N6-adenosine-methyltransferase PROTAC and methods of use thereof

Abstract

The present disclosure relates to proteolytic targeting chimera (PROTAC) that modulate N6-adenosine-methyltransferase and methods of use thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of European Patent Application No. 22207794.3 filed on November 16, 2022 and International Application No. PCT / EP2022 / 063350 filed on May 17, 2022, which are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to proteolysis targeting chimeras (PROTACs) that modulate N6-adenosine-methyltransferase and methods of use thereof. Background Art

[0004] Gene expression is regulated at the transcriptome (the messenger RNA obtained by transcription of the genome) level through dynamic levels of mRNA modification. 6 The conversion of A) is the most common internal post-transcriptional modification (also called epitranscriptome modification) in eukaryotic mRNA. This methylation event usually occurs within the DRACH (D = A, G, U; R = A, G; H = A, C, U) consensus sequence motif. 6 A levels may vary in different tissues, developmental states, or in response to cellular stress. 6 A affects the structure of RNA and its ability to form protein-RNA interactions, and therefore, it regulates the processing, translation, and stability of cellular transcripts. 6 A is involved in controlling embryonic development and stem cell differentiation, regulating the mammalian circadian clock and modulating stress responses (eg, heat shock).

[0005] m 6 The dynamic level of A is regulated by the interaction between erasers and writer proteins. 6 A writing molecule has been known for twenty years, but m 6 The discovery of the A-specific erasing proteins FTO (ALKBH9) and ALKBH5 finally demonstrated the reversibility of the modification and its regulatory role. 6A demethylases belong to the AlkB family of dioxygenases whose enzymatic reactions are dependent on Fe(II) and 2-oxoglutarate (2OG). The core writer molecular complex is formed by two methyltransferase-like proteins, METTL3 and METTL14, which depend on the recruitment of additional cofactors by the mRNA substrate, including WTAP and RBM15. The METTL3-METTL14 complex transfers a methyl group from S-adenosylmethionine (SAM) to adenosine within the consensus sequence 5'-GGACU-3'. Only METTL3 has a complete SAM binding site, while METTL14 has a degenerate SAM binding site that is non-functional. Individual depletion of METTL3 or METTL14 reduces m 6 More importantly, dysregulation of METTL3 has recently been associated with specific tumors, such as acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma. 6 A modification showed broad antiviral effects. Therefore, small molecule modulators of METTL3-METTL14 writing molecules have potential therapeutic uses in cancer and viral infections.

[0006] Based on the prior art mentioned above, the purpose of the present invention is to provide means and methods for regulating m6A modification levels using PROTAC therapeutic agents to achieve the purpose of regulating gene expression for cancer therapy. This purpose is achieved by the subject matter of the independent claims of this specification. Summary of the invention

[0007] In one aspect, the present disclosure relates to bifunctional compounds that have found utility in degrading and inhibiting N6-adenosine-methyltransferase. In some embodiments, the bifunctional compounds target the 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 bind to METTL14. In some embodiments, the bifunctional compounds modulate, inhibit and / or degrade METTL14.

[0008] The first aspect of the present disclosure relates to a compound of general formula (A)

[0009]

[0010] The second aspect of the present disclosure relates to a compound of general formula (U)

[0011]

[0012] A third aspect of the present disclosure relates to a compound according to the first or second aspect for use as a medicament. A fourth aspect of the present disclosure relates to a compound according to the first or second aspect for use in the treatment of cancer.

[0013] In another embodiment, the present disclosure is directed to a pharmaceutical composition comprising at least one compound of the present disclosure or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shown A) Design of METTL3 inhibitor 2 from hit compound 1. The formed bonds are depicted in red. 50 The values ​​are based on the time-resolved Förster ( ) Biochemical assay of TR-FRET. B) Superposition of compounds 1 (grey, X-rays from METTL3, PDB code: 7NHI) and 2 (cyan, drawn in Pymol).

[0015] Figure 2 Design of compound 8 starting from 5 is shown, with newly formed bonds drawn in red. A) Inhibitor 5 (cyan) with relevant residues (gray carbon atoms) in the METTL3 binding site. Major intermolecular interactions are shown (yellow dashed lines, PDB code: 7O08). B) Superposition of inhibitors 5 (cyan) and 7 (green, PDB code: 7O09). C) Superposition of inhibitors 5 (cyan) and 8 (yellow, PDB code: 7O0L), and the interaction of the lactam with the Gln550 side chain amide.

[0016] Figure 3 Shown are A) unusual interactions of the fluorine atom of compound 20 with the Pro397 amide π-system (PDB code: 7O29). B) Vander Waals contacts between the fluorine atom of inhibitor 21 and the side chains of Ser511 and Tyr406 (PDB code: 7O2E). Figure 4TR-FRET dose response curves measured for compounds 1, 22 and SAH (n≥3) and the chemical structure of lead compound 22 are shown. B) InCELL pulse dose response curves at 46°C for HEK293T cells (n=3). D) Dose response curves for m6A / A reduction in polyadenylated RNA fractions in MOLM-13 (n=5) and PC-3 (n=3) cell lines measured by UPLC-MS / MS.

[0017] Figure 5 Results of thermal shift assays are shown. The first derivative of the melting curves of METTL3 / METTL14 for inhibitor 22 or SAH is shown.

[0018] Figure 6 Results of thermal shift assays are shown. The first derivative of the melting curves of METTL1 for inhibitor 22 or SAH is shown. Compound 22 did not shift the melting temperature of METTL1.

[0019] Figure 7 Exemplary compounds.

[0020] Figure 8 Exemplary substitution patterns for R.

[0021] Fig. 9 Shown is the degradation of METTL3 protein measured by western blot after treatment with various PROTAC molecules at 2 μM concentration for 24 h.

[0022] Fig.10 Shown is the METTL14 protein measured by western blot after treatment with various PROTAC molecules at 2 μM concentration for 24 h.

[0023] Fig.11 Shown is the correlation between METTL3 and METTL14 degradation by various PROTAC molecules measured by western blotting. DETAILED DESCRIPTION

[0024] Terms and Definitions For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural, and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth will control.

[0025] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof are intended to be equivalent and open-ended, and one or more items following any of these words are not intended to be an exhaustive listing of such one or more items, nor are they intended to be limited to the listed one or more items. For example, an article "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only these), or may contain not only components A, B, and C, but also one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and grammatical equivalents thereof include disclosure of embodiments that "consist essentially of" or "consist of."

[0026] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, as well as any other stated or intervening value in that range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0027] Reference herein to "about" a value or parameter includes (and describes) variations to the value or parameter itself. For example, reference to "about X" includes description of "X." As used herein, including in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc.) and chemical methods.

[0029] The term METTL3 in the context of the present specification relates to the catalytic subunit of N6-adenosine-methyltransferase (UniprotID: Q86U44).

[0030] The term METTL14 in the context of the present specification relates to the N6-adenosine-methyltransferase non-catalytic subunit (Uniprot ID: Q9HCE5).

[0031] C1-C6 alkyl in the context of this specification refers to a saturated straight or branched hydrocarbon having 1, 2, 3, 4, 5 or 6 carbon atoms. In certain embodiments, the alkyl is substituted, meaning that, for example, one or more CH2 moieties may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR, wherein R is methyl, ethyl or propyl; amino bridge).

[0032] The term C3-C7 cycloalkyl in the context of this specification relates to a saturated hydrocarbon ring having 3, 4, 5, 6 or 7 carbon atoms, wherein in certain embodiments one carbon-carbon bond may be unsaturated. Non-limiting examples of C3-C7 cycloalkyl moieties include cyclopropyl (-C3H5), cyclobutyl (-C4H7), cyclopentenyl (C5H9) and cyclohexenyl (C6H 11 ) moiety. In certain embodiments, the cycloalkyl is substituted. In certain embodiments, the cycloalkyl is substituted with one C1 to C4 unsubstituted alkyl moiety. In certain embodiments, the cycloalkyl is substituted with more than one C1 to C4 unsubstituted alkyl moiety.

[0033] The term heterocycle in the context of the present specification relates to cycloalkyl groups wherein at least one ring atom or several ring atoms are replaced by nitrogen, oxygen and / or sulfur atoms.

[0034] The term heterobicyclic in the context of the present specification relates to two directly linked cycloalkyl groups, wherein at least one ring atom or several ring atoms are replaced by a nitrogen, oxygen and / or sulfur atom.

[0035] The term heterocycloalkyl in the context of the present specification relates to cycloalkyl groups wherein at least one ring atom or several ring atoms are replaced by nitrogen, oxygen and / or sulfur atoms.

[0036] When used herein in the narrowest sense, the term unsubstituted C n Alkyl-related part -C n H 2n - (if used as a bridge between molecular parts) or -C n H 2n+1 (if used in the context of a terminal part).

[0037] The term unsubstituted C n Alkyl and substituted C nAlkyl includes straight chain alkyl groups containing or attached to a cyclic structure, such as cyclopropane, cyclobutane, cyclopentane or cyclohexane moieties having straight chain alkyl substitutions, which are unsubstituted or substituted depending on the context in which they are noted or mentioned. The total number of carbons and (where appropriate) N, O or other heteroatoms in a straight chain or cyclic structure is summed to n.

[0038] When used in the context of a chemical formula, the following abbreviations may be used: Me is methyl CH3, Et is ethyl -CH2CH3, Prop is propyl -(CH2)2CH3 (n-propyl, n-pr) or -CH(CH3)2 (isopropyl, i-pr), but is butyl -C4H9, -(CH2)3CH3, -CHCH3CH2CH3, -CH2CH(CH3)2 or -C(CH3)3.

[0039] The term substituted alkyl in its broadest sense refers to an alkyl group as defined above in the broadest sense, which is covalently bonded to an atom which is not carbon or hydrogen, in particular to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may be bonded to one or several other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in the broadest sense), if applicable. In a narrow sense, substituted alkyl refers to alkyl as defined above in the broadest sense, wherein one or several carbon atoms are substituted by a group selected from the group consisting of amine NH2, alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl)NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl)OCOR, carbonyl O and its ketal or acetal (OR)2, nitrile CN, isonitrile NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluorine F, chlorine Cl, bromine Br, iodine I, phosphates PO3H2, PO3R2, phosphates OPO3H2 and OPO3R2, sulfhydryl SH, sulfoalkyl SR, sulfoxide SOR, sulfonyl S02R, sulfonamide SO2NHR, sulfate S03H and sulfate S03R, wherein R is further defined in the description.

[0040] The term hydroxy-substituted group refers to a group modified by one or several hydroxyl groups OH. The term amino-substituted group refers to a group modified by one or several amino groups NH2. The term carboxyl-substituted group refers to a group modified by one or several carboxyl groups COOH. Non-limiting examples of amino-substituted alkyl groups 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(CH2NHMe)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(NH2)(CH2)2NH2, -CH2CH(NHMe)(CH2)2NHMe, -CH2CH(NHEt)(CH2)2NHEt, -CH2CH(CH2NH2)2, -CH2CH(CH2NHMe)2, and -CH2CH(CH2NHEt)2 for terminal portions; and -CH2CHNH2-, -CH2CHNHMe-, -CH2CHNHEt- for amino-substituted alkyl portions bridging two other portions.

[0041] Non-limiting examples of hydroxy-substituted alkyl groups include -CH2OH, -(CH2)2OH, -(CH2)3OH, -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 portions; As well as -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-, which are used to bridge the hydroxyl substituted alkyl moieties of two other moieties.

[0042] Haloalkyl refers to an alkyl group as defined above substituted with one or more halo groups, for example trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl and the like.

[0043] The term sulfonyl substituted group refers to a group modified with one or several sulfonyl groups -SO2R or derivatives thereof, wherein R is further defined in the description.

[0044] The term sulfonamide-substituted groups refers to groups modified by one or several sulfonamide groups -SO2NHR or -NHSO2R or derivatives thereof, wherein R is further defined in the description. The term amine-substituted groups refers to groups modified by one or several amine groups -NHR or -NR2 or derivatives thereof, wherein R is further defined in the description.

[0045] The term carbonyl substituted group refers to a group modified with one or several carbonyl groups -COR or derivatives thereof, wherein R is further defined in the description.

[0046] Ester refers to the group -CO2R, where R is further defined in the description.

[0047] An ether is a group that has an oxygen between two saturated carbon atoms.

[0048] Amide refers to the group -CONHR, where R is further defined in the description.

[0049] Ethylene glycol refers to the group -(CH2-CH2-O) n -or-(O-CH2-CH2) n-, where n is further defined in the description.

[0050] Alkyne refers to the group -CΞC- (triple bond between two carbon atoms).

[0051] Alkenylene refers to the group -CH=CH- (a double bond between two carbon atoms).

[0052] The term halogen-substituted group refers to a group modified by one or several halogen atoms (independently) selected from F, Cl, Br, I.

[0053] The term fluorine-substituted alkyl refers to an alkyl group as defined above modified by one or several fluorine groups F. Non-limiting examples of fluorine-substituted alkyl groups include -CH2F, -CHF2, -CF3, -(CH2)2F, -(CHF)2H, -(CHF)2F, -C2F5, -(CH2)3F, -(CHF)3H, -(CHF)3F, -C3F7, -(CH2)4F, -(CHF)4H, -(CHF)4F and -C4F9.

[0054] Non-limiting examples of hydroxy and fluoro substituted alkyl groups include -CHFCH2OH, -CF2CH2OH, -(CHF)2CH2OH, -(CF2)2CH2OH, -(CHF)3CH2OH, -(CF2)3CH2OH, -(CH2)3OH, -CF2CH(OH)CH3, -CF2CH(OH)CF3, -CF(CH2OH)CHFCH3, and -CF(CH2OH)CHFCF3.

[0055] The term aryl in the context of this specification refers to a cyclic aromatic C5-C 10 Examples of aryl groups include, but are not limited to, phenyl and naphthyl.

[0056] Alkylaryl in the context of this specification relates to an alkyl group substituted with an aryl moiety. Specific examples are ethylphenyl, propylphenyl, butylphenyl and their higher homologues. Substituted alkylaryl groups may be substituted with indicated substituents on the alkyl portion of the moiety (if chemically feasible) or on the aryl portion.

[0057] Heteroaryl is an aryl group containing one or more nitrogen, oxygen and / or sulfur atoms. Examples of heteroaryl include, but are not limited to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazine, quinoline, benzofuran and indole. Heteroaryl also encompasses bicyclic heteroaryl. The aryl or heteroaryl in the context of this specification may also be substituted by one or more alkyl groups. The alkyl heteroaryl in the context of this specification refers to an alkyl group substituted by a heteroaryl moiety. As used herein, the term pharmaceutical composition refers to a compound of the present disclosure or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the present disclosure is provided in a form suitable for topical, parenteral or injection administration. As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. and combinations thereof, as known to those skilled in the art (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

[0058] As used herein, the terms treating or treatment of any disease or condition (e.g., cancer) refer in one embodiment to ameliorating the disease or condition (e.g., slowing or arresting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In another embodiment, "treating" or "treatment" refers to modulating the disease or condition physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters), or both. Methods for Assessing Treatment and / or Prevention of Disease

[0059] PROTAC Compounds

[0060] In one aspect, PROTAC compounds that inhibit and / or degrade the heterodimeric complex METTL3-METTL14 are disclosed herein. In one aspect, PROTAC compounds are disclosed herein, comprising (i) an active compound targeting the METTL3-METTL14 complex (see, e.g., the paragraph starting with the subtitle "PROTAC active compound"), and (ii) an E3 ligase binder (see, e.g., the paragraph starting with the subtitle "E3 ligase binder"). In some embodiments, the PROTAC compound further comprises a handle (see, e.g., the paragraph starting with the subtitle "Handle"). In some embodiments, the PROTAC compound further comprises a linker (see, e.g., the paragraph starting with the subtitle "Linker").

[0061] The first aspect of the present disclosure relates to a compound of general formula (A)

[0062]

[0063] in

[0064] -NR 31 R 32 is selected from optionally substituted 3-12 membered heterocycloalkyl (e.g.,

[0065] );

[0066] - Each R 2 Independently selected from halogen (e.g. F, Cl), C1-C3 alkyl and C1-C3 haloalkyl (e.g. CF3, CHF2, CH2F);

[0067] - n is an integer selected from 0, 1, 2, 3 and 4;

[0068] - the handle is a linker moiety comprising 3 to 10 atoms with an atomic mass ≥ 12 (C, N, O, S) or consisting essentially of 3 to 10 atoms with an atomic mass ≥ 12 (C, N, O, S); - the linker is a linker moiety comprising 3 to 50 atoms with an atomic mass ≥ 12 or consisting essentially of 3 to 50 atoms with an atomic mass ≥ 12;

[0069] - An E3 ligase binder is a moiety that binds an E3 ligase.

[0070] The first aspect of the present disclosure relates to a compound of general formula (A)

[0071]

[0072] in

[0073] -NR 31 R 32 Selected from

[0074]

[0075] - Each R 2 Independently selected from F, Cl, CF3, CHF2, CH2F;

[0076] - n is an integer selected from 0, 1, 2, 3 and 4;

[0077] - the handle is a linking moiety comprising or essentially consisting of 3 to 10 atoms with an atomic mass ≥ 12 (C, N, O, S);

[0078] - the linker is a linker moiety comprising or essentially consisting of 3 to 50 atoms with an atomic mass ≥ 12;

[0079] - An E3 ligase binder is a moiety that specifically binds to an E3 ligase.

[0080] In another aspect, the present disclosure relates to a compound of formula (A-1):

[0081]

[0082] in

[0083] -Z 1 and Z 2 Independently selected from N, CH and CR 2 ;

[0084] -X is O or NH;

[0085] -Y is CH2, C=O or SO2;

[0086] -R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl and heterocycle, in particular R 1 is unsubstituted or substituted heteroaryl;

[0087] -R 2 Selected from F, Me, Cl, OH, NH2, Br, CF3, CHF2, CH2F;

[0088] - n is an integer selected from 0, 1, 2, 3 and 4;

[0089] -R 3 is a substituted alkylamine;

[0090] -U and V are independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other is -(CH2)3-;

[0091] - the handle is a linking moiety comprising or essentially consisting of 3 to 10 atoms with an atomic mass ≥ 12 (C, N, O, S);

[0092] - the linker is a linker moiety comprising or essentially consisting of 3 to 50 atoms with an atomic mass ≥ 12;

[0093] - An E3 ligase binder is a moiety that binds an E3 ligase.

[0094] In another aspect, the compound of formula (A-1) has the structure of formula (A-1a),

[0095]

[0096] In another aspect, the compound of formula (A-1) has the structure of formula (A-1b),

[0097]

[0098] In some embodiments, R 1 have The structure of

[0099] - Each R 4 Independently selected from

[0100] secondary amines which are substituted by alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and / or heterocycle,

[0101] ·halogen;

[0102] and / or two R 4 together form an unsubstituted or substituted heteroaryl or heterocyclic ring; and

[0103] - m is an integer selected from 0, 1, 2 and 3.

[0104] In some embodiments of Formula (A), (A-1), (A-1a) or (A-1b), R 3 for

[0105]

[0106] in

[0107] - s is an integer selected from 1 and 2, more particularly s is 1;

[0108] -R 31 and R 32 together form a heterocyclic or heterobicyclic ring which is unsubstituted or substituted by alkyl, halogen and / or hydroxy, or R 31 and R 32Independently selected from hydrogen and unsubstituted or hydroxy and / or halogen substituted alkyl or cycloalkyl.

[0109] In some embodiments of Formula (A), (A-1), (A-1a) or (A-1b), R 3 With -CH2-NR 31 R 32 In some embodiments, NR 31 R 32 Selected from

[0110] In some embodiments of Formula (A), (A-1), (A-1a) or (A-1b), NR 31 R 32 Selected from

[0111]

[0112] wherein v is an integer selected from 0, 1 and 2, and each R N are independently selected from hydroxy, halogen and C1-C4 alkyl, or two R N This forms a C3-C6 cycloalkyl group.

[0113] In some embodiments, for In certain embodiments, each R 2 is F. In certain embodiments, n is an integer selected from 0, 1 and 2. In certain embodiments, n is 2. In certain embodiments, the handle is a linking moiety comprising 4 to 8 atoms of atomic mass ≥12 or consisting essentially of 4 to 8 atoms of atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 4 to 30 atoms of atomic mass ≥12 or consisting essentially of 4 to 30 atoms of atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 5 to 20 atoms of atomic mass ≥12 or consisting essentially of 5 to 20 atoms of atomic mass ≥12.

[0114] The following sections further describe various groups of the disclosed PROTAC compounds (e.g., R 1 , R 2 , R 3 , R 31 , R 32 , R 4 , handle and joint).

[0115] E3 ligase binders

[0116] An E3 ligase binder is a molecule that specifically binds to an E3 ligase. In certain embodiments,

[0117] The E3 ligase was cereblon (UniProt-ID: Q96SW2).

[0118] In certain embodiments, the E3 ligase binder has formula (B)

[0119]

[0120] in

[0121] -Ox is CH2 or C=O;

[0122] -T is selected from F, Cl;

[0123] - k is an integer selected from 0, 1, and 2;

[0124] - Indicates a bond to a linker.

[0125] In certain embodiments, k is an integer selected from 0, 1. In certain embodiments, k is 0. In certain embodiments, T is F.

