(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`19) World Intellectual Propert
`=
`
`ower Organization “pens
`
`=
`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:
`
`PCT/EP2020/060467
`
`(22) International Filing Date:
`
`(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
`
`R5
`
`Re
`
`RR?
`
`R4
`
`NC
`
`po
`o
`
`N
`
`we
`
`3
`
`R
`
`(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-
`
`sis: (I).
`
`(D.
`
`(RY
`
`
`==N
`
`LDN —
`Qqu
`
`1
`
`
`
`
`
`wo2020/212350A.ITNIMININIIKITICITTIMMAAATA
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`
`SUBSTITUTED CYANOPYRROLIDINES WITH ACTIVITY AS USP30 INHIBITORS
`
`FIELD OF THE INVENTION
`
`The present invention rclatcs to a class of substitutcd-cyanopyrrolidincs with activity as inhibitors of
`
`the deubiquitylating enzyme ubiquitin C-terminal hydrolase 30, also known as ubiquitin specific
`
`peptidase 30 (USP30), uses thereof, processes for the preparation thereof and composition containing
`
`said inhibitors. These inhibitors have utility in a variety of therapeutic areas, including conditions
`
`involving mitochondrial dysfunction, cancer and fibrosis.
`
`All documents cited or relied upon below are expressly incorporated herein by reference.
`
`BACKGROUNDOF THE INVENTION
`
`Ubiquitin is a small protein consisting of 76 aminoacidsthat is important for the regulation of protein
`
`function in the cell. Ubiquitylation and deubiquitylation are enzymatically mediated processes by
`
`which ubiquitin is covalently bound or cleaved from a target protcin by dcubiquitylating cnzymcs
`
`(DUBs), of which there are approximately 100 DUBs in human cells, divided into sub-families based
`
`on sequence homology. The USP family are characterised by their common Cys and His boxes which
`
`contain Cys and His residues critical for their DUB activities. The ubiquitylation and deubiquitylation
`
`processes have been implicated in the regulation of many cellular functions including cell cycle
`
`progression, apoptosis, modification of cell surface receptors, regulation of DNA transcription and
`
`DNArepair. Thus, the ubiquitin system has been implicated in the pathogenesis of numerous disease
`
`states
`
`including inflammation, viral
`
`infection, metabolic dysfunction, CNS disorders,
`
`and
`
`oncogcncsis.
`
`Ubiquitin is a master regulator of mitochondrial dynamics. Mitochondria are dynamic organelles
`
`whose biogenesis, fusion and fission events are regulated by the post-translational regulation via
`
`ubiquitylation of many key factors such as mitofusins. In humans, USP30 is a 517 aminoacid protein
`
`which 1s found in the mitochondrial outer membrane (Nakamuraet al, 2008, Mol Biol 19:1903-11). It
`
`is the sole deubiquitylating enzyme bearing a mitochondrial addressing signal and has been shown to
`
`deubiquitylate a number of mitochondrial proteins.
`
`It has been demonstrated that USP30 opposes
`
`parkin-mediated mitophagy and that reduction of USP30 activity can rescue parkin-mediated defects
`
`in mitophagy (Bingol et al, 2015, Nature 510:370-5; Gersch et al, 2017, Nat Struct Mol Biol 24(11):
`
`920-930; Cunningham et al, 2015, Nat Cell Biol 17(2): 160-169). USP30 inactivation can also
`
`increase mitochondrial protein import, potentially through ubiquitylation of TOM proteins (Jacoupy
`
`et al, 2019, Sci Rep 9(1): 11829). A small proportion of USP30 has been localized to peroxisomes,
`
`which are generated through fusion of mitochondrial and ER vesicles, with USP30 potentially
`
`antagonizing the Pex2/pexophagy pathway (Riccio et al, 2019, J Cell Biol 218(3): 798-807). The
`
`E3 Ub ligase March5 and the deubiquitinase USP30 associate with the translocase and regulate
`
`mitochondrial import, and while MarchS opposes mitochondrial import and directs degradation of
`
`1
