SUBSTITUTED CYANOPYRROLIDINES WITH ACTIVITY AS USP30 INHIBITORS

Abstract

The present invention relates to a class of substituted-cyanopyrrolidines with activity as inhibitors of the deubiquitylating enzyme USP30, having utility in a variety of therapeutic areas, including conditions involving mitochondrial dysfunction, cancer and fibrosis: (I).

##STR00001##

Claims

1. A compound of formula (I), ##STR00020## or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein: R.sup.1 is selected from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)fluoroalkyl and CH.sub.2OCH.sub.3; R.sup.2, R.sup.3 and R.sup.4 are each independently selected from hydrogen and fluorine; and R.sup.5 is selected from hydrogen, fluorine, (C.sub.1-C.sub.4)alkoxy and cyclopropoxy.

2. The compound according to claim 1, wherein R.sup.1 is selected from methyl, CH.sub.2F, CHF.sub.2, CF.sub.3 and CH.sub.2OCH.sub.3.

3. The compound according to claim 2, wherein R.sup.1 is methyl.

4. The compound according to claim 1, wherein R.sup.2, R.sup.3 and R.sup.4 are each hydrogen.

5. The compound according to claim 1, wherein R.sup.5 is selected from hydrogen, fluorine, methoxy and cyclopropoxy.

6. The compound according to claim 5, wherein R.sup.5 is hydrogen.

7. The compound according to claim 1, which is selected from: N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide; N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide; N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide; and 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide; or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

8-10. (canceled)

11. A method for the treatment or prevention of a condition involving mitochondrial dysfunction, cancer, or fibrosis, comprising the step of administering an effective amount of a compound according to claim 1, or tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, to a patient in need thereof.

12. The method according claim 11, wherein the condition involving mitochondrial dysfunction is selected from a CNS disorder; neurodegenerative disease; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis; Huntington's disease; ischemia; stroke; dementia with Lewy bodies; frontotemporal dementia; multiple sclerosis; mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome; materially-inherited diabetes and deafness; Leber's hereditary optic neuropathy; cancer; neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome; Danon disease; diabetes; diabetic nephropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; psychiatric diseases, schizophrenia; multiple sulfatase deficiency; mucolipidosis II; mucolipidosis III; mucolipidosis IV; GM1-gangliosidosis; neuronal ceroid-lipofuscinoses; Alpers disease; Barth syndrome; beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome; CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency; Leigh disease or syndrome; Leigh Syndrome French Canadian variant; lethal infantile cardiomyopathy; Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrogenase deficiency; myoclonic epilepsy and ragged-red fiber syndrome; mitochondrial cytopathy; mitochondrial recessive ataxia syndrome; mitochondrial DNA depletion syndrome; myoneurogastrointestinal disorder and encephalopathy; Pearson syndrome; pyruvate dehydrogenase deficiency; pyruvate carboxylase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; and very long-chain acyl-CoA dehydrogenase deficiency; peroxisomal disorders; methylmalonic acidemia; and age-dependent decline in cognitive function and muscle strength.

13. The method, according to claim 12, wherein the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, ischemia, stroke, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia; and Parkinson's disease related to mutations in α-synuclein, parkin, PINK1, GBA, and LRRK2, and autosomal recessive juvenile Parkinson's disease where parkin is mutated.

14. The method, according to claim 12, wherein the neurodegenerative disease is Leigh syndrome or disease, X-linked Leigh's disease, Leigh Syndrome French Canadian Variant, and/or the symptoms associated with Leigh's disease.

15. The method, according to claim 11, wherein the cancer is selected from breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, bone, liver, soft tissue, cancers of tissue organs, cancers of the blood cells, CML, AML, mantle cell lymphoma, neuroblastoma, melanoma, soft tissue sarcoma, liposarcoma, fibroblastic sarcoma, leiomyosarcoma, hepatocellular carcinoma, osteosarcoma, oesophageal cancer, leukaemia, lymphoma, multiple myeloma, metastatic carcinoma, osteosarcoma, chondosarcoma, Ewing's sarcoma, nasopharyngeal carcinoma, colorectal cancer, colorectal cancer, non-small cell lung carcinoma, cancer where apoptotic pathways are dysregulated, and cancer where proteins of the BCL-2 family are mutated, or over or under expressed.

16. The method, according to claim 11, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with the accumulation of extracellular matrix constituents that occurs following trauma, inflammation, tissue repair, immunological reactions, cellular hyperplasia, and neoplasia.

17. The method according to claim 16, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with major organ diseases, fibroproliferative disorders, and scarring associated with trauma.

18. The method according to claim 17, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with interstitial lung disease, liver cirrhosis, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis, kidney disease, acute kidney injury, chronic kidney disease, delayed kidney graft function, heart or vascular disease, diseases of the eye, systemic and local scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, chemotherapeutics drug-induced fibrosis, radiation-induced fibrosis, accidental injury and burns, retroperitoneal fibrosis, and peritoneal fibrosis/peritoneal scarring.

19. The method according to claim 18, wherein the fibrosis associated with interstitial lung disease is selected from sarcoidosis, silicosis, drug reactions, infections, collagen vascular diseases, rheumatoid arthritis, systemic sclerosis, scleroderma, pulmonary fibrosis, idiopathic pulmonary fibrosis, usual interstitial pneumonitis, interstitial lung disease, cryptogenic fibrosing alveolitis, bronchiolitis obliterans, and bronchiectasis.

20. The method according to claim 18, wherein the kidney disease is acute kidney injury or chronic kidney disease.

21. A pharmaceutical composition comprising a compound of formula (I) as defined in claim 1, or a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, together with one or more pharmaceutically acceptable excipients.

22. A compound, which is selected from formulae (II) and (III): ##STR00021## wherein PG is a protecting group and R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined for the compound of formula (I) in any one of claims 1 to 7, a tautomer thereof, or a salt of said compound or tautomer; and wherein the protecting group is preferably selected from tert-butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyl carbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethoxycarbonyl, 4-nitrobenzenesulfonyl and 2-nitrophenyl sulfenyl.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0033] 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.

[0034] It is generally desirable to maximise the potency of a drug molecule against the target enzyme in relevant assays in order to lower the 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.

[0035] 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 aims to 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.

[0036] To extend target binding knowledge to downstream pharmacodynamics, assessment of TOM20 (an outer mitochondrial membrane protein) ubiquitylation may be made.

[0037] 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 has yet 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 has selectivity 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, UCHL5, YOD1, SENP2, SENP6, TRABID, BAP1, Cezanne, MINDY2/FAM63B, OTU1, OTUD3, OTUD5, OTUD6A, OTUD6B, OTUB1/UBCH5B, OTUB2, CYLD, VCPIP, AMSH-LP, JOSD1, JOSD2, USP1/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, USP4? and USP48. Preferably, compounds of the invention have good selectivity for USP30 over one or more of these DUB enzymes.

[0038] 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, S, V and Z). Preferably, compounds of the invention have good selectivity for USP30 over one or more of these enzymes.

[0039] 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 it passes from the GI tract into the systemic circulation. Once a drug is in the systemic circulation the rate of metabolism is also important in determining the time of residence of the drug in the body.

[0040] 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.

[0041] 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.

[0042] 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 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.

[0043] 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, C.sub.max, C.sub.min and T.sub.max values.

[0044] The treatment of CNS disorders, including Alzheimer's disease, Parkinson's disease, and other 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 penetration properties and provide suitable residence time 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 utilizing a MDR1-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.

[0045] There is also a need for compounds that 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.

[0046] Toxicology and safety studies may also be conducted to identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses 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-human primate, or other suitable species).

[0047] The bacterial reverse mutation assay (Ames Test) may be used to evaluate the mutagenic properties of compounds of the invention, commonly by using the bacterial strain Salmonella typhimurium, which is mutant for the biosynthesis of the amino acid histidine.

[0048] 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).

[0049] 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 the hERG current causes QT interval prolongation resulting in potentially fatal ventricular tachyarrhythmia (Torsades de Pointes). Typically, assay data may be generated from an automated patch-clamp assay platform.

[0050] The present invention is therefore 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.

[0051] The compounds of the present invention have been found to demonstrate one or more of the above identified properties that are both significant and unexpected. For instance, all of the Examples of the present invention are highly potent against USP30 as measured in the biochemical assay described herein. All of the Examples of the present invention are significantly more selective for USP30 over other DUBs and cathepsins.

[0052] The significant and unexpected properties 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.

[0053] According to a first aspect, the present invention provides a compound of formula (I)

##STR00003##

[0054] a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein:

[0055] R.sup.1 is selected from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)fluoroalkyl and CH.sub.2OCH.sub.3;

[0056] R.sup.2, R.sup.3 and R.sup.4 are each independently selected from hydrogen and fluorine; and

[0057] R.sup.5 is selected from hydrogen, fluorine, (C.sub.1-C.sub.4)alkoxy and cyclopropoxy.

[0058] The compound of formula (I) exists as a single stereoisomer with the absolute stereochemistry shown.

[0059] Alkyl and alkoxy groups may be straight or branched and contain 1 to 4 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and sec-butyl. Examples of alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy and t-butoxy.

[0060] Fluoroalkyl groups may contain one or more fluorine substituents. Examples are fluoromethyl, difluoromethyl and trifluoromethyl.

[0061] Unless otherwise indicated, the term substituted means substituted by one or more defined groups. In the case where groups may be selected from more than one alternative, the selected groups may be the same or different. The term ‘independently’ means that where more than one substituent is selected from more than one possible substituent, those substituents may be the same or different.

[0062] Preferred embodiments of the compound of formula (I) are defined below.

[0063] Preferably, R.sup.1 is selected from methyl, CH.sub.2F, CHF.sub.2, CF.sub.3 and CH.sub.2OCH.sub.3.

[0064] More preferably, R.sup.1 is selected from methyl, CH.sub.2F and CH.sub.2OCH.sub.3.

[0065] Most preferably, R.sup.1 is methyl.

[0066] Preferably, R.sup.2 is hydrogen.

[0067] Preferably, R.sup.3 is hydrogen.

