N-cyanopyrrolidine compounds having activity as usp30 inhibitors

Abstract

The present invention relates to a class of N-cyanopyrrolidine compounds having activity as inhibitors of the deubiquitinase enzyme, ubiquitin USP30, which have utility in a variety of therapeutic areas, including disorders involving mitochondrial dysfunction, cancer and fibrosis.

Description

Invention Field

[0001] This invention relates to a class of N-cyanopyrrolidine compounds having activity as inhibitors of the deubiquitinating enzyme ubiquitin C-terminal hydrolase 30 (also known as ubiquitin-specific peptidase 30 (USP30)), their uses, methods of preparation, and compositions comprising said inhibitor. These inhibitors are applicable in a variety of therapeutic areas, including conditions involving mitochondrial dysfunction, cancer, and fibrosis.

[0002] All references cited or relied upon below are explicitly incorporated herein by reference. Background of the Invention

[0004] Ubiquitins are small proteins composed of 76 amino acids, crucial for regulating protein function in cells. Ubiquitination and deubiquitination are enzymatically mediated processes through which ubiquitin covalently binds to target proteins or is cleaved from them by deubiquitinating enzymes (DUBs). Approximately 100 DUBs exist in human cells, divided into subfamilies based on sequence homology. The USP family is characterized by its shared Cys and His boxes, containing Cys and His residues essential for their DUB activity. Ubiquitination and deubiquitination 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. Therefore, the ubiquitin system is involved in the pathogenesis of many disease states, including inflammation, viral infections, metabolic disorders, CNS diseases, and tumorigenesis.

[0005] Ubiquitin is a major regulator of mitochondrial dynamics. Mitochondria are dynamic organelles whose biogenesis, fusion, and fission events are regulated post-translationally through the ubiquitination of many key factors, such as mitochondrial fusion proteins. In humans, USP30 is a 517-amino acid protein found in the outer mitochondrial membrane (Nakamura et al., 2008, Mol Biol 19:1903-11). It is the only deubiquitinating enzyme carrying a mitochondrial addressing signal and has been shown to deubiquitinate many mitochondrial proteins. USP30 has been shown to antagonize perkinin-mediated mitophagy, and reduced USP30 activity can salvage perkinin-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 may also increase mitochondrial protein import through the ubiquitination of TOM protein (Jacoupy et al., 2019, Sci Rep 9(1):11829). A small proportion of USP30 is localized to peroxisomes, which are produced through the fusion of mitochondria and ER vesicles. USP30 potentially antagonizes the Pex2 / peroxisome phagocytic pathway (Riccio et al., 2019, J Cell Biol 218(3): 798-807). The E3 Ub ligase March5 and the deubiquitinase USP30 bind to translocases and regulate mitochondrial importation, while March5 resists mitochondrial importation and directs substrate degradation, and USP30 deubiquitinates substrates to promote their importation (Phu et al., 2020, Molecular Cell 77, 1107-1123).

[0006] Mitochondrial dysfunction can be defined as a reduction in mitochondrial content (mitochondria autophagy or mitochondrial biogenesis), decreased mitochondrial activity, and reduced oxidative phosphorylation, but it can also be defined as the regulation of reactive oxygen species (ROS) production. Therefore, mitochondrial dysfunction plays a role in a vast number of aging processes and pathological conditions.

[0007] For example, Parkinson's disease (PD) affects approximately 10 million people worldwide (Parkinson's Disease Foundation) and is characterized by the loss of dopaminergic neurons in the substantia nigra. The exact underlying mechanisms of PD remain unclear; however, mitochondrial dysfunction is increasingly recognized as a key determinant of susceptibility to dopaminergic neurons in PD and is characteristic of both familial and sporadic cases, as well as toxin-induced Parkinsonian syndromes. Parkinin is one of many proteins involved in early-onset PD. While most PD cases are associated with defects in α-synuclein, 10% of Parkinson's disease cases are associated with specific gene defects, one of which is present in the ubiquitin E3 ligase parkin. Parkin and PTEN-induced putative kinase 1 (PINK1) work together to ubiquitinate mitochondrial membrane proteins in damaged mitochondria, leading to mitophagy. Dysregulation of mitophagy results in increased oxidative stress, which has been described as characteristic of PD. Therefore, inhibition of USP30 may be a potential strategy for treating PD. For example, inhibiting USP30 could treat PD patients who compensate for mutations in parkin that lead to reduced activity.

