RU2848457C1 - N-cyanopyrrolidines with activity of usp30 inhibitors - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula (I), which is selected from compounds of formula (I)(i) and formula (I)(ii), or a pharmaceutically acceptable salt thereof, having activity of inhibitors of deubiquitylation USP30 enzyme. In formulas (I)(i) and (I)(ii) R¹ is selected from (C₁-C₄)alkyl, (C₁-C₄) fluoroalkyl and CH₂OCH₃; R² selected from (C₁-C₄)alkyl, CF₃ and cyclopropyl and R³, R⁴ and R⁵, each independently selected from hydrogen and halogen. Invention also relates to the use of said compounds for the treatment or prevention of a disorder or condition associated with USP30 activity, or for treating or preventing a condition involving mitochondrial dysfunction, cancer or fibrosis, which are associated with USP30 activity, a pharmaceutical composition having USP30 activity inhibition activity containing said compounds, and to intermediate compounds in their synthesis. EFFECT: disclosed are N-cyanopyrrolidines with activity of USP30 inhibitors.

31 cl, 1 tbl, 19 ex

Description

FIELD OF INVENTION

The present invention relates to a class of N-cyanopyrrolidines with inhibitory activity against the deubiquitylating enzyme ubiquitin C-terminal hydrolase 30, also known as ubiquitin-specific peptidase 30 (USP30), their use, methods for their preparation, and compositions containing said inhibitors. These inhibitors are useful in a variety of therapeutic areas, including conditions involving mitochondrial dysfunction, cancer, and fibrosis.

All documents cited or mentioned below are expressly incorporated into this specification by reference.

PRIOR ART

Ubiquitin is a small protein consisting of 76 amino acids that is important for regulating protein function in the cell. Ubiquitylation and deubiquitylation are enzymatically mediated processes by which ubiquitin is covalently bound to or cleaved from a target protein by deubiquitylating enzymes (DUBs), of which approximately 100 DUBs exist in human cells and are divided into subfamilies based on sequence homology. The USP family is characterized by their shared Cys and His boxes, which contain Cys and His residues critical for the activity of their DUBs. Ubiquitylation and deubiquitylation processes are involved in the regulation of many cellular functions, including cell cycle progression, apoptosis, modification of cell surface receptors, regulation of DNA transcription, and DNA repair. Thus, the ubiquitin system is involved in the pathogenesis of numerous disease states, including inflammation, viral infection, metabolic dysfunction, CNS disorders, and oncogenesis.

Ubiquitin is a master regulator of mitochondrial dynamics. Mitochondria are dynamic organelles whose biosynthesis, fusion, and fission are regulated post-translationally through the ubiquitylation of many key factors, such as mitofusins. In humans, USP30 is a 517-amino acid protein located in the mitochondrial outer membrane (Nakamura et al, 2008, Mol Biol 19:1903-11). It is the only deubiquitylating enzyme that carries a mitochondrial targeting signal and has been shown to deubiquitinate a wide range of mitochondrial proteins. It has been demonstrated that USP30 antagonizes Parkin-mediated mitophagy, and that downregulation of USP30 rescues Parkin-mediated defects in mitophagy (Bingol et al, 2015, Nature 510:370-5; Gersch et al, 2017, Nat Struct Mol Biol 24(11): 920-930; Cunningham et al, 2015, Nat Cell Biol 17(2): 160-169). USP30 inactivation can also increase mitochondrial protein import, potentially through ubiquitination of TOM proteins (Jacoupy et al, 2019, Sci Rep 9(1): 11829). A small fraction of USP30 is localized to peroxisomes, which are formed by the fusion of mitochondrial and ER vesicles, with USP30 potently antagonizing the Peroxiphagy/Pex2 pathway (Riccio et al, 2019, J Cell Biol 218(3): 798–807). The ubiquitin ligase March5 and deubiquitinase EP30 associate with the translocase and regulate mitochondrial import, and while March5 antagonizes mitochondrial import and directs substrate degradation, USP30 deubiquitylates substrates to promote their import (Phu et al, 2020, Molecular Cell 77, 1107–1123).

Mitochondrial dysfunction can be defined as a decrease in mitochondrial content (mitophagy or mitochondrial biogenesis), a reduction in mitochondrial activity and oxidative phosphorylation, and a modulation of reactive oxygen species (ROS) production. Consequently, mitochondrial dysfunction plays a role in a wide range of aging processes and pathologies.

For example, Parkinson's disease affects approximately 10 million people worldwide (Parkinson's Disease Foundation) and is characterized by the loss of dopaminergic neurons in the substantia nigra. The precise mechanisms underlying PD (Parkinson's disease) are unclear; however, mitochondrial dysfunction is increasingly recognized as a key determinant of dopaminergic neuronal sensitivity in PD and is a hallmark of both familial and sporadic disease, as well as toxin-induced parkinsonism. Parkin is one of several proteins associated with early-onset PD. Although most cases of PD are associated with defects in alpha-synuclein, 10% of Parkinson's cases are associated with specific genetic defects, one of which is in parkin, an E3 ubiquitin ligase. Parkin and protein kinase PTEN-induced putative kinase 1 (PINK1) are jointly involved in the ubiquitination of mitochondrial membrane proteins from damaged mitochondria, leading to mitophagy. Dysregulation of mitophagy leads to increased oxidative stress, which has been described as a characteristic of PD. Therefore, inhibition of USP30 may represent a potential strategy for the treatment of PD. For example, PD patients with Parkin mutations leading to decreased activity may benefit from USP30 inhibition.

Depletion of USP30 has been reported to enhance mitophagic mitochondrial clearance and parkin-induced cell death. USP30 has also been shown to regulate BAX/BAK-dependent apoptosis independent of parkin overexpression. Depletion of USP30 sensitizes cancer cells to BH-3 mimetics, such as ABT-737, without the need for parkin overexpression. Thus, the antiapoptotic role of USP30 has been demonstrated, making USP30 a potential target for anticancer therapy.

The ubiquitin-proteasome system has attracted interest as a target for cancer treatment following the approval of the proteasome inhibitor bortezomib (Velcade®) for the treatment of multiple myeloma. Long-term treatment with bortezomib is limited due to associated toxicity and drug resistance. However, therapeutic strategies that target specific aspects of the ubiquitin-proteasome pathway upstream of the proteasome, such as DUB, are predicted to be better tolerated (Bedford et al., 2011, Nature Rev 10:29-46).

Fibrosing diseases, including renal, hepatic, and pulmonary fibrosis, are a leading cause of morbidity and mortality and can affect all tissues and organs. Fibrosis is thought to result from acute or chronic stress on a tissue or organ and is characterized by extracellular matrix deposition, decreased vascular/tubular/ductal/airway patency, and impaired function, ultimately leading to organ failure. Many fibrosing conditions are caused by lifestyle or environmental factors; however, some fibrosing conditions may be initiated by genetic triggers or are truly considered idiopathic (i.e., without a known cause). Some fibrosing diseases, such as idiopathic pulmonary fibrosis (IPF), can be treated with a non-specific kinase inhibitor (nintedanib) or drugs without a clearly characterized mechanism of action (pirfenidone). Other treatments for organ fibrosis, such as renal or liver fibrosis, relieve pressure on the organ itself (e.g., beta-blockers for cirrhosis, angiotensin receptor blockers for chronic kidney disease). Attention to lifestyle factors, such as glucose control and diet, can also influence the course and severity of the disease.

Mitochondrial dysfunction is implicated in a number of fibrotic diseases, with oxidative stress following dysfunction being a key pathogenic mediator, along with decreased ATP (adenosine triphosphate) production. In preclinical models, disruption of the mitophagy pathway (through mutation or knockout of either Parkin or PINK1) exacerbates pulmonary fibrosis and renal fibrosis, with evidence of increased oxidative stress.

Kurita et al., 2017, Respiratory Research 18:114, revealed that the accumulation of profibrotic myofibroblasts is a crucial process for fibrotic remodeling in IPF. Recent evidence suggests the involvement of autophagy/mitophagy, part of the lysosomal degradation machinery, in the pathogenesis of IPF, and that mitophagy is involved in myofibroblast differentiation through reactive oxygen species (ROS)-mediated regulation of mitochondrial activation of platelet-derived growth factor receptor (PDGFR). Kurita's results showed that pirfenidone induces PARK2-mediated mitophagy and also inhibits the development of pulmonary fibrosis in conditions of deficient mitophagy, which may at least partially explain the anti-fibrotic mechanisms of IPF treatment.

Williams et al., 2015, Pharmacol Res. December; 102: 264–269, discuss the role of PINK 1-parkin-mediated autophagy in protecting against alcohol- and acetaminophen-induced liver injury by removing damaged mitochondria through mitophagy. It is suggested that pharmacological stabilization of USP8 or inactivation of USP15 and USP30 may be potential therapeutic targets for upregulating parkin-induced mitophagy and, in turn, may protect against drug-induced liver injury. However, it is noted that DUBs are regulated both transcriptionally and post-translationally, which may complicate the development of drugs to target these specific enzymes, and, furthermore, phosphorylated ubiquitin has been shown to be resistant to DUBs. The authors conclude that upregulation of PTNK1 stabilization or kinase activity may be a more effective target than DAB inhibition.

Williams et al, 2015, Biomolecules 5, 2619–2642, and Williams et al, 2015, Am J Physiol Gastrointest Liver Physiol 309: G324–G340, review the mechanisms involved in the regulation of mitochondrial homeostasis in the liver and how these mechanisms may protect against alcohol-induced liver disease.

Luciani et al. (2020) Nat. Commun. 11:970 report that dysregulation of the mitochondrial network in terminally differentiated cells contributes to the development of a wide range of disorders, including methylmalonic acidemia (MMA). MMA is one of the most common inherited metabolic disorders caused by deficiency of mitochondrial methylmalonyl-coenzyme A mutase (MMUT). MMUT deficiency induces metabolic and mitochondrial changes that are exacerbated by abnormalities in PINK1/Parkin-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria, which trigger epithelial stress and, ultimately, cellular damage. A link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction, and epithelial stress is suggested, and potential therapeutic prospects for MMA are proposed.

Kluge et al, Bioorganic & Medicinal Chemistry Letters, 2018, 28 2655-2659, report that selective USP30 inhibitors accelerate mitophagy.

A series of N-cyano-substituted heterocycle derivatives are disclosed as inhibitors of deubiquitylating enzymes in PCT applications WO 2016/046530 (US 15/513125, US 15/894025, US 16/448066), WO 2016/156816 (US 15/558632, US 16/297937, US 16/419558, US 16/419747, US 16/788446), WO 2017/009650 (US 15/738900), WO 2017/093718 (US 15/776149), WO 2017/103614 (US 15/781615), WO 2017/149313 (US 16/078518), WO 2017/109488 (US 16/060299), WO 2017/141036 (US 16/070936), WO 2017/163078 (US 16/087515), WO 2017/158381 (US 16/080229), WO 2017/158388 (US 16/080506), WO 2018/065768 (US 16/336685), WO 2018/060742 (US 16/336202), WO 2018/060689 (US 16/334836), WO 2018/060691 (US 16/336363), WO 2018/220355 (US 16/615040), WO 2018/234755 (US 16/615709), WO 2020/212350, WO 2020/212351 and WO 2021/043870 and PCT WO 2021/064166, each of which is incorporated herein by reference in its entirety. PCT application WO 2019/171042 (US 16/977019), the entire contents of which are incorporated herein by reference, discloses the use of N-cyanopyrrolidines as USP30 inhibitors for the treatment of fibrosing diseases.

Falgueyret et al., 2001, J. Med. Chem. 44, 94-104, and PCT application WO 01/77073 relate to cyanopyrrolidines as inhibitors of cathepsins K and L with potential use in the treatment of osteoporosis and other conditions associated with bone resorption. PCT application WO 2015/179190 relates to jV-acylethanolamine hydrolyzing acid amidase inhibitors with potential use in the treatment of ulcerative colitis and Crohn's disease. PCT application WO 2013/030218 relates to quinazolin-4-one compounds as inhibitors of ubiquitin-specific proteases, such as USP7, with potential utility in the treatment of cancer, neurodegenerative diseases, inflammatory disorders, and viral infections. PCT applications WO 2015/017502 and WO 2016/019237 relate to Bruton tyrosine kinase inhibitors with potential utility in the treatment of diseases such as autoimmune disease, inflammatory disease, and cancer. PCT applications WO 2009/026197, WO 2009/129365, WO 2009/129370, and WO 2009/129371 relate to cyanopyrrolidines as cathepsin C inhibitors with potential utility in the treatment of COPD (chronic obstructive pulmonary disease). US Patent Application US 2008/0300268 relates to polyaromatic compounds as inhibitors of the tyrosine kinase receptor PDGFR. PCT applications WO 2019/222468, WO 2019/071073, WO 2020/036940, and WO 2020/072964, Rusilowicz-Jones et al, 2020, bioRxiv 2020.04.16.044206 (20 April 2020) and Tsefou et al, bioRxiv 2021.02.02.429344 (2 February 2021) relate to cyanamide-containing compounds as USP30 inhibitors. Yue et al, 2014, Cell Research, 24, 482–496, relate to a diterpenoid derivative of 15-oxospiramylactone as a USP30 inhibitor that induces mitochondrial fusion.

PCT application WO 2015/183987 relates to pharmaceutical compositions comprising deubiquitinase inhibitors and human serum albumin for use in methods of treating cancer, fibrosis, an autoimmune disease or condition, an inflammatory disease or condition, a neurodegenerative disease or condition, or an infection. Deubiquitinases, including UCHL5/UCH37, USP4, USP9X, USP11, and USP15, are noted to be involved in regulating the TGF-beta signaling pathway, the disruption of which leads to neurodegenerative and fibrotic diseases, autoimmune dysfunction, and cancer.

PCT application WO 2006/067165 relates to a method for treating fibrotic diseases using indolinone kinase inhibitors. PCT application WO 2007/119214 relates to a method for treating early-stage pulmonary fibrosis with an endothelin receptor antagonist. PCT application WO 2012/170290 relates to a method for treating fibrotic diseases using THC (tetrahydrocannabinolic) acids. PCT application WO 2018/213150 relates to sulfonamide USP30 inhibitors with potential utility in the treatment of conditions involving mitochondrial defects. Larson-Casey et al., 2016, Immunity 44, 582-596, examine macrophage kinase Akt1-mediated mitophagy, apoptosis resistance, and pulmonary fibrosis. Tang et al., 2015, Kidney Diseases 1, 71-79, examine the potential role of mitophagy in renal pathophysiology.

There is a need for safe, alternative, and/or improved methods and compositions for the treatment or prevention of conditions involving mitochondrial dysfunction, cancer, and fibrosis, as well as various symptoms and conditions associated with them. Without being bound by any particular theory or mechanism, the compounds of the present invention are believed to inhibit the USP30 enzyme, which, in turn, upregulates Parkinson-induced mitophagy.

Acute kidney injury (AKI) is defined as an acute decline in kidney function occurring within 7 days or less, with severity of injury staged based on elevated serum creatinine (SCr) and decreased urine output, as described in the Kidney Disease Improving Global Outcomes (KDIGO) guidelines. AKI occurs in approximately 13.3 million people per year, 85% of whom live in developing countries, and is thought to contribute to approximately 1.7 million deaths each year (Mehta et al, 2015, Lancet 385 (9987): 2616–2643). AKI more than likely leads to irreversible kidney damage (i.e., chronic kidney disease; CKD) and may also lead to damage to non-renal organs. AKI is a major public health concern, particularly given the sheer number of patients who develop CKD, progressive CKD, end-stage renal disease, and cardiovascular events. AKI has been found to be prevalent in patients hospitalized with COVID-19 and is significantly associated with in-hospital mortality. Mitochondrial damage and dysfunction have been reported as a potential pathophysiological mechanism and therapeutic target (Kellum et al, Nephrol Dial Transplant (2020) 35:1652–1662).

AKI and CKD are considered a continuum within the same disease spectrum (Chawla et al, 2017, Nat Rev Nephrol 13(4): 241–257). Patients who have undergone coronary artery bypass grafting (CABG) are at high risk of kidney injury. There is a clear unmet medical need for the development of therapeutic products to treat and/or prevent AKI.

The kidney is a site of high metabolic demand, with high rates of mitophagy demonstrated in vivo (McWilliams et al, 2018, Cell Metab 27(2):439–449 e435). Renal proximal tubular epithelial cells (RPTECs), a cell type with a significant requirement for ATP for solute/ion exchange, are rich in mitochondria and are the primary effector cells of acute kidney injury (AKI) in the kidney. Mitochondrial dysfunction has been implicated in the mechanisms of AKI/CKD, both by multiple lines of evidence from preclinical models of AKI and CKD and by data demonstrating abnormal mitochondrial phenotypes in patient biopsies (Emma et al, 2016, Nat Rev Nephrol 12(5): 267–280; Eirin et al, 2017, Handb Exp Pharmacol 240: 229–250). In addition, primary mitochondrial disease often presents with renal features such as focal segmental glomerulosclerosis (Kawakami et al, 2015, J Am Soc Nephrol 26(5): 1040–1052) in patients with MELAS/MIDD (mitochondrial encephalopathy with lactic acidosis/maternally inherited diabetes mellitus and deafness syndrome) and primary tubular pathologies in patients with coenzyme Q deficiency. Mutations in mtDNA (mitochondrial DNA) can cause maternally inherited tubulointerstitial disease (Connor et al, 2017, PLoS Genet 13(3): e1006620).

