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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/10—Spiro-condensed systems
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/08—Bridged systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/10—Spiro-condensed systems
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D498/10—Spiro-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

• the bifunctional compounds are useful as modulators of targeted ubiquitination, especially with respect to Switch/Sucrose Non Fermentable (SWI/SNF)-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2) (i.e., BRAHMA or BRM), which are degraded and/or otherwise inhibited by bifunctional compounds according to the present disclosure.
• SWI/SNF Switch/Sucrose Non Fermentable
• SMARCA2 Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 2
• E3 ubiquitin ligases (of which hundreds are known in humans) confer substrate specificity for ubiquitination, and therefore, are more attractive therapeutic targets than general proteasome inhibitors due to their specificity for certain protein substrates.
• the development of ligands of E3 ligases has proven challenging, in part due to the fact that they must disrupt protein-protein interactions.
• recent developments have provided specific ligands which bind to these ligases. For example, since the discovery of nutlins, the first small molecule E3 ligase inhibitors, additional compounds have been reported that target E3 ligases but the field remains underdeveloped.
• E3 ligase mouse double minute 2 homolog MDM2
• target MDM2 i.e., human double minute 2 or HDM2
• E3 ligases J. Di, et al. Current Cancer Drug Targets (2011), 11(8), 987-994.
• VHL von Hippel-Lindau
• VCB von Hippel-Lindau
• VHL Hypoxia Inducible Factor 1D
• VEGF vascular endothelial growth factor
• cytokine erythropoietin a transcription factor that upregulates genes such as the pro-angiogenic growth factor VEGF and the red blood cell inducing cytokine erythropoietin in response to low oxygen levels.
• VHL Von Hippel Lindau
• E3 ligase The first small molecule ligands of Von Hippel Lindau (VHL) to the substrate recognition subunit of the E3 ligase were generated, and crystal structures were obtained confirming that the compound mimics the binding mode of the transcription factor HIF-1 ⁇ , the major substrate of VHL.
• Bifunctional compounds such as those that are described in U.S.
• Patent Application Publications 2015-0291562 and 2014-0356322 function to recruit endogenous proteins to an E3 ubiquiuin ligase for degradation.
• the publications describe bifunctional or proteolysis targeting chimeric (PROTAC) compounds, which find utility as modulators of targeted ubiquitination of a variety of polypeptides and other proteins, which are then degraded and/or otherwise inhibited by the bifunctional compounds.
• PROTAC proteolysis targeting chimeric
• the Switch/Sucrose Non Fermentable is a multi-subunit complex that modulates chromatic structure through the activity of two mutually exlusive helicase/ATPase catalytic subunits SWI/SNF-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2, BRAHMA or BRM) and SWI/SNF-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 4 (SMARCA4 or BRG1).
• the core and the regulatory subunits couple ATP hydrolysis to the perturbation of histone-DNA contacts, thereby providing access points to transcription factors and cognate DNA elements that facilitate gene activation and repression.
• Mutations in the genes encoding the twenty canonical SWI/SNF subunits are observed in nearly 20% of all cancers with the highest frenquency of mutations observed in rhabdoid tumors, female cancers (including ovarian, uterine, cerical and endometrial), lung adenocarcinoma, gastric adenocarcinoma, melanoma, esophageal, and feanal clear cell carcinoma.
• SMARCA2 and SMARCA4 have been reported as having different roles in cancer.
• SMARCA4 is frequently mutated in primary tumors, while SMARCA2 inactivation is infrequent in tumor development.
• numerous types of cancer have been shown to be SMARCA4-related (e.g., cancers having a SMARCA4-mutation or a SMARCA4-deficiency, such as lack of expression), including, e.g., lung cancer (such as non-small cell lung cancer).
• SMARCA2 has been demonstrated as one of the top essential genes in SMARCA4- related or–mutant cancer cell lines.
• SMARCA4-deficient patient populations or cells depend exclusively on SMARCA2 activity—i.e., there is a greater incorporation of SMARCA2 into the complex to compensate for the SMARCA4 deficiency.
• SMARCA2 may be targeted in SMARCA4-related/deficient cancers.
• the co-occurrence of the deficiency of the expression of two (or more) genes that leads to cell death is known as, synthetic lethality. Accordingly, synethetic lethality can be leveraged in the treatment of certain SMARCA2/SMARCA4-related cancers.
• SMARCA2/SMARCA4-related cancers There is an ongoing need for effective treatment for diseases that are treatable by inhibiting or degrading SMARCA2 (i.e., BRAHMA or BRM).
• the present disclosure describes bifunctional compounds which function to recruit endogenous proteins to an E3 ubiquitin ligase for degradation, and methods of using the same.
• the present disclosure provides bifunctional or proteolysis targeting chimeric (PROTAC) compounds, which find utility as modulators of targeted ubiquitination of a variety of polypeptides and other proteins, which are then degraded and/or otherwise inhibited by the bifunctional compounds as described herein.
• An advantage of the compounds provided herein is that a broad range of pharmacological activities is possible, consistent with the degradation/inhibition of targeted polypeptides from virtually any protein class or family.
• the description provides methods of using an effective amount of the compounds as described herein for the treatment or amelioration of a disease condition, such as cancer, e.g., SMARCA4-related/deficient cancer, such as lung cancer or non-small cell lung cancer.
• a disease condition such as cancer, e.g., SMARCA4-related/deficient cancer, such as lung cancer or non-small cell lung cancer.
• the disclosure provides bifunctional or PROTAC compounds, which comprise an E3 ubiquitin ligase binding moiety (i.e., a ligand for an E3 ubquitin ligase or “ULM” group), and a moiety that binds a target protein (i.e., a protein/polypeptide targeting ligand or “PTM” group) such that the target protein/polypeptide is placed in proximity to the ubiquitin ligase to effect degradation (and inhibition) of that protein.
• E3 ubiquitin ligase binding moiety i.e., a ligand for an E3 ubquitin ligase or “ULM” group
• a target protein i.e., a protein/polypeptide targeting ligand or “PTM” group
• the ULM ubiquitination ligase modulator
• VHL Von Hippel-Lindau E3 ubiquitin ligase binding moiety
• the structure of the bifunctional compound can be depicted as: [0012]
• the respective positions of the PTM and ULM moieties as well as their number as illustrated herein is provided by way of example only and is not intended to limit the compounds in any way.
• the bifunctional compounds as described herein can be synthesized such that the number and position of the respective functional moieties can be varied as desired.
• the bifunctional compound further comprises a chemical linker (“L”).
• the structure of the bifunctional compound can be depicted as: where PTM is a protein/polypeptide targeting moiety, L is a linker, e.g., a bond or a chemical group coupling PTM to ULM, and ULM is a Von Hippel-Lindau E3 ubiquitin ligase (VHL) binding moiety (VLM).
• PTM is a protein/polypeptide targeting moiety
• L is a linker, e.g., a bond or a chemical group coupling PTM to ULM
• ULM is a Von Hippel-Lindau E3 ubiquitin ligase (VHL) binding moiety (VLM).
• VHL Von Hippel-Lindau E3 ubiquitin ligase binding moiety
• VLM is Von Hippel-Lindau E3 ubiquitin ligase binding moiety that binds to VHL E3 ligase.
• the compounds as described herein comprise multiple independently selected ULMs, multiple PTMs, multiple chemical linkers or a combination thereof.
• VLM can be hydroxyproline or a derivative thereof.
• other contemplated VLMs are included in U.S. Patent Application Publication No. 2014/03022523, which as discussed above, is incorporated herein in its entirety.
• “L” is a bond.
• the linker “L” is a connector with a linear non-hydrogen atom number in the range of 1 to 20.
• the connector “L” can contain, but not limited to the functional groups such as ether, amide, alkane, alkene, alkyne, ketone, hydroxyl, carboxylic acid, thioether, sulfoxide, and sulfone.
• the linker can contain aromatic, heteroaromatic, cyclic, bicyclic and tricyclic moieties. Substitution with halogen, such as Cl, F, Br and I can be included in the linker. In the case of fluorine substitution, single or multiple fluorines can be included.
• VLM is a derivative of trans-3-hydroxyproline, where both nitrogen and carboxylic acid in trans-3-hydroxyproline are functionalized as amides.
• the description provides therapeutic compositions comprising an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier.
• the therapeutic compositions modulate protein degradation and/or inhibition in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating disease states or conditions which are modulated through the degraded/inhibited protein.
• the therapeutic compositions as described herein may be used to effectuate the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., cancer (including at least one of SWI/SNF associated cancer, a cancer with a SMARCA4 mutation, a cancer with a SMARCA4-deficiency, or a combination thereof), such as lung cancer (e.g., non- small cell lung cancer).
• a disease e.g., cancer (including at least one of SWI/SNF associated cancer, a cancer with a SMARCA4 mutation, a cancer with a SMARCA4-deficiency, or a combination thereof)
• lung cancer e.g., non- small cell lung cancer.
• the present disclosure provides a method of ubiquitinating/degrading a target protein in a cell.
• the method comprises administering a bifunctional compound as described herein comprising a VLM, preferably linked through a linker moiety, as otherwise described herein, wherein the VLM is coupled to the PTM through a linker to target a protein for degradation.
• a bifunctional compound as described herein comprising a VLM, preferably linked through a linker moiety, as otherwise described herein, wherein the VLM is coupled to the PTM through a linker to target a protein for degradation.
• Degradation of the target protein will occur when the target protein is placed in proximity to the E3 ubiquitin ligase, thus resulting in degradation/inhibition of the effects of the target protein and the control of protein levels.
• the control of protein levels afforded by the present disclosure provides treatment of a disease state or condition, which is modulated through the target protein by lowering the level of that protein in the cells of a patient.
• the description provides methods for treating or ameliorating a disease, disorder or symptom thereof in a subject or a patient, e.g., an animal such as a human, comprising administering to a subject in need thereof a composition comprising an effective amount, e.g., a therapeutically effective amount, of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject.
• the description provides methods for identifying the effects of the degradation of proteins of interest in a biological system using compounds according to the present disclosure.
• Exemplary PROTACs comprise a protein targeting moiety (PTM; darkly shaded rectangle), a ubiquitin ligase binding moiety (ULM; lightly shaded triangle), and optionally a linker moiety (L; black line) coupling or tethering the PTM to the ULM.
• PTM protein targeting moiety
• ULM ubiquitin ligase binding moiety
• L linker moiety
• the E3 ubiquitin ligase is complexed with an E2 ubiquitin-conjugating protein, and either alone or via the E2 protein catalyzes attachment of ubiquitin (dark circles) to a lysine on the target protein via an isopeptide bond.
• the poly-ubiquitinated protein (far right) is then targeted for degradation by the proteosomal machinery of the cell.
• DETAILED DESCRIPTION [0025] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.
• compositions and methods that relate to the surprising and unexpected discovery that an E3 ubiquitin ligase protein (e.g., Von Hippel-Lindau E3 ubiquitin ligase (VHL)) ubiquitinates a target protein once it and the target protein are placed in proximity by a bifunctional or chimeric construct that binds the E3 ubiquitin ligase protein and the target protein.
• E3 ubiquitin ligase protein e.g., Von Hippel-Lindau E3 ubiquitin ligase (VHL)
• VHL Von Hippel-Lindau E3 ubiquitin ligase
• the present disclosure provides such compounds and compositions comprising an E3 ubiquintin ligase binding moiety (“ULM”) coupled to a protein target binding moiety (“PTM”), which result in the ubiquitination of a chosen target protein, which leads to degradation of the target protein by the proteasome (see Figure 1).
• the present disclosure also provides a library of compositions and the use thereof.
• the present disclosure provides compounds which comprise a ligand, e.g., a small molecule ligand (i.e., having a molecular weight of below 2,000, 1,000, 500, or 200 Daltons), which is capable of binding to a ubiquitin ligase, such as VHL.
• the compounds also comprise a moiety that is capable of binding to target protein, in such a way that the target protein is placed in proximity to the ubiquitin ligase to effect degradation (and/or inhibition) of that protein.
• Small molecule can mean, in addition to the above, that the molecule is non-peptidyl, that is, it is not generally considered a peptide, e.g., comprises fewer than 4, 3, or 2 amino acids.
• the PTM, ULM or PROTAC molecule can be a small molecule.
• a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• “or” should be understood to have the same meaning as “and/or” as defined above.
• At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
• one or more of the present compounds described herein are coadministered in combination with at least one additional bioactive agent, especially including an anticancer agent.
• the co-administration of compounds results in synergistic activity and/or therapy, including anticancer activity.
• compound refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and derivatives, including prodrug and/or deuterated forms thereof where applicable, in context.
• Deuterated small molecules contemplated are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium.
• the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds.
• the term also refers, in context to prodrug forms of compounds which have been modified to facilitate the administration and delivery of compounds to a site of activity. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described.
• ubiquitin ligase refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation.
• an E3 ubiquitin ligase protein that alone or in combination with an E2 ubiquitin- conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome.
• E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins.
• the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth.
• Polyubiquitination marks proteins for degradation by the proteasome.
• Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin. Further complicating matters, different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome.
• alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., C 1- 8 means one to eight carbons).
• an alkyl group provided herein is assumed to have one to twelve carbons, one to eight carbons, one to six carbons, or one to four carbons.
• alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t- butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
• Alkyl groups may be optionally substituted as provided herein.
• the alkyl group is a C 1-6 alkyl; in some embodiments, it is a C 1- 4 alkyl.
• a substituent may be optionally substituted with one or more of: halo, cyano, C 1-6 alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, halo(C 1-6 )alkyl, C 1-6 alkoxy, halo(C 1-6 alkoxy), C 1-6 alkylthio, C 1-6 alkylamino, NH 2 , NH(C 1-6 alkyl), N(C 1-6 alkyl) 2 , NH(C 1-6 alkoxy), N(C 1-6 alkoxy) 2 , -C(O)NHC 1-6 alkyl, -C(O)N(C 1-6 alkyl) 2 , -C(O)NH2, -C(O)C 1-6 alkyl, -C(O) 2 C 1-6 alkyl, -NHCO(C 1-6 alkyl), -N(C 1-6 alkyl)CO(C 1-6 alkyl
• each of the above optional substituents are themselves optionally substituted by one or two groups.
• cycloalkyl refers to a C 3-12 cyclic alkyl group, and includes bridged and spirocycles (e.g., adamantine).
• cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, bicyclo[4.1.0]heptanyl, spiro[3.3]heptanyl, and spiro[3.4]octanyl.
• the cycloalkyl group is a C 3-6 cycloalkyl.
• alkenyl refers to C 2-12 alkyl group wherein at least two of the carbon atoms are sp2 hybridized and form a carbon-carbon double bond between them.
• An alkenyl group provided herein may contain more than one carbon-carbon double bond, but one is preferred.
• the alkyl portion of an alkenyl group provided herein may be substituted as provided above.
• the alkenyl group is a C 2-6 alkenyl.
• alkenyl group is a C 2-6 alkenyl.
• alkenyl refers to C 2-12 alkyl group wherein at least two of the carbon atoms are sp hybridized and form a carbon-carbon triple bond between them.
• alkynyl group provided herein may contain more than one carbon-carbon triple bond, but one is preferred.
• the alkyl portion of an alkynyl group provided herein may be substituted as provided above.
• the alkynyl group is a C 2-6 alkynyl.
• halo by itself or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, but preferably fluorine or chlorine.
• halo(C 1-x alkyl) refers to an alkyl that has 1-x carbon atoms and that is substituted with one or more (e.g.1, 2, 3, 4, 5, or 6) halo groups.
• the term includes an alkyl group having 1-6 carbon atoms that is substituted with one or more halo groups.
• halo(C 1 -C 6 alkyl) include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, and 2,2,2-trifluoroethyl.
• halo(C 1- x alkoxy) refers to an alkoxy group that has 1-x carbon atoms and that is substituted with one or more (e.g. 1, 2, 3, 4, 5, or 6) halo groups.
• the term includes an alkoxy group having 1-6 carbon atoms that is substituted with one or more halo groups.
• halo(C 1 -C 6 alkyl) include fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, and 2,2,2-trifluoroethoxy.
• heteroalkyl refers to a straight- or branched-chain alkyl group, e.g. having from 2 to 14 carbons, such as 2 to 10 carbons in the chain, one or more of which has been replaced by a heteroatom selected from S, O, P and N.
• exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl amides, alkyl sulfides, and the like.
• the group may be a terminal group or a bridging group.
• reference to the normal chain when used in the context of a bridging group refers to the direct chain of atoms linking the two terminal positions of the bridging group.
• aryl refers to a single all carbon aromatic ring or a multiple condensed all carbon ring system wherein at least one of the rings is aromatic.
• an aryl group has 6 to 12 carbon atoms.
• Aryl includes a phenyl radical.
• Aryl also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) having about 9 to 12 carbon atoms in which at least one ring is aromatic and wherein the other rings may be aromatic or not aromatic.
• Such multiple condensed ring systems are optionally substituted with one or more (e.g., 1, 2 or 3) oxo groups on any carbocycle portion of the multiple condensed ring system.
• the rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements.
• aryl groups include, but are not limited to, phenyl, indenyl, naphthyl, 1, 2, 3, 4-tetrahydronaphthyl, and the like.
• heteroaryl refers to a single aromatic ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; “heteroaryl” also includes multiple condensed ring systems that have at least one such aromatic ring, which multiple condensed ring systems are further described below.
• heteroaryl includes single aromatic rings of from about 1 to 6 carbon atoms and about 1- 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. The sulfur and nitrogen atoms may also be present in an oxidized form provided the ring is aromatic.
• heteroaryl ring systems include but are not limited to pyridyl, pyrimidinyl, oxazolyl or furyl.
• “Heteroaryl” also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a heteroaryl group, as defined above, is condensed with one or more rings selected from heteroaryls (to form for example a naphthyridinyl such as 1,8-naphthyridinyl), heterocycles, (to form for example a 1, 2, 3, 4-tetrahydronaphthyridinyl such as 1,2,3,4-tetrahydro-1,8- naphthyridinyl), carbocycles (to form for example 5,6,7,8-tetrahydroquinolyl) and aryls (to form for example indazolyl) to form the multiple condensed ring system.
• heteroaryls to form for example a naphthyridiny
• a heteroaryl (a single aromatic ring or multiple condensed ring system) has about 1-20 carbon atoms and about 1-6 heteroatoms within the heteroaryl ring.
• a heteroaryl (a single aromatic ring or multiple condensed ring system) can also have about 5 to 12 or about 5 to 10 members within the heteroaryl ring.
• Multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the condensed ring.
• the rings of a multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements.
• the individual rings of the multiple condensed ring system may be connected in any order relative to one another.
• the point of attachment of a multiple condensed ring system (as defined above for a heteroaryl) can be at any position of the multiple condensed ring system including a heteroaryl, heterocycle, aryl or carbocycle portion of the multiple condensed ring system.
• the point of attachment for a heteroaryl or heteroaryl multiple condensed ring system can be at any suitable atom of the heteroaryl or heteroaryl multiple condensed ring system including a carbon atom and a heteroatom (e.g., a nitrogen).
• heteroaryls include but are not limited to pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, quinazolyl, 5,6,7,8- tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazole and 3b,4,
• heteroaryl refers to a single aromatic ring containing at least one heteroatom.
• the term includes 5- membered and 6-membered monocyclic aromatic rings that include one or more heteroatoms.
• Non-limiting examples of heteroaryl include but are not limited to pyridyl, furyl, thiazole, pyrimidine, oxazole, and thiadiazole.
• heterocyclyl or “heterocycle” as used herein refers to a single saturated or partially unsaturated ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems that have at least one such saturated or partially unsaturated ring, which multiple condensed ring systems are further described below.
• the term includes single saturated or partially unsaturated rings (e.g., 3, 4, 5, 6 or 7-membered rings) from about 1 to 6 carbon atoms and from about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring.
• the ring may be substituted with one or more (e.g., 1, 2 or 3) oxo groups and the sulfur and nitrogen atoms may also be present in their oxidized forms.
• exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl.
• heterocycle also includes multiple condensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings) wherein a single heterocycle ring (as defined above) can be condensed with one or more groups selected from heterocycles (to form for example a 1,8-decahydronapthyridinyl ), carbocycles (to form for example a decahydroquinolyl) and aryls to form the multiple condensed ring system.
• a heterocycle a single saturated or single partially unsaturated ring or multiple condensed ring system
• Such multiple condensed ring systems may be optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocycle or heterocycle portions of the multiple condensed ring.
• the rings of the multiple condensed ring system can be connected to each other via fused, spiro and bridged bonds when allowed by valency requirements. It is to be understood that the individual rings of the multiple condensed ring system may be connected in any order relative to one another.
• a heterocycle (a single saturated or single partially unsaturated ring or multiple condensed ring system) has about 3-20 atoms including about 1-6 heteroatoms within the heterocycle ring system.
• the point of attachment of a multiple condensed ring system can be at any position of the multiple condensed ring system including a heterocycle, aryl and carbocycle portion of the ring. It is also to be understood that the point of attachment for a heterocycle or heterocycle multiple condensed ring system can be at any suitable atom of the heterocycle or heterocycle multiple condensed ring system including a carbon atom and a heteroatom (e.g., a nitrogen).
• the term heterocycle includes a C 2-20 heterocycle.
• heterocycle includes a C 2-7 heterocycle.
• the term heterocycle includes a C 2-5 heterocycle.
• heterocycle includes a C 2-4 heterocycle.
• exemplary heterocycles include, but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4- tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-ox
• heterocycle refers to a monocyclic, saturated or partially unsaturated, 3-8 membered ring having at least one heteroatom.
• the term includes a monocyclic, saturated or partially unsaturated, 4, 5, 6, or 7 membered ring having at least one heteroatom.
• Non-limiting examples of heterocycle include aziridine, azetidine, pyrrolidine, piperidine, piperidine, piperazine, oxirane, morpholine, and thiomorpholine.
• the term “9- or 10-membered heterobicycle” as used herein refers to a partially unsaturated or aromatic fused bicyclic ring system having at least one heteroatom.
• the term 9- or 10-membered heterobicycle includes a bicyclic ring system having a benzo ring fused to a 5-membered or 6-membered saturated, partially unsaturated, or aromatic ring that contains one or more heteroatoms.
• heteroatom is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). The nitrogen and sulfur can be in an oxidized form when feasible.
• chiral refers to molecules which have the property of non- superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
• stereoisomers refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
• a crossed line “ ” indicates a mixture of E and Z stereoisomers.
• a wavy line “ ” or a dashed line “----” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.
• “Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another.
• Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can separate under high resolution analytical procedures such as electrophoresis and chromatography. [0059] "Enantiomers" refer to two stereoisomers of a compound which are non- superimposable mirror images of one another. [0060] Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.
• the compounds of the invention can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention.
• Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s).
• d and l or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
• a compound prefixed with (+) or d is dextrorotatory.
• these stereoisomers are identical except that they are mirror images of one another.
• a specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
• racemic mixture and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• a bond in a compound formula herein is drawn in a non-stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities.
• a bond in a compound formula herein is drawn in a defined stereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge)
• the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer depicted unless otherwise noted.
• the compound may be at least 51% the absolute stereoisomer depicted.
• the compound may be at least 80% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 97% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 98% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% the absolute stereoisomer depicted. [0062] As used herein, the term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier.
• proton tautomers also known as prototropic tautomers
• proton tautomers include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations.
• Valence tautomers include interconversions by reorganization of some of the bonding electrons.
• solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
• hydrate refers to the complex where the solvent molecule is water.
• protecting group refers to a substituent that is commonly employed to block or protect a particular functional group on a compound.
• an “amino-protecting group” is a substituent attached to an amino group that blocks or protects the amino functionality in the compound.
• Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9- fluorenylmethylenoxycarbonyl (Fmoc).
• a "hydroxy-protecting group” refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality.
• Suitable protecting groups include acetyl and silyl.
• a "carboxy-protecting group” refers to a substituent of the carboxy group that blocks or protects the carboxy functionality.
• Common carboxy-protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2- (trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2- (diphenylphosphino)-ethyl, nitroethyl and the like.
• P.G.M For a general description of protecting groups and their use, see P.G.M.
• the term "pharmaceutically acceptable salts” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
• base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
• salts derived from pharmaceutically-acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like.
• Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine,
• acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
• pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
• salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19).
• Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
• the neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
• the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
• the present invention provides compounds which are in a prodrug form.
• prodrug refers to those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
• prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
• Prodrugs of the invention include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues, is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of a compound of the present invention.
• the amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, methyl- alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, methionine sulfone and tert-butylglycine.
• prodrugs are also encompassed.
• a free carboxyl group of a compound of the invention can be derivatized as an amide or alkyl ester.
• compounds of this invention comprising free hydroxy groups can be derivatized as prodrugs by converting the hydroxy group into a group such as, but not limited to, a phosphate ester, hemisuccinate, dimethylaminoacetate, or phosphoryloxymethyloxycarbonyl group, as outlined in Fleisher, D. et al., (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs Advanced Drug Delivery Reviews, 19:115.
• Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups.
• Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group can be an alkyl ester optionally substituted with groups including, but not limited to, ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed.
• Prodrugs of this type are described in J. Med. Chem., (1996), 39:10.
• More specific examples include replacement of the hydrogen atom of the alcohol group with a group such as (C 1-6 )alkanoyloxymethyl, 1-((C 1-6 )alkanoyloxy)ethyl, 1- methyl-1-((C 1-6 )alkanoyloxy)ethyl, (C 1-6 )alkoxycarbonyloxymethyl, N-(C 1- 6)alkoxycarbonylaminomethyl, succinoyl, (C 1-6 )alkanoyl, alpha-amino(C 1- 4)alkanoyl, arylacyl and alpha-aminoacyl, or alpha-aminoacyl-alpha-aminoacyl, where each alpha-aminoacyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH) 2 , -P(O)(O(C 1- 6)alkyl) 2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the
• prodrug derivatives see, for example, a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol.42, p.309-396, edited by K. Widder, et al. (Academic Press, 1985); b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and Application of Prodrugs," by H. Bundgaard p.113-191 (1991); c) H. Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992); d) H.
• a "metabolite” refers to a product produced through metabolism in the body of a specified compound or salt thereof. Such products can result for example from the oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound.
• Metabolite products typically are identified by preparing a radiolabelled (e.g., 14 C or 3 H) isotope of a compound of the invention, administering it parenterally in a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples.
• a detectable dose e.g., greater than about 0.5 mg/kg
• the metabolite structures are determined in conventional fashion, e.g., by MS, LC/MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well known to those skilled in the art. The metabolite products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the compounds of the invention.
• patient or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided.
• patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc.
• patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.
• effective is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application.
• the description provides compounds comprising an E3 ubiquitin ligase binding moiety (“ULM”) that is a Von Hippel-Lindae E3 ubiquitin ligase (VHL) binding moiety (VLM).
• ULM E3 ubiquitin ligase binding moiety
• VHL Von Hippel-Lindae E3 ubiquitin ligase binding moiety
• the ULM is coupled to a target protein binding moiety (PTM) via a chemical linker (L) according to the structure: (A) PTM-L-ULM wherein L is a bond or a chemical linker group, ULM is a E3 ubiquitin ligase binding moiety, and PTM is a target protein binding moiety.
• the present disclosure provides bifunctional or multifunctional compounds (e.g., PROTACs) useful for regulating protein activity by inducing the degradation of a target protein.
• the compound comprises a VLM coupled, e.g., linked covalently, directly or indirectly, to a moiety that binds a target protein (i.e., a protein targeting moiety or a “PTM”).
• the VLM and PTM are joined or coupled via a chemical linker (L).
• the VLM binds VHL, and the PTM recognizes a target protein and the interaction of the respective moieties with their targets facilitates the degradation of the target protein by placing the target protein in proximity to the ubiquitin ligase protein.
• An exemplary bifunctional compound can be depicted as: PTM—VLM.
• the bifunctional compound further comprises a chemical linker (“L”).
• the bifunctional compound can be depicted as: PTM—L—VLM, wherein the PTM is a protein/polypeptide targeting moiety, the L is a chemical linker, and the VLM is a VHL binding moiety.
• the ULM (e.g., VLM) shows activity or binds to the E3 ubiquitin ligase (e.g., VHL) with an IC 50 of less than about 200 ⁇ M.
• the IC 50 can be determined according to any method known in the art, e.g., a fluorescent polarization assay.
• the bifunctional compounds described herein demonstrate an activity with an IC 50 of less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 mM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 ⁇ M, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 nM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 pM.
• the ULMs are identical.
• the compound comprising a plurality of ULMs (e.g., ULM, ULM’, etc.), at least one PTM coupled to a ULM directly or via a chemical linker (L) or both.
• the compound comprising a plurality of ULMs further comprises multiple PTMs.
• the PTMs are the same or, optionally, different.
• the respective PTMs may bind the same protein target or bind specifically to a different protein target.
• the compound may comprise a plurality of ULMs and/or a plurality of ULM’s.
• the compound comprising at least two different ULMs, a plurality of ULMs, and/or a plurality of ULM’s further comprises at least one PTM coupled to a ULM or a ULM’ directly or via a chemical linker or both.
• a compound comprising at least two different ULMs can further comprise multiple PTMs.
• the PTMs are the same or, optionally, different.
• the respective PTMs may bind the same protein target or bind specifically to a different protein target.
• the description provides the compounds as described herein including their enantiomers, diastereomers, solvates and polymorphs, including pharmaceutically acceptable salt forms thereof, e.g., acid and base salt forms.
• Exemplary VLMs [0085]
• the compounds as described herein include a means for binding an E3 ubiquitin ligase, e.g., Von Hippel-Lindau E3 ubiquitin ligase.
