Methods and compositions for the treatment of cancer

Abstract

The present disclosure describes small molecule inhibitors that reduce YAP1 protein levels and therefore inhibit all YAP1 driven processes, including TEAD-mediated transcription. The present disclosure further provides methods of treating cancer utilizing such small molecule inhibitors.

Description

TITLE OF THE INVENTIONMETHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 685,824, filed August 22, 2024, the entire disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] This present disclosure relates to the field of cancer therapeutics, and more specifically to methods and compositions for reducing YES -associated protein 1 (YAP1) protein levels and TEA domain family (TE AD) -regulated transcriptional activity.BACKGROUND OF THE INVENTION

[0003] YAP1 is a potent amplified oncogene in multiple cancer types, including but not limited to glioblastoma, liver, colorectal, lung, breast, ovarian, pancreatic, and prostate cancer. YAP1 is also known to contribute to resistance to a number of small molecule inhibitors, chemotherapies, and radiotherapy. YAP1 is a downstream effector of Hippo signaling and a driver for approximately 150,000 tumors annually. Currently available commercial YAP1 inhibitors mediate their function by targeting YAP1 / TEAD binding, however, YAP1 also serves additional functions that are independent of transcriptional activation by TEAD. Thus, there is a continuing need for inhibitors of YAP1 that are capable of inhibiting all of YAP1 functions in the tumor microenvironment. The present disclosure describes small molecule inhibitors that reduce YAP1 protein levels and therefore inhibit all YAP1 driven processes, including TEAD-mediated transcription. This provides a significant advance in the art as compared to the YAP1 inhibitors currently known, which only regulate TEAD-mediated transcription.1US_ACTIVE\130819540W-1SUMMARY OF THE INVENTION

[0004] In one aspect, the present disclosure provides a compound of Formula I or a derivative thereof:Ri and R2 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, -CONH2, - CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, or -CN, X is a substituted methylene, a nitrogen, or an oxygen, R3 and R4 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, or a substituted alkyl, or R3 and R4, when attached to the same carbon, can form a carbocyclic ring, Q is CH or N, and is an aryl. In certain embodiments, the compound may have a formula seclected from the group consisting of:2US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-17US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1derivative of any thereof.

[0005] In another aspect the present disclosure provides a compound having a formula selected from the group consisting of Formula II, Formula II, and Formula IV, or a derivative of any thereof:amine spacer is selected from the group consisting of:9US_ACTIVE\130819540W-1Ri and R2 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, -CONH2, - CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, or -CN, X is a substituted methylene, a nitrogen, or an oxygen, R3 and R4 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, or a substituted alkyl, or R3 and R4, when attached to the same carbon, can form a carbocyclic ring,Q is CH or N, Ar is an aryl, linker is an alkyl, a branched alkyl, a repeating ethylene glycol chain, an ethylene diamine, or a substituted bicyclic amine, and Rs is a small molecule E3 -ligase ubiquitin-recruiting ligand.

[0006] In one embodiment, a compound of the present disclosure may have the formula ofFormula II or a derivative thereofIn another embodiment, a compound of the present disclosure may have the formula of Formula III or a derivative thereof10US_ACTIVE\130819540W-1In yet another embodiment, a compound of the present disclosure may have the formula of Formula IV or a derivative thereof. In still yet another embodiment, a compound of the present disclosure may have a formula selected from the group consisting of:1 1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1or a derivative of any thereof.

[0007] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a compound provided by the present disclosure and a pharmaceutically acceptable carrier, buffer, or diluent.

[0008] In another embodiment, the present disclosure provides a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound of the present disclosure to the subject. In yet another embodiment, the subject is afflicted with or at risk of developing cancer. In still yet another embodiment, the subject is afflicted with or at risk of developing brain cancer, breast cancer, ovarian cancer, prostate cancer, salivary cancer, lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, bladder cancer, skin cancer, head and neck squamous cell carcinoma, or genitourinary cancer. The administering, in one embodiment, comprises local, systemic, regional, systemic, or continual administration. The administering, in another embodiment, comprises oral administration, intravenous administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, topical administration, intradermal administration, intratracheal administration, intramuscular administration, subcutaneous administration, or inhalation. In yet another embodiment, the methods of the present disclosure may further comprise administering a second therapy to the subject. Non-limiting examples of second therapies that may be administered to the subject include a chemotherapy, a radiotherapy, a targeted therapy, a small molecule inhibitor, an immunotherapy, and surgery.16US_ACTIVE\130819540W-1In still yet another embodiment, the subject is a mammalian subject. The subject, in one embodiment, is a human subject.

[0009] In yet another aspect, the present disclosure provides a method of reducing YAP1 protein levels in a subject in need thereof, the method comprising administering a therapeutically effective amount a compound of Formula I, Formula II, Formula III, or Formula IV to the subject, wherein amine spacer is selected from the group consisting of:17US_ACTIVE\130819540W-1Ri and R2 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, -CONH2, - CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, or-CN, X is a substituted methylene, a nitrogen, or an oxygen, R3 and R4 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, or a substituted alkyl, or R3 and R4, when attached to the same carbon, can form a carbocyclic ring, Q is CH or N, Ar is an aryl, linker is an alkyl, a branched alkyl, a repeating ethylene glycol chain, an ethylene diamine, or a substituted bicyclic amine, and Rs is a small molecule E3-ligase ubiquitin-recruiting ligand. In one embodiment, the subject is afflicted with or at risk of developing cancer. In another embodiment, the subject is afflicted with or at risk of developing brain cancer, breast cancer, ovarian cancer, prostate cancer, salivary cancer, lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, bladder cancer, skin cancer, head and neck squamous cell carcinoma, or genitourinary cancer. In yet another embodiment, the administering comprises local, systemic, regional, systemic, or continual administration. In still yet another embodiment, the administering comprises oral administration, intravenous administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, topical administration, intradermal administration, intratracheal administration, intramuscular administration, subcutaneous administration, or inhalation. The18US_ACTIVE\130819540W-1methods of the present disclosure may further comprise, in one embodiment, administering a second therapy to the subject. In another embodiment, the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, a targeted therapy, a small molecule inhibitor, an immunotherapy, and surgery. In yet another embodiment, the subject is a mammalian subject. In still yet another embodiment, the subject is a human subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0011] FIG. 1 shows the core structure of compounds containing enriched fragments from the HTS screen conducted to identify compounds that degrade YAP1 protein. FIG. 1, Panel A shows the core structure commonly found in the screened hits. FIG. 1, Panel B shows the top 4 compounds containing the enriched fragment, ranked based on the measured intensity. FIG. 1 , Panel C shows the structure of the top 4 compounds containing the enriched fragment.

[0012] FIG. 2 shows the results of a luciferase assay for the analysis of YAP1 degraders. The tested compounds demonstrated a significant reduction in YAP1 activity. *p<0.05, **p<0.01, ***p<0.001.

[0013] FIG. 3 shows the IC50 results of proliferation assays for the analysis of YAP1 degraders shown in Fig 2.

[0014] FIG.4 demonstrates that CIDD-0166370 degrades YAP1 in YAP dependent cell lines. FIG. 4, Panel A shows western blot analysis for total YAP1 protein in the YAP 1 -dependent cell lines OVCAR5 and H292 treated with escalating doses of CIDD-0166370. FIG. 4, Panel B shows western blot analysis for total YAP1 protein in the YAP 1 -dependent cell lines sF268 and H2052 treated with escalating doses of CIDD-0166370. FIG. 4, Panel C shows western blot analysis for total YAP1 protein in the YAP 1 -dependent cell line NCI-H1693 and the YAPl-resistant cell line NCI-H1975 treated with escalating doses of CIDD-0166370.

[0015] FIG. 5 demonstrates that CIDD-0166370 degrades YAP1 in a time dependent manner. Western blot analysis for total YAP1 protein in the YAP 1 -dependent cell line NCI-H1693 treated with protein synthesis inhibitor CHX alone for 12 h and 24 h and CIDD-0166370 for 24 h.19US_ACTIVE\130819540W-1

[0016] FIG. 6 demonstrates that CIDD-0166370 degrades YAP1 via the ubiquitin-proteasome pathway. Western blot analysis for total YAP1 protein in the YAP 1 -dependent cell lines H292, SF268, H2052 and NCI-H1693 treated with IpM CIDD-0166370 in the presence and absence of proteasome inhibitor (MG132).

[0017] FIG. 7 demonstrates in vivo activity of CIDD-0166370. Low doses of CIDD-0166370 (1 mg / kg and 5 mg / kg) attenuated tumor growth in H292 mice xenograft models. ***p<0.001.

[0018] FIG. 8 shows pharmacokinetic (PK) analysis of CIDD-0166370 in plasma. PK analyses was performed on mice treated with a single i.p. injection of 5 mg / kg CIDD-0166370 and then scarified at 1 of 8 timepoints (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 24 h).

[0019] FIG. 9 shows pharmacokinetic (PK) bioanalysis of CIDD-0166370 in tissue. PK analyses was performed on tissues (kidney, liver, small intestine) collected from mice at the 30 min, 1 h, and 4 h timepoints following treatment with a single i.p. injection of 5 mg / kg CIDD-0166370.

[0020] FIG. 10 shows the results of a luciferase assay for the analysis of analogs of CIDD- 0166370. The tested compounds demonstrated significant reduction in YAP1 activity. *p<0.05, **p<0.01, ***p<0.001.

[0021] FIG. 11 shows the IC50 results of proliferation assays for the analysis of analogs of CIDD-0166370 shown in Fig 10.

[0022] FIG. 12 demonstrates that PROTACs degrade YAP1. The tested PROTACs CIDD- 0162288 and CIDD-0162289 (RLP-88 and RLP-89, respectively) degraded YAP1.

[0023] FIG. 13 demonstrates in vivo activity of CIDD-0162288. Low doses of CIDD-0162288 (1 and 5 mg / Kg) attenuated tumor growth in H292 mice xenograft models. ***p<0.001.

[0024] FIG. 14 shows the results of a luciferase assay for the analysis of YAP1 PROTACs. The tested compounds demonstrated significant reduction in YAP1 activity. *p<0.05, **p<0.01, ***p<0.001.

[0025] FIG. 15 shows the IC50 results of proliferation assays for the analysis of YAP1 PROTACs shown in Fig 14.

