Compositions and methods for treating MALT1-related diseases

Abstract

In particular, the present disclosure provides MALT1 inhibitors for use in reducing signaling by Jun / Fos and / or NF-[kappa] B and / or mTor pathway (e.g., mTORc1 and / or mTORc2) activation in cells having a function of obtaining alleles acting by MALT1. The disclosure also provides treatment of patients having these functionally obtained alleles by administering a MALT1 inhibitor. In a use, the MALT1 inhibitor is administered to a patient suffering from BENNTA.

Description

BACKGROUND

[0001] BENTA, B cell expansion with NF-κΒ and T cell dysfunction, is a rare genetic disorder of the immune system caused by mutations in the gene CARD11 (Caspase Recruitment Domain Family Member 11). The disease is characterized by high levels of certain B cells starting in infancy (B cell lymphocytosis), enlarged spleen (splenomegaly), enlarged lymph nodes (lymphadenopathy), immune deficiency, and increased risk of lymphoma.

[0002] Currently, there are few available treatment options for people with BENTA. Treatment options primarily address infections associated with BENTA and / or B cell cancers associated with BENTA. New medical treatments are needed to address the underlying cause of BENTA, rather than just treating the symptoms of BENTA. It is an object of the present disclosure to provide methods of treating the underlying genetic cause of BENTA. SUMMARY

[0003] The present disclosure relates to the use of MALT1 inhibitors for reducing Jun / Fos and / or NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) activation caused by gain-of-function mutations acting through MALT1 (e.g., CARD11 or CARD14 gain-of-function mutants). Such gain-of-function mutations cause diseases such as B cell expansion with NF-κΒ and T cell dysfunction (BENTA), A20 haploinsufficiency, CARD14 generalized pustular psoriasis, HOIL1 hypomorphism, and NF-κΒ gain-of-function syndrome. MALT1 inhibitors include, for example, compounds 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4. MALT1 inhibitors also include compounds of Formula 1:

[0004]

[0005] or a pharmaceutically acceptable salt thereof, wherein:

[0006] R 1 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, C3-6cycloalkyl, and 5-10 membered heterocyclyl, wherein the C1-6alkyl, the C3-6cycloalkyl, and the 5-10 membered heterocyclyl can be optionally substituted on one or more available carbons by one, two, three or more substituents, each substituent being independently selected from R 1a , wherein if the 5-10 membered heterocyclyl contains a substitutable ring nitrogen atom, the ring nitrogen atom can be optionally substituted by R 1bsubstituted, and wherein if said 5-10 membered heterocyclyl contains a substitutable ring sulfur atom, said ring sulfur atom can be optionally substituted with two O atoms;

[0007] R 2 is CH3or CF3;

[0008] R 3 is hydrogen; or

[0009] R 3 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, C3-7cycloalkyl, 5-6 membered heterocyclyl, 5-6 membered heterocyclyl-C1-3alkyl 5-6 membered heterocyclyl-O-, phenyl, and 5-6 membered heteroaryl, any of which can be optionally substituted with one, two, or three substituents each independently selected from R 3a ;

[0010] R 4 is C1-6alkyl;

[0011] R 1a is independently at each occurrence selected from the group consisting of cyano, halogen, hydroxyl, oxo, C1-6alkyl, -C(O)OR A , -C(O)N(R A )2, -N(R A )2, C1-6alkoxy, 5-6 membered heterocyclyl, and 5-6 membered heteroaryl, wherein said C1-6alkyl is optionally substituted with -N(R A )2, and wherein if said 5-6 membered heterocyclyl contains a substitutable ring nitrogen atom, said ring nitrogen atom can be optionally substituted with R p ;

[0012] R 1b is selected from the group consisting of C1-6alkyl, -C(O)OR A , -C(O)C1-6alkyl, -C(O)C3-6cycloalkyl, -C(O)N(R A )2, and -S(O)2C1-6alkyl;

[0013] R 3a is independently at each occurrence selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, C1-4haloalkoxy, hydroxyl, C1-4alkenyl, cyano, azido, -NR C R D, C3-6cycloalkyl, C1-4alkoxy, 5-6 membered heterocyclyl-O-, 5-6 membered heterocyclyl, and phenyl, wherein C3-6cycloalkyl, 5-6 membered heterocyclyl-O-, 5-6 membered heterocyclyl, and phenyl are optionally substituted with one, two, or three substituents each independently selected from the group consisting of R p ;

[0014] R p independently at each occurrence is selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, hydroxyl, C1-4alkoxy, C1-4alkoxyC1-4alkyl, NR C R D and aminoC1-3alkyl;

[0015] R A independently at each occurrence is selected from the group consisting of hydrogen, C1-6alkyl, -C(O)C1-6alkyl, and -C(O)OC1-6alkyl;

[0016] R B is selected from the group consisting of C1-6alkyl, C1-6cycloalkyl, and -C(O)OC1-6alkyl;

[0017] R C and R D independently at each occurrence is selected from the group consisting of hydrogen, C1-6alkyl, haloC1-6alkyl, and C-3-4cycloalkyl, or

[0018] R C and R D together with the nitrogen atom to which they are attached form a 4-6 membered heterocyclyl or 4-6 membered heteroaryl, wherein the 4-6 membered heterocyclyl or the 4-6 membered heteroaryl can contain an additional nitrogen atom or oxygen atom, and is optionally substituted with one or two fluorines; and

[0019] T is 0 or 1. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A diagram showing the pathway of MALT1 signaling to NF-κB is shown.

[0021] Figure 2 A diagram showing the pathway of MALT1 signaling to NF-κB with a CARD11 gain-of-function allele is shown. This diagram also shows the inhibition of CARD11 stimulation by a MALT1 inhibitor.

[0022] Figure 3A diagram showing the pathway of MALT1 signaling to NF-κΒ with a CARD14 gain-of-function allele. This diagram also shows the inhibition of CARD14 stimulation by MALT1 inhibitors. DETAILED DESCRIPTION

[0023] Before various embodiments are described in detail, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described and, therefore, can vary in accordance with the scope of the claims. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present teachings will be limited only by the appended claims.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0025] As will be apparent to those of ordinary skill in the art in light of the present disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0026] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide.

[0027] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0028] DEFINITIONS

[0029] Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described herein. Additionally, general principles of organic chemistry, and 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, 5thEd., 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, 3rdEd., Cambridge University Press, Cambridge, 1987.

[0030] The compounds described herein can comprise one or more asymmetric centers and thus can exist in various isomeric forms, e.g., enantiomeric and / or diastereomeric forms. 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 separated 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 the preferred isomer can be prepared by asymmetric synthesis. See, e.g., 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. 2.68 in Eliel, E. L., Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972. The present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0031] As used herein, the term "enantiopure compound" contains substantially no other enantiomer or stereoisomer of the compound (i.e., an enantiomeric excess), in other words, the "S" form of the compound contains substantially no "R" form of the compound and is thus in enantiomeric excess relative to the "R" form. The term "enantiomerically pure" or "enantiopure" means that a compound contains more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, or more than 99.9% by weight of an enantiomer. In one aspect, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0032] In the compositions provided herein, enantiomerically pure compounds can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound. In certain aspects, the enantiomerically pure R-compound in such compositions can comprise, for example, at least about 95% by weight R-compound and at most about 5% by weight S-compound, based on the total weight of the compound. For example, a pharmaceutical composition comprising an enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound. In certain aspects, the enantiomerically pure S-compound in such compositions can comprise, for example, at least about 95% by weight S-compound and at most about 5% by weight R-compound, based on the total weight of the compound. In certain aspects, the active ingredient can be formulated with little or no excipient or carrier.

[0033] The compounds described herein can also comprise one or more isotopic substitutions. For example, H can be in any isotopic form, including 3 H, H (D or deuterium), and l2 H, H (D or deuterium), and 13 H, H (D or deuterium), and 14 H, H (D or deuterium), and 16 H, H (D or deuterium), and 18 H, H (D or deuterium), and l5 H, H (D or deuterium), and 19 H, H (D or deuterium), and and the like.

[0034] When a range of values is listed, it is contemplated that each value and sub-range within the range is encompassed. For example, "Ci-6 alkyl" is intended to encompass Ci, C2, C3, C4, C5, C6, Ci-4, Ci-3, Ci-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

[0035] As used herein, the term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group (a "Ci-20 alkyl") having, for example, 1 to 20 carbon atoms. In some aspects, the alkyl group has 1 to 10 carbon atoms ("Ci-io alkyl"). In some aspects, the alkyl group has 1 to 9 carbon atoms ("Ci-9 alkyl"). In some aspects, the alkyl group has 1 to 8 carbon atoms ("Ci-8 alkyl"). In some aspects, the alkyl group has 1 to 7 carbon atoms ("Ci-7 alkyl"). In some aspects, the alkyl group has 1 to 6 carbon atoms ("Ci-6 alkyl"). In some aspects, the alkyl group has 1 to 5 carbon atoms ("Ci-5 alkyl"). In some aspects, the alkyl group has 1 to 4 carbon atoms ("C1-4 alkyl"). In some aspects, the alkyl group has 1 to 3 carbon atoms ("C1-3 alkyl"). In some aspects, the alkyl group has 1 to 2 carbon atoms ("C1-2 alkyl"). In some aspects, the alkyl group has 1 carbon atom ("C1 alkyl"). Examples of Ci-6 alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.

[0036] As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbon group ("C2-20alkenyl") having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds). In certain aspects, the alkenyl group does not contain any triple bonds. In some aspects, the alkenyl group has from 2 to 10 carbon atoms ("C2-10alkenyl"). In some aspects, the alkenyl group has from 2 to 9 carbon atoms ("C2-9alkenyl"). In some aspects, the alkenyl group has from 2 to 8 carbon atoms ("C2-8alkenyl"). In some aspects, the alkenyl group has from 2 to 7 carbon atoms ("C2-7alkenyl"). In some aspects, the alkenyl group has from 2 to 6 carbon atoms ("C2-6alkenyl"). In some aspects, the alkenyl group has from 2 to 5 carbon atoms ("C2-5alkenyl"). In some aspects, the alkenyl group has from 2 to 4 carbon atoms ("C2-4alkenyl"). In some aspects, the alkenyl group has from 2 to 3 carbon atoms ("C2-3alkenyl"). In some aspects, the alkenyl group has 2 carbon atoms ("C2alkenyl"). The one or more carbon-carbon double bonds can be internal (as in 2-butenyl) or terminal (as in 1-butenyl). Examples of C2-4alkenyl include ethenyl (C2), 1 -propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6alkenyl include the foregoing C2-4alkenyl as well as pentenyl (C5), pentadienyl (C5), hexenyl (Ce), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.

[0037] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon radical ("C2-20alkynyl") having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds). In certain aspects, the alkynyl group does not contain any double bonds. In some aspects, the alkynyl group has from 2 to 10 carbon atoms ("C2-10alkynyl"). In some aspects, the alkynyl group has from 2 to 9 carbon atoms ("C2-9alkynyl"). In some aspects, the alkynyl group has from 2 to 8 carbon atoms ("C2-8alkynyl"). In some aspects, the alkynyl group has from 2 to 7 carbon atoms ("C2-7alkynyl"). In some aspects, the alkynyl group has from 2 to 6 carbon atoms ("C2-6alkynyl"). In some aspects, the alkynyl group has from 2 to 5 carbon atoms ("C2-5alkynyl"). In some aspects, the alkynyl group has from 2 to 4 carbon atoms ("C2-4alkynyl"). In some aspects, the alkynyl group has from 2 to 3 carbon atoms ("C2-3alkynyl"). In some aspects, the alkynyl group has 2 carbon atoms ("C2alkynyl"). The one or more carbon-carbon triple bonds can be internal (as in 2-butynyl) or terminal (as in 1-butynyl). Examples of C2-4alkynyl include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6alkynyl include the foregoing C2-4alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like.

[0038] As used herein, the terms "alkylene," "alkenylene," "alkynylene," "cycloalkylene," "heterocyclylene," "heteroarylene," and "phenylene" refer to divalent radicals of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl (e.g., saturated and partially saturated), heteroaryl, and phenyl groups, respectively. When a range or number of carbons is provided for a particular "alkylene," "alkenylene," or "alkynylene," it is understood that the range or number refers to the entire linear carbon chain. "Alkylene," "alkenylene," and "alkynylene" groups can be substituted or unsubstituted by one or more substituents as described herein.

[0039] As used herein, the term "aryl" refers to a group ("C6-14 aryl") of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 p-electrons shared in a cyclic array) having from 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system. In some aspects, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some aspects, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some aspects, the aryl group has fourteen ring carbon atoms ("C14 aryl"). "Aryl" also includes ring systems in which an aryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, where the point of attachment is on the aryl ring, and in such cases the number of carbon atoms designates the number of carbon atoms in the aryl ring system.

[0040] Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, (chrysene), coronene, fluoranthene, fluorene, hexa- peri hexyl, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octalene, ovalene, penta-2,4- diene, pentacene, pentalene, periflanthene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene. In particular, aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.

[0041] As used herein, the term "heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in common in the ring 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 ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon atom or a nitrogen atom, as valence permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl rings, wherein the point of attachment is on the heteroaryl ring, and in which case the number of ring members continues from the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in which case the numbering of the ring members indicates the numbering in the fused polycyclic (aryl / heteroaryl) ring system, wherein a bicyclic heteroaryl group (e.g., indolyl, quinolinyl, carbazolyl, etc.) having one ring that does not contain a heteroatom, the point of attachment can be on either ring, i.e., the ring bearing a heteroatom (e.g., 2-indolyl) or the ring not containing a heteroatom (e.g., 5-indolyl).

[0042] In some aspects, a heteroaryl 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 aspects, a heteroaryl 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 aspects, a heteroaryl is a 5-6 membered 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 heteroaryl"). In some aspects, a 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects, a 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects, a 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0043] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Examples of representative heteroaryl groups include the following:

[0044]

[0045] wherein each Z is selected from the group consisting of carbonyl, N, NR65, O, and 8; and R65is independently hydrogen, Ci-8alkyl, C3-10carbocyclyl, 4-10 membered heterocyclyl, C6-C10aryl, and 5-10 membered heteroaryl.

[0046] As used herein, the term "carbocyclyl" or "carbocyclic" refers to a non-aromatic ring system having 3 to 10 ring carbon atoms ("C3-10 carbocyclyl") and zero heteroatoms in the non-aromatic ring system, in some aspects, a carbocyclyl group has 3 to 8 ring carbon atoms ("C3-8 carbocyclyl"). In some aspects, a carbocyclyl group has 3 to 7 ring carbon atoms ("C3-7 carbocyclyl"). In some aspects, a carbocyclyl group has 3 to 6 ring carbon atoms ("C3-6 carbocyclyl"). In some aspects, a carbocyclyl group has 5 to 10 ring carbon atoms ("C5-10 carbocyclyl"). Exemplary C3-6 carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-8 carbocyclyl groups include, but are not limited to, the foregoing C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclooctenyl (C8), bicyclo[2.2.2]octenyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include, but are not limited to, the foregoing C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-l-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decyl (C10), and the like. As illustrated by the foregoing examples, in certain embodiments, a carbocyclyl group is a monocyclic ("monocyclic carbocyclyl") or contains a fused, bridged, or spiro ring system, such as a bicyclic ring system ("bicyclic carbocyclyl") and can be saturated or can be partially unsaturated. "Carbocyclic" also includes ring systems in which a carbocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups, where the point of attachment is on the carbocyclyl ring, and in such cases, the number of carbons refers to the number of carbons in the carbocyclic ring system.