• the ULM is VLM and comprises a chemical structure selected from the group ULM-a: wherein: a dashed line indicates the attachment of at least one PTM, another ULM or VLM (i.e., VLM’), or a chemical linker moiety coupling at least one PTM, a ULM’ or a VLM’ to the other end of the linker;
• R Y3 , R Y4 of Formula ULM-a are each independently selected from the group of H, linear or branched C 1-6 alkyl, optionally substituted by 1 or more halo, optionally substituted C 1-6 alkoxyl (e.g., optionally substituted by 0-3 R P groups);
• R P of Formula ULM-a is 0, 1, 2, or 3 groups,
• R P is modified to form a prodrug, including by an ester or ether linkage.
• T is selected from the group of an optionally substituted alkyl, –(CH 2 ) n - group, wherein each one of the methylene groups is optionally substituted with one or two substituents selected from the group of halogen, methyl, optionally substituted alkoxy, a linear or branched C 1 -C 6 alkyl group optionally substituted by 1 or more halogen, C(O) NR 1 R 1a , or NR 1 R 1a or R 1 and R 1a are joined to form an optionally substituted heterocyclyl, or -OH groups or an amino acid side chain optionally substituted; and n is 0 to 6, often 0, 1, 2, or 3, preferably 0 or 1.
• W 4 of Formula ULM-a is wherein R 14a, R 14b, are each independently selected from the group of H, haloalkyl (e.g., fluoroalkyl), optionally substituted alkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted amide, optionally substituted alkyl-amide, optionally substituted alkyl-cyano, optionally substituted alkyl-phosphate, optionally substituted heteroalkyl, optionally substituted alkyl-heterocycloalkyl, optionally substituted alkoxy- heterocycloalkyl, COR26, alkyl-COR26, CONR27aR27b, NHCOR26, or NHCH 3 COR26; and the other of R 14a and R 14b is H; or R 14a, R 14b, together with the carbon atom to which they are attached, form an optionally substituted 3 to 5
• R 14a and R 14b is H;
• W 5 of Formula ULM-a is selected from the group of an optionally substituted phenyl, an optionally substituted napthyl or an optionally substituted 5-10 membered heteroaryl
• R15 of Formula ULM-a is selected from the group of H, halogen, CN, C ⁇ CH, OH, NO 2 , N R 14a R 14b , OR 14a , CONR 14a R 14b , NR 14a COR 14b , SO 2 NR 14a R 14b , NR 14a SO 2 R 14b , optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted haloalkoxy, optionally subsituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, or optionally substituted heterocyclyl;
• W 4 substituents for use in the present disclosure also include specifically (and without limitation to the specific compound disclosed) the W 4 substituents for use in the present disclosure also include specifically (and without limitation to
• W 4 substituents may be used in conjunction with any number of W 3 substituents which are also disclosed herein.
• ULM-a is optionally substituted by 0-3 R P groups in the pyrrolidine moiety.
• the W 3 , W 4 of Formula ULM-a can independently be covalently coupled to a linker which is attached one or more PTM groups.
• ULM is VHL and is represented by the structure: wherein: W 3 of Formula ULM-b is selected from the group of an optionally substituted aryl, optionally substituted heteroaryl, or R 9 and R 10 of Formula ULM-b are independently hydrogen, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted hydroxyalkyl, optionally substituted heteroaryl, or haloalkyl, or R9, R10, and the carbon atom to which they are attached form an optionally substituted cycloalkyl; R 11 of Formula ULM-b is selected from the group of an optionally substituted heterocyclyl, optionally substituted alkoxy, optionally substituted heteroaryl, optionally substituted aryl, R12 of Fo r
• fluoroalkyl optionally substituted alkyl, optionally substitute alkoxy, aminomethyl, alkylaminomethyl, alkoxymethyl, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted amide, optionally substituted alkyl-amide, optionally substituted alkyl-cyano, optionally substituted alkyl-phosphate, optionally substituted heteroalkyl, optionally substituted alkyl-heterocycloalkyl, optionally substituted alkoxy-heterocycloalkyl, COR26, alkyl-COR26, CONR27aR27b, CH 2 NHCOR26, or (CH 2 )N(CH 3 )COR26; and the other of R14a and R14b is H; or R14a, R14b, together with the carbon atom to which they are attached, form an optionally substituted 3 to 6 membered cycloalkyl, heterocycloalky, spirocycloalkyl or spir
• R 15 of Formula ULM-b is wherein R 17 is H, halo, optionally substituted C 3-6 cycloalkyl, optionally substituted C 1-6 alkyl, optionally substituted C 1- 6alkenyl, and C 1-6 haloalkyl; and Xa is S or O.
• R 17 of Formula ULM-b is selected from the group methyl, ethyl, isopropyl, and cyclopropyl.
• R15 of Formula ULM-b is selected from the group consisting of:
• R 11 of Formula ULM-b is selected from the group consisting of:
• R 14a, R 14b of Formula ULM-b are each independently selected from the group of H, optionally substituted haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted amide, optionally substituted alkyl-amide, optionally substituted alkyl-cyano, optionally substituted alkyl-phosphate, optionally substituted heteraolkyl, optionally substituted alkyl-heterocycloalkyl, optionally substituted alkoxy- heterocycloalkyl, COR 26 , alkyl-COR 26 , CH 2 OR 30 , CH 2 NHR 30, CH 2 NCH 3 R 30 , CONR 27a R 27b , CH 2 CONR 27a R 27b , CH 2 NHCOR 26 , or CH 2 NCH 3 COR 26 ; and the other of R 14a and R 14b is H;
• R 15 of Formula ULM-b is selected from H, halogen, CN, C ⁇ CH, OH, NO 2 , NR27aR27b, OR27a, CONR27aR27b, NR27aCOR27b, SO 2 NR 27a R 27b , NR 27a SO 2 R 27b , optionally substituted alkyl, optionally substituted haloalkyl (e.g.
• optionally substituted fluoroalkyl optionally substituted haloalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, or optionally substituted heterocyclyl
• optional substitution of the said aryl, heteroaryl, cycloalkyl and heterocycloalkyl includes CH 2 OR 30 , CH 2 NHR 30, CH 2 NCH 3 R 30 , CONR 27a R 27b , CH 2 CONR 27a R 27b , CH 2 NHCOR26, CH 2 NCH 3 COR26 or wherein R26, R27, R30 and R14a are as described above.
• R 14a, R 14b of Formula ULM-b are each independently selected from the group of H, optionally substituted haloalkyl, optionally substituted alkyl, CH 2 OR30, CH 2 NHR30, CH 2 NCH 3 R30, CONR27aR27b, CH 2 CONR27aR27b, CH 2 NHCOR 26 , or CH 2 NCH 3 COR 26 ; and the other of R 14a and R 14b is H; or R 14a, R 14b, together with the carbon atom to which they are attached, form an optionally substituted 3- to 6- membered spirocycloalkyl or spiroheterocyclyl, wherein the spiroheterocyclyl is not epoxide or aziridine, the said spirocycloalkyl or spiroheterocycloalkyl itself being optionally substituted with an alkyl, a haloalkyl, or -COR
• R14a of Formula ULM-f is H, haloalkyl, optionally substituted alkyl, methyl, fluoromethyl, hydroxymethyl, ethyl, isopropyl, or cyclopropyl;
• R9 of Formula ULM-f is H;
• R10 of Formula ULM-f is H, ethyl, isopropyl, tert-butyl, sec-butyl, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
• p of Formula ULM-f is 0, 1, 2, 3, or 4;
• each R 18 of Formula ULM-f is independently halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, haloalkoxy or a linker;
• the ULM is selected from the following structures:
• the phenyl ring in ULM-a1 through ULM -a15, ULM -b1 through ULM-b12, ULM-c1 through ULM-c15 and ULM-d1 through ULM-d9 is optionally substituted with fluorine, lower alkyl and alkoxy groups, and wherein the dashed line indicates the site of attachment of at least one PTM, another ULM (ULM’) or a chemical linker moiety coupling at least one PTM or a ULM’ or both to ULM-a.
• the phenyl ring in ULM-a1 through ULM-a15, ULM-b1 through ULM-b12, ULM-c1 through ULM-c15 and ULM-d1 through ULM-d9 can be functionalized as the ester to make it a part of the prodrug.
• the hydroxyl group on the pyrrolidine ring of ULM-a1 through ULM-a15, ULM-b1 through ULM-b12, ULM-c1 through ULM-c15 and ULM-d1 through ULM-d9, respectively, comprises an ester-linked prodrug moiety.
• the ULM and where present, ULM’ are each independently a group according to the chemical structure: ' or a pharmaceutically acceptable salt thereof, wherein: R 1’ of ULM-g is an optionally substituted C 1 -C 6 alkyl group, an optionally substituted - (CH 2 )nOH, an optionally substituted -(CH 2 )nSH, an optionally substituted (CH 2 )n-O-(C 1- C 6 )alkyl group, an optionally substituted (CH 2 ) n -WCOCW-(C 0 -C 6 )alkyl group containing an epoxide moiety WCOCW where each W is independently H or a C1-C3 alkyl group, an optionally substituted -(CH 2 ) n COOH, an optionally substituted -(CH 2 ) n C(0)-(Ci-C 6 alkyl), an optionally substituted -(CH 2 )
• Rs of ULM-g is a C1-C6 alkyl group, an optionally substituted aryl, heteroaryl or heterocyclyl group or a -(CH 2 ) m N(R")2 group;
• R 3 of ULM-g is an optionally substituted alkyl, an optionally substituted -((LfcV
• R IN and R2 N of ULM-g are each independently H, C1-C6 alkyl which is optionally substituted with one or two hydroxyl groups and up to three halogen groups or an optionally substituted -(CH 2 ) n -Aryl, -(QLVHeteroaryl or -(CH 2 ) n -Heterocyclyl group;
• the ULM, and when present, ULM’ are each independently according to the chemical structure: 1 ' wherein: any one or more of R 1’ , R 2’ and R 3’ of ULM-I are optionally modified to bind a linker group to which is further covalently bonded to the PTM group when PTM is not ULM’, or when PTM is ULM’, any one or more of R 1’ , R 2’ , R 3’ of each of ULM and ULM’ are optionally modified to be covalently bonded to each other directly or through a linker group, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof.
• R 1’ of ULM-g through ULM-i is preferably a hydroxyl group or a group which may be metabolized to a hydroxyl or carboxylic group, such that the compound represents a prodrug form of an active compound.
• Exemplary preferred R 1’ groups include, for example, -(CH 2 ) n OH, (CH 2 ) n -O-(C 1 -C 6 )alkyl group, - (CH 2 )nCOOH, -(CH 2 O)nH, an optionally substituted -(CH 2 )nOC(O)-(C 1 -C 6 alkyl), or an optionally substituted -(CH 2 )nC(O)-O-(C 1 -C 6 alkyl), wherein n is 0 or 1.
• R 1’ is or contains a carboxylic acid group, a hydroxyl group or an amine group, the hydroxyl group, carboxylic acid group or amine (each of which may be optionally substituted), may be further chemically modified to provide a covalent link to a linker group to which the PTM group (including a ULM’ group) is bonded;
• R 2’ of ULM-g through ULM-i is preferably an optionally substituted -NH-T-Aryl, an optionally substituted –N(CH 3 )-T-Aryl, an optionally substituted -NH-T-Heteroaryl group, an optionally substituted –N(CH 3 )-T-Heteroaryl, an optionally substituted -
• T may also be a –(CH 2 O)n- group, a –(OCH 2 )n- group, a –(CH 2 CH 2 O)n- group, a – (OCH 2 CH 2 )n- group, all of which groups are optionally substituted.
• Preferred Aryl groups for R 2’ of ULM-g through ULM-i include optionally substituted phenyl or naphthyl groups, preferably phenyl groups, wherein the phenyl or naphthyl group is connected to a PTM (including a ULM’ group) with a linker group and/or optionally substituted with a halogen (preferably F or Cl), an amine, monoalkyl- or dialkyl amine (preferably, dimethylamine), F, Cl, OH, COOH, C 1 -C 6 alkyl, preferably CH 3 , CF 3 , OMe, OCF 3 , NO 2 , or CN group (each of which may be substituted in ortho-, meta- and/or para- positions of the phenyl ring, preferably para-), an optionally substituted phenyl group (the phenyl group itself is optionally connected to a PTM group, including a ULM’, with a linker group), and/or optionally substituted
• R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl);
• R SS of ULM-g through ULM-i is H, CN, NO 2 , halo (preferably F or Cl), optionally substituted C 1 -C 6 alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O-(C 1 -C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted -C(O)(C 1- C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);
• R URE of ULM-g through ULM-i is
• hrough ULM-i is a group, where R PRO and n of ULM-g through ULM-i are the same as above.
• Preferred heteroaryl groups for R 2’ of ULM-g through ULM-i include an optionally substituted quinoline (which may be attached to the pharmacophore or substituted on any carbon atom within the quinoline ring), an optionally substituted indole, an optionally substituted indolizine, an optionally substituted azaindolizine, an optionally substituted benzofuran, including an optionally substituted benzofuran, an optionally substituted isoxazole, an optionally substituted thiazole, an optionally substituted isothiazole, an optionally substituted thiophene, an optionally substituted pyridine (2-, 3, or 4-pyridine), an optionally substituted imidazole, an optionally substituted pyrrole, an optionally substituted diazole, an optionally substituted triazo
• CF 3 optionally substituted O(C 1 -C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group –C ⁇ C-R a where R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl), each of which groups may be optionally connected to a PTM group (including a ULM’ group) via a linker group.
• O(C 1 -C 6 alkyl) preferably substituted with one or two hydroxyl groups or up to three halo groups
• R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl)
• PTM group including a ULM’ group
• Preferred heterocyclylheterocyclyl groups for R 2’ of ULM-g through ULM-i include tetrahydrofuran, tetrahydrothiene, tetrahydroquinoline, piperidine, piperazine, pyrrollidine, morpholine, oxane or thiane, each of which groups may be optionally substituted, or a group according to the chemical structure: preferably, group, wherein: R PRO of ULM-g through ULM-i is H, optionally substituted C 1 -C 6 alkyl or an optionally substituted aryl, heteroaryl or heterocyclyl group; R PRO1 and R PRO2 of ULM-g through ULM-i are each independently H, an optionally subsituted C 1 -C 3 alkyl group or together form a keto group and each n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (often 0 or 1), each of which groups may be optionally connected to
• R 2’ substituents of ULM-g through ULM-i also include specifically (and without limitation to the specific compound disclosed) the R 2’ substituents which are found in the identified compounds disclosed herein (which includes the specific compounds which are disclosed in the present specification, and the figures which are attached hereto). Each of these R 2’ substituents may be used in conjunction with any number of R 3’ substituents which are also disclosed herein.
• R 3’ of ULM-g through ULM-i is preferably an optionally substituted -NH-T-Aryl, an optionally substituted –N(C 1 -C 3 alkyl)-T-Aryl, an optionally substituted -NH-T-Heteroaryl group, an optionally substituted –N(C 1 -C 3 alkyl)-T-Heteroaryl, an optionally substituted -NH-T- Heterocyclyl, or an optionally substituted –N(C 1 -C 3 alkyl)-T-Heterocyclyl, wherein T is an optionally substituted –(CH 2 )n- group, wherein each one of the methylene groups may be optionally substituted with one or two substituents, preferably selected from halogen, a C 1 -C 3 alkyl group or the sidechain of an amino acid as otherwise described herein, preferably methyl, which may be optionally substituted; and n is 0 to 6, often 0, 1,
• T may also be a –(CH 2 O)n- group, a –(OCH 2 )n- group, a –(CH 2 CH 2 O)n- group, a – (OCH 2 CH 2 ) n - group, each of which groups is optionally substituted.
• Preferred aryl groups for R 3’ of ULM-g through ULM-i include optionally substituted phenyl or naphthyl groups, preferably phenyl groups, wherein the phenyl or naphthyl group is optionally connected to a PTM group (including a ULM’ group) via a linker group and/or optionally substituted with a halogen (preferably F or Cl), an amine, monoalkyl- or dialkyl amine (preferably, dimethylamine), an amido group (preferably a –(CH 2 )m-NR1C(O)R2 group where m, R1 and R2 are the same as above), a halo (often F or Cl), OH, CH 3 , CF 3 , OMe, OCF 3 , NO 2 , ,CN or a S(O) 2 R S group (R S is a a C 1 -C 6 alkyl group, an optionally substituted aryl, heteroaryl or heterocycl
• said substituent phenyl group is an optionally substituted phenyl group (i.e., the substituent phenyl group itself is preferably substituted with at least one of F, Cl, OH, SH, COOH, CH 3 , CF 3 , OMe, O CF 3 , NO 2 , CN or a linker group to which is attached a PTM group (including a ULM’ group), wherein the substitution occurs in ortho-, meta- and/or para- positions of the phenyl ring, preferably para-), a naphthyl group, which may be optionally substituted including as described above, an optionally substituted heteroaryl (preferably an optionally substituted isoxazole including a methylsubstituted isoxazole, an optionally substituted oxazole including a methylsubstituted oxazole, an optionally substituted thiazole including a methyl substituted thiazole, an optionally substituted pyrrole including a
• PTM group including a ULM’ group
• Preferred Heteroaryl groups for R 3’ of ULM-g through ULM-i include an optionally substituted quinoline (which may be attached to the pharmacophore or substituted on any carbon atom within the quinoline ring), an optionally substituted indole (including dihydroindole), an optionally substituted indolizine, an optionally substituted azaindolizine (2, 3 or 4-azaindolizine) an optionally substituted benzimidazole, benzodiazole, benzoxofuran, an optionally substituted imidazole, an optionally substituted isoxazole, an optionally substituted oxazole (preferably methyl substituted), an optionally substituted diazole, an optionally substituted triazole, a tetrazole, an optionally substituted
• R SS of ULM-g through ULM-i is H, CN, NO 2 , halo (preferably F or Cl), optionally substituted C 1 -C 6 alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O-(C 1 -C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted -C(O)(C 1 - C6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups); R URE of ULM-g through ULM-i is H
• CF 3 optionally substituted O(C 1 -C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted acetylenic group –C ⁇ C-R a where R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl).
• R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl).
• Each of said heteroaryl groups may be optionally connected to a PTM group (including a ULM’ group) via a linker group.
• Preferred heterocyclyl groups for R 3’ of ULM-g through ULM-i include tetrahydroquinoline, piperidine, piperazine, pyrrollidine, morpholine, tetrahydrofuran, tetrahydrothiophene, oxane and thiane, each of which groups may be optionally substituted or a group according to the chemical structure: wherein: R PRO of ULM-g through ULM-i is H, optionally substituted C 1 -C 6 alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclyl group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene
• R 3’ substituents of ULM-g through ULM-i also include specifically (and without limitation to the specific compound disclosed) the R 3’ substituents which are found in the identified compounds disclosed herein (which includes the specific compounds which are disclosed in the present specification, and the figures which are attached hereto). Each of these R 3’ substituents may be used in conjunction with any number of R 2’ substituents, which are also disclosed herein.
• S c of ULM-g through ULM-i is CHR SS , NR URE , or O;
• R HET of ULM-g through ULM-i is H, CN, NO 2 , halo (preferably Cl or F), optionally substituted C 1 -C 6 alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g.
• R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl);
• R PRO of ULM-g through ULM-i is H, optionally substituted C 1 -C 6 alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclyl group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine
• R SS of ULM-g through ULM-i is H, CN, NO 2 , halo (preferably F or Cl), optionally substituted C 1 -C 6 alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O-(C 1 -C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted -C(O)(C 1 - C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups); R URE of ULM-g through ULM-i is H
• R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl);
• R PRO of ULM-g through ULM-i is H, optionally substituted C 1 -C 6 alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclyl group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine
• R 3 ’ of ULM-g through ULM-i is –(CH 2 )n-Aryl, – (CH 2 CH 2 O) n -Aryl, –(CH 2 ) n -HET or –(CH 2 CH 2 O) n -HET, wherein: said Aryl of ULM-g through ULM-i is phenyl which is optionally substituted with one or two substitutents, wherein said substituent(s) is preferably selected from -(CH 2 ) n OH, C 1 -C 6 alkyl which itself is further optionally substituted with CN, halo (up to three halo groups), OH, -(CH 2 )nO(C 1 -C 6 )alkyl, amine, mono- or di-(C 1 -C 6 alkyl) amine wherein the alkyl group on the amine is optionally substituted with 1 or 2 hydroxyl groups or up to three halo (preferably
• S c of ULM-g through ULM-i is CHR SS , NR URE , or O;
• R HET of ULM-g through ULM-i is H, CN, NO 2 , halo (preferably Cl or F), optionally substituted C 1 -C 6 alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups (e.g.
• R PRO of ULM-g through ULM-i is H, optionally substituted C 1 -C 6 alkyl or an optionally substituted aryl (phenyl or napthyl), heteroaryl or heterocyclyl group selected from the group consisting of oxazole, isoxazole, thiazole, isothiazole, imidazole, diazole, oximidazole, pyrrole, pyrollidine, furan, dihydrofuran, tetrahydrofuran, thiene, dihydrothiene, tetrahydrothiene, pyridine, piperidine, piperazine, morph
• R a is H or a C 1 -C 6 alkyl group (preferably C 1 -C 3 alkyl);
• R SS of ULM-g through ULM-i is H, CN, NO 2 , halo (preferably F or Cl), optionally substituted C 1 -C 6 alkyl (preferably substituted with one or two hydroxyl groups or up to three halo groups), optionally substituted O-(C 1 -C 6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups) or an optionally substituted -C(O)(C 1- C6 alkyl) (preferably substituted with one or two hydroxyl groups or up to three halo groups);
• R URE of ULM-g through ULM-i is
• R PRO of ULM-g through ULM-i is H, optionally substituted C 1 -C 6 alkyl or an optionally substituted aryl, heteroaryl or heterocyclyl group;
• R PRO1 and R PRO2 of ULM-g through ULM-i are each independently H, an optionally subsituted C 1 -C 3 alkyl group or together form a keto group;
• each m’ of ULM-g through ULM-i is independently 0 or 1; and each n of ULM-g through ULM-i is independently 0, 1, 2, 3, 4, 5, or 6 (preferably 0 or 1), wherein each of said compounds, preferably on said Aryl or H
• preferred compounds include those according to the chemical structure: wherein: R 1’ of ULM-i is OH or a group which is metabolized in a patient or subject to OH; R 2’ of ULM-i is a —NH-CH 2 -Aryl-HET (preferably, a phenyl linked directly to a methyl substituted thiazole); R 3’ of ULM-i is a –CHR CR3’ -NH-C(O)-R 3P1 group or a –CHR CR3’ -R 3P2 group; R CR3’ of ULM-i is a C 1- C4 alkyl group, preferably methyl, isopropyl or tert-butyl; R 3P1 of ULM-i is C 1 -C 3 alkyl (preferably methyl), an optionally substituted oxetane group (preferably methyl substituted, a –(CH 2 ) n OCH 3 group where n
• J is O
• R 7 is H
• each R 14 is H
• o is 0.
• J is O
• R7 is H
• each R14 is H
• R 15 is optionally substituted heteroaryl
• o is 0.
• R11 is optionally substituted heterocyclyl or [00132]
• M is nd R11 is each R 18 is independently H, halo, optionally substituted alkoxy, cyano, optionally substituted alkyl, haloalkyl, or haloalkoxy; and p is 0, 1, 2, 3, or 4.
• each R 14 is independently substituted with at least one of H, hydroxyl, halo, amine, amide, alkoxy, alkyl, haloalkyl, or heterocyclic.
• R15 of ULM-j is a group according to O CN, or a haloalkyl, and each R 18 is independently H, halo, optionally substituted alkoxy, cyano, aminoalkyl, amidoalkyl, optionally substituted alkyl, haloalkyl, or haloalkoxy; and p is 0, 1, 2, 3, or 4.
• R 16 of ULM-k is defined is as above for ULM-j; and R 17 of ULM-k is H, halo, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, and haloalkyl.
• R17 of ULM-k is alkyl (e.g., methyl) or cycloalkyl (e.g., cyclopropyl).
• R11 of ULM-j or ULM-k is selected from the group consisting of: O
• ULM (or when present ULM’) is a group according to the chemical structure:
• X of ULM-l is O or S; Y of ULM-l is H, methyl or ethyl; R 17 of ULM-l is H, methyl, ethyl, hydoxymethyl or cyclopropyl; M of ULM-l is is optionally substituted aryl, optionally substituted heteroaryl, o R 9 of ULM-l is H; R10 of ULM-l is H, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted hydroxyalkyl, optionally substituted thioalkyl or cycloalkyl; R11 of ULM-l is optionally substituted heteroaromatic, optionally substituted heterocyclyl, optionally substituted aryl or ; R12 of ULM-l is H or optionally substituted alkyl; and R 13 of ULM-l is H, optionally substituted alkyl, optionally substituted alkyl,
• Y of ULM-m is H, methyol or ethyl
• R 9 of ULM-m is H
• R 10 is isopropyl, tert-butyl, sec-butyl, cyclopentyl, or cyclohexyl
• R11 of ULM-m is optionally substituted amide, optionally substituted isoindolinone, optionally substituted isooxazole, optionally substituted heterocyclyls.
• ULM and where present, ULM’ are each independently a group according to the chemical structure: wherein: R17 of ULM-n is methyl, ethyl, or cyclopropyl; and R9, R10, and R11 of ULM-n are as defined above. In other instances, R9 is H; and R 10 of ULM-n is H, alkyl, or or cycloalkyl (preferably, isopropyl, tert-butyl, sec-butyl, cyclopentyl, or cyclohexyl).
• ULM and where present, ULM’ are each independently a group according to the chemical structure: ULM-o ULM-p or a pharmaceutically acceptable salt thereof, wherein: R1 is H, optionally substituted alkyl or optionally substituted cycloalkyl; R3 is an optionally substituted 5-6 membered heteroaryl; W 5 is optionally substituted phenyl, optionally substituted napthyl or optionally substituted pyridinyl; one of R14a and R14b is H, optionally substituted alkyl, optionally substituted haloalkyl (e.g., fluoroalkyl), optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted heterolkyl, optionally substituted alkyl-heterocycloalkyl, optionally substituted alkoxy-heterocycloalkyl, COR26, CONR 27a R 27b ,
• each R28 is independently H, halogen, CN, optionally substituted aminoalkyl, optionally substituted amidoalkyl, optionally substituted haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted heteroalkyl, optionally substituted alkylamine, optionally substituted hydroxyalkyl, amine, optionally substituted alkynyl, or optionally substituted cycloalkyl; o is 0, 1 or 2; and p is 0, 1, 2, 3, or 4. [00146] In any of the aspects or embodiments described herein, the ULM is of the formula:
• each of X 4 , X 5 , and X 6 is selected from CH and N, wherein no more than 2 are N;
• R 1 is C1-6 alkyl;
• R 3 is the same as defined for ULM-o and ULM-p one of R 14a and R 14b is H, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted amide, optionally substituted alkyl-amide, optionally substituted alkyl-cyano, optionally substituted alkyl-phosphate, optionally substituted heteraolkyl, optionally substituted alkyl-heterocycloalkyl, optionally substituted alkoxy- heterocycloalkyl, COR 26 , CONR 27a R 27b , NHCOR 26 , or NHCH 3 COR 26 ; and the other of R 14a and R 14b is H; or R 14a and
• R 14a and R 14b are selected from: H, C 1- 4 alkyl, C 1- 4 cycloalkyl, C 1- 4 haloalkyl, C 1- 4 hydroxyalkyl, C 1- 4 alkyloxyalkyl, C 1- 4 alkyl-NR27aR27b and CONR27aR27b.
• at least one of R 14a and R 14b is H (e.g., both R 14a and R 14b are H).
• R 14a and R 14b is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted heterolkyl, optionally substituted alkyl-heterocycloalkyl, optionally substituted alkoxy-heterocycloalkyl, COR 26 , CONR 27a R 27b , NHCOR 26 , or NHCH 3 COR 26 .
• one of R 14a and R 14b is optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkoxy, optionally substituted hydroxyl alkyl, optionally substituted alkylamine, optionally substituted heterolkyl, optionally substituted alkyl- heterocycloalkyl, optionally substituted alkoxy-heterocycloalkyl, COR 26 , CONR 27a R 27b , NHCOR 26 , or NHCH 3 COR 26 ; and the other of R 14a and R 14b is H.
• R 14a and R 14b together with the carbon atom to which they are attached form wherein R 23 is selected from H, C 1-4 alkyl, - C(O)C 1- 4alkyl.
• R 23 is selected from H, C 1-4 alkyl, - C(O)C 1- 4alkyl.
• ULM and where present, ULM’ are each independently a group according to the chemical structure: ULM-q ULM-r or a pharmaceutically acceptable salt thereof, wherein: X is CH or N; and R 1 , R 3 , R 14a , R 14b , and R 15 of ULM-q and ULM-r are the same as defined for ULM-o and ULM- p.
• the ULM (or when present, ULM’) as described herein may be a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate or polymorph thereof.
• the ULM (or when present, ULM’) as described herein may be coupled to a PTM directly via a bond or by a chemical linker.
• the ULM moiety is selected from the group consisting of:
• the compounds as described herein include a means for chemically coupling the PTM to the ULM, e.g., one or more PTMs chemically linked or coupled to one or more ULMs (e.g., at least one of VLM) via a chemical linker (L).
• a chemical linker L
• the linker group L is a group comprising one or more covalently connected structural units (e.g., - A L 1... (A L ) q - or –(A L ) q -), wherein A L 1 is a group coupled to PTM, and (A L ) q is a group coupled to ULM.