[0008] Reports indicate that USP30 depletion enhances mitophagic clearance of mitochondria and also enhances perkinin-induced cell death. USP30 has also been shown to regulate Bax / Bak-dependent apoptosis in a manner independent of perkinin overexpression. USP30 depletion sensitizes cancer cells to BH-3 mimics such as ABT-737 without requiring perkinin overexpression. Therefore, the anti-apoptotic effect of USP30 has been demonstrated, making it a potential target for anticancer therapy.

[0009] After approving the proteasome inhibitor bortezomib Following its use in the treatment of multiple myeloma, the ubiquitin-proteasome system has gained attention as a target for cancer therapy. Prolonged treatment with bortezomib is limited by its associated toxicities and resistance. However, therapeutic strategies that predict and target specific aspects of the ubiquitin-proteasome pathway upstream (e.g., DUB) are better tolerated (Bedford et al., 2011, Nature Rev 10:29-46).

[0010] Fibrotic diseases, including kidney, liver, and lung fibrosis, are leading causes of morbidity and mortality and can affect all tissue and organ systems. Fibrosis is considered a result of acute or chronic stress on a tissue or organ, characterized by extracellular matrix deposition, reduced vascular / tubule / duct / airway patency, and impaired function, ultimately leading to organ failure. Many fibrotic conditions are promoted by lifestyle or environmental factors; however, some fibrotic conditions can be triggered by genetic triggers or are actually considered idiopathic (i.e., without a known cause). Some fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), can be treated with nonspecific kinase inhibitors (nintedanib) or drugs with poorly characterized mechanisms of action (pirfenidone). Other treatments for organ fibrosis (e.g., kidney or liver fibrosis) reduce stress on the organ itself (e.g., beta-blockers for cirrhosis, angiotensin receptor blockers for chronic kidney disease). Note that lifestyle factors (e.g., glucose and dietary control) can also influence the course and severity of the disease.

[0011] Mitochondrial dysfunction is involved in many fibrotic diseases, and downstream of oxidative stress, this dysfunction is a key pathogenic mediator, along with reduced ATP production. In preclinical models, disruption of the mitophagy pathway (through mutations or knockouts of Parkinson's protein or PINK1) exacerbates pulmonary and renal fibrosis, with evidence of increased oxidative stress.

[0012] Kurita et al., 2017, *Respiratory Research* 18:114, reported that the accumulation of pro-fibrotic myofibroblasts is a key process in fibrotic remodeling during IPF. Recent findings suggest that autophagy / mitochondrial autophagy (part of the lysosomal degradation mechanism) is involved in the pathogenesis of IPF, and that mitophagy is associated with myofibroblast differentiation by regulating mitochondrial reactive oxygen species (ROS)-mediated platelet-derived growth factor receptor (PDGFR) activation. Kurita's results suggest that pirfenidone induces PARK2-mediated mitophagy and inhibits pulmonary fibrosis development in the presence of insufficient mitophagy, which could at least partially explain the anti-fibrotic mechanism of IPF treatment.

[0013] Williams et al., 2015, Pharmacol Res. Dec; 102: 264-269, discussed the role of Pink 1-perkinin-mediated autophagy in protecting against alcohol- and acetaminophen-induced liver injury by removing damaged mitochondria via mitophagy. It suggests that pharmacological stabilization of USP8 or inactivation of USP15 and USP30 may be potential therapeutic targets for upregulating perkinin-induced mitophagy, thereby protecting against drug-induced liver injury. However, noting that DUB is regulated both transcriptionally and posttranslatively, this may make drug development for targeting these specific enzymes challenging; furthermore, phosphorylated ubiquitin was shown to be resistant to DUB. The authors concluded that upregulating PINK1 stabilization or kinase activity may be a more effective target than inhibiting DUB.

[0014] Williams et al., 2015, Biomolecules 5, 2619-2642 and Williams et al., 2015, Am JPhysiol Gastrointest Liver Physiol 309: G324–G340 reviewed the mechanisms involved in regulating mitochondrial homeostasis in the liver and how these mechanisms protect against alcohol-induced liver disease.

[0015] Luciani et al., 2020, Nat. Commun. 11, 970, reported that dysregulation of the mitochondrial reticulum in terminally differentiated cells leads to a broad spectrum of diseases, including methylmalonic acidemia (MMA). MMA is one of the most common inherited metabolic disorders caused by a deficiency of mitochondrial methylmalonyl-CoA mutase (MMUT). MMUT deficiency induces metabolic and mitochondrial alterations, which are exacerbated by abnormalities in Pink1 / Parkinin-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cellular damage. This study demonstrates an association between primary MMUT deficiency, diseased mitochondria, impaired mitophagy, and epithelial stress, and offers potential therapeutic prospects for MMA.

