USP28 inhibitors

Abstract

The present invention relates to compounds of Formula (I): (I) or a pharmaceutically acceptable salt thereof useful for treatment of a disease or disorder associated with USP28 and methods of inhibiting USP28.

Description

USP28 INHIBITORSFIELD

[0001] The present invention relates to USP28 inhibitors useful for the treatment of cancers, autoimmune diseases, and infectious diseases.BACKGROUND

[0002] Ubiquitin is a small protein consisting of 76 amino acids that is important in the regulation of protein function in the cell. Ubiquitination and deubiquitination are enzyme mediated processes by which ubiquitin is bound to or unbound from a target protein. Deubiquitination by deubiquinating enzymes (DUBs) allows ubiquitin to be recycled and restores the function of the deubiquitinated enzymes.

[0003] Ubiquitin-specific protease 28 (USP28) is a cysteine isopeptidase of the USP sub-family of DUBs that exerts its function through regulating the stability of a number of cellular proteins and has been characterized as a tumor-promoting factor found to stabilize many oncoproteins. Variations in USP28 have been identified in multiple cancer types, including breast cancer, acute myeloid leukemia (AML), ovarian cancer, and colorectal cancer. Additionally, USP28 overexpression has been correlated with poor prognosis in patients suffering from glioblastoma, non-small cell lung carcinoma, and bladder cancers.

[0004] Certain small molecule USP28 inhibitors and their uses are described in US Patent 10,913,753 and US 2023 / 0365583, but no small molecule USP28 inhibitors have been clinically evaluated or approved for treatment. Due to its major role in the deubiquitination and stabilization of oncoproteins, epigenetic drivers, and immunomodulatory proteins among other cellular factors necessary for immune response and tumor initiation and growth in humans, there remains a critical need for potent small molecule inhibitors of USP28 for treatment of cancers, autoimmune diseases, infectious diseases, and other USP28 mediated disorders.SUMMARY OF THE INVENTION

[0005] An aspect of the invention is to provide USP28 inhibitors of Formula I:Iwhere:R1is Ci - C4alkyl;R2is Cs - Cio heterocyclyl substituted with -C(O)R3;R3is Ci - C4 alkoxy, C5 - Cio heterocyclyl, or -NHR4;R4is C5 - Cio heterocyclyl; or a pharmaceutically acceptable salt thereof.

[0006] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl.

[0007] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl and R2is piperazin- 1-yl substituted with -C(O)R3.

[0008] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl and R2is piperazin- 1-yl substituted with -C(O)R3where R3is Ci - C4 alkoxy.

[0009] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl and R2is piperazin- 1-yl substituted with -C(O)R3where R3is C5 - Cio heterocyclyl.

[0010] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl and R2is piperazin- 1-yl substituted with -C(O)R3where R3is piperazin- 1-yl.

[0011] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl and R2is piperazin- 1-yl substituted with -C(O)R3where R3is -NHR4.

[0012] Another aspect of the invention is to provide USP28 inhibitors of Formula I, or a pharmaceutically acceptable salt thereof, wherein R1is propyl and R2is piperazin- 1-yl substituted with -C(O)R3where R3is -NHR4and R4is piperidin-4-yl.

[0013] Another aspect of the present invention provides a pharmaceutical composition comprising a USP28 inhibitor compound of Formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0014] A further aspect of the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof for use in therapy.

[0015] Another aspect of the present invention provides a method for treating cancer susceptible to inhibition of USP28 comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0016] Another aspect of the present invention provides a method for treating an autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0017] Another aspect of the present invention provides a method for treating a viral infection comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0018] Another aspect of the present invention provides a method for treating a bacterial infection comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0019] A further aspect of the present invention provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of disorders susceptible to inhibition of USP28.

[0020] Another aspect of the invention is to provide USP28 inhibitors of Formulawhere R5is piperidin-l-yl, piperidin-4-yl, morpholin-4-yl, piperazin- 1-yl, or 8- azabicyclo[3.2.1]octan-3-yl, or a pharmaceutically acceptable salt thereof.

[0021] Another aspect of the invention is to provide a USP28 inhibitor of Formula II or a pharmaceutically acceptable salt thereof where R5is piperidin-l-yl.

[0022] Another aspect of the invention is to provide a USP28 inhibitor of Formula II or a pharmaceutically acceptable salt thereof where R5is piperidin-4-yl.

[0023] Another aspect of the invention is to provide a USP28 inhibitor of Formula II or a pharmaceutically acceptable salt thereof where R5is morpholin-4-yl.

[0024] Another aspect of the invention is to provide a USP28 inhibitor of Formula II or a pharmaceutically acceptable salt thereof where R5is piperazin- 1-yl.

[0025] Another aspect of the invention is to provide a USP28 inhibitor of Formula II or a pharmaceutically acceptable salt thereof where R5is 8-azabicyclo[3.2.1]octan-3-yl.