[0047] As used herein, the term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic or bridged cyclic (e.g., adamantyl) hydrocarbon group having 3-12, 3-8, 4-8, or 4-6 carbons as referred to herein, e.g., a "cycloalkyl" group derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane.

[0048] As used herein, the term "C3-6 monocyclic cycloalkyl" or "monocyclic C3-6 cycloalkyl" refers to a saturated 3- to 7-membered monocyclic hydrocarbon ring system. 3- to 7-membered monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. When specified as optionally substituted or substituted, substituents on the cycloalkyl group (e.g., in the case of an optionally substituted cycloalkyl group) can be present at any substitutable position and include, for example, the position to which the cycloalkyl group is attached.

[0049] As used herein, the term "heterocyclyl" or "heterocycle" refers to a radical of a 3- to 10-membered nonaromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3-10 membered heterocyclyl"). A heterocyclyl radical containing one or more nitrogen atoms can be attached to the rest of the molecule via a carbon or nitrogen atom, as valence permits. The heterocyclyl radical can be monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiro ring system, such as a bicyclic ring system ("bicyclic heterocyclyl"), and can be saturated or can be partially unsaturated. A heterocyclyl bicyclic ring system can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems wherein a heterocyclyl ring as defined above is fused to one or more carbocyclyl rings, wherein the point of attachment is on either the carbocyclyl or the heterocyclyl ring; or wherein a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring, and in this case the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclyl group," "heterocyclic moiety," and "heterocyclic radical" can be used interchangeably.

[0050] In some embodiments, the heterocyclyl is a 4-7 membered nonaromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("4-7 membered heterocyclyl"). In some aspects, the heterocyclyl is a 5-10 membered nonaromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5-10 membered heterocyclyl").

[0051] In some aspects, the heterocyclyl is a 5-8 membered nonaromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("3-8 membered heterocyclyl"). In some aspects, the heterocyclyl is a 5-6 membered nonaromatic 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 aspects, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some aspects, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0052] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, aziridinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.

[0053] 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, 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 C5 aryl ring (also referred to herein as 5,6-bicyclic heterocycles) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocycles) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0054] Examples of such saturated or partially unsaturated heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, pyrrolidinyl, pyridonyl, pyrrolidonyl, piperidinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, oxetanyl, azetidinyl, and tetrahydropyrimidinyl. When designated as optionally substituted or substituted, substituents on a heterocyclyl group (e.g., in the case of an optionally substituted heterocyclyl group) can be present at any substitutable position and include, for example, the position at which the heterocyclyl group is attached.

[0055] As used herein, the term“hetero” when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group has been replaced with a nitrogen, oxygen, or sulfur heteroatom. Hetero can apply to any of the hydrocarbyl groups described above, such as alkyl (e.g., heteroalkyl); carbocyclyl (e.g., heterocyclyl); aryl (e.g., heteroaryl); and similar groups having 1 to 5, particularly 1 to 3, heteroatoms.

[0056] As used herein, the term“cyano” means -CN.

[0057] As used herein, the terms“halo” and“halogen” as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), and iodine (iodo, -I). In certain aspects, the halo group is fluorine or chlorine.

[0058] As used herein, the term“alkyl” means a straight or branched hydrocarbon chain radical, saturated and non-saturated, monovalent, having 1 to 20 carbon atoms, particularly 1 to 10 carbon atoms, more particularly 1 to 6 carbon atoms, and even more particularly 1 to 3 carbon atoms. In certain aspects, the alkyl group is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl, or the like. In certain aspects, the alkyl group is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, or the like. In certain aspects, the alkyl group is methyl, ethyl, propyl, isopropyl, butyl, or the like. In certain aspects, the alkyl group is methyl, ethyl, or the like. In certain aspects, the alkyl group is methyl.

[0059] As used herein, the term“alkoxy” as used herein means an alkyl group connected to another moiety through an oxygen atom (-O(alkyl)). Non-limiting examples include, for example, methoxy, ethoxy, propoxy, and butoxy.

[0060] As used herein, the term“fluoroalkoxy” is a haloalkyl group connected to another moiety through an oxygen atom, such as, but not limited to, -OCHCFH2or -OCF3.

[0061] As used herein, the term“oxo” means -C=O.

[0062] As used herein, 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 that upon substitution produces a stable compound, e.g., a compound that 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 is substituted, the substituents can be either the same or different at each position.

[0063] A nitrogen atom can be substituted or unsubstituted, and include primary, secondary, tertiary, and quaternary nitrogen atoms, as valence permits. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR, -N(R)2, -CN, -C(=O)R, -C(=O)N(R)2, -CO2-R, -N SO2R3, -C(=NR)R, -C(=NR)N(R)2, -SO2N(R)2, -SO2R, -SOR, -C(=S)N(R)2, -C(=O)SR, -C(S)SR-, -P(=O)2R, -P(=O)(R)2, -P(=O)2N(R)2, -P(=O)(NR)2, C1-10alkyl, C1-10perhaloalkyl, C2-10alkenyl, C2-10alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-11aryl, and 5-14 membered heteroaryl, or two Rccgroups attached to a nitrogen atom are optionally 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 RQQgroups.

[0064] As used herein, the term "coding sequence" is defined to mean a portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0065] As used herein, the term "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of a compound of the application can vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and condition of the subject. Effective amounts include therapeutic and prophylactic treatments.

[0066] As used herein, the term "wild type" is defined to mean a form that is found primarily in nature. For example, a wild type polypeptide or polynucleotide sequence is a sequence that exists primarily in organisms, can be isolated from a source in nature, and has not been intentionally modified by manipulation by humans.

[0067] As used herein, the terms "recombinant" or "engineered" or "non-naturally occurring" can be used interchangeably and are defined to mean a polypeptide or nucleic acid that is modified in a manner that does not occur in nature or is produced or derived through the use of recombinant technology and / or manipulation from synthetic materials. Non-limiting examples include, among others, a recombinant cell that expresses a gene not found therein in nature (non-recombinant form), or that expresses a natural gene under control of a different regulatory sequence.

[0068] As used herein, the terms "percent sequence identity" and "percent homology" are used interchangeably and are defined as meaning a comparison between polynucleotides or polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percent identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Alternatively, the percent identity can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences or the number of positions at which a nucleic acid base or amino acid residue aligns with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Those skilled in the art will appreciate that there are a number of established algorithms that are useful for aligning two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv Appl Math. 2:482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J Mol Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 supplement)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990; and Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1977, respectively.Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. BLAST for nucleotide sequences can use the BLASTN program with default parameters: e.g., wordlength (W) = 11, expectation (E) = 10, M = 5, N = -4, and a comparison of both strands. BLAST for amino acid sequences can use the BLASTP program with default parameters: e.g., wordlength (W) = 3, expectation (E) = 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Exemplary determinations of sequence identity and percent sequence identity also can use the BESTFIT or GAP programs in the GCG Wisconsin Software Package (Accelrys, Madison WI), using default parameters.

[0069] As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0070] As used herein, "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio. 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, incorporated herein by reference in its entirety. Pharmaceutically acceptable salts of the compounds of this application 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 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 such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, bisulfite, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, 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 and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(Ci-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, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0071] As used herein, the term "reference sequence" is defined as a defined sequence used as a basis for sequence comparisons. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or is the full-length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) comprise a sequence that is analogous to a portion of the other sequence (i.e., share a portion of the complete sequence) and (2) can further comprise a sequence that is divergent from the other sequence, sequence comparisons between two (or more) polynucleotide or polypeptide sequences are typically performed by comparing sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In one aspect, a "reference sequence" can be based on a primary amino acid sequence, wherein the reference sequence is a sequence to which one or more alterations can be made to the primary sequence.

[0072] As used herein, the term "substantial identity" means that two polynucleotide or polypeptide sequences are typically one of the following: at least 80% identical, at least 85% identical, and 89% to 95% identical, more typically at least 99% identical over a comparison window of at least 20 residues, usually over a window of at least 30-50 residues, wherein the percentage of sequence identity is determined by comparing the reference sequence to the sequence including deletions or additions that are within 20% of the reference sequence in the comparison window. In a specific embodiment applying to polypeptides, the term "substantial identity" means that two polypeptide sequences to be identified share at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity) when optimally aligned, as determined using the program GAP or BESTFIT with standard parameters (i.e., default parameters). Preferably, non-identical residue positions differ by conservative amino acid substitutions.

[0073] As used herein, the term "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with the disease, disorder, or condition. A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0074] As used herein, the term "amino acid substitution" or "amino acid difference" is defined to mean a change in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence, which is the primary translation product starting with the methionine start codon. The position of the amino acid difference is generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, "a residue difference at position X compared to the primary translation product starting with the methionine start codon" refers to a change in the amino acid residue at the position of the polypeptide corresponding to position X of the wild-type protein. Thus, if the reference polypeptide of the primary translation starting with the methionine start codon product of the wild-type gene has a valine at position X, then "an amino acid substitution" or "a residue difference at position X compared to the reference sequence" refers to an amino acid substitution of any residue other than valine at the position of the polypeptide corresponding to position X of the reference sequence. In most instances herein, a particular amino acid substitution or amino acid residue difference at a position is indicated as "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is the one-letter identifier of the amino acid found in the engineered polypeptide (that is, the residue that differs from the reference polypeptide). In one aspect, when more than one amino acid can occur at a specified residue position, the alternative amino acids can be listed in the form XnY / Z, where Y and Z represent the alternative amino acid residues. In some instances, the present disclosure also provides for a particular amino acid difference represented by the conventional notation "AnB", where A is the one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the one-letter identifier of the residue substitution in the sequence of the engineered polypeptide. Furthermore, in some instances, the polypeptides of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of specified positions that are changed relative to the reference sequence.

[0075] As used herein, the term "conservative amino acid substitution" or "conservative amino acid difference" is defined to mean a change in an amino acid at a residue position to a different residue with a similar side chain, and thus typically involves the substitution of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids. By way of example and not limitation, amino acids with aliphatic side chains can be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); amino acids with hydroxyl side chains are substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); amino acids with aromatic side chains are substituted with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); amino acids with basic side chains are substituted with another amino acid with a basic side chain (e.g., lysine and arginine); amino acids with acidic side chains are substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and hydrophobic or hydrophilic amino acids are substituted with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below.

[0076] Table 1

[0077]

[0078] As used herein, the term "non-conservative substitution" or "non-conservative amino acid difference" is defined to mean a change in an amino acid at a residue position to a different residue with substantially different side chain properties. Non-conservative substitutions can use amino acids between the defined groups rather than within, and affect (a) the structure of the peptide backbone in the region of the substitution (e.g., with proline for glycine), (b) the charge or hydrophobicity of the region, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0079] As used herein, the term "deletion" is defined to mean a modification of a polypeptide by removing one or more amino acids from a reference polypeptide, or a modification of a nucleic acid by removing one or more nucleotides from a reference nucleic acid. For example, a deletion can comprise removing 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids, that make up a reference polypeptide. A deletion can be to an internal portion and / or a terminal portion of a polypeptide. In various embodiments, a deletion can comprise a contiguous segment, or can be non-contiguous.

[0080] As used herein, the term "insertion" is defined to mean a modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide, or a modification of a nucleic acid by the addition of one or more nucleic acids. The insertion can be in an internal portion of the polypeptide, or can be at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a contiguous segment of amino acids, or can be separated by one or more of the amino acids in the reference polypeptide.

[0081] As used herein, the term "specificity" is used in reference to a biocatalyst or enzyme, which is defined to mean the discrimination of a biocatalyst for a substrate compound.

[0082] As used herein, the term "relative specificity" is defined to mean the specificity of a biocatalyst or enzyme for one substrate compound relative to another or other substrate compound.

[0083] As used herein, the term "stringent hybridization conditions" is defined to mean hybridization at a temperature of 42°C in 5X SSC in 50% formamide and washing filters in 0.2X SSC at 60°C. (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate.) Stringent hybridization conditions also encompass low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium lauryl sulfate at 50°C; hybridization with denaturants (e.g., formamide) at 42°C, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 / 750 mM NaCl, 75 mM sodium citrate; or 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, washing in 0.2X SSC (sodium chloride / sodium citrate) at 42°C and in 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1X SSC containing EDTA at 55°C.

[0084] As defined herein, the term "heterologous" polynucleotide or polypeptide is defined to mean any polynucleotide or polypeptide that does not naturally occur in the host cell. Thus, the term includes polynucleotides removed from the host cell, subjected to laboratory manipulation, and then reintroduced into the host cell. In some embodiments, the introduced polynucleotide expresses a heterologous polypeptide.

[0085] As used herein, the term "control sequence" is defined to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the disclosure. Each control sequence can be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a pre-pro sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and a transcription and translation termination signal (where appropriate). A linker can be provided for the control sequences, in order to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0086] As used herein, the term "operably linked" is defined to mean a configuration in which the control sequences are suitably positioned with respect to the polynucleotide of interest, i.e., in functional relationship to the polynucleotide of interest, such that the control sequences direct or modulate the expression of the polynucleotide of interest and / or polypeptide.

[0087] As used herein, the term "promoter sequence" is defined to mean a nucleic acid sequence recognized by a host cell for the initiation of expression of a polynucleotide of interest, such as a coding sequence or gene. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding proteins either homologous or heterologous to the host cell.

[0088] MALT1 inhibitors

[0089] Mucosa-associated lymphoid tissue lymphoma transmembrane inducer 1 (MALT1). MALT1 is a caspase-like protease that plays a role in BCL10-induced NF-κΒ activation and / or mTOR pathway signaling (e.g., mTORcl and / or mTORc2) activation and / or Jun / Fos activation. The protein can be a component of the CARMA1-BCL10-MALT1 (CBM) signalosome and other signalosomes (e.g., involving CARD11 or CARD14) that trigger NF-κΒ signaling, mTor pathway signaling (e.g., mTORcl and / or mTORc2), and Jun / Fos signaling, leading to lymphocyte activation upon antigen-receptor stimulation. Biallelic loss-of-function mutations in this gene cause immunodeficiency 12 (IMD12).

