WO2024123819A2

WO2024123819A2 - Small molecule degraders of c-src kinase

Info

Publication number: WO2024123819A2
Application number: PCT/US2023/082583
Authority: WO
Inventors: Wuxiang MAO; Matt SOELLNER
Original assignee: University of Michigan System
Current assignee: University of Michigan System
Priority date: 2022-12-05
Filing date: 2023-12-05
Publication date: 2024-06-13
Anticipated expiration: 2025-06-05
Also published as: EP4629981A2; WO2024123819A3

Abstract

Provided herein are proteolysis-targeting chimera capable of degrading c-Src and methods for the prevention and/or treatment of cancer therewith.

Description

SMALL MOLECULE DEGRADERS OF C-SRC KINASE

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority to U.S. Provisional Patent Application Serial Number 63/386,084, filed December 5, 2022, which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under grant 5R01GM125881 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

BACKGROUND

Over 400 diseases have been linked to aberrant protein kinase activity, including diabetes, abnormal inflammation, and many forms of cancer, including breast cancer [Ref. 1; incorporated by reference in its entirety]. Many kinases have been nominated as putative disease targets based on gene knockdown studies; however, pharmacological inhibition has not always recapitulated the knockdown studies.

Proteolysis targeting chimeras (PROTACs) have recently emerged as a compelling technology to degrade a target protein using a small molecule entity [Ref. 2; incorporated by reference in its entirety], PROTACs are bifunctional molecules that connect a ligand of a target protein to an E3 ubiquitin ligase (E3L). The degradation of the target protein is initiated when the PROTAC molecule forms a ternary complex between the target protein and the E3L, and the E3L ubiquitinates the target protein. Upon ubiquitination, the target protein is degraded by the 26S protcosomc. In other words, PROTACs represent a pharmacological knockdown system that leverages the naturally occurring ubiquination machinery. c-Src, a tyrosine kinase, was the first proto-oncogene discovered and is frequently over- expressed in cancers [Refs. 3-5; incorporated by reference in its entirety]. While the mechanisms remain poorly understood [Ref. 6; incorporated by reference in its entirety], the extent of c-Src over-expression typically correlates with the metastatic potential of the malignant tumor and inhibition of c-Src has been shown to decrease breast cancer metastases in mice [Ref. 4; incorporated by reference in its entirety], c-Src was nominated as a promising target in several cancers, including triple-negative breast cancer (TNBC) and basal bladder cancers, based on genetic knockdown studies. Upon knockdown (e.g., with siRNA), TNBC and basal bladder cancers exhibit decreased proliferation and invasion properties. Unfortunately, studies with small molecule inhibitors of c-Src (including: dasatinib, bosutinib, and ponatinib), failed to recapitulate the phenotype observed from genetic knockdown of c-Src, and were not successful in the clinic.

SUMMARY

In some embodiments, provided herein are compositions comprising a proto-oncogene tyrosine-protein kinase Src (c-Src) ligand covalently tethered to a ubiquitin E3 ligase (E3L) ligand. In some embodiments, the c-Src ligand is dasatinib, bosutinib, ponatinib, or a dasatinib derivative, bosutinib derivative, or ponatinib derivative capable of binding to c-Src. In some embodiments, the c-Src ligand is of the formula:

; wherein X is N or CH, wherein Q is H or Cl; wherein A is -L-phenyl, wherein L is -O- or -NHC(O)-, and wherein phenyl is optionally further substituted. In some embodiments, the phenyl of -L-phenyl is substituted at the meta position. In some embodiments, the phenyl of -L-phenyl is substituted with -CF3). In some embodiments, the E3L ligand is selected from thalidomide, pomalidomide, lenalidomide, iberdomide, (S,R,S)-AHPC-Me hydrochloride, (S,R,S)-AHPC-Me dihydrochloride, cereblon modulator 1, thalidomide-propargyl, (S,R,S)-AHPC-propargyl, (S,R,S)-AHPC hydrochloride, CC-885, thalidomide-O— COOH, lenalidomide hemihydrate, thalidomide fluoride, thalidomide- OH, lenalidomidc-Br, thalidomide D4, lenalidomide hydrochloride, (S,R,S)-AHPC-Mc, clAPl ligand 1, TD-106, E3 ligase Ligand 8, E3 ligase Ligand 9, E3 ligase Ligand 10, E3 ligase Ligand 13, E3 ligase Ligand 14, E3 ligase Ligand 18, BC-1215, VHL ligand 1 (VHL-1), VHL ligand 2 (VHL-2), VHL Ligand 8 (VHL-8), VH032, VH032-cyclopropane-F, VH032 thiol, VH-298, VL-

269, VL-285, LCL161, hydroxyproline-based ligands, HIE- 1α-derived (R)-hydroxyproline, Nutlin carboxylic acid, (4R,5S)-Nutlin carboxylic acid, (S,R,S)-AHPC-Boc, AR antagonist 1, NV03, (S,R,S)-AHPC TFA, (S,R,S)-AHPC, β-Naphthoflavone-CH2-Br,β-Naphthoflavone- CH2-OH, Bestatin-amido-Me, MV-l-NH-Me, (S,S,S)-AHPC hydrochloride, and clAPl ligand 2. In some embodiments, the E3L ligand is selected from:

. In some embodiments, the c-Src ligand is covalently tethered to the E3L ligand by a linker. In some embodiments, the linker is alkyl or heteroalkyl chain of 1-25 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or ranges therebetween) in length which may be optionally substituted. In some embodiments, the compound is selected from:

In some embodiments, provided herein are methods of degrading a c-Src protein comprising contacting the c-Src protein with a composition comprising a compound described herein. In some embodiments, the c-Src protein is within a cell or a subject.

In some embodiments, provided herein are methods of treating or preventing cancer in a subject, the method comprising administering a pharmaceutical composition comprising a compounds herein to the subject. In some embodiments, the pharmaceutical composition is co- administered with one or more additional therapies for the treatment or prevention of cancer. In some embodiments, the one or more additional therapies are selected from a chemotherapeutic, an immunotherapeutic, surgery, and radiation.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Library of putative c-Src PROTACs using dasatinib and varied E3 ligase ligands. Figure 2. c-Src PROTACs require binding to both E3 ligase and c-Src for cellular degradation of c-Src.

Figure 3. Conformation-selective PROTACs with kinome-wide selectivity.

Figure 4A-D. Conformation- selective PROTACs have differential ability to degrade c- Src and possess differential selectivity of degradation. (A) Conformation- selective PROTACs have differential abilities to degrade c-Src and Bcr-Abl in cell lines. (B) c-Src DC50 values for conformation-selective PROTACs in CAL148 cells. (C) Time-course for cellular degradation of c-Src. (D) Broad proteome screening using RPPA to determine degradation selectivity.

Figure 5A-D. Conformation- selective PROTACs have differential properties in cancer cell models. (A) In MDA-MB-231 cells, conformation-selective inhibitors have differential growth inhibition activity. (B) In HT29 cells, p38-based degraders have selective activity. (C) PROTAC and inhibitor washout resulting in non-physiological levels of c-Src. (D) c-Src PROTAC is advantageous over c-Src inhibitor in CAL51 proliferation assay.

Figure 6A-B. P38-alpha and p38-beta degradation by DAS-DFGO-5-oCRBN: (A) Blots, (B) graph.

Figure 7. Blot of PLC-gamma after treatment with DAS-DFGO-5-oCRBN.

Figure 8. BT-549 cells were implanted in the mammary fat pad of nude mice. After tumors were palpable, mice were divided into three groups and treated (via intraperitoneal injection) every other day with either vehicle (10% DMSO, 90% PEG-400), 40-6-19 (active c- Src PROTAC, 15 mg/kg in vehicle), or 40-6-19-2 (inactive c-Src PROTAC control, 15 mg/kg in vehicle). Treatment was performed for 28 days, after which tumors were monitored for one additional week. Tumor volumes were determined via caliper measurements (n=3).

Figure 9A-C. (A) Time course for c-Src degradation. CAL148 cells are treated with 100 nM of either 40-6-19, 40-6-19-2, or Dasatinib and c-Src levels are measured to determine the kinetics for protein degradation. (B) Determination of duration of action for c-Src PROTACs. CAL148 cells are treated with 100 nM 40-6-19, 40-6-19-2, or Dasatinib for 18 hours and then cells are washed every 24 hours with media containing no compound. Every 24 hours c-Src protein levels are measured. c-Src levels do not return to normal until 4 days after treatment and washout. (C) The mechanism of c-Src PROTACs requires binding c-Src and cereblon.