• the linker (L) to ULM e.g., VLM, ILM, CLM, or MLM
• coupling is a stable L-ULM connection.
• any subsequent heteroatom when a linker (L) and a ULM is connected via a heteroatom, any subsequent heteroatom, if present, is separated by at least one single carbon atom (e.g., -CH 2 -), such as with an acetal or aminal group.
• the heteroatom when a linker (L) and a ULM is connected via a heteroatom, the heteroatom is not part of a ester.
• the linker group L is a bond or a chemical linker group represented by the formula –(A L )q-, wherein A is a chemical moiety and q is an integer from 1-100, and wherein L is covalently bound to the PTM and the ULM, and provides for sufficient binding of the PTM to the protein target and the ULM to an E3 ubiquitin ligase to result in target protein ubiquitination.
• q of the linker is an integer greater than or equal to 0. In certain embodiments, q is an integer greater than or equal to 1. [00159] In certain embodiments, e.g., where q of the linker is greater than 2, (A L ) q is a group which is to A L 1 and (A L )q wherein the units A L couple a PTM to a ULM. [00160] In certain embodiments, e.g., where q of the linker is 2, (A L ) q is a group which is connected to A L 1 and to a ULM or PTM.
• the structure of the linker group L is –A L 1–, and A L 1 is a group which is connected to a ULM moiety and a PTM moiety.
• the unit A L of linker (L) comprises a group represented by a general structure selected from the group consisting of: -NR(CH 2 )n-(lower alkyl)-, -NR(CH 2 )n-(lower alkoxyl)-, -NR(CH 2 )n-(lower alkoxyl)-OCH 2 -, - NR(CH 2 ) n -(lower alkoxyl)-(lower alkyl)-OCH 2 -, -NR(CH 2 ) n -(cycloalkyl)-(lower alkyl)- OCH 2 -, -NR(CH 2 )n-(hetero cycloalkyl)-, -NR(CH 2 CH 2 O)n-(lower alkyl)-O-CH 2 -, - NR(CH 2 CH 2 O)n-(hetero cycloalkyl)-O-CH 2 -, - NR(CH 2 CH 2 O
• the unit A L of linker (L) comprises a group represented by a general structure selected from the group consisting of: -N(R)-(CH 2 ) m -O(CH 2 ) n -O(CH 2 ) o -O(CH 2 ) p -O(CH2) q -O(CH 2 ) r -OCH 2 -, -O-(CH2)m-O(CH 2 )n-O(CH 2 )o-O(CH 2 )p-O(CH 2 )q-O(CH 2 )r-OCH 2 -, -O-(CH2)m-O(CH 2 )n-O(CH 2 )o-O(CH 2 )p-O(CH 2 )q-O(CH 2 )r-O-; -N(R)-(CH 2 ) m -O(CH 2 ) n -O(CH 2 ) o -O(CH 2 )q-O(CH 2
• m, n, o, p, q, and r of the linker are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; when the number is zero, there is no N-O or O-O bond R of the linker is H, methyl and ethyl; X of the linker is H and F where m of the linker can be 2, 3, 4, 5
• each n and m of the linker can independently be 0, 1, 2, 3, 4, 5, 6.
• the unit A L of linker (L) is selected from the group consisting of:
• the unit A L of linker (L) is selected from the group consisting of: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; O N N O N N N m n m n N N O m n o m N n N N O O N m m n O n o O N N N N N N n m n m O O N N N N m n o o
• the unit A L of linker (L) is selected from the group consisting of: ;
• the linker unit or linker (L) comprises a group represented by a structure selected from the group consisting of: -O-(CH 2 )m-O(CH 2 )n-O(CH 2 )o-O(CH 2 )p-O(CH 2 )q-O(CH 2 )r-O(CH 2 )s-O(CH 2 )t-; -O-( CH 2 )m-O(CH 2 )n-O(CH 2 )o-O(CH 2 )p-O(CH 2 )q-O(CH 2 )r-O(CH 2 )s-O-; -(CH 2 ) m -O(CH 2 ) n -O(CH 2 ) o -O(CH 2 ) p -O(CH 2 ) q -O(CH 2 ) r -O(CH 2 ) s-O(CH 2 ) t -; -CH
• the linker (L) is selected from the group consisting of: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; [00169]
• the linker (L) is selected from the group consisting of: [00170]
• the linker (L) comprises a structure selected from, but not limited to the structure shown below, where a dashed line indicates the attachment point to the PTM or ULM moieties: wherein: W L1 and W L2 are each independently absent, a 4-8 membered ring with 0-4 heteroatoms, optionally substituted with R Q , each R Q
• the linker (L) comprises a structure selected from, but not limited to the structure shown below, where a dashed line indicates the attachment point to the PTM or ULM moieties: , wherein: W L1 and W L2 are each independently absent, aryl, heteroaryl, cyclic, heterocyclyl, C 1-6 alkyl and optionally one or more C atoms are replaced with O or N, C 1-6 alkenyl and optionally one or more C atoms are replaced with O, C 1-6 alkynyl and optionally one or more C atoms are replaced with O, bicyclic, biaryl, biheteroaryl,or biheterocyclyl, each optionally substituted with R Q , each R Q is independently a H, halo, OH, CN, CF 3 , hydroxyl, nitro, C ⁇ CH, C 2-6 alkenyl, C 2-6 alkynyl, optionally substituted linear or branched C 1 -C
• the linker group is optionally substituted (poly)ethyleneglycol having between 1 and about 100 ethylene glycol units, between about 1 and about 50 ethylene glycol units, between 1 and about 25 ethylene glycol units, between about 1 and 10 ethylene glycol units, between 1 and about 8 ethylene glycol units and 1 and 6 ethylene glycol units, between 2 and 4 ethylene glycol units,or optionally substituted alkyl groups interdispersed with optionally substituted, O, N, S, P or Si atoms.
• the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocyclyl group.
• the linker may be asymmetric or symmetrical.
• the linker group may be any suitable moiety as described herein.
• the linker is a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, between 1 and about 10 ethylene glycol units, about 2 about 6 ethylene glycol units, between about 2 and 5 ethylene glycol units, between about 2 and 4 ethylene glycol units.
• the present disclosure is directed to a compound which comprises a PTM group as described above, which binds to a target protein or polypeptide (e.g., SMARCA2, BRAHMA or BRM), which is ubiquitinated by a ubiquitin ligase and is chemically linked directly to the ULM group or through a linker moiety L, or PTM is alternatively a ULM’ group which is also a ubiquitin ligase binding moiety, which may be the same or different than the ULM group as described above and is linked directly to the ULM group directly or through the linker moiety; and L is a linker moiety as described above which may be present or absent and which chemically (covalently) links ULM to PTM, or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate or polymorph thereof.
• a target protein or polypeptide e.g., SMARCA2, BRAHMA or BRM
• PTM is alternatively a ULM’ group which
• the linker group L is a group comprising one or more covalently connected structural units independently selected from the group consisting of:
• the X is selected from the group consisting of O, N, S, S(O) and SO 2 ; n is integer from 1 to 5;
• R L1 is hydrogen or alkyl, is a mono- or bicyclic aryl or heteroaryl optionally substituted with 1–3 substituents selected from alkyl, halogen, haloalkyl, hydroxy, alkoxy or cyano; is a mono- or bicyclic cycloalkyl or a heterocyclyl optionally substituted with 1–3 substituents selected from alkyl, halogen, haloalkyl, hydroxy, alkoxy or cyano; and the phenyl ring fragment can be optionally substituted with 1, 2 or 3 substituents selected from the grou consisting of alkyl, halogen, haloalkyl, hydroxy, alkoxy and cyan
• the linker group L comprises up to 10 covalently connected structural units, as described above.
• the ULM group and PTM group may be covalently linked to the linker group through any group which is appropriate and stable to the chemistry of the linker, in preferred aspects of the present dislcosure, the linker is independently covalently bonded to the ULM group and the PTM group preferably through an amide, ester, thioester, keto group, carbamate (urethane), carbon or ether, each of which groups may be inserted anywhere on the ULM group and PTM group to provide maximum binding of the ULM group on the ubiquitin ligase and the PTM group on the target protein to be degraded.
• the target protein for degradation may be the ubiquitin ligase itself).
• the linker may be linked to an optionally substituted alkyl, alkylene, alkenyl or alkynyl group, an aryl group or a heterocyclyl group on the ULM and/or PTM groups.
• the PTM group is a moiety, which binds to target proteins, such as Switch/Sucrose Non Fermentable (SWI/SNF)-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2) or BRM.
• target proteins such as Switch/Sucrose Non Fermentable (SWI/SNF)-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2) or BRM.
• SMARCA2 or BRM protein specifically (binds to the target protein SMARCA2, BRAHMA or BRM).
• the compounds as described herein include a means for binding a target protein, e.g., Brm.
• the disclosure provides a bifunctional compound having a means for binding Brm, and a means for binding VHL and a means for chemically coupling the means for binding Brm to the means for binding VHL.
• the compositions described below exemplify some of the members of small molecule target protein binding moieties.
• Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target SMARCA2.
• binding moieties are linked to the ubiquitin ligase binding moiety preferably through a linker in order to present a target protein (to which the protein target moiety is bound) in proximity to the ubiquitin ligase for ubiquitination and degradation.
• Any protein e.g., SMARCA2, BRAHMA or BRM
• SMARCA2, BRAHMA or BRM which can bind to a protein target moiety or PTM group and acted on or degraded by a ubiquitin ligase is a target protein according to the present disclosure.
• the present disclosure may be used to treat a number of disease states and/or conditions; including any disease state and/or condition in which proteins are dysregulated (e.g., SMARCA4- deficiency/mutation) and where a patient would benefit from the degradation and/or inhibition of proteins, such as SMARCA2, BRAHMA or BRM.
• the description provides therapeutic compositions comprising an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier, additive or excipient, and optionally an additional bioactive agent.
• the therapeutic compositions modulate protein degradation in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating disease states or conditions which are modulated through the degraded protein.
• the therapeutic compositions as described herein may be used to effectuate the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., cancer such as at least one of a SWI/SNF associated cancer, a SMARCA4-mutation associated cancer, a SMARCA4-deficient cancer, or a cancer with decreased expression of SMARCA4 relative to normal SMARCA4 expression (e.g., decreased expression relative to the expression of non-mutated SMARCA4 or SMARCA4 in a similarly situated non-cancerous cell with wildtype SMARCA4), including lung cancer or non- small cell lung cancer.
• a disease e.g., cancer such as at least one of a SWI/SNF associated cancer, a SMARCA4-mutation associated cancer, a SMARCA4-de
• the disease is at least one of SWI/SNF associated cancer, a cancer with a SMARCA4 mutation, a cancer with a SMARCA4- deficiency, or a combination thereof, which may be lung cancer or a non-small cell lung cancer.
• the therapeutic compositions as described herein may be used to effectuate the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., cancer such as at least one of a SWI/SNF associated cancer, a SMARCA2- associated cancer or a cancer with normal or over-expression of SMARCA2.
• the present disclosure relates to a method for treating a disease state or ameliorating the symptoms of a disease or condition in a subject in need thereof by degrading a protein or polypeptide through which a disease state or condition is modulated comprising administering to said patient or subject an effective amount, e.g., a therapeutically effective amount, of at least one compound as described hereinabove, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and optionally an additional bioactive agent, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject.
• the method according to the present disclosure may be used to treat a large number of disease states or conditions including cancer, by virtue of the administration of effective amounts of at least one compound described herein.
• the disease state or condition may be a disease caused by a microbial agent or other exogenous agent such as a virus, bacteria, fungus, protozoa or other microbe or may be a disease state, which is caused by overexpression of a protein, which leads to a disease state and/or condition.
• a microbial agent or other exogenous agent such as a virus, bacteria, fungus, protozoa or other microbe
• the description provides methods for identifying the effects of the degradation of proteins of interest in a biological system using compounds according to the present disclosure.
• target protein is used to describe a protein or polypeptide, which is a target for binding to a compound according to the present disclosure and degradation by ubiquitin ligase hereunder.
• Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. These binding moieties are linked to at least one ULM group (e.g. VLM) through at least one linker group L.
• ULM group e.g. VLM
• linker group L e.g. L1
• the protein target may be used in screens that identify compound moieties which bind to the protein and by incorporation of the moiety into compounds according to the present disclosure, the level of activity of the protein may be altered for therapeutic end result.
• protein target moiety or PTM is used to describe a small molecule which binds to a target protein or other protein or polypeptide of interest, such as SMARCA2 or BRM, and places/presents that protein or polypeptide in proximity to an ubiquitin ligase such that degradation of the protein or polypeptide by ubiquitin ligase may occur.
• the compositions described below exemplify some of the members of the small molecule target proteins.
• the PTM of the present disclosure has a chemical structure represented by:
• W PTM1 is an optionally substituted 5-6-membered aryl or heteroaryl ring (e.g., a 5-6 member aryl or heteroaryl substituted with 0, 1, 2, or 3 substituents selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, phosphate, amino, alkylamino, cyano or combination thereof);
• W PTM2 is an optionally substituted 5-6-membered aryl or heteroaryl ring (e.g., a 5-6 membered aryl or heteroaryl substituted with 0, 1, 2, or 3 substituents selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, amino, alkylamino and cyano);
• WPTM3 is an optionally substituted 3-9-membered aryl or heteroaryl ring (e.g., an optionally substituted 5-6-membered aryl or heteroaryl ring, or a 3-9 or 5-6 member
• WPTM5 is absent (such that WPTM3 is connected directly to L (linker) or ULM) or an optionally substituted alkyl, an optionally substituted 5-6-membered cycloalkyl, heterocycle, aryl or heteroaryl ring (e.g.
• W PTM5 is a piperidine.
• WPTM1 comprises a phosphate substitution.
• the PTM of the PROTAC of the present disclosure is represented by Formula I, wherein at least one of: WPTM1 is an optionally substituted phenyl or a pyridyl (e.g., substituted as described herein, such as a phenyl substituted with a hydroxy or phosphate substituent with or without an additional optional substituent selected as described herein, e.g., substituted with 0, 1, 2, or 3 substituents selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, amino, alkylamino, cyano or combination thereof); W PTM2 is an optionally substituted 6-membered heteroaryl ring (e.g., substituted as described herein, such as a pyridazine substituted with amino group); WPTM3 is an optionally substituted 5-6-membered heteroaryl (e.g., a pyrazole, pyrrole, imidazole
• WPTM3 is a pyrazole or a 6-8-membered heterocyclyl (e.g., a piperazine or a diazabicyclooctane).
• the PTM of the present disclosure has a chemical structure represented by: wherein: WPTM1, WPTM2, and WPTM5 are as described in any other aspect or embodiment described herein (e.g., W PTM5 may or may not be present, such that WPTM4 may be connected directly to L (linker) or the ULM); WPTM4 is an optionally substitute 3-7 cycloalkyl or heterocyclyl (e.g., optionally substituted 5-7 cycloalkyl or heterocyclyl or a 5-7 cycloalkyl or heterocyclyl substituted with 0, 1, 2, or 3 substituents selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, amino, alkylamino and cyano) that is fused with the WPTM2 ring; and is the attachment point to the, linker, ULM group, ULM’group, VLM group, VLM’ groupgroup.
• WPTM1, WPTM2, and WPTM5 are as described in any other aspect
• the PTM of the present disclosure is represented by Formula II, wherein WPTM1, WPTM2 and WPTM5 are as described in any of the aspects or embodiment described herein, and W PTM4 is a piperazine ring.
• WPTM2 and WPTM4 of Formula II taken together constitute a dihydropirazino[2,3-e]pyridazine as shown: .
• the PTM of the present disclosure has a chemical structure represented by: wherein: WPTM1 and WPTM2 are as described in any aspect or embodiment described herein; WPTM6 and WPTM7 are independently an optionally 4-7 cycloalkyl or heterocyclyl (e.g., each is independently a 4-7 cycloalkyl or heterocyclyl substituted with 0, 1, or 2 substituents selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, amino, alkylamino and cyano), and the rings of WPTM6 and WPTM7 are fused or linked via a spiro connection; and is the attachment point to the, linker, ULM group, ULM’group, VLM group, VLM’ group.
• WPTM1 and WPTM2 are as described in any aspect or embodiment described herein
• WPTM6 and WPTM7 are independently an optionally 4-7 cycloalkyl or heterocyclyl (e.g., each is independently a
• the PTM of the present disclosure has a chemical structure represented by formula III, wherein WPTM1 and WPTM2 are each independently selected as described in any aspect or embodiment described herein (e.g., WPTM1 is a phenyl substituted with a hydroxy substituent with or without an additional optional substituent selected as described herein, W PTM2 is a pyridazine substituted with amino group), and WPTM6 and WPTM7 are a spirocyclic ring system, for example, a spirocyclic ring selected from: .
• WPTM1 and WPTM2 are each independently selected as described in any aspect or embodiment described herein (e.g., WPTM1 is a phenyl substituted with a hydroxy substituent with or without an additional optional substituent selected as described herein, W PTM2 is a pyridazine substituted with amino group), and WPTM6 and WPTM7 are a spirocyclic ring system, for example,
• WPTM1, WPTM2, and WPTM5 are as described in any other aspect or embodiment described herein;
• R PTM1 and R PTM2 are individually a H, halo, HO, C 1 -C 3 alkyl, C 1 -C 3 haloalky, or C 1 -C 3 alkoxy; and is the attachment point to the, linker, ULM group, ULM’group, VLM group, VLM’ group.
• the PTM of the present disclosure has the chemical structure represented by Formula IV, wherein at least one of: WPTM1 is a phenyl substituted with a hydroxy or phosphate substituent with or without an additional optional substituent selected as described herein; W PTM2 is a pyridazine substituted with amino group; W PTM5 is absent, a pyrazole ring, or a pyridine ring; or a combination thereof.
• WPTM1 is a phenyl substituted with a hydroxy or phosphate substituent with or without an additional optional substituent selected as described herein
• W PTM2 is a pyridazine substituted with amino group
• W PTM5 is absent, a pyrazole ring, or a pyridine ring; or a combination thereof.
• W PTM3 is absent or an optionally substituted 5-6-membered heteroaryl, an optionally substituted 4-9 cycloalkyl or heterocyclyl ring, an optionally substituted bridged bicycloalkyl and bridged biheterocyclyl ring; and WPTM5 is an optionally substituted 5-6-membered heteroaryl or aryl, e.g., pyridine, or pyridazine.
• W PTM5 is phenyl, pyridine, pyrimidine or pyrazine.
• the PTM of the present disclosure is represented by: Formula Vb or a pharmaceutically acceptable salt thereof, wherein: W PTM3 is an optionally substituted 5-6-membered heteroaryl, an optionally substituted 4-9 cycloalkyl or heterocyclyl ring, an optionally substituted bridged bicycloalkyl and bridged biheterocyclyl ring; WPTM5 is an optionally substituted 5-6-membered heteroaryl or aryl, e.g., pyridine, or pyridazine; Rv is 0, 1, 2 or 3 substituents independently selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, phosphate, amino, alkylamino, cyano or a combination thereof.
• the hydroxyl group is modified with a phosphate group (i.e., a phosphoester group).
• a phosphate group i.e., a phosphoester group.
• WPTM1 and WPTM2 are as described in any other aspect or embodiment described herein (e.g., WPTM5 may or may not be present, such that WPTM4 may be connected directly to L (linker) or the ULM);
• W PTM3 is absent or an optionally substituted 5-7 cycloalkyl or heterocyclyl (e.g., 5-7 cycloalkyl or heterocyclyl substituted with 0, 1, 2, or 3 substituents selected from hydroxy, halogen, alkoxy, alkyl, haloalkyl, amino, alkylamino and cyano) that is fused with the W PTM2 ring;
• WPTM4 is an optionally substituted 3-7-membered aryl or heteroaryl ring (e.g., optionally substituted 5-7 cycloalkyl or heterocyclyl, or a 3-7 or a 5-6 membered aryl or heteroaryl substituted with 0, 1, 2, or 3 substituents selected from hydroxy, hal
• WPTM5 is absent (such that WPTM3 is connected directly to L (linker) or ULM) or an optionally substituted alkyl, an optionally substituted 5-6-membered cycloalkyl, heterocycle, aryl or heteroaryl ring (e.g.
• the PTM of the present disclosure has a chemical structure represented by: wherein: W PTM1 , W PTM2 , W PTM3 , and W PTM4 are as described in any other aspect or embodiment described herein; LPTM is C1-C6 alkyl optionally substituted with an C1-C4 alkyl or C 1 -C 3 alkoxy; and is the attachment point to the, linker, ULM group, ULM’group, VLM group, VLM’ group. [00207] In any aspect or embodiment described herein, the PTM is selected from:
• the PTM is selected from:
• the PTM is selected from the group consisting of:
• compositions described herein exemplify some of the members of these types of small molecule target protein binding moieties.
• Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of these compositions, as well as other small molecules that may target a protein of interest. References which are cited herein below are incorporated by reference herein in their entirety.
• compositions comprising combinations of an effective amount of at least one bifunctional compound as described herein, and one or more of the compounds otherwise described herein, all in effective amounts, in combination with a pharmaceutically effective amount of a carrier, additive or excipient, represents a further aspect of the present disclosure.
• the present disclosure includes, where applicable, the compositions comprising the pharmaceutically acceptable salts, in particular, acid or base addition salts of compounds as described herein.
• the acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful according to this aspect are those which form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)]salts, among numerous others.
• non-toxic acid addition salts i.e., salts containing pharmacologically acceptable anions
• Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the compounds or derivatives according to the present disclosure.
• the chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non- toxic base salts with such compounds.
• Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (eg., potassium and sodium) and alkaline earth metal cations (eg, calcium, zinc and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
• pharmacologically acceptable cations such as alkali metal cations (eg., potassium and sodium) and alkaline earth metal cations (eg, calcium, zinc and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
• pharmacologically acceptable cations such as alkali metal cations (eg., potassium and sodium) and alkaline earth metal
• Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q.I.D.) and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal, sublingual and suppository administration, among other routes of administration.
• Enteric coated oral tablets may also be used to enhance bioavailability of the compounds from an oral route of administration.
• the most effective dosage form will depend upon the pharmacokinetics of the particular agent chosen as well as the severity of disease in the patient.
• Administration of compounds according to the present disclosure as sprays, mists, or aerosols for intra-nasal, intra-tracheal or pulmonary administration may also be used.
• compositions comprising an effective amount of compound as described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient.
• Compounds according to the present disclosure may be administered in immediate release, intermediate release or sustained or controlled release forms. Sustained or controlled release forms are preferably administered orally, but also in suppository and transdermal or other topical forms. Intramuscular injections in liposomal form may also be used to control or sustain the release of compound at an injection site.
• the compositions as described herein may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled-release formulations.
• Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as prolamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
• ion exchangers alumina, aluminum stearate, lecithin
• serum proteins such as human serum albumin
• buffer substances such as phosphates, glycine, sorb
• compositions as described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
• parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
• the compositions are administered orally, intraperitoneally or intravenously.
• Sterile injectable forms of the compositions as described herein may be aqueous or oleaginous suspension.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.
• a non-toxic parenterally- acceptable diluent or solvent for example as a solution in 1, 3-butanediol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or di-glycerides.
• Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.
• the pharmaceutical compositions as described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added.
• useful diluents include lactose and dried corn starch.
• the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
• the pharmaceutical compositions as described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient, which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
• suitable non-irritating excipient include cocoa butter, beeswax and polyethylene glycols.
• compositions as described herein may also be administered topically. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-acceptable transdermal patches may also be used.
• the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
• the compounds may be coated onto a stent which is to be surgically implanted into a patient in order to inhibit or reduce the likelihood of occlusion occurring in the stent in the patient.
• the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
• Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
• the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride.
• the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
• the pharmaceutical compositions as described herein may also be administered by nasal aerosol or inhalation.
• compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
• benzyl alcohol or other suitable preservatives to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
• the amount of compound in a pharmaceutical composition as described herein that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host and disease treated and the particular mode of administration.
• compositions should be formulated to contain between about 0.05 milligram to about 750 milligrams or more, more preferably about 1 milligram to about 600 milligrams, and even more preferably about 10 milligrams to about 500 milligrams of active ingredient, alone or in combination with at least one other compound according to the present disclosure.
• a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease or condition being treated.
• a patient or subject in need of therapy using compounds according to the methods described herein can be treated by administering to the patient (subject) an effective amount of the compound according to the present disclosure including pharmaceutically acceptable salts, solvates or polymorphs, thereof optionally in a pharmaceutically acceptable carrier or diluent, either alone, or in combination with other known therapeutic agents as otherwise identified herein.
• These compounds can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, including transdermally, in liquid, cream, gel, or solid form, or by aerosol form.
• the active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication, without causing serious toxic effects in the patient treated.
• a preferred dose of the active compound for all of the herein-mentioned conditions is in the range from about 10 ng/kg to 300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient/patient per day.
• a typical topical dosage will range from 0.01-5% wt/wt in a suitable carrier.
• the compound is conveniently administered in any suitable unit dosage form, including but not limited to one containing less than 1mg, 1 mg to 3000 mg, preferably 5 to 500 mg of active ingredient per unit dosage form.
• An oral dosage of about 25-250 mg is often convenient.
• the active ingredient is preferably administered to achieve peak plasma concentrations of the active compound of about 0.00001-30 mM, preferably about 0.1-30 ⁇ M. This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient. Oral administration is also appropriate to generate effective plasma concentrations of active agent.
• the concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
• the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
• Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents.
• the active compound or pharmaceutically acceptable salt thereof can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
• a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
• the active compound or pharmaceutically acceptable salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as anti-cancer agents, including pembrolizumab, among others.
• one or more compounds according to the present disclosure are coadministered with another bioactive agent, such as an anti-cancer agent or a would healing agent, including an antibiotic, as otherwise described herein.
• Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
• antibacterial agents such as benzyl alcohol or methyl parabens
• antioxidants such as ascorbic acid or sodium bisulfite
• chelating agents such
• the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• preferred carriers are physiological saline or phosphate buffered saline (PBS).
• PBS physiological saline or phosphate buffered saline
• the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
• Liposomal suspensions may also be pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811 (which is incorporated herein by reference in its entirety).
• liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound are then introduced into the container.
• appropriate lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol
• the description provides therapeutic compositions comprising an effective amount of a compound as described herein or salt form thereof, and a pharmaceutically acceptable carrier.
• the therapeutic compositions modulate protein degradation in a patient or subject, for example, an animal such as a human, and can be used for treating or ameliorating disease states or conditions which are modulated through the degraded protein.
• the terms “treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a patient for which the present compounds may be administered, including the treatment of any disease state or condition which is modulated through the protein to which the present compounds bind.
• Disease states or conditions including cancer such as lung cancer, including non-small cell lung cancer, which may be treated using compounds according to the present disclosure are set forth hereinabove.
• the description provides therapeutic compositions as described herein for effectuating the degradation of proteins of interest for the treatment or amelioration of a disease, e.g., cancer.
• the disease is multiple myeloma.
• the description provides a method of ubiquitinating/ degrading a target protein in a cell.
• the method comprises administering a bifunctional compound as described herein comprising, e.g., a ULM and a PTM, preferably linked through a linker moiety, as otherwise described herein, wherein the ULM is coupled to the PTM and wherein the ULM recognizes a ubiquitin pathway protein (e.g., an ubiquitin ligase, such as a VHL E3 ubiquitin ligase) and the PTM recognizes the target protein such that degradation of the target protein will occur when the target protein is placed in proximity to the ubiquitin ligase, thus resulting in degradation/inhibition of the effects of the target protein and the control of protein levels.
• a bifunctional compound as described herein comprising, e.g., a ULM and a PTM, preferably linked through a linker moiety, as otherwise described herein, wherein the ULM is coupled to the P
• control of protein levels afforded by the present disclosure provides treatment of a disease state or condition, which is modulated through the target protein by lowering the level of that protein in the cell, e.g., cell of a patient.
• the method comprises administering an effective amount of a compound as described herein, optionally including a pharamaceutically acceptable excipient, carrier, adjuvant, another bioactive agent or combination thereof.
• the description provides methods for treating or ameliorating a disease, disorder or symptom thereof in a subject or a patient, e.g., an animal such as a human, comprising administering to a subject in need thereof a composition comprising an effective amount, e.g., a therapeutically effective amount, of a compound as described herein or salt form thereof, and a pharmaceutically acceptable excipient, carrier, adjuvant, another bioactive agent or combination thereof, wherein the composition is effective for treating or ameliorating the disease or disorder or symptom thereof in the subject.
• the description provides methods for identifying the effects of the degradation of proteins of interest in a biological system using compounds according to the present disclosure.
• the present disclosure is directed to a method of treating a human patient in need for a disease state or condition modulated through a protein where the degradation of that protein will produce a therapeutic effect in the patient, the method comprising administering to a patient in need an effective amount of a compound according to the present disclosure, optionally in combination with another bioactive agent.
• the disease state or condition may be a disease caused by a microbial agent or other exogenous agent such as a virus, bacteria, fungus, protozoa or other microbe or may be a disease state, which is caused by overexpression of a protein, which leads to a disease state and/or condition [00249]
• the term “disease state or condition” is used to describe any disease state or condition wherein protein dysregulation (i.e., the amount of protein expressed in a patient is elevated) occurs and where degradation of one or more proteins in a patient may provide beneficial therapy or relief of symptoms to a patient in need thereof. In certain instances, the disease state or condition may be cured.
