N6-Adenosine-methyltransferase PROTAC and methods of using the same

Abstract

The present disclosure relates to a target protein degrading chimera (PROTAC) that modulates N6-adenosine-methyltransferase and methods of using the same.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of European Patent Application No. 22207794.3, filed on November 16, 2022, and International Application PCT / EP2022 / 063350, filed on May 17, 2022, and incorporates these in their entirety herein by reference.

[0002] The present disclosure relates to target protein - degrading chimeras (PROTACs) that modulate N6 - adenosine - methyltransferase and methods of using the same.

Background Art

[0003] Gene expression is regulated at the level of the transcriptome (messenger RNA obtained by transcription of the genome) by the dynamic levels of mRNA modification. The conversion from adenosine to N6 - methyladenosine (m 6 A) is the most common post - transcriptional internal modification in eukaryotic mRNA (also called epitranscriptome modification). This methylation event usually occurs within the consensus sequence motif of DRACH (D = A, G, U; R = A, G; H = A, C, U). The m 6 A level can vary between different tissues, developmental states, or in response to cellular stress. At the molecular level, the introduction of m 6 A affects the structure of RNA and its ability to form protein - RNA interactions, and as a result, regulates the processing, translation, and stability of cellular transcripts. Consequently, m 6 A is involved in the control of embryonic development processes and stem cell differentiation, the regulation of the mammalian circadian clock, and the modulation of stress responses, such as heat shock.

[0004] m 6 The dynamic level of m 6 A is regulated by the interaction between eraser and writer proteins. m 6The discovery of the A-specific eraser protein FTO (ALKBH9) and ALKBH5 finally demonstrated the reversibility of the modification and its regulatory role. These m 6 A demethylase belongs to the dioxygenase AlkB family, and its enzymatic reaction depends on Fe(II) and 2-oxoglutaric acid (2OG). The core writer complex is formed by two methyltransferase-like proteins, METTL3 and METTL14, and depends on additional cofactors for mRNA substrate recruitment, including WTAP and RBM15. The METTL3-METTL14 complex transfers a methyl group from S-adenosylmethionine (SAM) to adenosine within the consensus sequence of 5'-GGACU-3'. Only METTL3 has an intact SAM binding site, and METTL14 has a degenerate SAM binding site that is non-functional. When METTL3 or METTL14 is individually depleted, the m 6 A level decreases in HeLa cells. More importantly, the deregulation of METTL3 has only recently been found to be associated with certain tumors, such as acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma. Furthermore, m 6 A broad antiviral effect has been shown by inhibiting the modification. Therefore, small molecule modulators of the METTL3-METTL14 writer have potential therapeutic applications in cancer and viral infections.

[0005] Based on the above state of the art, an object of the present invention is to provide means and methods for using the PROTAC therapeutic modality to adjust the level of m6A modification for the purpose of regulating gene expression for cancer treatment. This object is achieved by the subject matter of the independent claims of this specification. Summary of the Invention Means for Solving the Problems

[0006] In one aspect, the present disclosure relates to bifunctional compounds that have been found to be useful for degrading and inhibiting N6-adenosine-methyltransferase. In some embodiments, the bifunctional compound targets the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compound binds to the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compound modulates the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compound inhibits and degrades the heterodimeric complex METTL3-METTL14. In some embodiments, the bifunctional compound targets METTL3. In some embodiments, the bifunctional compound binds to METTL3. In some embodiments, the bifunctional compound modulates, inhibits, and / or degrades METTL3. In some embodiments, the bifunctional compound targets METTL14. In some embodiments, the bifunctional compound binds to METTL14. In some embodiments, the bifunctional compound modulates, inhibits, and / or degrades METTL14.

[0007] The first aspect of the present disclosure is a compound of general formula (A)

[0008]

Chemical formula

[0009] relates to. The second aspect of the present disclosure is a compound of general formula (U)

[0010]

Chemical formula

[0011] relates to. The third aspect of the present disclosure relates to the compounds described in the first or second aspect for use as a medicament.

[0012] The fourth aspect of the present disclosure relates to the compounds described in the first or second aspect for use in the treatment of cancer. In another embodiment, the present disclosure relates to a pharmaceutical composition comprising at least one of the compounds 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

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0014] Detailed Description Terms and Definitions For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural and vice versa. If any of the definitions described below conflict with any document incorporated herein by reference, the definitions described below shall prevail.

[0015] As used herein, the terms "comprising", "having", "containing", and "including", and other similar forms, and their grammatical equivalents, have the same meaning and are intended to be open-ended, such that one or more items following any one of these words are not intended to mean that the listing of such one or more items is exhaustive or limited to only the one or more items listed. For example, a molded article "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. Thus, "comprises" and its similar forms, and their grammatical equivalents, are intended to and are understood to include the disclosure of "consisting essentially of" or "consisting of" embodiments.

[0016] When a range of values is indicated, unless otherwise clearly indicated in the context, each intervening value between the upper and lower limits of that range and any other stated value or intervening value within that specified range to one tenth of the unit of the lower limit is included in the present disclosure, it being understood that there may be excluded certain limitations within the specified range. When the stated range includes one or both of the limits, ranges excluding one or both of the included limits are also included in the present disclosure.

[0017] As used herein, the term "about" when used in reference to a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, the description of "about X" includes the description of "X".

[0018] As used in this specification, including the appended claims, the singular forms "a", "or", and "the" include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, 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 the fields of cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (generally see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Edition, John Wiley & Sons, Inc.) as well as for chemical methods.

[0019] As used in the context of this specification, the term METTL3 refers to the N6-adenosine-methyltransferase catalytic subunit (Uniprot ID: Q86U44). As used in the context of this specification, the term METTL14 refers to the N6-adenosine-methyltransferase non-catalytic subunit (Uniprot ID: Q9HCE5).

[0020] C in the context of this specification 1 ~C 6 Alkyl means a saturated straight-chain or branched hydrocarbon having 1, 2, 3, 4, 5, or 6 carbon atoms. In some embodiments, the alkyl is substituted, and being substituted means, for example, one or more CH 2It means that the moiety may be exchanged with oxygen (ether bridge) or nitrogen (NH, or NR where R is methyl, ethyl, or propyl; amino bridge).

[0021] The term C in the context of this specification 3 ~C 7 Cycloalkyl refers to a saturated hydrocarbon ring having 3, 4, 5, 6, or 7 carbon atoms, and in some embodiments, one carbon-carbon bond may be unsaturated. C 3 ~C 7 Non-limiting examples of cycloalkyl moieties include cyclopropanyl (-C 3 H 5 ), cyclobutanil (-C 4 H 7 ), cyclopentenyl (C 5 H 9 ) and cyclohexenyl (C 6 H 11 ) moieties. In some embodiments, cycloalkyl is substituted. In some embodiments, cycloalkyl is substituted by one C 1 ~C 4 unsubstituted alkyl moiety. In some embodiments, cycloalkyl is substituted by two or more C 1 ~C 4 unsubstituted alkyl moieties.

[0022] The term heterocyclic in the context of this specification refers to cycloalkyl in which at least one ring atom or several ring atoms are substituted by nitrogen, oxygen and / or sulfur atoms. The term heterobicycle in the context of this specification refers to two directly linked cycloalkyls in which at least one ring atom or several ring atoms are substituted by nitrogen, oxygen and / or sulfur atoms.

[0023] The term heterocycloalkyl in the context of this specification refers to cycloalkyl in which at least one ring atom or several ring atoms are substituted by nitrogen, oxygen and / or sulfur atoms.

[0024] When used in the narrowest sense in this specification, the term unsubstituted C n alkyl, when used as a crosslink between parts of a molecule, is -C n H 2n- part, or when used in the context of a terminal part, is -C n H 2n+1 with respect to the part.

[0025] The term unsubstituted C n alkyl and substituted C n alkyl includes linear alkyls that contain or are linked to cyclic structures such as cyclopropane, cyclobutane, cyclopentane or cyclohexane moieties, and are unsubstituted or substituted, with linear alkyl substitution, depending on the context of the annotation or reference. The total number of carbons and, where appropriate, N, O or other heteroatoms in the linear or cyclic structure is n.

[0026] When used in the context of chemical formulas, the following abbreviations can be used. Me is methyl CH 3 and Et is ethyl -CH 2 CH 3 and Prop is propyl -(CH 2 ) 2 CH 3 (n-propyl, n-pr) or -CH(CH 3 ) 2 (isopropyl, i-pr), and but is butyl -C 4 H 9 , -(CH 2 ) 3 CH 3 , -CHCH 3 CH 2 CH 3 , -CH 2 CH(CH 3 ) 2 or -C(CH 3 ) 3 .

[0027] In the broadest sense, a substituted alkyl refers to the broadest alkyl as defined above that is covalently bonded to an atom other than carbon or hydrogen, particularly an atom selected from N, O, F, B, Si, P, S, Cl, Br, and I, which itself may, if applicable, be bonded to one or several other atoms of this group, to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in the broadest sense). In a narrower sense, a substituted alkyl refers to an amine NH 2 , 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, fluoride F, chloride Cl, bromide Br, iodide I, phosphonate PO 3 H 2 , PO 3 R 2 , phosphate OPO 3 H 2 and OPO 3 R 2 , sulfhydryl SH, sulfalkyl SR, sulfoxide SOR, sulfonyl SO 2 R, sulfanilamide SO 2 NHR, sulfate SO 3 H and sulfate ester SO 3 R (R is further defined in the embodiments for carrying out the invention), which refers to the broadest alkyl as defined above substituted at one or several carbon atoms by a group selected from these.

[0028] The term hydroxyl substituent refers to a group modified by one or several hydroxyl groups OH. The term amino substituent refers to a group modified by one or several amino groups NH 2 .

[0029] The term "carboxyl substituent" refers to a group modified by one or several carboxyl groups COOH. Non-limiting examples of amino-substituted alkyls include, for the terminal part, -CH 2 NH 2 、-CH 2 NHMe, -CH 2 NHEt, -CH 2 CH 2 NH 2 、-CH 2 CH 2 NHMe, -CH 2 CH 2 NHEt, -(CH 2 ) 3 NH 2 、-(CH 2 ) 3 NHMe, -(CH 2 ) 3 NHEt, -CH 2 CH(NH 2 )CH 3 、-CH 2 CH(NHMe)CH 3 、-CH 2 CH(NHEt)CH 3 、-(CH 2 ) 3 CH 2 NH 2 、-(CH 2 ) 3 CH 2 NHMe, -(CH 2 ) 3 CH 2 NHEt, -CH(CH 2 NH 2 )CH 2 CH 3 、-CH(CH 2 NHMe)CH 2 CH 3 、-CH(CH 2 NHEt)CH 2 CH 3 、-CH 2 CH(CH 2 NH 2 )CH 3 、-CH 2 CH(CH 2 NHMe)CH 3 、-CH2 CH(CH 2 NHEt)CH 3 、-CH(NH 2 )(CH 2 ) 2 NH 2 、-CH(NHMe)(CH 2 ) 2 NHMe、-CH(NHEt)(CH 2 ) 2 NHEt、-CH 2 CH(NH 2 )CH 2 NH 2 、-CH 2 CH(NHMe)CH 2 NHMe、-CH 2 CH(NHEt)CH 2 NHEt、-CH 2 CH(NH 2 )(CH 2 ) 2 NH 2 、-CH 2 CH(NHMe)(CH 2 ) 2 NHMe、-CH 2 CH(NHEt)(CH 2 ) 2 NHEt、-CH 2 CH(CH 2 NH 2 ) 2 、-CH 2 CH(CH 2 NHMe) 2 and -CH 2 CH(CH 2 NHEt) 2 are included. For the amino - substituted alkyl moieties that cross - link the other two moieties, -CH 2 CHNH 2 -, -CH 2 CHNHMe -, -CH 2 CHNHEt - are included.

[0030] Non - limiting examples of hydroxy - substituted alkyl for the terminal moiety include -CH 2 OH, -(CH 2 ) 2 OH, -(CH 2 )3 OH, -CH 2 CH(OH)CH 3 , -(CH 2 ) 4 OH, -CH(CH 2 OH)CH 2 CH 3 , -CH 2 CH(CH 2 OH)CH 3 , -CH(OH)(CH 2 ) 2 OH, -CH 2 CH(OH)CH 2 OH, -CH 2 CH(OH)(CH 2 ) 2 OH and -CH 2 CH(CH 2 OH) 2 are mentioned. For the hydroxyl-substituted alkyl moieties that crosslink the other two moieties, -CHOH-, -CH 2 CHOH-, -CH 2 CH(OH)CH 2 -, -(CH 2 ) 2 CHOHCH 2 -, -CH(CH 2 OH)CH 2 CH 2 -, -CH 2 CH(CH 2 OH)CH 2 -, -CH(OH)(CH 2 CH)OH-, -CH 2 CH(OH)CH 2 OH, -CH 2 CH(OH)(CH 2 ) 2 OH and -CH 2 CHCH 2 OHCHOH- are mentioned.

[0031] As defined above, haloalkyl refers to an alkyl group substituted by one or more halo groups, such as trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0032] The term sulfoxyl substituent refers to a group modified by one or several sulfoxyl groups -SO 2 R, or a derivative thereof, where R is further defined in the context of the embodiments for carrying out the invention.

[0033] The term sulfonamide substituent refers to a group modified by one or several sulfonamide groups -SO 2 NHR or -NHSO 2 R, or a derivative thereof, where R is further defined in the context of the embodiments for carrying out the invention.

[0034] The term amine substituent refers to a group modified by one or several amine groups -NHR or -NR 2 and a derivative thereof, where R is further defined in the context of the embodiments for carrying out the invention.

[0035] The term carbonyl substituent refers to a group modified by one or several carbonyl groups -COR, or a derivative thereof, where R is further defined in the context of the embodiments for carrying out the invention.

[0036] Ester refers to a -CO 2 R group, where R is further defined in the context of the embodiments for carrying out the invention. Ether refers to a group having one oxygen between two saturated carbon atoms.

[0037] Amide refers to a -CONHR group, where R is further defined in the context of the embodiments for carrying out the invention. Ethylene glycol refers to -(CH 2 -CH 2 -O)n - or -(O-CH 2 -CH 2 ) n - group, and n is further defined in the form for carrying out the invention.

[0038] Alkylyne refers to a group of -C≡C- (triple bond between two carbon atoms). Alkylene refers to a group of -CH=CH- (double bond between two carbon atoms).

[0039] The term halogen substituent refers to a group modified by one or several halogen atoms (independently) selected from F, Cl, Br, I. The term fluoro-substituted alkyl refers to an alkyl as defined above, modified by one or several fluoride groups F. Non-limiting examples of fluoro-substituted alkyl include -CH 2 F, -CHF 2 , -CF 3 , -(CH 2 ) 2 F, -(CHF) 2 H, -(CHF) 2 F, -C 2 F 5 , -(CH 2 ) 3 F, -(CHF) 3 H, -(CHF) 3 F, -C 3 F 7 , -(CH 2 ) 4 F, -(CHF) 4 H, -(CHF) 4 F and -C 4 F 9 are included.

[0040] Non-limiting examples of hydroxyl-substituted and fluoro-substituted alkyl include -CHFCH 2 OH, -CF 2 CH 2 OH, -(CHF) 2 CH 2 OH, -(CF 2 ) 2 CH2 OH, -(CHF) 3 CH 2 OH, -(CF 2 ) 3 CH 2 OH, -(CH 2 ) 3 OH, -CF 2 CH(OH)CH 3 , -CF 2 CH(OH)CF 3 , -CF(CH 2 OH)CHFCH 3 , and -CF(CH 2 OH)CHFCF 3 are included.

[0041] The term aryl in the context of this specification means a cyclic aromatic C 5 ~C 10 hydrocarbon. Examples of aryl include, but are not limited to, phenyl and naphthyl.

[0042] The term alkylaryl in the context of this specification relates to an alkyl group substituted by an aryl moiety. Specific examples include ethylphenyl, propylphenyl, butylphenyl and their higher homologs. A substituted alkylaryl may, if chemically feasible, be substituted by the substituents shown on the alkyl portion thereof or by the substituents shown on the aryl portion of that moiety.

[0043] Heteroaryl is an aryl containing one or several 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. An aryl or heteroaryl in the context of this specification may further be substituted by one or more alkyl groups.

[0044] As used in the context of this specification, the term alkyl heteroaryl relates 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, together with at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition according to the present disclosure is provided in a form suitable for topical administration, parenteral administration or administration by injection.

[0045] As used herein, the term pharmaceutically acceptable carrier includes any solvent, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, absorption delaying agent, salt, preservative, drug, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetening agent, flavoring agent, coloring agent, etc., and combinations thereof known to those skilled in the art (see, e.g., Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

[0046] As used herein, treating or treatment of any disease or disorder (e.g., cancer) refers, in one embodiment, to ameliorating the disease or disorder (e.g., delaying or halting or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may be undetectable in a patient. In yet another embodiment, "treating" or "treatment" refers to modulating a disease or disorder, either physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for evaluating the treatment and / or prevention of a disease.

[0047] PROTAC compound 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 that comprise (i) an active compound that targets the METTL3-METTL14 complex (see, e.g., the paragraph beginning with the subtitle "Active Compounds of PROTACs"), and (ii) an E3 ligase binding molecule (see, e.g., the paragraph beginning with the subtitle "E3 Ligase Binding Molecules"). In some embodiments, the PROTAC compounds further comprise a handle (see, e.g., the paragraph beginning with the subtitle "Handle"). In some embodiments, the PROTAC compounds further comprise a linker (see, e.g., the paragraph beginning with the subtitle "Linker").

[0048] The first aspect of the present disclosure is of general formula (A)

[0049] [Chemical formula]

[0050] [wherein, -NR 31 R 32 is an optionally substituted 3- to 12-membered heterocycloalkyl (e.g.,

[0051] [Chemical formula]

[0052] selected from; - Each R 2 is independently selected from the group consisting of halogen (e.g., F, Cl), C 1 ~C 3 alkyl, and C 1 ~C 3 haloalkyl (e.g., CF 3 , CHF 2 , CH 2 F); - n is an integer selected from 0, 1, 2, 3, and 4; - The handle is a connecting part containing or consisting essentially of 3 to 10 atoms (C, N, O, S) with an atomic mass ≥ 12; - The linker is a linker part containing or consisting essentially of 3 to 50 atoms with an atomic mass ≥ 12; - The E3 ligase-binding molecule is a part that binds to the E3 ligase] of the compound.

[0053] The first aspect of the present disclosure is of a compound of general formula (A)

[0054]

Chemical formula

[0055] [wherein, - NR 31 R 32 is

[0056]

Chemical formula

[0057] selected from; - Each R 2 is independently selected from the group consisting of F, Cl, CF 3 , CHF 2 , CH 2 F; - n is an integer selected from 0, 1, 2, 3 and 4; - The handle is a connecting part containing or consisting essentially of 3 to 10 atoms (C, N, O, S) with an atomic mass ≥ 12; - The linker is a linker part containing or consisting essentially of 3 to 50 atoms with an atomic mass ≥ 12; - The E3 ligase-binding molecule is a part that specifically binds to the E3 ligase] of the compound.

[0058] In another aspect, the present disclosure is of a compound of general formula (A-1)

[0059] [Chemical formula]

[0060] [wherein, -Z 1 and Z 2 are independently selected from N, CH, and CR 2 ; -X is O or NH; -Y is CH 2 , C=O, or SO 2 ; -R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl, and heterocyclic ring, and in particular, R 1 is unsubstituted or substituted heteroaryl; -R 2 is selected from F, Me, Cl, OH, NH 2 , Br, CF 3 , CHF 2 , CH 2 F; -n is an integer selected from 0, 1, 2, 3, and 4; -R 3 is a substituted alkylamine; -U and V are independently selected from -CH 2 - and -(CH 2 ) 2 -, or one of U and V is -CH 2 - and the other is -(CH 2 ) 3 -; -The handle is a connecting moiety containing or consisting essentially of 3 to 10 atoms with an atomic mass ≥ 12 (C, N, O, S); -The linker is a linker moiety containing or consisting essentially of 3 to 50 atoms with an atomic mass ≥ 12; -The E3 ligase binding molecule is a moiety that binds to the E3 ligase] relates to the compound of.

[0061] In another aspect, the compound of formula (A-1) is of formula (A-1a)

[0062]

Chem.

[0063] and has the structure of In another aspect, the compound of formula (A-1) is of formula (A-1b)

[0064]

Chem.