Figure 10. Quantitative mass spectrometry proteomics reveals 40-6-19 is a selective degrader for c-Src. Only c-Src and EPHB2, a kinase also bound by Dasatinib, are degraded by 40-6-19. Figure 11 . 40_6_19 plasma concentration-time profile over time for mice following IV, IP and PO administration (n=3).

Figure 12A-C. Extracted Mass Chromatograms of Blank Plasma Sample. (A) Blank Plasma. (B) 40-6-19 calibration standard (1000 ng/mL) spiked in blank plasma. (C) Sample IV- 5MIN-1: 40-6-19 concentration 1010 ng/mL.

Figure 13. Calibration Curve for plasma, constructed in a 40-6-19 concentration range of 2-5000 ng/mL r = 0.9973.

DEFINITIONS

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a PROTAC compound” is a reference to one or more PROTAC compounds and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited fcaturc(s), clcmcnt(s), method stcp(s), etc. and any additional feature(s), clcmcnt(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.

As used herein, the term “proteolysis targeting chimera” (“PROTAC”) refers to a compound comprising two functional moieties, a target (e.g., c-Src) binding moiety and a degradation moiety (e.g., E3 ligase (E3L) ligand), tethered together, for example, by a suitable linker. PROTACs bind to a target molecule (e.g., c-Src) and signal for degradation of the target molecule (e.g., by recruitment of the E3 ligase, resulting in ubiquitination and subsequent degradation of the target protein by the proteasome). PROTACs may inhibit the activity of the target through their binding to a target active site (e.g., as with a conventional enzyme inhibitor) or may bind to the target without significant inhibition of activity. In some embodiments, the compounds described herein are PROTACs or c-Src PROTACs.

As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.

As used herein, the term “subject at risk for a disease,” for example, “a subject at risk for cancer” refers to a subject with one or more risk factors for developing the disease (e.g., cancer). Depending upon the specific disease, risk factors may include, but are not limited to, gender, age, genetic predisposition, environmental exposures, infections, and previous incidents of diseases, lifestyle, etc.

As used herein, the term “effective amount” refers to the amount of a composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdcrmal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) (e.g., c-Src degrader and one or more additional therapeutics) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety. As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. As is known to those of skill in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2- sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NW4+, wherein W is Cl -4 alkyl, and the like.

DETAILED DESCRIPTION

In some embodiments, provided herein are bifunctional PROTAC degraders of c-Src kinase. In some embodiments, methods are provided for the treatment of diseases, such as cancer (e.g., breast cancer (e.g., triple-negative breast cancer (TNBC), etc.), basal bladder cancers, etc.), by administration of the PROTAC compounds herein to a subject in need thereof. In some embodiments, the PROTACs herein effectively remove the target protein from cancer cells via “chemical knockdown”.

Experiments were conducted during development of embodiments herein to survey- several E3 ligase ligands as potential conjugates with dasatinib. Ligands for cereblon and VHL led to degradation of c-Src. Dasatinib is a pan-kinase inhibitor with c-Src and Abl kinases being the most potent targets. It was found that short alkyl linkers between dasatinib and the E3 ligase ligand afforded higher selectivity selectivity for c-Src over Bcr-Abl. The geometry between the E3 ligase ligand and dasatinib, which was varied using 3- and 4-amino thalidomide, also impacted selectivity for c-Src over Bcr-Abl. Using DAS-5-oCRBN, a potent c-Src degrader, as the template a matched pair of conformation-selective PROTACs was synthesized. The conformation-selective PROTACs, DAS-CHO-5-oCRBN, and DAS-DFGO-5-oCRBN, stabilize the closed and open global kinase conformations, respectively. Differential ability to degrade and inhibit c-Src was observed. The closed conformation degrader, DAS-CHO-5-oCRBN is a competent degrader of c-Src (DC₅₀ = 53 nM), but the open conformation degrader DAS-DFGO-5-oCRBN does not degrade c-Src. Both conformation-selective PROTACs are weak cellular binders of c-Src. Thus, Both DAS-5- oCRBN and DAS-CHO-5-oCRBN have potent anti-proliferative activity in c-Src dependent cell lines. These compounds represent highly valuable tools to study the differential impact of c-Src inhibition (using dasatinib) versus degradation (using DAS-CHO-5-oCRBN) versus dual inhibition/degradation (using DAS-5-oCRBN). Using dasatinib and DAS-CHO-5-oCRBN, RPPA was used to examine signaling changes in CAL148 cells to differentiate differences between cellular inhibition (dasatinib) and degradation (DAS-CHO-5-oCRBN). It was found that LC3A/B proteins were selectively upregulated via c-Src degradation, and several proteins known to interact with c-Src were downregulated when c-Src is degraded.

While DAS-DFGO-5-oCRBN does not degrade c-Src, it is a potent and selective degrader of p38a ( DC₅₀ < 3 nM) and p38b (DC₅₀ = xx nM) kinases. Consistent with its ability to degrade p38 kinases, in kinome profiling, DAS-DFGO-5-oCRBN potently binds p38a and p38b kinases. Notably, the other dasatinib-based PROTACs, DAS-5-oCRBN and DAS-CHO-5- oCRBN do not bind p38 kinases. The DC₅₀ values for DAS-DFGO-5-oCRBN are among the most potent p38a/b degraders reported and potent anti-proliferative activity was observed in p38 growth-dependent cell lines.

Using conformation-selective ligands, experiments have demonstrated that the conformation of the target protein can impact proteasomal degradation. c-Src is degraded when in the closed conformation but is not degraded when bound by a PROTAC in the open, extended conformation.

Over 200 putative tyrosine kinase (TK) PROTACs have been synthesized and tested during experiments conducted during development of embodiments herein. These TK PROTACs utilize dasatinib (an FDA-approved dual Abl/Src inhibitor) and dasatinib analogs [Ref. 6; incorporated by reference in its entirety], as the c-Src-binding element. Several E3L ligands, including pomalidomide (binder of cereblon E3L), VHL peptide (binder of Von Hippel Lindau E3L), and nutlin (binder of HDM2 E3L) we utilized. A large number (>30) of linkers have been explored for connecting the E3L ligand and the c-Src-binding clement. Dasatinib and the Dasatinib analogs inhibit diverse kinases (mostly tyrosine kinases), including c-Src. PROTAC selectivity has previously been shown to be modulated by linker length and linker composition. As has been demonstrated for other kinase PROTACs, pan-kinase inhibitors can be turned into selective PROTACs via optimal selection of target ligand, linker, and E3L ligand. Experiments were conducted during development of embodiments herein to evaluate the library of TK PROTACs against both c-Src and c-Abl kinases, using ELISA assays. Several TK PROTACs have been idenbtified that are efficient and selective cellular degraders of c-Src kinase in TNBC and basal bladder cancer cell lines. Despite dasatinib being a potent Src and Abl inhibitor, PROTACs have been designed and tested that selectively degrade Src over Abl. The c-Src PROTACs herein inhibit the growth of TNBC and basal bladder cell lines, proportional to their ability to degrade c-Src. Furthremore, the c-Src PROTACs herein display phenotypes consistent with c-Src knockdown in cancer cell lines.

Triple-negative breast cancer and c-Src

Despite the shared traits (ER-, PR-, HER2-), TNBC comprises a highly diverse group of cancers [7,8], FDA-approved c-Src inhibitors (e.g., dasatinib, bosutinib, saracatinib) have been tested against diverse TNBC cell lines and a wide range of activities have been reported [Ref. 7 ; incorporated by reference in its entirety]. In vivo, FDA-approved c-Src inhibitors have not shown efficacy against TNBC [Refs. 7,9; incorporated by reference in its entirety]. It is contemplated that the disconnect between preclinical studies validating c-Src as an ideal anti-TNBC target and the lack of efficacy in vivo, is attributable to a number of factors. Target validation of c-Src has mainly been performed using genetic techniques that ablate the entire c-Src gene, while pharmacological intervention with small molecules (e.g., dasatinib) inhibits only the catalytic function of the enzyme [Ref. 10; incorporated by reference in its entirety]. A recent study demonstrated a differential localization of c-Src in TNBCs versus non-TNBCs [Ref. 11; incorporated by reference in its entirety]. While both subtypes of breast cancer had an equal distribution of c-Src in the cytosol, membrane-bound c-Src was detected in 78% of TNBCs versus 38% of non-TNBCs. These data indicate that c-Src’s spatial positioning likely plays a role in the development of aggressive breast cancers [Ref. 11 ; incorporated by reference in its entirety]. The lack of in vitro efficacy for FDA-approved c-Src inhibitors across diverse TNBC cell lines suggests that inhibition of c-Src alone may not be adequate to obtain robust anti-TNBC activity. Thus, provided herein are small molecule degraders of c-Src, using the PROTACs herein methodology, that provide pharmacologically active compounds that degrade c-Src.

