Sunitinib-based selective inhibitors of g protein-coupled receptor kinase 5, compositions, and methods of use

Abstract

Selective and potent inhibitors of G protein-coupled receptor kinase 5 (GRK5), a pharmaceutical composition comprising same, and a method of use, such as in the treatment of heart disease or cancer.

Description

70774-02 / / 1165.202WO1 SUNITINIB-BASED SELECTIVE INHIBITORS OF G PROTEIN-COUPLED RECEPTOR KINASE 5, COMPOSITIONS, AND METHODS OF USE 5 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Appl. No. 63 / 665,628, filed June 28, 2024, which is incorporated by reference as if fully set forth herein. STATEMENT OF GOVERNMENT SUPPORT 10

[0002] This invention was made with government support under contract HL071818 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present disclosure relates to selective inhibitors of G protein-coupled 15 receptor kinase (GRK) 5, compositions comprising same, and methods of administering the compounds or compositions to a subject in need of GRK5 inhibition. BACKGROUND

[0004] G protein-coupled receptors (GPCRs) modulate cellular events in 20 response to extracellular signals. GPCR-related kinases (GRK) selectively recognize and phosphorylate activated GPCRs, leading to their desensitization and internalization, a process critical for maintaining cellular homeostasis. The seven known GRKs (GRK1-7) are classified by structural and sequence similarities into three subfamilies – GRK1 (GRK1 and 7), GRK2 (GRK2 and 3), 25 and GRK4, (GRK4, 5 and 6) (Pitcher et al., Ann Rev Biochem 67(1): 653-692 (1998)). GRK1 and 7 are expressed primarily in the retina, whereas GRK4 is primarily expressed in the testes and GRK2, 3, 5 and 6 are more ubiquitously expressed (Ferguson, Pharmacol Rev 53(1): 1-24 (2001)). Of these kinases, GRK2 and GRK5 are the two isoforms found in the highest concentration in 30 cardiovascular tissue. The GRK5 subfamily is part of a larger superfamily that includes the catalytic domains of serine / threonine kinases protein tyrosine kinases, RIO kinases, aminoglycoside phosphotransferase, choline kinase, and phosphatidylinositide 3-kinases.

[0005] GRK2 and GRK5 are considered therapeutic targets for various disease 35 states, such as cancer, inflammation, Parkinson’s disease, Alzheimer’s disease, heart failure and hypertrophic cardiomyopathy (Jiang et al., Cell Death Dis 9(3): 295 (2018); Belmonte et al., Circ Res 111(8): 957-958 (2012); Brinks et al., Today Dis Mech 7(2): e129-e134 (2010); Lymperopoulos et al., Nat Med 13(3): 315-323 170774-02 / / 1165.202WO1 (2007); and Nogues et al., Semin Cancer Biol 48: 78-90 (2018)). GrK5 is unique among the GRKs because it undergoes a Ca2+-calmodulin-dependent nuclear localization event. Once translocated to the nucleus, GRK5 can phosphorylate histone deacetylase 5 (HDAC5), which is responsible for increasing transcription 5 of genes associated with hypertrophic cardiomyopathy. In studies where GRK5 was knocked down, cardiomyocytes were protected from hypertrophic cardiomyopathy (Huang et al., Front Biosci Landmark Ed 16: 3047-3060 (2011)). The influence of GRK5 in progressive heart failure and hypertrophic cardiomyopathy remains unclear, in part because GRK2 can also mediate 10 hypertrophic responses (Schlegel et al., PLoS One 12(7): e0182110 (2017); Lieu et al., Exp Opin Ther Targets 23(3): 201-214 (2019)), and there are no available GRK5-selective and GRK5 subfamily-selective compounds to test mechanisms of action.

[0006] GRK5 is also involved in the progression of several human cancers. 15 Malignant cells can hijack the normal physiological functions of GPCRs to proliferate autonomously and evade immune detection (Dorsam et al., Nat Rev Cancer 7: 79-94 (2007)). In vivo experiments have validated the therapeutic benefit of eliminating GRK5 in suppressing multiple types of cancer growth (Chakraborty et al., Cancer Res 74: 3489-3500 (2014); Jiang et al. (2018), supra). 20

[0007] Accordingly, GRK5- and GRK5-subfamily- (e.g., GRK4, GRK5 and GRK6) selective compounds are needed. In view of the foregoing, it is an object of the present disclosure to provide compounds that selectively inhibit G protein- coupled receptor kinase (GRK) 5. Such compounds can be administered to subjects, such as subjects with heart disease or cancer, in need of such inhibition. 25 This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein. SUMMARY 30270774-02 / / 1165.202WO1 5and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers 10 thereof.

[0009] Also provided is a compound having a structure selected from: 370774-02 / / 1165.202WO1and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

[0010] Further provided is a compound having a structure selected from:470774-02 / / 1165.202WO15 and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

[0011] Still further provided is a compound having a structure selected from:and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers 10 thereof.

[0012] Still further provided is a compound having a structure selected from:, 570774-02 / / 1165.202WO1 ,, 670774-02 / / 1165.202WO1770774-02 / / 1165.202WO1 or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

[0013] In view of the above, also provided is a pharmaceutical composition. The pharmaceutical composition comprises an above-described compound and a 5 pharmaceutically acceptable carrier or excipient.

[0014] A method of inhibiting a G protein-coupled receptor kinase (GRK) 5 in a subject is also provided. The method comprises administering an above- described compound or an above-described pharmaceutical composition in an amount effective to inhibit GRK5. In embodiments of the method, the subject has 10 cancer, such as multiple myeloma. In other embodiments of the method, the subject has heart disease, such as heart failure or cardiac hypertrophy. DESCRIPTION

[0015] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in 15 conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0016] The present disclosure is based on the discovery of G protein-coupled receptor kinase (GRK) 5 inhibitors. Thus, provided is a compound having a structure selected from: 20

[0017] Also provided is a compound having a structure selected from:870774-02 / / 1165.202WO1and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.970774-02 / / 1165.202WO1and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers 5 thereof.

[0019] Further provided is a compound having a structure selected from: 101070774-02 / / 1165.202WO1and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 5

[0020] Still further provided is a compound having a structure selected from:and pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

[0021] Still further provided is a compound having a structure selected from: 10 ,, 1170774-02 / / 1165.202WO11270774-02 / / 1165.202WO1 5or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

[0022] The above compounds can be synthesized in accordance with methods 10 known in the art and exemplified herein. See, e.g., the Examples section provided herein. 1370774-02 / / 1165.202WO1

[0023] The compounds provided, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, have at least a two-fold, at least a ten- fold, at least a 100-fold, at least a 150-fold, at least a 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at least a 750-fold, at least a 1,000-fold, at 5 least a 2,000-fold, at least a 3,000-fold or at least a 5,000 fold selectivity for G protein-coupled receptor kinase 5 (GRK5) over GRK2.

[0024] Thus, for example, compounds provided, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, have a two-fold to 5,000- fold, two-fold to 100-fold, two-fold to 50-fold, 10-fold to 100-fold, 10-fold to 190-10 fold, 10-fold to 150-fold, 50-fold to 160-fold, 50-fold to 200-fold, 100-fold to 500- fold, 100-fold to 1,000-fold, 250-fold to 1,000-fold, 500-fold to 900-fold, 300-fold to 600-fold, 500-fold to 3,000-fold, 500-fold to 1,000-fold, 500-fold to 2,000-fold, or 50-fold to 150-fold selectivity for GRK5 over GRK2.

[0025] The compounds provided, or pharmaceutically acceptable salts, hydrates,15 tautomers, and optical isomers thereof, have at least a two-fold, at least a ten- fold, at least a 100-fold, at least a 150-fold, at least a 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at least a 750-fold, at least a 1,000-fold, at least a 2,000-fold, at least a 3,000-fold or at least a 5,000 fold selectivity for GRK5 over GRK6. 20

[0026] Thus, for example, compounds provided, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, have a two-fold to 5,000- fold, , two-fold to 100-fold, two-fold to 50-fold, 10-fold to 100-fold, 10-fold to 190- fold, 10-fold to 150-fold, 50-fold to 160-fold, 50-fold to 200-fold, 100-fold to 500- fold, 100-fold to 1,000-fold, 250-fold to 1,000-fold, 500-fold to 900-fold, 300-fold 25 to 600-fold, 500-fold to 3,000-fold, 500-fold to 1,000-fold, 500-fold to 2,000-fold, or 50-fold to 150-fold selectivity for GRK5 over GRK6.

[0027] The compound can be a pharmaceutically acceptable salt. Examples of acceptable salts include, without limitation, alkali metal (for example, sodium, potassium or lithium) or alkaline earth metals (for example, calcium) salts; 30 however, any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable. Similarly, “pharmaceutically acceptable salt” refers to those salts with counter ions, which may be used in pharmaceuticals. Such salts may include, without limitation, (1) acid addition salts, which can be obtained by reaction of the free base of the parent compound 35 with inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, and the like, or with organic acids, such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane 1470774-02 / / 1165.202WO1 sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or 5 coordinates with an organic base, such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are well-known to those skilled in the art, and any such pharmaceutically acceptable salts are contemplated.

[0028] Acceptable salts can be obtained using standard procedures known in the 10 art, including (without limitation) reacting a sufficiently acidic compound with a suitable base affording a physiologically acceptable anion. Suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative, albeit nonlimiting, examples include the acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, camsylate, citrate, edisylate, 15 esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, 20 saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts. Suitable base salts of the compounds can be formed from bases that form non- toxic salts. Illustrative, albeit nonlimiting, examples include the arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc 25 salts. Hemi-salts of acids and bases, such as hemi-sulphate and hemi-calcium salts, also can be formed.

[0029] The solvate can be any pharmaceutically acceptable solvate. When the solvent is water, the solvate is a hydrate.

[0030] One of ordinary skill in the art will further appreciate that the above 30 compounds can be “deuterated,” meaning one or more hydrogen atoms can be replaced with deuterium. As deuterium and hydrogen have nearly the same physical properties, deuterium substitution is the smallest structural change that can be made.

[0031] The compounds, in some embodiments, can contain one or more 35 asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R) or (S). Unless stated otherwise, it is intended that all stereoisomeric forms of 1570774-02 / / 1165.202WO1 the compounds are contemplated. When the conjugates contain alkene double bonds, and unless specified otherwise, it is intended that both E and Z geometric isomers (e.g., cis or trans) are included. Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended 5 to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring.

[0032] Accordingly, use of the term “compound” is intended to encompass, for 10 example, stereoisomers, geometric isomers, and tautomers. In addition, the “compound” can contain one or more asymmetric centers, thereby giving rise to enantiomers, diastereomers, and other stereoisomeric forms (e.g., R and S). When double bonds are present, E and Z (e.g., cis and trans) geometric isomers are possible. All isomers, including racemic, tautomeric, and optically pure forms 15 are encompassed. Also encompassed are positional isomers, such as ortho, meta and para structural isomers.

[0033] Further provided is a pharmaceutical composition comprising an above- described compound and a pharmaceutically acceptable carrier or excipient. The term "composition" generally refers to any product comprising more than one 20 ingredient, including the compound. It is to be understood that the compositions can be prepared from isolated compounds or from salts, solutions, hydrates, solvates, and other forms of the compounds. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups can form complexes with water and / or various solvents, in the various physical forms of 25 the compound. It is also to be understood that the compositions can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms of the compounds, and the compositions can be prepared from various hydrates and / or solvates of the compounds. Accordingly, such pharmaceutical compositions can include each of, or any combination of, or 30 individual forms of, the various morphological forms and / or solvate or hydrate forms of the compounds.

[0034] Any pharmaceutically acceptable carriers and excipients as known in the art can be used. A pharmaceutically acceptable carrier can include a solvent, dispersion medium, a coating, an antibacterial and / or antifungal agent(s), an 35 isotonic and / or absorption delaying agent(s), and the like, and combinations thereof, that are physiologically compatible. The carrier can be suitable for parenteral administration, e.g., a sterile aqueous solution or dispersion or a sterile 1670774-02 / / 1165.202WO1 powder for the extemporaneous preparation of a sterile injectable solution or dispersion.

[0035] Examples of various ingredients include, but are not limited to, a color additive, a preservative, and a stabilizer. More specific examples include crystal 5 cellulose, calcium carmellose, sodium carmellose, hydropropylcellulose, hydroxypropylmethylcellulose, ethylcellulose, and magnesium stearate. Such compositions can be manufactured in accordance with methods in the art and described, for example, in Remington, The Science and Practice of Pharmacy, 22nd edition. Supplementary active compounds can also be incorporated into the 10 compositions.

[0036] Oral dosage units can be tablets or capsules, for example. Other compositions for oral administration include elixirs, syrups, and the like.

[0037] Solutions of the active composition can be aqueous, optionally mixed with a nontoxic surfactant and / or can contain carriers or excipients, such as salts, 15 carbohydrates and buffering agents (preferably at a pH of from 3 to 9), but, for some applications, they can be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle, such as sterile, pyrogen-free water or phosphate-buffered saline. For example, dispersions can be prepared in glycerol, liquid PEGs, triacetin, and mixtures 20 thereof and in oils. Under ordinary conditions of storage and use, these preparations can further contain a preservative to prevent the growth of microorganisms.

[0038] Excipients can include suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium 25 alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, which can be a naturally occurring phosphatide, for example, lecithin; a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxcycetanol; 30 a condensation product of ethylene oxide with a partial ester derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate; or a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example, polyoxyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for 35 example ascorbic acid, ethyl, n-propyl, or p-hydroxybenzoate; or one or more coloring agents. 1770774-02 / / 1165.202WO1

[0039] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water can provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Additional excipients, for example, coloring agents, can also be 5 present.

[0040] Suitable emulsifying agents can be naturally occurring gums, for example, gum acacia or gum tragacanth; naturally occurring phosphatides, for example, soybean lecithin; and esters including partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan mono-oleate, and condensation 10 products of the said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. Isotonic agents, for example, sugars, polyalcohols, such as mannitol or sorbitol, or sodium chloride can be included in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, such as 15 monostearate salts and gelatin.

[0041] Liquid formulations can include suspensions and solutions. Such formulations can comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and / or suspending agents. Liquid formulations can also be 20 prepared by the reconstitution of a solid.

[0042] The compound can be formulated as a pharmaceutical composition and administered to a subject, such as a mammal, e.g., a human, in a variety of forms adapted to the chosen route of administration as discussed above. For example, the composition can be administered as an oral dosage unit, an injectable 25 composition (i.e., for subcutaneous or intravenous injection), or an infusion. See, e.g., Remington, supra.

[0043] An effective amount of the compound, or the pharmaceutical composition comprising the compound, can be determined in accordance with methods known in the art (e.g., animal models, human data, and human data for compounds that 30 are used in a similar manner). The amount can be determined by taking into consideration various factors, such as the potency of the conjugate, body weight, mode of administration, the type and location of fracture, and its causation. The effective amount can range from about 0.1 µg / kg / day, such as 0.5 µg / kg / day, 0.7 µg / kg / day, or 0.01 mg / kg / day up to about 1,000 mg / kg / day. Intravenous doses 35 can be several orders of magnitude lower. The compound / composition can be administered more than once, such as daily (1-3 or more times per day), weekly (including 1-3 or more times on a given day), bi-weekly (including 1-3 or more 1870774-02 / / 1165.202WO1 times on a given day), monthly (including 1-3 or more times on a given day), or bimonthly (including 1-3 or more times on a given day).

[0044] Prodrugs of the compounds described in the disclosure are also contemplated. The term “prodrug” means a derivative of a compound that can 5 hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound of the invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable 10 carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Specific prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those 15 described by Burger’s Medicinal Chemistry and Drug Discovery, 6th ed. (Donald J. Abraham ed., 2001, Wiley), and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).

[0045] A method of inhibiting a G protein-coupled receptor kinase (GRK) 5 in a subject is also provided. The method comprises administering an above- 20 described compound or an above-described pharmaceutical composition in an amount effective to inhibit GRK5. In embodiments of the method, the subject has cancer, such as multiple myeloma. In other embodiments of the method, the subject has heart disease, such as heart failure or cardiac hypertrophy.

[0046] “Administering” includes all means of introducing the conjugate or the 25 pharmaceutical composition comprising same. Useful dosages of the compounds can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods of extrapolating effective dosages in mice and other animals to human subjects are known in the art. Indeed, the dosage of the compounds can vary significantly depending on the condition of the subject, the 30 age of the subject, the type of disease the subject is experiencing or at risk of experiencing, the particular compounds used, how advanced the pathology is, the route of administration of the compounds and the possibility of co-usage of other therapeutic treatments or additional drugs in combination therapies. The amount of the composition required to inhibit GRK5 will vary not only with the 35 particular application, but also with the salt selected (if applicable) and the characteristics of the subject (such as, for example, age, condition, sex, the 1970774-02 / / 1165.202WO1 subject’s body surface area and / or mass, tolerance to drugs) and will ultimately be at the discretion of the attendant physician, clinician, or otherwise.