[0090] MALT1 inhibitors include compounds of Formula I:

[0091]

[0092] or a pharmaceutically acceptable salt thereof, wherein:

[0093] R 1 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, C3-6cycloalkyl, and 5-10 membered heterocyclyl, wherein the C1-6alkyl, the C3-6cycloalkyl, and the 5-10 membered heterocyclyl can be optionally substituted on one or more available carbons by one, two, three or more substituents each independently selected from R 1a , and wherein if the 5-10 membered heterocyclyl contains a substitutable ring nitrogen atom, the ring nitrogen atom can be optionally substituted by R 1b , and wherein if the 5-10 membered heterocyclyl contains a substitutable ring sulfur atom, the ring sulfur atom can be optionally substituted by two O atoms;

[0094] R 2 is CH3or CF3;

[0095] R 3 is hydrogen; or

[0096] R 3 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, C3-7cycloalkyl, 5-6 membered heterocyclyl, 5-6 membered heterocyclyl-C1-3alkyl 5-6 membered heterocyclyl-O-, phenyl, and 5-6 membered heteroaryl, any of which can be optionally substituted by one, two or three substituents each independently selected from R 3a ;

[0097] R 4 is C1-6alkyl;

[0098] R 1a is independently at each occurrence selected from the group consisting of cyano, halogen, hydroxyl, oxo, C1-6alkyl, -C(O)OR A , -C(O)N(R A )2, -N(R A )2, C1-6alkoxy, 5-6 membered heterocyclyl, and 5-6 membered heteroaryl, wherein the C1-6alkyl is optionally substituted by N(R A )2, and wherein if the 5-6 membered heterocyclyl contains a substitutable ring nitrogen atom, the ring nitrogen atom can be optionally substituted by R p ;

[0099] R 1b is selected from the group consisting of C1-6alkyl, -C(O)OR A , -C(O)C1-6alkyl, -C(O)C3-6cycloalkyl, -C(O)N(R A)2and -S(O)2C1-6alkyl;

[0100] R 3a independently at each occurrence, is selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, C1-4haloalkoxy, hydroxyl, C1-4alkenyl, cyano, azido, -NR C R D , C3-6cycloalkyl, C1-4alkoxy, 5-6 membered heterocyclyl-O-, 5-6 membered heterocyclyl, and phenyl, wherein C3-6cycloalkyl, 5-6 membered heterocyclyl-O-, 5-6 membered heterocyclyl, and phenyl are optionally substituted with one, two, or three substituents each independently selected from R p ;

[0101] R p independently at each occurrence, is selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, hydroxyl, C1-4alkoxy, C1-4alkoxyC1-4alkyl, NR C R D and aminoC1-3alkyl;

[0102] R A independently at each occurrence, is selected from the group consisting of hydrogen, C1-6alkyl, -C(O)C1-6alkyl, and -C(O)OC1-6alkyl;

[0103] R B is selected from the group consisting of C1-6alkyl, C1-6cycloalkyl, and -C(O)OC1-6alkyl;

[0104] R C and R D independently at each occurrence, is selected from the group consisting of hydrogen, C1-6alkyl, haloC1-6alkyl, and C-3-4cycloalkyl, or

[0105] R C and R D together with the nitrogen atom to which they are attached form a 4-6 membered heterocyclyl or a 4-6 membered heteroaryl, wherein the 4-6 membered heterocyclyl or the 4-6 membered heteroaryl can contain an additional nitrogen atom or an oxygen atom, and is optionally substituted with one or two fluorines; and

[0106] T is 0 or 1.

[0107] MALT1 inhibitors also include, for example,

[0108]

[0109]

[0110] Other compounds useful as MALT1 inhibitors can be found in WO 2022 / 081967 (PCT / US21 / 55173), which is incorporated by reference in its entirety for all purposes.

[0111] Still other compounds useful as MALT1 inhibitors can be found in WO 2023 / 192506 (PCT / US2023 / 016941), which is incorporated by reference in its entirety for all purposes. Other MALT1 inhibitors include inhibitors of the following formula:

[0112]

[0113] or stereoisomers and / or pharmaceutically acceptable salts thereof, wherein:

[0114] R 1 is C 1-6 alkyl or C 1-3 haloalkyl, wherein the C 1-6 alkyl can be optionally substituted with -O-C 1-3 alkyl;

[0115] R 2 is aryl or 5-6 membered heteroaryl, wherein the aryl can be optionally substituted with cyano;

[0116] R 4 is -C(O)OH or 5-6 membered heteroaryl;

[0117] m is 0 or 1; and

[0118] n is 0 or 1.

[0119] Other MALT1 inhibitors include, for example:

[0120]

[0121] MALT1 inhibitors include pharmaceutically acceptable salts, pharmaceutically acceptable esters, tautomeric forms, polymorphs, and prodrugs of such compounds. In some aspects, MALT1 inhibitors include pharmaceutically acceptable addition salts, pharmaceutically acceptable esters, solvates of addition salts (e.g., hydrates), tautomeric forms, polymorphs, enantiomers, mixtures of enantiomers, stereoisomers, or mixtures of stereoisomers (pure or as racemic or non-racemic mixtures), of the compounds described herein (e.g., compounds of Formula I); compounds of the formula as named herein.

[0122] Synthesis of MALT1 inhibitors

[0123] A mixture of tert-butyl N-{2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5- a]pyrimidin-6-yl}carbamate [INT 1-e] (420 mg, 1.28 mmol) in 4 N HC1 / dioxane (5 mL) was stirred at 2.5 °C for 2 hours. LCMS showed the reaction was complete and a new peak with desired MS was detected (Rt = 0.611 min, m / z: 227.8 [M+H]+). The mixture was concentrated under reduced pressure to give 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine hydrochloride [INT 1.1] as a solid (400 mg, crude).

[0124] (R)-2-chloro-7-(1-methoxyethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine [INT 1.3] can be prepared by the same synthetic route outlined in INT 1.1 using (2R)-2-methoxypropanoic acid as the starting material, m / z: C8H 11 C1N 50 The [M+H]+ calculated for C8H

[0125] To a solution of 4-bromobenzaldehyde [INT 2-a] (100 g, 541 mmol, 1.0 eq) in toluene (500 mL) was added (R)-2-methylpropane-2-sulfinamide (72.1 g, 595 mmol, 1.5 eq) at 25 °C. The mixture was stirred at 25 °C for 15 min. To the above reaction was then added NaOH (21.6 g, 541 mmol, 1.0 eq) and the mixture was stirred at 2.5 °C for 12 h. To the mixture was added Na2SO4(50 g) and stirred for 20 min. The four reaction mixtures were combined and filtered through celite to give a filtrate which was concentrated in vacuo to give the crude product as an oil. The crude product was dissolved in petroleum ether (1.0 L) and stirred at -50 °C for 1.0 h, filtered to give (R,E)-N-(4-bromo-benzylidene)-2-methylpropane-2-sulfinamide [INT 2-b] as a solid (620 g, 2.15 mol, 99.5% yield).

[0126] To a solution of (R,E)-N-(4-bromobenzylidene)-2-methylpropane-2-sulfmamide [INT 2-b] (2.06 g, 715 mmol, 1.0 equiv) and tetrabutylammonium acetate (216 g, 715 mmol, 21.8 mL, 1.0 equiv) in DMF (1.4 L) was added TMSCF3 (259 g, 1.82 mol, 2.5 equiv) at 0 °C. The mixture was stirred at 5 °C for 1.5 h. This procedure was repeated 2 times and the three reaction mixtures were combined for work-up. The mixture was poured into saturated NH4Cl solution (1.3.0 L) and stirred for 10 min to get a suspension. The suspension was filtered to get a filter cake and eluted with water (5.0 L). The filter cake was triturated with MTBE / petroleum ether (v / v = 1:4, 2.0 L) to get the product as a solid and the mother liquor was concentrated in vacuo to get the crude product as an oil which was purified by column chromatography with silica gel using petroleum ether / ethyl acetate (10 / 1-1 / 1) to get (R)-N-((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2-sulfmamide [INT 2.1] as a solid (389 g, 1.09 mol, 50.6% yield).1H NMR (400 MHz, CDCb) d = 1.25 (s, 9H), 3.64 (d, J = 6.40 Hz, 1H), 4.79-4.83 (m, 1H), 7.32 (d, I = 8.40 Hz, 2H), 7.56 (d, I = 6.40 Hz, 2H).

[0127] (S)-N-((R)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2-sulfmamide [INT 14-a] was prepared using (S)-(-)-2-methyl-2-propanesulfmamide following the same synthetic route outlined for (R)-N-((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2-sulfmamide [INT 2.1].

[0128] To a mixture of (S)-N-((R)-l-(4-bromophenyl)-2,2,2-trifluoroethyl)-2- methylpropane-2-sulfmamide [INT 14-a] (8 g, 22.3 mmol) in MeOH (60 mL) was added 4 M HC1 in dioxane (20 mL). The mixture was stirred at 20 °C for 1.5 h. The mixture was concentrated under reduced pressure to give the crude product. The mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic layers were washed with 1 M HC1 (50 mL x 2). The aqueous phase was basified with 2 N NaOH to pH = 9-10, and extracted with CH2Cl2(50 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the crude product (R)-l-(4-bromophenyl)-2,2,2-trifluoroethan-l-amine [INT 14-b] (3.50 g, 13.7 mmol, 61.8% yield) as a yellow solid, m / z: [M+H]+ calculated for C8H8BrF3N 254.0, 256.0; found 254.1.

[0129] To a mixture of tetrahydro-2H-thiopyran-4-carboxylic acid 1,1-dioxide [INT 4-a] (1.68 g, 9.44 mmol), EDCI (2.26 g, 11.8 mmol) and HOBt (1.59 g, 11.8 mmol) in CH2Cl2(20 ml) was added (R)-l-(4-bromophenyl)-2,2,2-trifluoroethan-l-amine [INT 14-b] (2 g, 7.87 mmol). The mixture was stirred at 25 °C for 16 h. The reaction was quenched by the addition of water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50 mL x 2), dried over anhydrous Na2SO4and concentrated under reduced pressure to give the crude product, which was purified by flash chromatography on silica gel (EtOAc / PE = 0 / 1 to 1 / 5) to give (R)-N-(l-(4-bromophenyl)-2,2,2-trifluoroethyl)tetrahydro-2H-thiopyran-4- carboxamide 1,1-dioxide [INT 14-c] (2.20 g, 5.31 mmol, 67.4% yield) as a white solid. m / z: C 14 H 16 [M+H]+ calculated for C8H8BrF3NO3S 414.0, 416.0; found 416.2.

[0130] To a solution of (R)-N-(l-(4-bromophenyl)-2,2,2-trifluoroethyl)tetrahydro-2H- thiopyran-4-carboxamide 1,1-dioxide [INT 4-c] (1 g, 2.41 mmol) in DMF (10 mL) was added Cs2CO3(1.57 g, 4.82 mmol) and the reaction mixture was stirred at 25 °C for 1 h. Then methyl iodide (1.02 g, 7.23 mmol) was added at 0 °C and the reaction was stirred at 25 °C for 2 h. The reaction was quenched by the addition of water (50 mL) and then extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50 mL x 2), dried over anhydrous Na2SO4and concentrated under reduced pressure to give the crude product which was purified by flash chromatography on silica gel (PE / EtOAc = 1 / 0 to 1 / 1) to give (R)-N-(l-(4-bromophenyl)-2,2,2-trifluoroethyl)-N-methyltetrahydro-2H- thiopyran-4-carboxamide 1,1-dioxide [INT 14.1] (700 mg, 1.63 mmol, 67.9% yield) as colorless oil. m / z: C 15 H 18 [M+H]+calcd for BrF3NO3S 428.0, 430.0; found 430.1.

[0131] (R)-2-methylpropane-2-sulfonamide (12.1 g, 100 mmol) and 4-bromo-2- methylbenzaldehyde [INT 15-a] (10 g, 50.2 mmol) were dissolved in tetrahydrofuran (50 mL) and titanium ethoxide (34.2 g, 150 mmol) was added. The mixture was stirred at 25 °C for 10 h. The reaction mixture was then poured into water (500 mL) and extracted with EtOAc (3 x 300 mL). The organic extracts were combined, dried over Na2SO4and evaporated in vacuo. The residue was purified by flash chromatography (hexane / MTBE = 1 / 0 to 0 / 1) to give (R,E)-N-(4-bromo-2-methylbenzylidene)-2-methylpropane-2- sulfonamide [INT 15-b] (10.4 g, 34.5 mmol, 68.8% yield) as a yellow solid.1H NMR (400 MHz, CDCh) δ 8.76 (1H), 7.75 (d, J = 8.7 Hz, 1H), 7.46-7.37 (m, 2H), 2.56 (s, 3H), 1.24 (s, 9H).

[0132] (R)-2-methylpropane-2-sulfonamide (18.1 g, 150 mmol) and 4-bromo-3- methylbenzaldehyde [INT 15-e] (15 g, 75.3 mmol) were dissolved in tetrahydrofuran (100 mL) and titanium ethoxide (51.3 g, 225 mmol) was added. The mixture was stirred at 60 °C for 10 hours. The reaction mixture was then poured into water (500 mL) and extracted with MTBE (3 x 300 mL). The organic extracts were re-extracted with water (3 x 200 mL), dried over Na2S04and evaporated in vacuo to give (R,E)-N-(4-bromo-3-methylbenzylidene)-2-methylpropane-2- sulfonamide [INT 15-f] as a yellow solid (15.2 g, 50.2 mmol, 66.9% yield). 1 H NMR (400 MHz, CDC13) δ 8.49 (s, 1H). 7.67 (d, J = 2.2 Hz, 1H), 7.65-7.52 (m, 1H), 7.51-7.43 (m, 1H). 2.43 (s, 3H), 1.23 (s, 9H).

[0133] (R,E)-N-(4-bromo-3-methylbenzylidene)-2-methylpropane-2-sulfonamide [INT 15-f] (21 g, 69.4 mmol) and tetrabutylammonium triphenyl difluorosilicate (56.1 g, 104 mmol) were dissolved in THF (500 ml). Trifluoromethyltrimethylsilane (49.3 g, 347 mmol) was added dropwise at -80 °C. The mixture was stirred at -30 °C for 30 minutes before adding aqueous NH4CI (300 mL). The mixture was extracted with EtOAc (2 x 300 ml). The organic phase was dried over sodium sulfate and evaporated in vacuo at 45 °C. The residue was purified by flash chromatography to obtain (R)-N-((S)-1-(4-bromo-3-methylphenyl)-2,2,2- trifluoroethyl)-2-methylpropane-2-sulfonamide [INT 15-g] as a white solid (18.5 g, 49.7 mmol, 71.7% yield), m / z: C 13 H 18 [M+H]+calcd for BrF3NOS 372.0; found 372.0. 1 H NMR (500 MHz, CDC13) δ 7.56 (d, J = 8.2 Hz, 1H), 7.30-7.26 (m, 1H). 7.14-7.08 (m, 1H), 4.75 (p, J = 7.1 Hz, 1H), 3.58 (d, J = 6.4 Hz, 1H), 2.41 (s, 3H), 1.25 (s, 9H).

