`19) World Intellectual Propert
`=
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`ower Organization “pens
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`=
`International Bureau
`——
`(43) International Publication Date —
`22 October 2020 (22.10.2020) WIPO! PCT
`
`UID IOAN MIANANTA
`(10) International Publication Number
`WO 2020/212350 Al
`
`1) International Patent Classification:
`CO7D 413/12 (2006.01)
`A61P 35/00 (2006.01)
`A61K 31/421 (2006.01)
`(21) International Application Number:
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`PCT/EP2020/060467
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`(22) International Filing Date:
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`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`1905371.9
`
`14 April 2020 (14.04.2020)
`English
`English
`
`16 April 2019 (16.04.2019)
`
`GB
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FL FR, GB, GR, HR, HU,IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR,NE, SN, TD, TG).
`Published:
`— with international search report (Art. 21(3))
`
`(71) Applicant: MISSION THERAPEUTICS LIMITED
`[GB/GB]; Babraham Hall, Babraham, Cambridge CB22
`3AT (GB).
`
`(72) Inventors: THOMPSON, Paul William; c/o Mission
`Therapeutics Limited, Babraham Hall, Babraham, Cam-
`bridge CB22 3AT (GB). LUCKHURST,Christopher An-
`drew; c/o Mission Thcrapcutics Limited, Babraham Hall,
`Babraham, Cambridge CB22 3AT (GB). KEMP, Mark
`Ian; c/o Mission Therapeutics Limited, Babraham Hall,
`Babraham, Cambridge CB22 3AT (GB). JONES, Alison,
`c/o Mission Therapeutics Limited, Babraham Hall, Babra-
`ham, Cambridge CB22 3AT (GB).
`
`(74) Agent: TLIP LTD; 14 King Street, Leeds Yorkshire LS1
`2HL (GB).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO,AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,
`DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT, HN,
`HR, HU,ID, IL, IN,IR,IS, JO, JP. KE, KG,KH, KN, KP,
`KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NL, NO, NZ,
`OM,PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW.
`
`(54) Title: SUBSTITUTED CYANOPYRROLIDINES WITH ACTIVITY AS USP30 INHIBITORS
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`(57) Abstract: The present invention relates to a class of substitut-
`ed-cyanopyrrolidines with activity as inhibitors of the deubiquitylating
`enzyme USP30, having utility in a variety of therapeutic areas, includ-
`ing conditions involving mitochondrial dysfunction, cancer and fibro-
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`sis: (I).
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`WO 2020/212350
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`PCT/EP2020/060467
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`SUBSTITUTED CYANOPYRROLIDINES WITH ACTIVITY AS USP30 INHIBITORS
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`FIELD OF THE INVENTION
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`The present invention rclatcs to a class of substitutcd-cyanopyrrolidincs with activity as inhibitors of
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`the deubiquitylating enzyme ubiquitin C-terminal hydrolase 30, also known as ubiquitin specific
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`peptidase 30 (USP30), uses thereof, processes for the preparation thereof and composition containing
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`said inhibitors. These inhibitors have utility in a variety of therapeutic areas, including conditions
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`involving mitochondrial dysfunction, cancer and fibrosis.
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`All documents cited or relied upon below are expressly incorporated herein by reference.
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`BACKGROUNDOF THE INVENTION
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`Ubiquitin is a small protein consisting of 76 aminoacidsthat is important for the regulation of protein
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`function in the cell. Ubiquitylation and deubiquitylation are enzymatically mediated processes by
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`which ubiquitin is covalently bound or cleaved from a target protcin by dcubiquitylating cnzymcs
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`(DUBs), of which there are approximately 100 DUBs in human cells, divided into sub-families based
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`on sequence homology. The USP family are characterised by their common Cys and His boxes which
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`contain Cys and His residues critical for their DUB activities. The ubiquitylation and deubiquitylation
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`processes have been implicated in the regulation of many cellular functions including cell cycle
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`progression, apoptosis, modification of cell surface receptors, regulation of DNA transcription and
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`DNArepair. Thus, the ubiquitin system has been implicated in the pathogenesis of numerous disease
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`states
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`including inflammation, viral
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`infection, metabolic dysfunction, CNS disorders,
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`and
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`oncogcncsis.
