WO2024107763A1 - N6-adenosine-methyltransferase protacs and methods of use thereof - Google Patents

N6-adenosine-methyltransferase protacs and methods of use thereof Download PDF

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Publication number
WO2024107763A1
WO2024107763A1 PCT/US2023/079694 US2023079694W WO2024107763A1 WO 2024107763 A1 WO2024107763 A1 WO 2024107763A1 US 2023079694 W US2023079694 W US 2023079694W WO 2024107763 A1 WO2024107763 A1 WO 2024107763A1
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alkyl
heterocycloalkyl
cycloalkyl
pharmaceutically acceptable
substituted
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PCT/US2023/079694
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French (fr)
Inventor
Annalisa INVERNIZZI
Francesco ERRANI
Frantisek ZALESAK
Rajiv K. BEDI
Ivan CORBESKI
Elena BOCHENKOVA
Marcin HEROK
Amedeo Caflisch
Michael John Hartshorn
Valeria ROMANUCCI
Gordon Saxty
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University Of Zurich
Scaffold Therapeutics, Inc.
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Publication of WO2024107763A1 publication Critical patent/WO2024107763A1/en

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    • 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
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • genes are regulated at the level of the transcriptome (messenger RNA obtained through transcription of the genome) through dynamic levels of mRNA modifications.
  • the conversion of adenosine to N6-methyladenosine (m 6 A) is the most common internal post-transcriptional modification (also called epitranscriptomic modification) in eukaryotic mRNA.
  • the m 6 A level can vary among different tissues, development states or in response to cellular stresses.
  • m 6 A On the molecular level introduction of the m 6 A affects the structure of RNA and its ability to form protein-RNA interactions, and as a consequence it modulates processing, translation, and stability of the cellular transcripts. As a consequence, m 6 A is implicated in controlling embryonic development processes and stem cell differentiation, regulating the mammalian circadian clock, and modulating stress response, e.g., heat shock.
  • the dynamic level of m 6 A is regulated by the interplay of erasers and writer proteins. While the m 6 A writer has been known for two decades, the discovery of m 6 A-specific eraser proteins FTO (ALKBH9) and ALKBH5 has ultimately demonstrated the reversibility of the modification and its regulatory role. These m 6 A demethylases belong to the dioxygenase AlkB family whose enzymatic reaction depends on Fe(II) and 2- oxoglutaric acid (2OG). The core writer complex is formed by two methyltransferase -like proteins, METTL3 and METTL14, which rely on additional cofactors for mRNA substrate recruitment, including WTAP and RBM15.
  • the METTL3-METTL14 complex transfers a methyl group from S-adenosylmethionine (SAM) to the adenosine within the consensus sequence of 5’-GGACU-3’.
  • SAM S-adenosylmethionine
  • the individual depletion of METTL3 or METTL14 reduces the level of m 6 A in HeLa cells. More importantly, deregulation of METTL3 has recently been linked to specific tumors, such as acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma. Moreover, inhibiting m 6 A modification shows a broad antiviral effect. Therefore, small -molecule modulators of the METTL3-METTL14 writer has potential therapeutic use in cancer and viral infection.
  • the objective of the present invention is to provide means and methods to use the PROTAC therapeutic modalities to modulate the levels of m6A modification with the goal of regulating gene expression for cancer therapy. This objective is attained by the subject-matter of the independent claims of the present specification.
  • the present disclosure relates to proteolysis targeting chimeras (PROTACs) that modulate N6- adenosine-methyltransferase and methods of using the same.
  • the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase.
  • the bifimctional compounds target a heterodimeric complex METTL3-METTL14.
  • the bifimctional compounds bind to the heterodimeric complex METTL3-METTL14.
  • the bifimctional compounds modulate the heterodimeric complex METTL3-METTL14.
  • the bifimctional compounds inhibit and degrade the heterodimeric complex METTL3- METTL14. In some embodiments, the bifimctional compounds target METTL3. In some embodiments, the bifimctional compounds bind to METTL3. In some embodiments, the bifimctional compounds modulate, inhibit, and/or degrade METTL3. In some embodiments, the bifunctional compounds target METTL14 via the METTL3-METTL14 complex. In some embodiments, the bifimctional compounds modulate, inhibit, and/or degrade METTL14.
  • the present disclosure relates to a bifimctional compound having a structure of Formula (I), or a pharmaceutically acceptable salt thereof,
  • MBM is a moiety that binds to METTL3
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • L is a linker that imparts rigidity, wherein L has a structure of Formula (V),
  • CS-Galkynyl cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; or two R La are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R;
  • each R a is independently C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, G-Galkcnyl.
  • G-Galkynyl cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each R b is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, G-Galkcnyl.
  • G-Galkynyl cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, G-Galkcnyl.
  • Ci-C 3 heteroalkyl or CYC, cycloalkyl.
  • the present disclosure relates to a bifimctional compound having a structure of Formula (lib*), or a pharmaceutically acceptable salt thereof,
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • L is a covalent linker
  • R 1 , U, V, X, Y, Z 1 , Z 2 , n, R 2 , R 13 and R 3 have the meanings defined in Formula (II).
  • the present disclosure relates to a bifimctional compound having a structure of Formula (lie*), or a pharmaceutically acceptable salt thereof,
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • L is a covalent linker
  • R 1 , U, V, X, Y, Z 1 , Z 2 , n, R 2 , R 13 and R 3 have the meanings defined in Formula (II).
  • the present disclosure relates to bifimctional compounds for use as a medicament.
  • the present disclosure relates to bifimctional compounds for use in treatment of cancer.
  • the cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a bifimctional compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure relates to a method of treating disease, the method comprising administering to a subject in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound disclosed herein.
  • the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6 -adenosine -methyltransferase, of the general formula (A-I)
  • the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase, of the general formula (A-II)
  • Fig. 1 shows A) Design of METTL3 inhibitor 2 from hit compound 1. The bonds formed are depicted in red. The IC50 values refer to the biochemical assay based on time resolved-Forster resonance energy transfer (TR-FRET). B) Superimposition of compounds 1 (grey, from X- ray with METTL3, PDB code: 7NHI) and 2 (cyan, drawn in Pymol).
  • Fig. 2 shows design of compound 8 starting from 5, the newly formed bonds are depicted in red.
  • Inhibitor 5 in the METTL3 binding site with relevant residues (carbon atoms in grey). The main intermolecular interactions are displayed (yellow dashed lines, PDB code: 7008). B) Superimposition of inhibitors 5 (cyan) and 7 (green, PDB code: 7009). C). Superimposition of inhibitors 5 (cyan) and 8 (yellow, PDB code: 7O0L), and interactions of the lactam with the side chain amide of Gln550.
  • Fig. 3 shows A) Unusual interaction of the fluorine atom of compound 20 with Pro397 amide 71- system, PDB code: 7029. B) Van der Waals contacts between the fluorine atom of inhibitor 21 and the side chains of Ser511 and Tyr406, PDB code: 7O2E.
  • Fig. 4 shows TR-FRET dose response curves (n > 3) measured for compounds 1, 22 and SAH and chemical structure of the lead compound 22.
  • B) InCELL Pulse dose response curve (n 3) in HEK293T cells at 46 °C.
  • Fig. 5 shows thermal shift assay results. Shown are the first derivative of the melting curves of
  • Fig. 6 shows thermal shift assay results. Shown are the first derivative of the melting curves of
  • Fig. 7 Exemplary compounds.
  • Fig . 9 shows the degradation of METTL3 protein after treatment with 2 pM concentration of various
  • PROTAC molecules for 24h measured with Western blot.
  • Fig. 10 shows the degradation of METTL14 protein after treatment with 2 pM concentration of various PROTAC molecules for 24h, measured with Western blot.
  • Fig. 11 shows the correlation between METTL3 and METTL14 degradation of various PROTAC molecules measured by Western blot.
  • Carboxyl refers to -COOH.
  • Cyano refers to -CN.
  • Alkyl refers to a straight-chain, or branched-chain saturated hydrocarbon monoradical having from one to about ten carbon atoms, more preferably one to six carbon atoms. Examples include, but are not limited to methyl, ethyl, n-propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3-methyl-l- butyl, 2-methyl-3 -butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 -methyl- 1 -pentyl, 4-methyl-l -pentyl, 2- methyl -2 -pentyl, 3 -methyl -2 -pentyl, 4-methyl -2 -pentyl, 2,2-dimethyl-l -butyl, 3, 3 -dimethyl- 1 -butyl, 2 -ethyl - 1 -butyl,
  • a numerical range such as “Ci- G, alkyl” or “Ci-ealkyl”, means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated.
  • the alkyl is a Ci-ioalkyl.
  • the alkyl is a Ci-ealkyl.
  • the alkyl is a G --alkyl.
  • the alkyl is a Ci-4alkyl.
  • the alkyl is a Ci-3alkyl.
  • an alkyl group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • the alkyl is optionally substituted with oxo, halogen, -CN, -COOH, -COOMe, -OH, -OMe, -NH2, or -NO2.
  • the alkyl is optionally substituted with halogen, -CN, -OH, or -OMe.
  • the alkyl is optionally substituted with halogen.
  • Alkenyl refers to a straight-chain, or branched-chain hydrocarbon monoradical having one or more carbon-carbon double-bonds and having from two to about ten carbon atoms, more preferably two to about six carbon atoms.
  • C2-C6 alkenyl or “C2-6alkenyl”
  • alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated.
  • an alkenyl group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • the alkenyl is optionally substituted with oxo, halogen, -CN, -COOH, -COOMe, -OH, -OMe, -NH2, or -NO2.
  • the alkenyl is optionally substituted with halogen, -CN, -OH, or -OMe.
  • the alkenyl is optionally substituted with halogen.
  • Alkynyl refers to a straight-chain or branched-chain hydrocarbon monoradical having one or more carboncarbon triple-bonds and having from two to about ten carbon atoms, more preferably from two to about six carbon atoms. Examples include, but are not limited to ethynyl, 2-propynyl, 2-butynyl, 1,3-butadiynyl and the like.
  • a numerical range such as “C2-C6 alkynyl” or “C2-6alkynyl”, means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkynyl” where no numerical range is designated.
  • an alkynyl group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • the alkynyl is optionally substituted with oxo, halogen, -CN, -COOH, COOMe, -OH, -OMe, - NH2, or -NO2.
  • the alkynyl is optionally substituted with halogen, -CN, -OH, or - OMe.
  • the alkynyl is optionally substituted with halogen.
  • Alkylene refers to a straight or branched divalent hydrocarbon chain. Unless stated otherwise specifically in the specification, an alkylene group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkylene is optionally substituted with oxo, halogen, -CN, -COOH, COOMe, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkylene is optionally substituted with halogen, -CN, -OH, or -OMe. In some embodiments, the alkylene is optionally substituted with halogen.
  • Alkoxy refers to a radical of the formula -Oalkyl where alkyl is as defined above. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkoxy is optionally substituted with halogen, -CN, -COOH, COOMe, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkoxy is optionally substituted with halogen, -CN, -OH, or -OMe. In some embodiments, the alkoxy is optionally substituted with halogen.
  • Aryl refers to a radical derived from a hydrocarbon ring system comprising 6 to 30 carbon atoms and at least one aromatic ring.
  • the aryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the aryl is bonded through an aromatic ring atom) or bridged ring systems.
  • the aryl is a 6- to 10-membered aryl.
  • the aryl is a 6-membered aryl (phenyl).
  • Aryl radicals include, but are not limited to, aryl radicals derived from the hydrocarbon ring systems of anthrylene, naphthylene, phenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
  • an aryl may be optionally substituted, for example, with halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • the aryl is optionally substituted with halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or -NO2.
  • the aryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe. In some embodiments, the aryl is optionally substituted with halogen.
  • Cycloalkyl refers to a partially or fully saturated, monocyclic, or polycyclic carbocyclic ring, which may include fused (when fused with an aryl or a heteroaryl ring, the cycloalkyl is bonded through a non-aromatic ring atom), spiro, or bridged ring systems. In some embodiments, the cycloalkyl is fully saturated.
  • Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to fifteen carbon atoms (e.g., C3-C15 fully saturated cycloalkyl or C3-C15 cycloalkenyl), from three to ten carbon atoms (e.g., C3-C10 fully saturated cycloalkyl or C3-C10 cycloalkenyl), from three to eight carbon atoms (e.g., C3-C8 fully saturated cycloalkyl or C3-C8 cycloalkenyl), from three to six carbon atoms (e.g., C3-C6 fully saturated cycloalkyl or C3-C6 cycloalkenyl), from three to five carbon atoms (e.g., C3-C5 fully saturated cycloalkyl or C3-C5 cycloalkenyl), or three to four carbon atoms (e.g., C3-C4 fully saturated cyclo
  • the cycloalkyl is a 3 - to 10-membered fully saturated cycloalkyl or a 3- to 10-membered cycloalkenyl. In some embodiments, the cycloalkyl is a 3- to 6-membered fully saturated cycloalkyl or a 3 - to 6-membered cycloalkenyl. In some embodiments, the cycloalkyl is a 5 - to 6-membered fully saturated cycloalkyl or a 5- to 6-membered cycloalkenyl.
  • Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyls include, for example, adamantyl, norbomyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cisdecalin, trans-decalin, bicyclo [2.1.1] hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl.
  • Partially saturated cycloalkyls include, for example cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl.
  • a cycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • a cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or -NO2.
  • a cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe.
  • the cycloalkyl is optionally substituted with halogen.
  • Halo or “halogen” refers to bromo, chloro, fluoro or iodo. In some embodiments, halogen is fluoro or chloro. In some embodiments, halogen is fluoro.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2- difluoroethyl, 3 -bromo-2 -fluoropropyl, 1,2-dibromoethyl, and the like.
  • “Hydroxyalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more hydroxyls. In some embodiments, the alkyl is substituted with one hydroxyl. In some embodiments, the alkyl is substituted with one, two, or three hydroxyls. Hydroxyalkyl include, for example, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, or hydroxypentyl. In some embodiments, the hydroxyalkyl is hydroxymethyl.
  • Non-limiting examples of hydroxyalkyl include -CH2OH, -(CH2)2OH, -(QLhOH, -CH2CH(OH)CH3, - (CH 2 ) 4 OH, -CH(CH 2 OH)CH 2 CH 3 , -CH 2 CH(CH 2 OH)CH 3 , -CH(OH)(CH 2 ) 2 OH, -CH 2 CH(OH)CH 2 OH, - CH 2 CH(OH)(CH 2 ) 2 OH and -CH 2 CH(CH 2 OH) 2 for terminal moieties and -CHOH-, -CH 2 CHOH-, - CH 2 CH(OH)CH 2 -, -(CH 2 ) 2 CHOHCH 2 -, -CH(CH 2 OH)CH 2 CH 2 -, -CH 2 CH(CH 2 OH)CH 2 -, -CH 2 CH(CH 2 OH)CH 2 -, -CH 2 CH(CH 2 OH)CH 2 -,
  • Aminoalkyl refers to an alkyl radical, as defined above, that is substituted by one or more amines. In some embodiments, the alkyl is substituted with one amine. In some embodiments, the alkyl is substituted with one, two, or three amines. Aminoalkyl include, for example, aminomethyl, aminoethyl, aminopropyl, aminobutyl, or aminopentyl. In some embodiments, the aminoalkyl is aminomethyl. Non-limiting examples of aminoalkyl include -CH 2 NH 2 , -CH 2 NHMe, -CH 2 NHEt,
  • Heteroalkyl refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, phosphorus, or combinations thereof.
  • a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl.
  • a heteroalkyl is a C 1 -C 6 heteroalkyl wherein the heteroalkyl is comprised of 1 to 6 carbon atoms and one or more atoms other than carbon, e.g., oxygen, nitrogen (e.g.
  • heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl.
  • heteroalkyl are, for example, -CH 2 OCH 3 , -CH 2 CH 2 OCH 3 , - CH 2 CH 2 OCH 2 CH 2 OCH 3 , -CH(CH 3 )OCH 3 , -CH 2 NHCH 3 , -CH 2 N(CH 3 ) 2 , -CH 2 CH 2 NHCH 3 , or - CH 2 CH 2 N(CH 3 ) 2 .
  • a heteroalkyl is optionally substituted for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 .
  • a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF 3 , -OH, or - OMe. In some embodiments, the heteroalkyl is optionally substituted with halogen.
  • Heterocycloalkyl refers to a 3- to 24-membered partially or fully saturated ring radical comprising 2 to 23 carbon atoms and from one to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, silicon, and sulfur. In some embodiments, the heterocycloalkyl is fully saturated. In some embodiments, the heterocycloalkyl comprises one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the heterocycloalkyl comprises one to three heteroatoms selected from the group consisting of nitrogen and oxygen. In some embodiments, the heterocycloalkyl comprises one to three nitrogens. In some embodiments, the heterocycloalkyl comprises one or two nitrogens.
  • the heterocycloalkyl comprises one nitrogen. In some embodiments, the heterocycloalkyl comprises one nitrogen and one oxygen.
  • the heterocycloalkyl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom), spiro, or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocycloalkyl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized.
  • heterocycloalkyls include, but are not limited to, heterocycloalkyls having from two to fifteen carbon atoms (e.g., C2-C15 fully saturated heterocycloalkyl or C2-C15 heterocycloalkenyl), from two to ten carbon atoms (e.g., C2-C10 fully saturated heterocycloalkyl or C2-C10 heterocycloalkenyl), from two to eight carbon atoms (e.g., C2-C8 fully saturated heterocycloalkyl or C2-C8 heterocycloalkenyl), from two to seven carbon atoms (e.g., C2-C7 fully saturated heterocycloalkyl or C2-C7 heterocycloalkenyl), from two to six carbon atoms (e.g., C2-C6 fully saturated heterocycloalkyl or C2- C7 heterocycloalkenyl), from two to five carbon atoms (e.g., C2-C5 fully saturated heterocycloalkyl or C2-C5 heterocycloal
  • heterocycloalkyl radicals include, but are not limited to, aziridinyl, azetidinyl, oxetanyl, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2- oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydro
  • heterocycloalkyl also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides, and the oligosaccharides.
  • heterocycloalkyls have from 2 to 10 carbons in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring).
  • the heterocycloalkyl is a 3- to 8-membered heterocycloalkyl.
  • the heterocycloalkyl is a 3 - to 7-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 3- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 4- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 5- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 3- to 8-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 3- to 7-membered heterocycloalkenyl.
  • the heterocycloalkyl is a 3- to 6-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 4- to 6-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 5 - to 6-membered heterocycloalkenyl.
  • a heterocycloalkyl may be optionally substituted as described below, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • the heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or -NO2.
  • the heterocycloalkyl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe. In some embodiments, the heterocycloalkyl is optionally substituted with halogen.
  • Heteroaryl refers to a 5 - to 14-membered ring system radical comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, and sulfur, and at least one aromatic ring.
  • the heteroaryl comprises one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur.
  • the heteroaryl comprises one to three heteroatoms selected from the group consisting of nitrogen and oxygen.
  • the heteroaryl comprises one to three nitrogens.
  • the heteroaryl comprises one or two nitrogens.
  • the heteroaryl comprises one nitrogen.
  • the heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the heteroaryl is bonded through an aromatic ring atom) or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized.
  • the heteroaryl is a 5 - to 10-membered heteroaryl.
  • the heteroaryl is a 5 - to 6-membered heteroaryl.
  • the heteroaryl is a 6-membered heteroaryl.
  • the heteroaryl is a 5-membered heteroaryl.
  • examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolin
  • a heteroaryl may be optionally substituted, for example, with halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • the heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or - N0 2 .
  • the heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, - OH, or -OMe. In some embodiments, the heteroaryl is optionally substituted with halogen.
  • an optionally substituted group may be un-substituted (e.g., - CH2CH3), fully substituted (e.g., -CF2CF3), mono-substituted (e.g., -CH2CH2F) or substituted at a level anywhere in-between fully substituted and mono-substituted (e.g., -CH2CHF2, -CH2CF3, -CF2CH3, - CFHCHF2, etc.).
  • any substituents described should generally be understood as having a minimum and maximum molecular weight of about 750-1,700 daltons, and more typically, about 900-1,250 daltons.
  • one or more when referring to an optional substituent means that the subject group is optionally substituted with one, two, three, four, or more substituents. In some embodiments, the subject group is optionally substituted with one, two, three or four substituents. In some embodiments, the subject group is optionally substituted with one, two, or three substituents. In some embodiments, the subject group is optionally substituted with one or two substituents. In some embodiments, the subject group is optionally substituted with one substituent. In some embodiments, the subject group is optionally substituted with two substituents.
  • an “effective amount” or “therapeutically effective amount” refers to an amount of a compound administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.
  • treat include alleviating, abating, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
  • METTL3 in the context of the present specification relates to N6-adenosine-methyltransferase catalytic subunit (e.g., see Uniprot ID: Q86U44).
  • METTL14 in the context of the present specification relates to N6-adenosine-methyltransferase non-catalytic subunit (e.g., see Uniprot ID: Q9HCE5).
  • One aspect of the disclosure relates to a bifiinctional compound having a structure of Formula (I), or a pharmaceutically acceptable salt thereof, MBM-L-EBM
  • MBM is a moiety that binds to METTL3
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • L is a linker, wherein L has a structure of Formula (V),
  • each R a is independently C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or R c and R d are taken together with the atom to which they are attached to form a heterocyclo
  • L is a linker that imparts rigidity.
  • R 11 is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In some embodiments, R 11 is hydrogen or C 1 -C 6 alkyl. In some embodiments, R 11 is hydrogen.
  • R La is oxo or C 1 -C 6 alkyl optionally substituted with one or more R. In some embodiments, R La is C 1 -C 6 alkyl. In some embodiments, R La is oxo.
  • two R La are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R. In some embodiments, two R La are taken together to form a 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl, each of which is unsubstituted or substituted.
  • each R RLa is independently halogen, -CN, -NO2, -OH, -OR a , -NR c R d , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl.
  • each R a is independently C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocycloalkyl, Ci- C6alkylene(cycloalkyl), or Ci-C6alkylene(heterocycloalkyl), wherein each alkyl, alkylene, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • each R a is independently C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, Ci- Ceaminoalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, or heterocycloalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • each R a is independently C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, Ci- Ceaminoalkyl, or C 1 -C 6 heteroalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • each R a is independently C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl.
  • each R a is independently C 1 -C 6 alkyl or C 1 -C 6 haloalkyl. In some embodiments of a compound disclosed herein, each R a is independently C 1 -C 6 alkyl.
  • each R b is independently hydrogen, C 1 -C 6 alkyl, Ci- Cehaloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocycloalkyl, Ci- C6alkylene(cycloalkyl), or Ci-C6alkylene(heterocycloalkyl), wherein each alkyl, alkylene, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • each R b is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, Ci- Cehydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, or heterocycloalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • each R b is independently hydrogen, C 1 -C 6 alkyl, Ci- Cehaloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • each R b is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, Ci- Cehydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl.
  • each R b is independently hydrogen, C 1 -C 6 alkyl, or C 1 -C 6 haloalkyl. In some embodiments of a compound disclosed herein, each R b is independently hydrogen or C 1 -C 6 alkyl. In some embodiments of a compound disclosed herein, each R b is hydrogen. In some embodiments of a compound disclosed herein, each R b is independently C 1 -C 6 alkyl.
  • R c and R d are each independently hydrogen, Ci- C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocycloalkyl, Ci-C6alkylene(cycloalkyl), or Ci-C6alkylene(heterocycloalkyl), wherein each alkyl, alkylene, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, cycloalkyl, or heterocycloalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R.
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, Ci- Cehaloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl. In some embodiments of a compound disclosed herein, R c and R d are each independently hydrogen, C 1 -C 6 alkyl, or C 1 -C 6 haloalkyl. In some embodiments of a compound disclosed herein, R c and R d are each independently hydrogen or Ci- Cealkyl. In some embodiments of a compound disclosed herein, R c and R d are each hydrogen. In some embodiments of a compound disclosed herein, R c and R d are each independently C 1 -C 6 alkyl.
  • R c and R d are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R. In some embodiments of a compound disclosed herein, R c and R d are taken together with the atom to which they are attached to form a heterocycloalkyl.
  • each R is independently halogen, -CN, -OH, -NH 2 , -NHCi-C 3 alkyl, -N(Ci-C 3 alkyl) 2 , Ci-C 3 alkyl, Ci-C 3 alkoxy, Ci-C 3 haloalkyl, Ci-C 3 haloalkoxy, Ci-C 3 hydroxyalkyl, Ci-C 3 aminoalkyl, Ci-C 3 heteroalkyl, or G-G, cycloalky I: or two R on the same atom form an oxo.
  • each R is independently halogen, -CN, -OH, -NH 2 , Ci-C 3 alkyl, Ci-C 3 alkoxy, Ci-C 3 haloalkyl, or Ci-C 3 haloalkoxy; or two R on the same atom form an oxo.
  • each R is independently halogen, -CN, -OH, -NH 2 , Ci-C 3 alkyl, or Ci-C 3 haloalkyl; or two R on the same atom form an oxo.
  • each R is independently halogen or Ci-C 3 alkyl.
  • the structure of Formula (I) has a structure of Formula (II),
  • Z 1 and Z 2 are independently selected from N, CH and CR 2 ;
  • X is O or NH
  • U and V are each independently selected from -CH 2 - and -(CH 2 ) 2 -, or one of U and V is -CH 2 - and the other one is -(CH 2 ) 2 -
  • R 1 is an unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted cycloalkyl, or unsubstituted or substituted heterocycloalkyl;
  • R 2 is selected from halogen (e.g., F, Cl), -CN, -NO 2 , -OH, -OR a , -SF5, -SH, -SR a , -NR c R d (e.g., NH 2 ), C 1 -C 6 alkyl (e.g., methyl), C 1 -C 6 haloalkyl (e.g., CF3, CHF 2 , CH 2 F), C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, and C 1 -C 6 heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; n is selected from 0, 1, 2, 3, and 4;
  • R 13 is selected from a bond, -O-, -S-, -NR 13N -, or CR 13C R 13C ;
  • R 3 is C 1 -C 6 alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; each of which is unsubstituted or substituted with one or more R 12 ; each R 12 is independently halogen, -CN, -NO 2 , -OH, -OR a , -SH, -SR a , -NR c R d , C 1 -C 6 alkyl, Ci- Cehaloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C2-G, alkenyl. CS-Galkynyl.
  • each R 13C is independently hydrogen, halogen, -CN, -NO 2 , -OH, -OR a , -SH, -SR a , -NR c R d , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; or two R 13C are
  • R 1 is unsubstituted or substituted with 1, 2, 3, or 4 R 14 . In some embodiments, R 1 is unsubstituted or substituted with 1 or 2 R 14 .
  • each R 14 is independently halogen, -CN, -NO 2 , -OH, -OR a , -NR c R d , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl.
  • each R 14 is independently halogen, -CN, -NO 2 , -OH, -OR a , amino, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, or C 1 -C 6 heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, or heteroalkyl is unsubstituted or substituted with one or more R.
  • R 14 is C 1 -C 6 aminoalkyl.
  • R 14 is -NHCH3.
  • R 1 is a monocyclic group. In some embodiments, R 1 is a bicyclic group.
  • the structure of Formula (II) has a structure of Formula (Ila),
  • the structure of Formula (II) has a structure of Formula (Illa),
  • -L-EBM is attached to the pyrimidine of Formula (Illa) through a R 14 group.
  • the structure of Formula (II) has a structure of Formula (lib)
  • the structure of Formula (II) has a structure of Formula (Illb),
  • the structure of Formula (II) has a structure of Formula (lie),
  • R 13 is selected from some embodiments, R 13 is , In some embodiments,
  • R 3 is 4-6 membered heterocycloalkyl, wherein R 3 is unsubstituted or substituted with 1, 2 or 3 R 12 . In some embodiments, R 3 is 6 membered heterocycloalkyl, wherein R 3 is unsubstituted or substituted with 1, 2 or 3 R 12 . In some embodiments, R 3 unsubstituted or substituted with 1, 2 or 3 R 12 . In some embodiments, R 3 is monocyclic heterocycloalkyl. In some embodiments, . In some embodiments, R 3 is bicyclic heterocycloalkyl.
