Chemical matters for targeting leukemic complex proteins

Abstract

The present disclosure relates to compounds, compositions, and methods for treating diseases or conditions mediated by aberrant leukemic complex proteins. (I) (II)

Description

CHEMICAL MATTERS FOR TARGETING LEUKEMIC COMPLEX PROTEINSRELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63 / 560,936, filed March 4, 2024, which is incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0002] Mixed lineage leukemia 1 (MLL1, also known as MLL or KMT2A) is an important transcription factor and histone-H3 lysine-4 (H3K4) methyltransferase. It is a regulator for transcription of important genes (e.g., Hox genes) for embryonic development and hematopoiesis. Dysregulation of MLL1 leads to overexpression of certain Hox genes and eventually leukemia initiation. Chromosome translocations involving MLL1 cause approximately 75% of acute leukemia in infants and 5-10% in children and adults, with a poor prognosis in all three groups (Li et al., J. Hematol. Oncol., 2021, 74:56). It is characterized by the expression of fusion genes involving the histone methyltransferase MLL1 and a variety of fusion partners. MLL and its associated fusion partners are anchored to chromatin through Menin. This proximity allows for histone methyltransferase proteins (HMTs) recruited by fusion partners to aberrantly alter histones, leading to active transcription of genes driving leukemia progression.

[0003] Onco-MLLl consists of the N-terminal DNA-interacting domains of MLL1 fused with any one of more than 70 fusion partners, among which transcription cofactors AF4, AF9 and its paralog ENL, and ELL are the most frequent. Wild-type (WT)- and onco-MLLl involve numerous protein-protein interactions, which play critical roles in regulating gene expression in normal physiology and leukemia. Targeted therapeutics against oncogenic MLL 1 -fusion proteins are needed.SUMMARY OF THE INVENTION

[0004] A first aspect of the present disclosure is directed to a compound having a structure represented by formula I or II:or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: the Degron binds cereblon (CRBN) or Von Hippel-Lindau (VHL) tumor suppressor; and the Linker represents a moiety that covalently connects the amide and the Degron, and which comprises at least 3 alkylene units.

[0005] A second aspect of the present disclosure is directed to a compound having a structure represented by formula I’ or II’:(ir), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: the Degron binds cereblon (CRBN) or Von Hippel-Lindau (VHL) tumor suppressor; and the Linker represents a moiety that covalently connects the amide and the Degron, and which comprises at least 2 alkylene units.

[0006] Another aspect of the present disclosure is directed to a pharmaceutical composition containing the compound of formula I, II, I’ or IL, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0007] Another aspect of the present disclosure is directed to intermediates for making the compounds of formulas I, II, I’ and II’ .

[0008] In another aspect of the present disclosure, methods of making the compounds and their intermediates are provided.

[0009] A further aspect of the present disclosure is directed to a method of treating a cancer that involves mixed lineage leukemia (MLL)-fusion protein, that includes administering the compound of formula I, II, I’ or II’, or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof.

[0010] Previous reports demonstrate that Menin functions as an essential oncogenic co-factor for MLL-r, making it a potential target for treatment of MLL-r leukemia. More recent reports show that the small molecule inhibitor VTP-50469 reduces MLL-r leukemia burden in mice by specifically targeting Menin, and in so doing, interrupting the protein-protein interaction between Menin and MLL1 (Krivtsov et al., Cancer Cell, 2019, 36(6 / 660-673). Applicant has discovered that VTP-50469 when bound to Menin in a Menin-MLLl fusion, may actually overlap with MLL1. Presently disclosed compounds, also known in the art as Proteolysis Targeting Chimeras (PROTACs), contain a structurally modified VTP-50469 that facilitates docking of the compound into this fusion. As shown in working examples, PROTACs of the present disclosure exhibit much broader activity in terms of recruiting MLL1, as well as Menin, for targeted degradation. In so doing, they may deplete the full function of the MLL1 protein, and not just its physical interaction with Menin. Therefore, the disclosed compounds may offer a wider spectrum of clinical benefits that includes cancers that involve any aberrant MLL-fusions.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic showing MLL and and its associated fusion partners are anchored to chromatin through Menin and this proximity allows for histone methyltransferase proteins (HMTs) recruited by fusion partners to aberrantly alter histones, leading to active transcription of genes driving leukemia progression.

[0012] FIG. 2 is an immunoblot showing degradation of MLL-AF4 in MV4-11 cells at 48 h.

[0013] FIG. 3 is an immunoblot showing degradation of MLL proteins and Menin in MV4-11 cells at 48 h

[0014] FIG. 4 is an immunoblot showing degradation of MLL 1 in MV4-11 cells at 24 h

[0015] FIG. 5 is an immunoblot showing degradation in MV4-11 cells at 24 h

[0016] FIG. 6 is an immunoblot showing degradation of MLL proteins MV4-11 cells at 16 h.

[0017] FIG. 7A-FIG. 7C is a series of graphs showing cell killing after 48 h. RS4-11 cells are shown in FIG. 7A and FIG. 7B. M0LM13 cells are shown in FIG. 7B.

[0018] FIG. 8 is a series of graphs showing cell killing after 48 h in indicated cell lines.

[0019] FIG. 9 is a series of graphs showing cell killing after 72 h in indicated cell lines.

[0020] FIG. 10 is a series of graphs showing cell killing after 96 h in indicated cell lines.

[0021] FIG. 11 is a series of graphs showing cell killing after 96 h in M0LM13 cells.

[0022] FIG. 12A is an immunoblot showing degradation of MENIN. FIG. 12B is an immunoblot showing that compound 1 decreased MENIN stabilization compared to VTP50469 in a CETSA assay. FIG. 12C is an immunoblot showing that compound 1 increased stabilization of MLL compared to VTP-50469 in a CETSA assay.

[0023] FIG. 13 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0024] FIG. 14 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0025] FIG. 15 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0026] FIG. 16 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0027] FIG. 17 is a series of immublots showing biotin and MLL protein conjugation with disclosed covalent biotin probes.

[0028] FIG. 18 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0029] FIG. 19 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 12 and 24 h.

[0030] FIG. 20 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0031] FIG. 21 is an immunoblot showing degradation of MLL proteins in MV4-11 cells at 24 h.

[0032] FIG. 22 is an immunoblot showing degradation of IKZF1 / 3 in MV4-11 cells at 24 h.

[0033] FIG. 23 is an immunoblot showing degradation of IKZF1 / 3 in MV4-11 cells at 24 h.

[0034] The compounds (VTP) and (POM) referenced in various figures have the followingDETAILED DESCRIPTION

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0036] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0037] Unless stated otherwise, the term “about” means within 10% (e.g, within 5%, 2%, or 1%) of the particular value modified by the term “about.”