[0134] (R)-N-((S)-1-(4-bromo-3-methylphenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2- sulfinamide [INT 15-g] (10 g, 26.8 mmol) was dissolved in THF (200 mL). Lithium(l+)bis(trimethylsilyl)amide (74.3 mL, 80.3 mmol) was added at 0°C. The mixture was stirred at 0°C for 20 min. Methyl iodide (22.7 g, 160 mmol) was added. The mixture was stirred at 20°C for 10 h before an aqueous NH4CI solution (200 mL) was added. The mixture was extracted with EtOAc (2 x 200 mL). The organic phase was dried over sodium sulfate and evaporated in vacuo at 45°C to obtain the crude product (R)-N-((S)-1-(4-bromo-3-methylphenyl)-2,2,2-trifluoroethyl)-N,2-dimethylpropane-2- sulfonamide [INT 15-h] as a brown oil (9.09 g, 23.5 mmol, 88.2% yield). m / z: C 14 H 20 [M+H]+calcd for BrF3NOS 386.0; found 386.0. 1 H NMR (400 MHz, CDC13) δ 7.57 (d, J = 8.3 Hz, 1H). 7.34 (s, 1H), 7.17 (d, J = 8.6 Hz, 1H), 5.03 (q, J = 8.5 Hz, 1H), 2.50-2.41 (m, 6H), 1.27 (s, 9H).

[0135] (R)-N-((S)-1-(4-bromo-3-methylphenyl)-2,2,2-trifluoroethyl)-N,2-dimethylpropane-2- sulfinamide [INT 15-h] (10.7 g, 27.7 mmol) was dissolved in methanol (20 mL) before hydrogen chloride (4 M in 1,4-dioxane, 100 mL, 2.54 mol) was added. The mixture was stirred at 20°C for 10 h before evaporation in vacuo at 50°C. MTBE (100 mL) was added. The formed solid was filtered and washed with MTBE (50 mL) to obtain (S)-1-(4-bromo-3-methylphenyl)-2,2,2-trifluoro-N-methylethan-1-amine hydrochloride [INT 15.2] as a beige solid (5.41 g, 16.9 mmol, 61.3% yield), m / z: C 10 H 12 [M+H]+calcd for BrF3N 282.0, 284.0; found 284.0. 1HNMR (500 MHz, DMSO-d6) δ 10.51 (s, 2H), 7.76 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 8.3, 2.2 Hz, 1H), 5.42 (s, 1H), 2.43 (s, 3H), 2.37 (s, 3H).

[0136] Phosphorous oxychloride (788 mg, 5.14 mmol) was added to [(1S)-1-(4-bromo-2- methylphenyl)-2,2,2-trifluoroethyl](methyl)amine hydrochloride [INT 15.1] (500 mg, 1.56 mmol) and 1,1-dioxo-1 lambda*6*-thiopyran-3-carboxylic acid [INT 4-a] (833 mg, 4.68 mmol) in pyridine (3 mL) at 0 °C. The reaction mixture was stirred overnight. Aqueous NaHC03solution (3 mL) was added and the mixture was extracted with EtOAc (3 x 10 mL) and washed with NaHS03(3 x 10 mL). The combined organic layers were dried over anhydrous NaS04and evaporated under reduced pressure. The crude product was purified by HPLC (see conditions below) to give N-[(1S)-1-(4-bromo-2-methylphenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda*6*-thiopyran-3-carboxamide [INT 16.1] as a yellow solid (1.1 g, 1.56 mmol, 100% yield). m / z: C 6 A solution of thiane-4-carboxylic acid [INT 4-a] (833 mg, 4.68 mmol) in pyridine (3 mL) was added phosphorous oxychloride (788 mg, 5.14 mmol) at 0 °C. The reaction mixture was stirred overnight. Aqueous NaHC03solution (3 mL) was added and the mixture was extracted with EtOAc (3 x 10 mL) and washed with NaHS03(3 x 10 mL). The combined organic layers were dried over anhydrous NaS04and evaporated under reduced pressure. The crude product was purified by HPLC (see conditions below) to give N-[(1S)-1-(4-bromo-2-methylphenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda*6*-thiopyran-3-carboxamide [INT 16.1] as a yellow solid (1.1 g, 1.56 mmol, 100% yield). m / z: C 6 Thiane-4-carboxamide [INT 16.3] (132 mg, 0.2985 mmol, 19.1% yield), m / z: C 16 H 20 [M+H]+calcd for BrF3NO3S 442.0, 444.0; found 444.0.

[0137] [(1S)-1-(4-Bromo-3-methylphenyl)-2,2,2-trifluoroethyl](methyl)amine hydrochloride [INT 15.2] (0.5 g, 1.56 mmol) and 1,1-dioxo-1 lambda*6*-thiopyran-3-carboxylic acid [INT 4-a] (833 mg, 4.68 mmol) were mixed in pyridine (2 mL). Phosphorous oxychloride (788 mg, 5.14 mmol) was added. The mixture was stirred at 90 °C for 10 hours. EtOAc (20 mL) was added and the mixture was washed with aqueous NaHS03solution (3 x 5 mL). The organic phase was dried over sodium sulfate and evaporated in vacuum at 45 °C to give the crude product N-[(1S)-1-(4-bromo-3-methylphenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda*6*-thiopyran-3-carboxamide [INT 16.2] as a yellow solid (1.1 g, 1.56 mmol, 100% yield). m / z: C 6 A solution of thiane-4-carboxylic acid [INT 4-a] (833 mg, 4.68 mmol) in pyridine (3 mL) was added phosphorous oxychloride (788 mg, 5.14 mmol) at 0 °C. The reaction mixture was stirred overnight. Aqueous NaHC03solution (3 mL) was added and the mixture was extracted with EtOAc (3 x 10 mL) and washed with NaHS03(3 x 10 mL). The combined organic layers were dried over anhydrous NaS04and evaporated under reduced pressure. The crude product was purified by HPLC (see conditions below) to give N-[(1S)-1-(4-bromo-2-methylphenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda*6*-thiopyran-3-carboxamide [INT 16.1] as a yellow solid (1.1 g, 1.56 mmol, 100% yield). m / z: C 64-Thiazide-4-carboxamide [INT 17.3] (656 mg, 1.48 mmol), m / z: C 16 H 20 The calculated value of [M+H]+ in BrF3NO3S is 442.0; the measured value is also 442.0.

[0138] To N-[(1R)-1-(4-bromophenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1λ 6 1,4-thiazide-4-carboxamide [INT 14.1] (50 mg, 116 μmol) and 2-chloro-7-[(1S)H-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidine-6-amine hydrochloride [INT 1.1] (31.6 mg, 139 μmol) were added to a solution of dioxane (2 mL) with Pd2(dba)3 (10.6 mg, ). Cs₂CO₃ (113 mg, 348 μmol) and xantphos (13.4 mg, 23.2 μmol) were added. The reaction mixture was stirred at 100 °C under N₂ for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC (column: Boston Green ODS 150*30 mm*5 pm, table: 24-64% B (A = water (0.05% ammonium hydroxide)), B = acetonitrile), flow rate: 30 mL / min, UV detector 220 nm) to obtain N-[(1R)-1-[4-({2-chloro-7-[(1S)-1-methoxyethyl]-[L2,4]triazolo[1,5-a]pyrimidin-6-yl}amino)phenyl]-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1λ 6 1,26-Thiazide-4-carboxamide [compound 1.26] (16.4 mg, 28.5 pmol, 24.6% yield), m / z: C 23 H 27 The calculated value of [M+H]+ in ClF3N6O4S is 575.1; the measured value is 575.3. 1HNMR (400MHZ, DMSO-d6) δ = 8.83 (s, 1H), 8.07-7.97 (m, 1H), 7.29-7.17 (m, 2H), 7.03-6.92 (m, 2H), 6.49-6.05 (m, 1H), 5.16 (q, J = 6. 8Hz,1H),3,26-3.19(m,2H),3.16(s,3H),3.13-3.08(m,2H),2.90(s,3H),2.65(s,1H),2.10-1.95(m,4H),1.59(d,J=6.8Hz,3H).

[0139] 2-Chloro-7-[(1R)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidine-6-amine hydrochloride [INT1.3] (60 mg, N-[(1R)-1-(4-bromophenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1λ 6 4-Thiazide-4-carboxamide [1MT 14.1] (97.2mg, A mixture of Pd2(dba)3 (20.7 mg, 22.7 μmol), Xantphos (26.2 mg, 45.4 μmol), and Cs2CO3 (221 mg, 681 μmol) in dioxane (2 ml) was stirred at 100 °C under a N2 atmosphere for 3 hours. The mixture was concentrated under reduced pressure to obtain a crude product, which was purified by rapid chromatography using silica gel (methanol / dichloromethane = 0 / 1 to 1 / 20). The obtained product was purified by preparative HPLC (column: YMCTriart C18 250*50mm*7um, table: 26-66% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 60mL / min, UV detector 220nm) and preparative TLC (SiO2, dichloromethane:methanol {=20:1) to obtain N-[(1R)-1-[4-({2-chloro-7-[(1R)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl}amino)phenyl]-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1λ] as a white dry powder. 6 1,27-Thiazide-4-carboxamide [compound 1.27] (5.20 mg, 9.04 μmol, 4.0% yield), m / z: C 23 H 27 The calculated value of [M+H]+ in ClF3N6O4S is 575.1; the measured value is 575.3. 1H NMR (400 MHz, CD3OD) δ = 8.86 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.52 (q, J = 9.2 Hz, 1H), 5.36 (q, J = 6.8 Hz, 1H), 3.36 (s, 3H), 3.25-3.10 (m, 5H), 3.02-2.75 (m, 3H), 2.35-2.12 (m, 4H), 1.64 (d, J = 6.8 Hz, 3H).

[0140] N-[(1S)-1-(4-bromo-2-methylphenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda 6 - thiazane-4-carboxamide [INT 16.3] (100 mg, 0.2260 mmol), 2-chloro-7-[(1S)-1- methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine [free base of INT 1.1] (51.4 mg, 226 pmol), C S2 A mixture of 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine [free base of INT 1.1] (51.4 mg, 226 pmol), xantphos (26.1 mg, 45.2 pmol) and Pd2(dba)3(20.6 mg, 22.6 pmol) in dioxane (3 mL) was purged with argon. Then N-[(1S)-1-(4-bromo-2-methylphenyl)-2,2,2- trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda 6 - thiazane-4-carboxamide [compound 1.30] (12.5 mg, 0.02128 mmol, 9.4% yield), m / z: C 24 H 29 [M+H]+calcd for C19H21CIF3N6O4S 589.2; found 589.0. 1H NMR (500 MHz, DMSO-d6) δ = 8,80 (s, 1H), 7.90 (s, 1H), 7.29 (d, J = 8.3 Hz, 1H) 6.87-6.75 (m, 2H), 6.43 (q, J = 9.1 Hz, 1H), 5.13 (q, J = 6.8 Hz, 1H), 3.27-3.14 (m, 2H), 3.15 (s, 3H), 3.12-3.04 (m, 3H), 2.79 (s, 3H), 2.15-2.03 (m, 2H). 2.01 (s, 3H), 1.99-1.94 (m, 2H), 1.57 (d, J = 6.7 Hz, 3H).

[0141] N-[(1S)-1-(4-bromo-3-methylphenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 6 - thiazane-4-carboxamide [INT 17.3] (0.1 g, 0.2260 mmol), 2-chloro-7-[(1S)-1- methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine [free base of INT 1.1] (51.4 mg, 226 pmol), Cs2C03(220 mg, 678 pmol) and xantphos (13.0 mg, 22.6 pmol) were mixed in dioxane (2 mL) and the reaction mixture was degassed with argon for 5 minutes. Pd2(dba)3(10.3 mg, 11.3 pmol) was added. The reaction mixture was then degassed with argon for 5 minutes and stirred at 100 °C for 10 hours. The reaction mixture was cooled to room temperature and the solids were filtered off. The filtrate was purified by HPLC (see conditions below) to give N-[(1S)-1-[4-({2-chloro-7-[(1S)-1-methoxyethyl]- [1,2,4]triazolo[1,5-a]pyrimidin-6-yl}amino)-3-methylphenyl]-2,2,2-trifluoroethyl]-N- methyl-1,1-dioxo-1 6 - thiazane-4-carboxamide [compound 1.33] (20.7 mg, 0.03515 mmol, 15.5% yield), m / z: C 24 H 29 [M+H]+calculated for C1F3N6O4S 589.2; found 589.0. 1H NMR (600 MHz, DMSO-d6) δ = 8.74 (s, 1H), 7.2.4 (s, 1H), 7.15 (s, 1H), 7.03 (d, J = 8, 6 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.41 (q, J = 9.4 Hz, 1H), 5.16 (q, J = 6.7 Hz, 1H), 3.21 (d, J = 13.9 Hz, 3H), 3.18-3.05 (m, 5H), 2.89 (s, 3H), 2.34-2.28 (m, 3H), 2.11-1.95 (m, 4H), 1.54 (d, J = 6.7 Hz, 3H).

[0142] 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine hydrochloride salt (Intermediate Synthesis of 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine hydrochloride salt (Intermediate

[0143]

[0144] Synthesis of (S)-tert-butyl 4-methoxy-3-oxopentanoate (INT1-b):

[0145] A solution of (S)-2-methoxypropanoic acid [INT 1-a] (20 g, 192 mmol) in anhydrous tetrahydrofuran (342 mL) was cooled to 0 °C. Carbonyldiimidazole (30.6 g, 189 mmol) was added in portions at 0 °C and the mixture was stirred at this temperature for 1.25 h. In a separate flask, to a solution of 3-(tert-butoxy)-3-oxopropanoic acid (46.1 g, 288 mmol) in anhydrous tetrahydrofuran (342 mL) at 0 °C was added 1-methylethylmagnesium chloride (1+) (249 mL, 499 mmol, 2 M in THF) and the mixture was stirred at room temperature for 1.25 h. Then, this solution was added to the acylimidazole solution at 0 °C through a cannula and the resulting mixture was stirred at room temperature overnight. The reaction mixture was cooled to 0 °C and quenched by the addition of a 10% aqueous citric acid solution, extracted with EtOAc, washed with saturated aqueous NaHC03, dried over anhydrous Na2S04and concentrated under reduced pressure to give the crude product which was purified by flash chromatography on silica gel (acetone / hexane = 0 / 1 to 1 / 9) to give (S)-tert-butyl 4-methoxy-3-oxopentanoate [INT 1-b] as an oil (30.0 g, 148 mmol, 55.6%).