[0026] FIG. 16 demonstrates that CIDD-0166579 degrades YAP1 in YAP dependent cell lines. FIG. 16, top panel shows western blot analysis for total YAP1 protein in the YAP1- dependent cell line OVCAR5. FIG. 16, bottom panel shows western blot analysis for total YAP1 protein in the YAP 1 -dependent cell line H292.20US_ACTIVE\130819540W-1

[0027] FIG. 17 demonstrates that CIDD-0166579 degrades YAP1 via the ubiquitin- proteasome pathway. Western blot analysis of total YAP1 protein in the YAP 1 -dependent cell lines OVCAR5 and H292 treated with IpM CIDD-0166579 in the presence and absence of proteasome inhibitor (MG132).

[0028] FIG. 18, demonstrates in vivo activity of CIDD-0166579. Low doses of CIDD- 0166579 (1 and 5 mg / Kg) attenuated tumor growth in H292 mice xenograft models. **p<0.01.

[0029] FIG. 19 shows the results of a luciferase assay for additional YAP1 degraders tested. *p<0.05, **p<0.01, ***p<0.001.DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure provides methods and compositions for treating cancer. YAP1 is a potent amplified oncogene in multiple cancer types and is known to contribute to resistance to inhibitors of the MAPK pathway. YAP1 is a downstream effector of Hippo signaling and a driver for approximately 150,000 tumors annually. In the canonical core Hippo signaling network, NF2 / merlin activates MST1 / 2, which in turn activates LATS1 / 2 by phosphorylation. Active LATS1 / 2 phosphorylates YAP1 leading to its cytoplasmic retention and inhibition of downstream target gene transcription. When upstream kinases are inactive, YAP1 translocates to the nucleus and activates gene expression through interaction with the TEAD family of DNA binding transcription factors, leading to cell growth and proliferation. YAP1 is essential for cancer initiation, progression, and / or metastasis, however it is dispensable for normal tissue homeostasis suggesting the importance of inhibiting YAP1 activity to block cancer progression. YAP1 is a bona-fide target for anticancer compounds in the subset of cancers expressing high YAP1, or in cancers harboring NF2 mutations. In addition, YAP1 has been implicated in mediating resistance to 5-FU in colorectal and esophageal cancer, Kras- inactivation in pancreatic and colon cancers, EGFR inhibitors in non-small cell lung and ovarian carcinoma, and cisplatin in ovarian and head and neck cancers. It also has been implicated in radiotherapy resistance in head and neck cancer, urothelial cancer, and medulloblastoma. Consistent with these reports, recent genome-wide screening data (Achilles / Avana data from Broad / Novartis) provides genetic evidence of YAP1 dependency among a significant number of cell lines across various tumor types. However, YAP1 does not harbor any enzymatic activity or easily discernible binding pockets that can be used for the discovery of small molecules. Considering that YAP1 has been shown to be regulated by21US_ACTIVE\130819540W-1proteasomal mediated degradation, one approach is to identify small molecules that promote YAP1 degradation.

[0031] YAP1 inhibitors currently known in the art target the YAP1 / TEAD binding surface, however, YAP1 serves additional functions that are independent of transcriptional activation by TEAD. Thus, there is a continuing need in the art for inhibitors of YAP1 that are capable of inhibiting all of YAP1 functions in the tumor microenvironment. The present disclosure describes small molecule inhibitors that reduce YAP1 protein levels and therefore inhibit all YAP1 driven processes, including TEAD-mediated transcription. This provides a significant advance in the art as compared to the YAP1 inhibitors currently known in the art, which only regulate TEAD-mediated transcription.A. Small Molecule Inhibitors of YAP1

[0032] In certain aspects, the present disclosure provides YAP1 inhibitors, degraders, and PROTACs (Proteolysis Targeting Chimeras) useful as therapeutics for the treatment of a variety of cancer types and tumors, including but not limited to brain, breast, ovarian, prostate, and salivary tumors.

[0033] In some embodiments, the present disclosure provides inhibitors, degraders, and PROTAC compounds of general Formula I, Formula II, Formula III, and Formula IV or pharmaceutically acceptable salts thereof:22US_ACTIVE\130819540W-1

[0034] In particular embodiments, the amine spacer group is a cyclic or bicyclic amine, or cyclic or bicyclic diamine containing 3-10 carbon atoms, optionally substituted by one or more R3 and / or R4 groups. In certain embodiments, the optionally substituted cyclic or bicyclic amine and cyclic or bicyclic diamine groups may be attached to a quinazoline moiety and a heterocyclic aryl moiety through the nitrogen atoms as depicted below. Some non-limiting representative examples of cyclic and bicyclic amines and cyclic and bicyclic diamines spacers are shown below.

[0035] In one embodiment, Ri and R2 are independently hydrogen, halogen, alkyl, branched alkyl, -CONH2, -CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, -CN. In another embodiment, X is a substituted methylene, nitrogen or oxygen, including -NH-C(O)-, -C(O)-NH-, -C(O)-O- , -O-C(O)-, -0-CH2-, -NH-CH2-, -CH2-NH-, -CH2-0-, -C(O)-NH-CH2-triazole-CH2-, -NH- C(O)- CH2-triazole-CH2-, -C(O)-CH2-, -CH2-C(O)-, -C(O)-NH-CH2-, -CH2-NH-C(O)-, and the like. In yet another embodiment, R3 and R4 are independently hydrogen, halogen, alkyl, branched alkyl (C1-C4), substituted alkyl (C1-C4). In still yet another embodiment, R3 and R4, when attached to the same carbon, can form a carbocyclic (spirocyclic) ring (C3-C6). Q, in one embodiment, is CH or N. Ar is aryl. Unless otherwise indicated, as used herein, the term “aryl” includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl (Ph), naphthyl, indenyl, indanyl and fluorenyl. “Aryl” encompasses fused ring groups wherein at least one ring is aromatic. Unless otherwise23US_ACTIVE\130819540W-1indicated, as used herein, “heteroaryl” refers to aromatic groups containing one or more heteroatoms, preferably from one to three heteroatoms, selected from O, S and N. A multicyclic group containing one or more heteroatoms wherein at least one ring of the group is aromatic is a “heteroaryl” group. The aryl group can be optionally substituted with 1-3 Ri and / or R2 groups.

[0036] In certain embodiments, in a compound of Formula II, the X-linker and / or X-R5 group may be optionally connected to any position of the quinazoline ring. In particular embodiments, in a compound of Formula III, the X-linker and / or X-R5 group may be optionally connected to any position of the amine spacer represented by the cyclic or bicyclic amines and cyclic or bicyclic diamines described herein. In further embodiments, in compound of Formula IV, the X-linker and / or X-R5 group may be optionally connected to any position of the aryl group (Ar).

[0037] As used herein the term “linker” refers to an alkyl chain (Cl -Cl 2), a branched alkyl chain (Cl -CIO), a repeating ethylene glycol chain, or an ethylene diamine. Non-limiting examples of a linker include (-O-CH2-CH2-O-)n, wherein n is 1-8, (-NH-CH2-CH2-NH-)n, wherein n is 1-8, and substituted cyclic amines such as:n=0-6.

[0038] In some embodiments, R5 may be a suitable small molecule E3-ligase ubiquitin- recruiting ligand, such as a CRBN ligands (such as A below), or a VHL ligand (such as B and C below). In certain embodiments, the Rs group ligands, such as A, B and C below, can be optionally substituted to the linkers and X groups on any atom in the structures.24US_ACTIVE\130819540W-1

[0039] Additional exemplary compounds of the present disclosure are provided in Table 1.Table 1: Exemplary YAP1 Inhibitors25US_ACTIVE\130819540\V-126US_ACTIVE\130819540\V-127US_ACTIVE\130819540\V-128US_ACTIVE\130819540\V-129US_ACTIVE\130819540\V-130US_ACTIVE\130819540\V-131US_ACTIVE\130819540\V-132US_ACTIVE\130819540\V-133US_ACTIVE\130819540\V-134US_ACTIVE\130819540\V-135US_ACTIVE\130819540\V-136US_ACTIVE\130819540\V-137US_ACTIVE\130819540\V-138US_ACTIVE\130819540\V-139US_ACTIVE\130819540\V-140US_ACTIVE\130819540\V-141US_ACTIVE\130819540\V-142US_ACTIVE\130819540\V-143US_ACTIVE\130819540\V-144US_ACTIVE\130819540\V-145US_ACTIVE\130819540\V-146US_ACTIVE\130819540\V-147US_ACTIVE\130819540\V-148US_ACTIVE\130819540\V-149US_ACTIVE\130819540\V-150US_ACTIVE\130819540\V-151US_ACTIVE\130819540\V-152US_ACTIVE\130819540\V-153US_ACTIVE\130819540\V-154US_ACTIVE\130819540\V-155US_ACTIVE\130819540\V-156US_ACTIVE\130819540\V-157US_ACTIVE\130819540\V-158US_ACTIVE\130819540\V-159US_ACTIVE\130819540\V-1

[0040] Pharmaceutically acceptable salts of compounds of the present disclosure, including those of Formula I, II, III and / or IV, include the acid or base addition salts thereof. All reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the resulting salt may vary from completely ionized to almost non-ionized. Suitable non-toxic,60US_ACTIVE\130819540\V-1acid-addition pharmaceutically acceptable salts include, but are not limited to, the acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mandelates mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, salicylate, saccharate, stearate, succinate, sulfonate, stannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

[0041] Suitable non-toxic, base-addition pharmaceutically acceptable salts include, but are not limited to the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), which is incorporated herein by reference.

[0042] Included within the scope of the present invention are all stereoisomers, geometric isomers, and tautomeric forms of the compounds of the present disclosure, including compounds of Formula I, II, III and / or IV. Compounds of the present disclosure include compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof.

[0043] The present disclosure includes all pharmaceutically acceptable isotopically labeled compounds of the present disclosure, including compounds of Formula I, II, III and / or IV, wherein one or more atoms may be replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. Non-limiting examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,35S,18F, and36C1. Non-limiting examples of radioactively labeled forms include compounds labeled with tritium, phosphorous-32, iodine-129, carbon-11, and fluorine-18. Certain isotopically labeled compounds, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. In some embodiments, substitution with heavier isotopes such as deuterium (2H) can afford certain therapeutic advantages such as greater metabolic stability, increased in vivo half-life, or reduced dosage requirements. Isotopically labeled compounds of the present disclosure and61US_ACTIVE\130819540W-1prodrugs thereof can generally be prepared by carrying out the methods provided in the present disclosure and by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.

