USP7 activators

Abstract

The present invention provides USP7 activators of Formula (I): (I).

Description

USP7 ACTIVATORSFIELD

[0001] The present invention relates to USP7 activators useful for the treatment of disorders associated with haploinsufficiency of USP7 such as Hao-Fountain Syndrome.BACKGROUND

[0002] Hao-Fountain Syndrome is a neurodevel opmental disorder due to haploinsufficiency of ubiquitin specific peptidase 7 (USP7). Patients suffering from this disorder experience developmental delay, intellectual disability, and speech impairment. The syndrome leads to autism spectrum disorder, epilepsy, gastrointestinal disorders, and deafness in some patients due to malformation of the cochlea. USP7 activators are needed for the treatment of Hao-Fountain Syndrome and other disorders associated with USP7 haploinsufficiency.

[0003] Certain substituted 7 - oxo-4, 5,6,7 -tetrahydro- 1 -thia-3 b, 6- diazacyclopenta [a] indene- 5 - carboxamides and 7-oxo-4,5,6,7-tetrahydro-l-oxa-3b,6-diazacyclopenta[a]indene-5-carboxamides are described in a library of potential physiologically active compounds (WO 2005 / 10585, US 2007 / 0191337), but their ability to activate USP7 has heretofore not been appreciated.SUMMARY OF THE INVENTION

[0004] An aspect of the present invention is a method for activating USP7 in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Formula:where:R1is methyl or ethyl;R2is cyclohexyl, cycloheptyl, or cyclooctyl;R3is -(CH2)n-R5;R4is H or methyl;R5is H, Ci - C3 alkoxy, morpholin-l-yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, or -N(Ci - C3 alkyl)2;n is 1 to 5; andX is O or S; or a pharmaceutically acceptable salt thereof.

[0005] Another aspect of the present invention provides a method for treating disorders associated with USP7 haploinsufficiency comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0006] Another aspect of the present invention provides a method for treating Hao-Fountain Syndrome comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0007] 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 associated with USP7 haploinsufficiency.

[0008] 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 Hao-Fountain Syndrome.

[0009] Another aspect of the invention is a compound of Formula I for use in the treatment of a disorder associated with USP7 haploinsufficiency.

[0010] Another aspect of the invention is a compound of Formula I for use in the treatment of Hao-Fountain Syndrome.

[0011] Another aspect of the invention is to provide USP7 activators of Formula:where:R1is methyl or ethyl;R2is Ci - Cs alkyl, benzyl, phenyl optionally substituted with Ci - C3 alkyl, or Ce - C10 cycloalkyl optionally substituted with hydroxy;R3is -(CH2)n-R5;R4is H, methyl, or hydroxymethyl;R5is H, OH, Ci - C3 alkoxy, CHO, morpholin-l-yl, azetidin- 1 -yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, pyrazol-l-yl, -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or -NH(CH2)2-R6;R6is -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or NHC(O)(Ci - C3 alkyl); and n' is 1 to 5; or a pharmaceutically acceptable salt thereof provided that when R2is cyclohexyl, cycloheptyl, or cyclooctyl then R5is OH, CHO, azetidin- 1-yl, pyrazol-l-yl, -NH(Ci - C3 alkyl), or -NH(CH2)2-R6’.

[0012] Another aspect of the invention is to provide USP7 activators of Formula II, or a pharmaceutically acceptable salt thereof, wherein R1is methyl.

[0013] Another aspect of the invention is to provide USP7 activators of Formula II, or a pharmaceutically acceptable salt thereof, wherein R1is methyl and R4is H.

[0014] Another aspect of the invention is to provide USP7 activators of Formula II, or a pharmaceutically acceptable salt thereof, wherein R1is methyl, R4is H, and n’ is 2 to 5.