Regarding mitochondrial quality control in renal injury (Tang et al, 2018, Autophagy 14(5): 880–897), it was demonstrated that renal injury was exacerbated after ischemic AKI in both PINK1 KO and PARK2 KO mice, suggesting that PINK1/PARKIN-mediated mitophagy plays a protective role after IRI (ischemic reperfusion injury) in the kidney. Furthermore, Parkin/PINK1 mitophagy protects against cisplatin-induced renal injury (Wang et al, 2018, Cell Death Dis 9(11): 1113). Limited CKD models are available to study mitophagy, and supporting evidence for mitochondrial quality control in fibrosis comes from studies of fibrosing lung diseases such as COPD and IPF. Parkin knockout animals exhibit exacerbated pulmonary fibrosis in response to bleomycin (Kobayashi et al, 2016, J Immunol, 197:504–516). Similarly, airway epithelial cells from parkin knockout (KO) animals exhibit exacerbated fibrotic and senescent responses to cigarette smoke (Araya et al, 2019, Autophagy 15(3): 510–526).

Preclinical models are available for the investigation of potential new therapeutics due to their ability to simulate fibrotic pathology (e.g., collagen deposition) consistent with the human condition. Preclinical models can be toxin-mediated (e.g., bleomycin for pulmonary and skin fibrosis), surgical (e.g., an ischemia/reperfusion injury model and a unilateral ureteral obstruction model for acute tubulointerstitial fibrosis), and genetic (e.g., diabetic (db/db) mice for diabetic neuropathy). For example, both previously cited examples of IPF treatments (nintedanib and pirfenidone) show efficacy in the bleomycin model of pulmonary fibrosis.

Accordingly, there is a need for compounds that are USP30 inhibitors for the treatment or prevention of conditions for which USP30 inhibition is indicated. Specifically, there is a need for USP30 inhibitors that possess suitable and/or improved properties to maximize efficacy against the target disease.

SUMMARY OF THE INVENTION

The present invention relates to a compound of formula (I), which is selected from compounds of formula (I)(i) and formula (I)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

The present invention also relates to the use of compounds of formula (I), in particular in the treatment of conditions involving mitochondrial dysfunction, cancer and fibrosis, as well as to methods for their preparation and pharmaceutical compositions containing said compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to USP30 inhibitors that possess suitable and/or improved properties to maximize efficacy against a target disease. Such properties include, for example, potency, selectivity, physicochemical properties, ADME (absorption, distribution, metabolism, and excretion) properties, including PK (pharmacokinetic) profile, and safety profile.

It is generally desirable to maximize the potency of a drug molecule against a target enzyme in relevant assays to reduce the effective dosage required to administer to patients. Compounds of the invention can be tested for affinity to USP30 using the in vitro fluorescence polarization (FP) biochemical assay described herein.

USP30 is a transmembrane protein located in the outer membrane of mitochondria, which are energy-producing organelles present within cells. Therefore, the ability to demonstrate cellular activity in vitro is advantageous, as it is one of a number of components that may exhibit greater ability to interact with a target under its physiological conditions, i.e., where a USP30 inhibitor compound is able to penetrate cells. The USP30 cellular Western blot (WB) assay described herein is designed to test the activity of anti-USP30 compounds in cells using an irreversible activity probe to monitor USP30 activity. Similar to the cellular Western blot assay, target interaction (ex vivo) assessment can be performed in brain or kidney tissue samples from animals administered the compound.

To extend target binding data to subsequent pharmacodynamics, TOM20 (mitochondrial outer membrane protein) ubiquitylation can be assessed.

As a rule, it is important for a drug to be as selective as possible for its desired target enzyme; additional activities increase the potential for side effects. The precise physiological role of many DUBs has not yet been fully determined; however, regardless of the role these DUBs may or may not play, it is a reasonable fundamental rule of medicinal chemistry to ensure that any drug exhibits selectivity over related mechanistic targets of unknown physiological function. Representative examples of DUB enzymes for which the compounds of the present invention can be screened 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, USP47 and USP48. Preferably, the compounds of the invention have good selectivity for USP30 relative to one or more of these DUB enzymes.

In addition to selectivity for other DUB enzymes, it is important for the drug to have low affinity for other targets. Pharmacological profiling can be performed against panels of targets to assess potential and minimize potential off-target effects. Examples of targets against which compounds of the present invention can be screened include the industry-standard Eurofins-Cerep SafetyScreen44 panel, which includes 44 targets representative of GPCR (G protein-coupled receptor) receptors, transporters, ion channels, nuclear receptors, and kinase and non-kinase enzymes. Preferably, compounds of the invention have low affinity for the targets of this screening panel. Other examples of targets for which a compound of the present invention can be screened include the kinases of the Thermo Fisher SelectScreen kinase profiling panel, which includes 39 targets as a representative selection of kinase enzymes. Preferably, the compounds of the invention have low affinity for the targets of this screening panel. Additionally, examples of a particular class of enzymes for which the compounds of the present invention can be screened include cathepsins (e.g., cathepsin A, B, C, H, K, L, L2, S, V, and Z). Preferably, the compounds of the invention exhibit good selectivity for USP30 over one or more of these enzymes.

There is also a need for compounds with favorable pharmacokinetic properties to be suitable for orally administered drugs. Orally administered drugs must have good bioavailability, meaning they can easily pass through the gastrointestinal tract (GI) and not undergo extensive metabolism upon passage from the GI tract into the systemic circulation. Once the drug enters the systemic circulation, the rate of metabolism is also important in determining the drug's residence time in the body.

Thus, it is clear that drug molecules must readily pass through the gastrointestinal tract and be slowly metabolized in the body. The Caco-2 assay is a widely accepted model for predicting the ability of a given molecule to pass through the gastrointestinal tract. Most drug metabolism typically occurs in the liver, and in vitro assays using whole-cell hepatocytes (animal or human) are widely used methods for measuring the susceptibility of a given molecule to hepatic metabolism. Such assays are designed to predict in vivo clearance based on the calculated hepatocyte clearance value.

Compounds that have good permeability in the Caco-2 model and are stable relative to hepatocytes are predicted to have good oral bioavailability (good absorption throughout the gastrointestinal tract and minimal extraction of the compound during its passage through the liver) and a long residence time in the body, which is sufficient for the drug to be effective.

Compound solubility is an important factor in achieving the desired drug concentration in the systemic circulation for the expected pharmacological response. Low aqueous solubility is a challenge in the development of new chemical compounds, and for absorption, the drug must be present as a solution at the site of absorption. The kinetic solubility of a compound can be measured using turbidimetric solubility analysis, the data from which can also be used in conjunction with permeability data in the Caco-2 model to predict dose-dependent intestinal absorption in humans.

Other parameters that can be measured using standard assays and that are indicative of the exposure profile of a compound include, for example, plasma stability (measurement of half-life),

AUC, C _max , C _min and T _max values in blood.

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. Therefore, there is a need for USP30 inhibitors that exhibit effective blood-brain penetration properties and provide an appropriate residence time in the brain to be effective. The likelihood of a compound crossing the blood-brain barrier can be measured in an in vitro permeability assay using a cell monolayer of MDR ^-1 -MDCK (Madin-Darby canine kidney cells transfected with MDR-1, resulting in overexpression of the human efflux transporter P-glycoprotein). Additionally, exposure can also be measured directly in the brain and in plasma using in vivo animal models.

There is also a need for compounds with a favorable safety profile that can be measured by various standard in vitro and in vivo assays. Parallel screening for cellular toxicity can be used to analyze the antiproliferative/cytotoxic effect in a specific cell line (e.g., HCT116) by fluorometrically detecting the conversion of resasurin (alamarBlue™) to resofurin in response to mitochondrial activity.

Toxicology and safety studies may also be conducted to identify potential target organs for adverse effects and to determine the therapeutic index for establishing starting doses in clinical trials. Regulatory requirements typically require that studies be conducted in at least two laboratory animal species: one rodent (rat or mouse) and one non-rodent (rabbit, dog, non-human primate, or other suitable species).

A bacterial reverse mutation assay (Ames test) can be used to evaluate the mutagenic properties of a compound of the invention, typically using a bacterial strain of Salmonella typhimurium that is mutant for the biosynthesis of the amino acid histidine.

Micronucleus analysis can be used to determine whether a compound is genotoxic by assessing the presence of micronuclei. Micronuclei can contain chromosomal fragments resulting from DNA breaks (clastogens) or entire chromosomes resulting from mitotic dysfunction (aneugens).

Prognostic hERG analysis provides valuable information about the potential binding of test compounds to the potassium channel and the potential for QT prolongation on echocardiography. Inhibition of hERG current causes QT prolongation, leading to potentially fatal ventricular tachyarrhythmia (Torsades de Pointes). This analysis can typically be obtained using an automated patch-clamp analysis platform.

Thus, the present invention relates to USP30 inhibitors that possess suitable and/or improved properties to maximize efficacy against the target disease. These properties include, for example, potency, selectivity, physicochemical properties, ADME (absorption, distribution, metabolism, and excretion) properties, including the PK (pharmacokinetic) profile, and a safety profile.

The compounds of the present invention were found to exhibit one or more of the above-identified properties, which are both significant and unexpected. For example, the compounds of Examples 1-12 of the present invention exhibit a potent effect on USP30, as measured in the biochemical assay described herein. All of the compounds of these Examples (1-12) of the present invention are significantly more selective for USP30 relative to other DABs and cathepsins.

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 associated with USP30 activity.

According to a first aspect of the present invention, there is provided a compound of formula (I), which is selected from compounds of formula (I)(i) and formula (I)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

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

Alkyl groups can be straight or branched and contain 1-4 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and sec-butyl.

Halogen means fluorine, chlorine, bromine, or iodine, particularly fluorine or chlorine. Fluoroalkyl groups may contain one or more fluorine substituents. Examples include fluoromethyl, difluoromethyl, and trifluoromethyl.

Unless otherwise specified, the term "substituted" means substituted by one or more specified groups. If groups can be selected from more than one alternative, the selected groups may be the same or different. The term "independently" means that if more than one substituent is selected from more than one possible substituent, these substituents may be the same or different.

Preferred embodiments of the compound of formula (I) for use in the present invention are defined below.

Preferably, R ¹ is selected from methyl, CH ₂ F, CHF ₂ , CF ₃ and CH ₂ OCH ₃ .

More preferably, R ¹ is selected from methyl, CH ₂ F and CH ₂ OCH ₃ .

Most preferably, R ¹ is selected from methyl and CH ₂ OCH ₃ .

Preferably, R ² is selected from methyl, CF ₃ and cyclopropyl.

Most preferably, R ² is selected from methyl and cyclopropyl.

Preferably, R ³ is selected from hydrogen, chlorine and fluorine.

More preferably, R ³ is selected from hydrogen and fluorine.

Most preferably, R ³ is hydrogen.

Preferably, R ⁴ and R ⁵ are each independently selected from hydrogen and fluorine.

Most preferably, each of R ⁴ and R ⁵ is hydrogen.

According to one aspect of the invention, the compound of formula (I) has formula (I)(i):

According to another aspect of the invention, the compound of formula (I) has formula (I)(ii):

According to a first preferred aspect of the invention there is provided a compound of formula (IA)(i):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

Preferred embodiments of the compound of formula (IA)(i) are defined below.

Preferably, R ¹ is selected from methyl, CH ₂ F, CHF ₂ , CF ₃ and CH ₂ OCH ₃ .

More preferably, R ¹ is selected from methyl, CH ₂ F and CH ₂ OCH ₃ .

Most preferably, R ¹ is selected from methyl and CH ₂ OCH ₃ .

Preferably, R ² is selected from methyl, CF ₃ and cyclopropyl.

Most preferably, R ² is selected from methyl and cyclopropyl.

Preferably, R ³ is selected from hydrogen, chlorine and fluorine.

More preferably, R ³ is selected from hydrogen and fluorine.

Most preferably, R ³ is hydrogen.

Preferably, R ⁴ and R ⁵ are each independently selected from hydrogen and fluorine.

Most preferably, each of R ⁴ and R ⁵ is hydrogen.

Preferred compounds of formula (IA)(i) according to the present invention are selected from:

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; and

5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

or their pharmaceutically acceptable salts.

The most preferred compounds of formula (IA)(i) according to the present invention are selected from:

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; and

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or their pharmaceutically acceptable salts.

According to a second preferred aspect of the invention there is provided a compound of formula (IB)(i):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

Preferred embodiments of the compound of formula (IB)(i) are defined below.

Preferably, R ¹ is selected from methyl, CH ₂ F, CHF ₂ , CF ₃ and CH ₂ OCH ₃ .

More preferably, R ¹ is selected from methyl, CH ₂ F and CH ₂ OCH ₃ .

Most preferably, R ¹ is CH ₂ OCH ₃ .

Preferably, R ² is selected from methyl, CF ₃ and cyclopropyl.

Most preferably, R ² is methyl.

Preferably, R ³ is selected from hydrogen, chlorine and fluorine.

More preferably, R ³ is selected from hydrogen and fluorine.

Most preferably, R ³ is hydrogen.

Preferably, R ⁴ and R ⁵ are each independently selected from hydrogen and fluorine.

Most preferably, each of R ⁴ and R ⁵ is hydrogen.

Preferred compounds of formula (IB)(i) according to the present invention are selected from:

4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; and

4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or their pharmaceutically acceptable salts.

According to a third preferred aspect of the invention there is provided a compound of formula (IA)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

Preferred embodiments of the compound of formula (IA)(ii) are defined below.

Preferably, R ¹ is selected from methyl, CH ₂ F, CHF ₂ , CF ₃ and CH ₂ OCH ₃ .

More preferably, R ¹ is selected from methyl, CH ₂ F and CH ₂ OCH ₃ .

Most preferably, R ¹ is selected from methyl and CH ₂ OCH ₃ .

Preferably, R ² is selected from methyl, CF ₃ and cyclopropyl.

Most preferably, R ² is selected from methyl and cyclopropyl.

Preferably, R ³ is selected from hydrogen, chlorine and fluorine.

More preferably, R ³ is selected from hydrogen and fluorine.

Most preferably, R ³ is hydrogen.

Preferably, R ⁴ and R ⁵ are each independently selected from hydrogen and fluorine.

Most preferably, each of R ⁴ and R ⁵ is hydrogen.

Preferred compounds of formula (IA)(ii) according to the present invention are selected from:

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; and

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or their pharmaceutically acceptable salts.

According to a fourth preferred aspect of the invention there is provided a compound of formula (IB)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

Preferred embodiments of the compound of formula (IB)(ii) are defined below.

Preferably, R ¹ is selected from methyl, CH ₂ F, CHF ₂ , CF ₃ and CH ₂ OCH ₃ .

More preferably, R ¹ is selected from methyl, CH ₂ F and CH ₂ OCH ₃ .

Most preferably, R ¹ is CH ₂ OCH ₃ .

Preferably, R ² is selected from methyl, CF ₃ and cyclopropyl.

Most preferably, R ² is methyl.

Preferably, R ³ is selected from hydrogen, chlorine and fluorine.

More preferably, R ³ is selected from hydrogen and fluorine.

Most preferably, R ³ is hydrogen.

Preferably, R ⁴ and R ⁵ are each independently selected from hydrogen and fluorine.

Most preferably, each of R ⁴ and R ⁵ is hydrogen.

Preferred compounds of formula (IB)(ii) according to the present invention are selected from:

4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; and

4-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or their pharmaceutically acceptable salts.

Pharmaceutically acceptable salts of the compounds of formula (I) include their acid addition salts and base addition salts (including disalts).

Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include 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, methyl sulfate, 2-napsylate, nicotinate, nitrate, orotate, palmate, phosphate, saccharate, stearate, succinate sulfate, D- and L-tartrate, and tosylate.

Suitable base salts are formed from bases that form nontoxic salts. Examples include aluminum, ammonium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts.

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

A pharmaceutically acceptable salt of a compound of formula (I) can be readily prepared by mixing solutions of the compound of formula (I) and the desired acid or base, as appropriate. The salt can precipitate from the solution and can be collected by filtration or isolated by evaporation of the solvent.

Pharmaceutically acceptable solvates according to the invention include hydrates and solvates wherein the crystallization solvent may be substituted with an isotope, for example D ₂ O, acetone-d ₆ , DMSO-d ₆ .

Also included within the scope of the invention are clathrates, drug-host inclusion complexes where, unlike 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).

In the following, all references to compounds of formula (I) include references to their salts and to solvates and clathrates of the compounds of formula (I) and their salts.

The invention encompasses all polymorphs of the compounds of formula (I) as defined below.

Also included within the scope of the invention are so-called "prodrugs" of compounds of formula (I). Thus, certain derivatives of compounds of formula (I), which themselves possess little or no activity, when metabolized after administration into or to the body, are converted into compounds of formula (I) possessing the desired activity. Such derivatives are called "prodrugs."

Prodrugs according to the invention can be obtained, for example, by replacing the corresponding functional groups present in the compounds of formula (I) with certain moieties known to those skilled in the art as "promoieties", as described, for example, in "Design of Prodrugs" by H Bundgaard (Elsevier, 1985).

Finally, some compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I).

Certain derivatives of the 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.

The present invention includes within its scope all tautomeric forms of the compounds of formula (I).

Conventional methods for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemic salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). Alternatively, the racemate (or racemic precursor) can be reacted with a suitable optically active compound, for example, an alcohol, or, if the compound of formula (I) contains an acidic or basic moiety, with a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture can be separated by chromatography and/or fractional crystallization, and one or both diastereoisomers can be converted to the corresponding pure enantiomer(s) by methods known to those skilled in the art. The chiral compounds of the invention (and their chiral precursors) can 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 0-50% by volume isopropanol, typically 2% to 20%, and 0-5% by volume alkylamine, typically 0.1% diethylamine. Concentration of the eluate gives the enriched mixture. The present invention encompasses crystalline forms of the compounds of formula (I), including their racemates and racemic mixtures (conglomerates). Stereoisomeric conglomerates can be separated by conventional methods 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)).

Compounds of formula (I) contain two chiral centers on the carbon atoms of the pyrrolidine ring, which are substituted by R ¹ and amide, and these stereocenters can exist in either the (R) or (S) configuration. The designation of the absolute configuration (R) and (S) for stereoisomers according to IUPAC nomenclature depends on the nature of the substituents and the application of the rule of precedence. Thus, compounds of formula (I) can exist in four stereoisomeric forms.