[0016] Kluge et al., Bioorganic & Medicinal Chemistry Letters, 2018, 282655-2659, reported that selective inhibitors of USP30 accelerate mitophagy.

[0017] Apply for WO 2016 / 046530 (US 15 / 513125, US 15 / 894025, US 16 / 448066), WO2016 / 156816 (US 15 / 558632, US 16 / 297937, US 16 / 419558, US 16 / 419747, US) in PCT 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 Several series of N-cyano-substituted heterocyclic derivatives as deubiquitinase inhibitors are disclosed in 2018 / 234755 (US 16 / 615709), WO 2020 / 212350, WO 2020 / 212351, WO 2021 / 043870 and PCT / EP2021 / 064166, each of which is expressly incorporated herein by reference. PCT application WO 2019 / 171042 (US 16 / 977019) discloses the use of N-cyanopyrrolidine compounds as USP30 inhibitors for the treatment of fibrotic diseases, which is also expressly incorporated herein by reference.

[0018] Falgueyret et al., 2001, J. Med. Chem. 44, 94-104 and PCT application WO 01 / 77073 relate to cyanopyrrolidine compounds as inhibitors of cathepsins K and L, with potential use in the treatment of osteoporosis and other bone resorption-related conditions. PCT application WO 2015 / 179190 relates to N-acylethanolamine hydrolase 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 use in the treatment of cancer, neurodegenerative diseases, inflammatory conditions, and viral infections. PCT applications WO 2015 / 017502 and WO 2016 / 019237 relate to inhibitors of Bruton's tyrosine kinase, which have potential use in the treatment of diseases such as autoimmune diseases, inflammatory diseases, and cancer. PCT applications WO 2009 / 026197, WO 2009 / 129365, WO 2009 / 129370, and WO 2009 / 129371 relate to cyanopyrrolidine compounds as cathepsin C inhibitors, which have potential use in the treatment of COPD. 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 (April 20, 2020), and Tsefou et al., bioRxiv 2021.02.02.429344 (February 2, 2021) relate to cyanamide-containing compounds as USP30 inhibitors. Yue et al., 2014, Cell Research, 24, 482-496, relate to the diterpenoid derivative 15-oxospironolone as a USP30 inhibitor that induces mitochondrial fusion.

[0019] PCT application WO 2015 / 183987 relates to a pharmaceutical composition comprising a deubiquitinase inhibitor and human serum albumin, used in the treatment of cancer, fibrosis, autoimmune diseases or conditions, inflammatory diseases or conditions, neurodegenerative diseases or conditions, or infections. It should be noted that deubiquitinases, including UCHL5 / UCH37, USP4, USP9X, USP11, and USP15, are said to be involved in the regulation of the TGF-β signaling pathway, the disruption of which causes neurodegenerative and fibrotic diseases, autoimmune dysfunction, and cancer.

[0020] PCT application WO 2006 / 067165 relates to a method for treating fibrotic diseases using dihydroindole kinase inhibitors. PCT application WO 2007 / 119214 relates to a method for treating early pulmonary fibrosis using endothelin receptor antagonists. PCT application WO 2012 / 170290 relates to a method for treating fibrotic diseases using THC acid. PCT application WO 2018 / 213150 relates to sulfonamide USP30 inhibitors with potential utility in treating conditions involving mitochondrial defects. Larson-Casey et al., 2016, Immunity 44, 582-596, discuss macrophage Akt1 kinase-mediated mitophagy, apoptosis resistance, and pulmonary fibrosis. Tang et al., 2015, Kidney Diseases 1, 71-79, review the potential role of mitophagy in renal pathophysiology.

[0021] 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 the various symptoms and conditions associated therewith. While not wishing to be bound by any particular theory or mechanism, it is believed that the compounds of the present invention act as inhibitors of the enzyme USP30, which in turn upregulates perkinin-induced mitophagy.

[0022] Acute kidney injury (AKI) is defined as a sudden decline in kidney function occurring within 7 days or less, graded based on an increase in serum creatinine (SCr) and a decrease in urine output, as described in the Kidney Disease Improving Global Outcomes (KDIGO) guidelines. AKI occurs in approximately 13.3 million people annually, 85% of whom live in developing countries, and is estimated to cause approximately 1.7 million deaths annually (Mehta et al., 2015, Lancet 385(9987): 2616-2643). AKI is more likely to result in permanent kidney damage (i.e., chronic kidney disease, CKD) and can also lead to damage to non-renal organs. AKI is a significant public health concern, especially when considering the absolute number of patients experiencing incidental CKD, progressive CKD, end-stage renal disease, and cardiovascular events. AKI was found to be prevalent in patients hospitalized for COVID-19 and was closely associated with in-hospital mortality. Mitochondrial damage and dysfunction have been reported as potential pathophysiological mechanisms and therapeutic targets (Kellum et al., Nephrol Dial Transplant (2020) 35: 1652–1662).

