DEGRADATION OF CYCLIN-DEPENDENT KINASE 4/6 (CDK4/6) BY CONJUGATION OF CDK4/6 INHIBITORS WITH E3 LIGASE LIGAND AND METHODS OF USE

RELATED APPLICATION

Tliis application claims priority to, and the benefit of, U.S. Provisional Application No. 62/326,571, filed on April 22, 2016, the entire contents of which are hereby incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant number R01

CA179483 awarded by The National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Ubiquitin-Proteasome Pathway (UPP) is a critical pathway that regulates proteins and degrades misfolded or abnormal proteins. UPP is central to multiple cellular processes, and if defective or unbalanced, leads to pathogenesis of a variety of diseases. The covalent attachment of ubiquitin to specific protein substrates is achieved through the action of E3 ubiquitin ligases. These ligases comprise over 500 different proteins and are categorized into multiple classes defined by the structural element of their E3 functional activity. For example, cereblon (CRBN) interacts with damaged DNA binding protein 1 and forms an E3 ubiquitin ligase complex with Cullin 4 in which the proteins recognized by CRBN are ubiquitinated and degraded by proteasomes. Various immunomodulatory drugs (IMiDs), e.g. thalidomide and lenalidomide, binds to CRBN and modulates CRBN's role in the ubiquitination and degradation of protein factors involved in maintaining regular cellular function.

Bifunctional compounds composed of a target protein-binding moiety and an E3 ubiquitin ligase-binding moiety have been shown to induce proteasome-mediated degradation of selected proteins. These drug-like molecules offer the possibility of temporal control over protein expression, and could be useful as biochemical reagents for the treatment of diseases.

Cy elm-dependent kinase is a kinase family integrating multiple signaling pathways to control either cell cycle or gene transcription. CDKl, 2, 4 and 6 are the critical enzymes that dri ve cell cycle transition. For example, CDKl is a key determinant of mitotic progression, CDK2 regulates DNA replication in S phase, and CDK4/6 drives the cell cycle from GO or Gl to S phase by phosphorylation on Rb protein to activate expression of genes involved in cell cycle control , CDK7, 9 and 12 are known enzymes that regulate the transcription instead of directly promoting cell cycles, CDK7 is the enzymatic component of TFIIH complex which is responsible for regulating transcription initiation, and CDK9 and CDK12 regulate transcription elongation and processing.

Deregulation of CDKs has been shown to have a significant impact on the cell state and is frequently identified as oncogenic. Numerous selective or pan-CDK small molecule inhibitors have been identified, however, most of the known inhibitors have failed in clinic trials due to the lack of high systemic drug concentration. More recently, the development of a CDK7 covaient inhibitor, THZl , has demonstrated that irreversible binders are superior to reversible CD binders.

Alternative strategies to inhibit cyclin-dependent kinases, such as CDK4 and CDK6, are needed. At present, suitable compounds with alternative mechanisms of action targeting CDK4 and CD 6 are not available. The present application addresses the need.

SUMMARY

The present application relates to novel bifunctional compounds, which function to recruit targeted proteins to E3 ubiquitin ligase for degradation, and methods of preparation and uses thereof. The bifunctional compound is of Formula X:

wherein:

the Targeting Ligand is capable of binding to a targeted protein, such as a cyclin- dependent kinase (e.g. , CDK4 and/or CDK6);

the Linker is a group that covalently binds to the Targeting Ligand and the Degron; and

the Degron is capabl e of binding to a ubiquitin ligase, such as an E3 ubiquitin ligase (e.g. , cereblon).

The present application also relates to targeted degradation of proteins through the use of bifunctional compounds, including bifunctional compounds that link an E3 ubiquitin ligase-bindiiig moiety to a ligand that binds the targeted proteins. The present application also relates to a bifunctional compound of Formula ]

, or

Targeting Ligand

or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein:

Ri, R.2, RJ, A, A', B, X, and n are each as defined herein; the Linker is a group that covalently binds to ^— f and the Degron;

the Degron i s capabl e of binding to a ubiquitin ligase, such as an E3 ubiquitin ligase (e.g. , cereblon); and

the Targeting Ligand is capable of binding to a targeted protein, such as CDK4 and/or

CDK6.

The present application further relates to a Degron of Formula D l : or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, Z, RB, Ri4, Ris, Ri6, v, and q are each as defined herein.

The present appl LO:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein pi , p2, p3, W, Q, and are each as defined herein, the Linker is covalently bonded to a Degron via the ¾ next to Q, and covalently bonded to the Targeting Ligand via the next to Ζ·, .

The present application also relates to a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound of the application, or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable earner. Another aspect of the present application relates to a method of inhibiting a kinase (e.g. , CDK4 and/or CDK6). The method comprises administering to a subject in need thereof an effective amount of a Afunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a

pharmaceutical composition of the application.

Another aspect of the present application relates to a method of modulating (e.g. , decreasing) the amount of a kinase (e.g. , CD 4 and/or CDK6). The method comprises administering to a subject in need thereof a therapeutically effective amount of a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application.

Another aspect of the present application relates to a method of treating or preventing a disease (e.g. , a disease in which CDK4 and/or CDK6 plays a role). The method comprises administering to a subject in need thereof an effective amount of a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application. In one aspect, the disease is a kinase (e.g., CDK4 and/or CDK6) mediated disorder, in one aspect, the disease is a proliferative disease (e.g. , a proliferative disease in which CDK4 and/or CDK6 plays a role).

Another aspect of the present application relates to a method of treating or preventing cancer in a subject, wherein the cancer cell comprises an activated CDK.4 and/or an activated CDK6 or wherein the subject is identified as being in need of inhibition of CDK.4 and/or CDK6 for the treatment or prevention of cancer. The method comprises administering to the subject an effective amount of a bifunctional compound of the application, or a

pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application.

Another aspect of the present application relates to a kit comprising a bifunctional compound capable of inhibiting CDK4 and/or CDK6 activity, selected from a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the present application relates to a kit comprising a bifunctional compound capable of modulating (e.g. , decreasing) the amount of CDK.4 and/or CDK6, selected from a bifuiictionai compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. Another aspect of the present application relates to a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the applicati on, for use in the manufacture of a medicament for inhibiting a kinase (e.g. , CD 4 and/or CDK6) or for modulating (e.g. , decreasing) the amount of a kinase (e.g., CDK4 and/or CDK6).

Another aspect of the present application relates to a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application, for use in the manufacture of a medicament for treating or preventing a disease (e.g. , a disease in which CDK4 and/or CDK6 plays a role). In one aspect, the disease is a kinase (e.g., CDK4 and/or CD 6) mediated disorder. In one aspect, the disease is a proliferative disease (e.g. , a proliferative disease in which CDK4 and/or CDK6 plays a role).

Another aspect of the present application relates to a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application, for use in the ma facture of a medicament for treating or preventing cancer in a subject, wherein the cancer cell comprises an activated CDK4 and/or an activated CDK6 or wherein the subject is identified as being in need of inhibition of CD 4 and/or CDK6 for the treatment or prevention of cancer.

Another aspect of the present application relates to a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application, for use in inhibiting a kinase (e.g. , CDK4 and/or CDK6) or modulating (e.g. , decreasing) the amount of a kinase (e.g. , CDK4 and/or CDK6).

Another aspect of the present application relates to a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application, for use in treating or preventing a disease (e.g. , a disease in which CDK4 and/or CDK6 plays a role). In one aspect, the disease is a kinase (e.g., CDK4 and/or CDK6) mediated disorder. In one aspect, the disease is a proliferative disease (e.g., a proliferative disease in which CDK.4 and/or CDK6 plays a role).

Another aspect of the present application relates to a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or a pharmaceutical composition of the application, for use in treating or preventing cancer in a subject, wherein the cancer cell comprises an activated CDK4 and/or activated CDK6 or wherein the subject is identified as being in need of inhibition of CDK4 and/or CDK6 for the treatment or prevention of cancer.

The present application provides inhibitors of CDK4 and/or CDK6 that are therapeutic agents in the treatment or prevention of diseases such as cancer and metastasis.

The present application further provides compounds and compositions with an improved efficacy and/or safety profile relative to known CD 4 and CDK6 inhibitors. The present application also provides agents with no vel mechanisms of action toward CDK4 and CDK6 kinases in the treatment of various types of diseases including cancer and metastasis.

The compounds and methods of the present application address unmet needs in the treatment of diseases or disorders in which pathogenic or oncogenic endogenous proteins (e.g. , CDK4 and/or CDK6) play a role, such as cancer.

The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now- described. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural 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 to which this disclosure belongs.

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. The references cited herein are not admitted to be prior art to the application. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a western blot showing the levels of CDK.4, CDK6, and tubulin at various time points in Jurkat cells treated with DMSO or 5 μΜ of Compound 1-3. A decrease in CD 6 levels over time was observed while CD 4 and tubulin levels were unaffected.

FIG.2 is a western blot showing the levels of CDK4, CDK6, and tubulin in wild-type

(WT) or cereblon knockout (CRBN-/-) Molt4 cells treated with various concentrations of Compound 1-3. Cells treated with Compound 1-3 showed CDK6 degradation in wild-type ceils while CD 4 was unaffected. Compound 1-3 is dependent on the presence of CRBN as seen by a rescue of CDK6 levels in the CRBN-/- cells.

FIG.3A-FIG. 3G are western blots showing levels of CDK4, CDK6, and actin in

Jurkat cells treated for 4 hours with various concentrations of Compound 1-1 (FIG. 3 A), Compound 1-20 (FIG. 3B), Compound 1-5 (FIG. 3C), Compound 1-11 (FIG. 3D), Compound 1-12 (FIG. 3E), Compound 1-13 (FIG. 3F), or Compound 1-14 (FIG. 3G).

FIG. 4 is a western blot showing the levels of CDK.4, CDK6, Rb, pRb, and actin in wild-type (WT) or cereblon knockout (CRBN-/-) Molt4 cells treated with the indicated compounds of the present application.

FIG.5 is a western blot showing the levels of CDK4, CDK.6, and tubulin in Jurkat ceils treated for 4 hours with various concentrations of Compound 1-3 or Compound 1-1.

FIG. 6A-FIG. 6D are graphs showing the viability of v arious cells as a function of the concentrations of palbociclib, Compound 1-1, or Compound 1-3 in IGROV1 (FIG. 6A), SKOV (FIG. 6B), MCAS (FIG. 6C), and MDA MB-453 (FIG. 6D),

DETAILED DES CRIPTION

Compounds of the Application

The present application relates to bifunctional compounds having utility as modulators of ubiquitination and proteosomal degradation of targeted proteins, especially compounds comprising a moiety capable of binding to a polypeptide or a protein that is degraded and/or otherwise inhibited by the bifunctional compounds of the present application. In particular, the present application is directed to compounds which contain a moiety, e.g., a small molecule moiety (i.e. , having a molecular weight of below 2,000, 1 ,000, 500, or 200 Daltons), such as a thalidomide-like moiety, which is capable of binding to an E3 ubiquitin iigase, such as cereblon, and a iigand that is capable of binding to a target protein, in such a way that the target protein is placed in proximity to the ubiquitin ligase to effect degradation (and/or inhibition) of that protein.

In one embodiment, the present application provides a Afunctional compound of Formula X:

wherein:

the Targeting Ligand is capable of binding to a targeted protein, such as CD 4 and

CDK6;

the Linker is a group that covalently binds to the Targeting Ligand and the Degron; and

the Degron is capable of binding to a ubiquitin ligase, such as an E3 ubiquitin ligase (e.g. , cereblon).

In one embodiment, the present application provides a compound of Formula I:

or

Targeting Ligand

or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein:

R], R2, Rs, A, A ^! , B, X, and n are each as defined herein;

X

the Linker is a group that covalently binds to and the Degron;

the Degron is capable of binding to a ubiquitin ligase, such as an E3 ubiquitin ligase (e.g. , cereblon); and

the Targeting Ligand is capable of binding to a targeted protein, such as CDK4 and/or

CDK6.

The present application further relates to a Degron of Formula Dl :

(Dl), or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, Z, R33, R , Ris, Ri6, q, and v are each as defined herein.

The present appl L0: , or an enantiomer, diastereomer, or stereoisomer thereof, wherein pi, p2, p3, W, Q, and Zi are

_A_

each as defined herein, the Linker is covalently bonded to a Degron via the ^ next to Q, and covalently bonded to the Targeting Ligand via the ^ next to Zi.

Targeting Ligand (TL) (or target protein moiety or target protein ligand or ligand) small molecule which is capable of binding to a target protein of interest, such as CD 4 and/or CDK6.

In one embodiment, a Targeting Ligand is a compound of Formula TL-I:

or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein:

A is absent or ( ^' { R sb:

' i : or O;

X is N or CH;

X ₂ is N or CRs;

each Ri is independently (C1-C4) alky! or {(VC haloalkyl;

R?. is H, (C1-C4) alkyl, (C1-C4) haloalkyl, halogen, OH, or NH?.;

R3 is (Ce-Cio) aryl or a monocyclic or bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, wherein the aryl and heteroaryl are optic

substituted with one or more R?: or R:2 and R3 together with the carbon atoms to which they are attached form a 5- or 6- membered heterocvcioalk}-] comprising one or two heteroatoms selected from N, O, and S, wherein the heterocycloalkyl is optionally substituted with one or more Rg; or R2 and R3 together with the carbon atoms to which they are attached form a 5- or 6-membered heteroaiyl comprising one or two heteroatoms selected from N, O, and S, wherein the heteroaryl is optionally substituted with one or more R9;

each 4 is independently H or (C 1-C4) alkyi;

Ri is H or (C1-C4) alkyl;

each Re is independently (C1-C4) alkyl, (C1-C4) haloalkyl, (C 1-C4) alkoxy, (C1-C4) haloaikoxy, halogen, OH, or NH ₂ ;

each R ₇ is independently (C1-C4) alkyl, (Ci -CY) haloalkyl, (C i-C ₄ ) alkoxy, (Ci -CY) haloaikoxy, halogen, OH, or NH2; or

each .8 is independently (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, (C1-C4) haloaikoxy, halogen, C(0)(C] -C4> alkyl, C(0)NH ₂ , C(0)NH(Ci-C ₄ ) alkyl, C(0)N((C]-C ₄ ) alkyl)?,, (C3-C7) cycloalkyl, or heterocycloalkyl, or two Rs together with the carbon to which they are attached form C(O);

each R9 is independently (C1-C4) alkyl, (C1-C4) haloalkyl, (C 1-C4) alkoxy, (C1-C4) haloaikoxy, halogen, C(0)(Ci-C4> alkyl, ( i O)Ni l . C(0)NH(Ci-C ₄ ) alky !. CiO sNuiViYs alkyl)2, (C3-C7) cycloalkyl, or heterocycloalkyl; and

n and t are independently 0, 1 , 2, or 3, wherein the Targeting Ligand is bonded to the Linker via the ¾ next to — f

In some embodiments, A is absent. In other embodiments, A is CH2.

In some embodiments, A' is Nils. In other embodiments, A' is O. In other embodiments, A' is NH or O. In other embodiments, A' is NH. . In other embodiments. B is . in other embodiments, B is

In some embodiments, X is N. In other embodiments, X is CH.

In some embodiments, X2 is N. In other embodiments, X2 is CH.

In some embodiments, each Rj is independently methyl, ethyl, propyl, or i-propyl. In other embodiments, each Ri is independently methyl or ethyl. In other embodiments, each Ri is independently methyl. In other embodiments, each Ri is independently (C 1-C4) haloalkyl (i.e. , CF3, CHF2, CH2CF3, or CF2CF3).

In some embodiments, R2 is H, (C1-C3) alkyi, (C1-C3) haloalkyl, halogen, OH, or NH ₂ . In other embodiments, R2 is (C1-C ) alky], (C1-C3) haloalkyl, halogen, OH, or NH2. In other embodiments, R2 is (C1-C3) alkyl, (C 1-C3) haloalkyl, or halogen. In other embodiments, R2 is halogen, OH, or NH2. In other embodiments, R2 is (C1-C3) alkyl or (C1-C3) haloalkyl. In other embodiments, R2 is (C1-C3) alkyl or halogen. In other embodiment, R2 is halogen. In other embodiment, R2 is methyl or F. In other embodiments, R2 is F.

In some embodiments, R3 is (Ce-Cso) aryl optionally substituted with one or more R ₇ .

In other embodiments, R3 is a monocyclic or bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R ₇ . In some embodiments, R3 is (Ce-Cio) aryl substituted with one or more R ₇ . In other embodiments, R3 is a monocyclic or bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, substituted with one or more R7. In other embodiments, R3 is a monocyclic heteroaryl comprising one to three heteroatoms selected from N, O, and S, optionally substituted with one or more R ₇ . In other embodiments, R3 is a bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R ₇ . In other embodiments, R3 is a monocyclic heteroaryl comprising one to three heteroatoms selected from N, O, and S, substituted with one or more R ₇ . In other

embodiments, R3 is a bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, substituted with one or more R7.

In some embodiments, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heterocycloalkvl comprising one or two heteroatoms selected from N, O, and S, opiionaily substituted with one or more Rs. In some embodiments, R2 and R3 together with the carbon atoms to which they are attached form a 6-membered

heterocycloalkyl comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more Rs. In some embodiments, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heterocycloalkyl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more Rs. In some embodiments, R2 and R3 together with the carbon atoms to which they are attached form a 6- membered heterocycloalkyl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more Rs.

In other embodiments, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatoms selected from , O, and S, optionally substituted with one or more R9. In other embodiments, R?, and R3 together with the carbon atoms to which they are attached form a 6-membered heteroaryl comprising one or two heteroatoms selected from , O, and S, optionally substituted with one or more R9. In other embodiments, R?, and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more R9. In other embodiments, R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heteroaryl comprising one or two heteroatoms selected from N, (), and S, substituted with one or more R9.

In some embodiments, each R4 is independently H or (C1-C3) alkyl (e.g. , methyl, ethyl, propyl, or i-propyl). In other embodiments, each R ₄ is independently H, methyl or ethyl. In other embodiments, each R ₄ is independently methyl or ethyl. In other

embodiments, each R ₄ is independently H or methyl. In other embodiments, at least one R ₄ is methyl. In other embodiments, each K s is i I

In some embodiments, Rs is H or (Ci -Cs) alkyl (e.g., methyl, ethyl, propyl, or 1- propyi). In other embodiments, Rs is H, methyl or eth l. In other embodiments, Rs is methyl or ethyl. In other embodiments, Rs is H or methyl. In other embodiments, Rs is methyl. In other embodiments, Rs is H.

In some embodiments, each Re is independently (C1-C3) alkyl, (C1-C3) haloalkyl, (Ci- C3) alkoxy, (C1-C3) haloalkoxy, halogen, OH, or NH2. In other embodiments, each Re is independently (C1-C3) alkyl, (C1-C3) haloalkyl, (C1-C3) alkoxy, (C1-C3) haloalkoxy, or halogen. In other embodiments, each e is independently halogen, OH, or NH ₂ . In other embodiments, each Re is independently (C1-C3) alkyl, (C1-C3) haloalkyl, or halogen. In other embodiments, each Re is independently (C1-C3) alkyl or halogen. In other embodiments, each R.6 is independently methyl, ethyl, propyl, iso-propyl, or halogen. In other

embodiments, each Re is independently methyl, ethyl, propyl, iso-propyl, or F.

In some embodiments, each R ₇ is independently (C1-C3) alkyl, (C1-C3) haloalkyl, (Ci- C3) alkoxy, (Ci-C j) haloalkoxy, halogen, OH, or NH2. In other embodiments, each R? is independently (C1-C3) alkyl, (C1-C3) haloalkyl, (C1-C3) alkoxy , (C1-C3) haloalkoxy, or halogen. In other embodiments, each R ₇ is independently halogen, OH, or NH2. In other embodiments, each R? is independently (C1-C3) alkyl, (C1-C3) haloalkyl, or halogen. In other embodiments, each R? is independently (C1-C3) alkyl or halogen. In other embodiments, each R? is independently methyl, ethyl, propyl, iso-propyl, or halogen. In other

embodiments, each R ₇ is independently methyl, ethyl, propyl, iso-propyl, or F.

In some embodiments, each Rs is independently (Ci -C j) alkyl, (C1-C3) haloalkyl, (C i- C3) alkoxy, (C1-C3) haloalkoxy, halogen, C(0)(Ci-C ₃ ) alkyl, C(0)NH?., C(0)NH(Ci-Cs) alkyl, C(0)N((Ci-C3) alkyl)?, (Cs-Ce) cycloalkyl, or heterocycloalky . In other embodiments, each Rs is independently (Ci-Cs) alkyl, (C1-C3) haloalkyl, halogen, C(0)(Ci-C3) alkyl, C(0)NH?, C(0)NH(Ci-C3) alkyl, C(())N((Ci-C3) alkyl)?., (Cs-Ce) cycloalkyl, or

heterocycloalkyi. In other embodiments, each Rs is independently (C1-C3) alkyl, (C1-C3) haloalkyl, halogen, C(0)(Ci-C3) alkyl, C(0)NH?, C(0)NH(Ci-C ₃ ) alkyl, C(0)N((Ci-C ₃ ) alkyl)?., or (C3-C&) cycloalkyl. In other embodiments, each Rs is independently (C1-C3) alkyl, C(0)(Ci-C3) alkyl, C(0)NH ₂ , C(0)NH(Ci-C ₃ ) alkyl, C(0)N((Ci-C ₃ ) alkyl)?, or (Cs-Ce) cycloalkyl. In other embodiments, each Rs is independently (C1-C3) alkyl, C(0)(Ci-C3) alkyl, or (C3-C6) cycloalkyl. In other embodiments, each Rs is independently (C1-C3) alkyl, C(0)(Ci-C3) alkyl, or (Cs-Ce) cycloalkyl. In some embodiments, two Rs together with the carbon to which they are attached form C(O).

