WO2020168172A1 - Conjugate compounds for the degradation of raf - Google Patents

Conjugate compounds for the degradation of raf Download PDF

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Publication number
WO2020168172A1
WO2020168172A1 PCT/US2020/018260 US2020018260W WO2020168172A1 WO 2020168172 A1 WO2020168172 A1 WO 2020168172A1 US 2020018260 W US2020018260 W US 2020018260W WO 2020168172 A1 WO2020168172 A1 WO 2020168172A1
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Prior art keywords
raf
linker component
conjugate compound
mmol
degrading conjugate
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PCT/US2020/018260
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French (fr)
Inventor
Robert Zamboni
Ryan HENNING
Xian Alan Ji
Tyler Smith
Thumkunta Jagadeeswar Reddy
Stephen Claridge
Yves Leblanc
Elham AKBARIROMANI
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Zamboni Chem Solutions Inc.
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Publication of WO2020168172A1 publication Critical patent/WO2020168172A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom 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
    • A61K47/545—Heterocyclic compounds
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • 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

Definitions

  • KRAS is a key component of the canonical MAPK signaling pathway and is the most frequently mutated oncogene across all human cancers. There are no approved therapies that directly target KRAS and treatment for these cancers remains a great unmet medical need.
  • KRAS is difficult to drug due to a lack of known binding pockets suitable for small molecule binding and pM affinity for its natural substrate, GTP.
  • mutations often arise in other components of the MAPK pathway that lead to cancer and other RAS-associated diseases known as RASopathies.
  • the RAF family of kinases including ARAF, BRAF, and CRAF, are the immediate downstream KRAS effectors that propagate downstream tumorigenic signals in cancer cells. Attempts were made to develop RAF inhibitors for KRAS-driven tumors, yet the compounds were discovered to cause paradoxical activation of RAF kinases due to allosteric activation of RAF homo- and hetero-dimers.
  • the linker component is as described herein.
  • compositions, methods for treating diseases, and kits using the RAF-Degrading Conjugate Compounds described are provided herein as pharmaceutical compositions, methods for treating diseases, and kits using the RAF-Degrading Conjugate Compounds described.
  • FIG. 1 shows CRAF degradation in Calu6 cells over time after addition of 12.5 mM of Ref. Comp. 1.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 2 shows CRAF degradation in Calu6 cells over time after addition of 1 mM of Compound 1.009. The values of the y-axis are displayed as a measure of
  • FIG. 3 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.032.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 4 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.034.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 5 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.039.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 6 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.035.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 7 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.038.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 8 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.037.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 9 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.033.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 10 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.036.
  • the values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
  • FIG. 11 shows CRAF degradation in Calu6 cells after 42 hours of incubation with various amounts of Compound 1.009.
  • the values of the y-axis are displayed as percent CRAF degradation. See, for example, Biological Example 2 for further information on data analysis. DETAILED DESCRIPTION OF THE INVENTION
  • the present disclosure provides, inter alia, RAF-Degrading Conjugate Compounds that are useful in the treatment of cancer and other RAF related diseases. Without being bound to any particular theory, it is believed that the currently described conjugates effectively permeate the cell membrace, possess improved bioavailability, and are useful in treating cancer and other RAF related diseases by lowering the relative amount of RAF protein in a cell.
  • the conjugate compounds of the present disclosure target RAF for destruction via recruitment of E3 ubiquitin ligases resulting in ubiquitination and
  • 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. Ci- 8 means one to eight carbons).
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n- octyl, and the like.
  • Halogen or“halo” refer to fluorine, chlorine, bromine and iodine.
  • Cycloalkyl refers to a saturated monocyclic or bridged ring assembly containing from 3 to 8 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bridged cycloalkyl rings include, for example, norbornane, and adamantane. Cycloalkyl groups can be substituted or unsubstituted.
  • Heterocycloalkyl refers to a saturated ring system having from 3 to 8 ring members and from 1 to 4 N atoms.
  • Heterocycloalkyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8 ring members. Any suitable number of nitrogen atoms can be included in the heterocycloalkyl groups, such as 1 , 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4.
  • the heterocycloalkyl group can include groups such as aziridine, azetidine, pyrrolidine, piperidine, piperazine, azepane, 1 ,3-diazepane, azocane, and 1 ,5-diazocane. Heterocycloalkyl groups can be substituted or unsubstituted.
  • heteroaryl refers to a monocyclic aromatic ring assembly containing 6 ring atoms, where from 1 to 3 of the ring atoms are nitrogen atoms.
  • Heteroaryl groups can include any number nitrogen atoms can be included in the heteroaryl groups, such as 1, 2, or 3, or 1 to 2, 1 to 3, or 2 to 3.
  • the heteroaryl group can include groups such as pyridine, pyrazine, pyrimidine, pyridazine, and triazine (1,2,3-, 1,2,4- and 1 ,3,5-isomers). Heteroaryl groups can be substituted or unsubstituted.
  • Spirocyclic moiety refers to a pair of saturated rings having a single atom in common containing from 5 to 11 ring atoms, or the number of atoms indicated. Spirocyclic moieties can include any number of carbons, such as C5-6, C7-8, Cs-9, C9-10, Cio-11. Additionally, spirocyclic moieties can include 0 to 2 nitrogen atoms. Thus, spriocylclic moieties can be cycloalkyl moieties, or heterocycloalkyl moieties (or a combination of one each).
  • Spriocyclic moieties include, but are not limited to spiro[5.5]undecane, spiro[4.5] decane, spiro[4.4]nonane, spiro[3.5]nonane, spiro[3.3]heptane, spiro[3.4]octane, 7-azaspiro[3.5]nonane, 3- azaspiro[5.5]undecane, and 8-azaspiro[4.5]decane.
  • Spirocylic moieties can be substituted or unsubstituted.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention.
  • a stereochemical depiction it is meant to refer the compound in which one of the isomers is present and substantially free of the other isomer.
  • ‘Substantially free of another isomer indicates at least an 80/20 ratio of the two isomers, more preferably 90/10, or 95/5 or more. In some embodiments, one of the isomers will be present in an amount of at least 99%.
  • the terms“subject”,“patient” or“individual” are used herein interchangeably to include a human or animal.
  • the animal subject may be a mammal, a primate (e.g a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
  • a primate e.g a monkey
  • livestock animal e.g., a horse, a cow, a sheep, a pig, or a goat
  • a companion animal e.g., a dog, a cat
  • a laboratory test animal e.g., a mouse, a rat, a guinea pig, a bird
  • selective CRAF inhibitor describes a CRAF inhibitor that preferentially binds to CRAF over other members of the RAF family including ARAF and BRAF.
  • a selective CRAF inhibitor has at least a 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower IC50 value when binding CRAF as compared to binding to ARAF or BRAF.
  • the fold difference described herein is for the inhibitor compounds when not covalently linked to a linking component or when part of a RAF-Degrading Conjugate Compound.
  • linking atoms refers to the number of atoms that separate a Ligand for RAF and a Degradation Signaling Agent in a RAF-Degrading Conjugate Compound.
  • the number of linking atoms is not the total number of atoms in the Linker Component. Rather, it is the number of atoms between a Ligand for RAF and a Degradation Signaling Agent.
  • the Linker Component is unbranched or does not contain any cyclic moieties, the number of total atoms in the Linker Component may be the number of linking atoms.
  • the linker component includes a carbonyl
  • the oxygen atom of the carbonyl is not a“linking atom.”
  • the number of linking atoms in a given cyclic moiety is the fewest number of ring vertices needed to traverse the ring. For example, when a six membered ring is included in a Linker Component and the points of attachment to the remainder of the Linker Component, to the Ligand for RAF, or the Degradation Signaling Agent are para to one another (i.e., a 1,4-linkage), the number of linking carbons will be the same regardless of the direction of traversal. The diagram below illustrates this point:
  • the present disclosure provides a RAF-Degrading Conjugate Compound comprising a Ligand for RAF covalently attached via a Linker Component to a Degradation Signaling Agent.
  • a Ligand for RAF is any compound that effectively binds to a RAF protein.
  • Ligands for RAF include, but are not limited to, modified versions of sorafenib, Hah lOd, PF-04880594, LY3009120, PLX4720, RAF709, Vemurafenib, MLN 2480, and LHX254. Select positions of the compounds shown in Table 1 may be modified to
  • the Ligand for RAF maintains sufficient affinity for its target.
  • the Ligand for RAF is a selective CRAF inhibitor.
  • Selective CRAF inhibitors include sorafenib and Hah et al. lOd.
  • a selective CRAF inhibitor is an compound that has an CRAF IC50 value that is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower than a BRAF IC50 value, when measured under the same conditions. In some embodiments, a selective CRAF inhibitor is an compound that has an CRAF IC 50 value that is at least 5-fold, 10-fold, 20-fold, 50- fold, or 100-fold lower than a ARAF IC 50 value, when measured under the same conditions. In some embodiments, a selective CRAF inhibitor is a compound that has an CRAF IC 50 value that is at least 10-fold lower than a BRAF IC 50 value, when measured under the same conditions. In some embodiments, a selective CRAF inhibitor is a compound that has an CRAF IC50 value that is at least 10-fold lower than a ARAF IC50 value, when measured under the same conditions.
  • the Ligand for RAF is a selective B/C RAF inhibitor.
  • Selective B/C RAF inhibitors include RAF709.
  • the selective B/C RAF inhibitor has the formula shown below
  • X is N or CH.
  • select positions of the formula shown above may be modified to accommodate covalent linkage with the Linker Component, yet not obstruct target binding of the Ligand for RAF.
  • the B/C RAF inhibitor is modified to accommodate covalent linkage with the Linker Component as shown below
  • RAF709 is modified to accommodate covalent linkage with the Linker Component as shown below
  • LXH254 is modified to accommodate covalent linkage with the Linker Component as shown below
  • sorafenib is modified to accommodate covalent linkage with the
  • LY3009120 is modified to accommodate covalent linkage with the Linker Component as shown below
  • LY3009120 is modified to accommodate covalent linkage with the Linker Component as shown below
  • MLN2480 is modified to accommodate covalent linkage with the Linker Component as shown below
  • PF-04880594 is modified to accommodate covalent linkage with the Linker Component as shown below
  • PF-04880594 is modified to accommodate covalent linkage with the Linker Component as shown below
  • PF-04880594 is modified to accommodate covalent linkage with the Linker Component as shown below
  • the Ligands for RAF may be modified to provide appropriate linker chemistry between the Ligand for RAF and the Linker Component.
  • Appropriate linker chemistry includes, but is not limited to, nucleophilic substitution, amide formation, and click chemistry.
  • the Ligand for RAF is modified to include a functional group that facilitates linkage between the
  • Linker Component and the Ligand for RAF include, but are not limited to a halogen, amine, hydroxyl, carboxylic acid, ester, alkyne, and azide. It is understood that functional group included in the modified Ligand for RAF will depend on the identity of the reactive group of the Linker Component with which it will be reacted. Based on the disclosure provided herein, a person of skill in the art can appropriately choose desirable functional groups.
  • generating the Ligand for RAF includes using a synthetic precursor of an existing ligand.
  • a synthetic precursor of sorafenib includes a terminal carboxylic acid moiety at the 2-position of the pyridine (sorafenib acid). Sorafenib has a terminal N-methyl at this position.
  • the synthetic precursor of sorafenib, sorafenib acid provides appropriate linker chemistry with the Linker Component.
  • the synthetic precursor of sorafenib (sorafenib acid) can be reacted with a reactive group of the Linker Compound such as an amine or hydroxyl to form the RAF- Degrading Conjugate Compound of the present disclosure.
  • a reactive group of the Linker Compound such as an amine or hydroxyl
  • Synthetic precursors used in RAF- Degrading Conjugate Compounds of the present disclosure maintain sufficient affinity for the target of interest. Based on the disclosure provided herein, a person of skill in the art could readily prepare synthetic precursors of the above mentioned Ligands for RAF that facilitate covalent attachment with the Linker Component, yet maintain sufficient affinity for its target.
  • generating the Ligand for RAF includes adding a functional group to the Ligand for RAF.
  • the functional group is added to an aromatic ring of the Ligand for RAF.
  • the functional group is added to an alkyl portion of the Ligand for RAF.
  • sorafenib is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of Linker Component.
  • Hah et al. lOd is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of Tanker Component.
  • RAF709 is modified as shown below to accommodate appropriate linker chemistry with the Link Component, where Z is the remainder of the Linker Component
  • a selective B/C RAF inhibitor of the formula shown below is modified to accommodate appropriate linker chemistry with the Link Component, where Z is the remainder of the Linker Component and X is N or CH
  • Degradation Signaling Agents of the present disclosure include compounds or peptides that induce degradation of the targeted RAF protein.
  • Degradation Signaling Agents degrade RAF by binding to or recruiting one or more degradation proteins.
  • the degradation proteins are usually associated with the proteasome, the ubiquitin-proteasome pathway, or lysosomal proteolysis.
  • Degradation Signaling Agents include, but are not limited to E3 ligase recognition agents, hydrophobic tagging agents, proteasome recognition agents, and lysosomal recognition peptides.
  • the Degradation Signaling Agents binds to a degradation protein or a component of a degradation protein complex. In some embodiments, the bound
  • Degradation Signaling Agent activates the degradation protein or degradation protein complex.
  • the bound Degradation Signaling Agent does not significantly alter the activity of the degradation protein or degradation protein complex.
  • the degradation protein or degradation protein complex is an E3 ubiquitin ligase or an E3 ubiquitin ligase complex.
  • the E3 ubiquitin ligase or component of the E3 ubiquitin ligase complex targeted is MDM2, cIAPl, VHL protein, CBRN, or SCFP- trcp .
  • the Degradation Signaling Agents recruits a degradation protein or degradation protein complex by binding to or associating with the RAF protein causing misfolding of the RAF protein.
  • the Degradation Signaling Agents recruits a degradation protein or degradation protein complex without misfolding the RAF protein.
  • the degradation protein or degradation protein complex is the proteasome. In some embodiments, the degradation protein or degradation protein complex is a chaperone protein. In some embodiments, the chaperone protein is hsc70. i) E3 Ligase Recognition Agents [0061] The E3 ligase recognition agent is any compound or peptide that effectively binds to an
  • the E3 ligase recognition agent is an E3 ubiquitin ligase ligand.
  • the E3 ubiquitin ligase ligand is a modified version of Pomalidomide, Nutlin-3, VHL Ligand, methyl bestatin, a VHL binding peptide, a SCF
  • the VHL binding peptide is HIF-la-VHL binding peptide or hydroxy proline-HIF-la VHL binding peptide.
  • the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is PI . In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P2. [0066] In some embodiments, pomalidomide is modified to accommodate covalent linkage with the Linker Component as shown below
  • the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P3. In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P4. In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P5. In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P6.
  • each of (PI) to (P6), the phenyl ring that includes the point of attachment to the Linker Component can be further substituted with an additional substituent selected from the group consisting of Ci- 8 alkyl, halogen, and phenyl.
  • P2 is substituted with X to form P2a and P2b
  • VHL Ligand is modified to accommodate covalent linkage with the Linker Component as shown below
  • VHL Ligand is modified to accommodate covalent linkage with the Linker Component as shown below
  • IAP Ligand is modified to accommodate covalent linkage with the Linker Component as shown below
  • attachment at the position shown above for the IAP Ligand provides stereoisomerism at the attachment carbon of the IAP Ligand.
  • a stereochemically-enriched form of the IAP Ligand shown above is prepared:
  • Hydrophobic tagging is a process for manipulating the folding of a protein to cause misfolding by contacting a hydrophobic tagging agent with a protein. Misfolded proteins are recognized by the cell and selectively targeted for degradation.
  • hydrophobic tagging agents are incorporated into the RAF-Degrading Conjugate Compounds of the present disclosure, the Ligands for RAF bring the hydrophobic tagging agent in close proximity to the targeted RAF, increasing the RAF -hydrophobic tagging agent relative concentration.
  • the misfolded protein-hydrophobic tagging agent complex is recognized by molecular chaperones which target the protein for degradation.
  • hydrophobic tagging agents are generally small molecules that associate with the hydrophobic surfaces of proteins.
  • hydrophobic tagging agents include one or more alkyl portions and/or one or more polyethylene glycol units, an amide group, and an optional terminal adamantane group.
  • hydrophobic tagging agents are HyT36 or HyT13, the structures of which are shown in Table 3, below. Select positions of the compounds shown in Table 3 may be modified to accommodate covalent linkage with the Linker Component and do not obstruct target binding of the hydrophobic tagging agent. When covalently attached to the linker component, the hydrophobic tagging agent maintains sufficient affinity for its target.
  • Suitable hydrophobic Tagging Agents include those described in US 2014/0302523, the contents of which are herein incorporated by reference for all purposes.
  • the Degradation Signaling Agent is a proteasome recognition agent.
  • Proteasome recognition agents are compounds that bind to or recruit the proteasome.
  • proteasome recognition agents When proteasome recognition agents are included in the RAF-Degrading Conjugate Compounds of the present disclosure, they localize target proteins to proteasome. Without being bound to any particular theory, it is believed that proteasome recognition agents induce target protein degradation via direct proteasome recruitment and do not include ubiquitination steps. [0077] In some embodiments, the proteasome recognition agent is BocsArginine
  • Lysosomal recognition peptides are peptides that include a signaling motif for targeted lysosomal degradation. Without being bound to any particular theory, it is believed that the lysosomal recognition peptide, when incorporated into an RAF-Degrading Conjugate Compound of the present disclosure, will mark the targeted RAF protein for degradation via Chaperone- mediated autophagy.
  • the lysosomal recognition peptide includes the amino acid motif KFERQ, where each letter is an amino acid. In some embodiments, the lysosomal recognition peptide is the sequence shown in Table 4.
  • the Degradation Signaling Agents may be modified to provide appropriate linker chemistry between the Degradation Signaling Agent and the Linker Component.
  • Appropriate linker chemistry includes, but is not limited to, nucleophilic substitution, amide formation, and click chemistry.
  • the Degradation Signaling Agents is modified to include a functional group that facilitates linkage between the Linker Component and the Degradation Signaling Agents.
  • Functional groups that facilitate linkage include, but are not limited to a halogen, amine, hydroxyl, carboxylic acid, ester, alkyne, and azide.
  • generating the Degradation Signaling Agents includes using a synthetic precursor.
  • a synthetic precursor of pomalidomide include a fluoro substituent at the 4-position of the isoindoline ring (fluoro pomalidomide). Pomalidomide has an amine group at this position.
  • the synthetic precursor of pomalidomide, fluoro pomalidomide provides appropriate linker chemistry with the Linker Component.
  • the synthetic precursor of pomalidomide (fluoro pomalidomide) can be reacted with a reactive group of the Linker Compound such as an amine or hydroxyl to form the RAF-Degrading Conjugate Compound of the present disclosure.
  • Synthetic precursors used in RAF-Degrading Conjugate Compounds of the present disclosure substantially maintain their affinity for binding the target of interest. Based on the disclosure provided herein, a person of skill in the art could readily devise synthetic schemes to prepare synthetic precursors of the above mentioned Degradation Signaling Agents that readily facilitate covalent attachment with the Linker Component and maintain sufficient affinity for its target.
  • generating the Degradation Signaling Agents includes adding a functional group to the Degradation Signaling Agents.
  • the functional group is added to an aromatic ring of the Degradation Signaling Agents.
  • the functional group is added to an alkyl portion of the Degradation Signaling Agents.
  • the Degradation Signaling Agent is subsequently covalently linked to the Linker Component or the RAF-Degrading Conjugate Compound, the Degradation Signaling Agent maintains sufficient affinity for its target.
  • the Degradation Signaling Agent when the Degradation Signaling Agent is a peptide, the Degradation Signaling Agent is covalently linked to the Linker Component by amide formation at the N- or C- terminus of the peptide sequence.
  • the HIF-Ia VHL binding peptide may be linked to the Linger Component, as shown below, where Z is the remainder of Linker Component.
  • the Degradation Signaling Agent is a peptide
  • the Degradation Signaling Agent is covalently linked to the Liner Component at a side-chain.
  • nutlin-3 is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of Linker Component.
  • methylbestatin is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of the linker component.
  • the chloro substituent in HyT13 or HyT36 is used in a nucleophilic substitution reaction to covalently link the Linker Component and the Degradation Signaling Agent as shown below, where Z is the remainder of the linker component.
  • the appropriate linker chemistry between the Degradation Signaling Agent and the Linker Component is already present and the Degradation Signaling Agent is not modified.
  • the carboxylic acid of BocsArginine is used to form a covalent linkage with the Linker Component, where Z is the remainder of the linker component.
  • the Linker Component of the present disclosure is included to provie sufficient separation between the Ligand for RAF and the Degradation Signaling Agent so that both the Ligand for RAF and the Degradation Signaling Agent can perform its desired function.
  • Two variables that are often included in Linker Components are structural features, such as cyclic moieties or points of unsaturation, that help maintain certain conformations between the Ligand for RAF and the Degradation Signaling Agent, and the number of linking atoms.
  • the number of linking atoms in the Linker Component can vary depending on the particular Lingand for RAF and Degradation Signaling Agent being used. In some
  • the number of linking atoms is from about 4 to about 23 atoms. In some embodiments, the number of linking atoms is from about 5 to 15 atoms. In some embodiments, the number of linking atoms is from about 5 to 10 atoms. In some embodiments, the number of lining atoms is from about 9 to 15 linking atoms. In some embodiments, the number of linking atoms is from about 16 to 21 linking atoms. [0093] It can be advantageous to include one or more cyclic moieties into the Linker
  • the Linker Component comprises 1 to 4 cyclic moieties. In some embodiment, the Linker Component includes 1 to 3 cyclic moieties. In some embodiments, the Linker Component includes 1 to 2 cyclic moieties. In some embodiments, the Linker Component includes 2 cyclic moieties. In some embodiments, the Linker Component includes 1 cyclic moiety.
  • Cyclic moieties as referred to above include a C3-8 cycloalkyl, a triazole, a phenyl, a 6- to 8-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms, a six-membered heteroaryl comprising 1 to 2 nitrogen atoms, and C7-11 spirocylic moieties comprising 0 to 2 nitrogen atoms.
  • cyclic moieties are selected from the group consisting of a triazole, a phenyl, a 6- to 8-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms. When two or more cyclic moieties are present the cyclic moieties are sometimes the same, sometimes they are different.
  • Linker Components can also includee points of unsaturation.
  • the Linker Component includes one or more double or triple bonds.
  • the Linker Component includes one double bond.
  • the Linker Componenet includes one triple bond.
  • the Linker Component includes two double bonds.
  • the Linker Componenet includes two triple bonds.
  • the Linker Component comprises 5 to 10 linking atoms and 1 or 2 cyclic moieties, wherein each cyclic moiety is independently selected from the group consisting of a triazole, a phenyl, and a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms.
  • the Linker Component comprising 9 to 15 linking atoms and two cyclic moieties, wherein the cyclic moieties are independently selected from the group consisting of a triazole, a phenyl, and a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms.
  • the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is unsubstituted.
  • the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is substituted.
  • the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is substituted with 1 to 2 substituents selected from C M alkyl and halo. In some embodiments, the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is substituted with 1 to 2 methyl substituents.
  • the 6- to 8-membered heterocycloalkyl includes both monocylic and bridged ring assemblies. In some embodiments the 6- to 8-membered heterocycloalkyl is bridged. In some embodimnets, the 6- to 8-membered heterocycloalkyl is monocyclic.
  • Linker Components are single pre-synthesized molecular entities that are covalently attached to a Ligand for RAF and a Degradation Signaling Agent (either simultaneously or in an order of most synthetic convenience).
  • Linker Components are made from separate molecules entities (segments of a linker component) which are covalently attached to a Ligand for RAF or a Degradation Signaling Agent. After each segment of the linker component is covalently attached to a respective Ligand for RAF and Degradation Signaling Agent, an additional reaction is performed to covalently link the two segments of the Linker Component to form a RAF-Degrading Conjugate Compound.
  • each segment of the Linker component will have an additional reactive group that facilitates covalent attachment between the two segments of the Linker Component.
  • Reactive groups that facilitate covalent attachment to the Ligand for RAF and the Degradation Signaling Agent depend on the specific Ligand for RAF and Degradation Signaling Agent used, but as discussed above, suitable covalent linking chemistries include, but are not limited to, nucleophilic substitution, amide formation, and click chemistry. Exemplary linker chemistries are shown in Table 5, below. In the table below, R 1 is the remainder of the Ligand for RAF or the remainder of the Degradation Signaling Agent, and R 2 is the remainder of the Linker Component. A person of skill in the art will recognize that the functional groups of R 1 and R 2 may switch without departing from the scope of the present disclosure.
  • the first and second reactive group are different functional groups. In some embodiments, the first and second reactive groups are the same functional group.
  • nucleophilic substitution reaction with an amine and iodine
  • additional nucleophiles and leaving groups may be used in the nucleophilic substitution reaction.
  • Suitable nucleophiles include, but are not limited to, hydroxyl and thiol groups.
  • Suitable leaving groups include, but are not limited to Cl, Br, and OTs.
  • the Ligand for RAF or Degradation Signaling Agent contain a reactive group that readily facilitates covalent attachment to the Linker Component.
  • the Ligand for RAF and/or Degradation Signaling Agent is modified to include a suitable functional group that readily facilitates covalent attachment.
  • a sorafenib component (sorafenib acid) is covalently attached to the Linker Component via amide formation. This is achieved by reacting sorafenib acid (comprising a carboxylic acid at the 2-position of the pyridine ring) with an amine group from the Linker Component.
  • both the Ligand for RAF and the Degradation Signaling Agent maintain sufficient affinity for their targets to carry out the desired effect.
  • Linker Components are made from separate molecular entities which are first covalently attached to a Ligand for RAF or a
  • the two segments of the Linker Component are covalently attached after each entity has been pre-attached to a respective Ligand for RAF or Degradation Signaling Agent.
  • Suitable functional groups that facilitate covalent attachment of the molecular entities include the exemplary linker chemistry shown in Table 5 and the groups discussed supra.
  • the two segments of the linker component are covalently attached using azide-alkyne cycloaddition.
  • the two segments of the linker component are covalently attached using amid formation chemistry.
  • Linking units of the Linker Component are generally non-reactive moieties such as alkyl groups.
  • the alkyl groups include one or more ether linkages.
  • the alkyl groups are linear.
  • the alkyl groups are branched.
  • the first reactive group is an amine group and the second reactive group is a carboxylic acid group.
  • the Linker Component includes a triazine.
  • the Linker Component comprises polyethylene glycol.
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has a formula
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has the formula a). In some embodiments, the Linker Component has the formula y). In some embodiments, the Linker Component has the formula z). In some embodiments, the Linker Component has the formula aa). In some embodiments, the Linker Component has the formula bb). In some embodiments, the Linker Component has the formula cc). In some embodiments, the Linker Component has the formula dd). In some embodiments, the Linker Component has the formula ee). In some embodiments, the Linker Component has the formula ft).
  • the RAF-Degrading Conjugate Compounds are generated by covalently linking a Ligand for RAF, a Degradation Signaling Agent, and a Linker Component. Covalent linkages between each component may be achieved using the linker chemistry and the modifications for the Ligands for RAF and the Degradation Signaling Agents described in the preceding sections.
  • the covalently linked Ligands for RAF and the Degradation Signaling Agents maintain sufficient binding affinity for their targets to carry out the desired effect.
  • the RAF-Degrading Conjugate Compound includes a Ligand for RAF that is RAF709, and a Degradation Signaling Agent that is pomalidomide. [0117] In some embodiments, the RAF-Degrading Conjugate Compound includes a Ligand for RAF that is RAF709, and a Degradation Signaling Agent that is VHL Ligand.
  • the RAF-Degrading Conjugate Compound includes a Ligand for RAF that is RAF709, and a Degradation Signaling Agent that is Inhibitor of Apoptosis Protein (IAP) Ligand.
  • RAF709 has a structure
  • X is N or CH and the wavy line indicates the site of attachment of the Linker Component.
  • X is N.
  • X is CH.
  • pomalidomide has a structure selected from the group consisting of
  • VHL Ligand has a structure
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has a formula selected from the group consisting of
  • Linker Component has a formula selected from the group consisting of
  • the Linker Component has a formula selected from the group consisting of
  • the Linker Component has the formula a). In some embodiments, the Linker Component has the formula y). In some embodiments, the Linker Component has the formula z). In some embodiments, the Linker Component has the formula aa). In some embodiments, the Linker Component has the formula bb). In some embodiments, the Linker Component has the formula cc). In some embodiments, the Linker Component has the formula dd). In some embodiments, the Linker Component has the formula ee). In some embodiments, the Linker Component has the formula ff).
  • the RAF-Degrading Conjugate Compound is selected from the Examples.
  • compositions for degrading RAF in humans and animals will typically contain a pharmaceutical carrier or diluent.
  • composition as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions for the administration of the compounds of this invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy and drug delivery. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients.
  • the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self emulsifications as described in U.S. Patent No. 6,451,339, hard or soft capsules, syrups, elixirs, solutions, buccal patch, oral gel, chewing gum, chewable tablets, effervescent powder and effervescent tablets.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, antioxidants and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example PVP, cellulose, PEG, starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated, enterically or otherwise, by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • emulsions can be prepared with a non-water miscible ingredient such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters and the like.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxy-ethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbit
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl, p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
  • the pharmaceutical compositions of the invention may also be in the form of oil-in water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG and surfactants.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG and surfactants.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • 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-butane diol.
  • the 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 diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions of the present invention may also be administered in the form of suppositories for rectal administration of the drug.
  • suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • the compounds can be administered via ocular delivery by means of solutions or ointments. Still further, transdermal delivery of the subject compounds can be accomplished by means of iontophoretic patches and the like.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. As used herein, topical application is also meant to include the use of mouth washes and gargles.
  • the compounds of the invention may be formulated for depositing into a medical device, which may include any of variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets or other device that can be deployed or permanently implanted within a body lumen.
  • a medical device which may include any of variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets or other device that can be deployed or permanently implanted within a body lumen.
  • a medical device may include any of variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets or other device that can be deployed or permanently implanted within a body lumen.
  • the presently disclosed RAF-Degrading Conjugate may include any of variety of conventional grafts, stent
  • Compound may be deposited within a medical device, such as a stent, and delivered to the treatment site for treatment of a portion of the body.
  • a medical device such as a stent
  • Stents have been used as delivery vehicles for therapeutic agents (i.e., drugs).
  • Intravascular stents are generally permanently implanted in coronary or peripheral vessels.
  • Stent designs include those of U.S. Pat. Nos. 4,733,655 (Palmaz), 4,800,882 (Gianturco), or 4,886,062 (Wiktor).
  • Such designs include both metal and polymeric stents, as well as self-expanding and balloon-expandable stents.
  • Stents may also used to deliver a drug at the site of contact with the vasculature, as disclosed in U.S. Pat. No. 5,102,417 (Palmaz) and in International Patent Application Nos.
  • WO 91/12779 Medtronic, Inc.
  • WO 90/13332 Cedars-Sanai Medical Center
  • U.S. Pat. Nos. 5,419,760 Narciso, Jr.
  • U.S. Pat. No. 5,429,634 Narciso, Jr.
  • Stents have also been used to deliver viruses to the wall of a lumen for gene delivery, as disclosed in U.S. patent application Ser. No. 5,833,651 (Donovan et al.).
  • the term“deposited” means that the RAF-Degrading Conjugate Compound is coated, adsorbed, placed, or otherwise incorporated into the device by methods known in the art.
  • the conjugate may be embedded and released from within (“matrix type”) or surrounded by and released through (“reservoir type”) polymer materials that coat or span the medical device.
  • the conjugate may be entrapped within the polymer materials or coupled to the polymer materials using one or more the techniques for generating such materials known in the art.
  • the conjugate may be linked to the surface of the medical device without the need for a coating by means of detachable bonds and release with time, can be removed by active mechanical or chemical processes, or are in a permanently immobilized form that presents the conjugate at the implantation site.
  • the RAF-Degrading Conjugate Compound may be incorporated with polymer compositions during the formation of biocompatible coatings for medical devices, such as stents.
  • the coatings produced from these components are typically homogeneous and are useful for coating a number of devices designed for implantation.
  • the polymer may be either a biostable or a bioabsorbable polymer depending on the desired rate of release or the desired degree of polymer stability, but a bioabsorbable polymer is preferred for this embodiment since, unlike a biostable polymer, it will not be present long after implantation to cause any adverse, chronic local response.
  • Bioabsorbable polymers that could be used include, but are not limited to, poly(L-lactic acid), polycaprolactone, polyglycolide (PGA), poly(lactide-co-glycobde) (PLLA/PGA), poly(hydroxybutyrate), poly(hydroxybutyrate- co-valerate), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(D-lactic acid), poly(L-lactic acid), poly(D,L-lactic acid), poly(D,L-lactide) (PLA) , poly (L-lactide) (PLLA), poly(glycobc acid-co-trimethylene carbonate) (PGA/PTMC), polyethylene oxide (PEO), polydioxanone (PDS), polyphosphoester, polyphosphoester urethane, poly(amino acids), cyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate), copoly(ether-esters) (e
  • biostable polymers with a relatively low chronic tissue response such as polyurethanes, silicones, and polyesters could be used and other polymers could also be used if they can be dissolved and cured or polymerized on the medical device such as polyolefins, polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers and copolymers, vinyl halide polymers and copolymers, such as polyvinyl chloride;
  • polyvinylpyrrolidone polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics, such as polystyrene, polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; pyran copolymer; polyhydroxy-propyl-methacrylamide -phenol;
  • polyhydroxyethyl-aspartamide-phenol polyethyleneoxide -polylysine substituted with palmitoyl residues
  • polyamides such as Nylon 66 and polycaprolactam
  • alkyd resins polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins, polyurethanes; rayon; rayon- triacetate; cellulose, cellulose acetate, cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and carboxymethyl cellulose.
  • Polymers and semipermeable polymer matrices may be formed into shaped articles, such as valves, stents, tubing, prostheses and the like.
  • the RAF-Degrading Conjugate Compound is coupled to a polymer or semipermeable polymer matrix that is formed as a stent or stent-graft device.
  • polymers are applied to the surface of an implantable device by spin coating, dipping or spraying. Additional methods known in the art can also be utilized for this purpose. Methods of spraying include traditional methods as well as microdeposition techniques with an inkjet type of dispenser. Additionally, a polymer can be deposited on an implantable device using photo-patterning to place the polymer on only specific portions of the device. This coating of the device provides a uniform layer around the device which allows for improved diffusion of various analytes through the device coating.
  • the RAF-Degrading Conjugate Compound is formulated for release from the polymer coating into the environment in which the medical device is placed.
  • the conjugate is released in a controlled manner over an extended time frame (e.g., months) using at least one of several well-known techniques involving polymer carriers or layers to control elution. Some of these techniques were previously described in U.S. Patent
  • the reagents and reaction conditions of the polymer compositions can be manipulated so that the release of the RAF-Degrading Conjugate Compound from the polymer coating can be controlled.
  • the diffusion coefficient of the one or more polymer coatings can be modulated to control the release of the conjugate from the polymer coating.
  • the diffusion coefficient of the one or more polymer coatings can be controlled to modulate the ability of an analyte that is present in the environment in which the medical device is placed (e.g.
  • Yet another embodiment of the invention includes a device having a plurality of polymer coatings, each having a plurality of diffusion coefficients. In such embodiments of the invention, the release of the conjugate from the polymer coating can be modulated by the plurality of polymer coatings.
  • the release of the RAF-Degrading Conjugate Compound from the polymer coating is controlled by modulating one or more of the properties of the polymer composition, such as the presence of one or more endogenous or exogenous compounds, or alternatively, the pH of the polymer composition.
  • the properties of the polymer composition such as the presence of one or more endogenous or exogenous compounds, or alternatively, the pH of the polymer composition.
  • certain polymer compositions can be designed to release a conjugate in response to a decrease in the pH of the polymer composition.
  • certain polymer compositions can be designed to release the conjugate in response to the presence of hydrogen peroxide.
  • RAF-Degrading Conjugate Compounds are useful in treating or preventing many disease or conditions including, but not limited to, cancer and RASopathies.
  • the diseases or conditions are mediated, at least in part, by RAF.
  • cancer can be treated or prevented by administering one or more RAF-Degrading Conjugate Compounds.
  • Cancer generally includes any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites.
  • Non-limiting examples of different types of cancer suitable for treatment using the compositions of the present invention include ovarian cancer, breast cancer, lung cancer (such as non-small-cell lung carcinoma), bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, colon cancer, anal cancer, colorectal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, rectal cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, renal cancer (i.e., renal cell carcinoma), cancer of the central nervous system, skin cancer, choriocarcinomas, head and neck cancers, bone cancer, osteogenic sarcomas, fibrosarcoma, neuroblastoma, glioma, melanoma, leukemia (e.g acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia
  • the cancer is melanoma, an epithelial cancer (e.g prostate cancer, ovarian cancer, breast cancer), or a blood cancer (e.g., leukemia, lymphoma, multiple myeloma).
  • an epithelial cancer e.g prostate cancer, ovarian cancer, breast cancer
  • a blood cancer e.g., leukemia, lymphoma, multiple myeloma.
  • the cancer is melanoma or colorectal cancer.
  • the RAF-Degrading Conjugate Compounds of the present disclosure are useful in treating genetically defined cancers, irrespective or of tissue origin.
  • Genetically defined cancers include, but are not limited to, those mediated, at least in part by mutant KRAS, HRAS, or NRAS proteins.
  • the mutations in these proteins include, but are not limited to, codons 12, 13, and 61, including misssense mutations to any of the 20 naturally occurring amino acids.
  • KRAS mutations include, but are not limited to, G12D, G12V, G13D, and G13C; HRAS mutations include, but are not limited to, G12V, Q61R, Q61L, and G13R; and NRAS mutations include, but are notlimited to Q61R, Q61K, G12D, and G13D.
  • Genetically defined cancers also include, but are not limited to, cancers mediated, at least in part, by mutant RAF proteins.
  • the mutated RAF protein is BRAF.
  • the BRAF mutation is V600E.
  • RASopathies can be treated or prevented by administering one or more RAF-Degrading Conjugate Compounds.
  • RASopathies are developmental syndromes involving dysregulation of the RAS/MPAK pathway.
  • Non-limiting examples of RASopathies include neurofibromatosis type 1, capillary malformation-arteriovenous malformation syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Noonan syndrome, Costello syndrome, Legius syndrome, LEOPARD syndrome.
  • the RASopathy is Noonan syndrome, Costello syndrome,
  • the RASopathy is Noonan syndrome or LEOPARD syndrome.
  • the present disclosure includes a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the RAF- Degrading Conjugate Compound and an additional cancer therapeutic agent.
  • Cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, and endocrine therapies.
  • RAF-Degrading Conjugate Compound and the additional cancer therapeutic agent are administered simultaneously.
  • the RAF-Degrading Conjugate Compound and the additional cancer therapeutic agent are administered sequentially.
  • the RAF-Degrading Conjugate Compounds and the additional therapeutic agent, when administered simultaneously are formulated in a single pharmaceutical composition.
  • the RAF- Degrading Conjugate Compounds and the additional therapeutic agent, when administered simultaneously are two separate compositions.
  • Chemotherapeutic agents are well known in the art and include, but are not limited to, anthracenediones (anthraquinones) such as anthracyclines (e.g., daunombicin (daunomycin; mbidomycin), doxorubicin, epimbicin, idambicin, and valmbicin), mitoxantrone, and pixantrone; platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin); tamoxifen and metabolites thereof such as 4-hydroxytamoxifen (afimoxifene) and N-desmethyl-4-hydroxytamoxifen (endoxifen); taxanes such as paclitaxel (taxol) and docetaxel; alkylating agents (e.g., anthracenediones (anthraquino
  • the chemotherapeutic agent is an MEK inhibitor.
  • MEK inhibitors are small molecules or biologies that bind to and inhibit or decrease the normal function of MEK proteins.
  • MEK inhibition is particularly useful in melanoma as well as RAS and RAF mediated cancers. Any known MEK inhibitor can be used in combination with the RAF-Degrading Conjugate Compounds of the present disclosure.
  • the MEK inhibitor targets MEK1, MEK2, or both MEK1 and 2.
  • the MEK inhibitors is a small molecule.
  • the MEK inhibitor is trametinib, pimasertib, selumertinib, PD- 0325901, Refametinib, TAK733, MEK162, R05126766, WX-554, R04987655, CD-0931, or AZD8330.
  • Radiotherapeutic agents are well known in the art and can comprise external-beam radiation therapy and/or internal radiation therapy.
  • External beam radiation therapy delivers radioactive beams of high energy X-rays and/or gamma rays to a patient’s tumor
  • internal radiation therapy delivers radioactive atoms to a patient’s tumor.
  • Both external beam radiation therapy and internal radiation therapy are used to suppress tumor growth or kill cancer cells by delivering a sufficient quantity of radioactivity to the target site.
  • the radiotherpaeutic agent comprises a radioactive atom and is complexed with a biologic or synthetic agent to increase delivery to the target site.
  • biologic or synthetic agents are known in the art.
  • Suitable radioactive atoms for use with the RAF-Degrading Conjugate Compounds of the present disclosure include any of the radionuclides described herein, or any other isotope which emits enough energy to destroy a targeted tissue or cell.
  • radiotherapeutic agents may be coupled to targeting moieties, such as antibodies, to improve the localization of radiotherapeutic agents to cancerous or infected cells.
  • radioactivity is intended to include any nuclide that exhibits radioactivity.
  • A“nuclide” refers to a type of atom specified by its atomic number, atomic mass, and energy state, such as carbon 14 ( 14 C).