[0023] AKI and CKD are considered continuums on the same disease spectrum (Chawla et al., 2017, Nat Rev Nephrol 13(4): 241-257). Patients undergoing coronary artery bypass grafting (CABG) are at high risk of kidney injury. There is a significant unmet medical need in developing pharmaceutical products for the treatment and / or prevention of AKI.

[0024] The kidney is a metabolically demanding site, exhibiting a high rate of mitochondrial autophagy in vivo (McWilliams et al., 2018, Cell Metab 27(2): 439-449e435). Proximal tubular epithelial cells (RPTECs), a cell type with significant ATP requirements for solute / ion exchange, are rich in mitochondria and are the primary effector cells in acute kidney injury (AKI). Mitochondrial dysfunction is involved in AKI / CKD mechanisms, both confirmed by multiple lines of evidence from preclinical AKI and CKD models and by data demonstrating abnormal mitochondrial phenotypes in patient biopsies (Emma et al., 2016, Nat RevNephrol 12(5): 267-280; Eirin et al., 2017, Handb Exp Pharmacol 240: 229-250). In addition, primary mitochondrial diseases often manifest as renal symptoms in patients with MELAS / MIDD, such as focal segmental glomerulosclerosis (Kawakami et al., 2015, J Am Soc Nephrol 26(5): 1040-1052), and also as primary tubular lesions in patients with coenzyme Q deficiency. Mutations in mtDNA can cause maternally inherited tubulointerstitial diseases (Connor et al., 2017, PLoS Genet 13(3): e1006620).

[0025] Mitochondrial quality control in kidney injury (Tang et al., 2018, Autophagy 14(5): 880-897) demonstrated that in both Pink1 KO and Park2 KO mice, kidney injury was exacerbated after ischemic AKI, suggesting that Pink1 / Parkinin-mediated mitophagy plays a protective role in renal IRI. Furthermore, Parkin / PINK1 mitophagy protects against cisplatin-induced kidney injury (Wang et al., 2018, Cell Death Dis 9(11): 1113). Limited CKD models are available for mitophagy studies, and supporting evidence for mitochondrial quality control in fibrosis comes from studies of fibrotic lung diseases such as COPD and IPF. Parkin knockout animals showed exacerbated pulmonary fibrosis in response to bleomycin (Kobayashi et al., 2016, JImmunol, 197: 504-516). Similarly, airway epithelial cells from Parkin knockout (KO) animals showed an enhanced fibrotic and aging response to cigarette smoke (Araya et al., 2019, Autophagy 15(3): 510-526).

[0026] Preclinical models can be used to investigate potential novel therapeutics by mimicking fibrotic pathology (e.g., collagen deposition) consistent with human conditions. Preclinical models can be toxin-mediated (e.g., bleomycin for pulmonary and skin fibrosis), surgical (e.g., ischemia / reperfusion injury models and unilateral ureteral obstruction models for acute tubulointerstitial fibrosis), and genetic (e.g., diabetic (db / db) mice for diabetic nephropathy). For example, two previously given examples of specific IPF treatments (nintedanib and pirfenidone) have shown efficacy in bleomycin-induced pulmonary fibrosis models.

[0027] Therefore, there is a need for compounds that act as USP30 inhibitors for the treatment or prevention of conditions in which USP30 is adapted to be inhibited. In particular, there is a need for USP30 inhibitors that possess suitable and / or improved properties to maximize efficacy against the target disease. Invention Overview

[0029] This invention relates to compounds of formula (I), selected from formula (I)(i) and formula (I)(ii):

[0030]

[0031] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0032] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0033] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0034] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0035] The present invention also relates to the use of compounds of formula (I), particularly their use in the treatment of conditions involving mitochondrial dysfunction, cancer and fibrosis, as well as methods for their preparation and pharmaceutical compositions comprising said compounds. Invention Details

[0037] This invention relates to USP30 inhibitors having suitable and / or improved properties to maximize efficacy against target diseases. These properties include, for example, potency, selectivity, physicochemical properties, ADME (absorption, distribution, metabolism, and excretion) properties, including PK (pharmacokinetic) characteristics, and safety characteristics.

[0038] It is generally desirable to maximize the potency of a drug molecule against the target enzyme in relevant assays in order to reduce the effective / efficacy dose administered to the patient. The USP30 affinity of the compounds of this invention can be tested using the in vitro biochemical fluorescence polarization (FP) assay described in this application.