Basal bladder cancer and c-Src.

Preclinical studies suggested c-Src as a potential therapeutic target in basal metastatic invasive bladder cancer (MIBC); however, FDA-approved inhibitors of c-Src (e.g., dasatinib, bosutinib) have not been successful in the clinic. Experiments conducted during development of embodiments herein have identified functional, non-catalytic signaling that c-Src promotes in basal MIBC. It is contemplated that the many non-catalytic functions of c-Src are relevant to MIBC progression. These non-catalytic functions occur via protein-protein of c-Src with other signaling proteins, including ADAM15, FAK, and TRIM29. Degradation of c-Src with the TK PROTACs herein provides inhibition of both c-Src’s catalytic and non-catalytic activities.

PROTACs and E3L ligands

In some embodiments, provided herein are bifunctional compounds which function to recruit endogenous proteins to an E3 ubiquitin ligase for degradation, and methods of using the same. In particular, the present disclosure provides bifunctional or proteolysis targeting chimeric (PROTAC) compounds, which find utility as modulators of targeted ubiquitination of a variety of polypeptides and other proteins, which are then degraded and/or otherwise inhibited by the bifunctional compounds as described herein. An advantage of the compounds provided herein is that a broad range of pharmacological activities is possible, consistent with the degradation/inhibition of targeted polypeptides from virtually any protein class or family. In addition, the description provides methods of using an effective amount of the compounds as described herein for the treatment or amelioration of a disease condition, such as cancer.

Bifunctional compounds such as those that are described in U.S. Patent Application Publications 2015/0291562 and 2014/0356322 (herein incorporated by reference in their entireties), function to recruit endogenous proteins to an E3 ubiquiuin ligase (E3L) for degradation of a target bound to the E3L. In particular, the publications describe bifunctional or proteolysis targeting chimeric (PROTAC) compounds, which find utility as modulators of targeted ubiquitination of a variety of polypeptides and other proteins, which are then degraded and/or otherwise inhibited by the bifunctional compounds.

E3 ubiquitin ligases (of which hundreds are known in humans) confer substrate specificity for ubiquitination, and therefore, are more attractive therapeutic targets than general proteasome inhibitors due to their specificity for certain protein substrates. Recent developments have provided specific ligands which bind to these ligases. For example, since the discovery of nutlins, the first small molecule E3 ligase inhibitors, additional compounds have been reported. For example, since the discovery of Nutlins, the first small molecule E3 ligase mouse double minute 2 homolog (MDM2) inhibitors, additional compounds have been reported that target MDM2 (e.g., human double minute 2 or HDM2) E3 ligases (J. Di, et al. Current Cancer Drug Targets (2011), 11(8), 987-994; herein incorporated by reference in its entirety).

One E3 ligase with exciting therapeutic potential is the von Hippel-Lindau (VHL) tumor suppressor, the substrate recognition subunit of the E3 ligase complex VCB, which also consists of elongins B and C, Cul2 and Rbxl. The primary substrate of VHL is Hypoxia Inducible Factor 1α (HIF-1α), a transcription factor that upregulates genes such as the pro-angiogenic growth factor VEGF and the red blood cell inducing cytokine erythropoietin in response to low oxygen levels. The first small molecule ligands of Von Hippel Lindau (VHL) to the substrate recognition subunit of the E3 ligase were generated, and crystal structures were obtained confirming that the compound mimics the binding mode of the transcription factor HIF-1α, the major substrate of VHL.

Cereblon is a protein that in humans is encoded by the CRBN gene. CRBN orthologs are highly conserved from plants to humans, which underscores its physiological importance. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC1). This complex ubiquitinates a number of other proteins.

Inhibitors of Apotosis Proteins (IAPS) are a protein family involved in suppressing apoptosis. The human IAP family includes 8 members, and numerous other organisms contain IAP homologs. IAPs contain an E3 ligase specific domain and baculoviral IAP repeat (BIR) domains that recognize substrates and promote their ubiquitination. IAPs promote ubiquitination.

In some embodiments, provided herein are bifunctional (PROTAC) compounds comprising comprise an E3 ubiquitin ligase ligand (or E3 ligase binding moiety), and a moiety that binds a target protein (e.g., a tyrosine kinase (e.g., c-Src, etc.), etc.) such that the target protein is placed in proximity to the ubiquitin ligase to effect degradation (inhibition) of that protein. In some embodiments, the Ligase ligand is a Von Hippel-Lindau E3 ubiquitin ligase binding moiety (e.g., hydroxyproline, hydroxyproline derivatives, or binding moieties described in U.S. Patent Application Pub. No. 2014/03022523 (herein incorporated by reference in its entirety)), a cereblon E3 ubiquitin ligase binding moiety (e.g., thalidomide, lenalidomide, pomalidomide, analogs thereof, isosteres thereof, derivatives thereof, or binding moieties described in U.S. Patent Application Publication US 2015/0291562 (herein incorporated by reference in its entirety)), a mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase binding moiety (e.g., binding moieties described in U.S. patent application Ser. No. 15/206,497 (herein incorporated by reference in its entirety)), or an IAP E3 ubiquitin ligase binding moiety. Suitable ligands for binding the aforementioned E3 ubiquitin ligases, as well as other known E3 ubiquitin ligases, are understood in the field and described in, for example, U.S. Pub. Nos. 2015/0291562, 2014/0356322, 2018/0256586, 2018/0228907, 2018/0193470, 2018/0179183, 2018/0134684; 2017/0327469; herein incorporated by reference in their entireties. The compounds and formulas within the scope of embodiments herein are not limited to specific ligase ligand structures described herein, or incorporated by reference, but include ligase ligands understood in the field.

In some embodiments, c-Src PROTACs herein include a moiety that binds to an E3 ubiquitin ligase, for example, as a ligand for the E3 ubiquitin ligase. Ligands for E3 ubiquitin ligases for use in preparing PROTACs are known in the art. (See, e.g., An et al., "Small- molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs," EBioMedicine. 2018 October; 36: 553-562; and Gu et al., "PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery," Bioessays. 2018 April; 40(4) :e 1700247, the contents of which are incorporated herein by reference in their entireties).

In some embodiments, the E3L ligand moiety of an c-Src PROTAC compound herein is a moiety that binds to an E3 ubiquitin ligase selected from Von Hippel-Lindau (VHL) E3 ubiquitin ligase, cereblon (CRBN) E3 ubiquitin ligase, inhibitor of apoptosis protein (IAP) E3 ubiquitin ligase, and mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase. In certain embodiments, the E3L ligand is a moiety derived from thalidomide, pomalidomide, lenalidomide, iberdomide, (S,R,S)-AHPC-Me hydrochloride, (S,R,S)-AHPC-Me dihydrochloride, ccrcblon modulator 1, thalidomidc-propargyl, (S,R,S)-AHPC-propargyl, (S,R,S)-AHPC hydrochloride, CC-885, thalidomide-O— COOH, lenalidomide hemihydrate, thalidomide fluoride, thalidomide-OH, lenalidomide-Br, thalidomide D4, lenalidomide hydrochloride, (S,R,S)-AHPC-Me, clAPl ligand 1, TD-106, E3 ligase Ligand 8, E3 ligase Ligand 9, E3 ligase Ligand 10, E3 ligase Ligand 13, E3 ligase Ligand 14, E3 ligase Ligand 18, BC-1215, VHL ligand 1 (VHL-1), VHL ligand 2 (VHL-2), VHL Ligand 8 (VHL-8), VH032, VH032-cyclopropane-F, VH032 thiol, VH-298, VL-269, VL-285, LCL161, hydroxyproline- based ligands, HIF- 1α-derived (R)-hydroxyproline, Nutlin carboxylic acid, (4R,5S)-Nutlin carboxylic acid, (S,R,S)-AHPC-Boc, AR antagonist 1, NV03, (S,R,S)-AHPC TEA, (S,R,S)- AHPC,β- Naphthoflavone-CH2-Br, βN- aphthoflavone-CH2-OH, Bestatin-amido-Me, MV-1- NH-Me, (S,S,S)-AHPC hydrochloride, and clAPl ligand 2.