  • Radioactivity refers to the radiation, including alpha particles, beta particles, nucleons, electrons, positrons, neutrinos, and gamma rays, emitted by a radioactive substance.
  • radionuclides suitable for use in the present invention include, but are not limited to, fluorine 18 ( 18 F), fluorine 19 ( 19 F), phosphorus 32 ( 32 P), scandium 47 ( 47 Sc), cobalt 55 ( 55 Co), copper 60 ( 60 Cu), copper 61 ( 61 Cu), copper 62 ( 62 Cu), copper 64 ( 64 Cu), gallium 66 ( 66 Ga), copper 67 ( 67 Cu), gallium 67 ( 67 Ga), gallium 68 ( 68 Ga), rubidium 82 ( 82 Rb), yttrium 86 ( 86 Y), yttrium 87 ( 87 Y), strontium 89 ( 89 Sr), yttrium 90 ( 90 Y), rhodium 105 ( 105 Rh), silver 1 1 1 ( m Ag), indium 1 1 1 ( m In), iodine 124 ( 124 I), iodine 125 ( 125 I), iodine 131 ( 131 I), tin 1 17
  • the“m” in 1 17m Sn and 99m Tc stands for the meta state.
  • naturally- occurring radioactive elements such as uranium, radium, and thorium, which typically represent mixtures of radioisotopes, are suitable examples of radionuclides.
  • 67 Cu, 131 1, 177 Lu, and 186 Re are beta- and gamma-emitting radionuclides.
  • 212 Bi is an alpha- and beta-emitting radionuclide.
  • 21 'At is an alpha-emitting radionuclide.
  • 32 P, 47 Sc, 89 Sr, 90 Y, 105 Rh, m Ag, 117m Sn, 149 Pm, 153 Sm, 166 Ho, and 188 Re are examples of beta-emitting radionuclides.
  • 67 Ga, 11 'in, 99m Tc, and 201 T1 are examples of gamma-emitting radionuclides.
  • 55 Co, 60 Cu, 61 Cu, 62 Cu, 66 Ga, 68 Ga, 82 Rb, and 86 Y are examples of positron-emitting radionuclides.
  • 64 Cu is a beta- and positron-emitting radionuclide. 3.
  • Endocrine therapy is the manipulation of the endocrine system through the
  • Endocrine therapy is particularly useful in certain types of cancer, including breast cancer. Any known hormone antagonist or modulator may be used in combination with the RAF-Degrading Conjugate Compounds of the present disclosure.
  • Useful Endocrine therapies include, but are not limited to, aromatase inhibitors (e.g . letrozole), megestrol acetate, flutamide, tamoxifen, raloxifene, lasofoxifene, apeledoxifene, thaledoxifene/conjugated estrogens, and combinations thereof.
  • kits and systems can be prepared according to the present invention, depending upon the intended user of the kit and system and the particular needs of the user.
  • the present disclosure provides a kit that includes one or more RAF-Degrading Conjugate Compounds.
  • the present disclosure provides a kit that includes one or more RAF-Degrading Conjugate Compounds and one or more therapeutic agents selected from a chemotherapeutic agent, a radiotherapeutic agent, an endocrine therapy.
  • kits described herein include a label describing a method of administering one or more RAF-Degrading Conjugate Compounds and/or one or more additional cancer therapeutic agents described herein. Some of the kits described herein include a label describing a method of treating a disease or disorder described herein.
  • compositions of the present invention including but not limited to, compositions comprising one or more RAF-Degrading Conjugate Compounds and one or more additional cancer therapeutic agents described herein may, if desired, be presented in a bottle, jar, vial, ampoule, tube, or other container-closure system approved by the Food and Drug Administration (FDA) or other regulatory body, which may provide one or more dosages containing the compounds.
  • the package or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice indicating approval by the agency.
  • the kit may include a formulation or composition as described herein, a container closure system including the formulation or a dosage unit form including the formulation, and a notice or instructions describing a method of use as described herein.
  • the kit includes a container which is compartmentalized for holding the various elements of a formulation (e.g ., the dry ingredients and the liquid ingredients) or composition, instructions for making the formulation or composition, and instructions for administering the formulation or composition in a subject.
  • a formulation e.g ., the dry ingredients and the liquid ingredients
  • the kit may include the pharmaceutical preparation in dehydrated or dry form, with instructions for its rehydration (or reconstitution) and
  • Kits with unit doses of the compounds described herein, e.g. in oral, rectal, transdermal, or injectable doses (e.g. , for intramuscular, intravenous, or subcutaneous injection), are provided.
  • injectable doses e.g. , for intramuscular, intravenous, or subcutaneous injection
  • an informational package insert describing the use and attendant benefits of the composition may be included in addition to the containers containing the unit doses.
  • Some embodiments of the present invention include packages that include one or more RAF-Degrading Conjugate Compounds and one or more additional cancer therapeutic agents described herein.
  • the compounds of this invention may be prepared in light of the specification using steps generally known to those of ordinary skill in the art. Those compounds may be analyzed by known methods, including but not limited to LC-MS (liquid chromatography mass
  • LRMS values were recorded on Waters micromass ZQ using direct injection of the samples in either methanol or acetonitrile.
  • Analytical HPLC was carried out on Waters alliance using Agilent, Zorbax-SB-CN, 3.5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100%) contains ammonium acetate buffer; flow rate, 1.5 mL/min, run time, 20 min]
  • HPLC Method A Column, Agilent, Zorbax Eclipse XDB-C8, 5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100%) contains ammonium acetate buffer; flow rate, 1.5 mL/min, run time, 20 min.
  • HPLC Method B Column, Agilent, Zorbax-SB-CN, 3.5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100%) contains ammonium acetate buffer; flow rate, 0.1 mL/min, run time, 20 min.
  • HPLC Method C Column, Agilent, Zorbax SB-C18, 5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100% for 8 min, 100% for 2 min) contains 0.1% TFA as buffer; flow rate, 1.5 mL/min, run time, 10 min.
  • HPLC Prep method Column, Phenomenex, Synergi, 4p, Max-RP 80A, AX; 250 x 21.2 mm, mobile phase, acetonitrile in water (10-100%, 25 min, water contains 0.2% HCO2H buffer); flow rate, 15 mL/min.
  • Example A Synthesis of /V-(6'-(( 14-hydroxy-3,6,9, 12-tetraoxatetradecyl)oxy)-2-methyl-5'- morpholino- [3 ,3 -bipyridin] -5-yl)-3 -(trifluoromethyl)benzamide
  • Example 1 Synthesis of /V-(6'-(( 14-((2-( 1 -(hydroxymcthyl)-2,6-dioxopipcridin-3-yl)- 1 - oxoisoindolin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.001)
  • Step I tert- butyl (2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolm-4-yl) carbonate:
  • Step II tert- butyl (2-(2,6-dioxo-l-((2-(trimethylsilyl)ethoxy)methyl)piperidin-3-yl)-l- oxoisoindolin-4-yl) carbonate
  • Step III 3-(4-hydroxy-l-oxoisomdolm-2-yl)-l-((2-(trimethylsilyl)ethoxy)methyl)piperidine- 2,6-dione
  • Step IV V-(6'-((14-((2-(2,6-dioxo-l-((2-(trimcthylsilyl)cthoxy)mcthyl)pipcridin-3- yl)-l-oxoisoindolin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-
  • the flask was purged with nitrogen/vacuum (3 cycles) and the reaction mixture was stirred at rt for 18 h under inert argon atmosphere with vigorous stirring.
  • the reaction mixture was diluted with water (2 mL) then extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine (2 mL), dried (Na2S04), and concentrated.
  • Example 3 Synthesis of /V-(6'-(2-(2-(2-(( 1 -(7-((2-(2,6-dioxopipcridin-3-yl)- 1 -oxoisoindolin- 4-yl)oxy)heptyl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)-2-methyl-5'- morphohno-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.003)
  • Example 4 Synthesis of ,V-(6’-(( 14-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin-4-yl)oxy)- 3,6,9, 12-tetraoxatetra-decyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ⁇ Compound 1.004).
  • V-(6'-(( 14-hydroxy-3,6,9, 12-tctraoxatctradccyl)oxy)-2-mcthyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in Example A.
  • Step I V-(6'-((14-iodo-3,6,9,12-tctraoxatctradccyl)oxy)-2-mcthyl-5'-morpholino-[3,3'- bipyridm]-5-yl)-3-(trifluoromethyl)benzamide
  • Example 5 Synthesis of /V-(6'-(2-(2-(2-(( 1 -(7-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin- 5-yl)oxy)heptyl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.005)
  • Example 6 Synthesis of /V-(6'-(2-(2-(2-(( 1 -(7-((2-(2,6-dioxopipcridin-3-yl)-3-oxoisoindolin- 4-yl)oxy)heptyl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (Compound 1.006)
  • Example 7 Synthesis of /V-(6'-(( 14-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin-4-yl)oxy)- 3,6,9, 12-7V-(6'-((14-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)oxy)-3,6,9, 12- tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ⁇ Compound 1.007).
  • A-(6'-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in Example A. [0211] To a solution of 3-(6-hydroxy-l-oxoisoindolin-2-yl)piperidine-2,6-dione (45 mg, 0.173 mmol) in THF (1 mL) was added PPh3 (100 mg, 0.432 mmol).
  • Example 8 Synthesis of /V-(6'-(( 14-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin-5-yl)oxy)- 3,6,9, 12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.008 )
  • Example 9 Synthesis of ,V-(6’-(2-(4-( 2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.009).
  • Step I tert- butyl 4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin] -6-yl)oxy)ethyl)-piperazine-l -carboxylate :
  • Example 10 Synthesis of N-(6'-(( 1 -(( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5-yl)- lH-l,2,3-triazol-4-yl)methyl)piperidin-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)- 3-(trifluoro-methyl)benzamide ( Compound 1.010).
  • Step I A'-(2-mcthvl-5'-morpholino-6'-((l-(prop-2-vn-l-yl)pipcridin-4-vl)oxy)-[3,3'- bipyridm]-5-yl)-3-(trifluoromethyl)benzamide: [0216] To a stirred solution of l-(prop-2-yn-l-yl)piperidin-4-ol (453 mg, 3.26 mmol) in 1 ,4- dioxane (5.0 mL) was added 60% sodium hydride (130 mg, 3.26 mmol), after stirred at rt for 30 min, /V-(6'-fliioro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromcthyl)bcnzamidc (300 mg, 0.652 mmol) was added in one portion, it was placed on pre-heated oil
  • Step II V-(6'-((l-((l-(2-(2,6-dioxopipcridin-3-yl)-l,3-dioxoisoindolin-5-yl)-l H-l ,2,3-triazol-
  • Example 11 Synthesis of / V- ( 6’ - ( 2 - ( ( 4 - ( ( ( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)methyl)benzyl)oxy)ethoxy)-2-methyl-5'-morpholino-[3 ,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.011)
  • Example 12 Synthesis of /V-(6'-((4-( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5-yl)- 1 H- 1 ,2,3 -triazol-4-yl)benzyl)oxy)-2-methyl-5 '-morpholino- [3,3 '-bipyridin] -5-yl)-3 - (trifluoromethyl)benzamide ( Compound 1.012)
  • Step I V-(6'-((4-cthvnvlbcnzvl)oxy)-2-mcthvl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide:
  • Example 13 Synthesis of N-(6'-(2-((l-((l-(2-(2,6-dioxopiperidin-4-yl)-l,3-dioxoisoindolin-5- yl)- 1 H- 1 ,2,3-triazol-4-yl)methyl)piperidin-4-yl)oxy)ethoxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide. ( Compound 1.013).
  • Example 14 Synthesis of /V-(6'-(2-( 1 -( 2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5-yl)- 1 H- 1 ,2,3 -triazol-4-yl)ethoxy) -2-m ethyl-5 '-morpholino- [3 ,3 '-bipyridin] -5-yl)-3 - (trifluoromethyl)benzamide ( Compound 1.014).
  • Example 15 Synthesis of /V-(6'-(2-( 1 -( 2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperidin-4-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.015 )
  • Step I tert- butyl 4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperidine-l-carboxylate
  • Step II 4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]- 6-yl)oxy)-ethyl)piperidin-l-ium chloride:
  • Example 16 Synthesis of /V-(6'-(( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperidin-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.016).
  • Step III Compound 1.016.
  • Example 17 Synthesis of /V-(6'-((l-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5- yl)piperidin-4-yl) methoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.017):
  • Step I Synthesis of tert- butyl 4-(((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl) benzamido)-[3,3'-bipyridm]-6-yl)oxy)methyl)piperidine-l-carboxylate
  • Title compound (150 mg, 70%) was prepared as a yellow solid from / ⁇ ?r/-butyl 4- (hydroxymethyl)piperidine- 1 -carboxylate (351 mg, 4.34 mmol) as described in step I of
  • Step II 4-(((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)methyl)piperidin-l-ium chloride
  • Example 18 Synthesis of V-(6’-((4-((4-((4-(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperazin-l-yl)methyl)benzyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.018 )
  • Step I tert- butyl 4-(4-(hydroxymethyl)benzyl)piperazine-l-carboxylate
  • Step II tert- butyl 4-(4-(((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)methyl)benzyl)piperazine-l-carboxylate.
  • the reaction mixture was cooled to rt and poured into water (30 mL).
  • the mixture was extracted with DCM (3 x 50 mL) and the combined organic extracts were dried over anhydrous Na 2 S0 4 , filtered and concentrated.
  • the residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-20%) as eluant to afford the title compound (51 mg, 28%) as a yellow solid.
  • Example 19 The synthesis of /V-(6'-(2-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin- 5-yl)piperazin-l-yl)ethoxy)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.019).
  • Step I tert- butyl 4-(2-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethoxy)ethyl)piperazine-l-carboxylate
  • Example 20 Synthesis of /V-(5'-(dimcthylamino)-6'-(2-(4-(2-(2,6-dioxopipcridin-3-yl)- 1,3- dioxoisoindolin-5-yl)piperazin-l-yl)ethoxy)-2-methyl-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.020 )
  • Step I V-(5'-(dimcthylamino)-6'-fluoro-2-mcthyl-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide
  • WO201653771 5-bromo-2-fluoro-/V,/V-dimcthylpyridin-3-aminc
  • 2M aqueous Na 2 C0 3 6.85 mL, 13.7 mmol
  • PdCl2(dppf).DCM 224 mg, 0.27 mmol
  • the reaction mixture was cooled to rt.
  • the mixture was diluted with DCM and brine and the aqueous phase was extracted with DCM (2 x 20 mL).
  • the organic extracts were collected, dried over anhydrous Na 2 S0 4 , filtered and concentrated.
  • Step II tert- butyl 4-(2-((5-(dimethylamino)-2'-methyl-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate [0241] To a solution of /er/-butyl 4-(2-hydroxyethyl)piperazine-l-carboxylate (338 mg, 2.01 mmol) in dry dioxane (4 mL) was added NaH (80.4 mg, 2.01 mmol. 60% on mineral oil) and the reaction mixture was stirred at rt for 30 min.
  • the reaction mixture was cooled to rt and poured into water (10 mL).
  • the mixture was extracted with DCM (2 x 10 mL) and the organic extracts were combined, dried over anhydrous Na 2 S0 4 , then filtered and concentrated.
  • the resultant solid was triturated with Et20 and purified by reverse phase Cl 8 column using a gradient of acetonitrile in 0.2% TFA in water (5-100%) to afford the title compound as a yellow solid (8 mg, 13%).
  • Step I (25,4/?)-l-((/?)-2-(4-bromobutanamido)-3,3-dimcthvlbutanoyl)-4-hydroxy- V-((S)-l- (4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide: [0244] To a stirred solution of 4-bromobutyric acid (300 mg, 0.624 mmol) in DMF (8.0 mL) was sequentially added (2S,4R)- 1 -((R)-2-amino-3,3-dimcthylbutanoyl)-4-hydroxy-/V-((S)- 1 -(4- (4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (300 mg, 0.624 mmol) and HATU (285 mg, 0.748 mmol) followed by DIPEA (0.223 mL, 1.28
  • Step I A'-(2-mcthyl-5'-morpholino-6'-(2-(4-(prop-2-yn-l-yl)pipcrazin-l-yl)cthoxy)-[3,3'- bipyridm]-5-yl)-3-(trifluoromethyl)benzamide
  • the title compound (145 mg, 43.9%) was prepared as off-white solid from N-(&- fluoro-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (250 mg, 0.543 mmol) and 2-(4-(prop-2-yn-l-yl)piperazin-l-yl)ethanol (457 mg, 2.71 mmol) as described in Example 9.
  • Step II Compound 1.022 [0248] In RBF, 7V-(2-methyl-5'-morpholino-6'-(2-(4-(prop-2-yn- 1 -yl)piperazin- 1 -yl)ethoxy)-
  • Example 23 (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(6-(4-(((4-((2-((2'-mcthyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethoxy)methyl)benzyl)oxy)m ethyl)- 1 H- 1 ,2,3-triazol- 1 -yl)hcxanamido)butanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.023)
  • Step III V-(2-mcthyl-5'-morpholino-6'-(2-((4-((prop-2-yn-l- yloxy)methyl)benzyl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide:
  • Step I Synthesis of 4-(prop-2-yn-l-yloxy)but-2-yn-l-ol.
  • Step III Synthesis of 2-((4-(prop-2-yn-l-yloxy)but-2-yn-l-yl)oxy)ethanol.
  • Step IV Synthesis of V-(2-mcthyl-5'-morpholino-6'-(2-((4-(prop-2-yn-l-yloxy)but-2-yn-l- yl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide.
  • Step I (2,V,4/?)-l-((/?)-2-(3-azidopropanamido)-3,3-dimcthvlbutanoyl)-4-hyd roxy- V-((A)-l- (4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide
  • Example 28 Synthesis of / ⁇ -((/?)- 1 -((2S, 7/?)-4-hydroxy-2-(((.S')- 1 -(4-(4-methylthiazol-5 - yl)phenyl)ethyl)carbamoyl)pyrrolidin- 1 -yl)-3 ,3 -dimethyl- 1 -oxobutan-2-yl)-6-(4-(2-((2'-methyl- 5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l- yl)pyridazine-3 -carboxamide ( Compound 1.028).
  • Step I methyl 6-(4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-yl)pyridazme-3-carboxylate: [0264] In a sealed tube, to a stirred mixture of l-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l,4-diium chloride (150 mg, 0.233 mmol) (Ref.
  • Step II of Example 9) and methyl 6-chloropyridazine-3-carboxylate (43 mg, 0.247 mmol) in acetonitrile (2.0 mL) was added DIPEA (172 pL, 0.988 mmol), it was heated at 82 °C for 1 h [35% of SM left], further heated for 3 h [ no progress], additional amount of methyl 6-chloropyridazine-3-carboxylate (43 mg) was added, heated for 1 h, additional amount of 6-chloropyridazine-3-carboxylate (33 mg) was added [Monitored by HPLC], after 1 h, cooled to rt, diluted with water (5 mL), extracted with ethyl acetate ( 3 x 15 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated.
  • DIPEA 172 pL, 0.988 mmol
  • Step II 4-(6-carboxypyridazin-3-yl)-l-(2-((2'-methyl-5-morpholmo-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridm]-6-yl)oxy)ethyl)piperazm-l-ium chloride:
  • Step I /er/-butyl 2-(4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin- 1 -yl)acetate:
  • step II of Example 9) and /er/-butyl 2-bromoacetate (24.4 mg, 0.125 mmol) in DMF (1 mL) was added potassium bicarbonate (50 mg, 0.5 mmol), heated overnight at 80 °C (HPLC showed the consumption of SM), cooled to rt, diluted with ethyl acetate, filtered off, concentrated.
  • the residue was purified on 25 g S1O2 cartridge using methanol in DCM (0 to 20%) as eluant to afford the title compound (40 mg, 56.1%) as light brown oil.
  • Rf 0.39 (10% MeOH in DCM).
  • Step I ethyl 3-(4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazm-l-yl)cyclobutanecarboxylate:
  • Step II 3-(4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-yl)cyclobutanecarboxylic acid:
  • Example 31 Synthesis of /V-(6'-(2-(4-(3-(4-((/?)-2-((.S')-2-(4-(4-fluorobcnzoyl)thiazol-2- yl)pyrrolidin- 1 -yl)- 1 -(( > S)-2-(methylamino)propanamido)-2-oxoethyl)piperidine- 1 - carbonyl)cyclobutyl)piperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.031 )
  • Step I Synthesis of (9H-fluoren-9-yl)methyl((S)-l-(((R)-2-((S)-2-(4-(4-fluorobenzoyl)thiazol- 2-yl)pyrrolidin-l-yl)-l-(l-(3-(4-(2-((2'-methyl-5-morpholmo-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazm-l- yl)cyclobutanecarbonyl)piperidm-4-yl)-2-oxoethyl)amino)-l-oxopropan-2- yl)(methyl)carbamate [0273] To a solution of 3-(4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-
  • WO 2017/182418 in DMF (0.8 mL) was added sequentially the HATU (0.053 mg, 0.137 mmol) and the Hunig's base (0.096 mL, 0.547 mmol). The reaction was followed by tic with 20 % MeOH in DCM. The reaction mixture was extracted with ethyl acetate and saturated sodium bicarbonate. The organic phase was collected, dried over sodium sulfate, filtered and evaporated.
  • Step I tert-butyl 4-(2-hydroxyethyl)-2-methylpiperazine-l-carboxylate : [0275] 2-Bromoethanol (1.87 g, 15.0 mmol) was added to a mixture of tert-butyl 2- methylpiperazine- 1 -carboxylate (2.00 g, 9.99 mmol) and potassium carbonate (4.13 g, 29.9 mmol) in acetonitrile (15 mL). The mixture was heated at 82 °C for 6hrs (follow reaction by tic using KMn04 100 % EA). The reaction mixture was filtered and evaporated.
  • the mixture was purified on 80 g column with hexane to 100 % EA, to provide tert-butyl 4-(2-hydroxyethyl)-2- methylpiperazine- 1 -carboxylate (1.20 g, 49 %). !
  • Step II tert-butyl 2-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridiii]-6-yl)oxy)ethyl)piperazme-l-carboxylate :
  • reaction mixture was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier.
  • residue was purified on 40 g S1O2 cartridge with 20 % EA in hexane to 100 % EA then EA to 5 % MeOH in EA to provide tert-butyl 2-methyl-4-(2-((2'-methyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l- carboxylate .
  • Step IV Compound 1.032: [0278] To a mixture of 2-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3-
  • Step I tert-butyl 4-(2-hydroxyethyl)-2,3-dimethylpiperazine-l-carboxylate : [0279] 2-Bromoethanol (0.612 g, 4.90 mmol) was added to a mixture of tert-butyl 2,3- dimethylpiperazine- 1 -carboxylate (0.700 g, 3.27 mmol) and potassium carbonate (1.35 g, 9.80 mmol) in acetonitrile (15 mL). The mixture was heated at 80 °C for 18hrs (follow reaction by tic using KMn04 100 % EA). The reaction mixture was filtered and evaporated.
  • the mixture was purified on 80 g column with hexane to 100 % EA, to provide tert-butyl 4-(2 -hydroxy ethyl)-2, 3- dimethylpiperazine-1 -carboxylate (0.286 g, 33 %).
  • Example 34 N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-5-yl)-2- methylpiperazin- 1 -yl)ethoxy)-2-methyl-5 '-morpholino- [3 ,3 '-bipyridin] -5-yl)-3- (trifluoromethyl)benzamide (Compound 1.034).
  • Step I tert-butyl 4-(2-hydroxyethyl)-3-methylpiperazine-l-carboxylate:
  • Step II tert-butyl 3-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate
  • reaction mixture was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier.
  • residue was purified on 40 g S1O2 cartridge with 20 % EA in hexane to 100 % EA then EA to 5 % MeOH in EA to provide tert-butyl 3-methyl-4-(2-((2'-methyl-5-morpholino-
  • Step III 3-methyl-4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-ium chloride:
  • Example 35 Synthesis ofN-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-5-yl)- 2,2-dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ⁇ Compound 1.035 ).
  • Step I tert-butyl 4-(2-hyd roxycthyl)-3 , 3-di methyl piperazine- 1 -carboxylate:
  • Step II tert-butyl 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridm]-6-yl)oxy)ethyl)piperazine-l-carboxylate: [0288] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-3,3-dimethylpiperazine-l- carboxylate (0.177 g, 0.684 mmol) in 1 ,4-dioxane (5.0 mL) was added 60% sodium hydride (0.045.6 g, 1.14 mmol) in one portions, after stirred at rt for 30 min, / ⁇ -( 6'-fl uoro-2-m cth yl- 5 morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (0.210
  • Step III 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethyl)piperazin- 1-ium chloride :
  • Step IV ( Compound 1.035 ).
  • Example 36 N-(6'-(2-(5-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,5- diazabicyclo[2.2.1]heptan-2-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-
  • Step I tert-butyl 5-(2-hydroxyethyl)-2,5-diazabicyclo[2.2.1]heptane-2- carboxylatecarboxylate:
  • reaction mixture was placed on pre-heated oil bath at 80 °C for 2 hrs, the reaction mixture was cooled to rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL), the combined organic layers were washed with water, brine, dried over Na 2 S0 4 , concentrated. The residue was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier.
  • acetylated product was purified on 40 g S1O2 cartridge using a gradient of (0% EtOAc to 100% EtOAc-Hexanes then MeOH-EtOAc (0% to 15%) to afford tert-butyl 5-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl) benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (120 mg, 30%) as a pale yellow solid.
  • StepIII 5-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridiii]- 6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptan-2-ium chloride :
  • Example 37 N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,5- dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.037).
  • Step I tert-butyl 4-(2-hydroxyethyl)-2,5-dimethylpiperazine-l-carboxylate:
  • reaction mixture was placed on pre-heated oil bath at 80 °C for 2 hrs.
  • the reaction mixture was cooled into rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL), the combined organic layers were washed with water, brine, dried over Na 2 S0 4 , concentrated.
  • the residue was dissolved in pyridine (1 mL) followed by addition of AC2O (1 mL). The reaction mixture stirred for 1 hrs to convert unreacted alcohol to acetylated product.
  • Step IV Compound 1.037'.
  • Example 38 N-(6'-(2-(3-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.038).
  • Step I tert-butyl 6-(2-hydroxyethyl)-3,6-diazabicyclo[3.1.1]heptane-3-carboxylate
  • StepII tert-butyl 6-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)-3,6-diazabicyclo[3.1.1]heptane-3-carboxylate:
  • reaction mixture was placed on pre -heated oil bath at 80 °C for 2 hrs.
  • the reaction mixture was cooled to rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL), the combined organic layers were washed with water, brine, dried over Na 2 S0 4 , concentrated.
  • the residue was dissolved in pyridine (1 mL) followed by addition of AC2O (1 mL). The reaction mixture stirred for 1 hrs to convert unreacted alcohol to acetylated product.
  • Step III 6-(2-((2 '-methyl-5-morpholino-5 '-(3-(trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] - 6-yl)oxy)ethyl)-3,6-diazabicyclo[3.1.1]heptan-3-ium chloride:
  • Example 39 N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,6- dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.039 ).
  • Step I tert-butyl 4-(2-hydroxyethyl)-3,5-dimethylpiperazine-l-carboxylate:
  • reaction mixture was cooled into rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL). The combined organic layers were washed with water, brine, dried over Na 2 S0 4 , concentrated. In order to have easier purification, the residue was dissolved in pyridine (1 mL) followed by addition of AC2O (1 mL). The reaction mixture stirred for 1 hrs to convert unreacted alcohol to acetylated product.
  • Step III 3,5-dimethyl-4-(2- ((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin- 1 -ium chloride:
  • Calu-6 cells were stably transfected with a CRAF fusion protein with a fluorescent protein attached to the C-terminus of CRAF in order to monitor protein levels via fluorescence.
  • Cells were seeded into a 96-well plate and allowed to adhere overnight. The next day cells are treated with compounds at the doses indicated in the table below and analyzed over time. Data was collected by monitoring Fluorescent CRAF protein levels an IncuCyte S3 Live Cell Analysis System (Essen BioScience). The fluorescent CRAF fusion protein was excited at 440- 480 nm and the emission was measured at 504-544 nm.
  • Calu6_CRAF fusion protein cells are imaged and analyzed using an Incucyte S3 live cell analysis system (Essen Biosciences). Fluorescence signal is due to expression of the fluorescent protein covalently fused to the C-terminus of CRAF and degradation is assessed by measuring fluorescence/confluence (-cell number) after treatment with compounds of the present disclosure.
  • Phase contrast images are analyzed using Incucyte S3 software. Percent confluence is calculated using a phase contrast mask to quantify the area of the image occupied by the
  • GCU Green Corrected Units
  • Reference Compound 1 (Ref. Comp. 1) (WO2018/200981) is included for comparison:
  • the reported Dmax (%) represents the maximum precent of total fluorescent CRAF that was degraded by the given compounds.
  • FIG. 1 shows the CRAF degradation over time after addition of Ref. Comp. 1 at 12.5 mM. The percent D max occurred at 1.5 hr and was 23.3 ⁇ 1.33 %.
  • FIG. 2 shows the CRAF degradation over time after addition of Compound 1.009 at 1 mM. The percent Dmax occurred at 34.5 hr and was 34.4 ⁇ 1.27 %.
  • FIG. 3 shows the CRAF degradation over time after addition of Compound 1.032 at the indicated concentrations.
  • the percent D max occurred at 32 hr and was 24.6 ⁇ 1.0 %.
  • FIG. 4 shows the CRAF degradation over time after addition of Compound 1.034 at the indicated concentrations.
  • the percent D max occurred at 26 hr and was 23.7 ⁇
  • FIG. 5 shows the CRAF degradation over time after addition of Compound 1.039 at the indicated concentrations.
  • the percent D max occurred at 32 hr and was 26.2 ⁇
  • FIG. 6 shows the CRAF degradation over time after addition of Compound 1.035 at the indicated concentrations.
  • the percent D max occurred at 36 hr and was 30.6 ⁇
  • FIG. 7 shows the CRAF degradation over time after addition of Compound 1.038 at the indicated concentrations.
  • the percent D max occurred at 26 hr and was 19.1 ⁇
  • FIG. 8 shows the CRAF degradation over time after addition of Compound 1.037 at the indicated concentrations.
  • the percent D max occurred at 35 hr and was 26.5 ⁇
  • FIG. 9 shows the CRAF degradation over time after addition of Compound 1.033 at the indicated concentrations.
  • the percent D max occurred at 35 hr and was 26.1 ⁇
  • FIG. 10 shows the CRAF degradation over time after addition of Compound 1.036 at the indicated concentrations.
  • the percent D max occurred at 24 hr and was 27.7 ⁇ 0.9 %.
  • 1 mM or less of Compounds 1.009 and 1.032-1.039 provide a faster degradation rate and a higher or substantially similar magnitude of CRAF reduction as compared to 12 mM of Ref. Comp. 1.
  • FIG. 11 shows the dose response curve of Compound 1.009 at an endpoint of 42 hours by plotting the percent CRAF degradation at different concentrations of Compound 1.009.
  • the DC50 was determined to be 104.4 ⁇ 5.2 nM, and the D max was 33.5% of - 100X overexpressed CRAF.
  • Compound 1.009 also showed minimal cytotoxicity in this assay (as determined by observing a low background and high signal to noise ratio.

Abstract

The present disclosure provides, inter alia, RAF-Degrading Conjugate Compounds that are useful in the treatment of cancer and other RAF related diseases. Also provided are, pharmaceutical compositions, methods of treatment, and kits comprising a RAF-Degrading Conjugate Compound.

Description

CONJUGATE COMPOUNDS FOR THE DEGRADATION OF RAF
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. 1 19(e) of U.S. Provisional Application No. 62/806,557, filed on February 15, 2019, which is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER
PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] NOT APPLICABLE
BACKGROUND OF THE INVENTION
[0004] KRAS is a key component of the canonical MAPK signaling pathway and is the most frequently mutated oncogene across all human cancers. There are no approved therapies that directly target KRAS and treatment for these cancers remains a great unmet medical need.
KRAS is difficult to drug due to a lack of known binding pockets suitable for small molecule binding and pM affinity for its natural substrate, GTP. In addition, mutations often arise in other components of the MAPK pathway that lead to cancer and other RAS-associated diseases known as RASopathies. [0005] The RAF family of kinases, including ARAF, BRAF, and CRAF, are the immediate downstream KRAS effectors that propagate downstream tumorigenic signals in cancer cells. Attempts were made to develop RAF inhibitors for KRAS-driven tumors, yet the compounds were discovered to cause paradoxical activation of RAF kinases due to allosteric activation of RAF homo- and hetero-dimers. Therefore, development of non-activating RAF inhibitors is an attractive strategy to block aberrant MAPK signaling. [0006] Despite interest in this target, there remains a need in the art for a potent and specific inhibitor of RAF that effectively inactivates or eliminates MAPK signaling. The present disclosure addresses this need and provides related advantages as well.
BRIEF SUMMARY OF THE INVENTION [0007] Provided herein are RAF-Degrading Conjugate Compounds comprising a Ligand for
RAF covalently attached via a Linker Component to a Degradation Recognition Agent. In certain embodiment, the linker component is as described herein.
[0008] In still other aspects, provided herein as pharmaceutical compositions, methods for treating diseases, and kits using the RAF-Degrading Conjugate Compounds described. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows CRAF degradation in Calu6 cells over time after addition of 12.5 mM of Ref. Comp. 1. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0010] FIG. 2 shows CRAF degradation in Calu6 cells over time after addition of 1 mM of Compound 1.009. The values of the y-axis are displayed as a measure of
fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0011] FIG. 3 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.032. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0012] FIG. 4 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.034. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0013] FIG. 5 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.039. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0014] FIG. 6 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.035. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0015] FIG. 7 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.038. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0016] FIG. 8 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.037. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0017] FIG. 9 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.033. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0018] FIG. 10 shows CRAF degradation in Calu6 cells over time after addition of the indicated amount of Compound 1.036. The values of the y-axis are displayed as a measure of fluorescence/confluence. See, for example, Biological Example 1 for further information on data analysis.
[0019] FIG. 11 shows CRAF degradation in Calu6 cells after 42 hours of incubation with various amounts of Compound 1.009. The values of the y-axis are displayed as percent CRAF degradation. See, for example, Biological Example 2 for further information on data analysis. DETAILED DESCRIPTION OF THE INVENTION
I. INTRODUCTION
[0020] The present disclosure provides, inter alia, RAF-Degrading Conjugate Compounds that are useful in the treatment of cancer and other RAF related diseases. Without being bound to any particular theory, it is believed that the currently described conjugates effectively permeate the cell membrace, possess improved bioavailability, and are useful in treating cancer and other RAF related diseases by lowering the relative amount of RAF protein in a cell.
[0021] In some embodiments, the conjugate compounds of the present disclosure target RAF for destruction via recruitment of E3 ubiquitin ligases resulting in ubiquitination and
proteasomal degradation.
Definitions
[0022] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.
[0023] The term "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. Ci-8 means one to eight carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n- octyl, and the like.
[0024] The terms“Halogen” or“halo” refer to fluorine, chlorine, bromine and iodine.
[0025] The term“Cycloalkyl” refers to a saturated monocyclic or bridged ring assembly containing from 3 to 8 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bridged cycloalkyl rings include, for example, norbornane, and adamantane. Cycloalkyl groups can be substituted or unsubstituted.
[0026] The term“Heterocycloalkyl” refers to a saturated ring system having from 3 to 8 ring members and from 1 to 4 N atoms. Heterocycloalkyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8 ring members. Any suitable number of nitrogen atoms can be included in the heterocycloalkyl groups, such as 1 , 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The heterocycloalkyl group can include groups such as aziridine, azetidine, pyrrolidine, piperidine, piperazine, azepane, 1 ,3-diazepane, azocane, and 1 ,5-diazocane. Heterocycloalkyl groups can be substituted or unsubstituted.
[0027] The term“6- membered Heteroaryl” refers to a monocyclic aromatic ring assembly containing 6 ring atoms, where from 1 to 3 of the ring atoms are nitrogen atoms. Heteroaryl groups can include any number nitrogen atoms can be included in the heteroaryl groups, such as 1, 2, or 3, or 1 to 2, 1 to 3, or 2 to 3. The heteroaryl group can include groups such as pyridine, pyrazine, pyrimidine, pyridazine, and triazine (1,2,3-, 1,2,4- and 1 ,3,5-isomers). Heteroaryl groups can be substituted or unsubstituted.
[0028] The term“Spirocyclic moiety” refers to a pair of saturated rings having a single atom in common containing from 5 to 11 ring atoms, or the number of atoms indicated. Spirocyclic moieties can include any number of carbons, such as C5-6, C7-8, Cs-9, C9-10, Cio-11. Additionally, spirocyclic moieties can include 0 to 2 nitrogen atoms. Thus, spriocylclic moieties can be cycloalkyl moieties, or heterocycloalkyl moieties (or a combination of one each). Spriocyclic moieties include, but are not limited to spiro[5.5]undecane, spiro[4.5] decane, spiro[4.4]nonane, spiro[3.5]nonane, spiro[3.3]heptane, spiro[3.4]octane, 7-azaspiro[3.5]nonane, 3- azaspiro[5.5]undecane, and 8-azaspiro[4.5]decane. Spirocylic moieties can be substituted or unsubstituted.
[0029] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. When a stereochemical depiction is shown, it is meant to refer the compound in which one of the isomers is present and substantially free of the other isomer. ‘Substantially free of another isomer indicates at least an 80/20 ratio of the two isomers, more preferably 90/10, or 95/5 or more. In some embodiments, one of the isomers will be present in an amount of at least 99%.
[0030] The terms“a,”“an,” or“the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to“the agent” includes reference to one or more agents known to those skilled in the art, and so forth.
[0031] The terms“subject”,“patient” or“individual” are used herein interchangeably to include a human or animal. For example, the animal subject may be a mammal, a primate ( e.g a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
[0032] The term“selective CRAF inhibitor” as used herein describes a CRAF inhibitor that preferentially binds to CRAF over other members of the RAF family including ARAF and BRAF. A selective CRAF inhibitor has at least a 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower IC50 value when binding CRAF as compared to binding to ARAF or BRAF. The fold difference described herein is for the inhibitor compounds when not covalently linked to a linking component or when part of a RAF-Degrading Conjugate Compound.
[0033] As used herein term“linking atoms” refers to the number of atoms that separate a Ligand for RAF and a Degradation Signaling Agent in a RAF-Degrading Conjugate Compound. The number of linking atoms is not the total number of atoms in the Linker Component. Rather, it is the number of atoms between a Ligand for RAF and a Degradation Signaling Agent. When the Linker Component is unbranched or does not contain any cyclic moieties, the number of total atoms in the Linker Component may be the number of linking atoms. However, when the linker component includes a carbonyl, for example, the oxygen atom of the carbonyl is not a“linking atom.” Additionally, when a cyclic moiety is present in the Linker Component, the number of linking atoms in a given cyclic moiety is the fewest number of ring vertices needed to traverse the ring. For example, when a six membered ring is included in a Linker Component and the points of attachment to the remainder of the Linker Component, to the Ligand for RAF, or the Degradation Signaling Agent are para to one another (i.e., a 1,4-linkage), the number of linking carbons will be the same regardless of the direction of traversal. The diagram below illustrates this point:
Figure imgf000008_0001
(exemplary number of linking atoms with para attachment)
However, when a six membered ring is includes points of attachment that are meta to one another (i.e., a 1,3-linkage), the direction of traversal of the ring will result in different numbers of linking atoms. As described above, in such instances, the proper calculation to determine the number of linking atoms is the direction of traversal that will result in the fewest number of linking atoms. In the diagram below illustrates the calculation of the number of linking atoms in this instance:
Figure imgf000008_0002
(exemplary number of linking atoms with meta attachment) Like the meta example above, when five membered rings are included in the Linker Component, the number of linking atoms will depend on the direction of traversal of the ring. Again, the the proper calculation to determine the number of linking atoms is the direction of traversal that will result in the fewest number of linking atoms. The diagram below illustrates the calculation of the number of linking atoms in this instance:
Figure imgf000008_0003
(exemplary number of linking atoms with a five-membered ring) Exemplary calculations for determining the number of linking atoms in specific examples included in the disclosure are provided below, the“wavy-lines” distinguish the segments between the Ligand for RAF, the Linker Component, and the Degradation Signaling Agent:
Figure imgf000009_0001
II. DESCRIPTION OF THE EMBODIMENTS
A. RAF-Degrading Conjugate Compounds
[0034] In certain aspects, the present disclosure provides a RAF-Degrading Conjugate Compound comprising a Ligand for RAF covalently attached via a Linker Component to a Degradation Signaling Agent.
1. Ligands for RAF
[0035] A Ligand for RAF is any compound that effectively binds to a RAF protein.
Accordingly, Ligands for RAF include, but are not limited to, modified versions of sorafenib, Hah lOd, PF-04880594, LY3009120, PLX4720, RAF709, Vemurafenib, MLN 2480, and LHX254. Select positions of the compounds shown in Table 1 may be modified to
accommodate covalent linkage with the Linker Component, yet not obstruct target binding of the Ligand for RAF. When covalently attached to the linker component, the Ligand for RAF maintains sufficient affinity for its target.
Table 1: Ligands for RAF
Figure imgf000010_0001
[0036] In some embodiments, the Ligand for RAF is a selective CRAF inhibitor. Selective CRAF inhibitors, include sorafenib and Hah et al. lOd.