[0015] Another aspect of the invention is to provide USP7 activators of Formula II, or a pharmaceutically acceptable salt thereof, wherein R1is methyl, R4is H, n is 2 to 5, and R5is morpholin-l-yl, azetidin- 1-yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or -NH(CH2)2-R6’.

[0016] Another aspect of the invention is to provide USP7 activators of Formula II, or a pharmaceutically acceptable salt thereof, wherein R1is methyl, R2is phenyl optionally substituted with Ci - C3 alkyl, R4is H, n’ is 2 to 5, and R5is morpholin-l-yl, azetidin- 1 -yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, -NH(Ci - C3 alkyl), -N(Ci - C3 alky 1)2, or -NH(CH2)2-R6.

[0017] Another aspect of the invention is to provide USP7 activators of Formula II, or a pharmaceutically acceptable salt thereof, wherein R1is methyl, R2is Ce - C10 cycloalkyl, R4is H, n is 2 to 5, and R5is morpholin-l-yl, azetidin- 1-yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or -NH(CH2)2-R6’.

[0018] Another aspect of the present invention provides a pharmaceutical composition comprising a USP7 activator compound of Formula II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0019] A further aspect of the present invention provides a compound of Formula II or a pharmaceutically acceptable salt thereof for use in therapy.

[0020] Another aspect of the present invention provides a method for treating disorders associated with USP7 haploinsufficiency comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0021] Another aspect of the present invention provides a method for treating Hao-Fountain Syndrome comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0022] 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 associated with USP7 haploinsufficiency.

[0023] 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 Hao-Fountain Syndrome.DESCRIPTION OF THE DRAWINGS

[0024] To assist those of ordinary skills in the relevant art in making and using the subject matter hereof, reference is made to the appended drawing, wherein:

[0025] FIGURE 1 demonstrates USP7 activation by A-Cycloheptyl-7-(2-(dimethylamino)ethyl)- 6-methyl-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide (Example 1) as assessed by measuring covalent labeling by a ubiquitin probe.DETAILED DESCRIPTION

[0026] 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.

[0027] The term “Ci - C3 alkyl” is taken to mean a straight or branched alkyl chain of from 1 to 3 carbon atoms and includes methyl, ethyl, propyl, and isopropyl. Similarly, the term “Ci - Cs alkyl” is taken to mean a straight or branched alkyl chain of from 1 to 8 carbon atoms and includes ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl, hexyl, heptyl, octyl and the like.

[0028] The term “Ci - C3 alkoxy” is taken to mean an oxygen atom bonded to a Ci - C3 alkyl moiety and includes methoxy, ethoxy, propoxy, isopropoxy and the like.

[0029] The term “Ce - C10 cycloalkyl” is taken to mean a carbocyclic ring of from 6 to 10 carbon atoms and includes cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl, as well as all bridged fused bicyclic and spirofused isomers thereof such as bicyclo[4.4.0]decyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[4.2.0]decyl, spiro[2.4]heptyl, spiro[5.4]decyl, adamantyl and the like.

[0030] The term “cyclohexyl” is taken to mean

[0031] The term “cycloheptyl” is taken to mean ^ZZ^.

[0032] The term “cyclooctyl” is taken to mean ^ZZZ

[0033] The moiety “-N(Ci - C3 alkyl)2” is taken to mean a nitrogen atom substituted with two Ci - C3 alkyl groups independently selected from methyl, ethyl, propyl, and isopropyl.

[0034] The term “patient” means mammal and “mammal” includes, but is not limited to, a human.

[0035] “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 activate USP7 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.

[0036] The terms “treatment”, “treat”, and “treating” are meant to include the full spectrum of pharmaceutical intervention for a patient suffering from USP7 haploinsufficiency, such as administration of a USP7 activator 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.

[0037] 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).

[0038] 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.

[0039] 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).

[0040] It will be understood that compounds of Formula I and Formula II have a chiral center and may be depicted as single stereoisomers as illustrated below:(S)-enantiomer Racemate (R)-enantiomer

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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).