In some embodiments, each R9 is (Ci-Cs) alkyl, (C1-C3) haloalkyl, (C 1-C3) alkoxy, (C 1-C3) haloalkoxy, halogen, C ^' i GK CVO ) alkyl, C(Q)NH?, C{0 )\ i R C ViVs alkyl,

C(0)N((Ci-C3) alkyl)?, {( ^' ;··( ·.} cy cloalkyl, or heterocycloalkyi. In other embodiments, each R9 is (C 1-C3) alkyl, (C1-C3) haloalkyl, halogen, C(0)(Ci-C3) alkyl, C(0)NH?, C(0)NH(Ci- C3) alkyl, C(0)N((Ci-C3) alkyl)?, (C3-C6) cycloalkyl, or heterocycloalkyi. In other embodiments, each R9 is (C1-C3) alkyl, (C1-C3) haloalkyl, halogen, C(0)(Ci-C3) alkyl, C(0)NH?, C(0)NH(Ci-C3) alkyl, C(0)N((Ci-C ₃ ) alkyl)?, or (Cs-Ce) cycloalkyl. In other embodiments, each R, is (C1-C3) alkyl, C(0)(Ci-C3) alkyl, C(0)NH?, C(0)NH(Ci-C ₃ ) alkyl, C(0)N((Ci -C3) alkyl)?, or (C3-C6) cycloalkyl. In other embodiments, each R9 is (C 1-C3) alkyl, C(0)NH?, C{ () )N! ! (( ^' ] ·{ ^' = ) alkyl, C(())N((Ci-C3) alkyl)?, or (Cs-Ce) cycloalkyl. In other embodiments, each R ₉ is C(0)NH ₂ , C«»\ ^' l l« ^' i -( ^' : ) alkyl, ( ^' ί ())Νί (( i -C alkyl)?., or (C3-C6) cycloalkyl.

In some embodiments, t is 0. In other embodiments, t is 1. In other embodiments, t is 2. In other embodiments, t is 3. In other embodiments, t is 0 or 1. In other embodiments, t is 1 or 2. In other embodiments, t is 0, 1 or 2. In other embodiments, t is 1 , 2 or 3.

In some embodiments, n is 0. In other embodiments, n is 1 , In other embodiments, n is 2. In other embodiments, ii is 3. In other embodiments, n is 0 or 1. In other embodiments, n is 1 or 2. In other embodiments, n is 0, 1 or 2. In other embodiments, n is 1 , 2 or 3.

Any of the groups described herein for any of A, A', B, X, X ₂ , Ri, R2, R3, R ₄ , Rs, R ₆ , R7, Rs, R9, n, and t can be combined with any of the groups described herein for one or more of the remainder of A, A', B, X, X2, Ri , R2, R3, R ₄ , Rs, Re, R7, Rs, Rs, n, and t, and may further be combined with any of the groups described herein for the Linker.

For a Targeting Ligand of Formula TL-I:

(1) In one embodiment, X is and A is absent.

(2) In one embodiment, X is N and A is CH ₂ .

(3) In one embodiment, X is N and A' is NRs.

(4) In one embodiment, X is N, A is absent, and A' is NRs.

(5) In one embodiment, X is N, A is CH2, and A' is Nils.

(6) In one embodiment. X is N and B is

(7) In one embodiment, X is N, A is abse nt, and B is

(8) In one embodiment, X is N, A is CH2, and B is

(9) In one embodiment, X is N and B is

(10) In one embodiment, X is N, A is abse nt, and B is (1 1) In one embodiment, X is N, A is CH ₂ , and B is

(12) In one embodiment, X is N and B i s

(13) In one embodiment, X is N, A is abse nt, and B is

(14) In one embodiment, X is N. A is CH2. and B is

(15) In one embodiment, X is N and B is

(16) in one embodiment, X is N, A is abse nt, and B is

(17) In one embodiment, X is N, A is CH2, and B is

(18) In one embodiment, X is N. A' is N 5. and B is

In one embodiment, X is N, A is absent, A' is N Rs, and B

(20) In one embodiment, X is N. A is CH2. A' is R . and B is

(21) In one embodiment, X is N, A' is NRs, and B is 1

(22) In one embodiment, X is N, A is absent, A' is NR ₅ , and B i

(23) in one embodiment, X is N, A is CH ₂ , A' is NRs and B is

(24) In one embodiment. X is N. A' is NRs. and B

(25) in one embodiment, X is N, A is absent, A' is NR s, and B

(26) in one embodiment, X is N, A is Ctb, A' is NRs and B is

(27) In one embodiment, X is N, A' is NRs, and B is

(28) In one embodiment, X is N. A is absent, A" is NR s, and B i

(29) In one embodiment, X is N, A is CJ¾ A' is NR and B is

(30) In one embodiment, X is N, A' is NRs, B is

(31 ) in one embodiment, X is N, A is absent, A' is NRs, B is _; and : is H. (32) In one embodiment, X is N, A is CH?„ A' is Nils, B is _; and Rs is

(33) In one embodiment, X is N, A' is NRs, B is

In one embodiment, X is N, A is absent, A ^! is NR , and Rs

(35) In one embodiment, X is N, A is CH?„ A' is Nils, B is and Rs is

(36) In one embodiment, X is N, A' is NRs, B is

(37) In one embodiment, X is N, A is absent, A' i s NRs, B is and Rs is H.

(38) In one embodiment, X is N, A is i ^' i k A' is NRs, B is _¾ and Rs is

In one embodiment, X is N, A' is NRs, B is

(40) In one embodiment, X is N, A is absent, A' i s NRs, B is and Rs is H. (41 ) In one embodiment, X is N, A is CH ₂ , A' is NRs, B is , and R.5 is

In one embodiment, the compound of Formula TL-I is of Formula TL-Ia or TL-Ib:

wherein A', B, X, Ri, R?„ R:¾, and n are each as defined above in Formula TL-1.

For a Targeting Ligand of Formula TL-Ia or TL-Ib:

(1 ) In one embodiment, X is N and A' is NRs.

(2) In one embodiment, X is N and B is

(3) In one embodiment, X is N and B is

In one embodiment. X is and B is

(5) In one embodiment, X is N and B is

(6) In one embodiment, X is N, A' is N

(7) In one embodiment, X is N, A' is NR s, and B is

In one embodiment, X is N, A' is NR (9) In one embodiment, X is N, A' is NRs, and

(10) In one embodiment, X is N, A' is NRs, and

(1 1) In one embodiment, X is N, A' is NRs, B is _; and Rs is H.

(12) In one embodiment, X is N, A ^! is NRs, B is

(13) In one embodiment, X is N, A' is NRs, B is

(14) In one embodiment, X is N, A' is NRs, B is

(15) In one embodiment, X is N, A' is NRs, B is , and R is H.

A', B, X, Ri, R ₂ , R ₃ , Re, n, and t can each be selected from any of the groups and combined as described above in Formula TL -I,

In another embodiment, the compound of Formula TL-I is of Formula TL-Ic, TL-ld, TL-Ie, or TL-If:

wherein X, Ri, R3, Re, n, and t are each as defined above in Formula TL-I.

For a Targeting Ligand of Formula TL-Ic, TL-Id, TL-Ie, or TL-If:

(1) In one embodiment, X is N.

(2) In one embodiment, n is 0.

(3) In one embodiment, t is 0.

(4) In one embodiment, n is 0 and t is 0.

(5) In one embodiment, R2 is halogen.

(6) In one embodiment, R2 is F.

(7) In one embodiment, n is 0 and R2 is halogen.

(8) In one embodiment, n is 0 and R2 is F.

(9) In one embodiment, n is 0, t is 0, and R2 is halogen.

(10) In one embodiment, n is 0, t is 0, and R2 is F.

(1 1) In one embodiment, n is 0, t is 0, X is N, and R2 is halogen.

(12) In one embodiment, n is 0, t is 0, X is N, and R2 is F.

(13) In one embodiment, R3 is bicyclic heteroaryl comprising one to four heteroatoms seiected from N, O, and S, optionally substituted with one or more R ₇ .

(14) In one embodiment, R3 is bicyclic heteroaryl comprising one to four heteroatoms seiected from , O, and S, substituted with one or more R ₇ .

(15) In one embodiment, R2 is halogen and R:< is bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R-.

(16) In one embodiment, R2 is halogen and 3 is bicy clic heteroaryl comprising one to four heteroatoms selected from N, O, and S, substituted with one or more R ₇ .

(17) In one embodiment, n is 0, R2 is halogen, and R3 is bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R ₇ . (18) In one embodiment, n is 0, Rs is halogen, and R3 is tricyclic heteroaryl comprising one to four heteroaioms selected from N, O, and S, substituted with one or more R -.

(19) In one embodiment, n is 0, R2 is halogen, R3 is bicyclic heteroaiyl comprising one to four heteroaioms selected from N, O, and S, optionally substituted with one or more R ₇ , and X is N.

(20) In one embodiment, n is 0, R2 is halogen, R3 is bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, substituted with one or more R7, and X is N.

(21) In one embodiment, n is 0, t is 0, R2 is halogen, and R3 is bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R7.

(22) In one embodiment, n is 0, t is 0, R2 is halogen, and R3 is bicyclic heteroaiyl comprising one to four heteroatoms selected from N, O, and S, substituted with one or

(23) In one embodiment, n is 0, t is 0, R2 is halogen, R3 is bicyclic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R7, and X is N.

(24) In one embodiment, n is 0, t is 0, R2 is halogen, R3 is bicyclic heteroaryl comprising one to four heteroaioms selected from N, O, and S, substituted with one or more R7, and X is N.

(25) In one embodiment, each R7 is independently (C1-C4) alkyl or halogen.

(26) In one embodiment, n is 0, t is 0, R2 is halogen, and R3 is bicyclic heteroaiyl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R7, and each R7 is independently (C 1-C4) alkyl or halogen.

(27) In one embodiment, n is 0, t is 0, R2 is halogen, and R:< is bicyclic heteroaryl comprising one to four heteroaioms selected from N, O, and S, substituted with one or more R7, and each R7 is independently (C]-C ₄ ) alkyl or halogen.

(28) In one embodiment, n is 0, t is 0, R2 is halogen, and R3 is bicy clic heteroaryl comprising one to four heteroatoms selected from N, O, and S, optionally substituted with one or more R?, each R? is independently (C1-C4) alkyl or halogen, and X is N.

(29) In one embodiment, n is 0, t is 0, R2 is halogen, and R3 is bicyclic heteroaiyl comprising one to four heteroatoms selected from N, (), and S, substituted with one or more R7, each R7 is independently (C1-C4) alkyl or halogen, and X is N. (30) In one embodiment, R? and Rs together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatoms selected from N, Q, and S, optionally substituted with one or more Re.

(31) In one embodiment, R?. and 3 together with the carbon atoms to which they are attached form a 5-membered heteroaryi comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more R9.

(32) In one embodiment, X is and R?. and R3 together with the carbon atoms to which they are attached form a 6-membered heterocy cloalkyl comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more Re.

(33) In one embodiment, X is N and R2 and Ra together with the carbon atoms to which they are attached form a 5-membered heteroaryi comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more R9.

(34) In one embodiment, R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more Rs.

(35) In one embodiment, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryi comprising one or two heteroatoms selected from N, O, and S, substituted with one or more R9.

(36) In one embodiment, X is N and R ₂ and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatoms selected from , O, and S, substituted with one or more Rs.

(37) In one embodiment, X is N, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryi comprising one or two heteroatoms selected from N, O, and S, substituted with one or more R9.

(38) In one embodiment, n is 0 and R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more Rs.

(39) In one embodiment, n is 0 and R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaiyi comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more R9.

(40) In one embodiment, n is 0 and R ₂ and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatoms selected from N, (), and S, substituted with one or more Rs. (41) In one embodiment, n is 0 and R ₂ and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatorns selected from N, O, and S, substituted with one or more R9.

(42) In one embodiment, X is N, n is 0, and 1 ?. and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatorns selected from N, O, and S, optionally substituted with one or more Rs.

(43) In one embodiment, X is N, n is 0, and R> and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatorns selected from N, O, and S, optionally substituted with one or more Rs>.

(44) In one embodiment, X is N, n is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatorns selected from N, O, and S, substituted with one or more Rs.

(45) In one embodiment, X is N, n is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heieroatoms selected from N, O, and S, substituted with one or more R9.

(46) In one embodiment, n is 0, t is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatorns selected from N, O, and S, optionally substituted with one or

(47) In one embodiment, n is 0, t is 0, and R?. and Rs together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatorns selected from N, O, and S, optionally substituted with one or more Rs>.

(48) In one embodiment, n is 0, t is 0, and R2 and R:< together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatorns selected from N, O, and S, substituted with one or more Rs.

(49) In one embodiment, n is 0, t is 0, and R?. and Rs together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heieroatoms selected from N, O, and S, substituted with one or more R9.

(50) In one embodiment, X is N, n is 0, t is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroaioms selected from N, O, and S, optionally substituted with one or more Rs.

(51) In one embodiment, X is N, n is 0, t is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatoms selected from N, G, and S, optionally substituted with one or more R9.

(52) In one embodiment, X is , n is 0, t is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocy cloalkyl comprising one or two heteroatoms selected from N, O, and S, substituted with one or

(53) In one embodiment, X is N, n is 0, t is 0, and R?. and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more R9.

(54) In one embodiment each Rs is independently (Ci-CY) alkyl, CXO)(Ci -C ₄ ) aikyl, (C3-C7) cycloalkyl, or two Rs together with the carbon to which they are attached form C(O).

(55) In one embodiment, each R9 is independently C(0)NH ₂ , C(0)N((Ci-C ₄ ) alkyl)?., or (C3-C7) cycloalkyl.

(56) In one embodiment, n is 0, t is 0, R ₂ and R:< together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyl comprising one or two heteroatoms selected from , O, and S, optionally substituted with one or more Rs, and each Rg is independently (C1-C ) alkyl, C(0)(Ci-C4) alkyl, (C3-C7) cycloalkyl, or two Rs together with the carbon to which they are attached form C(O).

(57) In one embodiment, n is 0, t is 0, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroaioms selected from N, (), and S, optionally substituted with one or more R9, and each R9 is independently C(0)NH ₂ , C(0)N((Ci-C ₄ ) alkyl)?, or (C3-C7) cycloalkyl.

(58) In one embodiment, n is 0, t is 0, R ₂ and R3 together with the carbon atoms to which they are attached form a 6-membered heterocy cloalkyl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more Rs, and each Rs is independently (C1-C4) alkyl, C(0)(Ci-C ₄ ) alkyl, (C3-C7) cycloalkyl, or two Rs together with the carbon to which they are attached form C(O).

(59) In one embodiment, n is 0, t is 0, R ₂ and R:< together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more R9, and each R9 is independently C(0)NH ₂ , C(0)N((C.-C4) alky]) ₂ , or (C3-C7) cycloalkyl.

(60) In one embodiment, X is N, n is 0, t is 0, R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyi comprising one or two heteroatoms selected from N, G, and S, optionally substituted with one or more Rg and each Rs is independently (C1-C4) alkyl, C(0)(Ci-C ₄ ) alkyl, (C3-C7) cycloalkyl, or two s together with the carbon to which they are attached form C(O).

(61) In one embodiment, X is N, n is 0, t is 0, R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaryl comprising one or two heteroatoms selected from N, O, and S, optionally substituted with one or more R9, and each R is independently C(0)NH ₂ , C(0)N((Ci-C4) alkyl)?., or (C3-C7) cycloalkyl.

(62) In one embodiment, X is N, n is 0, t is 0, R2 and R3 together with the carbon atoms to which they are attached form a 6-membered heterocycloalkyi comprising one or tw ^? o heteroatoms selected from N, O, and S, substituted with one or more Rs, and each Rg is independently (C1-C4) alkyl, C(0)(Ci-Gi) alkyl, (C3-C7) cycloalkyl, or two Rs together with the carbon to which they are attached form C(O).

(63) In one embodiment, X is N, n is 0, t is 0, and R2 and R3 together with the carbon atoms to which they are attached form a 5-membered heteroaiyl comprising one or two heteroatoms selected from N, O, and S, substituted with one or more R9, and each R9 is independently C(0)NH ₂ , C(0)N((Ci-C ₄ ) alkyl or (C3-C7) cycloalkyl. X, Ri, R2, R3, Rb, R7, Rg, R9, n, and t can each be selected from any of the groups and combined as described above in Formula TL-I.

In another embodiment, the compound of Formula TL-I is of Formula TL-Ig, TL-Ih r TL-Ii:

or

wherein X, Ri, R ₇ , Rs, R9, and n are each as defined above in Formula TL-I.

For a Targeting Ligand of Formula TL-ig, TL-ih, or TL-Ii :

(1) In one embodiment, X is N.

(2) In one embodiment, n is 0.

(3) In one embodiment, t is 0.

(4) In one embodiment, n is 0 and t is 0.

(5) In one embodiment, R2 is halogen.

(6) In one embodiment, R2 is F.

(7) In one embodiment, n is 0 and R2 is halogen,

(8) In one embodiment, n is 0 and R2 is F.

(9) In one embodiment, n is 0, t is 0, and R2 is halogen.

(10) In one embodiment, n is 0, t is 0, and R2 is F,

(11) In one embodiment, n is 0, t is 0, X is N, and R2 is halogen.

(12) In one embodiment, n is 0, t is 0, X is N, and R2 is F.

(13) In one embodiment, each R7 is independently (C1-C4) alk l or halogen.

(14) In one embodiment, n is 0, t is 0, R2 is halogen, and each R ₇ is independently (C1-C4) alkyl or halogen.

(15) In one embodiment, n is 0, t is 0, R2 is halogen, each R? is independently (Ci- C4) alkyl or halogen, and X is N.

(16) In one embodiment, each Rs is independently (C 1-C4) alkyl, C(0)(Ci-C ₄ ) alkyl, or (C3-C7) cycloaikyl.

(17) In one embodiment, each R9 is independently C(0)NH ₂ , C(0)N((C 1-C4) aikyl or (C3-C7) cycloaikyl. (18) In one embodiment n is 0, t is 0, and each Rs is independently (C1-C4) alkyl, C(0)(Ci-C ₄ ) alky] , or (C3-C7) cycioalky] ,

(19) In one embodiment, n is 0, t is 0, and each R9 is independently C(0)NH ₂ , C(0)N((Ci-C4) alk>'i) ₂ , or (C3-C7) cycloalkyl.

X, Ri, R ₂ , Re, R?, Rs, R9, n, and t can each be selected from any of the groups and combined as described above in Formula TL-I.

Degrosi

The Degron serves to link a targeted protein, through a Linker and a Targeting Ligand, to a ubiquitin ligase for proteosomal degradation. In one embodiment, the Degron is capable of binding to a ubiquitin ligase, such as an F3 ubiquitin ligase. In one embodiment, the Degron is capable of binding to cerebion.

In one embodiment, he Degron is of Formula Dl :

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

Y is a bond, (CH ₂ )i -5, (CH ₂ )o-6-0, (CH ₂ )o..6-C(0)NRi], (CH ₂ )o..6~NRnC(0), (CH ₂ )o-6-

R11 is H or Ci-C ₆ alkyl;

Ri ₂ is 00, alkyl or C(0)-Ci-C6 alkyl;

each Rr, is independently H or C1-C3 alkyl;

each Ri4 is independently C1-C3 alkyl;

Ri5 is H, deuterium, C1-C3 alkyl, F, or CI;

each i6 is independently halogen, OH, Ci-C& alkyl, or Ci-Ce alkoxy:

q is 0, 1, or 2; and

v is 0, 1, 2, or 3, wherein the Degron is covaiently bonded to the Linker via ^ .

In one embodiment, Z is C(O).

In one embodiment, Z is C(Ri3) ₂ ; and each R13 is H. In one embodiment, X is C(Ri3) ₂ ; and one of R13 is H, and the other is C1-C3 alkyl selected from methyl, ethyl, and propyl. In one embodiment, Z is C( i and each R13 is independently selected from methyl, ethyl, and propyl.

In one embodiment, Y is a bond.

In one embodiment, Y is a bond, O, or NH.

In one embodiment, Y is NH.

In one embodiment, Y is (CH ₂ ) _1; (CH ₂ ) ₂ , (CH ₂ )3, (CH ₂ ) ₄ , ( CI 1: ): . or (CH ₂ )e. In one embodiment, Y is { ( ^* ! 1 ^■ } >. (CH ₂ ) ₂ , or { ( ^' I b ) ;. In one embodiment, Y is (CH ₂ )i or (CH ₂ ) ₂ .

In one embodiment, Y is O, CH2-O, (CH^-O, (CH ₂ ) ₃ -0, (CH ₂ )4-0, (CH ₂ )s-0, or (CH ₂ ) ₆ -0. In one embodiment, Y is O, CH ₂ -0, (CH ₂ ) ₂ -0, or (CH ₂ )3-0. In one embodiment, Y is O or CH ₂ -0. In one embodiment, Y is O.

In one embodiment, Y is C(0)NRn, CH ₂ -C(G)NRn, ( CU ^> ):- ; (0)\ R r _: . (CH ₂ )3- C(0)NRii, (CH2)4-C(0)NRii, (CH ₂ )s-C(0)NRn, or (CH ₂ )6-C(0)NRn. In one embodiment,

Y is C(0)NRii, CH ₂ -C(G)NRn, (CH ₂ ) ₂ -C(G)NRn, or (CH ₂ )3~C(0)NRii, In one embodiment, Y is C(0)NRn or CH?~C(0)NRi i . In one embodiment, Y is C(0)NRn.