[0068] Preferably, R.sup.4 is hydrogen.

[0069] Preferably, R.sup.5 is selected from hydrogen, fluorine, methoxy and cyclopropoxy.

[0070] More preferably, R.sup.5 is selected from hydrogen and methoxy.

[0071] Most preferably, R.sup.5 is hydrogen.

[0072] According to one preferred embodiment of the invention:

[0073] R.sup.1 is methyl;

[0074] R.sup.2, R.sup.3 and R.sup.4 are each independently selected from hydrogen and fluorine; and

[0075] R.sup.5 is selected from hydrogen and methoxy.

[0076] According to a further preferred embodiment of the invention:

[0077] R.sup.1 is CH.sub.2F;

[0078] R.sup.2, R.sup.3 and R.sup.4 are each independently selected from hydrogen and fluorine; and

[0079] R.sup.5 is selected from hydrogen and methoxy.

[0080] According to a further preferred embodiment of the invention:

[0081] R.sup.1 is CH.sub.2OCH.sub.3;

[0082] R.sup.2, R.sup.3 and R.sup.4 are each independently selected from hydrogen and fluorine; and

[0083] R.sup.5 is selected from hydrogen and methoxy.

[0084] Preferred compounds of formula (I) of the present invention are selected from: [0085] N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide; [0086] N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide; [0087] N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide; [0088] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide; and [0089] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide;

[0090] a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

[0091] The most preferred compound of formula (I) of the present invention is:

[0092] N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

[0093] Pharmaceutical acceptable salts of the compounds of formula (I) include the acid addition and base salts (including di-salts) thereof.

[0094] Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate, camsylate, citrate, edisylate, esylate, fumarate, gluceptate, gluconate, glucuronate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate, malonate, mesylate, methylsulfate, 2-napsylate, nicotinate, nitrate, orotate, palmate, phosphate, saccharate, stearate, succinate sulfate, D- and L-tartrate, and tosylate salts.

[0095] Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, ammonium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

[0096] For a review on suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, Weinheim, Germany (2002).

[0097] A pharmaceutical acceptable salt of a compound of formula (I) may be readily prepared by mixing together solutions of the compound of formula (I) and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

[0098] Pharmaceutical acceptable solvates in accordance with the invention include hydrates and solvates wherein the solvent of crystallization may be isotopically substituted, e.g. D.sub.2O, acetone-d.sub.6, DMSO-d.sub.6.

[0099] Also, within the scope of the invention are clathrates, drug-host inclusion complexes wherein, in contrast to the aforementioned solvates, the drug and host are present in non-stoichiometric amounts. For a review of such complexes, see J. Pharm Sci, 64 (8), 1269-1288 by Haleblian (August 1975).

[0100] Hereinafter all references to compounds of formula (I) include references to salts thereof and to solvates and clathrates of compounds of formula (I) and salts thereof.

[0101] The invention includes all polymorphs of the compounds of formula (I) as hereinbefore defined.

[0102] Also, within the scope of the invention are so-called “prodrugs” of the compounds of formula (I). Thus, certain derivatives of compounds of formula (I) which have little or no pharmacological activity themselves can, when metabolised upon administration into or onto the body, give rise to compounds of formula (I) having the desired activity. Such derivatives are referred to as “prodrugs”.

[0103] Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as “pro-moieties” as described, for example, in “Design of Prodrugs” by H Bundgaard (Elsevier, 1985). Finally, certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I).

[0104] Certain derivatives of compounds of formula (I) which contain a nitrogen atom may also form the corresponding N-oxide, and such compounds are also within the scope of the present invention.

[0105] Included within the scope of the present invention are all tautomeric forms of the compounds of formula (I).

[0106] Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person. Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture. The present invention includes all crystal forms of the compounds of formula (I) including racemates and racemic mixtures (conglomerates) thereof. Stereoisomeric conglomerates may be separated by conventional techniques known to those skilled in the art—see, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel and S. H. Wilen (Wiley, New York, 1994).

[0107] The compounds of formula (I) contain two chiral centres at the carbon atoms of the pyrrolidine ring that are substituted by R.sup.1 and the amide and said stereocentres could exist in either the (R) or (S) configuration. The designation of the absolute configuration (R) and (S) for stereoisomers in accordance with IUPAC nomenclature is dependent on the nature of the substituents and application of the sequence-rule procedure. The compounds of formula (I) could, therefore, exist in four stereoisomeric forms.

[0108] The compounds of formula (I) of the present invention exist as a single stereoisomer. The pyrrolidine carbon atom of the amide substituent exists as the (R)-stereocentre, whereas the designation of the pyrrolidine carbon atom of the R.sup.1 group is dependent on the nature of the substituent. The compound of formula (I) is isolated as a single stereoisomer and may exist with a stereoisomeric excess of at least 60%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, for example 96%, 97%, 98%, 99%, or 100%.

[0109] Additional chiral centres may exist in the compounds of formula (I) within the R.sup.1 substituent itself. Included within the scope of the present invention are all such stereoisomeric forms of the compounds of formula (I).

[0110] The present invention also includes all pharmaceutically acceptable isotopic variations of a compound of formula (I). An isotopic variation is defined as one in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature.

[0111] Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as .sup.2H and .sup.3H, carbon, such as .sup.13C and .sup.14C, nitrogen, such as .sup.15N, oxygen, such as .sup.17O and .sup.18O, phosphorus, such as .sup.32P, sulfur, such as .sup.35S, fluorine, such as .sup.18F, and chlorine, such as .sup.36Cl.

[0112] Substitution of the compounds of the invention with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

[0113] Certain isotopic variations of the compounds of formula (I), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, and .sup.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0114] Isotopic variations of the compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using appropriate isotopic variations of suitable reagents.

[0115] The compounds of formula (I) are inhibitors of the deubiquitylating enzyme USP30.

[0116] According to a further aspect, the present invention provides a compound of formula (I) as defined herein, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer for use as a medicament.

[0117] According to a further aspect, the present invention provides a method of treatment or prevention of a disorder or condition where inhibition of USP30 is known, or can be shown, to produce a beneficial effect, in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I) as defined herein, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer. In one preferred embodiment of all aspects of the invention, the disorder or condition is a CNS indication. In a further preferred embodiment of all aspects of the invention, the disorder or condition is a peripheral indication.

[0118] According to a further aspect, the present invention provides the use of a compound of formula (I) as defined herein, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, in the manufacture of a medicament for the treatment or prevention of a disorder or condition where inhibition of USP30 is known, or can be shown, to produce a beneficial effect. The manufacture of a medicament may include, inter alia, the chemical synthesis of the compound of formula (I) or a salt thereof, or the preparation of a composition or formulation comprising the compound or salt, or the packaging of any medicament comprising the compound. In one preferred embodiment of all aspects of the invention, the disorder or condition is a CNS indication. In a further preferred embodiment of all aspects of the invention, the disorder or condition is a peripheral indication.

[0119] The disorder or condition benefiting from USP30 activity is selected from a condition involving mitochondrial dysfunction, cancer and fibrosis.

[0120] In one preferred embodiment of all aspects of the invention, the disorder or condition benefiting from USP30 activity is a condition involving mitochondrial dysfunction. The condition involving mitochondrial dysfunction may be a CNS indication or a peripheral indication.

[0121] Mitochondrial dysfunctions result from defects of the mitochondria, which are specialized compartments present in every cell of the body except red blood cells. When mitochondria fail, less and less energy is generated within the cell and cell injury or even cell death will follow. If this process is repeated throughout the body the life of the subject in whom this is happening is severely compromised. Diseases of the mitochondria appear most often in organs that are very energy demanding such as the brain, heart, liver, skeletal muscles, kidney and the endocrine and respiratory system.

[0122] The condition involving mitochondrial dysfunction may be selected from a condition involving a mitophagy defect, a condition involving a mutation in mitochondrial DNA, a condition involving mitochondrial oxidative stress, a condition involving a defect in mitochondrial membrane potential, mitochondrial biogenesis, a condition involving a defect in mitochondrial shape or morphology, and a condition involving a lysosomal storage defect.

[0123] In particular, the condition involving mitochondrial dysfunction may be selected from a neurodegenerative disease; multiple sclerosis (MS); mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome; materially-inherited diabetes and deafness (MIDD); Leber's hereditary optic neuropathy (LHON); cancer (including, for example, breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, bone or other cancers of tissue organs and cancers of the blood cells, such as lymphoma and leukaemia, multiple myeloma, metastatic carcinoma, osteosarcoma, chondosarcoma, Ewing's sarcoma, nasopharyngeal carcinoma, colorectal cancer, and non-small cell lung carcinoma); neuropathy, ataxia, retinitis pigmentosa, maternally inherited Leigh syndrome (NARP-MILS); Danon disease; diabetes; diabetic nephropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; psychiatric diseases, for example schizophrenia; multiple sulfatase deficiency (MSD); mucolipidosis II (ML II); mucolipidosis III (ML III); mucolipidosis IV (ML IV); GM1-gangliosidosis (GM1); neuronal ceroid-lipofuscinoses (NCL1); Alpers disease; Barth syndrome; beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency (LCHAD); Leigh disease or syndrome; Leigh Syndrome French Canadian (LSFC) variant; lethal infantile cardiomyopathy (LIC); Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrogenase deficiency (MCAD); myoclonic epilepsy and ragged-red fiber (MERRF) syndrome; mitochondrial cytopathy; mitochondrial recessive ataxia syndrome; mitochondrial DNA depletion syndrome; myoneurogastointestinal disorder and encephalopathy; Pearson syndrome; pyruvate dehydrogenase deficiency; pyruvate carboxylase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency; very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency; peroxisomal disorders; methylmalonic acidemia; and age-dependent decline in cognitive function and muscle strength.

[0124] The condition involving mitochondrial dysfunction may be a CNS disorder, for example a neurodegenerative disease.

[0125] Neurodegenerative diseases include, but are not limited to, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, ischemia, stroke, dementia with Lewy bodies, multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and frontotemporal dementia.