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`Ubiquitin is a master regulator of mitochondrial dynamics. Mitochondria are dynamic organelles
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`whose biogenesis, fusion and fission events are regulated by the post-translational regulation via
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`ubiquitylation of many key factors such as mitofusins. In humans, USP30 is a 517 aminoacid protein
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`which 1s found in the mitochondrial outer membrane (Nakamuraet al, 2008, Mol Biol 19:1903-11). It
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`is the sole deubiquitylating enzyme bearing a mitochondrial addressing signal and has been shown to
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`deubiquitylate a number of mitochondrial proteins.
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`It has been demonstrated that USP30 opposes
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`parkin-mediated mitophagy and that reduction of USP30 activity can rescue parkin-mediated defects
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`in mitophagy (Bingol et al, 2015, Nature 510:370-5; Gersch et al, 2017, Nat Struct Mol Biol 24(11):
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`920-930; Cunningham et al, 2015, Nat Cell Biol 17(2): 160-169). USP30 inactivation can also
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`increase mitochondrial protein import, potentially through ubiquitylation of TOM proteins (Jacoupy
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`et al, 2019, Sci Rep 9(1): 11829). A small proportion of USP30 has been localized to peroxisomes,
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`which are generated through fusion of mitochondrial and ER vesicles, with USP30 potentially
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`antagonizing the Pex2/pexophagy pathway (Riccio et al, 2019, J Cell Biol 218(3): 798-807). The
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`E3 Ub ligase March5 and the deubiquitinase USP30 associate with the translocase and regulate
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`mitochondrial import, and while MarchS opposes mitochondrial import and directs degradation of
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`substrates, USP30 deubiquitinates substrates to promote their import (Phu et al, 2020, Molecular Cell
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`77, 1107-1123).
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`Mitochondrial dysfunction can be defined as diminished mitochondrial content (mitophagy or
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`mitochondrial biogenesis), as a decrease in mitochondrial activity and oxidative phosphorylation, but
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`also as modulation of reactive oxygen species (ROS) generation. Hence a role for mitochondrial
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`dysfunctions in a very large number of aging processes and pathologies.
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`For example, Parkinson’s disease affects around 10 million people worldwide (Parkinson’s Disease
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`Foundation) and is characterised by the loss of dopaminergic neurons in the substantia nigra. The
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`exact mechanisms underlying PD are unclear; however mitochondrial dysfunction is increasingly
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`appreciated as a key determinant of dopaminergic neuronal susceptibility in PD and is a feature of
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`both familial and sporadic disease, as well as in toxin-induced Parkinsonism. Parkin is one of a
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`numberof protcins that have becn implicated with carly onsct PD. Whilc most PD cascsare linked to
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`defects in alpha-synuclein, 10% of Parkinson’s cases are linked to specific genetic defects, one of
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`which is in the ubiquitin E3 ligase parkin. Parkin and the protem kinase PTEN-induced putative
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`kinase
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`(PINK1) collaborate to ubiquitylate mitochondrial membrane proteins of damaged
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`mitochondria resulting in mitophagy. Dysregulation of mitophagy results in increased oxidative
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`stress, which has been described as a characteristic of PD.
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`Inhibition of USP30 could therefore be a
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`potential strategy for the treatment of PD. For example, PD patients with parkin mutations leading to
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`reduced activity could be therapeutically compensated by inhibition of USP30.
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`It has been reported that depletion of USP30 enhances mitophagic clearance of mitochondria and also
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`enhances parkin-induced cell death. USP30 has also been shown to regulate BAX/BAK-dependent
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`apoptosis independently of parkin overexpression. Depletion of USP30 sensitises cancer cells to BH-
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`3 mimetics such as ABT-737, without the need for parkin overexpression. Thus, an anti-apoptotic
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`role has been demonstrated for USP30 and USP30 is therefore a potential target for anti-cancer
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`therapy.