In one embodiment, Y is NRj 3 C(O), CH2-NR1 iC(O), (CI { ^• •) - -X : iC(O), (CH ₂ )3-

NRiiC(O), (CH ₂ )4-NRiiC(0), (CH2)5-NRiiC(0), or (CH ₂ ) ₆ -NRiiC(G). In one embodiment,

Y is NRiiC(O), CH2-NR- iC(O), iCH ₂ ) ₂ ~NRiiCiO), or (CH ₂ )3-NR¾ iC(0). In one embodiment, Y is NRuC(O) or CH ₂ -NRnC(0). In one embodiment, Y is NR] j C(0).

In one embodiment, R11 is H. In one embodiment, R11 is selected from methyl, ethyl, propyl, butyl, i-butyl, t-butyl, pentyl, i-pentyl, and hexyl. In one embodiment, R11 is C1-C3 alkyl selected from methyl, ethyl, and propyl

In one embodiment, Y is NH, CH2-NH, (CH ₂ ) ₂ -NH, (CH ₂ )3-NH, (CH ₂ )4-NH, { ( ^' i ! · ) =·· NH, or (CH ₂ )6-NH. In one embodiment, Y is NH, CH2-NH, (CH ₂ )2-NH, or (CH ₂ )3-NH. In one embodiment, Y is NH or CH2-NH In one embodiment, Y is NH.

In one embodiment, Y is NR12, CH2-NR12, (CHa^-NRu, i ( ^' i b ) ^; -\ R i \ (CHa^-NRu,

(CH2)5-NRi ₂ , or (CH ₂ )e-NR ₁₂ . In one embodiment, Y is NRi¾ CH ₂ -NRi¾ (CH ₂ ) ₂ -NRi ₂ , or (CH ₂ )3-NRi2. In one embodiment, Y is NR12 or CH2-NR12. In one embodiment, Y is NR12.

In one embodiment, R12 is selected from methyl, ethyl, propyl, butyl, i-butyl, t-butyl, pentyl, i-pentyl, and hexyl. In one embodiment, R12 is C1-C3 alkyl selected from methyl, ethyl, and propyl.

In one embodiment, R12 is selected from C(0)-methyl, C(0)-ethyl, C(0)-propyl, C(0)-butyl, C(Q)-i-butyi, C(0)-t-butyl, C(0)-pentyl, CXO)-i-pentyl, and C(G)-hexyl. In one embodiment, R12 is C(0)-Ci-C3 alkyl selected from C(0)-methyl, C(0)-ethyl, and C(O)- propyl. In one embodiment, Rr, is H.

In one embodiment, R13 is C1-C3 alkyl selected from methyl, ethyl, and propyl. In one embodiment, R13 is methyl.

In one embodiment, q is 0.

In one embodiment, q is 1.

In one embodiment, q is 2.

In one embodiment, each R-,4 is independently CVC3 alkyl selected from methyl, ethyl, and propyl.

In one embodiment, v is 0.

In one embodiment, v is 1.

In one embodiment, v is 2.

In one embodiment, v is 3.

In one embodiment, each Rie is independently selected from halogen (e.g., F, CI, Br, and I), OH, Ci-Ce alkyl (e.g., methyl, ethyl, propyl, butyl, i-butyl, t-butyl, pentyi, i-pentyl, and hexyl), and Ci-Ce alkoxy (e.g. , methoxy, ethoxy, propoxy, butoxy, i-butoxy , t-butoxy, and pentoxy). In a further embodiment, each Rie is independently selected from F, CI, OH, methyl, ethyl, propyl, butyl, i-butyl, t-butyl, methoxy, and ethoxy.

In one embodiment, is H, deuterium, or C1-C3 alkyl. In another embodiment, R15 is H or C1-C3 alkyl. In a further embodiment, R15 is in the (S) or (R) configuration. In a further embodiment, R15 is in the (S) configuration. In one embodiment, the compound comprises a racemic mixture of (Aj- is and (ii)-Ris.

In one embodiment, R15 is H.

In one embodiment, R15 is deuterium.

In one embodiment, R15 is C1-C3 alkyl selected from methyl, ethyl, and propyl. I one embodiment, is methyl.

In one embodiment, R15 is F or CI. In a further embodiment, R15 is in the (S) or (R) configuration. In a further embodiment, R15 is in the (R) configuration. In one embodiment, the compound comprises a racemic mixture of (<S)-Ris and (i?)-Ris. In one embodiment, R15 is F.

Any of the groups described herein for any of Y, Z, R11, R12, R13, R14, R15, Rie, q and v can be combined with any of the groups described herein for one or more of the remainder of Y, Z, Rn, R12, Ri3, Ri4, Ris, Rie, q and v, and may further be combined with any of the groups described herein for the Linker.

For a Degron of Formula Dl : (1) In one embodiment, Z is C(O) and Y is a bond.

(2) In one embodiment, Z is C(O) and Y is NH.

(3) In one embodiment, Z is C(O) and Y is (CH ₂ )o-6-0. In a further embodiment, Y is

(4) In one embodiment, Z is C(O); Y is a bond; and q and v are each 0.

(5) In one embodiment, Z is C(O); Y is NH; and q and v are each 0.

(6) In one embodiment, z is C(0); Y is a bond; and RB is H.

(7) In one embodiment, z is C(O); Y is a bond; and Ris is H.

(8) In one embodiment, z is C(O); Ύ is NH; and Ris is H.

(9) In one embodiment, z is C(0); Y is NH; and Ris is H.

(10) In one embodiment, Ζ is C(O); Y is a bond; and RB is H; and Ris is H.

(1 1 ) In one embodiment, Ζ is C(O); Y is NH; and RB is H; and Ris is H.

(12) In one embodiment, Ζ is C(O); Y is (CH ₂ )o-6-0; and R13 is H. In a further embodiment, Y is O.

(13) In one embodiment, Z is C(O); Y is (CH ₂ )o-6-0; and R15 is H. In a further embodiment, Y is O.

(14) In one embodiment, Z is C(O); Y is id ί.· )· ,-ϋ: RB is H; and R15 is H. In a further embodiment, Y is O.

(15) In one embodiment, q and v are each 0; and Y, Z, RB, R35, and Ri6 are each as defined in any of (1) - (3) and (6) - (14).

In one embodiment, the Degron is of Formula Dla or Dlb:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein Y, R14, Ri6, q, and v are each as defined above in Formula Dl , and can be selected from any moieties or combinations thereof described above.

In one embodiment, the Degron is of Formula D2:

or an enantiomer, diastereomer, or stereoisomer thereof, wherein:

each Ri? is independently C1-C3 alkyl;

q' is 0, 1, 2, 3 or 4; and

Rig is H or C1-C3 alkyl,

wherein the Degron is covalently bonded to another moiety (e.g. , a compound, or a Linker) via ^ .

In one embodiment, q' is 0.

In one embodiment, q' is 1.

In one embodiment, q' is 2.

In one embodiment, q' is 3.

In one embodiment, each Rs? is independently C1-C3 alkyl selected from methyl, ethyl, and propyl.

In one embodiment, Ris is methyl, ethyl, or propyl. In one embodiment, Rie is methyl.

In one embodiment, the Degron is of Formula D2a or D2b:

The Linker is a bond or a carbon chain that serves to link a Targeting Ligand with a Degron. In one embodiment, the carbon chain optionally comprises one, two, three, or more heteroaioms selected from N, O, and S. In one embodiment, the carbon chain comprises only saturated chain carbon atoms. In one embodiment, the carbon chain optionally comprises two or more unsaturated chain carbon atoms (e.g. , c===c ₀ r c^≡c ). in one embodiment, one or more chain carbon atoms in the carbon chain are optionally substituted with one or more substituents (e.g., oxo, Ci-Ce all y!, C2-C6 alkenyl, C2-C0 alkynyl, C1-C3 alkoxy, OH, halogen, NH ₂ , NHiCi-Cs alkyl), N(Ci~Cj alkyl) ₂ , CN, Cs-Cs cycloalkyl, heierocyclvl, phenyl, and heteroaryl).

In one embodiment, the Linker comprises at least 5 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises less than 25 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises less than 20 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 chain atoms (e.g. , C, (), N, and S). In one embodiment, the Linker comprises 5, 6, 7, 8, 9, 10, 11, 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 5, 7, 9, 11, 13, 15, 17, or 19 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 5, 7, 9, or 11 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 11, 13, 15, 17, or 19 chain atoms (e.g., C, O, N, and S). In one embodiment, the Linker comprises 1 1 , 13, 15, 17, 19, 21, or 23 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 6, 8, 1 0, 12, .14, 16, 18, or 20 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 6, 8, 10, or 12 chain atoms (e.g. , C, O, N, and S). In one embodiment, the Linker comprises 12, 14, 16, 18, or 20 chain atoms (e.g., C, O, N, and S).

In one embodiment, the Linker comprises from 1 1 to 19 chain atoms (e.g. , C, O, N, and S).

In one embodiment, the Linker is a carbon chain optionally substituted with non- bulky substituents (e.g., oxo, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C3 alkoxy, OH, halogen, M 1 ^■ ■. NH(CI-CJ alkyl), \{C ^' ;~C ; alk l and CN). In one embodiment, the non- bulky substitution is located on the chain carbon atom proximal to the Degron (i.e. , the carbon atom is separated from the carbon atom to which the Degron is bonded by at least 3, 4, or 5 chain atoms in the Linker). In one embodiment, the non-bulky substitution is located on the chain carbon atom proximal to the Targeting Ligand (i.e., the carbon atom is separated from the carbon atom to which the Degron is bonded by at least 3, 4, or 5 chain atoms in the Linker).

In one embodiment, the Linker is of Formula LO: or an enantiomer, diastereomer, or stereoisomer thereof, wherein

i is an integer selected from 0 to 12;

p2 is an integer selected from 0 to 12;

p3 is an integer selected from 1 to 6;

each W is independently absent, CH?,, O, S, NH, or NR19;

Zi is absent, C(0), O i ( i())NI I. CH ₂ , O, NH, or NR _i9 ;

each Ri9 is independently C1-C3 alkyi; and

Q is absent or NHC(0)CH ₂ , wherein the Linker is covalently bonded to a Degron via the ^ next to Q, and covalently bonded to a Targeting Ligand via the ^ next to Zi.

In one embodiment, the total number of chain atoms in the Linker is less than 30. In a further embodiment, the total number of chain atoms in the Linker is less than 20.

For a Linker of Formula L0:

In one embodiment, pi is an integer selected from 0 to 10.

In one embodiment, pi is an integer selected from 1 to 10.

In one embodiment, pi is selected from 1, 2, 3, 4, 5, and 6.

In one embodiment, pi is 0, 1, 3, or 5.

In one embodiment, pi is 0, I, 2, or 3.

In one embodiment, i is 0.

In one embodiment, pi is 1.

In one embodiment, pi is 2.

In one embodiment, i is 3.

In one embodiment, p2 is an integer selected from 0 to 10.

In one embodiment, p2 is selected from 0, 1, 2, 3, 4, 5, and 6.

In one embodiment, p2 is 0, I, 2, or 3.

In one embodiment, p2 is 0.

In one embodiment, p2 is 1.

In one embodiment, p2 is 2.

In one embodiment, p3 is an integer selected from 1 to 5.

In one embodiment, p3 is 2, 3, 4, or 5.

In one embodiment, p3 is 0, 1, 2, or 3.

In one embodiment, p3 is 0. In one embodiment, p3 is 1.

In one embodiment, p3 is 2.

In one embodiment, at least one W is CH .

In one embodiment, at least one W is O.

In one embodiment, at least one W is S.

In one embodiment, at least one W is NH.

In one embodiment, at least one W is NRj ' and Rj is C 1-C3 alkyl selected from methyl, ethyl, and propyl.

In one embodiment, each W is O.

In one embodiment, Q is absent.

In one embodiment, Q is NHC(0)CH ₂ .

In one embodiment, Zi is absent.

In one embodiment, Zi is CHb,

In one embodiment, Zi is O,

In one embodiment, Zi is C(O).

In one embodiment, Zi is CH ₂ C(0)NH.

In one embodiment, Zi is NR19; and R19 is C1-C3 alkyl selected from methyl, ethyl, and propyl

In one embodiment, Zi is part of the Targeting Ligand that is bonded to the Linker, namely, Zi is formed from reacting a functional group of the Targeting Ligand with the Linker.

In one embodiment, Q is absent.

In one embodiment, Q is NHC(0)CH ₂ .

In one embodiment, pi is 1, 2, 3, or 4. In one embodiment, pi is 1. In one embodiment, pi is 2. In one embodiment, pi is 3. In one embodiment, pi is 4.

In one embodiment, pi is 1 and Zi is absent.

In one embodiment, pi is 2 and Zi is absent.

In one embodiment, pi is 3 and Zi is absent.

In one embodiment, p3 is 1 and Zi is absent.

In one embodiment, p3 is 2 and Zi is absent.

In one embodiment, p3 is 3 and Zi is absent.

In one embodiment, pi is 1, Zi is absent, and Q is absent.

In one embodiment, pi is 2, Zi is absent, and Q is absent.

In one embodiment, pi is 3, Zi is absent, and Q is absent. In one embodiment, p3 is 1, Zi is absent, and Q is absent.

In one embodiment, p3 is 2, Zi is absent, and Q is absent.

In one embodiment, p3 is 3, Zi is absent, and Q is absent.

In one embodiment, pi is 1, Zi is absent, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 2, Zi is absent, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 3, Zi is absent, and Q is NHC(0)CH ₂ .

In one embodiment, p3 is 1, Zj is absent, and Q is NHC(0)CH ₂ .

In one embodiment, p3 is 2, Zi is absent, and Q is NHC(0)CH ₂ .

In one embodiment, p3 is 3, Zi is absent, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 1, Zi is absent, and p3 is 1.

In one embodiment, pi is 2, Zi is absent, and p3 is 1.

In one embodiment, pi is 3, Zi is absent, and p3 is 1.

In one embodiment, i is 1, Z] is absent, and p3 is 2.

In one embodiment, pi is 2, Zj is absent, and p3 is 2.

In one embodiment, pi is 3, Zi is absent, and p3 is 2,

In one embodiment, pi is 1 , Zi is absent, and p3 is 3.

In one embodiment, pi is 2, Zi is absent, and p3 is 3.

In one embodiment, pi is 3, Zi is absent, and p3 is 3.

In one embodiment, pi is 1, Zi is absent, p3 is 1, and Q is absent.

In one embodiment, pi is 2, Zi is absent, p3 is 1, and Q is absent.

In one embodiment, pi is 3, Zi is absent, p3 is 1, and Q is absent.

In one embodiment, pi is 1, Zi is absent, p3 is 2, and Q is absent.

In one embodiment, pi is 2, Zi is absent, p3 is 2, and Q is absent.

In one embodiment, pi is 3, Zi is absent, p3 is 2, and Q is absent.

In one embodiment, pi is 1, Zi is absent, p3 is 3, and Q is absent.

In one embodiment, pi is 2, Zi is absent, p3 is 3, and Q is absent.

In one embodiment, pi is 3, Zi is absent, p3 is 3, and Q is absent.

In one embodiment, pi is 1, Zj is absent, p3 is 1, and Q is NHC(0)CH2.

In one embodiment, pi is 2, Zi is absent, p3 is 1, and Q is NHC(0)CH ₂ . In one embodiment, pi is 3, Zi is absent, p3 is 1 , and Q is NHC(0)CH ₂ .

In one embodiment, pi is 1, Zi is absent, p3 is 2, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 2, Zi is absent, p3 is 2, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 3, Zi is absent, p3 is 2, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 1, Zi is absent, p3 is 3, and Q is NHC(0)CH ₂ . In one embodiment, pi is z, Zi is absent, p3 is 3, and Q is NHC(G)CH ₂ .

In one embodiment, pi is 3, Zi is absent, p3 is 3, and Q is NHC(0)CH ₂ .

In one embodiment, pi is 1, Zi is absent, p3 is 1, and p2 is 0.

In one embodiment, pi iS 2, Zi is absent, p3 is 1, and p2 is 0.

In one embodiment, pi is Zi is absent, p3 is 1, and p2 is 0.

In one embodiment, i is 1, Zi is absent, p3 is 2, and p2 is 0.

In one embodiment, pi is Z] is absent, p3 is 2, and p2 is 0.

In one embodiment, pi is 3, Zi is absent, p3 is 2, and p2 is 0.

In one embodiment, pi is 1 , Zi is absent, p3 is 3, and p2 is 0,

In one embodiment, pi is 2, Zi is absent, p3 is 3, and p2 is 0.

In one embodiment, pi iS 3, Zi is absent, p3 is 3, and p2 is 0.

In one embodiment, pi is 1, Zi is absent, p3 is 1, p2 is 0, and Q is absent.

In one embodiment, i is 2. Zi is absent, p3 is 1, p2 is 0, and Q is absent.

In one embodiment, pi is 3, Zi is absent, p3 is 1, p2 is 0, and Q is absent.

In one embodiment, pi is 1, Zi is absent, p3 is 2, p2 is 0, and Q is absent.

In one embodiment, pi is 2, Zi is absent, p3 is 2, p2 is 0, and Q is absent.

In one embodiment, pi is 3, Zi is absent, p3 is 2, p2 is 0, and Q is absent.

In one embodiment, pi iS 1, Zi is absent, p3 is 3, p2 is 0, and Q is absent.

In one embodiment, pi is 2, Zi is absent, p3 is 3, p2 is 0, and Q is absent.

In one embodiment, pi is Zi is absent, p3 is 3, p2 is 0, and Q is absent.

In one embodiment, pi is 1, Zi is absent, p3 is 1, p2 is 0, and Q is M !( ^' (())( ^' ! !.·

In one embodiment, pi is Zi is absent, p3 is 1, p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi is 3, Zi is absent, p3 is 1 , p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi is 1, Zi is absent, p3 is 2, p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi iS 2, Zi is absent, p3 is 2, p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi is Zi is absent, p3 is 2, p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi is 1, Zi is absent, p3 is 3, p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi is Zi is absent, p3 is 3, p2 is 0, and Q is M !( ^' (())( ^' ! I ·

In one embodiment, pi is 3, Zi is absent, p3 is 3, p2 is 0, and Q is NHC(0)CH ₂

In one embodiment, pi is 1 and Zi is C(O).

In one embodiment, pi is 1, Zi is C(0), and p3 is 2.

In one embodiment, pi iS 1, Zi is C(O), and p2 is 0.

In one embodiment, pi is 1, Zi is (·(()). p3 is 2, and p2 is 0.

In one embodiment, i is 3 and Zi is C(0), In one embodiment, pi is 3, Zi is C(O), and p3 is 2,

In one embodiment, pi is 3, Zi is C(0), and p2 is 0.

In one embodiment, pi is 3, Zi is C(0), p3 is 2, and p2 is 0,

In one embodiment, pi is 5 and Zj is C(O).

In one embodiment, pi is 5, Zi is C(O), and p3 is 2.

In one embodiment, pi is 5, Zi is C(O), and p2 is 0,

In one embodiment, pi is 5, Zj is C(O), p3 is 2, and p2 is 0.

In one embodiment, p3 is 3, Zi is absent, and pi is 0.

In one embodiment, pi is 5, and Zi is absent.

In one embodiment, pi is 5, Zi is absent, and p3 is 2.

In one embodiment, pi is 5, Zi is absent, and p2 is 0.

In one embodiment, pi is 5, Zi is absent, p3 is 2, and p2 is 0.

In one embodiment, i is 1 and Zi is Π I ίί))\Ί 1.

In one embodiment, pi is 1, Zj is CH2.C(Q)NH, and p3 is 2.

In one embodiment, pi is 1, Zi is CH ₂ C(0)NH, and Q is absent.

In one embodiment, pi is 1 , Zi is CH ₂ C(0)NH, p3 is 2, and Q is absent.

In one embodiment, pi is 1, Zi is Π 1 ^> ( ίί ⁾ )Μ 1. p3 is 2, p2 is 0, and Q is absent.

In one embodiment, pi is 2 and Zj is CH2.C(0)NH.

In one embodiment, pi is 2, Zi is CH ₂ C(0)NH, and p3 is 2.

In one embodiment, pi is 2, Zi is CH ₂ C(0)NH, and Q is absent.

In one embodiment, pi is 2, Zi is CH2C(Q)NH, p3 is 2, and Q is absent.

In one embodiment, pi is 3, Zi is CH ₂ C(0)NH, p3 is 2, p2 is 0, and Q is absent.

In one embodiment, pi is 3 and Zi is CH ₂ C(0)NH.

In one embodiment, pi is 3, Zi is Π 1 ^> ( ίί))\ 1. and p3 is 2.

In one embodiment, pi is 3, Zi is CH ₂ C(0)NH, and Q is absent.

In one embodiment, pi is 3, Zi is CH ₂ C(0)NH, p3 is 2, and Q is absent.

In one embodiment, pi is 3, Zi is CH ₂ C(0)NH, p3 is 2, p2 is 0, and Q is absent.

In one embodiment, Z, Q, pi, p2, and/or p3 are as defined and combined above, and each W is O.

In one embodiment, pi is 2, Zi is absent, p3 is 2, p2 is 0, Q is absent, and each W is

O.

In one embodiment, pi is 2, Zi is absent, p3 is 2, p2 is 0, Q is NHC(0)CH ₂ , and each

W is (). In one embodiment, pi is 3, Zi is absent, p3 is 2, p2 is 0, Q is absent, and each W is

O.

In one embodiment, pi is 3, Zi is absent, p3 is 2, p2 is 0, Q is NHC(0)CH ₂ , and each

W is O.

