Patent Application
20240390340
This disclosure provides compounds as well as their compositions and methods of use. The compounds modulate KRAS activity and are useful in the treatment of various diseases including cancer.
Ras proteins are part of the family of small GTPases that are activated by growth factors and various extracellular stimuli. The Ras family regulates intracellular signaling pathways responsible for growth, migration, survival and differentiation of cells. Activation of Ras proteins at the cell membrane results in the binding of key effectors and initiation of a cascade of intracellular signaling pathways within the cell, including the RAF and PI3K kinase pathways. Somatic mutations in RAS may result in uncontrolled cell growth and malignant transformation while the activation of RAS proteins is tightly regulated in normal cells (D. Simanshu, et al., Cell, 2017, 170(1), 17-33).
The Ras family is comprised of three members: KRAS, NRAS and HRAS. RAS mutant cancers account for about 25% of human cancers. KRAS is the most frequently mutated isoform accounting for 85% of all RAS mutations whereas NRAS and HRAS are found mutated in 12% and 3% of all Ras mutant cancers respectively (D. Simanshu, et al., Cell, 2017, 170(1), 17-33). KRAS mutations are prevalent amongst the top three most deadly cancer types: pancreatic (97%), colorectal (44%), and lung (30%) (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51). The majority of RAS mutations occur at amino acid residue 12, 13, and 61. The frequency of specific mutations varies between RAS gene isoforms and while G12 and Q61 mutations are predominant in KRAS and NRAS respectively, G12, G13 and Q61 mutations are most frequent in HRAS. Furthermore, the spectrum of mutations in a RAS isoform differs between cancer types. For example, KRAS G12D mutations predominate in pancreatic cancers (51%), followed by colorectal adenocarcinomas (45%) and lung cancers (17%) while KRAS G12V mutations are associated with pancreatic cancers (30%), followed by colorectal adenocarcinomas (27%), and lung adenocarcinomas (23%) (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51). In contrast, KRAS G12C mutations predominate in non-small cell lung cancer (NSCLC) comprising 11-16% of lung adenocarcinomas, and 2-5% of pancreatic and colorectal adenocarcinomas (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51). Genomic studies across hundreds of cancer cell lines have demonstrated that cancer cells harboring KRAS mutations are highly dependent on KRAS function for cell growth and survival (R. McDonald, et al., Cell, 2017, 170(3), 577-92). The role of mutant KRAS as an oncogenic driver is further supported by extensive in vivo experimental evidence showing mutant KRAS is required for early tumor onset and maintenance in animal models (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51).
Taken together, these findings indicate that KRAS mutations play a critical role in human cancers. Development of inhibitors targeting KRAS, including mutant KRAS, will therefore be useful in the clinical treatment of diseases that are characterized by involvement of KRAS, including diseases characterized by the involvement or presence of a KRAS mutation.
The present disclosure provides, inter alia, a compound of Formula (I):
The present disclosure further provides a pharmaceutical composition comprising a compound of the disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.
The present disclosure further provides methods of inhibiting KRAS activity, which comprises administering to an individual a compound of the disclosure, or a pharmaceutically acceptable salt thereof. The present disclosure also provides uses of the compounds described herein in the manufacture of a medicament for use in therapy. The present disclosure also provides the compounds described herein for use in therapy.
The present disclosure further provides methods of treating a disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of a compound of the disclosure, or a pharmaceutically acceptable salt thereof.
The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims.
For the terms “e.g.,” and “such as,” and grammatical equivalents thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.
The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
The term “about” means “approximately” (e.g., plus or minus approximately 10% of the indicated value).
In an aspect, provided herein is a compound having Formula (I):
In some embodiments, each R7 is independently selected from H, D, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, halo, OH, and C1-3 alkoxy, provided that if one R7 is OH, the other R7 is selected from H, D, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, and C2-3 alkynyl.
In an embodiment, the compound of Formula (I) is a compound of Formula (I-A):
In another embodiment, the compound of Formula (I) is a compound of any one of the Formulae (I-B), (I-C), (I-D), (I-E), and (I-F):
In yet another embodiment, the compound of Formula (I) is a compound of any one of the Formulae (I-G) and (I-H):
In still another embodiment, the compound of Formula (I) is a compound of any one of the Formulae (I-I), (I-J), (I-K), (I-L), or (I-M):
In an embodiment, the compound of Formula (I) is a compound of Formula (I-N):
In another embodiment, R1 is H.
In yet another embodiment, R2 is selected from C1-3 alkyl (e.g., methyl or ethyl), C1-3 haloalkyl, halo, and CN, wherein the C1-3 alkyl forming R2 is optionally substituted with 1 or 2 substituents (or 1 substituent) independently selected from R2A. In still another embodiment, R2 is C1-3 alkyl (e.g., methyl or ethyl) optionally substituted with 1 or 2 substituents (or 1 substituent) independently selected from R2A. In an embodiment, R2 is selected from C1-3 alkyl, halo, CN, and 2-cyanoethyl (i.e., CH2CH2CN). In another embodiment, R2 is 2-cyanoethyl (i.e., CH2CH2CN).
In an embodiment, each R2A is independently selected from halo and CN. In another embodiment, each R2A is CN.
In yet another embodiment, each Ra2 is H or C1-3 alkyl (e.g., methyl).
In still another embodiment, Cy1 is C6-10 aryl optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy1. In an embodiment, Cy1 is phenyl optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy1. In another embodiment, Cy1 is naphthalenyl (e.g., naphthalene-1-yl or naphthalene-2-yl) optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy1. In yet another embodiment, Cy1 is 6-10 membered heteroaryl optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy1. In still another embodiment, Cy1 is 2-chloro-3-methylphenyl. In an embodiment, Cy1 is 2,3-dichlorophenyl.
In another embodiment, each RCy1 is independently selected from D, C1-3 alkyl (e.g., methyl or ethyl), C1-3 haloalkyl (e.g., halomethyl, e.g., trifluoromethyl), halo, CN, and ORaCy1 In yet another embodiment, each RCy1 is independently selected from D, C1-3 alkyl (e.g., methyl or ethyl), C1-3 haloalkyl (e.g., halomethyl, e.g., trifluoromethyl), or halo.
In still another embodiment, each RaCy1 is independently selected from H and C1-3 alkyl (e.g., methyl or ethyl).
In an embodiment, each RbCy1, RcCy1, and RdCy1 is independently selected from H and C1-3 alkyl (e.g., methyl or ethyl).
In another embodiment, R3 is halo. In yet another embodiment, R3 is fluoro.
In still another embodiment, R4 is H. In an embodiment, R4 is C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, wherein the C1-3 alkyl, C2-3 alkenyl, and C2-3 alkynyl forming R4 are each optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4B.
In another embodiment, R4 is C1-3 alkyl (e.g., methyl or ethyl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4B. In yet another embodiment, R4 is methyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4B. In still another embodiment, R4 is methyl. In an embodiment, R4 is ethyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4B. In another embodiment, R4 is ethyl. In yet another embodiment, R4 is 1-hydroxyethyl.
In still another embodiment, R4 is 4-6 membered heterocycloalkyl (or 4-membered, or 5-membered-, or 6-membered heterocycloalkyl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4A. In an embodiment, R4 is azetidin-1-yl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4A. In another embodiment, R4 is 2-methyl-2-(N,N-dimethylamino)-2-methylazetidin-1-yl.
In yet another embodiment, R4 is 5-6 membered heteroaryl (or 5-membered or 6-membered heteroaryl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4A. In still another embodiment, R4 is 6 membered heteroaryl (e.g., pyridyl, such as 2-pyridyl, 3-pyridyl, or 4-pyridyl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) independently selected from R4A. In an embodiment, R4 is 6 membered heteroaryl (e.g., pyridyl, such as 2-pyridyl, 3-pyridyl, or 4-pyridyl) optionally substituted with 2-hydroxypropyl or with C(O)NRc4BRc4D (such as C(O)NH2, C(O)NHMe, or C(O)NMe2). In another embodiment, R4 is ORa4.
In yet another embodiment, each R4A is independently selected from C1-3 alkyl, NRc4BRc4D (such as NH2, NHMe, or NMe2), or C(O)NRc4BRc4D (such as C(O)NH2, C(O)NHMe, or C(O)NMe2), wherein the C1-3 alkyl forming R4A is optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) each independently selected from R4B. In still another embodiment, each R4A is independently selected from C1-3 alkyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents, or 1 substituent) each independently selected from R4B. In an embodiment, each R4A is R4B.
In another embodiment, R5 is selected from H, D, C1-3 alkyl, C1-3 haloalkyl, cyclopropyl, halo, CN, and ORa5; wherein the C1-3 alkyl and cyclopropyl forming R5 are each optionally substituted with 1 or 2 substituents (or 1 substituent) independently selected from C1-3 alkyl and R5A. In yet another embodiment, R5 is H. In still another embodiment, R5 is C1-3 alkyl (such as methyl or ethyl). In an embodiment, R5 is C1-3 haloalkyl (such as trifluoromethyl). In another embodiment, R5 is halo (such as fluoro or chloro).
In yet another embodiment, R6 is C1-3 alkyl (such as methyl or ethyl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E.
In still another embodiment, R6 is C3-10 cycloalkyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E. In an embodiment, R6 is C3-7 cycloalkyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E. In another embodiment, R6 is C3-6 cycloalkyl (such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E. In yet another embodiment, R6 is cyclobutyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6EIn still another embodiment, R6 is cyclopropyl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E.
In an embodiment, R6 is cyclopropyl. In another embodiment, R6 is 1-fluorocycloprop-1-yl. In yet another embodiment, R6 is 1-methylcycloprop-1-yl.
In still another embodiment, R6 is C6-10 aryl (such as phenyl) optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E. In an embodiment, R6 is 5-10 membered heteroaryl optionally substituted with 1, 2, or 3 substituents (or 1 or 2 substituents or 1 substituent) independently selected from R6E. In another embodiment, R6 is OR6A. In yet another embodiment, R6 is NR6BR6C.
In yet another embodiment, each R7 is H or D or C1-3 alkyl (such as methyl). In still another embodiment, each R7 is H or C1-3 alkyl (such as methyl). In an embodiment, each R7 is H. In another embodiment, each R7 is C1-3 alkyl (such as methyl). In yet another embodiment, two R7 together with the carbon atom to which they are both attached, form a spiro C3-5 cycloalkyl (such as cyclopropyl) ring. In some embodiments, each R7 is independently selected from H, D, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, C2-3 alkynyl, halo, OH, C1-3 alkoxy, and C1-3 alkyl-C1-3 alkoxy, provided that if one R7 is OH, the other R7 is selected from H, D, C1-3 alkyl, C1-3 haloalkyl, C2-3 alkenyl, and C2-3 alkynyl.
In still another embodiment, each R8 is H or D or C1-3 alkyl (such as methyl). In an embodiment, each R8 is H or C1-3 alkyl (such as methyl). In another embodiment, R8 is C1-3 alkyl (such as methyl). In yet another embodiment, each R8 is H.
In an embodiment, each R9 is H or D or C1-3 alkyl (such as methyl). In another embodiment, each R9 is H or C1-3 alkyl (such as methyl). In yet another embodiment, each R9 is H. In still another embodiment, each R9 is C1-3 alkyl (such as methyl). In an embodiment, two R9 together with the carbon atom to which they are both attached, form a spiro C3-5 cycloalkyl (such as cyclopropyl) ring.
In another embodiment, X is O. In yet another embodiment, X is a bond directly connecting the groups to which X is attached.
In still another embodiment, Y is C1-3 alkylene (such as CH2, CH2CH2 or CH2CH2CH2) optionally substituted by 1, 2, 3, 4, 5, or 6 D, 1, 2, or 3 halo, or oxo. In an embodiment, Y is C1-3 alkylene (such as CH2, CH2CH2 or CH2CH2CH2). In another embodiment, Y is a bond directly connecting the groups to which Y is attached.
In yet another embodiment, the moiety X—Y is a bond directly connecting the groups to which X, and Y are attached.
In still another embodiment, Cy2 is C3-7 cycloalkyl optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy2. In an embodiment, Cy2 is 4-10 membered heterocycloalkyl optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy2. In another embodiment, Cy2 is C6-10 aryl (such as phenyl) optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy2. In yet another embodiment, Cy2 is C6-10 aryl (such as phenyl) optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy2. In still another embodiment, Cy2 is 5-10 membered heteroaryl optionally substituted with 1, 2, 3, or 4 substituents (or 1, 2, or 3 substituents, or 1 or 2 substituents, or 1 substituent) independently selected from RCy2.
In an embodiment, Cy2 is selected from phenyl, pyridinyl, pyrazolyl, thiazolyl, isothiazolyl, and triazolyl, all of which are optionally substituted with 1, 2, or 3 substituents independently selected from RCy2. In another embodiment, Cy2 is selected from pyridinyl, pyrimidinyl, isoxazolyl, and cyclobutyl, all of which are optionally substituted with 1, 2, or 3 substituents independently selected from RCy2.
In another embodiment, each RCy2 is independently selected from D, C1-4 alkyl, C1-4 haloalkyl, OC1-3 alkyl, OH, halo, CN, C3-6 cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, NH2, NH(C1-3 alkyl), and N(C1-3 alkyl)2; wherein cycloalkyl is optionally substituted with C1-3 alkyl or C1-3 haloalkyl.
In yet another embodiment, each RCy2 is independently selected from halo, C3-6 cycloalkyl, OC1-3 alkyl.
In some embodiments of the compound Formula (I), or a pharmaceutically acceptable salt thereof:
In some embodiments of the compound of Formula (I), or a pharmaceutically acceptable salt thereof,
In other embodiments, the compound of Formula (I) is selected from the compounds listed in Table 1, and pharmaceutically acceptable salts thereof.
In other embodiments, the compound of Formula (I) is selected from the compounds listed in Table 2, and pharmaceutically acceptable salts thereof.
In other embodiments, the compound of Formula (I) is in the form of a pharmaceutically acceptable salt. In other embodiments, the compound of Formula (I) is in the form of a free base or free acid, or other than in the form of a salt.
In another aspect, provided herein is a pharmaceutical composition comprising a compound of Formula (I), or any of the embodiments thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiply dependent form). Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. Thus, it is contemplated as features described as embodiments of the compounds of Formula (I) can be combined in any suitable combination.
At various places in the present specification, certain features of the compounds are disclosed in groups or in ranges. It is specifically intended that such a disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-6 alkyl” is specifically intended to individually disclose (without limitation) methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.
The term “n-membered,” where n is an integer, typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.
At various places in the present specification, variables defining divalent linking groups may be described. It is specifically intended that each linking substituent include both the forward and backward forms of the linking substituent. For example, —NR(CR′R″)n— includes both —NR(CR′R″)n— and —(CR′R″)nNR— and is intended to disclose each of the forms individually. Where the structure requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl” or “aryl” then it is understood that the “alkyl” or “aryl” represents a linking alkylene group or arylene group, respectively.
The term “substituted” means that an atom or group of atoms formally replaces hydrogen as a “substituent” attached to another group. The hydrogen atom is formally removed and replaced by a substituent. A single divalent substituent, e.g., oxo, can replace two hydrogen atoms. The term “optionally substituted” means unsubstituted or substituted. The term “substituted,” unless otherwise indicated, refers to any level of substitution, e.g., mono-, di-, tri-, tetra- or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. It is to be understood that substitution at a given atom is limited by valency. It is to be understood that substitution at a given atom results in a chemically stable molecule.
The term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons present in a chemical moiety. The term is intended to include each and every member in the indicated range. Thus, Cn-m includes each member in the series Cn, Cn+1, . . . Cm−1, and Cm. Examples include C1-4 (which includes C1, C2, C3, and C4), C1-6 (which includes C1, C2, C3, C4, C5, and C6) and the like.
The term “alkyl” employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chained or branched. The term “Cn-m alkyl,” refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C—H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and the like.
The term “alkenyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkene with one C—H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. The term “Cn-m alkenyl” refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl and the like.
The term “alkynyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C—H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. The term “Cn-m alkynyl” refers to an alkynyl group having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.
The term “alkylene,” employed alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two C—H bond replaced by points of attachment of the alkylene group to the remainder of the compound. The term “Cn-m alkylene” refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, methylene, ethan-1,2-diyl, ethan-1,1-diyl, propan-1,3-diyl, propan-1,2-diyl, propan-1,1-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl and the like.
The term “alkoxy,” employed alone or in combination with other terms, refers to a group of formula —O-alkyl, wherein the alkyl group is as defined above. The term “Cn-m alkoxy” refers to an alkoxy group, the alkyl group of which has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. The term “Cn-m dialkoxy” refers to a linking group of formula —O—(Cn-m alkyl)-O—, the alkyl group of which has n to m carbons. Example dialkoxy groups include —OCH2CH2O— and OCH2CH2CH2O—. In some embodiments, the two O atoms of a Cn-m dialkoxy group may be attached to the same B atom to form a 5- or 6-membered heterocycloalkyl group.
The term “amino,” employed alone or in combination with other terms, refers to a group of formula —NH2, wherein the hydrogen atoms may be substituted with a substituent described herein. For example, “alkylamino” can refer to —NH(alkyl) and —N(alkyl)2.
The term “carbonyl,” employed alone or in combination with other terms, refers to a —C(═O)— group.
The term “carboxy,” employed alone or in combination with other terms, refers to a group of formula —C(═O)OH.
The terms “halo” or “halogen,” used alone or in combination with other terms, refers to fluoro, chloro, bromo and iodo. In some embodiments, “halo” refers to a halogen atom selected from F, Cl, or Br. In some embodiments, halo groups are F.
The term “haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term “Cn-m haloalkyl” refers to a Cn-m alkyl group having n to m carbon atoms and from at least one up to {2(n to m)+1} halogen atoms, which may either be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CH2F, CCl3, CHCl2, C2Cl5 and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.
The term “haloalkoxy,” employed alone or in combination with other terms, refers to a group of formula —O-haloalkyl, wherein the haloalkyl group is as defined above. The term “Cn-m haloalkoxy” refers to a haloalkoxy group, the haloalkyl group of which has n to m carbons. Example haloalkoxy groups include trifluoromethoxy and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
The term “oxo” or “oxy” refers to an oxygen atom as a divalent substituent, forming a carbonyl group when attached to carbon, or attached to a heteroatom forming a sulfoxide or sulfone group, or an N-oxide group. In some embodiments, heterocyclic groups may be optionally substituted by 1 or 2 oxo (═O) substituents.
The term “oxidized” in reference to a ring-forming N atom refers to a ring-forming N-oxide.
The term “oxidized” in reference to a ring-forming S atom refers to a ring-forming sulfonyl or ring-forming sulfinyl.
The term “aromatic” refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e., having (4n+2) delocalized π (pi) electrons where n is an integer).
The term “aryl,” employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2 fused rings). The term “Cn-m aryl” refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g., phenyl, naphthyl, and the like. In some embodiments, aryl groups have from 6 to about 10 carbon atoms. In some embodiments, aryl groups have 6 carbon atoms. In some embodiments, aryl groups have 10 carbon atoms. In some embodiments, the aryl group is phenyl. In some embodiments, the aryl group is naphthyl.
The term “heteroaryl” or “heteroaromatic,” employed alone or in combination with other terms, refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-14 ring atoms including carbon atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-10 ring atoms including carbon atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. In other embodiments, the heteroaryl is an eight-membered, nine-membered or ten-membered fused bicyclic heteroaryl ring. Example heteroaryl groups include, but are not limited to, pyridinyl (pyridyl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, azolyl, oxazolyl, isoxazolyl, thiazolyl, imidazolyl, furanyl, thiophenyl, quinolinyl, isoquinolinyl, naphthyridinyl (including 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3- and 2,6-naphthyridine), indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzisoxazolyl, imidazo[1,2-b]thiazolyl, purinyl, and the like. In some embodiments, the heteroaryl group is pyridone (e.g., 2-pyridone). In some embodiments, the heteroaryl group is selected from, pyridinyl, imidazolyl, thiazolyl, triazolyl, pyrazolyl, oxazolyl, isothiazolyl, quinolinyl, benzothiazolyl, indazolyl, pyrimidinyl, pyrazinyl, pyrazolopyridinyl, thiadiazolyl, benzoisothiazolyl, dihydropyranopyridinyl, dioxooxazolidinyl, 2-oxo-2,3-dihydro-1H-benzoimidazolyl, 2,3-dihydrofuro[2,3-b]pyridin-4-yl, 2,4,5,6-tetrahydrocyclopenta[c]-2-yl, tetrazolyl, thienopyridinyl, and pyridonyl.
A five-membered heteroaryl ring is a heteroaryl group having five ring atoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independently selected from N, O and S. Exemplary five-membered ring heteroaryls include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
A six-membered heteroaryl ring is a heteroaryl group having six ring atoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independently selected from N, O and S.
Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, isoindolyl, and pyridazinyl.
The term “cycloalkyl,” employed alone or in combination with other terms, refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic or polycyclic), including cyclized alkyl and alkenyl groups. The term “Cn-m cycloalkyl” refers to a cycloalkyl that has n to m ring member carbon atoms. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6 or 7 ring-forming carbons (C3-7). In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl group is a C3-6 monocyclic cycloalkyl group. Ring-forming carbon atoms of a cycloalkyl group can be optionally oxidized to form an oxo or sulfido group. Cycloalkyl groups also include cycloalkylidenes. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, e.g., benzo or thienyl derivatives of cyclopentane, cyclohexane and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, bicyclo[1.1.1]pentanyl, bicyclo[2.1.1]hexanyl, and the like. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, the cycloalkyl group is tetrahydronaphthalenyl (e.g., 1,2,3,4-tetrahydronaphthalenyl). In some embodiments, the cycloalkyl group is selected from cyclohexyl, cyclopentyl, cyclopropyl, and cyclobutyl.
The term “heterocycloalkyl,” employed alone or in combination with other terms, refers to a non-aromatic ring or ring system, which may optionally contain one or more alkenylene groups as part of the ring structure, which has at least one heteroatom ring member independently selected from nitrogen, sulfur, oxygen and phosphorus, and which has 4-10 ring members, 4-7 ring members, or 4-6 ring members. Included within the term “heterocycloalkyl” are monocyclic 4-, 5-, 6- and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can include mono- or bicyclic (e.g., having two fused or bridged rings) or spirocyclic ring systems. In some embodiments, the heterocycloalkyl group is a monocyclic group having 1, 2 or 3 heteroatoms independently selected from nitrogen, sulfur and oxygen. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally oxidized to form an oxo or sulfido group or other oxidized linkage (e.g., C(O), S(O), C(S) or S(O)2, N-oxide etc.) or a nitrogen atom can be quaternized. The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the heterocycloalkyl ring, e.g., benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Examples of heterocycloalkyl groups include 2,5-diazobicyclo[2.2.1]heptanyl; pyrrolidinyl; hexahydropyrrolo[3,4-b]pyrrol-1(2H)-yl; 1,6-dihydropyridinyl; morpholinyl; azetidinyl; piperazinyl; and 4,7-diazaspiro[2.5]octan-7-yl. In some embodiments, heterocycloalkyl is selected from dihydropyranopyridinyl, dioxooxazolidinyl, 2-oxo-2,3-dihydro-1H-benzoimidazolyl, 2,3-dihydrofuro[2,3-b]pyridin-4-yl, 2,4,5,6-tetrahydrocyclopenta[c]-2-yl, tetrahydrofuranyl, tetrahydropyranyl, and pyrrolidinyl.
At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an azetidine ring may be attached at any position of the ring, whereas an azetidin-3-yl ring is attached at the 3-position.
The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C═N double bonds and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms.
Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. One method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, e.g., optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as—camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of—methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane and the like.
Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.
When the compounds described herein contain a chiral center, unless otherwise indicated, the compounds can be any of the possible stereoisomers. In some embodiments, the compounds provided herein have the (R)-configuration. In other embodiments, the compounds have the (S)-configuration. In compounds with more than one chiral centers, each of the chiral centers in the compound may be independently (R) or (S), unless otherwise indicated. In compounds with a single chiral center, the stereochemistry of the chiral center can be (R) or (S). In compounds with two chiral centers, the stereochemistry of the chiral centers can each be independently (R) or (S) so the configuration of the chiral centers can be (R) and (R), (R) and (S); (S) and (R), or (S) and (S). In compounds with three chiral centers, the stereochemistry each of the three chiral centers can each be independently (R) or (S) so the configuration of the chiral centers can be (R), (R) and (R); (R), (R) and (S); (R), (S) and (R); (R), (S) and (S); (S), (R) and (R); (S), (R) and (S); (S), (S) and (R); or (S), (S) and (S).
Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified (e.g., in the case of purine rings, unless otherwise indicated, if a compound name or structure described the 9H tautomer, it would be understood that the 7H tautomer is also encompassed).
Compounds of the invention can exist in the form of atropisomers (i.e., conformational diastereoisomers) that can be stable at ambient temperature and separable, e.g., by chromatography. For example, compounds of the invention in which Cy1 is 2,3-dichlorophenyl, or any of the embodiments thereof, can exist in the form of atropisomers in which the conformation of the phenyl relative to the remainder of the molecule is as shown by the partial formulae Formula (II-A) or Formula (II-B) below. Reference to the compounds described herein or any of the embodiments is understood to include all such atropisomeric forms of the compounds, including, without limitation, the atropisomeric forms represented by Formula (II-A) or Formula (II-B) below. Without being limited by any theory, it is understood that, for a given compound, the atropisomer represented by Formula (II-A) is generally more potent as an inhibitor of KRAS (including G12C, G12D or G12V mutated forms of KRAS) than the atropisomer represented by Formula (II-B).
Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the invention can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced or substituted by deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 deuterium atoms.
Substitution with heavier isotopes such as deuterium can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. (A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm. 2015, 58, 308-312). Unless otherwise stated, when a position is designated specifically as “D” or “deuterium,” the position is understood to have deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium). In embodiments, the compounds provided herein have an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).
The term “compound” is intended, unless otherwise specified, to include all stereoisomers, including without limitation geometric isomers, configurational isomers, conformational isomers, rotational isomers, and atropisomers, of the structures depicted, including each of the embodiments thereof. The term is also intended to refer to compounds described herein regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.
All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated. When in the solid state, the compounds described herein and salts thereof may occur in various forms and may, e.g., take the form of solvates, including hydrates. The compounds may be in any solid state form, such as a polymorph or solvate, so unless clearly indicated otherwise, reference in the specification to compounds and salts thereof should be understood as encompassing any solid state form of the compound.
In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. “Substantially isolated” means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, e.g., a composition enriched in the compounds of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds of the invention, or salt thereof.
The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The expressions “ambient temperature” and “room temperature” are understood in the art, and refer generally to a temperature, e.g., a reaction temperature, that is about the temperature of the room in which the reaction is carried out, e.g., a temperature from about 20° C. to about 30° C.
The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein, including any of the embodiments thereof. The term “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the non-toxic salts of the parent compound formed, e.g., from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol or butanol) or MeCN are preferred. Lists of suitable salts are found in A. R. Gennaro (Ed.), Remington's Pharmaceutical Sciences, 17th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, S. M. Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19, S. Gaisford in A. Adejare (Ed.), Remington, The Science and Practice of Pharmacy, 23rd Ed., (Elsevier, 2020), Chapter 17, pp. 307-14; S. M. Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19, T. S. Wiedmann, et al., Asian J. Pharm. Sci., 2016; 11, 722-34. D. Gupta et al., Molecules, 2018, 23(7), 1719; P. H. Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002) and in P. H. Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, 2nd Ed. (Wiley, 2011). In some embodiments, the compounds described herein include the N-oxide forms.
Compounds of the present disclosure, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, such as those in the Schemes below.
The reactions for preparing compounds of the invention can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.
Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups is described, e.g., in Kocienski, Protecting Groups, (Thieme, 2007); Robertson, Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th Ed. (Wiley, 2007); Peturssion et al., “Protecting Groups in Carbohydrate Chemistry,” J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).
Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry or by chromatographic methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC).
Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) (“Preparative LCMS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6(6), 874-883) and normal phase silica chromatography.
The Schemes below provide general guidance in connection with preparing the compounds of the invention. One skilled in the art would understand that the preparations shown in the Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention.
Compounds of formula 1-13 can be prepared via the synthetic route as outlined in Scheme 1. Compound 1-2 can be prepared by treating 1-1 with reagents such as Me2SO4. The bromo of 1-2 can be converted to Cy1 via transition metal mediated coupling or other suitable methods to obtain 1-3. Halogenation of starting material 1-3 with an appropriate reagent, such as N-bromo-succinimide (NBS), affords intermediate 1-4 (Hal is a halide, such as F, Cl, Br, or I). Intermediate 1-4 can then be subjected to hydrolysis followed by treatment with reagent such as triphosgene to afford compound 1-5. Intermediate 1-5 can then react with ketoester 1-6 to deliver compound 1-7. Coupling of halogen of 1-7 (Hal), under standard Heck Cross-Coupling conditions, or standard Suzuki Cross-Coupling conditions, gives 1-8. Compound 1-9 can be prepared by treating 1-8 with an appropriate chlorinating reagent such as POCl3. A SNAr reaction of intermediate 1-9 with Boc-protected amine 1-10 can be carried out to generate compound 1-11. Subsequent hydrolysis of the ester of 1-11 affords 1-12, which can then be treated with NIS and an appropriate base (eg. K3PO4) to afford the corresponding iodide 1-13. Intermediate 1-13 is a versatile intermediate that can be converted to compounds of this invention via a number of different strategies. For certain compounds in this invention, the route in Scheme 2 can be followed.
Sonogashira coupling of iodide 1-13 with pyrrolidine 2-1 (optionally protected with a protecting group, such as tert-butylimethylsilyl group or other suitable protecting group) followed by base-promoted cyclization affords 2-2. A variety of transformations can then be performed with 2-2 after deprotection of protecting group PG (only if 2-1 is protected) to afford compounds of general structure 2-3. Such transformations can include alkylation with the appropriate aliphatic electrophile, transition-metal catalyzed cross-coupling reaction, strong base mediated SNAr reaction with the appropriate aryl electrophile, decarboxylative coupling under electrochemical conditions, Mitsunobu reaction, installation of an azide and click chemistry with the corresponding alkyne and an appropriate copper or ruthenium catalyst, or any other suitable transformation. Finally, removal of Boc-group of 2-3, under standard acidic conditions then affords compound of general structure 2-4.
For other compounds of the invention, intermediate 1-13 can undergo Sonogashira coupling with a pre-functionalized pyrrolidine 3-1, which can be prepared using similar strategies to those outlined for the conversion of 2-2 to 2-3 in Scheme 2. Cyclization then affords intermediate 2-3 directly. Removal of Boc-group of 2-3 as above then affords compound 2-4.