[0026] Another aspect of the present invention provides a pharmaceutical composition comprising a USP28 inhibitor compound of Formula II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0027] A further aspect of the present invention provides a compound of Formula II or a pharmaceutically acceptable salt thereof for use in therapy.

[0028] Another aspect of the present invention provides a method for treating cancer susceptible to inhibition of USP28 comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0029] Another aspect of the present invention provides a method for treating an autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0030] Another aspect of the present invention provides a method for treating a viral infection comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0031] Another aspect of the present invention provides a method for treating a bacterial infection comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0032] A further aspect of the present invention provides the use of a compound of Formula II or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of disorders susceptible to inhibition of USP28.DETAILED DESCRIPTION

[0033] Terms used herein but not separately defined are taken to have their normal and customary meaning as understood by one of ordinary skill in the art.

[0034] The term “Ci - C4 alkyl” is taken to mean a straight or branched alkyl chain of from 1 to 4 carbon atoms and includes methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and the like.

[0035] The term “Ci - C4 alkoxy” is taken to mean a straight or branched alkyl chain of from 1 to 4 carbon atoms attached to an oxygen atom and includes methoxy, ethoxy, propoxy, isopropoxy, n- butoxy, sec-butoxy, isobutoxy, tert-butoxy, and the like.

[0036] The term “Cs - Cio heterocyclyl” is taken to mean a saturated ring of 5 to 10 members comprising carbon atoms and from 1 to 3 heteroatoms independently selected from N, O, and S, and includes pyrrolidinyl, piperidinyl, homopiperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl, azepanyl, diazepanyl, azocanyl, as well as all bridged fused bicyclic and spirofused isomers thereof such as tropanyl, 3,8-diazabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1]octanyl, 2,5- diazabicyclo[2.2.1]heptanyl, 3,6-diazabicyclo[3.1.1]heptyl, hexahydropyrrolo[3,2-b]pyrrol-l(2H)- yl, hexahydropyrrolo[3,4-b]pyrrol-5(lH)-yl, 2,6-diazaspiro[3,3]heptanyl, octahydro-5H-pyrrolo[3,4- c]pyridinyl, 2,5-diazaspiro[3,4]octanyl, 2,7-diazaspiro[4,4]nonanyl, 2,7-diazaspiro[3,5]nonanyl, 2,7- diazaspiro[4,5]decanyl, 9-oxa-3,7-diazabicyclo[3.3.1]nonanyl, 6-oxa-2,9-diazaspiro[4,5]decanyl, and the like.

[0037] The term “patient” means mammal and “mammal” includes, but is not limited to, a human.

[0038] “Therapeutically effective amount” means the dosage of a compound of Formula I or a pharmaceutically acceptable salt thereof or a compound of Formula II, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a compound of Formula II, or a pharmaceutically acceptable salt thereof, necessary to inhibit USP28 in a patient in need thereof. Anticipated dosages of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a compound of Formula II, or a pharmaceutically acceptable salt thereof, are in the range of 1 mg / patient / day to 2000 mg / patient / day. The exact dosage required to treat a patient and the duration of treatment will be determined by a physician in view of the stage and severity of the disease as well as the specific needs and response of the individual patient. Dosage administration may be adjusted to provide an optimal therapeutic benefit to an individual patient and to manage or avoid drug-related toxicities. For example, in addition to single daily dosing, multiple smaller daily doses or administration on a staggered daily, weekly, or monthly schedule may be appropriate.

[0039] The terms “treatment”, “treat”, and “treating” are meant to include the full spectrum of pharmaceutical intervention for a patient in need of USP28 inhibition, such as administration of a USP28 inhibitor of the present invention to alleviate, slow, or reverse one or more of a patient’s symptoms or to delay progression of the disorder even if the disorder is not actually eliminated.

[0040] Compounds of Formula I or a pharmaceutically acceptable salt thereof or compounds of Formula II or a pharmaceutically acceptable salt thereof are preferably formulated as a pharmaceutical composition using a pharmaceutically acceptable carrier and administered by a variety of routes. Preferably, such compositions are for oral administration. Such pharmaceutical compositions andmethods for preparing them are well known in the art. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (L.V. Allen ed., Pharmaceutical Press, 22ndEdition, 2012).

[0041] A compound of Formula I or a pharmaceutically acceptable salt thereof or a compound of Formula II or a pharmaceutically acceptable salt thereof may be administered either simultaneously with, or before, or after, one or more other therapeutic agents. When said compounds or pharmaceutically acceptable salts thereof are administered with one or more therapeutic agents, they may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other therapeutic agent or agents. Where one or more additional therapeutic agents are administered, the administration of each therapeutic agent may be simultaneous, separate, or sequential.

[0042] Compounds of Formula I and Formula II are capable of reacting with a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Such pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, for example, P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SCIENCES, (VCHA / Wiley - VCH, 2002); S.M. Berge, et al., Pharmaceutical Salts, 66 JOURNAL OF PHARMACEUTICAL SCIENCES 1 (1977).