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`The ubiquitin-proteasome system has gained interest as a target for the treatment of cancer following
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`the approval of the proteasome inhibitor bortezomib (Velcade®) for the treatment of multiple
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`myeloma. Extended treatment with bortezomib is limited by its associated toxicity and drug
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`resistance. However, therapeutic strategies that target specific aspects of the ubiquitin-proteasome
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`pathway upstream of the proteasome, such as DUBs,are predicted to be better tolerated (Bedford et
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`al, 2011, Nature Rev 10:29-46).
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`Fibrotic diseases, including renal, hepatic and pulmonary fibrosis, are a leading cause of morbidity
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`and mortality and can affect all tissues and organ systems. Fibrosis is considered to be the result of
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`acute or chronic stress on the tissue or organ, characterized by extracellular matrix deposition,
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`reduction of vascular/tubule/duct/airway patency and impairment of function ultimately resulting in
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`organ failure. Many fibrotic conditions are promoted by lifestyle or environmental factors; however,
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`a proportion of fibrotic conditions can be initiated through genetic triggers or indeed are considered
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`idiopathic (i.e. without a known cause). Certain fibrotic disease, such as idiopathic pulmonary
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`fibrosis (IPF), can be treated with non-specific kinase inhibitor (nintedanib) or drugs without a well-
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`characterized mechanism of action (pirfenidone). Other treatments for organ fibrosis, such as kidney
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`or liver fibrosis, alleviate pressure on the organ itself (e.g. beta blockers for cirrhosis, angiotensin
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`receptor blockers for chronic kidney disease). Attention to lifestyle factors, such as glucose and diet
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`control, may also influence the course and severity of disease.
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`Mitochondrial dysfunction has been implicated in a numberoffibrotic diseases, with oxidative stress
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`downstream of dysfunction being the key pathogenic mediator, alongside decreased ATP production.
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`In preclinical models, disruption of the mitophagy pathway (through mutation or knockout of either
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`parkin or PINK1) exacerbates lung fibrosis and kidney fibrosis, with evidence of increased oxidative
`stress.
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`Kurita et al, 2017, Respiratory Research 18:114, discloses that accumulation of profibrotic
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`myofibroblasts is a crucial process for fibrotic remodelling in IPF. Recent findings are said to show
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`participation of autophagy/mitophagy, part of the lysosomal degradation machinery,
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`in IPF
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`pathogenesis, and that mitophagy has been implicated in myofibroblast differentiation through
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`regulating mitochondrial reactive oxygen species (ROS)-mediated platelet-derived growth factor
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`receptor (PDGFR)activation. Kurita’s results suggested that pirfenidone induces PARK2-mediated
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`mitophagy and also inhibits lung fibrosis developmentin the setting of insufficient mitophagy, which
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`mayat least partly explain the anti-fibrotic mechanisms for IPF treatment.
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`Williams et al, 2015, Pharmacol Res. December; 102: 264-269, discuss the role of PINK1-Parkin-
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`mediated autophagy in protecting against alcohol and acetaminophen-induced liver
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`injury by
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`removing damaged mitochondria via mitophagy.
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`It is suggested that pharmacological stabilization of
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`USP8 or inactivation of USP15 and USP30 may be potential therapeutic targets for upregulating
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`Parkin-induced mitophagy and in tum protect against drug-induced liver injury. However, it is noted
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`that the DUBs are regulated both transcriptionally and post-translationally, which may make drug
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`development for targeting these specific enzymes challenging, and in addition, phosphorylated
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`ubiquitin was shown to be resistant to DUBs. The authors conclude that upregulating PINK1
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`stabilization or kinase activity may be a more effective target than inhibiting DUBs.
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`Williamset al, 2015, Biomolecules 5, 2619-2642, and Williamset al, 2015, Am J Physiol Gastrointest
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`Liver Physiol 309: G324—-G340,
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`review mechanisms involved in regulation of mitochondrial
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`homeostasis in the liver and how these mechanisms may protect against alcohol-inducedliver disease.