[0039] USP30 is a transmembrane protein located in the outer membrane of mitochondria, energy-producing organelles within the cell. Therefore, the ability to demonstrate cellular activity in vitro is advantageous, as it is one of many components that can indicate a greater capacity to bind to a target in its physiological environment (i.e., where a USP30 inhibitor compound can penetrate the cell). The USP30 Western blot (WB) assay described in this application aims to test the activity of a compound against USP30 in cells by monitoring USP30 activity using an irreversible active probe. Similar to the Western blot assay, target binding can be assessed (in vitro) from brain or kidney tissue samples from animals to which the compound has been administered.

[0040] To extend target-binding knowledge to downstream pharmacodynamics, TOM20 (mitochondrial outer membrane protein) ubiquitination can be evaluated.

[0041] Generally, it is important that the drug has the highest possible selectivity for its desired target enzyme; other activities may lead to side effects. The exact physiological functions of many DUBs are not yet fully determined; however, regardless of what these DUBs may or may not have, ensuring that any drug is selective for the relevant mechanism target with an unknown physiological function is a reasonable medicinal chemistry approach. Representative examples of DUB enzymes that can be used to screen the compounds of this invention 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, and USP1 / UA. F1, 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 present invention exhibit good selectivity for USP30 relative to one or more of these DUB enzymes.

[0042] In addition to selectivity relative to other DUB enzymes, it is important that the drug has low affinity for other targets and that pharmacological profiling can be performed on the target group to assess the likelihood of potential off-target effects and minimize them. Examples of targets that can be used to screen the compounds of the present invention are those in the industry-standard Eurofins-CerepSafetyScreen44 group, which includes 44 targets as a representative selection of GPCR receptors, transport proteins, ion channels, nuclear receptors, and kinases and non-kinases. Preferably, the compounds of the present invention have no meaningful affinity for the targets in this screening group. Other examples of targets that can be used to screen the compounds of the present invention are kinases in the Thermo Fisher SelectScreen kinase profiling group, which includes 39 targets as a representative selection of kinases. Preferably, the compounds of the present invention have no meaningful affinity for the targets in this screening group. Additionally, examples of specific enzyme classes that can be used to screen the compounds of the present invention are cathepsins (e.g., cathepsins A, B, C, H, K, L, L2, S, V, and Z). Preferably, the compounds of the present invention have good selectivity for USP30 relative to one or more of these enzymes.

[0043] Compounds also need to possess favorable pharmacokinetic properties to make them suitable for oral administration. Orally administered drugs should have good bioavailability; this is the ability to easily cross the gastrointestinal (GI) tract and not undergo extensive metabolism as they pass through the GI tract into systemic circulation. Once a drug enters systemic circulation, the metabolic rate is also important in determining the duration of drug residence in the body.

[0044] Therefore, it is clearly advantageous for drug molecules to have the property of readily crossing the gastrointestinal tract and being metabolized only slowly in vivo. The Caco-2 assay is a widely accepted model for predicting the ability of a given molecule to cross the gastrointestinal tract. The majority of the metabolism of drug molecules typically occurs in the liver, and in vitro assays using whole-cell hepatocytes (animal or human) are a widely accepted method for measuring the sensitivity of a given molecule to metabolism in the liver. These assays aim to predict in vivo clearance from clearance values ​​calculated from hepatocytes.

[0045] Compounds with good Caco-2 flux and hepatocyte stability are predicted to have good oral bioavailability (good absorption via the gastrointestinal tract and minimal extraction of the compound as it passes through the liver) and long residence time in the body, which is sufficient to make the drug effective.

[0046] The solubility of a compound is a crucial factor in determining the drug concentration required to achieve the desired pharmacological response in systemic circulation. Low water solubility is a challenge in the formulation development of novel chemical entities, and drugs must exist in solution form at the absorption site for absorption. The kinetic solubility of a compound can be measured using turbidimetric solubility assays, and the resulting data can be combined with Caco-2 permeability data to predict dose-dependent human intestinal absorption.

[0047] Other parameters that can be measured using standard assays that indicate the exposure curve of a compound include, for example, plasma stability (half-life measurement), blood AUC, C... max C min and T max value.

[0048] Treatment of CNS diseases (including Alzheimer's disease, Parkinson's disease, and other diseases described in this application) requires drug molecules to target the brain, which necessitates adequate penetration of the blood-brain barrier. Therefore, effective blood-brain penetration properties and the provision of adequate residence time in the brain are required for effective USP30 inhibitors. The probability that a compound can cross the blood-brain barrier can be measured by in vitro flux assays using an MDR1-MDCK cell monolayer (MDR-1-transfected Madin-Darby canine kidney cells, resulting in overexpression of the human efflux transporter P-glycoprotein). Alternatively, exposure can be measured directly in the brain and plasma using in vivo animal models.