  • R 1 is unsubstituted or substituted 6 membered heteroaryl. In some embodiments, R 1 is unsubstituted or substituted pyrimidine. In some embodiments, R 1 is a bicyclic heteroaryl.
  • L 1 attaches to the MBM and L 2 attaches to the EBM.
  • L 2 attaches to the MBM and L 1 attaches to the EBM.
  • each of L 1 , L 2 , and L k is independently selected from a bond, C 1 -C 6 alkylene, and Ci- G, heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more R La .
  • each of L 1 , L 2 , and L k is independently selected from a bond, C1-C4 alkylene, and C1-C4 heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more R La .
  • each of L 1 , L 2 , and L k is independently selected from a bond,
  • each of RL 1 and RL k is independently selected from , 6-14 membered cycloalkyl, 6-14 membered heterocycloalkyl, 6-14 membered aryl, and 6-14 membered heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more R RLa .
  • RL 1 is unsubstituted or substituted monocyclic heterocycloalkyl.
  • RL 1 is unsubstituted or substituted 5-6 membered monocyclic heterocycloalkyl.
  • RL 1 is . , .
  • RL 1 is unsubstituted or substituted bicyclic heterocycloalkyl. In some embodiments, RL 1 is a spiro bicyclic ring. In some embodiments, RL 1 is a fused bicyclic ring. In some embodiments, RL 1 is some embodiments, RL 1 is unsubstituted or substituted phenyl. In some embodiments, RL 1 is unsubstituted or substituted monocyclic heteroaryl. In some embodiments, RL k is unsubstituted or substituted monocyclic heterocycloalkyl. In some embodiments, , . In some embodiments, RL k is unsubstituted or substituted bicyclic heterocycloalkyl. In some embodiments, some embodiments, RL k is unsubstituted or substituted phenyl. In some embodiments, RL k is unsubstituted or substituted monocyclic heteroaryl.
  • the bifunctional compound has a structure of
  • the bifunctional compound has a structure of Formula (lib*), or a pharmaceutically acceptable salt thereof,
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • L is a covalent linker
  • R 1 , U, V, X, Y, Z 1 , Z 2 , n, R 2 , R 13 and R 3 have the meanings defined in Formula (II).
  • the bifunctional compound has a structure of Formula (lie*), or a pharmaceutically acceptable salt thereof,
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • L is a covalent linker
  • R 1 , U, V, X, Y, Z 1 , Z 2 , n, R 2 , R 13 and R 3 have the meanings defined in Formula (II).
  • the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase, of the general formula (A-I)
  • NR 31 R 32 is selected from an optionally substituted 3-12 membered heterocycloalkyl each R 2 is independently selected from the group consisting of halogen (e.g., F, Cl), C1-C3 alkyl, and Ci- C3 haloalkyl (e.g., CF3, CHF2, CH2F); n is an integer selected from 0, 1, 2, 3, and 4;
  • halogen e.g., F, Cl
  • C1-C3 alkyl e.g., C1-C3 alkyl
  • Ci- C3 haloalkyl e.g., CF3, CHF2, CH2F
  • n is an integer selected from 0, 1, 2, 3, and 4;
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S);
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C2-C6alkenyl, C 2 -C 6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or R c and R d are taken together with the atom to which they are attached to form a heterocycloalky
  • the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase, of the general formula (A-II)
  • R 13 is selected from a bond, -O-, -S-, -NR 13N -, or CR 13C R 13C ;
  • R 3 is C 1 -C 6 alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; each of which is unsubstituted or substituted with one or more R 12 ; each R 12 is independently halogen, -CN, -NO2, -OH, -OR a , -SH, -SR a , -NR c R d , C 1 -C 6 alkyl, Ci- Cehaloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C2- Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl
  • halogen e.g., F, Cl
  • Ci- C3 haloalkyl e.g.,
  • EBM is a moiety that binds to an E3 ubiquitin ligase
  • Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S);
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C2-C6alkenyl, C 2 -C 6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or R c and R d are taken together with the atom to which they are attached to form a heterocycloalky
  • NR 31 R 32 is selected from
  • each R 2 is independently selected from the group comprising F, Cl, CF3, CHF2, CH2F;
  • - n is an integer selected from 0, 1, 2, 3, and 4;
  • - Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S);
  • Linker is a linker moiety comprising or essentially consisting of 3 to 50 atoms of atomic mass >12.
  • each R 2 is F.
  • n is an integer selected from 0, 1, and 2.
  • n is 2.
  • Handle is a connecting moiety comprising or essentially consisting of 4 to 8 atoms of atomic mass >12.
  • Spacer is a linker moiety comprising or essentially consisting of 4 to 30 atoms of atomic mass >12.
  • Spacer is a linker moiety comprising or essentially consisting of 5 to 20 atoms of atomic mass >12.
  • a bifunctional compound disclosed herein has a MBM of Formula (XI) wherein the attachment point is not shown.
  • a bifunctional compound disclosed herein has a MBM of Formula (Xia) wherein the attachment point is not shown.
  • a bifunctional compound disclosed herein has a MBM of Formula (Xlb) wherein the attachment point is not shown.
  • Z 1 and Z 2 are independently selected from N, CH and CR 2 ;
  • X is O or NH;
  • R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl, and a heterocycle, particularly R 1 is unsubstituted or substituted heteroaryl;
  • R 2 is selected from F, Me, Cl, OH, NH 2 , Br, CF 3 , CHF 2 , CH 2 F; n is an integer selected from 0, 1, 2, 3, and 4;
  • R 3 is a substituted alkylamine
  • U and V are independently selected from -CH 2 - and -(CH 2 ) 2 -, or one of U and V is -CH 2 - and the other one is -(CH 2 ) 3 -.
  • a bifunctional compound disclosed herein has a MBM of Formula (U) wherein the attachment point is not shown,
  • R 2 is selected from the group comprising F, Cl, CF 3 , CHF 2 , CH 2 F;
  • - n is an integer selected from 0, 1, 2, 3, and 4;
  • R 5 is selected from an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and a heterocycloalkyl.
  • R 2 is F.
  • n is an integer selected from 0, 1, and 2.
  • n is 2.
  • R 5 is selected from -Ci-6-alkyl, -Ci-6-alkyl-aryl, -Ci-6-alkyl- heteroaryl, -Ci-6-heteroalkyl-heteroaryl, and -Ci-6-heteroalkyl-aryl.
  • R 5 is selected from -Ci-6-alkyl, -Ci-6-alkyl-aryl, -Ci-6-alkyl-heteroaryl, -Ci-6-heteroalkyl-heteroaryl,-Ci-6-heteroalkyl-aryl, - Ci-6-alkyl-cycloalkyl, -Ci-6-alkyl-heterocycloalkyl, -Ci-6-heteroalkyl-heterocycloalkyl, and -Ci-6-heteroalkyl- cycloalkyl.
  • R 5 is selected from an alkyl, an alkylaryl, and a cycloalkyl.
  • the moiety R 1 is selected from R 1 , R 1 , R 1
  • R 1 is unsubstituted or substituted heteroaryl. In certain embodiments, R 1 is unsubstituted or substituted with a moiety selected from
  • R 1 is unsubstituted or substituted with a moiety selected from
  • a bifunctional compound disclosed herein has a MBM of Formula (XII) wherein the attachment point is not shown, wherein
  • each R 14 is independently selected from
  • - n2 is an integer selected from 0, 1, 2, and 3.
  • a bifimctional compound disclosed herein has a MBM of Formula (XIII) wherein
  • R 5 is selected from an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and a heterocycle;
  • R 6 is selected from halogen and hydrogen.
  • a bifimctional compound disclosed herein has a MBM of Formula (XIV) wherein
  • R 6 is selected from halogen and hydrogen
  • - W is selected from N and CH.
  • At least one of Z 1 and Z 2 is CH or CR 2 . In certain embodiments, both Z 1 and Z 2 are CH or CR 2 .
  • R 3 is substituted with one or several moieties selected independently from alkyl-, hydroxy-, amino-, amine-, halogen-, cycloalkyl-, and heterocycle- moieties.
  • R 3 is substituted C1-C4 alkylamine. In certain embodiments, R 3 is substituted C1-C2 alkylamine.
  • - s is an integer selected from 1 and 2, more particularly s is 1 ;
  • R 31 and R 32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, halogen-, and/or hydroxyl-substituted, or
  • R 31 and R 32 are independently selected from hydrogen and unsubstituted or hydroxy-, and/or halogen-substituted alkyl or cycloalkyl. In certain embodiments, R 31 and/or R 32 are unsubstituted or substituted with alkyl-, hydroxy-, halogen-, cycloalkyl-, heterocycle- and/or -groups. In certain embodiments, R 31 and/or R 32 are independently selected from H and unsubstituted or hydroxy-, and/or halogen-substituted alkyl and cycloalkyl. In certain embodiments, R 31 and R 32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, hydroxy-, and/or halogen-substituted.
  • NR 31 R 32 is selected from v being an integer selected from 0, 1 and 2 and each R N being independently selected from hydroxyl, halogen, and C1-C4 alkyl, or two R N form a C3-C6 cycloalkyl. from , ,
  • n is an integer selected from 0, 1, and 2. In certain embodiments, n is 2. In certain embodiments, R 2 is selected from F, Cl and OH. In certain embodiments, R 2 is F. R 2 can be bound to any of the carbon atoms of the aryl-or heteroaryl -ring. Thus, it can also be bound to Z 1 or Z 2 if they are carbon atoms.
  • R 5 is selected from an alkyl, an alkylaryl, and a cycloalkyl. In certain embodiments, R 5 is selected from methyl and methylphenyl.
  • An E3 ligase binder is a molecule which specifically binds an E3 ligase or a component of the E3 ligase complex.
  • the E3 ligase binder binds to cereblon (CRBN, UniProt-ID: Q96SW2).
  • the E3 ligase binder binds to Von Hippel- Lindau (VHL, UniProt-ID: P40337).
  • the E3 ligase binder binds to mouse double minute 2 homolog (MDM2, UniProt-ID: Q00987).
  • the E3 ligase binder binds to an inhibitor of apoptosis protein (IAP, UniProt-ID: P98170).
  • EBM is of the formula (B) wherein
  • - T is selected from the group comprising F, Cl; - k is an integer selected from the group comprising 0, 1, 2;
  • k is an integer selected from the group comprising 0, 1. In certain embodiments, k is 0. In certain embodiments, T is F.
  • EBM has a structure according to Table A. TABLE A
  • EBM suitable for bifunctional compounds of the instant disclosure are further illustrated in Alesa Bricelj, et al., “E3 Ligase Ligands in Successful PROTACs: An Overview of Syntheses and Linker Attachment Points,” front Chem. 2021; 9: 707317, which is hereby incorporated by reference in its entirety.
  • L is a linker, wherein L has a structure of Formula (V), wherein, k is 0, 1, 2, or 3; each of L 1 , L 2 , and L k is independently selected from a bond, -O-, -S-, -NR 11 -, Ci-Cs alkylene, and Ci-Cs heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa.
  • V Formula
  • CS-G alkynyl. cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R;
  • R c and R d are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 aminoalkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or R c and R d are taken together with the atom to which they are attached to form a heterocyclo
  • L is a linker that imparts rigidity. In some embodiments, L comprises 5-40 atoms in length. In some embodiments, L comprises 8-30 atoms in length. In some embodiments, L comprises 10-20 atoms in length. In some embodiments, L comprises 15-20 atoms in length. In some embodiments, L comprises 20-25 atoms in length. In some embodiments, L comprises 12-25 atoms in length.
  • each of L 1 , L 2 , and L k is independently no more than 12 atoms in length. In some embodiments, each of L 1 , L 2 , and L k is independently no more than 11 atoms in length.
  • each of L 1 , L 2 , and L k is independently no more than 10 atoms in length.
  • each of L 1 , L 2 , and L k is independently no more than 9 atoms in length.
  • each of L 1 , L 2 , and L k is independently no more than 8 atoms in length. In some embodiments, each of L 1 , L 2 , and L k is independently no more than 7 atoms in length. In some embodiments, each of L 1 , L 2 , and L k is independently no more than 6 atoms in length. In some embodiments, each of L 1 , L 2 , and L k is independently no more than 5 atoms in length.
  • a covalent linker L in some embodiments, does not imparts rigidity.
  • the linker is O . In some embodiments, the linker is In some embodiments, the linker is embodiments, the linker some embodiments, the linker is embodiments, the linker is some embodiments, the linker embodiments, the linker some embodiments, the linker is some embodiments, the linker i
  • Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S). In certain embodiments, Handle is a connecting moiety comprising or essentially consisting of 4 to 8 atoms of atomic mass >12.
  • the Handle comprises or essentially consists of 1, 2, 3, or 4 chemical moieties selected from the group comprising alkyl, amine, phenyl, and carbonyl. In some embodiments, the Handle comprises 1, 2, 3, or 4 chemical moieties selected from alkyl, amine, phenyl, carbonyl, cycloalkyl, or heterocycloalkyl.
  • the Handle is selected from the group comprising the following formulas: wherein
  • Mid is selected from the group comprising C1-C3 alkyl, heterocycloalkyl, or cycloalkyl, and phenyl.
  • Mid comprises C1-C3 alkyl, phenyl, heterocycloalkyl, or cycloalkyl. In some embodiments, Mid comprises C1-C3 alkyl or phenyl.
  • the Handle is selected from the group comprising the following formulas:
  • the Handle is selected from:
  • pacer In certain embodiments, embodiments, In certain embodiments, pacer
  • Spacer is a linker moiety comprising or essentially consisting of 3 to 50 atoms of atomic mass >12 (C, N, O, S). In certain embodiments, Spacer is a linker moiety comprising or essentially consisting of 4 to 30 atoms of atomic mass >12. In certain embodiments, the Spacer is a linker moiety comprising or essentially consisting of 5 to 20 atoms of atomic mass >12.
  • the Spacer comprises or essentially consists of 1, 2, 3, 4, 5, 6, or 7 chemical moieties independently selected from the group comprising alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkylene, alkylyne, ethylene glycol, carbonyl, ether, ester, amine, amide, sulfonamide, wherein the chemical moieties are each independently unsubstituted or substituted with C1-C3 alkyl, halogen, CN, NO2, hydroxyl, amine, sulfate, phosphate, and/or carboxyl.
  • the Spacer comprises or essentially consists of 1, 2, 3, or 4 chemical moieties selected from the group comprising alkyl, ethylene glycol, carbonyl, piperazine, aryl, amine, triazole.
  • the Spacer is selected from the group comprising the following formulas: wherein - Lin is selected from the group comprising C3-C20 alkyl, C3-C20 alkyl-triazole, oligo(ethylene glycol).
  • the Spacer is selected from the group comprising the following formulas: wherein
  • - p is selected from 2, 3, 4, and 5;
  • - q is selected from 7, 8, 9, 10, 11, 12, and 13;
  • - r is selected from 11, 12, 13, 14, 15, 16, and 17;
  • - s is selected from 7, 8, 9, 10, 11, 12, and 13;
  • - t is selected from 3, 4, 5, 6, 7, 8, and 9;
  • - u is selected from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • the Spacer is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S), and (T), wherein
  • - p is selected from 2, 3, 4, and 5;
  • - q is selected from 7, 8, 9, 10, 11, 12, and 13;
  • - r is selected from 11, 12, 13, 14, 15, 16, and 17;
  • - s is selected from 7, 8, 9, 10, 11, 12, and 13;
  • - t is selected from 3, 4, 5, 6, 7, 8, and 9; and u is selected from 7, 8, 9, 10, 11, 12, and 13.
  • the Spacer is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S), and (T), wherein
  • - p is selected from 2, 3, 4, and 5;
  • - q is selected from 7, 8, 9, 10, 11, 12, and 13;
  • - r is selected from 11, 12, 13, 14, 15, 16, and 17;
  • - s is selected from 7, 8, 9, 10, 11, 12, and 13;
  • - t is selected from 3, 4, 5, 6, 7, 8, and 9;
  • - u is selected from 7, 8, 9, 10, 11, 12, 13, 14 and 15.
  • the Spacer is a peptide. In certain embodiments, the Spacer is a peptide consisting of proteinogenic amino acids.
  • the EMB is of the formula (B) or selected from Table A;
  • - Linker L has a structure of Formula (V) or comprises a Handle and a Spacer,
  • the Handle is of formula (F), (G), (H), or (J); and the Spacer is of formula (O); (P); (Q); (R); (S); or (T).
  • the compound comprises the definitions of Handle, Spacer, and E3 ligase binder (one row is one combination) according to Table 1 :
  • PROTAC compounds of this disclosure are illustrated in TABLES 2 and 3.
  • the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase.
  • the bifunctional compounds target a heterodimeric complex METTL3-METTL14.
  • the bifunctional compounds bind to the heterodimeric complex METTL3-METTL14.
  • the bifunctional compounds modulate the heterodimeric complex METTL3-METTL14.
  • the bifunctional compounds inhibit and degrade the heterodimeric complex METTL3-METTL14.
  • the bifunctional compounds target METTL3.
  • the bifunctional compounds bind to METTL3.
  • the bifunctional compounds modulate, inhibit, and/or degrade METTL3.
  • the bifunctional compounds target METTL14.
  • the bifunctional compounds modulate, inhibit, and/or degrade METTL14.
  • the present disclosure relates to bifunctional compounds for use as a medicament.
  • the present disclosure relates to bifunctional compounds for use in treatment of cancer.
  • the cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.
  • the present disclosure relates to a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure relates to a method of treating disease, the method comprising administering to a subject in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound disclosed herein.
  • the disclosure relates to a compound according to the first or second aspect for use as a medicament.
  • the disclosure relates to a compound according to the first or second aspect for use in treatment of cancer.
  • the cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.
  • a method or treating cancer in a patient in need thereof comprising administering to the patient a compound according to the above description.
  • a dosage form for the prevention or treatment of cancer comprising a non -agonist ligand or antisense molecule according to any of the above aspects or embodiments of the disclosure.
  • any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug.
  • Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion.
  • Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate.
  • compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
  • the compounds described herein may be artificially enriched in one or more particular isotopes.
  • the compounds described herein may be artificially enriched in one or more isotopes that are not predominantly found in nature.
  • the compounds described herein may be artificially enriched in one or more isotopes selected from deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C).
  • the compounds described herein are artificially enriched in one or more isotopes selected from 2 H, n C, 13 C, 14 C, 15 C, 12 N, 13 N, 15 N, 16 N, 16 O, 17 O, 14 F, 15 F, 16 F, 17 F, 18 F, 33 S, 34 S, 35 S, 36 S, 35 C1, 37 C1, 79 Br, 81 Br, 131 I, and 125 I.
  • the abundance of the enriched isotopes is independently at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% by molar.
  • the compound is deuterated in at least one position.
  • the compounds disclosed herein have some or all of the 3 H atoms replaced with 2 H atoms.
  • the compounds described herein exist as geometric isomers. In some embodiments, the compounds described herein possess one or more double bonds. The compounds presented herein include all cis, trans, syn, anti,
  • Z isomers as well as the corresponding mixtures thereof. In some situations, the compounds described herein possess one or more chiral centers and each center exists in the R configuration, or S configuration. The compounds described herein include all diastereomeric, enantiomeric, and epimeric forms as well as the corresponding mixtures thereof.
  • mixtures of enantiomers and/or diastereoisomers, resulting from a single preparative step, combination, or interconversion are useful for the applications described herein.
  • the compounds described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers.
  • dissociable complexes are preferred.
  • the diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and are separated by taking advantage of these dissimilarities.
  • the diastereomers are separated by chiral chromatography, or preferably, by separation/resolution techniques based upon differences in solubility.
  • the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • Tautomers are compounds that are interconvertible by migration of a hydrogen atom, accompanied by a switch of a single bond and adjacent double bond. In bonding arrangements where tautomerization is possible, a chemical equilibrium of the tautomers will exist. All tautomeric forms of the compounds disclosed herein are contemplated. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH.
  • Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository.
  • parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms.
  • a pharmaceutically acceptable carrier and/or excipient may be present.
  • compositions comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
  • the compound of the present disclosure is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.
  • the pharmaceutical composition can be formulated for oral administration, parenteral administration, or rectal administration.
  • the pharmaceutical compositions of the present disclosure can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
  • the dosage regimen for the compounds of the present disclosure will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired.
  • the compounds of the disclosure may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
  • the pharmaceutical composition or combination of the present disclosure can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg.
  • the therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
  • compositions of the present disclosure can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892). Method of Manufacture and Method of Treatment
  • the disclosure further encompasses, as an additional aspect, the use of a compound as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of cancer.
  • the disclosure encompasses methods of treatment of a patient having been diagnosed with a disease associated with cancer.
  • This method entails administering to the patient an effective amount of a compound as identified herein, or its pharmaceutically acceptable salt, as specified in detail herein.
  • the application further encompasses the following items:
  • - Z 1 and Z 2 are independently selected from N, CH and CR 2 ;
  • - X is O or NH, particularly X is NH;
  • R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl, and a heterocycle, particularly R 1 is unsubstituted or substituted heteroaryl;
  • R 2 is selected from F, Me, Cl, OH, NH 2 , Br, CF 3 , CHF 2 , CH 2 F;
  • - n is an integer selected from 0, 1, 2, 3, and 4, particularly n is an integer selected from 0, 1, and 2;
  • R 3 is a substituted alkylamine
  • - U and V are independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other one is -(CH2) 3 -, particularly U and V are both -CH2- or are both -(012)2-.
  • R 1 is unsubstituted or substituted with a moiety selected from
  • R N is selected from a C 1 -C 6 alkyl, a C4-C6 cycloalkyl, an aryl, and a heteroaryl, an alkylaryl, and an alkylheteroaryl; • a halogen, particularly Cl or F; and
  • R 3 is substituted with one or several moieties selected independently from alkyl-, hydroxy-, amino-, amine-, halogen-, cycloalkyl-, and heterocycle- moieties.
  • R 3 is C1-C4 alkylamine, particularly R 3 is C1-C2 alkylamine.
  • each R 14 is independently selected from
  • a secondary amine substituted with an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and/or a heterocycle, particularly substituted with an alkyl, an alkylaryl, or a cycloalkyl;
  • R 5 is selected from an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and a heterocycle, particularly R 5 is selected from an alkyl, an alkylaryl, and a cycloalkyl;
  • R 6 is selected from halogen and hydrogen.
  • R 6 is selected from halogen and hydrogen
  • - W is selected from N and CH.
  • - s is an integer selected from 1 and 2, more particularly s is 1 ;
  • R 31 and R 32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, halogen-, and/or hydroxyl-substituted, or
  • R 31 and R 32 are independently selected from hydrogen and unsubstituted or hydroxy-, and/or halogen-substituted alkyl or cycloalkyl, particularly R 31 and R 32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, halogen-, and/or hydroxyl-substituted.
  • NR 31 R 32 is selected from with v being an integer selected from 0, 1 and 2 and each R N being independently selected from hydroxyl, halogen, and C1-C4 alkyl, or two R N form a C3-C6 cycloalkyl.
  • R 2 is selected from F, Cl and OH, particularly R 2 is F.
  • a compound according to any of the preceding items for use as a medicament for use as a medicament.
  • SAM S-Adenosyl methionine
  • RNA methyltransferases conducted protein thermal shift assay.
  • the authors expressed and purified METTL1 protein that is a writer of 7- methylguanosine mark on tRNA, mRNA, and miRNAs and serves as a representative closely related protein.
  • the authors employed as positive control S-adenosyl-L-homocysteine (SAH), a by-product of RNA methyltransferase catalytic activity and a natural binder, which showed AT m of 2.8 °C and 3.5 °C at 100 pM for METTL3/METTL14 and METTL1, respectively ( Figure 5 and 6).
  • SAH S-adenosyl-L-homocysteine
  • the enhanced thermal stabilization of METTL3 by compound 22 allowed the authors to study its cellular target engagement in two orthogonal assays based on protein thermal denaturation.
  • the binding of 22 was evaluated in InCELL Pulse assay where enhanced ProLabel® (ePL) enzyme fragment fused to the N-terminus of the truncated METTL3 (residues 354-580) was expressed in HEK293T cells. After the incubation of these cells with inhibitor 22 for 1 h at 37°C, cells were heated at 46°C for 3 min, and the non -aggregated METTL3- ePL protein was quantified using luminescence-based assay ( Figure 4B).
  • ePL enhanced ProLabel®
  • Table 5 Derivatization from the spiro scaffold.
  • Table 6 Optimization of the aminopyrimidine ring.
  • Table 7 Fluorine scan on the phenyl ring.
  • Scheme 1 Synthesis route to the spirocycle intermediate (33). Reagents and conditions: (a) MeNO 2 , NH3, MeOH, 25 °C, 17 h; (b) (i) CbzCl, NaHCO 3 , DCM/H 2 O, 0-25 °C, 17 h; (ii) NiCl 2 .6H 2 O, NaBH 4 , MeOH, N 2 , 0-25 °C, 1 h, 32 % over three steps; (c) ethyl 2-bromoacetate, Et 3 N, DCM, 25 °C, 2 h; (d) Pd/C, NEU HCOO" , zPrOH, 80 °C, 4 h, 55 % over two steps.
  • Scheme 5 General synthetic route for compounds 20-22. Reagents and conditions: (a) For 47 and 49: 1- bromo-4-(bromomethyl)-2 -fluorobenzene (47) or 4-bromo-l-(bromomethyl)-2 -fluorobenzene (49), 4,4- dimethylpiperidine hydrochloride, K2CO3, DMF, 25 °C, 17 h, 98 % (47) / 99 % (49).
  • Scheme 17 (d) (i) TEA (3 eq), EtOH, reflux, 24 h; (ii) 38% HC1, MeOH, 24 h; (e) HATU (1.1 eq), DIPEA (5 eq), DMF, rt, 5 h.
  • Scheme 18 (f) (i) Propargylamine (3 eq), TEA (3 eq), EtOH, reflux, 5 h; (g) Sodium ascorbate (1.1 eq), CuSO 4 (0.24 eq), THF, 40 °C, 24 h.
  • Scheme 19 (h) (i) TEA (3 eq), EtOH, reflux, 5 h; (ii) TFA (10 eq), DCM, rt, 12 h; (i) HATU (1.1 eq), DIPEA
  • the reaction mixture was stirred at 25 °C for 17 h and diluted with a saturated aqueous NaHCO 3 solution.
  • the aqueous layer was extracted three times with DCM and the combined organic layers were washed once with water, once with brine, dried over MgSCL, fdtered and concentrated under reduced pressure, to afford the impure desired product (12.4 g, 29 mmol), which was engaged in the next step without further purification.
  • l-(4-Bromobenzyl)-4-fluoro-4-methylpiperidine (55): To a stirred solution of tert-butyl 4-hydroxy-4-methylpiperidine-l -carboxylate (500 mg, 2.32 mmol) in dry DCM (7 mb), at 0°C under a nitrogen atmosphere, DAST (1.5 eq., 3.48 mmol, 460 pL) was added. The mixture was stirred at 25 °C for 3 h and quenched by adding saturated aqueous NaHCCE solution. The two phases were separated and the aqueous layer was extracted two times with DCM. The combined organic layers were washed with brine, dried over MgSCE, filtered and concentrated under reduced pressure to afford the desired product, which was engaged in the next step without further purification.
  • METTL3 protein degradation was monitored using Western blot.
  • Cells were treated with the indicated concentration of PROTACs (or DMSO, control) for 24h, 37°C with 5% CO2. Samples were then collected and lysed with RIPA buffer with protease inhibitors (11697498001, Roche). After SDS-PAGE, proteins were transferred to a nitrocellulose membrane, blocked (with 5% milk, 0.5% BSA in TBST buffer) and incubated overnight with primary antibodies.
  • GAPDH (#2118, Cell Signaling, 1 :4000), P-actin (ab8226, Abeam, 1 :2000), METTL3 (abl95352, Abeam, 1 :1000), METTL14 (ab220031, Abeam, 1 : 1000).
  • Membranes were scanned using LI-COR Odyssey DLx Imager after incubation with appropriate secondary antibodies (anti-mouse IgG IRDye® 680RD (926-68072, LI-COR, 1 : 10000), Goat anti -Rabbit IgG IRDye® 800CW (926-32211, LI-COR, 1 : 10000)). Densitometry was performed in Image Studio Lite software and analysis in GraphPad Prism 9.