[0038] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group that that minimum number of heteroatoms. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure.

[0039] With respect to compounds of the present disclosure, and to the extent the following terms are used herein to further describe them, the following definitions apply.

[0040] As used herein, the term “alkyl” refers to a saturated linear or branched-chain monovalent hydrocarbon radical. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds of formula I, II, I’ or II’, the alkyl radical is a Co-Ce, C0-C5, Co- C3, C1-C6, C1-C5, C1-C4 or C1-C3 group (wherein Co alkyl refers to a bond). In some embodiments, an alkyl group is a C1-C6 alkyl group.

[0041] As used herein, the term “alkylene” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation. The alkylene chain may be attached to the rest of the molecule through a single bond and to the radical group through a single bond. In some embodiments, the alkylene group contains one to 15 carbon atoms (C1-C15 alkylene).

[0042] As used herein, the term “carbocycle” (also "carbocyclyl") refers to a group that used alone or as part of a larger moiety, contains a saturated, partially unsaturated, or aromatic ring system having 3 to 12 carbon atoms, that is alone or part of a larger moiety (e.g, an alkcarbocyclic group). The term carbocyclyl includes mono-, bi-, tri-, fused, bridged, and spiro-ring systems, and combinations thereof. In one embodiment, carbocyclyl includes 3 to 10 carbon atoms (C3-C10). Representative examples of monocyclic carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, phenyl, and naphthalene.

[0043] As used herein, the term "heterocyclyl" refers to a "carbocyclyl" that used alone or as part of a larger moiety, contains a saturated, partially unsaturated or aromatic ring system, wherein one or more (e.g, 1, 2, 3, 4, or 5) carbon atoms have been replaced with a heteroatom or heteroatomcontaining group (e.g., O, N, N(O), S, S(O), or S(O)2). The term heterocyclyl includes mono-, bi- , tri-, fused, bridged, and spiro-ring systems, and combinations thereof. In some embodiments, a heterocyclyl group includes 3-12 ring atoms and includes monocycles, bicycles, tricycles and spiro ring systems, wherein the ring atoms are carbon, and one to 5 ring atoms is a heteroatom such as nitrogen, sulfur or oxygen. In some embodiments, heterocyclyl includes 3- to 7-membered monocycles having one or more heteroatoms selected from O, N, and S. Representative examples of heterocyclyls include pyrrolidinyl, dihydro- IH-pyrrolyl, dihydrofuranyl, tetrahydropyranyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, pyrrolinyl, and indolinyl.

[0044] As used herein, the term "heteroaryl" used alone or as part of a larger moiety (e.g., "heteroarylalkyl" (also “heteroaralkyl”), or "heteroarylalkoxy" (also “heteroaralkoxy”)) refers to a monocyclic, bicyclic or tricyclic ring system having 5 to 10 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom. Representative examples of heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, imidazopyridyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotri azolyl, benzoimidazolyl, and indolyl.

[0045] Unless stated otherwise, and to the extent not further defined for any particular group(s) in the compounds of formula I, II, I’ and II’, any of the groups described herein may be substituted or unsubstituted. To the extent not disclosed otherwise for any particular group(s), representative examples of substituents may include alkyl (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, Ci), substituted alkyl (e.g., substituted C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, Ci), alkoxy (e.g., C1-C6, Ci- Cs, C1-C4, C1-C3, C1-C2, Ci), substituted alkoxy (e.g., substituted C1-C6, C1-C5, C1-C4, C1-C3, Ci- C2, Ci), haloalkyl (e.g., CF3), alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkenyl (e.g., substituted C2-C6, C2-C5, C2-C4, C2-C3, C2), alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkynyl (e.g., substituted C2-C6, C2-C5, C2-C4, C2-C3, C2), cyclic (e.g., C3-C12, C5-C6), substituted cyclic (e.g., substituted C3-C12, C5-C6), carbocyclic (e.g., C3-C12, C5-C6), substituted carbocyclic (e.g., substituted C3-C12, C5-C6), heterocyclic (e.g., 3- to 12-membered, 5-to 6- membered), substituted heterocyclic (e.g., substituted 3- to 12-membered, 5-to 6-membered), aryl (e.g., benzyl and phenyl), substituted aryl (e.g., substituted benzyl or substituted phenyl), heteroaryl (e.g., pyridyl or pyrimidyl), substituted heteroaryl (e.g., substituted pyridyl or substituted pyrimidyl), aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl), halo, hydroxyl, aryloxy (e.g., C6-C12, Ce), substituted aryloxy (e.g., substituted C6-C12, Ce), alkylthio (e.g., C1-C6), substituted alkylthio (e.g., substituted C1-C6), arylthio (e.g., C6-C12, Ce), substituted arylthio (e.g., substituted C6-C12, Ce), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfmamide, substituted sulfmamide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid, and peptide groups. In some embodiments, the substituent(s) can be selected from the group consisting of halo (e.g., F, Cl, Br, I), alkylhalo (e.g., CF3, CHF2, CH2F, CCI3, CHCI2,CH2CI), cyano, hydroxy, (C1-C6) alkyl, (C1-C6) alkoxy, hydroxy(C1-C6) alkyl, amino, amino(Ci- Ce) alkyl, amino-di(C1-C6) alkyl, and (C3-C7) cycloalkyl.

[0046] In one aspect, compounds of the disclosure are represented by Formula I or II:or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: the Degron binds cereblon (CRBN) or Von Hippel-Lindau (VHL); and the Linker represents a moiety that covalently connects the amide and the Degron, and which comprises at least 3 alkylene units.

[0047] In another aspect, compounds of the disclosure are represented by Formula I’ or II’ :or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: the Degron binds cereblon (CRBN) or Von Hippel-Lindau (VHL); and the Linker represents a moiety that covalently connects the amide and the Degron, and which comprises at least 2 alkylene units.

[0048] In some embodiments, Formula I is of Formula la:

[0049] In some embodiments, Formula I’ is of Formula la’:

[0050] In some embodiments with respect to any of the compounds of formulas I, II, I’ and II’, the degron binds cereblon and is represented by any one of the following structures:wherein,X is CH2 or C(O); R1 is absent, CH2, C=C, NH, O, or 5- to 11-membered mono- or bicyclic heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O, and S (e.g. piperidinyl, piperazinyl, 2,6-diazaspiro[3.3]heptanyl, 2,7-diazaspiro[3.5]nonane);X’ is absent or C(O);Y is N or CH; andR2 is an optional substituent.

[0051] In some embodiments, the degron is of formula

[0052] In some embodiments, X is C(O). In some embodiments, X is CH2.

[0053] In some embodiments, R1 is absent. In some embodiments, R1 is NH. In some embodiments, R1 is O. In some embodiments, R1 is C=C.