[0044] In certain embodiments, the compounds of the present disclosure may form prodrugs. In certain embodiments, the compounds of the present disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, or similar groups as the prodrug forming moieties. For instance, the hydroxymethyl position may form mono-, di-, or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources and any such method known in the art may be used to prepare prodrugs of the compounds of the present disclosure.

[0045] In certain embodiments, the compounds of the present disclosure can be present as a solvate. In some embodiments, the solvent used to prepare the solvate is an aqueous solution and may be referred to as a hydrate. The compounds of the present disclosure, in certain embodiments, can be present as a hydrate. In certain embodiments, a hydrate may be obtained by crystallization from a solvent or from aqueous solution. In particular embodiments, one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds of the present disclosure to form solvates and hydrates. Unless otherwise specified, the present disclosure includes all such possible solvates.

[0046] As used herein the term “derivative” when used in reference to the compounds of the present disclosure refers to pharmaceutically acceptable salts, prodrugs, isotopically labeled forms, deuterated forms, radioactively labeled forms, isomers, solvates, and any combination thereof of the compounds described herein.B. Methods of Treatment and Therapeutic Compositions

[0047] In certain aspects, the present disclosure provides methods, pharmaceutical compositions, and therapeutic compositions for the treatment of cancer. In one embodiment, the pharmaceutical and therapeutic compositions of the present disclosure reduce YAP1 protein levels in a subject. In certain embodiments, the methods of the present disclosure may comprise administering a second therapy selected from the group consisting of a chemotherapy, a radiotherapy, a targeted therapy, a small molecule inhibitor, an immunotherapy, and surgery.

[0048] In some embodiments, a compound of the present disclosure may be combined with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier,”62US_ACTIVE\130819540W-1“pharmaceutically acceptable adjuvant,” or “adjuvant” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with a therapeutic agent, or other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or pharmaceutical compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an agent may be added to a therapeutic composition or pharmaceutical composition to modify for example the cellular target, cellular localization, or cellular uptake of a therapeutic agent as described herein. Such an agent may include any excipient, diluent, carrier, or adjuvant that is acceptable for pharmaceutical use. Such an agent may be non- naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the therapeutic or pharmaceutical composition.

[0049] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a composition comprising a therapeutic agent of the present disclosure. In one embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor growth or tumor progression in a subject. Tn another embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor size in a subject. In still yet another embodiment, the composition is capable of reducing YAP1 protein levels in a subject.

[0050] A compound or composition of the present disclosure is meant to encompass a composition suitable for administration to a subject, such as a mammal, particularly a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the composition are pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, intraarticular, intraarterial, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalation, vaginal, intraosseous, trans nasal, injection, microneedle, topical, and transdermal. The appropriate dosage of a composition, as described herein, may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, clinical history, response to the treatment, and the discretion of the attending physician. In some embodiments, therapeutic compositions provided by the present disclosure may include63US_ACTIVE\130819540W-1various "unit doses." A unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some respects, a unit dose comprises a single administrable dose.

[0051] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0052] As used herein, “subject” or “patient” refers to animals, including humans, who are treated with the inhibitors, therapeutic compounds, or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs and the like. In particular embodiments, a subject in need of therapy may be any subject who comprises a cancer cell as described herein. In another embodiment, the subject may be afflicted with or at risk of developing a cancer as described herein. Nonlimiting examples of such diseases or conditions include brain cancer, breast cancer, ovarian cancer, prostate cancer, salivary cancer, lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, bladder cancer, skin cancer, head and neck squamous cell carcinoma, and genitourinary cancer.

[0053] A composition, as described herein, may include, in particular embodiments, a combination of therapeutic agents. In some embodiments, a composition as described here may be administered as a single composition or as more than one composition. Different compositions as provided herein, in certain embodiments, may be administered by the same route of administration or by different routes of administration.

[0054] A pharmaceutical composition of the present disclosure may comprise, in some embodiments, a targeting molecule. In one embodiment, the targeting molecule may be cellspecific or tissue-specific. Numerous such targeting molecules are known in the art and any such targeting molecule may be used according to the present disclosure. In certain embodiments, a composition of the present disclosure may be modified with or conjugated to64US_ACTIVE\130819540W-1a peptide, a protein, a colloidal molecule, or a polymer to facilitate delivery or adsorption. The pharmaceutical composition of the present disclosure, in some embodiments, may be serum-free, endotoxin-free, or sterile.

[0055] In certain embodiments, the compositions and methods for treating an individual described herein may be combined with any other composition or method of treatment known in the art. The compositions and methods may be administered in any suitable manner known in the art. For example, a first and a second therapeutic agent or inhibitor may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, a first and a therapeutic agent or inhibitor may be administered in separate compositions. In certain embodiments, a first and a second cancer treatment or inhibitor may be administered in the same composition.

[0056] Non-limiting examples of additional treatment modalities that may be included in combination with the compositions and methods provided herein include a therapeutic agent or surgery. In specific embodiments, the methods and compositions of the present disclosure may be combined with other therapies directed towards the treatment of cancer as described herein.

[0057] The term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word "comprising" or other open language in the claims, the words "a" and "an" denote "one or more," unless specifically noted otherwise. The terms "comprise," "have," and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that "comprises," "has," or "includes" one or more components is not limited to possessing only those components and covers other unlisted components.

[0058] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the65US_ACTIVE\130819540W-1disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0059] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0060] The following examples are included to illustrate embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0061] The compounds of the present disclosure may be prepared by any suitable method known in the art. In some embodiments, the compounds of the present disclosure may be prepared by methods described in the Examples, together with synthetic methods known in the art of organic chemistry, or modifications and derivatizations that are familiar to those of ordinary skill in the art.

[0062] In certain embodiments, preferred methods for preparation of the compounds of the present disclosure include, but are not limited to, those described in the Examples. During any of the synthetic sequences described herein, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups, such as those described in T. W. Greene, Protective Groups in Organic Chemistry, John Wiley & Sons, 1981; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Chemistry, John Wiley & Sons, 1991, which are hereby incorporated by reference.66US_ACTIVE\130819540W-1

[0063] The following schemes and examples are exemplary of the processes for making compounds of general Formula I, II, III, and / or IV. It is to be understood, however, that the disclosure, as fully described herein and as recited in the claims, is not intended to be limited by the details of the following examples. In certain embodiments, compounds of Formula I, Formula II, Formula IV, and / or their pharmaceutically acceptable salts, can be prepared according to exemplary reactions Schemes 1-5 described below. Isolation and purification of the products may be accomplished by standard procedures, which are known to a chemist of ordinary skill in the art.EXAMPLE 1: PRIMARY HIGH-THROUGHPUT SCREEN TO IDENTIFY SMALL MOLECULES THAT DEGRADE YAP1 IN CANCER CELLS

[0064] Primary image-based high-throughput screening (HTS) was used to identify small molecules that degrade YAP1 protein in OVCAR5-mCherry-YAPl-GFP cells. The screening was performed by seeding OVCAR5-mCherry-YAPl-GFP cells in a 384-well plate (-1000 cells / well) for 24 h followed by treatment with either vehicle (DMSO) or drug (ChemBridge drug library - 50,000 drugs compounds selected from a stock of over 480,000 compounds) at a dose of 10 p M for 72 h. Next, an !NCell6000 confocal imaging platform was used to image, analyze, and score YAP1-GFP and mCherry expression in control and drug-treated cells. Various assay quality control measures were utilized including a reproducibility plot to identify several compounds with a core structure that degraded YAP1 protein. The HTS screening results were further validated by Western blot analysis for YAP1 protein in OVCAR5 treated with the selected compounds for 24 h (data not shown).

[0065] To identify a core structure, fragments that were enriched in all hits in the original screen that produced reproducible results were identified. The chemistry and data analytics libraries of Pipeline Pilot (BIO VIA, Server 2018) were used to establish a structure-activity relationship (SAR) using the fluorescent read-out of the high throughput screen to rank activity. The data was evaluated using two different approaches, which were used collectively to identify a common core structure and rationalize activity based on functional modifications. The first method uses Bayesian enrichment of extended connectivity fingerprints, as previously described in Chen et al. J Chem Inf Model, 52:792-803, 2012. The second approach uses a matched molecular pairs filter to identify repetitive core structures found with-in active molecules as described in Griffen et al. J. Med. Chem., 54, 22, 7739-7750, 2011. Exemplary results of the primary screen are shown in Table 2.67US_ACTIVE\130819540W-1Table 2: Exemplary Results of Primary Screen for YAP1 Inhibitors68US_ACTIVE\130819540W-169US_ACTIVE\130819540W-170US_ACTIVE\130819540W-171US_ACTIVE\130819540W-172US_ACTIVE\130819540W-173US_ACTIVE\130819540W-174US_ACTIVE\130819540W-175US_ACTIVE\130819540W-176US_ACTIVE\130819540W-1

[0066] Both of the methods of analysis identified the same core structure (FIG. 1, Panel A), and a SAR table was built by ranking all the tested molecules containing this common core (FIG. 1, Panel B; FIG. 1, Panel C). Based on these fragments, hits were ranked, and the top 2 compounds were identified (41086740 and 60069350) that harbored the core structure and produced reproducible results in the screen (absolute range of 0.007 for 41086740 and 0.04 for 60069350).EXAMPLE 2: SYNTHESIS OF YAP1 LIGANDS WHEN THE CARBOXYLATE HANDLE TO INSTALL THE LINKER AND E3 LIGAND IS ON THE 7-POSITION OF THE QUINAZOLINE

[0067] Scheme 1 exemplifies the synthesis of YAP1 ligands, wherein the carboxylate handle to install the linker and E3 ligand is on the 7-position of the quinazoline.77US_ACTIVE\130819540W-1Scheme 1.

[0068] In reference to Scheme 1, alkylation of quinazoline 1 with N-BOC-protected piperazine 2 in the presence of iPrOH and DIEA at reflux temperatures produces the desired compound 3. Deprotection of the tert-butylcarbamate (N-BOC) under HC1 conditions produces the desired secondary amine 4. Reductive amination of 4 with aldehyde 5 cleanly produces the desired compound 6. Hydrolysis of the methyl ester in 6 under mild LiOH conditions cleanly produces the desired carboxylic acid 7.