In one embodiment, pi is 3, Zi is absent, p3 is 2, p2 is 0, Q is absent, and each W is

O.

In one embodiment, pi is 3, Zj is CH ₂ CXO)NH, p3 is 2, p2 is 0, Q is HC(0)CH ₂ , and each W is O.

In one embodiment, pi is 3, Zi is CH ₂ C(0)NH, p3 is 2, p2 is 0, Q is absent, and each

W is O.

In one embodiment, Z, Q, pi, p2, and/or p3 are as defined above, and each W is absent.

In one embodiment, pi is 1, Zi is absent, p3 is 2, p2 is 0, Q is absent, and W is absent. In one embodiment, pi is 1, Zj is absent, p3 is 2, p2 is 0, Q is NHC(0)CH ₂ , and W is absent.

In one embodiment, the Linker-Targeting Ligand (TL) has the structure selected from Table L:

Table L:

wherein Q, TL, W, pi, and p3 are each as described above.

Any one of the Degrons described herein can be covalently bound to any one of the Linkers described herein. Any one of the Targeting Ligands described herein can be covalently bound to any one of the Linkers described herein.

In one embodiment, the present application relates to the Degron-Linker (DL), wherein the Degron is of Formula Dl, and the Linker is selected from LI - LI 4. In one embodiment, the Degron is of Formula Dla or Dlb, and the Linker is selected from LI - LI 4. In one embodiment, the Degron is of Formula Dla or Dlb, and the Linker is LI, L3, L5, L7, L8, Ll l or L12. In one embodiment, the Degron is of Formula Dla or Dlb, and the Linker is L2, L4, L6, L9, LI 0, L13 or LI 4. In one embodiment, the Degron is of Formula Dl, and the Linker is LI , L3, L5, L7, L8, Ll l or L12. In one embodiment, the Degron is of Formula Dl, and the Linker is L2, L4, L6, L9, L10, LI 3 or L14. In one embodiment, the Degron is of Formula Dl a or Dlb, and the Linker is LI, L2, L3, or L4. In one embodiment, the Degron is of Formula Dla or Dlb, and the Linker is L5, L6, L7, or L8, In one embodiment, the Degron is of Formula Dla or Dlb, and the Linker is L9, L10, LI 1, or L12. In one embodiment, the Degron is of Formula Dl a or Dlb, and the Linker is Ll l , L12, LI 3, or LI 4.

In one embodiment, the present application relates to the Degron-Linker (DL), wherein the Degron is of Formula D2, and the Linker is selected from L I - L14. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is selected from LI - L14. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is LI, L3, L5, L7, L8, L I 1 or L12. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is L2, L4, L6, L9, L10, L13 or LI 4. In one embodiment, the Degron is of Formula D2, and the Linker is LI , L3, L5, L7, L8, Ll l or L12. In one embodiment, the Degron is of Formula D2, and the Linker is L2, L4, L6, L9, L10, LI 3 or LI 4. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is LI, L2, L3, or L4. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is L5, L6, L7, or L8. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is L9, L10, LI 1 , or LI 2. In one embodiment, the Degron is of Formula D2a or D2b, and the Linker is Ll l, LI 2, LI 3, or L I 4.

In one embodiment, the Linker is designed and optimized based on SA (structure- activity relationship) and X-ray crystallography of the Targeting Ligand with regard to the location of attachment for the Linker.

In one embodiment, the optimal Linker length and composition vary by the Targeting Ligand and can be estimated based upon X-ray structure of the Targeting Ligand bound to its target. Linker length and composition ca be also modified to modulate metabolic stability and pharmacokinetic (PK) and pharmacodynamics (PD) parameters.

Some embodiments of present application relate to the Afunctional compounds having the following structures in Table A:

Table A

Some of the foregoing compounds can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., stereoisomers and/or diastereomers.

Accordingly, compounds of the application may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. In one embodiment, the compounds of the application are enantiopure compounds. In another embodiment, mixtures of stereoisomers or diastereomers are provided.

Furthermore, certain compounds, as described herein, may have one or more double bonds that can exist as either the Z or E isomer, unless otherwise indicated. The application additionally encompasses the compounds as individual Z/E isomers substantially free of other E/Z isomers and alternatively, as mixtures of various isomers.

In one embodiment, the present application relates to compounds that target proteins, such as such as CDK4 and/or CDK6 for degradation, which have numerous advantages over inhibitors of protein function (e.g., kinase activity) and can a) overcome resistance in certain cases; b) prolong the kinetics of drug effect by destroying the protein, thus requiring resynthesis of the protein even after the compound has been metabolized; c) target ail functions of a protein at once rather than a specific catalytic activity or binding event; d) expand the number of drug targets by including all proteins that a ligand can be developed for, rather than proteins whose activity (e.g. , kinase activity) can be affected by a small molecule inhibitor, antagonist or agonist; and e) have increased potency compared to inhibitors due to the possibility of the small molecule acting catalytically.

Some embodiments of the present application relate to degradation or loss of 30% to 100% of the target protein. Some embodiments relate to the loss of 50-100% of the target protein. Other embodiments relate to the loss of 75-95% of the targeted protein.

A bifunctional compound of the present application (e.g. , a bifunctional compound of any of the formulae described herein, or selected from any bifunctional compounds described herein) is capable of modulating (e.g., decreasing) the amount of a targeted protein (e.g. , CD 4 and/or CDK6). A bifunctional compound of the present application (e.g. , a bifunctional compound of any of the formulae described herein, or selected from any bifunctional compounds described herein) is also capable of degrading a targeted protein (e.g. , CDK4 and/or CDK6) through the UPP pathway. Accordingly, a bifunctional compound of the present application (e.g. , a bifunctional compound of any of the formulae described herein, or selected from any bifunctional compounds described herein) is capable of treating or preventing a disease or disorder in which CDK4 and/or CDK6 plays a role. A bifunctional compound of the present application (e.g. , a bifunctional compound of any of the formulae described herein, or selected from any bifunctional compounds described herein) is also capable of treating or preventing a disease or disorder in which CDK4 and/or CDK6 play s a role or in which CDK4 and/or CDK6 is deregulated (e.g. , overexpressed).

Modulation of CDK.4 and/or CDK6 through UPP-mediated degradation by a bifunctional compound of the application, such as those described herein, provides a novel approach to the treatment, prevention, or amelioration of diseases or disorders in which CDK.4 and/or CDK6 plays a role including, but not limited to, cancer and metastasis, inflammation, arthritis, systemic lupus erthematosus, skin-related disorders, pulmonary disorders, cardiovascular disease, ischemia, neurodegenerative disorders, liver disease, gastrointestinal disorders, viral and bacterial infections, central nervous system disorders, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, and peripheral neuropathy. Further, modulation of CDK4 and/or CD 6 through UPP-mediated degradation by a Afunctional compound of the application, such as those described herein, also provides a new paradigm for treating, preventing, or ameliorating diseases or disorders in which CDK4 and/or CDK6 is deregulated.

In one embodiment, a bifunctionai compound of the present application (e.g. , a Afunctional compound of any of the formulae described herein, or selected from any bifunctionai compounds described herein) is more efficacious in treating a disease or condition (e.g. , cancer) than, or is capable of treating a disease or condition resistant to, the Targeting Ligand, when the Targeting Ligand is administered alone (i.e. , not bonded to a Linker and a Degron). In one embodiment, a bifunctionai compound of the present application (e.g. , a bifunctionai compound of any of the formulae described herein, or selected from any bifunctionai compounds described herein) is capable of modulating (e.g., decreasing) the amount of CDK4 and/or CDK6, and thus is useful in treating a disease or condition (e.g., cancer) in which CDK4 and/or CDK6 plays a role.

In one embodiment, the bifunctionai compound of the present application that is more efficacious in treating a disease or condition than, or is capable of treating a disease or condition resistant to, the Targeting Ligand, when the Targeting Ligand is administered alone (i.e., not bonded to a Linker and a Degron), is more potent in inhibiting the growth of cells (e.g. , cancer cells) or decreasing the viability of ceils (e.g., cancer cells), than the Targeting Ligand, when the Targeting Ligand is administered alone (i.e., not bonded to a Linker and a Degron). In one embodiment, the bifunctionai compound inhibits the growth of cells (e.g. , cancer cells) or decreases the viability of cells (e.g. , cancer cells) at an ICso that is lower than the ICso of the Targeting Ligand (when the Targeting Ligand is administered alone (i.e., not bonded to a Linker and a Degron)) for inhibiting the growth or decreasing the viability of the cells. In one embodiment, the ICso of the bifunctionai compound is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1 % of the ICso of the Targeting Ligand. In one embodiment, the ICso of the Afunctional compound is at most 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the ICso of the Targeting Ligand. In one embodiment, the ICso of the bifunctionai compound is at most 30%», 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0,2%, or 0.1% of the ICso of the Targeting Ligand. In one embodiment, the IC50 of the bifunctional compound is at most 10%, 8%, 5%», 4%, 3%, 2%, 1%, 0,8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the TCso of the Targeting Ligand. In one embodiment, the IC50 of the bifunctional compound is at most 5%, 4%, 3%, 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the IC50 of the Targeting Ligand. In one embodiment, the IC50 of the bifunctional compound is at most 2%, 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1 % of the IC50 of the Targeting Ligand. In one embodiment, the IC50 of the bifunctional compound is at most 1%, 0.8%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the ICso of the

Targeting Ligand. In one embodiment, the bifunctional compound inhibits the growth of cells (e.g. , cancer cells) or decreases the viabilit ' of cells (e.g. , cancer cells) at an Emax that is lower than the Emax of the Targeting Ligand (when the Targeting Ligand is administered alone (i.e. , not bonded to a Linker and a Degron)) for inhibiting the growth or decreasing the viability of the cells. In one embodiment, the Emax. of the bifunctional compound is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 5%, 4%, 3%, 2%, or 1% of the Emax of the Targeting Ligand. In one embodiment, the Emax of the bifunctional compound is at most 50%, 40%, 30%, 20%», 10%, 8%, 5%, 4%, 3%, 2%, or 1 % of the Emax of the Targeting Ligand. In one embodiment, the Emax of the bifunctional compound is at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the Emax of the Targeting Ligand.

In some embodiments, the inhibition of CDK4 and/or CDK6 activity is measured by

ICso.

In some embodiments, the inhibition of CDK4 and/or CDK6 activity is measured by

ECso.

Potency of the inhibitor can be determined by ECso value. A compound with a lower ECso value, as determined under substantially similar conditions, is a more potent inhibitor relative to a compound with a higher ECso value. In some embodiments, the substantially similar conditions comprise determining a CDK4-dependent phosphorylation level (e.g., in cells expressing a wild-type CD 4, a mutant CDK4, or a fragment of any thereof). In other embodiments, the substantially similar conditions comprise determining a CDK6-dependent phosphorylation level, in vitro or in vivo (e.g. , in ceils expressing a wild-type CDK6, a mutant CDK6, or a fragment of any thereof). In other embodiments, the substantially similar conditions comprise determining a CDK4-dependent phosphorylation level and a CDK6- dependent phosphorylation level, in vitro or in vivo (e.g. , in cells expressing a wild-type

CDK4 and/or CDK6, a mutant CDK4 and/or CDK6, or a fragment of any thereof).

Potency of the inhibitor can also be determined by ICso value. A compound with a lower ICso value, as determined under substantially similar conditions, is a more potent inhibitor relative to a compound with a higher IC50 value. In some embodiments, the substantially similar conditions comprise determining a CDK4-dependent phosphorylation level (e.g. , in cells expressing a wild-type CDK4, a mutant CDK4, or a fragment of any thereof). In other embodiments, the substantially similar conditions comprise determining a CD 6-dependent phosphorylation level, in vitro or in vivo (e.g., in cells expressing a wild- type CDK6, a mutant CDK6, or a fragment of any thereof). In other embodiments, the substantially similar conditions comprise determining a CDK4-dependent phosphorylation level and a CDK6-dependent phosphorylation level, in vitro or in vivo (e.g. , in cells expressing a wild-type CDK4 and/or CDK6, a mutant CDK4 and/or CDK6, or a fragment of any thereof).

In one embodiment, the bifunctional compounds of the present application are useful as anticancer agents, and thus may be useful in the treatment of cancer, by effecting tumor cell death or inhibiting the growth of tumor cells. In certain exemplary embodiments, the disclosed anticancer agents are useful in the treatment of cancers and other proliferative disorders, including, but not limited to breast cancer, cervical cancer, colon and rectal cancer, leukemia, lung cancer (e.g. , non-small cell lung cancer), melanoma, multiple myeloma, non- Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, leukemias (e.g. , myeloid, lymphocytic, myelocytic and lymphoblastic leukemias), malignant melanomas, and T-cell lymphoma.

A "selective CDK4 inhibitor," can be identified, for example, by comparing the ability of a compound to inhibit CDK4 kinase activity to its ability to inhibit the other members of the CDK kinase family or other kinases. For example, a substance may be assayed for its ability to inhibit CD 4 kinase activity, as well as CDK 1 , CDK2, CDK6, CDK 7 CDK8, CDK9, CDK 11, CDK.12, CDK.13, CDK 14, and other kinases. In some embodiments, the selectivity can be identified by measuring the EC so or ICso of the compounds.

In some embodiments, the bifunctional compounds of the present application containing a Target Ligand inhibit CDK4 more selectively over other cyclin-dependent kinases and/or other kinases than the Target Ligand alone (i.e. , a Target Ligand itself compared to the Target Ligand covalently bound to a Linker and a Degron). In certain embodiments, the bifunction al compounds of the application are about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99%) more selective at inhibiting CDK4 than the Target Ligand alone. In certain

embodiments, the bifunctional compounds of the application are about 10%, about 20%, about 30%, about 40%, or about 50%) more selective at inhibiting CDK4 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 20%, about 30%, about 40%, about 50% or about 60% more selective at inhibiting CDK4 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 30%, about 40%, about 50%), about 60% or about 70% more selective at inhibiting CDK4 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 40%, about 50%, about 60%, about 70%», or about 80% more selective at inhibiting CD 4 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 50%, about 60%, about 70%, about 80%, or about 90% more selective at inhibiting CDK4 than the

Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 60%, about 70%, about 80%, about 90%, or about 99% more selective at inhibiting CDK4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%), at least 90%, or at least 99% more selective at inhibiting CDK4 than the Target Ligand alone.

In other embodiments, the bifunctional compounds of the application are between about 10% and about 99% more selective at inhibiting CD 4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 10% and about 30% more selective at inhibiting CDK.4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 20% and about 40% more selective at inhibiting CDK4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 30% and about 50% more selective at inhibiting CDK.4 tha the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 40% and about 60% more selective at inhibiting CDK4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 50% and about 70% more selective at inhibiting CDK.4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 60% and about 80% more selective at inhibiting CDK4 than the Target Ligand alone. In other embodiments, the bifunction al compounds of the application are between about 70% and about 90% more selective at inhibiting CDK4 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 80% and about 99% more selective at inhibiting CDK4 than the Target Ligand alone. In some embodiments, the compounds of the present application are selective over other kinases. As used herein, "selective", "selective CDK4 inhibitor", or "selective CDK4 compound" refers to a compound, for example a bifunctional compound of the application, that effectively inhibits€D 4 kinase to a greater extent than any other kinase enzyme, particularly any enzyme from the Cyclic-dependent kinase family (e.g. , CDK1, CDK2, C.DK6, CDK7, CDK.8, C.DK9, CDKl l , CDK 12, CDKLL C.DK14, eta ).

In certain embodiments, the compounds of the application are CD 4 inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other kinases (e.g. , CDK1, CDK2, CD 6, CDK7, CDK.8, CDK9, CDK1 1 , CDK12, CDK.13, CDK.i etc.). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other kinases.

In certain embodiments, the compounds of the application are CDK4 inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other cyclin-dependent kinases (e.g., CDKL CDK2, CDK6, CDK7, CDKS, CDK9, CDKl l, CDK12, CDK13, CDK14, etc.). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other cyclin-dependent kinases.

A "selective CDK6 inhibitor," can be identified, for example, by comparing the ability of a compound to inhibit CDK6 kinase activity to its ability to inhibit the other members of the CDK kinase family or other kinases. For example, a substance may be assayed for its ability to inhibit CDK.6 kinase activity, as well as CDKL CDK2, CDK.4, C.DK7, CDKS, CDK.9, CDK1 1 , C.DK12, CDK.1 , CDKLL and other kinases, in some embodiments, the selectivity can be identified by measuring the ECso or ICso of the compounds.

In some embodiments, the bifunctional compounds of the present application containing a Target Ligand inhibit CDK6 more selectively over other cyclin-dependent kinases and/or other kinases than the Target Ligand alone (i.e. , a Target Ligand itself compared to the Target Ligand covalently bound to a Linker and a Degron). In certain embodiments, the bifunctional compounds of the applicati on are about 10%, about 20%, about 30%, about 40%, about 50%», about 60%, about 70%, about 80%, about 90%. or about 99% more selective at inhibiting CDK.6 than the Target Ligand alone. In certain

embodi ments, the bifuncti onal compounds of the appli cation are about 10%, about 20%, about 30%, about 40%, or about 50% more selective at inhibiting CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 20%, about 30%, about 40%, about 50% or about 60% more selective at inhibiting CDK6 than the Target Ligand alone. In certain embodiments, the biiunctional compounds of the application are about 30%, about 40%, about 50%, about 60% or about 70% more selective at inhibiting CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 40%, about 50%, about 60%, about 70%, or about 80% more selective at inhibiting CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 50%, about 60%, about 70%, about 80%, or about 90% more selective at inhibiting CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 60%, about 70%, about 80%, about 90%, or about 99% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% more selective at inhibiting CDK6 than the Target Ligand alone.

In other embodiments, the bifunctional compounds of the application are between about 10% and about 99% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 10% and about 30% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 20% and about 40% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 30% and about 50% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 40% and about 60% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodi ments, the bifuncti onal compounds of the appli cation are between about 50% and about 70% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 60% and about 80% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the applicati on are between about 70% and about 90% more selective at inhibiting CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 80% and about 99% more selective at inhibiting CDK6 tha the Target Ligand alone.

In some embodiments, the compounds of the present application are selective over other kinases. As used herein, '"selective", "selective CDK6 inhibitor', or "selective CDK6 compound" refers to a compound, for example a bifunctional compound of the application, that effectively inhibits CDK.6 kinase to a greater extent than any other kinase enzyme, particularly any enzyme from the Cyclic-dependent kinase family (e.g. , CD 1 , CDK2, CDKA CD 7, CDK8, CDK9, CDK1 L C.DK.12, CDK1 3, CDK 14, etc.).

In certain embodiments, the compounds of the application are CD 6 inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other kinases (e.g. , CDKL CDK2, CDK4, CD .7, C.DK8, CDK9, CD I L CD .12, CDK] 3, CDK14, etc. ). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other kinases.

In certain embodiments, the compounds of the application are CDK6 inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other cyclin-dependent kinases (e.g., CD 1, CDK2, CDK4, CDK7, CD 8, CD 9, CDK11, CDK12, CDK S 3, CDK.I4, etc.). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other cyclin-dependent kinases.

A ^" 'selective CDK4 and CDK6 inhibitor" or "selective CDK 4/6 inhibitor," can be identified, for example, by comparing the ability of a compound to inhibit CDK.4 and CDK.6 kinase activity to its ability to inhibit the other members of the CDK kinase family or other kinases. For example, a substance may be assayed for its ability to inhibit CDK4 and CDK6 kinase activity, as well as CDK1, CDK2, CDK7, CDK8, CDK9, CD 11, CDK12, CDK13, CDK14, and other kinases. In some embodiments, the selectivity can be identified by measuring the EC50 or IC50 of the compounds.

In some embodiments, the bifunctional compounds of the present application containing a Target Ligand inhibit CDK4 and CDK6 more selectively over other cyclin- dependent kinases and/or other kinases than the Target Ligand alone (i.e. , a Target Ligand itself compared to the Target Ligand covalently bound to a Linker a d a Degron). In certain embodiments, the bifunctional compounds of the application are about 10%, about 20%, about 30%», about 40%, about 50%, about 60%, about 70%», about 80%. about 90% or about 99% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 10%, about 20%, about 30%, about 40%, or about 50%) more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 20%, about 30%, about 40%, about 50% or about 60% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 30%, about 40%», about 50%), about 60% or about 70% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 40%, about 50%, about 60%, about 70%, or about 80% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In certain embodiments, the bifimctional compounds of the application are about 50%, about 60%, about 70%, about 80%, or about 90% more selective at inhibiting CD 4 and CDK6 than the Target Ligand alone. In certain embodiments, the bifunctional compounds of the application are about 60%, about 70%, about 80%, about 90%, or about 99% more selective at inhibiting CDK4 and CD 6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone.

In other embodiments, the bifunctional compounds of the application are between about 10% and about 99% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 10% and about 30% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 20% and about 40% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 30% and about 50% more selective at inhibiting CDK4 and CD 6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 40% and about 60% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 50% and about 70% more selective at inhibiting CD 4 and CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 60% and about 80% more selective at inhibiting CDK4 AND CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 70% and about 90% more selective at inhibiting CDK.4 and CDK6 than the Target Ligand alone. In other embodiments, the bifunctional compounds of the application are between about 80% and about 99% more selective at inhibiting CDK4 and CDK6 than the Target Ligand alone.

In some embodiments, the compounds of the present application are selective over other kinases. As used herein, "selective", "selective CDK4 and CDK6 inhibitor", or "selective CD 4 and CDK6 compound" refers to a compound, for example a bifunctional compound of the application, that effectively inhibits CDK4 and CDK6 kinase to a greater extent than any other kinase enzyme, particularly any enzyme from the Cyclic-dependent kinase faintly (e.g. , CD 1 , CDK2, CDK7, C D 8, CDK9, CD I L CD 12, CD 1 3,

In certain embodiments, the compounds of the application are CDK4 and CDK6 inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other kinases (e.g. , CDK1 , CDK2, CDK.7, CDK8, CDK9, CDK12, CDK.13, etc. ). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other kinases.