[0126] handle

[0127] In certain embodiments, the handle is a linking moiety comprising 3 to 10 atoms with an atomic mass ≥12 (C, N, O, S) or consisting essentially of 3 to 10 atoms with an atomic mass ≥12 (C, N, O, S). In certain embodiments, the handle is a linking moiety comprising 4 to 8 atoms with an atomic mass ≥12 or consisting essentially of 4 to 8 atoms with an atomic mass ≥12. In certain embodiments, the handle is a linking moiety comprising 5 to 10 atoms with an atomic mass ≥12 or consisting essentially of 5 to 10 atoms with an atomic mass ≥12. In certain embodiments, the handle is a linking moiety comprising 5 to 15 atoms with an atomic mass ≥12 or consisting essentially of 5 to 15 atoms with an atomic mass ≥12. In certain embodiments, the handle is a linking moiety comprising 10 to 15 atoms with an atomic mass ≥12 or consisting essentially of 10 to 15 atoms with an atomic mass ≥12. In certain embodiments, the handle is a linking moiety comprising or consisting essentially of 5 to 20 atoms with an atomic mass ≥12.

[0128] In certain embodiments, the handle comprises or consists essentially of 1, 2, 3, or 4 chemical moieties selected from the group consisting of alkyl, amine, phenyl, and carbonyl.

[0129] In certain embodiments, the handle is selected from the following formula:

[0130]

[0131] in

[0132] -Mid is selected from the group consisting of C1-C3 alkyl and phenyl.

[0133] In certain embodiments, the handle is of formula (X)

[0134]

[0135] wherein Mid is selected from C1-C3 alkyl and phenyl.

[0136] In certain embodiments, the handle is of formula (Y):

[0137]

[0138] In certain embodiments, the handle is selected from the following formula:

[0139]

[0140] Connectors

[0141] In certain embodiments, the linker is a linker moiety comprising 3 to 50 atoms (C, N, O, S) with an atomic mass ≥12 or consisting essentially of 3 to 50 atoms (C, N, O, S) with an atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 4 to 30 atoms with an atomic mass ≥12 or consisting essentially of 4 to 30 atoms with an atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 5 to 20 atoms with an atomic mass ≥12 or consisting essentially of 5 to 20 atoms with an atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 5 to 10 atoms with an atomic mass ≥12 or consisting essentially of 5 to 10 atoms with an atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 5 to 15 atoms with an atomic mass ≥12 or consisting essentially of 5 to 15 atoms with an atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising 5 to 25 atoms with an atomic mass ≥12 or consisting essentially of 5 to 25 atoms with an atomic mass ≥12. In certain embodiments, the linker is a linker moiety comprising or consisting essentially of 10 to 15 atoms with an atomic mass ≥12.

[0142] In certain embodiments, the linker and handle as combined are a linker moiety comprising 5 to 25 atoms of atomic mass ≥12 or consisting essentially of 5 to 25 atoms of atomic mass ≥12. In certain embodiments, the linker and handle as combined are a linker moiety comprising 4 to 35 atoms of atomic mass ≥12 or consisting essentially of 4 to 35 atoms of atomic mass ≥12. In certain embodiments, the linker and handle as combined are a linker moiety comprising 4 to 25 atoms of atomic mass ≥12 or consisting essentially of 4 to 25 atoms of atomic mass ≥12. In certain embodiments, the linker and handle as combined are a linker moiety comprising 15 to 25 atoms of atomic mass ≥12 or consisting essentially of 15 to 25 atoms of atomic mass ≥12. In certain embodiments, the linker and handle as combined are a linker moiety comprising 12 to 30 atoms of atomic mass ≥12 or consisting essentially of 12 to 30 atoms of atomic mass ≥12. In certain embodiments, the linker and handle as combined is a linker moiety comprising or consisting essentially of 16 to 22 atoms with an atomic mass ≥12.

[0143] In certain embodiments, the linker comprises or consists essentially of 1, 2, 3, 4, 5, 6, or 7 chemical moieties independently selected from alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkenylene, alkynylene, glycol, carbonyl, ether, ester, amine, amide, sulfonamide, wherein each chemical moiety is independently unsubstituted or substituted with C1-C3 alkyl, halogen, CN, NO2, hydroxyl, amine, sulfate, phosphate, and / or carboxyl.

[0144] In certain embodiments, the linker comprises or consists essentially of 1, 2, 3, or 4 chemical moieties selected from alkyl, ethylene glycol, carbonyl, piperazine, aryl, amine, triazole.

[0145] In certain embodiments, the linker is selected from the following formula:

[0146]

[0147]

[0148] -Lin is selected from C3-C 20 Alkyl, C3-C 20 Alkyl-triazole, oligo(ethylene glycol). In certain embodiments, the linker is selected from the following formula:

[0149]

[0150]

[0151] -p is selected from 2, 3, 4 and 5;

[0152] -q is selected from 7, 8, 9, 10, 11, 12 and 13;

[0153] -r is selected from 11, 12, 13, 14, 15, 16 and 17;

[0154] -s is selected from 7, 8, 9, 10, 11, 12 and 13;

[0155] -t is selected from 3, 4, 5, 6, 7, 8 and 9;

[0156] -u is selected from the group consisting of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0157] In certain embodiments, the linker is selected from the following formulae (O), (P), (Q), (R), (S), and (T), wherein

[0158] -p is selected from 2, 3, 4 and 5;

[0159] -q is selected from 7, 8, 9, 10, 11, 12 and 13;

[0160] -r is selected from 11, 12, 13, 14, 15, 16 and 17;

[0161] -s is selected from 7, 8, 9, 10, 11, 12 and 13;

[0162] -t is selected from 3, 4, 5, 6, 7, 8 and 9; and

[0163] -u is selected from 7, 8, 9, 10, 11, 12 and 13.

[0164] In certain embodiments, the linker is selected from the following formulae (O), (P), (Q), (R), (S), and (T), wherein

[0165] -p is selected from 2, 3, 4 and 5;

[0166] -q is selected from 7, 8, 9, 10, 11, 12 and 13;

[0167] -r is selected from 11, 12, 13, 14, 15, 16 and 17;

[0168] -s is selected from 7, 8, 9, 10, 11, 12 and 13;

[0169] -t is selected from 3, 4, 5, 6, 7, 8 and 9; and

[0170] -u is selected from 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0171] In certain embodiments, the linker is selected from the following formula:

[0172]

[0173] in

[0174] -p is selected from 2, 3, 4 and 5;

[0175] -q is selected from 7, 8, 9, 10, 11, 12 and 13;

[0176] -r is selected from 11, 12, 13, 14, 15, 16 and 17;

[0177] -s is selected from 7, 8, 9, 10, 11, 12 and 13;

[0178] -t is selected from 3, 4, 5, 6, 7, 8 and 9;

[0179] -u is selected from the group consisting of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0180] In certain embodiments, the linker is selected from the following formulae (O), (P), (Q), (R), (S), (T), and (V), wherein

[0181] -p is selected from 2, 3, 4 and 5;

[0182] -q is selected from 7, 8, 9, 10, 11, 12 and 13;

[0183] -r is selected from 11, 12, 13, 14, 15, 16 and 17;

[0184] -s is selected from 7, 8, 9, 10, 11, 12 and 13;

[0185] -t is selected from 3, 4, 5, 6, 7, 8 and 9; and

[0186] -u is selected from 7, 8, 9, 10, 11, 12 and 13.

[0187] In certain embodiments, the linker is selected from the following formulae (O), (P), (Q), (R), (S), (T), and (V), wherein

[0188] -p is selected from 2, 3, 4 and 5;

[0189] -q is selected from 7, 8, 9, 10, 11, 12 and 13;

[0190] -r is selected from 11, 12, 13, 14, 15, 16 and 17;

[0191] -s is selected from 7, 8, 9, 10, 11, 12 and 13;

[0192] -t is selected from 3, 4, 5, 6, 7, 8 and 9; and

[0193] -u is selected from 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0194] In certain embodiments, the linker is a peptide. In certain embodiments, the linker is a peptide composed of proteinogenic amino acids.

[0195] In certain embodiments, the linker comprises formula (W):

[0196] Where Lin is selected from C3-C 20 Alkyl, C3-C 20 Alkyl-triazoles and oligo(ethylene glycol).

[0197] In certain embodiments, the linker comprises formula (Z):

[0198] wherein z is selected from 4, 5, 6, 7, 8, 9 and 10.

[0199] Combination of features

[0200] In certain embodiments,

[0201] - the E3 ligase binder has formula (B); and

[0202] - the handle has the formula (F), (G), (H) or (J); and - the linker has the formula (O); (P); (Q); (R); (S) or (T).

[0203] In certain embodiments,

[0204] - the E3 ligase binder has formula (B); and

[0205] - the handle has the formula (X) or (Y); and

[0206] - the linker has the formula (O); (P); (Q); (R); (S); (T) or (V).

[0207] In certain embodiments,

[0208] - the E3 ligase binder has formula (B); and

[0209] - the handle has the formula (C), (D), (E); and

[0210] - The linker has the formula (W) or (Z).

[0211] In certain embodiments, the compound comprises the definitions of a handle, a linker, and an E3 ligase binder according to Table 1 (one row is one combination):

[0212] Table 1

[0213]

[0214]

[0215] Exemplary PROTAC compounds of the present disclosure are described in Tables 2 and 3.

[0216] Table 2

[0217]

[0218]

[0219]

[0220]

[0221] Table 3

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] Active compounds of PROTAC

[0296] The second aspect of the present disclosure relates to a compound of general formula (I)

[0297]

[0298] An alternative embodiment of the second aspect of the present disclosure relates to a compound of formula (Ia)

[0299]

[0300] An alternative embodiment of the second aspect of the present disclosure relates to a compound of formula (Ib)

[0301]

[0302] in

[0303] -Z 1 and Z 2 Independently selected from N, CH and CR 2 ;

[0304] -X is O or NH;

[0305] -Y is CH2, C=O or SO2;

[0306] -R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl and heterocycle, in particular R 1 is unsubstituted or substituted heteroaryl;

[0307] -R 2 Selected from F, Me, Cl, OH, NH2, Br, CF3, CHF2, CH2F;

[0308] - n is an integer selected from 0, 1, 2, 3 and 4;

[0309] -R 3 is a substituted alkylamine;

[0310] -U and V are independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other is -(CH2)3-.

[0311] 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 both -(CH2)2-.

[0312] In certain embodiments, the compound has the general formula (U)

[0313]

[0314] -NR 31 R 32 Selected from

[0315]

[0316] -R 2 Selected from F, Cl, CF3, CHF2, CH2F;

[0317] - n is an integer selected from 0, 1, 2, 3 and 4;

[0318] -R 5 is selected from the group consisting of alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and heterocycle.

[0319] In certain embodiments, R 2 is F. In certain embodiments, n is an integer selected from 0, 1 and 2. In certain embodiments, n is 2. In certain embodiments, R 5 is selected from the group consisting of alkyl, alkylaryl and cycloalkyl. 5 Selected from methyl and methylphenyl.

[0320] Center spiral ring

[0321] In certain embodiments, X is NH. In certain embodiments, Y is C═O.

[0322] In certain embodiments, part Selected from

[0323]

[0324] In certain embodiments, part Selected from

[0325]

[0326] Part R 1

[0327] In certain embodiments, R 1 is unsubstituted or substituted heteroaryl. In certain embodiments, R 1 is unsubstituted or substituted with a moiety selected from the following:

[0328] ·Secondary amine NHR N , where R N is selected from C1-C6 alkyl, C4-C6 cycloalkyl, aryl, and heteroaryl, alkylaryl and alkylheteroaryl;

[0329] Halogen, in particular Cl or F;

[0330] C1-C6 alkyl, C4-C6 cycloalkyl, aryl and heteroaryl.

[0331] In certain embodiments, R 1 is unsubstituted or substituted with a moiety selected from the following:

[0332] ·Secondary amine NHR N , where R N is selected from C1-C6 alkyl, C4-C6 cycloalkyl, aryl and heteroaryl;

[0333] Halogen, in particular Cl or F.

[0334] In certain embodiments, the compound has the general formula (II)

[0335]

[0336] in

[0337] -Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n have the same definitions as above;

[0338] - Each R 4 Independently selected from

[0339] secondary amines which are substituted by alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and / or heterocycle,

[0340] ·halogen;

[0341] And / or two R 4 together form an unsubstituted or substituted heteroaryl or heterocyclic ring;

[0342] - m is an integer selected from 0, 1, 2 and 3.

[0343] In certain embodiments, the compound has the general formula (III)

[0344]

[0345] in

[0346] -Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n have the same definitions as above;

[0347] -R 5 is selected from the group consisting of alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and heterocycle;

[0348] -R 6 is selected from halogen and hydrogen.

[0349] In certain embodiments, the compound has the general formula (IV)

[0350]

[0351] in

[0352] -Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n have the same definitions as above;

[0353] -R 6 is selected from halogen and hydrogen;

[0354] -W is selected from N and CH.

[0355] In certain embodiments, Z 1 and Z 2 At least one of them is CH or CR 2 In certain embodiments, Z 1 and Z 2 All are CH or CR 2 .

[0356] Part R 3

[0357] In certain embodiments, R 3 Substituted with one or several moieties independently selected from alkyl, hydroxy, amino, amine, halogen, cycloalkyl and heterocyclic moieties.

[0358] In certain embodiments, R 3 is a substituted C1-C4 alkylamine. 3 It is a substituted C1-C2 alkylamine.

[0359] In certain embodiments, R 3 for

[0360]

[0361] in

[0362] - s is an integer selected from 1 and 2, more particularly s is 1;

[0363] -R 31 and R 32 together form a heterocyclic or heterobicyclic ring which is unsubstituted or substituted by alkyl, halogen and / or hydroxy, or

[0364] -R 31 and R 32 Independently selected from hydrogen and unsubstituted or hydroxy and / or halogen substituted alkyl or cycloalkyl.

[0365] In certain embodiments, R 31 and / or R 32 is unsubstituted or substituted by alkyl, hydroxy, halogen, cycloalkyl, heterocycle and / or radicals. 31 and / or R 32 R is independently selected from H and unsubstituted or hydroxy and / or halogen-substituted alkyl and cycloalkyl. 31 and R 32 Together they form a heterocyclic or heterobicyclic ring which is unsubstituted or substituted by alkyl, hydroxy and / or halogen.

[0366] In certain embodiments, NR 31 R 32 Selected from

[0367]

[0368] wherein v is an integer selected from 0, 1 and 2, and each R N are independently selected from hydroxy, halogen and C1-C4 alkyl, or two R N This forms a C3-C6 cycloalkyl group.

[0369] In certain embodiments, -NR 31 R 32 Selected from

[0370]

[0371] In certain embodiments, NR31 R 32 Selected from

[0372]

[0373] In certain embodiments, NR 31 R 32 for In certain embodiments, NR 31 R 32 for In certain embodiments, NR 31 R 32 for In certain embodiments, NR 31 R 32 for Part R 2

[0374] In certain embodiments, n is an integer selected from 0, 1, and 2. In certain embodiments, n is 2. In certain embodiments, R 2 In certain embodiments, R 2 For F. R 2 can be bound to any carbon atom of an aryl or heteroaryl ring. 1 or Z 2 If it is a carbon atom, it can also combine with them.

[0375] Part R 5

[0376] In certain embodiments, R 5 is selected from the group consisting of alkyl, alkylaryl and cycloalkyl. 5 Selected from methyl and methylphenyl.

[0377] Uses of Compounds A third aspect of the present disclosure relates to a compound according to the first or second aspect for use as a medicament. A fourth aspect of the present disclosure relates to a compound according to the first or second aspect for use in the treatment of cancer.

[0378] In certain embodiments, the cancer is selected from renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.

[0379] Similarly, within the scope of the present disclosure is a method for treating cancer in a patient in need thereof, the method comprising administering to the patient a compound according to the above description.

[0380] Similarly, provided is a dosage form for preventing or treating cancer, comprising a non-agonist ligand or antisense molecule according to any one of the above aspects or embodiments of the present disclosure.

[0381] The technician knows that any medicine clearly mentioned can exist as a pharmaceutically acceptable salt of the medicine. Pharmaceutically acceptable salts include ionized drugs and counterions with opposite charges. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, benzenesulfonate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, enpolate, propionate dodecyl sulfate (estolate), fumarate, glucoheptonate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methylsulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, toluenesulfonate, triethyl iodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0382] Unless otherwise stated, the compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a specific 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.

[0383] For example, the compounds described herein can be artificially enriched in one or more specific isotopes. In some embodiments, the compounds described herein can be artificially enriched in one or more isotopes that are not primarily found in nature. In some embodiments, the compounds described herein can be artificially enriched in one or more isotopes selected from deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 In some embodiments, the compounds described herein may be artificially enriched with one or more selected from 2 H. 11 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 Cl, 37 Cl, 79 Br, 81 Br, 131 I and 125 In some embodiments, the abundance of the enriched isotope 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% on a molar basis. In some embodiments, the compound is deuterated at at least one position. In some embodiments, the compounds disclosed herein have 2 H atoms replace part or all of 1 H atom.

[0384] In some embodiments, the compounds described herein exist in the form of geometric isomers. In some embodiments, the compounds described herein have one or more double bonds. The compounds provided herein include all cis (cis), trans (trans), syn (syn), anti (anti), entgegen (E) and zusammen (Z) isomers and their corresponding mixtures. In some cases, the compounds described herein have one or more chiral centers, and each center exists in the R configuration or S configuration. The compounds described herein include all diastereomeric forms, enantiomeric forms and epimeric forms and their corresponding mixtures. In additional embodiments of the compounds and methods provided herein, the mixture of enantiomers and / or diastereomers produced by a single preparation step, combination or mutual conversion can be used for the applications described herein. In some embodiments, the compounds described herein are prepared into their single stereoisomers by the following manner: reacting the racemic mixture of the compound with an optically active resolving agent to form a pair of diastereomeric compounds, separating diastereomers, and recovering optically pure enantiomers. In some embodiments, dissociable complexes are preferred. In some embodiments, diastereomers have different physical properties (e.g., melting points, boiling points, solubility, reactivity, etc.) and are separated by taking advantage of these dissimilarities. In some embodiments, diastereomers are separated by chiral chromatography, or preferably, by separation / resolution techniques based on solubility differences. In some embodiments, the optically pure enantiomers and the resolving agent are then recovered by any practical means that do not result in racemization.

[0385] In some cases, compounds exist as tautomers. The compounds described herein include all possible tautomers within the formulas described herein. Tautomers are compounds that can be interconverted by the migration of hydrogen atoms, which is accompanied by the conversion of single bonds and adjacent double bonds. In the bonding arrangements in which tautomerism is possible, there will be a chemical equilibrium of tautomers. All tautomeric forms of the compounds disclosed herein are considered. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH.

[0386] The dosage form can be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or in the form of inhalation or suppository. Alternatively, parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection, can be used. Optionally, a pharmaceutically acceptable carrier and / or excipient may be present.

[0387] Pharmaceutical compositions and administration

[0388] Another aspect of the present 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 other embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. In certain embodiments of the present disclosure, the compounds of the present disclosure are generally formulated into pharmaceutical dosage forms that provide an easily controlled drug dosage and give patients a delicate and easy-to-handle product.

[0389] The pharmaceutical composition can be formulated for oral administration, parenteral administration or rectal administration. In addition, the pharmaceutical composition of the present disclosure can be prepared into solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories) or liquid form (including but not limited to solutions, suspensions or emulsions).

[0390] The dosage regimen of the compounds of the present disclosure will vary according to known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health condition, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and liver function, and the desired effect. In certain embodiments, the compounds of the present disclosure may be administered in a single daily dose, or the total daily dose may be administered in divided doses twice, three times, or four times a day.

[0391] In certain embodiments, for an object of about 50-70 kg, a pharmaceutical composition or combination of the present disclosure can be in the form of a unit dose of about 1-1000 mg of active ingredient. The therapeutically effective dose of a compound, pharmaceutical composition, or combination thereof depends on the species, body weight, age, and individual condition of the object, the disorder or disease treated, or its severity. A physician, clinician, or veterinarian of ordinary skill can easily determine the effective amount of each active ingredient required for preventing, treating, or inhibiting the progression of a disorder or disease.

[0392] The pharmaceutical compositions of the present disclosure may be subjected to conventional pharmaceutical operations, such as sterilization, and / or may contain conventional inert diluents, lubricants or buffers, and adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers. They may be produced by standard processes, such as by conventional mixing, granulation, dissolution or lyophilization 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 edition, 2013 (ISBN8123922892).

[0393] Manufacturing methods and treatment methods

[0394] As a further aspect, the present disclosure also encompasses the use of a compound identified herein as specified in detail above, or a pharmaceutically acceptable salt thereof, in a method for the manufacture of a medicament for the treatment or prevention of cancer.

[0395] Similarly, the present disclosure encompasses methods of treating a patient who has been diagnosed with a disease associated with cancer. The methods entail administering to the patient an effective amount of a compound identified herein, or a pharmaceutically acceptable salt thereof, as specified in detail herein.

[0396] Wherever alternatives to a single separable feature (e.g., ligand type or medical indication) are listed herein as "embodiments," it is understood that such alternatives may be freely combined to form discrete embodiments of the present disclosure disclosed herein. Thus, any alternative embodiment for a ligand type may be combined with any medical indication mentioned herein.