[0069] Synthesis of methyl 4-(4-(tert-butoxycarbonyl)piperazin-l-yl)quinazoline-7- carboxylate.Boc

[0070] In Scheme 1, to a solution of 1 (0.5 g, 2.25 mmol, 1 equiv.) and tert-butyl piperazine- 1-carboxylate, 2 (0.418 g, 2.25 mmol, 1 equiv.) in anhydrous isopropanol (5 mL), DIEA (1 mL, 5.63 mmol, 2.5 equiv.) was added and kept under reflux for 20 h. The reaction mixture was cooled to room temperature and solvent was evaporated. Crude product was concentrated78US_ACTIVE\130819540W-1and purified by silica gel chromatography on biotage using 0-10% methanol and dichloromethane to obtain the required compound 3 (yield: 68%). ' H NMR (400 MHz, CDCh): 5 8.82 (s, 1H), 8.61 (d, J = 1.6 Hz, 1H), 8.08 (dd, J = 8.7, 1.8 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 4.02 (s, 3H), 3.81 - 3.77 (m, 4H), 3.69 (dd, J = 4.7, 2.1 Hz, 4H), 1.52 (s, 9H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 373 [M+H]+.

[0071] Synthesis of methyl 4-(piperazin-l-yl)quinazoline-7-carboxylate (4).

[0072] In Scheme 1, to the solution of compound 3 (0.25 g, 0.67 mmol, 1 equiv.) in CH2CI2 (5 mL), 4 M HC1 (1.7 mL, 6.72 mmol, 10 equiv.) was added and stirred at room temperature for 20 h. The reaction mixture was basified with 1 M NH4OH and extracted with dichloromethane (2 x 10 mL). The combined organic layer was dried over sodium sulfate, concentrated, and purified on biotage using 4-5% methanol / dichloromethane (0.1% NH4OH) to obtain the corresponding free amine 4 (yield: 98%). ’H NMR (400 MHz, CDCh): 5 8.77 (s, 1H), 8.56 (d, J = 1.7 Hz, 1H), 8.03 (dd, J = 8.7, 1.7 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 3.99 (s, 3H), 3.84 - 3.72 (m, 4H), 3.13 - 3.02 (m, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 273 [M+H]+.

[0073] Synthesis of methyl 4-(4-((l-phenyl-lH-pyrazol-4-yl)methyl)piperazin-l- yl)quinazoline-7-carboxyIate (6).79US_ACTIVE\130819540W-1

[0074] In Scheme 1, to the solution of 4 (0.18 g, 0.66 mmol, 1 equiv.) and aldehyde, 5 (0.12 g, 0.72 mmol, 1.1 equiv.) in dichloroethane (3.3 mL) sodiumtriacetoxyborohydride (0.28 g, 1.32 mmol, 2 equiv.) was added followed by acetic acid (3 drops). The reaction mixture was allowed to stir at room temperature for 24 h. Solvent was evaporated and the crude compound was purified by silica gel chromatography on biotage using 0-8% methanol and dichloromethane to obtain the required compound 6 (yield: 69%).]H NMR (400 MHz, DMSO-de): 8 8.68 (s, 1H), 8.45 (s, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.97 (dd, J = 8.7, 1.8 Hz, 1H), 7.88 - 7.80 (m, 2H), 7.70 (s, 1H), 7.53 - 7.45 (m, 2H), 7.34 - 7.23 (m, 1H), 3.94 (s, 3H), 3.80 (t, J = 4.9 Hz, 4H), 3.55 (s, 2H), 2.62 (t, 7 = 4.9 Hz, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 429 [M+H]+.

[0075] Synthesis of 4-(4-((l-phenyl-lH-pyrazol-4-yl)methyl)piperazin-l-yl)quinazoline- 7-carboxylic acid (7).

[0076] In Scheme 1, to the solution of 6 (0.2 g, 0.45 mmol, 1 equiv.) in THF (9 mL) 1.2 M LiOH (6.5 mL, 6.75 mmol, 15 equiv.) was added and stirred at room temperature for 8 h. Solvent was evaporated, acidified to pH 6 with 1 M HC1, and extracted with 15% methanol and dichloromethane (3 10 mL). The combined organic layer was dried over sodium sulfate and concentrated to obtain acid 7. Crude compound was taken to next step (yield: 80%). 'H NMR (400 MHz, DMSO- d ) 8 8.61 (s, 1H), 8.45 (s, 1H), 8.21 (d, J = 1.6 Hz, 1H), 7.98 (dd, J = 8.6, 1.6 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 1.3 Hz, 1H), 7.82 (d, 7 = 1.1 Hz, 1H), 7.69 (s, 1H), 7.51 - 7.45 (m, 2H), 7.31 - 7.25 (m, 1H), 3.73 (t, J = 4.8 Hz, 5H), 3.54 (s, 2H), 2.62 (t, J = 4.9 Hz, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 413 [M- H]+.

[0077] Additional compounds synthesized following the procedure of Scheme 1 include: a) methyl 4-(4-((l-phenyl-lH-pyrazol-4-yl)methyl)piperazin-l-yl)quinazoline-6- carboxylate - 'H NMR (400 MHz, CDCh): 8 8.73 (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.29 (dd,80US_ACTIVE\130819540W-17 = 8.7, 1.9 Hz, 1H), 7.94 (s, 1H), 7.89 (d, 7 = 8.8 Hz, 1H), 7.73 - 7.62 (m, 3H), 7.51 - 7.40 (m, 2H), 7.36 - 7.27 (m, 1H), 3.96 (d, 7 = 4.7 Hz, 7H), 3.62 (s, 2H), 2.73 (s, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 429 [M+H]+;b) methyl 4-(4-(( 1 -benzyl- 1 H-pyrazol-4-yl)methyl)piperazin- 1 -yl)quinazoline- 6-carboxylate -]H NMR (400 MHz, CDCh): 5 8.74 (s, 1H), 8.64 (d, 7 = 1.9 Hz, 1H), 8.30 (dd, 7 = 8.8, 1.8 Hz, 1H), 7.90 (d, 7 = 8.8 Hz, 1H), 7.52 (s, 1H), 7.42 - 7.30 (m, 4H), 7.27 - 7.21 (m, 2H), 5.31 (s, 2H), 3.99 (s, 3H), 3.94 (t, 7 = 4.9 Hz, 4H), 3.53 (s, 2H), 2.67 (t, 7 = 4.8 Hz, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 443 [M+H]+; andc) Al,7V-dimethyl-4-(4-((l-phenyl-177-pyrazol-4-yl)methyl)piperazin-l- yl)quinazoline-7-carboxamide - H NMR (400 MHz, CDCh): 5 8.71 (s, 1H), 7.90 (t, 7 = 4.3 Hz, 2H), 7.85 (d, 7= 1.6 Hz, 1H), 7.70 - 7.63 (m, 3H), 7.51 - 7.37 (m, 3H), 7.28 (td, 7= 7.2,81US_ACTIVE\130819540W-12.0 Hz, 1H), 3.84 (t, 7 = 4.9 Hz, 4H), 3.59 (s, 2H), 3.15 (s, 3H), 3.01 (s, 3H), 2.69 (t, 7= 4.9 Hz, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 442 [M+H]+.EXAMPLE 3: SYNTHESIS OF YAP1 LIGANDS WHEN THE CARBOXYLATEHANDLE TO INSTALL THE LINKER AND E3 LIGASE IS ON EITHER THE 2- OR 5-POSITION OF THE PIPERAZINE RING

[0078] Scheme 2 exemplifies the synthesis of YAP1 ligands wherein the carboxylate handle to install the linker and E3 ligase is on either the 2- or 5-position of the piperazine ring (representative of the amine spacer group).Scheme 2.14, Ri = CO2CH3, R2= H 16, RT = CO2H, R2= H15, Ri = H, R2= CO2CH317, R-] = H, R2= CO2H

[0079] With reference to Scheme 2, alkylation of quinazoline 8 with N-BOC-protected piperazine 9 in the presence of iPrOH and HC1, cleanly produces either compound 10 (Ri =82US_ACTIVE\130819540W-1CO2CH3) or 11 (R2 = CO2CH3). Deprotection of the tert-butylcarbamate (N-BOC) under HC1 conditions produces the desired secondary amines 12 (Ri = CO2CH3) or 13 (R2 = CO2CH3). Reductive amination of 12 or 13 with aldehyde 5, cleanly produces the desired compounds 14 (Ri = CO2CH3) or 15 (R2 = CO2CH3). Hydroylsis of the methyl ester in either 14 or 15 under mild LiOH conditions cleanly produces the desired carboxylic acids 16 (Ri = CO2CH3) or 17 (R2= CO2CH3).

[0080] Synthesis of l-(tert-Butyl) 2-methyl 4-(quinazolin-4-yl)piperazine-l,2- dicarboxylate (10).Boc

[0081] In Scheme 2, compound 10 was synthesized following the same procedure used to synthesize compound 3 (Scheme 1, Example 1). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 373 [M+H]+.

[0082] Synthesis of l-(tert-Butyl) 3-methyl 4-(quinazolin-4-yl)piperazine-l,3- dicarboxylate (11).Boc

[0083] In Scheme 2, compound 11 was synthesized following the same procedure used to synthesize compound 3 (Scheme 1, Example 1). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 373 [M+H]+.

[0084] Synthesis of methyl 4-(quinazolin-4-yl)piperazine-2-carboxylate (12).83US_ACTIVE\130819540W-1Boc

[0085] In Scheme 2, compound 12 was synthesized following the same procedure used to synthesize compound 4 (Scheme I, Example 1). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 273 [M+H]+.

[0086] Synthesis of methyl l-(quinazolin-4-yl)piperazine-2-carboxylate (13).Boc

[0087] In Scheme 2, compound 13 was synthesized following the same procedure used to synthesize compound 4 (Scheme 1, Example 1). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 273 [M+H]+.

