COMPOUNDS FOR MUTANT KRAS PROTEIN DEGRADATION AND USES

THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates generally to bifunctional compounds that bind more preferentially to mutant KRAS proteins than to wild KRAS proteins and promote degradation of KRAS protein via recruitment of an E3 ubiquitin ligase, and uses of the compounds in the treatment of diseases associated with KRAS mutation.

BACKGROUND OF THE INVENTION

[0002] A proteolysis targeting chimera (PROTAC) technology was first described in 2001

(Sakamoto et al., "Protacs: chimeric molecules that target proteins to the Skpl-Cullin-F box complex for ubiquitination and degradation," Proceedings of the National Academy of

Sciences of the United States of America. 98 (15): 8554-9). PROTAC is a two-headed heterobifunctional molecule capable of removing unwanted proteins by inducing selective intracellular proteolysis. PROTACs consist of two protein binding moieties, one for binding an

E3 ubiquitin ligase and the other for binding a target protein. By binding both proteins,

PROTAC brings the target protein to E3 ligase, resulting in the ubiquitination of the target protein for subsequent degradation by the proteasome (Bondeson et al., "Lessons in PROTAC design from selective degradation with a promiscuous warhead." Cell Chem Biol. 25(1): 78-87,

2018). PROTAC technology has been used in several targets: AR, ER, STAT3, BTK, FLT-3, EGFR, BCR-ABL, BET, BRD7/9, CDK4/6, CK2, ALK, PI3K, MCL-1, PARP1 and c-MET.

[0003] RAS proteins are proto-oncogenes and are encoded by three RAS genes: HRAS, Kirsten ras sarcoma protein (KRas or KRAS), and NRAS. The RAS proteins function in signal transduction pathways controlling cell growth and differentiation as binary switches, transitioning from an inactive GDP-bound state to an active GTP-bound state. Mutations usually occur at codons 12, 13, and 61 that result in the impairment of the intrinsic GTPase activity of RAS proteins, or that prevent GAP binding, activate downstream signaling pathways and contribute to tumor formation and maintenance. (Chang et al., "Detection of N-, H-, and

KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-spedfic primer extension” Clin Biochem. 43(3):296-301, 2010.) Mutations of RAS genes are commonly found in numerous malignancies, including pancreatic (90%), colon

(45%), and lung cancers (35%). Many tumor types have been shown to be dependent on the continued expression of oncogenic mutant RAS in cell and animal models. Pharmacologically targeting RAS is difficult because of its picomolar affinity for GTP and GDP, and because it lades well-defined pockets for high-affinity small-molecule binding, and has therefore often been referred to as "undruggable" (Cox et al, "Drugging the undruggable Ras: mission possible?"

Nat Rev Drug Discov. 13(11): 828-851, 2014.).

[0004] Currently, AMG510 (Sotorasib), a small molecule inhibitor targeting KRAS ^G12C mutant, has passed clinical trials and was approved for marketing by the US FDA on May 28, 2021. The strategy of targeting the mutated KRAS proteins is shown to be therapeutically effective.

[0005] Michael J. Bond et al, "Targeted Degradation of Oncogenic KRAS ^G12C by VHL-Recruiting PROTACs” ACS Cent Sci. 2020, 6, 8, 1367-1375; US 2019/0315732 Al; W02019195609A2; and W02021/207172 Al discloses using KRAS ^G12C inhibitor as a Binder to produce a Degrader, thus providing a different therapeutic strategy from the inhibitor. However, both KRAS ^G12C inhibitor and KRAS ^G12C degrader are only effective for KRAS ^G12C -, but not for other variants, such as G12D,

G12V, G13D, G12R, G12S and Q61H.

[0006] An ongoing need exists in the art for effective treatments for KRAS related disease and disorders, such as pancreatic cancer, colon cancer, colorectal cancer, lung cancer, and non-small lung cancer, biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, and breast cancer.

SUMMARY OF THE INVENTION

[0007] The present disclosure describes hetero-bifunctional compounds that function to recruit KRAS, such as mutant or gain-of-function KRAS, to an E3 ubiquitin ligase for targeted ubiquitination and subsequent proteasomal degradation, and methods of making and using the same. In addition, the description provides methods of using an effective amount of a bifunctional compound of the present disclosure for the treatinent or amelioration of a disease condition, such as a KRAS-related disease or disorder, e.g., accumulation or overactivity of an KRAS protein or a mutated or gain-of function KRAS protein or a mis-folded

KRAS protein, pancreatic cancer, colon cancer, colorectal cancer, lung cancer, non-small cell lung cancer, biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, and breast cancer.

[0008] In one aspect, the present disclosure describes a bifunctional compound of formula

(I):

RB-Linker-ULM (I) or pharmaceutically acceptable salts, tautomers, stereoisomers, solvates, hydrates, polymorphs, isotopically enriched derivatives, or prodrugs thereof, wherein: ULM is an E3 ubiquitin ligase binding moiety or a chaperone complex binding moiety;

Linker is a group covalently binding to the RB and ULM moieties; and

RB is a RAS protein binding moiety, and is represented by formula RB-I: wherein:

X ₁ is H, NR ^X1 R ^X2 , SO ₂ NR ^X1 R ^X3 ,OR ^X1 , CHR ¹ R ^X2 NH(C=O)R ^X3 , or CN;

R ^X1 and R ^X2 are each independently H, OH, or a linear or branched Ci-« alkyl , optionally substituted with one or more halo, or R ^X1 and R ^X2 taken together with the atom they are attached to, form a 4-8 membered ring system containing 0-2 heteroatoms and optionally substituted with one or more C _1-4 alkyl; R ^X3 is a linear or branched C _1-10 alkyl optionally substituted with one or more halo, a linear or branched C _1-10 alkoxyl optionally substituted with one or more halo, or a 4-8 membered ring system containing 0-2 heteroatoms and optionally substituted with one or more halo, CF ₃ ,

NHz, OH, or CN;

W ₁ is a bond, a C _1-6 alkyl, alicyclic, heterocyclic, bicyclic, or biheterocydic, each optionally substituted by one, two or three R ^w1 , and each R ^W1 is independently H, halo, OH, NH ₂ , NMe ₂ ,

NEt ₂ , CN, a C _1-4 alkyl optionally substituted by one or more F, or a C _1-3 alkoxyl optionally substituted with one or more F ;

R ₁ is CH, C-Me, C-halide, or N; is aryl, heteroaryl or indazol, independently substituted with one or more halo, CF ₃ , OCF ₃ , O-R ^A1 -R ^A2 , hydroxyl, nitro, CN, C=CH, a linear or branched C _1-6 alkyl optionally substituted with one or more halo or C _1-6 alkoxyl, a linear or branched C _1-6 alkoxy1 optionally substituted with one or more halo, a C _{2- 6} alkenyl, a C _2-6 alkynyl, or a 4-7 membered saturated or partially unsaturated heterocyclic containing 1-2 heteroatoms,

R ^A1 is a C _1-6 alkyl,

R ^A2 is a heteroaryl containing 1-2 heteroatoms and optionally substituted with one or more halo;

X ₂ is CH ₂ or C=O; is a 4-8 membered alicydic system with 0-4 heteroatoms, optionally substituted with 0-4 R ^Q , each R ^Q is independently OH, or a linear or branched C _1-6 alkyl optionally substituted with one or more halo, or 2 R ^Q groups taken together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms;

Z ₁ is H, C _1-4 alkyl, C _1-4 alkoxyl, halide, OH, CN, or C _2-4 alkynyi; and the dashed line indicates the site of attachment to Linker.

[0009] The bifonctional compounds of formula (I) can induce the ubiquitination of KRAS protein and promote its degradation in cells. An advantage of the bifunctional compounds of formula (I) provided herein is that a broad range of pharmacological activities is possible.

[0010] In an additional aspect, the present disclosure provides pharmaceutical compositions comprising an effective amount of the bifunctional compound as described herein, and a pharmaceutically acceptable earner.

[0011] In another aspect, the present disclosure provides pharmaceutical compositions for use in preventing, ameliorating and/or treating a disease associated with KRAS mutation in a subject in need thereof, comprising an effective amount of the bifunctional compound, and one or more pharmaceutically acceptable excipients.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows tumor growth curve in Calu-1 implanted male NOD SCID mice.

[0013] FIG. 2 shows body weight changes in Calu-1 implanted male NOD SCID mice.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Definitions

[0015] In order for the present disclosure to be folly understood, the following detailed description is set forth. In the description, the following terms are employed:

[0016] It must be noted that, as used herein, the singular forms “a," "an" and "the" include piurai referents unless the context clearly dictates otherwise. Thus, unless otherwise required by context, singular terms shall include the plural and plural terms shall indude the singular.

[0017] Often, ranges are expressed herein as from "about" one particular value and/or to

"about" another particular value. When such a range is expressed, an embodiment includes the range from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of tire word "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to and independently of the other endpoint. As used herein, the term "about” refers to ± 20%, preferably ± 10%, and even more preferably ± 5%.

[0018] The term "and/or” is used to refer to both things or either one of the two mentioned.

[0019] The terms "treatment," “treating," and "treat" generally refer to obtaining a desired pharmacological and/or physiological effect. The effect maybe preventive in terms of completely or partially preventing a disease, disorder, or symptom thereof, and may be therapeutic in terms of a partial or complete cure for a disease, disorder, and/or symptoms attributed thereto. "Treatinent" used herein covers any treatment of a disease in a mammal, preferably a human, and indudes (1) suppressing development of a disease, disorder, or symptom thereof in a subject or (2) relieving or ameliorating the disease, disorder, or symptom thereof in a subject.

[0020] The term "preventing" or "prevention" is recognized in the art, and when used in relation to a condition, it includes administering, prior to onset of the condition, an agent to reduce the frequency or severity of or delay the onset of symptoms of a medical condition in a subject relative to a subject which does not receive the agent.

[0021] The terms "individual, " "subject," and “patient” herein are used interchangeably and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired.

[0022] The term "effective amount" of an active ingredient as provided herein means a sufficient amount of the ingredient to provide the desired regulation of a desired function. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the disease state, physical conditions, age, sex, spedes and weight of the subject, the specific identity and formulation of the composition, etc. Dosage regimens may be adjusted to induce the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

[0023] Unless otherwise specified, the term "alkyl" as used herein refers to a monovalent, saturated, straight or branched chain hydrocarbon radical containing 1 to 12 carbon atoms. Preferably, the alkyl is a C _1-4 alkyl group. More preferably, the alkyl is a C _1-6 alkyl group. The alkyl can be substituted or unsubstituted. Examples of a C _1-6 alkyl group indude, but are not limited to, methyi, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl (induding all isomeric forms), and hexyl (including all isomeric forms), heptyl (including all isomeric forms), and octyl (including all isomeric forms).

[0024] Heteroatoms such as oxygen, sulfur and nitrogen (in the form of tertiary amine moieties) may be present in the alkylene group, to provide a “heteroalkylene" group.

Examples of heteroalkylene radicals include, but are not limited to, -CH ₂ CH ₂ N(CH ₃ ) ₂ and -

CH ₂ CH ₂ OCH ₂ CH ₃ .

[0025] Unless otherwise specified, the term "alkoxy" as used herein refers to radicals of the general formula -O-(alkyl), wherein alkyl is as defined above. Exemplary alkoxy indudes, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-hexoxy.

[0026] As used herein, the term "aryl" refers to all-carbon monocyclic or fused-ring polycyclic aromatic groups having a conjugated pi-electron system. The aryl group may have 6 to 14 carbon atoms in the ring(s). Exemplary aryl includes, but is not limited to, phenyl, biphenyl and napthyl.

