically active agents include, but are not limited to, small organic molecules such as drug compounds (e.g., compounds approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
Also encompassed by the present disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a provided pharmaceutical composition or compound and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a provided pharmaceutical composition or compound. In some embodiments, a provided pharmaceutical composition or compound provided in the container and the second container are combined to form one unit dosage form. In some embodiments, a provided kits further includes instructions for use.
Compounds and compositions described herein are generally useful for the inhibition of PRMT5. In some embodiments, methods of treating PRMT5-mediated disorder in a subject are provided which comprise administering an effective amount of a compound described herein (e.g., a compound of Formula (A), e.g., Formula (I)), or a pharmaceutically acceptable salt thereof), to a subject in need of treatment. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the subject is suffering from a PRMT5-mediated disorder. In certain embodiments, the subject is susceptible to a PRMT5-mediated disorder.
As used herein, the term “PRMT5-mediated disorder” means any disease, disorder, or other pathological condition in which PRMT5 is known to play a role. Accordingly, in some embodiments, the present disclosure relates to treating or lessening the severity of one or more diseases in which PRMT5 is known to play a role.
In some embodiments, the present disclosure provides a method of inhibiting PRMT5 comprising contacting PRMT5 with an effective amount of a compound described herein (e.g., a compound of Formula (A), e.g., Formula (I)), or a pharmaceutically acceptable salt thereof. The PRMT5 may be purified or crude, and may be present in a cell, tissue, or subject. Thus, such methods encompass both inhibition of in vitro and in vivo PRMT5 activity. In certain embodiments, the method is an in vitro method, e.g., such as an assay method. It will be understood by one of ordinary skill in the art that inhibition of PRMT5 does not necessarily require that all of the PRMT5 be occupied by an inhibitor at once. Exemplary levels of inhibition of PRMT5 include at least 10% inhibition, about 10% to about 25% inhibition, about 25% to about 50% inhibition, about 50% to about 75% inhibition, at least 50% inhibition, at least 75% inhibition, about 80% inhibition, about 90% inhibition, and greater than 90% inhibition.
In some embodiments, provided is a method of inhibiting PRMT5 activity in a subject in need thereof comprising administering to the subject an effective amount of a compound described herein (e.g., a compound of Formula (A), e.g., Formula (I)), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
In certain embodiments, provided is a method of altering gene expression in a cell which comprises contacting a cell with an effective amount of a compound of Formula (A), e.g., Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the cell in culture in vitro. In certain embodiments, the cell is in an animal, e.g., a human. In certain embodiments, the cell is in a subject in need of treatment.
In certain embodiments, provided is a method of altering transcription in a cell which comprises contacting a cell with an effective amount of a compound of Formula (A), e.g., Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the cell in culture in vitro. In certain embodiments, the cell is in an animal, e.g., a human. In certain embodiments, the cell is in a subject in need of treatment.
In certain embodiments, a method is provided of selecting a therapy for a subject having a disease associated with PRMT5-mediated disorder or mutation comprising the steps of determining the presence of PRMT5-mediated disorder or gene mutation in the PRMT5 gene or and selecting, based on the presence of PRMT5-mediated disorder a gene mutation in the PRMT5 gene a therapy that includes the administration of a provided compound. In certain embodiments, the disease is cancer.
In certain embodiments, a method of treatment is provided for a subject in need thereof comprising the steps of determining the presence of PRMT5-mediated disorder or a gene mutation in the PRMT5 gene and treating the subject in need thereof, based on the presence of a PRMT5-mediated disorder or gene mutation in the PRMT5 gene with a therapy that includes the administration of a provided compound. In certain embodiments, the subject is a cancer patient.
In some embodiments, a provided compound is useful in treating a proliferative disorder, such as cancer, a benign neoplasm, an autoimmune disease, or an inflammatory disease. For example, while not being bound to any particular mechanism, PRMT5 has been shown to be involved in cyclin D1 dysregulated cancers. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth including CUL4 repression, CDT1 overexpression, and DNA re-replication. Further, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity. See, e.g., Aggarwal et al., Cancer Cell. (2010) 18(4):329-40. Additionally, PRMT5 has also been implicated in accelerating cell cycle progression through G1 phase and modulating regulators of G1; for example, PRMT5 may upregulate cyclin-dependent kinase (CDK) 4, CDK6, and cyclins D1, D2 and E1. Moreover, PRMT5 may activate phosphoinositide 3-kinase (PI3K)/AKT signaling. See, e.g., Wei et al., Cancer Sci. (2012) 103(9):1640-50. PRMT5 has been reported to play a role in apoptosis through methylation of E2F-1. See, e.g., Cho et al., EMBO J. (2012) 31:1785-1797; Zheng et al., Mol. Cell. (2013) 52:37-51. PRMT5 has been reported to be an essential regulator of splicing and affect the alternative splicing of ‘sensor’ mRNAs that can then lead to defects in downstream events such as apoptosis. See, e.g., Bezzi et al., Genes Dev. (2013) 27:1903-1916. PRMT5 has been reported to play a role in the RAS-ERK pathway. See, e.g., Andrew-Perez et al., Sci Signal. (2011) September 13; 4(190)ra58 doi: 10.1126/scisignal.2001936. PRMT5 has been reported to affect C/EBPb target genes through interaction with the Mediator complex and hence affect cellular differentiation and inflammatory response. See, e.g., Tsutsui et al., J. Biol. Chem. (2013) 288:20955-20965. PRMT5 has been shown to methylate HOXA9 essential for ELAM expression during the EC inflammatory response. See, e.g., Bandyopadhyay et al., Mol. Cell. Biol. (2012) 32:1202-1203. Thus in some embodiments, the inhibition of PRMT5 by a provided compound is useful in treating the following non-limiting list of cancers: breast cancer, esophageal cancer, bladder cancer, lung cancer, hematopoietic cancer, lymphoma, medulloblastoma, rectum adenocarcinoma, colon adenocarcinoma, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, brain tumors, hepatocellular carcinoma, renal cell carcinoma, melanoma, oligodendroglioma, ovarian clear cell carcinoma, and ovarian serous cystadenocarcinoma. See, e.g., Pal et al., EMBO J. (2007) 26:3558-3569 (mantle cell lymphoma); Wang et al., Mol. Cell Biol. (2008) 28:6262-77 (chronic lymphocytic leukemia (CLL)); and Tae et al., Nucleic Acids Res. (2011) 39:5424-5438.
In some embodiments, the inhibition of PRMT5 by a provided compound is useful in treating prostate cancer and lung cancer, in which PRMT5 has been shown to play a role. See, e.g., Gu et al., PLoS One 2012; 7(8):e44033; Gu et al., Biochem. J. (2012) 446:235-241. In some embodiments, a provided compound is useful to delay the onset of, slow the progression of, or ameliorate the symptoms of cancer. In some embodiments, a provided compound is administered in combination with other compounds, drugs, or therapeutics to treat cancer.
In some embodiments, compounds described herein are useful for treating a cancer including, but not limited to, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenström's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, and vulvar cancer (e.g., Paget's disease of the vulva).
In some embodiments, a provided compound is useful in treating a metabolic disorder, such as diabetes or obesity. For example, while not being bound to any particular mechanism, a role for PRMT5 has been recognized in adipogenesis. Inhibition of PRMT5 expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes, while overexpression of PRMT5 enhanced adipogenic gene expression and differentiation. See, e.g., LeBlanc et al., Mol Endocrinol. (2012) 26:583-597. Additionally, it has been shown that adipogenesis plays a pivotal role in the etiology and progression of diabetes and obesity. See, e.g., Camp et al., Trends Mol Med. (2002) 8:442-447. Thus in some embodiments, the inhibition of PRMT5 by a provided compound is useful in treating diabetes and/or obesity.
In some embodiments, a provided compound is useful to delay the onset of, slow the progression of, or ameliorate the symptoms of, diabetes. In some embodiments, the diabetes is Type 1 diabetes. In some embodiments, the diabetes is Type 2 diabetes. In some embodiments, a provided compound is useful to delay the onset of, slow the progression of, or ameliorate the symptoms of, obesity. In some embodiments, a provided compound is useful to help a subject lose weight. In some embodiments, a provided compound could be used in combination with other compounds, drugs, or therapeutics, such as metformin and insulin, to treat diabetes and/or obesity.
In some embodiments, a provided compound is useful in treating a blood disorder, e.g., a hemoglobinopathy, such as sickle cell disease or β-thalassemia. For example, while not being bound to any particular mechanism, PRMT5 is a known repressor of γ-globin gene expression, and increased fetal γ-globin (HbF) levels in adulthood are associated with symptomatic amelioration in sickle cell disease and β-thalassemia. See, e.g., Xu et al., Haematologica. (2012) 97:1632-1640; Rank et al. Blood. (2010) 116:1585-1592. Thus in some embodiments, the inhibition of PRMT5 by a provided compound is useful in treating a blood disorder, such as a hemoglobinopathy such as sickle cell disease or β-thalassemia.
In some embodiments, a provided compound is useful to delay the onset of, slow the progression of, or ameliorate the symptoms of, sickle cell disease. In some embodiments, a provided compound is useful to delay the onset of, slow the progression of, or ameliorate the symptoms of, β-thalassemia. In some embodiments, a provided compound could be used in combination with other compounds, drugs, or therapeutics, to treat a hemoglobinopathy such as sickle cell disease or β-thalassemia.
In some embodiments, a provided compound is useful in treating inflammatory and autoimmune disease. PRMT5 is reported to activate NFkB signaling pathway through the methylation of p65. PRMT5 is reported to interact with Death receptor 4 and Death receptor 5 contributing to TRAIL-induced activation of inhibitor or kB kinase (IKK) and nuclear factor-kB (NF-kB). See, e.g., Tanaka et al., Mol. Cancer. Res. (2009) 7:557-569; Wei et al., Proc. Nat'l. Acad. Sci. USA (2013) 110:13516-21.
The term “inflammatory disease” refers to those diseases, disorders or conditions that are characterized by signs of pain (dolor, from the generation of noxious substances and the stimulation of nerves), heat (calor, from vasodilatation), redness (rubor, from vasodilatation and increased blood flow), swelling (tumor, from excessive inflow or restricted outflow of fluid), and/or loss of function (functio laesa, which can be partial or complete, temporary or permanent. Inflammation takes on many forms and includes, but is not limited to, acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and/or ulcerative inflammation.
Exemplary inflammatory diseases include, but are not limited to, inflammation associated with acne, anemia (e.g., aplastic anemia, haemolytic autoimmune anaemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis, bronchitis, bursitis, chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type I diabetes mellitus, type 2 diabetes mellitus), a skin condition (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), endometriosis, Guillain-Barre syndrome, infection, ischaemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine headaches, tension headaches), ileus (e.g., postoperative ileus and ileus during sepsis), idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosis, morphea, myeasthenia gravis, myocardial ischemia, nephrotic syndrome, pemphigus vulgaris, pernicious aneaemia, peptic ulcers, polymyositis, primary biliary cirrhosis, neuroinflammation associated with brain disorders (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, reperfusion injury, regional enteritis, rheumatic fever, systemic lupus erythematosus, schleroderma, scierodoma, sarcoidosis, spondyloarthopathies, Sjogren's syndrome, thyroiditis, transplantation rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scarring, burns, physical injury), vasculitis, vitiligo and Wegener's granulomatosis.
In certain embodiments, the inflammatory disease is an acute inflammatory disease (e.g., for example, inflammation resulting from infection). In certain embodiments, the inflammatory disease is a chronic inflammatory disease (e.g., conditions resulting from asthma, arthritis and inflammatory bowel disease). The compounds may also be useful in treating inflammation associated with trauma and non-inflammatory myalgia. The compounds may also be useful in treating inflammation associated with cancer.
Exemplary autoimmune diseases, include, but are not limited to, arthritis (including rheumatoid arthritis, spondyloarthopathies, gouty arthritis, degenerative joint diseases such as osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondylitis, Behcet's disease, haemolytic autoimmune anaemias, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriatic, and juvenile arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin condition (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itch)), enuresis, eosinophilic disease, gastrointestinal disorder (e.g., selected from peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)), and disorders ameliorated by a gastroprokinetic agent (e.g., ileus, postoperative ileus and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies and other functional bowel disorders, such as non-ulcerative dyspepsia (NUD) and non-cardiac chest pain (NCCP, including costo-chondritis)).
In some embodiments, a provided compound is useful in somatic cell reprogramming, such as reprogramming somatic cells into stem cells. See, e.g., Nagamatsu et al., J Biol Chem. (2011) 286:10641-10648. In some embodiments, a provided compound is useful in germ cell development, and are thus envisioned useful in the areas of reproductive technology and regenerative medicine. See, e.g., Ancelin et al., Nat. Cell. Biol. (2006) 8:623-630.
In some embodiments, compounds described herein can prepared using methods shown in general Scheme 1, which describes ring opening of a chiral or racemic epoxide group to form the amino alcohol moiety linker. Further substitution of the tetrahydroisoquinoline ring and/or the G-containing ring can be carried out before or after the coupling reaction.