[0043] It will be understood that compounds of Formula I and Formula II may have a chiral center and may be depicted as single stereoisomers. For example, a compound of Formula I is illustrated below:(R) - enantiomer(S) - enantiomer

[0044] All stereoisomers of the compounds of Formula I and Formula II are contemplated within the scope of the present invention. As used herein, references to a single stereoisomer are meant to also include stereoisomeric mixtures including the named or depicted compound of Formula I or II. Herein, the Cahn-Ingold-Prelog designations of (R)- and (S)- may be used to refer to specific stereoisomers. Specific stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enriched starting materials. The specific stereoisomers of either starting materials, intermediates, or racemic mixtures including compounds of Formula I and II can be resolved by techniques well known in the art, such as those found in Stereochemistry of Organic Compounds, E. I. Eliel and S. H. Wilen (Wiley 1994) and Enantiomers, Racemates, and Resolutions, J., Jacques, A Collet, and S. H. Wilen (Wiley 1991), including chromatography on chiral stationary phases, enzymatic resolutions, or fractional crystallization or chromatography of diastereomers formed for that purpose, such as diastereomeric salts.

[0045] Compounds of the present invention are named according to IUPAC, and may also be named according to CAS, and other naming conventions may be used to unambiguously identify a compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof.

[0046] The compounds employed as initial starting materials in the synthesis of compounds of Formula I and Formula II are well known and, to the extent not commercially available, are readily synthesized using specific references provided, by standard procedures commonly employed by those of ordinary skill in the art or are found in general reference texts. Examples of known procedures and methods include those in general reference texts such as: COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH Publishers Inc., 1989); COMPENDIUM OF ORGANIC SYNTHETIC METHODS (Wiley Interscience, Volumes 1 - 10, 1974 - 2002); Michael B. Smith and Jerry March, ADVANCED ORGANIC CHEMISTRY, REACTIONS, MECHANISMS, AND STRUCTURE (Wiley Interscience, 5thed. 2001); Francis A. Carey and Richard J. Sundberg, ADVANCED ORGANIC CHEMISTRY, PART B, REACTIONS AND SYNTHESIS (Kluwer Academic / Plenum Publishers, 4thed. 2000), and references cited therein.

[0047] Certain intermediates described in the following preparations may contain one or more nitrogen protecting groups. It is understood that protecting groups may be varied as appreciated by one of skill in the art depending on the actual reaction conditions and the particular transformations to be performed. The protection and deprotection conditions are well known to the skilled artisan and are described in the literature (See for example "Greene's Protective Groups in Organic Synthesis", Fifth Edition, by Peter GM. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2014).

[0048] Compounds of Formula I or pharmaceutically acceptable salts thereof and compounds of Formula II or pharmaceutically acceptable salts thereof may be prepared by a variety of procedures known in the art, some of which are illustrated in the Schemes, Preparations, and Examples below which are provided to further illustrate the invention without limiting the scope of the invention in any way. The specific steps and methodology for each of the synthetic routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of the present invention or pharmaceutically acceptable salts thereof. The products of each step in the schemes below may be isolated by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents are as previously defined unless otherwise indicated.

[0049] Certain abbreviations are defined as follows: “AcOH” refers to acetic acid; “BOC” refers to tert-butoxycarbonyl”; “DCM” refers to dichloromethane; “DiPEA” refers to N,N- diisopropylethylamine; “DMF” refers to dimethylformamide; “equiv” refers to equivalent(s); “EtOAc” refers to ethyl acetate; “EtOH” refers for ethyl alcohol; “h” refers to hour(s); “HATU” refers to ( 1 - [bis(dimethylamino)methylene] - 1 H- 1 ,2,3 -triazolo[4, 5-b] pyridinium 3 -oxide hexafluorophosphate; “LC-MS” refers to liquid chromatography-mass spectrometry; “LDA” refers to lithium diisopropylamide; “MeOH” refers to methanol; “min” refers to minute(s); “Pd(dppf)C12.CH2C12” refers to (l,l'bis(diphenylphosphino)ferrocene)di chloropalladiumdichloromethane; “Pd(OAc)2” refers to palladium(II) acetate; “RT” refers to room temperature; “THF” refers to tetrahydrofuran; “XPhos” refers to dicyclohexyl[2’,4’,6’-tris(propan-2-yl)[l,r- biphenyl]-2-yl]phosphane.