[0054] In some embodiments, R1 is 5- to 11 -membered mono- or bicyclic heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O, and S. In some embodiments, Ri is a 6-membered mono-heterocyclyl. In some embodiments, R1 is piperazinyl.

[0055] In some embodiments, the degron is of formula

[0056] In some embodiments, X’ is absent.

[0057] In some embodiments, R2 is fluoro.

[0058] In some embodiments, R1 is absent. In some embodiments, R1 is NH. In some embodiments, R1 is O. In some embodiments, R1 is C=C.

[0059] In some embodiments, the degron is represented by any one of the following structures:

[0060] In some embodiments with respect to any of the compounds of formulas I, II, I’ and II’, the degron binds VHL and is represented by any one of the following structures:wherein:X’ is absent or C(O);each R2' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; each R4' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; each R5' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; R6' is H or -OH; nl is 0, 1, 2, 3, 4, 5, or 6; n2 is 0, 1, 2, 3, or 4; n3 is 0, 1, or 2; andY’ is absent, CH2, C=C, NH, or O.

[0061] In some embodiments, the degron is represented by:stereoisomer thereof. In some embodiments, the degron is represented

[0062] The linker (“L”) of formula I or II represents a moiety that covalently connects the amide and the Degron, and which comprises at least 3 alkylene units, which may or may not be contiguous. The linker (“L”) of formula I’ or II’ represents a moiety that covalently connects the amide and the Degron, and which comprises at least 2 alkylene units, which may or may not be contiguous.

[0063] In some embodiments, the at least 3 alkylene units may be interrupted by, and / or terminate at either or both termini in at least one of-O-, -S-, -N(R’)-, -C=C~, -C(O)-, -C(O)O-, -0C(0)-, -0C(0)0- -C(NOR’)-, -C(O)N(R’)-, -C(O)N(R’)C(O)-, -C(O)N(R’)C(O)N(R’)- , -N(R’)C(O)-, -N(R’)C(O)N(R’)-, -N(R’)C(O)O- -OC(O)N(R’)-, -C(NR’)-, -N(R’)C(NR’)- , -C(NR’)N(R’)-, -N(R’)C(NR’)N(R’)-, -OB(Me)O- -S(O)2- -OS(O)-, -S(O)O- -S(O)-, - OS(O)2- -S(O)2O-, -N(R’)S(O)2- -S(O)2N(R’)-, -N(R’)S(O)-, -S(O)N(R’)-, - N(R’)S(O)2N(R’)- -N(R’)S(O)N(R’)-, optionally substituted C.3-C12 carbocyclene, optionally substituted 3- to 12-membered heterocyclene, optionally substituted 5- to 12-membered heteroarylene or any combination thereof, wherein R’ is H or C1-C6 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

[0064] In some embodiments, the at least 2 alkylene units may be interrupted by, and / or terminate at either or both termini in at least one of-O-, -S-, -N(R’)-, C=C , C(O) , -C(O)O- , -OC(O)-, -OC(O)O-, -C(NOR’)-, -C(O)N(R’)-, -C(O)N(R’)C(O)-, -C(O)N(R’)C(O)N(R’)- , -N(R’)C(O)-, -N(R’)C(O)N(R’)-, -N(R’)C(O)O-, -OC(O)N(R’)-, -C(NR’)-, -N(R’)C(NR’)- , -C(NR’)N(R’)-, -N(R’)C(NR’)N(R’)-, -OB(Me)O-, -S(O)2- -OS(O)-, -S(O)O- -S(O)-, - OS(O)2- -S(O)2O- -N(R’)S(O)2- -S(O)2N(R’)-, -N(R’)S(O)-, -S(O)N(R’)-, - N(R’)S(O)2N(R’)-, -N(R’)S(O)N(R’)-, optionally substituted C3-C12 carbocyclene, optionally substituted 3- to 12-membered heterocyclene, optionally substituted 5- to 12-membered heteroarylene or any combination thereof, wherein R’ is H or C1-C6 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

[0065] “Carbocyclene” as used herein with respect to any of the compounds of formulas I, II, I’ and II’ refers to a bivalent carbocycle radical, which is optionally substituted.

[0066] “Heterocyclene” as used herein with respect to any of the compounds of formulas I, II, I’ and II’ refers to a bivalent heterocyclyl radical which may be optionally substituted.

[0067] “Heteroarylene” as used herein with respect to any of the compounds of formulas I, II, I’ and II’ refers to a bivalent heteroaryl radical which may be optionally substituted.

[0068] In some embodiments, the linker with respect to any of the compounds of formulas I, II, I’ and II’ includes an alkylene chain having 4-20 alkylene units and contains a terminal group such as C(O). In some embodiments, the linker includes an alkylene chain having 4-20 alkylene units, without any interrupting and / or terminating group. In some embodiments, the linker includes an alkylene chain having 4-11 alkylene units and contains a terminal group such as C(O). In some embodiments, the linker includes an alkylene chain having 4-11 alkylene units. In some embodiments, the linker includes an alkylene chain having 3-8 alkylene units and contains aterminal group such as C(O). In some embodiments, the linker includes an alkylene chain having 3-8 alkylene units.

[0069] In some embodiments with respect to the compounds of formulas I’ and II’, the linker includes includes an alkylene chain having 2-20 alkylene units and contains a terminal group such as C(O). In some embodiments, the linker includes an alkylene chain having 2-20 alkylene units, without any interrupting and / or terminating group. In some embodiments, the linker includes an alkylene chain having 2-11 alkylene units and contains a terminal group such as C(O). In some embodiments, the linker includes an alkylene chain having 2-11 alkylene units. In some embodiments, the linker includes an alkylene chain having 2-8 alkylene units and contains a terminal group such as C(O). In some embodiments, the linker includes an alkylene chain having 2 alkylene units.

[0070] In some embodiments, the linker is:

[0071] In some embodiments, compounds of the disclosure are represented by any one of the following structures:pharmaceutically acceptable salt or stereoisomer thereof.

[0072] In some embodiments, compounds of the disclosure are represented by any one of theacceptable salt or stereoisomer thereof.

[0073] In some embodiments, a compound of the disclosure is represented by:pharmaceutically acceptable salt or stereoisomer thereof.

[0074] In some embodiments, compounds of the disclosure include:ceptable salt or stereoisomer thereof.

[0075] In another aspect, compounds of the disclosure include:B and D may be used as intermediates in the synthesis of compounds of any one or more offormulas I, II, I’ and II’.

[0076] Compounds of the present disclosure may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. A pharmaceutically acceptable salt of the compounds of this disclosure can be formed, for example, by reaction of an appropriate free base of a compound of the invention and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, P. L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33:201-217 (1986); Bastin, R. J., et al., “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, :427-435 (2000); and Berge, S. M., etal., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66: 1-19 (1977).