In some embodiments, epoxide opening can be performed in the final step as shown in exemplary Schemes 2 and 3.








In some embodiments, an amide coupling step can be used to provide a key intermediate for further synthesis, as shown in exemplary Schemes 4-6.












In some embodiments of the compounds described herein, R12 or R13 is an amine. A non-limiting example of the synthetic sequence used to prepare such analogs is provided herein (see Scheme 7). In this example, an alcohol of Formula (Z-1) is oxidized under suitable conditions S1 to affect transformation into an intermediate ketone of Formula (Z-2). A ketone of Formula (Z-2) can be contacted with a primary or secondary amine under suitable conditions S2 to affect a reductive amination which can afford an amino compound of Formula (Z-3).




In some embodiments, the oxidation reaction S1 is carried out directly with a stoichiometeric oxidant. In some embodiments, the stoichiometric oxidant is pyridinium chlorochromate. In some embodiments, the stoichiometric oxidant is pyridinium dichromate. In some embodiments, the stoichiometric oxidant is Dess-Martin periodinane. In some embodiments, the stoichiometric oxidant is prepared in situ. In some embodiments, the stoichiometric oxidant is prepared in situ using sulfur trioxide pyridine complex and dimethylsulfoxide. In some embodiments, the stoichiometric oxidant is prepared in situ using oxallyl chloride and dimethylsulfoxide. In some embodiments, the stoichiometric oxidant is prepared in situ using a carbodiimide and dimethylsulfoxide. In some embodiments, the stoichiometric oxidant is prepared in situ using N-chlorosuccinimide and dimethylsulfide. In some embodiments, the oxidation reaction S1 is catalyzed. In some embodiments, the catalyst is (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl. In some embodiments, the catalyst is a ruthenium complex. In some embodiments, the catalyst is a palladium complex. In some embodiments, the catalyst is a copper complex. For examples of standard methods and conditions for alcohol oxidation, see Epstein et al., Chem. Rev. (1967) 67(3):247-260 and B. M. Trost ed. “Comprehensive Organic Synthesis”, (1991), Vol. 7, p 281-305.
In some embodiments, both the oxidation step S1 and reductive amination step S2 occur in one pot. In some embodiments, both the oxidation step S1 and the reductive amination step S2 are carried out using the same catalyst. In some embodiments, the catalyst is a rhodium complex. In some embodiments, the catalyst is a ruthenium complex. In some embodiments, the catalyst is an iridium complex.
In some embodiments, the reductive amination reaction S2 is carried out using a borohydride. In some embodiments, the reductive amination reaction S2 is carried out using sodium borohydride. In some embodiments, the reductive amination reaction S2 is carried out using sodium cyanoborohydride. In some embodiments, the reductive amination reaction S2 is carried out using sodium triacetoxyborohydride. In some embodiments, the reductive amination reaction S2 is carried out using a borane. In some embodiments, the reductive amination reaction S2 is carried out using a silyl hydride. In some embodiments, the reductive amination reaction S2 is carried out using hydrogen. In some embodiments, the reductive amination reaction S2 is carried out in two steps, by first contacting a ketone of (Z-2) with an amine to form an intermediate imine, and then reducing the intermediate imine under sufficient conditions to afford a compound of Formula (Z-3). In some embodiments, the reaction conditions S2 comprise addition of a protic acid. In some embodiments, the reaction conditions S2 comprise addition of an aprotic acid. In some embodiments, the reaction conditions S2 comprise in situ formation of the reducing agent. In some embodiments, the reaction conditions S2 comprise a catalyst. In some embodiments, the reaction conditions S2 comprise a transition metal catalyst. In some embodiments, the reaction conditions S2 comprise a palladium or nickel catalyst. In some embodiments, the reductive amination reaction S2 is stereoselective. In some embodiments, the stereoselective reductive amination reaction S2 is carried out in the presence of a chiral catalyst. For examples of standard methods and conditions for reductive aminations, see Gomez et al., Adv. Synth. Catal. (2002) 344(10):1037-1057 and Abdel-Magid et al., J. Org. Chem. (1996), 61:3849.
An alternative non-limiting synthetic sequence leading to the aforementioned amine analogs is described herein (see Scheme 8). The hydroxyl moiety of a compound of Formula (Z-4) can be transformed into a leaving group under sufficient conditions S3 to afford a compound of Formula (Z-5). The leaving group of a compound of Formula (Z-5) can be displaced with an amine under suitable conditions S4 to produce an amino compound of Formula (Z-6).