[0037] In some embodiments, a selective CRAF inhibitor is an compound that has an CRAF IC50 value that is at least 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold lower than a BRAF IC50 value, when measured under the same conditions. In some embodiments, a selective CRAF inhibitor is an compound that has an CRAF IC50 value that is at least 5-fold, 10-fold, 20-fold, 50- fold, or 100-fold lower than a ARAF IC50 value, when measured under the same conditions. In some embodiments, a selective CRAF inhibitor is a compound that has an CRAF IC50 value that is at least 10-fold lower than a BRAF IC50 value, when measured under the same conditions. In some embodiments, a selective CRAF inhibitor is a compound that has an CRAF IC50 value that is at least 10-fold lower than a ARAF IC50 value, when measured under the same conditions.
[0038] In some embodiments, the Ligand for RAF is a selective B/C RAF inhibitor. Selective B/C RAF inhibitors include RAF709. In some embodiments, the selective B/C RAF inhibitor has the formula shown below
Figure imgf000011_0001
wherein X is N or CH. As described above, select positions of the formula shown above may be modified to accommodate covalent linkage with the Linker Component, yet not obstruct target binding of the Ligand for RAF. For example, in some embodiments, the B/C RAF inhibitor is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000011_0002
where X is N or CH and the wavy line indicates the site of attachment of the Linker Component.
[0039] In some embodiments, RAF709 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000011_0003
where X is N or CH and the wavy line indicates the site of attachment of the Linker Component. [0040] In some embodiments, LXH254 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000012_0001
where the wavy line indicates the site of attachment of the Linker Component.
[0041] In some embodiments, sorafenib is modified to accommodate covalent linkage with the
Linker Component as shown below
Figure imgf000012_0002
where the wavy line indicates the site of attachment of the Linker Component.
[0042] In some embodiments, LY3009120 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000012_0003
where the wavy line indicates the site of attachment of the Linker Component.
[0043] In some embodiments, LY3009120 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000012_0004
where the wavy line indicates the site of attachment of the Linker Component. [0044] In some embodiments, MLN2480 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000013_0001
where the wavy line indicates the site of attachment of the Linker Component.
[0045] In some embodiments, PF-04880594 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000013_0002
where the wavy line indicates the site of attachment of the Linker Component.
[0046] In some embodiments, PF-04880594 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000013_0003
where the wavy line indicates the site of attachment of the Linker Component.
[0047] In some embodiments, PF-04880594 is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000014_0001
where the wavy line indicates the site of attachment of the Linker Component.
[0048] For the purposes of calculating the number of linking atoms in the Linker Component, the modified structures shown above are considered to be part of the Ligand for RAF. Preparing RAF -Degrading Conjugate Compounds
[0049] When preparing RAF-Degrading Conjugate Compounds, the Ligands for RAF may be modified to provide appropriate linker chemistry between the Ligand for RAF and the Linker Component. Appropriate linker chemistry includes, but is not limited to, nucleophilic substitution, amide formation, and click chemistry. Accordingly, in some embodiments, the Ligand for RAF is modified to include a functional group that facilitates linkage between the
Linker Component and the Ligand for RAF. Functional groups that facilitate linkage include, but are not limited to a halogen, amine, hydroxyl, carboxylic acid, ester, alkyne, and azide. It is understood that functional group included in the modified Ligand for RAF will depend on the identity of the reactive group of the Linker Component with which it will be reacted. Based on the disclosure provided herein, a person of skill in the art can appropriately choose desirable functional groups.
[0050] In some embodiments, generating the Ligand for RAF includes using a synthetic precursor of an existing ligand. For example, a synthetic precursor of sorafenib includes a terminal carboxylic acid moiety at the 2-position of the pyridine (sorafenib acid). Sorafenib has a terminal N-methyl at this position. In some embodiments, the synthetic precursor of sorafenib, sorafenib acid, provides appropriate linker chemistry with the Linker Component. As a non limiting example, the synthetic precursor of sorafenib (sorafenib acid) can be reacted with a reactive group of the Linker Compound such as an amine or hydroxyl to form the RAF- Degrading Conjugate Compound of the present disclosure. Synthetic precursors used in RAF- Degrading Conjugate Compounds of the present disclosure maintain sufficient affinity for the target of interest. Based on the disclosure provided herein, a person of skill in the art could readily prepare synthetic precursors of the above mentioned Ligands for RAF that facilitate covalent attachment with the Linker Component, yet maintain sufficient affinity for its target.
[0051] In some embodiments, generating the Ligand for RAF includes adding a functional group to the Ligand for RAF. In some embodiments, the functional group is added to an aromatic ring of the Ligand for RAF. In some embodiments the functional group is added to an alkyl portion of the Ligand for RAF. When a functional group is added to a Ligand for RAF, the Ligand for RAF maintains sufficient affinity for its target.
[0052] In some embodiments, sorafenib is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of Linker Component.
Figure imgf000015_0001
(Exemplary linkage of sorafenib to Linker Component)
[0053] In some embodiments, Hah et al. lOd is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of Tanker Component.
Figure imgf000015_0002
(Exemplary linkage of Hah et al 1 Od to Linker Component)
[0054] In some embodiments, RAF709 is modified as shown below to accommodate appropriate linker chemistry with the Link Component, where Z is the remainder of the Linker Component
Figure imgf000016_0001
[0055] In some embodiments, a selective B/C RAF inhibitor of the formula shown below is modified to accommodate appropriate linker chemistry with the Link Component, where Z is the remainder of the Linker Component and X is N or CH
Figure imgf000016_0002
2. Degradation Signaling Agents
[0056] Degradation Signaling Agents of the present disclosure include compounds or peptides that induce degradation of the targeted RAF protein. Generally, Degradation Signaling Agents degrade RAF by binding to or recruiting one or more degradation proteins. The degradation proteins are usually associated with the proteasome, the ubiquitin-proteasome pathway, or lysosomal proteolysis. Accordingly, Degradation Signaling Agents include, but are not limited to E3 ligase recognition agents, hydrophobic tagging agents, proteasome recognition agents, and lysosomal recognition peptides.
[0057] In some embodiments, the Degradation Signaling Agents binds to a degradation protein or a component of a degradation protein complex. In some embodiments, the bound
Degradation Signaling Agent activates the degradation protein or degradation protein complex.
In some embodiments, the bound Degradation Signaling Agent does not significantly alter the activity of the degradation protein or degradation protein complex.
[0058] In some embodiments, the degradation protein or degradation protein complex is an E3 ubiquitin ligase or an E3 ubiquitin ligase complex. In some embodiments, the E3 ubiquitin ligase or component of the E3 ubiquitin ligase complex targeted is MDM2, cIAPl, VHL protein, CBRN, or SCFP-trcp. [0059] In some embodiments, the Degradation Signaling Agents recruits a degradation protein or degradation protein complex by binding to or associating with the RAF protein causing misfolding of the RAF protein. In some embodiments, the Degradation Signaling Agents recruits a degradation protein or degradation protein complex without misfolding the RAF protein.
[0060] In some embodiments, the degradation protein or degradation protein complex is the proteasome. In some embodiments, the degradation protein or degradation protein complex is a chaperone protein. In some embodiments, the chaperone protein is hsc70. i) E3 Ligase Recognition Agents [0061] The E3 ligase recognition agent is any compound or peptide that effectively binds to an
E3 ubiquitin ligase or an E3 ubiquitin ligase complex. In some embodiments, the E3 ligase recognition agent is an E3 ubiquitin ligase ligand. In some embodiments, the E3 ubiquitin ligase ligand is a modified version of Pomalidomide, Nutlin-3, VHL Ligand, methyl bestatin, a VHL binding peptide, a SCF|3 TRCP targeting peptide, or an Inhibitor of Apoptosis Protein (IAP) ligand. In some embodiments, the VHL binding peptide is HIF-la-VHL binding peptide or hydroxy proline-HIF-la VHL binding peptide. Select positions of the compounds shown in Table 2 may be modified to accommodate covalent linkage with the Linker Component and do not obstruct target binding of the E3 Ligase Recognition Agent. When covalently attached to the linker component, the E3 Ligase Recognition Agent maintains sufficient affinity for its target. [0062] Without being bound to any particular theory, it is believed that bringing the RAF protein and the E3 ubiquitin ligase or an E3 ubiquitin ligase complex in close proximity z
Table 2: E3 Ubiquitin Ligase Ligands
Figure imgf000017_0001
Figure imgf000018_0002
[0063] The letters of the final entries in the second to last row and the left column of the last row (the peptide sequences) represent amino acids.
[0064] In some embodiments, pomalidomide is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000018_0001
where the wavy line indicates the site of attachment of the Linker Component. [0065] In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is PI . In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P2. [0066] In some embodiments, pomalidomide is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000019_0001
where the wavy line indicates the site of attachment of the Linker Component.
[0067] In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P3. In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P4. In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P5. In some embodiments, the modified pomalidomide structure used to accommodate covalent linkage with the Linker Component is P6. [0068] In some embodiments, each of (PI) to (P6), the phenyl ring that includes the point of attachment to the Linker Component can be further substituted with an additional substituent selected from the group consisting of Ci-8 alkyl, halogen, and phenyl. For example, in some embodiments, P2 is substituted with X to form P2a and P2b
Figure imgf000020_0001
Similar substitution patterns are possible for PI P6.
[0069] In some embodiments, VHL Ligand is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000020_0002
where the wavy line indicates the site of attachment of the Linker Component.
[0070] In some embodiments, VHL Ligand is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000020_0003
where the wavy line indicates the site of attachment of the Linker Component.
[0071] In some embodiments, IAP Ligand is modified to accommodate covalent linkage with the Linker Component as shown below
Figure imgf000021_0001
where the wavy line indicates the site of attachment of the Linker Component.
[0072] Typically, attachment at the position shown above for the IAP Ligand provides stereoisomerism at the attachment carbon of the IAP Ligand. Thus, in some embodiments, a stereochemically-enriched form of the IAP Ligand shown above is prepared:
Figure imgf000021_0002
where the wavy line indicates the site of attachment of the Linker Component. ii) Hydrophobic Tagging Agents
[0073] Hydrophobic tagging is a process for manipulating the folding of a protein to cause misfolding by contacting a hydrophobic tagging agent with a protein. Misfolded proteins are recognized by the cell and selectively targeted for degradation. When hydrophobic tagging agents are incorporated into the RAF-Degrading Conjugate Compounds of the present disclosure, the Ligands for RAF bring the hydrophobic tagging agent in close proximity to the targeted RAF, increasing the RAF -hydrophobic tagging agent relative concentration. Without being bound to any particular theory, it is believed that the misfolded protein-hydrophobic tagging agent complex is recognized by molecular chaperones which target the protein for degradation.
[0074] In some embodiments, hydrophobic tagging agents are generally small molecules that associate with the hydrophobic surfaces of proteins. Generally, hydrophobic tagging agents include one or more alkyl portions and/or one or more polyethylene glycol units, an amide group, and an optional terminal adamantane group. In some embodiments, hydrophobic tagging agents are HyT36 or HyT13, the structures of which are shown in Table 3, below. Select positions of the compounds shown in Table 3 may be modified to accommodate covalent linkage with the Linker Component and do not obstruct target binding of the hydrophobic tagging agent. When covalently attached to the linker component, the hydrophobic tagging agent maintains sufficient affinity for its target.
Table 3: Hydrophobic Tagging Agents
Figure imgf000022_0002
[0075] Suitable hydrophobic Tagging Agents include those described in US 2014/0302523, the contents of which are herein incorporated by reference for all purposes.
Hi) Proteasome Recognition Agents
[0076] In some embodiments, the Degradation Signaling Agent is a proteasome recognition agent. Proteasome recognition agents are compounds that bind to or recruit the proteasome.
When proteasome recognition agents are included in the RAF-Degrading Conjugate Compounds of the present disclosure, they localize target proteins to proteasome. Without being bound to any particular theory, it is believed that proteasome recognition agents induce target protein degradation via direct proteasome recruitment and do not include ubiquitination steps. [0077] In some embodiments, the proteasome recognition agent is BocsArginine
Figure imgf000022_0001
iv) Lysosomal Recognition Peptides [0078] Lysosomal recognition peptides are peptides that include a signaling motif for targeted lysosomal degradation. Without being bound to any particular theory, it is believed that the lysosomal recognition peptide, when incorporated into an RAF-Degrading Conjugate Compound of the present disclosure, will mark the targeted RAF protein for degradation via Chaperone- mediated autophagy.
[0079] In some embodiments, the lysosomal recognition peptide includes the amino acid motif KFERQ, where each letter is an amino acid. In some embodiments, the lysosomal recognition peptide is the sequence shown in Table 4.
Table 4: Lysosomal recognition peptide
Figure imgf000023_0001
v) Preparing RAF-Degrading Conjugate Compounds
[0080] When preparing RAF-Degrading Conjugate Compounds, the Degradation Signaling Agents may be modified to provide appropriate linker chemistry between the Degradation Signaling Agent and the Linker Component. As discussed above in Section A, Appropriate linker chemistry includes, but is not limited to, nucleophilic substitution, amide formation, and click chemistry. Accordingly, in some embodiments, the Degradation Signaling Agents is modified to include a functional group that facilitates linkage between the Linker Component and the Degradation Signaling Agents. Functional groups that facilitate linkage include, but are not limited to a halogen, amine, hydroxyl, carboxylic acid, ester, alkyne, and azide. It is understood that functional group included in the modified Degradation Signaling Agents will depend on the identity of the reactive group of the Linker Component with which it will be reacted. Based on the disclosure provided herein, a person of skill in the art can appropriately choose desirable functional groups.
[0081] In some embodiments, generating the Degradation Signaling Agents includes using a synthetic precursor. For example, a synthetic precursor of pomalidomide include a fluoro substituent at the 4-position of the isoindoline ring (fluoro pomalidomide). Pomalidomide has an amine group at this position. In some embodiments, the synthetic precursor of pomalidomide, fluoro pomalidomide, provides appropriate linker chemistry with the Linker Component. As a non-limiting example, the synthetic precursor of pomalidomide (fluoro pomalidomide) can be reacted with a reactive group of the Linker Compound such as an amine or hydroxyl to form the RAF-Degrading Conjugate Compound of the present disclosure.
Synthetic precursors used in RAF-Degrading Conjugate Compounds of the present disclosure substantially maintain their affinity for binding the target of interest. Based on the disclosure provided herein, a person of skill in the art could readily devise synthetic schemes to prepare synthetic precursors of the above mentioned Degradation Signaling Agents that readily facilitate covalent attachment with the Linker Component and maintain sufficient affinity for its target.
[0082] In some embodiments, generating the Degradation Signaling Agents includes adding a functional group to the Degradation Signaling Agents. In some embodiments, the functional group is added to an aromatic ring of the Degradation Signaling Agents. In some embodiments, the functional group is added to an alkyl portion of the Degradation Signaling Agents. When a functional group is added to a Degradation Signaling Agent and the Degradation Signaling
Agent is subsequently covalently linked to the Linker Component or the RAF-Degrading Conjugate Compound, the Degradation Signaling Agent maintains sufficient affinity for its target.
[0083] In some embodiments, when the Degradation Signaling Agent is a peptide, the Degradation Signaling Agent is covalently linked to the Linker Component by amide formation at the N- or C- terminus of the peptide sequence. For example, the HIF-Ia VHL binding peptide may be linked to the Linger Component, as shown below, where Z is the remainder of Linker Component.
VHL binding
Figure imgf000024_0001
peptide to Linker Component) [0084] In some embodiments, when the Degradation Signaling Agent is a peptide, the Degradation Signaling Agent is covalently linked to the Liner Component at a side-chain.
[0085] In some embodiments, nutlin-3 is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of Linker Component.
(Exemplary linkage of nutlin-3 to Linker
Figure imgf000025_0001
Component)
[0086] In some embodiments, methylbestatin is modified as shown below to accommodate appropriate linker chemistry with the Linker Component, where Z is the remainder of the linker component.
(Exemplary linkage of methylbestatin to
Figure imgf000025_0002
Linker Component)
[0087] In some embodiments, the chloro substituent in HyT13 or HyT36 is used in a nucleophilic substitution reaction to covalently link the Linker Component and the Degradation Signaling Agent as shown below, where Z is the remainder of the linker component.
Figure imgf000025_0003
omponent)
[0088] In some embodiments, the appropriate linker chemistry between the Degradation Signaling Agent and the Linker Component is already present and the Degradation Signaling Agent is not modified. [0089] In some embodiments, the carboxylic acid of BocsArginine is used to form a covalent linkage with the Linker Component, where Z is the remainder of the linker component.
(Exemplary linkage of B0C3 Arginine to Linker
Figure imgf000026_0001
Component)
[0090] For the purposes of calculating the number of linking atoms in the Linker Component, the modified structures shown in this subsection are considered to be part of the Degradation Signaling Agent.
3. Linker Components
[0091] The Linker Component of the present disclosure is included to provie sufficient separation between the Ligand for RAF and the Degradation Signaling Agent so that both the Ligand for RAF and the Degradation Signaling Agent can perform its desired function. Two variables that are often included in Linker Components are structural features, such as cyclic moieties or points of unsaturation, that help maintain certain conformations between the Ligand for RAF and the Degradation Signaling Agent, and the number of linking atoms.
[0092] The number of linking atoms in the Linker Component can vary depending on the particular Lingand for RAF and Degradation Signaling Agent being used. In some
embodiments, the number of linking atoms is from about 4 to about 23 atoms. In some embodiments, the number of linking atoms is from about 5 to 15 atoms. In some embodiments, the number of linking atoms is from about 5 to 10 atoms. In some embodiments, the number of lining atoms is from about 9 to 15 linking atoms. In some embodiments, the number of linking atoms is from about 16 to 21 linking atoms. [0093] It can be advantageous to include one or more cyclic moieties into the Linker
Component to increase the rigidity of the Linker Component. In some embodiments, the Linker Component comprises 1 to 4 cyclic moieties. In some embodiment, the Linker Component includes 1 to 3 cyclic moieties. In some embodiments, the Linker Component includes 1 to 2 cyclic moieties. In some embodiments, the Linker Component includes 2 cyclic moieties. In some embodiments, the Linker Component includes 1 cyclic moiety. [0094] Cyclic moieties, as referred to above include a C3-8 cycloalkyl, a triazole, a phenyl, a 6- to 8-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms, a six-membered heteroaryl comprising 1 to 2 nitrogen atoms, and C7-11 spirocylic moieties comprising 0 to 2 nitrogen atoms. In some embodiments cyclic moieties are selected from the group consisting of a triazole, a phenyl, a 6- to 8-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms. When two or more cyclic moieties are present the cyclic moieties are sometimes the same, sometimes they are different.
[0095] Linker Components can also inclue points of unsaturation. In some embodiments, the Linker Component includes one or more double or triple bonds. In some embodiments, the Linker Component includes one double bond. In some embodiments, the Linker Componenet includes one triple bond. In some embodiments, the Linker Component includes two double bonds. In some embodiments, the Linker Componenet includes two triple bonds.
[0096] In some embodiments, the Linker Component comprises 5 to 10 linking atoms and 1 or 2 cyclic moieties, wherein each cyclic moiety is independently selected from the group consisting of a triazole, a phenyl, and a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms.
[0097] In some embodiments, the Linker Component comprising 9 to 15 linking atoms and two cyclic moieties, wherein the cyclic moieties are independently selected from the group consisting of a triazole, a phenyl, and a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms. [0098] In some embodiments, the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is unsubstituted. In some embodiments, the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is substituted. In some embodiments, the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is substituted with 1 to 2 substituents selected from CM alkyl and halo. In some embodiments, the 6- to 8-membered heterocycloalkyl or the 6-membered heterocycloalkyl is substituted with 1 to 2 methyl substituents.
[0099] It is understood that the 6- to 8-membered heterocycloalkyl includes both monocylic and bridged ring assemblies. In some embodiments the 6- to 8-membered heterocycloalkyl is bridged. In some embodimnets, the 6- to 8-membered heterocycloalkyl is monocyclic.
[0100] The methods of making RAF-Degrading Conjugate Compounds with linker components are discussed below, but a person of skill in the art will readily recognize that a variety of synthetic schemes can be used. For example, in some embodiments, Linker
Components are single pre-synthesized molecular entities that are covalently attached to a Ligand for RAF and a Degradation Signaling Agent (either simultaneously or in an order of most synthetic convenience). In some embodiments, Linker Components are made from separate molecules entities (segments of a linker component) which are covalently attached to a Ligand for RAF or a Degradation Signaling Agent. After each segment of the linker component is covalently attached to a respective Ligand for RAF and Degradation Signaling Agent, an additional reaction is performed to covalently link the two segments of the Linker Component to form a RAF-Degrading Conjugate Compound. It is understood that when segments of the Linker Component are covalently attached after pre-attachment to a respective Ligand for RAF or Degradation Signaling Agent, each segment of the Linker component will have an additional reactive group that facilitates covalent attachment between the two segments of the Linker Component.
[0101] Reactive groups that facilitate covalent attachment to the Ligand for RAF and the Degradation Signaling Agent depend on the specific Ligand for RAF and Degradation Signaling Agent used, but as discussed above, suitable covalent linking chemistries include, but are not limited to, nucleophilic substitution, amide formation, and click chemistry. Exemplary linker chemistries are shown in Table 5, below. In the table below, R1 is the remainder of the Ligand for RAF or the remainder of the Degradation Signaling Agent, and R2 is the remainder of the Linker Component. A person of skill in the art will recognize that the functional groups of R1 and R2 may switch without departing from the scope of the present disclosure.
Table 5: Exemplary Linker Chemistry
Figure imgf000029_0001
[0102] In some embodiments, the first and second reactive group are different functional groups. In some embodiments, the first and second reactive groups are the same functional group.
[0103] Although Table 5 shows a nucleophilic substitution reaction with an amine and iodine, it is understood that additional nucleophiles and leaving groups may be used in the nucleophilic substitution reaction. Suitable nucleophiles include, but are not limited to, hydroxyl and thiol groups. Suitable leaving groups include, but are not limited to Cl, Br, and OTs.
[0104] In some embodiments, the Ligand for RAF or Degradation Signaling Agent contain a reactive group that readily facilitates covalent attachment to the Linker Component. In some embodiments, the Ligand for RAF and/or Degradation Signaling Agent is modified to include a suitable functional group that readily facilitates covalent attachment. As discussed above, in some embodiments, a sorafenib component (sorafenib acid) is covalently attached to the Linker Component via amide formation. This is achieved by reacting sorafenib acid (comprising a carboxylic acid at the 2-position of the pyridine ring) with an amine group from the Linker Component. In the RAF-Degrading Conjugate Compounds of the present disclosure, after attachment of the Linker Component to the Ligand for RAF and the Degradation Signaling Agent, both the Ligand for RAF and the Degradation Signaling Agent maintain sufficient affinity for their targets to carry out the desired effect.
[0105] As discussed above, in some embodiments, Linker Components are made from separate molecular entities which are first covalently attached to a Ligand for RAF or a
Degradation Signaling Agent. In such embodiments, the two segments of the Linker Component are covalently attached after each entity has been pre-attached to a respective Ligand for RAF or Degradation Signaling Agent. Suitable functional groups that facilitate covalent attachment of the molecular entities include the exemplary linker chemistry shown in Table 5 and the groups discussed supra. In some embodiments, the two segments of the linker component are covalently attached using azide-alkyne cycloaddition. In, some embodiments, the two segments of the linker component are covalently attached using amid formation chemistry.
[0106] Linking units of the Linker Component are generally non-reactive moieties such as alkyl groups. In some embodiments, the alkyl groups include one or more ether linkages. In some embodiments, the alkyl groups are linear. In some embodiments, the alkyl groups are branched. In some embodiments, the first reactive group is an amine group and the second reactive group is a carboxylic acid group. In some embodiments, the Linker Component includes a triazine.
[0107] In some embodiments, the Linker Component comprises polyethylene glycol.
[0108] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
wherein the wavy lines indicate sites of attachment of the Ligand for RAF and the Degradation Signaling Agent. [0109] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000033_0002
Figure imgf000034_0001
wherein the wavy lines indicate sites of attachment of the Ligand for RAF and the Degradation Signaling Agent. [0110] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000034_0002
wherein the wavy lines indicate sites of attachment of the Ligand for RAF and the Degradation Signaling Agent.
[0111] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000035_0001
Figure imgf000036_0001
wherein the wavy lines indicate sites of attachment of the Ligand for RAF and the Degradation Signaling Agent.
[0112] In some embodiments, the Linker Component has a formula
Figure imgf000036_0002
wherein the wavy lines indicate sites of attachment of the Ligand for RAF and the Degradation Signaling Agent. [0113] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000037_0001
wherein the wavy lines indicate sites of attachment of the Ligand for RAF and the
Degradation Signaling Agent.
[0114] In some embodiments, the Linker Component has the formula a). In some embodiments, the Linker Component has the formula y). In some embodiments, the Linker Component has the formula z). In some embodiments, the Linker Component has the formula aa). In some embodiments, the Linker Component has the formula bb). In some embodiments, the Linker Component has the formula cc). In some embodiments, the Linker Component has the formula dd). In some embodiments, the Linker Component has the formula ee). In some embodiments, the Linker Component has the formula ft).
4. Particular Embodiments of the Present Disclosure [0115] Provided below are specifically contemplated embodiments. The provided disclosure is intended exemplify contemplated embodiments and is not intended to be a comprehensive or limiting list. The RAF-Degrading Conjugate Compounds are generated by covalently linking a Ligand for RAF, a Degradation Signaling Agent, and a Linker Component. Covalent linkages between each component may be achieved using the linker chemistry and the modifications for the Ligands for RAF and the Degradation Signaling Agents described in the preceding sections. The covalently linked Ligands for RAF and the Degradation Signaling Agents maintain sufficient binding affinity for their targets to carry out the desired effect.
[0116] In some embodiments, the RAF-Degrading Conjugate Compound includes a Ligand for RAF that is RAF709, and a Degradation Signaling Agent that is pomalidomide. [0117] In some embodiments, the RAF-Degrading Conjugate Compound includes a Ligand for RAF that is RAF709, and a Degradation Signaling Agent that is VHL Ligand.
[0118] In some embodiments, the RAF-Degrading Conjugate Compound includes a Ligand for RAF that is RAF709, and a Degradation Signaling Agent that is Inhibitor of Apoptosis Protein (IAP) Ligand. [0119] In some embodiments, RAF709 has a structure
Figure imgf000038_0001
where each X is N or CH and the wavy line indicates the site of attachment of the Linker Component. In some embodiments, X is N. In some embodiments, X is CH. [0120] In some embodiments, pomalidomide has a structure selected from the group consisting of
Figure imgf000039_0001
wherein the wavy line indicates the site of attachment of the Linker Component.
[0121] In some embodiments, VHL Ligand has a structure
Figure imgf000039_0002
wherein the wavy line indicates the site of attachment of the Linker Component.
[0122] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000039_0003
Figure imgf000040_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
[0123] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000041_0002
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
[0124] In some embodiments, Linker Component has a formula selected from the group consisting of
Figure imgf000041_0001
Figure imgf000042_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent. [0125] In some embodiments, the Linker Component has a formula selected from the group consisting of
Figure imgf000043_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent. [0126] In some embodiments, the Linker Component has the formula a). In some embodiments, the Linker Component has the formula y). In some embodiments, the Linker Component has the formula z). In some embodiments, the Linker Component has the formula aa). In some embodiments, the Linker Component has the formula bb). In some embodiments, the Linker Component has the formula cc). In some embodiments, the Linker Component has the formula dd). In some embodiments, the Linker Component has the formula ee). In some embodiments, the Linker Component has the formula ff).
[0127] In some embodiments, the RAF-Degrading Conjugate Compound is selected from the Examples.
B. Pharmaceutical Compositions
[0128] In addition the conjugate compounds provided above, the compositions for degrading RAF in humans and animals will typically contain a pharmaceutical carrier or diluent.
[0129] The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[0130] The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy and drug delivery. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. [0131] The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self emulsifications as described in U.S. Patent No. 6,451,339, hard or soft capsules, syrups, elixirs, solutions, buccal patch, oral gel, chewing gum, chewable tablets, effervescent powder and effervescent tablets. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, antioxidants and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example PVP, cellulose, PEG, starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated, enterically or otherwise, by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
[0132] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Additionally, emulsions can be prepared with a non-water miscible ingredient such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters and the like.
[0133] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxy-ethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
[0134] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
[0135] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
[0136] The pharmaceutical compositions of the invention may also be in the form of oil-in water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
[0137] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG and surfactants.
[0138] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. 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-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
[0139] The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
Such materials include cocoa butter and polyethylene glycols. Additionally, the compounds can be administered via ocular delivery by means of solutions or ointments. Still further, transdermal delivery of the subject compounds can be accomplished by means of iontophoretic patches and the like. For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. As used herein, topical application is also meant to include the use of mouth washes and gargles.
[0140] The compounds of the invention may be formulated for depositing into a medical device, which may include any of variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets or other device that can be deployed or permanently implanted within a body lumen. As a particular example, it would be desirable to have devices and methods which can deliver compounds of the invention to the region of a body which has been treated by interventional technique. [0141] In exemplary embodiment, the presently disclosed RAF-Degrading Conjugate
Compound may be deposited within a medical device, such as a stent, and delivered to the treatment site for treatment of a portion of the body.
[0142] Stents have been used as delivery vehicles for therapeutic agents (i.e., drugs).
Intravascular stents are generally permanently implanted in coronary or peripheral vessels. Stent designs include those of U.S. Pat. Nos. 4,733,655 (Palmaz), 4,800,882 (Gianturco), or 4,886,062 (Wiktor). Such designs include both metal and polymeric stents, as well as self-expanding and balloon-expandable stents. Stents may also used to deliver a drug at the site of contact with the vasculature, as disclosed in U.S. Pat. No. 5,102,417 (Palmaz) and in International Patent Application Nos. WO 91/12779 (Medtronic, Inc.) and WO 90/13332 (Cedars-Sanai Medical Center), U.S. Pat. Nos. 5,419,760 (Narciso, Jr.) and U.S. Pat. No. 5,429,634 (Narciso, Jr.), for example. Stents have also been used to deliver viruses to the wall of a lumen for gene delivery, as disclosed in U.S. patent application Ser. No. 5,833,651 (Donovan et al.).
[0143] The term“deposited” means that the RAF-Degrading Conjugate Compound is coated, adsorbed, placed, or otherwise incorporated into the device by methods known in the art. For example, the conjugate may be embedded and released from within (“matrix type”) or surrounded by and released through (“reservoir type”) polymer materials that coat or span the medical device. In the later example, the conjugate may be entrapped within the polymer materials or coupled to the polymer materials using one or more the techniques for generating such materials known in the art. In other formulations, the conjugate may be linked to the surface of the medical device without the need for a coating by means of detachable bonds and release with time, can be removed by active mechanical or chemical processes, or are in a permanently immobilized form that presents the conjugate at the implantation site.
[0144] In one embodiment, the RAF-Degrading Conjugate Compound may be incorporated with polymer compositions during the formation of biocompatible coatings for medical devices, such as stents. The coatings produced from these components are typically homogeneous and are useful for coating a number of devices designed for implantation.
[0145] The polymer may be either a biostable or a bioabsorbable polymer depending on the desired rate of release or the desired degree of polymer stability, but a bioabsorbable polymer is preferred for this embodiment since, unlike a biostable polymer, it will not be present long after implantation to cause any adverse, chronic local response. Bioabsorbable polymers that could be used include, but are not limited to, poly(L-lactic acid), polycaprolactone, polyglycolide (PGA), poly(lactide-co-glycobde) (PLLA/PGA), poly(hydroxybutyrate), poly(hydroxybutyrate- co-valerate), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(D-lactic acid), poly(L-lactic acid), poly(D,L-lactic acid), poly(D,L-lactide) (PLA) , poly (L-lactide) (PLLA), poly(glycobc acid-co-trimethylene carbonate) (PGA/PTMC), polyethylene oxide (PEO), polydioxanone (PDS), polyphosphoester, polyphosphoester urethane, poly(amino acids), cyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate), copoly(ether-esters) (e.g., PEO/PLA), polyalkylene oxalates, polyphosphazenes and biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid, polyepsilon caprolactone, polyhydroxy butyric acid, poly orthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, cross linked or amphipathic block copolymers of hydrogels, and other suitable bioabsorbable poplymers known in the art. Also, biostable polymers with a relatively low chronic tissue response such as polyurethanes, silicones, and polyesters could be used and other polymers could also be used if they can be dissolved and cured or polymerized on the medical device such as polyolefins, polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers and copolymers, vinyl halide polymers and copolymers, such as polyvinyl chloride;
polyvinylpyrrolidone; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics, such as polystyrene, polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; pyran copolymer; polyhydroxy-propyl-methacrylamide -phenol;
polyhydroxyethyl-aspartamide-phenol; polyethyleneoxide -polylysine substituted with palmitoyl residues; polyamides, such as Nylon 66 and polycaprolactam; alkyd resins, polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins, polyurethanes; rayon; rayon- triacetate; cellulose, cellulose acetate, cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and carboxymethyl cellulose.
[0146] Polymers and semipermeable polymer matrices may be formed into shaped articles, such as valves, stents, tubing, prostheses and the like. [0147] In one embodiment of the invention, the RAF-Degrading Conjugate Compound is coupled to a polymer or semipermeable polymer matrix that is formed as a stent or stent-graft device.
[0148] Typically, polymers are applied to the surface of an implantable device by spin coating, dipping or spraying. Additional methods known in the art can also be utilized for this purpose. Methods of spraying include traditional methods as well as microdeposition techniques with an inkjet type of dispenser. Additionally, a polymer can be deposited on an implantable device using photo-patterning to place the polymer on only specific portions of the device. This coating of the device provides a uniform layer around the device which allows for improved diffusion of various analytes through the device coating.
[0149] In preferred embodiments, the RAF-Degrading Conjugate Compound is formulated for release from the polymer coating into the environment in which the medical device is placed. Preferably, the conjugate is released in a controlled manner over an extended time frame (e.g., months) using at least one of several well-known techniques involving polymer carriers or layers to control elution. Some of these techniques were previously described in U.S. Patent
Application 20040243225A1.
[0150] Moreover, as described for example in U.S. Patent No. 6,770,729, the reagents and reaction conditions of the polymer compositions can be manipulated so that the release of the RAF-Degrading Conjugate Compound from the polymer coating can be controlled. For example, the diffusion coefficient of the one or more polymer coatings can be modulated to control the release of the conjugate from the polymer coating. In a variation on this theme, the diffusion coefficient of the one or more polymer coatings can be controlled to modulate the ability of an analyte that is present in the environment in which the medical device is placed (e.g. an analyte that facilitates the breakdown or hydrolysis of some portion of the polymer) to access one or more components within the polymer composition (and for example, thereby modulate the release of the conjugate from the polymer coating). Yet another embodiment of the invention includes a device having a plurality of polymer coatings, each having a plurality of diffusion coefficients. In such embodiments of the invention, the release of the conjugate from the polymer coating can be modulated by the plurality of polymer coatings. [0151] In yet another embodiment, the release of the RAF-Degrading Conjugate Compound from the polymer coating is controlled by modulating one or more of the properties of the polymer composition, such as the presence of one or more endogenous or exogenous compounds, or alternatively, the pH of the polymer composition. For example, certain polymer compositions can be designed to release a conjugate in response to a decrease in the pH of the polymer composition. Alternatively, certain polymer compositions can be designed to release the conjugate in response to the presence of hydrogen peroxide.
C. Methods of Treatment
[0152] The presently disclosed RAF-Degrading Conjugate Compounds are useful in treating or preventing many disease or conditions including, but not limited to, cancer and RASopathies.
[0153] In some embodiments the diseases or conditions are mediated, at least in part, by RAF.
1. Cancer
[0154] In certain aspects, cancer can be treated or prevented by administering one or more RAF-Degrading Conjugate Compounds. Cancer generally includes any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites. Non-limiting examples of different types of cancer suitable for treatment using the compositions of the present invention include ovarian cancer, breast cancer, lung cancer (such as non-small-cell lung carcinoma), bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, colon cancer, anal cancer, colorectal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, rectal cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, renal cancer (i.e., renal cell carcinoma), cancer of the central nervous system, skin cancer, choriocarcinomas, head and neck cancers, bone cancer, osteogenic sarcomas, fibrosarcoma, neuroblastoma, glioma, melanoma, leukemia ( e.g acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, or hairy cell leukemia), lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, B-cell lymphoma, or Burkitt's lymphoma), and multiple myeloma. [0155] In some embodiments, the cancer is melanoma, an epithelial cancer ( e.g prostate cancer, ovarian cancer, breast cancer), or a blood cancer (e.g., leukemia, lymphoma, multiple myeloma).
[0156] In some embodiments, the cancer is melanoma or colorectal cancer.
[0157] In some embodiments, the RAF-Degrading Conjugate Compounds of the present disclosure are useful in treating genetically defined cancers, irrespective or of tissue origin. Genetically defined cancers include, but are not limited to, those mediated, at least in part by mutant KRAS, HRAS, or NRAS proteins. In some embodiments, the mutations in these proteins include, but are not limited to, codons 12, 13, and 61, including misssense mutations to any of the 20 naturally occurring amino acids. In some embodiments, KRAS mutations include, but are not limited to, G12D, G12V, G13D, and G13C; HRAS mutations include, but are not limited to, G12V, Q61R, Q61L, and G13R; and NRAS mutations include, but are notlimited to Q61R, Q61K, G12D, and G13D. Genetically defined cancers also include, but are not limited to, cancers mediated, at least in part, by mutant RAF proteins. In some embodiments the mutated RAF protein is BRAF. In some embodiments, the BRAF mutation is V600E.
2. RASopathies
[0158] In certain other aspects, RASopathies can be treated or prevented by administering one or more RAF-Degrading Conjugate Compounds. RASopathies are developmental syndromes involving dysregulation of the RAS/MPAK pathway. Non-limiting examples of RASopathies include neurofibromatosis type 1, capillary malformation-arteriovenous malformation syndrome, autoimmune lymphoproliferative syndrome, cardio-facio-cutaneous syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Noonan syndrome, Costello syndrome, Legius syndrome, LEOPARD syndrome.
[0159] In some embodiments, the RASopathy is Noonan syndrome, Costello syndrome,
Legius syndrome, LEOPARD syndrome.
[0160] In some embodiments, the RASopathy is Noonan syndrome or LEOPARD syndrome. D. Combination Therapy
[0161] The presently disclosed RAF-Degrading Conjugate Compounds can be used in combination with other cancer treatment agents to treat various types of cancer. Accordingly, in some aspects, the present disclosure includes a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the RAF- Degrading Conjugate Compound and an additional cancer therapeutic agent. Cancer therapeutic agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, and endocrine therapies.
[0162] It is contemplated that any of the above listed cancers can be used in the combination therapy described herein.
[0163] In embodiments where combination therapy is employed, the RAF-Degrading
Conjugate Compound and the additional cancer therapeutic agent are administered
simultaneously or sequentially. In some embodiments the RAF-Degrading Conjugate
Compound and the additional cancer therapeutic agent are administered simultaneously. In some embodiments the RAF-Degrading Conjugate Compound and the additional cancer therapeutic agent are administered sequentially. In some embodiments, the RAF-Degrading Conjugate Compounds and the additional therapeutic agent, when administered simultaneously, are formulated in a single pharmaceutical composition. In some embodiments, the RAF- Degrading Conjugate Compounds and the additional therapeutic agent, when administered simultaneously, are two separate compositions.
1. Chemotherapeutic Agents
[0164] Chemotherapeutic agents ( e.g ., anti-cancer agents) are well known in the art and include, but are not limited to, anthracenediones (anthraquinones) such as anthracyclines (e.g., daunombicin (daunomycin; mbidomycin), doxorubicin, epimbicin, idambicin, and valmbicin), mitoxantrone, and pixantrone; platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin); tamoxifen and metabolites thereof such as 4-hydroxytamoxifen (afimoxifene) and N-desmethyl-4-hydroxytamoxifen (endoxifen); taxanes such as paclitaxel (taxol) and docetaxel; alkylating agents (e.g., nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil); ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa, alkyl sulphonates such as busulfan, nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U), and streptozoein (streptozotocin), and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)); antimetabolites (e.g., folic acid analogues such as methotrexate (amethopterin), pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside), and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; 6-TG), and pentostatin (2'-deoxycofonnycin)); natural products (e.g., vinca alkaloids such as vinblastine (VLB) and vincristine, epipodophyllotoxins such as etoposide and teniposide, and antibiotics such as dactinomycin (actinomycin D), bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin Q); enzymes such as L-asparaginase; biological response modifiers such as interferon alpha); substituted ureas such as hydroxyurea; methyl hydrazine derivatives such as procarbazine (N-methylhydrazine; MIH); adrenocortical suppressants such as mitotane (o,r'-DDD) and aminoglutethimide; analogs thereof; derivatives thereof; and combinations thereof.
[0165] In some embodiments, the chemotherapeutic agent is an MEK inhibitor. MEK inhibitors are small molecules or biologies that bind to and inhibit or decrease the normal function of MEK proteins. MEK inhibition is particularly useful in melanoma as well as RAS and RAF mediated cancers. Any known MEK inhibitor can be used in combination with the RAF-Degrading Conjugate Compounds of the present disclosure.
[0166] In some embodiments, the MEK inhibitor targets MEK1, MEK2, or both MEK1 and 2. In some embodiments, the MEK inhibitors is a small molecule.
[0167] In some embodiments, the MEK inhibitor is trametinib, pimasertib, selumertinib, PD- 0325901, Refametinib, TAK733, MEK162, R05126766, WX-554, R04987655, CD-0931, or AZD8330.