[0047] The disclosure also relates to the following numbered embodiments that are listed in no particular order of importance: 5 1. A compound having a structure selected from: 102070774-02 / / 1165.202WO1or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers 5 thereof.or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 3. A compound having a structure selected from: 2170774-02 / / 1165.202WO1 5or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 10 4. A compound having a structure selected from:or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 2270774-02 / / 1165.202WO1 5. A compound having a structure selected from:, 2370774-02 / / 1165.202WO1 5or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 6. The compound of any preceding Embodiment, or pharmaceutically 10 acceptable salts, hydrates, tautomers, and optical isomers thereof, having at least a two-fold, at least a ten-fold, at least a 100-fold, at least a 150-fold, at least a 2470774-02 / / 1165.202WO1 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at least a 750- fold, at least a 1,000-fold, at least a 2,000-fold, at least a 3,000-fold or at least a 5,000 fold selectivity for G protein-coupled receptor kinase 5 (GRK5) over GRK2. 7. The compound of Embodiment 6, or pharmaceutically acceptable salts, 5 hydrates, tautomers, and optical isomers thereof, having a two-fold to 5,000-fold, two-fold to 100-fold, two-fold to 50-fold, 10-fold to 100-fold, 10-fold to 190-fold, 10-fold to 150-fold, 50-fold to 160-fold, 50-fold to 200-fold, 100-fold to 500-fold, 100-fold to 1,000-fold, 250-fold to 1,000-fold, 500-fold to 900-fold, 300-fold to 600- fold, 500-fold to 3,000-fold, 500-fold to 1,000-fold, 500-fold to 2,000-fold, or 50- 10 fold to 150-fold selectivity for GRK5 over GRK2. 8. The compound of any one of Embodiments 1-5, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, having at least a two-fold, at least a ten-fold, at least a 100-fold, at least a 150-fold, at least a 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at least a 750- 15 fold, at least a 1,000-fold, at least a 2,000-fold, at least a 3,000-fold or at least a 5,000 fold selectivity for GRK5 over GRK6. 9. The compound of Embodiment 8, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, having a two-fold to 5,000-fold, , two-fold to 100-fold, two-fold to 50-fold, 10-fold to 100-fold, 10-fold to 190-fold, 20 10-fold to 150-fold, 50-fold to 160-fold, 50-fold to 200-fold, 100-fold to 500-fold, 100-fold to 1,000-fold, 250-fold to 1,000-fold, 500-fold to 900-fold, 300-fold to 600- fold, 500-fold to 3,000-fold, 500-fold to 1,000-fold, 500-fold to 2,000-fold, or 50- fold to 150-fold selectivity for GRK5 over GRK6. 10. A pharmaceutical composition comprising a compound of any 25 preceding Embodiment, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, and at least one pharmaceutically acceptable carrier or excipient. 11. A method of inhibiting a G protein-coupled receptor kinase (GRK) 5 in a subject, which method comprises administering a compound of any one of 30 Embodiments 1-9 or a pharmaceutical composition of Embodiment 10 in an amount effective to inhibit GRK5, whereupon GRK5 in the subject is inhibited. 12. The method of Embodiment 11, wherein the subject has heart disease. 13. The method of Embodiment 11, wherein the heart disease is heart 35 failure or cardiac hypertrophy. 14. The method of Embodiment 11, wherein the subject has cancer. 2570774-02 / / 1165.202WO1 15. The method of Embodiment 11, wherein the cancer is multiple myeloma. Examples

[0048] The disclosure can be better understood by reference to the following 5 examples which are offered by way of illustration. The disclosure is not limited to the examples given herein. General

[0049] Unless stated otherwise, all reagents and solvents were purchased from commercial sources and used without purification. NMR spectra were recorded 10 with a 300, 400 or 500 MHz spectrometer for1H NMR, 100 or 125 MHz for13C NMR spectroscopy. Chemical shifts are reported relative to the residual signals of tetramethyl silane in DMSO-d6 for1H and13C NMR spectroscopy. Multiplicities are reported as follows: singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), quartet (q), multiplet (m). double-doublet (dd), double- 15 double-doublet (ddd), triple-doublet (td), triple-triplet (tt), double-quartet (dq) etc. HRMS were recorded by using TOF, Qt of, Orbitrap mass spectrometer. Column chromatography was performed with silica gel (100–200 mesh) as the stationary phase. All reactions were monitored by using TLC. Characterizations of new compounds were further established by using HRMS. 20 GRK5 Protein Expression and Purification

[0050] Human GRK5 (1-590) WT and D311N mutants were expressed in E. coli and purified through a series of chromatography from nickel-nitrilotriacetic acid affinity, HiTrap Q HP anion exchange chromatography column followed by HiTrap SP HP cation exchange chromatography column, and Superdex 200 Increase 25 size-exclusion chromatography as described previously (Beyett et al., Protein Expr Purif 168: 105547 (2020)). Bovine GRK5-C474S was purified from baculovirus infected insect cells as previously described (Rowlands et al., J Med Chem 64: 566-585 (2021)). GRK5 Kinase Inhibition Assays 30

[0051] GRK inhibition assays were performed in reaction buffer 20 mM HEPES pH 7.0, 2 mM MgCl2, 0.025% n-dodecyl-β-D-maltoside with 50 nM human GRK5 wild-type. GRK5 was incubated with 500 nM porcine brain tubulin (PurSolutions) and inhibitors. Reactions were started by the addition of 5 μM ATP supplemented with radioactive [γ-32P]-ATP (PerkinElmer Life Sciences) for 5-min reactions at 35 room temperature. Reactions were quenched with 4X SDS loading buffer, separated in SDS-PAGE, dried, and exposed with a phosphor-imaging screen, then quantified via a Personal Molecular Imager and Quantity One 1-D Analysis 2670774-02 / / 1165.202WO1 Software. Data were analyzed via GraphPad Prism, and three-parameter dose- dependent curves (Hill coefficient = 1) plotting phosphate transferred against inhibitor concentration were used for the calculation of IC50. At least three replications were obtained to calculate IC50values. 5 Protein Crystallization

[0052] GRK5D311N was mixed with MgCl2and Sgv to achieve a final concentration of 118 μM GRK5, 354 μM Sgv, and 118 μM MgCl2. Crystals were obtained in a hanging drop vapor diffusion apparatus by mixing 2 μl of protein mixture (in 20 mM HEPES pH 7.2, 100 mM NaCl, and 0.5 mM tris(2- 10 carboxyethyl)phosphine) with 2 μl well solution composed of 220 mM potassium citrate tribasic and 20% polyethylene glycol 3350 and suspended over well solution at 4 °C. Crystal soaking and harvesting

[0053] Crystals were allowed to grow for one week until stable sizes were 15 obtained and were transferred to a new hanging drop tray in a 4 μl suspended drop containing 20% PEG3350, 10% glycerol, and inhibitor at a final concentration of 1 mM (4% DMSO in final mixture), over 600 µl of reservoir buffer containing 25% PEG3350, 10% glycerol. Crystals started to develop yellow color from the inhibitor after 24 hours, and better electron density was achieved by 20 soaking for an additional two to three days. Finally, individual crystals were directly frozen by flash freezing on nylon loops in liquid nitrogen. Data Collection and Structure Determination

[0054] Diffraction data were collected at the Brookhaven National Laboratory on NSLS-II 17-ID-1(AMX) or NSLS-II 17-ID-2(FMX) at a wavelength of 0.9201 Å. 25 Data was collected at 1° angle per frame for a total of 180 frames. Automated- processed data from Fast Data Processing (Fast DP) was used to achieve molecular replacement in PHENIX Phaser-MR or through the Dimple pipeline in NSLS-II for data reduction, using as a search model the GRK5 structure from PDB entry 8UAP. Refinements were performed using phenix.refine alternating 30 with manual building and fitting in COOT. The final models were validated with MolProbity prior to deposition along with structure factors in the Protein Data Bank. Atomic figures were created with Pymol. Example 1: Synthesis of Compounds 1 and 6 (see Table 1 for structures): 2770774-02 / / 1165.202WO1ATU (1.1 equiv) IPEA (1.3 equiv) MF, rt, 12-14 hGRK inhibitors (1, 6) General Procedure for the synthesis of starting materials:

[0055] Synthesis of Compound C: To a stirred solution of compound 5- carboxyindoline A (438 mg, 2.47 mmol, 1.2 equiv) in dry DMF (4 mL) was added 5 with (R)-1-phenylethan-1-amine B (0.26 mL, 2. 06 mmol, 1 equiv), HATU (904 mg, 2.47 mmol, 1.2 equiv) and DIPEA (0.46 mL, 2.7 mmol, 1.3 equiv) under Ar atmosphere at 0oC and the mixture was stirred for 12 h at 23oC. After completion of the reaction (monitored by TLC), the mixture was quenched with saturated Na2CO3(10 mL) and the mixture was extracted with EtOAc (3 × 50 mL). The 10 combined organic layer was washed with brine (2 × 50 mL), dried over NaSO4, and concentrated under reduced presser. The crude mixture was purified by column chromatography (0–15% MeOH in DCM) to give compound C as a pale pink color solid in 85% yields.

[0056] Synthesis of compound E: To a stirred compound C (100 mg, 0.41 15 mmol, 1 equiv) and 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid D (151 mg, 0.48 mmol, 1.1 equiv) were dissolved in absolute EtOH (2 mL). To this solution piperidine (0.04 mL, 0.497 mmol, 1.2 equiv) was added and heated to 2870774-02 / / 1165.202WO1 reflux (90 °C) for 4 h. After completion, the reaction was cooled to room temperature and the product was filtered off, the collected orange solid compound E was washed with cold ether and dried under vacuum. Compound E was obtained in 78% yield and it was used for the next reaction without further 5 purification. Compound E was coupled with amine under the standard HATU coupling reagent to provide the respective amide derivatives. Representative procedures for the synthesis of compound 1 and compound 6 are shown below. General procedure for the synthesis of Inhibitors 1-104:

[0057] To a stirred solution of acid E (25 mg, 0.0597 mmol) in dry DMF (1.5 mL) 10 was added with commercially available amines (1.2 equiv), HATU (27.3 mg, 0.07 mmol, 1.2 equiv) and DIPEA (0.051 mL, 0.298 mmol, 5 equiv) under inert atmosphere at 0oC and the mixture was stirred for 6-12 h at 23oC. After completion of the reaction (monitored by TLC), the solvent was evaporated under reduced pressure and the crude mixture was diluted with water and extracted with 15 CH2Cl2(3 x 10 mL). The combined organic layer was dried over NaSO4and concentrated under reduced pressure. The resulting crude mixture was purified by column chromatography (0-10% MeOH in CH2Cl2) to give the final inhibitor.

[0058] (Z)-3-((4-(((S)-1-methoxypropan-2-yl)carbamoyl)-3,5-dimethyl-1H-pyrrol- 20 2-yl)methylene)-2-oxo-N-((R)-1-phenylethyl)indoline-5-carboxamide (1): Compound 1 was prepared following general procedure, using acid E (20 mg, 0.04 mmol) and commercially available (S)-1-methoxypropan-2-amine F was added (4.3 mg, 0.06 mmol) to provide brick red solid compound 1 (18 mg, 82% yield);1H NMR (400 MHz, DMSO-d6) 11.10 (s, 1H), 8.5 (d, J = 7.9 Hz, 1H), 8.25 25 (s, 1H), 7.67 (d, J = 4.8 Hz, 2H), 7.42-7.2 (m, 6H), 7.34 (t, J = 7.6 Hz, 2H), 5.17 (m, 1H), 4.12 (m, 1H), 3.41-3.22 (m, 2H), 3.41-3.22 (s, 3H), 2.82 (m.1H), 2.56 – 2970774-02 / / 1165.202WO1 2.42 (m, 6H), 1.51 (d, J = 7.0 Hz, 3H), 1.1 (brs, 3H). Chemical Formula of Compound 1: C29H32N4O4; ESI-MASS Calc: 500.24 found [M+H]+.

[0059] Z)-3-((4-((2-methoxypropyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2- 5 yl)methylene)-2-oxo-N-((R)-1-phenylethyl)indoline-5-carboxamide (6): Compound 6 was prepared following general procedure, using acid E (20 mg, 0.04 mmol) and commercially available racemic 2-methoxypropan-1-amine G (4.0 mg, 0.06 mmol) to provide brick red solid compound 6 (18 mg, 82% yield); 1H NMR (400 MHz, DMSO-d6) 11.14 (s, 1H), 8.5 (d, J = 7.9 Hz, 1H), 8.25 (brs, 10 1H), 7.8-7.67 (m, 3H), 7.45-7.2 (m, 5H), 6.9 (m, 1H), 5.22 (m, 1H), 3.5 (m, 1H), 3.41-3.22 (m, 2H), 3.41 (s, 3H), 2.52 – 2.42 (m, 6H), 1.51 (d, J = 7.0 Hz, 3H), 1.1 (d, J = 7.0 Hz, 3H). Chemical Formula of Compound 6: C29H32N4O4; ESI-MASS Calc: 500.24 found [M+H]+. Synthesis of Compounds 67 and 69: 3070774-02 / / 1165.202WO1General Procedure for the synthesis of starting materials:

[0060] (R)-N-(1-(4-fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide I: To a stirred compound 2- oxoindoline-5-carboxylic acid A (700 mg, 5.029 mmol, 1 5 equiv) in 7 mL of dry DMF was added (R)-1-(4- fluorophenyl)ethan-1-amine H (0.684 mL, 5.532 mmol, 1.1 equiv), HATU (2.1g, 5.531 mmol, 1.1 equiv) and DIPEA (1.14 mL, 6.5377 mmol, 1.3 equiv) under inert atmosphere at 0oC and the mixture was stirred for 12 h at 23oC. After completion of the reaction (monitored by TLC), the mixture was quenched with sat. Na2CO3(10 mL) and the mixture 10 was extracted with EtOAc (3 × 50 mL). The combined organic layer was washed with brine (2 × 50 mL) and then dried over Na2SO4, concentrated under a reduced pressure. The crude mixture was purified by column chromatography (0–15% MeOH in CH2Cl2) to give a pale pink color solid compound I in 79% yields (1.2 g).

[0061] (R,Z)-3-((3,5-dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-N-(1-(4- 15 fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide (K): To a stirred compound I (300 mg, 1.01 mmol, 1 equiv) and commercially available 3,5-dimethyl-4-nitro- 1H-pyrrole-2-carbaldehyde J (186 mg, 1.11 mmol, 1.1 equiv) was dissolved in abs. EtOH (4 mL). To this solution piperidine (0.11 mL, 1.21 mmol, 1.2 equiv) was added and heated to reflux (95 °C) for 4 h. After completion, the reaction was 20 cooled to room temperature and the product was filtered off, the collected orange solid compound was washed with cold ethanol and dried under vacuum. Amorphous orange solid, compound K was obtained in 58% yield (260 mg). 3170774-02 / / 1165.202WO1

[0062] (R,Z)-3-((4-amino-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-N-(1-(4- fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide L: To stirred solution of compound K (100 mg, 0.229 mmol, 1 equiv) in 2:1 EtOH / EtOAc (5 mL) at 23oC, Zn powder (204 mg, 3.12, mmol, 14 equiv) and AcOH (1.96 mL, 34.35 mmol, 150 5 equiv) were added. The turbid orange solution was stirred at 50 °C for 2 h. After completion, the reaction was cooled to room temperature and then EtOAc (20 mL) was added before basifying with sat. Na2CO3(pH = >10). The basified aqueous layer was extracted with EtOAc (3 × 30 mL) and then washed with water and brine (1 x 30 mL). The organic layer was dried over Na2SO4, and then the 10 solvent was removed under pressure to give product L as a red solid in quantitative yields (93 mg).1H NMR (400 MHz, DMSO) δ 10.85 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 1.4 Hz, 1H), 7.58 (dd, J = 8.1, 1.6 Hz, 1H), 7.44 – 7.40 (m, 3H), 7.15 – 7.10 (m, 2H), 6.87 (d, J = 8.1 Hz, 1H), 5.20 – 5.14 (m, 1H), 3.99 (s, 2H), 2.24 (s, 3H), 2.15 (s, 3H), 1.47 (d, J = 7.1 Hz, 3H). 15 General procedure for the synthesis of Inhibitors 1-104:

[0063] To a stirred solution of compound L (25 mg, 0.0597 mmol) in dry DMF (1.5 mL) was added with commercially available acids (1.2 equiv), HATU (27.3 mg, 0.0716 mmol, 1.2 equiv) and DIPEA (0.051 mL, 0.298 mmol, 5 equiv) under inert atmosphere at 0oC and the mixture was stirred for 6-12 h at 23oC. After 20 completion of the reaction (monitored by TLC), the solvent was evaporated under reduced pressure and the crude mixture was diluted with water and extracted with CH2Cl2(3 x 10 mL). The combined organic layer was dried over NaSO4and concentrated under reduced pressure. The resulting crude mixture was purified by column chromatography (0-10% MeOH in CH2Cl2) to give the final inhibitor. 253270774-02 / / 1165.202WO1

[0064] Methyl(R,Z)-3-((5-((5-((1-(4-fluorophenyl)ethyl)carbamoyl)-2- oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)amino)-3- oxopropanoate (67): Compound L was prepared following general procedure, using amine L and malonic acid M (11 mg, 0.09 mmol) to give brick red compound 5 67 (22 mg, 71% yield);1H NMR (400 MHz, DMSO) δ 11.05 (s, 1H), 9.36 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.74 – 7.59 (m, 2H), 7.50 – 7.38 (m, 2H), 7.18 – 7.08 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (t, J = 7.3 Hz, 1H), 3.65 (s, 3H), 3.43 (s, 2H), 2.18 (d, J = 6.1 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H).10

[0065] (Z)-3-((4-((S)-4-cyano-3-hydroxybutanamido)-3,5-dimethyl-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (69): Compound 69 was prepared following general procedure, using amine L (25 mg, 0.06 mmol) and acid N (12 mg, 0.09 mmol) to provide brick red solid compound 69 (21 mg, 65% yield);1H NMR (400 MHz, DMSO) δ 11.03 15 (s, 1H), 9.19 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.17 (d, J = 1.7 Hz, 1H), 7.83 – 7.55 (m, 2H), 7.50 – 7.33 (m, 2H), 7.13 (t, J = 8.9 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 5.53 (d, J = 5.1 Hz, 1H), 5.26– 5.08 (m, 1H), 4.19 (h, J = 5.3 Hz, 1H), 2.69 (dd, J = 17.3, 5.2 Hz, 2H), 2.55 (dd, J = 14.4, 7.7 Hz, 2H), 2.17 (d, J = 6.7 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H). 20 Table 1: Compound Structures3370774-02 / / 1165.202WO13470774-02 / / 1165.202WO13570774-02 / / 1165.202WO13670774-02 / / 1165.202WO13770774-02 / / 1165.202WO13870774-02 / / 1165.202WO1 4 5 5 5

[0066] Activity data are shown in Table 2. Table 2: Compound Activity Data5 Example 2 Introduction

[0067] G protein-coupled receptor (GPCR) kinases (GRKs) regulate a large and diverse family of cell surface receptors known as GPCRs by triggering their internalization and desensitization. These essential molecular events occur 10 across all organs and tissues in humans and other metazoans. GRKs 3970774-02 / / 1165.202WO1 phosphorylate the intracellular loops or C-terminal tails of active GPCRs, which then recruits cytosolic arrestin proteins that target the receptor for endocytosis, leading to loss of cellular sensitivity to extracellular signals. These signaling events are critical for maintenance of homeostatic cell functions. Uncontrolled 5 GRK activity and signaling abnormalities can lead to adverse effects on cell survival and contribute to diverse disease processes including heart failure and cancer. GRKs exist in seven different isoforms in humans and are classified in three groups (GRK1 / 7, GRK2 / 3, and GRK4 / 5 / 6) based on their homology. GRK1 / 7 are abundant in the retina, GRK4 in the testes, and GRK3 in the nasal 10 epithelium, whereas GRK2 and GRK5 are more ubiquitous. GRK6 is also expressed ubiquitously, including neuronal and immune cells, skeletal muscle, and pancreas. It also exists at lower levels in the heart, lung, kidney, placenta, and liver.