[0088] Synthesis of methyl l-((l-phenyl-lH-pyrazol-4-yl)methyl)-4-(quinazolin-4- yl)piperazine-2-carboxylate (14).84US_ACTIVE\130819540W-1

[0089] In Scheme 2, compound 14 was synthesized following the same procedure used to synthesize compound 6 (Scheme 1, Example 1).!H NMR (400 MHz, CDCh): 8 8.75 (d, J = 2.4 Hz, 1H), 7.94 - 7.87 (m, 3H), 7.77 - 7.71 (m, 1H), 7.71 - 7.65 (m, 3H), 7.46 (td, J = 7.8, 5.9 Hz, 3H), 7.30 (d, J = 7.4 Hz, 1H), 4.04 (dd, J = 12.9, 6.5 Hz, 1H), 4.01 - 3.86 (m, 2H), 3.75 (s, 3H), 3.81 (td, J = 7.7, 4.7 Hz, 2H), 3.73 (d, 7 = 3.3 Hz, 1H), 3.57 (dd, 7 = 6.5, 3.6 Hz, 1H), 3.30 (ddd, 7 = 10.6, 6.4, 3.5 Hz, 1H), 2.67 (ddd, 7 = 11.0, 6.8, 3.8 Hz, 1H); LCMS (ESI): > 95% purity at A = 254 nm. MS m / z, 429 |M+HJ+.

[0090] Synthesis of methyl 4-((l-phenyl-LH-pyrazol-4-yi)methyl)-l-(quinazolin-4- yl)piperazine-2-carboxylate (15).

[0091] In Scheme 2, compound 15 was synthesized following the same procedure used to synthesize compound 6 (Scheme 1, Example 1). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 429 [M+H]+.

[0092] Synthesis of l-((l-phenyl-lH-pyrazol-4-yl)methyl)-4-(quinazolin-4-yl)piperazine- 2-carboxylic acid (16).85US_ACTIVE\130819540W-1

[0093] In Scheme 2, compound 16 was synthesized following the same procedure used to synthesize compound 7 (Scheme 1, Example 1). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 413 [M-H]+.

[0094] Synthesis of 4-((l-phenyl-l / / -pyrazol-4-yl)methyl)-l-(quinazolin-4- yl)piperazine-2-carboxylic acid (17).

[0095] In Scheme 2, compound 17 was synthesized following the same procedure used to synthesize compound 7 (Scheme 1, Example 1). CMS (ESI): > 95% purity at = 254 nm. MS m / z, 413 [M-H]+.EXAMPLE 4: SYNTHESIS OF LINKERS AND E3 LIGANDS USED TO SYNTHESIZE PROTAC COMPOUNDS

[0096] PROTAC compounds (PROteolysis-TArgeting Chimeras) have been designed and developed to utilize the intracellular ubiquitin-proteasome system to efficiently degrade targeted proteins. PROTAC compounds that are heterobifunctional, consisting of a targeting substrate, a linker, and a ligand for E3 ubiquitin ligase, have been developed and reported for a number of biological targets. Although PROTACs are a relatively new area of research, lead compounds are being studied clinically, and reports are now emerging describing PROTAC designs that influence PCP and drug-like properties. Since the targeting element and E3 -ligase element are relatively defined in a PROTAC, structural manipulation of the linker provides an opportunity to not only change and optimize the binding orientation of the PROTAC, but also impact key physicochemical properties that directly impact the AB-MPS score and subsequently drug permeability, oral bioavailability, and in vivo activity. Scheme 3 exemplifies the synthesis of various linkers and E3 ligands used to synthesize PROTAC compounds.86US_ACTIVE\130819540W-1Scheme 3.

[0097] With reference to Scheme 3, alkylation of pomalidomide 18 with tertbutylbromoacetate in the presence of potassium carbonate in DMF produces the desired alkylated product 19. Alternatively (not depicted), 19 can be prepared by SNAR reaction of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline- 1,3-dione with tert-butyl glycinate under reflux conditions. Removal of the tert-butyl group under acid trifluoroacetic acid conditions cleanly provides carboxylic acid 20. Alkylation of 18 with the bis -chloroethylene glycol compound 21 produces the desired alkylated compound 22, which can be further converted to87US_ACTIVE\130819540W-1the corresponding azide 23 by reaction with sodium azide in DMF. Pomalidomide 18 can also be acylated with bromo-acid chloride 24 in refluxing THF to produce the desired bromide 25, which can be converted to the corresponding azide 26 by reaction with sodium azide in DMF. Finally, carboxylic acid 20 can be coupled with N-BOC diamine 27, under HATU conditions in the presence of DIPEA to produce the desired amide 28. Deprotection of the N-BOC group with TFA produces the desired amine 29.

[0098] Synthesis of tot- Butyl 2-(3-(4-amino-l,3-dioxoisoindolin-2-yl)-2,6- dioxopiperidin-l-yl)acetate (19).

[0099] In Scheme 3, to a solution of pomalidomide 18 (1 g, 3.66 mmol, 1 equiv.) in DMF (10 mL), K2CO3 (0.75 g, 5.49 mmol, 1.5 equiv.) was added followed by tert-butyl 2-bromoacetate (0.54 mL, 3.66 mmol, 1 equiv.). The reaction was stirred at room temperature overnight. Water was added to the reaction mixture water (10 mL), the precipitated solid was then filtered and purified on biotage using 0-6% methanol and dichloromethane to get the required compound 19 (yield: 96%). ’H NMR (400 MHz, DMSO-< / 6): 57.48 (dd, J= 8.5, 7.0 Hz, 1H), 7.06 - 6.98 (m, 2H), 6.52 (s, 2H), 5.17 (dd, J = 13.1, 5.2 Hz, 1H), 4.37 - 4.20 (m, 2H), 3.07 (ddd, J = 17.2, 13.8, 5.4 Hz, 1H), 2.89 - 2.79 (m, 1H), 2.66 (qd, J = 13.2, 4.4 Hz, 1H), 2. 15 - 2.06 (m, 1H), 1.40 (s, 9H); LCMS (ESI): > 95% purity at ! = 254 nm. MS m / z, 388.4 [M+H]+.

[0100] Synthesis of 2-(3-(4-Amino-l,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-l- yl)acetic acid (20).88US_ACTIVE\130819540W-1

[0101] In Scheme 3, to the solution of above compound 19 (0.45 g, 1.16 mmol, 1 equiv.) in anhydrous dichloromethane (5 mL), trifluoroacetic acid (0.3 mL, 3.48 mmol, 3equiv.) was added and stirred overnight at room temperature. Solvent was evaporated and crude compound was dried to obtain the corresponding acid 20 (yield: 95%). LCMS (ESI): > 95% purity at Z = 254 nm. MS m / z, 330 [M-H]+.

[0102] Synthesis of 4-amino-2-(l-(2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethyI)-2,6- dioxopiperidin-3-yl)isoindoli- -ne-1, 3-dione (22).

[0103] In Scheme 3, a solution of 18 (0.68 g, 2.49 mmol, 1 equiv.), 21 (2.31 g, 9.96 mmol, 4 equiv.), Nal ( 0.37 g, 2.49 mmol, 1 equiv.), and K2CO3 (1.03 g, 7.5 mmol, 3 equiv.) in DMF (12.5 mL) was heated to 100 °C and stirred for 24 h. The reaction mixture was allowed to come to room temperature, brine (3 x 10 mL) was added and extracted with ethyl acetate (3 x 10 mL). The combined organic layer was dried over sodium sulfate, concentrated, and purified on biotage using 0-5% methanol and dichloromethane to obtain the required product 22 (yield: 68%). H NMR (400 MHz, CDCh) 57.43 (dd, 7 = 8.3, 7.1 Hz, 1H), 7.16 (d, J = 7.1 Hz, 1H), 6.87 (d, J= 8.3 Hz, 1H), 5.23 (s, 2H), 5.01 - 4.89 (m, 1H), 4.16 - 3.93 (m, 2H), 3.75 (t, J= 5.9 Hz, 2H), 3.72 - 3.54 (m, 12H), 3.03 - 2.89 (m, 1H), 2.87 - 2.69 (m, 2H), 2.15 - 2.06 (m, 1H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 468 [M+H]+.

[0104] Synthesis of 4-Amino-2-(l-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-2,6- dioxopiperidin-3-yl)isoindoli- ne- 1,3-dione (23).

[0105] In Scheme 3, to a solution of 22 (0.79 g, 1.69 mmol, 1 equiv.) in DMF (3.4 mL), sodium azide (0.22 g, 3.38 mmol, 2 equiv.) was added and heated to 80 °C for 14 h. The reaction mixture was allowed to come to room temperature, brine (3 x 10 mL) added and extracted with ethyl acetate (3 x 10 mL). The combined organic layer was dried over sodium89US_ACTIVE\130819540W-1sulfate and concentrated to obtain the required product 23 (yield: 75%). ' H NMR (400 MHz, CDCh) 87.43 (dd, J = 8.3, 7. 1 Hz, 1H), 7.16 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 5.23 (s, 2H), 5.01 - 4.85 (m, 1H), 4.06 (dtd, J = 19.2, 13.2, 6.1 Hz, 2H), 3.70 - 3.58 (m, 12H), 3.38 (t, J = 5.1 Hz, 2H), 3.01 - 2.89 (m, 1H), 2.87 - 2.69 (m, 2H), 2.16 - 2.01 (m, 1H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 475 [M+H]+.

[0106] Synthesis of 5-bromo- / V-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)pentanamide (25).

[0107] To a solution of 18 (0.68 g, 2.5 mmol, 1 equiv.) in THF (10 mL), 5 -bromopentanoy 1 chloride 24 (0.22 g, 3.38 mmol, 2 equiv.) was added and heated to 80 °C for 14 h. The reaction mixture was allowed to come to room temperature, concentrated and purified on biotage using 0-5% methanol and dichloromethane to get the required product 25 (yield: 99%).1H NMR (400 MHz, CDCh) 6 9.42 (s, 1H), 8.82 (d, J = 8.5 Hz, 1H), 7.99 (s, 1H), 7.72 (dd, J = 8.5, 7.3 Hz, 1H), 7.56 (dd, 7 = 7.4, 0.8 Hz, 1H), 4.96 (dd, 7 = 12.2, 5.4 Hz, 1H), 3.45 (t, 7 = 6.3 Hz, 2H), 2.97 - 2.70 (m, 3H), 2.51 (t, 7 = 7.0 Hz, 2H), 2.23 - 2.10 (m, 1H), 2.06 - 1.86 (m, 4H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 436 [M+H]+.

[0108] Synthesis of 5-azido-N-(2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)pentanamide (26).

