# Patent

## Title
Pyrazole derivatives as arginine methyltransferase inhibitors and uses thereof

## Abstract

Described herein are compounds of Formula (I), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds described herein are useful for inhibiting arginine methyltransferase activity. Methods of using the compounds for treating arginine methyltransferase-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/US2014/029583, filed Mar. 14, 2014 which claims priority under 35 U.S.C. §119(e) to U.S. provisional patent application, U.S. Ser. No. 61/781,054, filed Mar. 14, 2013, the entire contents of each of which is 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., arginine methyltransferases), many of which are associated with specific genetic alterations that can cause human disease.
Disease-associated chromatin-modifying enzymes (e.g., arginine methyltransferases) play a role in diseases such as proliferative disorders, autoimmune disorders, muscular disorders, vascular disorders, metabolic disorders, and neurological disorders. Thus, there is a need for the development of small molecules that are capable of inhibiting the activity of arginine methyltransferases.


DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Arginine methyltransferases are attractive targets for modulation given their 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 arginine methyltransferases. Such compounds have the general Formula (I):




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R1, R2, R3, R6, R7, R8, and Rx are as defined herein.

In some embodiments, pharmaceutical compositions are provided which comprise a compound described herein (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.
In certain embodiments, compounds described herein inhibit activity of an arginine methyltransferase (RMT) (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, methods of inhibiting an arginine methyltransferase are provided which comprise contacting the arginine methyltransferase with an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. The RMT may be purified or crude, and may be present in a cell, tissue, or a subject. Thus, such methods encompass inhibition of RMT activity both in vitro and in vivo. In certain embodiments, the RMT is wild-type. In certain embodiments, the RMT is overexpressed. In certain embodiments, the RMT is a mutant. In certain embodiments, the RMT is in a cell. In certain embodiments, the RMT is in an animal, e.g., a human. In some embodiments, the RMT is expressed at normal levels in a subject, but the subject would benefit from RMT inhibition (e.g., because the subject has one or more mutations in an RMT substrate that causes an increase in methylation of the substrate with normal levels of RMT). In some embodiments, the RMT is in a subject known or identified as having abnormal RMT activity (e.g., overexpression).
In certain embodiments, methods of modulating gene expression in a cell are provided which comprise contacting a cell with an effective amount of a compound of 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 modulating transcription in a cell are provided which comprise contacting a cell with an effective amount of a compound of 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 an RMT-mediated disorder (e.g., a PRMT1-, PRMT3-, CARM1-, PRMT6-, or PRMT8-mediated disorder) are provided which comprise administering to a subject suffering from an RMT-mediated disorder an effective amount of a compound described herein (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the RMT-mediated disorder is a proliferative disorder. In certain embodiments, compounds described herein are useful for treating cancer. In certain embodiments, compounds described herein are useful for treating breast cancer, prostate cancer, lung cancer, colon cancer, bladder cancer, or leukemia. In certain embodiments, the RMT-mediated disorder is a muscular disorder. In certain embodiments, the RMT-mediated disorder is an autoimmune disorder. In certain embodiments, the RMT-mediated disorder is a neurological disorder. In certain embodiments, the RMT-mediated disorder is a vascular disorder. In certain embodiments, the RMT-mediated disorder is a metabolic disorder.
Compounds described herein are also useful for the study of arginine methyltransferases in biological and pathological phenomena, the study of intracellular signal transduction pathways mediated by arginine methyltransferases, and the comparative evaluation of new RMT 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.
“Radical” refers to a point of attachment on a particular group. Radical includes divalent radicals of a particular group.
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 (“C1-4alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2alkyl”). 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 C1-10alkyl.
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.
As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds), and optionally one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C2-20 alkenyl”). In certain embodiments, alkenyl does not comprise triple bonds. 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.
As used herein, “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds), and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-20 alkynyl”). In certain embodiments, alkynyl does not comprise double bonds. 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 (“C2-6alkynyl”). 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 C2-6 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.
“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.

“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 certain embodiments, a carbocyclyl group refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. 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 contain 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, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 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 selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms 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 thiiranyl. 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 three 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 14π 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 (“C1-4 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 10π 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 selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms 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 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, any one of the following formulae:
















In any of the monocyclic or bicyclic heteroaryl groups, the point of attachment can be any carbon or nitrogen atom, as valency permits.

“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, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “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, —SO2Raa, —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 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(C1-4 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, C610 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-chloroethoxy)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-benzodithiolan-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), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, 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, a-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.
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 arginine methyltransferase (RMT) inhibitors. In some embodiments, the present disclosure provides a compound of Formula (I):




or a pharmaceutically acceptable salt thereof,

wherein

X is N, Z is NR4, and Y is CR5; or
X is NR4, Z is N, and Y is CR5; or
X is CR5, Z is NR4, and Y is N; or
X is CR5, Z is N, and Y is NR4;
R1, R2, R6, R7, and R8 are independently selected from the group consisting of hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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, or —SO2N(RB)2;
each RA is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom;
each RB is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, and a nitrogen protecting group, or two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring;
R1 and R2 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring; or
R1 and R6 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring; or
R2 and R8 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring; or
R6 and R7 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring;
R3 is hydrogen, C1-4 alkyl, or C3-4 cycloalkyl;
R4 is hydrogen, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl; or optionally substituted C1-4 alkyl-Cy;
Cy is optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
R5 is hydrogen, halo, —CN, optionally substituted C1-4 alkyl, or optionally substituted C3-4 cycloalkyl; and
Rx is optionally substituted C1-4 alkyl or optionally substituted C3-4 cycloalkyl.
In some embodiments, R2 is not —CF3 or —CHF2. In some embodiments, R6 is not —CF3 or —CHF2. In some embodiments, R1 is not Ry, wherein Ry is —NRBC(O)RA, —NRBSO2RA, or —(CRzRz)nC(O)N(RB)2; wherein each Rz is independently hydrogen or fluoro; and n 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, R2, R3, R4, R5, R6, R7, R8, and Rx are as described herein.

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




or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4, R5, R6, R7, R8, and Rx are as described herein.

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




or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4, R5, R6, R7, R8, and Rx are as described herein.

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




or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, R4, R5, R6, R7, R8, and Rx are as described herein.

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




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R1, R3, and Rx are as described herein, and R1 is not hydrogen.

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




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R1, R2, R3, and Rx are as described herein, and R1 and R2 are not hydrogen.

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




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R2, R3, and Rx are as described herein, and R2 is not hydrogen.

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




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R2, R3, R6, and Rx are as described herein, and R2 and R6 are not hydrogen.

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




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R2, R8, R3, and Rx are as described herein, and R2 and R8 are not hydrogen.

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




or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R, R2, R3, R6, and Rx are as described herein, and R, R2, and R6 are not hydrogen.

In certain embodiments, a provided compound is any one of Formula (II-a) to (II-f):







or a pharmaceutically acceptable salt thereof, wherein R1, R3, R4, R5 and Rx are as described herein, and wherein neither of R1, R2, R6, or R8 is hydrogen. In certain embodiments, R3 is hydrogen. In certain embodiments, R3 is C1-4 alkyl (e.g., methyl). In certain embodiments, R4 is hydrogen or optionally substituted C1-6 alkyl (e.g., optionally substituted methyl). In certain embodiments, Rx is optionally substituted C1-4 alkyl (e.g., optionally substituted methyl). In certain embodiments, R5 is hydrogen. In certain embodiments, R1 is halo, —CN, optionally substituted alkyl, —ORA, —N(RB)2. In certain embodiments, R1 is halog, e.g, fluoro. In certain embodiments, R1 is —CN. In certain embodiments, R1 is optionally substituted C1-6alkyl. In certain embodiments, R1 is —N(RB)2. In certain embodiments, R1 is —ORA. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted alkyl, e.g., RA is unsubstituted C1-6 alkyl or C1-6 alkyl substituted with optionally substituted heterocyclyl, optionally substituted carbocyclyl, —OH, or alkoxy. In certain embodiments, R1 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R1 is —O—C1-6 alkyl-O—C1-6alkyl. In certain embodiments, R1 is —OCH2CH2OCH3 or —OCH2CH2CH2OCH3. In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R2 is halo (e.g., fluoro, chloro, or bromo). In certain embodiments, R2 is fluoro.

In certain embodiments, a provided compound is of any one of Formula (III-a) to (III-f):




or a pharmaceutically acceptable salt thereof, wherein R1, R3, R4, R5 and Rx are as described herein, and wherein neither of R1, R2, R6, or R8 is hydrogen. In certain embodiments, R3 is hydrogen. In certain embodiments, R3 is C1-4 alkyl (e.g., methyl). In certain embodiments, R4 is hydrogen or optionally substituted C1-6 alkyl (e.g., optionally substituted methyl). In certain embodiments, Rx is optionally substituted C1-4 alkyl (e.g., optionally substituted methyl). In certain embodiments, R5 is hydrogen. In certain embodiments, R1 is halo, —CN, optionally substituted alkyl, —ORA, —N(RB)2. In certain embodiments, R1 is halog, e.g, fluoro. In certain embodiments, R1 is —CN. In certain embodiments, R1 is optionally substituted C1-6alkyl. In certain embodiments, R1 is —N(RB)2. In certain embodiments, R1 is —ORA. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted alkyl, e.g., RA is unsubstituted C1-6 alkyl or C1-6 alkyl substituted with optionally substituted heterocyclyl, optionally substituted carbocyclyl, —OH, or alkoxy. In certain embodiments, R1 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R1 is —O—C1-6alkyl-O—C1-6alkyl. In certain embodiments, R1 is —OCH2CH2OCH3 or —OCH2CH2CH2OCH3. In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R2 is halo (e.g., fluoro, chloro, or bromo). In certain embodiments, R2 is fluoro.

In certain embodiments, a provided compound is any one of Formula (IV-a) to (IV-f):








or a pharmaceutically acceptable salt thereof, wherein R1, R3, R4, R5 and Rx are as described herein, and wherein neither of R1, R2, R6, or R8 is hydrogen. In certain embodiments, R3 is hydrogen. In certain embodiments, R3 is C1-4 alkyl (e.g., methyl). In certain embodiments, R4 is hydrogen or optionally substituted C1-6 alkyl (e.g., optionally substituted methyl). In certain embodiments, Rx is optionally substituted C1-4 alkyl (e.g., optionally substituted methyl). In certain embodiments, R5 is hydrogen. In certain embodiments, R1 is halo, —CN, optionally substituted alkyl, —ORA, —N(RB)2. In certain embodiments, R1 is halog, e.g, fluoro. In certain embodiments, R1 is —CN. In certain embodiments, R1 is optionally substituted C1-6alkyl. In certain embodiments, R1 is —N(RB)2. In certain embodiments, R1 is —ORA. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted alkyl, e.g., RA is unsubstituted C1-6 alkyl or C1-6 alkyl substituted with optionally substituted heterocyclyl, optionally substituted carbocyclyl, —OH, or alkoxy. In certain embodiments, R1 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R1 is —O—C1-6alkyl-O—C1-6alkyl. In certain embodiments, R1 is —OCH2CH2OCH3 or —OCH2CH2CH2OCH3. In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R2 is halo (e.g., fluoro, chloro, or bromo). In certain embodiments, R2 is fluoro.

In certain embodiments, a provided compound is any one of Formula (V-a) to (V-f):





or a pharmaceutically acceptable salt thereof, wherein R1, R3, R4, R5 and Rx are as described herein, and wherein neither of R1, R2, R6, or R8 is hydrogen. In certain embodiments, R3 is hydrogen. In certain embodiments, R3 is C1-4 alkyl (e.g., methyl). In certain embodiments, R4 is hydrogen or optionally substituted C1-6 alkyl (e.g., optionally substituted methyl). In certain embodiments, Rx is optionally substituted C1-4 alkyl (e.g., optionally substituted methyl). In certain embodiments, R5 is hydrogen. In certain embodiments, R1 is halo, —CN, optionally substituted alkyl, —ORA, —N(RB)2. In certain embodiments, R1 is halog, e.g, fluoro. In certain embodiments, R1 is —CN. In certain embodiments, R1 is optionally substituted C1-6alkyl. In certain embodiments, R1 is —N(RB)2. In certain embodiments, R1 is —ORA. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted alkyl, e.g., RA is unsubstituted C1-6 alkyl or C1-6 alkyl substituted with optionally substituted heterocyclyl, optionally substituted carbocyclyl, —OH, or alkoxy. In certain embodiments, R1 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R1 is —O—C1-6alkyl-O—C1-6alkyl. In certain embodiments, R1 is —OCH2CH2OCH3 or —OCH2CH2CH2OCH3. In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R2 is halo (e.g., fluoro, chloro, or bromo). In certain embodiments, R2 is fluoro.

As defined generally above, R1 is hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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, or —SO2N(RB)2, wherein R1 is not Ry, wherein Ry is —NRBC(O)RA, —NRBSO2RA, or —(CRzRz)nC(O)N(RB)2; wherein each Rz is independently hydrogen or fluoro; and n is 0, 1, 2, 3, or 4. In certain embodiments, R1 is hydrogen. In some embodiments, R1 is not hydrogen. In some embodiments, R1 is halo. In certain embodiments, R1 is fluoro. In certain embodiments, R1 is chloro. In some embodiments, R1 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl. In certain embodiments, R1 is optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, or optionally substituted C3-6 carbocyclyl. In certain embodiments, R1 is optionally substituted C1-6 alkyl. In certain embodiments, R1 is substituted C1-6 alkyl. In certain embodiments, R1 is —CF3, CHF2, or CH2F. In certain embodiments, R1 is —C1-6alkyl-carbocyclyl. In certain embodiments, R1 is —CH2-cyclopropyl or —CH2-cyclobutyl. In certain embodiments, R1 is unsubstituted C1-6 alkyl. In certain embodiments, R1 is methyl, ethyl, propyl, butyl, or pentyl. In certain embodiments, R1 is optionally substituted alkoxyalkyl. In certain embodiments, R1 is —CH2ORA, —CH2CH2ORA, or —CH2CH2CH2ORA. In certain embodiments, R1 is




In certain embodiments, R1 is isopropyl, isobutyl, or isoamyl. In certain embodiments, R1 is isobutyl. In some embodiments, R1 is —CN. In some embodiments, R1 is optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl. In some embodiments, R1 is —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)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, or —SO2N(RB)2. In certain embodiments, R1 is —N(RB)2. In certain embodiments, R1 is —NHRB. In certain embodiments, R1 is —NH2. In certain embodiments, R1 is —ORA. In certain embodiments, R1 is —OH. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl. In certain embodiments, R1 is —O-isobutylenyl. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted C1-6 alkyl. In certain embodiments, R1 is —ORA, wherein RA is unsubstituted C1-6 alkyl. In certain embodiments, R1 is —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R1 is —ORA, wherein RA is substituted C1-6 alkyl. In certain embodiments, R1 is —O—C1-6alkyl-O—C1-6alkyl. In certain embodiments, R1 is —OCH2CH2OCH3 or —OCH2CH2CH2OCH3. In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is —O-carbocyclyl. In certain embodiments, R1 is —O-cyclohexyl, —O-cyclopentyl, —O-cyclobutyl, or —O-cyclopropyl. In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is





In certain embodiments, R1 is —O—C1-6alkyl-carbocyclyl. In certain embodiments, R1 is —O—CH2-cyclobutyl or —O—CH2-cyclopropyl. In certain embodiments, R1 is —O—C1-6alkyl-heterocyclyl. In certain embodiments, R1 is —O—CH2-tetrahydropyranyl, —O—CH2-tetrahydrofuranyl, or —O—CH2-oxetanyl. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted heterocyclyl. In certain embodiments, R1 is —O-tetrahydropyranyl, —O-tetrahydrofuranyl, or —O-oxetanyl. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted aryl. In certain embodiments, R1 is —O-phenyl. In certain embodiments, R1 is unsubstituted —O-phenyl. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted heteroaryl. In certain embodiments, R1 is —ORA, wherein RA is optionally substituted pyrazole, imidazole, oxazole, thiazole, oxadiazole, thiadiazole, triazole, pyridine, pyrimidine, pyridizine, or pyrazine. In certain embodiments, R1 is —ORA, wherein RA is unsubstituted pyrazole, imidazole, oxazole, thiazole, oxadiazole, thiadiazole, triazole, pyridine, pyrimidine, pyridizine, or pyrazine. In certain embodiments, R1 is —ORA, wherein RA is unsubstituted pyrimidine. In certain embodiments, R1 is —N(RB)2, wherein each instance of RB is independently optionally substituted C1-6 alkyl; or wherein two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring. In certain embodiments, R1 is —N(RB)2, wherein each instance of RB is independently unsubstituted C1-6 alkyl. In certain embodiments, R1 is —N(RB)2, wherein each instance of RB is independently methyl, ethyl, propyl, or butyl. In certain embodiments, R1 is —N(RB)2, wherein each instance of RB is independently branched unsubstituted C1-6 alkyl. In certain embodiments, R1 is —N(RB)2, wherein each instance of RB is independently isopropyl, isobutyl, or tert-butyl. In certain embodiments, R1 is —N(RB)2, wherein one instance of RB is independently optionally substituted C1-6 alkyl; and the second instance of RB is hydrogen. In certain embodiments, R1 is —N(RB)2, wherein one instance of RB is independently branched unsubstituted C1-6 alkyl; and the second instance of RB is hydrogen. In certain embodiments, R1 is —N(RB)2, wherein one instance of RB is independently isopropyl, isobutyl, or tert-butyl; and the second instance of RB is hydrogen. In certain embodiments, R1 is —N(RB)2, wherein one instance of RB is independently optionally substituted C1-6 alkyl; and the second instance of RB is methyl. In certain embodiments, R1 is —N(RB)2, wherein one instance of RB is independently branched unsubstituted C1-6 alkyl; and the second instance of RB is methyl. In certain embodiments, R1 is —N(RB)2, wherein one instance of RB is independently isopropyl, isobutyl, or tert-butyl; and the second instance of RB is methyl.

As defined generally above, R2 is hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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, or —SO2N(RB)2, wherein R2 is not —CF3 or —CHF2. In certain embodiments, R2 is hydrogen. In some embodiments, R2 is not hydrogen. In some embodiments, R2 is halo. In certain embodiments, R2 is fluoro. In certain embodiments, R2 is chloro. In some embodiments, R2 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl. In certain embodiments, R2 is optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, or optionally substituted C3-6 carbocyclyl. In certain embodiments, R2 is optionally substituted C1-6 alkyl. In certain embodiments, R2 is substituted C1-6 alkyl. In certain embodiments, R2 is —C1-6alkyl-carbocyclyl. In certain embodiments, R2 is —CH2-cyclopropyl or —CH2-cyclobutyl. In certain embodiments, R2 is unsubstituted C1-6 alkyl. In certain embodiments, R2 is methyl, ethyl, propyl, butyl, or pentyl. In certain embodiments, R2 is isopropyl, isobutyl, or isoamyl. In certain embodiments, R2 is isobutyl. In some embodiments, R2 is —CN. In some embodiments, R2 is optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl. In some embodiments, R2 is —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, or —SO2N(RB)2. In certain embodiments, R2 is —N(RB)2. In certain embodiments, R2 is —NHRB. In certain embodiments, R2 is —NH2. In certain embodiments, R2 is —ORA. In certain embodiments, R2 is —OH. In certain embodiments, R2 is —ORA, wherein RA is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl. In certain embodiments, R2 is —O-isobutylenyl. In certain embodiments, R2 is —ORA, wherein RA is optionally substituted C1-6 alkyl. In certain embodiments, R2 is —ORA, wherein RA is unsubstituted C1-6 alkyl. In certain embodiments, R2 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R2 is —OCH(CH2CH3)2. In certain embodiments, R2 is —OCH(OH)CH(CH3)2. In certain embodiments, R2 is —ORA, wherein RA is substituted C1-6 alkyl. In certain embodiments, R2 is —O—C1-6alkyl-O—C1-6 alkyl. In certain embodiments, R2 is —OCH2CH2OCH3, —OCH2CH2CH2OCH3, —OCH2CH2OH, or —OCH2CH2OCH2CH2CH3. In certain embodiments, R2 is —OCH2CF3 or —OCH2CH2CH2CF3. In certain embodiments, R2 is —OCH(CH3)2, —OCH2CH(CH3)2, —OCH2CH2CH3, —OCH2CH2CH(CH3)2, —OCH2CH2CH2OCH3, or —OCH2CH2OCH3. In certain embodiments, R2 is —O—C1-6alkyl-carbocyclyl. In certain embodiments, R2 is —O—CH2-cyclobutyl or —O—CH2-cyclopropyl. In certain embodiments, R2 is —O—CH2-cyclopentyl or —O—CH2CH2-cyclohexyl. In certain embodiments, R2 is —O—C1-6alkyl-heterocyclyl. In certain embodiments, R2 is —O—CH2-tetrahydropyranyl or —O—CH2-oxetanyl. In certain embodiments, R2 is —O—C1-6alkyl-aryl. In certain embodiments, R2 is —O-benzyl or —OCH2CH2Ph. In certain embodiments, R2 is —O—C1-6alkyl-heteroaryl. In certain embodiments, R2 is —ORA, wherein RA is optionally substituted heterocyclyl. In certain embodiments, R2 is —O-tetrahydropyranyl or —O-oxetanyl. In certain embodiments, R2 is —ORA, wherein RA is optionally substituted aryl. In certain embodiments, R2 is —O-phenyl. In certain embodiments, R2 is —ORA, wherein RA is optionally substituted heteroaryl. In certain embodiments, R2 is —O-pyridyl. In certain embodiments, R2 is —O-2-pyridyl. In certain embodiments, R2 is —O-3-pyridyl. In certain embodiments, R2 is —O-4-pyridyl. In certain embodiments, R2 is —O-pyrimidinyl.
As defined generally above, R6 is hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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, or —SO2N(RB)2, wherein R6 is not —CF3 or —CHF2. In certain embodiments, R6 is hydrogen. In some embodiments, R6 is not hydrogen. In some embodiments, R6 is halo. In certain embodiments, R6 is fluoro. In certain embodiments, R6 is chloro. In some embodiments, R6 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl. In certain embodiments, R6 is optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, or optionally substituted C3-6 carbocyclyl. In certain embodiments, R6 is optionally substituted C1-6 alkyl. In certain embodiments, R6 is substituted C1-6 alkyl. In certain embodiments, R6 is —C1-6alkyl-carbocyclyl. In certain embodiments, R6 is —CH2-cyclopropyl or —CH2-cyclobutyl. In certain embodiments, R6 is unsubstituted C1-6 alkyl. In certain embodiments, R6 is methyl, ethyl, propyl, butyl, or pentyl. In certain embodiments, R6 is isopropyl, isobutyl, or isoamyl. In certain embodiments, R6 is isobutyl. In some embodiments, R6 is —CN. In some embodiments, R6 is optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl. In some embodiments, R6 is —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, or —SO2N(RB)2. In certain embodiments, R6 is —N(RB)2. In certain embodiments, R6 is —NHRB. In certain embodiments, R6 is —NH2. In certain embodiments, R6 is —ORA. In certain embodiments, R6 is —OH. In certain embodiments, R6 is —ORA, wherein RA is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclyl. In certain embodiments, R6 is —O-isobutylenyl. In certain embodiments, R6 is —ORA, wherein RA is optionally substituted C1-6 alkyl. In certain embodiments, R6 is —ORA, wherein RA is unsubstituted C1-6 alkyl. In certain embodiments, R6 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certain embodiments, R6 is —OCH(CH2CH3)2. In certain embodiments, R6 is —OCH(OH)CH(CH3)2. In certain embodiments, R6 is —ORA, wherein RA is substituted C1-6 alkyl. In certain embodiments, R6 is —O—C1-6alkyl-O—C1-6alkyl. In certain embodiments, R6 is —OCH2CH2OCH3, —OCH2CH2CH2OCH3, —OCH2CH2OH, or —OCH2CH2OCH2CH2CH3. In certain embodiments, R6 is —OCH2CF3 or —OCH2CH2CH2CF3. In certain embodiments, R6 is —O—C1-6alkyl-carbocyclyl. In certain embodiments, R6 is —O—CH2-cyclobutyl or —O—CH2-cyclopropyl. In certain embodiments, R6 is —O—CH2-cyclopentyl or —O—CH2CH2-cyclohexyl. In certain embodiments, R6 is —O—C1-6alkyl-heterocyclyl. In certain embodiments, R6 is —O—CH2-tetrahydropyranyl or —O—CH2-oxetanyl. In certain embodiments, R6 is —O—C1-6 alkyl-aryl. In certain embodiments, R6 is —O-benzyl or —OCH2CH2Ph. In certain embodiments, R6 is —O—C1-6alkyl-heteroaryl. In certain embodiments, R6 is —ORA, wherein RA is optionally substituted heterocyclyl. In certain embodiments, R6 is —O-tetrahydropyranyl or —O-oxetanyl. In certain embodiments, R6 is —ORA, wherein RA is optionally substituted aryl. In certain embodiments, R6 is —O-phenyl. In certain embodiments, R6 is —ORA, wherein RA is optionally substituted heteroaryl. In certain embodiments, R6 is —O-pyridyl. In certain embodiments, R6 is —O-2-pyridyl. In certain embodiments, R6 is —O-3-pyridyl. In certain embodiments, R6 is —O-4-pyridyl. In certain embodiments, R6 is —O-pyrimidinyl.
As defined generally above, R7 is hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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, or —SO2N(RB)2. In some embodiments, R7 is hydrogen. In some embodiments, R7 is halo. In some embodiments, R7 is chloro. In some embodiments, R7 is fluoro. In some embodiments, R7 is —CN. In some embodiments, R7 is optionally substituted C1-6 alkyl. In some embodiments, R7 is —CF3 or —CHF2. In some embodiments, R7 is methyl or ethyl. In some embodiments, R7 is isopropyl or isobutyl. In some embodiments, R7 is —ORA, —N(RB)2, —SRA. In some embodiments, R7 is —ORA. In some embodiments, R7 is —OCH3.
As defined generally above, R8 is hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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, or —SO2N(RB)2. In some embodiments, R8 is hydrogen. In some embodiments, R8 is halo. In some embodiments, R8 is chloro. In some embodiments, R8 is fluoro. In some embodiments, R is —CN. In some embodiments, R8 is optionally substituted C1-6 alkyl. In some embodiments, R8 is —CF3 or —CHF2. In some embodiments, R8 is methyl or ethyl. In some embodiments, R8 is isopropyl or isobutyl. In some embodiments, R8 is —ORA, —N(RB)2, —SRA. In some embodiments, R8 is —ORA. In some embodiments, R8 is —OCH3.
In some embodiments, at least one of R7 and R8 is not hydrogen. In some embodiments, R7 is hydrogen and R8 is not hydrogen. In some embodiments, R7 is not hydrogen and R8 is hydrogen. In some embodiments, R7 and R8 are not hydrogen. In some embodiments, R7 and R8 are hydrogen.
In some embodiments, R1 and R2 are taken together to form an optionally substituted carbocyclic, optionally substituted aryl, or optionally substituted heterocyclic ring. In some embodiments, R1 and R2 are taken together to form an optionally substituted carbocyclic ring. In some embodiments, R1 and R2 are taken together to form an optionally substituted aryl ring. In certain embodiments, R1 and R2 are taken together to form an optionally substituted benzene ring. In some embodiments, R1 and R2 are taken together to form an optionally substituted heterocyclic ring.
In some embodiments, R2 and R8 are taken together to form an optionally substituted carbocyclic, optionally substituted aryl, or optionally substituted heterocyclic ring. In some embodiments, R2 and R8 are taken together to form an optionally substituted carbocyclic ring. In some embodiments, R2 and R8 are taken together to form an optionally substituted aryl ring. In some embodiments, R2 and R8 are taken together to form an optionally substituted benzene ring. In some embodiments, R2 and R8 are taken together to form an optionally substituted heterocyclic ring.
In some embodiments, R6 and R7 are taken together to form an optionally substituted carbocyclic, optionally substituted aryl, or optionally substituted heterocyclic ring. In some embodiments, R6 and R7 are taken together to form an optionally substituted carbocyclic ring. In some embodiments, R6 and R7 are taken together to form an optionally substituted aryl ring. In some embodiments, R6 and R7 are taken together to form an optionally substituted benzene ring. In some embodiments, R6 and R7 are taken together to form an optionally substituted heterocyclic ring.
In some embodiments, R1 and R6 are taken together to form an optionally substituted carbocyclic, optionally substituted aryl, or optionally substituted heterocyclic ring. In some embodiments, R1 and R6 are taken together to form an optionally substituted carbocyclic ring. In some embodiments, R1 and R6 are taken together to form an optionally substituted aryl ring. In some embodiments, R1 and R6 are taken together to form an optionally substituted benzene ring. In some embodiments, R1 and R6 are taken together to form an optionally substituted heterocyclic ring.
As defined generally above, R3 is hydrogen, C1-4 alkyl, or C3-4 cycloalkyl. In certain embodiments, R3 is hydrogen. In certain embodiments, R3 is C1-4 alkyl. In certain embodiments, R3 is methyl. In certain embodiments, R3 is ethyl. In certain embodiments, R3 is propyl or butyl. In certain embodiments, R3 is cyclopropyl. In certain embodiment, R3 is cyclobutyl.
As defined generally above, R4 is hydrogen, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl; or optionally substituted C1-4 alkyl-Cy, wherein Cy is optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, R4 is hydrogen. In certain embodiments, R4 is optionally substituted C1-6 alkyl. In certain embodiments, R4 is unsubstituted C1-6 alkyl. In certain embodiments, R4 is methyl, ethyl, or isopropyl. In certain embodiments, R4 is substituted C1-6 alkyl. In certain embodiments, R4 is methoxyethyl. In certain embodiments, R4 is hydroxyethyl or propane-1,2-diol. In certain embodiments, R4 is optionally substituted C3-7 cycloalkyl. In certain embodiments, R4 is unsubstituted C3-7 cycloalkyl. In certain embodiments, R4 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In certain embodiments, R4 is optionally substituted 4- to 7-membered heterocyclyl. In certain embodiments, R4 is optionally substituted 4- to 7-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R4 is oxetane, tetrahydrofuran, or tetrahydropyran. In certain embodiments, R4 is optionally substituted C1-4 alkyl-Cy, wherein Cy is optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Cy is optionally substituted C3-7 cycloalkyl. In some embodiments, Cy is optionally substituted 4- to 7-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy is oxetane, tetrahydrofuran, or tetrahydropyran. In some embodiments, Cy is optionally substituted aryl. In some embodiments, Cy is optionally substituted phenyl. In some embodiments, Cy is unsubstituted phenyl. In some embodiments, Cy is optionally substituted heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy is optionally substituted 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, Cy is pyridyl. In some embodiments, R4 is