In certain embodiments, the E3L ligand of the c-Src PROTACs herein has a formula selected from:

* is the position of conjugation to a linker or c-Src ligand.

; wherein * is the position of conjugation to a linker or c-Src ligand.

In other embodiments, the E3L ligand moiety has a formula selected from:

; wherein * is the position of conjugation to a linker or c-Src ligand. Other E3L ligands understood in the field may find use in embodiments herein. c-Src inhibitors and ligands In certain embodiments, the c-Src ligand of the c-Src PROTACs herein has a formula selected from, for example:

In some embodiments, he c-Src ligand of the c-Src PROTACs herein has a formula of:

; wherein X is N or CH, wherein Q is H or Cl; wherein A is -L-phenyl, wherein L is -O- or -NHC(O)-, and wherein phenyl is optionally further substituted (e.g., with -CF3, -F, -Cl). In some embodiments, the phenyl of -L-phenyl is substituted at the meta position (e.g., with -CF_3, -F, -Cl). In some embodiments, the phenyl of -L- phenyl is substituted at the para position (e.g., with -CF3, -F, -Cl). Linkers In some embodiments, the E3L ligand and the c-Src ligand of the present compounds are tethered by a covalent bond or a suitable linker moiety. The PROTAC linker connects the functional moieties of a PROTAC, a c-Src protein binder and an E3 ligase recruiter. Linkers used in the development of the c-Src PROTACs herein include polyethylene glycol (PEG) linkers, Alkyl-Chain linkers, and Alkyl/ether linkers. Other linkers may include those linkers described in one or more of U.S. Publication Nos.2020/0140456; 2020/0102298; 2020/0085817; 2020/0022966; 2019/0275161; 2019/0263798; 2019/0262502; 2019/0194190; 2019/0151457; 2019/0151295; 2019/0106417; 2019/0076542; 2019/0076541; 2019/0076540; 2019/0076539; 2019/0071415; 2019/0016703; 2018/0327419; 2018/0186785; 2018/0134684; and 2018/0085465; the contents of which are incorporated herein by reference in their entireties. In some embodiments, the linker is any of the linkers provided in Example 3 herein. In some embodiments, the c-Src PROTAC compounds herein comprise a linker (L) which comprises an alkyl chain of formular (CH2)1-25. In some embodiments, the linker (L) comprises ((CH₂)₂)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the linker (L) comprises ((CH₂)₂)_nC(O)NH, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the linker (L) comprises one or more (CH2)2O groups. In some embodiments, the linker (L) comprises one or more CH₂, NHC(O), C(O)NH, alkenes, alkynes, C(O), NH, cycloalkanes, heterocycles, multi-ring systems, etc. In some embodiments, linker (L) comprises a formula selected from *--(CH2)m-- (CH₂CH₂O)_n--*, *--CH₂--C(O)--NH--(CH₂)_m--(CH₂CH₂O)--*, *--(CH₂CH₂O)_m--(CH₂)_n--C(O)-- *, *--CH₂--C(O)--NH--(CH₂CH₂O)_m--(CH₂)_n--C(O)-- -*, *--CH₂--C(O)--NH--(CH₂)_m-- (CH2CH2O)m--*, *--(CH2CH2O)m--(CH2)n--C(O)--*, wherein m and n are independently 0-20 (e.g., 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or ranges therebetween). In some embodiments, an c-Src PROTAC comprises a linker (L) selected from a covalent bond, -CH₂C(O)NH-, -(CH₂)₂C(O)NH-, -(CH₂)₃C(O)NH-,-(CH₂)₄C(O)NH-, -(CH₂)₅C(O)NH-, - (CH2)6C(O)NH-, -(CH2)7C(O)NH-, -(CH2)8C(O)NH-, -(CH2)9C(O)NH-, -(CH2)10C(O)NH-, - (CH₂)₁₁C(O)NH-, -(CH₂)₁₂C(O)NH-, -(CH₂)_1-8(O(CH₂)₂)_1-6(CH₂)_1-8C(O)NH-, -(CH₂)_1- ₈NHC(O)CH₂O- -(CH₂)_1-8C(O)NHCH₂O-, -C(O)(CH₂)_1-8NH-, and -(CH₂)_1-8-. In some embodiments, an c-Src PROTAC comprises an alkyl chain linker (L) with one or more hctcroatoms and/or substituents, such as: -NH-, -C(O), -NHC(O)-, C(O)NH-, -O-, -S-, -OH, -SH, -NH2, -Cl, -F, -Br, -I, -CH3, CF3, etc. In some embodiments, linker (L) comprises one or more cycloalkyl, heterocycle, aryl, or heteroaryl groups, such as:

In some embodiments, an c-Src PROTAC comprises a linker (L) comprises any combination of the linker components depicted or described herein. In some embodiments, an c- Src PROTAC comprises a linker (L) selected from those depicted in the c-Src PROTACs herein. Other linkers comprising any suitable combinations of the above or other linkers understood in the field may find use in embodiments herein.

Compounds

In some embodiments, c-Src PROTAC compounds within the scope herein are of the formula (c-Src ligand)-linker-(E3L ligand), wherein the c-Src ligand, linker, and E3L ligand are selected from those described herein. Exemplary c-Src PROTAC compounds within the scope herein include:

Pharmaceutical compositions

In certain embodiments, c-Src PROTAC compounds herein or salts thereof are combined with one or more additional agents to form pharmaceutical compositions. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Additional details about suitable excipients for pharmaceutical compositions described herein may be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference for such disclosure.

A pharmaceutical composition, as used herein, refers to a mixture of a c-Src PROTAC compound disclosed herein or salts thereof with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. In practicing the methods of treatment or use provided herein, therapeutically effective amounts of c-Src PROTAC compounds described herein are administered in a pharmaceutical composition to a mammal having a disease, disorder, or condition to be treated. In some embodiments, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The c-Src PROTAC compounds or salts thereof disclosed herein, can be used singly or in combination with one or more therapeutic agents as components of mixtures (as in combination therapy).

The pharmaceutical formulations described herein can be administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. Moreover, the pharmaceutical compositions described herein, which include a c-Src PROTAC compound described herein, can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, aerosols, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, and capsules.

One may administer the c-Src PROTAC compounds and/or compositions in a local rather than systemic manner, for example, via injection of the compound directly into an organ or tissue, often in a depot preparation or sustained release formulation. Such long-acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with organ specific antibody. The liposomes will be targeted to and taken up selectively by the organ. In addition, the drug may be provided in the form of a rapid release formulation, in the form of an extended-release formulation, or in the form of an intermediate release formulation. Pharmaceutical compositions including a compound described herein may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or compression processes.

In some embodiments, a pharmaceutical composition will include at least one c-Src PROTAC compound described herein, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In certain embodiments, compositions provided herein may also include one or more preservatives to inhibit microbial activity. Suitable preservatives include quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipients with one or more of the c-Src PROTAC compounds or salts thereof disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets, pills, or capsules. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

In some embodiments, the solid dosage forms disclosed herein may be in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid- disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder), a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”), solid dispersion, solid solution, bioerodible dosage form, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet, including but not limited to, a fast-melt tablet. Additionally, pharmaceutical formulations of the compounds described herein may be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation is administered in two, or three, or four, capsules or tablets.

The pharmaceutical solid dosage forms described herein can also include one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof. In still other aspects, using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of the compound described herein. In one embodiment, some or all of the particles of the compound described herein are coated. In another embodiment, some or all of the particles of the compound described herein are microencapsulated. In still another embodiment, the particles of the compound described herein are not microencapsulated and are uncoated.

Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).

The aqueous suspensions and dispersions described herein can remain in a homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The homogeneity should be determined by a sampling method consistent with regard to determining homogeneity of the entire composition. In one embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

In some embodiments, the pharmaceutical formulations described herein can be self- emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase can be added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. SEDDS may provide improvements in the bioavailability of hydrophobic active ingredients. Methods of producing self-emulsifying dosage forms include, but are not limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563.