[0027] As used herein, the term "cycloalkyl" refers to an all-carbon monocyclic or fused ring

(i.e., rings which share an adjacent pair of carbon atoms) group wherein one or more rings does not have a completely conjugated pi-electron system. Exemplary cydoalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantanyl, 1-methylcyclopropyl, 2-methylcyclopentyl, and 2-methylcydooctyl.

[0028] As used herein, the terms "heterocyclic ring" and “heterocydyl" are used interchangeably. The term "heterocyclic ring” or "heterocyclyl" refers to a mono-, bi-, or polycyclic structure having from 3 to 14 atoms, alternatively 3 to 12 atoms, alternatively 3 to

10 atoms, alternatively 3 to 8 atoms, alternatively 4 to 7 atoms, alternatively 5 or 6 atoms; wherein one or more atoms, for example 1, 2 or 3 atoms, are independently selected from the group consisting of N, O, and S,the remaining ring-constituting atoms being carbon atoms.

The ring structure may be saturated or unsaturated, but is not aromatic. Exemplary heterocyclic rings indude, but are not limited to, imidazolyl, imidazolinoyl, imidazolidinyl, quinolyl, isoqinolyl, indolyl, indazolyl, indazolinolyl, perhydropyridazyl, pyridazyl, pyridyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazinyl, quinoxolyl, piperidinyl, pyranyl, pyrazolinyl, piperazinyl, pyrimidinyl, pyridazinyl, morpholinyl, thiamorpholinyl, furyl, thienyl, triazolyl, thiazolyl, carbolinyl, tetrazolyl, thiazolidinyl, benzofuranoyl, thiamorpholinyl sulfone, oxazofyl, benzoxazolyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, isoxozolyl, isothiazolyl, furazanyl, tetrahydropyranyl, tetrahydrofuranyl, thiazolyl, thiadiazoyl, dioxolyl, dioxinyl, oxathiolyl, benzodioxolyl, dithiolyl, thiophenyl, tetrahydrothiophenyl, sulfolanyl, dioxanyl, dioxolanyi, tetahydrofurodihydrofuranyl, tetrahydropyranodihydrofuranyl, dihydropyranyl, tetradyrofurofuranyl, and tetrahydropyranofuranyl.

[0029] As used herein, the term "heteroaryl" refers to a mono-, bi- or tri-cydic aromatic radical containing 1 to 4 heteroatoms selected from S, N and O, and indudes radicals having two such monocyclic rings, or one such monocyclic ring and one monocyclic aryi ring, which are directly linked by a covalent bond. The heteroaryl group may have 5 to 14 ring-forming atoms, including 1 to 13 ring-forming carbon atoms, and 1 to 8 ring-forming heteroatoms each independently selected from 0, 5 and N. Exemplary heteroaryl includes, but is not limited to, thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyi, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

[0030] As used herein, the term "heterocycloalkyl” refers to a monocyclic or polycyclic

(induding 2 or more rings that are fused together, including spiro, fused, or bridged systems, for example, a bicyclic ring system), saturated or unsaturated, non-aromatic 4- to 15- membered ring system, induding 1 to 14 ring-forming carbon atoms and 1 to 10 ring-forming heteroatoms each independently selected from O, S and N. Examples of heterocycloalkyl include, but are not limited to, azetidinyl, tetrahydrofuran, dihydrofuran, dioxane, morpholine, etc.

[0031] As used herein, the terms "ring system" refers to one or more cycles of atoms (e.g., monocyclic and polycyclic), which may be saturated, unsaturated or aromatic, and the atoms may be carbons only or may include heteroatoms (e.g., N, S, O). For example, the ring system may include, but not limited to, aryl (e.g., phenyl, heterophony, naphthyl), alicydic, heterocydic, bicyclic (induding spiro, fused and bridged), and biheterocyclic structures.

[0032] As used herein, the terms "halide" and "halo" are used interchangeably and include fluoro, chloro, bromo and iodo.

[0033] Compounds

[0034] In one aspect, the description provides hetero-bifunctional compounds comprising an

E3 ubiquitin ligase binding moiety ("ULM”) covalently coupled to a protein targeting moiety

(RB) via a chemical linking group (Linker) according to the structure (I):

RB-Linker-ULM (I) or pharmaceutically acceptable salts, solvates, hydrates, polymorphs, tautomers, stereoisomers, isotopically enriched derivatives, or prodrugs thereof, wherein: Linker is a group covalently binding to the RB and ULM moieties, RB is a RAS protein binding moiety that binds to mutant or wild-type KRAS protein; and ULM is inclusive of all E3 ubiquitin ligase binding moiety or a chaperone complex binding moiety.

[0035] The bifunctional compound described herein interacts more preferentially with KRAS mutant proteins than with KRAS wild type protein. As described herein, the therapeutic effect may be a result of degradation, modulation, binding, or modification of KRAS protein by a compound described herein. Without wishing to be bound by any particular theory, the therapeutic effect may be the result of modulation, targeting, binding, or modification of an

E3 ubiquitin ligase by a compound described herein. The therapeutic effect may be a result of recruitment of an E3 ubiquitin ligase by modulation, targeting, binding, or modification of the

E3 ubiquitin ligase to an ubiquitinated RAS protein and marking it for proteasomal degradation, by a compound. Specifically, the bifunctional compound of formula (I) is useful for the treatment and/or prevention of diseases associated with RAS mutation in a subject in need thereof.

[0036] ULM

[0037] ULM is an E3 ubiquitin ligase binding moiety or a chaperone complex binding moiety, and can be a Cereblon E3 ubiquitin ligase binding moiety (CLM), a Von Hippel-Lindau (VHL) E3 ubiquitin ligase binding moiety (VLM), a DDB1- and CUL4-associated factor 16 (DCAF16) E3 ubiquitin ligase tending moiety (DIM), an IAP E3 ubiquitin ligase binding moiety (ILM), a mouse double minute 2 (MDM2) homolog E3 ubiquitin ligase binding moiety (MLM), a Kelch-like ECH- associated protein-1 (KEAP1) E3 ubiquitin ligase tending moiety, a DCAF15 E3 ubiquitin ligase binding moiety, an RNF4 E3 ubiquitin ligase binding moiety, an RNF114 E3 ubiquitin ligase binding moiety, an arylhydrocarbon receptor (AhR) E3 ubiquitin ligase binding moiety, or others E3 ubiquitin ligase binding moiety described in SIAS Discovery, 1-19, 2020. ULM is inclusive of all moieties that bind, can bind, or form covalent bond with any E3 ubiquitin ligase.

For example, in certain embodiments, ULM is capable of binding an E3 ubiquitin ligase, such as Cereblon or von Hippel-Lindau (VHL). In certain embodiments, ULM is capable of forming covalent bond with an E3 ubiquitin ligase, such as DCAF16. in certain embodiments, ULM is capable of binding to multiple different E3 ubiquitin ligases. In certain embodiments, ULM binds to Cereblon. in certain embodiments, ULM binds to VHL. in certain embodiments, ULM forms covalent bond with DCAF16.

[0038] Cereblon is an E3 ubiquitin ligase, and it forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and a regulator of cullinsl (ROC1).

This complex ubiquitinates a number of proteins. [0039] In certain embodiments, ULM is a Cereblon E3 lyase-binding moiety (CLM) selected from the group consisting of a thalidomide, lenalidomide, pomalidomide, analogs thereof, isosteres thereof, or derivatives thereof; preferably is of the following formula CLM-a and

CLM-b: wherein:

W is selected from CH ₂ , and C=O; and

Q ₁ , Q ₂ , Q ₃ , and Q ₄ are each independently C, N, or C-halide, and wherein one of Q ₁ , Q ₂ , Q ₃ , and Q ₄ covalently joins to Linker; wherein the dashed line indicates the site of attachment to Linker.

[0040] In certain embodiments, ULM is wherein the dashed line indicates the site of attachment to Linker; and

X _CLM is halide.

[0041] The von Hippel-Lindau (VHL) is an E3 ubiquitin ligase. VHL comprises the substrate recognition subunit/E3 ubiquitin ligase complex VCB, which includes etongins B and C, and a complex including Cullin-2 and Rbxl. The primary substrate of VHL is Hypoxia Inducible Factor

I (HIF-la), a transcription factor that up regulates genes, such as the pro-angiogenic growth factor VEGF and the red blood cell inducing cytokine, erythropoietin, in response to tow oxygen levels. VCB is a known target in cancer, chronic anemia, and ischemia. In an embodiment, ULM is a VHL E3 ubiquitin ligase binding moiety, and can be hydroxyproline or a derivative thereof.

[0042] In certain embodiments, ULM comprises a peptide backbone structure. In certain embodiments, ULM is of a chemical structure represented by: wherein:

R ₅ is H, or a linear or branched C _1-3 alkyl;

R ₆ is CN or a 5-membered heteroaryl having one or two heteroatoms selected from N, S, or O, optionally substituted with methyl (e.g, the dashed line indicates the site of attachment to the benzene ring);

Z ₂ is F or CN; and the dashed line indicates the site of attachment to Linker.

[0043] As described in Nature Chemical Biology, VOL 15, 2019, p73791og by Xiaoyu Zhang et al.,DDBl- and CUL4-associated factor 16 (DCAF16) is a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases. The DCAF16 protein has eight cysteine residues, and can covalently bound to DCAF16 binding moiety of a heterobifunctional degrader at a cysteine residue and then promote protein degradation.

[0044] In certain embodiments, ULM is a DCAF16 ligase-binding moiety (DLM), and is of a chemical structure represented by D16-a: wherein:

R ₇ is H, halide, C _1-4 alkyl, C _1-4 alkoxy;

Y ₁ is O, or S, or NH;

R ^m is a covalent electrophile, and is selected from the following group, the dashed line indicates the site of attachment to the nitrogen atom ; and the dashed line linked to Y indicates the site of attachment to Linker.

[0045] In certain embodiments, ULM is the DIM represented by: wherein the dashed line indicates the site of attachment to linker.

[0046] In certain embodiments, ULM binds an E3 ubiquitin ligase with a K _D value of less than about 10,000 nM, less than about 5,000 nM, less than about 1,000 nM, less than about 500 nM, less than about 100 nM, less than about 50 nM.

[0047] In certain embodiments, ULM binds Cereblon with a K _D value less than about 50 μM, less than about 10,000 nM, less than about 5,000 nM, less than about 1,000 nM, less than about 500 nM, less than about 100 nM, less than about 50 nM.

[0048] In certain embodiments, ULM binds VHL with a K _D value of less than about 50 μM, less than about 10,000 nM, less than about 5,000 nM, less than about 1,000 nM, less than about

500 nM, less than about 100 nM, less than about 50 nM.