In some embodiments, R4 is





In some embodiments, R4 is





As defined generally above, R5 is hydrogen, halo, —CN, optionally substituted C1-4 alkyl, or optionally substituted C3-4 cycloalkyl. In certain embodiments, R5 is hydrogen. In certain embodiments, R5 is halo. In certain embodiments, R5 is chloro. In certain embodiments, R5 is fluoro. In certain embodiments, R5 is —CN. In certain embodiments, R5 is optionally substituted C1-4 alkyl. In certain embodiments, R5 is unsubstituted C1-4 alkyl. In certain embodiments, R5 is methyl. In certain embodiments, R5 is ethyl. In certain embodiments, R5 is propyl or butyl. In certain embodiments, R5 is C1-4 alkyl substituted with one or more fluoro groups. In certain embodiments, R5 is —CF3. In certain embodiments, R5 is —CHF2. In certain embodiments, R5 is optionally substituted C3-4 cycloalkyl. In certain embodiments, R5 is cyclopropyl or cyclobutyl.
As defined generally above, Rx is optionally substituted C1-4 alkyl or optionally substituted C3-4 cycloalkyl. In certain embodiments, Rx is optionally substituted C1-4 alkyl. In certain embodiments, Rx is unsubstituted C1-4 alkyl. In certain embodiments, Rx is methyl. In certain embodiments, Rx is ethyl. In certain embodiments, Rx is isopropyl. In certain embodiments, Rx is propyl or butyl. In certain embodiments, Rx is substituted C1-4 alkyl. In certain embodiments, Rx is C1-4 alkyl substituted with hydroxyl or alkoxy. In certain embodiments, Rx is hydroxyethyl or methoxyethyl. In certain embodiments, Rx is optionally substituted C3-4 cycloalkyl. In certain embodiments, Rx is unsubstituted C3-4 cycloalkyl. In certain embodiments, Rx is cyclopropyl. In certain embodiments, Rx is cyclobutyl.
In some embodiments, for Formula (I-f), R2 and R6 are halo, and R1 is —ORA. In certain embodiments, R2 and R6 are chloro, and R1 is —ORA, wherein RA is optionally substituted alkyl or optionally substituted alkenyl. In certain embodiments, R2 and R6 are chloro, and R1 is —ORA, wherein RA is isopropyl, isobutyl, or cyclopropylmethyl.
In some embodiments, for Formula (I-f), R2 and R6 are optionally substituted alkyl, and R1 is —ORA. In some embodiments, R2 and R6 are independently isopropyl or isobutyl, and R1 is —ORA, wherein RA is optionally substituted alkyl or optionally substituted alkenyl.
In some embodiments, for Formula (I-d), R2 and R6 are optionally substituted alkyl. In some embodiments, R2 and R6 are independently isopropyl or isobutyl.
In some embodiments, for Formula (I-d), R2 is —ORA and R6 is optionally substituted alkyl. In some embodiments, R6 are isopropyl or isobutyl, and R2 is —ORA wherein RA is optionally substituted alkyl. In some embodiments, R6 are isopropyl or isobutyl, and R2 is —ORA, wherein RA is isopropyl or isobutyl.
As defined generally above, each RA is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom. In some embodiments, RA is hydrogen. In some embodiments, RA is optionally substituted alkyl. In some embodiments, RA is optionally substituted alkyl substituted with a Cy group to form optionally substituted alkyl-Cy, wherein Cy is described herein. In some embodiments, RA is optionally substituted alkenyl or optionally substituted alkynyl. In some embodiments, RA is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, RA is an oxygen protecting group when attached to an oxygen atom. In some embodiments, RA is not an oxygen protecting group. In some embodiments, RA is sulfur protecting group when attached to an sulfur atom. In some embodiments, RA is not a sulfur protecting group.
As defined generally above, each RB is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, and a nitrogen protecting group, or two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring. In some embodiments, RB is hydrogen. In some embodiments, RB is optionally substituted alkyl. In some embodiments, RB is optionally alkyl substituted with a Cy group to form optionally substituted alkyl-Cy, wherein Cy is described herein. In some embodiments, RB is optionally substituted alkenyl or optionally substituted alkynyl. In some embodiments, RB is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, RB is a nitrogen protecting group. In some embodiments, RB is not a nitrogen protecting group. In some embodiments, two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring.
In certain embodiments, a provided compound is a compound listed in Table 1A or Table 1B, or a pharmaceutically acceptable salt thereof.







TABLE 1A







Exemplary Compounds











LC- 




MS




m/z


Cmpd

(M +


No
Structure
H)





 1




249.1


 


 2




263.1


 


 3




263.2


 


 4




263.1


 


 5




307.3


 


 6




307.3


 


 7




291.3


 


 8




291.3


 


 9




277.3


 


 10




277.3


 


 11




361.3


 


 12




361.3


 


 13




303.3


 


 14




303.3


 


 15




305.3


 


 16




305.3


 


 17




270.3


 


 18




281.3


 


 19




303.1


 


 20




287.4


 


 21




290.2


 


 22




270.2


 


 23




275.2


 


 24




275.2


 


 25




275.2


 


 26




261.2


 


 27




261.2


 


 28




275.2


 


 29




273.4


 


 30




365.4


 


 31




365.3


 


 32




260.2


 


 33




260.2


 


 34




284.2


 


 35




261.1


 


 36




337.2


 


 37




337.3


 


 38




313.3


 


 39




329.2


 


 40




329.2


 


 41




338.3


 


 42




302.3


 


 43




316.3


 


 44




287.2


 


 45




349.3


 


 46




321.3


 


 47




321.3


 


 48




263.2


 


 49




281.4


 


 50




281.2


 


 51




281.2


 


 52




293.2


 


 53




309.2


 


 54




305.4


 


 55




305.4


 


 56




305.2


 


 57




295.2


 


 58




295.2


 


 59




351.3


 


 60




277.1


 


 61




335.1


 


 62




263.1


 


 63




277.1


 


 64




335.1


 


 65




293.1


 


 66




293.1


 


 67




289.1


 


 68




256.1


 


 69




249.1


 


 70




256.1


 


 71




249.1


 


 72




249.1


 


 73




231.1


 


 74




287.2


 


 75




277.1


 


 76




323.2


 


 77




305.1


 


 78




275.1


 


 79




319.1


 


 80




319.1


 


 81




305.1


 


 82




315.1


 


 83




270.1


 


 84




270.1


 


 85




367.0


 


 86




299.1


 


 87




291.2


 


 88




259.3


 


 89




283.2


 


 90




279.2


 


 91




279.1


 


 92




299.1


 


 93




315.0


 


 94




297.1


 


 95




265.0


 


 96




329.1


 


 97




307.1


 


 98




315.1


 


 99




267.1


 


100




256.1


 


101




273.1


 


102




265.1


 


103




299.0


 


104




271.1


 


105




315.0


 


106




283.1


 


107




279.1


 


108




263.2


 


109




261.2


 


110




270.2


 


111




267.2


 


112




273.2


 


113




323.1


 


114




287.0


 


115




275.0


 


116




271.1


 


117




333.0


 


118




283.0


 


119




287.0


 


120




295.1


 


121




283.2


 


122




265.2


 


123




283.1


 


124




283.1


 


125




295.2


 


126




279.3


 


127




295.1


 


128




347.3


 


129




263.2


 


130




261.3


 


131




245.3


 


132




283.2


 


133




279.2


 


134




279.1


 


135




333.0


 


136




302.1


 


137




299.0


 


138




297.0


 


139




310.1


 


140




256.1


 


141




317.0


 


142




279.1


 


143




330.1


 


144




307.1


 


145




315.1


 


146




295.1


 


147




290.1


 


148




317.1


 


149




274.3


 


150




303.3


 


151




279.2


 


152




263.2


 


153




261.3


 


154




329.2


 


155




275.3


 


156




283.2


 


157




295.2


 


158




270.2


 


159




263.1


 


160




279.2


 


161




245.1


 


162




245.2


 


163




316.1


 


164




344.2


 


165




287.2


 


166




275.1


 


167




305.1


 


168




323.1


 


169




263.1


 


170




287.1


 


171




279.1


 


172




360.2


 


173




302.1


 


174




313.2


 


175




345.1


 


176




303.1


 


177




303.1


 


178




331.1


 


179




319.1


 


180




399.1


 


181




321.1


 


182




297.0


 


183




296.0


 


184




310.0


 


185




296.0


 


186




374.2


 


187




289.1


 


188




307.1


 


189




371.1


 


190




433.1


 


191




358.1


 


192




316.0


 


193




358.1


 


194




291.0


 


195




305.0


 


196




316.0


 


197




302.0


 


198




316.1


 


199




317.1


 


200




323.1


 


201




337.1


 


202




348.0


 


203




323.0


 


204




337.1


 


205




341.1


 


206




351.0


 


207




351.0


 


208




351.0


 


209




333.1


 


210




321.1


 


211




323.0


 


212




337.0


 


213




331.4


 


214




371.4


 


215




373.4


 


216




373.3


 


217




393.0


 


218




343.3


 


219




343.3


 


220




351.2


 


221




362.1


 


222




270.1


 


223




314.1


 


224




341.1


 


225




341.1


 


226




394.2


 


227




380.2


 


228




367.1


 


229




329.1


 


230




367.2


 


231




355.1


 


232




394.2


 


233




408.2


 


234




362.1


 


235




287.1


 


236




319.1


 


237




355.1


 


238




333.1


 


239




367.1


 


240




394.2


 


241




408.2


 


242




329.2


 


243




317.3


 


244




317.3


 


245




394.3


 


246




394.4


 


247




394.4


 


248




375.3


 


249




375.4


 


250




361.3


 


251




360.3


 


252




331.3


 


253




361.4


 


254




357.4


 


255




371.4


 


256




385.3


 


257




328.3


 


258




314.0


 


259




341.1


 


260




345.1


 


261




362.1


 


262




379.2


 


263




394.2


 


264




408.2


 


265




371.0


 


266




347.2


 


267




362.1


 


268




394.1


 


269




408.1


 


270




355.1


 


271




357.2


 


272




345.2


 


273




371.1


 


274




394.2


 


275




317.1


 


276




347.1


 


277




379.2


 


278




394.2


 


279




355.1


 


280




305.1


 


281




357.2


 


282




383.1
















TABLE 1B







Exemplary Compounds









Cmpd

LC-MS m/z


No
Structure
(M + H)





283




317.05


 


284




359.15


 


285




345.15


 


286




347.15


 


287




333.15


 


288




315.05


 


289




319.05


 


290




331.1


 


291




363.1 


 


292




379.1 


 


293




301.05


 


294




289.05


 


295




302.1 


 


296




316.1 


 


297




355.1 


 


298




333.05


 


299




359.15


 


300




316.1 


 


301




319.1 


 


302




339.05


 


303




339  


 


304




315.05


 


305




333.05


 


306




317.05


 


307




329.05


 


308




317.1 


 


309




317.1 


 


310




347.15


 


311




361.35


 


312




339.1 


 


313




331.15


 


314




375.15


 


315




345.05


 


316




363.2 









In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, a provided compound inhibits wild-type PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8. In certain embodiments, a provided compound inhibits a mutant RMT. In certain embodiments, a provided compound inhibits PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8, e.g., as measured in an assay described herein. In certain embodiments, the RMT is from a human. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC50 less than or equal to 10 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC50 less than or equal to 1 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC50 less than or equal to 0.1 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC50 less than or equal to 0.01 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC30 less than or equal to 10 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC30 less than or equal to 12 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC30 less than or equal to 3 μM. In certain embodiments, a provided compound inhibits PRMT1 in a cell at an EC30 less than or equal to 12 μM. In certain embodiments, a provided compound inhibits PRMT1 in a cell at an EC30 less than or equal to 3 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC30 less than or equal to 1 μM. In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC30 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.
It will be understood by one of ordinary skill in the art that the RMT can be wild-type, or any mutant or variant.
The present disclosure provides pharmaceutical compositions comprising a compound described herein, e.g., a compound of 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 an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, the effective amount is an amount effective for treating an RMT-mediated disorder (e.g., a PRMT1-, PRMT3-, CARM1-, PRMT6-, and/or PRMT8-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 an RMT-mediated disorder.
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.
In some embodiments, a composition provided herein is sterilized.
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.
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 (I). In certain embodiments, the additional therapeutically active agent is not a compound of 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. In certain embodiments, an additional therapeutically active agent is prednisolone, dexamethasone, doxorubicin, vincristine, mafosfamide, cisplatin, carboplatin, Ara-C, rituximab, azacitadine, panobinostat, vorinostat, everolimus, rapamycin, ATRA (all-trans retinoic acid), daunorubicin, decitabine, Vidaza, mitoxantrone, or IBET-151.
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 RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In some embodiments, methods of treating an RMT-mediated disorder in a subject are provided which comprise administering an effective amount of a compound described herein (e.g., a compound of 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 RMT-mediated disorder. In certain embodiments, the subject is susceptible to a RMT-mediated disorder.
As used herein, the term “RMT-mediated disorder” means any disease, disorder, or other pathological condition in which an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) 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 an RMT is known to play a role.
In some embodiments, the present disclosure provides a method of inhibiting an RMT comprising contacting the RMT with an effective amount of a compound described herein (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt thereof. The RMT 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 RMT 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 an RMT does not necessarily require that all of the RMT be occupied by an inhibitor at once. Exemplary levels of inhibition of an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) 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 RMT activity in a subject in need thereof (e.g., a subject diagnosed as having an RMT-mediated disorder) comprising administering to the subject an effective amount of a compound described herein (e.g., a compound of Formula (I)), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
In certain embodiments, provided is a method of modulating gene expression in a cell which comprises contacting a cell with an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the cell is 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 modulating transcription in a cell which comprises contacting a cell with an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, the cell is 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 an RMT-mediated disorder or mutation comprising the steps of determining the presence of an RMT-mediated disorder or gene mutation in an RMT gene (e.g., a PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8 gene) or and selecting, based on the presence of an RMT-mediated disorder a gene mutation in the RMT 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 an RMT-mediated disorder or a gene mutation in the RMT gene and treating the subject in need thereof, based on the presence of a RMT-mediated disorder or gene mutation in the RMT 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 compound provided herein is useful in treating a proliferative disorder, such as cancer. For example, while not being bound to any particular mechanism, protein arginine methylation by PRMTs is a modification that has been implicated in signal transduction, gene transcription, DNA repair and mRNA splicing, among others; and overexpression of PRMTs within these pathways is often associated with various cancers. Thus, compounds which inhibit the action of PRMTs, as provided herein, are effective in the treatment of cancer.
In some embodiments, compounds provided herein are effective in treating cancer through the inhibition of PRMT1. For example, PRMT1 overexpression has been observed in various human cancers, including, but not limited to, breast cancer, prostate cancer, lung cancer, colon cancer, bladder cancer, and leukemia. In one example, PRMT1 specifically deposits an asymmetric dimethylarginine (aDMA) mark on histone H4 at arginine 3 (H4R3me2a), and this mark is associated with transcription activation. In prostate cancer, the methylation status of H4R3 positively correlates with increasing tumor grade and can be used to predict the risk of prostate cancer recurrence (Seligson et al., Nature 2005 435, 1262-1266). Thus, in some embodiments, inhibitors of PRMT1, as described herein, are useful in treating cancers associated with the methylation status of H4R3, e.g., prostate cancer. Additionally, the methylarginine effector molecule TDRD3 interacts with the H4R3me2a mark, and overexpression of TDRD3 is linked to poor prognosis for the survival of patients with breast cancer (Nagahata et al., Cancer Sci. 2004 95, 218-225). Thus, in some embodiments, inhibitors of PRMT1, as described herein, are useful in treating cancers associated with overexpression of TDRD3, e.g., breast cancer, as inhibition of PRMT1 leads to a decrease in methylation of H4R3, thereby preventing the association of overexpressed TDRD3 with H4R3me2a. In other examples, PRMT1 is known to have non-histone substrates. For example, PRMT1, when localized to the cytoplasm, methylates proteins that are involved in signal transduction pathways, e.g., the estrogen receptor (ER). The expression status of ER in breast cancer is critical for prognosis of the disease, and both genomic and non-genomic ER pathways have been implicated in the pathogenesis of breast cancer. For example, it has been shown that PRMT1 methylates ERα, and that ERα methylation is required for the assembly of ERα with SRC (a proto-oncogene tyrosine-protein kinase) and focal adhesion kinase (FAK). Further, the silencing of endogenous PRMT1 resulted in the inability of estrogen to activate AKT. These results suggested that PRMT1-mediated ERα methylation is required for the activation of the SRC-PI3K-FAK cascade and AKT, coordinating cell proliferation and survival. Thus, hypermethylation of ERα in breast cancer is thought to cause hyperactivation of this signaling pathway, providing a selective survival advantage to tumor cells (Le Romancer et al., Mol. Cell 2008 31, 212-221; Le Romancer et al., Steroids 2010 75, 560-564). Accordingly, in some embodiments, inhibitors of PRMT1, as described herein, are useful in treating cancers associated with ERα methylation, e.g., breast cancer. In yet another example, PRMT1 has been shown to be involved in the regulation of leukemia development. For example, SRC-associated in mitosis 68 kDa protein (SAM68; also known as KHDRBS1) is a well-characterized PRMT1 substrate, and when either SAM68 or PRMT1 is fused directly to the myeloid/lymphoid leukemia (MLL) gene, these fusion proteins can activate MLL oncogenic properties, implying that the methylation of SAM68 by PRMT1 is a critical signal for the development of leukemia (Cheung et al., Nature Cell Biol. 2007 9, 1208-1215). Accordingly, in some embodiments, inhibitors of PRMT1, as described herein, are useful in treating cancers associated with SAM68 methylation, e.g., leukemia. In still another example, PRMT1 is implicated in leukemia development through its interaction with AE9a, a splice isoform of AML1-ETO (Shia et al., Blood 2012 119:4953-62). Knockdown of PRMT1 affects expression of certain AE9a-activated genes and suppresses AE9a's self-renewal capability. It has also been shown that AE9a recruits PRMT1 to AE9a activated gene promoters, which leads to increased H4 Arg3 methylation, H3 Lys9/14 acetylation, and transcription activated. Accordingly, in some embodiments, inhibitors of PRMT1, as described herein, are useful in treating cancers associated with AML1-ETO, e.g., leukemia. Thus, without being bound by any particular mechanism, the inhibition of PRMT1, e.g., by compounds described herein, is beneficial in the treatment of cancer.
In some embodiments, compounds provided herein are effective in treating cancer through the inhibition of PRMT3. In one example, the DAL1 tumor suppressor protein has been shown to interact with PRMT3 and inhibits its methyltransferase activity (Singh et al., Oncogene 2004 23, 7761-7771). Epigenetic downregulation of DAL1 has been reported in several cancers (e.g., meningiomas and breast cancer), thus PRMT3 is expected to display increased activity, and cancers that display DAL1 silencing may, in some aspects, be good targets for PRMT3 inhibitors, e.g., those described herein. Thus, without being bound by any particular mechanism, the inhibition of PRMT3, e.g., by compounds described herein, is beneficial in the treatment of cancer.
In some embodiments, compounds provided herein are effective in treating cancer through the inhibition of PRMT4, also known as CARM1. For example, PRMT4 levels have been shown to be elevated in castration-resistant prostate cancer (CRPC), as well as in aggressive breast tumors (Hong et al., Cancer 2004 101, 83-89; Majumder et al., Prostate 2006 66, 1292-1301). Thus, in some embodiments, inhibitors of PRMT4, as described herein, are useful in treating cancers associated with PRMT4 overexpression. PRMT4 has also been shown to affect ERα-dependent breast cancer cell differentiation and proliferation (Al-Dhaheri et al., Cancer Res. 2011 71, 2118-2128), thus in some aspects PRMT4 inhibitors, as described herein, are useful in treating ERα-dependent breast cancer by inhibiting cell differentiation and proliferation. In another example, PRMT4 has been shown to be recruited to the promoter of E2F1 (which encodes a cell cycle regulator) as a transcriptional co-activator (Frietze et al., Cancer Res. 2008 68, 301-306). Thus, PRMT4-mediated upregulation of E2F1 expression may contribute to cancer progression and chemoresistance as increased abundance of E2F1 triggers invasion and metastasis by activating growth receptor signaling pathways, which in turn promote an antiapoptotic tumor environment (Engelmann and Piitzer, Cancer Res 2012 72; 571). Accordingly, in some embodiments, the inhibition of PRMT4, e.g., by compounds provided herein, is useful in treating cancers associated with E2F1 upregulation. Thus, without being bound by any particular mechanism, the inhibition of PRMT4, e.g., by compounds described herein, is beneficial in the treatment of cancer.
In some embodiments, compounds provided herein are effective in treating cancer through the inhibition of PRMT6. For example, PRMT6 has been reported to be overexpressed in a number of cancers, e.g., bladder and lung cancer (Yoshimatsu et al., Int. J. Cancer 2011 128, 562-573). Thus, in some embodiments, the inhibition of PRMT6, by compounds provided herein, is useful in treating cancers associated with PRMT6 overexpression. In some aspects, PRMT6 is primarily thought to function as a transcriptional repressor, although it has also been reported that PRMT6 functions as a co-activator of nuclear receptors. For example, as a transcriptional repressor, PRMT6 suppresses the expression of thrombospondin 1 (TSP1; also known as THBS1; a potent natural inhibitor of angiogenesis and endothelial cell migration) and p21 (a natural inhibitor of cyclin dependent kinase), thereby contributing to cancer development and progression (Michaud-Levesque and Richard, J. Biol. Chem. 2009 284, 21338-21346; Kleinschmidt et al., PLoS ONE 2012 7, e41446). Accordingly, in some embodiments, the inhibition of PRMT6, by compounds provided herein, is useful in treating cancer by preventing the repression of THBs1 and/or p21. Thus, without being bound by any particular mechanism, the inhibition of PRMT6, e.g., by compounds described herein, is beneficial in the treatment of cancer.
In some embodiments, compounds provided herein are effective in treating cancer through the inhibition of PRMT8. For example, deep-sequencing efforts of cancer genomes (e.g., COSMIC) have revealed that of all the PRMTs, PRMT8 is reported to be the most mutated. Of 106 sequenced genomes, 15 carry mutations in the PRMT8 coding region, and nine of these result in an amino acid change (Forbes et al., Nucleic Acids Res. 2011 39, D945-D950). Because of its high rate of mutation in cancer, PRMT8 is thought to contribute to the initiation or progression of cancer. Thus, without being bound by any particular mechanism, the inhibition of PRMT8, e.g., by compounds described herein, is beneficial in the treatment of 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 (e.g., “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 compound provided herein is useful in treating diseases associated with increased levels of circulating asymmetric dimethylarginine (aDMA), e.g., cardiovascular disease, diabetes, kidney failure, renal disease, pulmonary disease, etc. Circulating aDMA is produced by the proteolysis of asymmetrically dimethylated proteins. PRMTs which mediate aDMA methylation include, e.g., PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8. aDMA levels are directly involved in various diseases as aDMA is an endogenous competitive inhibitor of nitric oxide synthase (NOS), thereby reducing the production of nitric oxide (NO) (Vallance et al., J. Cardiovasc. Pharmacol. 1992 20 (Suppl. 12):S60-2). NO functions as a potent vasodilator in endothelial vessels, and as such inhibiting its production has major consequences on the cardiovascular system. For example, since PRMT1 is a major enzyme that generates aDMA, the dysregulation of its activity is likely to regulate cardiovascular diseases (Boger et al., Ann. Med. 2006 38:126-36), and other pathophysiological conditions such as diabetes mellitus (Sydow et al., Vasc. Med. 2005 10 (Suppl. 1):S35-43), kidney failure (Vallance et al., Lancet 1992 339:572-5), and chronic pulmonary diseases (Zakrzewicz et al., BMC Pulm. Med. 2009 9:5). Additionally, it has been demonstrated that the expression of PRMT1 and PRMT3 are increased in coronary heart disease (Chen et al., Basic Res. Cardiol. 2006 101:346-53). In another example, aDMA elevation is seen in patients with renal failure, due to impaired clearance of this metabolite from the circulation (Jacobi et al., Am. J. Nephrol. 2008 28:224-37). Thus, circulating aDMA levels is observed in many pathophysiological situations. Accordingly, without being bound by any particular mechanism, the inhibition of PRMTs, e.g., by compounds described herein, results in the decrease of circulating aDMA, which is beneficial in the treatment of diseases associated with increased levels of circulating aDMA, e.g., cardiovascular disease, diabetes, kidney failure, renal disease, pulmonary disease, etc. In certain embodiments, a compound described herein is useful for treating or preventing vascular diseases.
In some embodiments, a compound provided herein is useful in treating metabolic disorders. For example, PRMT1 has been shown to enhance mRNA levels of FoxO1 target genes in gluconeogenesis, which results in increased hepatic glucose production, and knockdown of PRMT promotes inhibition of FoxO1 activity and thus inhibition of hepatic gluconeogenesis (Choi et al., Hepatology 2012 56:1546-56). Additionally, genetic haploinsufficiency of Prmt1 has been shown to reduce blood glucose levels in mouse models. Thus, without being bound by any particular mechanism, the inhibition of PRMT1, e.g., by compounds described herein, is beneficial in the treating of metabolic disorders, such as diabetes. In some embodiments, a provided compound is useful in treating type I diabetes. In some embodiments, a provided compound is useful in treating type II diabetes.
In some embodiments, a compound provided herein is useful in treating muscular dystrophies. For example, PRMT1, as well as PRMT3 and PRMT6, methylate the nuclear poly(A)-binding protein (PABPN1) in a region located near its C-terminus (Perreault et al., J. Biol. Chem. 2007 282:7552-62). This domain is involved in the aggregation of the PABPN1 protein, and abnormal aggregation of this protein is involved in the disease oculopharyngeal muscular dystrophy (Davies et al., Int. J. Biochem. Cell. Biol. 2006 38:1457-62). Thus, without being bound by any particular mechanism, the inhibition of PRMTs, e.g., by compounds described herein, is beneficial in the treatment of muscular dystrophies, e.g., oculopharyngeal muscular dystrophy, by decreasing the amount of methylation of PABPN1, thereby decreasing the amount of PABPN1 aggregation.
CARM1 is also the most abundant PRMT expressed in skeletal muscle cells, and has been found to selectively control the pathways modulating glycogen metabolism, and associated AMPK (AMP-activated protein kinase) and p38 MAPK (mitogen-activated protein kinase) expression. See, e.g., Wang et al., Biochem (2012) 444:323-331. Thus, in some embodiments, inhibitors of CARM1, as described herein, are useful in treating metabolic disorders, e.g., for example skeletal muscle metabolic disorders, e.g., glycogen and glucose metabolic disorders. Exemplary skeletal muscle metabolic disorders include, but are not limited to, Acid Maltase Deficiency (Glycogenosis type 2; Pompe disease), Debrancher deficiency (Glycogenosis type 3), Phosphorylase deficiency (McArdle's; GSD 5), X-linked syndrome (GSD9D), Autosomal recessive syndrome (GSD9B), Tarui's disease (Glycogen storage disease VII; GSD 7), Phosphoglycerate Mutase deficiency (Glycogen storage disease X; GSDX; GSD 10), Lactate dehydrogenase A deficiency (GSD 11), Branching enzyme deficiency (GSD 4), Aldolase A (muscle) deficiency, β-Enolase deficiency, Triosephosphate isomerase (TIM) deficiency, Lafora's disease (Progressive myoclonic epilepsy 2), Glycogen storage disease (Muscle, Type 0, Phosphoglucomutase 1 Deficiency (GSD 14)), and Glycogenin Deficiency (GSD 15).
In some embodiments, a compound provided herein is useful in treating autoimmune disease. For example, several lines of evidence strongly suggest that PRMT inhibitors may be valuable for the treatment of autoimmune diseases, e.g., rheumatoid arthritis. PRMTs are known to modify and regulate several critical immunomodulatory proteins. For example, post-translational modifications (e.g., arginine methylation), within T cell receptor signaling cascades allow T lymphocytes to initiate a rapid and appropriate immune response to pathogens. Co-engagement of the CD28 costimulatory receptor with the T cell receptor elevates PRMT activity and cellular protein arginine methylation, including methylation of the guanine nucleotide exchange factor Vav1 (Blanchet et al., J. Exp. Med. 2005 202:371-377). PRMT inhibitors are thus expected to diminish methylation of the guanine exchange factor Vav1, resulting in diminished IL-2 production. In agreement, siRNA directed against PRMT5 was shown to both inhibit NFAT-driven promoter activity and IL-2 secretion (Richard et al., Biochem J. 2005 388:379-386). In another example, PRMT1 is known to cooperate with PRMT4 to enhance NFkB p65-driven transcription and facilitate the transcription of p65 target genes like TNFα (Covic et al., Embo. J. 2005 24:85-96). Thus, in some embodiments, PRMT1 and/or PRMT4 inhibitors, e.g., those described herein, are useful in treating autoimmune disease by decreasing the transcription of p65 target genes like TNFα. These examples demonstrate an important role for arginine methylation in inflammation. Thus, without being bound by any particular mechanism, the inhibition of PRMTs, e.g., by compounds described herein, is beneficial in the treatment of autoimmune diseases.
In some embodiments, a compound provided herein is useful in treating neurological disorders, such as amyotrophic lateral sclerosis (ALS). For example, a gene involved in ALS, TLS/FUS, often contains mutated arginines in certain familial forms of this disease (Kwiatkowski et al., Science 2009 323:1205-8). These mutants are retained in the cytoplasm, which is similar to reports documenting the role arginine methylation plays in nuclear-cytoplasmic shuffling (Shen et al., Genes Dev. 1998 12:679-91). This implicates PRMT, e.g., PRMT1, function in this disease, as it was demonstrated that TLS/FUS is methylated on at least 20 arginine residues (Rappsilber et al., Anal. Chem. 2003 75:3107-14). Thus, in some embodiments, the inhibition of PRMTs, e.g., by compounds provided herein, are useful in treating ALS by decreasing the amount of TLS/FUS arginine methylation.