[0126] In particular, the compounds of the invention may be useful in the treatment or prevention of Parkinson's disease, including, but not limited to, PD related to mutations in α-synuclein, parkin, PINK1, GBA, and LRRK2, and autosomal recessive juvenile Parkinson's disease (AR-JP) where parkin is mutated.

[0127] The compounds of the invention or pharmaceutical compositions thereof as described herein may be combined with one or more additional agents when used for the treatment or prevention of conditions involving mitochondrial dysfunction. The compounds may be combined with one or more additional agents selected from levodopa, a dopamine agonist, a monoamino oxygenase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, riluzole, amantadine, a cholinesterase inhibitor, memantine, tetrabenazine, an antipsychotic, diazepam, clonazepam, an antidepressant, and an anti-convulsant. The compounds may be combined with agents which reduce/remove pathogenic protein aggregates in neurodegenerative diseases, such as agents which reduce/remove alpha-synuclein in Parkinson's disease, multiple system atrophy or dementia with Lewy bodies; agents which reduce/remove Tau in Alzheimer's disease or progressive supranuclear palsy; agents which reduce/remove TDP-43 in ALS or frontotemporal dementia.

[0128] In another preferred embodiment of all aspects of the invention, the disorder or condition benefiting from USP30 activity is cancer. The cancer may be linked to mitochondrial dysfunction. Preferred cancers include, for example, breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, bone or other cancers of tissue organs and cancers of the blood cells, such as lymphoma and leukaemia, multiple myeloma, metastatic carcinoma, osteosarcoma, chondosarcoma, Ewing's sarcoma, nasopharyngeal carcinoma, colorectal cancer, colorectal cancer, and non-small cell lung carcinoma.

[0129] In particular, the compounds of the invention may be useful in the treatment or prevention of cancer where apoptotic pathways are dysregulated and more particularly where proteins of the BCL-2 family are mutated, or over or under expressed.

[0130] Fibrosis refers to the accumulation of extracellular matrix constituents that occurs following trauma, inflammation, tissue repair, immunological reactions, cellular hyperplasia, and neoplasia. Fibrotic disorders that may be treated by the compounds and compositions of the present invention include, inter alia, fibrosis/fibrotic disorders associated with major organ diseases, for example, interstitial lung disease (ILD), liver cirrhosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (hepatic fibrosis), kidney disease (renal fibrosis), acute kidney injury (AKI), chronic kidney disease (CKD), delayed kidney graft function, heart or vascular disease (cardiac fibrosis) and diseases of the eye; fibroproliferative disorders, for example, systemic and local scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, and Dupuytren's contracture; scarring associated with trauma, for example, surgical complications, chemotherapeutics drug-induced fibrosis (e.g. bleomycin-induced fibrosis), radiation-induced fibrosis, accidental injury and burns); retroperitoneal fibrosis (Ormond's disease); and peritoneal fibrosis/peritoneal scarring in patients receiving peritoneal dialysis, usually following renal transplantation. See, for example, Wynn et al, 2004, Nat Rev Immunol. August; 4(8): 583-594. The present invention therefore relates to methods of treatment or prevention, and compounds and compositions used in said methods, of fibrosis/fibrotic disorders of and/or associated with the major organs, including for example, the lung, liver, kidney, heart, skin, eye, gastrointestinal tract, peritoneum and bone marrow, and other diseases/disorders herein described.

[0131] The compounds may be combined with agents which are used as treatments for kidney disease, including anti-diabetic agents, cardiovascular disease agents, and novel agents targeting disease relevant pathways such as oxidative stress (including, but not limited to, the nrf2/keap-1 pathway) and anti-apoptotic pathways (including, but not limited to, anti p53 agents).

[0132] Interstitial lung disease (ILD) includes disorders in which pulmonary inflammation and fibrosis are the final common pathways of pathology, for example, sarcoidosis, silicosis, drug reactions, infections, and collagen vascular diseases, such as rheumatoid arthritis and systemic sclerosis (scleroderma). The fibrotic disorder of the lung includes, for example, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonitis (UIP), interstitial lung disease, cryptogenic fibrosing alveolitis (CFA), bronchiolitis obliterans, and bronchiectasis.

[0133] Idiopathic pulmonary fibrosis (IPF) is the most common type of ILD and has no known cause.

[0134] The compounds may be combined with agents which are treatments for IPF and potentially for ILD, including nintedanib and pirfenidone.

[0135] Liver cirrhosis has similar causes to ILD and includes, for example, cirrhosis associated with viral hepatitis, schistosomiasis and chronic alcoholism.

[0136] Kidney disease may be associated with diabetes, which can damage and scar the kidneys leading to a progressive loss of function, and also hypertensive diseases. Kidney fibrosis may occur at any stage of kidney disease, from chronic kidney disease (CKD), such as incident CKD and progressive CKD, through to end-stage renal disease (ESRD). Kidney fibrosis can develop as a result of cardiovascular disease such as hypertension or diabetes, both of which place immense strain on kidney function which promotes a fibrotic response. However, kidney fibrosis can also be idiopathic (without a known cause), and certain genetic mitochondrial diseases also present kidney fibrosis manifestations and associated symptoms.

[0137] Heart disease may result in scar tissue that can impair the ability of the heart to pump.

[0138] Diseases of the eye include, for example, macular degeneration and retinal and vitreal retinopathy, which can impair vision.

[0139] In a preferred embodiment, the present invention is directed to the treatment or prevention of idiopathic pulmonary fibrosis (IPF).

[0140] In another preferred embodiment, the present invention is directed to the treatment or prevention of kidney fibrosis.

[0141] In another preferred embodiment, the present invention is directed to the treatment or prevention of acute kidney injury (AKI), especially in high risk patients. Examples include post-surgical AKI, for example organ transplantation, such as due to ischemia reperfusion injury, delayed graft function; oncology, such as AKI due to chemotherapy; contrast medium-induced nephropathy, such as direct-tubular cytotoxicity, hemodynamic ischemia and osmotic effects; and acute interstitial nephritis, such as due to drugs or infection. A particular high risk patient sub-group are those undergoing cardiac surgery, for example, coronary artery bypass graft and/or valve surgery. There are established static risk factors for AKI such as age 65 years or over, insulin dependent diabetes, CKD (adults with an estimated glomerular filtration rate [eGFR] less than 60 ml/min/1.73 m2 are at particular risk), heart failure, liver disease, history of AKI.

[0142] In another preferred embodiment, the present invention is directed to the treatment or prevention of chronic kidney disease (CKD) stemming from such AKI, including for example, tubulointerstitial fibrosis and diabetic nephropathy.

[0143] Leigh Syndrome is a rare inherited neurometabolic disorder that affects the central nervous system. This progressive disorder begins in infants between the ages of three months and two years. Rarely, it occurs in teenagers and adults. Leigh Syndrome can be caused by mutations in nuclear DNA encoding for mitochondrial proteins, mutations in mitochondrial DNA (maternally inherited leigh syndrome—MILS), or by deficiencies of an enzyme called pyruvate dehydrogenase located on the short arm of the X Chromosome (X-linked Leigh syndrome). Symptoms of Leigh syndrome usually progress rapidly. The earliest signs may be poor sucking ability, and the loss of head control and motor skills. These symptoms may be accompanied by loss of appetite, vomiting, irritability, continuous crying, and seizures. As the disorder progresses, symptoms may also include generalized weakness, lack of muscle tone, and episodes of lactic acidosis, which can lead to impairment of respiratory and kidney function.

[0144] In maternally inherited Leigh syndrome (MILS), genetic mutations in mitochondrial DNA (at a high proportion of >90%) interfere with the energy sources that run cells in an area of the brain that plays a role in motor movements. Genetic mutations in mitochondrial DNA result in a chronic lack of energy in these cells, which in turn affects the central nervous system and causes progressive degeneration of motor functions. When the genetic mutations in mitochondrial DNA that causes MILS are less abundant (less than 90%), the condition is known as neuropathy ataxia and retinitis pigmentosa (NARP). There is also a form of Leigh's disease (called X-linked Leigh's disease) which is the result of mutations in a gene that produces another group of substances that are important for cell metabolism. A further variant of Leigh syndrome exists which is called French Canadian Variant, characterized by mutations in a gene called LRPPRC. Similar neurological symptoms are expressed as those for Leigh Syndrome, although Liver Steatosis is commonly also observed in the French Canadian Variant.

[0145] In a preferred embodiment, the present invention is directed to the treatment or prevention of Leigh syndrome or disease, including for example, X-linked Leigh's disease, Leigh Syndrome French Canadian Variant, and/or the symptoms associated with Leigh's disease.

[0146] The compounds may be combined with novel agents which may be used as treatments for mitochondrial disease, including, but not limited to, nicotinamide riboside.

[0147] References to ‘treatment’ includes means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis. The compounds of the invention are useful in the treatment of the diseases disclosed herein in humans and other mammals.

[0148] In another embodiment, the invention encompasses prophylactic therapy of the diseases disclosed herein and includes means to prevent or slow the appearance of symptoms of the named disorder or condition. The compounds of the invention are useful in the prevention of the diseases disclosed herein in humans and other mammals.

[0149] A patient in need of treatment or prevention may, for example, be a human or other mammal suffering from the condition or at risk of suffering from the condition.

[0150] According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt of said compound or tautomer, together with a pharmaceutically acceptable diluent or carrier.

[0151] Pharmaceutical compositions of the invention comprise any of the compounds of the invention combined with any pharmaceutically acceptable carrier, adjuvant or vehicle. Examples of pharmaceutically acceptable carriers are known to those skilled in the art and include, but are not limited to, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may be in the form of, for example, tablets, capsules, powders, granules, elixirs, lozenges, suppositories, syrups, and liquid preparations including suspensions and solutions. The term “pharmaceutical composition” in the context of this invention means a composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms.

