WO2024175998A1 - Benzenesulfonamide derivatives and uses thereof - Google Patents

Benzenesulfonamide derivatives and uses thereof Download PDF

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WO2024175998A1
WO2024175998A1 PCT/IB2024/000098 IB2024000098W WO2024175998A1 WO 2024175998 A1 WO2024175998 A1 WO 2024175998A1 IB 2024000098 W IB2024000098 W IB 2024000098W WO 2024175998 A1 WO2024175998 A1 WO 2024175998A1
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substituted
compound
unsubstituted
polypeptide
linker
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Patrick T. GUNNING
Jeffrey O'meara
Siawash AHMAR
Graham L. SIMPSON
David Alexander ROSA
Ji Sung Park
Giles Albert Brown
Jeffrey David ST. DENIS
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Dunad Therapeutics, Ltd.
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Abstract

Provided herein are para-substituted tetrafluoro-benzenesulfonamide compounds, pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of diseases, said compounds having reduced reactivity with glutathione.

Description

BENZENESULFONAMIDE DERIVATIVES AND USES THEREOF
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 63/486,621, filed February 23, 2023, U.S. Provisional Application No. 63/486,620, filed February 23, 2023, and U.S. Provisional Application No. 63/514,307, filed July 18, 2023, which are hereby incorporated by reference in their entirety herein.
BACKGROUND
[0002] Chemical modification is an important tool to alter structure and function of proteins. One way to achieve chemical modification of proteins is to use protein binders, such as covalent small molecule binder (e.g., inhibitors). As a result, protein binders (e.g., covalent small molecule binders (e.g., inhibitors of proteins)) are considered to be useful in multiple applications, including therapeutics.
BRIEF SUMMARY OF THE DISCLOSURE
[0003] Many covalent small molecule binders, specifically the warhead group of such compounds, are susceptible to off-target (e.g., covalent) modification, such as before reaching a target protein. In some instances, a warhead group of such a binder covalently modifies a protein (e.g., an off-target protein) or is metabolized, such as via nucleophilic metabolism by glutathione (GSH), and is neutralized before it has a chance to (e.g., covalently) modify its target protein. A warhead group that is more selective for a target protein and/or more stable to off-target (e.g., covalent) modification, such as modification via nucleophilic metabolism by glutathione (GSH), would help decrease off-target effects, such as resulting from covalent binding of the warhead to an off-target protein, and increase efficacy of (e.g., on-target effects of) covalent small molecule binders.
[0004] Provided herein are protein binders (e.g., covalent small molecule protein binders (e.g, inhibitors)). In some instances, the protein binders provided herein comprise a warhead group. In some instances, the warhead group directs covalent and/or irreversible interaction with a (e.g., cysteine residue of a) protein (e.g., described herein, such as a target protein). In some instances, the warhead group directs covalent and/or irreversible interaction with a (e.g., cysteine residue of a) target protein and is stable to off-target (e.g., covalent) modification, such as modification via nucleophilic metabolism by glutathione (GSH). In some instances, the warhead group directs covalent and/or irreversible interaction of a (e.g., cysteine residue of a) protein (e.g., described herein, such as a target protein) to a position ofthe warhead group that is ortho ormeta to a sulfur- containing group (e.g., sulfone, sulfide, sulfoxide, or the like). In some instances, the protein does not covalently and/or irreversibly interact with the warhead group at a para-position. In some instances, the protein covalently and/or irreversibly interacts with the warhead group at either an ortho-position or a meta-position, such as a position of the warhead that is ortho or meta relative to the sulfur containing group. In some instances, the warhead comprises a blocking group (e.g, hydrogen, CN, NO2, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, or the like). In some embodiments, the blocking group is at aposition of the warhead thatis para relative to the sulfur-containing group. In some embodiments, the blocking group directs covalent and/or irreversible interaction of a (e.g., cysteine residue of a) protein (e.g., described herein) to a position of the warhead that is ortho or meta relative to the sulfur-containing group.
[0005] In some instances, a compound provided herein (or a warhead group thereof) does not substantially bind to glutathione (GSH). In some instances, a compound provided herein is stable. In some instances, a compound provided herein (or a warhead group thereof) is stable to GSH. In some instances, a warhead of a compound provided herein is stable (e.g., to metabolism by GSH). In some instances, a warhead of a compound provided herein is stable to GSH. In some instances, a compound provided herein (or a warhead group thereof) does not bind to GSH. In some instances, a warhead of a compound provided herein does not bind to GSH. In some instances, a compound provided herein (or a warhead group thereof) has reduced reactivity with GSH. In some instances, a compound provided herein (or a warhead group thereof) forms covalent bond(s) with a polypeptide (e.g., a protein, such as Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), protooncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), a KRAS protein or a mutant thereof (e.g., KRAS G12C, KRAS Cl 18A, or KRAS G12C/C118 A)) without substantially bindingto GSH. In some instances, a compound provided herein (or a warhead group thereof) forms covalent b on d(s) with a sulfur atom of a cysteine residue of a target protein without substantially covalently binding to the sulfur-containing GSH. In some instances, a compound provided herein (or a warhead group thereof) is selective for a polypeptide, such as a (target) protein described herein, relative to GSH. In some instances, GSH has minimal to no reactivity with a compound described herein (or a warhead group thereof), such that GSH is unable to react with (e.g., a warhead of) a compound provided herein. In some instances, GSH has a binding affinity (e.g., Kd) with a compound described herein (or a warhead group thereof) of more than 0.1 pM (e.g., 1 pm, 10 pm, 100 pm, or 1000 pm or more). In some instances, GSH has a binding affinity (e.g., Kd) with a compound described herein (or a warhead group thereof) of more than 10 pm.
[0006] In some embodiments, the compound (or a warhead group thereof) is selective for the polypeptide (e.g., a protein, such as Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), protooncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), a KRAS protein or a mutant thereof (e.g., KRAS G12C, KRAS C118A, or KRAS G12C/C118A)). In some embodiments, the compound (or a warhead group thereof) is selective for the polypeptide (e.g., a protein, such as Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), proto-oncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), a KRAS protein or a mutant thereof (e.g., KRAS G12C, KRAS Cl 18A, or KRAS G12C/C118A)) relative to GSH. In some embodiments, the compound (or a warhead group thereof) is selective for (e.g, covalent) binding to the polypeptide relative to GSH. In some embodiments, the compound (or a warhead group thereof) is selective for the polypeptide relative to GSH at a ratio of at least 10:1 (e.g., 20:1 or more, 50: 1 or more, 100: 1 or more, 500:1 or more, 1000:1 or more). In some embodiments, the compound (or a warhead group thereof) is selective for the polypeptide relative to GSH at a ratio of about 10:1 to about 100: 1.
[0007] In some embodiments, the compound (or a warhead group thereof) is at least 2-fold (e.g., 2-fold or more, 5-fold or more, 10-fold or more, 25-fold or more) selective for the polypeptide relative to GSH.
[0008] In some embodiments, the compound has an IC50 for the polypeptide of at least 10 pM and a GSH half life of greater than about 10 minutes. In some embodiments, the compound has an IC50 for the polypeptide of at least 1 pM and a GSH half life of greater than about 10 minutes. In some embodiments, the compound has an IC50 of at least 0.1 pM and a GSH half life of greater than about 100 minutes.
[0009] In some embodiments, the compound (or a warhead group thereof) does not covalently bind with (e.g., the thiol of) GSH (e.g., at Y2 or a position ortho or meta to Y2). In some embodiments, the compound (or a warhead group thereof) does not covalently bind with (e.g., the thiol of) GSH.
[0010] Provided in some embodiments herein is a compound having a structure of Formula (A):
Figure imgf000005_0001
Formula (A) or a salt thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
Q1 is Rx or L-G;
Q2 is L-G or Y2;
Y2 is a blocking group (e.g., a group that directs (e.g., covalent and/or irreversible) binding (of a cysteine residue) of a protein to a position other than Y2);
Rx is substituted or unsubstituted alkyl or NRyRz;
RA, Ry, and Rz are each independently hydrogen or substituted or unsubstituted alkyl;
L is a linker; and
G is an organic residue; wherein one and only one of Q1 or Q2 is L-G.
[0011] In some embodiments, Q1 is not substituted or unsubstituted methoxy phenyl.
[0012] In some embodiments, Q2 is L-G. In some embodiments, Q1 is Rx.
[0013] In some embodiments, the compound represented by the structure of Formula (A) is represented by the structure of Formula (II), Formula (ILA), Formula (II-B), or Formula (ILC). [0014] Provided in some embodiments herein is a compound having a structure of Formula (II):
Figure imgf000006_0001
Formula (II) or a salt thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
RA is hydrogen or substituted or unsubstituted alkyl;
Rxa is substituted or unsubstituted alkyl or NRyRz;
Ry and Rz are each independently hydrogen or substituted or unsubstituted alkyl;
L is a linker; and G is a protein-binding ligand (e.g., a radical of a compound that interacts with a protein or a mutant thereof, comprising one or more cyclic group wherein the one or more cyclic groups are individually linked by one or more linker).
[0015] In some embodiments, Gis or comprises a protein-binding ligand selected from aBTK- , EGFR-, FGFR-, AURKA-, TEAD-, JAK3- KRAS-, and BMX-binding ligand. In some embodiments, Gis or comprises a BTK -binding ligand. In some embodiments, Gis or comprises a EGFR-bindingligand. In some embodiments, Gis or comprises aFGFR-bindingligand. In some embodiments, G is or comprises a AURKA-binding ligand. In some embodiments, G is or comprises a TEAD-binding ligand. In some embodiments, G is or comprises a JAK3 -binding ligand. In some embodiments, G is or comprises KRAS-binding ligand. In some embodiments, G is or comprises a BMX-binding ligand.
[0016] In some embodiments, the compound (e.g., covalently and/or irreversibly) interacts with aprotein (e g., AURKA, KIT, BTK, EGFR, FGFR, BMX, TEAD, JAK3, KRAS) or a mutant thereof (e.g., a cysteine residue of the protein or the mutant thereof) at a position ortho or meta to L.
[0017] Provided in some embodiments herein is a compound having a structure of Formula (II- B):
Figure imgf000007_0001
Formula (II-B) or a salt thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
RA is hydrogen or substituted or unsubstituted alkyl;
Rxa is alkyl or NRXRZ;
Ry and Rz are each independently hydrogen or sub stituted or unsubstituted alkyl; L is a linker; and
G is a KRAS-binding ligand (e.g., a radical of a compound that interacts with KRAS or a mutant thereof (e.g., KRAS G12C, KRAS C118A, or KRAS G12C/C118 A) (e.g., an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle)). [0018] In some embodiments, the compound (e.g., covalently and/or irreversibly) interacts with KRAS or a mutant thereof (e.g., a cysteine residue of KRAS or the mutant thereof) at a position ortho or meta to L.
[0019] Provided in some embodiments herein is a compound having a structure of Formula (II- C):
Figure imgf000008_0001
Formula (II-C) or a salt thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
RA is hydrogen or substituted or unsubstituted alkyl;
Rxa is alkyl or NRXRZ;
Ry and Rz are each independently hydrogen or sub stituted or unsubstituted alkyl; L is a linker; and
G is a JAK3-binding ligand (e.g., a radical of a compound that interacts with a JAK3 protein or a mutant thereof (e.g., an unsubstituted or substituted heterocycle)).
[0020] In some embodiments, the JAK3-binding ligand is an unsubstituted or substituted pyrrolopyrimidine, an unsubstituted or substituted pyrrolopyridine, an unsubstituted or substituted pyrazolopyrimidine, an unsubstituted or substituted pyrazolopyridine, or an unsubstituted or substituted benzimidazole. In some embodiments, the J AK3-binding ligand is an unsubstituted or substituted pyrrolopyrimidine. In some embodiments, the JAK3 -binding ligand is a pyrrolopyrimidine described herein, such as a 7H-pyrrolo[2,3-J]pyrimidine described herein. [0021] In some embodiments, X1 is absent.
[0022] In some embodiments, X1 is O.
[0023] In some embodiments, X2 is absent.
[0024] In some embodiments, X2 is O.
[0025] In some embodiments, X2 is NRA. In some embodiments, RA is hydrogen. In some embodiments, X2 is NH. [0026] In some embodiments, Rxais NR.VRZ. In some embodiments, Ry andRz are hydrogen. In some embodiments, Rxa is NEE.
[0027] In some embodiments, Rxa is substituted alkyl (e.g., alkyl substituted with oxo and amino, such as -C(O)NH2).
[0028] In some embodiments, Rxa is unsubstituted alkyl. In some embodiments, Rxa is Ci-C6 alkyl. In some embodiments, Rxa is methyl.
[0029] In some embodiments, X1 is absent and Rxa is methyl.
[0030] In some embodiments, X1 is absent, X2 is O, and Rxa is methyl.
[0031] In some embodiments, X1 is absent, X2 is absent, and Rxa is methyl.
[0032] In some embodiments, X1 is O and Rxa is methyl.
[0033] In some embodiments, X1 is O, Rxa is methyl, and X2 is O.
[0034] In some embodiments, X1 is O, Rxa is methyl, and X2 is absent.
[0035] In some embodiments, X1 is O, Rxa is methyl, and X2 is NH.
[0036] In some embodiments, at least one of X1 or X2 is O.
Figure imgf000009_0001
[0037] In some embodiments,
Figure imgf000009_0002
directs (e.g., covalently and/or irreversibly) binding
(e.g, of a cysteine residue) of a protein (e.g, AURKA, KIT, BTK, EGFR, FGFR, BMX, TEAD, JAK3, KRAS) or a mutant thereof to a position ortho or meta to L.
[0038] In some embodiments, G or G’ is a radical of a compound that interacts with KRAS or a mutant thereof.
[0039] In some embodiments, G or G’ comprises an optionally substituted cyclic group, optionally substituted with one or moreL’-G’, wherein each L’ individually selected from a linker and is connected to another G’.
[0040] In some embodiments, G or G’ comprises an optionally substituted cyclic group, optionally substituted with -(L’-G’)nL’-G’, wherein n is 0 to 4. In some embodiments, n is 1 to 3. [0041] In some embodiments, G or G’ is a radical of a compound that interacts with a protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS) or a mutant thereof.
[0042] In some embodiments, G or G’ is amino, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, or comprises one or more cyclic groups wherein the one or more cyclic groups are individually linked by one or more linker.
[0043] In some embodiments, G or G’ comprises one or more cyclic groups wherein the one or more cyclic groups are individually linked by one or more linker.
[0044] In some embodiments, G or G’ is or comprises an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle)). [0045] In some embodiments, G or G’ comprises a substituted or unsubstituted carbocycle. [0046] In some embodiments, G or G’ comprises a substituted or unsubstituted heterocycle. [0047] In some embodiments, G or G’ comprises one or more (e.g., one, two, or three) nitrogen atoms (e.g., within its (e.g., fused) ring system).
[0048] In some embodiments, G or G’ comprises one or more (e.g., fused) rings. In some embodiments, G or G’ is a substituted or unsubstituted quinazoline.
[0049] In some embodiments, G or G’ is a substituted or unsubstituted indole.
[0050] In some embodiments, G or G’ is an unsubstituted quinoline.
[0051] In some embodiments, G or G’ is a substituted or unsubstituted indazole.
[0052] In some embodiments, G or G’ is a substituted or unsubstituted indazole. In some embodiments, G or G’ is aromatic or partially aromatic.
[0053] In some embodiments, G or G’ comprises one or more (e.g., one, two, or three) substituted or unsubstituted (e.g., fused) aromatic ring(s). In some embodiments, G or G’ comprises two or more substituted or unsubstituted aromatic or partially aromatic rings.
[0054] In some embodiments, G or G’ is a substituted carbocycle. In some embodiments, G or G’ is a substituted phenyl.
[0055] In some embodiments, G or G’ comprises one or more (e.g., one, two, or three) substituted or unsubstituted (e.g., fused) heteroaromatic ring(s).
[0056] In some embodiments, G or G’ comprises two or more substituted or unsubstituted aromatic or partially aromatic rings, each aromatic or partially aromatic ring independently being a carbocycle or a heterocycle. In some embodiments, G or G’ comprises one or more substituted or unsubstituted carbocycle and one or more substituted or unsubstitued heterocycle, each of the one or more substituted or unsubstituted carbocycle and the one or more substituted or unsubstitued heterocycle independently being linked (e.g., fused) to a substituted or unsubstituted carbocycle or a substituted or unsubstitued heterocycle by a bond. In some embodiments, G or G’ comprises two (or more) substituted or unsubstituted heteroaromatic rings, the heteroaromatic rings being linked (e.g., fused) by a bond, each heteroaromatic ring being aromatic or partially aromatic. In some embodiments, the heteroaromatic rings are selected from the group consisting of benzimidazole, indolizine, quinoline, indazole, and pyrimidine.
[0057] In some embodiments, G or G’ is a substituted heterocycle. In some embodiments, G or G’ is a substituted or unsubstituted N-heterocycle.
[0058] In some embodiments, G or G’ is a substituted or unsubstituted 4-6 membered O- heterocycle.
[0059] In some embodiments, G or G’ is a substituted or unsubstituted pyridine or pyrimidine. [0060] In some embodiments, G or G’ is a substituted quinazoline. [0061] In some embodiments, G or G’ is a substituted pyrazolopyrimidine (e.g., a 1H- pyrazolo[3,4-d]pyrimidine).
[0062] In some embodiments, G or G’ is an unsubstituted pyrrolopyrimidine (e.g., a 7H- pyrrolo[2,3-J]pyrimidine).
[0063] In some embodiments, G or G’ is an unsubstituted benzothiophene or an unsubstituted thiophene.
[0064] In some embodiments, G or G’ is substituted isoxazole (e.g., 3, 5-dimethylisoxazole). [0065] In some embodiments, G or G’ is a substituted quinazoline, a substituted tetrahydropyridopyrimidine (e.g., 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine), a substituted quinoline, a substituted pyridopyrazinone (e.g., pyrido[2,3-b]pyrazin-3(4H)-one). In some embodiments, G is a substituted quinazoline. In some embodiments, G is a substituted tetrahydropyridopyrimidine (e.g., 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine). In some embodiments, G is a substituted quinoline. In some embodiments, G is a substituted pyridopyrazinone (e.g., pyrido[2,3-b]pyrazin-3(4H)-one).
[0066] In some embodiments, G or G’ has a structure shown in Table 1A, Table IB, or Table 1C.
[0067] In some embodiments, linker (e.g., L, L’, or L1) is a bond, -O-, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted sulfoxide, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamide, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl.
[0068] In some embodiments, the linker (e.g., L, L’, or L1) is a bond, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted sulfoxide, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamide, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. In some embodiments, the linker (e.g., L, L’, or L1) is amino, sub stituted or unsubstituted sulfoxide, sub stituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamide, substituted or unsubstituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl.
[0069] In some embodiments, the linker (e.g., L, L’, or L1) is a bond, -O-, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl.
[0070] In some embodiments, the linker (e.g., L, L’, or L1) is a bond, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl.
[0071] In some embodiments, the linker (e.g., L, L’, or L1) is amino, substituted or unsubstituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl. In some embodiments, the linker comprises substituted or unsubstituted Ci-C6 heteroalkylene.
[0072] In some embodiments, the linker (e.g., L, L’, orL1) comprises one or more N atom(s). [0073] In some embodiments, the linker (e.g., L, L’, or L1) is substituted or unsubstituted alkylamine. In some embodiments, the linker comprises a Ci-C6 alkylamine.
[0074] In some embodiments, the linker (e.g., L, L’, or L1) is amino.
[0075] In some embodiments, the linker (e.g., L, L’, or L1) is substituted alkylamine, the alkylamine being substituted with oxo (e.g., -NHC(O)-).
[0076] In some embodiments, the linker (e.g., L, L’, or L1) is -NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl.
[0077] In some embodiments, the linker (e.g., L, L’, or L1) is -NH- or -NCH3-. In some embodiments, the linker (e.g., L, L’, or L1) is substituted or unsubstituted alkylamine.
[0078] In some embodiments, the linker (e.g., L, L’, or L1) is -NR3R4-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl.
[0079] In some embodiments, the linker (e.g., L, L’, or L1) is -NR4-R3-, -NR4-R3-NR7a-, -NR4- R3-O-, or -C(O)NR4-R3-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl, R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, andR7aisH or substituted or un substituted alkyl (e.g., alkyl substituted with oxo). In some embodiments, the linker (e.g., L, L’, or L1) is -NH(heterocyclyl)-. In some embodiments, the linker (e.g., L, L’, or L1) is -NH(azetidinyl)-.
[0080] In some embodiments, the linker (e.g., L, L’, or L1) is methylamine, ethylamine, or propylamine.
[0081] In some embodiments, the linker (e.g., L, L’, or L1) is substituted or unsubstituted heterocyclyl.
[0082] In some embodiments, the linker (e.g., L or L’) is -NR5R6-NR7-, -NR5R6-C(O)-, - NR5R6-CH2NR7C(O)-, -C(O)NR5R6-NR7-, -C(O)NR5R6-O-, or -NR5R6-O-, where R5 and R6 are taken together to form a substituted or unsubstituted heterocyclyl, and R7 is H or substituted or unsubstituted alkyl (e.g., alkyl substituted with oxo). [0083] In some embodiments, the linker (e.g., L, L’, or L1) is -NR5R6-, where R5 and R6 are taken together to form a substituted orunsubstitutedheterocyclyl. In some embodiments, -NR5R6- is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl), substituted or unsubstituted piperidinyl, or substituted or unsubstituted azetidinyl. In some embodiments, - NR5R6- is substituted or unsubstituted piperidinyl or substituted or un substituted azetidinyl and - NR7- is -NH- or -NCH3-.
[0084] In some embodiments, the linker (e.g., L, L’, or L1) is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl).
[0085] In some embodiments, the linker (e.g., L, L’, or L1) comprises two N atom(s) (e.g., (substituted or unsubstituted) diaminoalkyl, (substituted or un substituted) diamino-cycloalkyl, (substituted or unsubstituted) amino-heterocyclyl (e.g., the heterocyclyl being nitrogen containing), (substituted or un substituted) heterocyclyl (e.g., containing 2 nitrogen atoms), the heterocyclyl being optionally fused or spirocyclic).
[0086] In some embodiments, the linker (e.g., L, L’, orL1) comprises one or more (e.g., fused or spirocyclic) rings.
[0087] In some embodiments, the linker (e.g., L, L’, or L1) is or comprises a spirocyclic ring such as l,7-diazaspiro[4.4]nonane or l,6-diazaspiro[3.3]heptane.
[0088] In some embodiments, the linker (e.g., L, L’, or L1) is or comprises substituted or unsubstituted phenyl (e.g., phenyl substituted with halo or cyano).
[0089] In some embodiments, the linker (e.g., L, L’, or L1) is -NH-(un substituted phenyl)-, - NH-(unsubstituted phenyl)-NH-, -NH-(un substituted phenyl)-C(O)-, -CH2NH-(un substituted phenyl)-C(O)-, -CH2-(unsubstituted phenyl)-NH-, -NHCH2-(unsubstituted phenyl)-C(O)-, - NHC(O)-(un substituted phenyl)-C(O)-, -C(O)NH-(substituted phenyl)-C(O)-, -C(O)NH- (substituted phenyl)-C(O)NH-, or -O-(unsubstituted phenyl)-C(O)NH-.
[0090] In some embodiments, the linker (e.g., L, L’, or L1) is a bond.
[0091] In some embodiments, the compound is represented by a structure of Table 2 A, Table
2B, Table 2C, or Table 2D.
[0092] Also provided herein are pharmaceutical compositions comprising the compounds described herein, and methods for using said compounds for the treatment of diseases. Provided herein are pharmaceutical compositions comprising the compounds provided herein. Also provided herein are pharmaceutical compositions that are at least partially stable to glutathione (GSH).
[0093] Also provided in some embodiments herein is a pharmaceutically acceptable composition comprising a compound disclosed herein, or a salt, solvate, tautomer, or regioisomer thereof, and one or more pharmaceutically acceptable excipients. [0094] In some embodiments, provided herein is a pharmaceutical composition comprising a compound represented by a structure provided herein, such as the structure of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or provided in Table 2A, Table 2B, Table 2C, or Table 2D.
[0095] Provided herein in some embodiments, is a pharmaceutical composition comprising a compound represented by a structure provided herein, such as the structure of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or provided in Table 2A, Table 2B, Table 2C, or Table 2D, wherein the compound is selective for a polypeptide (e.g., a protein) relative to glutathione (GSH).
[0096] In some embodiments, provided herein is a pharmaceutical composition that is (e.g., at least partially) stable to glutathione (GSH).
[0097] In some embodiments, the pharmaceutical composition is (e.g., at least partially) stable to glutathione (GSH) in the presence of a polypeptide.
[0098] Provided herein in some embodiments is a pharmaceutical composition comprising a compound represented by a structure provided herein, such as the structure of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or provided in Table 2A Table 2B, Table 2C, or Table 2D, the compound having reduced reactivity with GSH.
[0099] In some embodiments, disclosed herein is a protein modified with a compound disclosed herein, or a salt, solvate, tautomer, or regioisomer thereof, wherein the compound forms a covalent bond with a sulfur atom of a cysteine residue of the protein.
[00100] In some cases, the present disclosure provides a KRAS protein or an active fragment thereof modified with a compound described herein, or a salt, solvate, tautomer, or regioisomer thereof, wherein the compound forms a covalent bond with a sulfur atom of a cysteine residue of the KRAS protein or an active fragment thereof (e.g., a polypeptide thereof).
[00101] Also provided herein are methods of modifying polypeptides, such as proteins, by contactingthe polypeptides with the compounds provided herein. In some embodiments, provided herein are methods of selectively modifying polypeptides in the presence of glutathione with the compounds provided herein comprising contacting the polypeptides with the compound.
[00102] In some embodiments, disclosed herein is a method of modifying (e.g., attaching to and/or degrading) a polypeptide with a compound, comprising contactingthe polypeptide with a compound disclosed herein, or a salt, solvate, tautomer, or regioisomer thereof, to form a covalent bond with a sulfur atom of a cysteine residue of the polypeptide.
[00103] In some cases, the present disclosure provides a method of modifying (e.g., attaching to and/or degrading) KRAS protein or an active fragment thereof with a compound, comprising contacting the polypeptide with a compound described herein, or a salt, solvate, tautomer, or regioisomer thereof, to form a covalentbond with a sulfur atom of a cysteine residue of the KRAS protein or an active fragment thereof (e.g., polypeptide thereof).
[00104] In some embodiments, disclosed herein is a method of binding a compound to a polypeptide, comprising contacting the polypeptide with a compound disclosed herein, or a salt, solvate, tautomer, or regioisomer thereof.
[00105] In some cases, the present disclosure provides a method of binding a compound to KRAS protein or an active fragmentthereof, comprising contactingthe KRAS protein or an active fragment thereof (e.g., polypeptide thereof) with a compound described herein, or a salt or solvate or tautomer or regioisomer thereof.
[00106] In some embodiments, disclosed herein is a method of disrupting a polypeptide (e.g, the function thereof), comprising contactingthe polypeptide with a compound disclosed herein, or a salt, solvate, tautomer, or regioisomer thereof.
[00107] In some cases, the present disclosure provides a method of disrupting KRAS protein or an active fragmentthereof (e.g., a function thereof), comprising contactingthe KRAS protein or an active fragmentthereof (e.g., polypeptide thereof) with a compound described herein, or a salt or solvate or tautomer or regioisomer thereof.
[00108] Provided in some embodiments herein is a method of (e.g., selectively) modifying (e.g, covalently) a polypeptide (e.g., a protein) (e.g., intracellularly) with a compound (e.g., wherein the selectivity is for a sulfur-containing nucleophile of the protein over other (e.g., intracellular) sulfur-containing nucleophiles (e.g., in a biological system)), comprising contacting the polypeptide with the compound described herein, or a salt thereof. In some embodiments, the compound contacts the polypeptide intracellularly (e.g., in an individual).
[00109] Provided in some embodiments herein is a method of (e.g., selectively) modifying (e.g, covalently) KRAS (or a mutant thereof) (e.g., intracellularly) with a compound provided herein (e.g., wherein the selectivity is for a sulfur-containing nucleophile of KRAS (or the mutant thereof) over other (e.g., intracellular) sulfur-containing nucleophiles (e.g., in a biological system))), comprising contacting KRAS (or the mutant thereof) with the compound provided herein, or a salt thereof. In some embodiments, the compound contacts KRAS (or the mutant thereof) intracellularly (e.g., in an individual).
[00110] Provided in some embodiments herein is a method for (e.g., selectively) modifying a polypeptide (e.g., a protein) with a compound represented by a structure provided herein, such as the structure of Formula (A), Formula (II), Formula (II- A), Formula (II-B), or Formula (II-C), the method comprising contactingthe polypeptide with the compound, the compound having reduced reactivity with glutathione (GSH). [00111] Provided in some embodiments herein is a method for (e.g., selectively) modifying a polypeptide (e.g., a protein) in the presence of glutathione (GSH) with a compound represented by a structure provided herein, such as the structure of Formula (A), Formula (II), Formula (II- A), Formula (II-B), or Formula (II-C), the method comprising contacting the polypeptide with the compound (e.g., to form a covalent bond with (e.g., a sulfur atom of a cysteine residue of) the polypeptide) without the compound substantially covalently binding to GSH (e.g., as demonstrated by the lack of covalent binding of GSH to the compound).
[00112] Provided herein in some embodiments, is a method for (e.g., selectively) modifying a polypeptide (e.g., a protein) in the presence of glutathione (GSH) with a compound represented by a structure provided herein, such as the structure of Formula (A), Formula (II), Formula (II- A), Formula (II-B), or Formula (II-C), the method comprising contacting the polypeptide with the compound, wherein the compound is selective for the polypeptide relative to GSH.
[00113] In some embodiments, the polypeptide covalently binds to the compound (e.g., wherein the polypeptide comprises a thiol (e.g., a cysteine residue) that covalently binds to the compound). In some embodiments, the polypeptide contacts (e.g., covalently binds) the compound in the absence of covalent binding of GSH to the compound.
[00114] In some embodiments, the polypeptide comprises Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), proto-oncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), or KRAS.
[00115] In some embodiments, the method further comprises inhibiting, deactivating, or degrading the polypeptide.
INCORPORATION BY REFERENCE
[00116] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference for the specific purposes identified herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[00117] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
[00118] FIG. 1A illustrates an example of a warhead portion, a linker portion, and a proteinbinding ligand portion of a compound provided herein. [00119] FIG. IB illustratesan example of a warhead portion, a linker portion, andaKRAS- binding ligand portion of a compound provided herein.
[00120] FIG. 1C illustrates an example of a warhead portion, a linker portion, and a JAK3- binding ligand portion of a compound provided herein.
[00121] FIG. 2 shows the amino acid sequence of KRAS G12C.
[00122] FIG. 3 shows the amino acid sequence of KRAS G12C Commercial.
[00123] FIG. 4 shows the amino acid sequence of KRAS G12C Lite.
[00124] FIG. 5 shows an exemplary warhead reaction progress curveused to calculate 50% warhead reaction (WR50) values, as described herein.
DETAILED DESCRIPTION OF THE DISCLOSURE
[00125] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the cell" includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. In some embodiments, about is within 10% of the stated number or numerical range. In some embodiments, about is within 5% of the stated number or numerical range. In some embodiments, about is within 1% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, orprocess, orthe like, described herein, "consist of" or "consist essentially of" the described features.
Definitions
[00126] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.
[00127] “KRAS protein” refers to a wild-type KRAS protein or a mutant thereof.
[00128] “ KRAS-binding ligand” refers to a ligand binding to a KRAS protein ora mutant thereof, for example KRAS G12C, KRAS Cl 18 A, or KRAS G12C/C118A. [00129] “ Amino” refers to the -NH2 moiety.
[00130] “Cyano” refers to the -CN radical.
[00131] “Nitro” refers to the -NO2 radical.
[00132] “Hydroxy” or “hydroxyl” refers to the -OH moiety.
[00133] "Alkyl" generally refers to a non-aromatic straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, partially or fully saturated, cyclic or acyclic, having from one to fifteen carbon atoms (e.g., C1-C14 alkyl). Unless otherwise state, alkyl is saturated or unsaturated (e.g., an alkenyl, which comprises at least one carbon-carbon double bond). Disclosures provided herein of an “alkyl” are intended to include independent recitations of a saturated “alkyl,” unless otherwise stated. Alkyl groups described herein are generally monovalent, but may also be divalent (which may also be described herein as “alkylene” or “alkylenyl” groups). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., Ci-Ci2 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., Ci-C8 alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (e.g., C1-C5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C1-C4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (e.g., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., Ci-C2 alkyl). In other embodiments, an alkyl comprises one carbonatom (e.g., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (e.g., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (e.g., C5-C8 alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (e.g., C2-C5 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1 -propyl (w-propyl), 1 -methylethyl (/.w-propyl), 1 -butyl (//-butyl), 1 -methylpropyl (sec-butyl), 2-methylpropyl (Ao-butyl), 1, 1 -dimethyl ethyl (tert-butyl), 1 -pentyl (//-pentyl). The alkyl is attached to the rest of the molecule by a single bond. In general, alkyl groups are each independently substituted or un substituted. Each recitation of “alkyl” provided herein, unless otherwise stated, includes a specific and explicit recitation of an unsaturated “alkyl” group. Similarly, unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(O)N(Ra)2, - N(Ra)C(O)ORa, -OC(O)-N(Ra)2, -N(Ra)C(O)Ra, -N(Ra)S(O)tRa (where t is 1 or 2), -S(O)tORa (where t is 1 or 2), -S(O)tRa (where t is 1 or 2) and -S(O)tN(Ra)2 (where t is 1 or 2) where each Ra is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally sub stituted with halogen, hydroxy , methoxy , or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl). In certain embodiments, an alkyl includes alkenyl, alkynyl, cycloalkyl, carbocycloalkyl, cycloalkylalkyl, haloalkyl, and fluoroalkyl, as defined herein.
[00134] "Alkenyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from two to twelve carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms. In other embodiments, an alkenyl comprises two to four carbon atoms. The alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (z.e., vinyl), prop-l-enyl (z.e., allyl), but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -OC(O)- N(Ra)2, -N(Ra)C(O)Ra, -N(Ra)S(O)tRa (where t is 1 or 2), -S(O)tORa (where t is 1 or 2), -S(O)tRa (where t is 1 or 2) and -S(O)tN(Ra)2 (where t is 1 or 2) where each Rais independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).
[00135] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain linkingthe rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation and having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, zz-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group are through one carbon in the alkylene chain or through any two carbons within the chain. In certain embodiments, an alkylene comprises one to eight carbon atoms (e.g., Ci-C8 alkylene). In other embodiments, an alkylene comprises one to five carbon atoms (e.g., C1-C5 alkylene). In other embodiments, an alkylene comprises one to four carbon atoms (e.g., C1-C4 alkylene). In other embodiments, an alkylene comprises one to three carbon atoms (e.g., C1-C3 alkylene). In other embodiments, an alkylene comprises one to two carbon atoms (e.g., Ci-C2 alkylene). In other embodiments, an alkylene comprises one carbon atom (e.g., Ci alkylene). In other embodiments, an alkylene comprises five to eight carbon atoms (e.g., Cs-C8 alkylene). In other embodiments, an alkylene comprises two to five carbon atoms (e.g., C2-C5 alkylene). In other embodiments, an alkylene comprises three to five carbon atoms (e.g., C3-C5 alkylene). Unless stated otherwise specifically in the specification, an alkylene chain is optionally substituted by one or more of the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -ORa, -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -OC(O)-N(Ra)2, - N(Ra)C(O)Ra, -N(Ra)S(O)tRa (where t is 1 or 2), -S(O)tORa (where t is 1 or 2), -S(O)tRa (where t is 1 or 2) and -S(O)tN(Ra)2 (where t is 1 or 2) where each Ra is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).
[00136] "Alkoxy" refers to a radical bonded through an oxygen atom of the formula -O-alkyl, where alkyl is as defined above. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted, as defined above for an alkyl group.
[00137] "Alkoxyalkyl" refers to an alkyl moiety comprising at least one alkoxy substituent, where alkyl is as defined above. Unless stated otherwise specifically in the specification, an alkoxyalkyl group is optionally substituted, as defined above for an alkyl group.
[00138] "Alkylamino" refers to a moiety of the formula -NHRa or -NRaRb where Ra and Rb are each independently an alkyl group as defined above. Unless stated otherwise specifically in the specification, an alkylamino group is optionally substituted, as defined above for an alkyl group. [00139] "Alkylaminoalkyl" refers to an alkyl moiety comprising at least one alkylamino substituent. The alkylamino substituent can be on a tertiary, secondary or primary carbon. Unless stated otherwise specifically in the specification, an alkylaminoalkyl group is optionally substituted, as defined above for an alkyl group.
[00140] “ Aminoalkyl” refers to an alkyl moiety comprising at least one amino substituent. The amino substituent can be on a tertiary, secondary or primary carbon. Unless stated otherwise specifically in the specification, an aminoalkyl group is optionally substituted, as defined above for an alkyl group.
[00141] "Aryl" refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, z.e., it contains a cyclic, delocalized (4n+2) 7i-electron system in accordance with the Hiickel theory. The ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene. Unless stated otherwise specifically in the specification, the term "aryl" or the prefix "ar-" (such as in "aralkyl") is meant to include aryl radicals optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)- N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb- N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa (where t is 1 or 2), -Rb-S(O)tRa (where t is 1 or 2), -Rb-S(O)tORa (where t is 1 or 2) and -Rb-S(O)tN(Ra)2 (where t is 1 or 2), where each Ra is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally sub stituted with halogen, hydroxy , methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rb is independently a direct bond or a straight or branched alkylene or alkenylene chain, andRc is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated. [00142] “Arylene” ref ers to a divalent aryl group which links one part of the molecule to another part of the molecule. Unless stated specifically otherwise, an arylene is optionally substituted, as defined above for an aryl group.
[00143] "Aralkyl" refers to a radical of the formula -Rc-aryl where Rc is an alkylene chain as defined above, for example, methylene, ethylene, and the like. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.
[00144] The term “carbocycle” or “carbocyclic” refers to a ring or ring system where the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic group from a “heterocycle” or “heterocyclic” in which the ring backbone contains at least one atom which is differentfrom carbon. In some embodiments, carbocycles are monocyclic, bicyclic, polycyclic, spirocyclic or bridged compounds. Carbocycle includes aromatic and partially or fully saturated ring systems. In some embodiments, carbocycle comprises cycloalkyl and aryl. In some embodiments, a carboxycle provided herein is optionally substituted (e.g., carbocycle substituted with one or more carbocycle substitutent, each carbocycle substituent being independently selected from the group consisting of alkyl, oxo, halo, hydroxyl, heteroalkyl, alkoxy, aryl, and heteroaryl). In some embodiments, a heterocycle provided herein is optionally substituted (e.g, heterocycle substituted with one or more heterocycle substitutent, each heterocycle substituent being independently selected from the group consisting of alkyl, oxo, halo, hydroxyl, heteroalkyl, alkoxy, aryl, and heteroaryl).
[00145] "Cyclic ring" refers to a carbocycle or heterocycle, including aromatic, non-saturated, and saturated carbocycle and heterocycle. A "cyclic ring" is optionally monocyclic or polycyclic (e.g., bicyclic).
[00146] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which includes fused or bridged ring systems, having from three to fifteen carbon atoms. In certain embodiments, a cycloalkyl comprises three to ten carbon atoms. In other embodiments, a cycloalkyl comprises five to seven carbon atoms. The cycloalkyl is attached to the rest of the molecule by a single bond. Cycloalkyl is saturated (z.e., containing single C-C bonds only) or unsaturated (ie., containing one or more double bonds or triple bonds). Examples of monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated cycloalkyl is also referred to as "cycloalkenyl." Examples of monocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, the term "cycloalkyl" is meant to include cycloalkyl radicals that are optionally substituted by one or more substituents independently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb- N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa (where t is 1 or 2), -Rb-S(O)tRa (where t is 1 or 2), -Rb- S(O)tORa (where t is 1 or 2) and -Rb-S(O)tN(Ra)2 (where t is 1 or 2), where each Ra is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally sub stituted with halogen, hydroxy , methoxy , or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rb is independently a direct bond or a straight or branched alkylene or alkenylene chain, andRc is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.
[00147] "Cycloalkylalkyl" refers to a radical of the formula -Rc-cycloalkyl where Rc is an alkylene chain as defined above. The alkylene chain and the cycloalkyl radical is optionally substituted as defined above.
[00148] "Halo" or "halogen" refers to bromo, chloro, fluoro or iodo substituents. A “haloalkyl” refers to an alkyl radical, as described herein, that is substituted with one or more halo radical, such as described above.
[00149] "Fluoroalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1 -fluoromethyl-2-fluoroethyl, and the like. In some embodiments, the alkyl part of the fluoroalkyl radical is optionally substituted as defined above for an alkyl group.
[00150] The term “heteroalkyl” refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are substituted with a heteroatom (with the appropriate number of substituents orvalencies- for example, -CH2- may be replaced with -NH- or -O-). For example, each substituted carbon atom is independently substituted with a heteroatom, such as wherein the carbon is substituted with a nitrogen, oxygen, sulfur, or other suitable heteroatom. In some instances, each substituted carbon atom is independently substituted for an oxygen, nitrogen (e.g, -NH-, -N(alkyl)-, or -N(aryl)- or having another substituent contemplated herein), or sulfur (e.g, -S-, -S(=O)-, or -S(=O)2-). In some embodiments, a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. In some embodiments, a heteroalkyl is a C1-C18 heteroalkyl. In some embodiments, a heteroalkyl is a Ci-C12 heteroalkyl. In some embodiments, a heteroalkyl is a Ci-Ce heteroalkyl. In some embodiments, a heteroalkyl is a Ci- C4 heteroalkyl. In some embodiments, heteroalkyl includes alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, heterocyclyl, and heterocycloalkylalkyl, as defined herein. Unless stated otherwise specifically in the specification, heteroalkyl does not include alkoxy as defined herein. Unless stated otherwise specifically in the specification, a heteroalkyl group is optionally substituted as defined above for an alkyl group.
[00151] “Heteroalkylene” refers to a divalent heteroalkyl group defined above which links one part of the molecule to another part of the molecule. Unless stated specifically otherwise, a heteroalkylene is optionally substituted, as defined above for an alkyl group.
[00152] The term “heterocycle” or “heterocyclic” refers to heteroaromatic rings (also known as heteroaryls) and heterocycloalkyl rings (also known as heteroalicyclic groups) that includes at least one heteroatom selected from nitrogen, oxygen and sulfur, wherein each heterocyclic group has from 3 to 12 atoms in its ring system, and with the proviso that any ring does not contain two adjacent O or S atoms. In some embodiments, heterocycles are monocyclic, bicyclic, polycyclic, spirocyclic or bridged compounds. Non-aromatic heterocyclic groups (also known as heterocycloalkyls) include rings having 3 to 12 atoms in its ring system and aromatic heterocyclic groups include rings having 5 to 12 atoms in its ring system. The heterocyclic groups include benzo-fused ring systems. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydro thiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3- azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3 h-indolyl, indolin-2-onyl, isoindolin-1- onyl, isoindoline-1, 3-dionyl, 3,4-dihydroisoquinolin-l(2H)-onyl, 3,4-dihydroquinolin-2(lH)- onyl, isoindoline- 1,3-dithionyl, benzo[d]oxazol-2(3H)-onyl, lH-benzo[d]imidazol-2(3H)-onyl, benzo[d]thiazol-2(3H)-onyl, and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups are either C-attached (or C-linked) or TV-attached where such is possible. For instance, a group derived from pyrrole includes both pyrrol-l-yl (TV-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole includes imidazol-l-yl or imidazol-3-yl (both TV-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). The heterocyclic groups include benzo-fused ring systems. Non-aromatic heterocycles are optionally substituted with one or two oxo (=0) moieties, such as pyrrolidin-2-one. Heterocycle includes aromatic and partially or fully saturated ring systems. In some embodiments, at least one of the two rings of a bicyclic heterocycle is aromatic. In some embodiments, both rings of a bicyclic heterocycle are aromatic.
[00153] "Heterocycloalkyl" refers to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes fused or bridged ring systems. The heteroatoms in the heterocycloalkyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocycloalkyl radical is partially or fully saturated. The heterocycloalkyl is attached to the rest of the molecule through any atom of the ring(s). In some embodiments, heterocycloalkyl comprises 2-12 C atoms, 0-6 N atoms, 0-4 O atoms, and 0-4 S atoms. In some embodiments, heterocycloalkyl comprises 2-10 C atoms, 0-4 N atoms, 0-2 O atoms, and 0-2 S atoms. In some embodiments, heterocycloalkyl comprises 2-8 C atoms, 0-3 N atoms, 0-1 0 atoms, and 0-1 S atoms. In some embodiments, heterocycloalkyl is a saturated or partially unsaturated 3 -7 membered monocyclic, 6-1 Omembered bicyclic, or 13-16 membered polycyclic (e.g., tricyclic or tetracyclic) ring system having 1, 2, 3, or 4 heteroatom ring members each independently selected from N, O, and S. In some embodiments, heterocycloalkyl comprises 1 or 2 heteroatom ring members each independently selected from N, O, and S. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, the term "heterocycloalkyl" is meant to include heterocycloalkyl radicals as defined above that are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-ORa, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-0C(0)-N(Ra)2, -Rb- N(Ra)2, -Rb-C(O)Ra, -Rb-C(O)ORa, -Rb-C(O)N(Ra)2, -Rb-O-Rc-C(O)N(Ra)2, -Rb-N(Ra)C(O)ORa, -Rb-N(Ra)C(O)Ra, -Rb-N(Ra)S(O)tRa (where t is 1 or 2), -Rb-S(O)tRa (where t is 1 or 2), -Rb- S(O)tORa (where t is 1 or 2) and -Rb-S(O)tN(Ra)2 (where t is 1 or 2), where each Ra is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally sub stituted with halogen, hydroxy , methoxy , or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rb is independently a direct bond or a straight or branched alkylene or alkenylene chain, andRc is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.
[00154] "7V-heterocycloalkyl" or “TV-attached heterocycloalkyl” refers to a heterocycloalkyl radical as defined above containing at least one nitrogen and where the point of attachment of the heterocycloalkyl radical to the rest of the molecule is through a nitrogen atom in the heterocycloalkyl radical. An 7V-heterocycloalkyl radical is optionally substituted as described above for heterocycloalkyl radicals. Examples of such 7V-heterocycloalkyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1 -pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.
[00155] " C-heterocycloalkyl" or “C-attached heterocycloalkyl” refers to a heterocycloalkyl radical as defined above containing at least one heteroatom and where the point of attachment of the heterocycloalkyl radical to the rest of the molecule is through a carbon atom in the heterocycloalkyl radical. A C-heterocycloalkyl radical is optionally substituted as described above for heterocycloalkyl radicals. Examples of such C-heterocycloalkyl radicals include, but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.
[00156] "Heteroaryl" refers to a radical derived from a 3 - to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, the heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, ie., it contains a cyclic, delocalized (4n+2) ^-electron system in accordance with the Hiickel theory. Heteroaryl includes fused or bridged ring systems. The heteroatom(s) in the heteroaryl radical is optionally oxidized. One or more nitrogen atoms, if present, are optionally quatemized. The heteroaryl is attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3 -benzodi oxolyl, benzofuranyl, benzooxazolyl, benzo [d]thiazolyl, benzothiadiazo lyl, benzo[b][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,
5.6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,
5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,
1.6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,
5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1 -phenyl- IH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl,
5.6.7.8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,
6.7.8.9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, the term "heteroaryl" is meant to include heteroaryl radicals as defined above which are optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -Rb-0Ra, -Rb-0C(0)-Ra, -Rb-0C(0)-0Ra, -Rb-0C(0)-N(Ra)2, -Rb-N(Ra)2, -Rb- C(0)Ra, -Rb-C(0)0Ra, -Rb-C(0)N(Ra)2, -Rb-0-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb- N(Ra)C(0)Ra, -Rb-N(Ra)S(0)tRa (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)t0Ra (where t is 1 or 2) and -Rb-S(0)tN(Ra)2 (where t is 1 or 2), where each Ra is independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally sub stituted with halogen, hydroxy , methoxy , or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), each Rb is independently a direct bond or a straight or branched alkylene or alkenylene chain, andRc is a straight or branched alkylene or alkenylene chain, and where each of the above substituents is unsubstituted unless otherwise indicated.
[00157] “Heteroarylene” refers to a divalent heteroaryl group which links one part of the molecule to another part of the molecule. Unless stated specifically otherwise, a heteroarylene is optionally substituted, as defined above for a heteroaryl group.
[00158] In general, optionally substituted groups are each independently substituted or un sub stituted. Each recitation of an optionally substituted group providedherein, unless otherwise stated, includes an independent and explicit recitation of both an unsubstituted group and a substituted group (e.g., substituted in certain embodiments, and unsubstituted in certain other embodiments). Unless otherwise stated, a substituted group provided herein (e.g., substituted alkyl) is substituted by one or more substituent, each substituent being independently selected from the group consisting of halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -ORa -SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -C(0)N(Ra)2, -N(Ra)C(0)0Ra, -0C(0)-N(Ra)2, - N(Ra)C(0)Ra, -N(Ra)S(O)tRa (where t is 1 or 2), -S(O)tORa (where t is 1 or 2), -S(O)tRa (where t is 1 or 2) and -S(O)tN(Ra)2 (where t is 1 or 2), where each Ra is independently hydrogen, alkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (e.g., optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl). Unless described otherwise, a substituent of a substituted group or an optionally substituted group recited herein is not further substituted.
[00159] The compounds disclosed herein, in some embodiments, contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R)- or (5)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by this disclosure. When the compounds described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers (e.g., cis or trans .) Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring.
[00160] "Pharmaceutically acceptable salt" includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Exemplary pharmaceutically acceptable salts of the compounds described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
[00161] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p -toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzene sulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S.M. et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66:1-19 (1997)). Acid addition salts of basic compounds are, in some embodiments, prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.
[00162] "Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts are, in some embodiments, formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N, A-dibenzyl ethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N- methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.
[00163] "Pharmaceutically acceptable solvate" refers to a composition of matter that is the solvent addition form. In some embodiments, solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and are formed during the process of making with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein are conveniently prepared or formed during the processes described herein. The compounds provided herein optionally exist in either un solvated as well as solvated forms.
[00164] The term “individual,” “subject,” or “patient” encompasses mammals. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human.
[00165] As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient is still afflicted with the underlying disorder. For prophylactic benefit, the compositions are, in some embodiments, administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease has not been made.
Protein Binding
[00166] Chemical modification is an important tool to alter structure and function of proteins. One way to achieve chemical modification of proteins is to use protein binders (e.g., a (e.g., covalent) small molecule inhibitor). As a result, binders (e.g., covalent small molecule binders (e.g., inhibitors) of proteins) are considered to be useful in multiple applications, including therapeutics. Covalent binding (e.g., inhibition) of a target protein may minimize the required systemic drug exposure. In some embodiments, protein (e.g., functional) activity can only be restored by de novo protein synthesis, resulting in a prolonged therapeutic effect long after the compound is cleared from the blood. Strategically placing an electrophilic moiety on the protein binder (e.g., inhibitor) will allow it to undergo attack by a nucleophilic amino acid residue upon binding to the target protein, forming a reversible or irreversible bond that is much stronger than typical noncovalent interactions. However, the ability to form a covalent bond with the target enzyme has raised concerns about indiscriminate reactivity with off-target proteins, even though some of the most prescribed drugs are covalent irreversible binders. This led to the disfavor of covalent modifiers as drug candidates until the recent successful development of irreversible covalent kinase inhibitors ibrutinib and afatinib, which form an irreversible covalent bond between an acrylamide warhead and a nonconserved cysteine residue on the ATP-binding site but also with nontargeted cellular thiols. The ability to form covalent adducts with off-target proteins has been linked to an increased risk of unpredictable idiosyncratic toxicity along with the increased daily drug dose administered to patients. Accordingly, there is a need to reduce the risk of non-target covalent interactions by incorporating less reactive electrophilic moieties into binders (e.g., to form covalent small molecule binders (e.g., inhibitors). In some embodiments, described herein is a protein binder, such as a covalent small molecule binder (e.g., inhibitor). In some embodiments, described herein is a covalent small molecule protein binder which acts functionally as a protein. In some embodiments, described herein is a covalent small molecule binder which acts functionally as an inhibitor. In some embodiments, the warhead group directs covalent and/or irreversible interaction with a (e.g., cysteine residue of a) protein (e.g., described herein). In some embodiments, the warhead group directs covalent and/or irreversible interaction of a (e.g., cysteine residue of a) protein (e.g., described herein) to a position of the warhead group that is ortho or meta to a sulfur-containing group (e.g., sulfone, sulfide, sulfoxide, or the like). In some embodiments, the protein does not covalently and/or irreversibly interact with the warhead group at a para-position. In some embodiments, the protein covalently and/or irreversibly interacts with the warhead group at either an ortho -position or a meta-position, such as a position of the warhead that is ortho or meta relative to the sulfur-containing group. In some embodiments, described herein is a pharmaceutical composition comprising a protein binder (e.g., a covalent small molecule binder (e.g., inhibitor) and one or more pharmaceutically acceptable excipients. In other embodiments, a protein binder (e.g., covalent small molecule binder (e.g., inhibitor)) is used to treat or prevent a disease or condition in a subject in need thereof.
[00167] In some embodiments, a protein binder provided herein, such as a covalent small molecule binder (e.g., inhibitor) is a benzenesulfonamide derivative compound. In some embodiments, a benzenesulfonamide derivative compound as described herein is used to treat or prevent a disease or condition in a subject in need thereof.
[00168] In some instances, a protein binder provided herein, such as any compound provided herein, such as a compound of Table 2A, Table 2B, Table 2C, or Table 2D binds to, (e.g., covalently) interacts with, modulates (e.g., inhibits), destabilizes, imparts a conformational change, (functionally) disrupts a protein described herein, such as, for example, epidermal growth factor receptor (EGFR), Bruton's tyrosine kinase (BTK), Fibroblast Growth Factor Receptor (FGFR) (e.g., FGFR4), Aurora kinase A (AURKA), tyrosine-protein kinase KIT (KIT), Cytoplasmic tyrosine-protein kinase (BMX)), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g, of the compound)). In some instances, a protein binder provided herein binds to BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)). In some instances, a protein binder provided herein interacts with BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3 , KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)). In some instances, a protein binder provided herein covalently interacts with BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof. In some instances, a protein binder provided herein modulates BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g, of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)). In some instances, a protein binder provided herein inhibits BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur- containing group (e.g., of the compound)). In some instances, a protein binder provided herein destabilizes BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)). In some instances, a protein binder provided herein imparts a conformational change to BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)). In some instances, a protein binder provided herein disrupts BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g, by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)). In some instances, a protein binder provided herein functionally disrupts BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g, of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound)).
[00169] In some instances, an inhibitor is a protein binder that degrades and/or disrupts the functionality of a protein described herein, such as BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof (e.g., by interacting with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g, of the compound)). [00170] In some embodiments, a compound provided herein interacts with the protein at a position of a warhead (e.g., of the compound) that is ortho or meta relative to a sulfur-containing group (e.g., of the compound).
[00171] In some instances, a compound provided herein is an irreversible binder (e.g., inhibitor). In some instances, mass spectrometry, enzyme kinetics, discontinuous exposure (e.g., jump dilution), or any combination thereof are used to determine the amount a compound modifies a target protein. In some instances, mass spectrometry (e.g., of the protein drug target modified (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) in the presence of a compound provided herein) is used to determine if a compoundis an irreversible binder (e.g., inhibitor). In some instances, a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) modulated (e.g., inhibited) by a compound provided herein is subjected to mass spectral analysis (e.g., to assess the formation of permanent, irreversible covalent adducts). In some instances, analytical methods to examine peptide fragments (e.g., generated upon tryptic cleavage of a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) include, but are not limited to mass spectroscopy. In some instances, such methods identify permanent, irreversible covalent protein adducts (e.g., by observing a mass peak that corresponds to the mass of a control sample plus the mass of an irreversible adduct).
[00172] In some instances, such as when a protein described herein interacts (e.g., is bound (e.g, covalently and/or irreversibly bound)) with a compound provided herein, binding of a protein described herein leads to functional inhibition of the protein target (e.g., in a cellular environment).
[00173] In some embodiments, a compound provided herein (e.g., selectively) modifies (e.g, covalently) a polypeptide (e.g., a protein) (e.g., intracellularly) (e.g., wherein the selectivity is for a sulfur-containing nucleophile of the protein over other (e.g., intracellular) sulfur-containing nucleophiles (e.g., in a biological system)).
[00174] Provided in some embodiments herein is a method of (e.g., selectively) modifying (e.g, covalently) a polypeptide (e.g., a protein) (e.g., intracellularly) with a compound (e.g., wherein the selectivity is for a sulfur-containing nucleophile of the protein over other (e.g., intracellular) sulfur-containing nucleophiles (e.g., in a biological system)), comprising contacting the polypeptide with the compound described herein, or a salt thereof. In some embodiments, the compound contacts the polypeptide intracellularly (e.g., in an individual).
[00175] In some embodiments, a measure of (e.g., GSH or protein) modification is relative (e.g, GSH or protein) modification over a period of time. In some embodiments, a measure of (e.g., GSH or protein) modification is relative (e.g., GSH or protein) modification over a period of time of at least 0.5 minutes (e.g., at least 1 minutes, atleast 5 minutes, at least 10 minutes, at least 30 minutes). In some embodiments, a measure of (e.g. , GSH or protein) modificationis relative (e.g., GSH or protein) modification over a period of time of at most 200 minutes (e.g., at most 150 minutes, at most 100 minutes, at most 50 minutes). In some embodiments, the relative (e.g., GSH or protein) modification over a period of time is measured using a method described herein, such as a GSH or GST assay described in Example IV1.
[00176] In some embodiments, a compound provided herein comprises a group (e.g., a warhead) that irreversibly or covalently binds to a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof). In some instances, a warhead provided herein is a functional group that covalently binds (e.g., at a position ortho ormetaofthe warhead that is ortho or meta to a sulfur-containing group of the compound) to an amino acid residue (such as cysteine, lysine, histidine, or other residues capable of being covalently modified), present in or near the binding pocket of a target protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof). In some instances, a warhead provided herein irreversibly inhibits BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof. In some instances, a warhead provided herein covalently and irreversibly inhibits BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof either alone or in combination with L, L1, or L’ (e.g., warhead-L-).
[00177] In some embodiments, a compound provided herein interacts with a protein (e.g., described herein) or a mutant thereof at a position ortho or meta to L. In some embodiments, a compound provided herein covalently and/or irreversibly interacts with a protein (e.g., described herein) or a mutant thereof at a position ortho or meta to L. In some embodiments, a compound provided herein covalently and/or irreversibly interacts with a cysteine residue of a protein (e.g., described herein) or the mutant thereof at a position ortho or meta to L.
[00178] In some embodiments, a compound provided herein is selective for a KRAS protein or a mutant thereof, e.g., selective for KRAS G12C, KRAS C118A, or KRAS G12C/C118A. In some cases, provided herein are small molecule binders (e.g., inhibitors) that bind effectively to a KRAS protein (a.k.a., K-Ras) or a mutantthereof (e.g., KRAS G12C), e.g., by a covalentbond, which are useful for treating cancer, as mutant KRAS proteins are major drivers of human cancers. Also provided herein are pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of diseases such as cancers.
[00179] In some embodiments, provided herein is a compound that interacts with KRAS or a mutantthereof at a position other than Y2. In some embodiments, provided herein is a compound that covalently and/or irreversibly interacts with KRAS or a mutant thereof at a position other than Y2. In some embodiments, provided herein is a compound that covalently and/or irreversibly interacts with a cysteine residue of KRAS or the mutant thereof at a position other than Y2. In some embodiments, provided herein is a compound that covalently and/or irreversibly interacts with a cysteine residue of KRAS or the mutant thereof at a position ortho or meta to Y2.
[00180] In some embodiments, a compound provided herein irreversibly and covalently modifies KRAS G12C at cysteine-12 and/or cysteine-118 in the full-length protein.
[00181] In some embodiments, a compound provided herein interacts with a protein as described in the Examples, such as shown in Tables 3-9F.
[00182] In some embodiments, provided herein is a compound (e.g., of Formula (A), Formula (II), Formula (II- A), Formula (II-B), or Formula (II-C)), wherein the compound (e.g., of Formula (A), Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)) has a warhead, such as wherein the warhead is the part of the compound identified with a box around it in FIG. 1.
[00183] In some embodiments, the warhead comprises a para-activating group, such as a paraactivating group that directs binding to, disruption of, and/or modification of a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3 , KRAS, or a mutant thereof) either alone or in combination with the linker (e.g., L, L’, or L1).
[00184] In some embodiments, provided herein is a compound (e.g., of Formula (A), Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)), wherein the compound (e.g., of Formula (A), Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)) comprises a protein-binding ligand (e.g., G or G’) of any one of the structures of Table 1 A, Table IB, or Table 1C, such as wherein the protein-binding ligand (e.g., G or G’) is the part of the compound identified with a box around it in FIG. 1.
[00185] In some embodiments, a compound of any one of the formulas provided herein, such as Formula (A), Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C) has a G or G’ as described in any of the compounds of Table 2A, Table 2B, Table 2C, or Table 2D. For example, a compound of any one of the formulas (e.g., Formula (A), Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)) provided herein has a G or G’ as identified with a box in FIG. 1.
[00186] In some embodiments, a protein-binding ligand provided herein binds to, disrupts, and/or modifies a protein (e g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) either alone or in combination with a warhead radical provided herein and/or a linker (e.g., L, L’, or L1) provided herein. In some instances, a protein-binding ligand provided herein has activity such that a compound provided herein binds to, disrupts, and/or modifies a protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) at a concentration of about 10 mM or less (e.g, 500 uM or less, lOOuM orless, or 10 uM or less). In some instances, a protein binding ligand provided herein has activity such that a compound provided herein has Ki to a protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS or a mutant thereof) of about 250 uM or less (e.g., about 50 uMor less or about 1 uM or less).
[00187] In some embodiments, the linker (e.g., L, L’, or L1) is a non-releasable linker.
[00188] In some instances, the linker (e.g., L, L’, or L1) does not decompose (e.g., hydrolyze) or release the warhead radical (or a free form thereof), the radical of the protein-binding ligand (or a free form thereof), or any other portion of the compound (e.g., a radical of any Formula provided herein) (or a free form thereof)).
[00189] In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linkergroup, each linker group being independently selected from the group consisting of a bond, -O-, (substituted or un substituted) amino (e.g., -NH-, -NCH3-, methylamine, or dimethylamine), substituted or unsubstituted (e.g., acyclic (e.g., straight or branched) or cyclic) alkyl(ene) (e.g., straight unsubstituted alkyl (e.g., methylene, ethylene, or the like) or straight alkylene substituted with oxo, amino (e.g., -NH-, -NCH3-, or methylamine), heterocyclyl (e.g., (methylene)piperidinyl or piperazinyl), and/or aryl (e.g., (methylene) phenyl)), substituted or unsubstituted (e.g., acyclic (e.g., straight or branched) or cyclic) heteroalkyl(ene) (e.g., cyclic heteroalkylene (e.g., piperazinyl or 1,4-diazepanyl) substituted with alkyl (e.g., methyl) and/or oxo, or straight heteroalkylene substituted with oxo, heterocyclyl (e.g., azetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl), aryl (e.g., phenyl), and/or heteroaryl (e.g., substituted or unsubstituted oxazolyl, pyridinyl, imidazolyl, or pyrazolyl)), substituted or unsubstituted alkoxy (e.g., unsubstituted alkoxy (e.g., methoxy, ethoxy, or the like) or alkoxy substituted with oxo, amino (e.g., -NH-, - NCH3-, substituted (e.g., methylamine) or -NH-azetidinyl-), cycloalkyl (e.g., cyclobutyl substituted with amino (e.g., -NH-, -NCH3-, or methylamine)), and/or heterocyclyl (e.g., azetidinyl or pyrrolidinyl)), and substituted or unsubstituted aryl (e.g., aryl substituted with alkyl (e.g., methyl)).
[00190] In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linkergroup, each linker group being independently selected from the group consisting of -O-, substituted or unsubstituted amino, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, and substituted or unsubstituted alkoxy.
[00191] In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linkergroup, each linker group being independently selected from the group consisting of -O-, substituted or unsubstituted amino and substituted or unsubstituted heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linker group, each linker group being independently selected from the group consisting of -O-, substituted or unsubstituted amino and substituted or unsubstituted acyclic (e.g., straight or branched) heteroalkylene. In some embodiments, the linker (e.g., L, L’, orL1) comprises one or morelinker group, each linkergroup being independently selected from the group consisting of -O-, substituted or unsubstituted amino and substituted or un substituted cyclic heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linker group, each linker group being independently selected from the group consisting of -O-, substituted or unsubstituted amino and substituted or unsubstituted heterocyclyl.
[00192] In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linkergroup, each linker group being independently selected from the group consisting of substituted or unsubstituted amino and substituted or unsubstituted heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linker group, each linker group being independently selected from the group consisting of substituted or unsubstituted amino and substituted or unsubstituted acyclic (e.g., straight or branched) heteroalkylene. In some embodiments, the linker (e.g., L, L’, orL1) comprises one or morelinker group, each linker group being independently selected from the group consisting of substituted or unsubstituted amino and substituted or un substituted cyclic heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises one or more linker group, each linker group being independently selected from the group consisting of substituted or unsubstituted amino and substituted or unsubstituted heterocyclyl.
[00193] In some embodiments, the linker (e.g., L, L’, or L1) comprises -O-.
[00194] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted amino. In some embodiments, the linker (e.g., L, L’, or L1) is -NH-. In some embodiments, the linker (e.g., L, L’, or L1) comprises an alkylamine. In some embodiments, the linker (e.g., L, L’, or LI) comprises a Ci-C6 alkylamine.
[00195] In some embodiments, the linker (e.g., L, L’, or L1) is a bond.
[00196] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted alkylene. In some embodiments, the linker (e.g., L, L’, orL1) comprises substituted or unsubstituted Ci-Ce alkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted Ci-C6 alkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises unsubstituted Ci-C6 alkylene.
[00197] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or un substituted Ci-Ce heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted Ci-C6 heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises unsubstituted Ci-C6 heteroalkylene.
[00198] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted alkoxyalkyl. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted alkoxyalkyl. In some embodiments, the linker (e.g., L, L’, or L1) comprises unsubstituted alkoxyalkyl. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted alkoxyheteroalkyl. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted Ci-C6 alkoxyheteroalkyl. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted Ci-C6 alkoxyheteroalkyl.
[00199] In some embodiments, the linker (e.g., L, L’, or L1) comprises a carbonyl. In some embodiments, the linker (e.g., L, L’, or L1) comprises an ester. In some embodiments, the linker (e.g., L, L’, or L1) comprises a ketone. In some embodiments, the linker (e.g., L, L’, or L1) comprises an amide.
[00200] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted alkylene. In some embodiments, the linker (e.g., L, L’, orL1) comprises substituted or unsubstituted acyclic (e.g., straight or branched) alkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted cyclic alkylene.
[00201] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted acyclic (e.g., straight or branched) heteroalkylene. In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted cyclic heteroalkylene (e.g., heterocycyl).