[0065] and has the structure of In some embodiments, R 1 is

[0066]

Chem.

[0067] [wherein - each R 4 is · a secondary amine substituted with alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and / or heterocycle, · halogen and is independently selected from; and / or two R 4 together form an unsubstituted or substituted heteroaryl or heterocycle; - m is an integer selected from 0, 1, 2 and 3] and has the structure of

[0068] In some embodiments of formula (A), (A-1), (A-1a) or (A-1b), R 3 is

[0069]

Chem.

[0070] [Wherein, - s is an integer selected from 1 and 2, and more specifically, s is 1; - R 31 and R 32 together form an unsubstituted or alkyl-substituted, halogen-substituted and / or hydroxyl-substituted heterocyclic ring or heterobicyclic ring, or R 31 and R 32 are independently selected from hydrogen and unsubstituted or hydroxy-substituted and / or halogen-substituted alkyl or cycloalkyl].

[0071] In some embodiments of formula (A), (A-1), (A-1a) or (A-1b), R 3 has the structure of -CH 2 -NR 31 R 32 In some embodiments, NR 31 R 32 is

[0072]

Chemical formula

[0073] selected from. In some embodiments of formula (A), (A-1), (A-1a) or (A-1b), NR 31 R 32 is

[0074]

Chemical formula

[0075] selected from, v is an integer selected from 0, 1 and 2, and each R N is independently selected from hydroxyl, halogen and C 1 ~C 4 alkyl, or two R N are C 3 ~C 6It forms a cycloalkyl.

[0076] In some embodiments,

[0077]

Chemical formula

[0078] is

[0079]

Chemical formula

[0080] is. In some embodiments, each R 2 is F. In some embodiments, n is an integer selected from 0, 1, and 2. In some embodiments, n is 2. In some embodiments, the handle is a connecting portion comprising or consisting essentially of 4 to 8 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker portion comprising or consisting essentially of 4 to 30 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker portion comprising or consisting essentially of 5 to 20 atoms with an atomic mass ≥ 12.

[0081] Further embodiments of the various groups of the disclosed PROTAC compounds (e.g., R 1 , R 2 , R 3 , R 31 , R 32 , R 4 , the handle, and the linker) are further described in the following sections.

[0082] E3 ligase binding molecule An E3 ligase binding molecule is a molecule that specifically binds to an E3 ligase. In some embodiments, the E3 ligase is cereblon (UniProt-ID: Q96SW2).

[0083] In some embodiments, the E3 ligase binding molecule has the formula (B)

[0084] [Chemical formula]

[0085] [wherein, - Ox is CH 2 or C=O; - T is selected from the group consisting of F, Cl; - k is an integer selected from the group consisting of 0, 1, 2; -

[0086] [Chemical formula]

[0087] means a bond to a linker] is an E3 ligase binding molecule of In some embodiments, k is an integer selected from the group consisting of 0, 1. In some embodiments, k is 0. In some embodiments, T is F.

[0088] Handle In some embodiments, the handle is a connecting moiety comprising or consisting essentially of 3 to 10 atoms having an atomic mass ≥ 12 (C, N, O, S). In some embodiments, the handle is a connecting moiety comprising or consisting essentially of 4 to 8 atoms having an atomic mass ≥ 12. In some embodiments, the handle is a connecting moiety comprising or consisting essentially of 5 to 10 atoms having an atomic mass ≥ 12. In some embodiments, the handle is a connecting moiety comprising or consisting essentially of 5 to 15 atoms having an atomic mass ≥ 12. In some embodiments, the handle is a connecting moiety comprising or consisting essentially of 10 to 15 atoms having an atomic mass ≥ 12. In some embodiments, the handle is a connecting moiety comprising or consisting essentially of 5 to 20 atoms having an atomic mass ≥ 12.

[0089] In some embodiments, the handle comprises, or consists essentially of, one, two, three, or four chemical moieties selected from the group consisting of alkyl, amine, phenyl, and carbonyl.

[0090] In some embodiments, the handle has the formula:

[0091]

Chemical formula

[0092] [wherein, - Mid is selected from the group consisting of C 1 ~C 3 alkyl, and phenyl.] is selected from the group consisting of.

[0093] In some embodiments, the handle has the formula (X)

[0094]

Chemical formula

[0095] and Mid is selected from the group consisting of C 1 ~C 3 alkyl, and phenyl. In some embodiments, the handle has the formula (Y):

[0096]

Chemical formula

[0097] is. In some embodiments, the handle has the formula:

[0098]

Chemical formula

[0099] selected from the group comprising linker In some embodiments, the linker is a linker moiety comprising or consisting essentially of 3 to 50 atoms with an atomic mass ≥ 12 (C, N, O, S). In some embodiments, the linker is a linker moiety comprising or consisting essentially of 4 to 30 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker moiety comprising or consisting essentially of 5 to 20 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker moiety comprising or consisting essentially of 5 to 10 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker moiety comprising or consisting essentially of 5 to 15 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker moiety comprising or consisting essentially of 5 to 25 atoms with an atomic mass ≥ 12. In some embodiments, the linker is a linker moiety comprising or consisting essentially of 10 to 15 atoms with an atomic mass ≥ 12.

[0100] In some embodiments, the linker and the handle, when combined, are a linker moiety comprising or consisting essentially of 5 to 25 atoms with an atomic mass ≥ 12. In some embodiments, the linker and the handle, when combined, are a linker moiety comprising or consisting essentially of 4 to 35 atoms with an atomic mass ≥ 12. In some embodiments, the linker and the handle, when combined, are a linker moiety comprising or consisting essentially of 4 to 25 atoms with an atomic mass ≥ 12. In some embodiments, the linker and the handle, when combined, are a linker moiety comprising or consisting essentially of 15 to 25 atoms with an atomic mass ≥ 12. In some embodiments, the linker and the handle, when combined, are a linker moiety comprising or consisting essentially of 12 to 30 atoms with an atomic mass ≥ 12. In some embodiments, the linker and the handle, when combined, are a linker moiety comprising or consisting essentially of 16 to 22 atoms with an atomic mass ≥ 12.

[0101] In some embodiments, the linker comprises, consists essentially of, or consists of 1, 2, 3, 4, 5, 6, or 7 chemical moieties independently selected from the group consisting of alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkylene, alkynyl, ethylene glycol, carbonyl, ether, ester, amine, amide, sulfonamide, wherein the chemical moieties are each independently unsubstituted or substituted with C 1 ~C 3 alkyl, halogen, CN, NO 2 , hydroxyl, amine, sulfate, phosphate, and / or carboxyl.

[0102] In some embodiments, the linker comprises, consists essentially of, or consists of 1, 2, 3, or 4 chemical moieties selected from the group consisting of alkyl, ethylene glycol, carbonyl, piperazine, aryl, amine, triazole.

[0103] In some embodiments, the linker has the formula:

[0104]

Chemical formula

[0105] [wherein, - Lin is selected from the group consisting of C 3 ~C 20 alkyl, C 3 ~C 20 alkyl-triazole, oligo(ethylene glycol).] is selected from the group consisting of.

[0106] In some embodiments, the linker has the formula:

[0107]

Chemical formula

[0108] [wherein, - p is selected from 2, 3, 4, and 5; - q is selected from 7, 8, 9, 10, 11, 12, and 13; - r is selected from 11, 12, 13, 14, 15, 16, and 17; - s is selected from 7, 8, 9, 10, 11, 12, and 13; - t is selected from 3, 4, 5, 6, 7, 8, and 9; - u is selected from the group consisting of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20] and is selected from the group comprising.

[0109] In some embodiments, the linker is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S) and (T), - 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, and 13.

[0110] In some embodiments, the linker is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S) and (T), - p is selected from 2, 3, 4, and 5; - q is selected from 7, 8, 9, 10, 11, 12, and 13; - r is selected from 11, 12, 13, 14, 15, 16, and 17; - s is selected from 7, 8, 9, 10, 11, 12, and 13; - t is selected from 3, 4, 5, 6, 7, 8, and 9; - u is selected from 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0111] In some embodiments, the linker has the formula:

[0112]

Chemical Formula

[0113] [wherein, - p is selected from 2, 3, 4, and 5; - q is selected from 7, 8, 9, 10, 11, 12, and 13; - r is selected from 11, 12, 13, 14, 15, 16, and 17; - s is selected from 7, 8, 9, 10, 11, 12, and 13; - t is selected from 3, 4, 5, 6, 7, 8, and 9; - u is selected from the group consisting of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20].

[0114] In some embodiments, the linker is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S), (T), and (V), - 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, and 13.

[0115] In some embodiments, the linker is selected from the group consisting of the following formulas (O), (P), (Q), (R), (S), (T), and (V), - p is selected from 2, 3, 4, and 5; - q is selected from 7, 8, 9, 10, 11, 12, and 13; - r is selected from 11, 12, 13, 14, 15, 16, and 17; - s is selected from 7, 8, 9, 10, 11, 12, and 13; - t is selected from 3, 4, 5, 6, 7, 8, and 9; - u is selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0116] In some embodiments, the linker is a peptide. In some embodiments, the linker is a peptide consisting of proteinogenic amino acids.

[0117] In some embodiments, the linker has the formula (W):

[0118]

Chemical formula

[0119] comprising, where Lin is selected from the group consisting of C 3 ~C 20 alkyl, C 3 ~C 20 alkyl-triazole, and oligo(ethylene glycol). In some embodiments, the linker has the formula (Z):

[0120]

Chemical formula

[0121] comprising, where z is selected from 4, 5, 6, 7, 8, 9, and 10. Combination of features In some embodiments, - the E3 ligase binding molecule is an E3 ligase binding molecule of formula (B); - The handle is a handle of formula (F), (G), (H) or (J); - The linker is a linker of formula (O); (P); (Q); (R); (S); or (T).

[0122] In some embodiments, - The E3 ligase binding molecule is an E3 ligase binding molecule of formula (B); - The handle is a handle of formula (X) or (Y); - The linker is a linker of formula (O); (P); (Q); (R); (S), (T) or (V).

[0123] In some embodiments, - The E3 ligase binding molecule is an E3 ligase binding molecule of formula (B); - The handle is a handle of formula (C), (D), (E); - The linker is a linker of formula (W) or (Z).

[0124] In some embodiments, the compound comprises the definitions of the handle, linker, and E3 ligase binding molecule set forth in Table 1 (one combination per line).

[0125]

Table 1-1

[0126]

Table 1-2

[0127] Exemplary PROTAC compounds of the present disclosure are shown in Tables 2 and 3.

[0128]

Table 2-1

[0129]

Table 2-2

[0130]

Table 2-3

[0131]

Table 2-4

[0132]

Table 3-1

[0133]

Table 3-2

[0134]

Table 3-3

[0135]

Table 3-4

[0136]

Table 3-5

[0137]

Table 3-6

[0138]

Table 3-7

[0139]

Table 3-8

[0140]

Table 3-9

[0141]

Table 3-10

[0142]

Table 3-11

[0143]

Table 3-12

[0144]

Table 3-13

[0145]

Table 3-14

[0146]

Table 3-15

[0147]

Table 3-16

[0148]

Table 3-17

[0149]

Table 3-18

[0150]

Table 3-19

[0151]

Table 3-20

[0152]

Table 3-21

[0153]

Table 3-22

[0154]

Table 3-23

[0155]

Table 3-24

[0156]

Table 3-25

[0157]

Table 3-26

[0158]

Table 3-27

[0159]

Table 3-28

[0160]

Table 3-29

[0161]

Table 3-30

[0162]

Table 3-31

[0163]

Table 3-32

[0164]

Table 3-33

[0165]

Table 3-34

[0166]

Table 3-35

[0167]

Table 3-36

[0168]

Table 3-37

[0169]

Table 3-38

[0170]

Table 3-39

[0171]

Table 3-40

[0172]

Table 3-41

[0173]

Table 3-42

[0174]

Table 3-43

[0175]

Table 3-44

[0176]

Table 3-45

[0177]

Table 3-46

[0178]

Table 3-47

[0179]

Table 3-48

[0180]

Table 3-49

[0181]

Table 3-50

[0182]

Table 3-51

[0183]

Table 3-52

[0184]

Table 3-53

[0185]

Table 3-54

[0186]

Table 3-55

[0187]

Table 3-56

[0188]

Table 3-57

[0189]

Table 3-58

[0190]

Table 3-59

[0191]

Table 3-60

[0192]

Table 3-61

[0193]

Table 3-62

[0194]

Table 3-63

[0195]

Table 3-64

[0196]

Table 3-65

[0197]

Table 3-66

[0198]

Table 3-67

[0199]

Table 3-68

[0200]

Table 3-69

[0201]

Table 3-70

[0202]

Table 3-71

[0203]

Table 3-72

[0204]

Table 3-73

[0205] The active compound of PROTAC The second aspect of the present disclosure relates to a compound of general formula (I)

[0206]

Chemical formula

[0207] relates to An alternative aspect of the second aspect of the present disclosure relates to a compound of general formula (Ia)

[0208]

Chemical formula

[0209] relates to An alternative aspect of the second aspect of the present disclosure relates to a compound of general formula (Ib)

[0210]

Chemical formula

[0211] [wherein, -Z1 and Z 2 is independently selected from N, CH, and CR 2 ; - X is O or NH; - Y is CH 2 , C=O, or SO 2 ; - R 1 is an unsubstituted or substituted moiety selected from aryl, heteroaryl, cycloalkyl, and heterocycle, and in particular, R 1 is unsubstituted or substituted heteroaryl; - R 2 is F, Me, Cl, OH, NH 2 , Br, CF 3 , CHF 2 , CH 2 F; - n is an integer selected from 0, 1, 2, 3, and 4; - R 3 is a substituted alkylamine; - U and V are independently selected from -CH 2 - and -(CH 2 ) 2 -, or one of U and V is -CH 2 - and the other is -(CH 2 ) 3 -].

[0212] In some embodiments, X is NH. In some embodiments, Y is C=O. In some embodiments, n is an integer selected from 0, 1, and 2. In some embodiments, U and V are both -CH 2 - or both are -(CH 2 ) 2 -. In some embodiments, the compound has the general formula (U)

[0213]

Chemical formula

[0214] is a compound, - NR 31 R 32 is

[0215]

Chem.

[0216] selected from; - Each R 2 is F, Cl, CF 3 , CHF 2 , CH 2 F and is selected from the group containing; - n is an integer selected from 0, 1, 2, 3, and 4; - R 5 is selected from alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl, and heterocycle.

[0217] In some embodiments, R 2 is F. In some embodiments, n is an integer selected from 0, 1, and 2. In some embodiments, n is 2. In some embodiments, R 5 is selected from alkyl, alkylaryl, and cycloalkyl. In some embodiments, R 5 is selected from methyl and methylphenyl.

[0218] The central spiro ring In some embodiments, X is NH. In some embodiments, Y is C=O.

[0219] In some embodiments,

[0220]

Chem.

[0221] The moiety is

[0222] [Chemical formula]

[0223] selected from In some embodiments,

[0224] [Chemical formula]

[0225] The moiety is

[0226] [Chemical formula]

[0227] selected from R 1 moiety In some embodiments, R 1 is unsubstituted or substituted heteroaryl. In some embodiments, R 1 is unsubstituted or · secondary amine NHR N [wherein R N is selected from C 1 ~C 6 alkyl, C 4 ~C 6 cycloalkyl, aryl, and heteroaryl, alkylaryl, and alkylheteroaryl]; · halogen, especially Cl or F; · C 1 ~C 6 alkyl, C 4 ~C 6 cycloalkyl, aryl and heteroaryl substituted with a moiety selected from

[0228] In some embodiments, R 1 is unsubstituted or · secondary amine NHR N [wherein R N is C1 ~C 6 alkyl, C 4 ~C 6 selected from cycloalkyl, aryl, and heteroaryl]; · halogen, especially Cl or F substituted with a moiety selected from.

[0229] In some embodiments, the compound has the general formula (II)

[0230]

Chemical formula

[0231] [wherein, - Z 1 、Z 2 、X, Y, R 2 、R 3 、U, V and n are as defined above; - each R 4 is · a secondary amine substituted with alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and / or a heterocyclic ring, · halogen independently selected from; and / or two R 4 together form an unsubstituted or substituted heteroaryl or heterocyclic ring; - m is an integer selected from 0, 1, 2 and 3].

[0232] In some embodiments, the compound has the general formula (III)

[0233]

Chemical formula

[0234] [wherein, - Z 1 、Z 2 、X, Y, R 2, R 3 , U, V, and n are defined the same as described above; - R 5 is selected from alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl, and heterocyclic ring; - R 6 is selected from halogen and hydrogen] is a compound.

[0235] In some embodiments, the compound has the general formula (IV)

[0236]

Chemical formula

[0237] [wherein, - Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V, and n are defined the same as described above; - R 6 is selected from halogen and hydrogen; - W is selected from N and CH] is a compound.

[0238] In some embodiments, at least one of Z 1 and Z 2 is CH or CR 2 . In some embodiments, both Z 1 and Z 2 are CH or CR 2 . R 3 moiety In some embodiments, R 3 is substituted with one or several moieties independently selected from alkyl-, hydroxy-, amino-, amine-, halogen-, cycloalkyl- and heterocyclic moieties.

[0239] In some embodiments, R 3 is substituted C 1 ~C4 It is an alkylamine. In some embodiments, R 3 is a substituted C 1 ~C 2 alkylamine. In some embodiments, R 3 is

[0240] [Chemical formula]

[0241] [wherein, - s is an integer selected from 1 and 2, and more particularly, s is 1; - R 31 and R 32 together form a heterocyclic or heterobicyclic ring that is unsubstituted or alkyl-substituted, halogen-substituted, and / or hydroxyl-substituted, or - R 31 and R 32 are independently selected from hydrogen, and unsubstituted or hydroxy-substituted, and / or halogen-substituted alkyl or cycloalkyl].

[0242] In some embodiments, R 31 and / or R 32 is unsubstituted or substituted with an alkyl group, a hydroxy group, a halogen group, a cycloalkyl group, a heterocyclic group and / or a - group. In some embodiments, R 31 and / or R 32 are independently selected from H and unsubstituted or hydroxy-substituted, and / or halogen-substituted alkyl and cycloalkyl. In some embodiments, R 31 and R 32 together form a heterocyclic ring or a heterobicyclic ring that is unsubstituted or alkyl-substituted, hydroxy-substituted and / or halogen-substituted.

[0243] In some embodiments, NR 31 R 32is selected from

[0244]

Chemical formula

[0245] and v is an integer selected from 0, 1 and 2, and each R is independently selected from hydroxyl, halogen and C N ~C 1 ~C 4 alkyl, or two Rs N form C 3 ~C 6 cycloalkyl.

[0246] In some embodiments, -NR 31 R 32 is

[0247]

Chemical formula

[0248] selected from. In some embodiments, NR 31 R 32 is

[0249]

Chemical formula

[0250] selected from. In some embodiments, NR 31 R 32 is

[0251]

Chemical formula

[0252] That is. In some embodiments, NR 31 R 32 is

[0253]

Chem.

[0254] is. In some embodiments, NR 31 R 32 is

[0255]

Chem.

[0256] is. In some embodiments, NR 31 R 32 is

[0257]

Chem.

[0258] is. R 2 portion In some embodiments, n is an integer selected from 0, 1, and 2. In some embodiments, n is 2. In some embodiments, R 2 is selected from F, Cl, and OH. In some embodiments, R 2 is F. R 2 can be attached to any of the carbon atoms of an aryl ring or a heteroaryl ring. Thus, R 2 is Z 1 or Z 2 when Z 1 or Z 2 is a carbon atom, can also be attached to Z

[0259] R 5 portion In some embodiments, R 5 is selected from alkyl, alkylaryl, and cycloalkyl. In some embodiments, R 5 is selected from methyl and methylphenyl.

[0260] Use of this compound The third aspect of the present disclosure relates to the compounds described in the first or second aspect for use as a medicament.

[0261] The fourth aspect of the present disclosure relates to the compounds described in the first or second aspect for use in the treatment of cancer. In some embodiments, the cancer is selected from the group including renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.

[0262] Similarly, within the scope of the present disclosure is a method for treating cancer in a patient in need thereof, comprising the step of administering to the patient a compound as described above. Similarly, there is provided a dosage form for the prevention or treatment of cancer, comprising a non-agonist ligand or an antisense molecule according to any of the above aspects or embodiments of the present disclosure.