[0152] The compounds of the invention or pharmaceutical compositions thereof, as described herein, may be used alone or combined with one or more additional pharmaceutical agents. The compounds may be combined with an additional anti-tumour therapeutic agent, for example, chemotherapeutic drugs or inhibitors of other regulatory proteins. In one embodiment, the additional anti-tumour therapeutic agent is a BH-3 mimetic. In a further embodiment, BH-3 mimetics may be selected from but not limited to one or more of ABT-737, ABT-199, ABT-263, and Obatoclax. In a further embodiment, the additional anti-tumour agent is a chemotherapeutic agent. Chemotherapeutic agents may be selected from but not limited to, olaparib, mitomycin C, cisplatin, carboplatin, oxaliplatin, ionizing radiation (IR), camptothecin, irinotecan, topotecan, temozolomide, taxanes, 5-fluoropyrimidines, gemcitabine, and doxorubicin.

[0153] For the treatment or prevention of fibrotic disorders, for example, the compounds of the invention or pharmaceutical compositions thereof, as described herein, may be used alone or combined with one or more additional pharmaceutical agents selected from the group consisting of anticholinergic agents, beta-2 mimetics, steroids, PDE-IV inhibitors, p38 MAP kinase inhibitors, NK1 antagonists, LTD4 antagonists, EGFR inhibitors and endothelin antagonists.

[0154] In particular, the compounds of the invention or pharmaceutical compositions thereof, as described herein, may be used alone or combined with one or more additional pharmaceutical agents selected from the group consisting of general immunosuppressive drugs, such as a corticosteroid, immunosuppressive or cytotoxic agents, or antifibrotics, such as pirfenidone or a non-specific kinase inhibitor (e.g. nintedanib).

[0155] The pharmaceutical compositions of the invention may be administered in any suitably effective manner, such as oral, parenteral, topical, inhaled, intranasal, rectal, intravaginal, ocular and aural. Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in “Remington's Pharmaceutical Sciences”, 19th Edition (Mack Publishing Company, 1995).

[0156] Oral Administration

[0157] The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth.

[0158] Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, films (including muco-adhesive), ovules, sprays and liquid formulations.

[0159] Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

[0160] The compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986 by Liang and Chen (2001).

[0161] A typical tablet may be prepared using standard processes known to a formulation chemist, for example, by direct compression, granulation (dry, wet, or melt), melt congealing, or extrusion. The tablet formulation may comprise one or more layers and may be coated or uncoated.

[0162] Examples of excipients suitable for oral administration include carriers, for example, cellulose, calcium carbonate, dibasic calcium phosphate, mannitol and sodium citrate, granulation binders, for example, polyvinylpyrrolidine, hydroxypropylcellulose, hydroxypropylmethylcellulose and gelatin, disintegrants, for example, sodium starch glycolat and silicates, lubricating agents, for example, magnesium stearate and stearic acid, wetting agents, for example, sodium lauryl sulfate, preservatives, anti-oxidants, flavours and colourants.

[0163] Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted and programmed release. Details of suitable modified release technologies such as high energy dispersions, osmotic and coated particles are to be found in Verma et al, Pharmaceutical Technology On-line, 25 (2), 1-14 (2001). Other modified release formulations are described in U.S. Pat. No. 6,106,864.

[0164] Parenteral Administration

[0165] The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0166] Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

[0167] The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0168] The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by suitable processing, for example, the use of high energy spray-dried dispersions and/or by the use of appropriate formulation techniques, such as the use of solubility-enhancing agents.

[0169] Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled dual, targeted and programmed release.

[0170] Pharmaceutical compositions of the present invention also include compositions and methods known in the art for bypassing the blood brain barrier or can be injected directly into the brain. Suitable areas for injection include the cerebral cortex, cerebellum, midbrain, brainstem, hypothalamus, spinal cord and ventricular tissue, and areas of the peripheral nervous system including the carotid body and the adrenal medulla.

[0171] Dosage

[0172] The magnitude of an effective dose of a compound will, of course, vary with the nature of the severity of the condition to be treated and the route of administration. The selection of appropriate dosages is within the remit of the physician. The daily dose range is about 10 μg to about 100 mg per kg body weight of a human and non-human animal and in general may be around 10 μg to 30 mg per kg body weight per dose. The above dose may be given from one to three times per day.

[0173] For example, oral administration may require a total daily dose of from 5 mg to 1000 mg, such as from 5 to 500 mg, while an intravenous dose may only require from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. The total daily dose may be administered in single or divided doses. The skilled person will also appreciate that, in the treatment or prevention of certain conditions, compounds of the invention may be taken as a single dose on an “as required” basis (i.e. as needed or desired).

[0174] Synthetic Methodologies

[0175] Compounds of formula (I) may be prepared using methods as described below in the general reaction schemes and the representative examples. Where appropriate, the individual transformations within a scheme may be completed in a different order. The invention is illustrated by the following non-limiting examples in which the following abbreviations and definitions are used. Compounds were characterised by liquid chromatography-mass spectroscopy (LCMS) or .sup.1H NMR or both.

[0176] According to a further aspect, the present invention provides a process for the preparation of a compound of formula (I) comprising reacting a compound of formula (IV), where Y is OH with an amine of formula (V), where PG is a protecting group, such as BOC or Cbz, to give an amide of formula (III) (Scheme 1). The amide-coupling reaction can be performed using standard methodology, for example by reaction using a coupling reagent such as DCC, HATU, HBTU, EDC or via a mixed anhydride. Alternatively, the acid (IV), where Y is OH, can be converted into the acid chloride (IV), where Y is Cl, using SOCl.sub.2, PCl.sub.3, or PCl.sub.5, which can then be reacted with the amine (V), preferably in a suitable solvent in the presence of a suitable base. Alternatively, the compound (IV), where Y forms the ester, can be reacted directly with the amine (V), preferably in a suitable solvent.

[0177] The compound of formula (III) may be deprotected using standard methods to give amine (II) which may then be reacted with cyanogen bromide to give the corresponding compound of formula (I).

##STR00004##

[0178] In a further aspect, the present invention provides a compound, which is selected from formulae (II) and (III):

##STR00005##

[0179] wherein PG is a protecting group and R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined herein for the compound of formula (I) and preferred embodiments thereof, a tautomer thereof, or a salt of said compound or tautomer.

[0180] Protecting groups are preferably selected from tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), p-methoxybenzyl carbonyl (MeOZ), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), 4-nitrobenzenesulfonyl (Nosyl) and 2-nitrophenylsulfenyl (Nps). Most preferred are BOC and Cbz.

TABLE-US-00001 Abbreviations br s broad singlet (NMR signal) CO carbon monoxide d doublet (NMR signal) dba dibenzylacetone DCM dichloromethane DMF N,N-dimethylformamide DMSO dimethylsulfoxide ES electrospray EtOAc ethyl acetate h hour(s) H.sub.2 hydrogen m multiplet (NMR signal) MsCl methanesulfonyl chloride MeCN acetonitrile MeOH methanol min minute(s) N.sub.2 nitrogen NMP N-methylpyrrolidone rac racemic rt room temperature s singlet (NMR signal) SOCl.sub.2 thionyl chloride TBD 1,5,7-triazabicyclo[4.4.0]dec-5-ene TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TsCl 4-toluenesulfonyl chloride vol volumes

[0181] LCMS Methods

[0182] Method C

TABLE-US-00002 Mobile (A) 2 mM ammonium acetate & 0.1% formic phase acid in water (B) 0.1% formic acid in acetonitrile Instrument Waters ACQUITY H Class with PDA and SQ Detector Column BEH C18 (50 mm × 2.1 mm) 1.7 μm Flow rate 0.550 mL/min Column oven Ambient temperature Run time 3.0 min Gradient TIME Flow Rate (min) (mL/min) % A % B 0.01 0.55 98 2 0.30 0.55 98 2 0.60 0.55 50 50 1.10 0.55 25 75 2.00 0.60 0 100 2.70 0.60 0 100 2.71 0.55 98 2 3.00 0.55 98 2

[0183] Method H

TABLE-US-00003 Mobile (A) 5 mM Ammonium bicarbonate in water phase (B) 100% Acetonitrile Instrument Shimadzu Nexera UFLC with 2020 single quadrupole mass detector Column Waters X-Bridge C18 (50 × 4.6 mm) 3.5 μm Column oven Ambient temperature Flow rate 1.0 mL/min Run time 8.0 min Gradient TIME Flow Rate (min) (mL/min) % A % B 0.01 1.0 95 5 3.50 1.0 10 90 4.50 1.0 5 95 6.00 1.0 5 95 6.01 1.0 95 5 8.00 1.0 95 5

[0184] Method H1

TABLE-US-00004 Mobile (A) 5 mM Ammonium bicarbonate in water phase (B) 100% Acetonitrile Instrument Shimadzu Nexera High Pressure UHPLC and LCMS-2020 Column Waters XBridge C18 (50 × 4.6 mm) 3.5 μm Column oven Ambient temperature Flow rate 1.0 mL/min Run time 10.0 min Gradient TIME Flow Rate (min) (mL/min) % A % B 0.01 1.0 95 5 5.00 1.0 10 90 5.80 1.0 5 95 7.20 1.0 5 95 7.21 1.0 95 5 10.00  1.0 95 5

[0185] Method J

TABLE-US-00005 Mobile (A) 2 mM ammonium acetate & 0.1% formic phase acid in water (B) 0.1% formic acid in acetonitrile Instrument Waters ACQUITY H Class with PDA and SQ detector Column BEH C18 (50 mm × 2.1 mm) 1.7 μm Flow rate 0.450 mL/min Column oven Ambient temperature Run time 8.0 min Gradient TIME Flow Rate (min) (mL/min) % A % B 0.01 0.45 98 2 0.50 0.45 98 2 5.00 0.45 10 90 6.00 0.45 5 95 7.00 0.45 5 95 7.01 0.45 98 2 8.00 0.45 98 2

[0186] Method Y3 Method Used for Analytical Chiral SFC

TABLE-US-00006 Mobile (A) Liquid carbon dioxide Phase (B) 0.1% diethylamine in IPA:acetonitrile (50:50) Instrument Waters SFC investigator and PDA detector Column Chiralcel OJ-H (250 × 4.6 mm), 5 μm Flow rate 4.0 mL/min Run time 7.0 min Gradient TIME Flow Rate (min) (mL/min) % B start % B end 0 to 5 4.0 5 5 5 to 7 4.0 50 50