[0109] In Scheme 3, to a solution of 25 (1.1 g, 2.5 mmol, 1 equiv.) in DMF (5 mL), sodium azide (0.32 g, 5 mmol, 2 equiv.) was added and heated to 80 °C for 14 h. The reaction mixture90US_ACTIVE\130819540W-1was allowed to come to room temperature, brine (3 x 10 mL) added and extracted with ethyl acetate (3 x 10 mL). The combined organic layer was dried over sodium sulfate and concentrated to obtain the required product, 26 (yield: 75%). H NMR (400 MHz, CDCh) 8 9.42 (s, 1H), 8.82 (dd, 7 = 8.5, 0.8 Hz, 1H), 8.11 (s, 1H), 7.72 (dd, 7 = 8.5, 7.3 Hz, 1H), 7.56 (dd, 7 = 7.4, 0.8 Hz, 1H), 4.96 (dd, 7 = 12.2, 5.4 Hz, 1H), 3.35 (t, 7= 6.7 Hz, 2H), 2.95 - 2.71 (m, 3H), 2.51 (t, 7 = 7.3 Hz, 2H), 2.23 - 2.11 (m, 1H), 1.91 - 1.79 (m, 2H), 1.77 - 1.62 (m, 2H); LCMS (ESI): > 95% purity at A = 254 nm. MS m / z, 399 [M+HJ+.

[0110] Synthesis of tcrt-Butyl(4-(2-(3-(4-amino-l,3-dioxoisoindolin-2-yl)-2,6- dioxopiperidin-l-yl)acetamido)butyl) carbamate (28).

[0111] In Scheme 3, to this solution of acid 20 (1 g, 3.02 mmol, 1 equiv.) in DMF (10 mL), HATU (1.75 g, 3.32 mmol, 1.1 equiv.) was added. After stirring for 5 min at room temperature, DIPEA was added (5.26 mL, 30.2 mmol, 10 equiv.) followed by tert-butyl (4- aminobutyl)carbamate (0.68 g, 3.62 mmol, 1.2 equiv.) and stirred for 2 h. Water (10 mL) was added and extracted with 10% methanol and dichloromethane (2 x 10 mL). The combined organic layer was washed with brine (3 x 10 mL), dried over sodium sulfate, concentrated, and purified on biotage using 0-10% methanol and dichloromethane to get the required product 28 (yield: 73%). ‘H NMR (400 MHz, DMSO-ri6): 5 7.89 (t, J = 5.6 Hz, 1H), 7.47 (dd, J = 8.4, 7.0 Hz, 1H), 7.06 - 6.97 (m, 2H), 6.75 (t, J = 5.7 Hz, 1H), 6.52 (s, 2H), 5.15 (dd, 7 = 13.1, 5.3 Hz, 1H), 4.28 - 4.13 (m, 2H), 3.09 - 2.93 (m, 3H), 2.93 - 2.75 (m, 4H), 2.68 - 2.60 (m, 1H), 2.08 (dtd, J = 12.6, 5.1, 2.3 Hz, 1H), 1.36 (s, 12 H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 502.4 [M+H]+.91US_ACTIVE\130819540W-1

[0112] Synthesis of 2-(3-(4-amino-l,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-l-yl)-Af- (4-aminobutyl)acetamide (29).

[0113] In Scheme 3, to the solution of compound 28 (0.7 g, 1.35 mmol, 1 equiv.) in anhydrous dichloromethane (5 mL), trifluoroacetic acid (1.24 mL, 16.2 mmol, 12 equiv.) was added and stirred overnight at room temperature. Solvent was evaporated and crude compound was dried to obtain the corresponding amine 29 (yield: 95%). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 402.4 [M+H]+.EXAMPLE 5: SYNTHESIS OF PROTAC COMPOUNDS WHEN THE LINKER AND E3 LIGASE IS COUPLED TO THE 7-POSITION OF THE QUINAZOLINE RING

[0114] Scheme 4 exemplifies the synthesis of the final PROTAC compounds, wherein the linker and E3 ligase is coupled to the 7-position of the quinazoline ring.

[0115] With regard to Scheme 4, reaction of carboxylic acid 7 and amine 29, under HATU and DIEA conditions in DMF, produces the desired PROTAC compound. In Scheme 4, the compound CIDD-0162288 is exemplified. Carboxylic acid 7 can also be reacted with propargyl amine in the presence of HATU and DIPEA to produce intermediate alkyne 30 which can be reacted with azide 26 under CuSO4 conditions to provide the desired triazole PROTAC compound. In Scheme 4, the compound CIDD-0162266 is exemplified.92US_ACTIVE\130819540W-1Scheme 4.CIDD-016226693US_ACTIVE\130819540W-1

[0116] Synthesis of 2V-(4-(2-(3-(4-amino-l,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-l- yl)acetamido)butyl)-4-(4-(( 1 -phenyl- 17 / -pyrazol-4-yl)methyl)piperazin- 1 -yDquinazoline- 7-carboxamide (CIDD-0162288).

[0117] In Scheme 4, to the solution of acid 7 (30 mg, 0.036 mmol, 1 equiv.) in DMF (2 mL), HATU (14 mg, 0.036 mmol, 1.1 equiv.) was added. After stirring for 5 min at room temperature, DIEA was added (0.8 mL, 36.2 mmol, 10 equiv.) followed by 29 (0.03 g, 3.62 mmol, 1.2 equiv.). The reaction mixture was allowed to stir at room temperature for 2 h. Water (10 mL) was added and extracted with 10% methanol and dichloromethane (2 x 10 mL). The combined organic layer was washed with brine (3 x 10 mL), dried over sodium sulfate, concentrated, and purified on biotage using 0-10% methanol and dichloromethane to obtain the required product (yield: 65%).]H NMR (400 MHz, DMSO-rfc): 8 8.78 (t, J - 5.6 Hz, 1H), 8.65 (s, 1H), 8.44 (s, 1H), 8.25 (d, J = 1.8 Hz, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.95 - 7.87 (m, 2H), 7.87 - 7.77 (m, 2H), 7.69 (s, 1H), 7.47 (td, J = 8.5, 7. 1 Hz, 3H), 7.32 - 7.25 (m, 1H), 7.00 (dd, 7 = 9.3, 7.7 Hz, 2H), 6.52 (s, 2H), 5.15 (dd, J= 13.1, 5.3 Hz, 1H), 4.22 (d, J= 2.0 Hz, 2H), 3.85 - 3.71 (m, 4H), 3.54 (s, 2H), 3.30 - 3.24 (m, 2H), 3.17 - 2.95 (m, 3H), 2.79 (ddd, J = 17.4, 4.5, 2.5 Hz, 1H), 2.74 - 2.55 (m, 5H), 2.07 (ddd, 7 = 12.4, 6.4, 3.8 Hz, 1H), 1.59 - 1.38 (m, 4H); LCMS (ESI): > 95% purity at 7 = 254 nm. MS m / z, at 798.3 [M+H]+.

[0118] Synthesis of 4-(4-((l-phenyl-17f-pyrazol-4-yl)methyI)piperazin-l-yI)-lV-(prop-2- yn-l-yl)quinazoline-7-carboxamide (28).94US_ACTIVE\130819540W-1

[0119] In Scheme 4, to the solution of acid 7 (0.1 g, 0.24 mmol, 1 equiv.) in DMF (6 mL) HATU (0.11g, 0.29 mmol, 1.2 equiv.) was added. After stirring for 5 min at room temperature, DIEA was added (0.1 mL, 0.6 mmol, 2.5 equiv.) followed by propargylamine (0.02 mL, 0.29 mmol, 1.2 equiv.). The reaction mixture was allowed to stir at room temperature for 2 h. Water (10 mL) was added and extracted with 10% methanol and dichloromethane (2 x 10 mL). The combined organic layer was washed with brine (3 x 10 mL), dried over sodium sulfate, concentrated, and purified on biotage using 0-10% methanol and dichloromethane to obtain the required product (yield: 74%). LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 452 [M+H]+.

[0120] Synthesis of 2V-((l-(5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4- yl)amino)-5-oxopentyl)- 1H- 1,2, 3-triazol-4-yl)methyl)-4-(4-((l -phenyl- lH-pyrazol-4- yl)methyl)piperazin-l-yl)quinazoline-7-carboxamide (CIDD-0162266).

[0121] In Scheme 4, a solution of 30 (0.05 g, 0.12 mmol, 1 equiv.), azide, 26 (0.05 g, 0.12 mmol, 1 equiv.), copper sulfate pentahydrate (0.006 g, 0.024 mmol, 0.02 equiv.), and sodium ascorbate (0.005 g, 0.024 mmol, 0.02 equiv.) in THF (1.1 mL) was purged with nitrogen for 5 minutes, a few drops of water were added and stirred at room temperature for 24 h. Solvent was evaporated and crude compound was purified on biotage using 0-20% methanol and dichloromethane to obtain the required product CIDD-0162266 (yield: 94%).1H NMR (400 MHz, DMSO-uL ) 5 11.14 (s, 1H), 9.70 (s, 1H), 9.34 (t, J = 5.7 Hz, 1H), 8.66 (s, 1H), 8.46 - 8.39 (m, 2H), 8.28 (dd, J = 10.0, 1.8 Hz, 1H), 8.04 (d, J = 9.1 Hz, 2H), 7.91 (ddd, J = 8.8, 4.1, 1.8 Hz, 1H), 7.87 - 7.80 (m, 3H), 7.69 (s, 1H), 7.60 (d, 7 = 7.3 Hz, 1H), 7.53 - 7.44 (m, 2H), 7.33 - 7.25 (m, 1H), 5.14 (dd, 7 = 12.7, 5.4 Hz, 1H), 4.55 (d, 7 = 5.6 Hz, 2H), 4.38 (t, 7 = 7.0 Hz, 2H), 3.77 (d, 7 = 5.8 Hz, 5H), 3.55 (s, 2H), 2.89 (ddd, 7 = 16.7, 13.7, 5.4 Hz, 1H), 2.60 (d,95US_ACTIVE\130819540W-1J = 18.5 Hz, 6H), 2.09 - 2.01 (m, 1H), 1.92 - 1.83 (m, 2H), 1.59 (p, J = 7.5 Hz, 2H), 1.25 (d, J = 7.6 Hz, 1H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 850 [M+H]+.EXAMPLE 6: SYNTHESIS OF PROTAC COMPOUNDS WHEN THE LINKER AND E3 LIGASE IS COUPLED TO EITHER THE 2- OR 5-POSITION OF THE PIPERAZINE RING

[0122] Scheme 5 exemplifies the synthesis of PROTAC compounds, wherein the linker and E3 ligase is coupled to either the 2- or 5 -position of the piperazine ring.