In some embodiments, LG of Formula (Z-5) is a halide. In some embodiments, LG of Formula (Z-5) is bromine. In some embodiments, LG of Formula (Z-5) is iodine. In some embodiments, LG of Formula (Z-5) is a substituted or unsubstituted alkyl sulfonate. In some embodiments, LG of Formula (Z-5) is a substituted or unsubstituted aryl sulfonate. In some embodiments, LG of Formula (Z-5) is methyl sulfonate. In some embodiments, LG of Formula (A-5) is trifluoromethane sulfonate. In some embodiments, LG of Formula (Z-5) is a toluene sulfonate. In some embodiments, LG of Formula (Z-5) is a nitrobenzene sulfonate. In some embodiments, when LG of Formula (Z-5) is halide, conditions S3 comprise a phosphoryl halide. In some embodiments, when LG of Formula (Z-5) is halide, conditions S3 comprise a sulfuryl halide. In some embodiments, when LG of Formula (Z-5) is sulfonate, conditions S3 comprise a sulfonyl halide. In some embodiments, when LG of Formula (Z-5) is sulfonate, conditions S3 comprise a sulfonyl anhydride. For examples of standard methods and conditions for organohalide or sulfonate ester synthesis, see Lautens et al., Synthesis (2011) 2:342-346 or Marcotullio et al., Synthesis (2006) 16:2760-2766.
In some embodiments, conditions S4 are neutral. In some embodiments, conditions S4 comprise addition of a base. In certain embodiments of conditions S4, the base is either inorganic or organic. In certain embodiments of conditions S4, the base is inorganic. In certain embodiments of conditions S4, the base is organic. In certain embodiments of conditions S4, the base is a metal acetate, alkoxide, amide, amidine, carbonate, hydroxide, phenoxide, or phosphate. In certain embodiments of conditions S4, the base is sodium, potassium, or caesium carbonate. In certain embodiments of conditions S4, the base is sodium, potassium, or caesium bicarbonate. In certain embodiments of conditions S4, the base is 1,1,3,3-tetramethylguanidine, 1,4-diazabicyclo[2.2.2]octane, 1,8-bis(dimethylamino)naphthalene, 1,8-diazabicycloundec-7-ene, ammonia, diisopropylamine, imidazole, N,N-diisopropylethylamine, piperidine, pyridine, pyrrolidine, or triethylamine. In some embodiments of conditions S4, the solvent is a polar protic solvent. In some embodiments of conditions S4, the solvent is a polar aprotic solvent. In some embodiments of conditions S4, the reaction is performed in the absence of solvent. In some embodiments, conditions S4 comprise a catalyst. In some embodiments of conditions S4, the catalyst is an iodide salt. In some embodiments, both step S3 and the displacement step S4 occur in one pot. In some embodiments, the hydroxyl moiety of a compound of Formula (Z-4) is converted into a leaving group in situ. In some embodiments, the hydroxyl moiety of a compound of Formula (Z-4) is converted into a leaving group in situ using an azodicarboxylate and an aryl or alkyl phosphine. For examples of standard methods and conditions for amine syntheses through alkylation reactions, see Salvatore et. al, Tetrahedron (2001) 57:7785-7811.
EXAMPLES
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
Synthetic Methods
Intermediate Synthesis
2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline




To a solution of 1,2,3,4-tetrahydroisoquinoline (15 g, 0.11 mol) in MeCN (100 mL) was added K2CO3 (30.7 g, 0.23 mol) at 0° C. 2-(bromomethyl)oxirane (17 g, 0.12 mol) was added to the reaction after 1 h. The solution was stirred at 22° C. for 16 h at which time the solids were filtered and washed with MeCN. The solution was concentrated and the residue was used in the next step without further purification (17 g, Yield 78%). LCMS (m/z): 190.1 (M+1).
Compound 1
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)indolin-2-one




To a solution of indolin-2-one (50.0 mg, 0.378 mmol) in ethanol (5 mL) was added 2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (85.0 mg, 0.451 mmol). The mixture was heated under microwave conditions at 120° C. for 0.5 h, concentrated and purified by preparative HPLC purification. (40.0 mg, yield 33.0%) MS (ESI+) e/z: 323.1 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.30-7.23 (m, 2H), 7.14-7.07 (m, 4H), 7.06 (d, J=7.5 Hz, 1H), 7.01 (t, J=5.8 Hz, 1H), 4.94 (d, J=7.8 Hz, 2H), 4.31-4.18 (m, 1H), 3.97-3.86 (m, 1H), 3.82-3.72 (m, 1H), 3.71-3.64 (m, 2H), 2.90-2.73 (m, 4H), 2.73-2.59 (m, 2H).
Compound 3
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(m-tolyl)piperazin-2-one




Step 1: tert-butyl 3-oxopiperazine-1-carboxylate




Piperazin-2-one (10 g, 0.1 mol), Et3N (20.2 g, 0.2 mmol) and DCM (100 mL) were combined and cooled to 0° C. To the solution was added Boc2O (26.1 g, 0.12 mol) in DCM (250 ml), and the resulting mixture was warmed to room temperature and stirred for 12 h. The reaction mixture was concentrated and dissolved in ethyl acetate, washed with 1 N HCl, brine, dried over sodium sulfate, filtered and concentrated. The crude product was used without further purification. (19.5 g, yield 97.5%).
Step 2: tert-butyl 4-(oxiran-2-ylmethyl)-3-oxopiperazine-1-carboxylate




tert-butyl 3-oxopiperazine-1-carboxylate (400 mg, 2 mmol) was dissolved in THF (20 mL) and cooled to 0° C. NaH (72 mg, 3 mmol) was added to the solution followed by 2-(bromomethyl)oxirane (326 mg, 2.4 mmol), and the resulting mixture was stirred at 0° C. for 16 h. The reaction mixture was quenched by addition of ice water, extracted with ethyl acetate, washed with brine, dried over sodium sulfate and concentrated. The crude product was used without further purification. (400 mg, yield 78.1%).
Step 3: tert-butyl 4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-3-oxopiperazine-1-carboxylate




To a solution of tert-butyl 4-(oxiran-2-ylmethyl)-3-oxopiperazine-1-carboxylate (400 mg, 1.56 mmol) in EtOH (1 mL) was added 1,2,3,4-tetrahydroisoquinoline (207 mg, 1.56 mmol). The mixture was heated under microwave conditions at 120° C. for 0.5 h and concentrated. The crude product was used without further purification. (600 mg, yield 98%) MS (ESI+) e/z: 390.2 [M+1]+.
Step 4:1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)piperazin-2-one




To a solution of tert-butyl 4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxyl propyl)-3-oxopiperazine-1-carboxylate (200 mg, 0.69 mmol) in ethyl acetate (10 mL) was added HCl/ethyl acetate (4 mL). The reaction mixture was stirred at 20° C. overnight, concentrated and used in the next step without further purification. (100 mg, yield 50%) MS (ESI+) e/z: 290.2 [M+1]+.
Step 5:1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(m-tolyl)piperazin-2-one




A mixture of 1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)piperazin-2-one (50 mg, 0.17 mmol), 1-iodo-3-methylbenzene (55.3 mg, 0.25 mmol), Pd2(dba)3 (10 mg, 0.01 mmol), Xantphos (20 mg, 0.02 mmol) and t-BuONa (30 mg, 0.3 mmol) were placed in the three-neck reactor with a condenser and magnetic stirring bar, the system was degassed 3 times (N2) and dioxane (10 mL) was added. The mixture was heated at reflux for 16 h, cooled, filtered and concentrated. The residue was purified by preparative HPLC purification. (18.0 mg, yield 28.0%) MS (ESI+) e/z: 380.2 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.10 (br. s., 5H), 6.85-6.65 (m, 3H), 4.33-4.14 (m, 1H), 3.88-3.81 (m, 2H), 3.81-3.68 (m, 4H), 3.67-3.58 (m, 1H), 3.56-3.44 (m, 2H), 3.32-3.25 (m, 1H), 3.02-2.76 (m, 4H), 2.68-2.54 (m, 2H), 2.32 (s, 3H).
Compound 4
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(quinolin-8-yl)piperazin-2-one




To a solution of 1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl) piperazin-2-one (100 mg, 0.35 mmol) in 10 mL of dry toluene was added 8-bromoquinoline (108 mg, 0.52 mmol), Pd2(dba)3 (32 mg, 0.035 mmol), NINAP (44 mg, 0.07 mmol) and t-BuONa (101 mg, 1.05 mmol). The flask was degassed and charged with N2, then heated at 110° C. for 16 h. The reaction mixture was cooled, filtered, and concentrated. The residue was purified by preparative HPLC purification. (23 mg, yield 15.8%) MS (ESI+) e/z: 417.2 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 8.96-8.85 (m, 1H), 8.53-8.39 (m, 1H), 8.38-8.29 (m, 1H), 7.70-7.62 (m, 1H), 7.62-7.51 (m, 2H), 7.37-7.24 (m, 4H), 7.24-7.17 (m, 1H), 4.60-4.52 (m, 1H), 4.52-4.41 (m, 2H), 4.08-3.98 (m, 2H), 3.87-3.78 (m, 2H), 3.77-3.69 (m, 1H), 3.69-3.58 (m, 4H), 3.57-3.49 (m, 1H), 3.42-3.34 (m, 1H), 3.32-3.27 (m, 1H), 3.27-3.13 (m, 2H).
Compound 5
4-(3-chlorophenyl)-1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)piperazin-2-one