[00202] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted heterocycyl.
[00203] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or unsubstituted heterocycloalkylene. In some embodiments, the linker (e.g., L, L’, orL1) comprises substituted or unsubstituted C4-C7 heterocycloalkylene. In some embodiments, the linker (e.g, L, L’, orL1) comprises substituted C4-C7 heterocycloalkylene. In some embodiments, the linker (e.g, L, L’, orL1) comprisesunsubstitutedC4-C7heterocycloalkylene. In some embodiments, the linker (e.g., L, L’, orL1) comprises substituted or unsubstituted piperazine (e.g., piperazine substituted with methyl). In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted piperazine. In some embodiments, the linker (e.g., L, L’, or L1) comprises piperazine substituted with one or more methyl, alkoxy, alkoxyalkyl, alkoxyheteroalkyl, or heteroalkyl.
[00204] In some embodiments, the linker (e.g., L, L’, or L1) comprises substituted or un substituted alkoxy.
[00205] In some embodiments, provided herein is a compound (e.g., of Formula (A), Formula (II), Formula (ILA), Formula (II-B), or Formula (ILC)), wherein the compound (e.g., of Formula (A), Formula (II), Formula (ILA), Formula (II-B), or Formula (ILC)) comprises a linker (e.g., L, L’, or L1) of any one of the compounds of Table 2A, Table 2B, Table 2C, or Table 2D, such as wherein the linker (e.g., L, L’, or L1) is the part of the compound identified with a box around it in FIG. 1 .
[00206] In some instances, such as when then linker (e.g., L, L’, or L1) is substituted or unsubstituted heterocyclyl, the linker is part of the protein-binding ligand and/or the warhead. [00207] In some embodiments, a covalent small molecule protein binder (e.g., inhibitor) provided herein is a benzenesulfonamide derivative compound. In some embodiments, a benzenesulfonamide derivative compound as described herein is used to treat or prevent a disease or condition in a subject in need thereof.
[00208] In other embodiments, a pharmaceutical composition comprising a benzenesulfonamide derivative compound as described herein and one or more pharmaceutically acceptable excipients is used to treat or prevent a disease or condition in a subject in need thereof. [00209] In some embodiments, disclosed herein is a method of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of a benzenesulfonamide derivative compound as described herein.
[00210] In other embodiments, disclosed herein is a method of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a benzenesulfonamide derivative compound as described herein and one or more pharmaceutically acceptable excipients.
[00211] In some embodiments, disclosed herein is a protein modified with a benzenesulfonamide derivative compound as described herein, wherein the compound forms a covalent bond with a sulfur atom of a cysteine residue of the protein (e.g., as described elsewhere herein). In some embodiments, disclosedherein is a method of modifying (e.g., attachingto and/or degrading) a polypeptide with a benzenesulfonamide derivative compound as described herein, comprising contacting the polypeptide with the compound to form a covalent bond with a sulfur atom of a cysteine residue of the polypeptide (e.g., as described elsewhere herein). In some embodiments, disclosed herein is a method of binding a compound to a polypeptide, comprising contacting the polypeptide with a benzenesulfonamide derivative compound as described herein (e.g., as described elsewhere herein).
[00212] In one aspect, provided herein is a benzenesulfonamide derivative compound. In some embodiments, a benzenesulfonamide derivative compound is a protein-binding compound. In some embodiments, a benzenesulfonamide derivative compound is a protein-binding ligand inhibitory compound.
[00213] In some embodiments, the warhead comprises a para-activating group, such as a paraactivating group that directs binding to, disruption of, and/or modification of KRAS or a mutant thereof either alone or in combination with L. [00214] In some embodiments, the warhead is a selective warhead. In some embodiments, the warhead is a selective over other cysteine containing selectivity protein S0S1. In some embodiments, the warhead is selective for KRAS, such as over wild-type KRAS.
[00215] In some embodiments, the warhead covalently modifies KRAS (e.g., KRAS G12C (SEQ ID NO: 1 or SEQ ID NO: 2) and/or a mutant KRAS G12C Lite (SEQ ID NO: 3)).
Sequence of KRAS G12C:
MHHHHHHSSG RENLYFQGMT EYKLVVVGAC GVGKSALTIQ LIQNHFVDEY DPTIEDSYRK QVVIDGETCL LDILDTAGQE EYSAMRDQYM RTGEGFLCVF AINNTKSFED IHHYREQIKR VKDSEDVPMV LVGNKCDLPS RTVDTKQAQD LARSYGIPFI ETSAKTRQGV DDAFYTLVRE IRKHKEK (SEQ ID NO: 1)
[00216] Sequence of KRAS G12C Commercial:
HHHHHHSSG RENLYFQGMT EYKLVVVGAC GVGKSALTIQ LIQNHFVDEY DPTIEDSYRK QVVIDGETCL LDILDTAGQE EYSAMRDQYM RTGEGFLCVF AINNTKSFED IHHYREQIKR VKDSEDVPMV LVGNKCDLPS RTVDTKQAQD LARSYGIPFI ETSAKTRQGV DDAFYTLVRE IRKHKEK (SEQ ID NO:2)
[00217] Sequence of KRAS G12C Lite:
MHHHHHHSSG RENLYFQGMT EYKLVVVGAC GVGKSALTIQ LIQNHFVDEY DPTIEDCYRK QVVIDGETSL LDILDTAGQE EYSAMRDQYM RTGEGFLSVF AINNTKSFED IHHYREQIKR VKDSEDVPMV LVGNKSDLPS RTVDTKQAQD LARPYGIPFI ETSAKTRQGV DDAFYTLVRE IRKHKEK (SEQ ID NO:3)
[00218] KRAS G12C Lite (SEQ ID NO: 3) is FL KRAS mutated at all the cysteines except G12C (K-Ras(C51 S/C80L/C118S) describedin reference : Ostrem, J. M. L.; Shokat, K. M. Direct Small- Molecule Inhibitors of KRAS: From Structural Insights to Mechanism-Based Design. Nature Reviews Drug Discovery . Nature Publishing Group November 1, 2016, pp 771-785.
[00219] In some embodiments, the warhead does not covalently modify KRAS WT protein. In some embodiments, the warhead does not substantially covalently modify KRAS WT protein. [00220] In some embodiments, the binds to, disrupts, and/or modifies KRAS G12C (SEQ ID NO:
1 or SEQ ID NO: 2) and/or a mutant KRAS G12C Lite (SEQ ID NO: 3), such as in vitro, such as using differential scanning fluorimetry (DSF), such as described in the Examples.
[00221] In some embodiments, provided herein is a compound (e.g., of Formula (ILB)), wherein the compound (e.g., of Formula (II-B)) comprises a KRAS-binding ligand (e.g., G) of any one of the structures of Table IB, such as wherein the KRAS-binding ligand (e.g., G) is the part of the compound identified with a box around it in FIG. IB.
[00222] In some embodiments, a KRAS-binding ligand provided herein binds to, disrupts, and/or modifies KRAS either alone or in combination with a warhead radical provided herein and/or a linker provided herein. In some instances, a KRAS-binding ligand provided herein has activity such that a compound provided herein binds to, disrupts, and/or modifies KRAS (e.g., KRAS G12C) at a concentration of about 10 mM or less (e.g., 500 uM or less, 100 uM or less, or 10 uM or less). In some instances, a KRAS-binding ligand provided herein has activity such that a compound provided herein has Ki to KRAS (e.g., KRAS G12C) of about 250 uM or less (e.g., about 50 uM or less or about 1 uM or less).
[00223] In some embodiments, the warhead comprises a para-activating group, such as a paraactivating group that directs binding to, disruption of, and/or modification of JAK3 or a mutant thereof either alone or in combination with L.
[00224] In some embodiments, the warhead covalently modifies JAK3.
[00225] In some embodiments,thebindsto, disrupts, and/or modifies JAK3, such as in vitro, such as using differential scanning fluorimetry (DSF), such as described in the Examples.
[00226] In some embodiments, provided herein is a compound (e.g., of Formula (II-C)), wherein the compound (e.g., of Formula (II-C)) comprises a JAK3 -binding ligand (e.g., G) of a structure of Table 1 A or Table 1C, such as wherein the JAK3 -binding ligand (e.g., G) is the part of the compound identified with a box around it in FIG. 1C.
[00227] In some embodiments, a JAK3 -binding ligand provided herein binds to, disrupts, and/or modifies JAK3 either alone or in combination with a warhead radical provided herein and/or a linker provided herein. In some instances, a JAK3 -binding ligand provided herein has activity such that a compound provided herein binds to, disrupts, and/or modifies JAK3 at a concentration of about 10 mM or less (e.g., 500 uM or less, 100 uM or less, or 10 uM or less). In some instances, a JAK3 -binding ligand provided herein has activity such that a compound provided herein has Ki to JAK3 of about 250 uM or less (e.g., about 50 uM or less or about 1 uM or less).
[00228] Provided in some embodiments herein is a compound having a structure of Formula (A):
Figure imgf000042_0001
Formula (A). [00229] In some embodiments, X1 is absent or O. In some embodiments, X1 is O. In some embodiments, X2 is absent, O, orNRA. In some embodiments, X2 is absent. In some embodiments, X2 is O. In some embodiments, X2 is NRA. In some embodiments, Q1 is Rx or L-G. In some embodiments, Q1 is Rx. In some embodiments, Q1 is L-G. In some embodiments, Q2 is L-G or Y2. In some embodiments, Q2 is L-G. In some embodiments, Q2 is Y2. In some embodiments, Y2 is a blocking group. In some embodiments, a blocking group is a group that directs (e.g., covalent and/or irreversible) binding (e.g., of a cysteine residue) of a protein to a position other than Y2. In some embodiments, Rx is substituted or un substituted alkyl orNRXRz. In some embodiments, Rx is substituted or unsubstituted alkyl. In some embodiments, Rx is substituted alkyl. In some embodiments, Rx is unsubstituted alkyl. In some embodiments, Rx is NRyRz. In some embodiments, RA, Ry, and Rz are each independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, RAis hydrogen or unsubstituted alkyl. In some embodiments, RAis hydrogen. In some embodiments, RAi sun substituted alkyl. In some embodiments, Ry is hydrogen or substituted or unsubstituted alkyl. In some embodiments, Ry is hydrogen. In some embodiments, Ry is substituted alkyl. In some embodiments, Ry is unsubstituted alkyl. In some embodiments, Rz is hydrogen or substituted or unsubstituted alkyl. In some embodiments, Rz is hydrogen. In some embodiments, Rz is substituted alkyl. In some embodiments, Rz is unsubstituted alkyl. In some embodiments, L is a linker. In some embodiments, G is an organic residue. In specific embodiments, one and only one of Q1 or Q2 is L-G. In some embodiments, one of Q1 or Q2 is L-G.
[00230] In some embodiments, Rx is substituted or un substituted alkyl. In some embodiments, Rx is unsubstituted alkyl. In some embodiments, Rx is Ci-C6 alkyl. In some embodiments, Rx is methyl.
[00231] In some embodiments, Rx is substituted or unsubstituted aryl.
[00232] In some embodiments, Rx is substituted or unsubstituted heteroaryl.
In some embodiments, Rx is NRyRz. In some embodiments, Ry and Rz are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, Ry and Rz are each independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, Ry and Rz are hydrogen. In some embodiments, Ry and Rz are each independently substituted or unsubstituted alkyl. In some embodiments, Ry is substituted or unsubstituted alkyl and Rz is hydrogen.
[00233] In some embodiments, X1 is absent.
[00234] In some embodiments, X1 is O.
[00235] In some embodiments, at least one of X1 or X2 is O. [00236] In some embodiments, Q1 is not substituted or unsubstituted methoxy phenyl. In some instances, Q1 is not phenyl, such as specifically not methoxy phenyl.
[00237] In some embodiments, Q2 is not substituted or unsubstituted methoxy phenyl. In some instances, Q2 is not phenyl, such as specifically not methoxy phenyl.
[00238] In some embodiments, the compound (e.g., covalently and/or irreversibly) interacts with a protein or a mutant thereof (e.g., a cysteine residue of the protein or the mutant thereof) at a position other than Y2 (e.g., a position ortho or meta to Y2).
[00239] In some embodiments, Y2 is a group that directs (e.g., covalent and/or irreversible) binding (e.g., of a cysteine residue) of a protein (e.g., BTK, EGFR, FGFR, BMX, TEAD, JAK3, KRAS) or a mutant thereof to a position other than Y2 (e.g., a position ortho or meta to Y2).
[00240] In some embodiments, Y2 is not halo. In some embodiments, Y2 is not fluoro.
[00241] In some embodiments, Y2 is hydrogen, CN, NO2, hydroxy, substituted or unsubstituted amino (e.g., -NRxlRy1-, where Rxl is hydroxy and Ry is hydrogen), substituted or unsubstituted alkyl (e.g., fluoroalkyl), substituted or unsubstituted alkoxy (e.g., fluoro alkoxy), or substituted or unsubstituted heteroalkyl (e.g., alkylamine substituted with oxo and/or hydroxy)). In some embodiments, Y2 is hydrogen, CN, NO2, or substituted alkyl (e.g., alkyl substituted with halo (e.g., fluoroalkyl (e.g., methyl substituted with one, two, or three fluoro))).
[00242] In some embodiments, Y2 is hydrogen, CN, NO2, or CF3.
[00243] In some embodiments, Y2 is CN, NO2, or CF3.
In some embodiments, such as when Y2 is hydrogen, thiol, substituted thioether, substituted alkoxy (e.g., fluoroalkoxy), substituted amino, or NO2, the compound (e.g., covalently and/or irreversibly) interacts with a protein or a mutant thereof (e.g., a cysteine residue of the protein or the mutant thereof) at a position other than Y2 (e.g., a position ortho or meta to Y2).
[00244] Provided in some embodiments herein is a compound having a structure of Formula (II):
Figure imgf000044_0001
Formula (II).
[00245] In some embodiments, X1 is absent or O. In some embodiments, X2 is absent, O, or NRA. In some embodiments, RA is hydrogen or substituted or unsubstituted alkyl. In some embodiments, Rxa is substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or NRyRz. In some embodiments, Rxa is substituted or unsubstituted alkyl orNRfRz. In some embodiments, Ry and Rz are each independently hydrogen, substituted or unsubstituted alkyl, sub stituted or unsubstituted aryl, or substituted or un substituted heteroaryl. In some embodiments, R7 and Rz are each independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is a linker. In some embodiments, G is a proteinbinding ligand (e.g., a radical of a compound that interacts with a protein or a mutant thereof, comprising one or more cyclic group wherein the one or more cyclic groups are individually linked by one or more linker). In some embodiments, G is a KRAS-binding ligand. In some embodiments, the compound is a salt.
[00246] In some embodiments, X1 is absent.
[00247] In some embodiments, X1 is O.
[00248] In some embodiments, X2 is absent.
[00249] In some embodiments, X2 is O.
[00250] In some embodiments, X2 is NRA. In some embodiments, RA is hydrogen. In some embodiments, RA is substituted or unsubstituted alkyl. In some embodiments, X2 is NH.
[00251] In some embodiments, Rxais NR.VRZ. In some embodiments, Ry andRz are hydrogen. In some embodiments, Rxa is NH2.
[00252] In some embodiments, Rxa is substituted alkyl. In some embodiments, Rxa is alkyl substituted with oxo and amino. In some embodiments, Rxa is -C(O)NH2.
[00253] In some embodiments, Rxa is substituted or unsub stituted alkyl. In some embodiments, Rxa is unsubstituted alkyl. In some embodiments, Rxais Ci-C6 alkyl. In some embodiments, Rxa is methyl.
[00254] In some embodiments, X1 is O and Rxa is methyl.
[00255] In some embodiments, X1 is O, Rxa is methyl, and X2 is O.
[00256] In some embodiments, X1 is O, Rxa is methyl, and X2 is absent.
[00257] In some embodiments, X1 is O, Rxa is methyl, and X2 is NH.
[00258] In some embodiments, G or G’ is an organic residue. In some embodiments, G or G’ is a radical of a protein-binding ligand, such as a protein-binding ligand provided elsewhere herein. [00259] In some embodiments, G or G’ is a radical of a compound that interacts with a protein (e.g, BTK, EGFR, AURKA, FGFR, BMX, TEAD, JAK3, or KRAS) or a mutant thereof.
[00260] In some embodiments, G or G’ is or comprises a protein-binding ligand selected from a BTK-, EGFR-, FGFR-, AURKA-, BMX, TEAD-, JAK3 -binding ligand, or KRAS-binding ligand.
[00261] In some embodiments, G or G’ is a radical of a BTK -binding ligand. In some embodiments, G or G’ is a radical of an EGFR-binding ligand. In some embodiments, G or G’ is a radical of a FGFR-binding ligand. In some embodiments, G or G’ is a radical of a BMX-binding ligand. In some embodiments, G or G’ is a radical of an AURKA-binding ligand. In some embodiments, G or G’ is a radical of an TEAD-binding ligand. In some embodiments, G or G’ is a radical of an JAK3 -binding ligand. In some embodiments, G or G’ is a radical of a KRAS- binding ligand, such as a KRAS-binding ligand provided elsewhere herein.
[00262] In some embodiments, G is a radical of a KRAS-binding protein.
[00263] Provided in some embodiments herein is a compound having a structure of Formula (II-
B):
Figure imgf000046_0001
Formula (II-B)
[00264] In some embodiments, X1 is absent or O. In some embodiments, X2 is absent, O, or NRA. In some embodiments, RA is hydrogen or substituted or unsubstituted alkyl. In some embodiments, Rxa is alkyl or NRyRz. In some embodiments, Ry and Rz are each independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is a linker described herein. In some embodiments, G is a KRAS-binding ligand described herein, such as a radical of a compound that interacts with KRAS ora mutant thereof (e.g., KRAS G12C, KRAS Cl 18 A, or KRAS G12C/C118A). In some embodiments, the compound is a salt.
[00265] In some embodiments, Gis a radical of a JAK3 -binding ligand, such as a JAK3 -binding ligand provided elsewhere herein.
[00266] In some embodiments, G is a radical of a JAK3 -binding protein.
[00267] Provided in some embodiments herein is a compound having a structure of Formula (II-
C):
Figure imgf000046_0002
Formula (II-C)
[00268] In some embodiments, X1 is absent or O. In some embodiments, X2 is absent, O, or NRA. In some embodiments, RA is hydrogen or substituted or unsubstituted alkyl. In some embodiments, Rxa is alkyl or NRyRz. In some embodiments, Ry and Rz are each independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is a linker described herein. In some embodiments, Gis a JAK3 -binding ligand described herein, such as a radical of a compound that interacts with JAK3 or a mutant thereof. In some embodiments, the compound is a salt.
[00269] In some embodiments, the JAK3 -binding ligand is an un substituted or substituted heterocycle. In some embodiments, the JAK3 -binding ligand is an unsubstituted or substituted pyrrolopyrimidine, an unsubstituted or substitutedpyrrolopyridine, an un substituted or substituted pyrazolopyrimidine, an unsubstituted or substituted pyrazolopyridine, or an unsubstituted or substituted benzimidazole. In some embodiments, the JAK3 -binding ligand is an unsubstituted or substituted pyrrolopyrimidine. In some embodiments, the JAK3-binding ligand is an an unsubstituted or substituted pyrrolopyridine. In some embodiments, the JAK3 -binding ligand is an unsubstituted or substituted pyrazolopyrimidine. In some embodiments, the JAK3-binding ligand is an unsubstituted or substituted pyrazolopyridine. In some embodiments, the JAK3- bindingligand is an unsubstituted or substituted benzimidazole. In some embodiments, the JAK3- binding ligand is a pyrrolopyrimidine described herein, such as a 7H-pyrrolo[2,3-tZ]pyrimidine described herein. In some embodiments, the JAK3 -binding ligand is a pyrrolopyridine described herein, such as a lH-pyrrolo[2,3-b]pyridine described herein. In some embodiments, the JAK3- bindingligand is a pyrazolopyridine described herein, such as apyrazolo[l,5-a]pyridine described herein. In some embodiments, the JAK3 -binding ligand is attached to a linker described herein. [00270] In some embodiments, Ghas a structure represented by any oneofFormulas (IF)-(VII). [00271] In some embodiments, G or G’ has a structure represented in Table 1 A, Table IB, or Table 1C.
[00272] In some embodiments, G or G’ is or comprises one or more (e.g., unsaturated) unsubstituted or substituted carbocycle and/or one or more (e.g., unsaturated) unsubstituted or substituted heterocycle. In some embodiments, G or G’ is or comprises an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) un substituted or substituted heterocycle. In some embodiments, G or G’ consists of an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle. In some embodiments, G or G’ is (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substitutedheterocycle. In some embodiments, GorG’ is substituted or unsubstituted unsaturated carbocycle or substituted or unsubstituted unsaturated heterocycle. In some embodiments, G or G’ comprises one or more cyclic ring systems selected from substituted or unsubstituted unsaturated carbocycles and substituted or unsubstituted unsaturated heterocycles. In some embodiments, G or G’ comprises two or more cyclic ring systems selected from substituted or unsubstituted unsaturated carbocycles and substituted or unsubstituted unsaturated heterocycles. [00273] In some embodiments, G or G’ comprises one or more cyclic ring systems selected from substituted or unsubstituted carbocycles and substituted or unsubstituted heterocycles. In some embodiments, G or G’ comprises two or more cyclic ring systems selected from substituted or unsubstituted carbocycles and substituted or unsubstituted heterocycles.
[00274] In some embodiments, G or G’ is amino, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or alkoxy. In some embodiments, G or G’ is amino. In some embodiments, G or G’ is hydroxyl. In some embodiments, G or G’ is substituted or unsubstituted alkyl. In some embodiments, G or G’ is substituted or unsubstituted heteroalkyl. In some embodiments, G or G’ is alkoxy.
[00275] In some embodiments, G or G’ comprises one or more cyclic group. In some embodiments, Gor G’ comprises one or more cyclic groups wherein the one or more cyclic groups are individually linked by one or more linker (e.g., L, L’, orL1). In other words, Gor G’ comprises one or more cyclic groups, wherein each of the one or more cyclic group are individually linked to another of the one or more cyclic groups by a linker, such as a bond. For example, FIG. IB shows a G’ that comprises a cyclic group (e.g., a heterocyclyl described herein) linked to another cyclic group (e.g., a heteroaryl described herein) via a bond.
[00276] In some embodiments, G comprises an optionally substituted cyclic group, optionally substituted with one ormore L’-G’, wherein each L’ is individually selected from a linker and is connected to another G’ . In some embodiments, each L’ is a linker described elsewhere herein. In some embodiments, G’ comprises G as described elsewhere herein. In some instances, G’ and G are the same. In some instances, G’ and G are different. In some embodiments, G comprises an optionally substituted cyclic group, optionally substituted with -(L’-G’)nL’-G’, wherein n is 0 to 4. In some embodiments, n is 1 to 3. In some embodiments, n is 0 to 3.
[00277] In some embodiments, G is substituted with L’-G’. In some instances, G is substituted with one or more, such as 2 or 3, L’-G’ on a single G. In some embodiments G is substituted with 2 L’-G’, such as on a single G. In some embodiments, G is substituted with -(L’-G’)n-L’-G’. In some embodiments, G is substituted with one or more, such as 2 or 3, -(L’-G’)n-L’-G’ on a single G. In some embodiments, G is substituted with 2 -(L’-G’)n-L’-G’ on a single G.
[00278] In some embodiments, the two or more cyclic ring systems are connected via a bond. In some embodiments, the two or more cyclic ring systems are connected via one or more linker (e.g., L, L’, or LI) and/or bond. In some embodiments, the linker (e.g., L, L’, or L1) is -O-, -NR8- , -N(R8)2 +-, -S-, -S(=O)-, -S(=O)2-, -CH=CH-, =CH-, -C=C-, -C(=O)-, -C(=O)O-, -OC(=O)-, - OC(=O)O-, -C(=O)NR8-, -NR8C(=O)-, -OC(=O)NR8-, -NR8C(=O)O-, -NR8C(=O)NR8-, - NR8S(=O)2-, -S(=O)2NR8-, -C(=O)NR8S(=O)2-, -S(=O)2NR8C(=O)-, substituted or un substituted C1-C4 alkylene, substituted or un substituted Ci-C8 heteroalkylene, -(C1-C4 alkylene)-O-, -O-(Ci- C4 alkylene)-, -(C1-C4 alkylene)-NR8-, -NR8-(CI-C4 alkylene)-, -(C1-C4 alkylene)-N(R8)2 +-, or - N(R8)2+-(CI-C4 alkylene)-; and each R8 is independently hydrogen, substituted or unsubstituted Ci-C4 alkyl, substituted or unsubstituted C1-C4 haloalkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted orunsubstituted C2-C5 alkynyl, substituted orunsubstituted C3-C8 cycloalkyl, substituted orunsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[00279] In some embodiments, the cyclic ring system comprises substituted or unsubstituted monocyclic aryl or substituted orunsubstituted monocyclic heteroaryl. In some embodiments, the cyclic ring system comprises substituted or unsubstituted bicyclic aryl or substituted or unsubstituted bicyclic heteroaryl.
[00280] In some embodiments, G or G’ comprises one or more nitrogen atoms. In some embodiments, G or G’ comprises one or more nitrogen ring atoms. In some embodiments, G or G’ comprises one or more nitrogen atoms within its ring system. In some embodiments, G or G’ comprises one or more nitrogen atoms within its fused ring system. In some embodiments, G or G’ comprises one, two, or three nitrogen atoms (e.g., within its (e.g., fused) ring system).
[00281] In some embodiments, G or G’ comprises one or more (e.g., fused) rings. In some embodiments, G or G’ comprises one or more fused rings.
[00282] In some embodiments, G or G’ is substituted or unsubstituted naphthalene. In some embodiments, G or G’ is substituted naphthalene. In some embodiments, G or G’ is unsubstituted naphthalene.
[00283] In some embodiments, G or G’ is a substituted or unsubstituted indole. In some embodiments, G or G’ is a substituted indole. In some embodiments, G or G’ is an unsubstituted indole. In some embodiments, G or G’ is a substituted or unsubstituted indazole. In some embodiments, G or G’ is a substituted indazole. In some embodiments, GorG’ is an un substituted indazole. In some embodiments, G or G’ is a substituted or unsubstituted quinoline. In some embodiments, G or G’ is a substituted quinoline. In some embodiments, G or G’ is an unsubstituted quinoline.
[00284] In some embodiments, G or G’ is aromatic or partially aromatic. In some embodiments, G or G’ is partially aromatic. In some embodiments, G or G’ is partially aromatic.
[00285] In some embodiments, G or G’ comprises one or more substituted or unsubstituted aromatic ring(s). In some embodiments, G or G’ comprises one substituted or unsubstituted aromatic ring. In some embodiments, GorG’ comprises two substituted orunsubstituted aromatic rings. In some embodiments, G or G’ comprises three substituted orunsubstituted aromatic rings. [00286] In some embodiments, G or G’ comprises one or more (e.g., one, two, or three) substituted or unsubstituted heteroaromatic ring(s). [00287] In some embodiments, G or G’ comprises two or more substituted or unsubstituted aromatic or partially aromatic rings. In some embodiments, each aromatic or partially aromatic ring independently is a carbocycle or a heterocycle.
[00288] In some embodiments, G or G’ comprises one or more substituted or unsubstituted carbocycle and one or more substituted or unsubstitued heterocycle. In some embodiments, each of the one or more substituted or unsubstituted carbocycle and the one or more substituted or unsubstitued heterocycle are independently linked (e.g., fused) to a substituted or unsubstituted carbocycle or a sub stituted orunsubstitued heterocycle by aminoorabond. In some embodiments, G or G’ comprises one ormore substituted orunsubstituted carbocycle andoneormore substituted or unsubstitued heterocycle, each of the one or more substituted orunsubstituted carbocycle and the one or more substituted or unsubstitued heterocycle independently being linked (e.g., fused) to a substituted orunsubstituted carbocycle ora substituted orunsubstituedheterocycleby abond. [00289] In some embodiments, G or G’ comprises a substituted or unsubstituted carbocycle and a substituted or unsubstitued heterocycle, the substituted or unsubstituted carbocycle and the substituted or unsubstitued heterocycle being linked (e.g., fused) by a bond.
[00290] In some embodiments, G or G’ comprises two (or more) substituted or unsubstituted heteroaromatic rings, the heteroaromatic rings being linked (e.g., fused) by a bond, each heteroaromatic ring being aromatic or partially aromatic. In some embodiments, the heteroaromatic rings are selected from the group consisting of benzimidazole, indole, indolizine, pyridine, quinoline, indazole, pyrimidine, and pyridopyrimidinone (e.g., pyrido[2,3-d]pyrimidin- 2(lH)-one). In some embodiments, the heteroaromatic rings are selected from the group consisting of benzimidazole, indolizine, quinoline, indazole, and pyrimidine. In some embodiments, G or G’ is a substituted benzimidazole linked to an unsubstituted indolizine by a bond. In some embodiments, G or G’ is a substituted benzimidazole linked to an unsubstituted phenyl by a bond. In some embodiments, G or G’ is an unsubstituted quinoline linked to a substituted phenyl by a bond. In some embodiments, G or G’ is a substituted benzimidazole linked to an unsubstituted pyrimidine by abond. In some embodiments, G or G’ is a substituted quinoline linked to a substituted indazole by a bond.
[00291] In some embodiments, G or G’ has a structure of:
Figure imgf000050_0001
[00292] In some embodiments, G or G’ has a structure of:
Figure imgf000051_0001
[00293] In some embodiments, G or G’ has a structure of:
Figure imgf000051_0002
[00294] In some embodiments, G or G’ has a structure of:
Figure imgf000051_0003
[00295] In some embodiments, G or G’ has a structure of:
Figure imgf000051_0004
[00296] In some embodiments, G or G’ comprises a substituted or unsubstituted carbocycle. In some embodiments, G or G’ comprises an unsubstituted carbocycle. In some embodiments, G or G’ comprises a substituted carbocycle.
[00297] In some embodiments, G or G’ comprises a substituted or unsubstituted heterocycle. In some embodiments, G or G’ comprises a substituted heterocycle. In some embodiments, G or G’ comprises an unsubstituted heterocycle.
[00298] In some embodiments, G or G’ is a substituted carbocycle.
[00299] In some embodiments, G or G’ is a substituted phenyl.
[00300] In some embodiments, G or G’ is a substituted heterocycle.
[00301] In some embodiments, G or G’ is a substituted quinazoline or a substituted pyrazolopyrimidine. In some embodiments, G or G’ is a substituted quinazoline. In some embodiments, G or G’ is a substituted pyrazolopyrimidine. In some embodiments, G or G’ is a substituted lH-pyrazolo[3,4-d]pyrimidine.
[00302] In some embodiments, G or G’ is an unsubstituted or substituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-t/]pyrimidine). In some embodiments, G or G’ is an unsubstituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-J]pyrimidine). In some embodiments, G or G’ is an unsubstituted 7H-pyrrolo[2,3-J]pyrimidine.
[00303] In some embodiments, G or G’ is a substituted or unsubstituted 4-6 membered O- heterocycle. In some embodiments, G or G’ is an unsubstituted 5-6 membered O-heterocycle. In some embodiments, G or G’ is a substituted 5-6 membered O-heterocyle. In some embodiments, G or G’ is a substituted orunsubstitutedtetrahydro-2H-pyran. In some embodiments, G or G’ is a substituted or unsubstituted tetrahydrofuran.
[00304] In some embodiments, G or G’ is a substituted or un substituted N-heterocycle. In some embodiments, G or G’ is a substituted N-heterocycle. In some embodiments, G or G’ is an unsubstituted N-heterocycle. In some embodiments, G or G’ is a substituted or unsubstituted piperazine. In some embodiments, G or G’ is a substituted or unsubstituted piperidine. In some embodiments, G or G’ is substituted or unsubstituted pyridine. In some embodiments, G or G’ is substituted pyridine. In some embodiments, G or G’ is unsubstituted pyridine. In some embodiments, G or G’ is a substituted or un substituted pyrazole. In some embodiments, G or G’ is a substituted pyrazole. In some embodiments, G or G’ is an unsubstituted pyrazole. In some embodiments, G or G’ is a substituted or unsubstituted imidazole. In some embodiments, G or G’ is substituted imidazole. In some embodiments, G or G’ is unsubstituted imidazole. In some embodiments, G or G’ is substituted or unsubstituted pyrimidine. In some embodiments, G or G’ is substituted pyrimidine. In some embodiments, G or G’ is unsubstituted pyrimidine.
[00305] In some embodiments, G or G’ is a substituted or un substituted quinazoline. In some embodiments, G is a substituted quinazoline. In some embodiments, G or G’ is an unsubstituted quinazoline.
[00306] In some embodiments, G or G’ is or comprises a substituted or unsubsituted quinazoline. In some embodiments, G or G’ is or comprises a substituted or unsubstituted tetrahydropyridopyrimidine (e.g., 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine). In some embodiments, G or G’ is or comprises a substituted or unsubsituted quinoline. In some embodiments, G or G’ is or comprises a substituted or unsubstituted pyridopyrazinone (e.g., pyrido[2,3-b]pyrazin-3(4H)-one). In some embodiments, G or G’ is or comprises a substituted or unsubstituted benzimidazole. In some embodiments, G or G’ is or comprises a substituted or unsubstituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-t/]pyrimidine). In some embodiments, G or G’ is or comprises a substituted or unsubstituted pyrazolopyridine (e.g., a pyrazolo[l,5- a]pyridine). In some embodiments, Gor G’ is or comprises an unsubstitutedpyrrolopyridine (e.g, a lH-pyrrolo[2,3-Z>]pyridine). In some embodiments, G or G’ is or comprises a substituted pyrrolopyridine (e.g., a lH-pyrrolo[2,3-Z>]pyridine). [00307] In some embodiments, G or G’ is a substituted quinazoline, a substituted tetrahydropyridopyrimidine (e.g., 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine), a substituted quinoline, a substituted pyridopyrazinone (e.g., pyrido[2,3-b]pyrazin-3(4H)-one).
[00308] In some embodiments, G or G’ is a substituted tetrahydropyridopyrimidine (e.g., 5, 6, 7, 8- tetrahydropyrido[3,4-d]pyrimidine).
[00309] In some embodiments, G or G’ is a substituted quinazoline.
[00310] In some embodiments, G or G’ is a substituted tetrahydropyridopyrimidine (e.g., 5, 6, 7, 8- tetrahydropyrido[3,4-d]pyrimidine).
[00311] In some embodiments, G or G’ is a substituted quinoline.
[00312] In some embodiments, G or G’ is an unsubstituted or substituted benzothiophene or an unsubstituted or substituted thiophene. In some embodiments, G or G’ is an unsubstituted benzothiophene or an unsubstituted thiophene.
[00313] In some embodiments, G or G’ is a substituted or unsubstituted isoxazole (e.g., 3, 5- dimethylisoxazole). In some embodiments, G or G’ is a substituted isoxazole (e.g., 3, 5- dimethylisoxazole). In some embodiments, G or G’ is a substituted 3, 5-dimethylisoxazole.
[00314] In some embodiments, G or G’ is a substituted pyridopyrazinone (e.g., pyrido[2,3- b]pyrazin-3(4H)-one).
[00315] In some embodiments, G or G’ has a structure represented by Formula (II’):
Figure imgf000053_0001
Formula (IF).
[00316] In some embodiments,Rlx, R2x, R3x, R4x, R5x, R6x, and R7x are each independently selected from the group consisting of hydrogen, halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
[00317] In some embodiments,Rlx, R2x, R3x, R4x, R5x, R6x, andR7x are each independently selected from the group consisting of hydrogen, halogen, and substituted or unsubstituted alkyl.
[00318] In some embodiments, Rlx is hydrogen.
[00319] In some embodiments, R2x is halogen. In some embodiments, R2x is fluoro. [00320] In some embodiments, R3x is halogen. In some embodiments, R3x is chloro.
[00321] In some embodiments, R3x is substituted aryl. In some embodiments, R3x is aryl substituted with halogen. In some embodiments, R3x is aryl substituted with hydroxy. In some embodiments, R3x is aryl substituted with halogen and hydroxy. In some embodiments, R3x is aryl substituted with fluoro and hydroxy.
[00322] In some embodiments, R4x is substituted or unsubstituted alkyl. In some embodiments, R4x is methyl.
[00323] In some embodiments, R5x is hydrogen.
[00324] In some embodiments, R6x is hydrogen.
[00325] In some embodiments, R7x is substituted or unsubstituted alkyl. In some embodiments, R7x is isopropyl.
[00326] In some embodiments, Rlx is hydrogen, R2x is fluoro, R3x is aryl substituted with fluoro and hydroxy, R4x is methyl, R5x is hydrogen, R6x is hydrogen, and R7x is isopropyl.
[00327] In some embodiments, G or G’ has a structure represented by Formula (II’-A):
Figure imgf000054_0001
Formula (II’-A)
[00328] In some embodiments, Rlx is hydrogen, R2x is fluoro, R3x is chloro, R4x is methyl, R5x is hydrogen, R6x is hydrogen, and R7x is isopropyl.
[00329] In some embodiments, G or G’ has a structure represented by Formula (IF-B):
Figure imgf000054_0002
Formula (IF-B)
[00330] In some embodiments, G or G’ has a structure represented by Formula (IF-C):
Figure imgf000055_0001
Formula (IF-C)
[00331] In some embodiments, G or G’ has a structure represented by Formula (II’-D):
Figure imgf000055_0002
Formula (II’-D)
[00332] n some embodiments, G or G’ has a structure represented by Formula (III):
Figure imgf000055_0003
Formula (III)
[00333] In some embodiments, R8a is hydrogen, halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or un substituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, eachR9ais independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, each R10ais independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsub stituted alkoxy, substituted or unsub stituted aryl, and sub stituted or unsubstituted heteroaryl. In some embodiments, m is 0-6. In some embodiments, n is 0-7.
[00334] In some embodiments, R8a is hydrogen.
[00335] In some embodiments, n is 0 or 1.
[00336] In some embodiments, m is 0. [00337] In some embodiments, R10a is halogen, hydroxy, or unsubstituted alkoxy. In some embodiments, R10a is chloro, hydroxy, or OMe. In some embodiments, R10a is chloro. In some embodiments, R10a is hydroxy. In some embodiments, R10a is OMe.
[00338] In some embodiments, R8a is hydrogen, m is 0, n is 1, and R10a is chloro.
[00339] In some embodiments, G or G’ has a structure represented by Formula (III-A):
Figure imgf000056_0001
Formula (III-A)
[00340] In some embodiments, R8a is hydrogen, m is 0, and n is 0.
[00341] In some embodiments, G or G’ has a structure represented by Formula (III-B):
Figure imgf000056_0002
Formula (III-B)
[00342] In some embodiments, R8a is hydrogen, m is 0, n is 1, and R10a is hydroxy.
[00343] In some embodiments, G or G’ has a structure represented by Formula (III-C):
Figure imgf000056_0003
Formula (III-C)
[00344] In some embodiments, R8a is hydrogen, m is 0, n is 1, and R10a is -OMe.
[00345] In some embodiments, G or G’ has a structure represented by Formula (III-D):
Figure imgf000056_0004
Formula (III-D)
[00346] In some embodiments, G or G’ has a structure represented by Formula (IV):
Figure imgf000057_0001
Formula (IV)
[00347] In some embodiments, R11 and R12 are each independently hydrogen, halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsub stituted alkoxy, substituted or unsub stituted aryl, and substituted or un substituted heteroaryl. In some embodiments, each R13 is independently selected from the group consisting of halogen, hydroxy, substituted or un sub stituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, each R14 is independently selected from the group consisting of halogen, hydroxy, substituted or un sub stituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, o is 0-3. In some embodiments, p is 0-5.
[00348] In some embodiments, R11 is hydrogen.
[00349] In some embodiments, R12 is hydrogen.
[00350] In some embodiments, o is 0 or 1.
[00351] In some embodiments, R13 is halogen (e.g., chloro). In some embodiments, R13 is chloro. [00352] In some embodiments, each R14 is independently alkyl.
[00353] In some embodiments, each R14 is independently unsubstituted alkyl. In some embodiments, p is 2 and each R14 is methyl.
[00354] In some embodiments, R11 is hydrogen, R12 is hydrogen, o is 0, p is 2, and each R14 is methyl.
[00355] In some embodiments, G or G’ has a structure represented by Formula (IV-A):
Figure imgf000057_0002
Formula (IV-A) [00356] In some embodiments, R11 is hydrogen, R12 is hydrogen, o is 1, R13 is chloro, p is 2, and each R14 is independently methyl.
[00357] In some embodiments, G or G’ has a structure represented by Formula (IV-B):
Figure imgf000058_0001
Formula (IV-B)
[00358] In some embodiments, G or G’ has a structure represented by Formula (V):
Figure imgf000058_0002
Formula (V)
[00359] In some embodiments, R15 is hydrogen, halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or un substituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, each R16 is independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, each R17 is independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsub stituted alkoxy, substituted or unsub stituted aryl, and substituted or un substituted heteroaryl. In some embodiments, r is 0-3. In some embodiments, s is 0-5.
[00360] In some embodiments, R15 is hydrogen.
[00361] In some embodiments, r is 1 or 2.
[00362] In some embodiments, each R16 is independently halogen. In some embodiments, each R16 is independently chloro or fluoro.
[00363] In some embodiments, r is 1 and R16 is fluoro.
[00364] In some embodiments, R17 is independently halogen (e.g., fluoro) or hydroxyl. In some embodiments, R17 is independently fluoro or hydroxyl. [00365] In some embodiments, R15 is hydrogen, r is 1, R16 is chloro, s is 1, and R14 is fluoro.
[00366] In some embodiments, G or G’ has a structure represented by Formula (V-A):
Figure imgf000059_0001
Formula (V-A)
[00367] In some embodiments, G or G’ has a structure represented by Formula (VI):
Figure imgf000059_0002
[00368] In some embodiments, each R18 is independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, each R19 is independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or un substituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, each R20 is independently selected from the group consisting of halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or un substituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, R21 is hydrogen, halogen, hydroxy, substituted or un substituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some embodiments, tis 0-6. In some embodiments, u is 0-7. In some embodiments, v is 0-7.
[00369] In some embodiments, t is 0.
[00370] In some embodiments, u is 1. [00371] In some embodiments, R19 is halogen (e.g., chloro). In some embodiments, R19 is chloro.
[00372] In some embodiments, v is 0.
[00373] In some embodiments, R21 is unsubstituted alkyl (e.g., methyl). In some embodiments, R21 is methyl.
[00374] In some embodiments, t and v are 0, u is 1, R19 is chloro, and R21 is methyl.
[00375] In some embodiments, G or G’ has a structure represented by Formula (VI-A):
Figure imgf000060_0001
Formula (VI-A)
[00376] In some embodiments, G or G’ has a structure represented by Formula (VI-B):
Figure imgf000060_0002
Formula (VI-B)
[00377] In some embodiments, G or G’ has a structure represented by Formula (VI-C):
Figure imgf000060_0003
Formula (VI-C)
[00378] In some embodiments, G or G’ has a structure represented by Formula (VII):
Figure imgf000060_0004
Formula (VII)
[00379] In some embodiments, R22, R23, R24, R25, and R26 are each independently selected from the group consisting of hydrogen, halogen, hydroxy, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substitutedorunsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
[00380] In some embodiments, R22 is hydrogen, hydroxy, or substituted or unsubstituted heteroalkyl. In some embodiments, R22 is hydrogen. In some embodiments, R22 is hydroxy. In some embodiments, R22 is substituted or unsubstituted heteroalkyl.
[00381] In some embodiments, R23 is hydrogen or halogen. In some embodiments, R23 is hydrogen. In some embodiments, R23 is halogen. In some embodiments, R23 is hydrogen or chloro. [00382] In some embodiments, R24 is hydrogen or halogen. In some embodiments, R24 is hydrogen. In some embodiments, R24 is halogen. In some embodiments, R24 is chloro or bromo. In some embodiments, R24 is hydrogen, chloro or bromo.
[00383] In some embodiments, R25 is hydrogen, halogen, or substituted alkyl. In some embodiments, R25 is hydrogen. In some embodiments, R25 is halogen. In some embodiments, R25 is chloro. In some embodiments, R25 is substituted alkyl.
[00384] In some embodiments, R26 is hydrogen, halogen (e.g., chloro), unsubstituted alkoxy, or substituted alkyl. In some embodiments, R26 is hydrogen. In some embodiments, R26 is halogen. In some embodiments, R26 is chloro. In some embodiments, R26 is unsubstituted alkoxy. In some embodiments, R26 is substituted alkyl.
[00385] In some embodiments, R22 is hydrogen, R23 is hydrogen, R24 is chloro, R25 is hydrogen, and R26 is chloro.
[00386] In some embodiments, G or G’ has a structure represented by Formula (VII-A):
Figure imgf000061_0001
Formula (VII-A)
[00387] In some embodiments, R22 is hydrogen, R23 is chloro, R24 is hydrogen, R25 is hydrogen, and R26 is chloro.
[00388] In some embodiments, G or G’ has a structure represented by Formula (VII-B):
Figure imgf000061_0002
Formula (VII-B)
[00389] In some embodiments, GorG’ is substituted orunsubstitutedpyrrolopyridine(e.g., 1H- pyrrolo[2,3-Z>]pyridine). In some embodiments, G or G’ is substituted pyrrolopyridine. In some embodiments, G or G’ is lH-pyrrolo[2,3-Z>]pyridine substituted with substituted alkylamine, such as alkylamine substituted with oxo (e.g., -C(O)NH2). In some embodiments, Gor G’ is a JAK3- binding ligand that is a substituted or unsubstituted pyrrolopyridine (e.g., lH-pyrrolo[2,3- Z>]pyridine).
[00390] In some embodiments, G or G’ is a substituted or unsubstituted pyrazolopyrimidine (e.g., a lH-pyrazolo[3,4-d]pyrimidine). In some embodiments, G or G’ is a substituted pyrazolopyrimidine (e.g., a lH-pyrazolo[3,4-d]pyrimidine). In some embodiments, Gor G’ is a substituted lH-pyrazolo[3,4-d]pyrimidine. In some embodiments, Gor G’ is lH-pyrazolo[3,4- d]pyrimidine substituted with amino alkyl (e.g., -NHMe). In some embodiments, G or G’ is a JAK3 -binding ligand that is a substituted or unsubstituted pyrazolopyrimidine (e.g., a 1H- pyrazolo[3 ,4-d]pyrimidine).
[00391] In some embodiments, G or G’ has a structure represented by Formula (VII-B):
Figure imgf000062_0001
Formula (VII-C)
[00392] In some embodiments, G or G’ is unsubstituted or substituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-t/]pyrimidine). In some embodiments, G or G’ is unsubstituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-t/]pyrimidine). In some embodiments, G or G’ is unsubstituted 7H- pyrrolo[2,3-J]pyrimidine. In some embodiments, G or G’ is a JAK3 -binding ligand that is an unsubstituted or substituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-J]pyrimidine).
[00393] In some embodiments, G or G’ has a structure represented by Formula (VII-D):
Figure imgf000062_0002
Formula (VII-D)
[00394] In some embodiments, G or G’ is unsubstituted or substituted pyrazolopyrimidine (e.g, lH-pyrazolo[3,4-d]pyrimidine). In some embodiments, G or G’ is substituted pyrazolopyrimidine (e.g., lH-pyrazolo[3,4-d]pyrimidine), such as pyrazolopyrimidine (e.g., lH-pyrazolo[3,4- d]pyrimidine) substituted with substituted amino (e.g., amino substituted with alkyl (e.g., methyl) or unsubstituted or substituted heterocyclyl, such as pyrazole substituted with alkoxy). In some embodiments, G orG’ is substituted lH-pyrazolo[3,4-d]pyrimidine. In some embodiments, G or G’ is a JAK3 -binding ligand thatis an unsubstituted or substituted pyrazolopyrimidine (e.g., 1H- pyrazolo[3 ,4-d]pyrimidine). [00395] In some embodiments, G or G’ is substituted or unsubstituted pyrazolopyridine (e.g., a pyrazolofl ,5-a]pyridine). In some embodiments, G or G’ is unsubstituted pyrazolopyridine (e.g, a pyrazolofl, 5-a]pyridine). In some embodiments, G or G’ is un substituted a pyrazolofl, 5- a]pyridine. In some embodiments, G or G’ is a JAK3 -binding ligand that is an unsubstituted or substituted pyrazolopyridine (e.g., a pyrazolofl, 5-a]pyridine).
[00396] In some embodiments, G or G’ is unsubstituted or substituted benzimidazole. In some embodiments, G or G’ is substituted benzimidazole. In some embodiments, G or G’ is benzimidazole substituted with alkyl (e.g., methyl or fluoroalkyl).
[00397] In some embodiments, G’ is a radical of a protein-binding ligand, such as a proteinbinding ligand provided elsewhere herein.
[00398] In some embodiments, G’ is a radical of a compound that interacts with a protein (e.g., BTK, EGFR, FGFR, BMX, TEAD, JAK3, or KRAS) or a mutant thereof.
[00399] In some embodiments, G’ is or comprises a protein-binding ligand selected from a BTK-, EGFR-, FGFR-, TEAD-, JAK3-, KRAS-, or BMX-binding ligand.
[00400] In some embodiments, G’ is a radical of a BTK -binding ligand. In some embodiments, G’ is a radical of an EGFR-binding ligand. In some embodiments, G is a radical of a FGFR- binding ligand. In some embodiments, G’ is a radical of a BMX-binding ligand. In some embodiments, G’ is a radical of an AURKA-binding ligand. In some embodiments, G’ is a radical of a TEAD-binding ligand. In some embodiments, G’ is a radical of a JAK3 -binding ligand. In some embodiments, G’ is a radical of a KRAS-binding ligand.
[00401] In some embodiments, G’ has a structure represented in Table 1 A, Table IB, or Table 1C. In some embodiments, G’ is or comprises one or more (e.g., unsaturated) unsubstituted or substituted carbocycle and/or one or more (e.g., unsaturated) unsubstituted or substituted heterocycle. In some embodiments, G’ is or comprises an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle)). In some embodiments, G’ consists of an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle)). In some embodiments, G’ is (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle. In some embodiments, G’ is substituted or unsubstituted unsaturated carbocycle or substituted or unsubstituted unsaturated heterocycle. In some embodiments, G’ comprises one or more cyclic ring systems selected from substituted or unsubstituted unsaturated carbocycles and substituted or unsubstituted unsaturated heterocycles. In some embodiments, G’ comprises two or more cyclic ring systems selected from substituted or unsubstituted unsaturated carbocycles and substituted or unsubstituted unsaturated heterocycles. [00402] In some embodiments, G’ comprises one or more cyclic ring systems selected from substituted or unsubstituted carbocycles and substituted or unsubstituted heterocycles. In some embodiments, G’ comprises two or more cyclic ring systems selected from substituted or unsubstituted carbocycles and substituted or unsubstituted heterocycles.
[00403] In some embodiments, G’ comprises one or more cyclic group. In some embodiments, G’ comprises one or more cyclic groups wherein the one or more cyclic groups are individually linked by one or more linker (e.g., L, L’, or L1).
[00404] In some embodiments, G’ comprises one or more nitrogen atoms. In some embodiments, G’ comprises one or more nitrogen ring atoms. In some embodiments, G’ comprises one or more nitrogen atoms within its ring system. In some embodiments, G’ comprises one or more nitrogen atoms within its fused ring system. In some embodiments, G’ comprises one, two, or three nitrogen atoms (e.g., within its (e.g., fused) ring system).
[00405] In some embodiments, G’ comprises one or more (e.g., fused) rings. In some embodiments, G’ comprises one or more fused rings.
[00406] In some embodiments, G’ is aromatic or partially aromatic. In some embodiments, G’ is partially aromatic. In some embodiments, G’ is partially aromatic.
[00407] In some embodiments, G’ comprises one or more substituted or un substituted aromatic ring(s). In some embodiments, G’ comprises one substituted or unsubstituted aromatic ring. In some embodiments, G’ comprises two substituted or unsubstituted aromatic rings. In some embodiments, G’ comprises three substituted or un substituted aromatic rings.
[00408] In some embodiments, G’ comprises one or more (e.g., one, two, or three) substituted or unsubstituted heteroaromatic ring(s).
[00409] In some embodiments, G’ comprises a substituted or unsubstituted carbocycle. In some embodiments, G’ comprises an unsubstituted carbocycle. In some embodiments, G’ comprises a substituted carbocycle.
[00410] In some embodiments, G’ comprises a substituted orunsubstitutedheterocycle. In some embodiments, G’ comprises a substituted heterocycle. In some embodiments, G’ comprises an unsubstituted heterocycle.
[00411] In some embodiments, G’ is a substituted carbocycle.
[00412] In some embodiments, G’ is a substituted phenyl.
[00413] In some embodiments, G’ is a substituted heterocycle.
[00414] In some embodiments, G’ is a substituted quinazoline or a substituted pyrazolopyrimidine. In some embodiments, G’ is a substituted quinazoline. In some embodiments, G’ is a substituted pyrazolopyrimidine. In some embodiments, G’ is a substituted lH-pyrazolo[3,4-d]pyrimidine. [00415] Unless stated specifically otherwise herein, each instance of radical indicates that a hydrogen (i.e., a hydrogen radical (H*)) is removed from a free form of a compound provided herein, such as any protein-binding ligand (e.g., G or G’) or warhead described herein. In some instances, the removal of the hydrogen radical from the compound provided herein, such as any protein-binding ligand (e.g., G or G’) or warhead described herein, provides a radical of a proteinbinding ligand or a warhead that is taken together with any point of a linker provided herein (e.g, L, L1, orL’) to form a bond (e.g., between the linker and the radical of the protein-binding ligand or the warhead). In some instances, a carbon atom (e.g., of any protein-binding ligand (e.g., a substituted heterocycle or a substituted carbocycle) or warhead described herein) loses an H* to become a point of attachment to L. In some instances, >NH loses an H* to become >N-(point of attachment), such as >N-L-G, >N-L-warhead, >N-DG, or >N-warhead. In some instances, -OH loses an H* to become -O-(point of attachment), such as -O-L-G, -O-L-warhead, -O-G, or -O- warhead. In some instances, -S(=O)gH (where g is 1 or 2) loses an H* to become -S(=O)g-(point of attachment), such as -S(=O)g-L-G, -S(=O)g-L-warhead, -S(=O)g-G, or -S(=O)g-warhead. In some instances, the linker (e.g., L, L’, or L1) is a bond. In some instances, G-L- is a proteinbinding ligand.
[00416] In some embodiments, the protein-binding ligand (e.g., G or G’) has or comprises a structure shown in Table 1 A:
Table 1A
Figure imgf000065_0001
Figure imgf000066_0002
[00417] In some embodiments, the protein-binding ligand (e.g., G or G’) has or comprises a structure shown in Table IB:
Figure imgf000066_0001
Figure imgf000066_0003
Figure imgf000067_0002
[00418] In some embodiments, the protein-binding ligand (e.g., G or G’) has or comprises a structure shown in Table 1C:
Table 1C
Figure imgf000067_0001
Figure imgf000068_0005
[00419] In some embodiments, directs binding of aprotein (e.g., BTK, EGFR, FGFR,
Figure imgf000068_0004
BMX, KRAS, TEAD, JAK3) or a mutant thereof to a position ortho or meta to L. In some
Figure imgf000068_0003
K directs binding of a protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, KRAS,
TEAD, JAK3) or a mutant thereof to a position meta to L.
Figure imgf000068_0001
[00420] In some embodiments, K covalently and/or irreversibly directs binding of a cysteine residue of a protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, KRAS, TEAD,
JAK3) or a mutant thereof to a position ortho or meta to L. In some
Figure imgf000068_0002
covalently and/or irreversibly directs binding of a cysteine residue of a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, KRAS, TEAD, JAK3) or a mutant thereof to a position ortho to L.
Figure imgf000069_0001
In some embodiments, K covalently and/or irreversibly directs binding of a cysteine residue of a protein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, KRAS, TEAD, JAK3) or a mutant thereof to a position meta to L.
[00421] In some embodiments, L, L’, or L1 is a linker. In some embodiments, L, L’, or L1 is a bond, -O-, amino (e.g., -NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl), substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted sulfoxide (e.g., -S(=O)-), substituted or unsubstituted sulfonyl (e.g., -S(=O)2-), substituted or unsubstituted sulfonamide (e.g., - S(=O)2NR3R4-, where R3 is the attachment point to G, substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or un substituted heterocycyl andR4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, orR3 and R4 are taken togetherto form a substituted or unsubstituted heterocyclyl), substituted or un substituted heteroalkyl(ene) (e.g., alkylamine (e.g, -NR3R4-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl andR4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl)), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl (e.g, -NR5R6-, where R5 and R6 are taken together to form a substituted or unsubstituted heterocyclyl). In some embodiments, L, L’, or L1 is a bond, amino (e.g, -NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl), substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted sulfoxide (e.g, -S(=O)-), substituted or unsubstituted sulfonyl (e.g, - S(=O)2-), substituted or unsubstituted sulfonamide (e.g, -S(=O)2NR3R4-, where R3 is the attachment point to G, substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl andR4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, orR3 and R4 are taken togetherto form a substituted or unsubstituted heterocyclyl), substituted or unsubstituted heteroalkyl(ene) (e.g, alkylamine (e.g, -NR3R4-, where R3 is substituted or un substituted alkyl(ene), substituted or un substituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl)), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl (e.g., -NR5R6-, where R5 and R6 are taken together to form a substituted or unsubstituted heterocyclyl).
[00422] In some embodiments, L, L’, or L1 is -O-, amino (e.g., -NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl), substituted or unsubstituted sulfoxide (e.g., -S(=O)-), substituted or unsubstituted sulfonyl (e.g., -S(=O)2-), substituted or unsubstituted sulfonamide (e.g., -S(=O)2NR3R4-, where R3 is the attachment point to G, substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and Rc is hydrogen, substituted or un substituted alkyl, or substituted orunsubstituted carbocyclyl, orR3 and R4 are taken together to form a substituted or unsubstituted heterocyclyl), substituted or unsubstituted heteroalkyl(ene) (e.g., alkylamine (e.g., -NRbRc-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted orunsubstituted alkyl, or substituted orunsubstituted carbocyclyl)), or substituted or unsub stituted heterocyclyl (e.g., -NR5R6-, where R5 and R6 are taken together to form a sub stituted or unsubstituted heterocyclyl).
[00423] In some embodiments, L, L’, or L1 is amino (e.g., -NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl), substituted or unsubstituted sulfoxide (e.g., -S(=O)-), substituted or unsubstituted sulfonyl (e.g., -S(=O)2-), substituted orunsubstituted sulfonamide (e.g., -S(=O)2NR3R4-, where R3 is the attachment point to G, substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and Rc is hydrogen, substituted orunsubstitutedalkyl, or substituted orunsubstitutedcarbocyclyl, orR3 and R4 are taken together to form a substituted or unsubstituted heterocyclyl), substituted or unsubstituted heteroalkyl(ene) (e.g., alkylamine (e.g., -NRbRc-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted orunsubstituted alkyl, or substituted orunsubstituted carbocyclyl)), or substituted or unsub stituted heterocyclyl (e.g., -NR5R6-, where R5 and R6 are taken together to form a sub stituted or unsubstituted heterocyclyl).
[00424] In some embodiments, L, L’, or L1 is a bond, -O-, amino, substituted orunsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. In some embodiments, L or L’ is amino, substituted or unsubstituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl. In some embodiments, L, L’, or L1 is amino, -O-, substituted or unsubstituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl.
[00425] In some embodiments, L, L’, or L1 is a bond, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl. In some embodiments, L, L’, or L1 is amino, substituted or un substituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl.
[00426] In some embodiments, L, L’, or L1 is amino (e.g., -NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl), substituted or unsubstituted heteroalkyl(ene) (e.g., alkylamine (e.g., -NR3R4-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl)), or substituted or unsub stituted heterocyclyl (e.g., -NR5R6-, where R5 and R6 are taken together to form a sub stituted or unsubstituted heterocyclyl).
[00427] In some embodiments, L, L’, or L1 comprises one or more N atom(s).
[00428] In some embodiments, L, L’, or L1 is amino. In some embodiments, L, L’, or L1 is - NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or un sub stituted carbocyclyl. In some embodiments, L, L’, or L1 is -NH- or -NCH3-.
[00429] In some embodiments, L, L’, or L1 is substituted or un sub stituted alkylamine. In some embodiments, L, L’, or L1 is substituted alkylamine (e.g., alkylamine substituted with oxo, such as -NHC(O)-). In some embodiments, L, L’, or L1 is unsubstituted alkylamine. In some embodiments, L, L’, or L1 is methylamine, ethylamine, propylamine, or butylamine. In some embodiments, L, L’, or L1 is methylamine, ethylamine, or propylamine.
[00430] In some embodiments, the linker (e.g., L, L’, or L1) is -NR4-R3-, -NR4-R3-NR7a-, -NR4- R3-O-, or -C(O)NR4-R3-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl, R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, andR7aisH or substituted or un sub stituted alkyl (e.g., alkyl substituted with oxo). In some embodiments, L, L’, or L1 is -C(O)NH(unsub stituted alkyl)-. In some embodiments, L, L’, orL1 is -C(0)NH(m ethyl)-, -C(O)NH(ethyl)-, or-C(O)NH(propyl)-. In some embodiments, L, L’, or L1 is -NR2R3-, where R2 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl andR3 is hydrogen, substituted or unsub stituted alkyl, or substituted or unsubstituted carbocyclyl. [00431] In some embodiments, the linker (e.g., L, L’, or L1) is -NH(heterocyclyl)-. In some embodiments, the linker (e.g., L, L’, or L1) is -NH(azetidinyl)-.
[00432] In some embodiments, L, L’, orLHs substituted or unsubstituted sulfonamide. In some embodiments, L, L’, or L1 is -S(=O)2NR3R4-, where R3 is the attachment point to G, substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, or R3 and R4 are taken together to form a substituted or unsubstituted heterocyclyl.
[00433] In some embodiments, L, L’, orLHs substituted or unsubstituted heterocyclyl. In some embodiments, the linker (e.g., L, L’, or L1) is -NR5R6-, -NR5R6-NR7-, -NR5R6-C(O)-, -NR5R6- CH2NR7-, -NR5R6-CH2NR7C(O)-, -C(O)NR5R6-NR7-, -C(O)NR5R6-O-, -NR5R6-O-, or - C(O)NR5R6CH2-NR7-, where R5 and R6 are taken together to form a substituted or un substituted heterocyclyl, and R7 is H, substituted or unsubstituted alkyl (e.g., alkyl substituted with oxo), or alkoxy. In some embodiments, the linker (e.g., L, L’, or L1) is -NR5R6-NR7-, -NR5R6-C(O)-, - NR5R6-CH2NR7C(O)-, -C(O)NR5R6-NR7-, -C(O)NR5R6-O-, or -NR5R6-O-, where R5 and R6 are taken together to form a substituted or unsubstituted heterocyclyl, and R7 is H or substituted or unsubstituted alkyl (e.g., alkyl substituted with oxo). In some embodiments, L, L’, or L1 is - NR5R6-, where R5 and R6 are taken together to form a substituted or unsubstituted heterocyclyl. In some embodiments, L, L’, or L1 is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl), substituted or unsubstituted piperidinyl, substituted or un substituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted azepanyl, or diazepanyl (e.g., 1,4-diazepanyl). In some embodiments, -NR5R6- is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl), substituted or unsubstituted piperidinyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted azepanyl, or diazepanyl (e.g., 1,4-diazepanyl). In some embodiments, L, L’, or L1 is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl), substituted or unsubstituted piperidinyl, or substituted or unsubstituted azetidinyl. In some embodiments, - NR5R6- is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl), substituted or unsubstituted piperidinyl, or substituted or unsubstituted azetidinyl. In some embodiments, L, L’, or L1 is substituted or unsubstituted piperidinyl or substituted or unsubstituted azetidinyl and -NR7- is -NH-, -NCH3-, -NC(O)CH3-, or -NCH2CH2OCH3-. In some embodiments, -NR5R6- is substituted or un substituted piperidinyl or substituted or unsubstituted azetidinyl and -NR7-is -NH-, -NCH3-, -NC(O)CH3-, or-NCH2CH2OCH3-. In some embodiments, L, L’, or L1 is substituted or unsubstituted piperidinyl or substituted or unsubstituted azetidinyl and -NR7- is -NH- or -NCH3-. In some embodiments, -NR5R6- is substituted or un substituted piperidinyl or substituted or unsubstituted azetidinyl and -NR7- is -NH- or -NCH3-.
[00434] In some embodiments, the linker (e.g., L, L’, or L1) is substituted or unsubstituted piperazinyl. In some embodiments, the linker (e.g., L, L’, or L1) is piperazinyl substituted with methyl.
[00435] In some embodiments, L, L’, or L1 comprises two N atom(s). In some embodiments, L, L’, orL1 comprises oris (substituted or unsubstituted) diaminoalkyl, (substituted or un substituted) diamino-cycloalkyl, (substituted or un substituted) amino-heterocyclyl (e.g., the heterocyclyl being nitrogen containing), (substituted or unsubstituted) heterocyclyl (e.g., containing 2 nitrogen atoms). In some embodiments, the heterocyclyl is optionally fused or spirocyclic.
[00436] In some embodiments, L, L’, or L1 is or comprises -C(O)-diaminoalkyl-. In some embodiments, each amino group of the diaminoalkyl is independently unsubstituted or substituted. In some embodiments, each amino group of the diaminoalkyl is independently unsubstituted or substituted with methyl.
[00437] In some embodiments, L, L’, orL1 comprises one or more rings. In some embodiments, L, L’, or LI comprises one or more fused or spirocyclic rings.
[00438] In some embodiments, L, L’, or L1 is or comprises a spirocyclic ring. In some embodiments, L, L’, or L1 is or comprisesl,7-diazaspiro[4.4]nonane or 1,6- diazaspiro [3.3 ]heptane .
[00439] In some embodiments, L, L’, orL1 is or comprises substituted or un substituted phenyl. In some embodiments, L, L’, orL1 is or comprises substituted phenyl. In some embodiments, the phenyl is substituted with substitued with one or more substituent selected from the group consisting of halo, cyano, amino, alkylamino, heteroalkyl (e.g., substituted alkanolamine or substituted diaminoalkyl), -N-(substituted heterocyclyl), and substituted heterocyclyl. In some embodiments, the phenyl is substituted with substitued halo, cyano, amino, alkylamino, heteroalkyl (e.g., substituted alkanolamine or substituted diaminoalkyl), -N-(substituted heterocyclyl), or substituted heterocyclyl, such as wherein the substituted heterocyclyl is piperidinyl or pyrrolidinyl substituted with amino or alkylamine. In some embodiments, L, L’, or L1 is -NH-(unsubstituted phenyl)-, -NH-(unsubstitutedphenyl)-NH-, -NH-(unsubstitutedphenyl)- C(O)-, -CH2NH-(unsubstituted phenyl)-C(O)-, -CH2-(unsubstituted phenyl)-NH-, -NHCH2- (unsubstituted phenyl)-C(O)-, -NHC(O)-(unsubstitutedphenyl)-C(O)-, -NH-(substitutedphenyl)- C(O)-, -C(O)NH-(substituted phenyl)-C(O)-, -C(O)NH-(substituted phenyl)-C(O)NH-, -NH- (unsubstituted phenyl)-C(O)NH-, -O-(unsubstituted phenyl)-C(O)NH-, or-NH-(substituted phenyl)-C(O)NH-. In some embodiments, L, L’, or L1 is -NH-(un substituted phenyl)-, -NH- (unsubstituted phenyl)-NH-, -NH-(un substituted phenyl)-C(O)-, -CH2NH-(unsubstitutedphenyl)- C(O)-, -CH2-(unsubstituted phenyl)-NH-, -NHCH2-(unsubstituted phenyl)-C(O)-, -NHC(O)-
(unsubstituted phenyl)-C(O)-, -C(O)NH-(substituted phenyl)-C(O)-, -C(O)NH-(substituted phenyl)-C(O)NH-, or-O-(unsubstitutedphenyl)-C(O)NH-. In some embodiments, the substituted phenyl is phenyl substituted with halo or cyano.