[0068] The GRK family members are involved in various disease progression. 15 Hypertrophic cardiomyopathy is a leading cause of heart failure, a significant public health concern associated with considerable global morbidity and mortality rates. GRK2 and GRK5 are the most abundantly expressed GRKs in heart muscle, where they are known to regulate GPCRs including the β-adrenergic receptors. Overexpression of GRK2 and GRK5 is often observed in hearts 20 undergoing hypertrophy, a disease characterized by enlargement of the left ventricle, which leads to insufficient blood supply due to its loss of contractility. In addition, GRK5 can influence cardiac contractility genetically, in that nuclear translocated GRK5 can phosphorylate histone deacetylase 5 and lead to the upregulation of cardiac hypertrophy genes through myocyte enhancer factor 2. 25 GRK5 has also emerged as a key player in cancer biology through its regulation of chemokine receptors (e.g. CXCR4, ACKR3) and non-receptor substrates such as tumor suppressor p53 and moesin, which contribute to cancer proliferation, migration, and tumor growth in vivo in non-small cell lung cancer and prostate cancer. Because of the involvement of GRK5 in these disease pathologies, the 30 development of GRK5 inhibitors is an exciting avenue for treatment of heart failure and cancer. GRK6, a close homolog of GRK5, is highly expressed in tumor cells, particularly in multiple myeloma (MM) cell lines. Recent studies suggested that GRK6 is an important target against MM.

[0069] The identification of selective GRK5 inhibitors is important for reducing 35 off-target effects and toxicities. However, this is a challenging undertaking given the close homology and function of GRKs, particularly within the three groups of GRKs. Some reported strategies to overcome selectivity issues include allosteric 4070774-02 / / 1165.202WO1 inhibition and covalent inhibition. A series of potent and selective irreversible GRK5 inhibitors was described by incorporating a halo acetamide-based warhead on sunitinib, an FDA-approved drug targeting receptor tyrosine kinases (105, Scheme 1). These inhibitors were designed to form a covalent bond with a 5 Cys residue only found in GRK4 / 5 / 6 active sites. One of these inhibitors was the chloroacetamide derivative 106 which displayed an IC50value of 8.6 nM for GRK5 and 1.2 µM for GRK2 (1400-fold selectivity). Compound 106 was shown to form a covalent bond with GRK5. However, such highly reactive warheads could trigger potential safety and toxicity issues due to reactions with other kinase 10 targets or other proteins with exposed cysteines. A new class of highly selective and potent GRK5 inhibitors as exemplified by keto-amide derivative 3 was designed, displaying GRK5 IC50of 10 nM with >100,000-fold selectivity over GRK2. X-ray structural studies revealed the formation of a reversible covalent hemithioacetal intermediate P with GRK5-Cys474 (Scheme 2). Utilizing this 15 molecular insight, potent non-covalent GRK5 inhibitors with α-hydroxy amide derivatives were designed, albeit with reduced selectivity against GRK2. Compound 108a with (S)-configuration exhibited a GRK5 IC50of 33 nM but a selectivity greater than 120-fold over GRK2. The X-ray structure of inhibitor 108a- bound GRK5 was determined, and based upon this structural insight heterocyclic 20 carboxamide derivatives were investigated that would potentially interact with the active site tether (AST), a poorly conserved loop in GRKs that passes over the active site and contains the reactive Cys residue targeted by P. Upon optimization for GRK5 selectivity, the potency of these inhibitors against GRK6, a potential target for treatment of multiple myeloma, was explored. To this end, there has25 been reported one series of small molecule GRK6 inhibitors by Uehling and co- workers. Herein a new set of noncovalent GRK5 inhibitors that contain heterocyclic carboxamide functionalities are reported, along with the X-ray structures of some of these inhibitors in complex with GRK5. Surprisingly, some of these inhibitors also exhibited high selectivity for GRK6. The data aid further 30 optimization of inhibitor potency and selectivity for either GRK5 or GRK6. 4170774-02 / / 1165.202WO1Scheme 1. Structures of sunitinib and sunitinib-derived GRK5 inhibitorsScheme 2. The reversible covalent bond formation between compound 3 and 5 GRK5 is shown in structure P (X-ray structure of 107-GRK5 complex, PDB entry 8UAQ). This inspired the design of non-covalent inhibitors 108a and 109. 4270774-02 / / 1165.202WO1 Results and discussion

[0070] Structure-based design efforts were initially focused on the development of non-covalent reversible inhibitors due to a perceived concern for promiscuous 5 activity with off-target biological nucleophiles. The X-ray structure of 107-bound GRK5 showed evidence for the formation of a reversible covalent hemithioacetal intermediate as shown in P via nucleophilic attack by the Cys474 sulfur on the carbonyl center depicted in Scheme 2. From this structural insight, unreactive α- hydroxy amide derivatives such as compound 108a were designed, which is a 10 potent and selective GRK5 inhibitor. The X-ray structure of inhibitor 108a-bound GRK5 provides an explanation for enhanced GRK5 inhibitory activity of compound 108a, the (S)-isomer, relative to the compound 108b, the (R)-isomer. Based upon this structural insight, it was further speculated that heterocyclic carboxamide derivatives as represented in general structure 5 with appropriately 15 positioned heteroatoms and substituents may confer similar ligand interactions that could have higher potency and selectivity for GRK5 due to steric clashes with the GRK2 AST. Syntheses of GRK5 inhibitors 109a-i 20

[0071] The synthesis of selected heterocyclic carboxamide-derived GRK5 inhibitors is shown in Scheme 3. Indolinone derivative 110 and nitropyrrole aldehyde 111 were synthesized as described by us and others previously. In a slightly modified procedure, condensation of indolinone derivative 110 with aldehyde 111 in the presence of piperidine in ethanol at 0 °C for 4 h afforded the 25 corresponding nitro pyrrole-indolinone derivative in near quantitative yield. The resulting nitro group was reduced with powdered Zn in acetic acid in a mixture (2:1) of ethanol and ethyl acetate at 50 °C for 2 h to provide amine derivative 112 in 62% yield over two steps. This amine is not very stable and was utilized directly for the next reaction without further purification. Selected commercially available 30 heterocyclic and heteroaromatic carboxylic acids 113a-o were then coupled with pyrroloamine derivative 8 using HATU in DME in the presence of DIPEA at 0 °C to 23 °C for 12 h to furnish carboxamide derivatives 109a-o in good yield (44- 84%). Full structures of these GRK5 inhibitors are shown in Table 3. 4370774-02 / / 1165.202WO1Scheme 3. Synthesis of GRK5 inhibitors 109a-o Structure-activity relationship studies 5

[0072] Inhibitors were tested against tubulin phosphorylation by human GRK2 and GRK5 as described previously. In addition, the activity of test compounds against GRK6 was examined as it has been recognized as an efficacious target for the treatment of multiple myeloma. The results are shown in Table 3. Inhibitor 107 inhibited GRK6 with an IC50value of 22 nM, compared to its GRK5 IC50of 10 10 nM. Noncovalent inhibitor 108a with (S)-hydroxy configuration, exhibited similar GRK5 and GRK6 activity with IC50values 30 and 36 nM, respectively. Interestingly, isomeric inhibitor 108b with (R)-hydroxy configuration displayed GRK5 IC50of 4 μM compared to GRK6 IC50of 62 nM. The hydroxyl stereochemistry thus did not show major effect on GRK6 inhibition. The inhibitory 4470774-02 / / 1165.202WO1 properties of 2-furan and 3-furan carboxamide derivatives 109a and 109b was investigated as non-covalent inhibitors. Compound 109a exhibited an IC50of 900 nM for GRK5 and 140 µM for GRK2, (over 160-fold selectivity). Isomeric structure compound 109b with a 3-furan derivative showed enhancement of activity against 5 GRK5 with an IC50value of 56 nM (180-fold selectivity over GRK2). The results indicate that the position of the oxygen on the furan ring can have major effects on GRK5 inhibition. Compound 109b shows enhanced activity against GRK2, however, it shows similar selectivity as compound 109a. Both compounds 109a and 109b inhibited GRK6 with low nanomolar IC50values.2-thiophene derivative 10 109c was studied, which showed 300-fold reduction of activity against GRK5. Compound 109d with a dimethyl thiazole carboxamide also displayed a 900-fold reduction in GRK5 inhibition as well as 30-fold reduction in selectivity compared to 109b. Both 109c and 109d inhibited GRK6 more potently compared to GRK5. The effect of racemic 2-tetrahydrofuran carboxamide derivative 109e was 15 examined to compare the effect of the furan ring in inhibitor 109a. Interestingly, the tetrahydrofuran 1:1 diastereomeric mixture (due to racemic acid) showed reduction of GRK5 inhibition and selectivity over GRK2. Because 3-furan carboxamide 109b is significantly more potent than the 2-furan carboxamide derivative 109a, the effect of the corresponding 3-tetrahydrofuran derivatives 20 109f and 109g was examined. Compound 109f with 3(S)-configuration showed an approximate 5-fold better potency than 109g, but with less selectivity over GRK2. The effect of 3-dihydropyran carboxamide and 4-tetahydropyran carboxamide derivatives 109h and 109i, respectively, was also examined. However, both compounds showed significant reduction of potency and 25 selectivity compared to 109b. 45y6 tiK viR t G / .cebl 5sKeK R RSG GybyK ntivR oit itG al c / 2yelK reR oSG h psohp0)nilC5M Iμ(ubu t t) 6sMK niμ (R ag05Ga CIsroti )801)3biM hμ5.0(K0±).0 () 8.ni 05R03 ±f1( 033( 0±oaCIG0.0 10.0.4ytivitcO O aS) O ) d O(OR(nOOH O O H H aH N N H Ner N HuN Ht O O N H OcurN H7N HaNbt080H 80S1 1 1.er3uetlcO O burN H N HO tN Ha1 Tsr OotW i2 biF F h F02. nI5611 / / 20-47707y6 tiK viR tcG / ebl 5eK SR G ytiK vR itG c / e2lK e R S Gb5 2)K 05R CM μ G I()M6μ(K 0 R C5GI.0.0704) 0 0 )M56.. 3μ 0) 0 )3( ( K 0 R±±03( 6 (5±aC5IG9. 550 0. 701O O S O O O HN H N H NN H O N H O N H ON HaN He9b9 N Hcr0101 901utcuO O NHO rt N H N H1sr OotW i2 biF0hF F2. nI5611 / / 20-47707y6 tiK viR tcG / ebl 5eK SR G ytiK vR itG c / e2lK e R S Gb5 2 K 0)5R CM IμG ()M6μ(K0 R01406C5GI.00.0.08 )4) )M μ54(4( 30( K0 7 ..0)0 R3± 0± ± 3(aC5IG 05 8. 111.0S N O O HNO O R) (N HO OH NH NN HN HdO N H9 O01 N He9)Z0( N Hf90e1 1ruO tcN HO ON Hurt N H1sr OoF t FW i2 biF0h2. nI5611 / / 20-47707y6 tiK viR tcG / ebl 5eK SR G ytiK vR itG c / e2lK e R S Gb5 2)K 0C5R M μ G I()M6μ(K 0 R 5GCI094) )3M)μ5 ((K4. 3)(3(05R0 2 2G±5±6±CI.2a 0O O O ) R(O O O HN H N H NN H N HO N H O ON Hg9 N Hi0N Hh9 900e1 1 1rutcO O u O rt N H N H N H1sr OotW i2 biF h F F02. nI5611 / / 20-47707y6 tiK viR tcG / ebl 5eK SR G ytiK vR itG c / e2lK e R S Gb5 2)K 05R CM μ G I()M6μ(K 0 R C5GI0.0 005) 0M3) )μ5 .3( ( K0)36(05R±G03(7 ±22±3aCI.0 1O O ONO) OR(O ) S(OH NO HN N H O H NN HO N HN HkOeN Hj90r9 1 N Hlu 09t 10cO1urtON Hs O rN H N H1OotW i2 bihF02. nIF F5611 / / 20-47707y6 tiK viR tcG / ebl 5eK SR G ytiK vR itG c / e2lK e R S Gb5 2)K 05R CM μ G I()M6μ(K 0 R C5GI1 )) 5)3M( 20 3(μ5(K1..0± ) 9.05R0G±363( 00±aCI.0.0 7.1H N F N N N N O O HNO HN H NN H O N H O N H ON Hme9r0N Hu1n9 N Hot0910c1urtO sN HO O NH N H1 rOotW i2 bi0h2F. nIF F5611 / / 20-4770770774-02 / / 1165.202WO1

[0073] In Table 3,aIC50values for GRK5, GRK6, and GRK2 phosphorylation of the soluble substrate tubulin, with number of replicates shown in parentheses. bGRK2 / GRK5 indicates fold-selectivity for GRK5 over GRK2, and GRK5 / GRK6 the fold selectivity for GRK6 over GRK5. 5

[0074] Because compound 109b showed potent GRK5 activity, it was speculated that its furanyl oxygen may be involved in a polar interaction with the AST loop. It was proposed that 5-oxotetrahydrofuran carboxamide would position the carbonyl oxygen closer to the AST loop. The effect of both stereoisomers was examined. It was found that compound 109j with a (S)-configuration showed a 6-10 fold decrease in GRK5 inhibitory activity (IC50700 nM) compared to 3-(S)- tetrahydrofuryl carboxamide derivative 109f (IC50110 nM), and compound 109k (IC50140 µM) with a (R)-configuration showed over 15-fold reduction in potency compared to 5j. In general, inhibitors 109e-109k exhibited GRK6 inhibition in low nanomolar range, with less differences in potencies in response to modification 15 than observed with GRK5.

[0075] The effect of aromatic heterocyclic carboxamide derivatives was then examined. Compound 109l with a 6-benzoxazole aromatic heterocycle showed GRK5 inhibitory activity in the low micromolar range. Compound 109m with a 2- methyl benzimidazole aromatic heterocycle exhibited 10-fold enhancement of 20 GRK5 inhibitory activity (IC50300 nM). Surprisingly, compound 109m showed no appreciable GRK2 activity, thus displaying over 3000-fold selectivity. Compound 109n with a 2-imidazopyridine heterocycle displayed 5-fold improvement compared to 109m, with an IC50of 60 nM for GRK5. It also exhibited good selectivity over GRK2 (800-fold). Surprisingly, both compounds 109m and 109n 25 showed comparable GRK5 and GRK6 inhibitory activity. Compound 109o with fluoropyridine aromatic ring, exhibited low micromolar GRK5 activity and 180-fold selectivity over GRK2. X-ray Structure determination of compounds 108a, 109a, 109b, 109j, and 109n in complex with GRK5 30

[0076] To obtain molecular insight into ligand-binding site interactions, particularly with the AST loop, the inhibitors were soaked into crystals of GRK5 obtained by co-crystallizing sangivamycin (Sgv) with GRK5D311N, a catalytically inactive mutant that prevents the heterogeneous autophosphorylation that occurs in E. coli but retains the stability of the wild-type protein. Crystals soaked with 35 inhibitors were harvested after 72 to 96 hours. All resulting structures exhibited a similar “open” conformation of GRK5, with the kinase small and large lobes 11° more open than in the structure of GRK5 bound to Sgv (PDB entry 6PJX). All 5270774-02 / / 1165.202WO1 domains in GRK5 were resolved except for the N terminal helix (residues 1-25), a C-terminal portion of the AST (residues 475-491), and the extreme C terminus (residues 543-590). Sub-3 Å resolution was achieved for structures of GRK5 in complex with inhibitors 108a, 109a, 109j, and 109n (GRL019-21, GRL055-22, 5 GRL064-22, and GRL093-22, respectively) whereas 3.7 Å was achieved for the complex with 109b (GRL056-21). Structural comparison of bound inhibitors and structure-based SAR

[0077] Common interactions were observed for the fluorophenyl-indolinone cores of the compounds. All bind to the ATP binding pocket of GRK5, with the 10 indolinone core occupying the adenine site and forming two hydrogen bonds with backbone atoms in the kinase domain hinge (residues Thr264 and Met266). The fluorophenyl group engages the phosphate binding loop (P loop) via polar interactions between the fluoride and P loop backbone amides. An additional hydrogen bond is typically observed between the ethyl-amide group and the 15 catalytic Lys215 side chain. The AST region of the active site containing Cys474 is more dynamic in all of the crystal structures compared to that of the GRK5 complex with compound 3, consistent with loss of a covalent interaction. In the compounds reported here, variable moieties were attached to the pyrrole group with an amino carboxyl group. The carbonyl group of this moiety in each structure 20 orients towards the large lobe, away from the hinge of the kinase domain.