[0123] With regard to Scheme 5, reaction of carboxylic acid 16 or 17 with propargyl amine, under HATU and DIEA conditions in DMF, produces the desired alkynes 31 or 32. Alkyne 31 can be reacted with azide 26 under CuSO4 conditions to provide, for example, triazole PROTAC, CIDD-0160941. Similarly, alkynes 31 and 32 can be reacted under CuSO4 conditions with either azide 23 or 26 to produce, for example, PROTAC CIDD-0160942 or PROTAC CIDD-0162289.96US_ACTIVE\130819540W-1Scheme 5.

[0124] General procedure for the synthesis of compounds 31 and 32 in Scheme 5. To the solution of acid 16 / 17 (0.1 g, 0.24 mmol, 1 equiv.) in DMF (6 mL), HATU (0.12 g, 0.32mmol, 1.2 equiv.) was added. After stirring for 5 min at room temperature, DIEA was added (0.11 mL, 0.66 mmol, 2.5 equiv.) followed by propargylamine (0.02 mL, 0.32mmol, 1.2 equiv.).97US_ACTIVE\130819540W-1The reaction mixture was allowed to stir at room temperature for 2 h. Water (10 mL) was added and extracted with 10% methanol and dichloromethane (2 x 10 mL). The combined organic layer was washed with brine (3 x 10 mL), dried over sodium sulfate, concentrated, and purified on biotage using 0-10% methanol and dichloromethane to obtain the required product (yield: 94% for 31 and 34% for 32). LCMS (ESI): > 95% purity at A, = 254 nm. MS m / z, 452 [M+H]+.

[0125] General procedure for the synthesis of compounds CIDD-0160941, CIDD- 0160942, and CIDD-0162289. A solution of 31 / 32 (1 equiv.), azide, 26 (1 equiv.), copper sulfate pentahydrate (0.02 equiv.), and sodium ascorbate (0.02 equiv.) in THF (1.1 mL) was purged with nitrogen for 5 minutes, a few drops of water were added and stirred at room temperature for 24 h. Solvent was evaporated and crude compound was purified on biotage using 0-20% methanol and dichloromethane to obtain the required products (yield: 43% for CIDD-0160941, 55% for CIDD-0160942, and 43% for CIDD-0162289).

[0126] -((l-(5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)-5- oxopentyl)-lH-l,2,3-triazol-4-yl)methyl)-l-((l-phenyl-l / / -pyrazol-4-yl)methyl)-4- (quinazolin-4-yl)piperazine-2-carboxamide (CIDD-0160941).

[0127] ]H NMR (400 MHz, CDCh) 5 9.37 (s, 1H), 9.16 (s, 1H), 8.70 (d, J = 3.0 Hz, 1H), 8.02 (d, J = 5.9 Hz, 1H), 7.95 (t, J = 6.9 Hz, 2H), 7.86 (s, 1H), 7.78 - 7.62 (m, 5H), 7.60 (s, 1H), 7.58 - 7.40 (m, 5H), 7.26 (d, J= 14.8 Hz, 1H), 5.04 - 4.90 (m, 1H), 4.59 (dddd, J= 54.9,24.8, 15.2, 5.6 Hz, 2H), 4.37 (t, J = 7.0 Hz, 2H), 4.29 (d, J = 13.2 Hz, 1H), 4.09 (d, J = 13.1 Hz, 1H), 3.80 (dd, J = 13.8, 6.3 Hz, 1H), 3.65 (d, J = 9.1 Hz, 1H), 3.48 (s, 2H), 3.37 (dd, J =8.8, 3.7 Hz, 1H), 3.15 (d, J = 11.9 Hz, 1H), 2.88 (dd, J = 12.5, 2.6 Hz, 1H), 2.85 - 2.73 (m, 2H), 2.63 (t, 7 = 11.2 Hz, 1H), 2.44 (td, 7 = 7.3, 4.3 Hz, 2H), 2.15 (tt, 7= 6.5, 3.0 Hz, 1H), 2.0498US_ACTIVE\130819540W-1- 1.91 (m, 2H), 1.77 - 1.66 (m, 2H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 850 [M+H]+.

[0128] N-((l-(2-(2-(2-(2-(3-(4-amino-l,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-l- yl)ethoxy)ethoxy)ethoxy)ethyl)-lH-l,2,3-triazol-4-yl)methyl)-l-((l-phenyl-lH-pyrazol-4-yl)methyl)-4-(quinazolin-4-yl)piperazine-2-carboxamide (CIDD-0160942).

[0129] ]H NMR (400 MHz, CDCh) 5 8.71 (s, 1H), 8.05 (s, 1H), 7.94 (dd, J = 14.4, 8.4 Hz, 2H), 7.80 (d, 7 = 6.1 Hz, 1H), 7.77 - 7.68 (m, 5H), 7.60 (s, 1H), 7.45 (dt, 7 = 15.6, 7.4 Hz, 4H), 7.37 (d, 7 = 7.7 Hz, 1H), 7.26 (t, 7 = 7.4 Hz, 1H), 7.08 (d, 7 = 7.1 Hz, 1H), 6.84 (d, 7 = 8.3 Hz, 1H), 5.35 (s, 2H), 5.00 - 4.87 (m, 1H), 4.68 (dd, 7 = 15.2, 5.9 Hz, 1H), 4.49 (dt, 7 = 10.1, 4.8 Hz, 4H), 4.39 - 4.30 (m, 1H), 4.19 - 3.92 (m, 4H), 3.86 - 3.78 (m, 4H), 3.58 (dq, 7 = 8.5, 5.1 Hz, 6H), 3.43 (d, 7 = 13.5 Hz, 2H), 3.33 (dd, 7 = 8.9, 3.6 Hz, 1H), 3.14 (dt, 7 = 12.2, 3.4 Hz, 1H), 2.94 - 2.84 (m, 1H), 2.77 - 2.68 (m, 2H), 2.59 (Id, 7 = 11.3, 3.0 Hz, 1H), 2.05 (ddq, 7 = 9.3, 6.4, 3.1 Hz, 1H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 927 [M+H]+.99US_ACTIVE\130819540W-1

[0130] A'-((l-(5-((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindolin-4-yl)amino)-5- oxopentyl)- lfi-1, 2, 3-triazol-4-yl)methyl)-4-((l-phenyl-l / 7-pyrazol-4-yl)methyl)-l- (quinazolin-4-yl)piperazine-2-carboxamide (CIDD-0160289).

[0131] ]H NMR (400 MHz, DMSO-&) 8 11.2 (broad s, 1H), 9.68 (s, 1H), 8.65 (t, J= 5.8 Hz, 1H), 8.58 (s, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.38 (s, 1H), 8. 15 - 8.05 (m, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.84 (s, 1H), 7.68 - 7.58 (m, 3H), 7.49 - 7.45 (m, 3H), 7.27 (t, 7 = 7.4 Hz, 1H), 7.18 - 7.10 (m, 2H), 6.85 (s, 1H), 5.14 (dd, J = 12.7, 5.4 Hz, 1H), 4.95 (d, 7= 3.6 Hz, 1H), 4.4O (dd, 7 = 5.7, 3.2 Hz, 2H), 4.36 (t, 7 = 7.0 Hz, 3H), 4.15 (d, 7 = 13.7 Hz, 1 H), 3.75 (t, 7 = 1 1 .8 Hz, 1H), 3.50 (d, 7= 9.1 Hz, 2H), 3.12 (q, 7 = 6.7 Hz, 2H), 2.87 (dd, 7 = 12.4, 4.5 Hz, 2H), 2.66 (t, 7 = 7.3 Hz, 2H), 2.39 - 2.29 (m, 2H), 2.08 - 2.02 (m, 1H), 1.84 (t, 7 = 7.6 Hz, 2H), 1.57 (q, J = 7.6 Hz, 2H); LCMS (ESI): > 95% purity at X = 254 nm. MS m / z, 850 [M+H]+.EXAMPLE 7: TESTING YAP1 LIGANDS

[0132] To identify the compounds with high YAP1 degradative potential, an unbiased in vitro luciferase reporter assay screen was performed in HEK293 cells utilizing overexpression of GFP and YAP1. A synthetic promoter with eight TEAD binding sites upstream of Luciferase reporter (TEAD-Luc) was constructed in a lentiviral vector and stably expressing TEAD-Luc HEK293 cells were established. The screen was performed by transfecting the HEK293- TEAD-Luc cells with GFP or YAP1 in triplicate in a 96 well format. Renilla- luciferase was co-transfected with GFP / YAP1 plasmids to normalize for transfection and cell viability. 16 hours post transfection cells were treated with 1 nM to 1 M of each compound for 4 and 24 hours. Luciferase activity of each well was assayed and normalized to Renilla-luciferase data following treatment at the end of each timepoint. Luciferase assays performed for select YAP1 degraders are provided below in FIG. 2.100US_ACTIVE\130819540W-1

[0133] Cell proliferation assays, qPCR, and Western blots were then performed for each of the compounds that demonstrated high YAP1 degradative potential in FIG. 2. The IC50 of each compound in FIG. 2 was calculated based on cell proliferation assays (FIG. 3). To confirm these drugs inhibit YAP1 expression, their mechanism of action was investigated in several YAP1 dependent and independent cancer cell lines, identified from a publicly available database (DepMap). Western blots were performed to assess the efficacy of each compound in the following YAP1 dependent cell lines: NCI-H292 (lung cancer), NCI-H1693 (lung cancer), NCI-H2052 (lung cancer), SF268 (glioma), and OVCAR5 (ovarian cancer); and YAP1 independent cell lines: HUP-T3 (pancreatic cancer), NCI-H1975 (lung cancer), HCC2449 (lung cancer), and EFM19 (breast cancer). Each compound was tested individually at 1 nM, 10 nM, 100 nM, 1000 nM, and 10,000 nM concentrations at 4- and 24-hour time points. These time points were chosen to filter out those compounds that may affect YAP1 levels due to effect on transcription or other processes. Typically, degradation occurs within 4 hours and compounds that show activity during this time are likely to impact degradation. Of these compounds it was found that CIDD-0166370 in particular, functioned at post- transcriptional level and significantly degraded YAP1 protein in a dose-dependent manner in YAP1 dependent cell lines OVCAR 5, H292, sF268, H2052, and NCI-H1693 (FIG. 4, Panel A, Panel B, and Panel C).