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`substrates, USP30 deubiquitinates substrates to promote their import (Phu et al, 2020, Molecular Cell
`
`77, 1107-1123).
`
`Mitochondrial dysfunction can be defined as diminished mitochondrial content (mitophagy or
`
`mitochondrial biogenesis), as a decrease in mitochondrial activity and oxidative phosphorylation, but
`
`also as modulation of reactive oxygen species (ROS) generation. Hence a role for mitochondrial
`
`dysfunctions in a very large number of aging processes and pathologies.
`
`For example, Parkinson’s disease affects around 10 million people worldwide (Parkinson’s Disease
`
`Foundation) and is characterised by the loss of dopaminergic neurons in the substantia nigra. The
`
`exact mechanisms underlying PD are unclear; however mitochondrial dysfunction is increasingly
`
`appreciated as a key determinant of dopaminergic neuronal susceptibility in PD and is a feature of
`
`both familial and sporadic disease, as well as in toxin-induced Parkinsonism. Parkin is one of a
`
`numberof protcins that have becn implicated with carly onsct PD. Whilc most PD cascsare linked to
`
`defects in alpha-synuclein, 10% of Parkinson’s cases are linked to specific genetic defects, one of
`
`which is in the ubiquitin E3 ligase parkin. Parkin and the protem kinase PTEN-induced putative
`
`kinase
`
`|
`
`(PINK1) collaborate to ubiquitylate mitochondrial membrane proteins of damaged
`
`mitochondria resulting in mitophagy. Dysregulation of mitophagy results in increased oxidative
`
`stress, which has been described as a characteristic of PD.
`
`Inhibition of USP30 could therefore be a
`
`potential strategy for the treatment of PD. For example, PD patients with parkin mutations leading to
`
`reduced activity could be therapeutically compensated by inhibition of USP30.
`
`It has been reported that depletion of USP30 enhances mitophagic clearance of mitochondria and also
`
`enhances parkin-induced cell death. USP30 has also been shown to regulate BAX/BAK-dependent
`
`apoptosis independently of parkin overexpression. Depletion of USP30 sensitises cancer cells to BH-
`
`3 mimetics such as ABT-737, without the need for parkin overexpression. Thus, an anti-apoptotic
`
`role has been demonstrated for USP30 and USP30 is therefore a potential target for anti-cancer
`
`10
`
`15
`
`20
`
`25
`
`therapy.
`
`The ubiquitin-proteasome system has gained interest as a target for the treatment of cancer following
`
`the approval of the proteasome inhibitor bortezomib (Velcade®) for the treatment of multiple
`
`myeloma. Extended treatment with bortezomib is limited by its associated toxicity and drug
`
`resistance. However, therapeutic strategies that target specific aspects of the ubiquitin-proteasome
`
`pathway upstream of the proteasome, such as DUBs,are predicted to be better tolerated (Bedford et
`
`al, 2011, Nature Rev 10:29-46).
`
`Fibrotic diseases, including renal, hepatic and pulmonary fibrosis, are a leading cause of morbidity
`
`and mortality and can affect all tissues and organ systems. Fibrosis is considered to be the result of
`
`acute or chronic stress on the tissue or organ, characterized by extracellular matrix deposition,
`
`reduction of vascular/tubule/duct/airway patency and impairment of function ultimately resulting in
`
`30
`
`35
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`organ failure. Many fibrotic conditions are promoted by lifestyle or environmental factors; however,
`
`a proportion of fibrotic conditions can be initiated through genetic triggers or indeed are considered
`
`idiopathic (i.e. without a known cause). Certain fibrotic disease, such as idiopathic pulmonary
`
`fibrosis (IPF), can be treated with non-specific kinase inhibitor (nintedanib) or drugs without a well-
`
`characterized mechanism of action (pirfenidone). Other treatments for organ fibrosis, such as kidney
`