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`Series of derivatives of cyano-substituted heterocycles are disclosed as deubiquitylating enzyme
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`inhibitors in PCT applications WO 2016/046530 (US 15/513125, US 15/894025, US 16/448066),
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`WO 2016/156816 (US 15/558632, US 16/297937, US 16/419558, US 16/419747, US 16/788446),
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`WO 2017/009650
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`(US 15/738900), WO 2017/093718
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`(US 15/776149), WO 2017/103614
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`(US 15/781615), WO 2017/149313
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`(US 16/078518), WO 2017/109488
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`(US 16/060299),
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`WO 2017/141036 (US 16/087515),©WO 2017/158381(US 16/070936), WO 2017/163078
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`(US 16/080229), WO 2017/158388
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`(US 16/080506), WO 2018/065768
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`(US 16/336685),
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`WO 2018/060742
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`(US 16/336202), WO 2018/060689
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`(US 16/334836), WO 2018/060691
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`(US 16/336363), WO 2018/220355 (US 16/615040), and WO 2018/234755 (US 16/615709), each of
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`which are expressly incorporated herein by reference. PCT application WO 2019/171042, which is
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`expressly incorporated herein by reference, discloses the use of substituted-cyanopyrrolidines as
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`inhibitors of USP30 for the treatment of fibrotic diseases.
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`Falgueyret et al, 2001, J.Med.Chem. 44, 94-104, and PCT application WO 01/77073 refer to
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`cyanopyrrolidines as inhibitors of Cathepsins K and L, with potential utility in treating osteoporosis
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`and other bone-resorption related conditions.
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`PCT application WO 2015/179190 refers
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`to
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`N-acylethanolamine hydrolysing acid amidase inhibitors, with potential utility in treating ulcerative
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`colitis and Crohn’s disease. PCT application WO 2013/0302 18 refers to quinazolin-4-one compounds
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`as inhibitors of ubiquitin specific proteases, such as USP7, with potential utility in treating cancer,
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`neurodegenerative diseases,
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`inflammatory disorders
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`and viral
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`infections.
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`PCT applications
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`WO 2015/017502 and WO 2016/019237 refer to inhibitors of Bruton’s tyrosine kinase with potential
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`utility in treating discasc such as autoimmune discasc,
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`inflammatory discasc and canccr.
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`PCT
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`applications WO 2009/026197, WO 2009/129365, WO 2009/129370, and WO 2009/129371, refer to
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`cyanopyrrolidines as inhibitors of Cathepsin C with potential utility in treating COPD. United States
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`patent application US 2008/0300268 refers to polyaromatic compounds as inhibitors of tyrosine
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`kinase receptor PDGFR.
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`PCT applications WO 2019/222468 and WO 2019/071073 refer to
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`cyanamide-containing compounds as USP30 inhibitors.
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`PCT application WO 2015/183987, refers to pharmaceutical compositions comprising deubiquitinase
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`inhibitors and human serum albumin in methods oftreating cancer, fibrosis, an autoimmune disease
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`or condition, an inflammatory disease or condition, a neurodegenerative disease or condition or an
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`infection.
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`It is noted that deubiquitinases, including UCHL5/UCH37, USP4, USP9X, USP11 and
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`USPIS5, are said to have been implicated in the regulation of the TGF-beta signalling pathway, the
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`disruption of which gives rise to neurodegenerative and fibrotic diseases, autoimmune dysfunction
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`and cancer.
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`PCT application WO 2006/067165 refers to a method for treating fibrotic diseases using indolinone
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`kinase inhibitors. PCT application WO 2007/119214 refers to a method for treating carly stage
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`pulmonary fibrosis using an endothelin receptor antagonist. PCT application WO 2012/170290 refers
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`to a method for treating fibrotic diseases using THC acids. PCT application WO 2018/213150 refers
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`to sulfonamide USP30 inhibitors with potential utility in the treatment of conditions involving
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`mitochondnial defects. Larson-Casey et al, 2016, Immunity 44, 582-596, concerns macrophage Akt1
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`kinase-mediated mitophagy, apoptosis resistance and pulmonary fibrosis. Tang et al, 2015, Kidney
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`Diseases 1, 71-79, reviews the potential role of mitophagy in renal pathophysiology.