  • Table 10 shows METTL3(M3) and METTL14(M14) protein degradation in the MOLM-13, THP-1, N0M0-

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Abstract

The present disclosure relates to proteolysis targeting chimeras (PROTACs) that modulate N6-adenosine-5 methyltransferase and methods of using the same.

Description

N6-ADENOSINE-METHYLTRANSFERASE PROTACS AND METHODS OF USE THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 63/425,976, filed on November 16, 2022, and U.S. Provisional Patent Application No. 63/502,729, filed on May 17, 2023, which are incorporated herein by reference in their entireties.
BACKGROUND
Expression of genes is regulated at the level of the transcriptome (messenger RNA obtained through transcription of the genome) through dynamic levels of mRNA modifications. The conversion of adenosine to N6-methyladenosine (m6A) is the most common internal post-transcriptional modification (also called epitranscriptomic modification) in eukaryotic mRNA. This methylation event typically occurs within the DRACH (D=A, G, U; R=A, G; H=A, C, U) consensus sequence motif. The m6A level can vary among different tissues, development states or in response to cellular stresses. On the molecular level introduction of the m6A affects the structure of RNA and its ability to form protein-RNA interactions, and as a consequence it modulates processing, translation, and stability of the cellular transcripts. As a consequence, m6A is implicated in controlling embryonic development processes and stem cell differentiation, regulating the mammalian circadian clock, and modulating stress response, e.g., heat shock.
The dynamic level of m6A is regulated by the interplay of erasers and writer proteins. While the m6A writer has been known for two decades, the discovery of m6A-specific eraser proteins FTO (ALKBH9) and ALKBH5 has ultimately demonstrated the reversibility of the modification and its regulatory role. These m6A demethylases belong to the dioxygenase AlkB family whose enzymatic reaction depends on Fe(II) and 2- oxoglutaric acid (2OG). The core writer complex is formed by two methyltransferase -like proteins, METTL3 and METTL14, which rely on additional cofactors for mRNA substrate recruitment, including WTAP and RBM15. The METTL3-METTL14 complex transfers a methyl group from S-adenosylmethionine (SAM) to the adenosine within the consensus sequence of 5’-GGACU-3’. Only METTL3 has an intact SAM-binding site, while METTL14 possesses a degenerate SAM-binding site, which is not functional. The individual depletion of METTL3 or METTL14 reduces the level of m6A in HeLa cells. More importantly, deregulation of METTL3 has recently been linked to specific tumors, such as acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma. Moreover, inhibiting m6A modification shows a broad antiviral effect. Therefore, small -molecule modulators of the METTL3-METTL14 writer has potential therapeutic use in cancer and viral infection.
Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to use the PROTAC therapeutic modalities to modulate the levels of m6A modification with the goal of regulating gene expression for cancer therapy. This objective is attained by the subject-matter of the independent claims of the present specification.
SUMMARY
In one aspect, the present disclosure relates to proteolysis targeting chimeras (PROTACs) that modulate N6- adenosine-methyltransferase and methods of using the same. In one aspect, the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase. In some embodiments, the bifimctional compounds target a heterodimeric complex METTL3-METTL14. In some embodiments, the bifimctional compounds bind to the heterodimeric complex METTL3-METTL14. In some embodiments, the bifimctional compounds modulate the heterodimeric complex METTL3-METTL14. In some embodiments, the bifimctional compounds inhibit and degrade the heterodimeric complex METTL3- METTL14. In some embodiments, the bifimctional compounds target METTL3. In some embodiments, the bifimctional compounds bind to METTL3. In some embodiments, the bifimctional compounds modulate, inhibit, and/or degrade METTL3. In some embodiments, the bifunctional compounds target METTL14 via the METTL3-METTL14 complex. In some embodiments, the bifimctional compounds modulate, inhibit, and/or degrade METTL14.
In one aspect, the present disclosure relates to a bifimctional compound having a structure of Formula (I), or a pharmaceutically acceptable salt thereof,
MBM-L-EBM Formula (I) wherein,
MBM is a moiety that binds to METTL3;
EBM is a moiety that binds to an E3 ubiquitin ligase; and
L is a linker that imparts rigidity, wherein L has a structure of Formula (V),
Figure imgf000004_0001
Formula (V) wherein, k is 0, 1, 2, or 3; each of L1, L2, and Lk is independently selected from a bond, -O-, -S-, -NR11-, Ci-Cs alkylene, and Ci-Cs heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa; each of RLa is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-G, alkenyl. CS-Galkynyl. cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; or two RLa are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R;
R11 is hydrogen, -CN, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, CS-G, alkenyl. C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of RL1 and RLk is independently selected from
Figure imgf000005_0001
cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRLa: each of RRl a is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. G-Galkynyl. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. G-Galkynyl. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R; and each R is independently halogen, oxo, -CN, -OH, -SF5, -SH, -S(=O)Ci-C3alkyl, -S(=O)2Ci-C3alkyl, - S(=O)2NH2, -S(=O)2NHCi-C3alkyl, -S(=O)2N(Ci-C3alkyl)2, -S(=O)(=NCi-C3alkyl)(Ci-C3alkyl), - NH2, -NHCi-C3alkyl, -N(Ci-C3alkyl)2, -N=S(=O)(Ci-C3alkyl)2, -C(=O)Ci-C3alkyl, -C(=O)OH, - C(=O)OCi-C3alkyl, -C(=O)NH2, -C(=O)NHCi-C3alkyl, -C(=O)N(Ci-C3alkyl)2, -P(=O)(Ci-C3alkyl)2;
Ci-C3alkyl, Ci-C3alkoxy, Ci-C3haloalkyl, Ci-C3haloalkoxy, Ci-C3hydroxyalkyl, Ci-C3aminoalkyl,
Ci-C3heteroalkyl, or CYC, cycloalkyl.
In one aspect, the present disclosure relates to a bifimctional compound having a structure of Formula (lib*), or a pharmaceutically acceptable salt thereof,
Figure imgf000006_0002
Formula (lib*) wherein,
EBM is a moiety that binds to an E3 ubiquitin ligase;
L is a covalent linker; and
R1, U, V, X, Y, Z1, Z2, n, R2, R13 and R3 have the meanings defined in Formula (II).
In one aspect, the present disclosure relates to a bifimctional compound having a structure of Formula (lie*), or a pharmaceutically acceptable salt thereof,
Figure imgf000006_0001
Formula (lie*) wherein,
EBM is a moiety that binds to an E3 ubiquitin ligase;
L is a covalent linker; and
R1, U, V, X, Y, Z1, Z2, n, R2, R13 and R3 have the meanings defined in Formula (II).
In one aspect, the present disclosure relates to bifimctional compounds for use as a medicament.
In one aspect, the present disclosure relates to bifimctional compounds for use in treatment of cancer. In some embodiments, the cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.
In one aspect, the present disclosure relates to a pharmaceutical composition comprising a bifimctional compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
In one aspect, the present disclosure relates to a method of treating disease, the method comprising administering to a subject in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound disclosed herein. In one aspect, the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6 -adenosine -methyltransferase, of the general formula (A-I)
Figure imgf000007_0001
Formula (A-I).
In one aspect, the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase, of the general formula (A-II)
Figure imgf000007_0002
Formula (A-II).
DESCRIPTION OF FIGURES
Fig. 1 shows A) Design of METTL3 inhibitor 2 from hit compound 1. The bonds formed are depicted in red. The IC50 values refer to the biochemical assay based on time resolved-Forster resonance energy transfer (TR-FRET). B) Superimposition of compounds 1 (grey, from X- ray with METTL3, PDB code: 7NHI) and 2 (cyan, drawn in Pymol).
Fig. 2 shows design of compound 8 starting from 5, the newly formed bonds are depicted in red. A)
Inhibitor 5 (cyan) in the METTL3 binding site with relevant residues (carbon atoms in grey). The main intermolecular interactions are displayed (yellow dashed lines, PDB code: 7008). B) Superimposition of inhibitors 5 (cyan) and 7 (green, PDB code: 7009). C). Superimposition of inhibitors 5 (cyan) and 8 (yellow, PDB code: 7O0L), and interactions of the lactam with the side chain amide of Gln550.
Fig. 3 shows A) Unusual interaction of the fluorine atom of compound 20 with Pro397 amide 71- system, PDB code: 7029. B) Van der Waals contacts between the fluorine atom of inhibitor 21 and the side chains of Ser511 and Tyr406, PDB code: 7O2E.
Fig. 4 shows TR-FRET dose response curves (n > 3) measured for compounds 1, 22 and SAH and chemical structure of the lead compound 22. B) InCELL Pulse dose response curve (n = 3) in HEK293T cells at 46 °C. D) Dose response curves of m6A/A reduction in polyadenylated RNA fraction in MOLM-13 (n = 5) and PC-3 (n = 3) cell lines measured by UPLC-MS/MS.
Fig. 5 shows thermal shift assay results. Shown are the first derivative of the melting curves of
METTL3/METTL14 for inhibitor 22 or SAH.
Fig. 6 shows thermal shift assay results. Shown are the first derivative of the melting curves of
METTL1 for inhibitor 22 or SAH. Compound 22 does not shift the melting temperature of METTLE
Fig. 7 Exemplary compounds.
Fig. 8 Exemplary substitution patterns for R.
Fig . 9 shows the degradation of METTL3 protein after treatment with 2 pM concentration of various
PROTAC molecules for 24h, measured with Western blot.
Fig. 10 shows the degradation of METTL14 protein after treatment with 2 pM concentration of various PROTAC molecules for 24h, measured with Western blot.
Fig. 11 shows the correlation between METTL3 and METTL14 degradation of various PROTAC molecules measured by Western blot.
DETAILED DESCRIPTION
Definitions
In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the invention may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed invention.
Reference throughout this specification to “some embodiments” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The terms below, as used herein, have the following meanings, unless indicated otherwise:
;oxo” refers to =0. “Carboxyl” refers to -COOH.
“Cyano” refers to -CN.
“Alkyl” refers to a straight-chain, or branched-chain saturated hydrocarbon monoradical having from one to about ten carbon atoms, more preferably one to six carbon atoms. Examples include, but are not limited to methyl, ethyl, n-propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3-methyl-l- butyl, 2-methyl-3 -butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 -methyl- 1 -pentyl, 4-methyl-l -pentyl, 2- methyl -2 -pentyl, 3 -methyl -2 -pentyl, 4-methyl -2 -pentyl, 2,2-dimethyl-l -butyl, 3, 3 -dimethyl- 1 -butyl, 2 -ethyl - 1 -butyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl and hexyl, and longer alkyl groups, such as heptyl, octyl and the like. Whenever it appears herein, a numerical range such as “Ci- G, alkyl” or “Ci-ealkyl”, means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated. In some embodiments, the alkyl is a Ci-ioalkyl. In some embodiments, the alkyl is a Ci-ealkyl. In some embodiments, the alkyl is a G --alkyl. In some embodiments, the alkyl is a Ci-4alkyl. In some embodiments, the alkyl is a Ci-3alkyl. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkyl is optionally substituted with oxo, halogen, -CN, -COOH, -COOMe, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkyl is optionally substituted with halogen, -CN, -OH, or -OMe. In some embodiments, the alkyl is optionally substituted with halogen.
“Alkenyl” refers to a straight-chain, or branched-chain hydrocarbon monoradical having one or more carbon-carbon double-bonds and having from two to about ten carbon atoms, more preferably two to about six carbon atoms. The group may be in either the cis or trans conformation about the double bond(s), and should be understood to include both isomers. Examples include, but are not limited to ethenyl (-CH=CH2), 1 -propenyl (-CH2CH=CH2), isopropenyl [-C(CH3)=CH2], butenyl, 1,3-butadienyl and the like. Whenever it appears herein, a numerical range such as “C2-C6 alkenyl” or “C2-6alkenyl”, means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkenyl” where no numerical range is designated. Unless stated otherwise specifically in the specification, an alkenyl group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkenyl is optionally substituted with oxo, halogen, -CN, -COOH, -COOMe, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkenyl is optionally substituted with halogen, -CN, -OH, or -OMe. In some embodiments, the alkenyl is optionally substituted with halogen.
“Alkynyl” refers to a straight-chain or branched-chain hydrocarbon monoradical having one or more carboncarbon triple-bonds and having from two to about ten carbon atoms, more preferably from two to about six carbon atoms. Examples include, but are not limited to ethynyl, 2-propynyl, 2-butynyl, 1,3-butadiynyl and the like. Whenever it appears herein, a numerical range such as “C2-C6 alkynyl” or “C2-6alkynyl”, means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkynyl” where no numerical range is designated. Unless stated otherwise specifically in the specification, an alkynyl group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkynyl is optionally substituted with oxo, halogen, -CN, -COOH, COOMe, -OH, -OMe, - NH2, or -NO2. In some embodiments, the alkynyl is optionally substituted with halogen, -CN, -OH, or - OMe. In some embodiments, the alkynyl is optionally substituted with halogen.
“Alkylene” refers to a straight or branched divalent hydrocarbon chain. Unless stated otherwise specifically in the specification, an alkylene group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkylene is optionally substituted with oxo, halogen, -CN, -COOH, COOMe, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkylene is optionally substituted with halogen, -CN, -OH, or -OMe. In some embodiments, the alkylene is optionally substituted with halogen.
“Alkoxy” refers to a radical of the formula -Oalkyl where alkyl is as defined above. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkoxy is optionally substituted with halogen, -CN, -COOH, COOMe, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkoxy is optionally substituted with halogen, -CN, -OH, or -OMe. In some embodiments, the alkoxy is optionally substituted with halogen.
“Aryl” refers to a radical derived from a hydrocarbon ring system comprising 6 to 30 carbon atoms and at least one aromatic ring. The aryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the aryl is bonded through an aromatic ring atom) or bridged ring systems. In some embodiments, the aryl is a 6- to 10-membered aryl. In some embodiments, the aryl is a 6-membered aryl (phenyl). Aryl radicals include, but are not limited to, aryl radicals derived from the hydrocarbon ring systems of anthrylene, naphthylene, phenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, an aryl may be optionally substituted, for example, with halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the aryl is optionally substituted with halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the aryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe. In some embodiments, the aryl is optionally substituted with halogen.
“Cycloalkyl” refers to a partially or fully saturated, monocyclic, or polycyclic carbocyclic ring, which may include fused (when fused with an aryl or a heteroaryl ring, the cycloalkyl is bonded through a non-aromatic ring atom), spiro, or bridged ring systems. In some embodiments, the cycloalkyl is fully saturated. Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to fifteen carbon atoms (e.g., C3-C15 fully saturated cycloalkyl or C3-C15 cycloalkenyl), from three to ten carbon atoms (e.g., C3-C10 fully saturated cycloalkyl or C3-C10 cycloalkenyl), from three to eight carbon atoms (e.g., C3-C8 fully saturated cycloalkyl or C3-C8 cycloalkenyl), from three to six carbon atoms (e.g., C3-C6 fully saturated cycloalkyl or C3-C6 cycloalkenyl), from three to five carbon atoms (e.g., C3-C5 fully saturated cycloalkyl or C3-C5 cycloalkenyl), or three to four carbon atoms (e.g., C3-C4 fully saturated cycloalkyl or C3-C4 cycloalkenyl). In some embodiments, the cycloalkyl is a 3 - to 10-membered fully saturated cycloalkyl or a 3- to 10-membered cycloalkenyl. In some embodiments, the cycloalkyl is a 3- to 6-membered fully saturated cycloalkyl or a 3 - to 6-membered cycloalkenyl. In some embodiments, the cycloalkyl is a 5 - to 6-membered fully saturated cycloalkyl or a 5- to 6-membered cycloalkenyl. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbomyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cisdecalin, trans-decalin, bicyclo [2.1.1] hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Partially saturated cycloalkyls include, for example cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Unless stated otherwise specifically in the specification, a cycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, a cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, a cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe. In some embodiments, the cycloalkyl is optionally substituted with halogen.
“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo. In some embodiments, halogen is fluoro or chloro. In some embodiments, halogen is fluoro.
“Haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2- difluoroethyl, 3 -bromo-2 -fluoropropyl, 1,2-dibromoethyl, and the like.
“Hydroxyalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more hydroxyls. In some embodiments, the alkyl is substituted with one hydroxyl. In some embodiments, the alkyl is substituted with one, two, or three hydroxyls. Hydroxyalkyl include, for example, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, or hydroxypentyl. In some embodiments, the hydroxyalkyl is hydroxymethyl. Non-limiting examples of hydroxyalkyl include -CH2OH, -(CH2)2OH, -(QLhOH, -CH2CH(OH)CH3, - (CH2)4OH, -CH(CH2OH)CH2CH3, -CH2CH(CH2OH)CH3, -CH(OH)(CH2)2OH, -CH2CH(OH)CH2OH, - CH2CH(OH)(CH2)2OH and -CH2CH(CH2OH)2 for terminal moieties and -CHOH-, -CH2CHOH-, - CH2CH(OH)CH2-, -(CH2)2CHOHCH2-, -CH(CH2OH)CH2CH2-, -CH2CH(CH2OH)CH2-, - CH(OH)(CH2CHOH-, -CH2CH(OH)CH2OH, -CH2CH(OH)(CH2)2OH and -CH2CHCH2OHCHOH- for a hydroxyl substituted alkyl moiety bridging two other moieties.
“Aminoalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more amines. In some embodiments, the alkyl is substituted with one amine. In some embodiments, the alkyl is substituted with one, two, or three amines. Aminoalkyl include, for example, aminomethyl, aminoethyl, aminopropyl, aminobutyl, or aminopentyl. In some embodiments, the aminoalkyl is aminomethyl. Non-limiting examples of aminoalkyl include -CH2NH2, -CH2NHMe, -CH2NHEt,
-CH2CH2NH2, -CH2CH2NHMe, -CH2CH2NHEt, -(CH2)3NH2, -(CH2)3NHMe, -(CH2)3NHEt, -CH2CH(NH2)CH3, -CH2CH(NHMe)CH3, -CH2CH(NHEt)CH3, -(CH2)3CH2NH2, -(CH2)3CH2NHMe, -(CH2)3CH2NHEt, -CH(CH2NH2)CH2CH3, -CH(CH2NHMe)CH2CH3, -CH(CH2NHEt)CH2CH3, -CH2CH(CH2NH2)CH3, -CH2CH(CH2NHMe)CH3, -CH2CH(CH2NHEt)CH3, -CH(NH2)(CH2)2NH2, -CH(NHMe)(CH2)2NHMe, -CH(NHEt)(CH2)2NHEt, -CH2CH(NH2)CH2NH2, -CH2CH(NHMe)CH2NHMe, -CH2CH(NHEt)CH2NHEt, -CH2CH(NH2)(CH2)2NH2, -CH2CH(NHMe)(CH2)2NHMe, -CH2CH(NHEt)(CH2)2NHEt, -CH2CH(CH2NH2)2, -CH2CH(CH2NHMe)2 and -CH2CH(CH2NHEt)2 for terminal moieties and -CH2CHNH2-, -CH2CHNHMe-, -CH2CHNHEt- for an amino substituted alkyl moiety bridging two other moieties.
“Heteroalkyl” refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, phosphorus, or combinations thereof. A heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In one aspect, a heteroalkyl is a C1-C6 heteroalkyl wherein the heteroalkyl is comprised of 1 to 6 carbon atoms and one or more atoms other than carbon, e.g., oxygen, nitrogen (e.g. -NH-, -N(alkyl)-), sulfur, phosphorus, or combinations thereof wherein the heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. Examples of such heteroalkyl are, for example, -CH2OCH3, -CH2CH2OCH3, - CH2CH2OCH2CH2OCH3, -CH(CH3)OCH3, -CH2NHCH3, -CH2N(CH3)2, -CH2CH2NHCH3, or - CH2CH2N(CH3)2. Unless stated otherwise specifically in the specification, a heteroalkyl is optionally substituted for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, or - OMe. In some embodiments, the heteroalkyl is optionally substituted with halogen.
“Heterocycloalkyl” refers to a 3- to 24-membered partially or fully saturated ring radical comprising 2 to 23 carbon atoms and from one to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, silicon, and sulfur. In some embodiments, the heterocycloalkyl is fully saturated. In some embodiments, the heterocycloalkyl comprises one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the heterocycloalkyl comprises one to three heteroatoms selected from the group consisting of nitrogen and oxygen. In some embodiments, the heterocycloalkyl comprises one to three nitrogens. In some embodiments, the heterocycloalkyl comprises one or two nitrogens. In some embodiments, the heterocycloalkyl comprises one nitrogen. In some embodiments, the heterocycloalkyl comprises one nitrogen and one oxygen. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom), spiro, or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocycloalkyl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized. Representative heterocycloalkyls include, but are not limited to, heterocycloalkyls having from two to fifteen carbon atoms (e.g., C2-C15 fully saturated heterocycloalkyl or C2-C15 heterocycloalkenyl), from two to ten carbon atoms (e.g., C2-C10 fully saturated heterocycloalkyl or C2-C10 heterocycloalkenyl), from two to eight carbon atoms (e.g., C2-C8 fully saturated heterocycloalkyl or C2-C8 heterocycloalkenyl), from two to seven carbon atoms (e.g., C2-C7 fully saturated heterocycloalkyl or C2-C7 heterocycloalkenyl), from two to six carbon atoms (e.g., C2-C6 fully saturated heterocycloalkyl or C2- C7 heterocycloalkenyl), from two to five carbon atoms (e.g., C2-C5 fully saturated heterocycloalkyl or C2-C5 heterocycloalkenyl), or two to four carbon atoms (e.g., C2-C4 fully saturated heterocycloalkyl or C2-C4 heterocycloalkenyl). Examples of such heterocycloalkyl radicals include, but are not limited to, aziridinyl, azetidinyl, oxetanyl, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2- oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, 1,3-dihydroisobenzofuran-l-yl, 3-oxo- 1,3-dihydroisobenzofuran-l-yl, methyl-2-oxo-l,3-dioxol-4-yl, and 2-oxo-l,3-dioxol-4-yl. The term heterocycloalkyl also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides, and the oligosaccharides. In some embodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring). In some embodiments, the heterocycloalkyl is a 3- to 8-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 3 - to 7-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 3- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 4- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 5- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 3- to 8-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 3- to 7-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 3- to 6-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 4- to 6-membered heterocycloalkenyl. In some embodiments, the heterocycloalkyl is a 5 - to 6-membered heterocycloalkenyl. Unless stated otherwise specifically in the specification, a heterocycloalkyl may be optionally substituted as described below, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the heterocycloalkyl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, -OH, or -OMe. In some embodiments, the heterocycloalkyl is optionally substituted with halogen.
“Heteroaryl” refers to a 5 - to 14-membered ring system radical comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, and sulfur, and at least one aromatic ring. In some embodiments, the heteroaryl comprises one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl comprises one to three heteroatoms selected from the group consisting of nitrogen and oxygen. In some embodiments, the heteroaryl comprises one to three nitrogens. In some embodiments, the heteroaryl comprises one or two nitrogens. In some embodiments, the heteroaryl comprises one nitrogen. The heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the heteroaryl is bonded through an aromatic ring atom) or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized. In some embodiments, the heteroaryl is a 5 - to 10-membered heteroaryl. In some embodiments, the heteroaryl is a 5 - to 6-membered heteroaryl. In some embodiments, the heteroaryl is a 6-membered heteroaryl. In some embodiments, the heteroaryl is a 5-membered heteroaryl. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1- oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1 -phenyl- IH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, a heteroaryl may be optionally substituted, for example, with halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -COOH, COOMe, -CF3, -OH, -OMe, -NH2, or - N02. In some embodiments, the heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, - OH, or -OMe. In some embodiments, the heteroaryl is optionally substituted with halogen.
The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means either “alkyl” or “substituted alkyl” as defined above. Further, an optionally substituted group may be un-substituted (e.g., - CH2CH3), fully substituted (e.g., -CF2CF3), mono-substituted (e.g., -CH2CH2F) or substituted at a level anywhere in-between fully substituted and mono-substituted (e.g., -CH2CHF2, -CH2CF3, -CF2CH3, - CFHCHF2, etc.). It will be understood by those skilled in the art with respect to any group containing one or more substituents that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical and/or synthetically non -feasible. Thus, any substituents described should generally be understood as having a minimum and maximum molecular weight of about 750-1,700 daltons, and more typically, about 900-1,250 daltons.
The term “one or more” when referring to an optional substituent means that the subject group is optionally substituted with one, two, three, four, or more substituents. In some embodiments, the subject group is optionally substituted with one, two, three or four substituents. In some embodiments, the subject group is optionally substituted with one, two, or three substituents. In some embodiments, the subject group is optionally substituted with one or two substituents. In some embodiments, the subject group is optionally substituted with one substituent. In some embodiments, the subject group is optionally substituted with two substituents.
An “effective amount” or “therapeutically effective amount” refers to an amount of a compound administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.
The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating, or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
The term METTL3 in the context of the present specification relates to N6-adenosine-methyltransferase catalytic subunit (e.g., see Uniprot ID: Q86U44).
The term METTL14 in the context of the present specification relates to N6-adenosine-methyltransferase non-catalytic subunit (e.g., see Uniprot ID: Q9HCE5).
PROTAC compound
One aspect of the disclosure relates to a bifiinctional compound having a structure of Formula (I), or a pharmaceutically acceptable salt thereof, MBM-L-EBM
Formula (I) wherein,
MBM is a moiety that binds to METTL3;
EBM is a moiety that binds to an E3 ubiquitin ligase; and
L is a linker, wherein L has a structure of Formula (V),
Figure imgf000016_0001
Formula (V) wherein, k is 0, 1, 2, or 3; each of L1, L2, and Lk is independently selected from a bond, -O-, -S-, -NR11-, Ci-Cs alkylene, and Ci-Cs heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa; each of RLa is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-G, alkenyl. C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; or two RLa are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R;
R11 is hydrogen, -CN, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, CS-G, alkenyl. C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of RL1 and RLk is independently selected from
Figure imgf000016_0002
, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRLa: each of RRl a is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, G-Ghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R; and each R is independently halogen, oxo, -CN, -OH, -SF5, -SH, -S(=O)Ci-C3alkyl, -S(=O)2Ci-C3alkyl, - S(=O)2NH2, -S(=O)2NHCi-C3alkyl, -S(=O)2N(Ci-C3alkyl)2, -S(=O)(=NCi-C3alkyl)(Ci-C3alkyl), - NH2, -NHCi-C3alkyl, -N(Ci-C3alkyl)2, -N=S(=O)(Ci-C3alkyl)2, -C(=O)Ci-C3alkyl, -C(=O)OH, - C(=O)OCi-C3alkyl, -C(=O)NH2, -C(=O)NHCi-C3alkyl, -C(=O)N(Ci-C3alkyl)2, -P(=O)(Ci-C3alkyl)2, Ci-Csalkyl, C1-C3 alkoxy, Ci-Cdialoalkyl. C1-C3 haloalkoxy, C1-C3 hydroxyalkyl, C1-C3 aminoalkyl, C’l-Cdictcroalkyl. or C3-C6cycloalkyl.
In some embodiments, L is a linker that imparts rigidity.
In some embodiments, R11 is hydrogen, C1-C6alkyl, C1-C6hydroxyalkyl, C1-C6heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In some embodiments, R11 is hydrogen or C1-C6alkyl. In some embodiments, R11 is hydrogen.
In some embodiments, RLa is oxo or C1-C6alkyl optionally substituted with one or more R. In some embodiments, RLa is C1-C6alkyl. In some embodiments, RLa is oxo.
In some embodiments, two RLa are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R. In some embodiments, two RLa are taken together to form a 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl, each of which is unsubstituted or substituted.
In some embodiments, each RRLa is independently oxo or C1-C6alkyl optionally substituted with one or more R. In some embodiments, RRLa is C1-C6alkyl. In some embodiments, each RRLa is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R. In some embodiments, each RRLa is independently halogen, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -SH, -SRa, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, or heteroalkyl is unsubstituted or substituted with one or more R. In some embodiments, each RRLa is independently halogen, -CN, -NO2, -OH, -ORa, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl.
In some embodiments of a compound disclosed herein, each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, cycloalkyl, heterocycloalkyl, Ci- C6alkylene(cycloalkyl), or Ci-C6alkylene(heterocycloalkyl), wherein each alkyl, alkylene, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, Ci- Ceaminoalkyl, C1-C6heteroalkyl, cycloalkyl, or heterocycloalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, Ci- Ceaminoalkyl, or C1-C6heteroalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl. In some embodiments of a compound disclosed herein, each Ra is independently C1-C6alkyl or C1-C6haloalkyl. In some embodiments of a compound disclosed herein, each Ra is independently C1-C6alkyl.