[0045] 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. 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 tE 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. The reagents and starting materials are readily available to one of ordinary skill in the art.

[0046] Certain abbreviations are defined as follows: “h” refers to hour(s); “LC-MS” refers to liquid chromatography-mass spectrometry; “MeOH” refers to methanol; “EtOH” refers to ethanol.

[0047] The compounds of the present invention exemplified below were prepared essentially by the method described in Alexey P. Ilyin, et al., Eur. J. Org. Chem. 2005, 21, 4670-4679 as illustrated in Scheme 1, where variables R1, R2, R3, and R4are as previously defined and Y is a suitable ester forming moiety such as methyl, ethyl, isopropyl, tert-butyl, benzyl, and the like.Scheme 1

[0048] Briefly, an appropriate furo- or thieno-fused pyrrole-5-carboxylic acid ester (i) is reacted with an appropriate a-haloketone (ii), such as chloroacetone, l-bromo-2-butanone, or iodoacetone, in the presence of a suitable base, such as K2CO3, in a suitable solvent, such as 1,4-dioxane in the presence of a crown ether such as 18-crown-6 at reflux for up to 7 hours to provide intermediate ester (iii). Ester (iii) is subjected to appropriate ester cleavage conditions to provide the corresponding carboxylic acid (iv). The skilled person will appreciate that the specific ester cleavage conditions employed will depend upon the Y moiety. Where Y is methyl or ethyl, for example, base mediated ester cleavage, such as reaction with KOH in a suitable solvent such as EtOH at about 70°C for about 6 hours, would be appropriate. Intermediate (iv) is then reacted with an appropriate isonitrile (v) and a suitable amine (vi) in an appropriate solvent, such as MeOH, at 40°C for at least 5 h to provide a compound of the invention. Intermediate (i) is prepared from 3-bromofuran-2-carboxaldehyde (X =O) or 3-bromothiophene-2-carboxaldehyde (X = S) essentially by the method described in Hang Zhao, et al., Org. Process Res. Dev. 2014, 18, 198-204. The requisite carboxaldehyde are either commercially available or may be prepared by methods well known to the skilled artisan.Example 1 / V-Cycloheptyl-7-(2-(dimethylamino)ethyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0049] 'H NMR (500 MHz, DMSO-rfe) 5 8.22 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 6.68 (dd, J = 2.3, 0.9 Hz, 1H), 6.58 (d, J = 0.8 Hz, 1H), 4.67 (d, J = 12.4 Hz, 1H), 4.02 (d, J = 12.4 Hz, 1H), 3.72 (m, 1H), 3.65 - 3.43 (m, 2H), 2.48 - 2.38 (m, 2H), 2.23 (s, 6H), 1.60 (s, 3H), 1.52 - 1.23 (m, 12H).Example 2N-Cycloheptyl-6-methyl-7-(2-(methylamino)ethyl)-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0050] LCMS (ESI) [M+H]+calcd for C21H30N4O3+ m / z 387.2, found m / z 387.1Example 3 7-(2-((2-Acetamidoethyl)amino)ethyl)-N-cycloheptyl-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0051] 'H NMR (400 MHz, DMSO-de) 5 8.78 (d, J = 7.9 Hz, 1H), 7.82 (t, J = 5.4 Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H), 6.70 (dd, J = 2.2, 0.9 Hz, 1H), 6.59 (d, J = 0.8 Hz, 1H), 4.67 (d, J = 12.2 Hz, 1H),3.97 (m, 2H), 3.60 - 3.40 (m, 2H), 3.22 (m, 1H), 3.06 (m, 1H), 2.80 (m, 1H), 2.69 - 2.56 (m, 3H), 1.82 (s, 3H), 1.65 - 1.12 (m, 15H).Example 4 7-(2-(Dimethylamino)ethyl)- / V-(4-ethylphenyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0052] 'H NMR (400 MHz, MeOD) 5 7.56 (d, J = 2.3 Hz, 1H), 7.25 - 7.17 (d, J = 8.4, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.71 (s, 1H), 6.56 (d, J = 2.3 Hz, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.28 (m, 1H), 4.10 (d, 7 = 12.5 Hz, 1H), 3.62 - 3.51 (m, 1H), 2.79 (m, 1H), 2.65 - 2.51 (m, 3H), 2.33 (s, 6H), 1.84 (s, 3H), 1.16 (t, J = 7.6 Hz, 3H). LCMS (ESI) [M+H]+calcd for C23H28N4O3+ m / z 409.2, found m / z 409.1.Example 5 -Cycloheptyl-7-(2-((2-(dimethylamino)ethyl)amino)ethyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0053] LCMS (ESI) [M+H]+calcd for C24H37N5O3+ m / z 444.3, found m / z 444.3Example 62V-Cycloheptyl-7-(2-hydroxyethyl)-6-methyl-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2- a]pyrazine-6-carboxamide