[0049] Compounds with favorable safety profiles are also required, and these profiles can be measured using various standard in vitro and in vivo methods. Cytotoxicity-based reverse screening can be used with alamarblue in response to mitochondrial activity. TM The antiproliferative / cytotoxic effects of halogenated ...

[0050] Toxicological and safety studies can also be conducted to identify potential target organs for adverse effects and define therapeutic indices to set initial starting doses in clinical trials. Regulatory requirements typically necessitate studies in at least two laboratory animal species, one rodent (rats or mice) and one non-rodent (rabbit, dog, non-human primate, or other suitable species).

[0051] The mutagenic properties of the compounds of the present invention can be assessed using the bacterial reverse mutagenesis assay (Ames test), typically by using the bacterial strain *Salmonella typhimurium*, a mutant strain used for the biosynthesis of the amino acid histidine.

[0052] The small nucleus assay can be used to determine whether a compound is genotoxic by assessing the presence of a small nucleus. A small nucleus can contain chromosome fragments produced by DNA breakage (breakdown agents) or whole chromosomes produced by disruption of the mitotic apparatus (aneuploidy).

[0053] hERG predictive assays provide valuable information about the potential binding of the tested compound to potassium channels and QT prolongation on echocardiography. Inhibition of hERG currents causes QT interval prolongation, leading to potentially fatal ventricular tachyarrhythmias (torsades de pointes). Typically, assay data can be generated from automated patch-clamp measurement platforms.

[0054] Therefore, this invention relates to USP30 inhibitors having suitable and / or improved properties to maximize efficacy against the target disease. Such properties include, for example, potency, selectivity, physicochemical properties, ADME (absorption, distribution, metabolism, and excretion) properties, including PK (pharmacokinetic) characteristics, and safety characteristics.

[0055] The compounds of the present invention have been found to exhibit one or more of the properties identified above, which are significant and unexpected. For example, Examples 1-12 of the present invention are highly effective against USP30, as measured in the biochemical assays described in this application. All of these Examples (1-12) of the present invention are significantly more selective for USP30 compared to other DUBs and cathepsins.

[0056] The remarkable and unexpected properties of the compounds of this invention make them particularly suitable for the treatment and / or prevention of diseases associated with USP30 activity.

[0057] According to a first aspect, the present invention provides compounds of formula (I), selected from formula (I)(i) and formula (I)(ii):

[0058]

[0059] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0060] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0061] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0062] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0063] The compound of formula (I) exists as a single stereoisomer having the absolute stereochemistry shown.

[0064] Alkyl groups can be straight-chain or branched and contain 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and sec-butyl.

[0065] Halogens refer to fluorine, chlorine, bromine, or iodine, especially fluorine or chlorine. Fluoroalkyl groups may contain one or more fluorine substituents. Examples are fluoromethyl, difluoromethyl, and trifluoromethyl.

[0066] Unless otherwise stated, the term substitution means substitution by one or more defined groups. Where the groups can be selected from more than one alternative option, the selected groups can be the same or different. The term "independently" means that if more than one substituent is selected from more than one possible substituent, those substituents can be the same or different.

[0067] The preferred embodiments of the compound of formula (I) are defined as follows.

[0068] Preferred, R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

[0069] More preferably, R 1 Selected from methyl, CH2F and CH2OCH3.

[0070] Optimal choice, R 1Selected from methyl and CH2OCH3.

[0071] Preferred, R 2 Selected from methyl, CF3 and cyclopropyl.

[0072] Optimal choice, R 2 Selected from methyl and cyclopropyl.

[0073] Preferred, R 3 Selected from hydrogen, chlorine, and fluorine.

[0074] More preferably, R 3 Selected from hydrogen and fluorine.

[0075] Optimal choice, R 3 It is hydrogen.

[0076] Preferred, R 4 and R 5 They are each independently selected from hydrogen and fluorine.

[0077] Optimal choice, R 4 and R 5 Each is hydrogen.

[0078] According to one aspect of the invention, the compound of formula (I) has formula (I)(i):

[0079]

[0080] According to another aspect of the invention, the compound of formula (I) has formula (I)(ii):

[0081]

[0082] A first preferred aspect of the invention is a compound of formula (IA)(i):

[0083]

[0084] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0085] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0086] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0087] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0088] Preferred embodiments of the compounds of formula (IA)(i) are defined as follows.