`or liver fibrosis, alleviate pressure on the organ itself (e.g. beta blockers for cirrhosis, angiotensin
`
`receptor blockers for chronic kidney disease). Attention to lifestyle factors, such as glucose and diet
`
`control, may also influence the course and severity of disease.
`
`Mitochondrial dysfunction has been implicated in a numberoffibrotic diseases, with oxidative stress
`
`downstream of dysfunction being the key pathogenic mediator, alongside decreased ATP production.
`
`In preclinical models, disruption of the mitophagy pathway (through mutation or knockout of either
`
`parkin or PINK1) exacerbates lung fibrosis and kidney fibrosis, with evidence of increased oxidative
`stress.
`
`Kurita et al, 2017, Respiratory Research 18:114, discloses that accumulation of profibrotic
`
`myofibroblasts is a crucial process for fibrotic remodelling in IPF. Recent findings are said to show
`
`participation of autophagy/mitophagy, part of the lysosomal degradation machinery,
`
`in IPF
`
`pathogenesis, and that mitophagy has been implicated in myofibroblast differentiation through
`
`regulating mitochondrial reactive oxygen species (ROS)-mediated platelet-derived growth factor
`
`receptor (PDGFR)activation. Kurita’s results suggested that pirfenidone induces PARK2-mediated
`
`mitophagy and also inhibits lung fibrosis developmentin the setting of insufficient mitophagy, which
`
`mayat least partly explain the anti-fibrotic mechanisms for IPF treatment.
`
`Williams et al, 2015, Pharmacol Res. December; 102: 264-269, discuss the role of PINK1-Parkin-
`
`mediated autophagy in protecting against alcohol and acetaminophen-induced liver
`
`injury by
`
`removing damaged mitochondria via mitophagy.
`
`It is suggested that pharmacological stabilization of
`
`USP8 or inactivation of USP15 and USP30 may be potential therapeutic targets for upregulating
`
`Parkin-induced mitophagy and in tum protect against drug-induced liver injury. However, it is noted
`
`that the DUBs are regulated both transcriptionally and post-translationally, which may make drug
`
`development for targeting these specific enzymes challenging, and in addition, phosphorylated
`
`ubiquitin was shown to be resistant to DUBs. The authors conclude that upregulating PINK1
`
`stabilization or kinase activity may be a more effective target than inhibiting DUBs.
`
`Williamset al, 2015, Biomolecules 5, 2619-2642, and Williamset al, 2015, Am J Physiol Gastrointest
`
`Liver Physiol 309: G324—-G340,
`
`review mechanisms involved in regulation of mitochondrial
`
`homeostasis in the liver and how these mechanisms may protect against alcohol-inducedliver disease.
`
`Series of derivatives of cyano-substituted heterocycles are disclosed as deubiquitylating enzyme
`
`inhibitors in PCT applications WO 2016/046530 (US 15/513125, US 15/894025, US 16/448066),
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`WO 2016/156816 (US 15/558632, US 16/297937, US 16/419558, US 16/419747, US 16/788446),
`
`WO 2017/009650
`
`(US 15/738900), WO 2017/093718
`
`(US 15/776149), WO 2017/103614
`
`(US 15/781615), WO 2017/149313
`
`(US 16/078518), WO 2017/109488
`
`(US 16/060299),
`
`
`
`WO 2017/141036 (US 16/087515),©WO 2017/158381(US 16/070936), WO 2017/163078
`
`
`
`(US 16/080229), WO 2017/158388
`
`(US 16/080506), WO 2018/065768
`
`(US 16/336685),
`
`WO 2018/060742
`
`(US 16/336202), WO 2018/060689
`
`(US 16/334836), WO 2018/060691
`
`(US 16/336363), WO 2018/220355 (US 16/615040), and WO 2018/234755 (US 16/615709), each of
`
`which are expressly incorporated herein by reference. PCT application WO 2019/171042, which is
`
`expressly incorporated herein by reference, discloses the use of substituted-cyanopyrrolidines as
`
`10
`
`inhibitors of USP30 for the treatment of fibrotic diseases.
`
`Falgueyret et al, 2001, J.Med.Chem. 44, 94-104, and PCT application WO 01/77073 refer to
`