• Disease states or conditions which may be treated using compounds according to the present disclosure include, for example, asthma, autoimmune diseases such as multiple sclerosis, various cancers, ciliopathies, cleft palate, diabetes, heart disease, hypertension, inflammatory bowel disease, mental retardation, mood disorder, obesity, refractive error, infertility, Angelman syndrome, Canavan disease, Coeliac disease, Charcot–Marie–Tooth disease, Cystic fibrosis, Duchenne muscular dystrophy, Haemochromatosis, Haemophilia, Klinefelter's syndrome, Neurofibromatosis, Phenylketonuria, Polycystic kidney disease, (PKD1) or 4 (PKD2) Prader– Willi syndrome, Sickle-cell disease, Tay–Sachs disease, Turner syndrome.
• autoimmune diseases such as multiple sclerosis, various cancers, ciliopathies, cleft palate, diabetes, heart disease, hypertension, inflammatory bowel disease, mental retardation, mood disorder, obesity, refractive error,
• neoplasia or “cancer” is used throughout the specification to refer to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i.e., abnormal tissue that grows by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease.
• malignant neoplasms show partial or complete lack of structural organization and functional coordination with the normal tissue and most invade surrounding tissues, metastasize to several sites, and are likely to recur after attempted removal and to cause the death of the patient unless adequately treated.
• neoplasia is used to describe all cancerous disease states and embraces or encompasses the pathological process associated with malignant hematogenous, ascitic and solid tumors.
• Exemplary cancers which may be treated by the present compounds either alone or in combination with at least one additional anti-cancer agent include squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, and renal cell carcinomas, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, including Ewing's sarcoma, hemangiosarcoma, Kaposi's sar
• Additional cancers which may be treated using compounds according to the present disclosure include, for example, T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T-cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL and Philadelphia chromosome positive CML.
• T-ALL T-lineage Acute lymphoblastic Leukemia
• T-LL T-lineage lymphoblastic Lymphoma
• Peripheral T-cell lymphoma Peripheral T-cell lymphoma
• Adult T-cell Leukemia Pre-B ALL
• Pre-B Lymphomas Large B-cell Lymphoma
• Burkitts Lymphoma B-cell ALL
• Philadelphia chromosome positive ALL Philadelphia chromosome positive CML.
• bioactive agent is used to describe an agent, other than a compound according to the present disclosure, which is used in combination with the present compounds as an agent with biological activity to assist in effecting an intended therapy, inhibition and/or prevention/prophylaxis for which the present compounds are used.
• Preferred bioactive agents for use herein include those agents which have pharmacological activity similar to that for which the present compounds are used or administered and include for example, anti-cancer agents, antiviral agents, especially including anti-HIV agents and anti-HCV agents, antimicrobial agents, antifungal agents, etc.
• additional anti-cancer agent is used to describe an anti-cancer agent, which may be combined with compounds according to the present disclosure to treat cancer.
• agents include, for example, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor
• anti-HIV agent or “additional anti-HIV agent” includes, for example, nucleoside reverse transcriptase inhibitors (NRTI), other non-nucloeoside reverse transcriptase inhibitors (i.e., those which are not representative of the present disclosure), protease inhibitors, fusion inhibitors, among others, exemplary compounds of which may include, for example, 3TC (Lamivudine), AZT (Zidovudine), (-)-FTC, ddI (Didanosine), ddC (zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset), D4T (Stavudine), Racivir, L-FddC, L-FD4C, NVP (Nevirapine), DLV (Delavirdine), EFV (Efavirenz), SQVM (Saquinavir mesylate), RTV
• NRTI nucleo
• NNRTI NNRTI
• nevirapine nevirapine
• U-90152S/T delavirdine
• DMP-266 efavirenz
• UC-781 N-[4-chloro- 3-(3-methyl-2-butenyloxy)phenyl]-2methyl3-furancarbothiamide
• etravirine TMC125
• Trovirdine Ly300046.HCl
• MKC-442 emivirine, coactinon
• HI-236 HI-240, HI-280, HI-281, rilpivirine (TMC-278)
• MSC-127 HBY 097, DMP266, Baicalin (TJN-151) ADAM-II (Methyl 3’,3’-
• pharmaceutically acceptable salt is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds described herein which are presented to increase the solubility of the compound in the gastic juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds.
• Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids and bases well known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present disclosure.
• pharmaceutically acceptable derivative is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, amide other prodrug group), which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound.
• pharmaceutically acceptable derivative such as an ester, amide other prodrug group
• the synthetic realization and optimization of the bifunctional molecules as described herein may be approached in a step-wise or modular fashion. For example, identification of compounds that bind to the target molecules can involve high or medium throughput screening campaigns if no suitable ligands are immediately available.
• VLMs in hand, one skilled in the art can use known synthetic methods for their combination with or without a linker moiety.
• Linker moieties can be synthesized with a range of compositions, lengths and flexibility and functionalized such that the PTM and ULM groups can be attached sequentially to distal ends of the linker.
• a library of bifunctional molecules can be realized and profiled in in vitro and in vivo pharmacological and ADMET/PK studies.
• the final bifunctional molecules can be subject to iterative design and optimization cycles in order to identify molecules with desirable properties.
• protecting group strategies and/or functional group interconversions may be required to facilitate the preparation of the desired materials.
• ACN acetonitrile
• ADDP 1,1’-(azodicarbonyl)dipiperidine
• BAST N,N-bis(2-methoxyethyl)aminosulfur trifluoride
• BPO benzoyl peroxide
• Cbz Carbonylbezyloxy
• DAST diethylaminosulfur trifluoride
• DBE 1,2-dibromoethane
• DCM dichloromethane
• DEAD diethyl azodicarboxylate
• DIAD diisopropyl azodicarboxylate
• DIBAL disiobutylaluminium hydride
• DIEA or DIPEA diisopropylethylamine
• DMA N,N-diobutylaluminium hydride
• a compound having the WPTM5 moiety contains a linking group L which includes a nucleophilic group, such as an amino group.
• a compound having a good leaving group LG for instance, a perfluorosulfonyl group C 4 F 9 SO 3 -
• the coupling product reacts with a monoprotected amine molecule WPTM3, under palladium-catalyzed conditions to attach the WPTM3 fragment.
• Scheme 3 Z [00317] As shown in Scheme 3, a vinyl group is first introduced into a bis-halogenated derivative W PTM2 . The resulting product then undergoes the palladium-catalyzed Heck coupling reaction with a halogenated moiety WPTM2 bearing a linking group L’ which includes an optionally protected amino group. To introduce the W PTM1 moiety, the halide portion of W PTM2 - WPTM5 is coupled with the appropriate boronic acid under Suzuki conditions, and the resulting amine reacts with the ULM-containing aldehyde to afford the PTM-ULM coupling product.
• Scheme 4 illustrates exemplary coupling reactions that are utilized for connecting the PTM-binding moiety W PTM5 -W PTM3 -W PTM2 -W PTM1 with the ULM-containing portion. Such reactions may include reductive amination using sodium cyanoborohydride as a reducing agent or condensation coupling reactions between a carboxylic acid and a diamine. A person of ordinary skill in the art would be able to select appropriate reagents and conditions to carry out the desired transformations. [00320] Scheme 5
• the acetal-containing ULM moiety may undergo an acid- catalyzed hydrolysis under sufficiently mild conditions to form an aldehyde, which then reacts with the W PTM5 -W PTM3 -W PTM2 -W PTM2 fragment having an amino group-containing linker L’ under reductive amination conditions to form the PTM-ULM coupling product.
• a person of ordinary skill in the art would be able to select appropriate reagents and conditions to carry out the desired transformations.
• nucleophilic displacement of the fluorine atom in the moiety WPTM5 with a hydroxyl group-containing compound having R3 provides a WPTM5-L-R3 halide, which is coupled under Suzuki or Buchwald conditions to a monoprotected diamine W PTM5 .
• the WPTM3-WPTM5-L-R3 moiety is then reacted with bis-halogenated W PTM2 by a nucleophilic aromatic substitution to provide a monohalide.
• a subsequent Suzuki reaction with the appropriate boronic acid provides the WPTM1-WPTM2-WPTM3-WPTM5-L-R3 ester.
• the basic hydrolysis and coupling with a ULM portion bearing an amino group provides the PTM-ULM coupling product.
• One possible approach to synthesize exemplary compounds of the present disclosure is by following the general synthetic route detailed in the scheme below:
• exemplary compounds represented by Formula II can be prepared as described in the general synthetic scheme below where one skilled in the art would recognize that additional protection/deprotection steps may be required, dependending upon the specific chemical nature of the exemplary compound: [00334] In an embodiment, where X represents NH, the exemplary compound can be prepared according to one of the two schemes shown below below, depending on whether WPTM5 is present:
• Exemplary PTM represented by general Formula III can be prepared following the general approach described for compounds of Formula I when WPMT5 is not present.
• Examplary PTM represented by general Formula IV can be prepared according to the following general scheme: Z [00337] Example Sythesis of Examplary Compound 11 [00338] Step 1 [00339] To a mixture of tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)acetate (1.5g, 6.8 mmol) and TEA (2.07 g, 20.5 mmol) in DCM (5mL) was added TsCl (1.95 g, 10.23 mmol) at 0qC. The resulting mixture was warmed to room temperature and stirred for 3 hours.
• Step 2 A mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.11g,5.722 mmol), tert-butyl 2-(2-(2-(tosyloxy)ethoxy)ethoxy)acetate (2.14 g, 5.722 mmol) and Cs 2 CO 3 (3.73 g, 11.444mmol) in dry DMF (10 mL) was heated to 75 o C for 3 hours. The reaction mixture was then cooled to room temperature and diluted with EtOAc (30 mL).
• Step 3 A mixture of tert-butyl 2-(2-(2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrazol-1-yl)ethoxy)ethoxy)acetate (1.5 g, 3.79 mmol), 4-bromo-6-chloropyridazin-3-amine (1.1g, 5.69 mmol), PdCl2(dppf) (555mg, 0.758 mmol), tBuPHBF4 (441mg, 1.52 mmol) and Cs 2 CO 3 (3.09 g, 9.48 mmol) in dioxane (10 ml) and water (1ml) was heated to 100°C with
• Step 4 A suspension of tert-butyl 2-(2-(2-(4-(3-amino-6-chloropyridazin-4-yl)-1H-pyrazol-1- yl)ethoxy)ethoxy)acetate (800 mg, 2.02 mmol), (2-hydroxyphenyl)boronic acid (418 mg, 3.03 mmol), cesium carbonate (1.65 g, 5.05 mmol), PdCl2(dppf) (444 mg, 0.606mmol), and t Bu3PHBF4 (352 mg, 1.212 mmol) in dioxane (10 mL) and water (1 mL) was heated to 100qC under nitrogen for 3 hours.
• Step 5 To a solution of tert-butyl 2-(2-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)- 1H-pyrazol-1-yl)ethoxy)ethoxy)acetate (400 mg, 0.88mmol) in THF/H2O (5 mL, 2:1) was added LiOH (111 mg, 2.64mmol) at 0qC. The mixture was stirred at 0qC for 2 hours. The reaction solution was quenched with 1 M HCl.
• Step 6 To a solution of 2-(2-(2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)-1H- pyrazol-1-yl)ethoxy)ethoxy)acetic acid (200 mg crude), (2S,4R)-1-((S)-2-amino-3,3- dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (430 mg, 1 mmol), and DIPEA (516 mg, 4 mmol) in DMF (5mL) was added HATU (570 mg,1.5 mmol) at 0qC.
• the reaction mixture was stirred at room temperature for 30 minutes.
• the mixture was extracted with ethyl acetate (50 mL).
• the combined organic phases were washed with brine (8 mL x 2), dried (Na 2 SO 4 ), and filtered.
• the organic layer was concentrated under reduced pressure.
• Step 2 To a solution of 14-((4-bromopyridin-2-yl)oxy)-3,6,9,12-tetraoxatetradecan-1-ol (5.0 g, 12.7 mmol) in anhydrous THF (50 mL) was added 60% NaH (660 mg, 16.5 mmol) at 0qC. The reaction mixture was stirred at room temperature for 40 minutes. Then tert-butyl 2- bromoacetate (4.9 g, 25.4 mmol) was added dropwise to the mixture and stirred at room temperature overnight. The reaction mixture was quenched with 2N NH 4 Cl (10 mL) and extracted with EA (200 mL).
• Step 3 A mixture of tert-butyl 17-((4-bromopyridin-2-yl)oxy)-3,6,9,12,15- pentaoxaheptadecanoate (250 mg, 1.18 mmol), tert-butyl 3,8-diazabicyclo[3.2.1]octane-3- carboxylate (718 mg, 1.4 mmol), cesium carbonate (769 mg, 2.36 mmol), Pd2(dba)3 (110 mg, 0.12 mmol) and XantPhos (138 mg, 0.24 mmol) in dioxane (5 mL) in a seal tube was heated to 110qC under nitrogen overnight.
• Step 4 To a solution of tert-butyl 8-(2-((19,19-dimethyl-17-oxo-3,6,9,12,15,18- hexaoxaicosyl)oxy)pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (550 mg, 0.86 mmol) in MeOH (15mL) was added HCl in dioxane (6N in dioxane) (5 ml, 30 mmol) at rt. The reaction mixture was stirred at room temperature for 1 hour.
• Step 5 To a solution of crude methyl 17-((4-(3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl)oxy)-3,6,9,12,15-pentaoxaheptadecanoate (550 mg crude) in DMSO (5mL) were added 5- bromo-6-chloropyridazin-3-amine (526 mg, 2.71 mmol) and DIPEA (1.87 g, 14.5 mmol). The solution was stirred at 150qC overnight. The mixture was extracted with EA (60 mL). The organic phase was washed with water (8 mL) and brine (8 mL).
• Step 6 To a solution of methyl 17-((4-((1R,5S)-3-(3-amino-6-chloropyridazin-4-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)-3,6,9,12,15-pentaoxaheptadecanoate (325 mg, 0.52 mmol) and (2-hydroxyphenyl)boronic acid (93 mg, 0.68 mmol) in dioxane (12 mL) and water (1.2 mL) were added cesium carbonate (542 mg, 1.66 mmol), PdCl2(dppf) (73.2 mg, 0.1 mmol) and t-Bu3PHBF4 (58 mg, 0.2 mmol).
• Step 7 To a solution of crude 17-((4-((1R,5S)-3-(3-amino-6-(2-hydroxyphenyl)pyridazin-4- yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)-3,6,9,12,15-pentaoxaheptadecanoic acid (80 mg, 0.12 mmol) and (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (108 mg, 0.24 mmol) in DMF (5mL) were added DIPEA (124 mg, 0.96 mmol) and HATU (92 mg, 0.24 mmol) at 0qC.
• DIPEA 124 mg, 0.96 mmol
• HATU 92
• the reaction mixture was stirred at rt for 30 minutes.
• the mixture was extracted with EA (50 mL).
• the organic phase was washed with water (8 mL) and brine (8 mL).
• the organic layer was dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure.
• Step 2 To a solution of 2-(2-(2-(2-((4-bromopyridin-2-yl)oxy)ethoxy)ethoxy)acetic acid (3.0 g, 8.26 mmol) in MeOH (30 mL) was added SOCl 2 (4.0 g, 33.9 mmol) dropwise at 0qC. The reaction mixture was stirred at room temperature for 4 hours. The pH of the solution was adjusted to ⁇ 8 with saturated NaHCO 3 . The mixture was extracted with DCM (100 mL). The organic phase was washed with water (10 mL) and brine (10 mL).
• Step 2 To a solution of 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl bis(4- methylbenzenesulfonate) (3.5 g, 5.93 mmol) in anhydrous DMF (20 mL) were added 4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.15 g, 5.93 mmol) and Cs2CO3 (3.87 g, 11.86 mmol). The reaction mixture was stirred at 75qC for 0.5 hour. The mixture was cooled to rt and partitioned between EtOAc (200 mL) and water (20 mL).
• Step 3 To a solution of 17-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1- yl)-3,6,9,12,15-pentaoxaheptadecyl 4-methylbenzenesulfonate (0.3 g, 0.49 mmol) and (2S,4R)- 4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-3-methyl-2-(1-oxoisoindolin-2- yl)butanoyl)pyrrolidine-2-carboxamide (268 mg, 0.49 mmol) in DMF (5 mL) was added K 2 CO 3 (135 mg, 0.98 mmol).
• the mixture was stirred at 80qC under nitrogen atmosphere for 2 hours.
• the mixture was extracted with EA (80 mL).
• the organic phase was washed with water (10 mL) and brine (10 mL).
• the organic layer was dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure.
• Step 4 [00385] To a solution of the mixture of (2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1- oxoisoindolin-2-yl)butanoyl)-N-(4-(4-methylthiazol-5-yl)-2-((17-(4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazol-1-yl)-3,6,9,12,15-pentaoxaheptadecyl)oxy)benzyl)pyrrolidine-2- carboxamide and (1-(17-(2-(((2S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2- yl)butanoyl)pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)-3,6,9,12,15
• Step 1 Into a 250-mL round-bottom flask, was placed a solution of 4-(tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (1.5 g, 7.73 mmol, 1.00 equiv) in dioxane/H 2 O (1:1) (60 mL), 4- bromo-6-chloropyridazin-3-amine (1.7 g, 8.16 mmol, 1.20 equiv), Pd(PPh3)4 (800 mg, 0.69 mmol, 0.10 equiv), potassium carbonate (2.9 g, 20.98 mmol, 3.00 equiv).
• Step 2 Into a 10-mL sealed tube, was placed a solution of 6-chloro-4-(1H-pyrazol-4- yl)pyridazin-3-amine (390 mg, 1.99 mmol, 1.00 equiv) in dioxane (4 mL), [2- (methoxymethoxy)phenyl]boronic acid (546 mg, 3.00 mmol, 1.50 equiv), Pd(PPh3)4 (300 mg, 0.26 mmol, 0.20 equiv), a solution of potassium carbonate (552 mg, 3.99 mmol, 2.00 equiv) in water (2 mL). The resulting solution was stirred for 12 hours at 100°C in an oil bath.
• Step 3 Into a 50-mL round-bottom flask, was placed a solution of 6-[2- (methoxymethoxy)phenyl]-4-(1H-pyrazol-4-yl)pyridazin-3-amine (100 mg, 0.34 mmol, 1.00 equiv) in N,N-dimethylformamide (10 mL), 2-[2-(2-[[(4-methylbenzene)sulfonyl]oxy] ethoxy)ethoxy]ethan-1-ol (100 mg, 0.33 mmol, 1.00 equiv), potassium carbonate (91 mg, 0.66 mmol, 2.00 equiv).
• Step 4 Into a 100-mL round-bottom flask, was placed a solution of 2-[2-[2-(4-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-1H-pyrazol-1-yl)ethoxy]ethoxy]ethan-1-ol (100 mg, 0.23 mmol, 1.00 equiv) in dichloromethane (20 mL), 4-toluene sulfonyl chloride (66.0 mg, 0.35 mmol, 1.50 equiv), triethylamine (47 mg, 0.46 mmol, 2.00 equiv), 4-dimethylaminopyridine (10 mg, 0.08 mmol, 0.30 equiv).
• the product was purified by Chiral-Prep-HPLC with the following conditions: Column, CHIRAL ART Cellulose-SB, 2*25cm,5um; mobile phase, Hex--HPLC and ethanol-- HPLC (hold 50% ethanol--HPLC in 24 min); Detector, UV 220/254nm.
• Step 1 Into a 250-mL round-bottom flask, was placed (Z)-4-(benzyloxy)-N- hydroxybutcarbonimidoyl chloride (8.7 g, 38.21 mmol, 1.00 equiv), but-3-yn-1-ol (3.3 g, 47.08 mmol, 1.23 equiv), ethyl acetate (70 mL), water(70 mL), sodium bicarbonate (4.0 g, 47.61 mmol, 1.25 equiv).
• Step 2 Into a 100-mL round-bottom flask, was placed 2-[3-[3-(benzyloxy)propyl]-1,2- oxazol-5-yl]ethan-1-ol (550.0 mg, 2.10 mmol, 1.00 equiv), acetone (30 mL), Cr2O3 (100.0 mg), sulfuric acid (0.25 mL), water(1 mL). The resulting solution was stirred for 1 hour at 25qC. The resulting solution was diluted with water. The resulting solution was extracted with ethyl acetate and washed with saturated sodium chloride aqueous solution. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• Step 3 Into a 250-mL round-bottom flask, was placed ethyl 2-[3-[3-(benzyloxy)propyl]-1,2- oxazol-5-yl]acetate (8.0 g, 26.37 mmol, 1.00 equiv), ethanol (50 mL), sulfuric acid (0.1 mL). The resulting solution was stirred for 1.5 hours at 70qC. The resulting mixture was concentrated under vacuum.
• Step 4 Into a 250-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed ethyl 2-[3-[3-(benzyloxy)propyl]-1,2-oxazol-5-yl]acetate (4.5 g, 14.83 mmol, 1.00 equiv) and tetrahydrofuran (70 mL). This was followed by the addition of a solution of t-BuOK (2.0 g, 17.82 mmol, 1.20 equiv) in tetrahydrofuran (17.8 mL) dropwise with stirring at 0qC in 20 minutes.
• t-BuOK 2.0 g, 17.82 mmol, 1.20 equiv
• Step 5 Into a 250-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed ethyl 2-[3-[3-(benzyloxy)propyl]-1,2-oxazol-5-yl]-3- methylbutanoate (4.2 g, 12.16 mmol, 1.00 equiv) and dichloromethane (100 mL). This was followed by the addition of a solution of BBr3 (5.17 g, 20.64 mmol, 1.70 equiv) in dichloromethane (20.7 mL) dropwise with stirring at -78qC in 30 minutes.
• Step 6 Into a 100-mL round-bottom flask, was placed ethyl 2-[3-(3-hydroxypropyl)-1,2-oxazol-5-yl]-3-methylbutanoate (1.2 g, 4.70 mmol, 1.00 equiv), ethanol (20 mL), water (10 mL), sodium hydroxide (1.9 g, 47.50 mmol, 10.0 equiv). The resulting solution was stirred for 1 overnight at room temperature.
• Step 7 Into a 100-mL round-bottom flask, was placed 2-[3-(3-hydroxypropyl)-1,2-oxazol-5- yl]-3-methylbutanoic acid (800 mg, 3.52 mmol, 1.00 equiv), (2S,4R)-4-hydroxy-N-[[4-(4- methyl-1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide hydrochloride (1.24 g, 3.50 mmol, 1.00 equiv), N,N-dimethylformamide (15 mL), N-ethyl-N-isopropylpropan-2-amine (1.82 g, 14.08 mmol, 4.00 equiv), T 3 P (1.77 g, 1.20 equiv).
• the resulting solution was stirred for 2 hours at room temperature. The reaction was then quenched by water. The resulting solution was extracted with ethyl acetate and washed with saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (5:1). The collected fractions were combined and concentrated under vacuum.
• the resulting solution was stirred for 4 hours at room temperature.
• the resulting solution was extracted with dichloromethane and washed with water and saturated sodium chloride.
• the mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• Step 9 Into a 50-mL round-bottom flask, was placed 2-(2-hydroxyethoxy)ethan-1-ol (273.0 mg, 2.57 mmol, 5.00 equiv) and N,N-dimethylformamide (5 mL).
• Step 10 Into a 50-mL round-bottom flask, was placed (2S,4R)-4-hydroxy-1-[2-(3-[3-[2-(2- hydroxyethoxy)ethoxy]propyl]-1,2-oxazol-5-yl)-3-methylbutanoyl]-N-[[4-(4-methyl-1,3-thiazol- 5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (160 mg, 0.26 mmol, 1.00 equiv), dichloromethane (5 mL), 4-methylbenzene-1-sulfonyl chloride (59 mg, 0.31 mmol, 1.20 equiv), triethylamine (53 mg, 0.52 mmol, 2.00 equiv), 4-dimethylaminopyridine (6 mg, 0.05 mmol, 0.20 equiv).
• the resulting solution was stirred for 5 hours at room temperature.
• the resulting solution was extracted with dichloromethane and washed with water.
• the mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (12:1). The collected fractions were combined and concentrated under vacuum.
• the resulting solution was stirred for 1 overnight at 80qC.
• the resulting solution was extracted with ethyl acetate and washed with water. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• the resulting solution was stirred for 1 overnight at room temperature.
• the resulting solution was diluted with water.
• the pH value of the solution was adjusted to 8 with sodium carbonate.
• the resulting solution was extracted with ethyl acetate and washed with water. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• the crude product was purified by Prep-HPLC with the following conditions: Column: XBridge Shield RP18 OBD Column, 5um, 19*150mm; Mobile Phase A: Water (0.05%NH 3 H 2 O), Mobile Phase B: acetonitrile; Flow rate: 20 mL/min; Gradient: 34% B to 47% B in 8 min; 220 nm.
• Exemplary Compounds 25 and 26 were prepared using procedures analogous to those described above for Exemplary Compounds 22, 23 and 24.
• Exemplary Synthesis of Exemplary Compound 27 [00435] Step 1 [00436] Into a 50 mL round-bottom flask, was placed 2-[2-(2-hydroxyethoxy)ethoxy]ethan-1- ol (257 mg, 1.71 mmol, 3.00 equiv) and N,N-dimethylformamide (5 mL). This was followed by the addition of sodium hydride (34 mg, 1.42 mmol, 1.50 equiv) at 0qC in 10 minutes.
• Step 2 Into a 50 mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed 2-(2-[2-[(4-bromopyridin-2-yl)oxy]ethoxy]ethoxy)ethan-1-ol (500 mg, 1.63 mmol, 1.00 equiv), tert-butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (347 mg, 1.64 mmol, 1.00 equiv), Cs2CO3 (1599 mg, 4.91 mmol, 3.00 equiv), toluene (8 mL), Ruphos (69 mg, 0.05 equiv).
• the resulting solution was stirred for 5 hours at 100qC in an oil bath. The reaction was then quenched by the addition of 10 mL of water. The resulting solution was extracted with dichloromethane/MeOH and the organic layers combined. The resulting mixture was washed with saturate sodium chloride. The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• Step 3 Into a 50 mL round-bottom flask, was placed tert-butyl 8-(2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (419 mg, 0.96 mmol, 1.00 equiv) and 1M HCl in methanol (8 mL). The resulting solution was stirred for 2 hours at room temperature. The resulting mixture was concentrated under vacuum.
• Step 4 Into a 10 mL microwave tube, was placed 2-(2-[2-[(4-[3,8-diazabicyclo[3.2.1]octan- 8-yl]pyridin-2-yl)oxy]ethoxy]ethoxy)ethan-1-ol (319 mg, 0.95 mmol, 1.00 equiv), 4-bromo-6- chloropyridazin-3-amine (780 mg, 3.74 mmol, 4.00 equiv), DMSO (10 mL), DIEA (2 mL). The final reaction mixture was irradiated with microwave radiation for 3 hours at 130qC. The reaction was then quenched by the addition of 10 mL of water.
• Step 5 Into a 10 mL microwave tube purged and maintained under nitrogen atmosphere was placed 2-[2-[2-([4-[3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8- yl]pyridin-2-yl]oxy)ethoxy]ethoxy]ethan-1-ol (295 mg, 0.63 mmol, 1.00 equiv), [2- (methoxymethoxy)phenyl]boronic acid (223 mg, 1.23 mmol, 2.00 equiv), potassium carbonate (254 mg, 1.84 mmol, 3.00 equiv), dioxane (4 mL), water (1 mL), Pd(PPh3)4 (70 mg, 0.06 mmol, 0.10 equiv).
• the resulting solution was stirred overnight at 100qC. The reaction was then quenched by the addition of water. The resulting solution was extracted with dichloromethane/MeOH and the organic layers combined. The resulting mixture was washed with saturate sodium chloride. The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• the resulting solution was stirred for 2 hours at room temperature. The reaction was then quenched by the addition of water. The resulting solution was extracted with ethyl acetate and the organic layers combined. The resulting mixture was washed with saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• Step 7 Into a 50 mL round-bottom flask, was placed 2-[2-(2-[[4-(3-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl]oxy]ethoxy)ethoxy]ethyl 4-methylbenzene-1-sulfonate (87 mg, 0.12 mmol, 1.00 equiv), N,N- dimethylformamide (54 mg, 0.75 mmol, 1.00 equiv), (2S,4R)-4-hydroxy-1-[2-(3-hydroxy-1,2- oxazol-5-yl)-3-methylbutanoyl]-N-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methylpyrrolidine-2- carboxamide (79 mg, 0.16 m
• the resulting solution was stirred for 4 hours at room temperature.
• the resulting solution was extracted with dichloromethane/MeOH, and the organic layers combined.
• the resulting mixture was washed with saturated sodium chloride.
• the mixture was dried over anhydrous sodium sulfate.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (5:1). The collected fractions were combined and concentrated under vacuum.
• Step 8 Into a 50 mL round-bottom flask was placed (2S,4R)-1-[2-(3-[2-[2-(2-[[4-(3-[3- amino-6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin- 2-yl]oxy]ethoxy)ethoxy]ethoxy]-1,2-oxazol-5-yl)-3-methylbutanoyl]-4-hydroxy-N-[[4-(4- methyl-1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (131 mg, 0.13 mmol, 1.00 equiv), methanol (5 mL), and 1 M HCl in MeOH (1.5 mL).
• the resulting solution was stirred for 7 hours at room temperature.
• the resulting solution was diluted with 5 mL of H2O.
• the pH value of the solution was adjusted to 7 with aqueous Na2CO3 (2M).
• the resulting solution was extracted with dichloromethane, and the organic layers combined.
• the resulting mixture was washed with saturated sodium chloride.