[00440] In some embodiments, L, L’, or L1 is:
Figure imgf000074_0001
[00441] In some embodiments, L, L’, or L1 is a bond.
[00442] Provided in some embodiments herein is a compound having a structure of Formula (II- A):
G R1 F
Figure imgf000074_0002
F Dxa
Formula (II- A), or a salt or solvate thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
RA is hydrogen or substituted or unsubstituted alkyl;
Rxa is alkyl or NRXRZ;
Ry and Rz are each independently hydrogen or substituted or unsubstituted alkyl; L1 is a linker (e.g., a bond, substituted or unsubstituted alkyl (ene), substituted or unsubstituted sulfoxide (e.g., -S(=O)-), substituted or unsubstituted sulfonyl (e.g., -S(=O)2-), substituted or un substituted sulfonamide (e.g., -S(=O)2NR3R4-, where R3 is the attachment point to G, substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl and R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, or R3 and R4 are taken together to form a substituted or unsubstituted heterocyclyl), substituted or unsubstituted heteroalkyl(ene) (e.g., alkylamine), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl (e.g., azetidinyl)); and
R1 is hydrogen or substituted or unsubstituted alkyl; or L1 and R1 are taken together to form a substituted or unsubstituted heterocyclyl (e.g., substituted or unsub stitutedpiperazinyl (e.g., piperazinyl substituted with methyl)); G is a protein-binding ligand (e.g., a radical of a compound that interacts with a protein or a mutant thereof (e.g., epidermal growth factor receptor (EGFR), Bruton's tyrosine kinase (BTK), Fibroblast Growth Factor Receptor (FGFR) (e.g., FGFR4), Aurora kinase A (AURKA), tyrosine-protein kinase KIT (KIT), Cytoplasmic tyrosine-protein kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), or KRAS) (e.g., an (e.g., unsaturated) unsubstituted or substituted carbocycle or an (e.g., unsaturated) unsubstituted or substituted heterocycle)).
[00443] In some embodiments, provided herein is a compound (e.g., of Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)), wherein the compound (e.g., of Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)) comprises alinker(e.g., L) of any one of the compounds of Table 2C or Table 2D, such as wherein the linker (e.g., L) is the part of the compound identified with a box around it in FIG. 1 A, FIG. IB, or FIG. 1C.
[00444] In some instances, such as when L is substituted or unsubstituted heterocyclyl, L is part of G and/or the warhead.
[00445] In some embodiments, provided herein is a compound (e.g., of Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)), wherein the compound (e.g., of Formula (II), Formula (II-A), Formula (II-B), or Formula (II-C)) comprises a protein-binding ligand provided in Table 1 A, Table IB, or Table 1C, a linker described herein, and a warhead group described herein. [00446] In some embodiments, a compound provided herein has a structure shown in Table 2A, Table 2B, Table 2C, or Table 2D.
[00447] In some embodiments, a compound provided herein, such a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or provided in Table 2 A, Table 2B, Table 2C, or Table 2D, forms a bond, such as a covalent bond with a polypeptide provided herein. In some embodiments, the polypeptide covalently binds to the compound. In some embodiments, the polypeptide covalently binds to the compound, wherein the polypeptide comprises a thiol that covalently binds to the compound. In some embodiments, the polypeptide covalently binds to the compound, wherein the polypeptide comprises a thiol, such a thiol of a cysteine residue of the polypeptide, that covalently binds to the compound.
[00448] In some embodiments, a compound provided herein has reduced reactivity with GSH. In some embodiments, a compound provided herein has reduced reactivity with GSH (and GSH/GST catalyzed reactions) while still retaining the ability to bind to a (target) polypeptide. In some embodiments, a compound provided herein has increased stability to GSH, such as GSH metabolism in vitro and/or in vivo, such as in liver cells and whole blood. In some embodiments, a compound provided herein has a higher affinity to (e.g., and covalently modifying) the polypeptide (e.g., a target protein described herein) than GSH. In some embodiments, GSH has a binding affinity for a compound provided herein (e.g., Kd) of more than 0.1 pM (e.g., 1 μM, 10 μM, 100 μM, or 1000 pM or more). In some instances, GSH has a binding affinity for a compound provided herein (e.g., Kd) of more than 10 pM. In some instances, GSH has a binding affinity (e.g., Kd) for a compound provided herein of no less than 10 pM.
[00449] In some embodiments, a compound provided herein has reduced reactivity with glutathione (GSH). In some embodiments, a compound provided herein has a reduced reactivity with the sulfur (e.g., thiol) of glutathione (GSH), such as relative to that of a (e.g., sulfur- containing) polypeptide. In some embodiments, a compound provided herein is inert glutathione (GSH) metabolism. In some embodiments, a compound provided herein does not substantially covalently bind to glutathione (GSH). In some embodiments, the lack of substantial covalent binding of the compound to glutathione is demonstrated by the lack of binding of glutathione to the compound, such as measured by glutathione S-trasferase (GST) assay described herein, such as at Example IV.
[00450] Tables 9A-9F of the Examples demonstrate reactivity (expressed as IC50 and WR50 values) and selectivity of compounds described herein. For example, Tables 9 A-9F show, in some instances, that compounds described herein have reduced reactivity with glutathione (GSH), such as in comparison to a target protein, such as a target protein described herein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof). Additionally, Tables 9A-9F, in some instances, demonstrate that compounds described herein lack covalent binding to GSH. Moreover, Tables 9A-9F demonstrate, in some instances, that compounds described herein bind to and/or (e.g., covalently) modify a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof), without the compound substantially covalently binding to GSH. Furthermore, Tables 9A-9F demonstrate, in some instances, compounds that are selective for a target protein, such as a target protein described herein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutantthereof), relative to GSH. In some cases, the compounds provided herein contact, bind to, and/or modify a target protein, such as a target protein described herein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutantthereof), in the absence of any binding, including covalent binding, of GSH to the compounds. In some cases, compounds described herein are 10-fold or more selective for a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutantthereof), relative to GSH. In some cases, compoundsdescribed herein are 100-fold or more selective for a target protein, such as a target protein described herein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutantthereof), relative to GSH. In some cases, compounds described herein are 1 OOO-fold or more selective for a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof), relative to GSH.
[00451] In some embodiments, a compound provided herein is selective for the polypeptide (e.g., a target protein described herein) relative to glutathione (GSH). In some embodiments, a compound provided herein is selective for binding to the polypeptide (e.g., a target protein described herein) relative to GSH. In some embodiments, a compoundprovidedherein is selective for covalent binding to the polypeptide (e.g., a target protein described herein) relative to GSH. In some embodiments, compoundprovided herein is selective for the polypeptide (e.g., a target protein described herein) relative to GSH at a ratio of at least 10:1 (e.g., 10:1 or more, 20: 1 or more, 50:1 or more, 100:1 ormore, 500: 1 or more, 1000:1 ormore). In some embodiments, the compound is selective for the polypeptide (e.g., a target protein described herein) relative to GSH at a ratio of about 2: 1 to about 1000:1. In some embodiments, the compound is selective for the polypeptide (e.g., a target protein described herein) relative to GSH at a ratio of about 10: 1 to about 100:1. In some embodiments, a compound provided herein is atleast 2-fold more (e.g., 2- fold ormore, 5-fold or more, 10-fold or more, 25-fold or more) selective for the polypeptide (e.g, a target protein described herein) relative to GSH. In some embodiments, a compound provided herein is 2-fold to about 100-fold more selective for the polypeptide (e.g., a target protein described herein) relative to GSH. In some embodiments, a compound provided herein is 2-fold to about 50-foldmore selective for the polypeptide (e.g., a target protein described herein) relative to GSH.
[00452] In some embodiments, a compoundprovidedherein has a structure shown in Table 2A.
Table 2A
Figure imgf000078_0002
In some embodiments, a compound provided herein has a structure shown in Table 2B.
Table 2B
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
[00453] In some embodiments, a compound provided herein has a structure shown in Table 2C.
Table 2C
Figure imgf000104_0002
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0002
[00454] In some embodiments, a compound provided herein has a structure shown in Table 2D.
Table 2D
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
- Ill -
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
[00455] In some embodiments, a compound provided herein is stable to GSH modification, such as described herein. In some embodiments, a compound provided herein is stable to GSH modification and bind (e.g., covalently) to a polypeptide (e.g., a target protein). In some embodiments, a compound provided herein is stable to GSH modification and modifies a polypeptide (e.g., a target protein). In some embodiments, a compound provided herein (e.g, Compounds 205 and 206) is stable to GSH modification but does not (e.g., necessarily) bind (e.g, covalently) to a polypeptide (e.g., a target protein). In some embodiments, a compound provided herein (e.g., Compounds 205 and 206) is stable to GSH modification but does not (e.g., necessarily) modify a polypeptide (e.g., a target protein). In some embodiments, GSH stability is demonstrated in Table 7.
Preparation of Compounds
[00456] The compounds used in the reactions described herein are made according to organic synthesis techniques known to those skilled in this art, starting from commercially available chemicals and/or from compounds described in the chemical literature. "Commercially available chemicals" are obtained from standard commercial sources including Acros Organics (Pittsburgh, PA), Aldrich Chemical (Milwaukee, WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), Avocado Research (Lancashire, U.K.), BDHInc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester, PA), Crescent Chemical Co. (Hauppauge, NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, PA), Fisons Chemicals (Leicestershire, UK), Frontier Scientific (Logan, UT), ICN Biomedicals, Inc. (Costa Mesa, CA), Key Organics (Cornwall, U.K.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. Ltd. (Cornwall, U.K.), Parish Chemical Co. (Orem,UT), Pfaltz & Bauer, Inc. (Waterbury, CN), Poly organix (Houston, TX), Pierce Chemical Co. (Rockford, IL), Riedel de Haen AG (Hanover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, OR), Trans World Chemicals, Inc. (Rockville, MD), and Wako Chemicals USA, Inc. (Richmond, VA).
[00457] Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4thEd., Wiley-Interscience, New York, 1992. Additional suitable reference booksand treatise that detail the synthesis of reactants useful in the preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, Fuhrhop, J. and Penzlin G. "Organic Synthesis: Concepts, Methods, Starting Materials", Second, Revised and Enlarged Edition (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, RV. "Organic Chemistry, An Intermediate Text" (1996) Oxford University Press, ISBN 0-19-509618- 5; Larock, R. C. "Comprehensive Organic Transformations: A Guide to Functional Group Preparations" 2nd Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J. "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure" 4th Edition (1992) John Wiley & Sons, ISBN : 0-471 -60180-2; Otera, J. (editor) "Modern Carbonyl Chemistry" (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. "Patai's 1992 Guide to the Chemistry of Functional Groups" (1992) Interscience ISBN: 0-471-93022-9; Solomons, T. W. G. "Organic Chemistry" 7th Edition (2000) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J.C., "Intermediate Organic Chemistry" 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; "Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann's Encyclopedia" (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; "Organic Reactions" (1942-2000) John Wiley & Sons, in over 55 volumes; and "Chemistry of Functional Groups" John Wiley & Sons, in 73 volumes.
[00458] Specific and analogous reactants are optionally identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (contact the American Chemical Society, Washington, D.C. for more details). Chemicals that are known but not commercially available in catalogs are optionally prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services. A reference useful for the preparation and selection of pharmaceutical salts of the benzenesulfonamide derivative compounds described herein is P. H. Stahl & C. G. Wermuth "Handbook of Pharmaceutical Salts", Verlag Helvetica Chimica Acta, Zurich, 2002.
Pharmaceutical Compositions
[00459] In certain embodiments, the benzenesulfonamide derivative compound described herein is administered as a pure chemical. In other embodiments, the benzenesulfonamide derivative compound described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Remington: The Science andPractice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)).
[00460] Provided herein is a pharmaceutical composition comprising at least one benzenesulfonamide derivative compound as described herein, or a salt, solvate, tautomer, or regioisomer thereof, and one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject or the patient) of the composition. [00461] In some embodiments, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof. [00462] In some embodiments, provided herein is a pharmaceutical composition comprising a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof. [00463] In some embodiments, provided herein is a pharmaceutical composition comprising a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2A, Table 2B, Table 2C, or Table 2D ora pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof. In some embodiments, the compound has reduced reactivity with glutathione. In some embodiments, reduced reactivity is described elsewhere herein.
[00464] In some embodiments, provided herein is a pharmaceutical composition comprising a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2A, Table 2B, Table 2C, or Table 2D, ora pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof. In some embodiments, the compound is selective for a polypeptide relative to glutathione. In some embodiments, the compound is selective for a protein relative to glutathione. In some embodiments, selectivity for a polypeptide relative to glutathione is described elsewhere herein. [00465] In some embodiments, provided herein is a pharmaceutical composition that is (e.g., at least partially) stable to glutathione. In some embodiments, the pharmaceutical composition comprises a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof.
[00466] In some embodiments, the pharmaceutical compositions provided herein are stable to glutathione (GSH) in the presence of a polypeptide. In some embodiments, the pharmaceutical compositions are at least partially stable to glutathione (GSH) in the presence of a polypeptide. [00467] In some embodiments, provided herein is a method of preparing a pharmaceutical composition comprising mixing a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2 A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, and a pharmaceutically acceptable carrier.
[00468] In certain embodiments, the benzenesulfonamide derivative compound as described by Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2 A, Table 2B, Table 2C, or Table 2D is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as unreacted intermediates or synthesis by-products that are created, for example, in one or more of the steps of a synthesis method.
[00469] Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. In some embodiments, suitable nontoxic solid carriers are used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g, Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)).
[00470] In some embodiments, the compound as described by Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2 A, Table 2B, Table 2C, or Table 2D, or pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, is formulated for administration by injection. In some instances, the injection formulation is an aqueous formulation. In some instances, the injection formulation is a non-aqueous formulation. In some instances, the injection formulation is an oil-based formulation, such as sesame oil, or the like.
[00471] The dose of the composition comprising at least one compound as described herein differs depending upon the subject or patient's (e.g., human) condition. In some embodiments, such factors include general health status, age, and other factors.
[00472] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose andtreatmentregimen provides the composition^) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity. Optimal doses are generally determined using experimental models and/or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient. [00473] Oral doses typically range from about 1.0 mg to about 1000 mg, one to four times, or more, per day.
Methods of Polypeptide Modification
[00474] Provided herein are methods of modifying a polypeptide (e.g., a target protein described herein) in the presence of glutathione with a compound provided herein, such as in the ab sence of the compound reacting with glutathione or being metabolized by glutathione. In some embodiments, a polypeptide (e.g., a target protein described herein) is modified without reacting with or being metabolized in the presence of glutathione as demonstrated by a glutathione S- transferase (GST) assay described elsewhere herein (e.g., Example IV).
[00475] In some embodiments, the method comprises contactingthe polypeptide (e.g., a protein) with a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or represented in Table 2 A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises contactingthe polypeptide with the compound to form a covalent bond with the polypeptide. In some embodiments, the compounds provided herein have reduced reactivity to GSH. In some embodiments, the compounds provided herein are selective for the polypeptide over GSH.
[00476] In some embodiments, provided herein are methods of modifying a polypeptide (e.g, a target protein described herein) with a compoundprovided herein, such as a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or represented in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein are methods of selectively modifying a polypeptide with a compound provided herein. In some embodiments, the method comprises contacting the polypeptide with any of the compounds provided herein. In some embodiments, the compound has reduced reactivity with glutathione (GSH).
[00477] In some embodiments, provided herein are methods of modifying a polypeptide (e.g, a target protein described herein) with a compound provided herein in the presence of glutathione (GSH). In some embodiments, the method comprises contactingthe polypeptide with a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II-A), Formula (II- B), Formula (II-C), or represented in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable saltthereof . In some embodiments, the method comprises contacting the polypeptide with the compound (or a warhead group thereof) to form a covalent bond with the polypeptide. In some embodiments, the method comprises contacting the polypeptide with the compound (or a warhead group thereof) to form a covalent bond with a sulfur atom of a cysteine residue of the polypeptide. In some embodiments, the method comprises contacting the polypeptide with the compound (or a warhead group thereof) without the compound substantially covalently binding to GSH. In some embodiments, the method comprises contacting the polypeptide with the compound (or a warhead group thereof) to form a covalent bond polypeptide without the compound substantially covalently binding to GSH. In some embodiments, the method comprises contacting the polypeptide with the compound (or a warhead group thereof) to form a covalent bond with a sulfur atom of a cysteine residue of the polypeptide, without substantially covalently binding to GSH.
[00478] In some embodiments, provided herein is a method of modifying a polypeptide in the presence of glutathione (GSH) with a compoundprovided herein, such as a compound ofFormula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or represented in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is a method of selectively modifying a polypeptide in the presence of glutathione (GSH) with a compound provided herein, such as a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or represented in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises contacting the polypeptide with any of the compounds provided herein. In some embodiments, the compound is selective for the polypeptide relative to GSH. In some embodiments, the selectivity for the polypeptide relative to GSH is described elsewhere herein. [00479] In some embodiments, modifying herein comprises forming a bond. In some embodiments, modifying comprises forming a bond between the polypeptide and the compound. In some embodiments, modifying comprises forming a covalent bond. In some embodiments, modifying comprises forming a covalent bond between the polypeptide and the compound. In some embodiments, forming a bond comprises forming a non-covalent bond. In some embodiments, non-covalent bonding comprises electrostatic interactions, hydrogen bonding Van der Waals interactions, or depletion interactions. In some embodiments, formingabond comprises an electrostatic interaction. In some embodiments, forming a bond comprises forming a non- covalent bond between the compound and polypeptide and the compound.
[00480] In some embodiments, the method comprises contacting the compound (or a warhead group thereof) with the polypeptide to form a covalent bond with the polypeptide. In some embodiments, the method comprises forming a covalentbond with a heteroatom (e.g., a sulfur atom, a nitrogen atom, or an oxygen atom) of the polypeptide. In some embodiments, the method comprises forming a covalentbond with a sulfur atom of the polypeptide. In other embodiments, the method comprises forming a covalent bond with an oxygen or nitrogen atom of the polypeptide. In some embodiments, the method comprises forming a covalentbond with a sulfur atom of a cysteine residue of the polypeptide. In some embodiments, the polypeptide covalently binds to the compound (or a warhead group thereof). [00481] In some embodiments, the method comprises contacting the polypeptide with a compound (or a warhead group thereof) provided herein without the compound substantially covalently binding to glutathione (GSH). In some embodiment, the lack of substantial binding to GSH is demonstrated by the lack of covalentbinding of GSH to the compound (as measured using a GST assay described elsewhere herein). In some embodiments, the polypeptide contacts the compound (or a warhead group thereof) in the absence of covalent binding of GSH to the compound. In some embodiments, the polypeptide covalently binds to the compound (or a warhead group thereof) in the absence of covalent binding of GSH to the compound.
[00482] In some embodiments, glutathione (GSH) has a half life of at least 1 minute (e.g., 10 minutes or more, 30 minutes or more, 60 minutes or more, 100 minutes or more, or 200 minutes or more), such as in the presence of a compound provided herein. In some embodiments, GSH has a half life of at least 10 minutes in the presence of a compound provided herein. In some embodiments, GSH has a half life of at least 30 minutes in the presence of a compound provided herein. In some embodiments, GSH has a half life of at least 60 minutes in the presence of a compound provided herein. In some embodiments, GSH has a half life at least 100 minutes in the presence of a compound provided herein. In some embodiments, GSH has a half life of at least 200 minutes in the presence of a compound provided herein. In some embodiments, GSH has a half life of 1 minutes to 200 minutes, such as in the presence of a compound provided herein. In some embodiments, GSH has a half life of about 10 minutes to about 100 minutes, such as in the presence of a compound provided herein. In some embodiments, GSH half-life is measured using a glutathione S-transf erase (GST) assay described herein, such as in Example IV.
[00483] In some embodiments, a compound provided herein, such as a compound of Formula (A), Formula (I), Formula (II-B), Formula (II-C), orrepresentedin Table2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt thereof, has an IC50 for a polypeptide (e.g, a target protein described herein) of atleast 10 pM (e.g., 10 pM or less, 1 pM or less, 0.1 pM or less). In some embodiments, a compound provided herein has an IC50 for a polypeptide (e.g., a target protein described herein) of at least 10 pM (e.g., 10 pM or less, 1 pM or less, 0.1 pM or less, or 0.01 pM or less). In some embodiments, a compound provided herein has an IC50 for a polypeptide (e.g., a target protein described herein) of 1 pM or less. In some embodiments, a compound provided herein has an IC50 for a polypeptide (e.g., a target protein described herein) of 0.1 pM or less. In some embodiments, a compound provided herein has an IC50 for a polypeptide (e.g., a target protein described herein) of 0.01 pM or less. In some embodiments, a compound provided herein has an IC50 for a polypeptide (e.g., a target protein described herein) of about 0.01 pM to about 10 pM. [00484] In some embodiments, a compound provided herein, such as a compound of Formula (A), Formula (I), Formula (II-B), Formula (II-C), orrepresentedin Table2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt thereof has an IC50 for a polypeptide (e.g., a target protein described herein) of at least 10 pM and a GSH half life of greater than about 10 minutes (e.g., IC50 of at least 1 pM and a GSH half life of greater than about 10 minutes (e.g., IC50 of at least 0.1 pM and a GSH half life of greater than about 100 minutes)). In some embodiments, a compound provided herein has a an IC50 for a polypeptide(e.g., a target protein described herein) of 1 pM or less and a GSH half life of about 10 minutes or more. In some embodiments, a compound provided herein has a IC50 for a polypeptide (e.g., a target protein described herein) of 0.1 pM or less and a GSH half life of about 100 minutes or more. In some embodiments, a compound provided herein has a IC50 for a polypeptide (e.g., a target protein described herein) of 0.01 pM or less and a GSH half life of about 200 minutes or more. In some embodiments, a compound provided herein has an IC50 for a polypeptide (e.g., a target protein described herein) of about O.Ol pMto about 10 pM (e.g., about 0.1 pM to about 10 μM, about O. l pM to about 1 pM) and a GSH half life of about 10 minutes to about 200 minutes (e.g., about 30 minutes to about 100 minutes, e.g., about 50 minutes to about 100 minutes).
[00485] In some embodiments, the polypeptides described herein is a protein. In some embodiments, the polypeptide comprises natural and/or non-natural amino acid residues. In some embodiments, the polypeptide comprises natural amino acid residues. In some embodiments, the polypeptide comprises cysteine (e.g., a cysteine residue), such as a nucleophilic cysteine. In some embodiments, the polypeptide comprises methionine (e.g., a methionine residue). In some embodiments, the polypeptide comprises non-natural amino acids. In some embodiments, the polypeptide comprises non-natural amino acid residues. In some embodiments, the polypeptide comprises homocysteine. In some embodiments, the polypeptide comprises sulfur containing amino acids. In some embodiments, the polypeptide comprises a thiol, such as a nucleophilic thiol. [00486] In some embodiments, the polypeptides described herein comprise a nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a sulfur, oxygen, or nitrogen containing nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a sulfur containing nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a cysteine nucleophile. In some embodiments, the polypeptide comprises a cysteine residue, such as cysteine nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a methionine nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises an oxygen containing nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a hydroxyl containing nucleophile, such as a hydroxyl of a serine or a tyrosine residue. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a serine nucleophile. In some embodiments, the polypeptide (e.g., targetprotein provided herein) comprises a tyrosine nucleophile. In some embodiments, the polypeptide (e.g, target protein provided herein) comprises a nitrogen containing nucleophile. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises an amino containing nucleophile, such as an amino of a lysine residue. In some embodiments, the polypeptide (e.g., target protein provided herein) comprises a lysine nucleophile.
[00487] In some embodiments, a polypeptide provided herein is Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), proto-oncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), or KRAS. In some embodiments, the polypeptide is Bruton’s tyrosine kinase (BTK). In some embodiments, the polypeptide is epidermal growth factor receptor (EGFR). In some embodiments, the polypeptide is fibroblast growth factor receptor (FGFR). In some embodiments, the polypeptide is aurora kinase A (AURKA). In some embodiments, the polypeptide is protooncogene c-KIT (KIT). In some embodiments, the polypeptide is BMX non-receptor tyrosine kinase (BMX). In some embodiments, the polypeptide is transcriptional enhancer factor TEF (TEAD). In some embodiments, the polypeptide is Janus kinase 3 (JAK3). In some embodiments, the polypeptide is KRAS.
[00488] In some embodiments, the polypeptidereacts (e.g., forms abond with) with a compound provided herein. In some embodiments, the polypeptide reacts (e.g., forms a bond with) with a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B) Formula (II-C), or represented in Table 2A, Table 2B, Table 2C, or Table 2D.
[00489] In some embodiments, the thiol of the polypeptide reacts (e.g., forms a bond with) with a compound provided herein. In some embodiments, the thiol of the polypeptidereacts (e.g., forms a bond) with a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or represented in Table 2A, Table 2B, Table 2C, or Table 2D.
[00490] In some embodiments, the methods provided herein further comprise inhibiting deactivating, or degrading the polypeptide. In some embodiments, the method further comprises inhibiting the polypeptide. In some embodiments, the method further comprises deactivating the polypeptide. In some embodiments, the method further comprises degrading the polypeptide. In some instances, contacting of the polypeptidewith any of the compounds provided herein inhibits, deactivates, or degrades the polypeptide. In some embodiments, binding of the polypeptide inhibits, deactivates, or degrades the polypeptide. In some embodiments, covalent binding of the polypeptide, such as a covalent binding with a thiol of the polypeptide, inhibits, deactivates, or degrades the polypeptide. In some embodiments, the polypeptide is inhibited or deactivated by a factor of at least 2 (e.g., 2 or more, 3 or more, 4 or more, 5 or more).
[00491] In some embodiments, provided herein is a method of modifying (e.g., attaching to and/or degrading) a polypeptide with a benzenesulfonamide derivative compound as described herein, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, comprising contacting the polypeptide with the compound to form a covalent bond with a sulfur atom of a cysteine residue of the polypeptide.
[00492] In some embodiments, the method provides for selectively modifying a polypeptide with a compound. In some embodiments, the selectivity is for a sulfur-containing nucleophile of the protein over other (e.g., intracellular) sulfur-containing nucleophiles (e.g., in a biological system). In some embodiments, modifying comprises covalent modification of the polypeptide, such as a protein. In some embodiments, modifying a polypeptide occurs intracellularly.
[00493] In some embodiments, provided herein is a method of binding a compound to a polypeptide, comprising contacting the polypeptide with a benzenesulfonamide derivative compound as described herein, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomerthereof . In some embodiments, the protein isBTK. In some embodiments, the protein is EGFR. In some embodiments, the protein is FGFR. In some embodiments, the protein is AUKRA. In some embodiments, the protein is KIT. In some embodiments, the protein is BMX. In some embodiments, the protein is TEAD. In some embodiments, the protein is JAK3. In some embodiments, the protein is KRAS.
[00494] In some embodiments, provided herein is a method of disrupting a protein, or an active fragmentthereof (e.g., a function thereof), comprising contacting the protein or an active fragment thereof (e.g., polypeptide thereof) with a compound described herein, or a salt, solvate, tautomer, or regioisomer thereof.
Methods of Treatment
[00495] In some embodiments, provided herein is a compound of Formula (A), Formula (II), Formula (II- A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2 A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, for use in a method of treatment of the human or animal body.
[00496] In some embodiments, provided herein is a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-C), or a compound disclosed in Table 2A or Table 2B, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomerthereof, for use in a method of treatment of cancer or neoplastic disease.
[00497] In some embodiments, provided herein is ause of a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or a compound disclosed in Table 2 A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, in the manufacture of a medicament for the treatment of cancer or neoplastic disease.
[00498] In some embodiments, described herein is a method of treating cancer in a patient in need thereof comprising administering to the patient a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof.
[00499] In some embodiments, described herein is a method of treating cancer in a patient in need thereof comprising administering to the patient a compound disclosed in Table 2 A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof.
[00500] In some embodiments, also described herein is a method of treating cancer in a patient in need thereof comprising administering to the patient a pharmaceutical composition comprising a compound of Formula (A), Formula (II), Formula (II-A), Formula (II-B), Formula (II-C), or provided in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, and a pharmaceutically acceptable excipient.
[00501] In some embodiments, also described herein is a method of treating cancer in a patient in need thereof comprising administering to the patient a pharmaceutical composition comprising a compound disclosed in Table 2A, Table 2B, Table 2C, or Table 2D, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, and a pharmaceutically acceptable excipient. In some embodiments, the cancer is selected from chronic and acute myeloid leukemia. In some embodiments, the cancer is selected from chronic lymphocytic leukemia and small lymphocytic lymphoma.
[00502] Provided herein is the method wherein the pharmaceutical composition is administered orally. Provided herein is the method wherein the pharmaceutical composition is administered by injection.
[00503] In some embodiments, provided herein is a protein, or an active fragment thereof, modified with a benzenesulfonamide derivative compound as described herein, or a pharmaceutically acceptable salt, solvate, tautomer, or regioisomer thereof, wherein the compound forms a covalent bond with a sulfur atom of a cysteine residue of the protein. In some embodiments, the protein is BTK. In some embodiments, the protein is EGFR. In some embodiments, the protein is FGFR. In some embodiments, the protein is AURKA. In some embodiments, the protein is KIT. In some embodiments, the protein is BMX. In some embodiments, the protein is TEAD. In some embodiments, the protein is JAK3. In some embodiments, the protein is KRAS. [00504] In some embodiments, providedherein is a method of modifying(e.g., attachingto and/or degrading) a polypeptide with a benzenesulfonamide derivative compound as described herein, comprising contacting the polypeptide with the compound to form a covalent bond with a sulfur atom of a cysteine residue of the polypeptide.
[00505] In some embodiments, provided herein is a method of binding a compound to KRAS or an active fragment thereof (e.g., a polypeptide), comprising contacting the polypeptide with a benzenesulfonamide derivative compound as described herein.
[00506] Other embodiments and uses will be apparent to one skilled in the art in light of the present disclosures. The following examples are provided merely as illustrative of various embodiments and shall not be construed to limit the present disclosure in any way.
EXAMPLES
I. Chemical Synthesis
[00507] In some embodiments, the benzenesulfonamide derivative compounds disclosed herein are synthesized according to the following examples. As used below, and throughout the specification, the following abbreviations, unless otherwise indicated, shall be understood to have the following meanings:
°C degrees Celsius
5 chemical shift in parts per million downfield from tetramethylsilane
ACN acetonitrile bs or brs broad singlet
DCM dichloromethane (CH2C12) dd doublet of doublets
DMF dimethylformamide
DMSO dimethylsulfoxide
EA or EtOAc ethyl acetate
ESI electrospray ionization
Et ethyl
FA formic acid g gram(s) h/hr/hrs hour(s)
HPLC high performance liquid chromatography
Hz hertz coupling constant (in NMR spectrometry)
LCMS liquid chromatography mass spectrometry // micro m multiplet (spectral); meter(s); milli
M molar
M+ parent molecular ion
Me methyl
MHz megahertz min minute(s) mol mole(s); molecular (as in mol wt) mL milliliter
MS mass spectrometry nm nanometer(s)
NMR nuclear magnetic resonance pH potential of hydrogen; a measure of the acidity or basicity of an aqueous
PE petroleum ether
PMB para-methoxybenzyl
Rbf round bottom flask
RT retention time r.t. room temperature s singlet (spectral) t triplet (spectral)
T temperature
TFA trifluoroacetic acid
THF tetrahydrofuran
[00508] Exemplary compounds of the application are synthesized using the methods described herein, or other methods, which are known in the art. Unless otherwise noted, reagents and solvents are obtained from commercial suppliers.
[00509] Anhydrous solvents, methanol, acetonitrile, dichloromethane, tetrahydrofuran and dimethylformamide, are purchased from Sigma Aldrich and used directly from Sure-Seal bottles. Reactions are performed under an atmosphere of dry nitrogen in oven-dried glassware and are monitored for completeness by thin-layer chromatography (TLC) using silica gel (visualized by UV light, or developed by treatment with KMnO4 stain and ninhydrin stain) or by LC/MS. NMR spectra are recorded in Bruker Avance III spectrometer at23°C, unless otherwise stated, operating at 400 MHz for 1 H NMR and 376 MHz 19F NMR spectroscopy either in CDC13, CD3OD, CD3CN orDMSO-<76- Chemical shifts (d) are reported in parts per million (ppm) after calibration to residual isotopic solvent. Coupling constants (J) are reported in Hz. Mass spectrometry was performed with an Agilent G6110 A single quad mass spectrometer with an ESI source associated with an Agilent 1100 HPLC system. Before biological testing, inhibitor purity was evaluated by reversed-phase HPLC (rpHPLC). The following conditions were employed for analysis by rpHPLC:
[00510] Method I: Mobile phase is a linear gradient consisting of a changing solvent composition of 10 % to 90% ACN in H2O with 0.1 % TFA (v/v) over 9 minutes, followed by 5 minutes of 100% ACN. Method was run on a Welch Xtimate 5 pm Cl 8, 150 mm x 4.6 mm column; maintained at a temperature of 30°C; flow rate of 1.0 mL/min.
[00511] Method II: Mobile phase is a linear gradient consisting of a changing solvent composition of 10 % to 90% ACN in H2O with 0.1 % Ammonia (v/v) over 7 minutes, followed by 5 minutes of 100% ACN. Method was run on a Waters Atlantis 5 pm Cl 8, 150 mm x 4.6 mm column; maintained at a temperature of 30°C; flow rate of 1.0 mL/min.
[00512] Method III: Mobile phase is a linear gradient consisting of a changing solvent composition of 15 % to 100% ACN in H2O with 0.1 % TFA (v/v) over 15 minutes. Method was run on a Phenomenex Luna 5pm C18 150 mm x 4.6 mm column; column maintained at a temperature of 25°C; flow rate of 1.0 mL/min.
[00513] Additional analytical protcols referred to herein are provided in the table below:
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0002
[00514] For reporting HPLC data, percentage purity is given after the retention time for each condition. All biologically evaluated compounds are >95 % chemical purity as measured by HPLC.
Scheme 1: Synthesis of l,2,3,4,5-pentafluoro-6-(methylsulfonyl)benzene (A2)
Figure imgf000135_0001
Synthesis of Al:
[00515] A 2.5M solution of n-Butyllithium in hexanes (1.1 equiv) was added dropwise to a solution of 2,3,4,5,6-pentakis(fluoranyl)benzenethiol (1 .0 equiv) in THF (0.6 M) at -78°C under a positive pressure of nitrogen, and the reaction mixture was left to stir for 30 min. Then, this portion was cannulated to a solution of iodomethane (2.5 equiv) in THF (1.50 M) at -78°C and the reaction was stirred while gradually warming to rt for 2 hrs. The mixture was quenched with water, and the aqueous phase was extracted twice with ether. The organic layer was dried over sodium sulfate, filtered, and evaporated under reduced pressure to yield the product as yellow liquid, which was used in the next step without further purification (88% yield). 1HNMR (400 MHz, CDC13) δ 2.49 (d, J= 0.8 Hz, 3H). 19F NMR (376 MHz, CDC13) δ -133.60 - -133.81 (m, 2F), -153.83 - -154.12 (m, IF), -161.28 - -161.68 (m, 2F).
Synthesis of A2: l,2,3,4,5-pentafluoro-6-(methylsulfonyl)benzene
[00516] To a solution of methyl(perfluorophenyl)sulfane (1.0 eq) in DCM (0.17 M), 3- Chloroperbenzoic acid (3.0 equiv) was added in three portions at0°C. After addition, the reaction mixture was permitted to warm to rt and stirred for 12 hrs. The mixture was diluted with DCM, and the organic phase was washed three times with a saturated aqueous solution of NaHCO3, brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by flash column chromatography, eluting with a mixture of 1 :1 hexane/EtOAc, to afford the anticipated product as white solid (88%). 1HNMR (400 MHz, CDCI3) δ 3.37 (d, J= 4.1 Hz, 3H). 19F NMR (376 MHz, CDC13) 6 -135.70 - -136.62 (m, 2F), - 143.19 (tt, .7 = 21.0, 7.2 Hz, IF), -157.11 - -158.10 (m, 2F).
Scheme 2: Synthesis of (2-bromo-3,4,5,6-tetrafluorophenyl)(methyl)sulfane
Figure imgf000136_0001
Synthesis of Bl
[00517] To a stirred solution of (4-methoxyphenyl)methanethiol (35.0 mL, 253.4 mmol) in toluene (650 mL) were added l,4-dibromo-2,3,5,6-tetrafluorobenzene (65.0 g, 211.1 mmol) and DIPEA (73.0 mL, 422.3 mmol). The reaction mixture was purged with N2 for 30 minutes, followed by addition of Pd2(dba)s (5.2 g, 5.7 mmol) and xanthphos (6.1 g, 10.5 mmol) at room temperature. The reaction mixture was heated to 100°C for 16 h. After completion of reaction, the mixture was cooled to room temperature and the mixture diluted with water (5000 mL) and extracted with EtOAc (2 x 500 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by flash column chromatography, eluting with 4% EtOAc in hexane, to afford the title compound as a white solid (35.0 g, 91.9 mmol, 43% yield). 'H NMR (400 MHz, DMSO-d6) δ 7.15 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H), 4.14 (s, 2H), 3.71 (s, 3H). 19F NMR (376 MHz, DMSO-d6) δ - 131.88 - -131.98 (m, 2F), -133.75 - -133.84 (m, 2F).
Synthesis ofB2
[00518] (4-bromo-2,3,5,6-tetrafhrorophenyl)(4-methoxybenzyl)sulfane (31 .0 g, 81.4 mmol) was suspended in TFA (310 mL) at 0°C. The resulting mixture was heated to 70°C for 2 h. After completion of reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. The obtained residue was azeotropically distilled with DCM (5 X 100 mL) to afford the title compound as brown sticky oil (30.0 g, 106.00 mmol). The obtained material was used in the next step without further purification.
Synthesis ofB3: (4-bromo-2,3,5, 6-tetrajluorophenyl)(methyl)sulfane
[00519] To a stirred solution of 2-bromo-3,4,5,6-tetrafluorobenzenethiol (30.0 g, 114.9 mmol) in THF (300 mL) were added DIPEA (59.7 mL, 344.8 mmol) and iodomethane (10.8 mL, 172.4 mmol) at 0°C. The resulting mixture was stirred at room temperature for 1 h. After completion of reaction, the mixture was concentrated under reduced pressure. The obtained residue was diluted with water (2000 mL) and extracted with EtOAc (2 x 500 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography, eluting with 100% hexanes, to afford the title compound as a pale yellow liquid (20.0 g, 72.7 mmol, 63% yield). 1HNMR (400 MHz, CDC13) δ 2.54 (s, 3H). 19F NMR (376 MHz, CDC13) 6 -132.88 - -132.98 (m, 2F), -133.12 - -133.22 (m, 2F).
Scheme 3: Synthesis of tert-butyl ((4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)(oxo)-16- sulfanylidene)carbamate (C2)
Figure imgf000137_0001
Synthesis of Cl: (4-bromo-2 ,3 ,5 ,6-tetrafluorophenyl)(imino)(methyl)-X6-sulfanone
[00520] To a stirred solution of (4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (0.5 g, 1.8 mmol) in MeOH (5mL) were added ammonium carbamate (0.7 g, 9 mmol) and iodobenzene diacetate (2.92 g, 9 mmol) at O°C. The resulting reaction mixture was stirred atroom temperature for 16h. After completion of reaction, the mixture was concentrated under reduced pressure. The obtained crude material was diluted with water (20 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford title compound as a colorless liquid. (0.6 g, 1.97 mmol, Quantitative). ESI-MS: m/z 308.10, [M+H]+.
Synthesis of C2: tert-butyl ((4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)(oxo)-l6- sulfanylidene )carbamate
[00521] To a stirred solution of (4-bromo-2,3,5,6-tetrafluorophenyl)(imino)(methyl)- X6- sulfanone (0.6 g, 1.96 mmol) in DCM (6mL) was added potassium tert-butoxide (0.33 g, 2.95 mmol) at 0°C. The resulting reaction mixture was stirred at 0°C for 10 minutes before addition of di-tert-butyl carbamate (0.85 g, 3.95 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 h. More potassium tert-butoxide (0.33 g, 2.95 mmol) and di-tert-butyl carbamate (0.85 g, 3.95 mmol) was added at 0°C afterthe overnightperiod. The resulting reaction mixture was stirred at 80°C for a further 16 hours. After completion of reaction, the mixture was diluted with water (30 mL) and extracted with EtOAc (3 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound as brown solid (0.35 g, 0.50 mmol, 54% yield). 1HNMR (400 MHz, DMSO- d6) 5 3.60 (s, 3H), 1.30 (s, 9H). 19F NMR (376 MHz, DMSO-tL) δ -131 .49 - -131 .58 (m, 2F), - 137.23 - -137.32 (m, 2F). ESI-MS: m/z 308.1, 352.1.
Synthesis of tert-butyl (methyl(oxo)(perfluorophenyl)-l6-sulfaneylidene)carbamate
Figure imgf000138_0001
Step-1: Synthesis of l,2,3,4,5-pentafluoro-6-(methylsulfinyl)benzene
[00522] Methyl(perfluorophenyl)sulfane (6.500 g, 0.030 mol) was dissolved in Methanol (60 mL), reaction mixture was cooled at 0 °C. To the resulting reaction mixture, a solution of Oxone (10.745 g, 0.035 mol) in Water (30 mL) was added dropwise atO °C. After completion of addition, reaction mixture was allowed to stirred for 2 hours at room temperature. After completion, Methanol was evaporated under vacuum and the residue was diluted with Water (100 mL). The aqueous layer was extracted with Ethyl acetate (3 x 70 mL). The combined Ethyl acetate layers were dried over Sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 30% Ethyl acetate in Hexanes to obtain title compound as a white solid (5.500 g, 0.024 mol, 79%). LCMS (Method- C): Retention Time: 1.423 min, 93.25%, 220 nm, ES(+ve): 231.1 [M+H]+
Step-2: Synthesis of imino(methyl)(perfluorophenyl)-l6-sulfanone
[00523] l,2,3,4,5-pentafluoro-6-(methylsulfmyl)benzene (5.500 g, 0.024 mol) was dissolved in Eaton’s Reagent (60 mL) and the solution was cooled to 0 °C and stirred for 10 minutes. Sodium azide (4.680 g, 0.072 mol) was added portion wise into reaction mixture at 0 °C. The reaction mixture was then gradually brought to room temperature and subsequently heated to 50 °C for 30 minutes. After completion, the reaction mixture was gradually brought to room temperature and quenched with aq. Sodium bicarbonate solution (400 mL). The aqueous layer was extracted with Ethyl acetate (3 x 80 mL). The combined Ethyl acetate layers were dried over Sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 50% Ethyl acetate in Hexanes to obtain title compound as a white solid (4.200 g, 0.017 mol, 72%). LCMS (Method-C): Retention Time: 1 .400 min, 100%, 270 nm, ES(+ve): 246.1 [M+H]+
Step-3: Synthesis of tert-butyl (methyl(oxo)(perfluorophenyl)-l6-sulfaneylidene)car hamate [00524] Imino(methyl)(perfluorophenyl)-l6-sulfanone (4.0 g, 0.016 mol) was dissolved in THF (20 mL), the reaction mixture was cooled at 0 °C under Nitrogen atmosphere. Sodium hydride (60% suspensionin mineral oil) (2.0 g, 0.048 mol) was added portion wise at 0 °C into the reaction mixture under Nitrogen atmosphere and the reaction mixture was stirred for 30 minutes. Boc- anhydride (5.238 g, 0.024 mol) was added dropwise at O °C into reaction mixture. The reaction mixture was gradually brought to room temperature and stirred for 16 hours. After completion, the reaction mixture was quenched with cold water (50 mL) and the aqueous lay er was extracted with Ethyl acetate (3 x 50 mL). The combined Ethyl acetate layers were dried over Sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 80% Ethyl acetate in Hexanes to obtain title compound as a white solid (2.200 g, 6.377 mmol, 39%). 1HNMR (400 MHz, CDC13) δ 1 .46 (s, 9H), 3.45 (s, 3H).
Synthesis of l-bromo-2,3,5,6-tetrafluoro-4-(methylsulfonyl)benzene
Figure imgf000139_0001
[00525] (4-bromo-2,3,5,6-tetrafhrorophenyl)(methyl)sulfane (WO2022106897 A2) (1.0 eq) was dissolved in MeOH (10 vol) at room temperature and the solution was cooled to 0 °C. A mixture of Oxone (5.0 eq) in Water (5 vol) was added to the above reaction mixture. The reaction mixture was maintained at 0 °C for 10 minutes, gradually broughtto room temperature and stirred at room temperature for 15 hours. After completion, Methanol was evaporated under vacuum and the residue was diluted with Water. The aqueous layer was extracted with Ethyl acetate. The combined Ethyl acetate layers were dried over Sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography. Pure compound was eluted with Ethyl acetate in Hexanes to obtain desired compound. 'H NMR (400 MHz, DMSO- d6) δ 3.53 (s, 3H). 19F NMR (400 MHz, DMSO-d6) δ -131 .54 -131.60 (2F), -137.27 -137.33 (2F).
Synthesis of 2,3,5,6-tetrafluoro-4-(methylthio) benzoic acid
Figure imgf000139_0002
Step-1: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio) benzoic acid
[00526] To a stirred solution of 2,3,4,5,6-pentafluorobenzoic acid (5.0 g, 0.023 mol) in Methanol (50 mL) was added Sodium methoxide (6.300 g, 0.110 mol) and Sodium thiomethoxide (3.300 g, 0.047 mol) at room temperature under Nitrogen atmosphere The reaction mixture was heated at 80 °C for 16 hours. After completion, Methanol was evaporated under vacuum and the residue was diluted with 1N HC1 (100 mL). The aqueous layer was extracted with Ethyl Acetate (3 x 100 mL). The combined Ethyl Acetate layers were dried over Sodium sulfate and evaporated under reduced pressure. The resulting crude was purified by Reverse Phase column chromatography. Product was eluted at a gradient of 0-25% Formic acid (0.1M in Water) in Acetonitrile. Combined fraction was evaporated under reduced pressure to afford title compound as white solid (1 .500 g, 6.260 mmol, 26%). LCMS (Method-C): Retention time: 1.432 min, 230 nm, 98.22%, ES(-ve): 195.1 [M-CO2H]-
Synthesis of 4-bromo-2,3,5,6-tetrafluorobenzenesulfonyl chloride
Figure imgf000140_0001
Step-1: Synthesis of benzyl(4-bromo-2,3,5,6-tetrafluorophenyl)sulfane
[00527] Pheny Imethanethiol (3.8 mL 32.481 mmol) and DIPEA (11. 10 mL, 64.962 mmol) were sequentially added to a stirred solution of l,4-dibromo-2,3,5,6-tetrafluorobenzene (10.0 g, 32.481 mmol) in 1,4- Dioxane (100 mL) under an atmosphere of Nitrogen. Reaction mixture was purged with Nitrogen for 10 minutes and Pd2dba3 (0.594 g, 0.650 mmol) and Xantphos (0.940 g, 1.624 mmol) was added. The reaction mixture was purged again with Nitrogen for 5 minutes. The reaction vial was sealed and heated at 110 °C for 2 hours. After completion (as monitored by TLC), the reaction mixture was quenched with Water (100 mL). The aqueous layer was extracted with Ethyl acetate (2 x 100 mL). The combined organic layer was evaporated under reduced pressure to afford crude product as off white solid. The crude residue was purified by silica gel flash chromatography. Pure compound was eluted at 100% Hexanes to obtain title compound as off white solid (5.0 g, 14.240 mmol, 44%)1H. NMR (400 MHz, DMSO-d6) δ 4.20 (s, 2H), 7.22- 7.29 (m, 5H). Step-2: Synthesis of 4-bromo-2,3,5,6-tetrafluorobenzenesulfonyl chloride
[00528] To a stirred solution of Benzyl(4-bromo-2,3,5,6-tetrafluorophenyl)sulfane (5.0g 14.238 mmol) in Acetonitrile (50 mL), was added AcOH (35 mL) Water (15 mL) at 0 °C under Nitrogen atmposphere. The reaction mixture was stirred at 0 °C for 10 minutes. Trichloroisocyanuric acid (3.640 g, 15.662 mmol) was added portionwise to the above stirred reaction mixture at 0 °C. The reaction mixture was gradually brought to room temperature and stirred at room temperature for 1 hour or until complete consumption of starting material. After completion (as monitored by TLC), the reaction mixture was quenched with Water (50 mL). The aqueous layer was extracted with DCM (2 x 50 mL). The combined organic layer was evaporated under reduced pressure to afford crude title compound as colourless oil (2.5 g, crude) which was used in next step without further purification.
WHO: Synthesis of tert-butyl (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)carbamate
Figure imgf000141_0001
Step-1: Synthesis of tert-butyl (2,3,5,6-tetrafluorophenyl)carbamate
[00529] To a stirred solution of 2,3,5,6-tetrafhroroaniline (7.0g, 42.42mmol) in THF (70mL) was added a IM LiHMDS solution in THF (105mL, 106.06mmol) at-78 °C. The reaction mixture was stirred at -78 °C for Ih followed by addition of Boc anhydride (11.09g, 50.90mmol). The reaction mixture was then stirred at room temperature for 1 hour, diluted with aq. NH4C1 solution (400mL) and extracted with ethyl acetate (2 x 400mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude residue was purified by column chromatography, eluting with 30% ethyl acetate in hexane to afford the title compound as a white solid (5.82g, 21.95mmol, 52% yield). LCMS (ACQUITY PDA and QDA detector, UC05_FARl): m/z 264.0 (M-H)- (ESI -ve), RT = 2.33min, 36.5%, 200- 400nm.
1H NMR (400 MHz, DMSO) δ 9.37 (s, IH), 7.83-7.74 (m, IH), 1.44 (s, 9H).
Step-2: Synthesis of tert-butyl (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)carbamate
[00530] To a stirred solution of tert-butyl (2,3,5,6-tetrafluorophenyl)carbamate (5.60g 21 , 13mmol) in THF (60mL) was added 2.5M //-BuLi solution in hexane (22.0mL, 52.83mmol) at -78 °C. The reaction mixture was stirred at -78 °C for Ih followed by addition of S-methyl methanesulfonothioate (CAS No. 2949-92-0) (5.32g, 42.26mmol). The reaction mixture was then stirred at room temperature for 5 hours, diluted with aq. NH4C1 solution (150mL) and extracted with ethyl acetate (2 x 150mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude residue was purified by column chromatography, eluting with 10-15% ethyl acetate in hexane to afford the title compound as a white solid (5.0g, 16.06mmol, 76%yield). LCMS(ACQUITYPDA and QDA detector, UC05_FARl): m/z 310.1 (M-H)- (ESI -ve), RT = 2.58min, 63.7%, 200-400nm. iHNMR (400 MHz, CDC13) δ 2.50 (s, 3H), 1.59 (s, 9H).
WH2: Synthesis of (4-(bromomethyl)-2,3,5,6-tetrafluorophenyl)(methyl)sulfane
Figure imgf000142_0001
Step-1: Synthesis of (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)methanol
[00531] To a stirred solution of (2,3,5,6-tetrafluorophenyl)methanol (10g, 55.52 mmol) in THF (400 mL) was added2.5M n-butyllithium solution in hexane (55.51 mL, 138.81 mmol) at -78 °C. The reaction mixture was allowed to stir at -78 °C for Ih followed by addition of S-Methyl methanethiosulfonate (8.41g, 66.63 mmol) in THF (10 mL). The reaction mixture was slowly warmed up to room temperature and allowedto stir for 16 hours. The reaction mixture was diluted with an aqueous solution of NH4C1 solution (200 mL) and extracted with ethyl acetate (100 mL X 2). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtained the crude. The obtained crude was purified by flash chromatography, eluting with 10% ethyl acetate in hexane to afford the title compound as a lightyellow liquid (11.5g, 50.84 mmol, 91.57% yield)1H. NMR (400 MHz, DMSO-d6) δ 5.59 (t, J = 6.0Hz, IH), 4.56 (d, J = 5.6Hz, 2H), 2.52 (s, 3H). 19F NMR (400 MHz, DMSO) δ-136.00 -136.09 (2F), -144.35_-144.93 (2F).
Step-2: synthesis of (4-(bromomethyl)-2,3,5,6-tetrafluorophenyl)(methyl)sulfane
[00532] To a stirred solution of (2,3,5,6-tetrafhroro-4-(methylthio)phenyl)methanol (11.5g, 50.84 mmol) in DMF (100 mL) was added PBr3 (27.52g, 101.69 mmol) at 0 °C. The reaction mixture was allowed to stir at room temperature for 1 hour. The reaction mixture was poured into ice-cold water to precipitate out a white solid. The suspension was filtered under vacuum and dried to afford the title compound as a white solid (10.5g, 36.32 mmol, 71 .44% yield). 'HNMR (400 MHz, DMSO-d6) δ 4.74 (s,2H), 2.56 (s, 3H). 19F NMR (400 MHz, DMSO) δ -135.30_- 135.40 (2F), -142.85_-143.08 (2F).
WH3: Synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)acetic acid
Figure imgf000143_0001
Step-1: synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)acetic acid [00533] To a stirred solution of (4-(bromomethyl)-2,3,5,6-tetrafluorophenyl)(methyl)sulfane
(4.0g 13.84 mmol) (WH2) in formic acid (40 mL) was added potassium iodide (0.091g 0.55 mmol) at room temperature in an autoclave. The reaction mixture was purged with N2 gas for 20 minutes, followed by addition of cyclooctadiene rhodium chloride dimer (0.68g, 1.38 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 18h under CO pressure. The reaction mixture was cooled to room temperature and poured into ice-cold water to precipitate out the white solid. The suspension was filtered under vacuum and dried to afford the title compound as a white solid (2.6g, 10.23 mmol, 73.92% yield). XH NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 3.79 (s, 2H), 2.53 (s, 3H).19F NMR (400 MHz, DMSO) δ -136.22_-136.31 (2F), -142.58_- 142.68 (2F).
WH4: Synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)ethan-l-amine
Figure imgf000143_0002
Step-1: Synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)acetamide
[00534] To a stirred solution of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)acetic acid (1 .10g 4.33 mmol) (WH3) in DMF (15 mL) were added HATU (3 ,29g 8.66 mmol) and DIPEA (1 ,67g 12.99 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15minutes followed by the addition of NH4C1 (0.48g 8.66 mmol). The reaction mixture was then stirred at room temperature for 18 hours. The reaction mixture was poured into ice-cold water (100 mL) to precipitate the product. The obtained precipitate was filtered-off and dried under reduced pressure to afford the title compound as a light brown solid (0.84g, 3.32 mmol, 77% yield). LCMS (ACQUITY PDA and QDA detector, UC08_FARl): m/z 254.1 (M+H)+ (ESI +ve), RT = 1.78min, 94.2%, 200 - 400nm. !HNMR(400 MHz, DMSO) δ 7.67 (s, 1H), 7.21 (s, 1H), 3.61 (s, 2H), 2.52 (s, 3H). 19F NMR (376 MHz, DMSO) δ -136.70 (dd,J1 = 11.6 Hz, J2 = 24.9 Hz, 2F) -142.71 (dd,J1 = 11.7 Hz, J2 = 24.9 HZ, 2F).
Step-2: Synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)ethan-l-amine
[00535] To a stirred solution of 2-(2,3,5,6-tetrafhroro-4-(methylthio)phenyl)acetamide (0.63g, 2.49 mmol) in THF (8.0 mL) was added BH3-DMS (0.28g, 3.73 mmol) at 0 °C. The reaction mixture was stirred at 70 °C for 18 hours. The reaction mixture was cooled to room temperature and quenched with 1NHC1 (pH~5-6). The resulting suspension was extracted with ethyl acetate (2 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound as a colorless oil (0.16g, 0.66 mmol, 27% yield). LCMS (ACQUITY PDA and QDA detector, UC08_FARl): m/z 240.1 (M+H)+ (ESI +ve), RT = 1 ,37min, 96.3%, 200 - 400nm. 1HNMR (400 MHz, DMSO) δ 2.75-2.70 (m, 4H), 2.50 (s, 3H). 19F NMR (376 MHz, DMSO) δ -136.54 (dd, J1 = 11.7 Hz, J2 = 25.2 Hz, 2F), -143.71 (dd, J1 = 11.7 Hz, J2 = 25.2 Hz, 2F).
Step-3: Synthesis of tert-butyl (2,3,5,6-tetrafluoro-4-(methylthio)phenethyl)carbamate
[00536] To a stirred solution of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)ethan-l-amine (0.16g, 0.66 mmol) in dichloromethane (10 mL) were added TEA (0.10g, 1.0 mmol) and Boc- anhydride (0.17g, 0.80 mmol) atO °C. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound as a colorless oil (0.22g, 0.43 mmol, 97%yield). LCMS (ACQUITY PDA and QDA detector, UC08_FARl): m/z 284.0 (M- 56)+ (ESI +ve), RT = 2.63min, 98.1%, 200 - 400nm. 1HNMR(400 MHz, DMSO) δ 6.98 (t, J = 6.0 Hz, 1H), 3.18-3.13 (m, 2H), 2.84-2.81 (m, 2H), 2.49 (s, 3H), 1.31 (s, 9H). 19F NMR (376 MHz, DMSO) δ -136.80 (dd,J1 =11.6 Hz, J2 =25.0 Hz, 2F), -143.76 (m,J1 = 11.6 Hz, J2 = 25.1 Hz, 2F).
Step-4: Synthesis of tert-butyl (2,3,5, 6-tetrafluoro-4-(methylsulfonyl)phenethyl)carbamate [00537] To a stirred solution of tert-butyl (2, 3, 5, 6 -tetraflu or o4-
(methylthio)phenethyl)carbamate (0.22g, 0.65 mmol) in mixture of methanol, THF and water (8:1 :1, 6.0 mL) was added oxone (0.39g, 1.30 mmol) at O°C. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with a saturated, aqueous NaHCO3 solution (200 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated using n-pentane to afford the title compound as an off-white solid (0. 16g, 0.47 mmol, 66% yield). LCMS (ACQUITY PDA and QDA detector, UC08_FARl): m/z 373.2 (M+2) + (ESI +ve), RT = 1.91 min, 92.4%, 200 - 400 nm. 1HNMR (400 MHz, DMSO) δ7.01 (t, J = 5.6 Hz, 1H), 3.51 (s, 2H), 3.20-3.17 (m, 3H), 2.91- 2.89 (m, 2H), 1 .32 (s, 9H). 19F NMR (376 MHz, DMSO) δ -140.22 (dt, J =1 7.8 Hz, J2 = 15.8 Hz, 2H), -141.57 -141.76 (m, 2H).
Step-5: Synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)ethan-l-amine
[00538] To a stirred solution of tert-butyl (2,3,5,6-tetrafluoro4- (methylsulfonyl)phenethyl)carbamate (0.16g, 0.43 mmol) in dichloromethane (3.0 mL) was added 4M HC1 in 1,4-dioxane (1.0 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and azeotropically distilled with dichloromethane (3 x 10 mL) to obtain a crude residue. The obtained crude was triturated using n-pentane to afford the title compound as an off-white solid (0. 11g 0.40 mmol, 94% yield). LCMS (ACQUITY PDA and QDA detector, UC08_FARl): m/z 272.0 (M+H)+ (ESI +ve), RT- 0.63min, 42.1%, 200 - 400nm.
WH5: Synthesis of 3-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)propan-l-amine
Figure imgf000145_0001
Step-1: Synthesis of 2,3,5,6-tetrafluoro-4-(methylthio)benzaldehyde
[00539] To a stirred solution of (4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (WO2022106897 A2) (8g, 36.36 mmol) in THF (80ml) was added isopropylmagnesium chloride*LiCl solution (1 ,3Min THF) (29 mL, 47.27 mmol) -78°C. The resulting reaction mixture was stirred at -78°C for Ih followed by addition of DMF (3.7 mL, 54.50 mmol) at -78°C. The reaction mixture was stirred at -78°C for 3 hours. After completion of reaction, the reaction mixture was diluted with saturated NH4C1 solution (300 mL) and was extracted with ethyl acetate (3 x 100 mL). The combined organic portions were dried overNa2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluting with 10% ethyl acetate in hexane to afford the title compound as a colorless liquid (4.2g, 18.75 mmol, 64.47% yield). 1HNMR (400MHz, DMSO) δ 10.14 (s, IH), 2.50 (s, 3H). 19F NMR (376MHz, DMSO) δ -136.74 (dd, J1 = 12.1 Hz, J2 = 23.3 Hz, 2F), -146.50 (dd, J1 = 12.1 Hz, J2 = 23.0 Hz, 2F).
Step-2: Synthesis of (E)-3-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)acrylamide
[00540] To a stir ed solution of diethyl (2-amino-2-oxoethyl)phosphonate (3.4g, 17.85 mmol) in THF (40 mL) was added potassium tert-butoxide (2.9g, 26.78 mmol) at 0 °C. The reaction mixture was stirred at O°C for 15 minutes, followed by addition of 2,3,5, 6-tetrafluoro4- (methylthio)benzaldehyde (4.0g, 17.85 mmol) at 0°C and was allowed to stir for 10 minutes. After completion of reaction, the reaction mixture was diluted with water (200 mL) and was extracted with ethyl acetate (3 x lOO mL). The combined organic portions were dried overNa2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluting with 30% ethyl acetate in hexane to affordthetitle compoundas an off-white (2.1g, 7.92mmol, 44.38%yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl), m/z 226.0 (M+l) (ESI +ve), RT = 1.95 min, 81.8%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 7.88 (s, IH), 7.45 (s, IH), 7.37 (d, J = 16.0 Hz, IH), 6.91 (d, J= 16.0 Hz, IH), 2.57 (s, 3H). 19F NMR (376 MHz, DMSO) δ -136.32 (dd, J1 = 11.3 Hz, J2 = 23.3 Hz, 2F), -141.75 (dd, = 11.5 Hz, J2 = 23.1 Hz, 2F).
Step-3: Synthesis of 3-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)propan-l-amine
[00541] To a stirred solution of (E)-3-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)acrylamide (2.0g, 7.5 mmol) in THF (20 mL) was added BH3-DMS (2.1 mL, 22.64 mmol) at 0°C. The reaction mixture was stirred at 70°C for 3 hours. The reaction mixture was cooled to 0°C and to it was dropwise added methanol (2.5 mL). The resulting mixture was stirred at 70°C for 2 hours. After completion of reaction, the reaction mixture was evaporated under reduced pressure to obtain a crude residue. The obtained crude was purifiedby reversephasecolumn chromatography, eluting with 0.1% FA in water in acetonitrile to afford title compound as colorless liquid (0.6g 2.3 mmols, 31.42% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl), m/z 254.1 (M+l ) + (ESI +ve), RT = 1.48 min, 83.2%, 200 - 400nm. TI NMR (400 MHz, DMSO) δ 4.12 (s, IH), 2.73 (t, J = 7.6 Hz, 2H), 2.55 (t, J = 6.8 Hz, 2H), 2.50(s, 3H), 1.63-1.56 (m, 2H), 1.47 (s, 1H). 19F NMR (400 MHz, DMSO) δ -136.25 (dd, J1 = 11.6 Hz, J2 = 25.0 Hz, 2F), - 144.33 (dd, Jj = 11 .8 Hz, J2 = 25.0 Hz, 2F).
Step-4: Synthesis of tert-butyl (3-(2,3,5,6-tetrafluoroM-
(methylthio)phenyl)propyl)carbamate
[00542] To stirred solution of 3-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)propan-l-amine (0.6g, 2.37 mmol) in dichloromethane (6 mL) were added TEA (0.6 mL, 4.7 mmol) and Boc anhydride (0.775, 3.5 mmol) at 0 °C and reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 15% ethyl acetate in hexane to afford title compound as an off-white solid (0.6g, 1.69 mmol, 71.70% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl), m/z 298.1 (M-56) + (ESI +ve), RT = 2.75 min, 100.0%, 200 - 400nm. Tf NMR (400 MHz, DMSO) δ 6.91 (t, J= 5.2 Hz, 1H), 2.96 (
Figure imgf000147_0001
6.4 Hz, J2= 12.4 Hz, 2H), 2.69 (t, J = 7.6 Hz, 2H), 2.50 (s, 3H), 1.66 (t, J = 7.6 Hz, 2H), 1.37 (s, 9H). 19F NMR (400 MHz, DMSO) δ -136.21 (dd, J1 = 11.6 Hz, J2 = 25.0 Hz, 2F), -144.16 (dd, J1 = 11.8 Hz, J2 = 25.0 Hz, 2F).
Step-5: Synthesis of tert-butyl (3-(2,3,5,6-tetrafluoroM-
(methylsulfonyl)phenyl)propyl)carbamate
[00543] A solution of sodium tungsten (0.83g, 0.25 mmol) in water (1.8 mL) and acetic acid (0.12 mL) was stirred at 70°C for 1 hour. The reaction mixture was cooled to 0°C and to it were sequentially added H2O2 (3.0 mL, 1.5 mmol) and tert-butyl (3-(2,3,5,6-tetrafluoro4- (methylthio)phenyl)propyl)carbamate (0.18g, 0.50 mmol). The resulting reaction mixture was stirred at 70 °C for 4 days. The reaction mixture was diluted with aqueousNaHCO3 (20 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 40% ethyl acetate in hexane to afford title compound as an off-white solid (0.13g, 0.33 mmol, 66.23% yield). LCMS: (ACQUITY PDA and QDAdetector, UC08_FARl), m/z 330.0 (M-56) + (ESI+ve), RT = 2.29 min, 98.1%, 200 - 400nm. XH NMR (400 MHz, DMSO) δ 6.94 (t, J= 5.6 Hz, 1H), 3.50 (s, 3H), 2.98 (q,
Figure imgf000147_0002
= 6.4 Hz, J2= 12.4 Hz, 2H), 2.75 (t, J= 7.6 Hz, 2H), 1 .69 (t, J= 7.6 Hz, 2H), 1.37 (s, 9H). 19F NMR (400 MHz, DMSO) δ -139.52 > -139.74 (m, 2F), -142.05 (dd, J1 = 9.2 Hz, J2 = 21.8 Hz, 2F). Step-6: Synthesis of 3-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)propan-l-amine
[00544] To a stirred solution of tert-butyl (3-(2,3,5,6-tetrafluoro4- (methylsulfonyl)phenyl)propyl)carbamate (0.1g, 0.25 mmol) in dichloromethane (1 mL) was added 4MHC1 in l,4-dioxane (0.5 mL) at O°c. The resulting reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated using w-hexane to afford the title compound as a white solid (0.08g, 0.28 mmol, 95.80% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1), m/z 286.0 (M+l ) + (ESI +ve), RT = 1.06 min, 92.2%, 200 - 400nm. TINMR (400 MHz, DMSO) δ 8.03 (s, 2H), 3.51 (s, 2H), 2.86 (t, J= 7.2 Hz, 4H), 1.90-1.82 (m, 2H). 19F NMR (376 MHz, DMSO) : 5 - 139.40 (td, Jj = 6.3 Hz, J2 = 15.7 Hz, 2F), - 141.84 (td, Jj = 6.1 Hz, J2 = 15.5 Hz, 2F).