[0078] Compound 108a is a diastereomeric mixture (1:1) at the α-hydroxyfuranyl center. It was derived from the reversible covalent inhibitor (compound 3) after reduction of the α-carbonyl group and lacks the ability to form a covalent bond with Cys474. The furanyl group of 108a packs against the AST loop, occupying 25 a pocket formed between the AST and the hinge in a similar way to compound 3, with the hydroxyl group oriented towards the small lobe. In compounds 107 and 108a, the furan oxygen makes a hydrogen bond to the backbone amide nitrogen of Tyr473. Consistent with the higher potency of the (S)-isomer at the α chiral center, the structure of compound 108a (GRL-01921) is best fit with the (S) 30 configuration.

[0079] Compounds 109a and 109b, containing 2- and 3-furanyl side chains, as well as inhibitor 109j, containing a 5(S)-oxotetrahydrofuran carboxamide, are one carbon shorter than compound 108a. In the GRK5 complex with compound109b, the 3-furanyl group reaches toward the AST but the furan oxygen cannot form a 35 hydrogen bond. The furan oxygen in compound 109a in fact does not orient towards the AST. The potency increased from compound 109a (IC50= 0.9 μM) to compound 109b (IC50= 0.056 μM) by more than 10-fold. Therefore, the data 5370774-02 / / 1165.202WO1 support the idea that the furan oxygen in compound 109b could form a hydrogen bond with the AST. A caveat is that the low resolution of the R group and the flexible AST in this structure render this conclusion difficult to confirm.

[0080] In the GRK5 complex with 109j, the oxotetrahydrofuranyl group is flexible 5 and the center of the cyclic group flipped to a position parallel to the AST. Without the additional carbon in the linker that would allow for flexibilty, compounds such as this one are expected to have more steric clashes with the AST. Compound 5n contains a bulkier heterocyclic aromatic ring which was not well-resolved in the density maps but still seems to pack against the AST. Its relatively high 10 potency for GRK5 (IC50= 0.06 μM) proves its compatibility with the pocket. The 800-fold reduced potency for GRK2 however indicates that the bulky imidazopyridine group also contributes to improved selectivity, perhaps via unfavorable interactions with the GRK2 AST.

[0081] The selectivity of compounds 109m and 109n for GRK5 were 3000-fold 15 and 800-fold, respectively, over GRK2. This class of compounds can bind to GRK5 in an open conformation. To understand their selectivity against GRK2, compound 109n was manually docked into several available PDB structures of GRK2. In PDB entry 8JPB, the GRK2 kinase domain exhibits a more closed conformation. Based on the alignment, the imidazopyridine group of the ligands 20 would clash with the AST, although the kinase hinge can form hydrogen bonds with the indolinone core as in GRK5. In a structure of GRK2 that exhibits an open conformation (PDB entry 5UKM [37)], the hinge is incompatible with the core of the compound whereas the R group can avoid clashes with AST. Conclusions 25

[0082] GRK5 is a promising target for the treatment of cardiovascular disease and cancer. The series of inhibitors described herein contains pyrrolo-indolinone as the main scaffold as in the FDA approved, orally bioavailable anti-cancer drug, sunitinib. Compounds are synthesized efficiently using basic amide coupling with readily available carboxylic acids. A number of compounds including compound 30 109b and 109n exhibited potent GRK5 activity in low nanomolar range and also showed high selectivity over GRK2. To obtain further molecular insights into the ligand-binding site interactions, X-ray structures of GRK5 bound to compounds 108a, 109a, 109b, 109j, and 109n were determined. These structures provided direct insights into the potency and selectivity of these lead compounds. A major 35 conclusion is that steric hindrance imposed by the unique structure of the GRK2 AST is likely a key factor driving higher selectivity for GRK5. Compounds with less bulky heterocyclic groups or with a rotatable bond at the heterocyclic group 5470774-02 / / 1165.202WO1 are therefore more likely to inhibit GRK2 better, leading to lower selectivity. All compounds in this series showed nanomolar IC50for GRK6, suggesting that GRK6 may have a better spatial capacity for the modifications in these compounds and thus retains high affinity for all of them. It was speculated that 5 this could be due to the fact that the AST region of GRK6 adopts a distinct conformation in its open state (PDB entry 2ACX) wherein Tyr473 and Cys474 are positioned over 15 Å away from their positions in the GRK5 complexes determined here. Regardless of the mechanism, some surprisingly selective GRK6 inhibitors relative to both GRK5 and GRK2, such as 109d (500-fold and 10 3000-fold, respectively) and 109k (400-fold and 5000-fold, respectively) are described.

[0083] (R,Z)-3-((4-amino-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-N-(1-(4- fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide (112): To a stirred compound 110 (100 mg, 0.33 mmol, 1 equiv) and 3,5-dimethyl-4-nitro-1H-pyrrole- 15 2-carbaldehyde 111 (62 mg, 0.36 mmol, 1.1 equiv) was dissolved in abs. EtOH (3 mL). To this solution piperidine (0.04 mL, 0.4 mmol, 1.2 equiv) was added and heated to reflux (90 °C) for 4h. After completion, the reaction was cooled to room temperature and the product was filtered off, the collected orange solid compound was washed with cold ethanol and dried under vacuum. Amorphous golden 20 yellow solid, 65% yield (98 mg) was used as such for the next step without any purification.

[0084] To a round bottom flask were added above prepared nitro compound (95 mg, 0.212 mmol, 1 equiv) in 2:1 EtOH / EtOAc (4 mL). To this slurry, Zn powder (194 mg, 2.97 mmol, 14 equiv) and AcOH (1.90 mL, 31.77 mmol, 150 equiv) were 25 added. The turbid orange solution was stirred at 50 °C for 2 h. After completion, the reaction was cooled to room temperature and then EtOAc (20 mL) was added before basifying with sat. Na2CO3(pH >10). The basified aqueous layer was extracted with EtOAc (3 times) and then washed with water and brine (1time). The organic layer was dried over Na2SO4, and then the solvent was removed 30 under pressure to give the desired product as an amorphous red solid (85 mg, 95%).1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 3H), 8.54 (d, J = 8.0 Hz, 3H), 8.09 (d, J = 1.4 Hz, 3H), 7.58 (dd, J = 8.1, 1.6 Hz, 3H), 7.44 – 7.40 (m, 10H), 7.15 – 7.10 (m, 7H), 6.87 (d, J = 8.1 Hz, 3H), 5.20 – 5.14 (m, 3H), 3.99 (s, 5H), 2.24 (s, 9H), 2.15 (s, 9H), 1.47 (d, J = 7.1 Hz, 9H);13C NMR (100 MHz, DMSO-d6) δ 35 175.4, 169.6, 166.4, 141.7, 139.7, 133.6, 128.4 (d, J = 7.9 Hz, CF-C), 127.3, 126.3 (d, J = 23.6 Hz, CF-C), 125.3, 124.6, 123.3, 117.1, 116.5, 115.3, 115.1, 109.2, 5570774-02 / / 1165.202WO1 108.6, 48.1, 22.6, 11.6, 8.9; Molecular formula: C24H23FN4O2ESI-MS calc: 418.18 ESI-MS found: [M + 1] 419.0.

[0085] (R,Z)-N-(1-(4-fluorophenyl)ethyl)-3-((4-(furan-2-carboxamido)-3,5- dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindoline-5-carboxamide (109a): 5 To a stirred solution of amine 112 (20 mg, 0.05 mmol) in dry DMF (1.5 mL), commercially available 2-Furoic acid 9a (7 mg, 0.06 mmol), HATU (21.8 mg, 0.06 mmol) and DIPEA (42 μL, 0.24 mmol) were added under inert atmosphere at 0 oC. The resulting mixture was stirred at 0oC to 23oC for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude 10 mixture was diluted with water and extracted with CH2Cl2(3 times). The combined organic layer was dried over NaSO4and concentrated under reduced pressure. The resulting crude mixture was purified by column chromatography (2-5% MeOH in CH2Cl2) to yield compound 109a as an amorphous orange solid (19 mg, 77%).1H NMR (500 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.59 (s, 1H), 8.58 (d, J = 15 8.0 Hz, 1H), 8.20 (d, J = 1.7 Hz, 1H), 7.97 – 7.80 (m, 1H), 7.77 – 7.60 (m, 2H), 7.53 – 7.36 (m, 2H), 7.35 – 7.21 (m, 1H), 7.20 – 7.06 (m, 2H), 6.92 (d, J = 8.1 Hz, 1H), 6.67 (dd, J = 3.4, 1.8 Hz, 1H), 5.17 (m, 1H), 2.21 (d, J = 9.7 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 170.2, 161.4(d, J = 241.2Hz, CF-C), 160.5, 157.2, 148.1, 145.9, 141.7, 140.8, 132.8, 128.5(d, J = 8.3 Hz, 20 CF-C), 128.2, 128.0, 126.2, 125.9, 125.0, 124.7, 121.4, 117.8, 115.3(d, J = 21.0 Hz, CF-C), 114.6, 113.2, 112.4, 109.1, 48.3, 22.7, 12.2, 9.8; HRMS (ESI): m / z calcd for C29H26FN4O4[M+H]+ 513.1938 found 513.1927.

[0086] (R,Z)-N-(1-(4-fluorophenyl)ethyl)-3-((4-(furan-3-carboxamido)-3,5- dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindoline-5-carboxamide (109b): 25 Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and 3-Furoic acid 113b (7 mg, 0.06 mmol) provided compound 109b as red solid (20 mg, 82%);1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.37 (s, 1H), 8.43 – 8.14 (m, 2H), 7.77 (t, J = 1.7 Hz, 1H), 7.72 – 7.62 (m, 2H), 7.52 – 7.36 (m, 2H), 7.31 – 7.04 (m, 2H), 7.07 – 6.86 (m, 2H), 5.17 (p, J = 7.1 Hz, 1H), 30 2.22 (d, J = 6.4 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (101 MHz, DMSO-d6) δ 170.1, 166.2, 161.3(d, J = 241.9 Hz, CF-C), 161.0, 146.0, 144.5, 141.6, 140.7, 132.7, 128.3(d, J = 8.2 Hz, CF-C), 127.9(d, J = 3.8 Hz, CF-C), 126.1, 125.7, 124.8, 124.6, 122.9, 121.7, 117.7, 115.3, 115.1, 113.1, 109.5, 109.0, 48.1, 22.6, 12.1, 9.7; HRMS (ESI): m / z calcd for C29H26FN4O4[M+H]+ 513.1938 found 513.1930. 35

[0087] (R,Z)-3-((3,5-dimethyl-4-(thiophene-2-carboxamido)-1H-pyrrol-2- yl)methylene)-N-(1-(4-fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide (109c): Following the procedure as described for compound 109a, amine 112 5670774-02 / / 1165.202WO1 (20 mg, 0.05 mmol) and Thiophene 2-carboxylic acid 9c (8 mg, 0.06 mmol) provided compound 109c as an orange solid (17 mg, 67%);1H NMR (500 MHz, DMSO) δ 11.06 (s, 1H), 9.69 (s, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 7.96 (d, J = 3.7 Hz, 1H), 7.81 (d, J = 5.0 Hz, 1H), 7.67 (d, J = 6.5 Hz, 2H), 7.43 (dd, J 5 = 8.5, 5.5 Hz, 2H), 7.20 (t, J = 4.3 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.17 (t, J = 7.4 Hz, 1H), 2.23 (d, J = 8.0 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO DMSO-d6) δ 170.2, 166.3, 161.4(d, J = 241.6 Hz, CF-C), 160.7, 141.7, 140.8, 140.1, 132.8, 131.8, 129.3, 128.5(d, J = 8.4 Hz, CF-C), 128.0, 126.2, 125.9, 125.0, 124.7, 121.8, 117.8, 115.3(d, J = 21.3 Hz, CF-10C), 113.3, 109.1, 48.3, 22.7, 12.2, 9.8;HRMS (ESI): m / z calcd for C29H26FN4O3S [M+H]+ 529.1709 found 529.1693.

[0088] (R,Z)-N-(5-((5-((1-(4-fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3- ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2,5-dimethylthiazole-4- carboxamide (109d): Following the procedure as described for compound 109a, 15 amine 112 (20 mg, 0.05 mmol) and 2,5-dimethylthiazole-4-carboxylic acid 113d (9 mg, 0.06 mmol) provided compound 109d as an amorphous brick red solid (20 mg, 75%);1H NMR (500 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.42 (s, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.20 (s, 1H), 7.73 – 7.62 (m, 2H), 7.43 (dd, J = 8.5, 5.5 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.18 (m, 1H), 2.69 (s, 3H), 2.65 20 (s, 3H), 2.21 (d, J = 10.6 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 170.2, 166.4, 161.8, 161.4(d, J = 243.1 Hz, CF-C), 160.5, 142.7, 141.7, 140.7, 140.5, 133.0, 128.5(d, J = 8.0 Hz, CF-C), 128.3, 127.9, 126.1, 125.9, 124.9, 124.7, 122.1, 117.7, 115.3(d, J = 21.5 Hz, CF-C), 112.9, 109.1, 48.3, 22.7, 19.1, 12.8, 12.4, 9.9; HRMS (ESI): m / z calcd for C30H29FN5O3S [M+H]+ 558.1975 25 found 558.1964.

[0089] ((Z)-3-((3,5-dimethyl-4-(tetrahydrofuran-2-carboxamido)-1H-pyrrol-2- yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide (109e): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and tetrahydrofuran-2-carboxylic acid 113e (7 mg, 0.06 30 mmol) provided compound 109e as a red solid (18 mg, 72%);1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 9.03 (s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 4.1 Hz, 1H), 7.87 – 7.55 (m, 2H), 7.56 – 7.26 (m, 3H), 7.13 (t, J = 8.8 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.24 – 4.98 (m, 1H), 4.38 (dd, J = 8.2, 5.1 Hz, 1H), 3.99 (q, J = 6.9 Hz, 1H), 3.81 (q, J = 7.1 Hz, 1H), 2.16 (m, 7H), 1.91 (m, 3H), 1.48 (d, J = 35 7.2, 3H);13C NMR (126 MHz, DMSO-d6) δ 172.9, 170.2, 166.4, 161.4(d, J = 243.9 Hz, CF-C), 145.5, 141.7, 140.7, 132.8, 128.7, 128.5(d, J = 7.7 Hz, CF-C), 128.1, 127.9, 127.0, 126.5, 126.2, 125.9, 124.9, 124.7, 121.7, 117.6, 115.3(d, J = 21.0 5770774-02 / / 1165.202WO1 Hz, CF-C), 112.9, 109.2, 78.3, 69.2, 48.3, 30.9, 25.4, 22.7, 12.1, 9.6; HRMS (ESI): m / z calcd for C29H30FN4O4[M+H]+ 517.2251 found 517.2243.

[0090] (Z)-3-((3,5-dimethyl-4-((S)-tetrahydrofuran-3-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- 5 carboxamide (109f): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and (S)-tetrahydrofuran-3-carboxylic acid 113f (7 mg, 0.06 mmol) provided compound 109f as an orange solid (15 mg, 60%); Rf= 0.6 (MeOH : CH2Cl2= 9:1);1H NMR (500 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.20 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.90 – 7.55 (m, 2H), 10 7.60 – 7.36 (m, 2H), 7.37 – 7.05 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.4 Hz, 1H), 3.94 (t, J = 8.2 Hz, 1H), 3.80 – 3.65 (m, 3H), 3.15 (h, J = 7.0 Hz, 1H), 2.17 (d, J = 7.9 Hz, 6H), 2.13 – 2.06 (m, 3H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 172.5, 170.2, 166.4,162.4, 160.4, 141.7, 140.7, 132.4, 128.5(d, J = 8.0 Hz, CF-C), 127.9, 127.5, 126.1, 125.9, 124.9, 124.7, 122.3, 117.7, 15 115.3(d, J = 20.8 Hz, CF-C), 113.0, 109.1, 70.9, 68.2, 48.3, 44.3, 30.5, 22.7, 12.2, 9.7; HRMS (ESI): m / z calcd for C29H30FN4O4[M+H]+ 517.2251 found 517.2235.

[0091] (Z)-3-((3,5-dimethyl-4-((R)-tetrahydrofuran-3-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (109g): Following the procedure as described for compound 109a, 20 amine 112 (20 mg, 0.05 mmol) and (R)-tetrahydrofuran-3-carboxylic acid 113g (7 mg, 0.06 mmol) provided compound 109g as an orange solid (16 mg, 64%); 1H NMR (500 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.19 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.66 (dd, J = 8.1, 1.7 Hz, 1H), 7.63 (s, 1H), 7.43 (dd, J = 8.5, 5.5 Hz, 2H), 7.13 (t, J = 8.9 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.18 25 (p, J = 7.2 Hz, 1H), 3.95 (t, J = 8.2 Hz, 1H), 3.85 – 3.63 (m, 3H), 3.15 (h, J = 7.1 Hz, 1H), 2.17 (d, J = 7.3 Hz, 6H), 2.13 – 2.03 (m, 2H), 1.48 (d, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 172.5, 170.2, 166.4, 162.4(d, J = 241.4 Hz, CF-C), 141.7, 140.8, 132.4, 128.5(d, J = 8.1 Hz, CF-C), 128.0, 127.5, 126.1, 125.9, 124.9, 124.6, 122.3, 117.7, 115.3(d, J = 21.3 Hz, CF-C), 113.0, 109.1, 70.9, 68.2, 30 48.3, 44.4, 30.5, 22.7, 12.2, 9.7; HRMS (ESI): m / z calcd for C29H30FN4O4[M+H]+ 517.2251 found 517.2233.