In certain embodiments, the compounds of the application are CDK4 and CDK6 inhibitors that exhibit at least 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold selectivity over other cy din-dependent kinases (e.g. , CDKL CDK2, CDK7, C Dk X . CDK9, CD l L CDK12, CDK13, CD 14, etc. ). In various embodiments, the compounds of the application exhibit 1000-fold selectivity over other cy elm-dependent kinases.

Definitions

Listed below are definitions of various terms used in this application. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

The term "aikyl," as used herein, refers to saturated, straight or branched-chain hydrocarbon radicals containing, in certain embodiments, between one and six carbon atoms. Examples of Ci-Ce aikyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyi, n-butyl, tert-hutyl, neopentyl, and n-hexyl radicals.

The term "alkenyi," as used herein, denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Alkenyi groups include, but are not limited to, for example, ethenyl, propenyl, butenyi, l -methyl-2-buten-l-yl and the like.

The term "alkoxy" refers to an -O-alkyl radical.

The terms "hal," "halo," and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.

The term "aryl," as used herein, refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.

The term "aralkyi," as used herein, refers to an aikyl residue attached to an aryl ring. Examples include, but are not limited to, benzyl, phenethyl and the like. The term "cycloalkyl," as used herein, denotes a monovalent group derived from a monocyclic or poly cyclic saturated or partially unsaturated carbocyclic ring compound.

Examples of Cs-Cs cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl, cyciopentyl, cyclohexyl, cyclopentyl and cyciooctyl; and examples of C3-Cj 2-eycloalkyi include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1 j heptyl, and bicyclo [2.2.2] octyl. Also contemplated is a monovalent group derived from a monocyclic or poly cyclic carbocyclic ring compound having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Examples of such groups include, but are not limited to, cyclopropenyl, cycl obutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term "heteroaryl," as used herein, refers to a mono- or poly-cyclic (e.g. , bi-, or tricyclic or more) fused or non-fused, radical or ring system having at least one aromatic ring, having from five to ten ring atoms of which one ring atoms is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyi, pyrazolyl, imidazolyl, thiazolyl, oxazolyi, isooxazolyi, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxaimyl, and the like.

The term "heteroaraikyl," as used herein, refers to an alkyl residue attached to a heteroaryl ring. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term "heterocyclyl," or "heterocycloalkyl," as used herein, refers to a non- aromatic 3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused of non-fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii) each 5-nienibered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, and (iv) the nitrogen heteroatom may optionally be quaternized. Representative heterocycloalkyl groups include, but are not limited to, [i,3]dioxolane, pyrrolidinyl, pyrazolinyi, pyrazolidinyl, imidazolinyi, imidazolidinyl, piperidinyi, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidmy , isothiazolidinyl, and tetrahydrofuryi.

The term "alkylamino" refers to a group having the structure -NH(Ci-Ci2 alkyl), e.g., - NH(Ci-Cf, alky I s. where CVC 12 alkyl is as previously defined.

The term "dialkylamino" refers to a group having the structure -N(Ci-Ci2 alkyl)?,, e.g. , -NH(Ci-Ce alkyl), where Ci-Ci2 alkyl is as previously defined. The term "acyl" includes residues derived from acids, including but not limited to carboxylic acids, carbamic acids, carbonic acids, sulfonic acids, and phosphorous acids. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfmyls, aliphatic sulfmyls, aromatic phosphates and aliphatic phosphates. Examples of aliphatic carbonyls include, but are not limited to, acetyl, propionyl, 2-fluoroacetyl, butyryl, 2-hydroxy acetyl, and the like.

In accordance with the application, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted.

The terms "ha! . ^" "halo," and "halogen," as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.

As described herein, compounds of the application may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by- particular classes, subclasses, and species of the application. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." In general, the term "substituted", whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The terms "optionally substituted", "optionally substituted alkyl," "optionally substituted

"optionally substituted alkenyl," "optionally substituted alkynyl", "optionally substituted cycloalkyl," "optionally substituted cycloalkenyl," "optionally substituted aryl", "optionally substituted heteroaryi," "optionally substituted aralkyl", "optionally substituted heteroaralkyl," "optionally substituted heterocycloalkyl," and any other optionally substituted group as used herein, refer to groups that are substituted or unsubstituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to:

-F, -CI, -Br, -I, -OH, protected hydroxy, -NO2, -CN, -N i b. protected

amino, -NH-Ci-Ci ₂ -alkyl, -NH-C2-Ci2-alkenyl, -NH-C ₂ -C i ₂ -alkenyl, -NH -C3-Ci2-cycloa3kyl, -NH-aryl, -NH -heteroaryi, -NH -heterocycloalkyl, -dialkylamino, -diaryiamino,

-diheteroaryl amino, -O-Ci-C 12-alkyl, -O-C2-C 12-alkeny 1, -O-C2-C 12-alkeny 1, -0-C3-Ci2-cycloaikyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C(0)-Ci-C 32-alkyi, - C(O)- C2-Ci2-alkeny], -C(0)-C ₂ -C ₁₂ -a]kenyl, ~C(0)~C3~Ci2-cycloalkyl, -CiO in l . -C(O)- heteroaryl,

-C(0)-heterocycloaikyl, -CONH2, -CONH-C]-Ci ₂ -alkyi, -CONH-C ₂ -C _!2 -alkenyl,

-CONH-C ₂ -Ci ₂ -alkenyl, -CONH-Cs-Cu-cycloalkyl, -(ΌΝΠ-aryl. -CONH-heteroaiyl,

-CONH-heterocycloalkyl,-OC02-Ci-Ci2-alkyL -OC02-C2-Ci2-alkenyl, -OCG2-C2-C12- alkenyl,

-OC0 ₂ -C3-Ci ₂ -cycloalkyl, -OC0 ₂ -aiyl, -OC0 ₂ -heteroar l, -OCC -heterocycloalkyf, - OCONH2,

-OCONH-Ci-Cn-alkyl, -OCONH- C ₂ -Ci2-alkenyL -OCONH- C ₂ -C ₁₂ -a3kenyl,

-OCONH-C3-Ci ₂ -cycloalkyl, -OCONH-aiyl, -QCQNH-heteroaryL -OCONH- heterocycloalkyl,

-NHC(0)-Ci-Ci2-alkyL -NHC(0)-C2-Ci2-alkenyl, -NHC(0)-C ₂ -Ci2-alkenyl,

-NHC(0)-C3-Ci ₂ -cycloalkyl, -NHC(0)-aiyl, -NHC(0)-heteroaiyl, -NHC(0)- heterocycloalkyl,

-NHC0 ₂ -C ₁ -Ci ₂ -alkyl, -NHC0 ₂ -C ₂ -C ₁₂ -alkenyl, -NHC0 ₂ -C ₂ -Ci ₂ -alkenyl,

-NHC0 ₂ -C3-Ci2-cycloalkyl, -NHC0 ₂ -aryl, -NHC0 ₂ -heteroaryl, -M ICO - heterocycloalkyl, NHC(0) H ₂ , -NHC(0)NH-Ci-Ci ₂ -alkyl, -NHC(0)NH-C ₂ -C] ₂ -alkenyl,

-NHC(())NH-C2-Ci2-aIkenyl, -NHC(0)NH-C3-Ci2-cycioalkyl, -NHC(0)NH-aryl,

-NHC(0)NH-heteroaryl, NHC(0)NH-heterocydoalkyl, -NHC(S)NH ₂ ,

-NHC(S)NH-Ci-Ci2-alkyl, -NHC(S)NH-C2-Ci2-alkenyl,

-NHC(S)NH-C2-Ci2-alkenyl, -NHC(S)NH-C3-Ci2-cycloalkyl, -NHC (S )NH-ary 1,

-NHC(S)NH-heteroaiyl, -NHC(S)NH-heterocycloa]kyl ₅ -NHC(NH)NH ₂ ,

-NHC(NH)NH- C i-C _i2 ~al ky 1, -NHC(NH)NH-C ₂ -C 12-alkeny 1, -NHC(NH)NH-C ₂ -C 12-alkeny L ~NHC(NH)NH-C3-C]2-cycloalkyL -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl,

-NHC(NH)NHheterocycloalkyl, -NHC(NH)-C ₂ -Ci ₂ -alkenyl, -NHC(NH)-C ₂ -Ci2-alkenyl, -NHC(NH)-C3-Ci ₂ -cycloalkyl, -NHC(NH)-aryl,

-NHC(NH)-heteroai 'l, -NHC(NH)-heterocycloalkyl, -C(NH)NH-Ci-Ci ₂ -alkyl,

-C(NH)NH-C2-Ci2-alkenyl, -C(NH)NH-C ₂ -Ci ₂ -aikenyI, C(NH)NH-C3-Ci2-cycloalkyl, ~C(NH)NH-aryl, -C(NH)NH-heteroaryl, -( ( M DM lhcies ocx cloaikvL

-S(0)-Ci-Ci ₂ -alkyl,- S(0)-C ₂ -Ci ₂ -alkenyl,- S(0)-C2-C] ₂ -alkenyl,

-S(0)-C3-C] ₂ -cycloalkyl,- S(0)-aryl, -S(0)-heteroaiyl, -S(0)-heterocycloalkyl -SO2NH2, -S02NH-Ci-Ci2-aikyl, -SO-Ni !··( · ^■ ··( · -a! kens !. -SOAH-C '-C i.'-al keiivi.

-SO2NH-C3-C 12-cycloalkyj, -S02NH-aiyl, -S02NH-heteroaryi, -S0 ₂ NH-heterocycloalkyl, -NHSG ₂ -Ci-Ci2-alkyf, -NHS0 ₂ -C2-Ci2-alkenyl,- NHS0 ₂ -C2-Ci2-alkenyl,

-NHS0 ₂ -C3-Ci2-cycloa]kyl, -NHS0 ₂ -aryl, -NHSC -heteroaryl, -NHS0 ₂ -heterocycloalkyL -CH ₂ NH ₂ , -CH2SG2CH3, -atyi, -arylalkyl, -heteroaryl, -heteroaiylalkyi, -heterocycloalkyl, -C3-Ci2-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-Ci-Ci ₂ -alkyl, -S-C ₂ -Ci ₂ -alkenyl, -S-C ₂ -Ci ₂ -alkenyl, -S-C3-Ci ₂ -cycloalkyl, -S-aryl, -S-heteroar l, -S-heterocycloalkyl, or methylthiomethyl.

It is understood that the aryls, heteroaryls, alkyls, and the like can be substituted. The term "cancer" includes, but is not limited to, the following cancers: epidermoid Oral: buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung: bronchogenic carcinoma (squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous ceil carcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines

(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, coion- rectum, colorectal, rectum; Genitourinar ' tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional ceil carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages; Bone: osteogenic sarcoma

(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant ceil tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous

cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa- thecal ceil tumors, Sertoii-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, m eloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma) hairy cell; lymphoid disorders; Skm: malignant melanoma, basal ceil carcinoma, squamous cell carcinoma,

Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma,

dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions.

The term "CDK4" herein refers to cyclin-dependent kinase 4.

The term "CDK6" herein refers to cyclin-dependent kinase 6.

The term "subject" as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

"Treat", "treating" and "treatment" refer to a method of alleviating or abating a disease and/or its attendant symptoms.

As used herein, " ^• preventing" or "prevent" describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder.

The term "targeted protein(s)" is used interchangeably with "target protein(s)", unless the context clearly dictates otherwise. In one embodiment, a "targeted protein" is CD .

The term "subject" as used herein refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

The terms "disease(s)", "disorder(s)", and "condition(s)" are used interchangeably, unless the context clearly dictates otherwise. The term "therapeutically effective amount" of a bifunctional compound or pharmaceutical composition of the application, as used herein, means a sufficient amount of the bifunctional compound or pharmaceutical composition so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts a therapeutically effective amount of a bifunctional compo und or pharmaceutical composition of this application will be at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present application w ill be decided by the attending physician within the scope of sound medical judgment. The specific inhibitor}' dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; 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 specific compound employed; and like factors well known in the medical arts.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts of the compounds formed by the process of the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are

commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the application, or separately by reacting the free base or acid function with a suitable acid or base.

Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts: salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Other

pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,

camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate,

ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate,

hemisulfate, heptanoate, hexanoate, hydroiodide, 2 -hydroxy -ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanes ulfon ate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pi crate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, /7-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

As used herein, the term "pharmaceutically acceptable ester" refers to esters of the bi functional compounds formed by the process of the present application which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycioalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethyl succinates.

The term "pharmaceutically acceptable prodrugs" as used herein, refers to those prodrugs of the bifimctionai compounds formed by the process of the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwittenonie forms, where possible, of the compounds of the present application. "Prodrug", as used herein, means a compound which is convertible in vivo by metabolic means (e.g. , by hydrolysis) to afford any compound delineated by the formulae of the instant application. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et ai. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 1 13-191 (1991 ); Bundgaard, et al, Journal of Drug Deliver Reviews, 8: 1-38(1992); B undgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology," John Wiley and Sons, Ltd. (2002). This application also encompasses pharmaceutical compositions containing, and methods of treating disorders through administering, pharmaceutically acceptable prodrugs of bifunctional compounds of the application. For example, compounds of the application having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of the application. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma- aminobutyric acid, citrullme, homocysteine, homosenne, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alky! esters. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylammoacetates, and phosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 1 15. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyi and (acyloxy)ethyl ethers wherein the acyl group may be an alkyi ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in J. Med. Chem. 996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.

The application also provides for a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound of the application, or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable earner.

In another aspect, the application provides a kit comprising a bifunctional compound capable of inhibiting CD 4 activity selected from one or more compounds disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, optionally in combination with a second agent and instructions for use in treating cancer. In another aspect, the application provides a kit comprising a hifunctionai compound capable of inhibiting CDK6 activity selected from one or more compounds disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, optionally in combination with a second agent and instructions for use in treating cancer.

In another aspect, the application provides a kit comprising a bifunctional compound capable of inliibiting the activity CDK4 and/or CDK6 selected from one or more compounds disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, optionally in combination with a second agent and instructions for use in treating cancer.

In another aspect, the application provides a method of synthesizing a bifunctional compound disclosed herein.

The synthesis of the bifunctional compounds of the application can be found herein and in the Examples below.

Other embodiments are a method of making a bifunctional compound of any of the formulae herein using any one, or combination of, reactions delineated herein. The method can include the use of one or more intermediates or chemical reagents delineated herein.

Another aspect is an isotopicaily labeled bifunctional compound of any of the formulae delineated herein. Such compounds have one or more isotope atoms which may or may not be radioactive (e.g., ³ H, ² H, ¹⁴ C, ¹³ C, ¹⁸ F, ³⁵ S, ³² P, ¹²⁵ I, and ¹³¹ I) introduced into the bifunctional compound. Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.

A bifunctional compound of the application can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a bifunctional compound of the application can be prepared by reacting the free acid form of the bifunctional compound with a pharmaceutically acceptable inorganic or organic base.

Alternatively, the salt forms of the bifunctional compounds of the application can be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the bifunctional compounds of the application can be prepared from the corresponding base addition salt or acid addition salt from, respectively. For example, a bifunctional compound of the application in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g. , ammonium hydroxide solution, sodium hydroxide, and the like). A Afunctional compound of the application in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g. , hydrochloric acid, etc.).

Prodrugs of the Afunctional compounds of the application can be prepared by- methods known to those of ordinary skill in the art (e.g. , for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non-denvatized bifunctional compound of the application with a suitable carbamylating agent (e.g., 1,1-acyioxyaikylcarbanochloridate, para-nitrophenyl carbonate, or the like).

Protected derivatives of the bifunctional compounds of the application can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W.

Greene, "Protecting Groups in Organic Chemistry", 3rd edition, John Wiley and Sons, Inc., 1999.

Compounds of the present application can be conveniently prepared, or formed during the process of the application, as solvates (e.g. , hydrates). Hydrates of bifunctional

compounds of the present application can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxm, tetrahydrofuran or methanol.

Acids and bases useful in the methods herein are known in the art. Acid catalysts are any acidic chemical, which can be inorganic (e.g. , hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic (e.g. , camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium inflate) in nature. Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases are any basic chemical, which can be inorganic (e.g., sodium bicarbonate, potassium hydroxide) or organic (e.g. , triethylamine, pyridine) in nature. Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.

Combinations of substituents and variables envisioned by this application are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g. , therapeutic or prophylactic administration to a subject).

When any variable (e.g. , Rw) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more Ri moieties, then Ri ₄ at each occurrence is selected independently from the definition of R14. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds within a designated atom's normal valency.

In addition, some of the compounds of this application have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z- double isomeric forms, and other stereoisomenc forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)~, or as (D)~ or (L)- for amino acids. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be eis, trans, or a mixture of the two in any proportion. All such isomeric forms of such compounds are expressly included in the present application.

Optical isomers may be prepared from their respective optically active precursors by the procedures described herein, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated

crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al. , Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981).

"Isomerism" means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers".

Stereoisomers that are not mirror images of one another are termed "diastereoisomers", and stereoisomers that are non-superimposable mirror images of each other are termed

"enantiomers" or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chiraiity is termed a "racemic mixture".

A carbon atom bonded to four non-identical substituents is termed a "chiral center".

"Chiral isomer" means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed "diastereomeric mixture". When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Calm et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511 : ( ^' arm et al., Angew. Chem. 1966, 78, 413; Calm and Ingold, J. Chem. Soc. 1951 (London), 612; Calm et al., Expenentia 1956, 12, 81; Calm, J. Chem. Educ. 1964, 41 , 1 16).

"Geometric isomer" means the diastereomers that owe their existence to hindered rotation about double bonds. These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.

Furthermore, the structures and other compounds discussed in this application include all atropic isomers thereof. "Atropic isomers" are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques; it has been possible to separate mixtures of two atropic isomers in select cases.

^" Tautomer" is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solid form, usually one tautomer predominates. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs. Ring- chain tautomerism arises as a result of the aldehyde group (-CHO) in a sugar chain molecule reacting with one of the hydroxy groups (-OH) in the same molecule to give it a cyclic (ring- shaped) form as exhibited by glucose. Common tautomeric pairs are: ketone-enol, amide- nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings {e.g. , in nucleobases such as guanine, thymine and cytosine), amine-enamine and enamine-enamine. The compounds of this application may also be represented in multiple tautomeric forms, in such instances, the application expressly includes all tautomeric forms of the compounds described herein (e.g. , aikylation of a ring system may result in alkylation at multiple sites, the application expressly includes all such reaction products).

In the present application, the structural formula of the bifunctional compound represents a certain isomer for convenience in some cases, but the present application includes ail isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like. In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present application includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like.

Additionally, the compounds of the present application, for example, the salts of the bifunctional compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

"Solvate" means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate: and if the solvent is alcohol, the solvate formed is an alcohoiate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O.

The synthesized bifunctional compounds can be separated from a reaction mixture and further pun i led by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the bifunctional compounds of the formulae herein will be evident to those of ordinal}' skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction durations, etc. delineated herein are for purposes of illustration only and one of ordinary skill in the art will recognize that variation of the reaction conditions can produce the desired bridged macrocyclic products of the present application. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations,

VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic

Synthesis, 2d. Ed., John Wiley and Sons (199.1 ); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,

Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

The compounds of this application may be modified by appending various functionalities via any synthetic means delineated herein to enhance selecti ve biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g. , blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

The compounds of the application are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Method of Synthesizing the Compounds

Compounds of the present application can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. B., March, J., March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 ^th edition, John Wiley & Sons; New York, 2001; and Greene, T.W., Wuts, P.G. M., Protective Groups in Organic Synthesis, 3 ^rd edition, John Wiley & Sons: New York, 1999, incorporated by reference herein, are useful and recognized reference textbooks of organic synthesis known to those in the art. The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present application. The processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, ester or prodrug thereof. Suitable synthetic routes are depicted in the schemes below.

Those skilled in the art will recognize if a stereocenter exists in the compounds disclosed herein. Accordingly, the present application includes both possible stereoisomers (unless specified in the synthesis) and includes not only racemic compounds but the individual enantiomers and/or diastereomers as well. When a compound is desired as a single enantiomer or diastereomer, it may be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, an intermediate, or a starting material may be affected by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by E, L. Eliel,

S. H. Wilen, and L. N. Mander (Wiley -lnterscience, 1994).

The compounds of the present application can be prepared in a number of ways well kn own to those skilled in the art of organic synthesis. By way of example, compounds of the present application can be synthesized using the methods described below, together with sy nthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Preferred methods include but are not limited to those methods described below.

Compounds of the present application can be synthesized by following the steps outlined in General Scheme 1 which comprise different sequences of assembling

intermediates la, lb, Ic, Id, le, If, and Ig. Starting materials are either commercially available or made by known procedures in the reported literature or as illustrated.

wherein RB, RU, RJ S, Rie, W, pi, q, and v are as defined herein above.