Potential excipients for intranasal formulations include, for example, U.S. Pat. Nos.

4,476,116, 5,116,817 and 6,391,452. Formulations solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents. See, for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients The choice of suitable carriers is highly dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents may also be present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

For administration by inhalation, the compounds described herein may be in a form as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro tetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound described herein and a suitable powder base such as lactose or starch. Buccal formulations that include compounds described herein may be administered using a variety of formulations which include, but arc not limited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In addition, the buccal dosage forms described herein can further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. The buccal dosage form is fabricated so as to erode gradually over a predetermined time period, wherein the delivery of the compound is provided essentially throughout. Buccal drug delivery avoids the disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and/or first-pass inactivation in the liver. With regard to the bioerodible (hydrolysable) polymeric carrier, virtually any such carrier can be used, so long as the desired drug release profile is not compromised, and the carrier is compatible with the compounds described herein, and any other components that may be present in the buccal dosage unit. Generally, the polymeric carrier comprises hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the buccal mucosa. Other components may also be incorporated into the buccal dosage forms described herein include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.

Formulations suitable for transdermal administration of c-Src PROTAC compounds described herein may employ transdermal delivery devices and transdermal delivery patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery of the compounds described herein can be accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches can provide controlled delivery of the compounds described herein. The rate of absorption can be slowed by using rate-controlling membranes or by trapping the compound within a polymer matrix or gel. Conversely, absorption enhancers can be used to increase absorption. An absorption enhancer or carrier can include absorbable pharmaceutically acceptable solvents to assist passage through the skin. For example, transdermal devices are in the form of a bandage 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. Transdcrmal formulations may be administered using a variety of devices including but not limited to, U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and 6,946,144; incorporated by reference in their entireties.

Formulations suitable for intramuscular', subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection may also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, compounds described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally recognized in the field. For other parenteral injections, appropriate formulations may include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally recognized in the field.

Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In certain embodiments, delivery systems for pharmaceutical compounds may be employed, such as, for example, liposomes and emulsions. In certain embodiments, compositions provided herein also include an mucoadhesive polymer, selected from among, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, the compounds described herein may be administered topically and are formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

The compounds described herein may also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted. Generally, a c-Src PROTAC compound herein is administered in an amount effective for amelioration of, or prevention of the development of symptoms of, the disease or disorder (i.c., a therapeutically effective amount). Thus, a therapeutically effective amount can be an amount that is capable of at least partially preventing or reversing a disease or disorder. The dose required to obtain an effective amount may vary depending on the agent, formulation, disease or disorder, and individual to whom the agent is administered. Determination of effective amounts may also involve in vitro assays in which varying doses of agent are administered to cells in culture and the concentration of agent effective for ameliorating some or all symptoms is determined in order to calculate the concentration required in vivo. Effective amounts may also be based in in vivo animal studies.

An agent can be administered prior to, concurrently with and subsequent to the appearance of symptoms of a disease or disorder. In some embodiments, an agent is administered to a subject with a family history of the disease or disorder, or who has a phenotype that may indicate a predisposition to a disease or disorder, or who has a genotype which predisposes the subject to the disease or disorder.

In some embodiments, the compositions described herein are provided as pharmaceutical and/or therapeutic compositions. The pharmaceutical and/or therapeutic compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

The pharmaceutical composition described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compound. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple- dose rcclosablc containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multi dose containers, with an added preservative.

Dosing and administration regimes are tailored by the clinician, or others skilled in the pharmacological arts, based upon well-known pharmacological and therapeutic considerations including, but not limited to, the desired level of therapeutic effect, and the practical level of therapeutic effect obtainable. Generally, it is advisable to follow well-known pharmacological principles for administrating chemotherapeutic agents (e.g., it is generally advisable to not change dosages by more than 50% at time and no more than every 3-4 agent half-lives). For compositions that have relatively little or no dose-related toxicity considerations, and where maximum efficacy is desired, doses in excess of the average required dose are not uncommon. This approach to dosing is commonly referred to as the "maximal dose" strategy. In certain embodiments, the compounds are administered to a subject at a dose of about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg. When the compounds described herein are co- administered with another agent (e.g., as sensitizing agents), the effective amount may be less than when the agent is used alone. Dosing may be once per day or multiple times per day for one or more consecutive days.

Methods

The present disclosure provides compounds and methods for binding to c-Src and facilitating its degradation. In certain embodiments, the disclosure provides compounds that also inhibit c-Src activity upon binding. A full understanding of the mechanism of c-Src degradation/inhibition is not required to practice the invention.

Degradation and/or inhibition of c-Src by the compositions and methods herein may be assessed and demonstrated by a wide variety of ways known in the art. Non-limiting examples include measure (a) a decrease in c-Src activity; (b) a decrease in cell proliferation and/or cell viability; (c) a direct decrease in ASH1L levels, and/or (d) decrease in tumor volume and/or tumor volume growth rate. Kits and commercially available assays can be utilized for determining one or more of the above. In some embodiments, it is not necessary to understand the relative contribution of c-Src degradation and/or inhibition to the overall reduction in c-Src effect in order to practice the invention.

In some embodiments, the disclosure provides c-Src PROTAC compounds and methods for treating a subject suffering from a disease, comprising administering a c-Src PROTAC compound or salt thereof, for example, a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein to the subject. In certain embodiments, the disease is selected from a disease associated with c-Src expression (e.g., aberrant expression, overexpression, etc.) and/or activity (e.g., cancer). In certain embodiments, the disease is mediated by c-Src activity and/or expression (e.g., aberrant expression, overexpression, etc.). In certain embodiments, the disease is leukemia, hematologic malignancies, solid tumor cancer, glioma, other cancers, etc. In some embodiments, the disclosure provides a method for treating cancer in a subject, comprising administering a c-Src compound described herein or salt thereof, for example, a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein to the subject. In some embodiments, the cancer is mediated by a c-Src expression (e.g., aberrant expression, overexpression, etc.) and/or activity. In certain embodiments, the cancer is leukemia, breast cancer (e.g., triple-negative breast cancer, etc.), bladder cancer (e.g., a basal bladder cancer), prostate cancer, pancreatic cancer, lung cancer, thyroid cancer, liver cancer, skin cancer, a brain tumor, etc.

In certain embodiments, the disclosure provides method of treating a disease in a subject, wherein the method comprises determining if the subject has a c-Src-mediated condition (e.g., cancer) and administering to the subject a therapeutically effective dose of a c-Src-PROTAC compound or salt described herein, for example, a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein.

Certain embodiments are directed to administration of a c-Src-PROTAC compound or salt described herein, for example, a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein to a subject with a cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, c-Src PROTAC compounds are administered for the treatment, or prevention of acute myeloid leukemia, cancer in adolescents, adrenocortical carcinoma childhood, AIDS-related cancers, e.g., Lymphoma and Kaposi's Sarcoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumor, atypical teratoid, embryonal tumors, germ cell tumor, primary lymphoma, cervical cancer, childhood cancers, chordoma, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myleoproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumors, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, csthcsioncuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fibrous histiocytoma of bone, gall bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ (LOS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary, midline tract carcinoma, mouth cancer multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma of bone and osteosarcoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach (gastric) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, T- Cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, unusual cancers of childhood, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Viral-Induced cancer. In some embodiments, the method relates to the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin, e.g., psoriasis, restenosis, or prostate, e.g., benign prostatic hypertrophy (BPH). In some cases, the method relates to the treatment of leukemia, hematologic malignancy, solid tumor cancer, prostate cancer, e.g., castration-resistant prostate cancer, breast cancer, Ewing’s sarcoma, bone sarcoma, primary bone sarcoma, T-cell prolymphocyte leukemia, glioma, glioblastoma, liver cancer, e.g., hepatocellular carcinoma, or diabetes.

In some embodiments, c-Src PTOTAC compounds are administered for symptom reduction, to reduce the likelihood of occurrence of a condition, to treat an existing condition, and/or to reduce the likelihood of the spread of a condition (e.g., malignancy).