[0092] (R,Z)-3-((4-(5,6-dihydro-2H-pyran-3-carboxamido)-3,5-dimethyl-1H- pyrrol-2-yl)methylene)-N-(1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (109h): Following the procedure as described for compound 109a, 35 amine 112 (20 mg, 0.05 mmol) and 5,6-dihydro-2H-pyran-3-carboxylic acid 113h (7 mg, 0.06 mmol) provided compound 5h as an amorphous brick red solid (14 mg, 56%);1H NMR (500 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.11 (s, 1H), 8.58 (d, 5870774-02 / / 1165.202WO1 J = 7.9 Hz, 1H), 8.19 (s, 1H), 7.90 – 7.56 (m, 2H), 7.43 (dd, J = 8.4, 5.5 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 7.06 – 6.79 (m, 2H), 5.17 (p, J = 7.4 Hz, 1H), 4.39 – 4.15 (m, 2H), 3.69 (t, J = 5.5 Hz, 2H), 2.32 – 2.21 (m, 2H), 2.17 (d, J = 7.6 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 170.2, 166.4, 165.1, 5 161.4(d, J = 241.8 Hz, CF-C), 141.8, 140.8, 133.3, 132.9, 130.8, 128.5(d, J = 7.8 Hz, CF-C), 128.2, 128.0, 126.2, 125.9, 125.0, 124.7, 122.1, 117.8, 115.3(d, J = 21.0 Hz, CF-C), 113.0, 109.1, 64.8, 63.3, 48.3, 25.2, 22.8, 12.3, 9.8; HRMS (ESI): m / z calcd for C30H30FN4O4[M+H]+ 529.2251 found 529.2240.

[0093] (R,Z)-3-((3,5-dimethyl-4-(tetrahydro-2H-pyran-4-carboxamido)-1H-10 pyrrol-2-yl)methylene)-N-(1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (109i): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and tetrahydro-2H-pyran-4-carboxylic acid 113i (7.5 mg, 0.06 mmol) provided compound 109i as an orange solid (21 mg, 84%); 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 9.02 (s, 1H), 8.57 (d, J = 7.9 Hz, 15 1H), 8.17 (s, 1H), 7.81 – 7.56 (m, 2H), 7.48 – 7.35 (m, 2H), 7.12 (t, J = 8.9 Hz, 2H), 6.90 (d, J = 8.1 Hz, 1H), 5.16 (p, J = 7.0 Hz, 1H), 4.09 – 3.73 (m, 2H), 2.57 (dq, J = 10.2, 5.4 Hz, 1H), 2.16 (m, 7H), 1.83 – 1.55 (m, 4H), 1.47 (d, J = 7.0 Hz, 3H), 1.28 – 1.18 (m, 1H); 13C NMR (126 MHz, DMSO-d6) δ 173.9, 170.2, 166.4,161.4(d, J = 242.3 Hz, CF-C), 141.7, 140.7, 132.5, 128.5(d, J = 7.9 Hz, CF-C), 127.9, 20 127.7, 126.1, 125.9, 124.9, 124.6, 122.4, 117.7, 115.3(d, J = 21.6 Hz, CF-C), 112.9, 109.1, 66.9, 48.3, 41.3, 29.7, 22.7, 12.1, 9.7;HRMS (ESI): m / z calcd for C30H32FN4O4[M+H]+ 531.2407 found 531.2398.

[0094] (Z)-3-((3,5-dimethyl-4-((S)-5-oxotetrahydrofuran-2-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- 25 carboxamide (109j): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and (S)-5-oxotetrahydrofuran-2-carboxylic acid 113j (7.5 mg, 0.06 mmol) provided compound 109j as a golden yellow solid (18 mg, 72%);1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.50 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.85 – 7.54 (m, 2H), 7.51 – 7.34 (m, 30 2H), 7.27 – 7.07 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.1 Hz, 1H), 5.12 – 5.01 (m, 1H), 2.70 – 2.52 (m, 3H), 2.33 – 2.20 (m, 1H), 2.18 (d, J = 7.4 Hz, 6H), 1.47 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 177.4, 170.2, 169.3, 166.4, 161.4(d, J = 241.8 Hz, CF-C), 141.7, 140.8, 132.3, 128.5(d, J = 7.7 Hz, CF-C), 128.0, 127.5, 126.2, 125.8, 124.9, 124.7, 121.1, 117.9, 115.3(d, J = 20.6 Hz, 35 CF-C), 113.4, 109.1, 77.3, 48.3, 27.6, 26.6, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C29H28FN4O5[M+H]+ 531.2043 found 531.2024. 5970774-02 / / 1165.202WO1

[0095] (Z)-3-((3,5-dimethyl-4-((R)-5-oxotetrahydrofuran-2-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (109k): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and (R)-5-oxotetrahydrofuran-2-carboxylic acid 5 113k (7.5 mg, 0.06 mmol) provided compound 109k as a golden yellow solid (17 mg, 68%);1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.50 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.82 – 7.56 (m, 2H), 7.53 – 7.33 (m, 2H), 7.26 – 7.05 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.1 Hz, 1H), 5.06 (dd, J = 7.6, 5.4 Hz, 1H), 2.55 (h, J = 3.9 Hz, 3H), 2.29 – 2.20 (m, 1H), 2.18 (d, J 10 = 7.4 Hz, 6H), 1.47 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 177.4, 170.2, 169.3, 166.4, 161.4(d, J = 241.5 Hz, CF-C), 141.7, 140.8, 132.3, 128.5(d, J = 8.1 Hz, CF-C), 128.0, 127.5, 126.2, 125.8, 124.9, 124.7, 121.1, 117.9, 115.3(d, J = 21.0 Hz, CF-C), 113.4, 109.1, 77.3, 48.3, 27.6, 26.6, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C29H28FN4O5[M+H]+ 531.2043 found 531.2031. 15

[0096] (R,Z)-N-(5-((5-((1-(4-fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3- ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)benzo[d]oxazole-6- carboxamide (109l): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and benzo[d]oxazole-6-carboxylic acid 113l (9.3 mg, 0.06 mmol) provided compound 5l as a dark red solid (14 mg, 51%);1H NMR 20 (400 MHz, DMSO-d6) δ 11.06 (s, 1H), 9.79 (s, 1H), 8.90 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.39 (d, J = 1.6 Hz, 1H), 8.20 (d, J = 1.7 Hz, 1H), 8.06 (dd, J = 8.4, 1.6 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.72 – 7.61 (m, 2H), 7.50 – 7.30 (m, 2H), 7.24 – 7.08 (m, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 2.25 (d, J = 6.9 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 170.2, 166.4, 25 165.3, 161.4(d, J = 241.8 Hz, CF-C), 156.8, 149.6, 142.7, 141.7, 140.8, 132.9, 132.4, 128.6, 128.5(d, J = 7.9 Hz, CF-C), 128.0, 126.2, 125.9, 125.0, 125.0, 124.7, 122.3, 120.3, 117.8, 115.3(d, J = 21.4 Hz, CF-C), 114.3, 113.2, 111.1, 109.1, 48.3, 22.7, 12.3, 9.9;HRMS (ESI): m / z calcd for C32H27FN5O4[M+H]+ 564.2047 found 564.2041. 30

[0097] (R,Z)-N-(5-((5-((1-(4-fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3- ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-methyl-1H- benzo[d]imidazole-4-carboxamide (109m): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and 2-methyl-1H- benzo[d]imidazole-4-carboxylic acid 113m (10 mg, 0.06 mmol) provided 35 compound 5m as a red solid (12 mg, 44%);1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 11.07 (s, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.72 – 7.56 (m, 4H), 7.42 (dd, J = 8.4, 5.5 Hz, 3H), 7.30 (t, J = 7.8 6070774-02 / / 1165.202WO1 Hz, 1H), 7.13 (t, J = 8.9 Hz, 3H), 6.92 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.0 Hz, 1H), 2.61 (s, 3H), 2.29 (d, J = 7.3 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 170.2, 166.4, 164.1, 161.4(d, J = 244.4 Hz, CF-C), 153.4, 141.7, 141.5, 140.8, 135.1, 132.1, 128.5(d, J = 7.9 Hz, CF-C), 128.0, 126.9, 126.2, 125.9, 5 125.1, 124.7, 122.6, 122.4, 122.0, 121.5, 117.8, 115.3(d, J = 21.1 Hz, CF-C), 113.3, 109.1, 48.3, 22.7, 15.1, 12.6, 9.9; HRMS (ESI): m / z calcd for C33H30FN6O3[M+H]+ 577.2363 found 557.2354.

[0098] ((R,Z)-N-(5-((5-((1-(4-fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3- ylidene)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)-2-methylimidazo[1,2- 10 a]pyridine-3-carboxamide (109n): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and 2-methylimidazo[1,2- a]pyridine-3-carboxylic acid 113n (10 mg, 0.06 mmol) provided compound 109n as an orange solid (14 mg, 51%);1H NMR (500 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.09 (s, 1H), 8.95 (d, J = 7.1 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.22 (s, 1H), 7.83 15 – 7.65 (m, 2H), 7.60 (d, J = 9.0 Hz, 1H), 7.53 – 7.37 (m, 4H), 7.13 (t, J = 8.7 Hz, 3H), 7.04 (d, J = 7.0 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.18 (m,1H), 2.71 (s, 3H), 2.30 (d, J = 12.0 Hz, 6H), 1.49 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO- d6) δ 170.2, 166.4, 161.4(d, J = 242.8 Hz, CF-C), 146.0, 145.5, 141.7, 140.8, 133.0, 128.5(d, J = 7.8 Hz, CF-C), 128.2, 128.0, 127.6, 127.3, 126.2, 125.9, 125.1, 20 124.7, 121.8, 117.9, 116.6, 115.3(d, J = 21.0 Hz, CF-C), 113.6, 113.3, 109.1, 48.3, 22.7, 16.2, 12.4, 9.9; HRMS (ESI): m / z calcd for C33H30FN6O3[M+H]+ 577.2363 found 557.2353.

[0099] (R,Z)-3-((4-(6-fluoronicotinamido)-3,5-dimethyl-1H-pyrrol-2- yl)methylene)-N-(1-(4-fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide 25 (109o): Following the procedure as described for compound 109a, amine 112 (20 mg, 0.05 mmol) and 6-fluoronicotinic acid 113o (8 mg, 0.06 mmol) provided compound 109o as a brick red solid (13 mg, 50%);1H NMR (500 MHz, DMSO- d6) δ 11.07 (s, 1H), 9.87 (s, 1H), 8.84 (d, J = 2.5 Hz, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.51 (td, J = 8.2, 2.5 Hz, 1H), 8.21 (s, 1H), 7.67 (d, J = 7.8 Hz, 2H), 7.43 (dd, 30 J = 8.5, 5.5 Hz, 2H), 7.36 (dd, J = 8.5, 2.6 Hz, 1H), 7.13 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.18 (p, J = 7.2 Hz, 1H), 2.25 (d, J = 9.2 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO-d6) δ 171.7, 170.2, 166.4, 163.9, 163.4, 161.4(d, J = 241.2 Hz, CF-C), 148.2, 148.1, 142.2(d, J = 8.8 Hz, CF-C), 141.7, 140.8, 132.6, 129.1, 128.5(d, J = 8.0 Hz, CF-C), 128.0, 127.9, 126.2, 125.9, 125.0, 124.7, 35 121.7, 117.9, 115.3(d, J = 21.0 Hz, CF-C), 113.4, 110.2, 109.9, 109.1, 48.3, 22.7, 12.3, 9.8; HRMS (ESI): m / z calcd for C30H26F2N5O3[M+H]+ 542.2003 found 542.1996. 6170774-02 / / 1165.202WO1 Example 3 Syntheses of GRK Inhibitors 114a-v

[0100] The synthesis of various N-cyclic amine carboxamide derived GRK5 / GRK6 inhibitors are shown in Scheme 4. Pyrroloindoline amine derivative 5 115 was prepared from the known indoline derivative 116 as described by us previously. Selected optically active carboxylic acids 117a-o with N-cyclic amine functionalities are available commercially. Coupling these carboxylic acids with amine 115 using HATU in DME in the presence of excess of DIPEA at 0 °C to 23 °C for 12 h afforded carbonamide derivatives in good to excellent yields (52- 10 75%). As shown in Scheme 5 for the synthesis of amine derivatives, the corresponding Boc-derivatives 114c, 114d, 114i, 114o, 114p, and 114s were treated with trifluoroacetic acid in CH2Cl2at 0 °C to 23 °C for 2 h to provide inhibitors 114g, 114h, 114j, 114q, 114r, 114u, and 114v in excellent yield (81- 92%). Full structures of these inhibitors are shown in Table 4. 6270774-02 / / 1165.202WO1Scheme 4. Synthesis of GRK inhibitors containing N-cyclic carboxamide derivatives 6370774-02 / / 1165.202WO1Scheme 5. Synthesis of GRK inhibitors containing N-cyclic amine and N-cyclic acetamide derivatives 5 Structure-Activity Relationship Studies

[0101] The inhibitory potency of synthetic compounds containing the N- heterocyclic amide functionalities was evaluated against human GRK2, GRK5 and GRK6 using an in vitro assay, as reported previously. The structure and activity of these inhibitors are shown in Table 4. Compound 114a with (R)- 10 pyrrolidone functionality showed very potent GRK5 inhibitory activity with IC50value of 27 nM. In comparison, compound 114b with (S)-pyrrolidone functionality exhibited 30-fold reduction of GRK5 activity, displaying GRK5 IC50of 800 nM. Interestingly, both 114a and 114b showed very potent GRK6 activity (IC50values 20 nM and 28 nM), showing no stereochemical preference by the GRK6 binding 15 site. Both compounds showed only 3-fold difference in their activity against GRK2. However, compound 114a exhibited 26-fold selectivity over GRK2. The effect of (R)- and (S)-Boc pyrrolidine carboxamide derivatives was then investiaged. Compound 114d with (S)-Boc substituent exhibited over 100-fold enhanced GRK5 inhibitory activity over the (R)-Boc derivative 114c. Interestingly, 20 the corresponding N-acetyl derivatives 114e and 114f showed very potent inhibitory IC50values against GRK5 and GRK6, but selectivity among the GRKs was marginal. The removal of Boc groups from 114c and 114d resulted in pyrrolidine derivatives 114g and 114h with (S)- and (R)- configuration. Both 6470774-02 / / 1165.202WO1 compounds showed very good inhibitory activity against GRKs but likewise displayed little to no selectivity. These improvements of GRK5 IC50values over their Boc-derivatives (114c and 114d) indicated the bulky Boc group may not be able to fit as well in the catalytic site of these GRKs. On the other hand, sterically 5 less demanding acetamide derivatives 114e and 114f were well accommodated consistent with their very good activity against GRK5, GRK6, and GRK2, but only with marginal selectivity against GRK2. The (S)-Boc-2,5-dihydro-pyrrole derivative 114i exhibited sub-micromolar GRK5 and GRK6 IC50values and exhibited 130-fold selectivity against GRK2. The removal of Boc group resulted 10 in compound 114j which exhibited GRK5 and GRK6 IC50values of 30 nM and GRK2 activity of 480 nM, or 15-fold selectivity. The effect of 3(S)- and 3(R)-Boc- pyrrolidine derivatives was also examined. These derivatives 114k and 114i showed GRK5 and GRK6 inhibitory IC50values in low micromolar range. Interestingly, these compounds showed little to no activity against GRK2, and 15 thus compound 114i showed excellent selectivity against GRK2.