Scheme 1 shows a general synthesis route to pyrazole compounds of formula I, wherein R1 and R2′ are either the same as R1 and R2 or are substituents which serve as precursors for synthetic conversion to R1 and R2 wherein, R1, R2, R6, R7, R8, R3, Rx, X, Y and Z and are as defined above. In the first step iodopyrazole carboxaldehydes of general formula XI are allowed to react with mono-Boc protected ethylenediamines XII under reductive amination conditions (e.g. sodium cyanoborohydride and catalytic acid such as acetic acid) in an appropriate solvent such as methanol to give intermediates of general formula XIII. Suzuki reaction of the latter with boronic acids or boronic esters of general formula XIV in the presence of a palladium catalyst (e.g. PdCl2(dppf)) and a base (e.g. potassium carbonate) in an organic solvent (e.g. toluene) at elevated temperature yields intermediates of general formula XV. In a subsequent optional step or steps, either R1′ or R2′ or both R1′ and R2′ may be converted to yield R1 and R2 substituents present in final compounds of formula I. For example compounds wherein R2 is an alkoxy group can be synthesized by alkylating intermediates of formula XV where R2′ is OH with a suitable alkylbromide or iodide using an appropriate base (e.g. potassium carbonate) in a suitable organic solvent (e.g. tetrahydrofuran) at room or elevated temperature. In a final deprotection step the N-Boc protecting of XV can be removed under a variety of conditions such as with an acid (e.g. HCl, TFA) in a suitable organic solvent (e.g. ethanol, THF, dichloromethane) to give compounds of formula I.
In certain embodiments, iodopyrazole carboxaldehydes of general formula XI are prepared from suitable known pyrazole compound intermediates by established synthetic chemistry methods. Standard methods include direct iodination of a pyrazole 3-carboxylate and Sandmeyer reaction of a 3-amino pyrazole 4-carboxylate. In certain embodiments, iodopyrazole carboxaldehydes are derived from iodopyrazole carboxylates by reduction to a hydroxymethyl group followed by oxidation to carboxaldehyde. The mono-Boc protected ethylenediamines XII can be synthesized by standard methods known in the literature for derivatizing or preparing ethylenediamines. For example intermediates of formula XII may be prepared by treatment of the corresponding unprotected diamine precursorswith Boc2O and purifying the mixture of mono and dibocylated products. Phenyl boronic acids or esters of general formula XIV are either commercially available or can be prepared by standard method for example by boronylation of the corresponding commercially available phenylbromides.
In certain embodiments, pyrazole compounds of general formula II are prepared from iodopyrazole carboxaldehydes of general formula XXI as depicted in Scheme 2. In certain embodiments where R4 is hydrogen compounds of general formula II are equivalent to compounds of general formula III which are tautomers. In certain embodiments R4′ is a protecting group such as tetrahydropyranyl (THP) which maybe cleaved to hydrogen under acidic conditions in the final Boc-deprotection step. In one synthetic route iodopyrazole carboxaldehydes of general formula XXI can be prepared as depicted in Scheme 3.








In certain embodiments, iodopyrazole carboxaldehydes of general formula XXI are prepared as depicted in Scheme 4 which also provides iodopyrazole carboxyaldehydes of general formula XXXI. Thus, alkylation of intermediates of general formula XXX gives a mixture of pyrazole nitrogen alkylated isomers which are separated by chromatography to give pure isomers XXI and XXXI. In certain embodiments, pyrazole compounds of general formula III can be prepared from iodopyrazole carboxaldehydes of general formula XXXI as depicted in Scheme 5.








In certain embodiments, pyrazole compounds of general formula IV are prepared from iodopyrazole carboxaldehydes of general formula XLI as depicted in Scheme 6. In certain embodiments where R4 is hydrogen compounds of general formula IV are equivalent to compounds of general formula V which are tautomers. In certain embodiments where R4 in compounds of formula IV is hydrogen, R4′ in intermediate XLI may be a selected protecting group such as tetrahydropyranyl (THP) which can be cleaved to hydrogen under acidic conditions in the final Boc-deprotection step.




In certain embodiments, iodopyrazole carboxaldehydes of general formula XLI and LI can be prepared as depicted in Scheme 7. In certain embodiments an R4 group of iodopyrazole carboxaldehydes maybe introduced by alkylation of intermediates of formula XLVII. This reaction can give a mixture of intermediate compounds of formulas XLI and LI which may be separated by chromatography. The THP protected intermediates of formula XLVI can be used to prepare compounds of formula IV where R4═H as also depicted in Scheme 7.




In certain embodiments, pyrazole compounds of general formula V are prepared from iodopyrazole carboxaldehydes of general formula LI as depicted in Scheme 8.




In certain embodiments, phenyl boronic acids or esters of general formula XIV are commercially available or can be prepared from commercially available or known corresponding phenyl bromide precursors of general formula LX by standard methods. One such method to prepare pinacol boranes XIVd is depicted in Scheme 9.




In certain embodiments, compounds of formula XIV are prepared by multistep organic synthesis using known methods. For example bromides LX may be prepared from corresponding aniline intermediate compounds by Sandmeyer reaction. The required aniline intermediates can in turn be prepared from the corresponding nitrobenzenes by reduction of the nitro group under standard conditions (e.g. Pd/H2, SnCl2, Fe/HOAc).
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
General methods and experimental procedures for preparing and characterizing compounds of the present invention are set forth below. Wherever needed, reactions were heated using conventional hotplate apparatus or heating mantle or microwave irradiation equipment. Reactions were conducted with or without stirring, under atmospheric or elevated pressure in either open or closed vessels. Reaction progress was monitored using conventional techniques such as TLC, HPLC, UPLC, or LCMS using instrumentation and methods described below. Reactions were quenched and crude compounds isolated using conventional methods as described in the specific examples provided. Solvent removal was carried out with or without heating, under atmospheric or reduced pressure, using either a rotary or centrifugal evaporator.
Compound purification was carried out as needed using a variety of traditional methods including, but not limited to, preparative chromatography under acidic, neutral, or basic conditions using either normal phase or reverse phase HPLC or flash columns or Prep-TLC plates. Compound purity and mass confirmations were conducted using standard HPLC and/or UPLC and/or MS spectrometers and/or LCMS and/or GC equipment (e.g., including, but not limited to the following instrumentation: Waters Alliance 2695 with 2996 PDA detector connected with ZQ detector and ESI source; Shimadzu LDMS-2020; Waters Acquity H Class with PDA detector connected with SQ detector and ESI source; Agilent 1100 Series with PDA detector; Waters Alliance 2695 with 2998 PDA detector; AB SCIEX API 2000 with ESI source; Agilent 7890 GC). Exemplified compounds were dissolved in either MeOH or MeCN to a concentration of approximately 1 mg/mL and analyzed by injection of 0.5-10 μL into an appropriate LCMS system using the methods provided in the following table:























MS
MS






Flow

Heat
Detector




Mobile
Mobile
Rate

Block
Voltage


Method
Column
Phase A
Phase B
(mL/min)
Gradient Profile
Temp (° C.)
(kV)






















A
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.0
250
1.5



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



1.1 minutes, 100% to



3.0 × 50 mm



5% B in 0.2 minutes,







then stop


B
Gemini-NX
Water/0.04%
ACN
1
5% to 100% B in 2.0
200
0.75



3 μm C18
Ammonia


minutes, 100% B for



110A



1.1 minutes, 100% to







5% B in 0.1 minutes,







then stop


C
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.0
250
0.85



XR-ODS
FA
FA

minutes, 100% B for



1.6 μm



1.1 minutes, 100% to



2.0 × 50 mm



5% B in 0.1 minutes,







then stop


D
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.0
250
0.95



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



1.1 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


E
Waters
Water/0.05%
ACN/0.05%
0.9
5% to 100% B in 2.0
250
1.5



Xselect C18
FA
FA

minutes, 100% B for



3.5 μm



1.2 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


F
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 80% B in 3.25
200
0.95



XR-ODS
TFA
TFA

minutes, 80% B for



2.2 μm



1.35 minutes, 80% to



3.0 × 50 mm



5% B in 0.3 minutes,







then stop


G
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 70% B in 2.50
200
0.95



XR-ODS
TFA
TFA

minutes, 70% B for



2.2 μm



0.70 minutes, 70% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


H
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.20
250
0.95



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



1.00 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


I
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 1.20
250
0.95



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



1.00 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


J
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 70% B in 3.20
250
0.95



XR-ODS
TFA
TFA

minutes, 70% B for



2.2 μm



0.75 minutes, 70% to



3.0 × 50 mm



5% B in 0.35 minutes,







then stop


K
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 80% B in 3.00
250
1.5



XR-ODS
TFA
TFA

minutes, 80% B for 0.8



2.2 μm



minutes, 80% to 5% B



3.0 × 50 mm



in 0.1 minutes, then







stop


L
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 3.00
250
1.5



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



0.8 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


M
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.20
250
1.5



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



1.00 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


N
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 80% B in 2.20
250
1.5



XR-ODS
TFA
TFA

minutes, 80% B for 1.0



2.2 μm



minutes, 80% to 5% B



3.0 × 50 mm



in 0.1 minutes, then







stop


O
Zorbax
Water/0.05%
ACN/0.05%
1
5% to 70% B in 8.00
250
1.5



Eclipse Plus
TFA
TFA

minutes, 70% B for 2.0



C18



minutes, then stop



4.6 × 100 mm


P
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 65% B in 3.00
250
1.5



XR-ODS
TFA
TFA

minutes, 65% B for



2.2 μm



0.80 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


Q
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 60% B in 2.50
250
0.95



XR-ODS
TFA
TFA

minutes, 60% B for 0.7



2.2 μm



minutes, 60% to 5% B



3.0 × 50 mm



in 0.1 minutes, then







stop


R
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 50% B in 2.50
250
0.95



XR-ODS
TFA
TFA

minutes, 50% B for 0.7



2.2 μm



minutes, 50% to 5% B



3.0 × 50 mm



in 0.1 minutes, then







stop


S
XBridge
Water/0.05%
ACN/0.05%
1
5% to 95% B in 2.20
250
0.9



C18 3.5 μm
TFA
TFA

minutes, 95% B for



3.0 × 50 mm



1.00 minutes, 95% to







5% B in 0.1 minutes,







then stop


T
Shim-pack
Water/0.05%
ACN/0.05%
0.7
5% to 100% B in 2.0
250
0.85



XR-ODS
FA
FA

minutes, 100% B for



1.6 μm



1.1 minutes, 100% to



2.0 × 50 mm



5% B in 0.1 minutes,







then stop


U
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 40% B in 2.50
250
0.95



XR-ODS
TFA
TFA

minutes, 40% B for 0.7



2.2 μm



minutes, 40% to 5% B



3.0 × 50 mm



in 0.1 minutes, then







stop


V
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 60% B in 4.20
200
1.05



XR-ODS
TFA
TFA

minutes, 60% B for 1.0



2.2 μm



minutes, 60% to 5% B



3.0 × 50 mm



in 0.1 minutes, then







stop


W
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.20
200
0.95



XR-ODS
TFA
TFA

minutes, 100% B for



2.2 μm



1.00 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


X
Shim-pack
Water/0.05%
ACN/0.05%
0.7
5% to 100% B in 2.0
200
0.85



XR-ODS
FA
FA

minutes, 100% B for



1.6 μm



1.1 minutes, 100% to



2.0 × 50 mm



5% B in 0.1 minutes,







then stop


Y
Ecliplis Plus
Water/0.05%
ACN
1
5% to 100% B in 2.0
250
1



C18 3.5 μm
TFA


minutes, 100% B for



4.6 × 50 mm



1.0 minutes, 100% to







5% B in 0.1 minutes,







then stop


Z
Ecliplis Plus
Water/10
ACN/5%
1
5% to 100% B in 2.0
250
1.1



C18 3.5 μm
mM
water

minutes, 100% B for



4.6 × 50 mm
ammonium


1.0 minutes, 100% to




carbonate


5% B in 0.1 minutes,







then stop


A1
Shim-pack
Water/0.05%
ACN
1
5% to 100% B in 2.0
250
1



XR-ODS
TFA


minutes, 100% B for



2.2 μm



1.0 minutes, 100% to



3.0 × 50 mm



5% B in 0.1 minutes,







then stop


A2
Ecliplis Plus
Water/10
ACN
1
5% to 100% B in 2.0
250
0.95



C18 3.5 μm
mM


minutes, 100% B for



4.6 × 50 mm
ammonium


1.4 minutes, 100% to




acetate


5% B in 0.1 minutes,







then stop


A3
Acquity
Water/5
ACN/0.1%
0.55
5% B at 0.01 min up to



BEH C18
mM
FA

0.4 min, 35% B at 0.8



1.7 μm
ammonium


min, 55% B at 1.2 min,



2.1 × 50 mm
acetate/


100% B in 1.3 minutes,




0.1% FA


at 2.5 min up to 3.30







min, 5% B at 3.31 min







up to 4.0 min, then







stop


A4
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 30% B in 8.0
250
1.5



XR-ODS
TFA
TFA

minutes, 30% B for 2.0



3.0 × 50 mm



minutes, then stop


A5
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 100% B in 2.2
250
1.5



XR-ODS
TFA
TFA

minutes, 100% B for



3.0 × 50 mm



1.0 minutes, 100% to







5% B in 0.1 minutes,







then stop


A6
Atlantis
Water/0.05%
ACN/0.05%
0.8
95% to 60% B in 4.0
250
1.5



HILIC
TFA
TFA

minutes, 60% B for 4.0



3.0 × 100 mm



minutes, then stop


A7
Shim-pack
Water/0.05%
ACN/0.05%
1
5% B for 0.5 minutes,
250
1.5



XR-ODS
TFA
TFA

5% to 75% B at 2.2



3.0 × 50 mm



minutes, 100% B for







1.0 minutes, 100% to







5% B in 0.1 minutes,







then stop


A8
Zorbax SB-
Water/0.05%
ACN/0.05%
1.2
5% to 70% B in 10.0
250
1.05



C18
TFA
TFA

minutes, 70% B for 5.0



5 μm



minutes, then stop



4.6 × 150 mm


A9
Shim-pack
Water/0.05%
ACN/0.05%
1
5% to 40% B in 4.4
250
0.95



XR-ODS
TFA
TFA

minutes, 40% B for 0.9



3.0 × 50 mm



minutes, then stop


A10
Atlantis T3
Water/0.05%
ACN/0.05%
1
5% to 50% B in 8.0
200
1.05



3 μm
TFA
TFA

minutes, 50% B for 2.0



4.6 × 100 mm



minutes, then stop


A11
Shim-pack
Water/0.05%
ACN/0.05%
1
5% B for 0.5 minutes,
250
1.50



XR-ODS
TFA
TFA

5% to 100% B in 1.7



3.0 × 50 mm



minutes, 100% B for







1.0 minute, 100% to







5% B in 0.1 minute,







then stop









Compound structure confirmations were carried out using standard 300 or 400 MHz NMR spectrometers with NOe's conducted whenever necessary.
The following abbreviations are used herein:














Abbreviation
Meaning









ACN
acetonitrile



atm.
atmosphere



DCM
dichloromethane



DHP
dihydropyran



DIBAL
diisobutyl aluminum hydride



DIEA
diisopropyl ethylamine



DMF
dimethyl formamide



DMF-DMA
dimethyl formamide dimethyl acetal



DMSO
dimethyl sulfoxide



dppf
1,1′-bis(diphenylphosphino)ferrocene



EA
ethyl acetate



ESI
electrospray ionization



EtOH
ethanol



FA
formic acid



GC
gas chromatography



h
hour



Hex
hexanes



HMDS
hexamethyl disilazide



HPLC
high performance liquid chromatography



IPA
isopropanol



LCMS
liquid chromatography/mass spectrometry



MeOH
methanol



min
minutes



NBS
N-bromo succinimide



NCS
N-chloro succinimide



NIS
N-iodo succinimide



NMR
nuclear magnetic resonance



NOe
nuclear Overhauser effect



Prep.
preparative



PTSA
para-toluene sulfonic acid



Rf
retardation factor



rt
room temperature



RT
retention time



sat.
saturated



SGC
silica gel chromatography



TBAF
tetrabutyl ammonium fluoride



TEA
triethylamine



TFA
trifluoroacetic acid



THF
tetrahydrofuran



TLC
thin layer chromatography



UPLC
ultra performance liquid chromatography










Intermediate Synthesis
Synthesis of intermediate tert-butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate




Step 1: tert-butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate




A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (3.2 g, 10.45 mmol, 1.00 equiv), tert-butyl N-[2-(methylamino)ethyl]carbamate (2.2 g, 12.63 mmol, 1.21 equiv) and NaBH(OAc)3 (6.65 g, 31.38 mmol, 3.00 equiv) in dichloroethane (30 mL) was stirred for 2 h at room temperature. The reaction was quenched with 50 mL of saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted with 3×200 mL of dichloromethane. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 30-100% ethyl acetate in petroleum ether to give 4.05 g (83%) of tert-butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate as a light yellow oil. 1H NMR (300 MHz, CDCl3): δ 7.48 (s, 1H), 5.35-5.30 (m, 1H), 4.13-4.03 (m, 1H), 3.71-3.63 (m, 1H), 3.36 (s, 2H), 3.26-3.25 (m, 2H), 2.52-2.49 (m, 2H), 2.21 (s, 3H), 2.09-2.01 (m, 3H), 1.68-1.58 (m, 3H), 1.44 (s, 9H) ppm. LCMS (method C, ESI): RT=0.58 min, m/z=465.0 [M+H]+.
Synthesis of intermediate tert-butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino ethyl)(methyl)carbamate




Step 1: Ethyl 3-iodo-1H-pyrazole-4-carboxylate




To a stirred solution of ethyl 3-amino-1H-pyrazole-4-carboxylate (10 g, 64.45 mmol, 1.00 equiv) in 50% sulfuric acid (90 mL) at 5° C. was added dropwise a solution of NaNO2 (7.4 g, 107.25 mmol, 1.66 equiv) in water (15 mL). The reaction was stirred at 5° C. for another 30 min. A solution of KI (32.1 g, 193.37 mmol, 3.00 equiv) in water (15 mL) was added dropwise at 5° C. The reaction was allowed to stir at 5° C. for 1 h and then quenched by the addition of 50 mL of water. The precipitate was collected by filtration and then dissolved in 150 mL of ethyl acetate. The resulting solution was washed sequentially with 1×100 mL of saturated Na2SO3 solution, 1×100 mL of saturated sodium bicarbonate solution and 1×100 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to give 10.8 g (63%) of ethyl 3-iodo-1H-pyrazole-4-carboxylate as a yellow solid. 1H NMR (300 MHz, CDCl3): δ 8.18 (s, 1H), 4.38-4.29 (m, 2H), 1.41-1.33 (m, 3H) ppm. LCMS (method B, ESI): RT=1.36 min, m/z=267.0 [M+H]+.
Step 2: Ethyl 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate




A solution of ethyl 3-iodo-1H-pyrazole-4-carboxylate (10.8 g, 40.60 mmol, 1.00 equiv), 3,4-dihydro-2H-pyran (10 g, 118.88 mmol, 2.93 equiv) and TsOH (780 mg, 4.53 mmol, 0.11 equiv) in THF (100 mL) was stirred for 2 h at 60° C. The reaction mixture was cooled to room temperature and quenched by the addition of 100 mL of saturated sodium bicarbonate solution. The resulting solution was extracted with 2×80 mL of dichloromethane. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:20) to give 13 g (91%) of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 5.40-5.38 (m, 1H), 4.34-4.29 (m, 2H), 4.08-4.05 (m, 1H), 3.73-3.70 (m, 1H), 2.07-1.98 (m, 3H), 1.69-1.62 (m, 3H), 1.39-1.32 (m, 3H) ppm. LCMS (method C, ESI): RT=1.53 min, m/z=351.0 [M+H]+.
Step 3: 3-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylic acid




To a solution of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate (85 g, 242.75 mmol, 1.00 equiv) in THF (300 mL) and methanol (300 mL) was added a solution of LiOH (17.5 g, 730.69 mmol, 3.01 equiv) in water (400 mL). The resulting solution was stirred at room temperature overnight and then concentrated under vacuum to remove the organic solvent. The resulting solution was diluted with 400 mL of H2O and then acidified to pH 6.0 with IM hydrochloric acid. The mixture was extracted with 3×800 mL of dichloromethane. The combined organic layers was washed with 3×1000 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum to give 75 g (96%) of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid as an off-white solid. LCMS (method D, ESI): RT=1.23 min, m/z=323.0 [M+H]+.
Step 4: (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol




To a solution of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid (28 g, 86.93 mmol, 1.00 equiv) in anhydrous THF (300 mL) maintained under nitrogen at 5° C. was added a IM solution of BH3 in THF (300 mL) dropwise with stirring. The reaction was stirred overnight at room temperature and then quenched by the addition of 300 mL of saturated NH4Cl solution. The resulting mixture was extracted with 3×1000 mL of dichloromethane. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:1) to give 12.67 g (47%) of (3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanol as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 7.73 (s, 1H), 5.37-5.34 (m, 1H), 4.92 (s, 1H), 4.20 (d, J=3.6 Hz, 2H), 3.89-3.88 (m, 1H), 3.65-3.57 (m, 1H), 2.09-2.00 (m, 1H), 1.99-1.90 (m, 2H), 1.69-1.61 (m, 1H), 1.49-1.46 (m, 2H) ppm. LCMS (method A, ESI): RT=1.16 min, m/z=309.0 [M+H]+.
Step 5: 3-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde




Into a 250-mL 3-necked round-bottom flask purged and. To a stirred solution of oxalyl chloride (18.576 g, 146.35 mmol, 3.01 equiv) in anhydrous dichloromethane (300 mL) maintained under nitrogen at −78° C. was added DMSO (15.138 g, 193.75 mmol, 3.98 equiv) dropwise. The reaction mixture was stirred at −65° C. for 30 min. A solution of (3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanol (15.0 g, 48.68 mmol, 1.00 equiv) in dichloromethane (100 mL) was then added dropwise at −65° C. and the reaction was stirred for another 60 min at −65° C. Triethylamine (40.6 mL) was added dropwise at −65° C. and the reaction was stirred for 30 min at −65° C. The reaction was warmed to 0° C. then quenched by the addition of 100 mL of saturated NH4Cl solution. The resulting mixture was extracted with 3×400 mL of dichloromethane. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:20) to give 13.48 g (90%) of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde as a golden oil. 1H NMR (300 MHz, DMSO-d6): δ 9.69 (s, 1H), 8.57 (s, 1H), 5.49 (dd, J=2.7 Hz, 9.9 Hz, 1H), 3.95-3.91 (m, 1H), 3.68-3.62 (m, 1H), 2.11-2.01 (m, 3H), 1.69-1.62 (m, 3H) ppm. LCMS (method A, ESI): RT=1.35 min, m/z=307.0 [M+H]+.
Step 6: tert-Butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate




A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (21.5 g, 70.24 mmol, 1.00 equiv), tert-butyl N-methyl-N-(2-(methylamino)ethyl)carbamate (20 g, 106.23 mmol, 1.51 equiv) and NaBH(OAc)3 (29.8 g, 137.98 mmol, 1.96 equiv) in dichloroethane (300 mL) was stirred for 1 h at room temperature. The reaction was diluted with 300 mL of dichloromethane and then washed with 3×300 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-7% methanol in dichloromethane to give 31 g (92%) of tert-butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate as a yellow oil. 1H NMR (300 MHz, CDCl3): δ 7.62 (s, 1H), 5.34-5.30 (m, 1H), 4.06-4.02 (m, 1H), 3.68-3.62 (m, 1H), 3.42-3.38 (m, 4H), 2.85 (s, 4H), 2.62-2.53 (m, 2H), 2.47-2.46 (m, 2H), 2.13-1.97 (m, 3H), 1.74-1.69 (m, 3H), 1.46 (s, 9H) ppm. LCMS (method A, ESI): RT=1.17 min, m/z=479.0 [M+H]+.
Compound 2
N1-((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: (E)-2-(1-(4-fluorophenyl)ethylidene)hydrazinecarboxamide




To a solution of 1-(4-fluorophenyl)ethan-1-one (27.6 g, 199.80 mmol, 1.00 equiv) and hydrazinecarboxamide hydrochloride (22 g, 197.25 mmol, 0.99 equiv) in methanol (200 mL) was added a solution of sodium acetate (16.8 g, 204.79 mmol, 1.02 equiv) in water (80 mL) dropwise with stirring. The resulting solution was stirred at room temperature for 2 h. The solid was collected by filtration then air-dried to give 41.6 g of crude (E)-2-(1-(4-fluorophenyl)ethylidene)hydrazinecarboxamide as a white solid. LCMS (method A, ESI): RT=1.22 min, m/z=196.0 [M+H]+.
Step 2: 3-(4-Fluorophenyl)-1H-pyrazole-4-carbaldehyde




To a stirred solution of (E)-2-(1-(4-fluorophenyl)ethylidene)hydrazinecarboxamide (1 g, 5.12 mmol, 1.00 equiv) in N,N-dimethylformamide (5 mL) at 5° C. was added phosphorous oxychloride (5 mL) dropwise. The resulting solution was stirred at 70° C. for 2 h. The reaction mixture was cooled to 0° C. and quenched by the addition of 15 mL of ice/water mixture. The pH value of the solution was adjusted to 7 with 4N sodium hydroxide solution. The resulting mixture was stirred at 70° C. for an additional 1 h. The reaction mixture was cooled back to room temperature. The precipitate was collected by filtration and dried in a vacuum oven to give 600 mg (62%) of 3-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde as a gray solid. LCMS (method A, ESI): RT=1.23 min, m/z=191.0 [M+H]+.
Step 3: N-((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)-2,2-dimethoxy-N-methylethanamine




A solution of 3-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde (3 g, 15.78 mmol, 1.00 equiv) and (2,2-dimethoxyethyl)(methyl)amine (2.3 g, 19.30 mmol, 1.22 equiv) in acetic acid (1.89 g, 31.47 mmol, 2.00 equiv) and 1,2-dichloroethane (30 mL) was stirred at room temperature for 30 min. Sodium triacetoxyborohydride (10 g) was then added and the resulting solution was stirred at room temperature for 3 h. The resulting mixture was washed with 2×20 mL of 5% potassium carbonate solution and 2×20 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to give 3.29 g (71%) of N-((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)-2,2-dimethoxy-N-methylethanamine as a yellow oil. LCMS (method C, ESI): RT=0.63 min, m/z=294.0 [M+H]+.
Step 4: 2-(((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)acetaldehyde hydrochloride




A solution of N-((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)-2,2-dimethoxy-N-methylethanamine (3.92 g, 13.36 mmol, 1.00 equiv) in 6N hydrochloric acid (40 mL) was stirred at 80° C. for 2 h. The resulting mixture was concentrated under vacuum to yield 3.82 g of crude 2-(((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)acetaldehyde hydrochloride as a yellow solid. 1H NMR (300 MHz, DMSO) δ: 9.43 (s, 1H), 8.07 (s, 1H), 7.73-7.52 (m, 2H), 7.41-7.21 (m, 2H), 4.54-4.25 (m, 2H), 4.12-4.00 (m, 2H), 2.55 (s, 3H) ppm. LCMS (method C, ESI): RT=0.59 min, m/z=266.0 [M+H+H2O]+.
Step 5: N1-((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 2)




To a stirred solution of 2-([[3-(4-fluorophenyl)-1H-pyrazol-4-yl]methyl](methyl)amino)acetaldehyde (200 mg, 0.81 mmol, 1.00 equiv), methylamine hydrochloride (1 g, 14.81 mmol, 18.31 equiv) and acetic acid (126 mg, 2.10 mmol, 2.59 equiv) in 1,2-dichloroethane (20 mL) was added NaBH(OAc)3 (670 mg). The resulting solution was stirred at room temperature overnight and then quenched by the addition of 10 mL of water. The pH value of the solution was adjusted to 7 with saturated sodium carbonate solution. The resulting mixture was extracted with 2×50 mL of dichloromethane. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was initially purified on a silica gel column eluted with 10% MeOH in CH2Cl2 and the partially purified product was repurified by Prep-HPLC with the following conditions (1#-Pre-HPLC-005 (Waters)): Column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, Water with 10 mmol NH4HCO3 and CH3CN (18% CH3CN up to 58% in 10 min, up to 95% in 1 min, down to 18% in 2 min); Detector, UV 254/220 nm. The fractions containing the pure product were concentrated and then redissolved in 5 mL of 12N hydrochloric acid. The resulting solution was concentrated under vacuum to afford 100 mg (47%) of N1-((3-(4-fluorophenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride as a white solid. 1H NMR (300 MHz, D2O) δ: 7.91 (s, 1H), 7.50-7.45 (m, 2H), 7.25-7.19 (m, 2H), 4.41 (s, 2H), 3.21 (s, 4H), 2.60 (s, 3H), 2.57 (s, 3H) ppm. LCMS (method J, ESI): RT=1.01 min, m/z=263.1 [M+H]+.
Compound 19
N1-((3-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: 3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde




A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (6 g, 19.60 mmol, 1.00 equiv), [4-(propan-2-yloxy)phenyl]boronic acid (7.0 g, 38.89 mmol, 1.98 equiv), Pd(PPh3)4 (2.2 g, 1.90 mmol, 0.10 equiv) and sodium carbonate (6.2 g, 58.50 mmol, 2.98 equiv) in toluene (60 mL) and water (5 mL) was stirred under nitrogen at 98° C. overnight. The reaction was cooled to room temperature then diluted with 600 mL of ethyl acetate. The resulting mixture was washed with 3×300 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 3.0-6.5% ethyl acetate in petroleum ether to give 4.5 g (73%) of 3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde as yellow oil. 1H NMR (300 MHz, CDCl3) δ: 9.98 (s, 1H), 8.23 (s, 1H), 7.70-7.65 (m, 2H), 7.26 (s, 1H), 6.98-6.93 (m, 2H), 5.45-5.41 (m, 1H), 4.65-4.57 (m, 1H), 4.13-4.09 (m, 1H), 3.78-3.73 (m, 1H), 2.21-2.03 (m, 3H), 1.71-1.63 (m, 3H), 1.36 (d, J=6.0 Hz, 6H) ppm. LCMS (method D, ESI): RT=1.57 min, m/z=315.0 [M+H]+.
Step 2: tert-butyl 2-(((3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate




To a stirred solution of 3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (3.0 g, 9.54 mmol, 1.00 equiv) and tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate (2.2 g, 11.69 mmol, 1.22 equiv) in 1,2-dichloroethane (30 mL) at room temperature was added NaBH(OAc)3 (4.1 g, 19.34 mmol, 2.03 equiv) in portions. The resulting solution was stirred at room temperature overnight and then diluted with 300 mL of dichloromethane. The resulting mixture was washed with 3×100 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 1-3% MeOH in CH2Cl2 to give 4.0 g (86%) of tert-butyl 2-(((3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate as a yellow oil. LCMS (method D, ESI): RT=1.76 min, m/z=487.0 [M+H]+.
Step 3: N1-((3-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 19)




A solution of tert-butyl 2-(((3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (1.2 g, 2.47 mmol, 1.00 equiv) in 3N hydrochloric acid (30 mL) was stirred at 60° C. overnight. The resulting mixture was cooled to room temperature and washed with 3×50 mL of dichloromethane. The aqueous layer was concentrated under vacuum to give 0.8 g (86%) of N1-((3-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride as a yellow solid. 1H NMR (300 MHz, D2O) δ 7.92 (s, 1H), 7.40 (d, J=8.7 Hz, 2H), 7.04 (d, J=8.7 Hz, 2H), 4.69-4.62 (m, 1H), 4.41 (s, 2H), 3.25-3.10 (m, 4H), 2.58 (s, 3H), 2.53 (s, 3H), 1.24 (s, 3H), 1.22 (s, 3H) ppm. LCMS (method K, ESI): RT=1.27 min, m/z=303.1 [M+H]+.
Compound 199
N1-((3-(4-isopropoxy-3,5-dimethylphenyl)-1H-pyrazol-4-yl)methyl)-N-methylethane-1,2-diamine




Step 1: tert-butyl 2-(((3-(4-isopropoxy-3,5-dimethylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate




A mixture of tert-butyl N-[2-([[3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl](methyl)amino)ethyl]carbamate (500 mg, 1.08 mmol, 1.00 equiv), [3,5-dimethyl-4-(propan-2-yloxy)phenyl]boronic acid (672 mg, 3.23 mmol, 3.00 equiv), [1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (70 mg, 0.11 mmol, 0.10 equiv) and K3PO4 (680 mg, 3.21 mmol, 2.98 equiv) in ethylene glycol dimethyl ether (6 mL) and water (0.2 mL) was stirred overnight at 85° C. The reaction was cooled to room temperature, diluted with 30 mL of dichloromethane and washed with 3×20 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-10% of MeOH in CH2Cl2 to give 650 mg of tert-butyl N-[2-[([4-[3,5-dimethyl-4-(propan-2-yloxy)phenyl]-1-(oxan-2-yl)-1H-pyrrol-3-yl]methyl)(methyl)amino]ethyl]carbamate as a yellow oil. 1H-NMR (300 MHz, DMSO-d6): δ 7.81 (s, 1H), 7.46 (s, 1H), 7.42 (d, J=4.8 Hz, 1H), 5.40-5.30 (m, 1H), 4.21-4.09 (m, 1H), 4.01-3.88 (m, 2H), 3.47-3.41 (m, 1H), 3.35-3.30 (m, 1H), 3.10 (s, 1H), 2.40-2.32 (m, 1H), 2.18-2.08 (s, 9H), 2.08 (s, 1H), 1.61-1.50 (m, 1H), 1.37-1.35 (m, 4H), 1.21 (s, 15H), 1.05-1.03 (m, 2H) ppm. LCMS (method A, ESI): RT=1.37 min, m/z=501.0 [M+H]+.
Step 2: N1-((3-(4-isopropoxy-3,5-dimethylphenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine (Compound 199)




A solution of tert-butyl N-[2-[([4-[3,5-dimethyl-4-(propan-2-yloxy)phenyl]-1-(oxan-2-yl)-1H-pyrrol-3-yl]methyl)(methyl)amino]ethyl]carbamate (650 mg, 1.30 mmol, 1.00 equiv) in 3N hydrochloric acid (20 mL) was stirred at 60° C. overnight. The reaction mixture was cooled to room temperature and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions (2#-Waters 2767-2(HPLC-08)): Column, Xbridge Shield RP 18, Sum, 19*150 mm; mobile phase, water with 50 mmol CF3COOH and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to give 269 mg (38%) of N1-((3-(4-isopropoxy-3,5-dimethylphenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine as a light yellow oil. 1H-NMR (300 MHz, D2O): δ 7.88 (s, 1H), 7.17 (s, 2H), 4.42 (s, 2H), 4.35-4.26 (m, 1H), 3.30-3.10 (m, 4H), 2.57 (s, 3H), 2.23 (s, 6H), 1.23 (d, J=6.3 Hz, 6H) ppm. LCMS (method J, ESI): RT=1.57 min, m/z=317.1 [M+H]+.
Compound 220
N1-((5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: 3-(3-chloro-4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde




A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (600 mg, 1.96 mmol, 1.00 equiv), [3-chloro-4-(propan-2-yloxy)phenyl]boronic acid (671 mg, 3.13 mmol, 1.60 equiv), Pd(dppf)Cl2 (430 mg, 0.59 mmol, 0.30 equiv) and potassium carbonate (811 mg, 5.87 mmol, 2.99 equiv) in 1,4-dioxane (50 mL) and water (5 mL) was stirred under nitrogen at 105° C. for 8 h. The solid material was removed by filtration and the filtrate was extracted with ethyl acetate. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-16% ethyl acetate in petroleum ether to give 600 mg (88%) of 3-(3-chloro-4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde as a yellow oil. LCMS (method C, ESI): RT=0.98 min, m/z=371.0 [M+Na]+.
Step 2: 3-(3-chloro-4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde




A mixture of 3-(3-chloro-4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (600 mg, 1.72 mmol, 1.00 equiv) in a saturated methanolic HCl (50 mL) was stirred at 25° C. for 2 h. The resulting mixture was concentrated under vacuum to give 530 mg of crude 3-(3-chloro-4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde as a white solid. LCMS (method C, ESI): RT=0.86 min, m/z=265.0 [M+H]+.
Step 3: 5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde




To a mixture of 3-(3-chloro-4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde (530 mg, 1.76 mmol, 1.00 equiv) and potassium carbonate (828 mg, 5.99 mmol, 3.40 equiv) in acetonitrile (100 mL) was added iodomethane (852 mg, 6.00 mmol, 3.41 equiv) dropwise. The reaction was stirred at 70° C. for 8 h. The resulting mixture was cooled to room temperature then concentrated under vacuum. The residue was dissolved in 20 mL of H2O and then extracted with 3×30 mL ethyl acetate. The combined organic layers was washed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by Prep-HPLC with the following conditions: Column, XSelect CSH Prep C18 OBD Column, 5 μm, 19×150 mm; mobile phase, water with 10 mmol NH4HCO3 and MeCN (38.0% MeCN up to 45.0% in 15 min); Detector, UV 254/220 nm to afford 100 mg (18%) of 5-[3-chloro-4-(propan-2-yloxy)phenyl]-1-methyl-1H-pyrazole-4-carbaldehyde as colorless oil. 1H-NMR (300 MHz, DMSO-d6): δ 9.55 (s, 1H), 8.03 (s, 1H), 7.73 (d, J=2.1 Hz, 1H), 7.51 (dd, J=8.4 Hz, 2.1 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 4.85-4.77 (m, 1H), 3.78 (s, 3H), 1.33 (d, J=7.5 Hz, 6H) ppm. LCMS (method C, ESI): RT=0.92 min, m/z=279.0 [M+H]+ and 120 mg (22%) of the other regioisomer, 3-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde as a colorless oil. 1H-NMR (300 MHz, DMSO-d6): δ 9.83 (s, 1H), 8.51 (s, 1H), 7.96 (d, J=2.1 Hz, 1H), 7.82 (dd, J=8.4 Hz, 2.1 Hz, 1H), 7.23 (d, J=8.4 Hz, 1H), 4.77-4.69 (m, 1H), 3.94 (s, 3H), 1.32 (d, J=7.5 Hz, 6H) ppm.
Step 4: tert-butyl 2-(((5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate




To a solution of 5-[3-chloro-4-(propan-2-yloxy)phenyl]-1-methyl-1H-pyrazole-4-carbaldehyde (100 mg, 0.36 mmol, 1.00 equiv) and tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate (81 mg, 0.43 mmol, 1.20 equiv) in methanol (40 mL) was added acetic acid (0.2 mL) to adjust the solution pH to 7. NaBH3CN (31 mg, 0.49 mmol, 1.38 equiv) was added and the reaction mixture was stirred for 4 h at 65° C. The resulting mixture was concentrated under vacuum then dissolved in 30 mL of H2O. The resulting solution was extracted with 3×30 mL of ethyl acetate. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum to give 140 mg (87%) of tert-butyl 2-(((5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate as a colorless oil. LCMS (method C, ESI): RT=0.72 min, m/z=451.0 [M+H]+.
Step 5: N1-((5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 220)




A solution of tert-butyl 2-(((5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (140 mg, 0.31 mmol, 1.00 equiv) in dichloromethane (5 mL) and CF3COOH (10 mL) was stirred for 1 h at 25° C. The resulting mixture was concentrated under vacuum and the crude product was purified by Prep-HPLC with the following conditions: Column, XSelect CSH Prep C18 OBD Column, 5 μm, 19×150 mm; mobile phase, water with 0.05% TFA and MeCN (5.0% MeCN up to 35.0% in 10 min); Detector, UV 220/254 nm to yield 146.5 mg (82%) of N1-((5-(3-chloro-4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine trifluoroacetate as a colorless oil. 1H-NMR (300 MHz, D2O): δ 7.70 (s, 1H), 7.47 (s, 1H), 7.27 (s, 2H), 4.78-4.71 (m, 1H), 4.21 (s, 2H), 3.65 (s, 3H), 3.25-3.15 (m, 4H), 2.60 (s, 3H), 2.56 (s, 3H), 1.32 (s, 3H), 1.30 (s, 3H) ppm. LCMS (method M, ESI): RT=1.31 min, m/z=351.2 [M+H]+.
Compound 224
N1-((3-(3-fluoro-4-phenoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine




Step 1: 2-fluoro-4-nitro-1-phenoxybenzene




Sodium hydride (1.51 g, 62.92 mmol, 2.00 equiv) was added in small portions to a solution of phenol (3.25 g, 34.53 mmol, 1.10 equiv) in N,N-dimethylformamide (50 mL) maintained under nitrogen at 0-5° C. After stirring for 30 min at 0-5° C., 1,2-difluoro-4-nitrobenzene (5 g, 31.43 mmol, 1.00 equiv) was added. The reaction mixture was stirred overnight at room temperature and then diluted with 200 mL of water. The resulting solution was extracted with 3×100 mL of ethyl acetate. The combined organic layers was washed with 3×150 mL of brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified on a silica gel column eluted with 0-2% ethyl acetate in petroleum ether to afford 3.2 g (44%) of 2-fluoro-4-nitro-1-phenoxybenzene as a yellow solid. 1H-NMR (400 MHz, CDCl3): δ 8.10-8.07 (m, 1H), 7.99-7.94 (m, 1H), 7.46-7.41 (m, 2H), 7.30-7.26 (m, 1H), 7.12-7.04 (m, 2H), 7.02-6.93 (m, 1H) ppm.
Step 2: 3-fluoro-4-phenoxybenzenamine




To a solution of 2-fluoro-4-nitro-1-phenoxybenzene (3.2 g, 13.72 mmol, 1.00 equiv) and methanol (100 mL) was added 10% palladium on carbon (0.5 g) catalyst. The reaction mixture was stirred under 1 atmosphere of hydrogen at room temperature for 4 h. The catalyst was removed by filtration. The filtrate was concentrated under vacuum to give 2.65 g (95%) of 3-fluoro-4-phenoxybenzenamine as an off-white solid. 1H-NMR (300 MHz, CDCl3): δ 7.29-7.19 (m, 2H), 7.03-6.83 (m, 4H), 6.52-6.36 (m, 2H), 3.63 (s, 2H) ppm.
Step 3: 2-fluoro-4-iodo-1-phenoxybenzene




To a stirred solution of 3-fluoro-4-phenoxybenzenamine (2.6 g, 12.79 mmol, 1.00 equiv) in ethylene glycol dimethyl ether (35 mL) at room temperature was added a solution of 50% sulfuric acid (5 mL) in water (25 mL) dropwise. The reaction was cooled to −5° C. and a solution of sodium nitrite (1.33 g, 19.28 mmol, 1.51 equiv) in water (9 mL) was then added dropwise within 20 min. The resulting solution was stirred for 30 min at −5-0° C. A solution of sodium iodide (9.6 g, 64.00 mmol, 5.00 equiv) in water (25 mL) was added dropwise into the reaction mixture at −5° C. The resulting solution was stirred at −5-0° C. for 30 min then extracted with 3×100 mL of ethyl acetate. The combined organic layers was washed with 3×200 mL of saturated sodium sulfite solution, dried over anhydrous sodium sulfate and concentrated under vacuum to give 3.88 g (97%) of 2-fluoro-4-iodo-1-phenoxybenzene as a yellow oil. 1H-NMR (400 MHz, CDCl3): δ 7.53-7.49 (m, 1H), 7.42-7.30 (m, 2H), 7.13-7.08 (m, 1H), 6.99-6.96 (m, 2H), 6.78 (t, J=8.4 Hz, 2H) ppm.
Step 4: 2-(3-fluoro-4-phenoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane




A mixture of 2-fluoro-4-iodo-1-phenoxybenzene (1.2 g, 3.82 mmol, 1.00 equiv), Pd(dppf)Cl2.CH2Cl2 (312 mg, 0.38 mmol, 0.10 equiv), potassium acetate (1.12 g, 11.41 mmol, 2.99 equiv) and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (1.07 g, 4.21 mmol, 1.10 equiv) in 1,4-dioxane (10 mL) was stirred under nitrogen overnight at 95° C. The reaction was cooled to room temperature and then quenched by the addition of 100 mL of water. The resulting mixture was extracted with 3×100 mL of ethyl acetate. The combined organic layers was washed with 3×200 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-2% ethyl acetate in petroleum ether to give 710 mg (59%) of 2-(3-fluoro-4-phenoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a yellow oil. 1H-NMR (300 MHz, CDCl3): δ 7.64 (dd, J=1.5 Hz, J=11.1 Hz, 1H), 7.56 (d, J=8.1 Hz, 1H), 7.39-7.34 (m, 2H), 7.17-7.12 (m, 1H), 7.06-7.00 (m, 3H), 1.38 (s, 12H) ppm.
Step 5: tert-butyl 2-(((3-(3-fluoro-4-phenoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate




A mixture of tert-butyl N-[2-([[3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl](methyl)amino)ethyl]carbamate (380 mg, 0.82 mmol, 1.00 equiv), 2-(3-fluoro-4-phenoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (309 mg, 0.98 mmol, 1.20 equiv), [1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (54 mg, 0.08 mmol, 0.10 equiv) and K3PO4 (521 mg, 2.45 mmol, 3.00 equiv) in toluene (10 mL) and water (2.5 mL) was stirred overnight under nitrogen at 100° C. The reaction was cooled to room temperature and then quenched by the addition of 50 mL of water. The resulting mixture was extracted with 3×50 mL of ethyl acetate. The combined organic layers was washed with 3×100 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-10% ethyl acetate in petroleum ether to afford 197 mg (46%) of tert-butyl 2-(((3-(3-fluoro-4-phenoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate as a yellow oil. LCMS (method D, ESI): RT=1.35 min, m/z=525.0 [M+H]+.
Step 6: N1-((3-(3-fluoro-4-phenoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine trifluoroacetate (Compound 224)




A solution of tert-butyl 2-(((3-(3-fluoro-4-phenoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate (197 mg, 0.38 mmol, 1.00 equiv) in 4N hydrochloric acid (10 mL) was stirred overnight at 60° C. The resulting solution was cooled to room temperature and washed with 2×20 mL of dichloromethane. The aqueous layer was concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions (2#-Waters 2767-2(HPLC-08)): Column, XBridge Shield RP 18, 5 μm, 19×150 mm; mobile phase, water with 50 mmol TFA and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to give 158.5 mg (74%) of N1-((3-(3-fluoro-4-phenoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine trifluoroacetate as a white solid. 1H NMR (400 MHz, D2O) δ 7.95 (s, 1H), 7.46-7.36 (m, 3H), 7.27 (d, J=8.0 Hz, 2H), 7.220-7.13 (m, 2H), 7.07 (d, J=8.1 Hz, 2H), 4.47 (s, 2H), 3.28 (s, 4H), 2.63 (s, 3H) ppm. LCMS (method D, ESI): RT=1.43 min, m/z=341.1 [M+H]+.
Compound 225
N1-((3-(4-(4-fluorophenoxy)phenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine




Step 1: 1-fluoro-4-(4-nitrophenoxy)benzene




A mixture of 1-fluoro-4-nitrobenzene (4 g, 28.35 mmol, 1.00 equiv), 4-fluorophenol (3.177 g, 28.34 mmol, 1.00 equiv) and potassium carbonate (7.83 g, 56.24 mmol, 1.98 equiv) in N,N-dimethylformamide (50 mL) was stirred overnight at 120° C. The reaction was cooled to room temperature and then quenched by the addition of 200 mL of water. The resulting solution was extracted with 3×100 mL of ethyl acetate. The combined organic layers was washed with 3×150 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum to yield 6.4 g (97%) of 1-fluoro-4-(4-nitrophenoxy)benzene as a yellow solid. 1H-NMR (400 MHz, CDCl3): δ 8.22-8.18 (m, 2H), 7.16-7.04 (m, 4H), 7.00-6.97 (m, 2H) ppm.
Step 2: 4-(4-fluorophenoxy)benzenamine




A mixture of 1-fluoro-4-(4-nitrophenoxy)benzene (6.4 g, 27.44 mmol, 1.00 equiv) and 10% palladium on carbon (1 g) catalyst in methanol (200 mL) was stirred under 1 atmosphere of hydrogen at room temperature for 4 h. The catalyst was removed by filtration and the filtrate was concentrated under vacuum to give 5.4 g (97%) of 4-(4-fluorophenoxy)benzenamine as an off-white solid. 1H-NMR (300 MHz, CDCl3): δ 6.99-6.82 (m, 6H), 6.68 (d, J=8.7 Hz, 2H), 3.63 (br, 2H) ppm.
Step 3: 1-fluoro-4-(4-iodophenoxy)benzene




To a stirred solution of 4-(4-fluorophenoxy)benzenamine (3.4 g, 16.73 mmol, 1.00 equiv) in ethylene glycol dimethyl ether (45 mL) was added dropwise a solution of 50% sulfuric acid (7 mL) in water (34 mL) at room temperature. The reaction was cooled to −5° C. and a solution of sodium nitrite (1.73 g, 25.07 mmol, 1.50 equiv) in water (12 mL) was then added dropwise within 30 min. The resulting solution was stirred for 30 min at −5-0° C. then a solution of sodium iodide (12.6 g, 84.00 mmol, 5.02 equiv) in water (34 mL) was added dropwise at −5-0° C. After stirring for 30 min at −5-0° C., the solution was extracted with 3×200 mL of ethyl acetate. The combined organic layers was washed with 3×200 mL of saturated Na2SO3 solution, dried over anhydrous sodium sulfate and concentrated under vacuum to give 5.0 g (95%) of 1-fluoro-4-(4-iodophenoxy)benzene as a yellow solid. 1H-NMR (300 MHz, CDCl3): δ 7.62-7.57 (m, 2H), 7.08-6.93 (m, 4H), 6.77-6.70 (m, 2H) ppm.
Step 4: 2-(4-(4-fluorophenoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane




A mixture of 1-fluoro-4-(4-iodophenoxy)benzene (1.2 g, 3.82 mmol, 1.00 equiv), Pd(dppf)Cl2.CH2Cl2 (312 mg, 0.38 mmol, 0.10 equiv), potassium acetate (1.12 g, 11.41 mmol, 2.99 equiv) and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (1.07 g, 4.21 mmol, 1.10 equiv) in 1,4-dioxane (10 mL) was stirred under nitrogen at 95° C. overnight. The reaction was cooled to room temperature then quenched by the addition of 100 mL of water. The resulting solution was extracted with 3×100 mL of ethyl acetate. The combined organic layers was washed with 3×200 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-3% ethyl acetate in petroleum ether to give 682 mg (57%) of 2-[4-(4-fluorophenoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a yellow solid. 1H-NMR (300 MHz, CDCl3): δ 7.83-7.79 (m, 2H), 7.10-6.96 (m, 6H), 1.37 (s, 12H) ppm.
Step 5: tert-butyl 2-(((3-(4-(4-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate




A mixture of tert-butyl N-[2-([[3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl](methyl)amino)ethyl]carbamate (380 mg, 0.82 mmol, 1.00 equiv), 2-[4-(4-fluorophenoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (309 mg, 0.98 mmol, 1.20 equiv), [1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium (54 mg, 0.08 mmol, 0.10 equiv) and K3PO4 (521 mg, 2.45 mmol, 3.00 equiv) in toluene (10 mL) and water (2.5 mL) was stirred under nitrogen at 100° C. overnight. The reaction was cooled to room temperature then quenched by the addition of 50 mL of water. The resulting solution was extracted with 3×50 mL of ethyl acetate. The combined organic layers was washed with 3×100 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-10% ethyl acetate in petroleum ether to give 140 mg (33%) of tert-butyl 2-(((3-(4-(4-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate as a yellow oil. LCMS (method D, ESI): RT=1.35 min, m/z=525.0 [M+H]+.
Step 6: N1-((3-(4-(4-fluorophenoxy)phenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine (Compound 225)




A solution of tert-butyl 2-(((3-(4-(4-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate (140 mg, 0.27 mmol, 1.00 equiv) in 4N hydrochloric acid (10 mL) was stirred overnight at 60° C. The resulting solution was cooled to room temperature and washed with 2×20 mL of ethyl acetate. The aqueous layer was concentrated under vacuum and the crude product was purified by Prep-HPLC with the following conditions (2#-Waters 2767-2(HPLC-08)): Column, XBridge Shield RP 18, 5 μm, 19×150 mm; mobile phase, water with 50 mmol TFA and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to yield 73 mg (48%) of N1-((3-(4-(4-fluorophenoxy)phenyl)-1H-pyrazol-4-yl)methyl)-N1-methylethane-1,2-diamine trifluoroaceteate as a yellow oil. 1H-NMR (400 MHz, D2O): δ 7.92 (s, 1H), 7.48-7.44 (m, 2H), 7.16-7.06 (m, 6H), 4.44 (s, 2H), 3.30-3.18 (m, 4H), 2.61 (s, 3H) ppm. LCMS (method M, ESI): RT=1.26 min, m/z=341.1 [M+H]+.
Compound 229
N1-((3-(3,5-diisopropylphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: 4-bromo-2,6-diisopropylbenzenamine




To a stirred solution of 2,6-diisopropylaniline (14 g, 78.97 mmol, 1.00 equiv) in N,N-dimethylformamide (200 mL) at 0° C. was added a solution of NBS (14 g, 78.65 mmol, 1.00 equiv) in N,N-dimethylformamide (100 mL) dropwise in 60 min. The reaction was stirred for another hour at 0° C. then water (900 mL) was added. The resulting mixture was extracted with 300 mL of ethyl acetate. The combined organic layers was washed with 3×100 mL of saturated NH4Cl solution and 1×100 mL of water then dried over anhydrous sodium sulfate and concentrated under vacuum to give 20.05 g (99%) of 4-bromo-2,6-diisopropylaniline as a red oil. 1H NMR (400 MHz, DMSO-d6): δ 6.95 (s, 2H), 4.77 (s, 2H), 5.47-5.39 (m, 1H), 3.04-2.98 (m, 1H), 1.13 (d, J=6.8 Hz, 12H) ppm.
Step 2: 1-bromo-3,5-diisopropylbenzene




To a mixture of 4-bromo-2,6-bis(propan-2-yl)aniline (20.5 g, 80.02 mmol, 1.00 equiv) in 2N hydrochloric acid (210 mL) at −5° C. was added NaNO2 (13.8 g, 200.00 mmol, 2.50 equiv) in portions. The reaction was allowed stir at −5° C. for 10 min. Hypophosphorous acid (90 mL) was then added at −5° C. The reaction was warmed to room temperature and then stirred for 12 h. The resulting solution was extracted with 300 mL of ethyl acetate. The organic layer was concentrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:100) to give 12 g (62%) of 1-bromo-3,5-bis(propan-2-yl)benzene as a light brown oil. 1H NMR (300 MHz, CDCl3): δ 7.18 (s, 2H), 6.98 (s, 1H), 2.89-2.80 (m, 2H), 1.23 (d, J=6.9 Hz, 12H) ppm.
Step 3: 2-(3,5-diisopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane




A mixture 1-bromo-3,5-bis(propan-2-yl)benzene (2.41 g, 9.99 mmol, 1.00 equiv), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (3.05 g, 12.01 mmol, 1.20 equiv), potassium acetate (2.94 g, 30.00 mmol, 3.00 equiv) and PdCl2(dppf).CH2Cl2 (408 mg, 0.50 mmol, 0.05 equiv) in 1,4-dioxane (20 mL) was stirred under nitrogen for 12 h at 85° C. The resulting mixture was cooled to room temperature and concentrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:15) to afford 2.48 g (86%) of 2-(3,5-diisopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 7.34 (s, 2H), 7.23 (s, 2H), 2.92-2.83 (m, 2H), 1.29 (s, 12H), 1.13 (d, J=6.9 Hz, 12H) ppm.
Step 4: tert-butyl 2-(((3-(3,5-diisopropylphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate




A mixture of 2-(3,5-diisopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (288 mg, 1.00 mmol, 1.00 equiv), tert-butyl N-[2-([[3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl](methyl)amino)ethyl]-N-methylcarbamate (479 mg, 1.00 mmol, 1.00 equiv), K3PO4 (636 mg, 3.00 mmol, 3.00 equiv) and [1,1′-bis(di-tert-butylphosphino)ferrocene]dichloropalladium (65 mg, 0.10 mmol, 0.10 equiv) in ethylene glycol dimethyl ether (5 mL) was stirred under nitrogen in a 10-mL sealed tube for 12 h at 85° C. The resulting mixture was cooled to room temperature and concentrated under vacuum. The residue was purified on a silica gel column eluted with dichloromethane/methanol (39:1 with 0.1% triethylamine) to obtain 100 mg (20%) of tert-butyl N-[2-[([3-[3,5-bis(propan-2-yl)phenyl]-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl)(methyl)amino]ethyl]-N-methylcarbamate as a black solid. 1H NMR (300 MHz, DMSO-d6): δ 7.83 (s, 1H), 7.49 (s, 2H), 7.07 (s, 1H), 5.45-5.35 (m, 1H), 4.01-3.88 (m, 1H), 3.71-3.60 (m, 1H), 3.40 (s, 2H), 3.24 (s, 2H), 3.00-2.80 (m, 2H), 2.68 (s, 3H), 2.49-2.40 (m, 2H), 2.23 (s, 3H), 2.15-1.85 (m, 3H), 1.80-1.60 (m, 1H), 1.31 (s, 9H), 1.23 (d, J=6.9 Hz, 12H) ppm. LCMS (method D, ESI): RT=1.41 min, m/z=513.5 [M+H]+.
Step 5: N1-((3-(3,5-diisopropylphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 229)




A solution of tert-butyl N-[2-[([3-(3,5-diisopropylphenyl)-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl)(methyl)amino]ethyl]-N-methylcarbamate (180 mg, 0.35 mmol, 1.00 equiv) in a saturated hydrogen chloride solution in 1,4-dioxane (5 mL) was stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was triturated with 1×50 mL of ether to give 130 mg (92%) of ([3-[3,5-bis(propan-2-yl)phenyl]-1H-pyrazol-4-yl]methyl)(methyl)[2-(methylamino)ethyl]amine as a light yellow solid. 1H NMR (300 MHz, CD3OD): δ 8.25-8.22 (m, 1H), 7.31 (s, 1H), 7.27 (s, 2H), 4.70-4.37 (m, 2H), 3.60-3.35 (m, 4H), 3.04-2.94 (m, 2H), 2.72 (s, 3H), 2.71 (s, 3H), 1.31 (d, J=7.2 Hz, 12H) ppm. LCMS (method H, ESI): RT=1.34 min, m/z=329.1 [M+H]+.
Compound 238
N1-((3-(4-(3-methoxypropoxy)phenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: tert-butyl 2-(((3-(4-(3-methoxypropoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate




A mixture of [4-(3-methoxypropoxy)phenyl]boronic acid (200 mg, 0.95 mmol, 1.00 equiv), tert-butyl N-[2-([[4-iodo-1-(oxan-2-yl)-1H-pyrazol-3-yl]methyl](methyl)amino)ethyl]-N-methylcarbamate (379 mg, 0.79 mmol, 0.83 equiv), Pd(dppf)Cl2 (58 mg, 0.08 mmol, 0.08 equiv) and potassium carbonate (328 mg, 2.37 mmol, 2.49 equiv) in 1,4-dioxane (20 mL) and water (3 mL) was stirred under nitrogen at 100° C. overnight. The reaction was cooled to room temperature and then quenched by the addition of 50 mL of water. The resulting mixture was extracted with 3×100 mL of ethyl acetate. The combined organic layers was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with 0-10% ethyl acetate in petroleum ether to give 170 mg (35%) of tert-butyl 2-(((3-(4-(3-methoxypropoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl(methyl)carbamate as a light yellow oil. LCMS (method D, ESI): RT=1.31 min, m/z=517.0 [M+H]+.
Step 2: N1-((3-(4-(3-methoxypropoxy)phenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 238)




A solution of tert-butyl 2-(((3-(4-(3-methoxypropoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (150 mg, 0.29 mmol, 1.00 equiv) in 3N hydrochloric acid (15 mL) was stirred at room temperature overnight. The resulting mixture was concentrated under vacuum and the residue was purified by Prep-HPLC with the following conditions (1#-Pre-HPLC-005(Waters)): Column, XBridge Shield RP18 OBD Column, 5 μm, 19×150 mm; Mobile Phase, water with 10 mmol NH4HCO3 and CH3CN (18% CH3CN up to 58% in 10 min, up to 95% in 1 min, down to 18% in 2 min); Detector, UV 254/220 nm to give 76 mg (79%) of N1-((3-(4-(3-methoxypropoxy)phenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine as a colorless semi-solid. 1H NMR (300 MHz, DMSO-d6): δ 7.80-7.67 (m, 1H), 7.57-7.42 (m, 2H), 6.92 (d, J=8.4 Hz, 2H), 4.02 (t, J=6.3 Hz, 2H), 3.55-3.45 (m, 4H), 3.34-3.25 (m, 2H), 3.24 (s, 3H), 2.45-2.39 (m, 2H), 2.20 (s, 3H), 2.10 (s, 3H), 2.00-1.90 (m, 2H) ppm. LCMS (method N, ESI): RT=1.23 min, m/z=333.1 [M+H]+.
Compound 243
N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: Ethyl 3-iodo-1H-pyrazole-4-carboxylate




To a solution of ethyl 3-amino-1H-pyrazole-4-carboxylate (20 g, 129 mmol) in 50% H2SO4 (195 mL) was added drop wise NaNO2 (15.9 g, 230 mmol) solution in water (30 mL) at −10° C. and stirred for 1 h. A solution of KI (69.4 g 418 mmol) in water (33 mL) was added and the reaction stirred at −10° C. for 6 h. The reaction was then poured into water (200 mL) and extracted with DCM (100 mL×3). The combined extracts was washed with saturated NaHCO3 (100 mL×2), saturated sodium thiosulphate (100 mL), and concentrated in vacuo. The residue was purified by SGC using Hex/EA as eluent (0%-20%) to afford ethyl 3-iodo-1H-pyrazole-4-carboxylate (23 gm, yield: 67%).
Step 2: Ethyl 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate




A solution of ethyl 3-iodo-1H-pyrazole-4-carboxylate (27 g, 101.8 mmol), 3,4-DHP (25.71 g 305.6 mmol) and PTSA (1.94 g 10.18 mmol) in THF (100 mL) was heated at 60° C. for 5 h. Solvent was evaporated under vacuum and the residue redissolved in DCM (500 mL) was added. The solution was washed with sat.NaHCO3 (200 mL×2), dried, concentrated to afford crude ethyl 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate (35 g, yield: 98%). The crude material was used in the next synthetic step without further purification.
Step 3: 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylic acid




To a solution of ethyl 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate (5 g, 14.2 mmol) in (1:1) THF/MeOH (32 mL) was added a solution of LiOH (1.8 g, 4.28 mmol) in water (16 mL). The reaction was stirred at rt for 4 h then evaporated under vacuum and the residue was treated with water (25 mL) and washed with ethyl acetate (30 mL×2). The aqueous layer was isolated acidified to  ̃pH5 with 2N HCl and extracted with DCM (30 mL×3). The combined extracts were washed with brine (30 mL), dried and concentrated to afford 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylic acid (3.5 g, yield: 76%). LCMS (method B): RT=2.18 min, m/z=321.0 [M−H]−.
Step 4: (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol




A solution of 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylic acid (34 g, 105 mmol) in BH3-THF (340 mL, 340 mmol, IM in THF) was stirred at rt overnight. Solvent was evaporated under vacuum and the residue dissolved in DCM (340 mL), washed with water (340 mL×3), dried and concentrated. The residue was purified by SGC using Hex: EA (0%-70%) as eluent to afford (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol (27 g, yield: 83%). LCMS (method C): RT=1.74 min, m/z=309.1 [M+H]+.
Step 5: 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde
To a solution of oxalyl chloride (33.51 g, 22.8 mL, 263 mmol) in DCM (320 mL) at −65° C. under a dry N2 atm. was added DMSO (27.28 g, 24.8 mL, 350.3 mmol) dropwise. The reaction was stirred at −65° C. for 0.5 h then treated dropwise with a solution of (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol (27 g 87.66 mmol) in DCM (75 mL. The mixture was stirred at −65° C. for 1 h then treated with TEA (53.14 g, 73.2 mL, 525 mmol) then warmed to rt and stirred for 0.5 h. DCM (300 mL) was added and the mixture washed with 1N HCl (150 mL×2). The organic layer was dried and then concentrated under vacuum. The residue was purified by SGC using Hex: EA (0%-15%) as eluent to afford 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (22 g, yield: 82%). LCMS (method C): RT=1.97 min, m/z=307.0 [M+H]+.
Step 6: 3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde




To a solution of 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (8 g, 26.1 mmol) in 1,4-dioxane (240 mL) were added 4-isopropoxy phenylboronic acid (5.3 g, 27.36 mmol) and sat. NaHCO3 solution (50 mL). The reaction mixture was degassed under argon for 0.5 h. Tetrakis(triphenylphosphine)palladium (1.2 g, 1.03 mmol) was added under an argon atm. and the resultant suspension was heated for 8 h at 90° C. The reaction was poured into water (500 mL) and extracted with EtOAc (100 mL×3). The combined extracts was washed with brine (300 mL) dried and concentrated under vacuum. The residue was purified by SGC using Hex:EtOAc (0%-40%) as eluent to obtain 3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (7.4 g, Yield 90%).
Step 7: 3-(4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde hydrochloride




A solution of 3-(4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (7.4 g, 23.56 mmol) in 3M methanolic HCl (74 mL) was stirred at rt for 3 h. The resulting solution was concentrated and triturated using diethyl ether (50 mL×3) to afford 3-(4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde hydrochloride (5.3 g, yield 98%). LCMS (method C): RT=1.96 min, m/z=231.1 [M+H]+.
Step 8: 3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde




A mixture of 3-[4-(propan-2-yloxy)-phenyl]-1H-pyrazole-4-carbaldehyde hydrochloride (400 mg, 1.5 mmol) and K2CO3 (624 mg, 4.5 mmol) in acetonitrile (6 mL) was stirred at 0-5° C. for 1 h. Methyl iodide (310 mg, 2.2 mmol) was added and the mixture stirred at ambient temperature for 48 h. The reaction was poured into water (200 mL) and extracted with EtOAc (50 mL×3). The combined extracts were washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by prep-TLC using Hexane:EtOAc (210:90) as eluent to afford the 3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (82 mg, Yield: 19%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.94 (s, 1H), 8.00 (s, 1H), 7.68-7.64 (m, 2H), 7.02-6.97 (m, 2H), 4.69-4.59 (t, J=6.0 Hz, 1H), 4.00 (s, 3H), 1.38 (d, J=6.0 Hz, 6H) ppm. LCMS (method C RT=2.08 min, m/z=245.2 [M+H]+.
The regio isomeric compound, 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde was also isolated (88 mg, Yield 21%) as a white solid 1H NMR (400 MHz, CDCl3): δ 9.63 (s, 1H), 8.06 (s, 1H), 7.37-7.32 (m, 2H), 7.07-7.02 (m, 2H), 4.71-4.61 (m, 1H), 3.84 (s, 3H), 1.41 (d, J=6 Hz, 6H) ppm. LCMS (method C): RT=2.11 min, m/z=245.5 [M+H]+
Compound 244
N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride




Step 1: 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde




A mixture of 3-[4-(propan-2-yloxy)phenyl]-1H-pyrazole-4-carbaldehyde hydrochloride (400 mg, 1.5 mmol) and K2CO3 (624 mg, 4.5 mmol) in acetonitrile (6 ml) was stirred at 0-5° C. for 1 h. Methyl iodide (310 mg, 2.2 mmol) was added and stirred at ambient temperature for 48 h. The reaction was poured into water (200 mL), extracted with EtOAc (50 mL×3). The combined extracts were washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by prep-TLC using Hexane:EtOAc (21:9) as eluent to afford 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (88 mg, Yield 21%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.63 (s, 1H), 8.05 (s, 1H), 7.36-7.32 (m, 2H), 7.06-7.02 (m, 2H), 4.69-4.64 (m, 1H), 3.84 (s, 3H), 1.0 (d, J=6 Hz, 6H) ppm. LCMS (method C): RT=2.11 min, m/z=245.5 [M+H]+.
The regio isomeric compound, (3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde) (82 mg, Yield: 19%) was also isolated as white solid. 1H NMR (400 MHz, CDCl3): δ 9.94 (s, 1H), 8.00 (s, 1H), 7.68-7.64 (m, 2H), 7.02-6.97 (m, 2H), 4.69-4.59 (t, J=6.0 Hz, 1H), 4.00 (s, 3H), 1.38 (d, J=6.0 Hz, 6H) ppm. LCMS (method CRT=2.08 min, m/z=245.2 [M+H]+.
Step 2: tert-butyl 2-(((5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl(methyl)carbamate




To a solution of 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (88 mg, 0.36 mmol) in methanol (2 ml) was added TEA (145 mg, 1.4 mmol). ZnCl2 (7 mg, 0.05 mmol) and tert-butyl methyl(2-(methylamino)ethyl)carbamate (93 mg, 0.43 mmol) were added at rt and stirred for 45 min. NaCNBH3 (93 mg, 1.4 mmol) was added and stirred for 2 h at rt. The reaction was poured into water (100 mL) and extracted with EtOAc (50 mL×2). The combined extracts was washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by SGC using DCM:MeOH (0%-4%) as eluent to obtain tert-butyl 2-(((5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl(methyl)carbamate (50 mg, Yield 33%).
Step 3: N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (Compound 244)




A solution of tert-butyl 2-(((5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (50 mg, 0.2 mmol) in methanolic HCl was stirred for 2 h. The resulting solution was concentrated and triturated using diethyl ether (20 mL×2) to afford N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (41 mg, 96%). 1H NMR (400 MHz, DMSO-d6): δ 7.89 (s, 1H), 7.38 (d, =8.4 Hz, 2H), 7.08 (d, J=8.4 Hz, 2H), 4.78-4.65 (m, 1H), 4.22-4.10 (m, 2H), 3.68 (s, 3H), 3.43-3.10 (m, 6H), 2.51 (s, 6H), 1.31 (d, J=6 Hz, 6H) ppm. LCMS (method): RT=1.55 min, m/z=317.3 [M+H]+.
Compound 243
N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: N1-benzyl-N2-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




To a solution of 3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (82 mg, 0.32 mmol) in methanol (2 mL) was added TEA (129 mg, 1.2 mmol). ZnCl2 (5 mg, 0.05 mmol) and N-benzyl-N,N′-dimethylethane-1,2-diamine (71 mg, 0.38 mmol) were added at rt and stirred for 45 min. NaCNBH3 (87 mg, 1.2 mmol) was added and stirred for 2 h at rt. The reaction was poured into water (100 mL), extracted with EtOAc (50 mL×2). The combined extracts was washed with brine (100 mL), dried and concentrated under vacuum. The residue was purified by SGC using DCM:MeOH (0%-4%) as eluent to obtain N1-benzyl-N2-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (62 mg, Yield: 45%).
Step 2: N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 243)