WH6: Synthesis of (4-(3-aminopropyl)-2,3,5,6-tetrafluorophenyl)(imino)(methyl)-16- sulfanone
A i b t i
Figure imgf000148_0001
Figure imgf000148_0002
Step-1: Synthesis of tert-butyl (3-(2,3,5,6-tetrafluoro-4-(S- methylsulfonimidoyl)phenyl)propyl)carbamate
[00545] To a stirred solution of tert-butyl (3-(2,3,5,6-tetrafluoro4- (methylthio)phenyl)propyl)carbamate (0.2g, 0.56 mmol) (WH5 Step 4) in methanol (2 mL) were added ammonium carbamate (0.132g, 1.69 mmol) and iodobenzene diacetate (0.547g, 1.69 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours. The reaction mixture was diluted with water (70 mL) and extracted with ethyl acetate (3 x 40 mL). The combined organic portions were dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford title compound as an off-white solid (0.42g, 1.09 mmol, quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 385.2 (M+l) + (ESI +ve), RT = 1 .96 min, 64.0%, 200 - 400nm. 'HNMR (400MHz, DMSO) δ 11.97 (s, 1H), 5.51 (s, 1H), 3.34 (s, 3H), 2.97 (q, J1 = 6.4 Hz, J2 = 12.8 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 1.71-1.64 (m, 2H), 1.37 (s, 9H). 19F NMR (376 MHz, DMSO) δ -140.47 (dd,J1 = 11.3, J2 = 24.5 Hz, 2F), -142.97_-143. 12 (m, 2F).
Step-2: Synthesis of (4-(3-aminopropyl)-2,3,5,6-tetrafluorophenyl)(imino)(methyl)-16- sulfanone [00546] To a stirred solution of tert-butyl (3-(2,3,5,6-tetrafluoro-4-(S- methylsulfonimidoyl)phenyl)propyl)carbamate (0.42g, 1.09 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (4 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The obtained crude residue was triturated using n-hexane to afford the title compound as a brown sticky solid (0.41g, 1.43 mmol, quantitative yield). LCMS: (ACQUITYPDA and QDA detector, UC08 FAR1): m/z 285.1 (M+l) + (ESI +ve), RT = 0.48 min, 84.0%, 200 - 400nm. 'H NMR (400MHz, DMSO) δ 7.79 (s, 2H), 4.70 (br s, 1H), 3.34 (s, 3H), 2.90-2.81 (m, 4H), 1.87-1.79 (m, 2H). 19F NMR (376 MHz, DMSO) δ -140.22_-140.36 (m, 2F), -142.85 (dd, J1 = 12.3, J2 = 25.2 Hz, 2F).
WH7: Synthesis of 3-(2,3,5,6-tetrafluoro-4-(methylsulfinyl)phenyl)propan-l-amine
Figure imgf000149_0001
Step-1: Synthesis of tert-butyl (3-(2,3,5,6-tetrafluoroM-
(methylsulfinyl)phenyl)propyl)carbamate
[00547] To a stirred solution of tert-butyl (3-(2,3,5,6-tetrafluoro4- (methylthio)phenyl)propyl)carbamate (0.2g, 0.56 mmol) in dichloromethane (2 mL) was added m-CPBA (0.194g, 1.13 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 10 minutes, then diluted with aqueous NaHCO3 (20 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 40% ethyl acetate in hexane to afford title compound as an off- white solid (0.15g, 0.37 mmol, 66.96% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1), m/z 314.03 (M-56) + (ESI +ve), RT = 2.04 min, 87.3%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 6.93 (t, J = 5.2 Hz, 1H), 3.18 (s, 3H), 2.97 (q, J1 = 6.4 Hz, J2 = 12.4 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 1.70 (t, J = 6.8 Hz, 2H), 1.37 (s, 9H). 19F NMR (376 MHz, DMSO): 5 -142.00 (dd, J1 = 10.5, J2 = 22.0 Hz, 2F), -142.62 (dd, J1 = 11.4, J2 = 22.9 Hz, 2F).
Step-2: Synthesis of 3-(2,3,5,6-tetrafluoro-4-(methylsulfinyl)phenyl)propan-l-amine
[00548] To a stirred solution of tert-butyl (3-(2,3,5,6-tetrafluoro4- (methylsulfinyl)phenyl)propyl)carbamate (0.15g, 0.40 mmol) in dichloromethane (1.5 mL) was added trifluoroacetic acid (1.5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated with n-hexane to afford the title compound as a red sticky solid (0.16g, 0.59 mmol, quantitative yield). LCMS: (ACQUITY PDA and QD A detector, UC08 FAR1), m/z 270.0 (M+l) + (ESI +ve), RT = 0.81 min, 89.0%, 200 - 400nm. JH NMR (400 MHz, DMSO) δ 7.79 (br s, 2H), 3.19 (s, 3H), 2.89-2.81 (m, 4H), 1.84 (q, J1 = 8.0 Hz, J2= 15.2 Hz, 2H). 19F NMR (376 MHz, DMSO) δ -141 .76 (dd, J1 = 10.9, J2 = 22.4 Hz, 2F), -142.46 (dd, Ji = 11.5, J2 = 23.0 Hz, 2F).
WH8: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylsulfonyl)aniline
Figure imgf000150_0001
Step-1: Synthesis of tert-butyl (2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)carbamate
[00549] To a stirred solution of tert-butyl (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)carbamate (1 , 1g, 3.53 mmol) (WHO)in dichloromethane (11ml) was added m-CPBA (0.61g, 3.53 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 1 hour. To this reaction mixture another portion of m-CPBA (0.61g, 3.53 mmol) was added and again stirred for 1 hour, further m-CPBA (0.61g, 3.53 mmol) was added to the reaction and stirred for another 1 hour. After completion of reaction, the mixture was diluted with aqueous NaHCO3, extracted with ethyl acetate (2x50 mL). The combined organic phases were dried over anhydrous Na2SC>4, filtered and concentrated under reduced pressure. The resulting crude material was purified by flash column chromatography eluting with 25% ethyl acetate in hexane to afford title compound as an off-white solid (3.5g, 10.20 mmol, 58% yield). iHNMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 3.51 (s, 3H), 1.46 (s, 9H) 19F NMR (400 MHz, DMSO-d6) δ -139.78_-139.89 (2F), -144.14_-144.25 (2F). LCMS: Method-UC05_FAR1, RT- 2.212 ESI-MS: measured m/z 342.0 [M-l]+.
Step-2: Synthesis of 2,3,5,6-tetrafluoro-4-(methylsulfonyl)aniline
[00550] To a stirred solution of tert-butyl (2, 3, 5, 6 -tetraflu or o4-
(methylsulfonyl)phenyl)carbamate (3.5g, 10.20 mmol) in dichloromethane (35 mL) was added 4M HC1 in dioxane (35 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 6 hours. After completion of reaction, the reaction mixture was diluted with aqueous NaHCO3, extracted with ethyl acetate (3x50 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford title compound as an off-white solid (2.5g, 10.28 mmol, 100%). 1HNMR (400 MHz, DMSO-d6) δ 7.07 (s, 2H), 3.57 (s, 3H). 19F NMR (400 MHz, DMSO-d6) δ -142.45_-142.59 (2F), -161 21_- 161.32 (2F). LCMS: Method-UC05_FAR1, RT 1.440 ESI-MS: measured m/z 242.0 [M-l]+.
WH9: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)aniline
4M HCI in
Figure imgf000151_0001
Step-1: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)aniline
[00551] To a stirred solution of tert-butyl (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)carbamate (1 ,0g, 3.21 mmol) (WHO) in dichloromethane (10 mL) was added 4M HCI in l,4-dioxane (20 mL) at room temperature. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduce pressure and azeotropically distilled with dichloromethane (3 x 30 mL) to afford the title compound as a brown semi-solid (0.8g quantitative yield). Note: The obtained material was used without further purification. 'HNMR (400 MHz, DMSO) δ 6.36 (s, 2H), 2.32 (s, 3H). 19F NMR (377 MHz, DMSO) δ - 138.34 -138.57 (m, 2F), -160.67_-160.86 (m, 2F).
WH10: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)benzoic acid
Figure imgf000151_0002
Step-1: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)benzoic acid
[00552] To a stirred solution of 2, 3, 5, 6 -tetrafluorobenzoic acid (10.0g, 51.54 mmol) in anhydrous THF (100 mL) was added w-butyllithium (51.5 mL, 128.86 mmol, 2.5M in hexane) at -78 °C. The reaction mixture was stirred at -78 °C for 1 hour followed by addition of S-methyl methanesulfonothiolate (9.27g, 77.31 mmol). The resulting reaction mixture was stirred while gradually warmingto room temperature and allowed to stir at room temperature for 16 hours. The reaction mixture was diluted with IN aqueous HCI (300 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by triturating with w-hexane to afford the title compound as a white solid (10.0g, 41 .67 mmol, 81% yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 479 (M*2)- (ESI - ve), RT = 1.70 min, 95.9%, 200 - 400nm. JH NMR (400 MHz, DMSO) δ 2.64 (s, 3H). 19F NMR (400 MHz, DMSO) δ -134.62 (q, J= 10.4 Hz, 2F), -137.99_-138.08 (m, 2F).
WH11: Synthesis of 2,3,5,6-tetrafluoro-N-methoxy-N-methyl-4-(methylthio)benzamide
Figure imgf000152_0001
Step-1: Synthesis of 2,3,5,6-tetrafluoro-N-methoxy-N-methyl-4-(methylthio)benzamide [00553] To a stirred solution of 2,3,5,6-tetrafluoro-4-(methylthio) benzoic acid (2.0g, 8.33 mmol) in DMF (20 mL) were sequentially added HATU (3.79g, 9.97 mmol), DIPEA (3.22g, 24.9 mmol) and N,O-dimethyl hydroxylamine (1.61g, 16.6 mmol) at 0 °C. The resulting reaction mixture was allowed to stir at room temperature for 12 hours. The reaction mixture was diluted with ice cold water (200 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluting with 30% ethyl acetate in hexane to afford the title compound as a yellow liquid (1.66g 5.86 mmol, 70% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 284.04 (M+l) + (ESI +ve), RT = 2.20 min, 88.9%, 200 - 400nm1H. NMR (400 MHz, DMSO) δ 3.55 (s, 3H), 3.35 (s, 3H), 2.58 (s, 3H). 19F NMR (400 MHz, DMSO) δ -134.17_-134.27 (m, 2F), - 142.44_-142-48 (m, 2F).
WH12: Synthesis of l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-one
Figure imgf000152_0002
Step-1: Synthesis of l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-one
[00554] To a stirred solution of 2,3,5,6-tetrafluoro-N-methoxy-N-methyM- (methylthio)benzamide (1.6g, 5.65 mmol) (WHll) in anhydrous THF (10 mL) was addedmethyl magnesium bromide (3Min diethyl ether) (11.3 mL, 33.9 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with aqueous NH4C1 (100 mL) and extracted with ethyl acetate (3 x 75 mL). The combined organic portions were dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 20% ethyl acetate in hexane to afford the title compound as a yellow liquid (1.33g, 5.58 mmol, 99% yield). TI NMR (400 MHz, CDC13) δ 2.65 (s, 3H), 2.61 (s, 3H). 19F NMR (400 MHz, CDC13) 6 -134.38 (q, J = 12.8 Hz, 2F), -141.63 (q, J1= 12.4 Hz, 2F).
WH13: Synthesis of (E)-l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-one oxime
Figure imgf000153_0001
Step-1: Synthesis of (E)-l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-one oxime [00555] To a stirred solution of l-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)ethan-l-one (1 ,33g 5.58 mmol) (WH12) in ethanol (12 mL) was added NH2OH»HC1 (1.16g, 16.7 mmol) at room temperature. The resulting reaction mixture was stirred at 60 °C for 2 hours. The reaction mixture was diluted with ice-cold water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluting with 30% ethyl acetate in hexane to afford the title compound as an orange solid (1.3g, 5.13 mmol, 92% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 254.0 (M+l)+ (ESI +ve), RT = 2.25 min, 70.4%, 200 - 400nm. 'H NMR (400 MHz, DMSO) δ 11.98 (s, 1H), 2.55(s, 3H), 2.12 (s, 3H). 19F NMR (400 MHz, DMSO) δ - 135.38 -135.49 (m, 2F), -142.60 (d, J = 12.0 Hz, 2F).
WH14: Synthesis of l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-amine 01
Figure imgf000153_0002
Step-1: Synthesis of l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-amine
[00556] To a stirred solution of (E)-l-(2, 3, 5, 6 -tetrafl uoro-4-(m ethyl th io) phenyl) ethan-l-one oxime (0.75 g, 29.6 mmol) in a mixture of ethanol and water (8 : 1 , 8 mL) were added NH4C1 (1 ,25g 23.7 mmol) and zinc dust (1 ,54g, 23.7 mmol) at 0 °C. The resulting reaction mixture was stirred at 60 °C for 12 hours. The reaction mixture was filtrated through a celite bed and washed with ethanol (100 mL). The filtrate was concentrated under reduced pressure to obtain a residue. The obtained residue was suspended water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a red liquid (0.53g, 2.22 mmol, 75% yield). LCMS: (ACQUITYPDA and QDA detector, UC08_FARl): m/z 240.1 (M+l) + (ESI +ve), RT = 1 .40 min, 87.0%, 200 - 400nm.
WH15: Synthesis of 4-((4-((te/7-butoxyc:irbonyl) amino)butyl)thio)-2,3,5,6- tetrafluorobenzoic acid
Figure imgf000154_0001
Step-1: Synthesis of tert-butyl (4-((2,3,5,6-tetrafluorophenyl) thio) butyl) carbamate
[00557] To a stirred solution of 1,2,4, 5-tetrafluoro-3 -iodobenzene (2.5g, 9.05 mmol)in THF (25 mL) was added magnesium metal (0.33g, 13.58 mmol) at room temperature. The resulting reaction mixture was heated at 80 °C for 3 hours. The reaction mixture was cooled to 0 °C and sulfur (0.31g, 9.96 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled to 0 °C followed by addition of 4-(tBoc-amino)-l-butyl bromide (2.51g, 9.96 mmol). The reaction mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 26% ethyl acetate in hexane to afford the title compound as a brown oil (5.6g, 15.86 mmol, 88% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 298.1 (M-56)+ (ESI +ve), RT = 2.64 min, 74.8%, 200 - 400nm1H. NMR (400MHz, DMSO) δ 7.95-7.90 (m, 1H), 6.81 (d, J = 23.6 Hz, 1H), 3.52 (t, J= 13.6 Hz, 1H), 3.27 (t, J= 14.0 Hz, 1H), 3.18 (d, J= 3.6 Hz, 1H), 2.95-2.87 (m, 2H), 1.81-1.70 (m, 4H), 1.42-1.37 (m, 9H). 19F NMR (377 MHz, DMSO) δ -134.36_-134.48 (m, 2F), -138.35_- 138.48 (m, 2F).
Step-2: Synthesis of 4-((4-((ter?-butoxy carbonyl) amino)butyl)thio)-2, 3,5,6- tetrafluorobenzoic acid
[00558] To a stirred solution of tert-butyl (4-((2,3,5,6-tetrafluorophenyl)thio)butyl)carbamate (2.8g, 7.92 mmol) in anhydrous THF (28 mL) was added 2.5M n-butyllithium in hexane (6.34 mL, 15.8 mmol) at -78 °C . The reaction mixture was stirred at -78 °C f or 1 .5 h f ollowedby addition of dry ice. The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was acidified using dilute IN aqueous HC1 (200 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a brown liquid (4.8g, 12.09 mmol, 76% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 342.0 (Al- 56)+ (ESI +ve), RT = 2.06 min, 73.3%, 200 - 400nm1H. NMR (400 MHz, DMSO) δ 7.40 (br s, 1H), 3.16-3.15 (m, 2H), 2.92-2.87 (m, 2H), 1.76-1.75 (m, 2H), 1.49-1.47 (m, 2H), 1.44-1.35 (m, 9H). 19F NMR (377 MHz, DMSO 5 -134.60 (dd, .// = 12.6, J2 = 27.3 Hz, 2F), -143.16 (d, J= 15.7 Hz, 2F).
WH16: Synthesis of tert-butyl (4-((4-carbamoyl-2, 3,5,6- tetrafluorophenyl)thio)butyl)carbamate
Figure imgf000155_0001
Stepl : Synthesis of tert-butyl (4-((4-carbamoyl-2, 3,5,6- tetrafluorophenyl)thio)butyl)carbamate
[00559] To a stirred solution of 4-((4-((tert-butoxycarbonyl)amino)butyl)thio)-2,3,5,6- tetrafluorobenzoic acid (2.3g, 5.79 mmol) (WH15) in DMF (23 mL) was added HATU (3.3g 8.68 mmol) at 0 °C. The resulting reaction mixture was stirred at 0 °C for 20 minutes, followed by addition of DIPEA (2.24g, 17.37 mmol) andNH4Cl (0.46g, 8.68 mmol) at O °C. The reaction mixture was stirred at room temperature for 3 hours, diluted with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 47% ethyl acetate in hexane to afford the title compound as a brown semi-solid (0.27g, 0.68 mmol, 12% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_F ARI): m/z 297. 1 (M-100)+ (ESI +ve), RT = 2.11 min, 99.2%, 200 - 400nm. 1HNMR (400MHz, DMSO) δ 8.31 (s, 1H), 8.17 (s, 1H), 6.82 (t, J = 5.6 Hz, 1H), 2.97-2.96 (m, 2H), 2.89-2.88 (m, 2H), 1.46 (s, 4H), 1.36 (s, 9H). 19F NMR (377 MHz, DMSO): 5 -133.74 = 12.2, J2 = 25.9 Hz, 2F), -142.19 (dd,
Figure imgf000155_0002
= 11.8, J2 = 26.5 Hz, 2F).
WH17: Synthesis of tert-butyl (4-((4-cyano-2,3,5,6-tetrafluorophenyl)thio)butyl)carbamate
Figure imgf000155_0003
Step-1: Synthesis of tert-butyl (4-((4-cyano-2,3,5,6-tetrafluorophenyl)thio)butyl)carbamate [00560] To a stirred solution of tert-butyl (4-((4-carbamoyl-2,3,5,6- tetrafluorophenyl)thio)butyl)carbamate (0.12g, 0.30 mmol) in dichloromethane (2 mL) were added TEA (0.12g, 1.21 mmol) and trifluoroacetic acid (0.12g, 0.60 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 4 hours, diluted with water (70 mL) and extracted with dichloromethane (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a yellow liquid (0.3g, 0.79 mmol, quantitative yield). 1HNMR (400 MHz, DMSO) δ 6.82- 6.80 (m, 1H), 3.14-3.07 (m, 4H), 1.65-1.61 (m, 2H), 1 .54-1 .52 (m, 2H), 1.50 (s, 9H). 19F NMR (377 MHz, DMSO) δ -132.59 (td, .// = 5.3, J2 = 13.9 Hz, 2F), -134.09_-134.17 (m, 2F).
WH18: Synthesis of tert-butyl (4-((4-cyano-2, 3,5,6- tetrafluorophenyl)sulfonyl)butyl)carbamate
Figure imgf000156_0001
Step-1: Synthesis of tert-butyl (4-((4-cyano-2, 3,5,6- tetrafluorophenyl)sulfonyl)butyl)carbamate [00561] To a stirred solution of tert-butyl (4-((4-cyano-2,3 ,5,6- tetrafluorophenyl)thio)butyl)carbamate (0.15g, 0.39 mmol) (WH17) in methanol (2 mL) were added ammonium heptamolybdate tetrahydrate (0.046g, 0.03 mmol) andH2O2 (0.10g, 3. 17 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 16 hours, diluted with water (70 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a yellow semi-solid (0.31g, 0.76 mmol, 95%). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 355.1 (M-56)+ (ESI +ve), RT = 2.32 min, 100%, 200 - 400nm. JH NMR (400 MHz, DMSO) δ 6.86-6.84 (m, 1H), 3.63 (t, J = 16.0 Hz, 2H), 2.90 (q, = 6.4 Hz, J2 = 12.4Hz, 2H), 1.68-1.62 (m, 2H), 1.51-1.45 (m, 2H), 1.43 (s, 9H). 19F NMR (377 MHz, DMSO): 5 -130.73 (dd, J1 = 12.8 Hz, J2 = 24.2 Hz, 2F), -135.22 (dd, J1 = 12.3 Hz, J2 = 22.8 Hz, 2F).
WH19: Synthesis of 4-((4-aminobutyl)sulfonyl)-2,3,5,6-tetrafluorobenzonitrile
Figure imgf000156_0002
Step-1: Synthesis of 4-((4-aminobutyl)sulfonyl)-2,3,5,6-tetrafluorobenzonitrile
[00562] To a stirred solution of tert-butyl (4-((4-cyano-2,3,5,6- tetrafluorophenyl)sulfonyl)butyl)carbamate (0.29g, 0.71 mmol) (WH18) in dichloromethane (3 mL) was added trifluoroacetic acid (3 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was concentratedunder reducedpressureand azeotropically distilled with dichloromethane (3 x 10 mL)to afford the title compound as ayellow liquid (0.37g, 1.19 mmol, quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 311.0 (M+l)+ (ESI +ve), RT = 1 .32 min, 21.3%, 200 - 400nm. JH NMR (400 MHz, DMSO) δ 7.68 (s, 2H), 3.70 (t, J= 7.2 Hz, 1H), 2.81 (q,
Figure imgf000157_0001
= 6.8 Hz, J2 = 13.2 Hz, 1H), 1 .78-1 .72 (m, 2H), 1.69-1.64 (m, 2H). 19FNMR (377 MHz, DMSO) δ -130.59_-130.69 (m, 2F), -135.02_-135. l l (m, 2F).
WH20: Synthesis of 4-((3-aminopropyl)sulfonyl)-2,3,5,6-tetrafluorobenzamide
Figure imgf000157_0002
Step-la: Synthesis of 1,2,4, 5-tetrafluoro-3-iodo benzene
[00563] To a stirred solution of 1,2,4,5-tetrafluorobenzene (10.0g, 66.6 mmol) in diethyl ether (120 mL) was dropwise added 2.5M n-butyllithium in hexane (26.6 mL, 66.6 mmol) at -78 °C. The reaction mixture was stirred at -78 °C for 2h followed by addition of iodine (16.8g, 66.6 mmol) in THF (70 mL). The resulting reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with IN HCI (150 mL) and extracted with diethyl ether (3 x 100 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 100% hexane to afford the title compound as light pink liquid (23.0g, 83.62 mmol, 63% yield). 1HNMR (400MHz, CDC13) δ 7.22-7.14 (m, 1H). 19F NMR (377 MHz, CDC13): 6 -120.02_-120.13 (m, 2F), -136.63 -136.86 (m, 2F).
Step-1: Synthesis of tert-butyl (3-((2,3,5,6-tetrafluorophenyl)thio)propyl)carbamate [00564] To a stirred solution of l,2,4,5-tetrafluoro-3-iodobenzene (3.0g, 9.05 mmol) in anhydrous THF (10V) was added magnesium turnings (0.39g, 16.3 mmol) at room temperature. The reaction mixture was heated at 80°C for 3 hours. The reaction mixture was cooled to 0 °C followed by addition of sulfur powder (0.38g, 11.99 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was again cooled to 0 °C followed by addition of tert-butyl (3-bromopropyl)carbamate (2.6g, 10.9 mmol) in THF (5 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash chromatography, eluting with 20% ethyl acetate in hexaneto afford the title compound as a yellow oil (3.0g, 8.84 mmol, 81% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 284.0 (M-56)+ (ESI +ve), RT = 2.57 min, 88.6%, 200 - 400nm. TlNMR ^OOMHz, DMSO) 5 7.99-7.92(m, 1H), 6.91-6.84(m, 1H), 3.01-2.91 (m, 4H), 1.61-1.56 (m, 2H), 1.35(s, 9H). 19F NMR (377 MHz, DMSO) δ -134.32 (ddd, .// =8.2Hz, J2=l 1.9, J3=19.6Hz, 2F), -138.41 -138.67 (m, 2F).
Step-2: Synthesis of 4-((3-((tertebutoxycarbonyl)amino)propyl)thio)-2, 3,5,6- tetrafluorobenzoic acid
[00565] To a stirred solution of tert-butyl (3-((2,3,5,6-tetrafhrorophenyl)thio)propyl)carbamate (1 ,0g, 2.94 mmol) in anhydrous THF was added 1 ,6M n-butyllithium in hexane (7.3 mL, 11.79 mmol) at -78 °C. The resulting reaction mixture was stirred at -78 °C for 1.5h followed by addition of dry ice. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with aqueous citric acid solution (200 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a brown liquid (2.0g, 5.22 mmol, 88% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 284 (M- 100)+ (ESI +ve), RT = 1.99min, 90.5%, 200 - 400nm. 19F NMR (377 MHz, DMSO) δ -133.72 (dd, J! =11.8Hz, J2 =25.1HZ, 2F), -140.84 (dd, J1 =11.6Hz, J2 =25.0Hz, 2F).
Step-3: Synthesis of tert-butyl (3-((4-carbamoyl-2, 3,5,6- tetrafluorophenyl)thio)propyl)carbamate
[00566] To a stirred solution of 4-((3 -((tert-butoxy carbonyl)amino)propyl)thio)-2, 3,5,6- tetrafluorobenzoic acid (1.8g, 4.69 mmol) in THF (20 mL) were added HATU (3.5g, 9.39 mmol) and TEA (2.0ml, 14.0 mmol) at 0°C. The reaction mixture was allowed to stir at 0°C f or 15 minutes followed by addition of ammonium chloride (1 ,0g, 18.76 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentratedunder reduced pressure to obtain a crude residue. The obtained crude was purified by flash chromatography, eluting with 40% ethyl acetate in hexane to afford the title compound as a light brown solid (0.3g, 0.78 mmol, 17% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 327.1 (M-56)+ (ESI +ve), RT = 2.01 min, 96.8%, 200 - 400nm. 'HNMR (400MHz, DMSO) δ 8.31 (s, 1H), 8.17 (s, 1H), 6.87 (s, 1H), 3.02-2.94 (m, 4H), 1.59 (t, J = 7.2Hz, 2H), 1.36 (s, 9H). 19F NMR (377 MHz, DMSO) δ -133.68 (ddJ,1 =12.2HJZ1, =26.1HZ, 2F), -142.21 (ddJ, 1 =12.1HJz1, =12.1Hz, 2F).
Step-4: Synthesis of tert-butyl (3-((4-carbamoyl-2, 3,5,6- tetrafluorophenyl)sulfonyl)propyl)carbamate
[00567] To a stirred solution of tert-butyl (3-((4-carbamoyl-2,3,5,6- tetrafluorophenyl)thio)propyl)carbamate (0.25g, 0.654 mmol) in methanol (10V) were added ammoniu mmolybdate tetrahydrate (0.15g, 0.013 mmol) and H2O2 (0.7ml, 6.54 mmol, 30% in water) at 0°C. The resulting reaction mixture was stirred at room temperature for 35 hours. The reaction mixture was quenched with aqueous NaHCO3 solution (70 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a yellow semi solid (0.25g, 0.60 mmol, 92%). LCMS: (ACQUITYPDA and QDA detector, UC08_FARl): m/z 359.1 (M-56)+ (ESI +ve), RT = 1.81min, 88.8%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 10.21 (s, 1H), 8.38(d, J= 20.8 Hz, 2H), 6.93 (t, J= 4.8Hz, 1H), 3.57 (t, J= 7.6Hz, 2H), 3.05-3.00 (q, ./7 = 6.4Hz, J2 = 12.8Hz, 2H), 1.83-1.76 (m, 2H), 1.36 (s, 9H). 19F NMR (377 MHz, DMSO) δ - 136.70 (td, J;=5.0Hz, J1 =15.5HZ, 2F), -140.04 (td,J1 =5.3HzJ1, =15.9Hz, 2F).
Step-5: Synthesis of 4-((3-aminopropyl)sulfonyl)-2,3,5,6-tetrafluorobenzamide
[00568] To a stirred solution of tert-butyl (3-((4-carbamoyl-2,3,5,6- tetrafluorophenyl)sulfonyl)propyl)carbamate (0.25g, 0.603 mmol) in dichloromethane (3 mL) was added 4MHC1 in 1,4-dioxane (0.3 mL) at 0°C. The resulting reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentratedunder reducedpressureand azeotropically distilled with dichloromethane (3 x 10 mL)to afford the title compound as ayellow solid (0.300g, 0.95 mmol, quantitative yield). Note: The obtained material was used in next step without purification LCMS: (ACQUITY PDA and QDA detector, UC07_ ABR2): m/z 315 (M+H)+ (ESI +ve), RT = 0.97min, 93.58%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 8.46 (s, lH), 8.39 (s, 1H), 7.94 (s, 2H), 3.77-3.70 (q,J1 = 7.6Hz,J2= 15.6Hz2H), 2.93-2.89 (q, J1 =6.8Hz, J2 = 12.8Hz, 2H), 2.02-1.95 (m, 2H). 19F NMR (377 MHz, DMSO) δ -136.39 (td,J1 =5.2Hz, J7=15.6HZ, 2F), -139.81_-140.09 (m, 2F). WH21: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)benzamide
Figure imgf000160_0001
Step-1: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)benzamide
[00569] To a stirred solution of 2,3,5,6-tetrafhroro-4-(methylthio) benzoic acid (2.0g 8.33mmol) in DMF (20mL) were added HATU (6.33gm, 14.60mmol), DIPEA (3.22g 24.98mmol) and ammonium chloride (L33g, 24.98mmol) at O°C. The resulting reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water and extracted with ethyl acetate (3 x 70mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluted with 32% ethyl acetate in hexane to afford the title compound as a yellow solid (1.3g, 5.44mmol, 65% yield). 1HNMR (400 MHz, DMSO- tZ6) δ 8.31 (s, 1H), 8.17 (s, 1H), 2.05 (s, 3H). 19FNMR (400 MHz, DMSO-d6) δ -134.72_-134.82 (2F), -142.42_-142.52 (2F).
WH22: Synthesis 3-((perfluorophenyl)sulfonyl) propan-l-amine
Figure imgf000160_0002
Step-1: Synthesis of tert-butyl (3-((perfluorophenyl)thio)propyl) carbamate
[00570] To a stirred solution of l-bromo-2,3,4,5,6-pentafluorobenzene (1.4g, 4.76 mmol) in anhydrous THF (14 mL) was added Mg turnings (0.17g, 7.14 mmol) at room temperature under N2 atmosphere. The reaction mixture was heated at 70 °C for 2h (Reaction turned darkbrown in color). The reaction mixture was cooled 0°C followed by addition of sulfur powder (0.15g, 4.76 mmol). The resulting reaction mixture was stirred at 0°C for 1 h followed by addition of tert-butyl (3 -bromopropyl) carbamate (1 .13g, 4.76 mmol) in anhydrous THF (10 mL). The resulting reaction mixture was gradually warmed to room temperature and allowed to stir for 16 hours. The reaction mixture was diluted with IN HC1 solution (200 mL) and extracted with ethyl acetate (3 x 80 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purifiedby flash column chromatography, eluting with 21% ethyl acetate in hexane to afford the title compound as a yellow semi solid (0.65g, 1.82 mmol, 38% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 302.1 (M-56)+, (ESI+ve), RT-2.71min, 71.8%, 200 - 400nm 'HNMR (400 MHz, DMSO) 6 6.83 (d, .7=5.2Hz, 1H), 2.98 (q, J;=6.4Hz, J2=12.4Hz, 2H), 2.88 (t,J=6.8Hz, 2H), 1.61-1.54 (m, 2H), 1.35 (s, 9H). 19F NMR (377 MHz, DMSO) δ -133.24 (dd, J;=6.8Hz, J2 =25.7Hz, 2F), -153.63 (t, J =22.3 Hz, IF), -161.62 (td, J;=7.0Hz, J2 =25.5Hz, 2F).
Step-2: Synthesis of tert-butyl(3-((perfluorophenyl)sulfonyl) propyl) carbamate
[00571] To a stirred solution of tert-butyl (3-((perfluorophenyl)thio)propyl)carbamate(0.5g 1.4 mmol) in methanol (5 mL) were added (NH^MoyC^ (0.81g, 0.69 mmol) andH2O2 (0.2 mL, 5.6 mmol, 30% in water) at 0°C. The resulting reaction mixture was stirred at room temperature for 4 days. The reaction mixture was diluted with aqueous NaHCO3 (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluting with 23% ethyl acetate in hexane to afford the title compound as a white solid (0.15g, 0.385 mmol, 28%yield). LCMS: (ACQUITY PDA and QD A detector, UC08_FARl): m/z 334.1 (M-56)+ (ESI+ve), RT-2.26min, 83.2%, 200 - 400nm1HNMR (400MHz, DMSO) δ 6.90 (s, 1H), 3.54 (t, .7=8,0 Hz, 2H), 3.03-2.99 (q, J;=6.4Hz, J2=12.4HZ, 2H), 1.82-1.77 (m, 2H), 1.36 (s, 9H). 19F NMR (377 MHz, DMSO) δ -137.26 (d, J= 24.0 Hz, 2F), -145.16 (t, J= 22.6 Hz, IF), -159.37 (dd, J;=16.4 Hz, J2= 35.5 Hz, 2F).
Step-3: Synthesis 3-((perfluorophenyl)sulfonyl) propan-l-amine
[00572] To a stirred solution of tert-butyl (3 -((perfluorop henyl)sulfonyl)propyl)carbamate (0.14g, 0.359 mmol) in dichloromethane (1.4 mL) was added 4MHC1 in 1-4 dioxane (0.7 mL) at 0°C. The resulting reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduce pressure to obtain a residue. The obtained residue was azeotropically distilled with dichloromethane (2x15 mL) to obtain a crude residue. The obtained crude was triturated using w-pentane to afford the title compound as a yellow semi solid (0.13g 0.4 mmol, Quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC05 FAR1): m/z 290.0 (M+H)+ (ESI+ve), RT-1.25min, 83.2%, 200 - 400nm JH NMR (400 MHz, DMSO) δ 7.99 (brs, 2H), 3.73 (t, .7=7, 6 Hz, 2H), 2.91 (d, J= 5.6Hz, 2H), 2.03-1.96 (m, 2H). 19F NMR (377 MHz, DMSO) δ -136.74_-136.98 (m, 2F), -144.77 (tt, J;=6.9Hz, J2= 23.0Hz, IF), -158.96_-159.44 (m,2F).
WH24: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylthio)phenol
Figure imgf000162_0001
Step-1: Synthesis of 2,3,5, 6-tetrafhioro-4-(methylthio)phenol
[00573] To a stirred solution of 2,3,5,6-tetrafluorophenol (40g, 240.86mmol) in THF (400mL) was added 2.5M n-BuLi solution in n-Hexane (240.8ml, 602.15mmol) at -78°C. The reaction mixture was stirred at 78°C for Ih followed by addition of S-methyl methanesulfonothioate (31.11g, 240.86mmol). The reaction mixture was stirred at room temperature for 16h. The reaction mixture was diluted with aqueous solution of dil. HC1 Soln (700mL) and extracted with ethyl acetate (3x300mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure obtain the crude. The obtained crude was purified by flash column chromatography, product eluted with 2% ethyl acetate in hexane to afford the title compound as a brown solid (42g, 198.12mmol, 82% yield). LCMS: (ACQUITYPDA and QDA detector, UC05_F ARI): m/z211.0(M-H)- (ESI -ve), RT = 2.14 min, 98.9%,200 -400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity-II, HP07 TFAR1): RT = 6.26 min, 99.0%, 200- 400nm. 1HNMR(400MHz, DMSO) δ 11 .79 (s, IH), 2.38 (s, 3H). 19F NMR (377MHz, DMSO): 5-137. 11 -137.22 (m, 2F), -160.54_-160.65 (m, 2F).
INTI: Synthesis of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole
Figure imgf000162_0002
Step-1: Synthesis of N-methyl-2-nitro-5-(trifluoromethyl)aniline
[00574] To stirred solution of 2-fluoro-l-nitro-4-(trifluoromethyl)benzene (75.0g, 358.83 mmol) in THF (750 mL) was added K2CO3 (99.03g, 717.669 mmol) followed Methyl amine (2.0 M in THF) (358.83 mL, 717.669 mmol) at 0 °c and reaction mixture was stirred at room temperature for 2 hours. After completion of reaction, reaction mixture was diluted water (1000 mL) and extracted with ethyl acetate (3x400 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford title compound as a Yellow solid (77.0g, 349.92 mmol, 97% yield)1H. NMR (400MHz, DMSO-d6) 5 8.33(brs, 1H), 8.25 (d, J=9.2Hz, 1H), 7.23(s, 1H), 6.95- 6.92 (dd, Ji=l .6 Hz, J2=8.8 Hz 1H), 3.00 (d, J=4.8Hz, 3H).
Step-2: Synthesis of 4-bromo-N-methyl-2-nitro-5-(trifluoromethyl)aniline
[00575] To a stirred solution of N-methyl-2-nitro-5-(trifluoromethyl)aniline (28.5g, 129.54 mmol) in Acetic acid(280 mL), A-bromosuccinimide (28 ,5g, 160.13 mmol) was added in portion wise manner at room temperature and the re suiting reaction mixture was reflux for 16 hours. After completion of reaction, reaction mixture was slowly poured in to ice cold water(1000 mL). The obtained precipitate was filtered, dried under Vacuum. The resulting crude was purified by flash column chromatography eluting with 12% ethyl acetate in hexane to afford title compound as a yellow solid (86.0g, 288.62 mmol, 82% yield). JHNMR(400 MHz, DMSO-d6) δ 8.40(d, J =4.4 Hz, 1H), 8.37(s, 1H), 7.32(s, 1H), 3.00(d, J=4.8Hz, 3H).
Step-3: Synthesis of 4-bromo-Nl-methyl-5-(trifluoromethyl)benzene-l,2-diamine
[00576] To stirred solution of 4-bromo-N-methyl-2-nitro-5-(trifluoromethyl)aniline (81.0g 271.84 mmol) in THF :methanol:Water (270 :270:270 mL) were addedFe (75.89g, 1359.20 mmol) and NH4Cl (282.71g, 1359.20 mmol) at room temperature and reaction mixture was stirred at 70 °c for 3 hours. After completion of reaction, reaction mixture was diluted with ethyl acetate (500 mL) and filtered through celite Bed. The obtained filtrate was diluted with water (1000 mL) and extracted with 10% methanol in MDC (3x600 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography eluting with 52% ethyl acetate in hexane to afford title compound as a brown solid (56.0g, 208.97 mmol, 77%yield). 1HNMR (400 MHz, DMSO-d6) δ 6.83(s, 1H), 6.56(s, 1H), 5.46(brs, 2H), 5.14-5.1 l(brs, 1H), 2.73(d, J=5.2Hz, 3H). LCMS: Method-UC05_FAR1, RT- 2.290, ESI-MS: measured m/z 269.1-271.1 [M+2]+.
Step-4: Synthesis of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole
[00577] To stirred solution of 4-bromo-Nl-methyl-5-(trifluoromethyl)benzene-l,2-diamine (28.0g, 104.48 mmol) in trimethoxyorthoformate (280 mL) was added Cone. HC1 (28 mL) at rt. The resulting reaction mixture was stirred at room temperature for 3 hours. After completion of reaction, reaction mixture was diluted with aqueous NaHCCL solution (1000 mL) and extracted with ethyl acetate (3x500 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford title compound as a Pink solid (55 ,0g 197.86 mmol, 95% yield). 1HNMR (400 MHz, DMSO-d6) δ 8.47(s, 1H), 8.19(s, 1H), 8.15(s, 1H), 3.92(s, 3H). LCMS: Method-UC05_F ARI, RT-2.023, ESI-MS: measured m/z279.1 [M+l]+, 281.1 [M+3]+.HPLC: Method-HP07_trifluoroacetic acidRl, RT 5.616 (89.15%).
INT2: Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3- carboxylic acid
Figure imgf000164_0001
Step-1: Synthesis of ethyl 2-(3-bromo-2-methyl-114-pyridin-l-yl) acetate, bromide salt
[00578] To a stirred solution of ethyl 2-bromoacetate (58.25g, 348.79mmol) in THF (120mL) was added 3 -bromo-2 -methylpyridine (30.0g, 175.43mmol) at room temperature. The reaction mixture was stirred at 80 °C for 20 hours then cooled to room temperature. The obtained precipitate was filtered off, washed with hexane, and dried under vacuum to afford the title compound as an off-white solid (33.0g, quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 260.2 (M+H)+ (ESI +ve), RT = 0.96min, 99.4%, 200- 400nm. JH NMR (400 MHz, DMSO-d6) 5 9.11 (dd, J1 = 1.2 Hz, J2 = 6A Hz, 1H), 9.00 (dd, J1 = 1.2 Hz, J2 = 8.4 Hz, 1H), 8.06-8.02 (m, 1H), 5.89 (s, 2H), 4.25
Figure imgf000164_0002
= 1.Z, J2 = 14.4 Hz, 2H), 2.86 (s, 3H), 1.25 (t, J = 6.8 Hz, 3H).
Step-2: Synthesis of ethyl 8-bromoindolizine-3-carboxylate
[00579] A solution of dimethyl sulphate (132.44g, 1051.16mmol) and DMF (50.0mL) was mixed at room temperature and was stirred at 80 °C for 2h. The mixture was cooled to room temperature and added into a pre-stirred solution of ethyl 2-(3-bromo-2-methyl-114-pyridin-l-yl) acetate bromide salt (33.90g, 131 .39mmol) in DMF (50.0mL) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes followed by the addition of DIPEA (135.60g, 1051.16mmol) at 0 °C. The reaction mixture was stirred at room temperature for 30 minutes, poured into ice-cold water to precipitate out a solid. The obtained solid was filtered, washed with hexane and dissolvedin ethyl acetate (3 x 400mL). The organic portion was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude was purified by column chromatography, eluting with 30% ethyl acetate in hexane to afford the title compound as an off-white solid (11 ,70g, 43.82mmol, 33% yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 268.1 (M+H)+ (ESI +ve), RT = 2.82min, 99.4%, 200- 400nm. 1HNMR (400 MHz, DMSO-d6) δ 9.33 (d, J = 7.2 Hz, 1H), 7.54 (d, J = 4.4 Hz, 1H), 7.47 (d, J = 7.2 Hz, 1H), 6.91 (t, J=7.2 Hz, 1H), 6.64 (d, J = 4.8 Hz, 1H), 4.32 (q, ./, = 6.8 Hz, J2 = 14 Hz, 2H), 1.33 (t, J= 7.2 Hz, 3H).
Step-3: Synthesis of ethyl 8-(4,4,5,5-tetramethyl-l,3^-dioxaborolan-2-yl)indolizine-3- carboxylate
[00580] To a stirred solution of ethyl 8-bromoindolizine-3 -carboxylate (10.0g, 37.45mmol) in 1,4-dioxane (lOOOmL) were added potassium acetate (11.01g, 112.36mmol) and Bis(pinacolato)diboron (14.27g, 56.17mmol) at room temperature. The reaction mixture was purged with N2 gas for 15-20mins. followed by the addition of PdCl2(dppf)DCM (3.056g, 3.74mmol) at room temperature. The reaction mixture was stirred at 85 °C for 4 hours, cooled to room temperature and diluted with hexane (900mL). The resulting suspension was filtered through celite and washed with hexane (2 x 300mL). The filtrate was concentrated under reduced pressure and the obtained crude was purifiedby column chromatography, eluting with neat hexane to afford title compound as an off-white solid (14.0g, quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 316.4 (M+H)+ (ESI +ve), RT = 3.03min, 79.4%, 200- 400nm. 'HNMR (400 MHz, DMSO) δ 9.46 (d, J = 7.2 Hz, 1H), 7.53 (d, J = 4.4 Hz, 1H), 7.50 (dd, Ji = 1.2 Hz, J2 = 6.8 Hz, 1H), 7.00 (t, J= 6.8 Hz, 1H), 6.89 (d, J= 4.4 Hz, 1H), 4.30 (q, Jj = 7.2 Hz, J2 = 14.4 Hz, 2H), 1.34-1.31 (m, 15H).
Step-4: Synthesis of ethyl 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carboxylate
[00581] To a stirred solution of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole (4.0g, 36.36mmol) in mixture of 1,4-dioxane and water (8:2, lOOOmL) were added sodium carbonate (4.57g, 43.16mmol) and ethyl 8-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)indolizine-3 -carboxylate (6.79g, 21 .586mmol) at room temperature. The reaction mixture was purged with N2 gas for 10-15mins. followed by the addition of PdCl2(dppf) (1.05g, 1.43 mmol) at room temperature. The reaction mixture was stirred at 90°C for 4 hours, cooled to room temperature, diluted with water (400mL) and extracted with ethyl acetate (3 x 400mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude was purified by column chromatography, eluted with 100% ethyl acetate in hexane to afford title compound as an off-white solid (6.0g, 15.50mmol, 54% yield). LCMS: (ACQUITYPDA and QDA detector, UC05 FAR1): m/z 388.4 (M+H)+ (ESI +ve), RT = 2.37min, 98.6%, 200- 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity-II, HP07 TFAR1): RT = 6.42min, 100%, 200-400nm. 'HNMR (400 MHz, DMSO) δ 9.40 (d, J = 7.2 Hz, 1H), 8.51 (s, 1H), 8.22 (s, lH), 7.72 (s, 1H), 7.43 (d, J = 4.4 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.03 (d, .7= 6.8 Hz, 1H), 5.95 (d, J = 4.8 Hz, 1H), 4.38-4.29 (m, 2H), 3.99 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H).
19F NMR (377 MHz, DMSO) δ-55.02 (s, 3F).
Step-5: Synthesis of8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3- carboxylic acid
[00582] To a stirred solution of ethyl 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3 -carboxylate (1.0g, 2.58 mmol) in ethanol:Water (8:2, 10 mL) was added NaOH (0.310g, 7.75 mmol) at room temperature and reaction mixture was allowed to stirred at 70°C for 3 hours. After completion of reaction, the reaction mixture was cooled to room temperature, concentrated under reduce pressure and acidified with IN HC1 (25 mL), the obtained precipitate was filtered off, washed with n-hexane, dried under vacuum to afford title compound as an off- white solid (0.7g, 1 .94 mmol, 54% yield). iHNMR (400MHz,DMSO-d6) δ 12.46 (brs, 1H), 9.44 (d, J = 6.8 Hz, 1H), 8.64 (s, 1H), 8.27 (s, 1H), 7.76 (s, 1H), 7.40 (d, J = 4.4 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.00 (d, J = 6.8 Hz, 1H), 5.93 (d, J = 4.4 Hz, 1H), 4.02 (s, 3H). LCMS: Method- UC05 FAR1, RT- 1.775 ESI-MS: measured m/z 360.4 [M+l]+.
INT3: Synthesis of 8-(4-(trifluoromethyl)phenyl)quinoline-3-carboxylic acid
Figure imgf000166_0001
Step-1: Synthesis of 8-(4-(trifluoromethyl)phenyl)quinoline-3-carboxylic acid
[00583] To a stirred solution of 8-bromoquinoline-3-carboxylic acid (5.0g, 19.84 mmol) in mixture of 1,4-dioxane and water (8:2, 50 mL) were added sodium carbonate (6.3g, 59.52 mmol) and (4-(trifluoromethyl)phenyl)boronic acid (5.6g, 29.78 mmol) at room temperature. The reaction was purged with N2 for 15 minutes, followed by addition of PdCl2(dppf) (0.72g, 0.99 mmol) at room temperature and the reaction mixture was heated at 90°C for 5 hours. After completion of reaction, the reaction mixture was cooledto room temperature and filtrated through celite bed, eluting with 10% methanol: dichloromethane (300 mL). The obtained filtrate was concentrated under reduced pressure. The obtained recidue was diluted with water (200 mL) and back washed with ethyl acetate (80 mL X 3). The aqueous portions were acidified with 1MHC1 (~2-3 pH) and extracted with ethyl acetate (80 mL X 3). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtained the title compound as a grey solid (5.0g, 15.78 mmol, 79% yield). 1HNMR (400 MHz, DMSO-d6) δ 13.59 (brs, 1H), 9.29 (d, J=2.0Hz, 1H), 9.06 (d, J=2.0Hz, 1H), 8.28 (d, J=7.6Hz, 1H), 7.96 (d, J=6.4Hz, 1H), 7.89-7.79 (m, 1H). 19F NMR (400 MHz, DMSO-d6) δ -60.86 (3F).
LCMS: Method-UC08_FAR1, RT- 2.471 ESLMS: measured m/z 318.1 [M+l]+.
INT4: Synthesis of (4-aminophenyl)(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol- 5-yl)indolizin-3-yl)methanone
Figure imgf000167_0001
Step-1: Synthesis of 2-(3-bromo-2-methyl-114-pyridin-l-yl)-l-(4-nitrophenyl)ethan-l-one bromide salt
[00584] To a stirred solution of 2-bromo-l-(4-nitrophenyl)ethan-l-one (20.0g, 81 .95 mmol) in THF (200 mL) was added 3-bromo-2-methylpyridine (28.2g, 163.90 mmol) at room temperature. The reaction mixture was heated at 80°C for 32 hours. The reaction mixture was cooled to room temperature. The resulting suspension was filtered and washed with hexane. The resulting solid was dried under vacuum to afford title compound as an off-white solid (28.0g, 67.65 mmol, 83% yield). LCMS: (ACQUITYPDA and QD A detector, UC05_FARl): m/z 335.1 (M-79)+(ESI+ve), RT = 1 .32 min, 77.1%, 200 - 400nm1H. NMR (400 MHz, DMSO) δ 9.03-8.98 (m, 2H), 8.50 (d, J= 8.8Hz, 2H), 8.32 (d, J = 8.8Hz, 2H), 8.07-8.04 (m, 1H), 6.74 (s, 2H), 2.84 (s, 3H).
Step-2: Synthesis of (8-bromoindolizin-3-yl)(4-nitrophenyl)methanone
[00585] dimethyl sulphate (34.12g, 270.58 mmol) was dissolved in DMF (21 mL) at room temperature and heated at 80°C for 2 hours. The reaction mixturewas cooled to room temperature. In a separate vessel 2-(3-bromo-2-methyl-114-pyridin-l-yl)-l-(4-nitrophenyl)ethan-l-one bromide salt (14g, 33.82 mmol) was dissolved in in DMF (21 mL) at room temperature. The reaction mixture was cooled to 0 °c. A solution of dimethyl sulphate in DMF was added to the reaction mixture at 0 °c. The reaction mixture was stirred for 1 h at room temperature followed by addition of DIPEA (23.52 mL, 135.29 mmol) over a period of 1 hour. The reaction mixture was poured into ice-cold water to precipitate out the product. The obtained precipitate was filtered off and washed with hexane to afford the title compound as an orange solid (11g, 31.87 mmol, 94% yield). TI NMR (400 MHz, DMSO) δ 9.84 (d, J= 6.8 Hz, 1H), 8.39(d, J= 7.2 Hz, 2H), 8.02(d, J= 7.2 Hz, 2H), 7.75 (d, J= 7.2 Hz, 1H), 7.46-7.44 (m, 1H), 7.12(d, J=7.2 Hz, 1H), 6.79 (d, J= 4.8Hz, 1H).
Step-3: Synthesis of (4-nitrophenyl)(8-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)indolizin-3-yl)methanone
[00586] To a stirred solution of (8-bromoindolizin-3-yl)(4-nitrophenyl)methanone (2.5g, 7.24 mmol) in 1,4-dioxane (25 mL) were added potassium acetate (2.13g, 21.72 mmol) and bis(pinacolato)diboron (3.67g, 14.48 mmol) at room temperature. The reaction mixture was purged withN2 gas for 15-20 minutes followedby addition of PdCl2(dppf)dcm (0.59g, 0.72mmol) at room temperature. The reaction mixture was heated to 90°C for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (200 mL). The resulting suspension was filtered through celite bed and washed with ethyl acetate (100 mL). The obtained filtrate was concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, product eluting with 10-15% ethyl acetate in hexane to afford the title compound as a yellow solid (1 ,2g, 3.06 mmol, 42% yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 311 .2 (M+H)+ (ESI +ve), RT = 1.69 min, 91 .9%, 200 -400nm. JH NMR (400 MHz, DMSO) δ 9.99 (d, J= 6.4 Hz, 1H), 8.38 (d, J= 8.8 Hz, 2H), 7.99-7.97 (m, 2H), 7.75-7.73(m, 1H), 7.39 (d, J = 4.8Hz, 1H), 7.21 (t, J= 7.2Hz, 1H), 6.99 (d, J = 4.4Hz, 1H), 1.36 (s, 12H).
Step-4: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3- yl)(4- nitrophenyl) methanone
[00587] To a stirred solution of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole (17.01g, 61 .20 mmol) (INTI) in mixture of 1,4-dioxane and water (100:25, 125 mL) were added potassium carbonate (12.68g, 91.80 mmol) and (4-nitrophenyl)(8-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)indolizin-3-yl)methanone (12.0g, 30.60 mmol) at room temperature. The reaction mixture was purged with N2 gas for 15 minutes followed by PdCl2(dppf) (2.23g, 3.06 mmol) at room temperature. The reaction mixture was stirred at 90°C for 16 hours. The reaction mixture was cooled to room temperature and filtered through celite bed. The celite bed was washed with ethyl acetate (300 mL). The obtained filtrate was concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 100% ethyl acetate to afford the title compound as a yellow solid (5.0g, 10.76 mmol, 35% yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 465.2 (M+H)+ (ESI +ve), RT = 2.43 min, 94.6%, 200 - 400nm. 'HNMR (400 MHz, DMSO): 5 9.95-9.93 (m, 1H), 8.52 (s, 1H), 8.37 (d, J = 8.4 Hz, 2H), 8.26 (s, 1H), 8.04(d, J = 8.4 Hz, 2H), 7.77 (s, 1H), 7.32-7.30 (m, 3H), 6.09 (d, J = 4.8 Hz, 1H), 4.00 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.94 (s, 3F).
Step-5: Synthesis of (4-aminophenyl)(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizin-3-yl)methanone
[00588] To a stirred solution of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizin-3-yl)(4-nitrophenyl)methanone (5.0g, 10.76 mmol) in mixture of methanol (50 rnL) and ethyl acetate (50 mL) was added 10% Pd/C (2.5g, 50% wet) at room temperature. The resulting reaction mixture was purged with H2 gas for 6 hours. The reaction mixture was filtered through celite bed and washed with 10% methanol in dichloromethane (3x100 mL). The obtained filtrate was concentrated under reduced pressure to afford the title compound as an orange solid (4.0g, 9.20 mmol, 85% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 435.2 (M+H)+ (ESI +ve), RT = 1.99 min, 78.6%, 200 - 400nm.
INT4-A: Synthesis of 4-bromo-6-chloro-7-(2,6-dimethylphenyl)quinoline
Figure imgf000169_0001
Step-1: Synthesis of 4-bromo-6-chloro-7-(2,6-dimethylphenyl)quinoline
[00589] To a stirred solution of 6-chloro-7-(2,6-dimethylphenyl)quinolin-4-ol (5.0g, 17.66 mmol) in DMF (50 mL) (INT14) was added Phosphorus tribromide (7.15g, 26.50 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 1 hour. After completion of reaction, the reaction mixture was poured into ice. The obtained precipitates were filtered off and dissolved in ethyl acetate (3x200 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography, eluting with 10-12% ethyl acetate in hexane to afford title compound as ayellow solid (1.7g, 4.92 mmol, 28% yield). 1HNMR (400MHz, DMSO-de) δ 8.80 (d, J = 4.8 Hz, 1H), 8.33 (s, 1H), 8.06 (d, J = 4.4 Hz, 1H), 7.94 (s, 1H), 7.28-7 ,20(m, 3H), 1.95(s, 6H). LCMS: UC05 FAR1, RT- 3.136 ESI-MS: measured m/z 346.3, 348.2 [M+3] +, [M+l] +.
INT5: 4-bromo-6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)quinoline
Figure imgf000170_0001
Step-1: 4-bromo-6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)quinoline
[00590] To a stirred solution of 6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)quinolin-4-ol (5.0g, 15.57mmol) (INT9) in DMF (60mL) was added Phosphorus tribromide (10.51g 38.94mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 15 min. After completion of reaction, the reaction mixture was pouredin to an ice cold water. The obtained precipitate was filtered off. Isolated solid was dissolved in ethyl acetate (250mLx2). The combined organic phases were dried over anhydrous Na2SC>4, filtered and concentrated under reduced pressure. The resulting crude was purified by silica gel column chromatography, eluted with 28% ethyl acetate in hexane to afford title compound as a yellow solid (2.12g, 5.53mmol, 36% yield). 'HNMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 4.4 Hz, 1H), 8. 18-8. 16 (m, 2H), 7.62- 7.56 (m, 1H), 7.12- 7.02 (m, 2H), 3.78 (s, 3H). LCMS: UC05 FAR1, RT- 2.791 ESI-MS: measured m/z 384.2, +, 386.2 [M+l] ,[M+3] +■
INT6: tert-butyl 4-(4-bromo-6-chloroquinolin-7-yl)-5-methyl-lH-indazole-l-carboxylate
Synthesis of 4-bromo-6-chloro-7-(5-methyl-lH-indazol-4-yl) quinoline o o
Figure imgf000170_0002
Step-1: Synthesis of diethyl 2-(((3-bromo-4-chlorophenyl)amino)methylene)malonate
[00591] To a stirred solution of 3-bromo-4-chloroaniline (40.0g, 194.17mmol) in ethanol (400mL) was added diethyl 2-(ethoxymethylene) malonate (46.13g, 213.59mmol) at room temperature and reaction mixture was stirred at 80°C for 16h. The reaction mixture was cooled to room temperature and concentrated under reduce pressure. The obtained precipitates were triturated with //-hexane and dichloromethane, filtered under vacuum to afford the title compound as an off-white solid (133g 354.66mmol, 91% yield). 'HNMR (400MHz, DMSO-t/Q 8 10.59 (d, J = 13.2 Hz, 1H), 8.32 (d, J= 13.6 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.45 (dd, J1 = 2.4 Hz, J2 = 8.8 HZ, 1H), 4.21 (q, J1 = 7.2 Hz, J1 = 14.4 Hz, 2H), 4. 13 (q, J1 = 6.8 Hz, J2 = 14.0 Hz, 2H), 1 .25 (q,
Figure imgf000171_0001
= 7.2 Hz, J2 = 14.0 Hz, 6H). LCMS: Method-UC04_FAR1, RT- 2.484 ESI-MS: measured m/z 376.2 [M+l]+, 378.2 [M+3] +.
Step-2: Synthesis of ethyl 7-bromo-6-chloro-4-hydroxyquinoline-3-carboxylate
[00592] A suspension of diethyl 2-(((3-bromo-4-chlorophenyl)amino)methylene)malonate (13.2g, 35.20mmol) in Dowtherm (40mL) was heated at260°C (Pre-heated oil bath) for 16h. The reaction mixture was cooled to room temperature and diluted with //-hexane (140ml). The obtained precipitates were filtered off, washed with //-hexane, dried under vacuum to afford the title compound as an off-white solid (105.0g, 319.14mmol, 90% yield). 1HNMR (400 MHz, DMSO-t/Q 5 12.40 (s, 1H), 8.62 (s, 1H), 8.49 (s, 1H), 8.20 (s, 1H), 8.03 (s, 1H), 7.86 (d, J= 8.8 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 4.22 (q,
Figure imgf000171_0002
= 7.2 Hz, J2 = 14.4 Hz, 3H), 1.28 (t, J = 7.2 Hz, 6H). LCMS: Method-UC04_FAR1, RT1- 1.475 RT2- 1.532 ESI-MS: measured m/z 330.2 [M+l] +, 332.2 [M+3] +. Note: Isolated material was mixture of regioisomers.
Step-3: Synthesis of 7-bromo-6-chloro-4-hydroxyquinoline-3-carboxylic acid
[00593] To the stirred solution of ethyl 7-bromo-6-chloro-4-hydroxyquinoline-3-carboxylate (50.0g, 151.97mmol) in a mixture of ethanol:Water (8:2, 800mL) was added NaOH (30.39g 759.87mmol) at room temperature. The resulting reaction mixture was stirred at 100°C for 3h. The reaction mixture was acidified with IN solution of HC1 (190mL). The obtained precipitates were filtered off and dried under reduce vacuum to afford title compound as a yellow solid (100g 332.32mmol, Quantitative). 'HNMR (400 MHz, DMSO-//6) δ 13.43 (s, 1H), 8.97 (s, 1H), 8.36 (s, 1H), 8.21 (s, 1H). LCMS: Method-UC04_FAR1 , RT- 1.679ESI-MS: measured m/z 300.1 [M- 1]-, 302.2 [M-3]-.
Step-4: Synthesis of 7-bromo-6-chloroquinolin-4-ol
[00594] A suspension of 7 -bromo-6-chloro-4-hydroxyquinoline-3 -carboxylic acid (45.0g 149.55mmol) in a sulfolane (60mL) was heated at250°C (Pre-heated oil bath) for 3h. The reaction mixture was cooled to room temperature, was poured into a cold water and obtained precipitates were filtered off, dried under reduce vacuum to afford title compound as an off-white solid (75 ,0g 291.82mmol, Quantitative). 'HNMR (400 MHz, DMSO-t/6) δ 11.90 (s, 1H), 8.14 (s, 1H), 7.96 (s, 1H), 6.09 (d, J= 7.6 Hz, 1H).
LCMS: Method-UC04_FAR1, RT- 1 .335 ESI-MS: measured m/z 258.1 [M+ 1]+, 260. 1 [M+ 3]+. Step-5: Synthesis of 6-chloro-7-(5-methyl-lH-indazol-4-yl)quinolin-4-ol
[00595] To a stirred solution of 7-bromo-6-chloroquinolin-4-ol (8.5g, 33.07mmol) in mixture of l,4-dioxane:water (8:2, lOOOmL) were added sodium carbonate (14.0g, 132.29mmol) and (5- methyl-lH-indazol-4-yl)boronic acid (11.64g, 66.15mmol) at room temperature and reaction mixture was purged with N2 for 20 minutes followed by addition of Pd(pph3)4 (7.64g, 6.61mmol) at room temperature. The resulting reaction mixture was stirred at 90°C for 5h. After completion of reaction, the reaction mixture was cooled to room temperature, diluted with water (5 OOmL) and extracted with ethyl acetate (4x5 OOmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The obtained crude material was purified by reverse phase column chromatography, eluted with 40-50% acetonitrile in water to afford title compound as an off-white solid (8.0g, 0.25mmol, 25.88% yield). 'H NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 11.96 (br s, 1H), 8.21 (s, 1H), 8.01 (d, J= 7.2 Hz, 1H), 7.55-7.51 (m, 3H), 7.35 (d, J= 8.4 Hz, 1H), 6.13 (d, J= 7.2 Hz, lH), 2.15 (s, 3 H). LCMS: UC04 FAR1 , RT- 1.339 ESLMS: measured m/z 310.3 [M+1]+, 312.3 [M+3]+.
Step-6: Synthesis of 4-bromo-6-chloro-7-(5-methyl-lH-indazol-4-yl)quinoline
[00596] To a stirred solution of 6-chloro-7-(5-methyl-lH-indazol-4-yl)quinolin-4-ol (2.89g 9.0mmol) in DMF (30mL) was added phosphorus tribromide (6.07g, 22.50mmol) at 0°C and reaction mixture was stirred at room temperature for Ih. After completion ofreaction, the reaction mixture was poured into ice and obtained precipitates were filtered off and dissolved in ethyl acetate (3x1 OOmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography, eluted with 10-15% ethyl acetate in hexaneto afford title compound as a yellow solid (2.3g, 6.00mmol, 66% yield). 'HNMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 4.4 Hz, IH), 8.18-8.16 (m, 2H), 7.62-7.56 (m, IH), 7.11 (d, J= 8.8 Hz, IH), 7.05 (t, J= 8.8 Hz, lH), 3.78 (s, 3H). 19F NMR (400 MHz, DMSO-d6) δ -113.55 (IF), -117.30 (IF). LCMS: UC05 FAR1, RT- 2.799 ESLMS: measured m/z 384.2[M+1]+, 386.2 [M+3]+.
INT7 : 2-amino-5-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) indolizine-3- carbonyl)benzonitrile.
Figure imgf000173_0001
Step-1: Synthesis of 5-acetyl-2-fluorobenzonitrile
[00597] To a stirred solution of 5 -bromo-2 -fluorobenzonitrile (30.00g, 150.82mmol) and tributyl(l -ethoxyvinyl)stannane (65.50g, 180.99mmol) in toluene (300 mL), was purged with N2 for 15 minutes. To this reaction mixture were added Pd2dba3 (2.76g, 3.0 Immol) and Binap (4.10g 6.63mmol) atrt. The resulting reaction mixture was heated to 100°C for 5h. After completion of reaction, the reaction mixture was diluted with IN HC1 (500mL) and was stirred for 15 minutes at rt. The resulting reaction mixture was filtered through celite bed, washed with ethyl acetate (200mL). The obtained filtrate was extracted with ethyl acetate (2x300mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography, eluted with 25% ethyl acetate in hexane to afford the title compound as yellow solid (23 ,0g, 153.37mmol, yield- 93%). 'HNMR (400 MHz, DMSO-d6) 6 8.56-8.53 (dd, Ji=2.4Hz, J2=6.4Hz, 1H), 8.33-8.29 (m, 1H), 7.68 (t, J=9.2Hz, 1H), 2.62 (s, 3H).
Step-2: Synthesis of 5-(2-bromoacetyl)-2-fluorobenzonitrile
[00598] To a stirred solution of 5-acetyl-2 -fluorobenzonitrile (36.00g, 220.85mmol) in ethyl acetate (360mL), was added CuBr2 (98.50g, 441.17mmol) at room temperature. The resulting reaction mixture was stirred at 70°C for 6h. The reaction mixture was cool at room temperature and filtrated through celite bed, washed with ethyl acetate (100mL). The obtained filtrate was diluted with water (600mL), extracted with ethyl acetate (2x400mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The obtained crude material was purified by flash column chromatography, eluted with 17% ethyl acetate in hexane to afford the title compound as white solid (30.0g, 124.48mmol, 56%). 'H NMR (400 MHz, DMSO-d6) δ 8.63-8.61 (dd, Ji=3.2Hz, J2=6.0Hz, 1H), 8.38-8.34 (m, 1H), 7.73 (t, J=8.8Hz, 1H), 4.99 (s, 2H). Step-3: Synthesis of 5-(2-(3-bromo-2-methyl-114-pyridin-l-yl)acetyl)-2-fluorobenzonitrile, bromide salt
[00599] To a stirred solution of 5-(2-bromoacetyl)-2 -fluorobenzonitrile (30.00g, 124.48mmol) in THF (300mL) was added 3-bromo-2-methylpyridine (42.8g, 248.90mmol) at rt. The resulting reaction mixture was heated to 80°C for 48h. After completion of reaction, the reaction mixture was cooled to rt, the obtained precipitate was filtered, washed with hexane, dried under high vacuum to afford the title compound as white solid (40.00g, 120. 12mmol, 96% yield). 'H NMR (400 MHz, DMSO-tC) δ 9.01 (d, J=8.4Hz, 1H), 8.93 (d, J=6.0Hz, 1H), 8.76-8.74 (dd, Ji=2.0Hz, J2=6.0HZ, 1H), 8.45-8.41 (m, IH), 8.04 (t, J=6.8Hz, IH), 7.85 (t, .7=9, 2Hz, 1H), 6.64 (s, 2H), 2.81 (s, 3H). LCMS: Method-LC05_FAR1, RT 1.362, ESI+ MS: Measured m/z 335.2 [M+2]+.