2. Radiotherapeutic Agents
[0168] Radiotherapeutic agents are well known in the art and can comprise external-beam radiation therapy and/or internal radiation therapy. External beam radiation therapy delivers radioactive beams of high energy X-rays and/or gamma rays to a patient’s tumor, whereas internal radiation therapy delivers radioactive atoms to a patient’s tumor. Both external beam radiation therapy and internal radiation therapy are used to suppress tumor growth or kill cancer cells by delivering a sufficient quantity of radioactivity to the target site. In some embodiments, the radiotherpaeutic agent comprises a radioactive atom and is complexed with a biologic or synthetic agent to increase delivery to the target site. Such biologic or synthetic agents are known in the art. Suitable radioactive atoms for use with the RAF-Degrading Conjugate Compounds of the present disclosure include any of the radionuclides described herein, or any other isotope which emits enough energy to destroy a targeted tissue or cell. In some embodiments, radiotherapeutic agents may be coupled to targeting moieties, such as antibodies, to improve the localization of radiotherapeutic agents to cancerous or infected cells.
[0169] The term“radionuclide” is intended to include any nuclide that exhibits radioactivity.
A“nuclide” refers to a type of atom specified by its atomic number, atomic mass, and energy state, such as carbon 14 (14C). “Radioactivity” refers to the radiation, including alpha particles, beta particles, nucleons, electrons, positrons, neutrinos, and gamma rays, emitted by a radioactive substance. Examples of radionuclides suitable for use in the present invention include, but are not limited to, fluorine 18 (18F), fluorine 19 (19F), phosphorus 32 (32P), scandium 47 (47Sc), cobalt 55 (55Co), copper 60 (60Cu), copper 61 (61Cu), copper 62 (62Cu), copper 64 (64Cu), gallium 66 (66Ga), copper 67 (67Cu), gallium 67 (67Ga), gallium 68 (68Ga), rubidium 82 (82Rb), yttrium 86 (86Y), yttrium 87 (87Y), strontium 89 (89Sr), yttrium 90 (90Y), rhodium 105 (105Rh), silver 1 1 1 (mAg), indium 1 1 1 (mIn), iodine 124 (124I), iodine 125 (125I), iodine 131 (131I), tin 1 17m (1 17mSn), technetium 99m (99mTc), promethium 149 (149Pm), samarium 153 (153Sm), holmium 166 (166Ho), lutetium 177 (177Lu), rhenium 186 (186Re), rhenium 188 (188Re), thallium 201 (201T1), astatine 21 1 (21 1At), and bismuth 212 (212Bi). As used herein, the“m” in 1 17mSn and 99mTc stands for the meta state. Additionally, naturally- occurring radioactive elements such as uranium, radium, and thorium, which typically represent mixtures of radioisotopes, are suitable examples of radionuclides. 67Cu, 1311, 177Lu, and 186Re are beta- and gamma-emitting radionuclides. 212Bi is an alpha- and beta-emitting radionuclide. 21 'At is an alpha-emitting radionuclide. 32P, 47Sc, 89Sr, 90Y, 105Rh, mAg, 117mSn, 149Pm, 153Sm, 166Ho, and 188Re are examples of beta-emitting radionuclides. 67Ga, 11 'in, 99mTc, and 201T1 are examples of gamma-emitting radionuclides. 55Co, 60Cu, 61Cu, 62Cu, 66Ga, 68Ga, 82Rb, and 86Y are examples of positron-emitting radionuclides. 64Cu is a beta- and positron-emitting radionuclide. 3. Endocrine Therapies
[0170] Endocrine therapy is the manipulation of the endocrine system through the
administration of specific hormones or drugs which inhibit or decrease the production or activity of targeted hormones or alter the gene expression pattern of targeted cells. Endocrine therapy is particularly useful in certain types of cancer, including breast cancer. Any known hormone antagonist or modulator may be used in combination with the RAF-Degrading Conjugate Compounds of the present disclosure. Useful Endocrine therapies include, but are not limited to, aromatase inhibitors ( e.g . letrozole), megestrol acetate, flutamide, tamoxifen, raloxifene, lasofoxifene, bazedoxifene, bazedoxifene/conjugated estrogens, and combinations thereof.
E. Kits, Containers, Devices, and Systems
[0171] A wide variety of kits and systems can be prepared according to the present invention, depending upon the intended user of the kit and system and the particular needs of the user. In some aspects, the present disclosure provides a kit that includes one or more RAF-Degrading Conjugate Compounds. In other aspects, the present disclosure provides a kit that includes one or more RAF-Degrading Conjugate Compounds and one or more therapeutic agents selected from a chemotherapeutic agent, a radiotherapeutic agent, an endocrine therapy.
[0172] Some of the kits described herein include a label describing a method of administering one or more RAF-Degrading Conjugate Compounds and/or one or more additional cancer therapeutic agents described herein. Some of the kits described herein include a label describing a method of treating a disease or disorder described herein.
[0173] The compositions of the present invention, including but not limited to, compositions comprising one or more RAF-Degrading Conjugate Compounds and one or more additional cancer therapeutic agents described herein may, if desired, be presented in a bottle, jar, vial, ampoule, tube, or other container-closure system approved by the Food and Drug Administration (FDA) or other regulatory body, which may provide one or more dosages containing the compounds. The package or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice indicating approval by the agency. In certain aspects, the kit may include a formulation or composition as described herein, a container closure system including the formulation or a dosage unit form including the formulation, and a notice or instructions describing a method of use as described herein.
[0174] In some embodiments, the kit includes a container which is compartmentalized for holding the various elements of a formulation ( e.g ., the dry ingredients and the liquid ingredients) or composition, instructions for making the formulation or composition, and instructions for administering the formulation or composition in a subject.
[0175] In certain embodiments, the kit may include the pharmaceutical preparation in dehydrated or dry form, with instructions for its rehydration (or reconstitution) and
administration.
[0176] Kits with unit doses of the compounds described herein, e.g. in oral, rectal, transdermal, or injectable doses (e.g. , for intramuscular, intravenous, or subcutaneous injection), are provided. In such kits, an informational package insert describing the use and attendant benefits of the composition may be included in addition to the containers containing the unit doses.
[0177] Some embodiments of the present invention include packages that include one or more RAF-Degrading Conjugate Compounds and one or more additional cancer therapeutic agents described herein.
III. EXAMPLES
[0178] The following examples are offered to illustrate, but not to limit the claimed invention.
EXPERIMENTAL DETAILS
[0179] The following abbreviations are used in the examples below:
aq aqueous
CH3CN acetonitrile
CD3OD methanol-D4
CDCh chloroform-D
cone concentrate
CuS04 copper(II) sulfate CV column volume
DCM methylene chloride or dichloromethane
DIPEA diisopropylethyl amine
DMF dimethylformamide
DMSO dimethylsulfoxide
Eq. equivalent
EtOAc ethyl acetate
HATU 0-(7-azabcnzotriazol- 1 -yl),A, A, A",/V"- tetramethyluroniumhexafluorophosphate
h hour(s)
Hex hexanes
HPLC high performance liquid chromatography
LRMS low resolution mass spec
M molar
MeOH methanol
min minute(s)
NaCl sodium chloride
NaN3 sodium azide
Na2S04 sodium sulfate
rt room temperature
tR retention time
Si02 silica gel
THF tetrahydrofuran
TLC thin layer chromatography
[0180] The compounds of this invention may be prepared in light of the specification using steps generally known to those of ordinary skill in the art. Those compounds may be analyzed by known methods, including but not limited to LC-MS (liquid chromatography mass
spectrometry), HPLC (high performance liquid chromatography) and NMR (nuclear magnetic resonance). It should be understood that the specific conditions shown below are only examples, and are not meant to limit the scope of the conditions that can be used for making compounds of this invention. Instead, this invention also includes conditions that would be apparent to those skilled in that art in light of this specification for making the compounds of this invention.
Unless otherwise indicated, all variables in the following Examples are as defined herein.
[0181] LRMS values were recorded on Waters micromass ZQ using direct injection of the samples in either methanol or acetonitrile. Analytical HPLC was carried out on Waters alliance using Agilent, Zorbax-SB-CN, 3.5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100%) contains ammonium acetate buffer; flow rate, 1.5 mL/min, run time, 20 min]
[0182] HPLC Method A: Column, Agilent, Zorbax Eclipse XDB-C8, 5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100%) contains ammonium acetate buffer; flow rate, 1.5 mL/min, run time, 20 min.
[0183] HPLC Method B: Column, Agilent, Zorbax-SB-CN, 3.5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100%) contains ammonium acetate buffer; flow rate, 0.1 mL/min, run time, 20 min.
[0184] HPLC Method C: Column, Agilent, Zorbax SB-C18, 5 pm, 4.6 x 150 mm, mobile phase, acetonitrile in water (0 to 100% for 8 min, 100% for 2 min) contains 0.1% TFA as buffer; flow rate, 1.5 mL/min, run time, 10 min.
[0185] All reactions were carried out under inert atmosphere either 2 or Ar
[0186] HPLC Prep method: Column, Phenomenex, Synergi, 4p, Max-RP 80A, AX; 250 x 21.2 mm, mobile phase, acetonitrile in water (10-100%, 25 min, water contains 0.2% HCO2H buffer); flow rate, 15 mL/min.
[0187] The preparation of a particular intermediate used in some of the preparations below is described in Examples A-D.
Example A: Synthesis of /V-(6'-(( 14-hydroxy-3,6,9, 12-tetraoxatetradecyl)oxy)-2-methyl-5'- morpholino- [3 ,3 -bipyridin] -5-yl)-3 -(trifluoromethyl)benzamide
Figure imgf000059_0001
[0188] To a stirred solution of 3,6,9,12-tetraoxatetradecane-l,14-diol (5.18 g, 21.7 mmol) in 1,4-dioxane (15 mL) was added 60% sodium hydride (869 mg, 21.7 mmol) in two portions, after stirred at rt for 30 min, /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide (1000 mg, 2.17 mmol) was added in one portion, it was placed it on pre-heated oil bath and stirred at 82-85 °C for 3 h, The reaction mixture was cooled to rt, poured into water (40 mL), extracted with ethyl acetate (3 x 40 mL), combined extracts were dried (Na2S04), concentrated. The residue was purified on 40 g S1O2 cartridge using a gradient of methanol in DCM (0% to 15%) as eluant to afford the title compound (1.3 g, 88.2%) as gum. Rf = 0.3 (10% MeOH in DCM). !H NMR (400 MHz, CDCh) d 8.69 (d, J = 2.5 Hz, 1H), 8.54 (s, 1H), 8.20 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.09 - 8.07 (m, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.05 - 7.02 (m, 1H), 4.59 - 4.53 (m, 2H), 3.92 - 3.84 (m, 6H), 3.73 - 3.68 (m, 4H), 3.67 - 3.61 (m, 10H), 3.59 - 3.55 (m, 2H), 3.17 - 3.11 (m, 4H), 2.48 (s, 3H). LRMS, m/z, calculated (for C33H41F3N4O8) 678.29, found, 701.37 (M+Na)+, 677.3 (M-H)-.
Example B: Synthesis of 3-(6-((7-azidoheptyl)oxy)-l-oxoisoindolin-2-yl)piperidine-2,6-dione
Figure imgf000060_0001
Step I, 7-azidoheptyl 4-methylbenzenesulfonate
[0189] To a stirred solution of 7-azidoheptan-l-ol (4.50 g, 28.60 mmol, 1.0 equiv.) in DCM (90.0 mL) was added triethylamine (12.0 mL, 85.90 mmol) and -toluene sulfonyl chloride (5.46 g, 28.60 mmol) and the resulting reaction mixture was stirred at rt for 15 h. The progress of the reaction was monitored by TLC (40% EtOAC in hexanes). The reaction mixture was diluted with DCM (200 mL) and washed with water (2 x 100 mL), dried (Na2S04), filtered, and concentrated. The crude product was purified on silica gel (230-400 mesh size) column chromatography using a gradient of ethyl acetate in heptanes (10-30%) to afford the 7- azidoheptyl 4-methylbenzenesulfonate (5.1 g, 57.2%) as an oil. 1 H NMR (300 MHz, CDCb): d 7.85 - 7.78 (m, 2H), 7.40 - 7.28 (m, 2H), 4.03 (t, J = 6.4 Hz, 2H), 3.25 (t, J = 6.9 Hz, 2H), 2.46 (s, 3H), 1.73 - 1.49 (m, 4H), 1.41 - 1.19 (m, 6H). Step II, 3-(6-((7-azidoheptyl)oxy)-l-oxoisoindolin-2-yl)piperidine-2,6-dione
[0190] To a stirred solution of 3-(6-hydroxy-l-oxoisoindolin-2-yl) piperidine-2, 6-dione (500 mg, 1.92 mmol, 1.0 equiv.) (Ruchelman, A. L. et.al. Biorg. Med. Chem. Lett. 2013, 23, 360- 365) in DMF (10 mL) was added 7-azidoheptyl 4-methylbenzenesulfonate (778 mg, 2.50 mmol) and potassium carbonate (664 mg, 4.80 mmol) were added at rt. The reaction mixture was heated at 90°C for 15 h. The progress of the reaction was monitored by TLC (80% EtOAc in hexanes). Water (200 mL) was added and the product was extracted with ethyl acetate (3 x 200 mL). The combined organic extracts were washed with water (2 x 200 mL), brine (2 x 200 mL), dried (Na2S04), and concentrated under reduced pressure. The crude product was purified on silica gel column chromatography (230-400 mesh size) using a gradient of ethyl acetate in hexanes (10- 50% EtOAc) to afford 3-(6-((7-azidoheptyl)oxy)-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (90 mg, 11.7%) as an off-white solid. 'H NMR ^OO MHZ, DMSO-cfc): d 11.00 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.19 (m, 2H), 5.11 (m, 1H), 4.48 - 4.16 (m, 2H), 4.05 (t, J= 6.5 Hz, 2H), 3.31 (m, 2H), 3.01 - 2.81 (m, 1H), 2.59 (m, 1H), 2.46 - 2.22 (m, 1H), 2.1-1.2 (m, 11H). LRMS, m/z, calculated (for C20H25N5O4), 399.19, found, 399.9 (M+H)+.
Example C: Synthesis of 3-(4-((7-azidoheptyl)oxy)-l-oxoisoindolin-2-yl)piperidine-2, 6-dione
Figure imgf000061_0001
[0191] The title compound (100 mg, 13%) was prepared as off-white solid from 3-(4-hydroxy- l-oxoisoindolin-2-yl) piperidine-2, 6-dione (500 mg, 1.92 mmol) (Ruchelman, A. L. et.al. Biorg. Med. Chem. Lett. 2013, 23, 360-365) as described for 3-(6-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2, 6-dione. 1H NMR (300 MHz, DMSO-d6): d 1 1.03 (s, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.35-7.26 (m, 2H), 5.18 - 5.12 (m, 1H), 4.48 - 4.20 (m, 2H), 4.15 (t, J = 6.4 Hz, 2H), 3.36 (m, 2H), 2.95 (m, 1H), 2.61 (m, 1H), 2.52 - 2.38 (m, 1H), 2.08 - 1.91 (m, 1H), 1.77 (m, 2H), 1.65 - 1.33 (m, 8H). LRMS, m/z, calculated (for C20H25N5O4), 399.19, found, 399.9 (M+H)+.
Example D: Synthesis of 3-(5-((7-azidoheptyl)oxy)-l-oxoisoindolin-2-yl)piperidine-2, 6-dione
Figure imgf000062_0001
[0192] The title compound (210 mg, 27%) was prepared as off-white solid from 3-(5- hydroxy-l-oxoisoindolin-2-yl)piperidine-2,6-dione (500 mg, 1.92 mmol) (Ruchelman, A. L. et.al. Biorg. Med. Chem. Lett. 2013, 23, 360-365) as described for 3-(6-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2,6-dione. 'H NMR (300 MHz, DMSO-d6) d 10.98 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 5.08 (dd, J = 13.2, 5.0 Hz, 1H), 4.46 4.18 (m, 2H), 4.06 (t, J = 6.4 Hz, 2H), 3.30 (m, 2H), 3.05 2.76 (m, 1H), 2.58 (d, J = 18.2 Hz, 1H), 2.46 2.26 (m, 1H), 2.01-1.2 (m, 1 1H).
Example E: Synthesis of 3-(7-((7-azidoheptyl)oxy)-l-oxoisoindolin-2-yl)piperidine-2,6-dione
Figure imgf000062_0002
[0193] The title compound (50 mg, 6.5%) was prepared as off-white solid from 3-(7- hydroxy- 1 -oxoisoindolin-2-yl) piperidine-2, 6-dione (500 mg, 1.92 mmol) (Ruchelman, A. L. et.al. Biorg. Med. Chem. Lett. 2013, 23, 360-365) as described for 3-(6-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2, 6-dione. !H NMR (300 MHz, DMSO-d6): d 10.98 (s, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.06 (m, 2H), 5.00 (m, 1H), 4.49 4.16 (m, 2H), 4.09 (t, J = 6.4 Hz, 2H),
3.31 (m, 2H), 3.01 2.79 (m, 1H), 2.59 (d, J = 17.8 Hz, 1H), 2.46 2.24 (m, 1H), 2.01-1.2 (m, 11H).
[0194] Examples 1 to 20 report the synthesis of compounds using the general structure shown below:
Figure imgf000063_0001
Example 1: Synthesis of /V-(6'-(( 14-((2-( 1 -(hydroxymcthyl)-2,6-dioxopipcridin-3-yl)- 1 - oxoisoindolin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.001)
Figure imgf000063_0002
Step I, tert- butyl (2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolm-4-yl) carbonate:
[0195] To a stirred mixture of 3-(4-hydroxy-l-oxoisoindobn-2-yl)piperidine-2,6-dione (1000 mg, 3.84 mmol) [Ruchelman, et. al. BMCL, 2013, 23, 360-365] and DMAP (46.9 mg, 0.384 mmol) in pyridine (10 mL) was added Di-/er/-butyl carbonate (736 mg, 4.23 mmol), after stirred for 64 h (weekend), diluted with water (50 mL), and aq. 1 HC1 (pH 5-6), cooled with ice, resultant precipitate was filtered off, rinsed with water (50 mL), dried to afford the title compound (883 mg, 63.8%) as off-white solid. Rf = 0.45 (EίOAo^'H NMR (400 MHz, DMSO)
5 11.00 (s, 1H), 7.70 - 7.65 (m, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.51 (dd, J = 8.0, 0.9 Hz, 1H), 5.12 (dd, J = 13.3, 5.1 Hz, 1H), 4.44 (d, J = 17.4 Hz, 1H), 4.29 (d, J = 17.4 Hz, 1H), 2.96 - 2.84 (m, 1H), 2.59 (d, J = 17.4 Hz, 1H), 2.47 - 2.39 (m, 1H), 2.05 - 1.96 (m, 1H), 1.51 (s, 9H). LRMS, m/z, calculated (for C18H20N2O6), 360.13, found, 361.07 (M+H)+; 259.02 (M-H-BOC) .
Step II, tert- butyl (2-(2,6-dioxo-l-((2-(trimethylsilyl)ethoxy)methyl)piperidin-3-yl)-l- oxoisoindolin-4-yl) carbonate
[0196] To a cold (0 °C) stirred solution of /er/-butyl (2-(2,6-dioxopiperidin-3-yl)-l- oxoisoindolin-4-yl) carbonate (225 mg, 0.624 mmol) in DMF (2.5 mL) was added 60% sodium hydride (27.5 mg, 0.687 mmol), stirred for 30 min, (2-chloromethoxy)ethyl)trimethylsilane (115 mg, 0.687 mmol), slowly warmed up to rt during 1.5 h, diluted with ethyl acetate (10 mL), water (5 mL), acidified with aq HC1 (0.5 mL), organic layer was separated, aqueous solution was extracted with ethyl acetate (10 mL), combined extracts were washed with brine, dried
(Na2S04), and concentrated. The residue was purified on 25 g S1O2 cartridge using a gradient of ethyl acetate in hexanes (0 to 80%) as eluant to afford the title compound (200 mg, 65.3%) as a white solid. Rf = 0.6 (EtOAc). !H NMR (400 MHz, CDCb) d 7.75 (dd, J = 7.4, 0.7 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.42 (dd, J = 8.1, 0.8 Hz, 1H), 5.26 - 5.15 (m, 3H), 4.47 (d, J = 16.2 Hz, 1H), 4.32 (d, J = 16.2 Hz, 1H), 3.61 (dd, J = 11.0, 5.5 Hz, 2H), 3.06 - 2.95 (m, 1H), 2.93 - 2.81 (m, 1H), 2.40 - 2.26 (m, 1H), 2.23 - 2.10 (m, 1H), 1.55 (s, 9H), 0.99 - 0.88 (m, 2H), -0.01 (s, 9H).
Step III, 3-(4-hydroxy-l-oxoisomdolm-2-yl)-l-((2-(trimethylsilyl)ethoxy)methyl)piperidine- 2,6-dione
[0197] To a stirred solution of /er/-butyl (2-(2,6-dioxo-l-((2-(trimethylsilyl)ethoxy)methyl)- piperidin-3-yl)-l-oxoisoindolin-4-yl) carbonate (200 mg, 0.408 mmol) in DCM (3 mL) was added piperidine (1 mL), after stirred for 40 min, it was diluted with DCM (10 mL), water (10 mL), aq. IN HC1 (pH 4-5), organic solution was separated, aqueous solution was extracted with DCM (2 x 10 mL), combined organic extracts were washed with water, brine, dried ( a2S04), and concentrated. The residue was purified on 24 g S1O2 cartridge using a gradient of ethyl acetate in hexanes (0 to 100%) as eluant to afford the title compound (140 mg, 87.9%) as a white solid. Rf = 0.42 (EtOAc). lR NMR (400 MHz, CDCh) d 7.53 (s, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.32 - 7.26 (m, 1H), 6.97 (d, J = 7.9 Hz, 1H), 5.27 (d, J = 9.3 Hz, 1H), 5.23 - 5.16 (m, 2H), 4.44 (d, J = 16.4 Hz, 1H), 4.29 (d, J = 16.4 Hz, 1H), 3.71 - 3.61 (m, 2H), 3.08 - 2.95 (m, 1H), 2.94 - 2.79 (m, 1H), 2.39 - 2.23 (m, 1H), 2.21 - 2.10 (m, 1H), 0.99 - 0.89 (m, 2H), -0.00 (s, 9H). LRMS, m/z, calculated (for C19H26N2O5S1), 390.16, found, 389.11 (M-H) .
Step IV, V-(6'-((14-((2-(2,6-dioxo-l-((2-(trimcthylsilyl)cthoxy)mcthyl)pipcridin-3- yl)-l-oxoisoindolin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide
[0198] To a cold (ice) mixture of 3 -(4-hydroxy- l-oxoisoindolin-2-yl)-l-((2-(trimethylsilyl)- ethoxy)methyl)piperidine-2,6-dione (69 mg, 0.177 mmol) and triphenylphosphine (83.5 mg, 0.318 mmol) in THF (1.5 mL) was added a solution of/V-(6'-((14-hydroxy-3,6,9,12- tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)- benzamide {Example A) (120 mg, 0.177 mmol) and diisopropylazodicarboxylate (64 mg, 0.318 mmol) in THF (1.5 mL), after stirred for 1 h, cooling bath was removed, stirred at rt for 4 h, concentrated. The residue was purified on 25 g S1O2 cartridge using a gradient of methanol in ethyl acetate (0 to 10%) to afford /V-(6'-((14-((2-(2,6-dioxo-l-((2- (trimethylsilyl)ethoxy)methyl)piperidin-3-yl)-l-oxoisoindolin-4-yl)oxy)-3,6,9,12- tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)- benzamide (50 mg, 26.9%) as a gum. !H NMR (400 MHz, CDCb) d 8.80 - 8.76 (m, 1H), 8.73 (s, 1H), 8.24 (s, 1H), 8.15 (d, J = 8.1 Hz, 1H), 8.00 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.69 (s, 1H), 7.66 - 7.59 (m, 1H), 7.39 - 7.32 (m, 2H), 7.04 - 6.97 (m, 2H), 5.22 (d, J = 9.4 Hz, 1H), 5.18 - 5.10 (m, 2H), 4.59 - 4.51 (m, 2H), 4.40 (d, J = 16.8 Hz, 1H), 4.28 (d, J = 16.4 Hz, 1H), 4.25 - 4.17 (m, 2H), 3.94 - 3.80 (m, 8H), 3.73 - 3.55 (m, 14H), 3.16 - 3.07 (m, 4H), 2.99 (d, J = 17.5 Hz, 1H), 2.88 - 2.74 (m, 1H), 2.49 (s, 3H), 2.38 - 2.26 (m, 1H), 2.19 - 2.07 (m, 1H), 0.96 - 0.86 (m, 2H), -0.02 (9, 7H). 19F NMR (376 MHz, CDCb) d -63.04 (s). LRMS, m/z, calculated (for C52H65F3N60i2Si), 1050.44, found, 1051.58 (M+H)+.
Step V, Compound 1.001:
[0199] To a cold (0 °C) stirred solution of /V-(6'-((14-((2-(2,6-dioxo-l-((2-(trimethylsilyl)- ethoxy)methyl)piperidin-3-yl)-l-oxoisoindolin-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)oxy)-2- methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (25 mg, 0.0238 mmol) in DCM (0.3 mL) was added TFA (53 pL, 0.713 mmol), cooling bath was removed after 30 min, additional amount of TFA (53 pL) and ethanol (50 pL) was added, stirred at rt for 4 h, and concentrated. The residue was purified on 12 g Si(¾ cartridge using a gradient of methanol in ethyl acetate (0 to 30%) as eluant to afford the title compound (10 mg, 44.2%) as a white solid. Rf = 0.23 (10% MeOH in EtOAc). !H NMR (400 MHz, CDCb) d 8.91 (s, 1H), 8.29 (s, 1H), 8.19 (d, J = 7.6 Hz, 1H), 7.89 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 2.0 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.36 - 7.31 (m, 2H), 7.01 (dd, J = 6.4, 2.5 Hz, 1H), 6.98 (d, J = 2.0 Hz, 1H), 5.35 (d, J = 10.1 Hz, 1H), 5.25 (d, J = 10.0 Hz, 1H), 5.16 (dd, J = 13.6, 5.1 Hz, 1H), 4.66 - 4.49 (m, 2H), 4.37 (d, J = 16.8 Hz, 1H), 4.29 (d, J = 16.6 Hz, 1H), 4.26 - 4.16 (m, 2H), 3.94 - 3.81 (m, 8H), 3.74 - 3.58 (m, 12H), 3.12 - 3.05 (m, 4H), 3.03 - 2.95 (m, 1H), 2.88 - 2.76 (m, 1H), 2.49 (s, 3H), 2.41 - 2.29 (m, 1H), 2.21 - 2.1 1 (m, 1H). 19F NMR (376 MHz, CDCb) d - 63.06 (s). LRMS, m/z, calculated (for C47H53F3N6O12), 950.37, found, 973.57 (M+Na)+. HPLC, tR = 8.9 min (purity, 95%) (HPLC Method A).
Example 2: Synthesis of /V-(6'-(2-(2-(2-((l-(7-((2-(2,6-dioxopiperidin-3-yl)-3- oxoisoindolin-5-yl)oxy)heptyl)-lH- 1,2,3 -triazol-4-yl)methoxy)ethoxy)etho xy)ethoxy)-2- methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.002)
Figure imgf000066_0001
[0200] To a stirred solution of /V-(2-mcthyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 - yloxy)ethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (48.1 mg, 76.5 mmol) [as described in W02018/200981] in THF (1.4 mL) was added a solution of copper(II) sulfate pentahydrate (5.6 mg, 0.0224 mmol) in degassed water (0.35 mL) and sodium ascorbate (10.2 mg, 0.0512 mmol) in degassed water (0.35 mL) followed by 3-(6-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2,6-dione {Example B) (32.2 mg, 0.0806 mmol). The flask was purged with nitrogen/vacuum (3 cycles) and the reaction mixture was stirred at rt for 18 h under inert argon atmosphere with vigorous stirring. The reaction mixture was diluted with water (2 mL) then extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine (2 mL), dried (Na2S04), and concentrated. The residue was purified on a 25 g silica gold cartridge using a gradient of MeOH in DCM (0-15%, 20 CVs) followed by reverse phase column (C-18 40 g cartridge) using a gradient of acetonitrile in water (5-100%, 20 CVs, loading in DMSO) to afford the title compound (15.9 mg, 20.2% yield) as a white solid. Rf = 0.43 (10% MeOH in DCM). *H NMR (400 MHz, CDCh) d 8.97 (s, 1H), 8.74 (s, 1H), 8.57 (bs, 1H), 8.22 (s, 1H), 8.15 (d, J = 7.4 Hz, 1H), 8.1 1 (s, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.73 (s, 1H), 7.63 - 7.58 (m , 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.03 (s, 1H), 5.18 (dd, J = 12.8, 4.5 Hz, 1H), 4.65 (s, 2H), 4.58 (bs, 2H), 4.40 - 4.25 (m, 4H), 3.94 - 3.87 (m, 8H), 3.71 (bs, 2H), 3.68 - 3.59 (m, 6H), 3.17 - 3.09 (m, 4H), 2.95 - 2.74 (m, 2H), 2.49 (s, 3H), 2.29 - 2.25 (m, 1H), 2.25 - 2.16 (m, 1H), 1.92 - 1.82 (m, 2H), 1.79 - 1.70 (m, 2H), 1.48 - 1.28 (m, 6H). 19F NMR (376 MHz, CDCh) d -63.06 (s). LRMS, m/z, calculated (for C52H60F3N9O10), 1027.44; found 1050.64 (M+Na)+; found 1026.64 (MH)~. HPLC, tR = 9.55 min (purity, 97%). HPLC Method B.
Example 3: Synthesis of /V-(6'-(2-(2-(2-(( 1 -(7-((2-(2,6-dioxopipcridin-3-yl)- 1 -oxoisoindolin- 4-yl)oxy)heptyl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)-2-methyl-5'- morphohno-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.003)
Figure imgf000067_0001
[0201] /V-(2-mcthyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 -yloxycthoxy)cthoxy)cthoxy)-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in
WO2018/200981.
[0202] Title compound (38.9 mg, 47.5% yield) was prepared as a white solid from N-{ 2- methyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn-l-yloxyethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5- yl)-3-(trifluoromethyl)benzamide (50.1 mg, 0.0797 mmol) and 3-(4-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2,6-dione {Example C) (30.8 mg, 0.0771 mmol) as described in Example 2. Rf = 0.41 (10% MeOH in DCM). !H NMR (400 MHz, CDCh) d 8.84 (s, 1H), 8.81 (d, J = 2.1 Hz, 1H), 8.74 (bs, 1H), 8.22 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.40 -
7.34 (m, 2H), 7.01 (d, J = 2.1 Hz, 1H), 6.98 (dd, J = 6.6, 2.3 Hz, 1H), 5.18 (dd, J = 13.4, 5.1 Hz, 1H), 4.64 (s, 2H), 4.60 - 4.54 (m, 2H), 4.43 - 4.36 (m, 1H), 4.32 - 4.26 (m, 3H), 4.05 - 4.00 (m, 2H), 3.90 - 3.88 (m, 2H), 3.87 - 3.85 (m, 4H), 3.72 - 3.68 (m, 2H), 3.67 - 3.62 (m, 6H), 3.15 - 3.09 (m, 4H), 2.93 - 2.85 (m, 1H), 2.84 - 2.72 (m, 1H), 2.49 (s, 3H), 2.47 - 2.35 (m, 1H), 2.22 - 2.13 (m, 1H), 1.92 - 1.85 (m, 2H), 1.81 - 1.71 (m, 3H), 1.50 - 1.29 (m, 5H). 19F NMR (376
MHz, CDCh) d -63.08 (s). LRMS, m/z, calculated (for C52H60F3N9O10), 1027.44; found 1028.64 (M+Na)+; found 1026.71 (MH) . HPLC, tR = 9.68 min (purity, 92%). HPLC Method B.
Example 4: Synthesis of ,V-(6’-(( 14-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin-4-yl)oxy)- 3,6,9, 12-tetraoxatetra-decyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide {Compound 1.004).
Figure imgf000069_0001
[0203] V-(6'-(( 14-hydroxy-3,6,9, 12-tctraoxatctradccyl)oxy)-2-mcthyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in Example A.
Step I, V-(6'-((14-iodo-3,6,9,12-tctraoxatctradccyl)oxy)-2-mcthyl-5'-morpholino-[3,3'- bipyridm]-5-yl)-3-(trifluoromethyl)benzamide
[0204] To a solution of /V-(6'-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (1.1 g, 1.62 mmol) in DCM (30 mL) was added Imidazole (154 mg, 2.27 mmol), PPh3 (595 mg, 0.98 mmol), and Iodine (576 mg, 2.27 mmol) under argon flow at 0 °C, stirred at rt for 4 h. The solvent was evaporated and the crude was dissolved in DCM, filtered off the solids and the filtrate was concentrated and purified on silica gel (40 g) using a gradient of methanol in DCM (0 to 10 %) as eluant to afford the title compound (700 mg, 55%) as a yellow oil. Rf = 0.57 (5% MeOH in DCM). 'H NMR (400 MHz, CDCh) d 8.63 (d, J = 2.6 Hz, 1H), 8.20 - 8.05 (m, 3H), 7.98 - 7.81 (m, 2H), 7.76 (d, J = 2.1 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 4.64 - 4.54 (m, 2H), 4.02 - 3.84 (m, 6H), 3.79 - 3.70 (m, 4H), 3.66 (d, J = 1.7 Hz, 10H), 3.30 - 3.21 (m, 2H), 3.22 - 3.13 (m, 4H), 2.51 (s, 3H). LRMS, m/z, calculated (for (C33H40F3IN4O7), 788.189; found 811.22 (M+Na)+, 787.17 (M-H) . Step II, Compound 1.004
[0205] To a stirred mixture of 3-(4-hydroxy- 1 -oxoisoindolin-2-yl)piperidine-2,6-dione (40 mg, 0.154 mmol) and ,V-(6'-(( 14-iodo-3,6,9, 12-tctraoxatctradccyl)oxy)-2-mcthyl-5'-morpholino- [3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide in DMF (1 mL) was added potassium bicarbonate (23.1 mg), resultant reaction mixture was stirred at rt for 16 h, at 70 °C for 2 h, HPLC showed the presence of lots of iodide, additional amount of the phenol (40 mg) and KHCO3 (23 mg) was added, heated at 70 °C for 5 h, diluted with water (~5 mL), extracted with ethyl acetate (3x 10 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was purified on reverse phase column (Isco) using a gradient of acetonitrile in water (0 to 100%) to afford the title compound (1.004) (24 mg, 17%) as a white solid. 1 H NMR spectra showed a mixture of two compounds ( N and O-alkylated). To a stirred solution of product mixture and BOC2O (5.9 mg, 0.034 mmol) in pyridine (0.3 mL) was added spec of DMAP, stirred at rt for 1 h, diluted with methanol, and concentrated. The residue was purified on 42 g C- 18 silica gel cartridge using a gradient of acetonitrile in water (10 to 100%) as eluant to afford the title compound (1.004) (10.5 mg, 43.8%) as a white solid. 'H NMR (400 MHz, CDCb) d 8.82 (s, 1H), 8.77 (s, 1H), 8.67 (s, 1H), 8.25 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 2.2 Hz, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.38 - 7.33 (m, 2H), 7.02 - 6.97 (m, 2H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.61 - 4.51 (m, 2H), 4.40 (d, J = 16.6 Hz, 1H), 4.29 (d, J = 16.6 Hz, 1H), 4.25 - 4.18 (m, 2H), 3.93 - 3.81 (m, 8H), 3.75 - 3.58 (m, 12H), 3.16 - 3.06 (m, 4H), 2.92 - 2.84 (m, 1H), 2.82 - 2.69 (m, 1H), 2.48 (s, 3H), 2.41 - 2.25 (m, 1H), 2.22 - 2.12 (m, 1H). 19F NMR (376 MHz, CDCh) d -63.05 (s). LRMS, m/z, calculated (for C46H51F3N6O11), 920.36, found, 919.41 (M-H) . HPLC, tR = 8.9 min (purity, >99.9%). HPLC method A.
Example 5: Synthesis of /V-(6'-(2-(2-(2-(( 1 -(7-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin- 5-yl)oxy)heptyl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.005)
Figure imgf000070_0001
[0206] /V-(2-mcthyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 -yloxyethoxy)ethoxy)ethoxy)-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in
WO2018/200981. [0207] Title compound (22.8 mg, 45.6% yield) was prepared as a white solid from N-( 2- methyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn-l-yloxyethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5- yl)-3-(trifluoromethyl)benzamide (30.6 mg, 0.0487 mmol) and 3-(5-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2,6-dione (Example D) (19.7 mg, 0.0493 mmol) as described in Example 2. Rf = 0.45 (10% MeOH in DCM). ¾ NMR (400 MHz, CDCb) d 8.79 (d, J = 2.5 Hz, 1H), 8.48 (s, 1H), 8.20 (s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 8.07 (s, 1H), 8.01 (d, J = 2.5 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 2.1 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.02 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 8.7, 1.9 Hz, 1H), 6.90 (s, 1H), 5.16 (dd, J = 13.2, 5.0 Hz, 1H), 4.66 (s, 2H), 4.62 - 4.56 (m, 2H), 4.41 (d, J = 15.9 Hz, 1H), 4.35 - 4.24 (m, 3H), 3.99 (t, J = 6.3 Hz, 2H), 3.92 - 3.87 (m, 6H), 3.73 - 3.70 (m, 2H), 3.66 - 3.62 (m, 6H), 3.15 - 3.13 (m, 4H), 2.94 - 2.87 (m, 1H), 2.86 - 2.75 (m, 1H), 2.50 (s, 3H), 2.38 - 2.27 (m,
1H), 2.23 - 2.16 (m, 1H), 1.94 - 1.84 (m, 2H), 1.83 - 1.73 (m, 2H), 1.48 - 1.33 (m, 6H). 19F NMR (376 MHz, CDCb) d -63.10 (s). LRMS, m/z, calculated (for C52H60F3N9O10), 1027.44; found 1028.64 (M+H)+; found 1050.58 (M+Na)+; found 1026.64 (M-H) . HPLC, tR = 9.4 min (purity, 96%). HPLC Method B. Example 6: Synthesis of /V-(6'-(2-(2-(2-(( 1 -(7-((2-(2,6-dioxopipcridin-3-yl)-3-oxoisoindolin- 4-yl)oxy)heptyl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (Compound 1.006)
Figure imgf000071_0001
[0208] /V-(2-mcthyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 -yloxyethoxy)ethoxy)ethoxy)-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in
WO2018/200981.
[0209] Title compound (15.9 mg, 20.6% yield) was prepared as a white solid from N-{ 2- methyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn-l-yloxyethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5- yl)-3-(trifluoromethyl)benzamide (47.1 mg, 0.0794 mmol) and 3-(7-((7-azidoheptyl)oxy)-l- oxoisoindolin-2-yl)piperidine-2,6-dione {Example E) (27.2 mg, 0.0681 mmol) as described in Example 2 followed by reverse phase column. Rf = 0.43 (10% MeOH in DCM). ¾ NMR (400 MHz, CDCh) d 9.49 (s, 1H), 8.80 (d, J = 2.1 Hz, 1H), 8.63 (s, 1H), 8.26 (s, 1H), 8.18 (d, J = 2.2 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.59 (s, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.03 (d, J = 1.9 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 5.09 (dd, J = 13.3, 5.1 Hz, 1H), 4.63 (s, 2H), 4.59 - 4.53 (m, 2H), 4.40 (d, J = 16.3 Hz, 1H), 4.27 (d, J = 16.8 Hz, 1H), 4.23 (t, J = 7.5 Hz, 2H), 3.97 (t, J = 6.5 Hz, 2H), 3.91 - 3.85 (m, 6H), 3.71 - 3.69 (m, 2H), 3.64 - 3.62 (m, 6H), 3.12 (s, 4H), 2.89 - 2.83 (m, 1H), 2.81 - 2.72 (m, 1H), 2.48 (s, 3H), 2.38 - 2.27 (m, 1H), 2.17 - 2.11 (m, 1H), 1.84 - 1.77 (m,
2H), 1.73 - 1.66 (m, 2H), 1.35 - 1.31 (m, 2H), 1.31 - 1.19 (m, 4H). 19F NMR (376 MHz,
CDCh) d -63.10 (s). LRMS, m/z, calculated (for C52H60F3N9O10), 1027.44; found 1028.71 (M+H)+; found 1050.58 (M+Na)+; 1066.57 (M+K)+; 1026.71 (M-H) . HPLC, tR = 9.49 min (purity, 98%). HPLC Method B. Example 7: Synthesis of /V-(6'-(( 14-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin-4-yl)oxy)- 3,6,9, 12-7V-(6'-((14-((2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)oxy)-3,6,9, 12- tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide {Compound 1.007).