To a suspension of N1-benzyl-N2-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (62 mg, 0.15 mmol) and Pd/C (10 mg) in methanol was purged H2 gas for 2 h. The resulting mixture was filtered through celite and washed with methanol (5 mL×2). The combined filtrate was concentrated to afford N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (28 mg, 58%). 1H NMR (400 MHz, DMSO-d6): δ 8.12 (s, 1H), 7.48 (d, J=8.4 Hz, 2H), 6.98 (d, J=8.8 Hz, 2H), 4.71-4.61 (m, 1H), 4.44-4.27 (m, 2H), 3.90 (s, 3H), 3.50-3.18 (m, 5H), 3.11-3.04 (m, 1H), 2.65-2.45 (m, 4H) 1.29 (d, J=6 Hz, 6H) ppm. LCMS (method C): RT=1.76 min, m/z=317.3 [M+H]+.
Compound 244
N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride




Step 1: 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde




A mixture of 3-[4-(propan-2-yloxy)phenyl]-1H-pyrazole-4-carbaldehyde hydrochloride (400 mg, 1.5 mmol) and K2CO3 (624 mg, 4.5 mmol) in acetonitrile (6 ml) was stirred at 0-5° C. for 1 h. Methyl iodide (310 mg, 2.2 mmol) was added and stirred at ambient temperature for 48 h. The reaction was poured into water (200 mL), extracted with EtOAc (50 mL×3). The combined extracts were washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by prep-TLC using Hexane:EtOAc (21:9) as eluent to afford 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (88 mg, Yield 21%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.63 (s, 1H), 8.05 (s, 1H), 7.36-7.32 (m, 2H), 7.06-7.02 (m, 2H), 4.69-4.64 (m, 1H), 3.84 (s, 3H), 1.0 (d, J=6 Hz, 6H) ppm. LCMS (method C): RT=2.11 min, m/z=245.5 [M+H]+.
The regio isomeric compound, (3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde) (82 mg, Yield: 19%) was also isolated as white solid. 1H NMR (400 MHz, CDCl3): δ 9.94 (s, 1H), 8.00 (s, 1H), 7.68-7.64 (m, 2H), 7.02-6.97 (m, 2H), 4.69-4.59 (t, J=6.0 Hz, 1H), 4.00 (s, 3H), 1.38 (d, J=6.0 Hz, 6H) ppm. LCMS (method CRT=2.08 min, m/z=245.2 [M+H]+.
Step 2: tert-butyl 2-(((5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl(methyl)carbamate




To a solution of 5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazole-4-carbaldehyde (88 mg, 0.36 mmol) in methanol (2 ml) was added TEA (145 mg, 1.4 mmol). ZnCl2 (7 mg, 0.05 mmol) and tert-butyl methyl(2-(methylamino)ethyl)carbamate (93 mg, 0.43 mmol) were added at rt and stirred for 45 min. NaCNBH3 (93 mg, 1.4 mmol) was added and stirred for 2 h at rt. The reaction was poured into water (100 mL) and extracted with EtOAc (50 mL×2). The combined extracts was washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by SGC using DCM:MeOH (0%-4%) as eluent to obtain tert-butyl 2-(((5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl(methyl)carbamate (50 mg, Yield 33%).
Step 3: N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (Compound 244)




A solution of tert-butyl 2-(((5-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (50 mg, 0.2 mmol) in methanolic HCl was stirred for 2 h. The resulting solution was concentrated and triturated using diethyl ether (20 mL×2) to afford N1-((3-(4-isopropoxyphenyl)-1-methyl-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (41 mg, 96%). 1H NMR (400 MHz, DMSO-d6): δ 7.89 (s, 1H), 7.38 (d, =8.4 Hz, 2H), 7.08 (d, J=8.4 Hz, 2H), 4.78-4.65 (m, 1H), 4.22-4.10 (m, 2H), 3.68 (s, 3H), 3.43-3.10 (m, 6H), 2.51 (s, 6H), 1.31 (d, J=6 Hz, 6H) ppm. LCMS (method): RT=1.55 min, m/z=317.3 [M+H]+
Compound 253
N1-((3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride




Step 1: 3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazole-4-carbaldehyde




A mixture of 3-[4-(propan-2-yloxy)phenyl]-1H-pyrazole-4-carbaldehyde hydrochloride (400 mg, 1.5 mmol) and K2CO3 (655 mg, 4.6 mmol) in acetonitrile (6 ml) was stirred at 0-5° C. for 1 hr. 1-bromo-2-methoxyethane (195 mg, 1.4 mmol) was added and the reaction mixture was stirred at ambient temperature for 48 h. The reaction was poured into water (200 mL) and extracted with EtOAc (50 mL×3). The combined extracts was washed with brine (100 mL), dried and concentrated under vacuum (360 mg, yield 72%). The crude material was used in the next step without purification.
Step 2: tert-butyl 2-(((3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate




To a solution of 3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazole-4-carbaldehyde (360 mg, 1.25 mmol) in methanol (5 ml) was added TEA (757 mg, 7.5 mmol). ZnCl2 (5 mg, 0.05 mmol) and tert-butyl methyl(2-(methylamino)ethyl)carbamate (280 mg, 1.5 mmol) were added at rt and the reaction mixture was stirred for 45 min. NaCNBH3 (87 mg, 1.2 mmol) was added and the reaction mixture was stirred for 2 h at rt. The reaction was poured into water (100 mL) and extracted with EtOAc (50 mL×2). The combined extracts was washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by prep-TLC to afford two pure regioisomers. The higher eluting band provided regioisomer tert-butyl 2-(((3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (200 mg, 35%). 1HNMR of upper spot (400 MHz, CDCl3): δ 7.74-7.52 (m, 3H), 6.95 (d, J=8.4 Hz, 2H), 4.63-4.57 (m, 1H), 4.32 (s, 2H), 3.92 (s, 1H), 3.79 (t, J=8 Hz, 2H), 3.55 (s, 1H), 3.50-3.40 (m, 2H), 3.35-3.27 (m, 1H), 2.85-2.75 (m, 5H), 2.60-2.50 (m, 1H), 2.45-2.35 (m, 2H), 2.35-2.25 (m, 2H), 1.36-1.40 (m, 6H), 1.38 (s, 9H). LCMS (method C): RT=2.04 min, m/z=461.4 [M+H]+. The lower eluting band from the prep-TLC provided the other pyrazole regioisomer (68 mg, 12%).
Step 3: N1-((3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (Compound 253)




A solution of tert-butyl 2-(((3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (200 mg, 0.43 mmol) in methanolic HCl (5 mL) was stirred at rt for 1 h. The reaction mixture was concentrated and triturated with diethyl ether (20 mL) to afford N1-((3-(4-isopropoxyphenyl)-1-(2-methoxyethyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (150 mg, 87%). 1H NMR (400 MHz, MeOH-d4): δ 8.16 (s, 1H), 7.50 (d, J=8.8 Hz, 2H), 7.06 (d, J=8.4 Hz, 2H), 4.72-4.63 (m, 1H), 4.60-4.42 (m, 2H), 4.39 (t, J=5.2 Hz, 2H), 3.81 (t, J=5.2 Hz, 2H), 3.53-3.38 (m, 1H), 3.37 (s, 6H), 2.72 (s, 6H), 1.36 (d, J=6 Hz, 6H) ppm. LCMS (method C: RT=1.68 min, m/z=361.4 [M+H]+.
Compound 254
N1-((1-cyclobutyl-3-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride




Step 1: 1-cyclobutyl-3-(4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde




A mixture of 3-[4-(propan-2-yloxy)phenyl]-1H-pyrazole-4-carbaldehyde hydrochloride (250 mg, 0.9 mmol) and K2CO3 (626 mg, 4.5 mmol) in acetonitrile (5 ml) was stirred at 0-5° C. for 1 h. Bromocyclobutane (158 mg, 1.16 mmol) was added and the reaction mixture stirred at ambient temperature for 34 h. The reaction was poured into water (200 mL) and extracted with EtOAc (50 mL×3). The combined extracts was washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by SGC using Hexane:EtOAc (0%-40%) as eluent to afford 1-cyclobutyl-3-(4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde (218 mg, Yield: 71%) as a white solid. LCMS (method C): RT=2.54 min, m/z=285.2 [M+H]+.
Step 2: tert-butyl 2-(((1-cyclobutyl-5-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl) (methyl)amino)ethyl)(methyl)carbamate




To a solution of 1-cyclobutyl-3-(4-isopropoxyphenyl)-1H-pyrazole-4-carbaldehyde (218 mg, 0.76 mmol) in methanol (2 ml) was added TEA (206 mg, 2 mmol). ZnCl2 (5 mg, 0.07 mmol) and tert-butyl methyl(2-(methylamino)ethyl)carbamate (168 mg, 0.9 mmol) were added at rt and the reaction mixture was stirred for 45 min. NaCNBH3 (186 mg, 3 mmol) was added and the reaction mixture was stirred for 2 h at rt. The reaction was poured into water (100 mL) and extracted with EtOAc (50 mL×2). The combined extracts was washed with brine (100 mL), dried, and concentrated under vacuum. The residue was purified by SGC using DCM:MeOH (0%-5%) as eluent to obtain tert-butyl 2-(((1-cyclobutyl-5-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (132 mg, Yield 38%). LCMS (method C: RT=2.18 min, m/z=457.5 [M+H]+.
Step 3: N1-((1-cyclobutyl-3-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (Compound 254)




A solution of tert-butyl 2-(((1-cyclobutyl-5-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (132 mg, 0.28 mmol) in methanolic HCl was stirred for 2 h. The solvent was evaporated to afford N1-((1-cyclobutyl-3-(4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine hydrochloride (85 mg, 66%). 1H NMR (400 MHz, DMSO-d6): δ 8.30 (s, 1H), 7.49 (d, J=8 Hz, 2H), 6.98 (d, J=8 Hz, 2H), 4.92-4.81 (m, 1H), 4.72-4.62 (m, 1H), 4.45-4.25 (m, 2H), 3.39-3.26 (m, 4H), 3.16 (s, 1H), 2.60 (s, 3H), 2.43 (s, 3H), 2.33 (s, 5H), 1.86-1.79 (m, 2H), 1.30-1.28 (d, J=5.6 Hz, 6H) ppm. LCMS (method C: RT=1.70 min, m/z=357.4 [M+H]+.
Compound 265
N1-((3-(3,5-dichloro-4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: 2,6-dichloro-4-iodophenol




A solution of 2,6-dichlorophenol (10 g, 61.35 mmol, 1.00 equiv) and NIS (27.6 g, 245.33 mmol, 2.00 equiv) in methanol (100 mL) was stirred for 1 h at room temperature. The resulting mixture was concentrated under vacuum. The residue was redissolved in 50 mL of ethyl acetate and washed with 3×30 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with petroleum ether (100%) to give 10.5 g (59%) of 2,6-dichloro-4-iodophenol as a yellow solid. 1H-NMR (300 MHz, CDCl3): δ 9.83 (s, 1H), 7.27 (s, 2H) ppm.
Step 2: 1,3-dichloro-5-iodo-2-isopropoxybenzene




A mixture of 2-iodopropane (3.7 g, 21.77 mmol, 1.00 equiv), 2,6-dichloro-4-iodophenol (5 g, 17.31 mmol, 0.80 equiv) and potassium carbonate (9.2 g, 66.09 mmol, 3.04 equiv) in N,N-dimethylformamide (50 mL) was stirred for overnight at 50° C. The resulting solution was diluted with 300 mL of ethyl acetate then washed with 3×100 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with petroleum ether (100%) to give 4.6 g (64%) of 1,3-dichloro-5-iodo-2-isopropoxybenzeneas a light yellow oil. 1H-NMR (300 MHz, CDCl3): δ 7.60 (s, 2H), 4.63-4.54 (m, 1H), 1.34 (d, J=5.1 Hz, 6H) ppm.
Step 3: 2-(3,5-dichloro-4-isopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane




A mixture of 1,3-dichloro-5-iodo-2-(propan-2-yloxy)benzene (4.6 g, 13.90 mmol, 1.00 equiv), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (3.54 g, 13.94 mmol, 1.00 equiv), Pd(PPh3)2Cl2 (975 mg, 1.39 mmol, 0.10 equiv) and potassium acetate (4.1 g, 41.78 mmol, 3.01 equiv) in 1,4-dioxane (50 mL) was stirred under nitrogen at 100° C. overnight. The resulting solution was cooled to room temperature, diluted with 100 mL of ethyl acetate and washed with 3×100 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with petroleum ether (100%) to give 4.1 g (89%) of 2-(3,5-dichloro-4-isopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a yellow oil. 1H-NMR (300 MHz, CDCl3): δ 7.35 (s, 2H), 4.68-4.56 (m, 1H), 1.40 (d, J=7.5 Hz, 6H), 1.26 (s, 12H) ppm.
Step 4: tert-butyl 2-(((3-(3,5-dichloro-4-isopropoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl(methyl)carbamate




A mixture of tert-butyl 2-([3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl(methyl)amino)ethyl N-methylcarbamate (483 mg, 1.01 mmol, 1.00 equiv), 2-[3,5-dichloro-4-(propan-2-yloxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (500 mg, 1.51 mmol, 1.50 equiv), Pd(dppf)Cl2 (74 mg, 0.10 mmol, 0.10 equiv) and potassium carbonate (418 mg, 3.00 mmol, 2.98 equiv) in 1,4-dioxane (5 mL) and water (1 mL) was stirred under nitrogen in a 8-mL vial at 100° C. overnight. The resulting solution was cooled to room temperature and diluted with 20 mL of ethyl acetate. The resulting mixture was washed with 3×10 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:1) to give 210 mg (38%) of tert-butyl N-[2-[([3-[3,5-dichloro-4-(propan-2-yloxy)phenyl]-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl)(methyl)amino]ethyl]-N-methylcarbamate as a yellow oil. 1H-NMR (300 MHz, CDCl3): δ 7.72 (s, 1H), 7.54-7.53 (m, 2H), 5.38-5.36 (m, 1H), 4.66-4.63 (m, 1H), 4.13-4.05 (m, 2H), 3.70 (s, 2H), 3.41-3.36 (m, 4H), 2.88 (s, 3H), 2.54-2.53 (m, 2H), 2.26 (s, 3H), 2.10-2.04 (m, 2H), 1.72-1.67 (m, 2H), 1.43 (s, 9H), 1.38 (d, J=6.3 Hz, 6H) ppm. LCMS (method D, ESI): RT=1.50 min, m/z=555.0 [M+H]+.
Step 5: N1-((3-(3,5-dichloro-4-isopropoxyphenyl)-1H-pyrazol-4-yl)methyl)-N1,N2-dimethylethane-1,2-diamine ditrifluoroacetic acid salts (Compound 265)




A solution of tert-butyl N-[2-[([3-[3,5-dichloro-4-(propan-2-yloxy)phenyl]-1-(oxan-2-yl)-1H-pyrazol-4-yl]methyl)(methyl)amino]ethyl]-N-methylcarbamate (210 mg, 0.38 mmol, 1.00 equiv) in 3N hydrochloric acid (20 mL) was stirred at 40° C. overnight. The resulting solution was washed with 3×10 mL of dichloromethane. The aqueous layer was concentrated under vacuum and the crude product was purified by Prep-HPLC with the following conditions (2#-Waters 2767-2(HPLC-08)): Column, Xbridge Shield RP 18, 5 um, 19*150 mm; mobile phase, water with 50 mmol CF3COOH and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to yield 106.4 mg (47%) of ([3-[3,5-dichloro-4-(propan-2-yloxy)phenyl]-1H-pyrazol-4-yl]methyl)(methyl) [2-(methylamino)ethyl]amine bis(trifluoroacetic acid) salt as a yellow oil. 1H-NMR (300 MHz, D2O): δ 7.94 (s, 1H), 7.56 (s, 2H), 4.89-4.52 (m, 1H), 4.44 (s, 2H), 3.29 (s, 4H), 2.62 (d, J=4.5 Hz, 6H), 1.32 (d, J=6.3 Hz, 6H) ppm. LCMS (method D, ESI): RT=1.28 min, m/z=371.0 [M+H]+.
Compound 279
N1-((4-(4-(2-fluorophenoxy)phenyl)-1H-pyrazol-3-yl)methyl)-N1,N2-dimethylethane-1,2-diamine




Step 1: 4-(4-(2-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carbaldehyde




A mixture of 4-iodo-1-(oxan-2-yl)-1H-pyrazole-3-carbaldehyde (227 mg, 0.74 mmol, 1.00 equiv), Pd(dppf)Cl2 (54 mg, 0.07 mmol, 0.10 equiv), potassium carbonate (304 mg, 2.20 mmol, 2.97 equiv) and 2-[4-(2-fluorophenoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (280 mg, 0.89 mmol, 1.20 equiv) in 1,4-dioxane (20 mL) and water (2 mL) was stirred under nitrogen at 100° C. overnight. The resulting mixture was cooled to room temperature and concentrated under vacuum. The residue was purified on a silica gel column eluted with 1-9% ethyl acetate in petroleum ether to give 160 mg (59%) of 4-(4-(2-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carbaldehyde as a yellow oil. 1H NMR (300 MHz, CDCl3): δ 9.91 (s, 1H), 7.91 (s, 1H), 7.60 (d, J=2.1 Hz, 2H), 7.57-7.43 (m, 1H), 7.43-7.40 (m, 3H), 7.03 (d, J=2.1 Hz, 2H), 6.14-6.10 (m, 1H), 4.00-3.99 (m, 1H), 3.68-3.66 (m, 1H), 2.30-2.28 (m, 1H), 1.98-1.93 (m, 2H), 1.56-1.55 (m, 2H), 1.19-1.17 (m, 1H) ppm. LCMS (method D, ESI): RT=1.78 min, m/z=367.0 [M+H]+.
Step 2: tert-butyl 2-(((4-(4-(2-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)(methyl)amino)ethyl(methyl)carbamate




To a solution of 4-(4-(2-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carbaldehyde (160 mg, 0.44 mmol, 1.00 equiv) and tert-butyl N-methyl-N-[2-(methylamino)ethyl]carbamate (98.6 mg, 0.52 mmol, 1.20 equiv) in 1,2-dichloroethane (20 mL) was added NaBH(OAc)3 (280 mg, 1.32 mmol, 3.03 equiv). The reaction mixture was stirred at room temperature overnight then quenched by the addition of 50 mL of water. The reaction mixture was dried over anhydrous sodium sulfate and concentrated under vacuum to give 160 mg (68%) of tert-butyl 2-(((4-(4-(2-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate as a yellow oil. LCMS (method D, ESI): RT=0.99 min, m/z=539.0 [M+H]+.
Step 3: N1-((4-(4-(2-fluorophenoxy)phenyl)-1H-pyrazol-3-yl)methyl)-N1,N2-dimethylethane-1,2-diamine (Compound 279)




A solution of tert-butyl 2-(((4-(4-(2-fluorophenoxy)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)methyl)(methyl)amino)ethyl(methyl)carbamate (160 mg, 0.30 mmol, 1.00 equiv) in 4N hydrochloric acid (10 mL) was stirred at 60° C. for 2 h. The resulting mixture was cooled to room temperature and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions (2#-Waters 2767-2(HPLC-08)): Column, XBridge Shield RP 18, 5 μm, 19×150 mm; mobile phase, water with 50 mmol CF3COOH and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm to give 53.2 mg (31%) of N1-((4-(4-(2-fluorophenoxy)phenyl)-1H-pyrazol-3-yl)methyl)-N1,N2-dimethylethane-1,2-diamine trifluoroacetate as a yellow oil. 1H NMR (300 MHz, D2O): δ 7.89 (s, 1H), 7.40-7.03 (m, 8H), 4.56 (s, 1H), 3.40-3.23 (m, 4H), 2.73 (s, 3H), 2.64 (s, 3H) ppm. LCMS (method H, ESI): RT=1.34 min, m/z=355.1 [M+H]+.
Biological Methods
PRMT1 Biochemical Assay
General Materials.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, 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 36-50 was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was purified by high-performance liquid chromatography (HPLC) to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide (SEQ ID NO.:1).
Molecular Biology:
Full-length human PRMT1 isoform 1 (NM_001536.5) transcript clone was amplified from an HEK 293 cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (DYKDDDDK) (SEQ ID NO.:2) fused directly to Met 1 of PRMT1. The amplified gene was subcloned into pFastBacI (Life Technologies) modified to encode an N-terminal GST tag and a TEV cleavage sequence (MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLP YYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSK DFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPM CLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYF QGGNS)(SEQ ID NO.:3) fused to Asp of the Flag tag of PRMT1.
Protein Expression.
Recombinant baculovirus were generated according to Bac-to-Bac kit instructions (Life Technologies). Protein over-expression was accomplished by infecting exponentially growing High Five insect cell culture at 1.5×106 cell/ml with 1:100 ratio of virus. Infections were carried out at 27° C. for 48 hours, harvested by centrifugation, and stored at −80° C. for purification.
Protein Purification.
Expressed full-length human GST-tagged PRMT1 protein was purified from cell paste by glutathione sepharose affinity chromatography after equilibration of the resin with 50 mM phosphate buffer, 200 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT1 was eluted with 50 mM Tris, 2 mM glutathione, pH 7.8, dialysed in buffer A and concentrated to 1 mg/mL. The purity of recovered protein was 73%. 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:







GST-tagged PRMT1







(SEQ ID NO.: 4)







MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL


 


EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL


 


DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH


 


PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA


 


WPLQGWQATFGGGDHPPKSDENLYFQGGNSDYKDDDDKMAAAEAANCIME


 


NFVATLANGMSLQPPLEEVSCGQAESSEKPNAEDMTSKDYYFDSYAHFGI


 


HEEMLKDEVRTLTYRNSMFHNRHLFKDKVVLDVGSGTGILCMFAAKAGAR


 


KVIGIECSSISDYAVKIVKANKLDHVVTIIKGKVEEVELPVEKVDIIISE


 


WMGYCLFYESMLNTVLYARDKWLAPDGLIFPDRATLYVTAIEDRQYKDYK


 


IHWWENVYGFDMSCIKDVAIKEPLVDVVDPKQLVTNACLIKEVDIYTVKV


 


EDLTFTSPFCLQVKRNDYVHALVAYFNIEFTRCHKRTGFSTSPESPYTHW


 


KQTVFYMEDYLTVKTGEEIFGTIGMRPNAKNNRDLDFTIDLDFKGQLCEL


 


SCSTDYRMR






General Procedure for PRMT1 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% Tween 20, 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 PRMT1, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the PRMT1 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with PRMT1 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and peptide was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: PRMT1 was 0.5 nM, 3H-SAM was 200 nM, non-radiolabeled SAM was 1.5 uM, peptide was 20 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 300 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 once 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.
PRMT6 Biochemical Assay
General Materials.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), sodium butyrate 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 36-50 was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was purified by high-performance liquid chromatography (HPLC) to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide and contained a monomethylated lysine at position 44 (SEQ ID NO.:5).
Molecular Biology:
Full-length human PRMT6 (NM_018137.2) transcript clone was amplified from an HEK 293 cDNA library, incorporating a flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:6) fused directly to Ser 2 of PRMT6 and a 3′ sequence encoding a hexa His sequence (HHHHHH) (SEQ ID NO.: 17) fused directly to Asp 375. The amplified gene was subcloned into pFastBacMam (Viva Biotech).
Protein Expression.
Recombinant baculovirus were generated according to Bac-to-Bac kit instructions (Life Technologies). Protein over-expression was accomplished by infecting exponentially growing HEK 293F cell culture at 1.3×106 cell/ml with virus (MOI=10) in the presence of 8 mM sodium butyrate. Infections were carried out at 37° C. for 48 hours, harvested by centrifugation, and stored at −80° C. for purification.
Protein Purification.
Expressed full-length human Flag- and His-tagged PRMT6 protein was purified from cell paste by NiNTA agarose affinity chromatography after equilibration of the resin with buffer containing 50 mM Tris, 300 mM NaCl, 10% glycerol, pH 7.8 (Buffer N1-A). Column was washed with 20 mM imidazole in the same buffer and Flag-PRMT6-His was eluted with 150 mM imidazole. Pooled fractions were dialysed against buffer N1-A and further purified by anti-flag M2 affinity chromatography. Flag-PRMT6-His was eluted with 200 ug/ml FLAG peptide in the same buffer. Pooled fractions were dialysed in 20 mM Tris, 150 mM NaCl, 10% glycerol and 5 mM β-mercaptoethanol, pH 7.8. The purity of recovered protein was 95%.
Predicted Translations:







Flag-PRMT6-His


(SEQ ID NO.: 7)







MDYKDDDDKSQPKKRKLESGGGGEGGEGTEEEDGAEREAALERPRRTKRE


 


RDQLYYECYSDVSVHEEMIADRVRTDAYRLGILRNWAALRGKTVLDVGAG


 


TGILSIFCAQAGARRVYAVEASAIWQQAREVVRFNGLEDRVHVLPGPVET


 


VELPEQVDAIVSEWMGYGLLHESMLSSVLHARTKWLKEGGLLLPASAELF


 


IAPISDQMLEWRLGFWSQVKQHYGVDMSCLEGFATRCLMGHSEIVVQGLS


 


GEDVLARPQRFAQLELSRAGLEQELEAGVGGRFRCSCYGSAPMHGFAIWF


 


QVTFPGGESEKPLVLSTSPFHPATHWKQALLYLNEPVQVEQDTDVSGEIT


 