`cyanopyrrolidines as inhibitors of Cathepsins K and L, with potential utility in treating osteoporosis
`
`and other bone-resorption related conditions.
`
`PCT application WO 2015/179190 refers
`
`to
`
`N-acylethanolamine hydrolysing acid amidase inhibitors, with potential utility in treating ulcerative
`
`colitis and Crohn’s disease. PCT application WO 2013/0302 18 refers to quinazolin-4-one compounds
`
`as inhibitors of ubiquitin specific proteases, such as USP7, with potential utility in treating cancer,
`
`neurodegenerative diseases,
`
`inflammatory disorders
`
`and viral
`
`infections.
`
`PCT applications
`
`WO 2015/017502 and WO 2016/019237 refer to inhibitors of Bruton’s tyrosine kinase with potential
`
`utility in treating discasc such as autoimmune discasc,
`
`inflammatory discasc and canccr.
`
`PCT
`
`applications WO 2009/026197, WO 2009/129365, WO 2009/129370, and WO 2009/129371, refer to
`
`cyanopyrrolidines as inhibitors of Cathepsin C with potential utility in treating COPD. United States
`
`patent application US 2008/0300268 refers to polyaromatic compounds as inhibitors of tyrosine
`
`kinase receptor PDGFR.
`
`PCT applications WO 2019/222468 and WO 2019/071073 refer to
`
`cyanamide-containing compounds as USP30 inhibitors.
`
`PCT application WO 2015/183987, refers to pharmaceutical compositions comprising deubiquitinase
`
`inhibitors and human serum albumin in methods oftreating cancer, fibrosis, an autoimmune disease
`
`or condition, an inflammatory disease or condition, a neurodegenerative disease or condition or an
`
`infection.
`
`It is noted that deubiquitinases, including UCHL5/UCH37, USP4, USP9X, USP11 and
`
`USPIS5, are said to have been implicated in the regulation of the TGF-beta signalling pathway, the
`
`disruption of which gives rise to neurodegenerative and fibrotic diseases, autoimmune dysfunction
`
`and cancer.
`
`PCT application WO 2006/067165 refers to a method for treating fibrotic diseases using indolinone
`
`kinase inhibitors. PCT application WO 2007/119214 refers to a method for treating carly stage
`
`pulmonary fibrosis using an endothelin receptor antagonist. PCT application WO 2012/170290 refers
`
`to a method for treating fibrotic diseases using THC acids. PCT application WO 2018/213150 refers
`
`to sulfonamide USP30 inhibitors with potential utility in the treatment of conditions involving
`
`15
`
`20
`
`25
`
`30
`
`35
`
`4
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`mitochondnial defects. Larson-Casey et al, 2016, Immunity 44, 582-596, concerns macrophage Akt1
`
`kinase-mediated mitophagy, apoptosis resistance and pulmonary fibrosis. Tang et al, 2015, Kidney
`
`Diseases 1, 71-79, reviews the potential role of mitophagy in renal pathophysiology.
`
`There exists a need for safe, alternative, and/or improved methods and compositions for the treatment
`
`or prevention of conditions involving mitochondrial dysfunction, cancer and fibrosis, and the various
`
`symptoms and conditions associated therewith. While not wishing to be bound by any particular
`
`theory or mechanism, it is believed that the compounds of the present invention act to inhibit the
`
`enzyme USP30, which in tur upregulates Parkin-induced mitophagy.
`
`Acute Kidney Injury (AKI) is defined as an abrupt decrcasc in kidney function occurring over 7 days
`
`or less, with severity of injury staged based on increased serum creatinine (SCr) and decreased urine
`
`output as described in the Kidney Disease Improving Global Outcomes (KDIGO) guidelines. AKI
`
`occurs in about 13.3 million pcople per year, 85% of whom live in the developing world andit is
`
`thought to contribute to about 1.7 million deaths every year (Mehta et al, 2015, Lancet 385(9987):
`
`2616-2643). AKT more than likely results in permanent kidney damage(1.¢., chronic kidney disease;
`
`CKD) and may also result in damage to non-renal organs. AKIis a significant public health concern