The compounds of formula (I) according to the present invention exist as a single stereoisomer. The carbon atom of the pyrrolidine substituent of the amide exists as an (R)-stereocenter, while the designation of the pyrrolidine carbon atom of the R ¹ group depends on the nature of this substituent. The compound of formula (I) is isolated as a single stereoisomer and can 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%.

Additional chiral centers may exist in the compounds of formula (I) within the R ¹ substituent itself. The present invention includes within its scope all such stereoisomeric forms of the compounds of formula (I).

The present invention also encompasses all pharmaceutically acceptable isotopic variants of the compound of formula (I). An isotopic variant is defined as a variant 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 typically found in nature.

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

Substitution of the compounds of the invention with isotopes such as deuterium may provide some therapeutic advantages due to greater metabolic stability, such as increased half-life in vivo or reduced dosage requirements, and may therefore be preferable in some circumstances.

Certain isotopic variants of compounds of formula (I), such as those incorporating a radioactive isotope, are useful in tissue distribution studies of drugs or substrates. The radioactive isotopes tritium and ^14C are particularly useful for this purpose due to their ease of incorporation and readily available detection means.

Isotopic variations of compounds of formula (I) are typically prepared by standard methods known to those skilled in the art or by methods analogous to those described in the accompanying Examples and Preparations, using appropriate isotopic variations of suitable reagents.

Compounds of formula (I) are inhibitors of the deubiquitylating enzyme USP30.

According to a further aspect of the present invention there is provided 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.

According to a further aspect of the present invention, there is provided a method for the treatment or prevention of a disorder or condition for which inhibition of USP30 is known or can be shown to have 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.

According to another aspect of the present invention, there is provided 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 for which inhibition of USP30 is known or can be shown to have a beneficial effect. The manufacture of the medicament may include, inter alia, the chemical synthesis of a compound of formula (I) or a salt thereof, or the preparation of a composition or formulation containing the compound or salt, or the packaging of any medicament containing the compound.

According to another aspect of the present invention, there is provided a method of inhibiting USP30 in a patient, comprising administering to the patient a therapeutic amount of a compound of formula (I), as defined herein, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

The disorder or condition caused by USP30 activity is selected from a condition involving mitochondrial dysfunction, cancer, and fibrosis.

In one preferred embodiment of all aspects of the invention, the disorder or condition caused by USP30 activity is a condition involving mitochondrial dysfunction.

Mitochondrial dysfunctions arise from defects in the mitochondria, which are specialized compartments present in every cell in the body except red blood cells. When mitochondria fail, the cell produces less and less energy, leading to cell damage or even cell death. If this process is repeated throughout the body, the life of the affected individual is significantly compromised. Mitochondrial diseases most often occur in organs that require a high level of energy production, such as the brain, heart, liver, skeletal muscle, kidneys, and the endocrine and respiratory systems.

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 defect in lysosomal storage.

In particular, the condition in which mitochondrial dysfunction is involved may be selected from neurodegenerative disease; multiple sclerosis (MS); mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome (MELAS); maternally inherited diabetes and deafness (MIDD); Leber's hereditary optic neuropathy (LHON); cancer (including, for example, breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreatic, brain, melanoma, bone cancer or other tissue cancers and blood cell cancers such as lymphoma and leukemia, multiple myeloma, metastatic carcinoma, osteosarcoma, chondrosarcoma, 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 neuropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; psychiatric disorders such as schizophrenia; multiple sulfatase deficiency (MSD); Mucolipidosis II (ML II); Mucolipidosis III (ML III); Mucolipidosis IV (ML IV); GM1 gangliosidosis (GM1); Neuronal ceroid lipofuscinosis (NCL1); Alpers disease; Barth syndrome; Beta-oxidation defects; Carnitine acyl-carnitine deficiency; Carnitine deficiency; Creatinine deficiency syndromes; Coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; Complex V deficiency; COX (cyclooxygenase) deficiency; Chronic progressive external ophthalmoplegia syndrome (CPEO); CPTI (carnitine palmtoyltransferase I) deficiency; CPT II deficiency; Glutaraciduria type II; Kearns-Sayre syndrome; Lactic acidosis; Long-chain acyl-CoA dehydrogenase deficiency (LCHAD); Leigh disease or syndrome; French-Canadian variant of Leigh syndrome (LSFC); Lethal infantile cardiomyopathy (LIC); Luft disease; Medium-chain acyl-CoA dehydrogenase deficiency (MCAD); Myoclonic epilepsy and ragged red fiber syndrome (MERRF); Mitochondrial cytopathy; Mitochondrial recessive ataxia syndrome; Mitochondrial DNA depletion syndrome; Myoneurogastrointestinal disorder and encephalopathy; Pearson syndrome; Pyruvate dehydrogenase deficiency; Pyruvate carboxylase deficiency; POLG (mitochondrial polymerase gamma) mutations; Medium-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (M/SCHAD); very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency; peroxisomal disorders; methylmalonic acidemia; and age-related decline in cognitive function and muscle strength.

A condition involving mitochondrial dysfunction may represent a CNS disorder, such as a neurodegenerative disease.

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.

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 associated with mutations in α-synuclein, parkin, PINK1, GBA and LRRK2, and autosomal recessive juvenile Parkinson's disease (AR-JP), where parkin is mutated, truncated or deleted.

In particular, the compounds of the invention may be useful in the treatment or prevention of cognitive impairment associated with neurodegenerative and neuropsychiatric disorders, including, for example, cognitive impairment associated with Alzheimer's disease and Parkinson's disease, preclinical or prodromal forms of AD and PD, Huntington's disease, dementia with Lewy bodies, cognitive impairment associated with schizophrenia, mood disorders, bipolar and generalized depressive disorders.

The compounds of the invention or pharmaceutical compositions containing them, as described herein, can be combined with one or more additional agents when used to treat or prevent conditions involving mitochondrial dysfunction. The compounds can be combined with one or more additional agents selected from levodopa, a dopamine agonist, a monoamine oxygenase (MAO) B inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, an anticholinergic agent, riluzole, amantadine, a cholinesterase inhibitor, memantine, tetrabenazine, an antipsychotic, diazepam, clonazepam, an antidepressant, and an anticonvulsant. The compounds can be combined with agents that reduce/remove pathogenic protein aggregates in neurodegenerative diseases, such as agents that reduce/remove alpha-synuclein in Parkinson's disease, multiple system atrophy, or dementia with Lewy bodies; agents that reduce/remove TAI in Alzheimer's disease or progressive supranuclear palsy; agents that reduce/remove TDP-43 in ALS or frontotemporal dementia.

In another preferred embodiment of all aspects of the invention, the disorder or condition caused by the activity of USP30 is cancer. Cancer can be associated with mitochondrial dysfunction. Preferred cancers include, for example, breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreatic, brain, melanoma, bone or other tissue organ cancers and blood cell cancers such as lymphoma and leukemia, multiple myeloma, metastatic carcinoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, nasopharyngeal sarcoma, colorectal cancer, colorectal cancer and non-small cell lung carcinoma.

In particular, the compounds of the invention may be useful in the treatment or prevention of cancer in which apoptotic pathways are dysregulated, and more particularly where BCL-2 (B-cell lymphoma 2) family proteins are mutated or over- or under-expressed.

Fibrosis refers to the accumulation of extracellular matrix constituents that occurs as a result of injury, inflammation, tissue regeneration, immunological reactions, cellular hyperplasia, and neoplasia. Fibrosing disorders that can be treated with the compounds and compositions of the present invention include, but are not limited to, fibrosis/fibrosing disorders associated with diseases of vital organs, such as interstitial lung disease (ILD), liver cirrhosis, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) (liver fibrosis), kidney disease (renal fibrosis), acute kidney injury (AKI), chronic kidney disease (CKD), delayed kidney transplant function, cardiac or vascular disease (cardiac fibrosis), and eye diseases; fibroproliferative disorders such as systemic and focal sclerosis, keloids and hypertrophic scars, atherosclerosis, restenosis, and Dupuytren's contracture; scarring associated with trauma such as surgical complications, chemotherapeutic 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 after kidney transplantation (see, e.g., Wynn et al, 2004, Nat Rev Immunol. August; 4(8): 583-594). Thus, the present invention relates to methods for the treatment or prevention, and compounds and compositions used in said methods, of fibrosis/fibrosing disorders of organs and/or associated with major organs, including, for example, the lung, liver, kidney, heart, skin, eye, gastrointestinal tract, peritoneum and bone marrow, and other diseases/disorders described herein.

The compounds can be combined with agents that are used as therapeutic agents for the treatment of kidney disease, including antidiabetic agents, agents for the treatment of cardiovascular diseases, and new agents that target disease-related pathways such as oxidative stress (including, but not limited to, the nrf2/keap-1 pathway) and antiapopathic pathways (including, but not limited to, anti-p53 agents).

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

Idiopathic pulmonary fibrosis (IPF) is the most common type of ILD, and its cause is unknown.

The compounds can be combined with agents that are treatments for IPF and potentially ILD, including nintedanib and pirfenidone.

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

Kidney disease can be associated with diabetes, which can damage and scar the kidneys, leading to progressive loss of function, as well as hypertensive disorders. Renal fibrosis can occur at any stage of kidney disease, from acute kidney injury (AKD) following injury and chronic kidney disease (CKD), such as incidental CKD and progressive CKD, to end-stage renal disease (ESRD). Renal fibrosis can develop as a result of cardiovascular disease, such as hypertension, or diabetes, which both greatly limit kidney function, promoting a fibrotic response. However, renal fibrosis can also be idiopathic (without a known cause), and some genetic mitochondrial diseases also present with manifestations of renal fibrosis and associated symptoms.

Heart disease can cause scarring of tissue, which can impair the heart's ability to pump.

Eye diseases include macular degeneration and retinal and vitreous retinopathy, which can impair vision.

In a preferred embodiment, the present invention relates to the treatment or prevention of idiopathic pulmonary fibrosis (IPF).

In another preferred embodiment, the present invention relates to the treatment or prevention of renal fibrosis.

In another preferred embodiment, the present invention relates to the treatment or prevention of acute kidney injury (AKI), particularly in high-risk patients. Examples include post-surgical AKI, such as after organ transplantation, such as due to ischemia-reperfusion injury, delayed graft function; oncological, such as AKI due to chemotherapy; contrast medium-induced nephropathy, such as direct tubular cytotoxicity, hemodynamic ischemia and osmotic effects; acute interstitial nephritis, such as due to drug intake or infection; and COVID-19-induced AKI. A special subgroup of high-risk patients are patients who have undergone cardiac surgery, such as coronary artery bypass grafting and/or valve surgery. Statistical risk factors for the development of AKI have been established, such as age 65 years and older, insulin-dependent diabetes, CKD (the special risk group are adults with an estimated glomerular filtration rate [eGFR] less than 60 ml/min/1.73 ^m2 ), heart failure, liver disease, and a history of AKI.

In another preferred embodiment, the present invention relates to the treatment or prevention of chronic kidney disease (CKD) resulting from such AKI, including, for example, tubulointerstitial fibrosis and diabetic nephropathy.

In another preferred embodiment, the present invention relates to the treatment or prevention of liver diseases, including, but not limited to, NAFLD, NASH, liver cirrhosis, portal hypertension, acute liver failure, and hepatocellular carcinoma. Liver diseases such as NAFLD and NASH can be associated with various metabolic conditions, such as metabolic syndrome and type II diabetes, which also increases the risk of diabetes-associated pathologies, including diabetic retinopathy and peripheral neuropathies.

The compounds of the invention or pharmaceutical compositions thereof, as described herein, can be combined with one or more additional agents when used to treat or prevent conditions involving liver disease and metabolic dysfunction, including metformin, sulfonylureas, DPP-4 inhibitors, GLP-1 agonists, PPAR agonists, SGLT2 inhibitors, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin II receptor blockers (ARBs).

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. It rarely occurs in adolescents and adults. Leigh syndrome can be caused by mutations in the nuclear DNA encoding mitochondrial proteins, mutations in mitochondrial DNA (maternally inherited Leigh syndrome (MILS)) or a deficiency of an enzyme called pyruvate dehydrogenase, located on the short arm of the X chromosome (X-linked Leigh syndrome). Symptoms of Leigh syndrome typically progress rapidly. The earliest signs may include poor sucking ability, as well as loss of head control and motor skills. These symptoms may be accompanied by loss of appetite, vomiting, irritability, incessant crying, and seizures. As the disorder progresses, symptoms may also include generalized weakness, loss of muscle tone, and episodes of lactic acidosis, which can lead to impaired respiratory and renal function.

In maternally inherited Leigh syndrome (MILS), genetic mutations in mitochondrial DNA (at a high percentage >90%) interfere with the energy sources needed to power cells in the region of the brain that plays a role in motor function. These mutations in mitochondrial DNA lead to chronic energy shortages 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 cause MILS are less numerous (less than 90%), the condition is known as neuropathy, ataxia, and retinitis pigmentosa (NARP). There is also a form of Leigh disease (called X-linked Leigh disease), which results from mutations in a gene that produces another group of substances important for cellular metabolism. Another variant of Leigh syndrome, called the French-Canadian variant, is characterized by mutations in a gene called LRPPRC. Similar neurological symptoms are present as in Leigh syndrome, although the French Canadian variant usually also presents with hepatic steatosis.

In a preferred embodiment, the present invention relates to the treatment or prevention of Leigh syndrome or disease, including, for example, X-linked Leigh disease, French Canadian variant of Leigh syndrome and/or symptoms associated with Leigh disease.

The compounds can be combined with new agents that can be used to treat mitochondrial disease, including, but not limited to, nicotinamide riboside.

References to "treatment" include the meanings of alleviating, ameliorating, or eliminating the causal relationship of symptoms, either temporarily or permanently. The compounds of the invention are useful in treating the diseases disclosed herein in humans and other mammals.

In another embodiment, the invention encompasses prophylactic therapy for the diseases disclosed herein and includes preventing or delaying the onset of symptoms of the said disorder or condition. The compounds of the invention are useful in preventing the diseases disclosed herein in humans and other mammals.

The patient requiring treatment or prevention may be, for example, a human or other mammal suffering from the condition or at risk of developing the condition.

According to another aspect of the present invention, there is provided 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.

The pharmaceutical compositions of the invention comprise any of the compounds of the invention together with any pharmaceutically acceptable carrier, excipient, or diluent. Examples of pharmaceutically acceptable carriers are known to those skilled in the art and include, but are not limited to, preservatives, fillers, disintegrating agents, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, perfumes, antibacterial agents, antifungal agents, lubricating agents, and dispersing agents, depending on the nature of the route of administration and the dosage form. The compositions can be in the form of, for example, tablets, capsules, powders, granules, elixirs, syrups, and liquid preparations, including suspensions and solutions. The term "pharmaceutical composition" in the context of this invention means a composition containing an active agent and additionally containing one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, auxiliary substances, excipients, carriers, preservatives, fillers, disintegrating agents, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the administration route and dosage forms.

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

For the treatment or prevention of fibrosing disorders, for example, the compounds of the invention or pharmaceutical compositions thereof, as described herein, can be used 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.

In particular, the compounds of the invention or pharmaceutical compositions thereof, as described herein, can be used or combined with one or more additional pharmaceutical agents selected from the group consisting of conventional immunosuppressive drugs, such as corticosteroids, immunosuppressive or cytotoxic agents, or antibiotics, such as pirfenidone, or a non-specific kinase inhibitor (e.g., nintedanib).

The pharmaceutical compositions of the invention can be administered by any suitable effective route, such as oral, parenteral, topical, inhalational, intranasal, rectal, intravaginal, and aural. Pharmaceutical compositions suitable for delivering the compounds of the present invention and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in "Remington's Pharmaceutical Sciences," 19th Edition (Mack Publishing Company, 1995).

Oral administration

The compounds of the invention can be administered orally. Oral administration can involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration can be used, whereby the compound enters the bloodstream directly from the oral cavity.

Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particles, liquids or powders, lozenges (including liquid-filled), chewing gums, multi- and nano-particulates, gels, films (including mucoadhesive), ovules, sprays and liquid formulations.

Liquid formulations include suspensions, solutions, syrups, and elixirs. Such preparations can be used as fillers in soft or hard capsules and typically contain a carrier, such as water, ethanol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying and/or suspending agents. Liquid formulations can also be prepared by reconstituting a solid, such as from a sachet.

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

A typical tablet can be produced by standard processes familiar to a formulation chemist, such as direct compression, granulation (dry, wet, or melt), melt solidification, or extrusion. A tablet may contain one or more layers and may be coated or uncoated.

Examples of excipients suitable for oral administration include carriers such as cellulose, calcium carbonate, dibasic calcium phosphate, mannitol and sodium citrate, granulation binders such as polyvinylpyrrolidine, hydroxypropyl cellulose, hydroxypropyl methylcellulose and gelatin, disintegrating agents such as sodium starch glycolate and silicates, lubricating agents such as magnesium stearate and stearic acid, wetting agents such as sodium lauryl sulfate, preservatives, antioxidants, flavorings and coloring agents.

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

Parenteral administration

The compounds of the invention can also be administered directly into the bloodstream, 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 devices.

Parenteral formulations are usually aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but for certain uses they may be more suitably prepared as a sterile non-aqueous solution or as a dried form for use in combination with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral dosage forms under sterile conditions, for example by lyophilization, can be readily accomplished using standard pharmaceutical techniques known to those skilled in the art.

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

Parenteral dosage forms can be prepared for immediate and/or modified release. Modified-release dosage forms include those with delayed, sustained, pulsed, dual-controlled, targeted, and programmed release.

The pharmaceutical compositions of the present invention also include compositions and methods known in the art for bypassing the blood-brain barrier or for administration directly into the brain. Suitable injection sites include the cerebral cortex, cerebellum, midbrain, brainstem, hypothalamus, spinal cord, and ventricular tissue, as well as regions of the PNS, including the carotid body and adrenal medulla.