[0077] Compounds of the present disclosure may have at least one chiral center and thus may be in the form of a stereoisomer, which as used herein, embraces all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers which include the (R-) or (S-) configurations of the compounds), mixtures of mirror image isomers (physical mixtures of the enantiomers, and racemates or racemic mixtures) of compounds, geometric (cis / trans or E / Z, R / S) isomers of compounds and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The chiral centers of the compounds may undergo epimerization in vivo,' thus, for these compounds, administration of the compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Accordingly, the compounds of the present disclosure may be made and used in the form of individual isomers and substantially free of other isomers, or in the form of a mixture of various isomers, e.g., racemic mixtures of stereoisomers.

[0078] In some embodiments, a compound of the present disclosureis an isotopic derivative in that it has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound includes deuterium or multiple deuterium atoms. As used herein, the term “hydrogen”, i.e., H, refers to all isotopes of hydrogen, including protium (1H) and deuterium (2H). As used herein, the term “compound” embraces isotopic derivatives.

[0079] Compounds of the present disclosure may also be in the form of N-oxides, crystalline forms (also known as polymorphs), co-crystals, active metabolites of the compounds having the same type of activity, prodrugs, tautomers, and unsolvated as well as solvated (e.g., hydrated)forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, of the compounds. As used herein, the term “compound” embraces all these forms.Methods of Synthesis

[0080] In some aspects, the present disclosure is directed to a method for making a compound of any one of formulas I, II, I’ and II’, or a pharmaceutically acceptable salt or stereoisomer thereof. Broadly, the compounds or pharmaceutically acceptable salts or stereoisomers thereof, may be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present disclosure will be better understood in connection with the synthetic schemes that described in various working examples that illustrate non-limiting methods by which the compounds of the disclosure may be prepared.

[0081] In some aspects, the present disclosure is directed to a method for making an intermediate compound, or a pharmaceutically acceptable salt or stereoisomer thereof. Broadly, the intermediate compounds or pharmaceutically acceptable salts or stereoisomers thereof, may be prepared by any process known to be applicable to the preparation of chemically related compounds.Pharmaceutical Compositions

[0082] Another aspect of the present disclosure is directed to a pharmaceutical composition that includes the compound of any one of formulas I, II, I’ and IT, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as known in the art, refers to a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present disclosure to mammals. Suitable carriers may include, for example, liquids (both aqueous and non-aqueous alike, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases, that function to carry or transport the compound from one organ, or portion of the body, to another organ, or portion of the body. A carrier is “acceptable” in the sense of being physiologically inert to and compatible with the other ingredients of the formulation and not injurious to the subject (also referred to herein as “patient”). Depending on the type of formulation, the composition may also include one or more pharmaceutically acceptable excipients.

[0083] Broadly, compounds of formula I, II, I’ and IT, and their pharmaceutically acceptable salts and stereoisomers may be formulated into a given type of composition in accordance withconventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20thed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York), each of which is incorporated herein by reference in its entirety. The type of formulation depends on the mode of administration which may include enteral e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous (z.v.), intramuscular (i.m.'), and intrastemal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal).

[0084] In general, the most appropriate route of administration will depend upon a variety of factors including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). For example, parenteral (e.g., intravenous) administration may also be advantageous in that the compound may be administered relatively quickly such as in the case of a single-dose treatment and / or an acute condition.

[0085] In some embodiments, the compounds are formulated for intravenous administration (e.g., systemic intravenous injection).

[0086] Injectable preparations for parenteral administration may include sterile aqueous solutions or oleaginous suspensions. They may be formulated according to standard techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents (which along with diluents are embraced by the term “carrier”) that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved ordispersed in sterile water or other sterile injectable medium prior to use. The effect of the compound may be prolonged by slowing its absorption, which may be accomplished by the use of a liquid suspension or crystalline or amorphous material with poor water solubility. Prolonged absorption of the compound from a parenterally administered formulation may also be accomplished by suspending the compound in an oily vehicle.

[0087] In certain embodiments, compounds of formula I, II, I’ and II’ may be administered in a local rather than systemic manner, for example, via injection of the conjugate directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long-acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Injectable depot forms are made by forming microencapsule matrices of the compound in a biodegradable polymer, e. , polylactidepolyglycolides, poly(orthoesters) and poly(anhydrides). The rate of release of the compound may be controlled by varying the ratio of compound to polymer and the nature of the particular polymer employed. Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues. Furthermore, in other embodiments, the compound is delivered in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody. In such embodiments, the liposomes are targeted to and taken up selectively by the organ.

[0088] The compositions may include an excipient, representative examples of which include preservatives, antioxidants, buffering agents, solubilizing agents, and surfactants. Representative examples of preservatives that may be suitable include alcohols, quaternary amines, organic acids, parabens, and phenols. Representative examples of antioxidants that may be suitable include ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols, and chelating agents like EDTA and citric acid. Representative examples of buffering agents that may be suitable include citric, hydrochloric, and lactic acid buffers. Representative examples of solubilizing agents that may be suitable include quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.

[0089] In other embodiments, the compositions may be formulated for oral, buccal, sublingual administration (examples of which include tablets, lozenges and gels), inhalation, topical, ophthalmic and rectal administration.Dosage Amounts

[0090] The amount of the compound of any one of formulas I, TI, I’ and IT administered to a cancer patient may be effective in producing the desired therapeutic response in the patient. Therefore, the amount of the compound of formula I, II, I’ and II’, or a pharmaceutically acceptable salt or a stereoisomer thereof, may induce a positive modification in the disease or disorder to be treated, prevent development or progression of the disease or disorder, or alleviate to some extent, one or more of the symptoms of the disease or disorder being treated In a subject, or kills or inhibits the growth of cancer cells, or reduces the amount of mixed lineage leukemia (MLL)-fusion protein in cancer cells.

[0091] The total daily dosage of the compounds and usage thereof may be decided in accordance with standard medical practice, e.g., by the attending physician using sound medical judgment. The specific dose for any particular subject may depend upon a variety of factors including the disease or disorder being treated and the severity thereof (e.g., its present status); the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the compound; and like factors well known in the medical arts see, for example, Goodman and Gilman 's The Pharmacological Basis of Therapeutics, 10thEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001), which is incorporated herein by reference in its entirety.