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`There exists a need for safe, alternative, and/or improved methods and compositions for the treatment
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`or prevention of conditions involving mitochondrial dysfunction, cancer and fibrosis, and the various
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`symptoms and conditions associated therewith. While not wishing to be bound by any particular
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`theory or mechanism, it is believed that the compounds of the present invention act to inhibit the
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`enzyme USP30, which in tur upregulates Parkin-induced mitophagy.
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`Acute Kidney Injury (AKI) is defined as an abrupt decrcasc in kidney function occurring over 7 days
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`or less, with severity of injury staged based on increased serum creatinine (SCr) and decreased urine
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`output as described in the Kidney Disease Improving Global Outcomes (KDIGO) guidelines. AKI
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`occurs in about 13.3 million pcople per year, 85% of whom live in the developing world andit is
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`thought to contribute to about 1.7 million deaths every year (Mehta et al, 2015, Lancet 385(9987):
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`2616-2643). AKT more than likely results in permanent kidney damage(1.¢., chronic kidney disease;
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`CKD) and may also result in damage to non-renal organs. AKIis a significant public health concern
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`particularly when considering the absolute numberof patients developing incident CKD, progressive
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`CKD, end-stage renal disease and cardiovascular events.
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`AKI and CKD are viewed as a continuum on the same disease spectrum (Chawlaet al, 2017, Nat Rev
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`Nephrol 13(4): 241-257). Patients undergoing coronary artery bypass graft (CABG)are at high risk
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`for kidney injury. There is an obvious unmet medical need in the development of medicinal products
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`for the treatment and/or prevention of AKT.
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`The kidney is a site of high metabolic demand, with high mitophagy rates demonstrated in vivo
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`(McWilliams ct al, 2018, Cell Mctab 27(2): 439-449 c435). Renal Proximal Tubule Epithclial Cells
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`(RPTECs), a cell
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`type with significant ATP requirement for solute/ion exchange, are rich in
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`mitochondria and are the primary effector cells of Acute Kidney Injury (AKI)
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`in the kidney.
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`Mitochondrial dysfunction has been implicated in AKI/CKD mechanisms, both through multiple lines
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`of evidence from preclinical AKI and CKD models and also through data demonstrating abnormal
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`mitochondrial phenotypes in patient biopsies (Emma et al, 2016, Nat Rev Nephrol 12(5): 267-280;
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`Eirin et al, 2017, Handb Exp Pharmacol 240: 229-250). Furthermore, Primary mitochondrial disease
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`often manifest in renal symptoms, such as focal segmental glomerulosclerosis (Kawakamiet al, 2015,
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`J Am Soc Nephrol 26(5): 1040-1052) in patients with MELAS/MIDD, and also primary tubular
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`pathologies in patients with Coenzyme Q deficiencies. Mutations in mtDNA can cause matemally
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`inherited tubulointerstitial disease (Connoret al, 2017, PLoS Genet 13(3): ¢1006620).
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`Regarding mitochondnal quality control in renal injury (Tang et al, 2018, Autophagy 14(5): 880-897)
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`demonstrated that renal injury was exacerbated following ischemic AKI in both PINKI KO and
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`PARK2 KO mice, suggesting that PINK1/PARKIN-mediated mitophagy plays a protective role
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`following IRI in the kidney.
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`In addition, parkin/PINK1 mitophagy protects against cisplatin induced
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`kidney injury (Wanget al, 2018, Cell Death Dis 9(11): 1113). Limited models of CKD are available
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`for mitophagy investigation, supportive evidence for mitochondrial quality control in fibrosis comes
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`from studies on fibrotic lung conditions such as COPD and IPF. Parkin knockout animals show
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`exacerbated lung fibrosis in response to bleomycin (Kobayashiet al, 2016, J Immunol, 197:504-516).