[0187] Method Y4 Method Used for Analytical Chiral SFC

TABLE-US-00007 Mobile (A) Liquid carbon dioxide Phase (B) 0.1% diethylamine in IPA:acetonitrile (50:50) Instrument Waters SFC investigator and PDA detector Column Chiralpak IH (250 × 4.6 mm), 5 μm Flow rate 4.0 mL/min Run time 10.0 min Gradient TIME Flow Rate (min) (mL/min) % B start % B end 0 to 5  4.0 5 5 5 to 10 4.0 50 50

[0188] Method Y9 Method Used for Analytical Chiral SFC

TABLE-US-00008 Mobile (A) Liquid carbon dioxide Phase (B) 0.1% diethylamine in IPA:acetonitrile (50:50) Instrument Waters SFC investigator and PDA detector Column Chiralpak IB-N (250 × 4.6 mm), 5 μm Flow rate 4.0 mL/min Run time 10.0 min Gradient TIME Flow Rate (min) (mL/min) % B start % B end 0 to 5  4.0 5 5 5 to 10 4.0 50 50

Intermediate A

Ethyl 5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxylate

[0189] ##STR00006##

Step 1

3-Cyanobenzohydrazide

[0190] To a stirred solution of methyl 3-cyanobenzoate (CAS 13531-48-1, from CombiBlocks, 150.0 g, 930.5 mmol) in MeOH (1500 mL) was added NH.sub.2NH.sub.2.H.sub.2O (99% aq. solution, 114.3 mL, 2326.3 mmol) at 0° C. The mixture was stirred for 16 h at rt then concentrated under reduced pressure, diluted with water (2000 mL) and extracted with DCM (3×800 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to yield 3-cyanobenzohydrazide (138 g, 857.1 mmol, 92% yield).

[0191] LCMS: Method C, 1.23 min, MS: ES+ 162.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 10.01 (s, 1H), 8.22 (s, 1H), 8.13-8.15 (d, J=8.0 Hz, 1H), 8.00-8.02 (d, J=7.6 Hz, 1H), 7.70 (t, J=8.0 Hz, 1H), 4.60 (s, 2H).

Step 2

Ethyl 2-(2-(3-cyanobenzoyl) hydrazinyl)-2-oxoacetate

[0192] To a stirred solution of 3-cyanobenzohydrazide (131.0 g, 813.7 mmol) in DCM (1960 mL) was added K.sub.2CO.sub.3 (168.4 g, 1220.5 mmol). The mixture was stirred at rt for 15 min then cooled to 0° C. and ethyl chlorooxoacetate (203.6 mL, 1790.1 mmol) was added dropwise at 0° C. The mixture was stirred for 1 h at rt then diluted with water (5000 mL) and extracted with DCM (5×4000 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to yield ethyl 2-(2-(3-cyanobenzoyl)hydrazinyl)-2-oxoacetate (251.9 g). The crude material was used in the next step.

[0193] LCMS: Method C, 1.35 min, MS: ES+ 262.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 11.08 (s, 1H), 10.85 (s, 1H), 8.28 (s, 1H), 8.10-8.20 (m, 2H), 7.77 (t, J=8.0 Hz, 1H), 4.29-4.35 (m, 2H), 1.32 (t, 3H).

Step 3

Ethyl 5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxylate

[0194] To a stirred solution of ethyl 2-(2-(3-cyanobenzoyl) hydrazinyl)-2-oxoacetate (251.0 g, crude from Step 2) in DCM (4000 mL) was added TEA (269.8 mL, 1923.4 mmol) at 0° C. The mixture was stirred for 15 min at 0° C. then 4-toluenesulfonyl chloride (91.7 g, 480.8 mmol) was added at 0° C. The mixture was stirred for 1 h at rt then diluted with water (4000 mL) and extracted with DCM (2×5000 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography (25% EtOAc in hexane) to yield ethyl 5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxylate (105.6 g, 434.6 mmol, 53% yield over two steps). LCMS: Method C, 1.58 min, MS: ES+ 244.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 8.51 (s, 1H), 8.38-8.40 (d, J=8.0 Hz, 1H), 8.18-8.20 (d, J=8.0 Hz, 1H), 7.87 (t, J=8.0 Hz, 1H), 4.46-4.51 (m, 2H), 1.39 (t, 3H).

Intermediate B

Ethyl 5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxylate

[0195] ##STR00007##

Step (i)

Methyl 5-cyano-2-methoxybenzoate

[0196] To a stirred solution of 3-bromo-4-methoxybenzonitrile (CAS 117572-79-9, 2.0 g, 9.48 mmol) in MeOH (20 mL), in autoclave, was added NaOAc (3.88 g, 47.40 mmol) and the reaction mixture was degassed with N.sub.2 for 20 minutes. [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (1.54 g, 1.89 mmol) was added in to the reaction mixture at rt. The reaction mixture was heated at 70° C. for 36 h under CO (gas) pressure (20 kg/Cm.sup.2). The mixture was allowed to cool to rt and concentrated under reduced pressure. The residue was poured into water (50 mL) and extracted with EtOAc (3×70 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 40% EtOAc in n-hexanes) to yield methyl 5-cyano-2-methoxybenzoate (1.1 g, 5.75 mmol, 60% yield).

[0197] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 8.07 (s, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 3.92 (s, 3H), 3.81 (s, 3H).

Step (ii)

5-Cyano-2-methoxybenzohydrazide

[0198] To a stirred solution of methyl 5-cyano-2-methoxybenzoate (1.1 g, 5.75 mmol) in MeOH (11 mL) was added hydrazine hydrate (0.72 mL, 14.37 mmol) at 0° C. The mixture was allowed to warm to rt and stirred for 16 h, then concentrated under reduced pressure to yield 5-cyano-2-methoxybenzohydrazide (1.0 g, 5.23 mmol, 90% yield).

[0199] LCMS: Method C, 1.24 min, MS: ES+ 190.3.

Step (iii)

Ethyl 2-(2-(5-cyano-2-methoxybenzoyl)hydrazineyl)-2-oxoacetate

[0200] To a stirred solution of 5-cyano-2-methoxybenzohydrazide (1.0 g, 5.23 mmol) in DCM (10 mL) was added K.sub.2CO.sub.3 (1.44 g, 10.47 mmol) and followed by ethyl oxalyl chloride (1.16 mL, 10.47 mmol) dropwise at 0° C. The mixture was allowed to warm to rt and stirred for 1 h, then poured into water (50 mL) and extracted with DCM (3×150 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and concentrated to yield ethyl 2-(2-(5-cyano-2-methoxybenzoyl)hydrazineyl)-2-oxoacetate (1.2 g, 4.12 mmol, 78% yield). LCMS: Method J, 3.08 min, MS: ES+ 292.0.

Step (iv)

Ethyl 5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxylate

[0201] To a stirred solution of ethyl 2-(2-(5-cyano-2-methoxybenzoyl)hydrazineyl)-2-oxoacetate (1.2 g, 4.12 mmol) in DCM (12 mL) was added TEA (1.72 mL, 12.37 mmol) and TsCl (0.78 g, 4.12 mmol) in portions at 0° C. The mixture was allowed to warm at rt and stirred for 2 h, then poured into water (50 mL) and extracted with EtOAc (3×100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, and concentrated under reduced pressure, then the residue was purified using flash column chromatography (silica gel, 43% EtOAc in n-hexanes) to yield ethyl 5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxylate (0.64 g, 2.34 mmol, 56% yield).

[0202] LCMS: Method J, 3.94 min, MS: ES+ 274.0.

Example 1

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide

[0203] ##STR00008##

Step (i)

tert-Butyl (2R,4R)-4-(4-(3-cyanophenyl) picolinamido)-2-methylpyrrolidine-1-carboxylate

[0204] To a stirred solution of ethyl 5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxylate (0.15 g, 0.617 mmol, 1.0 eq) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 348165-63-9, from CombiBlocks, 0.123 g, 0.617 mmol, 1.0 eq) in THF (4.0 mL) was added TBD (0.103 g, 0.741 mmol, 1.2 eq) at 0° C. The mixture was stirred at rt for 1 h then poured into water (100 mL) and extracted with EtOAc (2×100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography (60-120#silica gel, 40% EtOAc in hexane) to yield teat-butyl (2R,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.115 g, 0.289 mmol, 47% yield).

[0205] LCMS: Method C, 1.69 min, MS: ES− 396.6 (M−1).

Step (ii)

5-(3-Cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt

[0206] To a stirred solution of tert-butyl (2R,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.115 g, 0.289 mmol, 1.0 eq) in DCM (5 mL) was added TFA (0.35 mL, 3 volumes) dropwise at 0° C. The mixture was stirred at rt for 40 min then concentrated under reduced pressure with DCM (2×50 mL) to give 5-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.10 g, 0.24 mmol, 84% yield). LCMS: Method C, 1.28 min, MS: ES+ 298.0.

Step (iii)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide

[0207] To a stirred solution of 5-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.10 g, 0.243 mmol, 1.0 eq) in THF (5 mL) was added K.sub.2CO.sub.3 (0.168 g, 1.22 mmol, 5 eq) portionwise at rt. The mixture was cooled to 0° C. and cyanogen bromide was added (0.026 g, 0.243 mmol, 1 eq) at 0° C. The mixture was stirred at rt for 40 min then poured into water (80 mL) and extracted with EtOAc (2×50 mL). The organic phases were combined, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography (60-120#silica gel, 35% EtOAc in hexane) to afford N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide (0.07 g, 0.217 mmol, 89% yield).

[0208] Melting point=152° C. to 154° C. [α].sub.D.sup.25=−31° (c=0.05 g/100 cm.sup.3, dichloromethane).

[0209] LCMS: Method H1, 3.15 min, MS: ES− 322.1; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 9.75-9.77 (d, J=6.8 Hz, 1H), 8.50 (s, 1H), 8.39-8.41 (d, J=8.0 Hz, 1H), 8.16-8.18 (d, J=8.0 Hz, 1H), 7.86 (t, J=8.0 Hz, 1H), 4.51-4.54 (m, 1H), 3.88-3.94 (m, 1H), 3.77-3.81 (m, 1H), 3.43-3.46 (m, 1H), 2.14-2.19 (m, 1H), 1.76-1.83 (m, 1H), 1.25-1.26 (d, J=6.4 Hz, 3H).