[0092] Compounds of formula I, II, I’ and IT, and their pharmaceutically acceptable salts and stereoisomers may be effective over a wide dosage range. In some embodiments, the total daily dosage (e.g., for adult humans) may range from about 0.001 to about 1600 mg, from 0.01 to about 1600 mg, from 0.01 to about 500 mg, from about 0.01 to about 100 mg, from about 0.5 to about 100 mg, from 1 to about 100-400 mg per day, from about 1 to about 50 mg per day, and from about 5 to about 40 mg per day, and in yet other embodiments from about 10 to about 30 mg per day. Individual dosages may be formulated to contain the desired dosage amount depending upon the number of times the compound is administered per day. By way of example, capsules may be formulated with from about 1 to about 200 mg of a compound (e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, and 200 mg). In some embodiments, individual dosages may be formulated to contain the desired dosage amount depending upon the number of times the compound is administered per day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed withoutcausing any harmful side effect, and therefore the above dosage range is not intended to limit the scope of the invention in any way.Methods of Use

[0093] In some aspects, the present disclosure is directed to methods of using compounds of formulas I, II, I’ and II’ for treating a cancer that involves a mixed lineage leukemia (MLL)-fusion protein, e.g., MLL-AF4, AF9. See, Marschalek, R., Ann. Lab. Med., 2016, 36(2 / 85-100; Abdel- Magid, A., ACS Med. Chem. Lett., 2018, 9(9 / 868-869; Lin, eta / ., Cancer Cell., 2016, 30(5):737- 749. The methods entail administration of a compound formula I, II, I’ or II’, or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof.

[0094] In some aspects, the present disclosure provides a compound of formula I, II, I’ or II’, or a pharmaceutically acceptable salt or stereoisomer thereof, for use in therapy. In some aspects, the present disclosure provides a compound of formula I, II, I’ or II’, or a pharmaceutically acceptable salt or stereoisomer thereof, for use in treating a cancer that involves mixed lineage leukemia (MLL)-fusion protein.

[0095] In some aspects, the present disclosure provides a compound of formula I, II, I’ or II’, or a pharmaceutically acceptable salt or stereoisomer thereof, for use in therapy, in particular for treating a cancer that involves mixed lineage leukemia (MLL)-fusion protein.

[0096] The present disclosure provides the use of a compound of formula I, II, I’ or II’, or a pharmaceutically acceptable salt or stereoisomer thereof, in the manufacture of a medicament for treating a cancer that involves mixed lineage leukemia (MLL)-fusion protein.

[0097] Without intending to be bound by any particular theory of operation, the compounds of formula I, II, I’ and IL are believed to promote the degradation of MLL, Menin, or MLL-fusion protein (FIG. 1). By conjugating MLL, Menin, or MLL-fusion protein ligands with an E3 ligase binder, these compounds recruit E3 ligase, and promote the degradation of MLL, Menin, or MLL- fusion protein.

[0098] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone to or suffering from the indicated disease or disorder. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. A subject “in need of’ treatment according to the present disclosure may be “suffering from or suspected of suffering from” a specific disease or disorder may have been positively diagnosed or otherwise presents with a sufficient number of risk factors or a sufficient number or combination of signs or symptoms suchthat a medical professional could diagnose or suspect that the subject was suffering from the disease or disorder. Thus, subjects suffering from, and suspected of suffering from, a specific disease or disorder are not necessarily two distinct groups.

[0099] Cancers that may be amenable to treatment with the disclosed compounds include carcinomas, sarcomas and blood-borne (hematological) cancers alike.

[0100] In some embodiments, the hematological cancer is leukemia, lymphoma, or myeloma.

[0101] In some embodiments, the leukemia is MLL-rearranged (MLL-r) leukemia.

[0102] In some embodiments, the MLL-r leukemia is MLL-r acute lymphocytic leukemia (ALL).

[0103] In some embodiments, the MLL-r leukemia is MLL-r acute myeloid leukemia (AML).

[0104] In some embodiments, the cancer is prostate, liver, breast, or ovarian cancer.

[0105] In some embodiments, the cancer is Ewing’s sarcoma.

[0106] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims.EXAMPLES

[0107] Example 1 : Synthesis of Compounds

[0108] Compounds of the disclosure were prepared using methods known to those skilled in the art in view of this disclosure, or by the illustrative methods shown in the General Schemes below. Suitable protecting can be employed in the synthesis, if needed. See, Wuts, P.G.M.; Greene, T.W., “Greene's Protective Groups in Organic Synthesis ', 5th Ed., J. Wiley & Sons, N Y, 2014. General Scheme 1Compound B Formula I

[0109] In General Scheme 1, compound A is reacted with compound B in an organic solvent to give a compound of Formula I. Suitable amine-to-amide coupling reagents and conditions, e.g., HATU / base, HBTU / base, or EDCl / HOBt / base, are well known in the art. See, Montalbetti and Falque, Tetrahedron, 2005, 67: 10827-10852.

[0110] N-( 12-((2-(2, 6-dioxopiperidin-3-yl)-l, 3-dioxoisoindolin-5-yl)amino)dodecyl)-2-( < 4-(7- ((4-(methylsulfonamido)cyclohexyl)methyl)-2, 7-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-

[0111] 5-((12-Aminododecyl)amino)-2-(2,6-dioxopiperi din-3 -yl)isoindoline- 1 ,3-dione (6.34 mg, 13.9 pmol), 2-((4-(7-((4-(methylsulfonamido)cyclohexyl)methyl)-2,7-diazaspiro[3.5]nonan- 2-yl)pyrimidin-5-yl)oxy)benzoic acid (7.36 mg, 13.9 pmol), HATU (5.81 mg, 15.29 pmol) and DIPEA (9.91 pL, 55.6 pmol) were dissolved in DMF (69.5 pL). The solution was stirred overnight under N2 atmosphere. After completion of the reaction, DMF was distilled under reduced pressure, and the residue was purified by flash chromatography (EtOAc / Hexane 0-100%) followed by (MeOH / DCM 0-20%) to give the title compound as a green solid. MS m / z [M + 1] 969.

[0112] The following compounds were prepared using a similar procedure as described for compound 1 : compound 3 - MS m / z [M + 1] 871 compound 4 - MS m / z [M + 1] 899 compound 5 - MS m / z [M + 1] 913 compound 6 - MS m / z [M + 1] 941 compound 7 - MS m / z [M + 1] 969 compound 9 - MS m / z [M + 1] 1042 compound 12 - MS m / z [M + 1] 954 compound 13 - MS m / z [M + 1] 996 compound 14 - MS m / z [M + 1] 1024compound 16 - MS m / z [M + 1] 968 compound 17 - MS m / z [M + 1] 982 compound 18 - MS m / z [M + 1] 1010 compound 21 - MS m / z [M + 1] 1070 compound 22 - MS m / z [M + 1] 1154 compound 26 - MS m / z [M + 1] 970Compound D Formula II

[0113] In General Scheme 2, compound C is reacted with compound D in an organic solvent to give a compound of Formula II.