[0054] 'H NMR (400 MHz, DMSO) 5 7.86 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H), 6.66 (dd, 7= 2.3, 0.9 Hz, 1H), 6.57 (d, 7 = 0.8 Hz, 1H), 4.91 (t, 7= 5.0 Hz, 1H), 4.66 (d, 7= 12.6 Hz, 1H), 4.06 (d, 7= 12.6 Hz, 1H), 3.65 - 3.47 (m, 5H), 1.57 (s, 3H), 1.54 - 1.20 (m, 12H).

[0055] LCMS (ESI) [M+H]+calcd for C20H27N3O4+ m / z 374.2, found m / z 374.0.Example 72V-Cycloheptyl-7-(3-hydroxypropyl)-6-methyl-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2- a]pyrazine-6-carboxamide

[0056] 'H NMR (400 MHz, DMSO) 5 7.72 (d, J = 2.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H), 6.64 (d, 7 = 2.2 Hz, 1H), 6.56 (s, 1H), 4.65 (d, 7 = 12.9 Hz, 1H), 4.49 (d, 7 = 5.9 Hz, 1H), 4.10 (d, 7 = 12.9 Hz, 1H), 3.65 (dt, 7= 15.1, 7.7 Hz, 2H), 3.43-3.22 (m, 3H), 1.71 (q, 7 = 7.0 Hz, 2H), 1.59 (s, 3H), 1.53 - 1.21 (m, 12H).

[0057] LCMS (ESI) [M+H]+calcd for C21H29N3O4+ m / z 388.2, found m / z 388.2.Example 8 -Benzyl-7-(2-(dimethylamino)ethyl)-6-methyl-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2- a]pyrazine-6-carboxamide

[0058] LCMS (ESI) [M+H]+calcd for C22H26N4O3+ m / z 395.2, found m / z 395.3Example 9 7-(2-(Dimethylamino)ethyl)-6-methyl-8-oxo- / V-(tetrahydro-27 / -pyran-4-yl)-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0059] 'H NMR (400 MHz, DMSO) 5 8.34 (d, 7 = 7.5 Hz, 1H), 7.72 (d, 7 = 2.3 Hz, 1H), 6.68 (d, 7 = 2.3 Hz, 1H), 6.58 (s, 1H), 4.66 (d, 7= 12.5 Hz, 1H), 4.04 (d, 7= 12.5 Hz, 1H), 3.78 - 3.66 (m, 3H), 3.60 (m, 1H), 3.54 - 3.43 (m, 1H), 3.22 (m, 2H), 2.50 - 2.38 (m, 2H), 2.22 (s, 6H), 1.61 (s, 3H), 1.54 - 1.19 (m, 4H).