Alternatively, if substitution at R4 is desired at a late stage, intermediate 4-13 can be prepared with thiomethyl serving as handle for late stage functionalization at this position. The synthesis of compounds of formula 4-13 is possible via the synthetic route outlined in Scheme 4. Halogenation of starting material 4-1 with an appropriate reagent, such as N-chloro-succinimide (NCS), affords intermediate 4-2 (Hal is a halide, such as F, Cl, Br, or I). Compound 4-3 can be prepared by treating 4-2 with reagents such as triphosgene. Intermediate 4-3 can then react with ester 4-4 to deliver the nitro compound 4-5, which can be treated with an appropriate reagent (e.g., POCl3) to afford compound 4-6. A SNAr reaction of intermediate 4-6 with Boc-protected amine 1-10 can be carried out to generate compound 4-7. The thiomethoxy group in 4-8 is then installed via SNAr reaction. Protection of the amino group affords intermediate 4-9, which can be reduced in the presence of reducing agents (e.g., Fe in acetic acid) to provide 4-10. The halogen of 4-10 (Hal) can optionally be converted to R2 via transition metal mediated coupling or other suitable method to obtain 4-11. Diazotization and reduction of the amino group in 4-11 affords iodide 4-12, which after protecting group (PG) removal provides 4-13.
Scheme 5 shows the conversion of intermediate 4-13 into compounds of formula 2-4. Intermediate 4-13 can undergo Sonogashira coupling with pyrrolidine 3-1 (optionally protected with a protecting group, such as tert-butylimethylsilyl group or other suitable protecting group) followed by cyclization to afford 5-1. The thiomethoxy group in 5-1 can then be converted to R4 via transition metal mediated coupling, or oxidation followed by base mediated SNAr displacement, or other suitable methods to obtain 2-2. Compound 2-4 can be prepared from the sequence starting from intermediate 2-2 as described in Scheme 3. An alkyne with general formula 3-1 (Scheme 4) can also be used instead of an alkyne of formula 2-1, in which case compounds of formula 2-4 are prepared by R4—X coupling and deprotection directly.
For the synthesis of particular compounds, the general schemes described above and specific methods described herein for preparing particular compounds can be modified. For example, the products or intermediates can be modified to introduce particular functional groups. Alternatively, the substituents can be modified at any step of the overall synthesis by methods know to one skilled in the art, e.g., as described by R. C. Larock, Comprehensive Organic Transformations: A Guide to Functional Group Preparations (Wiley, 1999); R. C. Larock, et al., Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 3rd Ed. Vols. 1-4 (Wiley, 2018); A. R. Katritzky, et al. (Eds.), Comprehensive Organic Functional Group Transformations, Vols. 1-6 (Pergamon Press, 1995), and A. R. Katritzky et al. (Eds.), Comprehensive Organic Functional Group Transformations II, Vols. 1-6 (Elsevier, 2nd Edition, 2005)
Starting materials, reagents and intermediates whose synthesis is not described herein are either commercially available, known in the literature, or may be prepared by methods known to one skilled in the art.
It will be appreciated by one skilled in the art that the processes described are not the exclusive means by which compounds of the invention may be synthesized and that a broad repertoire of synthetic organic reactions is available to be potentially employed in synthesizing compounds of the invention. The person skilled in the art knows how to select and implement appropriate synthetic routes. Suitable synthetic methods of starting materials, intermediates and products may be identified by reference to the literature, including reference sources such as: Advances in Heterocyclic Chemistry, Vols. 1-114 (Elsevier, 1963-2023); Journal of Heterocyclic Chemistry Vols. 1-60 (Journal of Heterocyclic Chemistry, 1964-2023); E. M. Carreira, et al. (Eds.) Science of Synthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010/1-4; 2011/1-4; 2012/1-4, 2013/1-4; 2014/1-4, 2015/1-2; 2016/1-3, 2017/1-3; 2018/1-4, 2019/1-3; 2020/1-3, 2021/1-3, 2022/1-3, 2023/1 (Thieme, 2001-2023); Houben-Weyl, Methoden der Organischen Chemie, 4th Ed. Vols. 1-67 (Thieme, 1952-1987); Houben-Weyl, Methoden der Organischen Chemie, E-Series. Vols. 1-23 (Thieme, 1982-2003); A. R. Katritzky, et al. (Eds.), Comprehensive Organic Functional Group Transformations, Vols. 1-6 (Pergamon Press, 1995); A. R. Katritzky et al. (Eds.), Comprehensive Organic Functional Group Transformations II, Vols. 1-6 (Elsevier, 2nd Edition, 2005); A. R. Katritzky et al. (Eds.); Comprehensive Heterocyclic Chemistry, Vols. 1-8 (Pergamon Press, 1984); A. R. Katritzky, et al. (Eds.); Comprehensive Heterocyclic Chemistry II, Vols. 1-10 (Pergamon Press, 1996); A. R. Katritzky, et al. (Eds.); Comprehensive Heterocyclic Chemistry III, Vols. 1-14 (Elsevier Science, 2008); D. St. C. Black, et al. (Eds.); Comprehensive Heterocyclic Chemistry IV, Vols. 1-14 (Elsevier Science, 2022); M. B. Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6th Ed. (Wiley, 2007); M. B. Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 8th Ed. (Wiley, 2020); B. M. Trost et al. (Ed.), Comprehensive Organic Synthesis, Vols. 1-9 (Pergamon Press, 1991); and Patai's Chemistry of Functional Groups, 100 Vols. (Wiley 1964-2022).
Compounds of the present disclosure, including the compounds of Formula (I), or any of the embodiments thereof, are useful for therapy as described in further detail below.
The present disclosure provides compounds of Formula (I), for use as a medicament, or for use in medicine. The present disclosure provides compounds of Formula (I), for use as a medicament, or for use in treating disease, as described in further detail below. The present disclosure also provides the use of compounds of Formula (I), or any of the embodiments thereof, as a medicament, or for treating disease, as described in further detail below. The present disclosure also provides the use of compounds of Formula (I), or any of the embodiments thereof, in the manufacture of medicament for treating disease, as described in further detail below.
Compounds of the present disclosure are KRAS inhibitors and, thus, are useful in treating diseases and disorders associated with activity of KRAS. For the uses described herein, any of the compounds of Formula (I), including any of the embodiments thereof, may be used.
In particular, compounds of the invention are KRAS inhibitors having activity against one or more mutant forms of KRAS, and, thus, are useful in treating diseases and disorders associated with the presence or activity of mutant forms of KRAS, such as G12C, G12D, and/or the G12V mutant forms of KRAS.
The Ras family is comprised of three members: KRAS, NRAS and HRAS. RAS mutant cancers account for about 25% of human cancers. KRAS is the most frequently mutated isoform in human cancers: 85% of all RAS mutations are in KRAS, 12% in NRAS, and 3% in HRAS (D. Simanshu, et al., Cell, 2017, 170(1), 17-33). KRAS mutations are prevalent amongst the top three most deadly cancer types: pancreatic (97%), colorectal (44%), and lung (30%) (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51). The majority of RAS mutations occur at amino acid residues/codons 12, 13, and 61; Codon 12 mutations are most frequent in KRAS. The frequency of specific mutations varied between RAS genes and G12D mutations are most predominant in KRAS whereas Q61R and G12R mutations are most frequent in NRAS and HRAS. Furthermore, the spectrum of mutations in a RAS isoform differs between cancer types. For example, KRAS G12D mutations predominate in pancreatic cancers (51%), followed by colorectal adenocarcinomas (45%) and lung cancers (17%) (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51). In contrast, KRAS G12C mutations predominate in non-small cell lung cancer (NSCLC) comprising 11-16% of lung adenocarcinomas (nearly half of mutant KRAS is G12C), as well as 2-5% of pancreatic and colorectal adenocarcinomas, respectively (A. D. Cox, et al. Nat. Rev. Drug. Discov., 2014, 13(11), 828-51). Using shRNA knockdown thousands of genes across hundreds of cancer cell lines, genomic studies have demonstrated that cancer cells exhibiting KRAS mutations are highly dependent on KRAS function for cell growth (R. McDonald, et al., Cell, 2017, 170(3), 577-92).
Taken together, these findings indicate that KRAS mutations play a critical role in human cancers. Development of inhibitors targeting KRAS, including mutant KRAS, will therefore be useful in the clinical treatment of diseases that are characterized by involvement of KRAS, including diseases characterized by the involvement or presence of a KRAS mutation.
Diseases that can be treated with the compounds of Formula (I) include cancers. The cancers can include adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor. In some embodiments, the cancer can be adenocarcinoma, adult T-cell leukemia/lymphoma, bladder cancer, blastoma, bone cancer, breast cancer, brain cancer, carcinoma, myeloid sarcoma, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioblastoma multiforme, glioma, gallbladder cancer, gastric cancer, head and neck cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, intestinal cancer, kidney cancer, laryngeal cancer, leukemia, lung cancer, lymphoma, liver cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, multiple myeloma, ocular cancer, optic nerve tumor, oral cancer, ovarian cancer, pituitary tumor, primary central nervous system lymphoma, prostate cancer, pancreatic cancer, pharyngeal cancer, renal cell carcinoma, rectal cancer, sarcoma, skin cancer, spinal tumor, small intestine cancer, stomach cancer, T-cell lymphoma, testicular cancer, thyroid cancer, throat cancer, urogenital cancer, urothelial carcinoma, uterine cancer, vaginal cancer, or Wilms' tumor.
The cancer types in which KRAS harboring G12C, G12V and G12D mutations are implicated and that can be treated using compounds of Formula (I), or any of the embodiments thereof, include, but are not limited to: carcinomas (e.g., pancreatic, colorectal, lung, bladder, gastric, esophageal, breast, head and neck, cervical skin, thyroid); hematopoietic malignancies (e.g., myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), chronic and juvenile myelomonocytic leukemia (CMML and JMML), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and multiple myeloma (MM)); and other neoplasms (e.g., glioblastoma and sarcomas). In addition, KRAS mutations were found in acquired resistance to anti-EGFR therapy (Knickelbein, K. et al. Genes & Cancer, (2015): 4-12). KRAS mutations were found in immunological and inflammatory disorders (Fernandez-Medarde, A. et al. Genes & Cancer, (2011): 344-358) such as Ras-associated lymphoproliferative disorder (RALD) or juvenile myelomonocytic leukemia (JMML) caused by one or more somatic mutations of KRAS or NRAS.
Compounds of the present disclosure, including any of the embodiments thereof, can inhibit the activity of the KRAS protein. For example, compounds of the present disclosure can be used to inhibit activity of KRAS in a cell or in an individual or patient in need of inhibition of the enzyme by administering an inhibiting amount of one or more compounds of the present disclosure to the cell, individual, or patient.
As KRAS inhibitors, the compounds of the present disclosure, or any of the embodiments thereof, are useful in the treatment of various diseases associated with abnormal expression or activity of KRAS. Compounds which inhibit KRAS will be useful in providing a means of preventing the growth or inducing apoptosis in tumors, or by inhibiting angiogenesis. It is therefore anticipated that compounds of the present disclosure will prove useful in treating or preventing proliferative disorders such as cancers. In particular, tumors with activating mutants of receptor tyrosine kinases or upregulation of receptor tyrosine kinases may be particularly sensitive to the inhibitors.
In an aspect, provided herein is a method of inhibiting KRAS activity, the method comprising contacting a compound of the instant disclosure with KRAS. In an embodiment, the contacting comprises administering the compound to a patient. In an embodiment, KRAS is characterized as having a somatic mutation of G12C. In another embodiment, KRAS is characterized as having a somatic mutation of G12D. In another embodiment, KRAS is characterized as having a somatic mutation of G12V.
In an aspect, provided herein is a method of inhibiting a KRAS protein harboring a G12C mutation, the method comprising contacting a compound of Formula (I), or any of the embodiments thereof, with KRAS.
In an aspect, provided herein is a method of inhibiting a KRAS protein harboring a G12D mutation, the method comprising contacting a compound of Formula (I), or any of the embodiments thereof, with KRAS harboring a G12D mutation.
In an aspect, provided herein is a method of inhibiting a KRAS protein harboring a G12V mutation, the method comprising contacting a compound of Formula (I), or any of the embodiments thereof, with KRAS harboring a G12V mutation.
In another aspect, provided herein is a method of treating a disease or disorder associated with inhibition of KRAS interaction, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In another aspect, provided herein is a method of treating a disease or disorder associated with activity of KRAS interaction, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In an embodiment, the disease or disorder is an immunological or inflammatory disorder. In another embodiment, the immunological or inflammatory disorder is Ras-associated lymphoproliferative disorder or juvenile myelomonocytic leukemia caused by one or more somatic mutations of KRAS. In an embodiment, the immunological or inflammatory disorder is caused by a somatic mutation of KRAS.
In another embodiment, the somatic mutation of KRAS is G12C. In another embodiment, the somatic mutation of KRAS is G12D. In another embodiment, the somatic mutation of KRAS is G12V.
In another embodiment, the immunological or inflammatory disorder is associated with activity of KRAS having a G12C mutation. In another embodiment, the immunological or inflammatory disorder is associated with activity of KRAS having a G12D mutation. In another embodiment, the immunological or inflammatory disorder is associated with activity of KRAS having a G12V mutation.
In yet another aspect, provided herein is a method of treating a disease or disorder associated with inhibiting a KRAS protein harboring a G12C mutation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In yet another aspect, provided herein is a method of treating a disease or disorder associated with inhibiting a KRAS protein harboring a G12D mutation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In another aspect, provided herein is a method of treating a disease or disorder associated with inhibiting a KRAS protein harboring a G12V mutation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In yet another aspect, provided herein is a method of treating a disease or disorder associated with activity of a KRAS protein harboring a G12C mutation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In yet another aspect, provided herein is a method of treating a disease or disorder associated with activity of a KRAS protein harboring a G12D mutation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In another aspect, provided herein is a method of treating a disease or disorder associated with activity of a KRAS protein harboring a G12V mutation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In another aspect, provided herein is also a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof.
In still another aspect, provided herein is also a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof, wherein the cancer is characterized by an interaction with a KRAS protein harboring a G12C mutation.
In still another aspect, provided herein is also a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof, wherein the cancer is characterized by an interaction with a KRAS protein harboring a G12D mutation.
In another aspect, provided herein is also a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof, wherein the cancer is characterized by an interaction with a KRAS protein harboring a G12V mutation.
In yet another aspect, provided herein is a method for treating a cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of any one of the compounds disclosed herein, or pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a method for treating a cancer in a patient comprising identifying that the patient is in need of treatment of a cancer and that abnormally proliferating cells of the cancer comprise KRAS having a G12C mutation, and administering to the patient a therapeutically effective amount of any one of the compounds disclosed herein, or pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a method for treating a cancer in a patient comprising identifying that the patient is in need of treatment of a cancer and that abnormally proliferating cells of the cancer comprise KRAS having a G12D mutation, and administering to the patient a therapeutically effective amount of any one of the compounds disclosed herein, or pharmaceutically acceptable salt thereof.
In another aspect, provided herein is a method for treating a cancer in a patient comprising identifying that the patient is in need of treatment of a cancer and that abnormally proliferating cells of the cancer comprise KRAS having a G12V mutation, and administering to the patient a therapeutically effective amount of any one of the compounds disclosed herein, or pharmaceutically acceptable salt thereof.
In an embodiment, the cancer is selected from carcinomas, hematological cancers, sarcomas, and glioblastoma. In another embodiment, the hematological cancer is selected from myeloproliferative neoplasms, myelodysplastic syndrome, chronic and juvenile myelomonocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, and multiple myeloma. In yet another embodiment, the carcinoma is selected from pancreatic, colorectal, lung, bladder, gastric, esophageal, breast, head and neck, cervical, skin, and thyroid. In another embodiment, the cancer is colorectal cancer, pancreatic cancer, or lung cancer. In yet another embodiment, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In still another embodiment, the cancer is non-small cell lung cancer (NSCLC).
In an embodiment, the cancer is metastatic.
In an aspect, provided herein is a method for treating a disease or disorder associated with inhibition of KRAS interaction or a mutant thereof, in a patient in need thereof, comprising the step of administering to the patient a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof, in combination with another therapy or therapeutic agent as described herein.
In an aspect, provided herein is a method for treating a disease or disorder associated with activity of KRAS interaction or a mutant thereof, in a patient in need thereof, comprising the step of administering to the patient a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof, in combination with another therapy or therapeutic agent as described herein.
In an embodiment, the cancer is selected from hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
In another embodiment, the lung cancer is selected from non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma, and pleuropulmonary blastoma.
In yet another embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In still another embodiment, the lung cancer is adenocarcinoma.
In an embodiment, the gastrointestinal cancer is selected from esophagus squamous cell carcinoma, esophagus adenocarcinoma, esophagus leiomyosarcoma, esophagus lymphoma, stomach carcinoma, stomach lymphoma, stomach leiomyosarcoma, exocrine pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumors, pancreatic vipoma, small bowel adenocarcinoma, small bowel lymphoma, small bowel carcinoid tumors, Kaposi's sarcoma, small bowel leiomyoma, small bowel hemangioma, small bowel lipoma, small bowel neurofibroma, small bowel fibroma, large bowel adenocarcinoma, large bowel tubular adenoma, large bowel villous adenoma, large bowel hamartoma, large bowel leiomyoma, colorectal cancer, gall bladder cancer, and anal cancer.
In an embodiment, the gastrointestinal cancer is colorectal cancer.
In another embodiment, the cancer is a carcinoma. In yet another embodiment, the carcinoma is selected from pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical skin carcinoma, and thyroid carcinoma.
In still another embodiment, the cancer is a hematopoietic malignancy. In an embodiment, the hematopoietic malignancy is selected from multiple myeloma, acute myelogenous leukemia, and myeloproliferative neoplasms.
In another embodiment, the cancer is a neoplasm. In yet another embodiment, the neoplasm is glioblastoma or sarcomas.
In certain embodiments, the disclosure provides a method for treating a KRAS-mediated disorder in a patient in need thereof, comprising the step of administering to the patient a compound according to the invention, or a pharmaceutically acceptable composition thereof.
In some embodiments, diseases and indications that are treatable using the compounds of the present disclosure include, but are not limited to hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers.
Exemplary hematological cancers include lymphomas and leukemias such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma, myeloproliferative diseases (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocytosis (ET), 8p11 myeloproliferative syndrome, myelodysplasia syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), multiple myeloma, cutaneous T-cell lymphoma, adult T-cell leukemia, Waldenstrom's Macroglubulinemia, hairy cell lymphoma, marginal zone lymphoma, chronic myelogenic lymphoma and Burkitt's lymphoma.
Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, harmatoma, lymphosarcoma, leiomyosarcoma, and teratoma.
Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-pavicellular carcinoma, bronchial adenoma and pleuropulmonary blastoma.
Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (exocrine pancreatic carcinoma, ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colorectal cancer, gall bladder cancer and anal cancer.
Exemplary genitourinary tract cancers include cancers of the kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma) and urothelial carcinoma.
Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.
Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumors
Exemplary nervous system cancers include cancers of the skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, meduoblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, neuro-ectodermal tumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma), neuroblastoma, Lhermitte-Duclos disease and pineal tumors.
Exemplary gynecological cancers include cancers of the breast (ductal carcinoma, lobular carcinoma, breast sarcoma, triple-negative breast cancer, HER2-positive breast cancer, inflammatory breast cancer, papillary carcinoma), uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), and fallopian tubes (carcinoma).
Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids.
Exemplary head and neck cancers include glioblastoma, melanoma, rhabdosarcoma, lymphosarcoma, osteosarcoma, squamous cell carcinomas, adenocarcinomas, oral cancer, laryngeal cancer, nasopharyngeal cancer, nasal and paranasal cancers, thyroid and parathyroid cancers, tumors of the eye, tumors of the lips and mouth and squamous head and neck cancer.
The compounds of the present disclosure can also be useful in the inhibition of tumor metastasis.
In addition to oncogenic neoplasms, the compounds of the invention are useful in the treatment of skeletal and chondrocyte disorders including, but not limited to, achrondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia (TD) (clinical forms TD I and TD II), Apert syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrate syndrome, Pfeiffer syndrome, and craniosynostosis syndromes. In some embodiments, the present disclosure provides a method for treating a patient suffering from a skeletal and chondrocyte disorder.
In some embodiments, compounds described herein can be used to treat Alzheimer's disease, HIV, or tuberculosis.
The term “8p11 myeloproliferative syndrome” refers to myeloid/lymphoid neoplasms associated with eosinophilia and abnormalities of FGFR1.
The term “cell” refers to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.
The term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” KRAS with a compound described herein includes the administration of a compound described herein to an individual or patient, such as a human, having KRAS, as well as, for example, introducing a compound described herein into a sample containing a cellular or purified preparation containing KRAS.
The terms “individual,” “subject,” or “patient,” are used interchangeably, and refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
The phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent such as an amount of any of the solid forms or salts thereof as disclosed herein that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. An appropriate “effective” amount in any individual case may be determined using techniques known to a person skilled in the art.
The phrase “pharmaceutically acceptable carrier or excipient” refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients or carriers are generally safe, non-toxic and neither biologically nor otherwise undesirable and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use. In one embodiment, each component is “pharmaceutically acceptable” as defined herein. See, e.g., P. Beringer, et al., (Eds.), Remington: The Science and Practice of Pharmacy, 21st Ed.; (Lippincott Williams & Wilkins: Philadelphia, Pa., 2005); A. Adejare (Ed.), Remington, The Science and Practice of Pharmacy, 23rd Ed., (Elsevier, 2020); R. C. Rowe et al., Eds., Handbook of Pharmaceutical Excipients, 6th Ed.; (Pharmaceutical Press, 2009); P. J. Shesky et al., Eds., Handbook of Pharmaceutical Excipients, 9th Ed.; (The Pharmaceutical Press, 2020); M. Ash, et al., (Eds.), Handbook of Pharmaceutical Additives, 3rd Ed.; (Gower Publishing Company: 2007); and M. Gibson (Ed.), Pharmaceutical Preformulation and Formulation, 2nd Ed. (CRC Press LLC, 2009).
The term “treating” or “treatment” refers to inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder (i.e., arresting further development of the pathology and/or symptomology) or ameliorating the disease; for example, ameliorating a disease, condition, or disorder in an individual who is experiencing or displaying the pathology or symptomology of the disease, condition, or disorder (i.e., reversing the pathology and/or symptomology) such as decreasing the severity of the disease.
The term “prevent,” “preventing,” or “prevention” comprises the prevention of at least one symptom associated with or caused by the state, disease or disorder being prevented.
Compounds of the invention, including the compounds of Formula (I), or any of the embodiments thereof, may be useful in therapy when used in combination with one or more additional pharmaceutical agents, as described in further detail below.
a. Cancer Therapies
Compounds of the present disclosure, including the compounds of Formula (I), or any of the embodiments thereof, may be useful in treatment of cancer when used in combination with one or more additional pharmaceutical agents, as described in further detail below.
Cancer cell growth and survival can be impacted by dysfunction in multiple signaling pathways. Thus, it is useful to combine different enzyme/protein/receptor inhibitors, exhibiting different preferences in the targets which they modulate the activities of, to treat such conditions. Targeting more than one signaling pathway (or more than one biological molecule involved in a given signaling pathway) may reduce the likelihood of drug-resistance arising in a cell population, and/or reduce the toxicity of treatment.
One or more additional pharmaceutical agents such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, immune-oncology agents, metabolic enzyme inhibitors, chemokine receptor inhibitors, and phosphatase inhibitors, as well as targeted therapies such as Bcr-Abl, Flt-3, EGFR, HER2, JAK, c-MET, VEGFR, PDGFR, c-Kit, IGF-1R, RAF, FAK, and CDK4/6 kinase inhibitors such as, for example, those described in WO 2006/056399 can be used in combination with the compounds of the present disclosure for treatment of KRAS-associated diseases, disorders or conditions. Other agents such as therapeutic antibodies can be used in combination with the compounds of the present disclosure for treatment of KRAS-associated diseases, disorders or conditions. The one or more additional pharmaceutical agents can be administered to a patient simultaneously or sequentially.
In some embodiments, the KRAS inhibitor is administered or used in combination with a BCL2 inhibitor or a CDK4/6 inhibitor.
The compounds as disclosed herein can be used in combination with one or more other enzyme/protein/receptor inhibitors therapies for the treatment of diseases, such as cancer and other diseases or disorders described herein. Examples of diseases and indications treatable with combination therapies include those as described herein.
Examples of cancers include solid tumors and non-solid tumors, such as liquid tumors, blood cancers. Examples of infections include viral infections, bacterial infections, fungus infections or parasite infections. For example, the compounds of the present disclosure can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, BCL2, CDK4/6, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IDH2, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K (alpha, beta, gamma, delta, and multiple or selective), CSF1R, KIT, FLK-II, KDR/FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, PARP, Ron, Sea, TRKA, TRKB, TRKC, TAM kinases (Axl, Mer, Tyro3), FLT3, VEGFR/Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf. In some embodiments, the compounds of the present disclosure can be combined with one or more of the following inhibitors for the treatment of cancer or infections. Non-limiting examples of inhibitors that can be combined with the compounds of the present disclosure for treatment of cancer and infections include an FGFR inhibitor (FGFR1, FGFR2, FGFR3 or FGFR4, e.g., pemigatinib (INCB54828), INCB62079), an EGFR inhibitor (also known as ErB-1 or HER-1; e.g., erlotinib, gefitinib, vandetanib, orsimertinib, cetuximab, necitumumab, or panitumumab), a VEGFR inhibitor or pathway blocker (e.g., bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab, lenvatinib, ziv-aflibercept), a PARP inhibitor (e.g., olaparib, rucaparib, veliparib or niraparib), a JAK inhibitor (JAK1 and/or JAK2; e.g., ruxolitinib or baricitinib; or JAK1; e.g., itacitinib (INCB39110), INCB052793, or INCB054707), an IDO inhibitor (e.g., epacadostat, NLG919, or BMS-986205, MK7162), an LSD1 inhibitor (e.g., GSK2979552, INCB59872 and INCB60003), a TDO inhibitor, a PI3K-delta inhibitor (e.g., parsaclisib (INCB50465) or INCB50797), a PI3K-gamma inhibitor such as PI3K-gamma selective inhibitor, a Pim inhibitor (e.g., INCB53914), a CSF1R inhibitor, a TAM receptor tyrosine kinases (Tyro-3, Axl, and Mer; e.g., INCB081776), an adenosine receptor antagonist (e.g., A2a/A2b receptor antagonist), an HPK1 inhibitor, a chemokine receptor inhibitor (e.g., CCR2 or CCR5 inhibitor), a SHP1/2 phosphatase inhibitor, a histone deacetylase inhibitor (HDAC) such as an HDAC8 inhibitor, an angiogenesis inhibitor, an interleukin receptor inhibitor, bromo and extra terminal family members inhibitors (for example, bromodomain inhibitors or BET inhibitors such as INCB54329 and INCB57643), c-MET inhibitors (e.g., capmatinib), an anti-CD19 antibody (e.g., tafasitamab), an ALK2 inhibitor (e.g., zilurgisertib); or combinations thereof.
In some embodiments, the compound or salt described herein is administered with a PI3Kδ inhibitor. In some embodiments, the compound or salt described herein is administered with a JAK inhibitor. In some embodiments, the compound or salt described herein is administered with a JAK1 or JAK2 inhibitor (e.g., baricitinib or ruxolitinib). In some embodiments, the compound or salt described herein is administered with a JAK1 inhibitor.
In some embodiments, the compound or salt described herein is administered with a JAK1 inhibitor, which is selective over JAK2.
Example antibodies for use in combination therapy include, but are not limited to, trastuzumab (e.g., anti-HER2), ranibizumab (e.g., anti-VEGF-A), bevacizumab (AVASTIN™, e.g., anti-VEGF), panitumumab (e.g., anti-EGFR), cetuximab (e.g., anti-EGFR), rituxan (e.g., anti-CD20), and antibodies directed to c-MET.
One or more of the following agents may be used in combination with the compounds of the present disclosure and are presented as a non-limiting list: a cytostatic agent, cisplatin, doxorubicin, taxotere, taxol, etoposide, irinotecan, camptosar, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS 214662, IRESSA™ (gefitinib), TARCEVA™ (erlotinib), antibodies to EGFR, intron, ara-C, adriamycin, cytoxan, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, oxaliplatin, leucovirin, ELOXATIN™ (oxaliplatin), pentostatine, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide 17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, navelbene, anastrazole, letrazole, capecitabine, reloxafine, droloxafine, hexamethylmelamine, avastin, HERCEPTIN™ (trastuzumab), BEXXAR™ (tositumomab), VELCADE™ (bortezomib), ZEVALIN™ (ibritumomab tiuxetan), TRISENOX™ (arsenic trioxide), XELODA™ (capecitabine), vinorelbine, porfimer, ERBITUX™ (cetuximab), thiotepa, altretamine, melphalan, trastuzumab, lerozole, fulvestrant, exemestane, ifosfomide, rituximab, C225 (cetuximab), Campath (alemtuzumab), clofarabine, cladribine, aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine, Smi1, fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP, and MDL-101,731.
The compounds of the present disclosure can further be used in combination with other methods of treating cancers, for example by chemotherapy, irradiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy or surgery. Examples of immunotherapy include cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccine, monoclonal antibody, bispecific or multi-specific antibody, antibody drug conjugate, adoptive T cell transfer, Toll receptor agonists, RIG-1 agonists, oncolytic virotherapy and immunomodulating small molecules, including thalidomide or JAK1/2 inhibitor, PI3Kδ inhibitor and the like. The compounds can be administered in combination with one or more anti-cancer drugs, such as a chemotherapeutic agent.
Examples of chemotherapeutics include any of: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, and zoledronate.
Additional examples of chemotherapeutics include proteasome inhibitors (e.g., bortezomib), thalidomide, revlimid, and DNA-damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.
Example steroids include corticosteroids such as dexamethasone or prednisone.
Example Bcr-Abl inhibitors include imatinib mesylate (GLEEVAC™), nilotinib, dasatinib, bosutinib, and ponatinib, and pharmaceutically acceptable salts. Other example suitable Bcr-Abl inhibitors include the compounds, and pharmaceutically acceptable salts thereof, of the genera and species disclosed in U.S. Pat. No. 5,521,184, WO 04/005281, and U.S. Pat. No. 7,745,437.
Example suitable Flt-3 inhibitors include midostaurin, lestaurtinib, linifanib, sunitinib, sunitinib, maleate, sorafenib, quizartinib, crenolanib, pacritinib, tandutinib, PLX3397 and ASP2215, and their pharmaceutically acceptable salts. Other example suitable Flt-3 inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 03/037347, WO 03/099771, and WO 04/046120.
Example suitable RAF inhibitors include dabrafenib, sorafenib, and vemurafenib, and their pharmaceutically acceptable salts. Other example suitable RAF inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO 05/028444.
Example suitable FAK inhibitors include VS-4718, VS-5095, VS-6062, VS-6063, B1853520, and GSK2256098, and their pharmaceutically acceptable salts. Other example suitable FAK inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.
Example suitable CDK4/6 inhibitors include palbociclib, ribociclib, trilaciclib, lerociclib, and abemaciclib, and their pharmaceutically acceptable salts. Other example suitable CDK4/6 inhibitors include compounds, and their pharmaceutically acceptable salts, as disclosed in WO 09/085185, WO 12/129344, WO 11/101409, WO 03/062236, WO 10/075074, and WO 12/061156.
In some embodiments, the compounds of the disclosure can be used in combination with one or more other kinase inhibitors including imatinib, particularly for treating patients resistant to imatinib or other kinase inhibitors.