• the crude product was purified by Prep-HPLC with the following conditions: Column, XBridge Shield RP18 OBD Column, 5um, 19*150mm; mobile phase, Water (0.05%NH 3 H 2 O) and acetonitrile (39.0% acetonitrile up to 50.0% in 9 min); Detector, UV 220nm.
• Exemplary Compounds 29 and 30 were prepared using procedures analogous to those described for Exemplary Compounds 27 and 28.
• Exemplary Synthesis of Exemplary Compound 31 [00453] Step 1 [00454] Into a 50 mL round-bottom flask, was placed 2-(oxan-2-yloxy)ethan-1-ol (4.5 g, 30.78 mmol, 1.00 equiv), tetrahydrofuran (10 mL), a solution of t-BuOK (3.6 g, 32.08 mmol, 2.00 equiv) in tetrahydrofuran (60 mL).
• Step 2 Into a 10-mL microwave tube purged and maintained under an inert atmosphere of nitrogen, was placed 2-[2-(prop-2-yn-1-yloxy)ethoxy]oxane (2.8 g, 15.20 mmol, 1.00 equiv), ZrCp2HCl (390 mg, 0.10 equiv), triethylamine (153 mg, 1.52 mmol, 0.10 equiv), pinacolborane (2.5 mL). The resulting solution was stirred overnight at 68 ⁇ in an oil bath.
• the reaction was then quenched by the addition of NH 4 Cl.
• the reaction mixture was cooled to 0qC with a water/ice bath.
• the resulting solution was extracted with ethyl acetate and the organic layers combined.
• the resulting mixture was washed with saturated sodium chloride.
• the mixture was dried over anhydrous sodium sulfate.
• the residue was applied onto a silica gel column eluting with ethyl acetate/petroleum ether (1:5). The collected fractions were combined and concentrated under vacuum.
• Step 3 Boc [00458] Into a 10-mL microwave tube purged and maintained under an inert atmosphere of nitrogen, was placed 4,4,5,5-tetramethyl-2-[(1E)-3-[2-(oxan-2-yloxy)ethoxy]prop-1-en-1-yl]- 1,3,2-dioxaborolane (1 g, 3.20 mmol, 1.00 equiv), Pd(PPh 3 ) 4 (800 mg, 0.69 mmol, 1.00 equiv), potassium carbonate (8 g, 57.88 mmol, 18.07 equiv), dioxane (2 g), tert-butyl 8-(2- bromopyridin-4-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (902 mg, 2.45 mmol, 3.00 equiv), water (252 mg, 0.10 equiv).
• the resulting solution was stirred for 1 overnight at 100qC in an oil bath. The reaction was then quenched by the addition of water. The resulting solution was extracted with ethyl acetate, and the organic layers combined. The resulting mixture was washed with saturate sodium chloride. The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• Step 4 Into a 50-mL round-bottom flask, was placed tert-butyl 8-[2-[(1E)-3-[2-(oxan-2- yloxy)ethoxy]prop-1-en-1-yl]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (639 mg, 1.35 mmol, 1.00 equiv), palladium on carbon (200 mg) and methanol (15 mL), and the mixture was stirred under hydrogen atmosphere overnight at room temperature. The solids were filtered off, and the filtrate was concentrated.
• Step 5 Into a 50-mL round-bottom flask, was placed tert-butyl 8-(2-[3-[2-(oxan-2- yloxy)ethoxy]propyl]pyridin-4-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (639 mg, 1.34 mmol, 1.00 equiv), methanol (15 mL), and hydrogen chloride gas was bubbled through the solution. The resulting solution was stirred for 3 hours at room temperature. The resulting mixture was concentrated under vacuum.
• Step 6 Into a 10-mL microwave tube, was placed 2-[3-(4-[3,8-diazabicyclo[3.2.1]octan-8- yl]pyridin-2-yl)propoxy]ethan-1-ol (387 mg, 1.85 mmol, 1.00 equiv), 4-bromo-6- chloropyridazin-3-amine (1.54 g, 7.39 mmol, 4.00 equiv), DMSO (8 mL), DIEA (1.53 mL, 5.00 equiv). The final reaction mixture was irradiated with microwave radiation for 3 hours at 130qC. The reaction was then quenched by the addition of water.
• Step 7 Into a 50-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed 2-(3-[4-[3-(3-amino-6-chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan- 8-yl]pyridin-2-yl]propoxy)ethan-1-ol (650 mg, 1.55 mmol, 1.00 equiv), [2- (methoxymethoxy)phenyl]boronic acid (566 mg, 3.11 mmol, 2.00 equiv), dioxane (8 mL), water (2 mL), potassium carbonate (644 mg, 4.66 mmol, 3.00 equiv), Pd(PPh 3 ) 4 (180 mg, 0.16 mmol, 0.10 equiv).
• the resulting solution was stirred overnight at room temperature. The reaction was then quenched by the addition of water. The resulting solution was extracted with dichloromethane, and the organic layers combined. The resulting mixture was washed with saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• Step 9 Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed methyl 2-(6-chloropyridin-3-yl)acetate (500 mg, 2.69 mmol, 1.00 equiv), tert-butyl piperazine-1-carboxylate (502 mg, 2.70 mmol, 1.00 equiv), Cs 2 CO 3 (2.63 g, 8.07 mmol, 3.00 equiv), toluene (10 mL), RuPhosPd (115 mg, 0.05 equiv). The resulting solution was stirred overnight at 100qC in an oil bath. The reaction was then quenched by the addition of water.
• Step 10 Into a 50-mL round-bottom flask, was placed tert-butyl 4-[5-(2-methoxy-2- oxoethyl)pyridin-2-yl]piperazine-1-carboxylate (319 mg, 0.95 mmol, 1.00 equiv), dichloromethane (8 mL), trifluoroacetic acid (2 mL). The resulting solution was stirred for 2 h at room temperature. The resulting mixture was concentrated under vacuum. This resulted in 223 mg (100%) of methyl 2-[6-(piperazin-1-yl)pyridin-3-yl]acetate as a yellow oil.
• Step 11 Into a 50-mL round-bottom flask, was placed methyl 2-[6-(piperazin-1-yl)pyridin-3- yl]acetate (101 mg, 0.43 mmol, 1.00 equiv), 2-3-[4-(3-3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl]propoxyethyl 4-methylbenzene-1-sulfonate (320 mg, 0.47 mmol, 1.10 equiv), acetonitrile (4 mL), potassium carbonate (298 mg, 2.16 mmol, 5.00 equiv), NaI (193 mg, 3.00 equiv).
• Step 12 Into a 50-mL round-bottom flask, was placed methyl 2-[6-[4-(2-[3-[4-(3-[3-amino-6- [2-(methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl]propoxy]ethyl)piperazin-1-yl]pyridin-3-yl]acetate (94 mg, 0.13 mmol, 1.00 equiv), methanol (5 mL), water (2 mL), and LiOH (15 mg, 0.64 mmol, 5.00 equiv).
• the resulting solution was stirred for 2 days at room temperature. The resulting mixture was concentrated under vacuum. The resulting solution was diluted with 10 mL of H 2 O. The resulting solution was extracted with dichloromethane, and the aqueous layers were combined and concentrated under vacuum. The residue was dissolved in 10 mL of methanol. The solids were filtered out. The resulting mixture was concentrated under vacuum.
• Exemplary Compound 50 was prepared using procedures described for Exemplary Compound 31.
• Exemplary Synthesis of Exemplary Compound 32 [00480] Step 1 [00481] Into a 250-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed a solution of 2-[2-(2-hydroxyethoxy)ethoxy]ethan-1-ol (10.0 g, 66.59 mmol, 1.00 equiv) in dichloromethane (100 mL), (diethyloxonio)trifluoroborate (1.9 g, 13.33 mmol, 0.20 equiv), and ethyl 2-diazoacetate (3.8 g, 0.50 equiv).
• Step 2 Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of ethyl 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]acetate (1.0 g, 4.23 mmol, 1.00 equiv) in dichloromethane (20 mL), 4-methylbenzene-1-sulfonyl chloride (970 mg, 5.09 mmol, 1.20 equiv), triethylamine (860.0 mg, 8.50 mmol, 2.00 equiv). The resulting solution was stirred for 5 hours at room temperature. The reaction was then quenched by the addition of water.
• Step 3 Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of ethyl 2-[2-[2-(2-[[(4- methylbenzene)sulfonyl]oxy]ethoxy)ethoxy]ethoxy]acetate (1.0 g, 2.56 mmol, 1.20 equiv) in N,N-dimethylformamide (20 mL), 6-bromopyridin-2-ol (370 mg, 2.13 mmol, 1.00 equiv), Cs 2 CO 3 (2.1 g, 6.45 mmol, 3.00 equiv). The resulting solution was stirred for 12 hours at 80qC.
• Step 1 Into a 250-mL round-bottom flask, was placed 1-bromo-4-ethylbenzene (5.5 g, 29.8 mmol, 1.0 equiv), CCl4 (100 mL), AIBN (490 mg, 3.0 mmol, 0.1 equiv), N-Bromosuccinimide (5.34 g, 30.0 mmol, 1.0 equiv). The resulting solution was stirred for 3 hours at 90°C. The resulting mixture was concentrated under vacuum.
• Step 2 Into a 100-mL round-bottom flask, was placed 6-[2-(methoxymethoxy)phenyl]-4- (1H-pyrazol-4-yl)pyridazin-3-amine (300 mg, 1.0 mmol, 1.0 equiv), N,N-dimethylformamide (5.0 mL), 1-bromo-4-(1-bromoethyl)benzene (400.0 mg, 1.5 mmol, 1.5 equiv), potassium carbonate (414 mg, 3.0 mmol, 3.0 equiv).
• Step 3 Into a 250-mL round-bottom flask, was placed prop-2-yn-1-ol (10 g, 178.4 mmol, 1.0 equiv), tetrahydrofuran (100.0 mL), sodium hydride (6.4 g, 266.7 mmol, 0.9 equiv), and tert- butyl 2-bromoacetate (28 g, 143.6 mmol, 0.8 equiv). The resulting solution was stirred for 3 hours at room temperature. The reaction was then quenched by the addition of 20 mL of ammonium chloride aqueous solution. The resulting mixture was concentrated under vacuum.
• Step 4 [00495] Into a 250-mL round-bottom flask, was placed tert-butyl 2-(prop-2-yn-1- yloxy)acetate (6.6 g, 38.8 mmol, 1.0 equiv), triethylamine (400.0 mg, 3.9 mmol, 0.1 equiv), pinacolborane (20.0 mL), ZrCp 2 HCl (1 g, 0.1 equiv). The resulting solution was stirred for 12 minutes at 60°C. The reaction was then quenched by the addition of 10 mL of ice/water. The resulting solution was extracted with ethyl acetate (30 mL x 3), and the organic layers were combined.
• Step 5 Into a 10-mL sealed tube, was placed 4-[1-[1-(4-bromophenyl)ethyl]-1H-pyrazol-4- yl]-6-[2-(methoxymethoxy)phenyl] pyridazin-3-amine (300 mg, 0.6 mmol, 1.0 equiv), tert-butyl 2-[[(2E)-3-(tetramethyl-1,3,2-dioxaborolan-2-yl)prop-2-en-1-yl]oxy]acetate (279 mg, 0.9 mmol, 1.5 equiv), Pd(PPh3)4 (72 mg, 0.06 mmol, 0.1 equiv), potassium carbonate (259 mg, 1.9 mmol, 3.0 equiv), dioxane (4.0 mL), and H2O (1.0 mL).
• Step 6 [00499] Into a 100-mL round-bottom flask, was placed tert-butyl 2-[[(2E)-3-[4-[1-(4-[3- amino-6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]-1H-pyrazol-1-yl)ethyl]phenyl]prop-2-en- 1-yl]oxy]acetate (93 mg, 0.2 mmol, 1.0 equiv), methanol (5.0 mL), palladium on carbon (100 mg). The resulting solution was stirred under hydrogen atmosphere for 1 hour at room temperature. The solids were filtered off. The resulting mixture was concentrated under vacuum.
• Step 7 Into a 100-mL round-bottom flask, was placed tert-butyl 2-(3-[4-[1-(4-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-1H-pyrazol-1-yl)ethyl]phenyl]propoxy)acetate (56 mg, 0.1 mmol, 1.0 equiv), dichloromethane (10.0 mg), trifluoroacetic acid (5 mL). The resulting solution was stirred for 2 hours at room temperature. The resulting mixture was concentrated under vacuum.
• Exemplary Synthesis of Exemplary Compound 34 was prepared according to the scheme below using procedures described for other examples above, as well as procedures known and appreciated to those skilled in the art.
• Exemplary Synthesis of Exemplary Compound 36 and Exemplary Compound 37 was prepared according to the scheme below using procedures described for other examples above, as well as as well as procedures known and appreciated to those skilled in the art.
• Step 1 Into a 500-mL round-bottom flask, was placed 2-(piperazin-1-yl)ethan-1-ol (26.0 g, 199.7 mmol, 1.0 equiv), dichloromethane (200.0 mL), triethylamine (40.4 g, 399.3 mmol, 2.0 equiv), benzyl carbonochloridate (40.8 g, 239.2 mmol, 1.2 equiv). The resulting solution was stirred for 2 h at 0°C. The reaction was then quenched by the addition of 10 mL of water.
• Step 2 Into a 100-mL round-bottom flask, was placed benzyl 4-(2-hydroxyethyl)piperazine- 1-carboxylate (5.3 g, 20.1 mmol, 1.0 equiv) and N,N-dimethylformamide (30.0 mL).
• Step 3 Into a 250-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed a solution of benzyl 4-[2-[(4-bromopyridin-2-yl)oxy]ethyl]piperazine-1- carboxylate (2.1 g, 5.0 mmol, 1.0 equiv), RuphosPd II (775.0 mg, 1.0 mmol, 0.2 equiv), and Cs 2 CO 3 (4.89 g, 15.0 mmol, 3.0 equiv) in PhMe (100 mL).
• Step 4 [00517] Into a 100-mL round-bottom flask, was placed tert-butyl8-[2-(2-[4- [(benzyloxy)carbonyl]piperazin-1-yl]ethoxy)pyridin-4-yl]-3,8-diazabicyclo[3.2.1] octane-3- carboxylate (670 mg, 1.2 mmol, 1.0 equiv) in methanol (10.0 mL). Then hydrogen chloride in 1,4-dioxane solution (4M, 10 mL) was added. The resulting solution was stirred for 5 hours at room temperature. The resulting mixture was concentrated under vacuum.
• Step 5 Into a 10-mL sealed tube, was placed benzyl 4-[2-[(4-[3,8-diazabicyclo[3.2.1]octan- 8-yl]pyridin-2-yl)oxy]ethyl]piperazine-1-carboxylate hydrochloride (200.0 mg, 0.4 mmol, 1.0 equiv), 4-bromo-6-chloropyridazin-3-amine (137.0 mg, 0.7 mmol, 1.5 equiv), methyl sulfoxide (3.0 mL), and N,N-diisopropylethylamine (1 mL).
• the final reaction mixture was irradiated with microwave radiation for 6 hours at 130°C.
• the reaction was then quenched by the addition of 1 mL of water.
• the resulting solution was extracted with ethyl acetate (20 mL x3), and the organic layers were combined and concentrated under vacuum. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 6 Into a 100-mL round-bottom flask, was placed benzyl 4-[2-([4-[3-(3-amino-6- chloropyridazin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl]pyridin-2-yl]oxy)ethyl]piperazine-1- carboxylate (390 mg, 0.7 mmol, 1.0 equiv), dioxane (8.0 mL), water (2.0 mL), potassium carbonate (279 mg, 2.0 mmol, 3.0 equiv), [2-(methoxymethoxy)phenyl]boronic acid (184.0 mg, 1.0 mmol, 1.50 equiv), Pd(PPh3)4 (78 mg, 0.07 mmol, 0.1 equiv).
• Step 7 To a solution of benzyl 4-(2-[[4-(3-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl]oxy]ethyl)piperazine-1-carboxylate (200 mg, 0.29 mmol, 1.00 equiv) in 10 mL of isopropanol was added palladium hydroxide (100 mg, 0.71 mmol, 2.42 equiv) under nitrogen atmosphere in a 100-mL round bottom flask.
• Step 8 Into a 100-mL round-bottom flask, was placed 4-(tetramethyl-1,3,2-dioxaborolan-2- yl)-1H-pyrazole (1.9 g, 10.0 mmol, 1.00 equiv), N,N-dimethylformamide (30 mL), ethyl 2- bromo-3-methylbutanoate (2.1 g, 10.0 mmol, 1.0 equiv), and cesium carbonate (10.0 g, 30.7 mmol, 3.0 equiv). The resulting solution was stirred for 12 hours at 90°C. The reaction was then quenched by the addition of 10 mL of water.
• Step 9 Into a 250-mL round-bottom flask, was placed ethyl 3-methyl-2-[4-(tetramethyl- 1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]butanoate (3.0 g, 9.3 mmol, 1.0 equiv), tetrahydrofuran (30 mL), water (30 mL), sodium perborate (3.5 g, 23.3 mmol, 2.5 equiv). The resulting solution was stirred for 12 hours at room temperature. The resulting mixture was concentrated under vacuum.
• Step 10 Into a 50-mL round-bottom flask, was placed ethyl 2-(4-hydroxy-1H-pyrazol-1-yl)-3- methylbutanoate (3.5 g, 16.5 mmol, 1.0 equiv), methanol (10 mL), water (10 mL), and sodium hydroxide (2.7 g, 67.5 mmol, 4.0 equiv). The resulting solution was stirred for 12 hours at room temperature. The pH value of the solution was adjusted to 2 with 2M HCl.
• the crude product was purified by prep-HPLC with the following conditions: Column, XBridge Shield RP18 OBD Column,, 5um,19*150mm; mobile phase, Water (0.1% FA) and acetonitrile (0.0% acetonitrile up to 4.0% in 2 min, up to 20.0% in 8 min); Detector, UV 254nm. This resulted in 1.5 g (49%) of 2- (4-hydroxy-1H-pyrazol-1-yl)-3-methylbutanoic acid as a white oil.
• Step 11 Into a 100-mL round-bottom flask, was placed 2-(4-hydroxy-1H-pyrazol-1-yl)-3- methylbutanoic acid (500 mg, 2.7 mmol, 1.0 equiv), N,N-dimethylformamide (20 mL), (2S,4R)- 4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide hydrochloride (900 mg, 2.5 mmol, 0.9 equiv), DIEA (1.1 g, 8.5 mmol, 3.0 equiv), T3P (2.6 g, 1.5 equiv).
• Step 12 Into a 5-mL sealed tube, was placed (2S,4R)-4-hydroxy-1-[2-(4-hydroxy-1H-pyrazol- 1-yl)-3-methylbutanoyl]-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2- carboxamide (300 mg, 0.60 mmol, 1.0 equiv), N,N-dimethylformamide (5.0 mL), 1,2- dibromoethane (222 mg, 1.2 mmol, 2.0 equiv), potassium carbonate (250 mg, 1.8 mmol, 3.0 equiv).
• the resulting solution was stirred for 12 hours at 70°C.
• the reaction was then quenched by the addition of 1 mL of water.
• the crude product was purified by Prep-HPLC with the following conditions: Column, XBridge Shield RP18 OBD Column,, 5um,19*150mm; mobile phase, water (10 mmol/L ammonium bicarbonate) and acetonitrile (35.0% acetonitrile up to 47.0% in 8 min); Detector, UV 254nm.
• Step 13 Into a 100-mL round-bottom flask, was placed 6-[2-(methoxymethoxy)phenyl]-4-(8- [2-[2-(piperazin-1-yl)ethoxy]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-amine (54.6 mg, 0.1 mmol, 1.0 equiv), acetonitrile (10.0 mL), potassium carbonate (41 mg, 0.30mmol, 3.0 equiv), sodium iodide (18 mg, 0.1 mmol, 1.2 equiv), (2S,4R)-1-2-[4-(2-bromoethoxy)-1H- pyrazol-1-yl]-3-methylbutanoyl-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol- 5yl)phenyl]ethy
• the resulting solution was stirred for 12 hours at 70°C. The reaction was then quenched by the addition of 3 mL of water. The resulting solution was extracted with ethyl acetate (30 mL x3), and the organic layers were combined and concentrated under vacuum. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10/1).
• Step 14 [00537] Into a 25-mL round-bottom flask, was placed (2S,4R)-1-[2-(4-[2-[4-(2-[[4-(3-[3- amino-6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin- 2-yl]oxy]ethyl)piperazin-1-yl]ethoxy]-1H-pyrazol-1-yl)-3-methylbutanoyl]-4-hydroxy-N-[(1S)- 1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (40 mg, 0.04 mmol, 1.0 equiv), methanol (8 mL), hydrogen chloride (150 mL).
• Step 1 Into a 250-mL round-bottom flask, was placed a solution of 2-bromo-4- fluoropyridine (7.8 g, 44.32 mmol, 1.05 equiv), tert-butyl 3,8-diazabicyclo[3.2.1]octane-3- carboxylate (9 g, 42.4 mmol, 1.00 equiv), DIEA (25 g, 193 mmol, 4.00 equiv) in NMP (120 mL). The resulting solution was stirred for 3 hours at 150qC. The reaction mixture was cooled.
• Step 2 Into a 250-mL round-bottom flask, was placed a mixture of [(prop-2-yn-1- yloxy)methyl]benzene (10 g, 68.4 mmol, 1.00 equiv), pinacolborane (10 mL), ZrCp2HCl (2 g), triethylamine (850 mg, 8.40 mmol, 0.12 equiv).
• Step 3 Into a 250-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed a solution of tert-butyl 8-(2-bromopyridin-4-yl)-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (4.0 g, 10.9 mmol, 1.00 equiv), 2-[(1E)-3- (benzyloxy)prop-1-en-1-yl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4.48 g, 16.3 mmol, 1.50 equiv), Pd(dppf) 2 Cl 2 dichloromethane (890 mg, 1.09 mmol, 0.10 equiv), and potassium carbonate (4.5 g, 32.6 mmol, 3.00 equiv) in dioxane/H 2 O (80/20 mL).
• Step 4 Into a 250-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed a solution of tert-butyl 8-[2-[(1E)-3-(benzyloxy)prop-1-en-1-yl]pyridin- 4-yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (12.9 g, 29.6 mmol, 1.00 equiv) in methanol/HOAc (200/0.5 mL). Palladium on carbon (6 g) was added under nitrogen atmosphere. The flask was then vacuumed and flushed with hydrogen.
• the reaction mixture was hydrogenated at 50qC for 20 hours under hydrogen atmosphere using a hydrogen balloon.
• the solids were filtered out through a celite pad, and the filtrate was concentrated under reduced pressure. This resulted in 10.2 g (99%) of tert-butyl 8-[2-(3-hydroxypropyl)pyridin-4-yl]-3,8- diazabicyclo[3.2.1]octane-3-carboxylate as a yellow oil.
• Step 5 Into a 100-mL round-bottom flask was placed tert-butyl 8-[2-(3- hydroxypropyl)pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (700 mg, 2.01 mmol, 1.00 equiv) in dichloromethane (30 mL), and Dess-Martin periodinane reagent (1.7 g, 2.00 equiv) was added at 0 ⁇ 5qC. The resulting solution was stirred for 6 hours at room temperature. The resulting mixture was concentrated under vacuum at 15qC.
• Step 6 Into a 50-mL round-bottom flask, was placed tert-butyl 8-[2-(3-oxopropyl)pyridin-4- yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (1.4 g, 4.05 mmol, 1.00 equiv), benzyl piperazine-1-carboxylate (1.07 g, 4.86 mmol, 1.20 equiv) in dichloromethane (20 mL). It was stirred for 20 min and NaBH(OAc) 3 (2.58 g, 12.2 mmol, 3.00 equiv) was added at 0qC.
• the resulting solution was stirred for 4 hours at room temperature. The reaction was then quenched by the addition of water/ice (50 mL). The resulting solution was extracted with dichloromethane (50 mL x 3) and the organic layers were combined and dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10/1).
• Step 7 Into a 100-mL round-bottom flask, was placed tert-butyl 8-[2-(3-[4- [(benzyloxy)carbonyl]piperazin-1-yl]propyl)pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octane-3- carboxylate (2.19 g, 3.98 mmol, 1.00 equiv) in methanol (20 mL). Hydrogen chloride gas was bubbled through the resulting solution while stirring for 1 h at room temperature. The resulting mixture was concentrated under vacuum.
• Step 8 Into a 20-mL microwave tube purged and maintained under an inert atmosphere of nitrogen was placed benzyl 4-[3-(4-[3,8-diazabicyclo[3.2.1]octan-8-yl]pyridin-2- yl)propyl]piperazine-1-carboxylate hydrochloride (1.79 g, 3.68 mmol, 1.00 equiv), 4-bromo-6- chloropyridazin-3-amine (2.47 g, 11.9 mmol, 3.00 equiv), DIEA (5.0 mL) in DMSO (10 mL). The resulting solution was stirred for 16 hours at 130qC in an oil bath.
• Step 9 Into a 50-mL round-bottom flask, was placed a solution of (2S,4R)-4-hydroxy-1-[2- (3-hydroxy-1,2-oxazol-5-yl)-3-methylbutanoyl]-N-[[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl]pyrrolidine-2-carboxamide (480 mg, 1.0 mmol, 1.0 equiv) in N,N- dimethylformamide (10 mL), potassium carbonate (300 mg, 2.2 mmol, 2.2 equiv) and 1,2- dibromoethane (300 mg, 1.6 mmol, 1.6 equiv).
• the resulting solution was stirred for 2 hours at 70°C in an oil bath. The reaction was then quenched by the addition of 20 mL of water. The resulting solution was extracted with of ethyl acetate (3x30 mL), and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column eluting with ethyl acetate/petroleum ether (1:0).
• Step 10 Into a 20-mL microwave tube purged and maintained under an inert atmosphere of nitrogen was placed 6-[2-(methoxymethoxy)phenyl]-4-(8-[2-[3-(piperazin-1-yl)propyl]pyridin-4- yl]-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-amine (92 mg, 0.17 mmol, 1.00 equiv) [prepared from benzyl 4-(3-[4-[3-(3-amino-6-chloropyridazin-4-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl]pyridin-2-yl]propyl)piperazine-1-carboxylate from step 8 according to the scheme below and using procedures described above for other examples], (2S,4R)-1-2-[3-(2-bromoethoxy)-1,2-o
• the resulting mixture was stirred for 12 hours at 70qC in an oil bath.
• the resulting solution was extracted with dichloromethane (100 mL) and the organic layers combined.
• the resulting mixture was washed with brine (30 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• Exemplary Synthesis of Exemplary Compound 40 [00562] Exemplary Compound 40 was prepared according to the scheme below using procedures described for other examples above as well as procedures appreciated by those skilled in the art. [00563] Exemplary Synthesis of Exemplary Compound 41 and Exemplar Compound 42 [00564] Exemplary Compounds 41 and 42 were prepared according to the scheme below using procedures described above for other exemplary compounds.
• Step 1 Into a 250-mL round-bottom flask, was placed a solution of 2-[2-(prop-2-yn-1- yloxy)ethoxy]ethan-1-ol (1 g, 6.94 mmol, 1.00 equiv) in dichloromethane (80 mL), triethylamine (2.8 g, 27.67 mmol, 3.00 equiv), 4-dimethylaminopyridine (270 mg, 2.21 mmol, 0.30 equiv), and 4-toluenesulfonyl chloride (1.9 g, 9.97 mmol, 1.50 equiv).
• Step 2 Into a 25-mL sealed tube, was placed a solution of 2-[2-(prop-2-yn-1- yloxy)ethoxy]ethyl 4-methylbenzene-1-sulfonate (1.4 g, 4.69 mmol, 1.00 equiv) in N,N- dimethylformamide (10 mL), potassium carbonate (1.6 g, 11.58 mmol, 3.00 equiv), sodium iodide (280 mg, 0.50 equiv), and tert-butyl 2-(piperazin-1-yl)acetate (898 mg, 4.48 mmol, 1.20 equiv). The resulting solution was stirred for 12 hours at 70qC in an oil bath.
• Step 3 Into a 25-mL sealed tube, was placed a solution of tert-butyl 2-(4-[2-[2-(prop-2-yn-1- yloxy)ethoxy]ethyl]piperazin-1-yl)acetate (910 mg, 2.79 mmol, 1.00 equiv) in tetrahydrofuran (10 mL), pinacolborane (630 mg, 2.00 equiv), ZrCp2HCl (70 mg, 0.10 equiv), and triethylamine (1 mL, 3.00 equiv). The resulting solution was stirred for 12 hours at 80qC in an oil bath. The resulting mixture was concentrated under vacuum.
• Step 4 Into a 100-mL round-bottom flask, was placed a solution of tert-butyl 3,8- diazabicyclo[3.2.1]octane-8-carboxylate (2.0 g, 9.42 mmol, 1.00 equiv) in dimethyl sulfoxide (40 mL), N,N-diisopropylethylamine (714.0 mg, 5.52 mmol, 4.00 equiv), 4-bromo-6- chloropyridazin-3-amine (3.0 g, 14.39 mmol, 1.50 equiv). The resulting solution was stirred for 3 hours at 130qC in an oil bath.
• the reaction was then quenched by the addition of water (90 mL).
• the resulting solution was extracted with ethyl acetate (80mL x 3) and the organic layers combined.
• the resulting mixture was washed with brine (100 mL x 1).
• the mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• the residue was applied onto a silica gel column with dichloromethane/methanol (10/1). The collected fractions were combined and concentrated under vacuum.