`
`particularly when considering the absolute numberof patients developing incident CKD, progressive
`
`CKD, end-stage renal disease and cardiovascular events.
`
`AKI and CKD are viewed as a continuum on the same disease spectrum (Chawlaet al, 2017, Nat Rev
`
`Nephrol 13(4): 241-257). Patients undergoing coronary artery bypass graft (CABG)are at high risk
`
`for kidney injury. There is an obvious unmet medical need in the development of medicinal products
`
`for the treatment and/or prevention of AKT.
`
`The kidney is a site of high metabolic demand, with high mitophagy rates demonstrated in vivo
`
`(McWilliams ct al, 2018, Cell Mctab 27(2): 439-449 c435). Renal Proximal Tubule Epithclial Cells
`
`(RPTECs), a cell
`
`type with significant ATP requirement for solute/ion exchange, are rich in
`
`mitochondria and are the primary effector cells of Acute Kidney Injury (AKI)
`
`in the kidney.
`
`Mitochondrial dysfunction has been implicated in AKI/CKD mechanisms, both through multiple lines
`
`of evidence from preclinical AKI and CKD models and also through data demonstrating abnormal
`
`mitochondrial phenotypes in patient biopsies (Emma et al, 2016, Nat Rev Nephrol 12(5): 267-280;
`
`Eirin et al, 2017, Handb Exp Pharmacol 240: 229-250). Furthermore, Primary mitochondrial disease
`
`often manifest in renal symptoms, such as focal segmental glomerulosclerosis (Kawakamiet al, 2015,
`
`J Am Soc Nephrol 26(5): 1040-1052) in patients with MELAS/MIDD, and also primary tubular
`
`pathologies in patients with Coenzyme Q deficiencies. Mutations in mtDNA can cause matemally
`
`inherited tubulointerstitial disease (Connoret al, 2017, PLoS Genet 13(3): ¢1006620).
`
`Regarding mitochondnal quality control in renal injury (Tang et al, 2018, Autophagy 14(5): 880-897)
`
`demonstrated that renal injury was exacerbated following ischemic AKI in both PINKI KO and
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`PARK2 KO mice, suggesting that PINK1/PARKIN-mediated mitophagy plays a protective role
`
`following IRI in the kidney.
`
`In addition, parkin/PINK1 mitophagy protects against cisplatin induced
`
`kidney injury (Wanget al, 2018, Cell Death Dis 9(11): 1113). Limited models of CKD are available
`
`for mitophagy investigation, supportive evidence for mitochondrial quality control in fibrosis comes
`
`from studies on fibrotic lung conditions such as COPD and IPF. Parkin knockout animals show
`
`exacerbated lung fibrosis in response to bleomycin (Kobayashiet al, 2016, J Immunol, 197:504-516).
`
`Similarly, airway epithelial cells from parkin knockout (KO) animals show exacerbated fibrotic and
`
`senescent responses to cigarette smoke (Arayaet al, 2019, Autophagy 15(3): 510-526).
`
`Preclinical models are available to study potential novel therapeutics, through their ability to model
`
`10
`
`fibrosis pathology (e.g. collagen deposition) consistent with the human condition. Preclinical models
`
`can be toxin-mediated (e.g. bleomycin for lung and skin fibrosis), surgical (e.g. 1ischemia/reperfusion
`
`injury model and unilateral ureter obstruction model for acute tubulointerstitial fibrosis), and genetic
`
`(e.g. diabetic (db/db) mice for diabetic nephropathy). For example, both examples previously given
`
`for indicated IPF treatments (nintedanib and pirfenidone) show efficacy in the bleomycin lung fibrosis
`
`15
`
`model.
`
`Accordingly, there is a need for compounds that are inhibitors of USP30 for the treatment or
`
`prevention of conditions where inhibition of USP30 is indicated. In particular, there exists a need for
`
`USP30 inhibitors that have suitable and/or improved properties in order to maximise efficacy against
`
`the target disease.
`
`20
`
`SUMMARYOF THE INVENTION
`
`The present invention is directed to compoundsof formula (1D:
`
`RS
`
`Ré
`
`R2
`
`4
`
`R
`
`N
`|
`
`fo