In some embodiments, the compounds of the disclosure can be used in combination with a chemotherapeutic in the treatment of cancer, and may improve the treatment response as compared to the response to the chemotherapeutic agent alone, without exacerbation of its toxic effects. In some embodiments, the compounds of the disclosure can be used in combination with a chemotherapeutic provided herein. For example, additional pharmaceutical agents used in the treatment of multiple myeloma, can include, without limitation, melphalan, melphalan plus prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Further additional agents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of an alkylating agent include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM). Additive or synergistic effects are desirable outcomes of combining a CDK2 inhibitor of the present disclosure with an additional agent.
The agents can be combined with the present compound in a single or continuous dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.
The compounds of the present disclosure can be used in combination with one or more other inhibitors or one or more therapies for the treatment of infections. Examples of infections include viral infections, bacterial infections, fungus infections or parasite infections.
In some embodiments, a corticosteroid such as dexamethasone is administered to a patient in combination with the compounds of the disclosure where the dexamethasone is administered intermittently as opposed to continuously.
The compounds of Formula (I) or any of the embodiments thereof as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be combined with another immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.
The compounds of Formula (I) or any of the embodiments thereof as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be used in combination with a vaccination protocol for the treatment of cancer. In some embodiments, the tumor cells are transduced to express GM-CSF. In some embodiments, tumor vaccines include the proteins from viruses implicated in human cancers such as Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). In some embodiments, the compounds of the present disclosure can be used in combination with tumor specific antigen such as heat shock proteins isolated from tumor tissue itself. In some embodiments, the compounds of Formula (I) or any of the embodiments thereof as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be combined with dendritic cells immunization to activate potent anti-tumor responses.
The compounds of the present disclosure can be used in combination with bispecific macrocyclic peptides that target Fe alpha or Fe gamma receptor-expressing effectors cells to tumor cells. The compounds of the present disclosure can also be combined with macrocyclic peptides that activate host immune responsiveness.
In some further embodiments, combinations of the compounds of the disclosure with other therapeutic agents can be administered to a patient prior to, during, and/or after a bone marrow transplant or stem cell transplant. The compounds of the present disclosure can be used in combination with bone marrow transplant for the treatment of a variety of tumors of hematopoietic origin.
The compounds of Formula (I) or any of the embodiments thereof as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be used in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful, include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas Aeruginosa.
Viruses causing infections treatable by methods of the present disclosure include, but are not limit to human papillomavirus, influenza, hepatitis A, B, C or D viruses, adenovirus, poxvirus, herpes simplex viruses, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, measles virus, herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.
Pathogenic bacteria causing infections treatable by methods of the disclosure include, but are not limited to, chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria.
Pathogenic fungi causing infections treatable by methods of the disclosure include, but are not limited to, Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
Pathogenic parasites causing infections treatable by methods of the disclosure include, but are not limited to, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.
When more than one pharmaceutical agent is administered to a patient, they can be administered simultaneously, separately, sequentially, or in combination (e.g., for more than two agents).
Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the “Physicians' Desk Reference” (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if set forth in its entirety.
b. Immune-Checkpoint Therapies
Compounds of the present disclosure can be used in combination with one or more immune checkpoint inhibitors for the treatment of diseases, such as cancer or infections. Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CBL-B, CD20, CD28, CD40, CD70, CD122, CD96, CD73, CD47, CDK2, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, HPK1, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, TLR (TLR7/8), TIGIT, CD112R, VISTA, PD-1, PD-L1 and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, TIGIT, and VISTA. In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and TGFR beta inhibitors.
In some embodiments, the compounds provided herein can be used in combination with one or more agonists of immune checkpoint molecules, e.g., OX40, CD27, GITR, and CD137 (also known as 4-1BB).
In some embodiments, the inhibitor of an immune checkpoint molecule is anti-PD1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1 or PD-L1, e.g., an anti-PD-1 or anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, cemiplimab, atezolizumab, avelumab, tislelizumab, spartalizumab (PDR001), cetrelimab (JNJ-63723283), toripalimab (JS001), camrelizumab (SHR-1210), sintilimab (1B1308), AB122 (GLS-010), AMP-224, AMP-514/MEDI-0680, BMS936559, JTX-4014, BGB-108, SHR-1210, MEDI4736, FAZ053, BCD-100, KN035, CS1001, BAT1306, LZM009, AK105, HLX10, SHR-1316, CBT-502 (TQB2450), A167 (KL-A167), STI-A101 (ZKAB001), CK-301, BGB-A333, MSB-2311, HLX20, TSR-042, or LY3300054. In some embodiments, the inhibitor of PD-1 or PD-L1 is one disclosed in U.S. Pat. No. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, or 10,308,644; U.S. Publ. Nos. 2017/0145025, 2017/0174671, 2017/0174679, 2017/0320875, 2017/0342060, 2017/0362253, 2018/0016260, 2018/0057486, 2018/0177784, 2018/0177870, 2018/0179179, 2018/0179201, 2018/0179202, 2018/0273519, 2019/0040082, 2019/0062345, 2019/0071439, 2019/0127467, 2019/0144439, 2019/0202824, 2019/0225601, 2019/0300524, or 2019/0345170; or PCT Pub. Nos. WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, or WO 2011161699, which are each incorporated herein by reference in their entirety. In some embodiments, the inhibitor of PD-L1 is INCB086550.
In some embodiments, the PD-L1 inhibitor is selected from the compounds in Table A, or a pharmaceutically acceptable salt thereof.
In some embodiments, the antibody is an anti-PD-1 antibody, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, sintilimab, AB122, AMP-224, JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, or TSR-042. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, or sintilimab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is cemiplimab. In some embodiments, the anti-PD-1 antibody is spartalizumab. In some embodiments, the anti-PD-1 antibody is camrelizumab. In some embodiments, the anti-PD-1 antibody is cetrelimab. In some embodiments, the anti-PD-1 antibody is toripalimab. In some embodiments, the anti-PD-1 antibody is sintilimab. In some embodiments, the anti-PD-1 antibody is AB122. In some embodiments, the anti-PD-1 antibody is AMP-224. In some embodiments, the anti-PD-1 antibody is JTX-4014. In some embodiments, the anti-PD-1 antibody is BGB-108. In some embodiments, the anti-PD-1 antibody is BCD-100. In some embodiments, the anti-PD-1 antibody is BAT1306. In some embodiments, the anti-PD-1 antibody is LZM009. In some embodiments, the anti-PD-1 antibody is AK105. In some embodiments, the anti-PD-1 antibody is HLX10. In some embodiments, the anti-PD-1 antibody is TSR-042. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD-1 monoclonal antibody is MGA012 (INCMGA0012; retifanlimab). In some embodiments, the anti-PD1 antibody is SHR-1210. Other anti-cancer agent(s) include antibody therapeutics such as 4-1BB (e.g., urelumab, utomilumab). In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is atezolizumab, avelumab, durvalumab, tislelizumab, BMS-935559, MEDI4736, atezolizumab (MPDL3280A; also known as RG7446), avelumab (MSB0010718C), FAZ053, KN035, CS1001, SHR-1316, CBT-502, A167, STI-A101, CK-301, BGB-A333, MSB-2311, HLX20, or LY3300054. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, or tislelizumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is durvalumab. In some embodiments, the anti-PD-L1 antibody is tislelizumab. In some embodiments, the anti-PD-L1 antibody is BMS-935559. In some embodiments, the anti-PD-L1 antibody is MEDI4736. In some embodiments, the anti-PD-L1 antibody is FAZ053. In some embodiments, the anti-PD-L1 antibody is KN035. In some embodiments, the anti-PD-L1 antibody is CS1001. In some embodiments, the anti-PD-L1 antibody is SHR-1316. In some embodiments, the anti-PD-L1 antibody is CBT-502. In some embodiments, the anti-PD-L1 antibody is A167. In some embodiments, the anti-PD-L1 antibody is STI-A101. In some embodiments, the anti-PD-L1 antibody is CK-301. In some embodiments, the anti-PD-L1 antibody is BGB-A333. In some embodiments, the anti-PD-L1 antibody is MSB-2311. In some embodiments, the anti-PD-L1 antibody is HLX20. In some embodiments, the anti-PD-L1 antibody is LY3300054.
In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule that binds to PD-L1, or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule that binds to and internalizes PD-L1, or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibitor of an immune checkpoint molecule is a compound selected from those in US 2018/0179201, US 2018/0179197, US 2018/0179179, US 2018/0179202, US 2018/0177784, US 2018/0177870, US 2019/0300524, and US 2019/0345170, or a pharmaceutically acceptable salt thereof, each of which is incorporated herein by reference in its entirety.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
In some embodiments, the inhibitor is MCLA-145.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, AGEN1884, or CP-675,206.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG3, e.g., an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525, INCAGN2385, or eftilagimod alpha (IMP321).
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD73. In some embodiments, the inhibitor of CD73 is oleclumab.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIGIT. In some embodiments, the inhibitor of TIGIT is OMP-31M32.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of VISTA. In some embodiments, the inhibitor of VISTA is JNJ-61610588 or CA-170.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of B7-H3. In some embodiments, the inhibitor of B7-H3 is enoblituzumab, MGD009, or 8H9.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR. In some embodiments, the inhibitor of KIR is lirilumab or IPH4102.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of A2aR. In some embodiments, the inhibitor of A2aR is CPI-444.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TGF-beta. In some embodiments, the inhibitor of TGF-beta is trabedersen, galusertinib, or M7824.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PI3K-gamma. In some embodiments, the inhibitor of PI3K-gamma is IPI-549.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD47. In some embodiments, the inhibitor of CD47 is Hu5F9-G4 or TTI-621.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD73. In some embodiments, the inhibitor of CD73 is MEDI9447.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD70. In some embodiments, the inhibitor of CD70 is cusatuzumab or BMS-936561.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM3, e.g., an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is INCAGN2390, MBG453, or TSR-022.
In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD20, e.g., an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of OX40, CD27, CD28, GITR, ICOS, CD40, TLR7/8, and CD137 (also known as 4-1BB).
In some embodiments, the agonist of CD137 is urelumab. In some embodiments, the agonist of CD137 is utomilumab.
In some embodiments, the agonist of an immune checkpoint molecule is an inhibitor of GITR. In some embodiments, the agonist of GITR is TRX518, MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323, MED11873, or MEDI6469. In some embodiments, the agonist of an immune checkpoint molecule is an agonist of OX40, e.g., OX40 agonist antibody or OX40L fusion protein. In some embodiments, the anti-OX40 antibody is INCAGN01949, MED10562 (tavolimab), MOXR-0916, PF-04518600, GSK3174998, BMS-986178, or 9B12. In some embodiments, the OX40L fusion protein is MEDI6383.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of CD40. In some embodiments, the agonist of CD40 is CP-870893, ADC-1013, CDX-1140, SEA-CD40, R07009789, JNJ-64457107, APX-005M, or Chi Lob 7/4.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of ICOS. In some embodiments, the agonist of ICOS is GSK-3359609, JTX-2011, or MEDI-570.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of CD28. In some embodiments, the agonist of CD28 is theralizumab.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of CD27. In some embodiments, the agonist of CD27 is varlilumab.
In some embodiments, the agonist of an immune checkpoint molecule is an agonist of TLR7/8. In some embodiments, the agonist of TLR7/8 is MEDI9197.
The compounds of the present disclosure can be used in combination with bispecific antibodies. In some embodiments, one of the domains of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3 or TGFβ receptor. In some embodiments, the bispecific antibody binds to PD-1 and PD-L1. In some embodiments, the bispecific antibody that binds to PD-1 and PD-L1 is MCLA-136. In some embodiments, the bispecific antibody binds to PD-L1 and CTLA-4. In some embodiments, the bispecific antibody that binds to PD-L1 and CTLA-4 is AK104.
In some embodiments, the compounds of the disclosure can be used in combination with one or more metabolic enzyme inhibitors. In some embodiments, the metabolic enzyme inhibitor is an inhibitor of IDO1, TDO, or arginase. Examples of IDO1 inhibitors include epacadostat, NLG919, BMS-986205, PF-06840003, IOM2983, RG-70099 and LY338196. Inhibitors of arginase inhibitors include INCB1158.
As provided throughout, the additional compounds, inhibitors, agents, etc. can be combined with the present compound in a single or continuous dosage form, or they can be administered simultaneously or sequentially as separate dosage forms.
When employed as pharmaceuticals, the compounds of the present disclosure can be administered in the form of pharmaceutical compositions. Thus, the present disclosure provides a composition comprising a compound of Formula (I), a compound as recited in any of the claims and described herein, or a pharmaceutically acceptable salt thereof, or any of the embodiments thereof, and at least one pharmaceutically acceptable carrier or excipient. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is indicated and upon the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, e.g., by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
This invention also includes pharmaceutical compositions which contain, as the active ingredient, the compound of the present disclosure or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the composition is suitable for topical administration. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, e.g., a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, e.g., up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.
In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention can be prepared by processes known in the art see, e.g., WO 2002/000196.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% silicon dioxide w/w.
In some embodiments, the composition is a sustained release composition comprising at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose and polyethylene oxide. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate and hydroxypropyl methylcellulose. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/or hydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel K00LV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105™).
In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to produce the composition.
The compositions can be formulated in a unit dosage form, each dosage containing from about 5 to about 1,000 mg (1 g), more usually about 100 mg to about 500 mg, of the active ingredient. In some embodiments, each dosage contains about 10 mg of the active ingredient. In some embodiments, each dosage contains about 50 mg of the active ingredient. In some embodiments, each dosage contains about 25 mg of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
The components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Particularly for human consumption, the composition is preferably manufactured or formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U.S. Food and Drug Administration. For example, suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U.S. Food and Drug Administration.
The active compound may be effective over a wide dosage range and is generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms and the like.
The therapeutic dosage of a compound of the present invention can vary according to, e.g., the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 g/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, e.g., about 0.1 to about 1000 mg of the active ingredient of the present invention.
The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.
Topical formulations can contain one or more conventional carriers. In some embodiments, ointments can contain water and one or more hydrophobic carriers selected from, e.g., liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and the like. Carrier compositions of creams can be based on water in combination with glycerol and one or more other components, e.g., glycerinemonostearate, PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be formulated using isopropyl alcohol and water, suitably in combination with other components such as, e.g., glycerol, hydroxyethyl cellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2 or at least about 5 wt % of the compound of the invention. The topical formulations can be suitably packaged in tubes of, e.g., 100 g which are optionally associated with instructions for the treatment of the select indication, e.g., psoriasis or other skin condition.
The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient and the like.
The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.
The therapeutic dosage of a compound of the present invention can vary according to, e.g., the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
Another aspect of the present invention relates to labeled compounds of the disclosure (radio-labeled, fluorescent-labeled, etc.) that would be useful not only in imaging techniques but also in assays, both in vitro and in vivo, for localizing and quantitating KRAS protein in tissue samples, including human, and for identifying KRAS ligands by inhibition binding of a labeled compound. Substitution of one or more of the atoms of the compounds of the present disclosure can also be useful in generating differentiated ADME (Adsorption, Distribution, Metabolism and Excretion). Accordingly, the present invention includes KRAS binding assays that contain such labeled or substituted compounds.
The present disclosure further includes isotopically-labeled compounds of the disclosure. An “isotopically” or “radio-labeled” compound is a compound of the disclosure where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). A “radio-labelled” compound can refer to an isotopically-labelled compound in which one or more atoms are replaced or substituted by an atom of an isotope that is radioactive.
Suitable isotopes that may be incorporated in compounds of the present disclosure include but are not limited to 2H (also written as D for deuterium), 3H (also written as T for tritium), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl, 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I and 131I. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced by deuterium atoms (e.g., one or more hydrogen atoms of a C1-6 alkyl group of Formula (I) can be optionally substituted with deuterium atoms, such as —CD3 being substituted for —CH3). In some embodiments, alkyl groups in Formula (I) can be perdeuterated.
One or more constituent atoms of the compounds presented herein can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of the hydrogen atoms in a compound can be replaced or substituted by deuterium atoms.
For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced by deuterium atoms (e.g., one or more hydrogen atoms of a C1-6 alkyl group of Formula (I) can be optionally substituted with deuterium atoms, such as —CD3 being substituted for-CH3). In some embodiments, alkyl groups in Formula (I) can be perdeuterated. The symbol D included in a chemical formula or as a substituent indicates that deuterium is incorporated in the position labelled at greater than natural abundance, and typically indicates an abundance of equal to or greater than 50%, preferably equal to or greater than 90% or equal to or greater than 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, or 99.99% relative to other forms of hydrogen.
Synthetic methods for including isotopes into organic compounds are known in the art (A. F. Thomas, Deuterium Labeling in Organic Chemistry, (Appleton-Century-Crofts, New York, N.Y., 1971); J. Atzrodt, et al., Angew. Chem. Int. Ed., 2007, 7744-65; J. R. Hanson, The Organic Chemistry of Isotopic Labelling, (Royal Society of Chemistry, 2011)). Isotopically labeled compounds can be used in various studies such as NMR spectroscopy, metabolism experiments, and/or assays.
Substitution with heavier isotopes, such as deuterium, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. (see e.g., A. Kerekes, et al., J. Med. Chem. 2011, 54(1), 201-10; R. Xu et al., J. Label. Compd. Radiopharm. 2015, 58, 308-12). In particular, substitution at one or more metabolism sites may afford one or more of the therapeutic advantages.
The radionuclide that is incorporated in the instant radio-labeled compounds will depend on the specific application of that radio-labeled compound. For example, for in vitro adenosine receptor labeling and competition assays, compounds that incorporate 3H, 14C, 82Br, 125I, 131I or 35S can be useful. For radio-imaging applications 11C, 18F, 125I, 123I, 124I, 131I, 75Br, 76Br or 77Br can be useful.
It is understood that a “radio-labeled” or “labeled compound” is a compound that has incorporated at least one radionuclide. In some embodiments, the radionuclide is selected from 3H, 14C, 125I, 35S and 82Br.
The present disclosure can further include synthetic methods for incorporating radio-isotopes into compounds of the disclosure. Synthetic methods for incorporating radio-isotopes into organic compounds are well known in the art, and an ordinary skill in the art will readily recognize the methods applicable for the compounds of disclosure.
A labeled compound of the invention can be used in a screening assay to identify and/or evaluate compounds. For example, a newly synthesized or identified compound (i.e., test compound) which is labeled can be evaluated for its ability to bind a KRAS protein by monitoring its concentration variation when contacting with the KRAS, through tracking of the labeling. For example, a test compound (labeled) can be evaluated for its ability to reduce binding of another compound which is known to bind to a KRAS protein (i.e., standard compound). Accordingly, the ability of a test compound to compete with the standard compound for binding to the KRAS protein directly correlates to its binding affinity. Conversely, in some other screening assays, the standard compound is labeled and test compounds are unlabeled. Accordingly, the concentration of the labeled standard compound is monitored in order to evaluate the competition between the standard compound and the test compound, and the relative binding affinity of the test compound is thus ascertained.
The present disclosure also includes pharmaceutical kits useful, e.g., in the treatment or prevention of diseases or disorders associated with the activity of KRAS, such as cancer or infections, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof. Such kits can further include one or more of various conventional pharmaceutical kit components, such as, e.g., containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.
The compounds of the Examples have been found to inhibit the activity of KRAS according to at least one assay described herein.
Experimental procedures for compounds of the invention are provided below. Preparatory LCMS purifications of some of the compounds prepared were performed on Waters mass directed fractionation systems. The basic equipment setup, protocols, and control software for the operation of these systems have been described in detail in the literature. See e.g., K. F. Blom, J. Combi. Chem., 2002, 4(4), 295-301; K. F. Blom, et al., J. Combi. Chem., 2003, 5(5), 670-683; and K. F. Blom, et al., J. Combi. Chem. 2004, 6(6), 874-83. The compounds separated were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity check.
The compounds separated were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity check under the following conditions: Instrument; Agilent 1100 series, LC/MSD, Column: Waters SUNFIRE® C18 5 μm particle size, 2.1×5.0 mm, Buffers: mobile phase A: 0.025% TFA in water and mobile phase B: MeCN; gradient 2% to 80% of B in 3 min. with flow rate 2.0 mL/min.
Some of the compounds prepared were also separated on a preparative scale by reverse-phase high performance liquid chromatography (RP-HPLC) with MS detector or flash chromatography (silica gel) as indicated in the Examples. Typical preparative reverse-phase high performance liquid chromatography (RP-HPLC) column conditions are as follows:
pH=2 purifications: Waters SUNFIRE® C18 5 μm particle size, 19×100 mm column, eluting with mobile phase A: 0.1% TFA in water and mobile phase B: MeCN; the flow rate was 30 mL/min., the separating gradient was optimized for each compound using the Compound Specific Method Optimization protocol as described in the literature [see K. F. Blom, et al., J. Combi. Chem. 2004, 6(6), 874-83]. Typically, the flow rate used with the 30×100 mm column was 60 mL/min.
pH=10 purifications: Waters XBRIDGE® C18 5 μm particle size, 19×100 mm column, eluting with mobile phase A: 0.15% NH4OH in water and mobile phase B: MeCN; the flow rate was 30 mL/min., the separating gradient was optimized for each compound using the Compound Specific Method Optimization protocol as described in the literature [See K. F. Blom, et al., J. Combi. Chem. 2004, 6(6), 874-83]. Typically, the flow rate used with 30×100 mm column was 60 mL/min.
The following abbreviations may be used herein: AcOH (acetic acid); Ac2O (acetic anhydride); aq. (aqueous); atm. (atmosphere(s)); BF3·OEt2 (boron trifluoride etherate); Boc (t-butoxycarbonyl); Boc2O (di-t-butyl dicarbonate); BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate); br (broad); Cbz (carboxybenzyl); calc. (calculated); Cs2CO3 (cesium carbonate); d (doublet); dd (doublet of doublets); DBU (1,8-diazabicyclo[5.4.0]undec-7-ene); DCM (dichloromethane); DIAD (N,N′-diisopropyl azidodicarboxylate); DIPEA (N,N-diisopropylethylamine); DIBAL (diisobutylaluminium hydride); DMF (N,N-dimethylformamide); DMSO (dimethylsulfoxide); DNP (dinitrophenol); eq. (equivalent(s)); Et (ethyl); EtOAc (ethyl acetate); Ex. (Example); FCC (flash column chromatography); g (gram(s)); h (hour(s)); HATU (N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate); HCl (hydrochloric acid); HPLC (high performance liquid chromatography); Hz (hertz); K2CO3 (potassium carbonate); KF (potassium fluoride); KI (potassium iodide); J (coupling constant); LiBH4 (lithium borohydride); LCMS (liquid chromatography-mass spectrometry); LiCl (lithium chloride); LDA (lithium diisopropylamide); m (multiplet); M (molar); mCPBA (3-chloroperoxybenzoic acid); MS (Mass spectrometry); Me (methyl); MeCN (acetonitrile); MeOH (methanol); mg (milligram(s)); MgSO4 (magnesium sulfate); min. (minutes(s)); mL (milliliter(s)); mmol (millimole(s)); Ms (mesyl); N (normal); N2 (nitrogen); NaBH4 (sodium borohydride); NADPH (nicotinamide adenine dinucleotide phosphate); NaHCO3 (sodium bicarbonate); NaIO4 (sodium metaperiodate); NaN3 (sodium azide); NaNO2 (sodium nitrite); Na2SO4 (sodium sulfate); Na2S2O3 (sodium thiosulfate); NCS (N-chlorosuccinimide); NEt3 (triethylamine); NH4Cl (ammonium chloride); NH4OH (ammonium hydroxide); nM (nanomolar); NMP (N-methylpyrrolidinone); NMR (nuclear magnetic resonance spectroscopy); OTf (trifluoromethanesulfonate); Pd(PPh3)4(tetrakis(triphenylphosphine)palladium(0)); Ph (phenyl); pM (picomolar); PPh3 (triphenylphosphine); prep. (preparative); RP-HPLC (reverse phase high performance liquid chromatography); r.t. (room temperature), s (singlet); sat. (saturated); Selectfluor (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)); t (triplet or tertiary); TBAF (tetrabutylammonium fluoride); TBDPS (tert-butyldiphenylsilyl); TBS (tert-butyldimethylsilyl); TBSCl (tert-butyldimethylsilyl chloride); TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl or (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl); tert (tertiary); tt (triplet of triplets); TFA (trifluoroacetic acid); THF (tetrahydrofuran); TMSOTf (trimethylsilyl trifluoromethanesulfonate); p Ts (tosyl); μg (microgram(s)); μL (microliter(s)); μM (micromolar); wt % (weight percent). Brine is sat. aq. sodium chloride. In vacuo is under vacuum.
1-Iodopyrrolidine-2,5-dione (21.15 g, 94 mmol) was added to a solution of 2-amino-4-bromo-3-fluorobenzoic acid (20 g, 85 mmol) in DMF (200 mL) and the reaction mixture was stirred at 80° C. for 3 h. The mixture was cooled with ice water and then water (500 mL) was added. The precipitate was filtered, washed with water, and dried to provide the sub-title compound as a solid. LCMS calc. for C7H5BrFINO2+ (M+H)+: m/z=359.9, 361.9; found 359.9, 361.9.
Triphosgene (9.07 g, 30.6 mmol) was added to a solution of 2-amino-4-bromo-3-fluoro-5-iodobenzoic acid (22 g, 61.1 mmol) in 1,4-dioxane (200 mL) and then the reaction mixture was stirred at 80° C. for 2 h. The reaction mixture was cooled with ice water and then filtered. The solid was washed with EtOAc to provide the sub-title compound as a solid. LCMS calc. for C8H3BrFINO3+ (M+H)+: m/z=385.8, 387.8; found 385.8, 387.8.
DIPEA (18.1 mL, 146 mmol) was added to a solution of ethyl 2-nitroacetate (11.6 mL, 146 mmol) and 7-bromo-8-fluoro-6-iodo-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (20 g, 73.0 mmol) in toluene (200 mL) at r.t. and the reaction mixture was stirred at 95° C. for 3 h. The reaction mixture was cooled, filtered, and washed with small amount of hexanes to provide the sub-title compound. LCMS calc. for C9H4BrFIN2O4+ (M+H)+: m/z=428.8, 430.8; found 428.8, 430.8.
DIPEA (8.14 mL, 46.6 mmol) was added to a mixture of 7-bromo-8-fluoro-6-iodo-3-nitroquinoline-2,4-diol (10 g, 23.31 mmol) in POCl3 (10.86 mL, 117 mmol) and the reaction mixture was stirred at 100° C. for 2 h. The solvent was removed under vacuum and then dried via azeotropic distillation with toluene (3×) to provide the crude material which was purified with FCC. LCMS calc. for C9H2BrCl2FIN2O2+ (M+H)+: m/z=464.8, 466.8; found 464.8, 466.8.
To a solution of Intermediate 1 (25 g, 53.7 mmol) and tert-butyl (1R,4R,5S)-5-amino-2-azabicyclo[2.1.1]hexane-2-carboxylate (10.6 g, 53.7 mmol) in MeCN (200 mL) was added DIPEA (14.0 mL, 81 mmol) and the reaction mixture was heated to 60° C. for 1 h. Ice chips and water (100 mL) were added and the suspension was stirred for 15 min. The solids were filtered, rinsed with water, and air dried under vacuum overnight. The solid obtained was suspended in MeCN (200 mL) and cooled to 0° C. A solution of sodium thiomethoxide (11.3 g, 161 mmol) in MeOH (30 mL) was slowly added and the reaction mixture was stirred at this temperature for 1 h. Ice and water were added, and the solid was filtered and air dried. The filtrate was extracted with EtOAc and combined with the solid. The combined product was used without purification. LCMS calc. for C20H22BrFIN4O4S+ (M+H)+: m/z=639.0, 641.0; found 639.1, 641.0.
To a solution of tert-butyl (1R,4R,5S)-5-((7-bromo-8-fluoro-6-iodo-2-(methylthio)-3-nitroquinolin-4-yl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (34.3 g, 53.7 mmol) in THF (200 mL), NEt3 (18.7 mL, 134 mmol), DMAP (0.66 g, 5.37 mmol), and Boc2O (23.4 g, 107 mmol) were added sequentially at r.t., and the reaction mixture was heated to 50° C. for 3 h. The reaction mixture was diluted with EtOAc and washed with sat. NaHCO3 and brine. The organic layer was dried over MgSO4, filtered, and concentrated. The product was used without purification. LCMS calc. for C21H22BrFIN4O6S+ (M−tBu+H)+: m/z=682.9, 684.9; found 682.9, 684.9.
A 1-L flask equipped with a mechanical stirrer was charged with tert-butyl (1R,4R,5S)-5-((7-bromo-8-fluoro-6-iodo-2-(methylthio)-3-nitroquinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (39.7 g, 53.7 mmol), MeOH (75 mL), water (75 mL), and THF (75 mL). Iron powder (15.0 g, 268 mmol) and NH4Cl (14.4 g, 268 mmol) were added, and the reaction mixture was stirred at 70° C. overnight. The reaction mixture was diluted with EtOAc and filtered through a pad of diatomaceous earth. The layers were separated and the organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The product was used in next step without further purification.
LCMS calc. for C21H24BrFIN4O4S+ (M−tBu+H)+: m/z=653.0, 655.0; found 653.0, 655.0.
To a solution of tert-butyl (1R,4R,5S)-5-((3-amino-7-bromo-8-fluoro-6-iodo-2-(methylthio)quinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (36.7 g, 51.7 mmol) in DMF (200 mL) was added Pd(PPh3)4 (12.0 g, 10.4 mmol), tetramethylammonium formate (30% w/w in water, 94 mL, 207 mmol), DIPEA (22.6 mL, 129 mmol), and acrylonitrile (49.3 mL, 0.129 mol). The headspace was purged with N2 and the reaction mixture was stirred at 80° C. overnight. The reaction mixture was cooled to r.t. and water was added. The crude mixture was extracted with EtOAc and the layers were separated. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The product was purified by FCC (5-40% EtOAc/DCM) to yield the title compound as a dark-brown solid (11.5 g, 18.1 mmol, 38% over 5 steps). LCMS calc. for C28H36BrFN5O4S+ (M+H)+: m/z=636.2 638.2; found 636.3, 638.2.
To a mixture of tert-butyl (1R,4R,5S)-5-((3-amino-7-bromo-6-(2-cyanoethyl)-8-fluoro-2-(methylthio)quinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (19 g, 29.8 mmol) in MeCN (200 mL) cooled to −20° C. was slowly added sulfuric acid (40% w/w in water, 14.1 mL, 85.6 mmol). To the mixture was slowly added a solution of NaNO2 (4.12 g, 59.7 mmol) in water (8 mL) over approximately 10 min to keep the internal temperature of the reaction mixture between −20° C. and −10° C. and stirring was continued for an additional 5 min. After this time, solution of KI (19.8 g, 119 mmol) in water (8 mL) was slowly added to the reaction mixture to keep reaction temperature between −10° C. and −5° C. and stirring was continued for an additional 10 min. Upon reaction completion, sat. aq. Na2S2O3 (50 mL) was added. The aq. layer was extracted with EtOAc (200 mL×3). Combined organic phase was washed with water (150 mL) followed by brine (150 mL), dried over MgSO4, and concentrated in vacuo. The product was used in the next step without purification. LCMS calc. for C24H26BrFIN4O4S+ (M−tBu+H)+: m/z=691.0, 693.0; found 691.0, 693.0.