[0049] RB

[0050] In certain embodiments, RB is represented by formula RB-I: wherein:

X ₁ is H, NR ^X1 R ^X2 , SO ₂ NR ^X1 R ^X2 , OR ^X1 , CHR ^X1 R ^X2 , NH(C=O)R ^X3 , or CN;

R ^X1 and R ^X2 are each independently H, OH, or C _1-4 alkyl (linear or branched, optionally substituted by 1 or more halo), or R ^X1 and R ^X2 taken together with the atom they are attached to, form a

4-8 membered ring system containing 0-2 heteroatoms (optionally substituted by 1 or more C _1-4 alkyl); R ^X3 is C _{1- 10} alky (llinear or branched, optionally substituted by 1 or more halo), C _{1- 10} alkoxy) (linear or branched, optionally substituted by 1 or more halo), or aryl or alicyclic (4-8 membered ring system containing 0-2 heteroatoms, optionally substituted by 1 or more halo, CF ₃ , NH ₂ , OH, or CN);

W ₁ is a bond, C _1-6 alkyl, alicyclic, heterocyclic, bicyclic, or biheterocyclic, each optionally substituted by 1, 2 or 3 R ^W1 ; and each R ^W1 is independently halo, C _1-4 alkyl (optionally substituted by 1 or more F), C _1-3 alkoxyl (optionally substituted by 1 or more F), OH, NH ₂ , NMe ₂ ,

NEt ₂ or CN;

R ₁ is CH, C-Me, C-halide, or N; is aryl or heteroaryl, independently substituted by 1 or more O-R ^A1 -R ^A2 , halo, CF ₃ , OCF ₃ , hydroxyl, nitro, CN, , C _1-6 alkyl (linear or branched, optionally substituted by 1 or more halo, C _1-6 alkoxyl), C _1-6 alkoxyl (linear or branched, optionally substituted by 1 or more hate),

C _2-6 alkenyl, C _2-6 alkynyl, or a 4-7 membered saturated or partially unsaturated heterocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,

R ^A1 is C _1-6 alkyl,

R ^A2 is heteroaryl containing 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and optionally substituted by 1 or more halo;

X ₂ is CH ₂ or C=0; is a 4-8 membered alicyclic with 0-4 heteroatoms, optionally substituted with 0-4 R ^Q , each R ^Q is independently OH, or C _1-6 alkyl (linear or branched, optionally substituted by 1 or more halo), or 2 R ^Q groups taken together with the atom they are attached to, form a 3-8 membered ring system containing 0-2 heteroatoms;

Z ₁ is H, C _1-4 alkyl, C _1-4 alkoxyl, halide, OH, CN, or C _2-4 alkynyl; and the dashed line indicates the site of attachment to Linker.

[0051] In certain embodiments, the heteroatom(s) in R ^X1 and R ^X3 is independently selected from nitrogen, oxygen, and sulfur. Preferably, the heteroatom(s) is nitorogen.

[0052] In certain embodiments, X ₁ is H, NR ^X1 R ^X2 , SO ₂ NR ^X1 R ^X2 , OR ^X1 , CHR ^X1 R ^X2 , NH(C=O)R ^X3 , or

CN; R ^X1 and R ^X2 are each independently H, or C _1-4 alkyl, or R ^X1 and R ^X2 taken together with the atom they are attached to, form a 4-8 membered aryl, alicyclic or bicyclic containing 0-2 nitrogens and optionally substituted by 1 or more C _1-4 alkyl; and R* ³ is C _{1- 10} alkyl (linear or branched, optionally substituted by 1 or more halo), C _{1- 10} alkyl (linear or branched, optionally substituted by 1 or more halo), or aryl or alicyclic (4-8 membered ring system containing 0-2 heteroatoms independently selected from nitrogen and oxygen, optionally substituted by 1 or more halo, CF ₃ , or NH ₂ ).

[0053] In certain embodiments, W ₁ is a bond or C _1-4 alkyl optionally substituted by C _1-3 alkoxyl. [0054] In certain embodiments, is phenyl, heterophenyl, naphthyl or indazol, independently substituted by 1 or more O-CH ₂ -R ^A2 , halo, CF ₃ , OCF ₃ , hydroxyl, CN, C _1-6 alkyl, C ₁ . 6 alkoxyl, R ^A2 is heteroaryl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and optionally substituted by 1 or more halo.

[0055] In certain embodiment, is phenyl or indazol, independently substituted by

1 or more O-CH ₂ -R ^A2 , halo, CF ₃ , OCF ₃ , CN, C _1-6 alkyl, C _1-6 alkoxyl, R ^A2 is heteroaryl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and optionally substituted by 1 or more halo.

[0056] In certain embodiments, the heteroatom(s) in is independently selected from nitorogen, oxygen, and sulfur. Preferably, the heteroatom(s) is nitrogen.

[0057] In certain embodiments, is a 4-6 membered alicyclic with 1-2 heteroatoms, optionally substituted with 0-4 R ^Q , each R ^Q is independently H or C _1-6 alkyl (linear or branched), or 2 R ^Q groups taken together with the atom they are attached to, form a 4-5 membered ring system containing 0-2 heteroatoms, and the heteroatom(s) is nitrogen.

[0058] In certain embodiments, formula RB-1 is selected from the group consisting of RB-1 to

RB-7:

wherein the dashed line indicates the site of attachment to Linker.

[0059] In certain embodiments, formula RB-I is represented by: wherein the dashed line indicates the site of attachment to Linker.

[0060] In some embodiments of the disclosure, the RB-I is selected from the group consisting of:

wherein the dashed line indicates the site of attachment to Unker.

[0061] In some embodiments of the disclosure, the bifunctional compound is selected from formula l-a to l-g:

[0062] In certain embodiments, the RB binding moiety binds KRAS protein with a K _D value of less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, or less than about 50 nM.

[0063] In certain embodiments, the RB binding moiety selectively binds mutant KRAS protein over wild type KRAS protein. In some embodiments, the compound of formula (I) selectively binds mutant KRAS protein over wild type KRAS protein. In certain embodiments, the selectivity is between about 2-fold and about 5-fold. In certain embodiments, the selectivity is between about 5-fold and about 10-fold. In certain embodiments, the selectivity is between about 10-fold and about 20-fold.

[0064] Linker

[0065] Linker is a divalent moiety linking RB and ULM; preferably, Linker covalently couples

RB to ULM. In certain embodiments, Linker comprises the following chemical structure wherein: the dashed line of Linker is the site of attachment to RB or ULM;

Y ^L2 is a bond, or an unsubstituted or substituted linear or branched C _1-4 alkyl or alkoxy (e.g., optionally substituted with a halogen, Cm alkyl, methyl, or ethyl);

W ^L3 is a bond, -C=OCH ₂ -, or a 3-7 membered ring system (e.g., 4-6 membered cycloalkyl, heterocydoalkyl or aryl) or an 8-12 membered spirocydk, each with 0-4 heteroatoms (e.g., 0-

4 heteroatoms independently selected from N, 0, and S) and optionally substituted with halogen or methyl;

Y ^L3 is a bond, or a C _1-32 alkyl alkenyl, or alkynyl, wherein one or more C atoms are optionally replaced with O, or NH, and each carbon is optionally substituted with a halogen, =0, methyl or ethyl, and each nitrogen is optionally substituted with methyl or ethyl;

Y ^L4 is a bond, O, or an unsubstituted or substituted linear or branched C _1-6 alkyl, wherein one or more carbons are optionally replaced with 0, NH, or NCH ₃ , and optionally substituted with a halogen or methyl;

W ^L4 is a 3-8 membered ring system (e.g., 4-6 membered cycloalkyl or heterocydoalkyl, aryl, or or a 5-8 membered spirocydk, each with 0-4 heteroatoms (e.g., 0-4 heteroatoms independently selected from N, O, and 5) and optionally substituted with halogen (e.g., F, Cl,

Br), or methyl; and

Y ^L5 is a bond or an unsubstituted or substituted C _1-6 alkyl, where one or more C atoms are optionally replaced with 0 and optionally substituted with a halo (e.g., F, Ci, Br), or methyl.

[0066] In certain embodiments, Y ^L2 is a bond or C _1-4 alkyl; preferably a bond or CH ₂ .

[0067] In certain embodiments, W ^L3 is a bond, -C=OCH ₂ -, a 4-6 membered heterocydoalkyl containing 0-2 nitrogen, or an 8-12 membered spirocyclic, each cylic containing 0-1 nitrogen.

[0068] In certain embodiments, Y ^L3 is a bond, or a C _1-8 alkyl or alkynyl, wherein one or more

C atoms are optionally replaced with O, and each carbon is optionally substituted with =0.

[0069] In certain embodiments, Y ^L4 is a bond, a bond or C _1-4 alkyl; preferably a bond or CH ₂ .

[0070] In certain embodiments, W ^L4 is an aryl optionally substituted with halogen (e.g., F, Cl,

Br), or a 4-6 membered heterocycloalkyl containing 0-2 nitrogen.

[0071] In certain embodiments, Y ^L5 is a bond or C _1-4 alkyl; preferably a bond or CH ₂ .

[0072] In certain embodiments, Linker is selected from the group consisting of

wherein the dashed line of Linker is the site of attachment to RB or ULM.

[0073] In certain embodiments, a ring system of Linker and a ring system of RB may share 1 or more atoms (carbon atoms or heteroatoms, preferably cartion atoms) to form a bicyclic structure. For example, the ring system of linker and the ring system of RB may share 1 atom to form a spirocyclic structure, share 2 atoms to form a fused bicyclic structure, or share 3 atoms to form a bridged bicyclic structure.

[0074] The RB group and ULM group may be covalently linked to Linker through any group which is appropriate and stable to the chemistry of the linker, in exemplary aspects of the present invention, the linker is independently covalently bonded to the RB group and the ULM group in certain embodiments through an amide, ester, thioester, keto group, carbamate

(urethane), carbon or ether, each of which groups may be inserted anywhere on the RB group and ULM group to provide maximum binding of the ULM group on the ubiquitin ligase and the

RB group on the target protein to be degraded.

[0075] WO 2021/222138 also discloses bifunctional compounds that bind to mutant KRAS proteins. In certain embodiments of the present disclosure, while not willing to be bound by any particular theory, it is believed that since Linker of is attached to of RB-I, and/or since the molecular weight of the bifunctional compound is relatively small (smaller than those of WO 2021/222138), the bifunctional compounds are provided with improved pharmacokinetic properties and improved therapeutic effect.

[0076] As used herein, the phrase "substituted or unsubstituted" means that substitution is optional. In the event a substitution is desired, then such substitution means that any number of hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the normal valence of the designated atom is not exceeded, and that the substitution results in a stable compound. For example, when a substituent is keto (i.e., =0), then 2 hydrogens on the atom are replaced. Examples of substituents for a "substituted" group are those found in the exemplary compounds and embodiments disclosed herein and can indude, for example, haloide, -OH, -CF ₃ , -CN, -NO ₂ alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, haloalkyl, alkylamino, aminoalkyl, dialkylamino, hydroxylalkyl, alkoxyalkyl, hydroxyalkoxy, alkoxyalkoxy, aminoalkoxy, alkylaminoalkoxy, alkylaminoalkyl, and aryl, and the like.

[0077] The term "pharmaceutically acceptable salts" as used herein refers to compounds according to the invention used in the form of salts derived from inorganic or organic acids and bases, included among add salts, for example, are tire following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectianate, persulfate, phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Salts derived from appropriate bases indude alkali metal (e.g. sodium), alkaline earth metal (e.g., magnesium), ammonium and NW ⁴⁺ (wherein W is C _1-4 alkyl).

[0078] As used herein, "prodrugs” are intended to include any covalently bonded canters that release the active parent drug according to formula (I) through in vivo physiological action, such as hydrolysis, metabolism and the like, when such prodrug is administered to a subject

The suitability and techniques involved in making and using prodrugs are well known by a person of ordinary skill in the art. Prodrugs of the compounds of formula (I) (parent compounds) can be prepared by modifying functional groups present in the compounds in such a way that the modifications are deaved, either in routine manipulation or in vivo, to the parent compounds. "Prodrugs" include the compounds of formula (I) wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when the prodrugs are administered to a subject, cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively.

Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds of formula (I) that indude biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. In certain embodiments, prodrugs of the compounds of formula (I) with carboxyl functional groups are the lower alkyl

(e.g., C ₁ -C ₅ ) esters of the carboxylic add. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule.

[0079] The compounds of the invention can exist as solvates. As used herein and unless otherwise indicated, the term "solvate" means a compound of formula (I), or a pharmaceutically acceptable salt thereof, that further includes a stoichiometric or non- stoichiometric amount of a solvent bound by non-covalent intermoiecular forces. If the solvent is water, the solvate may be conveniently referred to as a "hydrate," for example, a hemi-hydrate, a mono-hydrate, a sesqui-hydrate, a di-hydrate, a tri-hydrate, etc.

[0080] The term "tautomer" as used herein refers to compounds whose structures differ markedly in the arrangement of atoms, but which exist in easy and rapid equilibrium, and it is to be understood that compounds provided herein may be depicted as different tautomers, and when compounds have tautomeric forms, all tautomeric forms are intended to be within the scope of the invention, and the naming of the compounds does not exclude any tautomer.

Exemplary tautomerizations include, but are not limited to, amide-to-imide; enamine-to- imine; enamine-to-(a different) enamine tautomerizations; and keto-to-enol.

[0081] The term "stereoisomers” refer to compounds that have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

Stereoisomers indude diastereomers, enantiomers, conformers and the like.

[0082] The term "polymorph” refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and dectrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.

[0083] As used herein, "isotopically enriched derivatives” refers to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. "Isotopic enrichment" can be expressed in terms of the percentage of incorporation of an amount of a specific isotope at a given atom in a molecule in the place of tiie atom's natural isotopic abundance.

[0084] The bifunctional compound of the present invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans- forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and

L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and P-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof.

[0085] In certain embodiments, the bifunctional compound is selected from the compounds

(cpd) 1-72:

4-amino-N-(3-(5-((4-(4-((4-(2-(2,6-dioxopiperidin-3-Yf)-l ,3-dbxoisoindolin-5-yl)piperazin-l- yl)methyl)benzyl)piperazin-l-yl)methyl)-3-(4-(trifluorometho xy)phenyl)-lH-indol-l- yf)propyl)piperidine-4-carboxamide (cpd 1);

4-amino-N-(3-(5-((4-(4-((4-(2-{2,6-dioxopiperidin-3-yl)-l ,3-dioxoisoindoiin-5-yl)piperazin-l- yl)methyl)benzyl)piperazin-l-yl)methyl)-3-(4-(trifluorometho xy)phenyl)-lH-indol-l- yl)propyl)piperidine-4-carboxamide (cpd 2);

5-(4-((4-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy}pheny l)-lH-indol-5-yl)methyl)piperazin-l- yl)methyl)piperidin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 3);

5-(4-((4-((l-(3-aminopropyl)-3-(4-hydroxyphenyl)-lH-indol -5-yi)methyi)piperazin-l- yl)methyl)piperidin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 4);

5-(4-((l-((l-(3-aminopropyl)-3-(4-hydroxyphenyl)-lH-indol -5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dbxopiperidin-3-yl)isoindol ine-l, 3-dione (cpd 5);

5-(4-({l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny i)-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dbxopiperidin-3-yl)isoindol ine-l, 3-dione (cpd 6);

5-(4-({4-((l-(3-aminopropyl)-3-(4-fluorophenyl)-lH-indol- 5-yl)methyl)piperazin-l- yl)methyl)piperidin-l-Yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 7);

5-(4-({l-(3-aminopropyl)-3-(4-{trifluoromethoxy)phenyl)-l H-indol-5-yi)methyl)piperazin-l-yl)-2-

(2, 6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (cpd 8); l-(4-((l-(3-aminopropyl)-3-(4-{trifluoromethoxy)phenyl)-lH-i ndol-5-yi)methyl)piperazin-l-yl)-2-

((l-(2-chloroacetyl)-l,2,3,4-tetrahydroquinoiin-6-yl)oxy) ethan-l-one (cpd 9);

5-(4-({4-((l-(3-aminopropyl)-3-(4-methoxyphenyl)-lH-indol -5-yi)methyi)piperazin-l- yl)methyl)piperidin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 10); 4-(l-(3-aminopropyl)-5-((4-((4-(2-(2,6-dioxopiperidin-3-yl)- l,3-dioxoisoindolin-5-yl)piperazin-l- yl)methyl)piperidin-l-yl)methyl)-lH-indol-3-yl)benzonitrile (cpd 11);

5-(4-((l-((l-(3-aminopropyl)-3-(6-methoxypyridin-3-yl)-lH -indol-5-yl)methyl)piperidin-4- yl)metiiyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-Yl)isoind oline-l, 3-dione (cpdl2);

3-(l-(3-aminopropyl)-5-((4-((4-(2-(2,6-dioxopiperidin-3-y l)-l,3-dioxoisoindolin-5-yl)piperazin-l- yl)methyl)piperidin-l-yl)methyl)-lH-indol-3-yl)benzonitrile (cpd 13);

5-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-7-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 14);

5-(4-((l-((l-(3-aminopropyl)-3-(2-fiuoro-4-methoxyphenyl) -lH-indoi-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 15);

5-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-6-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 16);

5-(4-((l-((l-(3-aminopropyl)-3-(2-methoxypyridin-3-yl)-lH -indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 17);

5-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethyl)phenyi )-lH-indol-5-yl)methyl)pipendin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 18);

5-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-4-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 19);

4-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (cpd 20); tert-butyl (3-(5-((4-((4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindol in-5-yl)piperazin-l- yi)methyl)piperidin-l-yl)methyl)-3-(4-(trifluoromethoxy)phen yl)-lH-indol-l- yl)propyl)carbamate (cpd 21);

5-(3-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4- yl)oxy)prop-l-yn-l-yl)-2-(2,6-dioxopiperidin-3-yi)isoindolin e-l, 3-dione (cpd 22);

5-(3-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4- yl)oxY)propyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione (cpd 23);

5-(4-(2-(4-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)phe nyl)-lH-indol-5-yl)methyl)piperazin-l- yl)acetyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line- 1,3-dione (cpd 24);

5-(4-((l-((l-(3-aminopropyl)-3-(3-(trifluoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yi)isoindo iine-l, 3-dione (Cpd 25);

5-(4-((l-(l-(3-aminopropyl)-3-(4-(trifiuoromethoxy)phenyi )-lH-indole-5-carbonYl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (Cpd 26);

5-(4-((l-((l-(3-aminopropyl)-3-(3-hydroxynaphthalen-l-yl) -lH-indol-5-yi)methyi)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (Cpd 27);

5-(4-((l-((l-(azetidin-3-ylmethyl)-3-(4-(trifiuoromethoxy )phenyi)-lH-indol-5-yl)methyl)piperidin-

4-yl)methyl)piperazin-l-yl)-2-(2, 6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Cpd 28);

5-(4-((l-((l-(3-aminopropyl)-3-(5-diloro-6-methyl-lH-inda zol-4-yl)-lH-indol-5- yf)methyl)piperidin-4-yl)methyl)piperazin-l-yl)-2-(2,6-dioxo piperidin-3-yl)isoindoline-l,3- dione (Cpd 29);

5-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-Yf)isoindo line-l, 3-dione (Cpd 30);

(2S)-N-(2-(3-(4-((l-(3-aminopropyl)-3-(4-(trifiuoromethox y)phenyi)-lH-indol-5- yl)methyl)piperazin-l-yl)propoxy)-4-(4-methy!thiazol-5-yl)be nzyl)-l-((S)-2-(l- fluorocyclopropane-l-carboxamido)-3,3-dimethylbutanoyl)-4-hy droxypyrrolidine-2- carboxamide (Cpd 31);

5-(4-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)-6-fluo roisoindoline-l, 3-dione (Cpd 32);

2-(2,6-dioxopiperidin-3-yl)-5-(4-((l-((l-(3-(methylamino) propyl)-3-(4-(trifluoromethoxy)phenyl)- lH-indol-5-yl)methyl)piperidin-4-yl)methyl)piperazin-l-yl)is oindoline-l, 3-dione (Cpd 33);

5-(7-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)phenyl)-l H-indol-5-yi)methyl)-2,7- diazaspiro[3.5]nonan-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoind oline-l, 3-dione (Cpd 34);

5-(4-((l-((l-(3-aminopropyl)-3-(2-fiuoro-4-((3-fluoropyri din-2-yl)methoxy)phenyl)-lH-indol-5- yl)metfiyl)piperidin-4-^)meth^)piperazin-l-yl)-2-(2 _/ 6-dioxopiperidin-3-yl)isoindoline-l,3- dione (Cpd 35);

5-(4-(l-((l-(3-aminopropYl)-3-(4-(trifluoromethoxy)phenyl )-lH-indol-5-yl)methyl)azetidin-3- yl)piperazin-l-yl)-2-{2,6-dioxopiperidin-3-yi)isoindoiine-l, 3-dione (Cpd 36);

5-(4-((l-((l-(3-(dimethyiamino)propyl)-3-(4-(trifiuoromet hoxy)phenyl)-lH-indol-5- yl)methyl)piperidin-4-yl)methyl)piperazin-l-Yl)-2-(2,6-dioxo piperidin-3-yl)isoindoline-l,3- dione (Cpd 37);

5-(4-((l-((l-(5-aminopentyl)-3-(4-(trifuoromethoxy)phenyl )-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (Cpd 38);

5-(7-((l-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)pheny l)-lH-indol-5-Yl)methYl)piperidin-4- yi)methyl)-2,7-diazaspiro[3.5]nonan-2-yi)-2-(2,6-dioxopiperi din-3-yl)isoindoline- 1,3-dione (Cpd 39);

N-(3-(5-((4-((4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoiso indolin-5-yl)piperazin-l- yl)methyl)piperidin-l-yl)methyl)-3-(4-(trifiuoromethoxY)phen yi)-lH-indol-l- yf)propyl)acetamide (Cpd 40); 4-(5-({4-((4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindoli n-5-yl)piperazin-l-yl)methyl)piperidin- l-yl)methyl)-3-(4-(trifluoromethoxy)phenyl)-lH-indol-l-yl)bu tanenitrile (Cpd 41);

2-(2,6*dioxopiperidin-3-yl)-5-(4-{(l-((l-((l-methyl-lH-ii dazole-5-yl)methyl)-3-(4-

(trifluoromethoxy)phenyl)-lH-indol-5-yl)methyl)piperidin- 4-yl)methyl)piperazin-l- yf)isoindoline-l, 3-dione (Cpd 42);

2-(2,6-dioxopiperidin-3-yl)-5-(4-((l-((l-(3-methoxypropyl )-3-(4-(trifluoromethoxy)phenyl)-lH- indol-5-yl)methyl)piperidin-4-yi)methyi)piperazin-l-yl)isoin doline-l, 3-dione (Cpd 43);

2-(2,6-dioxopiperidin-3-yl)-5-(4-((l-((l-(3-(2,2,2-Triflu oroethoxy)propyl)-3-(4-

(trffluoromethoxy)phenyl)-lH-indol-5-yl)methyi)piperidin- 4-yl)methyl)piperazin-l- yl)isoindoline-13-dione (Cpd 44);

N-(3-(5-((4-((4-(2-{2,6-dioxopiperidin-3-yl)-l _J ,3-dioxoisoindolin-5-yl)piperazin-l- yi)methyl)piperidin-l-yl)methyl)-3-(4-(trifluoromethoxy)phen yl)-lH-indol-l-yl)propyl)-lH- pyrrole-2-carboxamide (Cpd 45);