Compound 5 was synthesized in by a procedure analogous to that employed for compound 3. (5.3 mg, yield 7.8%) MS (ESI+) e/z: 400.1 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.26-7.21 (m, 1H), 7.16-7.07 (m, 3H), 7.07-7.01 (m, 1H), 6.95-6.89 (m, 1H), 6.89-6.78 (m, 2H), 4.26-4.19 (m, 1H), 3.92-3.85 (m, 2H), 3.83-3.70 (m, 4H), 3.69-3.62 (m, 1H), 3.59-3.51 (m, 2H), 3.32-3.24 (m, 1H), 2.97-2.90 (m, 2H), 2.89-2.80 (m, 2H), 2.66-2.56 (m, 2H).
Compound 7
4-(4-chlorophenyl)-1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)piperazin-2-one




Compound 7 was synthesized in by a procedure analogous to that employed for compound 3. (14.6 mg, yield 21.5%) MS (ESI+) e/z: 400.1[M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.28-7.16 (m, 2H), 7.16-6.98 (m, 4H), 6.95-6.85 (m, 2H), 4.25-4.16 (m, 1H), 3.90-3.81 (m, 2H), 3.81-3.68 (m, 4H), 3.67-3.58 (m, 1H), 3.55-3.45 (m, 2H), 2.97-2.87 (m, 2H), 2.86-2.75 (m, 2H), 2.66-2.52 (m, 2H).
Compound 8
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(5-morpholinopyridin-3-yl)piperazin-2-one




Step 1: 4-(5-bromopyridin-3-yl)morpholine




To a solution of morpholine (3.67 g, 4.23 mmol), NMP (2 mL) and toluene (4 mL) was added 3,5-dibromopyridine (1.00 g, 4.20 mmol) and the mixture was heated under microwave conditions at 180° C. for 1 h. The solution was partitioned between DCM and water, the organic layer was washed with water, brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography eluting with (EtOAc: PE=1:5). (200 mg, yield 19.5%) MS (ESI+) e/z: 243.0 [M+1]+ LCMS.
Step 2: 1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(5-morpholinopyridin-3-yl)piperazin-2-one




A solution of 4-(5-bromopyridin-3-yl)morpholine (100 mg, 0.413 mmol), 1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)piperazin-2-one (119 mg, 0.413 mmol), X-phos (20 mg, 0.042 mmol), Pd(dba)2 (10 mg, 0.017 mmol) and NaOtBu (59.0 mg, 0.615 mmol) in dioxane (5 mL) was heated at 120° C. 1 h under microwave conditions. The crude reaction mixture was filtered, concentrated, and purified by preparative HPLC purification. (6.10 mg, yield 3.3%) 1H NMR (MeOD, 400 MHz), δ ppm: 7.76 (d, J=2.3 Hz, 1H), 7.72 (d, J=2.0 Hz, 1H), 7.17-7.07 (m, 3H), 7.07-7.01 (m, 1H), 6.89-6.80 (m, 1H), 4.28-4.18 (m, 1H), 3.93 (s, 2H), 3.89-3.82 (m, 4H), 3.82-3.72 (m, 4H), 3.71-3.63 (m, 1H), 3.59 (t, J=1.0 Hz, 2H), 3.32-3.28 (m, 1H), 3.27-3.16 (m, 4H), 2.99-2.90 (m, 2H), 2.90-2.81 (m, 2H), 2.68-2.58 (m, 2H).
Compound 11
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(2-fluorophenyl)piperazin-2-one




Compound 11 was synthesized in by a procedure analogous to that employed for compound 3. (15.7 mg, yield 24.1%) MS (ESI+) e/z: 384.2[M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.14-6.98 (m, 8H), 4.27-4.18 (m, 1H), 3.81-3.68 (m, 6H), 3.65-3.57 (m, 1H), 3.43-3.36 (m, 2H), 3.30-3.22 (m, 1H), 2.95-2.88 (m, 2H), 2.87-2.80 (m, 2H), 2.64-2.53 (m, 2H).
Compound 12
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(3-morpholinophenyl)piperazin-2-one




Step 1: 4-(3-bromophenyl)morpholine




To a solution of 3-bromoaniline (1.00 g, 5.81 mmol) in anhydrous DMF (20 mL) was added 1-bromo-2-(2-bromoethoxyl)ethane (1.62 g, 6.18 mmol) and DIPEA (2.25 g, 17.4 mmol). The solution was heated to 100° C. for 16 h, cooled and concentrated. The residue was purified by preparative HPLC purification. (230 mg, yield 16.4%) MS (ESI+) e/z: 242.0 [M+1]+.
Step 2: 1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(3-morpholinophenyl)piperazin-2-one




4-(3-bromophenyl)morpholine (100 mg, 0.413 mmol), 1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)piperazin-2-one (119 mg, 0.413 mmol), Xphos (20 mg, 0.042 mmol), Pd(dba)2 (10 mg, 0.017 mmol) and NaOtBu (59.0 mg, 0.615 mmol) and dioxane (5 mL) were combined in a three-neck flask with a condenser and magnetic stirring bar, the system was degassed (N2) and dioxane (10 mL) was added. The reaction mixture was heated at 100° C. for 16 h, filtered and concentrated. The residue was purified by preparative HPLC purification. (3.2 mg, yield 1.7%). 1H NMR (MeOD, 400 MHz), δ ppm: 7.21-7.15 (m, 1H), 7.15-7.07 (m, 3H), 7.07-7.00 (m, 1H), 6.58-6.45 (m, 3H), 4.22 (br. s., 1H), 3.86-3.82 (m, 6H), 3.78-3.69 (m, 4H), 3.68-3.59 (m, 1H), 3.56-3.46 (m, 2H), 3.31-3.26 (m, 1H), 3.16-3.12 (m, 4H), 2.98-2.90 (m, 2H), 2.90-2.78 (m, 2H), 2.60 (d, J=6.0 Hz, 2H).
Compound 15
1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(p-tolyl)piperazin-2-one




Compound 15 was synthesized in by a procedure analogous to that employed for compound 3. (14.9 mg, yield 23.1%) MS (ESI+) e/z: 380.2 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.14-6.98 (m, 6H), 6.89-6.80 (m, 2H), 4.26-4.13 (m, 1H), 3.84-3.65 (m, 6H), 3.65-3.55 (m, 1H), 3.49-3.38 (m, 2H),) 3.30-3.20 (m, 1H), 2.96-2.86 (m, 2H), 2.86-2.76 (m, 2H), 2.64-2.53 (m, 2H), 2.24 (s, 3H).
Compound 19
(R)-1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(phenylamino)pyridin-2(1H)-one




Step 1: (R)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline




A solution of 1,2,3,4-tetrahydroisoquinoline (10 g, 0.15 mol) in THF (100 mL) was cooled to 0° C. and potassium fluoride (22 g, 0.3 mol) was added. After 1 h, (R)-oxiran-2-ylmethyl 3-nitrobenzenesulfonate (21.4 g, 0.17 mol) was added; the mixture was warmed to 25° C. and stirred for 16 h. The solution was filtered, washed with THF and concentrated. The crude product was used in next step without further purification. (15 g, yield 53%) MS (ESI+) e/z: 190.1 [M+1]+.
Step 2: (R)-4-bromo-1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyridin-2(1H)-one




To a solution of 4-bromopyridin-2(1H)-one (1 g, 5.747 mmol) in EtOH (3 mL) was added (R)-2-(oxiran-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (1.087 g, 5.747 mmol). The mixture was heated under microwave conditions at 120° C. for 0.5 h and concentrated. The crude product was purified by column chromatography eluting with DCM/MeOH (10:1). (475 mg, yield 22.8%) MS (ESI+) e/z: 363.1 [M+1]+.
Step 3: (R)-1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-4-(phenylamino)pyridin-2(1H)-one




To a solution of (R)-4-bromo-1-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyridin-2(1H)-one (200 mg, 0.551 mmol) in anhydrous toluene (5 mL) was added aniline (51 mg, 0.551 mmol), xantphos (20 mg), Pd2(dba)3 (20 mg) and t-BuOK (123 mg, 1.102 mmol). The mixture was heated under microwave conditions at 140° C. for 1 h, filtered and concentrated. The crude product was purified by preparative HPLC purification. (18 mg, yield 8.7%) MS (ESI+) e/z: 376.1 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.45-7.35 (m, 3H), 7.24-7.18 (m, 2H), 7.17-7.08 (m, 4H), 7.07-7.02 (m, 1H), 6.11 (dd, J=2.6, 7.5 Hz, 1H), 5.99 (d, J=2.5 Hz, 1H), 4.33 (dd, J=3.3, 13.4 Hz, 1H), 4.27-4.15 (m, 1H), 3.73 (s, 2H), 3.63 (dd, J=8.0, 13.6 Hz, 1H), 2.96-2.89 (m, 2H), 2.88-2.82 (m, 2H), 2.66-2.58 (m, 2H).
Compound 31
4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2H-benzo[b][1,4]oxazin-3(4H)-one




Step 1: ethyl 2-(2-iodophenoxyl)acetate




To a solution of 2-iodophenol (1 g, 4.55 mmol) in CH3CN (20 mL) was added K2CO3 (0.93 g, 6.83 mmol) and ethyl 2-bromoacetate (0.835 g, 5.0 mmol). The reaction mixture was stirred at reflux for 2 h, cooled to room temperature, extracted with EtOAc, washed with brine, dried over sodium sulfate and concentrated. The crude product was used in the next step without further purification. (1.1 g, yield 79%) MS (ESI+) e/z: 307.1 [M+1]+.
Step 2: N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(2-iodophenoxyl)acetamide