Figure imgf000073_0001
[0210] A-(6'-((14-hydroxy-3,6,9,12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in Example A. [0211] To a solution of 3-(6-hydroxy-l-oxoisoindolin-2-yl)piperidine-2,6-dione (45 mg, 0.173 mmol) in THF (1 mL) was added PPh3 (100 mg, 0.432 mmol). The mixture was stirred at rt for 15 min followed by addition of diisopropylazodicarboxylate (75 mg, 0.207 mmol) at 0 °C. After 30 min at 0 °C, a solution of /V-(6'-(( 14-hydroxy-3,6,9, 12-tctraoxatctradccyl)oxy)-2-mcthyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (141 mg, 0.207 mmol) in THF (0.5 mL) was added to the reaction mixture and allowed to warm to rt and stirred for 16 h. The solvent was evaporated and the residue was purified on silica gel (24 g) using gradient of methanol in ethyl acetate (0 to 20%) followed by reverse phase column to provide a mixture of the desired compound (25 mg) along with an impurity. This impurity was separated by chemical transformation. To a solution of the mixture (21 mg, 0.0228 mmol) and BOC2O (5.16 mg, 0.0296 mmol) in pyridine (0.3 mL) was added spec of DMAP, stirred at rt for 1 h, diluted with methanol, and concentrated. The residue was purified on 42 g C- 18 cartridge using a gradient of acetonitrile in water (10 to 100%) as eluant to afford the title compound (1.007) (1 1.4 mg, 54.3%) as colorless oil. Rf = 0.43 (10% MeOH in DCM). *H NMR (400 MHz, CDCb) d 8.71 8.63 (m, 2H), 8.46 (s, 1H), 8.21 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 8.09 8.05 (m, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 1.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.25 7.22 (m, 1H), 7.12 (dd, J = 8.3, 2.3 Hz, 1H), 7.03 (d, J = 1.9 Hz, 1H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.61 - 4.54 (m, 2H), 4.36 (d, J = 15.9 Hz, 1H), 4.25 (d, J = 15.7 Hz, 1H), 4.15 - 4.05 (m, 2H), 3.95 - 3.78 (m, 8H), 3.76 - 3.58 (m, 12H), 3.18 - 3.08 (m, 4H), 2.93 - 2.84 (m, 1H), 2.84 - 2.72 (m, 1H), 2.48 (s, 3H), 2.40 - 2.25 (m, 2H), 2.23 - 2.12 (m, 1H). LRMS, m/z, calculated (for C46H51F3N6O11), 920.36, found, 921.65 (M+H)+; 943.59 (M+Na)+. HPLC, tR = 8.8 min (purity, >99.9%). HPLC method A.
Example 8: Synthesis of /V-(6'-(( 14-((2-(2,6-dioxopipcridin-3-yl )- 1 -oxoisoindolin-5-yl)oxy)- 3,6,9, 12-tetraoxatetradecyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.008 )
Figure imgf000074_0001
[0212] The title compound, 1.008(6.8 mg, 3.9% yield) was prepared as a pale yellow oil from ,V-(6'-(( 14-iodo-3,6,9, 12-tctraoxatctradccyl)oxy)-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)- 3-(trifluoromethyl)benzamide (150.0 mg, 0.190 mmol) and 3-(5-hydroxy-l-oxoisoindolin-2- yl)piperidine-2,6-dione (120.2 mg, 0.393 mmol) as described for Example 4. Rf = 0.44 (10% MeOH in DCM). lR NMR (400 MHz, CDCh) d 8.77 (d, J = 2.3 Hz, 1H), 8.71 (s, 1H), 8.24 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 2.1 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H), 7.67 - 7.61 (m, 2H), 7.00 (d, J = 1.8 Hz, 1H), 6.93 (m, 2H), 5.09 (dd, J = 13.3, 5.1 Hz, 1H), 4.57 - 4.55 (m, 2H), 4.37 (d, J = 15.9 Hz, 1H), 4.25 (d, J = 15.9 Hz, 1H), 4.18 - 4.15 (m, 2H), 3.91 - 3.85 (m, 8H), 3.67 - 3.62 (m, 12H), 3.12 - 3.10 (m, 4H), 2.90 - 2.84 (m, 1H), 2.78 - 2.69 (m, 1H), 2.48 (s, 3H), 2.35 - 2.24 (m, 1H), 2.19 - 2.11 (m, 1H). 19F NMR (376 MHz, CDCb) d -63.04 (s, J = 10.2 Hz). LRMS, m/z, calculated (for C46H51F3N6O11), 920.36; found 921.65 (M+H)+; found, 943.65 (M+Na)+; 919.65 (M-H) . HPLC, tR = 9.16 min (purity, 99%). HPLC Method B.
Example 9: Synthesis of ,V-(6’-(2-(4-( 2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.009).
Figure imgf000075_0001
Step I, tert- butyl 4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin] -6-yl)oxy)ethyl)-piperazine-l -carboxylate :
[0213] To a stirred solution of /er/-butyl 4-(2-hydroxyethyl)piperazine-l-carboxylate (1 g,
4.34 mmol) in 1 ,4-dioxane (6.5 mL) was added 60% sodium hydride (174 mg, 4.34 mmol) in two portions, after stirred at rt for 30 min, /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]- 5-yl)-3-(trifluoromethyl)benzamide (400 mg, 0.869 mmol) was added in one portion, it was placed on pre-heated oil bath at 82 °C for 1.5 h, The reaction mixture was cooled to rt, poured into water (5 mL), neutralized with 10% citric acid (pH 7-8), extracted with ethyl acetate (3 x 15 mL), combined extracts were washed with water, brine, dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge using a gradient of methanol in ethyl acetate (0% to 20%) to afford the title compound /er/-butyl 4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)-piperazine-l-carboxylate (320 mg, 54.9%) as a pale yellow solid. Rf = 0.37 (10% MeOH in EtOAc). !H NMR (400 MHz, CDCh) d 8.62 (d, J = 2.5 Hz, 1H), 8.17 - 8.13 (m, 2H), 8.10 (d, J = 7.8 Hz, 1H), 8.05 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 2.1 Hz, 1H), 4.56 (t, J = 5.8 Hz, 2H), 3.93 - 3.86 (m, 4H), 3.47 - 3.40 (m, 4H), 3.20 - 3.12 (m, 4H), 2.85 (t, J = 5.8 Hz, 2H), 2.57 - 2.52 (m, 4H), 2.51 (s, 3H), 1.46 (s, 9H). LRMS, m/z, calculated (for C34H41F3N6O5), 670.31, found, 671.63 M+H)+. Step II, l-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]- 6-yl)oxy)ethyl)piperazine-l,4-diium chloride:
[0214] To a stirred solution of /er/-butyl 4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (315 mg, 0.470 mmol) in MeOH (1.5 mL) was added a solution of HC1 (1.53 mL, 6.11 mmol, 4N) in dioxane, after stirred for 1 h, concentrated and dried to afford the title compound (300 mg, 99.3%) as a pale yellow solid. !H NMR (400 MHz, DMSO) d 1 1.63 (s, 1H), 9.78 (s, 2H), 9.25 (d, J = 2.0 Hz, 1H), 8.73 (s, 1H), 8.48 - 8.40 (m, 2H), 8.03 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 1.9 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 1.9 Hz, 1H), 4.86 - 4.75 (m, 2H), 3.81 - 3.65 (m, 6H), 3.58 - 3.38 (m, 6H), 3.13 - 3.04 (m, 4H), 2.66 (s, 3H). LRMS, m/z, calculated (for free base, C29H33F3N6O3), 570.26, found, 571.49 (M+H)+.
Step III, Compound 1.009:
[0215] To a mixture of l-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l ,4-diium chloride and 2-(2,6-dioxopiperidin-3-yl)-5- fluoroisoindoline-l ,3-dione in DMSO (500 pL) was added triethyl amine (35 pL, 0.247 mmol) and heated in a sealed tube at 80 °C for 20 h, cooled to rt, diluted with water, extracted with ethyl acetate (3 x 10 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was absorbed on the Celite, purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 20%) as eluant to afford the title compound (1.009) (10 mg, 14.7%) as a yellow solid. Rf = 0.37 (10% MeOH in DCM). !H NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.25 (s, 1H), 8.17 (d, J = 2.5 Hz, 1H), 8.15 (s, 1H), 8.12 - 8.06 (m, 2H), 7.84 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.71 - 7.62 (m, 2H), 7.28 (d, J = 2.2 Hz, 1H), 7.10 - 7.03 (m, 2H), 4.94 (dd, J = 12.3, 5.3 Hz, 1H), 4.61 (t, J = 5.8 Hz, 2H), 3.94 - 3.84 (m, 4H), 3.48 - 3.39 (m, 4H), 3.21 - 3.11 (m, 4H), 2.95 - 2.68 (m, 9H), 2.51 (s, 3H), 2.18 - 2.07 (m,lH). 19F NMR (376 MHz, CDCh) d -63.14 (s). LRMS, m/z, calculated (for C42H41F3N8O7), 826.31 , found, 849.78 (M+Na)+. HPLC, tR = 5.74 min (purity, 98.8%). HPLC method C. Example 10: Synthesis of N-(6'-(( 1 -(( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5-yl)- lH-l,2,3-triazol-4-yl)methyl)piperidin-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)- 3-(trifluoro-methyl)benzamide ( Compound 1.010).
Figure imgf000077_0001
Step I, A'-(2-mcthvl-5'-morpholino-6'-((l-(prop-2-vn-l-yl)pipcridin-4-vl)oxy)-[3,3'- bipyridm]-5-yl)-3-(trifluoromethyl)benzamide: [0216] To a stirred solution of l-(prop-2-yn-l-yl)piperidin-4-ol (453 mg, 3.26 mmol) in 1 ,4- dioxane (5.0 mL) was added 60% sodium hydride (130 mg, 3.26 mmol), after stirred at rt for 30 min, /V-(6'-fliioro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromcthyl)bcnzamidc (300 mg, 0.652 mmol) was added in one portion, it was placed on pre-heated oil bath at 82 °C for 1.5 h, The reaction mixture was cooled to rt, poured into water (5 mL), neutralized with 10% citric acid (pH 7-8), extracted with ethyl acetate (3 x 15 mL), combined extracts were washed with water, brine, dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge using a gradient of methanol in methylene chloride (0% to 30%) as eluant to afford the title compound (240 mg, 63.5%) as a yellow solid. RT = 0.4 (10% MeOH in CH2CI2). !H NMR (400 MHz, CDCb) d 8.61 (d, J = 2.5 Hz, 1H), 8.17 - 8.12 (m, 2H), 8.11 - 8.03 (m, 2H), 7.84 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 5.37 - 5.24 (m, 1H), 3.93 - 3.84 (m, 4H), 3.39 (d, J = 2.4 Hz, 2H), 3.21 - 3.12 (m, 4H), 2.90 - 2.77 (m, 2H), 2.65 - 2.54 (m, 2H), 2.51 (s, 3H), 2.27 (t, J = 2.4 Hz, 1H), 2.21 - 2.09 (m, 2H), 2.03 - 1.91 (m, 2H). LRMS, m/z, calculated (for C31H32F3N5O3), 579.25, found, 602.39 (M+Na)+.
Step II, V-(6'-((l-((l-(2-(2,6-dioxopipcridin-3-yl)-l,3-dioxoisoindolin-5-yl)-l H-l ,2,3-triazol-
4-yl)methyl)piperidin-4-yl)oxy)-2-methyl-5'-morpholmo-[3,3'-bipyridin]-5-yl)-3-
(trifluoromethyl)benzamide:
[0217] To a mixture of [3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (61 mg, 0.105 mmol), 5-azido-2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (40.9 mg, 0.137 mmol, (described in Example 11) in THF (2 mL) was added a solution of copper sulfate pentahydrate (5.26 mg, 0.021 mmol) in water (0.4 mL) and sodium ascorbate (4.17 mg, 0.021 mmol) in water (0.4 mL), stirred over weekend, additional amount of THF (2 mL) was added, stirred for 1 h, concentrated on rotavap, absorbed on the Celite and purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 40%) as eluant to afford the title compound 1.010 (67 mg, 72.4%) as a pale yellow solid. Rf = 0.2 (10% methanol in DCM). !H NMR (400 MHz, CDCb) d 8.61 (d, J = 2.4 Hz, 1H), 8.51 (s, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.34 (s, 1H), 8.22 (s, 1H), 8.17 - 8.12 (m, 2H), 8.09 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 5.35 - 5.27 (m, 1H), 5.04 (dd,
J = 12.3, 5.6 Hz, 1H), 4.01 (d, J = 13.5 Hz, 1H), 3.92 - 3.81 (m, 4H), 3.68 (d, J = 13.1 Hz, 1H), 3.20 - 3.09 (m, 4H), 2.95 - 2.81 (m, 3H), 2.81 - 2.72 (m, 2H), 2.71 - 2.56 (m, 2H), 2.49 (s, 3H), 2.25 - 2.01 (m, 4H), 1.98 - 1.90 (m, 1H). 19F NMR (376 MHz, CDCh) d -63.12 (s). LRMS, m/z, calculated (for C44H41F3N10O7), 878.31; found, 879.56 (M+H)+; 901.62 (M-H) . HPLC, tR = 5.78 min (purity, 99%). HPLC Method C.
Example 11: Synthesis of / V- ( 6’ - ( 2 - ( ( 4 - ( ( ( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)- 1 H- 1 ,2,3-triazol-4-yl)methoxy)methyl)benzyl)oxy)ethoxy)-2-methyl-5'-morpholino-[3 ,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide ( Compound 1.011)
Figure imgf000079_0001
Step I, Synthesis of 5-azido-2-(2,6-dioxopiperidm-3-yl)isomdolme-l,3-dione
[0218] To a solution of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (530 mg, 1.92 mmol) in dry DMSO (7 mL) was added sodium azide (162 mg, 2.49 mmol) and the mixture was stirred at rt overnight. A crude NMR showed remaining SM. Additional sodium azide (162 mg, 2.49 mmol) was added and the reaction mixture was heated to 70 °C for 30 min. The reaction mixture was poured into water (200 mL) and then the resultant precipitate was collected by filtration and dried under vacuum overnight to afford the title compound (450 mg, 78%). 1 H NMR (400 MHz, DMSO) 5 11.14 (s, 1H), 7.93 (d, .7= 8.1 Hz, 1H), 7.62 (d, J= 1.7 Hz, 1H), 7.55 (dd, J= 8.1, 2.1 Hz, 1H), 5.15 (dd, J= 12.8, 5.4 Hz, 1H), 2.95 - 2.83 (m, 1H), 2.68 - 2.54
(m, 2H), 2.10 - 2.01 (m, 1H). LRMS, m/z, calculated, 299.065; no ionization observed.
Step II, Compound 1.011
[0219] To a solution of /V-(2-mcthyl-5'-morpholino-6'-(2-((4-((prop-2-yn- 1 - yloxy)methyl)benzyl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide
(described in Step III of Example 23) (55 mg, 0.08 mmol) in THF (0.75 mL) was added 5- azido-2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (24.9 mg, 0.08 mmol) in THF (0.75 mL). A solution of copper sulfate pentahydrate (4.16 mg, 0.018 mmol) and sodium ascorbate (3.30 mg, 0.017 mmol) in water (0.75 mL) was added and the reaction mixture was stirred at rt overnight. The mixture was diluted with DCM (5 mL) and the aqueous phase was extracted with DCM (2 x 5 mL). The combined organic extracts were dried over anhydrous a2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-30%) to afford the title compound 1.011 (30.7 mg, 38%) as a yellow solid. *H NMR (400 MHz, CDCh) d 8.62 (d, J= 2.5 Hz, 1H), 8.30 (s, 1H), 8.26 (dd, J = 8.1, 1.9 Hz, 1H), 8.21 (d, J= 1.6 Hz, 1H), 8.16 (s, 1H), 8.14 (s, 1H), 8.12 (d, .7= 2.5 Hz, 1H), 8.09 (d, J= 6.0 Hz, 2H), 8.03 (d, J= 8.1 Hz, 1H), 7.83 (d, J= 7.8 Hz, 1H), 7.74 (d, J= 2.0 Hz, 1H), 7.65 (t, .7= 7.9 Hz, 1H), 7.39 - 7.32 (m, 4H), 7.05 (d, 7 = 2.0 Hz, 1H), 5.01 (dd, J= 12.5, 5.3 Hz, 1H), 4.78 (s, 2H), 4.66 (s, 2H), 4.63 (m, 4H), 3.93 - 3.89 (m, 2H), 3.85 - 3.80 (m, 4H),
3.14 (d , J= 4.3 Hz, 4H), 2.96 - 2.70 (m, 3H), 2.49 (s, 3H), 2.23 - 2.15 (m, 1H). LRMS, m/z, calculated, 959.321; found, 960.49 (M+H)+. HPLC, tR = 6.87 (purity >98%). HPLC method A.
Example 12: Synthesis of /V-(6'-((4-( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5-yl)- 1 H- 1 ,2,3 -triazol-4-yl)benzyl)oxy)-2-methyl-5 '-morpholino- [3,3 '-bipyridin] -5-yl)-3 - (trifluoromethyl)benzamide ( Compound 1.012)
Figure imgf000080_0001
Step I, V-(6'-((4-cthvnvlbcnzvl)oxy)-2-mcthvl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide:
[0220] To a stirred solution of (4-ethynylphenyl)methanol (215 mg, 1.63 mmol) in 1 ,4- dioxane (2.3 mL) was added 60% sodium hydride 65 mg, 1.63 mmol), after stirred for 30 min, /V-(6'-fliioro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromcthyl)bcnzamidc (150 mg, 0.326 mmol) was added in one portion, it was placed on pre-heated oil bath at 82 °C, stirred for 45 min under argon. The reaction mixture was cooled to rt, poured into water (10 mL), extracted with ethyl acetate (2 x 20 mL), combined extracts were washed with brine, and concentrated. The residue was absorbed on the Celite, purified on 25 g silica column using methanol in methylene chloride (0 to 10%) as eluant to afford the title compound (150 mg, 80.4%) as a yellow solid. Rf = 0.3 (5% MeOH in DCM). ¾ NMR (400 MHz, CDCh) d 8.63 - 8.55 (m, 2H), 8.18 - 8.13 (m, 2H), 8.09 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 1.9 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.43 (dd, J = 1 1.6, 5.7 Hz, 2H), 7.07 (d, J = 1.9 Hz, 1H), 5.50 (s, 2H), 3.91 - 3.82 (m, 4H), 3.18 - 3.11 (m, 4H), 3.10 (s, 1H),
2.48 (s, 3H). LRMS, m/z, calculated (for C32H27F3N4O3), 572.20; found, 573.32 (M+H)+.
Step II, Compound 1.012:
[0221] To a stirred suspension of 7V-(6'-((4-ethynylbenzyl)oxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (60 mg, 0.105 mmol), 5-azido-2-(2,6- dioxopiperidin-3-yl)isoindoline-l,3-dione (described in Example 11) (40.8 mg, 0.136 mmol) in THF (3.5 mL) was added a solution of copper sulfate pentahydrate (5.23 mg, 0.021 mmol) in water (0.4 mL) and sodium ascorbate (4.15 mg, 0.021 mmol) in water (0.4 mL), stirred over weekend, diluted with DCM and water, stirred for 30 min, organic solution was separated, aqueous solution was extracted with ethyl acetate (20 mL), combined organic solution was dried (Na2S04), and concentrated. The residue was absorbed on the Celite and purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 10%) as eluant to afford the title compound 1.012 (40 mg, 43%) as a white solid. Rf = 0.28 (5% methanol in DCM). !H NMR (400 MHz, DMSO) 5 1 1.16 (s, 1H), 10.67 (s, 1H), 9.65 (s, 1H), 8.86 (s, 1H), 8.54 - 8.48 (m, 2H), 8.32 (s, 1H), 8.28 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.06 (d, J = 2.2 Hz, 1H), 8.02 - 7.96 (m, 3H), 7.84 (d, J = 2.0 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 2.0 Hz, 1H), 5.51 (s, 2H), 5.22 (dd, J = 12.8, 5.3 Hz, 1H), 3.78 - 3.70 (m, 4H), 3.16 - 3.09 (m, 4H), 2.96 - 2.84 (m, 1H), 2.69 - 2.52 (m, 2H), 2.45 (s, 3H), 2.15 - 2.04 (m, 1H). 19F NMR (376 MHz, DMSO) d -61.48 (s). LRMS, m/z, calculated (for C45H36F3N9O7), 871.27; found, 872.57 (M+H)+. HPLC, tR = 6.97 min (purity, 98%). HPLC method C.
Example 13: Synthesis of N-(6'-(2-((l-((l-(2-(2,6-dioxopiperidin-4-yl)-l,3-dioxoisoindolin-5- yl)- 1 H- 1 ,2,3-triazol-4-yl)methyl)piperidin-4-yl)oxy)ethoxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide. ( Compound 1.013).
Figure imgf000082_0001
[0222] To a solution of /V-(2-mcthyl-5'-morpholino-6'-(2-(( 1 -(prop-2-yn- 1 -yl)pipcridin-4- yl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (prepared using a method similar to Step I in Example 22) (55 mg, 0.09 mmol) in THF (0.75 ml) was added 5-azido-2- (2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (described m Example 11) (26.4 mg, 0.09 mmol) in THF (0.75 mL). A solution of copper sulfate pentahydrate (4.40 mg, 0.018 mmol) and sodium ascorbate (3.49 mg, 0.018 mmol) in water (0.75 mL) was added and the reaction mixture was stirred at rt overnight. The mixture was diluted with DCM (5 mL) and the aqueous phase was extracted with DCM (2 x 5 mL). The combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-30%) to afford the title compound 1.013 (20 mg, 25%) as a yellow solid. ¾ NMR (400 MHz, CDCh) d 8.65 (s, 1H), 8.39 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.18 - 8.15 (m, 4H), 8.10 (d, J= 7.8 Hz, 1H), 8.04 (d , J= 8.1 Hz, 1H), 7.83 (d , J= 7.4 Hz, 1H), 7.75 (s, 1H), 7.65 (t, J= 7.6 Hz, 1H), 7.04 (s, 1H), 5.03 (s, 1H), 4.59 (s, 2H), 3.89 (s, 7H), 3.69 (d, J= 12.7 Hz, 1H), 3.48 (s, 1H), 3.17 (s, 4H), 2.87 - 2.78 (m, 5H), 2.50 (s, 3H), 2.39 (s, 2H), 2.18 (s, 1H), 1.94 (s, 3H) *missing exchangeable NH. LRMS, m/z, calculated, 922.337; found, 923.71 (M+H)+. HPLC, tR = 5.82 (purity >94%). HPLC method A.
Example 14: Synthesis of /V-(6'-(2-( 1 -( 2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5-yl)- 1 H- 1 ,2,3 -triazol-4-yl)ethoxy) -2-m ethyl-5 '-morpholino- [3 ,3 '-bipyridin] -5-yl)-3 - (trifluoromethyl)benzamide ( Compound 1.014).
Figure imgf000082_0002
[0223] /V-(6'-(but-3-yn- l -yloxy)-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide was prepared as described in WO2018/200981. [0224] The title compound, 1.014, (30 mg, 38%) was prepared as a yellow solid from of N-(6'- (but-3-yn-l-yloxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (50 mg, 0.098 mmol) and 5-azido-2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (29.3 mg, 0.098 mmol) (described in Example 11). !H NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.29 (dd, J = 8.2, 1.9 Hz, 1H), 8.21 (d, J = 1.6 Hz, 1H), 8.19 (s, 1H), 8.16 - 8.13 (m, 2H), 8.11 -
8.03 (m, 3H), 7.97 (s, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 5.02 (dd, J = 12.6, 5.4 Hz, 1H), 4.80 (t, J = 6.4 Hz, 2H), 3.86 3.79 (m, 4H), 3.42 (t, J = 6.2 Hz, 2H), 3.13 - 3.07 (m, 4H), 2.97 - 2.74 (m, 3H), 2.51 (s, 3H),
2.23 - 2.19 (m, 1H). LRMS, m/z, calculated, 809.253; found, 810.22 (M+H)+. HPLC, tR = 6.39 (purity, 99%). HPLC method A.
Example 15: Synthesis of /V-(6'-(2-( 1 -( 2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperidin-4-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.015 )
Figure imgf000083_0001
Step I, tert- butyl 4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperidine-l-carboxylate
[0225] To a stirred solution of /er/-butyl 4-(2-hydroxyethyl)piperidine-l-carboxylate (498 mg, 2.17 mmol) in 1 ,4-dioxane (3.5 mL) was added 60% sodium hydride (87 mg, 2.17 mmol) in two portions, after stirred at rt for 30 min, /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5- yl)-3-(trifluoromethyl)benzamide (400 mg, 0.869 mmol) was added in one portion, it was placed on pre -heated oil bath at 82 °C for 1.5 h, The reaction mixture was cooled to rt, poured into water (5 mL), extracted with ethyl acetate (3 x 15 mL), combined extracts were washed with water, brine, dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge using a gradient of ethyl acetate in hexanes (0% to 100%) as eluant to afford the title compound (240 mg, 82.5%) as a white solid. Rf = 0.2 (EtOAc). !H NMR (400 MHz, CDCh) d 8.63 (d, J = 2.5 Hz, 1H), 8.62 - 8.48 (m, 1H), 8.20 - 8.14 (m, 2H), 8.12 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 1.4 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 1.9 Hz, 1H), 4.46 (t, J =
6.7 Hz, 2H), 4.15 - 4.05 (m, 2H), 3.92 - 3.83 (m, 4H), 3.18 - 3.09 (m, 4H), 2.79 - 2.60 (m, 2H), 2.49 (s, 3H), 1.86 - 1.59 (m, 5H), 1.46 (s, 9H), 1.23 - 1.14 (m, 2H). LRMS, m/z, calculated (for C35H42F3N5O5), 669.31 , found, 692.54 M+Na)+; 668.59 (M-H) .
Step II, 4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]- 6-yl)oxy)-ethyl)piperidin-l-ium chloride:
[0226] To a stirred a solution of /er/-butyl 4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperidine-l-carboxylate (240 mg, 0.358 mmol) in dry methanol (1 mL) was added 4N HC1 in dioxane (0.9 mL, 3.58 mmol), stirred at rt for 1.5 h, concentrated, triturated with ether, dried to afford the title compound (226 mg, quant) as a pale yellow solid. This material was used as such in the next step. 1 H NMR (400 MHz, DMSO) 5 1 1.65 (s, 1H), 9.25 (d, J = 2.2 Hz, 1H), 8.97 - 8.89 (m, 1H), 8.75 (s, 1H), 8.73 - 8.64 (m, 1H), 8.47 - 8.42 (m, 2H), 8.03 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 2.1 Hz, 1H), 7.83 (t, J =
7.7 Hz, 1H), 7.35 (d, J = 2.1 Hz, 1H), 4.45 - 4.35 (m, 2H), 3.79 - 3.70 (m, 4H), 3.28 - 3.20 (m, 2H), 3.11 - 3.04 (m, 4H), 2.89 - 2.77 (m, 2H), 2.66 (s, 3H), 1.93 - 1.83 (m, 2H), 1.77 - 1.69 (m, 3H), 1.49 - 1.35 (m, 2H). LRMS, m/z, calculated (for C30H34F3N5O3), 569.26, found, 570.31 (M+H)+.
Step III, Compound 1.015:
[0227] 4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethyl)piperidin-l-ium chloride (100 mg, 0.165 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5- fluoroisoindoline-l ,3-dione (46 mg, 0.165 mmol) in DMSO (2 mL) was added triethyl amine (138 pL, 0.988 mmol) and heated in a sealed tube at 80 °C for 20 h, additional amount of 2-(2,6- dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (20 mg) was added, heated for 2 h, cooled to rt, diluted with water, extracted with ethyl acetate (3 x 10 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was purified on 25 g S1O2 cartridge using a gradient of methanol in ethyl acetate (0 to 10%) as eluant to afford the title compound 1.015 (45 mg, 35.2%) as a yellow solid. Rf = 0.15 (EtOAc). ¾ NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.19 - 8.13 (m, 3H), 8.12 - 8.06 (m, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.79 (d, J
= 2.0 Hz, 1H), 7.70 - 7.63 (m, 2H), 7.28 (d, J = 2.2 Hz, 1H), 7.09 - 7.02 (m, 2H), 4.94 (dd, J = 12.4, 5.2 Hz, 1H), 4.50 (t, J = 6.5 Hz, 2H), 3.98 (d, J = 13.0 Hz, 2H), 3.94 - 3.85 (m, 4H), 3.18 3.10 (m, 4H), 2.99 (t, J = 11.6 Hz, 2H), 2.93 - 2.69 (m, 3H), 2.51 (s, 3H), 2.16 - 2.07 (m, 1H), 1.95 (d, J = 12.2 Hz, 2H), 1.90 - 1.81 (m, 3H), 1.50 - 1.37 (m, 2H). LRMS, m/z, calculated (for C43H42F3N7O7), 825.31 , found, 826.67 (M+H)+. HPLC, tR = 7.02 min (purity, >99%). HPLC
Method C.
Example 16: Synthesis of /V-(6'-(( 1 -(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperidin-4-yl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.016).
Figure imgf000085_0001
[0228] The title compound 1.016 (21 mg) was prepared as described for Compound 1.009 ( Example 9) from ,V-boc-4-hydroxypipcridinc (328 mg).
Step I, tert- buty 4-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)piperidine-l-carboxylate. [0229] Ή NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.15 (d, J = 2.3 Hz, 2H), 8.09
(d, J = 7.7 Hz, 1H), 7.99 (s, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 2.1 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 5.39 (dt, J = 7.5, 3.7 Hz, 1H), 3.92 - 3.85 (m, 4H), 3.70 (d,
J = 8.3 Hz, 2H), 3.48 - 3.36 (m, 2H), 3.15 (d, J = 4.3 Hz, 4H), 2.52 (s, 3H), 2.09 - 1.99 (m, 2H),
1.89 - 1.77 (m, 2H), 1.49 (s, 9H). ). LRMS, m/z, calculated, 641.282; doesn’t ionize correctly.
Step II, N-(2-methyl-5'-morpholmo-6'-(piperidm-4-yloxy)-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide dihydrochloride
[0230] LRMS, m/z, calculated, 541.230; found, 542.40 (M+H)+.
Step III, Compound 1.016.
[0231] Ή NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.19 (dd, J = 8.1 , 4.6 Hz, 3H), 8.11 (d, J = 7.4 Hz, 2H), 7.84 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.71 (d, J = 8.5 Hz,
1H), 7.67 (t, J = 7.9 Hz, 1H), 7.36 - 7.31 (m, 1H), 7.1 1 (dd, J = 8.6, 2.3 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 5.54 - 5.46 (m, 1H), 4.95 (dd, J = 12.2, 5.2 Hz, 1H), 3.91 - 3.83 (m, 4H), 3.81 - 3.71 (m, 2H), 3.56 - 3.46 (m, 2H), 3.14 (s, 4H), 2.96 - 2.70 (m, 3H), 2.53 (s, 3H), 2.22 (s, 2H), 2.18 - 2.08 (m, 1H), 2.0 - 1.92 (m, 2H). LRMS, m/z, calculated, 797.228; found, 820.52 (M+Na)+ HPLC, tR = 6.72 (purity: 95%).
Example 17: Synthesis of /V-(6'-((l-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5- yl)piperidin-4-yl) methoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.017):
Figure imgf000086_0001
Step I, Synthesis of tert- butyl 4-(((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl) benzamido)-[3,3'-bipyridm]-6-yl)oxy)methyl)piperidine-l-carboxylate [0232] Title compound (150 mg, 70%) was prepared as a yellow solid from /<?r/-butyl 4- (hydroxymethyl)piperidine- 1 -carboxylate (351 mg, 4.34 mmol) as described in step I of
Example 9. ¾ NMR (400 MHz, CDCh) d 8.62 (d, J = 2.5 Hz, 1H), 8.33 (s, 1H), 8.20 - 8.13 (m, 2H), 8.10 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 1.8 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 1.9 Hz, 1H), 4.27 (d, J = 6.5 Hz, 2H), 3.94 - 3.73 (m, 4H), 3.21 - 3.08 (m, 4H), 2.86 - 2.69 (m, 2H), 2.50 (s, 3H), 2.13 - 2.05 (m, 1H), 2.04 (s, 2H), 1.88 - 1.75 (m, 2H), 1.46 (s, 9H), 1.38 - 1.27 (m, 2H). LRMS, m/z, calculated 655.298 for C34H40F3N5O5; found 678.45 (M+Na)+, 654.57 (M-H) .
Step II, 4-(((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)methyl)piperidin-l-ium chloride
[0233] The title compound was prepared from /er/-butyl 4-(((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)methyl)piperidine-l -carboxylate as described in step II of Example 9. !H NMR (400 MHz, DMSO) d 1 1.36 (s, 1H), 9.17 (s, 1H),
8.78 (s, 2H), 8.56 (s, 1H), 8.47 - 8.30 (m, 2H), 8.03 (d, J = 7.8 Hz, 1H), 7.92 - 7.75 (m, 2H),
7.34 (d, J = 1.9 Hz, 1H), 4.26 (d, J = 6.1 Hz, 2H), 3.80 - 3.71 (m, 2H), 3.54 (s, 3H), 3.15 (s, 1H), 3.10 - 2.99 (m, 2H), 2.91 (d, J = 1 1.4 Hz, 2H), 2.72 - 2.64 (m, 1H), 2.61 (s, 2H), 2.31 (s, 1H), 2.25 - 2.00 (m, 1H), 1.98 - 1.82 (m, 2H), 1.70 - 1.38 (m, 2H). LRMS, (m/z), calculated (for free base, C29H32F3N5O3), 555.246, found 556.36 (M+H)+, 554.37 (M-H) .
Step III, Compound 1.017
[0234] The title compound 1.017 (15 mg, 11%) was prepared as described in step III of
Example 9. ¾ NMR (400 MHz, CDCh) d 8.60 (d, J = 2.6 Hz, 1H), 8.21 - 8.13 (m, 2H), 8.06 (dd, J = 22.8, 16.3 Hz, 3H), 7.85 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.73 - 7.61 (m, 2H), 7.31 (d, J = 2.1 Hz, 1H), 7.13 - 7.01 (m, 2H), 4.94 (dd, J = 12.4, 5.3 Hz, 1H), 4.34 (d, J = 6.4 Hz, 2H), 4.04 (d, J = 13.0 Hz, 2H), 3.91 - 3.76 (m, 4H), 3.20 - 3.01 (m, 6H), 2.98 - 2.66 (m, 3H), 2.51 (s, 3H), 2.30 - 2.06 (m, 2H), 1.98 (d, J = 10.8 Hz, 2H), 1.54 (dd, J = 12.2, 8.9 Hz, 2H). LRMS, m/z, calculated (for C42H40F3N7O7), 811.29, found, 834.54 (M+Na)+, 810.60 (M-H) . HPLC, tR = 6.83 min (purity, 99%). HPLC Method C. Example 18: Synthesis of V-(6’-((4-((4-(2-(2,6-dioxopipcridin-3-yl )- 1 ,3-dioxoisoindolin-5- yl)piperazin-l-yl)methyl)benzyl)oxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.018 )
Figure imgf000088_0001
Step I, tert- butyl 4-(4-(hydroxymethyl)benzyl)piperazine-l-carboxylate
[0235] To a solution of 4-(hydroxymethyl)benzaldehyde (620 mg, 4.55 mmol) in dry DCM (7 mL) was added /er/-butyl piperazine- 1-carboxylate (1.02 g, 5.46 mmol) and anhydrous Na2S04 (~0.5 g) and NaBH(OAc)3 (1.16 g, 5.46 mmol) and the reaction mixture was stirred overnight at rt. The reaction mixture was diluted with DCM and then washed with brine (2 x 10 mL). The organic layer was collected, dried over anhydrous Na2S04, then filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-30%) to afford the title compound (1.1 g, 79%) as a colourless oil. 'H NMR (400 MHz, CDCh) d 7.32 (d, J = 2.0 Hz, 4H), 4.68 (s, 2H), 3.52 (s, 2H), 3.44 3.39 (m, 4H), 2.43 2.34 (m, 4H), 1.45 (s, 9H)* missing OH signal. LRMS, m/z, calculated, 306.194, found 307.36 (M+H)+.
Step II, tert- butyl 4-(4-(((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)methyl)benzyl)piperazine-l-carboxylate.
[0236] To a solution of /er/-butyl 4-(4-(hydroxymethyl)benzyl)piperazine- 1 -carboxylate (449 mg, 1.47 mmol) in dry dioxane (3 mL) was added NaH (58.6 mg, 1.47 mmol) and the reaction mixture was stirred for 30 min at rt. /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)- 3-(trifluoromethyl)benzamide (135 mg, 0.29 mmol) was added in one portion and the mixture was heated to 80 °C overnight. The mixture was diluted with EtOAc and washed with water (10 mL) and brine (10 mL). The organic phase was collected, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc in hexanes (0-100%) as eluant to afford the title compound (150 mg, 69%) as a brown solid. ¾ NMR (400 MHz, CDCh) d 8.63 (d, J = 2.6 Hz, 1H), 8.18 8.14 (m, 2H), 8.10 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 2.1 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 2.1 Hz, 1H), 5.51 (s, 2H), 3.92 3.83 (m, 4H), 3.53 (s, 2H), 3.47 3.40 (m, 4H), 3.21 3.09 (m, 4H), 2.53 (s, 3H), 2.46 2.35 (m, 4H), 1.45 (s, 9H).
Step III, Compound 1.018
[0237] To a solution of /er/-butyl 4-(4-(((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)methyl)benzyl)piperazine-l-carboxylate (150 mg, 0.2 mmol) in toluene (5 mL) and MeOH (5 mL) was added TFA (0.15 mL, 0.2 mmol) and the reaction mixture was stirred at rt for 6 h. The reaction mixture was concentrated and any residual TFA was removed by co-evaporation with toluene. The amine was used as such in the next step with no additional purification (175 mg, 100%). To a solution of /V-(2-methyl-5'- morpholino-6'-((4-(piperazin- 1 -ylmethyl)benzyl)oxy)-[3 ,3 '-bipyridin]-5-yl)-3 - (trifluoromethyl)benzamide bis(2,2,2-trifluoroacetate) (175 mg, 0.2 mmol) in dry DMSO (5.0 mL) was added TEA (0.17 mL, 1.2 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline- 1,3-dione (66.3 mg, 0.24 mmol) and the reaction mixture was heated to 80 °C for 6 h. The reaction mixture was cooled to rt and poured into water (30 mL). The mixture was extracted with DCM (3 x 50 mL) and the combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-20%) as eluant to afford the title compound (51 mg, 28%) as a yellow solid. *H NMR (400 MHz, CDCh) d 8.61 (d, J= 2.6 Hz, 1H), 8.17 (d, J= 2.6 Hz, 1H), 8.15 (s, 1H), 8.09 (d, J= 8.3 Hz, 1H), 7.94 (s, 1H), 7.85 (d, J= 8.7 Hz, 2H), 7.82 (d, J= 2.0 Hz, 1H), 7.69 (d, J= 8.4 Hz, 2H), 7.47 (d, J= 8.0 Hz, 2H), 7.38 (d, J= 8.0 Hz, 2H), 7.28 (d, J= 2.4 Hz, 1H), 7.09 (d, J= 2.1 Hz, 1H), 7.05 (dd, J= 8.7, 2.3 Hz, 1H), 5.53 (s, 2H), 4.94 (dd, J= 12.2, 5.4 Hz, 1H), 3.92 3.85 (m, 4H), 3.60 (s, 2H), 3.50 3.40 (m, 4H), 3.18 (d, J= 4.4 Hz, 4H), 2.93 2.71 (m, 3H), 2.66 2.59 (m, 4H), 2.53 (s, 3H), 2.13 - 2.10 (m, 1H). LRMS, m/z, calculated; 902.336, found 903.74 (M+H)+. HPLC, tR = 6.02 min (purity: 98%). HPLC method
C.
Example 19: The synthesis of /V-(6'-(2-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin- 5-yl)piperazin-l-yl)ethoxy)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.019).
Figure imgf000090_0001
Step I, tert- butyl 4-(2-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethoxy)ethyl)piperazine-l-carboxylate
[0238] To a solution of /er/-butyl 4-(2-(2-hydroxyethoxy)ethyl)piperazine-l-carboxylate (238 mg, 869 mmol) (from EP2149568) in dry dioxane (3 mL) was added NaH (34.7 mg, 0.869 mmol) and the reaction mixture was stirred at rt for 1 h. 7V-(6'-fluoro-2-methyl-5'-morpholino- [3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (80 mg, 174 mmol) was added in one portion and the mixture was heated to 80 °C for 3 h. The mixture was cooled to rt, and diluted with EtOAc (10 mL) and washed with water (10 mL). The organic phase was collected, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash
chromatography on silica gel using a gradient of MeOH in DCM (0-20%) as eluant to afford the title compound (100 mg, 80%) as a white foam. ¾ NMR (400 MHz, CDCh) d 8.73 (d, J = 2.5 Hz, 1H), 8.17 (s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.06 8.02 (m, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 2.1 Hz, 2H), 7.66 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 2.1 Hz, 2H), 4.65 4.59 (m, 2H), 3.92 - 3.88 (m, 6H), 3.71 - 3.73 (m, 2H), 3.42 - 3.37 (m, 4H), 3.20 - 3.12 (m, 4H), 2.60 (t, J =
5.6 Hz, 2H), 2.45 - 2.38 (m, 4H), 1.46 (s, 3H), 1.41 (s, 9H). LRMS, m/z, calculated, 714.335, found 715.52 (M+H)+.
Step II, Compound 1.019
[0239] To a solution of /er/-butyl 4-(2-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethoxy)ethyl)piperazine-l-carboxylate (100 mg, 0.14 mmol) in DCM (5 mL) and toluene (5 mL) was added TFA (0.2 mL, 2.74 mmol) and the reaction mixture was stirred at rt for 3 h. The reaction mixture was concentrated and toluene was added to remove residual TFA. The crude bis-TFA salt was used directly in the next step with no additional purification. To a solution of /V-(2-mcthyl-5'-morpholino-6'-(2-(2- (piperazin-l-yl)ethoxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide bis(2,2,2- trifluoroacetate (118 mg, 0.14 mmol) in dry DMSO (3.0 mL) was added 2-(2,6-dioxopiperidin- 3-yl)-5-fluoroisoindoline-l,3-dione (42.5 mg, 0.15 mmol) and TEA (0.12 mL, 0.84 mmol) and the reaction mixture was stirred at 80 °C for 6 h. The reaction mixture was cooled to rt and poured into water (20 mL) and the mixture was extracted with DCM (3 x 20 mL). The organic phase was collected, dried over Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-20%) as eluant to afford the title compound (3 mg, 3%) as a yellow solid. !H NMR (400 MHz, CDCh) d 8.54 (d, J = 2.6 Hz, 1H), 8.42 (bs, 1H), 8.30 (s, 1H), 8.18 (d, J = 2.5 Hz, 1H), 8.13 (bs, 1H), 8.07 (d, J =
7.9 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 6.98 (dd, J = 8.6, 2.3 Hz, 1H),
4.93 (dd, J = 12.2, 5.3 Hz, 1H), 4.71 - 4.61 (m, 2H), 3.92 - 3.89 (m, 6H), 3.73 (t, J = 5.4 Hz, 2H), 3.35 - 3.30 (m, 4H), 3.20 - 3.15 (m, 4H), 2.92 - 2.75 (m, 3H), 2.68 - 2.60 (m, 6H), 2.52 (s, 3H),
2.10 (dd, J = 10.4, 5.3 Hz, 1H). LRMS, m/z, calculated, 870.33, found 871.54 (M+H)+. HPLC, tR = 5.76 min (purity: 94.6%). HPLC method C.