[0102] The effect of a slightly larger ring cycle was also evaluated on whether it would interact with residues in the AST region. Compounds 114m and 114n with 2(S)- and 2(R)-Boc-piperidine core did not significantly improve activity against GRK5 and GRK6 compared to the corresponding pyrolidine derivatives 20 114c and 114d except 2(R)-isomer 114m showed GRK inhibitory activity IC50value of 900 nM with over 340-fold selectivity against GRK2. The corresponding 3(S)- and 3(R)-Boc- piperidine derivatives 114o and 114p improved activity against GRK5, showing IC50values of 90 and 410 nM, respectively. Particularly, 3(S)-isomer 114o showed stereochemical preference over 3(R)-isomer 114p, 25 showing GRK5 IC50value of 90 nM and 64-fold selectivity against GRK2. The selectivity over GRK6 however, was marginal. Compound 114p with 3(R)- configuration showed comparable GRK5 and GRK6 IC50values of 410 nM and 340 nM, respectively but exhibited excellent selectivity against GRK2, over 1500- fold. The removal of the Boc-group resulted in compounds 114q and 114r 30 showing GRK5 IC50value of 12 and 47 nM, respectively. These compounds displayed potent GRK5 inhibitory activity as well, but their selectivity against GRK2 was marginal. The effect of Boc-piperazine derivatives 114s and 114t with the incorporation of an additional ring nitrogen was then investigated. Compound 114s with 2(R)-configuration displayed IC50values of 81 and 60 nM against GRK535 and GRK6, respectively, over 5-fold improvement over the corresponding 3(R)- piperidine derivative 114p. Compound 114s with a basic amine showed over 5- fold improvement of GRK5 and GRK6 activity over compound 114p in which an 6570774-02 / / 1165.202WO1 NH group of 114s is replaced by a CH2 group. The results indicates that the NH group of 114s may be involved in hydrogen bonding interactions with GRK5. Compound 114t, with 2-(S)-configuration is nearly 10-fold less active than the compound 114s, however it is highly selective, showing over 1600-fold selectivity 5 against GRK2. Interestingly, compound 114t with a basic amine is less potent than compound 114o in which an NH group of 114t is replaced by a CH2 group with identical stereochemistry, indicating that the ring stereochemistry may have prevented hydrogen bonding interactions observed for compound 114s. Similar to what has been observed for other de-Boc derivatives, the resulting compounds 10 114u and 114v after removal of Boc-group exhibited improved potency against GRK5 and GRK6, but significantly reduced selectivity against GRK2. In essence, de-Boc derivatives containing piperidine and piperazine heterocycles (114q, 114r, 114u and 114v) irrespective of the ring stereochemistry or number of basic amines on the ring, show similar affinity to GRK5 or GRK6. Also, these de-Boc 15 derivatives are significantly less selective againsit GRK2 compared to their Boc counterparts (114o, 114p, 114s, and 114t). The six-membered heterocyclic ring, irrespective of ring stereochemistry, are suitably accommodated in the hydrophobic pocket adjacent to the AST loop. The X-ray structural studies will shed light into these important ligand-binding site interactions. 20 66]by[ti6 viK t R c0 5 4eG l / 1 3 3 0.e5 0SK R G .sK R Gybnoitalyrohpsohpnilubuttsniagasrotibihnifo]ytiviO O O O tcaN H N HN O N O) ) )R)Sd R(S( ( (nO O O O aeH N H N H N H Nrut N Ha4b1N H 4c4d4O1N H N HO1O1cO1 1 1 1urt N H N H N H N HS. er4uetlcbuO O artO O sN H N H N H N H1 T r) oR) ) (R(R) (R(OtW i2 bi0h2. nIF F F F5611 / / 20-47707]by[ti6 viK t R ceG1l / 2 1 3e5SK ]yb[ti5 vitcel / e2S)M μ2(0C5I)M6μ(0C5I)M μ( 50C5]a[ IO O NHN HN e e ) ) R M N ) M )SR(((S(O O O O HN H NH N H NN He4 N Hf41N Hg h4N H 41O11O1O11O1N H N HN H N HerutcurO O t O O NH1sN H N H N Hr) ) ) )Ro R(R(R( (OtW i2 bi0h2. nIF F F F5611 / / 20-47707]by[ti6 viK t R ceG2l / 1 1e5SK ]yb[tivitceleS)M μ(0C5I)M μ(0C5I.09)6M μ( 55.K0 ±9.5±4000 R320 09.0.±0 1]Ca[ IG .0.2N H)OOOS(O N NO) )S( H NR(O Oj4H N N HO11 H NkiN H 4N H 41O11O1)Z( N H) )N HeNZ(rZ( HutcuO rtON HO NH)N H1sr )RR() (R(OotW i2 bi0h2. nIF F F5611 / / 20-47707]by[ti6 viK t R c2. 7eG / 1 0 3 1le5SK ]yb[tivitceleS)M μ(0C5I)M μ(0C5I)M μ(0C5]a[ IOOOOOO ON N )S))N N O O N(R(R()SOOO O () OS(O HNe HH N N H NM lmH NN H 4 4m4n4O11 N N H HO O1111 N HO11 N HoO41e1) ))N HM Ze( N HZ(Z( N H)Z( N H)Z( N HrutcurtOO OO O sN H N N H H)N H)) eN H1 roR(R(R) (R() MR(OtW i2 bi0h2. nIFF FF F5611 / / 20-47707]by[ti6 viK t R ceG1 2 1 1l / e5SK ]yb[ti5 vitcel / e2S)M μ2(0C5I)M6μ(0C5I)M μ( 50C5]a[ IO OH N NO ON ) )S R)( (N R(O O e O e e MHM )R(H NN O HNMNqrH e 4N H 41H NM NHp4 N H1O O11e O1esMN H 4e1M1M )N He O1) )ZN HZ(M eZ( N H ()Z( N HrutcurtO O O N O sN H N HeHe ) e ) )RN H1 rOoMR(MR(M(e t M )R(W i2 bi0h2. nIF F F F5611 / / 20-47707]by[ti6 viK t R ceG7 3.1l / 0e5SK ]yb[tivitcel / eS)M μ(0C5I)M μ(0 R5G 9C5 0 0.620I.0 0.02 )5 7M μ1 10 0(5.0K0.00.± 0±00]C5R a[ IG6.4±010.6010.0O ON )S(H N O N H e H N HNM ) )S(Rt(N O e NH 4N O H H e M O11MH NeH NMN HOv)N H N Hue6Ze(e O6M r M ) )N HZ( N HutZ(curtO1sr eN HO O ) MRN(eN H) eH) OotMR(MR(W i2 bi0h2. nIF F F5611 / / 20-4770770774-02 / / 1165.202WO1

[0103] In Table 4,[a]IC50values for GRK5, GRK6, and GRK2 phosphorylation of the soluble substrate tubulin. Error bars are standard deviation from 3-4 independent experiments.[b]GRK2 / GRK5 indicates fold-selectivity for GRK5 over GRK2, and GRK5 / GRK6 the fold selectivity for GRK6 over GRK5. 5[c]This compound exhibited non-sigmoidal behavior with GRK6, suggesting that other inhibitory processes were occurring other than equilibrium binding. X-Ray Structure of Compound 114t in Complex with GRK5

[0104] Because wild-type human GRK5 expressed in E. coli undergoes heterogeneous hyperphosphorylation, a catalytically inactive mutant GRK5D11N10 which retains stability of protein, was used for co-crystallization. For X-ray structural studies, inhibitor 114t was soaked into crystals of GRK5D11Ngrown in the presence of sangivamycin (sgv) that were harvested under low salt conditions. The 2.78 Å crystal structure of 114t-bound GRK5 exhibits an ‘open’ conformation with the angle of opening between the kinase small lobe and the 15 kinase large lobe ~11° more than the structure of sgv-bound GRK5 (PDB:6PJX). Inhibitor 114t binds to the catalytic site of GRK5. The overall structure is consistent with other inhibitor-bound GRK5 structures where inhibitor 114t occupies the ATP binding site. The indoline heterocyclic core binds in the adenine subsite forming two strong hydrogen bonds with the GRK5 hinge backbone amide 20 NHs of Thr264 and Met266. The indoline ring occupies the hydrophobic pocket formed by Val247, Leu318 and Ser328.

[0105] The indoline amide carbonyl forms a hydrogen bond with the Lys215 side chain. The (R)-fluorophenylmethyl side chain of 114t nestles in the polyphosphate subsite, a hydrophobic pocket formed by Lys215, Glu199, Val200 25 and Len 217 under the P loop. The para-fluorine atom forms Van der Waals interactions with the backbone atoms of Gly198 and Glu199. The (3S)-Boc- piperazine carboxamide packed the hydrophobic pocket adjacent to the AST loop. There is no hydrogen bond interaction with the Cys474 in the AST loop. The density of this ligand core is not fully resolved possibly due to the bulky t-butyl 30 group extending out of the binding pocket. It appears that the tert-butyl group is exposed to the solvent.

[0106] In this series it was shown that the position of Boc-group and the 6-membered ring size are important for selectivity. To understand the origin of this selectivity, the X-ray structure of 114t-bound GRK5 and a docked model of 35 compound 114t with GRK2 was compared, either with its kinase domain relatively closed (PDB entry 8JPB) or open (PDB entry 5UKM). The Boc-piperazine heterocycle can be accommodated by GRK5, but the ligand would have a steric 7370774-02 / / 1165.202WO1 clash with the GRK2 AST in either representative structure. The removal of the bulky Boc groups in compounds 114o, 114p, 114s, and 114t provided de-Boc derivatives 114q, 114r, 114u, and 114v with better potency against GRK5, GRK6 and GRK2, but with significantly lower selectivity against GRK2, consistent with 5 the clash seen between the bulky Boc-group and the GRK2 AST region. Several compounds in this series were considerably more potent against GRK6 than GRK5 (e.g. 114c), possibly because the AST region of GRK6 adopts distinct configuration (PDB entry 2ACX) where its Cys474 and Tyr473 are ~15 Å away from the position of these GRK5 residues in the current X-ray structure. 10 Conclusion

[0107] GRK5 is a potential target for the treatment of heart failure because of its involvement as a regulator of pathological cardiac hypertrophy. GRK6, a close homolog of GRK5, is a promising target for human multiple myeloma. In this study, a series of very potent GRK5 inhibitors were designed, 15 including compounds 114s and 114t that exhibited high selectivity against GRK2. These inhibitors have N-heterocyclic carboxamide functionalities on a pyrroloindoline basic scaffold. A number of potent and relatively selective GRK6 inhibitors were also identified, including 114b and 114n that showed high selectivity against GRK2 and good selectivity against GRK5. Compounds 114s 20 and 114t also showed very potent GRK6 inhibitory activity and selectivity against GRK2. However, their selectivity against GRK5 was limited. Interestingly, the corresponding compounds 114u and 114v, lacking a bulky Boc-group, showed very potent GRK5, GRK6 inhibitory activity, but their selectivity against GRK2 was marginal. To assess the origin of potency and selectivity, inhibitor 114t-bound 25 GRK5 was co-crystallized and the X-ray structure at 3.7 Å resolution was determined. The X-ray structure provided insights into the ligand-binding site interactions responsible for potency and selectivity against GRK2. Among compounds examined, the piperazine carboxamide with the tert-butyloxycarbonyl group appears to fill in the hydrophobic pocket under the AST loop however, this 30 bulky alkyl group would impose steric clashes with the AST loop of GRK2 and raise selectivity against GRK2. Both stereoisomers 114u and 114v without the Boc-group, exhibited very potent GRK5 and GRK6 activity but little selectivity against GRK2. Experimental section 35 General

[0108] All reactions were carried out under an argon atmosphere in either flame or oven-dried (120oC) glassware. All reagents and chemicals were 7470774-02 / / 1165.202WO1 purchased from commercial suppliers and used without further purification unless otherwise noted. Anhydrous solvents were obtained as follows: dichloromethane and diisopropylamine (DIPA) were distilled over calcium hydride. All purification procedures were carried out with reagent grade solvents (purchased form VWR) 5 in air. TLC analysis was conducted using glass-backed Thin-Layer Silica Gel Chromatography Plates (60 Å, 250 µm thickness, F-254 indicator). Column chromatography was performed using 230-400 mesh, 60 Å pore diameter silica gel.1H,13C NMR spectra were recorded at room temperature on a Bruker AV800, DRX-500, ARX-400. Chemical shifts (δ values) are reported in parts per million, 10 and are referenced to the deuterated residual solvent peak. NMR data is reported as: δ value (chemical shift, J-value (Hz), integration, where s = singlet, d = doublet, t = triplet, q = quartet, brs = broad singlet). LRMS and HRMS spectra were recorded at the Purdue University Department of Chemistry Mass Spectrometry Center. 15 (Z)-3-((3,5-Dimethyl-4-((S)-5-oxopyrrolidine-2-carboxamido)-1H-pyrrol-2- yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide (114a): To a stirred solution of amine 115 (20 mg, 0.05 mmol) in dry DMF (1.5 mL), commercially available (S)-5-oxopyrrolidine-2-carboxylic acid 117a (8 mg, 0.06 mmol), HATU (21.8 mg, 0.06 mmol) and DIPEA (42 μL, 0.24 mmol) were 20 added under inert atmosphere at 0oC. The resulting mixture was stirred at 0oC to 23oC for 12 h. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude mixture was diluted with water and extracted with CH2Cl2(3 times). The combined organic layer was dried over NaSO4and concentrated under reduced pressure. The resulting crude mixture 25 was purified by column chromatography (2-5% MeOH in CH2Cl2) to yield compound 114a as an amorphous orange solid (16 mg, 62%). ):1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.27 (s, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.91 (s, 1H), 7.68 – 7.60 (m, 2H), 7.46 – 7.39 (m, 2H), 7.16 – 7.09 (m, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.16 (p, J = 7.3 Hz, 1H), 4.20 (dd, J = 8.7, 4.5 30 Hz, 1H), 2.44 – 2.32 (m, 1H), 2.27 – 2.09 (m, 8H), 2.00 (ddt, J = 15.0, 9.7, 5.2 Hz, 1H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO) δ 178.0, 172.4, 170.2, 166.4, 141.7, 140.8, 132.4, 128.5 (d, J = 8.0 Hz, CF-C), 127.9, 127.6, 126.2, 125.9, 124.9, 124.7, 121.7, 117.7, 115.3 (d, J = 20.9 Hz, CF-C), 113.1, 109.1, 56.3, 48.3, 29.8, 26.2, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C29H29FN5O4[M+H]+ 35 530.2204 found 530.2186.

[0109] (Z)-3-((3,5-Dimethyl-4-((R)-5-oxopyrrolidine-2-carboxamido)- 1H-pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- 7570774-02 / / 1165.202WO1 carboxamide (114b): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (R)-5-oxopyrrolidine-2-carboxylic acid 9b (8 mg, 0.06 mmol) provided compound 114b as an orange solid (16 mg, 65%); Rf= 0.20 (MeOH : CH2Cl2= 1:9);1H NMR (400 MHz, DMSO) δ 11.08 (s, 1H), 9.31 (s, 5 1H), 8.63 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 1.7 Hz, 1H), 7.94 (s, 1H), 7.72 – 7.57 (m, 2H), 7.42 (ddd, J = 10.1, 6.1, 3.3 Hz, 2H), 7.28 – 7.05 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.16 (p, J = 7.1 Hz, 1H), 4.19 (dd, J = 8.7, 4.5 Hz, 1H), 2.45 – 2.30 (m, 1H), 2.30 – 2.08 (m, 8H), 1.99 (ddt, J = 14.0, 9.7, 5.1 Hz, 1H), 1.47 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO) δ 177.8, 172.3, 170.2, 166.3, 161.4 (d, J = 10 241.8 Hz, CF-C), 141.7, 140.8, 132.4, 128.7, 128.5 (d, J = 8.0 Hz, CF-C), 128.0, 127.6, 126.20, 125.9, 124.9, 124.7, 121.8, 120.5, 117.8, 115.3 (d, J = 21.0 Hz, CF-C), 113.2, 109.1, 56.3, 48.3, 29.8, 26.3, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C29H29FN5O4[M+H]+530.2204 found 530.2195.

[0110] tert-Butyl(R)-2-((5-(((Z)-5-(((R)-1-(4-15 fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)pyrrolidine-1-carboxylate(114c): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (tert-butoxycarbonyl)-D-proline 117c (13 mg, 0.06 mmol) provided compound 114c as a brick red solid (17 mg, 58%); Rf= 0.50 (MeOH : 20 CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.15 (s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H), 7.83 – 7.58 (m, 2H), 7.53 – 7.35 (m, 2H), 7.13 (t, J = 8.9 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 4.23 (ddd, J = 18.7, 8.5, 3.1 Hz, 1H), 3.43 (t, J = 9.0 Hz, 1H), 2.29 – 2.15 (m, 7H), 1.85 (dq, J = 30.0, 7.4 Hz, 3H), 1.48 (d, J = 7.1 Hz, 3H), 1.39 (d, J = 15.9 Hz, 9H);13C NMR 25 (126 MHz, DMSO) δ 172.2, 172.1, 170.1, 166.4, 161.4 (d, J = 241.7 Hz, CF-C), 154.1, 153.8, 141.7, 140.7, 132.7, 132.3, 128.5 (d, J = 7.9 Hz, CF-C), 128.0, 127.8, 127.5, 126.1, 125.9, 124.9, 124.6, 122.2, 122.1, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.0, 112.9, 109.1, 79.1, 79.0, 60.2, 48.3, 47.1, 47.0, 32.2, 30.8, 28.6, 24.5, 23.6, 22.7, 12.2, 12.0, 9.7, 9.6; HRMS (ESI): m / z calcd for C34H39FN5O5[M+H]+30 616.2935 found 616.2921.

[0111] tert-Butyl(S)-2-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)pyrrolidine-1-carboxylate(114d): Following the procedure as described for compound 114a, amine 115 (20 mg, 35 0.05 mmol) and (tert-butoxycarbonyl)-L-proline 117d (13 mg, 0.06 mmol) provided compound 114d as a red solid (21 mg, 72%); Rf= 0.5 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.05 (s, 1H), 9.13 (d, J = 34.0 Hz, 1H), 7670774-02 / / 1165.202WO1 8.59 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H), 7.93 – 7.51 (m, 2H), 7.54 – 7.34 (m, 2H), 7.13 (dd, J = 10.1, 7.7 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.16 (h, J = 7.0 Hz, 1H), 4.23 (ddd, J = 18.9, 8.4, 3.1 Hz, 1H), 3.43 (t, J = 8.9 Hz, 1H), 2.33 – 2.13 (m, 7H), 1.97 – 1.74 (m, 4H), 1.48 (d, J = 7.1 Hz, 3H), 1.39 (d, J = 16.1 Hz, 9H);13C NMR 5 (126 MHz, DMSO) δ 172.2, 172.1, 170.1, 166.4, 161.4 (d, J = 241.8 Hz, CF-C), 154.1, 153.8, 141.7, 140.7, 132.7, 132.3, 128.3 (d, J = 8.1 Hz, CF-C), 128.0, 127.8, 127.5, 126.1, 125.9, 124.9, 124.6, 122.2, 122.1, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.0, 112.9, 109.1, 79.1, 79.0, 60.2, 48.3, 47.1, 47.0, 32.2, 30.8, 28.6, 24.5, 23.6, 22.7, 12.2, 12.0, 9.7, 9.6; HRMS (ESI): m / z calcd for C34H39FN5O5[M+H]+10 616.2935 found 616.2914.