In some embodiments of a compound disclosed herein, each Rb is independently hydrogen, C1-C6alkyl, Ci- Cehaloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, cycloalkyl, heterocycloalkyl, Ci- C6alkylene(cycloalkyl), or Ci-C6alkylene(heterocycloalkyl), wherein each alkyl, alkylene, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, Ci- Cehydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, cycloalkyl, or heterocycloalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, each Rb is independently hydrogen, C1-C6alkyl, Ci- Cehaloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, Ci- Cehydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl. In some embodiments of a compound disclosed herein, each Rb is independently hydrogen, C1-C6alkyl, or C1-C6haloalkyl. In some embodiments of a compound disclosed herein, each Rb is independently hydrogen or C1-C6alkyl. In some embodiments of a compound disclosed herein, each Rb is hydrogen. In some embodiments of a compound disclosed herein, each Rb is independently C1-C6alkyl. In some embodiments of a compound disclosed herein, Rc and Rd are each independently hydrogen, Ci- C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, cycloalkyl, heterocycloalkyl, Ci-C6alkylene(cycloalkyl), or Ci-C6alkylene(heterocycloalkyl), wherein each alkyl, alkylene, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, cycloalkyl, or heterocycloalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl, wherein each alkyl, cycloalkyl, and heterocycloalkyl is independently optionally substituted with one or more R. In some embodiments of a compound disclosed herein, Rc and Rd are each independently hydrogen, C1-C6alkyl, Ci- Cehaloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl. In some embodiments of a compound disclosed herein, Rc and Rd are each independently hydrogen, C1-C6alkyl, or C1-C6haloalkyl. In some embodiments of a compound disclosed herein, Rc and Rd are each independently hydrogen or Ci- Cealkyl. In some embodiments of a compound disclosed herein, Rc and Rd are each hydrogen. In some embodiments of a compound disclosed herein, Rc and Rd are each independently C1-C6alkyl.
In some embodiments of a compound disclosed herein, Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R. In some embodiments of a compound disclosed herein, Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl.
In some embodiments of a compound disclosed herein, each R is independently halogen, -CN, -OH, -NH2, - NHCi-C3alkyl, -N(Ci-C3alkyl)2, -C(=O)Ci-C3alkyl, -C(=O)OH, -C(=O)OCi-C3alkyl, -C(=O)NH2, - C(=O)NHCi-C3alkyl, -C(=O)N(Ci-C3alkyl)2, Ci-C3alkyl, Ci-C3alkoxy, Ci-C3haloalkyl, Ci-C3haloalkoxy, Ci-C3hydroxyalkyl, Ci-C3aminoalkyl, Ci-C3heteroalkyl, or C’,-G, cycloalky I: or two R on the same atom form an oxo. In some embodiments of a compound disclosed herein, each R is independently halogen, -CN, -OH, -NH2, -NHCi-C3alkyl, -N(Ci-C3alkyl)2, Ci-C3alkyl, Ci-C3alkoxy, Ci-C3haloalkyl, Ci-C3haloalkoxy, Ci-C3hydroxyalkyl, Ci-C3aminoalkyl, Ci-C3heteroalkyl, or G-G, cycloalky I: or two R on the same atom form an oxo. In some embodiments of a compound disclosed herein, each R is independently halogen, -CN, -OH, -NH2, Ci-C3alkyl, Ci-C3alkoxy, Ci-C3haloalkyl, or Ci-C3haloalkoxy; or two R on the same atom form an oxo. In some embodiments of a compound disclosed herein, each R is independently halogen, -CN, -OH, -NH2, Ci-C3alkyl, or Ci-C3haloalkyl; or two R on the same atom form an oxo. In some embodiments of a compound disclosed herein, each R is independently halogen or Ci-C3alkyl.
In some embodiments, the structure of Formula (I) has a structure of Formula (II),
Figure imgf000020_0001
Formula (II) wherein,
Z1 and Z2 are independently selected from N, CH and CR2;
X is O or NH;
Y is CH2, C=O, or SO2;
U and V are each independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other one is -(CH2)2-
R1 is an unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted cycloalkyl, or unsubstituted or substituted heterocycloalkyl;
R2 is selected from halogen (e.g., F, Cl), -CN, -NO2, -OH, -ORa, -SF5, -SH, -SRa, -NRcRd (e.g., NH2), C1-C6alkyl (e.g., methyl), C1-C6haloalkyl (e.g., CF3, CHF2, CH2F), C1-C6hydroxyalkyl, C1-C6aminoalkyl, and C1-C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; n is selected from 0, 1, 2, 3, and 4;
R13 is selected from a bond, -O-, -S-, -NR13N-, or CR13CR13C;
R3 is C1-C6alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; each of which is unsubstituted or substituted with one or more R12; each R12 is independently halogen, -CN, -NO2, -OH, -ORa, -SH, -SRa, -NRcRd, C1-C6alkyl, Ci- Cehaloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-G, alkenyl. CS-Galkynyl. cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more R; each R13C is independently hydrogen, halogen, -CN, -NO2, -OH, -ORa, -SH, -SRa, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; or two R13C are taken together to form an oxo; or two R13C are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R; and
R13N is hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, - C(=O)Ra-, -C(=O)ORb, or -S(=O)2Ra, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R. In some embodiments, R1 is unsubstituted or substituted with 1, 2, 3, or 4 R14, wherein each R14 is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, -OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, -NRbC(=O)NRcRd, -NRbC(=O)Ra, - NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, CS-G, alkenyl. C2- Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R. In some embodiments, R1 is unsubstituted or substituted with 1, 2, 3, or 4 R14. In some embodiments, R1 is unsubstituted or substituted with 1 or 2 R14. In some embodiments, each R14 is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, -OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, - -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl. In some embodiments, each R14 is independently halogen, -CN, -NO2, -OH, -ORa, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl. In some embodiments, each R14 is independently halogen, -CN, -NO2, -OH, -ORa, amino, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, or heteroalkyl is unsubstituted or substituted with one or more R. In some embodiments, R14 is C1-C6aminoalkyl. In some embodiments, R14is -NHCH3. In some embodiments, R1 is a monocyclic group. In some embodiments, R1 is a bicyclic group.
In some embodiments, the structure of Formula (II) has a structure of Formula (Ila),
Figure imgf000021_0001
Formula (Ila).
In some embodiments, the structure of Formula (II) has a structure of Formula (Illa),
Figure imgf000021_0002
Formula (Illa), wherein, NR31R32 are taken together to form a heterocycloalkyl, which is unsubstituted or substituted with one or more
Figure imgf000022_0001
each R14is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; and n2 is 0, 1, or 2.
In some embodiments, -L-EBM is attached to the pyrimidine of Formula (Illa) through a R14 group.
In some embodiments, the structure of Formula (II) has a structure of Formula (lib)
Figure imgf000022_0002
Formula (lib).
In some embodiments, the structure of Formula (II) has a structure of Formula (Illb),
Figure imgf000022_0003
Formula (Illb) wherein,
NR31R32 are taken together to form a heterocycloalkyl, which is unsubstituted or substituted with one
Figure imgf000022_0004
each R14is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, -
OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, -
NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; and n2 is 0, 1, 2 or 3.
In some embodiments, the structure of Formula (II) has a structure of Formula (lie),
Figure imgf000023_0001
In some embodiments, the structure of Formula (II) has a structure of Formula (IIIc),
Figure imgf000023_0002
wherein, each R14is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; and n2 is 0, 1, 2 or 3. In some embodiments, R13 is -O-. In some embodiments, R13 is -NR13N-. In some embodiments, R13 is - N((C=O)CH2CH3). In some embodiments, R13 is -CR13CR13C-. In some embodiments, R13 is -CH2-. In some embodiments, R13 is -CR13CR13C-, wherein two R13C are taken together to form a 3-6 membered cycloalkyl or
4-6 membered heterocycloalkyl, each of which is unsubstituted or substituted with one or more R. In some embodiments, R13 is selected from
Figure imgf000024_0001
some embodiments, R13 is
Figure imgf000024_0003
, In some embodiments,
Figure imgf000024_0002
In some embodiments, R3 is 4-6 membered heterocycloalkyl, wherein R3 is unsubstituted or substituted with 1, 2 or 3 R12. In some embodiments, R3 is 6 membered heterocycloalkyl, wherein R3 is unsubstituted or substituted with 1, 2 or 3 R12. In some embodiments, R3 unsubstituted or substituted with 1, 2 or 3 R12. In some embodiments, R3 is monocyclic heterocycloalkyl. In some embodiments,
Figure imgf000024_0004
Figure imgf000024_0005
. In some embodiments, R3 is bicyclic heterocycloalkyl.
In some embodiments,
Figure imgf000024_0006
In some embodiments, R1 is unsubstituted or substituted 6 membered heteroaryl. In some embodiments, R1 is unsubstituted or substituted pyrimidine. In some embodiments, R1 is a bicyclic heteroaryl.
In some embodiments, L1 attaches to the MBM and L2 attaches to the EBM. In some embodiments, L2 attaches to the MBM and L1 attaches to the EBM.
In some embodiments, each of L1, L2, and Lk is independently selected from a bond, C1-C6 alkylene, and Ci- G, heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa. In some embodiments, each of L1, L2, and Lk is independently selected from a bond, C1-C4 alkylene, and C1-C4 heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa. In some embodiments, each of L1, L2, and Lk is independently selected from a bond,
Figure imgf000024_0007
Figure imgf000025_0005
In some embodiments, each of RL1 and RLk is independently selected from
Figure imgf000025_0001
, 6-14 membered cycloalkyl, 6-14 membered heterocycloalkyl, 6-14 membered aryl, and 6-14 membered heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRLa. In some embodiments, RL1 is unsubstituted or substituted monocyclic heterocycloalkyl. In some embodiments, RL1 is unsubstituted or substituted 5-6 membered monocyclic heterocycloalkyl. In some embodiments, RL1 is
Figure imgf000025_0002
. , . In some embodiments, RL1 is unsubstituted or substituted bicyclic heterocycloalkyl. In some embodiments, RL1 is a spiro bicyclic ring. In some embodiments, RL1 is a fused bicyclic ring. In some embodiments, RL1 is
Figure imgf000025_0003
Figure imgf000025_0004
some embodiments, RL1 is unsubstituted or substituted phenyl. In some embodiments, RL1 is unsubstituted or substituted monocyclic heteroaryl. In some embodiments, RLk is unsubstituted or substituted monocyclic heterocycloalkyl. In some embodiments,
Figure imgf000026_0001
, . In some embodiments, RLk is unsubstituted or substituted bicyclic heterocycloalkyl. In some embodiments,
Figure imgf000026_0002
Figure imgf000026_0003
Figure imgf000026_0004
some embodiments, RLk is unsubstituted or substituted phenyl. In some embodiments, RLk is unsubstituted or substituted monocyclic heteroaryl.
In some embodiments, the bifunctional compound has a structure of
Figure imgf000026_0005
In some embodiments, the bifunctional compound has a structure of Formula (lib*), or a pharmaceutically acceptable salt thereof,
Figure imgf000026_0006
Formula (lib*) wherein,
EBM is a moiety that binds to an E3 ubiquitin ligase;
L is a covalent linker; and
R1, U, V, X, Y, Z1, Z2, n, R2, R13 and R3 have the meanings defined in Formula (II).
In some embodiments, the bifunctional compound has a structure of Formula (lie*), or a pharmaceutically acceptable salt thereof,
Figure imgf000027_0001
Formula (lie*) wherein,
EBM is a moiety that binds to an E3 ubiquitin ligase;
L is a covalent linker; and
R1, U, V, X, Y, Z1, Z2, n, R2, R13 and R3 have the meanings defined in Formula (II).
In one aspect, the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase, of the general formula (A-I)
Figure imgf000027_0002
Formula (A-I), wherein
NR31R32 is selected from an optionally substituted 3-12 membered heterocycloalkyl
Figure imgf000027_0003
Figure imgf000027_0004
each R2 is independently selected from the group consisting of halogen (e.g., F, Cl), C1-C3 alkyl, and Ci- C3 haloalkyl (e.g., CF3, CHF2, CH2F); n is an integer selected from 0, 1, 2, 3, and 4;
EBM is a moiety that binds to an E3 ubiquitin ligase;
Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S);
Spacer is a linker moiety comprising or essentially consisting of 3 to 50 atoms of atomic mass >12; each R14 is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, - NRcRd, -NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, Ci-C6alkyl, Ci-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; n2 is 0, 1, 2 or 3; each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R; and each Ris independently halogen, oxo, -CN, -OH, -SF5, -SH, -S(=O)Ci-C3alkyl, -S(=O)2Ci-C3alkyl, - S(=O)2NH2, -S(=O)2NHCi-C3alkyl, -S(=O)2N(Ci-C3alkyl)2, -S(=O)(=NCi-C3alkyl)(Ci-C3alkyl), - NH2, -NHCi-C3alkyl, -N(Ci-C3alkyl)2, -N=S(=O)(Ci-C3alkyl)2, -C(=O)Ci-C3alkyl, -C(=O)OH, - C(=O)OCi-C3alkyl, -C(=O)NH2, -C(=O)NHCi-C3alkyl, -C(=O)N(Ci-C3alkyl)2, -P(=O)(Ci- C3alkyl)2, Ci-Csalkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 haloalkoxy, C1-C3 hydroxyalkyl, Ci- Ckaminoalkyl. Ci-C3heteroalkyl, or O-Cecycloalkyl.
In one aspect, the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase, of the general formula (A-II)
Figure imgf000029_0001
Formula (A-II), wherein
R13 is selected from a bond, -O-, -S-, -NR13N-, or CR13CR13C;
R3 is C1-C6alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; each of which is unsubstituted or substituted with one or more R12; each R12 is independently halogen, -CN, -NO2, -OH, -ORa, -SH, -SRa, -NRcRd, C1-C6alkyl, Ci- Cehaloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2- Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more R; each R13Cis independently hydrogen, halogen, -CN, -NO2, -OH, -ORa, -SH, -SRa, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or C1-C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; or two R13C are taken together to form an oxo; or two R13C are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R;
R13N is hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, Ci- Ceheteroalkyl, -C(=O)Ra-, -C(=O)ORb, or -S(=O)2Ra, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; each R2 is independently selected from the group consisting of halogen (e.g., F, Cl), C1-C3 alkyl, and Ci- C3 haloalkyl (e.g., CF3, CHF2, CH2F); n is an integer selected from 0, 1, 2, 3, and 4;
EBM is a moiety that binds to an E3 ubiquitin ligase;
Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S);
Spacer is a linker moiety comprising or essentially consisting of 3 to 50 atoms of atomic mass >12; each R14 is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, - NRcRd, -NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, Ci-C6alkyl, Ci-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; n2 is 0, 1, 2 or 3; each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R; and each Ris independently halogen, oxo, -CN, -OH, -SF5, -SH, -S(=O)Ci-C3alkyl, -S(=O)2Ci-C3alkyl, - S(=O)2NH2, -S(=O)2NHCi-C3alkyl, -S(=O)2N(Ci-C3alkyl)2, -S(=O)(=NCi-C3alkyl)(Ci-C3alkyl), - NH2, -NHCi-C3alkyl, -N(Ci-C3alkyl)2, -N=S(=O)(Ci-C3alkyl)2, -C(=O)Ci-C3alkyl, -C(=O)OH, - C(=O)OCi-C3alkyl, -C(=O)NH2, -C(=O)NHCi-C3alkyl, -C(=O)N(Ci-C3alkyl)2, -P(=O)(Ci- C3alkyl)2, Ci-Csalkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 haloalkoxy, C1-C3 hydroxyalkyl, Ci- Ckaminoalkyl. Ci-C3heteroalkyl, or O-Cecycloalkyl.
In some embodiments,
NR31R32 is selected from
Figure imgf000031_0001
- each R2 is independently selected from the group comprising F, Cl, CF3, CHF2, CH2F;
- n is an integer selected from 0, 1, 2, 3, and 4;
- Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S);
- Linker is a linker moiety comprising or essentially consisting of 3 to 50 atoms of atomic mass >12.
In certain embodiments, each R2 is F. In certain embodiments, n is an integer selected from 0, 1, and 2. In certain embodiments, n is 2. In certain embodiments, Handle is a connecting moiety comprising or essentially consisting of 4 to 8 atoms of atomic mass >12. In certain embodiments, Spacer is a linker moiety comprising or essentially consisting of 4 to 30 atoms of atomic mass >12. In certain embodiments, Spacer is a linker moiety comprising or essentially consisting of 5 to 20 atoms of atomic mass >12.
In some embodiments, a bifunctional compound disclosed herein has a MBM of Formula (XI)
Figure imgf000031_0002
wherein the attachment point is not shown.
In some embodiments, a bifunctional compound disclosed herein has a MBM of Formula (Xia)
Figure imgf000031_0003
wherein the attachment point is not shown.
In some embodiments, a bifunctional compound disclosed herein has a MBM of Formula (Xlb)
Figure imgf000031_0004
wherein the attachment point is not shown. wherein
Z1 and Z2 are independently selected from N, CH and CR2; X is O or NH;
Y is CH2, C=O, or SO2;
R1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl, and a heterocycle, particularly R1 is unsubstituted or substituted heteroaryl;
R2 is selected from F, Me, Cl, OH, NH2, Br, CF3, CHF2, CH2F; n is an integer selected from 0, 1, 2, 3, and 4;
R3 is a substituted alkylamine;
U and V are independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other one is -(CH2)3-.
In certain embodiments, X is NH. In certain embodiments, Y is C=O. In certain embodiments, n is an integer selected from 0, 1, and 2. In certain embodiments, U and V are both -CH2- or are both -(CH2)2-.
In some embodiments, a bifunctional compound disclosed herein has a MBM of Formula (U)
Figure imgf000032_0001
wherein the attachment point is not shown,
- NR31R32 is selected from
Figure imgf000032_0002
- R2 is selected from the group comprising F, Cl, CF3, CHF2, CH2F;
- n is an integer selected from 0, 1, 2, 3, and 4;
- R5 is selected from an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and a heterocycloalkyl.
In certain embodiments, R2 is F. In certain embodiments, n is an integer selected from 0, 1, and 2. In certain embodiments, n is 2. In certain embodiments, R5 is selected from -Ci-6-alkyl, -Ci-6-alkyl-aryl, -Ci-6-alkyl- heteroaryl, -Ci-6-heteroalkyl-heteroaryl, and -Ci-6-heteroalkyl-aryl. In certain embodiments, R5 is selected from -Ci-6-alkyl, -Ci-6-alkyl-aryl, -Ci-6-alkyl-heteroaryl, -Ci-6-heteroalkyl-heteroaryl,-Ci-6-heteroalkyl-aryl, - Ci-6-alkyl-cycloalkyl, -Ci-6-alkyl-heterocycloalkyl, -Ci-6-heteroalkyl-heterocycloalkyl, and -Ci-6-heteroalkyl- cycloalkyl. In certain embodiments, R5 is selected from an alkyl, an alkylaryl, and a cycloalkyl.
In certain embodiments, X is NH. In certain embodiments, Y is C=O.
Figure imgf000033_0001
In certain embodiments, the moiety R1 is selected from R1 , R1 , R1
Figure imgf000033_0002
In certain embodiments, the moiety
Figure imgf000033_0004
selected from
Figure imgf000033_0003
In certain embodiments, R1 is unsubstituted or substituted heteroaryl. In certain embodiments, R1 is unsubstituted or substituted with a moiety selected from
• a secondary amine NHRN, wherein RN is selected from a C1-C6 alkyl, a C4-C6 cycloalkyl, an aryl, and a heteroaryl, an alkylaryl, and an alkylheteroaryl;
• a halogen, particularly Cl or F;
• a C1-C6 alkyl, a C4-C6 cycloalkyl, an aryl, and a heteroaryl.
In certain embodiments, R1 is unsubstituted or substituted with a moiety selected from
• a secondary amine NHRN, wherein RN is selected from a C1-C6 alkyl, a C4-C6 cycloalkyl, an aryl, and a heteroaryl;
• a halogen, particularly Cl or F.
In some embodiments, a bifunctional compound disclosed herein has a MBM of Formula (XII)
Figure imgf000033_0005
wherein the attachment point is not shown, wherein
- Z1, Z2, X, Y, R2, R3, U, V, and n have the same definitions as defined above;
- each R14 is independently selected from
• a secondary amine substituted with an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and/or a heterocycle,
• a halogen; and/or two R14 together form an unsubstituted or substituted heteroaryl or heterocycle;
- n2 is an integer selected from 0, 1, 2, and 3.
In some embodiments, a bifimctional compound disclosed herein has a MBM of Formula (XIII)
Figure imgf000034_0001
wherein
- Z1, Z2, X, Y, R2, R3, U, V, and n have the same definitions as defined above;
- R5 is selected from an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and a heterocycle;
- R6 is selected from halogen and hydrogen.
In some embodiments, a bifimctional compound disclosed herein has a MBM of Formula (XIV)
Figure imgf000034_0002
wherein
- Z1, Z2, X, Y, R2, R3, U, V, and n have the same definitions as defined above;
- R6 is selected from halogen and hydrogen;
- W is selected from N and CH.
In certain embodiments, at least one of Z1 and Z2 is CH or CR2. In certain embodiments, both Z1 and Z2 are CH or CR2.
In certain embodiments, R3 is substituted with one or several moieties selected independently from alkyl-, hydroxy-, amino-, amine-, halogen-, cycloalkyl-, and heterocycle- moieties.
In certain embodiments, R3 is substituted C1-C4 alkylamine. In certain embodiments, R3 is substituted C1-C2 alkylamine.
In certain embodiments,
Figure imgf000034_0003
wherein
- s is an integer selected from 1 and 2, more particularly s is 1 ;
- R31 and R32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, halogen-, and/or hydroxyl-substituted, or
- R31 and R32 are independently selected from hydrogen and unsubstituted or hydroxy-, and/or halogen-substituted alkyl or cycloalkyl. In certain embodiments, R31 and/or R32 are unsubstituted or substituted with alkyl-, hydroxy-, halogen-, cycloalkyl-, heterocycle- and/or -groups. In certain embodiments, R31 and/or R32 are independently selected from H and unsubstituted or hydroxy-, and/or halogen-substituted alkyl and cycloalkyl. In certain embodiments, R31 and R32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, hydroxy-, and/or halogen-substituted.
In certain embodiments, NR31R32 is selected from
Figure imgf000035_0001
v being an integer selected from 0, 1 and 2 and each RN being independently selected from hydroxyl, halogen, and C1-C4 alkyl, or two RN form a C3-C6 cycloalkyl.
Figure imgf000035_0002
from
Figure imgf000035_0003
, ,
In certain embodiments, n is an integer selected from 0, 1, and 2. In certain embodiments, n is 2. In certain embodiments, R2 is selected from F, Cl and OH. In certain embodiments, R2 is F. R2 can be bound to any of the carbon atoms of the aryl-or heteroaryl -ring. Thus, it can also be bound to Z1 or Z2 if they are carbon atoms.
In certain embodiments, R5 is selected from an alkyl, an alkylaryl, and a cycloalkyl. In certain embodiments, R5 is selected from methyl and methylphenyl.
EBM
An E3 ligase binder is a molecule which specifically binds an E3 ligase or a component of the E3 ligase complex. In certain embodiments, the E3 ligase binder binds to cereblon (CRBN, UniProt-ID: Q96SW2). In some embodiments, the E3 ligase binder binds to Von Hippel- Lindau (VHL, UniProt-ID: P40337). In some embodiments, the E3 ligase binder binds to mouse double minute 2 homolog (MDM2, UniProt-ID: Q00987). In some embodiments, the E3 ligase binder binds to an inhibitor of apoptosis protein (IAP, UniProt-ID: P98170). In certain embodiments, EBM is of the formula (B)
Figure imgf000036_0001
wherein
- Ox is CH2 or C=O;
- T is selected from the group comprising F, Cl; - k is an integer selected from the group comprising 0, 1, 2;
- I designates the bond to the Linker.
In certain embodiments, k is an integer selected from the group comprising 0, 1. In certain embodiments, k is 0. In certain embodiments, T is F.
In some embodiments, EBM has a structure according to Table A. TABLE A
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000038_0001
In some embodiments, EBM suitable for bifunctional compounds of the instant disclosure are further illustrated in Alesa Bricelj, et al., “E3 Ligase Ligands in Successful PROTACs: An Overview of Syntheses and Linker Attachment Points,” front Chem. 2021; 9: 707317, which is hereby incorporated by reference in its entirety.
Linker
In some embodiments, L is a linker, wherein L has a structure of Formula (V),
Figure imgf000039_0001
wherein, k is 0, 1, 2, or 3; each of L1, L2, and Lk is independently selected from a bond, -O-, -S-, -NR11-, Ci-Cs alkylene, and Ci-Cs heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa. each of RLa is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-G, alkenyl. CS-G, alkynyl. cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R;
R11 is hydrogen, -CN, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. C2- Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of RL1 and RLk is independently selected from , cycloalkyl, heterocycloalkyl, aryl, and
Figure imgf000039_0002
heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRLa; each of RRl a is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, - OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, - NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, - C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, Ci-Cghydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, G-Galkcnyl. G-G, alkynyl. cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; or two RLa are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R; each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), Ci-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R; and each R is independently halogen, oxo, -CN, -OH, -SF5, -SH, -S(=O)Ci-C3alkyl, -S(=O)2Ci-C3alkyl, - S(=O)2NH2, -S(=O)2NHCi-C3alkyl, -S(=O)2N(Ci-C3alkyl)2, -S(=O)(=NCi-C3alkyl)(Ci-C3alkyl), -NH2, - NHCi-C3alkyl, -N(Ci-C3alkyl)2, -N=S(=O)(Ci-C3alkyl)2, -C(=O)Ci-C3alkyl, -C(=O)OH, -C(=O)OCi- C3alkyl, -C(=O)NH2, -C(=O)NHCi-C3alkyl, -C(=O)N(Ci-C3alkyl)2, -P(=O)(Ci-C3alkyl)2, Ci-C3alkyl, Ci-Csalkoxy, Ci-C’,haloalkyl. C1-C3 haloalkoxy, C1-C3 hydroxyalkyl, Ci-Oami noalkyl, C’l-Cdictcroalkyl. or Cs-Cecycloalkyl.
In some embodiments, L is a linker that imparts rigidity. In some embodiments, L comprises 5-40 atoms in length. In some embodiments, L comprises 8-30 atoms in length. In some embodiments, L comprises 10-20 atoms in length. In some embodiments, L comprises 15-20 atoms in length. In some embodiments, L comprises 20-25 atoms in length. In some embodiments, L comprises 12-25 atoms in length.
In some embodiments, each of L1, L2, and Lk is independently no more than 12 atoms in length. In some embodiments, each of L1, L2, and Lk is independently no more than 11 atoms in length.
In some embodiments, each of L1, L2, and Lk is independently no more than 10 atoms in length.
In some embodiments, each of L1, L2, and Lk is independently no more than 9 atoms in length.
In some embodiments, each of L1, L2, and Lk is independently no more than 8 atoms in length. In some embodiments, each of L1, L2, and Lk is independently no more than 7 atoms in length. In some embodiments, each of L1, L2, and Lk is independently no more than 6 atoms in length. In some embodiments, each of L1, L2, and Lk is independently no more than 5 atoms in length.
In some embodiments, disclosed herein is a covalent linker L. In some embodiments, a covalent linker L does not imparts rigidity.
Figure imgf000041_0001
, wherein the Handle moiety is attached to the MBM, and the Spacer is attached to the EMB. In some
Figure imgf000041_0002
wherein the Handle moiety is attached to the EBM, and the Spacer is attached to the MBM.