The general way of preparing representati v e compounds of the present application (i. e. , Compound of Formula (I) shown above) using intermediates la, lb, le, Id, le, If, and Ig is outlined in General Scheme 1. Reaction of la with lb in the presence of a acetic acid/potassium acetate, optionally in a solvent, i.e. , tetrahydrofuran (THF) provides intermediate lc. Nucleophilic addition of Id to fluoride le in the presence of a base, i.e. , N,N-diisopropylethylamine (DIPEA), and in a solvent i.e. , dimethylformamide (DMF), provides intermediate le. Deprotection of le using a strong acid, i.e. , trifluoroacetic acid (T ^' FA) or hydrochloric acid (HC1), in a solvent, i. e. , dichloromethane (DCM) or dioxane, provides carboxyiic acid If. Coupling of acid If and Target Ligand Ig under standard coupling conditions using a coupling reagent, i.e. , l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and hydroxybenzotriazole, in a solvent, i.e. , DCM or DMF, provides bifunctionai compound of formula (I).

Biological Assays

Enzyme Degradation Assay

Wild-type or cereblon null cells are treated with a control or a bifunctionai compound of the application. After treatment, cells are washed and harvested by resuspending in buffer and lysed on ice 30 minutes. Lysaies are then cleared by centrifugation. Samples are boiled and equal amount of protein is loaded onto polyacrylamide gel. The gel is transferred to nitrocellulose and blotted for CDK6, CDK4 or Tubulin.

Western Blotting on CDK4/6

Cells are treated with a control or a bifunctionai compound of the application at various concentrations for a desired period of time. Cells are then lysed in a suitable buffer. Protein concentration may be measured with any appropriate assay known in the art.

Equivalent amounts of the samples are loaded on a polyacrylamide gel, transferred to nitrocellulose membranes, and immunoblotted with antibodies against CDK4 and CDK6 and a loading control, such as actin. Labeled secondary antibodies are added and washed. The signals from the label are detected.

Methods of the Application

Another aspect of the application provides a method of modulating a kinase, comprising contacting the kinase with a bifunctionai compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or with a pharmaceutical composition disclosed herein. In some embodiments, the kinase is CDK4. In other embodiments, the kinase is CDK6. In other embodiments, the kinase is CDK4 and CD 6.

In another aspect, the application provides a method of inhibiting a kinase, comprising contacting the kinase with a bifunctionai compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, or with a pharmaceutical composition disclosed herein. In some embodiments, the kinase is CDK4. In other embodiments, the kinase is CDK6. In other embodiments, the kinase is CDK4 and CDK6.

In another aspect, the application provides a method of inhibiting a kinase, the method comprising administering to a subject in need thereof an effective amount of a bifunctionai compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the kinase is CDK4. In other embodiments, the kinase is CDK6. In other embodiments, the kinase is CDK4 and CDK6.

In still another aspect, the application provides a method of modulating cy elm- dependent kinase 4 (CDK4), the method comprising administering to a subject in need thereof an effective amount of a bifunctionai compound disclosed herein, or a

pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In still another aspect, the application provides a method of modulating cyclin- dependent kinase 6 (CD 6), the method comprising administering to a subject in need thereof an effective amount of a bifunctionai compound disclosed herein, or a

pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In still another aspect, the application provides a method of modulating c clin- dependent kinase 4 (CDK4) and cycl in-dependent kinase 6 (CD 6), the method comprising administering to a subject in need thereof an effective amount of a bifunctionai compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug,

stereoisomer, or tautomer thereof.

In still another aspect, the application provides a method of modulating cycl independent kinase 4 (CDK4), the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctionai compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

In still another aspect, the application provides a method of modulating cy din- dependent kinase 6 (CD 6), the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctionai compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

In still another aspect, the application pro-, ides a method of modulating cyclin- dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6), the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a

pharmaceutically acceptable carrier.

Another aspect of the application provides a method of treating or preventing a disease, the method comprising administering to a subject in need thereof an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the disease is mediated by a kinase. In other embodiments, the kinase is CDK4. In other embodiments, the kinase is CDK6. In other embodiments, the kinase is CDK4 and CDK6.

Another aspect of the application provides a method of treating or preventing a disease, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier. In some embodiments, the disease is mediated by a kinase. In other embodiments, the kinase is CDK4. In other embodiments, the kinase is CDK6. In other embodiments, the kinase is CDK4 and CDK6.

In some embodiments, the disease is mediated by CDK4 (e.g. , CD 4 plays a role in the initiation or development of the disease). In other embodiments, the disease is mediated by CDK6 (e.g., CDK6 plays a role in the initiation or development of the disease). In other embodiments, the disease is mediated by CDK4 and CDK.6 (e.g. , CDK4 and CDK6 play a role in the initiation or development of the disease).

In certain embodiments, the disease or disorder is cancer or a proliferation disease. In further embodiments, the disease or disorder is lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas, myelomas, or solid tumors.

In other embodiments, the disease or disorder is inflammation, arthritis, rheumatoid arthritis, spondyiarthropathies, gouty arthritis, osteoarthritis, juvenile arthritis, and other arthritic conditions, systemic lupus erthematosus (SLE), skin-related conditions, psoriasis, eczema, bums, dermatitis, neuroinflammation, allergy, pain, neuropathic pain, fever, pulmonary disorders, lung inflammation, adult respiratory distress syndrome, pulmonary sarcoisosis, asthma, silicosis, chronic pulmonary inflammatory disease, and chronic obstructive pulmonary disease (COPD), cardiovascular disease, arteriosclerosis, myocardial infarction (including post-myocardial infarction indications), thrombosis, congestive heart failure, cardiac reperfusion injury, as well as complications associated with hypertension and/or heart failure such as vascular organ damage, restenosis, cardiomyopathy, stroke including ischemic and hemorrhagic stroke, reperfusion injury, renal reperfusion inj ury, ischemia including stroke and brain ischemia, and ischemia resulting from cardiac/coronary bypass, neurodegenerative disorders, liver disease and nephritis, gastrointestinal conditions, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viral and bacterial infections, sepsis, septic shock, gram negative sepsis, malaria, meningitis, HIV infection, opportunistic infections, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), pneumonia, herpes virus, myalgias due to infection, influenza, autoimmune disease, graft vs. host reaction and allograft rejections, treatment of bone resorption diseases, osteoporosis, multiple sclerosis, cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bone cancer, epithelial call-derived neoplasia (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small bowel cancer, stomach cancer, colon cancer, liver cancer, bladder cancer, pancreas cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell and/or basal cell cancers, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body, chronic myelogenous leukemia (CML), acute myeloid leukemia (AMI.,) and acute promyelocyte leukemia (APL), angiogenesis including neoplasia, metastasis, central nervous system disorders, central nervous system disorders having an inflammatory or apoptotic component, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, and peripheral neuropathy, or B-Ceil Lymphoma.

In further embodiments, the disease or disorder is inflammation, arthritis, rheumatoid arthritis, spondylarthropathies, gouty arthritis, osteoarthritis, juvenile arthritis, and other arthritic conditions, systemic lupus erthematosus (SLE), skin-related conditions, psoriasis, eczema, dermatitis, pain, pulmonary disorders, lung inflammation, adult respiratory distress syndrome, pulmonary sarcoisosis, asthma, chronic pulmonary inflammatory disease, and chronic obstructive pulmonary disease (COPD), cardiovascular disease, arteriosclerosis, myocardial infarction (including post-myocardial infarction indications), congestive heart failure, cardiac reperfusion injury, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, leukemia or lymphoma. Another aspect of the application pro vides a method of treating a kinase mediated disorder, the method comprising administering to a subject in need thereof an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. In some embodiments, the bifunctional compound is an inhibitor of CDK4. In other embodiments, the bifunctional compound is an inhibitor of CDK6. In other embodiments, the bifunctional compound is an inhibitor of CDK4 and CDK6. In other embodiments, the subject is administered an additional therapeutic agent. In other embodiments, the bifunctional compound and the additional therapeutic agent are administered simultaneously or sequentially.

In another aspect, the applicati on provides a method of treating a kinase mediated disorder, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier. In some embodiments, the bifunctional compound is an inhibitor of CDK4. In other embodiments, the bifunctional compound is an inhibitor of CDK6. In other embodiments, the bifunctional compound is an inhibitor of CDK4 and CDK6. In other embodiments, the subject is administered an additional therapeutic agent. In other embodiments, the pharmaceutical composition comprising a bifunciional compound and the additional therapeutic agent are administered simultaneously or sequentially.

In other embodiments, the disease or disorder is cancer. In further embodiments, the cancer is lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemias, lymphomas, myelomas, or solid tumors.

Another aspect of the present application relates to a method of treating or preventing a proliferative disease. The method comprises administering to a subject in need thereof an effecti ve amount of a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the present application relates to a method of treating or preventing a proliferative disease. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug,

stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

In another aspect, the application provides a method of treating or preventing cancer, wherein the cancer cell comprises activated CDK , comprising administering to a subject in need thereof an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the application provides a method of treating or preventing cancer, wherein the cancer cell comprises activated CDK4, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

In certain embodiments, the CDK4 activation is selected from mutation of CDK4, amplification of CDK4, expression of CDK4, and ligand mediated activation of CDK.4.

In another aspect, the application provides a method of treating or preventing cancer, wherein the cancer cell comprises activated CDK6, comprising administering to a subject in need thereof an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the application provides a method of treating or preventing cancer, wherein the cancer cell comprises activated CDK6, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable earner.

In certain embodiments, the CDK6 activation is selected from mutation of CDK6, amplification of CDK6, expression of CDK6, and ligand mediated activation of CDK.6.

In another aspect, the application provides a method of treating or preventing cancer, wherein the cancer cell comprises activated CDK4 and CDK6, comprising administering to a subject in need thereof an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

In another aspect, the application provides a method of treating or preventing cancer, wherein the cancer cell comprises activated CDK4 and CDK6, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptabie salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier. In certain embodiments, the CDK4 acti vation is selected from mutation of CDK4, amplification of CDK4, expression of CDK4, and ligand mediated activation of CDK4. in certain embodiments, the CDK6 activation is selected from mutation of CDK6, amplification of CDK6, expression of CDK6, and ligand mediated activation of CD 6. In certain embodiments, the CDK4 and CD 6 activation is selected from mutation of CD 4 and/or CDK6, amplification of CDK4 and/or CDK6, expression of CDK4 and/or CDK6, and ligand mediated activation of CDK4 and/or CDK6.

Another aspect of the application provides a method of treating or preventing cancer in a subject, wherein the subject is identified as being in need of CDK4 inhibition for the treatment of cancer, comprising administering to the subject an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptabie salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the application provides a method of treating or preventing cancer in a subject, wherein the subject is identified as being in need of CD 4 inhibition for the treatment of cancer, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

Another aspect of the application provides a method of treating or preventing cancer in a subject, wherein the subject is identified as being in need of CDK6 inhibition for the treatment of cancer, comprising administering to the subject an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptabie salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof.

Another aspect of the application provides a method of treating or preventing cancer in a subject, wherein the subject is identified as being in need of CD 6 inhibition for the treatment of cancer, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

Another aspect of the application provides a method of treating or preventing cancer in a subject, wherein the subject is identified as being in need of CDK4 and CDK.6 inhibition for the treatment of cancer, comprising administering to the subject an effective amount of a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. Another aspect of the application pro vides a method of treating or preventing cancer in a subject, wherein the subject is identified as being in need of CDK4 and CDK6 inhibition for the treatment of cancer, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier.

In certain embodiments, the application provides a method of treating any of the disorders described herein, wherein the subject is a human. In certain embodiments, the application provides a method of preventing any of the disorders described herein, wherein the subj ect i s a h uman .

In another aspect, the application provides a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the manufacture of a medicament for treating or preventing a disease in which CDK4 plays a role.

In another aspect, the application provides a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier, for use in the manufacture of a medicament for treating or preventing a disease in which CD 4 plays a role.

In another aspect, the application provides a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the manufacture of a medicament for treating or preventing a disease in which CDK6 plays a role.

In another aspect, the application provides a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier, for use in the manufacture of a medicament for treating or preventing a disease in which CDK6 plays a role.

In another aspect, the application provides a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in the manufacture of a medicament for treating or preventing a disease in which CDK4 and CD 6 play a role.

In another aspect, the application provides a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier, for use in the manufacture of a medicament for treating or preventing a disease in which CDK4 and CDK6 play a role.

In still another aspect, the application provides a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease in which CDK4 plays a role.

In still another aspect, the application provides a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically

acceptable carrier, for use in treating or preventing a disease in which CDK4 plays a role.

In still another aspect, the application provides a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease in which CDK6 plays a role.

In still another aspect, the application provides a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically

acceptable carrier, for use in treating or preventing a disease in which CDK6 plays a role.

In still another aspect, the application provides a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, for use in treating or preventing a disease in which CDK4 and CDK6 play a role.

In still another aspect, the application provides a pharmaceutical composition comprising a bifunctional compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof and a pharmaceutically acceptable carrier, for use in treating or preventing disease in which CDK4 and CD 6 play a role.

As inhibitors of CDK4 and/or CDK6 kinase, the bifunctional compounds and compositions of this application are particularly useful for treating or lessening the severity of a disease, condition, or disorder where a protein kinase is implicated in the disease, condition, or disorder. In one aspect, the present application provides a method for treating or lessening the severity of a disease, condition, or disorder where a protein kinase is implicated in the disease state. In another aspect, the present application provides a method for treating or lessening the severity of a kinase disease, condition, or disorder where inhibition of enzymatic activity is implicated in the treatment of the disease. In another aspect, this application provides a method for treating or lessening the severity of a disease, condition, or disorder with bifunctional compounds that inhibit enzymatic activity by binding to the protein kinase. Another aspect provides a method for treating or lessening the seventy of a kinase disease, condition, or disorder by inhibiting enzymatic activity of the kinase with a protein kinase inhibitor.

In some embodiments, said method is used to treat or prevent a condition selected from autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immunologically-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone related diseases, allergies, asthma, and Alzheimer's disease. In other embodiments, said condition is selected from a proliferative disorder and a neurodegenerative disorder.

One aspect of this application provides bifunctional compounds that are useful for the treatment of diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include, but are not limited to, a proliferative or

hyperproliferative disease, and a neurodegenerative disease. Examples of proliferative and hyperproliferative diseases include, without limitation, cancer. The term "cancer" includes, but is not limited to, the following cancers: breast; ovary: cervix; prostate; testis,

genitourinary tract; esophagus; larynx, glioblastoma; neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma; bone; colon; colorectal; adenoma; pancreas, adenocarcinoma; thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma; seminoma; melanoma; sarcoma; bladder carcinoma; liver carcinoma and biliary passages; kidney carcinoma;

myeloid disorders; lymphoid disorders, Hodgkin's, hair) _' - cells; buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx; small intestine; colonrectum, large intestine, rectum, brain and central nervous system; chronic myeloid leukemia (CML), and leukemia. The term "cancer" includes, but is not limited to, the following cancers: myeloma, lymphoma, or a cancer selected from gastric, renal, or and the following cancers: head and neck,

oropharangea!, non-small cell lung cancer (NSCLC), endometrial, hepatocarcinoma, Non- Hodgkins lymphoma, and pulmonary.

The term "cancer" refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas and the like. For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheral T- cell lymphomas, lymphomas associated with human T-cell lymphotrophic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute nonlymphoc tic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute

myelogenous leukemia, lymphomas, and multiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), Hodgkin's lymphoma,

Burkitt ly mphoma, adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodisplastic syndrome, childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal), genitourinary cancers (e.g. , prostate, bladder, renal, uterine, ovarian, testicular), lung cancer (e.g. , small-cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors related to Gorlin's syndrome (e.g. , medulloblastoma, meningioma, etc.), and liver cancer. Additional exemplary forms of cancer which may be treated by the subject bifuiictional compounds include, but are not limited to, cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, rectum carcinoma, cancer of the salivary gland, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

Additional cancers that the bifunctional compounds described herein may be useful in preventing, treating and studying are, for example, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditar ' non-polyposis colorectal cancer, or melanoma. Further, cancers include, but are not limited to, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma,

fibrosarcoma, Ewing sarcoma, and piasmocytoma. In one aspect of the application, the present application provides for the use of one or more bifunctional compounds of the application in the manufacture of a medicament for the treatment of cancer, including without limitation the various types of cancer disclosed herein. In some embodiments, the bifunctional compounds of this application are useful for treating cancer, such as colorectal, thyroid, breast, and lung cancer; and m eloproliferative disorders, such as polycythemia vera, thrombocythemia, myeloid metaplasia with

myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mast cell disease. In some embodiments, the bifunctional compounds of this application are useful for treating hematopoietic disorders, in particular, acute-myelogenous leukemia (AML), chronic- myelogenous leukemia (CML), acute-promyelocytic leukemia, and acute lymphocytic leukemia (ALL),

This application further embraces the treatment or prevention of cell proliferative disorders such as hyperplasias, dysplasias and pre-cancerous lesions. Dysplasia is the earliest form of pre-ca cerous lesion recognizable in a biopsy by a pathologist. The subject bifunctional compounds may be administered for the purpose of preventing said hyperplasias, dysplasias or pre-cancerous lesions from continuing to expand or from becoming cancerous. Examples of pre-cancerous lesions may occur in skin, esophageal tissue, breast and cervical intra-epithelial tissue.

Examples of neurodegenerative diseases include, without limitation,

Adrenoieukodystrophy (ALD), Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia. Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform

encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeidt- Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Levvy body dementia, Neuroborreliosis, Machado- Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease. Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff disease, Schiider's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease), Spinocerebellar" ataxia (multiple types with varying characteristics). Spinal muscular atrophy, Steele- Richardson-Olszewski disease, Tabes dorsalis, and Toxic encephalopathy.

Another aspect of this application provides a method for the treatment or lessening the severity of a disease selected from a proliferative or hyperproliterative disease, or a neurodegenerative disease, comprising administering an effective amount of a bifunctional compound, or a pharmaceutically acceptable composition comprising a bifunctional compound, to a subject in need thereof.

As inhibitors of CDK4 kinase and/or CDK6 kinase, the compounds and compositions of this application are also useful in biological samples. One aspect of the application relates to inhibiting protein kinase activity in a biological sample, which method comprises contacting said biol ogical sample with a bifunctional compound of the application or a composition comprising said bifunctional compound. The term "biological sample", as used herein, means an in vitro or an ex vivo sample, including, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof Inhibition of protein kinase activity in a biological sample is useful for a variety of purposes that are laiown to one of skill in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ- transplantation, and biological specimen storage.

Another aspect of this application relates to the study of CDK4 kinase and/or CDK6 kinase in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by such protein kinases; and the comparative evaluation of new protein kinase inhibitors. Examples of such uses include, but are not limited to, biological assays such as enzyme assays and cell-based assays.

The activity of the compounds and compositions of the present application as CDK4 and/or CDK6 inhibitors may be assayed in vitro, in vivo, or in a cell line. In vitro assays include assays that determine inhibition of either the kinase activity or ATPase activity of the activated kinase. Alternate in vitro assays quantitate the ability of the inhibitor to bind to the protein kinase and may be measured either by radio labelling the inhibitor prior to binding, isolating the inhibitor/kinase complex and determining the amount of radio l abel bound, or by running a competition experiment where new inhibitors are incubated with the kinase bound to laiown radioligands. Detailed conditions for assaying a compound utilized in this application as an inhibitor of various kinases are set forth in the Examples below.

In accordance with the foregoing, the present application further provides a method for preventing or treating any of the diseases or disorders described above in a subject in need of such treatment, which method comprises administering to said subject a therapeutically effective amount of a bifunctional compound of the application, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired. Pharmaceutical Compositions

in another aspect, the application provides a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound of the present application or an enantiomer, diastereomer, stereoisomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Bifunctional compounds of the application can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g. , orally, e.g. , in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, or topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a compound of the present application in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coaling methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g. , lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g. , silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethylenegivcol; for tableis also c) binders, e.g. , magnesium aluminum silicate, starch paste, gelatin, tragacanth,

methylceliulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g. , starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners, injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present application with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a ban dage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels w ell-know n in the ait. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

The pharmaceutical compositions of the present application comprise a

therapeutically effective amount of a compound of the present application formulated together with one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or

electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium h drogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pvrrolidone, poiyaerylates, waxes, polyethylenepolyoxy propylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil; saffiower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The pharmaceutical compositions of this application can be administered to humans and other animals orally, rectal ly, parenteral!}', intracisternally, intravaginally,

intraperitoneally, topically (as by powders, ointments, or drops), buccaliy, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol,

tetrahydrofurfuiyl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous, or oleaginous suspensions may be formulated according to the known art 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 parenteraily acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents 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.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may- depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenteraily administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this application with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g. , tableting lubricants and other tableting aids such a magnesium stearate and mierocrysta!line cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Dosage forms for topical or transdermal administration of a compound of this application include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this application.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this application, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of this application, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and poly amide powder, or mixtures of these substances. Sprays can additionally contain

customary propellants such as chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Compounds and compositions of the application can be administered in

therapeutically effective amounts in a combinational therapy with one or more therapeutic agents (pharmaceutical combinations) or modalities, e.g., an anti-proliferative, anti-cancer, immunomodulatory or anti -inflammatory agent. Where the compounds of the application are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth. Compounds and compositions of the application can be administered in therapeutically effective amounts in a combinational therapy with one or more therapeutic agents (pharmaceutical combinations) or modalities, e.g., anti -proliferative, anti-cancer, immunomodulatory or anti-inflammatory agent, and/or non-drug therapies, etc. For example, synergistic effects can occur with anti-proliferative, anti-cancer, immunomodulatory or anti-inflammatory substances. Where the compounds of the application are administered in conjunction with other therapies, dosages of the coadministered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.

Combination therapy includes the administration of the subject compounds in further combination with one or more other biologically active ingredients (such as, but not limited to, a second CDK4 inhibitor, a second CDK6 inhibitor, a second CDK4/6 inhibitor, a second and different antineoplastic agent, a second cyclin-dependent kinase inhibitor (i.e., CD 1, CD 2, CDK7, CDK8, CDK9, CDK1 1 , CDK 12, CDK13, CDK14, etc.) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the application can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the application. The compounds of the application can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy or treatment modality. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.