To a solution of ethyl 2-(2-iodophenoxyl)acetate (900 mg, 2.94 mmol) in EtOH (3 mL) was added 1-amino-3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-2-ol (677 mg, 3.24 mmol). The reaction mixture was heated under microwave conditions at 80° C. for 3 h, cooled and concentrated. The crude product was used in the next step without further purification. (1.3 g, yield 95%) MS (ESI+) e/z: 467.1 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.80 (dd, J=1.0, 7.8 Hz, 1H), 7.41-7.30 (m, 1H), 7.15-7.05 (m, 3H), 7.05-6.99 (m, 1H), 6.96 (d, J=8.0 Hz, 1H), 6.87-6.76 (m, 1H), 4.66-4.53 (m, 2H), 4.06 (quin, J=5.9 Hz, 1H), 3.77-3.63 (m, 2H), 3.58 (dd, J=4.5, 13.6 Hz, 1H), 3.38 (dd, J=6.4, 13.7 Hz, 1H), 2.98-2.86 (m, 2H), 2.86-2.76 (m, 2H), 2.66-2.57 (m, 2H).
Step 3: 4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2H-benzo[b][1,4]oxazin-3(4H)-one




To a solution of N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2-(2-iodophenoxy)acetamide (100 mg, 0.215 mmol) in dioxane (5 mL) was added Cs2CO3 (139 mg, 0.429 mmol), BINAP (13.3 mg, 0.0215 mmol) and Pd2(dba)3 (8.0 mg, 0.0215 mmol). The reaction mixture was heated at 120° C. for 16 h, cooled to room temperature, filtered, and concentrated. The crude produce was purified by preparative HPLC purification. (23.6 mg, yield 32.5%) MS (ESI+) e/z: 339.2 [M+1]+. 1H NMR (MeOD, 400 MHz), δ ppm: 7.41-7.32 (m, 1H), 7.15-7.06 (m, 3H), 7.06-6.94 (m, 4H), 4.67-4.50 (m, 2H), 4.29-4.13 (m, 2H), 4.07-3.94 (m, 1H), 3.77-3.64 (m, 2H), 2.95-2.83 (m, 3H), 2.83-2.76 (m, 1H), 2.73-2.62 (m, 2H).
LC-MS Conditions
Method A (LCMS-B (0-60AB_ELSD_2MIN))
Experiments performed on an Agilent 1200 HPLC (with a PDA detector and a ELSD detector) with Agilent 6100 MSD mass spectrometer using ESI as ionization source using an XtimateTM-C18 30*2.1 mm column and a 0.8 ml/minute flow rate. Acquire Time: 2 min, Wavelength: UV220, Oven Temp.: 50° C. The solvent system was a gradient starting with 100% water containing 0.038% TFA (solvent A) and 0% acetonitrile containing 0.02% TFA (solvent B), followed by a gradient up to 40% solvent A and 60% solvent B over the next 0.9 minutes. This was maintained for 0.6 minutes before returning to 100% solvent A over the next 0.5 minute. Total run time was 2 min.
Method B (LCMS-C(10-80_AB))
Experiments performed on an SHIMADZU 20A HPLC (with a PDA detector) with SHIMADZU 2010EV MSD mass spectrometer using ESI as ionization source using an XtimateTM-C18 30*2.1 mm column and a 1.2 ml/minute flow rate. The solvent system was a gradient starting with 90% water containing 0.038% TFA (solvent A) and 10% acetonitrile containing 0.02% TFA (solvent B), followed by a gradient up to 20% solvent A and 80% solvent B over the next 0.9 minutes. This was maintained for 0.6 minutes before returning to 90% solvent A and 10% solvent B over the next 0.5 minute. Total run time was 2 min.
Method C (LCMS-E(5-95AB_220&254 nm))
Experiments performed on an SHIMADZU 20A HPLC (with a PDA detector) with SHIMADZU 2010EV MSD mass spectrometer using ESI as ionization source using an Merk RP-18e 2*25 mm column and a 1.5 ml/minute flow rate. The solvent system was a gradient starting with 95% water containing 0.038% TFA (solvent A) and 5% acetonitrile containing 0.02% TFA (solvent B), followed by a gradient up to 5% solvent A and 95% solvent B over the next 0.7 minutes. This was maintained for 0.4 minutes before returning to 95% solvent A and 5% solvent B over the next 0.4 minute. Total run time was 1.5 min.
Method D (LCMS-A(0-30_AB))
Experiments performed on an SHIMADZU 20A HPLC (with a PDA detector) with SHIMADZU 2010EV MSD mass spectrometer using ESI as ionization source using an XtimateTM-C18 30*2.1 mm column and a 1.2 ml/minute flow rate. The solvent system was a gradient starting with 100% water containing 0.038% TFA (solvent A) and 0% acetonitrile containing 0.02% TFA (solvent B), followed by a gradient up to 70% solvent A and 30% solvent B over the next 0.9 minutes. This was maintained for 0.6 minutes before returning to 100% solvent A over the next 0.5 minute. Total run time was 2 min.
General HPLC Conditions (Acidic)


    Mobile phase A: 4 L H2O\1.5 ml TFA; Mobile phase B: 4 L ACN\0.75 ml TFA
    Column: HPLC-D: Innovation C18 UPLC Column 2.1×30 mm, 2.6 um
    
        HPLC-E: Xtimate C18 2.1*30 mm*3 um
        HPLC-H: Innovation C18 UPLC Column 2.1×30 mm, 2.6 um
    
    
    Column temperature: 50° C.; Wavelength: 220 nm & 254 nm & 215 nm

General HPLC Conditions (Basic)

    Mobile phase A: 4 L H2O\2 ml NH4OH; Mobile phase B: Acetonitrile
    Column: HPLC-B: XBridge C18 2.1*50 mm, 5 um
    
        HPLC-C: Xbridge shield RP18 2.1*50 mm, 5 u
    
    
    Column temperature: 30° C.; Wavelength: 220 nm & 254 nm & 215 nm

General HPLC Conditions (Neutral)

    Mobile phase A: H2O; Mobile phase B: Acetonitrile
    Column: HPLC-B: XBridge C18 2.1*50 mm, 5 um
    
        HPLC-C: Xbridge shield RP18 2.1*50 mm, 5 um
    
    
    Column temperature: 30° C.; Wavelength: 220 nm & 254 nm & 215 nm Method A (0-30AB_6MIN) Flow Rate: 0.8 ml/min Gradient: 0% B to 30% B in 4.2 min, holding 30% B for 1 min, 30% B to 0% B in 0.01 min, holding 0% B for 1.09 min and then end.

Method B (0-60AB_6MIN)

    Flow Rate: 0.8 ml/min
    Gradient: 0% B to 60% B in 4.2 min, holding 60% B for 1 min, 60% B to 0% B in 0.01 min, holding 0% B for 1.09 min and then end.

Method C (10-80AB_6MIN)

    Flow Rate: 0.8 ml/min
    Gradient: 10% B to 80% B in 4.2 min, holding 80% B for 1 min, 80% B to 10% B in 0.01 min, holding 10% B for 1.09 min and then end.

Chiral HPLC Conditions:

Method A (OJ-H):

Column: Chiralcel OJ-H 250×4.6 mm I.D., 5 um

Mobile phase: A/B=90/10, A:Hexane with 0.1% DEA, B: Ethanol

Flow rate: 0.5 mL/min

Wavelength: 220 nm

Method B (OD-H):

Column: Chiralcel OD-H 250×4.6 mm I.D., 5 um

Mobile phase: A/B=90/10, A: Hexane with 0.1% DEA, B: Ethanol

Flow rate: 0.5 mL/min

Wavelength: 220 nm

Method C (AD-H):

Column: Chiralpak AD-H 250×4.6 mm I.D., 5 um

Mobile phase: A/B=90/10, A: Hexane with 0.1% DEA, B: Ethanol

Flow rate: 0.5 mL/min

Wavelength: 220 nm

Method D (AS-H):

Column: Chiralpak OJ-H 250×4.6 mm I.D., 5 um

Mobile phase: A/B=90/10, A: Hexane with 0.1% DEA, B: Ethanol

Flow rate: 0.5 mL/min

Wavelength: 220 nm

Biological Assays

PRMT5 Biochemical Assay



General Materials.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, KCl, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Sigma-Aldrich at the highest level of purity possible. 3H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchased from PerkinElmer.
Substrates.
Peptide representative of human histone H4 residues 1-15 was synthesized with a C-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was high high-performance liquid chromatography (HPLC) purified to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Ac-SGRGKGGKGLGKGGA[K-Biot]-amide (SEQ ID NO.:3).
Molecular Biology:
Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by GatewayTM attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).
Protein Expression.
Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Protein Purification.
Expressed full-length human Flag-PRMT5/6His-MeP50 protein complex was purified from cell paste by NiNTA agarose affinity chromatography after a five hour equilibration of the resin with buffer containing 50 mM Tris-HCL, pH 8.0, 25 mM NaCl, and 1 mM TCEP at 4° C., to minimize the adsorption of tubulin impurity by the resin. Flag-PRMT5/6His-MeP50 was eluted with 300 mM Imidazole in the same buffer. The purity of recovered protein was 87%. Reference: Wasilko, D. J. and S. E. Lee: “TIPS: titerless infected-cells preservation and scale-up” Bioprocess J., 5 (2006), pp. 29-32.
Predicted Translations:









Flag-PRMT5



(SEQ ID NO.: 6)



MDYKDDDDKA AMAVGGAGGS RVSSGRDLNC VPEIADTLGA


 



VAKQGFDFLC MPVFHPRFKR EFIQEPAKNR PGPQTRSDLL


 



LSGRDWNTLI VGKLSPWIRP DSKVEKIRRN SEAAMLQELN


 



FGAYLGLPAF LLPLNQEDNT NLARVLTNHI HTGHHSSMFW


 



MRVPLVAPED LRDDIIENAP TTHTEEYSGE EKTWMWWHNF


 



RTLCDYSKRI AVALEIGADL PSNHVIDRWL GEPIKAAILP


 



TSIFLTNKKG FPVLSKMHQR LIFRLLKLEV QFIITGTNHH


 



SEKEFCSYLQ YLEYLSQNRP PPNAYELFAK GYEDYLQSPL


 



QPLMDNLESQ TYEVFEKDPI KYSQYQQAIY KCLLDRVPEE


 



EKDTNVQVLM VLGAGRGPLV NASLRAAKQA DRRIKLYAVE


 



KNPNAVVTLE NWQFEEWGSQ VTVVSSDMRE WVAPEKADII


 



VSELLGSFAD NELSPECLDG AQHFLKDDGV SIPGEYTSFL


 



APISSSKLYN EVRACREKDR DPEAQFEMPY VVRLHNFHQL


 



SAPQPCFTFS HPNRDPMIDN NRYCTLEFPV EVNTVLHGFA


 



GYFETVLYQD ITLSIRPETH SPGMFSWFPI LFPIKQPITV


 



REGQTICVRF WRCSNSKKVW YEWAVTAPVC SAIHNPTGRS


 



YTIG L


 



6His-MEP50



(SEQ ID NO.: 7)



MHHHHHHRKE TPPPLVPPAA REWNLPPNAP ACMERQLEAA


 



RYRSDGALLL GASSLSGRCW AGSLWLFKDP CAAPNEGFCS


 



AGVQTEAGVA DLTWVGERGI LVASDSGAVE LWELDENETL


 



IVSKFCKYEH DDIVSTVSVL SSGTQAVSGS KDICIKVWDL


 



AQQVVLSSYR AHAAQVTCVA ASPHKDSVFL SCSEDNRILL


 



WDTRCPKPAS QIGCSAPGYL PTSLAWHPQQ SEVFVFGDEN


 



GTVSLVDTKS TSCVLSSAVH SQCVTGLVFS PHSVPFLASL


 



SEDCSLAVLD SSLSELFRSQ AHRDFVRDAT WSPLNHSLLT


 



TVGWDHQVVH HVVPTEPLPA PGPASVTE






General Procedure for PRMT5/MEP50 Enzyme Assays on Peptide Substrates.
The assays were all performed in a buffer consisting of 20 mM Bicine (pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a polypropylene 384-well V-bottom plates (Greiner) using a Platemate Plus outfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of PRMT5/MEP50, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the PRMT5/MEP50 enzyme and the peptide was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with PRMT5/MEP50 for 30 min at 25 degrees Celsius, then a cocktail (10 ul) containing 3H-SAM was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: PRMT5/MEP50 was 4 nM, 3H-SAM was 75 nM, peptide was 40 nM, SAH in the minimum signal control wells was 100 uM, and the DMSO concentration was 1%. The assays were stopped by the addition of non-radioactive SAM (10 ul) to a final concentration of 600 uM, which dilutes the 3H-SAM to a level where its incorporation into the peptide substrate is no longer detectable. 50 ul of the reaction in the 384-well polypropylene plate was then transferred to a 384-well Flashplate and the biotinylated peptides were allowed to bind to the streptavidin surface for at least 1 hour before being washed three times with 0.1% Tween20 in a Biotek ELx405 plate washer. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled peptide bound to the Flashplate surface, measured as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
% Inhibition Calculation




  
    %
    ⁢
    
      
    
    ⁢
    inh
  
  =
  
    100
    -
    
      
        (
        
          
            
              dpm
              cmpd
            
            -
            
              dpm
              min
            
          
          
            
              dpm
              max
            
            -
            
              dpm
              min
            
          
        
        )
      
      ×
      100
    
  




Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit





  Y
  =
  
    Bottom
    +
    
      
        (
        
          Top
          -
          Bottom
        
        )
      
      
        (
        
          1
          +
          
            
              (
              
                X
                
                  IC
                  50
                
              
              )
            
            
              Hill
              ⁢
              
                
              
              ⁢
              Coefficient
            
          
        
      
    
  




Where top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.

Z-138 Methylation Assay

Z-138 suspension cells were purchased from ATCC (American Type Culture Collection, Manassas, Va.). RPMI/Glutamax medium, penicillin-streptomycin, heat inactivated fetal bovine serum, and D-PBS were purchased from Life Technologies, Grand Island, N.Y., USA. Odyssey blocking buffer, 800CW goat anti-rabbit IgG (H+L) antibody, and Licor Odyssey infrared scanner were purchased from Licor Biosciences, Lincoln, Nebr., USA. Symmetric di-methyl arginine antibody was purchased from EMD Millipore, Billerica, Mass., USA. 16% Paraformaldehyde was purchased from Electron Microscopy Sciences, Hatfield, Pa., USA.
Z-138 suspension cells were maintained in growth medium (RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin) and cultured at 37° C. under 5% CO2.
Cell Treatment, in Cell Western (ICW) for Detection of Symmetric Di-Methyl Arginine and DNA Content.
Z-138 cells were seeded in assay medium at a concentration of 50,000 cells per mL to a 384-well cell culture plate with 50 μL per well. Compound (100 nL) from 384 well source plates was added directly to 384 well cell plate. Plates were incubated at 37° C., 5% CO2 for 96 hours. After four days of incubation, 40 μL of cells from incubated plates were added to poly-D-lysine coated 384 well culture plates (BD Biosciences 356697). Plates were incubated at room temperature for 30 minutes then incubated at 37° C., 5% CO2 for 5 hours. After the incubation, 40 μL per well of 8% paraformaldehyde in PBS (16% paraformaldahyde was diluted to 8% in PBS) was added to each plate and incubated for 30 minutes. Plates were transferred to a Biotek 405 plate washer and washed 5 times with 100 μL per well of wash buffer (1×PBS with 0.1% Triton X-100 (v/v)). Next 30 μL per well of Odyssey blocking buffer were added to each plate and incubated 1 hour at room temperature. Blocking buffer was removed and 20 μL per well of primary antibody was added (symmetric di-methyl arginine diluted 1:100 in Odyssey buffer with 0.1% Tween 20 (v/v)) and plates were incubated overnight (16 hours) at 4° C. Plates were washed 5 times with 100 μL per well of wash buffer. Next 20 μL per well of secondary antibody was added (1:200 800CW goat anti-rabbit IgG (H+L) antibody, 1:1000 DRAQ5 (Biostatus limited) in Odyssey buffer with 0.1% Tween 20 (v/v)) and incubated for 1 hour at room temperature. The plates were washed 5 times with 100 μL per well wash buffer then 1 time with 100 μL per well of water. Plates were allowed to dry at room temperature then imaged on the Licor Odyssey machine which measures integrated intensity at 700 nm and 800 nm wavelengths. Both 700 and 800 channels were scanned.
Calculations:
First, the ratio for each well was determined by:




  (
  
    
      symmetric
      ⁢
      
        
      
      ⁢
      di
      ⁢
      
        -
      
      ⁢
      methyl
      ⁢
      
        
      
      ⁢
      Arginine
      ⁢
      
        
      
      ⁢
      800
      ⁢
      
        
      
      ⁢
      nm
      ⁢
      
        
      
      ⁢
      value
    
    
      DRAQ
      ⁢
      
        
      
      ⁢
      5
      ⁢
      
        
      
      ⁢
      700
      ⁢
      
        
      
      ⁢
      nm
      ⁢
      
        
      
      ⁢
      value
    
  
  )




Each plate included fourteen control wells of DMSO only treatment (minimum inhibition) as well as fourteen control wells for maximum inhibition treated with 3 μM of a reference compound (Background wells). The average of the ratio values for each control type was calculated and used to determine the percent inhibition for each test well in the plate. Reference compound was serially diluted three-fold in DMSO for a total of nine test concentrations, beginning at 3 μM. Percent inhibition was determined and IC50 curves were generated using triplicate wells per concentration of compound.