LLPSRDNPRRLRVLLRYKVGDQEEKTKDFAMEDHHHHHH






General Procedure for PRMT6 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% Tween 20, 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 PRMT6, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the PRMT6 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with PRMT6 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and peptide was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: PRMT6 was 1 nM, 3H-SAM was 200 nM, non-radiolabeled SAM was 250 nM, peptide was 75 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 400 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 once 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.
PRMT8 Biochemical Assay
General Materials.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), isopropyl-β-D-thiogalactopyranoside (IPTG), 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 31-45 was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was purified by high-performance liquid chromatography (HPLC) to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Biot-Ahx-KPAIRRLARRGGVKR-amide (SEQ ID NO.:8).
Molecular Biology:
Full-length human PRMT8 (NM_019854.4) isoform 1 transcript clone was amplified from an HEK 293 cDNA library and subcloned into pGEX-4T-1 (GE Life Sciences). The resulting construct encodes an N-terminal GST tag and a thrombin cleavage sequence (MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLP YYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSK DFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPM CLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS PEF) (SEQ ID NO.:9) fused directly to Met 1 of PRMT8.
Protein Expression.
E. coli (BL21(DE3) Gold, Stratagene) made competent by the CaCl2 method were transformed with the PRMT8 construct and ampicillin selection. Protein over-expression was accomplished by growing the PRMT8 expressing E. coli clone and inducing expression with 0.3 mM IPTG at 16° C. The culture was grown for 12 hours, harvested by centrifugation, and stored at −80° C. for purification.
Protein Purification.
Expressed full-length human GST-tagged PRMT8 protein was purified from cell paste by glutathione sepharose affinity chromatography after the resin was equilibrated with 50 mM phosphate buffer, 200 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT8 was eluted with 50 mM Tris, 2 mM glutathione, pH 7.8. Pooled fractions were cleaved by thrombin (10 U) and dialysed in buffer A. GST was removed by reloading the cleaved protein sample onto glutathione sepharose column and PRMT8 was collected in the flow-through fractions. PRMT8 was purified further by ceramic hydroxyapatite chromatography. The column was washed with 50 mM phosphate buffer, 100 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH 7.8 and PRMT8 was eluted by 100 mM phosphate in the same buffer. Protein was concentrated and buffer was exchanged to 50 mM Tris, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, pH 7.8 by ultrafiltration. The purity of recovered protein was 89%.
Predicted Translations:







GST-tagged PRMT8


(SEQ ID NO.: 10)







MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL


 


EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL


 


DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH


 


PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA


 


WPLQGWQATFGGGDHPPKSDLVPRGSPEFMGMKHSSRCLLLRRKMAENAA


 


ESTEVNSPPSQPPQPVVPAKPVQCVHHVSTQPSCPGRGKMSKLLNPEEMT


 


SRDYYFDSYAHFGIHEEMLKDEVRTLTYRNSMYHNKHVFKDKVVLDVGSG


 


TGILSMFAAKAGAKKVFGIECSSISDYSEKIIKANHLDNIITIFKGKVEE


 


VELPVEKVDIIISEWMGYCLFYESMLNTVIFARDKWLKPGGLMFPDRAAL


 


YVVAIEDRQYKDFKIHWWENVYGFDMTCIRDVAMKEPLVDIVDPKQVVTN


 


ACLIKEVDIYTVKTEELSFTSAFCLQIQRNDYVHALVTYFNIEFTKCHKK


 


MGFSTAPDAPYTHWKQTVFYLEDYLTVRRGEEIYGTISMKPNAKNVRDLD


 


FTVDLDFKGQLCETSVSNDYKMR






General Procedure for PRMT8 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% Tween 20, 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 PRMT8, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the PRMT8 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with PRMT8 for 30 min at room temperature, then a cocktail (10 ul) containing 3H-SAM and peptide was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: PRMT8 was 1.5 nM, 3H-SAM was 50 nM, non-radiolabeled SAM was 550 nM, peptide was 150 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 400 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 once 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.
PRMT3 Biochemical Assay
General Materials.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), isopropyl-β-D-thiogalactopyranoside (IPTG), 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 containing the classic RMT substrate motif was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was purified by high-performance liquid chromatography (HPLC) to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Biot-Ahx-GGRGGFGGRGGFGGRGGFG-amide (SEQ ID NO.:11).
Molecular Biology:
Full-length human PRMT3 (NM_005788.3) isoform 1 transcript clone was amplified from an HEK 293 cDNA library and subcloned into pGEX-KG (GE Life Sciences). The resulting construct encodes an N-terminal GST tag and a thrombin cleavage sequence (MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLP YYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSK DFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPM CLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS) (SEQ ID NO.: 12) fused directly to Cys 2 of PRMT3.
Protein Expression.
E. coli (BL21(DE3) Gold, Stratagene) made competent by the CaCl2 method were transformed with the PRMT3 construct and ampicillin selection. Protein over-expression was accomplished by growing the PRMT3 expressing E. coli clone and inducing expression with 0.3 mM IPTG at 16° C. The culture was grown for 12 hours, harvested by centrifugation, and stored at −80° C. for purification.
Protein Purification.
Expressed full-length human GST-tagged PRMT3 protein was purified from cell paste by glutathione sepharose affinity chromatography after equilibration of the resin with 50 mM phosphate buffer, 200 mM NaCl, 5% glycerol, 1 mM EDTA, 5 mM β-mercaptoethanol, pH6.5 (Buffer A). GST-tagged PRMT3 was eluted with 50 mM Tris, 2 mM glutathione, pH 7.1 and 50 mM Tris, 20 mM glutathione, pH 7.1. Pooled fractions were dialysed in 20 mM Tris, 50 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, pH7.5 (Buffer B) and applied to a Q Sepharose Fast Flow column. GST-tagged PRMT3 was eluted by 500 mM NaCl in buffer B. Pooled fractions were dialyzed in 25 mM phosphate buffer, 100 mM NaCl, 5% glycerol, 2 mM DTT, pH 6.8 (Buffer C) and loaded on to a ceramic hydroxyapatite column. GST-tagged PRMT3 eluted with 25-400 mM phosphate in buffer C. Protein was concentrated and buffer was exchanged to 20 mM Tris, 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH7.8 by ultrafiltration. The purity of recovered protein was 70%.
Predicted Translations:







GST-tagged PRMT3







(SEQ ID NO.: 13)







MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL


 


EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL


 


DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH


 


PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA


 


WPLQGWQATFGGGDHPPKSDLVPRGSCSLASGATGGRGAVENEEDLPELS


 


DSGDEAAWEDEDDADLPHGKQQTPCLFCNRLFTSAEETFSHCKSEHQFNI


 


DSMVHKHGLEFYGYIKLINFIRLKNPTVEYMNSIYNPVPWEKEEYLKPVL


 


EDDLLLQFDVEDLYEPVSVPFSYPNGLSENTSVVEKLKHMEARALSAEAA


 


LARAREDLQKMKQFAQDFVMHTDVRTCSSSTSVIADLQEDEDGVYFSSYG


 


HYGIHEEMLKDKIRTESYRDFIYQNPHIFKDKVVLDVGCGTGILSMFAAK


 


AGAKKVLGVDQSEILYQAMDIIRLNKLEDTITLIKGKIEEVHLPVEKVDV


 


IISEWMGYFLLFESMLDSVLYAKNKYLAKGGSVYPDICTISLVAVSDVNK


 


HADRIAFWDDVYGFKMSCMKKAVIPEAVVEVLDPKTLISEPCGIKHIDCH


 


TTSISDLEFSSDFTLKITRTSMCTAIAGYFDIYFEKNCHNRVVFSTGPQS


 


TKTHWKQTVFLLEKPFSVKAGEALKGKVTVHKNKKDPRSLTVTLTLNNST


 


QTYGLQ






General Procedure for PRMT3 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% Tween 20, 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 PRMT3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the PRMT3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with PRMT3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and peptide was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: PRMT3 was 0.5 nM, 3H-SAM was 100 nM, non-radiolabeled SAM was 1.8 uM, peptide was 330 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of potassium chloride (10 ul) to a final concentration of 100 mM. 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 once 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.
CARM1 Biochemical Assay
General Materials.
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), sodium butyrate 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 H3 residues 16-30 was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was purified by high-performance liquid chromatography (HPLC) to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Biot-Ahx-PRKQLATKAARKSAP-amide and contained a monomethylated arginine at position 26 (SEQ ID NO.: 14).
Molecular Biology:
Human CARM1 (PRMT4) (NM_199141.1) transcript clone was amplified from an HEK 293 cDNA library, incorporating a flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:6) fused directly to Ala 2 of CARM1 and 3′ sequence encoding a hexa His sequence (EGHHHHHH) (SEQ ID NO.: 15) fused directly to Ser 608. The gene sequence encoding isoforml containing a deletion of amino acids 539-561 was amplified subsequently and subcloned into pFastBacMam (Viva Biotech).
Protein Expression.
Recombinant baculovirus were generated according to Bac-to-Bac kit instructions (Life Technologies). Protein over-expression was accomplished by infecting exponentially growing HEK 293F cell culture at 1.3×106 cell/ml with virus (MOI=10) in the presence of 8 mM sodium butyrate. Infections were carried out at 37° C. for 48 hours, harvested by centrifugation, and stored at −80° C. for purification.
Protein Purification.
Expressed full-length human Flag- and His-tagged CARM1 protein was purified from cell paste by anti-flag M2 affinity chromatography with resin equilibrated with buffer containing 20 mM Tris, 150 mM NaCl, 5% glycerol, pH 7.8. Column was washed with 500 mM NaCl in buffer A and Flag-CARM1-His was eluted with 200 ug/ml FLAG peptide in buffer A. Pooled fractions were dialyzed in 20 mM Tris, 150 mM NaCl, 5% glycerol and 1 mM DTT, pH 7.8. The purity of recovered protein was 94.
Predicted Translations:







Flag-CARM1-His







(SEQ ID NO.: 16)







MDYKDDDDKAAAAAAVGPGAGGAGSAVPGGAGPCATVSVFPGARLLTIGD


 


ANGEIQRHAEQQALRLEVRAGPDSAGIALYSHEDVCVFKCSVSRETECSR


 


VGKQSFIITLGCNSVLIQFATPNDFCSFYNILKTCRGHTLERSVFSERTE


 


ESSAVQYFQFYGYLSQQQNMMQDYVRTGTYQRAILQNHTDFKDKIVLDVG


 


CGSGILSFFAAQAGARKIYAVEASTMAQHAEVLVKSNNLTDRIVVIPGKV


 


EEVSLPEQVDIIISEPMGYMLFNERMLESYLHAKKYLKPSGNMFPTIGDV


 


HLAPFTDEQLYMEQFTKANFWYQPSFHGVDLSALRGAAVDEYFRQPVVDT


 


FDIRILMAKSVKYTVNFLEAKEGDLHRIEIPFKFHMLHSGLVHGLAFWFD


 


VAFIGSIMTVWLSTAPTEPLTHWYQVRCLFQSPLFAKAGDTLSGTCLLIA


 


NKRQSYDISIVAQVDQTGSKSSNLLDLKNPFFRYTGTTPSPPPGSHYTSP


 


SENMWNTGSTYNLSSGMAVAGMPTAYDLSSVIASGSSVGHNNLIPLGSSG


 


AQGSGGGSTSAHYAVNSQFTMGGPAISMASPMSIPTNTMHYGSEGHHHHH


 


H






General Procedure for CARM1 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% Tween 20, 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 CARM1, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the CARM1 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with CARM1 for 30 min at room temperature, then a cocktail (10 ul) containing 3H-SAM and peptide was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: CARM1 was 0.25 nM, 3H-SAM was 30 nM, peptide was 250 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 300 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 once 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.







TABLE 2







Biochemical IC50












Cmpd No.
PRMT1
PRMT6
PRMT8
PRMT3
CARM1















1
A
A
B
C
C


2
A
A
B
D
—


3
A
A
B
E
—


4
B
A
C
E
B


5
C
B
D
E
E


6
D
B
—
E
E


7
B
A
D
E
C


8
C
B
D
E
D


9
—
—
B
D
C


10
—
—
B
E
C


11
—
—
E
E
E


12
—
B
E
E
D


13
C
B
E
E
C


14
B
A
C
E
B


15
D
B
E
E
D


16
C
A
E
E
D


17
A
A
A
C
C


18
A
A
B
D
B


19
A
A
B
—
B


20
A
A
A
D
B


21
A
A
B
C
C


22
B
A
C
E
C


23
B
B
D
E
C


24
A
A
B
E
C


25
A
A
B
E
C


26
B
A
C
E
C


27
A
A
B
E
C


28
A
A
A
B
B


29
B
A
C
E
D


30
D
B
E
E
D


31
B
B
D
E
C


32
—
A
C
C
C


33
B
A
C
C
C


34
—
A
B
E
C


35
—
B
E
E
E


36
—
C
E
E
E


37
A
A
A
B
A


38
C
B
D
E
E


39
—
A
B
E
C


40
B
B
D
E
E


41
—
D
E
E
E


42
—
A
C
C
C


43
B
A
C
E
D


44
—
A
B
D
C


45
A
A
B
D
A


46
C
D
E
E
E


47
—
A
A
B
A


48
—
A
C
E
D


49
B
A
B
D
D


50
B
A
C
E
D


51
A
A
B
D
B


52
A
A
B
D
C


53
D
B
E
E
E


54
B
B
D
E
E


55
B
A
D
E
D


56
D
C
E
E
E


57
A
A
B
D
C


58
A
A
B
D
B


59
D
B
D
E
D


60
B
A
C
E
E


61
E
E
E
E
E


62
A
A
B
D
B


63
C
C
D
E
E


64
E
C
E
E
E


65
C
B
D
E
D


66
C
C
D
E
D


67
A
A
A
D
B


68
A
A
B
D
B


69
A
A
C
E
D


70
B
A
C
E
D


71
A
A
B
E
B


72
B
A
D
E
D


73
B
A
B
E
C


74
A
A
B
E
D


75
B
C
C
E
E


76
D
C
E
E
E


77
B
B
C
D
D


78
B
A
C
E
E


79
E
E
E
E
E


80
E
E
E
E
E


81
B
B
D
E
C


82
A
B
C
C
D


83
A
A
B
E
C


84
E
E
E
E
E


85
B
B
D
E
E


86
A
A
B
D
D


87
B
B
D
E
D


88
B
A
C
D
D


89
A
A
C
D
D


90
A
A
C
D
D


91
B
B
D
E
E


92
A
A
B
D
B


93
A
A
A
D
C


94
A
A
B
D
B


95
A
A
B
C
B


96
A
A
B
D
A


97
A
A
A
D
B


98
A
A
B
B
B


99
A
A
C
E
D


100
A
A
B
D
D


101
A
B
B
E
D


102
A
A
B
D
C


103
A
A
B
E
C


104
A
A
B
E
C


105
A
A
B
D
D


106
B
A
D
E
E


107
A
A
B
D
C


108
A
A
C
D
C


109
B
B
C
E
D


110
A
A
B
D
D


111
A
A
B
D
B


112
A
A
B
D
C


113
C
B
D
E
E


114
A
A
A
D
B


115
A
A
C
D
D


116
A
A
B
C
C


117
A
A
A
C
B


118
A
A
B
C
B


119
A
A
B
D
B


120
B
A
D
E
D


121
A
A
B
C
B


122
A
A
B
D
B


123
A
A
B
D
C


124
A
A
B
C
B


125
A
A
B
D
C


126
A
A
B
C
B


127
A
A
B
D
C


128
D
C
E
E
E


129
B
A
B
D
C


130
B
A
B
D
C


131
A
A
B
D
C


132
A
A
B
D
C


133
A
A
B
D
D


134
A
A
B
D
C


135
A
A
C
E
E


136
A
A
B
E
C


137
A
A
B
D
B


138
A
A
B
E
C


139
E
E
E
E
E


140
B
C
D
D
E


141
A
A
B
D
C


142
A
A
B
D
C


143
B
B
C
D
D


144
A
A
B
E
D


145
A
A
B
D
D


146
A
A
C
D
D


147
C
A
E
E
E


148
B
A
B
E
E


149
A
A
B
D
C


150
A
A
B
E
C


151
B
B
C
E
D


152
A
A
B
D
D


153
A
A
B
D
C


154
B
B
D
E
E


155
A
A
B
D
E


156
B
A
C
E
D


157
C
B
D
E
E


158
C
B
E
E
E


159
A
A
B
E
E


160
B
B
D
E
E


161
B
A
C
E
D


162
A
A
B
D
B


163
B
A
C
E
—


164
B
A
C
E
—


165
A
A
A
C
—


166
A
A
B
D
—


167
A
A
B
D
—


168
A
A
B
D
—


169
B
A
C
E
—


170
A
A
B
D
—


171
B
C
C
E
—


172
B
A
D
D
—


173
B
A
D
—
—


174
A
A
B
—
—


175
A
A
—
—
—


176
A
A
—
—
—


177
A
A
—
—
—


178
A
A
—
—
—


179
A
A
—
—
—


180
B
A
D
—
—


181
A
A
—
—
—


182
A
A
B
—
—


183
C
B
D
—
—


184
C
C
D
—
—


185
C
B
D
—
—


186
B
A
B
—
—


187
B
A
C
—
—


188
B
A
C
—
—


189
B
A
C
—
—


190
B
A
C
—
—


191
B
B
D
—
—


192
B
A
C
—
—


193
A
B
B
—
—


194
A
A
B
—
—


195
A
A
B
—
—


196
B
A
C
—
—


197
B
A
C
—
—


198
C
B
E
—
—


199
A
B
B
—
—


200
A
A
A
—
—


201
A
A
B
—
—


202
A
A
B
—
—


203
A
A
B
—
—


204
A
A
A
—
—


205
A
A
B
—
—


206
A
A
B
—
—


207
A
A
B
—
—


208
A
A
B
—
—


209
A
A
B
—
—


210
A
A
B
—
—


211
A
A
B
—
—


212
A
A
A
—
—


213
A
A
—
—
B


214
A
B
—
—
B


215
A
B
—
—
B


216
A
A
—
—
B


217
A
A
—
—
B


218
A
B
—
—
B


219
A
A
—
—
B


220
A
A
B
—
—


221
A
A
B
—
—


222
C
C
D
—
—


223
E
E
—
—
—


224
A
A
B
—
—


225
A
A
A
—
—


226
A
A
D
—
—


227
A
A
B
—
—


228
A
A
B
—
—


229
A
B
—
—
—


230
A
A
—
—
—


231
A
A
—
—
—


232
A
A
—
—
—


233
A
A
—
—
—


234
A
A
—
—
—


235
A
B
—
—
—


236
A
A
—
—
—


237
A
A
—
—
—


238
A
A
—
—
—


239
A
A
—
—
—


240
A
A
—
—
—


241
A
A
—
—
—


242
B
C
—
—
—


243
A
B
—
—
B


244
A
A
—
—
A


245
A
B
—
—
—


246
A
B
—
—
C


247
B
B
—
—
C


248
B
B
—
—
D


249
B
B
C
—
D


250
B
D
C
—
E


251
B
C
—
—
C


252
A
B
—
—
B


253
B
B
—
—
C


254
A
B
—
—
B


255
A
A
—
—
C


256
B
B
C
—
B


257
B
B
—
—
E


258
A
B
—
—
C


259
A
A
—
—
—


260
A
B
—
—
—


261
A
A
—
—
—


262
A
A
—
—
—


263
A
A
—
—
—


264
A
A
—
—
—


265
A
A
—
—
—


266
B
A
—
—
—


267
A
A
—
—
—


268
A
A
—
—
—


269
A
A
—
—
—


270
A
A
—
—
—


271
A
B
—
—
—


272
B
C
—
—
—


273
A
A
—
—
—


274
A
A
—
—
—


275
A
A
—
—
—


276
E
C
—
—
—


277
A
A
—
—
—


278
A
A
—
—
—


279
A
A
—
—
—


280
A
A
—
—
—


281
A
B
—
—
—


282
A
A
—
—
—


283
A
A
A
—
—


284
A
A
A
—
—


285
A
A
A
—
—


286
A
A
A
—
—


287
A
A
A
—
—


288
A
A
A
—
—


289
A
A
A
—
—


290
A
A
A
—
—


291
A
A
A
—
—


292
A
A
A
—
—


293
A
A
A
—
—


294
A
A
A
—
—


295
A
A
A
—
—


296
A
A
A
—
—


297
A
A
A
—
—


298
A
A
A
—
—


299
A
A
A
—
—


300
A
B
A
—
—


301
A
A
A
—
—


302
A
A
B
—
—


303
A
A
A
—
—


304
A
A
A
—
—


305
A
A
A
—
—


306
A
A
A
—
—


307
A
A
A
—
—


308
A
A
A
—
—


309
A
A
A
—
—


310
A
A
A
—
—


311
A
A
A
—
—


312
A
A
A
—
—


313
A
B
A
—
—


314
A
A
B
—
—


315
A
A
B
—
—


316
A
A
A
—
—





“—” indicates on data provided.


For Table 2, “A” indicates an IC50 ≦0.100 μM, “B” indicates an IC50 of 0.101-1.00 μM, “C” indicates an IC50 of 1.01-3.00 μM, “D” indicates an IC50 of 3.01-10 μM, and IC50 ≧10.01 μM.