[0146] Synthesis of (S)-tert-butyl 2-amino-7-(1-methoxyethyl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (INT1-c): Synthesis of 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylic acid (INT1-d):

[0147] A solution of (S)-tert-butyl 4-methoxy-3-oxopentanoate [INT 1-b] (25 g, 123 mmol) and (dimethoxymethyl)dimethylamine (11.1 mL, 83.6 mmol) was heated at 120 °C for 1.5 h. The mixture was cooled to room temperature and 4H-1,2,4-triazole-3,5-diamine (12.1 g, 123 mmol) was added followed by ethanol (123 mL) and the mixture was heated at 85 °C for 1 h. Upon completion, the mixture was concentrated under reduced pressure and recrystallized with EtOH / water (1:1, 600 mL), filtered and the filter cake was washed with 30% EtOH / water followed by MTBE to give tert-butyl 2-amino-7-(1-methoxyethyl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (12.7 g, 43.2 mmol, 52%) as a solid. The filtrate was concentrated under reduced pressure to remove MTBE and the solid was filtered and washed with hexanes to give more tert-butyl 2-amino-7-(1-methoxyethyl)-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate [INT 1-c] (5.3 g, 18.0 mmol, 23%) as a solid. Total 18.0 g, 75% yield. Chiral HPLC showed 96.9% ee. 1 H NMR (400 MHz, CDC13) δ = 8.75 (s, 1H), 5.40 (q, J = 6.8 Hz, 1H), 4.95 (br s, 2H), 3.30 (s, 3H), 1.75 (d, J = 6.8 Hz, 3H), 1.62 (s, 9H).

[0148] Synthesis of N-{2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl}carbamic acid tert-butyl ester (INT1-e): Synthesis of 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine hydrochloride salt (Intermediate

[0149] To a mixture of 2-amino-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5- a]pyrimidine-6-carboxylic acid tert-butyl ester [INT 1-c] (1.2 g, 4.09 mmol) and copper (II) chloride dihydrate (173 mg, 1.02 mmol) in cone. HC1 (20 mL) was added a solution of sodium nitrite (338 mg, 4.90 mmol) in H2O (5 mL) at 5 °C using an ice bath and the mixture was stirred at 5 °C for 30 min. The mixture was then warmed to 25 °C and stirred for 16 h. Water (100 mL) was added and 1 N aqueous NaOH was added to adjust the pH to 3-4. The mixture was extracted with CHCl3:i-PrOH = 3:1 (100 mL x 3) and the combined organic layers were dried over anhydrous Na2SO4and concentrated under reduced pressure to give 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5- a]pyrimidine-6-carboxylic acid [INT 1-d] as a solid (962 mg, 92.4% yield). m / z: C9H 10 [M+H]+calcd for ClN4O3 257.0; found 256.9. 1 H NMR (400 MHz, DMSO-d6) d = 14.00 (br s, 1H), 9.07 (s, 1H), 5.39 (q, J = 6.4 Hz, 1H), 3.21 (s, 3H), 1.63 (d, J = 6.4 Hz, 3H).

[0150] Synthesis of 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6-amine hydrochloride salt (Intermediate Synthesis of tetrahydro-2H-thiopyran-4-carbonyl chloride 1,1-dioxide (Intermediate 4.1): Synthesis of (S)-N-(1-(4-bromophenyl)-2,2,2-trifluoroethyl)-N-methyltetrahydro-2H-thiopyran-4-carboxamide 1,1-dioxide (Intermediate 5.1)

[0151] To a solution of 2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5- a]pyrimidine-6-carboxylic acid [INT 1-d] (1.3 g, 5.06 mmol) in t-BuOH (10 mL) was added {[azido(phenoxy)phosphoryl]oxy}benzene (2.08 g, 7.58 mmol) and triethylamine (1.02 g, 10.1 mmol) and the mixture was stirred at 100 °C under N2atmosphere for 2 h. The mixture was concentrated under reduced pressure to give the crude product which was purified by flash chromatography on silica gel (EtOAc / petroleum ether = 1 / 10 to 1 / 5) to give tert-butyl N-{2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5- a]pyrimidin-6-yl}carbamate [INT 1-e] as a solid (420 mg, 25.4% yield). m / z: C 13 H 19[M+H]+calcd for C15H20N5O3, 328.1 ; found, 328.0. 1 H NMR (400 MHz, CDC13) δ = 9.62 (br s, 1H), 8.05 (s, 1H), 5.45 (q, J = 6.8 Hz, 1H), 3.48 (s, 3H), 1.63 (d, J = 6.8 Hz, 3H), 1.56 (s, 9H).

[0152] Synthesis of N-[(1S)-1-[4-({2-chloro-7-[(1S)-1-methoxyethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-6- yl}amino)phenyl]-2,2,2-trifluoroethyl]-N-methyl-1,1-dioxo-1 lambda6-thiane-4-carboxamide (also known as N- ((S)-1-(4-((2-chloro-7-((S)-1-methoxyethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)amino)phenyl)-2,2,2- trifluoroethyl)-N-methyltetrahydro-2H-thiopyran-4-carboxamide 1,1-dioxide) (Compound 1.1) Synthesis of 1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazol-4-amine

[0153] A mixture of tert-butyl N-{2-chloro-7-[(1S)-1-methoxyethyl]- [1,2,4]triazolo[1,5-a]pyrimidin-6-yl}carbamate [INT 1-e] (420 mg, 1.28 mmol) in 4 N HC1 / dioxane (5 mL) was stirred at 25 °C for 2 hours. LCMS showed the reaction was complete and a new peak with desired MS was detected (Rt = 0.611 min, m / z: 227.8 [M+H]+). The mixture was concentrated under reduced pressure to give 2-chloro-7-[(1S)-1-methoxyethyl]- [1,2,4]triazolo[1,5-a]pyrimidin-6-amine hydrochloride [INT 1.1] as a solid (400 mg, crude).

[0154] Synthesis of (R)-N-((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2-sulfenamide

[0155]

[0156] To a solution of tetrahydro-2H-thiopyran-4-carboxylic acid 1,1-dioxide [INT 4-a] (41.0 g, 230 mmol, 1.0 equiv) in DCM (410 mL) was added (COCl)2(58.4 g, 460 mmol, 40.3 mL, 2.0 equiv) and DMF (168 mg, 2.30 mmol, 177 μί, 0.01 equiv) at 0 °C under N2. The mixture was warmed to 20 °C and stirred at 20 °C for 2 hours. The suspension became clear, which indicated most of the starting material was consumed. The reaction mixture was concentrated in vacuum to give the crude product as a solid, which was concentrated by oil pump to remove the solvent residue to give tetrahydro-2H-thiopyran-4-carbonyl chloride 1,1-dioxide [INT 4.1] as a solid (46.5 g, crude).

[0157] Synthesis of methyl (1r,4r)-4-(chlorocarbonyl)cyclohexane-1-carboxylate Synthesis of methyl (1S,4r)-4-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)(methyl)carbamoyl)cyclohexane- 1-carboxylate

[0158]

[0159] To a solution of (S)-l-(4-bromophenyl)-2,2,2-trifluoro-N-methylethan-l-amine hydrochloride [INT 3.1] (39.0 g, 128 mmol, 1.0 eq, HC1) and TEA (45.7 g, 451 mmol, 62.8 mL, 3.5 eq) in DCM (200 mL) was added tetrahydro-2H-thiopyran-4-carbonyl chloride 1,1-dioxide [INT 4.1] (45.3 g, 231 mmol, 1.8 eq) at 0-10 °C. The mixture was stirred at 20 °C for 12 h. The mixture was separated to get the organic layer and the aqueous layer was extracted with DCM (100 mL). The combined organic layer was concentrated in vacuo to get the crude product as an oil. The crude product was purified by column chromatography with silica gel using petroleum ether / ethyl acetate (15 / 1 ~ 3 / 1) to get (S)-N-(l-(4-bromophenyl)-2,2,2-trifluoroethyl)-N-methyltetrahydro-2H-thiopyran-4- carboxamide 1,1-dioxide [INT 5.1] (26.0 g, 60.7 mmol, 47.4% yield, 100% purity) as a solid. 1 H NMR (400 MHz, CDC13) δ 2.25 - 2.37 (m, 1H), 2.38 - 2.40 (m, 3H), 2.88 - 3.00 (m, 6H), 3.30 - 3.31 (m, 1H), 3.22 - 3.45 (m, 1H), 6.56 - 6.63 (m, 1H), 7.23 (d, J = 8.00 Hz, 2H), 7.55 (d, J = 8.40 Hz, 2H).

[0160] SFC: Rt = 1.21 min, 100.0% ee; Column: Chiralpak AD-3 50 x 4.6 mm I.D., 3 um; Mobile phase: A: CO2, B: MeOH (0.05% IPAm, v / v); Flow rate: 3.4 mL / min; Column temperature: 35 °C.

[0161] LCMS: Rt = 2.431 min, 100% purity, m / z = 428.0, 430.0 (M+l)+. The gradient was 0.40 min 5% B, 0.4-3.0 min 5-95% B, hold 95% B 1.00 min, then 0.01 min 95-5% B at a flow rate of 1.0 ml / min. The mobile phase A was water with 0.037% trifluoroacetic acid and the mobile phase B was acetonitrile with 0.018% trifluoroacetic acid. The column used for the chromatography was Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and positive electrospray ionization. The MS range was 100-1000.

[0162] Synthesis of methyl (1r,4r)-4-{methyl[(1S)-2,2,2-trifluoro-1-(4-{[1-(pyridin-2-yl)-5-(trifluoromethyl)-1H- pyrazol-4-yl]amino}phenyl)ethyl]carbamoyl}cyclohexane-1-carboxylateFigure 1 Figure 2 Figure 3

[0163]

[0164] A mixture of 2-chloro-7-[(1 S)-1 -methoxyethyl]-[1,2,4]triazolo[1,5- a]pyrimidin-6-amine hydrochloride [INT 1.1 ] (460 mg, 1.74 mmol), N-[(1 S)-1 -(4- bromophenyl)-2,2,2-trifluoroethyl]-N-methyl-1,1 -dioxo-1 lambda6-thiophane-4- carboxamide [INT 5.1 ] (779 mg, 1.82 mmol), Pd2(dba)3(159 mg, 174 pmol), Xantphos (201 mg, 348 pmol) and Cs2CO3(1.70 g, 5.22 mmol) in dioxane (6 mL) was stirred at 100 °C for 4 h. The mixture was concentrated under reduced pressure to give a crude product which was purified by flash chromatography on silica gel (methanol / dichloromethane = 0 / 1 to 1 / 10) and preparative HPLC (column: YMC Triart C18 250*50 mm*7 pm, Table: 29-58% B (A = water (0.05% ammonia hydroxide v / v), B = acetonitrile), flow rate: 60 mL / min, UV detector 220 nm) to give N-[(1 S)-1 -[4-({2-chloro-7-[(1 S)-1 -methoxyethyl]- [1,2,4]triazolo[1,5-a]pyrimidin-6-yl}amino)phenyl]-2,2,2-trifluoroethyl]-N-methyl-1,1 - dioxo-1 lambda6-thiophane-4-carboxamide [compound 1.1 ] (211 mg, 368 pmol) as a dry powder. m / z: C 23 H 27 [M+H]+calcd for ClF3N6O4S 575.2; found 575.3. 1 H NMR (400 MHz, DMSO-d6) d = 8.80 (s, 1 H), 8.02-7.94 (m, 1 H), 7.26-7.12 (m, 2H), 6.99-6.89 (m, 2H), 6.45-6.02 (m, 1 H), 5.12 (q, J = 6.8 Hz, 1 H), 3.25-3.14 (m, 3H), 3.12 (s, 3H), 3.10-3.05 (m, 2H), 2.86 (s, 3H), 2.08-1.94 (m, 4H), 1.55 (d, J = 6.8 Hz, 3H). The chiral purity of compound 1.1 was determined to be at least 89%.

[0165] The synthesis of other MALT1 inhibitors is described in WO 2022 / 081967 (PCT / US21 / 55173), which is incorporated by reference in its entirety for all purposes.

[0166] Synthesis of MALT1 inhibitors of Formula Ia

[0167] Scheme 2

[0168]

[0169] The starting material G-2a is treated with a base (LiOH.H2O or NaOH) to provide a compound of Formula (B). R 2 is phenyl, pyridyl, or pyridazine; R 1 is CF3or (S)-methoxyethane; and m and n are each independently 0 or 1. 3' is methyl or benzyl.

[0170] ​

[0171]

[0172] To a solution of ethyl 2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutanoate (5 g, 20.8 mmol) and 2-hydrazinylpyridine [INT 1-iii] (2.26 g, 20.8 mmol) in EtOH (30 mL) was added triethylamine (2.10 g, 20.8 mmol). The mixture was stirred at 80 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give the crude product which was purified by flash chromatography with silica gel (EtOAc / PE = 0 / 1 to 10 / 1) to give ethyl 1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate [INT 1-jjj] (5.00 g, 17.5 mmol, 84.3% yield) as a yellow oil. m / z: C 12 H 11 [M+H]+calcd for F3N3O2 286.1; found 285.9. 1 H NMR (400 MHz, DMSO-d6) δ = 8.64-8.59 (m, 1H), 8.34 (s, 1H), 8.19-8.12 (m, 1H), 7.80 (dd, J = 0.8, 8.0 Hz, 1H), 7.68-7.63 (m, 1H), 4.33 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H).

[0173] To a mixture of ethyl 1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate [INT 1-jjj] (1 g, 3.50 mmol) in THF (6 mL) and H2O (2 mL) was added lithium hydroxide monohydrate (440 mg, 10.5 mmol). The reaction mixture was stirred at 15 °C for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was acidified with 1M HC1 to pH = 4. The reaction mixture was quenched by the addition of H2O (20 mL) and extracted with EtOAc (20 mL x 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid [INT 1-kkk] as a yellow solid (800 mg, 3.11 mmol, 88.8% yield). 1 H NMR (400 MHz, DMSO-d6) d = 13.43 (br s, 1H), 8.60 (td, J = 0.8, 4.8 Hz, 1H), 8.28 (s, 1H), 8.14 (dt, J = 2.0, 7.6 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.64 (ddd, J = 0.8, 4.8, 7.6 Hz, 1H).

[0174] A mixture of 1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid [INT 1-kkk] (400 mg, 1.55 mmol), triethylamine (784 mg, 7.75 mmol) and diphenyl phosphorazide (564 mg, 2.32 mmol) in t-BuOH (3 mL) and dioxane (3 mL) was stirred at 100 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give the crude product which was purified by flash chromatography with silica gel (petroleum ether / EtOAc = 1 / 0 to 1 / 1) to give tert-butyl N-[1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazol-4-yl]carbamate [INT 1-lll] as a white solid (470 mg, 1.43 mmol, 92.5% yield). m / z: C 14 H 16 [M+H]+ calculated for F3N4O2 329.1; found 328.9. 1 H NMR (400 MHz, DMSO-d6) d = 9.14 (br s, 1H), 8.56-8.51 (m, 1H), 8.06 (dt, J = 1.8, 7.6 Hz, 1H), 7.95 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.55-7.46 (m, 1H), 1.46 (s, 9H).

[0175] A solution of tert-butyl N-[l-(pyridin-2-yl)-5-(trifluoromethyl)-lH-pyrazol-4- yl]carbamate [INT 1-lll] (470 mg, 1.43 mmol) in 4 M HC1 / dioxane (4 mL) was stirred at 15 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give l-(pyridin-2-yl)-5-(trifluoromethyl)-lH-pyrazol-4-amine [INT 1.15] as a white solid (260 mg, 1.13 mmol, 79.7% yield). m / z: [M+H]+ calculated for C9H8F3N4 229.1; found 228.9. 1 H NMR (400 MHz, DMSO-d6) d = 8.48 (dd, J = 1.2, 4.8 Hz, 1H), 8.01 (dt, J = 1.6, 7.6 Hz, 1H), 7.76 - 7.72 (m, 2H), 7.65 (br s, 2H), 7.43 (dd, J = 4.8, 6.8 Hz, 1H).