[0397] The said application also covers the following provisions:

[0398] Terms

[0399] 1. A compound of general formula (I)

[0400]

[0401] -Z 1 and Z2 Independently selected from N, CH and CR 2 ;

[0402] -X is O or NH, in particular X is NH;

[0403] -Y is CH2, C=O or SO2, in particular Y is C=O;

[0404] -R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl and heterocycle, in particular R 1 is unsubstituted or substituted heteroaryl;

[0405] -R 2 Selected from F, Me, Cl, OH, NH2, Br, CF3, CHF2, CH2F;

[0406] - n is an integer selected from 0, 1, 2, 3 and 4, in particular n is an integer selected from 0, 1 and 2;

[0407] -R 3 is a substituted alkylamine;

[0408] -U and V are independently selected from -CH2- and -(CH2)2-, or one of U and V is -CH2- and the other is -(CH2)3-, in particular U and V are both -CH2- or both -(CH2)2-.

[0409] 2. The compound according to clause 1, wherein

[0410] -R 1 is unsubstituted or substituted with a moiety selected from the following:

[0411] ·Secondary amine NHR N , where R N is selected from C1-C6 alkyl, C4-C6 cycloalkyl, aryl, and heteroaryl, alkylaryl and alkylheteroaryl;

[0412] Halogen, in particular Cl or F; and

[0413] C1-C6 alkyl, C4-C6 cycloalkyl, aryl and heteroaryl.

[0414] 3. A compound according to any one of the preceding clauses, wherein

[0415] -R 3 Substituted with one or several moieties independently selected from alkyl, hydroxy, amino, amine, halogen, cycloalkyl and heterocyclic moieties.

[0416] 4. A compound according to any one of the preceding clauses, wherein R 3is a C1-C4 alkylamine, especially R 3 It is a C1-C2 alkylamine.

[0417] 5. A compound according to any one of the preceding clauses, wherein the compound has the general formula (II)

[0418]

[0419] in

[0420] -Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n have the same definitions as in clause 1; - each R 4 Independently selected from

[0421] secondary amines which are substituted by alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and / or heterocycle, in particular by alkyl, alkylaryl or cycloalkyl;

[0422] ·halogen;

[0423] And / or two R 4 together form an unsubstituted or substituted heteroaryl or heterocyclic ring;

[0424] - m is an integer selected from 0, 1, 2 and 3.

[0425] 6. A compound according to any one of the preceding clauses, wherein the compound has the general formula (III)

[0426]

[0427] in

[0428] -Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n have the same definitions as in clause 1; -R 5 is selected from the group consisting of alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and heterocycle,

[0429] Especially R 5 is selected from the group consisting of alkyl, alkylaryl and cycloalkyl;

[0430] -R 6 is selected from halogen and hydrogen.

[0431] 7. A compound according to any one of the preceding clauses 1 to 4, wherein the compound has the general formula (IV)

[0432]

[0433] in

[0434] -Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n have the same definitions as in clause 1; -R 6 is selected from halogen and hydrogen;

[0435] -W is selected from N and CH.

[0436] 8. A compound according to any one of the preceding clauses, wherein Z 1 and Z 2 At least one of them is CH or CR 2 , especially Z 1 and Z 2 All are CH or CR 2 .

[0437] 9. A compound according to any one of the preceding clauses, wherein R 3 for

[0438]

[0439] in

[0440] - s is an integer selected from 1 and 2, more particularly s is 1;

[0441] -R 31 and R 32 together form a heterocyclic or heterobicyclic ring which is unsubstituted or substituted by alkyl, halogen and / or hydroxy, or

[0442] R 31 and R 32 independently selected from hydrogen and unsubstituted or hydroxy and / or halogen-substituted alkyl or cycloalkyl,

[0443] Especially R 31 and R 32 Together they form a heterocyclic or heterobicyclic ring which is unsubstituted or substituted by alkyl, halogen and / or hydroxy.

[0444] 10. The compound according to clause 9, wherein NR 31 R 32 Selected from

[0445]

[0446] wherein v is an integer selected from 0, 1 and 2, and each R N are independently selected from hydroxy, halogen and C1-C4 alkyl, or two R NThis forms a C3-C6 cycloalkyl group.

[0447] 11. A compound according to any one of the preceding clauses 9 to 10, wherein NR 31 R 32 Selected from

[0448]

[0449] 12. A compound according to any one of the preceding clauses, wherein R 2 is selected from F, Cl and OH, in particular R 2 For F.

[0450] 13. A compound according to any of the preceding clauses for use as a medicament.

[0451] 14. A compound according to any one of the preceding clauses 1 to 12 for use in the treatment of cancer.

[0452] The present disclosure is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are intended to illustrate the present disclosure but not to limit its scope.

[0453] Example

[0454] All reagents were purchased from commercial suppliers and used as received. Reactions performed at elevated temperatures were performed in an oil bath. All reactions were analyzed by thin layer chromatography (coated with silica gel 60F 254 Flash column chromatography was performed on silica gel (0.040-0.063 mm). The spectra were recorded on AV2-400 MHz and AV600 Bruker spectrometers (400 MHz, 101 MHz and 600 MHz, 150 MHz, respectively) in DMSO or CDCl3. 1 H and 13 C{ 1 H} NMR spectra, chemical shifts are given in ppm and residual deuterated solvents 1 H and 13The multiplicities are abbreviated as follows: singlet (s), doublet (d), multiplet (m), and broad signal (bs). Purity was obtained by liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS): Acquity UPLC (Waters, Milford, USA) connected to an Acquity eλ diode array detector and a Synapt G2 HR-ESI-QTOF-MS (Waters, Milford, USA); 1 μL of sample was injected (c = about 10-100 μg / mL in the indicated solvent); Acquity BEH C18 HPLC column (1.7 μm particle size, 2 × 50 mm, Waters) was maintained at 30 °C; *eluted at a flow rate of 400 μL / min with A: H2O + 1% HCO2H and B: CH3CN + 0.1% HCO2H, linear gradient 5–98% B in 5 min, then isocratic elution for 1 min; *at 1.2 nm resolution and 20 points s -1 UV spectra were recorded from 200–600 nm; ESI: positive ionization mode, capillary voltage 3.0 kV, sampling cone 40 V, extraction cone 4 V, N2 cone gas 4 L / h, N2 desolvation gas 800 L / min, source temperature 120 °C; mass analyzer in resolution mode: mass range 100-2000 m / z, scan rate 1 Hz; the mass was calibrated to <2 ppm in the range 50–2'500 m / z using 5 mM HCO2Na in water, locked masses: m / z 195.0882 (caffein, 0.7 ng / mL) and 556.2771 (Leucine-enkephalin, 2 ng / mL).

[0455] General procedure for Buchwald-Hartwig coupling:

[0456] Under nitrogen atmosphere, to a stirred solution of the corresponding halide (1 eq.) in dioxane (0.3 M) was added the corresponding amine (1 eq.). Nitrogen was bubbled through the reaction for two minutes, and CsCO (1.2 eq.), 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 residue obtained was purified by flash column chromatography.

[0457] General procedure for deprotection of Boc group:

[0458] To a stirred solution of the corresponding Boc protected amine in MeOH (0.3 M) was added HCl (0.9 M, 37% in water). The reaction mixture was stirred at 25 °C for 4 h, and the reaction mixture was concentrated under reduced pressure. The obtained residue was used directly in the next step without further purification.

[0459] Using 4,6-dichloropyrimidine N General procedure for Ar:

[0460] 4,6-dichloro-pyrimidine (1.2 eq) and Et3N (1-4 eq) were added to a stirred solution of the corresponding amine (1 eq) or the hydrochloride of the amine (1 eq) in iPrOH (0.3 M). The reaction mixture was stirred in a microwave at 80 °C for 3 h and concentrated under reduced pressure. The crude residue was dissolved in nBuOH, washed three times with water, once with brine, dried over MgSO4 and concentrated under reduced pressure. The crude residue was co-evaporated with toluene several times to remove residual nBuOH and then purified by flash column chromatography.

[0461] Using chloropyrimidine derivatives S N General procedure for Ar:

[0462] The corresponding chloropyrimidine (1 equivalent) was dissolved in methylamine (0.1M, 8M in EtOH) or benzylamine (0.3M), and the reaction mixture was stirred in a microwave at 130°C for 3h (MeNH2) or at 140°C for 8h (BnNH2). The crude residue was concentrated under reduced pressure and purified by flash column chromatography. For the reaction with benzylamine, the crude residue was co-evaporated several times with water and then toluene to remove benzylamine before purification.

[0463] Embodiment 1:

[0464] The inventors' design started from one of their earlier inhibitors (1, Table 4), with the goal of simplifying the structure and reducing the molecular weight ( Figure 1 A). To this end, the position of the methylene group on the piperidine ring was changed from 1,3 to 1,4, removing the chiral center. In addition, according to the X-ray structure of 1 and METTL3, the deletion of the amide C=O group would allow the original vector to be retained ( Figure 1 B). These two modifications resulted in 2 and its two pyridine-containing derivatives 3 and 4, which not only exhibited similar potency (IC 50 =5.0, 4.6 and 5.8 μM, Table 4), and no chirality and reduced heavy atom counts, resulting in higher ligand efficiencies (LE = 0.23, 0.23 and 0.22, respectively). Because 4 had a better lipophilic ligand efficiency (LLE = 3.4, calculated with DataWarrior), its pyridine core was retained in the next optimization stage. Based on the crystal structure of the complex of METTL3 with inhibitor 1, the substitution of methylamine to benzylamine on the pyrimidine ring seemed to favor inhibition. This was proven to be correct, as the corresponding derivative 5 showed a 6-fold increase in potency (IC 50 =0.79 μM).

[0465] A notable feature of this inhibitor series is its linear shape and sp 3 The coupling of acyclic atom linkers makes it highly flexible. Rigidizing the structure is a feasible way to keep the ligand in its preferred conformation, which in turn can enhance the binding energy by reducing entropic penalties. Therefore, the inventors envisioned two different strategies to achieve this goal: establishing an amide connection between the piperidine and pyridine rings; or, depending on the conformation of compound 5, forming a spirocycle by connecting a tertiary alcohol to an aniline ( Figure 2 A). The two approaches led to opposite results, with amide derivative 6 losing its previous potency improvement (IC 50 =3.6 μM, Table 4), while spirocyclic ring 7 inhibited (IC 50 =0.28 μM, Table 5) and novelty are promising. The inventors managed to soak METTL3 with 5 and 7, and X-ray analysis showed strong structural overlap. The pyrimidine moiety forms two hydrogen bonds with the NH backbone of Asn549 and Ile378, while participating in π stacking with Phe534 and π interaction with the side chain of Asn549 ( Figure 2 A). The benzylamine group interacts with the Asp377 side chain and also forms a cation-π interaction with Arg379. At the opposite site of the binding pocket, the geminal dimethyl group fills the lipophilic pocket formed by Lys513, Pro514, Trp457 and Trp431 residues, while the charged piperidine forms a salt bridge with Asp395. The only difference between 5 and 7 is that for the latter, there is a lack of hydrogen bonding between the tertiary alcohol and the Gln550 side chain due to the conversion of the alcohol to an ether ( Figure 2 B). The inventors hypothesize that replacing the ether with a lactam could restore this interaction and even form an additional hydrogen bond due to the C=O group of the ligand and the NH2 amide of Gln550. The inventors obtained a strong potency improvement (IC 50 =0.037 μM), and the inventors' hypothesis was confirmed by two hydrogen bond interactions found in the crystal structure ( Figure 2 C). In addition, both LE and LLE were significantly improved (0.25 and 4.4, respectively, Table 5).

[0466] ADME properties, such as solubility, cell permeability, and metabolic stability, are critical for chemical probes, so they were considered early in the project. The inventors' newly synthesized inhibitors (5, 7-8) showed mixed results; however, they all exhibited moderate stability to enzymatic degradation after incubation with rat liver microsomes, with half-lives less than 12 minutes (Tables 4, 5). Therefore, the inventors focused on improving ADME properties while achieving good biochemical potency. The initial approach was to replace the pyridine nitrogen atom with a carbon atom to give 9, which has moderate permeability (9·10-6 cm·s -1 ) and surprisingly the solubility increased slightly (Table 5). However, the metabolic stability remained unchanged, so benzylamine was replaced by methylamine (10). In fact, the solubility and metabolic stability were significantly improved (108 μM and 107 min, respectively), and the LE and LLE values ​​were significantly improved (0.28 and 4.5, respectively), but at the expense of limited permeability (2·10 -6 cm·s -1 ) and the efficacy was reduced to 1 / 3.4. Starting from 10, two other possibilities of reducing the size of the inventor's molecule were explored: replacing spiropiperidine with spiroazetidine (11), and replacing spirolactam with spirourea (12). Unfortunately, the efficacy of both was significantly lost (to 1 / 5 and 1 / 20, respectively). However, the spiroazetidine part is still a potential alternative to help reduce molecular weight and improve the physicochemical properties in the later stage. Next, the direction of the inventor's strategy is to improve permeability. Lactam methylation in compound 13 caused a serious decrease in efficacy (down to 1 / 19), demonstrating the key role of lactam hydrogen bond interactions.

[0467] After a thorough optimization of the helical scaffold, the inventors turned their attention to the pyrimidine motif. Compared with 10, the addition of a second methyl group to the aniline (14) was highly detrimental to binding (0.97 and 0.089 μM, respectively, Table 6), probably due to the loss of hydrogen bonding to the Asp377 side chain, while substitution of the methyl group to an isopropyl group (15) showed a less pronounced reduction because the hydrogen bonding was retained (0.33 μM). These two modifications suggest that this position is available for branched sp 3 Carbon space is limited. Surprisingly, substitution with a cyclopropyl group (16) was not only well tolerated (0.084 μM), but also improved the three ADME properties (Table 6) and may be a promising alternative for lead optimization. S-adenosylmethionine (SAM) is the natural ligand of METTL3, which contains an adenosine scaffold that overlaps with the pyrimidine group of the inventors' inhibitor, so the inventors considered testing some bicyclic heteroaromatic modifications. Pyrrolopyrimidine 17 had a slight increase in potency compared to 10, but similarly had lower permeability and a larger efflux ratio in the Caco-2 assay (Table 6). N 3The interaction geometry between the pyrimidine atom and the Asn549 nitrogen backbone did not seem optimal, so the inventors considered removing this pyrimidine nitrogen atom to improve permeability and possibly suppress part of the desolvation penalty. The latter proved to be wrong, as pyrrolopyridine 18 showed a severe loss of binding (loss to 1 / 74). Incorporation of a chlorine atom between the two pyrimidine nitrogen atoms (19) was beneficial for potency (0.024 μM); however, solubility and metabolic stability were severely impaired (45 μM and 32 min, respectively), which prompted the inventors to look for different modifications.

[0468] Because the spiro-scaffold and pyrimidine moieties had already been optimized, the inventors considered the benzene ring as the next target area. Several publications have discussed the unique properties of fluorine atoms, which could translate into unexpected and promising results in drug design. Indeed, fluorine atoms are capable of producing unusual interactions, and aromatic fluorine atoms tend to increase permeability. Fluorine scanning of the benzene ring gave two new derivatives, 20 and 21. Both compounds showed similar improvements in binding compared to inhibitor 10 (0.038 and 0.032 μM, respectively); however, only 20 showed a significant increase in permeability (Table 7). The X-ray structures of each molecule in complex with METTL3 were solved and demonstrated that the fluorine in 21 exhibits hydrophobic contacts ( Figure 3 B), and the fluorine atom of 20 also has an unusual interaction with the nitrogen π system of Pro397 ( Figure 3 A) Inhibitor 20 is preferred because of its strong improvement in permeability and small efflux ratio (9.10 -6 cm·s -1 and 2), but the combination of two fluorine atoms quickly emerged as a key solution to achieve excellent potency while maintaining adequate ADME properties. Indeed, compound 22 exhibited single-digit nanomolar IC in the TR-FRET assay. 50 (0.008 μM) (Table 7 and Figure 4 A), high cell permeability (12·10 - 6 cm·s -1 ) and favorable LE and LLE values ​​(0.3 and 5.3, respectively), as well as acceptable metabolic stability (t 1 / 2=24min). To investigate the selectivity of compound 22 for other RNA methyltransferases, the inventors performed a protein thermal shift assay. The inventors expressed and purified the METTL1 protein, which is a writer molecule for the 7-methylguanosine marker on tRNA, mRNA, and miRNA and serves as a representative closely related protein. The inventors used S-adenosyl-L-homocysteine ​​(SAH) as a positive control, which is a byproduct and natural binder of RNA methyltransferase catalytic activity, and showed a ΔT of 2.8°C and 3.5°C for METTL3 / METTL14 and METTL1, respectively, at 100 μM. m ( Figure 5 and 6 Compared with the DMSO control, 100 μM of compound 22 can shift the melting temperature of METTL3 / METTL14 by 4.7°C ( Figure 5 and 6 ). In contrast, no shift was observed for METTL1 at up to 100 μM of compound 22, indicating no binding.

[0469] Compound 22 enhanced the thermal stability of METTL3, allowing the inventors to study its cellular target engagement in two orthogonal assays based on protein thermal denaturation. Binding of 22 was assessed in the InCELL Pulse assay, where the enhanced Fusions of the (ePL) enzyme fragment with the N-terminus of the truncated METTL3 (residues 354-580) were expressed in HEK293T cells. After incubation of these cells with inhibitor 22 at 37°C for 1 h, the cells were heated at 46°C for 3 min, and non-aggregated METTL3-ePL protein was quantified using a luminescence-based assay ( Figure 4 B). Compound 22 stabilizes the METTL3-ePL fusion protein and shows an EC 50 The concentration of 22 in the polyadenylated RNA was 2 μM in two different cancer cell lines, MOLM-13 (AML) and PC-3 (prostate cancer), after 16 hours of compound treatment. 6 The inventors found that 22 was able to reduce this ratio to 10-20% of the DMSO-treated control samples and had a certain degree of selectivity between the two cell lines (EC 50 = 0.7 and 2.5 μM, Figure 4 D).

[0470] The inventors have successfully improved the potency (up to 1000-fold), efficiency parameters, and ADME properties of a series of METTL3 inhibitors through protein crystallography-guided medicinal chemistry. The key feature is the rigidity achieved due to the design of the helical scaffold and the use of fluorine atoms at specific positions. The most potent inhibitor (compound 22) showed an IC of 2.1 in the TR-FRET assay. 50 The binding to the off-target METTL1 was not observed at concentrations up to 100 μM. Cellular target engagement of compound 22 was demonstrated using two different assays. In addition, for m 6 The reduction in A / A, as quantified by UPLC-MS / MS analysis, measured EC in MOLM-13 (leukemia) and PC-3 (prostate cancer) cell lines. 50 The values ​​were 0.7 μM and 2.5 μM. Therefore, compound 22 is a chemical probe to decipher the functional role of METTL3 / METTL14 and its relationship with hematological malignancies and solid tumors.

[0471]

[0472] Table 4: Early modifications to the initial scaffold.

[0473]

[0474] 1 : Time-resolved Forster resonance energy transfer (TR-FRET) assay (μM). 2 : g / mol. 3 : Ligand efficiency (kcal.mol -1 .Heavy atom count -1 ). 4 : Lipophilic ligand efficiency (pIC 50 -logP); 5 : μM; 6 :10 -6 cm·s -1 , (exclusion ratio), Caco-2 assay; 7 :Rat liver microsomes, t 1 / 2 ( min).

[0475]

[0476] Table 5: Derivation from screw scaffolds.

[0477]

[0478]

[0479] Table 6: Optimization of the aminopyrimidine ring.

[0480]

[0481]

[0482] Table 7: Fluorine scan on benzene ring.

[0483]

[0484]

[0485] Scheme 1: Synthesis of spirocyclic intermediate (33). Reagents and conditions: (a) MeNO2, NH3, MeOH, 25°C, 17h; (b) (i) CbzCl, NaHCO3, DCM / H2O, 0-25°C, 17h; (ii) NiCl2.6H2O, NaBH4, MeOH, N2, 0-25°C, 1h, 32% over three steps; (c) ethyl 2-bromoacetate, Et3N, DCM, 25°C, 2h; (d) Pd / C, NH 4+ HCOO - , iPrOH, 80°C, 4h, 55% over two steps.

[0486]

[0487] Scheme 2: Synthetic route to compounds 7-10 and 15-17, 19. Reagents and conditions: (a) 23 or 26, tert-butyl 1-oxa-4,9-diazaspiro[5.5]undecane-9-carboxylate (for 7) or 33, Pd Ruphos G4, Ruphos, Cs2CO3, dioxane, N2, 150°C, 17h, 93% (35); (b) (i) HCl (37% in water), MeOH, 25°C, 4h; (ii) for 7: N-benzyl-6-chloropyrimidin-4-amine (29), Et3N, iPrOH, 150°C, 8h, 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, 4h, 36%. For 19: 36, 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine, Et3N, iPrOH, 100-130°C, 6h, 42%; (c) HCl (37% in water), MeOH, 25°C, 4h, 36% over two steps from 23; (d) 4,6-dichloropyrimidine, Et3N, iPrOH, 80°C, 3h, MW, 27% / 63% over three steps from 26(34) (37); (e) For 8 and 9: BnNH2, 140°C, 8h, MW, 25% (8) / 5% over two steps from 36(9). For 10: MeNH2, EtOH, 130°C, 3h, MW, 3% over two steps from 36. For 15: iPrNH2, EtOH, 130°C, 8h, MW, 52%. For 16: cyclopropylamine, iPrOH, 130°C, 6h, MW, 25%.