[0263] Those skilled in the art will recognize that any drug mentioned in detail may exist as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts include the ionized drug and the oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylic acid, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

[0264] Unless otherwise indicated, the compounds described in this specification are shown in their natural isotope abundances or one or more atoms may be artificially enriched with specific isotopes having the same atomic number but different atomic masses or mass numbers from those predominantly found in nature. All isotope variations of the compounds of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure.

[0265] For example, the compounds described in this specification may be artificially enriched with one or more specific isotopes. In some embodiments, the compounds described in this specification may be artificially enriched with one or more isotopes that are not commonly found in nature. In some embodiments, the compounds described in this specification may be artificially enriched with one or more isotopes selected from deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). In some embodiments, the compounds described in this specification may be 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 125Artificially enriched with one or more isotopes selected from I. In some embodiments, the abundance of the enriched isotope is, independently, at least 1 mol%, at least 10 mol%, at least 20 mol%, at least 30 mol%, at least 40 mol%, at least 50 mol%, at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, or 100 mol%. In some embodiments, the compound is deuterated at least at one position. In some embodiments, the compounds disclosed herein are 1 Part or all of the H atoms are 2 Replaced by H atoms.

[0266] In some embodiments, the compounds described herein exist as geometric isomers. In some embodiments, the compounds described herein have one or more double bonds. The compounds shown herein include all cis isomers, trans isomers, syn isomers, anti isomers, entgegen (E) isomers and zusammen (Z) isomers and mixtures thereof corresponding thereto. In some situations, the compounds described herein have one or more chiral centers, each of which is present in the R configuration or the S configuration. The compounds described herein include all diastereomeric forms, enantiomeric forms, and epimeric forms and mixtures thereof corresponding thereto. In further embodiments of the compounds and methods provided herein, mixtures, combinations or tautomers of enantiomers and / or diastereoisomers obtained from a single preparation step are useful for the uses described herein. In some embodiments, the compounds described herein are prepared as individual stereoisomers of the compound by reacting a racemic mixture of the compound with an optically active resolving agent to form a set of diastereomeric compounds, separating the diastereomers, and recovering the optically pure enantiomers. In some embodiments, separable complexes are preferred. In some embodiments, the diastereomers have distinct physical properties (e.g., melting point, boiling point, solubility, reactivity, etc.) and are separated by taking advantage of these differences. In some embodiments, the diastereomers are separated by chiral chromatography or preferably by separation / resolution techniques based on differences in solubility. In some embodiments, the optically pure enantiomers are recovered with the resolving agent by any practical means that does not result in racemization.

[0267] In some situations, compounds exist as tautomers. The compounds described herein include all possible tautomers within the scope of the formulas described herein. Tautomers are compounds that are interconvertible by the movement of a hydrogen atom involving the switching of a single bond and an adjacent double bond. In bond arrangements where tautomerism is possible, a chemical equilibrium of tautomers exists. All tautomers of the compounds disclosed herein are contemplated. The exact ratio of tautomers depends on several factors including temperature, solvent, and pH.

[0268] The dosage form may be for enteral administration such as nasal administration, buccal administration, rectal administration, transdermal administration or oral administration, and may also be administered in the form of inhalation or as a suppository. Alternatively, parenteral administration such as subcutaneous injection, intravenous injection, intrahepatic injection or intramuscular injection may be used. In some cases, pharmaceutically acceptable carriers and / or excipients may be included.

[0269] Pharmaceutical Compositions and Administration 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 further embodiments, the composition comprises at least two pharmaceutically acceptable carriers as described herein.

[0270] In some embodiments of the present disclosure, the compounds of the present disclosure are generally formulated into pharmaceutical dosage forms to provide dosages that are readily controllable and to give the patient a product that is clear and easy to handle.

[0271] The pharmaceutical composition can be formulated for oral administration, parenteral administration, or rectal administration. Further, the pharmaceutical compositions of the present disclosure can be prepared in solid form (including but not limited to capsules, tablets, pills, granules, powders or suppositories), or in liquid form (including but not limited to solutions, suspensions or emulsions).

[0272] 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 status, medical condition and body 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 renal and hepatic functions of the patient, as well as the desired effect. In some embodiments, the compounds of the present disclosure may be administered once a day, and the total daily dosage may be divided and administered 2, 3, or 4 times a day.

[0273] In some embodiments, in the pharmaceutical compositions or combinations of the present disclosure, the active ingredient may be about 1 to 1000 mg per unit dosage for a subject weighing about 50 to 70 kg. The therapeutically effective dosage of the compound, pharmaceutical composition, or combination thereof depends on the species, body weight, age, individual condition, disorder or disease to be treated or its severity of the subject. A physician, clinician or veterinarian having ordinary skill can readily determine the respective effective amounts of the active ingredient necessary for the prevention, treatment or suppression of progression of the disorder or disease.

[0274] The pharmaceutical compositions of the present disclosure can be subjected to conventional pharmaceutical processes such as sterilization and / or can contain conventional inert diluents, lubricants or buffering agents, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifying agents and buffer solutions. The pharmaceutical compositions of the present disclosure can be produced by standard processes such as 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., Theory and Practice of Industrial Pharmacy, 4th Edition, 2013 (ISBN 8123922892).

[0275] Manufacturing and treatment methods The present disclosure further encompasses, as a further aspect, the use of the compounds identified herein or pharmaceutically acceptable salts thereof, as defined in detail above, for use in the manufacture of a medicament for the treatment or prevention of cancer.

[0276] Similarly, the present disclosure also encompasses a method for treating a patient diagnosed with a cancer-related disease. This method inevitably involves the step of administering to the patient an effective amount of a compound identified herein or a pharmaceutically acceptable salt thereof, as defined in detail herein.

[0277] For example, if alternatives for a separable single feature such as ligand type or medical application are described herein as "embodiments", it is understood that such alternatives may be freely combined to form individual embodiments of the present disclosure disclosed herein. Accordingly, any alternative embodiments for ligand type may be combined with any medical application mentioned herein.

[0278] The present use further encompasses the following items. Item 1. Compound of general formula (I)

[0279]

Chemical formula

[0280] which is a compound of - Z 1 and Z 2 are independently selected from N, CH and CR 2 ; - X is O or NH, in particular X is NH; - Y is CH 2 , C=O, or SO 2 and in particular Y is C=O; - R 1 is an unsubstituted moiety or a substituted moiety selected from aryl, heteroaryl, cycloalkyl, and heterocycle, in particular, R 1 is unsubstituted or substituted heteroaryl; - R 2 is F, Me, Cl, OH, NH 2 , Br, CF 3 , CHF 2, CH 2 selected from 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; - R 3 is a substituted alkylamine; - U and V are independently selected from -CH 2 - and -(CH 2 ) 2 -, or one of U and V is -CH 2 -, and the other is -(CH 2 ) 3 -, in particular, U and V are both -CH 2 -, or both are -(CH 2 ) 2 -, a compound.

[0281] 2. R 1 is unsubstituted or · secondary amine NHR N [wherein R N is selected from C 1 ~C 6 alkyl, C 4 ~C 6 cycloalkyl, aryl, and heteroaryl, alkylaryl, and alkylheteroaryl]; · halogen, in particular, Cl or F; and · C 1 ~C 6 alkyl, C 4 ~C 6 cycloalkyl, aryl and heteroaryl substituted with a moiety selected from, the compound according to item 1.

[0282] 3. R 3 is substituted with one or several moieties independently selected from alkyl-, hydroxy-, amino-, amine-, halogen-, cycloalkyl- and heterocyclic moieties, the compound according to item 1 or 2.

[0283] 4. R 3 is C1 ~C 4 is an alkylamine, especially R 3 is C 1 ~C 2 The compound according to any one of items 1 to 3, which is an alkylamine. 5. General formula (II)

[0284]

Chemical formula

[0285] [wherein, - Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V and n are defined in the same way as in item 1; - Each R 4 is · A secondary amine substituted with alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl and / or heterocyclic ring, especially a secondary amine substituted with alkyl, alkylaryl, or cycloalkyl; · Halogen is independently selected from; and / or two Rs 4 together form an unsubstituted or substituted heteroaryl or heterocyclic ring; - m is an integer selected from 0, 1, 2 and 3] The compound according to any one of items 1 to 4, which is a compound of

[0286] 6. General formula (III)

[0287]

Chemical formula

[0288] [wherein, - Z 1 , Z 2 , X, Y, R 2 , R 3, U, V, and n are defined the same as in item 1; - R 5 is selected from alkyl, alkylaryl, heteroalkylaryl, cycloalkyl, aryl, heteroaryl, and heterocycle, In particular, R 5 is selected from alkyl, alkylaryl, and cycloalkyl; - R 6 is selected from halogen and hydrogen] The compound according to any one of items 1 to 5, which is a compound of.

[0289] 7. General formula (IV)

[0290]

Chemical formula

[0291] [wherein, - Z 1 , Z 2 , X, Y, R 2 , R 3 , U, V, and n are defined the same as in item 1; - R 6 is selected from halogen and hydrogen; - W is selected from N and CH] The compound according to any one of items 1 to 4, which is a compound of.

[0292] 8. At least one of Z 1 and Z 2 is CH or CR 2 , In particular, Z 1 and Z 2 Both are CH or CR 2 The compound according to any one of items 1 to 7, which is a compound of.

[0293] 9. R 3 is

[0294]

Chemical formula

[0295] [wherein, - s is an integer selected from 1 and 2, and more specifically, s is 1; - R 31 and R 32 together form an unsubstituted, or alkyl-substituted, halogen-substituted, and / or hydroxyl-substituted heterocyclic ring or bicyclic ring, or R 31 and R 32 are independently selected from hydrogen, and unsubstituted or hydroxy-substituted and / or halogen-substituted alkyl or cycloalkyl, in particular, R 31 and R 32 together form an unsubstituted, or alkyl-substituted, halogen-substituted, and / or hydroxyl-substituted heterocyclic ring or bicyclic ring] is a compound according to any one of items 1 to 8.

[0296] 10. NR 31 R 32 is selected from

[0297]

Chemical formula

[0298] and v is an integer selected from 0, 1, and 2, and each R N is independently selected from hydroxyl, halogen, and C 1 ~C 4 alkyl, or two Rs N form C 3 ~C 6 cycloalkyl, is a compound according to item 9.

[0299] 11. NR 31 R 32 is

[0300] [Chemical formula]

[0301] The compound according to item 9 or 10, selected from 12.R 2 is selected from F, Cl and OH, especially R 2 is F, the compound according to any one of items 1 to 11.

[0302] 13. The compound according to any one of items 1 to 12 for use as a medicament. 14. The compound according to any one of items 1 to 12 for use in the treatment of cancer.

[0303] The present disclosure can be further illustrated by the following examples and figures, thereby drawing out further embodiments and advantages. These examples are meant to illustrate the present disclosure and are not intended to limit its scope. [Examples]

[0304] All reagents were used as purchased from commercial suppliers. The reactions were carried out at high temperature in an oil bath. All reactions were monitored by thin-layer chromatography (aluminum plates coated with silica gel 60 F 254 ). Flash column chromatography was performed on silica gel (0.040 - 0.063 mm). 1 H and 13 C{ 1 H} NMR spectra were recorded in DMSO or CDCl 3 on an AV2 - 400 MHz and an AV600 Bruker spectrometer (400 MHz, 101 MHz, and 600 MHz, 150 MHz, respectively), and the chemical shifts were shown in ppm, with the residual 1 H and 13Their calibrations were performed for the C signals. The abbreviations for multiplicity are as follows. Singlet (s), doublet (d), multiplet (m), and broad signal (bs). The purity was obtained by liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS). An Acquity UPLC (Waters, Milford, USA) was connected to an Acquity eλ diode array detector and a Synapt G2 HR-ESI-QTOF-MS (Waters, Milford, USA); 1 μL of the sample (concentration c = about 10 - 100 μg / mL in the indicated solvent) was injected; an Acquity BEH C18 HPLC column (particle size 1.7 μm, 2 × 50 mm, Waters) was maintained at 30 °C; * Solvent A: H 2 O + 1% HCO 2 H and Solvent B: CH 3 CN + 0.1% HCO 2 H were used, elution was carried out at a flow rate of 400 μL / min, and the ratio of Solvent B was linearly changed from 5% to 98% within 5 minutes and then maintained at a uniform concentration for 1 minute; * In the wavelength range from 200 nm to 600 nm, the UV spectrum was recorded at a resolution of 1.2 nm and 20 points / second (points s -1 ) ; ESI: positive ionization mode, capillary voltage 3.0 kV, sample cone 40 V, extraction cone 4 V, N 2 cone gas flow rate 4 L / h, N 2 desolvation gas flow rate 800 L / min, source temperature 120 °C; mass spectrometer in resolution mode: operating at a scan rate of 1 Hz for a mass range of 100 - 2,000 m / z; 5 mM HCO 2 Na aqueous solution was used, and mass calibration was performed with an accuracy of <2 ppm within 50 - 2’500 m / z. As lock masses, m / z 195.0882 (caffeine, 0.7 ng / mL) and m / z 556.2771 (leucine-enkephalin, 2 ng / mL) were used. General procedure for Buchwald-Hartwig coupling: In a nitrogen atmosphere, the corresponding amine (1 equivalent) was added to a stirred solution of the corresponding halide (1 equivalent) in dioxane (0.3 M). Nitrogen gas was bubbled through the reaction solution for 2 minutes, and Cs 2 CO 3 (1.2 equivalents), Ruphos Pd G4 (10 mol%) and Ruphos (10 mol%) were added. The reaction mixture was stirred at 150 °C for 17 hours, concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography.

[0305] General procedure for deprotection of the Boc group: HCl (0.9 M, 37% aq.) was added to a stirred solution of the corresponding Boc-protected amine in MeOH (0.3 M). The reaction mixture was stirred at 25 °C for 4 h, and the reaction mixture was concentrated under reduced pressure. The resulting residue was used directly in the next step without further purification.

[0306] General procedure for S N Ar using 4,6-dichloropyrimidine: 4,6-Dichloro-pyrimidine (1.2 equivalents) and Et 3 N (1 - 4 equivalents) were added to a stirred solution of the corresponding amine (1 equivalent) or amine hydrochloride (1 equivalent) in iPrOH (0.3 M). The reaction mixture was stirred at 80 °C for 3 h using microwave, and concentrated under reduced pressure. The crude residue was dissolved in nBuOH, washed 3 times with water and once with brine, dehydrated with MgSO 4 and concentrated under reduced pressure. The crude residue was co-evaporated several times with toluene to remove the remaining nBuOH, and then purified by flash column chromatography.

[0307] General procedure for S N Ar using a chloropyrimidine derivative: The corresponding chloropyrimidine (1 equivalent) was dissolved in methylamine (0.1 M, 8 M in EtOH) or benzylamine (0.3 M), and the reaction mixture was heated at 130 °C for 3 h (MeNH 2 ) or 140 °C for 8 h (BnNH 2It was stirred. The crude residue was concentrated under reduced pressure and purified by flash column chromatography. In the case of the reaction with benzylamine, the crude residue was co-evaporated several times in the order of water and toluene to remove benzylamine, and then purification was carried out.

[0308] Example 1: The inventors aimed to simplify the structure and reduce the molecular weight, and started the design based on one of the initial inhibitors (1, Table 4) prepared by the inventors (Figure 1A). For this purpose, the methylene position of the piperidine ring was changed from 1,3 to 1,4, and the chiral center was removed. Furthermore, according to the X-ray structure of 1 having METTL3, the deletion of the amide C=O group enables the maintenance of the original vector (Figure 1B). By these two modifications, 2 and its two pyridine-containing derivatives 3 and 4 were derived, and these showed similar potencies (IC 50 = 5.0 μM for 2, IC 50 = 4.6 μM for derivative 3, IC 50 = 5.8 μM for derivative 4, Table 4) as well as the absence of chirality and the reduction in the number of heavy atoms, and thus showed higher ligand efficiencies (LE = 0.23 for 2, LE = 0.23 for derivative 3, LE = 0.22 for derivative 4). Since 4 had better lipophilic ligand efficiency (LLE = 3.4, calculated by DataWarrior), its pyridine core was preserved in the next optimization step. According to the crystal structure of the complex of METTL3 with inhibitor 1, the substitution of the methylamine of the pyrimidine ring with benzylamine seemed to be beneficial for inhibition. This was proven correct as the corresponding derivative 5 showed a six-fold increase in potency (IC 50 = 0.79 μM).

[0309] A prominent feature of this inhibitor series is sp 3It has a linear shape and is bound to an acyclic atomic linker, and thus has very high flexibility. Immobilization of the structure is a feasible way to freeze the ligand in its preferred conformational state, and by this way, the binding energy can be increased by reducing the entropy penalty. Therefore, the inventors envisioned two different strategies to achieve this goal, which are to create an amide bond between the piperidine and pyridine rings, or to form a spiro ring by connecting the tertiary alcohol to aniline according to the conformation of compound 5' (Figure 2A). Two methods gave exactly opposite results. The amide derivative 6 lost its previous potency enhancement (IC 50 = 3.6 μM, Table 4), while the spirocycle 7 was promising in terms of inhibition (IC 50 = 0.28 μM, Table 5) and novelty. The inventors were able to soak 5 and 7 with METTL3, and X-ray analysis showed strong structural overlap. The pyrimidine moiety makes two hydrogen bonds with the NH backbone obtained from Asn549 and Ile378, participates in π-stacking with Phe534, and participates in π-interaction with the side chain of Asn549 (Figure 2A). The benzylamine group interacts with the side chain of Asp377 and also forms a cation-π interaction with Arg379. At the opposite site of the binding pocket, the gem dimethyl group blocks the lipophilic pocket formed by the Lys513, Pro514, Trp457 and Trp431 residues, and the charged piperidine forms a salt bridge with Asp395. The only difference between 5 and 7 is that in the latter case, the alcohol is converted to an ether, so there is no additional hydrogen bond between the tertiary alcohol and the side chain of Gln550 (Figure 2B). The inventors expected that by replacing the ether with a lactam, this interaction could be restored, and an additional hydrogen bond could be further created by the C=O group of the ligand and the NH 2 amide of Gln550. The inventors prepared the corresponding derivative 8 (IC 50For the compound with an EC50 value of 0.037 μM, a significant enhancement of efficacy was obtained. The hypothesis of the inventors was confirmed by two hydrogen bond interactions found in the crystal structure (Figure 2C). Furthermore, both LE and LLE (0.25 and 4.4 respectively, Table 5) were substantially improved.

[0310] ADME properties, such as solubility, cell permeability, and metabolic stability, are essential for chemical probes. Therefore, these properties were considered at an early stage of the project. For the newly synthesized inhibitors (5, 7 - 8) by the inventors, although the results were mixed, all of them showed a half-life shorter than 12 minutes in the incubation with rat liver microsomes, indicating that their stability against enzymatic degradation was mediocre (Tables 4 and 5). Therefore, the inventors focused on improving the ADME properties while obtaining better biochemical efficacy. The first approach was to replace the nitrogen atom of pyridine with a carbon atom. This substitution resulted in compound 9 with moderate permeability (9×10 -6 cm·s -1 −1), and unexpectedly, the solubility increased slightly (Table 5). However, since the metabolic stability did not change, benzylamine was replaced with methylamine (10). In fact, similar to the LE value and LLE value (0.28 and 4.5 respectively), the solubility and metabolic stability were significantly improved (108 μM and 107 minutes respectively), but in return, the permeability was limited (2×10 -6 cm·s -1 −1), and the efficacy decreased to one-fourth. From compound 10, as another attempt to reduce the molecular size developed by the inventors, two replacements were considered: replacing spiropiperidine with spiroazetidine (11), and replacing spiro lactam with spiro urea (12). Unfortunately, both of them showed a significant decrease in efficacy (one-fifth and one-twentieth respectively). Nevertheless, the spiroazetidine moiety remains a potential alternative that helps to reduce the molecular weight and improve the physicochemical properties at a later stage. Next, what the inventors focused on was to improve the permeability. Methylation of the lactam in compound 13 led to a serious decrease in efficacy (one-nineteenth), demonstrating the important role of the hydrogen bond interaction of the lactam.