Step-4: Synthesis of 5-(8-bromoindolizine-3-carbonyl)-2-fluorobenzonitrile
[00600] A mixture of Dimethyl sulphate (46.2mL, 480.0mmol) andDMF (37. ImL, 480.0mmol) was heated to 80°Cfor2h. Aftercooling, This obtained solution was added dropwiseto a separate solution of 5-(2-(3-bromo-2-methyl-114-pyridin-l-yl)acetyl)-2-fluoro benzonitrile, bromide salt (20.0g, 60.06mmol) in DMF (72mL) added at 0°C and was stirred for Ih at room temperature. To this reaction mixture DIPEA (82mL, 480.0mmol) was added dropwise atrt over a period of Ih. After completion of reaction, the reaction mixture was poured in ice-cold water. The obtained precipitate was filtered and dried under vacuum to afford title compound as yellow solid (10.0g 29.23mmol, 49% yield). 'HNMR (400 MHz, DMSO-d6) δ 9.78 (d, J=6.8Hz, IH), 8.33-8.31 (dd, ,/i=2.0Hz, ,72=6.0Hz, IH), 8.18-8.14 (m, IH), 7.73-7.69 (m, 2H), 7.54 (d, J=4.8Hz, IH), 7.09 (t, J=7.2Hz, IH), 6.77 (d, J=4.8Hz, IH). LCMS: Method-UC05_FAR1, RT 2.705 ESLMS: measured m/z 343.2 [M+l]+, 345.3 [M+3]+.
Step-5: Synthesis of 5-(8-bromoindolizine-3-carbonyl)-2-((4-methoxybenzyl)amino) benzonitrile
[00601] To a stirred solution of 5-(8-bromoindolizine-3-carbonyl)-2 -fluorobenzonitrile (10.0g 29.23mmol) inNMP(100mL), were added (4-methoxyphenyl)methanamine (4.2mL, 32.16mmol) and DIPEA (10.OmL, 58.47mmol) atroom temperature. The resultingreactionmixture was heated to 110°C for Ih. After completion ofreaction, the reaction mixture was poured in ice-cold water. The obtained precipitate was filtered and dried under vacuum to afford the title compound as off white solid (14.0g, 30.50mmol, quantitative). 1HNMR(400MHz, DMSO-tL) δ 9.64 (d, .7=7, 2 Hz, IH), 7.93 (d, J=1.6Hz, IH), 7.81-7.78 (dd, Ji=l ,6Hz, J2=8.8Hz, IH), 7.60-7.56 (m, 2H), 7.49 (d, J=4.8Hz, IH), 7.3 l(d, J=5.6Hz, 2H), 6.96 (t, J=7.2Hz, IH, IH), 6.91 (d, J=8.4Hz, 2H), 6.78 (d, J=8.8Hz, IH), 6.70 (d, J=4.4Hz, IH), 4.46 (d, J=6.0Hz, 2H), 3.79 (s,3H). LCMS: Method- UC05 FAR1, RT-2.975, ESI+ MS: measured m/z: 460.3 [M+l]+, 462.2 [M+3]+. Step-6: Synthesis of 2-((4-methoxybenzyl)amino)-5-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)benzonitrile
[00602] A solution of 5-(8-bromoindolizine-3-carbonyl)-2-((4-methoxybenzyl)amino) benzonitrile (14.0g, 30.5mmol), Bis(pinacolato)diboron (15.4g, 61.0mmol) and anhydrous potassium acetate (8.96g, 91.50mmol) in 1,4-dioxane (140mL), was purged with N2 for 15 minutes. To this reaction mixture was added PdCl2(dppf). dem (2.49g, 3.05mmol) at rt. The resulting reaction mixture was heated to 90°C for 2h. After completion of reaction, the mixture was cooled at room temperature. To this reaction mixture were added 5-bromo-l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazole (5.93g, 21.35mmol) and K2CO3 (12.6g, 91.50mmol) (INTI), and was purged withN2 for 15 minutes. Theresultingreaction mixture was heated to 90°C f or 36h. After completion of reaction, the reaction mixture was cooled to rt, filtrated through celite bed, washed with ethyl acetate (100mL), filtrate was concentrated under reduced pressure. The obtained crude material was purified by flash column chromatography, eluted with 68% ethyl acetate in hexane to afford the title compound as yellow solid (7.0g, 12.08mmol, 40% yield). JH NMR (400 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.51 (s, 1H), 8.24 (s, 1H), 7.92 (d, J=1.6Hz, 1H), 7.80 (d, J=8.8Hz, 1H), 7.74 (s, 1H), 7.50 (t, J=5.6Hz, 1H), 7.36 (d, J=4.8Hz, 1H), 7.32 (d, J=8.8Hz, 2H), 7.17-7. 14 (m, 2H), 6.90 (d,J=8.4Hz, 2H), 6.76 (d,./=9,2Hz, 1H), 6.01 (d,./=4,8Hz, 1H), 4.44 (d, J=5.6Hz, 2H), 4.00 (s, 3H), 3.71 (s, 3H). LCMS: Method- UC05_F ARI, RT- 2.561 ESI-MS: measured m/z 580.5 [M+l]+, 581.5 [M+2]+.
Step-7: Synthesis of 2-amino-5-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)benzonitrile
[00603] To a stirred solution of 2-((4-methoxybenzyl)amino)-5-(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3 carbonyl)benzonitrile (7.0g 12.08mmol) in mixture of TFA : Anisole (28mL:7mL) was stirred at room temperature for Ih. The reaction mixture was concentrated under reduced vacuum. The obtained crude material was diluted with saturated solution ofNaHCO3 (300mL) and extracted with ethyl acetate (2xl00mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The obtained crude material was triturated using diethyl ether to afford title compound as yellow solid. (4.6g, 10.02mmol, 83%). 'HNMR (400 MHz, DMSO-t/g) δ 9.78 (d, ,/=4.0Hz, 1 H), 8.52 (s, 1 H), 8.24 (s, 1 H), 7.86 (d, J= 1.6Hz, 1 H), 7.81 -7.78 (d, Ji=l .6Hz, J2=8.8Hz, IH), 7.75 (s, IH), 7.39 (d, J=4.8Hz, IH), 7.16(d, J=4.0Hz, 2H), 6.88 (d, J=8.8Hz, IH), 6.78 (s, 2H), 6.03 (d, J=4.4Hz, IH), 4.00 (s, 3H). LCMS: Method-UC05_FAR1, RT-2.099, ESI+ MS: measured m/z: 460.4 [M+l]+ 461.4 [M+2]+ INT8: (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3- yl)(piperazin-l-yl)methanone
Figure imgf000176_0001
Step-1: Synthesis of tert-butyl 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)piperazine-l-carboxylate
[00604] To a stirred solution of tert-butyl piperazine-1 -carboxylate (1.45g, 7.75mmol) in toluene (lOmL) were added TEA (0.782g, 7.75mmol) and trimethylaluminum (4.5mL, 9.04mmol, 2.0Min toluene) atO°C. The reaction mixture was stirred at 0°C for 30 min under N2 atm followed by addition of ethyl 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) indolizine-3- carboxylate (1 ,0g, 2.58mmol) in toluene (5mL). The reaction mixture was stirred at 90°C for Ih. The reaction mixture was cooled to room temperature and diluted with sat. NaHCCL (100mL). The resulting suspension was extracted with ethyl acetate (3x1 OOmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to affordthetitle compoundas abrown liquid (4.51g, 8.55mmol, Quantitative). 1HNMR(400 MHz, CDC13) δ 8.87 (d, J= 6.8 Hz, IH), 8.50 (5, IH), 8.21-8.20 (m, IH), 7.71 (s, IH), 7.10 (d, J= 4.4 Hz, IH), 6.93-6.83 (m, 2H), 5.89 (d, J= 4.0 Hz, IH), 3.89 (s, 3H), 3.69 (m, 4H), 3.43-3.42 (m, 4H), 1.38 (s, 9H). LCMS: UC05 FAR1, RT- 2.336 ESLMS: measured m/z 528.5 [M+l]+.
Step-2: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3- yl)(piperazin-l-yl)methanone
[00605] To a stirred solution of tert-butyl 4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)piperazine-l-carboxylate (4.5g, 8.53mmol) in dichloromethane (25mL) was added 4M HC1 in 1,4-dioxane (30mL) at room temperature. The reaction mixture was stirred at room temperature for 2h. The reaction mixture was concentrated under reduce pressure and basify using saturated aqueous NaHCOs (30mL). The resulting suspension was extracted with ethyl acetate (3x3 OOmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as a brown solid (2.5g, 5.85mmol, Quantitative). JH NMR (400 MHz, DMSO-t/Q 5 8.82 (d, J= 6.8 Hz, 1H), 8.49 (5, 1H), 8.21 (s, 1H), 7.71 (s, 1H), 7.04 (d, J = 4.4 Hz, 1H), 6.88- 6.80 (m, 2H), 5.88 (d, J = 4.0 Hz, 1H), 4.02 (s, 3H), 3.63 (br s, 4H), 2.74 (br s, 4H). LCMS: UC05 FAR1, RT- 1.499 ESI-MS: measured m/z 428.5, [M+l]+. INT9: 6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)quinolin-4-ol
Figure imgf000177_0001
INT10: (4-aminophenyl) (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) indolizin-3-yl) methanone
Synthesis of N-(4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3- carbonyl) phenyl) methanesulfonamide
Figure imgf000177_0002
Step-1: Synthesis of 2-(3-bromo-2-methyl-114-pyridin-l-yl)-l-(4-nitrophenyl)ethan-l-one bromide salt
[00606] To a stirred solution of 2-bromo-l-(4-nitrophenyl)ethan-l -one (20.0g, 81.95mmol) in THF (200mL) was added 3-bromo-2-methylpyridine (28.2g, 163.90mmol) at room temperature. The reaction mixture was heated at 80°C for 32h. The reaction mixture was cooled to room temperature. The resulting suspension was filtered and washed with hexane. The resulting solid was dried under vacuum to afford title compound as an off-white solid (28.0g, 67.65mmol, 83% yield). LCMS: (ACQUITYPDA and QD A detector, UC05_FARl): m/z 335.1 (M-79)+(ESI+ve), RT = 1 .32 min, 77.1%, 200 - 400nm1H. NMR (400 MHz, DMSO) δ 9.03-8.98 (m, 2H), 8.50 (d, J= 8.8Hz, 2H), 8.32 (d, J = 8.8Hz, 2H), 8.07-8.04 (m, 1H), 6.74 (s, 2H), 2.84 (s, 3H).
Step-2: Synthesis of (8-bromoindolizin-3-yl)(4-nitrophenyl)methanone
[00607] dimethyl sulphate (34.12g, 270.58mmol) was dissolved in DMF (21mL) at room temperature and heated at 80°C for 2h. The reaction mixture was cooled to room temperature. In a separate vessel 2-(3-bromo-2-methyl-114-pyridin-l-yl)-l-(4-nitrophenyl)ethan-l-one bromide salt (14g, 33.82mmol) was dissolved in in DMF (21mL) at room temperature. The reaction mixture was cooled to 0°C. A solution of dimethyl sulphate in DMF was added to the reaction mixture at 0°C. The reaction mixture was stirred for Ih at room temperature followed by addition of DIPEA (23.52mL, 135.29mmol) over a period of 1 h. The reaction mixture was poured into ice- cold water to precipitate out the product. The obtained precipitate was filtered off and washed with hexane to afford the title compound as an orange solid (11g, 31 .87mmol, 94% yield). JH NMR (400 MHz, DMSO) δ 9.84 (d, J= 6.8 Hz, IH), 8.39(d, J= 7.2 Hz, 2H), 8.02(d, J= 7.2 Hz, 2H), 7.75 (d, .7= 7.2 Hz, IH), 7.46-7.44 (m, IH), 7. 12(d, J= 7.2 Hz, IH), 6.79 (d, J= 4.8Hz, IH).
Step-3: Synthesis of (4-nitrophenyl)(8-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)indolizin-3-yl)methanone
[00608] To a stirred solution of (8-bromoindolizin-3-yl)(4-nitrophenyl)methanone (2.5g 7.24mmol) in 1,4-dioxane (25mL) were added potassium acetate (2.13g, 21.72mmol) and bis(pinacolato)diboron (3.67g, 14.48mmol) at room temperature. The reaction mixture was purged with N2 gas f or 15 -20 minutes f ollowedby addition of PdCl2(dppf)dcm (0.59g, 0.72mmol) at room temperature. The reaction mixture was heated to 90°C for 2h. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (200mL). The resulting suspension was filtered through celite bed and washed with ethyl acetate (100mL). The obtained filtrate was concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, product eluted with 10-15% ethyl acetate in hexane to afford the title compound as a yellow solid (1 ,2g, 3.06mmol, 42% yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 311 .2 (M+H)+ (ESI +ve), RT = 1.69 min, 91 .9%, 200 -400nm. JH NMR (400 MHz, DMSO) δ 9.99 (d, J= 6.4 Hz, IH), 8.38 (d, J= 8.8 Hz, 2H), 7.99-7.97 (m, 2H), 7.75-7.73(m, IH), 7.39 (d, J = 4.8Hz, IH), 7.21 (t, J= 7.2Hz, IH), 6.99 (d, J = 4.4Hz, IH), 1.36 (s, 12H).
Step-4: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3- yl)(4- nitrophenyl) methanone
[00609] To a stirred solution of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole (17.01g, 61.20mmol) in mixture of 1,4-dioxane and water (100:25, 125mL)were addedpotassium carbonate (12.68g, 91.80mmol) and (4-nitrophenyl)(8-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)indolizin-3-yl)methanone (12.0g, 30.60mmol) at room temperature. The reaction mixture was purged with N2 gas for 15 minutes followed by PdCl2(dppf) (2.23g, 3.06mmol) at room temperature. The reaction mixture was stirred at 90°C for 16h. The reaction mixture was cooled to room temperature and filtered through celite bed. The celite bed was washed with ethyl acetate (3 OOmL). The obtained filtrate was concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluted with 100% ethyl acetate to afford the title compound as a yellow solid (5 ,0g, 10.76mmol, 35% yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 465.2 (M+H)+ (ESI +ve), RT = 2.43 min, 94.6%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 9.95-9.93 (m, 1H), 8.52 (s, 1H), 8.37 (d, J = 8.4 Hz, 2H), 8.26 (s, 1H), 8.04(d, J = 8.4 Hz, 2H), 7.77 (s, 1H), 7.32-7.30 (m, 3H), 6.09 (d, J= 4.8 Hz, 1H), 4.00 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.94 (s, 3F).
Step-5: Synthesis of (4-aminophenyl)(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizin-3-yl)methanone
[00610] To a stirred solution of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizin-3-yl)(4-nitrophenyl)methanone (5.0g, 10.76mmol) in mixture of methanol (50mL) and ethyl acetate (50mL) was added 10% Pd/C (2.5g, 50% wet) atroom temperature. The resulting reaction mixture was purged with H2 gas for 6h. The reaction mixture was filtered through celite bed and washed with 10% methanol in dichloromethane (3xl00mL). The obtained filtrate was concentrated under reduced pressure to afford the title compound as an orange solid (4.0g 9.20mmol, 85% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 435.2 (M+H)+ (ESI +ve), RT = 1.99 min, 78.6%, 200 - 400nm.
INT11: l-(6-chloro-7-(2-fluorophenyl)quinazolin-4-yl)azetidin-3-amine
Figure imgf000179_0001
INT12: (S)-N4-(3-chloro-4-fluorophenyl)-7-((tetrahydrofuran-3-yl)oxy)quinazoline-4,6- diamine
Figure imgf000179_0002
INT13: 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3- carbonyl) benzoic acid
Figure imgf000180_0001
Step-1: Synthesis of tert-butyl (3-((2,3,5,6-tetrafluorophenyl)thio)propyl)carbamate
[00611] To a stirred solution of methyl 4 -acetylbenzoate (25.0g, 140.30mmol) in ethyl acetate (250mL) was added copper bromide (0.39g, 16.3mmol) at room temperature. The reaction mixture was stirred at 65°C temperature for 16h. After completion of the reaction, the reaction mixture was cooled to room temperature and filtrated through the celite bed. The obtained filtrate was diluted with water (500mL) and extracted with ethyl acetate (2 x 300mL). The combined organic lay er was dried over anhydrousNa2SO4, filtered, andconcentratedunderreducedpressure to obtain the crude. The obtained crude was triturated using w-Pentaneto afford the title compound as a yellow solid (35.5g, 138.69mmol, 97% yield). iH NMR (400MHz, DMSO) δ 8.20-8.02 (m, 4H), 3.85 (s, 2H), 3.82 (s, 3H).
Step-2: Synthesis of 3-bromo-l-(2-(4-(methoxycarbonyl)phenyl)-2-oxoethyl)-2- methylpyridin-l-ium bromide
[00612] To a stirred solution of methyl4-(2-bromoacetyl)benzoate (35.5g, 138.68mmol)in THF (355mL) was added 3 -bromo-2 -methylpyridine (59.64g, 346.7mmol) at room temperature. The reaction mixture was stirred at 70 °C for 16h. After completion of the reaction, the obtained precipitate was filtered off and dried under reduced pressure to afford the title compound as a brown solid (42.2g, 121.26mmol, 87% yield). LCMS (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 348.1 (M+H)+ (ESI +ve), RT = 1.38min, 96.9%, 200 - 400nm. JH NMR (400MHz, DMSO) δ 9.04
Figure imgf000180_0002
2.0 Hz, J2 = 12.0 Hz, 2H), 8.25-8.19 (m, 4H), 8.10-8.04 (m, 1H), 6.78 (s, 2H), 3.93 (s, 3H), 2.84 (s, 3H).
Step-3: Synthesis of methyl 4-(8-bromoindolizine-3-carbonyl)benzoate
[00613] A mixture of dimethyl sulphate (61.0g, 483.63 mmol) andDMF (35.3g, 4803.63mmol) was stirred at 80 °C for 2h. The resulting reaction mixture was cooled to room temperature and was added into a pre-stirred solution of 3-bromo-l-(2-(4-(methoxycarbonyl)phenyl)-2-oxoethyl)- 2-methylpyridin-l-ium bromide (21.1g, 60.45mmol) in DMF (73mL) at 0 °C. The reaction mixture was stirred at room temperature for Ih followed by the addition of DIPEA (84mL, 483.6mmol) at room temperature. The reaction mixture was stirred at room temperature for another Ih. and then poured into ice-cold water. The obtained precipitate was filtered off and dried under reduced pressure to afford the title compound as a yellow solid (12.0g, 33.61mmol, 55% yield). LCMS (ACQUITYPDA and QDA detector, UC08 FAR1): m/z 358. 1 (M+H)+ (ESI +ve), RT = 2.80min, 96.0%, 200 - 400nms. JH NMR (400MHz, DMSO) δ 8.32-8.14 (m, 3H), 7.95- 7.92 (m, 3H), 7.73 (br s, IH), 7.45 (br s, IH), 7.11-6.76 (m, IH), 2.89 (s, 3H).
Step-4: Synthesis of methyl 4-(8-(4,4,5,5-tetramethyl-l,3^-dioxaborolan-2-yl)indolizine-3- carbonyl)benzoate
[00614] To a stirred solution of methyl 4-(8-bromoindolizine-3-carbonyl)benzoate (11 ,0g 30.81 mmol) in 1 ,4-dioxane(l 1 .OmL) were added Bis(pinacolato)diboron (11.7g, 46.21 mmol) and potassium acetate (9.0g, 92.43 mmol) at room temperature. The reaction mixture was purged with N2 for 15 mins, followed by the addition of PdCl2(dppf)DCM (1.26g, 1.54mmol) at room temperature. The reaction mixture was stirred at 70 °C for 16 hours, cooled to room temperature and diluted with ethyl acetate (200mL). The resulting suspension was filtered through a celite bed and washed with ethyl acetate (100mL x 2). The obtained filtrate was concentrated under reduced pressure to afford the title compound as a brown semi-solid (22.50g, quantitative yield). LCMS: (ACQUITYPDA and QDAdetector, UC08 FAR1): m/z 406.3 (M+H)+ (ESI +ve), RT = 2.99min, 38.5%, 200 - 400nm.
Step-5: Synthesis of methyl 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)benzoate
[00615] To a stirred solution of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole (0.58g, 2.08mmol) in a mixture of 1,4-dioxane (9.2mL) and water (2.3mL) were added methyl 4- (8-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)indolizine-3-carbonyl)benzoate (3.80g 9.38mmol) andK2CO3 (0.57g, 4.17mmol) atroom temperature. The reaction mixture was purged with N2 for 15 mins, followed by the addition of PdCl2(dppf)DCM (0.04g, 0.05mmol) at room temperature. The reaction mixture was stirred at 100 °C for 2 hours, cooled to room temperature and filtered through celite. The filtrate was diluted with water (70mL) and extracted with ethyl acetate (2 x 50mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude residue was purified by column chromatography, eluted with 5% methanol in dichloromethane to afford the title compound as a yellow solid (0.5g, 1.05mmol, 50% yield). LCMS (ACQUITY PDA and QDA detector, UC08_ FAR1): m/z 478.2 (M+H)+ (ESI +ve), RT = 2.39min, 69.8%, 200 - 400nm. 1HNMR (400 MHz, DMSO) δ 9.93 (q, ./, = 2.8 Hz, J2 = 5.2 Hz, lH), 8.52 (s, IH), 8.25 (s, IH), 8.10 (d, J= 8.4 Hz, 2H), 7.90 (d, J= 8.4 Hz, 2H), 7.77 (s, 1H), 7.53 (d, J= 8.8 Hz, 1H), 7.31-7.26 (m, 2H), 6.07 (d, J = 4.4 Hz, lH), 4.00 (s, 3H), 3.91 (d, J= 9.2 Hz, 3H). 19F NMR (376 MHz, DMSO) δ -54.95 (s, 3F).
Step-6: Synthesis of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl) benzoic acid
[00616] To a stirred solution of methyl 4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)benzoate (0.5g, 1.05mmol) in THF (4.0mL), methanol (0.5mL) and water (0.5mL) was added lithium hydroxide (0.13g, 3.14mmol) at O °C. The reaction mixture was stirred at room temperature for 3 hours, acidified with IN aqueous HC1 (50mL) and extracted with ethyl acetate (3 x 30mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound as a white solid (0.2g, 0.42mmol, 40% yield). LCMS (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 464.2 (M+H)+ (ESI +ve), RT = 2.07min, 99.0%, 200 - 400nm. TI NMR (400 MHz, DMSO) δ 13.21 (br s, 1H), 9.95-9.93 (m, 1H), 8.53 (s, 1H), 8.26 (s, 1H), 8.08 (d, J= 8.0 Hz, 2H), 7.87 (d, J= 8.4 Hz, 2H), 7.77 (s, 1H), 7.32-7.25 (m, 3H), 6.07 (d, J = 4.8 Hz, 1H), 4.00 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.95 (s, 3F).
INTI 4: 6-chloro-7-(2,6-dimethylphenyl)quinolin-4-ol
Figure imgf000182_0001
Step-5: Synthesis of 6-chloro-7-(2,6-dimethylphenyl)quinolin-4-ol
[00617] To a stirred solution of 7-bromo-6-chloroquinolin-4-ol (1 ,0g, 3 ,89mmol) in a mixture of DME (8.0mL) (Int6 Step4), ethanol (3.0mL) and water (1.50mL) were added sodium carbonate (1.23g, 11.67mmol)and(2,6-dimethylphenyl)boronic acid (1.75g, 11.67 mmol) at room temperature. The reaction mixture was purged with N2 gas for 15mins. followed by the addition of Pd(PPh3)4 (0.45g, 0.39mmol) at room temperature. The reaction mixture was stirred at 100 °C for 3 hours, cooled to room temperature, and enriched with additional Pd(PPh3)4 (0.45g 0.39mmol) and (2,6-dimethylphenyl)boronic acid (1 ,75g, 11.67mmol). The reaction mixture was stirred at 100 °C for a further 16 hours, cooled to room temperature and diluted with ethyl acetate (100mL). The resulting reaction mixture was filtered through celite eluting with ethyl acetate (3 x 60 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude was purified by RFC, eluting with 30% acetonitrile in water to afford the title compound as a yellowish brown solid (1.90g, 6.71mmol, 86% yield). LCMS (ACQUITY PDA and QDA detector, o2h_LCMS_Method_A): m/z 284.1 (M+H)+ (ESI +ve), RT = 1.94min, 50.4%, 200-400nm.
INTI 5: Synthesis of N-((3R,6S)-6-methylpiperidin-3-yl)-7-((2-
(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
Figure imgf000183_0001
Step-1: Synthesis of benzyl (2S,5R)-2-methyl-5-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidine-l-carboxylate
[00618] To a stirred solution of benzyl (2S,5R)-5-amino-2-methylpiperidine-l -carboxylate (350 mg, 1.411 mmol) and 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidine (599 mg, 2.110 mmol) in n-Butanol (0.6 mL) was then added DIPEA (0.6 mL, 3.316 mmol) at 0 °C under Nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 16 hours. After completion of reaction (as confirmed by TLC), reaction mixture was cooled to room temperature, diluted with Water (20 mL), extracted with Ethyl acetate (2 x 20 mL). The organic extract was successively washed with Brine and Water and dried over Sodium sulphate to afford crude. The crude was purified by flash silica gel chromatography. Title compound was eluted at a gradient of 0-50% Ethyl acetate in Hexanes and obtained as a gummy solid (340 mg 0.686 mmol, 48%). LCMS (Method-C): Retention time: 1 .904 min, 98 %, 278 nm, ES(+ve): 497.7 [M+H]+
Step-2: Synthesis of N-((3R,6S)-6-methylpiperidin-3-yl)-7-((2-
(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
[00619] To a stirred solution of benzyl (2S,5R)-2-methyl-5-((7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidine-l-carboxylate (300 mg, 0.607 mmol) in MeOH was added Pd/C (10% Palladium on Carbon) under Nitrogen atmosphere and stirred reaction mixture at room temperature for 2 hours under H2 balloon. After completion (as confirmed by TLC), the reaction mixture was filtered through celite bed. The filtrate was concentrated under reduced pressure to afford crude as gummy solid (200 mg, crude). The crude was used in next step without further purification. LCMS (Method-C): Retention time:
1.673 min, 98 %, 278 nm, ES(+ve): 362.4 [M+H]+
INTI 6: Synthesis of 4-(pyrrolidin-3-yloxy)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H- pyrrolo[2,3-d]pyrimidine
Figure imgf000184_0001
Step-1: Synthesis of tert-butyl 3-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)oxy)pyrrolidine-l-carboxylate
[00620] To a stirred solution tert-butyl 3 -hydroxypyrrolidine- 1 -carboxylate (1.982 g, 10.60 mmol) in DMF (20 mL), NaH (60%) (0.848 g, 21.201 mmol) was added portionwise at 0 °C. The reaction mixture was stirred at room temperature under for 30 minutes. 4-chloro-7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (2.0 g, 7.067 mmol) was added into the reaction mixture at 0 °C and reaction mixture was stirred at room temperature for 3 hours. After completion, the reaction mixture was diluted with Water (100 mL) and the aqueous layer was extracted with Ethyl acetate (2 x 150 mL). The combined organic phases were dried over anhydrous Sodium sulfate and concentrated under vacuum. The resulting crude was purified by silica gel flash column chromatography. Pure compound was eluted at 40% Ethyl acetate in Hexanes to obtain title compound as off-white solid (1.5 g, 3.451 mmol, 48%). LCMS (Method- C): Retention Time: 2.138 min, 98.55%, 260 nm, ES(+ve): 435.49 [M+H] +
Step-2: Synthesis of 4-(pyrrolidin-3-yloxy)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H- pyrrolo[2,3-d]pyrimidine
[00621] In a 25 mL RBF, tert-butyl 3-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)oxy)pyrrolidine-l-carboxylate (1.5g, 3.451mmol) was dissolved in DCM (15 mL) and cooled to 0 °C. To the reaction mixture, 4.0 M HC1 in Dioxane (7.5 mL) was added dropwise and the reaction mixture was stirred for 1 hour at room temperature. After completion, the reaction mixture was azeotroped with Toluene (3 x 15 mL) to obtain title compound as an off- white solid(1.5 g crude). The crude productwas used in the next step without further purification. LCMS (Method-C): Retention Time: 1.519 min, 90.64%, 220 nm, ES(+ve): 335.2 [M+H] +
INT17: Synthesis of 4-(l,7-diazaspiro[4.4]nonan-7-yl)-7-((2-(trimethylsilyl)ethoxy)methyl)- 7H-pyrrolo [2,3-d]pyrimidine
Figure imgf000185_0001
Step-1: Synthesis of tert-butyl 7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-l,7- diazaspiro [4.4]nonane-l-carboxylate (Int-3)
[00622] To a stirred solution of tert-butyl l,7-diazaspiro[4.4]nonane-l -carboxylate (8.842 g 0.0391 mol) in w-BuOH (50.0 mL), DIPEA (23.3 mL, 0. 134 mol) was added at room temperature under Nitrogen atmosphere. The reaction mixture was stirred at room temperature for 10 minutes. 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (5.0 g, 0.0326 mol) was added and the reaction mixture was stirred at 100 °C for 16 hours. After completion, the reaction mixture was concentrated to give a crude residue which was diluted with Water (50 mL). The aqueous lay er was extracted with Ethyl acetate (2 x 50 mL). The combined organic layer was dried over anhydrous Sodium sulfate and concentrated under vacuum. The resulting crude was purified by silica gel flash column chromatography. Pure compound was eluted at 50% Ethyl acetate in Hexanes to obtain title compound as off-white solid (4.5 g, 0.013 mol, 40%). LCMS (Method-C): Retention Time: 1.434 min, 100%, 230 nm, ES(+ve): 343.9 [M+H] +
Step-2: Synthesis of tert-butyl 7-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-l,7-diazaspiro[4.4]nonane-l-carboxylate
[00623] NaH (60 % in mineral oil) (0.640 g, 0.016 mmol) was added portionwise to a stirred solution of tert-butyl 7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-l,7-diazaspiro[4.4]nonane-l- carboxylate (4.5 g, 0.013 mol) in DMF (45 mL) under Nitrogen gas at 0 °C. The reaction mixture was stirred at room temperature for 30 minutes. After 30 minutes, the reaction mixture was again cooled to 0 °C and SEMC1 (2.610 g, 0.016 mol) was added. The reaction mixture was stirred at room temperature for 2 hours. After completion, the reaction mixture was diluted with Water (100 mL) and aqueous layer was extracted with Ethyl acetate (2 x 150 mL). The combined organic layer was dried over anhydrous Sodium sulfate and concentrated under vacuum. The crude was used in the next step without further purification (3.0 g crude). LCMS (Method-C): Retention Time: 1.838 min, 100%, 225 nm, ES(+ve): 473.74 [M] +
Step-3: Synthesis of 4-(l,7-diazaspiro[4.4]nonan-7-yl)-7-((2-(trimethylsilyl)ethoxy)methyl)- 7H-pyrrolo [2,3-d]pyrimidine [00624] Prepared by the method of Intermediate 2, Step 2 using tert-butyl 7-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-l,7-diazaspiro[4.4]nonane-l- carboxylate (3.0 g, 6.333 mmol). Yield (1.5 g, crude). LCMS (Method-C): Retention Time: 1.387 min, 97.61%, 220 nm, ES(+ve): 374.1 [M+H] +
INT18: Synthesis of 4-(l,6-diazaspiro[3.3]heptan-6-yl)-7-((2-(trimethylsilyl) ethoxy) methyl)-7H-pyrrolo [2, 3-d] pyrimidine
Figure imgf000186_0001
Step-1: Synthesis of tert-butyl 6-(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)-l,6-diazaspiro[3.3]heptane-l-carboxylate
[00625] Prepared by the method of Intermediate 3, Step 1 using tert-butyl 1,6- diazaspiro[3.3]heptane-l-carboxylate(870 mg, 4.393 mmol). After completion (as monitored by TLC), the reaction mixture was filtered through a Buchner funnel, and the solid washed with Hexanes (20 mL) to obtain title compound as an off white solid (1.500 g, 3.370 mmol, 78%). LCMS (Method-C): Retention Time: 1.625 min, 98.57%, 280 nm, ES (+ve): 446.2 [M+H]+
Step-2: Synthesis of 4-(l,6-diazaspiro[3.3]heptan-6-yl)-7-((2-(trimethylsilyl) ethoxy) methyl)-7H-pyrrolo [2,3-d]pyrimidine
[00626] Prepared by the method of Example 2, Step 2 using 6-(7-((2- (trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-l,6-diazaspiro[3.3]heptane-l- carboxylate (500 mg, 1.123 mmol). The crude product was triturated with 10% Diethyl ether in Pentane to obtain title compound as an off white solid (350 mg, 1.014 mmol, 90%). LCMS (Method-C): Retention Time: 1.338 min, 87.38%, 280 nm, ES (+ve): 346.1 [M+H]+
INT19: Synthesis of l-(3-aminobenzyl)-N-methyl-lH-pyrazolo[3,4-d]pyrimidin-6-amine
Figure imgf000186_0002
Step-1: Synthesis of N-methyl-l-(3-nitrobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine [00627] To a stirred solution of 6-chloro-l-(3-nitrobenzyl)-lH-pyrazolo[3,4-d]pyrimidine (1.0 g, 3.452 mmol) in Isopropyl alcohol (10 mL) was added Triethyl amine (0.63 mL, 4.487 mmol) at O °C under Nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 2 minutes. Methyl amine (0.49 mL, 10.380 mmol) was then added portionwise at 0 °C. The reaction mixture was stirred at 90 °C for 12 hours. After completion (as monitored by TLC), the reaction mixture was quenched with Water (30 mL). The aqueous layer was extracted with Ethyl acetate (2 x 50 mL). The combined organic layer was evaporated under reduced pressure to afford crude title compound as yellow solid (0.900 g, crude) The crude was used in the next step without column purification. LCMS (Method-C): Retention Time: 1 .634 min, 94.30%, 290 nm, ES(+ve): 285.19 [M+H]+
Step-2: Synthesis of l-(3-aminobenzyl)-N-methyl-lH-pyrazolo[3,4-d]pyrimidin-6-amine
[00628] 10% Pd/C (0.300 g) was added to a stirred solution ofN-methyl-l-(3-nitrobenzyl)-lH- pyrazolo[3,4-d]pyrimidin-6-amine (0.500 g, 1.758 mmol) in Methanol (10 mL) at room temperature under Hydrogen gas atmosphere. The reaction mixture was stirred at room temperature for 16 hours or until complete consumption of starting material. After completion (as monitored by TLC), the reaction mixture was filtered through celite, washed with Methanol (2 x 15 ml). The filtrate was concentrated under vacuum to obtain crude title compound as a light yellow solid (0.300 g, crude). The crude was used in the next step without column purification or analysis.
INT20: Synthesis of (ls,4s)-Nl-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclohexane-l,4- diamine
Figure imgf000187_0001
Step-1: Synthesis of tert-butyl ((lS,4S)-4-((7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4- yl)amino)cyclohexyl)carbamate
[00629] Prepared by the method of Intermediate 3, Step 1 using tert-butyl ((l S,4S)-4- aminocyclohexyl)carbamate (1.400 g, 4.666 mmol). After concentration under reduced pressure, the crude residue was purified by silica gel flash chromatography. Title compound was eluted at a gradient of 20-100% Ethyl acetate in Hexanes to obtain title compound as light brown solid (1.400 g, 2.883 mmol, 93%). LCMS (Method-C): Retention Time: 1.759 min, 100%, 240 nm,
ES(+ve): 486.57 [M+H]+
Step-2: Synthesis of (lS,4S)-Nl-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclohexane-l,4- diamine
[00630] To a stirred solution of tert-butyl((l S,4S)-4-((7-tosyl-7H-pyrrolo[2,3-d]pyrimidin4- yl)amino)cyclohexyl)carbamate (0.500 g, 1.030 mmol) in DCM (5 mL), was added TFA (1.5 mL) at 0 °C under Nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 30 minutes and gradually brought to room temperature. The reaction was stirred at room temperature for 1 hour or until complete consumption of starting material. After completion, the reaction mixture was diluted with Toluene (10 mL) and co-evaporated with Toluene under reduced pressure to obtain a colorless viscous liquid. The crude was further triturated with 10% Diethyl ether in Pentane to obtain title compound as off white solid (0.500 g, crude). The crude was used in next step without column purification. LCMS (Method-C): Retention time 1.416 min, 100%, 230 nm, ES(+ve): 386.44 [M+H]+
INT21: Synthesis of N-methyl-N-((3R,4R)-4-methylpiperidin-3-yl)-7-(((trimethylsilyl) ethoxy) methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
Figure imgf000188_0001
Step-1
[00631] In a sealed tube vial, 4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidine (500 mg, 2.192 mmol) and tert-butyl (3R,4R)-4-methyl-3-(methylamino) piperidine- 1 -carboxylate (744 mg, 2.631 mmol) were dissolved in 5% aq Sodium bicarbonate (10 mL) at room temperature and heated at 120 °C for 16 hours. After completion (as monitored by TLC), the reaction mixture was diluted with Water (10 mL), extracted with DCM (3 x 10 mL). The organic layer died over Sodium sulfate, concentrated under reduced pressure to afford crude. The resulting crude was purified by normal phase column chromatography. Product was eluted at a gradient of 0-5% MeOH in DCM. Fractions containing pure compound were combined and concentrated under reduced pressure to obtain title compound as gummy solid (700 mg, 1.866 mmol, 72%). LCMS (Method-C): Retention Time: 1.42 min, 100%, 202 nm, ES (+ve): 376.26 [M-56+H]+ INT22: Synthesis of (lR,4R)-4-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)amino)cyclohexyl methanesulfonate
Figure imgf000189_0001
Step-1: Synthesis of (lR,4R)-4-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)amino)cyclohexan-l-ol
[00632] Prepared by the method of Intermediate 3, Step 1 using (lR,4R)-4-aminocyclohexan- l-ol (1.940 g, 16.911 mmol). After concentration under reduced pressure, the crude residue was purified by silica gel flash chromatography. Pure compound was eluted at a gradient of 80-100% Ethyl acetate in Hexanes and obtained as an off white semi solid (3.500 g, 13.330 mmol, 69%). LCMS (Method-H): Retention Time:2.782 min, 97.15%, 254 nm, ES+: 363.4 [M+H]+
Step-2: Synthesis of (lR,4R)-4-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-yl)amino)cyclohexyl methanesulfonate
[00633] To a stirred solution of tert-butyl (lR,4R)-4-((7-((2-(trimethylsilyl)ethoxy)methyl)-7H- pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclohexan-l-ol (3.0 g, 8.274 mmol) in DCM (30 mL), was added triethylamine (3.4 mL, 24.820 mmol) followed by Methane sulfonyl chloride (1.400 g, 12.411 mmol) at 0 °C under Nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 2 hours. After completion, the reaction mixture was diluted with cold water (50 mL) and extracted with Ethyl acetate (3 x 30 mL). Combined organic layer was dried over Sodium sulfate and concentrated under reduced pressure to obtain a colorless viscous liquid. The crude was purified by silica gel flash chromatography. Title compound was eluted at a gradient of 30-50% Ethyl acetate in Hexanes and obtained as an off white semi solid (3.200 g, 7.262 mmol, 88%). LCMS (Method-H): Retention time 3.286 min, 94.43%, 202 nm, ES+: 441.3 [M+H]+
INT23: Synthesis of 3-((6-((l-(2-methoxyethyl)-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d] py r imidin- 1 -y l)methyl)phenol
Figure imgf000189_0002
Step-1: Synthesis of 6-chloro-l-(3-methoxybenzyl)-lH-pyrazolo[3,4-d]py
Figure imgf000189_0003
[00634] To a stirred solution of 6-chloro-lH-pyrazolo[3,4-d]pyrimidine (5.0 g, 32.349 mmol) and 3-methoxyphenyl) methanol (4.470 g, 32.349 mmol) in THF (50 mL) under Nitrogen atmosphere was added PPh3 (392 mg, 48.520 mmol) at 10 °C. The reaction mixture was stirred at room temperature under Nitrogen for 10 minutes, and then cooled to 0 °C. DIAD (0.3 mL, 48.520 mmol) was added drop wise at 0 °C to 10 °C over a period of 5 minutes. The reaction mixture was gradually brought to room temperature and stirred at room temperature for 2 hours. After completion, the reaction mixture was diluted with Water (50 mL) and the aqueous layer was extracted with Ethyl acetate (3 x 50 mL). The combined organic phase was dried over Sodium sulfate and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography. Pure compound was eluted at 30% Ethyl acetate in Hexanes to obtain title compound as off white solid (6.500 g, 23.66 mmol, 73%). LCMS (Method-C): Retention time: 1.684 min, 100%, 220 nm, ES (+ve): 275.1 [M+H]+
Step-2: Synthesis of 3-((6-chloro-lH-pyrazolo[3,4-d] pyrimidin-l-yl)methyl)phenol
[00635] To a stirred solution of 6-chloro-l-(3-methoxybenzyl)-lH-pyrazolo[3,4-d]pyrimidine (3.0 g, 10.920 mmol) in DCM (30 mL) was added BBr3 (1.0 M in DCM) (32.8 mL, 32.760 mmol) at 0 °C under Nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 hours. After completion, the reaction mixture was treated with saturated aq. Sodium bicarbonate solution until pH = 8.0. The aqueous layer was extracted with DCM (3 x 30 mL). The combined organic phase was dried over Sodium sulfate and evaporated under reduced pressure to get crude. The obtained crude product was used for next step as starting material (1 .200 g, 4.600 mmol, 42%). LCMS (Method-C): Retention time: 1.449 min, 100%, 254 nm, ES (+ve): 261.1/263.1 [M+H]+
Step-3: Synthesis of 3-((6-((l-(2-methoxyethyl)-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)methyl)phenol
[00636] In a sealed tube vial 3-((6-chloro-lH-pyrazolo[3,4-d] pyrimidin-l-yl)methyl)phenol (150 mg, 0.575 mmol) was dissolvedin 1,4 Dioxane (1.5 mL) at room temperature. To the above stirred solution, 1 -(2 -meth oxy ethyl)- lH-pyrazol-4-amine (81.230 mg, 0.575 mmol) was added and stirred for another 5 minutes. DIPEA (0.3 mL, 1 .730 mmol) was added and the reaction vial was sealed and heated at 100 °C for 12 hours. After completion, the reaction mixture was concentrated under reduced pressure to yield a solid brown residue. The crude was purified by silica gel flash chromatography. Title compound was eluted at a gradient of 80-100%Ethyl acetate in Hexanes to afford light brown solid (80 mg, 0.219 mmol, 38%). LCMS (Method-C): Retention Time: 1.369 min, 96%, 254 nm, ES(+ve): 366.5 [M+H] + Scheme 4: Synthesis of Compound 1
Figure imgf000191_0001
General Procedure A:
Preparation of (S)-N4-(4-chloro-3-fluorophenyl)-N6-(2,3, 5, 6-tetrafluoro-4-(methylthio)phenyl)~ 7-(( tetrahydrofuran-3-yl)oxy)quinazoline-4, 6-diamine
[00637] To a stirred solution of (S)-N4-(3-chloro-4-fluorophenyl)-7-((tetrahydrofuran-3- yl)oxy)quinazoline-4, 6-diamine (0.54 g, 1.44 mmol) in THF (4 mL) were added (4-bromo- 2,3,5,6-tetrafhrorophenyl) (methyl)sulfane (0.4 g, 1.45 mmol) and Cs2CO3 (0.95 g, 2.9 mmol). The resulting reaction mixture was purged with N2 for 15 minutes, followed by addition of Pd2dba3 (0.13 g, 0.14 mmol) and xanthphos (0.084 g, 0.14 mmol) at room temperature. The resulting mixture was heated to 100°C for 16 hours. After reaction completion, the mixture was diluted with water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude material was purified by flash column chromatography, eluting with 85% EtOAc in hexanes, to afford the title compound as yellow solid (0.3 g, 0.52 mmol, 33% yield). 'H NMR (400 MHz, DMSO-t/g) δ 9.47 (s, 1H), 8.47 (s, 1H), 8.19 (s, 1H), 8.10 - 8.08 (m, 1H), 7.72 - 7.76 (m, 2H), 7.42 (t, J=9.2Hz, 1H), 7.21 (s, 1H), 5.32- 5.29 (m, 1H), 3.99- 3.95 (m, 1H), 3.88- 3.85 (m, 1H), 3.79- 3.75 (m, 2H), 2.50 (s, 3H), 2.34 - 2.27 (m, 1H), 2.08 - 2.20 (m, 1H). 19F NMR (376MHz, DMSO-t/g) δ -123.03 (s, IF), -136.66 - -136.75 (m, 2F), -148.75 - -148.84 (m, 2F). ESLMS: measured m/z 569.3, 571.3[M+H]+.
Preparation of (S)-N4-(3-chloro-4-fluorophenyl)-N6-(2,3,5,6-tetrafluoro-4-(methylsulfonyl) phenyl)-7-((tetrahydrofuran-3-yl)oxy)quinazoline-4, 6-diamine (Compound 1)
[00638] To a stirred solution of (S)-N4-(3-chloro-4-fhiorophenyl)-N6-(2,3,5,6-tetrafluoro4- (methylthio)phenyl)-7-((tetrahydrofuran-3-yl)oxy)quinazoline-4, 6-diamine (0.05 g, 0.088 mmol) in THF:MeOH:water (8:1 :1, 0.5 mL) was added oxone (0.13 g, 0.44 mmol) atO°C. The resulting reaction mixture was allowed to stir at room temperature for 16 hours. After completion of the reaction, the mixture was diluted with aqueous NaHCO? and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by flash column chromatography, eluting with 89% EtOAc in hexanes, to afford the title compound as a white solid (0.05 g, 0.083 mmol, 32% yield). 1HNMR (400 MHz, DMSO-d6) δ 9.77 (s, 1H), 9.11 (s, 1H), 8.57 (s, 1H), 8.21(s, 1H), 8.17- 8.15 (m, 1H), 7.79- 7.75 (m, 1H), 7.45 (t, J= 8.8Hz, 1H), 7.26 (s, 1H), 5.31- 5.28 (m, 1H), 3.91- 3.88 (m, 1H), 3.75- 3.70(m, 2H), 3.70- 3.59 (m, 1H), 3.45 (s, 3H), 2.34- 2.26 (m, 1H), 1.93- 1.87 (m, 1H). 19F NMR (376 MHz, DMSO-t/6) δ -122.75 (s, IF), -141.27 - -141.31 (m, 2F), - 153.34 (m, 2F). ESLMS: measured m/z 601.3 [M+H]+. HPLC (Method I): RT = 6.06 min., 97.7%.
Preparation of N4-(3-chloro-4-fluorophenyl)-N6-(2,3,5,6-tetrafluoroM-
(methylsulfinyl)phenyl)-7-(((S)-tetrahydrofuran-3-yl)oxy)quinazoline-4,6-diamine (Compound 2)
[00639] To a stirred solution of (S)-N4-(3-chloro-4-fhiorophenyl)-N6-(2,3,5,6-tetrafluoro4- (methylthio)phenyl)-7-((tetrahydrofuran-3-yl)oxy)quinazoline-4,6-diamine (0.06 g, 0. 1 mmol) in DCM (0.6 mL) was added oxone (0.16 g, 0.52 mmol) at O°C. The resulting mixture was stirred at room temperature for 16 h. After completion of reaction, the mixture was diluted with aqueous NaHCO3 and extracted with EtOAc (2 x 20 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by flash column chromatography, eluting with 89% EtOAc in hexanes, to afford the title compound as off-white solid (0.02 g, 0.034 mmol, 24% yield). 1HNMR (400 MHz, DMSO-dg) δ 9.63 (s, 1H), 8.40 (s, 1H), 8.53 (s, 1H), 8.16 - 8.13 (m, 1H), 8.02 (m, 1H), 7.77- 7.73 (m, 1H), 7.43 (t, J= 9.2Hz, 1H), 7.24 (s, 1H), 5.29 (m, 1H), 3.94- 3.90 (m, 1H), 3.78- 3.63 (m, 3H), 3.19 (s, 3H), 2.34- 2.25 (s, 1H), 1.97- 1.93 (s, 1H). 19F NMR (376 MHz, DMSO-d6) δ -122.99 (s, IF), -143.01 - -143.06 (s, 2F), -151.27 - -151.33 (s, 2F). ESI+MS: measured m/z 585.3 [M+H]+. HPLC
(Method I): RT = 5.89 min., 95.3%.
Scheme 5: Synthesis of Compound 3
Figure imgf000192_0001
Preparation of tert-butyl ((4-((4-((3-chloro-4-fluorophenyl)amino)-7-(((S)-tetrahydrofuran-3- yl)oxy)quinazolin-6-yl)amino)-2,3,5,6-tetrafluorophenyl)(methyl)(oxo)-r)^-sulfanylidene) carbamate
[00640] tert-butyl ((4-((4-((3-chloro-4-fluorophenyl)amino)-7-(((S)-tetrahydrofuran-3- yl)oxy)quinazolin-6-yl)amino)-2,3,5,6-tetrafluorophenyl)(methyl)(oxo)-X6-sulfanylidene) carbamate was prepared from tert-butyl ((4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)(oxo)-16- sulfanylidene)carbamate (C2, 0.35 g, 0.86 mmol) and (S)-N4-(3-chloro-4-fluorophenyl)-7- ((tetrahydrofuran-3-yl)oxy)quinazoline-4,6-diamine (0.32 g, 0.86 mmol) according to the protocol described in general procedure A and isolated as a brown solid (0.06 g, 0.085 mmol, 10% yield). XH NMR (400 MHz, DMSO-tL) δ 9.75 (s, 1H), 9.21 (s, 1H), 8.57 (s, 1H), 8.27 (s, 1H), 8.19 - 8.16 (dd, .// = 2.8 Hz, J2 = 6.8 Hz, 1H), 7.82 -7.78 (m, 1H), 7.44 (t, J= 9.2 Hz, 1H), 7.26 (s, 1H), 5.28 (brs, 1H), 3.91-3.87 (m, 1H), 3.72-3.61 (m, 2H), 3.53 (s, 3H), 2.33-2.24 (m, 1H), 1.87-1.84 (m, 1H), 1.32 (m, 9H). 19F NMR (376 MHz, DMSO-d6) δ -122.98 (s, IF), -141 .29 - - 141.36 (m, 2F), -154.71 - -154.78 (m, 2F). ESI+ MS: m/z 700.35 [M+H]+
Preparation of (4-((4-((3-chloro-4-fluorophenyl)amino)-7-(((S)-tetrahydrofuran-3- yl)oxy)quinazolin -6-yl)amino)-2, 3, 5, 6-tetrafluorophenyl)(imino)(methyl)-X6- sulfanone (Compound 3)
[00641] To a stirred solution of tert-butyl ((4-((4-((3-chloro-4-fhrorophenyl)amino)-7-(((S)- tetrahydrofuran-3-yl)oxy)quinazolin-6-yl)amino)-2,3,5,6-tetrafluorophenyl)(methyl)(oxo)-X6- sulfanylidene)carbamate (0.055 g, 0.078 mmol) in DCM (1 mL) was added 4NHC1 in Dioxane (0.5 mL) at O°C. The resulting mixture was stirred at room temperature for Ih. Upon completion, the reaction mixture was concentratedunder reduced pressure. The crudematerial was co-distilled with DCM (3 X 5mL). The obtained material was triturated with EtOAc (2 X 5 mL) to afford the title compound as brown solid (0.02 g, 0.033 mmol, 62% yield, HC1 salt). 1HNMR (400 MHz, DMSO-d6) δ 8.86 (s, lH), 8.78 (s, lH), 8.17 (s, 1H), 7.98- 7.96 (dd, .// =2.4 Hz, J2 = 6.8 Hz, 1H), 7.69-7.65 (m, 1H), 7.53 (t, J= 8.8 Hz, 1H), 7.38 (s, 1H), 5.30 (brs, 1H), 3.98- 3.79 (m, 6H), 3.36 (s, 3H). 19F NMR (376 MHz, DMSO-d6) δ -118.88 (s, IF), -140.68 - -140.77 (m, 2F), -147.68 - - 147.73 (m, 2F). ESLMS: m/z 600.3 [M+H]+. HPLC (Method I): RT = 5.63 min., 100% purity.
Scheme 6: Synthesis of Compound 207
Figure imgf000194_0001
Method 1:
General Procedure A-l
Preparation of (S)-6-chloro-7-(2-fluorophenyl)-4-(2-methyl-4-(2 , 3, 5, 6 -tetrafluor o-4-
(methylthio)phenyl)piperazin-l-yl)quinazoline
[00642] To a stirred solution of (4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (0.28mg, 1.02mmmol) in Dioxane (3mL) were added (S)-6-chloro-7-(2-fluorophenyl)-4-(2- methylpiperazin-l-yl)quinazoline (0.36 g, 1.02 mmol) and CS2CO3 (0.66 g, 2.04 mmol). The reaction mixture was purged with N2 for 15 minutes, followed by addition of Pd2dba3 (0.09 g, 0.10 mmol) and Xanthphos (0.059 g, O. lO mmol) atr.t. The reaction mixture was allowed to stir at 110 °C for 48h. After completion of reaction, the mixture was cooled to ambient temperature and diluted with water (40 mL). The resulting suspension was extracted with EtO Ac (2 x 40 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography, eluting with 25% EtO Ac in hexane to afford the title compound as a yellow solid (0.26 g, 0.36 mmol, 46% yield). 'HNMR (400 MHz, DMSO-d6) δ 8.70 (s, 1H), 8.12 (s, 1H), 7.86 (s, 1H), 7.58-7.55 (m, 1H), 7.52-7.48 (m, 1H), 7.41-7.36 (m, 2H), 4.80 (br s, 1H), 4.20 (d, J= 13.2 Hz, 2H), 3.81-3.78 (m, 2H), 3.57- 3.52 (m, 2H), 3.44 (br s, 1H), 2.45 (s, 3H), 1.51 (d, J = 6.8 Hz, 3H). ESI-MS: measured m/z 551.3 [M+H]+.
General Procedure B
Preparation of (S)-6-chloro-7-(2-fluorophenyl)-4-(2-methyl-4-(2,3,5,6-tetrafluoroM- (methylsulfonyl)phenyl)piperazin-l-yl)quinazoline
[00643] To a stirred solution of (S)-6-chloro-7-(2 -fhrorophenyl)-4-(2-methyl-4-(2, 3,5,6- tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)quinazoline (0.05 g, 0.09 mmol) in THF:MeOH:Water (0.8:0.2:0.2 mL) was added oxone (0.13 g, 0.45 mmol) at O °C. The resulting reaction mixture was stirred at room temperature for 16 h. After completion of reaction, the mixture was diluted with a saturated solution of NaHCO3 (30mL) and extracted with EtO Ac (3x1 OmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by Prep. TLC using (5% MeOH in DCM) to afford thetitle compound as a white solid (0.04 g, 0.06 mmol, 21 % yield). JH NMR (400 MHz, DMSO-tL) δ 8.72 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 7.60-7.55 (m, 1H), 7.52- 7.48 (m, 1H), 7.41-7.36 (m, 2H), 4.81-4.79 (m, 1H), 4.21 (d, J= 13.2 Hz, 1H), 3.82 (t, J= 11.6 Hz, 1H), 3.66-3.63 (m, 2H), 3.56.3.54 (m, 2H), 3.44 (s, 3H), 1.75 (d, J = 6.8 Hz, 3H). 19F NMR (376 MHz, DMSO-t/g) 6 -114.18 (s, IF), -140.59 - -140.65 (m, 2F), -150.40 - -150.46 (m, 2F). ESI-MS: measured m/z 583.3 [M+H]+, HPLC (Method I): RT = 6.43 min., 95.8% purity. Method 2:
Step-1: Synthesis of tert-butyl 4-(6-chloro-4-((2,3,5,6-tetrafluoro-4-(methylsulfonyl) phenyl)amino)quinolin-7 -yl)-5-methyl-l H-indazole-1 -carboxylate
[00644] To a stirred solution of 2,3,5,6-tetrafluoro-4-(methylsulfonyl)aniline (0.30g, 0.63mmol) (WH8) in THF (3mL) was added Cs2CO3 (0.62gm, 1.91mmol) at rt. The resulting reaction mixture was purged with N2 for 15 minutes followed by addition of Pd2dba3 (0.058g, 0.06mmol), xanthphos (0.036g, 0.06mmol) and tert-butyl 4-(4-bromo-6-chloroquinolin-7-yl)-5-methyl-lH- indazole-1 -carboxylate (0.18g, 0.76mmol) (INT6), at room temperature. The resulting reaction mixture was heated to 110°Cfor5h. After completion of reaction, the reaction mixture was filtered through celite bed, washed with ethyl acetate (50mL), concentrated under reduced pressure. The obtained crude material was purified by flash column chromatography, eluted with 39% ethyl acetate in hexane to afford title compound as off-white solid (0.17g, 0.26mmol, 42% yield). Tf NMR (400 MHz, DMSO-t/6) δ 12.12 (s, 1H), 8.53 (s, 1H), 8.09 (d, J= 8.8 Hz, 1H), 8.02 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.52 (s, 1H), 6.02 (d, J = 6.4 Hz, 1H), 3.48 (s, 3H), 2.22 (s, 3H), 1.67 (s, 9H). 19F NMR (400 MHz, DMSO-t/6) 6 -141 .30 -141.35 (2F), - 150.28_-150.33 (2F). LCMS: Method-UC05_FAR1, RT- 2.567 ESI-MS: measured m/z 635.4[M+1]+, 637.4[M+3]+.
Step-2: Synthesis of 6-chloro-7-(5-methyl-lH-indazol-4-yl)-N-(2,3,5,6-tetrafluoro-4-(methyl sulfonyl)phenyl)quinolin-4-amine
[00645] To a stirred solution of tert-butyl 4-(6-chloro-4-((2,3,5,6-tetrafluoro-4-(methylsulfonyl) phenyl)amino)quinolin-7-yl)-5 -methyl- 1 H-indazole-1 -carboxylate (0.08g, 0.12mmol) in 1,4- Dioxane (0.8mL) was added IN HC1 in Dioxane (1.3mL) at 0°C. The resulting reaction mixture was stirred at room temperature for 24h. After completion of reaction, the mixture was concentrated under reduced pressure. The obtained crude material was triturated with acetonitrile and ethyl acetate to afford title compound as yellow solid (0.035g, 0.06mmol, 52%). 'H NMR (400 MHz, MeOD) 6 9.00 (s, 1H), 8.77 (d, J = 6.8 Hz, 1H), 8.07 (s, 1H), 7.66-7.61 (m, 2H), 7.47 (d, J= 8.4 Hz, 1H), 7.11 (d, J= 6.4 Hz, 1H), 3.52 (s, 3H), 2.29 (s, 3H). 19F NMR (400 MHz, CDC13) δ -138.34 (2F), -143.23_-144.61 (2F). LCMS: Method-UC05_FAR1, RT- 1.900 ESI-MS: measured m/z 535.3 [M+l]+, 537.3 [M+3]+. HPLC: Method-HP07_TF ARI, RT- 5.653, 91.88%. Scheme 7: Synthesis of Compound 208 and Compound 209
Figure imgf000196_0001
Preparation of (S)-6-chloro-7-(2, 6-dimethylphenyl)-4-(2-methyl-4-(2, 3, 5, 6 -tetrafluor o-4- (methylthio) phenyl)piperazin-l -yl)quinoline
[00646] To a stirred solution of 6-chloro-7-(2,6-dimethylphenyl)-4-(2-methylpiperazin-l- yl)quinoline (0.16 g, 0.45 mmol) in 1,4-Dioxane (3 mL) were added cesium carbonate (0.44 g, 0.67 mmol) and (4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (0.18 g, 0.67 mmol) at room temperature. The resulting reaction mixture was purged with N2 for 15 minutes, followed by addition of Pd2(dba)s (0.041 g, 0.045 mmol) and xanthphos (0.026 g, 0.045 mmol) at room temperature. The resulting reaction mixture was stirred at 90°C for 16h. After completion of reaction, the mixture was cooled to ambient temperature and diluted with water (30 mL). The resulting suspension was extracted with EtOAc (3 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography, employing a mobile phase consisting of 10- 12% EtOAc in hexane, to afford the title compound as a yellow sticky solid (0.06 g, 0.12 mmol, 24% yield).1HNMR (400 MHz, DMSO-d6) δ 8.80 (d, J= 4.8 Hz, 1H), 8.27 (s, 1H), 7.79 (s, 1H), 7.26-7.18 (m, 4H), 3.89-3.59 (m, 6H), 3.05 (m, 1H), 2.46 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.08 (d, J= 6.0 Hz, 3H). ESI-MS: m/z 560.4 [M+H]+. Preparation of (S)-6-chloro-7-(2,6-dimethylphenyl)-4-(2-methyl-4-(2,3,5,6-tetrafluoroM- (methylsulfonyl) phenyl)piperazin-l-yl)quinoline
[00647] To a stirred solution of (S)-6-chloro-7-(2,6-dimethylphenyl)-4-(2-methyl-4-(2, 3,5,6- tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)quinoline (0.055 g, 0.09 mmol) in THF:MeOH:Water (8:1 :1, 1.4 mL) was added oxone (0.15 g, 0.49 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was diluted with saturated solution of NaHCCL (15 mL) and extracted with DCM (3 x 15 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography, eluting with 25-30% EtOAc in Hexane, to afford the title compound as a yellow solid (0.008 g, 0.013 mmol, 13% yield). 'HNMR (400 MHz, DMSO-d6) δ 8.81 (d, J = 4.8 Hz, 1H), 8.29 (s, 1H), 7.79 (s, 1H), 7.28-7.18 (m, 4H), 3.90-3.83 (m, 2H), 3.73-3.57 (m, 4H), 3.45 (s, 3H), 3.06 (m, 1H), 1.96 (s, 3H), 1 .95 (s, 3H), 1.06 (d, J= 6.0 Hz, 3H). 19F NMR (376 MHz, DMSO-d6) δ -140.59 - 140.63 (m, 2F), -150.20 - -150.26 (m, 2F). ESLMS: measured m/z 592.4 [M+H] +, HPLC (Method I) RT = 6.79 min., 95%, Chiral HPLC: RT = 4.50 min., 96.5%.
Preparation of 6-chloro-7-(2,6-dimethylphenyl)-4-((2S)-2-methyl-4-(2,3,5,6-tetrafluoroM- (methylsulfinyl)phenyl)piperazin-l-yl)quinoline
[00648] To a stirred solution of (S)-6-chloro-7-(2,6-dimethylphenyl)-4-(2-methyl-4-(2, 3,5,6- tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)quinoline (0.08 g, 0.14 mmol) in THF:MeOH:Water (8: 1 : 1, 2.7mL) was added Oxone (0.21 g, 0.71 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 h. Upon completion, the reaction mixture was diluted with aq. NaHCO3 (20 mL) and extracted with DCM (2 x 20 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The obtained crude material was purified by reverse phase column chromatography, eluting with 100% MeCN in water, to afford the title compound as an off-white solid (0.015 g, 0.026 mmol, 18%yield). 1HNMR (400 MHz, DMSO-d6) δ 8.80 (d, J= 5.2 Hz, 1H), 8.28 (s, 1H), 7.79 (s,lH), 7.26-7.18 (m, 4H), 3.89 (brs, 1H), 3.81- 3.78 (m, 1H), 3.66-3.57 (m, 4H), 3.17 (s, 3H), 3.04 (m, 1H), 1 .96 (d, J= 9.6 Hz, 6H), 1 .07 (d,J = 6.4 Hz, 3H). 19F NMR (376 MHz, DMSO- d6) 5 -142.57 - 142.62 (m, 2F), -149.87 - -149.95 (m, 2F). ESLMS: measured m/z 576.4 [M+H| +. HPLC (Method I) RT = 6.56 min., 100%.