In another aspect of the application, the compounds may be administered in combination with one or more separate pharmaceutical agents, e.g. , a chemotherapeutic agent, an immunotherapeutic agent, or an adjunctive therapeutic agent.

EXAMPLES

Analytical Methods, Materials, and instrumentation

All reactions were monitored Waters Acquity UPLC/MS system (Waters PDA βλ

Detector, QDa Detector, Sample manager - FL, Binary Solvent Manager) using Acquity UPLC® BEH C 18 column (2.1 x 50 mm, 1.7 μιη particle size): solvent gradient = 90% A at 0 rain, 1 % A at 1.8 min; solvent A = 0.1 % formic acid in Water; solvent B ^: = 0.1% formic acid in Acetomtrile; flow rate : 0.6 mL/min. Reaction products were purified by flash column chromatography using CombiFlash ^® Rf with Teledyne Isco Redi&p ^® Rf High Performance Gold or Silicycle SiliaSte/> ^rM High Performance columns (4 g, 12 g, 24 g, 40 g, or 80 g), Waters HPLC system using SunFire™ Prep C 18 column (19 x 100 mm, 5 μηι particle size): solvent gradient = 80% A at 0 min, 5%> A at 25 min: solvent A = 0.035% TFA in Water; solvent B = 0.035%» TFA in MeOH; flow rate : 25 mL/min (Method A), and Waters Acquity UPLC/MS system (Waters PDA βλ Detector, QDa Detector, Sample manager - FL, Binary Solvent Manager) using Acquity UPLC® BEH C I 8 column (2.1 x 50 mm, 1 .7 μηι particle size): solvent gradient = 80% A at 0 min, 5% A at 2 min; solvent A = 0.1% formic acid in Water; solvent B = 0.1% formic acid in Acetonitrile; flow rate : 0.6 mL/min (method B). The purity of all compounds was over 95% and was analyzed with Waters LC/MS system. Ή NMR was obtained using a 500 MHz Bruker Avance III. Chemical shifts are reported relative to dimethyl sulfoxide (δ ------ 2.50) for Tl NMR, Data are reported as (br ------ broad, s ------ singlet, d

= doublet, t = triplet, q = quartet, m = multiplet).

Abbreviations used in the following examples and elsewhere herein are:

atm atmosphere

br broad

DCM dichloromethane

DIEA NN-diisopropylethylamine

DMA N,N-dimethylacetamide

DMF iV,N-dimethylformamide

DMSO dimethyl sulfoxide

EDCI l -ethyl-3-(3-dimethylaminopropyl) carbodiimide

ESI electrospray ionization

EtOAc ethyl acetate

HC3 hydrochloric acid

h hour(s)

HATU bis(dim.ethylamino)m.ethylene]-lH-l,2,3~triazolo[4,5- ?]pyridinium 3- oxide hexafluoro-phosphate

HPLC high-performance liquid chromatography

LCMS liquid chromatography-mass spectrometry

m multiplet

MeOH methanol

MHz megahertz

min minutes

MS mass spectrometry

NMR nuclear magnetic resonance

Pd ₂ (dba)j tris(dibenz5'lideneacetone)dipalladiujn(0) pm parts per million

rt room temperature

TBAF tetra-n-butylammonium fluoride

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrof an

TLC thin layer chromatography

Xphos 2-dic clohexylphosphino-2',4',6'-triisopropylbiphenyl Example 1: Synthesis of N-(4-(4-(6-(6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-ylamino)pyridin-3-yl)piperaz in-l-yl)butyl)-2-(2' (2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-ylamino)acet amide (1-1)

Step 1: tert-butyl (4-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d] pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l- yl)butyl)carbamate (2-3)

To a solution of 6-ace T-8-cyc]opentyl-5-methyl-2-((5-(piperazin-l -yl)pyridin-2- yl)arrdno)pyrido[2,3-i ]pyrirnidin-7(8H)-one (Palbociclib, 2-1) (20 mg, 0.045 mmol) in acetone (0.5 mL) was added 4-bromobutyicarbamate (2-2, 17.2 mg, 0.068 mmol), followed by 2CO3 (12,3 mg, 0.09 mmol) and KI (1 ! .3mg, 0.068 mmol). The resulting mixture was then heated to reflux and stirred overnight. The reaction mixture was diluted with EtOAc and Η2Ο, extracted, and washed with brine. The organic layer was dried over anhydrous Na ₂ S04, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give Boc protected amine 2-3 as a yellow solid (22.3 mg, 80%). LCMS: m/z 620.3 [M+l]. Step 2: 6-acetyl-2-((5-(4-(4-amiiiobutyl)piperaziii-l-yl)pyridin-2-y l)amino)-8- cyclopentyl-5-methyIpyrido[2,3-d]pyrimidin-7(8H)-one Trifluoroacetic acid (2-4)

To a solution of tert-butyl (4-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d] pyrimidin-2-yl)arnino)pyridin-3-yl)piperazin-l-yl)butyl)carb amate (2-3, 22 mg, 0.035 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and the resulting mixture was stirred at rt for 2 h. Once the reaction was complete by LCMS, the reaction mixture was concentrated to provide the crude product 2-4 which was carried on to the next step without further purification.

Step 3: N-(4-(4-(6-(6-acetyl-8-cyclopentyl-5-methyI-7-oxo-7,8-dihydr opyrido[2,3- d]pyrimidin-2-ylamino)pyridin-3-yl)piperazin-l-yl)butyl)-2-( 2-(2,6-dioxopiperidin- 3-yl)-l ,3-dioxoisoindolin-4-ylamino)acetamide (1-1)

To a solution of 6-ace d-2-((5-(4-(4-arninobutyl)piperazin- l -yl)pyridin-2-yl)amino)- 8-cyclopent\d-5-methylpyrido[2,3-d]pyrirnidin-7(8H)-one (2-4) in DMF (0.5 mL) was added 2-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-ylamino )acetic acid (1 1.6 nig, 0.035 mmol) followed by EDCI (8.7 mg, 0.046 mmol), HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μΐ.,, 0.19 mmol) and the resulting mixture was stirred at rt overnight. The mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H Q) to give compound 1-1 as a yellow solid (8.7 mg, 30% over two steps). ^! H NMR (500 MHz, DYiSG-c/V.) δ 11.10 (s, IH), 10.28 (s, IH), 9.66 (s, IH), 8.97 (s, IH), 8.19 < i. ./ 5.8 Hz, I I I . 8.12 i d. J ------ 3.1 Hz, IH), 7,91 (d, ./ 9.1 Hz, ! ! ! ). 7.68-7.55 (m, 2H), 7.09 (dd, J = 7.1, 4.2 Hz, IH), 7.02-6.91 (m, IH), 6.88 (d, J = 8.6 Hz, IH), 5.83 { p. ./ 8.9 Hz, IH), 5.08 (dd, J = 12.7, 5.4 Hz, IH), 4.00-3.91 (m, IH), 3.86 (d, J = 12.7 Hz, 2H), 3.56 (d, J = 12.4 Hz, 2H), 3.22-3.10 (m, 6H), 3.03 (d, ,/ 8,6 Hz, 2H), 2.95-2.83 (m, IH), 2.43 (s, M l ). 2.32 (s, M l ). 2.25 (dq, ,/ 15.7, 8. 1 Hz, 2H), 2.09-1.98 (m, IH), 1 ,91 ( q. ./ 7.4 Hz, 2H), 1 , 83-1.72 (m, 2H), 1.72- 1.62 (m, IH), 1.59 (br, 2H), 1.47 (p, J = 7.2 Hz, IH), 1.28-1.22 (m, 2H). LCMS: m/z 832.3 [M+l ] .

Example 2ι Synthesis of N-(2-(2-(2-(4-(6-(6-acety I-8-cy clopen tyl-5-methyl-7-oxo-7,8- dihyd ropyri do [2,3-d] pyrimidin-2-ylamin o)py ridin-3-yl)piperazin- 1- yl)ethoxy)ethoxy)e hyl)-2-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- ylamino)acetamide (1-2)

(Siep 1 : tert-butyl (2-(2-(2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido [2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l- yl)ethoxy)ethoxy)ethyl)carbamate (2-7)

To a solution of 6-ace -8-cyc]opentyl-5-methyl-2-((5-(piperazin-l -yl)pyridin-2- yl)arnino)pyrido[2,3-i/]pyrirnidin-7(8H)-one (Paibociclib, 2-1) (20 mg, 0.045 mmol) in acetone (0.5 mL) was added tert-butyl 2-(2-(2-bromoethoxy)ethoxy)ethylcarbamate (2-6, 21 .2 mg, 0.068 mmol), followed by K2CO3 (12.3 mg, 0.09 mmol) and KI (1 1.3mg, 0,068 mmol) and the resulting mixture was then heated to reflux and stirred overnight. The reaction mixture was diluted with EtOAc and Η2Ο, extracted, and washed with brine. The organic layer was dried over anhydrous Na?,S04, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give Boc protected amine 2-7 as a yellow solid (26.0 mg, 85%). LCMS: m/z 679.4 [M+l j.

Step 2: 6-acetyl-2-((5-(4-(2-(2-(2-aminoethoxy)ethoxy)ethyl)piperazi n-l-yl)pyridin-2- yl)amino)-8-cyclopentyl-5-methylpyrido [2,3-d] pyrimidin-7(8H)-one Trifluoroacetic acid

To a solution of tert-butyl (2-(2-(2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-

7,8-dihydropyrido 12,3-d |pyrimidin-2-yi)a,mino)pyridin-3-yi)pipera,zin-l - yl)ethoxy)ethoxy)ethyl) carbamate (2-7, 22 mg, 0.035 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and and the resulting mixture was stirred at rt for 2 h. Once the reaction was complete by LCMS, the reaction mixture was concentrated to provide the crude product 2-8 which was carried on to the next step without further purification.

Step 3: N-(2-(2-(2-(4-(6-(6-acetyl-8-cyclopentyI-5-methyl-7-oxo-7,8- dihydropyrido[2,3- d]pyrimidin-2-ylamino)pyridin-3-yl)piperazin-l-yl)ethoxy)e hoxy)ethyl)-2-(2-(2,6- dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-ylamino)acetamide (1-2)

To a solution of 6-acetyl-2-((5-(4-(2-(2-(2-arrunoethoxy)ethoxy)ethy

y])pyridm-2-yl)ammo)-8-cydopentyl-5-m (2-8) in DMF (0.5 mL) was added 2-(2-(2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindolin-4- ylamino)acetic acid (11.6 mg, 0.035 mmol) was added, followed by EDCI (8.7 nig, 0.046 mmol), HOBT (6,6 mg, 0.049 mmol), and TEA (19 mg, 26 μΐ,, 0.19 mmol) and the resulting mixture was stirred at rt overnight. The mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H2O) to give compound 1-2 as a yellow solid (10.9 mg, 35% over two steps). Ή NMR (500 MHz, DMSQ-ife) δ 11.09 (s, 1H), 10.27 (s, 1H), 9.71 (s, 1H), 8.95 (s, IH), 8.21-8.16 (m, H i ). 8.12 (d, J = 3. 1 Hz, I I I ). 7.91 id. ./ 9 1 Hz, IH), 7.68-7,55 (m, 2H), 7.09 (dd, ,/ 7, 1 , 4.2 Hz, IH), 7,02-6.91 (m, I I I ). 6.88 (d, ,/ 8,6 Hz, 1H), 5.83 (p, J = 8.9 Hz, 1H), 5.08 {dd. ./ 12.7, 5.4 Hz, IH), 4.00-3.91 (m, 2H), 3.86 id. ./ 12.7 Hz, 2H), 3.61 -3.49 (m, 4H), 3.42-3.33 (m, 4H), 3.22-3.10 (m, 6H), 3.03 id. ./ 8.6 Hz, 2H), 2.95-2.83 (m, 2H), 2.43 (s, 3H), 2.32 (s, 3H), 2.25 (dq, ./ 15.7, 8.1 Hz, 2H), 2,09-1.98 (m, IH), 1.91 (q, J = 7.4 Hz, 2H), 1.83-1.72 (m, 2H), 1,72-1.62 (m, IH), 1.59 (q, J = 5,3, 4.5 Hz, 2H), LCMS: m/z 892.4 [M+l].

Example 3: Sysithesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)pipera zin-l-yl)-N^

(2-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)ethoxy)ethoxy)ethoxy) ethyl)acetamide (1-3)

Step 1 : tert-butyl 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydrop yrido[2,3- d]pyrimidiii-2-yl)amino)pyridiii-3-yl)piperaziii-l-yl)acetat e (2-10)

To a solution of 6-acetyl-8-cyclopent}4-5-methyl-2-((5-(piperazin-l-yl)pyridi n-2- y])amino)pyrido[2,3-i/]pyrimidin-7(8H)-one (Palbociclib, 2-1) (20 mg, 0.045 mmol) in DMF (0.5 mL) was added tert-butyl bromoacetate (2-9, 13.2 mg, 0.068 mmol), followed by K2CO3 (12.3 mg, 0.09 mmol) and the resulting mixture was stirred at rt overnight. The reaction mixture was diluted with EtOAc and Η2Ο, extracted, and washed with brine. The organic layer was dried with Na ₂ S04, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give t-butyl ester 2-1Θ as a yellow solid (22 mg, 85%).

Step 2: 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydrop yrido[2 - d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l-yl)acetic acid trifluoroacetic acid salt (2-11)

To a solution of tert-butyl 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dilty'dropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)pipe razin-l^ (2-10, 22 mg,

0.038 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and the resulting mixture was stirred at rt for 2 h. The reaction mixture was concentrated to provide the crude product 2-11 which was carried on to the next step without further purification.

Step 3: 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydrop yrido[2,3- d]pyrimidin-2-yl)amino)pyridiii-3-yI)piperazin-l-yI)-N-(2-(2 -(2-(2-((2-(2,6- dioxopiperidiii-3-yl)-l,3-dioxoisoindolin-4-yl)amino)ethoxy) ethoxy)ethoxy) ethyl)acetamide (1-3)

To a solution of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- cUhyclropyrido|X3-d]pyrirnicton-2-yl)amino acid trifluoroacetic acid salt (2-11) in DMF (0.5 ml.) was added 4-((2~(2~(2~(2- aminoethoxy)ethoxy)ethoxy)ethyl)arruno)-2-(2,6-dioxopiperidi n-3-yl)isom

(17 mg, 0.038 mmol) followed by EDC (8.7 mg, 0.046 mmol), HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μΕ, 0.19 mmol) and the resulting mixture was stirred at rt overnight. The reaction mixture was then filtered and purified by reverse phase HPLC (0-100% MeOH in H2O) to give compound 1-3 as a yellow solid (9.5 mg, 27% over two steps). ^[ H NMR (500 MHz, DY!SO- /,). δ 11.10 (s, I I I ). 10.35 (s, I I I ). 8.98 (s, I I I ). 8.75-8.60 (m, 1H), 8.11 (s, I I I ). 7.90 (d, ,/ 9.1 Hz, IH), 7.65-7,53 (m, 2H), 7.15 (d, 8.6 Hz, 11 1 ). 7.05 (d, ,/ 6,9 Hz, 1H), 6.60 (s, 1H), 5.84 (p, J = 8,9 Hz, IH), 5.06 (dd, J = 12,8, 5.4 Hz, H), 4,02 (s, 2H), 3.93-3.69 (m, IH), 3.63 (t, J = 5.4 Hz, 2H), 3.60-3.50 (m, 8H), 3.47 (t, J = 5.5 Hz, 4H), 3.36- 3.26 (m, 2H), 3.26-3.08 (m, IH), 2.89 (ddd. ./ 17.0, 13.8, 5.4 Hz, i l l ). 2.64-2,56 (m, i l l . 2,55 (s, IH), 2,43 (s, 3H), 2,33 (s, 3H), 2,29-2.17 (m, 2H), 2,08-1 ,98 (m, IH), 1.95-1 .85 (m, 2H), 1.84-1.72 (m, 2H), 1.65-1.53 (m, 2H). LCMS: mJz 936,4 [M+l] ,

Example 4: Synthesis of 7-cyclopentyl-2-(5-(4-(4-(2-(2-(2,6-dioxopiperidin-3-yI)-^ dioxoisoindolin-4-ylamino)acetamido)butyl)piperazin-l-yl)pyr idin-2-ylamino)-N,N^ dimethyl- 7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (1-5)

Step 1 : tert-butyl (4-(4-(6-((7-cycIopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2, 3- d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l-yl)butyl)car bamate (2-14)

To a solution of 7-(yciopentyl-N,N-dimethyl-2-(5-(piperazin-l-yl)pyridin-2- ylamino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Rebociclib, 2-13) (19.6 mg, 0.045 mmol) in acetone (0.5 mL) was added tert-butyl 4-bromobut lcarbamate (2-2, 1 7.2 mg, 0.068 mmol), followed by K2CO3 (12.3 mg, 0.09 mmol) and Kl (1 1.3mg, 0.068 mmol) and the resulting mixture was then heated to reflux and stirred overnight. The reaction mixture was diluted with EtOAc and H2O, extracted, and washed with brine. The organic layer was dried over anhydrous Na ₂ S04, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give Boc protected amine 2-14 as a yellow solid (23.5 mg, 86%). LCMS : m/z 606.4 [M+l] .

Step 2: 2-((5-(4-(4-amiiiobutyl)piperaziii-l-yl)pyridin-2-yl)amino)- 7-cyclopentyI-N,N- dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxainide Trifluoroacetic add salt (2-15) To a solution of tert-butyl (4-(4-(6-((7-cyclopentyl-6-(dimemylcarbamoyl)-7H- pyrrolo[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-l -yl)butyl)carb (2-14, 21.2 mg, 0.035 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and the resulting mixture was stirred at rt for 2 h. . Once the reaction was complete by LCMS, the reaction mixture was concentrated to provide the crude product 2-15 which was carried on to the next step without further purification.

Step 2: 7-cyclopentyl-2-(5-(4-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-l,3 -dioxoisoindolin-4- ylamino)acetamido)butyl)piperazin-l-yl)pyridin-2-ylamino)-N, N-dimethyl-7H- pyrroIo[2,3-d]pyrimidine-6-carboxaniide (1-5)

To a solution of 2-((5-(4-(4-aminobutyl)piperazin- l-yl)pyridin-2-yl)amino)-7- cyclopenty -N,N-dimethy1-7H-pyrrolo[2,3~d]pyrimidine-6-carboxamide trifluoroacetic acid salt (2-15) in DMF (0.5 mL) was added 2-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- ylamino)acetic acid (1 1.6 mg, 0.035 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol), HOBT (6,6 mg, 0.049 mmol), and TEA (1 9 mg, 26 μΐ,, 0.19 mmol) and the resulting mixture was stirred at rt overnight. The reaction mixture was then filtered and purified by reverse phase HPLC (0- 100% MeOH in H2O) to give compound 1-5 as a yellow solid (9.5 mg, 33% over two steps, Step 2 and 3). ^! H NMR (500 MHz, DMSO-is¾) 5 1 1 .10 (s, 1 H),

10.94 (br. I I I ). 9, 85 (br, I H i. 8.94 (s, 1H), 8.20 (t, J = 5, 8 Hz, I I I ). 8.00 (d, ,/ 3.0 Hz, 1H), 7.96-7.86 (m, M l ). 7.80-7.68 (m, 1H), 7.65-7.57 (m, IH), 7.13-7.04 (m, lH), 6.96 (t, J = 5.8 Hz, IH), 6.89 id. ./ 8.6 Hz, i l l ). 6.78 (s, i l l ). 5.14-5.01 (m, I I I ). 4.85-4.74 (m. I I I ). 4.17 (d, ./ 14.5 Hz, IH), 3.95 (d, 5.0 Hz, 2H), 3.82 id. ,/ 12.5 Hz, 2H), 3.59 i d. ./ 12,2 Hz, 2H), 3.16 (h, ,/ 6.2 Hz, 6H), 3.01 (s, 1H), 2.97-2.81 (m, 2H), 2.63-2,51 (m, 2H), 2.41-2.25 (m, 3H), 2,09- 1.87 (m, ?! ! }. 1.73-1.57 (m, 5H), 1 ,48 (p, ,/ 7.1 Hz, 2H). LCMS: m/z 819,4 [M+l].

Example 5: Synthesis of 7-cyclopentyl-2-(5-(4-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-l^- dioxoisoindolin-4-ylamino)acetamido)hutyl)piperazin-l-yl)py

dimethyl- 7H~pyrrolo[2^~d]pyrimidine~6~carboxamide (1-9)

Sep 7 : 6-(2-€hIoro-5-nnoropyrimidin-4-yl)-4-fii!oro-l-isopropyl-2 -metliyI-lH- benzo[d]imida¾oie (2-18)

To a suspension of 4-fluoro-l-isopropyl-2-methyl-6-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)-lH-benzo[d]imidazole (2-16, 318 nig, 1 mmol), 2,4-dichloro-5- fluoropyrimidine (2-17, 166 mg, 1 mmol), and Pd(PPrn)4 (115.6 mg, 0.1 mmol) in 6 mL of CH3CN was added 2 ml, of saturated Na ₂ C03 under an atmosphere of N ₂ . The mixture was heated to 85 °C and stirred for 8h. Then the reaction was cooled to room temperature, extracted with CHCh and isopropanol (V/V=4: 1) and the combined organic layers were washed with brine, dried over anhydrous Na2S€>4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give 6-(2-cUoro-5-fluoropyrimidin-4-yl)-4-fluoro-l-isopropyl-2-me thyl IH-benzoj djirnidazole 2-18 as a gray solid (277 mg, 86%). LCMS: m/z 323.1 [M+l].