[0311] After fully optimizing the spiro ring scaffold, the inventors focused on the pyrimidine motif. Adding one or more methyl groups to aniline (14) significantly impaired the binding compared to 10, presumably due to the loss of hydrogen bonding to the side chain of Asp377 (0.97 μM and 0.089 μM, respectively, Table 6). However, substitution from methyl to isopropyl (15) did not show a significant decrease (0.33 μM) because the hydrogen bonding was retained. These two modifications exemplified that the available space for branched sp 3 carbon was restricted. Surprisingly, substitution with a cyclopropyl group (16) was not only well-tolerated (0.084 μM), but also improved three ADME properties (Table 6), making it a promising alternative for lead optimization. Since S-adenosylmethionine (SAM) is the natural ligand of METTL3 containing an adenosine scaffold that overlaps with the pyrimidine group of the inventors' inhibitors, the inventors considered testing some bicyclic aromatic heterocyclic modifications. Pyrrolopyrimidine 17 showed a slight increase in potency compared to 10, but similarly had low permeability and a large efflux ratio in the Caco-2 assay (Table 6). N 3 Since the interaction geometry between the pyrimidine atom and the nitrogen backbone of Asn549 did not seem to be optimal, the inventors considered removing this pyrimidine nitrogen atom as it might increase permeability and suppress the effect of the partial desolvation penalty. The latter was proven to be incorrect as pyrrolopyridine 18 showed a significant loss of binding (a 74-fold decrease). Incorporating a chlorine atom between the two pyrimidine nitrogen atoms (19) was beneficial for potency (0.024 μM), but the solubility and metabolic stability were critically impaired (45 μM and 32 minutes, respectively), so the inventors had to look for different modifications.

[0312] Since the spiro ring scaffold and the pyrimidine moiety had already been optimized, the inventors examined the phenyl ring as the next target region. There are publications discussing the unique properties of fluorine atoms, which can lead to unexpected promising results in drug design. In fact, fluorine atoms can interact in a different way than usual, and aromatic fluorine atoms tend to enhance permeability. A fluorine scan was performed on the phenyl ring, and two new derivatives 20 and 21 were obtained. Compared to inhibitor 10, both compounds improved binding to a similar extent (0.038 μM and 0.032 μM, respectively), but permeability increased significantly only for 20 (Table 7). When the X-ray structures of the complexes with METTL3 were analyzed molecule by molecule, the fluorine in 21 showed hydrophobic contacts (Figure 3B), and it was also revealed that the fluorine atom in 20 was also making an unusual interaction with the nitrogen π-system of Pro397 (Figure 3A). Inhibitor 20 had a strongly improved permeability and a low efflux ratio (permeability 9·10 -6 cm·s -1 and 2), which was favorable. However, as the most important solution to achieve excellent potency and maintain appropriate ADME properties, the combination of both fluorine atoms quickly emerged. In fact, compound 22 showed a single-digit nanomolar IC 50 (0.008 μM) in the TR-FRET assay (Table 7 and Figure 4A), high cell permeability (12·10 -6 cm·s -1 ), as well as favorable values for LE and LLE (0.3 and 5.3, respectively), and acceptable metabolic stability (t 1 / 2 = 24 minutes).

[0313] To investigate the selectivity of compound 22 against other RNA methyltransferases in detail, the inventors performed a protein thermal shift assay. The inventors expressed and purified the METTL1 protein, which is a writer of 7-methylguanosine labels on tRNA, mRNA, and miRNA and serves as a representative related protein. The inventors used S-adenosyl-L-homocysteine (SAH), which is a by-product of the catalytic activity of RNA methyltransferase and a natural binder, as a positive control, and at 100 μM, ΔT m showed 2.8 °C for METTL3 / METTL14 and 3.5 °C for METTL1 (Figures 5 and 6). Compound 22 at 100 μM was able to shift the melting temperature of METTL3 / METTL14 by 4.7 °C compared to the DMSO control (Figures 5 and 6). In contrast, no shift was observed and no binding was shown for METTL1 even when compound 22 was increased up to 100 μM.

[0314] The inventors were able to study the cellular target engagement of compound 22 with its target protein in two orthogonal assays based on protein thermal denaturation by the promotion of the thermal stabilization of METTL3 by compound 22. The binding of 22 was evaluated in the InCELL Pulse assay, in which the promotion of the ProLabel® (ePL) enzyme fragment conjugated to the N-terminus (residues 354 - 580) of cleaved METTL3 was shown in HEK293T cells. After incubating these cells with the inhibitor 22 at 37 °C for 1 h, the cells were heated at 46 °C for 3 min, and the non-aggregated METTL3-ePL protein was quantified using a luminescence-based assay (Figure 4B). Compound 22 had an EC 50Stabilized the METTL3-ePL fusion protein at 2 μM. Thus, both experiments provided clear evidence of cell permeability and binding to the target protein intracellularly. Finally, to highlight the biological potential of 22 as an inhibitor of METTL3 enzyme activity, the inventors measured the m 6 A / A ratio in two different cancer cell lines, MOLM-13 (AML) cells and PC-3 (prostate cancer) cells, 16 hours after compound treatment. The inventors found that 22 was able to reduce this ratio to 10 - 20% of the DMSO-treated control sample and had some selectivity between the two cell lines (for MOLM-13, EC 50 = 0.7 μM and for PC-3, EC 50 = 2.5 μM, Figure 4D).

[0315] The inventors successfully improved the potency (1,000-fold), efficiency parameters, and ADME properties of a series of METTL3 inhibitors by medicinal chemistry guided by protein crystallography. The most important features are the rigidification as a result of the design of the spiro ring scaffold and the use of fluorine atoms at specific positions. The most potent inhibitor (compound 22) shows an IC 50 of 8 nM in the TR-FRET assay. Binding to the off-target METTL1 was not observed at concentrations up to 100 μM. The binding of compound 22 to the target protein intracellularly was demonstrated using two different assays. Furthermore, as quantified by UPLC-MS / MS analysis, for the decrease in m 6 A / A in polyadenylated RNA, an EC 50 value of 0.7 μM was measured in the MOLM-13 (leukemia) cell line and an EC 50 value of 2.5 μM was measured in the PC-3 (prostate cancer) cell line. Thus, compound 22 is a chemical probe for deciphering the functional roles of METTL3 / METTL14 and the involvement of METTL3 / METTL14 in hematological malignancies and solid tumors.

[0316] [Chemical Structure]

[0317]

Table 4

[0318]

Chem.

[0319]

Table 5

[0320]

Chem.

[0321]

Table 6

[0322]

Chem.

[0323]

Table 7

[0324]

Chem.

[0325] Scheme 1: Synthetic route of spirocycle intermediate (33). Reagents and conditions: (a) MeNO 2 , NH 3 , MeOH, 25 °C, 17 h; (b) (i) CbzCl, NaHCO 3 , DCM / H 2 O, 0~25 °C, 17 h; (ii) NiCl 2 .6H 2O, NaBH 4 , MeOH, N 2 , 0 - 25 °C, 1 h, through a three - step reaction to 32%; (c) Ethyl 2 - bromoacetate, Et 3 N, DCM, 25 °C, 2 h; (d) Pd / C, NH 4 +. HCOO - , iPrOH, 80 °C, 4 h, through a two - step reaction to 55%.

[0326]

Chem.

[0327] Scheme 2: Synthetic routes of 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 (in the case of 7) or 33, Pd Ruphos G4, Ruphos, Cs 2 CO 3 , dioxane, N 2 , 150 °C, 17 h, 93% (35);; (b) (i) HCl (37% aq.), MeOH, 25 °C, 4 h; (ii) In the case of 7: N - benzyl - 6 - chloropyrimidin - 4 - amine (29), Et 3 N, iPrOH, 150 °C, 8 h, MW, through a three - step reaction from 23 to 6%. In the case of 17: 4 - chloro - 7H - pyrrolo[2,3 - d]pyrimidine, 36 Pd Ruphos G4, Ruphos, LiHMDS, THF, N 2 , 65 °C, 4 h, 36%. In the case of 19: 36, 2,4 - dichloro - 7H - pyrrolo[2,3 - d]pyrimidine, Et 3 N, iPrOH, 100 - 130 °C, 6 h, 42%; (c) HCl (37% aq.), MeOH, 25 °C, 4 h, through a two - step reaction from 23 to 36%; (d) 4,6 - dichloro - pyrimidine, Et 3 N, iPrOH, 80 °C, 3 h, MW, through a three - step reaction from 26 (34) to 27% / 63% (37); (e) In the case of 8 and 9: BnNH 2 , 140 °C, 8 h, MW, 25% (8) / 36 through a two - step reaction to 5% (9). In the case of 10: MeNH2 , EtOH, 130 °C, 3 h, MW, 3% through a two-step reaction. In the case of 15: iPrNH 2 , EtOH, 130 °C, 8 h, MW, 52%. In the case of 16: cyclopropylamine, iPrOH, 130 °C, 6 h, MW, 25%.

[0328]

Chem.

[0329] Scheme 3: Synthetic route of intermediate 40. Reagents and conditions: (a) MeNO 2 , K 2 CO 3 , EtOH, 25 °C, 17 h; (b) DAST, DCM, N 2 , -78 °C, 3 h; (c) NH 3 , MeOH, 25 °C, 2 h, quantitative through a three-step reaction; (d) (i) CbzCl, NaHCO 3 , DCM / H 2 O, 0 - 25 °C, 17 h; (ii) NiCl 2 .6H 2 O, NaBH 4 , MeOH, N 2 , 0 - 25 °C, 1 h, 54% through a two-step reaction; (e) ethyl 2-bromoacetate, Et 3 N, DCM, 25 °C, 2 h; (f) Pd / C, NH4 +. HCOO - , iPrOH, 80 °C, 4 h, 32% through a two-step reaction.

[0330]

Chem.

[0331] Scheme 4: Synthetic route of compound 11. Reagents and conditions: (a) 40, Pd Ruphos G4, Ruphos, Cs 2 CO 3 , dioxane, N 2 , 150 °C, 17 h; (b) (i) HCl (37% aq.), MeOH, 25 °C, 4 h; (ii) 4,6-dichloro-pyrimidine, Et3 N, iPrOH, 80 °C, 7 h, MW: (c) MeNH 2 , EtOH, 130 °C, 3 h, MW, via a four-step reaction, 19%.

[0332]

Chem.

[0333] 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, K 2 CO 3 , DMF, 25 °C, 17 h, 98% (47) / 99% (49). For 52: (i) 4-bromo-2,5-difluorobenzoic acid, BH 3 . SMe 2 , THF, N 2 , 25 °C, 17 h, 83%; (ii) SOCl 2 , DMF, DCM, 25 °C, 3 h; (iii) 4,4-dimethylpiperidine hydrochloride, K 2 CO 3 , DMF, 25 °C, 17 h, 92%; (b) 33, Pd Ruphos G4, Ruphos, Cs 2 CO 3 , dioxane, N 2 , 150 °C, 17 h, 83% (50); (c) (i) HCl (37% aq.), MeOH, 25 °C, 4 h; (ii) 4,6-dichloropyrimidine, Et 3 N, iPrOH, 80 °C, 3 h, MW, 15% from 47 via a two-step reaction (48) / 58% from 50 via a two-step reaction (51); (d) MeNH 2 , EtOH, 130 °C, 3 h, MW, 47% (20) / 69% (21) / 56% (22) from 52 via a four-step reaction.

[0334] 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

[0335]

Chem.

[0336] The chloropyrimidine derivative 6-1 (100 mg, 0.21 mmol) was dissolved in ethanol (0.5 M), and then N-Boc-1,3-propanediamine 6-2 (108 mg, 0.62 mmol) and TEA (86.6 μL, 0.62 mmol) were 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 by flash column chromatography (SiO 2 ; DCM / MeOH = 9:1) to give a slightly yellow solid (117 mg, 91%). S N The impurity of Ar (117 mg, 0.19 mmol) was dissolved in MeOH (0.5 M), and 37% HCl (57.7 μL, 1.9 mmol) was added. The resulting reaction mixture was stirred at rt until completion (monitored by TLC). The reaction mixture was evaporated, dissolved in butanol, quenched with a saturated aqueous solution of Na 2 CO 3 and extracted with butanol (3 × 10 ml). The combined organic layers were dried over MgSO4, filtered, and evaporated to give the product (80 mg, 81%). 11H NMR (400 MHz, MeOD) δ 8.06 (d, J = 0.9 Hz, 1H), 7.38 (d, J = 8.2 Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 5.77 (d, J = 1.0 Hz, 1H), 4.01 (s, 2H), 3.86 (s, 2H), 3.83 (s, 2H), 3.63 - 3.56 (m, 2H), 3.53 (s, 2H), 3.43 (t, J = 6.5 Hz, 2H), 3.17 (d, J = 0.8 Hz, 1H), 2.98 (t, J = 7.0 Hz, 6H), 1.95 - 1.87 (m, 4H), 1.81 (ddd, J = 13.3, 8.6, 4.1 Hz, 2H), 1.58 (d, J = 5.9 Hz, 4H), 1.29 (d, J = 2.7 Hz, 3H), 1.02 (s, 6H). 13 13C NMR (101 MHz, DMSO) δ 167.6, 163.7, 162.1, 157.8, 148.6, 130.1, 129.2, 114.7, 65.4, 62.4, 53.3, 52.9, 51.9, 49.7, 38.8, 38.0, 37.9, 34.8, 29.6, 28.8, 28.6, 15.7. LRMS(ESI): m / z [M+H]+ theoretical value 520.36 (C29H44N8O), measured value 521.37.

[0337] Scheme 7: General procedure for amide coupling:

[0338]

Chem.

[0339] The carboxylic acid (1 equivalent) was dissolved in DMF (0.5 M), and the solution was cooled to 0 °C. DIPEA (5 equivalents) was added. After 10 minutes, HATU (1.1 equivalents) was added, and the solution was stirred for 30 minutes. Then, amine 6-3 (1 equivalent) was added. The resulting reaction mixture was stirred at rt until completion (while monitoring by TLC). Scheme 8: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)propyl)-3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propenamide

[0340] [Chemical Structure]

[0341] Compound 8-4 was prepared according to general amide coupling procedure 1 using amine 3 (159 mg, 0.306 mmol) and the corresponding pomalidomide carboxylic acid (132 mg, 0.306 mmol). The crude product was first purified using flash column chromatography (Al 2 O 3 ; DCM / MeOH = 90:10 - 80:20) and then 8-4 was obtained using semi-preparative HPLC (15 mg, 5%). 11H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.83 (t, J = 5.6 Hz, 1H), 7.59 - 7.56 (m, 1H), 7.13 - 7.03 (m, 3H), 6.89 (d, J = 8.4 Hz, 1H), 6.62 - 6.58 (m, 1H), 5.61 (s, 1H), 5.05 (dd, J = 12.7, 5.4 Hz, 1H), 3.80 - 3.75 (m, 1H), 3.65 (s, 1H), 3.59 (t, J = 6.0 Hz, 2H), 3.53 - 3.43 (m, 6H), 3.19 - 3.17 (m, 2H), 3.08 (q, J = 6.6 Hz, 2H), 2.87 (ddd, J = 16.7, 13.7, 5.4 Hz, 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). 13 13C NMR (126 MHz, DMSO) δ 172.8, 172.1, 170.1, 170.1, 169.0, 167.3, 167.1, 163.3, 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]+ theoretical value 935.50 (C49H65N11O8), measured value 936.51. Scheme 9: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-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)propenamide

[0342]

Chem.

[0343] Compound 9-5 was prepared according to general amide coupling procedure 1 using amine 3 (69 mg, 0.13 mmol) and the corresponding pomalidomide carboxylic acid (64 mg, 0.13 mmol). The crude product was first purified by flash column chromatography (Al 2 O 3 ; DCM / MeOH = 90:10 to 80:20) and then 9-5 was obtained using semi-preparative HPLC (28 mg, 22%). 11H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.84 (t, J = 5.6 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.14 - 7.03 (m, 3H), 6.89 (d, J = 8.2 Hz, 2H), 6.60 (m, 2H), 5.61 (s, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 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 = 6.6 Hz, 2H), 2.91 - 2.84 (m, 1H), 2.63 - 2.56 (m, 2 H), 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). 13 13C NMR (126 MHz, DMSO) δ 172.8, 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, 49.3, 48.6, 41.7, 38.4, 36.3, 36.2, 34.3, 31.0, 29.0, 28.3, 22.2, 21.2. LRMS(ESI): m / z [M+H]+ theoretical value 979.53 (C51H69N11O9), measured value 980.54.

[0344] Scheme 10: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-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

[0345]

Chem.

[0346] Compound 10-6 was prepared according to the general amide coupling procedure 1 using amine 3 (50 mg, 0.096 mmol) and the corresponding pomalidomide carboxylic acid (50 mg, 0.096 mmol). The crude product was first purified using flash column chromatography (Al 2 O 3 ; DCM / MeOH = 90:10 to 80:20) and then 10-6 was obtained using semi-preparative HPLC (20 mg, 20%). 11H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.60 - 7.55 (m, 1H), 7.16 - 7.04 (m, 3H), 6.89 (d, J = 8.4 Hz, 2H), 6.62 - 6,58 (m, 2H), 5.61 (s, 1H), 5.05 (dd, J = 12.8, 5.5 Hz, 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 = 6.5 Hz, 2H), 2.87 (ddd, J = 16.8, 13.8, 5.4 Hz, 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). 13 13C NMR (126 MHz, DMSO) δ 172.8, 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, 117.5, 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. LRMS(ESI): m / z [M+H]+ theoretical value 1023.55 (C53H73N11O10), measured value 1024.56. Scheme 11: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-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-pentaoxooctadecane-18-amide

[0347]

Chemical Structure

[0348] Compound 11-7 was prepared according to the general amide coupling procedure 1 using amine 3 (51 mg, 0.098 mmol) and the corresponding pomalidomide carboxylic acid (51 mg, 0.096 mmol). The crude product was first purified using flash column chromatography (Al 2 O 3 ; DCM / MeOH = 90:10 - 80:20) and then 11-7 was obtained using semi-preparative HPLC (13 mg, 12%). 11H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 8.25 (s, 1H), 7.98 (s, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.61 - 7.54 (m, 1H), 7.16 - 7.02 (m, 3H), 6.89 (d, J = 8.3 Hz, 2H), 6.60 (m, 2H), 5.61 (s, 1H), 5.05 (dd, J = 12.7, 5.4 Hz, 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 = 6.6 Hz, 2H), 2.87 (ddd, J = 16.9, 13.7, 5.4 Hz, 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). 13 13C NMR (126 MHz, DMSO) δ 173.3, 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. LRMS(ESI): m / z [M+H]+ theoretical value 1067.58 (C55H77N11O11), measured value 1068.59.

[0349] Scheme 12: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-(prop-2-yn-1-ylamino)isoindoline-1,3-dione

[0350] [Chemical formula]

[0351] After dissolving 4-fluoro-pomalidomide derivative 12-8 (320 mg, 1.16 mmol) in DMSO (0.5 M), propargylamine (64 mg, 1.16 mmol) and DIPEA (605 μl, 3.48 mmol) were added to the solution. The resulting reaction mixture was stirred at 130 °C until completion (while monitoring by TLC). NaHCO 3 The reaction mixture was quenched by adding a saturated aqueous solution and extracted with EtOAc (3 × 15 ml). The combined organic layers were dried over MgSO4, filtered, and evaporated. Product 12-9 was obtained as a yellow solid (280 mg, 78%). 1 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.57 (dd, J = 8.5, 7.1 Hz, 1H), 7.20 (d, J = 7.1 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 6.44 (t, J = 6.5 Hz, 1H), 4.92 (dd, J = 12.2, 5.4 Hz, 1H), 4.09 (dd, J = 6.1, 2.5 Hz, 2H), 2.95 - 2.67 (m, 3H), 2.27 (t, J = 2.4 Hz, 1H), 2.20 - 2.07 (m, 1H). 13 13C NMR (101 MHz, CDCl3) δ 170.81, 169.24, 168.10, 167.44, 145.56, 136.16, 132.43, 117.17, 112.77, 111.42, 79.11, 72.20, 48.96, 32.33, 31.41, 22.76. LRMS (ESI): m / z [M+H]+ theoretical value 311.09 (C16H13N3O4), measured value 312.09.