[0112] (Z)-3-((4-((R)-1-Acetylpyrrolidine-2-carboxamido)-3,5- dimethyl-1H-pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2- oxoindoline-5-carboxamide (114e): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and acetyl-D-proline 117e (9 15 mg, 0.06 mmol) provided compound 114e as an golden yellow solid (14 mg, 52%); Rf= 0.30 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.02 (s, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.18 (dd, J = 5.4, 1.6 Hz, 1H), 7.97 – 7.55 (m, 2H), 7.55 – 7.36 (m, 2H), 7.31 – 7.08 (m, 2H), 6.91 (dd, J = 8.2, 1.8 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 4.42 (ddd, J = 77.5, 8.5, 3.0 Hz, 1H), 3.82 – 3.59 20 (m, 1H), 3.48 (dq, J = 11.9, 7.4 Hz, 1H), 3.44 – 3.33 (m, 1H), 2.15 (d, J = 5.1 Hz, 6H), 2.00 (s, 3H), 1.96 – 1.77 (m, 3H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO) δ 171.9, 171.8, 170.2, 169.1, 166.4, 160.5, 141.7, 132.9, 128.5 (d, J = 7.9 Hz, CF-C), 128.0, 126.1, 125.9, 124.9, 124.7, 122.2, 117.9, 117.7, 115.3 (d, J = 21.1 Hz, CF-C), 112.9, 109.1, 61.0, 60.0, 48.3, 48.0, 46.8, 32.8, 30.5, 24.9, 25 23.1, 22.9, 22.7, 22.6, 12.0, 9.6; HRMS (ESI): m / z calcd for C31H33FN5O4[M+H]+558.2517 found 558.2495.

[0113] ((Z)-3-((4-((S)-1-Acetylpyrrolidine-2-carboxamido)-3,5- dimethyl-1H-pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2- oxoindoline-5-carboxamide (114f): Following the procedure as described for 30 compound 114a, amine 115 (20 mg, 0.05 mmol) and acetyl-L-proline 117f (9 mg, 0.06 mmol) provided compound 114f as golden yellow solid (15 mg, 58%); Rf= 0.30 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.02 (s, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.18 (dd, J = 5.4, 1.7 Hz, 1H), 7.83 – 7.56 (m, 2H), 7.55 – 7.34 (m, 2H), 7.13 (t, J = 8.9 Hz, 2H), 6.91 (dd, J = 8.1, 1.9 Hz, 1H), 5.17 35 (p, J = 7.2 Hz, 1H), 4.49 – 4.22 (m, 1H), 3.62 (td, J = 8.6, 4.2 Hz, 1H), 3.52 – 3.34 (m, 2H), 2.25 – 2.06 (m, 6H), 2.00 (s, 3H), 1.95 – 1.81 (m, 3H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO) δ 171.8, 170.2, 169.1, 166.4, 161.4 (d, J = 7770774-02 / / 1165.202WO1 241.6 Hz, CF-C), 141.7, 140.7, 132.9, 128.5 (d, J = 8.2 Hz, CF-C), 127.9, 127.6, 126.1, 125.9, 124.9, 124.7, 122.1, 121.7, 117.9, 117.7, 115.3 (d, J = 21.1 Hz, CF-C), 112.9, 109.1, 61.0, 60.0, 48.3, 48.0, 32.8, 30.5, 24.9, 23.1, 22.9, 22.7, 22.6, 12.0, 9.6; HRMS (ESI): m / z calcd for C31H33FN5O4[M+H]+558.2517 found 5 558.2502.

[0114] (Z)-3-((3,5-Dimethyl-4-((R)-pyrrolidine-2-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (114g): To a stirred solution of 114c (15 mg, 0.024 mmol) in dry DCM (1.5 mL), added TFA (0.048 mmol) under inert atmosphere at 0oC. The 10 resulting mixture was stirred at 0oC to 23oC for 2 h. After completion of the reaction, the solvent was evaporated under reduced pressure and washed with diethyl ether several times. The solid was dried in vacuum to yield compound 114g as an amorphous brick red solid (12 mg, 92%).1H NMR (500 MHz, DMSO) δ 11.08 (s, 1H), 9.78 (s, 1H), 9.36 (s, 1H), 8.94 – 8.36 (m, 2H), 8.20 (s, 1H), 7.92 15 – 7.57 (m, 2H), 7.42 (dd, J = 8.5, 5.5 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.17 (t, J = 7.2 Hz, 1H), 4.38 (s, 1H), 3.26 (d, J = 13.7 Hz, 2H), 2.43 (t, J = 7.0 Hz, 1H), 2.20 (d, J = 12.4 Hz, 6H), 1.98 (ddt, J = 19.5, 12.9, 6.8 Hz, 3H), 1.48 (d, J = 7.0 Hz, 3H), 1.24 (q, J = 7.0 Hz, 1H); 13C NMR (126 MHz,DMSO) δ 170.2, 168.0, 166.3, 161.4 (d, J = 241.8 Hz, CF-C), 141.7, 140.9, 131.8, 20 128.5 (d, J = 8.1 Hz, CF-C), 128.1, 127.1, 126.3, 125.8, 125.0, 124.7, 120.7, 118.0, 115.3 (d, J = 21.1 Hz, CF-C), 113.8, 109.1, 59.8, 48.3, 46.1, 30.4, 24.0, 22.7, 12.1, 9.6; HRMS (ESI): m / z calcd for C29H31FN5O3[M+H]+516.2411 found 516.2402.

[0115] (Z)-3-((3,5-Dimethyl-4-((S)-pyrrolidine-2-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- 25 carboxamide (114h): Following the general procedure for compound 114g, compound 114d (15 mg, 0.024 mmol) provided compound 114h as a red solid (11 mg, 90%); Rf= 0.2 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 9.80 (s, 1H), 9.39 (d, J = 37.0 Hz, 1H), 8.61 (d, J = 8.0 Hz, 1H), 8.20 (s, 1H), 7.87 – 7.61 (m, 2H), 7.54 – 7.35 (m, 2H), 7.13 (t, J = 8.7 Hz, 2H), 30 6.92 (d, J = 8.1 Hz, 1H), 5.41 – 4.78 (m, 1H), 4.39 (t, J = 7.5 Hz, 1H), 3.33 – 3.20 (m, 2H), 2.44 (d, J = 12.3 Hz, 1H), 2.20 (d, J = 12.4 Hz, 6H), 1.98 (ddt, J = 19.5, 12.9, 6.9 Hz, 3H), 1.48 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO) δ 170.2, 168.0, 166.3, 161.4 (d, J = 241.8 Hz, CF-C), 141.7, 140.9, 131.8, 128.5 (d, J = 8.0 Hz, CF-C), 128.1, 127.1, 126.3, 125.8, 125.0, 124.7, 120.7, 118.0, 115.3 (d, J = 35 21.1 Hz, CF-C), 113.8, 109.1, 59.8, 48.3, 46.1, 30.4, 24.0, 22.7, 12.1, 9.6; HRMS (ESI): m / z calcd for C29H31FN5O3[M+H]+516.2411 found 516.2395. 7870774-02 / / 1165.202WO1

[0116] tert-Butyl(S)-2-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-yl- idene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)-2,5-dihydro-1H-pyrrole-1-carboxylate (114i): Following the procedure as described for compound 114a, amine 115 (20 5 mg, 0.05 mmol) and (S)-1-(tert-butoxycarbonyl)-2,5-dihydro-1H-pyrrole-2- carboxylic acid 117g (13 mg, 0.06 mmol) provided compound 114i as an amorphous an orange solid (21 mg, 74%); Rf= 0.45 (MeOH:CH2Cl2= 0.5:9.5); 1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.22 (d, J = 43.7 Hz, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (s, 1H), 7.88 – 7.58 (m, 2H), 7.51 – 7.33 (m, 2H), 7.25 – 7.07 10 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 6.04 (ddt, J = 12.2, 6.4, 3.2 Hz, 1H), 5.87 (ddd, J = 6.5, 3.9, 2.0 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 5.09 – 4.89 (m, 1H), 4.57 – 4.01 (m, 2H), 2.17 (dd, J = 10.8, 7.3 Hz, 6H), 1.48 (d, J = 7.0 Hz, 3H), 1.41 (d, J = 16.4 Hz, 9H);13C NMR (126 MHz, DMSO) δ 170.2, 169.4, 166.4, 161.4 (d, J = 242.3 Hz, CF-C), 153.6, 153.5, 141.7, 140.8, 132.7, 132.2, 129.1, 128.7, 128.5 (d, 15 J = 8.1 Hz, CF-C), 128.0, 127.4, 127.1, 126.6, 126.1, 125.9, 124.9, 124.6, 121.9, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.2, 113.0, 109.1, 79.4, 68.2, 68.1, 54.1, 48.3, 28.5, 28.5, 22.7, 12.2, 12.0, 9.7, 9.6; HRMS (ESI): m / z calcd for C34H37FN5O5 [M+H]+614.2779 found 614.2757.

[0117] (Z)-3-((4-((S)-2,5-Dihydro-1H-pyrrole-2-carboxamido)-3,5-20 dimethyl-1H-pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2- oxoindoline-5-carboxamide (114j): Following the general procedure for compound 114g, compound 114i (15 mg, 0.024 mmol) provided compound 114j as an orange solid (11 mg, 88%); Rf= 0.2 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 9.94 (s, 1H), 8.98 (s, 1H), 8.60 (d, J = 7.9 Hz, 1H), 25 8.19 (d, J = 1.7 Hz, 1H), 7.80 – 7.59 (m, 2H), 7.59 – 7.25 (m, 2H), 7.22 – 7.06 (m, 2H), 6.92 (d, J = 8.1 Hz, 1H), 6.11 (d, J = 2.2 Hz, 2H), 5.36 – 5.02 (m, 2H), 4.28 – 3.95 (m, 2H), 2.20 (d, J = 13.2 Hz, 6H), 1.47 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO) δ 170.2, 166.3, 166.0, 161.4 (d, J = 242.2 Hz, CF-C), 141.7, 140.9, 131.8, 128.5 (d, J = 8.1 Hz, CF-C), 128.0, 127.0, 126.3, 125.8, 125.8, 125.0, 124.6, 30 120.6, 118.0, 115.3 (d, J = 21.1 Hz, CF-C), 113.9, 109.2, 66.9, 53.1, 48., 22.73, 12.0, 9.6; HRMS (ESI): m / z calcd for C29H29FN5O3[M+H]+514.2254 found 514.2245.

[0118] tert-Butyl(R)-3-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- 35 dimethyl-1H-pyrrol-3-yl)carbamoyl)pyrrolidine-1-carboxylate(114k): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (R)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid 117h (13 7970774-02 / / 1165.202WO1 mg, 0.06 mmol) provided compound 114k as a brick red solid (19 mg, 65%); Rf= 0.53 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.07 (s, 1H), 9.32 (s, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.30 (s, 1H), 7.84 – 7.57 (m, 2H), 7.44 (dd, J = 8.4, 5.5 Hz, 2H), 7.12 (t, J = 8.8 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J 5 = 7.3 Hz, 1H), 3.51 (dd, J = 10.6, 7.9 Hz, 1H), 3.37 (m, 2H), 3.24 (m, 1H), 3.20 – 3.12 (m, 1H), 2.17 (d, J = 2.3 Hz, 6H), 2.12 (q, J = 5.5 Hz, 1H), 2.01 (d, J = 5.9 Hz, 1H), 1.49 (d, J = 7.1 Hz, 3H), 1.39 (s, 9H);13C NMR (126 MHz, DMSO) δ 170.2, 166.4, 161.4 (d, J = 241.8 Hz, CF-C), 153.9, 141.7, 140.7, 132.4, 128.5 (d, J = 8.0 Hz, CF-C), 127.9, 127.5, 126.1, 125.9, 124.9, 124.7, 122.1, 117.7, 115.3 10 (d, J = 21.1 Hz, CF-C), 113.0, 109.1, 78.8, 48.9, 48.3, 45.9, 45.7, 43.8, 42.9, 29.8, 29.1, 28.6, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C34H39FN5O5[M+H]+616.2935 found 616.2893.

[0119] tert-Butyl(S)-3-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- 15 dimethyl-1H-pyrrol-3-yl)carbamoyl)pyrrolidine-1-carboxylate(114l): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (S)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid 117i (13 mg, 0.06 mmol) provided compound 114l as a brick red solid (21 mg, 71%); Rf =0.53 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.22 20 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.30 – 8.08 (m, 1H), 7.75 – 7.60 (m, 2H), 7.52 – 7.37 (m, 2H), 7.27 – 7.09 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 3.52 (dd, J = 10.6, 7.9 Hz, 1H), 3.48 – 3.34 (m, 2H), 3.25 (t, J = 9.5 Hz, 1H), 3.13 (dt, J = 15.1, 7.5 Hz, 1H), 2.17 (d, J = 7.5 Hz, 6H), 2.10 (td, J = 7.4, 5.1 Hz, 1H), 2.05 – 1.96 (m, 1H), 1.48 (d, J = 7.1 Hz, 3H), 1.39 (s, 9H);13C NMR (126 25 MHz, DMSO) δ 171.9, 170.2, 166.4, 161.4 (d, J = 241.4 Hz, CF-C), 153.8, 141.7, 140.7, 132.4, 128.5 (d, J = 7.9 Hz, CF-C), 128.0, 127.5, 126.1, 125.9, 124.9, 124.6, 122.1, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.1, 109.1, 78.7, 48.9, 48.3, 45.9, 45.7, 43.8, 42.9, 29.8, 29.1, 28.6, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C34H39FN5O5[M+H]+616.2935 found 616.2927. 30

[0120] tert-Butyl(R)-2-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)piperidine-1-carboxylate(114m): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid 117j (14 35 mg, 0.06 mmol) provided compound 114m as a dark red solid (19 mg, 65%); Rf= 0.5 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.11 (s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.90 – 7.58 (m, 8070774-02 / / 1165.202WO1 2H), 7.53 – 7.34 (m, 2H), 7.13 (dd, J = 10.0, 7.8 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 4.69 (d, J = 25.4 Hz, 1H), 3.81 (d, J = 12.8 Hz, 1H), 3.16 (d, J = 51.9 Hz, 1H), 2.17 (d, J = 7.5 Hz, 6H), 1.62 (d, J = 10.4 Hz, 3H), 1.48 (d, J = 7.0 Hz, 3H), 1.39 (s, 9H), 1.34 – 1.17 (m, 2H);13C NMR (126 MHz, DMSO) δ 5 170.2, 166.4, 161.4 (d, J = 242.9 Hz, CF-C), 141.7, 140.8, 132.5, 128.5 (d, J = 8.1 Hz, CF-C), 128.0, 127.7, 126.1, 125.9, 124.9, 124.7, 122.2, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.1, 109.1, 79.2, 48.3, 41.5, 28.5, 28.2, 24.7, 22.7, 20.2, 12.1, 9.7; HRMS (ESI): m / z calcd for C35H41FN5O5[M+H]+630.3092 found 630.3074.

[0121] tert-Butyl(S)-2-((5-(((Z)-5-(((R)-1-(4-10 fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)piperidine-1-carboxylate(114n): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (S)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid 117k (14 mg, 0.06 mmol) provided compound 114n as a red solid (18 mg, 60%); Rf= 0.6 15 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.11 (s, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.84 – 7.56 (m, 2H), 7.52 – 7.39 (m, 2H), 7.29 – 7.08 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 4.69 (d, J = 25.5 Hz, 1H), 3.81 (d, J = 12.9 Hz, 1H), 3.16 (d, J = 51.0 Hz, 1H), 2.17 (d, J = 7.4 Hz, 6H), 1.62 (d, J = 10.4 Hz, 3H), 1.48 (d, J = 7.0 Hz, 3H), 20 1.39 (s, 9H), 1.36 – 1.26 (m, 2H);13C NMR (126 MHz, DMSO) δ 170.2, 166.4, 161.4 (d, J = 241.8 Hz, CF-C), 141.7, 140.8, 132.5, 128.5 (d, J = 8.1 Hz, CF-C), 128.0, 127.7, 126.1, 125.9, 124.9, 124.7, 122.2, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.0, 109.1, 79.2, 48.3, 28.5, 24.7, 22.7, 20.2, 12.1, 9.7; HRMS (ESI): m / z calcd for C35H41FN5O5[M+H]+630.3092 found 630.3072. 25

[0122] tert-Butyl(S)-3-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)piperidine-1-carboxylate(114o): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (S)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid 117l (14 30 mg, 0.06 mmol) provided compound 114o as brick red solid (21 mg, 72%); Rf= 0.32 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.16 (s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H), 7.80 – 7.57 (m, 2H), 7.42 (dd, J = 8.6, 5.7 Hz, 2H), 7.13 (t, J = 8.9 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 1H), 4.01 (q, J = 7.1 Hz, 1H), 3.87 (d, J = 13.1 Hz, 1H), 3.01 35 – 2.53 (m, 5H), 2.16 (d, J = 7.6 Hz, 6H), 1.89 – 1.55 (m, 2H), 1.48 (d, J = 7.1 Hz, 3H), 1.40 (s, 9H);13C NMR (126 MHz, DMSO) δ 172.7, 171.7, 170.2, 166.4, 161.4 (d, J = 241.3 Hz, CF-C), 154.3, 141.7, 140.7, 132.4, 128.5 (d, J = 8.1 Hz, CF-C), 8170774-02 / / 1165.202WO1 127.9, 127.5, 126.1, 125.9, 124.9, 124.6, 122.1, 117.7, 115.3 (d, J = 21.0 Hz, CF-C), 113.0, 109.1, 79.2, 48.3, 43.4, 42.6, 28.5, 24.7, 22.7, 12.1, 9.6; HRMS (ESI): m / z calcd for C35H41FN5O5[M+H]+630.3092 found 630.3075.