To a mixture of tert-butyl (1R,4R,5S)-5-((7-bromo-6-(2-cyanoethyl)-8-fluoro-3-iodo-2-(methylthio)quinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (8 g, 14.6 mmol) in MeCN (20 mL) was added TFA (80 mL), and the mixture was stirred at 21° C. for 1 h. The reaction mixture was evaporated to dryness. The residue was diluted with THF (100 mL) and Et3N (20.4 mL, 146 mmol) was slowly added. After stirring for approximately 5 min, Boc2O (5.10 mL, 21.9 mmol) was added in one portion. The reaction mixture was left to stir at 21° C. for 1 h. The mixture was diluted with water (50 mL) and the aq. phase was extracted with EtOAc (100 mL×3). The combined organic phase was washed with water (100 mL) followed by brine (100 mL), dried over MgSO4 and concentrated. The product was purified by FCC (5-40% acetone/hexanes) to yield the title compound as a dark-brown solid (5.1 g, 36% over 2 steps). LCMS calc. for C23H26BrFIN4O2S+ (M+H)+: m/z=647.0, 649.0; found 647.0, 649.0.
To a solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-hydroxypyrrolidine-1,2-dicarboxylate (10.5 g, 42.8 mmol) in DMF (100 mL) at 0° C. was added imidazole (5.83 g, 86.0 mmol) followed by TBSCl (9.06 g, 55.7 mmol). The reaction mixture was warmed up to r.t. and stirred for 3 h. The reaction mixture was quenched by the addition of water (30 mL) and the aq. phase was extracted with EtOAc (3×200 mL). Combined organic phase was washed with water (150 mL) followed by brine (150 mL), dried over MgSO4, filtered, and concentrated. The mixture was filtered through a plug of diatomaceous earth and the filtrate was concentrated. The product was purified by FCC (5-40% EtOAc/hexanes) to yield the title compound as colorless oil (15 g, 41.7 mmol, 97%). LCMS calc. for C12H26NO3Si+ (M−CO2tBu+H)+: m/z=260.2; found 260.2.
To a solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1,2-dicarboxylate (15 g, 41.7 mmol) in THF (60 mL) at 0° C. was added LiBH4 (47.0 mL, 94.0 mmol, 2 M in THF) and the reaction mixture was warmed to r.t. and stirred for 1 h. The reaction mixture was evaporated to remove most of THF and then diluted with EtOAc (50 mL). To the solution was added water (10 mL) followed by careful addition of sat. aq. NaHCO3 (20 mL). The heterogeneous mixture was stirred vigorously for 5 min. The aq. phase was extracted with EtOAc (3×100 mL). Combined organic phase was washed with water (100 mL) followed by brine (100 mL), dried over MgSO4, filtered, and concentrated. The product was purified by FCC (5-50% EtOAc/hexanes) to yield the title compound as colorless oil (12 g, 41.7 mmol, 87%). LCMS calc. for C12H26NO4Si+ (M−tBu+H)+: m/z=276.2; found 276.2.
To a solution of tert-butyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (12 g, 36.2 mmol) in DCM (100 mL) at 0° C. was added Dess-Martin-periodinane (27.6 g, 65.2 mmol) and the reaction mixture was warmed to r.t. and stirred for 2 h. The reaction mixture was filtered through a pad of diatomaceous earth with DCM as eluent. The filtrate was washed with sat. aq. NaHCO3 (100 mL), sat. aq. Na2S2O3 (100 mL), water (100 mL), and brine (100 mL), dried over MgSO4, filtered, and concentrated. The product was purified by FCC (5-40% EtOAc/hexanes) to yield the title compound as colorless oil (8.5 g, 25.8 mmol, 72%). LCMS calc. for C11H24NO2Si+ (M−CO2tBu+H)+: m/z=230.2; found 230.2.
To a solution of tert-butyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-2-formylpyrrolidine-1-carboxylate (8.5 g, 25.8 mmol) in MeOH (70 mL) at 0° C. were added K2CO3 (7.13 g, 51.6 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (3.73 mL, 28.4 mmol) dropwise. The reaction mixture was allowed to warm to r.t. for 1 h and then concentrated. The crude residue was partitioned between water and EtOAc. The organic layer was washed with water and brine, dried over Na2SO4 and concentrated. The crude product was purified by FCC (5-40% EtOAc in hexanes) to provide the sub-title compound (5.5 g, 16.9 mmol, 66%). LCMS calc. for C13H24NO3Si+ (M−tBu+H)+: m/z=270.2; found 270.2.
To a solution of tert-butyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate (5.5 g, 16.9 mmol) in DCM (70 mL) at 0° C. was slowly added Et3N (18.8 mL, 135 mmol) followed by TMSOTf (10.7 mL, 59.1 mmol). The reaction mixture was allowed to warm to r.t. for 30 min and then sat. aq. NaHCO3 (20 mL) was added to quench the reaction. The aq. phase was extracted with EtOAc (3×70 mL). The combined organic phase was washed with water and brine, dried over Na2SO4, filtered, and concentrated. The residue was diluted with THF (50 mL), cooled to 0° C., and Et3N (7.0 mL, 50.7 mmol) was added followed by cyclopropanecarbonyl chloride (2.0 mL, 22.0 mmol). The reaction mixture was warmed to r.t. and stirred for an additional 20 min. Water (20 mL) was added to the reaction mixture and aq. phase was extracted with EtOAc (3×50 mL). The combined organic phase was dried over MgSO4, filtered, and concentrated. The product was purified by FCC (5-40% EtOAc/hexanes) to yield the title compound as colorless oil (4.5 g, 15.3 mmol, 91%). LCMS calc. for C16H28NO2Si+ (M+H)+: m/z=294.2; found 294.2.
To a mixture of Intermediate 2 (260 mg, 0.402 mmol) and Intermediate 3 (185 mg, 0.602 mmol) were added DMF (4 mL) and NEt3 (0.383 mL, 2.81 mmol), followed by Pd(PPh3)4 (93 mg, 0.080 mmol) and copper(I) iodide (45.9 mg, 0.241 mmol). The reaction flask was evacuated, back filled with N2, and then the reaction mixture was stirred at 80° C. for 2 h. After cooling to r.t., Cs2CO3 (654 mg, 2.01 mmol) was added and the reaction mixture was heated to 80° C. for 2 h. The reaction mixture was quenched with water and a small amount of sat. aq. NH4OH, then diluted with EtOAc and filtered through a pad of diatomaceous earth. The filtrate layers were separated and the organic layer was washed with water and brine, dried over Na2SO4 and concentrated. The crude product was purified by FCC (5-50% acetone in hexanes) to provide the sub-title compound (220 mg, 0.273 mmol, 68%). LCMS calc. for C39H52BrFN5O4SSi+ (M+H)+: m/z=812.3, 814.3; found 812.3, 814.3.
To a mixture of tert-butyl (1R,4R,5S)-5-(7-bromo-2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (220 mg, 0.271 mmol), (2,3-dichlorophenyl)boronic acid (103 mg, 0.541 mmol), KF (47.2 mg, 0.812 mmol) and bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (19.2 mg, 0.0271 mmol) were added 1,4-dioxane (2 mL)/water (0.5 mL) and the reaction flask was evacuated, back filled with N2, then the reaction mixture was stirred at 100° C. for 1 h. The reaction mixture was diluted with DCM and filtered through a plug of diatomaceous earth. The filtrate was concentrated and the crude product was purified by FCC (5-50% acetone in hexanes) to provide the sub-title compound (90 mg, 0.271 mmol, 38%). LCMS calc. for C45H55Cl2FN5O4SSi+ (M+H)+: m/z=878.3; found 878.3.
To a mixture of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (90 mg, 0.271 mmol), methylboronic acid (49.0 mg, 0.819 mmol), Pd(PPh3)4 (47.3 mg, 0.041 mmol) and copper(I) 3-methylsalicylate (77 mg, 0.358 mmol) was added 1,4-dioxane (1.5 mL) and the reaction flask was evacuated, back filled with N2, then the reaction mixture was stirred at 100° C. for 3 h. The reaction mixture was quenched with water and aq. 30% NH4OH, then diluted with EtOAc and filtered through a plug of diatomaceous earth. The layers of the filtrate were separated and the organic layer was washed with water and brine, dried over Na2SO4 and concentrated. The crude product was purified by FCC (5-50% acetone in hexanes) to provide the sub-title compound (50 mg, 0.102 mmol, 58%). LCMS calc. for C45H55Cl2FN5O4Si+ (M+H)+: m/z=846.3; found 846.3.
To a solution of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (50 mg, 0.059 mmol) in THF (1.0 mL) at 0° C. was added TBAF (1 M in THF, 0.059 mL, 0.059 mmol) and the reaction mixture was stirred at r.t. for 30 min. To the reaction mixture was added water (1 mL) and the aq. phase was extracted with EtOAc (3×2 mL). The combined organic phase was washed with brine (1.5 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by FCC (0-10% MeOH in DCM) to provide the title compound (40 mg, 0.059 mmol, 92%). LCMS calc. for C39H41Cl2FN5O4+ (M+H)+: m/z=732.3; found 732.3.
To a stirred solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-hydroxypyrrolidine-1,2-dicarboxylate (39.9 g, 163 mmol) in DCM (407 mL) at 0° C. was added imidazole (18.83 g, 277 mmol) followed by tert-butylchlorodiphenylsilane (44.4 mL, 171 mmol). The mixture was removed from ice and allowed to warm to r.t. overnight. The reaction mixture was diluted with water (400 mL) and the organic layer was retained. The aq. layer was further extracted with DCM (3×300 mL) and all organic layers were combined, washed with brine, and dried over MgSO4. Solvent was removed in vacuo and the crude product was used in the next step without further purification. LCMS calc. for C22H30NO3Si+ (M−Boc+H)+: m/z=384.2; found 384.2.
LiBH4 (104 mL, 208 mmol, 2M in THF) was added to a stirred solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)pyrrolidine-1,2-dicarboxylate (80.3 g, 166 mmol) in THF (415 mL, 0.4 M) at 0° C. at a rate that kept internal reaction temperature (monitored by thermocouple) <5° C. After complete addition of LiBH4 solution, the mixture was stirred for another h on ice and then the ice bath was removed. The mixture was allowed to warm to r.t. gradually and stirred overnight. The mixture was cooled in an ice bath and quenched by portion-wise addition of sat. aq. NH4Cl (caution: gas evolution, total volume of 100 mL sat. aq. NH4Cl added). After that, the mixture was diluted with water (100 mL) and brine (250 mL or more) to salt out THF and obtain a discrete organic layer. This organic layer was retained and the aq. layer was extracted with EtOAc (3×400 mL). Combined organic phase was washed with brine (100 mL), dried over MgSO4, and volatiles were removed in vacuo. The crude residue was dissolved in DCM, split into two batches, and purified by FCC (two runs, each 330-gram HP-Sil, 0-70% EtOAc/heptane over 15 CVs). Fractions containing the desired compound were combined and dried down to yield the sub-title compound (71.13 g, 156 mmol, 94% 2-step yield) as a clear, viscous oil. LCMS calc. for C22H30NO4Si+ (M−tBu+H)+: m/z=400.2; found 400.2.
To a stirred solution of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (60.43 g, 133 mmol) in DCM (265 mL, 0.5 M) at 0° C. was added trichloroisocyanuric acid (32.4 g, 139 mmol) followed by TEMPO (0.207 g, 1.326 mmol) with rapid stirring. The suspension markedly increased in turbidity within a few min. The ice bath was removed and the mixture was stirred for an additional 20 min. At this time, reaction monitoring by TLC (1:1 heptane/EtOAc, monitoring with KMnO4 and/or 2,4-DNP) indicated appearance of a higher Rf spot that stained by DNP in a manner consistent with an aldehyde (and disappearance of the lower Rf spot when stained with KMnO4). The mixture was filtered through a pad of diatomaceous earth with DCM as eluent. If precipitate was observed to form in the yellow oil, it would be filtered out and washed with DCM prior to FCC. The material was split into two batches and purified by FCC (two runs, 330-gram HP-Sil, 0-50% EtOAc/heptane over 15 CVs). Fractions containing the desired compound were combined and dried down to yield the sub-title compound (51.67 g, 114 mmol, 86% yield) as a light-yellow, viscous oil. LCMS calc. for C21H28NO2Si+ (M−Boc+H)+: m/z=354.2; found 354.2.
K2CO3 (22.12 g, 160 mmol) and dimethyl (1-diazo-2-oxopropyl)phosphonate (12.28 mL, 82 mmol) were added sequentially to a rapidly stirred solution of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-formylpyrrolidine-1-carboxylate (32.27 g, 71.1 mmol) in MeOH (237 mL) at 0° C. After addition of all reagents, the mixture was stirred on ice for 10 min. and then the ice bath was removed and the mixture was stirred for an additional 70 min. Reaction progress was monitored by TLC (1:1 heptane/EtOAc, KMnO4) based on the appearance of a higher-Rf, strongly staining spot. Upon reaction completion, the mixture was diluted with water (100 mL) and brine (300 mL) and extracted with EtOAc (3×300 mL). Organic layers were combined, dried over MgSO4, and dried in vacuo to yield the crude reaction product. The material was dissolved in DCM (50 mL) and purified by FCC (330 gram HP-Sil, 0-50% heptane/EtOAc over 15 CVs) to yield tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate (23.89 g, 53.1 mmol, 74.7% yield) as a clear yellow oil. LCMS calc. for C17H22NO3Si+ (M−tBu−Ph+H)+: m/z=316.1; found 316.1. 1H NMR (400 MHz, CDCl3) δ 7.66 (ddd, J=8.1, 5.0, 1.6 Hz, 4H), 7.43 (dddd, J=12.9, 8.3, 5.6, 2.1 Hz, 6H), 4.59 (s, 1H), 4.47 (p, J=4.6 Hz, 1H), 3.39 (s, 2H), 2.30-2.19 (m, 2H), 2.01 (dt, J=12.3, 5.7 Hz, 1H), 1.51 (s, 9H), 1.07 (s, 9H).
In a 40 mL vial tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate (2.6139 g, 5.81 mmol) was dissolved in DCM (14.53 mL) under N2. The mixture was cooled to −0° C. on an ice bath and NEt3 (6.48 mL, 46.5 mmol) was added at once. Once thermally equilibrated (internal temp monitored via a thermocouple), TMSOTf (4.20 mL, 23.25 mmol) was added dropwise to maintain an internal temperature <8° C. The mixture was stirred on ice for 30 min. with LCMS monitoring and then quenched by slow-addition of sat. aq. NaHCO3 (10 mL). The mixture was extracted with DCM (3×10 mL), washed with brine, and dried over MgSO4. Volatiles were removed in vacuo. The crude product was used in next step without further purification. LCMS calc. for C22H28NOSi+ (M−Boc+H)+: m/z=350.2; found 350.2.
The crude material from Intermediate 5, step 5 containing (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine was dissolved in DCM (10 mL) and cooled to 0° C. DIPEA (3.05 mL, 17.44 mmol) was added at once and the mixture was allowed to cool/thermally equilibrate (no change in temperature via thermocouple monitoring). Cyclopropanecarbonyl chloride (0.581 mL, 6.39 mmol) was added dropwise over 5 min., keeping the internal temperature of the reaction mixture below 8° C. The mixture was quenched with sat. aq. NaHCO3 (10 mL), extracted with DCM (3×10 mL), washed with brine, and dried over MgSO4. Volatiles were removed in vacuo and the residue was dissolved in DCM (4 mL) and purified by FCC (EtOAc/heptane) to yield the title compound as a clear/light-yellow oil. LCMS calc. for C26H32NO2Si+ (M+H)+: m/z=418.2; found 418.2.
To a 40 mL scintillation vial was charged with Intermediate 2 (800 mg, 1.121 mmol), Intermediate 5 (231 mg, 1.290 mmol), copper(I) iodide (214 mg, 1.121 mmol) and Pd(PPh3)4 (130 mg, 0.112 mmol). After which, to the mixture was added DMF (3.74 mL) followed by DIPEA (0.98 mL, 5.61 mmol) under N2. The reaction mixture was sparged with N2 via a needle and then heated to 90° C. for 1 h. The mixture was removed from heat, allowed to cool for 10 min., and then additional DMF (3.74 mL) and CS2CO3 (3654 mg, 11.21 mmol) were added to the mixture. The vial was recapped and heated at 90° C. for 3 h with rapid stirring. Cyclization progress was monitored by observing a peak-shift by reverse-phase LCMS (water/MeCN, 0.1% TFA, C18 stationary phase) [desired cyclized material elutes earlier than un-cyclized material].
After 3 h the material was removed from heat, cooled to r.t, and diluted with EtOAc (50 mL), 30% aq. NH4OH (30 mL), and brine (20 mL). The organic layer was retained and the aq. phase was further extracted with EtOAc (3×50 mL). Organic extracts were combined, washed with 1:1 brine/NH4OH (25 mL Vtot), dried over MgSO4, and dried in vacuo. The crude residue was dissolved in a minimal volume of DCM (<5 mL) and purified by FCC (0-100% acetone/heptane, 40G HP-Sil column). Fractions containing the desired compound were combined and dried in vacuo to yield the title compound (714.9 mg, 0.935 mmol, 83% yield) as a brownish-orange solid. LCMS calc. for C39H41Cl2FN5O4S+ (M+H)+: m/z=764.2; found 764.2.
In a 250 mL round-bottomed flask, 1-(tert-butyl) 2-methyl (2R,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (12 g, 48.9 mmol) and imidazole (5.00 g, 73.4 mmol) were dissolved in DCM (120 mL) at 0° C. TBS-Cl (11.06 g, 73.4 mmol) was added to the reaction mixture portion-wise to keep the internal temperature below 5° C. (monitored by thermocouple inserted into reaction mixture). After complete addition of TBS-Cl, the mixture was allowed to warm naturally to r.t. and stirred overnight. The following day, the mixture was quenched with MeOH (10 mL), diluted with water (100 mL), extracted with EtOAc (100 mL) and the organic layer was retained. The aq. layer was further extracted with EtOAc (3×100 mL). All organic layers were combined, washed with brine, dried over MgSO4, and volatiles were removed in vacuo. The residue was used directly in the subsequent step without further purification. LCMS calc. for C12H26NO3Si (M−Boc+H)+: m/z=260.2; found 260.1.
To the crude material of 1-(tert-butyl) 2-methyl (2R,4R)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1,2-dicarboxylate from Intermediate 7, Step 1 was added THF (122 mL). Upon cooling to 0° C., LiBH4 (48.9 mL, 98 mmol, 2 M in THF) was added over 1 min, upon which point the mixture was allowed to warm to r.t. and stirred overnight. The reaction mixture was quenched by slow addition of sat. aq. NH4Cl (20 mL), diluted with brine (80 mL), and extracted with EtOAc (3×150 mL). Organic layers were combined and dried over MgSO4 and solvent was removed in vacuo. The crude residue was purified by FCC (120 G HP-Sil column, 0-100% EtOAc/heptane over 20 CVs) to yield the title compound as a clear, viscous oil (13.45 grams, 40.6 mmol, 83% over two steps). LCMS calc. for C12H26NO4Si+ (M−tBu+H)+: m/z=276.2; found 276.2.
In a 40 mL vial tert-butyl (2R,4R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (500 mg, 1.508 mmol) and trichloroisocyanuric acid (386 mg, 1.659 mmol) were suspended in DCM (3.02 mL). The mixture was cooled to 0° C. and stirred rapidly. To the reaction mixture was then added TEMPO (5.89 mg, 0.038 mmol) and it was allowed to warm to r.t. and stir for 20 min. The mixture was filtered through diatomaceous earth, diluted with DCM (20 mL), washed with 1M HCl (5 mL), sat. aq. NaHCO3 (5 mL), and brine (5 mL). The organic layer was dried over MgSO4 and volatiles were removed in vacuo to yield a viscous yellow oil that was of suitable purity to be used directly in the next step (497 mg, 1.448 mmol, 96%). LCMS calc. for C12H24NO4Si+ (M−tBu+H)+: m/z=274.1; found 274.1.
To a solution of tert-butyl (2R,4R)-4-((tert-butyldimethylsilyl)oxy)-2-formylpyrrolidine-1-carboxylate (479 mg, 1.454 mmol) in MeOH (7.27 mL) at 0° C. was added K2CO3 (442 mg, 3.20 mmol) followed by dimethyl (1-diazo-2-oxopropyl)phosphonate (262 μL, 1.744 mmol) at once. The reaction mixture was allowed to warm to 21° C., stir for 1 h, and then water (10 mL), brine (10 mL), and EtOAc (15 mL) were added. The organic layer was retained, and the aq. layer extracted with EtOAc (3×10 mL). The organic layers were combined and washed with brine (5 mL), dried over MgSO4, and concentrated in vacuo. The crude product was purified by FCC (40 G HP-Sil, 0-50% EtOAc/heptane) to provide the title compound as a viscous light-yellow oil (154 mg, 0.473 mmol, 32.5% yield). LCMS calc. for C13H24NO3Si+ (M−tBu+H)+: m/z=270.2; found 270.1.
To a solution of tert-butyl (2R,4R)-4-((tert-butyldimethylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate (767 mg, 2.356 mmol) in DCM (11.78 mL) at 0° C. was slowly added NEt3 (2.63 mL, 18.85 mmol) followed by TMSOTf (1.703 mL, 9.42 mmol). The reaction mixture was allowed to warm to 21° C., stir for 30 min., and then sat. aq. NaHCO3 (5 mL) was added to quench the reaction. The mixture was diluted with water (5 mL), the organic layer was retained, and the aq. layer was extracted with DCM (3×10 mL). Organic layers were combined, washed with brine (7.5 mL), dried over MgSO4, and solvent was removed in vacuo. The residue was diluted with DCM (10 mL), cooled to 0° C., and DIPEA (1.235 mL, 7.07 mmol) was added followed by cyclopropanecarbonyl chloride (0.236 mL, 2.59 mmol) dropwise over 2 min. The reaction mixture was stirred at 0° C. for 30 min and then diluted with water (5 mL) and brine (5 mL). The organic layer was retained and the aq. layer further extracted with DCM (3×7.5 mL). The organic layers were combined, dried over MgSO4, and dried in vacuo. The crude residue was purified by FCC (40 G HP-Sil, 0-50% EtOAc/heptane) to provide the title compound as a colorless oil (627 mg, 2.136 mmol, 91% yield). LCMS calc. for C16H28NO2Si+ (M+H)+: m/z=294.2; found 294.2.
This compound was prepared according to the procedure described in Intermediate 4, Step 1, using Intermediate 7 instead of Intermediate 3. LCMS calc. for C39H52BrFN5O4SSi+ (M+H)+: m/z=812.3, 814.3; found 812.2, 814.2.
This compound was prepared according to the procedure described in Intermediate 4, Step 2, using tert-butyl (1R,4R,5S)-5-(7-bromo-2-((2R,4R)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(7-bromo-2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C45H55Cl2FN5O4SSi+ (M+H)+: m/z=878.3; found 878.2.
This compound was prepared according to the procedure described in Intermediate 4, Step 3, using tert-butyl (1R,4R,5S)-5-(2-((2R,4R)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C45H55Cl2FN5O4Si+ (M+H)+: m/z=846.3; found 846.2.
This compound was prepared according to the procedure described in Intermediate 4, Step 4, using tert-butyl (1R,4R,5S)-5-(2-((2R,4R)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C39H41Cl2FN5O4+ (M+H)+: m/z=732.3; found 732.2.
Methanesulfonyl chloride (6.54 μL, 0.084 mmol) was added to a solution of tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4R)-1-(cyclopropanecarbonyl)-4-hydroxypyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (41 mg, 0.056 mmol) and NEt3 (0.023 mL, 0.168 mmol) in DCM (2 mL) cooled to 0° C. under N2, stirring at 0° C. was continued for 1 h. The mixture was quenched by the addition of sat. aq. NaHCO3 (2 mL), diluted with water (2 mL), and extracted with DCM (3×5 mL). Organic extracts were combined, dried over MgSO4, and volatiles were removed in vacuo to afford the title compound as a white residue. (44.9 mg, 0.055 mmol, 99% yield). LCMS calc. for C40H43Cl2FN5O6S (M+H)+: m/z=810.2; found 810.2.
To a mixture of NaIO4 (10.14 g, 47.4 mmol, 2.8 eq.) in water (130 mL) was added ruthenium (IV) oxide (789 mg, 5.93 mmol, 0.35 eq.) The solution was stirred for 5 min. at r.t. To this reaction mixture was added a solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1,2-dicarboxylate (Intermediate 3, Step 1) (6.09 g, 16.94 mmol, 1.00 eq.) in EtOAc (75 mL), and the reaction mixture was stirred vigorously for 3 h at r.t. under an atmosphere of N2. The resulting solution was quenched with aq. NaHSO3, diluted with EtOAc, washed with water and brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The crude residue was purified by FCC (0-20% EtOAc/Hexanes) to afford the sub-title compound as a colorless oil which crystalized on standing (4.94 g, 4.63 mmol, 78%). LCMS calc. for C17H31NNaO6Si+ (M+Na)+: m/z=396.2; found 396.2.
Methylmagnesium bromide (3M in ether, 5.3 mL, 15.87 mmol, 1.20 eq.) was added dropwise to a solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-oxopyrrolidine-1,2-dicarboxylate (4.94 g, 13.23 mmol) in dry THF (100 mL) at −78° C. The solution was stirred for 30 min. at −78° C., then quenched with sat. aq. NH4Cl solution. The mixture was warmed to r.t., diluted with EtOAc, washed with water and brine, dried over anhydrous Na2SO4, and concentrated under vacuum. The crude residue was taken to the next step without further purification. LCMS calc. for C13H26NO3Si+ (M+H−CO2tBu−OH)+: m/z=272.2; found 272.2.
A solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-hydroxy-5-methylpyrrolidine-1,2-dicarboxylate (5.15 g, 13.22 mmol, assumed mass from Intermediate 9, Step 2) in DCM (88 mL) was cooled to 0° C. NEt3 (18.43 mL, 132 mmol, 10 eq.) was added followed by the dropwise addition of TMSOTf (14.33 mL, 79 mmol, 6 eq.). The reaction mixture was stirred at 0° C. for 3 h. The reaction mixture was quenched at 0° C. with 10:1 MeOH/AcOH (100 mL) and raised to r.t. To this solution was added NaBH3CN (1.66 g, 26.4 mmol, 2 eq.) and the reaction mixture was stirred at r.t. for 10 min. The reaction mixture was further quenched with sat. aq. NaHCO3 solution, extracted with EtOAc, dried over MgSO4, and concentrated under reduced pressure. The crude residue was taken to the next step without further purification. LCMS calc. for C13H28NO3Si+ (M+H)+: m/z=274.2; found 274.2.
A vial was charged with methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-methylpyrrolidine-2-carboxylate (3.61 g, 13.20 mmol, assumed mass from previous step), DCM (44 mL), and NEt3 (3.68 mL, 26.4 mmol, 2 eq.). The solution was cooled to 0° C. and cyclopropanecarbonyl chloride (1.44 mL, 15.84 mmol, 1.2 eq.) was added dropwise. The reaction mixture was warmed to r.t. and stirred for 1 h. The reaction mixture was quenched with sat. aq. NaHCO3 solution, extracted with DCM, dried over MgSO4, and concentrated under reduced pressure. The crude residue was purified by FCC (0-50% EtOAc/hexanes) to afford the sub-title compound as a mixture of diastereomers (2.36 g, 6.92 mmol, 52% over three steps). LCMS calc. for C17H32NO4Si+ (M+H)+: m/z=342.2; found 342.2.
To a solution of methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-5-methylpyrrolidine-2-carboxylate (2.6 g, 7.61 mmol) in tetrahydrofuran (38 mL) at 0° C. was added LiBH4 (0.2 g, 9.14 mmol, 1.2 eq.) and the reaction mixture was stirred at r.t. for 2 h. After that, reaction mixture was quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, dried over Na2SO4, and concentrated. Purification by FCC (0-100% EtOAc/hexanes) afforded the sub-title compound as a mixture of diastereomers (1.75 g, 5.59 mmol, 73%). LCMS calc. for C16H32NO3Si+ (M+H)+: m/z=314.2; found 314.2.
To a solution of ((3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-methylpyrrolidin-1-yl)(cyclopropyl)methanone (1.75 g, 5.58 mmol) in DCM (11 mL) cooled to 0° C. was added trichlorocyanuric acid (1.36 g, 5.86 mmol, 1.05 eq.) followed by TEMPO (8.72 mg, 0.056 mmol, 0.01 eq.). The reaction mixture was warmed to r.t. and stirred for 20 min. The reaction mixture was filtered, the filter cake washed with DCM, and the filtrate concentrated. The crude residue was purified by FCC (0-100% EA/hexanes) to afford the sub-title compound as a mixture of diastereomers (1.29 g, 4.15 mmol, 74%). LCMS calc. for C16H30NO3Si+ (M+H)+: m/z=312.2; found 312.2.
To a solution of (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-5-methylpyrrolidine-2-carbaldehyde (1.29 g, 4.14 mmol) in MeOH (20 mL) cooled to 0° C. was added K2CO3 (1.15 g, 8.28 mmol, 2 eq.) followed by dropwise addition of dimethyl (1-diazo-2-oxopropyl)phosphonate (0.75 mL, 4.97 mmol, 1.2 eq.). The reaction mixture was stirred rapidly and gradually warmed to r.t. over 3 h. The reaction mixture was quenched with sat. aq. NH4Cl and extracted with DCM. The combined organic fractions were dried over MgSO4, filtered, and concentrated. The crude residue was purified by FCC (0-100% EA/hexanes) to afford the sub-title compound as a mixture of diastereomers (0.9 g, 2.93 mmol, 71%). LCMS calc. for C17H30NO2Si+ (M+H)+: m/z=308.2; found 308.2.
A solution of ((3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-ethynyl-2-methylpyrrolidin-1-yl)(cyclopropyl)methanone (0.9 g, 2.94 mmol) in THF (15 mL) was cooled to 0° C. TBAF (1 M in THF, 3.3 mL, 1.1 eq.) was added dropwise and the reaction mixture was stirred at 0° C. for 1 h. The reaction mixture was quenched with sat. aq. NH4Cl and extracted with DCM. The combined organic fractions were dried over MgSO4, filtered, and concentrated.
The crude residue was purified by FCC (0-100% EA/hexanes) to afford the title compound as a single diastereomer (0.253 g, 45%). LCMS calc. for C11H16NO2+ (M+H)+: m/z=194.1; found 194.2. 1H NMR (500 MHz, DMSO-d6, single rotamer) δ 5.07 (d, J=3.6 Hz, 1H), 4.83 (td, J=7.7, 2.1 Hz, 1H), 3.95-3.91 (m, 1H), 3.86 (qd, J=6.7, 1.7 Hz, 1H), 3.42 (d, J=2.1 Hz, 1H), 2.25 (dd, J=7.7, 3.8 Hz, 1H), 2.14 (dd, J=7.7, 3.4 Hz, 1H), 1.96-1.91 (m, 1H), 1.09 (d, J=6.7 Hz, 3H), 0.83-0.67 (m, 4H).