[0352] Scheme 13: Preparation of N-(3-((6-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-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)undecanamide

[0353] [Chemical formula]

[0354] Compound 12-9 (100 mg, 0.32 mmol) was dissolved in THF (0.5 M), and then azidomethyl ester (77 mg, 0.32 mmol), copper(II) sulfate (26 mg, 0.16 mmol), and sodium ascorbate (70 mg, 0.35 mmol) were added to the solution. The resulting reaction mixture was stirred at 40 °C until completion (while monitoring by TLC), then evaporated, and the crude product was purified using flash column chromatography (SiO2; EtOAc / Hept = 1:1) to obtain a bright yellow solid (90 mg, 50%). The resulting methyl ester (90 mg, 0.16 mmol) was dissolved in THF (0.5 M), and 37% HCl (49 μL, 1.6 mmol) was added. The resulting reaction mixture was stirred at rt until completion (while monitoring by TLC), then evaporated, and product 13-10 was used in the next reaction without 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, 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 rt until completion (while monitoring by TLC). The reaction mixture was evaporated, and the crude product was purified using flash column chromatography (SiO 2 ; DCM / MeOH = 90:10~80:20) to obtain 13-11 (7 mg, 5%). 11H NMR (600 MHz, MeOD) δ 8.06 (t, J = 5.6 Hz, 1H), 8.00 (s, 1H), 7.92 (s, 1H), 7.52 (t, J = 8.5, 7.1, 4.4 Hz, 1H), 7.29 (d, 2H), 7.10 - 7.06 (m, 2H), 7.00 (t, J = 6.1 Hz, 1H), 6.97 (d, 2H), 5.68 (s, 1H), 5.11 - 5.02 (dd, 1H), 4.65 (d, J = 6.1 Hz, 2H), 4.36 (t, J = 7.0 Hz, 2H), 3.83 (s, 2H), 3.72 (m, 2H), 3.55 (m, J = 14.3, 4.0 Hz, 2H), 3.50 (m, J = 4.3 Hz, 2H), 3.26 (q, J = 6.6 Hz, 4H), 2.87 - 2.81 (m, 2H), 2.75 (dt, J = 5.1, 2.6 Hz, 1H), 2.72 (t, J = 1.1 Hz, 1H), 2.71 - 2.66 (m, 2H), 2.18 (t, J = 8.7, 6.0 Hz, 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 = 6.7 Hz, 2H), 1.60 (q, J = 7.1, 6.3 Hz, 4H), 1.48 (, J = 5.7 Hz, 4H), 1.32 - 1.29 (m, 16H), 0.97 (d, J = 1.6 Hz, 6H). 1313C NMR (151 MHz, MeOD) δ 176.55, 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, 146.62, 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, 54.42, 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]+ theoretical value 1040.61 (C56H77N14O6), measured value 1040.61.

[0355] Scheme 13: General scheme for amide coupling 2

[0356]

Chem.

[0357] The carboxylic acid (1 equiv) was dissolved in DMF (0.5 M), the solution was cooled to 0 °C, and DIPEA (5 equiv) was added. After 10 minutes, COMU (1.1 equiv) was added, and the solution was stirred for 30 minutes, then amine 6-3 (1 equiv) was added. The resulting reaction mixture was stirred at rt until completion (while monitoring by TLC). Scheme 14: Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)propyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanamide

[0358]

Chemical Structure

[0359] Compound 14-12 was prepared according to general amide coupling procedure 2 using amine 3 (63 mg, 0.12 mmol) and the corresponding carboxylic acid (50 mg, 0.12 mmol). The reaction mixture was evaporated and the crude product was purified by preparative thin layer chromatography (SiO 2 ; DCM / MeOH = 84:16) twice to give 14-12 (6 mg, 5%). 11H NMR (400 MHz, DMSO) δ 11.00 (s, 1H), 8.27 (s, 1H), 7.98 (s, 1H), 7.78 (t, J = 5.6 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 7.14 (d, J = 8.2 Hz, 2H), 6.91 (dd, J = 7.9, 3.9 Hz, 3H), 6.72 (d, J = 8.0 Hz, 1H), 6.64 (t, J = 5.8 Hz, 1H), 5.61 (s, 1H), 5.54 (t, J = 5.5 Hz, 1H), 5.10 (dd, J = 13.2, 5.1 Hz, 1H), 4.22 (d, J = 17.3 Hz, 1H), 4.12 (d, J = 17.2 Hz, 1H), 3.77 (m, 2H), 3.66 (s, 2H), 3.17 (m, J = 5.3 Hz, 2H), 3.08 (p, J = 6.6 Hz, 4H), 2.92 (ddd, J = 18.0, 13.4, 5.6 Hz, 2H), 2.69 - 2.57 (m, 2 H), 2.36 - 2.23 (m, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.72 (m, J = 13.4 Hz, 2H), 1.58 (td, J = 13.2, 12.8, 6.2 Hz, 6H), 1.47 (t, J = 7.1 Hz, 2H), 1.31 (m, 2H), 1.25 (d, J = 11.0 Hz, 12H), 0.88 (s, 6H). 1313C NMR (151 MHz, MeOD) δ 176.53, 174.82, 172.54, 172.46, 170.82, 164.62, 163.41, 163.31, 163.08, 158.30, 150.81, 145.26, 132.93, 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. LRMS (ESI): m / z [M+H]+ theoretical value 917.56 (C51H72N11O5), measured value 918.57. Scheme 15: Preparation of N-(3-((6-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)propyl)-11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecanamide

[0360]

Chemical Structure

[0361] Compound 15-13 was prepared according to general amide coupling procedure 2 using amine 3 (47 mg, 0.090 mmol) and the corresponding lenalidomide carboxylic acid (40 mg, 0.090 mmol). The reaction mixture was evaporated and the crude product was purified by flash column chromatography (SiO2 Purified using DCM / MeOH = 90:10~80:20) to obtain 15-13 (36 mg, 42%). 1 H NMR (500 MHz, DMSO) δ 11.00 (s, 1 H), 8.32 (s, 1H), 7.98 (s, 1H), 7.84 (t, J = 5.7 Hz, 1H), 7.43 - 7.34 (m, 2H), 7.27 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 8.3 Hz, 2H), 6.91 (d, J = 7.4 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.67 (m, J = 6.1 Hz, 1H), 5.62 (s, 1H), 5.57 (t, J = 5.5 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.23 (d, J = 17.2 Hz, 1H), 4.12 (d, J = 17.2 Hz, 1H), 3.81 - 3.74 (m, 2 H), 3.72 (s, 2H), 3.59 (m, J = 11.8, 5.1 Hz, 2H), 3.51 (m, J = 3.0 Hz, 2H), 3.45 (s, 2H), 3.17 (m, J = 8.8, 5.4 Hz, 2H), 3.08 (dq, J = 13.0, 6.5 Hz, 4H), 2.91 (ddp, J = 15.1, 9.8, 5.2, 4.7 Hz, 2H), 2.61 (dt, J = 17.5, 3.3 Hz, 2H), 2.29 (qd, J = 13.2, 4.7 Hz, 2H), 2.03 (t, J = 7.0 Hz, 2H), 1.70 (m, J = 10.5, 4.2 Hz, 2H), 1.59 (tt, J = 15.4, 8.3 Hz, 6H), 1.46 (m, J = 12.8, 11.0, 5.5 Hz, 4H), 1.34 (m, J = 7.0 Hz, 2H), 1.25 (m, J = 21.7, 6.4 Hz, 14 H), 0.94 (s, 5H). 1313C NMR (126 MHz, DMSO) δ 172.95, 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. LRMS(ESI): m / z [M+H]+ theoretical value 945.60 (C53H76N11O5), measured value 946.60.

[0362]

Chem.

[0363] Scheme 16: (a) Sodium ascorbate (1.1 equiv), CuSO4 (0.24 equiv), THF, 40 °C, 24 h; (ii) TFA (10 equiv), DCM, rt, 8 h; (b) (i) TEA (3 equiv), EtOH, reflux, 24 h; (ii) 38% HCl, MeOH, 24 h; (c) HATU (1.1 equiv), DIPEA (5 equiv), DMF, rt, 5 h.

[0364]

Chem.

[0365] Scheme 17: (d) (i) TEA (3 equiv), EtOH, reflux, 24 h; (ii) 38% HCl, MeOH, 24 h; (e) HATU (1.1 equiv), DIPEA (5 equiv), DMF, rt, 5 h.

[0366] [Chemical]

[0367] Scheme 18: (f)(i) propargylamine (3 eq), TEA (3 eq), EtOH, reflux, 5 h; (g) sodium ascorbate (1.1 eq), CuSO 4 (0.24 eq), THF, 40 °C, 24 h.

[0368] [Chemical]

[0369] 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.

[0370] [Chemical]

[0371] 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.

[0372] [Chemical]

[0373] Scheme 21: (l) sodium ascorbate (1.1 eq), CuSO 4 (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.

[0374] [Chemical]

[0375] Scheme 22: (n) DIPEA (3 equiv), DMSO, 80 °C, 24 h; (o) TFA (10 equiv), DCM, rt, 8 h b) HATU (1.1 equiv), DIPEA (5 equiv), DMF, rt, 8 h.

[0376] 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]undecan-2-one (Compound 23-2)

[0377]

Chem.

[0378] 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). Subsequently, 4,6-difluoropyrimidine (27 μL, 0.32 mmol) and TEA (150 μL, 1.01 mmol) were added to the stirred solution. The resulting reaction mixture was stirred at 80 °C (oil bath temperature) until completion (monitored by TLC). The volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 9:1) to give the desired product 112 mg (89%). 11H NMR (400 MHz, DMSO) δ 8.30 (d, J = 2.8 Hz, 1H), 8.22 (s, 1H), 7.15 (dd, J = 13.5, 6.6 Hz, 1H), 6.93 (dd, J = 11.5, 7.3 Hz, 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 = 5.3 Hz, 4H), 0.87 (s, 6H). 13 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]+ theoretical value 503.270 (C26H34F3N6O), measured value 503.276.

[0379] Scheme 24: General Procedure 1

[0380]

Chem.

[0381] Subsequently, amine (1.5 equiv) and TEA (4 equiv) were added to a solution of compound 23-2 (1 equiv) in EtOH (0.5 M) in a pressure vial. The resulting reaction mixture was stirred at 120 °C (oil bath temperature) until completion (while monitoring by TLC). 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]undecan-9-yl)pyrimidin-4-yl)amino)propyl)carbamate (Compound 25-1)

[0382]

Chem.

[0383] Compound 19 was prepared according to General Procedure 1 using Compound 23-2 (300 mg, 0.6 mmol) and tert-butyl (3-aminopropyl) carbamate (156 mg, 0.9 mmol). The volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10) to give the desired product (280 mg, 71%). 1 H NMR (400 MHz, DMSO) δ 8.18 (s, 1H), 7.97 (s, 1H), 7.17 (s, 1H), 6.94 (dd, J = 11.5, 7.3 Hz, 1H), 6.82 (t, J = 5.7 Hz, 1H), 6.61 (t, J = 5.7 Hz, 1H), 5.60 (s, 1H), 3.86 (d, J = 13.5 Hz, 2H), 3.60 (s, 2H), 3.27 (s, 2H), 3.22 - 3.12 (m, 2H), 2.96 (q, J = 6.6 Hz, 2H), 2.37 (s, 4H), 1.77 (dt, J = 14.2, 4.2 Hz, 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 657.404 (C34H51F2N8O3), found 657.405.

[0384] 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]undecan-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)carbamate (Compound 26-1)

[0385] [Chemical formula]

[0386] Compound 26-1 was prepared according to General Procedure 1 using Compound 23-2 (50 mg, 0.1 mmol) and 1-(N-Boc-aminomethyl)-3-(aminomethyl)benzene (35 mg, 0.15 mmol). Volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10) to give the desired product 51 mg (71%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.16 (s, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.23 - 7.18 (m, 3H), 7.12 (dd, J = 12.8, 6.6 Hz, 1H), 6.60 - 6.56 (m, 2H), 5.43 (s, 1H), 5.20 (s, 1H), 4.89 (s, 1H), 4.53 (d, J = 5.8 Hz, 2H), 4.30 (d, J = 6.0 Hz, 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, 6.5 Hz, 4H), 0.90 (s, 6H). 13 C NMR (101 MHz, CDCl 3) 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]+ theoretical value 719.420 (C39H53F2N8O3), measured value 719.422. 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]undecan-9-yl)pyrimidin-4-yl)amino)methyl)piperidine-1-carboxylate (Compound 27-1)

[0387] [Chemical formula]

[0388] Compound 18 (50 mg, 0.1 mmol) and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (42 mg, 0.15 mmol) were used to prepare Compound 21 according to General Procedure 1. Volatiles were removed in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10) to give the desired product 55 mg (79%). 1 H NMR (400 MHz, CDCl 3) δ 8.14 (s, 1H), 7.11 (dd, J = 12.9, 6.6 Hz, 1H), 6.67 (m, 1H), 6.58 (dd, J = 10.9, 7.1 Hz, 1H), 5.43 (s, 1H), 4.87 (s, 1H), 4.13 (s, 2H), 3.78 - 3.68 (m, 4H), 3.60 (m, 2H), 3.48 (s, 2H), 3.28 (s, 2H), 3.14 (t, J = 6.1 Hz, 2H), 2.70 (t, J = 12.7 Hz, 2H), 2.44 - 2.37 (m, 4H), 1.97 (m, 2H), 1.82 (m, 2H), 1.77 - 1.69 (m, 3H), 1.45 (s, 9H), 1.39 (t, J = 5.6 Hz, 4H), 1.28 - 1.11 (m, 2H), 0.91 (s, 6H). 13 C NMR (101 MHz, CDCl 3 ) 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(I): m / z [M+H]+ theoretical value 697.440 (C37H55F2N8O3), measured value 697.438.

[0389] Scheme 28: General Procedure 2

[0390]

Chemical Structure

[0391] Subsequently, TsCl (1.6 equivalents) and TEA (5 equivalents) were added to a stirred solution of the corresponding tert-butyl ester (1 equivalent) in DCM (0.5 M). The resulting reaction mixture was stirred at rt until completion (while monitoring by TLC). Volatiles were removed in vacuo, and the product was used in the next step without further purification.

[0392] Potassium phthalimide (2 equivalents) was added to a stirred solution of the corresponding tosylated tert-butyl ester in DMF (0.5 M). The resulting reaction mixture was stirred at rt until completion (while monitoring by TLC). Subsequently, volatiles were removed in vacuo, and the product was used in the next step without further purification.

[0393] The crude product was dissolved in methanol (0.5 M) and hydrazine hydrate 50 - 60% (3 equivalents). The resulting reaction mixture was stirred at rt until completion (while monitoring by TLC). DCM was added to the resulting mixture, and the formed precipitate was removed by filtration. The precipitate was concentrated in vacuo, and the resulting product was used in the next step without any further purification.

[0394] Subsequently, the corresponding tert-butyl ester (1 equivalent) and DIPEA (2 equivalents) were added to a stirred solution of 4-fluoro-salidomide (1 equivalent) in DMSO (0.5 M). The resulting reaction mixture was stirred at 130 °C (oil bath temperature) until completion (while monitoring by TLC).

[0395] 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)

[0396]

Chem.

[0397] Compound 29-1 was prepared according to General Procedure 2 using 4-fluoro-salidomide (82 mg, 0.3 mmol) and the corresponding tert-butyl ester (70 mg, 0.3 mmol). Subsequently, the volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 80:20 to 90:10) to give the desired product (64 mg, 57%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.1 (s, 1H), 7.52 (m, J = 8.4, 7.1 Hz, 1H), 7.12 (d, J = 7.1 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.47 (t, J 5.6 Hz, 1H), 4.85 (dd, J 12.4, 5.3 Hz, 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 = 6.6 Hz, 2H), 2.18 - 2.10 (m, 1H), 1.40 (s, 9H). 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)

[0398]

Chemical Structure

[0399] Compound 30-1 was prepared according to General Procedure 2 using 4-fluoro-salidomide (68 mg, 0.25 mmol) and the corresponding tert-butyl ester (69 mg, 0.25 mmol). Subsequently, the volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 80:20 to 90:10) to give the desired product (52 mg, 39%). 11H NMR (400 MHz, CDCl 3 ) δ 8.12 (s, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 6.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 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 = 6.2 Hz, 2H), 2.15 (m, 1H), 1.44 (s, 9H) Scheme 31: Preparation of tert-butyl 1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxapentadecane-15-oate (Compound 31-1)

[0400]

Chem.

[0401] Compound 31-1 was prepared according to General Procedure 2 using 4-fluoro-salidomide (53 mg, 0.2 mmol) and the corresponding tert-butyl ester (62 mg, 0.2 mmol). Subsequently, the volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 80:20~90:10) to give the desired product 64 mg (57%). 1 1H NMR (400 MHz, CDCl 3 ) δ 8.11 (s, 1H), 7.44 (dd, J = 7.32, 8.34 Hz, 1H), 7.04 (d, J = 7.11 Hz, 1H), 6.83 (d, J = 8.54 Hz, 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). 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-oate (Compound 32-1)

[0402] [Chemical formula]

[0403] Compound 32-1 was prepared according to General Procedure 2 using 4-fluoro-salidomide (56 mg, 0.2 mmol) and the corresponding tert-butyl ester (74 mg, 0.2 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 80:20 to 90:10) to yield 61 mg (49%) of the desired product. 1 H NMR (400 MHz, CDCl 3 ) δ 8.19 (s, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.13 (d, J = 7.0 Hz, 1H), 6.90 (d, J = 8.4 Hz, 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 = 6.4 Hz, 2H), 2.14 (m, 1H), 1.43 (m, 9H). Scheme 33: General Procedure 3

[0404] [Chemical formula]

[0405] Subsequently, the corresponding tert-butyl ester (1 equivalent) and DIPEA (10 equivalents) were added to a stirred solution of lenalidomide (1 equivalent) in NMP (0.5 M). The resulting reaction mixture was stirred at 110 °C (oil bath temperature) until completion (monitored by TLC).

[0406] Scheme 34: Preparation of tert-butyl 9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanoate (Compound 34-1)

[0407] [Chemical formula]

[0408] Compound 34-1 was prepared according to General Procedure 3 using lenalidomide (267 mg, 1.03 mmol) and the corresponding tert-butyl ester (303 mg, 1.03 mmol). Volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 70:30) to yield the desired product, 200 mg (41%). 1 H NMR (400 MHz, DMSO) δ 11.00 (s, 1H), 7.27 (t, J = 7.7 Hz, 1H), 6.92 (d, J = 7.3 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 5.55 (t, J = 5.5 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.26 - 4.08 (m, 2H), 3.10 (q, J = 6.6 Hz, 2H), 2.92 (ddd, J = 18.3, 13.6, 5.4 Hz, 1H), 2.73 - 2.57 (m, 2H), 2.36 - 2.22 (m, 2H), 2.16 (td, J = 7.3, 3.7 Hz, 2H), 2.08 - 1.97 (m, 1H), 1.56 (m, 2H), 1.47 (m, 2H), 1.38 (s, 9H), 1.25 (m, 8H). 1313C NMR (101 MHz, CDCl 3 ) 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]+ theoretical value 416.22 (C22H30N3O5), measured value 416.26.

[0409] Scheme 35: Preparation of tert-butyl 11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecanoate (Compound 35-1)

[0410]

Chemical Structure

[0411] Compound 35-1 was prepared according to General Procedure 3 using lenalidomide (245 mg, 0.95 mmol) and the corresponding tert-butyl ester (304 mg, 0.95 mmol). Subsequently, the volatiles were removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 70:30) to give the desired product (125 mg, 26%). 11H NMR (400 MHz, DMSO) δ 11.00 (s, 1H), 7.27 (t, J = 7.7 Hz, 1H), 6.95 - 6.88 (m, 1H), 6.73 (d, J = 8.0 Hz, 1H), 5.55 (t, J = 5.5 Hz, 1H), 5.10 (dd, J = 13.2, 5.1 Hz, 1H), 4.28 - 4.07 (m, 2H), 3.10 (q, J = 6.6 Hz, 2H), 2.92 (ddd, J = 18.0, 13.5, 5.3 Hz, 1H), 2.72 - 2.58 (m, 2H), 2.36 - 2.23 (m, 2H), 2.16 (t, J = 7.3 Hz, 2H), 2.07 - 1.98 (m, 1H), 1.57 (m, 2H), 1.46 (m, 2H), 1.38 (s, 9H), 1.24 (m, 14H). 13 13C 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]+ theoretical value 500.31 (C28H42N3O5), measured value 500.34. Scheme 36: Preparation of tert-butyl 15-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentadecanoate (Compound 36-1)

[0412]

Chemical Structure

[0413] Compound 36-1 was prepared according to General Procedure 3 using lenalidomide (264 mg, 1.02 mmol) and the corresponding tert-butyl ester (432 mg, 1.02 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 70:30) to yield 125 mg (22%) of the desired product. 1 H NMR (400 MHz, CDCl 3 ) δ 7.98 (s, 1H), 7.36 (t, J = 7.7 Hz, 1H), 6.80 (dd, J = 8.0, 0.9 Hz, 1H), 5.25 (dd, J = 13.2, 5.2 Hz, 1H), 4.36 - 4.09 (m, 2H), 4.05 (t, J = 6.8 Hz, 1H), 3.20 (t, J = 7.3 Hz, 2H), 2.92 - 2.78 (m, 2H), 2.36 - 2.26 (m, 2H), 2.20 (t, J = 7.5 Hz, 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.