Dosage

The effective dose of the compound will naturally vary depending on the nature and severity of the condition being treated and the route of administration. The selection of appropriate dosages is the responsibility of the treating physician. The daily dose ranges from 10 mcg to approximately 100 mg per kg of body weight in humans and non-human animals, and typically ranges from 10 mcg to 30 mg per kg of body weight per dose. This dose can be administered one to three times daily.

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

Synthesis methodologies

Compounds of formula (I) can be prepared using the methods described below in the general reaction schemes and in the representative examples. If appropriate, individual transformations in the scheme can be carried out in a different order. The invention is illustrated by the following non-limiting examples, in which the following abbreviations and definitions are used. The compounds were characterized by liquid chromatography-mass spectrometry (LCMS) or ¹ H NMR, or both.

According to another aspect of the present invention, there is provided a process for the preparation of a compound of formula (I)(i), comprising reacting a compound of formula (IV), wherein Y is OH, with an amine of formula (V)(i), wherein PG is a protecting group such as BOC or CBZ, to form an amide of formula (III)(i) (Scheme 1). The amide coupling reaction can be carried out using standard methodology, for example by reacting using a coupling reagent such as DCC, HATU, HBTU, EDC, or via a mixed anhydride. Alternatively, the acid (IV), wherein Y is OH, can be converted to the acid chloride of acid (IV), wherein Y is Cl, using SOCh, PCl3 or PCl, which can then be reacted with amine (V)(i), preferably in a suitable solvent in the presence of a suitable base. Alternatively, compound (IV), wherein Y forms an ester, can be reacted directly with amine (V)(i), preferably in a suitable solvent. The compound of formula (III)(i) can be deprotected by standard methods to form amine (II)(i), which can then be reacted with cyanogen bromide to yield the corresponding compound of formula (I)(i).

In a further aspect the present invention provides a compound which is selected from compounds of formulae (II)(i) and (III)(i):

wherein PG is a protecting group, preferably BOC or CBZ, and R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined herein for the compound of formula (I) and its preferred embodiments, its tautomer or a salt of said compound or tautomer.

When the compound of formula (I)(i) has the formula (IA)(i), then formulas (II)(i), (III)(i), and (IV) are formulas (IIA)(i), (IIIA)(i), and (IVA), respectively. When the compound of formula (I)(i) has the formula (IB)(i), then formulas (II)(i), (III)(i), and (IV) are formulas (IIB)(i), (IIIB)(i), and (IVB)(i), respectively.

According to another aspect of the present invention, there is provided a process for the preparation of a compound of formula (I)(ii), comprising reacting a compound of formula (IV), wherein Y is OH, with an amine of formula (V)(ii), wherein PG is a protecting group such as BOC or CBZ, to form an amide of formula (III)(ii) (Scheme 2). The amide coupling reaction can be carried out using standard methodology, for example by reacting using a coupling reagent such as DCC, HATU, HBTU, EDC, or via a mixed anhydride. Alternatively, the acid (IV), wherein Y is OH, can be converted to the acid chloride of acid (IV), wherein Y is Cl, using SOCl ₂ , PCl ₃ or PCl ₅ , which can then be reacted with amine (V)(ii), preferably in a suitable solvent in the presence of a suitable base. Alternatively, compound (IV), wherein Y forms an ester, can be reacted directly with amine (V)(ii), preferably in a suitable solvent. The compound of formula (III)(ii) can be deprotected by standard methods to form amine (II)(ii), which can then be reacted with cyanogen bromide to yield the corresponding compound of formula (I)(ii).

In another aspect of the present invention, there is provided a compound which is selected from compounds of formulas (II)(ii) and (III)(ii):

wherein PG is a protecting group, preferably BOC or CBZ, and R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined herein for the compound of formula (I) and its preferred embodiments, its tautomer or a salt of said compound or tautomer.

When the compound of formula (I)(ii) has the formula (IA)(ii), then formulas (II)(ii), (III)(ii) and (IV) are formulas (IIA)(ii), (IIIA)(ii) and (IVA), respectively. When the compound of formula (I)(ii) has the formula (IB)(ii), then formulas (II)(ii), (III)(ii) and (IV) are formulas (IIB)(ii), (IIIB)(ii) and (IVB)(ii), respectively.

The protecting groups are preferably selected from tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (MeOZ), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzial (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), 4-nitrobenzenesulfonyl (Nosyl) and 2-nitrophenylsulfenyl (Nps). The most preferred are BOC and Cbz.

Abbreviations

LCMS/HPLC/SFC Methods Method C

Method C1

Method C2

Method H

Method H1

Method J

Method F

Method P3

Method Y3 Method used for analytical chiral SFC

Method Y4 Method used for analytical chiral SFC

Method Y5 Method used for analytical chiral SFC

Method Y6 Method used for analytical chiral SFC

Method Y7 Method used for analytical chiral SFC

Method Y8 Method used for analytical chiral SFC

Method Y12 Method used for analytical chiral SFC

Method Y15 Method used for analytical chiral HPLC

Method Y16 Method used for analytical chiral SFC

Intermediate compound A

Ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate

(i) NaH, THF, 0°C, 2 h; (ii) phenyltrimethylammonium tribromide, THF, 0°C to rt, 16 h; ( _iii ) NaN(CHO) ₂ , MeCN, 70°C, 1 h, then conc. HCl, 70°C, 16 h; (iv) _K2CO3 , DCM, 0°C to rt, 2 h; (v) _POCl3 , 100°C, 7 h.

Stage (i)

3-Acetyl-4-cyclopropoxybenzonitrile

This reaction was performed in triplicate. NaH (60% in oil, 0.49 g, 12.27 mmol) was added portionwise to a stirred solution of cyclopropanol (CAS 16545-68-9, Synthonix, 0.71 g, 12.27 mmol) in THF (6 mL) at 0°C and stirred for 30 min. A solution of 3-acetyl-4-fluorobenzonitrile (CAS 267875-54-7, from Combi-blocks, 1.0 g, 6.13 mmol) in THF (4 mL) was added dropwise at 0 °C, and the mixture was stirred at 0 °C for 2 h. The reaction mixture was combined with two more identical batches and then poured into ice water (500 mL) and extracted with EtOAc (2×500 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 12% EtOAc in n-hexanes) to give 3-acetyl-4-cyclopropoxybenzonitrile (3.18 g, 15.82 mmol, 85% yield).

LCMS: m/z not supported; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.05 (dd, J=8.8, 2.4 Hz, 1H), 7.96 (d, J=2.0 Hz, 1H), 7.64 (d, J=8.8 Hz, 1H), 4.13-4.17 (m, 1H), 2.50 (s, 3H), 0.81-0.93 (m, 4H).

Stage (ii)

3-(2-Bromoacetyl)-4-cyclopropoxybenzonitrile

To a stirred solution of 3-acetyl-4-cyclopropoxybenzonitrile (3.18 g, 15.82 mmol) in THF (40 mL) was added phenyltrimethylammonium tribromide (5.94 g, 15.82 mmol) at 0 °C. This mixture was slowly warmed to rt and stirred for 16 h, then poured into water (200 mL) and extracted with EtOAc (2 × 200 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 3-(2-bromoacetyl)-4-cyclopropoxybenzonitrile (4.3 g, 15.41 mmol, 97% yield). This crude material was used directly in the next step.

LCMS: Method F, 6.61 min; MS: ER+: 297.0, 299.0 (M+18).

Stage (iii)

4-Cyclopropoxy-3-glycylbenzonitrile hydrochloride

To a stirred solution of 3-(2-bromoacetyl)-4-cyclopropoxybenzonitrile (4.30 g, 15.41 mmol) in acetonitrile (43 mL) was added sodium diformylamide (1.75 g, 18.49 mmol) and heated at 70 °C for 3 h. The residue was diluted with MeOH (43 mL) and conc. HCl (4.3 mL). This mixture was further heated at 70 °C for 16 h, then allowed to cool to rt. This mixture was concentrated under reduced pressure, and the residue was stirred with isopropyl alcohol (25 mL) until a precipitate formed. The solid was collected by filtration under reduced pressure to afford 4-cyclopropoxy-3-glycylbenzonitrile hydrochloride (5.0 g, quantitative yield).

LCMS: Method C, 1.30 min; MS: ER+: 217.4.

Stage (iv)

Ethyl 2-((2-(5-cyano-2-cyclopropoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate

To a stirred solution of 4-cyclopropoxy-3-glycylbenzonitrile HCl salt (5.0 g, 19.80 mmol) in DCM (50 mL) was added K ₂ CO ₃ (10.92 g, 79.2 mmol) at 0 °C. Ethyl oxalyl chloride (5.4 g, 4.43 mL, 39.60 mmol) was added dropwise at 0 °C. This mixture was allowed to warm to rt, stirred for 2 h, then poured into water (500 mL) and extracted with DCM (2×300 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give ethyl 2-((2-(5-cyano-2-cyclopropoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate (5.0 g, quantitative yield).

LCMS: Method C, 1.52 min; MS: ER+ 316.9.

Stage (v)

Ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate

A stirred solution of ethyl 2-((2-(5-cyano-2-cyclopropoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate (5.0 g, 15.81 mmol) in POCl ₃ (50 mL, 10 vol) was heated at 100 °C for 7 h. This mixture was cooled to rt, poured into ice water (500 mL), and extracted with EtOAc (2×200 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was suspended in MeOH (40 mL) and stirred at -78 °C for 15 min. The solid was collected by filtration under reduced pressure to give ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (1.0 g, 3.35 mmol, 21% yield over three steps). LCMS: Method C, 1.77 min; MS: ER+: 299.5.

Intermediate connection B

Ethyl 5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate

(i) Zn(CN) ₂ , Zn dust, _PdCl2 (dppf) complex, DCM, DMA, 120°C, 16 h; (ii) pyridinium tribromide, THF, 0°C to rt, 16 h; (iii) NaN(CHO) ₂ , MeCN, 80°C, 5 h, then conc. HCl _, 80°C, 16 h; (iv) _K2CO3 , DCM, 0°C to rt, 4 h; (v) _POCl3 , 100°C, 16 h.

Stage (i)

3-Acetyl-4-methoxybenzonitrile

To a stirred solution of 1-(5-bromo-2-methoxyphenyl)ethan-1-one (CAS 16740-73-1, from Combi-blocks, 60.0 g, 261.89 mmol) in DMA (600 mL) were added zinc cyanide (92.25 g, 785.67 mmol) and zinc dust (17.22 g, 261.89 mmol) at rt. The mixture was degassed with _N2 for 30 min, then [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with DCM (10.69 g, 13.09 mmol) was added. This mixture was heated at 120°C for 16 h. The mixture was allowed to cool to rt, filtered through Celite Hyflow™, and the filtrate was poured into ice water (800 mL) and extracted with EtOAc (2×1000 mL). The combined organic phases were washed with ice water (4×1000 mL), dried over _{Na2SO4 ,} and concentrated under reduced pressure. The residue was purified by column chromatography (60-120# silica gel, 15% EtOAc in n-hexanes) to afford 3-acetyl-4-methoxybenzonitrile (40.0 g, 228.57 mmol, 87% yield).

LCMS: Method C, 1.52 min; MS: ER+: 176.08.

Stage (ii)

3-(2-Bromoacetyl)-4-methoxybenzonitrile

To a stirred solution of 3-acetyl-4-methoxybenzonitrile (7.20 g, 41.14 mmol) in THF (72 mL) was added pyridinium tribromide (14.47 g, 45.25 mmol) at 0 °C. This mixture was slowly warmed to rt and stirred for 16 h, then poured into water (400 mL) and extracted with EtOAc (2 × 400 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 3-(2-bromoacetyl)-4-methoxybenzonitrile (13.0 g, quantitative yield). This crude material was used directly in the next step.

Stage (iii)

3-Glycyl-4-methoxybenzonitrile hydrochloride

To a stirred solution of 3-(2-bromoacetyl)-4-methoxybenzonitrile (10.5 g, 41.51 mmol) in acetonitrile (105 mL) was added sodium diformylamide (5.91 g, 62.26 mmol), and the mixture was heated at 80 °C for 8 h. The reaction mixture was cooled to rt, concentrated under reduced pressure, and the residue was diluted with MeOH (105 mL) and conc. HCl (10.5 mL). The mixture was heated at 80 °C for an additional 16 h, then allowed to cool to rt. The mixture was concentrated under reduced pressure, and the residue was stirred with isopropyl alcohol (40 mL) until a precipitate formed, which was collected by filtration under reduced pressure to give 3-glycyl-4-methoxybenzonitrile hydrochloride (9.0 g, 39.73 mmol, 95% yield).

LCMS: Method C2, 0.40 min; MS: ER+: 191.0.

Stage (iv)

Ethyl 2-((2-(5-cyano-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate

_To a stirred solution of 3-glycyl-4-methoxybenzonitrile hydrochloride HCl salt (9.0 g, 39.73 mmol) in DCM (90 mL) was added _K2CO3 (21.93 g, 158.92 mmol) at 0°C. Ethyl oxalyl chloride (10.84 g, 8.89 mL, 79.46 mmol) was added dropwise at 0°C. This mixture was allowed to warm to rt and stirred for 4 h. The mixture was poured into water (900 mL) and extracted with DCM (2×500 mL). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure to give ethyl 2-((2-(5-cyano-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate (9.0 g, 31.03 mmol, 78% yield).

LCMS: Method C, 1.34 min; MS: ER+ 291.1.

Stage (v)

Ethyl 5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate

A stirred solution of ethyl 2-((2-(5-cyano-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate (1.2 g, 4.14 mmol) in POCl ₃ (12 mL, 10 vol) was heated at 100 °C for 2 h. The mixture was cooled to rt, poured into ice water (200 mL), and extracted with EtOAc (3×100 mL). The combined organic phases were washed with saturated NaHCO ₃ solution (3×100 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 32% EtOAc in n-hexanes) to give ethyl 5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate (0.60 g, 2.20 mmol, 53% yield).

LCMS: Method C, 1.53 min; MS: ER+: 272.6.

Intermediate compound C

Ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxylate

(i) HATU, DIPEA, THF, 0°C to rt, 3 h; (ii) methylmagnesium bromide, THF, -10°C to 0°C, 4 h; (iii) phenyltrimethylammonium tribromide, MeCN, 0°C to rt, 9 h; (iv) NaN(CHO) ₂ , MeCN, 80°C, 1 h, then conc. HCl, 80°C, 3 h; (v) K ₂ CO ₃ , DCM, 0°C to rt, 3 h; (vi) POCl ₃ , 110°C, 16 h.

Stage (i)

5-Bromo-N-methoxy-N-methyl-2-(trifluoromethoxy)benzamide

To a stirred solution of 5-bromo-2-(trifluoromethoxy)benzoic acid (CAS 403646-47-9, from Combi-blocks, 10.0 g, 35.08 mmol) in THF (100 mL) was added DIPEA (13.57 g, 17.9 mL, 105.25 mmol) and HATU (20.0 g, 52.62 mmol) in portions at 0 °C. After 30 min, N,O-dimethylhydroxylamine HCl (4.45 g, 45.61 mmol) was added at 0 °C. This mixture was slowly warmed to rt and stirred for 3 h, then poured into ice water (300 mL) and extracted with EtOAc (3×150 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 5-bromo-N-methoxy-N-methyl-2-(trifluoromethoxy)benzamide (12.2 g, quantitative yield). This crude material was used directly in the next step.

LCMS: Method C, 1.69 min; MS: ER+: 327.8, 329.8.

Stage (ii)

1-(5-Bromo-2-(trifluoromethoxy)phenyl)ethan-1-one

To a stirred solution of 5-bromo-N-methoxy-N-methyl-2-(trifluoromethoxy)benzamide (12.2 g, 37.31 mmol) in THF (130 mL) was added methylmagnesium bromide (3 M in diethyl ether, 24.9 mL, 74.62 mmol) dropwise at -10 °C. This mixture was slowly warmed to 0 °C and stirred for 4 h, then poured into saturated NH ₄ Cl solution (300 mL) and extracted with EtOAc (3×200 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one (10.0 g, quantitative yield). This crude material was used directly in the next step.

LCMS: Method C, m/z not supported; ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.92 (s, 1H), 7.67-7.71 (m, 1H), 7.24 (d, J=8.0 Hz, 1H), 2.62 (s, 3H).

Stage (iii)

2-Bromo-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one

To a stirred solution of 1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one (10.0 g, 35.46 mmol) in acetonitrile (100 mL) was added phenyltrimethylammonium tribromide (13.33 g, 35.46 mmol) in portions at 0 °C. The mixture was slowly warmed to rt and stirred for 9 h, then poured into water (200 mL) and extracted with EtOAc (3×100 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 5% EtOAc in n-hexanes) to give 2-bromo-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one (11.5 g, 31.95 mmol, 91% yield in three steps).

LCMS: m/z no support; ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.93 (s, 1H), 7.73 (dd, J=8.8, 2.4 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H), 4.45 (s, 2H).

Stage (iv)

2-Amino-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one hydrochloride

To a stirred solution of 2-bromo-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one (10.0 g, 27.78 mmol) in acetonitrile (100 mL) was added sodium diformylamide (5.28 g, 55.58 mmol), and the mixture was heated at 80 °C for 1 h. The mixture was cooled to rt, concentrated under reduced pressure, and the residue was diluted with MeOH (100 mL) and conc. HCl (10.0 mL). The mixture was heated at 80 °C for 3 h, then allowed to cool to rt. The mixture was concentrated under reduced pressure and the residue was stirred with diethyl ether (4×30 mL) until a precipitate formed, which was collected by filtration under reduced pressure to give 2-amino-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one hydrochloride (9.41 g, quantitative yield). LCMS: Method C, 1.65 min; MS: ER+: 299.9, 301.9.