Figure imgf000041_0003
In some embodiments, the linker is O . In some embodiments, the linker is
Figure imgf000041_0004
In some embodiments, the linker is
Figure imgf000041_0005
embodiments, the linker
Figure imgf000041_0006
some embodiments, the linker is
Figure imgf000041_0007
embodiments, the linker is
Figure imgf000041_0008
Figure imgf000041_0009
some embodiments, the linker
Figure imgf000041_0010
embodiments, the linker
Figure imgf000042_0001
some embodiments, the linker is
Figure imgf000042_0003
some embodiments, the linker i
Figure imgf000042_0002
Handle
In certain embodiments, Handle is a connecting moiety comprising or essentially consisting of 3 to 10 atoms of atomic mass >12 (C, N, O, S). In certain embodiments, Handle is a connecting moiety comprising or essentially consisting of 4 to 8 atoms of atomic mass >12.
In certain embodiments, the Handle comprises or essentially consists of 1, 2, 3, or 4 chemical moieties selected from the group comprising alkyl, amine, phenyl, and carbonyl. In some embodiments, the Handle comprises 1, 2, 3, or 4 chemical moieties selected from alkyl, amine, phenyl, carbonyl, cycloalkyl, or heterocycloalkyl.
In certain embodiments, the Handle is selected from the group comprising the following formulas:
Figure imgf000042_0004
wherein
Mid is selected from the group comprising C1-C3 alkyl, heterocycloalkyl, or cycloalkyl, and phenyl.
In some embodiments, Mid comprises C1-C3 alkyl, phenyl, heterocycloalkyl, or cycloalkyl. In some embodiments, Mid comprises C1-C3 alkyl or phenyl.
In certain embodiments, the Handle is selected from the group comprising the following formulas:
Figure imgf000042_0005
Figure imgf000043_0001
In certain embodiments, the Handle is selected from:
Figure imgf000043_0002
Figure imgf000043_0003
In certain embodiments,
Figure imgf000043_0004
certain embodiments,
Figure imgf000043_0005
In certain embodiments,
Figure imgf000043_0006
embodiments,
Figure imgf000043_0007
In certain embodiments,
Figure imgf000044_0001
pacer
Figure imgf000044_0002
Figure imgf000044_0003
Spacer
In certain embodiments, Spacer is a linker moiety comprising or essentially consisting of 3 to 50 atoms of atomic mass >12 (C, N, O, S). In certain embodiments, Spacer is a linker moiety comprising or essentially consisting of 4 to 30 atoms of atomic mass >12. In certain embodiments, the Spacer is a linker moiety comprising or essentially consisting of 5 to 20 atoms of atomic mass >12.
In certain embodiments, the Spacer comprises or essentially consists of 1, 2, 3, 4, 5, 6, or 7 chemical moieties independently selected from the group comprising alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkylene, alkylyne, ethylene glycol, carbonyl, ether, ester, amine, amide, sulfonamide, wherein the chemical moieties are each independently unsubstituted or substituted with C1-C3 alkyl, halogen, CN, NO2, hydroxyl, amine, sulfate, phosphate, and/or carboxyl.
In certain embodiments, the Spacer comprises or essentially consists of 1, 2, 3, or 4 chemical moieties selected from the group comprising alkyl, ethylene glycol, carbonyl, piperazine, aryl, amine, triazole.
In certain embodiments, the Spacer is selected from the group comprising the following formulas:
Figure imgf000044_0004
wherein - Lin is selected from the group comprising C3-C20 alkyl, C3-C20 alkyl-triazole, oligo(ethylene glycol).
In certain embodiments, the Spacer is selected from the group comprising the following formulas:
Figure imgf000045_0001
Figure imgf000045_0003
Figure imgf000045_0002
wherein
- p is selected from 2, 3, 4, and 5;
- q is selected from 7, 8, 9, 10, 11, 12, and 13;
- r is selected from 11, 12, 13, 14, 15, 16, and 17;
- s is selected from 7, 8, 9, 10, 11, 12, and 13;
- t is selected from 3, 4, 5, 6, 7, 8, and 9;
- u is selected from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
In certain embodiments, the Spacer is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S), and (T), wherein
- p is selected from 2, 3, 4, and 5;
- q is selected from 7, 8, 9, 10, 11, 12, and 13;
- r is selected from 11, 12, 13, 14, 15, 16, and 17;
- s is selected from 7, 8, 9, 10, 11, 12, and 13;
- t is selected from 3, 4, 5, 6, 7, 8, and 9; and u is selected from 7, 8, 9, 10, 11, 12, and 13.
In certain embodiments, the Spacer is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S), and (T), wherein
- p is selected from 2, 3, 4, and 5;
- q is selected from 7, 8, 9, 10, 11, 12, and 13;
- r is selected from 11, 12, 13, 14, 15, 16, and 17;
- s is selected from 7, 8, 9, 10, 11, 12, and 13;
- t is selected from 3, 4, 5, 6, 7, 8, and 9; and
- u is selected from 7, 8, 9, 10, 11, 12, 13, 14 and 15.
In certain embodiments, the Spacer is a peptide. In certain embodiments, the Spacer is a peptide consisting of proteinogenic amino acids.
Combination of features
In certain embodiments,
- the EMB is of the formula (B) or selected from Table A;
- Linker L has a structure of Formula (V) or comprises a Handle and a Spacer,
- wherein the Handle is of formula (F), (G), (H), or (J); and the Spacer is of formula (O); (P); (Q); (R); (S); or (T).
In certain embodiments, the compound comprises the definitions of Handle, Spacer, and E3 ligase binder (one row is one combination) according to Table 1 :
TABLE 1
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Exemplary PROTAC compounds of this disclosure are illustrated in TABLES 2 and 3.
TABLE 2
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
TABLE 3
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Use of the compound
In one aspect, the present disclosure relates to bifunctional compounds, which find utility to degrade and inhibit N6-adenosine-methyltransferase. In some embodiments, the bifunctional compounds target a heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compounds bind to the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compounds modulate the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compounds inhibit and degrade the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compounds target METTL3. In some embodiments, the bifunctional compounds bind to METTL3. In some embodiments, the bifunctional compounds modulate, inhibit, and/or degrade METTL3. In some embodiments, the bifunctional compounds target METTL14. In some embodiments, the bifunctional compounds modulate, inhibit, and/or degrade METTL14.
In one aspect, the present disclosure relates to bifunctional compounds for use as a medicament.
In one aspect, the present disclosure relates to bifunctional compounds for use in treatment of cancer. In some embodiments, the cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma. In one aspect, the present disclosure relates to a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
In one aspect, the present disclosure relates to a method of treating disease, the method comprising administering to a subject in need thereof a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound disclosed herein.
In some embodiments, the disclosure relates to a compound according to the first or second aspect for use as a medicament.
In some embodiments, the disclosure relates to a compound according to the first or second aspect for use in treatment of cancer. In some embodiments, the cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.
Similarly, within the scope of the present disclosure is a method or treating cancer in a patient in need thereof, comprising administering to the patient a compound according to the above description.
Similarly, a dosage form for the prevention or treatment of cancer is provided, comprising a non -agonist ligand or antisense molecule according to any of the above aspects or embodiments of the disclosure.
The skilled person is aware that any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.
Unless otherwise stated, compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
For example, the compounds described herein may be artificially enriched in one or more particular isotopes. In some embodiments, the compounds described herein may be artificially enriched in one or more isotopes that are not predominantly found in nature. In some embodiments, the compounds described herein may be artificially enriched in one or more isotopes selected from deuterium (2H), tritium (3H), iodine-125 (125I) or carbon-14 (14C). In some embodiments, the compounds described herein are artificially enriched in one or more isotopes selected from 2H, nC, 13C, 14C, 15C, 12N, 13N, 15N, 16N, 16O, 17O, 14F, 15F, 16F, 17F, 18F, 33S, 34S, 35S, 36S, 35C1, 37C1, 79Br, 81Br, 131I, and 125I. In some embodiments, the abundance of the enriched isotopes is independently at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% by molar. In some embodiments, the compound is deuterated in at least one position. In some embodiments, the compounds disclosed herein have some or all of the 3H atoms replaced with 2H atoms.
In some embodiments, the compounds described herein exist as geometric isomers. In some embodiments, the compounds described herein possess one or more double bonds. The compounds presented herein include all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well as the corresponding mixtures thereof. In some situations, the compounds described herein possess one or more chiral centers and each center exists in the R configuration, or S configuration. The compounds described herein include all diastereomeric, enantiomeric, and epimeric forms as well as the corresponding mixtures thereof. In additional embodiments of the compounds and methods provided herein, mixtures of enantiomers and/or diastereoisomers, resulting from a single preparative step, combination, or interconversion are useful for the applications described herein. In some embodiments, the compounds described herein are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. In some embodiments, dissociable complexes are preferred. In some embodiments, the diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and are separated by taking advantage of these dissimilarities. In some embodiments, the diastereomers are separated by chiral chromatography, or preferably, by separation/resolution techniques based upon differences in solubility. In some embodiments, the optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
In some situations, compounds exist as tautomers. The compounds described herein include all possible tautomers within the formulas described herein. Tautomers are compounds that are interconvertible by migration of a hydrogen atom, accompanied by a switch of a single bond and adjacent double bond. In bonding arrangements where tautomerization is possible, a chemical equilibrium of the tautomers will exist. All tautomeric forms of the compounds disclosed herein are contemplated. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH.
Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present. Pharmaceutical Composition and Administration
Another aspect of the disclosure relates to a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
In certain embodiments of the disclosure, the compound of the present disclosure is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.
The pharmaceutical composition can be formulated for oral administration, parenteral administration, or rectal administration. In addition, the pharmaceutical compositions of the present disclosure can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
The dosage regimen for the compounds of the present disclosure will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the disclosure may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.
In certain embodiments, the pharmaceutical composition or combination of the present disclosure can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
The pharmaceutical compositions of the present disclosure can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892). Method of Manufacture and Method of Treatment
The disclosure further encompasses, as an additional aspect, the use of a compound as identified herein, or its pharmaceutically acceptable salt, as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of cancer.
Similarly, the disclosure encompasses methods of treatment of a patient having been diagnosed with a disease associated with cancer. This method entails administering to the patient an effective amount of a compound as identified herein, or its pharmaceutically acceptable salt, as specified in detail herein.
Wherever alternatives for single separable features such as, for example, a ligand type or medical indication, are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the disclosure disclosed herein. Thus, any of the alternative embodiments for a ligand type may be combined with any medical indication mentioned herein.
The application further encompasses the following items:
Items
1. A compound of the general formula (XI)
Figure imgf000168_0001
- Z1 and Z2 are independently selected from N, CH and CR2;
- X is O or NH, particularly X is NH;
- Y is CH2, C=O, or SO2, particularly Y is C=O;
- R1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl, and a heterocycle, particularly R1 is unsubstituted or substituted heteroaryl;
- R2 is selected from F, Me, Cl, OH, NH2, Br, CF3, CHF2, CH2F;
- n is an integer selected from 0, 1, 2, 3, and 4, particularly n is an integer selected from 0, 1, and 2;
- R3 is a substituted alkylamine;
- U and V are independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other one is -(CH2)3-, particularly U and V are both -CH2- or are both -(012)2-.
2. The compound according to item 1, wherein
- R1 is unsubstituted or substituted with a moiety selected from
• a secondary amine NHRN, wherein RN is selected from a C1-C6 alkyl, a C4-C6 cycloalkyl, an aryl, and a heteroaryl, an alkylaryl, and an alkylheteroaryl; • a halogen, particularly Cl or F; and
• a C1-C6 alkyl, a C4-C6 cycloalkyl, an aryl, and a heteroaryl. The compound according to any one of the preceding items, wherein
R3 is substituted with one or several moieties selected independently from alkyl-, hydroxy-, amino-, amine-, halogen-, cycloalkyl-, and heterocycle- moieties. The compound according to any one of the preceding items, wherein R3 is C1-C4 alkylamine, particularly R3 is C1-C2 alkylamine. The compound according to any one of the preceding items, wherein the compound is of the general formula (XII)
Figure imgf000169_0001
wherein
- Z1, Z2, X, Y, R2, R3, U, V, and n have the same definitions as in item 1;
- each R14 is independently selected from
• a secondary amine substituted with an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and/or a heterocycle, particularly substituted with an alkyl, an alkylaryl, or a cycloalkyl;
• a halogen; and/or two R14 together form an unsubstituted or substituted heteroaryl or heterocycle;
- n2 is an integer selected from 0, 1, 2, and 3. The compound according to any one of the preceding items, wherein the compound is of the general formula (XIII)
Figure imgf000169_0002
wherein
- Z1, Z2, X, Y, R2, R3, U, V, and n have the same definitions as in item 1;
- R5 is selected from an alkyl, an alkylaryl, a heteroalkylaryl, a cycloalkyl, an aryl, a heteroaryl and a heterocycle, particularly R5 is selected from an alkyl, an alkylaryl, and a cycloalkyl;
- R6 is selected from halogen and hydrogen.
7. The compound according to any one of the preceding items 1 to 4, wherein the compound is of the general formula (XIV)
Figure imgf000170_0001
wherein
- Z1, Z2, X, Y, R2, R3, U, V, and n have the same definitions as in item 1;
- R6 is selected from halogen and hydrogen;
- W is selected from N and CH.
8. The compound according to any one of the preceding items, wherein at least one of Z1 and Z2 is CH or CR2, particularly both Z1 and Z2 are CH or CR2.
R31
Xk> N ' R32
9. The compound according to any one of the preceding items, wherein R3 is J s wherein
- s is an integer selected from 1 and 2, more particularly s is 1 ;
- R31 and R32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, halogen-, and/or hydroxyl-substituted, or
R31 and R32 are independently selected from hydrogen and unsubstituted or hydroxy-, and/or halogen-substituted alkyl or cycloalkyl, particularly R31 and R32 together form a heterocycle or heterobicycle, which is unsubstituted or alkyl-, halogen-, and/or hydroxyl-substituted.
10. The compound according to item 9, wherein NR31R32 is selected from
Figure imgf000170_0002
with v being an integer selected from 0, 1 and 2 and each RN being independently selected from hydroxyl, halogen, and C1-C4 alkyl, or two RN form a C3-C6 cycloalkyl.
11. The compound according to any one of the preceding items 9 to 10, wherein NR31R32 is selected from
Figure imgf000171_0001
12. The compound according to any one of the preceding items, wherein R2 is selected from F, Cl and OH, particularly R2 is F.
13. A compound according to any of the preceding items for use as a medicament.
14. A compound according to any of the preceding items 1 to 12 for use in treatment of cancer.
The disclosure is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the disclosure but not to limit its scope.
Examples
General procedure for Buchwald-Hartwig coupling:
To a stirred solution of the corresponding halide (1 equiv.) in dioxane (0.3 M), under a nitrogen atmosphere, the corresponding amine (1 equiv.) was added. Nitrogen gas was bubbled through the reaction for two minutes and CS2CO3 (1.2 equiv), Ruphos Pd G4 (10 mol %) and Ruphos (10 mol %) were added. The reaction mixture was stirred at 150 °C for 17 h, concentrated under reduced pressure and the obtained residue was purified by flash column chromatography.
General procedure for Boc group deprotection:
To a stirred solution of the corresponding Boc protected amine in MeOH (0.3 M), HC1 (0.9 M, 37 % aq.) was added. The reaction mixture was stirred at 25 °C for 4 h and the reaction mixture was concentrated under reduced pressure. The obtained residue was directly engaged in the next step without further purification.
General procedure for S Ar with 4, 6-dichloropyrimidine :
To a stirred solution of the corresponding amine (1 eq.) or amine hydrochloride salt (1 equiv.) in zPrOH (0.3 M), 4,6-dichloro-pyrimidine (1.2 equiv.) and ETN (1-4 equiv.) were added. The reaction mixture was stirred at 80 °C for 3 h in the microwave and concentrated under reduced pressure. The crude residue was dissolved in wBuOH. washed three times with water, once with brine, dried over MgSCE and concentrated under reduced pressure. The crude residue was coevaporated with toluene several times to remove the residual wBuOH and then purified by flash column chromatography.
General procedure for S Ar with chloropyrimidine derivatives:
The corresponding chloropyrimidine (1 eq.) was dissolved in methylamine (0.1 M, 8 M in EtOH) or benzylamine (0.3 M) and the reaction mixture was stirred at 130 °C for 3 h (McNFE) or 140 °C for 8 h (BnNH2) in the microwave. The crude residue was concentrated under reduced pressure and purified by flash column chromatography. For reactions with benzylamine, the crude residue was coevaporated with water then toluene several times to remove the benzylamine before performing the purification.
Example 1:
The authors’ design started at the roots of one of the author’s early inhibitor (1, Table 4), with the aim to simplify the structure and reduce molecular weight (Figure 1A). For this purpose, changing the methylene position from 1,3 to 1,4 on the piperidine ring removes the chiral center. In addition, according to the X-ray structure of 1 with METTL3, the amide C=O group deletion would allow to keep the original vector (Figure IB). These two modifications led to 2 and its two pyridine containing derivatives 3 and 4, which exhibited not only similar potency compared to the parent molecule (IC50 = 5.0, 4.6, and 5.8 pM respectively, Table 4), but also no chirality and a reduced heavy atom count, hence higher ligand efficiency (LE = 0.23, 0.23, and 0.22, respectively). Because 4 had better lipophilic ligand efficiency (LLE = 3.4, calculated with DataWarrior), its pyridine core was conserved in the next optimization stage. According to the crystal structure of the complex of METTL3 with inhibitor 1, methylamine to benzylamine replacement on the pyrimidine ring seemed beneficial to inhibition. This proved to be true as the corresponding derivative 5 showed a 6-fold increase in potency (IC50 = 0.79 pM).
One striking feature of this inhibitor series is their linear shape coupled with sp3 acyclic atom linkers that makes them highly flexible. Rigidifying the structure is a viable way to freeze a ligand in its preferred conformation, which in turn can enhance the binding energy by reducing entropic penalties. Thus, the authors envisioned two different strategies to achieve this goal: either making an amide connection between the piperidine and the pyridine ring, or according to compound 5’s conformation, a spirocycle could be formed by connecting the tertiary alcohol with the aniline (Figure 2A). The two methods brought opposite results, the amide derivative 6 lost the previous potency boost (IC50 = 3.6 pM, Table 4) while spirocycle 7 was promising both in terms of inhibition (IC50 = 0.28 pM, Table 5) and novelty. The authors managed to soak both 5 and 7 with METTL3, and the X-ray analysis showed a strong structural overlap. The pyrimidine moiety is engaged in two hydrogen bonds with NH backbone from Asn549 and Ile378 while involved in 71-stacking with Phe534 and 7i interactions with Asn549 side chain (Figure 2A). The benzylamine group interacts with Asp377 side chain and also forms a cation -71 interaction with Arg379. On the opposite site of the binding pocket, the gem dimethyl group fdls a lipophilic pocket formed by Lys513, Pro514, Trp457 and Trp431 residues, whereas the charged piperidine forms a salt-bridge with Asp395. The sole difference between 5 and 7 is, for the latter, the missing hydrogen bond between the tertiary alcohol and Gln550 side chain due to the alcohol transformation into an ether (Figure 2B). The authors envisaged that replacing the ether by a lactam could restore this interaction and even make an additional hydrogen bond thanks to the C=O group of the ligand and the NH2 amide of Gln550. The authors obtained a strong potency boost for the corresponding derivative 8 (IC50 = 0.037 pM), and the author’s hypothesis was confirmed by the two hydrogen bond interactions found in the crystal structure (Figure 2C). Furthermore, both LE and LLE improved substantially (0.25 and 4.4 respectively, Table 5). ADME properties, such as solubility, cell permeability, and metabolic stability are essential for chemical probes, so they were considered early on in the project. The author’s newly synthesized inhibitors (5, 7-8) displayed mixed results; however, all of them displayed mediocre stability towards enzymatic degradation with half-lives lower than 12 minutes upon incubations with rat liver microsomes (Tables 4, 5). Therefore, the authors focused on improving ADME properties while getting better biochemical potency. The initial approach was to substitute the pyridine nitrogen atom by a carbon atom, yielding 9 with moderate permeability (9- 10“6 cm s 1) and, surprisingly, slightly increased solubility (Table 5). However, metabolic stability remained unchanged, so the benzylamine was replaced with methylamine (10). Indeed, solubility and metabolic stability were significantly improved (108 pM and 107 min, respectively) as well as LE and LLE values (0.28 and 4.5, respectively), but at the expense of limited permeability (2- 10’6 cm s 1) and a 3.4-fold potency reduction. From 10, two other possibilities of decreasing the size of the author’s molecules were pursued: replacing spiropiperidine with spiroazetidine (11) and spirolactam with spirourea (12). Unfortunately, both displayed a substantial loss in potency (5 and 20 fold, respectively). Yet, the spiroazetidine moiety remains a potential alternative helping to reduce molecular weight and to improve physicochemical properties at a later stage. Next, the author’s strategy was oriented towards permeability improvement. Lactam methylation in compound 13 resulted in a serious decrease in potency (19 fold), demonstrating the crucial role of the lactam hydrogen bond interactions.
After thorough spiro scaffold optimization, the authors turned their attention to the pyrimidine motif. Addition of one more methyl on the aniline (14) was highly detrimental to binding compared to 10 (0.97 and 0.089 pM, respectively, Table 6), probably due to loss of the hydrogen bond to the side chain of Asp377, while methyl to isopropyl substitution (15) showed a less pronounced reduction as the hydrogen bond is preserved (0.33 pM). These two modifications illustrated the limited space available for branched sp3 carbons at this position. Surprisingly, substitution with a cyclopropyl group (16) was not only well tolerated (0.084 pM), but it also improved the three ADME properties (Table 6) and could become a promising alternative for lead optimization. S-Adenosyl methionine (SAM) is the natural ligand of METTL3 that contains an adenosine scaffold overlapping with the pyrimidine group of the author’s inhibitors, thus the authors thought to test a few bicyclic heteroaromatic modifications. The pyrrolopyrimydine 17 had a slight increase in potency in comparison to 10, but similarly low permeability and a larger efflux ratio in the Caco-2 assay (Table 6). The interaction geometry between the N3 pyrimidine atom and Asn549 nitrogen backbone seemed not optimal, thus the authors thought to remove this pyrimidine nitrogen atom in order to improve permeability and possibly to suppress a partial desolvation penalty. The latter proved to be false since pyrrolopyridine 18 exhibited a severe binding loss (74 fold). Incorporation of a chlorine atom between the two pyrimidine nitrogen atoms (19) was beneficial for potency (0.024 pM); however, solubility and metabolic stability were critically impaired (45 pM and 32 min, respectively), which prompted the authors to look for different modifications.
Because the spiro scaffold and the pyrimidine moiety were already optimized, the authors considered the phenyl ring as the next target region. Several publications discuss the unique properties of fluorine atoms that can translate into unexpected and promising results in drug design. Indeed, fluorine atoms are able to make unusual interactions, and aromatic fluorine atoms tend to increase permeability. A fluorine scan was performed on the phenyl ring, affording two novel derivatives 20 and 21. Compared to the inhibitor 10, both compounds improved binding to a similar extent (0.038 and 0.032 pM, respectively); however, permeability was considerably increased only for 20 (Table 7). An X-ray structures in complex with METTL3 were solved for each molecule and revealed that the fluorine in 21 displays hydrophobic contacts (Figure 3B), whereas the fluorine atom of 20 is also engaged in an unusual interaction with the nitrogen n system of Pro397 (Figure 3A). Inhibitor 20 was preferable because of its strong improvement in permeability and small efflux ratio (9- 10’6 era s’1 and 2, respectively), but the combination of both fluorine atoms quickly emerged as the key solution to achieve excellent potency and to keep adequate ADME properties. Indeed, compound 22 exhibited single digit nanomolar IC50 (0.008 pM) in the TR-FRET assay (Table 7 and Figure 4A), high cell permeability (12- 10“6 em s’1), and favorable values of LE and LLE (0.3 and 5.3, respectively), as well as acceptable metabolic stability (ti/2 = 24 min).
To investigate the selectivity of compound 22 towards other RNA methyltransferases, the authors conducted protein thermal shift assay. The authors expressed and purified METTL1 protein that is a writer of 7- methylguanosine mark on tRNA, mRNA, and miRNAs and serves as a representative closely related protein. The authors employed as positive control S-adenosyl-L-homocysteine (SAH), a by-product of RNA methyltransferase catalytic activity and a natural binder, which showed ATm of 2.8 °C and 3.5 °C at 100 pM for METTL3/METTL14 and METTL1, respectively (Figure 5 and 6). Compound 22 at 100 pM was able to shift the melting temperature of METTL3/METTL14 by 4.7 °C compared to DMSO control (Figure 5 and 6). On the contrary, no shift was observed for METTL1 with compound 22 up to 100 pM indicating no binding.
The enhanced thermal stabilization of METTL3 by compound 22 allowed the authors to study its cellular target engagement in two orthogonal assays based on protein thermal denaturation. The binding of 22 was evaluated in InCELL Pulse assay where enhanced ProLabel® (ePL) enzyme fragment fused to the N-terminus of the truncated METTL3 (residues 354-580) was expressed in HEK293T cells. After the incubation of these cells with inhibitor 22 for 1 h at 37°C, cells were heated at 46°C for 3 min, and the non -aggregated METTL3- ePL protein was quantified using luminescence-based assay (Figure 4B). Compound 22 stabilized the METTL3-ePL fusion protein with an EC50 of 2 pM in HEK293T cells. Therefore, both experiments brought clear evidence of cell permeability and cellular target engagement. Finally, to highlight the biological potential of 22 as an inhibitor of METTL3 enzymatic activity, the authors measured m6A/A ratio in polyadenylated RNA in two distinct cancer cell lines, MOLM-13 (AML) and PC-3 (prostate cancer) cells after 16 hours of compound treatment. The authors found that 22 was able to reduce this ratio down to 10-20% of DMSO- treated control samples and with a certain degree of selectivity between the two cell lines (EC50 = 0.7 and 2.5 pM for MOLM-13 and PC-3 respectively, Figure 4D).
The authors successfully improved potency (by a factor of 1 000), efficiency parameters, and ADME properties of a series of METTL3 inhibitors by protein crystallography -guided medicinal chemistry. The key features were rigidification thanks to the design of spiro scaffolds and the use of fluorine atoms at specific positions. The most potent inhibitor (compound 22) shows an IC50 of 8 nM in a TR-FRET assay. No binding to the off-target METTL1 was observed at concentrations of up to 100 pM. Cellular target engagement of compound 22 was demonstrated using two different assays. Furthermore, for the reduction of m6A/A in polyadenylated RNA, as quantified by UPLC-MS/MS analysis, EC50 values of 0.7 pM and 2.5 pM were measured in MOLM-13 (leukemia) and PC-3 (prostate cancer) cell lines. Thus, compound 22 is a chemical probe to decipher the functional role of METTL3/METTL14 and its involvement in hematological malignancies and solid tumors.
Figure imgf000175_0001
Table 4: Early modifications of the original scaffold.
Figure imgf000175_0002
4: Time resolved-Forster resonance energy transfer (TR-FRET) assay (uM), 2: g/mol, 3: Ligand efficiency
(kcal.mol 1.heavy atom count 4). 4: Lipophilic ligand efficiency (pICso-logP); 5: uM; 6: 10~6 cm s4. (efflux ratio). Caco-2 experiment; 7: Rat liver microsomes, ti/2 (min).
Figure imgf000176_0001
Table 5: Derivatization from the spiro scaffold.
Figure imgf000176_0003
Figure imgf000176_0002
Table 6: Optimization of the aminopyrimidine ring.
Figure imgf000176_0004
Figure imgf000177_0003
Figure imgf000177_0001
Table 7: Fluorine scan on the phenyl ring.
Figure imgf000177_0004
Figure imgf000177_0002
33
Scheme 1: Synthesis route to the spirocycle intermediate (33). Reagents and conditions: (a) MeNO2, NH3, MeOH, 25 °C, 17 h; (b) (i) CbzCl, NaHCO3, DCM/H2O, 0-25 °C, 17 h; (ii) NiCl2.6H2O, NaBH4, MeOH, N2, 0-25 °C, 1 h, 32 % over three steps; (c) ethyl 2-bromoacetate, Et3N, DCM, 25 °C, 2 h; (d) Pd/C, NEU HCOO" , zPrOH, 80 °C, 4 h, 55 % over two steps.