A vial was charged with tert-butyl (1R,4R,5S)-5-((3-amino-7-bromo-6-(2-cyanoethyl)-8-fluoro-2-(methylthio)quinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (Intermediate 2, Step 4) (9 g, 14.14 mmol), (2,3-dichlorophenyl)boronic acid (3.78 g, 19.79 mmol, 1.4 eq.), KF (3.29 g, 56.6 mmol, 4 eq.), bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (1.5 g, 2.12 mmol, 0.15 eq.), 1,4-dioxane (80 mL) and water (15 mL). The reaction mixture was sparged with N2 for 5 min., and then heated to 95° C. for 30 min. The reaction mixture was cooled to r.t. and partitioned between water (100 mL) and EtOAc (100 mL). The layers were separated, then the organic layer was washed with brine (2×80 mL) and dried over Na2SO4, filtered, and concentrated. The crude residue was purified by FCC (0-50% EtOAc/DCM) to afford the sub-title compound (6.26 g, 8.93 mmol, 63%). LCMS calc. for C34H39Cl2FN5O4S+ (M+H)+: m/z=702.2; found 702.3.
This compound was prepared according to the procedure described in Intermediate 4, Step 3, using tert-butyl (1R,4R,5S)-5-((3-amino-6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoro-2-(methylthio)quinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C30H31Cl2FN5O4+ (M−t-Bu+H)+: m/z=614.2; found 614.2.
To a solution of tert-butyl (1R,4R,5S)-5-((3-amino-6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoro-2-methylquinolin-4-yl)(tert-butoxycarbonyl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (11 g, 16.4 mmol) in MeCN (160 mL) was added sulfuric acid (50 wt. %, 4.37 mL, 41 mmol, 2.5 eq.) dropwise at −20° C. Sat. aq. NaNO2 (2.26 g, 32.8 mmol, 2 eq.) was slowly added to maintain internal temperature between −20 and −15° C. After stirring for 30 min., sat. aq. KI (10.89 g, 65.6 mmol, 4 eq.) was slowly added to maintain internal temperature between −20 and −15° C. After stirring for 5 min., the reaction mixture was quenched with sat. aq. NaHCO3 and sat. aq. Na2S2O3 and diluted with DCM. The layers were separated and the aq. layer extracted with DCM. The combined organic fractions were dried over MgSO4, filtered, and concentrated. The crude residue was purified by FCC (0-100% EtOAc/hexanes) to afford the sub-title compound (10.45 g, 13.4 mmol, 82%). LCMS calc. for C34H37Cl2FIN4O4+ (M+H)+: m/z=781.1; found 781.3.
A flask was charged with tert-butyl (1R,4R,5S)-5-((tert-butoxycarbonyl)(6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoro-3-iodo-2-methylquinolin-4-yl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (10.45 g, 13.37 mmol), DCM (50 mL) and TFA (50 mL). The solution was stirred at r.t. for 2 h. The reaction mixture was concentrated. To the crude residue was added 50 mL DCM, and the solution was concentrated again. To the crude residue was added THF (50 mL), NEt3 (18.64 mL, 10 eq.) and Boc2O (4.38 g, 20.06 mmol, 1.5 eq.). The reaction mixture was stirred at r.t. for 1 h. The reaction mixture was quenched with sat. aq. NaHCO3, diluted with DCM, and the layers were separated. The aq. layer was extracted with DCM, and the combined organic extracts were dried over MgSO4, filtered, and concentrated. Purification by FCC (0-100% EtOAc/hexanes) afforded the sub-title compound (8.2 g, 12.0 mmol, 90%). LCMS calc. for C29H29Cl2FIN4O2+ (M+H)+: m/z=681.1; found 681.2.
To a solution of tert-butyl (1R,4R,5S)-5-((6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoro-3-iodo-2-methylquinolin-4-yl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (400 mg, 0.587 mmol), Intermediate 9 (125 mg, 0.646 mmol, 1.1 eq.), Pd(PPh3)4 (136 mg, 0.117 mmol, 0.2 eq.), and DIPEA (1.03 mL, 5.87 mmol, 10 eq.) in DMF (3 mL) was added copper (I) Iodide (45 mg, 0.235 mmol, 0.4 eq.). The reaction mixture was sparged with N2 for 5 min. then heated to 55° C. for 1 h. Then, Cs2CO3 (1.9 g, 5.87 mmol, 10 eq.) was added and the reaction mixture was stirred rapidly for 2 h at 100° C. The reaction mixture was cooled to r.t., quenched with sat. aq. NH4OH and extracted with DCM. The combined organic fractions were dried over MgSO4, filtered, and concentrated. The crude residue was purified by FCC (0-100% acetone/hexanes) to afford the title compound (251 mg, 0.336 mmol, 57%). LCMS calc. for C40H43Cl2FN5O4+ (M+H)+: m/z=746.3; found 746.4.
To a 40 mL scintillation vial was added tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (90 mg, 0.102 mmol) (Intermediate 4, Step 2), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2-((trimethylsilyl)oxy)propan-2-yl)pyridine (137 mg, 0.410 mmol), copper (I) 3-methylsalicylate (77 mg, 0.358 mmol), and Pd(PPh3)4 (59 mg, 0.051 mmol). After that, the mixture was dissolved in 1,4-dioxane (4.3 mL) under N2. The mixture was sparged with N2 for 2 min. and then the headspace was purged with N2. The mixture was stirred at 100° C. for 4 h, subsequently cooled to r.t., diluted with 1:1 NH4OH/brine (60 mL), and extracted with EtOAc (3×50 mL). Organic extracts were combined, washed with 1:1 NH4OH/brine (25 mL), dried over MgSO4, and solvent was removed in vacuo. The crude residue was purified by FCC (0-100% hexane/acetone) and fractions containing the desired compound were combined and dried in vacuo to yield the sub-title compound (35 mg, 0.036 mmol, 35% yield) as white solid. LCMS calc. for C52H62Cl2FN6O5Si+ (M+H)+: m/z=967.4; found 967.4.
To a solution of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (99 mg, 0.102 mmol) in THF (1.0 mL) at 0° C. was added TBAF (1 M in THF, 0.128 mL, 0.128 mmol) and the reaction mixture was stirred at r.t. for 30 min. To the reaction mixture was added water (1 mL) and the aq. phase was extracted with EtOAc (3×2 mL). The combined organic phase was washed with brine (1.5 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by FCC (0-10% MeOH in DCM) to provide the title compound (78 mg, 0.091 mmol, 90%). LCMS calc. for C46H48Cl2FN5O5(M+H)+: m/z=853.3; found 853.3.
This compound was prepared according to the procedure described in Step 1, Intermediate 11, using N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinamide instead of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2-((trimethylsilyl)oxy)propan-2-yl)pyridine. LCMS calc. for C46H47Cl2FN7O5(M+H)+: m/z=866.3; found 866.3.
This compound was prepared according to the procedure described in Step 2, Intermediate 11, using tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-4-(6-(dimethylcarbamoyl)pyridin-3-yl)-6-fluoro-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C46H47Cl2FN7O5(M+H)+: m/z=866.3; found 866.3.
This compound was prepared according to the procedure described in Step 3, Intermediate 4, using Intermediate 6 instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate, and isopropenylboronic acid pinacol ester instead of methylboronic acid. LCMS calc. for C41H43Cl2FN5O4+ (M+H)+: m/z=758.3; found 758.3.
This compound was prepared according to the procedure described in Step 5, Intermediate 3, using 1-methylcyclopropane-1-carbonyl chloride instead of cyclopropanecarbonyl chloride. LCMS calc. for C17H30Cl2NO2Si+ (M+H)+: m/z=308.2; found 308.2.
This compound was prepared according to the procedure described in Step 1, Intermediate 4, using Intermediate 14 instead of Intermediate 3. LCMS calc. for C46H57Cl2FN5O4SSi+ (M+H)+: m/z=892.3; found 892.3.
This compound was prepared according to the procedure described in Step 3, Intermediate 4, using tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(1-methylcyclopropane-1-carbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C48H59Cl2FN5O4Si+ (M+H)+: m/z=886.4; found 886.4.
This compound was prepared according to the procedure described in Step 4, Intermediate 4, using tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(1-methylcyclopropane-1-carbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(prop-1-en-2-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C42H45Cl2FN5O4+ (M+H)+: m/z=772.3; found 772.3.
This compound was prepared according to the procedure described in Step 8, Intermediate 9, using Intermediate 5 instead of ((3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-ethynyl-2-methylpyrrolidin-1-yl)(cyclopropyl)methanone. LCMS calc. for C10H14NO2+ (M+H)+: m/z=180.1; found 180.1.
A mixture of 2-amino-4-bromo-3-fluorobenzoic acid (28.0 g, 120 mmol), (2,3-dichlorophenyl)boronic acid (25.1 g, 132 mmol), bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (2.12 g, 3.00 mmol) and potassium phosphate (50.8 g, 239 mmol) in 1,4-dioxane (170 mL) and water (30 mL) was sparged with N2 and heated at 70° C. for 1 h. Once completed, the reaction mixture was cooled down to r.t. and poured into 1 N HCl (200 mL). The mixture was stirred for another 10 min, resulting in precipitation. The solids were collected on a fritted filter, washed with water followed by hexanes and dried under reduced pressure to afford the title compound in near quantitative yield. The crude product was used in next step without further purification. LCMS calc. for C13H9Cl2FNO2 (M+H)+: m/z=300.0; found 300.0.
To a solution of 3-amino-2′,3′-dichloro-2-fluoro-[1,1′-biphenyl]-4-carboxylic acid (35.8 g, 119 mmol) in DMSO (100 mL) was added N-bromosuccinimide (22.3 g, 125 mmol). The resulting mixture was heated at 50° C. for 1 h. Once completed, the reaction mixture was cooled down to r.t. and poured into ice water (400 mL). To the suspension, was added 20 mL sat. Na2S2O3. After stirring for 15 min, the solids were collected on a fritted filter, washed with water followed by hexanes and dried under reduced pressure to afford the title compound (43.0 g, 95% yield). The crude product was used in next step without further purification. LCMS calc. for C13H8BrCl2FNO2 (M+H)+: m/z=377.9, 379.9; found 378.0, 380.0.
To a solution of 3-amino-6-bromo-2′,3′-dichloro-2-fluoro-[1,1′-biphenyl]-4-carboxylic acid (38.6 g, 102 mmol) in THF (300 mL) was added triphosgene (10.6 g, 35.6 mmol) portionwise. After addition, the mixture was heated at 60° C. for 0.5 h. Once completed, the reaction mixture was cooled down to r.t. and poured into heptane (1000 mL). After stirring for 1 h, the solids were collected on a fritted filter, washed with hexanes and dried under reduced pressure to afford the title compound in near quantitative yield. The crude product was used in next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.10 (s, 1H), 7.85 (dd, J=7.9, 1.5 Hz, 1H), 7.58 (t, J=7.9 Hz, 1H), 7.43 (dd, J=7.9, 1.5 Hz, 1H).
To a stirring solution of (R)-2-(benzyloxy)propanoic acid (8.00 g, 44.4 mmol) in EtOAc (22 mL) was slowly added 1,1′-carbonyldiimidazole (7.92 g, 48.8 mmol), resulting in gas evolving. The mixture was stirred at r.t. for 1 h to afford a clear solution.
To a separate reaction vessel containing magnesium chloride (10.6 g, 111 mmol) and potassium 3-ethoxy-3-oxopropanoate (15.1 g, 89.0 mmol), was added NEt3 (19.8 mL, 142 mmol) and EtOAc (33 mL). The mixture was stirred vigorously at 60° C. for 1 h. Then the aforementioned clear solution was slowly added to the mixture. The resulting suspension was stirred at 80° C. for 3 h.
Upon completion, the reaction mixture was cooled to r.t. and acidified with 1 N HCl to pH<3 with stirring. The mixture was diluted with EtOAc and water. The organic phase was separated and the aq. phase was extracted two times with EtOAc. The combined organic layers were washed with sat. NaHCO3, water and brine, dried over Na2SO4, filtered and concentrated to afford title compound as an oil in near quantitative yield. The crude product was used in next step without further purification. LCMS calc. for C14H19O4(M+H)+: m/z=251.1; found 251.1.
To a solution of ethyl (R)-4-(benzyloxy)-3-oxopentanoate (43.4 g, 173 mmol) in DMSO (220 mL) were added Intermediate 17 (45.0 g, 111 mmol), sodium acetate (16.4 g, 200 mmol) and sodium chloride (11.7 g, 200 mmol). The mixture was heated at 65° C. for 18 h. Once completed, the reaction mixture was cooled to 0° C. with ice bath, diluted with water (300 mL) and acidified to pH 3-4 using 1 N HCl, resulting in precipitation. After stirring for 1 h at 0° C., the solids were collected on a fritted filter, washed with 1:1 mixture of MeCN and water and dried under reduced pressure to afford title compound (54.0 g, 82% yield). The crude product was used in next step without further purification. LCMS calc. for C27H22BrCl2FNO4 (M+H)+: m/z=592.0, 594.0; found 592.0, 594.0.
To a solution of ethyl 2-((R)-1-(benzyloxy)ethyl)-6-bromo-7-(2,3-dichlorophenyl)-8-fluoro-4-hydroxyquinoline-3-carboxylate (47.7 g, 80.0 mmol), bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (2.28 g, 3.21 mmol) in DMF (191 mL), acrylonitrile (13.3 mL, 201 mmol) and NEt3 (33.6 mL, 241 mmol) were added. The mixture was sparged with N2 and heated at 85° C. for 1 h. Once completed, the reaction mixture was diluted with brine and EtOAc. The organic phase was separated and aq. phase was extracted two more times with EtOAc. The combined organic layers was washed with brine five times and once with 1 N HCl, filtered and dried over Na2SO4. The volatiles were removed under reduced pressure to afford the title compound in near quantitative yield. The crude product was used in next step without further purification. LCMS calc. for C30H24Cl2FN2O4(M+H)+: m/z=565.1; found 565.1.
To a slurry of ethyl 2-((R)-1-(benzyloxy)ethyl)-6-((E)-2-cyanovinyl)-7-(2,3-dichlorophenyl)-8-fluoro-4-hydroxyquinoline-3-carboxylate (48.0 g, 85 mmol) and benzyltriethylammonium chloride (19.3 g, 85 mmol) in MeCN (210 mL) at 0° C., was added DIPEA (29.6 mL, 170 mmol). The reaction mixture was cooled to 0° C. and phosphoryl chloride (31.6 mL, 340 mmol) was added dropwise into the mixture. Then the mixture was heated at 60° C. for 2 h. Upon completion, the reaction mixture was cooled to r.t. and slowly poured into ice water (1000 mL). The mixture was extracted with EtOAc three times, dried over Na2SO4, filtered and concentrated to afford the title compound as a light brown solid (46.9 g, 95% yield). The crude product was used in next step without further purification. LCMS calc. for C30H23Cl3FN2O3(M+H)+: m/z=583.1; found 583.0.
A mixture of copper(II) acetate monohydrate (1.60 g, 8.03 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (xantphos) (5.58 g, 9.64 mmol) was stirred in toluene (50 mL) and tert-butanol (62 mL) at 60° C. for 0.5 h to afford a homogeneous solution. In a separate vessel, to a mixture of ethyl 2-((R)-1-(benzyloxy)ethyl)-4-chloro-6-((E)-2-cyanovinyl)-7-(2,3-dichlorophenyl)-8-fluoroquinoline-3-carboxylate (46.9 g, 80.0 mmol) and polymethylhydrosiloxane (102 g, 402 mmol) in toluene (150 mL) at 50° C., was added the previous copper-containing solution. The mixture was stirred at 50° C. for 1 h. Upon completion, the reaction mixture was filtered through diatomaceous earth and concentrated. The crude was purified using FCC (0-40% acetone/n-heptane) to afford the title compound (23.2 g, 49% yield). LCMS calc. for C30H25Cl3FN2O3(M+H)+: m/z=585.1; found 585.1.
To a solution of ethyl 2-((R)-1-(benzyloxy)ethyl)-4-chloro-6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoroquinoline-3-carboxylate (34.3 g, 58.6 mmol) in DMSO (110 mL), was added tert-butyl (1R,4R,5S)-5-amino-2-azabicyclo[2.1.1]hexane-2-carboxylate (20.9 g, 105 mmol), LiCl (4.97 g, 117 mmol) and DIPEA (30.6 mL, 176 mmol). The resulting mixture was heated at 100° C. for 18 h. Once completed, the reaction mixture was diluted with water and EtOAc. The organic phase was separated and aq. phase extracted with EtOAc two more times. The combined organic layers were washed with 1 N HCl and brine, dried over Na2SO4, filtered and concentrated. The crude was purified using FCC (0-40% acetone/n-heptane) to afford the title compound (33.6 g, 77% yield). LCMS calc. for C40H42Cl2FN4O5 (M+H)+: m/z=747.2; found 747.2.
To a solution of tert-butyl (1R,4R,5S)-5-((2-((R)-1-(benzyloxy)ethyl)-6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-3-(ethoxycarbonyl)-8-fluoroquinolin-4-yl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (33.6 g, 45 mmol) in THF (100 mL) and water (100 mL), was added NaOH (10.0 g, 250 mmol). The mixture was heated at 60° C. for 3 h. Once completed, the reaction mixture was cooled down to r.t. and acidified to pH 5 using 1 N HCl. The organic volatiles were removed under reduced pressure. The residue aq. phase was extracted with EtOAc three times. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a light yellow solid in near quantitative yield. The crude material was used in the next step without further purification. LCMS calc. for C38H38Cl2FN4O5(M+H)+: m/z=719.2; found 719.2.
To a solution of 2-((R)-1-(benzyloxy)ethyl)-4-(((1R,4R,5S)-2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexan-5-yl)amino)-6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoroquinoline-3-carboxylic acid (34.8 g, 48.3 mmol) in MeCN (242 mL), was added potassium phosphate (20.5 g, 97.0 mmol) and N-iodosuccinimide (19.6 g, 87.0 mmol). The mixture was stirred at r.t. for 1 h. Once completed, the reaction mixture was poured into sat. aq. Na2S2O3. After stirring for 10 min., the mixture was extracted with EtOAc three times. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was further purified with FCC (0-40% acetone/n-heptane) to afford the title compound as a light yellow solid (35.0 g, 90% yield). LCMS calc. for C37H37Cl2FIN4O3(M+H)+: m/z=801.1; found 801.1.
To a solution of Intermediate 18 (1.30 g, 1.62 mmol) in DCM (8 mL) cooled to −78° C. was slowly added BCl3 (1 M in DCM, 8.1 mL, 8.11 mmol). The mixture was warmed to r.t. and stirred for 2 h. The mixture was cooled back down to −78° C. and quenched with MeOH (10 mL). The mixture was then warmed back up to r.t. and concentrated under reduced pressure. An additional portion of MeOH (10 mL) was added and the mixture was concentrated once again to get rid of trace B(OMe)3. The crude material was then dried under high vacuum for an additional 2 h.
To the crude material was added DCM (10 mL) followed by Et3N (3.39 mL, 24.3 mmol) and DMAP (9.77 mg, 0.08 mmol). The mixture was stirred at r.t. for 5 min., then Boc2O (707 mg, 3.24 mmol) was added. The reaction mixture was stirred at the same temperature overnight and then concentrated under reduced pressure. The crude residue was partitioned between aq. 1 M HCl (15 mL) and EtOAc (15 mL). Organic layer was then filtered through a pad of silica gel with EtOAc as eluent and then concentrated under reduced pressure. The crude material was purified with FCC (5-40% EtOAc/Hexanes) to afford the title compound (802 mg, 1.13 mmol, 70% yield). LCMS calc. for C30H31Cl2FIN4O3 (M+H)+: m/z=711.1; found 711.1.
To a solution of 2-((R)-1-(benzyloxy)ethyl)-4-(((1R,4R,5S)-2-(tert-butoxycarbonyl)-2-azabicyclo[2.1.1]hexan-5-yl)amino)-6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoroquinoline-3-carboxylic acid (802 mg, 1.13 mmol) in DCM (5 mL) was added 3,4-dihydropyran (1.48 mL, 16.2 mmol) and p-TsOH (31 mg, 0.162 mmol) at r.t. The mixture was stirred at r.t. for 2 h and then concentrated under reduced pressure. The crude material was purified with FCC (5-40% EtOAc/DCM) to afford the title compound (719 mg, 0.904 mmol, 80% yield). LCMS calc. for C35H39Cl2FIN4O4(M+H)+: m/z=795.2; found 795.2.
This compound was prepared according to the procedure described in Step 1, Intermediate 4, using Intermediate 19 instead of Intermediate 2. LCMS calc. for C51H65Cl2FN5O6Si+ (M+H)+: m/z=960.4; found 960.4.
This compound was prepared according to the procedure described in Step 4, Intermediate 4, using tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C45H51Cl2FN5O6+ (M+H)+: m/z=846.3; found 846.3.
To 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-oxopyrrolidine-1,2-dicarboxylate (Step 1, Intermediate 9) (1.2 g, 3.21 mmol) in toluene (5 mL) was added titanocene dichloride (120 mg, 0.482 mmol) at r.t. To the mixture was then added dimethyltitanocene (5% in THF/Toluene, 45.6 mL, 9.64 mmol). The mixture was sparged with N2 for 10 min. and then heated to 77° C. for 5 h. The mixture was concentrated under reduced pressure and then hexane (20 mL) was added. The mixture was then filtered through a pad of diatomaceous earth, washed with hexane and concentrated under reduced pressure. The crude residue was purified by FCC (5-40% EtOAc/Hexanes) to afford the sub-title compound as a light-yellow oil (900 mg, 3.21 mmol, 75%). LCMS calc. for C14H26NO5Si+ (M−t-Bu+H)+: m/z=316.2; found 316.2.
To diethylzinc (1 M in hexane, 2.0 mL, 1.95 mmol) in DCM (3 mL) cooled to 0° C. was added TFA (0.136 mL, 1.78 mmol) in DCM (1.5 mL). The mixture was stirred at the same temperature for 30 min., upon which diiodomethane (0.172 mL, 2.13 mmol) in DCM (1.5 mL) was slowly added. The mixture was kept stirring at 0° C. for 20 min. To the reaction mixture was then slowly added 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-methylenepyrrolidine-1,2-dicarboxylate (220 mg, 0.592 mmol) in DCM (2 mL). The reaction mixture was stirred at 0° C. for 10 min. and then r.t. for 3h. To the mixture was then added sat. aq. NaHCO3 (10 mL). The aq. phase was extracted with DCM (10 mL×3). Combined organic phase was washed with brine, dried over MgSO4, filtered, and concentrated. The crude material was carried onto next step with no further purification. LCMS calc. for C14H28NO3Si+ (M+H)+: m/z=286.2; found 286.2.
This compound was prepared according to the procedure described in Step 6, Intermediate 5, using methyl (5R,7S)-7-((tert-butyldimethylsilyl)oxy)-4-azaspiro[2.4]heptane-5-carboxylate instead of (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine. LCMS calc. for C18H31NO4Si+ (M+H)+: m/z=353.2; found 353.2.
This compound was prepared according to the procedure described in Step 2, Intermediate 5, using methyl (5R,7S)-7-((tert-butyldimethylsilyl)oxy)-4-(cyclopropanecarbonyl)-4-azaspiro[2.4]heptane-5-carboxylate instead of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)pyrrolidine-1,2-dicarboxylate. LCMS calc. for C17H31NO3Si+ (M+H)+: m/z=325.2; found 325.2.
This compound was prepared according to the procedure described in Step 3, Intermediate 5, using ((5R,7S)-7-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-4-azaspiro[2.4]heptan-4-yl)(cyclopropyl)methanone instead of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-(hydroxymethyl)pyrrolidine-1-carboxylate. LCMS calc. for C17H29NO3Si+ (M+H)+: m/z=323.2; found 323.2.
This compound was prepared according to the procedure described in Step 4, Intermediate 5, using (5R,7S)-7-((tert-butyldimethylsilyl)oxy)-4-(cyclopropanecarbonyl)-4-azaspiro[2.4]heptane-5-carbaldehyde instead of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-formylpyrrolidine-1-carboxylate. LCMS calc. for C17H29NO3Si+(M+H)+: m/z=323.2; found 323.2.
This compound was prepared according to the procedure described in Step 5, Intermediate 10, using Intermediate 21 instead of Intermediate 9. LCMS calc. for C47H57Cl2FN5O4Si+ (M+H)+: m/z=871.4; found 871.4.
This compound was prepared according to the procedure described in Step 2, Intermediate 11, using tert-butyl (1R,4R,5S)-5-(2-((5R,7S)-7-((tert-butyldimethylsilyl)oxy)-4-(cyclopropanecarbonyl)-4-azaspiro[2.4]heptan-5-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C41H43Cl2FN5O4+ (M+H)+: m/z=758.3; found 758.3.
A solution of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate (13.0 g, 28.9 mmol) from Step 4, Intermediate 5 in THF (145 mL) was cooled to 0° C. TBAF (1 M in THF, 31.8 mL, 31.8 mmol) was added dropwise at the same temperature. The reaction mixture was then warmed to r.t. and stirred overnight. The reaction mixture was quenched with sat. aq. NH4Cl (20 mL) and extracted with DCM (20 mL×3). The reaction mixture was concentrated and directly purified by FCC (EtOAc/hexanes 0-100%) to afford the title compound (5.75 g, 28.9 mmol, 94%). LCMS calc. for C7H10NO3+ (M−tBu+H)+: m/z=156.1; found 156.1.
To a stirred solution of tert-butyl (2R,4S)-2-ethynyl-4-hydroxypyrrolidine-1-carboxylate (4 g, 18.9 mmol), 4-nitrobenzoic acid (3.22 g, 19.9 mmol), and PPh3 (7.45 g, 28.4 mmol) in THF (8 mL) cooled at 0° C. was added DIAD (5.52 ml, 28.4 mmol) dropwise. The reaction mixture was warmed to r.t. and stirred for 3 h. The reaction mixture was quenched with sat. aq. NaHCO3 (8 mL) and extracted with EtOAc (10 mL×3). Combined organic phase was concentrated and purified by FCC (EtOAc/hexanes 10-50%) to afford the title compound (5.0 g, 13.9 mmol, 73%). 1H NMR (500 MHz, CDCl3) δ 8.31 (s, 4H), 5.63 (m, 1H), 5.15-4.94 (m, 1H), 4.87-4.63 (m, 1H), 3.78 (m, 2H), 2.55-2.25 (m, 3H), 1.52 (s, 9H).
To a stirred solution of tert-butyl (2R,4R)-2-ethynyl-4-((4-nitrobenzoyl)oxy)pyrrolidine-1-carboxylate (5.0 g, 13.9 mmol) in THF (100 mL) and MeOH (50 mL) at r.t. was added NaOH (1M in H2O, 37.9 mL, 37.9 mmol). The reaction mixture was allowed to stir for 2 h at the same temperature, upon which point it was concentrated. The residue was dissolved in EtOAc (100 mL) and washed with sat. aq. NaHCO3 (50 mL). The organic phase was filtered over a short plug of silica and then concentrated. The residue was purified by FCC (EtOAc/hexanes 0-100%) to afford the title compound (2.50 g, 11.8 mmol, 85%). LCMS calc. for C7H10NO3+ (M−tBu+H)+: m/z=156.1; found 156.1.
This compound was prepared according to the procedure described for Intermediate 5, Step 5-Step 6, starting with tert-butyl (2R,4R)-2-ethynyl-4-hydroxypyrrolidine-1-carboxylate instead of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate. LCMS calc. for C10H14NO2+ (M+H)+: m/z=180.1; found 180.1.
To a mixture of Intermediate 19 (1.0 g, 1.26 mmol), Intermediate 23 (451 mg, 2.51 mmol), Pd(PPh3)4 (291 mg, 0.251 mmol), and copper(I) iodide (144 mg, 0.754 mmol) was added DMF (4 mL) at r.t. To the mixture was then added NEt3 (1.27 mL, 2.81 mmol). The reaction mixture was sparged with N2 for 5 min., and then stirred at 75° C. for 3 h. After being cooled to r.t., Cs2CO3 (2.05 g, 6.29 mmol) was added and the reaction mixture was heated to 90° C. for 3 h. The reaction mixture was quenched with sat. aq. NH4Cl (2 mL), extracted with EtOAc (3 mL×3), and concentrated. The residue was purified by FCC (MeOH/DCM 1-15%) to afford the title compound (500 mg, 0.590 mmol, 47%). LCMS calc. for C45H51Cl2FN5O6(M+H)+: m/z=846.3; found 846.3.
To a solution of methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-methylpyrrolidine-2-carboxylate (10.3 g, 37.5 mmol) from Intermediate 9, Step 3 in MeCN (80 mL) was added Et3N (20.5 mL, 150 mmol) followed by N (12.2 g, 46.9 mmol) at r.t. The reaction mixture was allowed to stir at r.t. for 3 h. The reaction mixture was quenched with H2O (80 mL), extracted with EtOAc (100 mL×3), and concentrated. The crude residue was purified by FCC (0-50% EtOAc/hexanes) to afford the sub-title compound as a mixture of diastereomers (7.83 g, 18.8 mmol, 50%). LCMS calc. for C17H36NO5Si2+ (M−(CH2)2+H)+: m/z=390.2; found 390.2.
This compound was prepared according to the procedure described for Intermediate 5, Step 2-Step 4, starting with 2-methyl 1-(2-(trimethylsilyl)ethyl) (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-methylpyrrolidine-1,2-dicarboxylate instead of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)pyrrolidine-1,2-dicarboxylate. LCMS calc. for C17H34NO3Si2+ (M−(CH2)2+H)+: m/z=356.2; found 356.2.
A solution of 2-(trimethylsilyl)ethyl (3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-ethynyl-2-methylpyrrolidine-1-carboxylate (2.10 g, 5.47 mmol) in THF (20 mL) was cooled to 0° C. TBAF (1 M in THF, 6.02 mL, 1.1 eq.) was added dropwise at the same temperature. The reaction mixture was then warmed to r.t. and stirred overnight, upon which point brine (10 mL) was added. The aq. phase was extracted with EtOAc (15 mL×3) and the combined organic phase was concentrated and directly purified by FCC (EtOAc/hexanes 0-100%) to afford the title compound as a single diastereomer that elutes as the second peak (590 mg, 28.9 mmol, 40%). LCMS calc. for C11H20NO3Si+ (M−(CH2)2+H)+: m/z=242.1; found 242.1. 1H NMR (500 MHz, DMSO-d6, single rotamer) δ 4.94 (d, J=4.0 Hz, 1H), 4.48-4.43 (m, 1H), 4.19-4.04 (m, 2H), 3.97-3.85 (m, 1H), 3.65 (qd, J=6.6, 2.3 Hz, 1H), 3.05 (d, J=2.1 Hz, 1H), 2.15-2.10 (m, 2H), 1.16 (d, J=6.7 Hz, 3H), 0.99 (t, J=8.0 Hz, 2H), 0.06 (s, 9H).
This compound was prepared according to the procedure described in Intermediate 24, using Intermediate 25 instead of Intermediate 23. LCMS calc. for C48H61Cl2FN5O7Si+(M+H)+: m/z=936.4; found 936.4.