[0134] Treatment with protein synthesis inhibitor CHX alone and in combination with CIDD- 0166370 resulted in a time-dependent decrease of YAP1 expression in NCI-H1693 cells (FIG 5). As the ubiquitin-proteasome pathway is the predominant mechanism for YAP1 degradation, YAP1 dependent cell lines H292, SF268, H2052 and NCI-H1693 treated with CIDD-0166370 in the presence or absence of a proteasome inhibitor (MG132) showed that CIDD-0166370-mediated degradation of YAP1 was significantly rescued when MG132 was used to inhibit proteasomes (FIG 6). CIDD-0166370 showed robust in vivo activity by attenuating tumor burden in xenograft mouse models (FIG. 7). Pharmacokinetic (PK) analysis of plasma (FIG 8) and tissue (kidney, liver, small intestine) (FIG 9) collected from mice treated with a single i.p. dose of CIDD-0166370 (5 mg / kg) showed adsorption range between 15 min and 2 h and elimination range between 4 and 24h.

[0135] Considering the YAP1 degradation potential CIDD-0166370, SAR data described in Example 1 was utilized to quickly design and synthesize nine CIDD-0166370 analogs for assessment. The same strategy described above was used to screen CIDD-0166370 analogs with high YAP1 degradative potential through an unbiased in vitro luciferase reporter assay101US_ACTIVE\130819540W-1screen, the results of which are provided in FIG. 10. The IC50 of these compounds was also calculated based on cell proliferation assays (FIG. 11).EXAMPLE 8: TESTING YAP1 PROTACS

[0136] Based on the initial SAR studies described in Example 1, this data was utilized to quickly design and synthesize a small number of PROTAC analogs for assessment, as represented by CIDD-0162288 and CIDD-0162289. The initial YAP1 SAR studies suggested that substitution on the quinazoline ring system and piperazine core was tolerated. Thus, the amide group was incorporated at the C-7 position of the quinazoline with a C-4 alkane linker to the CRBN ligand, producing CIDD-0162288. Similarly, the amide group was incorporated at the C-2 position of the piperazine connected to the triazole-C-4 alkane linker to the CRBN ligand, producing CIDD-0162289. As shown in FIG. 12, both CIDD-0162288 and CIDD- 0162289 show robust in vitro YAP1 protein degradation activity, even down to 100 nM. Furthermore, treatment with low dose (1 mg / kg and 5 mg / kg) of CIDD-0162288 showed robust in vivo activity by attenuating tumor burden in xenograft mouse models (FIG. 13). The same strategy described above was used to screen YAP1 PROTACs with high YAP1 degradative potential through and unbiased in vitro luciferase reporter assay screen provided in FIG. 14, and the IC50 of these compounds was calculated based on cell proliferation assays (FIG. 15).

[0137] Western blot analysis was performed for the PROTAC (CIDD-0166579) that demonstrated high YAP1 degradative potential. Results showed that CIDD-0166579 showed significantly degraded YAP1 protein up to 10 nM dosage at 24 h in YAP-dependent cell lines OVACAR 5 and H292 (FIG. 16). Western blots confirmed that CIDD-0166579-mediated degradation of YAP1 was significantly rescued when MG132 was used to inhibit proteasomes in the YAP-dependent cell lines OVACAR 5 and H292 (FIG 17). CIDD-0166579 also reduced tumor burden in mouse xenograft models at low doses (FIG. 18). YAP1 is one of the most important effectors of the Hippo pathway while also having crosstalk with other cancer promoting pathways. Phosphorylated and inactive YAP1 is retained in the cytoplasm of the cell where it is subsequently degraded by proteosomes. In contrast, activation of YAP1 (e.g., due to inactivation of the Hippo signaling or activation by other pathways like Wnt) results in its dephosphorylation, triggering its nuclear relocation and consequent activation of transcriptional responses.

[0138] The results provided herein demonstrate that the compounds of the present disclosure are YAP1 ligands that potently inhibit YAP1. These compounds have favorable102US_ACTIVE\130819540W-1physicochemical drug properties, and the data provided herein further demonstrates that we can utilize the compounds of the present disclosure to develop YAP1 PROTACs with potent YAP1 protein degradation activity.

[0139] The results of a luciferase assay performed with other YAP1 degraders is provided in FIG. 19.

[0140] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments or aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.103US_ACTIVE\130819540W-1

Claims

CLAIMS1. A compound of Formula I or a derivative thereof:, wherein amine spacer is selected from the group consisting of:Ri and R2 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, -CONH2, - CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, or -CN,X is a substituted methylene, a nitrogen, or an oxygen,104US_ACTIVE\130819540W-1Rs and R4 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, or a substituted alkyl, or Rs and R4, when attached to the same carbon, can form a carbocyclic ring,Q is CH or N, andAr is an aryl.

2. The compound of claim 1, having a formula selected from the group consisting of:105US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1noUS_ACTIVE\130819540W-1a derivative of any thereof.

3. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier, buffer, or diluent.

4. A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of the compound of claim 1 to the subject.

5. The method of claim 4, wherein said subject is afflicted with or at risk of developing cancer.

6. The method of claim 5, wherein said subject is afflicted with or at risk of developing brain cancer, breast cancer, ovarian cancer, prostate cancer, salivary cancer, lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma,1 1 1US_ACTIVE\130819540W-1hepatobiliary cancer, gynecological cancer, hematopoietic cancer, bladder cancer, skin cancer, head and neck squamous cell carcinoma, or genitourinary cancer.

7. The method of claim 4, wherein said administering comprises local, systemic, regional, systemic, or continual administration.

8. The method of claim 4, wherein said administering comprises oral administration, intravenous administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, topical administration, intradermal administration, intratracheal administration, intramuscular administration, subcutaneous administration, or inhalation.

9. The method of claim 4, the method further comprising administering a second therapy to said subject.

10. The method of claim 9, wherein the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, a targeted therapy, a small molecule inhibitor, an immunotherapy, and surgery.

11. The method of claim 4, wherein the subject is a mammalian subject.

12. The method of claim 11, wherein the subject is a human subject.

13. A compound having a formula selected from the group consisting of Formula II,Formula II, and Formula IV, or a derivative of any thereof:amine spacer is selected from the group consisting of:1 12US_ACTIVE\130819540W-1Ri and R2 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, -CONH2, -CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, or -CN,X is a substituted methylene, a nitrogen, or an oxygen,R3 and R4 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, or a substituted alkyl, or R3 and R4, when attached to the same carbon, can form a carbocyclic ring,Q is CH or N,Ar is an aryl, linker is an alkyl, a branched alkyl, a repeating ethylene glycol chain, an ethylene diamine, or a substituted bicyclic amine, andR5 is a small molecule E3-ligase ubiquitin-recruiting ligand.1 13US_ACTIVE\130819540W-114. The compound of claim 13, having the formula of Formula II or a derivative thereof16. The compound of claim 13, having the formula of Formula IV or a derivative thereof17. The compound of claim 13, having a formula selected from the group consisting of:1 14US_ACTIVE\130819540\V-1115US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1US_ACTIVE\130819540W-1or a derivative of any thereof.

18. A pharmaceutical composition comprising the compound of claim 13 and a pharmaceutically acceptable carrier, buffer, or diluent.

19. A method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of the compound of claim 13 to said subject.

20. The method of claim 19, wherein said subject is afflicted with or at risk of developing cancer.

21. The method of claim 20, wherein said subject is afflicted with or at risk of developing brain cancer, breast cancer, ovarian cancer, prostate cancer, salivary cancer, lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, bladder cancer, skin cancer, head and neck squamous cell carcinoma, or genitourinary cancer.

22. The method of claim 19, wherein said administering comprises local, systemic, regional, systemic, or continual administration.

23. The method of claim 19, wherein said administering comprises oral administration, intravenous administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, topical administration, intradermal administration, intratracheal administration, intramuscular administration, subcutaneous administration, or inhalation.1 19US_ACTIVE\130819540W-124. The method of claim 19, the method further comprising administering a second therapy to said subject.

25. The method of claim 24, wherein the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, a targeted therapy, a small molecule inhibitor, an immunotherapy, and surgery.

26. The method of claim 19, wherein the subject is a mammalian subject.

27. The method of claim 26, wherein the subject is a human subject.

28. A method of reducing YAP1 protein levels in a subject in need thereof, said method comprising administering a therapeutically effective amount a compound of Formula I, Formula II, Formula III, or Formula IV to said subject, wherein amine spacer is selected from the group consisting of:120US_ACTIVE\130819540W-1Ri and R2 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, -CONH2, -CONHCH3, -CON(CH3)2, -CO2H, -CO2CH3, or-CN,X is a substituted methylene, a nitrogen, or an oxygen,R3 and R4 are independently a hydrogen, a halogen, an alkyl, a branched alkyl, or a substituted alkyl, or R3 and R4, when attached to the same carbon, can form a carbocyclic ring,Q is CH or N,Ar is an aryl, linker is an alkyl, a branched alkyl, a repeating ethylene glycol chain, an ethylene diamine, or a substituted bicyclic amine, andR5 is a small molecule E3-ligase ubiquitin-recruiting ligand.

29. The method of claim 28, wherein said subject is afflicted with or at risk of developing cancer.

30. The method of claim 29, wherein said subject is afflicted with or at risk of developing brain cancer, breast cancer, ovarian cancer, prostate cancer, salivary cancer, lung cancer,121US_ACTIVE\130819540W-1colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, hematopoietic cancer, bladder cancer, skin cancer, head and neck squamous cell carcinoma, or genitourinary cancer.

31. The method of claim 28, wherein said administering comprises local, systemic, regional, systemic, or continual administration.

32. The method of claim 28, wherein said administering comprises oral administration, intravenous administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, topical administration, intradermal administration, intratracheal administration, intramuscular administration, subcutaneous administration, or inhalation.

33. The method of claim 28, the method further comprising administering a second therapy to said subject.

34. The method of claim 33, wherein the second therapy is selected from the group consisting of a chemotherapy, a radiotherapy, a targeted therapy, a small molecule inhibitor, an immunotherapy, and surgery.

35. The method of claim 28, wherein the subject is a mammalian subject.

36. The method of claim 35, wherein the subject is a human subject.122US_ACTIVE\130819540W-1

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