Example 20: Synthesis of /V-(5'-(dimcthylamino)-6'-(2-(4-(2-(2,6-dioxopipcridin-3-yl)- 1,3- dioxoisoindolin-5-yl)piperazin-l-yl)ethoxy)-2-methyl-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.020 )
Figure imgf000092_0001
Step I, V-(5'-(dimcthylamino)-6'-fluoro-2-mcthyl-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide [0240] To a solution of 5-bromo-2-fluoro-/V,/V-dimcthylpyridin-3-aminc (WO201653771) (1.0 g, 4.57 mmol) in dioxane (20 mL) was added /V-(6-methyl-5-(4, 4,5, 5-tetramethyl-l, 3,2- dioxaborolan-2-yl)pyridin-3-yl)-3-(trifluoromethyl)benzamide (2.23 g, 5.48 mmol), 2M aqueous Na2C03 (6.85 mL, 13.7 mmol) and PdCl2(dppf).DCM (224 mg, 0.27 mmol) and the reaction mixture was degassed with argon before being heated to 100 °C for 2 h. The reaction mixture was cooled to rt. The mixture was diluted with DCM and brine and the aqueous phase was extracted with DCM (2 x 20 mL). The organic extracts were collected, dried over anhydrous Na2S04, filtered and concentrated. The resultant solid was triturated with a 1 : 1 mixture of Et20 and hexanes to afford the title compound as a grey solid which was used directly in the next step with no additional purification (2.02 g, 106%) 'H NMR (400 MHz, CDCh) d 8.65 (d, J = 2.6 Hz, 1H), 8.16 (d, J = 2.4 Hz, 2H), 8.10 (d, J = 7.8 Hz, 1H), 8.02 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H),
7.66 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 2.0 Hz, 1H), 7.10 (dd, J = 10.1, 2.1 Hz, 1H), 2.95 (s, 6H), 2.51 (s, 3H). LRMS, m/z, calculated, 418.14, found 419.13, (M+H)+.
Step II, tert- butyl 4-(2-((5-(dimethylamino)-2'-methyl-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate [0241] To a solution of /er/-butyl 4-(2-hydroxyethyl)piperazine-l-carboxylate (338 mg, 2.01 mmol) in dry dioxane (4 mL) was added NaH (80.4 mg, 2.01 mmol. 60% on mineral oil) and the reaction mixture was stirred at rt for 30 min. /V-(5'-(dimcthylamino)-6'-fluoro-2-mcthyl-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (200 mg, 0.48 mmol) was added in one portion and the reaction mixture was heated to 80 °C for 2 h. The reaction mixture was cooled to rt, diluted with DCM and water then separated. The aqueous phase was extracted with DCM (3 x 10 mL) and the organic extracts were combined, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-10%) to afford an impure compound. The material was dissolved in the minimum EtOAc and allowed to stand for 10 min where upon a solid precipitated. To this suspension was added 10% Et20 in hexanes, the mixture was filtered and the collected solid was washed with additional 10% Et20 in hexanes and then dried in the air to give the title compound as an off-white solid (150 mg, 50%). ¾ NMR (400 MHz, CDCh) d 8.63 (d, J = 2.6 Hz, 1H), 8.15 (s, 1H), 8.12 (d, J = 2.5 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.88 7.83 (m, 2H), 7.71 (d, J = 2.1 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 4.58 (t, J = 6.1 Hz, 2H), 3.44 (dd, J = 8.0, 3.3 Hz, 4H), 2.90 (t, J = 6.1 Hz, 2H), 2.87 (s, 6H), 2.55 (s, 3H), 2.53 (s, 3H), 1.46 (s, 9H) (NH amide missing). LRMS, m/z, calculated, 628.30, found 629.41 (M+H)+.
Step III, Compound 1.020
[0242] To a solution of /er/-butyl 4-(2-((5-(dimethylamino)-2'-methyl-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (150 mg, 0.239 mmol) in MeOH (4 mL) was added 4M HC1 in dioxane (0.6 mL, 2.39 mmol) and the mixture was stirred at rt for 4 h. The reaction mixture was concentrated and the resultant solid was triturated with Et20 to afford crude /V-(5'-(dimethylamino)-2-methyl-6'-(2-(piperazin- 1 - yl)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide dihydrochloride as a yellowish solid (144 mg, 100%). To a solution of crude /V-(5'-(dimethylamino)-2-methyl-6'-(2-(piperazin- l-yl)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide dihydrochloride (75 mg, 0.12 mmol) in dry DMSO (1 mL) was added 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3- dione (41.3 mg, 0.15 mmol) and TEA (0.1 mL, 0.75 mmol) and the reaction mixture was heated to 85 °C for 5 days. The reaction mixture was cooled to rt and poured into water (10 mL). The mixture was extracted with DCM (2 x 10 mL) and the organic extracts were combined, dried over anhydrous Na2S04, then filtered and concentrated. The resultant solid was triturated with Et20 and purified by reverse phase Cl 8 column using a gradient of acetonitrile in 0.2% TFA in water (5-100%) to afford the title compound as a yellow solid (8 mg, 13%). 1 H NMR (400 MHz, CDCh) d 8.61 (d, J = 2.6 Hz, 1H), 8.15 (s, 2H), 8.09 (d, J = 7.9 Hz, 1H), 7.98 (s, 1H), 7.90 (s, 1H), 7.84 (s, 1H), 7.79 7.61 (m, 3H), 7.29 (d, J = 2.2 Hz, 1H), 7.12 6.98 (m, 2H), 4.94 (dd, J
= 12.3, 5.3 Hz, 1H), 4.63 (t, J = 5.8 Hz, 2H), 3.59 3.35 (m, 4H), 2.96 (t, J = 5.9 Hz, 2H), 2.92 2.80 (m, 7H), 2.82 2.66 (m, 6H), 2.53 (s, 3H), 2.23 2.08 (m, 1H). LRMS, m/z, calculated 784.294; found 785.44 (M+H)+. HPLC, tR = 4.86 min (purity, >99%). HPLC method C.
[0243] Examples 21 to 30 report the synthesis of compounds using the general structure shown below:
Figure imgf000094_0001
Example 21: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimethyl-2-(4-(4-((2-(2-(2-((2'-methyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethoxy)ethoxy)ethoxy)methyl)- 1 H- 1 ,2,3-triazol- 1 -yl)butanamido)butanoyl)-4-hydroxy- 7V-((S)-l-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.021)
Figure imgf000095_0001
Step I, (25,4/?)-l-((/?)-2-(4-bromobutanamido)-3,3-dimcthvlbutanoyl)-4-hydroxy- V-((S)-l- (4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide: [0244] To a stirred solution of 4-bromobutyric acid (300 mg, 0.624 mmol) in DMF (8.0 mL) was sequentially added (2S,4R)- 1 -((R)-2-amino-3,3-dimcthylbutanoyl)-4-hydroxy-/V-((S)- 1 -(4- (4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (300 mg, 0.624 mmol) and HATU (285 mg, 0.748 mmol) followed by DIPEA (0.223 mL, 1.28 mmol). The reaction mixture was stirred at rt for 20 h, diluted with water (10 mL), extracted with ethyl acetate (3 x 15 mL), Combined organic extracts were washed with brine (20 mL), dried (Na2S04), and concentrated. The residue was purified on 40 g gold Si(¾ cartridge using a gradient of methanol in DCM (0- 10%) to afford the title compound (220 mg, 59.4%) as a pale yellow solid. Rf = 0.45 (10% MeOH in DCM). ¾ NMR (400 MHz, CDCb) d 8.68 (s, 1H), 7.43 7.39 (m, 3H), 7.37 (d, J = 8.3 Hz, 2H), 6.20 (d, J = 8.5 Hz, 1H), 5.12 5.03 (m, 1H), 4.74 (t, J = 7.8 Hz, 1H), 4.55 4.49 (m, 2H), 4.14 4.06 (m, 1H), 3.64 3.56 (m, 3H), 2.62 2.54 (m, 1H), 2.53 (s, 3H), 2.47 2.36
(m, 2H), 2.15 2.01 (m, 2H), 1.54 1.41 (m, 4H), 1.06 (s, 9H).
Step II, (2S,4/?)-l-((/?)-2-(4-azidobutanamido)-3,3-dimcth lbutanoyl)-4-h droxy-A'-((S)-l- (4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidme-2-carboxamide: [0245] To a solution of (2S,4R)- 1 -((/?)-2-(4-bromobutanamido)-3,3-dimctliylbutanoyl)-4- hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5 -yl)phenyl)ethyl)pyrrolidine-2-carboxamide (220 mg, 0.371 mmol) in DMSO (3 mL) was added potassium iodide (86 mg, 0.519 mmol) followed by sodium azide (72.3 mg, 1.11 mmol), reaction mixture was stirred at rt for 18 h (Rf is same as SM), diluted with water (10 mL), extracted with ethyl acetate (3 x 15 mL), combined extracts were washed with brine, dried (Na2S04), concentrated and dried. The residue was purified on 25 g S1O2 cartridge using a gradient of methanol in methylene chloride (0 to 10%) as eluant to afford the title compound (170 mg, 82.5%) as a white solid. Rf = 0.45 (10% MeOH in DCM). !H NMR (400 MHz, CDCb) d 8.68 (s, 1H), 7.47 - 7.42 (m, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 6.42 (d, J = 8.6 Hz, 1H), 5.14 - 5.03 (m, 1H), 4.70 (t, J = 7.8 Hz, 1H), 4.58 (d, J = 8.7 Hz, 1H), 4.52 (brs, 1H), 4.01 (d, J = 11.3 Hz, 1H), 3.84 (brs, 1H), 3.67 - 3.53 (m, 2H), 3.36 - 3.30 (m, 1H), 2.52 (s, 3H), 2.50 - 2.43 (m, 1H), 2.42 - 2.34 (m, 1H), 2.33 - 2.25 (m, 1H), 2.1 1 - 2.01 (m, 2H), 1.94 - 1.83 (m, 1H), 1.48 (d, J = 6.9 Hz, 3H), 1.05 (s, 9H). LRMS, m/z,
Calculated (for C27H37N7O4S) 555.26, Found, 554.31 (M-H) ; 578.38 (M+Na)+.
Step III, Compound 1.021
[0246] In RBF, 7V-(2-methyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 - yloxy)ethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (50 mg,
0.0795 mmol), ( 2S 4R)- 1 -((/?)-2-(4-azidobutanamido)-3 ,3 -d imeth ylbutan oyl )-4-h ydroxy-/V-((.S')- l-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (51.3 mg, 0.092 mmol) in THF (1.5 mL) was added a solution of copper sulphate pentahydrate (4.61 mg, 0.0185 mmol) in water (0.4 mL) and sodium ascorbate (3.66 mg, 0.0185 mmol) in water (0.4 mL), stirred for 20 h, concentrated on rotavap, purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 40%) as eluant to afford the title compound (1.021, 26 mg, 23.8%) as a off-white solid. Rf = 0.4 (10% methanol in DCM). ¾ NMR (400 MHz, CDCb) d 8.90 (s, 1H), 8.79 (s, 1H), 8.67 (s, 1H), 8.23 (s, 1H), 8.16 (d, J = 7.5 Hz, 1H), 8.03 (s, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.58 (s, 1H), 7.41 - 7.30 (m, 4H), 7.02 (s, 1H), 6.67 (d, 1H), 5.10 - 5.00 (m, 1H), 4.81 - 4.71 (m, 1H), 4.69 - 4.26 (m, 8H), 4.11 - 4.02 (m, 1H), 3.93 - 3.82 (m, 5H),
3.74 - 3.53 (m, 10H), 3.18 - 3.09 (m, 4H), 2.51 (s, 3H), 2.50 (s, 3H), 2.27 - 2.04 (m, 4H), 1.80 - 1.72 (m, 2H), 1.45 (d, J = 7.0 Hz, 3H), 1.03 (s, 9H). LRMS, m/z, calculated (for
C59H72F3N11O10S), 1 183.51 ; found, 1182.93 (M-H) ; HPLC, tR = 6.3 min (purity, >99.5%).
HPLC Method C. Example 22: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(6-(4-((4-(2-((2'-mcthyl-5- morphobno-5'-(3-trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l- yl)m ethyl)- 1 H- 1 ,2,3 -triazol- 1 -yl )h cx an am i do )butanoyl )-4-hydrox y-/V-((.S')- 1 -(4-(4- methylthiazol-5 -yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.022 )
Figure imgf000097_0001
Step I, A'-(2-mcthyl-5'-morpholino-6'-(2-(4-(prop-2-yn-l-yl)pipcrazin-l-yl)cthoxy)-[3,3'- bipyridm]-5-yl)-3-(trifluoromethyl)benzamide [0247] The title compound (145 mg, 43.9%) was prepared as off-white solid from N-(&- fluoro-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (250 mg, 0.543 mmol) and 2-(4-(prop-2-yn-l-yl)piperazin-l-yl)ethanol (457 mg, 2.71 mmol) as described in Example 9. Rf = 0.5 (EtOAc). lR NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.29 (s, 1H), 8.16 - 8.11 (m, 2H), 8.08 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H), 4.54 (t, J = 5.7 Hz, 2H), 3.93 - 3.85 (m,
4H), 3.30 (d, J = 2.4 Hz, 2H), 3.19 - 3.12 (m, 4H), 2.85 (t, J = 5.7 Hz, 2H), 2.72 - 2.54 (m, 8H), 2.48 (s, 3H), 2.25 (t, J = 2.4 Hz, 1H). LRMS, m/z, calculated (for C32H35F3N6O3) 608.27, found, 607.5 (M-H) .
Step II, Compound 1.022 [0248] In RBF, 7V-(2-methyl-5'-morpholino-6'-(2-(4-(prop-2-yn- 1 -yl)piperazin- 1 -yl)ethoxy)-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (50 mg, 0.0795 mmol), (2S,4R)- 1 -((/?)-2-(6- azidohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-/V-((S)-l-(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide (51.3 mg, 0.092 mmol) in THF (1.5 mL) was added a solution of copper sulfate pentahydrate (4.61 mg, 0.0185 mmol) in water (0.4 mL) and sodium ascorbate (3.66 mg, 0.0185 mmol) in water (0.4 mL), stirred for 20 h, concentrated on rotavap, purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 40%) as eluant to afford the title compound (1.022, 66 mg, 52.6%) as a light yellow solid. Rf = 0.45 (20% methanol in DCM). !H NMR (400 MHz, CDCb) d 9.06 (s, 1H), 8.69 - 8.65 (m, 2H), 8.23 (s,
1H), 8.19 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.41 - 7.31 (m, 4H), 7.03 (d, J = 2.0 Hz, 1H), 6.26 (d, J = 8.9 Hz, 1H), 5.10 - 5.01 (m, 1H), 4.75 (t, J = 8.0 Hz, 1H), 4.66 (d, J = 8.9 Hz, 1H),
4.6O - 4.49 (m, 3H), 4.32 - 4.21 (m, 2H), 4.02 (d, J = 11.3 Hz, 1H), 3.89 - 3.82 (m, 4H), 3.67 (s, 2H), 3.59 (dd, J = 11.1 , 3.4 Hz, 1H), 3.13 (s, 4H), 2.83 (t, J = 5.7 Hz, 2H), 2.69 - 2.41 (m, 8H), 2.51 (s, 3H), 2.49 (s, 3H), 2.28 - 2.06 (m, 4H), 1.87 - 1.74 (m, 2H), 1.66 - 1.51 (m, 2H), 1.47 (d, J = 6.9 Hz, 3H), 1.33 - 1.21 (m, 2H), 1.03 (s, 9H). 19F NMR (376 MHz, CDCh) d -63.06 (s). LRMS, m/z, calculated (for C61H76F3N13O7S), 1 191.57; found, 1192.92 (M+H)+; 1 191.02 (M- H)~; HPLC, tR = 5.82 min ( purity, 98.3. HPLC Method C.
Example 23: (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(6-(4-(((4-((2-((2'-mcthyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethoxy)methyl)benzyl)oxy)m ethyl)- 1 H- 1 ,2,3-triazol- 1 -yl)hcxanamido)butanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.023)
[0249] To a cold (0 °C) stirred solution of (4-((prop-2-yn-l-yloxy)methyl)phenyl)methanol (1.5 g, 8.51 mmol) (Ref: The Royal Society of Chemistry 2017 Org. Biomol. Chem., 2017, 15, 8091-8101) in DMF (15 mL) was added 60% sodium hydride in oil (409 mg, 10.2 mmol) in two portions, after stirred for 30 min, neat 2-(2-bromoethoxy)tetrahydro-2H-pyran (2.14 g, 10.2 mmol) was added, slowly warmed up to rt, stirred for 16 h, diluted with water (30 mL), extracted with ethyl acetate (3 x 30 mL), combined extracts were washed with brine, dried (Na2S04), concentrated and dried (Na2S04). The residue was purified on 100 g S1O2 cartridge using a gradient of ethyl acetate in hexanes (0 to 100%) as eluant to afford the title compound (1.0 g, 38.6%) as colorless liquid. In addition, (4-((prop-2-yn-l-yloxy)methyl)phenyl)methanol (800 mg, 53%) was also isolated. Rf = 0.3 (30% EtOAc in Hexanes). !H NMR (400 MHz, CDCb) d 7.34 (s, 4H), 4.67 - 4.63 (m, 1H), 4.63 - 4.55 (m, 4H), 4.17 (d, J = 2.4 Hz, 2H), 3.92 - 3.84 (m, 2H), 3.67 - 3.61 (m, 3H), 3.54 - 3.46 (m, 1H), 2.46 (t, J = 2.4 Hz, 1H), 1.91 - 1.47 (m, 6H).
Step II, 2-((4-((prop-2-yn-l-yloxy)methyl)benzyl)oxy)ethanol:
[0250] To a stirred solution of 2-(2-((4-((prop-2-yn- 1 - yloxy)methyl)benzyl)oxy)ethoxy)tetrahydro-2H-pyran (1.0 g, 3.29 mmol) in methanol (10 mL) was added pyridinium /;- to 1 ucn csul fon ate (83 mg, 0.329 mmol), stirred at rt for 2 h, additional amount of the PPTS (83 mg, 0.329 mmol) was added, stirred at rt for 2 h, concentrated, diluted with water (5 mL), extracted with ethyl acetate ( 3 x 15 mL), combined extracts were dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge using a gradient of ethyl acetate in hexanes (0 to 100%) as eluant to afford the title compound (600 mg, 82.9%) as a colorless liquid. Rf = 0.15 (30% EtOAc-hexanes). LRMS, m/z, calculated for (C13H16O3),
220.1 1, found, 243.22 (M+Na)+. ¾ NMR (400 MHz, CDCb) d 7.38 - 7.30 (m, 4H), 4.61 (s,
2H), 4.56 (s, 2H), 4.17 (d, J = 2.2 Hz, 2H), 3.79 - 3.74 (m, 2H), 3.61 - 3.57 (m, 2H), 2.47 (t, J = 2.2 Hz, 1H), 2.06 - 1.98 (m, 1H).
Step III, V-(2-mcthyl-5'-morpholino-6'-(2-((4-((prop-2-yn-l- yloxy)methyl)benzyl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide:
[0251] To a stirred solution of 2-((4-((prop-2-yn-l-yloxy)methyl)benzyl)oxy)ethanol (443 mg, 2.01 mmol) in 1 ,4-dioxane (3.2 mL) was added 60% sodium hydride (80.4 mg, 2.01 mmol), after stirred at rt for 30 min, /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide (185 mg, 0.402 mmol) was added in one portion, it was placed on pre-heated oil bath at 82 °C for 1 h, The reaction mixture was cooled to rt, poured into water (10 mL), acidified with 10% aq. citric acid (pH 5-6), extracted with ethyl acetate (3 x 15 mL), combined extracts were washed with water, brine, dried (Na2S04), concentrated. The residue was purified on 40 g Si(¾ cartridge using a gradient of ethyl acetate in hexanes (0% to 100%; 100%) to afford the title compound (150 mg, 56.5%) as off-white solid. Rf = 0.23 (EtOAc). 'H NMR (400 MHz, CDCh) d 8.65 - 8.62 (m, 2H), 8.15 (s, 1H), 8.11 - 8.07 (m, 2H), 7.80 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.35 - 7.29 (m, 4H), 7.03 (d, J = 2.0 Hz, 1H), 4.64 - 4.58 (m, 6H), 4.16 (d, J = 2.4 Hz, 2H), 3.93 - 3.87 (m, 2H), 3.87 - 3.81 (m, 4H), 3.17 - 3.11 (m, 4H), 2.47 (s and t, 4H). 19F NMR (376 MHz, CDCb) d -63.10 (s). LRMS, m/z, calculated (for C32H35F3N6O3) 660.26, found, 659.44 (M-H) .
Step IV, Compound 1.023 :
[0252] In RBF, /V-(2-mcthyl-5'-morpholino-6'-(2-((4-((prop-2-yn- 1 -yloxy)methyl)benzyl)- oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (55 mg, 0.083 mmol), ( 2S,4R )- 1 -((/?)-2-(6-azidohcxanamido)-3 ,3-dimcthylbutanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol- 5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (48.6 mg, 0.0832 mmol) in THF (1.5 mL) was added a solution of copper sulfate pentahydrate (4.16 mg, 0.0166 mmol) in water (0.4 mL) and sodium ascorbate (3.30 mg, 0.0166 mmol) in water (0.4 mL), stirred for 20 h, diluted with water (5 mL), extracted with methylene chloride (3 x lOmL), dried ( a2S04), concentrated on rotavap, purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 30%) as eluant to afford (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(6-(4-(((4-((2-((2'-mcthyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethoxy)methyl)benzyl)oxy)m ethyl)- 1 H- 1 ,2,3-triazol- 1 -yl)hcxanamido)-butanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide (70 mg, 67.6%) as off-white solid. Rf = 0.18 (5% methanol in DCM). !H NMR (400 MHz, CDCb) d 9.46 (s, 1H), 8.67 (s, 1H), 8.60 (d, J = 2.5 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 8.27 (s, 1H), 8.18 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H),
7.72 (d, J = 2.0 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.51 (s, 1H), 7.41 - 7.33 (m, 4H), 7.31 - 7.24 (m, 4H), 7.04 (d, J = 2.0 Hz, 1H), 6.34 (d, J = 8.8 Hz, 1H), 5.12 - 5.03 (m, 1H), 4.75 - 4.62 (m, 6H), 4.60 (s, 2H), 4.56 (s, 2H), 4.51 (brs, 1H), 4.24 (t, J = 7.1 Hz, 2H), 3.98 (d, J = 11.5 Hz, 1H), 3.90 - 3.82 (m, 6H), 3.59 (dd, J = 11.2, 3.2 Hz, 1H), 3.17 - 3.10 (m, 4H), 2.51 (s, 3H), 2.47 (s, 3H), 2.46 2.42 (m, 1H), 2.24 2.07 (m, 3H), 1.83 1.66 (m, 2H), 1.58 1.49 (m, 2H), 1.48 (d, J = 6.9 Hz, 3H), 1.28 1.18 (m, 2H), 1.03 (s, 9H). 19F NMR (376 MHz, CDCh) d -63.01 (s). LRMS, m/z, calculated (for CesHreFsNiiOgS), 1243.55, found, 1267.01 (M+Na)+; 1243.11 (M- H) ; HPLC, tR = 6.8 min (purity, >99%). HPLC Method C. Example 24: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(6-(4-(((4-(2-((2'-mcthyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethoxy)but-2-yn-l- yl)oxy)m ethyl)- 1 H- 1 ,2,3 -triazol- 1 -yl )h cx an am ido )butan oyl )-4-h ydroxy-/V-( (.S)- 1 -(4-(4- methylthiazol-5 -yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.024 )
Figure imgf000101_0001
Step I, Synthesis of 4-(prop-2-yn-l-yloxy)but-2-yn-l-ol.
[0253] To a solution of but-2-yne-l,4-diol (15.0 g, 174 mmol) in dry DMSO (85 mL) was added solid KOH (9.77 g, 174 mmol) and propargyl bromide (3.88 mL, 34.8 mmol, 80% w/v solution in toluene) and the reaction mixture was stirred rt for 2 h. The mixture was poured into water and extracted twice with DCM. The aqueous phase was acidified to pHl and extracted twice with DCM. The organic extracts were combined, dried over anhydrous Na2S04, then filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc in hexanes (0-50%) to afford the title compound (2.3 g, 11%) as an oil. 'H NMR (400 MHz, CDCh) d 4.33 4.30 (m, 4H), 4.25 (d, J = 2.4 Hz, 2H), 2.46 (t, J = 2.4 Hz,
1H), 1.65 (s, 1H). LRMS, m/z, calculated, 124.052; no ionization observed. Step II, Synthesis of 2-(2-((4-(prop-2-yn-l-yloxy)but-2-yn-l-yl)oxy)ethoxy)tetrahydro-2H- pyran.
[0254] To a solution of 4-(prop-2-yn-l-yloxy)but-2-yn-l-ol (1.3 g, 10.5 mmol) in dry DMF (12 mL) at 0 °C was added NaH (503 mg, 12.6 mmol, 60% on mineral oil) and the mixture was stirred for 30 min. 2-(2-bromoethoxy)tetrahydro-2H-pyran (2.63 g, 12.6 mmol) in DMF (2 mL) was added and the reaction mixture was stirred at rt for 6 h. The mixture was quenched with water and extracted with EtOAc (4 x 25 mL). The organic phase was collected, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc in hexanes (0-60%) to afford the title compound (1.6 g, 61%) as colourless oil. ¾ NMR (400 MHz, CDCh) d 4.67 4.62 (m, 1H), 4.31 (t, J = 1.8 Hz, 2H), 4.29 4.25 (m, 4H), 3.93 3.84 (m, 2H), 3.72 (dd, J = 5.3, 4.2 Hz, 2H), 3.66 3.60 (m, 1H), 3.55 3.48 (m, 1H), 2.45 (t, J = 2.4 Hz, 1H), 1.90 1.79 (m, 1H), 1.79 1.69 (m, 1H), 1.66 1.48 (m, 4H). LRMS, m/z, calculated, 252.136; no ionization observed.
Step III, Synthesis of 2-((4-(prop-2-yn-l-yloxy)but-2-yn-l-yl)oxy)ethanol.
[0255] To a solution of 2-(2-((4-(prop-2-yn- 1 -yloxy)but-2-yn- 1 -yl)oxy)ethoxy)tetrahydro-2H- pyran (1.0 g, 3.96 mmol) in MeOH (20 mL) was added PPTS (249 mg, 0.99 mmol) and the reaction mixture was stirred at rt for 3 h. Solid NaHCCL was added and the reaction mixture was concentrated. The crude material was dissolved in EtOAc and washed with water (2 x 10 mL). The organic phase was collected, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc in hexanes (0-100%) as eluant to afford the title compound (504 mg, 76%) as a colourless oil. 1 H NMR (400 MHz, CDCh) d 4.31 (t, J = 1.7 Hz, 2H), 4.25 (t, J = 2.1 Hz, 4H), 3.77 (s, 2H), 3.64 (dd, J = 5.1, 3.9 Hz, 2H), 2.45 (dd, J = 3.0, 1.8 Hz, 1H), 2.00 (s, 1H). LRMS, m/z, calculated, 168.078; no ionization observed.
Step IV, Synthesis of V-(2-mcthyl-5'-morpholino-6'-(2-((4-(prop-2-yn-l-yloxy)but-2-yn-l- yl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide.
[0256] To a solution of 2-(2-((4-(prop-2-yn- 1 -yloxy)but-2-yn- 1 -yl)oxy)ethoxy)tetrahydro-2H- pyran (338 mg, 2.01 mmol) in dry dioxane (4 mL) was added NaH (80.4 mg, 2.01 mmol. 60% on mineral oil) and the reaction mixture was stirred at rt for 30 min. /V-(6'-fluoro-2-mcthyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (338 mg, 2.01 mmol) was added in one portion and the reaction mixture was heated to 80°C for 2 h. The reaction mixture was poured into water and acidified to pH 4-5 with 10% citric acid solution. The mixture was extracted with DCM (3 x 20 mL) and the combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc in hexanes (0-100%) to afford the title compound (140 mg, 7%) as a solid. ¾ NMR (400 MHz, DMSO) 5 10.66 (s, 1H), 8.85 (d, J = 2.4 Hz, 1H), 8.38 - 8.23 (m, 2H), 8.02 (dd, J = 15.1 , 5.0 Hz, 2H), 7.80 (dd, J = 14.8, 4.9 Hz, 2H), 7.22 (d, J = 1.9 Hz, 1H), 4.53 - 4.45 (m, 2H), 4.32 (s, 2H), 4.26 (s, 2H), 4.19 (d, J = 2.4 Hz, 2H), 3.87 - 3.82 (m, 2H),
3.75 (s, 4H), 3.49 (t, J = 2.3 Hz, 1H), 3.10 (s, 4H), 2.44 (s, 3H). LRMS, m/z, calculated,
608.224; found, 609.43 (M+H)+
Step V, Compound 1.024
[0257] To a solution of /V-(2-mcthyl-5'-morpholino-6'-(2-((4-(prop-2-yn- 1 -yloxy)but-2-yn- 1 - yl)oxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (55 mg, 0.09 mmol) in THF (0.75 mL) was added (2S,4R)- 1 -((/?)-2-(6-azidohcxanamido)-3,3-dimcthylbutanoyl)-4-hydroxy- /V-((.S')- 1 -(4-(4-mcthylthiazol-5-yl)phcnyl)cthyl)pyrrolidinc-2-carboxamidc (52.8 mg, 0.09 mmol) in THF (0.75 mL). A solution of copper sulfate pentahydrate (4.51 mg, 0.018 mmol) and sodium ascorbate (3.58 mg, 0.018 mmol) in water (0.5 mL) was added and the reaction mixture was stirred at rt overnight. The mixture was diluted with DCM (5 mL) and the aqueous phase was extracted with DCM (2 x 5 mL). The combined organic extracts were dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-30%) as eluant to afford the title compound (65 mg, 60%) as a solid. ¾ NMR (400 MHz, CDCh) d 9.25 (s, 1H), 8.70 (brs, 1H), 8.67 (s, 1H), 8.30 (s, 1H), 8.22 - 8.18 (m, 2H), 7.82 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.47 (s, 1H), 7.42 - 7.32 (m, 5H), 7.02 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 8.8 Hz, 1H), 5.12 - 5.02 (m, 1H), 4.73 (t, J = 8.1 Hz, 1H), 4.69 (d, J = 8.9 Hz, 1H), 4.67 - 4.61 (m, 4H), 4.54 (brs, 1H), 4.30 (s, 2H), 4.20 (s, 2H), 4.17 - 4.1 1 (m, 1H), 4.03 (d, J = 11.6 Hz, 1H), 3.96 (t, J = 4.8 Hz, 2H), 3.91 - 3.86 (m, 4H), 3.62 (dd, J = 11.2, 3.1 Hz, 1H), 3.49 (s, 2H), 3.18 - 3.12 (m, 4H), 2.52 (s and m, 4H), 2.48 (s, 3H), 2.27 - 2.18 (m, 1H), 2.18 - 2.07 (m, 2H), 1.82 - 1.51 (m, 4H), 1.48 (d, J = 6.9 Hz, 3H), 1.29 - 1.16 (m, 2H), 1.04 (s, 9H). LRMS, m/z, calculated 1 191.52, found 1192.98 (M+H)+, HPLC, tR = 11.68 min (purity: >99%). HPLC method A
Example 25: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(2-(4-((2-(2-(2-((2'-mcthyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethoxy)ethoxy)ethoxy)methyl)- 1 H- 1 ,2,3-triazol- 1 -yl)acctamido)butanoyl)-4-hydroxy-/V- ((>Sr)-l-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.025 )
Figure imgf000104_0001
[0258] /V-(2-mcthyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 -yloxyethoxy)ethoxy)ethoxy)-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in
WO2018/200981.
[0259] The title compound 1.025 (41 mg, 41%) was prepared from V-(2-mcthyl-5'- morpholino-6'-(2-(2-(2-(prop-2-yn-l-yloxyethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide (55 mg, 0.09 mmol) (2S,4R)- 1 -((/?)-2-(2-azidoacetamido)-3,3- dimcthylbutanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-mcthylthiazol-5-yl)phcnyl)cthyl )pyrrolidinc-2- carboxamide (46.2 mg, 0.09 mmol) as described in Example 24. ¾ NMR (400 MHz, CDCh) d 8.77 (d, J = 2.3 Hz, 1H), 8.67 (bs, 2H), 8.21 (bs, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 2.3 Hz, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.38 (d, J = 8.3 Hz, 3H), 7.32 (d, J = 8.3 Hz, 2H), 7.16 (s, 1H), 7.03 (d, J = 2.0 Hz, 1H), 5.15 (d, J = 16.1 Hz, 1H), 5.09 - 5.01 (m, 1H), 4.95 (d, J = 16.0 Hz, 1H), 4.69 - 4.65 (m, 1H), 4.64 (s, 2H), 4.59 - 4.57 (m, 3H), 4.47 (bs, 1H), 3.94 - 3.82 (m, 7H), 3.73 - 3.70 (m, 2H), 3.63 - 3.59 (m, 8H), 3.13 (s, 4H), 2.51 (s, 3H), 2.48 (s, 3H), 2.40 (s, 1H), 2.09 - 2.01 (m, 1H), 1.44 (d, J = 6.9 Hz, 3H), 0.98 (s, 9H). LRMS, m/z, calculated 1155.58, found 1 178.98 (M+Na+). HPLC, tR = 6.27 (purity: >99%). HPLC method C.
Example 26: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(3-(4-((2-(2-(2-((2'-mcthyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethoxy)ethoxy)ethoxy)methyl)- 1 H- 1 ,2,3-triazol- 1 -yl)propanamido)butanoyl)-4-hydroxy- N-((S)- 1 -(4-(4-mcthylthiazol-5-yl)phcnyl)cthyl)pyrrolidinc-2-carboxamidc. ( Compound 1.026)
Figure imgf000105_0001
Step I, (2,V,4/?)-l-((/?)-2-(3-azidopropanamido)-3,3-dimcthvlbutanoyl)-4-hyd roxy- V-((A)-l- (4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide
[0260] 3-azidopropanoic acid (28.5 mg, 0.247 mmol) was dissolved in dry DMF (2.6 mL). HATU (94.1 mg, 0.247 mmol) and DIPEA (0.087 mL, 0.495 mmol) were added and the reaction mixture was stirred for 10 minutes at 0°C. (2S,4R)- 1 -((/?)-2-amino-3,3-dimcthylbutanoyl )-4- hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5 -yl)phenyl)ethyl)pyrrolidine-2-carboxamide (100 mg, 0.225 mmol) was added in one portion to the reaction mixture which was then stirred at rt for 24 h. The reaction mixture was diluted with water (6 mL) and extracted with EtOAc (3 x 10 mL). The combined extracts were washed with a saturated solution of NaHC03 (5 mL) followed by brine (5 mL) and dried over anhydrous Na2S04. The mixture was filtered and concentrated. The residue was purified by flash chromatography on silica gel using a gradient of MeOH in DCM (0-20%) to afford the title compound (71 mg, 58%) as a brown solid. ¾ NMR (400 MHz,
CDC13) 5 8.67 (s, 1H), 7.37 (dd, J = 13.9, 7.0 Hz, 5H), 6.53 (d, J = 8.2 Hz, 1H), 5.15 5.00 (m, 1H), 4.67 (t, J = 7.7 Hz, 1H), 4.63 4.46 (m, 2H), 4.02 (d, J = 11.2 Hz, 1H), 3.68 3.49 (m, 4H), 2.51 (s, 3H), 2.42 (s, 3H), 2.12 2.00 (m, 1H), 1.47 - 1.46 (d, J = 6.8 Hz, 3H), 1.05 (s, 9H). LCMS, m/z, calculated 541.247, found 564.42 (M+Na+).
Step II, Compound 1.026
[0261] 7V-(2-methyl-5'-morpholino-6'-(2-(2-(2-(prop-2-yn- 1 -yloxyethoxy)ethoxy)ethoxy)-
[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide was prepared as described in
WO2018/200981.
[0262] The title compound (35 mg, 34%) was prepared from /V-(2-mcthyl-5'-morpholino-6'-(2- (2-(2-(prop-2-yn-l-yloxyethoxy)ethoxy)ethoxy)-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide (55 mg, 0.09 mmol) and (2S,4R)- 1 -((/?)-2-(3-azidopropanamido)- 3 ,3-dim ethylbutanoyl)-4-hydroxy-/V-((>Sr)- 1 -(4-(4-methylthiazol-5 -yl)phenyl)ethyl)pyrrolidine-2- carboxamide (47.4 mg, 0.09 mmol) as described in Example 24. !H NMR (400 MHz, CDCh) d 8.85 (s, 2H), 8.66 (s, 1H), 8.23 (s, 1H), 8.16 (d, J= 7.2 Hz, 1H), 7.91 (s, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.70 (s, 1H), 7.65 7.57 (m, 2H), 7.37 - 7.33 (m, 3H), 7.31 (d, J= 8.1 Hz, 2H), 7.01 (s, 1H), 6.67 (m, 1H), 5.03 - 5.01 (m, 1H), 4.60 - 4.58 (m, 8H), 4.40 (s, 1H), 3.95 - 3.86 (m, 7H), 3.70 (s, 2H), 3.62 (s, 7H), 3.53 (d, J= 8.2 Hz, 1H), 3.13 (s, 4H), 2.86 (s, 1H), 2.72 (s, 1H), 2.51 - 2.49 (m, 6H), 2.34 (s, 1H), 2.09 (s, 1H), 1.44 - 1.42 (m, 3H), 0.98 (s, 9H). LRMS, m/z, calculated 1 169.497, found 1171.07 (M+H)+ HPLC, tR = 6.27 (purity: 99%). HPLC method A.
Example 27: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(6-(4-((4-((2'-mcthyl-5-morpholino- 5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)piperidin-l-yl)methyl)-lH- 1,2,3- triazol- 1 -yl)hcxanamido)butanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5 -yl)phenyl) ethyl)pyrrobdine-2-carboxamide ( Compound 1.027)
Figure imgf000106_0001
[0263] The title compound (67 mg, 54.7%) was prepared as a beige solid from 7V-(2-methyl-5'- morpholino-6'-((l-(prop-2-yn-l-yl)piperidin-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide (61 mg, 0.105 mmol) (Ref. step I of Example 10), (2S,4R)- 1 -((R)-2- (6-azidohexanamido)-3 ,3 -dimcthylbutanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5 - yl)phenyl)ethyl)pyrrolidine-2-carboxamide (61.4 mg, 0.105 mmol) as described in Example 24. Rf = 0.14 (10% methanol in DCM). ¾ NMR (400 MHz, CDCb) d 9.22 (s, 1H), 8.66 (s, 1H), 8.61 (d, J = 2.4 Hz, 1H), 8.28 - 8.22 (m, 2H), 8.16 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.51 (s, 1H), 7.42 - 7.33 (m, 4H), 7.02 (d, J =
2.0 Hz, 1H), 6.32 (d, J = 8.9 Hz, 1H), 5.26 (brs, 1H), 5.12 - 5.01 (m, 1H), 4.76 (t, J = 8.0 Hz, 1H), 4.68 (d, J = 9.0 Hz, 1H), 4.53 (brs, 1H), 4.33 - 4.23 (m, 2H), 4.01 (d, J = 1 1.1 Hz, 1H), 3.88 - 3.78 (m, 4H), 3.72 (s, 2H), 3.63 - 3.54 (m, 1H), 3.18 - 3.07 (m, 4H), 2.78 (brs, 2H), 2.57 - 2.46 (m, 2H), 2.5 (s, 3H), 2.49 (s, 3H), 2.27 - 2.05 (m, 5H), 1.99 1.73 (m, 5H), 1.69 - 1.52 (m, 2H), 1.48 (d, J = 6.9 Hz, 3H), 1.32 - 1.21 (m, 2H), 1.03 (s, 9H). 19F NMR (376 MHz, CDCb) d -
63.04 (s). LRMS, m/z, calculated (for C60H73F3N12O7S), 1162.54; found, 1185.89 (M+Na)+;
1163.91 (M+H)+. HPLC, tR = 5.99 min (purity, 98.8% @ 254 and 98.5% @ 234). HPLC Method C.