Figure imgf000178_0001
Scheme 2: Synthesis route to compounds 7-10 and 15-17, 19. Reagents and conditions: (a) 23 or 26, tertbutyl l-oxa-4,9-diazaspiro[5.5]undecane-9-carboxylate (for 7) or 33, Pd Ruphos G4, Ruphos, CS2CO3, dioxane, N2, 150 °C, 17 h, 93 % (35);; (b) (i) HC1 (37 % aq.), MeOH, 25 °C, 4 h; (ii) For 7: JV-benzyl-6- chloropyrimidin-4-amine (29), Et2N, zPrOH, 150 °C, 8 h, MW, 6 % over three steps from 23. For 17: 4- chloro-7H-pyrrolo[2,3-d]pyrimidine, 36, Pd Ruphos G4, Ruphos, LiHMDS, THF, N2, 65 °C, 4 h, 36 %. For 19: 36, 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine, Et2N, zPrOH, 100-130 °C, 6 h, 42 %; (c) HC1 (37 % aq.), MeOH, 25 °C, 4 h, 36 % over two steps from 23; (d) 4,6-dichloro-pyrimidine, Et2N, zPrOH, 80 °C, 3 h, MW, 27 % over three steps from 26 (34) / 63 % (37); (e) For 8 and 9: BnNH2, 140 °C, 8 h, MW, 25 % (8) / 5 % over two steps from 36 (9). For 10 : MeNH2, EtOH, 130 °C, 3 h, MW, 3 % over two steps from 36. For 15: /PrNH2, EtOH, 130 °C, 8 h, MW, 52 %. For 16: cyclopropylamine, zPrOH, 130 °C, 6 h, MW, 25 %.
Figure imgf000180_0001
Figure imgf000181_0001
Scheme 4: Synthesis route to compound 11. Reagents and conditions: (a) 40, Pd Ruphos G4, Ruphos, CS2CO3,
5 dioxane, N2, 150 °C, 17 h; (b) (i) HC1 (37 % aq.), MeOH, 25 °C, 4 h; (ii) 4,6 -dichloro-pyrimidine, EtsN, zPrOH, 80 °C, 7 h, MW: (c) MeNH2, EtOH, 130 °C, 3 h, MW, 19 % over four steps.
Figure imgf000182_0001
48: R1 = F, R2 = H 51:R1 = H, R2 = F
Scheme 5: General synthetic route for compounds 20-22. Reagents and conditions: (a) For 47 and 49: 1- bromo-4-(bromomethyl)-2 -fluorobenzene (47) or 4-bromo-l-(bromomethyl)-2 -fluorobenzene (49), 4,4- dimethylpiperidine hydrochloride, K2CO3, DMF, 25 °C, 17 h, 98 % (47) / 99 % (49). For 52: (i) 4-bromo-2,5- difluorobenzoic acid, BH3 SMe2, THF, N2, 25 °C, 17 h, 83 % °C; (ii) SOC12, DMF, DCM, 25 °C, 3 h; (iii) 4,4- dimethylpiperidine hydrochloride, K2CO3, DMF, 25 °C, 17 h, 92 %; (b) 33, Pd Ruphos G4, Ruphos, Cs2CO3, dioxane, N2, 150 °C, 17 h, 83 % (50); (c) (i) HC1 (37 % aq.), MeOH, 25 °C, 4 h; (ii) 4,6-dichloropyrimidine, Et3N, iPrOH, 80 °C, 3 h, MW, 15 % over three steps from 47 (48) / 58 % over two steps from 50 (51); (d) MeNH2, EtOH, 130 °C, 3 h, MW, 47 % (20) / 69 % (21) / 56 % over four steps from 52 (22).
Scheme 6: Preparation of 9-(6-((3-aminopropyl)amino)pyrimidin-4-yl)-4-(4-((4,4- dimethylpiperidin-l-yl)methyl)phenyl)-l,4,9-triazaspiro[5.5]undecan-2-one
Figure imgf000183_0001
Chloropyrimidine derivative 6-1 (100 mg, 0.21 mmol) was dissolved in ethanol (0.5 M), to a stirred solution was subsequently added A-Boc- 1,3 -propanediamine 6-2 (108 mg, 0.62 mmol) and TEA (86.6 pL, 0.62 mmol). The resulting reaction mixture was stirred under reflux until completion (monitored by TLC). The reaction mixture was evaporated and the crude product was purified using flash column chromatography (SiCh; DCM/MeOH = 9 : 1) and obtained as a slightly yellow solid (117 mg, 91%). The impure product of SxAr (117 mg, 0.19 mmol) was dissolved in MeOH (0.5 M) and HC1 37 %(57.7 pL, 1.9 mmol) was added. The resulting reaction mixture was stirred at rt until completion (monitored by TLC). The reaction mixture was evaporated, taken up with butanol, quenched with a saturated aqueous solution of Na2CO3 and extracted with butanol (3x 10ml). The combined organic layers were dried over MgSO4, filtrated and evaporated. The product was obtained as a slightly yellow solid (80 mg, 81%).
XH NMR (400 MHz, MeOD) 5 8.06 (d, J = 0.9 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 5.77 (d, J = 1.0 Hz, 1H), 4.01 (s, 2H), 3.86 (s, 2H), 3.83 (s, 2H), 3.63 - 3.56 (m, 2H), 3.53 (s, 2H), 3.43 (t, J = 6.5 Hz, 2H), 3.17 (d, J = 0.8 Hz, 1H), 2.98 (t, J = 7.0 Hz, 6H), 1.95 - 1.87 (m, 4H), 1.81 (ddd, J = 13.3, 8.6, 4.1 Hz, 2H), 1.58 (d, J = 5.9 Hz, 4H), 1.29 (d, J = 2.7 Hz, 3H), 1.02 (s, 6H) 13C NMR (101 MHZ, DMSO) 5 167.6, 163.7, 162.1, 157.8, 148.6, 130.1, 129.2, 114.7, 65.4, 62.4, 53.3, 52.9, 51.9, 49.7, 38.8, 38.0, 37.9, 34.8, 29.6, 28.8, 28.6, 15.7 .
LRMS (ESI) m/z: [M + H]+ calcd for C29H44N8O; 520.36 found, 521.37.
Scheme 12: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(prop-2-yn-l-ylamino)isoindoline- 1, 3-dione
Figure imgf000184_0001
4-Fluoro-pomalidomide derivative 12-8 (320 mg, 1.16 mmol) was dissolved in DMSO (0.5 M), to the solution were subsequently added propargyl amine (64 mg, 1.16 mmol) and DIPEA (605 pl, 3.48 mmol). The resulting reaction mixture was stirred at 130°C until completion (monitored by TLC). The reaction mixture was quenched by addition of a saturated aqueous solution of NaHCOs and extracted into EtOAc (3x 15ml). The combined organic layers were dried over MgSO4, filtrated, and evaporated. Product 12-9 was obtained as a yellow solid (280 mg, 78%).
XH NMR (400 MHz, CDC13) 5 8.00 (s, 1H), 7.57 (dd, J = 8.5, 7.1 Hz, 1H), 7.20 (d, J = 7.1 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 6.44 (t, J = 6.5 Hz, 1H), 4.92 (dd, J = 12.2, 5.4 Hz, 1H), 4.09 (dd, J = 6.1, 2.5 Hz, 2H), 2.95 - 2.67 (m, 3H), 2.27 (t, J = 2.4 Hz, 1H), 2.20 - 2.07 (m, 1H).
13C NMR (101 MHz, CDC13) 5 170.81, 169.24, 168.10, 167.44, 145.56, 136.16, 132.43, 117.17, 112.77, 111.42, 79.11, 72.20, 48.96, 32.33, 31.41, 22.76.
LRMS (ESI) m/z: [M + H]+ calcd for C16H13N3O4; 311.09 found, 312.09.
Figure imgf000185_0001
Scheme 16: (a) Sodium ascorbate (1.1 eq), CuSO4 (0.24 eq), THF, 40 °C, 24 h; (ii) TFA (10 eq), DCM, rt, 8 h; (b) (i) TEA (3 eq), EtOH, reflux, 24 h; (ii) 38% HC1, MeOH, 24 h; (c) HATU (1.1 eq), DIPEA (5 eq), DMF, rt, 5 h.
Figure imgf000186_0001
Scheme 17: (d) (i) TEA (3 eq), EtOH, reflux, 24 h; (ii) 38% HC1, MeOH, 24 h; (e) HATU (1.1 eq), DIPEA (5 eq), DMF, rt, 5 h.
Figure imgf000186_0002
Scheme 18: (f) (i) Propargylamine (3 eq), TEA (3 eq), EtOH, reflux, 5 h; (g) Sodium ascorbate (1.1 eq), CuSO4 (0.24 eq), THF, 40 °C, 24 h.
Figure imgf000187_0001
Scheme 19: (h) (i) TEA (3 eq), EtOH, reflux, 5 h; (ii) TFA (10 eq), DCM, rt, 12 h; (i) HATU (1.1 eq), DIPEA
(5 eq), DMF, rt, 8h.
Figure imgf000187_0002
Scheme 20: (j) (i) TEA (3 eq), EtOH, reflux °C, 24 h; (ii) TFA (10 eq), DCM, rt, 12 h; (k) HATU (1.1 eq), DIPEA (5 eq), DMF, rt, 8h.
Figure imgf000188_0001
Scheme 21: (1) Sodium ascorbate (1.1 eq), CuSCU (0.24 eq), THF, 40 °C, 24 h; (ii) TFA (10 eq), DCM, rt, 8 h (m) HATU (1.1 eq), DIPEA (5 eq), DMF, rt, 8h.
Figure imgf000188_0002
Scheme 22: (n) DIPEA, (3 eq), DMSO, 80 °C, 24 h; (o) TFA (10 eq), DCM, rt, 8 h b) HATU (1.1 eq), DIPEA (5 eq), DMF, rt, 8h. Synthesis schemes from literature incorporated by reference herein:
Figure imgf000189_0001
Also included by reference herein are references Front. Chem. 2021, 9, 707317 (e.g., see schemes 1-20 and figure 3) and Org. Biomol. Chem., 2021, 19, 166-170 (e.g., see click chemistry procedures, and methods of making the triazole in Formula J) for general procedures and methods of making starting materials. tert- Butyl 4-(aminomethyl)-4-(((benzyloxy)carbonyl)amino)piperidine-l-carboxylate (32):
To a stirred solution of MeNCh (1.3 equiv. 130 mmol, 7 mL) in NH3 (53 mL, 7 N in MeOH), tert-butyl 4-oxopiperidine-l -carboxylate (20 g, 100 mmol) was added
Figure imgf000190_0001
portionwise. The reaction mixture was stirred at 25 °C for 17 h and concentrated under reduced pressure. The crude residue was diluted with DCM and water. The two phases were separated and the aqueous layer was extracted two times with DCM. The combined organic layers were dried over MgSCL, filtered and concentrated under reduced pressure to afford the desired product, which was engaged in the next step without further purification.
To a stirred solution of the corresponding amine (100 mmol) in dichloromethane (130 mL), a solution of K2CO3 (2 equiv., 200 mmol, 27.6 g) in water (130 mL) was added. The reaction mixture was cooled to 0 °C and CBzCl (1.1 equiv., 110 mmol, 15.6 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 17 h and the two phases were separated. The aqueous layer was extracted two times with DCM. The combined organic layers were washed once with brine, dried over MgSCL, filtered and concentrated reduced pressure to afford the desired product, which was engaged in the next step without further purification.
To a stirred solution of the corresponding nitroalkane (100 mmol) in dry MeOH (450 mL), under a nitrogen atmosphere, at 0 °C, NiCh.6H2O (1 equiv., 100 mmol, 27.3 g) was added, followed by NaBH4 (5 equiv., 500 mmol, 18.9 g) portionwise to avoid strong H2 evolution. Caution when adding NaBfL. the reaction is highly exothermic and produce hydrogen gas. The reaction mixture was stirred at 25 °C for 1 h and quenched by adding saturated aqueous NaHCCF solution. The mixture was filtered through a pad of Celite, the filtrate was concentrated under reduced pressure and the obtained residue was diluted with water. The aqueous layer was extracted three times with DCM and the combined organic layers were washed once with brine, dried over MgSCL, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM/MeOH/NH4OH = 100:0:0 to 100:3:0 to 100:3: 1 to 100:5: 1 to 100: 10: 1 to 100: 15: 1) to afford the desired product as a white solid (11.7 g, 32 % yield overthree steps). LRMS (ESI) m/z calcd for [Ci9H3oN304]+: 364.2 found: 364.3 tert- Butyl 2-oxo-l,4,9-triazaspiro[5.5]undecane-9-carboxylate (33):
To a stirred solution of tert-butyl 4-(aminomethyl)-4- (((benzyloxy)carbonyl)amino)piperidine-l -carboxylate (14.5 g, 40 mmol) in DCM (133 mL) at 0 °C, EhN (0.8 equiv., 32 mmol, 4.4 mL) and ethyl 2 -bromoacetate (0.7
Figure imgf000190_0002
equiv., 28 mmol, 3.1 mL) were added. The reaction mixture was stirred at 25 °C for 2 h and diluted with saturated aqueous NaHCCL solution. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed once with brine, dried over MgSCE, filtered and concentrated under reduced pressure, to afford the impure desired product, which was engaged in the next step without further purification.
To a stirred solution of the corresponding Cbz protected amine (40 mmol) in zPrOH (400 mb), Pd/C (5 mol %, 2 mmol, 2.1 g, 10 % wt) and ammonium formate (6 equiv., 240 mmol, 15 g) were added portionwise. The reaction mixture was stirred at 80 °C for 4 h, cooled to 25 °C, filtered through a pad of celite and concentrated under reduced pressure. The obtained residue was dissolved in DCM, the organic layer was washed once with water, once with brine, dried over MgSCf. filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM/MeOH = 100:5 to 100:8 to 100: 10 to 100: 15) to afford the desired product as a white solid (4.14 g, 55 % yield over two steps). LRMS (ESI) m/z calcd for C26H47N6O6]+ = [2M+H]+: 539.4 found: 539.4
2V-Benzyl-6-(4-(6-((4,4-dimethylpiperidin-l-yl)methyl)pyridin-3-yl)-l-oxa-4,9-diazaspiro[5.5]undecan-
9-yl)pyrimidin-4-amine (7):
Figure imgf000191_0002
for Boc group deprotection. After evaporation, the crude residue was triturated in acetone and the resulting precipitate was filtered, washed with acetone and dried to afford the impure desired product, which was engaged in the next step without further purification.
To a stirred solution of the corresponding amine (1 equiv.) in zPrOH (0.3 M), 29 (1.5 equiv.) and EbN (4 equiv.) were added. The reaction mixture was stirred at 150 °C for 8 h in the microwave and concentrated under reduced pressure. The reaction was diluted with water and the aqueous layer was extracted three times with DCM. The combined organic layers were washed five times with water, once with brine, dried over MgSCE, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM/MeOH = 100:5 to 100:8 to 100: 10 to 100: 13 to 100:20) to afford the desired product as a brown solid (6 % yield over three steps). Mp: 61-62°C; HRMS (ESI): m/z: calcd for [C32H44N?O]+: 542.3607 found: 542.3602.
2V-Benzyl-6-chloropyrimidin-4- amine (29) :
N ^N To a stirred solution of 4,6-dichloro-pyrimidine (5 g, 33.6 mmol) in zPrOH (100 mb), benzylamine (1.2 equiv., 40.3 mmol, 4.4 mb) and EhN (1.2 equiv., 40.3 mmol, 5.59
Figure imgf000191_0001
mb) were added. The reaction mixture was stirred at 25 °C for 3 d and concentrated under reduced pressure. The crude residue was triturated in water, filtered and dried to afford the desired product as a beige solid (7.21 g, 98 % yield). LRMS (ESI) m/z calcd for [CHHHC1N3]+: 220.1 found: 220.1
9-(6-(Benzylamino)pyrimidin-4-yl)-4-(6-((4,4-dimethylpiperidin-l-yl)methyl)pyridin-3-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (8):
Figure imgf000192_0001
calcd for [C32H43N8O]+: 555.3560 found: 555.3554.
9-(6-(Benzylamino)pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-l,4,9- tnazaspiro[5.5]undecan-2-one (9):
Figure imgf000192_0002
pressure to afford the desired product, which was engaged in the next step without further purification.
Compound 9 was obtained following the general procedure for SNAr with chloropyrimidine derivatives (chromatography: DCM/MeOH = 100:0 to 100:8 in 20 min, 100:8 for 10 min, 100:8 to 100: 10 in 10 min). The obtained impure product was triturated in water, filtered, washed once with water to afford the desired product as a pale yellow solid (5 % yield over two steps). Mp: 228-231°C; HRMS (ESI): m/z: calcd for [C33H44N7O]+: 554.3607 found: 554.3602.
4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-9-(6-(methylamino)pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (10):
Figure imgf000192_0003
100: 12 in 20 min, 100: 12 for 10 min, 100: 12 to 100: 15 in 10 min, 100: 15 for 10 min). White solid (3 % yield over two steps). Mp: 235-236°C; HRMS (ESI): m/z: calcd for [C27H40N7O]+: 478.3294 found: 478.3289.
4-(4-((4,4-Dimethylpiperidin-l-yl)methyl)phenyl)-9-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (17):
Figure imgf000193_0001
, , , ,
1.39 mmol, 1.39 mb, 1 M THF) were added. The reaction mixture was stirred at 65 °C for 4 h, cooled down to 25 °C and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM/MeOH = 100:3 to 100:5 to 100:8 to 100: 10 to 100: 15) to afford the desired product as a yellow solid (37 mg, 36 % yield). Mp: 250-252 °C; HRMS (ESI): m/z: calcd for [C28H38N7O]+: 488.3138. found: 488.3132.
9-(2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-l,4,9- triazaspiro[5.5]undecan-2-one (19):
Figure imgf000193_0002
both in the microwave. The reaction mixture was concentrated under reduced pressure and the crude residue was triturated in water. The resulting precipitate was fdtered, washed with water and dried to afford the desired impure product, which was further purified by flash column chromatography (DCM/MeOH = 100:0 to 100: 10 in 15 min, 100: 10 for 10 min) to afford the desired product as a beige solid (46 mg, 42 % yield). Mp: 199- 201 °C; HRMS (ESI): m/z: calcd for [C28H37C1N7O]+: 522.2748 found: 522.2731.
9-(6-(Cyclopropylamino)pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-l,4,9- triazaspiro[5.5]undecan-2-one (16):
Figure imgf000193_0003
4-(4-((4,4-Dimethylpiperidin-l-yl)methyl)phenyl)-9-(6-(isopropylamino)pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (15):
Figure imgf000194_0003
l-(4-Bromobenzyl)-4,4-dimethylpiperidine (23):
Intermediate 23 was obtained following the general procedure for dimethylpiperidine
Figure imgf000194_0001
alkylation (chromatography: EtOAc/heptane = 0: 10 to 3:7). Yellow oil, 99 % yield.
LRMS (ESI) m/z calcd for [Ci4H2iBrN]+: 282.1 found: 282.1.
5-Chloro-2-((4,4-dimethylpiperidin-l-yl)methyl)pyridine (26):
To a stirred solution of (5-chloropyridin-2-yl)methanol (2.43 g, 17 mmol) in DCM
Figure imgf000194_0002
(40 mL), SOC12 (1.5 equiv., 25.5 mmol, 1.85 mL) and DMF (1 drop) were added. The reaction mixture was stirred at 25 °C for 2 h and concentrated under reduced pressure to afford the chloroalkane, which was engaged in the next step without further purification.
Intermediate 26 was obtained following the general procedure for dimethylpiperidine alkylation but the reaction mixture was stirred at 70 °C for 3 h (column chromatography: EtOAc/heptane = 1:9 to 3:7). Yellow solid, 86 % yield over two steps. LRMS (ESI) m/z calcd for [Ci3H2oClN2]+: 239.1 found: 239.2
9-(6-Chloropyrimidin-4-yl)-4-(6-((4,4-dimethylpiperidin-l-yl)methyl)pyridin-3-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (34): er
Figure imgf000194_0004
purification. The corresponding amine was obtained following the general procedure for Boc group deprotection. After evaporation, the crude residue was triturated in acetone and the resulting precipitate was filtered, washed with acetone and dried to afford the impure desired product, which was engaged in the next step without further purification.
Intermediate 34 was obtained following the general procedure for S\Ar with 4,6-dichloropyrimidine (column chromatography: DCM/MeOH = 100:2 to 100:5 to 100.8 to 100: 10 to 100: 15 to 100:20) to afford the desired product as a yellow solid (123 mg, 27 % yield over three steps). LRMS (ESI) m/z calcd for [C25H35C1N7O]+: 484.3 found: 484.3. tert- Butyl 4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-2-oxo-l,4,9-triazaspiro[5.5]undecane-9- carboxylate (35):
Figure imgf000195_0001
4-(4-((4,4-Dimethylpiperidin-l-yl)methyl)phenyl)-l,4,9-triazaspiro[5.5]undecan-2-one hydrochloride up
Figure imgf000195_0002
intermediate 32). LRMS (ESI) m/z calcd for [C22H35N4O]+: 371.3 found: 371.3.
9-(6-Chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-l,4,9- triazaspiro[5.5]undecan-2-one (37):
Figure imgf000195_0003
the desired product as a brown solid (63 % yield). LRMS (ESI) m/z calcd for [C26H36C1N6O]+: 483.3 found: 483.3.
4-(4-((4,4-Dimethylpiperidin-l-yl)methyl)-2-fluorophenyl)-9-(6-(methylamino)pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (20):
Figure imgf000195_0004
496.3195. 4-(4-((4,4-Dimethylpiperidin-l-yl)methyl)-3-fluorophenyl)-9-(6-(methylamino)pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (21):
Figure imgf000196_0002
or [ 27 39 7 ] : . oun : . .
4-(4-((4,4-Dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)-9-(6-(methylamino)pyrimidin-4-yl)- l,4,9-triazaspiro[5.5]undecan-2-one (22, UZH2):
Figure imgf000196_0003
for Boc group deprotection. The impure desired product was engaged in the next step without further purification.
The corresponding chloropyrimidine was obtained following the general procedure for SNAr with 4,6- dichloropyrimidine. The impure desired product was engaged in the next step without further purification.
UZH2 was obtained following the general procedure for SNAr with chloropyrimidine derivatives (chromatography: DCM/MeOH = 100:0 to 100: 10 in 15 min, to 100: 10 for 10 min, 100: 10 to 100: 12 in 10 min. White solid, 56 % yield over four steps. Mp: 214-216; HRMS (ESI): m/z: calcd for [C27H3sF2N7O]+: 514.3106 found: 514.3100. l-(4-Bromo-3-fluorobenzyl)-4,4-dimethylpiperidine (47):
Intermediate 47 was obtained following the general procedure for dimethylpiperidine alkylation (column chromatography: EtOAc/heptane = 1:9). Colorless oil, 98 % yield.
Figure imgf000196_0001
LRMS (ESI) m/z calcd for [Ci4H2oBrFN]+: 300.1 found: 300.1.
9-(6-Chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2-fluorophenyl)-l,4,9- triazaspiro[5.5]undecan-2-one (48):
Figure imgf000196_0004
The corresponding amine was obtained following the general procedure for Boc group deprotection. The impure desired product was engaged in the next step without further purification.
Intermediate 48 was obtained following the general procedure for SNAr with 4,6-dichloropyrimidine (column chromatography: DCM/MeOH = 100:3 to 100:5 to 100.8 to 100: 10) to afford the desired product as a white solid (15 % yield over three steps). LRMS (ESI) m/z calcd for ICAMv'ClFNeOl : 501.3 found: 501.3. l-(4-Bromo-2-fluorobenzyl)-4,4-dimethylpiperidine (49):
Intermediate 49 was obtained following the general procedure for dimethylpiperidine
Figure imgf000197_0001
alkylation. Colorless liquid, 99 % yield. LRMS (ESI) m/z calcd for [Ci4H2oBrFN]+:
300.1 found: 300.1. tert- Butyl 4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-3-fluorophenyl)-2-oxo-l,4,9- triazaspiro[5.5]undecane-9-carboxylate (50):
Figure imgf000197_0003
z 27 42 4 3 : . u . .
9-(6-Chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-3-fluorophenyl)-l,4,9- triazaspiro[5.5]undecan-2-one (51):
Figure imgf000197_0004
with 4,6-dichloropyrimidine. Instead of chromatography, after evaporation of the crude mixture, the residue was triturated in water. The obtained precipitate was filtered, washed with water and dried to afford the desired product as a brown solid (58 % yield over two steps). LRMS (ESI) m/z calcd for ICSefL A’IFNeOl : 501.3 found: 501.3. l-(4-Bromo-2,5-difluorobenzyl)-4,4-dimethylpiperidine (52):
To a stirred solution of 4-bromo-2,5-difluorobenzoic acid (1 g, 4.2 mmol) in dry THF (10 mb), under a nitrogen atmosphere, BH3.SMe2 (2 equiv., 8.4 mmol, 4.2 mb, 2 M
Figure imgf000197_0002
THF) was added. The reaction mixture was stirred for 17 h at 25 °C, cooled down to 0 °C and quenched by the addition of a saturated aqueous Na2CO3 solution. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed once with brine, dried over MgSCL and concentrated under reduced pressure to afford the desired product as a brown solid (789 mg, 83 % yield).
To a stirred solution of the corresponding alcohol (789 mg, 3.54 mmol) in DCM (10 mL), SOCh (1.5 equiv., 5.3 mmol, 385 pL) and DMF (1 drop) were added. The reaction mixture was stirred at 25 °C for 3 h and concentrated under reduced pressure to afford the desired chloroalkane, which was engaged in the next step without further purification.
To a stirred solution of the corresponding chloroalkane (425 mg, 1.76 mmol) in dimethylformamide (5 mL), 4,4-dimethylpiperidine hydrochloride (1 equiv., 1.76 mmol, 263 mg) and K2CO3 (2 equiv. 3.52 mmol, 486 mg) were added. The reaction mixture was stirred at 25 °C for 3 days and concentrated under reduced pressure. The obtained residue was purified by flash column chromatography (EtOAc/heptane = 3: 100 to 10: 100) to afford the desired product as a colorless liquid (514 mg, 92 %). LRMS (ESI) m/z calcd for [Ci4Hi9BrF2N]+: 318.1 found: 318.1
8-(4-((4,4-Dimethylpiperidin-l-yl)methyl)phenyl)-2-(6-(methylamino)pyrimidin-4-yl)-2,5,8- triazaspiro[3.5]nonan-6-one (11):
Figure imgf000198_0002
The corresponding amine was obtained following the general procedure for Boc group deprotection. The impure desired product was engaged in the next step without further purification.
The corresponding chloropyrimidine was obtained following the general procedure for SNAr with 4,6- dichloropyrimidine. Due to 40 derivative impurities still present, 7 equivalents of the pyrimidine and 7 equivalents of EhN were used and the reaction was heated for 7 h at 80 °C in the microwave. The impure desired product was engaged in the next step without further purification.
Compound 11 was obtained following the general procedure for SNAr with chloropyrimidine derivatives (chromatography: DCM/MeOH = 100:0 to 100: 10 in 15 min, 100: 10 for 5 min, 100: 10 to 100: 13 in 5 min, 100: 13 for 5 min). Yellow solid, 19 % yield over four steps. Mp: Decomposition; HRMS (ESI): m/z: calcd for [C25H36N7O]+: 450.2981 found: 450.2976. tert- Butyl 3-amino-3-(nitromethyl)azetidine-l-carboxylate (38):
H2N To a stirred solution of tert-butyl 3 -oxoazetidine -1 -carboxylate (10.65 g, 62 mmol) in
EtOH (31 mL), MeNCE (13 mL) and K2CO3 (1 mol %, 0.62 mmol, 86 mg) were added.
Figure imgf000198_0001
The reaction mixture was stirred at 25 °C for 17 h and filtered. The filtrate was concentrated under reduced pressure to afford the desired product, which was engaged in the next step without further purification.
To a stirred solution of the corresponding alcohol (62 mmol) in dry DCM (250 mL), under a nitrogen atmosphere and cooled to -78 °C, DAST (1.2 eq., 74.4 mmol, 9.8 mL) was added dropwise. The cooling bath was removed and the reaction mixture was stirred for 3 h, cooled to 0 °C and quenched slowly by the addition of a saturated aqueous NaHCCL solution. The aqueous layer was extracted three times with DCM, washed once with brine, dried over MgSCL, filtered and concentrated under reduced pressure to obtain the desired product, which was engaged in the next step without further purification.