[0176] ​

[0177]

[0178] To a solution of 4-bromobenzaldehyde [INT 2-a] (100 g, 541 mmol, 1.0 equiv) in toluene (500 mL) was added (R)-2-methylpropane-2-sulfmamide (72.1 g, 595 mmol, 1.1 equiv) at 25 °C. The mixture was stirred at 25 °C for 15 min. To the above reaction was then added NaOH (21.6 g, 541 mmol, 1.0 equiv) and the mixture was stirred at 25 °C for 12 h. Na2S04(50 g) was added to the mixture and stirred for 20 min. Four reaction mixtures were combined and filtered through celite to give a filtrate which was concentrated in vacuo to give the crude product as an oil. The crude product was dissolved in petroleum ether (1.0 L) and stirred at -50 °C for 1.0 h, filtered to give (R,E)-N-(4-bromo-benzylidene)-2-methylpropane-2-sulfmamide [INT 2-b] as a solid (620 g, 2.15 mol, 99.5% yield).

[0179] To a solution of (R,E)-N-(4-bromobenzylidene)-2-methylpropane-2-sulfmamide [INT 2-b] (206 g, 715 mmol, 1.0 equiv) and tetrabutylammonium acetate (216 g, 715 mmol, 218 mL, 1.0 equiv) in DMF (1.4 L) was added TMSCF3(259 g, 1.82 mol, 2.5 equiv) at 0 °C. The mixture was stirred at 5 °C for 1.5 h. The procedure was repeated 2 times and the three reaction mixtures were combined for work-up. The mixture was poured into saturated NH4Cl solution (13.0 L) and stirred for 10 min to give a suspension. The suspension was filtered and the filter cake was washed with water (5.0 L). The filter cake was triturated with MTBE / petroleum ether (v / v = 1 :4, 2.0 L) to give the product as a solid and the mother liquor was concentrated in vacuo to give the crude product as an oil, which was purified by column chromatography with silica gel using petroleum ether / ethyl acetate (10 / 1-1 / 1) to give (R)-N-((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2-sulfmamide [INT 2.1] as a solid (389 g, 1.09 mol, 50.6% yield). 1 H NMR (400 MHz, CDC13) δ = 1.25 (s, 9H), 3.64 (d, J = 6.40 Hz, 1H), 4.79-4.83 (m, 1H), 7.32 (d, J = 8.40 Hz, 2H), 7.56 (d, J = 6.40 Hz, 2H).

[0180] Synthesis of (S)-1-(4-bromophenyl)-2,2,2-trifluoro-N-methylethan-1-amine

[0181]

[0182] To a solution of LiHMDS (1.0 M, 838 mL, 3.0 eq) was added (R)-N-((S)-1-(4- bromophenyl)-2,2,2-trifluoroethyl)-2-methylpropane-2-sulfmide [INT 2.1] (100 g, 279 mmol, 1.0 eq) at 0-10 °C and the resulting mixture was stirred at 0-10 °C for 0.5 h. To the above mixture was added Mel (119 g, 838 mmol, 52.1 mL, 3.0 eq) at 0-10 °C and the reaction was stirred at 25 °C for 1 h. The procedure was repeated 2 times, the three combined reaction mixtures were poured into saturated aqueous NH4Cl (3.0 L) and diluted with EtOAc (1.0 L). The mixture was separated to get the organic layer and the aqueous layer was extracted with EtOAc (500 mL). The combined organic layers were washed with saturated NaCl (1.0 L) and dried over Na2S04, filtered and concentrated in vacuo to give the crude product as an oil. The crude product was purified by column chromatography with silica gel using petroleum ether / ethyl acetate (15 / 1-1 / 1) to give (R)-N-((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-N,2-dimethylpropane-2- sulfmide [INT 3-b] as an oil (161 g, 432.5 mmol, 51.6% yield).

[0183] To a mixture of (R)-N-((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)-N,2-dimethylpropane- 2-sulfmide [INT 3-b] (202 g, 543 mmol, 1.0 eq) in EtOAc (600 mL) was added HCl / EtOAc (4.0 M, 2.02 L, 14.9 eq) slowly. The above mixture was stirred at 20 °C for 1 h. The reaction mixture was filtered to give a solid, which was eluted with EtOAc (200 mL) and the mother liquor was concentrated in vacuo to give a solid. The solid was purified by column chromatography with silica gel using petroleum ether / ethyl acetate (10 / 1-1 / 0) and combined with the filter cake and concentrated with an oil pump at 45 °C for 1 h to remove the solvent residue to give (S)-1-(4-bromophenyl)-2,2,2-trifluoro-N-methylethan-1- amine hydrochloride [INT 3.1] as a solid (115 g, 378 mmol, 69.6% yield, 100% purity, HC1). 1 H NMR (400 MHz, DMSO-d6) d = 2.45 (s, 3H), 5.51 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.40 Hz, 2H), 10.59 (s, 2H).

[0184] SFC: Rt = 0.776 min, 99.98% ee; Column: Chiralpak AD-3, 100 x 4.6 mm, I.D., 3 μm; Mobile phase: A: CO2, B: MeOH (0.05% IPAm); Gradient: A: B = 97:3; Flow rate: 3 mL / min; Column temperature: 35 °C.

[0185] LCMS: Rt = 1.755 min, purity 100.0%, m / z = 268.0, 270.0 (M+1)+ The gradient was 5% B over 0.40 min, 5-95% B over 0.4-3.0 min, hold 95% B 1.00 min then 0.01 min 95-5% B at a flow rate of 1.0 ml / min. The mobile phase A was water with 0.037% trifluoroacetic acid and the mobile phase B was acetonitrile with 0.018% trifluoroacetic acid. The column used for the chromatography was a Kinetex C18 50*2.1 mm column (5 μm particles). The detection method was diode array (DAD) and positive electrospray ionisation. The MS range was 100-1000.

[0186] ​

[0187]

[0188] To a mixture of (1r,4r)-4-(methoxycarbonyl)cyclohexane-1 -carboxylic acid [INT 4-d] (1.45 g, 7.78 mmol) in dichloromethane (10 mL) was added slowly oxalyl chloride (2.93 g, 23.3 mmol) and DMF (56.8 mg, 778 μmol) and the mixture was stirred at 40 °C for 2 hours. The mixture was concentrated under reduced pressure to give crude methyl (1r,4r)-4-(chlorocarbonyl)cyclohexane-1 -carboxylate [INT 4.4] (1.59 g, 7.76 mmol) as a yellow gum.

[0189] ​ ​

[0190]

[0191] To a mixture of (1r,4r)-methyl 4-(chloroformyl)cyclohexane-1-carboxylate [INT 4.4] (1.59 g, 7.76 mmol) and Et3N (2.65 g, 26.2 mmol) in dichloromethane (6 mL) was added a solution of [(1S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl](methyl)amine hydrochloride [INT 3.1] (1.6 g, 5.25 mmol) in dichloromethane (6 mL) and the mixture was stirred at 25 °C for 16 h. Water (30 mL) was added and the mixture was extracted with dichloromethane (30 mL x 2). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a crude product which was purified by flash chromatography with silica gel (EtOAc / petroleum ether = 1 / 10 to 1 / 5) to give (1S,4r)-methyl 4-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)(methyl)carbamoyl)cyclohexane-1-carboxylate [INT 5.4] (1.10 g, 2.52 mmol, 32.5% yield) as a yellow oil. m / z: C 18 H 22 [M+H]+calcd for BrF3NO3, 436.1, 438.1; found, 438.0.

[0192] ​ ​

[0193]

[0194] To a solution of 1-(pyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazol-4-amine [INT 1.15] (100 mg, 438 µmol), (1r,4r)-4-{[(1S)-1-(4-bromophenyl)-2,2,2- trifluoroethyl](methyl)carbamoyl}cyclohexane-1-carboxylate [INT 5.4] (191 mg, 438 µmol), Cs2CO3 (426 mg, 1.31 mmol) and xantphos (50.6 mg, 87.6 µmol) in dioxane (3 mL) was added Pd2(dba)3 (40.1 mg, 43.8 µmol) and the reaction mixture was stirred at 100 °C under N2 for 2 h. The reaction mixture was concentrated under reduced pressure to give the crude product which was purified by flash chromatography with silica gel (EtOAc / PE = 0 / 1 to 1 / 3) to give (1r,4r)-4-{methyl[(1S)-2,2,2-trifluoro-1-(4-{[1-(pyridin-2-yl)-5- (trifluoromethyl)-1H-pyrazol-4-yl]amino}phenyl)ethyl]carbamoyl}cyclohexane-1- carboxylate [INT 6.4] (170 mg, 291 µmol, 66.6 % yield) as a yellow oil. m / z: C 27 H 28 [M+H]+ calculated for F6N5O3 584.2; found 584.1.

[0195] The synthesis of MALT1 inhibitors is also described in WO 2023 / 192506 (PCT / US23 / 16941), which is incorporated by reference in its entirety for all purposes.

[0196] Pharmaceutical compositions and administration

[0197] The compounds provided according to the present disclosure are generally administered in the form of pharmaceutical compositions. The present disclosure provides pharmaceutical compositions containing as active ingredients one or more of the compounds described or a pharmaceutically acceptable salt or ester thereof and one or more pharmaceutically acceptable excipients, carriers (including inert solid diluents and fillers), diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers and adjuvants. Such compositions can also contain buffers, such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical compositions can be administered alone or in combination with other therapeutic agents. Such compositions are prepared in a manner well known in the medical arts (see, e.g., Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd edition (G. S. Banker and C. T. Rhodes, Eds.), which are incorporated by reference in their entirety for all purposes.

[0198] Suitable pharmaceutically acceptable excipients are well known to those skilled in the art. Examples of pharmaceutically acceptable excipients include phosphate buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), aqueous solutions containing an inorganic acid salt such as hydrochloride, hydrobromide, phosphate, or sulfate, saline, solutions of glycols or ethanol, and organic acid salts such as acetate, propionate, malonate, or benzoate. Adjuvants such as wetting agents or emulsifiers and pH buffers can also be used. Pharmaceutically acceptable excipients described in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991), which is incorporated by reference herein in its entirety for all purposes, can be used as appropriate. The compositions can be formulated to be suitable for oral administration or parenteral administration (e.g., injection or infusion). The compositions can include formulation additives such as suspending, preserving, stabilizing and / or dispersing agents and preservatives to extend the shelf-life of the composition during storage.

[0199] The pharmaceutical composition can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although an appropriate dosage can be determined through clinical trials.

[0200] The pharmaceutical composition can be administered in single or multiple doses by any of the accepted modes of agent administration known to have similar efficacy, for example, as described in those patents and patent applications incorporated by reference, including rectally, buccally, sublingually, intranasally, and transdermally, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or for example, by impregnation or coating of devices such as stents, or arterial insertion cylindrical polymers.

[0201] One mode of administration is parenteral, particularly by injection. The novel compositions of the present disclosure can be incorporated in forms for administration by injection including aqueous or oleaginous suspensions or emulsions with sesame oil, corn oil, cottonseed oil or peanut oil, and elixirs, mannitol, dextrose or sterile aqueous solutions and similar pharmaceutical vehicles. Saline solutions are also commonly used for injection, but are not preferred for use in the context of the present invention. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, etc. (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be employed. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0202] Sterile injectable solutions are prepared by incorporating the compound according to the present disclosure in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile- filtered solution thereof.

[0203] Oral administration is another route of administration for the compounds according to the present disclosure. Administration can be by capsule or enteric-coated tablet, among others. In the manufacture of a pharmaceutical composition of at least one of the compounds described herein, active ingredient is typically diluted by an excipient and / or enclosed in a suitable carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be in solid, semisolid, or liquid materials, as described above, which act as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

[0204] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginic acid, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propylhydroxybenzoates; sweetening agents; and flavoring agents.

[0205] The compositions of the present application can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release pharmaceutical delivery systems for oral administration include osmotic pump systems and dissolution systems containing polymer-coated reservoirs or a drug-polymer matrix formulation. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345. Another formulation for use in the methods of the present application employs transdermal delivery devices (“patches”). Such transdermal patches can be used to provide continuous or discontinuous infusion of the compounds of the present application in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known

[0206] The compositions are preferably formulated in a unit dosage form. The term "unit dosage form" refers to physically discrete units suitable for use in administering a unit dosage to a human subject and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, along with suitable pharmaceutical excipients (e.g., tablets, capsules, ampoules). The compounds are generally administered in a pharmaceutically effective amount. Preferably, for oral administration, each dosage unit contains from 1 mg to 2 g of a compound described herein, and for parenteral administration, preferably from 0.1 mg to 700 mg of a compound described herein. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0207] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that there are no significant variations in the active ingredient concentration within the composition. When such preformulation compositions are treated or combined with excipients, they result in a homogeneous combination with the active ingredient either dispersed predominantly in a solid phase matrix or with the active ingredient dispersed primarily in the liquid phase.

[0208] Tablets or pills of the disclosure can be coated or otherwise compounded to provide a dosage form with a prolonged or sustained action or to protect the active ingredient from the acidic conditions of the stomach. For example, a tablet or pill can comprise an inner dosage component and an outer dosage component, the latter being in the form of an envelope surrounding the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, ethanol, and cellulacetate.

[0209] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of inert gases for inhalation. Nebulized solutions can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

[0210] MALT1-Related Diseases

[0211] Mucosa-associated lymphatic tissue lymphoma translocase 1 (MALT1). MALT1 is a caspase-like protease that plays a role in BCL10-induced NF-κΒ activation and / or mTor pathway (e.g., mTORcl and / or mTORc2) activation and / or Jun / Fos activation. The protein is a component of the CARMA1-BCL10-MALT1 (CBM) signaling complex that triggers NF-κΒ signaling and / or mTor pathway (e.g., mTORcl and / or mTORc2) signaling and / or Jun / Fos signaling to induce lymphocyte activation following antigen-receptor stimulation. Many MALT1 -related genetic diseases cause constitutive or hyperactive activation of NF-κΒ and / or hyperactivation of mTor pathway (e.g., mTORcl and / or mTORc2) and / or hyperactivation of Jun / Fos and / or hyperactivation of other signaling pathways by increasing the activation signals through MALT1 or decreasing the downregulation signals through MALT1.

[0212] B-cell expansion with NF-κΒ and T-cell impairment (BENTA) is a rare genetic disorder of the immune system caused by mutations in the gene CARD11 (caspase recruitment domain family member 11). The disease can be characterized by high levels of certain B-cells starting in infancy (B-cell lymphocytosis), enlarged spleen (splenomegaly), enlarged lymph nodes (lymphadenopathy), immune deficiency, and / or increased risk of lymphoma.