[0414] Scheme 37: General Procedure 4

[0415]

Chemical Structure

[0416] Sodium azide (1.2 equiv) was added to a stirred solution of the tert-butyl ester (1 equiv) in DMF (0.5 M). The resulting reaction mixture was stirred at rt until complete (monitored by 1 1H NMR). The volatiles were then removed in vacuo, the residue was dissolved in DCM and filtered through filter paper. The product was used in the next step without further purification.

[0417] Compound 12-9 (1 equiv) was dissolved in THF (0.5 M), and then the corresponding tert-butyl ester (1 equiv), anhydrous CuSO 4 (0.5 equiv) and sodium ascorbate (1.1 equiv) were added respectively. The resulting reaction mixture was stirred at 40 °C (oil bath temperature) until complete (monitored by TLC).

[0418] 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)

[0419]

Chemical Structure

[0420] Compound 38-1 was prepared according to General Procedure 4 using compound 12-9 (80 mg, 0.26 mmol) and the corresponding tert-butyl ester (73 mg, 0.26 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 40:60 to 50:50) to give the desired product 85 mg (55%). 1 1H NMR (400 MHz, CDCl 3) δ 7.99 (s, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.45 (s, 1H), 7.14 (d, J = 7.1 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.68 (t, J = 6.0 Hz, 1H), 4.92 (dd, J = 12.2, 5.4 Hz, 1H), 4.64 (d, J = 6.0 Hz, 2H), 4.31 (t, J = 7.3 Hz, 2H), 2.95 - 2.67 (m, 3H), 2.19 (t, J = 7.5 Hz, 2H), 2.13 (m, 1H), 1.88 (t, J = 7.2 Hz, 2H), 1.44 (s, 9H), 1.33 - 1.21 (m, 14H). 13 C NMR (101 MHz, CDCl 3 ) 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]+ theoretical value 595.32 (C31H43N6O6), measured value 595.35.

[0421] 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)

[0422]

Chemical Structure

[0423] Compound 12-9 (85 mg, 0.27 mmol) and the corresponding tert-butyl ester (70 mg, 0.27 mmol) were used to prepare Compound 39-1 according to General Procedure 4. The volatiles were then removed in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 40:60 - 50:50) to give the desired product (73 mg, 47%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.06 (s, 1H), 7.49 (dd, J = 8.5, 7.2 Hz, 1H), 7.44 (s, 1H), 7.14 (d, J = 7.2, 0.6 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.67 (t, J = 5.9 Hz, 1H), 4.92 (dd, J = 12.1, 5.4 Hz, 1H), 4.64 (d, J = 6.0 Hz, 2H), 4.31 (t, J = 7.9, 6.5 Hz, 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, CDCl 3 ) 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]+ calcd for 567.29 (C29H39N6O6), found 597.30.

[0424] Scheme 40: General Procedure 5

[0425]

Chem.

[0426] Subsequently, the corresponding tert-butyl ester (1 equivalent) and DIPEA (3 equivalents) were added to a stirred solution of 4-fluoro-salidomide (1 equivalent) in DMSO (0.5 M). The resulting reaction mixture was stirred at 130 °C (oil bath temperature) until completion (monitored by TLC).

[0427] Scheme 41: Preparation of tert-butyl 8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoate (Compound 41-1)

[0428]

Chem.

[0429] Compound 41-1 was prepared according to General Procedure 5 using 4-fluoro-salidomide (258 mg, 0.93 mmol) and the corresponding tert-butyl ester (201 mg, 0.93 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 2:3) to yield the desired product, 251 mg (57% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.07 (s, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.22 (t, J = 5.7 Hz, 1H), 4.91 (dd, J = 12.1, 5.3 Hz, 1H), 3.25 (td, J = 7.1, 5.6 Hz, 2H), 2.96 - 2.67 (m, 3H), 2.20 (t, J = 7.5 Hz, 2H), 2.17 - 2.09 (m, 1H), 1.70 - 1.53 (m, 4H), 1.43 (s, 9H), 1.37 - 1.30 (m, 5H). Scheme 42: Preparation of tert-butyl 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoate (Compound 42-1)

[0430]

Chem.

[0431] Compound 42-1 was prepared according to General Procedure 5 using 4-fluoro-salidomide (516 mg, 1.9 mmol) and the corresponding tert-butyl ester (350 mg, 1.9 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 2:3) to yield the desired product 623 mg (75% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 7.89 (s, 1H), 7.42 (dd, J = 8.5, 7.1 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.16 (t, J = 5.7 Hz, 1H), 4.84 (dd, J = 12.1, 5.4 Hz, 1H), 3.20 (td, J = 7.1, 5.6 Hz, 2H), 2.89 - 2.59 (m, 3H), 2.17 (t, J = 7.4 Hz, 2H), 2.10 - 2.04 (m, 1H), 1.68 - 1.53 (m, 4H), 1.37 (s, 9H). Scheme 43: Preparation of tert-butyl 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoate (Compound 43-1)

[0432]

Chem.

[0433] Compound 43-1 was prepared according to General Procedure 5 using 4-fluoro-thalidomide (55 mg, 0.2 mmol) and the corresponding tert-butyl ester (34 mg, 0.2 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / Hept = 2:3) to yield 80 mg (96% yield) of the desired product. 1 H NMR (400 MHz, CDCl 3 ) δ 8.02 (s, 1H), 7.50 (dd, J = 8.6, 7.1 Hz, 1H), 7.10 (d, J = 7.0 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 6.29 (t, J = 5.8 Hz, 1H), 4.96 - 4.84 (m, 1H), 3.33 (q, J = 6.6 Hz, 2H), 2.93 - 2.69 (m, 3H), 2.35 (t, J = 7.1 Hz, 2H), 2.17 - 2.10 (m, 1H), 1.94 (p, J = 7.1 Hz, 2H), 1.45 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) 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 416.181 (C21H26N3O6), found 416.182.

[0434] Scheme 44: General Procedure 6

[0435]

Chemical Structure

[0436] Subsequently, 1-Cbz-piperazine (1 eq) and DIPEA (2 eq) were 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 completion (monitored by TLC). Subsequently, the volatiles were removed in vacuo and the residue was extracted with EtOAc (3×). The combined organic layers were dried over MgSO 4 and filtered, and concentrated under reduced pressure. The product was used in the next step without further purification.

[0437] The piperazine derivative (1 eq) was added to a pre-prepared mixture of iPrOH (0.5 M) and Pd / C (0.5 eq), and then ammonium formate (6 eq) was added. The resulting reaction mixture was stirred at 40 °C (oil bath temperature) until completion (monitored by TLC). The reaction mixture was evaporated and extracted with DCM (3×). The combined organic layers were dried over MgSO 4 and filtered, and evaporated. The product was used in the next step without further purification.

[0438] Subsequently, the piperazine derivative (1 eq) and DIPEA (3 eq) were added to a stirred solution of 4-fluoro-salidomide (1 eq) in DMSO (0.5 M). The resulting reaction mixture was stirred at 130 °C (oil bath temperature) until completion (monitored by TLC).

[0439] 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)

[0440]

Chemical formula

[0441] Compound 45-1 was prepared according to General Procedure 6 using 4-fluoro-thalidomide (60 mg, 0.2 mmol) and the corresponding tert-butyl ester (55 mg, 0.2 mmol). The volatiles were then removed in vacuo and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / MeOH = 95:5) to yield 68 mg (61%) of the desired product. 1 H NMR (400 MHz, DMSO) δ 11.08 (s, 1H), 7.70 (dd, J = 8.4, 7.1 Hz, 1H), 7.34 (t, J = 8.3 Hz, 2H), 5.09 (dd, J = 12.8, 5.4 Hz, 1H), 2.87 (ddd, J = 18.2, 13.8, 5.3 Hz, 1H), 2.61 (m, 1H), 2.39 - 2.27 (m, 2H), 2.17 (t, J = 7.3 Hz, 2H), 2.07 - 1.97 (m, 1H), 1.47 (m, 4H), 1.39 (s, 9H), 1.25 (m, 10H). 13 C 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]+ theoretical value 555.320 (C30H43N4O6), measured value 555.318. Scheme 46: General Procedure 7

[0442]

Chemical Structure

[0443] To a stirred solution of the corresponding tert-butyl ester (1 equiv) in DCM (0.5 M), TFA (10 equiv) was added. The resulting reaction mixture was stirred at rt until complete (monitored by TLC). The volatiles were then removed in vacuo.

[0444] Scheme 47: General Procedure 8

[0445]

Chem.

[0446] The Boc-protected amine (1 equiv) was dissolved in MeOH (0.25 M), and then 4 M HCl in dioxane (10 equiv) was added to the stirred solution. The resulting reaction mixture was stirred at rt until complete (monitored by TLC) and evaporated.

[0447] Scheme 48: General Procedure 9

[0448]

Chem.

[0449] To a cooled solution of the carboxylic acid (1 equiv) in DMF (0.5 M) (water-ice bath), DIPEA (4 equiv) was added and the reaction mixture was stirred at the same temperature for 10 min. After addition of HATU or COMU (1.1 equiv), the solution was stirred for a further 30 min, then the amine (1 equiv) was added. The resulting reaction mixture was stirred at rt until complete (monitored by TLC), concentrated in vacuo and purified using flash column chromatography. Preparation of Scheme 49.4-(4-(9-(4-(((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-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)

[0450] [Chemical formula]

[0451] Compound 49-1 was prepared according to the general amide coupling 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 by flash column chromatography (SiO 2 ; EtOAc / MeOH = 85:15 to 80:20) to yield the desired product (65 mg, 62%). 11H NMR (400 MHz, MeOD) δ 7.99 (s, 1H), 7.67 (dd, J = 8.4, 7.2 Hz, 1H), 7.38 (d, J = 7.1 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.18 (dd, J = 13.0, 6.6 Hz, 1H), 6.87 (dd, J = 11.2, 7.2 Hz, 1H), 5.69 (s, 1H), 5.09 (dd, J = 12.5, 5.5 Hz, 1H), 4.54 (d, J = 13.3 Hz, 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 = 12.9 Hz, 1H), 2.86 (ddd, J = 17.7, 14.2, 5.1 Hz, 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, 3.8 Hz, 2H), 2.15 - 2.07 (m, 1H), 1.98 (m, 2H), 1.91 (s, 1H), 1.89 - 1.76 (m, 5H), 1.59 (m,f 5H), 1.44 (t, J = 5.6 Hz, 5H), 1.37 (s, 8H), 0.93 (s, 6H). 1313C NMR (101 MHz, MeOD) δ 173.2, 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]+ theoretical value 1077.620 (C58H79F2N12O6), measured value 1077.623.

[0452] 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]undecan-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanamide (Compound 50-1)

[0453]

Chemical Structure

[0454] 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 by flash column chromatography (SiO 2 ; EtOAc / MeOH = 90:10 - 80:20) to give the desired product (25 mg, 61%). 11H NMR (400 MHz, DMSO) δ 11.01 (s, 1H), 8.35 (t, J = 6.0 Hz, 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 = 7.5 Hz, 1H), 6.97 - 6.86 (m, 2H), 6.71 (d, J = 8.0 Hz, 1H), 5.69 (s, 1H), 5.60 (t, J = 5.6 Hz, 1H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.41 (d, J = 6.2 Hz, 2H), 4.22 (s, 2H), 4.18 (dd, J = 45.9, 17.1 Hz, 2H), 3.83 (d, J = 13.3 Hz, 2H), 3.58 (s, 2H), 3.47 - 3.37 (m, 4H), 3.25 (s, 2H), 3.09 (q, J = 6.6 Hz, 2H), 2.92 (ddd, J = 18.1, 13.5, 5.4 Hz, 1H), 2.67 - 2.57 (m, 1H), 2.40 - 2.22 (m, 4H), 2.11 (t, J = 7.4 Hz, 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). 1313C NMR (101 MHz, DMSO) δ 172.9, 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]+ theoretical value 1016.568 (C56H72F2N11O5), measured value 1016.566.

[0455] Scheme 51. N-(3-(((6-(4-(4-((4,4-Dimethylpiperidin-1-yl)methyl)-2,5-difluorophenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecane-9-yl)pyrimidin-4-yl)amino)methyl)benzyl)-11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoindolin-4-yl)amino)undecanamide (Compound 51-1)

[0456] [Chemical formula]

[0457] Using the corresponding amine (18.6 mg, 0.04 mmol) and carboxylic acid (26 mg, 0.04 mmol), Compound 51-1 was prepared according to General Procedure 9 (COMU). The reaction mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10~80:20) to give the desired product 32 mg (73%). 11H NMR (400 MHz, MeOD) δ 8.06 (s, 1H), 7.47 - 7.38 (m, 1H), 7.33 - 7.25 (m, 3H), 7.22 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H), 6.98 (dd, J = 11.4, 7.2 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 5.69 (s, 1H), 5.13 (dd, J = 13.3, 5.1 Hz, 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, 8.1 Hz, 2H), 3.49 - 3.43 (m, 1H), 3.41 (s, 2H), 3.26 - 3.16 (m, 6H), 2.90 (ddd, J = 18.5, 13.3, 5.1 Hz, 1H), 2.81 - 2.73 (m, 1H), 2.46 (qd, J = 13.1, 4.7 Hz, 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). 1313C NMR (101 MHz, MeOD) δ 174.8, 173.3, 171.1, 171.0, 168.4, 161.2, 143.8, 141.1, 139.4, 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]+ theoretical value 1044.600 (C58H76F2N11O5), measured value 1044.600.

[0458] 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]undecan-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)

[0459] [Chemical formula]

[0460] Using the corresponding amine (18.2 mg, 0.03 mmol) and carboxylic acid (15 mg, 0.03 mmol), Compound 52-1 was prepared according to General Procedure 9 (COMU). The reaction mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10~80:20) to give the desired product 10 mg (31%). 1 1H NMR (500 MHz, CDCl3 ) δ 10.24 (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 = 7.1 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.81 (s, 1H), 6.67 (t, J = 6.0 Hz, 1H), 6.58 (dd, J = 10.9, 7.0 Hz, 1H), 6.03 (t, J = 6.0 Hz, 1H), 5.37 (s, 1H), 4.89 (dd, J = 12.2, 5.4 Hz, 1H), 4.62 (d, J = 5.9 Hz, 2H), 4.41 (dd, J = 11.9, 5.8 Hz, 3H), 4.31 (td, J = 6.9, 2.5 Hz, 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, 6.5 Hz, 2H), 1.87 (m, 4H), 1.75 (m, 3H), 1.57 (t, J = 7.8, 7.1 Hz, 2H), 1.45 (m, 2H), 1.26 (m, 16H), 0.94 (s, 4H), 0.88 (t, J = 6.7 Hz, 2H). 13 C NMR (101 MHz, CDCl 3) δ 173.2, 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, 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]+ theoretical value 1111.580 (C59H73F2N14O6), measured value 1111.581.

[0461] 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]undecane-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)undecanamide (Compound 53-1)

[0462]

Chemical Structure

[0463] Using the corresponding amine (17 mg, 0.03 mmol) and carboxylic acid (15 mg, 0.03 mmol), Compound 27 was prepared according to General Procedure 9 (COMU). The reaction mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10 to 80:20) to give the desired product 12 mg (37%). 1 1H NMR (400 MHz, CDCl 3) δ 10.08 (bs, 1H), 8.16 (s, 1H), 7.50 - 7.43 (m, 2H), 7.21 (s, 1H), 7.16 (m, 2H), 7.10 (d, J = 7.2 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.83 (s, 1H), 6.68 (t, J = 5.9 Hz, 1H), 6.58 (dd, J = 10.9, 7.1 Hz, 1H), 6.07 (s, 1H), 5.93 (bs, 1H), 5.40 - 5.32 (m, 2H), 4.90 (dd, J = 11.9, 5.3 Hz, 1H), 4.62 (d, J = 5.9 Hz, 2H), 4.42 (m, 3H), 4.31 (t, J = 7.0 Hz, 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), 2.48 (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 (101 MHz, CDCl 3 ) δ 173.2, 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, 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, 29.3, 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]+ theoretical value 1139.610 (C61H77F2N14O6), measured value 1139.608.

[0464] Scheme 54. Preparation of N-(3-((6-(4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-9-yl)pyrimidin-4-yl)amino)propyl)-15-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)pentadecanamide (Compound 54-1)

[0465]

Chemical Structure

[0466] 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 by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10 to 80:20) to give the desired product (120 mg, 53%). 11H NMR (400 MHz, MeOD) δ 8.01 (s, 1H), 7.35 - 7.31 (m, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.00 - 6.87 (m, 3H), 6.71 (d, J = 8.0 Hz, 1H), 5.71 (s, 1H), 5.04 (dd, J = 13.3, 5.1 Hz, 1H), 4.19 (d, J = 6.4 Hz, 2H), 4.13 (d, J = 7.4 Hz, 2H), 3.78 (d, J = 3.7 Hz, 4H), 3.70 - 3.60 (m, 2H), 3.45 (s, 2H), 3.19 - 3.14 (m, 3H), 3.10 (t, J = 7.2 Hz, 2H), 3.05 - 2.94 (m, 2H), 2.81 (ddd, J = 18.4, 13.4, 5.4 Hz, 1H), 2.68 (ddd, J = 17.6, 4.7, 2.5 Hz, 1H), 2.38 (qd, J = 13.2, 4.7 Hz, 1H), 2.13 - 2.04 (m, 3H), 1.83 (td, J = 8.1, 6.6, 3.4 Hz, 2H), 1.73 (ddd, J = 20.3, 9.7, 5.4 Hz, 3H), 1.53 (tt, J = 14.5, 6.5 Hz, 9H), 1.35 - 1.15 (m, 26H), 0.95 (d, J = 14.2 Hz, 7H). 13 13C NMR (101 MHz, MeOD) δ 175.2, 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]+ theoretical value 1002.670 (C57H84N11O5), measured value 1002.664.

[0467] 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]undecan-9-yl)pyrimidin-4-yl)amino)propyl)-9-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)nonanamide (Compound 55-1)

[0468]

Chem.

[0469] Using the corresponding amine (85 mg, 0.14 mmol) and carboxylic acid (60 mg, 0.24 mmol), Compound 55-1 was prepared according to General Procedure 9 (COMU). The reaction mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / MeOH = 90:10 - 80:20) to give the desired product 100 mg (72%). 1 H NMR (400 MHz, CDCl 3) δ 8.14 (s, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.11 (dd, J = 12.9, 6.6 Hz, 1H), 7.02 (s, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.57 (dd, J = 10.9, 7.1 Hz, 1H), 6.36 (s, 1H), 5.74 (s, 1H), 5.43 (s, 1H), 5.20 (dd, J = 13.2, 5.2 Hz, 1H), 4.20 (dd, J = 65.9, 15.8 Hz, 2H), 3.71 (d, J = 7.1 Hz, 4H), 3.60 - 3.53 (m, 2H), 3.50 (s, 2H), 3.29 (q, J = 6.9 Hz, 4H), 3.25 (s, 2H), 3.18 (q, J = 7.1, 6.6 Hz, 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). 13 C NMR (101 MHz, CDCl 3) δ 173.8, 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]+ theoretical value 954.552 (C51H70F2N11O5), measured value 954.552.