`
`4
`Nar)
`
`NC
`
`3
`
`R
`
`6
`
`
`==N
`
`N
`
`aw
`
`'
`
`()
`
`25
`
`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.
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`The present invention is also directed to uses of the compounds of formula (1), particularly in the
`
`treatment of conditions involving mitochondrial dysfunction, cancer and fibrosis, and also processes
`
`for the preparation thereof and pharmaceutical compositions containing said compounds.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present invention is directed to USP30 inhibitors that have suitable and/or improved properties in
`
`order to maximise efficacy against the target disease. Such properties include, for example, potency,
`
`selectivity, physicochemical properties, ADME(absorption, distribution, metabolism and excretion)
`
`properties, including PK (pharmacokinetic) profile, and safety profile.
`
`It is generally desirable to maximise the potency of a drug molecule against the target enzyme in
`
`relevant assays in order to lowerthe effective/efficacious dosage that is to be administered to patients.
`
`Compounds of the invention may be tested for USP30 affinity using the in vitro biochemical
`
`fluorescence polarization (FP) assay described herein.
`
`USP30 is a transmembrane protein located in the outer membrane of mitochondria, which are energy-
`
`producing organelles present inside cells. Therefore, being able to demonstrate cellular activity in
`
`vitro is advantageous, as this is one of a number of components that may indicate a greater ability to
`
`engage the target in its physiological setting, i.e. where the USP30 inhibitor compound is able to
`
`penetrate cells. The USP30 cellular western blot (WB) assay described herein aimsto test the activity
`
`of compounds against USP30 in cells using an irreversible activity probe to monitor USP30 activity.
`
`Analogously to the cellular western blot assay, target engagement assessment (ex vivo) may be
`
`carried out in either brain or kidney tissue samples from compound-dosed animals using the assay
`
`described herein.
`
`To extend target binding knowledge to downstream pharmacodynamics, assessment of TOM20 (an
`
`outer mitochondrial membraneprotein) ubiquitylation may be made.
`
`In general, it is important for a drug to be as selective as possible for its desired target enzyme;
`
`additional activities give rise to the possibility of side effects. The exact physiological role of many
`
`DUBs hasyet to be fully determined, however, irrespective of whatever role these DUBs may or may
`
`not play, it is a sound medicinal chemistry precept to ensure that any drug hasselectivity over related
`
`mechanistic targets of unknown physiological function. Representative examples of DUB enzymes
`
`for which the compounds of the present invention may be screened against are UCHL1, UCHL3,
`
`UCHLS, YOD1, SENP2, SENP6, TRABID, BAP1, Cezanne, MINDY2/FAM63B, OTU1, OTUD3,
`
`OTUDS5, OTUD6A, OTUD6B, OTUB1/UBCH5B, OTUB2, CYLD, VCPIP, AMSH-LP, JOSD1,
`
`JOSD2, USPI1/UAF1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP11,
`
`USP12/UAF1, USP13, USP14, USP15, USP16, USP19, USP20, USP21, USP22, USP24, USP25,
`
`USP28, USP32, USP34, USP35, USP36, USP45, USP46/UAF1, USP47 and USP48. Preferably,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`compounds of the invention have good selectivity for USP30 over one or more of these DUB
`
`enzymes.
`
`Aside from selectivity over other DUB enzymes,it is important for a drug to have low affinity for
`
`other targets, and pharmacological profiling may be performed against panels of targets to assess the
`
`potential for, and to minimise, potential off-target effects. Examples of targets for which the
`
`compounds of the present invention may be screened against are those of the industry standard
`
`Eurofins-Cerep SafetyScreen44 panel, which includes 44 targets as a representative selection of
`
`GPCR receptors, transporters, ion channels, nuclear receptors, and kinase and non-kinase enzymes.
`
`Preferably, compounds of the invention have insignificant affinity against targets of this screening
`