To a mixture of Intermediate 19 (1.17 g, 1.47 mmol), Intermediate 9 (298 mg, 1.54 mmol), Pd(PPh3)4 (340 mg, 0.294 mmol), and copper(I) iodide (112 mg, 0.588 mmol) was added DMF (9 mL) at r.t. To the mixture was then added NEt3 (1.61 mL, 11.8 mmol). The reaction mixture was sparged with N2 for 5 min., and then stirred at 75° C. for 3 h. After being cooled to r.t., CS2CO3 (2.40 g, 7.35 mmol) was added and the reaction mixture was heated to 90° C. for 3 h. The reaction mixture was added brine (15 mL) followed by aq. 30% NH4OH (15 mL), and extracted with EtOAc (30 mL×3). The combined organic phase was washed with brine (20 mL), and concentrated. The residue was purified by FCC (acetone/hexanes 0-100%) to afford the title compound (600 mg, 1.47 mmol, 47%). LCMS calc. for C46H53Cl2FN5O6+ (M+H)+: m/z=860.3; found 860.3.
To a solution of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-5-oxopyrrolidine-1,2-dicarboxylate (7.77 g, 20.80 mmol) from Intermediate 9, Step 1 in THF (41.6 mL) at −78° C. was added lithium triethylborohydride (1 M in THF, 21.84 mL, 21.84 mmol) over 5 min. The reaction mixture was stirred for 1 h at −78° C., after which it was quenched with sat. aq. NaHCO3 (15 mL) followed by addition of 30% aq. H2O2 (0.1 mL). The resulting mixture was allowed to warm to r.t. All the volatiles were removed in vacuo. The residue was diluted with DCM (30 mL), the resulting organic phase was washed with brine (15 mL), dried over MgSO4, then concentrated in vacuo. After separation, the organic layer was dried over MgSO4 and concentrated in vacuo. The residue was dissolved in DCM (50 mL) and NEt3 (4.35 mL, 31.2 mmol), Ac2O (2.94 mL, 31.2 mmol), and DMAP (37.9 mg, 0.31 mmol) were sequentially added. The mixture was stirred at r.t. overnight. To the mixture was added sat. aq NaHCO3 (10 mL). The layers were separated and the aqueous layer was extracted with DCM (3×20 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was filtered through a pad of silica with 10% EtOAc in hexanes (200 mL) as eluent and concentrated in vacuo to afford the sub-title compound (6.9 g, 16.6 mmol, 80%). The crude product was used in next step without further purification. LCMS calc. for C12H24NO3Si (M−CO2tBu−OAc+H)+: m/z=258.2; found 258.2.
To a solution of 1-(tert-butyl) 2-methyl (2R,4S)-5-acetoxy-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1,2-dicarboxylate (9.2 g, 22.0 mmol) in dry Et2O (100 mL) at −78 C was added BF3·OEt2 (3.30 mL, 26.0 mmol) and allyltrimethylsilane (14.88 mL, 94 mmol). The reaction mixture was stirred for 15 min at −78° C. and then allowed to warm to r. t. over 1 h, after which the solution was stirred for an additional 2 h at r.t. The reaction mixture was quenched by the addition of sat. aq. NaHCO3 (20 mL). The layers were separated and the aqueous layer was extracted with Et2O (2×50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was filtered through a pad of silica with 10% EtOAc in hexanes (200 mL) as eluent and concentrated in vacuo. The residue was diluted with DCM (100 mL) and cooled to 0° C. To it was slowly added TFA (16.03 mL, 208 mmol). The reaction mixture was stirred at 6° C. overnight. Sat. aq NaHCO3 was carefully and slowly added at 0° C. The layers were separated and the aqueous phase was extracted with DCM (2×50 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuo to afford the sub-title compound (6.0 g, 20.03 mmol, 91%). The crude product was used in next step without further purification. LCMS calc. for C15H30NO3Si+ (M+H)+: m/z=300.2; found 300.2.
The sub-title compound was prepared according to the procedure described in Step 6, Intermediate 5, using methyl (2R,4S)-5-allyl-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylate instead of (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine. LCMS calc. for C19H34NO4Si+ (M+H)+: m/z=368.2; found 368.2.
A vial was charged with methyl (2R,4S)-5-allyl-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)pyrrolidine-2-carboxylate (4 g, 10.88 mmol), a stir bar, THF (43.5 mL) and water (10.9 mL). To this suspension was added osmium tetroxide (4 wt. % in water, 3.46 mL, 0.544 mmol). The mixture was stirred for 5 min and then NaIO4 (11.64 g, 54.4 mmol) was added. After stirring at r.t. for 1 h, the reaction mixture was diluted with water (50 mL) and extracted with DCM (2×60 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude material was purified by FCC (0-50% EtOAc/hexanes) to afford the sub-title compound (1.15 g, 3.11 mmol, 29%) as the 2nd peak that elutes after the undesired diastereomer. LCMS calc. for C18H32NO5Si+ (M+H)+: m/z=370.2; found 370.2.
A vial was charged with methyl (2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-5-(2-oxoethyl)pyrrolidine-2-carboxylate (1.15 g, 3.11 mmol) followed by the addition of methanol (16 mL). After that, the solution was cooled to 0° C. and was then added NaBH4 (0.235 g, 6.22 mmol). The reaction mixture was stirred for 1 h, after which point it was quenched with sat. aq. NaHCO3 (10 mL). The aqueous layer was extracted with DCM (3×30 mL). The combined organic extracts were filtered through a plug of MgSO4 and concentrated in vacuo. The crude material was purified by FCC (0-50% EtOAc/hexanes) to afford the sub-title compound (0.954 g, 2.57 mmol, 83%). LCMS calc. for C18H34NO5Si+(M+H)+: m/z=372.2; found 372.2.
To a round bottom flask was added methyl (2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-5-(2-hydroxyethyl)pyrrolidine-2-carboxylate (1.32 g, 3.55 mmol) followed by THF (35 mL). After that, 1-nitro-2-selenocyanatobenzene (1.2 g, 5.32 mmol) was added. The headspace of the round bottom flask was briefly purged with nitrogen, and then tributylphosphine (1.3 mL, 5.3 mmol) was added. The reaction mixture was allowed to stir at r.t. for 15 min. The reaction mixture was quenched with 1 M aq. NaOH (7.1 mL, 7.1 mmol) and then allowed to stir for 30 min. The mixture was diluted with water (10 mL) and the aqueous phase was extracted with DCM (3×20 mL). The combined organic extracts were filtered through a plug of MgSO4 and concentrated in vacuo. The residue was dissolved in THF (35 mL) treated with 30% aq. H2O2 (3.6 mL, 35.5 mmol). The mixture was stirred at r.t. for 2.5 h. The reaction mixture was quenched with sat. aq. Na2SO3 (15 mL) and extracted with DCM (3×20 mL). The combined organic extracts were washed with sat. aq. Na2SO3 (10 mL), water (10 mL), and brine (10 mL), then dried over MgSO4 and concentrated in vacuo. The crude residue was purified by FCC (0-50% EtOAc/hexanes) to afford the sub-title compound (0.982 g, 2.78 mmol, 78%). LCMS calc. for C18H32NO4Si+ (M+H)+: m/z=354.2; found 354.1.
The title compound was prepared according to the procedure described for Intermediate 9, Step 5-Step 8, starting with methyl (2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-5-vinylpyrrolidine-2-carboxylate instead of methyl (2R,4S)-4-((tert-butyldimethylsilyl)oxy)-1-(cyclopropanecarbonyl)-5-methylpyrrolidine-2-carboxylate. LCMS calc. for C12H16NO2+ (M+H)+: m/z=206.1; found 206.1.
The title compound was prepared according to the general procedure described for Intermediate 18, starting with (R)-2-((4-methoxybenzyl)oxy)propanoic acid instead of (R)-2-(benzyloxy)propanoic acid. LCMS calc. for C38H39Cl2FIN4O4+ (M+H)+: m/z=831.3; found 831.3.
The following examples can be isolated as separate atropisomers listed in Table 2 using techniques well known in the art of organic synthesis such as chiral column chromatography.
To a 40 mL scintillation vial was added Intermediate 6 (500 mg, 0.654 mmol), Cs2CO3 (1598 mg, 4.90 mmol), copper(I) iodide (93 mg, 0.490 mmol), 3-iodo-2-methoxypyridine (768 mg, 3.27 mmol), and 3,4,7,8-tetramethyl-1,10-phenanthroline (232 mg, 0.981 mmol). To the mixture was then added toluene (13.076 mL) under N2. The mixture was sparged with N2 for 2 min., after which the vial headspace was purged with N2. The mixture was stirred rapidly (1000 RPM) via magnetic stirring and heated overnight at 110° C. The mixture was filtered over diatomaceous earth (washed with 50 mL EtOAc) and then diluted with NH4OH (20 mL), brine (20 mL), and water (10 mL). Organic extracts were set aside and the aq. phase was further extracted with EtOAc (3×40 mL). Organic extracts were combined, washed with 1:1 NH4OH/brine (100 mL), dried over MgSO4 and solvent was removed in vacuo. The crude residue was purified by FCC (heptane/acetone 0-100% over 20 CVs, 80 G HP-Sil column) and fractions containing the desired compound were concentrated in vacuo to yield the title compound (499 mg, 0.572 mmol, 88% yield) as a light-orange solid. LCMS calc. for C45H46Cl2FN6O5S (M+H)+: m/z=871.3; found 871.3.
To a 40 mL scintillation vial was added tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-3-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (499 mg, 0.572 mmol), methylboronic acid (343 mg, 5.72 mmol), copper (I) 3-methylsalicylate (430 mg, 2.00 mmol), and Pd(PPh3)4 (198 mg, 0.172 mmol) followed by 1,4-dioxane (3.8 mL) under N2. The mixture was sparged with N2 for 2 min. and then the headspace was purged with N2. The mixture was stirred at 100° C. for 4 h, subsequently cooled to r.t., diluted with 1:1 NH4OH/brine (60 mL), and extracted with EtOAc (3×50 mL). Organic extracts were combined, washed with 1:1 NH4OH/brine (25 mL), dried over MgSO4, and solvent was removed in vacuo. The crude residue was purified by FCC (0-100% heptane/acetone over 20 CVs, 40 G HP-Sil) and fractions containing the desired compound were combined and dried in vacuo to yield the sub-title compound (421 mg, 0.501 mmol, 88% yield) as a light-brown solid. LCMS calc. for C45H46Cl2FN6O5 (M+H)+: m/z=839.3; found 839.2.
In a 40 mL vial tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-3-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (421 mg, 0.501 mmol) was dissolved in TFA (2.507 mL) and DCM (2.507 mL) and stirred at r.t. for 30 min. Volatiles were removed in vacuo and the residue was dissolved in MeCN (4 mL) and purified by preparative LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as peak 1 and peak 2 (atropisomers). Fractions containing peaks 1 and 2 were frozen on dry ice independently and lyophilized for two days to yield the title compound(s) as TFA salts (white amorphous powder).
Diastereomer 1. Peak 1. LCMS calc. for C40H38Cl2FN6O3+ (M+H)+: m/z=739.2; found 739.2. This is the desired (potent) diastereoisomer. 1H NMR (500 MHz, DMSO-d6) δ 9.33 (s, 1H), 8.15 (s, 1H), 8.12 (s, 1H), 7.86 (dd, J=8.1, 1.5 Hz, 1H), 7.78 (dd, J=4.9, 1.5 Hz, 1H), 7.59 (t, J=7.9 Hz, 1H), 7.46 (dd, J=7.7, 1.5 Hz, 1H), 7.35 (dd, J=7.9, 1.6 Hz, 1H), 6.92 (dd, J=7.8, 5.0 Hz, 1H), 6.82 (s, 1H), 5.57 (d, J=2.9 Hz, 1H), 5.40 (dd, J=8.7, 4.0 Hz, 1H), 4.98-4.93 (m, 1H), 4.86 (d, J=6.0 Hz, 1H), 4.41 (dd, J=11.5, 5.8 Hz, 1H), 4.07 (dd, J=11.5, 3.9 Hz, 1H), 3.96-3.90 (m, 1H), 3.07-2.96 (m, 1H), 2.93-2.81 (m, 5H), 2.74-2.62 (m, 2H), 2.34-2.24 (m, 2H), 2.09-2.02 (m, 1H), 1.57 (d, J=9.1 Hz, 1H), 0.96-0.70 (m, 4H).
Diastereomer 2. Peak 2. LCMS calc. for C40H38Cl2FN6O3+ (M+H)+: m/z=739.2; found 739.3.
In a 50 mL round-bottomed flask tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-hydroxypyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (Intermediate 6, 811.6 mg, 1.061 mmol), 4-iodo-2-methoxypyridine (1247 mg, 5.31 mmol), copper(I) iodide (152 mg, 0.796 mmol), 3,4,7,8-tetramethyl-1,10-phenanthroline (376 mg, 1.592 mmol), and Cs2CO3 (2593 mg, 7.96 mmol) were suspended in toluene (21.23 mL, 0.05M) under N2. The mixture was stirred rapidly and heated to 115° C. for 4 h. After this time, the mixture was cooled, diluted with MeCN (100 mL), sonicated, and then filtered through a pad of diatomaceous earth. The filtrate was concentrated in vacuo, dissolved in EtOAc (30 mL), washed with a 1:1 mixture of NH4OH/brine (30 mL Vtot), dried over MgSO4, and dried in vacuo. The residue was dissolved in DCM (6 mL) and purified by FCC (40-gram HP-Sil, 0-100% acetone/heptane over 20 column volumes). Fractions containing the desired compound were pooled and dried in vacuo to provide the sub-title compound (750 mg, 0.860 mmol, 81% yield) as an orange solid. LCMS calc. for C45H46Cl2FN6O5S (M+H)+: m/z=871.3, 873.3; found 871.2, 873.2.
To a 40 mL scintillation vial was added tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-3-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(methylthio)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (750 mg, 0.860 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (723 mg, 4.30 mmol), copper (I) 3-methylsalicylate (554 mg, 2.58 mmol), and Pd(PPh3)4 (199 mg, 0.172 mmol). After that, the mixture was dissolved in 1,4-dioxane (4.3 mL) under N2. The mixture was sparged with N2 for 2 min. and then the headspace was purged with N2. The mixture was stirred at 100° C. for 3 h, subsequently cooled to r.t., diluted with 1:1 NH4OH/brine (60 mL), and extracted with EtOAc (3×50 mL). Organic extracts were combined, washed with 1:1 NH4OH/brine (25 mL), dried over MgSO4, and solvent was removed in vacuo. The crude residue was purified by FCC (0-100% heptane/acetone over 20 CVs, 40 G HP-Sil) and fractions containing the desired compound were combined and dried in vacuo to yield the sub-title compound (400 mg, 0.462 mmol, 54% yield) as a light-brown solid. LCMS calc. for C47H48Cl2FN6O5(M+H)+: m/z=865.3; found 865.2.
In a 40 mL vial tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-4-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(prop-1-en-2-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (400.9 mg, 0.463 mmol), osmium tetroxide (294 μL, 0.046 mmol, 4 wt. % in water), and NaIO4 (495 mg, 2.315 mmol) were dissolved in a THF (3704 μL) and water (926 μL). The vial was capped under ambient air and the mixture was stirred rapidly for 2 h. The reaction mixture was quenched by the addition of sat. aq. Na2S2O3 (2 mL), diluted with water (2 mL), and extracted with DCM (3×10 mL). The organic layers were combined, washed with brine, dried over MgSO4, and dried in vacuo. The crude residue was purified by FCC (0-100% heptane/acetone over 15 CVs, 40 G HP-Sil column). Fractions containing the desired compound were combined and dried in vacuo to yield the sub-title compound (268 mg, 0.309 mmol, 66.7% yield) as a light-brown solid. LCMS calc. for C46H46Cl2FN6O6+ (M+H)+: m/z=867.3; found 867.2.
In a 40-mL vial containing tert-butyl (1R,4R,5S)-5-(4-acetyl-8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-4-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (267.8 mg, 0.309 mmol) was added DCM (1286 μL), followed in order by RuCl(p-cymene)[(R,R)-Ts-DPEN] (39.3 mg, 0.062 mmol) and 5:2 formic acid/NEt3 (257 μL) under N2 at 40° C. After ca. 2-3 min. of stirring at r.t., bubbles were apparent, emanating from the stirring vortex of the red-black solution. After 5 h of stirring at 40° C., the starting material had been consumed. Sat. aq. NaHCO3 (5 mL) was added and the mixture was extracted with DCM (3×10 mL). The organic layer was washed with brine (5 mL), dried over MgSO4, and dried in vacuo. The crude residue was dissolved in DCM (2 mL) and TFA was added (2 mL) while stirring at r.t. After 30 min., volatiles were removed in vacuo and the crude residue was dissolved in MeCN and purified by preparative LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound(s) as peak 1 and peak 2 (atropisomers). Fractions containing peaks 1 and 2 were frozen on dry ice independently and lyophilized for two days to yield the title compound(s) as TFA salts (white amorphous powder).
Diastereomer 1. Peak 1. LCMS calc. for C41H40Cl2FN6O4+ (M+H)+: m/z=769.2; found 769.2. This is the desired (potent) diastereoisomer. 1H NMR (500 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.20-8.09 (m, 2H), 8.03 (d, J=5.9 Hz, 1H), 7.84 (dd, J=8.0, 1.5 Hz, 1H), 7.58 (t, J=8.0 Hz, 1H), 7.50-7.45 (m, 1H), 6.92 (s, 1H), 6.67 (dd, J=6.0, 2.2 Hz, 1H), 6.42 (d, J=2.2 Hz, 1H), 5.59-5.52 (m, 1H), 5.40 (dd, J=8.8, 4.8 Hz, 1H), 5.26 (q, J=6.6 Hz, 1H), 5.16-5.11 (m, 1H), 4.86 (d, J=5.9 Hz, 1H), 4.29 (dd, J=12.0, 5.2 Hz, 1H), 4.18 (dd, J=12.0, 2.9 Hz, 1H), 3.93-3.87 (m, 1H), 3.85 (s, 3H), 3.81-3.74 (m, 1H), 3.48-3.35 (m, 1H), 3.09-3.00 (m, 1H), 2.90-2.79 (m, 2H), 2.78-2.60 (m, 2H), 2.36-2.25 (m, 2H), 2.10-2.04 (m, 1H), 1.64-1.53 (m, 4H), 0.95-0.78 (m, 4H).
Diastereomer 2. Peak 2. LCMS calc. for C41H40Cl2FN6O4+ (M+H)+: m/z=769.2; found 769.3.
To a solution of Intermediate 10 (10 mg, 0.013 mmol) in DMF (0.3 mL) was added NaH (60% in mineral oil, 1.07 mg, 0.027 mmol) at r.t. The reaction mixture was stirred for 5 min. and then 3-(chloromethyl)-5-methylisothiazole (2.18 mg, 0.015 mmol) was added at once. The mixture was allowed to stir for 1 h, and then water (0.20 mL) was carefully added at r.t. The aq. phase was extracted with EtOAc (3×2 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was dissolved in MeCN (0.5 mL) and TFA (2 mL) was added dropwise. The reaction mixture was stirred at r.t. for 30 min and diluted with MeCN, which was then purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C40H40Cl2FN6O2S (M+H)+: m/z=757.2; found 757.4. This is the desired diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C40H40Cl2FN6O2S (M+H)+: m/z=757.2; found 757.4.
In a 40 mL vial Intermediate 8 (44.9 mg, 0.055 mmol) and NaN3 (36.0 mg, 0.554 mmol) were suspended in DMF (2 mL) under N2. The mixture was stirred at 70° C. for 3 h and then cooled to 21° C. The mixture was diluted with water (5 mL) and extracted with EtOAc (3×10 mL). Organic extracts were combined and washed with 1:1 brine/10% aq. LiCl (3×10 mL), dried over MgSO4, and volatiles removed in vacuo. The residue was dried under high-vacuum overnight to remove traces of DMF, providing the title compound as an off-white residue (41.8 mg, 0.055 mmol, 100% yield). LCMS calc. for C39H40Cl2FN8O3(M+H)+: m/z=757.3; found 757.2.
In a 1 dram vial tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-azido-1-(cyclopropanecarbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (7 mg, 9.24 μmol), 3,3-dimethylbut-1-yne (2.277 mg, 0.028 mmol), tris((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amine (0.980 mg, 1.848 μmol), and Na ascorbate (3.66 mg, 0.018 mmol) were dissolved in 1:1 t-BuOH/water (1 mL Vtot) open to air. Copper(II) sulfate pentahydrate (0.018 mL, 1.848 μmol) was added to the reaction mixture and it was stirred at 21° C. for 5 min. After that, volatiles were removed in vacuo and the residue was treated with TFA (0.4 mL) for 30 min. The mixture was diluted with 1:1 MeOH/MeCN (4 mL) and purified by preparative LCMS (pH 10.0, XBRIDGE® C18 column, eluting with a gradient of MeCN/water containing 0.15% NH4OH, at flow rate of 60 mL/min) to afford the title compound as peak 1 and peak 2 (atropisomers). Fractions containing peaks 1 and 2 were frozen on dry ice independently and lyophilized for two days to yield the title compound(s) as free bases (white amorphous powder).
Diastereomer 1. Peak 1. LCMS calc. for C40H42Cl2FN8O (M+H)+: m/z=739.3; found 739.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C40H42Cl2FN8O+ (M+H)+: m/z=739.3; found 739.2.
To a solution of Intermediate 4 (6 mg, 8.19 μmol) and 2-fluoropyridine (1.59 mg, 0.016 mmol) in THF (0.5 mL) cooled at 0° C. was slowly added NaOtBu (2.36 mg, 0.025 mmol). The reaction mixture was allowed to warm to r.t. and stirred for additional 1 h. After that, sat. aq. NH4Cl (0.5 mL) was slowly added to quench the reaction, followed by the addition of EtOAc (1 mL). The organic phase was separated and the aq. phase was extracted with EtOAc (1.5 mL×3). Combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was diluted with MeCN (1 mL) followed by slow addition of TFA (3 mL). The reaction mixture was stirred at r.t. for 1 h and diluted with MeCN, which was then purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C39H36Cl2FN6O2(M+H)+: m/z=709.2; found 709.2. This is the desired diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C39H36Cl2FN6O2(M+H)+: m/z=709.2; found 709.2.
Examples 6-33 were prepared according to the procedure described for Example 3 with the corresponding electrophile and intermediate listed in Table B.
This compound was prepared according to the general procedure described for Example 3, using Intermediate 15 instead of Intermediate 10, and 2-(bromomethyl)-5-fluorobenzonitrile instead of 3-(chloromethyl)-5-methylisothiazole. LCMS calc. for C50H49Cl2F2N6O4+ (M+H)+: m/z=905.3; found 905.3.
This compound was prepared according to the procedure described for Example 2, Step 2, using tert-butyl (1R,4R,5S)-5-(2-((2R,4S)-4-((2-cyano-4-fluorobenzyl)oxy)-1-(1-methylcyclopropane-1-carbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(prop-1-en-2-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-4-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-(prop-1-en-2-yl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate. LCMS calc. for C49H47Cl2F2N6O5+ (M+H)+: m/z=907.3; found 907.3.
This compound was prepared according to the procedure described for Example 2, Step 3, using tert-butyl (1R,4R,5S)-5-(4-acetyl-2-((2R,4S)-4-((2-cyano-4-fluorobenzyl)oxy)-1-(1-methylcyclopropane-1-carbonyl)pyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate instead of tert-butyl (1R,4R,5S)-5-(4-acetyl-8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-((2-methoxypyridin-4-yl)oxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate.
Diastereomer 1. Peak 1. LCMS calc. for C44H41Cl2F2N6O3+ (M+H)+: m/z=809.3; found 809.3. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C44H41Cl2F2N6O3+ (M+H)+: m/z=809.3; found 809.3.
In a 1 dram vial the appropriate intermediate (25 mg), the appropriate electrophile (5 eq.), copper(I) iodide (0.75 eq.), 3,4,7,8-tetramethyl-1,10-phenanthroline (1.5 eq.), and Cs2CO3 (10 eq.) were suspended in toluene (1 mL) under N2. The mixture was stirred rapidly and heated to 115° C. for 4 h. After this time, the mixture was cooled, diluted with MeCN (2 mL), sonicated, and then filtered through a pad of diatomaceous earth. The filtrate was concentrated in vacuo, dissolved in EtOAc (5 mL), washed with a 1:1 mixture of NH4OH/sat. NaCl (10 mL Vtot), dried over MgSO4, and dried in vacuo. The crude product was dissolved in 1:1 TFA/MeCN (1 mL), then stirred at r.t. for 30 min. The reaction mixture was concentrated, then diluted with MeCN (5 mL), and filtered. The crude solution was purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder. Compounds and data are listed in Table C.
To a 40 mL scintillation vial was added Intermediate 27 (0.390 g, 0.453 mmol), Cs2CO3 (1.107 g, 3.40 mmol), copper(I) iodide (0.065 g, 0.340 mmol), 5-fluoro-4-iodo-2-methoxypyridine (0.860 g, 3.40 mmol), and 3,4,7,8-tetramethyl-1,10-phenanthroline (0.161 g, 0.680 mmol). To the mixture was then added toluene (15 mL) under N2. The mixture was sparged with N2 for 5 min., after which the vial headspace was purged with N2. The mixture was stirred rapidly (1500 RPM) via magnetic stirring and heated overnight at 120° C. The mixture was diluted with 30% aq. NH4OH (10 mL) and water (10 mL). The biphasic mixture was shaken rapidly for 1 min. The layers were separated and the aq. phase was further extracted with DCM (3×20 mL). The organic extracts were combined, filtered over a plug of MgSO4 and the solvent was removed in vacuo. The crude residue was purified by FCC (acetone/hexanes 0-100%) to afford the title compound (245 mg, 0.453 mmol, 55% yield) LCMS calc. for C52H57Cl2F2N6O7(M+H)+: m/z=985.4; found 985.5.
To a 40 mL scintillation vial was added tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S,5R)-1-(cyclopropanecarbonyl)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-methylpyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (245 mg, 0.248 mmol) and DCM (5 mL). The reaction mixture was cooled to 0° C., and a solution of TFA (2.3 mL, 30 mmol, 66 eq.) dissolved in DCM (2.5 mL) was added dropwise slowly. The reaction mixture was stirred at 0° C. for 6 h. The reaction mixture was quenched with sat. aq. NaHCO3 (50 mL) and extracted with 10% MeOH in DCM (50 mL×2). The combined organic extracts were dried over a plug of MgSO4 and concentrated. The crude residue was purified by FCC ((10:1 MeOH: 30% aq. NH4OH)/DCM 0-10%) to afford the title compound (0.095 g, 0.118 mmol, 48% yield). This material was further purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C42H41Cl2F2N6O4+ (M+H)+: m/z=801.3; found 801.3. This is the desired (potent) diastereoisomer. 1H NMR (500 MHz, DMSO-d6) δ 8.10, (s, 1H), 8.09 (d, J=5.8 Hz, 1H), 7.84 (dd, J=7.9, 1.5 Hz, 1H), 7.58 (t, J=7.9 Hz, 1H), 7.47 (dd, J=7.9, 1.5 Hz, 1H), 6.86 (s, 1H), 6.63 (d, J=5.8 Hz, 1H), 5.51 (d, J=2.9 Hz, 1H), 5.23 (t, J=8.3 Hz, 1H), 5.17 (q, J=6.5 Hz, 1H), 5.08 (d, J=4.6 Hz, 1H), 4.84 (d, J=5.9 Hz, 1H), 4.72 (q, J=6.9 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 1H), 3.79-3.72 (m, 1H), 3.44-3.37 (m, 1H), 3.08-2.98 (m, 1H), 2.92-2.78 (m, 3H), 2.71-2.61 (m, 1H), 2.27 (d, J=8.9 Hz, 1H), 2.21 (ddd, J=13.8, 8.3, 4.6 Hz, 1H), 2.11-2.04 (m, 1H), 1.52 (d, J=6.5 Hz, 3H), 1.51 (d, J=8.9 Hz, 1H), 1.42 (d, J=6.9 Hz, 3H), 0.97-0.89 (m, 1H), 0.87-0.80 (m, 1H), 0.80-0.73 (m, 1H).
Diastereomer 2. Peak 2. LCMS calc. for C42H41Cl2F2N6O4+ (M+H)+: m/z=801.3; found 801.3.
This compound was prepared according to the procedure described in General procedure for Examples 36-50, using Intermediate 26 and 5-fluoro-4-iodo-2-methoxypyridine as electrophile. LCMS calc. for C54H65Cl2F2N6O8Si+ (M+H)+: m/z=1061.4; found 1061.4.
To a solution of tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-2-((2R,4S,5R)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-methyl-1-((2-(trimethylsilyl)ethoxy)carbonyl)pyrrolidin-2-yl)-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (58 mg, 0.055 mmol) in THF (1.0 mL) at 0° C. was added TBAF (1 M in THF, 68 μL, 0.068 mmol) and the reaction mixture was stirred at 60° C. for 30 min. To the reaction mixture was added water (1 mL) and the aq. phase was extracted with EtOAc (3×2 mL). The combined organic phase was washed with brine (1.5 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by FCC (0-10% MeOH in DCM) to provide the sub-title compound (35 mg, 0.055 mmol, 70%). LCMS calc. for C48H53Cl2F2N6O6+ (M+H)+: m/z=917.3; found 917.3.
To a solution of tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-2-((2R,4S,5R)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-methylpyrrolidin-2-yl)-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (7 mg, 7.63 μmol), HATU (5.80 mg, 0.015 mmol), and 1-methylcyclopropane-1-carboxylic acid (3.82 mg, 0.038 mmol) dissolved in CH3CN (0.5 mL) was added Et3N (10.6 μL, 0.076 mmol) dropwise at r.t. The reaction mixture was allowed to stir for 1 h. To it was added sat. aq. NaHCO3 (1 mL) and the aq. phase was extracted with EtOAc (3×2 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was dissolved in MeCN (0.5 mL) and kept below −20° C. To it was then added TFA (2 mL) dropwise over 5 min., after which it was allowed to warm to r.t. and stir for additional 15 min. The reaction mixture was diluted with more MeCN, and then directly purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C43H43Cl2F2N6O4+ (M+H)+: m/z=815.3; found 815.3. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C43H43Cl2F2N6O4+ (M+H)+: m/z=815.3; found 815.3.
A solution of 2,3-difluoro-4-iodopyridine (240 mg, 0.996 mmol) in MeOH (5 mL) was added sodium methoxide (25% wt. in MeOH, 0.307 mL, 1.195 mmol). The reaction mixture was stirred at r.t. for 2 h, quenched with sat. aq. NaHCO3 (3 mL), and extracted with EtOAc (3 mL×2). The combined organic phase was concentrated to afford the sub-title compound and carried to next step without further purification. LCMS calc. for C6H6FINO+ (M+H)+: m/z=252.9; found 253.0.
This compound was prepared according to the procedure described in General procedure for Examples 36-50, using Intermediate 27 and 3-fluoro-4-iodo-2-methoxypyridine as electrophile.
Diastereomer 1. Peak 1. LCMS calc. for C41H39Cl2F2N6O4+ (M+H)+: m/z=787.2; found 787.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C41H39Cl2F2N6O4+ (M+H)+: m/z=787.2; found 787.2.