3-((4-(l-((l-(3-aminopropyl)-3-{4-(trifiuoromethoxy)pheny l)-lH-indol-5-yl)methyl)piperidin-4-yl)-

3-fiuorophenyl)amino)piperidine-2, 6-dione (Cpd 46);

N-{3-(5-({4-{(4-(2-{2,6-dioxopiperidin-3-yl)-l,3-dioxoiso indoiin-5-yl)piperazin-l- yl)methyl)piperidin-l-yl)methyl)-3-(4-(trifluoromethoxY)phen Yl)-lH-indol-l-yl)propyl)-3-

(trifluoromethyl)benzamide (Cpd 47);

N-(3-(5-((4-((4-(2-{2,6-dioxopiperidin-3-yl)-l _J ,3-dioxoisoindolin-5-yl)piperazin-l- yl)methyl)piperidin-l-yl)methyl)-3-(4-(trifluoromethoxy)phen yl)-lH-indol-l-yl)propyl)-4- fluorobenzamide (Cpd 48);

3-((4-(4-({l-((l-(3-aminopropyl)-3-(4-(trifiuoromethoxy)p henyl)-lH-indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-3-fluorophenyl)amino)piperidine-2, 6-dione (Cpd 49);

5-(4-((l-((l-butyl-3-(4-(trifiuoromethoxy)phenyl)-lH-indo l-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)-2-(2, 6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Cpd 50);

2-(2,6-dioxopiperidin-3-yl)-5-(4-((l-((3-(4-(trifluoromet hoxy)phenyl)-lH-indoi-5- yl)methyl)piperidin-4-yl)methyl)piperazin-l-yi)isoindoiine-l , 3-dione (Cpd 51);

5-(4-((l-((l-((2,3-dihydrobenzo[b][l,4]dioxin-2-yl)methyl )-3-(4-(trifluoromethoxy)phenyi)-lH- indol-5-yl)methyl)piperidin-4-yl)methyl)piperazin-l-yi)-2-(2 ,6-dioxopiperidin-3-yi)isoindoline-

1,3-dione (Cpd 52);

2-(2,6-dioxopiperidin-3-yl)-5-(4-((l-((l-(pyridin-2-ylmet hyl)-3-(4-(trifluoromethoxy)phenyl)-lH- indol-5-yl)methyl)piperidin-4-yi)methyi)piperazin-l-yl)isoin doline-l, 3-dione (Cpd 53);

3-((4-(l‘-{(l-(3-aminopropyl)-3-(4-(trifluoromethoxy)ph enyl)-lH-indol-5-yl)methyl)-[4,4'- bipiperidin]-l-yl)-3-fluorophenyl)amino)piperidine-2, 6-dione (Cpd 54); 3-((4-(4-((l-((l-butyl-3-(4-(trifluoromethoxY)phenyl)-lH-ind ol-5-yl)rnethyl)piperidin-4- yl)methyl)piperazin-l-yl)-3-fiuorophenyl)amino)piperidine-2, 6-dione (Cpd 55);

5-(4-((l-t(l-((l,3-dioxolan-2-yl)methYl)-3-(4-(trifluorom ethoxy)phenyl)-lH-indol-5- yl)methyl)piperidin-4-yl)methyl)piperazin-l-yi)-2-(2,6-dioxo piperidin-3-Yl)isoindoline-l,3- dione (Cpd 56);

2-(2,6-dioxopiperidin-3-yl)-5-(4-((l-((l-(4-methylpentyl) -3-(4-(trifluoromethoxy)phenyl)-lH- indol-5-yl)methyl)piperidin-4-Yi)methYi)piperazin-l-yl)isoin doline-l, 3-dione (Cpd 57);

3-((4-(4-((l-((l-{3-(dimethylamino)propyi)-3-(4-(trifluor omethoxy)phenyl)-lH-indol-5- yl)methyl)piperidin-4-Yi)methYl)piperazin-l-yl)-3-fluorophen yl)amino)piperidine-2, 6-dione

(Cpd 58);

5-(l'-{(l-(3-aminopropyl)-3-(4-(trifluoromethoxy)phenYl)- lH-indol-5-yl)methyl)-[4,4’- bipiperidinj-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione (Cpd 59);

3-((4-(l'-((l-(3-(dimethylamino)propyi)-3-(4-(trifluorome thoxY)phenyl)-lH-indol-5-Yl)methyl)-

[4, 4'-bipipendin]-l-yl)-3-fluorophenyl)amino)piperidine-2, 6-dione (Cpd 60);

4-amino-N-(3-{5-((4-{(4-(4-((2,6-dioxopiperidin-3-yl)amin o)-2-fluorophenyl)piperazin-l- yl)methyl)piperidin-l-yl)methyl)-3-(4-(trifluoromethoxy)phen yl)-lH-indol-l- yl)propyl)piperidine-4-carboxamide (Cpd 61);

5-((4-((4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxo-2,3-dihy dro-lH-inden-5-Yl)piperazin-l- yl)methyl)piperidin-l-yl)methYl)-N,N-dimethyi-3-(4-(trifiuor omethoxy)phenyl)-lH-indole-l- sulfonamide (Cpd 62);

3-({3-fluoro-4-(4-((l-((l-(4-methylpentyl)-3-(4-(trifluor omethoxy)phenyl)-lH-indol-5- yl)methyl)piperidin-4-Yl)methyl)piperazin-l-yl)phenYl)amino) piperidine-2, 6-dione (Cpd 63);

3-((4-fluoro-3-(4-((l-((3-(4-{trifiuoromethoxy)phenyl)-lH -indol-5-yl)methyl)piperidin-4- yl)methyl)piperazin-l-yl)phenyl)amino)piperidine-2, 6-dione (Cpd 64);

3-((3-fluoro-4-(l'-((3-(4-(trifiuoromethoxy)phenyl)-lH-in dol-5-yl)methyl)-[4,4'-bipiperidinj-l- yl)phenyl)amino)piperidine-2, 6-dione (Cpd 65);

5-(5-({l-(3-aminopropyl)-3-(4-(trifluoromethoxy)phenyl)-l H-indol-5- yi)methyl)hexahydropyrrolo[3,4-c]pYrrol-2(lH)-yl)-2-{2,6-dio xopiperidin-3-yl)isoindoline-l,3- dione (Cpd 66);

3-((4-(5-((l-(3-aminopropyl)-3-(4-(trifiuoromethoxy)pheny l)-lH-indol-5- yl)methyl)hexahydropYrroio[3,4-c]pyrrol-2(lH)-yl)-3-fiuoroph enyl)amino)piperidine-2,6- dione (Cpd 67);

5-(4-((l-((l-(3,3-dimethoxypropyl)-3-(4-(trifluoromethoxy )phenyl)-lH-indol-5- yi)methyl)piperidin-4-yl)methYl)piperazin-l-yl)-2-(2,6-dioxo piperidin-3-yl)isoindoline-l,3- dione (Cpd 68);

5-(5-((l-(3-(dimethylamino)propyl)-3-(4-(trifluoromethoxY )phenyl)-lH-indol-5- yl)methyl)hexahydropyrrolo[3,4-c]pyrrol-2(lH)-yl)-2-(2,6-dio xopiperidin-3-yl)isoindoline-l,3- dione (Cpd 69);

5-(l'-((l-(3-aminopropyl)-3-(4-(tiifluoromethoxy)|rfren^) -lH-indol-5-yl)methyl)-[3,3 ^, biazetidin]- l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Cpd 70);

5-(l ^, -((l-(3-(dimethyiamino)propyl)-3-(4-(trifluoromethoxY) phenyi)-lH-indol-5-yl)methyl)-[4,4 ^, - bipiperidin]-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (Cpd 71);

(2R,4S)-l-{(R)-2-(2-(4-((l-((l-(3-aminopropyl)-3-(4-(trif luoromethoxy)phenyl)-lH-indol-5- yl)methyl)piperidin-4-yl)methyl)piperazin-l-yl)acetamido)-3, 3-dimethylbutanoyl)-4-hydroxy- N-((R)-l-(4-(4-methylthiazoi-5-yl)phenyl)ethyl)pyrroiidine-2 -carboxamide (Cpd 72); or pharmaceutically acceptable salts, solvates, hydrates, polymorphs, tautomers, stereoisomers, isotopically enriched derivatives, or prodrugs thereof.

[0086] In certain embodiments, the bifunctional compound as described therein binds KRAS protein with a K _D value of less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 1 μM, less than about 500 nM, less than about 100 nM, less than about 50 nM.

[0087] In certain embodiments, the bifunctional compound as described therein selectively binds mutant KRAS protein over wild type KRAS protein, in some embodiments, the compound of Formula (I) selectively binds mutant KRAS protein over wild type KRAS protein.

In certain embodiments, the selectivity is between about 2-fold and about 5-fold. In certain embodiments, the selectivity is between about 5-fold and about 10-fold. In certain embodiments, tire selectivity is between about 10-fold and about 20-fold.

[0088] In certain embodiments, the bifunctional compound as described therein binds an E3 ubiquitin ligase with a K _D value of less than about 50 μM, less than about 10,000 nM, less than about 5,000 nM, less than about 1,000 nM, less than about 500 nM, less than about 100 nM, less than about 50 nM.

[0089] In certain embodiments, the bifunctional compound as described tirerein promotes tire degradation of mutant KRAS protein up to 10%, up to 20%, up to 30%, up to 40%, up to

50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100% at a concentration of 20 μM or less, 10 μM or less, 5 μM or less, 1,000 nM or less, 500 nM or less, 100 nM or less, 50 nM or less, 10 nM or less.

[0090] Pharmaceutical Compositions and Use [0091] The bifunctional compound as described therein can be therapeutically administered as the neat chemical, but it may be useful to administer the compounds as a pharmaceutical composition or formulation. Thus, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the bifunctional compound as described therein or pharmaceutically acceptable salts, tautomers, stereoisomers, solvates, hydrates, polymorphs, isotopically enriched derivatives, or prodrugs thereof, and one or more pharmaceutically acceptable excipients.

[0092] The pharmaceutical compositions can be administered in a variety of dosage forms including, but not limited to, a solid dosage form or a liquid dosage form, an oral dosage form, a parenteral dosage form, an intranasal dosage form, a suppository, a lozenge, a troche, buccal, a controlled release dosage form, a pulsed release dosage form, an immediate release dosage form, an intravenous solution, a suspension or combinations thereof. The pharmaceutical compositions can be administered, for example, by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

[0093] An "excipient" generally refers to a substance, often an inert substance, added to a pharmacological composition or otherwise used as a vehicle to further facilitate administration of a compound. Examples of excipients include, but are not limited to, inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents, preservatives, effervescent mixtures, and adsorbents.

Suitable inert diluents include, but are not limited to, sodium and calcium carbonate, sodium and calcium phosphate, lactose, and the like. Suitable disintegrating agents include, but are not limited to, starches, such as corn starch, cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, and the like. Binding agents may include, but are not limited to, magnesium aluminum silicate, starches such as com, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like. A lubricating agent, if present, will generally be magnesium stearate and calcium stearate, stearic acid, talc, or hydrogenated vegetable oils. If desired, the tablet may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. The compositions can also be formulated as chewable tablets, for example, by using substances such as mannitol in the formulation.

[0094] The term "therapeutically effective amount" refers to the amount of the bifunctional compound as described therein, alone or in combination with an anticancer agent which, upon single or multiple dose administration to the subject, provides the desired effect in the subject under treatment.