[0060] LCMS (ESI) [M+H]+calcd for C20H28N4O4+ m / z 389.2, found m / z 389.2.Example 102V-Cycloheptyl-7-(2-methoxyethyl)-6-methyl-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2- a]pyrazine-6-carboxamide

[0061] LCMS (ESI) [M+H]+calcd for C21H29N3O4+ m / z 388.2, found m / z 386.2Example 112V-Cycloheptyl-6-methyl-8-oxo-7-(3-oxopropyl)-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2- a]pyrazine-6-carboxamide

[0062] LCMS (ESI) [M+H]+calcd for C21H27N3O4+ m / z 386.2, found m / z 386.1Example 127-(2-(Dimethylamino)ethyl)- / V-((lr,4r)-4-hydroxycyclohexyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0063] 'H NMR (400 MHz, DMSO) 5 8.19 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H), 6.67 (dd, = 2.2, 0.9 Hz, 1H), 6.56 (d, J = 0.9 Hz, 1H), 4.66 (d, J = 12.4 Hz, 1H), 4.02 (d, J = 12.5 Hz, 1H), 3.70 (m, 1H), 3.45 (m, 2H), 3.25 (s, 1H), 3.15 (s, 3H), 2.46 - 2.36 (m, 2H), 2.21 (s, 6H), 1.67 (s, 2H), 1.60 (s, 3H), 1.41 - 1.15 (m, 6H).

[0064] LCMS (ESI) [M+H]+calcd for C22H32N4O4+ m / z 417.3, found m / z 417.2.Example 132V-Cycloheptyl-6-methyl-8-oxo-7-(2-(piperidin-l-yl)ethyl)-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0065] LCMS (ESI) [M+H]+calcd for C25H37N4O3+ 441.29 m / z, found m / z 441.23Example 14 -Cycloheptyl-7-(2-(dimethylamino)ethyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrothieno[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0066] 'H NMR (500 MHz, CDCh) 5 7.36 (d, J = 5.4 Hz, 1H), 7.18 (s, 1H), 6.92 (d, J = 5.4 Hz,1H), 6.44 (d, J = 8.0 Hz, 1H), 4.79 (d, J = 12.5 Hz, 1H), 4.07 (d, J = 12.5 Hz, 1H), 4.05 - 3.92 (m, 2H), 3.82 (m, 1H), 3.42 (dt, J = 12.4, 7.5 Hz, 1H), 3.30 (dt, J = 12.8, 6.6 Hz, 1H), 2.99 (s, 6H), 1.74 (s, 3H), 1.64 - 1.29 (m, 12H).Example 15 JV-Cycloheptyl-6-methyl-8-oxo-7-(2-(pyrrolidin-l-yl)ethyl)-5,6,7,8- tetrahydrothieno[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0067] LCMS (ESI) [M+H]+calcd for C24H35N4O2S+ m / z 443.25, found m / z 443.09Example 16N-((lr,3r,5r,7r)-Adamantan-2-yl)-7-(2-(dimethylamino)ethyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0068] LCMS (ESI) [M+H]+calcd for C25H35N4O3+ m / z 439.27, found m / z 438.57Example 17 2V-Cycloheptyl-7-(5-(dimethylamino)pentyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0069] 'H NMR (500 MHz, DMSO) 5 7.71 (d, J = 2.1 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 6.64 (dd, 7 = 2.2, 1.0 Hz, 1H), 6.56 (d, J = 0.9 Hz, 1H), 4.60 (d, 7 = 12.8 Hz, 1H), 4.10 (d, 7 = 12.8 Hz, 1H), 3.75 - 3.49 (m, 2H), 3.16 - 3.07 (m, 1H), 2.17 (t, 7= 7.3 Hz, 2H), 2.10 (s, 6H), 1.63 - 1.21 (m, 21 H).