[0488]

[0489] Scheme 3: Synthesis route of intermediate 40. Reagents and conditions: (a) MeNO2, K2CO3, EtOH, 25°C, 17h; (b) DAST, DCM, N2, -78°C, 3h; (c) NH3, MeOH, 25°C, 2h, quantitative over three steps; (d) (i) CbzCl, NaHCO3, DCM / H2O, 0-25°C, 17h; (ii) NiCl2.6H2O, NaBH4, MeOH, N2, 0-25°C, 1h, 54% over two steps; (e) ethyl 2-bromoacetate, Et3N, DCM, 25°C, 2h; (f) Pd / C, NH4 + . HCOO - , iPrOH, 80°C, 4h, 32% after two steps.

[0490]

[0491] Scheme 4: Synthesis route of compound 11. Reagents and conditions: (a) 40, Pd Ruphos G4, Ruphos, Cs2CO3, dioxane, N2, 150°C, 17h; (b) (i) HCl (37% in water), MeOH, 25°C, 4h; (ii) 4,6-dichloro-pyrimidine, Et3N, iPrOH, 80°C, 7h, MW; (c) MeNH2, EtOH, 130°C, 3h, MW, 19% over four steps.

[0492]

[0493] 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-1-(bromomethyl)-2-fluorobenzene (49), 4,4-dimethylpiperidine hydrochloride, K2CO3, DMF, 25°C, 17h, 98% (47) / 99% (49). For 52: (i) 4-bromo-2,5-difluorobenzoic acid, BH3·SMe2, THF, N2, 25°C, 17h, 83%; (ii) SOCl2, DMF, DCM, 25°C, 3h; (iii) 4,4-dimethylpiperidine hydrochloride, K2CO3, DMF, 25°C, 17h, 92%; (b) 33, Pd Ruphos G4, Ruphos, Cs2CO3, dioxane, N2, 150°C, 17h, 83% (50); (c) (i) HCl (37% in water), MeOH, 25°C, 4h; (ii) 4,6-dichloropyrimidine, Et3N, iPrOH, 80°C, 3h, MW, 15% over three steps from 47 (48) / 58% over two steps from 50 (51); (d) MeNH2, EtOH, 130°C, 3h, MW, 47% (20) / 69% (21) / 56% over four steps from 52 (22).

[0494] Scheme 6: Preparation of 9-(6-((3-aminopropyl)amino)pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one

[0495]

[0496] The chloropyrimidine derivative 6-1 (100 mg, 0.21 mmol) was dissolved in ethanol (0.5 M), and N-Boc-1,3-propanediamine 6-2 (108 mg, 0.62 mmol) and TEA (86.6 μL, 0.62 mmol) were subsequently added to the stirred solution. 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 (SiO2; DCM / MeOH=9:1) to obtain a slightly yellow solid (117 mg, 91%). The impure S NThe product of Ar (117mg, 0.19mmol) was dissolved in MeOH (0.5M) and HCl 37% (57.7 μL, 1.9mmol) was added. The obtained reaction mixture was stirred at room temperature until completion (monitored by TLC). The reaction mixture was evaporated, absorbed with butanol, quenched with saturated NaCO aqueous solution and extracted with butanol (3x 10ml). The organic layer merged was dried over MgSO, filtered and evaporated to obtain product (80mg, 81%).

[0497] 1 H NMR (400MHz, MeOD) δ8.06(d,J=0.9Hz,1H),7.38(d,J=8.2Hz,2H),7.01(d,J=8.5Hz,2H),5. 77(d,J=1.0Hz,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=6.5Hz,2H),3.17(d,J=0.8Hz,1H),2.98(t,J=7.0Hz,6H),1.95–1.87(m,4H),1. 81(ddd,J=13.3,8.6,4.1Hz,2H),1.58(d,J=5.9Hz,4H),1.29(d,J=2.7Hz,3H),1.02(s,6H).

[0498] 13 C NMR (101MHz, DMSO) δ167.6,163.7,162.1,157.8,148.6,130.1,129.2,114.7,65 .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.

[0499] LRMS (ESI) m / z: [M+H]+ calculated for C29H44N8O 520.36, found 521.37.

[0500] Scheme 7: General procedure for amide coupling

[0501]

[0502] The carboxylic acid (1 eq) was dissolved in DMF (0.5 M), the solution was cooled to 0 °C and DIPEA (5 eq) was added. After 10 min, HATU (1.1 eq) was added and the solution was stirred for 30 min before the addition of amine 6-3 (1 eq). The resulting reaction mixture was stirred at room temperature until completion (monitored by TLC).

[0503] Scheme 8: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)acrylamide

[0504]

[0505] Compound 8-4 was prepared using amine 3 (159 mg, 0.306 mmol) and the corresponding pomalidomide carboxylic acid (132 mg, 0.306 mmol) according to the general amide coupling procedure 1. The crude product was first purified using flash column chromatography (Al2O3; DCM / MeOH=from 90:10 to 80:20) and then semi-preparative HPLC to obtain 8-4 (15 mg, 5%).

[0506] 1 H NMR (500MHz, DMSO) δ11.09(s,1H),8.25(s,1H),7.98(s,1H),7.83(t,J=5.6Hz,1H),7.59–7.56(m,1H),7.13–7.03(m,3H),6.89(d,J=8.4Hz,1H ),6.62–6.58(m,1H),5.61(s,1H),5.05(dd,J=12.7,5.4Hz,1H),3.80–3 .75(m,1H),3.65(s,1H),3.59(t,J=6.0Hz,2H),3.53–3.43(m,6H),3.19 -3.17(m,2H),3.08(q,J=6.6Hz,2H),2.87(ddd,J=16.7,13.7,5.4Hz,1H),2.64–2.50(m,2H),2.30 –2.28(m,3H),2.04–1.99(m,,1H),1.90(s,1H),1.74–1.57(m,4H),1.29–1,23(m,3H),0.87(s,3H).

[0507] 13C NMR (126MHz, DMSO) δ172.8,172.1,170.1,170.1,169.0,167.3,167.1,16 3.3,161.7,157.4,148.2,146.4,136.3,132.1,129.7,128.6,117.5,114. 3,110.7,109.3,69.7,69.6,68.9,66.9,61.9,52.8,52.4,51.4,49.2,48. 6,41.7,38.3,38.0,36.3,36.2,34.3,31.0,29.1,28.3,22.2,22.1,21.1. LRMS (ESI) m / z: [M+H]+ calculated for C49H65N11O8 935.50, found 936.51.

[0508] Scheme 9: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)acrylamide

[0509]

[0510] Compound 9-5 was prepared using amine 3 (69 mg, 0.13 mmol) and the corresponding pomalidomide carboxylic acid (64 mg, 0.13 mmol) according to the general amide coupling procedure 1. The crude product was first purified by flash column chromatography (Al2O3; DCM / MeOH=from 90:10 to 80:20) and then by semi-preparative HPLC to obtain 9-5 (28 mg, 22%).

[0511] 1H NMR (500MHz, DMSO) δ11.09(s,1H),8.25(s,1H),7.98(s,1H),7.84(t,J=5.6Hz,1H),7.57(t,J=7.8Hz,1H),7. 14–7.03(m,3H),6.89(d,J=8.2Hz,2H),6.60(m,2H),5.61(s,1H),5.05(dd,J=12.8,5.4Hz,1H),3.79–3.77(m, 2H),3.65(s,1H),3.60–3.45(m,9H),3.19–3.17(m,2H),3.08(q,J=6.6Hz,2H),2.91–2.84(m,1H),2.63–2.56( m,2H),2.30–2.26(m,3H),2.04–2.00(m,1H),1.90(s,1H),1.74–1.57(m,4H),1.29–1.23(m,3H),0.87(s,3H).

[0512] 13 C NMR (126MHz, DMSO) δ172.8,170.1,170.1,169.0,167.3,167.1,163.3,161.7,157.4,148.1,146.4,136.3,132.1,129.7,128.7,117.5,114.3 ,110.7,109.3,69.8,69.7,69.6,68.9,66.9,62.0,52.8,52.4,51.4,4 9.3,48.6,41.7,38.4,36.3,36.2,34.3,31.0,29.0,28.3,22.2,21.2.

[0513] LRMS (ESI) m / z: [M+H]+ calculated for C51H69N11O9 979.53, found 980.54.

[0514] Scheme 10: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecan-15-amide

[0515]

[0516] Compound 10-6 was prepared using amine 3 (50 mg, 0.096 mmol) and the corresponding pomalidomide carboxylic acid (50 mg, 0.096 mmol) according to the general amide coupling procedure 1. The crude product was first purified by flash column chromatography (Al2O3; DCM / MeOH=from 90:10 to 80:20) and then by semi-preparative HPLC to obtain 10-6 (20 mg, 20%).

[0517] 1 H NMR (500MHz, DMSO) δ11.09(s,1H),8.25(s,1H),7.98(s,1H),7.84(t,J=5.7Hz,1H),7.60–7.55(m,1H),7.16–7.04(m,3H ),6.89(d,J=8.4Hz,2H),6.62–6,58(m,2H),5.61(s,1H),5.05(dd,J=12.8,5.5Hz,1H),3.79–3.76(m,2H),3.65(s,1H), 3.62–3.51(m,5H),3.48–3.45(m,8H),3.19–3.16(m,2H),3.08(q,J=6.5Hz,2H),2.87(ddd,J=16.8,13.8,5.4Hz,1H),2. 63–2.56(m,2H),2.31–2.28(m,4H),2.04–1.99(m,1H),1.90(s,1H),1.75-1.57(m,6H),1.30–1.23(m,4H),0.87(s,4H).

[0518] 13 C NMR (126MHz, DMSO) δ172.8,172.1,170.1,170.1,169.0,167.3,167.1,163.3,161. 7,157.4,149.8,148.2,146.4,136.3,134.7,132.1,132.0,129.8,123.4,122.1,1 17.5,114.3,110.7,109.3,69.8,69.8,69.8,69.7,69.5,68.9,66.9,61.9,52.8,52.4,51.4,49.2,48.6,41.7,38.3,38.0,36.3,36.2,34.3,31.0,29.1,28.3,22.2.

[0519] LRMS (ESI) m / z: [M+H]+ calculated for C53H73N11O10 1023.55, found 1024.56.

[0520] Scheme 11: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecane-18-amide

[0521]

[0522] Compound 11-7 was prepared using amine 3 (51 mg, 0.098 mmol) and the corresponding pomalidomide carboxylic acid (56 mg, 0.096 mmol) according to the general amide coupling procedure 1. The crude product was first purified by flash column chromatography (Al2O3; DCM / MeOH = from 90:10 to 80:20) and then by semi-preparative HPLC to obtain 11-7 (13 mg, 12%).

[0523] 1 H NMR (500MHz, DMSO) δ11.09(s,1H),8.25(s,1H),7.98(s,1H),7.84(t,J=5.7Hz,1H),7.61–7.54(m,1H),7.16–7.02(m,3H),6.89(d,J=8.3Hz,2 H),6.60(m,2H),5.61(s,1H),5.05(dd,J=12.7,5.4Hz,1H),3.80–3.75 (m,2H),3.65(s,2H),3.61–3.52(m,5H),3.48–3.45(m,10H),3.18–3.17 8m.2H9,3.08(q,J=6.6Hz,2H),2.87(ddd,J=16.9,13.7,5.4Hz,1H),2.63–2.53(m,2H),2.30–2.26(m, 4H),2.04–1.99(m,1H),1.90(s,1H),1.75–1.57(m,5H),1.28–1,23(m,4H),1.23(s,1H),0.87(s,4H).

[0524] 13C NMR (126MHz, DMSO) δ173.3,172.6,170.6,170.5,169.4,167.8,167.6,163.7,162.1 ,157.8,148.6,146.9,136.7,134.9,132.6,132.0,130.2,129.2,123.5,117.9,114 .7,111.2,109.7,70.3,70.2,70.2,70.1,70.00,69.4,67.3,62.4,53.3,52.9,51.9,49.7,49.1,49.0,42.2,38.8,38.4,36.8,36.7,34.8,31.5,29.5,28.7,22.6,21.6.

[0525] LRMS (ESI) m / z: [M+H]+ calculated for C55H77N11O11 1067.58, found 1068.59.

[0526] Scheme 12: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(prop-2-yn-1-ylamino)isoindoline-1,3-dione

[0527]

[0528] 4-Fluoro-pomalidomide derivative 12-8 (320 mg, 1.16 mmol) was dissolved in DMSO (0.5 M), and propargylamine (64 mg, 1.16 mmol) and DIPEA (605 μL, 3.48 mmol) were subsequently added to the solution. The resulting reaction mixture was stirred at 130 ° C until completion (monitored by TLC). The reaction mixture was quenched by adding saturated NaHCO3 aqueous solution and extracted into EtOAc (3 x 15 ml). The combined organic layer was dried over MgSO4, filtered and evaporated. The product 12-9 was obtained as a yellow solid (280 mg, 78%).

[0529] 1 H NMR (400MHz, CDCl3) δ8.00(s,1H),7.57(dd,J=8.5,7.1Hz,1H),7.20(d,J=7.1Hz,1H),7.03(d,J=8.6Hz,1H),6.44(t,J=6.5Hz ,1H),4.92(dd,J=12.2,5.4Hz,1H),4.09(dd,J=6.1,2.5Hz,2H),2.95–2.67(m,3H),2.27(t,J=2.4Hz,1H),2.20–2.07(m,1H).

[0530] 13 C NMR (101MHz, CDCl3) δ170.81,169.24,168.10,167.44,145.56,136.16,13 2.43,117.17,112.77,111.42,79.11,72.20,48.96,32.33,31.41,22.76.

[0531] LRMS (ESI) m / z: [M+H]+ calculated for C16H13N3O4 311.09, found 312.09.

[0532] Scheme 13: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-11-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)undecaneamide

[0533]

[0534] Compound 12-9 (100 mg, 0.32 mmol) is dissolved in THF (0.5 M), and then methyl azido (77 mg, 0.32 mmol), ketone sulfate (II) (26 mg, 0.16 mmol) and sodium ascorbate (70 mg, 0.35 mmol) are added to the solution. The resulting reaction mixture is stirred at 40 ° C until completion (monitored by TLC), then evaporated and the crude product is purified by flash column chromatography (SiO2; EtOAc / heptane = 1: 1) and a bright yellow solid (90 mg, 50%) is obtained. The resulting methyl ester (90 mg, 0.16 mmol) is dissolved in THF (0.5 M) and HCl 37% (49 μL, 1.6 mmol) is added. The resulting reaction mixture is stirred at room temperature until completion (monitored by TLC), then evaporated and the product 13-10 is used for the next reaction without any further purification. Carboxylic acid 13-10 (83 mg, 0.15 mmol) was dissolved in DMF (0.5 M), the solution was cooled to 0 ° C and DIPEA (134 μL, 0.768 mmol) was added. After 10 minutes, HATU (65 mg, 0.169 mmol) was added and the solution was stirred for 30 minutes, and then amine 6-3 (80 mg, 0.15 mmol) was added. The resulting reaction mixture was stirred at room temperature until completion (monitored by TLC). The reaction mixture was evaporated and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH=from 90:10 to 80:20) and 13-11 (7 mg, 5%) was obtained.

[0535] 1H NMR(600MHz,MeOD)δ8.06(t,J=5.6Hz,1H),8.00(s,1H),7.92(s,1H),7.52(t,J=8.5,7.1,4.4Hz,1H),7.29(d,2H),7.10–7.06(m,2H),7.00(t,J=6.1Hz,1H),6.97(d,2H),5.68(s,1H),5.11–5.02(dd,1H),4.65(d,J=6.1Hz,2H),4.36(t,J=7.0Hz,2H),3.83(s,2H),3.72(m,2H),3.55(m,J=14.3,4.0Hz,2H),3.50(m,J=4.3Hz,2H),3.26(q,J=6.6Hz,4H),2.87–2.81(m,2H),2.75(dt,J=5.1,2.6Hz,1H),2.72(t,J=1.1Hz,1H),2.71–2.66(m,2H),2.18(t,J=8.7,6.0Hz,3H),2.13–2.07(m,1H),2.07–2.02(m,1H),1.94–1.88(m,2H),1.88–1.80(m,2H),1.78(m,J=6.7Hz,2H),1.60(q,J=7.1,6.3Hz,4H),1.48(,J=5.7Hz,4H),1.32–1.29(m,16H),0.97(d,J=1.6Hz,6H)。

[0536] 13C NMR(151MHz,MeOD)δ176.55,174.75,171.72,170.93,170.55,169.20,164. 62,163.44,163.31,163.08,162.85,158.30,150.59,147.53,147.48,146.6 2,137.15,137.00,133.96,132.70,128.76,124.10,118.38,118.20,115.82,112.52,112.43,112.28,112.05,57.91,57.76,57.62,57.48,57.33,54.4 2,53.33,52.40,51.39,50.65,50.24,41.63,39.71,39.02,38.32,38.00,37.20,33.11,32.25,31.75,31.71,31.18,30.80,30.77,30.51,30.34,30.32 , 30.30, 30.27, 30.23, 30.18, 30.15, 29.95, 29.91, 29.14, 27.33, 27.02, 23.81, 18.58, 17.62, 17.50, 17.37, 17.24, 17.12, 14.71, 14.58, 14.33, 14.20. LRMS (ESI) m / z: [M+H]+ calculated for C56H77N14O6 1040.61, found 1040.61.

[0537] Scheme 13: General Scheme 2 for Amide Coupling

[0538]

[0539] The carboxylic acid (1 eq) was dissolved in DMF (0.5 M), the solution was cooled to 0 °C and DIPEA (5 eq) was added. After 10 min, COMU (1.1 eq) was added and the solution was stirred for 30 min before the addition of amine 6-3 (1 eq). The resulting reaction mixture was stirred at room temperature until completion (monitored by TLC).

[0540] Scheme 14: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanamide

[0541]

[0542] Compound 14-12 was prepared using amine 3 (63 mg, 0.12 mmol) and the corresponding carboxylic acid (50 mg, 0.12 mmol) according to the general amide coupling procedure 2. The reaction mixture was evaporated, and the crude product was purified using two consecutive preparative thin layer chromatography (SiO2; DCM / MeOH=84:16) to obtain 14-12 (6 mg, 5%).

[0543] 1 H NMR (400MHz, DMSO) δ11.00(s,1H),8.27(s,1H),7.98(s,1H),7.78(t,J=5.6H z,1H),7.27(t,J=7.7Hz,1H),7.14(d,J=8.2Hz,2H),6.91(dd,J=7.9,3.9Hz,3 H),6.72(d,J=8.0Hz,1H),6.64(t,J=5.8Hz,1H),5.61(s,1H),5.54(t,J=5.5H z,1H),5.10(dd,J=13.2,5.1Hz,1H),4.22(d,J=17.3Hz,1H),4.12(d,J=17.2H z,1H),3.77(m,2H),3.66(s,2H),3.17(m,J=5.3Hz,2H),3.08(p,J=6.6Hz,4H) ,2.92(ddd,J=18.0,13.4,5.6Hz,2H),2.69–2.57(m,2H),2.36–2.23(m,2H),2 .04(t,J=7.4Hz,2H),1.72(m,J=13.4Hz,2H),1.58(td,J=13.2,12.8,6.2Hz,6 H), 1.47 (t, J = 7.1Hz, 2H), 1.31 (m, 2H), 1.25 (d, J = 11.0Hz, 12H), 0.88 (s, 6H).

[0544] 13C NMR(151MHz,MeOD)δ176.53,174.82,172.54,172.46,170.82,164.62,163.41,163.31,163.08,158.30,150.81,145.26,13 2.93,132.90,130.63,128.03,115.77,113.78,111.81,62.43,57.76,57.62,57.48,57.33,54.40,54.28,53.58,52.27,50. 27,47.38,44.52,41.61,39.83,39.69,39.55,39.41,39.27,37.98,37.94,37.21,35.59,33.10,32.45,31.45,30.82,30.79,30.76,30.66,30.51,30.38,30.33,30.20,30.13,29.07,28.12,27.04,27.01,24.29,23.77,17.62,17.37,14.58,14.47.

[0545] LRMS (ESI) m / z: [M+H]+ calculated for C51H72N11O5 917.56, found 918.57.