Scheme 6: Synthesis ofCompound 210,Compound211,Compound 212,andCompound 213
Figure imgf000198_0001
Absolute stereochemistry is arbitrarily assigned
Preparation of 6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)-4-((S)-2-methyl-4-(2,3,5,6- tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)quinoline
[00649] To a stirred solution of 6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)-4-((S)-2- methylpiperazin-l-yl)quinoline (0.40 g, 0.99 mmol) in THF (20 mL) were added cesium carbonate (0.97 g, 2.97 mmol) and (4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (0.32 g, 1.19 mmol) at room temperature. The reaction mixture was purged with N2 for 15 minutes, followed by addition of Pd2(dba)s (0.091 g, 0.099 mmol) and xanthphos (0.057 g, 0.099 mmol) at room temperature. The resulting reaction mixture was stirred at 86°C for 32 h. The resulting suspension was cooled to ambient temperature, diluted with water (100 mL) and extracted with EtOAc (3 x lOOmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by silica gel column chromatography, eluting with 30% EtOAc in hexane, to afford the title compound as a yellow solid (0.18 g, 0.29 mmol, 17%). 'HNMR (400 MHz, DMSO-d6) δ 8.84 (d, J = 4.8 Hz, 1H), 8.08 (d, J=1 2Hz, 1H), 7.37-7.35 (m, 1H), 7.10-7.00(m, 3H), 3.90 (brs, 2H), 3.81 - 3.77 (m, 4H), 3.41- 3.37 (m, 3H), 3.03-3.08 (m, 1H), 2.49 (s, 3H), 1 .07 (d, .7=6, 4 Hz, 3H). 19FNMR(376 MHz, DMSO- d6) 5 -113.60 - -113.49 (m, IF), -118.29 - -118.18 (m, IF), -136.66 - -136. 16 (m, 2F), -150.33 - - 150.20(m, 2F). ESLMS: measured m/z 598.3 [M+H]+. Preparation of 2-(6-chloro-8-fluoro-4-((S)-2-niethyl-4-(2,3,5,6-tetrafluoro-4- (methylthio)phenyl) piperazin-1 -yl)quinolin- 7 -yl)-3 -fluorophenol
[00650] To the stirred solution of 6-chloro-8-fluoro-7-(2-fluoro-6-methoxyphenyl)-4-((S)-2- methyl-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)quinoline (0.30 g, 0.50 mmol) in DCM (3 mL) was added BBr3 (0. 18 g, 0.75 mmol) at 0°C. The resulting solution was stirred at room temperature for 3h. Upon completion, the mixture was diluted with a saturated solution of NaHCCf (50 mL) and extracted with DCM (3 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as an off-white solid (0.25 g, 0.43 mmol, 86% yield). 1HNMR (400 MHz, DMSO-t/g) 5 10.23 (brs, 1H), 8.84 (d, J=4.4Hz, 1H), 8.07 (s, lH), 7.36 (m, 2H), 6.87-6.80 (m, 2H), 3.90 (bra, 1H), 3.71 (brs, 1H), 3.57-3.50 (m, 4H), 3.06 (brs, 1H), 2.45 (s, 3H), 1 .06 (d, J= 5.6 Hz, 3H). 19F NMR (376 MHz, DMSO-d6) δ -113.63 - -113.67 (m, IF), -117.99 - -118.17 (m, IF), -136.57 - - 136.66(m, 2F), -150.25 - -150.33 (m, 2F). ESI-MS: measured m/z 584.3 [M+l]+, HPLC (Method I): RT1 6.95 min., 8.75%, RT2 7.14 min., 88.61% (indicates a mixture of atropisomers)
Preparation of 2-(6-chloro-8-fluoro-4-((S)-2-methyl-4-(2,3,5,6-tetrafluoroM- (methylsulfonyl) phenyl)piperazin-l-yl)quinolin-7-yl)-3-fluorophenol
[00651] 2 -(6 -chloro-8 -fluoro-4-((S)-2 -methyl-4 -(2, 3,5, 6-tetrafluoro-4 -(methyl sulfonyl) phenyl)piperazin-l-yl)quinolin-7-yl)-3-fluorophenol was prepared from 2-(6-chloro-8-fluoro-4- ((S)-2-methyl-4 -(2,3,5, 6-tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)quinolin-7-yl)-3- fluorophenol (0.050 g, 0.085 mmol) accordingto the protocol described in general procedure B and isolated as an white solid (0.060g, 0.097mmol, 38% yield) using reverse-phase chromatography (isocratic; 51% ACN in water). 1 H NMR (400 MHz, DMSO-tC) δ 10.27 (s, 1H), 8.84 (d, J= 4.8Hz, 1H), 8.09 (s, 1H), 7.36- 7.34 (m, 2H), 6.87- 6.79 (m, 2H), 3.91-3.83 (m, 2H), 3.70-3.58 (m, 4H), 3.50 (s, 3H), 3.06 - 3.03 (m, 1H), 1 .04 (d, J=6.0Hz, 3H). 19F NMR (376 MHz, DMSO-t/g) 6 -113.64 - -113.68 (m, IF), -117.99 - -118.17 (m, IF), -140.60 - -140.64 (m, 2F), - 150.24 - -150.29 (m, 2F). ESLMS: measured m/z 616.3 [M+l]+. HPLC (Method I): RT = 6.28 min, 92.5%, Chiral HPLC: RT = 4.98 min, 34.40% (FR-1), RT = 6.50 min., 43.1% (FR-2). (Indicates a mixture of atropisomers)
The atropisomers were separated using the chiral SFC conditions detailed in the following protocol:
Figure imgf000199_0001
Figure imgf000200_0001
2-(6-chloro-8-fluoro-4-((S)-2-methyl-4-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl) piperazin-l-yl)quinolin-7-yl)-3-fluorophenol
[00652] Isolatedasa white solid(0.007g, 0.011 mmol, 17% yield). JHNMR(400MHz,DMSO- tZ6) δ 10.99 (s, 1H), 8.83 (d, J = 4.8 Hz, lH), 8.07 (s, 1H), 7.34 (d,J = 4.8 Hz, 1H), 7.28 (brs, 1H), 6.81 (brs, 1H), 6.70 (brs, 1H), 3.58-3.91 (m, 6H), 3.45 (s, 3H), 3.06- 3.04 (brs, 1H), 1.03 (d, ,/=6.4Hz, 3H). 19F NMR (376 MHz, DMSO-d6) δ -113.89 (s, IF), -118.19 (s, IF), -140.60 - - 140.65 (m, 2F), -150.25 --150.29 (m,2F). ESI-MS: measured m/z616.3 [M+H]+. HPLC (Method I): RT = 6.23 min., 100%. Chiral HPLC: RT = 4.85 min, 95.4%.
2-(6-chloro-8-fluoro-4-((S)-2-methyl-4-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl) piperazin-l-yl)quinolin-7-yl)-3-fluorophenol
[00653] Isolated as an off-white solid (0.007 g, 0.011 mmol, 17% yield). 1HNMR (400 MHz, DMSO-fiQ 5 10.99 (s, 1H), 8.83 (d, J = 4.8Hz, 1H), 8.07 (s, 1H), 7.34 (d, J= 4.8Hz, 1H), 7.28 (brs, 1H), 6.81 (brs, 1H), 6.70 (brs, 1H), 3.58 - 3.91 (m, 6H), 3.45 (s, 3H), 3.06- 3.04 (brs, 1H), 1.03 (d, J= 6.4Hz, 3H). 19F NMR (376 MHz, DMSO-d6) δ -113.89 (s, IF), -118.19 (s, IF), - 140.60 - 140.65(m, 2F), -150.25 - -150.29 (m, 2F). ESI-MS: measured m/z 616.3 [M+H]+. HPLC (Method I): RT = 6.26 min., 98.6%. Chiral HPLC: RT = 6.36 min., 97.6%.
General Procedure C
Synthesis of 7-(8-chloronaphthalen-l-yl)-2-((l-methylpyrrolidin-2-yl)methoxy)-4-(4- (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)-5,6,7,8-tetrahydropyrido[3,4- djpyrimidine (Compound 214)
Figure imgf000201_0001
[00654] To a flask charged with (S)-7-(8-chloronaphthalen-l-yl)-2-((l-methylpyrrolidin-2- yl)methoxy)-4-(piperazin-l-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine (0.12 g, 0.24 mmol, 1 eq.), methyl(perfluorophenyl)sulfane (0.052 g, 0.24 mmol, 1 eq.), and DMSO (2 mL) was added potassium carbonate (0.037 g, 0.27 mmol, 1.1 eq.) atrt. The solution was stirred and heated at 70 °C for 19 hrs. Once complete, the flask was cooled to rt and the mixture was quenched with water, and the aqueous phase extracted with DCM. The organic lay er was washed three times with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by reverse-phase flash column chromatography, eluting 5:95 ACN/Milli- Q water with 0.1% formic acid, to afford the anticipated product as an off-white solid (10 mg, 0.013 mmol, 5% yield). Tf NMR (400 MHz, CDC13) δ 7.77 (d, J = 7.2 Hz, 1H), 7.63 (t, J = 6 9 Hz, 1H), 7.54 (d, J= 6.8 Hz, 1H), 7.46 (t, J= 7.5 Hz, 1H), 7.36 (d, J= 7.4 Hz, 1H), 7.35 - 7.22 (m, lH), 4.45 (dd, J = 17.7, 6.3 Hz, 2H), 4.18 (ddd, J = 13.9, 10.6, 6.7Hz, 1H), 3.89 (dd, J= 17.6, 5.3 Hz, 1H), 3.72 (dd, J= 13.4, 6.3 Hz, 2H), 3.58 (t, J = 7.2 Hz, 3H), 3.41 (dt, J= 32.5, 10.8 Hz, 5H), 3.14 (p, J= 6.6 Hz, 3H), 2.74 (d, J= 8.9 Hz, 1H), 2.52 (d, J= 5.2 Hz, 3H), 2.48 (d, J= 5.2 Hz, 2H), 2.33 (t, J=7.8 Hz, 1H), 2.15 - 2.04 (m, 2H), 1.87 (s, 1H), 1.84 - 1.75 (m, 2H). 19FNMR (376 MHz, CDC13) δ -135.62 - -136.70 (m, 2F), -150.35 (dd, J = 22.8, 8.4 Hz, 2F). ESI-MS: measured m/z 687.3 [M+H]+. HPLC (Method III): RT = 13.57 min., 93%.
Synthesis of (S)-7-(8-chloronaphthalen-l-yl)-2-((l-methylpyrrolidin-2-yl)methoxy)-4-(4- (2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)piperazin-l-yl)-5,6,7,8-tetrahydropyrido[3,4- djpyrimidine (Compound 215)
Figure imgf000201_0002
[00655] (S)-7-(8-chloronaphthalen-l-yl)-2-((l-methylpyrrolidin-2-yl)methoxy)-4-(4-(2,3,5,6- tetrafluoro-4-(methylsulfonyl)phenyl)piperazin-l-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine was prepared from (S)-7-(8-chloronaphthalen-l-yl)-2-((l-methylpyrrolidin-2-yl)methoxy)-4- (piperazin-l-yl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine (0.15 g, 0.304 mmol, 1 eq.) and l,2,3,4,5-pentafluoro-6-(methylsulfonyl)benzene (0.075 g, 0.304 mmol, 1.0 eq.) according to the protocol described in general procedure C and isolated as an off-white solid (0.018 g, 0.024 mmol, 8 % yield). 'HNMR (400 MHz, CDC13) δ 7.83 - 7.74 (m, 1H), 7.64 (dd, J= 8.3, 4.0 Hz, 1H), 7.60 - 7.51 (m, 1H), 7.47 (td, J = 7.9, 3.7 Hz, 1H), 7.36 (td,J = 7.9, 3.6 Hz, 1H), 7.25 (d, J= 7.4 Hz, 1H), 4.60 - 4.37 (m, 1H), 4.32 - 4.14 (m, 1H), 3.89 (d, J = 17.8 Hz, 1H), 3.73 (dd, J= 10.7, 5.8 Hz, 2H), 3.65 - 3.42 (m, 5H), 3.32 (d, J = 3.5 Hz, 2H), 3. 16 (d, J = 9.0 Hz, 2H), 2.78 (s, 1H), 2.54 (d, J= 3.7 Hz, 3H), 2.30 (t, J= 7.5 Hz, 1H), 2.10 (dq, J= 9.4, 4.6, 4.0 Hz, 3H), 1.95 - 1.73 (m, 2H), 1.28 (d, J= 3.6 Hz, 3H), 1.04 - 0.76 (m, 2H). 19F NMR (470 MHz, CDC13) 6 - 138.97 (d, J= 14.2 Hz, 2F), -149.47 (d, J= 17.2 Hz, 2F). ESI+MS: measured m/z 719.2 [M+H]+. HPLC (Method III): RT = 9.55 min., 97.5%.
General Procedure D:
Synthesis of 7-(8-chloronaphthalen-l-yl)-4-(4-(2,3,5,6-tetrafluoro-4- (methylsulfonyl)phenyl)piperazin-l-yl)-5,6,7,8-tetrahydropyrido [3, 4-d] pyrimidine (Compound 216)
Figure imgf000202_0001
[00656] To a stirred solution of 7-(8-chloranyl-l-naphthyl)-4-piperazin-l-yl-6,8-dihydro-5H- pyrido[3,4-d]pyrimidine (140 mg, 368.53 μmol) in THF (1 mL) at -78°C was added n- butyllithium (2.5 M, 162.15 pL) dropwise under a positive pressure of N2. The resulting solution was stirred for 20 min before being added to an ice cold (0°C) solution of 1,2, 3, 4, 5- pentakis(fluoranyl)-6-methylsulfonyl-benzene (136.07 mg, 552.80 μmol) in THF (1 mL) via cannula. The reaction was permitted to warm gradually to room temperature over 5 hours. Once the reaction was deemed complete, water was added to quench the mixture. The resultingbiphasic mixture was extracted with EtOAc (3x). The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by normal phase FCC (40%-l 00% EtOAc in Hexanes) to afford the desired product as an off-white solid (24 mg, 38.41 pmol, 10% yield). 1HNMR (400 MHz, CDC13) δ 8.63 (s, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.27 - 7.25 (m, 1H), 4.51 (d, J = 17.7 Hz, 1H), 3.93 (d, J = 17.7 Hz, 1H), 3.73 - 3.67 (m, 2H), 3.60 - 3.48 (m, 7H), 3.30 (s, 3H), 3.24 - 3.13 (m, 2H), 2.64 (d, J = 14.8 Hz, 1H) . 19F NMR (376 MHz, CDC13) 6 -138.89 - -139.01 (m, 2F), -149.41 - -149.45 (m, 2F). ESI+MS: measured m/z 606.1 [M+H]+. HPLC (Method III): RT = 11.12 min., 97.4%.
Synthesis of 7-(naphthalen-l-yl)-4-(4-(2,3,5,6-tetrafluoro-4- (methylsulfonyl)phenyl)piperazin-l-yl)-5,6,7,8-tetrahydropyrido [3, 4-d] pyrimidine (Compound 217)
Figure imgf000203_0001
[00657] 7-(naphthalen-l-yl)-4-(4-(2,3,5,6-tetrafhroro-4-(methylsulfonyl)phenyl)piperazin-l-yl)-
5.6.7.8-tetrahydropyrido[3,4-d]pyrimidine was prepared from 7-(l-naphthyl)-4-piperazin-l-yl-
6.8-dihydro-5H-pyrido[3,4-d]pyrimidine (130 mg, 376.33 μmol) and 1, 2, 3,4,5- pentakis(fluoranyl)-6-methylsulfonyl-benzene (138.95 mg, 564.50 μmol) according to the protocol describedin general procedure D and isolated as an off-white solid (48.5 mg, 84.85 pmol, 23% yield). 'HNMR (400 MHz, CDC13) δ 8.66 (s, 1H), 8.23 - 8.21 (m, 1H), 7.88 - 7.86 (m, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.53 - 7.48 (m, 2H), 7.45 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 7.4 Hz, 1H), 4.36 (s, 2H), 3.66 - 3.57 (m, 8H), 3.41 (br, 2H), 3.30 (s, 3H), 2.97 (br, 2H). 19F NMR (376 MHz, CDC13) δ -138.87 - -138.96 (m, 2F), -149.41 - -149.45 (m, 2F). ESI+MS: measured m/z 572.1 [M+H]+. HPLC (Method III): RT = 10.8 min., 96.9%.
Synthesis of 7-(8-chloranyl-l-naphthyl)-4-[4-[2,3,5,6-tetrakis(fluoranyl)-4-methylsulfinyl- phenyl]piperazin-l-yl]-6,8-dihydro-5H-pyrido[3,4-d]pyrimidine (Compound 218)
Figure imgf000204_0001
[00658] To a stirred solution of 7-(8-chloranyl-l-naphthyl)-4-[4-[2,3,5,6-tetrakis(fluoranyl)-4- methylsulfanyl-phenyl]piperazin-l-yl]-6,8-dihydro-5H-pyrido[3,4-d]pyrimidine (27.4 mg, 47.73 μmol) in a mixture of THF/Water (1 mL) (1/1 ratio) was added Oxone (22.01 mg, 71.60 μmol) at room temperature. The mixture was heated at 60°C for 12 hours. Once complete, the reaction was cooled to r.t. and the mixture diluted with water andEtOAc. The organic phase was removed, and the remaining aqueous phase extracted a further 3x with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified via reverse phase column chromatography (ACN/H20/0.1% Formic acid, gradient from 10-100% ACN in H2O) to afford the anticipated product as an off- white solid (9 mg, 14.49 pmol, 30% yield). 'HNMR (400 MHz, CDC13) δ 8.63 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.27 - 7.25 (m, 2H), 4.51 (d, J = 17.7 Hz, 1H), 3.94 (d, J = 17.6 Hz, 1H), 3.72 - 3.68 (m, 3H), 3.61 - 3.45 (m, 8H), 3.30 - 3.16 (m, 1H), 3.14 (s, 3H). 19F NMR (376 MHz, CDC13) 5 -141.07 - -141.15 (m, 2F), -149.22 - -149.26 (m, 2F). ESI+MS: measured m/z 590.1 [M+H]+. HPLC (Method III): RT = 9.84 min., 95.1%.
Synthesis of Compound 219 and Compound 220
Figure imgf000204_0002
Preparation of l-(6-chloro-7-(2, 6-dimethylphenyl)quinolin-4-yl)-N-(2, 3, 5, 6 -tetrafluor o-4-
(methylthio)phenyl)azetidin-3-amine
[00659] l-(6-chloro-7-(2,6-dimethylphenyl)quinolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-
(methylthio)phenyl)azetidin-3 -amine was prepared from l-(6-chloro-7-(2,6- dimethylphenyl)quinolin-4-yl)azetidin-3-amine (0.37 g, 1.90 mmol) according to the protocol described in general procedure A-l and isolated as a yellow solid (0.2 g, 0.37 mmol, 34% yield). JH NMR (400 MHz, DMSO-tL) δ 8.47 (d, J=5.2Hz, 1H), 8.12 (s, 1H), 7.62 (s, 1H), 7.26- 7.22 (m, 1H), 7.19- 7.17 (m, 2H), 6.86 (brs, 1H), 6.41 (d, J=5.2Hz, 1H), 4.73 (d, J=4.4Hz, 3H), 4.45 (d, ./=4, 8 Hz, 2H), 2.37 (s, 3 H), 1.95 (s, 6H). 19FNMR(376 MHz, DMSO-d6) 6 -137.39 - -137.49 (m, 2F), -159.14 - -159.24 (m, 2F). ESI-MS: measured m/z 532.4 [M+H]+.
General Procedure E:
Preparation of l-(6-chloro-7-(2,6-dimethylphenyl)quinolin-4-yl)-N-(2,3,5,6-tetrafluoroM- (methylsulfonyl)phenyl)azetidin-3-amine
[00660] To a stirred solution of l-(6-chloro-7-(2,6-dimethylphenyl)quinolin-4-yl)-N-(2,3,5,6- tetrafluoro-4-(methylthio)phenyl)azeti din-3 -amine (0.15 g, 0.28 mmol) in DCM (1.5 mL) was added oxone (0.43 g, 1.41 mmol) at 0°C. The resulting mixture was stirred at room temperature for 16 h. Another portion of oxone (0.43 g, 1.41 mmol) was added at room temperature and the reaction allowed to stir for further 22 h. The reaction mixture was diluted with aqueous NaHCO3, extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by Prep. TLC using 0.3% MeOH in DCM with ammonia to afford l-(6-chloro-7-(2,6- dimethylphenyl)quinolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)azetidin-3- amine as off white solid (0.040 g, 0.071 mmol, 19% yield). 1HNMR (400 MHz, DMSO-tL) 6 8.48 (d, J=5.2Hz, lH), 8.11 (s, lH), 7.63 (s, 1H), 7.57- 7.55 (m, 1H), 7.26- 7.22 (m, lH), 7.18 (d, J=7.6Hz, 2H), 6.42 (d, .7=5, 6 Hz, 1H), 4.80 (brs, 1H), 4.73 (t, J=8.4Hz, 2H), 4.52- 4.49 (m, 2H), 3.39 (s, 3H) 1.95 (s, 6H). 19FNMR (400 MHz, DMSO-d6) 6 -141.66 - -136.71 (m, 2F), -160.02 - -160.07 (m,2F). ESI-MS: measured m/z 564.3 [M+H]+. HPLC (Method I): RT =6.39 min, 99.2% purity.
Preparation of l-(6-chloro-7-(2,6-dimethylphenyl)quinolin-4-yl)-N-(2,3,5,6-tetrafluoroM- (methylsulfinyl) phenyl)azetidin-3-aminephenyl)pyrimidine-2,4-diamine
[00661] l-(6-chloro-7-(2,6-dimethylphenyl)quinolin-4-yl)-N-(2,3,5,6-tetrafluoro-4- (methylsulfinyl) phenyl)azetidin-3-aminephenyl)pyrimidine-2,4-diamine was isolated from the same mixture giving rise to 1 -(6-chloro-7-(2,6-dimethylphenyl)quinolin-4-yl)-N-(2, 3,5,6- tetrafluoro-4-(methylsulfinyl) pheny l)azeti din-3 -aminephenyl)pyrimidine-2,4-diamine in 15% yield (0.025 g, 0.045 mmol). 'HNMR (400 MHz, DMSO-d6) 6 8.47 (d, J= 5.2Hz, 1H), 8.11 (s, 1H), 7.62 (s, 1H), 7.28- 7.22 (m, 2H), 7.18 (d, J=7.2Hz, 2H), 6.41 (d, J=5.2Hz, 1H), 4.74- 4.71 (m, 3H), 4.49 - 4.48 (m, 2H), 3.12 (s, 3H) 1.95 (s, 6H). 19FNMR (400 MHz, DMSO-d6) 6 -143.46 - -143.50 (m, 2F), -159.58 - -159.63 (m, 2F). ESI-MS: measured m/z 548.4 [M+H]+. HPLC (Method I): RT = 6.23 min., 97.1% purity.
Synthesis of Compound 221 and Compound 222
Figure imgf000206_0001
Preparation of l-( 6-chloro-7 -(2-jluorophenyl)quinazolin-4-yl)-N-(2 , 3, 5, 6 -tetrafluor o-4-
(methylthio)phenyl)azetidin-3-amine
[00662] l-(6-chloro-7-(2-fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-
(methylthio)phenyl)azetidin-3 -amine was prepared from l-(6-chloro-7-(2- fluorophenyl)quinazolin-4-yl)azetidin-3 -amine (0.3 g, 0.91 mmol) according to the protocol described in general procedure A-l and isolated as a white solid (0.23 g, 0.44 mmol, 38% yield). JH NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.05 (s, 1H), 7.74 (s, 1H), 7.59 - 7.53 (m, 1H), 7.50 - 7.46 (m, 1H), 7.40 - 7.34 (m, 2H), 6.90 (d, J= 6 Hz, 1H), 4.85 (br s, 2H), 4.73, (br s, 1H), 4.61 (s, 2H), 2.37 (s, 3H). 19H NMR (376 MHz, DMSO-d6) 6 -114.29 (s, IF), -137.39 - -137.44 (m, 2F), -159.15 - -159.20 (m, 2F). ESI-MS: measured m/z 523.3 [M+H]+.
Preparation of l-(6-chloro-7-(2-fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoroM- (methylsulfonyl)phenyl)azetidin-3-amine
[00663] l-(6-chloro-7-(2-fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-
(methylsulfonyl)phenyl)azeti din-3 -amine was prepared from l-(6-chloro-7-(2- fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)azetidin-3-amine (0.028 g, 0.05 mmol) accordingto the protocol described in general procedureE and isolated as an white solid (0.02 g, 0.03 mmol, 10% yield). 'HNMR (400 MHz, DMSO-d6) 6 8.54 (s, 1H), 8.04 (s, 1H), 7.75 (s, 1H), 7.59-7.55 (m, 2H), 7.48-7.46 (m, 1H), 7.40-7.34 (m, 2H), 4.83 (br s, 3H), 4.66 (s, 2H) 3.39 (s, 3H). 19F NMR (376 MHz, DMSO-d6) 6 -114.30 (s, IF), -141.62 - - 141.67 (m, 2F), -159.98 --160.03 (m, 2F). ESI-MS: measured m/z 555.3 [M+H]+. HPLC (Method I): RT = 5.98 min., 96.8% purity.
Preparation of l-(6-chloro-7-(2-fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoroM- (methylsulfinyl) phenyl)azetidin-3-amine
[00664] l-(6-chloro-7-(2-fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-
(methylsulfinyl) phenyl)azetidin-3-amine was isolated from the same mixture l-(6-chloro-7-(2- fluorophenyl)quinazolin-4-yl)-N-(2,3,5,6-tetrafluoro-4-(methylsulfmyl) phenyl)azetidin-3-amine in 6% yield (0.013 g, 0.03 mmol). 'HNMR (400 MHz, DMSO-d6) 6 8.54 (s, 1H), 8.05 (s, 1H), 7.75 (s, 1H), 7.75-7.46 (m, 2H), 7.40-7.35 (m, 2H), 7.34 (br s, 1H), 4.86 (br s, 3H), 4.66 (br s, 1H), 3.13 (s, 3H). 19F NMR (400 MHz, DMSO-d6) 6 -114.30 (s, IF), -143.43 - -143.47 (m, 2F), - 159.54 - -159.59 (m, 2F). ESI-MS: measured m/z 539.3 [M+H]+, HPLC (Method i): RT = 5.78 min., 99.5%.
Preparation of (S)-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-(2-methyl-4-(2,3,5,6- tetrafluoro-4-(methylsulfonyl)phenyl)piperazin-l-yl)pyrido[2,3-d]pyrimidin-2(lH)-one (Compound 223)
[00665] (S)-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-(2-methyl-4-(2,3,5,6-tetrafluoro-4-
(methylsulfonyl)phenyl)piperazin-l-yl)pyrido[2,3-d]pyrimidin-2(lH)-one was prepared using similar protocols as described herein for Compounds 1-16 (m/z 657.4 [M+H]+).
Synthesis of 7-chloro-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-((l-(2, 3,5,6- tetrafluoro-4-(methylsulfinyl)phenyl)azetidin-3-yl)amino)pyrido [2,3-d]pyrimidin-2(lH)- one (Compound 230)
Figure imgf000207_0001
Step-1: Synthesis of tert-butyl (l-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)azetidin-3- yl)carbamate
[00666] To a stirred solution of te/7-butyl azetidin-3-ylcarbamate (0.97g, 5.67mmol)in anhydrous THF (13mL) were added CS2CO3 (4.6g, 14.18mmol) and (4-bromo-2, 3,5,6- tetrafluorophenyl)(methyl)sulfane (WO2022106897 A2) (1.3g, 4.72mmol) at room temperature. The reaction mixture was purged with N2 for 15 min followed by addition of Pd2(dba)3 (0.43g 0.47mmol) and xantphos (0.27g, 0.47mmol) at room temperature. The resulting reaction mixture was stirred at 80°C for 6h. The reaction mixture was cooled to room temperature, diluted with water (100mL) and extracted with ethyl acetate (3 x 80mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by silica gel column chromatography, eluted with 23-25% ethyl acetate in n- hexane to afford the title compound as ayellow solid (1.7g, 4.64mmol, 90%yield). 'H NMR (400 MHz, DMSO-t/6) δ 7.46 (d, J= 1 .6 Hz, 1H), 4.43-4.42 (m, 3H), 4.07 (br s, 2H), 2.34 (s, 3H), 1.39 (s, 9H). 19F NMR (400 MHz, DMSO-tC) 6 -137.50 -137.71 (2F), -162.25_-162.36 (2F).
Step-2: Synthesis of tert-butyl (l-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)azetidin-3- yl)carbamate
[00667] To a stirred solution of tert-butyl (l-(2,3,5,6-tetrafhroro-4-(methylthio)phenyl)azetidin- 3-yl)carbamate (1 .7g, 4.6mmol) in dichloromethane (15mL) was added TFA (17ml) at 0°C. The reaction mixture was allowed to stirred at 0°C for 1 h. The reaction mixture was concentratedunder reduced pressure to afford the title compound as a yellow liquid. (1 ,7g, 7.5mmol, Quantitative). JH NMR (400 MHz, DMSO-t/6) 4.60-4.51 (m, 1 H), 4.19-4.16 (m, 1 H), 3.66- 3.51 (m, 2H), 3.48- 3.33 (m, 1H), 2.34 (s, 3H). 19F NMR (400 MHz, DMSO-t/6) 6 137.31 -137.84 (2F), -158.47_- 158.75 (2F).
Step-3: Synthesis of 7-chloro-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-((l-(2, 3,5,6- tetrafluoro-4-(methylthio) phenyl)azetidin-3-yl)amino)pyrido[2,3-d]pyrimidin-2(lH)-one
[00668] To a stirred solution of l-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)azetidin-3-amine (1.7g, 6.39mmol) in MDC (17mL) was added TEA (1 ,9g, 19.1 mmol). The reaction mixture was allowed to stirred at room temperature for 1 h followed by addition of 4, 7-dichloro-6-fluoro-l -(2- isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(lH)-one (2.8, 7.66mmol) at room temperature for 4h. The reaction was diluted with water (1 OOmL) and extracted with ethyl acetate (3xl00mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by silica gel column chromatography, eluted with 90% ethyl acetate in w-hexane to afford the title compound as a yellow solid (0.23g, 0.38mmol, 9% yield). 'HNMR (400 MHz, DMSO-t/6) 9.30 (d, J= 5.6 Hz, 1H), 8.87 (d, J= 8.8 Hz ,1H), 8.47 (d, J= 4.8 Hz, 1H), 7.26 (d, J= 4.8 Hz, 1H), 4.98-5.10 (m, 1H), 4.70-4.68 (m, 2H), 4.39-4.37 (m, 2H), 2.45-2.62 (m, 1H), 2.49 (s, 3H) 1 .91 (s, 3H), 1 .02 (d, J= 6.8 Hz, 3H), 0.98 (d, J= 6.8 Hz, 3H). 19F NMR (400 MHz, DMSO-t/6) -128.11 -128.14 (IF), -137.33 -137.55 (2F), 162.23_-162.35 (2F).
Step-4: Synthesis of 7-chloro-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-((l-(2, 3,5,6- tetrafluoro-4-(methylsulfinyl) phenyl)azetidin-3-yl)amino)pyrido[2,3-d]pyrimidin-2(lH)- one
[00669] To a stirred solution of 7-chloro-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-((l- (2,3,5,6-tetrafhroro-4-(methylthio)phenyl)azetidin-3 -yl)amino)pyrido[2,3-d]pyrimidin-2(lH)- one (0.05g, 0.08mmol) in MDC (5mL) was added m-CPBA (0.021g, 0.12mmol) at 0°C. The reaction mixture was allowed to stirred at room temperature for Ih. The reaction mixture was quenched with sat. solution of NaHCO3 (80mL) extracted with ethyl acetate (3x60mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by reverse phase column chromatography, eluted with 40-45% acetonitrile in water to afford thetitle compound as an off-white solid (0.04g 0.06mmol, 19% yield). 'HNMR (400 MHz, DMSO-t/6) 9.37 (s, IH), 8.88 (d, J= 8.8 Hz ,lH), 8.46 (d, .7= 4.8 Hz, IH), 7.25 (d, J = 4.0 Hz, IH), 5.00 (br s, IH), 4.77 (br s, 2H), 4.46 (br s, 2H), 3.12 (s, 3H), 2.60.2.70 (m, 1H) 1.93 (s, 3H), 1.06 (d, J = 6.8 Hz, 3H), 0.98 (d, J= 6.4 Hz, 3H). 19F NMR (400 MHz, DMSO-t/6) -128.14 (IF), -143.57 -143.61 (IF), 163.07 -163.12 (IF). LCMS: Method-UC05_FAR1 RT- 1.850 ESI-MS: measured m/z 613.4 [M+l]+. HPLC: Method- HP07-TFARl .amx, RT 5.492, 97.27%.
Synthesis of 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-l-(2-isopropyl-4-methylpyridin-3-yl)-4- ((S)-2-methyl-4-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)piperazin-l-yl)pyrido[2,3- d]pyrimidin-2(lH)-one (Compound 236 and Compound 235)
Figure imgf000209_0001
Step-IA: Synthesis of tert-butyl (S)-2-methyl-4-(2,3,5,6-tetrafluoro-4- (methylthio)phenyl)piperazine-l-carboxylate
[00670] To a stirred solution of tert-butyl(S)-2-methylpiperazine-l-carboxylate(2.0g, 9.98mmol) in 1,4-dioxane (20mL) were added (4-bromo-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (WO2022106897 A2) (3.0g, 10.98mmol) and Cs2CO3(9.73g, 29.95mmol) at room temperature. The reaction mixture was purged with nitrogen for 15 minutes followed by addition of xantphos (1.15g, 1.99mmol) and Pd2(dba)s (0.91g, 0.99mmol). The resulting reaction mixture was stirred at 90°C for 4h. The reaction mixture was cooled to room temperature and filtered through celite bed. The celite bed was washed with dichloromethane (1 OOmL). The filtrate was combined and evaporated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluted with 10% ethyl acetate in hexane to afford the title compound as a white solid (1.5g, 3.80mmol, 38%yield). LCMS: (ACQUITY PD A and QDA detector, UC05_FARl): m/z 295.3 (M-100)+ (ESI +ve), RT = 3.07 min, 62.4%, 200 - 400nm. Step-2A: Synthesis of N2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)pyridine-2,5-diamine [00671] To a stirred solution of tert-butyl (S)-2-methyl-4-(2,3,5,6-tetrafluoro4- (methylthio)phenyl)piperazine-l-carboxylate amine (1.5g, 3.80mmol) in 1,4-dioxane (15. OmL) was added 4M HC1 in 1,4-dioxane at 0°C. The resulting reaction mixture was stirred at room temperature for 2h. The reaction mixture was concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated using diethyl ether to afford the title compound as a brown solid (1.2g, 4.07mmol, quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC05_FARl): m/z 295.3 (M+H)+ (ESI +ve), RT = 1.59 min, 94.5%, 200 - 400nm.
Step-3A: Synthesis of (S)-7-chloro-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-(2- methyl-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)pyrido [2,3-d]pyrimidin- 2(lH)-one
[00672] To a stirred solution of (S)-3 -methyl- 1 -(2,3,5, 6 -tetraflu oro4-
(methylthio)phenyl)piperazine (1.2g, 4.07mmol) in THF (12mL) was added TEA (1.23g 12.23mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 Ominutes followed by addition of 4, 7-dichloro-6-fluoro-l-(2 -isopropyl -4-methylpyridin-3-yl) pyrido [2,3 -d] pyrimidin-2(lH)-one (1 ,79g, 4.89mmol). The resulting reaction mixture was stirred at room temperature for 2h. The reaction mixture was diluted with water (1 OOmL) and extracted with ethyl acetate (2 x 75mL). The combined organic layer was dried and concentrated under reduced pressure to obtain a crude residue. The obtained crude material was purified by flash column chromatography, eluted with 35% ethyl acetate in hexane to afford the title compound as a yellow solid (1.2g, 1.92mmol, 52% yield). LCMS: (ACQUITY PDA and QDA detector, UC05 FAR1): m/z 625.4 (M+H)+ (ESI +ve), RT = 2.67 min, 93.4%, 200 - 400nm. 'H NMR (400 MHz, DMSO) δ 8.49 (d, J = 4.8 Hz, 1H), 8.42 (t,J= 8.0 Hz, 1H), 7.29-7.26 (m, 1H), 4.93 (br s, 1H), 4.35-4.20 (m, 1H), 3.80-3.70 (m, 1H), 3.53-3.41 (m, 3H), 2.52-2.51 (m, 2H), 2.45 (s, 3H), 1.95 (d, J = 3.6 Hz, 3H), 1.52 (t, J= 6.4 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.8 Hz, 3H). 19F NMR (400 MHz, DMSO) δ -127.67 (d, J= 18.6 Hz, IF), -136.55 (d, J = 25.2 Hz, IF), - 150.32 (d, J = 25.6 Hz, IF).
Step-1: Synthesis of 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-l-(2-isopropyl-4- methylpyridin-3-yl)-4-((S)-2-methyl-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)piperazin- l-yl)pyrid°[2,3-d]pyrimidin-2(lH)-one
[00673] To a stirred solution of (S)-7-chloro-6-fluoro-l-(2-isopropyl-4-methylpyridin-3-yl)-4-(2- methyl-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)piperazin-l-yl)pyrido[2,3-d]pyrimidin- 2(lH)-one (0.4g, 0.64mmol) in l,4-dioxane:water (3:1, 12mL) were added (2-fluoro-6- hydroxyphenyl) boronic acid (0.199g, 1.28mmol) and Na2CO3 (0.204g, 1.92mmol) at room temperature. The resulting reaction mixture was purged with N2 for 15 minutes followed by addition of Pd(dppf)Cl2. dichloromethane complex (0.053g, 0.064mmol). The reaction mixture was heated at 100°C for Ih under microwave irradiation. After completion of the reaction, The reaction mixture was diluted with water (100mL) and extracted with dichloromethane (3x5 OmL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by revers phase chromatography, product eluted with 58-60% acetonitrile in water to afford title compound as a white solid (0.4g, 0.57mmol, 44% yield). 1HNMR (400 MHz, DMSO) δ 10.22 (s, IH), 8.34-8.28 (m, IH), 7.28-7. 17 (m, IH), 7.04 (d, J=4 Hz, IH), 6.96 (td, J1 = 2.0 Hz, J2 = 8.4 Hz, IH), 6.86- 6.85 (m, 2H), 4.82 (brs, IH), 4.29-4.17 (m, IH), 3.84-3.74 (m, IH), 3.57-3.37 (m, 4H), 2.45 (s, 3H), 1.81 (d, J= 0.8Hz, 3H), 1.54-1.48 (m, 4H), 1 .04 (d, 22.4Hz, 3H), 0.75-0.73 (m, 3H). 19F NMR (400MHz, DMSO) δ-111 .22 -111.23 (IF), -115.61 -115.63 (IF), -128.62 (IF), -136.55 - 136.64 (IF), -145.81 -145.93 (IF), -150.40_-150.48(lF). LCMS: UCO5_FRA1, RT-2.61 & 2.77 ESI-MS: measured m/z 701.4 [M+l] +. (duplication of peak observed due to mixture of atropisomers).
Step-2: Synthesis of 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-l-(2-isopropyl-4- methylpyridin-3-yl)-4-((S)-2-methyl-4-(2,3,5,6-tetrafluoro-4-
(methylsulfonyl)phenyl)piperazin-l-yl)pyrido [2,3-d]pyrimidin-2(lH)-one
[00674] To a stirred solution of 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-l-(2-isopropyl-4- methylpyridin-3-yl)-4-((S)-2-methyl-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)piperazin-l- yl)pyrido[2,3-d]pyrimidin-2(lH)-one (0.06g, 0.08mmol) in methanol :THF: Water (1.2:9.61.2 mL) was added Oxone (0.052g, 0.17mmol) at room temperature. The resulting reaction mixture was stirred at room temperature for 8h. The reaction mixture was enriched with Oxone (0.052g 0.17mmol) at defined interval of 8h thrice. The mixture was diluted with saturated aqueous solution of NaHC03 (100mL) and extracted with ethyl acetate (2x50mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting crude was purified by revers phase column chromatography, eluted with 47-50% acetonitrile in water to afford the title compound as a white solid (0.04g, 0.054mmol, 16% yield). JH NMR (400 MHz, DMSO-d6) δ 8.40-8.32 (m, 1H), 8.28 (q, J= 4.8 Hz, 1H), 7.20
Figure imgf000211_0001
8.4 Hz, J2= 15.2 Hz, IH), 7.04 (d,J = 4.8 Hz, IH), 6.99-6.94 (dt, Ji =2.4 Hz, J2 = 8.8 Hz, 1H), 6.86- 6.79 (m, 1H), 4.83 (br s, 1H), 4.26-4.19 (m, 1H), 3.91-3.57 (m, 5H), 3.52 (s, 3H), 1.81 (s, 3H), 1.50 (d, .7= 6.8 Hz, 1.5H), 1.45 (d,J = 6.4 Hz, 1.5H), 1.00 (d, 1 = 6.8 Hz, 3H), 0.75-0.73 (m, 3H). 19F-NMR (400MHz, DMSO) δ -111 .21 -111.22 (IF), -140.60_-140.64 (IF), -145.80_-445.90 (2F), -150.41_-150.48 (2F). LCMS: Method-UC05 FRA1, RT 2.321 ESLMS: measured m/z 733.4 [M+l]+. HPLC: Method-HP07_TFAR1 , RT 6.312, 98.18%. Mixture of atropisomers were separated by preparative HPLC on a CHIRALPAK IG 250X50 mm 5um column eluting with liquid CO2 and 70:30 isopropyl alcohol :acetonitrile.
Synthesis of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl) phenyl)-2,3,5,6-tetrafluoro-4-(methylsulfonyl)benzamide
Figure imgf000212_0001
Step-1 A: Synthesis of 3-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)aniline
[00675] To a stirred solution of 5-bromo-l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazole (1.0g, 3.58mmol) (INTI) in mixture of 1,4-dioxane (9mL) and water (ImL) were added 3- (4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)aniline (0.94g, 4.30mmol) and anhydrous K2CO3 (1.48g, 10.75mmol) at room temperature. The reaction mixture was purged with N2 for 15 minutes followed by addition of PdCl2(dppf) (0. 14g, 0.17mmol) at room temperature. The reaction mixture was heated at 100°C for 16h. The reaction mixture was diluted with water (120mL) and extracted with ethyl acetate (3 x 50mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as black gummy solid (1.9g, Quantitative Yield). LCMS (ACQUITYPDA and QDA detector, UC08 FAR1): m/z 292.1 (M+H)+ (ESI +ve), RT-1.41min, 90.9%, 200 - 400nm.1HNMR (400MHz, DMSO) δ 9.38 (s, 1H), 8.47 (s, 1H), 7.77 (s, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.45 (d, J= 8.0 Hz, 1H), 7.39 (br s, 2H), 7.36 (s, 1H), 4.11 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.06 (s, 3F). Note: The obtained material was used in next step without further purification.
Step-2A: Synthesis of 3-cyano-4-fluoro-N-(3-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)phenyl) benzamide
[00676] To a stirred solution of 3-cyano-4-fluorobenzoic acid (0.68g, 4. 12mmol) in DMF (lOmL) were added HATU (1.94g, 5.15mmol) and DIPEA (1.32g, 10.30mmol) at 0°C. The reaction mixture was stirred at 0°C for 15min. followed by addition of 3-(l-methyl-6-(trifluoromethyl)- lH-benzo[d]imidazol-5-yl)aniline (1 ,0g, 3.43mmol). The reaction mixture was allowed to stir at room temperature for Ih. The reaction mixture was diluted with cold water (1 OOmL) and extracted with ethyl acetate (3 x 50mL). The combined organic layer was dried over anhydrous Na2SO4 filtrate was concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluted with 80% ethyl acetate in hexane to afford the title compound as brown solid (0.5g, 1.14mmol, 33% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 439.2(M+H)+ (ESI +ve), RT = 2.13 min, 96.4%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 10.52 (s, 1H), 8.53 (dd, J; = 2.4Hz, J2=6A Hz, 2H), 8.48 (s, IH), 8.35-8.31 (m, lH), 8.15 (s, IH), 7.83 (d, J = 9.6 Hz, 2H), 7.72 (t, J = 9.2 Hz, IH), 7.60 (s, IH), 7.43 (t, J= 8.0 Hz, IH), 7.11 (d, J = 7.2 Hz, IH), 3.97 (s, 3H). 19F NMR (376 MHz, DMSO) δ - 53.42 (s, 3F), -104.54 (s, IF).
Step-1 : Synthesis of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4-(methylthio)benzamide
[00677] To a stirred solution of 2,3,5,6-tetrafhroro-4-(methylthio)benzamide (0.11g, 0.47mmol) (WH21) in DMSO (2mL) were added Cs2CO3 (0.38g, 1. 17mmol) and 2-fhroro-5-(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3-carbonyl)benzonitrile (0. 18g
0.39mmol) at room temperature. The reaction mixture was heated at 100°C for 16h. The reaction mixture was diluted with ice-cold water (50mL) to precipitate out the product. The obtained precipitate was filtered under vacuum and washed with cold water. The resulting solid was dried under reduced vacuum to afford the title compound as a yellow solid (0.13g, 0.19mmol, 49% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 682.2 (M+H)+ (ESI +ve), RT = 2.61 min, 95.5%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 11.52 (brs, IH), 9.94-9.88 (m, IH), 8.52 (s, IH), 8.25 (s, IH), 8.18 (s, IH), 8.08 (d, J= 8.8Hz, IH), 8.00 (d, J= 8.5Hz, IH), 7.77 (s, IH), 7.58 (d, J= 7.6Hz, IH), 7.42 (d, J = 4.8 Hz, 2H), 7.27-7.24 (m, 2H), 6.08 (d, J = 4.4Hz, 1H), 4.O1 (s, 3H), 2.59 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.93 (s, 3F), -134.77 (d, J = 14.9 Hz, 2F), -142.07 (s, 2F).
Step-2: Synthesis of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4-(methylsulfonyl)benzamide
[00678] To a stirred solution of Na2WO4 (0.03g, 0.08 mmol) in H2O (ImL) was added acetic acid (O. lmL) at room temperature. The reaction mixture was heated to 70°C for Ih. The reaction mixture was cooled to room temperature followed by addition of (N-(2-cyano4-(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro- 4-(methylthio)benzamide (0. 13g, 0.19mmol) in methanol (ImL) and H2O2 (0.02mL, 0.57mmol, 30% in water) at room temperature. The reaction mixture was allowed to stirred at room temperature for 16h. The reaction mixture was diluted with saturated NaHCO3 solution (30mL) and extracted with ethyl acetate (3 x lOmL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentratedunder reduced pressure to obtain a crude residue. The obtained crude was purified by column chromatography, eluted with 80% ethyl acetate in hexane to afford the title compound a yellow solid (0.02g, 0.03mmol, 15% yield). LCMS (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 714.1 (M+H)+ (ESI +ve), RT = 2.37 min, 95.1%, 200 - 400nm. HPLC (Agilent Technologies. 1260 Series, Infinity-II, HP07 TFAR1): RT = 6.45 min, 95.15%, 200-400nm. 'HNMR (400 MHz, DMSO) δ 11 .66 (s, 1H), 9.91 (d, J= 2.8 Hz, 1H), 8.53 (s, 1H), 8.28 (d, J= 8.8Hz, 2H), 8.17 (d, J = 8.4 Hz, 1H), 7.94 (d, J= 6.8 Hz, 1H), 7.77 (s, 1H), 7.42 (d, J= 4.8 Hz, 1H), 7.30 (s, 2H), 6.10 (d, J= 4.8 Hz, 1H), 4.O1 (s, 3H), 3.60 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.93 (s, 3F), -136.84_-137.02 (m, 2F), -139.23_- 139.40 (m, 2F).
Synthesis of N-(2,6-difluoro-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4-(methylsulfonyl)benzamide (Compound 238) and N-(2,6-difluoro-4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl) phenyl)-2,3,5,6-tetrafluoro-4- (methylsulfinyl)benzamide (Compound 224)
Figure imgf000214_0001
Step-1: Synthesis of 2-bromo-l-(3,4,5-trifluorophenyl)ethan-l-one
[00679] To a stirred solution of l-(3,4,5-trifluorophenyl)ethan-l-one (5.0g, 28.73mmol) in ethyl acetate (50mL) was added CuBr2 (12.8g, 57.47mmol) at room temperature. The reaction mixture was heated to 80°C for 5h. The reaction mixture was cooled to room temperature and filtrated through celite bed. The celite bed was washed with ethyl acetate (100mL). The obtained filtrate was diluted with water (200mL) and extracted with ethyl acetate (2x lOOmL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the title compound as yellow liquid (7.0g, 27.66 mmol, 96%yield). 1HNMR(400 MHz, DMSO-t/Q 8 8.02-7.96 (m, 2H), 4.96 (s, 2H).
19F NMR (376 MHz, DMSO): 6 -133.00 (d, J = 22.2 Hz, 2F), -152.65 (t, J = 21.3 Hz, IF).
Step-2: Synthesis of 2-(3-bromo-2-methyl-114-pyridin-l-yl)-l-(3,4,5-trifluorophenyl)ethan- 1-one, bromide salt
[00680] To a stirred solution of 2-bromo-l-(3,4,5-trifluorophenyl)ethan-l-one (7.0g, 27.66mmol) in toluene (70mL) was added 3 -brom o-2 -methylpyridine (9.5g, 55.33mmol) at room temperature. The resulting reaction mixture was heated to 90°C for 48h. The reaction mixture was cooled to room temperature to precipitate out the product. The suspension was filtered under vacuum and washed with hexane. The obtained material was dried under high vacuum to afford the title compound as gray solid (7.0g, 16.55mmol, 60% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 344.1 (M+H)+ (ESI +ve), RT = 1.46 min, 93.9%, 200 - 400nm. TI NMR (400 MHz, DMSO): 69.01 (d, J = 7.2 Hz, 1H), 8.96-8.94 (m, 1H), 8.14-8.09 (m, 2H), 8.06- 8.03 (m, 1H), 6.63 (s, 2H), 2.81 (s, 3H). 19F NMR (376 MHz, DMSO): 6 -133.01 (d, J = 21.2 Hz, 2F), -151.21 (t, J = 21.4 Hz, IF).
Step-3: Synthesis of (8-bromoindolizin-3-yl)(3,4,5-trifluorophenyl)methanone
[00681] dimethyl sulphate (15.7mL, 163.26mmol) was dissolved in DMF (12.6mL, 163.26mmol) at room temperature and heated at 80°C for 2h. The reaction mixture was cooled to room temperature. In a separate vessel 2-(3-bromo-2-methyl-114-pyridin-l-yl)-l-(3,4,5- trifluorophenyl)ethan-l-one, bromide salt (7.0g, 20.40mmol) was dissolved in DMF (24mL) at room temperature. The reaction mixture was cooled to 0°C. A solution of dimethyl sulphate in DMF was added to the reaction mixture at 0°C. The reaction mixture was stirred for Ih at room temperature followed by addition of DIPEA (6.97mL, 40.81 mmol) over a period of Ih. The reaction mixture was poured in ice-cold water (500mL). The obtained precipitate was filtered and dried under vacuum to afford the title compound as yellow solid (2.0g, 5.66mmol, 28% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 354.1 (M+H)+ (ESI +ve), RT = 2.92 min, 93.0%, 200 - 400nm. 'HNMR (400 MHz, DMSO): 6 9.76 (d, J= 6.8 Hz, IH), 7.77- 7.69 (m, 3H), 7.55 (d, J=4.8Hz, IH), 7.09 (t, J=7.2Hz, IH), 6.77 (d, J=4.8Hz, IH). 19FNMR(376 MHz, DMSO): 6 -133.91 (d, J = 22.3 Hz, 2F), -157.39 (t, J = 22.3 Hz, IF).
Step-4: Synthesis of (8-bromoindolizin-3-yl)(3,5-difluoro-4-((4- methoxybenzyl)amino)phenyl)methanone
[00682] To a stirred solution of (8-bromoindolizin-3-yl)(3,4,5-trifluorophenyl)methanone (2.0g 5.66mmol) in NMP (20mL) were added (4-methoxyphenyl)methanamine (0.85g, 6.23 mmol) and DIPEA (2.9mL, 16.99mmol) at room temperature. The resulting reaction mixture was heated to 110°C for 36h. The reaction mixture was poured in ice-cold water (200mL). The obtained precipitate was filtered and dried under vacuum to afford the title compound as off white solid (2.1g, 4.46mmol, 79% yield). LCMS: (ACQUITY PDA and QDA detector, UCO4_FAR1): m/z 471 .2 (M+H)+ (ESI +ve), RT = 2.82 min, 93.2%, 200 - 400nm. 'HNMR (400 MHz, DMSO): 5 9.63 (d, .7= 7.2 Hz, 1H), 7.61 (d, J=6.8Hz, 1H), 7.52 (d, J=4.8Hz, 1H), 7.38-7.33 (dd, Ji=2.0Hz, J2=8.4Hz, 2H), 7.25 (d, J=8.8Hz, 2H), 6.98 (t, .7=7, 2 Hz, 1H), 6.89-6.87 (dd, Ji=2.0Hz, J2=6.8Hz, 2H), 6.71 (d, .7=4.8 Hz, 2H), 4.45 (d, J=6.8Hz, 2H), 3.71 (s, 3 H) . 19F NMR (376 MHz, DMSO) δ -128.09 (s, 2F).
Step-5: Synthesis of (3,5-difluoro-4-((4-methoxybenzyl)amino)phenyl)(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3-yl)methanone
[00683] To a stirred solution of (8-bromoindolizin-3-yl)(3,5-difluoro-4-((4- methoxybenzyl)amino)phenyl)methanone (2.0g, 4.25mmol) in 1,4-dioxane (20mL) were added bis(pinacolato)diboron (2.1g, 8.51mmol) and anhydrous potassium acetate (0.99g, 12.76mmol) at room temperature. The reaction mixture was purged with N2 gas for 15 min followed by addition of PdCl2(dppf).dcm (0.34g, 0.42mmol) at room temperature. The reaction mixture was heated at 90°C for 2h. The reaction mixture was cooled to room temperature followed by addition of 5- bromo-l-methyl-6-(trifhioromethyl)-lH-benzo[d]imidazole (0.82g, 2.97mmol) (INTI) and K2CO3 (1.76g, 12.76mmol) atroom temperature. The reaction mixture was heated at 90°C for 16h. The reaction mixture was cooled to room temperature and filtrated through celite bed. The celite bed was washed with ethyl acetate (200mL). The obtained filtrate was concentratedunder reduced pressure to obtain a crude residue. The obtained crude was purified by column chromatography, eluted with 60% ethyl acetate in hexane to afford the title compound as yellow solid (1.0g 1 ,69mmol, 40% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 591.2 (M+H)+ (ESI +ve), RT = 2.73 min, 100%, 200 - 400nm. 1HNMR (400 MHz, DMSO) δ 9.78- 9.73 (m, 1H), 8.52 (s, lH), 8.24 (s, 1H), 7.74 (s, 1H), 7.40-7.37 (m, 3H), 7.26-7.15 (m, 4H), 6.89- 6.85 (m, 2H), 6.60 (brs, 1H), 6.01 (d, J= 4.8 Hz, 1H), 4.44 (d, J=6.8Hz, 2H), 4.00 (s, 3H), 3.70 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.93 (s, 3F), -128.13 (s, 2F).
Step-6: Synthesis of (4-amino-3,5-difluorophenyl)(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizin-3-yl)methanone
[00684] (3,5-difluoro-4-((4-methoxybenzyl)amino)phenyl)(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizin-3 -yl)methanone(l ,0g, 1 .69mmol) was dissolvedin in mixture of TFA and anisole (8mL:2mL) at room temperature. The reaction mixture was stirred at room temperature for Ih. The reaction mixture was concentrated under reduced pressure to obtain a residue. The obtained residue was diluted with saturated solution of NaHCO3 (300mL) and extracted with ethyl acetate (2x80mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated using diethyl ether to afford the title compound as an off white solid. (0.4g 0.85mmol, 50% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 471.2 (M+H)+ (ESI +ve), RT = 2.25 min, 98.9%, 200 - 400nm. TINMR (400 MHz, DMSO) δ 9.76 (t, J=8.0Hz, 1H), 8.52 (s, 1H), 8.25 (s, 1H), 7.75 (s, 1H), 7.43-7.39 (m, 3H), 7.18-7. 15 (m, 2H), 6.04- 6.03 (m, 3H), 4.07 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.92 (s, 3F), -131.45 (s, 2F).
Step-7 : Synthesis of N-(2,6-difluoro-4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4- (methylthio)benzamide
[00685] To a stirred solution of (4-amino-3,5-difluorophenyl)(8-(l-methyl-6-(trifluoromethyl)- lH-benzo[d]imidazol-5-yl)indolizin-3-yl)methanone (0.28g, 0.59mmol) in of pyridine (2.8mL) were added 2,3,5,6-tetrafhioro-4-(methylthio)benzoic acid (0.1g, 0.47mmol) (WH10) and POC13 (0.16mL, 1.19mmol) at 0°C. The reaction mixture was stirred at room temperature for Ih. The reaction mixture was diluted with water (100mL) and extracted with ethyl acetate (2x50mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The obtained crude was purified by reverse phase chromatography, eluted with 48% acetonitrile in water using 0.1% formic acid as modifier to afford the title compound as yellow solid (0.1g, 0.14mmol, 24% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 693.3 (M+H)+ (ESI +ve), RT = 2.66 min, 83.6%, 200 - 400nm. JHNMR(400 MHz, DMSO) δ 11.08 (s, IH), 9.91-8.89(m, lH), 8.53 (s, IH), 8.26 (s, IH), 7.76 (s, IH), 7.65 (d, J=8.0Hz, 2H), 7.45 (d,J=4.8Hz, IH), 7.31-7.28 (m, 2H), 6.09 (d,J=4.8Hz, IH), 4.01 (s, 3H), 2.57 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.92 (s, 3F), -116.06 (d, J = 7.5 Hz, 2F), -134.24_- 134.40 (m, 2F), -141.82 (dd, Jj = 11.7 Hz, J2 = 25.4 Hz, 2F).
Step-8: Synthesis of N-(2,6-difluoro-4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4- (methylsulfonyl)benzamide (V0003306) and N-(2,6-difluoro-4-(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)-2, 3,5,6- tetrafluoro-4-(methylsulfinyl)benzamide (V3306-SO)
[00686] To a stirred solution of Na2WO4:2H2O (0.02g, 0.07mmol) in H2O (1.2mL) was added 2- 3 drops of acetic acid at room temperature. The reaction mixture was heated at 65°C for Ih. The reaction mixture was cooled to 0°C followed by addition of N-(2,6-difluoro4-(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro- 4-(methylthio)benzamide (0.12g, 0.17mmol) in methanol (1.2mL) andH202(0.06mL, 0.52mmol, 30% in water). The reaction mixture was heated to 65°C for 5h. The reaction mixture was diluted with water(80mL) and extracted with ethyl acetate (2x30mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by Prep. TLC using mobile phase 4% methanol in dichloromethane to afford the title compounds.
Synthesis of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3- carbonyl)-N-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)benzamide (Compound 240) and (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3-yl)(4-(((2,3,5,6- tetrafluoro-4-(methylsulfonyl)phenyl)amino)methyl) phenyl)methanone (Compound 242)
Figure imgf000218_0001
Step-1: Synthesis of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)-N-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)benzamide.
[00687] To a stirred solution of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3 -carbonyl) benzoic acid (0.2g, 13.84mmol) (INT13) in pyridine (2. OmL) were added 2,3,5,6-tetrafhroro-4-(methylsulfonyl)aniline (0.15g, 0.64mmol) (WH8)and POCI3 (0.08mL, 0.86mmol) at 0°C. The reaction mixture was stirred at room temperature for 16h. The reaction mixture was diluted with water (150mL) and extracted with ethyl acetate (3 x 50mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue that was purified by preparative HPLC on a WATERS XBRIDGE (250mm x 19mm x 5pm) column eluting with aqueoues 0.1% formic acid and acetonitrile to afford title compound as a yellow solid (0.025g, 0.04mmol, 4% yield). LCMS: (ACQUITYPDA and QDAdetector, UC08 FAR1): m/z 689.3 (M+l)+ (ESI+ve), RT = 2.31 min, 100%, 200 - 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity -II, HP07 TF ARI): RT = 6.39 min, 99.4%, 200-400nm. 1HNMR (400MHz, DMSO) δ 11.14 (s, 1H), 9.95 (t, J= 4.0 Hz, 1H), 8.52 (s, 1H), 8.26 (s, 1H), 8.16 (d, J= 8.4 Hz, 2H), 7.92 (d, J= 9.2 Hz, 2H), 7.77 (s, 1H), 7.34-7.27 (m, 3H), 6.09 (s, d, J= 4.8 Hz, 1H), 4.01 (s, 3H), 3.57 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.91 (s, 3F), -139.53 (s, 2F), -142.49 (s, 2F).
Step-2: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin- 3-yl)(4-(((2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)amino)methyl)phenyl)methanone. [00688] To a stirred solution of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)-N-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)benzamide (0.12g 0.17mmol) in THF (1.2mL) was added Borane THF (0.26g, 3.13mmol) at 0°C. The reaction mixture was stirred at 70°C for 4 days. The reaction mixture was cooled to room temperature and diluted with dilute HC1 (75mL) and extracted with ethyl acetate (3 x 50mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by preparative HPLC on a Xtimate C18 (250mm x 21.2mm x 5pm) column eluting with aqueoues 0.1% formic acid and acetonitrile. to afford the title compound as a green solid (0.026g, 0.04mmol, 11% yield). LCMS: (ACQUITY PDA and QD A detector, UC08 FAR1 ): m/z 675.2 (M+l )+ (ESI +ve), RT = 2.38 min, 95.4%, 200 - 400nm HPLC: (Agilent Technologies. 1260 Series, Infinity -II, HP07 TF ARI): RT = 6.40 min, 95.4%, 200-400nm. 1HNMR (400MHz, DMSO) δ 10.97 (s, IH), 8.47 (s, IH), 8.17 (s, IH), 8.04 (d, J= 6.8 Hz, IH), 7.98 (d, J= 8.0 Hz, 2H), 7.68 (s, IH), 7.48 (d, J= 8.0 Hz, 2H), 6.68 (t, J= 6.4 Hz, IH), 6.55-6.54 (m, 2H), 5.81 (d, J = 3.6 Hz, IH), 4.41 (s, 2H), 3.98 (s, 3H), 3.55 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.85 (s, 3F), -139.67 (s, 2F), -142.59 (s, 2F).
Synthesis of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) indolizine-3-carbonyl) phenyl)-2,3,5,6-tetrafluoro-4-(S-methylsulfonimidoyl) benzamidec (Compound 268)
Figure imgf000219_0001
Step-1 : Synthesis of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl) indolizine-3-carbonyl) phenyl)-2,3,5,6-tetrafluoro-4-(methylthio)benzamide
To a stirred solution of 2,3,5, 6-tetrafluoro-4-(methylthio)benzamide (0.4g, 0.83mmol) (WH21) [00689] in dimethylacetamide (2mL) was added 2-fluoro-5-(8-(l-methyl-6-(trifluoromethyl)- lH-benzo[d]imidazol-5-yl)indolizine-3-carbonyl)benzonitrile (0.38g, 0.83mmol) at room temperature. The resulting reaction mixture was stirred at 130°C for Ih under microwave irradiation. The reaction mixture was cooled to room temperature and diluted with water. The resulting suspension was extracted with ethyl acetate (3 x 30mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purifiedby flash column chromatography, eluted with 45% ethyl acetate in hexane to afford the title compound as ayellow solid (0.6g, 0.88mmol, 53 %y ield). JH NMR (400 MHz, DMSO-t/6) δ 11.51 (s, IH), 9.92-9.90 (m, IH), 8.52 (s, IH), 8.27-8.27 (m, 2H), 8.16 (d, .7= 8.0 Hz, IH), 7.89 (d, J= 8.4Hz, 1H), 7.77 (s, IH), 7.42 (d, J = 4.8 Hz, IH), 7.30- 7.27 (m, 2H), 6.09 (d, J= 4.0 Hz, IH), 4.00 (s, 3H), 2.61 (s, 3H). 19F NMR (400 MHz, DMSO- d6) 5 -54.91_-55.02 (3F), -134.39_-134.49 (2F), -141.46_-141.56 (2F). LCMS: Method- UC08 FAR1, RT- 2.622 ESI-MS: measured m/z 682.2 [M+l] +.
Step-2: Synthesis of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4-(S-methylsulfonimidoyl)benzamide [00690] To a stirred solution of N-(2-cyano-4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)-2,3,5,6-tetrafluoro-4-
(methylthio)benzamide (0.55g, 0.80mmol) in methanol (5.5mL) were added iododbenzene diacetate (0.77gm, 2.42mmol) and ammonium carbamate (0. 18g, 2.42mmol) atO°C. The resulting reaction mixture was stirred to 0°C for 15min. The reaction mixture was concentrated under reduced pressure. The resulting residue was diluted with water (150mL) and extracted with ethyl acetate (3 x 30mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by reverse phase chromatography, eluted with 36% acetonitrile in water to enrich the title compound as a yellow solid. The obtained enriched material was purified by preparative HPLC on a WATERS XB RIDGE (250mm x 19mm x 5 pm) column eluting with water and acetonitrile, to afford the title compound as a yellow solid (0.055g, 0.08mmol, 10% yield). 1HNMR (400 MHz, DMSO-t/6) 6 11.63 (s, 1H), 9.92-9.90 (m, 1H), 8.52 (s, 1H), 8.28-8.26 (m, 2H), 8.16 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.42 (d, J = 4.4 Hz, 1H), 7.30-7.28 (m, 2H), 6.09 (d, .7=4.8 Hz, 1H), 5.79 (s, 1H), 4.00 (s, 3H), 3.41 (s, 3H). 19F NMR (400 MHz, DMSO-t/6) 5 -54.91 (3F), -137.97_-138.07 (2F), -140.14_- 140.24 (2F). LCMS: Method-UC08_FAR1, RT- 2.208 ESI-MS: measured m/z 713. 1 [M+l]+, measured m/z 713.1 [M+l]+. HPLC: HP07 TFAR1, RT 6.637, 100%.
Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo [d]imidazol-5-yl)-N-(2, 3,5,6- tetrafluoro-4-(methyl sulfonyl)phenethyl)indolizine-3-carboxamide (Compound 250)
Figure imgf000220_0001
Step-1: Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(2, 3,5,6- tetrafluoro-4-(methylsulfonyl)phenethyl)indolizine-3-carboxamide
[00691] To a stirred solution of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3 -carboxylic acid (0. 11g, 0.31 mmol) (INT2) in DMF (4.0 mL) were added HATU (0.23g, 0.61 mmol) and DIPEA (0. 11g, 0.92 mmol) at 0 °C. The reaction mixture was allowed to stirred at 0 °C for lOmins. followed by the addition of 2-(2,3,5,6-tetrafluoro4- (methylsulfonyl)phenyl)ethan-l -amine (0.099g, 0.37 mmol) (WH4). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with cold water (200 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by RFC, eluting with 30% acetonitrile in water to afford the title compound as an off-white solid (0.020g, 0.032 mmol, 8% yield). LCMS (ACQUITYPDA and QDA detector, UC08_FARl): m/z 613.2 (M+H)+ (ESI +ve), RT = 2.19min, 99.2%, 200 - 400nm. HPLC Agilent Technologies. 1260 Series, Infinity -II, HP05 trifluoroacetic acidRl): RT = 6.54min, 98.3%, 200-400nm. 1HNMR (400 MHz, DMSO) δ 9.57 (d, J = 7.2 Hz, 1H), 8.50 (s, 1H), 8.43-8.40 (m, 1H), 8.21 (s, 1H), 7.72 (s, 1H), 7.38 (d, J = 4.0 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.87 (d, J = 6.4 Hz, 1H), 5.89 (d, J = 4.4 Hz, 1H), 3.99 (s, 3H), 3.60-3.50 (m, 2H), 3.51 (s, 3H), 3.07 (br s, 2H). 19F NMR (376 MHz, DMSO) δ -54.66 (s, 3F), -139.85 (dd, J1 = 9.3Hz, J2 = 21.8 Hz, 2F), -141.57 (d, J = 12.5 Hz, 2F).
Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin-3-yl)(4- ((2,3,5,6-tetrafluoro-4-(methylsulfinyl)benzyl)amino)phenyl)methanone (Compound 227)
Figure imgf000221_0001
Step-1: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin- 3-yl)(4-((2,3,5,6-tetrafluoro-4-(methylthio)benzyl)amino)phenyl)methanone
[00692] To a stirred solution of (4-(bromomethyl)-2,3,5,6-tetrafluorophenyl)(methyl)sulfane (0.49g, 1.73 mmol) (WH2) in DMF (5 mL) were added KI (0.02g, 0.11 mmol), K2CO3 (0.47g 3.45 mmol) and (4-aminophenyl)(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizin-3-yl)methanone (0.5g, 1 .15 mmol) (INT4) atroom temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured in to ice cold water (50 mL) to precipitate out the product. The obtained precipitate was filtered and washed with water (20ml). The obtained solid was dried under high vacuum to obtain a crude residue. The obtained crude was purified by reverse phase column chromatography, eluting with 75-80% acetonitrile in water using 0.1% formic acid as modifier to afford the title compound as a yellow solid (0.39g, 0.61 mmol, 53% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 643.2 (M+H)+(ESI +ve), RT = 2.75 min, 75.9%, 200 - 400nm. 1HNMR (400MHz, DMSO) 5 9.76 (t, J=4.0Hz, 1H), 8.51 (s, 1H), 8.24 (s, 1H), 7.75 (s, 1H), 7.68(d, J=8.8Hz, 2H), 7.32(d, J=4.4Hz, 1H), 7.14-7.11(m, 2H), 6.97 (t, J=5.6Hz, 1H), 6.75(d, J=8.8Hz, 2H), 6.00(d, J=4.8Hz, 1H), 4.48(d, J=5.6Hz, 2H), 4.00 (s, 3H), 2.52 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.92(s, 3F), -135.87 (dd, J1 = 12.0Hz, J2 = 25.2Hz, 2F), -143.05 (dd, J1 = 11.9Hz, J2 = 25.2Hz, 2F).