Stage (v)

Ethyl 2-((2-(5-bromo-2-(trifluoromethoxy)pent)-2-oxoethyl)amino)-2-oxoacetate

To a stirred solution of 2-amino-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethan-1-one HCl salt (9.4 g, 28.19 mmol) in DCM (94 mL) was added K ₂ CO ₃ (11.67 g, 84.56 mmol) at 0 °C. Ethyl oxalyl chloride (5.77 g, 4.73 mL, 42.28 mmol) was added dropwise at 0 °C. The mixture was allowed to warm to rt, stirred for 3 h, then poured into water (150 mL) and extracted with DCM (3 x 120 mL). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure to give ethyl 2-((2-(5-bromo-2-(trifluoromethoxy)phenyl)-2-oxoethyl)amino)-2-oxoacetate (3.4 g, 8.56 mmol, 31% yield over two steps).

LCMS: Method C, 2.09 min; MS: ER- 396.0, 398.0.

Stage (vi)

Ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxylate

A stirred solution of ethyl 2-((2-(5-bromo-2-(trifluoromethoxy)phenyl)-2-oxoethyl)amino)-2-oxoacetate (3.4 g, 8.56 mmol) in POCl ₃ (17 mL, 5 vol) was heated at 110 °C for 16 h. The mixture was cooled to rt, poured into ice water (250 mL), and extracted with EtOAc (3×100 mL). The combined organic phases were washed with saturated NaHCO ₃ solution (3×100 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 18% EtOAc in n-hexanes) to give ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxylate (1.3 g, 3.43 mmol, 40% yield).

LCMS: Method C, 2.39 min; MS: ER+: 379.8, 381.8.

Intermediate connection D

Ethyl 5-(5-cyano-4-fluoro-2-methoxyphenyl)oxazole-2-carboxylate

(i) trifluoromethanesulfonic acid, NBS, MeCN, -30°C to rt, 18 h; (ii) _K2CO3 , MeI, DMF, 0°C to rt, 3 h; (iii) tributyl(1-ethoxyvinyl)tin, _PdCl2 ( _{PPh3 ) 2} , 1,4-dioxane, 100°C, 3 h, then NBS, TNT:water, 0°C, 10 min; (iv) NaN(CHO) ₂ , MeCN, 80°C, 2 h, then conc. HCl, 80°C, 16 h; (v) _ethyl chlorooxoacetate, _K2CO3 , DCM, 0°C to rt, 2 h; (vi) _POCl3 , 100°C, 5 h.

Stage (i)

5-Bromo-2-fluoro-4-hydroxybenzonitrile

To a stirred solution of 2-fluoro-4-hydroxybenzonitrile (CAS 82380-18-5, from Combi-blocks, 8.0 g, 58.39 mmol) in acetonitrile (80 mL) was added trifluoromethanesulfonic acid (10.51 g, 6.18 mL, 70.07 mmol) dropwise at -30 °C. After 10 min, N-bromosuccinimide (10.39 g, 58.39 mmol) was gradually added at -30 °C. This mixture was slowly warmed to rt and stirred for 18 h, then poured into saturated NaHCO ₃ solution (150 mL) and extracted with EtOAc (2 x 150 mL). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure to give 5-bromo-2-fluoro-4-hydroxybenzonitrile (5.08 g, 23.63 mmol, 40% yield). This crude material was used directly in the next step.

LCMS: Method C, 1.58 min; MS: ER-: 214.0, 216.0.

Stage (ii)

5-Bromo-2-fluoro-4-methoxybenzonitrile

To a stirred solution of 5-bromo-2-fluoro-4-hydroxybenzonitrile (5.08 g, 23.63 mmol) in DMF (30 mL) was added K ₂ CO ₃ (6.52 g, 47.26 mmol) at 0 °C. After 10 min, methyl iodide (5.03 g, 2.21 mL, 35.45 mmol) was added dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 3 h, then poured into water (100 mL) and extracted with EtOAc (2×100 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 2% EtOAc in n-hexanes) to give 5-bromo-2-fluoro-4-methoxybenzonitrile (5.70 g, quantitative yield).

LCMS: m/z no support; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.30 (d, J=7.2 Hz, 1H), 7.46 (d, J=12.0 Hz, 1H), 4.01 (s, 3H).

Stage (iii)

5-(2-Bromoacetyl)-2-fluoro-4-methoxybenzonitrile

A stirred solution of 5-bromo-2-fluoro-4-methoxybenzonitrile (5.70 g, 24.89 mmol) and tributyl(1-ethoxyvinyl)tin (CAS 97674-02-7, from Combi-blocks, 13.46 g, 37.34 mmol) in 1,4-dioxane (70 mL) was degassed with _N2 for 15 min, then bis(triphenylphosphino)palladium(II) dichloride (0.87 g, 1.24 mmol) was added at rt. This mixture was heated at 100 °C for 3 h. This mixture was cooled to 0 °C and THP:water (2:1, 120 mL) was added. N-Bromosuccinimide (8.85 g, 49.78 mmol) was added at 0 °C. The mixture was stirred at 0°C for 10 min, then poured into water (100 mL) and extracted with EtOAc ( ₂ ×100 mL). The combined organic phases were dried over _Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 2% EtOAc in n-hexanes) to give 5-(2-bromoacetyl)-2-fluoro-4-methoxybenzonitrile (6.3 g, 23.25 mmol, 98% yield over two steps).

LCMS: m/z no support; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.27 (dd, J=7.6, 2.4 Hz, 1H), 7.54 (dd, J=12.0, 2.4 Hz, 1H), 4.84 (s, 2H), 4.05 (s, 3H).

Stage (iv)

2-Fluoro-5-glycyl-4-methoxybenzonitrile hydrochloride

To a stirred solution of 5-(2-bromoacetyl)-2-fluoro-4-methoxybenzonitrile (6.30 g, 23.25 mmol) in acetonitrile (65 mL) was added sodium diformylamide (25.85 g, 27.9 mmol), and the mixture was heated at 80 °C for 2 h. The mixture was cooled to rt, concentrated under reduced pressure, and the residue was diluted with MeOH (65 mL) and conc. HCl (6.5 mL). The mixture was heated at 80 °C for 16 h, then allowed to cool to rt. The mixture was concentrated under reduced pressure, and the residue was stirred with isopropyl alcohol (100 ml) until a precipitate formed, which was collected by filtration under reduced pressure to give 2-fluoro-5-glycyl-4-methoxybenzonitrile hydrochloride (5.3 g, quantitative yield).

LCMS: Method H, 1.96 min; MS: ER+: 209.1.

Stage (v)

Ethyl 2-((2-(5-cyano-4-fluoro-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate

To a stirred solution of 2-fluoro-5-glycyl-4-methoxybenzonitrile HCl salt (5.30 g, 21.67 mmol) in DCM (60 mL) was added K ₂ CO ₃ (11.96 g, 86.68 mmol) at 0 °C. Ethyl oxalyl chloride (5.89 g, 4.83 mL, 43.34 mmol) was added dropwise at 0 °C. The mixture was allowed to warm to rt, stirred for 2 h, then poured into water (150 mL) and extracted with EtOAc (2×150 mL). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure to give ethyl 2-((2-(5-cyano-4-fluoro-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate (4.20 g, 13.63 mmol, 58% yield over two steps). LCMS: Method C, 1.50 min; MS: ER+ 309.4.

Stage (vi)

Ethyl 5-(5-cyano-4-fluoro-2-methoxyphenyl)oxazole-2-carboxylate

A stirred solution of ethyl 2-((2-(5-cyano-4-fluoro-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoacetate (4.2 g, 13.63 mmol) in POCl ₃ (42 mL, 10 vol) was heated at 100 °C for 5 h. This mixture was cooled to rt, poured into ice water (100 mL) and extracted with EtOAc (2 × 150 mL). The combined organic phases were washed with saturated NaHCO ₃ solution (2 × 100 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was suspended in MeOH (30 mL) and stirred at -78 °C for 15 min. The solid was collected by filtration under reduced pressure to give ethyl 5-(5-cyano-4-fluoro-2-methoxyphenyl)oxazole-2-carboxylate (1.60 g, 5.51 mmol, 40% yield).

LCMS: Method C, 1.63 min; MS: ER+: 291.2.

Example 1

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

(i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K ₂ CO ₃ , CNBr, THF, 0°C to rt.

Stage (i)

tert-Butyl-(2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylam

To a stirred solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.60 g, 2.01 mmol) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 348165-63-9, 0.60 g, 3.02 mmol) in THF (10 mL) was added TBD (0.42 g, 3.02 mmol) portionwise at 0 °C. The mixture was warmed to rt and stirred for 1 h, then poured into water (70 mL) and extracted with EtOAc (2×70 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 30% EtOAc in n-hexanes) to give tert-butyl (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.32 g, 0.71 mmol, 35% yield).

LCMS: Method C, 1.86 min; MS: ER- 451.4.

Stage (ii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.31 g, 0.68 mmol) in DCM (7 mL) was added TFA (0.93 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 45 min, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.3 g, 0.64 mmol, 94% yield).

LCMS: Method C, 1.38 min; MS: ER +353.3.

Stage (iii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.3 g, 0.64 mmol) in THF (8 mL) was added K ₂ CO ₃ (0.27 g, 1.93 mmol) at rt, and the mixture was stirred for 10 min. Cyanogen bromide (0.07 g, 0.64 mmol) was added at 0 °C, and the mixture was stirred at rt for 45 min, then poured into water (70 mL) and extracted with EtOAc (2×70 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 40% EtOAc in n-hexanes) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide (0.1 g, 0.27 mmol, 42% yield).

LCMS: Method H, 2.96 min; MS: ER+ 378.0; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.36 (d, J=6.4 Hz, 1H), 8.27 (s, 1H), 7.98 (d, J=8.4 Hz, 1H), 7.68-7.70 (m, 2H), 4.51-4.53 (m, 1H), 4.21-4.23 (m, 1H), 3.89-3.95 (m, 1H), 3.77-3.81 (m, 1H), 3.43-3.46 (m, 1H), 2.16-2.18 (m, 1H), 1.76-1.83 (m, 1H), 1.26 (d, J=6.4 Hz, 3H), 0.92 (s, 4H). Chiral SFC: Method Y5, 5.18 min.

Example 2

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

(i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K ₂ CO ₃ , CNBr, THF, 0°C to rt.

Stage (i)

tert-Butyl (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate

To a stirred solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.50 g, 1.67 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.46 g, 2.01 mmol) in THF (8 mL) was added TBD (0.35 g, 2.51 mmol) portionwise at 0 °C. The mixture was warmed to rt and stirred for 3 h, then poured into water (100 mL) and extracted with EtOAc (2×80 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 55% EtOAc in n-hexanes) to give tert-butyl (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.32 g, 0.66 mmol, 39% yield).

LCMS: Method C1, 1.35 min; MS: ER+ 483.4.

Stage (ii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.32 g, 0.66 mmol) in DCM (8 mL) was added TFA (0.96 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.32 g, 0.64 mmol, 97% yield).

LCMS: Method C1, 1.06 min; MS: ER+ 383.4.

Stage (iii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.32 g, 0.64 mmol) in THF (8 mL) was added K ₂ CO ₃ (0.27 g, 1.93 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.07 g, 0.64 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (80 mL) and extracted with EtOAc (2×80 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 70% EtOAc in n-hexanes) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5Sr)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.10 g, 0.24 mmol, 38% yield).

LCMS: Method H, 2.94 min; MS: ER+ 408.2; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.32 (d, J=6.4 Hz, 1H), 8.26 (d, J=1.6 Hz, 1H), 7.98 (d, J=8.8 Hz, 1H), 7.69-7.70 (m, 2H), 4.47-4.59 (m, 1H), 4.19-4.27 (m, 1H), 4.00-4.10 (m, 1H), 3.70-3.74 (m, 1H), 3.39-3.51 (m, 3H), 3.36 (s, 3H), 2.11-2.18 (m, 1H), 1.98-2.03 (m, 1H), 0.92 (s, 4H). Chiral SFC: Method Y3, 3.56 min.

Step (iii) alternative synthesis

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

Under N ₂ atmosphere, to a stirred solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (600.0 g, 1209.67 mmol) in THF (6 L) was added K ₂ CO ₃ (500.80 g, 3629.01 mmol) at rt under N ₂ atmosphere and stirred for 15-20 min. A solution of cyanogen bromide (153.87 g, 1451.60 mmol) in THF (1.2 L) was added dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h at rt. The reaction progress was monitored by TLC (mobile phase: 50% EtOAc in n-hexanes, product R _f 0.05; 10% MeOH/DCM, product R _f 0.4). This mixture was quenched with water (3 L) and extracted with EtOAc (3×6 L). The combined organic phases were washed with brine (1.5 L), dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure to give crude 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (505 g). This crude material (505 g) was suspended in IPA (5 L, 10 vol) and heated at 80°C to give a clear The mixture was allowed to cool slowly to rt and then at 0°C until a crystalline solid formed, which was collected by filtration under reduced pressure, washed with cold IPA (505 mL), and dried under vacuum at 50°C to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)-pyrrolidin-3-yl)oxazole-2-carboxamide as a white crystalline solid (400.0 g, 982.80 mmol).

The material obtained from recrystallization from IPA (400 g) was suspended in IPA (10 L, 25 vol), activated carbon (100 g) was added, and the mixture was heated at 80°C for 1 h. The hot mixture was filtered through Celite Hyflow™ and washed with hot IPA (800 mL, 2 vol). The filtrate was allowed to cool slowly to rt. and then at 0°C to form a crystalline solid, which was collected by suction filtration, washed with cold IPA (400 L, 1 vol) and dried under vacuum at 50°C to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide as a white crystalline solid (320.0 g, 786.24 mmol, 63% yield).

LCMS: Method H1, 2.81 min, MS: ER+ 408.2; HPLC: Method P3, 24.22 min; Chiral HPLC: Method Y, 28.97 min; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.29 (d, J=6.8 Hz, 1H), 8.22 (d, J=2.0 Hz, 1H), 7.95 (dd, J=8.8, 2.4 Hz, 1H), 7.67 (d, J=8.8 Hz, 1H), 7.65 (s, 1H), 4.48-4.58 (m, 1H), 4.18-4.22 (m, 1H), 4.01-4.07 (m, 1H), 3.70-3.74 (m, 1H), 3.40-3.51 (m, 3H), 3.35 (s, 3H), 2.12-2.18 (m, 1H), 1.97-2.03 (m, 1H), 0.88-0.96 (m, 4H); Chiral HPLC: Method Y15, 15.11 min; 99.8% ee (enantiomeric excess); HPLC: Method Y26, 27.87 min; DSC maximum (melting point) = 132.7°C; High-Resolution MS: ED: ED- 406.1521 (calculated exact mass 407.1594). The X-ray powder diffraction data in the table below was obtained on a Bruker AXS D8 Advance: Cu,kα: Kα1(Å): 1.540598; Kα2(Å): 1.544426; Kα2/Kα1=0.50.

Alternative recrystallization

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

Crude 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (134.4 g, 98.9% pure by LC) was suspended in ethanol (1340 mL, 10 vol) and heated to 55-65 °C until a brown solution formed. This mixture was allowed to cool to 40-45 °C and seeded with 135 mg of a similar smaller batch, and the mixture was stirred at 40-45 °C for 30 min. The mixture was cooled to rt and stirred overnight. The beige suspension was filtered and the solid collected, washed with ethanol (2 x 260 mL), the filter cake collected, then dried at 60°C overnight to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide as an off-white solid (116.2 g, 86% yield from crude material).

MS: ER+ 408.2; ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 8.24 (d, J=2.0 Hz, 1H), 7.66 (dd, J=8.8, 2.0 Hz, 1H), 7.56 (s, 1H), 7.47 (d, J=8.4 Hz, 1H), 4.68-4.75 (m, 1H), 3.97-4.00 (m, 2H), 3.83-3.85 (m, 1H), 3.60-3.66 (m, 1H), 3.45-3.53 (m, 2H), 3.42 (s, 3H), 2.24-2.32 (m, 1H), 2.09-2.15 (m, 1H), 0.89-1.02 (m, 4H); residual solvents according to NMR results = EtOH (2260 ppm); HPLC purity 99.6%; DSC (differential scanning colorimetry): maximum temperature (melting point) = 86.96°C.

The compound of Example 2 was prepared as an aqueous suspension containing 0.1% (w/v) Tween-80 and 0.5% (w/v) hydroxypropyl methylcellulose.

Example 3

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

(i) TBD, THF, 0°C to RT; (ii) TFA, DCM, 0°C to RT; (iii) K ₂ CO ₃ , CNBr, THF, from 0°C to room temperature.

Stage (i)

tert-Butyl (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylate

To a stirred solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.15 g, 0.50 mmol) and tert-butyl (2S,4R)-4-amino-2-(fluoromethyl)pyrrolidine-1-carboxylate (CAS 1207853-03-9, from Angene, 0.11 g, 0.50 mmol) in THF (4 mL) was added TBD (0.07 g, 0.50 mmol) portionwise at 0 °C. This mixture was warmed to rt and stirred for 8 h, then poured into water (40 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 51% EtOAc in n-hexanes) to give tert-butyl (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylate (0.09 g, 0.19 mmol, 37% yield).

LCMS: Method C, 1.76 min; MS: ER- 469.1.

Stage (ii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylate (0.09 g, 0.19 mmol) in DCM (5 mL) was added TFA (0.9 mL, 10 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.16 g, quantitative yield).

LCMS: Method C, 1.35 min; MS: ER- 368.9.

Stage (iii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.15 g, 0.32 mmol) in THF (5 mL) was added K ₂ CO ₃ (0.13 g, 0.96 mmol) at rt, and the mixture was stirred for 10 min. Cyanogen bromide (0.03 g, 0.32 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (40 mL) and extracted with EtOAc (2×40 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 69% EtOAc in n-hexanes) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-(3R,5S)-1-cyano-5-(fluoromethyl)-pyrrolidin-3-yl)oxazole-2-carboxamide (0.01 g, 0.03 mmol, 16% yield over two steps).