Example 28: Synthesis of /¥-((/?)- 1 -((2S, 7/?)-4-hydroxy-2-(((.S')- 1 -(4-(4-methylthiazol-5 - yl)phenyl)ethyl)carbamoyl)pyrrolidin- 1 -yl)-3 ,3 -dimethyl- 1 -oxobutan-2-yl)-6-(4-(2-((2'-methyl- 5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l- yl)pyridazine-3 -carboxamide ( Compound 1.028).
Figure imgf000107_0001
Step I, methyl 6-(4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-yl)pyridazme-3-carboxylate: [0264] In a sealed tube, to a stirred mixture of l-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l,4-diium chloride (150 mg, 0.233 mmol) (Ref. Step II of Example 9) and methyl 6-chloropyridazine-3-carboxylate (43 mg, 0.247 mmol) in acetonitrile (2.0 mL) was added DIPEA (172 pL, 0.988 mmol), it was heated at 82 °C for 1 h [35% of SM left], further heated for 3 h [ no progress], additional amount of methyl 6-chloropyridazine-3-carboxylate (43 mg) was added, heated for 1 h, additional amount of 6-chloropyridazine-3-carboxylate (33 mg) was added [Monitored by HPLC], after 1 h, cooled to rt, diluted with water (5 mL), extracted with ethyl acetate ( 3 x 15 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was absorbed on the Celite, purified on 25 g S1O2 cartridge using a gradient of methanol in DCM to afford the title compound (105 mg, 63.7%) as a white solid. Rf = 0.33 (10% MeOH in DCM). ¾ NMR (400 MHz, CDCh) d 8.68 - 8.61 (m, 2H), 8.22 - 8.16 (m, 2H), 8.12 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 9.6 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 9.6 Hz, 1H), 4.60 (t, J = 5.8 Hz, 2H), 3.95 (s, 3H), 3.91 - 3.85 (m, 4H), 3.83 - 3.75 (m, 4H), 3.21 - 3.12 (m, 4H), 2.91 (t, J = 5.8 Hz, 2H), 2.77 - 2.69 (m, 4H), 2.50 (s, 3H). 19F NMR (376 MHz, CDCh) d -63.16 (s). LRMS, m/z, calculated (for
C35H37F3N8O5), 706.28; found, 707.5 (M+H)+; 729.51 (M+Na)+.
Step II, 4-(6-carboxypyridazin-3-yl)-l-(2-((2'-methyl-5-morpholmo-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridm]-6-yl)oxy)ethyl)piperazm-l-ium chloride:
[0265] To a stirred solution of methyl 6-(4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-yl)pyridazine-3- carboxylate (102 mg, 0.144 mmol) in methanol (0.6 mL) was added aq. LiOH (188 pL, 0.188 mmol), stirred at rt for 4 h, acidified with aq. IN HC1, concentrated to afford the title compound (contains lithium chloride) (105 mg, quant.) as a beige solid. *H NMR (400 MHz, DMSO) d 12.09 (brs, 1H), 1 1.87 (s, 1H), 9.30 (d, J = 2.1 Hz, 1H), 8.86 (s, 1H), 8.52 - 8.45 (m, 2H), 8.02 (d, J = 7.8 Hz, 1H), 7.98 - 7.92 (m, 2H), 7.82 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 9.6 Hz, 1H), 7.46 (d, 1H), 4.90 - 4.82 (m, 2H), 4.74 - 4.62 (m, 2H), 3.81 - 3.58 (m, 10H), 3.42 - 3.27 (m, 2H), 3.12 - 3.05 (m, 4H), 2.69 (s, 3H). LRMS, m/z, calculated (for parent, C34H35F3N8O5), 692.27, found, 691.56 (M-H)~. HPLC, tR = 5.38 min (purity, >98%). HPLC Method C. Step III, Compound 1.028:
[0266] 4-(6-carboxypyridazin-3-yl)-l-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)- benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride (60 mg, 0.082 mmol) and ( R )- 1 -((2S, 7/?)-4-hydroxy-2-(((.S')- 1 -(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin- l-yl)-3 ,3-dimethyl- l-oxobutan-2-aminium chloride (39.6 mg, 0.082 mmol) in DMF (0.8 mL) was sequentially added HATU (46.9 mg, 0.123 mmol) and DIPEA (86 pL, 0.494 mmol) at rt, after 5 h, quenched with water (3 mL), filtered off the solids, rinsed with water, dried under high vacuum, dissolved in methanol-DCM, absorbed on the Celite and purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 20%) as eluant to afford the title compound 1.028 (47 mg, 51%) as a white solid. Rf = 0.35 (10% MeOH in DCM). !H NMR (400 MHz, CDCh) d 8.66
(s, 1H), 8.62 (d, J = 2.2 Hz, 1H), 8.56 (d, J = 7.9 Hz, 1H), 8.26 - 8.17 (m, 2H), 8.12 (d, J = 7.9 Hz, 1H), 7.84 - 7.73 (m, 4H), 7.62 (t, J = 7.9 Hz, 1H), 7.37 (t, J = 7.9 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.07 (s, 1H), 6.89 (d, J = 9.6 Hz, 1H), 5.08 - 4.97 (m, 1H), 4.79 (t, J = 7.9 Hz, 1H), 4.67 - 4.56 (m, 3H), 4.52 (s, 1H), 4.18 (d, J = 10.7 Hz, 1H), 3.93 - 3.85 (m, 4H), 3.80 - 3.71 (m, 4H), 3.62 (d, J = 9.2 Hz, 1H), 3.20 - 3.09 (m, 4H), 2.96 - 2.88 (m, 2H), 2.79 - 2.67 (m, 4H),
2.51 (s, 3H), 2.49 (s, 3H), 2.47 - 2.39 (m, 1H), 2.09 - 1.98 (m, 1H), 1.47 (d, J = 6.9 Hz, 3H), 1.12 (s, 9H). 19F NMR (376 MHz, CDCb) d -63.10 (s). LRMS, m/z, calculated (for
C57H65F3N12O7S), 1 118.48; found, 1 119.87 (M+H)+; 1141.83 (M+Na)+; 117.6 (M-H)\ HPLC, tR = 6.03 min (purity, >98%). HPLC Method C. Example 29: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(2-(4-(2-((2'-mcthyl-5-morpholino-5'- (3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l- yl)acctamido)butanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-mcthylthiazol-5-yl)phcnyl)cthyl)pyrrolidinc- 2-carboxamide {Compound 1.029)
Figure imgf000110_0001
Step I, /er/-butyl 2-(4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin- 1 -yl)acetate:
[0267] To a mixture of l-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l ,4-diium chloride (67 mg, 0.,104 mmol) (Ref. step II of Example 9) and /er/-butyl 2-bromoacetate (24.4 mg, 0.125 mmol) in DMF (1 mL) was added potassium bicarbonate (50 mg, 0.5 mmol), heated overnight at 80 °C (HPLC showed the consumption of SM), cooled to rt, diluted with ethyl acetate, filtered off, concentrated. The residue was purified on 25 g S1O2 cartridge using methanol in DCM (0 to 20%) as eluant to afford the title compound (40 mg, 56.1%) as light brown oil. Rf = 0.39 (10% MeOH in DCM). 1H NMR (400 MHz, CDCh) d 8.63 (d, J = 2.5 Hz, 1H), 8.16 (s, 1H), 8.13 (d, J = 2.5 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 8.05 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 4.55 (t, J = 5.8 Hz, 2H), 3.92 - 3.85 (m, 4H), 3.19 - 3.14 (m, 4H), 3.1 1 (s, 2H), 2.85 (t, J = 5.8 Hz, 2H), 2.72 - 2.54 (m, 8H), 2.51 (s, 3H), 1.46 (s, 9H). LRMS, m/.z, calculated (for C35H43F3N6O5), 684.32, found, 683.59 (M-l) .
Step II, l-(carboxymethyl)-4-(2-((2'-methyl-5-morpholino-5'-(3-
(trifluoromethyl)benzamido)-[3,3'-bipyridm]-6-yl)oxy)ethyl)piperazme-l,4-diium chloride:
[0268] To a stirred solution of /er/-butyl 2-(4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-yl)acetate (40 mg, 0.058 mmol) in methanol (200 pL) was added aq. LiOH (117 pL, 0.177), stirred for 16 h (HPLC showed 40% conversion), aq 4N NaOH (50 pL, 4M) was added, stirred for 3 h, additional amount of aq NaOH (50 pL, 4M) was added, stirred for 3 h, additional amount of aq. NaOH (50 pL, 4M) was added, stirred for 18 h (HPLC showed complete consumption of SM), acidified with aq. IN HC1 (0.9 mL), concentrated, suspended in dry methanol (2 mL) and dry DCM (4 mL), filtered through 0.4 micron filter, rinsed with methanol-DCM (1 :2, 6 mL), concentrated, purified on 43 g C18 S1O2 cartridge using a gradient of acetonitrile in water (10 to 100%) as eluant to afford the title compound (12 mg, 29.3%) as a white solid. 1 H NMR (400 MHz, CD3OD) 5 8.80 (d, J = 2.4 Hz, 1H), 8.30 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 2.4 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 2.0 Hz, 1H), 4.58 (t, J = 5.3 Hz, 2H), 3.89 - 3.81 (m, 4H), 3.60 (s, 2H), 3.36 - 3.31 (m, 4H), 3.19 - 3.12 (m, 4H), 3.02 - 2.91 (m, 6H), 2.48 (s, 3H). LRMS, m/z, Calculated (for free base,
C31H35F3N6O5), 628.26, found, 629.4 (M+H)+. HPLC, tR = 5.3 min (purity, >99%). HPLC Method C.
Step III, Compound 1.029:
[0269] l-(carboxymethyl)-4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l ,4-diium chloride (12 mg, 0.082mmol) and ( 2S,4R )- 1 -((i?)-2-amino-3 ,3 -dimethylbutanoyl)-4-hydroxy-/V-((>S)- 1 -(4-(4-methylthiazol-5- yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (9.05 mg, 0.0188 mmol) in DMF (0.4 mL) was sequentially added HATU (10 mg,, 0.026 mmol) and DIPEA (18 pL, 0.103 mmol) at rt for 4.5 h, it was directly loaded onto 43 g of Cl 8 S1O2 cartridge using a gradient of acetonitrile in water (10% to 100%) as eluant afforded 12 mg of the impure and 4.5 mg of the pure product; 12 mg of the product was repurified on 12 g S1O2 cartridge using a gradient of methanol in DCM (0 to 40%) as eluant to afford the title compound 1.029 (6.0 mg) as a white solid. Rf = 0.2 (10% MeOH in DCM). ¾ NMR (400 MHz, CDCb) d 8.75 - 8.68 (m, 1H), 8.66 (s, 1H), 8.61 (d, J = 2.3 Hz, 1H), 8.27 - 8.18 (m, 2H), 8.14 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.44 - 7.34 (m, 5H), 7.05 (d, J = 2.0 Hz, 1H), 5.13 - 5.02 (m, 1H), 4.75 (t, J = 8.0 Hz, 1H), 4.63 - 4.48 (m, 4H), 4.14 (d, J = 1 1.5 Hz, 1H), 3.90 - 3.84 (m, 4H), 3.60 (dd, J = 11.3, 3.4 Hz, 1H), 3.21 - 3.08 (m, 4H), 2.99 (d, J = 3.2 Hz, 2H), 2.90 - 2.83 (m, 2H), 2.75 - 2.54 (m, 8H), 2.52 (s, 3H), 2.50 (s, 3H), 2.15 - 2.05 (m, 2H), 1.49 (d, J = 6.9 Hz, 3H), 1.07 (s, 9H). 19F NMR (376 MHz, CDCh) d -63.01 (s). LRMS, m/z, calculated (for C54H65F3N10O7S), 1054.47; found, 1077.79 (M+Na)+; 1053.86 (M-H) . HPLC, tR = 5.85 min (purity, 97%). HPLC Method C.
Example 30: Synthesis of (2S,4R)- 1 -((/?)-3,3-dimcthyl-2-(3-(4-(2-((2'-mcthyl-5-morpholino- 5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l- yl)cyclobutanccarboxamido)butanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5 - yl)phenyl)ethyl)pyrrolidine-2-carboxamide ( Compound 1.030):
Figure imgf000112_0001
Step I, ethyl 3-(4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazm-l-yl)cyclobutanecarboxylate:
[0270] To a stirred suspension of l-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l,4-diium chloride (100 mg, 0.155 mmol) (Ref. step II of Example 9) and ethyl 3-oxocyclobutanecarboxylate (26.5 mg, 0.186 mmol) in THF (1 mL) was added DIPEA (81 pL, 0.466 mmol), after 5 min, sodium triacetoxyborohydride (49.4 mg, 0.233 mmol) (8.15 am) was added in one portion, stirred overnight at rt, quenched with water, extracted with ethyl acetate (2 x 15 mL), combined extracts were dried (Na2S04), and concentrated. The residue was purified on 25 g S1O2 cartridge using a gradient of methanol in DCM (0 to 40%) as eluant to afford the title compound (50 mg, 46%) as a white solid. Rf = 0.3 (10% MeOH in DCM). ¾ NMR (400 MHz, CDCh) d 9.08 (s, 1H), 8.66 (d, J = 2.3 Hz, 1H), 8.22 (d, J = 2.3 Hz, 1H), 8.19 (s, 1H), 8.15 - 8.09 (m, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 4.55 (t, J = 5.5 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.87 - 3.82 (m, 4H), 3.13 - 3.06 (m, 4H), 2.89 (t, J = 5.5 Hz, 2H), 2.80 - 2.66 (m, 5H), 2.55 - 2.37 (m, 5H), 2.36 - 2.25 (m, 3H), 2.23 - 2.13 (m, 2H),
2.00 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H). LRMS, m/z, calculated (for C36H43F3N6O5), 696.32, found, 697.53 (M+H)+.
Step II, 3-(4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-yl)cyclobutanecarboxylic acid:
[0271] To a stirred solution of ethyl 3-(4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethyl)piperazin- 1 - yl)cyclobutanecarboxylate (50 mg, 0.072 mmol) in methanol (0.5 mL) was added a solution of aq. LiOH (144 pL, 0.144 mmol), stirred overnight at rt (HPLC showed >95% of the product), acidified with aq. IN HC1, concentrated to afford the title compound (48 mg, quant) as a white solid. This material was used as such in the next step without further purification. LRMS, m/z, calculated (for C34H39F3N6O5), 668.29, found, 667.44 (M-H) . HPLC, tR = 5.32 min ( purity, 95%). HPLC Method C.
Step III, Compound 1.030 :
[0272] 3-(4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethyl)piperazin-l-yl)cyclobutanecarboxylic acid (48 mg, 0.0718 mmol) and (2S,4R)- 1 - ((/?)-2-amino-3 ,3 -dimcthylbutanoyl)-4-hydroxy-/V-((.S')- 1 -(4-(4-methylthiazol-5 - yl)phenyl)ethyl)pyrrolidine-2-carboxamide hydrochloride (34.5 mg, 0.0718 mmol in DMF (0.8 mL) was sequentially added HATU (40.9 mg, 0.108 mmol) and DIPEA (75 pL, 0.431 mmol) at rt for 16 h, diluted with water (5 mL), extracted with ethyl acetate (3x 10 mL), combined extracts were washed with bicarbonate solution, dried ( a2S04), concentrated. The residue was absorbed on the Celite, purified on 25 g S1O2 cartridge using a gradient of methanol in ethyl acetate (0 to 60%) as a eluant to afford the title compound 1.030 (26 mg, 33.1%) as a white solid. Rf = 0.2 (35% MeOH in DCM).‘H NMR (400 MHz, CDCh) d 9.40 (s, 1H), 8.67 (s, 1H), 8.56 (d, J = 2.5 Hz, 1H), 8.38 (d, J = 2.4 Hz, 1H), 8.26 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.42 - 7.33 (m, 5H), 7.04 (d, J = 2.0 Hz, 1H), 6.50 (d, J = 8.9 Hz, 1H), 5.14 - 5.02 (m, 1H), 4.79 - 4.68 (m, 2H), 4.57 - 4.49 (m, 3H), 4.00 (d, J = 11.4 Hz, 1H), 3.90 - 3.83 (m, 4H), 3.65 - 3.56 (m, 1H), 3.20 - 3.08 (m, 4H), 2.83 (t, J = 5.8 Hz, 2H), 2.70 - 2.53 (m, 5H), 2.52 (s, 3H), 2.49 (s, 3H), 2.41 - 1.93 (m, 11H), 1.49 (d, J = 6.9 Hz, 3H), 1.04 (s, 9H). 19F NMR (376 MHz, CDCh) d -63.01 (s). LRMS, m/z, calculated (for C57H69F3N10O7S), 1094.50; found, 1095.88 (M+H)+, 11 17.75 (M+Na)+. HPLC, tR = 5.76 min (purity, >99%). HPLC Method C.
Example 31: Synthesis of /V-(6'-(2-(4-(3-(4-((/?)-2-((.S')-2-(4-(4-fluorobcnzoyl)thiazol-2- yl)pyrrolidin- 1 -yl)- 1 -((>S)-2-(methylamino)propanamido)-2-oxoethyl)piperidine- 1 - carbonyl)cyclobutyl)piperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.031 )
Figure imgf000114_0001
Step I, Synthesis of (9H-fluoren-9-yl)methyl((S)-l-(((R)-2-((S)-2-(4-(4-fluorobenzoyl)thiazol- 2-yl)pyrrolidin-l-yl)-l-(l-(3-(4-(2-((2'-methyl-5-morpholmo-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazm-l- yl)cyclobutanecarbonyl)piperidm-4-yl)-2-oxoethyl)amino)-l-oxopropan-2- yl)(methyl)carbamate [0273] To a solution of 3-(4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-
[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-yl)cyclobutanecarboxylic acid (described in Example
30) (0.061 g, 0.091 mmol) and (9H-fluoren-9-yl)methyl ((5)-l-(((5)-2-((5)-2-(4-(4- fluorobenzoyl)thiazol-2-yl)pyrrolidin- 1 -yl)-2-oxo- 1 -(piperidin-4-yl)ethyl)amino)- 1 -oxopropan- 2-yl)(methyl)carbamate (0.066 g, 0.0912 mmol) (70 mg of the HC1 salt, Ref. WO 2017/182418) in DMF (0.8 mL) was added sequentially the HATU (0.053 mg, 0.137 mmol) and the Hunig's base (0.096 mL, 0.547 mmol). The reaction was followed by tic with 20 % MeOH in DCM. The reaction mixture was extracted with ethyl acetate and saturated sodium bicarbonate. The organic phase was collected, dried over sodium sulfate, filtered and evaporated. The mixture was purified on a 12 g column with DCM to 10 % MeOH in DCM to provide (9H-fluoren-9- yl)m ethyl (( S )- 1 -(((S)-2-((.S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin- 1 -yl)- 1 -( 1 -(3-(4-(2- ((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethyl)piperazin- 1 -yl)cyclobutanecarbonyl)piperidin-4-yl)-2-oxoethyl)amino)- 1 - oxopropan-2-yl)(methyl)carbamate (0.060 g, 0.044 mmol). LRMS, m/z, calculated, 1373.57; found, 1374.65 (M+H)+ and 1396.67 (M+Na)+.
Step II, Compound 1.031
[0274] To (9H-fluoren-9-yl)methyl ((S)-l-(((.S)-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-l-yl)-l-(l-(3-(4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-yl)cyclobutanecarbonyl)piperidin-4-yl)-2- oxoethyl)amino)-l-oxopropan-2-yl)(methyl)carbamate (0.060 g, 0.044 mmol) at 0 °C was added 1 mL of 20 % piperidine in DMF and stir at rt. After a period of 15 min, hexane was added to the mixture and the hexane decanted, the procedure was repeated three times. Water was then added to the DMF followed by extraction with ethyl acetate several times, dried over sodium sulfate, filtered and evaporated. The crude mixture was purified on a 12 g column with DCM to 25 % MeOH in DCM and fractions followed by HPLC. The desired fractions were combined and evaporated followed by the addition of DCM. The DCM solution was filtered on a 45 m filter to provide the title compound ( 0.032g, 0.028 mmol). LRMS, m/z, calculated, 1151.50; found,
1152.67 (M+H)+ and 1174.59 (M+Na)+. ¾ NMR (600 MHz, DMSO) 5 11.14 (s, 1H), 9.68 (s, 1H), 9.28 9.13 (m, 2H), 9.00 (s, 1H), 8.51 (d, J = 5.4 Hz, 1H), 8.44 (d, J = 8.3 Hz, 1H), 8.11 (s, 1H), 7.93 (s, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.69 7.55 (m, 5H), 7.40 (s, 1H), 7.17 (d, J = 6.9 Hz, 3H), 5.14 (dd, J = 12.7, 5.0 Hz, 1H), 4.50 (d, J = 4.8 Hz, 2H), 4.31 (t, J = 6.9 Hz, 2H), 2.88 (dd, J = 22.0, 9.0 Hz, 1H), 2.65 2.53 (m, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.10 2.01 (m, 1H), 1.86 1.77 (m, 2H), 1.69 1.57 (m, 2H), 1.33 1.26 (m, 2H). LRMS, m/z, calculated, 900.236; found, 924.736 (M+Na)+. HPLC, tR = 4.77 min (purity: 94%). Example 32: Synthesis ofN-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-5-yl)-3- methylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.032).
Figure imgf000116_0001
Step I, tert-butyl 4-(2-hydroxyethyl)-2-methylpiperazine-l-carboxylate : [0275] 2-Bromoethanol (1.87 g, 15.0 mmol) was added to a mixture of tert-butyl 2- methylpiperazine- 1 -carboxylate (2.00 g, 9.99 mmol) and potassium carbonate (4.13 g, 29.9 mmol) in acetonitrile (15 mL). The mixture was heated at 82 °C for 6hrs (follow reaction by tic using KMn04 100 % EA). The reaction mixture was filtered and evaporated. The mixture was purified on 80 g column with hexane to 100 % EA, to provide tert-butyl 4-(2-hydroxyethyl)-2- methylpiperazine- 1 -carboxylate (1.20 g, 49 %).!H NMR (400 MHz, CDCh) d 4.24 (s, 1H), 3.84 (d, J = 13.0 Hz, 1H), 3.61 (t, J = 5.2 Hz, 2H), 3.08 (td, J = 12.7, 3.2 Hz, 1H), 2.87 2.69 (m, 2H), 2.65 (d, J = 1 1.3 Hz, 1H), 2.58 2.37 (m, 1H), 2.26 (dd, J = 11.3, 3.9 Hz, 1H), 2.14 1.97 (m, 1H), 1.46 (s, 9H), 1.28 1.15 (m, 3H). LRMS, m/z 245.53 (M+H)+. Step II, tert-butyl 2-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridiii]-6-yl)oxy)ethyl)piperazme-l-carboxylate :
[0276] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-2-methylpiperazine-l-carboxylate (0.398 g, 1.63 mmol) in 1,4-dioxane (3.0 mL) was added 60% sodium hydride (0.065 g, 1.63 mmol) in one portions, after stirred at rt for 30 min, /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (0.150 g, 0.326 mmol) was added in one portion, it was placed on pre -heated oil bath at 80 °C for 2 hrs, The reaction mixture was cooled to rt, poured into water and extracted with ethyl acetate , the organic phase was collected, dried (Na2S04), and concentrated.. The reaction mixture was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier. After evaporation , the residue was purified on 40 g S1O2 cartridge with 20 % EA in hexane to 100 % EA then EA to 5 % MeOH in EA to provide tert-butyl 2-methyl-4-(2-((2'-methyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l- carboxylate . ¾ NMR (400 MHz, CDCh) d 8.61 (d, J = 2.5 Hz, 1H), 8.15 (s, 2H), 8.09 (d, J = 7.8 Hz, 1H), 7.98 (s, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 4.54 (t, J = 5.4 Hz, 2H), 4.33 - 4.12 (m, 1H), 3.87 (m, 4H), 3.80 (s, 1H), 3.12 (d, J = 34.0 Hz, 4H), 3.04 (t, J = 12.4 Hz, 1H), 2.89 (d, J = 1 1.9 Hz, 1H), 2.83 - 2.59 (m, 3H), 2.51 (s, 3H), 2.29 (dd, J = 1 1.2, 3.9 Hz, 1H), 2.20 - 2.01 (m, 2H), 1.46 (s, 11H), 1.24 - 1.09 (d, 3H). LRMS, m/z 585.38 (M-100+H)+.
StepIII, 2-methyl-4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-ium chloride :
[0277] To a stirred solution of tert-butyl 2-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (0.200 g, 0.292 mmol) in MeOH (2.0 mL) was added a solution of HC1 (0.95mL, 3.80 mmol, 4N) in 1,4- dioxane, after stirred for 2 hrs, concentrated and dried to afford the title compound 2-methyl-4- (2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethyl)piperazin-l-ium chloride (0.200 g, 100%) which was used as such for the next step . LRMS, m/z 585.38 (M+H)+.
Step IV, Compound 1.032: [0278] To a mixture of 2-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3-
(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride (0.150 g, 0.257 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (0.085 g, 0.308 mmol) in DMSO (1.5 mL) was added triethyl amine (0.156 g, 1.54 mmol) and potassium carbonate (0.071g, 0.513 mmol). The mixture was heated at 100 °C for 18 hrs, cooled to rt, diluted with water, extracted with ethyl acetate (3 x 10 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge with EA to 10 % MeOH in EA followed by a second purification with DCM to 10 % MeOH in DCM to afford the title compound (1.032) (12 mg, 6%) . !H NMR (400 MHz, CDCb) d 8.59 (t, J = 9.2 Hz, 1H), 8.25 - 8.00 (m, 5H), 7.84 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 1.9 Hz, 1H), 7.66 (t, J =
8.1 Hz, 2H), 7.23 (s, 1H), 7.07 (d, J = 1.9 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 4.93 (dd, J = 11.9, 4.7 Hz, 1H), 4.61 (d, J = 5.3 Hz, 2H), 4.19 (s, 1H), 3.89 (d, J = 4.2 Hz, 4H), 3.57 (d, J = 10.2 Hz, 1H), 3.33 - 3.05 (m, 6H), 3.04 - 2.60 (m, 7H), 2.53 (s, 3H), 2.35 (d, J = 8.7 Hz, 1H), 2.18 - 2.07 (m, 1H), 1.20 (d, J = 6.5 Hz, 3H). LRMS, m/z, 863.33(M+Na)+. HPLC (purity, 95.0%). Example 33: Synthesis ofN-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-5-yl)- 2,3-dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.033).
Figure imgf000118_0001
Step I, tert-butyl 4-(2-hydroxyethyl)-2,3-dimethylpiperazine-l-carboxylate : [0279] 2-Bromoethanol (0.612 g, 4.90 mmol) was added to a mixture of tert-butyl 2,3- dimethylpiperazine- 1 -carboxylate (0.700 g, 3.27 mmol) and potassium carbonate (1.35 g, 9.80 mmol) in acetonitrile (15 mL). The mixture was heated at 80 °C for 18hrs (follow reaction by tic using KMn04 100 % EA). The reaction mixture was filtered and evaporated. The mixture was purified on 80 g column with hexane to 100 % EA, to provide tert-butyl 4-(2 -hydroxy ethyl)-2, 3- dimethylpiperazine-1 -carboxylate (0.286 g, 33 %). ' H NMR (400 MHz, CDCh) d 3.99 (s, 1H), 3.81 (d, J= 12.9 Hz, 1H), 3.68 (m, 1H), 3.55 (m, 1H), 3.15 - 2.98 (m, 2H), 2.89 (d , J= 10.8 Hz, 1H), 2.50 (m, 1H), 2.20 - 2.04 (m, 2H), 1.46 (s, 9H), 1.09 (m, 3H), 1.02 (m, 3H).
StepII, tert-butyl 2,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate :
[0280] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-2,3-dimethylpiperazine-l- carboxylate (0.253 g, 0.977 mmol) in 1 ,4-dioxane (4.0 mL) was added 60% sodium hydride (0.062 g, 1.56 mmol) in one portions, after stirred at rt for 30 min, /V- ( 6 ' - fl uo ro- 2 - m c t h y 1 - 5 morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (0.180 g, 0.391 mmol) was added in one portion, it was placed on pre -heated oil bath at 80 °C for 1 h, The reaction mixture was cooled to rt, poured into water and extracted with ethyl acetate , the organic phase was collected, dried (Na2S04), and concentrated.. The reaction mixture was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier. After evaporation , the residue was purified on 40 g S1O2 cartridge with hexane to 100 % EA then EA to 5 %MeOH in EA to provide tert-butyl 2,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3 ,3'-bipyridin]-6-yl)oxy)ethyl)piperazine- 1 -carboxylate( 0.130 g, 72 %) . ¾ NMR (400 MHz, CDCh) 5 8.61 (d, .7= 2.5 Hz, 1H), 8.15 (s, 2H), 8.08 (m, 1H),
7.89 (s, 1H), 7.85 (d , J= 7.7 Hz, 1H), 7.78 (d , J= 2.0 Hz, 1H), 7.67 (t, J= 8.0 Hz, 1H), 7.06 (d, J= 1.9 Hz, 1H), 4.55 (s, 1H), 4.48 (s, 1H), 3.89 (m, 4H), 3.86 - 3.74 (m, 1H), 3.20 (m, 2H), 3.13 (m, 2H), 2.96 (m, 2H), 2.48 (s, 3H), 2.28 (m, 1H), 1.46 (s, 9H), 1.07 (2d, 6H).
StepIII, 2,3-dimethyl-4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethyl)piperazin- 1-ium chloride :
[0281] To a stirred solution of tert-butyl 2,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3 ,3'-bipyridin]-6-yl)oxy)ethyl)piperazine- 1 -carboxylate (0.130 g, 0.186 mmol) in MeOH (2.0 mL) was added a solution of HC1 (0.90mL, 3.80 mmol, 4N) in 1,4- dioxane, after stirred for 1 h, concentrated and dried to afford the title compound 2,3-dimethyl-4- (2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethyl)piperazin-l-ium chloride which was used as such for the next step . LRMS, m/z 599.98 (M+H)+.
Step IV, Compound 1.033:
[0282] To a mixture of 2,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride (0.150 g, 0.251 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (0.083 g, 0.301 mmol) in DMSO (1.5 mL) was added triethyl amine (0.152 g, 1.52 mmol) and potassium carbonate (0.069g, 0.501 mmol). The mixture was heated at 130 °C in a seal tube for two days, cooled to rt, diluted with water, extracted with ethyl acetate (3 x 10 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge with EA to 10 % MeOH in EA then to 10 % MeOH in EA to afford the title compound (1.033) (29 mg, 13%) . *H NMR (400 MHz, CDCh) d 8.59 (s, 1H), 8.18 (s, 1H), 8.15 (s, 1H),
8.09 (d, J = 8.3 Hz, 1H), 8.00 (m, 1H), 7.96 (m, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.67 (m, 2H), 7.23 (s, 1H), 7.08 (s, 1H), 7.00 (d, J = 8.7 Hz, 1H), 4.93 (m, 1H), 4.64 (m, 1H), 4.53 (m, 1H), 3.89 (m, 5H), 3.55 (m, 1H), 3.34 (m, 1H), 3.21 (m, 4H), 3.14 (m, 2H), 2.93 - 2.69 (m, 6H), 2.61 (m, 1H), 2.52 (s, 3H), 2.12 (m, 1H), 1.19 (d, J = 6.4 Hz, 3H), 1.08 (d, J = 6.4 Hz, 3H).. LRMS, m/z, 855.47(M+Na)+. HPLC (purity, 94.0%).
Example 34: N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-5-yl)-2- methylpiperazin- 1 -yl)ethoxy)-2-methyl-5 '-morpholino- [3 ,3 '-bipyridin] -5-yl)-3- (trifluoromethyl)benzamide (Compound 1.034).
Figure imgf000121_0001
Step I, tert-butyl 4-(2-hydroxyethyl)-3-methylpiperazine-l-carboxylate:
[0283] 2-Bromoethanol (1.87 g, 15.0 mmol) was added to a mixture of tert-butyl 3- methylpiperazine- 1 -carboxylate (2.00 g, 9.99 mmol) and potassium carbonate (4.14 g, 30.0 mmol) in acetonitrile (15 mL). The mixture was heated at 82 °C for 6hrs (followed reaction by tic, eluted TLC in 100 % EA used KMn04 stain). The reaction mixture was filtered and evaporated. The mixture was purified on 80 g column with hexane to 100 % EA, to provide tert- butyl 4-(2-hydroxyethyl)-3-methylpiperazine-l -carboxylate (1.00 g, 41%). ' H NMR (400 MHz, CDCh) d 3.68 3.52 (m, 4H), 3.26 (s, 1H), 3.0-2.85 (m, 2H), 2.84-2.76 (m, 1H), 2.58-2.48 (m, 1H), 2.40-2.20 (m, 2H), 1.45 (s, 9H), 1.04 (d, J= 6.3 Hz, 3H). LRMS, m/z 243.38 (M-H) .
Step II, tert-butyl 3-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate
[0284] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-3-methylpiperazine-l -carboxylate (0.207 g, 0.847 mmol) in 1,4-dioxane (6.0 mL) was added 60% sodium hydride (0.065 g, 1.63 mmol) in one portions, after stirred at rt for 30 min, /V-(6'-fluoro-2-mcthyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (0.30 g, 0.652 mmol) was added in one portion, it was placed on pre -heated oil bath at 80 °C for 2hrs, The reaction mixture was cooled to rt, poured into water and extracted with ethyl acetate , the organic phase was collected, dried (Na2S04), and concentrated.. The reaction mixture was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier. After evaporation ,the residue was purified on 40 g S1O2 cartridge with 20 % EA in hexane to 100 % EA then EA to 5 % MeOH in EA to provide tert-butyl 3-methyl-4-(2-((2'-methyl-5-morpholino-
5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate. 1 H
NMR (400 MHz, CDCb) d 8.61 (d, J= 2.5 Hz, 1H), 8.15 (s, 2H), 8.09 (d, J= 8.0 Hz, 1H), 7.89 (s, 1H), 7.85 (d , J= 8.0 Hz, 1H), 7.78 (d , J= 2.0 Hz, 1H), 7.67 (t, J= 7.8 Hz, 1H), 7.06 (d, J = 2.1 Hz, 1H), 4.52 (m, 2H), 3.89 (m, 4H), 3.72 (d, J= 13.5 Hz, 2H), 3.16 (m, 6H), 2.90 (m, 1H), 2.76 (m, 2H), 2.51 (m, 4H), 2.43 (s, 1H), 1.46 (s, 9H), 1.09 (d, J= 6.2 Hz, 3H). LRMS, m/z 683.53 (M-H) +.
Step III, 3-methyl-4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridm]-6-yl)oxy)ethyl)piperazin-l-ium chloride:
[0285] To a stirred solution of tert-butyl 3-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (0.22 g, 0.321 mmol) in MeOH (2.0 mL) was added a solution of HC1 (0.83 mL, 3.21 mmol, 4N) in p- dioxane, after stirred for 1 h, the mixture was concentrated and dried to afford the title compound 3-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride which was used as such for the next step. LRMS, m/z 585.44 (M+H)+.
Step IV, (Compound 1.034).
[0286] 3-methyl-4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride (0.220 g, 0.354 mmol) and 2-(2,6- dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (0.117 g, 0.425 mmol) in DMSO (1.5 mL) was added triethyl amine (0.215 g, 2.13 mmol). The mixture was heated at 120 °C in a seal tube for 24h, cooled to rt, diluted with water, extracted with ethyl acetate (3 x 10 mL), combined extracts were washed with brine, dried (Na2S04), and concentrated. The residue was purified on 40 g S1O2 cartridge with hexane to 100% then to 10 % MeOH in EA to afford the title compound (80 mg, 26.9%). ¾ NMR (400 MHz, CDCb) d 8.60 (d, J= 2.3 Hz, 1H), 8.16 (m, 2H), 8.09 (d, J= 8.4 Hz, 1H), 7.94 (s, 1H), 7.90 (s, 1H), 7.85 (d, J= 7.9 Hz, 1H), 7.79 (m, 1H), 7.68 (m, 2H), 7.06 (m, 2H), 4.93 (m, 1H), 4.56 (m, 2H), 3.89 (m, 4H), 3.64 (m, 2H), 3.15 (m, 7H), 2.95 - 2.59 (m, 7H), 2.52 (s, 3H), 2.15 (m, 1H), 1.21 (d, J= 6.1 Hz, 3H). LRMS, m/z, 841.47(M+H)+. HPLC (purity, 94.04%).
Example 35 : Synthesis ofN-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-5-yl)- 2,2-dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide {Compound 1.035 ).
Figure imgf000123_0001
Step I, tert-butyl 4-(2-hyd roxycthyl)-3 , 3-di methyl piperazine- 1 -carboxylate:
[0287] 2-Bromoethanol (0.87 g, 7.0 mmol) was added to a mixture of tert-butyl 3,3- dimethylpiperazine- 1 -carboxylate (1.00 g, 4.67 mmol) and potassium carbonate (1.93 g, 14.0 mmol) in acetonitrile (15 mL). The mixture was heated at 80°C for 18hrs (followed reaction by tic, eluted TLC in 100 % EA used KMn04 stain). The reaction mixture was filtered and evaporated. The mixture was purified on 80 g column with hexane to 100 % EA, to provide tert- butyl 4-(2-hydroxyethyl)-3, 3 -dimethylpiperazine-1 -carboxylate (0.210 g, 17.4 %). ' H NMR (400 MHz, CDCh) d 3.52 (t, J= 5.4 Hz, 2H), 3.44 (s, 2H), 3.17 (m, 2H), 2.53 (t, J= 5.2 Hz, 4H), 1.46 (s, 9H), 1.01 (s, 6H).
Step II, tert-butyl 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridm]-6-yl)oxy)ethyl)piperazine-l-carboxylate: [0288] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-3,3-dimethylpiperazine-l- carboxylate (0.177 g, 0.684 mmol) in 1 ,4-dioxane (5.0 mL) was added 60% sodium hydride (0.045.6 g, 1.14 mmol) in one portions, after stirred at rt for 30 min, /¥-( 6'-fl uoro-2-m cth yl- 5 morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (0.210 g, 0.456 mmol) was added in one portion, it was placed on pre -heated oil bath at 90 °C for 2 h, The reaction mixture was cooled to rt, poured into water and extracted with ethyl acetate , the organic phase was collected, dried (Na2S04), and concentrated.. The reaction mixture was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier. After evaporation , the residue was purified on 40 g S1O2 cartridge with hexane to 100 % EA then EA to 5 % MeOH in EA to provide tert-butyl 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate ( 0.220 g, 69 %) .‘H NMR (400 MHz, CDCh) d 8.61 (d, J= 2.5 Hz, 1H), 8.14 (m, 2H), 8.09 (d, J= 8.0 Hz, 1H), 7.91 (s, 1H), 7.85 (d, J= 7.7 Hz, 1H), 7.78 (d, J= 1.9 Hz, 1H), 7.67 (t, J= 7.7 Hz, 1H), 7.06 (d, J= 2.0 Hz, 1H), 4.44 (t, J= 6.1 Hz, 2H), 3.88 (m, 4H), 3.44 (m, 2H), 3.15 (m, 6H), 2.80 (t, J= 6.2 Hz, 2H), 2.67 (m, 2H), 2.51 (s, 3H), 1.46 (s, 9H), 1.03 (s, 6H).
Step III, 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethyl)piperazin- 1-ium chloride :
[0289] To a stirred solution of tert-butyl 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (0.220 g, 0.315 mmol) in MeOH (2.0 mL) was added a solution of HC1 (0.780mL, 3.15 mmol, 4N) in 1 ,4- dioxane, after stirred for 1 h, the mixture was concentrated and dried to afford the title compound 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin- 1-ium chloride which was used as such for the next step . LRMS, m/z 599.42 (M+H) +.
Step IV, ( Compound 1.035 ).
[0290] To a mixture of 3,3-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride (0.180 g, 0.283 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (0.094 g, 0.340 mmol) in DMSO (1.0 mL) was added triethylamine (0.172 g, 1.70 mmol). The mixture was heated at 120 °C in a sealed tube for 24 h, cooled to rt, diluted with water, extracted with ethyl acetate (3 x 10 mL), combined extracts were washed with brine, dried (TteSCL), and concentrated. The residue was purified on 40 g S1O2 cartridge with Hexane to 100% EA then to 10 % MeOH in EA to afford the title compound (57mg, 23.5%) H NMR (400 MHz, CDCb) d 8.59 (d, J= 2.5 Hz, 1H), 8.16 (m, 2H), 8.09 (d, J= 7.8 Hz, 1H), 8.00 (s, 1H), 7.94 (s, 1H), 7.85
(d , J= 7.7 Hz, 1H), 7.80 (d , J= 1.8 Hz, 1H), 7.67 (m, 2H), 7.25 (m, 1H), 7.08 (m, 1H), 7.02 (m, 1H), 4.93 (m, 1H), 4.49 (t, J= 6.2 Hz, 2H), 3.90 (m, 4H), 3.43 (m, 2H), 3.18 (m, 6H), 2.93 - 2.65 (m, 8H), 2.51 (s, 3H), 2.21 - 2.08 (m, 1H), 1.14 (s, 6H). LRMS, m/z, 855.46(M+H)+. HPLC (purity, 92.90 %). Example 36: N-(6'-(2-(5-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,5- diazabicyclo[2.2.1]heptan-2-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-
(trifluoromethyl)benzamide ( Compound 1.036 ).