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`Similarly, airway epithelial cells from parkin knockout (KO) animals show exacerbated fibrotic and
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`senescent responses to cigarette smoke (Arayaet al, 2019, Autophagy 15(3): 510-526).
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`Preclinical models are available to study potential novel therapeutics, through their ability to model
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`fibrosis pathology (e.g. collagen deposition) consistent with the human condition. Preclinical models
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`can be toxin-mediated (e.g. bleomycin for lung and skin fibrosis), surgical (e.g. 1ischemia/reperfusion
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`injury model and unilateral ureter obstruction model for acute tubulointerstitial fibrosis), and genetic
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`(e.g. diabetic (db/db) mice for diabetic nephropathy). For example, both examples previously given
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`for indicated IPF treatments (nintedanib and pirfenidone) show efficacy in the bleomycin lung fibrosis
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`model.
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`Accordingly, there is a need for compounds that are inhibitors of USP30 for the treatment or
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`prevention of conditions where inhibition of USP30 is indicated. In particular, there exists a need for
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`USP30 inhibitors that have suitable and/or improved properties in order to maximise efficacy against
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`the target disease.
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`SUMMARYOF THE INVENTION
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`The present invention is directed to compoundsof formula (1D:
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`a tautomerthereof, or a pharmaceutically acceptable salt of said compoundor tautomer, wherein:
`R!is selected from (C\-C,)alkyl, (Ci-C,)fluoroalkyl, CH2OCH3 and CH:N(CHs)2;
`R’ is selected from hydrogen and methyl; and
`R?, R*, R° and R°®are each independently selected from hydrogen, deuterium andfluorine.
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`The present invention is also directed to uses of the compounds of formula (1), particularly in the
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`treatment of conditions involving mitochondrial dysfunction, cancer and fibrosis, and also processes
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`for the preparation thereof and pharmaceutical compositions containing said compounds.
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`DETAILED DESCRIPTION OF THE INVENTION
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`The present invention is directed to USP30 inhibitors that have suitable and/or improved properties in
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`order to maximise efficacy against the target disease. Such properties include, for example, potency,
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`selectivity, physicochemical properties, ADME(absorption, distribution, metabolism and excretion)
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`properties, including PK (pharmacokinetic) profile, and safety profile.
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`It is generally desirable to maximise the potency of a drug molecule against the target enzyme in
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`relevant assays in order to lowerthe effective/efficacious dosage that is to be administered to patients.
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`Compounds of the invention may be tested for USP30 affinity using the in vitro biochemical
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`fluorescence polarization (FP) assay described herein.
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`USP30 is a transmembrane protein located in the outer membrane of mitochondria, which are energy-
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`producing organelles present inside cells. Therefore, being able to demonstrate cellular activity in
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`vitro is advantageous, as this is one of a number of components that may indicate a greater ability to
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`engage the target in its physiological setting, i.e. where the USP30 inhibitor compound is able to
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`penetrate cells. The USP30 cellular western blot (WB) assay described herein aimsto test the activity
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`of compounds against USP30 in cells using an irreversible activity probe to monitor USP30 activity.
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`Analogously to the cellular western blot assay, target engagement assessment (ex vivo) may be
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`carried out in either brain or kidney tissue samples from compound-dosed animals using the assay
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`described herein.
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`To extend target binding knowledge to downstream pharmacodynamics, assessment of TOM20 (an
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`outer mitochondrial membraneprotein) ubiquitylation may be made.