TABLE 2a







Biochemical IC50 (Numerical, μM)*












Cmpd No.
PRMT1
PRMT6
PRMT8
PRMT3
CARM1















1
0.01831
0.01103
0.25139
2.64514
1.34918


2
0.0576
0.0961
0.50705
4.47947
—


3
0.0581
0.027
0.95966
25.54303
—


4
0.15222
0.07611
2.42787
>10
0.74151


5
1.18543
0.46706
4.2012
>10
>10


6
3.45579
0.31168
—
>50
>10


7
0.26524
0.03832
3.24288
26.00988
2.66419


8
1.17848
0.28691
7.20411
>50
3.46464


9
—
—
0.39588
3.15985
2.8052


10
—
—
0.69261
13.37611
1.18758


11
—
—
>10
>10
>10


12
—
0.19141
>10
>50
4.84095


13
1.4988
0.27086
>10
>10
2.24194


14
0.12272
0.02774
1.37716
23.32833
0.96977


15
4.38091
0.52599
>10
>10
3.40172


16
2.07127
0.05786
>10
>50
6.73823


17
0.01378
0.01276
0.08914
2.78966
1.56113


18
0.03623
0.01363
0.48267
7.43065
0.65622


19
0.00581
0.0106
0.65007
—
0.10766


20
0.00412
0.00779
0.05768
3.53874
0.27801


21
0.05786
0.01224
0.52027
2.29825
2.09431


22
0.16634
0.01185
2.23592
>10
1.37373


23
0.19422
0.18257
3.2898
>10
1.76379


24
0.0856
0.02621
0.88819
>10
1.21003


25
0.02091
0.0109
0.54961
>10
1.56333


26
0.19713
0.02252
1.50473
>10
2.34333


27
0.09773
0.01852
0.71238
>10
1.70988


28
0.01111
0.0091
0.09551
0.20236
0.33668


29
0.17797
0.06172
2.31427
>10
6.3012


30
3.55551
0.56481
>50
>10
3.68313


31
0.77916
0.10962
6.31461
>10
1.83668


32
—
0.0222
1.53202
2.75966
1.13394


33
0.15263
0.03791
1.43231
1.88567
1.37801


34
—
0.01129
0.77282
>10
1.37348


35
—
0.45665
24.81929
>10
>10


36
—
2.43855
22.43451
>10
>10


37
0.00274
0.00329
0.05758
0.24996
0.01519


38
1.82987
0.3524
9.9001
>10
>10


39
—
0.03785
0.59599
>10
2.10972


40
0.70049
0.67971
6.76377
>10
>10


41
—
8.1435
40.02827
>10
>10


42
—
0.04762
1.54014
2.54801
2.23662


43
0.33759
0.05194
2.26253
>10
4.95998


44
—
0.03945
0.52204
3.43562
1.08566


45
0.00834
0.00754
0.3083
5.5337
0.04292


46
2.50134
3.22498
11.91838
>10
>10


47
—
0.00476
0.09084
0.52989
0.02831


48
—
0.04406
2.16658
>10
5.61754


49
0.16497
0.03927
0.74859
4.4269
5.03815


50
0.14931
0.04277
1.65935
>10
4.32122


51
0.0129
0.00995
0.18427
5.00886
0.62499


52
0.01263
0.00892
0.21926
5.24549
1.50058


53
3.1645
0.96808
12.84662
>10
>10


54
0.56921
0.1816
7.49517
>10
>10


55
0.19043
0.08697
3.18794
>10
4.19952


56
5.84573
1.90486
34.8817
>10
>10


57
0.00707
0.01032
0.19587
3.34757
1.31319


58
0.00573
0.00614
0.1701
3.16393
0.5817


59
5.85844
0.62727
8.86177
>10
4.5853


60
0.17018
0.05324
1.53515
>10
>10


61
>10
>10
>10
>10
>10


62
0.01393
0.00787
0.36166
4.13469
0.6721


63
1.54013
1.15196
8.03029
>10
>10


64
>10
1.57353
>10
>10
>10


65
1.94161
0.42255
8.6118
>10
8.92836


66
2.41119
1.03695
8.74785
>10
3.79101


67
0.00339
0.00723
0.0324
4.57692
0.24679


68
0.00604
0.0067
0.40352
4.36197
0.80182


69
0.0511
0.03422
2.04439
>10
3.916


70
0.20028
0.01891
2.48706
>10
3.63056


71
0.01879
0.00816
0.34733
>10
0.71505


72
0.27837
0.06292
3.73012
>10
7.03597


73
0.12624
0.02592
0.95863
>10
2.90415


74
0.0149
0.03781
0.18504
>10
3.53476


75
0.61625
1.63714
2.50738
>10
>10


76
6.39224
2.73221
>10
>10
>10


77
0.21459
0.10117
1.65896
6.28526
4.09191


78
0.32816
0.06649
1.49223
>10
>10


79
>10
>10
>10
>10
>10


80
>10
>10
>10
>10
>10


81
0.43483
0.10456
3.45369
>10
1.62937


82
0.09606
0.12919
2.46984
1.59224
9.01462


83
0.03027
0.05278
0.7102
>10
1.13312


84
>10
>10
>10
>10
>10


85
0.23373
0.70087
4.2587
>10
>10


86
0.02403
0.00916
0.74024
5.82395
4.19169


87
0.17182
0.10263
3.11178
>10
4.37776


88
0.12774
0.02904
1.88507
8.0799
5.21538


89
0.06736
0.01346
1.72983
9.564
5.97689


90
0.04135
0.01017
1.38237
6.96934
6.92214


91
0.27231
0.23691
6.90002
>10
>10


92
0.00569
0.00645
0.15227
3.13932
0.68957


93
0.00464
0.01069
0.07614
3.61276
1.83222


94
0.00797
0.00802
0.19824
5.37344
0.87319


95
0.00784
0.00561
0.20654
2.38746
0.39208


96
0.00441
0.00709
0.17083
3.13505
0.07655


97
0.0026
0.00735
0.08898
3.50253
0.51073


98
0.00584
0.01537
0.15629
0.68358
0.43869


99
0.09023
0.0279
2.19956
>10
7.60576


100
0.012
0.01123
0.29003
7.25842
4.13226


101
0.02031
0.1358
0.57
>10
4.76468


102
0.01134
0.00902
0.31099
6.15828
1.06465


103
0.01024
0.01698
0.36552
>10
1.48038


104
0.01565
0.04531
0.30078
>10
1.96452


105
0.00885
0.05558
0.12969
9.83012
3.05664


106
0.33579
0.04978
3.11349
>10
>10


107
0.04407
0.01497
0.91206
8.00064
2.85799


108
0.03129
0.00949
1.00418
9.91363
1.53088


109
0.24429
0.21712
2.07608
>10
5.21438


110
0.02163
0.01652
0.53964
7.20857
8.76846


111
0.01886
0.01177
0.79894
5.3912
0.45843


112
0.08097
0.08474
0.33455
3.68077
2.43503


113
1.48906
0.59428
8.51814
>10
>10


114
0.00575
0.0162
0.04752
3.20459
0.33198


115
0.04605
0.02545
1.10242
8.32267
3.53002


116
0.00484
0.0073
0.12385
2.78172
1.22069


117
0.00576
0.00487
0.08784
2.30296
0.6022


118
0.00294
0.00318
0.13578
1.0372
0.11351


119
0.01615
0.08801
0.33586
9.60309
0.44119


120
0.29113
0.06585
4.12854
>10
8.14576


121
0.01066
0.00748
0.21754
2.87757
0.21568


122
0.02428
0.00899
0.5481
5.01068
0.40543


123
0.0439
0.01713
0.53156
3.92469
1.86629


124
0.0137
0.00715
0.29427
2.21725
0.54524


125
0.00838
0.00665
0.30784
4.32487
1.43584


126
0.00681
0.00494
0.23169
2.55169
0.10224


127
0.04614
0.01407
0.54787
5.64979
1.1152


128
6.50204
2.36637
>10
>10
>10


129
0.12092
0.05276
0.98983
9.91198
2.4226


130
0.13548
0.06335
0.64527
6.45834
2.41349


131
0.02964
0.01211
0.35966
3.5716
1.18201


132
0.02388
0.00991
0.36461
3.98181
2.11467


133
0.02479
0.01014
0.14155
4.09836
4.27211


134
0.01083
0.01077
0.25079
5.4422
2.6012


135
0.04815
0.02894
1.31801
>10
>10


136
0.02746
0.01285
0.49618
>10
2.07478


137
0.01919
0.00842
0.65634
7.57844
0.84923


138
0.02122
0.04454
0.13114
>10
2.69989


139
>10
>10
>10
>10
>10


140
0.39891
3.00992
8.2652
5.21145
>10


141
0.00816
0.00779
0.30465
3.47077
1.37423


142
0.02973
0.01622
0.27273
4.8291
1.29051


143
0.11624
0.10723
1.74997
8.37365
5.84815


144
0.00579
0.04909
0.17086
>10
4.61685


145
0.02012
0.05996
0.16673
7.18055
4.91912


146
0.04631
0.01254
1.1227
9.00035
5.76826


147
2.86453
0.05359
>10
>10
>10


148
0.12075
0.06818
0.8088
>10
>10


149
0.06099
0.06255
0.91041
9.0201
1.29874


150
0.07149
0.02897
0.5662
>10
2.41844


151
0.12212
0.11242
1.3651
>10
8.3151


152
0.04716
0.02267
0.98268
9.62799
6.87419


153
0.02182
0.01163
0.13197
4.0976
2.06154


154
0.66025
0.37535
6.16843
>10
>10


155
0.05992
0.02226
0.97733
8.55798
>10


156
0.24267
0.04312
1.70766
>10
6.74133


157
1.54228
0.50401
5.82164
>10
>10


158
2.32659
0.1153
>10
>10
>10


159
0.07186
0.02731
0.87907
>10
>10


160
0.16745
0.19099
3.0129
>10
>10


161
0.18732
0.09645
2.09976
>10
6.13327


162
0.01699
0.00771
0.27308
3.51996
0.61151


163
0.11526
0.06259
1.43324
>10
—


164
0.10686
0.04179
2.18379
>10
—


165
0.00536
0.01279
0.07894
1.73131
—


166
0.00727
0.01558
0.1907
3.44937
—


167
0.00928
0.01741
0.28412
3.73808
—


168
0.00434
0.01098
0.12828
3.911
—


169
0.30722
0.08498
2.99169
>10
—


170
0.01144
0.05928
0.16008
7.33108
—


171
0.92864
2.41966
2.62818
>10
—


172
0.22727
0.02635
3.263
3.41202
—


173
0.16749
0.01495
3.07931
—
—


174
0.01193
0.01503
0.22833
—
—


175
0.0105
0.01246
—
—
—


176
0.01155
0.06726
—
—
—


177
0.01003
0.03419
—
—
—


178
0.00828
0.00722
—
—
—


179
0.01733
0.01344
—
—
—


180
0.64771
0.02053
5.78964
—
—


181
0.00496
0.0173
—
—
—


182
0.00475
0.00548
0.271
—
—


183
2.41503
0.26045
4.81626
—
—


184
1.60305
2.38214
3.03468
—
—


185
1.1231
0.4067
4.36969
—
—


186
0.11737
0.01329
0.87342
—
—


187
0.14629
0.07758
1.69855
—
—


188
0.16261
0.03743
2.88541
—
—


189
0.47511
0.02946
2.98732
—
—


190
0.38743
0.0201
1.15388
—
—


191
0.65535
0.4354
4.10845
—
—


192
0.12132
0.03459
2.00675
—
—


193
0.06338
0.24682
0.84543
—
—


194
0.01421
0.01535
0.37413
—
—


195
0.01143
0.00924
0.40156
—
—


196
0.2104
0.02866
2.57875
—
—


197
0.678
0.06717
2.10351
—
—


198
2.17311
0.32166
>10
—
—


199
0.01507
0.10319
0.4591
—
—


200
0.00252
0.00229
0.05717
—
—


201
0.00558
0.00924
0.15845
—
—


202
0.00427
0.0029
0.18652
—
—


203
0.00428
0.00242
0.18504
—
—


204
0.00315
0.00114
0.06928
—
—


205
0.00244
0.00153
0.286
—
—


206
0.00707
0.02142
0.34909
—
—


207
0.00616
0.0033
0.62665
—
—


208
0.00416
0.00293
0.27917
—
—


209
0.00529
0.00405
0.4833
—
—


210
0.00879
0.01613
0.18216
—
—


211
0.00608
0.02051
0.11656
—
—


212
0.00646
0.00907
0.04034
—
—


213
0.0266
0.08211
—
—
0.20009


214
0.0519
0.12362
—
—
0.16823


215
0.08221
0.32294
—
—
0.29918


216
0.02115
0.02769
—
—
0.28518


217
0.01869
0.04211
—
—
0.61998


218
0.05836
0.13173
—
—
0.41355


219
0.01842
0.03077
—
—
0.14239


220
0.01393
0.02313
0.32142
—
—


221
0.00647
0.00672
0.32833
—
—


222
1.26137
1.15907
8.88239
—
—


223
>10
>10
—
—
—


224
0.00251
0.00198
0.34598
—
—


225
0.00319
0.00133
0.03582
—
—


226
0.02681
0.01376
7.25064
—
—


227
0.02432
0.01149
0.65987
—
—


228
0.00555
0.00822
0.31641
—
—


229
0.05968
0.20115
—
—
—


230
0.00343
0.00219
—
—
—


231
0.00408
0.00297
—
—
—


232
0.02503
0.01656
—
—
—


233
0.02935
0.0182
—
—
—


234
0.00775
0.00474
—
—
—


235
0.00835
0.17592
—
—
—


236
0.00917
0.01473
—
—
—


237
0.00206
0.00213
—
—
—


238
0.03507
0.01703
—
—
—


239
0.00251
0.00101
—
—
—


240
0.03816
0.01458
—
—
—


241
0.05216
0.03408
—
—
—


242
0.37569
>1
—
—
—


243
0.01772
0.28614
—
—
0.30135


244
0.00868
0.04508
—
—
0.09251


245
0.03527
0.2235
—
—
—


246
0.06151
0.31824
—
—
2.43357


247
0.14747
0.80504
—
—
2.82426


248
0.21512
0.83133
—
—
3.75283


249
0.10939
0.42838
1.50481
—
9.62827


250
0.73659
4.2591
2.53587
—
>10


251
0.27479
2.37302
—
—
2.97719


252
0.02584
0.10207
—
—
0.53136


253
0.11293
0.74825
—
—
2.26375


254
0.02933
0.20142
—
—
0.4129


255
0.01682
0.07514
—
—
1.16976


256
0.13247
0.92061
2.64726
—
0.57071


257
0.1346
0.21282
—
—
>10


258
0.04391
0.1728
—
—
1.70385


259
0.00132
0.0008
—
—
—


260
0.02393
0.24335
—
—
—


261
0.00294
0.00244
—
—
—


262
0.00563
0.00146
—
—
—


263
0.00435
0.00192
—
—
—


264
0.02588
0.00318
—
—
—


265
0.00567
0.01565
—
—
—


266
0.17571
0.033
—
—
—


267
0.00347
0.00156
—
—
—


268
0.00458
0.002
—
—
—


269
0.05954
0.00273
—
—
—


270
0.00279
0.00124
—
—
—


271
0.05241
0.86351
—
—
—


272
0.15578
>1
—
—
—


273
0.00856
0.03163
—
—
—


274
0.01927
0.00757
—
—
—


275
0.00863
0.02091
—
—
—


276
>1
>1
—
—
—


277
0.02018
0.00166
—
—
—


278
0.04963
0.00517
—
—
—


279
0.00346
0.00187
—
—
—


280
0.0043
0.01018
—
—
—


281
0.01123
0.2409
—
—
—


282
0.00221
0.00475
—
—
—


283
0.00665
0.02556
0.04283
—
—


284
0.01334
0.04048
0.09016
—
—


285
0.01479
0.03324
0.06917
—
—


286
0.02652
0.03205
0.05533
—
—


287
0.01276
0.02477
0.02159
—
—


288
0.00897
0.01429
0.01422
—
—


289
0.02114
0.03647
0.04601
—
—


290
0.00828
0.01149
0.01061
—
—


291
0.01559
0.02773
0.0254
—
—


292
0.02726
0.08318
0.00852
—
—


293
0.01351
0.02884
0.02805
—
—


294
0.01272
0.02463
0.02749
—
—


295
0.02867
0.06486
0.03365
—
—


296
0.02878
0.03296
0.05762
—
—


297
0.00337
0.01206
0.00668
—
—


298
0.00787
0.0269
0.01634
—
—


299
0.01975
0.0159
0.05503
—
—


300
0.0279
0.17654
0.06471
—
—


301
0.00643
0.02529
0.02353
—
—


302
0.03344
0.04974
0.12258
—
—


303
0.02794
0.02397
0.05468
—
—


304
0.00659
0.02411
0.02244
—
—


305
0.00636
0.0276
0.01829
—
—


306
0.0071
0.02326
0.01852
—
—


307
0.00589
0.01971
0.01404
—
—


308
0.00707
0.01307
0.0141
—
—


309
0.0067
0.0152
0.01296
—
—


310
0.0145
0.03239
0.01697
—
—


311
0.0216
0.05032
0.05092
—
—


312
0.01162
0.01946
0.09085
—
—


313
0.0349
0.11317
0.06012
—
—


314
0.01823
0.03858
0.10988
—
—


315
0.02348
0.01062
0.1081
—
—


316
0.0175
0.09669
0.04303
—
—





*For Table 2a, numerical values represent data from a single experiment or an average of multiple experiments.






RKO Methylation Assay

RKO adherent cells were purchased from ATCC (American Type Culture Collection), Manassas, Va., USA. DMEM/Glutamax medium, penicillin-streptomycin, heat inactivated fetal bovine serum, 0.05% trypsin 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. Mono-methyl arginine antibody was purchased from Cell Signaling Technology, Danvers, Mass., USA. Methanol was purchased from VWR, Franklin, Mass., USA. 10% Tween 20 was purchased from KPL, Inc., Gaithersburg, Md., USA. DRAQ5 was purchased from Biostatus Limited, Leicestershire, UK.
RKO adherent cells were maintained in growth medium (DMEM/Glutamax medium 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 Mono-Methyl Arginine and DNA Content.
RKO cells were seeded in assay medium at a concentration of 20,000 cells per mL to a poly-D-lysine coated 384 well culture plate (BD Biosciences 356697) with 50 μL per well. Compound (100 nL) from a 96-well source plate was added directly to 384 well cell plate. Plates were incubated at 37° C., 5% CO2 for 72 hours. After three days of incubation, plates were brought to room temperature outside of the incubator for ten minutes and blotted on paper towels to remove cell media. 50 μL of ice cold 100% methanol was added directly to each well and incubated for 30 min at room temperature. After 30 min, plates were transferred to a Biotek EL406 plate washer and washed 2 times with 100 μL per well of wash buffer (1×PBS). Next 60 μL per well of Odyssey blocking buffer (Odyssey Buffer with 0.1% Tween 20 (v/v)) 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 (mono-methyl arginine diluted 1:200 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 2 times 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:




  (
  
    
      monomethyl
      ⁢
      
        
      
      ⁢
      Argimine
      ⁢
      
        
      
      ⁢
      800
      ⁢
      
        
      
      ⁢
      nm
      ⁢
      
        
      
      ⁢
      value
    
    
      DRAQS
      ⁢
      
        
      
      ⁢
      700
      ⁢
      
        
      
      ⁢
      nm
      ⁢
      
        
      
      ⁢
      value
    
  
  )




Each plate included fourteen control wells of DMSO only treatment (minimum activation) as well as fourteen control wells for maximum activation treated with 20 μM of a reference compound. The average of the ratio values for each control type was calculated and used to determine the percent activation 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 20 μM. Percent activation was determined and EC30 curves were generated using triplicate wells per concentration of compound.




  
    Percent
    ⁢
    
      
    
    ⁢
    Activation
  
  =
  
    100
    -
    
      (
      
        
          (
          
            
              
                
                  
                    
                      (
                      
                        Individual
                        ⁢
                        
                          
                        
                        ⁢
                        Test
                        ⁢
                        
                          
                        
                        ⁢
                        Sample
                        ⁢
                        
                          
                        
                        ⁢
                        Ratio
                      
                      )
                    
                    -
                  
                
              
              
                
                  
                    (
                    
                      Minimum
                      ⁢
                      
                        
                      
                      ⁢
                      Activation
                      ⁢
                      
                        
                      
                      ⁢
                      Ratio
                    
                    )
                  
                
              
            
            
              
                
                  
                    
                      (
                      
                        Maximum
                        ⁢
                        
                          
                        
                        ⁢
                        Activation
                        ⁢
                        
                          
                        
                        ⁢
                        Ratio
                      
                      )
                    
                    -
                  
                
              
              
                
                  
                    (
                    
                      Minimum
                      ⁢
                      
                        
                      
                      ⁢
                      Activation
                      ⁢
                      
                        
                      
                      ⁢
                      Ratio
                    
                    )
                  
                
              
            
          
          )
        
        *
        100
      
    
  











TABLE 3







In Cell Western data










Cmpd No.
EC30














1
B



2
C



3
C



4
C



5
C



6
C



7
B



8
C



9
C



10
C



11
C



12
C



13
C



14
B



15
C



16
C



17
C



18
C



19
A



20
A



21
C



22
C



23
C



24
C



25
C



26
C



27
C



28
B



29
C



30
C



31
C



32
C



33
C



34
C



35
C



36
C



37
A



38
C



39
C



40
C



41
C



42
C



43
C



44
C



45
A



46
C



47
A



48
C



49
C



50
C



51
C



52
C



53
C



54
C



55
C



56
C



57
C



58
C



59
C



60
C



61
C



62
C



63
C



64
C



65
C



66
C



67
C



68
C



69
B



70
C



71
B



72
C



73
C



74
B



75
C



76
C



77
C



78
B



79
C



80
C



81
C



82
C



83
B



84
C



85
C



86
B



87
C



88
C



89
B



90
B



91
C



92
A



93
A



94
A



95
A



96
A



97
A



98
A



99
B



100
A



101
B



102
A



103
B



104
B



105
A



106
C



107
C



108
C



109
C



110
C



111
C



112
C



113
C



114
A



115
C



116
A



117
A



118
A



119
B



120
C



121
A



122
B



123
A



124
A



125
A



126
A



127
B



128
C



129
B



130
B



131
A



132
A



133
A



134
A



135
B



136
C



137
B



138
A



139
C



140
C



141
A



142
A



143
C



144
A



145
A



146
B



147
C



148
B



149
B



150
B



151
B



152
B



153
A



154
C



155
B



156
B



157
C



158
C



159
B



160
C



161
C



162
A



163
C



164
C



165
A



166
A



167
A



168
A



169
C



170
A



171
C



172
C



173
C



174
A



175
A



176
A



177
A



178
A



179
A



180
C



181
C



182
A



183
C



184
A



185
C



186
C



187
B



188
C



189
C



190
B



191
C



192
C



193
B



194
C



195
B



196
C



197
C



198
C



199
A



200
A



201
A



202
A



203
A



204
A



205
A



206
A



207
A



208
A



209
A



210
A



211
A



212
A



213
C



214
C



215
C



216
C



217
C



218
C



219
C



220
A



221
A



222
C



223
C



224
A



225
A



226
C



227
C



228
A



229
B



230
A



231
A



232
B



233
C



234
A



235
A



236
A



237
A



238
A



239
A



240
B



241
B



242
C



243
A



244
A



245
A



246
A



247
B



248
C



249
B



250
C



251
C



252
A



253
B



254
A



255
A



256
C



257
C



258
C



259
A



260
B



261
A



262
A



263
A



264
B



265
A



266
C



267
A



268
B



269
C



270
A



271
C



272
C



273
A



274
A



275
A



276
C



277
C



278
B



279
A



280
A



281
B



282
A



283
A



284
A



285
A



286
A



287
A



288
A



289
A



290
A



291
A



292
A



293
A



294
A



295
A



296
A



297
A



298
A



299
A



300
A



301
A



302
A



303
A



304
A



305
A



306
A



307
A



308
A



309
A



310
A



311
A



312
A



313
A



314
A



315
A



316
A







For Table 3, “A” indicates an EC30 ≦ 3.00 μM, “B” indicates an EC30 of 3.01-12.00 μM, and “C” indicates an EC30 ≧ 12.01 μM.













TABLE 3a







In Cell Western data (Numerical, μM)*










Cmpd No.
EC30














1
7.396



2
1.12



3
16.165



4
12.102



5
>20



6
>20



7
5.758



8
>20



9
—



10
—



11
—



12
—



13
>20



14
3.35



15
>20



16
>20



17
>20



18
—



19
0.067



20
0.18



21
—



22
—



23
—



24
—



25
—



26
—



27
12.255



28
10.198



29
—



30
—



31
—



32
—



33
—



34
—



35
—



36
—



37
0.05



38
—



39
—



40
—



41
—



42
—



43
—



44
—



45
0.454



46
—



47
0.104



48
—



49
—



50
—



51
—



52
—



53
—



54
—



55
—



56
—



57
—



58
—



59
—



60
—



61
—



62
>20



63
>20



64
—



65
—



66
—



67
0.507



68
0.775



69
7.986



70
>20



71
3.716



72
—



73
14.734



74
6.336



75
>20



76
—



77
12.468



78
10.562



79
>20



80
>20



81
>20



82
>20



83
6.747



84
—



85
>20



86
5.626



87
>20



88
14.481



89
11.002



90
10.445



91
>20



92
0.956



93
1.112



94
1.784



95
1.225



96
0.92



97
0.184



98
2.85



99
11.771



100
2.98



101
5.985



102
2.316



103
3.668



104
3.921



105
2.278



106
—



107
—



108
—



109
—



110
—



111
—



112
—



113
—



114
0.607



115
—



116
1.407



117
1.292



118
0.314



119
4.537



120
—



121
2.074



122
3.476



123
2.964



124
1.225



125
1.574



126
1.229



127
7.013



128
—



129
7.378



130
3.915



131
2.561



132
2.399



133
2.906



134
1.094



135
8.051



136
>20



137
4.182



138
2.28



139
>20



140
>20



141
0.583



142
2.098



143
>20



144
0.284



145
1.949



146
7.157



147
>20



148
7.296



149
4.32



150
6.657



151
9.73



152
5.777



153
1.387



154
>20



155
10.578



156
9.562



157
>20



158
>20



159
8.564



160
17.728



161
14.853



162
1.336



163
>20



164
18.536



165
0.66



166
0.434



167
0.845



168
0.066



169
>20



170
1.393



171
>20



172
>20



173
>20



174
0.26



175
0.174



176
0.882



177
0.295



178
0.954



179
0.03



180
—



181
0.046



182
0.179



183
—



184
2.413



185
>20



186
12.797



187
7.073



188
12.323



189
>20



190
8.394



191
>20



192
>20



193
9.722



194
17.038



195
7.183



196
>20



197
>20



198
>20



199
2.818



200
0.596



201
0.255



202
0.941



203
1.719



204
0.801



205
1.69



206
1.164



207
2.312



208
0.696



209
0.557



210
0.268



211
0.523



212
0.238



213
0.708



214
2.247



215
7.467



216
0.726



217
2.148



218
5.736



219
0.919



220
0.211



221
0.413



222
>20



223
>20



224
0.27



225
0.688



226
>20



227
15.037



228
0.279



229
8.708



230
0.206



231
0.23



232
7.142



233
17.343



234
0.759



235
1.557



236
0.852



237
0.114



238
2.553



239
1.278



240
6.45



241
10.434



242
>20



243
0.809



244
0.142



245
0.627



246
1.077



247
8.256



248
>20



249
9.532



250
>20



251
18.721



252
1.825



253
8.002



254
2.243



255
2.66



256
>20



257
>20



258
>20



259
0.532



260
6.071



261
0.327



262
1.231



263
1.293



264
3.517



265
0.503



266
>20



267
0.694



268
3.693



269
14.871



270
1.06



271
>20



272
>20



273
1.298



274
3.045



275
0.122



276
>20



277
>20



278
8.232



279
0.637



280
1.721



281
3.516



282
0.383



283
0.379



284
0.856



285
0.620



286
0.812



287
0.202



288
0.076



289
0.419



290
0.021



291
0.154



292
0.098



293
0.336



294
0.224



295
0.116



296
0.478



297
0.080



298
0.104



299
0.877



300
0.611



301
0.128



302
0.976



303
0.712



304
0.155



305
0.193



306
0.100



307
0.109



308
0.065



309
0.024



310
0.032



311
0.227



312
0.824



313
0.894



314
0.510



315
0.843



316
0.063







*For Table 3a, numerical values represent data from a single experiment or an average of multiple experiments.






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 compound of Formula (I):




or a pharmaceutically acceptable salt thereof,
wherein:
X is N, Z is NR4, and Y is CR5; or
X is NR4, Z is N, and Y is CR5;
R1, R2, R6, R7, and R8 are independently selected from the group consisting of hydrogen, halo, —CN, —NO2, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 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 alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, and a sulfur protecting group when attached to a sulfur atom;
each RB is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and a nitrogen protecting group, or two RB groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring;
R1 and R2 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring; or
R1 and R6 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring; or
R2 and R8 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring; or
R6 and R7 are optionally taken together to form an optionally substituted carbocyclic, optionally substituted heterocyclic, or optionally substituted aryl ring;
R3 is hydrogen, C1-4 alkyl, or C3-4 cycloalkyl;
R4 is hydrogen, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, or optionally substituted C1-4 alkyl-Cy;
Cy is optionally substituted C3-7 cycloalkyl, optionally substituted 4- to 7-membered heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;
R5 is hydrogen, halo, —CN, optionally substituted C1-4 alkyl, or optionally substituted C3-4 cycloalkyl;
Rx is optionally substituted C1-4 alkyl or optionally substituted C3-4 cycloalkyl;
provided that R2 is not —CF3 or —CHF2,
provided that R6 is not —CF3 or —CHF2; and
provided that R1 is not —NRBC(O)RA, —NRBSO2RA, or —(CRzRz)nC(O)N(RB)2; wherein each Rz is independently hydrogen or fluoro; and n is 0, 1, 2, 3, or 4.

2. The compound of claim 1, wherein the compound is of Formula (II):




or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein the compound is of Formula (I-a):




or a pharmaceutically acceptable salt thereof, wherein R1 is not hydrogen.

4. The compound of claim 1, wherein the compound is of Formula (I-b):




or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are not hydrogen.

5. The compound of claim 1, wherein R1 is not hydrogen.

6. The compound of claim 5, wherein R1 is —ORA.

7. The compound of claim 6, wherein R1 is —ORA, wherein RA is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclic.

8. The compound of claim 7, wherein R1 is —ORA, wherein RA is optionally substituted C1-6 alkyl.

9. The compound of claim 8, wherein R1 is —O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl.

10. The compound of claim 8, wherein R1 is —ORA, wherein RA is substituted C1-6 alkyl.

11. The compound of claim 10, wherein R1 is —O—C1-6alkyl-carbocyclyl or —O—C1-6alkyl-heterocyclyl.

12. The compound of claim 1, wherein R6 is hydrogen, —ORA, halo, —CN, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted carbocyclic.

13. The compound of claim 12, wherein R6 is halo.

14. The compound of claim 13, wherein R6 is fluoro or chloro.

15. The compound of claim 1, wherein R2 is hydrogen.

16. The compound of claim 1, wherein R7 and R8 are hydrogen.

17. The compound of claim 1, wherein R3 is C1-4 alkyl.

18. The compound of claim 17, wherein R3 is methyl.

19. The compound of claim 1, wherein R3 is hydrogen, methyl, ethyl, propyl, butyl, cyclopropyl, or cyclobutyl.

20. The compound of claim 1, wherein R4 is hydrogen or optionally substituted C1-6 alkyl.

21. The compound of claim 1, wherein R5 is hydrogen or optionally substituted C1-4 alkyl.

22. The compound of claim 1, wherein Rx is methyl, ethyl, propyl, butyl, isopropyl, hydroxyethyl methoxyethyl, cyclopropyl, or cyclobutyl.

23. The compound of claim 22, wherein Rx is methyl.

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































































































































and pharmaceutically acceptable salts thereof.

25. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

26. A kit or packaged pharmaceutical comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof, and instructions for use thereof.

27. The compound of claim 1, wherein the compound is of Formula (II-a) or (II-b):




or a pharmaceutically acceptable salt thereof;
wherein:
R1 is optionally substituted alkoxyalkyl, —ORA, —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, or —NRBC(O)ORA; and
R2 is halogen.

28. The compound of claim 27, wherein R1 is —ORA, —N(RB)2, or optionally substituted alkoxyalkyl.

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




























and pharmaceutically acceptable salts thereof.

30. A pharmaceutical composition comprising a compound of claim 24 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

31. A pharmaceutical composition comprising a compound of claim 29 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.