Step 2: tert-butyl 4-((6-((5-fluoro-4-(4-fluoro-l-isopropyl-2-methyl-lH- benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)meth yl)piperazine-l- carboxylate (2-20)

To a suspension of 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-l-isopropyl-2- methyl-lH-benzo[d]imidazole (2-18, 258 mg, 0.8 mmol), /erf-butyl 4~((6~aminopyridin-3- yl)methyl)piperazine-l-carboxylate (2-19, 350.6 mg, 1.2 mmol) and Cs ₂ COs (782 mg, 2.4 mmol) in 5 mL of -BuOH were added Pd2(dba)3 (73.3 mg, 0.08 mmol) and Xantphos (23 mg, 0.04 mmol) under an atmosphere of N ₂ . The mixture was heated to i l0°C and stirred for 8h. The mixture was then cooled to room temperature, extracted with CHCta and isopropanoi (V7V=4: 1), and the combined organic layers were washed with brine, dried over anhydrous Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give fe/ -butyl 4-((6-(5- fluoro-4-(4-fluoro-l-isopropyl-2-memyl-lH-benzo| ^" d]inudazol-6-yl)pyrimidin-2- ylamino)pyridin-3-yl)methyl)piperazine-l-carboxyiate 2-20 as a white solid (403 mg, 87%). 5 ( VI : m/z 579,3 [M+ 1 ] .

Step 3: 5-fluoro-4-(4-fluoro-l-isopropyl-2-methyl-lH-benzo[d]imidazo l-6-yl)-N-(5- (piperazin-l-ylmethyl)pyridin-2-yl)pyrimidin-2-amine Trifluoroacetic acid salt (2-21)

To a solution of tert-butyl 4~((6~((5-fluoro~4~(4 ]uoro-l~isopropy

benzo[d]iniidazol-6-yl)pyTiiTHdin-2-yl)ainiiio)pyridm-3-y l)niethyi)

(2-20, 57.9 mg, 0.1 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and the resulting mixture was stirred at rt for 2 h. Once the reaction was complete by LCMS, the reaction mixture was concentrated to provide the crude product 2-21 which was earned on to the next step without further purification.

Step 4: tert-butyl (4-(4-((6-((5-fluoro-4-(4-fluoro-l-isopropyl-2-methyl-lH-ben zo[dl imidazol-6-yI)pyrimids -2-yl)ammo)pyridiis~3-yl)metbyI)pipera¾m-l- yl)butyl)carbamate (2-22)

To a solution of 5-fluoro-4-(4-fluoro-l-isopropyl-2-methyl-lH-benzo[d]imidazo l-6- y])-N-(5-(piperazin-l-ylmethyl)pyridin-2-y])pyrimidin-2-amin e trifluoroacetic acid salt (2- 22) in 3 ml, of acetone, was added fert-butyl 4-bromobut>'lcarbamate (2-2, 50 mg, 0.2 mmol), 2CQ3 (41.4 mg, 0.3 mmol) and KI (33.2 mg, 0.2 mmol) and the resulting mixture was heated to reflux and stirred overnight. The reaction mixture was then cooled to room temperature and extracted with CHCh and isopropanoi (V/V=4/l). The combined organic layers were washed with brine, dried over anhydrous Na ₂ S04, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (0-10% MeOH in DCM) to give /erf-butyl 4-(4-((6-(5-fluoro-4-(4-fiuoro-l-isopropyl-2- methyl-lH-benzo[d]imidazol-6-yl)pyrimidin-2-ylammo)pyridm

yl)butylcarbamate 2-22 as a gray solid (50.7 mg, 78%). LCMS: mJz 650.4 [M+l]. Step 5: N-(5-((4-(4-aminobutyl)piperazin-l-yl)methyl)pyridin-2-yl)-5 -fluoro-4-(4-fluoro- l-tsopropyI~2~methyI-lH-beiszoId]tmida¾oI~6~yI)pyrimidisi-2 -amssie Trifluoroacetic add salt (2-23)

To a solution of tert-butyl (4-(4-((6-((5-fluoro-4-(4-fluoro-l-isopropyl-2-methyl-lH- benzo[ d| imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)m

yl)butyl)carbamate (2-22, 32.5 nig, 0.05 mmol) in DCM (0.5 mL) was added TFA (0,5 mL) and the resulting mixture was stirred at rt for 2 h. Once the reaction was complete by LCMS, the reaction mixture was concentrated to provide the crude product 2-23 which was carried on to the next step without further purification.

Step 6: 7-cyclopentyl-2-(5-(4-(4-(2-(2-(2,6-dioxopiperidin-3-yl)-l,3 -dioxoisoindolin-4- ylamino)acetamido)butyl)piperazin-l-yl)pyridin-2-ylamino)-N, N-dimethyl-7H- pyrrolo[2,3-d]pyrimidine-6-carboxamide (1-9)

To a solution of N-(5-((4-(4-aminobutyl)pipera^

fluoro-l-isopropyl-2-methy -lH~beiizo|d]iiriidazol-6-y )pyrimidin~2-airiine trifluoroacetic aad salt in 1 mL. of DMF was added 2-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- ylamino)acetic acid (16.6 mg, 0.05 mmol), followed by EDCT (1 1.5 mg, 0.06 mmol), HOBT (8.1 mg, 0.06 mmol), and TEA (19 mg, 26 μΕ, 0.19 mmol) and the resulting mixture was stirred at rt overnight. The reaction mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H?0) to give compound 1-9 as a yellow solid (12.9 mg, 30% over two steps). 'H MR (500 MHz, DMSO~<%) δ 1 1.10 (s, IH), 10.77 (s, IH), 8.78 (dd, J= 3.7, 1.7 Hz, IH), 8.33 (d, ./ 2.1 Hz, IH), 8.29 (d, ./ 1.3 Hz, IH), 8.23 - 8.11 (m, IH), 7.97 - 7.87 (m, IH), 7.78-7.65 (m, IH), 7.64-7.53 (m, IH), 7.08 (dd, ./ 7.0, 4.6 Hz, IH), 7.04-6.90 (m, IH), 6.86 (d, ./ 8,6 Hz, IH), 5. 1 1-5,02 (m, 2H), 4.91 -4.82 (m, 2H), 4,27-4,06 (m, 21 1 ). 3.93 (d, J = 3.4 Hz, 2H), 3,69 (d, J= 9.0 Hz, IH), 3,48 (s, IH), 3,28-2.94 (m, 6H), 2.95-2.82 (m, 2H), 2.67 i(s, 3H), 2.63-2.52 (m, 3H), 2.04-1.95 (m, 2H), 1.64 (s, 3H), 1.53 (s, 3H), 1.61-.152 (m, 2H), 1.49-1.37 (m, 2H). LCMS: m/z 863.4 | M ! |.

Example 6: Synthesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)am^

dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-y])amino)ethox y)ethyl)acetamide (1-11)

To a solution of the t-butyl ester obtained from Example 3 (22 mg, 0.038 mmol) in DCM (0.5 mL) was added TFA (0.5 mL). The mixture was stirred at room temperature for 2 h. The mixture was then concentrated and dissolved in DMF (0.5 mL). 4-((2-(2- anTinoemoxy)emyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoli ne-l ,3-dione (14 mg, 0.038 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol), HOST (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μί,, 0.19 mmol). The mixture was again stirred at room temperature overnight. The mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H2O) to give compound 1-11 as a yellow solid (7.9 mg, 25% over two steps). ¾ NMR (500 MHz, DMSO-ife) δ 11.11 (s, 1 H), 10.33 (s, 1 H), 8.97 (s, 1 H), 8.68 ( L ./ 5.6 Hz, ! ! ! ). 8.10 (d, J = 3.0 Hz, IH), 7.90 (d, J == 9.1 Hz, 1H), 7.64 - 7.52 (m, 2H), 7, 16 (d, J = 8.6 Hz, 1H), 7,06 (d, ./ 7.0 Hz, i l l ). 6,60 (s, IH), 5,83 (p, «/= 8.9 Hz, IH), 5.06 (dd, «/ = 12.9, 5,4 Hz, 11 1 ). 4.02 (s, 2H), 3.64 (t, ./ 5.5 Hz, 2H), 3.54 (t, ./ = 5.6 Hz, 2H), 3.49 (q, ./ 4.8, 4.4 Hz, 2H), 3.36 (qd, ,/ 5.8, 5.4, 2,2 Hz, 2H), 3.24 - 3.02 (m, IH), 2.94 - 2.81 (m, IH), 2.62 - 2.54 i ns. IH), 2,43 is, 3H), 2,32 (s, 3H), 2,29-2.19 (m, 2H), 2.16-2.00 (m, IH), 1.95 - 1.84 (m, 2H), 1.78 (q, J = 11 ,7, 10.6 Hz, 2H), 1.64 - 1.53 (m, 2H). LCMS: m/z 833,4 [M+l].

Example 7: Synthesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyri^

(2,6~dsoxopiperidiF8~3~yI)~ 1 -dioxossoffl

To a solution of the t-butyl ester obtained from Example 3 (22 mg, 0.038 mmol) in DCM (0.5 mL) was added TFA (0.5 mL). The mixture was stirred at room temperature for 2 h. The mixture was then concentrated and dissolved in DMF (0.5 mL). 4-((2-(2~(2~ ainiiioethoxy)ethoxy)eth}4)aniino)-2"(2,6-dioxopiperidiii-3- yl)isoindoline- 1 ,3-dione (15.4 mg, 0.038 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol), HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μΕ, 0.19 mmol). The mixture was again stirred at room temperature overnight. The mixture was filtered and purified by reverse phase HPLC (0- 100% MeOH in H2O) to give compound 1-12 as a yellow solid (10.2 mg, 30% over two steps). ^l H NMR (500 MHz, DMSO-ifc) δ 11.09 (s, H), 10.35 (s, H), 8.97 (s, H), 8.66 (t, J = 5.6 Hz, IH), 8, 10 (d, J = 3.0 Hz, IH), 7.89 (d, «/= 9.1 Hz, 1H), 7.63 - 7.54 (m, 2H), 7.15 (d, ./ 8.6 Hz, IH), 7.05 (d, ./ 7.0 Hz, i l l ). 6.60 (t, ./ 5.6 Hz, IH), 5.83 (p, ./ 8.9 Hz, i l l ). 5.06 (dd, J ------ 12.8, 5.5 Hz, I H), 4.01 (s, 2H), 3.63 (t, ./ 5.4 Hz, 2H), 3.59 (dd, ,/ 6.3,

3,6 Hz, 2H), 3.56 (dd, ./ 6,3, 3.7 Hz, 2H), 3,48 (t, J ------- 5.4 Hz, 4H), 3.32 (q, ./ 5.6 Hz, 2H), 2,90 - 2.86 (m, IH), 2.63 - 2.56 (m, IH), 2,43 (s, 3H), 2,32 (s, 3H), 2,28 - 2, 19 (m, 2H), 2.13 - 2.01 (m, IH), 1.90 (h, ./ 7.0 Hz, 2H), 1.83 - 1.74 (m, 2H), 1.64 - 1.54 (m, 2H). LCMS: m/z 892.4 [M+l ].

Example 8: Synthesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyri

To a solution of the t-butyl ester obtained from Example 3 (22 mg, 0.038 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and stirred at room temperature for 2 h. The mixture was then concentrated and dissolved in DMF (0.5 mL). 4-((6-aminohexyl)amino)-2- (2,6-dioxopiperidin-3-yl)isoindoline-l,3-dione (14.1 mg, 0.038 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol), HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μί..0.19 mmol). The mixture was again stirred at room temperature overnight. The mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H2O) to give compound 1-13 as a yellow solid (9.2 mg, 28% over two steps). ^! H NMR (500 MHz, OMSO-de) δ 11.09 (s, ill).10.33 (s, ill).8.97 (s, ill).8.54 (t, ./= 5.6 Hz, 1H), 8.10 (d, J = 3.0 Hz, ill).7.90 (d, ./ 9.1 FIz, 1H), 7.59 (ddd, ,/ 8.6, 4.7, 1.8 Hz, 2H), 7.10 (d,■/ 8.6 Hz, 1H), 7.03 (d, ./ 7.0 Hz, 111).6,53 (s. 111).5,83 (p, J ------ 8.9 Hz, lis).5.05 hid.,/ 12.8, 5.4 Hz, III).4.00 (s, 2H), 3.30

(q, ./ 6.3 Hz, 3H), 3.16 (q, J= 6.5 Hz, 211).2.94 - 2.83 (111, ill).2,62 - 2.55 (m, ill).2.43 (s, 3H), 2.32 (s, 3H), 2.29 - 2.21 (m, 2H), 2.03 (ddq, J= 10.5, 5.4, 3.0, 2.6 Hz, 1H), 1.90 (h, J ------ 7.0 Hz, 2II).1.81 - 1.70 (m, 2H), 1.65 - 1.52 (m, 4FI), 1.46 (p. J ------ 7.0 Hz, 2H), 1.41 - 1.26 (m, 411) LCMS: rn/z 860.4 [M+l].

Example 9: Synthesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyI-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridm^

dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-y])amino)-3,6, 9,12,lS-

To a solution of the t-butyl ester obtained from Example 3 (22 mg, 0,038 mrnol) in DCM (0.5 mL) was added TFA (0.5 mL) and stirred at room temperature for 2 h. The mixture was then concentrated and dissolved in DMF (0.5 mL). 4-((17-amino-3, 6,9,12,15- pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l,3-dione (20.4 mg, 0.038 mrnol) was added, followed by EDCI (8,7 mg, 0.046 mrnol), HOBT (6,6 mg, 0.049 mrnol), and TEA (19 mg, 26 μΐ.,, 0.19 mrnol). The mixture was again stirred at room temperature overnight. The mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H2O) to give compound 1-14 as a yellow solid (9,2 mg, 28% over two steps). ³ H NM (500 MHz, DMSO-cfe) δ 11.09 (s, 1H), 10.37 (s, 1H), 8.97 (s, 1H), 8.67 is.../ 5.6 H/.1H), 8.10 (d,J=3.0Hz, HI}.7.89 (d, J =9.0 Hz, 1H), 7.65 - 7.51 (ni, 2H), 7.18-7.09 (m, 1H), 7.04 id../ 7.0 Hz, i 11 L 6.60 is. i 11 K 5.83 (p. J ------ 8.9 Hz, 1H), 5.05 (dd, ./ 12.8, 5.4 Hz, 111).

3.62 (t, ,/ 5.4 Hz, 2H), 3.56 (dd, 5.6, 2,9 Hz, 21 \) 3.52 (s, 3H), 3.51 - 3,49 (m, 18H), 3.33 (q, J = 5,5 Hz, 2H), 2.88 (ddd, J= 16.9, 13.8, 5.4 Hz, 1H), 2.59 (dt, J= 17.4, 3.2 Hz, 1H), 2.43 (s, 3H), 2.32 (s, 3H), 2.29 - 2.17 (ni, 2H), 2.02 (dtd, ./ 13.0, 5.3, 2.2 Hz, ill).

1,89 id../ 8.2 Hz, 2! I ).1,83 - 1.72 (m, 2H), 1.68-1.51 (m, 2H). LCMS: m/z 1024.5 |M !|

Example 10: Synthesis of 2-(4-(6-((6-acetyl-8-cyclopentyl-5-methyI-7- dihydropyrido[2,3-d]pyrimidin-2-yl)amino)p

dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)aniino)-3,6 ,9,12,lS- peniaoxaheptadeeyI)aceiam e (1-20)

To a suspension of dimethyl 4-hydroxyphthalate (l . lg, 5 mmol) and K · ⁽ . ^' () . (1.38g, 10 mmol) in 20 mL of anhydrous DMF was added tert-b yl (4-broniobutyl)carbamate (1.88g, 7.5 mmol) dropwise at room temperature. The mixture was heated to 80°C and kept stirring overnight, and then cooled to room temperature, before being diluted with water and extracted with EtOAc three times. The combined organic layers were dried over anhydrous Na?.S04, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (ISCO, 40g silica column, 0 to 5% MeOH/DCM 30min gradient) to give dimethyl 4-(4~((tert-butoxycarbonyi)amino)butoxy)phthalate (1.7g, 90%). LCMS: m/z 382.2 [M+l].

Dimethyl 4-(4-((tert-butoxy carbonyl)amino)butoxy)phthaIate (1.7g, 4.5 mmol) was dissolved in 10 mL of MeOH, aqueous 3M NaOH (4.5 mL, 13.5 mmol) was then added, and the mixture was heated to 80°C for 22 hours. The mixture was cooled to room temperature, diluted with 50 ml DCM and 20 mL 0.5M HC1. The layers were separated and the organic layer was washed with 25 mL water. The aqueous layers were combined and extracted three times with 50 ml. chloroform. The combined organic layers were dried over anhydrous NazSOt, filtered and condensed to give 1.59g of the acid that was carried forward without further purification. LCMS: m/z 354.1 M+l].

The resultant acid (1.59g, 4.5 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (0.74 g, 4.5 mmol) were dissolved in pyridine (11.7 ml, 0.25 M) and heated to 110°C for 17h. The mixture was cooled to room temperature and concentrated under reduced pressure to give crude tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5- yl)oxy)butyl)carbamate as a black sludge that was carried forward without further purification. I . ( VIS: rr, :: -! -!6.2 | V! · i |.

The crude tert-butyl (4-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-5- yi)oxy)butyl)carbamate was dissolved in 20mL TFA and heated to 50°C for 2.5 hours. The mixture was cooled to room temperature, diluted with MeOH, and concentrated under reduced pressure. The material was purified by preparative HPLC to give 5-(4- aminobutoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l,3-dion e as a grey solid (l.Og, 60% over 3 steps). LCMS: /z 346.2 | \ · 1 I

To a solution of the t-butyl ester obtained from Example 3 (22 mg, 0.038 mmol) in DCM (0.5 mL) was added TFA (0.5 mL) and stirred at room temperature for 2 h. The mixture was concentrated and dissolved in DMF (0.5 mL). 5-(4-aminobutoxy)-2-(2,6- dioxopiperidin-3-yl)isoindoline-l,3-dione (13.11 nig, 0.038 mmol) was added, followed by EDCI (8.7 mg, 0.046 mmol), HOBT (6.6 mg, 0.049 mmol), and TEA (19 mg, 26 μΐ,, 0.19 mmol). The mixture was stirred at room temperature overnight. The mixture was filtered and purified by reverse phase HPLC (0-100% MeOH in H2O) to give compound Ι-2Θ as a yellow solid (9.5 mg, 30% over two steps). ^! H NMR (500 MHz, DYlSi V.) 5 11.11 (s, IH), 10.33 (s, I I I . 8.97 (s, I I I . 8.62 ( ;. ./ 5.6 i !/. IH), 8.1 1 id. 3.0 ! !/. I I I ). 7.88 ( dd. J 24,8, 8.7 Fiz, 2H), 7,59 (dd, J = 9.2, 3.1 Hz, IH), 7.44 (d, 2.3 Hz, 1H), 7.35 (dd, ,/ 8.4, 2.3 Hz, 1H), 5.83 (p, J = 8.9 Hz, IH), 5.12 (dd, J = 12.9, 5.4 Hz, IH), 4.20 (q, J = 6.7 Hz, 2H), 4.03 (s, 2H), 3.25 (q, J = 6.6 Hz, 2H), 2.89 (s, 2H), 2.73 (s, IH), 2.60 (dt, J = 18.0, 3.4 Hz, IH), 2.54 (s, 2H), 2.43 (s, 3H), 2.32 (s, 3H), 2.25 (ύύ\. 13.0, 10.8, 4.7 Fiz, IH), 2.05 (dtd, ./ = 12.9, 5,2, 2.1 Hz, i l l ). 1,89 (d, J = 8.5 Hz, 2H), 1.85-1.72 (m, 5H), 1.67 - 1.52 (m, 5H). LCMS: m/z 833.4 [M+l].

Example 11: Biochemical Studies

Enzyme Degradation Assay

Jurkat cell or Molt4 wild-type or cereblon null cells were treated with a control or a bifunctional compound of the application. After treatment, cells were washed and harvested by resuspending in buffer and lysed on ice 30 minutes. Lysates were then cleared by centnfugation. Samples were boiled and equal amount protein is loaded onto gel. Gel was transferred to nitrocellulose and blotted for CDK6, CDK4 or Tubulin. As can be seen in FIG. 1, a decrease in CDK6 levels over time was observed while CDK4 and tubulin levels were unaffected. Cells treated with Compound 1-3 show degradation of CDK6 in the wild-type cells while CDK4 was unaffected (FIG. 2). Compound 1-3 is dependent on the presence of CRBN as seen by a rescue of CDK6 levels in the CRBN-/- cells.

Western Blotting on CDK4/6

Jurkat ceils were treated with the indicated compounds at the indicated concentrations for the indicated amount of time. Cell s were then lysed in M-PER buffer (Thermo Scientific) containing protease/phosphatase inhibitor cocktail (Roche). Protein concentration was measured using a BCA assay (Pierce). Equivalent amounts of each samples were loaded on 4-12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membranes, and

immunoblotted with antibodies against CDK4, CDK6, and Actin (Cell Signaling). IRDye® 800-labeled goat anti-rabbit IgG and IRDye® 680-labeled goat anti-mouse IgG (LI-COR) secondary antibodies were purchased for LI-COR, and membranes were detected on an Odyssey detection system (LI-COR Biosciences). The results are shown in FIG. 3A-FIG. 3G, FIG.4, FIG.5, and FIG.6A-FIG.6D.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the present application.

All patents, patent applications, and literature references cited herein are hereby expressly incorporated by reference.