• Step 5 Into a 250-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of tert-butyl 3-(3-amino-6-chloropyridazin-4-yl)- 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (1.7 g, 5.00 mmol, 1.00 equiv) in dioxane/water (50/10 mL), potassium carbonate (2.1 g, 15.19 mmol, 3.00 equiv), (2-hydroxyphenyl)boronic acid (1.04 g, 7.54 mmol, 1.50 equiv), tetrakis(triphenylphosphine)palladium(0) (578 mg, 0.50 mmol, 0.10 equiv).
• the resulting solution was stirred for 2 hours at 100qC in an oil bath. The reaction was then quenched by the addition of water (60 mL). The resulting solution was extracted with ethyl acetate (40 mL x 3) and the organic layers combined. The resulting mixture was washed with brine (60 mL x 1). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10/1). The collected fractions were combined and concentrated under vacuum.
• Step 6 Into a 250-mL round-bottom flask, was placed a solution of tert-butyl 3-[3-amino-6- (2-hydroxyphenyl)pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (1.62 g, 4.08 mmol, 1.00 equiv) in dioxane (50 mL). To the above hydrogen chloride (gas) was introduced in. The resulting solution was stirred for 5 hours at room temperature. The resulting mixture was concentrated under vacuum.
• Step 7 Into a 100-mL round-bottom flask, was placed a solution of 2-(6-amino-5-[3,8- diazabicyclo[3.2.1]octan-3-yl]pyridazin-3-yl)phenol (400 mg, 1.35 mmol, 1.00 equiv) in 1- methyl-2-pyrrolidinone (30 mL), N,N-diisopropylethylamine (693 mg, 5.36 mmol, 4.00 equiv), 2-bromo-4-fluoropyridine (355.0 mg, 2.02 mmol, 1.50 equiv). The resulting solution was stirred for 2 hours at 130qC in an oil bath.
• the reaction was then quenched by the addition of water (40 mL).
• the resulting solution was extracted with ethyl acetate (30 mL x 3), and the organic layers were combined.
• the resulting mixture was washed with brine (40 mL x 1).
• the mixture was dried over anhydrous sodium sulfate and concentrated under vacuum.
• the residue was applied onto a silica gel column eluting with ethyl acetate/petroleum ether (4/1). The collected fractions were combined and concentrated under vacuum.
• Step 8 [00581] Into a 20-mL sealed tube was placed a solution of tert-butyl 2-[4-[2-(2-[[(2E)-3- (tetramethyl-1,3,2-dioxaborolan-2-yl)prop-2-en-1-yl]oxy]ethoxy)ethyl]piperazin-1-yl]acetate (628 mg, 1.38 mmol, 1.00 equiv) in dioxane/water (5:1 mL), tetrakis(triphenylphosphine)palladium (108 mg, 0.15 mmol, 0.10 equiv), potassium carbonate (286 mg, 2.07 mmol, 1.50 equiv), 2-(6-amino-5-((1R,5S)-8-(2-bromopyridin-4-yl)-3,8- diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-yl)phenol
• the resulting solution was stirred for 2 hours at 80qC in an oil bath.
• the resulting mixture was concentrated under vacuum.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1). The collected fractions were combined and concentrated under vacuum.
• Step 9 Into a 100-mL round-bottom flask, was placed a solution of tert-butyl 2-[4-[2-(2- [[(2E)-3-(4-[3-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8- yl]pyridin-2-yl)prop-2-en-1-yl]oxy]ethoxy)ethyl]piperazin-1-yl]acetate (200 mg, 0.29 mmol, 1.00 equiv) in 2-propanol (30 mL) and PtO 2 (10 mg, 0.10 equiv).
• Step 10 Into a 100-mL round-bottom flask, was placed a solution of tert-butyl 2-[4-(2-[2-[3- (4-[3-[3-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl]pyridin-2- yl)propoxy]ethoxy]ethyl)piperazin-1-yl]acetate (190 mg, 0.27 mmol, 1.00 equiv) in dioxane (20 mL). Into the above hydrogen chloride (gas) was introduced. The resulting solution was stirred for 2 hours at room temperature.
• hydrogen chloride gas
• Step 11 Into a 100-mL round-bottom flask, was placed 2-[4-(2-2-[3-(4-3-[3-amino-6-(2- hydroxyphenyl)pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-ylpyridin-2- yl)propoxy]ethoxyethyl)piperazin-1-yl]acetic acid (160 mg, 0.25 mmol, 1.00 equiv), N,N- diisopropylethylamine (0.3 mL, 0.30 equiv), a solution of (2S,4R)-1-[(2S)-2-amino-3,3- dimethylbutanoyl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2- carboxamide hydrochloride (139 mg, 0.30 mmol, 1.
• the resulting solution was stirred for 2 hours at room temperature.
• the resulting mixture was concentrated under vacuum.
• the crude product (100 mL) was purified by prep-HPLC with the following conditions: Column, XBridge Prep C18 OBD Column, 5um,19*150mm; mobile phase, Water(10 mmol/L ammonium bicarbonate) and acetonitrile (26.0% acetonitrile up to 49.0% in 8 min); Detector, UV 254nm.
• Step 1 Into a 50-mL round-bottom flask, was placed a solution of 2-(3-methoxy-1,2-oxazol- 5-yl)-3-methylbutanoic acid (2.0 g, 10.0 mmol, 1.0 equiv) in N,N-dimethylformamide (20 mL), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophospate (4.1 g, 10.8 mmol, 1.1 equiv), N,N-diisopropylethylamine (6.0 g, 46.4 mmol, 4.6 equiv), and (2S,4R)-4- hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]eth
• the resulting solution was stirred for 30 minutes at room temperature. The reaction was then quenched by the addition of 30 mL of water. The resulting solution was extracted with of ethyl acetate (30 mL x 3), and the organic layers were combined and concentrated under vacuum. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 3 Into a 50-mL round-bottom flask, was placed a solution of (2S,4R)-4-hydroxy-1-[2- (3-hydroxy-1,2-oxazol-5-yl)-3-methylbutanoyl]-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5- yl)phenyl]ethyl]pyrrolidine-2-carboxamide (1.9 g, 3.8 mmol, 1.0 equiv) in CH 3 CN (20 mL), potassium carbonate (3.5 g, 25.3 mmol, 6.6 equiv), sodium iodide (800.0 mg, 5.3 mmol, 1.4 equiv), and 1,2-dibromoethane (1 g, 5.3 mmol, 1.4 equiv).
• Step 4 Into a 50-mL round-bottom flask, was placed a solution of (2S,4R)-1-[2-[3-(2- bromoethoxy)-1,2-oxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3- thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (200 mg, 0.3 mmol, 1.0 equiv) in CH3CN (20 mL), potassium carbonate (400 mg, 2.9 mmol, 8.8 equiv), sodium iodide (80 mg, 0.53 mmol, 1.6 equiv), and 6-[2-(methoxymethoxy)phenyl]-4-(8-2-[2-(piperazin-1-yl)ethoxy]pyridin- 4-yl-3,8-diazabicyclo[3.2.1]oc
• Step 1 Into a 100-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed methyl 2-(6-chloropyridin-3-yl)acetate (1.86 g, 10.0 mmol, 1.00 equiv), toluene (50 mL), Cs 2 CO 3 (10.0 g, 30.0 mmol, 3.00 equiv), 1-benzylpiperazine (2.11 g, 12.0 mmol, 1.2 equiv), and RuPhosPd (0.39 g, 0.05 equiv). The resulting solution was stirred for 10 hours at 100°C in an oil bath.
• Step 2 Into a 100-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed tetrahydrofuran (30 mL), lithium aluminium hydride (0.34 g, 3.00 equiv). This was followed by the addition of a solution of methyl 2-[6-(4-benzylpiperazin- 1-yl)pyridin-3-yl]acetate (1.0 g, 3.01 mmol, 1.00 equiv) in tetrahydrofuran (5 mL) dropwise with stirring at 0°C in 5 minutes. The resulting solution was stirred for 10 hours at 25°C.
• Step 3 Into a 50-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed 2-[6-(4-benzylpiperazin-1-yl)pyridin-3-yl]ethan-1-ol (750 g, 2.5 mmol, 1.00 equiv), N,N-dimethylformamide (20 mL), sodium hydride (60%) (200 mg, 8.4 mmol, 2.00 equiv) at 0°C. The resulting solution was stirred for 0.5 hour at 0°C.
• Step 4 Into a 25-mL sealed tube purged and maintained with an inert atmosphere of nitrogen, was placed 1-benzyl-4-[5-[2-(prop-2-yn-1-yloxy)ethyl]pyridin-2-yl]piperazine (1.0 g, 3.00 mmol, 1.00 equiv), tetrahydrofuran (15 mL), pinacolborane (2 mL), ZrCp2HCl (0.077 g, 0.10 equiv), triethylamine (0.030 g, 0.10 equiv).
• Step 5 Into a 100-mL round-bottom flask, was placed 1-benzyl-4-[5-(2-[[(2E)-3- (tetramethyl-1,3,2-dioxaborolan-2-yl)prop-2-en-1-yl]oxy]ethyl)pyridin-2-yl]piperazine (928 mg, 2.00 mmol, 1.00 equiv), dichloromethane (25 mL). This was followed by the addition of 1- chloroethyl chloroformate (568.0 mg, 3.97 mmol, 2.00 equiv) dropwise with stirring at 0°C. The resulting solution was stirred for 1 hour at 25°C.
• Step 6 Into a 100-mL 3-neck round-bottom flask purged and maintained under an inert atmosphere of nitrogen was placed N-(2-aminoethyl)-N-(2-chloroethyl)-5-(2-[[(2E)-3- (tetramethyl-1,3,2-dioxaborolan-2-yl)prop-2-en-1-yl]oxy]ethyl)pyridin-2-amine (410 mg, 1.00 mmol, 1.00 equiv) in dichloromethane, N,N-diisopropylethylamine (645 mg, 4.99 mmol, 5.00 equiv), and tert-butyl 2-bromoacetate (585 mg, 3.00 mmol, 3.00 equiv) was added.
• the resulting solution was stirred for 3 hours at 80°C in an oil bath.
• the reaction mixture was cooled to 25°C.
• the reaction was then quenched by the addition of water (5mL).
• the resulting solution was extracted with ethyl acetate (20 mL x 2), and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated under vacuum.
• Step 1 Into a 100-mL round-bottom flask, was placed benzyl 3-hydroxyazetidine-1- carboxylate (3.11 g, 15.01 mmol, 1.00 equiv), dichloromethane (30 mL), tert-butyl 2- bromoacetate (4.37 g, 22.40 mmol, 1.50 equiv), sodium hydroxide(37%aq.) (30 mL), and TBAC (4.17 g, 1.00 equiv) at 0qC. The resulting solution was stirred for 3 hours at 25qC.
• Step 2 Into a 250-mL round-bottom flask, was placed benzyl 3-[2-(tert-butoxy)-2- oxoethoxy]azetidine-1-carboxylate (3.22 g, 10.02 mmol, 1.00 equiv), ethanol (100 mL), palladium on carbon (0.322 g). The resulting solution was stirred for 12 hours under H 2 atmosphere at 25qC. The solids were filtered out. The resulting mixture was concentrated under vacuum. This resulted in 1.75 g (93%) of tert-butyl 2-(azetidin-3-yloxy)acetate as a colorless oil.
• Step 3 Into a 50-mL round-bottom flask, was placed tert-butyl 2-(azetidin-3-yloxy)acetate (374 mg, 2.00 mmol, 1.00 equiv), N,N-dimethylformamide (15 mL), potassium carbonate (828 mg, 5.99 mmol, 3.00 equiv), and 1,2-dibromoethane (1.86 g, 9.90 mmol, 5.00 equiv). The resulting solution was stirred for 12 hours at 25qC. The reaction was then quenched by the addition of water (10mL).
• Step 1 Into a 100-mL round-bottom flask, was placed 2-(3-methoxy-1,2-oxazol-5-yl)-3- methylbutanoic acid (2.1 g, 10.5 mmol, 1.00 equiv), ethanol (20.0 g, 434 mmol, 41.2 equiv), sulfuric acid (2.0 g, 20.4 mmol, 1.9 equiv). The resulting solution was stirred for 12 hours at 70°C in an oil bath. The resulting mixture was concentrated under vacuum.
• Step 2 Into a 50-mL round-bottom flask was placed ethyl 2-(3-methoxy-1,2-oxazol-5-yl)-3- methylbutanoate (1.9 g, 8.4 mmol, 1.0 equiv) and sulfuryl chloride (20 mL). The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. The reaction was then quenched by the addition of 30 mL of water.
• Step 3 Into a 50-mL round-bottom flask, was placed a solution of ethyl 2-(4-chloro-3- methoxy-1,2-oxazol-5-yl)-3-methylbutanoate (1.5 g, 5.7 mmol, 1.0 equiv) in methanol/H2O (5/5 mL), and lithium hydroxide (300 mg, 12.5 mmol, 2.2 equiv). The resulting solution was stirred overnight at 50°C in an oil bath. The pH value of the solution was adjusted to 6 with 1M HCl. The resulting solution was extracted with ethyl acetate (30 ml x 3), and organic layers were combined and concentrated under vacuum.
• the resulting solution was stirred for 30 minutes at room temperature. The reaction was then quenched by the addition of 30 mL of water. The resulting solution was extracted with ethyl acetate (30 ml x 3), and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 5 Into a 50-mL round-bottom flask, was placed (2S,4R)-1-[2-(4-chloro-3-methoxy-1,2- oxazol-5-yl)-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5- yl)phenyl]ethyl]pyrrolidine-2-carboxamide (275 mg, 0.5 mmol, 1.0 equiv) and 4M hydrogen chloride solution in dioxane (25 mL). The resulting solution was stirred for 96 hours at 60°C in an oil bath. The resulting mixture was concentrated under vacuum.
• Step 1 Into a 250-mL round-bottom flask, was placed a solution of 3- (benzyloxy)cyclobutan-1-one (6.0 g, 34.05 mmol, 1.0 equiv) in ethanol (100 mL). This was followed by the addition of sodium borohydride (1.25 g, 33.94 mmol, 1.00 equiv) in several batches at 0°C. The resulting solution was stirred for 3 hours at 0°C. The reaction was then quenched by the addition of water (100 mL).
• Step 2 Into a 500-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen was placed a solution of (1s,3s)-3-(benzyloxy)cyclobutan-1-ol (8.0 g, 44.89 mmol, 1.10 equiv) in N,N-dimethylformamide (100 mL).
• Step 3 Into a 500-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed a solution of 4-bromo-2-[(1s,3s)-3-(benzyloxy)cyclobutoxy]pyridine (8.4 g, 25.13 mmol, 1.30 equiv) in toluene (200 mL), tert-butyl 3,8-diazabicyclo[3.2.1]octane-3- carboxylate (4.11 g, 19.36 mmol, 1.00 equiv), sodium tert-butoxide (3.2 g, 33.2 mmol, 1.5 equiv), xantphos (2.32 g, 4.01 mmol, 0.20 equiv), and Pd2(dba)3CH 2 Cl2 (430.0 mg, 0.40 mmol, 0.02 equiv).
• the resulting solution was stirred overnight at 100°C. The reaction was then quenched by the addition of water (100 mL). The resulting solution was extracted with ethyl acetate (80 mL x 2), and the organic layers were combined. The resulting mixture was washed with brine (100 mL x 2). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied onto a silica gel column eluting with ethyl acetate/hexane (1:1).
• Step 4 To a solution of tert-butyl 8-[2-[(1s,3s)-3-(benzyloxy)cyclobutoxy]pyridin-4-yl]-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (7.0 g, 15.03 mmol, 1.00 equiv) in 150 mL ethanol was added palladium hydroxide (7.0 g, 49.85 mmol, 3.32 equiv) and acetic acid (2 mL) under nitrogen atmosphere in a 250 ml round bottom flask. The flask was then vacuumed and flushed with hydrogen.
• reaction mixture was hydrogenated at 50°C for 3 days under hydrogen atmosphere using a hydrogen balloon, then filtered through a Celite pad and concentrated under reduced pressure. This resulted in 5.4 g (96%) of tert-butyl 8-[2-[(1s,3s)-3-hydroxycyclobutoxy]pyridin-4- yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate as a light yellow oil.
• Step 5 Into a 100-mL round-bottom flask, was placed a solution of tert-butyl 8-[2-[(1s,3s)-3- hydroxycyclobutoxy]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (5.0 g, 13.32 mmol, 1.00 equiv) in the mixed solvent of ethyl acetate and methanol (10:1) (50 mL). Hydrogen chloride (gas) was passed through the solution. The resulting mixture was stirred for 3 hours at room temperature and concentrated under reduced pressure.
• Hydrogen chloride gas
• Step 6 Into a 20-mL pressure tank reactor purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (1s,3s)-3-[(4-[3,8-diazabicyclo[3.2.1]octan-8-yl]pyridin-2- yl)oxy]cyclobutan-1-ol hydrochloride (1.0 g, 3.21 mmol, 1.00 equiv) in dimethyl sulfoxide (10 mL).
• N,N-diisopropylethylamine (5 mL) and 4-bromo-6-chloropyridazin-3-amine (3.01 g, 14.44 mmol, 4.00 equiv) were added.
• the resulting solution was stirred for 16 hours at 130qC.
• the reaction was then quenched by the addition of water (50 mL).
• the resulting solution was extracted with ethyl acetate (3x30 mL), and the organic layers combined.
• the resulting mixture was washed with brine (3x50 mL).
• the mixture was dried over anhydrous sodium sulfate.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 7 Into a 20-mL pressure tank reactor purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (1s,3s)-3-([4-[3-(3-amino-6-chloropyridazin-4-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl]pyridin-2-yl]oxy)cyclobutan-1-ol (800 mg, 1.99 mmol, 1.00 equiv) in the mixed solvent of dioxane and water (4:1) (15 mL), [2-(methoxymethoxy)phenyl]boronic acid (544 mg, 2.99 mmol, 1.50 equiv), potassium carbonate (824 mg, 6.0 mmol, 3.00 equiv), and Pd(PPh3)4 (230 mg, 0.20 mmol, 0.10 equiv).
• the resulting solution was stirred for 2 hours at 90 oC. The reaction was then quenched by the addition of water (30 mL). The resulting solution was extracted with ethyl acetate (50 mL x 2), and the organic layers combined. The resulting mixture was washed with brine (50 mL x 2). The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 8 Into a 50-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of triphenylphosphine (351 mg, 1.34 mmol, 2.00 equiv) in tetrahydrofuran (distilled) (30 mL). This was followed by the addition of diisopropyl azodicarboxylate (270 mg, 1.34 mmol, 2.00 equiv) dropwise with stirring at 0°C while a white precipitate appeared.
• triphenylphosphine 351 mg, 1.34 mmol, 2.00 equiv
• diisopropyl azodicarboxylate 270 mg, 1.34 mmol, 2.00 equiv
• the resulting solution was stirred for 1 hour at 80°C. The reaction was then quenched by the addition of water. The resulting solution was extracted with ethyl acetate (30 mL x 3), and the organic layers were combined. The resulting mixture was washed with brine (30 mL x 2). The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 10 Into a 25-mL round-bottom flask, was placed a solution of 6-[2- (methoxymethoxy)phenyl]-4-(8-[2-[(1r,3r)-3-[(6-[3-[(tert-butyldimethylsilyl)oxy]prop-1-en-1- yl]pyridin-3-yl)oxy]cyclobutoxy]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3- amine (120 mg, 0.16 mmol, 1.00 equiv) in tetrahydrofuran (2 mL), tetrabutylammonium fluoride (1M in tetrahydrofuran) (3.2 mL, 2.00 equiv).
• Step 11 Into a 50-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of tributylphosphine (149 mg, 0.74 mmol, 10.00 equiv) in tetrahydrofuran (20 mL). This was followed by the addition of a solution of tetramethylazodicarboxamide (126 mg, 0.73 mmol, 10.00 equiv) in tetrahydrofuran (5 mL) dropwise with stirring at -5°C.
• tributylphosphine 149 mg, 0.74 mmol, 10.00 equiv
• tetramethylazodicarboxamide 126 mg, 0.73 mmol, 10.00 equiv
• the resulting solution was stirred overnight at 50°C. The reaction was then quenched by the addition of water. The resulting solution was extracted with ethyl acetate (30 mL x 3), and the organic layers were combined. The resulting mixture was washed with brine (50 mL). The organic phase was evaporated under reduced pressure and applied onto a silica gel column eluting with dichloromethane/methanol (10:1).
• Step 12 Into a 25-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (2S,4R)-4-hydroxy-N-[[4-(4-methyl-1,3-thiazol-5- yl)phenyl]methyl]-1-(3-methyl-2-[3-[(3-[5-[(1r,3r)-3-[[4-(3-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl]oxy]cyclobutoxy]pyridin-2-yl]prop-2-en-1-yl)oxy]-1,2-oxazol-5-yl]butanoyl)pyrrolidine-2- carboxamide (23 mg, 0.02 mmol, 1.00 equiv) in
• the resulting solution was stirred for 2 hours at room temperature.
• the reaction mixture was cooled to 0°C with a water/ice bath.
• the pH value of the solution was adjusted to 7 with NH 4 HCO 3 .
• the resulting solution was extracted with dichloromethane (30 mL x 3), and the organic layers were combined.
• the resulting mixture was washed with brine (50 mL x 3). The mixture was dried over anhydrous sodium sulfate.
• the crude product was purified by Prep- HPLC with the following conditions: Column, XBridge Prep C18 OBD Column, 5um, 19*150mm; mobile phase, Water (10 mmoL/L NH4HCO3) and ACN (41.0% ACN up to 62.0% in 8 min); Detector, UV 254nm.
• Step 2 To a solution of sodium hydride (6.26 g, 156 mmol, 1.3 eq) in dimethyl formamide (240 mL) was added benzyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (35 g, 132 mmol, 1.1 eq) at 0°C for 0.5 hour. Then 4-bromo-2-fluoro-pyridine (21.18 g, 120 mmol, 1 eq) was added into the reaction and stirred at 25 °C for 1 h.
• Step 3 A vial was charged with benzyl 4-[2-[(4-bromo-2-pyridyl)oxy]ethyl]piperazine-1- carboxylate (18.15 g, 43 mmol, 1 eq), tert-butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (11 g, 51 mmol, 1.2 eq), [2-(2-aminophenyl)phenyl]-chloro-palladium dicyclohexyl-[2-(2,6- diisopropoxyphenyl)phenyl]phosphane (1.68 g, 2.16 mmol, 0.05 eq), cesium carbonate (28.14 g, 86.36 mmol, 2 eq) was added into toluene (350 mL).
• the mixture was purged with nitrogen for 5 minutes and then heated to 110°C for 12 hours.
• the reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (300 mL x 3). The combined organic layers were washed with brine (200 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue.
• Step 4 To a mixture of tert-butyl 8-[2-[2-(4-benzyloxycarbonylpiperazin-1-yl)ethoxy]-4- pyridyl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (18.3 g, 33.17 mmol, 1 eq) in methanol (150 mL) was added hydrochloric acid/methanol (4 M, 360 mL, 43.41 eq). Then the reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to give the compound without purification.
• Step 5 A mixture of benzyl 4-[2-[[4-(3,8-diazabicyclo[3.2.1]octan-8-yl)-2- pyridyl]oxy]ethyl]piperazine-1-carboxylate (19 g, 42.08 mmol, 1 eq), 4-bromo-6-chloro- pyridazin-3-amine (10.52 g, 50.49 mmol, 1.2 eq) in dimethylsulfoxide (190 mL) was added diisopropylethylamine (54.38 g, 420.76 mmol, 10 eq). Then the reaction mixture was stirred at 130°C for 5 hours.
• Step 6 To the mixture of benzyl 4-[2-[[4-[3-(3-amino-6-chloro-pyridazin-4-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl]-2-pyridyl]oxy]ethyl]piperazine-1-carboxylate (10.6 g, 18.30 mmol, 1 eq), (2-hydroxyphenyl)boronic acid (5.05 g, 36.61 mmol, 2 eq) in dioxane (160 mL) and water (25 mL) was added tetrakis[triphenylphosphine]palladium(0) (2.12 g, 1.83 mmol, 0.1 eq) and potassium carbonate (5.06 g, 36.61 mmol, 2 eq).
• reaction mixture was stirred at 110°C for 2 hours.
• the reaction mixture was diluted with water (200 mL), and then extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with brine (150 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue.
• the residue was purified by prep-HPLC (basic condition: column: Phenomenex Gemini C18250*50 mm*10 um; mobile phase: [water (0.05% ammonia hydroxide v/v)- acetonitrile]; B%: 50%-80%, 20MIN 60%min) to give desired compound.
• Step 7 To the solution of benzyl 4-[2-[[4-[3-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]- 3,8-diazabicyclo[3.2.1]octan-8-yl]-2-pyridyl]oxy]ethyl]piperazine-1-carboxylate (7 g, 10.99 mmol, 1 eq) in tetrahydrofuran (100 mL) was added palladium hydroxide on activated carbon catalyst (0.7 g, 0.49 mmol, 0.04 eq) under nitrogen. Then the reaction mixture was stirred at 60°C under hydrogen (50 Psi) for 48 hours.
• Step 8 To a solution of methyl 2-(3-hydroxyisoxazol-5-yl)-3-methyl-butanoate (3.5 g, 17.57 mmol, 1 eq) in N,N-dimethylformamide (40 mL) was added 2-bromo-1,1-diethoxy-ethane (5.19 g, 26.35 mmol, 1.5 eq) and potassium carbonate (4.86 g, 35.14 mmol, 2 eq). The reaction mixture was stirred at 70°C for 12 hours. The reaction mixture was quenched by the addition water 50 mL, and then diluted with water 100 mL and extracted with ethyl acetate (80 mL x 3).
• Step 9 To a solution of methyl 2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butanoate (4.38 g, 12.5 mmol, 1 eq) in methanol (30 mL) and water (15 mL) was added lithium hydroxide monohydrate (2.10 g, 50 mmol, 4 eq). The reaction mixture was stirred at 40°C for 2 hours. The pH was adjusted to 4 ⁇ 5 with 1M hydrogen chloride, and reaction mixture was extracted with ethyl acetate (50 mL x 2).
• Step 10 To a solution of 2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butanoic acid (3.84 g, 11.99 mmol, 1 eq) in N,N-dimethylformamide (20 mL) was added O-(7-azabenzotriazol- 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.47 g, 14.38 mmol, 1.2 eq). The reaction mixture was stirred at 20°C for 0.5 hour.
• Step 11 [00691] (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butanoyl]-4-hydroxy- N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (7 g, 11.39 mmol, 1 eq) was separated by SFC. The condition was column: DAICEL CHIRALPAK AD (250 mm*30 mm,10 um); mobile phase: [0.1%NH3H2O IPA]; B%: 35%-35%, 2.4min: 550 min.
• Step 12 To a solution of (2S,4R)-1-[(2R)-2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl- butanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2- carboxamide (2 g, 3.25 mmol, 1 eq) in tetrahydrofuran (40 mL) was added sulfuric acid (1 M, 40 mL, 12.3 eq). The solution was heated to 50 °C for 7 h.
• the mixture was extracted with ethyl acetate (30 mL x 3).
• the combined organic layer was washed with brine (50 mL) and dried over sodium sulfate.
• the mixture was then filtered, and the filtrate was concentrated in vacuum.
• the crude product was used directly in next step.
• Step 13 [00696] To a solution of (2S,4R)-4-hydroxy-1-[(2R)-3-methyl-2-[3-(2-oxoethoxy)isoxazol-5- yl]butanoyl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (930 mg, 1.38 mmol, 1 eq) and 2-[6-amino-5-[8-[2-(2-piperazin-1-ylethoxy)-4-pyridyl]-3,8- diazabicyclo[3.2.1]octan-3-yl]pyridazin-3-yl]phenol (691 mg, 1.38 mmol, 1 eq) in methanol (15 mL) and dichloromethane (5 mL) was added acetic acid (82 mg, 1.38 mmol, 1 eq).
• Step 2 Into a 100 mL round-bottom flask to a solution of 1-(6-bromopyridin-3-yl)ethan-1-ol (2 g, 9.90 mmol, 1 equiv) and triethylamine (3 g, 0.03 mmol) in dichloromethane (30 mL) was added methanesulfonyl chloride (1.3 g, 11.35 mmol, 1.15 equiv) at 0 ⁇ . The resulting mixture was stirred for 16 hours at room temperature. The resulting mixture was extracted with dichloromethane (30 mL x 3).
• Step 3 Into a 25 mL sealed tube 1-(6-bromopyridin-3-yl)ethyl methanesulfonate(1.4 g, 1.2 equiv), 6-[2-(methoxymethoxy)phenyl]-4-(1H-pyrazol-4-yl)pyridazin-3-amine (350 mg, 1 equiv), and potassium carbonate (490 mg, 3.0 equiv) were added to N,N-Dimethylformamide (15 mL) at room temperature. The resulting mixture was stirred for 2 h at 60 ⁇ under nitrogen atmosphere. The aqueous layer was extracted with dichloromethane (50 mL x 3).
• Step 2 [00711] To a solution of 4-[1-[2-(2-bromoethoxy)ethyl]pyrazol-4-yl]-6-[2- (methoxymethoxy)phenyl]pyridazin-3-amine (100 mg, 0.22 mmol, 1 eq) and tert-butyl piperazine-1-carboxylate (83 mg, 0.44 mmol, 2 eq) in acetonitrile (2 mL) was added N,N- diisopropylethylamine (86.49 mg, 0.66 mmol, 3 eq). Then the reaction mixture was stirred at 100°C for 12 hours.
• Exemplary compounds 165 and 124 were prepared using analogous procedures.