[0050] Compounds of Formula I and Formula II may be prepared from the substituted 3- aminothieno[2,3-b]pyridine-2-carboxylic acid (i) as illustrated in Scheme 1 where R1and R2are as previously defined.Scheme 1

[0051] The carboxylic acid (i) is reacted with an appropriate amine under standard amide coupling conditions known to a skilled artisan and include a coupling agent, such as HATU, and a suitable base, such as DiPEA, in an appropriate solvent, such as DMF.PREPARATIONSPreparation 1Heptane-2, 4-dione

[0052] To a solution of LDA (9.3 mL, 69.6 mmol) in anhydrous ether (75 mL) at -78 °C was added methyl / i-propyl ketone (6.2 mL, 58 mmol) dropwise, followed by the dropwise addition of ethyl formate (4.7 mL, 58 mmol). After the reaction mixture was stirred for 5 h, it was allowed to warm to RT. MeOH (50 mL) was added to the reaction mixture, and the crude was concentrated in vacuo to give the title compound.Preparation 22-Oxo-6-propyl-l,2-dihydropyridine-3-carbonitrile

[0053] A solution of heptane-2, 4-dione (1.0 equiv), 2-cyanoacetamide (1.05 equiv) in H2O (IM) was stirred for 6 min at RT. A solution of piperidine (1.0 equiv), AcOH (1.0 equiv) in H2O (5.0 equiv) was added dropwise to the reaction mixture. The reaction mixture was heated to reflux and stirred for 2 h. Then, the reaction mixture was cooled to RT and adjusted to pH 4 using 4M HC1. The resulting solid was filtered, washed with H2O, and dried in vacuo to give the title compound.Preparation 32-Chloro-6-propylnicotinonitrile

[0054] A solution of 2-oxo-6-propyl-l,2-dihydropyridine-3-carbonitrile (1.0 equiv) and POCh (3 mL) in 1,4-di oxane (5 mL) was heated to 90 °C and stirred for 12 h. The reaction mixture was cooled to RT, poured into ice water, adjusted pH around 7, and extracted with EtOAc. The combined organics were dried over Na2SO4, filtered, and concentrated to give the title compound.Preparation 4Ethyl 3-amino-6-propylthieno[2,3-Z>]pyridine-2-carboxylate

[0055] To a solution of 2-chloro-6-propylnicotinonitrile (1.0 equiv) in DMF (0.2 M) at RT was added K2CO3 (3.0 equiv), followed by ethyl thioglycolate (1.2 equiv). After the reaction mixture was stirred under reflux for 5 h, it was cooled to RT and diluted with H2O. The resulting precipitate was filtered and washed with water to give the title compound.Preparation 5 3-Amino-6-propylthieno[2,3-Z>]pyridine-2-carboxylic acid

[0056] To a solution of ethyl 3-amino-6-propylthieno[2,3-Z?]pyridine-2-carboxylate (200 mg) in EtOH (4 mL) was added 2M NaOH solution (1.5 mL). The reaction mixture was refluxed for 4 h under stirring. After completion, the resulting mixture was concentrated in vacuo, and the pH was adjusted to around 6 with IM HC1 solution. The resulting precipitate was filtered and dried to afford the title compound.

[0057] The compounds in the table below were prepared from the corresponding ester essentially as described in Preparation 5.1 -r - Butyl 2-methyl 4-(4-(2-(((benzyloxy)carbonyl)amino)ethyl)phenyl)- piperazine- 1,2-

[0058] 1 -tert-butyl 2-methyl piperazine- 1,2-di carboxylate (2.0 equiv), CS2CO3 (2.0 equiv),Pd(OAC)2 (0.2 equiv), and XPhos (0.2 equiv) were added to a solution of benzyl 4- bromophenethylcarbamate (1.0 equiv) and toluene (0.2 M) at room temperature. The reaction mixture was heated to 100 °C for 12 hours and was then allowed to cool to room temperature. The cooled reaction mixture was filtered through a pad of Celite and the filtrate was diluted with saturated aqueous NH4CI, and extracted with DCM. The combined organic phases were washed with saturated aqueous NaCl, dried with Na2SO4, filtered, concentrated under reduced pressure, and subjected to flash silica gel chromatography, eluting with a gradient of EA / Hex 0-40% to provide the title compound.

[0059] The compounds in the following table were prepared essentially as described in Preparation 10 beginning with the appropriate amine.Preparation 14 tert-Butyl 3-(4-(2-(((benzyloxy)carbonyl)amino)ethyl)phenyl)-8-azabicyclo[3.2. l]oct-2-ene-8- carboxylate

[0060] To a solution of benzyl 4-bromophenethylcarbamate (0.5 g, 1.5 mmol) in 1,4-dioxane (9 mL) and H2O (1 mL) at RT under N2 atmosphere, were added K2CO3 (0.62 g, 4.5 mmol), A-boc-8- azabicyclo[3.2. l]oct-2-ene-2-boronic acid, pinacol ester (0.55 g, 1.64 mmol), and Pd(dppf)Ch (0.22 g, 0.3 mmol). The reaction mixture was heated to 100 °C and stirred for 2 h. Once complete, the reaction was cooled to RT, and concentrated in vacuo. The reaction mixture was diluted with H2O(10 mL) and extracted with EtOAc (90 mL). The combined organics were washed with brine, dried over Na2SO4, filtered, concentrated in vacuo, and purified via flash silica gel chromatography (0-60% EtOAc in hexanes) to give the title compound.