Determining whether a tumor or cancer expresses (e.g., overexpresses, aberrantly expresses, etc.) c-Src can be undertaken by assessing the nucleotide sequence encoding c-Src or by assessing the amino acid sequence of c-Src. Methods for detecting a c-Src nucleotide sequence are known by those of skill in the art. These methods include, but are not limited to, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assays, real- time PCR assays, PCR sequencing, mutant allele- specific PCR amplification (MASA) assays, direct sequencing, primer extension reactions, electrophoresis, oligonucleotide ligation assays, hybridization assays, TaqMan assays, SNP genotyping assays, high resolution melting assays and microarray analyses. Methods for detecting c-Src protein are known by those of skill in the art. These methods include, but are not limited to, detection using a binding agent, e.g., an antibody, specific for c-Src, protein electrophoresis and Western blotting, and direct peptide sequencing.

Methods for determining whether a tumor or cancer expresses (e.g., overexpresses, aberrantly expresses, etc.) c-Src or is mediated by c-Src activity can use a variety of samples. In some embodiments, the sample is taken from a subject having a tumor or cancer. In some embodiments, the sample is taken from a subject having a cancer or tumor. In some embodiments, the sample is a fresh tumor/cancer sample. In some embodiments, the sample is a frozen tumor/cancer sample. In some embodiments, the sample is a formalin-fixed paraffin- embedded sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.

In certain embodiments, the disclosure provides a method of inhibiting c-Src activity in a sample (e.g., by facilitating the degradation of c-Src, by inhibiting c-Src activity, by degrading c- Src and inhibition c-Src activity, etc.), comprising administering the compound or salt described herein to said sample comprising c-Src. In some embodiments, the disclosure provides a method of degrading c-Src protein in a sample, comprising administering the compound or salt described herein to said sample comprising c-Src.

The disclosure provides methods for treating a disease by administering a c-Src PROTAC compound or salt thereof, for example a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein to a subject suffering from the disease, wherein the compound binds c-Src, inhibits c-Src activity, and/or facilitates degradation of c-Src by engaging/recruiting the E3 ubiquitin ligase complex. The disclosure provides methods for preventing or reducing the likelihood of occurrence (e.g., first occurrence, reoccurrence, etc.) of a disease by administering a c-Src PROTAC compound or salt thereof, for example a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein to a subject at risk from the disease, wherein the compound binds c-Src, inhibits c-Src activity, and/or facilitates degradation of c-Src by engaging/recruiting the E3 ubiquitin ligase complex.

In certain embodiments, the c-Src PROTAC compound covalently binds to c-Src. In certain embodiments, the compound noncovalently binds to c-Src. In certain embodiments, the compound covalently binds to the E3 ubiquitin ligase complex. In certain embodiments, the compound noncovalently binds to the E3 ubiquitin ligase complex.

The compositions containing the c-Src PROTAC compounds or salts thereof described herein can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the symptoms of the disease. Amounts effective for this use will depend on the severity and course of the disease, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating clinician.

In prophylactic applications, compositions containing the c-Src PROTAC compounds or salts thereof described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a "prophylactically effective amount or dose." In this use, the precise amounts also depend on the patient's state of health, weight, and the like. When used in a patient, effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating clinician.

Provided herein are methods for combination therapies in which an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes are used in combination with a c-Src PROTAC compound or salt thereof, for example a compound formula of:

with suitable substituents are described or exemplified herein, a compound described or exemplified herein, or any combination of c-Src ligand, linker and E3L ligand described or exemplified herein. In one aspect, such therapy includes but is not limited to the combination of one or more compounds of the invention with chemotherapeutic agents, targeted agents, therapeutic antibodies, and radiation treatment, to provide a synergistic or additive therapeutic effect.

In general, the compositions described herein and, in embodiments where combinational therapy is employed, other agents do not have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, have to be administered by different routes. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the clinician. The initial administration can be made according to established protocols recognized in the field, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the clinician.

The pharmaceutical agents which make up the combination therapy disclosed herein (e.g., a c-Src PROTAC containing composition and a second therapeutic agent) may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical agents may also be administered sequentially, with either therapeutic agent being administered by a regimen calling for two-step administration. The two- step administration regimen may call for sequential administration of the active agents or spaced- apart administration of the separate active agents. The time period between the multiple administration steps may range from, a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half- life and kinetic profile of the pharmaceutical agent. Circadian variation of the target molecule concentration may also determine the optimal dose interval.

The c-Src PROTAC compounds described herein also may be used in combination with procedures that may provide additional or synergistic benefit to the patient.

When the c-Src PROTAC compounds and pharmaceutical compositions herein are used for treating cancer, they may be co-administered with one or more chemotherapeutics. Many chemotherapeutics arc presently known in the art and can be used in combination with the compounds herein. In some embodiments, the chemotherapeutic is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzyme inhibitors, topoisomerase inhibitors, protein-protein interaction inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens. The c-Src PROTAC compounds and pharmaceutical compositions herein may also be co-administered with various immunotherapeutic (e.g., monoclonal antibodies, immune checkpoint inhibitors, bispecific engagers, CAR-T cells, etc.) that are understood in the field. In some embodiments, the c-Src PROTAC compounds and pharmaceutical compositions herein are co-administered with surgery, radiation, or other non- drug-related treatments.

EXPERIMENTAL

Example 1

Experiments were conducted during development of embodiments herein to identify PROTACs for c-Src. A library of putative c-Src degraders as constructed. Dasatinib, a potent c- Src/Abl kinase inhibitor, was selected as the base c-Src-directed ligand. Dasatinibwas conjugated to varied ligands for four E3 ligases (cereblon, VHL, MDM2, and IAP) using varied linkers (Figure 1). The panel of putative c-Src degraders was evaluated for their ability to degrade c-Src in a cellular context. CAL148 cells were treated with 100 nM compound for 18 hours and the amount of c-Src was quantified using a c-Src total protein ELISA. ELISA was developed to quantify c-Src levels, which proved to be robust and reproducible. Using the c-Src total protein ELISA, it was determined that dasatinib-based PROTACs with ligands for ccrcblon and VHL yield significant degradation of c-Src. No degradation was observed for dasatinib PROTACs using ligands for MDM2 and IAP. Using a biochemical assay for c-Src kinase activity, all dasatinib PROTACs were potent inhibitors of c-Src (IC50 < 100 μM at 1 mM ATP) except for the MDM2 targets PROTACs.

Dasatinib is a pan-tyrosine kinase inhibitor, however, previously reported kinase-directed PROTACs have reported selective degradation. In addition to c-Src, dasatinib is a potent inhibitor of Abl kinase and thus, degradation of Abl represents a useful initial measure of selectivity. An ELISA for Bcr-Abl was developed and the amount of Bcr-Abl degraded by PROTACs in the initial library was determined after treatment of KCL-22 cells with 100 nM compound for 18 hours. Using the ELISA for Bcr-Abl, varied degrees of selectivity for c-Src over Bcr-Abl were observed across the panel of dasatinib-based PROTACs. DAS-5-oCRBN was the most selective c-Src PROTAC (78% of c-Src was degraded in CAL148 cells, but only 21% of Bcr-Abl was degraded in CAL148 cells). It was observed that that the cereblon ligand trajectory (3-amino- versus 4-amino-thalidomide) can dramatically impact selectivity (Table 1).

Table 1. c-Src biochemical inhibitory activity and cellular degradation of c-Src and Bcr- Abl.

To confirm that the observed degradation is mediated by ubiquitinylation and proteosomal degradation, the amount of c-Src remaining was measured in the presence and absence of 1 p M bortezomib (proteosome inhibitor), 1 pM DGY-06-116 (an irreversible inhibitor of c-Src), and 1 pM pomalidomide (a ligand of cereblon). It was found that each of these conditions prevents degradation of c-Src by 100 nM DAS-5-oCRBN, confirming that degradation is mediated by ubiquitinylation and proteosomal degradation (Figure 2).

Conformation- selective analogs of dasatinib modulate the global conformation of c-Src. DAS-CHO-II is an αC-helix out analog of dasatinib that stabilizes the closed global conformation of c-Src. DAS-DFGO-II is a DFG-out analog of dasatinib that stabilizes the open global conformation of c-Src. DAS-CHO-5-oCRBN and DAS-DFGO-5-oCRBN were synthesized using the optimal linker and thalidomide orientation as found in DAS-5-oCRBN (Figure 3). In biochemical activity assays, the conformation- selective inhibitors are potent inhibitors of c-Src (Table 2).

Table 2. Conformation selective PROTACs and their inactive controls have differential ability to inhibit c-Src in cells and differential degradation of c-Src.

