WO2020168172A1 - Composés conjugués pour la dégradation de raf - Google Patents

Composés conjugués pour la dégradation de 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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raf
linker component
conjugate compound
mmol
degrading conjugate
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PCT/US2020/018260
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English (en)
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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/fr

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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.

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Abstract

La présente invention concerne, entre autres, des composés conjugués de dégradation de RAF qui sont utiles dans le traitement du cancer et d'autres maladies associées à RAF. L'invention concerne également des compositions pharmaceutiques, des méthodes de traitement et des kits comprenant un composé conjugué de dégradation de RAF.
PCT/US2020/018260 2019-02-15 2020-02-14 Composés conjugués pour la dégradation de raf WO2020168172A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022060996A1 (fr) * 2020-09-18 2022-03-24 Kinnate Biopharma Inc. Inhibiteurs de 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
EP4051386A4 (fr) * 2019-10-30 2024-05-22 Dana Farber Cancer Inst Inc Agents de dégradation à petites molécules d'hélios et procédés d'utilisation

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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

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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

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Cited By (7)

* 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
EP4051386A4 (fr) * 2019-10-30 2024-05-22 Dana Farber Cancer Inst Inc Agents de dégradation à petites molécules d'hélios et procédés d'utilisation
WO2022060996A1 (fr) * 2020-09-18 2022-03-24 Kinnate Biopharma Inc. Inhibiteurs de 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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