[0546] Scheme 15: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecaneamide

[0547]

[0548] Compound 15-13 was prepared using amine 3 (47 mg, 0.090 mmol) and the corresponding lenalidomide carboxylic acid (40 mg, 0.090 mmol) according to the general amide coupling procedure 2. The reaction mixture was evaporated, and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 90:10 to 80:20) to obtain 15-13 (36 mg, 42%). 1HNMR(500MHz,DMSO)δ11.00(s,1H),8.32(s,1H),7.98(s,1H),7.84(t,J=5.7Hz,1H),7.43–7.34(m,2H),7.27(t,J=7.7Hz,1H),6.97(d,J=8.3Hz,2H),6.91(d,J=7.4Hz,1H),6.72(d,J=8.1Hz,1H),6.67(m,J=6.1Hz,1H),5.62(s,1H),5.57(t,J=5.5Hz,1H),5.10(dd,J=13.3,5.1Hz,1H),4.23(d,J=17.2Hz,1H),4.12(d,J=17.2Hz,1H),3.81–3.74(m,2H),3.72(s,2H),3.59(m,J=11.8,5.1Hz,2H),3.51(m,J=3.0Hz,2H),3.45(s,2H),3.17(m,J=8.8,5.4Hz,2H),3.08(dq,J=13.0,6.5Hz,4H),2.91(ddp,J=15.1,9.8,5.2,4.7Hz,2H),2.61(dt,J=17.5,3.3Hz,2H),2.29(qd,J=13.2,4.7Hz,2H),2.03(t,J=7.0Hz,2H),1.70(m,J=10.5,4.2Hz,2H),1.59(tt,J=15.4,8.3Hz,6H),1.46(m,J=12.8,11.0,5.5Hz,4H),1.34(m,J=7.0Hz,2H),1.25(m,J=21.7,6.4Hz,14H),0.94(s,5H)。

[0549] 13 C NMR(126MHz,DMSO)δ172.95,172.16,171.27,168.94,166.91,163.23,161.64,158.00,157.75,157.32,143.80,132.04,129.23,126.46,118.54,116.15,113.76,111.71,109.88,53.29,52.42,51.88,50.73,48.60,47.66,45.80,42.75,41.55,38.01,36.23,35.47,34.18,31.25,29.04,28.94,28.93,28.82,28.69,28.55,27.77,26.67,25.34,22.83,22.10,18.61,18.01,16.74。

[0550] LRMS (ESI) m / z: [M+H]+ calculated for C53H76N11O5 945.60, found 946.60.

[0551]

[0552] 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% HCl, MeOH, 24 h; (c) HATU (1.1 eq.), DIPEA (5 eq.), DMF, rt, 5 h.

[0553]

[0554] Scheme 17: (d) (i) TEA (3 eq.), EtOH, reflux, 24 h; (ii) 38% HCl, MeOH, 24 h; (e) HATU (1.1 eq.), DIPEA (5 eq.), DMF, rt, 5 h.

[0555]

[0556] 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.

[0557]

[0558] 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, 8 h.

[0559]

[0560] 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, 8 h.

[0561]

[0562] Scheme 21: (l) Sodium ascorbate (1.1 eq.), CuSO4 (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, 8 h.

[0563]

[0564] Scheme 22: (n) DIPEA, (3 eq.), DMSO, 80°C, 24h; (o) TFA (10 eq.), DCM, rt, 8h b) HATU (1.1 eq.), DIPEA (5 eq.), DMF, rt, 8h.

[0565] Scheme 23: Preparation of 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-9-(6-fluoropyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecane-2-one (Compound 23-2)

[0566]

[0567] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-1,4,9-triazaspiro[5.5]undecan-2-one 23-1 (100 mg, 0.27 mmol) was dissolved in iPrOH (0.5 M). 4,6-difluoropyrimidine (27 μL, 0.32 mmol) and TEA (150 μL, 1.01 mmol) were then added to the stirred solution. The resulting reaction mixture was stirred at 80 ° C (oil bath temperature) until complete (monitored by TLC). The volatiles were removed in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH=9:1) to provide 112 mg of the desired product (89%). 1 H NMR (400MHz, DMSO) δ8.30(d,J=2.8Hz,1H),8.22(s,1H),7.15(dd,J=13.5,6.6Hz,1H),6.93(dd,J=11.5,7.3Hz,1H),6.58(s,1H),4.00(s, 2H),3.60(s,2H),3.58–3.47(m,2H),3.42(s,2H),3.28(s,2H),2.33(s,3H),1.83(m,2H),1.68(m,2H),1.30(t,J=5.3Hz,4H),0.87(s,6H). 13C NMR (101 MHz, DMSO) δ 172.5, 170.1, 167.1, 164.9, 164.8, 158.6, 158.4, 156.0, 152.0, 149.7, 107.1, 106.8, 85.9, 85.6, 55.4, 54.5, 53.3, 53.0, 49.5, 46.1, 38.7, 35.1, 28.6. LRMS (ESI) m / z: [M+H]+ calcd. for C26H34F3N6O 503.270, found 503.276.

[0568] Scheme 24: General Procedure 1

[0569]

[0570] To a solution of compound 23-2 (1 eq) in EtOH (0.5 M) in a pressure vial was then added amine (1.5 eq) and TEA (4 eq). The resulting reaction mixture was stirred at 120 °C (oil bath temperature) until completion (monitored by TLC).

[0571] Scheme 25: Preparation of tert-butyl (3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)carbamate (Compound 25-1)

[0572]

[0573] Compound 19 was prepared using compound 23-2 (300 mg, 0.6 mmol) and tert-butyl (3-aminopropyl)carbamate (156 mg, 0.9 mmol) according to General Procedure 1. The volatiles were removed in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH=90:10) to afford 280 mg of the desired product (71%). 1H NMR (400MHz, DMSO) δ8.18(s,1H),7.97(s,1H),7.17(s,1H),6.94(dd,J=11.5,7.3Hz,1 H),6.82(t,J=5.7Hz,1H),6.61(t,J=5.7Hz,1H),5.60(s,1H),3.86(d,J=13.5Hz,2H),3 .60(s,2H),3.27(s,2H),3.22–3.12(m,2H),2.96(q,J=6.6Hz,2H),2.37(s,4H),1.77(d t,J=14.2,4.2Hz,2H),1.70–1.52(m,5H),1.37(s,9H),1.34–1.28(m,4H),0.88(s,6H). 13 C NMR (101 MHz, DMSO) δ 166.5, 163.2, 161.6, 157.3, 155.7, 117.9, 106.5, 106.3, 77.5, 54.7, 52.8, 52.7, 49.0, 38.0, 37.7, 34.6, 29.5, 28.3, 28.1. LRMS (ESI) m / z: [M+H]+ calculated for C34H51F2N8O3 657.404, found 657.405.

[0574] Scheme 26: Preparation of tert-butyl (3-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)carbamate (Compound 26-1)

[0575]

[0576] Compound 26-1 was prepared using compound 23-2 (50 mg, 0.1 mmol) and 1-(N-Boc-aminomethyl)-3-(aminomethyl)benzene (35 mg, 0.15 mmol) according to General Procedure 1. The volatiles were removed in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH=90:10) to provide 51 mg of the desired product (71%). 1H NMR(400MHz, CDCl3)δ8.16(s,1H),7.30(t,J=7.5Hz,1H),7.23–7.18(m,3H),7.12(dd,J=1 2.8,6.6Hz,1H),6.60–6.56(m,2H),5.43(s,1H),5.20(s,1H),4.89(s,1H),4.53(d,J=5.8H z,2H),4.30(d,J=6.0Hz,2H),3.73–3.66(m,4H),3.54(m,2H),3.50(s,2H),3.27(s,2H),2. 41(s,3H),1.93(m,2H),1.77(m,2H),1.44(s,8H),1.40(t,J=7.4,6.5Hz,4H),0.90(s,6H). 13 C NMR (101 MHz, CDCl3) δ 167.7, 163.2, 162.5, 157.9, 155.9, 139.7, 138.6, 129.1, 126.5, 126.2, 126.1, 105.8, 105.5, 81.3, 56.4, 54.7, 53.5, 53.0, 49.7, 45.7, 44.5, 40.4, 38.5, 35.2, 29.7, 28.4, 28.4. LRMS (ESI) m / z: [M+H]+ calcd. for C39H53F2N8O3 719.420, found 719.422.

[0577] Scheme 27: Preparation of tert-butyl 4-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)piperidine-1-carboxylate (Compound 27-1)

[0578]

[0579] Compound 21 was prepared using compound 18 (50 mg, 0.1 mmol) and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (42 mg, 0.15 mmol) according to General Procedure 1. The volatiles were removed in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH=90:10) to afford 55 mg of the desired product (79%). 1H NMR(400MHz, CDCl3)δ8.14(s,1H),7.11(dd,J=12.9,6.6Hz,1H),6.67(m,1H),6.58(dd,J=10.9, 7.1Hz,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=6.1Hz,2H),2.70(t,J=12.7Hz,2H),2.44–2.37(m,4H),1.97(m,2H),1.8 2(m,2H),1.77–1.69(m,3H),1.45(s,9H),1.39(t,J=5.6Hz,4H),1.28–1.11(m,2H),0.91(s,6H). 13 C NMR (101 MHz, CDCl3) δ 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.

[0580] Scheme 28: General Procedure 2

[0581]

[0582] To a stirred solution of the corresponding tert-butyl ester (1 eq.) in DCM (0.5 M) was then added TsCl (1.6 eq.) and TEA (5 eq.). The resulting reaction mixture was stirred at room temperature until complete (monitored by TLC). The volatiles were removed in vacuo and the product was taken on to the next step without further purification.

[0583] To the stirred solution of the corresponding tosylated tert-butyl ester in DMF (0.5 M) was added potassium phthalimide (2 equivalents). The resulting reaction mixture was stirred at room temperature until complete (monitored by TLC). The volatiles were then removed in vacuo and the product was used in the next step without further purification.

[0584] The crude product was dissolved in methanol (0.5M) and hydrazine hydrate 50-60% (3 equivalents). The resulting reaction mixture was stirred at room temperature until complete (monitored by TLC). DCM was added to the resulting mixture and the formed precipitate was removed by filtration. The filtrate was concentrated in vacuo and the resulting product was used in the next step without any further purification.

[0585] To a stirred solution of 4-fluoro-thalidomide (1 eq.) in DMSO (0.5 M) was then added the corresponding tert-butyl ester (1 eq.) and DIPEA (2 eq.). The resulting reaction mixture was stirred at 130 °C (oil bath temperature) until completion (monitored by TLC). Scheme 29: Preparation of tert-butyl 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propanoate (Compound 29-1)

[0586]

[0587] Compound 29-1 was prepared using 4-fluoro-thalidomide (82 mg, 0.3 mmol) and the corresponding tert-butyl ester (70 mg, 0.3 mmol) according to General Procedure 2. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = from 80:20 to 90:10) to provide 64 mg of the desired product (57%). 1 H NMR (400MHz, CDCl3) δ8.1(s,1H),7.52(m,J=8.4,7.1Hz,1H),7.12(d,J=7.1Hz,1H),6.97(d,J=8.5Hz,1H),6.47(t,J 5.6Hz,1H),4.85(dd,J 12.4,5.3Hz,1H),3.74(m,4H),3.65(m,4H),3.46(m,2H),2.89(m,1H),2.86(m,2H),2.53(t,J=6.6Hz,2H),2.18–2.10(m,1H),1.40(s,9H).

[0588] Scheme 30: Preparation of tert-butyl 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)propanoate (Compound 30-1)

[0589]

[0590] Compound 30-1 was prepared using 4-fluoro-thalidomide (68 mg, 0.25 mmol) and the corresponding tert-butyl ester (69 mg, 0.25 mmol) according to General Procedure 2. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = from 80:20 to 90:10) to afford 52 mg of the desired product (39%). 1 H NMR (400MHz, CDCl3) δ8.12(s,1H),7.55(t,J=7.6Hz,1H),7.13(d,J=6.8Hz,1H),6.94(d,J=8.3Hz,1H),6.51(s, 1H),4.94(m,1H),3.73(m,13H),3.50(m,2H),2.92–2.70(m,3H),2.50(t,J=6.2Hz,2H),2.15(m,1H),1.44(s,9H)

[0591] Scheme 31: Preparation of tert-butyl 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecan-15-ate (Compound 31-1)

[0592]

[0593] Compound 31-1 was prepared using 4-fluoro-thalidomide (53 mg, 0.2 mmol) and the corresponding tert-butyl ester (62 mg, 0.2 mmol) according to General Procedure 2. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = from 80:20 to 90:10) to give 64 mg of the desired product (57%). 1 H NMR (400MHz, CDCl3) δ8.11(s,1H),7.44(dd,J=7.32,8.34Hz,1H),7.04(d,J=7.11Hz,1H),6.83(d,J=8.54Hz,1H),6 .42(m,1H),4.8(s,1H),3.58(m,18H),3.42–3.35(m,2H),2.72(s,3H),2.45–2.39(m,2H),1.98(m,1H),1.43(m,9H).

[0594] Scheme 32: Preparation of tert-butyl 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaoctadecane-18-ate (Compound 32-1)

[0595]

[0596] Compound 32-1 was prepared using 4-fluoro-thalidomide (56 mg, 0.2 mmol) and the corresponding tert-butyl ester (74 mg, 0.2 mmol) according to General Procedure 2. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = from 80:20 to 90:10) to give 61 mg of the desired product (49%). 1 H NMR (400MHz, CDCl3) δ8.19(s,1H),7.46(t,J=7.7Hz,1H),7.13(d,J=7.0Hz,1H),6.90(d,J=8.4Hz,1H),6.50(m,1H) ,4.92(m,1H),3.76–3.58(m,18H),3.46(m,2H),2.94–2.73(m,3H),2.52(t,J=6.4Hz,2H),2.14(m,1H),1.43(m,9H).

[0597] Scheme 33: General Procedure 3

[0598]

[0599] To a stirred solution of lenalidomide (1 eq) in NMP (0.5 M) was then added the corresponding tert-butyl ester (1 eq) and DIPEA (10 eq). The resulting reaction mixture was stirred at 110° C. (oil bath temperature) until complete (monitored by TLC).

[0600] Scheme 34: Preparation of tert-butyl 9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanoate (Compound 34-1)

[0601]

[0602] Compound 34-1 was prepared using lenalidomide (267 mg, 1.03 mmol) and the corresponding tert-butyl ester (303 mg, 1.03 mmol) according to General Procedure 3. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane=70:30) to afford 200 mg of the desired product (41%). 1H NMR (400MHz, DMSO) δ11.00 (s, 1H), 7.27 (t, J = 7.7Hz, 1H), 6.92 (d, J = 7.3Hz, 1H), 6.73 (d, J = 8. 0Hz,1H),5.55(t,J=5.5Hz,1H),5.10(dd,J=13.3,5.1Hz,1H),4.26–4.08(m,2H),3.10(q,J=6 .6Hz,2H),2.92(ddd,J=18.3,13.6,5.4Hz,1H),2.73–2.57(m,2H),2.36–2.22(m,2H),2.16(t d,J=7.3,3.7Hz,2H),2.08–1.97(m,1H),1.56(m,2H),1.47(m,2H),1.38(s,9H),1.25(m,8H). 13 C NMR (101 MHz, CDCl3) δ 173.9, 172.2, 170.2, 166.3, 147.5, 131.6, 129.8, 125.72, 116.7, 116.2, 80.3, 55.4, 48.2, 43.6, 35.2, 31.1, 29.3, 28.4, 27.7, 27.2, 26.4, 24.3. LRMS (ESI) m / z: [M+H]+ calcd. for C22H30N3O5 416.22, found 416.26.

[0603] Scheme 35: Preparation of tert-butyl 11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecanoate (Compound 35-1)

[0604]

[0605] Compound 35-1 was prepared using lenalidomide (245 mg, 0.95 mmol) and the corresponding tert-butyl ester (304 mg, 0.95 mmol) according to General Procedure 3. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane=70:30) to afford 125 mg of the desired product (26%). 1H NMR (400MHz, DMSO) δ11.00(s,1H),7.27(t,J=7.7Hz,1H),6.95–6.88(m,1H),6.73(d,J=8.0 Hz,1H),5.55(t,J=5.5Hz,1H),5.10(dd,J=13.2,5.1Hz,1H),4.28–4.07(m,2H),3.10(q,J= 6.6Hz,2H),2.92(ddd,J=18.0,13.5,5.3Hz,1H),2.72–2.58(m,2H),2.36–2.23(m,2H),2.1 6(t,J=7.3Hz,2H),2.07–1.98(m,1H),1.57(m,2H),1.46(m,2H),1.38(s,9H),1.24(m,14H). 13 C NMR (101 MHz, CDCl3) δ 173.7, 172.5, 170.3, 165.9, 148.1, 131.7, 130.1, 125.6, 117.2, 116.4, 80.5, 56.3, 49.1, 43.6, 35.4, 31.3, 30.8, 29.7, 29.1, 28.2, 27.7, 27.3, 26.7, 23.3. LRMS (ESI) m / z: [M+H]+ calcd. for C28H42N3O5 500.31, found 500.34.

[0606] Scheme 36: Preparation of tert-butyl 15-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentadecanoate (Compound 36-1)

[0607]

[0608] Compound 36-1 was prepared using lenalidomide (264 mg, 1.02 mmol) and the corresponding tert-butyl ester (432 mg, 1.02 mmol) according to General Procedure 3. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane=70:30) to give 125 mg of the desired product (22%). 1H NMR (400MHz, CDCl3) δ7.98(s,1H),7.36(t,J=7.7Hz,1H),6.80(dd,J=8.0,0.9Hz,1H),5.25(dd,J=13.2,5.2Hz,1H),4.36–4.09(m,2H),4.05(t,J=6 .8Hz,1H),3.20(t,J=7.3Hz,2H),2.92–2.78(m,2H),2.36–2.26(m,2H),2 .20(t,J=7.5Hz,2H),1.69–1.53(m,6H),1.44(s,9H),1.31–1.22(m,18H). 13 C NMR (101 MHz, DMSO) δ 174.4, 173.1, 170.5, 165.6, 149.7, 131.2, 129.5, 126.2, 117.1, 116.3, 79.8, 56.1, 48.4, 43.8, 36.7, 33.4, 30.7, 29.5, 29.5, 29.2, 28.4, 28.1, 27.7, 27.2, 26.9, 26.7, 25.5, 24.8. LRMS (ESI) m / z: [M+H]+ calculated for C32H50N3O5 556.38, found 556.41.

[0609] Scheme 37: General Procedure 4

[0610]

[0611] To a stirred solution of tert-butyl ester (1 eq.) in DMF (0.5 M) was added sodium azide (1.2 eq.). The resulting reaction mixture was stirred at room temperature until complete (by 1 H NMR monitoring). The volatiles were then removed in vacuo, taken up in DCM and filtered through filter paper. The product was used in the next step without further purification.

[0612] Compound 12-9 (1 equivalent) was dissolved in THF (0.5 M), and then the corresponding tert-butyl ester (1 equivalent), anhydrous CuSO4 (0.5 equivalent) and sodium ascorbate (1.1 equivalent) were added respectively. The resulting reaction mixture was stirred at 40° C. (oil bath temperature) until complete (monitored by TLC).

[0613] Scheme 38: Preparation of tert-butyl 11-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)undecanoate (Compound 38-1)

[0614]

[0615] Compound 38-1 was prepared using compound 12-9 (80 mg, 0.26 mmol) and the corresponding tert-butyl ester (73 mg, 0.26 mmol) according to General Procedure 4. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = from 40:60 to 50:50) to provide 85 mg of the desired product (55%). 1 H NMR (400MHz, CDCl3) δ7.99 (s, 1H), 7.49 (dd, J = 8.5, 7.1Hz, 1H), 7.45 (s, 1H), 7.14 ( d,J=7.1Hz,1H),7.00(d,J=8.5Hz,1H),6.68(t,J=6.0Hz,1H),4.92(dd,J=12.2,5. 4Hz,1H),4.64(d,J=6.0Hz,2H),4.31(t,J=7.3Hz,2H),2.95–2.67(m,3H),2.19(t, J=7.5Hz,2H),2.13(m,1H),1.88(t,J=7.2Hz,2H),1.44(s,9H),1.33–1.21(m,14H). 13 C NMR (101 MHz, CDCl3) δ 173.2, 172.5, 171.0, 164.9, 163.4, 142.7, 140.8, 132.5, 131.6, 122.7, 121.8, 119.8, 113.4, 82.3, 52.5, 49.8, 38.3, 35.1, 30.3, 29.7, 29.2, 28.6, 27.7, 26.8, 26.1, 23.2. LRMS (ESI) m / z: [M+H]+ calcd. for C31H43N6O6 595.32, found 595.35.

[0616] Scheme 39: Preparation of tert-butyl 9-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)nonanoate (Compound 39-1)

[0617]

[0618] Compound 39-1 was prepared using compound 12-9 (85 mg, 0.27 mmol) and the corresponding tert-butyl ester (70 mg, 0.27 mmol) according to General Procedure 4. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = from 40:60 to 50:50) to give 73 mg of the desired product (47%).1 H NMR(400MHz, CDCl3)δ8.06(s,1H),7.49(dd,J=8.5,7.2Hz,1H),7.44(s,1H),7.14(d, J=7.2,0.6Hz,1H),7.00(d,J=8.5Hz,1H),6.67(t,J=5.9Hz,1H),4.92(dd,J=12.1,5. 4Hz,1H),4.64(d,J=6.0Hz,2H),4.31(t,J=7.9,6.5Hz,2H),2.94–2.67(m,3H),2.21– 2.15(m,2H),2.15–2.09(m,1H),1.92–1.83(m,1H),1.43(s,9H),1.30–1.22(m,11H). 13 C NMR (101 MHz, CDCl3) δ 173.8, 172.3, 170.3, 165.5, 164.8, 143.4, 141.0, 131.4, 130.3, 123.1, 121.4, 120.6, 111.5, 80.25, 51.7, 50.2, 37.3, 35.1, 30.5, 29.2, 28.6, 27.7, 26.4, 24.3. LRMS (ESI) m / z: [M+H]+ calculated for C29H39N6O6 567.29, found 597.30.