To determine the kinase binding profile for each of the three conformation-selective PROTACs, kinome-wide profiling was performed in cell lysate (500 nM PROTAC against 176 diverse kinases, Luceome Biotechnologies, Tucson, AZ). Consistent with the conformation- selective dasatinib inhibitor analogs, the conformation-selective PROTACs have similar kinome profiles (Figure 3). The DAS-DFGO-II-based PROTAC, DAS-DFGO-5-oCRBN, was the most promiscuous, consistent with the DFG-out conformation ligands being less selective. DAS-CHO- 5-oCRBN was the most selective, consistent with the αC -helix out conformation being less conserved across the kinome.

Using the c-Src and Bcr-Abl ELIS As, it was found that DAS-CHO-5-oCRBN selectively degrades c-Src over Bcr-Abl while DAS-DFGO-5-oCRBN does not degrade c-Src nor Bcr-Abl (Figure 4A). Thus, degradation was observed when c-Src is stabilized in the closed global conformation, but not in the open conformation. Each of these ligands is a potent inhibitor of c- Src. This is the first example of conformation- selective PROTACs where the conformation stabilized dictates target degradation.

The concentration-dependence was determined for the three conformation- selective PROTACs with respect to c-Src degradation. In CAL 148 cells (18 h treatment), the DC₅₀ of DAS-5-oCRBN = 2.8 nM and the DC₅₀ of DAS-CHO-5-oCRBN = 53 nM (Figure 4B). No degradation of c-Src was observed at any concentration of DAS-DFGO-5-oCRBN. The timecourse for degradation in CAL148 cells (treated with 100 nM PROTAC) was determined and it was found that the D_max for both DAS-5-oCRBN and DAS-CHO-5-oCRBN = 18 h.

The kinome-wide profiling indicates that DAS-5-oCRBN, DAS-CHO-5-oCRBN, and DAS-DFGO-5-oCRBN bind to kinases other than c-Src and Abl (Figure 3). Thus, broader profiling of putative targets degraded by each of the conformation- selective PROTACs was performed. After treatment of CAL148 cells (100 nM, 18 h), reverse-phase protein arrays (RPPA, MD Anderson) were used to quantify the level remaining of 394 cancer-related proteins (Figure 4D). In the RPPA panel, DAS-5-oCRBN significantly (>50% degradation) degraded Csk and c-Src. DAS-CHO-5-oCRBN only significantly degraded c-Src out of the 394 proteins surveyed. DAS-DFGO-5-oCRBN did not degrade c-Src (consistent with our ELISA experiments) but did degrade p38 kinase and PLCg. DAS-DFGO-II inhibits p38 kinases, and p38 kinases were found to be targets of DAS-DFGO-5-oCRBN in the kinome profiling (Figure 3). Using western blots, it was conformed that DAS-DFGO-5-oCRBN causes the proteosome- mcdiatcd degradation of p38a and p38b kinases (no degradation was observed with p38g and p38d). No degradation of PLCg by DAS-DFGO-5-oCRBN was observed. This indicates that the PLCg was a false positive in the RPPA. Thus, DAS-DFGO-5-oCRBN was found to be a selective degrader of p38 across the 394 proteins studied. These data further highlight that modulating protein conformation (here kinase conformation) not only impacts kinome-wide selectivity, but also modulate which proteins are degraded.

As controls for cell proliferation assays, N-methyl thalidomide analogs were synthesized for each of the conformation-selective PROTACs. The N-methyl analogs of thalidomide have been reported to not bind cereblon and thus do not cause proteosome-based degradation. Using NanoBRET intracellular target engagement assays (Promega, Madison, WI) in HEK293 cells, it was found that the N-methyl analogs did not bind cereblon. Consistent with not binding cereblon, no c-Src degredation was observed resulting from treatment of CAL148 cells with the N-methyl analogs. Using a NanoBRET intracellular target engagement assay for c-Src (Promega, Madison, WI), it was determined the cellular EC₅₀ of binding c-Src for each conformation- selective PROTAC and inactive control). All of the conformation- selective parent inhibitors are potent cellular inhibitors of c-Src. DAS-5-oCRBN and it’s inactive control (DAS-5-oCRBN- NMe) are also potent inhibitors of c-Src. Meanwhile, the two conformation- selective PROTACs (DAS-CHO-5-oCRBN and DAS-DFGO-5-oCRBN) and their inactive controls are all poor cellular inhibitors of c-Src. Thus, DAS-5-oCRBN represents a potent degrader and inhibitor of c- Src, while DAS-CHO-5-oCRBN is a potent degrader but not a cellular inhibitor of c-Src.

DAS-5-oCRBN and DAS-CHO-5-oCRBN thus represent useful compounds to understand the cellular effects of c-Src degradation versus inhibition. In MDA-MB-231 cells, known to be dependent on both c-Src activity and knockdown, both DAS-5-oCRBN and DAS- CHO-5-oCRBN are potent inhibitors of cellular proliferation (GI₅₀ = 6 and 50 nM, respectively). The corresponding inactive control compounds, DAS-5-oCRBN-NMe and DAS-CHO-5- oCRBN-NMe, have GI₅₀ values of 50 and >10,000 nM, respectively. From these data, it was observed that either cellular inhibition or degradation leads to growth inhibition of MDA-MB- 231 cells. The DAS-CHO-5-oCRBN is a potent degrader (DC₅₀ = 53 nM) matches its ability to inhibit cellular proliferation ( GI₅₀ = 50 nM). DAS-DFGO-5-oCRBN is a selective degrader of p38 kinase. Thus, the impact of DAS- DFGO-5-oCRBN on the cellular proliferation of HT29 cells was measured, which known to be dependent on p38 kinase (Figure 5B). It was found that DAS-DFGO-5-oCRBN is a potent growth inhibitor (GI₅₀ = 200 nM) while the inactive control compound, DAS-DFGO-5-oCRBN- NMe exhibited ten-fold weaker growth inhibition (GI₅₀ = 2,620 nM). DAS-5-oCRBN, which does not degrade p38 kinase had no growth inhibition of HT29 cells (Figure 5B, GI₅₀ > 10,000 nM).

Using the ELISA, it was found that 100 nM DAS-5-oCRBN has a D_max = 78% at 18 h in CAL148 cells (Figure 4C). Next, the protein resynthesis rate of c-Src after DAS-5-oCRBN treatment and washout was determined. CAL148 cells were treated with 100 nM DAS-5-oCRBN and then the compound was washed out after 18 h. C-Src levels were monitored using the ELISA to determine the c-Src resynthesis rate. Four days (96 h) was required to regain normal cellular c- Src (figure 5C). It was observed that treatment with c-Src inhibitors (including dasatinib) increased cellular c-Src levels and thus a washout study was performed with dasatinib to determine the timecourse for normalization of c-Src levels following inhibitor washout. It was found that c-Src levels were immediately increased (to a maximum of 200%) upon treatment with 100 nM dasatinib and required 4 days post dasatinib washout to return to baseline levels (Figure 5C).

Experiments were conducted during development of embodiments herein to determine whether the pharmacodynamic differences between c-Src inhibitors (e.g., dasatinib) and c-Src degraders (e.g., DAS-5-oCRBN) would impact cellular growth proliferation of cancer cells. In the NanoBRET target engagement assays, dasatinib is a 5x better inhibitor of c-Src compared to DAS-5-oCRBN. However, in MDA-MB-231 growth proliferation assays, 2x better activity was observe for DAS-5-oCRBN over dasatinib (GI₅₀ = 6 and 12 nM, respectively). Thus, despite being weaker kinase inhibitors, degraders can be better inhibitors of cellular proliferation.

Reverse-phase protein arrays (RPPA, MD Anderson) were used to elucidate signaling changes between c-Src inhibition (dasatinib) and c-Src degradation (DAS-CHO-5-oCRBN) in CAL148 cells. DAS-CHO-5-oCRBN is a potent degrader (DC₅₀ = 53 nM) without inhibiting c- Src in cells (EC50 > 10,000 nM). C-Src degradation led to significantly decreased (>50% reduction) of c-Src (54% reduction) and LC3A/B (55% reduction) protein levels (relative to c- Src inhibition). C-Src levels are decreased by PROTAC induced proteolysis. LC3A/B are autophagsome proteins previously reported to be involved in Src family kinase signaling (PNAS 2018 115, E12407-E12416; incorporated by reference in its entirety). Degradation of c-Src also led to significant upregulation (>50% increase) of several proteins (relative to c-Src inhibition). Increased proteins include Bak, pSer-79 ACC, PARG, JNK2, pSer-1981 ATM, pThr-246 PRAS40, SGK3, and pSer-897 EphA2. c-Src is known to be directly involved in signaling networks with JNK2, ATM, and EPHA2.