Step-2: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizin- 3-yl)(4-((2,3,5,6-tetrafluoro-4-(methylsulfinyl)benzyl)amino)phenyl)methanone
[00693] To a stirred solution of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizin-3-yl)(4-((2,3,5,6-tetrafluoro-4-(methylthio)benzyl)amino)phenyl)methanone (0.05g 0.07 mmol)in THF (1 mL) was addedm-CPBA(0.026g, 0.15 mmol)at0°C. The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with saturated solution of NaHCO3 (25 mL) and extracted with ethyl acetate (2x25 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by reverse phase column chromatography, eluting with 50-55% acetonitrile in water using 0.1% formic acid as modifier to afford the title compound as a yellow solid (0.06g, 0.09 mmol, 23% yield). LCMS: (ACQUITYPDA and QDA detector, UC08_FARl): m/z 659.2 (M+H)+ (ESI +ve), RT = 2.27 min, 97.7%, 200 - 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity-II, o2h_HP07_trifluoroacetic acidRl): RT = 6.35 min, 97.9%, 200-400nm. 'HNMR (400MHz, DMSO) δ 9.84 (d, J=7.2Hz, 1H), 8.20 (s, 1H), 8.1 l(s,lH), 7.75 (t, J=7.6Hz, 3H), 7.32(d, J=4.4Hz, lH), 7.14(d, J=6.8Hz, 1H), 7.05 (t, J=7.2Hz, 1H), 6.79(d, J=8.4Hz, 2H), 6.06(d, J=4.8Hz, 1H), 5.52 (t, J=6.4Hz, 1H), 4.60(d, J=6.4Hz, 2H), 3.97 (s, 3H), 3.11 (s, 3H). 19FNMR (376 MHz, CD3CN) δ -56.87 (s, 3F), -142.80
-143.14 (m, 4F).
Synthesis of N-(2,3,5,6-tetrafluoro-4-(S-methylsulfonimidoyl)phenyl)-8-(4-
(trifluoromethyl)phenyl)quinoline-3-carboxamide (Compound 274)
Figure imgf000222_0001
Step-1: Synthesis of 2-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)-l-(8-(4-
(trifluoromethyl)phenyl)quinolin-3-yl)ethan-l-one
[00694] To a stirred solution of 2,3,5, 6-tetrafhioro-4-(methylthio)aniline (0.29g, 0.94 mmol)
(WH9) in pyridine (3 mL) were sequentially added 8-(4-(trifluoromethyl)phenyl)quinoline-3- carboxylic acid (0.3g, 0.94 mmol) (INT3) and POC13 (0.28g, 1.89 mmol) at 0 °C. The resulting reaction mixture was allowed to stirred at room temperature for 1 hour. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purifiedby normal phase column chromatography, eluting with 15% ethyl acetate in hexane to afford the title compound as an off-white solid (0.3g 0.59 mmol, 41% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 511.1 (M+l)+ (ESI +ve), RT = 2.90 min, 99.4%, 200 - 400nm. 1HNMR (400 MHz, DMSO) δ 11.07 (s, 1H), 9.36 (d, J = 2.0 Hz, 1H), 9.13 (d, J = 2.4 Hz, 1H), 8.28-8.26 (m, 1H), 8.02 (dd,J1 = 1 .2 Hz, J2= 7.2 Hz, 1H), 7.93-7.84 (m, 5H), 2.57 (s, 3H). 19F NMR (377 MHz, DMSO) δ -60.84 (s, 3F), -135.83 (dd, J) = 9.6 Hz, J2 = 25.4 Hz, 2F), -144.64 (dd, J1 = 9.6 Hz, J2 = 25.4 Hz, 2F).
Step-2: Synthesis of N-(2,3,5,6-tetrafluoro-4-(S-methylsulfonimidoyl)phenyl)-8-(4- (trifluoromethyl)phenyl) quinoline-3-carboxamide
[00695] To a stirred solution of 2-(2,3,5,6-tetrafhioro-4-(methylthio)phenyl)-l-(8-(4- (trifluoromethyl)phenyl)quinolin-3 -yl)ethan-l-one(0.25g, 0.49 mmol)in methanol (2.5 mL) were added iododbenzenediacetate (0.47gm, 1.47 mmol) and ammonium carbamate (0.11g, 1.47mmol) at 0 °C. The resulting reaction mixture was stirred at 0 °C for 5 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue. The obtained residue was suspended in water (50 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by normal phase column chromatography, eluting with 60% ethyl acetate in hexane to afford the title compound as an off-white solid (0.15g, 0.28 mmol, 57% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 542. 1 (M+l)+ (ESI +ve), RT = 2.46 min, 97.4%, 200 - 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity- II, HP07_trifluoroacetic acidRl): RT = 8.63 min, 98.5%, 200-400nm. JH NMR (400 MHz, DMSO) δ 11.26 (s, 1H), 9.36 (d, J = 2.0 Hz, 1H), 9.14 (d, J = 2.0 Hz, 1H), 8.29-8.27 (m, 1H), 8.02 (dd,J1 = 1.2 Hz, J2 = 7.2 Hz, 1H), 7.93-7.85 (m, 5H), 5.62 (s, 1H), 3.41 (s, 3H). 19F NMR (377 MHz, DMSO) δ -60.85 (s, 3F), -140.08_-14017 (m, 2F), -143.17 -143.38 (m, 2F).
Synthesis of imino(methyl)(2,3,5,6-tetrafluoro-4-(((4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)amino)methyl)phenyl)-16-sulfanone (Compound 271)
Figure imgf000224_0001
Step-1: Synthesis of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) indolizin- 3-yl) (4-((2,3,5,6-tetrafluoro-4-(methylthio)benzyl) amino) phenyl)methanone
[00696] To a stirred solution of (4-aminophenyl) (8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl) indolizin-3-yl) methanone (0.50g, 1 .15 mmol) (INTI 0) in DMF (5 mL) was added K2CO3 (0.47g, 3.45 mmol) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, followed by the addition of (4-(bromomethyl)-2, 3,5,6- tetrafluorophenyl)(methyl)sulfane (1.33g, 4.60 mmol) (WH2). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ice-cold water (200 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The obtained crude was purified by reverse phase flash chromatography and eluting with 56% acetonitrile in water to afford the title compound as a yellow solid (0.38g, 0.59 mmol, 51% yield). LCMS (ACQUITY PDA and QDA detector, o2h_LCMS_Method_A): m/z 643.3 (M+H)+ (ESI+ve), RT-2.54min, 68.8%, 200 - 400nm. 1HNMR(400 MHz, DMSO) δ 9.76 (t, J = 4.0 Hz, 1H), 8.50 (s, 1H), 8.23 (s, 1H), 7.74 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 4.8 Hz, 1H), 7.11 (d, J = 4.8 Hz, 2H), 6.95 (t, J = 5.6 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 5.99 (d, J = 4.8 Hz, 1H), 4.46 (d, J = 5.6 Hz, 2H), 4.01 (s, 3H), 2.50 (s, 3H). 19F NMR (377 MHz, DMSO) δ -54.95 (s, 3F), -135.62_ -135.96 (m, 2F), -142.35_ -143.26 (m, 2F).
Step-2: Synthesis of tert-butyl (4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)phenyl)(2,3,5,6-tetrafluoro-4-(methylthio)benzyl)carbamate
[00697] To a stirred solution of (8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizin-3-yl)(4-((2,3,5,6-tetrafluoro-4-(methylthio)benzyl)amino)phenyl)methanone (0. 19g 0.29 mmol) in THF (1.9 mL) were added TEA (0. 17g, 1.77 mmol), boc anhydride (0.19g, 0.88 mmol) and DMAP (0.007g, 0.05 mmol) at room temperature. The reaction mixture was heated at 80 °C for 30 minutes. The reaction mixture was diluted with cold water (100 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by reverse phase chromatography, product eluting with 42% acetonitrile in water to afford the title compound as a white solid (0.28g, 0.37 mmol, 64% yield). LCMS (ACQUITY PDA and QDA detector, o2h_LCMS_Method_A): m/z 743.3 (M+H)+ (ESI+ve), RT-2.88min, 68.4%, 200 - 400nm. 'H NMR (400 MHz, DMSO) δ 9.88 (t, J = 4.4 Hz, 1H), 8.51 (s, 1H), 8.24 (s, 1H), 7.75-7.73 (m, 3H), 7.39 (d, J = 8.4 Hz, 2H), 7.24-7.20 (m, 3H), 6.05 (d, J = 4.8 Hz, 1H), 5.29 (s, 1H), 5.06 (s, 2H), 3.99 (s, 3H), 2.50 (s, 3H), 1.37 (s, 9H). 19F NMR (377 MHz, DMSO) δ -54.95 (s, 3F), -135.58 -136.40 (m, 2F), -142.40_-142.94 (m, 2F).
Step-3: Synthesis of tert-butyl (4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)phenyl)(2,3,5,6-tetrafluoro-4-(S- methylsulfonimidoyl)benzyl)carbamate
[00698] To a stirred solution of tert-butyl (4-(8-(l-methyl-6-(trifhroromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)(2,3,5,6-tetrafluoro-4-
(methylthio)benzyl)carbamate (0.03g, 0.04 mmol) in methanol (1 mL) were added ammonium carbamate (0.012g, 0.16 mmol) and lodobenzene diacetate (0.051, 0.16 mmol) at 0 °C. The reaction mixture was stirred atroom temperature for 10 minutes. The reaction mixture was diluted with cold water (100ml) and extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound a yellow solid (0.40g, 0.52 mmol, Quantitative yield). Note: The obtained material was used in the next step without purification. LCMS (ACQUITY PDA and QDA detector, o2h_LCMS_Method_A): m/z 774.3 (M+H)+ (ESI+ve), RT-2.36min, 50.7%, 200 - 400nm.
Step-4: Synthesis of imino(methyl)(2,3,5,6-tetrafluoro-4-(((4-(8-(l-methyl-6- (trifluoromethyl)-lH-benzo[d] imidazol-5-yl)indolizine-3- carbonyl)phenyl)amino)methyl)phenyl)-16-sulfanone
[00699] To a stirred solution of tert-butyl (4-(8-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)indolizine-3-carbonyl)phenyl)(2,3,5,6-tetrafluoro-4-(S- methylsulfonimidoyl)benzyl)carbamate (0.40g, 0.51 mmol) in l,4 dioxane (4 mL) was added 4 M HC1 in l,4-dioxane (4 mL) at 0 °C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. The obtained crude was purified by purified by preparative HPLC on a Gemini 5um NX-C18 110 A (250mm x 20mm x 5 pm) column eluting with 0.1% aqueous formic acid and acetonitrile, to afford the title compound as a yellow solid (0.03g, 0.04 mmol, 9% yield). LCMS (ACQUITY PDA and QDA detector, o2h_LCMS_Method_A): m/z 674.2 (M+H)+ (ESI +ve), RT = 2.10 min, 100%, 200 - 400nm. HPLC (Agilent Technologies. 1260 Series, Infinity -II, o2h_HPLC_Method_A): RT = 7.57 min, 100%, 200-400nm. XH NMR (400 MHz, DMSO) δ 9.76 (t, J = 4.4 Hz, 1H), 8.50 (s, 1H), 8.23 (s, 1H), 7.74 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 4.4 Hz, 1H), 7.12 (d, J = 4.4 Hz, 2H), 7.02 (t, J = 6.4 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.00 (d, J = 4.4 Hz, 1H), 5.56 (s, 1H), 4.54 (d, J = 6.0 Hz, 2H), 3.99 (s, 3H), 3.33 (s, 3H). 19F NMR (377 MHz, DMSO) δ -54.94 (s, 3F), - 139.89 -139.98 (m, 2F), -141.65_-141.76 (m, 2F).
Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(3-(2, 3,5,6- tetrafluoro-4-(methylsulfonyl)phenyl)propyl)indolizine-3-carboxamide (Compound 248)
Figure imgf000226_0001
Step-1: Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(3- (2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)propyl)indolizine-3-carboxamide
[00700] To a stirred solution of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3 -carboxylic acid (0.094g, 0.26 mmol) (INT2) in DMF (1 mL) were added HATU (0.15g, 0.39 mmol) and DIPEA (0.1 mL, 0.78 mmol) at 0°C. The reaction mixture stirred at 0 °C for 30 minutes, followed by addition of 3-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenyl)propan- l-amine(0.075g, 0.26 mmol) (WH5). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into ice-cold water (100 mL). The obtained solid was filtered through Buchner funnel and dried under vacuum to obtain a crude residue. The obtained crude was purified by reverse phase column chromatography, eluting with 48% acetonitrile in 0.1% FA in water to afford title compound as light a green solid (0.018g, 0.028 mmol, 10.93% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 F ARI), m/z 627.2 (M+l) + (ESI +ve), RT = 2.22 min, 100%, 200 - 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity-II, HP07_trifluoroacetic acidSl.amx), RT = 9.22min, 98.0%, 200 - 400nm. 1HNMR (400 MHz, DMSO) δ 9.62 (d, J= 7.2 Hz, 1H), 8.50 (s, 1H), 8.28 (t, J= 5.6 Hz, 1H), 8.21 (s, 1H), 7.71 (s, 1H), 7.46 (d, J = 4.4 Hz, 1H), 6.92 (t,J= 6.8 Hz, 1H), 6.86 (d, J= 6.4 Hz, 1H), 5.90 (d, J = 4.4 Hz, 1H), 4.02 (s, 3H), 3.48 (s, 3H), 3.32-3.29 (m, 2H), 2.86 (t, J= 7.2 Hz, 2H), 1.89 (q, = 6.8 Hz, J 2= 14.0 Hz, 2H). 19F NMR (400 MHz, DMSO) δ -54.98 (s, 3F), -139.62 (dd, J} = 9.5 Hz, J2 = 22.1 Hz, 2F), -141.93 (dd, Jj = 9.6 Hz, J2 = 21.9 Hz, 2F). Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(3-(2, 3,5,6- tetrafluoro-4-(S-methylsulfonimidoyl)phenyl)propyl)indolizine-3-carboxamide (Compound 272)
Figure imgf000227_0001
Step-1: Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(3- (2,3,5,6-tetrafluoro-4-(S-methylsulfonimidoyl)phenyl)propyl)indolizine-3-carboxamide
[00701] To a stirred solution of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3 -carboxylic acid (0.288g, 0.80 mmol) (INT2) in DMF (4 mL) were added HATU (0.574g, 1.50 mmol) and DIPEA (0.85 mL, 5.02 mmol) at 0 °C. The reaction mixture stirred at 0 °C for 30 minutes. followed by addition of (4-(3-aminopropyl)-2, 3,5,6- tetrafluorophenyl)(imino)(methyl)-16-sulfanone (0.40g, 1 .00 mmol) (WH6). The reaction mixture was stirred at room temperature for 16 hours. After completion of reaction, the reaction mixture was diluted with ice-cold water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrousNa2SO4, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by reverse phase column chromatography, eluting with 60% acetonitrile in 0.1% FA in water to afford the title compound as an off-white solid (0.09g, 0.14 mmol, 12.90% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 626.1 (M+l) + (ESI+ve), RT = 2.04min, 97.7%, 200 -400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity -II, HP07_trifluoroacetic acidRl): RT = 5.81 min, 97.2%, 200-400nm. 'HNMR (400 MHz, DMSO) δ 9.62 (d, J = 6.8 Hz, 1H), 8.50 (s, 1H), 8.29 (t, J = 5.6 Hz, 1H), 8.21 (s, 1H), 7.71 (s, 1H), 7.47 (d, J = 4.4 Hz, 1H), 6.92 (t, J = 6.8 Hz, 1H), 6.86 (d, J = 6.8 Hz, 1H), 5.90 (d, J = 4.4 Hz, 1H), 5.51 (s, 1H), 3.99 (s, 3H), 3.31 (s, 43), 2.83 (t, J = 7.2 Hz, 2H), 1 .86 (t, = 7.2 Hz, 2H). 19F NMR (377 MHz, DMSO) δ -54.98 (s, 3F), -140.00 (dd,J1 = 12.0, J2 = 24.1 HZ, 2F), -142.93 (dd, J1 = 11.5, J2 = 23.2 Hz, 2F).
Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(3-(2, 3,5,6- tetrafluoro-4-(methyl sulfinyl)phenyl)propyl)indolizine-3-carboxamide(Compound 229)
Figure imgf000227_0002
Step-1: Synthesis of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(3- (2,3,5,6-tetrafluoro-4-(methylsulfinyl)phenyl)propyl)indolizine-3-carboxamide
[00702] To a stirred solution of 8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3 -carboxylic acid (0.16g, 0.44 mmol) (INT2) in DMF (2 mL) were added HATU (0.254g, 0.66 mmol) and DIPEA (0.2 mL, 1.33 mmol) at 0 °C. The reaction mixture stirred at 0 °C for 30 minutes. followed by addition of 3-(2,3,5,6-tetrafluoro4- (methylsulfinyl)phenyl)propan-l-amine (0.119g, 44 mmol) (WH7). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured in to ice cold water (100 mL). The obtained solid was filtered through Buchner funnel and dried under vacuum to obtain a crude residue. The obtained crude was purified by reverse phase column chromatography eluting with 48% acetonitrile in 0.1% FA in water to afford title compound as an off-white solid (0.02g, 0.032 mmol, 7.22%). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl), m/z 611.17 (M+1) + (ESI +ve), RT = 2.09min, 100.0%, 200 - 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity-II, HP07_trifluoroacetic acidRl), RT = 5.97 min, 100.0%, 200 - 400nm. JH NMR (400 MHz, DMSO) δ 9.61 (d, J = 6.8 Hz,lH), 8.50 (s, 1H), 8.27 (t, J = 5.2 Hz, 1H), 8.21 (s, 1H), 7.71 (s, 1H), 7.46 (d, J = 4.4 Hz, 1H), 6.92 (t, J = 6.8 Hz, 1H), 6.86 (d, J = 6.8 Hz, 1H), 5.89 (d, J = 4.4 Hz, 1H), 3.99 (s, 3H), 3.15 (s, 3H), 2.83 (t, J = 7.2 Hz, 2H), 1.88 (q, J1 = 6.8 Hz, J2= 14.0 Hz, 2H). 19FNMR (376 MHz, DMSO) δ -54.99 (s, 3F), -142.00 (dd, J1 = 12.0, J2 = 24.1 Hz, 2F), -142.46 (dd,J1 = 11 .5, J2 = 23.2 Hz, 2F).
Synthesis of N-(l-(2,3,5,6-tetrafluoro-4-(methyl sulfonyl) phenyl) ethyl)-8-(4- (trifluoromethyl) phenyl) quinoline-3-carboxamidess (Compound 33)
Figure imgf000228_0001
Step-1: Synthesis of N-(l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethyl)-8-(4- (trifluoromethyl)phenyl)quinoline-3-carboxamide
[00703] To a stirred solution of l-(2,3,5,6-tetrafluoro-4-(methylthio) phenyl) ethan-l-amine (0.24g, 1.02 mmol) (WH14) in DMF (2.5ml) was added 8-(4-(trifluoromethyl)phenyl)quinoline- 3 -carboxylic acid (0.25 g, 0.78 mmol)(INT3), TEA (0.23 g, 2.36 mmol) and T3P (0.37g, 1. 18 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 40% ethyl acetate in hexane to afford the title compound as a white solid (0.37g, 0.69 mmol, 68% yield). LCMS: (ACQUITY PDA and QD A detector, UC08 FAR1): m/z 539.2 (M+l)+ (ESI +ve), RT = 3.02 min, 85.8%, 200 - 400nm. 'HNMR (400 MHz, DMSO) 5 9.48 (d, J = 5.2 Hz, 1H), 9.23 (s, 1H), 8.95 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 7.96-7.79 (m, 6H), 5.45-5.43 (m, 1H), 2.51 (s, 3H), 1.67 (d, J = 6.4 Hz, 3H). 19F NMR (400 MHz, DMSO) δ -60.80 (q, J = 22 Hz, 3F), -136.15 (q, J = 11.6 Hz, 2F), -143.98 -143.53 (m, 2F).
Step-2: Synthesis of N-(l-(2,3,5,6-tetrafluoro-4-(methyl sulfonyl) phenyl) ethyl)-8-(4- (trifluoromethyl) phenyl) quinoline-3-carboxamide
[00704] To a stirred solution of N-(l-(2,3,5,6-tetrafluoro-4-(methylthio)phenyl)ethyl)-8-(4- (trifluoromethyl)phenyl) quinoline-3-carboxamide (0.3g, 0.55 mmol) in methanol (3 mL) were added H2O2 (0.07g, 2.23 mmol) and hexammonium heptamolybdate (0.06g, 0.05 mmol) at 0 °C. The resulting reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with NaHCO3 solution (30 mL) and extracted with diethyl ether (2 x 20 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by flash column chromatography, eluting with 70% ethyl acetate in hexane to afford the title compound as a white solid (0.045g, 0.08 mmol, 14% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 571 .1 (M+l)+ (ESI +ve), RT = 2.70 min, 99.8%, 200-400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity -II, HP07_trifluoroacetic acidRl): RT = 7.47 min, 100%, 200- 400nm. 'HNMR (400 MHz, DMSO) δ 9.56 (d, J = 5.6 Hz, 1H), 9.24 (d, J = 2.0 Hz, 1H), 8.96 (d, J = 2.0 Hz , 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 6.8 Hz, 1H), 7.90-7.80 (m, 5H), 5.47-5.44 (m, 1H), 3.53 (s, 3H), 1 .69 (d, J = 7.2 Hz, 3H). 19F NMR (400 MHz, DMSO) δ -60.82 (s, 3H), - 139.31 (q, J = 9.6 Hz, 2H), -141 .86 -141.96 (m, 2H).
Synthesis of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)indolizine-3- carbonyl)-N-(2,3,5,6-tetrafluoro-4-sulfamoylphenyl)benzamide (Compound 267)
Figure imgf000230_0001
Step-1 A: Synthesis of 2,3,5, 6-tetrafluoro-4-((4-methoxybenzyl)amino)benzene sulfonamide [00705] To a stirred solution of 2,3,4,5,6-pentafhiorobenzenesulfonamide (0.5g, 2.02 mmol) (WH23) in THF (1.7 mL) were added (4-methoxyphenyl)methanamine (0.24g, 1.82 mmol) and K2CO3 (0.20g, 0.55 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (70 mL) and extracted with ethyl acetate (3 x 50 mL). The combine organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtained the crude. The obtained crude was purified by column chromatography, eluting with 24% ethyl acetate in hexane to afford the title compound as a white solid (0.36g, 0.99 mmol, 49% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1 ): m/z 363.1 (M-l )- (ESI -ve), RT = 2.1 Omin, 100%, 200 - 400nm. 1 H NMR. (400 MHz, DMSO) δ 7.94 (s, 2H), 7.32 (br s, 1H), 7.22 (d, J= 8.4 Hz, 2H), 6.89 (d, J= 8.4 Hz, 2H), 4.45 (d, J= 6.8 Hz, 2H), 3.71 (s, 3H). 19F NMR (376 MHz, DMSO) δ -142.05_-142.18 (m, 2F), -159.64 (d, J= 8.4 Hz, 2F).
Step-2A: Synthesis of 4-amino-2,3,5,6-tetrafluorobenzenesulfonamide
[00706] A solution of 2,3,5, 6-tetrafluoro-4-((4-methoxybenzyl)amino)benzen esulfonamide (0.5g 2.02 mmol) in trifluoroacetic acid (1.6 mL) was stirred at room temperature for 1 hour. The reaction mixture was basified with aqueous NaHCO3 solution (50 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound as a brown semi-solid (0.28g, 1.17 mmol, 84% yield). LCMS: (ACQUITYPDA and QDA detector, UC08_FARl): m/z 243.0 (M-l)- (ESI -ve), RT = 1 ,06min, 100%, 200 - 400nm. 1 H NMR (400 MHz, DMSO) δ 7.92 (s, 2H), 6.76 (s, 2H). 19F NMR (376 MHz, DMSO) δ -142.64_-142.80 (m, 2F), -161.64 -161.80 (m, 2F).
Step-1: Synthesis of tert-butyl (3-((2,3,5,6-tetrafluorophenyl)thio)propyl)carbamate
[00707] To a stirred solution of methyl 4-acetylbenzoate (25.0g, 140.30 mmol) in ethyl acetate (250 mL) was added copper bromide (0.39g, 16.3 mmol) at room temperature. The reaction mixture was stirred at 65°C for 16 hours. The reaction mixture was cooled to room temperature and filtrated through the celite bed. The obtained filtrate was diluted with water (500 mL) and extracted with ethyl acetate (2 x 300 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated using //-Pentane to afford the title compound as a yellow solid (35.5g, 138.69 mmol, 97% yield). TINMR ^OOMHz, DMSO) δ 8.20-8.02 (m, 4H), 3.85 (s, 2H), 3.82 (s, 3H).
Step-2: Synthesis of 3-bromo-l-(2-(4-(methoxycarbonyl)phenyl)-2-oxoethyl)-2- methylpyridin-l-ium bromide
[00708] To a stirred solution of methyl 4-(2-bromoacetyl)benzoate (35.5g, 138.68 mmol) in THF (355 mL) was added 3-bromo-2-methylpyridine (59.64g, 346.7 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 16 hours. The obtained precipitate was filtered off and dried under reduced pressure to afford the title compound as a brown solid (42.2g, 121.26 mmol, 87% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 350.0 (M+2)+ (ESI +ve), RT = 1 ,38min, 96.9%, 200 - 400nm. 1HNMR (400MHz, DMSO) δ 9.04 (q, =2.0 Hz, J2 = 12.0 Hz, 2H), 8.25-8.19 (m, 4H), 8.10-8.04 (m, 1H), 6.78 (s, 2H), 3.93 (s, 3H), 2.84 (s, 3H).
Step-3: Synthesis of methyl 4-(8-bromoindolizine-3-carbonyl)benzoate
[00709] A mixture of dimethyl sulphate (61 ,0g, 483.63 mmol) and DMF (35.3g, 4803.63 mmol) was stirred at 80°C for 2 hours. The resulting reaction mixture was cooled to room temperature and was added into a pre-stirred solution of 3-bromo-l-(2-(4-(methoxycarbonyl)phenyl)-2- oxoethyl)-2-methylpyridin- 1-ium bromide (21.1g, 60.45 mmol) in DMF (73 mL) at 0 °C. The reaction mixture was stirred at room temperature for Ih followed by the addition of DIPEA (84 mL, 483.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into ice-cold water. The obtained precipitate was filtered off and dried under reduced pressure to afford the title compound as a yellow solid (12.0g, 33.61 mmol, 55% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 358.1 (M+l)+ (ESI +ve), RT =2.80min, 96.0%, 200 - 400nms. 1HNMR (400MHz, DMSO) δ 8.32-8.14 (m, 3H), 7.95-7.92 (m, 3H), 7.73 (br s, IH), 7.45 (br s, IH), 7.11-6.76 (m, IH), 2.89 (s, 3H). Step-4: Synthesis of methyl 4-(8-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)indolizine-3- carbonyl)benzoate
[00710] To a stirred solution of methyl 4-(8-bromoindolizine-3-carbonyl)benzoate (11 ,0g, 30.81 mmol) in 1,4-dioxane (11 .0 mL) were added Bis(pinacolato)diboron (11 ,7g, 46.21 mmol) and potassium acetate (9.0g, 92.43 mmol) at room temperature. The reaction mixture was purged with N2 for 15 mins, followed by the addition of PdCl2(dppf)dichloromethane (1.26g, 1.54 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 16 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (200 mL). The resulting suspension was filtered through the celite bed and washed with ethyl acetate (100 mL x 2). The obtained filtrate was concentrated under reduced pressure to afford the title compound as a brown semisolid (22.50g, 55.53 mmol, quantitative yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 406.3 (M+l)+ (ESI +ve), RT = 2.99min, 38.5%, 200 - 400nm.
Step-5: Synthesis of methyl 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)benzoate
[00711] To a stirred solution of 5-bromo-l-methyl-6-(trifhroromethyl)-lH-benzo[d]imidazole (0.58g, 2.08 mmol) (INTI) in a mixture of 1,4-dioxane (9.2 mL) and water (2.3 mL) were added methyl 4-(8-(4,4,5,5-tetramethyl-l ,3,2-dioxaborolan-2-yl)indolizine-3-carbonyl)benzoate (3 ,80g 9.38 mmol) and K2CO3 (0.57g, 4. 17 mmol) at room temp erature . The reaction mixture was purged with N2 for 15 mins, followedby the addition of PdCl2(dppf)dichloromethane (0.04g, 0.05 mmol) at room temperature. The reaction mixture was stirred at 100°C for 2 hours. The reaction mixture was cooled to room temperature and filtered through the celite bed. The obtained filtrate was diluted with water (70 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by column chromatography, eluting with 5% methanol in dichloromethane to afford the title compound as a yellow solid (0.5g, 1 .05 mmol, 50% yield). LCMS: (ACQUITY PDA and QDAdetector, UC08_ ABR2): m/z 478.2 (M+H)+ (ESI +ve), RT = 2.39min, 69.8%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 9.93
Figure imgf000232_0001
2.8 Hz, J 2 = 5.2 Hz, 1H), 8.52 (s, 1H), 8.25 (s, 1H), 8.10 (d, J= 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.77 (s, 1H), 7.53 (d, J= 8.8 Hz, 1H), 7.31-7.26 (m, 2H), 6.07 (d, J= 4.4 Hz, lH), 4.00 (s, 3 H), 3.91 (d, J = 9.2 Hz, 3H). 19F NMR (376 MHz, DMSO) δ -54.95 (s, 3F).
Step-6: Synthesis of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)benzoic acid [00712] To a stirred solution of methyl 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol- 5-yl)indolizine-3-carbonyl)benzoate (0.5g, 1 .04 mmol) in THF (4.0 mL), methanol (0.5 mL) and water (0.5 mL) was added lithium hydroxide (0.13g, 3.14 mmol) at 0°C . The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was acidified using IN HC1 (50 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentratedunder reduced pressure to afford the title compound as a white solid (0.2g, 0.42 mmol, 40% yield). LCMS: (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 464.2 (M+l)+ (ESI +ve), RT = 2.07min, 99.0%, 200 - 400nm. 'H NMR (400 MHz, DMSO) δ 13.21 (br s, 1H), 9.95-9.93 (m, 1H), 8.53 (s, 1H), 8.26 (s, 1H), 8.08 (d , J= 8.0 Hz, 2H), 7.87 (d, J= 8.4 Hz, 2H), 7.77 (s, 1H), 7.32-7.25 (m, 3H), 6.07 (d, J = 4.8 Hz, 1H), 4.00 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.95 (s, 3F).
Step-7: Synthesis of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)-N-(2,3,5,6-tetrafluoro-4-sulfamoylphenyl)benzamide
[00713] To a stirred solution of 4-(8-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)indolizine-3-carbonyl)benzoic acid (0.17g, 0.36 mmol) in DMF (1.7 mL) were added DIPEA (0.14g, 1.10 mmol) and HATU (0.20g, 0.55 mmol) at O °C. The reaction mixture was stirred for lOmins. at 0 °C followed by the addition of 4-amino-2,3,5,6-tetrafluorobenzenesulfonamide (0.17g, 0.73 mmol). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with water (70 mL) and extracted with ethyl acetate (3 x 50 mL). The combine organic portions were dried over anhydrous Na2SO4, filtered, and concentratedunder reduced pressure to obtain a crude residue. The obtained crude was purified by preparativeHPLC on a Shim-Pack Waters X-B ridge (250mm x 19mm 5pm) column eluting with 0.1% aqueous formic acid and acetonitrile, using following method to afford title compound as a yellow solid (0.051g, 0.08 mmol, 20% yield). LCMS: (ACQUITY PDA and QDA detector, UC08_FARl): m/z 688.2 (M-l)- (ESI -ve), RT = 2.16min, 99.5%, 200 - 400nm. HPLC: (Agilent Technologies. 1260 Series, Infinity-II, HP07_trifluoroacetic acidRl): RT = 6.26min, 99.2%, 200-400nm. TI NMR (400 MHz, DMSO) δ 9.94-9.92 (m, 1H), 8.58 (s, 1H), 8.27 (s, 1H), 8.03 (s, J= 8.0 Hz, 2H), 7.84 (d , J= 8.0 Hz, 2H), 7.78 (s, 1H), 7.28 (d, J= 2.4 Hz, 3H), 7.00 (br s, 2H), 6.06 (d, J= 4.4 Hz, 1H), 4.01 (s, 3H). 19F NMR (376 MHz, DMSO) δ -54.99 (s, 3F), -141 .27 (d, J= 17.9 Hz, 2F), -161.86 (s, 2F).
Synthesis of N-(3-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)phenyl)-4-
(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenoxy)benzamide (Compound 247)
Figure imgf000234_0001
Step-1: Synthesis of ethyl 3-nitro-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)benzoate [00714] To a stirred solution of 2,3,5,6-tetrafluoro-4-(methylthio)phenol (2.0g, 9.42 mmol) (WH24) in THF (20 mL) was added K2CO3 (3 ,9g, 28.2 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for lOmins followed by the addition of ethyl 4 -flu oro-3 -nitrobenzo ate (2.0g, 9.42 mmol). The reaction mixture was stirred at 65 °C for 16 hours. The reaction mixture was cooled to room temperature and diluted with water (300 mL). The resulting suspension was extracted with ethyl acetate (2 x 100 mL). The combined organic portions were dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by column chromatography, eluting with 10% ethyl acetate in hexane to afford the title compound as a colourless oil (1.5g, 3.7 mmol, 39% yield). 1HNMR (400 MHz, DMSO) 5 8.58 (dd, Ji = 2.0 Hz, J2 = 5.2 Hz, 1H), 8.18 (dd, Jj = 2.4 Hz, J2= 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 4.36 (
Figure imgf000234_0002
6.8 Hz, J2 = 14.0 Hz, 2H), 2.56 (s, 3H), 1 .35-1 .32 (m, 3H). 19F NMR (376 MHz, DMSO) δ -133.92 (dd, Ji = 7.7 Hz, J2 = 25.1 Hz, 2F), -154.65
Figure imgf000234_0003
= 8.2 Hz, J2 = 24.6 Hz, 2F).
Step-2: Synthesis of ethyl 3-amino-4-(2,3,5,6-tetrafluoro -4-(methylthio)phenoxy) benzoate
[00715] To a stirred solution of ethyl 3-nitro-4-(2,3,5,6-tetrafluoro-4- (methylthio) phenoxy) benzoate (1.50g, 3.70 mmol) in mixture of ethanol (15 mL) and water (2.0 mL) were added Zinc dust (1.93g, 29.61 mmol) and ammonium chloride (1.58g, 29.61 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was filtered through the celite bed and washed with ethanol (200 mL). The filtrate was concentrated under reduced pressure to obtain a residue. The obtained residue was suspended in water (250 mL) and extracted with ethyl acetate (2x 80 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the title compound as a yellow solid (1.2g, 3.2 mmol, 86% yield). LCMS (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 376.1 (M+H)+ (ESI +ve), RT = 2.65min, 72.8%, 200 - 400nm. 'HNMR (400 MHz, DMSO): 5 7.43 (d, J= 2.0 Hz, 1H), 7.08-7.05 (m, 1H), 6.83 (d, J= 8.4 Hz, 1H), 5.55 (s, 2H), 4.26 (q, = 6.8 Hz, J2 = 14.0 HZ, 2H), 2.54 (s, 3H), 1 .29 (t, J= 7.2 Hz, 3H). 19F NMR (376 MHz, DMSO) δ -134.79 (dd, Ji = 8.2 Hz, J2 = 25.0 Hz, 2F) -155.18 (dd, Ji = 7.7
Figure imgf000235_0001
= 25.1 Hz, 2F).
Step-3: Synthesis of ethyl 4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)benzoate
[00716] To a stirred solution of ethyl 3-amino-4-(2,3,5,6-tetrafluoro4- (methylthio)phenoxy)benzoate (1.20g, 3.2 mmol) in 1,4-dioxane (12 mL) was added Isoamyl nitrite (2.1 mL, 1 .6 mmol) at room temperature. The reaction mixture was stirred at 100 °C for 2 hours. The reaction mixture was cooled to room temperature and diluted with water (150 mL). The resulting suspension was extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by column chromatography, eluting with 10% ethyl acetate in hexane to afford the title compound as a yellow solid (0.81g, 2.25 mmol, 70% yield). 'HNMR (400 MHz, DMSO) δ 7.98 (d, J= 8.8 Hz, 2H), 7.28 (d, J = 8.8 Hz, 2H), 4.30 (q, Ji = 6.8 HZ, J2 = 14.0 Hz, 2H), 2.55 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). 19F NMR (376 MHz, DMSO) δ -134.45 (dd, Jr = 8.2 Hz, J2 = 25.2 Hz, 2F), -154.64 (dd, Jr = 8.0 Hz, J2= 25.1Hz, 2F).
Step-4: Synthesis of 4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)benzoic acid
[00717] To a stirred solution of ethyl 4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)benzoate (0.81g, 2.25 mmol) in mixture of THF, ethanol and water (8: 1 : 1, 8.1 mL) was added lithium hydroxide (0.28g, 6.75 mmol) at 0 °C. The reaction mixture was stirred at 70 °C for 4 hours. The reaction mixture was cooled to room temperature and concentratedunderreducepressureto obtain a residue. The obtained residue was acidified with IN HC1 (100 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was triturated using w-pentane to afford the title compound as a light-yellow solid (0.67g, 2.01 mmol, 90% yield). LCMS: (ACQUITYPDA and QDA detector, UC08 FAR1): m/z 331 .0 (M-H)- (ESI -ve), RT = 2.47min, 73.2%, 200- 400nm. TI NMR (400 MHz, DMSO) δ 12.99 (s, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.25 (d, J= 9.2 Hz, 2H), 2.55 (s, 3H). 19F NMR (376 MHz, DMSO) δ -134.48 (dd, Ji = 8.3 Hz, J2 = 25.1 HZ, 2F), -154.66 (dd, Ji = 7.8 Hz, J2 = 25.2 Hz, 2F).
Step-5: Synthesis N-(3-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)phenyl)-4- (2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)benzamide
[00718] To a stirred solution of 4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)benzoic acid (0.2g 0.6 mmol) and 3-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)aniline (0.19g, 0.6 mmol) in pyridine (2.0 mL) (See Compound 237) was added POC13 (0.2 mL, 1.81 mmol) at O °C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with aqueous NaHCO3 solution (100 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by RFC, eluting at 50% acetonitrile in water using 0.1% formic acid as a modifier to afford the title compound as an off-white solid (0.07g, 0.115 mmol, 10% yield). LCMS (ACQUITY PDA and QDA detector, UC08 FAR1): m/z 606.2 (M+H)+ (ESI +ve), RT = 2.70min, 98.9%, 200 - 400nm. 'HNMR (400 MHz, DMSO) δ 10.34 (s, 1H), 8.48 (s, 1H), 8.14 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.82 (s, 2H), 7.60 (s, 1H), 7.40 (t, J= 8.4 Hz, 1H), 7.30 (d, J= 8.8 Hz, 2H), 7.07 (d, J= 7.6 Hz, 1H), 4.O2 (s, 3H), 2.55 (s, 3H). 19F NMR (376 MHz, DMSO) δ -53.43 (s, 3F), -134.56 (dd, = 8.6 Hz, J2 = 24.8 Hz, 2F), -154.73 (dd, J) = 8.0 Hz, J2 = 25.3Hz, 2F).
Step-6: N-(3-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl) phenyl)-4-(2, 3,5,6- tetrafluoro-4-(methylsulfonyl) phenoxy) benzamide
[00719] To a stirred solution of N-(3-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5- yl)phenyl)-4-(2,3,5,6-tetrafluoro-4-(methylthio) phenoxy) benzamide (0.025g, 0.041 mmol) in mixture of THF, methanol and water (8:1 :1, 0.25 mL) was added oxone (0.038g, 0.12 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic portions were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude residue. The obtained crude was purified by Prep. TLC to afford the title compound as an off-white solid (0.03g, 0.047 mmol, 57% yield). LCMS (ACQUITY PDA and QDA detector, UC08_FARl): m/z 638.1 (M+H)+ (ESI +ve), RT = 2.35min, 95.0%, 200 - 400nm. HPLC (Agilent Technologies. 1260 Series, Infinity-II, HP07_trifluoroacetic acidRl): RT = 6.37min, 95.8%, 200-400nm. 1HNMR (400 MHz, DMSO) δ 10.37 (s, 1H), 8.47 (s, 1H), 8.14 (s, 1H), 8.01 (d, J= 8.8 Hz, 2H), 7.82 (s, 2H), 7.60 (s, 1H), 7.43-7.36 (m, 3H), 7.08 (d, J= 7.6 Hz, 1H), 3.97 (s, 3H), 3.55 (s, 3H). 19F NMR (376 MHz, DMSO) δ -53.41 (s, 3F), -138.20 (dd, Ji = 14.7Hz, J2 = 20.9 Hz, 2F), -148.30 _-157.05 (m, 2F).
Example 2: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylsulfinyl)-N-phenyl aniline (Compound 199)
Figure imgf000236_0001
Step-1: Synthesis of l-bromo-2,3,5,6-tetrafluoro-4-(methylsulfinyl) benzene
[00720] (4-bromo-2,3,5,6-tetrafhrorophenyl)(methyl)sulfane (WO2022106897 A2) (1.0 g, 3.630 mmol) was dissolved in MeOH (10.0 mL) at RT and the solution was cooled to 0°C. To the above solution, a mixture of Oxone (2.23 g, 7.27 mmol) in Water (5.0 mL) was added. The reaction mixture was maintained at 0°C for 10 minutes, gradually brought to RT and stirred at RT for 30 minutes. After completion, Methanol was evaporated under vacuum and the residue was diluted with Water (5.0 mL). The aqueous layer was extracted by Ethyl Acetate (3 x 20.0 mL). The combined Ethyl Acetate layers were dried overNa2SO4 and evaporated under reduced pressure. The crude residue was purified by flash silica gel chromatography (Normal-Phase 100-200 silica at 0 to 30% EtOAc in Hexanes) to give l-bromo-2,3,5,6-tetrafluoro-4-(methylsulfmyl) benzene as White solid (722 mg, 68%). LCMS (Method-C): Retention Time: 1.511 min, 97%, 225 nm, ES (+ve): 291.0/292.9 [M+H] +
Step-2: Synthesis of 2,3,5, 6-tetrafluoro-4-(methylsulfinyl)-N-phenyl aniline
[00721] To a stirred solution of l-bromo-2,3,5,6-tetrafluoro-4-(methylsulfmyl) benzene (250 mg 0.85 mmol) and Cyclohexanamine (102 mg, 1 .030 mmol) in Toluene (5.0 mL) was added K3PO4 (546 mg, 2.570 mmol) at room temperature. The reaction mixture was purged with N2 gas for 10 minutes. Pd2dba3 (40 mg, 0.040 mmol) and Xantphos (50 mg, 0.080 mmol) were added and the reaction mixture was again purged with N2 gas for 5 minutes. The reaction mixture was heated at 100°C for 16h. After completion, the reaction mixture was diluted with Water (5.0 mL) and the aqueous layer was extracted with Ethyl acetate (3 x 5 mL). The combined organic phase was dried overNa2SO4 and evaporated under reduced pressure to get crude product. The crude product was purified by column chromatography (Normal-Phase 100-200 silica at 0 to 50% EtOAc in Hexanes) to give the pure product as a brown solid (20.0 mg, 15%). 1HNMR (400 MHz, DMSO- d6) δ 1.07-1.10 (m, 1H), 1.23-1.33 (m, 4H), 1.56-1.59 (m, 1H), 1.70-1.72 (m, 2H), 1.86 (s, 2H), 3.08-3.09 (m, 3H), 3.53 (s, lH), 6.20 (s, 1H). 19F NMR (400 MHz, DMSO-d6) δ -143.62_-143.66 (2F), -158.90 -158.95 (2F). LCMS (Method-C): Retention time: 1.739 min, 99%, 285 nm, ES(+ve): 310.52 [M+H]+. HPLC (Method-59): Retention Time: 5.406, 97.8%, 285 nm.
Example 4: Synthesis of benzyl 4-((2,3,5,6-tetrafluoro-4-
(methylsulfonyl)phenoxy)methyl)piperidine-l-carboxylate (Compound 125)
Figure imgf000237_0001
Step-1: Synthesis of benzyl 4-((2,3,5,6-tetrafluoro-4- (methylthio)phenoxy)methyl)piperidine-l-carboxylate
[00722] To a stirred solution of 2,3,5, 6-tetrafluoro-4-(methylthio)phenol (Example 1, Step 1) (0.250 g, 1.179 mmol) in DMF (2.5 mL) was added K2CO3 (0.488 g, 3.537 mmol), under N2 at RT. benzyl 4-(bromomethyl)piperidine-l -carboxylate (479 mg, 1.535 mmol) was then added and the reaction mixture was heated at 100 °C under microwave condition for Ih. After completion, the reaction mixture was diluted with water (3 mL) and the aqueous layer was extracted with ethyl acetate (3x3 mL). The combined organic phase was dried over Na2SO4 and evaporated under reduced pressure. The crude residue was purified by flash silica gel chromatography. Pure compound was eluted at 5-8% EtOAc/Hexanes and obtained as light yellow solid. (0.404 g, 0.912 mmol, 77%). LCMS (Method-C): Retention time 2.61 min, 93% (ES+): 444.3 [M+H]+.
Step-2: Synthesis of benzyl 4-((2,3,5,6-tetrafluoro-4- (methylsulfonyl)phenoxy)methyl)piperidine-l-carboxylate
[00723] Prepared by the method of Example 1, Step 2 using benzyl 4 -((2, 3, 5, 6 -tetraflu oro4- (methylthio)phenoxy)methyl)piperidine-l -carboxylate (0.150 g, 0.339 mmol). The crude residue was purified by flash silica gel chromatography. Pure compound was eluted at 25% EtOAc/Hexanes and obtained as off-white solid (0. 100g, 0.210 mmol, 62%). 1HNMR(400 MHz, DMSO-d6) δ 7.32-7.39 (m, 5H), 5.07 (s, 2H), 4.28 (d, J= 6.4 Hz, 2H), 4.05 (d, J= 13.2 Hz, 2H), 3.52 (s, 3H), 2.84 (br s, 2H), 1.99 (br s, 1H), 1.75 (d, J = 12.4 Hz, 2H), 1.15-1.24 (m, 2H). 19F NMR (400 MHz, DMSO-d6) δ -139.90_-139.95 (2F), -155.73 -155.78 (2F). LCMS (METHODCS): Retention time 1 .802 min, 100% (ES+): 476.4 [M+H]+. HPLC (Method_59): Retention time 5.693 min, 97.07 %.
Example 13: Synthesis of N-((lS,4S)-4-(2,3,5,6-tetrafluoro-4-(methylsulfonyl)phenoxy) cyclohexyl)-7H-pyrrolo [2,3-d]pyrimidin-4-amine (Compound 264)
Figure imgf000238_0001
Step-1: Synthesis of N-((lS,4S)-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)cyclohexyl)-7- ((2-(trimethylsilyl) ethoxy) methyl)-7H-pyrrolo [2, 3-d] pyrimidin-4-amine
[00724] Intermediate 8 (2.800 g, 6.349 mmol) was dissolved in DMF (30 mL ) at room temperature. To the above stirred solution was added 2,3,5,6-tetrafhroro-4-(methylthio)phenol (Example 1, Step 1) (0.900 g, 4.232 mmol) and the mixture was stirred at room temperature for another 5 minutes. Cs2CO3 (3.400 g, 10.580 mmol) was then added and the reaction vial was sealed and heated at 70 °C for 16 hours. After completion (as monitored by TLC), the reaction mixture was concentrated under reduced pressure to obtain a light yellow viscous liquid. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 15% THF in Hexanes to obtain title compound as an off white solid (1 .100 g, 1 .975 mmol, 29%). LCMS (Method-C): Retention time 2.087 min, 80.81%, 270 nm, ES+: 557.2 [M+H]+
Step-2: Synthesis of N-((lS,4S)-4-(2,3,5,6-tetrafluoro-4- (methylsulfonyl)phenoxy)cyclohexyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo [2,3- d] pyrimidin-4-amine
[00725] Prepared by the Method of Example 1, Step 2 usingN-((l S,4S)-4-(2,3,5,6-tetrafluoro4- (methylthio)phenoxy)cyclohexyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3- d]pyrimidin-4-amine (1.0 g, 1.796 mmol). The resulting crude was purified by silica gel flash chromatography and pure compound was eluted at a gradient 40-45% Ethyl acetate in Hexanes to afford title compound as a colorless liquid (0.300 g, 0.510 mmol, 28%). LCMS (Method-H): Retention time 3.712 min, 85.97%, 202 nm ES+: 589.2 [M+H]+
Step-3: Synthesis of N-((lS,4S)-4-(2,3,5,6-tetrafhioro-4-
(methylsulfonyl)phenoxy)cyclohexyl)-7H-pyrrolo[2,3-d] pyrimidin-4-amine
[00726] Prepared by a method broadly similar to Example 5, Step 2 usingN-((l S,4S)-4-(2,3,5,6- tetrafluoro-4-(methylsulfonyl)phenoxy)cyclohexyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H- pyrrolo[2,3-d] pyrimidin-4-amine (300 mg, 0.5096 mmol). The resulting crude was purified by Reverse Phase column chromatography. Pure compound was eluted at a gradient of 55-65% Ammonia (0.1 Min Water) in Acetonitrile to obtain the title compound as off white solid (20 mg 0.043 mmol, 9%). 1HNMR (400 MHz, DMSO) δ 1.81-1.84 (m, 6H), 2.05-2.08 (m, 2H), 3.43 (s, 3H), 4.18 (m, 1H), 4.79 (m, 1H), 6.63 (m, 1H), 7.05 (m, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.08 (s, 1H), 11.45 (s, 1H). 19F NMR (400 MHz, DMSO-d6) δ -139.81 -139.86 (2F), -154.49_-154.54 (2F). LCMS (Method-H): Retention time 2.587 min, 95.69%, 202 nm, ES (+ve): 459.0 [M+H]+. HPLC (Method-59): Retention Time: 4.299 min, 96.39%, 275nm.
Example 14: Synthesis of N-(l-(2-methoxyethyl)-lH-pyrazol-4-yl)-l-(3-(2,3,5,6-tetrafluoro- 4-(methylsulfonyl)phenoxy)benzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine (Compound 251)
Figure imgf000240_0001
[00727] Prepared by a method broadly similar to Example 7, Step 1 using Intermediate 9 (80 mg 0.219 mmol) and l-bromo-2,3,5,6-tetrafluoro-4-(methylsulfonyl)benzene (67.220 mg, 0.219 mol). The resulting crude was purified by reverse phase column chromatography. Product was eluted at a gradient of 0-60% Ammonia (0.1 M in Water) in Acetonitrile. Combined fraction was lyophilized to afford title compound as off white solid (30 mg, 0.050 mmol, 23%). 1HNMR(400 MHz, DMSO-d6) δ 3.27 (s, 3H), 3.76 (s, 3H), 4.56 (d, J = 4.4 Hz, 2H), 4.81 (s, 2H), 5.53 (s, 2H), 6.81-6.83 (m, 2H), 6.91-6.95 (m, 2H), 7.24 (d, J= 7.6 Hz, 1H), 7.63 (s, 1H), 7.97 (s, 1H), 8.08 (s, 1H), 8.81 (s, 1H). 19F NMR (400 MHz, DMSO-d6) δ -154.00_-154.04 (2F), -138.14 -138.19 (2F). LCMS (Method-J): Retention Time: 3.721 min, 94.98%, 202 nm, ES (+ve) δ 91.8 [M+H| +. HPLC (Method-59): Retention Time: 4.852 min, 97.46%, 230 nm.
Example 15: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2, 3,5,6- tetrafluoro-4-(methylsulfinyl)benzamide (Compound 234)
Figure imgf000240_0002
Step-1: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2,3,5,6- tetrafluoro-4-(methylthio)benzamide
[00728] In a 50 mL RBF previously equipped with a magnetic stirrer and Nitrogen balloon was taken l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine (679 mg, 3.122 mmol) and 2, 3,5,6- tetrafluoro-4 -(methyl thio) benzoic acid (500 mg , 2.082 mmol) in DMF (5 mL) and cooled at 0 °C. To the reaction mixture, triethylamine (1.45 mL, 10.409 mmol) was added and reaction mixture was stirred for 30 minutes at 0 °C. After 30 minutes, T3P (50% solution in Ethyl acetate) (1.986 g, 3.122 mmol) was added and reaction mixture was stirred at room temperature for 30 minutes. After completion, the reaction mixture was diluted with Water (30 mL) and aqueous layer was extracted with Ethyl acetate (3 x 30 mL). The combined organic layer was dried over anhydrous Sodium sulfate filtered and the solvent was removed in vacuo. The crude residue was purified by silica gel flash chromatography and pure compound was eluted at 5% Methanol in Dichloromethane to obtain title compound as off white solid (90 mg, 0.204 mmol, 10%). LCMS (Method-C): Retention time: 1.429 min, 220 nm, 100%, ES(+ve): 440.16 [M+H]+
Step-2: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2, 3,5,6- tetrafluoro-4-(methylsulfinyl)benzamide
[00729] Prepared by the method of Example 1, Step 2 usingN-(l-(7H-pyrrolo[2,3-d]pyrimidin- 4-yl)piperidin-4-yl)-2,3,5,6-tetrafhroro-4-(methylthio)benzamide (90 mg, 0.204 mmol). The resulting crude was purified by Reverse Phase column chromatography. Product was eluted at 40% NH3 (0. IM in Water) in Acetonitrile. Fractions containing pure compound were combined and lyophilized to obtain title compound as (17 mg, 0.037 mmol, 18%). 'H NMR. (400 MHz, DMSO-d6) δ 1.48-1.50 (m, 2H), 1.97-2.00 (m, 2H), 3.18 (s, 3H), 3.23 (s, 2H), 4.13 (bs, 1H), 4.53-4.57 (m, 2H), 6.61 (s, lH), 7.19(s, lH), 8.15 (s, 1H), 9.04 (d, J = 6.8 Hz, 1H), 11.71 (s, 1H). 19F NMR (400 MHz, DMSO-d6) δ -141.04 -141.11 (2F), -140.09_-140.12 (2F). LCMS (Methodes Retention time: 1.286 min, 220 nm, 93.08%, ES(+ve): 456.40 [M+H]+. HPLC (Method-59): Retention Time: 3.146 min, 90.97%, 215 nm.
Example 16: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2, 3,5,6- tetrafluoro-4-(methylsulfinyl)benzenesulfonamide (Compound 233)
Figure imgf000241_0001
Step-1: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-4-bromo- 2,3,5,6-tetrafluorobenzenesulfonamide
[00730] 1- (7H-pyrrolo [2, 3-d] pyrimidin-4-yl) piperidin-4-amine (1.500 g, 6.903 mmol) was dissolved in dry THF (15 mL) at room temperature under Nitrogen atmosphere. The solution was cooled to 0 °C. A solution of NaOH (0.828 g, 20.710 mmol) in Water (4.5 mL) was added to the above reaction mixture and stirred at 0 °C for 5 minutes. A solution of 4-bromo-2, 3,5,6- tetrafluorobenzen esulfonyl chloride (2.486 g, 7.593 mmol) in dry THF (12.5 mL) was then added dropwise at 0 °C. The reaction mixture was gradually brought to room temperature and stirred at room temperature for 1 hour. After completion (as monitored by TLC), the reaction mixture was quenched with Water (30 mL). The aqueous layer was extracted with DCM (2 x 50 mL). The combined organic layer was evaporated under reduced pressure to afford crude product as brown solid. The crude residue was purified by silica gel flash chromatography. Pure compound was eluted at 1.5% MeOH in DCM to obtain title compound as yellow-brown solid (2.400 g, 4.720 mmol, 62%). LCMS (Method-C): Retention Time: 1.607 min, 88.92%, 220nm, ES(+ve): 508.0/510.0 [M+H]+
Step-2: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2, 3,5,6- tetrafluoro-4-(methylthio)benzenesulfonamide
[00731] To a stirred solution of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-4-bromo- 2,3,5,6-tetrafluorobenzenesulfonamide (1.0 g, 1.967 mmol) in dry THF (10 mL) under Argon atmosphere was added MeMgBr (2.0 M solution in THF) (1.48 mL, 2.951 mmol) at -15 °C and stirred for Ih at -15 °C. Sulfur powder (0.189 g, 5.901 mmol) was then added portion wise at -15 °C. Reaction mixture was gradually brought to room temperature and stirred at room temperature for 3 hours. After completion (as monitored by TLC), the reaction mixture was quenched with Water (30 mL). The aqueous layer was extracted with DCM (2 x 50 mL). The combined organic layer was evaporated under reduced pressure to afford crude product as yellow solid. The crude residue was purified by silica gel flash chromatography. Pure compound was eluted at 2% MeOH in DCM to obtain title compound as yellow brown solid (0.800 g, 1.680 mmol, 86%). LCMS (Method-H): Retention Time: 2.943 min, 100%, 202 nm, ES(+ve): 476.0 [M+H] +
Step-3: Synthesis of N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2, 3,5,6- tetrafluoro-4-(methylsulfinyl)benzenesulfonamide
[00732] N-(l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)-2,3,5,6-tetrafluoro-4-
(methylthio)benzenesulfonamide (0.800 g, 1.967 mmol) was dissolved in Methanol (5.6 mL) at room temperature. The solution was cooled to 0 °C. A solution of Oxone (0.907 g, 2.951 mmol) in Water (2.4 ml) was added dropwise to the above reaction mixture. The reaction mixture was gradually brought to room temperature and stirred at room temperature for 5 hours. After completion (as monitored by TLC), the reaction mixture was quenched with aq. Sodium Bicarbonate solution (30 mL). The aqueous layer was extracted with DCM (2 x 50 mL). The combined organic layer was evaporated under reduced pressure to afford crude product as off white solid. The crude residue was purified by silica gel flash chromatography. Pure compound was eluted at 2.5% MeOH in DCM to obtain title compound as off white solid (0.250 g, 0.508 mmol, 30%). 'HNMR (400 MHz, DMSO-d6): 6 1.42-1.51 (m, 2H), 1.83-1.86 (m, 2H), 3.16 (t, J = 12.0 Hz, 2H), 3.23 (s, 3H), 3.63-3.65 (m, IH), 4.56-4.59 (m, 2H), 6.57 (s, IH), 7.18 (s, IH), 8.13 (s, IH), 8.98 (d, .7 = 7.6 Hz, IH), 11.71 (s, IH). 19F NMR (400 MHz, DMSO-d6): 6 -139.02_- 139.12 (2F), 5 -136.89 -136.99 (2F). LCMS (Method-H): Retention Time: 2.343 min, 100%, 202 nm, ES(+ve): 492.2 [M+H]+. HPLC (Method-60): Retention Time: 3.062 min, 96.81%, 290 nm.
Example 17: Synthesis of (4-(4-(7-(8-chloronaphthalen-l-yl)-2-(((S)-l-methylpyrrolidin-2- yl)methoxy)-5,6,7,8-tetrahydropyrido [3,4-d]pyrimidin-4-yl)piperazin-l-yl)-2,3,5,6- tetrafluorophenyl)(imino)(methyl)-16-sulfanone(Compound 270)
Figure imgf000243_0001
[00733] To a microwave vial charged with azanylidene-methyl-oxidanylidene-[2,3,4,5,6- pentakis(fluoranyl)phenyl]-{6}-sulfane (82.0 mg, 334 μmol), (7R)-7-(8-chloranyl-l-naphthyl)-2- [[(l S,2S)-l-methylpyrrolidin-2-yl]methoxy]-4-piperazin-l-yl-6,8-dihydro-5H-pyrido[3,4- d]pyrimidine (247 mg, 502 μmol) and ACN (3.7 mL) was added potassium carbonate (116 mg 836 μmol) atrt. The solution was stirred and heated at 100 °C in the microwave for 3 hrs. The vial was cooled down to room temperature, water was added and extracted with DCM (3x). The combined organic layers were washed with saturatedbrine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resultant solid was purified by reverse phase HPLC to afford the title compound as off-white solid (11 .0 mg, 15.2 pmol, 4.5%). 1HNMR (400 MHz, CDC13) δ 8.46 (s, 1H), 7.78 (d, J= 8.3 Hz, 1H), 7.64 (d, J= 8.2 Hz, 1H), 7.55 (dd, J= 7.6, 1.8 Hz, 1H), 7.52 - 7.44 (m, 1H), 7.37 (dd, J= 8.4, 4. 1 Hz, 1H), 7.26 (d, J= 7.8 Hz, 1H), 4.63 (dd, J = 11.6, 6. 1 Hz, 1H), 4.47 - 4.29 (m, 2H), 3.88 (dd, J= 17.7, 3.5 Hz, 1H), 3.74 (t, J= 8.8 Hz, 2H),
3.59 (s, 3H), 3.49 (s, 2H), 3.41 (d, J = 11.9 Hz, 4H), 3.16 (d, J = 9.2 Hz, 3H), 2.74- 2.70 (m, 3H),
2.59 (s, 3H), 2.22 (s, 2H), 2.03 (s, 1H), 1.93 (s, 2H). 19F NMR (470 MHz, cdcl3) 6 -141 .03 (d, J= 17.8 Hz), -149.49 (d, J = 18.2 Hz).
Example 18: Synthesis 2-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N- (2,3,5,6-tetrafluoro-4-(methylsulfinyl)benzyl)pyrimidine-5-carboxamide (Compound 231)
Figure imgf000244_0001
Step-1: Synthesis of [l-methyl-6-[tris(fluoranyl)methyl]benzimidazol-5-yl]boronic acid
[00734] In an oven-dried microwave vial was added 5-bromo-l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazole(500mg, 1.79 mmol), XPhos Pd G2 (117 mg, 143 μmol), XPhos (137 mg, 287 μmol), tetrahydroxydiboron (482 mg, 5.38 mmol), potassium Acetate (527 mg, 5.38 mmol, 336 pL). The vial was sealed and then evacuated and backfilled with Argon. To the reaction mixture, degassed ethanol was added 0.1M (18 mL) and the resultant mixture was degassed for 10-15 minutes. The reaction mixture was then stirred at 80 °C overnight. The reaction mixture was cooled to room temperature, filtered through a thin pad of Celite (eluting with 50 mL EtOAc), and concentrated under reduced pressure. The reaction mixture was purified using reverse phase Biotage Isolera eluting in gradient from 5-95 % MeCN in water containing 0.1% v/v formic acid to afford the title compound (509 mg, mmol, >98%).1HNMR(400MHz, DMSO) δ 11.00 (s, 2H), 8.55 (s, 1H), 8.32 (s, 1H), 7.88 (d, J= 6.0 Hz, 2H), 3.90 (s, 3H).
Step-2: Synthesis of 2-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)pyrimidine- 5-carboxylic acid
[00735] In a microwave vial, (l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)boronic acid (0.1 g, 409.87 μmol), 2-chloropyrimidine-5-carboxylic acid (77.98 mg, 491.85 μmol), and Pd(PPh3)4 (23.68 mg, 20.49 μmol) were combined. The resulting mixture was purged w/ argon, capped and toluene (1 mL), ethanol (1 mL) and water (0.5 mL) were added. The mixture was stirred at room temperature after which sodium carbonate (2 M, 409.87 pL) was added under argon. The resulting mixture was then stirred at 100 °C overnight after which it was cooled and filtered through a pad of Celite. The filtrate was then concentrated and separated on Cl 8 by Biotage Isolera eluting in a mixture of ACN and Mili-Q water to afford the title compound (0.07 g, 217 pmol, 53% yield).1HNMR (400 MHz, DMSO) δ 9.30 (s, 2H), 8.52 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H), 8.04 (s, 1H), 4.00 (s, 3H). 19F NMR (376 MHz, DMSO) δ -53.32 (3F).
Step-3: 2"(l-methyl"6“(triflMoroniethyl)-lH“benzo[d|imidazol"5“yI)“N“(2,3}5,6“tetraflMorO" 4“(methyIsisifiny0benzyl)pyrimidine-5-carboxamide 2-(l-methyl-6-(trifluoromethyl)-lH- benzo[d]imidazol-5-yl)pyrimidine-5-carboxylic acid
[00736] (0.035 g, 109μmol) in THF (0.4 mL) was stirred under N2 and triethylamine was added (27.5 mg, 271 pmol, 37.9 pL) followed by TBTU (38.4 mg, 119 μmol). The reaction mixture was stirred for 30 minute sand (2,3,5,6-tetrafluoro-4-(methylthio)phenyl)methanamine— methane (1/1) (24.46 mg, 108.61 μmol) was added. The reaction mixturewas stirred overnight, quenched w/ water and extracted w/ DCM three times. The collected organic lay er was dried w/ sodium sulfate, filtered and evaporated under reduced pressure. The crude mixture was separated on Cl 8 by Biotage Isolera eluting in a mixture of Mili-Q water (+0.1% FA) and ACN (+0.1% FA) to afford the title compound (0.015g, 28.3 pmol, 26%). XH NMR (400 MHz, CD3CN) δ 9.18 (s, 2H), 8.47 (s, 2H), 7.84 (s, 1H), 4.73 (d, J = 5.4 Hz, 2H), 3.97 (s, 3H), 2.54 (s, 3H). 19F NMR (376 MHz, CD3CN) δ -55.25 (3F), -137.55 (dd, J = 22.6, 11.8 Hz, 2F), -144.39 (dd, J = 22.8, 11.9 Hz, 2F).
Step-4: Synthesis of 2-(l-methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N-(2, 3,5,6- tetrafluoro-4-(methylsulfinyl)benzyl)pyrimidine-5-carboxamide
[00737] In a micro wave vial, 2-( 1 -m ethy 1 -6 -( tri fl u orometh yl)~ 1 H-benzo [d]imidazol-5 -y I >N- (2,3,5, 6-te traflu oro-4-(m ethy 1 sulfinyl )b enzy I )py ri m i din e- 5 -carb ox amid e (21.0 mg, 39.7 μmol) was dissolved in THF (0.4 mL) , MeOH (0.4 mL) and water (0.4 mL) under N2. The mixture was then cooled to 0 °C and oxone monopersulfate (60.96 mg, 198.33 μmol) was added. The reaction was stirred overnight at room temperature after which saturated Na2CO3 solution was added and the resultant mixture extracted with EtOAc. The organic layers were combined and dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was then purified by preparative HPLC to afford the title compound sulfoxide (3.1 mg, 5.5 pmol, 14%) as well as the corresponding sulfone 2-(l -methyl-6-(trifluoromethyl)-lH-benzo[d]imidazol-5-yl)-N- (2,3,5,6-tetrafluoro-4-(methylsulfonyl)benzyl)pyrimidine-5-carboxamide (4.7 mg, 8.62 pmol, 22%). !H NMR (400 MHz, MeOD) δ 9.26 (s, 2H), 8.46 (s, 1H), 8.17 (s, 1H), 8.07 (s, 1H), 4.82 (s, 2H), 4.05 (s, 3H), 3.25 (s, 3H). 19F NMR (376 MHz, MeOD) δ -56.34 (3F), -142.55 (td, J = 15.7, 5.5 Hz, 2F), -142.95 - -143.12 (m, 2F).