Figure imgf000125_0001
Step I, tert-butyl 5-(2-hydroxyethyl)-2,5-diazabicyclo[2.2.1]heptane-2- carboxylatecarboxylate:
[0291] A mixture of tert-butyl 2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (1.00 g, 4.79 mmol), bromoethanol (0.50 mL, 7.19 mmol), potassium carbonate (1.99 g, 14.1 mmol) in acetonitrile (10 mL) was stirred at 100 °C overnight. The reaction mixture was cooled to rt, filtered, then rinsed with acetonitrile. The filtrate was concentrated and purified on 40 g S1O2 cartridge using (0% to 100 %) EtOAc-Hexanes then (0% to 15%) MeOH-EtOAc to obtain tert- butyl 5-(2-hydroxyethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (770 mg, 66%) as a pale yellow oil. ¾ NMR (400 MHz, CDCb) d 4.52 - 4.17 (m, 1H), 3.55 (t, J= 5.2 Hz, 2H), 3.50 - 3.35 (m, 3H), 3.20 (t , J= 9.2 Hz, 1H), 3.01 - 2.85 (m, 1H), 2.78 - 2.45 (m, 3H), 1.93 - 1.65 (m, 2H), 1.45 (s, 9H).
StepII, tert-butyl 5-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin] -6-yl)oxy)ethyl)-2,5-diazabicyclo [2.2.1] heptane-2-carboxylate:
[0292] To a stirred solution of tert-butyl 5-(2-hydroxyethyl)-2,5-diazabicyclo[2.2.1]heptane-2- carboxylate (199 mg, 0.821 mmol) in 1,4-dioxane (3 mL) was added 60% sodium hydride (70 mg, 1.65 mmol) in two portions, after stirred at rt for 5 min, N-(6'-fluoro-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (270 mg, 0.586 mmol) was added under inert atmosphere. The reaction mixture was placed on pre-heated oil bath at 80 °C for 2 hrs, the reaction mixture was cooled to rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL), the combined organic layers were washed with water, brine, dried over Na2S04, concentrated. The residue was treated with pyridine and acetic anhydride in order to acetylate the excess of alcohol and make purification easier. The acetylated product was purified on 40 g S1O2 cartridge using a gradient of (0% EtOAc to 100% EtOAc-Hexanes then MeOH-EtOAc (0% to 15%) to afford tert-butyl 5-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl) benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (120 mg, 30%) as a pale yellow solid. lR NMR (400 MHz, CDCb) d 8.63 (d, J = 7.6 Hz, 1H), 8.22 - 8.03 (m, 4H), 7.83 (d, J= 7.6 Hz, 1H), 7.77 (s, 1H), 7.66 (t, J= 7.6 Hz, 1H), 7.06 (s, 1H), 4.49 (t, J= 5.7 Hz, 2H), 4.39 - 4.22 (m, 1H), 4.01 - 3.80 (m, 4H), 3.72 - 3.48 (m, 2H), 3.28 - 2.95 (m, 8H), 2.77 (s, 1H), 2.50 (s, 3H), 1.91 - 1.63 (m, 2H), 1.45 (s, 9H).
StepIII, 5-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridiii]- 6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptan-2-ium chloride :
[0293] To a solution of tert-butyl 5-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptane-2- carboxylate (120 mg, 0.176 mmol) in MeOH (1 mL) was added HC1 (4 N in 1,4-dioxane, 2.20 mL, 8.79 mmol). The reaction mixture was stirred at room temperature for 15 min. The solvents were evaporated to afford 5-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptan-2-ium chloride as a pale yellow solid which was Used as such for next step. HPLC (94.07% purity)
Step IV, Compound 1.036 :
[0294] To a mixture of 5-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)-2,5-diazabicyclo[2.2.1]heptan-2-ium chloride (220 mg, 0.346 mmol) , 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (210 mg, 0.762 mmol) in DMSO (4 mL) was added Et;,N (4 mL, 17 mmol). The reaction mixture was stirred at 130 °C in sealed tube for 3 days. The mixture cooled at room temperature, water (6 mL) was added and product was extracted with EtOAc (4X 10 mL). The combined organic phase was washed with water, dried over Na2S04, concentrated. The residue was purified on 24 g S1O2 cartridge (gold) using 0% to 100 % EtOAc-Hexanes then 0% to 10 % MeOH-EtOAc, to afford N-(6'-(2-(5-(2- (2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethoxy)- 2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (30 mg, 16%) as a yellow solid containing a major isomer. ¾ NMR (400 MHz, CDCb) d 8.67 (s, 1H), 8.38 (d, J = 19.6 Hz, 2H), 8.17 (s, 1H), 8.15 - 8.02 (m, 2H), 7.82 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 24.0 Hz, 1H), 7.69 - 7.55 (m, 2H), 7.04 (d, J = 1.5 Hz, 1H), 6.94 (s, 1H), 6.68 (d, J = 7.9 Hz, 1H), 4.97 - 4.80 (m, 1H), 4.61 - 4.41 (m, 2H), 4.40 (s, 1H), 3.98 - 3.71 (m, 5H), 3.62 - 3.38 (m, 2H), 3.30 - 2.93 (m, 7H), 2.92 - 2.58 (m, 4H), 2.48 (s, 3H), 2.21 - 2.05 (m, 2H), 2.03 - 1.91 (m, 1H). LRMS, m/z, 839.53(M+H)+. HPLC (purity, 97.7%).
Example 37: N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,5- dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.037).
Figure imgf000128_0001
Step I, tert-butyl 4-(2-hydroxyethyl)-2,5-dimethylpiperazine-l-carboxylate:
[0295] 2-bromoethanol (0.660 ml, 11.3 mmol) was added to cold mixture of tert-butyl 2,5- dimethylpiperazine- 1 -carboxylate (1.00 g, 4.67 mmol) and NaH (60%, 280 mg, 7 mmol) in DMF (6 mL). The mixture was heated to 90 °C 3 hrs. Water was added then extracted with EtOAc (4X20 mL) the combined organic layers were washed with water, dried over Na2S04, concentrated. The residue was purified on 40 g silica gel cartridge using EtOAc-Hexanes (0% to 100%) then MeOH-EtOAc (0% to 15%) to obtain tert-butyl 4-(2-hydroxyethyl)-2,5- dimethylpiperazine- 1 -carboxylate (500 mg, 41%)as a pale yellow oil and as a mixture of cis and trans isomers which was used as such for next step.
StepII, tert-butyl 2,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate:
[0296] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-2,5-dimethylpiperazine-l- carboxylate (210 mg, 0.814 mmol) in 1,4-dioxane (3 mL) was added 60% sodium hydride (65 mg, 1.65 mmol) in two portions. The mixture was stirred at rt for 5 min, then N-(6'-fluoro-2- methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl) benzamide (250 mg, 0.543 mmol) was added under inert atmosphere. The reaction mixture was placed on pre-heated oil bath at 80 °C for 2 hrs. The reaction mixture was cooled into rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL), the combined organic layers were washed with water, brine, dried over Na2S04, concentrated. In order to have easier purification, the residue was dissolved in pyridine (1 mL) followed by addition of AC2O (1 mL). The reaction mixture stirred for 1 hrs to convert unreacted alcohol to acetylated product. The mixture was concentrated, then purified on 40 g S1O2 cartridge using a gradient of (0% to 100% EtOAc-Hexanes then methanol in ethyl acetate (0% to 15%) to afford tert-butyl 2,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (200 mg, 53%) as a pale yellow solid. lR NMR (400 MHz, CDCh) d 8.61 (d, J = 2.6 Hz, 1H), 8.15 (s,
2H), 8.09 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 7.9 Hz, 2H), 7.81 (d, J = 25.0 Hz, 2H), 7.67 (t, J = 8.0 Hz, 1H), 7.06 (s, 1H), 4.63 - 4.46 (m, 2H), 4.32 - 4.17 (m, 1H), 3.89 (d, J = 4.4 Hz, 4H), 3.17 (dd, J = 28.3, 10.3 Hz, 5H), 2.94 (d, J = 39.4 Hz, 2H), 2.86 - 2.68 (m, 2H), 2.52 (s, 3H), 2.40 (d, J = 12.3 Hz, 1H), 1.46 (s, 9H), 1.19 (d, J = 6.7 Hz, 3H), 1.1 1 (d, J = 6.2 Hz, 1H), 0.98 (d, J = 6.6 Hz, 2H). LRMS, m/z 721.54 (M+Na)+.
StepIII, 2,5-dimethyl-4-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] -6-yl)oxy)ethyl)piperazin- 1-ium chloride :
[0297] To a mixture of tert-butyl 2,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3
(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (200 mg, 0.286 mmol) in MeOH (1 mL) was added HC1 in 1,4-dioxane (4 N, 3.5 mL,14.3 mmol). The reaction mixture was stirred at rt for 1 h. The completion of reaction was confirmed with TLC (5% MeOH-EtOAc) and HPLC. The solvents were evaporated to dryness to afford 2,5-dimethyl- 4-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6- yl)oxy)ethyl)piperazin- 1-ium chloride (220 mg, 120%) pale yellow solid which is used as such for next step. LRMS, m/z 599.52 (M+H)+.
Step IV, Compound 1.037'.
[0298] To a mixture of 2,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin- 1-ium chloride (220 mg, 0.346 mmol) , 2-(2,6-dioxopiperidin-3-yl)-5- fluoroisoindoline-l,3-dione (210 mg, 0.762 mmol) in DMSO (4 mL) was added EίbN (4 mL, 17 mmol). The reaction mixture was stirred for 3 days at 130 °C in a sealed tube. The mixture was cooled at room temperature, water (3 mL) was added and product was extracted with EtOAc (4X 10 mL). The combined organic phase was washed with water, dried over Na2S04, concentrated, the residue was purified on 40 g silica gel cartridge using 0% to 100 % EtOAc-Hexanes then 0% to 10 % MeOH-EtOAc, then using second purification on 24 g S1O2 cartridge (gold) to obtain major isomer, N-(6'-(2-(4-(2-(2,6- dioxopiperi din-3 -yl)- 1,3- dioxoisoindolin-5-yl)-2,5-dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'- morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide 10 mg, 3%) as a yellow solid. 'IT NMR (400 MHz, CDCh) d 8.68 (s, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 8.17 (s, 1H), 8.1 1 (d , J = 7.7 Hz, 2H), 7.84 (d, J= 7.9 Hz, 1H), 7.79 (d, J= 1.9 Hz, 1H), 7.77 - 7.62 (m, 2H), 7.23 (s, 1H), 7.13 - 6.93 (m, 2H), 5.00 - 4.86 (m, 1H), 4.76 - 4.47 (m, 2H), 4.28 - 4.06 (m, 1H), 3.94 - 3.78 (m, 4H), 3.57 - 3.31 (m, 2H), 3.26 - 2.97 (m, 6H), 2.98 - 2.64 (m, 6H), 2.53 (s, 3H), 2.16 - 2.05 (m, 1H), 1.39 - 1.03 (m, 6H). LRMs, m/z, 855.52 (M+H)+, 853.60 (M-H) . HPLC (purity
89.56%, cis or trans, 9.32% other isomers).
Example 38 : N-(6'-(2-(3-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-3,6- diazabicyclo[3.1.1]heptan-6-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.038).
Figure imgf000130_0001
Step I, tert-butyl 6-(2-hydroxyethyl)-3,6-diazabicyclo[3.1.1]heptane-3-carboxylate
[0299] A mixture of tert-butyl 3,6-diazabicyclo[3.1.1]heptane-3-carboxylate (1.00 g, 4.79 mmol), bromoethanol (0.50 mL, 7.19 mmol), potassium carbonate (1.99 g, 14.1 mmol) in acetonitrile (10 mL) was stirred at 100 °C overnight. The reaction mixture was cooled to rt, filtered, rinsed with acetonitrile. The filtrate was concentrated and purified on 40 g S1O2 cartridge using 0% to 100 % EtOAc-Hexanes then 0% to 15% MeOH-EtOAc to obtain tert-butyl 6-(2-hydroxyethyl)-3,6-diazabicyclo[3.1.1]heptane-3-carboxylate (770 mg, 66%) as a pale yellow oil. !H NMR (400 MHz, CDCb) d 4.08 (bs, 2H), 3.69 - 3.51 (m, 2H), 3.43 - 2.99 (m, 2H), 2.93 - 2.58 (m, 4H), 2.45 (m, J = 14.1, 6.1 Hz, 1H), 1.61 (d, J = 8.1 Hz, 1H), 1.45 (s, 9H).
StepII, tert-butyl 6-(2-((2'-methyl-5-morpholmo-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)-3,6-diazabicyclo[3.1.1]heptane-3-carboxylate:
[0300] To a stirred solution of tert-butyl 6-(2-hydroxyethyl)-3,6-diazabicyclo[3.1.1]heptane-3- carboxylate (491 mg, 2.03 mmol) in 1 ,4-dioxane (3 mL) was added 60% sodium hydride (94.0 mg, 2.35 mmol) in two portions. The mixture was stirred at rt for 30 min, then N-(6'-fluoro-2- methyl-5'-morphobno-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide (360 mg, 0.326 mmol) was added under inert atmosphere. The reaction mixture was placed on pre -heated oil bath at 80 °C for 2 hrs. The reaction mixture was cooled to rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL), the combined organic layers were washed with water, brine, dried over Na2S04, concentrated. In order to have easier purification, the residue was dissolved in pyridine (1 mL) followed by addition of AC2O (1 mL). The reaction mixture stirred for 1 hrs to convert unreacted alcohol to acetylated product. The mixture was concentrated, then purified on 40 g S1O2 cartridge using a gradient of (0% to 100% EtOAc-Hexanes then methanol in ethyl acetate (0% to 15%) to afford tert-butyl 6-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy) ethyl)-3,6-diazabicyclo[3.1.1]heptane-3- carboxylate (210 mg, 39%) as a pale yellow solid. !H NMR (400 MHz, CDCb) d 8.61 (d, J = 2.5 Hz, 1H), 8.15 (s, 2H), 8.09 (d, J = 8.2 Hz, 1H), 7.92 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 1.9 Hz, 1H), 4.54 (t, J = 5.6 Hz, 2H), 4.16 - 4.02 (m, 3H), 3.95 - 3.78 (m, 4H), 3.24 - 3.09 (m, 5H), 3.07 - 2.91 (m, 4H), 2.51 (s, 3H), 2.44 - 2.30 (m, 1H), 1.69 (d, J = 8.0 Hz, 1H), 1.45 (s, 9H). LRMS, m/z, 683.47 (M+H)+, 681.61 (M-H)
Step III, 6-(2-((2 '-methyl-5-morpholino-5 '-(3-(trifluoromethyl)benzamido)- [3 ,3 '-bipyridin] - 6-yl)oxy)ethyl)-3,6-diazabicyclo[3.1.1]heptan-3-ium chloride:
[0301] To a solution of tert-butyl 6-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)-3,6-diazabicyclo[3.1.1]heptane-3- carboxylate (210 mg, 0.308 mmol) in MeOH (lmL) was added HC1 in 1,4-dioxane (4 N, 4 mL) the mixture was stirred at room temperature for 20 min. The completion of reaction was confirmed by HPLC. The solvents were evaporated to dryness to afford 6-(2-((2'-methyl-5- morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)-3,6- diazabicyclo[3.1.1] heptan-3-ium chloride (200 mg, 105%) as a pale yellow solid. Step IV, Compound 1.038:
[0302] To a mixture of 6-(2-((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl) oxy)ethyl)-3,6-diazabicyclo[3.1.1]heptan-3-ium chloride (150 mg, 0.242 mmol) , 2-(2,6-dioxopiperidin-3- yl)-5-fluoroisoindoline-l ,3-dione (167 mg, 0.606 mmol) in DMSO (4 mL) was added Et;,N (3.94 mL, 17 mmol). The reactiom mixture stirred for 3hrs at 130 °C in a sealed tube. The mixture cooled at room temperature, water 3 mL was added and product was extracted with EtOAc (4X 10 mL). The combined organic phase was washed with water, dried over Na2S04, and concentrated. The residue was purified on 40 g silica gel cartridge (gold ) using 0 to 100 % EtOAc-Hexanes then 0 to 10 % MeOH-EtOAc to afford N-(6'- (2-(3-(2- (2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)ethoxy)- 2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (40 mg, 20%) as a yellow solid.‘H NMR (400 MHz, CDCh) d 8.69 (s, 1H), 8.30 - 8.01 (m, 5H), 7.84 (d, J= 7.5 Hz, 1H), 7.73 (s, 1H), 7.66 (t, J= 7.6 Hz, 1H), 7.59 (d, J= 8.0 Hz, 1H), 7.03 (s, 1H), 6.84 (s,
1H), 6.58 (d, J= 7.5 Hz, 1H), 5.00 - 4.80 (m, 1H), 4.57 - 4.31 (m, 4H), 3.96 - 3.75 (m, 4H),
3.28 (d, J= 10.9 Hz, 2H), 3.18 - 2.98 (m, 6H), 2.94 - 2.58 (m, 6H), 2.50 (s, 3H), 2.10 (d, J= 7.9 Hz, 2H). LRMS, m/z, 861.50 (M+Na)+, 837.63 (M-H) . HPLC (purity 95%)
Example 39: N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5-yl)-2,6- dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3- (trifluoromethyl)benzamide ( Compound 1.039 ).
Figure imgf000133_0001
Step I, tert-butyl 4-(2-hydroxyethyl)-3,5-dimethylpiperazine-l-carboxylate:
[0303] 2-bromoethanol (0.8 ml, 1 1.3 mmol) was added to cold mixture of tert-butyl 3,5- dimethylpiperazine- 1 -carboxylate (1.62 g, 7.56 mmol) and NaH (60%, 454 mg, 2.45 mmol) in 1,4-dioxane (4 mL). The mixture was heated to 90 °C 18 hrs. The solvent was evaporated and the residue was purified on 40 g silica gel cartridge using 0% to 100% (EtOAc-Hexanes) then 0% to 15% MeOH-EtOAc to obtain tert-butyl 4-(2- hydroxyethyl)-3,5-dimethylpiperazine-l- carboxylate (1.1 g, 56%) pale yellow oil. 1H NMR (400 MHz, CDCh) d 3.81 (bs, 2H), 3.57 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 6.2 Hz, 2H), 2.70 2.46 (m, 4H), 1.46 (s, 9H), 1.14 1.00 (m, 6H). Step II, tert-butyl 3,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3-
(trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazme-l-carboxylate:
[0304] To a stirred solution of tert-butyl 4-(2-hydroxyethyl)-3,5-dimethylpiperazine-l- carboxylate (982mg, 3.80 mmol) in 1,4-dioxane (5 mL) was added 60% sodium hydride (152 mg, 3.80 mmol) in two portions. The mixture was stirred at rt for 30 min, then N-(6'-fluoro-2- methyl-5'-morpholino-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl) benzamide (350 mg, 0.326 mmol) was added under inert atmosphere. The reaction mixture was placed on pre -heated oil bath at 80 C for 2 hrs. The reaction mixture was cooled into rt, poured into water (5 mL), extracted with EtOAc (4 X 20 mL). The combined organic layers were washed with water, brine, dried over Na2S04, concentrated. In order to have easier purification, the residue was dissolved in pyridine (1 mL) followed by addition of AC2O (1 mL). The reaction mixture stirred for 1 hrs to convert unreacted alcohol to acetylated product. The mixture was concentrated and purified on 40 g S1O2 cartridge using a gradient of (0% to 100% EtOAc-Hexanes then methanol in ethyl acetate (0% to 15%) to afford tert-butyl 3,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl) oxy)ethyl)piperazine- 1 -carboxylate (300 mg, 56%) as a pale yellow solid. !H NMR (400 MHz, CDCh) d 8.61 (d, J = 2.6 Hz, 1H), 8.15 (s, 2H), 8.09 (d, J = 7.6 Hz, 1H), 7.95 - 7.81 (m, 2H), 7.79 (d, J = 2.0 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 4.48 (s, 2H), 3.94 - 3.78 (m, 6H), 3.15 (dd, J = 15.2, 8.2 Hz, 6H), 2.81 - 2.64 (m, 2H), 2.63 - 2.53 (m, 2H), 2.52 (s, 3H), 1.46 (s, 9H), 1.17 (t, J = 17.4 Hz, 6H). LRMS, m/z, 46 (M+H)+, 697.54 (M-H)-
Step III, 3,5-dimethyl-4-(2- ((2'-methyl-5-morpholino-5'-(3-(trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin- 1 -ium chloride:
[0305] To a solution of tert-butyl 3,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)- [3,3'-bipyridin]-6-yl)oxy)ethyl)piperazine-l-carboxylate (280 mg) in MeOH (1 mL) was added HC1 (4M, 1.3 mL in 1 ,4-dioxane). The mixture was stirred at rt for 1.5 h, and concentrated to obtain 3,5-dimethyl-4-(2- ((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'-bipyridin]-6-yl)oxy)ethyl)piperazin-l- ium chloride (250 mg, 98%) as a pale yellow solid. LRMS, m/z, 599.42 (M+H)+, 597.47 (M-H)~
Step IV, Compound 1.039 :
[0306] To a solution of 3,5-dimethyl-4-(2-((2'-methyl-5-morpholino-5'-(3- (trifluoromethyl)benzamido)-[3,3'- bipyridin]-6-yl)oxy)ethyl)piperazin-l-ium chloride (250 mg) in DMSO (3 mL) was added EίbN (0.33 mL, 2.36 mmol), followed by addition of 2-(2,6- dioxopiperidin-3-yl)-5-fluoroisoindoline-l,3-dione (141 mg, 2.36 mmol). The reaction mixture was heated to 120 °C overnight. The reaction mixture was cooled to rt, water (5 mL) was added and extracted with EtOAc (4X15 mL). The combined organic layers were washed with water, brine, dried over Na2S04 and concentrated under vacuum. The residue was purified on 40 g silica gel using 0 to 10% MeOH-EtOAc to obtain N-(6'-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-5-yl)-2,6- dimethylpiperazin-l-yl)ethoxy)-2-methyl-5'-morpholino-[3,3'- bipyridin]-5-yl)-3-(trifluoromethyl) benzamide (120 mg, 35.7%) as a yellow solid. 1 H NMR (400 MHz, CDCh) d 8.62 (d, 7 = 2.5 Hz, 1H), 8.20 8.11 (m, 2H), 8.09 (d , 7 = 7.8 Hz, 1H),
8.00 (d, 7 = 14.7 Hz, 2H), 7.85 (d, = 7.7 Hz, 1H), 7.79 (d, 7= 2.0 Hz, 1H), 7.72 7.61 (m, 2H), 7.25 (d, 7= 2.3 Hz, 1H), 7.07 (d, 7= 2.0 Hz, 1H), 7.02 (dd, 7= 8.6, 2.2 Hz, 1H), 4.93 (dd, 7 = 12.3, 5.2 Hz, 1H), 4.52 (t, 7= 6.8 Hz, 2H), 3.93 3.82 (m, 4H), 3.72 (d, 7= 12.0 Hz, 2H), 3.21 (t, 7= 6.8 Hz, 2H), 3.16 3.07 (m, 4H), 2.98 2.64 (m, 7H), 2.51 (s, 3H), 2.15 2.07 (m, 1H), 1.30 (d, 7= 5.9 Hz, 6H). LRMs, m/z, 877.47 (M+Na)+, 853.67 (M-H) . HPLC (purity, 95.6%).
Biochemical & Biological Data
Biological Example 1 - The currently disclosed compound suprisingly improve CRAF degradation at lower efficacious concentrations in the Degradation Assay
Materials and Methods
[0307] Calu-6 cells were stably transfected with a CRAF fusion protein with a fluorescent protein attached to the C-terminus of CRAF in order to monitor protein levels via fluorescence. Cells were seeded into a 96-well plate and allowed to adhere overnight. The next day cells are treated with compounds at the doses indicated in the table below and analyzed over time. Data was collected by monitoring Fluorescent CRAF protein levels an IncuCyte S3 Live Cell Analysis System (Essen BioScience). The fluorescent CRAF fusion protein was excited at 440- 480 nm and the emission was measured at 504-544 nm.
[0308] Calu6_CRAF fusion protein cells are imaged and analyzed using an Incucyte S3 live cell analysis system (Essen Biosciences). Fluorescence signal is due to expression of the fluorescent protein covalently fused to the C-terminus of CRAF and degradation is assessed by measuring fluorescence/confluence (-cell number) after treatment with compounds of the present disclosure.
[0309] Phase contrast images are analyzed using Incucyte S3 software. Percent confluence is calculated using a phase contrast mask to quantify the area of the image occupied by the
Calu6_CRAF fusion protein cells.
[0310] Fluorescent images are analyzed using Incucyte S3 software. Green Corrected Units (GCU) is calculated using a fluorescence mask to quantify the area of the image occupied by green fluorescent signal and the intensity of the signal. [0311] The following compounds are tested in this assay:
[0312] Reference Compound 1 (Ref. Comp. 1) (WO2018/200981) is included for comparison:
Figure imgf000136_0003
[0313] Compound 1.009:
Compound 1,009
Figure imgf000136_0001
[0314] Compound 1.032:
Figure imgf000136_0004
[0315] Compound 1.034:
Figure imgf000136_0002
[0316] Compound 1.039:
Figure imgf000136_0005
[0317] Compound 1.035
Figure imgf000137_0001
[0318] Compound 1.038
Figure imgf000137_0002
[0319] Compound 1.037
Figure imgf000137_0003
[0320] Compound 1.033
Figure imgf000137_0004
[0321] Compound 1.036
Figure imgf000137_0005
Results
[0322] The results for each test compound are summarized in Table 6, below.
Table 6: Degradation Assay Results Summary
Figure imgf000137_0006
Figure imgf000138_0001
[0323] The reported Dmax (%) represents the maximum precent of total fluorescent CRAF that was degraded by the given compounds.
[0324] The reported DCso (mM) reports the concentration at which 50% of the degradation effect was observed for the given compounds.
[0325] FIG. 1 shows the CRAF degradation over time after addition of Ref. Comp. 1 at 12.5 mM. The percent Dmax occurred at 1.5 hr and was 23.3 ± 1.33 %. [0326] FIG. 2 shows the CRAF degradation over time after addition of Compound 1.009 at 1 mM. The percent Dmax occurred at 34.5 hr and was 34.4 ± 1.27 %.
[0327] FIG. 3 shows the CRAF degradation over time after addition of Compound 1.032 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 32 hr and was 24.6 ± 1.0 %.
[0328] FIG. 4 shows the CRAF degradation over time after addition of Compound 1.034 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 26 hr and was 23.7 ±
2.2 %.
[0329] FIG. 5 shows the CRAF degradation over time after addition of Compound 1.039 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 32 hr and was 26.2 ±
1.3 %.
[0330] FIG. 6 shows the CRAF degradation over time after addition of Compound 1.035 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 36 hr and was 30.6 ±
2.7 %.
[0331] FIG. 7 shows the CRAF degradation over time after addition of Compound 1.038 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 26 hr and was 19.1 ±
0.4 %.
[0332] FIG. 8 shows the CRAF degradation over time after addition of Compound 1.037 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 35 hr and was 26.5 ±
2.6 %.
[0333] FIG. 9 shows the CRAF degradation over time after addition of Compound 1.033 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 35 hr and was 26.1 ±
1.3 %.
[0334] FIG. 10 shows the CRAF degradation over time after addition of Compound 1.036 at the indicated concentrations. In the 1 mM trial, the percent Dmax occurred at 24 hr and was 27.7 ± 0.9 %. [0335] Surprisingly, 1 mM or less of Compounds 1.009 and 1.032-1.039 provide a faster degradation rate and a higher or substantially similar magnitude of CRAF reduction as compared to 12 mM of Ref. Comp. 1.
Biological Example 2 - Degradation Assay Dose Response Materials and Methods
[0336] The Calu6 CRAF degradation assay described above was used to determine the dose response of Compound 1.009. The percent CRAF Degradation after 42 hours of incubation with Compound 1.009 at various concentrations was determined as the percentage of
fluorescence/confluence levels relative to DMSO control. Further parameters such as Dmax, endpoint DC so, Hillslope, R square were determined using GraphPad Prism Version 7.04
Results
[0337] FIG. 11 shows the dose response curve of Compound 1.009 at an endpoint of 42 hours by plotting the percent CRAF degradation at different concentrations of Compound 1.009. The DC50 was determined to be 104.4 ± 5.2 nM, and the Dmax was 33.5% of - 100X overexpressed CRAF. Compound 1.009 also showed minimal cytotoxicity in this assay (as determined by observing a low background and high signal to noise ratio.
[0338] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:
1. A RAF-Degrading Conjugate Compound comprising a Ligand for RAF covalently attached via a Linker Component to a Degradation Signaling Agent, wherein
the Linker Component comprises 5 to 10 linking atoms and 1 or 2 cyclic moieties,
wherein each cyclic moiety is independently selected from the group consisting of C3-8 cycloalkyl, a triazole, a phenyl, a 6- to 8-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms, a six-membered heteroaryl comprising 1 to 2 nitrogen atoms, and C7-11 spirocylic moieties comprising 0 to 2 nitrogen atoms; or the Linker Component comprising 9 to 15 linking atoms and two cyclic moieties, wherein the cyclic moieties are independently selected from the group consisting of C3-8 cycloalkyl, a triazole, a phenyl, a 6- to 8-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms, a six-membered heteroaryl comprising 1 to 2 nitrogen atoms, and C7-11 spirocylic moieties comprising 0 to 2 nitrogen atoms.
2. The RAF-Degrading Conjugate Compound of claim 1, wherein the Linker Component comprises 5 to 10 linking atoms and 1 or 2 cyclic moieties, wherein each cyclic moiety is independently selected from the group consisting of a triazole, a phenyl, and a 6- membered heterocycloalkyl comprising 1 to 2 nitrogen atoms.
3. The RAF-Degrading Conjugate Compound of claim 1 , wherein the Linker Component comprising 9 to 15 linking atoms and two cyclic moieties, wherein the cyclic moieties are independently selected from the group consisting of a triazole, a phenyl, and a 6- membered heterocycloalkyl comprising 1 to 2 nitrogen atoms
4. The RAF-Degrading Conjugate Compound of any one of claims 1 to 3, wherein when two cyclic moieties are present, each cyclic moiety is different.
5. The RAF-Degrading Conjugate Compound of claim 1 or claim 4, wherein one of the cyclic moieties of the Linker Component is directly attached to the Degradation Signaling Agent.
6. The RAF-Degrading Conjugate Compound of any one of claims 1 to 5, wherein the Linker Component comprises a tertiary amine that is directly attached to the Degradation Signaling Agent is a tertiary amine.
7. The RAF-Degrading Conjugate Compound of any one of claims 1 to 6, wherein at least one cyclic moiety of the Linker Component is a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms, and the 6-membered heterocycloalkyl comprises a 1 ,4- linkage to the remainder of the Linker Component, to the Ligand for RAF, or the Degradation Signaling Agent.
8. The RAF-Degrading Conjugate Compound of any one of claims 1 to 6, wherein at least one cyclic moiety of the Linker Component is a triazole or a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms.
9. The RAF-Degrading Conjugate Compound of claim 8, wherein the triazole or the 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms of the Linker Component is covalently attached to the Degradation Signaling Agent.
10. The RAF-Degrading Conjugate Compound of claim 9, wherein the atom of the Linker Component that is covalently attached to the Degradation Signaling Agent is an amine.
11. The RAF-Degrading Conjugate Compound of any one of claims 1 to 10, wherein the 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms is a piperidine or a piperazine.
12. The RAF-Degrading Conjugate Compound of claim 1, wherein the Linker Component is selected from the group consisting of
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
13. The RAF-Degrading Conjugate Compound of claim 1, wherein the Linker Component is selected from the group consisting of
e
Figure imgf000144_0002
ff)
Figure imgf000145_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
14. The RAF-Degrading Conjugate Compound of any one of claims 1 to 13, wherein the Ligand for RAF is RAF709.
15. The RAF-Degrading Conjugate Compound of any one of claims 1 to 14, wherein the E3 ligase recognition agent is pomalidomide.
16. The RAF-Degrading Conjugate Compound of claim 14 or claim 15, wherein RAF709 has a structure selected from the group consisting of
Figure imgf000145_0002
where each X is N or CH and the wavy line indicates the site of attachment of the Linker Component.
17. The RAF-Degrading Conjugate Compound of claim 16, wherein X is N.
18. The RAF-Degrading Conjugate Compound of any one of claims 15 to 17, wherein pomalidomide has a structure selected from the group consisting of
Figure imgf000146_0001
wherein the wavy line indicates the site of attachment of the Linker Component.
19. A RAF-Degrading Conjugate Compound comprising a Degradation Signaling Agent covalently attached via a Linker Component to a Ligand for RAF, wherein the Linker Component comprises 5 to 10 linking atoms.
20. The RAF-Degrading Conjugate Compound of claim 19, wherein the Linker Component comprises at least one cyclic moiety selected from the group consisting of a C3-8 cycloalkyl, a triazole, a phenyl, a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms, a six-membered heteroaryl comprising 1 to 2 nitrogen atoms, and C7-11 spirocylic moieties comprising 0 to 2 nitrogen atoms.
21. The RAF-Degrading Conjugate Compound of claim 19, wherein the Linker Component comprises at least one cyclic moiety selected from the group consisting of a triazole, a phenyl, and a 6-membered heterocycloalkyl comprising 1 to 2 nitrogen atoms.
22. The RAF-Degrading Conjugate Compound of claim 20 or claim 21, wherein the cyclic moiety of the Linker Component is directly attached to the Degradation Signaling Agent.
23. The RAF-Degrading Conjugate Compound of any one of claims 19 to claim 22, wherein the Linker Component comprises a tertiary amine that is directly attached to the Degradation Signaling Agent.
24. The RAF-Degrading Conjugate Compound of claim 19, wherein the Linker Component is selected from the group consisting of
Figure imgf000147_0001
Figure imgf000148_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
25. The RAF-Degrading Conjugate Compound of claim 19, wherein the Linker Component is selected from the group consisting of
e
Figure imgf000148_0002
ff)
Figure imgf000149_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation
Signaling Agent.
26. The RAF-Degrading Conjugate Compound of any one of claims 19 to 25, wherein the Ligand for RAF is RAF709.
27. The RAF-Degrading Conjugate Compound of any one of claims 19 to 26, wherein the E3 ligase recognition agent is pomalidomide.
28. The RAF-Degrading Conjugate Compound of claim 26 or claim 27, wherein RAF709 has a structure selected from the group consisting of
Figure imgf000149_0002
where each X is N or CH and the wavy line indicates the site of attachment of the Linker Component.
29. The RAF-Degrading Conjugate Compound of claim 28, wherein X is N.
30. The RAF-Degrading Conjugate Compound of any one of claims 27 to 29, wherein pomalidomide has a structure selected from the group consisting of
Figure imgf000150_0001
wherein the wavy line indicates the site of attachment of the Linker Component.
31. A RAF-Degrading Conjugate Compound comprising a Ligand for RAF covalently attached via a Linker Component to a Degradation Signaling Agent, wherein
the Ligand for RAF is selected from the group consisting of sorafenib RAF709, LHX254, Hah 10, PF-04880594, LY3009120, and PLX4720;
the Degradation Signaling Agent is an E3 ligase recognition agent; and
the Linker component comprises 5 to 22 linking atoms.
32. A RAF-Degrading Conjugate Compound of claim 31, wherein the Ligand for RAF is RAF709.
33. A RAF-Degrading Conjugate Compound of claim 31 or claim 32, wherein the E3 ligase recognition agent is pomalidomide.
34. A RAF-Degrading Conjugate Compound of claim 31 or claim 32, wherein the E3 ligase recognition agent is a small molecule VHL ligand.
35. A RAF-Degrading Conjugate Compound of claim 31 or claim 32, wherein the E3 ligase recognition agent is Inhibitor of Apoptosis Protein (IAP) Ligand.
36. The RAF-Degrading Conjugate Compound of any one of claims 32 to 35, wherein RAF709 has a structure selected from the group consisting of
Figure imgf000151_0001
where each X is N or CH and the wavy line indicates the site of attachment of the Linker Component.
37. The RAF-Degrading Conjugate Compound of claim 36, wherein X is N.
38. The RAF-Degrading Conjugate Compound of claim 33 or claim 36, wherein pomalidomide has a structure selected from the group consisting of
Figure imgf000151_0002
wherein the wavy line indicates the site of attachment of the Linker Component.
39. The RAF-Degrading Conjugate Compound of claim 34 or claim 36, wherein VHL Ligand has a structure
Figure imgf000152_0001
wherein the wavy line indicates the site of attachment of the Linker Component.
40. The RAF-Degrading Conjugate Compound of claim 35 or claim 36, wherein IAP Ligand has a structure
Figure imgf000152_0002
wherein the wavy line indicates the site of attachment of the Linker Component.
41. The RAF-Degrading Conjugate Compound of any one of claims 32 to 40, wherein the Linker Component has a formula selected from the group consisting of
Figure imgf000152_0003
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation
Signaling Agent.
42. The RAF-Degrading Conjugate Compound of any one of claims 32 to 40, wherein the Linker Component is selected from the group consisting of
Figure imgf000155_0002
Figure imgf000156_0001
wherein the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
43. A RAF-Degrading Conjugate Compound wherein the Ligand for RAF has the structure
Figure imgf000156_0002
wherein X is N or CH and the wavy line indicates the site of attachment of the Linker
Component;
the Degradation Signaling Agent is selected from the group consisting of
Figure imgf000157_0001
wherein the wavy line indicates the site of attachment of the Linker Component; and the linker component is selected from the group consisting of
Figure imgf000157_0002
Figure imgf000158_0001
WO 2020/168172
Figure imgf000160_0001
the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
44. A RAF-Degrading Conjugate Compound wherein the Ligand for RAF has the structure
Figure imgf000160_0002
wherein X is N or CH and the wavy line indicates the site of attachment of the Linker Component;
the Degradation Signaling Agent is selected from the group consisting of
Figure imgf000161_0001
wherein the wavy line indicates the site of attachment of the Linker Component; and the linker component is selected from the group consisting of
Figure imgf000161_0002
Figure imgf000162_0001
the wavy line on the left indicates the site of attachment to the Ligand for RAF and the wavy line on the right of the structure indicates the site of attachment to the Degradation Signaling Agent.
45. The RAF-Degrading Conjugate Compound of claim 43 or claim 44, wherein the Degradation Signaling Agent is
Figure imgf000162_0002
46. The RAF-Degrading Conjugate Compound of claim 43 or claim 44, wherein the Degradation Signaling Agent is
Figure imgf000162_0003
47. A RAF-Degrading Conjugate Compound selected from the Examples &
Tables.
48. A pharmaceutical composition comprising the compound of any one of claims 1 to 47 and a pharmaceutically acceptable excipient or carrier.
49. A method of treating a disease or disorder selected from the group consisting of a cancer and a RASopathy comprising administering to a subject in need thereof a therapeutically effective amount of the RAF-Degrading Conjugate Compound of any one of claims 1 to 47.
50. A method of claim 49, wherein the disease or condition is a cancer.
51. A method of claim 50, wherein the cancer is selected from the group consisting of is melanoma, an epithelial cancer ( e.g prostate cancer, ovarian cancer, breast cancer), or a blood cancer (e.g., leukemia, lymphoma, multiple myeloma).
52. A method of claim 49, wherein the disease or condition is an RASopathy.
53. A method of claim 52, wherein the RASopathy is selected from the group consisting of Noonan syndrome, Costello syndrome, Legius syndrome, and LEOPARD syndrome.
54. A method of claims 50 or 51, further comprising administering an additional cancer therapeutic agent.
55. A method of claim 54, wherein the additional cancer therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, and an endocrine therapy.
56. A kit comprising a RAF-Degrading Conjugate Compound.
57. The kit of claim 56, further comprising a label with instructions for use.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022060996A1 (en) * 2020-09-18 2022-03-24 Kinnate Biopharma Inc. Inhibitors of raf kinases
US11377431B2 (en) 2020-10-12 2022-07-05 Kinnate Biopharma Inc. Inhibitors of RAF kinases
US11667634B2 (en) 2019-05-03 2023-06-06 Kinnate Biopharma Inc. Inhibitors of RAF kinases
US11918587B2 (en) 2021-04-23 2024-03-05 Kinnate Biopharma Inc. Treatment of cancer with a RAF inhibitor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140356322A1 (en) * 2012-01-12 2014-12-04 Yale University Compounds & Methods for the Enhanced Degradation of Targeted Proteins & Other Polypeptides by an E3 Ubiquitin Ligase
US20180346457A1 (en) * 2017-04-28 2018-12-06 Quartz Therapeutics, Inc. Raf-degrading conjugate compounds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140356322A1 (en) * 2012-01-12 2014-12-04 Yale University Compounds & Methods for the Enhanced Degradation of Targeted Proteins & Other Polypeptides by an E3 Ubiquitin Ligase
US20180346457A1 (en) * 2017-04-28 2018-12-06 Quartz Therapeutics, Inc. Raf-degrading conjugate compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PUBCHEM 15 December 2018 (2018-12-15), Database accession no. CID 377306921 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11667634B2 (en) 2019-05-03 2023-06-06 Kinnate Biopharma Inc. Inhibitors of RAF kinases
WO2022060996A1 (en) * 2020-09-18 2022-03-24 Kinnate Biopharma Inc. Inhibitors of raf kinases
US11407737B2 (en) 2020-09-18 2022-08-09 Kinnate Biopharma Inc. Inhibitors of RAF kinases
US11377431B2 (en) 2020-10-12 2022-07-05 Kinnate Biopharma Inc. Inhibitors of RAF kinases
US11746095B2 (en) 2020-10-12 2023-09-05 Kinnate Biopharma Inc. Inhibtors of RAF kinases
US11918587B2 (en) 2021-04-23 2024-03-05 Kinnate Biopharma Inc. Treatment of cancer with a RAF inhibitor

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