[0070] LCMS (ESI) [M+H]+calcd for C25H39N4O3+ m / z 443.30, found m / z 443.53.Example 18 / V-Cycloheptyl-7-(2-(dimethylamino)ethyl)-6-ethyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0071] 'H NMR (500 MHz, DMSO) 5 8.31 (d, 7 = 7.8 Hz, 1H), 7.72 (d, 7 = 2.2 Hz, 1H), 6.72 (dd, 7 = 2.3, 0.8 Hz, 1H), 6.57 (d, 7 = 0.8 Hz, 1H), 4.67 (d, 7 = 12.6 Hz, 1H), 4.13 (d, 7 = 12.7 Hz, 1H), 3.69 - 3.57 (m, 2H), 3.42 - 3.36 (m, 1H), 2.59 (m, 1H), 2.48 - 2.43 (m, 1H), 2.20 (s, 6H), 2.12 - 2.05 (m, 1H), 1.91 (dq, 7 = 14.2, 7.1 Hz, 1H), 1.60 - 1.32 (m, 12H), 0.92 (t, 7 = 7.4 Hz, 3H).

[0072] LCMS (ESI) [M+H]+calcd for C23H35N4O3+ m / z 415.27, found m / z 414.78.Example 197-(2-(Azetidin-l-yl)ethyl)- / V-cycloheptyl-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0073] 'H NMR (500 MHz, DMSO) 5 8.63 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 2.2 Hz, 1H), 6.69 (dd, 7= 2.3, 0.9 Hz, 1H), 6.58 (s, 1H), 4.66 (d, J = 12.2 Hz, 1H), 3.96 (d, J = 12.3 Hz, 1H), 3.80 (m, 1H), 3.25 - 3.15 (m, 6H), 2.59 (m, 2H), 1.96 (m, 2H), 1.56 - 1.28 (m, 15H).

[0074] LCMS (ESI) [M+H]+calcd for C23H33N4O3+ m / z 413.25, found m / z 413.43.Example 20 2V-Cycloheptyl-7-(3-(dimethylamino)propyl)-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0075] 'H NMR (500 MHz, DMSO) 5 7.71 (t, J = 2.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 6.64 (d, J= 2.2 Hz, 1H), 6.56 (s, 1H), 4.61 (d, J = 12.7 Hz, 1H), 4.11 (d, J = 12.7 Hz, 1H), 3.67 - 3.55 (m, 2H), 3.16 (m, 1H), 2.21 (m, 2H), 2.12 (s, 6H), 1.69 (dd, J = 8.8, 5.5 Hz, 2H), 1.57 (s, 3H), 1.49 (m, 4H), 1.43 - 1.21 (m, 8H).

[0076] LCMS (ESI) [M+H]+calcd for C23H35N4O3+ m / z 415.27, found m / z 415.02.Example 212V-Cycloheptyl-6-methyl-7-(2-morpholinoethyl)-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2- a]pyrazine-6-carboxamide

[0077] LCMS (ESI) [M+H]+calcd for C24H35N4O4+ m / z 443.27, found m / z 442.5Example 22 7-(2-(Dimethylamino)ethyl)-6-methyl-8-oxo- / V-(2,4,4-trimethylpentan-2-yl)-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0078] LCMS (ESI) [M+H]+calcd for C23H37N4O3+ m / z 417.29, found m / z 417.11Example 237-(2-(lH-Pyrazol-l-yl)ethyl)- / V-cycloheptyl-6-methyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0079] LCMS (ESI) [M+H]+calcd for C23H30N5O3+ m / z 424.23, found m / z 423.91Example 24 -Cycloheptyl-7-(2-(dimethylamino)ethyl)-2,6-dimethyl-8-oxo-5,6,7,8- tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6-carboxamide

[0080] 1H NMR (500 MHz, CDCh) 5 6.73 (s, 1H), 6.35 (d, J = 8.1 Hz, 1H), 6.04 (s, 1H), 4.60 (d, J = 12.5 Hz, 1H), 3.99 (m, 1H), 3.95 - 3.82 (m, 2H), 3.79 (m, 1H), 3.38 (m, 1H), 3.25 (m, 1H), 2.96 (br, 6H), 2.39 (d, J= 1.0 Hz, 3H), 1.80 - 1.20 (m, 15H).