LCMS: Method H, 2.96 min; MS: ER+ 396.1; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.39 (d, J=6.8 Hz, 1H), 8.25 (s, 1H), 7.98 (d, J=7.2 Hz, 1H), 7.68-7.70 (m, 2H), 4.41-4.69 (m, 3H), 4.10-4.26 (m, 2H), 3.61-3.80 (m, 1H), 3.43-3.53 (m, 1H), 2.12-2.25 (m, 1H), 1.95-2.07 (m, 1H), 0.92 (s, 4H). Chiral SFC: Method Y4, 4?96 min.

Example 4

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

(i) TBD, THF, 0°C to RT; (ii) TFA, DCM, 0°C to RT; (iii) K ₂ CO ₃ , CNBr, THF, from 0°C to room temperature.

Stage (i)

tert-Butyl-(2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylam

To a stirred solution of ethyl 5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate (0.5 g, 1.84 mmol) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 348165-63-9, 0.37 g, 1.84 mmol) in THF (10 mL) was added TBD (0.38 g, 2.76 mmol) portionwise at 0 °C. The mixture was warmed to rt and stirred for 2 h, then poured into water (100 mL) and extracted with EtOAc (2×70 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 30% EtOAc in n-hexanes) to give tert-butyl (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.30 g, 0.70 mmol, 38% yield).

LCMS: Method C, 1.61 min; MS: ER- 425.1.

Stage (ii)

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.30 g, 0.70 mmol) in DCM (6 mL) was added TFA (0.9 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.30 g, 0.68 mmol, 97% yield).

LCMS: Method C, 1.22 min; MS: ER+ 327.1.

Stage (iii)

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

To a stirred solution of 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.30 g, 0.68 mmol) in THF (7 mL) was added K ₂ CO ₃ (0.28 g, 2.04 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.07 g, 0.68 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 1% MeOH in DCM) to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide (0.10 g, 0.28 mmol, 42% yield).

LCMS: Method H, 2.73 min; MS: ER+ 352.2; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.35 (d, J=6.8 Hz, 1H), 8.25 (s, 1H), 7.95 (d, J=7.2 Hz, 1H), 7.84 (s, 1H), 7.41 (d, J=8.4 Hz, 1H), 4.47-4.57 (m, 1H), 4.08 (s, 3H), 3.89-3.94 (m, 1H), 3.76-3.80 (m, 1H), 3.42-3.49 (m, 1H), 2.16-2.20 (m, 1H), 1.76-1.83 (m, 1H), 1.26 (d, J=6.4 Hz, 3H). Chiral SFC: Method Y6, 3.76 min.

Example 5

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

(i) TBD, THF, 0°C to RT; (ii) TFA, DCM, 0°C to RT; (iii) K ₂ CO ₃ , CNBr, THF, from 0°C to room temperature.

Stage (i)

tert-Butyl (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate

To a stirred solution of ethyl 5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate (0.80 g, 2.94 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.67 g, 2.94 mmol) in THF (20 mL) was added TBD (0.61 g, 4.41 mmol) in portions at 0 °C. This mixture was stirred at 0 °C for 6 h, then poured into water (50 mL) and extracted with EtOAc (2×70 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 50% EtOAc in n-hexanes) to give tert-butyl (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.25 g, 0.55 mmol, 18% yield).

LCMS: Method C1, 1.29 min; MS: ER+ 457.2.

Stage (ii)

5-(5-Cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.25 g, 0.55 mmol) in DCM (5 mL) was added TFA (2.5 mL, 10 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 2 h, then concentrated under reduced pressure to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.32 g, quantitative yield).

LCMS: Method C1, 1.00 min; MS: ER+ 357.3.

Stage (1H)

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

To a stirred solution of 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.31 g, 0.66 mmol) in THF (15 mL) was added K ₂ CO ₃ (0.27 g, 1.98 mmol) at rt and after stirring for 10 min, cyanogen bromide (0.07 g, 0.66 mmol) was added to this mixture at 0 °C. The mixture was stirred at 0 °C for 0.5 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 60% EtOAc in n-hexanes) to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.11 g, 0.29 mmol, 52% yield over two steps).

LCMS: Method H, 2.66 min; MS: ER+ 382.2; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.34 (d, J=6.8 Hz, 1H), 8.25 (s, 1H), 7.96 (d, J=8.0 Hz, 1H), 7.85 (s, 1H), 7.43 (d, J=8.8 Hz, 1H), 4.47-4.58 (m, 1H), 4.09 (s, 3H), 4.00-4.10 (m, 1H), 3.70-3.74 (m, 1H), 3.41-3.53 (m, 3H), 3.36 (s, 3H), 2.08-2.20 (m, 1H), 1.95-2.06 (m, 1H). Chiral SFC: Method Y7, 5.42 min.

Example 6

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

(i) AgOTf, EtOAc, 80°C; (ii) LiOH, _{H 2} O, H ₂ O, THF, 0°C to rt; (iii) POCl ₂ , pyridine, 0°C to rt; (iv) TFA, DCM, 0°C to rt; (v) K ₂ CO ₃ , CNBr, THF, 0°C to rt.

Stage (i)

Ethyl 4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate

To a stirred solution of ethyl 5-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate (3.5 g, 13.83 mmol) and ethyl oxamate (CAS 617-36-7, from Sigma-Aldrich, 4.86 g, 41.51 mmol) in EtOAc (35 mL) was added silver triflate (CAS 2923-28-6, from Combi-blocks, 10.66 g, 41.51 mmol) at rt. This mixture was heated at 80 °C for 24 h, then poured into water (120 mL), filtered through Celite Hyflow™, and the filtrate was extracted with EtOAc (3×70 mL). The combined organic phases were dried over _{anhydrous Na2SO4} and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 16% EtOAc in n-hexanes) to give ethyl 4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate (0.53 g, 1.95 mmol, 14% yield).

LCMS: Method C, 1.65 min; MS: ER+ 273.1.

Stage (ii)

4-(5-Cyano-2-methoxyphenyl)oxazole-2-carboxylic acid

To a stirred solution of ethyl 4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylate (0.50 g, 1.84 mmol) in THP:water (1:1, 10 mL) was added lithium hydroxide monohydrate (0.23 g, 5.51 mmol) portionwise at 0 °C. The mixture was stirred at 0 °C for 2 h, then poured into water (70 mL), acidified with 1 N HCl and extracted with DCM (3 x 70 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give 4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylic acid (0.15 g, 0.61 mmol, 33% yield). LCMS: Method C2, 0.81 min; MS: ER+ 245.1.

Stage (iii)

tert-Butyl-(2S,4R)-4-(4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylam

To a stirred solution of 4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxylic acid (0.16 g, 0.65 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.15 g, 0.65 mmol) in pyridine (3 mL) was added POCl ₃ (0.3 g, 0.18 mL, 1.97 mmol) dropwise at 0 °C. This mixture was stirred at 0 °C for 10 min, then poured into water (50 mL) and extracted with EtOAc (2×30 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 22% EtOAc in n-hexanes) to give tert-butyl (2S,4R)-4-(4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.18 g, 0.39 mmol, 60% yield).

LCMS: Method C, 1.70 min; MS: ER- 455.1.

Stage (iv)

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

To a stirred solution of tert-butyl (2S,4R)-4-(4-(5-cyano-2-methoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.17 g, 0.37 mmol) in DCM (5 mL) was added TFA (0.85 mL, 5 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.25 g, quantitative yield).

LCMS: Method C, 1.32 min; MS: ER+ 357.1.

Stage (v)

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

To a stirred solution of 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.24 g, 0.51 mmol) in THF (5 mL) was added K ₂ CO ₃ (0.21 g, 1.53 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.04 g, 0.41 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (3×50 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 71% EtOAc in n-hexanes) to give 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.05 g, 0.14 mmol, 36% yield over two steps).

LCMS: Method H, 2.80 min; MS: ER- 380.1; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.25 (d, J=6.8 Hz, 1H), 8.72 (s, 1H), 8.44 (d, J=1.6 Hz, 1H), 7.89 (dd, J=8.8, 2.0 Hz, 1H), 7.37 (d, J=8.8 Hz, 1H), 4.47-4.58 (m, 1H), 4.07 (s, 3H), 4.00-4.18 (m, 1H), 3.69-3.73 (m, 1H), 3.43-3.50 (m, 3H), 3.34 (s, 3H), 2.09-2.19 (m, 1H), 1.96-2.02 (m, 1H). Chiral SFC: Method Y3, 3.49 min.

Example 7

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

(i) TBD, THF, 0°C to rt; (ii) Zn(CN) ₂ , Zn, _Pd2 (dba) ₃ , phosphine ligand, DMA, 140°C; (iii) TFA, DCM, 0°C to rt; (iv) _{K2CO3 ,} CNBr, THF, 0°C to rt.

Stage (i)

tert-Butyl-(2S,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylam

To a stirred solution of ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxylate (0.70 g, 1.84 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.42 g, 1.84 mmol) in THF (8 mL) was added TBD (0.38 g, 2.77 mmol) in portions at 0 °C. This mixture was stirred at 0 °C for 2 h, then poured into water (100 mL) and extracted with EtOAc (3×70 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 25% EtOAc in n-hexanes) to give tert-butyl (2S,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.37 g, 0.66 mmol, 35% yield).

LCMS: Method C, 2.00 min; MS: ER+ 564.4, 566.4.

Stage (ii)

tert-Butyl-(2S,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.35 g, 0.62 mmol) in DMA (3 mL) were added zinc cyanide (0.18 g, 1.55 mmol), zinc dust (0.02 g, 0.31 mmol), and 1,1'-ferrocenediyl bis(diphenylphosphine) (0.07 g, 0.12 mmol) at rt. The mixture was degassed with _N2 for 15 min, then trms(dibenzylideneacetone)dipalladium(0) (0.11 g, 0.12 mmol) was added. The mixture was heated under microwave irradiation at 140 °C for 1 h, then allowed to cool to rt, poured into ice water (70 mL), and extracted with EtOAc (3×70 mL). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 22% EtOAc in n-hexanes) to afford tert-butyl (2S,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.25 g, 0.5 mmol, 80% yield).

LCMS: Method C, 1.83 min; MS: ER+: 511.4.

Stage (iii)

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2S,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.25 g, 0.49 mmol) in DCM (3 mL) was added TFA (1.25 mL, 5 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 2 h, then concentrated under reduced pressure to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.39 g, quantitative yield).

LCMS: Method C, 1.38 min; MS: ER+ 411.1.

Stage (iv)

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.39 g, 0.74 mmol) in THF (5 mL) was added K ₂ CO ₃ (0.31 g, 2.23 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.06 g, 0.59 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (70 mL) and extracted with EtOAc (3×70 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 40% EtOAc in n-hexanes) to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.14 g, 0.33 mmol, 66% yield over two steps).

LCMS: Method H, 3.03 min; MS: ER- 434.1; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.40 (d, J=6.8 Hz, 1H), 8.51 (d, J=1.6 Hz, 1H), 8.12 (dd, J=8.8, 2.0 Hz, 1H), 7.86 (s, 1H), 7.82 (d, J=8.4 Hz, 1H), 4.51-4.55 (m, 1H), 3.99-4.08 (m, 1H), 3.69-3.74 (m, 1H), 3.42-3.51 (m, 3H), 3.34 (s, 3H), 2.11-2.17 (m, 1H), 1.97-2.03 (m, 1H). Chiral SFC: Method Y4, 3.57 min.

Example 8

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

(i) TBD, THF, 0°C to rt; (ii) Zn(CN) ₂ , Zn, _Pd2 (dba) ₃ , phosphine ligand, DMA, 140°C; (iii) TFA, DCM, 0°C to rt; (iv) _{K2CO3 ,} CNBr, THF, 0°C to rt.

Stage (i)

tert-Butyl-(2R,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylam

To a stirred solution of ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxylate (0.40 g, 1.05 mmol) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 348165-63-9, 0.23 g, 1.16 mmol) in THF (8 mL) was added TBD (0.22 g, 1.57 mmol) portionwise at 0 °C. The mixture was allowed to warm to rt and stirred for 3 h, then poured into water (50 mL) and extracted with EtOAc (3×60 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 32% EtOAc in n-hexanes) to give tert-butyl (2R,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.14 g, 0.26 mmol, 24% yield).

LCMS: Method C, 2.01 min; MS: ER+ 534.2, 536.2.

Stage (ii)

tert-Butyl-(2R,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-methylpyrrolidip-1-carboxylate

To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.13 g, 0.24 mmol) in DMA (1.3 mL) were added zinc cyanide (0.07 g, 0.61 mmol), zinc dust (0.01 g, 0.12 mmol), and 1,1'-ferrocenediyl bis(diphenylphosphine) (0.03 g, 0.05 mmol) at rt. The mixture was degassed with _N2 for 10 min, then trms(dibenzylideneacetone)dipalladium(0) (0.04 g, 0.05 mmol) was added. The mixture was heated under microwave irradiation at 140 °C for 1 h, then allowed to cool to rt, poured into water (20 mL), and extracted with EtOAc (3×30 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 38% EtOAc in n-hexanes) to afford tert-butyl (2R,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)-oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.05 g, 0.10 mmol, 42% yield).

LCMS: Method C, 1.88 min; MS: ER+ 425.6 (M-56).

Stage (iii)

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.05 g, 0.1 mmol) in DCM (2 mL) was added TFA (0.15 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.08 g, quantitative yield).

LCMS: Method C, 1.38 min; MS: ER+ 381.2.

Stage (iv)

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.08 g, 0.16 mmol) in THF (4 mL) was added K ₂ CO ₃ (0.07 g, 0.48 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.02 g, 0.16 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (20 mL) and extracted with EtOAc (3×25 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 47% EtOAc in n-hexanes) to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide (0.02 g, 0.05 mmol, 47% yield over two steps).

LCMS: Method H, 3.02 min; MS: ER- 404.0; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.43 (d, J=6.8 Hz, 1H), 8.51 (s, 1H), 8.11 (d, J=8.4 Hz, 1H), 7.84 (s, 1H), 7.82 (d, J=8.4 Hz, 1H), 4.48-4.57 (m, 1H), 3.89-3.94 (m, 1H), 3.76-3.80 (m, 1H), 3.42-3.46 (m, 1H), 2.15-2.18 (m, 1H), 1.76-1.83 (m, 1H), 1.25 (d, J=6.4 Hz, 3H). Chiral SFC: Method Y8, 5.02 min.

Example 9

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

(i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K ₂ CO ₃ , CNBr, THF, 0°C to rt.

Stage (i)

tert-Butyl-(2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylam

To a stirred solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.50 g, 1.72 mmol) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 348165-63-9, 0.27 g, 1.38 mmol) in THF (10 mL) was added TBD (0.29 g, 2.07 mmol) portionwise at 0 °C. The mixture was warmed to rt and stirred for 2 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 40% EtOAc in n-hexanes) to give tert-butyl (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.3 g, 0.67 mmol, 38% yield).

LCMS: Method C, 1.78 min; MS: ER+ 389.3 (M-56).

Stage (ii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.3 g, 0.67 mmol) in DCM (7 mL) was added TFA (0.9 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.29 g, quantitative yield).

LCMS: Method C, 1.39 min; MS: ER+ 345.3.

Stage (iii)

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

To a stirred solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.29 g, 0.63 mmol) in THF (7 mL) was added K ₂ CO ₃ (0.26 g, 1.89 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.07 g, 0.63 mmol) was added at 0 °C. The mixture was stirred at rt. for 1 h, then poured into water (70 ml) until a precipitate formed, and the solid was collected by filtration under reduced pressure to give 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide (0.13 g, 0.35 mmol, 52% yield over two steps).

LCMS: Method H, 2.75 min; MS: ER+ 370.1; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.31 (d, J=6.4 Hz, 1H), 8.28-8.31 (m, 1H), 7.79-7.82 (m, 1H), 7.49-7.52 (m, 1H), 4.47-4.59 (m, 1H), 4.08 (s, 3H), 3.85-3.99 (m, 1H), 3.76-3.80 (m, 1H), 3.42-3.45 (m, 1H), 2.12-2.23 (m, 1H), 1.73-1.87 (m, 1H), 1.24-1.29 (m, 3H). Chiral SFC: Method Y5, 4.98 min.

Example 10

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

Stage (i)

Ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate

To a stirred solution of 3-(2-bromoacetyl)-4-cyclopropoxybenzonitrile (0.70 g, 2.50 mmol) and ethyl oximate (CAS 617-36-7, from Sigma-Aldrich, 0.66 g, 3.75 mmol) in EtOAc (10 mL) was added silver triflate (CAS 2923-28-6, from Combi-blocks, 0.96 g, 3.75 mmol) at rt. This mixture was heated at 80 °C for 16 h, combined with another identical batch and then poured into water (100 mL), filtered through Celite Hyflow™, and the filtrate was extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 10% EtOAc in n-hexanes) to give ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.35 g, 1.17 mmol, 23% yield).

LCMS: Method H1, 3.40 min; MS: ER+ 299.0.

Stage (ii)

tert-Butyl-(2S,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylam

To a stirred solution of ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.32 g, 1.07 mmol) and tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.25 g, 1.07 mmol) in DMF (7 mL) was added TBD (0.18 g, 1.28 mmol) portionwise at 0 °C. The mixture was allowed to warm to rt and stirred for 2 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 30% EtOAc in n-hexanes) to give tert-butyl (2S,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.10 g, 0.22 mmol, 20% yield).

LCMS: Method H1, 3.51 min; MS: ER- 481.3.

Stage (iii)

4-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazol-2-carboxamide TFA salt

To a stirred solution of tert-butyl (2S,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.1 g, 0.22 mmol) in DCM (5 mL) was added TFA (1 mL, 10 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.14 g, quantitative yield).

LCMS: Method J, 3.61 min; MS: ER+ 383.0.

Stage (iv)

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

To a stirred solution of 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.14 g, 0.28 mmol) in THF (5 mL) was added K ₂ CO ₃ (0.12 g, 0.84 mmol) at rt and stirred for 5 min. Cyanogen bromide (0.03 g, 0.28 mmol) was added at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (30 mL) and extracted with EtOAc (2×30 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 45% EtOAc in n-hexanes) to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.04 g, 0.09 mmol, 47% yield over two steps).