Example 2

N-((3R,5S)-1-Cyano-5-(methoxymethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide

[0210] ##STR00009##

Step (i)

tert-Butyl (2S,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(methoxymethyl) pyrrolidine-1-carboxylate

[0211] To a stirred solution of ethyl 5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxylate (0.15 g, 0.62 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.14 g, 0.62 mmol) in THF (3.0 mL) was added TBD (0.13 g, 0.92 mmol) at 0° C. The mixture was stirred at 0° C. for 10 min then poured into water (50 mL) and extracted with EtOAc (3×50 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 50% EtOAc in n-hexanes) to yield tert-butyl (2S,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(methoxymethyl) pyrrolidine-1-carboxylate (0.07 g, 0.17 mmol, 28% yield).

[0212] LCMS: Method C, 1.62 min, MS: ES− 426.1.

Step (ii)

5-(3-Cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt

[0213] To a stirred solution of tert-butyl (2S,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.07 g, 0.16 mmol) in DCM (3 mL) was added TFA (0.35 mL, 5 vol) dropwise at 0° C. The mixture was stirred at rt for 2 h then concentrated under reduced pressure to give 5-(3-cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.12 g, quantitative yield). LCMS: Method C, 1.27 min, MS: ES+ 328.0.

Step (iii)

N-((3R,5S)-1-Cyano-5-(methoxymethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide

[0214] To a stirred solution of 5-(3-cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.11 g, 0.25 mmol) in THF (5 mL) was added K.sub.2CO.sub.3 (0.1 g, 0.75 mmol) in portions at rt. After 10 min, cyanogen bromide (0.02 g, 0.19 mmol) was added at 0° C. The mixture was stirred at 0° C. for 45 min then poured into water (50 mL) and extracted with EtOAc (3×40 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 70% EtOAc in n-hexanes) to afford N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide (0.02 g, 0.07 mmol, 43% yield over two steps).

[0215] LCMS: Method H, 2.47 min, MS: ES− 351.1; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 9.75 (d, J=6.4 Hz, 1H), 8.50 (s, 1H), 8.40 (d, J=8.0 Hz, 1H), 8.17 (d, J=7.6 Hz, 1H), 7.86 (t, J=8.0 Hz, 1H), 4.48-4.58 (m, 1H), 3.98-4.08 (m, 1H), 3.70-3.74 (m, 1H), 3.43-3.50 (m, 3H), 3.32 (s, 3H), 2.09-2.14 (m, 1H), 1.96-2.02 (m, 1H); Chiral SFC: Method Y4, 4.30 min.

Example 3

N-((3R,5S)-1-Cyano-5-(fluoromethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide

[0216] ##STR00010##

Step (i)

tert-Butyl (2S,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(fluoromethyl) pyrrolidine-1-carboxylate

[0217] To a stirred solution of ethyl 5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxylate (0.15 g, 0.62 mmol) and tert-butyl (2S,4R)-4-amino-2-(fluoromethyl)pyrrolidine-1-carboxylate (CAS 1207853-03-9, from Angene, 0.11 g, 0.49 mmol) in THF (5.0 mL) was added TBD (0.1 g, 0.74 mmol) at 0° C. The mixture was allowed to warm to rt and stirred for 1 h then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 50% EtOAc in n-hexanes) to yield tert-butyl (2S,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylate (0.1 g, 0.25 mmol, 40% yield).

[0218] LCMS: Method C, 1.66 min, MS: ES− 414.1.

Step (ii)

5-(3-Cyanophenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt

[0219] To a stirred solution of tert-butyl (2S,4R)-4-(5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylate (0.1 g, 0.24 mmol) in DCM (5 mL) was added TFA (1.0 mL, 10 vol) dropwise at 0° C. The mixture was stirred at rt for 1 h then concentrated under reduced pressure to give 5-(3-cyanophenyl)-N-((3R,5S)-5-(fluoromethyl)-pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.12 g, quantitative yield). LCMS: Method C, 1.31 min, MS: ES+ 316.2.

Step (iii)

N-((3R,5S)-1-Cyano-5-(fluoromethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide

[0220] To a stirred solution of 5-(3-cyanophenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.12 g, 0.28 mmol) in THF (5 mL) at rt was added K.sub.2CO.sub.3 (0.12 g, 0.84 mmol) in portions and stirred for 10 min. The mixture was cooled to 0° C. and cyanogen bromide (0.03 g, 0.28 mmol) was added and the mixture was allowed to warm to rt and stirred for 1 h. The mixture was poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 70% EtOAc in n-hexanes) to afford N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)-5-(3-cyanophenyl)-1,3,4-oxadiazole-2-carboxamide (0.03 g, 0.10 mmol, 41% yield over two steps).

[0221] LCMS: Method H, 2.51 min, MS: ES− 338.9; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 9.81 (d, J=6.4 Hz, 1H), 8.51 (s, 1H), 8.40 (d, J=8.0 Hz, 1H), 8.18 (d, J=7.6 Hz, 1H), 7.87 (t, J=8.0 Hz, 1H), 4.43-4.67 (m, 3H), 4.13-4.26 (m, 1H), 3.75-3.79 (m, 1H), 3.46-3.53 (m, 1H), 2.13-2.23 (m, 1H), 2.01-2.06 (m, 1H); Chiral SFC: Method Y3, 3.57 min.

Example 4

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)-1,3,4-oxadiazole-2-carboxamide

[0222] ##STR00011##

Step (i)

tert-Butyl (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(methoxy methyl)pyrrolidine-1-carboxylate

[0223] To a stirred solution of ethyl 5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxylate (0.29 g, 1.06 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.24 g, 1.06 mmol) in THF (2.9 mL) was added TBD (0.18 g, 1.27 mmol) at 0° C. The mixture was allowed to warm to rt and stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (3×150 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 70% EtOAc in n-hexanes) to yield tert-butyl (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.16 g, 0.35 mmol, 33% yield). LCMS: Method J, 4.34 min, MS: ES− 455.6.

Step (ii)

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt

[0224] To a stirred solution of tert-butyl (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.16 g, 0.35 mmol) in DCM (1.6 mL) was added TFA (1.6 mL, 10 vol) dropwise at 0° C. The mixture was stirred at rt for 1 h then concentrated under reduced pressure to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl) pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.15 g, quantitative yield). LCMS: Method J, 2.92 min, MS: ES+ 357.4.

Step (iii)

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide

[0225] To a stirred solution of 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.15 g, 0.32 mmol) in THF (1.5 mL) was added K.sub.2CO.sub.3 (0.13 g, 0.95 mmol) in portions at rt and stirred for 10 min. Cyanogen bromide (0.04 g, 0.38 mmol) was added at 0° C. and the mixture was stirred at rt for 1 h. The mixture was poured into water (50 mL) and extracted with EtOAc (3×150 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 90% EtOAc in n-hexanes) to afford 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)37yrrolidine-3-yl)-1,3,4-oxadiazole-2-carboxamide (0.06 g, 0.17 mmol, 48% yield over two steps).

[0226] LCMS: Method H, 2.38 min, MS: ES+ 383.1; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 9.74 (d, J=6.4 Hz, 1H), 8.35 (s, 1H), 8.16 (d, J=7.2 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 4.48-4.57 (m, 1H), 4.02 (br s, 4H), 3.69-3.73 (m, 1H), 3.43-3.51 (m, 3H), 3.33 (s, 3H), 2.10-2.17 (m, 1H), 1.96-2.02 (m, 1H); Chiral SFC: Method Y9, 5.42 min.

Example 5

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide

[0227] ##STR00012##

Step (i)

tert-Butyl (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxamido)-2-methyl pyrrolidine-1-carboxylate

[0228] To a stirred solution of ethyl 5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxylate (0.31 g, 1.13 mmol) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 348165-63-9, 0.23 g, 1.13 mmol) in THF (6.0 mL) was added TBD (0.19 g, 1.36 mmol) at 0° C. The mixture was allowed to warm to rt and stirred for 1 h, then poured into water (30 mL) and extracted with EtOAc (3×50 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 65% EtOAc in n-hexanes) to yield tert-butyl (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.13 g, 0.30 mmol, 27% yield).

[0229] LCMS: Method J, 4.45 min, MS: ES+ 426.2.

Step (ii)

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide

[0230] To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)-1,3,4-oxadiazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.13 g, 0.30 mmol) in DCM (1.3 mL) was added TFA (1.3 mL, 10 vol) dropwise at 0° C. The mixture was stirred at rt for 1 h then concentrated under reduced pressure to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.13 g, qauntitative yield).

[0231] LCMS: Method J, 2.85 min, MS: ES+ 328.0.

Step (iii)

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide

[0232] To a stirred solution of 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide TFA salt (0.13 g, 0.29 mmol) in THF (1.3 mL) was added K.sub.2CO.sub.3 (0.12 g, 0.88 mmol) in portions at rt and stirred for 10 min. Cyanogen bromide (0.04 g, 0.35 mmol) was added at 0° C. and the mixture was stirred at rt for 1 h. The mixture was poured into water (40 mL) and extracted with EtOAc (3×100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 70% EtOAc in n-hexanes) to afford 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-1,3,4-oxadiazole-2-carboxamide (0.01 g, 0.03 mmol, 11% yield over two steps). LCMS: Method H, 2.57 min, MS: ES+ 353.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm: 9.77 (d, J=6.0 Hz, 1H), 8.35 (d, J=1.6 Hz, 1H), 8.16 (d, J=8.8 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 4.48-4.55 (m, 1H), 4.02 (s, 3H), 3.88-3.93 (m, 1H), 3.76-3.80 (m, 1H), 3.43-3.46 (m, 1H), 2.15-2.18 (m, 1H), 1.75-1.82 (m, 1H), 1.25 (d, J=6.4 Hz, 3H); Chiral SFC: Method Y4, 4.65 min.