[0081] LCMS (ESI) [M+H]+calcd for C23H35N4O3+ 415.27 m / z, found m / z 415.02.Example 25A-Cycloheptyl-6,7-dimethyl-8-oxo-5,6,7,8-tetrahydrofuro[2',3':4,5]pyrrolo[l,2-a]pyrazine-6- carboxamideUbiquitin-Rhodamine Biochemistry

[0083] pET28aLIC-TJSP7 (His-tagged, aa 208-560) (“USP7”) was prepared essentially as described in: V area, A. et al..Identification and validation of selective deubiquitinase inhibitors. Cell Chem. Biol. 28 (12), 16 December 2021, 1758-1771.

[0084] Enzyme was tested with a Ubiquitin-Rhodamine assay with either test compounds at various concentrations or DMSO. USP7 (25 nM) was incubated with test compound or DMSO for 30 min at room temperature in 50 mM Tris pH 8.0, 50 mM NaCl, 5 mM TCEP, 0.002% Tween20. Ubiquitin- Rhodamine (Bio-Techne) was added to a final concentration of 500 nM to initiate the reaction. The enzyme initial velocity was collected over a 20 min period with half minute intervals using a CLARIOstar microplate reader (BMG Labtech) at excitation: 487 nm and emission: 535 nm. The linear regression of the initial velocity (RFU / min) was extracted and plotted against test compound concentrations, using GraphPad Prism. Initial velocities were normalized to the enzyme’s baseline activity with DMSO (n=2). The results of these experiments are shown in Table 1.Table 1Ubiquitin-probe labeling experiment

[0085] USP7 covalent labeling by ubiquitin probe was assessed by gel with and without Example 1. To 4 pM USP7 in PBS buffer was added either Example 1 at various concentrations or DMSO.Ubiquitin-proparglyamine (UbiQ, CAT#UbiQ-076) was added to a final concentration of 40 pM. Reactions were incubated at room temperature for 2 hours and quenched with 10% 2-mercaptoethanol (BME) in LDS Sample Buffer. The SDS-PAGE gel was run with BOLT MES buffer at 175V for 30 minutes, then stained with InstantBlue Coomassie Protein Stain (Abeam). The gel was destained with milliQ water and imaged on Amersham Imager 600. The results of this experiment are shown in Figure 1 where [MS-1] is the concentration of the compound of Example 1 in pM.

Claims

CLAIMSWe claim:

1. A method for activating USP7 in a patient in need thereof comprising administering a therapeutically effective amount of a compound of Formula:where:R1is methyl or ethyl;R2is cyclohexyl, cycloheptyl, or cyclooctyl;R3is -(CH2)n-R5;R4is H or methyl;R5is H, Ci - C3 alkoxy, morpholin-l-yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, or -N(Ci - C3 alkyl)2; n is 1 to 5; andX is O or S; or a pharmaceutically acceptable salt thereof.

2. A method for treating disorders associated with USP7 haploinsufficiency comprising administering to a patient in need thereof a therapeutically effective amount of a compound ofFormulawhere:R1is methyl or ethyl;R2is cyclohexyl, cycloheptyl, or cyclooctyl;R3is -(CH2)n-R5;R4is H or methyl;R5is H, Ci - C3 alkoxy, morpholin-l-yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, or -N(Ci - C3 alkyl)2; n is 1 to 5; andX is O or S; or a pharmaceutically acceptable salt thereof.

3. The use of a compound of Formulawhere:R1is methyl or ethyl;R2is cyclohexyl, cycloheptyl, or cyclooctyl;R3is -(CH2)n-R5;R4is H or methyl;R5is H, Ci - C3 alkoxy, morpholin-l-yl, pyrrolidin-l-yl, piperidin-l-yl, homopiperidin-l-yl, or -N(CI - C3alkyl)2; n is 1 to 5; andX is O or S; or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of disorders associated with USP7 haploinsufficiency.