[0123] tert-Butyl(R)-3-((5-(((Z)-5-(((R)-1-(4- 5 fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)piperidine-1-carboxylate(114p): Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (R)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid 117m (14 mg, 0.06 mmol) provided compound 114p as a brick red solid (22 mg, 75%); Rf10 = 0.43 (MeOH : CH2Cl2= 0.5:9.5);1H NMR (500 MHz, DMSO) δ 9.16 (s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 7.76 – 7.58 (m, 2H), 7.47 – 7.38 (m, 2H), 7.23 – 7.09 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.4 Hz, 1H), 4.15 – 3.98 (m, 1H), 3.87 (d, J = 13.2 Hz, 1H), 2.80 (d, J = 56.5 Hz, 2H), 2.44 (dq, J = 11.0, 3.5 Hz, 1H), 2.16 (d, J = 7.5 Hz, 6H), 1.94 (s, 1H), 1.78 – 1.55 (m, 2H), 15 1.48 (d, J = 7.1 Hz, 3H), 1.40 (s, 9H);13C NMR (126 MHz, DMSO) δ 172.6, 170.1, 166.4, 161.4 (d, J = 241.9 Hz, CF-C), 154.3, 141.7, 140.7, 132.4, 128.5 (d, J = 8.1 Hz, CF-C), 128.0, 127.6, 126.1, 125.9, 124.9, 124.6, 122.1, 117.7, 115.3 (d, J = 21.1 Hz, CF-C), 113.0, 109.1, 79.2, 48.3, 42.65, 28.5, 28.2, 24.7, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C35H40FN5O5Na [M+Na]+652.2911 found 652.2909. 20

[0124] (Z)-3-((3,5-Dimethyl-4-((S)-piperidine-3-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (114q): Following the general procedure for compound 114g, compound 114o (15 mg, 0.023 mmol) provided compound 114q as red solid (10 mg, 82%); Rf= 0.2 (MeOH : CH2Cl2= 1:9);1H NMR (400 MHz, DMSO) δ 11.05 25 (s, 1H), 9.36 (s, 1H), 8.59 (d, J = 7.9 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 7.77 – 7.56 (m, 2H), 7.52 – 7.37 (m, 2H), 7.26 – 7.07 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.16 (p, J = 7.1 Hz, 1H), 3.02 (d, J = 61.7 Hz, 4H), 2.72 – 2.53 (m, 2H), 2.17 (d, J = 8.2 Hz, 6H), 1.77 (d, J = 48.3 Hz, 3H), 1.47 (d, J = 7.0 Hz, 3H);13C NMR (126 MHz, DMSO) δ 171.8, 170.2, 166.4, 161.4 (d, J = 241.3 Hz, CF-C), 141.7, 140.8, 30 132.2, 128.5 (d, J = 8.2 Hz, CF-C), 128.0, 127.4, 126.1, 125.9, 124.9, 124.7, 121.6, 117.9, 115.3 (d, J = 21.0 Hz, CF-C), 113.3, 109.1, 48.3, 44.9, 43.6, 39.1, 27.0, 22.7, 21.6, 12.1, 9.7; HRMS (ESI): m / z calcd for C30H33FN5O3[M+H]+530.2567 found 530.2558.

[0125] (Z)-3-((3,5-Dimethyl-4-((R)-piperidine-3-carboxamido)-1H-35 pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (114r): Following the general procedure for compound 114g, compound 114p (15 mg, 0.023 mmol) provided compound 114r as a brick red 8270774-02 / / 1165.202WO1 solid (11 mg, 86%); Rf= 0.2 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.06 (s, 1H), 9.37 (s, 1H), 8.86 – 8.42 (m, 3H), 8.18 (s, 1H), 7.86 – 7.59 (m, 2H), 7.42 (dd, J = 8.5, 5.5 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.3 Hz, 1H), 3.30 (d, J = 12.7 Hz, 1H), 3.22 – 3.02 (m, 2H), 2.88 5 (d, J = 48.3 Hz, 3H), 2.17 (d, J = 10.5 Hz, 6H), 2.06 (d, J = 9.3 Hz, 1H), 1.83 (s, 1H), 1.71 (t, J = 9.0 Hz, 2H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO) δ 171.7, 170.2, 166.4, 161.4 (d, J = 241.8 Hz, CF-C), 141.7, 140.8, 132.2, 128.5 (d, J = 8.2 Hz, CF-C), 128.0, 127.4, 126.1, 125.9, 124.9, 124.7, 121.6, 117.9, 115.3 (d, J = 21.0 Hz, CF-C), 113.3, 109.1, 48.3, 44.8, 43.5, 39.0, 27.0, 22.7, 21.5, 12.1, 10 9.6; HRMS (ESI): m / z calcd for C30H33FN5O3[M+H]+530.2567 found 530.2561.

[0126] tert-Butyl(R)-3-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)piperazine-1-carboxylate(114s): Following the procedure as described for compound 114a, amine 115 (20 mg, 15 0.05 mmol) and (R)-4-(tert-butoxycarbonyl)piperazine-2-carboxylic acid 117n (14 mg, 0.06 mmol) provided compound 114s as dark red solid (19 mg, 64%); Rf= 0.3 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.13 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.94 – 7.57 (m, 2H), 7.53 – 7.36 (m, 2H), 7.22 – 7.04 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (p, J = 7.2 Hz, 20 1H), 3.96 (d, J = 63.8 Hz, 2H), 3.60 (d, J = 12.4 Hz, 1H), 3.34 (dd, J = 9.6, 3.9 Hz, 1H), 3.13 – 2.84 (m, 2H), 2.60 (t, J = 11.9 Hz, 1H), 2.17 (d, J = 8.7 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H), 1.40 (s, 9H);13C NMR (126 MHz, DMSO) δ 170.5, 170.2, 166.4, 161.4 (d, J = 241.8 Hz, CF-C), 154.3, 141.7, 140.7, 132.5, 128.5 (d, J = 8.1 Hz, CF-C), 128.0, 127.7, 126.1, 125.9, 124.9, 124.7, 121.9, 117.8, 115.3 (d, J = 25 20.8 Hz, CF-C), 113.0, 109.1, 79.3, 58.2, 48.3, 44.1, 28.5, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C34H40FN6O5[M+H]+631.3044 found 631.3044.

[0127] tert-Butyl(S)-3-((5-(((Z)-5-(((R)-1-(4- fluorophenyl)ethyl)carbamoyl)-2-oxoindolin-3-ylidene)methyl)-2,4- dimethyl-1H-pyrrol-3-yl)carbamoyl)piperazine-1-carboxylate(114t): 30 Following the procedure as described for compound 114a, amine 115 (20 mg, 0.05 mmol) and (S)-4-(tert-butoxycarbonyl)piperazine-2-carboxylic acid 117o (14 mg, 0.06 mmol) provided compound 114t as a brick red solid (18 mg, 60%); Rf= 0.3 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 9.14 (s, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.18 (s, 1H), 7.95 – 7.58 (m, 2H), 7.43 (dd, J = 8.4, 35 5.5 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 6.91 (d, J = 8.1 Hz, 1H), 5.17 (q, J = 7.5 Hz, 1H), 3.90 (s, 1H), 3.60 (d, J = 12.5 Hz, 1H), 3.53 – 3.32 (m, 2H), 3.16 – 2.76 (m, 3H), 2.73 – 2.55 (m, 1H), 2.17 (d, J = 8.6 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H), 1.40 8370774-02 / / 1165.202WO1 (s, 9H);13C NMR (126 MHz, DMSO) δ 170.5, 170.2, 166.4, 161.4 (d, J = 241.5 Hz, CF-C), 154.3, 141.7, 140.8, 132.5, 128.5 (d, J = 8.0 Hz, CF-C), 128.0, 127.7, 126.17, 125.9, 124.9, 124.6, 121.9, 117.8, 115.3 (d, J = 21.1 Hz, CF-C), 113.0, 109.1, 79.4, 58.2, 48.3, 44.1, 28.5, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for 5 C34H40FN6O5[M+H]+631.3044 found 631.3044.

[0128] (Z)-3-((3,5-Dimethyl-4-((R)-piperazine-2-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (114u): Following the general procedure for compound 114g, compound 114s (15 mg, 0.023 mmol) provided compound 114u as red solid (10 10 mg, 81%); Rf= 0.1 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) δ 11.08 (s, 1H), 9.76 (s, 1H), 8.60 (d, J = 7.6 Hz, 1H), 8.19 (s, 1H), 7.93 – 7.56 (m, 2H), 7.42 (dd, J = 8.4, 5.5 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.18 (q, J = 7.4 Hz, 1H), 4.15 (d, J = 42.3 Hz, 1H), 3.66 (s, 1H), 3.30 (d, J = 15.9 Hz, 2H), 3.12 (dd, J = 25.8, 13.4 Hz, 3H), 2.20 (d, J = 12.5 Hz, 6H), 1.48 (d, J = 15 7.1 Hz, 3H);13C NMR (126 MHz, DMSO) δ 170.2, 166.3, 161.4 (d, J = 245.3 Hz, CF-C), 141.7, 140.9, 132.0, 128.5 (d, J = 8.1 Hz, CF-C), 128.1, 127.2, 126.3, 125.8, 125.0, 124.7, 120.7, 118.0, 115.3 (d, J = 20.8 Hz, CF-C), 113.7, 109.1, 54.7, 48.3, 43.9, 22.7, 12.1, 9.7; HRMS (ESI): m / z calcd for C29H32FN6O3 [M+H]+531.2520 found 531.2511. 20

[0129] (Z)-3-((3,5-Dimethyl-4-((S)-piperazine-2-carboxamido)-1H- pyrrol-2-yl)methylene)-N-((R)-1-(4-fluorophenyl)ethyl)-2-oxoindoline-5- carboxamide (114v): Following the general procedure for compound 114g, compound 114t (15 mg, 0.023 mmol) provided compound 114v as a brick red solid (11 mg, 89%); Rf= 0.1 (MeOH : CH2Cl2= 1:9);1H NMR (500 MHz, DMSO) 25 δ 11.09 (s, 1H), 9.92 (s, 1H), 9.25 (s, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.20 (s, 1H), 7.77 – 7.59 (m, 2H), 7.55 – 7.36 (m, 2H), 7.13 (t, J = 8.6 Hz, 2H), 6.92 (d, J = 8.1 Hz, 1H), 5.60 – 5.05 (m, 1H), 4.27 (s, 1H), 3.81 (d, J = 13.1 Hz, 1H), 3.41 (s, 2H), 3.18 (dt, J = 27.4, 13.2 Hz, 3H), 2.20 (d, J = 13.2 Hz, 6H), 1.48 (d, J = 7.1 Hz, 3H);13C NMR (126 MHz, DMSO) δ 170.2, 166.3, 161.4 (d, J = 241.6 Hz, CF-C), 30 141.7, 140.9, 131.9, 128.5 (d, J = 8.0 Hz, CF-C), 128.1, 127.1, 126.3, 125.8, 125.0, 124.6, 120.4, 118.0, 115.3 (d, J = 21.1 Hz, CF-C), 113.9, 109.1, 54.3, 48.3, 43.3, 42.1, 41.3, 22.7, 12.0, 9.6; HRMS (ESI): m / z calcd for C29H32FN6O3[M+H]+531.2520 found 531.2511. Determination of X-ray structures of GRK5 and inhibitor 114t complex: 35

[0130] Human GRK5 (residues 1-590) D311N mutant were expressed in E. coli Rosetta (DE3) and purified as described previously.

[0038] GRK5D311Nwas mixed with MgCl2and Sgv to achieve a final concentration of 118 μM GRK5, 354 8470774-02 / / 1165.202WO1 μM Sgv, and 118 μM MgCl2. Crystals were obtained in a hanging drop vapor diffusion apparatus over a condition of 220 mM potassium citrate tribasic and 20% polyethylene glycol 3350 at 4 °C. Crystals were allowed to grow for one week until stable sizes were obtained and were transferred to a new hanging drop 5 tray in a 4 μl suspended drop containing 20% PEG3350, 10% glycerol, and inhibitor at a final concentration of 1 mM (4% DMSO in final mixture) as described previously.[38,39]Finally, individual crystals were directly frozen by flash freezing on nylon loops in liquid nitrogen.

[0131] Diffraction data were collected at the Brookhaven National 10 Laboratory on NSLS-II 17-ID-1(AMX) at a wavelength of 0.9201 Å, with 1° angle per frame for a total of 180 frames. Automated-processed data from Fast Data Processing (Fast DP) was used to achieve molecular replacement in PHENIX Phaser-MR , using as a search model the GRK5 structure from PDB entry 8UAP. Refinements were performed using phenix.refine alternating with manual building 15 and fitting in COOT.

[0046] The final models were validated with MolProbity prior to deposition along with structure factors in the Protein Data Bank

[0047] Atomic figures were created with Pymol.

[0048] Crystallographic statistics are reported in Supplemental Table 1. Coordinates for inhibitor 114t (GRL-098-22) were deposited in the Protein Data Bank

[0047] with accession code 9BRK. 20

[0132] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents 25 thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and can include modification thereto and permutations thereof. 30

[0133] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub- ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or 35 “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The 8570774-02 / / 1165.202WO1 statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0134] In this document, the terms “a,” “an,” or “the” are used to include 5 one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to 10 be interpreted as limiting. Further, information that is relevant to a section heading can occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those 15 documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0135] In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a 20 temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed 25 process.

[0136] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each 30 individual publication were specifically and individually indicated to be incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls. 35

[0137] The invention illustratively described herein may be suitably practiced in the absence of any element(s) or limitation(s), which is / are not specifically disclosed herein. Thus, for example, each instance herein of any of 8670774-02 / / 1165.202WO1 the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. Likewise, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods 5 and / or steps of the type, which are described herein and / or which will become apparent to those ordinarily skilled in the art upon reading the disclosure. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated.

[0138] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill 10 in the art. The following terms and phrases shall have the meaning indicated.

[0139] The term “about,” when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the 15 numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., + / - 5 % to 15% of the recited value, such as within 10%, within 5%, or within 1% of a stated value or stated limit of a range) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term “substantially” can allow for a 20 degree of variability in a value or range, for example, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range.

[0140] In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of 25 description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. 87

Claims

70774-02 / / 1165.202WO1 What is claimed is:8870774-02 / / 1165.202WO1or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 5 2. A compound having a structure selected from:or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

3. A compound having a structure selected from: 8970774-02 / / 1165.202WO1 5or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof. 10 4. A compound having a structure selected from:9070774-02 / / 1165.202WO1 or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof.

5. A compound having a structure selected from: , , ,, 9170774-02 / / 1165.202WO1 , ,, 9270774-02 / / 1165.202WO1or pharmaceutically acceptable salts, hydrates, tautomers, and optical 5 isomers thereof.

6. The compound of any preceding claim, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, having at least a two-fold, at least a ten-fold, at least a 100-fold, at least a 150-fold, at least 10 a 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at least a 750-fold, at least a 1,000-fold, at least a 2,000-fold, at least a 3,000-fold or at least a 5,000 fold selectivity for G protein-coupled receptor kinase 5 (GRK5) over GRK2. 15 7. The compound of claim 6, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, having a two-fold to 5,000-fold, two-fold to 100-fold, two-fold to 50-fold, 10-fold to 100-fold, 10-fold to 190- fold, 10-fold to 150-fold, 50-fold to 160-fold, 50-fold to 200-fold, 100-fold to 500-fold, 100-fold to 1,000-fold, 250-fold to 1,000-fold, 500-fold to 900- 20 fold, 300-fold to 600-fold, 500-fold to 3,000-fold, 500-fold to 1,000-fold, 500-fold to 2,000-fold, or 50-fold to 150-fold selectivity for GRK5 over GRK2. 9370774-02 / / 1165.202WO1 8. The compound of any one of claims 1-5, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, having at least a two-fold, at least a ten-fold, at least a 100-fold, at least a 150-fold, at least a 200-fold, at least a 300-fold, at least a 400-fold, at least a 500-fold, at 5 least a 750-fold, at least a 1,000-fold, at least a 2,000-fold, at least a 3,000-fold or at least a 5,000 fold selectivity for GRK5 over GRK6.

9. The compound of claim 8, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, having a two-fold to 5,000-fold, ,10 two-fold to 100-fold, two-fold to 50-fold, 10-fold to 100-fold, 10-fold to 190- fold, 10-fold to 150-fold, 50-fold to 160-fold, 50-fold to 200-fold, 100-fold to 500-fold, 100-fold to 1,000-fold, 250-fold to 1,000-fold, 500-fold to 900- fold, 300-fold to 600-fold, 500-fold to 3,000-fold, 500-fold to 1,000-fold, 500-fold to 2,000-fold, or 50-fold to 150-fold selectivity for GRK5 over 15 GRK6.

10. A pharmaceutical composition comprising a compound of any preceding claim, or pharmaceutically acceptable salts, hydrates, tautomers, and optical isomers thereof, and at least one 20 pharmaceutically acceptable carrier or excipient.

11. A method of inhibiting a G protein-coupled receptor kinase (GRK) 5 in a subject, which method comprises administering a compound of any one of claims 1-9 or a pharmaceutical composition of claim 10 in an 25 amount effective to inhibit GRK5, whereupon GRK5 in the subject is inhibited.

12. The method of claim 11, wherein the subject has heart disease. 30 13. The method of claim 11, wherein the heart disease is heart failure or cardiac hypertrophy.

14. The method of claim 11, wherein the subject has cancer. 35 15. The method of claim 11, wherein the cancer is multiple myeloma. 94