[0619] Scheme 40: General Procedure 5

[0620]

[0621] To a stirred solution of 4-fluoro-thalidomide (1 eq.) in DMSO (0.5 M) was then added the corresponding tert-butyl ester (1 eq.) and DIPEA (3 eq.). The resulting reaction mixture was stirred at 130 °C (oil bath temperature) until complete (monitored by TLC). Scheme 41: Preparation of tert-butyl 8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoate (Compound 41-1)

[0622]

[0623] Compound 41-1 was prepared using 4-fluoro-thalidomide (258 mg, 0.93 mmol) and the corresponding tert-butyl ester (201 mg, 0.93 mmol) according to General Procedure 5. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = 2:3) to provide 251 mg of the desired product (57% yield). 1H NMR (400MHz, CDCl3) δ8.07(s,1H),7.49(dd,J=8.5,7.1Hz,1H),7.08(d,J=7.0Hz,1H),6.87(d,J=8.5Hz,1H),6.22(t,J=5.7Hz,1H),4.91(dd,J=12.1,5 .3Hz,1H),3.25(td,J=7.1,5.6Hz,2H),2.96–2.67(m,3H),2.20(t,J=7.5Hz ,2H),2.17–2.09(m,1H),1.70–1.53(m,4H),1.43(s,9H),1.37–1.30(m,5H).

[0624] Scheme 42: Preparation of tert-butyl 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoate (Compound 42-1)

[0625]

[0626] Compound 42-1 was prepared using 4-fluoro-thalidomide (516 mg, 1.9 mmol) and the corresponding tert-butyl ester (350 mg, 1.9 mmol) according to General Procedure 5. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = 2:3) to afford 623 mg of the desired product (75% yield). 1 H NMR (400MHz, CDCl3) δ7.89(s,1H),7.42(dd,J=8.5,7.1Hz,1H),7.02(d,J=7.0Hz,1H),6.81(d,J=8.5Hz,1H),6.16(t,J=5.7Hz,1H),4.84(dd, J=12.1,5.4Hz,1H),3.20(td,J=7.1,5.6Hz,2H),2.89–2.59(m,3H),2.17(t,J=7.4Hz,2H),2.10–2.04(m,1H),1.68–1.53(m,4H),1.37(s,9H).

[0627] Scheme 43: Preparation of tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyrate (Compound 43-1)

[0628]

[0629] Compound 43-1 was prepared using 4-fluoro-thalidomide (55 mg, 0.2 mmol) and the corresponding tert-butyl ester (34 mg, 0.2 mmol) according to General Procedure 5. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / heptane = 2:3) to afford 80 mg of the desired product (96% yield). 1 H NMR (400MHz, CDCl3) δ8.02(s,1H),7.50(dd,J=8.6,7.1Hz,1H),7.10(d,J=7.0Hz,1H),6.93(d,J=8.5Hz,1H),6.29(t,J=5.8Hz,1H),4.9 6–4.84(m,1H),3.33(q,J=6.6Hz,2H),2.93–2.69(m,3H),2.35(t,J=7.1Hz,2H),2.17–2.10(m,1H),1.94(p,J=7.1Hz,2H),1.45(s,9H). 13 C NMR (101 MHz, CDCl3) δ 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]+ calculated for C21H26N3O6 416.181, found 416.182.

[0630] Scheme 44: General Procedure 6

[0631]

[0632] 1-Cbz-piperazine (1 eq.) and DIPEA (2 eq.) were then added to a stirred solution of the corresponding tert-butyl ester (1 eq.) in MeCN (0.5 M). The resulting reaction mixture was stirred at 85 ° C (oil bath temperature) until complete (monitored by TLC). The volatiles were then removed in vacuo and the residue was extracted into EtOAc (3x). The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. The product was taken to the next step without further purification.

[0633] The piperazine derivative (1 eq) was added to a previously prepared mixture of iPrOH (0.5 M) and Pd / C (0.5 eq), followed by the addition of ammonium formate (6 eq). The resulting reaction mixture was stirred at 40 °C (oil bath temperature) until complete (monitored by TLC). The reaction mixture was evaporated and extracted into DCM (3x). The combined organic layers were dried over MgSO4, filtered and evaporated. The product was taken on to the next step without further purification.

[0634] To a stirred solution of 4-fluoro-thalidomide (1 eq) in DMSO (0.5 M) was then added the piperazine derivative (1 eq) and DIPEA (3 eq). The resulting reaction mixture was stirred at 130° C. (oil bath temperature) until completion (monitored by TLC).

[0635] Scheme 45. Preparation of tert-butyl 9-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazin-1-yl)nonanoate (Compound 45-1)

[0636]

[0637] Compound 45-1 was prepared using 4-fluoro-thalidomide (60 mg, 0.2 mmol) and the corresponding tert-butyl ester (55 mg, 0.2 mmol) according to General Procedure 6. The volatiles were then removed in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / MeOH=95:5) to afford 68 mg of the desired product (61%). 1 H NMR (400MHz, DMSO) δ11.08(s,1H),7.70(dd,J=8.4,7.1Hz,1H),7.34(t,J=8.3Hz,2H),5.09(dd,J=12.8,5.4Hz,1H),2.87(ddd,J=18.2 ,13.8,5.3Hz,1H),2.61(m,1H),2.39–2.27(m,2H),2.17(t,J=7.3Hz,2H),2.07–1.97(m,1H),1.47(m,4H),1.39(s,9H),1.25(m,10H). 13C NMR (101 MHz, DMSO) δ 173.3, 172.8, 170.5, 167.5, 166.8, 150.2, 134.1, 116.9, 79.8, 58.3, 53.2, 51.0, 49.3, 40.7, 40.6, 40.4, 35.2, 31.4, 29.3, 29.1, 28.8, 28.2, 27.3, 26.7, 25.1. LRMS (ESI) m / z: [M+H]+ calcd. for C30H43N4O6 555.320, found 555.318.

[0638] Scheme 46: General Procedure 7

[0639]

[0640] To a stirred solution of the corresponding tert-butyl ester (1 eq.) in DCM (0.5 M) was added TFA (10 eq.). The resulting reaction mixture was stirred at room temperature until complete (monitored by TLC). The volatiles were then removed in vacuo.

[0641] Scheme 47: General Procedure 8

[0642]

[0643] The Boc protected amine (1 eq) was dissolved in MeOH (0.25 M) and to the stirred solution was added HCl 4 M in dioxane (10 eq). The resulting reaction mixture was stirred at room temperature until completion (monitored by TLC) and evaporated.

[0644] Scheme 48: General Procedure 9

[0645]

[0646] To a cooled solution of carboxylic acid (1 eq.) in DMF (0.5 M) (ice-water bath) was added DIPEA (4 eq.) and the reaction mixture was stirred at the same temperature for 10 min. After addition of HATU or COMU (1.1 eq.), the solution was stirred for another 30 min and then the amine (1 eq.) was added. The resulting reaction mixture was stirred at room temperature until completion (monitored by TLC), concentrated in vacuo and purified using flash column chromatography.

[0647] Scheme 49. Preparation of 4-(4-(9-(4-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)piperidin-1-yl)-9-oxononyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Compound 49-1)

[0648]

[0649] Compound 49-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (58 mg, 0.09 mmol) and carboxylic acid (60 mg, 0.09 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / MeOH = from 85:15 to 80:20) to provide 65 mg of the desired product (62%). 1 H NMR (400MHz, MeOD) δ7.99(s,1H),7.67(dd,J=8.4,7.2Hz,1H),7.38(d,J=7.1Hz,1H),7.31(d,J=8.4Hz,1H),7.18(dd,J=13.0,6.6Hz,1H),6.87(dd,J=11. 2,7.2Hz,1H),5.69(s,1H),5.09(dd,J=12.5,5.5Hz,1H),4.54(d,J=13.3Hz, 1H),4.03–3.96(m,1H),3.92(m,2H),3.72(s,2H),3.62–3.57(s,2H),3.42–3 .33(m,7H),3.18(m,2H),3.08(t,J=12.9Hz,1H),2.86(ddd,J=17.7,14.2,5. 1Hz,1H),2.77(m,5H),2.66–2.57(m,1H),2.54(m,4H),2.51–2.44(m,2H),2. 39(td,J=7.4,3.8Hz,2H),2.15–2.07(m,1H),1.98(m,2H),1.91(s,1H),1.89 –1.76(m,5H),1.59(m,f5H),1.44(t,J=5.6Hz,5H),1.37(s,8H),0.93(s,6H). 13C NMR(101MHz,MeOD)δ173.2,172.6,170.2,168.9,167.5,166.7,163.4,162.0,156.9,150.0,135.5,134.1,123.3,117.5,115.1,58.3,53.9,53.2 ,52.8,52.3,50.1,49.1,45.9,45.7,41.6,40.3,37.6,36.2,34.5,32.8 ,30.8,30.4,29.5,29.4,29.0,28.9,28.9,27.7,27.1,25.9,25.3,22.3. LRMS (ESI) m / z: [M+H]+ calculated for C58H79F2N12O6 1077.620, found 1077.623. Scheme 50. Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanamide (Compound 50-1)

[0650]

[0651] Compound 50-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (25 mg, 0.04 mmol) and carboxylic acid (17 mg, 0.04 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / MeOH = from 90:10 to 80:20) to provide 25 mg of the desired product (61%). 1H NMR (400MHz, DMSO) δ11.01 (s, 1H), 8.35 (t, J = 6.0Hz, 1H), 8.18 (s, 1H), 7.98 ( s,1H),7.31–7.20(m,3H),7.20–7.12(m,3H),7.08(d,J=7.5Hz,1H),6.97–6.8 6(m,2H),6.71(d,J=8.0Hz,1H),5.69(s,1H),5.60(t,J=5.6Hz,1H),5.10(dd, J=13.3,5.1Hz,1H),4.41(d,J=6.2Hz,2H),4.22(s,2H),4.18(dd,J=45.9,17. 1Hz,2H),3.83(d,J=13.3Hz,2H),3.58(s,2H),3.47–3.37(m,4H),3.25(s,2H) ),3.09(q,J=6.6Hz,2H),2.92(ddd,J=18.1,13.5,5.4Hz,1H),2.67–2.57(m,1 H),2.40–2.22(m,4H),2.11(t,J=7.4Hz,2H),2.06–1.97(m,1H),1.81–1.72(m ,2H),1.69–1.60(m,1H),1.60–1.44(m,3H),1.38–1.20(m,14H),0.87(s,6H). 13 C NMR(101MHz,DMSO)δ172.9,172.1,171.3,168.9,166.5,163.2,161.6,157.4, 143.8,139.8,132.0,129.2,128.1,126.5,126.0,125.5,125.5,111.7,109.8 ,69.8,59.8,54.7,52.8,52.6,51.5,49.0,45.8,43.6,42.7,41.9,38.1,35.3,34.6,31.2,28.9,28.8,28.7,28.5,28.1,26.7,25.3,22.8,21.1,20.8,14.1. LCMS (ESI) m / z: [M+H]+ calculated for C56H72F2N11O5 1016.568, found 1016.566.

[0652] Scheme 51. N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecaneamide (Compound 51-1)

[0653]

[0654] Compound 51-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (18.6 mg, 0.04 mmol) and carboxylic acid (26 mg, 0.04 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 90:10 to 80:20) to provide 32 mg of the desired product (73%). 1 H NMR(400MHz,MeOD)δ8.06(s,1H),7.47–7.38(m,1H),7.33–7.25(m,3H),7.22 (d,J=7.8Hz,1H),7.17(d,J=7.5Hz,1H),7.03(d,J=7.5Hz,1H),6.98(dd,J=11 .4,7.2Hz,1H),6.79(d,J=8.0Hz,1H),5.69(s,1H),5.13(dd,J=13.3,5.1Hz, 1H),4.48(s,2H),4.34(s,2H),4.30–4.26(m,3H),3.94–3.86(m,2H),3.76(s, 2H),3.75–3.69(m,2H),3.64(s,1H),3.57(dd,J=10.8,8.1Hz,2H),3.49–3.4 3(m,1H),3.41(s,2H),3.26–3.16(m,6H),2.90(ddd,J=18.5,13.3,5.1Hz,1H) ,2.81–2.73(m,1H),2.46(qd,J=13.1,4.7Hz,1H),2.23–2.14(m,3H),1.97–1 .89(m,2H),1.78(m,2H),1.70–1.57(m,7H),1.39–1.36(m,16H),1.05(s,4H). 13C NMR(101MHz,MeOD)δ174.8,173.3,171.1,171.0,168.4,161.2,143.8,141.1,1 39.4,131.6,129.2,128.5,126.6,126.1,125.9,125.8,112.4,110.4,54.4,53 .0,52.2,51.9,48.6,46.1,44.6,43.1,42.5,42.4,40.5,35.7,35.1,34.3,31.0,29.4,29.2,29.1,28.9,28.8,28.8,27.3,26.8,25.6,22.9,17.3,15.9,11.8. LRMS (ESI) m / z: [M+H]+ calcd for C58H76F2N11O5 1044.600, found 1044.600.

[0655] Scheme 52. Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-9-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)nonanamide (Compound 52-1)

[0656]

[0657] Compound 52-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (18.2 mg, 0.03 mmol) and carboxylic acid (15 mg, 0.03 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 90:10 to 80:20) to provide 10 mg of the desired product (31%). 1H NMR(500MHz,CDCl3)δ10.24(bs,1H),8.16(s,1H),7.51–7.41(m,2H),7.29(m,1H),7.22–7.13(m,3H),7.11(d,J=7.1Hz,1H),6.98(d,J=8.5Hz,1H),6.81(s,1H),6.67(t,J=6.0Hz,1H),6.58(dd,J=10.9,7.0Hz,1H),6.03(t,J=6.0Hz,1H),5.37(s,1H),4.89(dd,J=12.2,5.4Hz,1H),4.62(d,J=5.9Hz,2H),4.41(dd,J=11.9,5.8Hz,3H),4.31(td,J=6.9,2.5Hz,2H),3.68(m,4H),3.50(m,2H),3.28(s,2H),2.89–2.83(m,1H),2.75(m,2H),2.56(bs,2H),2.12(t,J=7.1,6.5Hz,2H),1.87(m,4H),1.75(m,3H),1.57(t,J=7.8,7.1Hz,2H),1.45(m,2H),1.26(m,16H),0.94(s,4H),0.88(t,J=6.7Hz,2H)。 13C NMR (101MHz, CDCl3) δ173.2,171.8,169.4,169.2,167.8,167.6,146.2,139.3,1 38.6,136.2,132.4,129.0,126.7,126.1,125.9,121.5,117.2,112.3,110.8,53 .4,52.9,50.4,49.6,49.0,45.5,43.2,40.4,38.8,36.6,35.0,31.9,31.5,30.0,29.7,29.33,29.0,29.0,29.0,28.6,28.3,27.2,26.2,25.6,22.8,22.7,14.1. LRMS (ESI) m / z: [M+H]+ calcd. 1111.580 for C59H73F2N14O6, found 1111.581. Scheme 53. Preparation of N-(3-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-11-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)undecaneamide (Compound 53-1)

[0658]

[0659] According to General Procedure 9 (COMU), compound 27 was prepared using the corresponding amine (17 mg, 0.03 mmol) and carboxylic acid (15 mg, 0.03 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 90:10 to 80:20) to afford 12 mg of the desired product (37%). 1H NMR (400MHz, CDCl3) δ10.08(bs,1H),8.16(s,1H),7.50–7.43(m,2H),7.21(s,1H),7.16(m,2H),7.10(d,J=7.2Hz,1H),6.97(d,J=8.5Hz,1H), 6.83(s,1H),6.68(t,J=5.9Hz,1H),6.58(dd,J=10.9,7.1Hz,1H),6.07(s,1H),5.93(bs,1H),5.40–5.32(m,2H),4.90(dd,J=11.9,5.3Hz,1H) ,4.62(d,J=5.9Hz,2H),4.42(m,3H),4.31(t,J=7.0Hz,2H),3.71(s,2H ),3.65(m,2H),3.59–3.45(m,4H),3.27(s,2H),2.90–2.69(m,3H),2.4 8(bs,2H),2.26–2.07(m,3H),2.02(m,2H),1.93–1.83(m,4H),1.80–1. 71(m,2H),1.58(m,4H),1.42(m,4H),1.28–1.25(m,12H),0.92(s,6H). 13 C NMR (101MHz, CDCl3) δ173.2,171.8,169.4,169.2,167.8,167.6,146.2,139.3, 138.6,136.2,132.4,129.0,126.7,126.1,125.9,121.5,117.2,112.3,110.8,5 3.4,52.9,50.4,49.6,49.0,45.5,43.2,40.4,38.8,36.6,35.0,31.9,31.5,30.0,29.7,29.3,29.0,29.0,29.0,28.6,28.3,27.2,26.2,25.6,22.8,22.7,14.1. LRMS (ESI) m / z: [M+H]+ calcd. for C61H77F2N14O6 1139.610, found 1139.608.

[0660] Scheme 54. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triaza-spiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-15-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentadecanamide (Compound 54-1)

[0661]

[0662] Compound 54-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (117 mg, 0.224 mmol) and the corresponding carboxylic acid (112 mg, 0.224 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 90:10 to 80:20) to provide the desired product (120 mg, 53%). 1 H NMR (400MHz, MeOD) δ8.01(s,1H),7.35–7.31(m,2H),7.21(t,J=7.8Hz,1H),7.00–6.87(m,3H),6.71(d,J=8.0Hz,1H),5.71(s,1H),5.04(dd,J=13. 3,5.1Hz,1H),4.19(d,J=6.4Hz,2H),4.13(d,J=7.4Hz,2H),3.78(d,J=3. 7Hz,4H),3.70–3.60(m,2H),3.45(s,2H),3.19–3.14(m,3H),3.10(t,J=7. 2Hz,2H),3.05–2.94(m,2H),2.81(ddd,J=18.4,13.4,5.4Hz,1H),2.68(d dd,J=17.6,4.7,2.5Hz,1H),2.38(qd,J=13.2,4.7Hz,1H),2.13–2.04(m,3 H),1.83(td,J=8.1,6.6,3.4Hz,2H),1.73(ddd,J=20.3,9.7,5.4Hz,3H), 1.53(tt,J=14.5,6.5Hz,9H),1.35–1.15(m,26H),0.95(d,J=14.2Hz,7H). 13 C NMR(101MHz,MeOD)δ175.2,173.3,171.1,171.0,150.2,143.8,132.3,131.6,129.2,126.6,119.0,114.3,112.4,110.4,59.6,52.6,52.4 ,52.2,50.4,46.1,43.1,40.6,38.7,36.2,35.8,35.2,34.1,31.0,29 .2,29.2,29.1,29.0,28.9,28.8,28.4,27.4,26.8,25.6,22.9,22.1. LRMS (ESI) m / z: [M+H]+ calculated for C57H84N11O5 1002.670, found 1002.664.

[0663] Scheme 55. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanamide (Compound 55-1)

[0664]

[0665] Compound 55-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (85 mg, 0.14 mmol) and carboxylic acid (60 mg, 0.24 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / MeOH = from 90:10 to 80:20) to provide 100 mg of the desired product (72%). 1 H NMR (400MHz, CDCl3) δ8.14(s,1H),7.32(t,J=7.7Hz,1H),7.20(d,J=7.5Hz,1H),7.11(dd,J=12.9,6.6Hz,1H),7.02(s,1H),6.76(d,J=8.1Hz,1H) ,6.57(dd,J=10.9,7.1Hz,1H),6.36(s,1H),5.74(s,1H),5.43(s,1H),5.20(dd,J=13.2,5.2Hz,1H),4.20(dd,J=65.9,15.8Hz,2H),3.71(d,J=7. 1Hz,4H),3.60–3.53(m,2H),3.50(s,2H),3.29(q,J=6.9Hz,4H),3.25(s ,2H),3.18(q,J=7.1,6.6Hz,2H),2.85–2.75(m,2H),2.47–2.38(m,4H),2 .33–2.19(m,3H),2.19–2.07(m,4H),1.98–1.86(m,2H),1.84–1.69(m,2H ),1.66–1.53(m,4H),1.44–1.36(m,5H),1.35–1.21(m,7H),0.90(s,6H). 13C NMR (101MHz, CDCl3) δ173.8,172.2,171.1,170.3,168.3,163.1,162.3,157.4,156.2 ,152.0,149.9,143.4,137.6,132.0,129.8,126.3,118.6,113.3,112.5,105.9,105. 7, 56.6, 56.5, 54.7, 53.5, 53.1, 51.9, 51.0, 49.8, 45.2, 43.7, 40.3, 38.9, 38.6, 38.3, 38.3, 36.9, 36.5, 35.3, 31.8, 29.8, 29.3, 29.2, 29.0, 28.9, 28.5, 26.8, 25.7, 23.7. LCMS (ESI) m / z: [M+H]+ calcd for C51H70F2N11O5 954.552, found 954.552.