• Exemplary Synthesis of Exemplary Compound 64 [00716] To a solution of pyridin-4-ol (3.20 g, 33.66 mmol, 1.5 eq) and 3- benzyloxycyclobutanol (4 g, 22.44 mmol, 1 eq) in tetrahydrofuran (200 mL) was added triphenylphosphine (7.06 g, 26.93 mmol, 1.2 eq) and diisopropyl azodicarboxylate (5.45 g, 26.93 mmol, 1.2 eq) in one portion at 10 °C under nitrogen.
• Step 2 To a solution of 4-(3-benzyloxycyclobutoxy)pyridine (4.2 g, 16.45 mmol, 1 eq) in toluene (65 mL) was added benzyl bromide (2.81 g, 16.45 mmol, 1 eq). The mixture was stirred at 80°C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove toluene. The crude product was triturated with petroleum ether (80 mL).
• Step 3 To a solution of 1-benzyl-4-(3-benzyloxycyclobutoxy)pyridine-1-ium bromide (6.5 g, 15.25 mmol, 1 eq) in ethanol (120 mL) was added sodium borohydride (3.46 g, 91.47 mmol, 6 eq) at 0°C. The mixture was stirred at 15°C for 4 hours. The reaction mixture was concentrated under reduced pressure to remove ethanol.
• Step 4 To a solution of 1-benzyl-4-(3-benzyloxycyclobutoxy)-3,6-dihydro-2H-pyridine (4.5 g, 12.88 mmol, 1 eq) in tetrahydrofuran (95 mL) and ethanol (70 mL) was added palladium on activated carbon catalyst (0.5 g, 10% purity) under nitrogen atmosphere. The suspension was degassed and purged with hydrogen for 3 times. The mixture was stirred under hydrogen (50 Psi) at 25°C for 24 hours and then at 35°C for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated.
• Step 5 To a solution of 3-[(1-benzyl-4-piperidyl)oxy]cyclobutanol (1.1 g, 4.21 mmol, 1 eq) in methanol (10 mL) was added palladium hydroxide (591 mg) and di-tert-butyl dicarbonate ester (1.84 g, 8.42 mmol, 2 eq) under nitrogen atmosphere. The suspension was degassed and purged with hydrogen for 3 times. The mixture was stirred under hydrogen (50 Psi) at 25°C for 12 hours. The reaction mixture was filtered and the filter was concentrated.
• Step 6 A mixture of tert-butyl 4-(3-hydroxycyclobutoxy)piperidine-1-carboxylate (1 g, 3.69 mmol, 1 eq), 4-bromo-2-fluoro-pyridine (778 mg, 4.42 mmol, 1.2 eq), cesium carbonate (2.40 g, 7.37 mmol, 2 eq) in acetonitrile (10 mL) was degassed and purged with nitrogen for three times, and then the mixture was stirred at 90°C for 12 hours under nitrogen atmosphere. The reaction mixture was filtered and concentrated to afford crude product.
• Step 7 To a solution of tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (10 g, 47.11 mmol, 1 eq) in dichloromethane (140 mL) was added CbzCl (9.64 g, 56.53 mmol, 8.04 mL, 1.2 eq) and triethylamine (8.58 g, 84.79 mmol, 11.80 mL, 1.8 eq). The mixture was stirred at 20 °C for 20 hours.
• Step 9 Cbz A mixture of benzyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (784 mg, 3.18 mmol, 1 eq), tert-butyl 4-[3-[(4-bromo-2-pyridyl)oxy]cyclobutoxy]piperidine-1-carboxylate (1.36 g, 3.18 mmol, 1 eq), [2-(2-aminophenyl)phenyl]-chloropalladium; dicyclohexyl-[2-(2,6- diisopropoxyphenyl)phenyl]phosphane (148 mg, 0.19 mmol, 0.06 eq), and cesium carbonate (2.07 g, 6.37 mmol, 2 eq) in toluene (27 mL) was degassed and purged with nitrogen for three times, and then the mixture was stirred at 110°C for 12 hours under nitrogen atmosphere
• the reaction mixture was diluted with water (30 mL), and extracted with ethyl acetate (40 mL) two times. The combined organic layers were washed with saturated aqueous sodium chloride (30 mL) two times, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give crude product.
• Step 10 To a solution of benzyl 8-[2-[3-[(1-tert-butoxycarbonyl-4- piperidyl)oxy]cyclobutoxy]-4-pyridyl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (1.35 g, 2.28 mmol, 1 eq) in a mixture of tetrahydrofuran (27 mL) and ethanol (27 mL) was added palladium hydroxide on activated carbon catalyst (320 mg, 10% purity) under nitrogen. The suspension was degassed under vacuum and purged with hydrogen several times.
• Step 11 A mixture of tert-butyl 4-[3-[[4-(3,8-diazabicyclo[3.2.1]octan-8-yl)-2- pyridyl]oxy]cyclobutoxy]piperidine-1-carboxylate (932 mg, 2.03 mmol, 1 eq), 4-bromo-6- chloro-pyridazin-3-amine (508 mg, 2.44 mmol, 1.2 eq), N,N-diisopropylethylamine (2.63 g, 20.32 mmol, 3.54 mL, 10 eq) in dimethylsulfoxide (30 mL) was degassed and purged with nitrogen for three times, and then the mixture was stirred at 130°C for 3 hours under nitrogen atmosphere.
• reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL) two times. The combined organic layers were washed with saturated aqueous sodium chloride (30 mL) two times, dried over anhydrous sodium sulfate and concentrated to afford crude product. The residue was purified by semi-preparative reverse phase HPLC (column: Phenomenex Gemini C18250*5010 um; mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN]; B%: 45%-70%, 24 MIN; 53% min).
• Step 12 A mixture of tert-butyl 4-[3-[[4-[3-(3-amino-6-chloro-pyridazin-4-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl]-2-pyridyl]oxy]cyclobutoxy]piperidine-1-carboxylate (772 mg, 1.32 mmol, 1 eq), (2-hydroxyphenyl)boronic acid (218 mg, 1.58 mmol, 1.2 eq), tetrakis[triphenylphosphine]palladium(0) (152 mg, 0.13 mmol, 0.1 eq), potassium carbonate (364 mg, 2.63 mmol, 2 eq) in a mixture of dioxane (12 mL) and water (2 mL) was degassed and purged with nitrogen for three times, and then the mixture was stirred at 90°C for 10 hours under nitrogen atmosphere.
• reaction mixture was concentrated to afford the crude product.
• residue was purified by semi-preparative reverse phase HPLC (column: Kromasil 250*50 mm*10 um; mobile phase: [water (0.1%trifluoroacetic acid)-ACN]; B%: 20ACN%-50ACN%, 20 minmin).
• Step 13 To a solution of tert-butyl 4-[3-[[4-[3-[3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl]- 3,8-diazabicyclo[3.2.1]octan-8-yl]-2-pyridyl]oxy]cyclobutoxy]piperidine-1-carboxylate (500 mg, 0.78 mmol, 1 eq) in dichloromethane (5 mL) was added hydrochloric acid/dioxane (4 M, 5 mL, 25.75 eq). The mixture was stirred at 20°C for 2 hours. The mixture was concentrated to afford the crude product.
• Step 14 To a solution of 2-[6-amino-5-[8-[2-[3-(4-piperidyloxy)cyclobutoxy]-4-pyridyl]-3,8- diazabicyclo[3.2.1]octan-3-yl]pyridazin-3-yl]phenol trihydrochloride (400 mg, 612 umol, 1 eq,) and (2S,4R)-4-hydroxy-1-[(2R)-3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butanoyl]-N-[(1S)-1- [4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (331 mg, 0.61 mmol, 1 eq) in a mixture of dichloromethane (16 mL) and methanol (16 mL) was added sodium acetate (201 mg, 2.45 m
• Exemplary compound 63 and 146 were prepared using analogous procedures.
• Exemplary Synthesis of Exemplary Compound 67 and 68 [00746] To a stirred solution of NaOH (10 g, 0.250 mol, 2.7 eq. ) in water (500 mL) was added benzyl alcohol (10 g, 92.47 mmol, 1 equiv), 1,4-dibromobutane (39.93 g, 184.9 mmol, 2.00 equiv) and Bu 4 NHSO 4 (0.78 g, 2.297 mmol, 0.02 equiv) at room temperature .
• Step 2 To a solution of 2-(2-hydroxyethoxy)ethan-1-ol (14.18 g, 133.7 mmol, 5 equiv) in DMF (100 mL) was added sodium hydride (60% in oil, 2.15 g, 2.5 eq.) at 0 o C under nitrogen atmosphere. After 15 minutes to the reaction mixture was added [(4- bromobutoxy)methyl]benzene (6.5 g, 26.7 mmol, 1 equiv). The resulting mixture was stirred for 3 h at 25 o C and then quenched by the addition of water (250 mL). The resulting mixture was extracted with EA (3 x 200 mL).
• Step 3 To a stirred solution of 2-[2-[4-(benzyloxy)butoxy]ethoxy]ethan-1-ol (2.4 g, 8.94 mmol, 1 equiv) and DMAP (218 mg, 1.79 mmol, 0.2 equiv) in DCM (50 mL) was added TsCl (5.1 g, 26.83 mmol, 3 equiv) and TEA (2.7 g, 26.83 mmol, 3 equiv) at room temperature. The resulting mixture was stirred overnight at 40 o C. The reaction mixture was diluted with DCM (100 mL) and washed with 2x50 mL of brine.
• Step 4 To a solution of 2-[2-[4-(benzyloxy)butoxy]ethoxy]ethyl 4-methylbenzene-1- sulfonate (3.5 g, 8.28 mmol, 1 equiv) in 100 mL EtOH was added 10% Pd/C (3.5 g) and CH 3 COOH (0.95 mL, 15.88 mmol, 2.01 equiv) under nitrogen atmosphere. The flask was vacuumed and flushed with hydrogen. The resulting mixture was hydrogenated at 40°C overnight under hydrogen atmosphere using a hydrogen balloon, then filtered through a Celite pad and concentrated under reduced pressure.
• Step 5 To a stirred solution of methyl 2-(3-hydroxy-1,2-oxazol-5-yl)-3-methylbutanoate (1.5 g, 7.53 mmol, 1 equiv) and 4-(2-[2-[(4-methylbenzenesulfonyl)oxy]ethoxy]ethoxy)butan-1-ol (3.0 g, 9.04 mmol, 1.2 equiv) in acetone (50 mL) was added Cs2CO3 (4.9 g, 15.04 mmol, 2.00 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 4 hours at 60 o C and then concentrated under reduced pressure.
• Step 6 To a stirred solution of methyl 2-(3-[2-[2-(4-hydroxybutoxy)ethoxy]ethoxy]-1,2- oxazol-5-yl)-3-methylbutanoate (550 mg, 1.53 mmol, 1 equiv) in DCM (20 mL) was added Dess-Martin periodinane (1298 mg, 3.06 mmol, 2 equiv) at 0 o C. The resulting mixture was stirred for 2 hours at 30 o C and then quenched by the addition of saturated sodium thiosulfate solution. The resulting mixture was extracted with DCM (3 x 20 mL).
• Step 2 Into a 500-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (1s,3s)-3-(benzyloxy)cyclobutan-1-ol (8.0 g, 44.89 mmol, 1.10 equiv) in N,N-dimethylformamide (100 mL).
• Step 3 Into a 500-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 4-bromo-2-[(1s,3s)-3-(benzyloxy)cyclobutoxy]pyridine (8.4 g, 25.13 mmol, 1.30 equiv) in toluene (200 mL), tert-butyl 3,8-diazabicyclo[3.2.1]octane-3- carboxylate (4.11 g, 19.36 mmol, 1.00 equiv), sodium tert-butoxide (3.2 g, 33.2 mmol, 1.5 equiv), xantphos (2.32 g, 4.01 mmol, 0.20 equiv), Pd 2 (dba) 3 CH 2 Cl 2 (430.0 mg, 0.40 mmol, 0.02 equiv).
• the resulting solution was stirred overnight at 100°C. The reaction was then quenched by the addition of water (100 mL). The resulting solution was extracted with ethyl acetate (80 mL x 2), and the organic layers were combined. The resulting mixture was washed with of brine (100 mL x 2). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied onto a silica gel column eluting with ethyl acetate/hexane (1:1).
• Step 4 To a solution of tert-butyl 8-[2-[(1s,3s)-3-(benzyloxy)cyclobutoxy]pyridin-4-yl]-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (7.0 g, 15.03 mmol, 1.00 equiv) in 150 mL ethanol was added palladium hydroxide catalyst (7.0 g, 49.85 mmol, 3.32 equiv) and acetic acid (2 mL) under nitrogen atmosphere in a 250 ml round bottom flask. The flask was then vacuumed and flushed with hydrogen.
• reaction mixture was hydrogenated at 50 °C for 3 days under hydrogen atmosphere using a hydrogen balloon, then filtered through a Celite pad and concentrated under reduced pressure. This resulted in 5.4 g (96%) of tert-butyl 8-[2-[(1s,3s)-3- hydroxycyclobutoxy]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate as a light yellow oil.
• Step 5 Into a 100-mL round-bottom flask, was placed a solution of tert-butyl 8-[2-[(1s,3s)-3- hydroxycyclobutoxy]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octane-3-carboxylate (5.0 g, 13.32 mmol, 1.00 equiv) in the mixed solvent of ethyl acetate and methanol (10:1) (50 mL). Hydrogen chloride (g) was passed through. The resulting mixture was stirred for 3 hours at room temperature and concentrated under reduced pressure.
• Step 6 Into a 20-mL pressure tank reactor purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (1s,3s)-3-[(4-[3,8-diazabicyclo[3.2.1]octan-8-yl]pyridin-2- yl)oxy]cyclobutan-1-ol hydrochloride (1.0 g, 3.21 mmol, 1.00 equiv) in dimethyl sulfoxide (10 mL).
• N,N-Diisopropylethylamine (5 mL) and 4-bromo-6-chloropyridazin-3-amine (3.01 g, 14.44 mmol, 4.00 equiv) were added.
• the resulting solution was stirred for 16 hours at 130°C.
• the reaction was then quenched by the addition of water (50 mL).
• the resulting solution was extracted with ethyl acetate (3x30 mL) and the organic layers combined.
• the resulting mixture was washed with brine (3x50 mL).
• the mixture was dried over anhydrous sodium sulfate.
• the residue was applied onto a silica gel column with dichloromethane/methanol (10:1).
• Step 7 Into a 20-mL pressure tank reactor purged and maintained with an inert atmosphere of nitrogen, was placed a solution of (1s,3s)-3-([4-[3-(3-amino-6-chloropyridazin-4-yl)-3,8- diazabicyclo[3.2.1]octan-8-yl]pyridin-2-yl]oxy)cyclobutan-1-ol (800.0 mg, 1.99 mmol, 1.00 equiv) in the mixed solvent of dioxane and water (4:1) (15 mL), [2- (methoxymethoxy)phenyl]boronic acid (543.5 mg, 2.99 mmol, 1.50 equiv), potassium carbonate (823.9 mg, 6.0 mmol, 3.00 equiv), Pd(PPh 3 ) 4 (230.0 mg, 0.20 mmol, 0.10 equiv).
• the resulting solution was stirred for 2 hours at 90oC. The reaction was then quenched by the addition of water (30 mL). The resulting solution was extracted with ethyl acetate (50 mL x 2) and the organic layers combined. The resulting mixture was washed with brine (50 mL x 2). The mixture was dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column with dichloromethane/methanol (10:1).
• Step 8 To a stirred solution of 6-bromopyridin-3-ol (12.70 g, 72.9 mmol, 1.00 equiv) and imidazole (7.45 g, 109.5 mmol, 1.50 equiv) in DMF (100 mL) were added TBSCl (16.50 g, 109.47 mmol, 1.50 equiv) in portions at 0°C under nitrogen atmosphere. The resulting mixture was stirred for 3 hours at room temperature under nitrogen atmosphere. The reaction was quenched with water (100 mL) at room temperature, and the resulting mixture was extracted with EtOAc (50 mL x 3).
• Step 9 To a stirred mixture of 2-bromo-5-[(tert-butyldimethylsilyl)oxy]pyridine (4.00 g, 13.88 mmol, 1.00 equiv) and NaI (10.40 g, 69.38 mmol, 5.00 equiv) in 1,4-dioxane (20 mL) were added CuI (0.26 g, 1.365 mmol, 0.10 equiv) and methyl[2-(methylamino)ethyl]amine (0.12 g, 1.361 mmol, 0.10 equiv) dropwise at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 5 hours at 110°C under nitrogen atmosphere.
• reaction mixture was filtered, and the filter cake was rinsed with 1,4-dioxane.
• the filtrate was concentrated under reduced pressure.
• the residue was purified by silica gel column chromatography eluting with hexane/EtOAc (5:1) to afford 5-[(tert-butyldimethylsilyl)oxy]-2- iodopyridine (4.2 g, 90%) as an off-white solid.
• Step 10 To a stirred solution of 5-[(tert-butyldimethylsilyl)oxy]-2-iodopyridine (4.20 g, 12.53 mmol, 1.00 equiv) in THF (20 mL) were added TBAF (1 mol/L, 27 mL, 27 mmol, 2.00 equiv) dropwise at 0°C under hydrogen atmosphere. The resulting mixture was stirred for 2 hours at room temperature under nitrogen atmosphere. The reaction was quenched with water (50 mL) at room temperature. The resulting mixture was extracted with EtOAc (50 mL x 3).
• Step 11 To a solution of PPh 3 (523 mg, 2.00 mmol, 2.00 equiv) in THF (10 mL) was added DIAD (403 mg, 2.00 mmol, 2.00 equiv), (1s,3s)-3-[[4-(3-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl)pyridin-2- yl]oxy]cyclobutan-1-ol (504 mg, 1.00 mmol, 1 equiv) and 6-iodopyridin-3-ol (265 mg, 1.20 mmol, 1.20 equiv) at 0°C under nitrogen atmosphere.
• the resulting mixture was stirred for 5 minutes at 0 o C, heated up to 50°C and stirred for 2 hours at 50°C under nitrogen atmosphere.
• the reaction was quenched by the addition of water (20 mL) at room temperature, and the resulting mixture was extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure.
• Step 12 To a solution of (2S,4R)-4-hydroxy-1-[2-(3-hydroxy-1,2-oxazol-5-yl)-3- methylbutanoyl]-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2- carboxamide (780 mg, 1.56 mmol, 1.00 equiv) in acetone (40 mL) was added Cs 2 CO 3 (1019 mg, 3.13 mmol, 2.00 equiv), and 3-bromoprop-1-yne (558 mg, 4.69 mmol, 3.00 equiv) at room temperature.
• Step 13 To a stirred mixture of 6-[2-(methoxymethoxy)phenyl]-4-(8-[2-[(1r,3r)-3-[(6- iodopyridin-3-yl)oxy]cyclobutoxy]pyridin-4-yl]-3,8-diazabicyclo[3.2.1]octan-3-yl)pyridazin-3- amine (224 mg, 0.317 mmol, 1.00 equiv) and CuI (6.0 mg, 0.032 mmol, 0.10 equiv) in DMF (5 mL) was added TEA (96 mg, 0.950 mmol, 3.00 equiv), and Pd(PPh3) 2 Cl2 (22.2 mg, 0.032 mmol, 0.10 equiv) at room temperature under nitrogen atmosphere.
• di-tert-butyl dicarbonate ester (5.45 g, 24.99 mmol, 1.0 eq) was added, and the mixture and stirred at 0 °C for 30 minutes followed by 20 °C for 2 hours.
• the mixture was quenched with saturated aqueous sodium bicarbonate (20 mL), diluted with water (50 mL), extracted with dichloromethane (100 mL x 2), washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated.
• Exemplary compound 75 was prepared using analogous procedures.
• Exemplary Synthesis of Exemplary Compound 116 [00797] To a solution of 1-(6-bromo-3-pyridyl)ethanone (11.88 g, 59.41 mmol, 1.2 eq) and tetraethoxytitanium (22.58 g, 99.01 mmol, 2 eq) in tetrahydrofuran (50 mL) was added (R)-2- methylpropane-2-sulfinamide (6 g, 49.50 mmol, 1 eq) under nitrogen. The reaction mixture was stirred at 70°C for 12 hours.
• reaction mixture was quenched by addition water (30 mL), and then further diluted with water (100 mL), filtered and extracted with ethyl acetate (80 mL ⁇ 3). The combined organic layers were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to produce the desired compound (R,E)- N-(1-(6-bromopyridin-3-yl)ethylidene)-2-methylpropane-2-sulfinamide (16 g, 43.27 mmol, 87% yield) as a light yellow solid.
• Step 2 To a solution of (R,E)-N-(1-(6-bromopyridin-3-yl)ethylidene)-2-methylpropane-2- sulfinamide (16 g, 52.77 mmol, 1 eq) in tetrahydrofuran (160 mL) was added L-selectride (1 M, 158 mL, 3 eq) at 0°C. The reaction mixture was warmed up and stirred at 20°C for 3 hours. The reaction mixture was quenched by addition water (100 mL), and then further diluted with water (100 mL), filtered and extracted with ethyl acetate (100 mL ⁇ 3).
• Step 3 To a solution of N-[(1S)-1-(6-bromo-3-pyridyl)ethyl]-2-methyl-propane-2-sulfinamide (2.6 g, 8.52 mmol, 1 eq) in dichloromethane (20 mL) was added hydrochloric acid/methanol (4 M, 21.3 mL, 10 eq). The reaction mixture was stirred at 20°C for 0.15 hours.
• Step 4 To a solution of (1S)-1-(6-bromo-3-pyridyl)ethanamine hydrochloride (1.9 g, 8.00 mmol, 1 eq,) in dichloromethane (20 mL) was added triethylamine (2.43 g, 24.00 mmol, 3.34 mL, 3 eq) at 0°C for 0.5 hour followed by di-tert-butyl dicarbonate (2.62 g, 12.00 mmol, 2.76 mL, 1.5 eq) at 0°C for 0.5 hours.
• reaction mixture was stirred at 20°C for 12 hours.
• the reaction mixture was quenched by the addition of water (20 mL), and then further diluted with water 60 mL, and extracted with ethyl acetate (80 mL ⁇ 3).
• Step 6 To a solution of 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (617 mg, 2.74 mmol, 1.5 eq) and tert-butyl N-[(1S)-1-(6-bromo-3-pyridyl)ethyl]carbamate (550 mg, 1.83 mmol, 1 eq) in dioxane (12 mL) and water (2 mL) was addded 1,1'- bis(diphenylphosphino)ferrocene-palladium(II)dichloride (134 mg, 0.18 mmol, 0.1 eq) and potassium carbonate (505 mg, 3.65 mmol, 2 eq).
• reaction mixture was stirred at 100°C for 12 hours.
• the reaction mixture was quenched by addition water (10 mL), and then diluted with water (30 mL), filtered and extracted with ethyl acetate (30 mL ⁇ 3).
• Step 2 To a solution of tert-butyl N-[(1S)-1-(6-formyl-3-pyridyl)ethyl]carbamate (550 mg, 2.20 mmol, 1 eq) and 1-(1-isocyanoethylsulfonyl)-4-methyl-benzene (506 mg, 2.42 mmol, 1.1 eq) in methanol (10 mL) was added potassium carbonate (607 mg, 4.39 mmol, 2 eq). The reaction mixture was stirred at 70°C for 12 hours.
• tert-butyl N-[(1S)-1-[6-(4-methyloxazol-5-yl)-3-pyridyl]ethyl]carbamate (570 mg, 1.65 mmol, 75% yield, 87% purity) was obtained as a white solid.
• tert-Butyl N-[(1S)-1-[6-(4-methyloxazol-5-yl)-3-pyridyl]ethyl]carbamate was converted to the final compound as described for exemplary compound 76.
• Exemplary compounds 161, 229, and 230 were prepared using analogous procedures.
• Step 2 [00821] Into a 250-mL round-bottom flask, was placed 1-[4-[(tert- butyldimethylsilyl)oxy]phenyl]ethan-1-one (5 g, 20.0 mmol, 1 equiv) in MeOH (100 mL), to which was added sodium borohydride (1.5 g, 39.94 mmol, 2 equiv) in portions at room temperature. The resulting solution was stirred for 5 h at room temperature, and then concentrated under reduced pressure. The residue was applied onto a silica gel column eluting with ethyl acetate/petroleum ether (1/1).
• Step 3 Into a 250-mL round-bottom flask, was placed 1-[4-[(tert- butyldimethylsilyl)oxy]phenyl]ethan-1-ol (3.8 g, 15.05 mmol, 1 equiv) and TEA (6.1 g, 60.21 mmol, 4 equiv) in dichloromethane (100 mL), to which was added methanesulfonyl chloride (2.1 g, 18.07 mmol, 1.20 equiv) slowly at room temperature.
• Step 4 Into a 250-mL round-bottom flask, was placed 1-[4-[(tert- butyldimethylsilyl)oxy]phenyl]ethyl methanesulfonate (5.0 g, 15.13 mmol, 1 equiv), 2- (cyclopenta-1,4-dien-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3.5 g, 18.17 mmol, 1.20 equiv), and K2CO3 (6.3 g, 45.38 mmol, 3 equiv) in DMF (150 mL) under nitrogen atmosphere. The resulting mixture was stirred overnight at 90°C.
• 1-[4-[(tert- butyldimethylsilyl)oxy]phenyl]ethyl methanesulfonate 5.0 g, 15.13 mmol, 1 equiv
• Step 2 Into a 500-mL 3-necked round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed 1-(6-fluoropyridin-3-yl)ethan-1-ol (4.06 g, 28.76 mmol, 1 equiv) in dichloromethane (200 mL). The solution was cooled to 0 ⁇ in water/ice bath, and PPh 3 (11.28 g, 43.01 mmol, 1.50 equiv) and NBS (7.66 g, 43.04 mmol, 1.50 equiv) were added sequentially at 0 ⁇ . The resulting solution was stirred for 2 h at room temperature.
• Step 3 Into a 100-mL round-bottom flask, was placed a solution of 5-(1-bromoethyl)-2- fluoropyridine (3.06 g, 15.00 mmol, 1 equiv), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)- 1H-pyrazole (3.20 g, 16.49 mmol, 1.10 equiv), and K2CO3 (4.15 g, 30.03 mmol, 2.00 equiv) in DMF (50 mL). The resulting mixture was stirred for 2 hours at 60 ⁇ in an oil bath.
• Step 4 Into a 100-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed 2-fluoro-5-[1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrazol-1-yl]ethyl]pyridine (2.54 g, 8.01 mmol, 1 equiv, 4-bromo-6-chloropyridazin-3-amine (2.00 g, 9.60 mmol, 1.20 equiv), Na2CO3 (2.12 g, 20.00 mmol, 2.50 equiv), and Pd(dppf)Cl 2 .CH 2 Cl 2 (327 mg, 0.40 mmol, 0.05 equiv) in dioxane (50 mL) and H 2 O (8 mL).
• Step 5 Into a 30-mL sealed tube purged and maintained with an inert atmosphere of nitrogen, was placed 6-chloro-4-[1-[1-(6-fluoropyridin-3-yl)ethyl]-1H-pyrazol-4-yl]pyridazin-3-amine (765 mg, 2.40 mmol, 1 equiv), [2-(methoxymethoxy)phenyl]boronic acid (524 mg, 2.88 mmol, 1.20 equiv), K2CO3 (829 mg, 6.00 mmol, 2.50 equiv), and Pd(dppf)Cl2.CH 2 Cl2 (196 mg, 0.24 mmol, 0.10 equiv) in dioxane (18 mL) and H2O (3 mL).
• Step 6 Into a 50-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed ethane-1,2-diol (1.48 g, 23.84 mmol, 10.03 equiv) in DMF (15mL). The solution was cooled to 0 ⁇ in water/ice bath and to this was added NaH (0.76 g, 19.00 mmol, 7.99 equiv, 60%) in portions at 0 ⁇ .
• Step 7 Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of tetramethylazodicarboxamide (861 mg, 5.00 mmol, 10.00 equiv) and tributylphosphine (1.01 g, 4.99 mmol, 9.98 equiv) in THF (15 mL) at 0 ⁇ .
• the resulting solution was stirred for 10 minutes at 0 ⁇ , and then stirred for an additional 2 hours at 50 ⁇ in an oil bath.
• the reaction mixture was cooled to room temperature and diluted with water (150 mL).
• the resulting mixture was extracted with ethyl acetate (150 mL), and the organic layer was washed with brine (150 mL x 2), dried over anhydrous sodium sulfate and concentrated under vacuum.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (10/1).
• the obtained product was further purified by Prep-HPLC with the following conditions: Column, XBridge Prep OBD C18 Column, 19 x 150 mm, 5 um; mobile phase, water (with 10 mmol/L NH 4 HCO 3 ) and acetonitrile, 35% ACN up to 53% in 8 min.
• Exemplary compound 157 was preparing using analogous procedures.
• Exemplary Synthesis of Exemplary Compound 100 [00846] Step 1 [00847] Into a stirred mixture of ethanol (350 mL, 7.60 mmol, 0.04 equiv) and water (60 mL, 3.33 mmol, 0.02 equiv) at room temperature was added anhydrous sodium acetate (18 g, 264.71 mmol, 1.47 equiv) in portions under nitrogen atmosphere. The resulting mixture was stirred for 16 hours at 70 ⁇ under nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. The resulting mixture was diluted with water (500 mL).
• Step 2 Into a 1 L round-bottom flask were added 1-(4-bromophenyl)-2-hydroxyethan-1-one (32 g, 148.8 mmol, 1 equiv), methylene chloride (500 mL), imidazole (30 g, 440.7 mmol, 2.96 equiv), 4-dimethylaminopyridine (1.8 g, 14.73 mmol, 0.10 equiv) and tert-butyldimethylsilyl chloride (26.8 g, 177.8 mmol, 1.19 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for 2 hours at room temperature under nitrogen atmosphere.