LCMS: Method H1, 2.97 min; MS: ER- 406.2; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.24 (d, J=6.8 Hz, 1H), 8.52 (s, 1H), 8.43 (d, J=2.0 Hz, 1H), 7.92 (dd, J=8.8, 2.0 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 4.48-4.56 (m, 1H), 4.15-4.23 (m, 1H), 4.0-4.08 (m, 1H), 3.68-3.72 (m, 1H), 3.42-3.50 (m, 3H), 3.32 (s, 3H), 2.10-2.15 (m, 1H), 1.96-2.01 (m, 1H), 0.86-0.97 (m, 4H).

Chiral HPLC: Method Y15, 10.0 min.

Example 11

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

Stage (i)

tert-Butyl-(2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylam

To a stirred solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.25 g, 0.83 mmol) and tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5, 0.19 g, 0.83 mmol) in toluene (5 mL) was added TBD (0.12 g, 0.83 mmol) portionwise 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 anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 35% EtOAc in n-hexanes) to give tert-butyl (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.08 g, 0.16 mmol, 19% yield).

LCMS: Method C1, 1.41 min; MS: ER+ 483.4.

Stage (ii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt

To a stirred solution of tert-butyl (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.07 g, 0.15 mmol) in DCM (1 mL) was added TFA (0.22 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.10 g, quantitative yield).

LCMS: Method C1, 1.06 min; MS: ER+ 383.2.

Stage (iii)

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.10 g, 0.20 mmol) in THF (5 mL) was added K ₂ CO ₃ (0.08 g, 0.60 mmol) at rt and stirred for 5 min. Cyanogen bromide (0.02 g, 0.20 mmol) was added at 0 °C. This mixture was stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 45% EtOAc in n-hexanes) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.02 g, 0.04 mmol, 29% yield, over two steps).

LCMS: Method H1, 2.94 min; MS: ER+ 408.2; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.18 (d, J=6.8 Hz, 1H), 8.24 (s, 1H), 7.96 (d, J=8.4 Hz, 1H), 7.67-7.69 (m, 2H), 4.50-4.61 (m, 1H), 4.15-4.25 (m, 1H), 3.88-3.97 (m, 1H), 3.63-3.72 (m, 1H), 3.46-3.60 (m, 2H), 3.37-3.41 (m, 4H), 2.27-2.38 (m, 1H), 1.80-1.90 (m, 1H), 0.83-0.98 (m, 4H). Chiral SFC: Method Y12, 3.53 min.

Example 12

4-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

Stage (i)

tert-Butyl-(2R,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylam

To a stirred solution of ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxylate (0.40 g, 1.34 mmol) [Example 10; Step (i)] and tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5, 0.31 g, 1.34 mmol) in toluene (5 mL) was added TBD (0.19 g, 1.34 mmol) portionwise at 0 °C. The mixture was allowed to warm to rt and stirred for 4 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 40% EtOAc in n-hexanes) to give tert-butyl (2R,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.22 g, 0.46 mmol, 34% yield). LCMS: Method C1, 1.42 min, MS: ER+ 483.3.

Stage (ii)

4-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt

To a stirred solution of tert-butyl (2R,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazole-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.22 g, 0.46 mmol) in DCM (5 mL) was added TFA (0.66 mL, 3 vol) dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 1 h, then concentrated under reduced pressure to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.28 g, quantitative yield).

LCMS: Method C1, 1.09 min. MS: ER+ 383.2.

Stage (iii)

4-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

To a stirred solution of 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide TFA salt (0.28 g, 0.56 mmol) in THF (5 mL) was added K ₂ CO ₃ (0.23 g, 1.69 mmol) at rt and stirred for 5 min. Cyanogen bromide (0.06 g, 0.56 mmol) was added at 0 °C. This mixture was stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 65% EtOAc in n-hexanes) to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide (0.06 g, 0.14 mmol, 31% yield, over two steps).

LCMS: Method HI, 3.01 min; MS: ER+ 408.2; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.10 (d, J=7.6 Hz, 1H), 8.52 (s, 1H), 8.38 (s, 1H), 7.91 (d, J=8.4, 2.0 Hz, 1H), 7.64 (d, J=8.8 Hz, 1H), 4.51-4.56 (m, 1H), 4.16-4.23 (m, 1H), 3.88-3.97 (m, 1H), 3.63-3.67 (m, 1H), 3.46-3.56 (m, 2H), 3.38-3.41 (m, 4H), 2.29-2.36 (m, 1H), 1.83-1.90 (m, 1H), 0.92 (br s, 4H). Chiral SFC: Method Y16, 5.11 min.

Example 13

5-(5-Cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

The title compound can be prepared by a method similar to that of Example 1 using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in Step (i).

Example 14

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

The title compound can be prepared by a method similar to that of Example 4 using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in Step (i).

Example 15

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

The title compound can be prepared by a method similar to that of Example 5 using tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5) in Step (i).

Example 16

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

The title compound can be prepared by a method similar to that of Example 6 using tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5) in Step (iii).

Example 17

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide

The title compound can be prepared by a method similar to that of Example 7 using tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5) in Step (i).

Example 18

5-(5-Cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

The title compound can be prepared in a manner similar to that of Example 8 using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in Step (i).

Example 19

5-(5-Cyano-4-fluoro-2-methoxyphenyl)-H-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide

The title compound can be prepared by a method similar to that of Example 9 using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in Step (i).

Biological activity of the compounds according to the invention

Abbreviations:

TAMRA carboxytetramethylrhodamine

PCR polymerase chain reaction

PBS phosphate-buffered saline

EDTA ethylenediaminetetraacetic acid

Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol

NP-40 Nonidet P-40, octylphenoxypolyethoxyethanol

BSA bovine serum albumin

PNS peripheral nervous system

BH3 Bcl-2 homology domain 3

PTEN phosphatase and tensin homologue

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

DMSO dimethyl sulfoxide

YFP yellow fluorescent protein

VME vinyl methyl ether

ON hemagglutinin

Ahx aminohexanoic acid

Biochemical analysis IC ₅₀ in relation to USP30

Plates were prepared with 21-fold final concentration dilutions (2100 µM for a final concentration of 100 µM) in 50% DMSO in a 96-well V-bottom polypropylene plate (Greiner #651201). A typical 8-point dilution series is 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 1 µL of 50% DMSO or the 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 a final assay concentration of 4 nM, and 10 μl of the diluted USP30 was added to the compound. The enzyme and compound were incubated for 30 min at room temperature. Reactions were initiated by adding 50 nM TAMRA-labeled peptide linked to ubiquitin via an isopeptide bond as a fluorescence polarization substrate. Reactions were read immediately after substrate addition and subsequent 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). Excitation λ 540 nm; emission λ 590 nm.

Activity of the illustrated compounds in the biochemical assay IC ₅₀ in relation to USP30:

Comparison examples

Activity of the reference compounds in the biochemical analysis IC ₅₀ in relation to USP30:

Non-target pharmacology

Example 2 compound was subjected to pharmacological profiling in the Eurofins CEREP SafetyScreen44 panel. At a single concentration of 10 μM, less than 50% inhibition of binding or enzyme activity was observed for all targets in the panel. Example 1 compound has a low probability of off-target interactions due to its low affinity for the targets in this assay.

Safety pharmacology

Example 2 compound was evaluated for its effect on the hERG potassium channel in stably expressed CHO cells at concentrations ranging from 0.01 to 30 μM. Example 2 compound yielded a 35% inhibition value of TOKahERG amplitude at 30 μM, indicating little potential for QT interval effects.

Genetic toxicology

Example 2 compound was evaluated in a bacterial reverse mutation assay (Ames) and an in vitro micronucleus assay. All in vitro tests were performed with and without exogenous metabolic activation using concentrations up to those limited by cytotoxicity or insolubility. Example 2 compound did not induce mutations when tested up to 1000 μg/well with and without metabolic activation in the reverse mutation assay in Salmonella typhimurium strains TA98, TA 100, TA1535, and TA97a and in Escherichia coli strain WP2 uvrA pKM101.

Induction of chromosome damage was assessed using an in vitro micronucleus assay in TK6 cells. Example 2 was negative for micronuclei induction when incubated for 3 hours in the presence of exogenous metabolic activation followed by 27 hours of recovery, as well as when incubated for 27 hours in the absence of exogenous metabolic activation followed by 27 hours of recovery.

Cell-based analysis of interaction with the endogenous target USP30

HeLa cells stably expressing YFP-parkin were seeded in 6-well dishes. Immediately after attachment, cells were treated with the appropriate test compounds at the appropriate concentrations or vehicle control for 1 h at 37°C, 5% _CO2 . Whole-cell lysates were prepared by scraping cells into cold PBS, centrifuging, and lysis in lysis buffer (50 mM Tris base, pH 7.5, 50 mM NaCl, 1% NP-40/Igepal CA-630, 2 mM MgCl2, ₁₀ % glycerol, 5 mM beta-mercaptoethanol, Complete EDTA-free minitablets (Roche), PhosStop tablets (Roche)) for 10 min. A 20 μg protein equivalent from the cleared cell lysate was incubated with a final concentration of 2.5 μM HA-Ahx-Ahx-Ub-VME probe at room temperature. The reaction was stopped by the addition of 5x SDS loading buffer, and proteins were isolated by SDS-PAGE and Western blotting. USP30 was detected using sheep anti-USP30 antibody S746D (MRC PPU Reagents and Services) and horseradish peroxidase-conjugated rabbit anti-sheep IgG (H+L) secondary antibody (Thermo #31480) and visualized using ECL reagent (GE #RPN2109) on a GE LAS4000 imager. Target interaction was measured by quantifying bands corresponding to USP30 and USP30 bound to the Ub-VME probe and expressing this proportion compared to the vehicle-treated control.

TOM20 ubiquitylation analysis

Human cell lines can be sensitized with mitochondrial depolarizing agents (ionophores (e.g., CCCP, valinomycin), mitochondrial complex inhibitors (oligomycin, antimycin A)) to induce TOM20 ubiquitylation, which is then further promoted in the presence of USP30 inhibitors. TOM20 ubiquitylation is then assessed by Western blotting of cell lysates, with detection of the TOM20 ubiquitylation adduct being possible due to an 8 kDa increase in molecular mass for each molecule of added ubiquitin, resulting in a stepped TOM20 immunoreactive band. TOM20 ubiquitylation levels can be quantified using chemiluminescence densitometry of the stepped immunoreactive bands.

In vitro cytotoxicity (Cell Tox): Measured in human colorectal carcinoma HCT116 cells using alamarBlue™ as the assay endpoint. Compound cytotoxicity was measured over a 96-hour period of continuous exposure.

Additional research

log P: partition coefficient; a measurement of lipophilicity.

log D: partition coefficient; a measurement of lipophilicity.

TPSA: Topological polar surface area.

Turbidimetric solubility: A solution of the test compound prepared in DMSO is diluted in aqueous buffer. Turbidimetry is used as an endpoint by measuring absorbance at 620 nm.

FaSSIF: Fasting simulated intestinal fluid measured at pH 6.5.

Hep Cl in mouse: in vitro clearance of hepatocytes in mouse cells.

Hep Cl in humans: in vitro clearance of hepatocytes in human cells.

f _u,p in plasma: The free fraction of a compound in a blood plasma preparation, determined by in vitro equilibrium dialysis.

f _u,br in brain: Free fraction of the compound in a brain homogenate preparation, determined by in vitro equilibrium dialysis.

Cl _u : in vitro clearance. Cl _u , as defined herein, is the scaled clearance, which is in turn calculated from the intrinsic clearance. Intrinsic clearance is the predicted clearance due to hepatic metabolic reactions, determined by incubating the compound in a hepatocyte preparation. The lower the value in mL/min/kg, the more stable the compound.

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

Oral F: Oral bioavailability.

MDR1-MDCK (Madin-Darby canine kidney cell monolayer) (in vitro) permeability assay.

WT-MDCK (wild type) permeability in vitro.

Kp _u,u is the ratio of unbound drug in the brain to unbound drug in plasma and may serve as an indicator of potential for treatment of peripheral and/or CNS indications.

The example compounds possess beneficial properties, demonstrating potential superiority over other compounds. For example, for Examples 1 and 2, the observed intravenous plasma clearances of 41 and 20 ml/min/kg, as measured in mice, are low, indicating valuable plasma stability, and the compounds have very good oral bioavailability of 37 and 47%, respectively.

Comparative data

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

Efficacy relative to USP30

The compounds of Examples 1-11 of the present invention are significantly more potent against USP30 than the compounds of Comparative Examples A, B, C, D, and E, as measured in a biological assay. The compound of Example 12 is significantly more potent against USP30 than the compounds of Comparative Examples B, C, D, and E. For example, the compounds of Examples 1-11 are 6.8-34 times more potent than the compound of Comparative Example A, 16-155 times more potent than the compounds of Comparative Examples B, C, and D, and at least 440 times more potent than the compound of Comparative Example E.

Selectivity for USP30 relative to other DUBs

The data presented demonstrate that the compounds of Examples 1, 2, 4, 6, 7, 11, and 12 are significantly more selective for USP30 relative to nine DUBs (USP2, USP6, USP10, USP15, USP16, USP21, USP25, USP28, and USP46) compared to the compound of Comparative Example A. These compounds are at least 110-7820 times more potent against USP30 than against each of these nine DUBs (the compound of Example 7 was not tested against USP15). This is a significant advantage in selectivity over the compound of Comparative Example A, which is only 2.2 times more potent.

Selectivity for USP30 over UCHL1

The presented data demonstrate that the compounds of Examples 1, 2, 4, 6, 7, 11 and 12 are significantly more selective for USP30 relative to UCHL1 compared to the compounds of Comparative Examples B, C, D and E. The compounds of Examples 1, 2, 4, 6, 7, 11 are more than 30,000 times more effective against USP30 than against UCHL1, and the compound of Example 12 is more than 4412 times more effective, while the compounds of Comparative Examples B, C, D and E are only 3.0, 1.3, 0.8 and 1.5 times more effective, respectively; 'C' is more selective for UCHL1.

Selectivity for USP30 relative to cathepsins B, K, L, S and V

The presented data demonstrate that the compounds of Examples 1, 2, 4, 6, 7, 11, and 12 are significantly more selective for USP30 relative to cathepsins (B, K, L, S, and V) compared to the compound of Comparative Example A. The compounds of these examples are all more than 1540 times more potent against USP30 than against each of the cathepsins. This is a significant advantage in selectivity over the compound of Comparative Example A, which is only 11.6 times more potent against cathepsin K.

The above-identified advantages of the compounds of the invention over the comparative examples of the prior art are both significant and unexpected. This superiority, both individually and in combination, makes the compounds of the invention particularly suitable for use in the treatment or prevention of diseases associated with USP30 activity.

Preclinical in vitro model

(a) Human renal proximal tubule cell cisplatin injury model modified for testing renal protective agents.

Primary human RTCs are isolated from kidneys deemed unsuitable for transplantation.

Isolated cells were seeded onto 96-well Transwell inserts (surface area = 0.143 ^cm2 ) at a density of 20,000 cells per insert. The medium was refreshed 24 hours after the initial seeding, as well as on days 3 and 5 of culture.

Monolayers exhibited TEER (transepithelial electrical resistance) values in the range of 80-120 ^Ω.cm2 before use in experiments.

Human PTC monolayers were pre-exposed to the compound of Example 2 (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 μM) for 1 hour on both the apical and basolateral sides of the cell layer, after which the cells were exposed to cisplatin (20 μM). The cells were then exposed to both the compound of Example 2 (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 μM) and cisplatin (20 μM) on both the apical and basolateral sides for 24 and 48 hours. In addition, negative controls (Example 2 compound only, 0.5% DMSO for Example 2 compound, 0.2% dimethylformamide (DMF) cisplatin solvent control, positive control (cisplatin 20 μM), and cisplatin plus cisplatin uptake inhibitor (dolutegravir 100 μM) were included in parallel. Experiments were performed in triplicate in human cell monolayers (n=3) and from three different donors (N=3).

Cell viability assays based on ATP production and LDH release were performed on test compound-treated monolayers in 96-well Transwell inserts.

Example 2 protected against cisplatin-induced ATP loss in a concentration-dependent manner. Example 2 also reduced the cisplatin-induced increase in LDH. These data demonstrate that Example 2 can protect against cisplatin-induced toxicity to proximal tubular epithelial cells.

Preclinical in vivo models

The compounds of the invention can be tested for efficacy in representative disease models in vivo using standard test methods from the literature, including, for example:

(a) The bleomycin-induced pulmonary fibrosis model, which is the leading in vivo preclinical model of idiopathic pulmonary fibrosis [Kobayashi et al, 2016, J Immunol, 197(2):504–516].

(b) Diet-induced model of NAFLD (non-alcoholic fatty liver disease) and glucose homeostasis [Nishida et al, 2013, Lab Invest; Feb;93(2):230-41].

(c) The MPTP (1-methyl-phenyl-1,2,3,6-tetrahydropyridine)-induced Parkinson's disease model, which is a widely used paradigm to study neurodegeneration in the brain dopaminergic system that is triggered by chemically induced mitochondrial dysfunction [Karuppagouner et al, 2014, Sci Rep. 2014 May 2;4:4874].

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

(e) Aged rodent model: Effects on the hippocampus, cognitive and motor function [Kobilo et al, 2014, Learn Mem. Jan 17;21(2): 119-26; Creed et al, 2019, Neuroscience. Jun 15;409:169-179; Van Skike et al, 2020, Aging Cell. 19;e13057].

(f) Unilateral ureteral obstruction (UUO) model of renal disease [Chevalier et al, 2009, Kidney Int 75(11): 1145-1152].