[0213] CARD11 encodes a membrane-associated guanylate kinase (MAGUK), a class of proteins that act as molecular scaffolds that assemble multiprotein complexes at specific regions of the plasma membrane. The protein is also a member of the CARD protein family, which is defined by the possession of a characteristic caspase-associated recruitment domain (CARD). The CARD domain of the protein specifically interacts with BCL10, a protein known to act as a positive regulator of apoptosis, NF-κΒ activation, mTor pathway (e.g., mTORcl and / or mTORc2) activation. When expressed in cells, this protein activates NF-κΒ, mTor pathway (e.g., mTORcl and / or mTORc2), and induces phosphorylation of BCL10.

[0214] BENTA disease is inherited in an autosomal dominant manner, or can be caused by de novo mutations in the CARD11 gene. BENTA disease is caused by "gain-of-function" mutations in the gene CARD11, which provides instructions for the production of the CARD11 protein. These gain-of-function mutations cause the CARD11 protein to be overactive. The CARD11 protein is required for activation of the NF-κΒ and / or mTor pathways (e.g., mTORcl and / or mTORc2) and / or activation of Jun / Fos in both B cells and T cells, which are critical for a healthy immune response. In BENTA disease, development and differentiation of B cells can also be partially impaired. BENTA disease is inherited in an autosomal dominant manner. For a person to have BENTA disease, only one copy of the two copies of CARD11 needs to be abnormal.

[0215] In the CARD11 gain-of-function alleles, the MALT1 signaling pathway becomes constitutively active, and the Jun / Fos and / or NF-κΒ and / or mTor pathways (e.g., mTORcl and / or mTORc2) activation in immune cells occurs continuously. This leads to a lymphoproliferative syndrome known as BENTA. BENTA patients have elevated levels of inflammation and are predisposed to recurrent infections, autoimmunity, lymphoma, and hemophagocytic lymphohistiocytosis (HLH). HLH is a rare but potentially fatal condition in which certain white blood cells (histiocytes and lymphocytes) accumulate in organs and damage them (including the bone marrow, liver, and spleen), and destroy other blood cells. BENTA-associated mutations can be located within the N-terminal portion of CARD11, which contains the CARD, LATCH, and coiled-coil domains. These domains are responsible for CARD11 oligomerization and recruitment of BCL10 and MALT1, making this region a hot spot for CARD11 mutations. CARD11 mutants can spontaneously cluster to form active signaling clusters with BCL10, MALT1, and active IKK, triggering constitutive NF-κΒ activation and / or mTor pathways (e.g., mTORcl and / or mTORc2) activation and / or Jun / Fos activation in the absence of antigen receptor stimulation. Examples of BENTA-associated mutations include, for example, the following CARD11 mutations: R30G, Q or W, C49Y or S or E or F or N, F115I, T117P, G123S or D, G126R, T128M, F130I or C, E134G.

[0216] BENTA patients typically have a mild immune deficiency and are susceptible to recurrent sinus and lung infections, as well as infections with viruses such as molluscipoxvirus, Epstein-Barr virus, or BK virus. BENTA patients also have an increased risk of developing lymphoma (e.g., large B-cell lymphoma).

[0217] Most B cells in the blood of BENTA patients are naive, mature B cells, with elevated levels of a subtype of B cells known as transitional B cells. Laboratory studies also show poor B cell differentiation and immunoglobulin or antibody secretion. Serum IgM is low in most patients, and total IgG and IgA are typically at the lower end of the normal range. Some patients have a poor immune response to certain vaccines. T cell counts in people with BENTA are within or slightly above the normal range, but T cells can respond poorly to certain foreign pathogens, hence the word “disabled” as part of the BENTA acronym.

[0218] Other genetic diseases are associated with increased signaling through the MALT1 pathway. For example, A20 haploinsufficiency, HOIL1 hypomorphic allele disease, CARD14 generalized pustular psoriasis, and NF-κΒ gain-of-function syndrome.

[0219] A20 haploinsufficiency is an autosomal dominant genetic disease caused by a pathogenic mutation in the tumor necrosis factor (TNF)-a-induced protein 3 gene. As a result, production of nuclear factor (NF)-κΒ regulatory protein A20 encoded by the TNFAIP3 gene is insufficient. Protein A20 (also known as TNAP3) is encoded by TNFAIP3 and plays a crucial role in the negative regulation of inflammation and immunity.

[0220] HOIL1 or HOIP deficiency is associated with immune disorders involving symptoms of autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. MALT1 paracaspase is a novel negative regulator of LUBAC through proteolytic cleavage of HOIL1. Linear ubiquitin chain assembly complex (LUBAC) is composed of HOIL1, HOIP, and SHARPIN, which catalyzes linear ubiquitination of target proteins - a post-translational modification that is critical for NF-κΒ activation. HOIL1 or HOIP deficiency is caused by loss-of-function mutations in HOIL1.

[0221] Other diseases associated with MALT1 include, for example, autoinflammatory conditions (a state of severe, recurrent, or persistent inflammation without apparent cause); hyperinflammatory conditions (a state of severe or persistent inflammation far greater than the triggering stimulus); autoimmune or autoinflammatory or hyperinflammatory states due to a genetic mutation causing a "gain-of-function" (including partial gain-of-function) state in a protein that normally activates or promotes activation in a cellular signaling pathway, where the protein in that pathway is a target of the MALT1 protease; and autoimmune or autoinflammatory or hyperinflammatory states due to a genetic mutation causing a "loss-of-function" (including partial loss-of-function) state in a protein that normally inhibits or promotes inhibition in a cellular signaling pathway, where the protein in that inhibitory pathway is a target of the MALT1 protease.

[0222] Methods of use

[0223] The compounds and compositions described herein are generally useful for modulating MALT1 and can be used to treat diseases or disorders, particularly those susceptible to modulation of MALT1 proteolytic and / or autoproteolytic activity. In some aspects, the compounds and compositions described herein can be used to inhibit MALT1. In some aspects, the compounds and compositions of the present disclosure are contemplated to be useful for treating diseases, disorders, or conditions characterized by dysregulation of NF-κΒ and / or mTor pathways (e.g., mTORcl and / or mTORc2) and / or Jun / Fos activation, such as genetic diseases, autoimmune or immune and inflammatory disorders, allergic disorders, respiratory disorders, and neoplastic disorders. These disease states or conditions, including those above, can be treated by administering to a patient having the disease, disorder, or condition a MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) disclosed herein.

[0224] Other autoimmune and inflammatory conditions that can be treated with a MALT1 inhibitor include, for example, arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn's disease, celiac disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allograft rejection, Bechet's disease, uveitis, psoriasis, psoriatic arthritis, BENTA disease, A20 haploinsufficiency, HOIL1 or HOIP deficiency, dermatomyositis, dermatitis, atopic dermatitis, dermatomyositis, acne vulgaris, myasthenia gravis, hidradenitis suppurativa, Grave's disease, Hashimoto thyroiditis, Sjogren's syndrome, and a vesicular condition (e.g., pemphigus vulgaris), antibody-mediated vasculitis syndromes, including ANCA-associated vasculitis, Henoch-Schoenlein Purpura, IKBKG gain-of-function (also known as NEMO), IKB gain-of-function, immune complex-driven diseases (lupus, rheumatoid arthritis, IgA nephritis, IgA vasculitis, IgG vasculitis), and immune complex vasculitis (primary or secondary to infection or cancer).

[0225] As shown in ​ , MALT1 is a key component in the signaling pathway that leads to NF-κΒ activation and / or mTor pathway (e.g., mTORcl and / or mTORc2) activation and / or Jun / Fos activation of immune cells (e.g., B cells, T cells, macrophages, and / or neutrophils). As shown in ​ , CARD11 mutations can lead to gain-of-function that activates NF-κΒ through MALT1 signaling and / or activates mTor pathway (e.g., mTORcl and / or mTORc2) and / or activates Jun / Fos to produce BENTA in a patient.

[0226] Using the MALT1 inhibitors disclosed herein (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can suppress or prevent CARD11 alleles that produce gain-of-function and BENTA from activating NF-κΒ. Providing MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4) to cells (e.g., immune cells such as B cells, T cells, macrophages, and / or neutrophils) with NF-κΒ activation from CARD11 alleles that produce gain-of-function and BENTA through the MALT1 signaling complex can reduce NF-κΒ activation and decrease the activation state of the cells. In some cases, MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can restore the cells to a quiescent state.

[0227] Patients with CARD11 gain-of-function mutations (e.g., BENTA) can be treated by administering the MALT1 inhibitors disclosed herein (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4). MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can be administered at a suitable dose (e.g., 25-1000 mg / day) in a suitable formulation (e.g., an oral dosage form). MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) produce a therapeutic effect by reversing some or all of the effects of CARD11 gain-of-function alleles in patients (e.g., BENTA patients). CARD11 gain-of-function mutants also reduce the killing activity of natural killer cells. The MALT1 inhibitors disclosed herein for treating viral infections (e.g., EBV or CMV, members of the herpes family) can reverse this effect on NK cells. These infections can be very severe or life-threatening in BENTA patients.

[0228] As shown in ​ CARD14 gain-of-function mutants in CARD14 generalized pustular psoriasis interact with MALT1 in a similar manner to CARD11 mutants in BENTA.

[0229] The production of NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) and / or Jun / Fos activation by CARD14 gain-of-function and CARD14 generalized pustular psoriasis can be mitigated or prevented by using the MALT1 inhibitors disclosed herein (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4). Providing a MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4) to a cell (e.g., an immune cell such as a B cell, a T cell, a macrophage, and / or a neutrophil) having NF-κΒ activation from a CARD14 allele that produces gain-of-function and CARD14 generalized pustular psoriasis through the MALT1 signaling complex can reduce NF-κΒ activation, and / or reduce mTor pathway (e.g., mTORcl and / or mTORc2) activation, and / or reduce Jun / Fos activation, thereby reducing the activation state of the cell. In some cases, the MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can restore the cell to a quiescent state.

[0230] Patients having CARD14 skin inflammation (e.g., generalized pustular psoriasis, pityriasis rubra pilaris) can be treated by administering a MALT1 inhibitor disclosed herein (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4). The MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can be administered at a suitable dose (e.g., 25-1000 mg / day) in a suitable formulation (e.g., an oral dosage form). The MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) produces a therapeutic effect by reversing some or all of the effects of the CARD14 gain-of-function allele in a patient having CARD14 generalized pustular psoriasis.

[0231] The production of functional gain-of-function and A20 haploinsufficient tumor necrosis factor (TNF)-alpha-induced protein 3 allele activation of the Jun / Fos and / or NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) can be mitigated or prevented by using the MALT1 inhibitors disclosed herein (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4). Providing a MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) to a cell (e.g., an immune cell such as a B cell, a T cell, a macrophage, and / or a neutrophil) having a tumor necrosis factor (TNF)-alpha-induced protein 3 allele that produces a functional gain-of-function and A20 haploinsufficiency by the MALT1 signaling complex can reduce NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) and / or Jun / Fos activation, thereby reducing the activation state of the cell. In some cases, the MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can restore the cell to a quiescent state.

[0232] A patient having A20 haploinsufficiency can be treated by administering a MALT1 inhibitor disclosed herein (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4). The MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can be administered at a suitable dose (e.g., 25-1000 mg / day) in a suitable formulation (e.g., an oral dosage form). The MALT1 inhibitor (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) produces a therapeutic effect by reversing some or all of the effects of the tumor necrosis factor (TNF)-alpha-induced protein 3 gain-of-function allele in a patient having A20 haploinsufficiency.

[0233] By using the MALT1 inhibitors disclosed herein (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4), the unwanted activation of the Jun / Fos and / or NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) by the MALT1 signaling complex from any source (e.g., genetic and / or receptor stimulation) can be suppressed or prevented. Providing the MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) to cells (e.g., immune cells such as B cells, T cells, macrophages, and / or neutrophils) with unwanted activation of the Jun / Fos and / or NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) by the MALT1 signaling complex can reduce NF-κΒ activation and decrease the activation state of the cells. In some cases, the MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can restore the cells to a resting state.

[0234] In general, by using the MALT1 inhibitors disclosed herein (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4), the unwanted activation of the Jun / Fos and / or NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) by the MALT1 signaling complex from any source (e.g., genetic and / or receptor stimulation) can be suppressed or prevented. Providing the MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) to cells (e.g., immune cells such as B cells, T cells, macrophages, and / or neutrophils) with unwanted activation of the Jun / Fos and / or NF-κΒ and / or mTor pathway (e.g., mTORcl and / or mTORc2) by the MALT1 signaling complex can reduce NF-κΒ activation and decrease the activation state of the cells. In some cases, the MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) can restore the cells to a resting state.

[0235] The compounds of the compositions described herein can be administered in combination with another agent or therapy. A subject to be administered a compound disclosed herein can have a disease, disorder, or condition that would benefit from treatment with another agent or therapy, or a symptom thereof. The compounds of the compositions described herein can be administered simultaneously with, prior to, or after one or more other therapeutic agents. The compounds of the compositions described herein can be administered separately from the other agents by the same or different route of administration, or together in the same pharmaceutical composition as the other agents. The compounds described herein can be administered as the sole active ingredient, or in conjunction with (e.g., as an adjuvant to) other drugs, such as immunosuppressants or immunomodulators or other anti-inflammatory agents, or chemotherapeutic agents (e.g., malignant cell antiproliferative agents) used to treat or prevent acute or chronic rejection of allogeneic or xenogeneic transplants, or inflammatory or autoimmune disorders. For example, the compounds of the application can be used in combination with a calcineurin inhibitor, such as cyclosporin A or FK 506; an mTOR inhibitor, such as rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, biolimus-7 or biolimus-9; an ascomycin having immunosuppressive properties, such as ABT-281, ASM 981; a corticosteroid; cyclophosphamide; azathioprine; methotrexate; leflunomide; mizoribine; mycophenolic acid or salt; mycophenolate mofetil; or an IL-1 beta inhibitor.

[0236] CARD11 gain-of-function mutants (alleles) that produce BENTA can be subjected to appropriate genetic testing using well-known methods. For example, such methods include high-throughput genomic sequencing, exome sequencing, FISH techniques, microarrays, other hybridization techniques, PCR-related diagnostics, other nucleic acid amplification techniques, CRISPR diagnostics, denaturing HPLC, gene expression profiling, pharmacokinetic testing of CARD11 and / or levels of CARD11 / MALT1 / NF-κB-related metabolites.

[0237] Various features and embodiments of the present disclosure are demonstrated in the following representative examples, which are intended to be illustrative and not limiting. However, those of skill in the art will readily understand that the particular methods and results discussed are merely illustrative of the present application as described more fully in the claims below. The present disclosure is not limited to the specific procedures, materials, etc. as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0238] Example

[0239] Example 1: MALT1 inhibitors reverse gain-of-function CARD11 variants

[0240] MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) are incubated with T cells that lack endogenous CARD11 and carry transiently transfected CARD11 cDNA that carries a gain-of-function mutation. An optional variation is to co-transfect a copy of the wild-type CARD11 gene with the CARD11 gain-of-function mutant at a 50:50 ratio. The T cells include a reporter gene (kB-GFP) that serves as a readout of NFKB signaling. The expression of NF-kB-GFP at baseline and after anti-CD3 or PMA / ionomycin stimulation is measured.