To a scintillation vial was added Intermediate 20 (20 mg, 0.024 mmol), NaOt-Bu (22.7 mg, 0.236 mmol), 2-bromothiazole (38.7 mg, 0.236 mmol), copper(I) iodide (4.50 mg, 0.024 mmol), and N,N′-bis(2-phenylethyl)ethanediamide (7.00 mg, 0.024 mmol). To the mixture was then added 1,4-dioxane (0.5 mL) and the mixture was sparged with N2 for 5 min. The mixture was heated and stirred at 60° C. overnight. The mixture was diluted with MeCN (10 mL) and MeOH (10 mL), filtered, and concentrated. The crude material was dissolved in MeCN and purified by preparative LCMS (XBRIDGE® C18 column, eluting with a gradient of MeCN/water containing 0.37% NH4OH, at flow rate of 60 mL/min) to afford the desired Boc-protected intermediate. The Boc-protected intermediate was then diluted with MeCN (0.5 mL) followed by addition of TFA (2 mL). The reaction mixture was stirred at r.t. for 1 h and diluted with MeCN, which was then purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt.
Diastereomer 1. Peak 1. LCMS calc. for C38H36Cl2FN6O3S+ (M+H)+: m/z=745.2; found 745.2. This is the desired (potent) diastereoisomer. 1H NMR (500 MHz, DMSO-d6) δ 9.30 (s, 1H), 8.12 (s, 1H), 7.84 (dd, J=8.1, 1.5 Hz, 1H), 7.57 (t, J=7.9 Hz, 1H), 7.49-7.44 (m, 1H), 7.18 (t, J=3.0 Hz, 1H), 7.11 (m, 1H), 6.85 (s, 1H), 5.57-5.53 (M, 1H), 5.46-5.37 (m, 2H), 5.17 (m, 1H), 4.88-4.84 (m, 1H), 4.35 (ddd, J=17.5, 11.9, 5.6 Hz, 1H), 4.21 (dd, J=11.8, 3.7 Hz, 1H), 3.95-3.90 (m, 1H), 3.85-3.78 (m, 1H), 3.45-3.30 (m, 1H), 3.07-2.98 (m, 1H), 2.70-2.62 (m, 1H), 2.36-2.28 (m, 2H), 2.10-2.03 (m, 1H), 1.57-1.52 (m, 3H), 0.95-0.78 (m, 4H).
Diastereomer 2. Peak 2. LCMS calc. for C38H36Cl2FN6O3S+ (M+H)+: m/z=745.2; found 745.2.
Examples 55-62 were prepared according to the procedure described for Example 5 with the corresponding electrophile and intermediate listed in Table D.
This compound was prepared according to the procedure described for Example 5, using 2,4-difluoro-6-methylpyridine instead of 2-fluoropyridine and Intermediate 20 instead of Intermediate 4.
Diastereomer 1. Peak 1. LCMS calc. for C41H39Cl2F2N6O3+ (M+H)+: m/z=771.2; found 771.2. This is the desired (potent) diastereoisomer. 1H NMR (600 MHz, DMSO-d6) δ 9.27-9.24 (m, 1H), 8.14-8.11 (m, 2H), 7.84 (dd, J=8.1, 1.6 Hz, 1H), 7.57 (t, J=7.8 Hz, 1H), 7.47 (dd, J=7.7, 1.6 Hz, 1H), 6.86-6.83 (m, 2H), 6.59 (dd, J=10.3, 2.1 Hz, 1H), 5.57-5.52 (m, 1H), 5.51-5.45 (m, 1H), 5.39 (dd, J=8.6, 4.1 Hz, 1H), 5.19 (q, J=6.6 Hz, 1H), 4.86 (d, J=5.9 Hz, 1H), 4.35 (dd, J=11.6, 5.9 Hz, 1H), 4.03 (dd, J=11.5, 4.0 Hz, 1H), 3.93-3.88 (m, 1H), 3.85-3.77 (m, 1H), 3.44-3.40 (m, 1H), 3.05-2.99 (m, 1H), 2.88-2.81 (m, 2H), 2.75-2.68 (m, 1H), 2.67-2.61 (m, 1H), 2.36 (s, 3H), 2.34-2.26 (m, 2H), 2.09-2.02 (m, 1H), 1.55 (d, J=6.4 Hz, 3H), 0.94-0.89 (m, 1H), 0.86-0.79 (m, 1H).
Diastereomer 2. Peak 2. LCMS calc. for C41H39Cl2F2N6O3+ (M+H)+: m/z=771.2; found 771.2.
This compound was prepared according to the procedure described for Example 5, using 4-fluoro-2-nitropyridine instead of 2-fluoropyridine and Intermediate 20 instead of Intermediate 4.
Diastereomer 1. Peak 1. LCMS calc. for C41H39Cl2F2N6O3+ (M+H)+: m/z=771.2; found 771.2. This is the desired (potent) diastereoisomer. 1H NMR (600 MHz, DMSO-d6) δ 9.24-9.17 (m, 1H), 8.24 (dd, J=9.2, 5.8 Hz, 1H), 8.11 (s, 1H), 8.04 (s, 1H), 7.84 (dd, J=8.1, 1.5 Hz, 1H), 7.58 (t, J=7.9 Hz, 1H), 7.47 (dd, J=7.6, 1.5 Hz, 1H), 7.02 (dd, J=8.1, 5.8, 2.2 Hz, 1H), 6.87-6.81 (m, 2H), 5.52 (d, J=2.9 Hz, 1H), 5.37 (d, J=4.8 Hz, 1H), 5.26 (t, J=8.4 Hz, 1H), 5.16 (q, J=6.6 Hz, 1H), 4.85 (d, J=6.0 Hz, 1H), 4.62 (q, J=6.9 Hz, 1H), 3.85-3.79 (m, 1H), 3.77 (dt, J=6.0, 2.9 Hz, 1H), 3.06-2.98 (m, 1H), 2.91-2.77 (m, 3H), 2.69-2.61 (m, 1H), 2.28 (d, J=8.8 Hz, 1H), 2.22 (ddd, J=13.7, 8.4, 4.8 Hz, 1H), 2.03 (ddd, J=12.6, 7.8, 4.8 Hz, 1H), 1.53-1.51 (m, 1H) 1.52 (d, J=6.6 Hz, 3H), 1.45 (d, J=6.9 Hz, 3H), 0.96-0.88 (m, 2H), 0.83-0.76 (m, 2H).
Diastereomer 2. Peak 2. LCMS calc. for C41H39Cl2F2N6O3+ (M+H)+: m/z=771.2; found 771.2.
This compound was prepared according to the procedure described for Example 5, using 2-chloro-3-fluoro-4-nitropyridine instead of 2-fluoropyridine and Intermediate 27 instead of Intermediate 4. LCMS calc. for C51H54Cl3FN7O8+ (M+H)+: m/z=1016.3; found 1016.3.
To a solution of tert-butyl (1R,4R,5S)-5-(2-((2R,4S,5R)-4-((2-chloro-4-nitropyridin-3-yl)oxy)-1-(cyclopropanecarbonyl)-5-methylpyrrolidin-2-yl)-8-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (20 mg, 0.020 mmol) dissolved in DMF (0.25 mL) was added TBAF (1 M in THF, 39 μL, 0.039 mmol) dropwise at r.t. The reaction mixture was allowed to stir at r.t. for 1 h, after which brine (1 mL) was added. The aq. phase was extracted with EtOAc (2 mL×3). The combined organic phase was dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in MeCN (0.5 mL) and kept below −20° C. To it was then added TFA (2 mL) dropwise over 5 min., after which it was allowed to warm to r.t. and stir for additional 15 min. The reaction mixture was diluted with more MeCN, and then directly purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C41H38Cl3F2N6O3+ (M+H)+: m/z=805.2; found 805.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C41H38Cl3F2N6O3+ (M+H)+: m/z=805.2; found 805.2.
To a vial was added 2-bromo-4-fluoropyridine (100 mg, 0.568 mmol), 3-fluoroazetidine-HCl salt (76 mg, 0.682 mmol), Ruphos-Pd-G3 (88 mg, 0.114 mmol), followed by Cs2CO3 (741 mg, 2.273 mmol). To the mixture was then added tBuOH (3.0 mL). The vial was sealed with a teflon screw-cap, evacuated and backfilled with N2. The mixture was heated to 85° C. for 4 h. The reaction mixture was concentrated and purified with FCC (10% MeOH in DCM) to give the sub-title compound (110 mg, 0.369 mmol, 65% yield). LCMS calc. for C8H9F2N2+ (M+H)+: m/z=171.1; found 171.1.
This compound was prepared according to the general procedure described for Example 5, using 2-fluoro-6-(3-fluoroazetidin-1-yl)pyridine instead of 2-fluoropyridine. LCMS calc. for C42H39Cl2F2N7O2+ (M+H)+: m/z=782.2; found 782.2.
Examples 66-69 were prepared according to procedures described for Example 4, Step 1-Step 2 and are listed in Table E.
NaN3 (3.12 mg, 0.048 mmol) was added to a solution of the corresponding intermediate (0.016 mmol) in DMF (1 mL), and the resulting mixture was stirred at 70° C. for 5 h. After cooling down to r.t., the reaction mixture was diluted with EtOAc and washed with water for 3 times followed by brine. The organic phase was dried over Na2SO4, and solvent was removed under vacuum to afford the crude azide. To the crude azide was added the corresponding alkyne (0.048 mmol), and Cp*Ru(PPh3)2C1 (2.52 mg, 3.17 μmol). The resulting mixture was suspended in toluene (1 mL) and then sparged with N2 for 10 min. The reaction mixture was stirred at 105° C. for 3 h. Upon completion, toluene was removed under vacuum.
To the crude residue was added DCM (1 mL) and TFA (1 mL), and the reaction mixture was stirred at r.t. for 30 min. The reaction mixture was concentrated, then diluted with MeCN (5 mL), and filtered. The crude solution was purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder.
A mixture of Intermediate 8 (10 mg, 0.012 mmol), 4-fluoro-1H-pyrazole (3.18 mg, 0.037 mmol) and Cs2CO3 (12.06 mg, 0.037 mmol) in DMF (0.4 mL) was stirred at 70° C. with LCMS monitoring. Upon completion, the reaction mixture was diluted with EtOAc, washed with water (×3) and brine, dried over Na2SO4, and concentrated under vacuum.
To the crude residue, DCM (1 mL) and TFA (1 mL) were added, and the reaction mixture was stirred at r.t. for 30 min. The reaction mixture was concentrated then diluted with MeCN (5 mL) and filtered. The crude solution was purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as a TFA salt in the form of a white amorphous powder. LCMS calc. for C37H34Cl2F2N7O+ (M+H)+: m/z=700.2; found 700.2.
This compound was prepared according to the procedure described for Example 66, using 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine instead of 4-fluoro-1H-pyrazole. LCMS calc. for C41H38Cl2FN8O2+ (M+H)+: m/z=763.3; found 763.3.
DIAD (0.020 mL, 0.102 mmol) was added to a mixture of tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4R)-1-(cyclopropanecarbonyl)-4-hydroxypyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (from Step 4, Intermediate 8), (15 mg, 0.020 mmol), 2H-1,2,3-triazole (7.07 mg, 0.102 mmol), and PPh3 (26.8 mg, 0.102 mmol) in THF (1 mL). The resulting mixture was stirred at r.t. with LCMS monitoring. Upon completion, the volatiles were removed under vacuum.
To the crude residue was added DCM (1 mL) and TFA (1 mL), and the reaction mixture was stirred at r.t. for 30 min. The reaction mixture was concentrated then diluted with MeCN (5 mL) and filtered. The crude solution was purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the product as a TFA salt in the form of a white amorphous powder. LCMS calc. for C36H34Cl2FN8O+ (M+H)+: m/z=683.2; found 683.2.
This compound was prepared according to the procedure described for Example 68, using 5-(pyrrolidin-1-yl)-2H-tetrazole instead of 2H-1,2,3-triazole. LCMS calc. for C39H40Cl2FN10O+ (M+H)+: m/z=753.3; found 753.3.
To a solution of Intermediate 26 (20 mg, 0.024 mmol) in THF (1 mL), 6-methoxypyridin-3-ol (11.82 mg, 0.094 mmol) and PPh3 (24.78 mg, 0.094 mmol) were added, followed by dropwise addition of DEAD 40% in toluene (0.037 mL, 0.094 mmol). The mixture was heated at 50° C. for 4 h. After the reaction mixture was cooled to r.t., it was concentrated to dryness. The residue was dissolved in MeCN (1 mL) and then 4 M aq. HCl (1 mL) was added. The mixture was stirred at 50° C. 2 h. The crude solution was diluted with more MeCN and directly purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C41H39Cl2FN6O4+ (M+H)+: m/z=769.7; found 769.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C41H39Cl2FN6O4+ (M+H)+: m/z=769.7; found 769.2.
DIAD (0.046 mL, 0.236 mmol) was added to a mixture of Intermediate 24 (20 mg, 0.0224 mmol), 5-cyclopropylisoxazol-3-ol (29.6 mg, 0.236 mmol), and PPh3 (61.9 mg, 0236 mmol) in THF (1 mL) at 0° C. The resulting mixture was stirred at r.t. for 3 h. Upon completion, the crude mixture was diluted with MeOH (3 mL) and MeCN (3 mL), filtered, and purified by prep.-LCMS (pH 10.0, XBRIDGE® C18 column, eluting with a gradient of MeCN/water containing 0.15% NH4OH, at flow rate of 60 mL/min) to afford the Boc-protected intermediate.
The purified Boc-protected intermediate was dissolved in MeCN (3 mL), cooled to −40° C., and TFA (2 mL) was added dropwise. The reaction mixture was allowed to warm to r.t. and stir for additional 2 h. The mixture was then diluted with more MeCN and directly purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C41H40Cl2FN6O4+ (M+H)+: m/z=769.3; found 769.3. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C41H40Cl2FN6O4+ (M+H)+: m/z=769.3; found 769.3.
A 10 mL ElectraSyn vial with a stir bar was charged with 1-methylcyclobutane-1-carboxylic acid (45.65 mg, 0.400 mmol), Intermediate 16 (215 mg, 1.20 mmol), 2,4,6-collidine (0.158 mL, 1.20 mL), tetrabutylammonium hexafluorophosphate (465 mg, 1.20 mmol), silver (V) hexafluroantimonate (206 mg, 0.600 mmol), 3A molecular sieves (150 mg, 0.400 mmol), and DCM (6 mL). The ElectraSyn vial was equipped with a graphite anode and a graphite cathode that were inserted into the reaction mixture. After pre-stirring for 15 min., the reaction mixture was electrolyzed at a constant current of 10 mA for 3 h at r.t. The ElectraSyn cap was then removed, and the electrodes were rinsed with DCM. The reaction mixture was filtered through a pad of SiliaMetS Thiol®, rinsed with MeCN (6 mL), and concentrated. After that, Et2O was added to the residue and the slurry was stirred for 30 min. The suspension was filtered and the filtrate was concentrated. The residue was then subjected to FCC (MeOH in DCM 0-10%) to afford the sub-title compound. LCMS calc. for C15H22NO2+ (M+H)+: m/z=248.2; found 248.2.
A 40 mL scintillation vial was charged with tert-butyl (1R,4R,5S)-5-((6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoro-3-iodo-2-methylquinolin-4-yl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (from Intermediate 10, Step 3) (50 mg, 0.073 mmol), cyclopropyl((2R,4S)-2-ethynyl-4-(1-methylcyclobutoxy)pyrrolidin-1-yl)methanone (27.2 mg, 0.110 mmol), copper(I) iodide (14.0 mg, 0.073 mmol) and Pd(PPh3)4 (12.7 mg, 0.011 mmol). DMF (1.5 mL) and DIPEA (0.128 mL, 0.734 mmol) were added under N2. The reaction mixture was sparged with N2 via needle and then heated to 90° C. for 1 h. After that, Cs2CO3 (120 mg, 0.367 mmol) was added to the mixture. The vial was recapped and heated at 60° C. for 24 h with rapid stirring. The reaction mixture was diluted with water (10 mL) and DCM (10 mL). The organic layer was separated and the aq. layer was extracted with DCM (10 mL×2). Combined organic phase was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The obtained residue was purified by FCC (MeOH in DCM 0-10%). Fractions containing the desired Boc-protected compound were then combined and concentrated.
To the crude residue was added MeCN (8 mL) and 4M HCl in 1,4-dioxane (2.0 mL, 8.0 mmol), and the reaction mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated then diluted with MeCN (5 mL) and filtered. The crude solution was purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C39H41Cl2FN5O2+ (M+H)+: m/z=700.3; found 700.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C39H41Cl2FN5O2+ (M+H)+: m/z=700.3; found 700.2.
This compound was prepared according to the procedure described for Step 1, Example 78, using 2,2-difluoro-2-phenylacetic acid instead of 1-methylcyclobutane-1-carboxylic acid. LCMS calc. for C17H18Cl2F2NO2+ (M+H)+: m/z=306.1; found 306.1.
This compound was prepared according to the procedure described for Step 2, Example 78, using cyclopropyl((2R,4S)-4-(difluoro(phenyl)methoxy)-2-ethynylpyrrolidin-1-yl)methanone instead of cyclopropyl((2R,4S)-2-ethynyl-4-(1-methylcyclobutoxy)pyrrolidin-1-yl)methanone.
Diastereomer 1. Peak 1. LCMS calc. for C41H37Cl2F3N5O2+ (M+H)+: m/z=758.2; found 758.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C41H37Cl2F3N5O2+ (M+H)+: m/z=758.2; found 758.2.
To a mixture of cis-cyclobutane-1,2-diol (1.00 g, 11.4 mmol) and imidazole (3.25 g, 47.7 mmol) was added DCM (12 mL) at r.t. Then a solution of thionyl chloride (1.66 mL, 22.7 mmol) in DCM (4 mL) and NEt3 (4.75 mL, 34.0 mmol) in DCM (4 mL) were added dropwise at 0° C. The reaction mixture was stirred at r.t. overnight. Water (20 mL) was added to the reaction mixture and the resulting solution was extracted with DCM (20 mL×3). The combined organic layers were dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure and filtered through a short pad of silica gel with DCM. The filtrate was concentrated and dissolved in MeCN (4 mL). The solution was cooled down to 0° C., after which NaIO4 (2.91 g, 13.6 mmol), ruthenium(III) chloride (24.0 mg, 0.114 mmol), and pre-chilled water (4.0 mL) were sequentially added to the reaction mixture. The mixture was stirred at r.t. for 5 h. Upon completion of reaction, the reaction mixture was extracted with EtOAc (4 mL×3) and dried over anhydrous Na2SO4. The residue was purified by FCC (100% DCM) to afford the sub-title compound (1.5 g, 10 mmol, 88% yield). 1H NMR (400 MHz, DMSO-d6) 5.62-5.56 (m, 2H), 2.56-2.34 (m, 4H).
To a vial of Intermediate 16 (1.5 g, 8.37 mmol) in DMF (10 mL) was treated with NaH (60% wt. in mineral oil, 480 mg, 12 mmol) at 0° C. After the mixture was stirred for 30 min. at r.t., cis-2,4-dioxa-3-thiabicyclo[3.2.0]heptane 3,3-dioxide (1.26 g, 8.37 mmol) was added dropwise to the solution at r.t. The mixture was stirred for 1 h then quenched with aq. sat. NH4Cl (5 mL). Water (10 mL) was added. The mixture was washed with EtOAc (10 mL×2). The aq. layers were concentrated under reduced pressure to remove DMF and water. The remaining organic material was dissolved in MeCN (20 mL), and filtered to remove salts. The filtrate was treated with 4M HCl in 1,4-dioxane (5 mL) and stirred for 1 h. The resultant mixture was diluted with MeCN, and purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as peak 1 (desired diastereomer) and peak 2. All desired fractions were dissolved in MeCN (3 mL), washed with sat. aq. NaHCO3 (2 mL), filtered, and concentrated to afford the sub-title compound (peak 1,175 mg, 8.4%) as free base. LCMS calc. for C14H20NO3 (M+H)*: m/z=250.1; found 250.1.
To a 40 mL scintillation vial was added tert-butyl (1R,4R,5S)-5-((6-(2-cyanoethyl)-7-(2,3-dichlorophenyl)-8-fluoro-3-iodo-2-methylquinolin-4-yl)amino)-2-azabicyclo[2.1.1]hexane-2-carboxylate (from Intermediate 10, Step 3) (232 mg, 0.341 mmol), trans-cyclopropyl((2R,4S)-2-ethynyl-4-(2-hydroxycyclobutoxy)pyrrolidin-1-yl)methanone (85 mg, 0.341 mmol), copper(I) iodide (64.9 mg, 0.341 mmol), and Pd(PPh3)4 (59.1 mg, 0.051 mmol). The mixture was dissolved in DMF (4 mL) under N2 followed by addition of DIPEA (0.596 mL, 3.41 mmol). The mixture was sparged with N2 via a needle for 5 min., and subsequently heated to 90° C. for 1 h. After that, Cs2CO3 (120 mg, 0.367 mmol) was added and the mixture was stirred at 60° C. for 24 h. Upon completion, the reaction mixture was diluted with water (10 mL) and DCM (10 mL). The layers were separated and the aq. layer was extracted with DCM (10 mL×2). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was then subjected to FCC (MeOH in DCM 0-10%) to afford the sub-title compound. LCMS calc. for C43H47Cl2FN5O5(M+H)+: m/z=802.3; found 802.4.
To a solution of trans-tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S)-1-(cyclopropanecarbonyl)-4-(2-hydroxycyclobutoxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-methyl-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (from Step 3) in DMF (10 mL) was added NaH (60% wt. in mineral oil, 0.068 g, 1.705 mmol) at r.t. After stirring at r.t. for 10 min., iodomethane-d3 (0.424 mL, 6.82 mmol) was added. The solution was further stirred for 2 h at r.t. The mixture was quenched by sat. aq. NH4Cl solution and extracted with DCM (20 mL×3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was diluted with MeCN (8 mL), filtered, and purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the desired Boc-protected intermediate as TFA salt. The purified TFA salt was re-dissolved in MeCN (8 mL), and HCl (4 M in 1,4-dioxane, 2 mL) was added. The reaction mixture was stirred at r.t. for 2 h, diluted with more MeCN, and purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C39H38D3Cl2FN5O3+ (M+H)+: m/z=719.3; found 719.3. This is the desired (potent) diastereoisomer. 1H NMR (500 MHz, DMSO-d6) 9.26 (brs, 1H), 8.12 (s, 1H), 8.05 (brs, 1H), 7.84 (dd, J=8.1, 1.6 Hz, 1H), 7.57 (dd, J=8.1, 7.7 Hz, 1H), 7.45 (dd, J=7.7, 1.6 Hz, 1H), 6.65 (s, 1H), 5.52 (s, 1H), 5.23 (dd, J=8.4, 3.9 Hz, 1H), 4.85 (d, J=6.0 Hz, 1H), 4.17-4.06 (m, 2H), 3.90-3.71 (m, 4H), 3.67-3.59 (m, 1H), 3.43-3.36 (m, 1H), 3.06-2.95 (m, 1H), 2.90-2.76 (m, 5H), 2.69-2.61 (m, 1H), 2.50-2.42 (m, 1H), 2.31 (d, J=9.0 Hz, 1H), 2.08-1.87 (m, 4H), 1.56 (d, J=9.0 Hz, 1H), 1.32-1.13 (m, 2H), 0.95-0.84 (m, 3H), 0.82-0.74 (m, 1H).
Diastereomer 2. Peak 2. LCMS calc. for C39H38D3Cl2FN5O3+ (M+H)+: m/z=719.3; found 719.3.
To a scintillation vial was added 1-(tert-butyl) 2-methyl (R)-4-methylenepyrrolidine-1,2-dicarboxylate (1.44 g, 5.97 mmol) followed by THF (20 mL). The solution was cooled to 0° C. and then LiAlH4 (2 M in THF, 4.48 mL, 8.95 mmol) was added over 4 min. After stirring at 0° C. for 1 h, the reaction was quenched by sequential addition of MeOH (1 mL) and water (5 mL). The resulting slurry was acidified to pH=7 with 1 M HCl (5 mL). The aqueous phase was extracted with EtOAc (25 mL). The organic layer was dried over MgSO4, filtered, then concentrated to give the sub-title compound (956 mg, 4.48 mmol, 75% yield). The crude product was carried to the next step without further purification. LCMS calc. for C7H12NO3+(M−tBu+2H)+: m/z=158.1; found 158.1.
To a 250 mL round bottom flask was added tert-butyl (R)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carboxylate (0.960 g, 4.50 mmol) followed by addition of DMF (15.0 mL). To the solution was then added tert-butyldiphenylchlorosilane (1.270 ml, 4.95 mmol) and imidazole (0.919 g, 13.50 mmol). The mixture was stirred at r.t. for 48 h. The mixture was diluted with EtOAc (250 mL), then washed with 5% aq. LiCl (300 mL) and brine (300 mL). The organic layer was dried over Na2SO4, filtered, concentrated, and purified by FCC (0-10% EtOAc/hexanes) to afford the sub-title compound (1.48 g, 3.28 mmol, 73%) as a clear oil. LCMS calc. for C22H30NOSi+ (M−CO2tBu+2H)+: m/z=352.2; found 352.2.
To a solution of tert-butyl (R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carboxylate (1.48 g, 3.28 mmol) in DCM (11 mL) was added mCPBA (1.836 g, 8.19 mmol) at 0° C. The mixture was allowed to warm up to r.t. and stirred overnight. The reaction mixture was quenched with sat. aq. Na2S2O3 (10 mL) and stirred for 5 min. The aqueous layer was extracted with DCM (2×75 mL). The organic layers were combined and washed with sat. aq. NaHCO3 (250 mL) and then brine (250 mL). The organic layer was dried over Na2SO4, filtered, then concentrated in vacuo. The crude oil was purified by FCC (0-20% EtOAc/hexanes) to afford the sub-title compound (520 mg, 1.11 mmol, 34%) as a first peak that elutes before the undesired diastereomer. LCMS calc. for C22H30NO2Si+ (M−CO2tBu+2H)+: m/z=368.2; found 368.2.
To a 40 mL scintillation vial containing tert-butyl (3S,6R)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-1-oxa-5-azaspiro[2.4]heptane-5-carboxylate (520 mg, 1.11 mmol) was added lithium triethylborohydride (1 M in THF, 2.2 mL, 2.22 mmol) dropwise at 0° C. The mixture was allowed to warm to r.t. and stirred for an additional 1.5 h. After that, the mixture was cooled to 0° C. and water (5 mL) was slowly added. The resulting slurry was partitioned between water (30 mL) and EtOAc (30 mL). The aqueous layer was saturated with K2CO3. The aqueous phase was extracted with EtOAc (3×50 mL). The organic layers were combined, dried over Na2SO4, filtered, then concentrated. The resulting residue was filtered through a small plug of silica with EtOAc (50 mL) as the eluent, then concentrated to afford the sub-title compound (531 mg, 1.13 mmol, 100% yield) as a white foam. LCMS calc. for C22H32NO2Si+ (M−CO2tBu+2H)+: m/z=370.2; found 370.3.
The sub-title compound was prepared according to the procedure described for Intermediate 5, Step 5-Step 6, starting with tert-butyl (2R,4S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxy-4-methylpyrrolidine-1-carboxylate instead of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-ethynylpyrrolidine-1-carboxylate. LCMS calc. for C26H36NO3Si+ (M+H)+: m/z=438.3; found 438.3.
To a 40 mL scintillation vial were added ((2R,4S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxy-4-methylpyrrolidin-1-yl)(cyclopropyl)methanone (236 mg, 0.539 mmol) and THF (2.5 mL). The solution was cooled to 0° C. in an ice water bath followed by the addition of benzyl bromide (96 μL, 0.809 mmol). To the solution was added potassium bis(trimethylsilyl)amide (1 M in THF, 1.1 mL, 1.10 mmol) dropwise. After that, the reaction mixture was added sat. aq. NaHCO3 (5 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (3×5 mL). The organic layers were combined, dried over Na2SO4, filtered, then concentrated to afford the sub-title compound (224 mg, 0.425 mmol, 79%). The material was carried forward to the next step without further purification. LCMS calc. for C33H42NO3Si+ (M+H)+: m/z=528.3; found 528.3.
The sub-title compound was prepared according to the procedure described for Intermediate 9, Step 8, starting with ((2R,4S)-4-(benzyloxy)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-methylpyrrolidin-1-yl)(cyclopropyl)methanone instead of ((3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-ethynyl-2-methylpyrrolidin-1-yl)(cyclopropyl)methanone. LCMS calc. for C17H24NO3+ (M+H)+: m/z=290.2; found 290.2.
The sub-title compound was prepared according to the procedure described for Intermediate 9, Step 6-Step 7, starting with ((2R,4S)-4-(benzyloxy)-2-(hydroxymethyl)-4-methylpyrrolidin-1-yl)(cyclopropyl)methanone instead of ((3S,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-methylpyrrolidin-1-yl)(cyclopropyl)methanone. LCMS calc. for C18H22NO2+ (M+H)+: m/z=284.2; found 284.2.
This compound was prepared according to the procedure described for Example 78, Step 2, using ((2R,4S)-4-(benzyloxy)-2-ethynyl-4-methylpyrrolidin-1-yl)(cyclopropyl)methanone instead of cyclopropyl((2R,4S)-2-ethynyl-4-(1-methylcyclobutoxy)pyrrolidin-1-yl)methanone.
Diastereomer 1. Peak 1. LCMS calc. for C42H41Cl2FN5O2+ (M+H)+: m/z=736.3; found 736.3. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C42H41Cl2FN5O2+ (M+H)+: m/z=736.3; found 736.3.
The sub-title compound was prepared according to the procedure described in Intermediate 4, Step 1, using Intermediate 29 instead of Intermediate 2 and Intermediate 28 instead of Intermediate 3. LCMS calc. for C50H53Cl2FN5O6+ (M+H)+: m/z=908.3; found 908.5.
The sub-title compound was prepared according to the procedure described in the general procedure for Examples 36-50, using tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S,5R)-1-(cyclopropanecarbonyl)-4-hydroxy-5-vinylpyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((R)-1-((4-methoxybenzyl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate as the appropriate intermediate and 5-fluoro-4-iodo-2-methoxypyridine as the electrophile. LCMS calc. for C56H57Cl2F2N6O7(M+H)+: m/z=1033.4; found 1033.3.
A vial was charged with tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S,5R)-1-(cyclopropanecarbonyl)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-vinylpyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((R)-1-((4-methoxybenzyl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (20 mg, 0.019 mmol), 10% palladium on carbon (4.12 mg, 3.87 μmol), and MeOH (500 μL). The solution was briefly sparged with H2 in a balloon and stirred under the H2 balloon at r.t. overnight. The reaction mixture was filtered and concentrated in vacuo. The residue was dissolved in MeCN (0.5 mL) and kept below −20° C. TFA (2 mL) was then added dropwise over 5 min., after which the mixture was allowed to warm to r.t. and stirred for an additional 15 min. The reaction mixture was diluted with MeCN and purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford each diastereoisomer of the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C43H43Cl2F2N6O4+ (M+H)+: m/z=815.3; found 815.4. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C43H43Cl2F2N6O4+ (M+H)+: m/z=815.3; found 815.4.
A vial was charged with tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S,5R)-1-(cyclopropanecarbonyl)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-vinylpyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((R)-1-((4-methoxybenzyl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (186 mg, 0.180 mmol) from Example 82, Step 2, THF (3 mL) and water (720 μL). To this suspension was added osmium tetroxide (4% in water, 57.2 μL, 8.99 μmol). The reaction was stirred at r.t. for 5 min., and then NaIO4 (192 mg, 0.899 mmol) was added. After stirring for 1 h, the reaction mixture was diluted with water (10 mL) and DCM (10 mL) and then filtered. The layers were separated using a hydrophobic frit. The organic layer was dried over Na2SO4, filtered, then concentrated. The crude material was purified FCC (0-50% acetone/hexanes) to afford the sub-title compound (186 mg, 180 mmol, 100% yield). LCMS calc. for C55H55Cl2F2N6O8(M+H)+: m/z=1035.5; found 1035.5.