[0666] Scheme 56. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecaneamide (Compound 56-1)

[0667]

[0668] Compound 56-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (40 mg, 0.07 mmol) and carboxylic acid (30 mg, 0.07 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; EtOAc / MeOH = from 90:10 to 80:20) to provide 37 mg of the desired product (56%). 1H NMR(400MHz,DMSO)δ11.00(s,1H),10.87(s,1H),8.36(s,1H),8.29(s,1H),7. 99(t,J=5.5Hz,1H),7.73(dd,J=13.4,6.8Hz,1H),7.27(t,J=7.7Hz,1H),7.05( dd,J=11.7,7.4Hz,1H),6.91(d,J=7.4Hz,1H),6.72(d,J=8.0Hz,1H),5.94(s,1 H),5.10(dd,J=13.2,5.1Hz,1H),4.24(d,J=4.5Hz,2H),4.18(dd,J=46.2,17.3 Hz,2H),3.69(s,2H),3.40–3.33(m,3H),3.30(d,J=6.2Hz,2H),3.21–3.01(m, 8H),2.92(ddd,J=18.0,13.4,5.2Hz,1H),2.68–2.57(m,1H),2.29(qd,J=13.4, 4.9Hz,1H),2.13–1.97(m,3H),1.91–1.70(m,5H),1.66(t,J=6.8Hz,2H),1.60– 1.51(m,2H),1.51–1.39(m,4H),1.38–1.16(m,17H),0.99(s,3H),0.93(s,3H). 13 C NMR (101MHz, DMSO) δ173.4,172.8,171.7,169.4,166.9,158.8,158.5,144.2,141.0,132.5,129.7,127.0,118.9,116.0,112.2,110. 4,53.3,52.7,52.0,48.2,46.3,43.2,36.4,35.9,35.1,31.7,31.5,29.5,29.4,29.4,29.3,29.2,29.0,28.0,27.1,25.8,23.5,23.3. LCMS (ESI) m / z: [M+H]+ calculated for C53H74F2N11O5 982.583, found 982.584.

[0669] Scheme 57. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-9-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)nonanamide (Compound 57-1)

[0670]

[0671] Compound 57-1 was prepared according to General Procedure 9 (HATU) using the corresponding amine (67 mg, 0.13 mmol) and the corresponding carboxylic acid (66 mg, 0.13 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 90:10 to 80:20) to provide 100 mg of the desired product (76%). 1 H NMR (400MHz, MeOD) δ7.88(d,J=0.8Hz,1H),7.81(s,1H),7.41(dd,J=8.5,7.1Hz,1H),7.28(d,J=8.7Hz,2H),6.97(t,J=7.9Hz,2H),6.92–6.88( m,2H),5.58(d,J=1.0Hz,1H),4.94(dd,J=12.5,5.4Hz,1H),4.52(s,2H),4.24(t,J=7.0Hz,2H),4.01(s,2H),3.76(s,2H),3.69(q,J=5.6,3.7Hz ,2H),3.49–3.43(m,2H),3.42(s,2H),3.25(s,1H),3.19–3.11(m,4H),2 .98(s,3H),2.76–2.67(m,1H),2.64(dd,J=4.3,2.4Hz,1H),2.62–2.56( m,1H),2.05(d,J=7.5Hz,2H),1.79–1.64(m,8H),1.48(dt,J=19.1,6.5Hz,7H),1.27(dd,J=7.2,4.5Hz,4H),1.16(d,J=3.3Hz,8H),0.93(s,6H). 13C NMR(101MHz,MeOD)δ175.0,173.2,170.2,169.2,167.8,163.2,162.0,156 .9,150.0,146.0,145.1,135.8,132.5,132.1,122.7,117.0,114.2,111.1, 110.6,60.0,54.4,52.9,52.5,50.5,49.9,40.2,38.2,37.5,36.5,35.7,35 .5,34.1,30.8,29.7,28.8,28.7,28.5,28.3,27.5,25.8,25.5,22.4,11.8. LRMS (ESI) m / z: [M+H]+ calculated for C54H73N14O6 1013.580, found 1013.584.

[0672] Scheme 58. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-9-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)nonanamide (Compound 58-1)

[0673]

[0674] Compound 58-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (34.9 mg, 0.063 mmol) and carboxylic acid (32.0 mg, 0.063 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 100:5 to 100:20) to provide 15 mg of the desired product (22.3%). 1H NMR (400MHz, CDCl3) δ10.38(bs,1H),8.14(s,1H),7.47(dd,J=8.5,7.1Hz,1H),7.13(dd,J=12.8,6.7Hz,1H),7.11(d,J=7.1H z,1H),6.98(d,J=8.5Hz,1H),6.90(s,1H),6.70(t,J=5.9Hz,1H),6.58(dd,J=10.9,7.2Hz,1H),6.28(bs,1H),5.79(bs,1H),5 .44(s,1H),4.91(dd,J=12.1,5.4Hz,1H),4.63(d,J=5.9Hz,2H),4.32(t,J=7.0Hz,2H),3.75–3.49(m,8H),2.87–2.81(m,3H) ,2.49(bs,4H),2.17–2.05(m,3H),1.95–1.91(m,8H),1.60–1.51(m,2H),1.45–1.38(m,4H),1.28–1.17(m,10H),0.92(s,6H). 13 C NMR (101MHz, CDCl3) δ207.1,173.8,171.8,169.8,169.6,168.1,167.7,162.5,162.3, 158.7,157.2,152.3,146.3,145.2,136.4,132.5,121.8,118.9,117.3,112.4,110.9,1 05.9,105.6,63.9,56.7,54.5,53.5,53.0,50.5,49.7,49.1,40.3,38.9,38.3,36.8,36.5,35.4,32.1,31.6,31.1,30.1,29.8,29.5,29.3,28.8,28.4,28.4,26.2,25.6,23.0. LRMS (ESI) m / z: [M+H]+ calcd. for C54H71F2N14O6 1049.564, found 1049.565. Scheme 59. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undec-9-yl)pyrimidin-4-yl)amino)propyl)-11-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)undecaneamide (Compound 59-1)

[0675]

[0676] Compound 59-1 was prepared according to General Procedure 9 (COMU) using the corresponding amine (33.08 mg, 0.059 mmol) and carboxylic acid (32.0 mg, 0.059 mmol). The reaction mixture was concentrated in vacuo and the crude product was purified using flash column chromatography (SiO2; DCM / MeOH = from 100:10 to 100:20) to provide 14 mg of the desired product (21%). 1 H NMR (400MHz, CDCl3) δ10.38(bs,1H),8.14(s,1H),7.47(dd,J=8.5,7.1Hz,1H),7.13(dd,J=12.8,6.7Hz,1H),7.11(d,J=7.1H z,1H),7.05(s,1H),6.98(d,J=8.5Hz,1H),6.70(t,J=5.9Hz,1H),6.58(dd,J=10.9,7.2Hz,1H),6.28(bs,1H),5.79(bs,1H),5 .44(s,1H),4.91(dd,J=12.1,5.4Hz,1H),4.63(d,J=5.9Hz,2H),4.32(t,J=7.0Hz,2H),3.75–3.49(m,8H),2.87–2.81(m,3H) ,2.49(bs,4H),2.17–2.05(m,3H),1.95–1.91(m,8H),1.60–1.51(m,2H),1.45–1.38(m,4H),1.28–1.17(m,14H),0.92(s,6H). 13 C NMR (101MHz, CDCl3) δ207.1,173.9,171.9,169.5,168.2,167.7,162.9,162.3,158.7,157.2, 156.3,152.3,149.9,146.3,145.1,137.8,136.3,132.5,121.7,118.8,118.6,117.3,112.4,1 LRMS (ESI) m / z: [M+H]+ calcd for C56H75F2N14O6 1077.596, found 1077.593.

[0677] Synthesis schemes from literature incorporated herein by reference:

[0678]

[0679] Regarding general procedures and methods for preparing starting materials, reference Front. Chem. 2021, 9, 707317 (see, for example, Schemes 1-20 and Figure 3 ) and Org.Biomol.Chem., 2021, 19, 166-170 (see, for example, click chemistry procedures and methods for preparing triazoles in Formula J).

[0680] Tert-butyl 4-(aminomethyl)-4-(((benzyloxy)carbonyl)amino)piperidine-1-carboxylate (32):

[0681]

[0682] To a stirred solution of MeNO2 (1.3 eq., 130 mmol, 7 mL) in NH3 (53 mL, 7 N in MeOH) was added tert-butyl 4-oxopiperidine-1-carboxylate (20 g, 100 mmol) in portions. 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 twice with DCM. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure to obtain the desired product, which was used in the next step without further purification.

[0683] To a stirred solution of the corresponding amine (100mmol) in dichloromethane (130mL) was added a solution of K2CO3 (2 equivalents, 200mmol, 27.6g) in water (130mL). The reaction mixture was cooled to 0°C and CBzCl (1.1 equivalents, 110mmol, 15.6L) was added dropwise. The reaction mixture was stirred at 25°C for 17h and the two phases were separated. The aqueous layer was extracted twice with DCM. The combined organic layers were washed once with brine, dried over MgSO4, filtered and concentrated under reduced pressure to obtain the desired product, which was used in the next step without further purification. NiCl2.6H2O (1 equivalent, 100mmol, 27.3g) was added to a stirred solution of the corresponding nitroalkane (100mmol) in anhydrous MeOH (450mL) under a nitrogen atmosphere at 0°C, followed by addition of NaBH4 (5 equivalents, 500mmol, 18.9g) in batches to avoid strong H2 evolution. Be careful when adding NaBH4, the reaction is highly exothermic and produces hydrogen. The reaction mixture is stirred at 25 ° C for 1h and quenched by adding saturated NaHCO3 aqueous solution. The mixture is filtered through a celite pad, the filtrate is concentrated under reduced pressure, and the obtained residue is diluted with water. The aqueous layer is extracted three times with DCM, and the combined organic layer is washed once with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue is 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 obtain the desired product (11.7g, 32% yield over three steps) as a white solid. LRMS (ESI) m / z [C 19 H 30 N3O4] + Calculated value: 364.2, measured value: 364.3

[0684] 2-Oxo-1,4,9-triazaspiro[5.5]undecane-9-carboxylic acid tert-butyl ester (33):

[0685]

[0686] To a stirred solution of tert-butyl 4-(aminomethyl)-4-(((benzyloxy)carbonyl)amino)piperidine-1-carboxylate (14.5 g, 40 mmol) in DCM (133 mL) was added EtN (0.8 eq., 32 mmol, 4.4 mL) and ethyl 2-bromoacetate (0.7 eq., 28 mmol, 3.1 mL) at 0°C. The reaction mixture was stirred at 25°C for 2 h and quenched with saturated NaHCO aqueous solution. The aqueous layer was extracted three times with EtOAc, and the combined organic layers were washed once with brine, dried over MgSO, filtered and concentrated under reduced pressure to give the impure desired product, which was used in the next step without further purification.

[0687] Pd / C (5 mol%, 2 mmol, 2.1 g, 10 wt%) and ammonium formate (6 eq., 240 mmol, 15 g) were added in batches to a stirred solution of the corresponding Cbz protected amine (40 mmol) in iPrOH (400 mL). The reaction mixture was stirred at 80 ° C for 4 h, cooled to 25 ° C, filtered through a celite pad 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 MgSO4, 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 give the desired product (4.14 g, 55% yield over two steps) as a white solid. LRMS (ESI) m / z C 26 H 47 N6O6] + =[2M+H] + Calculated value: 539.4, Measured value: 539.4

[0688] N-Benzyl-6-(4-(6-((4,4-dimethylpiperidin-1-yl)methyl)pyridin-3-yl)-1-oxa-4,9-diazaspiro[5.5]undec-9-yl)pyrimidin-4-amine (7):

[0689]

[0690] The corresponding Boc-protected amine was obtained according to the general procedure of Buchwald-Hartwig coupling (chromatography: EtOAc / heptane=7:3 to 9:1 to EtOAc / MeOH=100:0 to 100:1 to 100:5). The impure desired product was used directly in the next step without further purification. The corresponding amine was obtained according to the general procedure of Boc group deprotection. After evaporation, the crude residue was ground in acetone, and the resulting precipitate was filtered, washed with acetone and dried to give the impure desired product, which was used in the next step without further purification.

[0691] To a stirred solution of the corresponding amine (1 eq.) in iPrOH (0.3 M) was added 29 (1.5 eq.) and Et3N (4 eq.). The reaction mixture was stirred at 150 °C in a microwave for 8 h 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 MgSO4, 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 give the desired product as a brown solid (6% over three steps). Mp: 61-62 °C; HRMS (ESI) m / z [C 32 H 44 N7O] + Calculated value: 542.3607, measured value: 542.3602.

[0692] N-Benzyl-6-chloropyrimidin-4-amine (29):

[0693]

[0694] Benzylamine (1.2 eq., 40.3 mmol, 4.4 mL) and Et3N (1.2 eq., 40.3 mmol, 5.59 mL) were added to a stirred solution of 4,6-dichloro-pyrimidine (5 g, 33.6 mmol) in iPrOH (100 mL). The reaction mixture was stirred at 25 °C for 3 d and concentrated under reduced pressure. The crude residue was ground in water, filtered and dried to give the desired product (7.21 g, 98% yield) as a beige solid. LRMS (ESI) m / z [C 11 H 11 ClN3] + Calculated value: 220.1, Measured value: 220.1

[0695] 9-(6-(Benzylamino)pyrimidin-4-yl)-4-(6-((4,4-dimethylpiperidin-1-yl)methyl)pyridin-3-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (8):

[0696]

[0697] According to the S NThe general procedure of Ar obtained compound 8 (chromatography: DCM / MeOH=100:5 to 100:10 to 100:15 to 100:20 to 100:30). The impure product obtained was triturated in diethyl ether, filtered, washed twice with diethyl ether, once with water, and once with diethyl ether to give the desired product as a white solid (14 mg, 25% yield). Mp: 208-209 ° C; HRMS (ESI) m / z [C 32 H 43 N8O] + Calculated value: 555.3560, measured value: 555.3554.

[0698] 9-(6-(Benzylamino)pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (9):

[0699]

[0700] According to the S N The general procedure for Ar afforded the corresponding chloropyrimidine. Instead of chromatography, after evaporation, the crude residue was triturated in water, filtered and washed once with water. The obtained sticky solid was dissolved in MeOH and concentrated under reduced pressure to give the desired product, which was used in the next step without further purification.

[0701] According to the S N General procedure of Ar obtained compound 9 (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 impure product obtained was triturated in water, filtered, and washed once with water to give the desired product as a light yellow solid (5% yield over two steps). Mp: 228-231 ° C; HRMS (ESI) m / z [C 33 H 44 N7O] + Calculated value: 554.3607, measured value: 554.3602.

[0702] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-9-(6-(methylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (10):

[0703]

[0704] The corresponding chloropyrimidine batch was identical to the one used for compound 9.

[0705] According to the S N Ar General Procedure Compound 10 was obtained (chromatography: DCM / MeOH = 100:0 to 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 [C 27 H 40 N7O] + Calculated value: 478.3294, measured value: 478.3289.

[0706] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-9-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (17):

[0707]

[0708] Under nitrogen atmosphere, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1 equivalent, 0.21mmol, 32mg) was added to a solution of 36 (100mg, 0.21mmol) in anhydrous THF (700 μL) stirred. Nitrogen was bubbled through the reaction for two minutes, and Ruphos Pd G4 (5mol%, 0.011mmol, 8.9mg), Ruphos (5mol%, 0.011mmol, 5.1mg) and LiHMDS (6.6 equivalents, 1.39mmol, 1.39mL, 1M THF) were added. The reaction mixture was stirred at 65°C for 4h, cooled 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 obtain the desired product (37mg, 36% yield) as a yellow solid. Mp: 250-252℃; HRMS(ESI)m / z[C 28 H 38 N7O] + Calculated value: 488.3138, measured value: 488.3132.

[0709] 9-(2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (19):

[0710]

[0711] 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (1.2 eq., 0.25 mmol, 47 mg) and Et3N (4 eq., 0.84 mmol, 116 μL) were added to a stirred solution of 36 (100 mg, 0.21 mmol) in iPrOH (1 mL). The reaction mixture was stirred at 100 °C for 3 h and at 130 °C for another 3 h, both in a microwave. The reaction mixture was concentrated under reduced pressure and the crude residue was triturated in water. The resulting precipitate was filtered, washed with water and dried to give the impure desired 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 give the desired product as a beige solid (46 mg, 42% yield). Mp: 199-201 °C; HRMS (ESI) m / z [C 28 H 37 ClN7O] + Calculated value: 522.2748, measured value: 522.2731.

[0712] 9-(6-(Cyclopropylamino)pyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (16):

[0713]

[0714] According to the S N General procedure of Ar obtained compound 16 (chromatography: DCM / MeOH = 100:0 to 100:10 (in 15 min), 100:10 (for 10 min), 100:10 to 100:15 (in 15 min)). Brown solid (25% yield). Mp: 172-174 ° C; HRMS (ESI) m / z [C 29 H 42 N7O] + Calculated value: 504.3451, measured value: 504.3445.

[0715] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-9-(6-(isopropylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (15):

[0716]

[0717] According to the S NGeneral procedure of Ar obtained compound 15 (chromatography: DCM / MeOH = 100:0 to 100:8 (in 15 min), 100:8 (for 10 min), 100:8 to 100:10 (in 10 min)). Beige solid (52% yield). Mp: 175-176 ° C; HRMS (ESI) m / z [C 29 H 44 N7O] + Calculated value: 506.3607, measured value: 506.3602.

[0718] 1-(4-Bromobenzyl)-4,4-dimethylpiperidine (23):

[0719]

[0720] Intermediate 23 was obtained following the general procedure for dimethylpiperidinyl alkylation (chromatography: EtOAc / heptane = 0:10 to 3:7). Yellow oil, 99% yield. LRMS (ESI) m / z [C 14 H 21 BrN] + Calculated value: 282.1, measured value: 282.1.

[0721] 5-Chloro-2-((4,4-dimethylpiperidin-1-yl)methyl)pyridine (26):

[0722]

[0723] To a stirred solution of (5-chloropyridin-2-yl)methanol (2.43 g, 17 mmol) in DCM (40 mL) was added SOCl (1.5 eq., 25.5 mmol, 1.85 mL) and DMF (1 drop). The reaction mixture was stirred at 25 °C for 2 h and concentrated under reduced pressure to give the chloroalkane, which was used in the next step without further purification.

[0724] Intermediate 26 was obtained following the general procedure for dimethylpiperidinyl 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 [C 13 H 20 ClN2] + Calculated value: 239.1, measured value: 239.2

[0725] 9-(6-chloropyrimidin-4-yl)-4-(6-((4,4-dimethylpiperidin-1-yl)methyl)pyridin-3-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (34):

[0726]

[0727] The corresponding Boc-protected amine was obtained according to the general procedure of Buchwald-Hartwig coupling (chromatography: DCM / MeOH=100:2 to 100:5 to 100:8 to 100:12 to 100:15 to 100:20). The impure desired product was used directly in the next step without further purification. The corresponding amine was obtained according to the general procedure of Boc group deprotection. After evaporation, the crude residue was ground in acetone, and the resulting precipitate was filtered, washed with acetone and dried to give the impure desired product, which was used in the next step without further purification.

[0728] According to the S N The general procedure for Ar afforded intermediate 34 (column chromatography: DCM / MeOH = 100:2 to 100:5 to 100.8 to 100:10 to 100:15 to 100:20) to give the desired product (123 mg, 27% yield over three steps) as a yellow solid. LRMS (ESI) m / z [C 25 H 35 ClN7O] + Calculated value: 484.3, found value: 484.3. tert-Butyl 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecane-9-carboxylate (35):

[0729]

[0730] Intermediate 35 was obtained following the general procedure of Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:5 to 100:8 to 100:12 to 100:20). Beige solid, 93% yield.

[0731] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one hydrochloride (36):

[0732]

[0733] Intermediate 36 was obtained following the general procedure for deprotection of the Boc group. After evaporation, the crude residue was triturated in acetone and the resulting precipitate was filtered, washed with acetone and dried to give the desired product as a beige solid (50% yield over two steps from intermediate 32). LRMS (ESI) m / z [C 22 H 35 N4O] +Calculated value: 371.3, measured value: 371.3.

[0734] 9-(6-chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (37):

[0735]

[0736] According to the S N The general procedure of Ar afforded intermediate 37. Instead of chromatography, after evaporation of the crude mixture, the residue was triturated in water. The resulting precipitate was filtered, washed with water and dried to give the desired product as a brown solid (63% yield). LRMS (ESI) m / z [C 26 H 36 ClN6O] + Calculated value: 483.3, measured value: 483.3.

[0737] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2-fluorophenyl)-9-(6-(methylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (20):

[0738]

[0739] According to the S N Ar General Procedure Compound 20 was obtained (chromatography: DCM / MeOH = 100:0 to 100:8 (in 20 min), 100:8 (for 15 min)). White solid (47% yield). Mp: 202-204 °C; HRMS (ESI) m / z [C 27 H 39 FN7O] + Calculated value: 496.3200, measured value: 496.3195.

[0740] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-3-fluorophenyl)-9-(6-(methylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (21):

[0741]

[0742] According to the S NAr General Procedure Compound 21 was obtained (chromatography: DCM / MeOH = 100:0 to 100:8 (in 20 min), 100:8 (for 5 min), 100:8 to 100:12 (in 10 min), 100:12 (for 5 min)). Yellow solid, 69% yield. Mp: 210-211 °C; HRMS (ESI) m / z [C 27 H 39 FN7O] + Calculated value: 496.3200, measured value: 496.3195.