[0061] The compound in the following table was prepared essentially as described in Preparation 14.Preparation 16 tert-Butyl 4-(4-(2-(((benzyloxy)carbonyl)amino)ethyl)phenyl)piperazine-l -carboxylate

[0062] To a solution of benzyl 4-bromophenethylcarbamate (1.0 equiv) in toluene (0.2 M), were added 1 -boc-piperazine (2.0 equiv), CS2CO3 (2.0 equiv), Pd(OAc)2 (0.2 equiv), and Xphos (0.2 equiv) at RT. The reaction mixture was heated to 100 °C and stirred for 12 h. Then, the reaction mixture was allowed to cool to RT, and filtered through a pad of diatomaceous earth. The filtrate was diluted with saturated aqueous NH4CI and extracted with DCM. The combined organics were washed with NaCl saturated solution, dried over Na2SO4, filtered, concentrated in vacuo and further purified via flash silica gel chromatography (0-40% EtOAc in hexanes) to give the title compound.

[0063] The compounds in the following table were prepared essentially as described in Preparation 16.Preparation 19I -rm- Butyl 2-methyl 4-(4-(2-aminoethyl)phenyl)- piperazine- 1,2-di carboxylate

[0064] A mixture of I -rm- Butyl 2-methyl 4-(4-(2-(((benzyloxy)carbonyl)amino)ethyl)phenyl)- piperazine-l,2-dicarboxylate (1.0 equiv) and 10% Pd / C (0.1 equiv) in MeOH (0.2 M) was stirred at room temperature under a hydrogen atmosphere (balloon) for 12 hours. The reaction mixture was then filtered through Celite and the filtrate concentrated under reduced pressure to provide the title compound.

[0065] The compounds in the following table were prepared essentially as described in Preparation 19.Preparation 23 r -Butyl 3-(4-(2-aminoethyl)phenyl)-8-azabicyclo[3.2.1]octane-8-carboxylate

[0066] To a solution of tert-butyl 3-(4-(2-(((benzyloxy)carbonyl)amino)ethyl)phenyl)-8- azabicyclo[3.2.1]oct-2-ene-8-carboxylate (0.33 g, 0.71 mmol) in MeOH (10 m ), was added 10% Pd / C (100 mg). The reaction mixture was sparged with a H2 filled balloon and stirred at RT for 12 h. Once complete, the reaction mixture was filtered through diatomaceous earth and concentrated in vacuo to afford the title product as a colorless oil.

[0067] The compounds in the following table were prepared essentially as described in Preparation23.Preparation 281 -(tert-Butyl) 2-methyl 4-(4-(2-(3-amino-6-propylthieno[2,3-Z?]pyridine-2- carboxamido)ethyl)phenyl)piperazine- 1 ,2-di carboxylate

[0068] A solution of 3-amino-6-propylthieno[2,3-Z?]pyridine-2-carboxylic acid (1.0 equiv), 1-tert- butyl 2-methyl 4-(4-(2-aminoethyl)phenyl)- piperazine- 1 ,2-dicarboxy late (1.5 equiv), HATU (1.2 equiv), and DiPEA (3.0 equiv) in DMF (0.2 M) was stirred for 2 h at RT. The reaction mixture was poured into H2O and extracted with DCM. The combined organic phases were washed with NaCl saturated solution, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was subjected to flash silica gel chromatography to provide the title compound.

[0069] The compounds in the following table were prepared essentially as described in Preparation28.Preparation 32 4-(4-(2-(3-Amino-6-propylthieno[2,3-Z?]pyridine-2-carboxamido)ethyl)phenyl)-l-(tert- butoxy carbony I )pi perazine-2-N-( l -(r -butoxycarbony I )piperazin-4-y I (carboxamide

[0070] A solution of 4-(4-(2-(3-amino-6-propylthieno[2,3-Z?]pyridine-2- carboxamido)ethyl)phenyl)-l-(tert-butoxycarbonyl)piperazine-2-carboxylic acid (1.0 equiv), l-(tert- butoxycarbonyl)piperazine (1.5 equiv), 1- HATU (1.2 equiv), and DIPEA (3.0 equiv) in DMF (0.2M) was stirred for 2 hours at room temperature. The reaction mixture was then poured into water and extracted three times with DCM (x3). The combined organics were washed with saturated aqueousNaCl, dried with Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to flash silica gel chromatography eluting with EA / Hexane to provide the title compound.