[0095] The bifunctional compound as described therein is effective in the treatment or amelioration of diseases associated with KRAS mutation. Degradation of the target protein will occur when the target protein is placed in proximity to the E3 ubiquitin ligase, thus resulting in degradation/inhibition of the effects of the target protein and the control of protein levels. The control of protein levels afforded by the present disclosure provides treatment of a disease state or condition, which is modulated through the target protein by lowering the level of that protein in the cells of a patient. The "diseases associated with KRAS mutation" may be cancers, autoimmune diseases, infectious diseases, or blood vessel proliferative disorders. Cancers may be lung cancer (e.g., non-smail cell lung cancer), colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, blader cancer, gastric cancer, renal cancer, salivary gland cancer, ovarian cancer, uterine body cancer, cervical cancer, oral cancer, skin cancer, brain cancer, lymphoma, leukemia, biliary tract malignancies, endometrial cancer, cervical cancer, or myeloid leukemia.

[0096] The bifunctional compound as described therein can be administered as the sole active agent, or administered separately, sequentially or together with one or more additional anticancer agents. The term "anticancer agents" as used herein, unless otherwise indicated, refers to agents capable of inhibiting or preventing the growth of neoplasms, or checking the maturation and proliferation of malignant (cancer) cells. Anticancer agents suitable for use in combination with the compounds of formula (I) include, but are not limited to, targeted cancer drugs, such as trastuzumab, ramucirumab, vismodegib, sonidegib, bevacizumab, everolimus, tamoxifen, toremifene, fulvestrant, anastrozole, exemestane, lapatinib, letrozole, pertuzumab, ado-trastuzumab emtansine, palbociclib, cetuximab, panitumumab, ziv- aflibercept, regorafenib, Imatinib mesylate, lanreotide acetate, sunitinib, regorafenib, denosumab, alitretinoin, sorafenib, pazopanib, temsirolimus, everolimus, tretinoin, dasatinib, nilotinib, bosutinib, rituximab, alemtuzumab, ofatumumab, obinutuxumab, ibrutinib, idelalisib, blinatumomab, soragenib, crizotinib, erlotinib, gefitinib, afatinib dimaleate, ceritnib, ramucirumab, nivolumab, pembrolizumab, osimertinib, and necitumumab; an alkylating agent, such as busulfan, chlorambucil, cyclophosphamide, iphosphamide, melphalan, nitrogen mustard, streptozocin, thiotepa, uracil nitrogen mustard, triethylenemelamine, temozolomide, and 2-chloroethyl-3-sarcosinamide-l-nitrosourea (SarCNU); an antibiotic or piant alkaloid, such as actinomycin-D, bleomycin, cryptophycins, daunorubicin, doxorubicin, idarubicin, irinotecan, L-asparaginase, mitomycin-C, mitramycin, navelbine, paclitaxel, docetaxel, topotecan, vinblastine, vincristine, teniposide (VM-26), and etoposide (VP-16); a hormone or steroid, such as 5a-reductase inhibitor, aminoglutethimide, anastrozole, bicalutamide, chlorotrianisene, diethylstilbestrol (DES), dromostanolone, estramustine, ethinyl estradiol, flutamide, fluoxymesterone, goserelin, hydroxyprogesterone, letrozole, leuprolide, medroxyprogesterone acetate, megestrol acetate, methyl prednisolone, methyltestosterone, mitotane, nilutamide, prednisolone, arzoxifene (SERM-3), tamoxifen, testolactone, testosterone, triamicnolone, and zoladex; a synthetic, such as all-trans retinoic acid, carmustine (BCNU), carboplatin (CBDCA), lomustine (CCNU), cis- diaminedichloroplatinum (cisplatin), dacarbazine, gliadel, hexamethylmelamine, hydroxyurea, levamisole, mitoxantrone, o,p‘- dichlorodiphenyldichloroethane (o,p'-DDD) (also known as lysodren or mitotane), oxaliplatin, porfimer sodium, procarbazine, and imatinib mesylate (Gleevec*); an antimetabolite, such as chlorodeoxyadenosine, cytosine arabinoside, 2'- deoxycoformycin, fludarabine phosphate, 5-fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (5-

FUdR), gemcitabine, camptothecin, 6-mercaptopurine, methotrexate, 4- methylthioamphetamine (4-MTA), and thioguanine; and a biologic, such as alpha interferon,

BCG (Bacillus Calmette-Guerin), granulocyte colony stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interleukin-2, and herceptin.

[0097] Furthermore, the present disclosure provides a method for preventing, ameliorating and/or treating diseases associated with KRAS mutation in a subject in need thereof comprising administering the bifunctional compound as described therein or a pharmaceutical composition comprising the same, to the subject.

[0098] Furthermore, the present disclosure provides a therapeutically effective amount of tiie bifunctional compound for manufacturing a medicament for preventing, ameliorating and/or treating a disease associated with KRAS mutation in a subject in need thereof.

[0099] In an additional aspect, the present disclosure provides a method of ubiquitinating and degrading a target protein in a cell by contacting the target protain with the bifunctional compound of formula (I) as described herein.

[0100] In order for the invention described herein to be more folly understood, the following examples are set forth. The examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

[0101] EXAMPLES

[0102] ABBREVIATIONS [0103] ACN: Acetonitrile; AcOH: Acetic acid; Boc: tert-butoxycarbonyl; DCM:

Dichloromethane DMA: Dimethylacetamide; DMF: Dimethylformamide; DMSO: Dimethyl Sulfoxide; DMAC/DMA: Dimethylacetamide; DIPEA/DIEA: N,N-Diisopropylethylamine; EtOAc/EA: Ethyl Acetate; EtOH: Ethanol; FA: Formic Acid; HPLC: High pressure liquid chromatography; LAH: Lithium aluminium hydride; LCMS or LC-MS: Liquid Chromatography/

Mass Spectrometry; NBS: N-Bromosuccinimide; NCS: N-Chlorosuccinimide; NMR: Nuclear

Magnetic Resonance; NMP: N-Methyl-2-pyrrolidone; MeOH: Methanol; MPLC: Medium pressure liquid chromatography; RT or r.t. : Room temperature; TEA: Triethylamine; THF:

Tetrahydrofuran; TFA: Trifluoracetic acid; TLC: Thin layer chromatography.

[0104] General Synthetic approach

[0105] The synthetic realization and optimization of the bifunctional molecules as described herein may be approached in a stepwise or modular fashion. With RBs and ULMs in hand, one skilled in the art can use known synthetic methods for their combination with or without a chemical linking group(s). Chemical linking group(s) can be synthesized with a range of compositions, lengths and flexibility and functionalized such that the RB and ULM groups can be attached sequentially to distal ends of Linker. In some instances, protecting group strategies and/or functional group interconversions (FGIs) may be required to facilitate the preparation of the desired materials. Such chemical processes are well known to the synthetic organic chemist and many of these may be found in texts or books.

[0106] Example ISynthesls of cpd 1

[0107] Cpd 1 can be prepared using the synthetic schemes and procedures described in detail below.

[0108] Synthesis of (B)

[0109] Sodium triacetoxyborohydride (NaBH(OAc) ₃ , 32.3 mg, 0.415 mmol, 2 eq) was added to a solution of 2-(2,6-dioxopiperidin-3-^)-5-(piperazin-l-yl) isoindoline-1, 3-dione (compound

A, 100 mg, 0.228 mmol, 1.1 eq) and tert-butyl 4-(4-formylbenzyl)piperazine-l-carboxylate (63.0 mg, 0.207 mmol, 1.0 eq) in DMF (2.2 ml) and stirred for 2 hours. Upon completion, the solvent was removed by vacuum, and the crude material was purified by silica gel chromatography (0 to 5% MeOH in DCM) to yield the Boc-intermediate (89.2 mg, 62%). To a solution of the Boc-intermediate was added TEA (155 μL, 2.02 mmol, 15eq). The reaction mixture was stirred at RT for 2 h. Upon completion, the solvent was removed by rotary evaporator, and the crude material was purified by silica gel chromatography (Oto 20% MeOH in DCM) to yield compound (B), 2-(2,6-djoxopiperidin-3-yi)-5-(4-(4-(piperazin-l- ylmethyl)benzyl)piperazin-l-yl)isoindoline-l, 3-dione (71.5 mg, 99%). ¹ H NMR (600 MHz,

DMSO-d ₆ ) 6 11.10 (s, 1H), 8.68 (s, 2H), 7.75 (s, 1H), 7.42-7.32 (m, 6H), 5.09 (dd, J = 12.9, 5.4

Hz, 1H), 4.38-4.22 (m, 3H), 3.59 (s, 2H), 3.11 (s, 4H), 2.92-2.85 (m, 1H), 2.62 - 2.52 (m, 6H), 2.06 - 1.98 (m, 1H). LC-MS (m/z): [M] ⁺ calcd for 530.63, found 531.52.

[0110] Synthesis of (D)

[0111] Compound B (71.5 mg, 0.135 mmol, 1 eq) was added to a solution of tert-butyl (3-(5- formyl-3-(4-(trifluoromethoxy)phenyl)-lH-indol-l-yl)propyl)c arbamate (compound C, 62.4 mg, 0.135 mmol, 1 eq) in DMF (1.3 ml) and stirred for 30 minutes. Sodium triacetoxyborohydride (NaBH(OAc) ₃ , 57.2 mg, 0.27 mmol, 2eq) was added and stirred for an additional 16 hours. Upon completion, the solvent was removed by vacuum, and the crude material was purified by silica gel chromatography (0 to 7% MeOH in DCM) to yield compound

(D), tert-butyl (3-(5-((4-{4-((4-(2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-5-yl)piperazin- l-yl)methyl)benzyf)piperazin-l-yl)methyl)-3-(4-(trifluoromet hoxy)phenyf)-lH-indol-l- yl)propyl)carbamate (66.0 mg, 50%). NMR (600 MHz, DMSO-d ₆ ) 611.09 (s, 1H), 7.82-7.73 (m, 3H), 7.67 (d, J = 8.5 Hz, 1H), 749 (s, 1H), 7.46 - 7.41 (m, 2H), 7.32 (d, J = 2.2 Hz, 1H), 7.30

- 7.20 (m, 5H), 7.18 (s, 1H), 7.00 (t, J = 5.6 Hz, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 4.21 (t, J =

6.9 Hz, 2H), 3.49 (s, 2H), 3.43 (m, 5H), 2.96 - 2.85 (m, 3H), 2.62 - 2.52 (m, 2H), 2.47 (br, 4H), 2.36 (br, 4H), 2.04 - 1.98 (m, 1H), 1.91-1.88 (m, 4H), 1.37 (s, 9H). LC-MS (m/z): [M] ⁺ calcd for

C ₅₃ H ₅₉ F ₃ N ₈ O ₇ , 977.10, found 977.59.

[0112] Synthesis of cpd 1

[0113] To a solution of the compound D (63 mg, 0.0644 mmol) in CH ₂ Cl ₂ (0.2 ml) was added

TFA (100 μL, 1.29 mmol, 20eq). The reaction mixture was stirred at RT for 2 h. Upon completion, the solvent was removed by rotary evaporator, and the crude material was purified by silica gel chromatography (0 to 20% MeOH in DCM) to yield cpd 1, 5-(4-(4-((4-((l- (3-aminopropyl)-3-(4-(trifluoromethoxy)phenyi)-lH-indol-5-yl )methyl)piperazin-l- yl)methyl)benzyl)piperazin-l-yl)-2-(2,6-dioxopiperidin-3-yl) isoindoline-l, 3-dione (55.9 mg, 98%). ¹ H NMR (600 MHz, DMSO-d ₆ ) 611.10 (s, 1H), 7.89-7.69 (m, 8H), 7.46-7.34 (m, 8H), 5.08

(q, J = 6.0 Hz, 1H), 4.45-4.11 (m, 7H), 3.56 (br, 2H), 3.17 (s, 4H), 3.09-3.07 (br, 2H), 2.91-2.89

(m, 2H), 2.82-2.81 (m, 2H), 2.61-2.57 (m, 2H), 2.32 (br, 2H), 2.08 (m, J = 7.2 Hz, 2H), 2.02 (t, J - 5.4 Hz, 1H). LC-MS (m/z): [M] * calcd for C ₄₈ H ₅₁ F ₃ N ₈ O ₅ 876.39, found 877.38.