[0241] By flow cytometry, the baseline activation of NF-kB-GFP of T cells carrying GOF variants of CARD11 is elevated and remains higher after stimulation. MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) inhibit the effect of the CARD11 gain-of-function mutant, causing a decrease in NF-kB-GFP expression.

[0242] Example 2: Clinical trial of MALT1 inhibitors against BENTA

[0243] MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) are used to treat patients with BENTA. MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) are formulated in an oral dosage form and administered to patients once daily at a starting dose of 25 mg / day. Alternatively, the daily dose is escalated from 25 mg / day to 1000 mg / day.

[0244] Patients receiving MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) show a decrease in Jun / Fos and / or NF-kB and / or mTor pathway (e.g., mTORcl and / or mTORc2) activation and their BENTA symptoms are improved.

[0245] Example 3: MALT1 inhibitors reverse mTor pathway (e.g., mTORcl and / or mTORc2) activation by gain-of-function CARD11 variants

[0246] Primary T cells are stimulated for activation and cultured in the presence of IL-2. For stimulation, cells are pre-incubated with anti-CD3s and anti-CD28 on ice and washed. Cells are stimulated by cross-linking the anti-CD3 antibody with a secondary antibody (e.g., cross-linking with anti-mouse IgG for 10 minutes) in complete T cell media and then fixed with pre-warmed fixation buffer. Cells are then washed with FACS buffer and permeabilized with pre-chilled to -20°C permeabilization buffer and incubated on ice for 30 minutes. Cells are washed and then stained with the following antibodies or appropriate isotype control anti-pAkt (Ser473) and anti-pS6 (Ser240 / 244).

[0247] MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) are incubated with primary T cells after activation. MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) inhibit mTor pathway (e.g., mTORcl and / or mTORc2) activation.

[0248] Example 4: MALT1 inhibitors reverse gain-of-function of CARD14 variants

[0249] MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) are incubated with epithelial cells (e.g., HEK293 cells) that lack endogenous CARD14 and carry transiently transfected CARD14 cDNA that carries a gain-of-function mutation. The epithelial cells include a reporter gene (K B-GFP) that serves as a readout of NFKB signaling. The expression of NF-KB-GFP at baseline and after anti-CD3 or PMA / ionomycin stimulation is measured.

[0250] The baseline activation of NF-KB-GFP of epithelial cells carrying gain-of-function variants of CARD14 is elevated and remains high after stimulation by flow cytometry. MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) inhibit the effect of the CARD14 gain-of-function mutants, causing a decrease in NF-KB-GFP expression.

[0251] Example 5: MALT1 inhibitors reduce MALT1 cleavage of HOIL1

[0252] Recombinant full-length human MALT1 protein was expressed and purified. C-terminal Myc-FLAG tagged full-length human HOIL1 was obtained from Origene. HOIL1 protein (0.05 pg / pL) was incubated with different concentrations of MALT1 in assay buffer (200 mM Tris-HCl, 0.8 M sodium citrate, 0.1 mM EGTA, 0.05% CHAPS, 1 mM DTT, pH 7.4) at 37 °C for 2 hours. Cleavage of HOIL1 was analyzed by gel electrophoresis using 4-12% Bis-Tris-SDS-polyacrylamide (PAGE) gradient gels (Life Technologies). Presence of small fragments was confirmed by immunoblotting using an N-terminal antibody (anti-N-terminal HOIL1; HPA024185; Sigma). Total HOIL1 was observed using a C-terminal directed antibody (anti-FLAG, clone M2, Sigma). These assays were performed with both cleavable HOIL1 and as a negative control, non-cleavable HOIL1 (where Lys substituted Arg165).

[0253] MALT1 inhibitors (e.g., compounds 1.1, 1.26, 1.27, 1.30, 1.33, 2.1, and / or 2.4) were added to the reaction mixture for HOIL1 cleavage, and cleavage of HOIL1 was reduced.

[0254] Example 6: MALT1 inhibitors reduce NF-κΒ activity of CARD11 gain-of-function mutants in Jurkat cells

[0255] Jurkat cells were engineered with E134G, C49Y, or G123D CARD11 gain-of-function (GOF) mutants. Jurkat cells include a reporter (κΒ-GFP) that serves as a readout for NFκΒ signaling.

[0256] Jurkat cells carrying GOF E134G, C49Y, or G123D CARD11 have elevated baseline activation of NF-κΒ-GFP. MALT1 inhibitors (e.g., compounds 1.1 or 2.4) were incubated with the above engineered Jurkat cells. MALT1 inhibitors (e.g., compounds 1.1 or 2.4) inhibited the effect of CARD11 gain-of-function mutants, causing a reduction in NF-κΒ-GFP expression for all three GOF CARD11 mutants. These MALT1 inhibitors reduced NF-κΒ-GFP expression in a dose-dependent manner from about 100 nM to 5 mM for both compound 1.1 or compound 2.4.

[0257] Jurkat cells harboring GOF E134G, C49Y, or G123D CARD11 were also assessed for cleavage of CYLD as a measure of proteolytic function of MALT1. In the absence of inhibitor, Jurkat cells with GOF CARD11 mutants cleave CYLD. MALT1 inhibitors (e.g., Compound 1.1 or 2.4) inhibit this CYLD cleavage in a dose-dependent manner in the range of about 100 nM to 10 µM.

[0258] Example 7: MALT1 inhibitors reduce NF-κΒ activity of CARD11 gain-of-function mutants in B cells B cells are obtained from patients with BENTA (B-cell expansion with NF-κΒ and T cell inactivation). Both resting and activated (CD40L+IL-4) B cells are treated with MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4).

[0259] Changes in steady-state CARD11-induced NF-κΒ signaling are measured at different time points over about 24 hours by monitoring CARD11 aggregation and nuclear accumulation of p65 or directly quantifying active NF-κΒ complexes in nuclear lysates (TransAm assay). MALT1 substrate cleavage will also be monitored over time.

[0260] Changes in expression of known NF-κΒ-dependent genes (e.g., BCL2, NFKB2, c-FLIP, cyclin D1) that are elevated in BENTA B cells are assessed by quantitative PCR and / or immunoblotting of whole cell lysates - / + MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4). Alternatively, bulk RNA sequencing can be performed on BENTA B cells - / + MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4) to assess changes in the whole B cell transcriptome.

[0261] CARD11 gain-of-function is known to promote survival and proliferation of B cells, which can lead to an excess number of B cells, such as in BENTA or B-cell lymphoma. MALT1 inhibitors reduce this survival. Healthy or BENTA B cells are assessed for survival and proliferation in response to a combination of stimuli (e.g., anti-IgM / IgD, rCD40L / IL-4 / IL-21, phorbol myristate acetate, etc.) - / + MALT1 inhibitors (e.g., Compound 1.1, 1.26, 1.27, 1.30, 1.33, and / or 2.4).

[0262] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0263] While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the scope of the application of this disclosure.

Claims

1. A method for reducing NF-κΒ activation caused by a CARD11 gain-of-function allele, the method comprising the steps of: obtaining a cell having the CARD11 gain-of-function allele; and exposing the cell to a MALT1 inhibitor, thereby reducing the NF-κΒ activation.

2. A method for treating a patient having a CARD11 gain-of-function mutation, the method comprising the steps of: obtaining the patient having BENTA; and administering to the patient a therapeutically effective amount of a MALT1 inhibitor, thereby reducing NF-κΒ activation in the patient.

3. The method of claim 3, wherein the patient is a BENTA patient.

4. A method for reducing NF-κΒ activation caused by a CARD14 gain-of-function allele, the method comprising the steps of: obtaining a cell having the CARD14 gain-of-function allele; and exposing the cell to a MALT1 inhibitor, thereby reducing the NF-κΒ activation.

5. A method for treating a patient having CARD14 skin inflammation, the method comprising the steps of: obtaining the patient having CARD14 generalized pustular psoriasis; and administering to the patient a therapeutically effective amount of a MALT1 inhibitor, thereby reducing NF-κΒ activation in the CARD14 generalized pustular psoriasis patient.

6. The method of claim 4, wherein the CARD14 skin inflammation is pustular psoriasis or pityriasis rubra pilaris.

7. A method for reducing NF-κΒ activation caused by a tumor necrosis factor (TNF)-alpha induced protein 3 gain-of-function allele, the method comprising the steps of: obtaining a cell having the tumor necrosis factor (TNF)-alpha induced protein 3 gain-of-function allele; and exposing the cell to a MALT1 inhibitor, thereby reducing the NF-κΒ activation.

8. A method for treating a patient having an A20 haploinsufficiency, the method comprising the steps of: obtaining the patient having the A20 haploinsufficiency; and administering to the patient a therapeutically effective amount of a MALT1 inhibitor, thereby reducing NF-κΒ activation in the A20 haploinsufficiency patient.

9. A method for reducing NF-κΒ activation caused by a gain-of-function allele acting on MALT1, the method comprising the steps of: obtaining a cell having the gain-of-function allele; and exposing the cell to a MALT1 inhibitor, thereby reducing the NF-κΒ activation.

10. A method for reducing NF-κΒ activation caused by a CARD11 gain-of-function allele, the method comprising the steps of: obtaining a cell having the CARD11 gain-of-function allele; and exposing the cell to a MALT1 inhibitor, thereby reducing mTORcl or mTORc2 activation.

11. A method for reducing NF-κΒ activation caused by a CARD11 gain-of-function allele, the method comprising the steps of: obtaining a cell having the CARD11 gain-of-function allele; and exposing the cell to a MALT1 inhibitor, thereby increasing Jun / Fos inactivation.

12. The method of any one of claims 1-11, wherein the cell is an immune cell.

13. The method of claim 12, wherein the immune cell is a B cell, a T cell, a macrophage, or a neutrophil.

14. The method of claim 13, wherein the immune cell is a B cell.

15. The method of any one of claims 1-11, wherein the MALT1 inhibitor is a compound of Formula 1, or a pharmaceutically acceptable salt thereof, wherein: T is 0 or 1.

16. The method of claim 15, wherein the MALT1 inhibitor comprises Compound 1.1, Compound 1.26, Compound 1.27, Compound 1.30, or Compound 1.33, or a combination of the foregoing. R 1 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, C3-6cycloalkyl, and 5-10 membered heterocyclyl, wherein said C1-6alkyl, said C3-6cycloalkyl, and said 5-10 membered heterocyclyl can optionally be substituted on one or more available carbons by one, two, three or more substituents, each independently selected from R 1a , and wherein if said 5-10 membered heterocyclyl contains a substitutable ring nitrogen atom, said ring nitrogen atom can optionally be substituted by R 1b , and wherein if said 5-10 membered heterocyclyl contains a substitutable ring sulfur atom, said ring sulfur atom can optionally be substituted by two O atoms; R 2 is CH3or CF3; R 3 is hydrogen; or R 3 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, C3-7cycloalkyl, 5-6 membered heterocyclyl, 5-6 membered heterocyclyl-C1-3alkyl 5-6 membered heterocyclyl-O-, phenyl, and 5-6 membered heteroaryl, any of which can be optionally substituted with one, two, or three substituents each independently selected from R 3a ; R 4 is Ci-6alkyl; R 1a independently at each occurrence is selected from the group consisting of cyano, halogen, hydroxyl, oxo, Ci-6alkyl, -C(0)OR A , -C(0)N(R A )2, -N(R A )2, Ci-6alkoxy, 5-6 membered heterocyclyl, and 5-6 membered heteroaryl, wherein said Ci-6alkyl is optionally substituted with -N(R A )2, and wherein if said 5-6 membered heterocyclyl contains a substitutable ring nitrogen atom, said ring nitrogen atom can be optionally substituted with R p ; R 1b selected from the group consisting of Ci-6alkyl, -C(0)OR A , -C(0)Ci-6alkyl, -C(0)C3-6cycloalkyl, -C(0)N(R A )2, and -S(0)2Ci-6alkyl; R 3a independently at each occurrence are selected from the group consisting of halogen, Ci-4alkyl, Ci-4haloalkyl, Ci-4alkoxy, Ci-4haloalkoxy, hydroxyl, Ci-4alkenyl, cyano, azido, -NR C R D , C3-6cycloalkyl, Ci1-4alkoxy, 5-6 membered heterocyclyl-O-, 5-6 membered heterocyclyl, and phenyl, wherein C3-6cycloalkyl, 5-6 membered heterocyclyl-O-, 5-6 membered heterocyclyl, and phenyl are optionally substituted with one, two, or three substituents each independently selected from R p ; R p independently at each occurrence are selected from the group consisting of halogen, Ci-4alkyl, Ci-4haloalkyl, hydroxyl, Ci-4alkoxy, Ci-4alkoxyCi-4alkyl, NR C R D and aminoCi-3alkyl; R A is independently selected, at each occurrence, from the group consisting of hydrogen, Ci-6alkyl, -C(0)Ci-6alkyl, and -C(0)OCi-6alkyl; R B Ci-6alkyl, C1-6cycloalkyl, and -C(O)OC1-6alkyl; R C and R D is independently selected at each occurrence from the group consisting of hydrogen, Ci-6alkyl, haloCi-6alkyl and C3-4cycloalkyl, or R C and R D together with the nitrogen atom to which they are attached form a 4-6 membered heterocyclyl or a 4-6 membered heteroaryl, wherein said 4-6 membered heterocyclyl or said 4-6 membered heteroaryl can contain an additional nitrogen atom or an oxygen atom and is optionally substituted with one or two fluorine; and 17. The method of claim 16, wherein the MALT1 inhibitor comprises Compound 1.

1.

18. The method of claim 16, wherein the MALT1 inhibitor comprises Compound 1.

26.

19. The method of claim 16, wherein the MALT1 inhibitor comprises Compound 1.

27.

20. The method of claim 16, wherein the MALT1 inhibitor comprises Compound 1.

30.

21. The method of claim 16, wherein the MALT1 inhibitor comprises Compound 1.

33.

22. The method of any one of claims 1-11, wherein the MALT1 inhibitor is a compound of Formula la, or a stereoisomer and / or pharmaceutically acceptable salt thereof, wherein: m is 0 or 1; and n is 0 or 1.

23. The method of claim 22, wherein the MALT1 inhibitor is Compound 2.1 or Compound 2.

4.

24. The method of claim 23, wherein the MALT1 inhibitor is Compound 2.

1.

25. The method of claim 23, wherein the MALT1 inhibitor is Compound 2.

4. R 1 is Ci-6alkyl or Ci-3haloalkyl, wherein said Ci-6alkyl can optionally be substituted with -O-Ci-3alkyl; R 2 is aryl or 5-6 membered heteroaryl, wherein the aryl can optionally be substituted with cyano; R 4 is -C(O)OH or 5-6 membered heteroaryl; ​ ​ ​ ​ ​

Images