[0089] Preferred, R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

[0090] More preferably, R 1 Selected from methyl, CH2F and CH2OCH3.

[0091] Optimal choice, R 1 Selected from methyl and CH2OCH3.

[0092] Preferred, R 2 Selected from methyl, CF3 and cyclopropyl.

[0093] Optimal choice, R 2 Selected from methyl and cyclopropyl.

[0094] Preferred, R 3 Selected from hydrogen, chlorine, and fluorine.

[0095] More preferably, R 3 Selected from hydrogen and fluorine.

[0096] Optimal choice, R 3 It is hydrogen.

[0097] Preferred, R 4 and R 5 They are each independently selected from hydrogen and fluorine.

[0098] Optimal choice, R 4 and R 5 Each is hydrogen.

[0099] The preferred compounds of formula (IA)(i) of the present invention are selected from:

[0100] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0101] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0102] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0103] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0104] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0105] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0106] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and

[0107] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0108] Or its pharmaceutically acceptable salt.

[0109] The most preferred compounds of formula (IA)(i) of the present invention are selected from:

[0110] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and

[0111] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0112] Or its pharmaceutically acceptable salt.

[0113] A second preferred aspect of the invention is a compound of formula (IB)(i):

[0114]

[0115] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0116] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0117] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0118] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0119] The preferred embodiments of the compound of formula (IB)(i) are defined as follows.

[0120] Preferred, R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

[0121] More preferably, R 1 Selected from methyl, CH2F and CH2OCH3.

[0122] Optimal choice, R 1 It is CH2OCH3.

[0123] Preferred, R 2 Selected from methyl, CF3 and cyclopropyl.

[0124] Optimal choice, R 2 It is a methyl group.

[0125] Preferred, R 3 Selected from hydrogen, chlorine, and fluorine.

[0126] More preferably, R 3 Selected from hydrogen and fluorine.

[0127] Optimal choice, R 3 It is hydrogen.

[0128] Preferred, R 4 and R 5 They are each independently selected from hydrogen and fluorine.

[0129] Optimal choice, R 4 and R 5 Each is hydrogen.

[0130] The preferred compounds of formula (IB)(i) of the present invention are selected from:

[0131] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; and

[0132] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0133] Or its pharmaceutically acceptable salt.

[0134] A third preferred aspect of the invention is a compound of formula (IA)(ii):

[0135]

[0136] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0137] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0138] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0139] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0140] Preferred embodiments of the compounds of formula (IA)(ii) are defined as follows.

[0141] Preferred, R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

[0142] More preferably, R 1 Selected from methyl, CH2F and CH2OCH3.

[0143] Optimal choice, R 1 Selected from methyl and CH2OCH3.

[0144] Preferred, R 2 Selected from methyl, CF3 and cyclopropyl.

[0145] Optimal choice, R 2 Selected from methyl and cyclopropyl.

[0146] Preferred, R 3 Selected from hydrogen, chlorine, and fluorine.

[0147] More preferably, R 3 Selected from hydrogen and fluorine.

[0148] Optimal choice, R 3 It is hydrogen.

[0149] Preferred, R 4 and R 5 They are each independently selected from hydrogen and fluorine.

[0150] Optimal choice, R 4 and R 5 Each is hydrogen.

[0151] The preferred compounds of formula (IA)(ii) of the present invention are selected from:

[0152] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0153] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0154] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0155] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0156] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0157] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0158] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and

[0159] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0160] Or its pharmaceutically acceptable salt.

[0161] A fourth preferred aspect of the invention is a compound of formula (IB)(ii):

[0162]

[0163] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0164] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0165] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0166] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0167] Preferred embodiments of the compounds of formula (IB)(ii) are defined as follows.

[0168] Preferred, R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

[0169] More preferably, R 1 Selected from methyl, CH2F and CH2OCH3.

[0170] Optimal choice, R 1 It is CH2OCH3.

[0171] Preferred, R 2 Selected from methyl, CF3 and cyclopropyl.

[0172] Optimal choice, R 2 It is a methyl group.

[0173] Preferred, R 3 Selected from hydrogen, chlorine, and fluorine.

[0174] More preferably, R 3 Selected from hydrogen and fluorine.

[0175] Optimal choice, R 3 It is hydrogen.

[0176] Preferred, R 4 and R 5 They are each independently selected from hydrogen and fluorine.

[0177] Optimal choice, R 4 and R 5 Each is hydrogen.

[0178] The preferred compounds of formula (IB)(ii) of the present invention are selected from:

[0179] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; and

[0180] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0181] Or its pharmaceutically acceptable salt.