  
    Percent
    ⁢
    
      
    
    ⁢
    Inhibition
  
  =
  
    100
    -
    
      (
      
        
          (
          
            
              
                (
                
                  Individual
                  ⁢
                  
                    
                  
                  ⁢
                  Test
                  ⁢
                  
                    
                  
                  ⁢
                  Sample
                  ⁢
                  
                    
                  
                  ⁢
                  Ratio
                
                )
              
              -
              
                (
                
                  Background
                  ⁢
                  
                    
                  
                  ⁢
                  Avg
                  ⁢
                  
                    
                  
                  ⁢
                  Ratio
                
                )
              
            
            
              
                (
                
                  Minimum
                  ⁢
                  
                    
                  
                  ⁢
                  Inhibition
                  ⁢
                  
                    
                  
                  ⁢
                  Ratio
                
                )
              
              -
              
                (
                
                  Background
                  ⁢
                  
                    
                  
                  ⁢
                  Average
                  ⁢
                  
                    
                  
                  ⁢
                  Ratio
                
                )
              
            
          
          )
        
        *
        100
      
      )
    
  




Z-138 Proliferation Assay

Z-138 suspension cells were purchased from ATCC (American Type Culture Collection, Manassas, Va.). RPMI/Glutamax medium, penicillin-streptomycin, heat inactivated fetal bovine serum were purchased from Life Technologies, Grand Island, N.Y., USA. V-bottom polypropylene 384-well plates were purchased from Greiner Bio-One, Monroe, N.C., USA. Cell culture 384-well white opaque plates were purchased from Perkin Elmer, Waltham, Mass., USA. Cell-Titer Glo® was purchased from Promega Corporation, Madison, Wis., USA. SpectraMax M5 plate reader was purchased from Molecular Devices LLC, Sunnyvale, Calif., USA.
Z-138 suspension cells were maintained in growth medium (RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine serum and cultured at 37° C. under 5% CO2. Under assay conditions, cells were incubated in assay medium (RPMI 1640 supplemented with 10% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin) at 37° C. under 5% CO2.
For the assessment of the effect of compounds on the proliferation of the Z-138 cell line, exponentially growing cells were plated in 384-well white opaque plates at a density of 10,000 cells/ml in a final volume of 50 μl of assay medium. A compound source plate was prepared by performing triplicate nine-point 3-fold serial dilutions in DMSO, beginning at 10 mM (final top concentration of compound in the assay was 20 M and the DMSO was 0.2%). A 100 nL aliquot from the compound stock plate was added to its respective well in the cell plate. The 100% inhibition control consisted of cells treated with 200 nM final concentration of staurosporine and the 0% inhibition control consisted of DMSO treated cells. After addition of compounds, assay plates were incubated for 5 days at 37° C., 5% CO2, relative humidity>90%.
Cell viability was measured by quantitation of ATP present in the cell cultures, adding 35 μl of Cell Titer Glo® reagent to the cell plates. Luminescence was read in the SpectraMax M5 microplate reader. The concentration of compound inhibiting cell viability by 50% was determined using a 4-parametric fit of the normalized dose response curves.
Results for certain compounds described herein are shown in Table 2.







TABLE 2







Biological Assay Results













Biochemical

Proliferation



Cmpd No
IC50
ICW EC50
EC50
















1
B
B
**



2
D
—
—



3
D
—
—



4
A
B
C



5
A
C
—



6
A
B
**



7
A
B
D



8
A
B
C



9
B
B
D



10
—
E
—



11
—
E
—



12
A
A
C



13
—
E
—



14
—
E
—



15
B
B
**



16
C
—
—



17
C
—
—



18
C
—
—



19
B
B
**



20
B
C
—



21
C
—
—



22
C
—
—



23
C
—
—



24
C
—
—



25
C
—
—



26
C
—
—



27
E
—
—



28
C
—
—



29
B
B
D



30
C
—
—



31
B
B
**



32
*
—
—



33
B
C
**



34
C
—
—







For Table 2,



“A” indicates an IC50 or EC50 <0.100 μM,



“B” indicates an IC50 or EC50 of 0.101-1.000 μM,



“C” indicates an IC50 or EC50 of 1.001-10.000 μM,



“D” indicates an IC50 or EC50 of 10.001-50 μM, and



“E” indicates an IC50 or EC50 >50 μM.



“—” indicates no data.



“*” indicates an IC50 or EC50 >10 μM.



“**” indicates an IC50 or EC50 >20 μM.






Other Embodiments
The foregoing has been a description of certain non-limiting embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

## Claims

1. A method of treating a PRMT5-mediated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I):




or a pharmaceutically acceptable salt thereof,
wherein:
 represents a single or double bond;
Ring A is an optionally substituted 6-membered monocyclic heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an optionally substituted 6-membered monocyclic heteroaryl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur;
R1 is hydrogen, Rz, or —C(O)Rz, wherein Rz is optionally substituted C1-6 alkyl;
Y is O;
R5, R6, R7, and R8 are independently hydrogen, halo, or optionally substituted aliphatic;
each Rx is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR′, and —N(R′′)2;
R′ is hydrogen or optionally substituted aliphatic;
each R′′ is independently hydrogen or optionally substituted aliphatic, or two R′′ are taken together with their intervening atoms to form a heterocyclic ring; and
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits;

wherein, and unless otherwise specified,
heterocyclyl or heterocyclic refers to a radical of a 3-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur;
carbocyclyl or carbocyclic refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms and zero heteroatoms in the non-aromatic ring system;
aryl refers to a radical of a monocyclic or polycyclic aromatic ring system having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system; and
heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur;
each instance of aliphatic is independently an alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl group;
each instance of optionally substituted is independently unsubstituted or substituted, and each instance of substituted is, when on a carbon atom, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, N(Rbb)2, —SII, —SRaa, —C(═O)Raa, —CO2H, —CHO, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups, or two geminal hydrogens on the carbon atom are replaced with the group ═O;
each instance of substituted is, when on a nitrogen, independently —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —SO2N(Rcc)2, —SO2Rcc, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rcc groups attached to a nitrogen atom are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Raa is, independently, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Raa groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rbb is, independently, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —SO2N(Rcc)2, —SO2Rcc, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rbbgroups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rcc is, independently, hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rcc groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rdd is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —N(Rff)2, —SH, —SRcc, —C(═O)Ree, —CO2H, —CO2Rcc, —OC(═O)Ree, —OCO2Ree, —C(═O)N(Rff)2, —OC(═O)N(Rff)2, —NRffC(═O)Rcc, —NRffCO2Ree, —NRffC(═O)N(Rff)2, —NRffSO2Ree, —SO2N(Rff)2, —SO2Ree, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rgg groups;
each instance of Ree is, independently, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein each alkyl, alkenyl, and alkynyl, and alkynyl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
each instance of Rff is, independently, hydrogen, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein each alkyl, alkenyl, and alkynyl, is independently substituted with 0, 1, or 2 Rgg groups; and
each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OC1-6 alkyl, —N(C1-6 alkyl)2, —SH, —SC1-6 alkyl, —C(═O)(C1-6 alkyl), —CO2H, —CO2(C1-6 alkyl), —OC(═O)(C1-6 alkyl), —OCO2(C1-6 alkyl), —C(═O)NH2, —C(═O)N(C1-6 alkyl)2, —OC(═O)NH(C1-6 alkyl), —NHC(═O)(C1-6 alkyl), —N(C1-6 alkyl)C(═O)(C1-6 alkyl), —NHCO2(C1-6 alkyl), —NHC(═O)N(C1-6 alkyl)2, —NHC(═O)NH(C1-6 alkyl), —NHC(═O)NH2, —NHSO2(C1-6 alkyl), —SO2N(C1-6 alkyl)2, —SO2NH(C1-6 alkyl), —SO2NH2, —SO2C1-6 alkyl, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl;

wherein:
the PRMT5-mediated disorder is cancer, a metabolic disorder, or a blood disorder;
the metabolic disorder is diabetes or obesity; and
the blood disorder is sickle cell anemia or β-thalessemia.