[0743] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-9-(6-(methylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (22):

[0744]

[0745] The corresponding Boc-protected amine was obtained following the general procedure of Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:3 to 100:5 to 100:8).The impure desired product was used directly in the next step without further purification.

[0746] The corresponding amine was obtained following the general procedure for deprotection of the Boc group. The impure desired product was used directly in the next step without further purification.

[0747] According to the S N The general procedure for Ar afforded the corresponding chloropyrimidines. The impure desired product was used directly in the next step without further purification.

[0748] According to the S N General procedure for Ar UZH2 was obtained (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 [C 27 H 38 F2N7O] + Calculated value: 514.3106, measured value: 514.3100.

[0749] 1-(4-Bromo-3-fluorobenzyl)-4,4-dimethylpiperidine (47):

[0750]

[0751] Intermediate 47 was obtained according to the general procedure of dimethylpiperidinyl alkylation (column chromatography: EtOAc / heptane = 1:9). Colorless oil, 98% yield. LRMS (ESI) m / z [C 14 H 20 BrFN] + Calculated value: 300.1, measured value: 300.1.

[0752] 9-(6-chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2-fluorophenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (48):

[0753]

[0754] The corresponding Boc-protected amine was obtained following the general procedure of Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:3 to 100:5 to 100:8).The impure desired product was used directly in the next step without further purification.

[0755] The corresponding amine was obtained following the general procedure for deprotection of the Boc group. The impure desired product was used directly in the next step without further purification.

[0756] According to the S N The general procedure for Ar afforded intermediate 48 (column chromatography: DCM / MeOH = 100:3 to 100:5 to 100.8 to 100:10) to give the desired product as a white solid (15% yield over three steps). LRMS (ESI) m / z [C 26 H 35 ClFN6O] + Calculated value: 501.3, measured value: 501.3.

[0757] 1-(4-Bromo-2-fluorobenzyl)-4,4-dimethylpiperidine (49):

[0758]

[0759] Intermediate 49 was obtained following the general procedure for dimethylpiperidinyl alkylation. Colorless liquid, 99% yield. LRMS (ESI) m / z [C 14 H 20 BrFN] + Calculated value: 300.1, found value: 300.1. tert-Butyl 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-3-fluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecane-9-carboxylate (50):

[0760]

[0761] Intermediate 50 was obtained following the general procedure of Buchwald-Hartwig coupling (chromatography: DCM / MeOH=100:5 to 100:8 to 100:12 to 100:16). Brown solid (83% yield). LRMS (ESI) m / z [C 27 H 42 FN4O3] + Calculated value: 489.3, measured value: 489.4.

[0762] 9-(6-chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-3-fluorophenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (51):

[0763]

[0764] The corresponding amine was obtained following the general procedure for deprotection of the Boc group. The impure desired product was used directly in the next step without further purification.

[0765] According to the S N The general procedure of Ar afforded intermediate 51. Instead of chromatography, after evaporation of the crude mixture, the residue was triturated in water. The resulting precipitate was filtered, washed with water and dried to give the desired product as a brown solid (58% yield over two steps). LRMS (ESI) m / z [C 26 H 35 ClFN6O] + Calculated value: 501.3, found: 501.3. 1-(4-bromo-2,5-difluorobenzyl)-4,4-dimethylpiperidine (52):

[0766]

[0767] Under nitrogen atmosphere, to a stirred solution of 4-bromo-2,5-difluorobenzoic acid (1 g, 4.2 mmol) in anhydrous THF (10 mL) was added BH3.SMe2 (2 equiv., 8.4 mmol, 4.2 mL, 2M THF). The reaction mixture was stirred at 25 °C for 17 h, cooled to 0 °C and quenched by adding saturated Na2CO3 aqueous solution. The aqueous layer was extracted three times with EtOAc, and the combined organic layers were washed once with brine, dried over MgSO4 and concentrated under reduced pressure to give the desired product (789 mg, 83% yield) as a brown solid.

[0768] To a stirred solution of the corresponding alcohol (789 mg, 3.54 mol) in DCM (10 mL) was added SOCl (1.5 eq., 5.3 mmol, 385 μL) and DMF (1 drop). The reaction mixture was stirred at 25 °C for 3 h and concentrated under reduced pressure to give the desired chloroalkane, which was used in the next step without further purification.

[0769] 4,4-Dimethylpiperidine hydrochloride (1 eq., 1.76 mmol, 263 mg) and K2CO3 (2 eq., 3.52 mmol, 486 mg) were added to a stirred solution of the corresponding chloroalkane (425 mg, 1.76 mmol) in dimethylformamide (5 mL). 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 give the desired product (514 mg, 92%) as a colorless liquid. LRMS (ESI) m / z [C 14 H 19 BrF2N] + Calculated value: 318.1, Measured value: 318.1

[0770] 8-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-(6-(methylamino)pyrimidin-4-yl)-2,5,8-triazaspiro[3.5]nonan-6-one (11):

[0771]

[0772] The corresponding Boc protected amine was obtained following the general procedure of Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:5 to 100:8 to 100:11 to 100:15).The impure desired product was used directly in the next step without further purification.

[0773] The corresponding amine was obtained following the general procedure for deprotection of the Boc group. The impure desired product was used directly in the next step without further purification.

[0774] According to the S N The general procedure of Ar afforded the corresponding chloropyrimidine. Since the 40 derivative impurity was still present, 7 equivalents of pyrimidine and 7 equivalents of Et3N were used, and the reaction was heated in a microwave at 80°C for 7 h. The impure desired product was used directly in the next step without further purification.

[0775] According to the S NAr General procedure to obtain compound 11 (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 [C 25 H 36 N7O] + Calculated value: 450.2981, measured value: 450.2976.

[0776] tert-Butyl 3-amino-3-(nitromethyl)azetidine-1-carboxylate (38):

[0777]

[0778] MeNO2(13mL) and K2CO3(1mol%, 0.62mmol, 86mg) are added to the solution of tert-butyl 3-oxoazetidine-1-formate (10.65g, 62mmol) in EtOH (31mL) under stirring. The reaction mixture is stirred for 17h at 25°C and filtered. The filtrate is concentrated under reduced pressure to obtain the desired product, which is used in the next step without further purification. Under a nitrogen atmosphere and cooled to -78°C, DAST (1.2 equivalents, 74.4mmol, 9.8mL) is added dropwise to the solution of the corresponding alcohol (62mmol) in anhydrous DCM (250mL) under stirring. The cooling bath is removed and the reaction mixture is stirred for 3h, cooled to 0°C and slowly quenched by adding saturated NaHCO3 aqueous solution. The water layer is extracted three times with DCM, washed once with brine, dried over MgSO4, filtered and concentrated under reduced pressure to obtain the desired product, which is used in the next step without further purification.

[0779] The corresponding nitromethylene group (62 mmol) was dissolved in ammonia (17.7 mL, 7N in MeOH) and the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give the desired product as an orange solid (15.67 g, quantitative yield over three steps). tert-Butyl 3-(aminomethyl)-3-(((benzyloxy)carbonyl)amino)azetidine-1-carboxylate (39):

[0780] To a stirred solution of 38 (62 mmol) in dichloromethane (100 mL) was added a solution of NaHCO (2 eq., 124 mmol, 10.42 g) in water (100 mL). The reaction mixture was cooled to 0 °C and CbzCl (1 eq., 62 mmol, 8.8 L) 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 twice with DCM. The combined organic layers were washed once with brine, dried over MgSO, filtered and concentrated under reduced pressure to give the desired product, which was used in the next step without further purification.

[0781] Under nitrogen atmosphere, at 0 ℃, NiCl2.6H2O (1 eq., 62mmol, 16.9g) was added to a stirred solution of the corresponding nitroalkane (62mmol) in anhydrous MeOH (300mL), followed by addition of NaBH4 (5 eq., 310mmol, 11.7g) in portions to avoid strong H2 evolution. Be careful when adding NaBH4, the reaction is highly exothermic and produces hydrogen. The reaction mixture was stirred at 25 ℃ for 1h and quenched by adding saturated NaHCO3 aqueous solution. The mixture was filtered through a celite pad, the filtrate was concentrated under reduced pressure, and the obtained residue was diluted with brine and saturated Na2CO3 aqueous solution. The aqueous layer was extracted three times with DCM, and the combined organic layer was washed once with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM / MeOH / NH4OH = 100:3:0 to 100:3:1 to 100:5:1 to 100:8:1 to 100:12:1 to 100:20:1) to give the desired product (11.3 g, 54% yield over two steps) as a white solid. LRMS (ESI) m / z [C 34 H 51 N6O8] + =[2M+H] + Calculated value: 671.4, found value: 671.4. tert-Butyl 6-oxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (40):

[0782]

[0783] To a stirred solution of 39 (11.3 g, 33.7 mmol) in DCM (110 mL) was added EtN (1 eq., 33.7 mmol, 4.7 mL) and ethyl 2-bromoacetate (1 eq., 33.7 mmol, 3.7 mL) at 0°C. The reaction mixture was stirred at 25°C for 17 h and diluted with saturated aqueous NaHCO. The aqueous layer was extracted three times with DCM, and the combined organic layers were washed once with water, once with brine, dried over MgSO, filtered and concentrated under reduced pressure to give the impure desired product (12.4 g, 29 mmol), which was used in the next step without further purification.

[0784] Pd / C (5 mol%, 1.5 mmol, 1.6 g, 10 wt%) and ammonium formate (6 equivalents, 174 mmol, 11 g) were added in batches to a stirred solution of the corresponding Cbz protected amine (29 mmol) in iPrOH (240 mL). The reaction mixture was stirred at 80 ° C for 4 h, cooled to 25 ° C, filtered through a celite pad and concentrated under reduced pressure. The obtained residue was distributed between DCM and water, the two phases were separated, and the aqueous layer was extracted three times with DCM. The combined organic layer was washed once with water, once with brine, dried over MgSO4, 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 obtain the desired product (2.6 g, 32% yield over two steps) as a white solid. LRMS (ESI) m / z [C7H 12 N3O3] + =[M-tBu+2H] + Calculated value: 186.1, measured value: 186.2

[0785] 4-(4-((4-fluoro-4-methylpiperidin-1-yl)methyl)phenyl)-9-(6-(methylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (53):

[0786]

[0787] According to the S N The general procedure of Ar gave compound 53 (chromatography: DCM / MeOH = 100:0 to 100:7 (in 20 min), 100:7 (for 5 min), 100:7 to 100:8 (in 5 min), 100:8 (for 10 min)). Pale yellow solid, 53% yield. Mp: 203-205 °C; HRMS (ESI) m / z [C 26 H 37FN7O] + Calculated value: 482.3044, measured value: 482.3038.

[0788] 9-(6-Chloropyrimidin-4-yl)-4-(4-((4-fluoro-4-methylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (54)

[0789]

[0790] The corresponding Boc protected amine was obtained following the general procedure of Buchwald-Hartwig coupling (chromatography: EtOAc / heptane = 7:3 to 9:1 to EtOAc / MeOH = 100:3 to 100:5 to 100:8 to 100:10). The impure desired product was used directly in the next step without further purification.

[0791] The corresponding amine was obtained following the general procedure for deprotection of the Boc group. The impure desired product was used directly in the next step without further purification.

[0792] According to the S N General procedure for Ar afforded intermediate 54 (column chromatography: DCM / MeOH = 100:3 to 100:5 to 100.8) to give the desired product as a white solid (12% yield over three steps).

[0793] 1-(4-Bromobenzyl)-4-fluoro-4-methylpiperidine (55):

[0794]

[0795] DAST (1.5 equivalents, 3.48 mmol, 460 μL) was added to a stirred solution of tert-butyl 4-hydroxy-4-methylpiperidine-1-carboxylate (500 mg, 2.32 mmol) in anhydrous DCM (7 mL) at 0 ° C under a nitrogen atmosphere. The mixture was stirred for 3 h at 25 ° C and quenched by adding a saturated aqueous solution of NaHCO. The two phases were separated and the aqueous layer was extracted twice with DCM. The combined organic layers were washed with brine, dried over MgSO, filtered and concentrated under reduced pressure to obtain the desired product, which was used in the next step without further purification.

[0796] The corresponding amine was obtained following the general procedure for deprotection of the Boc group. The impure desired product was used directly in the next step without further purification.

[0797] Intermediate 55 was obtained following the general procedure of dimethylpiperidine alkylation. (Column chromatography: EtOAc / heptane = 1:9 to 3:7 to 1:1). Yellow oil, 77% yield over three steps.

[0798] 4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-9-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (56):

[0799]

[0800] 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (1.2 eq., 0.38 mmol, 58 mg) and Et3N (4 eq., 1.24 mmol, 172 μL) were added to a stirred solution of 36 (150 mg, 0.31 mmol) in iPrOH (1.5 mL). The reaction mixture was stirred at 50 °C for 3 h and at 70 °C for another 2 h, both in a microwave. The reaction mixture was concentrated under reduced pressure and the crude residue was ground in water. The resulting precipitate was filtered, washed with water, washed with DCM and dried to give the impure desired 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 give the desired product (33 mg, 22% yield) as a yellow solid. Mp: 252-254℃; HRMS(ESI)m / z[C 27 H 37 N8O] + Calculated value: 489.3090, measured value: 489.3085

[0801] Table 8: IC50 data of N6-adenosine-methyltransferase

[0802]

[0803]

[0804]

[0805] Table 9: IC50 data of exemplary PROTAC compounds for N6-adenosine-methyltransferase

[0806]

[0807]

[0808]

[0809]

[0810] Reader-based HTRF assay for in vitro METTL3 inhibition by homogeneous time-resolved fluorescence (HTRF) measurement of the binding of modified oligoribonucleotides to m 6 A reader specifically binds to YTHDC1 to quantify m in oligoribonucleotide substrates after METTL3-METTL14 catalysis 6 The levels of A. Test compounds that inhibit METTL3 reduce m 6 A levels and thereby reduce the HTRF signal. The two-step protocol for the METTL3-METTL14 assay consists of a reaction step and a subsequent detection step.

[0811] In the reaction step, METTL3-METTL14 (final concentration 3 nM) methylated 5′-biotinylated ssRNA (5′-AAGAACCGGACUAAGCU-3′ (Microsynth)) (final concentration 50 nM). The co-substrate SAM (Cisbio, 62SAHZLD) was added as the last component and the methylation reaction was initiated. The final reaction volume was 15 μL in 20 mM Tris-HCl, pH 7.5, 0.01% (w / v) bovine serum albumin (BSA). The reaction was incubated at room temperature (RT) for 40 min and then detected by adding 5 μL of detection buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 200 mM KF, 0.05% (w / v) BSA, 25 nM GST-labeled m 6 A reader YTHDC1 (345-509), 3 nM XL665-conjugated streptavidin (Cisbio, 610SAXLB), 1x anti-GST Eu 3+ The labeled antibody (from 400x stock (Cisbio, 61GSTKLB)) was used to stop. At room temperature and in the dark, the cells were allowed to grow by m 6 A reader captures m 6A modified RNA and biotinylated RNA captured by streptavidin for 3h, then the TR-FRET signal was measured using a Tecan Spark plate reader (Tecan). After the HTRF donor was excited with ultraviolet light at 320nm, the plate reader recorded the emission at 620 and 665nm with a delay of 100μs. This time-resolved measurement eliminates the transient background fluorescence of the signal. The emission signal is read within an integration time of 400μs. The ratio of 665 / 620nm signals (HTRF ratio) allows correction of well-to-well variations and medium absorbance of colored compounds. The response curve is plotted in GraphPad Prism8.4 and fitted with nonlinear regression "log (inhibitor) to normalized response variable slope", from which the IC50 value is determined. Each compound is measured in triplicate.

[0812] The results of IC50 values ​​are shown in Table 9.

[0813] Screening PROTAC activity by Western blot

[0814] METTL3 (and METTL14) protein degradation was monitored using Western blotting. Cells were treated with the indicated concentrations of PROTAC (or DMSO, control) at 37°C and 5% CO2 for 24 h. Samples were then collected and lysed with RIPA buffer (11697498001, Roche) containing protease inhibitors. After SDS-PAGE, proteins were transferred to nitrocellulose membranes, blocked (with 5% milk, 0.5% BSA in TBST buffer) and incubated with primary antibodies overnight. The following antibodies were used: GAPDH (#2118, Cell Signaling, 1:4000), β-actin (ab8226, Abcam, 1:2000), METTL3 (ab195352, Abcam, 1:1000), METTLS14 (ab220031, Abcam, 1:1000). Incubate with appropriate secondary antibodies (anti-mouse IgG 680RD (926-68072, LI-COR, 1:10000), goat anti-rabbit IgG After incubation with 800CW (926-32211, LI-COR, 1:10000), the membrane was scanned using a LI-COR Odyssey DLx Imager. Densitometry was performed in Image Studio Lite software and analyzed in GraphPad Prism 9.

[0815] Fig. 9 Shown is the degradation of METTL3 protein measured by western blot after treatment with various PROTAC molecules at 2 μM concentration for 24 h.

[0816] Fig.10 Shown is the METTL14 protein measured by western blot after treatment with various PROTAC molecules at 2 μM concentration for 24 h.

[0817] Fig.11 Shown is the correlation between METTL3 and METTL14 degradation by various PROTAC molecules measured by western blotting.

Claims

1. A compound of general formula (A) or a pharmaceutically acceptable salt thereof, in -NR 31 R 32 Selected from - Each R 2 Independently selected from F, Cl, CF3, CHF2, CH2F, in particular each R 2 is F; - n is an integer selected from 0, 1, 2, 3 and 4, in particular n is an integer selected from 0, 1 and 2, more in particular n is 2; The handle is a linking moiety comprising or essentially consisting of 3 to 10 atoms (C, N, O, S) with an atomic mass ≥ 12, in particular 4 to 8 atoms with an atomic mass ≥ 12; - the linker is a linker moiety comprising or essentially consisting of 3 to 50 atoms with an atomic mass ≥ 12, particularly 4 to 30 atoms with an atomic mass ≥ 12, more particularly 5 to 20 atoms with an atomic mass ≥ 12; - An E3 ligase binder is a moiety that specifically binds to an E3 ligase.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the E3 ligase binder has formula (B): in -Ox is CH2 or C=O; - T is selected from F, Cl, in particular T is F; - k is an integer selected from 0, 1, 2, in particular 0, 1, more particularly k is 0; - represents a bond to the linker.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein k is 0.

4. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the handle comprises or consists essentially of 1, 2, 3 or 4 chemical moieties selected from alkyl, amine, phenyl and carbonyl.

5. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the handle is selected from the following formula: in -Mid is selected from the group consisting of C1-C3 alkyl and phenyl.

6. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the handle is selected from the following formula:

7. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein the handle is of formula (X): Wherein Mid is selected from C1-C3 alkyl and phenyl.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the handle is of formula (Y):

9. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the linker comprises or consists essentially of 1, 2, 3, 4, 5, 6 or 7 chemical moieties independently selected from alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkenylene, alkynylene, ethylene glycol, carbonyl, ether, ester, amine, amide, sulfonamide, wherein each of the chemical moieties is independently unsubstituted or substituted with C1-C3 alkyl, halogen, CN, NO2, hydroxyl, amine, sulfate, phosphate and / or carboxyl; In particular wherein the linker comprises or essentially consists of 1 , 2, 3 or 4 chemical moieties selected from alkyl, ethylene glycol, carbonyl, piperazine, aryl, amine, triazole.

10. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the linker is selected from the following formula: in -Lin is selected from C3-C 20 Alkyl, C3-C 20 Alkyl-triazole, oligo(ethylene glycol).

11. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the linker is selected from the following formula: in -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 or 20.

12. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, 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 the group consisting of 11, 12, 13, 14, 15, 16 and 17; s is selected from 7, 8, 9, 10, 11, 12 and 13; t is selected from the group consisting of 3, 4, 5, 6, 7, 8, and 9; and u is selected from 7, 8, 9, 10, 11, 12 and 13.

13. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein the linker comprises formula (W): Where Lin is selected from C3-C 20 Alkyl, C3-C 20 Alkyl-triazoles and oligo(ethylene glycol).

14. The compound according to claim 13 or a pharmaceutically acceptable salt thereof, wherein the linker comprises formula (Z): wherein z is selected from 4, 5, 6, 7, 8, 9 and 10.

15. The compound according to any one of the preceding claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide, in particular a peptide consisting of proteinogenic amino acids.

16. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein - said E3 ligase binder has formula (B) as defined in claim 2; and - the handle is as defined in claim 6; and - The linker has the definition as claimed in claim 11 or 12.

17. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein - said E3 ligase binder has formula (B) as defined in claim 2; and - the handle is as defined in claim 7 or 8; and - The linker has the definition as claimed in claim 11 or 12.

18. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein - said E3 ligase binder has formula (B) as defined in claim 2; and - the handle is as defined in claim 5; and - The linker has the definition as claimed in claim 13 or 14.

19. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the compound comprises the following definitions of the handle, linker and E3 ligase binder:

20. A compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, wherein NR 31 R 32 for 21. A compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, wherein NR 31 R 32 for 22. A compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, wherein NR 31 R 32 for Especially NR 31 R 32 for 23. A compound selected from Table 2 and Table 3 or a pharmaceutically acceptable salt thereof.

24. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, for use as a medicament.

25. A compound according to any one of the preceding claims 1 to 23, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

26. The compound for use according to claim 25, or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.

Images