A vial was charged with tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S,5S)-1-(cyclopropanecarbonyl)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-formylpyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((R)-1-((4-methoxybenzyl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (50 mg, 0.048 mmol) followed by addition of MeOH (480 μL). The solution was cooled to 0° C., NaBH4 (3.65 mg, 0.097 mmol) was added and the mixture was stirred at 0° C. for 1 h. The reaction was quenched with sat. aq. NaHCO3 (2 mL). The aqueous layer was extracted with DCM (2×5 mL), and the combined organic fractions were filtered through a small plug of MgSO4, then concentrated in vacuo to afford the sub-title compound (42.6 mg, 0.041 mmol, 85%). The crude product was used in the next step without further purification. LCMS calc. for C55H57Cl2F2N6O8+ (M+H)+: m/z=1037.4; found 1037.3.
A vial was charged with tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2R,4S,5R)-1-(cyclopropanecarbonyl)-4-((5-fluoro-2-methoxypyridin-4-yl)oxy)-5-(hydroxymethyl)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((R)-1-((4-methoxybenzyl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (10 mg, 9.63 μmol) and DMF (0.3 mL). The solution was cooled to 0° C. and then treated with NaH (60% dispersion in mineral oil, 0.462 mg, 0.012 mmol). The mixture was stirred at 15 min., after which point methyl iodide (1.205 μL, 0.019 mmol) was added. The solution was stirred at 0° C. for 1 h and then quenched with sat. aq. NH4Cl (1 mL). The layers were separated and the aqueous layer was extracted with DCM (3×2 mL). The combined organic fractions were filtered over a plug of MgSO4 and concentrated in vacuo. The residue was dissolved in MeCN (0.5 mL) and kept below −20° C. TFA (2 mL) was added dropwise over 5 min. to the mixture, which was then allowed to warm to r.t. and stirred for an additional 15 min. The reaction mixture was diluted with MeCN and purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the each diastereomer of the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C43H43Cl2F2N6O5+ (M+H)+: m/z=831.3; found 831.4. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C43H43Cl2F2N6O5+ (M+H)+: m/z=831.3; found 831.4.
To a solution of 1-(tert-butyl) 2-methyl (R)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylate (12 g, 52.8 mmol) in acetone (90 mL) and water (30 mL) cooled to 0° C. were added 4-methylmorpholine-N-oxide (15.5 g, 132 mmol) and potassium osmate(VI) dihydrate (1.56 g, 4.22 mmol). The reaction mixture was vigorously stirred at 40° C. overnight. The mixture was concentrated in vacuo, then diluted with EtOAc (120 mL) and washed with water (50 mL). The aqueous phase was back-extracted with EtOAc (3×50 mL). The combined organic phase was washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by FCC (0-15% MeOH/DCM) to afford the sub-title compound (10 g, 38.3 mmol, 73%). LCMS calc. for C6H12NO4+ (M−CO2tBu+2H)+: m/z=162.1; found 162.1.
To a solution of 1-(tert-butyl) 2-methyl (2R,3S,4R)-3,4-dihydroxypyrrolidine-1,2-dicarboxylate (13.8 g, 52.8 mmol) in acetone (150 mL) were added 2,2-dimethoxypropane (32.5 mL, 264 mmol) and p-toluenesulfonic acid monohydrate (1.51 g, 7.92 mmol) at r.t. The reaction mixture was allowed to stir at r.t. for 3 h, then Et3N (6 mL) was added to quench the reaction. The mixture was concentrated in vacuo, then purified by FCC (0-10% MeOH/DCM) to afford the sub-title compound (12.1 g, 40.2 mmol, 76%). LCMS calc. for C10H16NO6+ (M−tBu+2H)+: m/z=246.1; found 246.1.
The sub-title compound was prepared according to the procedure described for Intermediate 5, Step 2, starting with 5-(tert-butyl) 4-methyl (3aS,4R,6aR)-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-4,5-dicarboxylate instead of 1-(tert-butyl) 2-methyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)pyrrolidine-1,2-dicarboxylate. LCMS calc. for C8H16NO3+ (M−CO2tBu+2H)+: m/z=174.1; found 174.1.
To a solution of oxalyl chloride (4.6 mL, 52.0 mmol) in DCM (50 mL) cooled to −78° C. was slowly added a solution of DMSO (6.6 mL, 92 mmol) in DCM (20 mL). The reaction mixture was allowed to stir at −78° C. for 45 min. Then, a solution of tert-butyl (3aS,4S,6aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (7.9 g, 28.9 mmol) in DCM (20 mL) was added at −78° C. and the mixture was stirred at −78° C. for 2.5 h. Et3N (28 mL, 202 mmol) was added at −78° C., stirring was continued at −78° C. for 30 min., then the solution was allowed to warm to r.t. The mixture was diluted with DCM (100 mL), sequentially washed with 1 M aq. HCl (30 mL), sat. aq. NaHCO3 (30 mL), and brine (30 mL). The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by FCC (0-70% EtOAc/hexanes) to give the sub-title compound (6.3 g, 28.9 mmol, 80%). LCMS calc. for C9H14NO5+ (M−tBu+2H)+: m/z=216.1; found 216.1.
The sub-title compound was prepared according to the procedure described for Intermediate 5, Step 4-Step 6, starting with tert-butyl (3aS,4R,6aR)-4-formyl-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate instead of tert-butyl (2R,4S)-4-((tert-butyldiphenylsilyl)oxy)-2-formylpyrrolidine-1-carboxylate. LCMS calc. for C13H18NO3+ (M+H)+: m/z=236.1; found 236.1.
To a solution of cyclopropyl((3aS,4S,6aR)-4-ethynyl-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrol-5-yl)methanone (2.0 g, 8.50 mmol) in MeOH (10 mL) at r.t. were added ethylene glycol (0.95 mL, 17.0 mmol) and p-toluenesulfonic acid monohydrate (0.32 g, 1.70 mmol). The reaction mixture was allowed to stir at r.t. for 16 h, then Et3N (3 mL) was added to quench the reaction. The mixture was concentrated in vacuo, then purified by FCC (0-10% MeOH/DCM) to afford the sub-title compound (1.15 g, 5.89 mmol, 69%). LCMS calc. for C10H14NO3+ (M+H)+: m/z=196.1; found 196.1.
To a solution of cyclopropyl((2S,3S,4R)-2-ethynyl-3,4-dihydroxypyrrolidin-1-yl)methanone (840 mg, 4.30 mmol) in THF (8 mL) at r.t. was added 2-fluoropyridine (585 mg, 6.02 mmol) followed by sodium tert-butoxide (1.2 g, 12.9 mmol). The reaction mixture was stirred vigorously at r.t. for 3 h. After that, the reaction was quenched by the addition of sat. aq. NH4Cl (5 mL). The aqueous phase was extracted with EtOAc (3×10 mL) The combined organic phase was washed with brine (10 mL) and concentrated in vacuo. The crude material was purified by FCC (0-100% EtOAc/hexanes) to afford the sub-title compound (800 mg, 2.94 mmol, 68%). LCMS calc. for C15H17N2O3+ (M+H)+: m/z=273.1; found 273.1.
The sub-title compound was prepared according to the procedure described for Intermediate 24, using cyclopropyl((2S,3S,4R)-2-ethynyl-3-hydroxy-4-(pyridin-2-yloxy)pyrrolidin-1-yl)methanone instead of Intermediate 23. LCMS calc. for C50H54Cl2FN6O7 (M+H)+: m/z=939.3; found 939.3.
A vial was charged with tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2S,3S,4R)-1-(cyclopropanecarbonyl)-3-hydroxy-4-(pyridin-2-yloxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (12 mg, 0.013 mmol) and MeCN (0.5 mL). The solution was then cooled to −40° C. and TFA (2 mL) dropwise over 5 min. while maintaining the reaction mixture below −20° C. After complete addition, the reaction mixture was allowed to warm to r.t. and stirred for an additional 15 min. The reaction mixture was diluted with MeCN and purified by prep.-LCMS (SUNFIRE® C18 column, eluting with a gradient of acetonitrile/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the diasteromers of the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C40H38Cl2FN6O4+ (M+H)+: m/z=755.2; found 755.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C40H38Cl2FN6O4+ (M+H)+: m/z=755.2; found 755.2.
A vial was charged with tert-butyl (1R,4R,5S)-5-(8-(2-cyanoethyl)-2-((2S,3S,4R)-1-(cyclopropanecarbonyl)-3-hydroxy-4-(pyridin-2-yloxy)pyrrolidin-2-yl)-7-(2,3-dichlorophenyl)-6-fluoro-4-((1R)-1-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-pyrrolo[3,2-c]quinolin-1-yl)-2-azabicyclo[2.1.1]hexane-2-carboxylate (20 mg, 0.021 mmol) from Example 84, Step 8 and DCM (0.5 mL). The solution was then cooled to −78° C. and SELECTFLUOR® (1 M in DCM, 0.17 mL, 0.170 mmol) was added slowly. The reaction mixture was allowed to warm to r.t. and stirred overnight. After that, the reaction mixture was quenched by slow addition of sat. aq. NaHCO3 (1 mL). The aqueous phase was extracted with EtOAc (3×1.5 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was dissolved in MeCN (0.5 mL) and TFA (2 mL) was slowly added over 5 min. while maintaining the reaction mixture below −20° C. After complete addition, the reaction mixture was allowed to warm to r.t. and stirred for an additional 15 min. The reaction mixture was diluted with MeCN and purified by prep-LCMS (SUNFIRE® C18 column, eluting with a gradient of MeCN/water containing 0.1% TFA, at flow rate of 60 mL/min) to afford the diasteroeomers of the title compound as a TFA salt in the form of a white amorphous powder.
Diastereomer 1. Peak 1. LCMS calc. for C40H37Cl2F2N6O3+ (M+H)+: m/z=757.2; found 757.2. This is the desired (potent) diastereoisomer.
Diastereomer 2. Peak 2. LCMS calc. for C40H37Cl2F2N6O3+ (M+H)+: m/z=757.2; found 757.2.
Additional Example compounds within the scope of the present disclosure were also prepared and are listed in Table G below.
The inhibitor potency of the exemplified compounds was determined in a fluorescence based guanine nucleotide exchange assay, which measures the exchange of bodipy-GDP (fluorescently labeled GOP) for GppNHp (Non-hydrolyzable GTP analog) to generate the active state of KRAS in the presence of SOS1 (guanine nucleotide exchange factor). Inhibitors were serially diluted in DMSO and a volume of 0.1 μL was transferred to the wells of a black low volume 384-well plate. 5 μL/well volume of bodipy-loaded KRAS G12D diluted to 2.5 nM in assay buffer (25 mM Hepes pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 0.01% Brij-35) was added to the plate and pre-incubated with inhibitor for 4 h at r.t. Appropriate controls (enzyme with no inhibitor or with a G12D inhibitor) were included on the plate. The exchange was initiated by the addition of a 5 μL/well volume containing 1 mM GppNHp and 300 nM SOS1 in assay buffer. The 10 μL/well reaction concentration of the bodipy-loaded KRAS G12D, GppNHp, and SOS1 were 2.5 nM, 500 μM, and 150 nM, respectively. The reaction plates were incubated at r.t. for 2 h, a time estimated for complete GDP-GTP exchange in the absence of inhibitor. For the KRAS G12V mutant, similar guanine nucleotide exchange assays were used with 2.5 nM as final concentration for the bodipy loaded KRAS proteins and 3 h incubation after adding GppNHp-SOS1 mixture. A cyclic peptide described to selectively bind G12D mutant (Sakamoto et al., BBRC 484.3 (2017), 605-611) or internal compounds with confirmed binding were used as positive controls in the assay plates. Fluorescence intensities were measured on a PheraStar plate reader instrument (BMG Labtech) with excitation at 485 nm and emission at 520 nm.
Either GraphPad prism or Genedata Screener SmartFit was used to analyze the data. The IC50 values were derived by fitting the data to a four parameter logistic equation producing a sigmoidal dose-response curve with a variable Hill coefficient.
The KRAS G12D and KRAS G12V exchange assay IC50 data are provided in Tables H and I below. The symbol “†” indicates IC50≤100 nM, “††” indicates IC50>100 nM but ≤1 μM; and “†††” indicates IC50 is >1 μM but ≤5 μM, “††††” indicates IC50 is >5 μM but ≤10 μM. “NA” indicates IC50 not available. For certain compounds that were isolated as more atropisomers and/or diastereoisomers, the value for the most potent isomer is quoted.
MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-1H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-1H838 (KRAS WT; ATCC® CRL-5844) cells are cultured in RPMI 1640 media supplemented with 10% FIBS (Gibco/Life Technologies). Eight hundred cells per well in RPMI 1640 media supplemented with 2% FIBS are seeded into white, clear bottomed 384-well Costar tissue culture plates containing 50 nL dots of test compounds (final concentration is a 1:500 dilution, with a final concentration in 0.2% DMSO). Plates are incubated for 3 days at 37° C., 5% CO2. At the end of the assay, 25 μL/well of CellTiter-Glo reagent (Promega) is added. Luminescence is read after 15 min. with a PHERAstar (BMG). Data are analyzed in Genedata Screener using SmartFit for IC50 values.
MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are purchased from ATCC and maintained in RPMI 1640 media supplemented with 10% FBS (Gibco/Life Technologies). The cells are plated at 5000 cells per well (8 μL) into Greiner 384-well low volume, flat-bottom, and tissue culture treated white plates and incubated overnight at 37° C., 5% CO2. The next morning, test compound stock solutions are diluted in media at 3× the final concentration and 4 μL are added to the cells, with a final concentration of 0.1% of DMSO. The cells are incubated with the test compounds for 4 h (G12C and G12V) or 2 h (G12D) at 37° C., 5% CO2. 4 μL of 4× lysis buffer with blocking reagent (Cisbio) are added to each well and plates are rotated gently (300 rpm) for 30 min. at room temperature. 4 μL per well of Cisbio anti Phospho-ERK 1/2 d2 is mixed with anti Phospho-ERK 1/2 Cryptate (1:1), and added to each well, incubated overnight in the dark at room temperature. Plates are read on the Pherastar plate reader at 665 nm and 620 nm wavelengths. Data are analyzed in Genedata Screener using SmartFit for IC50 values.
MIA PaCa-2 cells (KRAS G12C; ATCC® CRL-1420), HPAF-II (KRAS G12D; ATCC® CRL-1997) and YAPC (KRAS G12V; DSMZ ACC382) are maintained in RPMI 1640 with 10% FBS (Gibco/Life Technologies). For MIA PaCa-2 assay, cells are seeded into 96 well tissue culture plates (Corning #3596) at 25000 cells per well in 100 μL media and cultured for 2 days at 37° C., 5% CO2 before the assay. For HPAF-II and YAPC assay, cells are seeded in 96 well tissue culture plates at 50000 cells per well in 100 μL media and cultured for 1 day before the assay. Whole Blood are added to the 1 μL dots of compounds (prepared in DMSO) in 96 well plates and mixed gently by pipetting up and down so that the concentration of the compound in blood is 1× of desired concentration, in 0.5% DMSO. The media is aspirated from the cells and 50 μL per well of whole blood with test compound is added and incubated for 4 h for MIA PaCa and YAPC assay; or 2 h for HPAF-II assay, respectively at 37° C., 5% CO2. After dumping the blood, the plates are gently washed twice by adding PBS to the side of the wells and dumping the PBS from the plate onto a paper towel, tapping the plate to drain well. 50 μL/well of 1× lysis buffer #1 (Cisbio) with blocking reagent (Cisbio) and Benzonase nuclease (Sigma Cat #E1014-5KU, 1:10000 final concentration) is then added and incubated at room temperature for 30 min. with shaking (250 rpm). Following lysis, 16 μL of lysate is transferred into 384-well Greiner small volume white plate using an Assist Plus (Integra Biosciences, NH). 4 μL of 1:1 mixture of anti Phospho-ERK 1/2 d2 and anti Phospho-ERK 1/2 Cryptate (Cisbio) is added to the wells using the Assist Plus and incubated at room temperature overnight in the dark. Plates are read on the Pherastar plate reader at 665 nm and 620 nm wavelengths. Data are analyzed in Genedata Screener using SmartFit for IC50 values.
The 96-Well Ras Activation ELISA Kit (Cell Biolabs Inc; #STA441) uses the Raf1 RBD (Rho binding domain) bound to a 96-well plate to selectively pull down the active form of Ras from cell lysates. The captured GTP-Ras is then detected by a pan-Ras antibody and HRP-conjugated secondary antibody.
MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are maintained in RPMI 1640 with 10% FBS (Gibco/Life Technologies). The cells are seeded into 96 well tissue culture plates (Corning #3596) at 25000 cells per well in 100 μL media and cultured for 2 days at 37° C., 5% CO2 so that they are approximately 80% confluent at the start of the assay. The cells are treated with compounds for either 4 h or overnight at 37° C., 5% CO2. At the time of harvesting, the cells are washed with PBS, drained well and then lysed with 50 μL of the 1× Lysis buffer (provided by the kit) plus added Halt Protease and Phosphatase inhibitors (1:100) for 1 h on ice.
The Raf-1 RBD is diluted 1:500 in Assay Diluent (provided in kit) and 100 μL of the diluted Raf-1 RBD is added to each well of the Raf-1 RBD Capture Plate. The plate is covered with a plate sealing film and incubated at room temperature for 1 h on an orbital shaker. The plate is washed 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. 50 μL of Ras lysate sample (10-100 μg) is added per well in duplicate. A “no cell lysate” control is added in a couple of wells for background determination. 50 μL of Assay Diluent is added to all wells immediately to each well and the plate is incubated at room temperature for 1 h on an orbital shaker. The plate is washed 5 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. 100 μL of the diluted Anti-pan-Ras Antibody is added to each well and the plate is incubated at room temperature for 1 h on an orbital shaker. The plate is washed 5 times as previously. 100 μL of the diluted Secondary Antibody, HRP Conjugate is added to each well and the plate is incubated at room temperature for 1 h on an orbital shaker. The plate is washed 5 times as previously and drained well. 100 μL of Chemiluminescent Reagent (provided in the kit) is added to each well, including the blank wells. The plate is incubated at room temperature for 5 min. on an orbital shaker before the luminescence of each microwell is read on a plate luminometer. The % inhibition is calculated relative to the DMSO control wells after a background level of the “no lysate control” is subtracted from all the values. IC50 determination is performed by fitting the curve of inhibitor percent inhibition versus the log of the inhibitor concentration using the GraphPad Prism 7 software.
The cellular potency of compounds is determined by measuring phosphorylation of KRAS downstream effectors extracellular-signal-regulated kinase (ERK), ribosomal S6 kinase (RSK), AKT (also known as protein kinase B, PKB) and downstream substrate S6 ribosomal protein.
To measure phosphorylated extracellular-signal-regulated kinase (ERK), ribosomal S6 kinase (RSK), AKT and S6 ribosomal protein, cells (details regarding the cell lines and types of data produced are further detailed in Table J) are seeded overnight in Corning 96-well tissue culture treated plates in RPMI medium with 10% FBS at 4×104 cells/well. The following day, cells are incubated in the presence or absence of a concentration range of test compounds for 4 h at 37° C., 5% CO2. Cells are washed with PBS and lysed with 1× lysis buffer (Cisbio) with protease and phosphatase inhibitors (Thermo Fisher, 78446). 10 or 20 μg of total protein lysates is subjected to SDS-PAGE and immunoblot analysis using following antibodies: phospho-ERK1/2-Thr202/Tyr204 (#9101L), total-ERK1/2 (#9102L), phosphor-AKT-Ser473 (#4060L), phospho-p90RSK-Ser380 (#11989S) and phospho-S6 ribosomal protein-Ser235/Ser236 (#2211S) are from Cell Signaling Technologies (Danvers, MA).
MIA-PaCa-2 (KRAS G12C), H358 (KRAS G12C), HPAF-II (KRAS G12D), AGS (KRAS G12D), SW480 (KRAS G12V) or YAPC(KRAS G12V) human cancer cells are obtained from the American Type Culture Collection and maintained in RPMI media supplemented with 10% FBS. For efficacy studies experiments, 5×106 cells are inoculated subcutaneously into the right hind flank of 6- to 8-week-old BALB/c nude mice (Charles River Laboratories, Wilmington, MA, USA). When tumor volumes are approximately 150-250 mm3, mice are randomized by tumor volume and compounds are orally administered. Tumor volume is calculated using the formula (L×W2)/2, where L and W refer to the length and width dimensions, respectively. Tumor growth inhibition is calculated using the formula (1−(VT/VC))×100, where VT is the tumor volume of the treatment group on the last day of treatment, and VC is the tumor volume of the control group on the last day of treatment. Two-way analysis of variance with Dunnett's multiple comparisons test is used to determine statistical differences between treatment groups (GraphPad Prism). Mice are housed at 10-12 animals per cage, and are provided enrichment and exposed to 12 h light/dark cycles. Mice whose tumor volumes exceeded limits (10% of body weight) are humanely euthanized by CO2 inhalation. Animals are maintained in a barrier facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. All of the procedures are conducted in accordance with the US Public Service Policy on Human Care and Use of Laboratory Animals and with Incyte Animal Care and Use Committee Guidelines.
Caco-2 cells are grown at 37° C. in an atmosphere of 5% CO2 in DMEM growth medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, penicillin (100 U/mL), and streptomycin (100 μg/mL). Confluent cell monolayers are subcultured every 7 days or 4 days for Caco-2 by treatment with 0.05% trypsin containing 1 μM EDTA. Caco-2 cells are seeded in 96-well Transwell plates. The seeding density for Caco-2 cells is 14,000 cells/well. DMEM growth medium is replaced every other day after seeding. Cell monolayers are used for transport assays between 22 and 25 days for Caco-2 cells.
Cell culture medium is removed and replaced with HBSS. To measure the TEER, the HBSS is added into the donor compartment (apical side) and receiver compartment (basolateral side). The TEER is measured by using a REMS Autosampler to ensure the integrity of the cell monolayers. Caco-2 cell monolayers with TEER values 300 Ω·cm2 are used for transport experiments. To determine the Papp in the absorptive direction (A-B), solution of test compound (50 μM) in HBSS is added to the donor compartment (apical side), while HBSS solution with 4% BSA is added to the receiver compartment (basolateral side). The apical volume was 0.075 mL, and the basolateral volume is 0.25 mL. The incubation period is 120 min. at 37° C. in an atmosphere of 5% CO2. At the end of the incubation period, samples from the donor and receiver sides are removed and an equal volume of MeCN is added for protein precipitation. The supernatants are collected after centrifugation (3000 rpm, Allegra X-14R Centrifuge from Beckman Coulter, Indianapolis, IN) for LCMS analysis. The permeability value is determined according to the equation:
The Caco-2 data are provided in Table K below. The symbol “+” indicates a Caco-2 value of ≤0.5, “++” indicates a Caco-2 value of >0.5 but ≤1; and “+++” indicates a Caco-2 value of >1. “NA” indicates IC50 not available.
The whole blood stability of the exemplified compounds is determined by LCMS/MS. The 96-Well Flexi-Tier™ Block (Analytical Sales & Services, Inc, Flanders, NJ) is used for the incubation plate containing 1.0 mL glass vials with 0.5 mL of blood per vial (pooled gender, human whole blood sourced from BIOIVT, Hicksville, NY or similar). Blood is pre-warmed in water bath to 37° C. for 30 min. A 96-deep well analysis plate is prepared with the addition of 100 μL ultrapure water/well. 50 μL chilled ultrapure water/well is added to 96-deep well sample collection plate and covered with a sealing mat. 1 μL of 0.5 M compound working solution (DMSO:water) is added to the blood in incubation plate to reach final concentrations of 1 μM, mixed by pipetting thoroughly and 50 μL is transferred 50 into the T=0 wells of the sample collection plate. Blood is allowed to sit in the water for 2 min. and then 400 μL stop solution/well is added (MeCN containing an internal standard). The incubation plate is placed in the Incu-Shaker CO2 Mini incubator (Benchmark Scientific, Sayreville, NJ) at 37° C. with shaking at 150 rpm. At 1, 2 and 4 h, the blood samples are mixed thoroughly by pipetting and 50 μL is transferred into the corresponding wells of the sample collection plate. Blood is allowed to sit in the water for 2 min. and then 400 μL of stop solution/well is added. The collection plate is sealed and vortexed at 1700 rpm for 3 min. (VX-2500 Multi-Tube Vortexer, VWR International, Radnor, PA), and samples are then centrifuged in the collection plate at 3500 rpm for 10 min. (Allegra X-14R Centrifuge Beckman Coulter, Indianapolis, IN). 100 μL of supernatant/well is transferred from the sample collection plate into the corresponding wells of the analysis plate. The final plate is vortexed at 1700 rpm for 1 min. and analyze samples by LCMS/MS. The peak area ratio of the 1, 2, and 4 h samples relative to T=0 is used to determine the percent remaining. The natural log of the percent remaining versus time is used determine a slope to calculate the compounds half-life in blood (t1/2=0.693/slope).
For in vitro metabolic stability experiments, test compounds are incubated with human liver microsomes at 37° C. The incubation mixture contains test compounds (1 μM), NADPH (2 mM), and human liver microsomes (0.5 mg protein/mL) in 100 mM phosphate buffer (pH 7.4). The mixture is pre-incubated for 2 min at 37° C. before the addition of NADPH. Reactions are commenced upon the addition of NADPH and quenched with ice-cold MeOH at 0, 10, 20, and 30 min. Terminated incubation mixtures are analyzed using LCMS/MS system. The analytical system consisted of a Shimadzu LC-30AD binary pump system and SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, MD) coupled with a Sciex Triple Quad 6500+ mass spectrometer from Applied Biosystems (Foster City, CA). Chromatographic separation of test compounds and internal standard is achieved using a Hypersil Gold C18 column (50×2.1 mm, 5 μM, 175 Å) from ThermoFisher Scientific (Waltham, MA). Mobile phase A consists of 0.1% formic acid in water, and mobile phase B consists of 0.1% formic acid in MeCN. The total LCMS/MS runtime can be 2.75 min. with a flow rate of 0.75 mL/min. Peak area integrations and peak area ratio calculations are performed using Analyst software (version 1.6.3) from Applied Biosystems.
The in vitro intrinsic clearance, CLint, in vitro, is calculated from the t1/2 of test compound disappearance as CLint, in vitro=(0.693/t1/2)×(1/Cprotein), where Cprotein is the protein concentration during the incubation, and t1/2 is determined by the slope (k) of the log-linear regression analysis of the concentration versus time profiles; thus, t1/2=ln 2/k. The CLint, in vitro values are scaled to the in vivo values for human by using physiologically based scaling factors, hepatic microsomal protein concentrations (45 mg protein/g liver), and liver weights (21 g/kg body weight). The equation CLint=CLint, in vitro×(mg protein/g liver weight)×(g liver weight/kg body weight) is used. The in vivo hepatic clearance (CLH) is then calculated by using CLint and hepatic blood flow, Q (20 mL·min−1·kg−1 in humans) in the well-stirred liver model disregarding all binding from CLH=(Q×CLint)/(Q+CLint). The hepatic extraction ratio was calculated as CLH divided by Q.
For in vivo pharmacokinetic experiments, test compounds are administered to male Sprague Dawley rats or male and female Cynomolgus monkeys intravenously or via oral gavage. For intravenous (IV) dosing, test compounds are dosed at 0.5 to 1 mg/kg using a formulation of 10% dimethylacetamide (DMAC) in acidified saline via IV bolus for rat and 5 min or 10 min IV infusion for monkey. For oral (PO) dosing, test compounds are dosed at 1.0 to 3.0 mg/kg using 5% DMAC in 0.5% methylcellulose in citrate buffer (pH 2.5). Blood samples are collected at predose and various time points up to 24 h postdose. All blood samples are collected using EDTA as the anticoagulant and centrifuged to obtain plasma samples. The plasma concentrations of test compounds are determined by LCMS methods. The measured plasma concentrations are used to calculate PK parameters by standard noncompartmental methods using Phoenix® WinNonlin software program (version 8.0, Pharsight Corporation).
In rats and monkeys, cassette dosing of test compounds are conducted to obtain preliminary PK parameters.
In vivo pharmacokinetic experiments with male beagle dogs may be performed under the conditions described above.
This assay is designed to characterize an increase in CYP inhibition as a test compounds is metabolized over time. Potential mechanisms for this include the formation of a tight-binding, quasi-irreversible inhibitory metabolite complex or the inactivation of P450 enzymes by covalent adduct formation of metabolites. While this experiment employs a 10-fold dilution to diminish metabolite concentrations and therefore effects of reversible inhibition, it is possible (but not common) that a metabolite that is an extremely potent CYP inhibitor could result in a positive result.
The results are from a cocktail of CYP specific probe substrates at 4 times their Km concentrations for CYP2C9, 2C19, 2D6 and 3A4 (midazolam) using human liver microsomes (HLM). The HLMs can be pre-incubated with test compounds at a concentration 10 μM for 30 min in the presence (+N) or absence (−N) of a NADPH regenerating system, diluted 10-fold, and incubated for 8 min in the presence of the substrate cocktail with the addition of a fresh aliquot of NADPH regenerating system. A calibration curve of metabolite standards can be used to quantitatively measure the enzyme activity using LCMS/MS. In addition, incubations with known time dependent inhibitors, tienilic acid (CYP2C9), ticlopidine (CYP2C19), paroxetine (CYP2D6), and troleandomycin (CYP3A4), used as positive controls are pre-incubated 30 min with or without a NADPH regenerating system.
The analytical system consists of a Shimadzu LC-30AD binary pump system and SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, MD) coupled with a Sciex Triple Quad 6500+ mass spectrometer from Applied Biosystems (Foster City, CA). Chromatographic separation of test compounds and internal standard can be achieved using an ACQUITY UPLC BEH 130A, 2.1×50 mm, 1.7 μm HPLC column (Waters Corp, Milford, MA). Mobile phase A consists of 0.1% formic acid in water, and mobile phase B consists of 0.1% formic acid in MeCN. The total LCMS/MS runtime will be 2.50 min. with a flow rate of 0.9 mL/min. Peak area integrations and peak area ratio calculations are performed using Analyst software (version 1.6.3) from Applied Biosystems.
The percentage of control CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activity remaining following preincubation of the compounds with NADPH is corrected for the corresponding control vehicle activity and then calculated based on 0 min. as 100%. A linear regression plot of the natural log of % activity remaining versus time for each isozyme is used to calculate the slope. The −slope is equal to the rate of enzyme loss, or the Kobs.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including without limitation all patent, patent applications, and publications, cited in the present application is incorporated herein by reference in its entirety.
This application is claims priority to U.S. Provisional Application No. 63/496,856 filed Apr. 18, 2023, and U.S. Provisional Application No. 63/580,831 filed Sep. 6, 2023, the entire content of which is incorporated herein.
| Number | Date | Country | |
|---|---|---|---|
| 63580831 | Sep 2023 | US | |
| 63496856 | Apr 2023 | US |