UUO induces renal injury characterized by tubular cell damage, interstitial inflammation, and fibrosis. It serves as a model of irreversible postrenal 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. Therefore, the UUO model provides researchers with information beyond obstruction (Chevalier et al., 2009, Kidney Int 75(11): 1145–1152).

Example 2 compound was evaluated in the UUO model to determine the ability of the compound to attenuate progressive tubulointerstitial fibrosis and chronic kidney disease (CKD).

On Day 1 of the study, adult C57BL/6 mice were dosed by oral gavage according to one of the following dosing regimens: vehicle, 1.5, or 5 mg/kg of Example 2 BID (twice daily). Two hours after dosing on Day 1 of the study, mice underwent surgery to ligate the left ureter at two points. Successful UUO was later confirmed by determining renal pelvic dilation due to hydronephrosis. Animals were dosed according to their assigned regimen for 10 days, at which time kidneys were harvested for histopathology and protein/RNA assessment. Picrosirius Red staining was performed to assess the extent of collagen deposition, and IHC was used to assess the relative expression of smooth muscle actin α (α-SMA).

The results demonstrated that both 1.5 and 5 mg/kg of Example 2 (p.o.) administered BID statistically reduced collagen deposition, as evidenced by a decrease in Picrosirius red staining in ligated kidneys. Evaluation of α-SMA staining revealed that oral administration of 5 and 1.5 mg/kg of Example 2 BID resulted in a statistically reduced α-SMA levels in UUO-injured kidneys compared to vehicle-treated controls.

(g) AKI can be caused by bilateral renal pedicle clamping, resulting in ischemia reperfusion injury (IRI), leading to severe loss of renal function, tubular injury, and inflammation [Lu et al. 2012. J Nephrol. 25(5):738–45].

Preventive administration:

Example 2 was administered to C57BL/6 mice at 5 and 1.5 mg/kg BID (p.o.) and compared with vehicle treatment from Day -1 to Day +21. On Day 0, mice were anesthetized, and their left renal pedicle was clamped for 45 min and then released to induce IRI. On Day +21, kidneys were harvested. Morphology and fibrosis were assessed.

Body weight was similar between groups and remained constant throughout the observation period. Masson's trichrome staining revealed significantly lower tubular atrophy and collagen content in the outer medulla in animals treated with Example 2 (both dose levels) on Day +21. Fibronectin expression in the cortex and outer medulla was significantly reduced in animals treated with Example 2 (both dose levels) on Day +21.

Compound Example 2 demonstrated efficacy in this model of IR-induced CKD. Daily treatment demonstrated significant benefits in attenuating tubular atrophy and reducing fibrosis.

Post-injury administration:

On Day 0 (zero), C57BL/6 mice were anesthetized and their left renal pedicle was clamped for 45 min and then released to induce IRI. Mice were then treated with either vehicle or Example 2 compound; 5 mg/kg (p.o.) BID for 21 days, with the first treatment beginning 5 hours after IRI surgery (i.e., administration of therapeutic agents). Mice were monitored, and kidneys were harvested on Day +21. Kidney sections were quantified for relative cellular morphology and fibrosis using blinded histological scoring methods.

Body weight was similar between groups and remained constant throughout the observation period. Fibronectin staining in the cortex was significantly reduced in mice treated with Example 2 on Day +21.

Compound Example 2 demonstrated partial efficacy in this IR-induced CKD model at a therapeutic dose. Initial treatment after the onset of ischemia-reperfusion injury showed significant benefits in attenuating cortical fibrosis.

Claims of the invention

The present invention relates to the following:

1. A compound of formula (I) which is selected from compounds of formula (I)(i) and formula (I)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer, where:

R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ;

R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and

R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen.

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

3. The compound according to claim 2, wherein R ¹ is selected from methyl and CH ₂ OCH ₃ .

4. A compound according to any one of claims 1 to 3, wherein R ² is selected from methyl, CF ₃ and cyclopropyl.

5. The compound according to claim 4, wherein R ² is selected from methyl and cyclopropyl.

6. A compound according to any one of claims 1-5, wherein R ³ is selected from hydrogen and fluorine.

7. The compound according to claim 6, wherein R ³ is hydrogen.

8. A compound according to any one of claims 1 to 7, wherein each of R ⁴ and R ⁵ is hydrogen.

9. A compound according to any one of claims 1 to 8, having formula (I)(i):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer.

10. The compound according to claim 9, having the formula (IA)(i):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer.

11. The compound according to claim 10, which is selected from:

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; and

5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

12. The compound according to claim 9, having the formula (IB)(i):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer.

13. The compound according to claim 12, which is selected from:

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

4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

14. A compound according to any one of claims 1 to 8, having formula (I)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer.

15. The compound according to claim 14, having the formula (IA)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer.

16. The compound according to claim 15, which is selected from:

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide;

5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; and

5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

17. The compound according to claim 14, having the formula (IB)(ii):

its tautomer or a pharmaceutically acceptable salt of the said compound or tautomer.

18. The compound of claim 17, which is selected from: 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; and

4-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide;

or a pharmaceutically acceptable salt thereof.

19. A compound according to any one of claims 1-18, its tautomer or a pharmaceutically acceptable salt of said compound or tautomer for use as a medicinal product.

20. A compound according to any one of claims 1-18, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer for use in the treatment or prevention of a condition involving mitochondrial dysfunction, cancer, or fibrosis.

21. The use of a compound according to any one of claims 1 to 18, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer in the manufacture of a medicament for use in the treatment or prevention of a condition involving mitochondrial dysfunction, cancer, or fibrosis.

22. A method for treating or preventing a condition involving mitochondrial dysfunction, cancer or fibrosis, comprising the step of administering an effective amount of a compound according to any one of claims 1-18, its tautomer or a pharmaceutically acceptable salt of said compound or tautomer to a patient in need thereof.

23. The compound, use or method of claims 20-22, wherein the condition involving mitochondrial dysfunction is selected from a CNS disorder; a 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 episode syndrome; maternally inherited diabetes and deafness; Leber's hereditary optic neuropathy; neuropathy, ataxia, maternally inherited retinitis pigmentosa Leigh syndrome; Danon disease; diabetes; diabetic neuropathy; metabolic disorders; heart failure; Ischemic heart disease leading to myocardial infarction; Psychiatric disorders, schizophrenia; Multiple sulfatase deficiency; Mucolipidosis II; Mucolipidosis III; Mucolipidosis IV; GM1 gangliosidosis; Neuronal ceroid lipofuscinosis; Alpers disease; Barth syndrome; Beta-oxidation defects; Carnitine acyl-carnitine deficiency; Carnitine deficiency; Creatine deficiency syndromes; Coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; Complex V deficiency; COX (cyclooxygenase) deficiency; Chronic progressive external ophthalmoplegia syndrome; CPT I (carnitine palmitoyltransferase I) deficiency; CPT II deficiency; Glutaraciduria type II; Kearns-Sayre syndrome; Lactic acidosis; Long-chain acyl-CoA dehydrogenase deficiency; Leigh disease or syndrome; French-Canadian variant of Leigh syndrome; Lethal infantile cardiomyopathy; Luft disease; 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 (mitochondrial polymerase gamma) mutations; Medium/short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; and Very-long-chain acyl-CoA dehydrogenase deficiency; Peroxisomal disorders; methylmalonic acidemia; mevalonate kinase deficiency; age-related decline in cognitive function and muscle strength; and cognitive impairment associated with all neurodegenerative and neuropsychiatric disorders.

24. The compound, use or method of claim 23, 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 associated with mutations in α-synuclein, parkin, PINK1, GBA and LRRK2, and autosomal recessive juvenile Parkinson's disease or early-onset Parkinson's disease (EOPD), wherein parkin or PINK1 is mutated, truncated or deleted.

25. The compound, use or method of claim 23, wherein the neurodegenerative disease is Leigh syndrome or disease, X-linked Leigh disease, French Canadian variant of Leigh syndrome and/or symptoms associated with Leigh disease.

26. The compound, use or method according to paragraphs. 20-22, wherein the cancer is selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma cancer, bone cancer, liver cancer, soft tissue cancer, organ cancer, blood cell cancer, CML (chronic myelogenous leukemia), AML (acute myeloid leukemia), mantle cell lymphoma, neuroblastoma, melanoma, soft tissue sarcoma, liposarcoma, fibroblastic sarcoma, leiomyosarcoma, hepatocellular carcinoma, osteosarcoma, esophageal cancer, leukemia, lymphoma, multiple myeloma, metastatic carcinoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, nasopharyngeal carcinoma, colorectal cancer, non-small cell lung carcinoma, cancers in which apoptotic pathways are dysregulated, and cancers in which BCL-2 (B-cell lymphoma 2) family proteins are mutated or over- or under-expressed.

27. The compound, use or method of claims 20-22, wherein fibrosis is selected from fibrosis or a fibrosing disorder associated with the accumulation of extracellular matrix constituents that occurs as a result of trauma, inflammation, tissue regeneration, immunological reactions, cellular hyperplasia and neoplasia.

28. The compound, use or method of claim 27, wherein the fibrosis is selected from fibrosis or fibrosing disorder associated with diseases of vital organs, fibroproliferative disorders and scarring associated with trauma.

29. The compound, use, or method of claim 28, wherein the fibrosis is selected from fibrosis or a fibrosing 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 disease, acute kidney injury, chronic kidney disease, delayed kidney transplant function, cardiovascular disease, eye diseases, systemic and local sclerosis, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, fibrosis induced by chemotherapeutic drugs, fibrosis induced by radiation, accidental injury and burns, retroperitoneal fibrosis, and peritoneal fibrosis/peritoneal scarring.

30. The compound, use, or method of claim 29, 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, obliterating bronchiolitis, and bronchiectasis.

31. The compound, use, or method of claim 29, wherein the kidney disease is acute kidney disease, acute kidney injury, or chronic kidney disease.

32. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 18, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, together with one or more pharmaceutically acceptable excipients.

33. A compound that is selected from compounds of formulas (II)(i), (III)(i), (II)(ii) and (III)(ii):

its tautomer or a salt of the said compound or tautomer;

wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined for the compound of formula (I) in any one of claims 1 to 18; and PG is a protecting group which is preferably selected from tert-butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzylcarbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethoxycarbonyl, 4-nitrobenzenesulfonyl and 2-nitrophenylsulfenyl.

Claims (76)

1. A compound of formula (I) which is selected from compounds of formula (I)(i) and formula (I)(ii): , or a pharmaceutically acceptable salt of the said compound, wherein R ¹ is selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )fluoroalkyl and CH ₂ OCH ₃ ; R ² is selected from (C ₁ -C ₄ )alkyl, CF ₃ and cyclopropyl; and R ³ , R ⁴ and R ⁵ , each independently, are selected from hydrogen and halogen. 2. The compound according to claim 1, wherein R ¹ is selected from methyl, CH ₂ F, CHF ₂ , CF ₃ and CH ₂ OCH ₃ . 3. The compound according to claim 2, wherein R ¹ is selected from methyl and CH ₂ OCH ₃ . 4. A compound according to any one of claims 1 to 3, wherein R ² is selected from methyl, CF ₃ and cyclopropyl. 5. The compound according to claim 4, wherein R ² is selected from methyl and cyclopropyl. 6. A compound according to any one of claims 1-5, wherein R ³ is selected from hydrogen and fluorine. 7. The compound according to claim 6, wherein R ³ is hydrogen. 8. A compound according to any one of claims 1 to 7, wherein each of R ⁴ and R ⁵ is hydrogen. 9. A compound according to any one of claims 1 to 8, having formula (I)(i): (I)(i), or a pharmaceutically acceptable salt of the said compound. 10. A compound according to claim 9, having the formula (IA)(i): (IA)(i), or a pharmaceutically acceptable salt of the said compound. 11. The compound according to claim 10, which is selected from: 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 S )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 S )-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-(( 3R , 5S )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R ,5 S )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R ,5 R )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide and 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; or a pharmaceutically acceptable salt thereof. 12. The compound according to claim 9, having the formula (IB)(i): (IB)(i), or a pharmaceutically acceptable salt of the said compound. 13. The compound according to claim 12, which is selected from: 4-(5-cyano-2-methoxyphenyl)-N-(( 3R , 5S )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazol-2-carboxamide and 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 S )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; or a pharmaceutically acceptable salt thereof. 14. A compound according to any one of claims 1 to 8, having formula (I)(ii): (I)(ii), or a pharmaceutically acceptable salt of the said compound. 15. The compound according to claim 14, having the formula (IA)(ii): (IA)(ii), or a pharmaceutically acceptable salt of the said compound. 16. The compound according to claim 15, which is selected from: 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 S )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R ,5 S )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R ,5 R )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R ,5 S )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide; 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3 R ,5 S )-1-cyano-5-methylpyrrolidin-3-yl)oxazole-2-carboxamide and 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-(fluoromethyl)pyrrolidin-3-yl)oxazole-2-carboxamide; or a pharmaceutically acceptable salt thereof. 17. The compound according to claim 14, having the formula (IB)(ii): (IB)(ii), or a pharmaceutically acceptable salt of the said compound. 18. The compound according to claim 17, which is selected from: 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazole-2-carboxamide and 4-(5-cyano-2-methoxyphenyl)-N-((3 R ,5 R )-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)oxazol-2-carboxamide; or a pharmaceutically acceptable salt thereof. 19. The use of a compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt of said compound for the treatment or prevention of a disorder or condition associated with USP30 activity. 20. The use of a compound according to any one of claims 1-18 or a pharmaceutically acceptable salt of said compound for the treatment or prevention of a condition involving mitochondrial dysfunction, cancer or fibrosis that is associated with USP30 activity. 21. The use according to claim 20, wherein the condition involving mitochondrial dysfunction is selected from a CNS disorder; a 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 episode syndrome; maternally inherited diabetes and deafness; Leber's hereditary optic neuropathy; neuropathy, ataxia, maternally inherited retinitis pigmentosa Leigh syndrome; Danon disease; diabetes; diabetic neuropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; Psychiatric disorders, schizophrenia; Multiple sulfatase deficiency; Mucolipidosis II; Mucolipidosis III; Mucolipidosis IV; GM1 gangliosidosis; Neuronal ceroid lipofuscinosis; Alpers disease; Barth syndrome; Beta-oxidation defects; Carnitine acyl-carnitine deficiency; Carnitine deficiency; Creatine deficiency syndromes; Coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; Complex V deficiency; COX (cyclooxygenase) deficiency; Chronic progressive external ophthalmoplegia syndrome; CPT I (carnitine palmitoyltransferase I) deficiency; CPT II deficiency; Glutaraciduria type II; Kearns-Sayre syndrome; Lactic acidosis; Long-chain acyl-CoA dehydrogenase deficiency; Leigh disease or syndrome; French-Canadian variant of Leigh syndrome; lethal infantile cardiomyopathy; Luft disease; 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 (mitochondrial polymerase gamma) mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; very-long-chain acyl-CoA dehydrogenase deficiency; peroxisomal disorders; methylmalonic acidemia; mevalonate kinase deficiency; age-related decline in cognitive function and muscle strength; and cognitive impairment associated with all neurodegenerative and neuropsychiatric disorders. 22. The use according to claim 21, 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 associated with mutations in α-synuclein, parkin, PINK1, GBA and LRRK2, and autosomal recessive juvenile Parkinson's disease or early-onset Parkinson's disease (EOPD), wherein parkin or PINK1 is mutated, truncated or deleted. 23. The use according to claim 21, wherein the neurodegenerative disease is Leigh syndrome or disease, X-linked Leigh disease, French Canadian variant of Leigh syndrome and/or symptoms associated with Leigh disease. 24. The use according to claim 20, wherein the cancer is selected from breast, ovarian, prostate, lung, kidney, stomach, colon, testicle, head and neck, pancreatic, brain, melanoma, bone, liver, soft tissue, tissue organ cancers, blood cell cancers, CML (chronic myelogenous leukemia), AML (acute myeloid leukemia), mantle cell lymphoma, neuroblastoma, soft tissue sarcoma, liposarcoma, fibroblastic sarcoma, leiomyosarcoma, hepatocellular carcinoma, osteosarcoma, esophageal cancer, leukemia, lymphoma, multiple myeloma, metastatic carcinoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, nasopharyngeal carcinoma, colorectal cancer, non-small cell lung carcinoma, cancers in which apoptotic pathways are dysregulated, and cancers in which BCL-2 (B-cell lymphoma 2) family proteins are mutated or over- or underexpressed. 25. The use according to claim 20, wherein fibrosis is selected from fibrosis or a fibrosing disorder associated with the accumulation of extracellular matrix constituents that occurs as a result of trauma, inflammation, tissue regeneration, immunological reactions, cellular hyperplasia and neoplasia. 26. The use according to claim 25, wherein the fibrosis is selected from fibrosis or fibrosing disorder associated with diseases of vital organs, fibroproliferative disorders and scarring associated with trauma. 27. The use according to claim 26, wherein fibrosis is selected from fibrosis or fibrosing disorder associated with interstitial lung disease, liver cirrhosis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, kidney disease, acute kidney disease, acute kidney injury, chronic kidney disease, delayed kidney transplant function, cardiovascular disease, eye diseases, systemic and local scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, fibrosis caused by chemotherapeutic drugs, fibrosis caused by radiation, accidental injury and burns, retroperitoneal fibrosis and peritoneal fibrosis/peritoneal scarring. 28. The use according to claim 27, 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, obliterating bronchiolitis and bronchiectasis. 29. The use according to claim 27, wherein the kidney disease is acute kidney disease, acute kidney injury or chronic kidney disease. 30. A pharmaceutical composition having the activity of inhibiting USP30 activity, comprising an effective amount of a compound of formula (I) as defined in any one of claims 1 to 18, or a pharmaceutically acceptable salt of said compound, together with one or more pharmaceutically acceptable excipients. 31. A compound that is selected from compounds of formulas (II)(i), (III)(i), (II)(ii) and (III)(ii): or salts of the specified compound; where R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined for the compound of formula (I) in any one of claims 1 to 18, and PG is tert -butyloxycarbonyl.