• Step 3 Into a 1 L 3-neck round-bottom flask were added 1-(4-bromophenyl)-2-[(tert- butyldimethylsilyl)oxy]ethan-1-one (27 g, 81.99 mmol, 1 equiv), tetrahydrofuran (500 mL) and (S)-2-methylpropane-2-sulfinamide (19.5 g, 160.9 mmol, 1.96 equiv) at room temperature. To the stirred solution was added Ti(i-PrO)4 (70 g, 246.5 mmol, 3.01 equiv) dropwise at room temperature under nitrogen atmosphere.
• Ti(i-PrO)4 70 g, 246.5 mmol, 3.01 equiv
• the resulting mixture was stirred for 16 hours at 70 ⁇ under nitrogen atmosphere. The mixture was allowed to cool down to room temperature. The resulting mixture was filtered, and the filter cake was washed with ethyl acetate (200 mL x 2). The combined organic layers were washed with brine (200 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure.
• Step 4 Into a 500 mL 3-neck round-bottom flask were added (S)-N-[(1Z)-1-(4- bromophenyl)-2-[(tert-butyldimethylsilyl)oxy]ethylidene]-2-methylpropane-2-sulfinamide(15 g, 34.68 mmol, 1 equiv) and tetrahydrofuran (150 mL) at room temperature. To the stirred solution was added BH 3 .THF (52 mL, 52 mmol, 1.5 equiv) dropwise at -70 ⁇ under nitrogen atmosphere. The resulting mixture was stirred for 1 hour at -70 ⁇ under nitrogen atmosphere.
• BH 3 .THF 52 mL, 52 mmol, 1.5 equiv
• the reaction was quenched with methanol at room temperature.
• the resulting mixture was filtered, and the filter cake was washed with ethyl acetate (100 mL x 2).
• the filtrate was washed with brine (50 mL x 2), dried over anhydrous sodium sulfate and concentrated under reduced pressure.
• Step 5 Into a 100-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed (S)-N-[(1R)-1-(4-bromophenyl)-2-[(tert-butyldimethylsilyl)oxy]ethyl]-2- methylpropane-2-sulfinamide (2.6 g, 5.98 mmol, 1.00 equiv), dioxane (30 mL), 4-methyl-5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-thiazole (2 g, 8.88 mmol, 1.48 equiv), potassium carbonate (2.5 g, 18.09 mmol, 3.02 equiv), water (5 mL), and Pd(dppf)Cl2.CH 2 Cl2 (440 mg, 0.51 mmol, 0.09 equiv).
• Step 6 Into a 250-mL round-bottom flask, was placed (S)-N-[(1R)-2-[(tert- butyldimethylsilyl)oxy]-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]-2-methylpropane-2- sulfinamide (2.6 g, 5.74 mmol, 1.00 equiv) and hydrogen chloride in dioxane (100 mL, 4M). The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum.
• Step 7 Into a 250-mL round-bottom flask, was placed ethyl 2-(3-hydroxy-1,2-oxazol-5-yl)-3- methylbutanoate (1 g, 4.69 mmol, 1 equiv), Cs 2 CO 3 (4.5 g, 13.81 mmol, 3.0 equiv), and 1,2- dibromoethane (2.60 g, 13.84 mmol) in acetone (100 mL).
• Step 2 A flask was charged with tert-butyl N-[(1S)-1-[4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)phenyl]ethyl]carbamate (1.90 g, 5.47 mmol, 1.1 eq), 2-bromo-1-methyl- imidazole (800 mg, 4.97 mmol, 1 eq), (1,1'-bis(diphenylphosphino)ferrocene) palladium(II) dichloride (181 mg, 0.24 mmol, 0.05 eq), sodium carbonate (1.05 g, 9.94 mmol, 2 eq), dioxane (18 mL) and water (3 mL).
• Step 3 To a solution of tert-butyl N-[(1S)-1-[4-(1-methylimidazol-2- yl)phenyl]ethyl]carbamate (850 mg, 2.82 mmol, 1 eq) in dioxane (2 mL) was added hydrogen chloride solution (4 M in dioxane, 10 mL, 14.18 eq). The solution was stirred at 20 °C for 1 h. The solvent was removed in vacuum. (1S)-1-[4-(1-methylimidazol -2-yl)phenyl]ethanamine (670 mg, crude, hydrochloride) was obtained as a grey solid.
• Step 4 To a solution of (2S,4R)-1-tert-butoxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (651 mg, 2.82 mmol, 1 eq) in N,N-dimethylformamide (2 mL) was added 1- hydroxybenzotriazole (456 mg, 3.38 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (648 mg, 3.38 mmol, 1.2 eq).
• Step 1 Into a 250-mL round-bottom flask, was placed a solution of 1-(6-fluoropyridin-3- yl)ethan-1-one (4.17 g, 29.97 mmol, 1 equiv) in EtOH (60 mL). The solution was cooled to 0 ⁇ in a water/ice bath, and the solution of sodium borohydride (2.27 g, 61.65 mmol, 2.06 equiv) in H2O (10 mL) was added dropwise. The resulting mixture was stirred for 0.5 h at room temperature.
• Step 2 Into a 500-mL 3-necked round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed 1-(6-fluoropyridin-3-yl)ethan-1-ol (4.06 g, 28.76 mmol, 1 equiv) in dichloromethane (200 mL). The solution was cooled to 0 ⁇ in water/ice bath, and PPh3 (11.28 g, 43.01 mmol, 1.50 equiv) and NBS (7.66 g, 43.04 mmol, 1.50 equiv) were added sequentially at 0 ⁇ . The resulting solution was stirred for 2 h at room temperature.
• Step 3 Into a 100-mL round-bottom flask, was placed a solution of 5-(1-bromoethyl)-2- fluoropyridine (3.06 g, 15.00 mmol, 1 equiv), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)- 1H-pyrazole (3.20 g, 16.49 mmol, 1.10 equiv), and K 2 CO 3 (4.15 g, 30.03 mmol, 2.00 equiv) in DMF (50 mL). The resulting mixture was stirred for 2 h at 60 ⁇ in an oil bath.
• Step 4 Into a 100-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed 2-fluoro-5-[1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- pyrazol-1-yl]ethyl]pyridine (2.54 g, 8.01 mmol, 1 equiv, 4-bromo-6-chloropyridazin-3-amine (2.00 g, 9.60 mmol, 1.20 equiv), Na2CO3 (2.12 g, 20.00 mmol, 2.50 equiv), and Pd(dppf)Cl2.CH 2 Cl2 (327 mg, 0.40 mmol, 0.05 equiv) in dioxane (50 mL) and H2O (8 mL).
• Step 5 Into a 30-mL sealed tube purged and maintained with an inert atmosphere of nitrogen, was placed 6-chloro-4-[1-[1-(6-fluoropyridin-3-yl)ethyl]-1H-pyrazol-4-yl]pyridazin-3-amine (765 mg, 2.40 mmol, 1 equiv), [2-(methoxymethoxy)phenyl]boronic acid (524 mg, 2.88 mmol, 1.20 equiv), K 2 CO 3 (829 mg, 6.00 mmol, 2.50 equiv), and Pd(dppf)Cl 2 .CH 2 Cl 2 (196 mg, 0.24 mmol, 0.10 equiv) in dioxane (18 mL) and H 2 O (3 mL).
• Step 6 Into a 50-mL round-bottom flask purged and maintained under an inert atmosphere of nitrogen, was placed ethane-1,2-diol (1.48 g, 23.84 mmol, 10.03 equiv) in DMF (15mL). The solution was cooled to 0 ⁇ in water/ice bath and to this was added NaH (0.76 g, 19.00 mmol, 7.99 equiv, 60%) in portions at 0 ⁇ .
• the resulting solution was stirred for 10 min at 0 ⁇ , and then stirred for an additional 2 h at 50 ⁇ in an oil bath.
• the reaction mixture was cooled to room temperature and diluted with water (150 mL).
• the resulting mixture was extracted with ethyl acetate (150 mL), and the organic layer was washed with brine (150 mL x 2), dried over anhydrous sodium sulfate and concentrated under vacuum.
• the residue was applied onto a silica gel column eluting with dichloromethane/methanol (10/1).
• the obtained product was further purified by Prep-HPLC with the following conditions: Column, XBridge Prep OBD C18 Column, 19 x 150 mm, 5 um; mobile phase, water (with 10 mmol/L NH4HCO3) and acetonitrile, 35% ACN up to 53% in 8 min.
• Exemplary compound 157 was prepared using analogous procedures.
• Exemplary Synthesis of Exemplary Compound 82 [00895] Into a 100-mL round-bottom flask, was placed methyl 2-(3-hydroxy-1,2-oxazol-5-yl)- 3-methylbutanoate (498 mg, 2.50 mmol, 1 equiv), 2-(2-bromoethoxy)oxane (627.3 mg, 3.00 mmol, 1.20 equiv), Cs2CO3 (1.63 g, 5.00 mmol, 2.00 equiv), and NaI (37.5 mg, 0.25 mmol, 0.10 equiv) in acetone (20 mL).
• Step 3 Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed methyl 2-[3-(2-hydroxyethoxy)-1,2-oxazol-5-yl]-3-methylbutanoate (375 mg, 1.54 mmol, 1.20 equiv) in DMF (6 mL). The solution was cooled to 0 ⁇ in a water/ice bath, and NaH (128.4 mg, 3.21 mmol, 2.50 equiv, 60%) was added.
• Methyl 2-[3-[2-([5-[1-(4-[3-amino-6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]- 1H-pyrazol-1-yl)ethyl]pyridin-2-yl]oxy)ethoxy]-1,2-oxazol-5-yl]-3-methylbutanoate was converted to the final compound using procedures analogous to those described for other examples above.
• Exemplary compound 163 was prepared using analogous procedures.
• Step 2 Into a 100-mL round-bottom flask, was placed a solution of tert-butyl 2-[2-(oxan-2- yloxy)ethoxy]-7-azaspiro[3.5]nonane-7-carboxylate (1.0 g, 2.71 mmol, 1 equiv) in dioxane (15 mL), to which was added hydrogen chloride in 1,4-dioxane solution (4.0 M, 15 mL). The resulting solution was stirred for 16 h at room temperature, and then concentrated under reduced pressure.
• Step 3 Into a 100-mL round-bottom flask, was placed 2-[7-azaspiro[3.5]nonan-2- yloxy]ethan-1-ol (600 mg, 3.24 mmol, 1 equiv), Et3N (983 mg, 9.72 mmol, 3 equiv) in DCM (30.0 mL), to which was added CbzCl (663 mg, 3.89 mmol, 1.2 equiv) at room temperature. The resulting mixture was stirred for 16 h at room temperature.
• Step 4 Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed benzyl 2-(2-hydroxyethoxy)-7-azaspiro[3.5]nonane-7-carboxylate (400 mg, 1.25 mmol, 1 equiv), Et 3 N (380 mg, 3.76 mmol, 3 equiv) in DCM (20 mL), to which was added MsCl (287 mg, 2.50 mmol, 2.00 equiv) at 0 °C. The resulting mixture was stirred for 1 h at 0 °C in a water/ice bath.
• Step 5 Into a 100-mL round-bottom flask, was placed a solution of benzyl 2-[2- (methanesulfonyloxy)ethoxy]-7-azaspiro[3.5]nonane-7-carboxylate (300 mg, 0.75 mmol, 1 equiv), 6-[2-(methoxymethoxy)phenyl]-4-[1-oxa-4,9-diazaspiro[5.5]undecan-9-yl]pyridazin-3- amine (291 mg, 0.75 mmol, 1 equiv), K 2 CO 3 (313 mg, 2.26 mmol, 3 equiv), NaI (113 mg, 0.75 mmol, 1 equiv) in MeCN (15 mL).
• Step 6 Into a 100-mL round-bottom flask, was placed a solution of benzyl 2-[2-(9-[3-amino- 6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]-1-oxa-4,9-diazaspiro[5.5]undecan-4-yl)ethoxy]- 7-azaspiro[3.5]nonane-7-carboxylate (230 mg, 0.33 mmol, 1 equiv) in THF (5 mL), to which was added lithium triethylborohydride (1.0 M in THF, 1.65 mmol, 5.0 eq) at 0 °C.
• Step 4 [00924] To a solution of 1-(4-tetrahydropyran-2-yloxybut-2-ynyl)-4-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)pyrazole (2 g, 5.78 mmol, 1 eq), 4-bromo-6-chloro-pyridazin-3-amine (1.20 g, 5.78 mmol, 1 eq) in dioxane (60 mL) and water (20 mL) was added sodium carbonate (1.53 g, 14.44 mmol, 2.5 eq) and (1,1'-bis(diphenylphosphino)ferrocene) palladium(II) dichloride (422 mg, 0.58 mmol, 0.1 eq) under nitrogen.
• Step 5 To a solution of 6-chloro-4-[1-(4-tetrahydropyran-2-yloxybut-2-ynyl)pyrazol-4- yl]pyridazin-3-amine (1.6 g, 4.60 mmol, 1 eq) in methanol (15 mL) was added hydrochloric acid solution (4 M in methanol, 15 mL, 13.04 eq). The reaction mixture was stirred at 20°C for 1 hour. Saturated sodium bicarbonate (50 mL) was added to the mixture to adjust pH to about 6, after which the resulting mixture was extracted with ethyl acetate (30 mL x 3).
• Step 6 To a solution of 4-[4-(3-amino-6-chloro-pyridazin-4-yl)pyrazol-1-yl]but-2-yn-1-ol (1.0 g, 3.79 mmol, 1 eq), triphenylphosphine (1.19 g, 4.55 mmol, 1.2 eq) in tetrahydrofuran (20 mL) was added carbon tetrabromide (1.51 g, 4.55 mmol, 1.2 eq). The reaction mixture was stirred at 20°C for 1 hour. The mixture was concentrated in vacuum.
• Step 7 To a solution of 4-[1-(4-bromobut-2-ynyl)pyrazol-4-yl]-6-chloro-pyridazin-3-amine (1 g, 1.87 mmol, 1 eq) and tert-butyl piperazine-1-carboxylate (696 mg, 3.74 mmol, 2 eq) in acetonitrile (10 mL) was added N,N-diisopropylethylamine (724 mg, 5.60 mmol, 1.0 mL, 3 eq). The mixture was stirred at 80°C for 12 hours.
• Step 8 To a solution of tert-butyl 4-[4-[4-(3-amino-6-chloro-pyridazin-4-yl)pyrazol-1-yl]but- 2-ynyl]piperazine-1-carboxylate (500 mg, 1.16 mmol, 1 eq), (2-hydroxyphenyl)boronic acid (192 mg, 1.39 mmol, 1.2 eq) in dioxane (10 mL) and water (2 mL) was added potassium carbonate (400 mg, 2.89 mmol, 2.5 eq) and tetrakis[triphenylphosphine]palladium(0) (134 mg, 0.12 mmol, 0.1 eq) under nitrogen.
• (2-hydroxyphenyl)boronic acid 192 mg, 1.39 mmol, 1.2 eq
• potassium carbonate 400 mg, 2.89 mmol, 2.5 eq
• tetrakis[triphenylphosphine]palladium(0)
• Step 2 [00939] Into a 30-mL vial, was placed methyl 4-(1-bromoethyl)benzoate (1.2 g, 4.94 mmol, 1 equiv), 6-[2-(methoxymethoxy)phenyl]-4-(1H-pyrazol-4-yl)pyridazin-3-amine (1.2 g, 3.95 mmol, 0.80 equiv), K 2 CO 3 (2.0 g, 14.81 mmol, 3.00 equiv), NaI (739.9 mg, 4.94 mmol, 1.00 equiv) in DMF (15 mL).
• Step 3 Into a 100-mL round-bottom flask, was placed a solution of methyl 4-[1-(4-[3-amino- 6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]-1H-pyrazol-1-yl)ethyl]benzoate (600 mg, 1.31 mmol, 1 equiv) in THF (20 mL), to which was added LiAlH 4 (495.6 mg, 13.06 mmol, 10.00 equiv) at 0°C. The resulting mixture was stirred for 3 hours at room temperature.
• Step 4 Into a 100-mL round-bottom flask, was placed a solution of [4-[1-(4-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-1H-pyrazol-1-yl)ethyl]phenyl]methanol (310 mg, 0.72 mmol, 1 equiv) in DCM (10.0 mL, 117 mmol, 218 equiv), to which was added manganese dioxide (1.2 g, 14.37 mmol, 20 equiv) at room temperature. The resulting mixture was stirred for 16 hours at room temperature.
• Step 5 Into a 100-mL round-bottom flask, was placed 4-[1-(4-[3-amino-6-[2- (methoxymethoxy)phenyl]pyridazin-4-yl]-1H-pyrazol-1-yl)ethyl]benzaldehyde (300.0 mg, 0.70 mmol, 1 equiv), benzyl piperazine-1-carboxylate (185 mg, 0.84 mmol, 1.2 equiv), HOAc (0.1 mL, 1.75 mmol, 2.50 equiv) in DCM (10 mL).
• Step 2 A mixture of ethyl 5-bromothiazole-4-carboxylate (3.49 g, 14.77 mmol, 0.9 eq), tert- butyl N-[(1S)-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl]carbamate (5.70 g, 16.41 mmol, 1 eq), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (120 mg, 0.16 mmol, 0.01 eq), potassium carbonate (4.54 g, 32.83 mmol, 2 eq) in mixture of dioxane (130 mL) and water (22 mL) was degassed and purged with nitrogen for three times, and then the mixture was stirred at 80°C for 10 hours under nitrogen atmosphere.
• Step 3 A solution of ethyl 5-[4-[(1S)-1-(tert-butoxycarbonylamino)ethyl]phenyl]thiazole-4- carboxylate (4 g, 10.63 mmol, 1 eq) in tetrahydrofuran (60 mL) was cooled to -78 °C under nitrogen atmosphere. Then diisobutylaluminium hydride THF solution (1 M, 11.69 mL, 1.1 eq) was added dropwise at -78°C. The reaction mixture was stirred at -78°C for 0.5 hour. Methanol (2 mL) was added to quench the reaction, followed by water (50 mL).
• Step 4 To a solution of dimethylamine hydrochloride (368 mg, 4.51 mmol, 0.41 mL, 3 eq) in dichloromethane (30 mL) was added triethylamine (609 mg, 6.02 mmol, 0.84 mL, 4 eq), a solution of tert-butyl N-[(1S)-1-[4-(4-formylthiazol-5-yl)phenyl]ethyl]carbamate (500 mg, 1.50 mmol, 1 eq) in dichloromethane (10 mL), and sodium triacetoxyborohydride (1.28 g, 6.02 mmol, 4 eq), and the mixture was stirred at 20 °C for 2 hours.
• Step 2 To a solution of (5-bromothiazol-4-yl)methanol (980 mg, 5.05 mmol, 1 eq) in THF (30 mL) was added sodium hydride (404 mg, 10.10 mmol, 2 eq) at 0 °C under nitrogen. The mixture was stirred at 0°C for 30 minutes, and iodomethane (3.58 g, 25.25 mmol, 1.57 mL, 5 eq) was then added at 0°C. The mixture was stirred at 15°C for 2 hours under nitrogen.
• Step 3 [00970] A flask was charged with tert-butyl N-[(1S)-1-(4-bromophenyl)ethyl]carbamate (1.4 dioxaborolane (1.42 g, 5.60 mmol, 1.2 eq), (1,1'-bis(diphenylphosphino)ferrocene)palladium(II) dichloride (170 mg, 0.23 mmol, 0.05 eq), potassium acetate (915 mg, 9.33 mmol, 2 eq) and dioxane (30 mL). The mixture was purged with nitrogen for 10 minutes, then heated to 80°C for 1 hour.
• Step 4 To a mixture of 5-bromo-4-(methoxymethyl)thiazole (900 mg, 4.33 mmol, 1 eq) and tert-butyl N-[(1S)-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]ethyl]carbamate (2.25 g, 6.49 mmol, 1.5 eq) in dioxane (30 mL) and water (5 mL) was added potassium carbonate (1.20 g, 8.65 mmol, 2 eq) and [1,1'- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (316 mg, 0.43 mmol, 0.1 eq) in one portion at 15°C under nitrogen.
• Step 4 To a solution of 5-tetrahydropyran-2-yloxypent-2-ynyl 4-methylbenzenesulfonate (2.5 g, 7.39 mmol, 1 eq), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.72 g, 8.86 mmol, 1.2 eq) in acetonitrile (20 mL) was added potassium carbonate (2.04 g, 14.77 mmol, 2 eq). Then the reaction mixture was stirred at 80°C for 12 hours.
• Exemplary compound 171 was prepared using analogous procedures.
• Exemplary Synthesis of Exemplary Compound 172 [00996] To a solution of tert-butyl N-[(1S)-1-[4-(4-formylthiazol-5-yl)phenyl]ethyl]carbamate (2 g, 6.02 mmol, 1 eq) and 1-diazo-1-dimethoxyphosphoryl-propan-2-one (1.16 g, 6.02 mmol, 1 eq) in methanol (20 mL) was added potassium carbonate (1.66 g, 12.03 mmol, 2 eq) at 0°C. The reaction mixture was stirred at 15°C for 16 hours.
• Step 2 To a solution of tert-butyl N-[(1S)-1-[4-(4-ethynylthiazol-5- yl)phenyl]ethyl]carbamate (400 mg, 1.22 mmol, 1 eq), dimethylamine (1.37 g, 12.18 mmol, 1.54 mL, 10 eq) and triformol (362 mg, 4.02 mmol, 3.3 eq) in dioxane (8 mL) was added cuprous iodide (232 mg, 1.22 mmol, 1 eq) at 0 °C.
• reaction mixture was degassed and charged with nitrogen for 3 times and then stirred at 30°C for 4 hours.
• the reaction solution was concentrated under vacuum to get the residue.
• the residue was purified by silica gel column chromatography (0-8% methanol in dichloromethane) to obtain tert-butyl N-[(1S)-1-[4-[4-[3- (dimethylamino)prop-1-ynyl]thiazol-5-yl]phenyl]ethyl]carbamate (274 mg, 0.71 mmol, 58% yield) as a light yellow gum.
• Step 2 To a mixture of 4-bromo-2-(2,2-diethoxyethoxy)pyridine (10 g, 34.46 mmol, 1 eq) and benzyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (8.49 g, 34.46 mmol, 1 eq) in toluene (200 mL) was added cesium carbonate (22.46 g, 68.93 mmol, 2 eq) and [2-(2- aminophenyl)phenyl]-chloro-palladium dicyclohexyl-[2-(2,6- diisopropoxyphenyl)phenyl]phosphane (1.61
• Step 3 To a solution of benzyl 8-[2-(2,2-diethoxyethoxy)-4-pyridyl]-3,8- diazabicyclo[3.2.1]octane-3-carboxylate (13.1 g, 28.76 mmol, 1 eq) in tetrahydrofuran (300 mL) and ethanol (300 mL) was added 10% palladium hydroxide on carbon (4.04 g, 2.88 mmol, 0.1 eq) under nitrogen. The suspension was degassed under vacuum and purged with hydrogen several times. The mixture was stirred under hydrogen (50 psi) at 60°C for 12 hours.
• Step 2 Into a 30-mL sealed tube purged and maintained with an inert atmosphere of nitrogen, was placed 1-(4-bromophenyl)-2,2-difluoroethan-1-one (3.5 g, 14.9 mmol, 1.0 equiv), (S)-2- methylpropane-2-sulfinamide (3.6 g, 29.8 mmol, 2.0 equiv) and tetrakis(propan-2-yloxy)titanium (12.7 g, 44.7 mmol, 3.0 equiv) in THF (10 mL) under nitrogen atmosphere. The resulting mixture was stirred for 14 hours at 80°C in an oil bath under nitrogen atmosphere.
• Step 3 Into a 250-mL 3-neck round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed (S)-N-[(1Z)-1-(4-bromophenyl)-2,2-difluoroethylidene]-2- methylpropane-2-sulfinamide (2.8 g, 8.3 mmol, 1 equiv) and a solution of BH 3 -THF (40.0 mL, 40.0 mmol, 1.0 M, 4.8 equiv) in THF (40.0 mL) at 0°C.
• Step 4 Into a 30-mL sealed tube purged and maintained with an inert atmosphere of nitrogen, was placed (S)-N-[(1R)-1-(4-bromophenyl)-2,2-difluoroethyl]- 2-methylpropane-2-sulfinamide (1.3 g, 3.8 mmol, 1.0 equiv), 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1,3- thiazole (1.25 g, 5.5 mmol, 1.5 equiv), K2CO3 (1.5 g, 11.3 mmol, 3.0 equiv) and Pd(dppf)Cl2 .
• Step 2 A mixture of tert-butyl N-[(1S)-1-[4-[4-[(1,3-dioxoisoindolin-2-yl)methyl]thiazol-5- yl]phenyl]ethyl]carbamate (600 mg, 1.29 mmol, 1 eq) and hydrazine hydrate (661 mg, 12.94 mmol, 0.64 mL, 98% purity, 10 eq) in ethanol (24 mL) was degassed and purged with nitrogen three times, and then the mixture was stirred at 70°C for 3 hours under nitrogen atmosphere. The reaction mixture was filtered, and the filter cake was washed with ethanol (30 mL).
• Step 3 To a solution of tert-butyl N-[(1S)-1-[4-[4-(aminomethyl)thiazol-5- yl]phenyl]ethyl]carbamate (618 mg, 1.85 mmol, 1 eq) and triethylamine (563 mg, 5.56 mmol, 0.77 mL, 3 eq) in dichloromethane (30 mL) was added acetic anhydride (227 mg, 2.22 mmol, 0.21 mL, 1.2 eq) at 0 °C. The mixture was stirred at 15 °C for 3 h.
• tert-Butyl N-[(1S)-1-[4-[4-(3-hydroxyprop-1-ynyl)thiazol-5- yl]phenyl]ethyl]carbamate was converted to tert-butyl (S)-(1-(4-(4-(3-acetamidoprop-1-yn-1- yl)thiazol-5-yl)phenyl)ethyl)carbamate using procedures described above for exemplary compound 174.
• aqueous phase was extracted with ethyl acetate (20 mL ⁇ 3).
• the combined organic phase was washed with brine (15 mL ⁇ 2), dried with anhydrous sodium sulfate, filtered and concentrated under vacuum.
• Step 2 To tert-butyl 2-(2,2-diethoxyethoxy)acetate (0.7 g, 2.82 mmol, 1 eq) in a mixture of THF (3 mL), methanol (3 mL) and water (6 mL) was added lithium hydroxide monohydrate (1.18 g, 28.19 mmol, 10 eq) in one portion at 15°C under nitrogen. The mixture was stirred at 2 hours. The pH was adjusted to 6 with 1 M hydrochloric acid. The aqueous phase was extracted with ethyl acetate (35 mL ⁇ 4).
• Step 3 To a mixture of (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)- 1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (0.2 g, 0.45 mmol, 1 eq) and 2-(2,2-diethoxyethoxy)acetic acid (87 mg, 0.45 mmol, 1 eq) in N,N-dimethylformamide (10 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (222 mg, 0.59 mmol, 1.3 eq) and triethylamine (137 mg, 1.35 mmol, 0.19 mL, 3 eq) in one portion at 15°C under nitrogen.
• 6-bromopyridin-3-ol (2.07 g, 11.891 mmol, 2.0 equiv) dropwise over 5 minutes at 0°C under nitrogen atmosphere.
• the resulting mixture was stirred for 5 minutes at 0 o C, then heated up to 50 o C and stirred for 2 hours at 50 o C.
• the reaction was quenched with water (25 mL), and the resulting mixture was extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (50mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure.
• Step 3 To a stirred solution of 6-[2-(methoxymethoxy)phenyl]-4-(8-[2-[(1r,3r)-3-([6-[(1E)-3- [(tert-butyldimethylsilyl)oxy]prop-1-en-1-yl]pyridin-3-yl]oxy)cyclobutoxy]pyridin-4-yl]-3,8- diazabicyclo[3.2.1]octan-3-yl)pyridazin-3-amine (2.10 g, 2.79 mmol, 1.00 equiv) in THF (20 mL) was added TBAF (1 mol/L, 8.3 mL, 8.3 mmol, 3.0 equiv) dropwise at 0 o C under nitrogen atmosphere.
• Step 4 To a solution of tributylphosphine (634 mg, 3.14 mmol, 10.00 equiv) and tetramethylazodicarboxamide (588 mg, 3.14 mmol, 10.00 equiv) in THF was added (2S,4R)-4- hydroxy-1-[2-(3-hydroxy-1,2-oxazol-5-yl)-3-methylbutanoyl]-N-[(1S)-1-[4-(4-methyl-1,3- thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (172 mg, 0.345 mmol, 1.10 equiv) and (2E)-3-[5-[(1r,3r)-3-[[4-(3-[3-amino-6-[2-(methoxymethoxy)phenyl]pyridazin-4-yl]-3,8- diazabicyclo[3.2.1]octan
• Exemplar compounds 186, 206, and 207 were preparing using analogous procedures.
• Exemplary Synthesis of Exemplary Compounds 189 [001060] To a solution of tert-butyl 4-(3-hydroxycyclobutoxy)piperidine-1-carboxylate (2 g, 7.37 mmol, 1 eq) in N,N-dimethylformamide (40 mL) was added sodium hydride (353 mg, 8.84 mmol, 60% purity, 1.2 eq) at 0°C. The mixture was stirred at 25°C for 1 hour.

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