[0114] Example 2 Synthesis of cpd 2

[0115] Cpd 2 can be prepared using the synthetic schemes and procedures described in detail below.

[0116] Synthesis of (E)

[0117] l-(tert-butoxycarbonyi)-4-((tert-butoxycarbonyl)amino)piperi dine-4-carboxyiic acid(14 mg, 0.04 mmol) was added with HBTU (21 mg, 0.056 mmol), DMF (0.4 ml) and DIPEA

(0.026mL, 0.15 mmol), and the solution was stirred under Ar atmosphere for 10 min at 25°C.

5-(4-(4-((4-((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)ph enyl)-lH-indol-5-yl) methyl)piperazin-l-yl)methyi)benzyl)piperazin-l-yl)-2-(2,6-d ioxopiperidin-3-yl)isoindoline- l,3-dione(33 mg, 0.038 mmol)was added, then the resulting mixture was stirred for 2 h at

25°C. The reaction mixture was diluted to 10 ml with acetonitrile and purified via preparative HPLC and concentrated to provide the title compound (8 mg, 18%). LC-MS (m/z): [M] ⁺ calcd for C ₆₄ H ₇₇ F ₃ N ₁₀ O ₁₀ , 1203.37, found 1203.75. NMR (600 MHz, DMSO-d ₆ ) S 11.11 (s, 1H), 9.98

(s, 1H), 9.42 (s, 1H), 8.03 (s, 1H), 7.89 (s, 1H), 7.77 (m, 3H), 7.69 (s, 1H), 7.46 (m, 6H), 7.37 -

7.27 (m, 2H), 7.07 (s, 1H), 5.10 (dd, J = 12.9, 5.4 Hz, 1H), 4.42 (d, J = 42.2 Hz, 4H), 4.24 (s, 4H),

3.61 (s, 4H), 3.10 (m, 4H), 2.95 - 2.88 (m, 2H), 2.07 - 1.96 (m, 3H), 1.96 - 1.82 (m, 4H), 1.80 -

1.73 (m, 2H), 1.39 (s, 9H).

[0118) Synthesis of cpd 2

[0119] To a solution of tert -butyl 4-((tert-butoxycarbonyl)amino)-4-((3-(5- ((4-(4-((4-(2-(2,6- dioxopipe ridin-3-yl)-l,3-dioxoisoindolin-5-yl)piperazin-l-yl)methyl)b enzyl)piperazin-l- yl)methyl)-3-(4-(trifluoromethoxy)phenyl)-lH-indol-l-yl)prop yl)carbamoyl)piperidine-l- carboxylate(5 mg, 0.04 mmol) in CH ₂ CI ₂ (0.1 ml) was added TFA (0.05 mL, 0.62 mmol). The reaction mixture was stirred at RT for 1 h. The mixture was neutralized with NaHCO _3(sat) and diluted with CH ₂ CI ₂ . The CH ₂ CI ₂ was dried with MgSO«, concentrated, and the crude material was concentrated to get cpd 2, 4-amino-N-{3-(5-((4-(4-((4-(2-(2,6-dioxo piperidin-3-yl)-l,3- dioxoisoindolin-5-yl)piperazin-l-yl)methyl)benzyl)piperazin- l-yl) methyl)-3-{4-

(trifluoromethoxy)phenyl)-lH-indol-l-yi)propyl)piperidine -4-carboxamide(4 mg, 96%). ¹ H

NMR (600 MHz, Methanol-d«) 6 8.02 (s, 1H), 7.81 - 7.76 (m, 1H), 7.73 - 7.68 (m, 1H), 7.60 (d,

J = 8.0 Hz, 1H), 7.38 (m, 4H), 7.26 (dd, J = 8.6, 2A Hz, 1H), 5.36 (ddd, J = 5.7, 4.4, 1.1 Hz, 2H),

5.09 (dd, J = 12.8, 5.5 Hz, 1H), 4.64 (s, 6H), 4.34 (t, J = 6.9 Hz, 1H), 3.77 (d, J = 25.0 Hz, 2H), 3.69

- 3.65 (m, 1H), 3.53 (s, 2H), 3.30- 3.26 (m, 2H), 2.92-2.71 (m, 5H), 2.23 - 2.19 (m, 2H), 2.16

- 2.10 (m, 2H), 2.07 - 2.03 (m, 4H), 1.66 - 1.59 (m, 4H). LC-MS (m/z): [M] ⁺ cald for

C ₅₄ H ₆₁ F ₃ N ₁₀ O ₆ , 1003.14, found 1003.59.

[0120] Example 3 Synthesis of cpds 3 to 72

[0121] Cpd 3 can be prepared using the synthetic schemes and procedures described in detail below.

[0122] Synthesis of (G)

[0123] Compound F (70 mg, 0.147 mmol, 1 eq) was added to a solution of tert-butyl (3-{5- formyl-3-{4-(trifluoromethoxy)phenyl}-lH-indol-l-yl)propyl)c arbamate (compound C, 67.9 mg, 0.147 mmol, 1 eq) in DMF (1.4 ml) and stirred for 30 minutes. Sodium triacetoxyborohydride (NaBH(OAc) ₃ , 62.3mg, 0.294 mmol, 2 eq) was added and stirred for an additional 16 hours. Upon completion, the solvent was removed by vacuum, and the crude material was purified by silica gel chromatography (Oto 5% MeOH in DCM) to yield compound

(G) tert-butyl (3-(5-((4-((l-(2-(2,6-dioxopiperidin-3-yl)-l,3- dioxoisoindolin-5-yl)piperidin-4- yi)methyl)piperazin-l-yl)methyl)-3-(4-(trifluoromethoxy)phen yl)-lH-indol-l- yl)propyl)carbamate (52.8 mg, 41%). ¹ H NMR (600 MHz, Met hanol-d,) 57.92 (d, J= 1.5 Hz, 1H),

7.79 - 7.74 (m, 2H), 7.64 (d, J = 8.5 Hz, 1H), 7.60 (s, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.36 - 7.29 (m,

3H), 7.27 (dd, J = 8.5, 1.6 Hz, 1H), 7.18 (dd, J = 8.7, 2.4 Hz, 1H), 5.05 (dd, J = 12.7, 5.5 Hz, 1H), 4.28 (t, J = 6.9 Hz, 2H), 4.04 - 3.95 (m, 4H), 3.07 (t, J = 6.7 Hz, 2H), 2.99 - 2.92 (m, 3H), 2.91 -

2.80 (m, 4H), 2.76 - 2.65 (m, 4H), 2.32 (d, J = 6.7 Hz, 2H), 2.13 - 2.06 (m, 1H), 2.03 (p, J = 6.8 Hz, 2H), 1.94 (s, 3H), 1.88-1.85 (m, 3H), 1.44 (s, 9H), 1.30 - 1.22 (m, 2H). LC-MS (m/z): [M] * calcd for C47H54F3N7O7, 885.99, found 886.45.

[0124] Synthesis of cpd 3

[0125] To a solution of the compound G (40 mg, 0.0451 mmol) in CH ₂ CI ₂ (0.15 ml) was added

TFA (69.1 pl, 0.903 mmol, 20eq). The reaction mixture was stirred at RT for 2 h. Upon completion, the solvent was removed by rotary evaporator, and the crude material was purified by silica gel chromatography (Oto 20% MeOH in DCM) to yield cpd 3, 5-(4-((4-((l-(3- aminopropyl)-3-(4-{trifiuoromethoxy)phenyi)-lH-indol-5-yl)me thyl)piperazin-l- yl)methyl)piperidin-l-yl)-2-(2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (13.0 mg, 37%). ¹ H

NMR (600 MHz, MeOD-d«) 6 7.84 (s, 1H), 7.76-7.74 (m, 2H), 7.63 (d, J = 8.6 Hz, 1H), 7.55 (s,

1H), 7.47 (d, J - 8.6 Hz, 1H), 7.33-7.31 (m, 3H), 7.23 (dd, J = 1.4, 8.5 Hz, 1H), 7.18 (dd, J = 2.4, 8.7 Hz, 1H), 5.05 (dd, J = 5.5, 12.7 Hz, 1H), 4.61 (br, 3H), 4.31 (t, J = 6.9 Hz, 2H), 4.01 (d, J = 13.1

Hz, 2H), 3.66 (br, 2H), 2.97-2.93 (m, 2H), 2.85-2.81 (m, 1H), 2.74-2.68 (m, 4H), 2.54 (br, 6H), 2.23 (d, J = 6.8 Hz, 2H), 2.10-2.04 (m, 3H), 1.88-1.82 (m, 3H), 1.28-1.26 (m, 3H). LC-MS (m/z): [M] ⁺ calcd for C ₄₂ H ₄₆ F ₃ N ₇ O ₅ , 785.87, found 786.36.

[0126] Example 4 Synthesis of cpd 24

[0127] Cpd 24 can be prepared using the synthetic schemes and procedures described in detail below.

[0128] Synthesis of (H-2)

[0129] To a solution of the compound C (400 mg, 0.865 mmol, 1.1 eq) and Ethyl piperazinoacetate (129.2 uL, 0.786 mmol, 1.0 eq) in DMF (3.93 ml) was added Sodium triacetoxyborohydride (NaBH(OAc) ₃ , 733.3 mg, 3.46 mmol, 4 eq) and stirred for 18 hours in room temprature. Upon completion, the solvent was removed by vacuum, and the crude material was purified by silica gel chromatography (0 to 5% MeOH in DCM) to yield the ester- intermediate (H-2) Ethyl 2-(4-((l-{3-((tert-butoxycarbonyi)amino)propyi)-3- (4-

(trifluoromethoxy)phenyl)-lH-indol-5-yl)methyl)piperazin- l-yi)acetate (357.5 mg, 67%). LC- MS (m/z): [M]+ calcd for C ₃₂ 4 ₁₉ F ₃ N ₄ O ₅ , 618.70, found 619.07.

[0130] Synthesis of (H-3)

[0131] To a solution of the compound H-2 (70 mg, 0.113 mmol, 1.0 eq) in THF/ EtOH (0.6 mL/

0.2 ml) was added Lithium hydroxide (LiOH, 14.2 mg, 0.339 mmol, 3.0 eq)in H ₂ O (0.2 ml) and stirred for 4 hours at RT. Upon completion, the mixture was adjust pH to 4-5 with IN HCI in an ice bath. Collected the solids by filtration and wash with H ₂ O to yield (H-3), 2-(4-((l-(3-((tert- butoxycarbonyl)amino)propyl)-3-(4-(trifluoromethoxy)phenyl)- lH-indol-5- yl)methyl)piperazin-l-yl)acetic acid (37.3 mg, 55.9%). LC-MS (m/z): [M] * calcd for C ₃₀ H ₃₇ F ₃ N ₄ O ₅ ,

590.64, found 590.92.