Example 2

Determination of Optimal Linker Lengths Using Src Degraders with Semi-Rigid Linkers

Example 3

Investigation of the Effect of Different Rigid Linkers on the Src Degraders

Example 5

Investigation of fluorine substitution and Different Linking Positions of the Cereblon ligand on the Src Degraders

Example 6

DC₅₀ and D_max Values of Selected Compounds in the CAL148 Cell Line

Example 7

Assessment of Plasma Exposure of Selected compounds in Mice

Example 8

In vivo pharmacokinetic study of Compound 40-6-19

Chemical Formula: C₄₁H₄₄CIN₁₁O₅S Exact Mass: 837.29361 Molecular Weight: 838.38500 Experiments were conducted during development of embodiments herein to evaluate the pharmacokinetic characteristics of 40-6-19 in CD-I mice following IP, IV and PO injection. The drug at 1 mg/mL in PBS containing 15% DMSO and 25% PEG-400 was given by IV injection (3mg/kg) and oral (PO) 10mg/kg. The drug at 1.5 mg/mL in PBS containing 15% DMSO and 25% PEG-400 was given by IP injection (7.5mg/kg) and IP injection (15mg/kg). At the given time points (0.083h, 0.167h, 0.25h, 0.5h, Ih, 2h, 4h, 7h, 16h and 24h), blood samples were collected using heparinized calibrated pipettes. Samples were centrifuged at 15000 rpm for 10 min. Subsequently, plasma was collected from the upper layer. The plasma was frozen at -80°C for later analysis.

Table 3. Summary of actual intravenous (IV) intraperitoneal (IP)and oral (PO) administration dosages by animal weight and sampling time points by mouse.

The chromatographic conditions showed that the blank plasma and IS (ERD1233A) have no interference to the 40_6_19 determination (Figure 5A).

Plasma Calibration curve

The analytical curve was constructed using nine non-zero standards with 40_6_19 concentration ranging from 2 to 5000 ng/mL in the blank plasma samples. A blank sample (matrix sample processed without internal standard) was used to exclude contamination. The linear regression analysis of 40_6_19 was performed by plotting the peak area ratio (y) against the 40_6_19 concentration (x) in ng/mL. The linearity of the relationship between peak area ratio and concentration was demonstrated by the correlation coefficients (R=0.9973) obtained for the linear regression of 40_6_19 (***Figure 6).

Quality control (QC) samples

The accuracy and precision were evaluated at three concentration levels (5, 2000, and 5000 ng/mL for plasma) with three individual replicates at each concentration in plasma. The QC stock solution was prepared from separate weighing. QC samples were prepared at 3 levels (5, 2000, and 5000 ng/mL for plasma). QC samples were run before, in the middle and after running the samples. At least 50% of QCs at each level were within 15% of their nominal concentration.

The intra-batch precision was calculated and expressed as relative standard deviation (RSD %). The data indicated that the assay method was reliable and reproducible.

LC-MS/MS Conditions

Chromatographic Conditions:

Column: 5 cm x 2.1 ram I.D., packed with 3.5 μm

XBridge C 18

Mobile Phase A; 0.1% formic acid in purified deionized water

Mobile Phase B: 0.1 % formic acid in acetonitrile

Results

Accuracy and Intra-batch Precision for 40-6-19 in mouse plasma:

Means and SD of 40_6_19 concentration (ng/mL) in plasma samples after IV administration (3mg/kg):

Means and SD of 40_6_19 concentration (ng/mL) in plasma samples PO administration (lOmg/kg):

Means and SD of 40_6_19 concentration (ng/mL) in plasma samples IP administration (7.5mg/kg):

Means and SD of 40_6_19 concentration (ng/mL) in plasma samples IP administration (15mg/kg):

BLQ: below limit of quantitation

BLQ and * is outlier and was not included in the calculation

40_6_19 pharmacokinetic parameters in plasma following IV administration

PK parameters were estimated using non-compartmental analysis with Phoenix/WINONLIN. Cmax = Maximum observed concentration, Tmax = Time to reach Cmax, AUC(O-tldc) = Area under the concentration-time curve from time zero to time of last detectable concentration, AUC(O-inf) = Area under the concentration-time curve from time zero to infinite, CL = Systemic

10 clearance, CL_F: Apparent clearance, Vss: Volume of distribution at steady state, Vz_F: Volume of distribution associated with the terminal elimination phrase, Terminal elimination half-life (t½) was calculated based on data points (>= 3) in the terminal phase with correlation of coefficient > 0.90, %F = bioavailability REFERENCES

The following references, some of which are cited above by number, are herein incorporated by reference in their entireties.

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Claims

1. A composition comprising a proto-oncogene tyrosine-protein kinase Src (c-Src) ligand covalently tethered to a ubiquitin E3 ligase (E3L) ligand.

2. The composition of claim 1, wherein the c-Src ligand is dasatinib, bosutinib, ponatinib, or a dasatinib derivative, bosutinib derivative, or ponatinib derivative capable of binding to c-Src.

3. The composition of claim 2, wherein the c-Src ligand is of the formula:

; wherein X is N or CH, wherein Q is H or Cl; wherein A is -L-phenyl, wherein L is -O- or -NHC(O)-, and wherein phenyl is optionally further substituted.

4. The composition of claim 3, wherein the phenyl of -L-phcnyl is substituted at the meta position.

5. The composition of claim 4, wherein the phenyl of -L-phenyl is substituted with -CF3).

6. The composition of claim 1, wherein the E3L ligand is selected from thalidomide, pomalidomide, lenalidomide, iberdomide, (S,R,S)-AHPC-Me hydrochloride, (S,R,S)-AHPC-Me dihydrochloride, cereblon modulator 1, thalidomide-propargyl, (S,R,S)-AHPC-propargyl, (S,R,S)-AHPC hydrochloride, CC-885, thalidomide-O— COOH, lenalidomide hemihydrate, thalidomide fluoride, thalidomide-OH, lenalidomide-Br, thalidomide D4, lenalidomide hydrochloride, (S,R,S)-AHPC-Me, clAPl ligand 1, TD-106, E3 ligase Ligand 8, E3 ligase Ligand 9, E3 ligase Ligand 10, E3 ligase Ligand 13, E3 ligase Ligand 14, E3 ligase Ligand 18, BC-1215, VHL ligand 1 (VHL-1), VHL ligand 2 (VHL-2), VHL Ligand 8 (VHL-8), VH032, VH032-cyclopropanc-F, VH032 thiol, VH-298, VL-269, VL-285, LCL161, hydroxyprolinc- based ligands, HIF- 1α-derived (R)-hydroxyproline, Nutlin carboxylic acid, (4R,5S)-Nutlin carboxylic acid, (S,R,S)-AHPC-Boc, AR antagonist 1, NV03, (S,R,S)-AHPC TFA, (S,R,S)- AHPC,β- Naphthoflavone-CH2-Br, βN- aphthoflavone-CH2-OH, Bestatin-amido-Me, MV-1- NH-Me, (S,S,S)-AHPC hydrochloride, and clAPl ligand 2.

7. The composition of claim 1, wherein the E3L ligand is selected from:

8. The composition of claim 1 , wherein the c-Src ligand is covalently tethered to the E3L ligand by a linker.

9. The composition of claim 8, wherein the linker is alkyl or heteroalkyl chain of 1-25 atoms in length which may be optionally substituted.

10. The composition of claim 1, wherein the compound is selected from:

11. A method of degrading a c-Src protein comprising contacting the c-Src protein with a composition of one of claims 1-10.

12. The method of claim 11, wherein the c-Src protein is within a cell or a subject.

13. A method of treating or preventing cancer in a subject, the method comprising administering a composition of one of claims 1-10 to the subject.

14. The method of claim 13, wherein the composition is co-administered with one or more additional therapies for the treatment or prevention of cancer.

15. The method of claim 14, wherein the one or more additional therapies are selected from a chemotherapeutic, an immunotherapeutic, surgery, and radiation.

16. Use of the composition of any of claims 1-10 in the manufacture of a medicament for degrading a c-Src protein.

17. Use of the composition of any of claims 1-10 in the manufacture of a medicament for the treatment of cancer.