The corresponding nitromethylene (62 mmol) was dissolved in ammonia (17.7 mL, 7 N in MeOH) and the reaction mixture was stirred for 2 h at 25 °C. The reaction mixture was concentrated under reduced pressure to afford the desired product as an orange solid (15.67 g, quantitative yield over three steps). tert- Butyl 3-(aminomethyl)-3-(((benzyloxy)carbonyl)amino)azetidine-l-carboxylate (39):
T° a stirred solution of 38 (62 mmol) in dichloromethane (100 mL), a solution of
NaHCCL (2 equiv., 124 mmol, 10.42 g) in water (100 mL) was added. The reaction mixture was cooled to 0 °C and CbzCl (1 equiv., 62 mmol, 8.8 mL) was
Figure imgf000199_0001
added dropwise. The reaction mixture was stirred at 25 °C for 17 h and the two phases were separated. The aqueous layer was extracted two times with DCM. The combined organic layers were washed once with brine, dried over MgSCL, filtered and concentrated reduced pressure to afford the desired product, which was engaged in the next step without further purification.
To a stirred solution of the corresponding nitroalkane (62 mmol) in dry MeOH (300 mL), under a nitrogen atmosphere at 0 °C, NiCh.6H2O (1 equiv., 62 mmol, 16.9 g) was added, followed by NaBH4 (5 equiv., 310 mmol, 11.7 g) portionwise to avoid strong H2 evolution. Caution when adding NaBfL. the reaction is highly exothermic and produce hydrogen gas. The reaction mixture was stirred at 25 °C for 1 h and quenched by adding saturated aqueous NaHCCL solution. The mixture was filtered through a pad of Celite, the filtrate was concentrated under reduced pressure and the obtained residue was diluted with brine and a saturated aqueous NazCCL solution. The aqueous layer was extracted three times with DCM and the combined organic layers were washed once with brine, dried over MgSCL, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM/McOH/NfLOH = 100:3:0 to 100:3: 1 to 100:5: 1 to 100:8: l to 100: 12: l to 100:20: 1) to afford the desired product as a white solid (11.3 g, 54 % yield overtwo steps). LRMS (ESI) m/z calcd for [CsiHsiNsOsT = [2M+H]+: 671.4 found: 671.4. tert- Butyl 6-oxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (40): To a stirred solution of 39 (11.3 g, 33.7 mmol) in DCM (110 mL) at 0 °C, Et3N (1 equiv., 33.7 mmol, 4.7 mL) and ethyl 2 -bromoacetate ( 1 equiv., 33.7 mmol, 3.7 mL) were added. The reaction mixture was stirred at 25 °C for 17 h and diluted with a saturated aqueous
Figure imgf000200_0001
NaHCO3 solution. The aqueous layer was extracted three times with DCM and the combined organic layers were washed once with water, once with brine, dried over MgSCL, fdtered and concentrated under reduced pressure, to afford the impure desired product (12.4 g, 29 mmol), which was engaged in the next step without further purification.
To a stirred solution of the corresponding Cbz protected amine (29 mmol) in zPrOH (240 mL), Pd/C (5 mol %, 1.5 mmol, 1.6 g, 10 % wt) and ammonium formate (6 equiv., 174 mmol, 11 g) were added portionwise. The reaction mixture was stirred at 80 °C for 4 h, cooled to 25 °C, filtered through a pad of Celite and concentrated under reduced pressure. The obtained residue was partitioned between DCM and water, the two phases were separated and the aqueous layer was extracted three times with DCM. The combined organic layers were washed once with water, once with brine, dried over MgSCE. filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM/MeOH = 100:5 to 100:8 to 100: 10 to 100: 15 to 100:20) to afford the desired product as a white solid (2.6 g, 32 % yield over two steps). LRMS (ESI) m/z calcd for [C7Hi2N3O3]+ = [M-tBu+2H]+: 186.1 found: 186.2
4-(4-((4-Fluoro-4-methylpiperidin-l-yl)methyl)phenyl)-9-(6-(methylamino)pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (53):
Figure imgf000200_0002
. . .
9-(6-Chloropyrimidin-4-yl)-4-(4-((4-fluoro-4-methylpiperidin-l-yl)methyl)phenyl)-l,4,9- triazaspiro[5.5]undecan-2-one (54)
Figure imgf000200_0003
w ou ur er pur ica on.
The corresponding amine was obtained following the general procedure for Boc group deprotection. The impure desired product was engaged in the next step without further purification. Intermediate 54 was obtained following the general procedure for SNAr with 4,6-dichloropyrimidine (column chromatography: DCM/MeOH = 100:3 to 100:5 to 100.8) to afford the desired product as a white solid (12 % yield over three steps). l-(4-Bromobenzyl)-4-fluoro-4-methylpiperidine (55): To a stirred solution of tert-butyl 4-hydroxy-4-methylpiperidine-l -carboxylate (500
Figure imgf000201_0001
mg, 2.32 mmol) in dry DCM (7 mb), at 0°C under a nitrogen atmosphere, DAST (1.5 eq., 3.48 mmol, 460 pL) was added. The mixture was stirred at 25 °C for 3 h and quenched by adding saturated aqueous NaHCCE solution. The two phases were separated and the aqueous layer was extracted two times with DCM. The combined organic layers were washed with brine, dried over MgSCE, filtered and concentrated under reduced pressure to afford the desired product, which was engaged in the next step without further purification.
The corresponding amine was obtained following the general procedure for Boc group deprotection. The impure desired product was engaged in the next step without further purification.
Intermediate 55 was obtained following the general procedure for dimethylpiperidine alkylation, (column chromatography: EtOAc/heptane = 1:9 to 3:7 to 1: 1). Yellow oil, 77 % yield over three steps.
4-(4-((4,4-dimethylpiperidin-l-yl)methyl)phenyl)-9-(lH-pyrazolo[3,4-d]pyrimidin-4-yl)-l,4,9- triazaspiro[5.5]undecan-2-one (56):
Figure imgf000201_0002
w v . x u w u u pressure and the crude residue was triturated in water. The resulting precipitate was fdtered, washed with water, washed with DCM and dried to afford the desired impure product, which was further purified by flash column chromatography (DCM/MeOH = 100:0 to 100: 10 in 20 min, 100: 10 for 10 min) to afford the desired product as a yellow solid (33 mg, 22 % yield). Mp: 252-254 °C; HRMS (ESI): m/z: calcd for |CAH^NxO| : 489.3090 found: 489.3085
Scheme 23: General Procedure A
Figure imgf000202_0001
To a cooled solution (water-ice bath) of carboxylic acid (1 eq) in DMF (0.5 M) was added DIPEA (4 eq), and the reaction mixture was stirred at the same temperature for 10 minutes. After addition of HATU or COMU (1.1 eq), the solution was stirred for an additional 30 minutes after which the amine (1 eq) was added. The resulting reaction mixture was stirred at rt until completion (monitored by TLC), concentrated in vacuo and purified using flash column chromatography.
Scheme 24: Preparation of 4-((4-(4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5- difluorophenyl)- 2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)-piperidin-l- yl)-4-oxobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Compound 24-1).
Figure imgf000202_0002
Compound 24-1 was prepared following the General Procedure A (COMU) using the corresponding amine (53 mg, 0.08 mmol) and carboxylic acid (30 mg, 0.08 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc/MeOH = from 90 : lO to 80 : 20) providing 32 mg of the desired product (41% yield). ’H NMR (400 MHz. McOD) 5 7.99
(bs, 1H), 7.55 (dd, J = 8.6, 7.1 Hz, 1H), 7.18 (dd, J= 12.9, 6.6 Hz, 1H), 7.10 (d, J= 8.5 Hz, 1H), 7.03 (d, J= 7.1 Hz, 1H), 6.88 (dd, J= 11.2, 7.3 Hz, 1H), 5.66 (s, 1H), 5.05 (dd, J= 12.4, 5.4 Hz, 1H), 4.53 (d, J= 13.1 Hz, 1H), 3.97 - 3.90 (m, 4H), 3.72 (s, 2H), 3.60 (s, 2H), 3.43 - 3.35 (m, 6H), 3.14 - 2.99 (m, 4H), 2.90 - 2.60 (m, 4H), 2.63 - 2.45 (m, 7H), 2.44 - 2.38 (m, 2H), 2.11 - 2.08 (m, 1H), 1.97 - 1.93 (m, 4H), 1.84 - 1.70 (m, 4H), 1.43 (t, J= 5.6 Hz, 4H), 1.12 - 1.05 (m, 2H), 0.93 (s, 6H). 13C NMR (101 MHz, MeOD) 5 174.7, 173.1, 171.7, 170.7, 170.3, 169.3, 164.8, 163.4, 158.3, 148.2, 137.3, 134.0, 120.1, 119.9, 118.1,
111.9, 111.2, 107.4, 107.1, 56.2, 55.3, 54.6, 53.7, 47.3, 46.9, 43.0, 42.8, 41.8, 39.0, 37.4, 35.9, 32.2, 31.6, 31.2, 31.0, 30.8, 29.1, 25.9, 23.8. LCMS (ESI) m/z: [M + H]+ calcd for C49H62F2N1106; 938.484 found, 938.492.
Scheme 25: Preparation of 4-((6-(4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5- difluorophenyl)-2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidin-l- yl)-6-oxohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Compound 25-1)
Figure imgf000203_0001
Compound 25-1 was prepared following the General Procedure A (COMU) using the corresponding amine (26 mg, 0.042 mmol) and carboxylic acid (17 mg, 0.042 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc/MeOH = 2:0.6) providing 32 mg of the desired product (79 %). ’H NMR (400 MHz, MeOD) 5 7.89 (bs, 1H), 7.43 (dd, J = 8.5, 7.1 Hz, lH), 7.12 (dd, J= 12.9, 6.7 Hz, 1H), 6.94 (d, J= 8.5 Hz, 1H), 6.91 (d, J= 7.1 Hz, 1H) 6.82 (dd, J= 11.3, 7.2 Hz, 1H), 5.58 (s, 1H), 4.94 (dd, J= 13.1, 6.0 Hz, 1H), 4.42 (d, J= 13.3 Hz, 1H), 3.88 - 3.76 (m, 4H), 3.82 (s, 2H), 3.72 (s, 2H), 3.64 (s, 2H), 3.32 - 3.28 (m, 4H), 3.24 (d, J= 5.6 Hz, 2H), 3.11 - 3.03 (m, 2H), 2.95 (t, J= 12.4 Hz, 1H), 2.71 - 2.60 (m, 6H), 2.53 - 2.47 (m, 1H), 2.34 - 2.28 (m, 2H), 2.01 - 1.96 (m, 1H), 1.88 - 1.85 (m, 2H), 1.74 - 1.68 (m, 5H), 1.61 - 1.54 (m, 4H), 1.42 - 1.38 (m, 6H), 1.09 - 0.99 (m, 2H), 0.87 (s, 6H). 13C NMR (101 MHz, MeOD) 5 165.2, 164.3, 162.2, 161.3, 160.7, 159.8, 155.3,
153.9, 151.0, 148.8, 148.6, 141.5, 138.8, 127.8, 124.4, 110.7, 108.5, 102.3, 101.5, 97.9, 97.6, 46.5, 45.4, 45.1, 44.1, 40.9, 40.7, 37.8, 37.6, 33.7, 33.5, 32.2, 28.8, 28.1, 26.3, 24.5, 22.7, 22.2, 21.3, 20.5, 19.5, 18.1,
16.9, 14.3. LCMS (ESI) m/z: [M + H]+ calcd for C51H66F2N1106; 966.451 found, 966.545. Scheme 26: Preparation of 4-((8-(4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluoro phenyl)-2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidin-l-yl)-8- oxooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Compound 26-1)
Figure imgf000204_0001
Compound 26-1 was prepared following the General Procedure A (COMU) using the corresponding amine (50 mg, 0.08 mmol) and carboxylic acid (33 mg, 0.08 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiCh; EtOAc/MeOH = from 85 : 15 to 80 : 20) providing 25 mg of the desired product (32%). JH NMR (400 MHz, MeOD) 5 7.99 (s, 1H), 7.53 (dd, J = 8.6, 7.1 Hz, 1H), 7.18 (dd, J = 13.0, 6.6 Hz, 1H), 7.02 (dd, J = 7.8, 5.4 Hz, 2H), 6.88 (dd, J = 11.2, 7.2 Hz, 1H), 5.68 (s, 1H), 5.04 (dd, J = 12.5, 5.5 Hz, 1H), 4.53 (d, J = 13.2 Hz, 1H), 3.94 (m, 3H), 3.72 (s, 2H), 3.63 (m, 2H), 3.39 (m, 4H), 3.17 (d, J = 6.4 Hz, 2H), 3.05 (t, J = 12.8 Hz, 1H), 2.85 (ddd, J = 17.6, 14.2, 5.0 Hz, 1H), 2.76 (m, 1H), 2.74 - 2.68 (m, 1H), 2.58 (m, 5H), 2.38 (td, J = 7.4, 4.9 Hz, 2H), 2.10 (dtd, J = 12.9, 4.9, 2.3 Hz, 1H), 1.98 (m, 2H), 1.81 (m, 6H), 1.67 (t, J = 6.8 Hz, 2H), 1.60 (t, J = 7.1 Hz, 2H), 1.44 (t, J = 5.7 Hz, 6H), 1.38 (m, 5H), 1.32 - 1.28 (m, 2H), 0.94 (s, 6H).13C NMR (101 MHz, MeOD) 5 173.2, 172.6, 170.3, 169.4, 168.9, 167.9, 163.4, 162.0, 156.9, 146.9, 135.9, 132.5, 116.6, 110.3, 109.6, 106.0, 105.7, 54.8, 53.8, 53.2, 52.3, 49.1, 48.8, 45.9, 45.7, 41.9, 41.6, 40.3, 37.5, 36.1, 34.5, 32.7, 30.8, 30.3, 29.4, 28.8, 28.8, 28.7, 27.7, 26.4, 25.1, 22.4. LRMS (ESI) m/z: [M + H]+ calcd for C53H70F2N1106; 994.550 found, 994.549.
Scheme 27: Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)- 2- oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-4-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)butanamide (Compound 27-1)
Figure imgf000204_0002
Compound 27-1 was prepared following the General Procedure A (COMU) using the corresponding amine (55 mg, 0.08 mmol) and carboxylic acid (30 mg, 0.08 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiCh; EtOAc/MeOH = 90 : 10) providing 47 mg of the desired product (59% yield). XH NMR (400 MHz, CDCE) 5 10.85 (s, 1H), 8.16 (s, 1H), 7.43 (dd, J= 8.5, 7.1 Hz, 1H), 7.25 - 7.17 (m, 3H), 7.15 - 7.07 (m, 2H), 7.04 (d, J= 7.1 Hz, 1H), 6.92 - 6.84 (m, 2H), 6.57 (dd, J= 10.9, 7.1 Hz, 1H), 6.26 (t, J= 5.9 Hz, 1H), 6.09 - 6.04 (m, 1H), 5.77 (s, 1H), 5.34 (s, 1H), 4.89 (dd, J= 12.2, 5.3 Hz, 1H), 4.38 (dd, J= 13.1, 5.7 Hz, 4H), 3.71 (s, 2H), 3.66 - 3.50 (m, 4H), 3.33 (q, J= 6.5 Hz, 2H), 3.25 (s, 2H), 2.90 - 2.69 (m, 3H), 2.46 (s, 4H), 2.31 (t, J= 6.9 Hz, 2H), 2.15 - 2.06 (m, 1H), 2.05 - 1.96 (m, 2H), 1.94 - 1.83 (m, 2H), 1.81 - 1.70 (m, 4H), 1.45 - 1.38 (m, 4H), 0.92 (s, 6H). 13C NMR (101 MHz, CDC13) 5 172.0, 171.9, 170.4, 169.8, 168.2, 167.8, 162.5, 157.6, 147.0, 139.1, 138.8, 136.4, 132.6, 129.1, 127.0, 126.7, 126.3, 116.9, 111.8, 110.3, 56.7, 54.3, 53.5, 53.0, 49.8, 49.1, 45.7, 43.6, 41.8, 40.3, 35.2, 33.3, 31.7, 29.9, 28.5, 24.8, 23.0, 1.2. LCMS (ESI) m/z: [M + H]+ calcd for C51H60F2N11O6; 960.469 found, 960.469.
Scheme 28: Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)-2- oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-6-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)hexanamide (Compound 28-1)
Figure imgf000205_0001
Compound 28-1 was prepared following the general amide coupling procedure 4 using the corresponding amine (124 mg, 0.19 mmol) and pomalidomide carboxylic acid (95 mg, 0.19 mmol). The reaction mixture was evaporated and the crude product was purified using flash column chromatography (SiO2;
EtOAc/MeOH = from 85 : 15 to 80 : 20) affording 113 mg of the desired product (60%). ’H NMR (400 MHz, MeOD) 5 7.98 (s, 1H), 7.51 (t, 1H), 7.30 - 7.23 (m, 2H), 7.23 - 7.18 (m, 1H), 7.19 - 7.12 (m, 2H), 7.00 (dd, J = 7.9, 3.0 Hz, 2H), 6.86 (dd, J = 11.2, 7.3 Hz, 1H), 5.62 (s, 1H), 5.03 (dd, J = 12.4, 5.5 Hz, 1H), 4.45 (s, 2H), 4.34 (s, 2H), 3.89 - 3.81 (m, 2H), 3.70 (s, 2H), 3.60 (s, 2H), 2.90 - 2.62 (m, 3H), 2.54 (s, 3H), 2.24 (t, J = 7.3 Hz, 2H), 2.13 - 2.03 (m, 1H), 1.97 - 1.87 (m, 2H), 1.82 - 1.71 (m, 2H), 1.71 - 1.60 (m, 4H), 1.43 (t, J = 4.9, 3.7 Hz, 6H), 1.36 - 1.27 (m, 1H), 0.93 (s, 6H). 13C NMR (101 MHz, MeOD) 5 174.49, 173.29, 170.28, 169.41, 168.89, 167.92, 163.13, 162.00, 156.95, 146.84, 139.15, 135.87, 132.47, 128.35, 125.93, 125.70, 116.62, 110.40, 53.17, 52.28, 49.07, 48.78, 42.52, 41.89, 40.35, 37.57, 35.47, 34.35, 30.80, 28.56, 27.67, 26.08, 25.25, 22.39. LRMS (ESI) m/z: [M + H]+ calcd for C58H79F2N12O6; 988.50 found,
988.56.
Figure imgf000206_0001
To a solution of Intermediate 1 (1 eq) in a corresponding solvent (0.5 M) was subsequently added corresponding amine (1.5 eq) and TEA (4 eq). The resulting reaction mixture was stirred at 120°C (the temperature of oil bath until completion, monitored by TLC). Scheme 30: General Procedure C
Figure imgf000206_0002
To a stirred solution of corresponding Boc protected amine (1 eq) in MeOH (0.5 M) was added 4M HCI in dioxane (10 eq). The resulting reaction mixture was stirred at rt until full completion (monitored by TLC). The volatiles were then removed in vacuo and the product was used in the next step without further purification.
Scheme 31: General Procedure D
Figure imgf000206_0003
To a stirred solution of 4-fluoro-thalidomide (1 eq) in DMSO (0.5 M) was subsequently added corresponding tert-butyl ester (1 eq) and DIPEA (3 eq). The resulting reaction mixture was stirred at 130°C until full completion (monitored by TLC).
Scheme 32: General Procedure E
Figure imgf000207_0001
To a stirred solution of corresponding tert-butyl ester (1 eq) in DCM (0.5 M) was added TFA (10 eq). The resulting reaction mixture was stirred at rt until full completion (monitored by TLC). The volatiles were then removed in vacuo.
Scheme 33: General Procedure F
Figure imgf000207_0002
To a cooled solution (water-ice bath) of corresponding carboxylic acid, synthesized following General Procedure E, (1 eq) in DMF (0.5 M) was added DIPEA (4 eq), and the reaction mixture was stirred at the same temperature for 10 minutes. After addition of HATU or COMU (1.1 eq), the solution was stirred for an additional 30 minutes after which the amine (1 eq) was added. The resulting reaction mixture was stirred at rt until completion (monitored by TLC), concentrated in vacuo and purified using flash column chromatography .
Scheme 34: Preparation of tert-butyl 4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)- 2,5 difluoro phenyl) -2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidine-l- carboxylate (Intermediate 2)
Figure imgf000208_0001
Intermediate 2 was prepared following the General Procedure B using Intermediate 1 (50 mg, 0.1 mmol) and tert-butyl 4 -(aminomethyl)piperidine-l -carboxylate (42 mg, 0.15 mmol) in zPrOH. After the reaction completion, the volatiles were removed in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM/MeOH = 90 : 10) providing 55 mg of the desired product (79 %). 1H NMR (400 MHz, CDC13) 5 8.14 (s, 1H), 7.11 (dd, J = 12.9, 6.6 Hz, 1H), 6.67 (m , 1H), 6.58 (dd, J = 10.9, 7.1 Hz, 1H), 5.43 (s, 1H), 4.87 (s, 1H), 4.13 (s, 2H), 3.78 - 3.68 (m, 4H), 3.60 (m, 2H), 3.48 (s, 2H), 3.28 (s, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.70 (t, J = 12.7 Hz, 2H), 2.44 - 2.37 (m, 4H), 1.97 (m, 2H), 1.82 (m, 2H), 1.77 - 1.69 (m, 3H), 1.45 (s, 9H), 1.39 (t, J = 5.6 Hz, 4H), 1.28 - 1.11 (m, 2H), 0.91 (s, 6H).13C NMR (101 MHz, CDC13) 5 167.8, 163.4, 162.4, 157.8, 156.0, 154.8, 152.2, 137.3, 118.4, 118.2, 105.9, 105.6, 80.7, 79.5, 56.5, 54.7, 53.4, 53.0, 49.8, 47.2, 40.2, 38.6, 36.4, 35.4, 30.0, 28.5, 28.4. LRMS (ESI) m/z: [M + H]+ calcd for C37H55F2N8O3; 697.440 found, 697.438.
Scheme 35: Preparation of tert-butyl (3-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)- 2,5- difluorophenyl)-2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)4-(2- aminoethyl)piperazin)carbamate (Intermediate 3)
Figure imgf000208_0002
Intermediate 3 was prepared following the General Procedure B using Intermediate 1 (50 mg, 0.1 mmol) and tert-butyl 4-(2-aminoethyl)piperazine-l -carboxylate (34 mg, 0.15 mmol) in DMSO. After the reaction completion, the volatiles were removed in vacuo and the crude product was purified using flash column chromatography (SiCh; DCM/MeOH = 90: 10) providing 91 mg of the desired product (86 %). ’H NMR (400 MHz, CDCh) 5 8.16 (s, 1H), 7.26 (s, 1H), 7.14 (dd, J = 12.7, 6.5 Hz, 1H), 6.78 (s, 1H), 6.58 (dd, J = 10.8, 7.2 Hz, 1H), 5.45 (s, 1H), 5.36 (s, 1H), 3.77-3.68 (m, 4H), 3.67-3.58 (m, 2H), 3.53 (s, 2H), 3.44 (t, J = 4.6 Hz, 4H), 3.35-3.24 (m, 4H), 2.51-2.37 (m, 8H), 1.96 (m, 2H), 1.84 (m, 2H), 1.77 (m, 2H), 1.46 (s, 9H), 1.41 (t, J = 5.3 Hz, 4H), 0.92 (s, 6H). 13C NMR (125 MHz, CDCh) 5 172.6, 166.8, 166.1, 162.5, 161.0, 160.5, 159.0, 155.7, 153.8, 142.2, 142.1, 122.9, 122.7, 84.1, 60.3, 59.3, 58.1, 57.2, 56.5, 56.4, 53.2, 44.4, 43.7, 41.8, 41.7, 38.7, 31.9. LRMS (ESI) m/z: [M + H]+ calcd for C37H56F2N9O3; 712.447, found: 712.902.
Scheme 36: Preparation of tert-butyl 8-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino) octanoate (Intermediate 4)
Figure imgf000209_0001
Intermediate 4 was prepared following the General Procedure D using 4-fluoro-thalidomide (258 mg, 0.93 mmol) and the corresponding tert-butyl ester (201 mg, 0.93 mmol). The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiCE; EtOAc/Hept = 2:3) providing 251 mg of the desired product (57% yield). ’H NMR (400 MHz, CDCI3) 5 8.07 (s, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.22 (t, J = 5.7 Hz, 1H), 4.91 (dd, J = 12.1, 5.3 Hz, 1H), 3.25 (td, J = 7.1, 5.6 Hz, 2H), 2.96 - 2.67 (m, 3H), 2.20 (t, J = 7.5 Hz, 2H), 2.17 - 2.09 (m, 1H), 1.70 - 1.53 (m, 4H), 1.43 (s, 9H), 1.37 - 1.30 (m, 5H).
Scheme 37: Preparation of tert-butyl 6-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino) hexanoate (Intermediate 5)
Figure imgf000209_0002
Intermediate 5 was prepared following the General Procedure D using 4-fluoro-thalidomide (516 mg, 1.9 mmol) and the corresponding tert-butyl ester (350 mg, 1.9 mmol). The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiCE; EtOAc/Hept = 2:3) providing 623 mg ofthe desired product (75% yield). 'HNMR (400 MHz, CDCh) 5 7.89 (s, 1H), 7.42 (dd, J= 8.5, 7.1 Hz, 1H), 7.02 (d, J= 7.0 Hz, 1H), 6.81 (d, J= 8.5 Hz, 1H), 6.16 (t, J= 5.7 Hz, 1H), 4.84 (dd, J= 12.1, 5.4 Hz, 1H), 3.20 (td, J= 7.1, 5.6 Hz, 2H), 2.89 - 2.59 (m, 3H), 2.17 (t, J= 7.4 Hz, 2H), 2.10 - 2.04 (m, 1H), 1.68 - 1.53 (m, 4H), 1.37 (s, 9H).
Scheme 38: Preparation of tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin- 4-yl)amino) butanoate (Intermediate 6)
Figure imgf000210_0001
Intermediate 6 was prepared following the General Procedure D using 4-fluoro-thalidomide (55 mg, 0.2 mmol) and the corresponding tert-butyl ester (34 mg, 0.2 mmol). The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc/Hept = 2:3) providing 80 mg of the desired product (96% yield). ‘HNMR (400 MHz, CDCh) 5 8.02 (s, 1H), 7.50 (dd, J= 8.6, 7.1 Hz, 1H), 7.10 (d, J= 7.0 Hz, 1H), 6.93 (d, J= 8.5 Hz, 1H), 6.29 (t, J= 5.8 Hz, 1H), 4.96 - 4.84 (m, 1H), 3.33 (q, J= 6.6 Hz, 2H), 2.93 - 2.69 (m, 3H), 2.35 (t, J= 7.1 Hz, 2H), 2.17 - 2.10 (m, 1H), 1.94 (p, J= 7.1 Hz, 2H), 1.45 (s, 9H). 13C NMR (101 MHz, CDCh) 5 172.4, 171.1, 169.6, 168.4, 167.7, 147.0, 136.3, 132.7, 116.8, 111.8, 110.2, 80.8, 49.0, 42.0, 32.7, 31.6, 28.3, 24.8, 22.9. LRMS (ESI) m/z: [M + H]+ calcd for C21H26N3O6; 416.181 found, 416.182.