[0114] Methyl 2-((4-(7-(((lr,4r)-4-(5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)pentanamido)cyclohexyl)methyl)-2, 7 -diazaspiro [ 3.5 ]nonan-2-yl)pyrimi din-5- yl)oxy)benzoate (8)

[0115] 5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)pentanoic acid (6.35 mg, 16.4 pmol), methyl 2-((4-(7-(((lr,4r)-4-aminocyclohexyl)methyl)-2,7-diazaspiro[3.5]nonan- 2-yl)pyrimidin-5-yl)oxy)benzoate (7.59 mg, 16.4 pmol), HATU (6.84 mg, 18 pmol) and DIPEA (11.684 pL, 65.4 pmol) were dissolved in DMF (82 pL). The solution was stirred overnight under N2 atmosphere. After completion of the reaction, DMF was distilled under reduced pressure and the residue was purified by flash chromatography (EtOAc / Hexane 0-100%) followed by (MeOH / DCM 0-15%) give the title compound as a green solid. MS m / z [M + 1] 835.

[0116] The following compound was prepared using a similar procedure as described for compound 8: compound 11 - MS m / z [M + 1] 1009 compound 15 - MS m / z [M + 1] 863 compound 19 - MS m / z [M + 1] 891 compound 20 - MS m / z [M + 1] 919 compound 23 - MS m / z [M + 1] 1049 compound 24 - MS m / z [M + 1] 1132 compound 25 - MS m / z [M + 1] 975 compound 27 - MS m / z [M + 1] 1136

[0117] 2-((4-(7-(( 4-(Methylsulfonamido)cyclohexyl)methyl)-2, 7-diazaspiro[ 3.5 ]nonan-2- yl)pyrimidin-5-yl)oxy)benzoic acid

[0118] tert-Butyl 2-(5-bromopyrimidin-4-yl)-2, 7-diazaspiro[3.5]nonane-7-carboxylate

[0119] 5 -Bromo-4-chloropyrimidine (1.00 equiv ), tert-butyl 2, 7-di azaspiro [3.5]nonane-7- carboxylate (0.95 equiv.) and diisopropylethylamine (3.00 equiv.) were dissolved in 0.2 M of isopropanol, and heated to 75-85°C for 5 hours. The reaction mixture was concentrated in vacuo and the residue was purified by flash chromatography to give the title compound.

[0120] 2-((4-(7-(tert-Butoxycarbonyl)-2, 7-diazaspiro[3.5]nonan-2-yl)pyrimidin-5- yl)oxy)benzoic acid

[0121] 2-Hydroxybenzoic acid (1.00 equiv.), Zc / 7-butyl 2-(5-bromopyrimidin-4-yl)-2,7- diazaspiro[3.5]nonane-7-carboxylate (1.03 equiv.), cesium carbonate (1.30 equiv.), were dissolved in 0.2 M of dimetylacetamide, and heated and stirred at 120°C for 4 days. The reaction mixture was cooled to room temperature and quenched with 3 times of water compared to solvent amount. The precipitate was collected and purified by flash chromatography to give the title compound.

[0122] 2-((4-(7-(((lr,4r)-4-(Methylsulfonamido)cyclohexyl)methyl)-2, 7- diazaspiro[ 3.5 ]nonan-2-yl)pyrimidin-5-yl)oxy) benzoic acid

[0123] 2-((4-(7-( / erZ-Butoxycarbonyl)-2,7-diazaspiro[3.5]nonan-2-yl)pyrimidin-5- yl)oxy)benzoic acid (1.00 equiv.) was dissolved in 0.2 M of 3: 1 dichloromethane / trifluoroacetic acid solution and stirred for 2 hours. The solution was concentrated in vacuo and neutralized with excess tri ethylamine. 2 times of methanol compared to solvent amount was added and the solution was concentrated in vacuo. ((lr,4r)-4-(Methylsulfonamido)cyclohexyl)methyl 4- methylbenzenesulfonate (1.10 equiv.), KI (1.05 equiv.), and K2CO3 (3.00 euiv.) were dissolved in 0.2 M of .'V-methyl 2-pyrrolidone. The slurry was heated to 65-75°C and stirred for 16 hours. The solution was cooled to 0-10°C and 3 times of water compared to solvent amount was added while maintaining a batch temperature of about less than 20°C. The mixture was extracted with isopropyl acetate, washed with water, and the organic phase was concentrated in vacuo and the residue was purified by flash chromatography to give the title compound.

[0124] ((lr, 4r)-4-(Methylsulfonamido)cyclohexyl)methyl 4-methylbenzenesulfonate

[0125] The synthesis of ((lr,4r)-4-(methylsulfonamido)cyclohexyl)methyl 4- methylbenzenesulfonate was followed by the synthetic method described in Krivtsov etal., Cancer Cell, 2019, 36 61:660-673.

[0126] Methyl 2-((4-(7-(((lr,4r)-4-aminocyclohexyl)methyl)-2, 7-diazaspiro[3.5]nonan-2- yl)pyrimidin-5-yl)oxy)benzoate

[0127] tert-Butyl 2-(5-(2-(methoxycarbonyl)phenoxy)pyrimidin-4-yl)-2, 7- diazaspiro[ 3.5 Jnonane -7 -carboxylate

[0128] Methyl 2-hydroxybenzoate (1.00 equiv.), R1 -butyl 2-(5-bromopyrimidin-4-yl)-2,7- diazaspiro[3.5]nonane-7-carboxylate (1.03 equiv ), cesium carbonate (1.30 equiv.), were dissolved in 0.2 M of dimetylacetamide, and the mixture was heated and stirred at 120°C over 4 days. The reaction mixture was cooled to room temperature and quenched with 3 times of water comparedto solvent. The precipitate was collected and purified by flash chromatography to give the title compound.

[0129] Methyl 2-((4-(7-(((lr,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)methyl)-2, 7- diazaspiro[ 3.5 ]nonan-2-yl)pyrimidin-5-yl)oxy) benzoate

[0130] R1 -Butyl 2-(5-(2-(methoxycarbonyl)phenoxy)pyrimidin-4-yl)-2,7- diazaspiro[3.5]nonane-7-carboxylate (1.00 equiv.) was dissolved in 0.2 M of 3: 1 dichloromethane / trifluoroacetic acid solution and stirred for 2 hours. The solution was concentrated in vacuo and neutralized with excess triethylamine. 2 times of methanol compared to solvent amount was added and the solution was concentrated in vacuo. (( l r,4r)-4-( R1 - butoxycarbonyl)amino)cyclohexyl)methyl 4-methylbenzenesulfonate (1.10 equiv.), KI (1.05 equiv.), and K2CO3 (3.00 equiv.) were dissolved in 0.2 M ofN-methyl 2-pyrrolidone. The slurry was heated to 65-75°C and stirred for 16 hours. The reaction mixture was cooled to 0-10°C and 3 times of water compared to solvent amount was added while maintaining a batch temperature of about less than 20°C. The mixture was extracted with isopropyl acetate, washed with water, and the organic phase was concentrated in vacuo and the residue was purified by flash chromatography to give the title compound.