[0071] The compounds in the following table were prepared essentially as described in Preparation32 beginning with the appropriate carboxylic acids and amines.EXAMPLESExample 13-Amino-A-(4-(3-(piperazine-l-carbonyl)piperazin-l-yl)phenethyl)-6-propylthieno[2,3-Z?]pyridine-

[0072] A solution of 4M HC1 in dioxane (5 equiv) is added to a solution of 4-(4-(2-(3-amino-6- propylthieno[2,3-Z?]pyridine-2-carboxamido)ethyl)phenyl)-l-(tert-butoxycarbonyl)piperazine-2-N- (l-(tert-butoxycarbonyl)piperazin-4-yl)carboxamide (1 equiv) in MeOH (1 M) and the reaction mixture was stirred at RT for 30 min. The reaction mixture was then concentrated in vacuo, and purified by reverse phase high pressure column chromatography to give the title compound.

[0073] XH NMR (500 MHz, Chloroform-^ / ) 5 7.82 (d, J= 8.3 Hz, 1H), 7.16 (s, 1H), 7.14 (d, J = 8.0 Hz, 2H), 6.87 (d, J = 8.0 Hz, 2H), 3.97 - 3.87 (m, 1H), 3.75 - 3.42 (m, 8H), 3.19 (d, J = 13.4 Hz, 1H), 3.05 (t, J = 12.4 Hz, 1H), 2.95 - 2.80 (m, 7H), 2.70 - 2.59 (m, 2H), 1.99 (s, 1H), 1.78 (h, J= 7.4 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H).

[0074] 13C NMR (126 MHz, CDCh) 5 203.33, 169.69, 165.47, 163.71, 150.24, 130.69, 129.68,129.23, 124.46, 119.05, 117.27, 77.31, 77.06, 76.81, 55.21, 52.91, 51.33, 46.45, 44.69, 40.95, 40.60, 35.08, 29.71, 23.25, 13.89.

[0075] LCMS (ESI) [M+H]+calculated for C28H38N7O2S+m / z 536.72, found m / z 536.54.

[0076] The compounds in the following table were prepared essentially as described in Example 1.Example 8A-(4-(8- Azabicyclo[3.2.1] octan-3 -yl)phenethyl)-3 -amino-6-propylthieno[2, 3 -b\ pyridine-2- carboxamide

[0077] A solution of tert-butyl 3-(4-(2-aminoethyl)phenyl)-8-azabicyclo[3.2.1]octane-8- carboxylate (1.5 equiv), 3-amino-6-propylthieno[2,3-Z?]pyridine-2-carboxylic acid (1.0 equiv), HATU (1.2 equiv), DiPEA (3.0 equiv) in DMF (0.2M) was stirred for 12 h at RT. Once completed, the reaction mixture was poured into H2O, extracted with EtOAc. The combined organics were washed with NaCl saturated solution, dried over Na2SO4, filtered, concentrated in vacuo, and purifiedvia flash silica gel chromatography (0-80 % EtOAc in hexanes). The flash chromatography fractions were collected, concentrated in vacuo, then the residue was dissolved in MeOH (I M) and 4M HC1 in dioxane was added and the reaction mixture was stirred at RT for 30 min. Once the reaction was completed, the reaction mixture was concentrated in vacuo, and purified by reverse phase high pressure column chromatography to give the title compound. LC-MS (ESI) (m / z) 450 (M+H)Example 93-Amino-A-(4-(piperidin-4-yl)phenethyl)-6-propylthieno[2,3-Z?]pyridine-2-carboxamide

[0078] A solution of tert-butyl 4-(4-(2-aminoethyl)phenyl)piperidine-l -carboxylate (1.5 equiv), 3- amino-6-propylthieno[2,3-Z?]pyridine-2-carboxylic acid (1.0 equiv), HATU (1.2 equiv), DiPEA (3.0 equiv) in DMF (0.2M) was stirred for 2 h at RT. Once completed, the reaction mixture was poured into H2O and extracted with EtOAc. The combined organics were washed with NaCl saturated aqueous solution, dried over Na2SO4, filtered, concentrated in vacuo, and purified via flash silica gel chromatography (0-80 % EtOAc in hexanes). The flash chromatography fractions were collected, concentrated in vacuo, then the residue was dissolved in MeOH (I M) and 4M HC1 in dioxane was added and the reaction mixture was stirred at RT for 30 min. Once the reaction was completed, the reaction mixture was concentrated in vacuo, and purified by reverse phase high pressure column chromatography to give the title compound.

[0079] 'H NMR (500 MHz, DMSO-de) 5 8.32 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.22 (d, J= 8.2 Hz, 2H), 7.17 (d, J= 8.1 Hz, 2H), 3.47 - 3.34 (m, 4H), 3.00 (q, J= 12.4 Hz, 2H), 2.81 (dd, J = 8.6, 6.6 Hz, 4H), 1.93 (d, J = 13.1 Hz, 2H), 1.82 - 1.69 (m, 4H), 0.93 (t, J = 7.3 Hz, 3H).