[0470] 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]undecan-9-yl)pyrimidin-4-yl)amino)propyl)-11-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)undecanamide (Compound 56-1)

[0471] [Chemical Structure]

[0472] Using the corresponding amine (40 mg, 0.07 mmol) and carboxylic acid (30 mg, 0.07 mmol), Compound 56-1 was prepared according to General Procedure 9 (COMU). The reaction mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; EtOAc / MeOH = 90:10~80:20) to give the desired product 37 mg (56%). 11H NMR (400 MHz, DMSO) δ 11.00 (s, 1H), 10.87 (s, 1H), 8.36 (s, 1H), 8.29 (s, 1H), 7.99 (t, J = 5.5 Hz, 1H), 7.73 (dd, J = 13.4, 6.8 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 7.05 (dd, J = 11.7, 7.4 Hz, 1H), 6.91 (d, J = 7.4 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 5.94 (s, 1H), 5.10 (dd, J = 13.2, 5.1 Hz, 1H), 4.24 (d, J = 4.5 Hz, 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 = 6.2 Hz, 2H), 3.21 - 3.01 (m, 8H), 2.92 (ddd, J = 18.0, 13.4, 5.2 Hz, 1H), 2.68 - 2.57 (m, 1H), 2.29 (qd, J = 13.4, 4.9 Hz, 1H), 2.13 - 1.97 (m, 3H), 1.91 - 1.70 (m, 5H), 1.66 (t, J = 6.8 Hz, 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). 1313C NMR (101 MHz, DMSO) δ 173.4, 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]+ theoretical value 982.583 (C53H74F2N11O5), measured value 982.584.

[0473] Scheme 57. Preparation of N-(3-((6-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-2-oxo-1,4,9-triazaspiro[5.5]undecan-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)

[0474] [Chemical formula]

[0475] Using the corresponding amine (67 mg, 0.13 mmol) and the corresponding carboxylic acid (66 mg, 0.13 mmol), Compound 57-1 was prepared according to General Procedure 9 (HATU). The reaction mixture was concentrated in vacuo, and the crude product was purified by flash column chromatography (SiO 2 ; DCM / MeOH = 90:10~80:20) to give the desired product 100 mg (76%). 11H NMR (400 MHz, MeOD) δ 7.88 (d, J = 0.8 Hz, 1H), 7.81 (s, 1H), 7.41 (dd, J = 8.5, 7.1 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 6.97 (t, J = 7.9 Hz, 2H), 6.92 - 6.88 (m, 2H), 5.58 (d, J = 1.0 Hz, 1H), 4.94 (dd, J = 12.5, 5.4 Hz, 1H), 4.52 (s, 2H), 4.24 (t, J = 7.0 Hz, 2H), 4.01 (s, 2H), 3.76 (s, 2H), 3.69 (q, J = 5.6, 3.7 Hz, 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, 2.4 Hz, 1H), 2.62 - 2.56 (m, 1H), 2.05 (d, J = 7.5 Hz, 2H), 1.79 - 1.64 (m, 8H), 1.48 (dt, J = 19.1, 6.5 Hz, 7H), 1.27 (dd, J = 7.2, 4.5 Hz, 4H), 1.16 (d, J = 3.3 Hz, 8H), 0.93 (s, 6H). 1313C NMR (101 MHz, MeOD) δ 175.0, 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]+ theoretical value 1013.580 (C54H73N14O6), measured value 1013.584.

[0476] 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]undecan-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)

[0477]

Chemical Structure

[0478] 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 by flash column chromatography (SiO 2 ; DCM / MeOH = 100:5 to 100:20) to give the desired product 15 mg (22.3%). 1 1H NMR (400 MHz, CDCl 3) δ 10.38 (bs, 1H), 8.14 (s, 1H), 7.47 (dd, J = 8.5, 7.1 Hz, 1H), 7.13 (dd, J = 12.8, 6.7 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.90 (s, 1H), 6.70 (t, J = 5.9 Hz, 1H), 6.58 (dd, J = 10.9, 7.2 Hz, 1H ), 6.28 (bs, 1H ), 5.79 (bs, 1H ), 5.44 (s, 1H), 4.91 (dd, J = 12.1, 5.4 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 4.32 (t, J = 7.0 Hz, 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 (101 MHz, CDCl 3 ) δ 207.1, 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, 105.9, 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]+ theoretical value 1049.564 (C54H71F2N14O6), measured value 1049.565.

[0479] 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]undecan-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)undecanamide (Compound 59-1)

[0480]

Chem.

[0481] 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 by flash column chromatography (SiO 2 ; DCM / MeOH = 100:10 to 100:20) to afford the desired product as 14 mg (21%). 1 H NMR (400 MHz, CDCl 3) δ 10.38 (bs, 1H), 8.14 (s, 1H), 7.47 (dd, J = 8.5, 7.1 Hz, 1H), 7.13 (dd, J = 12.8, 6.7 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 7.05 (s, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.70 (t, J = 5.9 Hz, 1H), 6.58 (dd, J = 10.9, 7.2 Hz, 1H ), 6.28 (bs, 1H ), 5.79 (bs, 1H ), 5.44 (s, 1H), 4.91 (dd, J = 12.1, 5.4 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 4.32 (t, J = 7.0 Hz, 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 (101 MHz, CDCl3) δ 207.1, 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, 110.9, 105.9, 105.6, 63.9, 56.6, 54.6, 53.5, 53.0, 50.6, 49.7, 49.1, 40.3, 38.9, 38.4, 36.9, 36.5, 35.3, 31.6, 31.1, 30.2, 29.8, 29.5, 29.4, 29.1, 29.1, 29.1, 28.7, 28.4, 26.3, 25.8, 22.9. LRMS(ESI): m / z [M+H]+ theoretical value 1077.596 (C56H75F2N14O6), measured value 1077.593. Synthetic schemes from the documents incorporated by reference in this specification:

[0482] [Chemical formula]

[0483] References regarding general procedures and methods for preparing starting materials, 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, the procedures of click chemistry and the method for preparing the triazole in Formula J) are also incorporated herein by reference.

[0484] tert - Butyl 4 - (aminomethyl) - 4 - ((((benzyloxy)carbonyl)amino)piperidine - 1 - carboxylate (32):

[0485] [Chemical formula]

[0486] MeNO 2 (1.3 equivalents, 130 mmol, 7 mL) of NH 3 (53 mL, 7N in MeOH) was added portionwise to a stirred solution of tert - butyl 4 - oxopiperidine - 1 - carboxylate (20 g, 100 mmol). 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 - phase was separated, and the aqueous layer was extracted twice with DCM. The combined organic layers were dried over MgSO 4 and filtered, and concentrated under reduced pressure to give the desired product, which was used in the next step without further purification.

[0487] To a stirred solution of the corresponding amine (100 mmol) in dichloromethane (130 mL), K 2 CO 3(2 equivalents, 200 mmol, 27.6 g) of an aqueous solution of water (130 mL) was added. The reaction mixture was cooled to 0 °C, and CBzCl (1.1 equivalents, 110 mmol, 15.6 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 17 h, and the two phases were separated. The aqueous layer was extracted twice with DCM. The combined organic layers were washed once with brine, dehydrated with MgSO 4 and filtered, and concentrated under reduced pressure to produce the desired product, which was used in the next step without further purification.

[0488] To a stirred solution of the corresponding nitroalkane (100 mmol) in dry MeOH (450 mL) at 0 °C under a nitrogen atmosphere, NiCl 2 .6H 2 O (1 equivalent, 100 mmol, 27.3 g) was added, followed by NaBH 4 (5 equivalents, 500 mmol, 18.9 g), which was added portionwise to avoid vigorous evolution of H 2 . Care must be taken when adding NaBH 4 as the reaction is highly exothermic and generates hydrogen gas. The reaction mixture was stirred at 25 °C for 1 h and quenched by adding saturated NaHCO 3 aqueous solution. The mixture was filtered through a pad of celite, the filtrate was concentrated under reduced pressure, and the resulting residue was diluted with water. The aqueous layer was extracted three times with DCM, the combined organic layers were washed once with brine, dehydrated with MgSO 4 and filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM / MeOH / NH 4 OH = 100:0:0~100:3:0~100:3:1~100:5:1~100:10:1~100:15:1) to produce the desired product as a white solid (11.7 g in 32% yield via a three-step reaction). LRMS (ESI): m / z calculated 364.2 ([C 19 H 30 N 3 O 4 + ), found 364.3.

[0489] ​tert-Butyl 2-oxo-1,4,9-triazaspiro[5.5]undecane-9-carboxylate (33):

[0490] [Chemical formula]

[0491] 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) at 0 °C was added Et 3 N (0.8 equiv, 32 mmol, 4.4 mL) and ethyl 2-bromoacetate (0.7 equiv, 28 mmol, 3.1 mL). The reaction mixture was stirred at 25 °C for 2 h and diluted with saturated NaHCO 3 aqueous solution. The aqueous layer was extracted three times with EtOAc, and the combined organic layers were washed once with brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure to give the desired impurities, which were used in the next step without further purification.

[0492] To a stirred solution of the corresponding Cbz-protected amine (40 mmol) in iPrOH (400 mL) were added Pd / C (5 mol%, 2 mmol, 2.1 g, 10% wt) and ammonium formate (6 equiv, 240 mmol, 15 g) in small portions. The reaction mixture was stirred at 80 °C for 4 h, cooled to 25 °C, filtered through a pad of celite, and concentrated under reduced pressure. The resulting residue was dissolved in DCM, and the organic layer was washed once with water, once with brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM / MeOH = 100:5~100:8~100:10~100:15) to give the desired product as a white solid (4.14 g in 55% yield after a two-step reaction). LRMS (ESI): m / z [2M+H] + Theoretical value 539.4 (C 26 H 47 N 6 O 6 ) + ), found 539.4.

[0493] N-Benzyl-6-(4-(6-((4,4-dimethylpiperidin-1-yl)methyl)pyridin-3-yl)-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)pyrimidin-4-amine (7):

[0494]

Chem.

[0495] According to the general procedure of the backward-Hartwig coupling, the corresponding Boc-protected amine was obtained (chromatography: EtOAc / heptane = 7:3 to 9:1 to EtOAc / MeOH = 100:0 to 100:1 to 100:5). The desired impurities were used in the next step without further purification.

[0496] According to the general procedure for the deprotection of the Boc group, the corresponding amine was obtained. After evaporation, the crude residue was triturated with acetone, and the resulting precipitate was filtered, washed with acetone, and dried to obtain the desired impurities, which were used in the next step without further purification.

[0497] To a stirred solution of the corresponding amine (1 equiv) in iPrOH (0.3 M), 29 (1.5 equiv) and Et 3 N (4 equiv) were added. The reaction mixture was stirred at 150 °C for 8 h in a microwave and concentrated under reduced pressure. The reaction was diluted with water, and the aqueous layer was extracted 3 times with DCM. The combined organic layers were washed 5 times with water, once with brine, dehydrated over MgSO 4 and 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% yield after a three-step reaction). Mp: 61 - 62 °C; HRMS (ESI): m / z calcd for 542.3607 ([C 32 H 44 N 7 O] +) Measured value: 542.3602. N-Benzyl-6-chloropyrimidin-4-amine (29):

[0498]

Chem.

[0499] To a stirred solution of 4,6-dichloro-pyrimidine (5 g, 33.6 mmol) in iPrOH (100 mL) were added benzylamine (1.2 equiv, 40.3 mmol, 4.4 mL) and Et 3 N (1.2 equiv, 40.3 mmol, 5.59 mL). The reaction mixture was stirred at 25 °C for 3 d and concentrated under reduced pressure. The crude residue was triturated with water, filtered, and dried to afford the desired product as a beige solid (7.21 g, 98% yield). LRMS (ESI) m / z calcd for 11 H 11 ClN 3 + ) Measured value: 220.1. 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):

[0500]

Chem.

[0501] General procedure for S N Ar using the chloropyrimidine derivative (Chromatography: DCM / MeOH = 100:5~100:10~100:15~100:20~100:30) gave compound 8. The obtained impurities were triturated with diethyl ether, filtered, and washed twice with ether, once with water, and once with ether to afford the desired product as a white solid (14 mg, 25% yield). Mp: 208~209 °C; HRMS (ESI): m / z calcd for 32 H 43 N​8 O] + )), measured value 555.3554. 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):

[0502]

Chem.

[0503] The corresponding chloropyrimidine was obtained according to the general procedure of S N Ar. Instead of chromatography, after evaporation, the crude residue was triturated with water, filtered, and washed once with water. The resulting 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.

[0504] S N Ar using the chloropyrimidine derivative, compound 9 was obtained (chromatography: DCM / MeOH = 100:0 to 100:8 for 20 minutes, 100:8 for 10 minutes, 100:8 to 100:10 for 10 minutes). The resulting impurities were triturated with water, filtered, and washed once with water to give the desired product as a pale yellow solid (after a two-step reaction, yield 5%). Mp: 228 - 231 °C; HRMS(ESI): m / z calculated value 554.3607 ([C 33 H 44 N 7 O] + )), measured value 554.3602. 4-(4-((4,4-Dimethylpiperidin-1-yl)methyl)phenyl)-9-(6-(methylamino)pyrimidin-4-yl)-1,4,9-triazaspiro[5.5]undecan-2-one (Compound 10):

[0505]

Chem.

[0506] The corresponding chloropyrimidine batch was the same as that used for Compound 9. S using chloropyrimidine derivatives N According to the general procedure for Ar (chromatography: DCM / MeOH = 100:0 to 100:12 for 20 minutes, 100:12 for 10 minutes, 100:12 to 100:15 for 10 minutes, 100:15 for 10 minutes), Compound 10 was obtained. White solid (through a two-step reaction, yield 3%). Mp: 235 - 236 °C; HRMS (ESI): m / z theoretical value 478.3294 ([C 27 H 40 N 7 O] + ), measured value 478.3289. 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):

[0507]

Chemical formula

[0508] Under a nitrogen atmosphere, 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1 equivalent, 0.21 mmol, 32 mg) was added to a stirred solution of 36 (100 mg, 0.21 mmol) in dry THF (700 μL). Nitrogen gas was bubbled through this reaction solution for 2 minutes, and then Ruphos Pd G4 (5 mol%, 0.011 mmol, 8.9 mg), Ruphos (5 mol%, 0.011 mmol, 5.1 mg), and LiHMDS (6.6 equivalents, 1.39 mmol, 1.39 mL, 1 M THF) were added. The reaction mixture was stirred at 65 °C for 4 h, 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 give the desired product as a yellow solid (37 mg, yield 36%). Mp: 250 - 252 °C; HRMS (ESI): m / z theoretical value 488.3138 ([C 28 H38 N 7 O] + )、Measured value 488.3132. 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):

[0509]

Chem.

[0510] To a stirred solution of 36 (100 mg, 0.21 mmol) in iPrOH (1 mL) were added 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (1.2 equiv, 0.25 mmol, 47 mg) and Et 3 N (4 equiv, 0.84 mmol, 116 μL). The reaction mixture was stirred by microwave at 100 °C for 3 h and then at 130 °C for 3 h. The reaction mixture was concentrated under reduced pressure, and the crude residue was triturated with water. The resulting precipitate was filtered, washed with water, and dried to give the desired impurity, which was further purified by flash column chromatography (DCM / MeOH = 100:0 to 100:10 for 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 calcd for 28 H 37 N 7 O] + )、Measured value 522.2731. 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):

[0511]

Chem.

[0512] S using chloropyrimidine derivative N Compound 16 was obtained according to the general procedure of Ar (chromatography: DCM / MeOH = 100:0 to 100:10 for 15 minutes, 100:10 for 10 minutes, 100:10 to 100:15 for 15 minutes). Brown solid (yield 25%). Mp: 172 - 174 °C; HRMS(ESI): m / z theoretical value 504.3451 ([C 29 H 42 N 7 O] + ), measured value 504.3445. 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):

[0513]

Chemical Structure

[0514] S using chloropyrimidine derivative N Compound 15 was obtained according to the general procedure of Ar (chromatography: DCM / MeOH = 100:0 to 100:8 for 15 minutes, 100:8 for 10 minutes, 100:8 to 100:10 for 10 minutes). Beige solid (yield 52%). Mp: 175 - 176 °C; HRMS(ESI): m / z theoretical value 506.3607 ([C 29 H 44 N 7 O] + ), measured value 506.3602. 1-(4-Bromobenzyl)-4,4-dimethylpiperidine (23):

[0515]

Chemical Structure

[0516] Intermediate 23 was obtained according to the general procedure for the alkylation of dimethylpiperidine (chromatography: EtOAc / heptane = 0:10 to 3:7). Yellow oil, 99% yield. LRMS (ESI): m / z calculated 282.1 ([C 14 H 21 BrN] + ), found 282.1. 5-Chloro-2-((4,4-dimethylpiperidin-1-yl)methyl)pyridine (26):

[0517]

Chemical Structure

[0518] (5-Chloropyridin-2-yl)methanol (2.43 g, 17 mmol) in stirred DCM (40 mL) was treated with SOCl 2 (1.5 equiv, 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.

[0519] Intermediate 26 was obtained according to the general procedure for the alkylation of dimethylpiperidine, 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 calculated 239.1 ([C 13 H 20 ClN 2 + ), found 239.2. 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):

[0520]

Chemical Structure

[0521] ​According to the general procedure for the Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:2 to 100:5 to 100:8 to 100:12 to 100:15 to 100:20), the corresponding Boc-protected amine was obtained. The desired impurities were used in the next step without further purification.

[0522] According to the general procedure for the deprotection of the Boc group, the corresponding amine was obtained. After evaporation, the crude residue was triturated with acetone, and the resulting precipitate was filtered, washed with acetone, and dried to obtain the desired impurities, which were used in the next step without further purification.

[0523] S using 4,6-dichloropyrimidine N According to the general procedure for Ar (column chromatography: using DCM / MeOH = 100:2, 100:5, 100.8, 100:10, 100:15, 100:20 as the eluent in sequence), intermediate 34 was obtained, and the desired product was produced as a yellow solid (through a three-step reaction, 123 mg in a yield of 27%). LRMS (ESI): m / z theoretical value 484.3 ([C 25 H 35 ClN 7 O] + ), measured 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):

[0524]

Chemical formula

[0525] According to the general procedure for the Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:5 to 100:8 to 100:12 to 100:20), intermediate 35 was obtained. Beige solid, yield 93%. 4-(4-((4,4-Dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one hydrochloride (36):

[0526]

Chem.

[0527] According to the general procedure for the deprotection of the Boc group, intermediate 36 was obtained. After evaporation, the crude residue was triturated with acetone, and the resulting precipitate was filtered, washed with acetone, and dried to produce the desired product as an off-white solid (50% yield over two steps from intermediate 32). LRMS (ESI): m / z calcd 371.3 ([C 22 H 35 N 4 O] + ), found 371.3. 9-(6-Chloropyrimidin-4-yl)-4-(4-((4,4-dimethylpiperidin-1-yl)methyl)phenyl)-1,4,9-triazaspiro[5.5]undecan-2-one (37):

[0528]

Chem.

[0529] Intermediate 37 was obtained according to the general procedure for S N Ar using 4,6-dichloropyrimidine. Instead of chromatography, the crude mixture was evaporated and the residue was triturated with water. The resulting precipitate was filtered, washed with water, and dried to produce the desired product as a brown solid (63% yield). LRMS (ESI): m / z calcd 483.3 ([C 26 H 36 ClN 6 O] + ), found 483.3. 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):

[0530]

Chem.

[0531] General procedure for S using chloropyrimidine derivative N According to the general procedure of Ar (chromatography: DCM / MeOH = 100:0 to 100:8 for 20 minutes, 100:8 for 15 minutes), compound 20 was obtained. White solid (yield 47%). Mp: 202 - 204 °C; HRMS (ESI): m / z theoretical value 496.3200 ([C 27 H 39 FN 7 O] + ), measured value 496.3195. 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):

[0532]

Chem.

[0533] General procedure for S using chloropyrimidine derivative N According to the general procedure of Ar (chromatography: DCM / MeOH = 100:0 to 100:8 for 20 minutes, 100:8 for 5 minutes, 100:8 - 100:12 for 10 minutes, 100:12 for 5 minutes), compound 21 was obtained. Yellow solid, yield 69%. Mp: 210 - 211 °C; HRMS (ESI): m / z theoretical value 496.3200 ([C 27 H 39 FN 7 O] + ), measured value 496.3195. 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):

[0534]

Chem.

[0535] According to the general procedure for the Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:3 to 100:5 to 100:8), the corresponding Boc-protected amine was obtained. The desired impurities were used in the next step without further purification.

[0536] According to the general procedure for the deprotection of the Boc group, the corresponding amine was obtained. The desired impurities were used in the next step without further purification. The corresponding chloropyrimidine was obtained according to the general procedure for S N Ar. The desired impurities were used in the next step without further purification.