[0131] Methyl 2-((4-(7-(((lr,4r)-4-aminocyclohexyl)methyl)-2, 7-diazaspiro[3.5Jnonan-2- yl)pyrimidin-5-yl)oxy)benzoate

[0132] Methyl 2-((4-(7-(((lr,4r)-4-(( / cr / -butoxycarbonyl)amino)cyclohexyl)methyl)-2,7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzoate was dissolved in 0.2 M of 3: 1 dichloromethane / trifluoroacetic acid solution and stirred for 2 hours. After that, the solution was concentrated in vacuo and neutralized with excess triethylamine. 2 times of methanol compared to solvent amount was added and the solution was concentrated in vacuo. The residue was purified by flash chromatography to give the title compound.

[0133] Example 2: Sythesis of Covalent Biotin Probes

[0134] tert-Butyl (2-(2-((4-(7-( ( ( Ir, 4r)-4-(methylsulfonamido)cyclohexyl)methyl)-2, 7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzamido)ethyl)carbamate

[0135] 2-((4-(7-(((lr,4r)-4-(methylsulfonamido)cyclohexyl)methyl)-2,7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzoic acid (2 mg, 3.78 pmol), tert- butyl (2- aminoethyl)carbamate (0.8 mg, 4.91 pmol), HATU (2.2 mg, 5.67 pmol) and DIPEA (10 pL) were dissolved in DMF (200 pL) and the reaction mixture was stirred overnight under N2 atmosphere. The reaction mixture was then concentrated in vacuo and the residue was purified by flash chromatography (EtOAc / MeOH 0-20%) to give the title compound as a colorless solid. MS m / z [M + 1] 672.

[0136] N-( 2-aminoethyl)-2-((4-(7-( ( ( Ir, 4r)-4-(methylsulfonamido)cyclohexyl)methyl)-2, 7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzamide

[0137] tert-Butyl (2-(2-((4-(7-(((lr,4r)-4-(methylsulfonamido)cyclohexyl)methyl)-2,7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzamido)ethyl)carbamate was dissolved in a TFA / DCM 1 :3 mixture solution and stirred for 2 h and monitored by LC-MS. After completion of the reaction, the reaction mixture was concentrated in vacuo to give the title compound which was used directly in the next step. MS m / z [M + 1] 572.

[0138] N-( 2-(3-(3-(but-3-yn-l-yl)-3H-diazirin-3-yl)propanamido)ethyl)-2-( ( 4-( 7-( ((lr, 4r)-4- (methylsulfonamido)cyclohexyl)methyl)-2, 7-diazaspiro[ 3.5 ]nonan-2-yl)pyrimidin-5- yl) oxy) benzamide

[0139] N-(2-aminoethyl)-2-((4-(7-(((lr,4r)-4-(methylsulfonamido)cyclohexyl)methyl)-2,7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzamide (2.5 mg, 3.72 pmol), 3-(3-(but-3-yn-l- yl)-3H-diazirin-3-yl)propanoic acid (0.8 mg, 4.84 pmol), HATU (2.2 mg, 5.67 pmol) and DIPEA (10 pL) were dissolved in DMF (200 pL) and the reaction mixture was stirred overnight under N2 atmosphere. The reaction mixture was then concentrated in vacuo and the residue was purified by flash chromatography (EtOAc / MeOH 0-20%) to give the title compound as a white solid. MS m / z [M + 1] 720.

[0140] Methyl 2-((4-(7-(((lr,4r)-4-aminocyclohexyl)melhyl)-2, 7 -diazaspiro [3.5] nonan-2- yl)pyrimidin-5-yl)oxy)benzoate

[0141] Methyl 2-((4-(7-(((lr,4r)-4-((tert-butoxycarbonyl)amino)cyclohexyl)methyl)-2,7- diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzoate was dissolved in a TFA / DCM 1 :3 mixture solution and stirred for 2 h and monitored by LC-MS. After completion of the reaction, the reaction mixture was concentrated in vacuo to give the title compound which was used directly in the next step. MS m / z [M + 1] 465.

[0142] Methyl 2-((4-(7-(((lr,4r)-4-(3-(3-(but-3-yn-l-yl)-3H-diazirin-3- yl)propanamido)cyclohexyl)methyl)-2, 7-diazaspiro[3.5]nonan-2-yl)pyrimidin-5-yl)oxy)benzoate

[0143] Methyl 2-((4-(7-(((lr,4r)-4-aminocyclohexyl)methyl)-2,7-diazaspiro[3.5]nonan-2- yl)pyrimidin-5-yl)oxy)benzoate (2.0 mg, 3.54 pmol), 3-(3-(but-3-yn-l-yl)-3H-diazirin-3- yl)propanoic acid (0.8 mg, 4.60 pmol), HATU (2.2 mg, 5.67 pmol) and DIPEA (10 pL) were dissolved in DMF (200 pL) and the reaction mixture was stirred overnight under N2 atmosphere. The reaction mixture was then concentrated in vacuo and the residue was purified by flashchromatography (EtOAc / MeOH 0-20%) to give the title compound as a white solid. MS m / z [M + 1] 614.

[0144] Example 3 : Biological Data

[0145] Cell Culture

[0146] MV4-11 cells were purchased from ATCC®. These cells were cultured in RPMI-1640 with 10% FBS and 1% penicillin / streptomycin and maintained at 37°C in 5% CO2. Cell line identity was confirmed by short tandem repeats (STR) profiling.

[0147] Western Blotting

[0148] 1E6 MV4-11 cells were plated into 6-well tissue culture plates in 2mL RPMI and treated with indicated concentrations of compound or DMSO control. After the indicated period of time, cells were collected, washed 1 time in IX PBS, and lysed in RIPA buffer. Protein concentration was measured using the Bradford protein assay, and 20-30 pg total protein were loaded into each well of the gels. Gels were run and proteins were transferred to polyvinylidene difluoride (PVDF) 0.45 pM membranes. Membranes were blocked for at least 1 hr shaking at room temperature and incubated overnight at 4°C with the indicated antibodies. Membranes were washed and incubated with secondary antibodies the next day and imaged on a LI-COR imaging system (FIG. 2-FIG. 6, FIG. 12A-FIG. 12C, FIG. 13-FIG. 16, and FIG. 18-FIG. 23).

[0149] Cell Viability Assays

[0150] The CellTiter-Glo® assay was performed as per the manufacturer’s instructions (Promega™). Briefly, 1,000 cells per well were plated into 96-well plates and treated with a range of compound concentrations. Cell viability was measured at the noted time points based on luminescence by the CellTiter-Glo® assay (Promega™) and read on an EnVision® 2104 (PerkinElmer) according to the manufacturer’s protocol (FIG. 7-FIG. 11, FIG. 13-FIG. 16, and FIG. 18-FIG. 23). The data indicated that the tested compounds out-performed the controls, VTP / POM.