`panel. Further examples of targets for which the compounds of the present invention may be screened
`
`against are kinases of the Thermo Fisher SelectScreen kinase profiling panel, which includes 39
`
`targets as a representative selection of kinase enzymes. Preferably, compounds of the invention have
`
`insignificant affinity against targets of this screening panel. Additionally, examples of a particular
`
`enzyme class for which the compounds of the present invention may be screened against are the
`
`cathepsins (e.g. cathepsin A, B, C, H, K, L, L2, 8, V and Z). Preferably, compoundsof the invention
`
`have good selectivity for USP30 over one or more of these enzymes.
`
`There is also a need for compounds that have favourable pharmacokinetic properties such that they
`
`are suitable for oral administration. An orally administered drug should have good bioavailability;
`
`that is an ability to readily cross the gastrointestinal (GI) tract and not be subject to extensive
`
`metabolism as 1t passes from the GItract into the systemic circulation. Once a drugis in the systemic
`
`circulation the rate of metabolism is also important in determining the time of residence of the drug in
`
`the body.
`
`Thus, it is clearly favourable for drug molecules to have the properties of being readily able to cross
`
`the GI tract and being only slowly metabolised in the body. The Caco-2 assay is a widely accepted
`
`model for predicting the ability of a given molecule to cross the GI tract. The majority of metabolism
`
`of drug molecules generally occurs in the liver, and in vitro assays using whole cell hepatocytes
`
`(animal or human) are widely accepted methods for measuring the susceptibility of a given molecule
`
`towards metabolism in the liver. Such assays aim to predict in vivo clearance from the hepatocyte
`
`calculated clearance value.
`
`Compounds which have good Caco-2 flux and are stable towards hepatocytes are predicted to have
`
`good oral bioavailability (good absorption across the GI tract and minimal extraction of compound as
`
`it passes through the liver) and a long residence time in the body that is sufficient for the drug to be
`
`efficacious.
`
`The solubility of a compound is an important factor in achieving a desired concentration of drug in
`
`systemic circulation for the anticipated pharmacological response. Low aqueous solubility is a
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`problem encountered with formulation development of new chemical entities and to be absorbed a
`
`drug must be present in the form of solution at the site of absorption. The kinetic solubility of a
`
`compound may be measured using a turbidimetric solubility assay, the data from which may also be
`
`used in conjunction with Caco-2 permeability data to predict dose dependent human intestinal
`
`absorption.
`
`Other parameters that may be measured using standard assays that are indicative of a compound’s
`
`exposure profile include, for example plasma stability (half-life measurement), blood AUC, Cmax, Cmin
`
`and Tmax values.
`
`The treatment of CNS disorders,
`
`including Alzhcimcr’s discasc, Parkinson’s discasc, and othcr
`
`disorders described herein, requires drug molecules to target the brain, which requires adequate
`
`penetration of the blood brain barrier. There is, therefore, a need for USP30 inhibitors that possess
`
`effective blood brain penctration propcrtics and provide suitable residcnce timc in the brain to be
`
`efficacious. The probability that a compound can cross the blood brain barrier may be measured by an
`
`in vitro flux assay utilizmg a MDRI-MDCK cell monolayer (Madin-Darby Canine Kidney cells
`
`transfected with MDR-1! resulting in overexpression of the human efflux transporter P-glycoprotein).
`
`Additionally, exposure may also be measured directly in brain and plasma using in vivo animal
`
`models.
`
`There is also a need for compoundsthat have a favourable safety profile, which may be measured by a
`
`variety of standard in vitro and in vivo methods. A cell toxicity counter-screen may be used to assay
`
`the anti-proliferative/cytotoxic effect in a particular cell line (e.g. HCT116) by fluorometric detection