[0132] Synthesis of (H-4) [0133] To a solution of H-3 (37.0 mg, 0.0626 mmol) in DMF (03 ml) was added with HBTU

(28.5 mg, 0.0751 mmol, 1.2 eq) and DIPEA (0.016 mL, 0.113 mmol, 1.8 eq), and the solution was stirred under Ar atmosphere for 10 min at RT. 2 -(2, 6-dioxopiperidin-3-yl)-5-(piperazin-l- yl)isoindoline-13-dione (23.6 mg, 0.0689 mmol, 1.1 eq) was added, then the resulting mixture was stirred for 16 h at RT. Upon completion, the solvent was removed by vacuum, and the crude material was purified by silica gel chromatography (0 to 6% MeOH in DCM) to yield compound (H-4), tert-butyl (3-(5-((4-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoind olin-5- yl)piperazin-l-yl)-2-oxoethyl)piperazin-l-yi)methyi)-3-(4-(t rifluoromethoxy)phenyl)-lH-indol- l-yl)propyl)carbamate (21.3 mg, 37.2%). LC-MS (m/z): [M] ⁺ calcd for C ₄₇ H ₅₃ F ₃ N ₈ O ₈ , 914.98, found 915.26

[0134] Synthesis of cpd 24

[0135] Compound H-4 (20 mg, 0.0219 mmol) was added 4N HCI in dioxane (0.24 ml). The reaction mixture was stirred at RT for 16 h. The reaction mixture was diluted to 1 ml with acetonitrile and purified via preparative HPLC and concentrated to provide cpd 24, 5-(4-(2-(4-

((l-(3-aminopropyl)-3-(4-(trifluoromethoxy)phenyl)-lH-ind ol-5-yl)methyl)piperazin-l- yl)acetyl)piperazin-l-yl)-2-{2,6-dioxopiperidin-3-yl)isoindo line-l, 3-dione (4.4 mg, 24.7%). LC- MS (m/z): [Mp calcd for C ₄₂ H ₄₅ F ₃ N ₈ O ₆ , 814.87, found 815.66. ¹ H NMR (500 MHz, Methanol-d ₄ ,)

68.04 (d, J = 1.6 Hz, 1H), 7.79 - 7.73 (m, 2H), 7.71 (d, 1 = 8.5 Hz, 1H), 7.68 - 7.61 (m, 2H), 7.41

- 7.32 (m, 4H), 7.25 (dd, J = 8.6, 2.4 Hz, 1H), 5.08 (dd, J = 12.6, 5.5 Hz, 1H), 4.48 - 433 (m, 4H),

3.79 - 3.67 (m, 4H), 3.56 - 3.45 (m, 6H), 3.03 - 2.91 (m, 3H), 2.91-2.81 (m, 2H), 2.79 - 2.65 (m,

2H), 2.24 (p, J = 7.1 Hz, 2H), 2.15-2.07 (m, 1H).

[0136] Example 5 Synthesis of cpd 26

[0137] Cpd 26 can be prepared using the synthetic schemes and procedures described in detail below.

[0138] Synthesis of (J) [0139] To a solution of compound F (90 mg, 0.188 mmol, 1.0 eq.), HBTU (82 mg, 0.216 mmol) and DIPEA (49 mg, 0.376 mmol) in DMF (1.25 ml) was added compound I (121 mg, 0.226 mmol,

1.2 eq.) and stirred for 16 hours. Upon completion, the solvent was removed by vacuum, and the crude material was purified by silica gel chromatography (0 to 5% MeOH in DCM) to yield compound (J) tert-butyl (3-(5-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindoli n-5- yl)piperazin-l-yl)methyl)piperidine-l-carbonyl)-3-(4-(triflu oromethoxy)phenyl)-lH-indol-l- yl)propyl)carbamate (39 mg, 80%).LC-MS (m/z): [M] + calcd for C ₄₇ H ₅₂ F ₃ N ₁₇ O ₈ , 899.97, found

900.59.

[0140] Synthesis of cpd 26

[0141] To a solution of the compound J (55 mg, 0.061 mmol) in ethyl acetate (0.61 ml) was added TFA (1.23 ml, 20eq). The reaction mixture was stirred at RT for 2 h. Upon completion, the solvent was removed by rotary evaporator, and the crude material was purified by silica gel chromatography (0 to 20% MeOH in DCM) to yield cpd 26, 5-(4-((l-(l-(3-aminopropyl)-3-

(4-(trifluoromethoxy)phenyl)-lH-indole-5-carbonyl)piperid in-4-yl)methyl)piperazin-l-yl)-2- (2, 6-dioxopiperidin-3-yl)isoindoline-l, 3-dione (39 mg, 80%). ¹ H NMR (500 MHz, Methanol-d*)

6 7.99 (d, J = 1.1 Hz, 1H), 7.82 - 7.75 (m, 3H), 7.73 (s, 1H), 7.71 - 7.65 (m, 1H), 7.51 (d, J - 2.4

Hz, 1H), 7.43 - 7.35 (m, 4H), 5.12 (dd, J = 12.5, 5.5 Hz, 1H), 4.46 (t, J = 6.9 Hz, 2H), 4.19 (d, J =

13.9 Hz, 2H), 3.82 - 3.75 (m, 2H), 3.48 (t, J = 12.9 Hz, 2H), 3.33 - 3.28 (m, 2H), 3.24 (d, J = 6.9

Hz, 2H), 3.03 - 2.96 (m, 2H), 2.95 - 2.84 (m, 1H), 2.82 - 2.68 (m, 2H), 2.39 - 2.25 (m, 3H), 2.19

- 2.10 (m, 1H), 2.04 (s, 1H), 1.92 - 1.88 (m, 1H), 1.51 - 1.36 (m, 2H), 1.27 (t, J = 7.2 Hz, 1H). LC- MS (m/z): [M]* calcd for C ₄₂ H ₄₄ F ₃ N ₇ O ₆ , 799.85, found 800.43.

[0142] Example compound 4-72 in Table 1 were prepared in an analogous manner to compound 3, employing the corresponding aldehyde starting materials, like cpd C, and amine, like cpd F.

0143] Table 1 _

Example 6 Biological activity

[0145] Various RAS-PROTACs were tested for their specificity and abilities for degrading the targeted protein. Brief descriptions of different assays are described below.

[0146] Western blot to evaluate the cellular potencies of RAS-PROTACs in KRAS protein degradation

[0147] For western blot experiments, BT-474 (KRAS ^WT ) and MIA PaCa-2(KRAS ^G12C ) cells were cultured in DMEM medium with 10% FBS. BxPC3 (KRAS ^WT ) and KLM-1 (KRAS ^G12D ) cells were cultured in DMEM medium with 10% FBS. MDA-MB-231 (KRAS ^G13D ) cells were cultured in L15 medium with 10% FBS. BT-474, MIA PaCa-2, BxPC3 and MDA-MB-231 were established from the American Type Culture Collection. KLM-1 were established from the ExPASy. On the assay day, two hundred thousand cells were pretreated with each of the test compounds for 24 hours. After 24 hours, the whole cell lysate was harvested by adding 2x SDS Sample Buffer.

Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane.

Protein expression was detected using immunoblot with various primary antibodies and secondary antibodies following standard protocols. Antibody against KRAS was purchased from Abeam (Cambridge, UK). Anti-rabbit IgG, HRP-linked secondary antibodies was purchased from Cell Signaling Technology (Danvers, MA). Antibody against actin was purchased from Millipore (Burlington, MA). Immunoblots were revealed by chemiluminescence (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher, Waltham, MA) and detected by ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA). Band intensities of western blot were also quantified by ChemiDoc™ MP Imaging System. Relative intensities of bands corresponding to the drug treatment group were compared to those of the untreated group.

[0148] The primary screening of bifunctional compounds 1-21 degraded KRAS protein in

Calu-1, H358, H441, KLM-1, MDA-MB-231 and HCT-116 cell line were as shown in Tables 2 which tested under the conditions described above.

[0149] Table 2

[0150] Example 7 Anti- pro l lferathon activity [0151] As noted above, KRAS-PROTACs of the invention may be used to treat diseases or disorders harboring a specific KRAS mutation. The diseases may be cancers, autoimmune diseases, infectious diseases, or blood vessel proliferative disorders. The cancers may be lung cancer (e.g., non-small cell lung cancer), colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, gastric cancer, renal cancer, salivary gland cancer, ovarian cancer, uterine body cancer, cervical cancer, oral cancer, skin cancer, brain cancer, lymphoma, leukemia, biliary tract malignancies, endometrial cancer, cervical cancer, or myeloid leukemia. Inhibitions of cell growth by KRAS-PROTACs of the invention were measured using CellTiter™-96 assay. The cytotoxicities of KRAS-PROTACs were evaluated in lung cancer cell lines, breast cancer cell lines and pancreatic cancer cell lines with different RAS mutations. The results were as shown in Table 3.

[0152] Table 3

[0153] Example 6 Xenograft mode! of Ras-PROTAC (Lung cancer)

[0154] The aim of this study was to evaluate the in vivo anti-tumor efficacy of Ras-PROTAC in

Calu-1 human lung cancer xenograft model in male NOD SCID mice.

[0155] Test articles Cpd 6 and corresponding vehicle were formulated and given intra-tumor

(IT) injection to mice once daily for consecutive 14 days. The Calu-1 cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO ₂ in air. The tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA treatment The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. Male NOD SCID mice at age of 6-7 weeks were purchased from BioLasco Taiwan Co., LTD. and quarantined for one week. Six mice were housed in each cage. All animals were hosted in the animal facility with a 12-h light/12-h dark cycle at 19-25°C. Animals had free access to rodent pellet foods and water ad libitum. Calu-1 cells were subcutaneously (SC) implanted (5 x 10 ⁶ cells in 1:1 PBS/matrigel mixture at 0.1 ml per mouse) into the right flank of male NOD SCID mice. When the average tumor volume had reathed 170 mm ³ , the mice were randomly divided into 2 groups (N=6 per group). Vehide or Cpd 6 (10 mg/kg) were given intra-tumor (IT) injection once daily for consecutive 14 days. The tumor volumes, body weights, mortality, and signs of overt toxicity were monitored and recorded three times weekly for 28 days. Tumor volumes (mm ³ ) were measured three times per week using calipers and calculated according to the formula: Tumor Volume = (w ² x/)/2, where w = width and I = length in diameter (mm) of the tumor. The percentages of tumor growth inhibition (TGI) were calculated using the following formula: %TGI = [1 - (T/C)] x 10096, where T and C represent the mean tumor volumes of the treatment group and the control group, respectively. The results are shown in Table 4 and FIGs. 1 and 2. Student's t test was applied for comparison between the vehicle and test article-treated groups. Differences are considered significant at *P<0.05. Animals were weighed three times weekly until the completion of the study. The body weight changes were calculated as percentage increases in tiie body weights, as compared with the initial body weights.

[0156] Table 4

[0157] FIG. 1 shows tumor growth curve in Calu-1 implanted male NOD SCID mice. Test articles Cpd 6 at 10 mg/kg significantly reduced Calu-1 tumor growth from Day 3 to Day 28, with the TGI values ranged from 56% to 93%> and was considered as very high efficacious to

Calu-1 xenografts.

[0158] FIG. 2 shows body weight changes in Calu-1 implanted male NOD SCID mice. No body weight loss was observed throughout the experiment.

[0159] The KRAS-PROTACs of the invention are promising new therapeutics for cancer patients with KRAS mutation.

[0160] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, tiie specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims.

Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded, even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0161] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.