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`In general, it is important for a drug to be as selective as possible for its desired target enzyme;
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`additional activities give rise to the possibility of side effects. The exact physiological role of many
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`DUBs hasyet to be fully determined, however, irrespective of whatever role these DUBs may or may
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`not play, it is a sound medicinal chemistry precept to ensure that any drug hasselectivity over related
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`mechanistic targets of unknown physiological function. Representative examples of DUB enzymes
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`for which the compounds of the present invention may be screened against are UCHL1, UCHL3,
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`UCHLS, YOD1, SENP2, SENP6, TRABID, BAP1, Cezanne, MINDY2/FAM63B, OTU1, OTUD3,
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`OTUDS5, OTUD6A, OTUD6B, OTUB1/UBCH5B, OTUB2, CYLD, VCPIP, AMSH-LP, JOSD1,
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`JOSD2, USPI1/UAF1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP11,
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`USP12/UAF1, USP13, USP14, USP15, USP16, USP19, USP20, USP21, USP22, USP24, USP25,
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`USP28, USP32, USP34, USP35, USP36, USP45, USP46/UAF1, USP47 and USP48. Preferably,
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`compounds of the invention have good selectivity for USP30 over one or more of these DUB
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`enzymes.
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`Aside from selectivity over other DUB enzymes,it is important for a drug to have low affinity for
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`other targets, and pharmacological profiling may be performed against panels of targets to assess the
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`potential for, and to minimise, potential off-target effects. Examples of targets for which the
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`compounds of the present invention may be screened against are those of the industry standard
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`Eurofins-Cerep SafetyScreen44 panel, which includes 44 targets as a representative selection of
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`GPCR receptors, transporters, ion channels, nuclear receptors, and kinase and non-kinase enzymes.
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`Preferably, compounds of the invention have insignificant affinity against targets of this screening
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`panel. Further examples of targets for which the compounds of the present invention may be screened
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`against are kinases of the Thermo Fisher SelectScreen kinase profiling panel, which includes 39
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`targets as a representative selection of kinase enzymes. Preferably, compounds of the invention have
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`insignificant affinity against targets of this screening panel. Additionally, examples of a particular
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`enzyme class for which the compounds of the present invention may be screened against are the
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`cathepsins (e.g. cathepsin A, B, C, H, K, L, L2, 8, V and Z). Preferably, compoundsof the invention
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`have good selectivity for USP30 over one or more of these enzymes.
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`There is also a need for compounds that have favourable pharmacokinetic properties such that they
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`are suitable for oral administration. An orally administered drug should have good bioavailability;
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`that is an ability to readily cross the gastrointestinal (GI) tract and not be subject to extensive
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`metabolism as 1t passes from the GItract into the systemic circulation. Once a drugis in the systemic
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`circulation the rate of metabolism is also important in determining the time of residence of the drug in
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`the body.
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`Thus, it is clearly favourable for drug molecules to have the properties of being readily able to cross
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`the GI tract and being only slowly metabolised in the body. The Caco-2 assay is a widely accepted
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`model for predicting the ability of a given molecule to cross the GI tract. The majority of metabolism
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`of drug molecules generally occurs in the liver, and in vitro assays using whole cell hepatocytes
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`(animal or human) are widely accepted methods for measuring the susceptibility of a given molecule
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`towards metabolism in the liver. Such assays aim to predict in vivo clearance from the hepatocyte
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`calculated clearance value.
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`Compounds which have good Caco-2 flux and are stable towards hepatocytes are predicted to have
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`good oral bioavailability (good absorption across the GI tract and minimal extraction of compound as
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`it passes through the liver) and a long residence time in the body that is sufficient for the drug to be
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`efficacious.
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`The solubility of a compound is an important factor in achieving a desired concentration of drug in
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`systemic circulation for the anticipated pharmacological response. Low aqueous solubility is a
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`problem encountered with formulation development of new chemical entities and to be absorbed a
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`drug must be present in the form of solution at the site of absorption. The kinetic solubility of a
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`compound may be measured using a turbidimetric solubility assay, the data from which may also be
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`used in conjunction with Caco-2 permeability data to predict dose dependent human intestinal
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`absorption.
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`Other parameters that may be measured using standard assays that are indicative of a compound’s
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`exposure profile include, for example plasma stability (half-life measurement), blood AUC, Cmax, Cmin
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`and Tmax values.