[0080] LCMS (ESI) [M+H]+calculated for C24H3 IN4OS+m / z 423.60, found m / z 423.69.Example 103-Amino-A-(4-(piperazin-l-yl)phenethyl)-6-propylthieno[2,3-Z?]pyridine-2-carboxamide

[0081] A solution of tert-butyl 4-(4-(2-aminoethyl)phenyl)piperazine-l -carboxylate (1.5 equiv), 3- amino-6-propylthieno[2,3-Z?]pyridine-2-carboxylic acid (1.0 equiv), HATU (1.2 equiv), DiPEA (3.0equiv) in DMF (0.2M) was stirred for 2 h at RT. Once completed, the reaction mixture was poured into H2O and extracted with EtOAc. The combined organics were washed with NaCl saturated aqueous solution, dried over Na2SO4, filtered, concentrated in vacuo, and purified via flash silica gel chromatography (0-80 % EtOAc in hexanes). The flash chromatography fractions were collected, concentrated in vacuo, then the residue was dissolved in MeOH (I M) and 4M HC1 in dioxane was added and the reaction mixture was stirred at RT for 30 min. Once the reaction was completed, the reaction mixture was concentrated in vacuo, and purified by reverse phase high pressure column chromatography to give the title compound.

[0082] LCMS (ESI) [M+H]+calculated for C23H3oN5OS+m / z 424.59, found m / z 424.33.Example 113-Amino-A-(4-morpholinophenethyl)-6-propylthieno[2,3-Z>]pyridine-2-carboxamide

[0083] A solution of 2-(4-morpholinophenyl)ethan-l -amine (1.5 equiv), 3-amino-6- propylthieno[2,3-Z?]pyridine-2-carboxylic acid (1.0 equiv), HATU (1.2 equiv), DiPEA (3.0 equiv) in DMF (0.2M) was stirred for 2 h at RT. Once completed, the reaction mixture was poured into H2O and extracted with EtOAc. The combined organics were washed with NaCl saturated aqueous solution, dried over Na2SO4, filtered, concentrated in vacuo, and purified via reverse phase high pressure column chromatography to give the title compound.

[0084] LCMS (ESI) [M+H]+calculated for C23H29N4O2S+m / z 425.57, found m / z 424.98.Example 123-Amino- / V-(4-(piperidin-l-yl)phenethyl)-6-propylthieno[2,3-Z>]pyridine-2-carboxamide

[0085] A solution of 2-(4-(piperidin-l-yl)phenyl)ethan-l -amine (1.5 equiv), 3-amino-6- propylthieno[2,3-Z?]pyridine-2-carboxylic acid (1.0 equiv), HATU (1.2 equiv), DiPEA (3.0 equiv) in DMF (0.2M) was stirred for 2 h at RT. Once completed, the reaction mixture was poured into H2O and extracted with EtOAc. The combined organics were washed with NaCl saturated aqueoussolution, dried over Na2SO4, filtered, concentrated in vacuo, and purified via reverse phase high pressure column chromatography to give the title compound.

[0086] XH NMR (500 MHz, Chloroform-7) 5 7.84 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 3.68 - 3.57 (m, 2H), 3.15 (t, J = 5.5 Hz, 4H), 2.88 (dd, 7 = 8.4, 7.0 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H), 1.81 (qd, 7= 7.5, 1.6 Hz, 2H), 1.74 (s, 4H), 1.59 (p, 7= 6.0 Hz, 2H), 1.00 (t, 7 = 7.4 Hz, 3H).

[0087] 13C NMR (126 MHz, CDCh) 5 165.49, 163.65, 158.61, 144.47, 129.46, 129.22, 124.48, 119.01, 117.02, 77.32, 77.07, 76.82, 51.00, 41.05, 40.59, 35.08, 25.81, 24.23, 23.25, 13.89.

[0088] LCMS (ESI) [M+H]+calculated for C24H3 IN4OS+m / z 423.60, found m / z 423.32.Biological AssaysUSP28 Preparation

[0089] USP28 catalytic domain plasmid (human, aa 149-703 in pETDUET SUMO) (Genewiz) was transformed into E. coli BL21 (DE3) cells and overexpressed. Cells were grown at 37°C in media supplemented with ampicillin to an OD of 0.9, induced with 100 pg / mL isopropyl- 1 -thio-D- galactopyranoside (IPTG), cooled to and incubated overnight at 16°C. The bacterial cell pellet was collected by centrifugation and stored at -80°C. Cell pellets were sonicated in lysis buffer (25 mM Tris pH 8.0, I M NaCl, and 10 mM BME) supplemented with 10 mg / mL phenylmethanesulfonylfluoride (PMSF) and the resulting lysate was centrifuged. Lysate supernatant was added to a gravity column containing Ni-NTA beads (Qiagen), which was mixed for 1 hour at 4°C. Flowthrough was discarded, and beads were washed with lysis buffer supplemented with 25 mM imidazole. Tagged protein was eluted from beads in buffer containing 25 mM Tris pH 8.0, 200 mM NaCl, 10 mM BME, and 300 mM imidazole. The elution was dialyzed and His- SUMO tag cleaved overnight at 4°C in buffer containing 25 mM Tris pH 8.5, 200 mM NaCl, 10 mM BME supplemented with 200 pg / mL SUMO protease. The dialyzed elution was then added to a gravity column containing Ni-NTA beads, and the flowthrough containing protein without tag was collected following incubation of beads for two minutes. This flowthrough was then incubated on beads again and collected two additional times. The sample was then concentrated, centrifuged and purified by size-exclusion chromatography in buffer containing 25 mM HEPES pH 7.5, 200 mM NaCl, and 1 mM DTT. Fractions were pooled, concentrated, and frozen at 80°C.Ubiquitin-Rhodamine 110 Assay