[0233] Biological Activity of Compounds of the Invention

Abbreviations

[0234] TAMRA carboxytetramethylrhodamine

[0235] PBS phosphate buffered saline

[0236] EDTA ethylenediaminetetraacetic acid

[0237] Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol

[0238] NP-40 Nonidet P-40, octylphenoxypolyethoxyethanol

[0239] BSA bovine serum albumin

[0240] SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0241] DMSO Dimethyl sulfoxide

[0242] YFP Yellow fluorescent protein

[0243] VME Vinyl methyl ester

[0244] HA Hemagglutinin

[0245] Ahx Aminohexanoic acid

[0246] USP30 Biochemical IC.sub.50 Assay

[0247] Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 411 of 50% DMSO or diluted compound was added to the plate. USP30 (Boston Biochem #E582) was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to achieve a final assay concentration of 4 nM, and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2-hour incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.

[0248] Activity of exemplary compounds in USP30 biochemical IC.sub.50 assay:

TABLE-US-00009 Example IC.sub.50 (nM) 1 29 (n = 7) 2 37 (n = 2) 3 13 (n = 4) 4 29 (n = 2) 5 34 (n = 2)

Reference Examples

[0249] Activity of Compounds in USP30 Biochemical IC.sub.50 Assay:

TABLE-US-00010 Reference USP30 Example Structure Origin IC.sub.50 (nM) A [00013] WO 2016/156816 Example 221 70 B [00014] WO 2016/156816 Example 8 44 C [00015] WO 2016/156816 Example 70 26 D [00016] WO 2016/156816 Example 160 41 E [00017] WO 2016/046530 Example 1 310 F [00018] WO 2016/046530 Example 11 700 G [00019] WO 2016/046530 Example 88 4400

[0250] Off-Target Pharmacology

[0251] Activity in DUB Biochemical IC.sub.50 Assays:

TABLE-US-00011 DUB Example 1 UCHL1, USP2, USP4, USP8, USP10, IC.sub.50 (μM) ≥ 5.8 USP12/UAF1, USP15, USP16, USP21, (>200 × more selective for USP22, USP25, USP28, USP35, USP30) USP36, USP46/UAF1.

[0252] Example 1 was subject to pharmacological profiling in the Eurofins CEREP SafetyScreen44 panel. At a single concentration of 10 μM, less than 25% inhibition of binding or enzyme activity was observed against all targets in the panel.

[0253] Example 1 has low probability for off-target interactions due to the low affinity for targets in these screening panels.

[0254] Safety Pharmacology

[0255] Example 1 was evaluated for effects on the hERG potassium channel, in stably expressed CHO cells at concentrations between 0.01 and 30 μM. Example 1 produced maximum inhibition values of 7.7% and 18.8%, respectively, of the hERG current amplitude at 30 μM indicating little propensity for affecting the QT interval.

[0256] Genetic Toxicology

[0257] Example 1 was assessed in the bacterial reverse mutation assay (Ames) and in vitro micronucleus assay. All in vitro tests were conducted with and without exogenous metabolic activation using concentrations up to those limited by cytotoxicity or insolubility. Example 1 did not induce mutations when tested up to 100 μg/well (equivalent to 5000 μg/plate) with and without metabolic activation in the reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535 and TA97a and the Escherichia Coli strain WP2 uvrA pKM101.

[0258] Induction of chromosome damage was assessed using the in vitro micronucleus assay in TK6 cells. Example 1 was negative for induction of micronuclei when incubated for 3 hours in the presence of exogenous metabolic activation followed by 27 hours recovery, and also when incubated for 27 hours in the absence of exogenous metabolic activation followed by 27 hours recovery.

[0259] TOM20-Ubiquitylation Assay

[0260] Human cell lines can be challenged with mitochondrial depolarizing agents (ionophores (eg. CCCP, valinomycin), mitochondrial complex inhibitors (oligomycin, antimycin A)) to induce ubiquitylation of TOM20, which is then further promoted in the presence of USP30 inhibitors. TOM20 ubiquitylation is subsequently assessed through western blotting of the cell lysates, with TOM20 ubiquitylation adduct detection possible due to an 8 kDa molecule weight increase for each molecule of ubiquitin added, resulting in laddering of a TOM20 immunoreactive band. TOM20-ubiquitylation levels can be quantified using chemiluminescence densitometry of laddered immunoreactive bands.

[0261] USP30 Endogenous Cellular Target Engagement Assay

[0262] Hela cells stably overexpressing YFP-Parkin were seeded into 6 well dishes. Once adhered, cells were treated with appropriate concentrations of test compounds or vehicle control for 1 hour at 37° C., 5% CO.sub.2. Whole cell lysates were prepared by scraping the cells into cold PBS, centrifuging and lysing in lysis buffer (50 mM Tris-base, pH 7.5, 50 mM NaCl, 1% NP-40/Igepal CA-630, 2 mM MgCl.sub.2, 10% Glycerol, 5 mM beta-mercaptoethanol, cOmplete mini tablets EDTA free (Roche), PhosStop tablets (Roche)) for 10 mins. The equivalent of 20 μg of protein from the cleared cell lysate was incubated with a final conc of 2.5 μM HA-Ahx-Ahx-Ub-VME probe at room temperature. The reaction was stopped by addition of 5×SDS sample loading buffer and proteins separated by SDS PAGE and western blotting. USP30 was detected using an anti-USP30 Sheep S746D antibody (MRC PPU Reagents and Services) and a rabbit anti sheep secondary IgG (H+L) horseradish peroxidase conjugated (Thermo #31480) and visualised using ECL reagent (GE #RPN2109) on a GE LAS4000 imager. Target engagement was measured by quantitation of the bands corresponding to USP30 and USP30 bound to the Ub-VME probe and expression of this proportion compared to vehicle treated control.

[0263] USP30 Kidney Tissue Target Engagement Assay

[0264] 50 to 100 mg of tissue was homogenised in 3× volume of lysis buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 0.1% NP-40/Igepal CA-630, 0.5% CHAPS, 2 mM MgCl.sub.2, 5 mM beta-mercaptoethanol, cOmplete mini tablets EDTA free (Roche), PhosStop tablets (Roche)) using a Retch Mixer Mill (MM400). Lysate containing 60 μg protein was incubated with a final conc of 6 μM of HA-Ahx-Ahx-Ub-VME probe for 15 mins at room temperature. The reaction was stopped by addition of 5×SDS sample loading buffer and proteins separated by SDS-PAGE and western blotting. USP30 was detected using an anti-USP30 Sheep S746D antibody (MRC PPU Reagents and Services) and a rabbit anti sheep secondary IgG (H+L) horseradish peroxidase conjugated (Thermo #31480) and visualised using ECL reagent (GE #RPN2109) on a GE LAS4000 imager. Target engagement was measured by quantitation of the bands corresponding to USP30 and USP30 bound to the Ub-VME probe and expression of this proportion compared to vehicle treated control.

[0265] In Vitro Cytotoxicity (Cell Tox): Measured in HCT116 human colorectal carcinoma cells using alamarBlue as the assay endpoint. Compound cytotoxicity was measured over a period of 96-hour continual compound exposure.

[0266] Further Studies

[0267] log P: partition coefficient; lipophilicity measurement.

[0268] log D: distribution co-efficient; lipophilicity measurement.

[0269] TPSA: topological polar surface area.

[0270] Turbidimetric solubility: Test compound solution prepared in DMSO diluted into aqueous buffer.

[0271] Turbidimetry is used as the end-point by measuring absorbance at 620 nm.

[0272] FaSSIF: simulated intestinal fluid in fasted state measured at pH 6.5.

[0273] Hep Cl mouse: in vitro hepatocyte clearance in mouse cells.

[0274] Hep Cl human: in vitro hepatocyte clearance in human cells.

[0275] Plasma f.sub.u,p: The free fraction of a compound in plasma preparation determined by in vitro equilibrium dialysis. It is understood that only unbound (free) compound is capable of engaging with the target.

[0276] Brain f.sub.u,br: The free fraction of a compound in brain tissue homogenate preparation determined by in vitro equilibrium dialysis. It is understood that only unbound (free) compound is capable of engaging with the target.

[0277] Cl.sub.u: in vitro clearance. Cl.sub.u as defined here is the scaled clearance, in turn calculated from the intrinsic clearance. The intrinsic clearance is the predicted clearance due to hepatic metabolic reactions, determined from incubation of a compound in a hepatocyte preparation. The lower the value in mL/min/kg, the more stable the compound.

[0278] Cl in vivo clearance: Pharmacokinetic measurement of the volume of plasma (or any matrix) from which a substance is completely removed per unit time. The lower the value in mL/min/kg, the more stable the compound.

[0279] Oral F: Oral Bioavailability.

[0280] MDR1-MDCK (Madin-Darby Canine Kidney cell monolayer) (in vitro) flux assay.

[0281] Cell TE WB: USP30 endogenous cellular target engagement western blot (WB) assay. Assays the activity of compounds against USP30 in cells using an irreversible activity probe to monitor USP30 activity.

[0282] TE ex vivo: USP30 kidney tissue target engagement assay.

[0283] Kp.sub.u,u is the ratio of unbound drug in brain to unbound drug in plasma and may be indicative of potential for treating peripheral and/or CNS indications.

TABLE-US-00012 Studies Example 1 Physicochemical Log D measured at pH 7.4 1.3 TPSA 119 Turbidimetric solubility (μM) 65 FaSSIF (μM) measured at pH 6.5 415 Hepatocyte Cl mouse scaled Cl.sub.u (mL/min/kg) 45 Hepatocyte Cl human scaled Cl.sub.u (mL/min/kg) 6 Stability Mouse plasma t.sub.1/2 (min) >2000 MDR1-MDCK Efflux Ratio (A-B (P.sub.app flux 10.sup.−6 cm/s)) 81 (A-B 0.9) Binding Mouse Plasma f.sub.u, p/Brain f.sub.u, br 0.54/0.26 PK mouse 2 mg/kg iv Cl blood (mL/min/kg) 23 PK mouse 10 mg/kg Oral F (%) 48 TE ex vivo Mouse kidney 10 mg/kg PO: 1, 4, 8, 94, 89, 79, 8 12 h post-dose TE % occupancy TOM20-Ub 1.5-fold antimycin A/oligomycin mitophagy 0.035 (n = 5) gain trigger EC1.5x (μM) (0.015-0.075) Unbound plasma Cmax/TOM20-Ub cell potency (10 mg/kg PO dose - mouse) 82-fold Kp.sub.u, u 2 mg/kg IV dose - mouse 0.01

[0284] Example 1 possess beneficial properties demonstrating potential superiority over other compounds. For instance, the observed IV plasma clearance of 23 mL/min/kg, as measured in the mouse, is low, demonstrating valuable plasma stability, and the compound has very good oral bioavailability of 48%.