2. A method of treating a PRMT5-mediated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (II):




or a pharmaceutically acceptable salt thereof,
wherein:
R1 is hydrogen, Rz, or —C(O)Rz, wherein Rz is optionally substituted C1-6 alkyl;
Y is O;
each Rx is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR′, and —N(R′′)2;
R′ is hydrogen or optionally substituted aliphatic;
each R′′ is independently hydrogen or optionally substituted aliphatic, or two R′′ are taken together with their intervening atoms to form a heterocyclic ring;
G is NR2, CR3R4, O or S;
R2 is selected from the group consisting of optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —C(O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(═NRB)RA, —C(═NRB)N(RB)2, —C(═S)RA, —C(═S)N(RB), —S(═O)RA, —SO2RA, and —SO2N(RB)2;
R3 is selected from the group consisting of hydrogen, halo, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA, —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2;
each RA is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
each RB is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring;
R4 is selected from the group consisting of hydrogen, halo, and optionally substituted aliphatic;
each Ry is independently selected from the group consisting of halo, —CN, —NO2, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA, —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2;
p is 1;
m is 0, 1, 2, 3, or 4; and
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits;

wherein, and unless otherwise specified,
heterocyclyl or heterocyclic refers to a radical of a 3-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur;
carbocyclyl or carbocyclic refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms and zero heteroatoms in the non-aromatic ring system;
aryl refers to a radical of a monocyclic or polycyclic aromatic ring system having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system; and
heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur;
each instance of aliphatic is independently an alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl group;
each instance of optionally substituted is independently unsubstituted or substituted, and each instance of substituted is, when on a carbon atom, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —N(Rbb)2, —SH, —SRaa, —C(═O)Raa, —CO2H, —CHO, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups, or two geminal hydrogens on the carbon atom arc replaced with the group ═O;
each instance of substituted is, when on a nitrogen, independently —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —SO2N(Rcc)2, —SO2Rcc, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rcc groups attached to a nitrogen atom arc joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Raa is, independently, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Raa groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rbb is, independently, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —SO2N(Rcc)C2, —SO2Rcc, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rbb groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rcc is, independently, hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rcc groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rdd is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —N(Rff)2, —SH, —SRee, —C(═O)Ree, —CO2H, —CO2Ree, —OC(═O)Ree, —OCO2Ree, —C(═O)N(Rff)2, —OC(═O)N(Rff)2, —NRffC(═O)Ree, —NRffCO2Ree, —NRffC(═O)N(Rff)2, —NRffSO2Ree, —SO2N(Rff)2, —SO2Ree, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rgg groups;
each instance of Rcc is, independently, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein each alkyl, alkenyl, and alkynyl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
each instance of Rff is, independently, hydrogen, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein each alkyl, alkenyl, and alkynyl is independently substituted with 0, 1, or 2 Rgg groups; and
each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OC1-6 alkyl, —N(C1-6 alkyl)2, —SH, —SC1-6 alkyl, —C(═O)(C1-6 alkyl), —CO2H, —CO2(C1-6 alkyl), —OC(═O)(C1-6 alkyl), —OCO2(C1-6 alkyl), —C(═O)NH2, —C(═O)N(C1-6 alkyl)2, —OC(═O)NH(C1-6 alkyl), —NHC(═O)(C1-6 alkyl), —N(C1-6 alkyl)C(═O)(C1-6 alkyl), —NHCO2(C1-6 alkyl), —NHC(═O)N(C1-6 alkyl)2, —NHC(═O)NH(C1-6 alkyl), —NHC(═O)NH2, —NHSO2(C1-6 alkyl), —SO2N(C1-6 alkyl)2, —SO2NH(C1-6 alkyl), —SO2NH2, —SO2C1-6 alkyl, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl;

wherein:
the PRMT5-mediated disorder is cancer, a metabolic disorder, or a blood disorder;
the metabolic disorder is diabetes or obesity; and
the blood disorder is sickle cell anemia or β-thalessemia.

3. The method of claim 2, wherein the compound is of Formula (III):




or a pharmaceutically acceptable salt thereof.

4. The method of claim 2, wherein the compound is of Formula (IV):




or a pharmaceutically acceptable salt thereof.

5. A method of treating a PRMT5-mediated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (V):




or a pharmaceutically acceptable salt thereof;
wherein:
each Rx is independently selected from the group consisting of halo, —CN, optionally substituted aliphatic, —OR′, and —N(R′′);
R′ is hydrogen or optionally substituted aliphatic;
each R′′ is independently hydrogen or optionally substituted aliphatic, or two R′′ are taken together with their intervening atoms to form a heterocyclic ring;
each Ry is independently selected from the group consisting of halo, —CN, —NO2, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —ORA, —N(RB)2, —SRA, —C(═O)RA, —C(O)ORA, —C(O)SRA, —C(O)N(RB)2, —C(O)N(RB)N(RB)2, —OC(O)RA, —OC(O)N(RB)2, —NRBC(O)RA, —NRBC(O)N(RB)2, —NRBC(O)N(RB)N(RB)2, —NRBC(O)ORA, —SC(O)RA, —C(═NRB)RA, —C(═NNRB)RA; —C(═NORA)RA, —C(═NRB)N(RB)2, —NRBC(═NRB)RB, —C(═S)RA, —C(═S)N(RB)2, —NRBC(═S)RA, —S(O)RA, —OS(O)2RA, —SO2RA, —NRBSO2RA, and —SO2N(RB)2;
each RA is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;
each RB is independently selected from the group consisting of hydrogen, optionally substituted aliphatic, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, or two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring;
m is 0, 1, 2, 3, or 4; and
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits;

wherein, and unless otherwise specified,
heterocyclyl or heterocyclic refers to a radical of a 3-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur;
carbocyclyl or carbocyclic refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms and zero heteroatoms in the non-aromatic ring system;
aryl refers to a radical of a monocyclic or polycyclic aromatic ring system having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system; and
heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur;
each instance of aliphatic is independently an alkyl, alkenyl, alkynyl, cycloalkyl, or cycloalkenyl group;
each instance of optionally substituted is independently unsubstituted or substituted, and each instance of substituted is, when on a carbon atom, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —N(Rbb)2, —SH, —SRaa, —C(═O)Raa, CO2H, —CHO, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2, —OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2, —C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, C1-10 alkyl, C1-10 perhaloalkyl, C1-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups, or two geminal hydrogens on the carbon atom are replaced with the group ═O;
each instance of substituted is, when on a nitrogen, independently —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —SO2N(Rcc)2, —SO2Rcc, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rcc groups attached to a nitrogen atom are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Raa is, independently, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Raa groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rbb is, independently, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —SO2N(Rcc)2, —SO2Rcc, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rbb groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rcc is, independently, hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, or two Rcc groups are joined to form a monocyclic 3-6 membered heterocyclyl or 5-6 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rdd groups;
each instance of Rdd is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —N(Rff)2, —SH, —SRcc, —C(═O)Ree, —CO2H, —CO2Ree, —OC(═O)Ree, —OCO2Ree, —C(═O)N(Rff)2, —OC(═O)N(Rff)2, —NRffC(═O)Ree, —NRffCO2Ree, —NRffC(═O)N(Rff)2, —NRffSO2Ree, —SO2N(Rff)2, —SO2Ree, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, C2-6 alkynyl, monocyclic C3-6 carbocyclyl, monocyclic 3-6 membered heterocyclyl, C6aryl, or 5-6 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, or 2 Rgg groups;
each instance of Rcc is, independently, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein each alkyl, alkenyl, and alkynyl, is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
each instance of Rff is, independently, hydrogen, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl, wherein each alkyl, alkenyl, and alkynyl is independently substituted with 0, 1, or 2 Rgg groups; and
each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —OC1-6 alkyl, —N(C1-6 alkyl)2, —SH, —SC1-6 alkyl, —C(═O)(C1-6 alkyl), —CO2H, —CO2(C1-6 alkyl), —OC(═O)(C1-6 alkyl), —OCO2(C1-6 alkyl), —C(═O)NH2, —C(═O)N(C1-6 alkyl)2, —OC(═O)NH(C1-6 alkyl), —NHC(═O)(C1-6 alkyl), —N(C1-6 alkyl)C(═O)(C1-6 alkyl), —NHCO2(C1-6 alkyl), —NHC(═O)N(C1 alkyl)2, —NHC(═O)NH(C1-6 alkyl), —NHC(═O)NH2, —NHSO2(C1-6 alkyl), —SO2N(C1-6 alkyl)2, —SO2NH(C1-6 alkyl), —SO2NH2, —SO2C1-6 alkyl, C1-6 alkyl, C1-6 perhaloalkyl, C2-6 alkenyl, or C2-6 alkynyl;

wherein:
the PRMT5-mediated disorder is cancer, a metabolic disorder, or a blood disorder;
the metabolic disorder is diabetes or obesity; and
the blood disorder is sickle cell anemia or β-thalessemia.

6. The method of claim 1, wherein R1 is hydrogen.

7. The method of claim 1, wherein n is 0.

8. The method of claim 1, wherein n is 1.

9. The method of claim 2, wherein m is 0.

10. The method of claim 2, wherein G is NR2, O, or CR3R4, wherein R4 is hydrogen.

11. The method of claim 10, wherein O is NR2, and R2 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aliphatic, —C(═O)RA, or —SO2RA.

12. The method of claim 1, wherein the compound is selected from the group consisting of:













and pharmaceutically acceptable salts thereof.

13. The method of claim 1, wherein the disorder is cancer, and the cancer is breast cancer, esophageal cancer, bladder cancer, lymphoma, medulloblastoma, rectum adenocarcinoma, colon adenocarcinoma, gastric cancer, liver cancer, adenoid cystic carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, renal cell carcinoma, oligodendroglioma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, hematopoietic cancer, lung cancer, prostate cancer, melanoma, or pancreatic cancer.

14. The method of claim 1, wherein the disorder is a metabolic disorder, and the metabolic disorder is diabetes or obesity.

15. The method of claim 1, wherein the disorder is a blood disorder, and the blood disorder is sickle cell anemia or β-thalessemia.

16. The method of claim 1, wherein the compound is selected from the group consisting of:




and pharmaceutically acceptable salts thereof.