4. A compound of Formulawhere:R1is methyl or ethyl;R2is cyclohexyl, cycloheptyl, or cyclooctyl;R3is -(CH2)n-R5;R4is H or methyl;R5is H, Ci - C3 alkoxy, morpholin- 1 -yl, pyrrolidin- 1 -yl, piperidin-l-yl, homopiperidin-l-yl, orN(Ci - C3 alky 1)2; n is 1 to 5; andX is O or S; or a pharmaceutically acceptable salt thereof for use in the treatment of a disorder associated with USP7 haploinsufficiency.

5. A method or use of any of Claims 1 - 5 where the disorder is Hao-Fountain Syndrome.

6. A method or use of any of Claims 1 - 5 where R1is methyl, R4is H, and n is 2 to 5.

7. A method or use of any of Claims 1 - 6 where R5is morpholin- 1 -yl, pyrrolidin- 1 -yl, piperidin- 1-yl, homopiperidin-l-yl, or -N(Ci - C3 alkyl)2.

8. A compound of Formulawhere:R1is methyl or ethyl;R2is Ci - Cs alkyl, benzyl, phenyl optionally substituted with Ci - C3 alkyl, or Ce - C10 cycloalkyl optionally substituted with hydroxy;R3is -(CH2)n-R5;R4is H, methyl, or hydroxymethyl;R5is H, OH, Ci - C3 alkoxy, CHO, morpholin-l-yl, azetidin-l-yl, pyrrolidin-l-yl, piperidin- 1-yl, homopiperidin-l-yl, pyrazol-l-yl, -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or -NH(CH2)2-R6;R6is -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or NHC(O)(Ci - C3 alkyl); andN’ is 1 to 5; or a pharmaceutically acceptable salt thereof provided that when R2is cyclohexyl, cycloheptyl, or cyclooctyl then R5is OH, CHO, azetidin-l-yl, pyrazol-l-yl, -NH(Ci - C3 alkyl), or -NH(CH2)2-R6.

9. A compound of Claim 8 or a pharmaceutically acceptable salt thereof wherein R1is methyl.

10. A compound of any of Claims 8 or 9 or a pharmaceutically acceptable salt thereof wherein R4is H.

11. A compound of any of Claims 8 - 10 or a pharmaceutically acceptable salt thereof wherein n’ is 2 - 5.

12. A compound of any of Claims 8 - 11 or a pharmaceutically acceptable salt thereof wherein R5is morpholin-l-yl, azetidin-l-yl, pyrrolidin-l-yl, piperidin- 1 -yl, homopiperidin-l-yl, -NH(Ci - C3 alkyl), -N(Ci - C3 alkyl)2, or -NH(CH2)2-R6’.

13. A compound of any of Claims 8 - 12 or a pharmaceutically acceptable salt thereof wherein R2is phenyl optionally substituted with Ci - C3 alkyl.

14. A compound of any of Claims 8 - 12 or a pharmaceutically acceptable salt thereof wherein R2is Ce - C10 cycloalkyl.

15. A pharmaceutical composition comprising a compound of any of Claims 8 - 14 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

16. A compound of any of Claims 8 - 14 or a pharmaceutically acceptable salt thereof for use in therapy.

17. The use of a compound of any of Claims 8 - 14 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a disorder associated with USP7 haploinsufficiency.

18. The use of a compound of any of Claims 8 - 14 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of Hao-Fountain Syndrome.

19. A method for treating disorders associated with USP7 haploinsufficiency comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of Claims 8 - 14 or a pharmaceutically acceptable salt thereof.

20. A method for treating Hao - Fountain Syndrome comprising administering to a patient in need thereof an effective amount of a compound of any of Claims 8 - 14 or a pharmaceutically acceptable salt thereof.

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