`
`of rezasurin (alamarBlue) to resofurin in response to mitochondrial activity.
`
`Toxicology and safety studies may also be conducted to identify potential target organs for adverse
`
`effects and define the Therapcutic Index to sct the initial starting doscs in clinical trials. Regulatory
`
`requirements generally require studies to be conducted in at least two laboratory animal species, one
`
`rodent (rat or mouse) and one nonrodent (rabbit, dog, non-humanprimate, or other suitable species).
`
`The bactenal reverse mutation assay (Ames Test) may be used to evaluate the mutagenic properties of
`
`compoundsof the invention, commonly by using the bacterial strain Salmonclla typhimurium, which
`
`is mutant for the biosynthesis of the aminoacid histidine.
`
`The micronucleus assay may be used to determine if a compound is genotoxic by evaluating the
`
`presence of micronuclei. Micronuclei may contain chromosome fragments produced from DNA
`
`breakage (clastogens) or whole chromosomes produced by disruption of the mitotic apparatus
`
`(aneugens).
`
`The hERG predictor assay provides valuable information about
`
`the possible binding of test
`
`compounds to the potassium channel and potential QT prolongation on echocardiogram. Inhibition of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2020/212350
`
`PCT/EP2020/060467
`
`the hERG current causes QT interval prolongation resultng im potentially fatal ventricular
`
`tachyarrhythmia (Torsades de Pointes). Typically, assay data may be generated from an automated
`
`patch-clamp assay platform.
`
`The present invention is therefore directed to USP30 inhibitors that have surtable and/or improved
`
`properties in order to maximise efficacy against the target disease.
`
`Such properties include, for
`
`example, potency,
`
`selectivity, physicochemical properties, ADME (absorption, distribution,
`
`metabolism and excretion) properties, including PK (pharmacokinetic) profile, and safety profile.
`
`The compounds of the present invention have been found to demonstrate one or more of the above
`
`identificd advantagcs over refercnee cxamplcs from the prior art sharing some structural similarity
`
`that are both significant and unexpected. For instance, all of the Examples of the present invention
`
`are significantly more potent for USP30 than the Reference Examples as measured in the biochemical
`
`assay described hercin. All of the Examples of the present invention arc significantly more sclective
`
`for USP30 over other DUBs.
`
`The significant and unexpected superiority of the compounds of the present invention make them
`
`particularly suitable for use in the treatment and/or prevention of diseases linked to UP30 activity.
`
`According to a first aspect, the present invention provides a compound of formula(I):
`
`10
`
`15
`
`R5
`
`R6
`
`R2
`
`R4
`
`N
`|
`
`VA

`
`4
`Na)
`
`NC
`
`3
`
`R
`
`4
`
`N
`
`=n
`
`zw
`
`'
`
`)
`
`20
`
`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

Accessing this document will incur an additional charge of $.

After purchase, you can access this document again without charge.

Accept $ Charge

This document could not be displayed.

We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.

You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.

Set your membership status to view this document.

With a Docket Alarm membership, you'll get a whole lot more, including:

  • Up-to-date information for this case.
  • Email alerts whenever there is an update.
  • Full text search for other cases.
  • Get email alerts whenever a new case matches your search.

Become a Member

One Moment Please

The filing “” is large (MB) and is being downloaded.

Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!

If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document

We are unable to display this document, it may be under a court ordered seal.

If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.


Access Government Site

We are redirecting you
to a mobile optimized page.

We are unable to display this document.

PTO Denying Access

Refresh this Document
Go to the Docket