Example 19: Generic experimental procedure Suzuki-Reaction
[00738] The aryl halide (1.00 equiv.), aryl/heteroarylboronic acid (1.5 equiv.), and solvent ( Toluene or 1,4-Dioxane) (4.0 mL for 100 mg aryl halide) were added to a 20-mL pressure vial equipped with a stir bar in air. The reaction mixture was degassed with Nitrogen followed by base (CsF or Na2CO3) (3.0 equiv.) and [Pd(dppf)C12.DCM] (0.1 equiv.) were introduced. Then the pressure vail was sealed and heated at 100 oC for 16 h with vigorous stirring. The mixture was then filtered through a pad of celite (washing with Ethyl acetate), the filtrate concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel or by preparative HPLC. Combined fraction was lyophilized to get the target compounds. The targets were characterized and confirmed by using LCMS/GCMS, 1H NMR and 19F NMR data.
Example 20: Generic experimental procedure Suzuki-Reaction
[00739] The aryl halide (1.00 equiv.), aryl/heteroarylboronic acid (1.5 equiv.), and solvent (Toluene or 1,4-Dioxane) (4.0 mL for 100 mg aryl halide) were added to a 20-mL pressure vial equipped with a stir bar in air. The reaction mixture was degassed with Nitrogen followed by base (CsF or Na2CO3) (3.0 equiv.) and Pd(dppf)C12.DCM] (0.1 equiv.) were introduced. Then the pressure vail was sealed and heated at 100 oC for 16 h with vigorous stirring. The mixture was then filtered through a pad of celite (washing with Ethyl acetate), the filtrate concentrated under reduced pressure. The residue was then purified by column chromatography on silica gel or by preparative HPLC. Combined fraction was lyophilized to get the target compounds. The targets were characterized and confirmed by using LCMS/GCMS, 1H NMR and 19F NMR data.
Example 21: Synthesis of (4-((6,7-dimethoxyquinolin-4-yl)oxy)-2, 3,5,6- tetrafluorophenyl)(imino)(methyl)-16-sulfanone (Compound 276)
Figure imgf000247_0001
Step-1: Synthesis of methyl(perfluorophenyl)sulfane
[00740] In a 100 mL two neck dried round bottom flask under nitrogen atmosphere, 2, 3, 4,5,6- pentafluorobenzenethiol (3 ,0 g, 15 mmol) was dissolved in DCM (30 ml) and cooled to -20 °C. To the reaction mixture, was added triethylamine (4.2 ml, 30 mmol), followed by drop wise addition of methyl iodide (0.93 ml, 15 mmol) at -20 °C. The reaction was stirred at the same temperature for 10 min and progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was quenched with water and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried over anhydrous sodium sulphate and concentrated over rotary evaporator under reduced pressure. The resultant residue was purified by flash chromatography (Isolera) using petroleum ether as an eluting solvent system to provide the title compound (1. 1 g, 5.1 mmol, 34 % yield) as colorless liquid. 1HNMR (4OO MHz, CDC13) δ: 2.50 (s, 3H). 19F NMR (400 MHz, CDC13 ) δ: -133.58 (q, J = 10.3 Hz, 2F), -153.78 (t, J = 20.8 Hz, IF), -161.27 (m, J = 6.1 Hz, 2F)
Step 2: Synthesis of 6,7-dimethoxy-4-(2,3,5,6-tetrafluoro-4-(methylthio)phenoxy)quinoline [00741] In a 100 mL two neck dried round bottom flask under nitrogen atmosphere, 6,7- dimethoxyquinolin-4-ol (100 mg, 0.487 mmol) was dissolved in DMF. Methyl(perfluorophenyl)sulfane (104 mg, 0.49 mmol) and K2CO3 (101 mg, 0.731 mmol) were added subsequently, and the reaction was stirred at 25 °C for 16 h. Reaction progress was monitored by TLC. After completion, the reaction mixture was diluted with ethyl acetate (20 ml x 2) and water (20 x 2). Combined organic layers were dried over anhydrous sodium sulfate and concentrated on a rotary evaporator. The resultant resodie was purified by flash chromatography (Isolera) using ethyl acetate- pet ether as an eluent. The title compound eluted in 30-40% ethyl acetate- petroleum ether to afford 6,7-dimethoxy-4-(2,3,5,6-tetrafluoro4- (methylthio)phenoxy)quinoline (0.06 g, 0.15 mmol, 30.8%)asagum. 1HNMR (400MHz, DMSO ) δ7.92 (d, J = 7.8 Hz, lH), 7.59 (s, lH), 6.54 (s, 1H), 6.21 (d, J = 7.8 Hz, 1H), 3.88 (s, 3H), 3.76 (s, 3H), 2.67 (s, 3H). 19F NMR (377 MHz, DMSO) δ -134.16 (q, J= 10.4 Hz, 2F), -146.97 (q, J= 10.3 Hz, 2F).
Step 3: Synthesis of (4-((6,7-dimethoxyquinolin-4-yl)oxy)-2, 3,5,6- tetrafluorophenyl)(imino)(methyl)-16-sulfanone
[00742] In a 25 mL dry round bottom flask under nitrogen atmosphere, 6,7-dimethoxy-4-(2,3,5,6- tetrafluoro-4-(methylthio)phenoxy)quinoline (60 mg, 0.150 mmol) was dissolved in MeOH (10 ml). To this reaction mixture, phenyl-1-iodanediyl diacetate (110 mg, 0.35 mmol) and ammonium carbonate (22 mg, 0.23 mmol) were added subsequently. The resulting reaction mixture was stirred at 25°C for 16 h. Progress of the reaction was monitored by TLC. After completion of reaction, solvent was evaporated under vacuum and the resultant residue was purified by preparative HPLC (0.1% Formic Acid in water and 0.05% Formic Acid in Acetonitrile) to provide the title compound (7. 5 mg, 0.017 mmol, 12 % yield). 1HNMR (400 MHz, DMSO ) δ 7.92 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 6.54 (s, 1H), 6.21 (d, J= 7.8 Hz, 1H), 3.88 (s, 3H), 3.76 (s, 3H), 2.67 (s, 3H). 19F NMR (376 MHz, DMSO) δ -138.15 (m, J= 17.4 Hz, 2F), -144.73 (m, J= 12.1 Hz, 2F).
[00743] Additional compounds described herein are made using methods similar to those described above. For example, the compounds provided in Table B and Table C were prepared usingmethods similar to those described above. The synthetic methods described above exemplify how to synthesize compounds described herein.
Table B
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Table C
Figure imgf000251_0002
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
IL Biological Evaluation
Examples Bl: In Vitro Cell Viability Studies
[00744] Anti-cancer efficacy of exemplary compounds of this application is assessed in vitro in different cancer cell lines. Cell viability is examined following treatment at various concentration of inhibitor (0.097656-50pM) using a cell Titer-Blue cell viability assay. 1X104 cells (NHF cells)/well are plated in 96-well assay plates in culture medium. All cells are grown in DMEM, IMDM and RPMI-1640 supplemented with 10% FBS. After 24hrs, test compounds and vehicle controls are added to appropriate wells so the final volume is 1 OOpl in each well. The cells are cultured for the desired test exposure period (72hrs) at 37°C and 5%CO2. The assay plates are removed from 37°C incubator and 20pl/well of CellTiter-Blue® Reagent is added. The plates are incubated using standard cell culture conditions for 1-4 hours and the plates are shaken for 10 seconds and record fluorescence at 560/590nm.
Intact mass analysis
[00745] The covalent modification ofthe proteins with the compounds are evaluated using intact mass analysis by liquid chromatography -mass spectrometry instrument (LC-MS/MS).
[00746] The reaction solution (20 pL) is prepared in 96-well plate and contained the protein (2 pM), the compound (10 - 100 pM), HEPES buffer (20 mM, pH 8), 137mMNaCl, 5% DMSO, 1 mM TCEP, 1 mM EDTA, and 5 mM MgCh.. The reaction is allowed to proceed for 24 h at 25 °C. Thereafter, the plate was centrifuged for 1 min at 5,000 *g, and the supernatant was (0.5 pL) was directly injected into the LC/MS/MS for intact protein mass analysis.
[00747] The LC-MS/MS instrument comprises of a Waters G2-XS quadrupole-time of flight (QTof) mass spectrometer and a Waters Acuity Lclass Ultra-High Performance Liquid Chromatography (UPLC) system. The Lclass UPLC system includes a binary solvent manager (BSM), and a Acquity sample manager (SM). The mobile phase consisted of: A) 0.1% (v/v) formic acid in MilliQ water; B) 0.1% (v/v) formic acid acetonitrile. Gradients are run over 5 min and proceeded as follows: A:B, 85 :15, 0.0 - 0.7 min, 85 :15 -> 15:85, 0.7 - 1.5 min, 10:90, 1.5 - 4 min, 10:90 85 :15, 4 - 4.5 min, 85: 15, 4.5 - 5 min. The analytical column is a Waters BEH C4 column 1.7 pm (50 ^ 1 mm) column with pore sizes of 300 A. The TOF MS data is collected in positive ion mode (m/z of 400-2000 Da) using MassLynx software (Waters).
[00748] The spectral deconvolution is performed using UNIFI software (Waters). The added mass of the protein upon covalent modification to cysteine residues are specified. Multiple modification of up to 8 cysteine is allowed. All the adducts with signal intensities of <2% of the base peak are ignored. The % modification is calculated as the adduct signal intensity over the total intensities of the protein peaks and the adducts.
Peptide mapping
[00749] The site(s) of compounds covalent modification on proteins are identified using a peptide mapping analysis by liquid chromatography-mass spectrometry instrument (LC-MS/MS). [00750] The reaction solution (100 pL) is prepared in a 1 ,5-mL Eppendorf tube and contained protein (2-10 pM), the compound (10-100 pM), HEPES buffer (20 mM, pH 8), ), 137 mMNaCl, 5% DMSO, 1 mM TCEP, 1 mM EDTA, and 5 mMMgCh. The reaction is allowed to proceed for 5 - 24 h at 25 °C or 37 °C. Thereafter, the reaction is quenched by the addition of 500 pL of cold acetone and incubated at -20 °C for 2 h. Then, the tube is centrifuged for 10 min at 10,000 *g, and the supernatant is discarded. The pellet is washed by adding 200 pL of cold acetone and centrifugation at 10,000 *g for 10 min. The pellet is re-dissolved in 50 pL of ammonium bicarbonate solution (ABC, 100 mM, pH 7.9) containing 8 M urea. The tube is centrifuged for 10 min at 10,000 *g, and the supernatant is transferred to a new tube. The protein is first reduced by adding 1 .25 pL of 200 mMDTT and incubation at 37 °C for 30 min, then alkylated by adding 1.5 pL of 400 mM iodoacetamide incubation at room temperature for another 20 min. Then the solution is diluted 8 times in ammonium bicarbonate. Sequencing-grade trypsin (Promega) is added at an enzyme-to-protein ratio of 1 :50, and the tube is incubated overnight at 37 °C. After digestion, the solution is acidified by trifluoracetic acid at 0.1%, and tubes are centrifuged at 10,000 xg for 10 min. The supernatant is transferred to an autosampler vial, and 2 pL is injected into the LC-MS/MS for peptide mapping analysis.
[00751] The LC-MS/MS instrument comprises of a Waters G2-XS quadrupole-time of flight (QTof) mass spectrometer and a Waters Acuity M-class Ultra-High Performance Liquid Chromatography (UPLC) system. The M-class UPLC system includes a micro binary solvent manager (pBSM), a micro sample manager (pSM), and an lonKey (iKey) separation system. The mobile phase consisted of: A) 0.1% (v/v) formic acid in MilliQ water; B) 0.1% (v/v) formic acid acetonitrile. Gradients are run over20 min and proceeded as follows: A:B, 97:3, 0.0 - 1 min, 97:3
Figure imgf000259_0001
, , , , , , 97:3, 17.5 -
20 min. The analytical column is a Waters BEH Cl 8 iKey 1 .7 pm (50 x 0.15 mm) column with pore sizes of 150 A. The TOF MSE data is collected in positive ion mode (m/z of 350-2000 Da) using MassLynx software (Waters).
[00752] The peptide mapping analysis is performed using UNIFI software (Waters). Carb amidomethyl (+57 Da) and the compound mass addition upon covalent modification are specified as variable modification to cysteine residues.
[00753] A similar protocol was used to determine binding to lysine residues, such as lysine residues of AURKA. The peptide mapping analysis is performed using UNIFI software (Waters). Carb amidomethyl (+57 Da) and the compound mass addition upon covalent modification are specified as variable modification to lysine residues.
Sample preparation for differential scanning fluorimetry
[00754] KRas proteins at 0.05 mg/ml in buffer Hepes, 147 mMNaCl, 2% glycerol, 5 mMMgCh, 1 mM EDTA, 10 pM GDP, pH 8.0 are incubated in the presence desired concentration of compounds (5% DMSO final concentration) at 37°C for 5 hours. After incubation, Sypro Orange to a final concentration of 5X is added to each sample. 20 pl aliquots of samples are transferred PCR tubes, sealed with caps, and run in a BioRad CFX96 RTPCR thermocycler. In the instrument, the samples are heated at a rate of 1 °C/min from 25 to 90 degrees. Fluorescence readings are taken every 1 degree. The data generated is exported and analyzed using GraphPad-Prism software. The denaturation curves are fit using a Boltzmann sigmoidal equation to calculate melting temperatures (Tm).
Sample preparation for isothermal denaturation
[00755] Protein samples are prepared by diluting stock protein to 0.2 mg/ml in buffer (20 mM Hepes, 147 mM HaCl, 2% glycerol, 5 mM MgCl2, 1 mM EDTA, 1 mM TCEP, pH 8.0). Compounds and controls are added to the desired concentrations keeping DMSO constant at 5%. Sypro Orange to a final concentration of 5Xis added to all samples and 50 pL transferred to a 384 well plate (black, flat bottom). A citation instrument is used to monitor the kinetic of protein unfolding by setting the temperature at 44°C and collecting datapoints every 2 minutes for 18 hours using excitation wavelength of 470 nm and emission 580 nm. The data generated is then plotted and analyzed using GraphPad Prism and fit to a single exponential function to calculate rates of unfolding and half lives.
HTRF assay for KRAS:CRAF protein-protein interaction assay
KRAS
KRAS G12C::cRaf PPI assay protocol
[00756] Interaction between GTP analog (GppNHP) loaded biotinylated Kras G12C protein (Reaction Biology Corporation, aa 2-169) and GST-tagged cRAF protein (Reaction Biology Corporation, aa 2-303) was monitored using HTRF assay. Compounds were tested in a 10 concentration IC50 mode with 3 -fold serial dilutions.7.5 uL of 2x Kras solution in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgC12, 0.5 mM TCEP, 0.005% NP40; 45 nM final assay concentration) were delivered to assay well. Compounds were dispensed using a Beckman Coulter ECHO liquid handler and incubated with Kras protein for 30 minutes at room temperature. Final DMSO concentrationin assay was 1%. 7.5 uLof a mixture containing 2x GST- cRAF, MAb Anti GST-Tb cryptate (Cisbio 61GSTTLB) and Streptavidin-XL665 (Cisbio 610SAXLB) (final assay concentrations were 10 nM, 0.67 nM and 11 .25 nM, respectively) was then added to assay wells and the HTRF signal was measured 2 hours later. Measurements were performedusingaBMGLABTECH PHERAstar with TR-FRET module (Ex/Em 337/665 nm and 337/620 nm). Observed signal was converted to percent binding relative to DMSO control. IC50 determinations were performedin GraphPad 4.0 software using Sigmoidal dose response (variable slope) equation.
HTRF Assay for Tyrosine Kinases (BTK, BMX, EGFR, FGFR4)
BTK [00757] Kinase activity is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC). For BTK, the IX kinase buffer is supplemented with 10 mMMnC12, 5 mMMgC12, 1 pMATPyS, ImM TCEP and 100 fold diluted Supplementary Enzyme Buffer. BTK is purchased fromPromega. 0. 111 ng/pLBTK(1.42 nM)is preincubated inthe absenceor presence of inhibitor at room temperature for 3 hours. Reaction with substrate is then initiated by adding biotinylated substrate and ATP and the reaction is allowed to proceed for 45 min. Final concentration of substrate in the reaction mixture is 1 pM and ATP is 28 pM (reported Km value). The reaction is terminated by adding 62.5 nM SA-XL665 and 100-fold diluted europium labelled antibody (Eu- Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase.
BMX
[00758] Kinase activity is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC). The buffer used is IX kinase buffer supplemented with 2 mM MnC12, 5 mM MgC12, ImM TCEP and 100X diluted Supplementary Enzyme Buffer. BMX is purchased from Promega. 0.333 ng/pL BMX (3.03 nM) is preincubated in the absence or presence of inhibitor at room temperature for 3 hours. The reaction with substrate is then initiated by adding biotinylated substrate and ATP. The concentration of substrate in the reaction mixture is 0.5 pM and ATP is 26 pM (reported Km value). Reaction is terminated by adding 31 .25 nM SA-XL665 and 100 fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase.
EGFR
[00759] Kinase activity is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC). The buffer used is IX kinase buffer supplemented with 2 mM MnC12, 5 mM MgC12, ImM TCEP and 100X diluted Supplementary Enzyme Buffer. EGFR is purchased from Promega. 0.041 ng/pL EGFR (0.46 nM) is preincubated in the absence or presence of inhibitor at room temperature for 3 hours. Reaction with substrate is then initiated by adding biotinylated substrate and ATP. The concentration of substrate in the reaction mixture is 0.5 pM and ATP is 1.57 pM (reported Km value). The reaction is terminated by adding31.25 nMSA-XL665 and 100 fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase. FGFR4
[00760] Kinase activity is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC). The buffer used is IX kinase buffer supplemented with 2 mM MnC12, 5 mM MgC12, 1 mM TCEP and 100X diluted Supplementary Enzyme Buffer. FGFR-4 is purchased from Promega. 0.333 ng/pL FGFR4 (5.12 nM) is preincubated in the absence or presence of inhibitor at room temperature for 3 hours. Reaction with substrate is then initiated by adding biotinylated substrate and ATP. The concentration of substrate in the reaction mixture is 0.5 pM and ATP is 113 pM (reported Km value). The reaction is terminated by adding 31.25 nM SA-XL665 and 100 fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase.
JAK3
[00761] JAK3 Protocol 1 : Kinase activity is monitored using a Lance® Ultra TR-FRET platform from Revvity (TRF0121-M). The bufferused is IX kinase buffer supplemented with 50mM HEPES pH 7.4, ImM EGTA, 10mM MgC12, 50uM TCEP, 0.01% Tween-20. JAK3 is purchased from Carna Bioscience. JAK3 (0.25 nM) is preincubated in the absence or presence of inhibitor at room temperature for 30 min. Reaction with substrate is then initiated by adding LANCE Ultra ULight™-JAK-l (Tyrl 023) Peptide and ATP and incubated for 60 min at room temperature. The concentration of substratein the reaction mixture is 50 nM and the concentration of ATP is 2 uM (reported Km value). The reaction is terminated by adding40 mMEDTA solution. Europium-labeled anti-phosphotyrosine (PT66) antibody (2 nM) is added to the reaction and incubated for 60 min at room temperature. After 60 min of incubation, the fluorescence emission is measured at 615 nm and 665 nm. The ratio of Em 665 nm to Em 615 nm is proportional to the amount of substrate phosphorylated by the kinase.
[00762] JAK3 Protocol 2: Kinase activity was monitored using a HTRF® KinEASE-
TK kit from Cisbio (62TK0PEC). The buffer used was 1 X kinase buffer supplemented with 2 mM MnCh, 5 mM MgCh, ImM TCEP and 100X diluted Supplementary Enzyme Buffer (SEB). 0.2 nM JAK-3 (Promega) was preincubated in the absence or presence of compound at room temperature for 30 minutes. The preincubation times for time dependent tests were done for the following time points: 2 mins, 5 mins, 20 mins, 60 mins and 120 mins. Reaction with substrate was then initiated by adding 0.5 uM biotinylated substrate and 1 .434 uM ATP. The reaction was terminated after 45 minutes by adding 31.25 nM SA-XL665 and 100-fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission was measured at 620 nm and 665 nm using a Tecan Infinite Ml 000 PRO. The ratio of Em 665 nm to Em 620 nm was proportional to the amount of substrate phosphorylated by the kinase.
Inhibitor treatment
[00763] For initial screening, enzymes are pre incubated with compounds at 2 different concentrations (1 and 10 pM) and 0.5% DMSO. Compounds of interest arising from the 2 dose screen are tested together with a known inhibitor (such as Ibrutinib) in a multipoint dose response format with concentrations of compound ranging from 24 pM to 25 pM.
Time-dependent inhibition
BTK
[00764] For the time-dependent inhibition experiments, the activity of BTK kinases is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC) as described above. Compounds showing higher potency during dose response analysis are further evaluated for their change in inhibitory potency over time. Ibrutinib is used as a positive control. The selected compounds are pre incubated with the kinase at 11 different concentrations, ranging from 24 pM to 25 pM final compound concentrations. The pre incubation times are 0, 1, 3, 5, 10, 15, 30 and 45 min. After the preincubation, reactionis initiated by addingbiotinylated substrate and ATP and the reaction is allowed to proceed for 45 min. The concentration of substrate in the reaction mixture is 1 pM and ATP is 28 pM (reported Km value). The reaction is terminated by adding 62.5 nM SA-XL665 and lOOfold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase. Product formation vs pre-incubation time data are fitted to a one phase exponential decay equation using GraphPad Prism. The same product formation vs inhibitor concentration data are also fitted to a log(antagonist) vs. response — Variable slope equation in GraphPad Prism to provide IC50 values at different pre incubation times.
EGFR
[00765] For the time -dep end ent inhibition experiments, the activity of EGFR kinase is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC) as described above. Compounds showing higher potency during dose response analysis against EGFR, are further evaluated fortheir change in inhibitory potency overtime. Ibrutinib is used as a positive control. The selected compounds are pre incubated with the kinase at 11 different concentrations, ranging from 24 pM to 25 pM final compound concentrations. The pre incubation times are 0, 2, 5, 10, 15, 30, 45 and 60 min. After the preincubation, the reaction is initiated by addingbiotinylated substrate and ATP and the reaction is allowed to proceed for 45 min. The concentration of substrate in the reaction mixture is 0.5 pM andATP is 1.57 pM (reported Km value). The reaction is terminated by adding 31 .25 nM SA-XL665 and 100 fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase. Product formation vs preincubation time data are fitted to a one phase exponential decay equation using GraphPad Prism. The same product formation vs inhibitor concentration data are also fitted to a log(antagonist) vs. response — Variable slope equation in GraphPad Prism to provide IC50 values at different pre incubation times.
Cell viability assays (Ramos RAI, A549, K562, MIA PaCa-2)
Ramos RAI
[00766] Ramos RA 1 cells (ATCC) are cultured in RPMI-1640 media (Wisent) supplemented with 10% heat-inactivated FB Sand l% penicillin/streptomycin. Cells are seeded in 96-well plates at 57,000 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Serially -diluted compounds or DMSO alone are added to cells and incubated at 37°C, 5% CO2 for 24 hours. Cell viability is measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’ s protocol. The luminescence signal of each treated well is normalized to the DMSO control well and the medium-only background is subtracted. Cell viability curves and IC50 values are visualized using Prism (GraphPad).
A549
[00767] A549 cells (ATCC) are cultured in Dulbecco’s Modified Eagle’s Medium (Wisent) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells are seeded in 96-well plates at 17,500 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Serially -diluted compounds or DMSO alone are added to cells and incubated at 37°C, 5% CO2 for 24 hours. Cell viability is measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’ s protocol. The luminescence signal of each treated well is normalized to the DMSO control well and the medium-only background is subtracted. Cell viability curves andIC50 values are visualized using Prism (GraphPad).
K562
[00768] K562 cells (ATCC) are cultured in Iscove's Modified Dulbecco’s Medium (IMDM) (Wisent) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells are seeded in 96- well plates at 57,000 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Serially -diluted compounds or DMSO alone are added to cells and incubated at 37°C, 5% CO2 for 24 hours. Cell viability is measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’s protocol. The luminescence signal of each treated well is normalized to the DMSO control well and the medium-only background is subtracted. Cell viability curves and IC50 values are visualized using Prism (GraphPad).
MIA PaCa-2
[00769] MIA PaCa-2 cells are cultured in Dulbecco’s Modified Eagle’s Medium (Wisent) supplemented with 10% FBS, 2.5% horse serum, and 1 % penicillin/streptomycin. Cells are seeded in 96-well plates at a density of 2,500 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Serially -diluted compounds or DMSO alone are added to cells and incubated at 37°C, 5% CO2 for 72 hours. Cell viability is measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer’s protocol. The luminescence signal of each treated well is normalized to the DMSO control well and the media-only background is subtracted. Cell viability curves and IC50 values are visualized using Prism (GraphPad).
ERK1/2 Phosphorylation and Total KRAS assays
MIA PaCa-2 cell lysis and immunoassay to detect KRas and phospho-ERKl /2
[00770] MIA PaCa-2 cells are cultured in Dulbecco’s Modified Eagle’s Medium (Wisent) supplemented with 10% FBS, 2.5% horse serum, and 1 % penicillin/streptomycin. Cells are seeded in 6-well plates at 500,000 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Cells are treated with the indicated concentrations of compound or DMSO alone and incubated at37°C, 5% CO2 for 6 or 24 hours. Conditioned media is discarded, and adherent cells are washed with PBS before they are scraped. Cell suspensions are centrifugated at 2,000 RPM for 5 minutes at 4°C and supernatants are discarded. Cell pellets are resuspended with IX RIPA buffer (Millipore) and incubated on ice for 15 minutes. Protein lysates are extracted by centrifugating at 20,000 RPM for 20 minutes at 4°C. Proteins are separated and total KRas protein levels are quantified by Simple Western Immunoassay (ProteinSimple) using the 2-40 kDa Separation Module for Jess according to the manufacturer’s protocol, using a protein concentration of 0.5 pg/pl and antibodies targeting KRas (clone# 4F3, Sigma) and GAPDH (clone# 14C10, Cell Signaling) diluted to 1 :10 and 1 :1600, respectively, where the latter is used as a loading control. A 3 : 1 ratio of rabbit to mouse HRP-conjugated secondary antibodies is used for detection. KRas protein levels are quantified relative to GAPDH loading levels and subsequently normalized to the DMSO control. Phosphorylated ERK 1/2 levels are quantified by Simple Western Immunoassay (ProteinSimple) using the Protein Normalization Assay Module for Jess according to the manufacturer’s protocol using a protein concentration of 0.5 pg/pl and an antibody targeting phosphor-ERKl/2 (clone# D13.14.4E, Cell Signalling) diluted to 1 :10. Phosphorylated ERK1/2 levels are normalized to the Jess Protein Normalization Reagent loading control and subsequently to the DMSO control.
[00771] MIA PaCa-2 cells are cultured in Dulbecco’s Modified Eagle’s Medium (Wisent) supplemented with 10% FBS, 2.5% horse serum, and 1% penicillin/streptomycin. Cells are seeded in 96-well plates at a density of 25,000 cells/well and incubated at 37°C, 5% CO2 for 24 hours. The next day, cells are starved in cell culture media containing 1% FBS only at 37°C, 5% CO2 for 16 hours. Following starvation, serially-diluted compounds or DMSO alone are added to cells and incubated at 37°C, 5% CO2 for 1 or 3 hours. Prior to cell lysis, 25 ng/ml human epidermal growth factor (hEGF) (Sigma) is added to the cells and incubated at 37°C, 5% CO2 for 10 minutes. Conditioned media is discarded, and adherent cells lysed and basal ERK1/2 phosphorylation levels are measured using the Phospho-ERK (Thr202/Tyr204) Cellular HTRF kit (Perkin Elmer) according to the manufacturer’s protocol. The fluorescent signal of the acceptor antibody at a wavelength of 665 nm is normalized to that of the donor antibody at 620 nm. HTRF ratios are plotted and relative IC50 values are obtained using Prism (GraphPad).
Cell lysis and immunoassays
Ramos RAI cell lysis and immunoassay to detect BTK and phospho-BTK
[00772] Ramos RA 1 cells (ATCC) are cultured in RPMI-1640 media (Wisent) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Cells are seeded in 6-well plates at 1.71 x 106 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Cells are treated with compound at the indicated concentrations or DMSO alone and incubated at 37°C, 5% CO2 for 24 hours. Cell suspensions are centrifugated at 1,400-1,600 RPM for 5 minutes at room temperature and supernatants are discarded. Cell pellets are resuspended with 1XRIPA buffer (Millipore) and incubated on ice for 15 minutes. Protein lysates are extracted by centrifugating at 20,000 RPM for 20 minutes at 4°C. Proteins are separated and total BTK protein levels are quantified by Simple Western Immunoassay (Protein Simple) usingthe 12-230 kDa Jess Separation Module according to the manufacturer’s protocol, using a protein concentration of 0.25 pg/pl and antibodies targeting BTK (clone# D3H5, Cell Signalling) and P-Actin (clone# AC-15, Santa Cruz) diluted to 1 :400 and 1 :10, respectively, where the latter is used as a loading control. An equal ratio of rabbit and mouse HRP-conjugated secondary antibodies is used for detection. BTK protein levels are quantified relative to P-Actin loading levels and subsequently normalized to the DMSO control. Phosphorylated BTK levels are quantifiedusingthe Protein Normalization Assay Module for Jess (Protein Simple) accordingto the manufacturer’s protocol, using a protein concentration of 1.5 pg/pl and an antibody targeting pBTK (Y223) (Cell Signalling) diluted to 1 :50. Phosphorylated BTK levels are quantified relative to the Protein Normalization Reagent and subsequently normalized to the DMSO control.
A549 cell lysis and immunoassay to detect total EGFR and phospho-EGFR
[00773] A549 cells (ATCC) are cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Wisent) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells are seeded in 6-well plates at 530,000 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Cells are starved in DMEM containing 1% FBS for 4 hours before they are treated with the indicated concentrations of compound orDMSO alone and incubated at 37°C, 5% CO2 for 24 hours. Prior to cell harvest and lysis, 50 ng/ml human epidermal growth factor (hEGF) (Sigma) is added to the cells and incubated at 37°C, 5% CO2 for 10 minutes. Conditioned media is discarded, and adherent cells are washed with PBS before they are scraped on ice. Cell suspensions are centrifugated at 2,000 RPM for 5 minutes at 4°C and supernatants are discarded. Cell pellets are resuspended with IX RIPA buffer (Millipore) and incubated on ice for 15 minutes. Protein lysates are extracted by centrifugating at20, 000 RPMfor 20 minutes at4°C. Proteins are separated and total EGFR protein levels are quantified by Simple Western Immunoassay (Protein Simple) using the Protein Normalization Assay Module for Jess according to the manufacturer’s protocol, using a protein concentration of 0.125 pg/pl and an antibody targeting EGFR (clone# EP38Y, Abcam)diluted to 1 :400. Phosphorylated EGFR protein levels are quantified with the same assay using a protein concentration of 1 pg/pl and an antibody targeting pEGFR (Y1068) (Cell Signalling) diluted to 1 :25. EGFR and phosphorylated EGFR levels are normalized to the Jess Protein Normalization Reagent loading control and subsequently to the DMSO control.
K562 cell lysis and immunoassay to detect /3-Tubulin
[00774] K562 cells (ATCC) are cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Wisent) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells are seeded in 6-well plates at 1 ,71xl06 cells/well and incubated at 37°C, 5% CO2 for 16 hours. Cells are treated with compounds at the indicated concentrations orDMSO alone and incubated at 37°C, 5% CO2 for 24 hours. Cell suspensions are centrifugated at 1,400 RPMfor 5 minutes at room temperature and supernatants are discarded. Cell pellets are resuspended with IX RIPA buffer (Millipore) and incubated on ice for 15 minutes. Protein lysates are extracted by centrifugating at 20,000 RPM for 20 minutes at 4°C. Proteins are separated and total P-Tubulin levels are quantified by Simple Western Immunoassay (ProteinSimple) using the Protein Normalization Assay Module for Jess (ProteinSimple) accordingto the manufacturer’s protocol, using a protein concentration of 0.05 pg/pl and an antibody targeting P-Tubulin (Abeam) diluted to 1 :800. P-Tubulin levels are quantified relative to the Protein Normalization Reagent and subsequently normalized to the DMSO control.
Time-dependent inhibition
BTK
[00775] For the time-dependent inhibition experiments, the activity of BTK kinases is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC) as described above. Compounds showing higher potency during dose response analysis are further evaluated for their change in inhibitory potency over time. Ibrutinib is used as a positive control. The selected compounds are pre incubated with the kinase at 11 different concentrations, ranging from 24 pM to 25 pM final compound concentrations. The pre incubation times are 0, 1, 3, 5, 10, 15, 30 and 45 min. After the preincubation, reactionis initiated by addingbiotinylated substrate and ATP and the reaction is allowed to proceed for 45 min. The concentration of substrate in the reaction mixture is 1 pM and ATP is 28 pM (reported Km value). The reaction is terminated by adding 62.5 nM SA-XL665 and lOOfold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase. Product formation vs pre-incubation time data are fitted to a one phase exponential decay equation using GraphPad Prism. The same product formation vs inhibitor concentration data are also fitted to a log(antagonist) vs. response — Variable slope equation in GraphPad Prism to provide IC50 values at different pre incubation times.
EGFR
[00776] For the time -dep end ent inhibition experiments, the activity of EGFR kinase is monitored using a HTRF® KinEASE-TK kit from Cisbio (62TK0PEC) as described above. Compounds showing higher potency during dose response analysis against EGFR, are further evaluated fortheir change in inhibitory potency overtime. Ibrutinib is used as a positive control. The selected compounds are pre incubated with the kinase at 11 different concentrations, ranging from 24 pM to 25 pM final compound concentrations. The pre incubation times are 0, 2, 5, 10, 15, 30, 45 and 60 min. After the preincubation, the reaction is initiated by addingbiotinylated substrate and ATP and the reaction is allowed to proceed for 45 min. The concentration of substrate in the reaction mixture is 0.5 pM andATP is 1.57 pM (reported Km value). The reaction is terminated by adding 31 .25 nM SA-XL665 and 100 fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase. Product formation vs pre- incubation time data are fitted to a one phase exponential decay equation using GraphPad Prism. The same product formation vs inhibitor concentration data are also fitted to a log(antagonist) vs. response — Variable slope equation in GraphPad Prism to provide IC50 values at different pre incubation times.
Jump dilution experiment
BTK
[00777] Compounds, along with ibrutinib as a control compound (used at 10 times their IC50 concentrations) are pre incubated with BTK (at 142 nM or 100-fold the normal assay concentration) for 1 .5 h at room temperature. Sample containing only DMSO vehicle is used as a positive (full activity) control while sample with no enzyme is used as a negative (zero activity) control. After the preincubation, the mixture is diluted 100-fold into a reaction mixture containing 1 pM biotinylated substrate and 28 pM ATP and the reaction is allowed to proceed for various time points (2, 5, 10, 15, 20, 30, 45 and 60 min), at which the reaction is terminated by adding 1 pL of 0.5 M EDTA. 62.5 nM SA-XL665 and 100-fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer, is added as detection mixture. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase. EGFR
[00778] Compounds (used at 10 times their IC50 concentrations) are pre incubated with EGFR (at 46 nM or 100-fold the normal assay concentration) for 1.5 h at room temperature. Sample containing only DMSO vehicle is used as a positive (full activity) control while sample with no enzyme is used as a negative (zero activity) control. After the preincubation, the mixture is diluted 100-fold into a reaction mixture containing 0.5 pM biotinylated substrate and 1.57 pM ATP and the reaction is allowed to proceed for various time points (2, 5, 10, 15, 30, 40, 45 and 60 min), at which the reaction is terminated by adding 1 pL of 0.5 MEDTA. 31.25 nM SA-XL665 and 100- fold diluted europium labelled antibody (Eu-Ab), diluted in IX detection buffer, is added as detection mixture. After 60 min of incubation, the fluorescence emission is measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm is proportional to the amount of substrate phosphorylated by the kinase.
[00779] In some instances, Table 3 demonstrates the (e.g., binding) activity of a compound provided herein.
[00780] In some instances, Table 3 demonstrates the extent to which a compound binds to BTK. In some instances, in vitro binding to BTK shows the extent to which a compound binds to BTK. In some instances, Table 3 shows the extent to which a compound binds to BTK and inhibits phosphorylation of a peptide substrate. In some instances, a compound demonstrates strong binding to BTK when the remaining BTK activity is low. In some instances, in vitro BTK inhibition is shown in Table 3.
Table 3
Figure imgf000269_0001
Figure imgf000270_0001
n.d.: not determined
A: >90%; B: 50-90%; C: <50%
[00781] In some instances, Table 4 demonstrates the extent to which a compound binds to BTK. In some instances, in vitro BTK inhibition shows the extent to which a compound binds to BTK. In some instances, Table 4 shows the extent to which a compound binds to BTK and inhibits phosphorylation of a peptide substrate across a dose response. In some instances, in vitro BTK inhibition is shown in Table 4.
Table 4
Figure imgf000270_0002
n.d.: not determined a: <1000 nM; b: >1000 nM
[00782] In some instances, Table 5 demonstrates the extent to which a compound binds to EGFR. In some instances, in vitro binding to EGFR shows the extent to which a compound binds to EGFR. In some instances, Table 5 shows the extent to which a compound binds to EGFR and inhibits phosphorylation of a peptide substrate. In some instances, a compound demonstrates strong binding to EGFR when the remaining EGFR activity is low. In some instances, in vitro EGFR inhibition is shown in Table 5.
Table 5 μM,
Figure imgf000270_0003
A: >90%; B: 50-90%; C: <50% [00783] In some instances, Table 6 demonstrates the extent to which a compound binds to EGFR. In some instances, in vitro EFGR inhibition shows the extentto which a compoundbinds to EGFR. In some instances, Table 6 shows the extent to which a compound binds to EGFR and inhibits phosphorylation of a peptide substrate across a dose response. In some instances, in vitro EGFR inhibition is shown in Table 6.
Table 6
Figure imgf000271_0001
a: <1000 nM; b: >1000 nM
III. Preparation of Pharmaceutical Dosage Forms
Example Pl : Solution for injection
[00784] The active ingredient is a compound of Table 2A or Table 2B, or a pharmaceutically acceptable saltthereof . A solution for intraperitoneal administration is prepared by mixing 1-1000 mg of active ingredient with 10-50 mLof a solventmixmadeupby 25% dim ethylacetamide, 50% propylene glycol and 25% Tween 80. Filter through millipore sterilizing filter and then distribute in 1 mL amber glass ampoules, performing all the operations under sterile conditions and under nitrogen atmosphere. 1 mL of such solution is mixed with 100 or 200 mL of sterile 5% glucose solution before using intraperitoneally.
IV. Half Life Measurements
Example IV1 : Measurement of GSH and GST Half Lives
[00785] The measurement of glutathione (GSH) and glutathione S-transf erase (GST) half lives was measured via UV-Vis. Test articles are diluted from 5 mM DMSO stock in IMDM buffer containing 10% FBS and 5 mM reduced glutathione (GSH), for a final concentration of the test article of 25 pM. A parallel set of samples is incubated in the absence of GSH.
[00786] At the specified time points, an aliquot is extracted from each sample and quenched over 2 volumes of acetonitrile. After the last time point, all samples are centrifuged at 3500 rpm for 15 minutes, and the supernatant analyzed by UHPLC.
[00787] Percentage remaining is calculated for each sample, from which reaction rate constant and half life are obtained by applying a pseudo first order reaction kinetic model.
[00788] GSH Method A = 3 time point assay (5 OuM compound, 5 mM GSH, 10% FBS, 37°C). [00789] GSH Method B = Full-curve method (5 OuM compound, 5mM GSH, 10% FBS, 37°C) [00790] GSH method C = 3 time point assay(25uM compound, 5mM GSH, 10% FBS, 37°C) Chemical Stability in the Presence of GSH and GST
[00791] Test articles are diluted from 5 mMDMSO stock in IMDM buffer containing 10% FBS, 5 mM reduced glutathione (GSH) and the appropriate amount of glutathione S-transferase (GST; Sigma-Aldrich, PN: G6511) to yield a final activity of 3 U/mL, for a final concentration of the test article of 25 pM.
[00792] At the specified time points, an aliquot is extracted from each sample and quenched over 3 volumes of acetonitrile containing internal standard. After the last time point, all samples are centrifuged at 3500 rpm for 15 minutes, and the supernatant analyzed by LC-MS after appropriate dilution.
[00793] Percentage remaining is calculated for each sample after normalization against internal standard, from which reaction rate constant and half life are obtained by applying a pseudo first order reaction kinetic model.
[00794] The half lives measured are described in Table 7 for selected compounds. In some instances, the data provided in Table 7 demonstrates relative modification of GST or GSH (e.g., in the presence of a compound provided herein) over a period of time.
Table 7
Figure imgf000272_0001
Figure imgf000273_0001
n.d. not determined
Example IV2: Measurement of Whole Blood Half Lives
Whole Blood Stability by LC-MSD
[00795] Test articles are diluted from 10 mMDMSO stock in acetonitrile then to 5 pM in human, mouse, rat whole blood.
[00796] Duplicate samples are incubated for up to 90 min at 37°C and 800 rpm.
Individual samples are quenched by addition of over 3 volumes of acetonitrile containing internal standard. After the last time point, all samples are centrifuged at 3500 rpm for 15 minutes, and the supernatant analyzed by LC-MSD after appropriate dilution.
[00797] Percentage remaining is calculated for each sample after normalization against internal standard, from which reaction rate constant and half life are obtainedby applying a pseudo first order reaction kinetic model.
[00798] The whole blood metabolism half life of selected compounds can be found in Table 8. In some instances, the whole blood half -lives provided in Table 8 represent relative modification of a compound (e.g., by GSH) over a period of time.
Table 8
Figure imgf000274_0001
WRso values and the WR50/IC50 ratio
[00799] The intrinsic reactivity of a given warhead described herein with an adventitious thiol, such as glutathione (GSH), can be expressed as a second order rate constant. However, that second order rate constant can also be used to calculate the proportion of a warhead that would remain, at a set observation time, in its reaction with an equimolar concentration of GSH. This percentage would vary accordingto the concentration of warhead used in the reaction, insofar as the rate of the reaction shows first order dependence on both the concentration of warhead and the concentration of GSH. This provides a sigmoidal curve of percent remaining warhead versus initial concentration of warhead to be prepared, for any given second order rate constant and at any given observation time. The inflection point of this curve reflects the concentration of warhead at which 50% will have disappeared at the set observation time, or the “50% Warhead Reaction” concentration, defined as WR50. For example, see FIG. 5.
[00800] By way of comparison, the IC50 value for a time-dependent irreversible inhibitor, in some instances, reflects the concentration of inhibitor that is necessary to decrease by 50% the endpoint concentration of product formed by an enzyme that has been pre- incubated/incubated with the inhibitor and (then) substrate for set period of time. This IC50 value is related to the binding affinity of the inhibitor, but also the rate constant for the reaction of bound inhibitor with the enzyme.
[00801] Both WR50 and IC50 report concentrations that are required to generate 50% relative reactivity. As such, a comparison of IC50 and WR50 values reflects the relative reactivity of a warhead with the target enzyme and with GSH, respectively. The ratio of WR50/IC50 ratio therefore represents the intrinsic efficiency of on-target reactivity versus off-target reactivity for a given inhibitor.
Second order rate constants
[00802] The reaction of an irreversible inhibitor (aka ‘warhead’, WH) with glutathione anion (GS ) can be represented as the following kinetic scheme:
Figure imgf000275_0003
[00803] whose rate law can be written as: v = k2 [WH][GS-]
[00804] Experimentally, 25 pM of various compounds described herein were incubated at 37 °C in IMDM (pH 7.4) in the presence of a large excess concentration (5 mM) of glutathione (GSH). Under these pseudo-first order conditions, the disappearance of WH was followed over time and fitted to a mono-exponential curve, giving the pseudo-first order rate constant:
Figure imgf000275_0001
[00805] The concentration of glutathione anion (GS-) can be calculated by multiplying the total concentration of GSH by the fraction of thiolate, which in turn can be calculated from the p/fa of GSH (8.7) and the pH of the solution (7.4) as follows:
Figure imgf000275_0002
[00806] Corrected second order rate constants were then calculated by dividing the observed pseudo-first order rate constants by the concentration of glutathione anion:
Figure imgf000276_0001
Reaction progress curves
[00807] Once a second order rate constant has been measured, a reaction progress curve can be constructed as a means of comparing reactivities. Under equimolar conditions, wherein the initial concentrations of WH and GSH are equal, the integrated second order rate law can be written as:
Figure imgf000276_0004
[00808] from which the following explicit equation can be written, for the concentration of WH that would be observed at time Z0bs:
Figure imgf000276_0002
[00809] Expressing remaining warhead (WH) as a percentage of the initial concentration ([WH]0) gives the following equation:
Figure imgf000276_0005
[00810] This equation can be used to generate a plot of the percent remaining warhead that would be observed at time Cbs, depending on the initial concentration of warhead, [WH]0 (e.g., see FIG. 5). The initial concentration of warhead that leads to 50% warhead remaining at the time of observation, WR50, can thus be taken as a measure of reactivity, for comparison with IC50 curves.
WR50 Values
[00811] The value of WR50 can also be calculated directly from the second order rate constant and time of observation. Knowing that [WH]0 = WR50 when the observed concentration of WH is 50% of the initial concentration at Cbs, the following can be written:
Figure imgf000276_0003
[00812] from which, the following can be shown:
Figure imgf000276_0006
[00813] Thus, once the observation time has been set, the WR50 for a compound described herein can be calculated from any measured second order rate constant. [00814] For example, provided in Tables 9A-9F below are WR50 values for compounds described herein. The data provided below shows that the compounds described herein have reduced reactivity with glutathione (GSH), such as in comparison to a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof). Additionally, the data provided below demonstrates that the compounds described herein lack covalent binding to GSH. Moreover, the data provided below demonstrates thatthe compounds described hereinbind to and/or (e.g., covalently) modify a target protein, such as a target protein described herein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or amutantthereof) withoutthe compound substantially covalently binding to GSH. Furthermore, the data provided below demonstrates compounds that are selective for a target protein, such as a target protein described herein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3 , KRAS, or a mutant thereof) relative to GSH. In some cases, the compounds provided herein contact, bind to, and/or modify a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) in the absence of any binding, including covalent binding, of GSH to the compounds. In some cases, compounds described herein are 10-fold or more selective for a target protein, such as a target protein described herein (e g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) relative to GSH. In some cases, compounds described herein are 100-fold or more selective for a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) relative to GSH. In some cases, compounds described herein are 1000-fold or more selective for a target protein, such as a target protein described herein (e.g, BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, KRAS, or a mutant thereof) relative to GSH.
EGFR
Table 9A
Figure imgf000277_0001
EGFR assay - HTRF® KinEASE-TK kit 3h pre-incubation with compound, 45 mins enzymatic reaction time, 60 min detection mix = Total 285min
GSH Method A = 3 time point assay (50uM compound, 5mM GSH, 10% FBS, 37°C)
GSH Method B = Full-curve method (50uM compound, 5mM GSH, 10% FBS, 37°C)
JAK3 Table 9B
Figure imgf000278_0001
JAK3 assay - HTRF® KinEASE-TK kit 30min pre-incub ation with compound, 45 mins enzymatic reaction time, 60 min detection mix - total = 135mins
GSH method C = 3 time point assay Syngene (25uM compound, 5mM GSH, 10% FBS, 37°C) nd: not determined
FGFR4 and cKIT
Table 9C
Figure imgf000278_0002
FGFR4 assay - HTRF® KinEASE-TK kit 30min pre-incub ation with compound, 45 mins enzymatic reaction time, 60 min detection mix - total = 135mins cKIT assay - HTRF® KinEASE-TK kit30min pre-incub ation with compound, 45 mins enzymatic reaction time, 30 min detection mix - total = 105mins nd: not determined
KRAS Table 9D
Figure imgf000279_0001
Figure imgf000280_0001
nd: not determined
TEAD
Table 9E
Figure imgf000280_0002
TEAD IMA luM TEAD4, lOuM compound, 25°C, 4h nd: not determined
GSH/GST
Table 9F
Figure imgf000280_0003
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
nd: not determined
[00815] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMS What is claimed is:
1. A compound having a structure of Formula (A):
Figure imgf000284_0001
Formula (A) or a salt thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
Q1 is Rx or L-G;
Q2 is L-G or Y2;
Y2 is a blocking group (e.g., a group that directs (e.g., covalent and/or irreversible) binding (of a cysteine residue) of a protein to a position other than Y2);
Rx is substituted or unsubstituted alkyl or NRyRz;
RA, Ry, and Rz are each independently hydrogen or substituted or un substituted alkyl;
L is a linker; and
G is an organic residue; wherein one and only one of Q1 or Q2 is L-G.
2. The compound of claim 1 , wherein Q1 is not substituted or unsubstituted methoxy phenyl.
3. The compound of claim 1, wherein Y2 is hydrogen, CN, CF3, or NO2.
4. The compound of any one of the preceding claims, wherein Q2 is L-G.
5. The compound of any one of the preceding claims, wherein Q1 is Rx.
6. A compound having a structure of Formula (II):
Figure imgf000284_0002
Formula (II) or a salt thereof, wherein,
X1 is absent or O;
X2 is absent, O, or NRA;
RA is hydrogen or substituted or unsubstituted alkyl;
Rxa is substituted or unsubstituted alkyl or NRXRZ;
Ry and Rz are each independently hydrogen or substituted or unsubstituted alkyl;
L is a linker; and
G is a protein-binding ligand (e.g., a radical of a compound that interacts with a protein or a mutant thereof, comprising one or more cyclic group wherein the one or more cyclic groups are individually linked by one or more linker).
7. The compound of any one of claims 1-6, wherein X1 is absent.
8. The compound of any one of claims 1-6, wherein X1 is O.
9. The compound of any one of claims 1-8, wherein X is absent.
10. The compound of any one of claims 1-8, wherein X is O.
11 . The compound of any one of claims 1-8, wherein X is NRA.
12. The compound of any one of claims 1-11, wherein RA is hydrogen (e.g., NH).
13. The compound of any one of claims 1-12, wherein Rxa is unsubstituted alkyl.
14. The compound of any one of claims 1-12, wherein Rxa is Ci-C6 alkyl.
15. The compound of any one of claims 1-12, wherein Rxa is methyl.
16. The compound of any one of claims 1-5 and 8-15, wherein X1 is O and Rxa is methyl.
17. The compound of any one of claims 1-5 and 8-15, wherein X1 is absent and Rxa is methyl.
18. The compound of claim 17, wherein X is NH.
19. The compound of any one of the preceding claims, wherein at least one of X1 or X2 is O.
20. The compound of any one of the preceding claims, wherein X1 is absent, X2 is O, and Rxa is methyl.
1 2
21 . The compound of any one of the preceding claims, wherein X is absent, X is absent, and Rxa is methyl.
22. The compound of any one of the preceding claims, wherein X1 is O and Rxa is methyl.
1 2
23. The compound of any one of the preceding claims, wherein X is O, X is O, and Rxa is methyl.
24. The compound of any one of claims 1-12, wherein Rxa is substituted alkyl (e.g., alkyl substituted with oxo and amino, such as -C(O)NH2).
25. The compound of any one of the preceding claims, wherein the compound(e.g., covalently and/or irreversibly) interacts with a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, KRAS, TEAD, JAK3) or a mutant thereof (e.g., a cysteine residue of the protein or the mutant thereof) at a position ortho or meta to L.
Figure imgf000286_0001
26. The compound of any one of the preceding claims, wherein K directs (e.g., covalently and/or irreversibly) binding (e.g., of a cysteine residue) of a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, or KRAS) or a mutant thereof to a position ortho or meta to L.
27. The compound of any one of the preceding claims, wherein G comprises an optionally substituted cyclic group, optionally substituted with one or more L’-G’, wherein eachL’ is individually selected from a linker and is connected to another G’.
28. The compound of any one of the preceding claims, wherein G comprises an optionally substituted cyclic group, optionally substituted with -(L’-G’)n-L’-G’, wherein n is 0 to 4.
29. The compound of claim 28, wherein n is 1 to 3.
30. The compound of any one of the preceding claims, wherein G or G’ is a radical of a compound that interacts with a protein (e.g., BTK, EGFR, FGFR, AURKA, KIT, BMX, TEAD, JAK3, or KRAS) or a mutant thereof.
31 . The compound of any one of the preceding claims, wherein G or G’ is amino, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, or comprises one or more cyclic groups wherein the one or more cyclic groups are individually linked by one or more linker.
32. The compound of any one of the preceding claims, wherein G or G’ comprises one or more cyclic group wherein the one or more cyclic groups are individually linked by one or more linker.
33. The compound of any one of the preceding claims, wherein G or G’ comprises a sub stituted or unsubstituted carbocycle.
34. The compound of any one of the preceding claims, wherein G or G’ is a substituted carbocycle.
35. The compound of any one of the preceding claims , wherein G or G’ is a sub stituted phenyl.
36. The compound of any one of the preceding claims, wherein G or G’ comprises a sub stituted or unsubstituted heterocycle.
37. The compound of any one of claim 36, wherein G or G’ comprises one or more (e.g., one, two, or three) nitrogen atoms (e.g., within its (e.g., fused) ring system).
38. The compound of any one of the preceding claims, wherein G or G’ comprises one or more (e.g., fused) rings.
39. The compound of any one ofthe preceding claims, wherein G or G’ is aromatic orpartially aromatic.
40. The compound of any one of the preceding claims, wherein G or G’ comprises one or more (e.g., one, two, or three) substituted or unsubstituted (e.g., fused) aromatic ring(s).
41 . The compound of any one of the preceding claims, wherein G or G’ comprises one or more (e.g., one, two, or three) substituted or unsubstituted heteroaromatic ring(s).
42. The compound of any one ofthe preceding claims, wherein G or G’ comprises two or more substituted or unsubstituted aromatic or partially aromatic rings, each aromatic or partially aromatic ring independently being a carbocycle or a heterocycle.
43. The compound of any one of the preceding claims, wherein G or G’ comprises one or more substituted or unsubstituted carbocycle and one or more substituted or unsubstitued heterocycle, each of the one or more substituted or unsubstituted carbocycle and the one or more substituted or unsubstitued heterocycle independently being linked (e.g., fused) to a substituted or unsubstituted carbocycle or a substituted or unsubstitued heterocycle by a bond.
44. The compound of any one of the preceding claims, wherein G or G’ comprises a substituted or un substituted carbocycle and a substituted or unsubstitued heterocycle, the substituted or unsubstituted carbocycle and the substituted or unsubstitued heterocycle being linked (e.g., fused) by a bond.
45. The compound of any one of the preceding claims, wherein G or G’ has a structure of:
Figure imgf000287_0001
46. The compound of claim 44, wherein G or G’ has a structure of:
Figure imgf000287_0002
47. The compound of any one of claims 1-43, wherein G or G’ comprises two (or more) substituted or unsubstituted heteroaromatic rings, the heteroaromatic rings being linked (e.g., fused) by a bond, each heteroaromatic ring being aromatic or partially aromatic.
48. The compound of claim 47, wherein the heteroaromatic rings are selected from the group consisting of benzimidazole, indolizine, quinoline, indazole, and pyrimidine.
49. The compound of claim 47 or 48, wherein G or G’ has a structure of:
Figure imgf000288_0001
50. The compound of claim 47 or 48, wherein G or G’ has a structure of:
Figure imgf000288_0002
51. The compound of claim 47 or 48, wherein G or G’ has a structure of:
Figure imgf000288_0003
52. The compound of any one of the preceding claims, wherein G or G’ is a substituted heterocycle.
53. The compound of any one of the preceding claims, wherein G or G’ is a substituted or unsubstituted N-heterocycle.
54. The compound of claim 52 or 53, wherein G or G’ is a substituted pyrazolopyrimidine (e.g., a lH-pyrazolo[3,4-d]pyrimidine).
55. The compound of claim 54, wherein G or G’ has a structure of:
Figure imgf000288_0004
56. The compound of claim 52 or 53, wherein G or G’ is an unsubstituted pyrrolopyrimidine (e.g., a 7H-pyrrolo[2,3-J]pyrimidine).
57. The compound of claim 56, wherein G or G’ has a structure of:
Figure imgf000289_0001
58. The compound of claim 52, wherein G or G’ is an unsubstituted benzo thiophene or an unsubstituted thiophene.
59. The compound of claim 52, wherein G or G’ is substituted isoxazole (e.g., 3, 5- dimethylisoxazole).
60. The compound of any one of the preceding claims, wherein G or G’ is a substituted quinazoline.
61. The compound of any one of the preceding claims, wherein G or G’ is a substituted or unsubstituted indazole.
62. The compound of any one of the preceding claims, wherein G or G’ is a substituted or unsubstituted pyridine or pyrimidine.
63. The compound of any one of the preceding claims, wherein G or G’ has a structure of:
Figure imgf000289_0002
64. The compound of any one of the preceding claims, wherein G or G’ is a substituted quinazoline, a substituted tetrahydropyridopyrimidine (e.g., 5,6,7,8-tetrahydropyrido[3,4- d]pyrimidine), a substituted quinoline, a substituted pyridopyrazinone (e.g., pyrido[2,3- b]pyrazin-3(4H)-one).
65. The compound of any one of the preceding claims, wherein G or G’ is a substituted quinazoline.
66. The compound of any one of the preceding claims, wherein G or G’ has a structure of:
Figure imgf000289_0003
67. The compound of any one of the preceding claims, wherein G or G’ is a substituted tetrahydropyridopyrimidine (e.g., 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine).
68. The compound of claim 67, wherein G has a structure of:
Figure imgf000290_0001
69. The compound of any one of the preceding claims, wherein G or G’ is a substituted quinoline.
70. The compound of claim 69, wherein G has a structure of:
Figure imgf000290_0002
71. The compound of any one of the preceding claims, wherein G or G’ is a substituted pyridopyrazinone (e.g., pyrido[2,3-b]pyrazin-3(4H)-one).
72. The compound of claim 71, wherein G or G’ has a structure of:
Figure imgf000290_0003
73. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is a bond, -O-, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted sulfoxide, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamide, substituted or unsubstituted heteroalkyl(ene), substituted or un substituted carbocyclyl, or substituted or unsubstituted heterocyclyl.
74. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is a bond, -O-, amino, substituted or unsubstituted alkyl(ene), substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocyclyl.
75. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is amino, substituted or unsubstituted sulfoxide, substituted or unsubstituted sulfonyl, substituted or un substituted sulfonamide, substituted or unsubstituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl.
76. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is amino, substituted or unsubstituted heteroalkyl(ene), or substituted or unsubstituted heterocyclyl.
77. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) comprises one or more N atom(s).
78. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is amino.
79. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is - NR2-, where R2 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl.
80. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is - NH- or -NCH3-.
81. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is substituted or unsubstituted alkylamine.
82. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is substituted alkylamine, the alkylamine being substituted with oxo (e.g., -NHC(O)-).
83. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is - NR4-R3-, -NR4-R3-NR7a-, -NR4-R3-O-, or -C(O)NR4-R3-, where R3 is substituted or unsubstituted alkyl(ene), substituted or unsubstituted heteroalkyl(ene), substituted or unsubstituted carbocyclyl, or substituted or unsubstituted heterocycyl, R4 is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted carbocyclyl, and R7a is H or substituted or unsubstituted alkyl (e.g., alkyl substituted with oxo).
84. The compound of any one of the preceding claims, wherein L or L’ is -NH(heterocyclyl)-
85. The compound of any one of the preceding claims, wherein L or L’ is -NH(azetidinyl)-.
86. The compound of any one of the preceding claims, wherein L or L’ is - C(O)NH(un substituted alkyl)-.
87. The compound of any one of the preceding claims, wherein L orL’ is -C(O)NH(methyl)- , -C(O)NH(ethyl)-, or -C(O)NH(propyl)-.
88. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is methylamine, ethylamine, or propylamine.
89. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is substituted or unsubstituted heterocyclyl.
90. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) is - NR5R6-NR7-, -NR5R6-C(O)-, -NR5R6-CH2NR7C(O)-, -C(O)NR5R6-NR7-, -C(O)NR5R6- O-, or -NR5R6-O-, where R5 and R6 are taken together to form a substituted or unsubstituted heterocyclyl, and R7 is H or substituted or unsubstituted alkyl (e.g., alkyl substituted with oxo).
91 . The compound of claim 90, wherein -NR5R6- is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl), substituted or unsubstituted piperidinyl, or substituted or unsubstituted azetidinyl.
92. The compound of claim 90, wherein -NR5R6- is substituted or unsubstituted piperidinyl or substituted or unsubstituted azetidinyl and -NR7- is -NH- or -NCH3-.
93. The compound of any one of the preceding claims, wherein L is substituted or unsubstituted piperazinyl (e.g., piperazinyl substituted with methyl).
94. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) comprises two N atom(s) (e.g., (substituted or unsubstituted) diaminoalkyl, (substituted or unsubstituted) diamino-cycloalkyl, (substituted or unsubstituted) amino-heterocyclyl (e.g, the heterocyclyl being nitrogen containing), (substituted or un substituted) heterocyclyl (e.g., containing2 nitrogen atoms), the heterocyclyl being optionally fused or spirocyclic).
95. The compound of any one of the preceding claims, wherein the linker (e.g., L or L’) comprises one or more (e.g., fused or spirocyclic) rings.
96. The compound of any one of the preceding claims, wherein L or L’ is or comprises a spirocyclic ring, such as l,7-diazaspiro[4.4]nonane or l,6-diazaspiro[3.3]heptane.
97. The compound of any one of the preceding claims, wherein L or L’ is or comprises substituted or unsubstituted phenyl (e.g., phenyl substituted with halo or cyano).
98. The compound of claim 97, wherein L or L’ is -NH-(unsubstituted phenyl)-, -NH- (un substituted phenyl)-NH-, -NH-(unsubstituted phenyl)-C(O)-, -CH2NH-(unsubstituted phenyl)-C(O)-, -CH2-(unsubstituted phenyl)-NH-, -NHCH2-(unsubstituted phenyl)-C(O)- , -NHC(O)-(un substituted phenyl)-C(O)-, -C(O)NH-(substituted phenyl)-C(O)-, - C(O)NH-(substituted phenyl)-C(O)NH-, or -O-(unsubstituted phenyl)-C(O)NH-.
99. The compound of claim 97 or 98, wherein L or L’ is:
Figure imgf000293_0001
100. The compound of any one of the preceding claims, wherein the linker (e.g., L or
L’) is a bond.
101. The compound of any one of the preceding claims, wherein the compound does not covalently bind with (e.g., the thiol of) GSH.
102. A compound of Table 2A, Table 2B, Table 2C, or Table 2D.
103. A pharmaceutically acceptable composition comprising a compound of any one of the preceding claims, or a salt thereof, and one or more pharmaceutically acceptable excipients.
104. A protein modified with a compound of any one of the preceding claims, or a salt thereof, wherein the compound forms a covalentbond with a sulfur atom of a cysteine residue of the protein.
105. A method of modifying (e.g., attaching to and/or degrading) a polypeptide with a compound, comprising contacting the polypeptide with a compound of any one of the preceding claims, ora salt thereof, to form a covalentbond with a sulfur atom of a cysteine residue of the polypeptide.
106. A method of binding a compound to a polypeptide (e.g., a protein), comprising contacting the polypeptide with a compound of any one of the preceding claims, or a salt thereof.
107. A method of disrupting a polypeptide (e.g., the function thereof), comprising contacting the polypeptide with a compound of any one of the preceding claims, or a salt thereof.
108. A method of modifying a polypeptide with a compound, comprising contactingthe polypeptide with the compound of any one of the preceding claims, or a salt thereof.
109. The method of any one of claims 105-107, wherein the compound contacts the polypeptide intracellularly (e.g., in an individual).
110. A method for (e.g., selectively) modifying a polypeptide (e.g., a protein) with a compound of any one of claims 1-102, the method comprising contacting the polypeptide with the compound, the compound having reduced reactivity with glutathione (GSH).
111. A method for (e.g., selectively) modifying a polypeptide (e.g., a protein) in the presence of glutathione (GSH) with a compound of any one of claims 1-102, the method comprising contacting the polypeptide with the compound (e.g., to form a covalent bond with (e.g., a sulfur atom of a cysteine residue of) the polypeptide) without the compound substantially covalently binding to GSH (e.g., as demonstrated by the lack of covalent binding of GSH to the compound).
112. A method for (e.g., selectively) modifying a polypeptide (e.g., a protein) in the presence of glutathione (GSH) with a compound of any one of claims 1-102, the method comprising contacting the polypeptide with the compound, wherein the compound is selective for the polypeptide relative to GSH.
113. The method of any one of the preceding claims, wherein the polypeptide covalently binds to the compound (e.g., wherein the polypeptide comprises a thiol (e.g., a cysteine residue) that covalently binds to the compound).
114. The method of any one of the preceding claims, wherein the polypeptide contacts (e.g., covalently binds) the compound in the absence of covalent binding of GSH to the compound.
115. The method of any one of the preceding claims, wherein the compound is selective for (e.g., covalent) binding to the polypeptide relative to GSH.
116. The method of any one of the preceding claims, wherein the compound is selective for the polypeptide relative to GSH at a ratio of at least 10: 1 (e.g., 20: 1 or more, 50:1 or more, 100:1 or more, 500: 1 or more, 1000: 1 or more).
117. The method of any one of the preceding claims, wherein the compound has an IC50 for the polypeptide of at least 10 pM and a GSH half life ofgreater than about 10 minutes (e.g., IC50 of at least 1 pM and a GSH half life of greater than about 10 minutes (e.g., IC50 of at least 0. 1 pM and a GSH half life of greater than about 100 minutes)).
118. The method of any one of the preceding claims, wherein the compound is at least 2-fold (e.g., 2-fold or more, 5-fold or more, 10-fold or more, 25 -fold or more) selective for the polypeptide relative to GSH.
119. The method of any one of the preceding claims, wherein the polypeptide is Bruton’s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), proto-oncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), KRAS, or a mutant thereof.
120. The method of any one of the preceding claims, further comprising inhibiting, deactivating, or degrading the polypeptide.
121. A pharmaceutical composition comprising a compound of any one of claims 1- 102, the compound having reduced reactivity with GSH.
122. A pharmaceutical composition comprising a compound of any one of claims 1- 102, wherein the compound is selective for a polypeptide (e.g., a protein) relative to glutathione (GSH).
123. A pharmaceutical composition that is (e.g., at least partially) stable to glutathione (GSH), the composition comprising a compound of any one of claims 1-102.
124. The pharmaceutical composition of any one of claims 121-123, wherein the pharmaceutical composition is (e.g., at least partially) stable to glutathione (GSH) in the presence of a polypeptide.
125. The pharmaceutical composition of any one of claims 121-124, wherein the compound is selective for the polypeptide over GSH.
126. The pharmaceutical composition of any one of claims 121-125, wherein the compoundis selective for the polypeptide relative to GSH at a ratio of atleast 10:1 (e.g., 20:1 or more, 50:1 or more, 100:1 or more, 500:1 or more, 1000:1 or more).
127. The pharmaceutical composition of any one of claims 121-126, wherein the compoundis selective for the polypeptide relative to GSH ata ratio of about 10:1 to about 100:1.
128. The pharmaceutical composition of any one of claims 121-127, wherein the polypeptide comprises Bruton’ s tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), aurora kinase A (AURKA), protooncogene c-KIT (KIT), BMX non-receptor tyrosine kinase (BMX), transcriptional enhancer factor TEF (TEAD), Janus kinase 3 (JAK3), KRAS, or a mutant thereof.
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