[0285] The high thermodynamic solubility of Example 1 of 415 μM, as measured in simulated intestinal fluid (fasted state), is potentially indicative of greater absorption through the gut into the hepatic portal vein. Higher solubility also gives a greater probability that, on dose escalation, where all other factors are equal, solubility-limited plateauing of systemic exposure can be avoided.

TABLE-US-00013 Compound Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 DUB USP30   0.029     0.037   0.013   0.029     0.034 IC.sub.50 (μM) UCHL1 >300    >300 200.5  >300   >300 DUB selectivity 10300    8100 15400    10300    8800 preference for USP30 v UCHL1 DUB USP2, USP10, USP16, 5.8-202   6.9->300 4.4->300  10.6->300  12.4->300  IC.sub.50 (μM) USP21,USP22, USP25, USP28, USP35, USP46 DUB selectivity 200-6900 186-8100 338-23000  365-10300 360-8800 preference for USP30 v 9 DUBs Cathepsin Cathepsin B 154.2  >300 29.4 110.5 >300 IC.sub.50 (μM) Cathepsin K 26.2  113 18.8  44.5    24.5 Cathepsin L 30.9    32.8  9.9  54.5 >300 Cathepsin S >300    >300 256.2  226.3 >300 Cathepsin V 176   >300 42.9 223.2  162 B, K, L, S, V 26.2-300.sup.  32.8-300.sup.  9.9-256.2 44.5-226.3 24.5-300.sup.  Selectivity preference for USP30 v  900-10300 880-8100 760-19700 1500-7800  720-8800 cathepsins Cell Tox HCT116 EC.sub.50 (μM) >30   — >30   >30   — Cell Tox EC.sub.50/USP30 IC.sub.50 >1000    — >2300    >1000    — Cell TE Endogenous USP30   0.064     0.089   0.037   0.087     0.072 WB EC.sub.50 (μM)

TABLE-US-00014 Compound Ref. Ex. A Ref. Ex. B Ref. Ex. C Ref. Ex. D DUB USP30 0.07 0.044 0.026 0.041 IC.sub.50 (μM) USP2, USP10, USP16, 0.19-9.9 0.35-10.2 0.025-8.9  0.19-5.1  USP21,USP22, USP25, (excl. (excl. USP28, USP35, USP46 USP22) USP2, 46) DUB selectivity   2.7-141  8-232 0.96-342   4.6-124 preference for USP30 v 9 DUBs Cathepsin Cathepsin B 4.5 2.9 4.8 — IC.sub.50 (μM) Cathepsin K 0.79 1.1 2.4 — Cathepsin L 2.8 0.79 1.5 2.3 Cathepsin S 8.8 2.3 5.9 6.9 Cathepsin V 5.8 4.2 4.0 3.0 B, K, L, S, V 0.79-8.8 0.79-4.2  1.5-5.9 2.3-6.9 Selectivity preference for USP30 v  11-126 18-95  58-227  56-168 cathepsins Compound Ref. Ex. E Ref. Ex. F Ref. Ex. G DUB USP30 0.31 0.70 4.4 IC.sub.50 (μM) UCHL1 0.25 3.2 6.8 DUB selectivity 0.8 4.6 1.5 preference for USP30 v UCHL1 Cathepsin Cathepsin B 5.3 9.8 — IC.sub.50 (μM) Cathepsin K 2.7 0.86 — Cathepsin L 1.0 0.24 — Cathepsin S — 2.1 — Cathepsin V — 0.82 — B, K, L, S, V 1.0-5.3 0.24-9.8 — Selectivity preference for USP30 v 3.2-17  0.34-14  — cathepsins

[0286] Comparative Data

[0287] Reference Examples A, B, C, D, E, F and G are known DUB inhibitors that have been identified as active as inhibitors of USP30 and share some structural similarity with the compounds of the present invention, possessing the cyanamide structural feature. Reference Examples E, F and G are disclosed in WO 2016/046530 as having UCHL1 inhibitory activity.

[0288] Potency for USP30

[0289] All of Examples 1 to 5 of the present invention are significantly more potent against USP30 than Reference Examples A, E, F and G, as measured in the biochemical assay. For instance, Example 1 is 2.4, 10.7, 24 and 152-fold more potent than Reference Examples A, E, F and G, respectively. Example 3 is significantly more potent against USP30 than all Reference Examples, being 5.4, 3.4, 2, 3.2, 24, 54 and 338-fold more potent than Reference Examples A, B, C, D, E, F and G, respectively.

[0290] Selectivity for USP30 over USP2, USP10, USP16, USP21, USP22, USP25, USP28, USP35 and USP46

[0291] All of Examples 1 to 5 are significantly more selective for USP30 over nine DUBs compared to Reference Examples A, B, C and D (USP2, USP10, USP16, USP21, USP22, USP25, USP28, USP35 and USP46; subject to incomplete data for C and D). Examples 1 to 5 are, at a minimum, 186-fold more potent against USP30 than against each of the nine DUBs. This is a significant advantage over A, B, C and D, which against one of the nine DUBs are 2.7, 8, 0.96 and 4.6-fold more potent, respectively (C is equipotent).

[0292] Selectivity for USP30 Over UCHL1

[0293] All of Examples 1 to 5 are significantly more selective for USP30 over UCHL1 compared to Reference Examples E, F and G. Examples 1 to 5 are at least 8100-fold more potent against USP30 than UCHL1, whereas E, F and G are only 0.8, 4.6 and 1.5-fold more potent, respectively (E is equipotent). Examples 1 to 5 are therefore, at a minimum, 1700-fold more selective for USP30 over UCHL1 than E, F and G.

[0294] Selectivity for USP30 Over Cathepsins B, K, L, S and V

[0295] All of Examples 1 to 5 are significantly more selective for USP30 over the cathepsins (B, K, L, S and V) compared to Reference Examples A, B, C, D, E and F (subject to incomplete data for D and E). Examples 1 to 5 are, at a minimum, 720-fold more potent against USP30 than against each of the cathepsins. This is a significant advantage over A, B, C, D, E and F, which against one cathepsin are 11, 18, 58, 56, 3.2 and 0.34-fold more potent, respectively.

[0296] For instance, Example 1 is 27, 59, 18, 19, 333 and 3135-fold more selective for USP30 over cathepsin L than A, B, C, D, E and F, respectively.

[0297] The above-identified advantages of the compounds of the invention over the reference examples of the prior art are both significant and unexpected. On their own, and in particular in combination, this superiority makes the compounds of the invention particularly suitable for use in the treatment or prevention of diseases linked to UP30 activity.

[0298] Preclinical In Vivo Models

[0299] Compounds of the invention may be tested for efficacy in representative in vivo disease models, using standard study procedures from the published literature, including, for example:

[0300] (a) Bleomycin-induced lung fibrosis model, which is a leading preclinical in vivo model of Idiopathic Pulmonary Fibrosis. [Kobayashi et al, 2016, J Immunol, 197(2):504-516]

[0301] (b) Diet-induced model of NAFLD and glucose homeostasis. [Nishida et al, 2013, Lab Invest; February; 93(2):230-41]

[0302] (c) MPTP Model of Parkinson's Disease, which is a commonly used paradigm for looking at neurodegeneration in the dopaminergic system of the brain which is triggered by chemically-induced mitochondrial dysfunction. [Karuppagouner et al, 2014, Sci Rep. 2014 May 2; 4:4874]

[0303] (d) Ndufs4KO Leigh syndrome model. [Kruse et al, 2008, Cell Metab. April; 7(4):312-20]

[0304] (e) Aged mice model: effects on cognitive and motor function. [Kobilo et al, 2014, Learn Mem. Jan. 17; 21(2):119-26; Creed et al, 2019, Neuroscience. June 15; 409:169-179]

[0305] (f) The unilateral ureteral obstructive kidney disease model (UUO). [Chevalier et al, 2009, Kidney Int 75(11): 1145-1152]

[0306] UUO causes renal injury characterised by tubular cell injury, interstitial inflammation and fibrosis. It serves as a model of irreversible post-renal acute kidney injury (AKI). Experimental UUO has illustrated the molecular mechanisms of apoptosis, inflammation and fibrosis, all of which are key processes in renal injury, regardless of the primary insult. Consequently, the UUO model provides investigators information beyond obstruction.

[0307] Example 1 was assessed in the UUO model to determine the ability of the compound to attenuate progressive tubulointerstitial fibrosis and chronic kidney disease (CKD).

[0308] On day 1 of the study, adult C57BL/6 mice were dosed by oral gavage according to one of the following dosing regimens; Vehicle or 5 mg/kg Example 1 BID. Two hours post dosing on day 1 study mice underwent surgery to ligate the left ureter at two points. Successful UUO surgery was later confirmed by observation of dilation of renal pelvis due to hydronephrosis. The animals were dosed according to their prescribed regimen for 10 days at which point kidneys were harvested, or histopathology assessment and for protein/RNA assessment. Picrosirius Red staining was performed to assess the extent of collagen deposition and IHC was employed to assess relative α-Smooth Muscle Actin (α-SMA) expression.

[0309] Results demonstrated that 5 mg/kg Example 1 (p.o.) dosed BID, statistically reduced collagen deposition as evidenced by reduced picrosirius red staining in ligated kidneys. Assessment of α-SMA staining, revealed that oral dosing of 5 mg/kg Example 1 BID also statistically reduced α-SMA levels in UUO injured kidneys when compared to vehicle treated controls.

[0310] (g) The ischemia-induced acute kidney injury model (AKI).