[0090] USP28 was tested with a ubiquitin-rhodamine kinetic assay with either test compound at various concentrations or DMSO. Test compound was plated onto low volume 384-well plates (Corning® catalog number: 3820) using a Hewlett-Packard D300e Digital Dispenser. Enzyme (USP28cat: 10 nM) in 50 mM Tris pH 8.0, 50 mM NaCl, 5 mM TCEP, 0.002% Tween20 was added to the compound plate using a Multidrop™ Combi Reagent Dispenser. Enzyme was incubated with test compounds for 30 min at room temperature. Ubiquitin-Rhodamine (Bio-Techne) was added to a final concentration of 250 nM via Multidrop™ Combi Reagent Dispenser. The enzyme’s initial velocity was collected over a 30 minute period at minute intervals using a FlexStation III Microplate reader at excitation: 485 nm and emission: 535 nm. The initial velocity (RFU / min) were extracted from the SoftMax Pro data analysis software and normalized to control columns on plate. Normalized initial velocities were plotted as a function of test compound concentrations, using GraphPad Prism, to determine IC50. The results of these experiments are shown in Table 1.Table 1

Claims

CLAIMSWe claim:

1. A compound of Formula I:where:R1is Ci - C4alkyl;R2is Cs - Cio heterocyclyl substituted with -C(O)R3;R3is Ci - C4 alkoxy, C5 - Cio heterocyclyl, or -NHR4;R4is C5 - Cio heterocyclyl; or a pharmaceutically acceptable salt thereof.

2. A compound of Claim 1 or a pharmaceutically acceptable salt thereof where R1is propyl.

3. A compound of any of Claims 1 - 2 or a pharmaceutically acceptable salt thereof where R2is piperazin- 1-yl or morpholin-4-yl substituted with -C(O)R3.

4. A compound of Claim 3 or a pharmaceutically acceptable salt thereof where R3is Ci - C4 alkoxy.

5. A compound of Claim 3 or a pharmaceutically acceptable salt thereof where R3is Cs - Cio heterocyclyl.

6. A compound of Claim 5 or a pharmaceutically acceptable salt thereof where R3is piperazin- 1-yl.

7. A compound of Claim 3 or a pharmaceutically acceptable salt thereof where R3is -NHR4.

8. A compound of Claim 7 or a pharmaceutically acceptable salt thereof where R4is piperidin- 4-yl.

9. A pharmaceutical composition comprising a compound of any of Claims 1 - 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. A compound of any of Claims 1 - 8 or a pharmaceutically acceptable salt thereof for use in therapy.

11. A method for treating cancer susceptible to inhibition of USP28 comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of Claims 1 - 8 or a pharmaceutically acceptable salt thereof.

12. A method for treating an autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of Claims 1 - 8 or a pharmaceutically acceptable salt thereof.

13. A compound of any of Claims 1 - 8 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of disorders susceptible to inhibition of USP28.

14. A compound of Formula II:where R5is piperidin-l-yl, piperidin-4-yl, morpholin-4-yl, piperazin- 1-yl, or 8- azabicyclo[3.2.1]octan-3-yl, or a pharmaceutically acceptable salt thereof.

15. A compound of Claim 14 or a pharmaceutically acceptable salt thereof where R5is piperidin- 1-yl.

16. A compound of Claim 14 or a pharmaceutically acceptable salt thereof where R5is piperidin-4-yl.

17. A compound of Claim 14 or a pharmaceutically acceptable salt thereof where R5is morpholin- 4-yl.

18. A compound of Claim 14 or a pharmaceutically acceptable salt thereof where R5is piperazin- 1-yl.

19. A compound of Claim 14 or a pharmaceutically acceptable salt thereof where R5is 8- azabicyclo[3.2.1]octan-3-yl.

20. A pharmaceutical composition comprising a compound of any of Claims 14 - 19 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

21. A compound of any of Claims 14 - 19 or a pharmaceutically acceptable salt thereof for use in therapy.

22. A method for treating cancer susceptible to inhibition of USP28 comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of Claims 14 - 19 or a pharmaceutically acceptable salt thereof.

23. A method for treating an autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of Claims 14 - 19 or a pharmaceutically acceptable salt thereof.

24. The use of a compound of any of Claims 14 - 19 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of disorders susceptible to inhibition of USP28.

Images