[0151] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications (including any specific portions thereof that are referenced) are herein incorporated by reference to the sameextent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0152] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A compound having a structure represented by formula I or II:or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: the Degron binds cereblon (CRBN) or Von Hippel-Lindau (VHL) tumor suppressor; and the Linker represents a moiety that covalently connects the amide and the Degron, and which comprises at least 3 alkylene units.

2. The compound of claim 1, wherein the Degron binds CRBN.

3. The compound of claim 2, wherein the Degron is represented by:stereoisomer thereof, wherein,X is CH2or C(0); R1 is absent, CH2, C=C, NH, 0, or 5- to 11-membered mono- or bi-cyclic heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O, and S;X’ is absent or C(O);Y is N or CH; andR2 is an optional substituent.

4. The compound of claim 3, wherein X is C(O).

5. The compound of claim 3 or 4, wherein R1 is NH.

6. The compound of claim 3 or 4, wherein R1 is piperazinyl.

7. The compound of claim 1, wherein the Degron binds VHL.

8. The compound of claim 7, wherein the Degron is represented by:wherein:X’ is absent or C(O); each R2' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; each R4' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; each R5’ is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy;R6' is H or -OH; nl is 0, 1, 2, 3, 4, 5, or 6; n2 is 0, 1, 2, 3, or 4; n3 is 0, 1, or 2; andY’ is absent, CH2, C=C, NH, or O.

9. The compound of claim 8, wherein the Degron is represented by:stereoisomer thereof.

10. The compound of any one of claims 1-9, wherein the at least 3 alkylene units may be interrupted by, and / or terminate at either or both termini in at least one of -O-, -S-, -N(R')-, - C=C- -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -C(O)N(R')C(O)-, - C(O)N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)N(R')-, -N(R')C(O)O- -OC(O)N(R')-, - C(NR')-, -N(R')C(NR')-, -C(NR')N(R')-, -N(R')C(NR')N(R')-, -OB(Me)O-, -S(O)2- -OS(O)- , -S(O)O- -S(O)-, -OS(O)2- -S(O)2O- -N(R')S(O)2- -S(O)2N(R')-, -N(R')S(O)-, - S(O)N(R')-, -N(R')S(O)2N(R')-, -N(R')S(O)N(R')-, C.3-12 carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene or any combination thereof, wherein R' is H or C1-C6 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

11. The compound of claim 10, wherein the linker comprises 4 to 20 alkylene units.

12. The compound of claim 11, wherein the linker comprises 4 to 11 alkylene units.

13. The compound of claim 10, wherein the linker comprises 3 to 8 alkylene units.

14. The compound of claim 10, wherein the linker is:

15. The compound of claim 1, which is:pharmaceutically acceptable salt or stereoisomer thereof.

16. The compound of claim 1, which is:acceptable salt or stereoisomer thereof.

17. A compound having a structure represented by formula I’ or II’:or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: the Degron binds cereblon (CRBN) or Von Hippel-Lindau (VHL) tumor suppressor; and the Linker represents a moiety that covalently connects the amide and the Degron, and which comprises at least 2 alkylene units.

18. The compound of claim 17, wherein the Degron binds CRBN.

19. The compound of claim 18, wherein the Degron is represented by:stereoisomer thereof, wherein,X is CH2or C(0); R1 is absent, CH2, C=C, NH, 0, or 5- to 11-membered mono- or bi-cyclic heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O, and S;X’ is absent or C(O);Y is N or CH; andR2 is an optional substituent.

20. The compound of claim 19, wherein X is C(O).

21. The compound of claim 19 or 20, wherein R1 is NH.22 The compound of claim 19 or 20, wherein R1 is piperazinyl.

23. The compound of claim 17, wherein the Degron binds VHL.

24. The compound of claim 23, wherein the Degron is represented by:wherein:X’ is absent or C(O); each R2' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; each R4' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy; each R3' is independently halogen, -OH, C1-C6 alkyl, or C1-C6 alkoxy;R6' is H or -OH; nl is 0, 1, 2, 3, 4, 5, or 6; n2 is 0, 1, 2, 3, or 4; n3 is 0, 1, or 2; andY’ is absent, CH2, C=C, NH, or O.

25. The compound of claim 24, wherein the Degron is represented by:stereoisomer thereof.

26. The compound of any one of claims 17-25, wherein the at least 2 alkylene units may be interrupted by, and / or terminate at either or both termini in at least one of -O-, -S-, -N(R')-, - C=C- -C(O)-, -C(O)O- -OC(O)-, -OC(O)O- -C(NOR')-, -C(O)N(R')-, -C(O)N(R')C(O)-, - C(O)N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, - C(NR')-, -N(R')C(NR')-, -C(NR')N(R')-, -N(R')C(NR')N(R')-, -OB(Me)O- -S(O)2- -OS(O)- , -S(O)O- -S(O)-, -OS(O)2- -S(O)2O- -N(R')S(O)2- -S(O)2N(R')-, -N(R')S(O)-, - S(O)N(R')-, -N(R')S(O)2N(R')-, -N(R')S(O)N(R')-, C3-12 carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene or any combination thereof, wherein R' is H or C1-C6 alkyl, wherein the interrupting and the one or both terminating groups may be the same or different.

27. The compound of claim 26, wherein the linker comprises 2 to 20 alkylene units.

28. The compound of claim 27, wherein the linker comprises 2 to 11 alkylene units.

29. The compound of claim 28, wherein the linker comprises 2 to 8 alkylene units.

30. The compound of claim 17, which is:(15), or a pharmaceutically acceptable salt or stereoisomer thereof.

31. A compound which is:stereoisomer thereof.

32. A pharmaceutical composition, comprising the compound of any one of claims 1-30, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

33. The method of treating a cancer that involves mixed lineage leukemia (MLL)-fusion protein, comprising administering to a subject in need thereof, the compound of any one of claims 1-30, or a pharmaceutically acceptable salt or stereoisomer thereof.

34. The method of claim 33, wherein the cancer is leukemia.

35. The method of claim 34, wherein the leukemia is MLL-rearranged (MLL-r) leukemia.

36. The method of claim 35, wherein the MLL-r leukemia is MLL-r acute lymphocytic leukemia (ALL).

37. The method of claim 35, wherein the MLL-r leukemia is MLL-r acute myeloid leukemia (AML).

38. The method of claim 33, wherein the cancer is prostate, liver, breast, or ovarian cancer.

39. The method of any one of claims 33-38, wherein the subject is a human.

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