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`The treatment of CNS disorders,
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`including Alzhcimcr’s discasc, Parkinson’s discasc, and othcr
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`disorders described herein, requires drug molecules to target the brain, which requires adequate
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`penetration of the blood brain barrier. There is, therefore, a need for USP30 inhibitors that possess
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`effective blood brain penctration propcrtics and provide suitable residcnce timc in the brain to be
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`efficacious. The probability that a compound can cross the blood brain barrier may be measured by an
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`in vitro flux assay utilizmg a MDRI-MDCK cell monolayer (Madin-Darby Canine Kidney cells
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`transfected with MDR-1! resulting in overexpression of the human efflux transporter P-glycoprotein).
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`Additionally, exposure may also be measured directly in brain and plasma using in vivo animal
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`models.
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`There is also a need for compoundsthat have a favourable safety profile, which may be measured by a
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`variety of standard in vitro and in vivo methods. A cell toxicity counter-screen may be used to assay
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`the anti-proliferative/cytotoxic effect in a particular cell line (e.g. HCT116) by fluorometric detection
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`of rezasurin (alamarBlue) to resofurin in response to mitochondrial activity.
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`Toxicology and safety studies may also be conducted to identify potential target organs for adverse
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`effects and define the Therapcutic Index to sct the initial starting doscs in clinical trials. Regulatory
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`requirements generally require studies to be conducted in at least two laboratory animal species, one
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`rodent (rat or mouse) and one nonrodent (rabbit, dog, non-humanprimate, or other suitable species).
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`The bactenal reverse mutation assay (Ames Test) may be used to evaluate the mutagenic properties of
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`compoundsof the invention, commonly by using the bacterial strain Salmonclla typhimurium, which
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`is mutant for the biosynthesis of the aminoacid histidine.
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`The micronucleus assay may be used to determine if a compound is genotoxic by evaluating the
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`presence of micronuclei. Micronuclei may contain chromosome fragments produced from DNA
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`breakage (clastogens) or whole chromosomes produced by disruption of the mitotic apparatus
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`(aneugens).
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`The hERG predictor assay provides valuable information about
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`the possible binding of test
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`compounds to the potassium channel and potential QT prolongation on echocardiogram. Inhibition of
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`the hERG current causes QT interval prolongation resultng im potentially fatal ventricular
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`tachyarrhythmia (Torsades de Pointes). Typically, assay data may be generated from an automated
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`patch-clamp assay platform.
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`The present invention is therefore directed to USP30 inhibitors that have surtable and/or improved
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`properties in order to maximise efficacy against the target disease.
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`Such properties include, for
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`example, potency,
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`selectivity, physicochemical properties, ADME (absorption, distribution,
`
`metabolism and excretion) properties, including PK (pharmacokinetic) profile, and safety profile.
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`The compounds of the present invention have been found to demonstrate one or more of the above
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`identificd advantagcs over refercnee cxamplcs from the prior art sharing some structural similarity
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`that are both significant and unexpected. For instance, all of the Examples of the present invention
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`are significantly more potent for USP30 than the Reference Examples as measured in the biochemical
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`assay described hercin. All of the Examples of the present invention arc significantly more sclective
`
`for USP30 over other DUBs.
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`The significant and unexpected superiority of the compounds of the present invention make them
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`particularly suitable for use in the treatment and/or prevention of diseases linked to UP30 activity.
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`According to a first aspect, the present invention provides a compound of formula(I):
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`R5
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`R6
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`R2
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`R4
`
`N
`|
`
`VA
`©
`
`4
`Na)
`
`NC
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`3
`
`R
`
`4
`
`N
`
`=n
`
`zw
`
`'
`
`)
`
`20
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`a tautomerthereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein:
`R'is selected from (Ci-Ca)alkyl, (Ci-C,4)fluoroalkyl, CH2OCH; and CH»N(CHs3)»;
`R’is selected from hydrogen and methy]; and
`R’*, R*, R° and R° are each independently selected from hydrogen, deuterium andfluorine.
`
`The compound of formula (J) exists as a single stereois