[0537] S using the chloropyrimidine derivative N Ar general procedure (chromatography: DCM / MeOH = 100:0 to 100:10 for 15 minutes, ~100:10 for 10 minutes, 100:10 to 100:12 for 10 minutes), UZH2 was obtained. White solid, through a four-step reaction, yield 56%. Mp: 214 - 216; HRMS(ESI): m / z theoretical value 514.3106 ([C 27 H 38 F 2 N 7 O] + ), measured value 514.3100. 1-(4-Bromo-3-fluorobenzyl)-4,4-dimethylpiperidine (47):

[0538]

Chem.

[0539] Intermediate 47 was obtained according to the general procedure for the alkylation of dimethylpiperidine (chromatography: EtOAc / heptane = 1:9). Colorless oil, yield 98%. LRMS (ESI): m / z calculated 300.1 ([C 14 H 20 BrFN] + ), found 300.1. 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):

[0540]

Chemical Structure

[0541] The corresponding Boc-protected amine was obtained according to the general procedure for the backward-Hartwig coupling (chromatography: DCM / MeOH = 100:3 to 100:5 to 100:8). The desired impurities were used in the next step without further purification.

[0542] The corresponding amine was obtained according to the general procedure for the deprotection of the Boc group. The desired impurities were used in the next step without further purification. S using 4,6-dichloropyrimidine N Ar general procedure (column chromatography: DCM / MeOH = 100:3 to 100:5 to 100.8 to 100:10) to obtain Intermediate 48 and the desired product as a white solid (yield 15% after a three-step reaction). LRMS (ESI): m / z calculated 501.3 ([C 26 H 35 ClFN 6 O] + ), found 501.3. 1-(4-Bromo-2-fluorobenzyl)-4,4-dimethylpiperidine (49):

[0543]

Chem.

[0544] Intermediate 49 was obtained according to the general procedure for the alkylation of dimethylpiperidine. Colorless liquid, yield 99%. LRMS (ESI): m / z calculated 300.1 ([C 14 H 20 BrFN] + ), found 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):

[0545]

Chem.

[0546] Intermediate 50 was obtained according to the general procedure for the Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:5~100:8~100:12~100:16). Brown solid (yield 83%). LRMS (ESI): m / z calculated 489.3 ([C 27 H 42 FN 4 O 3 + ), found 489.4. 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):

[0547]

Chem.

[0548] The corresponding amine was obtained according to the general procedure for the deprotection of the Boc group. The desired impurities were used in the next step without further purification. ​S using 4,6-dichloropyrimidine N Intermediate 51 was obtained according to the general procedure of Ar. Instead of chromatography, the crude mixture was evaporated and the residue was triturated with water. The resulting precipitate was filtered, washed with water and dried to give the desired product as a brown solid (58% yield via a two-step reaction). LRMS (ESI): m / z calcd 501.3 ([C 26 H 35 ClFN 6 O] + ), found 501.3. 1-(4-Bromo-2,5-difluorobenzyl)-4,4-dimethylpiperidine (52):

[0549]

Chemical formula

[0550] Under a nitrogen atmosphere, BH 3 .SMe 2 (2 equiv, 8.4 mmol, 4.2 mL, 2 M THF) was added to a stirred solution of 4-bromo-2,5-difluorobenzoic acid (1 g, 4.2 mmol) in dry THF (10 mL). The reaction mixture was stirred at 25 °C for 17 h, cooled to 0 °C and quenched by the addition of saturated Na 2 CO 3 aqueous solution. The aqueous layer was extracted three times with EtOAc, the combined organic layers were washed once with brine, dried over MgSO 4 and concentrated under reduced pressure to give the desired product as a brown solid (789 mg, 83% yield).

[0551] To a stirred solution of the corresponding alcohol (789 mg, 3.54 mmol) in DCM (10 mL) were added SOCl 2 (1.5 equiv, 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.

[0552] To a stirred solution of the corresponding chloroalkane (425 mg, 1.76 mmol) in dimethylformamide (5 mL) were added 4,4-dimethylpiperidine hydrochloride (1 equiv, 1.76 mmol, 263 mg) and K 2 CO 3 (2 equiv 3.52 mmol, 486 mg). The reaction mixture was stirred at 25 °C for 3 days and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc / heptane = 3:100 to 10:100) to yield the desired product as a colorless solid (514 mg, 92%). LRMS (ESI): m / z calcd for 318.1 ([C 14 H 19 BrF 2 N] + ), found 318.1. 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):

[0553] [Chemical Structure Diagram]

[0554] The corresponding Boc-protected amine was obtained according to the general procedure for the Buchwald-Hartwig coupling (chromatography: DCM / MeOH = 100:5 to 100:8 to 100:11 to 100:15). The desired impurities were used in the next step without further purification.

[0555] The corresponding amine was obtained according to the general procedure for the deprotection of the Boc group. The desired impurities were used in the next step without further purification. The corresponding chloropyrimidine was obtained according to the general procedure for S N Ar using 4,6-dichloropyrimidine. Due to the remaining impurities of 40 derivatives, 7 equivalents of pyrimidine and 7 equivalents of Et 3Using N, the reactants were heated at 80 °C for 7 h with microwave. The desired impurities were used in the next step without further purification.

[0556] S using chloropyrimidine derivatives N Compound 11 was obtained according to the general procedure for Ar (chromatography: DCM / MeOH = 100:0 to 100:10 for 15 min, 100:10 for 5 min, 100:10 to 100:13 for 5 min, 100:13 for 5 min). Yellow solid, through a four-step reaction, yield 19%. Mp: decomposition; HRMS (ESI): m / z calculated value 450.2981 ([C 25 H 36 N 7 O] + )), measured value 450.2976. tert-Butyl 3-amino-3-(nitromethyl)azetidine-1-carboxylate (38):

[0557]

Chemical formula

[0558] To a stirred solution of tert-butyl 3-oxoazetidine-1-carboxylate (10.65 g, 62 mmol) in EtOH (31 mL), MeNO 2 (13 mL) and K 2 CO 3 (1 mol%, 0.62 mmol, 86 mg) were added. The reaction mixture was stirred at 25 °C for 17 h and filtered. The filtrate was concentrated under reduced pressure to give the desired product, which was used in the next step without further purification.

[0559] Under a nitrogen atmosphere, a stirred solution of the corresponding alcohol (62 mmol) in dry DCM (250 mL) was cooled to -78 °C, and DAST (1.2 equiv, 74.4 mmol, 9.8 mL) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred for 3 h, cooled to 0 °C, and quenched slowly by adding saturated NaHCO 3 aqueous solution. The aqueous layer was extracted three times with DCM, washed once with brine, and dried over MgSO 4It was dehydrated, filtered, and concentrated under reduced pressure to obtain the desired product, which was used in the next step without further purification.

[0560] The corresponding nitromethylene (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 obtain the desired product as a pale yellow solid (quantitative yield 15.67 g via a three-step reaction).

[0561] tert-Butyl 3-(aminomethyl)-3-(((benzyloxy)carbonyl)amino)azetidine-1-carboxylate (39):

[0562]

Chemical formula

[0563] To a stirred solution of 38 (62 mmol) in dichloromethane (100 mL), an aqueous solution of NaHCO 3 (2 equivalents, 124 mmol, 10.42 g) in water (100 mL) was added. The reaction mixture was cooled to 0 °C, and CbzCl (1 equivalent, 62 mmol, 8.8 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 17 h, and the two phases were separated. The aqueous layer was extracted twice with DCM. The combined organic layers were washed once with brine, dried over MgSO 4 filtered, and concentrated under reduced pressure to produce the desired product, which was used in the next step without further purification.

[0564] To a stirred solution of the corresponding nitroalkane (62 mmol) in dry MeOH (300 mL) at 0 °C under a nitrogen atmosphere, NiCl 2 .6H 2 O (1 equivalent, 62 mmol, 16.9 g) was added, followed by NaBH 4 (5 equivalents, 310 mmol, 11.7 g), which was added portionwise to avoid vigorous H 2 evolution. NaBH 4When adding, sufficient care must be taken as the reaction is highly exothermic and generates hydrogen gas. The reaction mixture was stirred at 25 °C for 1 h and quenched by adding saturated NaHCO 3 aqueous solution. The mixture was filtered through a pad of celite, the filtrate was concentrated under reduced pressure, and the resulting residue was diluted with brine and saturated Na 2 CO 3 aqueous solution. The aqueous layer was extracted 3 times with DCM, the combined organic layers were washed once with brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM / MeOH / NH 4 OH = 100:3:0 to 100:3:1 to 100:5:1 to 100:8:1 to 100:12:1 to 100:20:1), and the desired product was obtained as a white solid (11.3 g, 54% yield via a two-step reaction). LRMS (ESI): m / z [2M+H] + Theoretical value 671.4 ([C 34 H 51 N 6 O 8 + ), found 671.4.

[0565] tert-Butyl 6-oxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (40):

[0566]

Chemical Structure

[0567] To a stirred solution of 39 (11.3 g, 33.7 mmol) in DCM (110 mL) at 0 °C was added Et 3 N (1 equiv, 33.7 mmol, 4.7 mL) and ethyl 2-bromoacetate (1 equiv, 33.7 mmol, 3.7 mL). The reaction mixture was stirred at 25 °C for 17 h and diluted with saturated NaHCO 3 aqueous solution. The aqueous layer was extracted 3 times with DCM, the combined organic layers were washed once with water and once with brine, dried over MgSO 4 ​It was dehydrated, filtered, and concentrated under reduced pressure to produce the desired impurities (12.4 g, 29 mmol), which were used in the next step without further purification.

[0568] To a stirred solution of the corresponding Cbz-protected amine (29 mmol) in iPrOH (240 mL), Pd / C (5 mol%, 1.5 mmol, 1.6 g, 10% wt) and ammonium formate (6 equiv, 174 mmol, 11 g) were added portionwise. The reaction mixture was stirred at 80 °C for 4 h, cooled to 25 °C, filtered through a pad of celite, and concentrated under reduced pressure. The resulting residue was partitioned between DCM and water, the two phases were separated, and the aqueous layer was extracted three times with DCM. The combined organic layers were washed once with water and once with brine, and dried over MgSO 4 dried, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (DCM / MeOH = 100:5~100:8~100:10~100:15~100:20) to give the desired product as a white solid (2.6 g, 32% yield over two steps). LRMS (ESI): m / z [M-tBu+2H] + Theoretical 186.1 ([C 7 H 12 N 3 O 3 )] + )), found 186.2.

[0569] 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):

[0570]

Chemical Structure

[0571] S using a chloropyrimidine derivative NAccording to the general procedure for Ar (chromatography: DCM / MeOH = 100:0 to 100:7 for 20 minutes, 100:7 for 5 minutes, 100:7 to 100:8 for 5 minutes, 100:8 for 5 minutes), compound 53 was obtained. Pale yellow solid, yield 53%. Mp: 203 - 205 °C; HRMS(ESI): m / z theoretical value 482.3044([C 26 H 37 FN 7 O] + ), measured value 482.3038.

[0572] 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)

[0573]

Chemical Structure

[0574] According to the general procedure for the backward-Hartwig coupling, the corresponding Boc-protected amine was obtained (chromatography: EtOAc / heptane = 7:3 to 9:1 to EtOAc / MeOH = 100:3 to 100:5 to 100:8 to 100:10). The desired impurities were used in the next step without further purification.

[0575] According to the general procedure for the deprotection of the Boc group, the corresponding amine was obtained. The desired impurities were used in the next step without further purification. S using 4,6-dichloropyrimidine N According to the general procedure for Ar of column chromatography (DCM / MeOH = 100:3 to 100:5 to 100.8), intermediate 54 was obtained and the desired product was produced as a white solid (yield 12% through a three-step reaction). 1-(4-Bromobenzyl)-4-fluoro-4-methylpiperidine (55):

[0576]

Chemical Structure

[0577] To a stirred solution of tert-butyl 4-hydroxy-4-methylpiperidine-1-carboxylate (500 mg, 2.32 mmol) in dry DCM (7 mL) at 0 °C under a nitrogen atmosphere, DAST (1.5 eq, 3.48 mmol, 460 μL) was added. The mixture was stirred at 25 °C for 3 h and quenched by adding saturated NaHCO 3 aqueous solution. 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 4 and filtered, and concentrated under reduced pressure to give the desired product, which was used in the next step without further purification.

[0578] According to the general procedure for the deprotection of the Boc group, the corresponding amine was obtained. The desired impurities were used in the next step without further purification. Intermediate 55 was obtained according to the general procedure for the alkylation of dimethylpiperidine. (Column chromatography: EtOAc / heptane = 1:9 to 3:7 to 1:1). Yellow oil, via a three-step reaction, 77% yield.

[0579] 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):

[0580]

Chemical Structure

[0581] To a stirred solution of 36 (150 mg, 0.31 mmol) in iPrOH (1.5 mL), 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (1.2 eq, 0.38 mmol, 58 mg) and Et 3N (4 equivalents, 1.24 mmol, 172 μL) was added. The reaction mixture was stirred by microwave at 50 °C for 3 h and then at 70 °C for 2 h. The reaction mixture was concentrated under reduced pressure, and the crude residue was triturated with water. The resulting precipitate was filtered, washed with water, washed with DCM, and dried to produce the desired impurities, which were further purified by flash column chromatography (DCM / MeOH = 100:0 to 100:10 for 20 min, 100:10 for 10 min) to produce the desired product as a yellow solid (33 mg, yield 22%). Mp: 252 - 254 °C; HRMS (ESI): m / z theoretical value 489.3090 ([C 27 H 37 N 8 O] + ), found 489.3085.

[0582]

Table 8-1

[0583]

Table 8-2

[0584]

Table 8-3

[0585]

Table 9-1

[0586]

Table 9-2

[0587]

Table 9-3

[0588]

Table 9-4

[0589]

Table 9-5

[0590] Plate reader-based HTRF assay for METTL3 inhibition in vitro The modification oligoribonucleotide's specific binding to the m 6 A reader YTHDC1 was measured, and the m 6 A level in the oligoribonucleotide substrate after the reaction was catalyzed by METTL3-METTL14 was quantified. The test compound that inhibits METTL3 causes a decrease in the m 6 A level, resulting in a decrease in the HTRF signal. The two-step protocol of the METTL3-METTL14 assay consists of a reaction step and a subsequent detection step.

[0591] In the reaction step, METTL3-METTL14 (final concentration 3 nM) methylates 5'-biotinylated ssRNA (5'-AAGAACCGGACUAAGCU-3' (Microsynth)) (final concentration 50 nM). As the last component, the co-substrate SAM (Cisbio, 62SAHZLD) was added to initiate the methylation reaction. The final reaction volume was 15 μL with 20 mM Tris-HCl, pH 7.5, 0.01% (w / v) bovine serum albumin (BSA). After incubating the reaction at room temperature (RT) for 40 minutes, 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-tagged m 6 A reader YTHDC1 (345 - 509), 3 nM XL665-conjugated streptavidin (Cisbio, 610SAXLB), 1× anti-GST Eu 3+ labeled antibody (from 400× stock solution (Cisbio, 61GSTKLB))) was added to stop it. m 6m by A leader 6 After the capture of modified RNA by A and the capture of biotinylated RNA by streptavidin were allowed to proceed for 3 h by RT in the dark, the TR-FRET signal was measured using a Tecan Spark plate reader (Tecan). After excitation of the HTRF donor with UV light at 320 nm, the luminescence at 620 nm and 665 nm was recorded by the plate reader with a 100 μs delay. In this time-resolved measurement, the short-lived background fluorescence of the signal was excluded. The luminescence signal was read over an integration time of 400 μs. The 665 / 620 nm signal ratio (HTRF ratio) makes it possible to correct for the variation between wells and the medium absorbance by the colored compounds. The reaction curves were plotted with GraphPad Prism 8.4 and fitted with a non-linear regression of "log(inhibitor) vs. normalized response - variable slope", from which the IC50 values were determined. Each compound was measured in triplicate.

[0592] The results of the IC50 values are shown in Table 9. Screening of PROTAC activity by Western blot The degradation of METTL3 (and METTL14) protein was monitored using Western blot. Cells were treated with the indicated concentration of PROTAC (or control DMSO) for 24 h at 37 °C, CO 2It was treated at 5%. Subsequently, the samples were collected and lysed with RIPA buffer containing a protease inhibitor (11697498001, Roche). After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane, blocked (5% milk, 0.5% BSA in TBST buffer), and incubated overnight with the primary antibodies. The following antibodies were used. GAPDH (#2118, Cell Signaling, 1:4000), β-actin (ab8226, Abcam, 1:2000), METTL3 (ab195352, Abcam, 1:1000), METTL14 (ab220031, Abcam, 1:1000). After incubation with the appropriate secondary antibodies (anti-mouse IgG IRDye® 680RD (926-68072, LI-COR, 1:10000), goat anti-rabbit IgG IRDye® 800CW (926-32211, LI-COR, 1:10000)), the membrane was scanned using a LI-COR Odyssey DLx imager. Densitometry was performed with Image Studio Lite software and the analysis was performed with GraphPad Prism 9.

[0593] Figure 9 shows the degradation of METTL3 protein after treatment with various PROTAC molecules at a concentration of 2 μM for 24 h, measured using Western blot. Figure 10 shows METTL14 protein after treatment with various PROTAC molecules at a concentration of 2 μM for 24 h, measured using Western blot.

[0594] Figure 11 shows the correlation between METTL3 degradation and METTL14 degradation of various PROTAC molecules, measured using Western blot.

Claims

1. General formula (A) 【Chemistry 1】 [In the formula, - NR ３１ R ３２ teeth, 【Chemistry 2】 Selected from; - Each R ２ F, Cl, CF ３ CHF ２ , and CH ２ Independently selected from the group containing 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; - The handle is a connecting portion containing 3 to 10 atoms (C, N, O, S) with atomic mass ≥ 12; - The linker is a linker portion containing 3 to 50 atoms with an atomic mass ≥ 12; - The E3 ligase-binding molecule is the part that specifically binds to E3 ligase. Compounds of or pharmaceutically acceptable salts thereof.

2. The E3 ligase-binding molecule is given by formula (B) 【Transformation 3】 [In the formula, - Ox is CH ２ Or C = O; - T is selected from the group including F and Cl; - k is an integer selected from the group including 0, 1, and 2; 【Chemistry 4】 This means linking to the linker. The compound according to claim 1, which is an E3 ligase-binding molecule, or a pharmaceutically acceptable salt thereof.

3. The handle is as follows: 【Transformation 5】 [In the formula, - Mid is C １ ~ C ３ selected from the group including alkyl and phenyl] A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the group comprising the above.

4. The handle is as follows: 【Transformation 6】 A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the group comprising the above.

5. The handle is, formula (X): 【Transformation 7】 And, Mid is C １ ~C ３ A compound according to claim 1, selected from the group comprising alkyl and phenyl, or a pharmaceutically acceptable salt thereof.

6. The linker is as follows: 【Transformation 8】 [In the formula, - Lin is C ３ ~C ２０ Alkyl, C ３ ~C ２０ [Selected from the group including alkyl-triazoles and oligo(ethylene glycol)] A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the group comprising the above.

7. The linker is as follows: 【Chemistry 9】 [In the formula, - 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. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the group comprising the above.

8. 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; The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein u is selected from 7, 8, 9, 10, 11, 12, and 13.

9. The linker, formula (W): 【Chemistry 10】 It includes Lin, C ３ ~C ２０ Alkyl, C ３ ~C ２０ A compound according to claim 1, selected from the group comprising alkyl-triazoles and oligo(ethylene glycol), or a pharmaceutically acceptable salt thereof.

10. Linker, equation (Z): 【Chemistry 11】 A compound according to claim 1 or a pharmaceutically acceptable salt thereof, comprising [wherein z is selected from 4, 5, 6, 7, 8, 9, and 10].

11. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, comprising the following definitions of handle, linker and E3 ligase-binding molecule. Table 1-1 Table 1-2 Table 1-3

12. NR ３１ R ３２ but, 【Chemistry 12】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

13. A compound selected from Table 3, or a pharmaceutically acceptable salt thereof. 【Request Item 14】 【Chemistry 13-1】 【Chemistry 13-2】 【Chemistry 13-3】 【Chemistry 13-4】 【Chemistry 13-5】 A compound selected from, or a pharmaceutically acceptable salt thereof.

15. A composition comprising the compound described in claim 1 or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, A composition wherein the cancer is selected from the group including renal cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, and lung adenocarcinoma.

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