Scheme 38: Preparation of tert-butyl (2-(4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5- difluorophenyl)-2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidin-l- yl)-2-oxoethyl)carbamate (Intermediate 7)
Figure imgf000210_0002
Intermediate 7 was prepared following the General Procedure F using the corresponding amine (108 mg, 0.17 mmol) and N-Boc-glycine (30 mg, 0.17 mmol). After the reaction completion, the reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2;
DCM/MeOH = 95:5) providing 75 mg of the desired product (58%). ’H NMR (400 MHz, MeOD) 5 7.99 (d, J= 0.8 Hz, 1H), 7.26 (dd, J= 12.9, 6.7 Hz, 1H), 6.93 (dd, J= 11.3, 7.3 Hz, 1H), 5.72 - 5.68 (m, 1H), 4.49 (d, J= 13.2 Hz, 1H), 3.95 - 3.82 (m, 6H), 3.75 (s, 2H), 3.41 (s, 3H), 3.22 - 3.15 (m, 2H), 3.06 (t, J= 12.9 Hz, 1H), 2.79 (s, 4H), 2.71 - 2.61 (m, 1H), 2.03 - 1.92 (m, 2H), 1.89 - 1.76 (m, 5H), 1.52 (t, J= 5.8 Hz, 4H), 1.45 (s, 9H), 1.32 - 1.09 (m, 4H), 0.98 (s, 6H). 13C NMR (101 MHz, MeOD) 5 168.7, 167.9, 163.4, 162.0, 157.0, 156.9, 156.7, 119.1, 118.8, 106.1, 105.8, 79.2, 54.6, 54.4, 53.4, 53.2, 52.2, 49.0, 48.45, 45.9, 44.4, 42.4, 42.0, 41.6, 40.3, 36.8, 36.1, 34.4, 29.9, 29.3, 27.5, 27.3, 11.8. LRMS (ESI) m/z: [M + H]+ calcd for C39H58F2N9O4; 754.457 found, 754.458. Scheme 39: Preparation of 4-((2-(4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5- difluorophenyl)-2-oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidin-l- yl)-2-oxoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Compound 39-1)
Figure imgf000211_0001
The deprotection of Intermediate 7 was achieved following the General Procedure E. After the reaction completion, the volatiles were removed in vacuo and the crude product Intermediate 8 was used in the next reaction step without further purification.
Compound 39-1 was prepared following the General Procedure F from 40 mg of Intermediate 8 (0.052 mmol, 1 eq) and 14 mg of 4-fluoro-thalidomide (1 eq). The reaction was stirred at 80°C for 24 hours, evaporated and the crude product was purified using flash column chromatography (SiO2; EtOAc/MeOH = from 90: 10 to 80:20) providing 23 mg of the desired product (48%). JH NMR (400 MHz, DMSO) 5 11.14 (s, 1H), 8.22 (s, 1H), 8.03 (s, 1H), 7.68 - 7.61 (m, 1H), 7.24 - 7.08 (m, 4H), 6.97 (dd, J= 11.5, 7.4 Hz, 1H), 6.82 (s, 1H), 5.70 (s, 1H), 5.11 (dd, J= 12.9, 5.4 Hz, 1H), 4.43 (d, J= 12.8 Hz, 1H), 4.21 (dd, J= 9.5, 4.5 Hz, 1H), 3.91 (d, J= 15.9 Hz, 3H), 3.64 (s, 2H), 3.48 (s, 2H), 3.31 (s, 2H), 3.17 (s, 2H), 3.05 (t, J= 12.4 Hz, 1H), 2.93 (ddd, J= 17.3, 14.1, 5.7 Hz, 1H), 2.76 - 2.60 (m, 2H), 2.38 (d, J= 6.7 Hz, 4H), 2.12 - 2.01 (m, 1H), 1.91 - 1.63 (m, 9H), 1.27 - 1.01 (m, 8H), 0.92 (s, 6H). 13C NMR (126 MHz, DMSO) 5 172.9, 170.1, 168.8, 167.4, 166.6, 166.0, 163.5, 158.0, 157.8, 157.3, 151.3, 149.4, 145.4, 136.2, 132.0, 118.5, 118.3, 116.2, 110.8, 109.5, 106.5, 106.5, 106.3, 106.3, 69.8, 54.7, 52.8, 52.7, 49.0, 48.6, 43.7, 41.6, 40.4, 35.5, 34.6, 31.0, 29.9, 29.3, 29.1, 29.0, 28.1, 22.2, 14.0. LRMS (ESI) m/z: [M + H]+ calcd for C47H57F2N1106; 910.453 found, 910.454. Scheme 40: 5-((4-(4-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)-2-oxo-l,4,9- triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidin-l-yl)-4-oxobutyl)amino)-2-(2,6- dioxopiperidin-3-yl)isoindoline-l, 3-dione (Compound 40-1)
Figure imgf000212_0001
The reaction sequence started with the General Procedure D using 5-fluoro-thalidomide (813 mg, 2.95 mmol) and the corresponding tert-butyl ester (469 mg, 2.95 mmol). The volatiles were then removed in vacuo and the crude product was used in the next synthetic step without further purification. To a stirred solution of the tert-butyl ester (1 eq) in DCM (0.5 M) was added TFA (10 eq). The resulting reaction mixture was stirred at rt until full completion. The free carboxylic acid was used in the next step without further purification, (monitored by TLC). Compound 40-1 was prepared following the General Procedure F (COMU) using the corresponding amine (79 mg, 0.125 mmol) and carboxylic acid (44 mg, 0.125 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiCh; DCM/MeOH = 2:0.1 to 2:0.2), followed by the second flash column chromatography (SiCh; EtOAc/MeOH = 2:0.4) providing 7 mg of the desired product (0.2 % after three steps). ‘H NMR (400 MHz, MeOD) 5 7.9 (bs, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 12.9, 6.6 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.91 (dd, J = 11.3, 7.2 Hz, 1H), 6.86 (dd, J = 8.4, 2.2 Hz, 1H), 5.67 (s, 1H), 5.03 (dd, J = 12.8, 5.5 Hz, 1H), 4.60 (s, 4H), 4.54 (d, J = 13.7 Hz, 1H), 3.97 - 3.90 (m, 4H), 3.74 - 3.72 (m, 4H), 3.48 - 3.43 (m, 4H), 3.15 - 3.12 (m, 2H), 3.06 - 3.01 (m, 1H), 2.88 - 2.80 (m, 2H), 2.75 - 2.59 (m, 8H), 2.55 - 2.46 (m, 2H), 2.08 (ddt, J = 13.0, 5.6, 2.8 Hz, 1H), 2.00 - 1.91 (m, 4H), 1.86 - 1.79 (m, 4H), 1.50 - 1.46 (m, 4H), 1.14 - 1.06 (m, 2H), 0.96 (s, 6H). 13C NMR (151 MHz, MeOD) 5 174.7, 173.2, 171.8, 170.2, 169.6, 169.3, 164.9, 164.7, 163.2, 160.0, 158.4, 158.3, 156.1, 153.1, 151.8, 140.6, 136.0, 126.6, 120.2, 118.2, 116.8, 107.4, 107.2, 106.7, 56.1, 55.0, 54.6, 53.6, 50.4, 50.3, 49.6, 46.9, 43.4, 43.0, 41.7, 38.5, 37.5, 40.0, 35.8, 32.2, 31.6, 31.3, 30.8, 29.0, 25.6, 23.8. LCMS (ESI) m/z: [M + H]+ calcd for C49H62F2N1106; 938.485 found, 938.495.
Scheme 41: Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)- 2- oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)4-(2-aminoethyl)piperazin)-4-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)butanamide (Compound 41-1)
Figure imgf000213_0001
Compound 41-1 was prepared following the General Procedure F (HATU) using the corresponding amine (74 mg, 0.11 mmol) and carboxylic acid (40 mg, 0.11 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiCE; EtOAc/MeOH = 90 : 10) providing 61 mg of the desired product (59% yield). 'HNMR (500 MHz, MeOD) 5 8.57 (s, 1H), 8.03 (s, 1H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.22 (dd, J =12.9, 6.7 Hz, 1H), 7.12 (d, J = 8.5, 1H), 7.06 (d, J = 7.0, 1H), 6.92 (dd, J =11.1, 7.3 Hz, 1H), 5.73 (s, 1H), 5.07 (dd, J =12.5, 5.4 Hz, 1H), 3.96 (m, 2H), 3.76 (s, 2H), 3.71 (s, 2H), 3.66-3.53 (m, 4H), 3.50-3.37 (m, 9H), 2.91-2.61 (m, 8H), 2.59 (t, J = 6.2, 2H), 2.53 (t, J = 6.8, 1H), 2.48 (m, 4H), 2.12 (m, 1H), 1.99 (m, 4H), 1.84 (m, 2H), 1.49 (t, J = 5.6, 4H), 1.21 (s, 3H), 0.97 (s, 6H). 13C NMR (101 MHz, MeOD) 5 173.2, 171.9, 170.2, 169.3, 168.8, 167.9, 163.0, 162.1, 158.9, 156.9, 156.5, 152.1, 149.7, 148.6, 146.8, 139.0, 138.9, 135.9, 132.6, 118.7, 118.5, 116.8, 110.5, 109.8, 106.0, 105.7, 81.2,
56.3, 54.8, 53.7, 53.2, 52.8, 52.4, 52.3, 49.1, 48.8, 45.3, 41.4, 41.3, 40.3, 37.7, 37.3, 34.5, 30.8, 29.5, 27.6,
24.4, 22.4. LCMS (ESI) m/z: [M + H]+ calcd for C49H63F2Ni2O6; 953.469, found: 953.584.
Scheme 42: Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)- 2- oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)4-(2-aminoethyl)piperazin)-6-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)hexanamide (Compound 42-1)
Figure imgf000213_0002
Compound 42-1 was prepared following the General Procedure F (HATU), the corresponding amine (74 mg, 0.11 mmol) and carboxylic acid (42 mg, 0.11 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiCh; EtOAc/MeOH = 90 : 10) providing 53 mg of the desired product (50% yield). 1 H NMR (500 MHz, MeOD) 5 8.58 (s, 1H), 8.02 (s, 1H), 7.92 (s, 1H), 7.56 (dd, J = 8.2, 7.3 Hz, 1H), 7.20 (dd, J =12.9, 6.5 Hz, 1H), 7.06 (dd, J =10.4, 8.7 Hz, 2H), 6.90 (dd, J =11.1, 7.2 Hz, 1H), 5.74 (s, 1H), 5.06 (dd, J =12.4, 5.4 Hz, 1H), 3.97 (m, 2H), 3.75 (s, 2H), 3.66-3.54 (m, 6H), 3.49-3.36 (m, 8H), 2.91-2.67 (m, 3H), 2.60 (t, J =6.1, 2H); 2.59-2.46 (m, 7H), 2.44 (t, J =7.0, 2H), 2.12 (m, 1H), 2.01 (m, 2H), 1.84 (m, 2H), 1.70 (m, 4H), 1.54-1.42 (m, 6H), 1.31 (s, 3H), 0.93 (s, 6H). 13C NMR (101 MHz, MeOD) 5 173.2, 172.5, 170.2, 169.4, 168.9, 167.9, 163.1, 162.1, 158.9, 156.9, 156.5, 146.9, 138.7, 135.9, 132.5, 118.6, 118.4, 116.6, 110.4, 109.6, 106.0, 105.7, 78.1, 56.3, 54.8, 53.9, 53.2, 52.9, 52.4, 52.3, 49.1, 48.8, 45.4, 41.8, 41.3, 40.3, 37.7, 37.6, 34.5, 32.4, 30.8, 29.3, 28.6, 27.7, 26.2, 24.8, 22.4. LCMS (ESI) m/z: [M + H]+ calcd for C|HMF2N|2O(1: 981.527, found: 981.584.
Scheme 43: Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-l-yl)methyl)-2,5-difluorophenyl)- 2- oxo-l,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)4-(2-aminoethyl)piperazin)-8-((2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)octanamide (Compound 43-1)
Figure imgf000214_0001
Compound 43-1 was prepared following the General Procedure F (HATU) using the corresponding amine (74 mg, 0.11 mmol) and carboxylic acid (45 mg, 0.11 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiCE; EtOAc/MeOH = 90 : 10) providing 73 mg of the desired product (66% yield). 'HNMR (500 MHz, DMSO-de) 5 8.56 (s, 1H), 8.03 (s, 1H), 7.56 (dd, J = 8.3, 7.0 Hz, 1H), 7.27 (dd, J =12.6, 6.4 Hz, 1H), 7.05 (dd, J = 8.5, 6.3 Hz, 2H), 6.95 (dd, J =11.1, 7.2 Hz, 1H), 5.74 (s, 1H), 5.07 (dd, J =12.6, 5.5 Hz, 1H), 3.95 (m, 2H), 3.90 (s, 2H), 3.77 (s, 2H), 3.70-3.55 (m, 4H), 3.50-3.39 (m, 8H), 2.89-2.71 (m, 6H), 2.66 (s, 1H), 2.63 (t, J = 6.2, 2H), 2.53 (m, 4H), 2.40 (t, J = 7.4, 1H), 2.13 (m, 1H), 2.00 (m, 2H), 1.85 (m, 2H), 1.70 (m, 2H), 1.62 (m, 2H), 1.55 (t, J =
5.3, 4H), 1.50-1.37 (m, 9H), 1.31 (s, 6H), 1.01 (s, 6H). 13C NMR (125 MHz, DMSO-d6) 5 173.2, 172.8,
170.3, 169.4, 168.7, 167.9, 163.0, 162.1, 158.9, 156.8, 156.5, 151.8, 146.9, 135.9, 132.5, 119.1, 116.6,
110.4, 109.6, 106.1, 105.9, 56.4, 54.6, 53.3, 53.2, 53.0, 52.4, 52.2, 49.0, 48.8, 45.4, 42.0, 41.3, 40.3, 39.0, 37.7, 36.7, 34.5, 32.5, 30.8, 29.4, 28.7, 27.5, 26.3, 25.0, 22.4. LCMS (ESI) m/z: [M + H]+ calcd for C53H71F2N12O6: 1009.559, found: 1009.712. Example 2: Screening PROTACs activity with Western blot
METTL3 (and METTL14) protein degradation was monitored using Western blot. Cells were treated with the indicated concentration of PROTACs (or DMSO, control) for 24h, 37°C with 5% CO2. Samples were then collected and lysed with RIPA buffer with protease inhibitors (11697498001, Roche). After SDS-PAGE, proteins were transferred to a nitrocellulose membrane, blocked (with 5% milk, 0.5% BSA in TBST buffer) and incubated overnight with primary antibodies. The following antibodies were used: GAPDH (#2118, Cell Signaling, 1 :4000), P-actin (ab8226, Abeam, 1 :2000), METTL3 (abl95352, Abeam, 1 :1000), METTL14 (ab220031, Abeam, 1 : 1000). Membranes were scanned using LI-COR Odyssey DLx Imager after incubation with appropriate secondary antibodies (anti-mouse IgG IRDye® 680RD (926-68072, LI-COR, 1 : 10000), Goat anti -Rabbit IgG IRDye® 800CW (926-32211, LI-COR, 1 : 10000)). Densitometry was performed in Image Studio Lite software and analysis in GraphPad Prism 9.
Table 10 shows METTL3(M3) and METTL14(M14) protein degradation in the MOLM-13, THP-1, N0M0-
1, KASUMI-1, PC3 and DU145 cell lines after 24-hour treatment with a 2 pM PROTACs solution (Measured by Western-Blot assay)
Table 10
Figure imgf000215_0001
Table 8: IC50 data for N6-adenosine-methyltransferase
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Table 9. Exemplary PROTAC compounds IC50 data for N6-adenosine-methyltransferase
The activity for representative examples is shown in the following table, wherein:
A: 0< IC50 < 0.1 pM; B: 0.1 pM < IC50 < 1 pM; C: IC50 > 1 pM
Figure imgf000219_0001

Claims

CLAIMS A bifunctional compound having a structure of Formula (I), or a pharmaceutically acceptable salt thereof,
MBM-L-EBM
Formula (I) wherein,
MBM is a moiety that binds to METTL3;
EBM is a moiety that binds to an E3 ubiquitin ligase; and
L is a linker that imparts rigidity, wherein L has a structure of Formula (V),
Figure imgf000220_0001
Formula (V) wherein, k is 0, 1, 2, or 3; each of L1, L2, and Lk is independently selected from a bond, -O-, -S-, -NR11-, Ci-Cs alkylene, and Ci-Cs heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa; each of RLa is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, - OC(=O)ORb, -OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, - S(=O)(=NRb)Rb, -NRcRd, -NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, - NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; or two RLa are taken tougher to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R;
R11 is hydrogen, -CN, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -C(=O)Ra, -C(=O)ORb, - C(=O)NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of RL1 and RLk is independently selected from
Figure imgf000221_0001
cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRL:I; each of RRLa is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, - OC(=O)ORb, -OC(=O)NRcRd, -SF5, -SH, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, - S(=O)(=NRb)Rb, -NRcRd, -NRbC(=O)NRcRd, -NRbC(=O)Ra, -NRbC(=O)ORb, - NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, -C(=O)ORb, -C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R; each Ra is independently C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), C1-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; each Rb is independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), C1-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R;
Rc and Rd are each independently hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, Ci-C6alkylene(cycloalkyl), Ci-C6alkylene(heterocycloalkyl), C1-C6alkylene(aryl), or Ci-C6alkylene(heteroaryl), wherein the alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is independently optionally substituted with one or more R; or Rc and Rd are taken together with the atom to which they are attached to form a heterocycloalkyl optionally substituted with one or more R; and each R is independently halogen, oxo, -CN, -OH, -SF5, -SH, -S(=O)Ci-C3alkyl, -S(=O)2Ci- C3alkyl, -S(=O)2NH2, -S(=O)2NHC1-C3alkyl, -S(=O)2N(C1-C3alkyl)2, -S(=O)(=NCi- C3alkyl)(C1-C3alkyl), -NH2, -NHC1-C3alkyl, -N(C1-C3alkyl)2, -N=S(=O)(C1-C3alkyl)2, - C(=O)C1-C3alkyl, -C(=O)OH, -C(=O)OC1-C3alkyl, -C(=O)NH2, -C(=O)NHC1-C3alkyl, -C(=O)N(C1-C3alkyl)2, -P(=O)(C1-C3alkyl)2, C1-C3alkyl, C1-C3alkoxy, C1-C3haloalkyl, C1-C3haloalkoxy, C1-C3hydroxyalkyl, C1-C3aminoalkyl, C1-C3heteroalkyl, or C3- Cecycloalkyl. The bifunctional compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (I) has a structure of Formula (II),
Figure imgf000222_0001
Formula (II) wherein,
Z1 and Z2 are independently selected from N, CH and CR2;
X is O or NH;
Y is CH2, C=O, or SO2;
U and V are each independently selected from -CH2- and -(CH2)2-, or one of U and V is - CH2- and the other one is -(CH2)3-
R1 is an unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted cycloalkyl, or unsubstituted or substituted heterocycloalkyl;
R2 is selected from halogen (e.g., F, Cl), -CN, -NO2, -OH, -ORa, -SF5, -SH, -SRa, -NRcRd (e.g., NH2), C1-C6alkyl (e.g., methyl), C1-C6haloalkyl (e.g., CF3, CHF2, CH2F), C1-C6hydroxyalkyl, C1-C6aminoalkyl, and C1-C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; n is selected from 0, 1, 2, 3, and 4;
R13 is selected from a bond, -O-, -S-, -NR13N-, or CR13CR13C;
R3 is C1-C6alkyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; each of which is unsubstituted or substituted with one or more R12; each R12 is independently halogen, -CN, -NO2, -OH, -ORa, -SH, -SRa, -NRcRd, Ci- Cealkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, C1-C6heteroalkyl, C2-Cealkenyl, C2-Cealkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more R; each R13C is independently hydrogen, halogen, -CN, -NO2, -OH, -ORa, -SH, -SRa, -NRcRd, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, or Ci- C6heteroalkyl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R; or two R13C are taken together to form an oxo; or two R13C are taken together to form a cycloalkyl or heterocycloalkyl, each of which is unsubstituted or substituted with one or more R; and
R13N is hydrogen, C1-C6alkyl, C1-C6haloalkyl, C1-C6hydroxyalkyl, C1-C6aminoalkyl, Ci- C6heteroalkyl, -C(=O)Ra-, -C(=O)ORb, or -S(=O)2Ra, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, and heteroalkyl is unsubstituted or substituted with one or more R. The bifunctional compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein R1 is unsubstituted or substituted with 1, 2, 3, or 4 R14, wherein each R14 is independently halogen, oxo, -CN, -NO2, -OH, -ORa, -OC(=O)Ra, -OC(=O)ORb, -OC(=O)NRcRd, -SF5, -SH, - SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRcRd, -S(=O)(=NRb)Rb, -NRcRd, -NRbC(=O)NRcRd, - NRbC(=O)Ra, -NRbC(=O)ORb, -NRbS(=O)2Ra, -N=S(=O)(Rb)2, -C(=O)Ra, -C(=O)ORb, - C(=O)NRcRd, -P(=O)(Rb)2, C1-C6alkyl, Ci-C6haloalkyl, Ci-C6hydroxyalkyl, Ci-C6aminoalkyl, C1-C6heteroalkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, wherein the alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted with one or more R. The bifunctional compound of claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (II) has a structure of Formula (Ila),
Figure imgf000223_0001
Formula (Ila). The bifunctional compound of any one of claims 2 to 4, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (II) has a structure of Formula (Illa),
Figure imgf000224_0001
Formula (Illa), wherein,
NR31R32 are taken together to form a heterocycloalkyl, which is unsubstituted or substituted with one or more
Figure imgf000224_0002
R14 is defined above; and n2 is 0, 1, or 2. The bifunctional compound of claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (II) has a structure of Formula (lib),
Figure imgf000224_0003
Formula (lib). The bifunctional compound of any one of claims 2, 3 or 6, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (II) has a structure of Formula (Illb),
Figure imgf000224_0004
Formula (Illb) wherein,
NR31R32 are taken together to form a heterocycloalkyl, which is unsubstituted or substituted with one or more
Figure imgf000224_0005
R14 is defined above; and n2 is 0, 1, or 2. The bifunctional compound of claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (II) has a structure of Formula (lie),
Figure imgf000225_0001
Formula (lie). The bifunctional compound of any one of claims 2, 3 or 8, or a pharmaceutically acceptable salt thereof, wherein the structure of Formula (II) has a structure of Formula (IIIc),
Figure imgf000225_0002
Formula (IIIc) wherein,
R14 is defined above; and n2 is 0, 1, or 2. The bifunctional compound of any one of claims 2 to 4, 6, 8, or 9, or a pharmaceutically acceptable salt thereof, wherein R13 is -O-. The bifunctional compound of any one of claims 2 to 4, 6, 8, or 9, or a pharmaceutically acceptable salt thereof, wherein R13 is -NR13N-. The bifunctional compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein R13 is -N((C=O)CH2CH3). The bifunctional compound of any one of claims 2 to 4, 6, 8, or 9, or a pharmaceutically acceptable salt thereof, wherein R13 is -CR13CR13C-. The bifunctional compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein R13 is -CH2-.
15. The bifunctional compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein R13 is -CR13CR13C-, wherein two R13C are taken together to form a 3-6 membered cycloalkyl or 4-6 membered heterocycloalkyl, each of which is unsubstituted or substituted with one or more R.
16. The bifunctional compound of any one of claims 2 to 4, 6 to 15, or a pharmaceutically acceptable salt thereof, wherein R3 is 4-6 membered heterocycloalkyl, wherein R3 is unsubstituted or substituted with 1, 2 or 3 R12.
17. The bifunctional compound of claim 16, or a pharmaceutically acceptable salt thereof, wherein R3 is monocyclic heterocycloalkyl.
18. The bifunctional compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000226_0001
19. The bifunctional compound of claim 16, or a pharmaceutically acceptable salt thereof, wherein
R3 is bicyclic heterocycloalkyl. 0. The bifunctional compound of claim 19, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000226_0002
1. The bifunctional compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein R1 is unsubstituted or substituted 6 membered heteroaryl. 2. The bifunctional compound of claim 21, or a pharmaceutically acceptable salt thereof, wherein R1 is pyrimidine. 3. The bifunctional compound of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein L1 attaches to the MBM and L2 attaches to the EBM. 4. The bifunctional compound of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein each of L1, L2, and Lk is independently selected from a bond, C1-C6 alkylene, and C1-C6 heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa. 5. The bifunctional compound of any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, wherein each of L1, L2, and Lk is independently selected from a bond, C1-C4 alkylene, and C1-C4 heteroalkylene, wherein the alkylene and heteroalkylene is unsubstituted or substituted with one or more RLa. The bifunctional compound of any one of claims 1 to 25, or a pharmaceutically acceptable salt
Figure imgf000227_0001
The bifunctional compound of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, wherein each of L1, L2, and Lk is independently selected from a bond, -NH-(CH2)3-, -
Figure imgf000227_0002
The bifunctional compound of any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, wherein each of RL1 and RLk is independently selected from
Figure imgf000227_0003
, 6-14 membered cycloalkyl, 6-14 membered heterocycloalkyl, 6-14 membered aryl, and 6-14 membered heteroaryl, wherein each of the cycloalkyl, heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRLa. The bifunctional compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, wherein each of RL1 and RLk is independently selected from 6-10 membered heterocycloalkyl, 6-10 membered aryl, and 6-10 membered heteroaryl, wherein each of the heterocycloalkyl, aryl, and heteroaryl is unsubstituted or substituted with one or more RRLa. The bifunctional compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein RL1 is unsubstituted or substituted monocyclic heterocycloalkyl.
31. The bifunctional compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000228_0001
32. The bifunctional compound of claim 30, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000228_0002
33. The bifunctional compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein RL1 is unsubstituted or substituted bicyclic heterocycloalkyl.
34. The bifunctional compound of claim 33, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000228_0003
35. The bifunctional compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein RL1 is unsubstituted or substituted phenyl.
36. The bifunctional compound of any one of claims 1 to 29, or a pharmaceutically acceptable salt thereof, wherein RL1 is unsubstituted or substituted monocyclic heteroaryl.
37. The bifunctional compound of any one of claims 1 to 36, or a pharmaceutically acceptable salt thereof, wherein RLk is unsubstituted or substituted monocyclic heterocycloalkyl or unsubstituted or substituted bicyclic heterocycloalkyl.
38. The bifunctional compound of any one of claims 1 to 36, or a pharmaceutically acceptable salt thereof, wherein RLk is unsubstituted or substituted phenyl.
39. The bifunctional compound of any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, wherein k is 0 or 1.
40. The bifunctional compound of any one of claims 1 to 39, or a pharmaceutically acceptable salt thereof, wherein EBM has a structure of
Figure imgf000229_0001
Figure imgf000230_0001
41. The bifunctional compound of any one of claims 1 or 23 to 40, or a pharmaceutically acceptable salt thereof, wherein the bifunctional compound has a structure of:
Figure imgf000231_0001
42. A bifunctional compound having a structure of Formula (lib*), or a pharmaceutically acceptable salt thereof,
Figure imgf000231_0002
Formula (lib*) wherein,
EBM is a moiety that binds to an E3 ubiquitin ligase;
L is a covalent linker; and
R1, U, V, X, Y, Z1, Z2, n, R2, R13 and R3 have the meanings defined in Formula (II).
43. A bifunctional compound having a structure of Formula (lie*), or a pharmaceutically acceptable salt thereof,
Figure imgf000231_0003
Formula (lie*) wherein,
EBM is a moiety that binds to an E3 ubiquitin ligase;
L is a covalent linker; and
R1, U, V, X, Y, Z1, Z2, n, R2, R13 and R3 have the meanings defined in Formula (II).
44. A compound selected from Table 2 and Table 3, or a pharmaceutically acceptable salt thereof.
45. A compound of any one of claims 1 to 44 for use as a medicament.
46. A compound of any one of claims 1 to 44 for use in treatment of cancer. The compound for use according to claim 46, wherein said cancer is selected from the group comprising renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma. A pharmaceutical composition comprising a compound of any one of claims 1 to 44, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. A method of treating a disease or condition, comprising administering to a subject in need thereof, a compound of any one of claims 1 to 44, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 48.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020201773A1 (en) * 2019-04-05 2020-10-08 Storm Therapeutics Ltd Mettl3 inhibitory compounds
WO2021011634A1 (en) * 2019-07-15 2021-01-21 Kymera Therapeutics, Inc. Protein degraders and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020201773A1 (en) * 2019-04-05 2020-10-08 Storm Therapeutics Ltd Mettl3 inhibitory compounds
WO2021011634A1 (en) * 2019-07-15 2021-01-21 Kymera Therapeutics, Inc. Protein degraders and uses thereof

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Title
ZENG CHENGWU, HUANG WANXU; LI YANGQIU; WENG HENGYOU: "Roles of METTL3 in cancer: mechanisms and therapeutic targeting", JOURNAL OF HEMATOLOGY & ONCOLOGY, BIOMED CENTRAL LTD, LONDON UK, vol. 13, no. 1, 1 December 2020 (2020-12-01), London UK , XP093175116, ISSN: 1756-8722, DOI: 10.1186/s13045-020-00951-w *

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