[0182] Pharmaceutically acceptable salts of compounds of formula (I) include their acid addition salts and basic salts (including disalts).

[0183] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates, camphorsulfonates, citrates, ethanedisulfonates, ethanesulfonates, fumarates, glucoheponicates, glucurons, glucuronates, phenacetinates, hydrochlorides / chlorides, hydrobromides / bromines, hydroiodates / iodides, hydrogen phosphates, hydroxyethyl sulfonates, D-lactates and L-lactates, malates, maleates, malonates, methanesulfonates, methyl sulfates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, palmitates, phosphates, glycosides, stearates, succinate sulfates, D-tartrates and L-tartrates, and toluenesulfonates.

[0184] Suitable base salts are formed from bases that form non-toxic salts. Examples include aluminum salts, ammonium salts, arginine salts, N,N'-dibenzylethylenediamine salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts.

[0185] For a review of suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, Weinheim, Germany (2002).

[0186] Pharmaceutically acceptable salts of compounds of formula (I) can be readily prepared by mixing a solution of the compound with the desired acid or base (as the case may be). The salt can be precipitated from the solution and collected by filtration, or it can be recovered by evaporating the solvent.

[0187] Pharmaceutically acceptable solvates according to the invention include hydrates and solvates, wherein the solvent for crystallization may be isotopically substituted, such as D2O, acetone-d6, DMSO-d6.

[0188] Furthermore, within the scope of this invention are cage-like inclusion complexes and drug-host inclusion complexes, wherein, unlike the solvates described above, the drug and host are present in non-stoichiometric amounts. For a review of such complexes, see Haleblian’s J. Pharm Sci, 64(8), 1269-1288 (August 1975).

[0189] All references to compounds of formula (I) below include references to their salts, as well as solvates and cage-like inclusion compounds of compounds of formula (I) and their salts.

[0190] This invention includes all polymorphs of compounds of formula (I) as defined above.

[0191] Furthermore, within the scope of this invention are so-called "prodrugs" of compounds of formula (I). Thus, certain derivatives of compounds of formula (I) that themselves have little or no pharmacological activity can, upon being administered or metabolized in the body, produce compounds of formula (I) with the desired activity. Such derivatives are referred to as "prodrugs".

[0192] The prodrug according to the invention can be prepared, for example, by using suitable functional groups present in the compound of formula (I), which are known to those skilled in the art as “pre-parts” as described in, for example, H. Bundgaard’s “Design of Prodrugs” (Elsevier, 1985).

[0193] Ultimately, some compounds of formula (I) can be used as prodrugs for other compounds of formula (I).

[0194] Certain derivatives of compounds of formula (I) containing nitrogen atoms can also form corresponding N-oxides, and such compounds are also within the scope of this invention.

[0195] All tautomer forms of compounds of formula (I) are included within the scope of this invention.

[0196] Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemic mixture (or racemic mixture of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). Alternatively, the racemic mixture (or racemic precursor) can be reacted with a suitable optically active compound (e.g., an alcohol), or, in the case where the compound of formula (I) contains an acidic or basic moiety, with a base or acid (e.g., 1-phenylethylamine or tartaric acid). The resulting mixture of diastereomers can be separated by chromatography and / or fractional crystallization, and one or both diastereomers can be converted to the corresponding pure enantiomers by methods well known to those skilled in the art. The chiral compounds (and their chiral precursors) of the present invention can be obtained enantiomerically enriched on an asymmetric resin using chromatography (typically HPLC), with a mobile phase consisting of hydrocarbons (typically heptane or hexane) containing 0-50% by volume propan-2-ol (typically 2% to 20%) and 0-5% by volume alkylamines (typically 0.1% diethylamine). Concentrating the eluent yields the enriched mixture. The present invention includes all crystalline forms of compounds of formula (I), including their racemic forms and racemic mixtures (aggregates). Stereoimeric aggregates can be separated using conventional techniques known to those skilled in the art—see, for example, "Stereochemistry of Organic Compounds" by Elle Liel and S. Wilen (Wiley, New York, 1994).

[0197] The compound of formula (I) is subjected to R 1 The substituted pyrrolidine ring contains two chiral centers on its carbon atoms, and the amide and the stereocenter can exist in either (R) or (S) configurations. According to IUPAC nomenclature, the absolute configurations (R) and (S) of the stereoisomers are designated based on the nature of the substituents and the application of the cis-rule procedure. Therefore, compounds of formula (I) can exist in four stereoisomeric forms.

[0198] The compound of formula (I) of the present invention exists as a single stereoisomer. The pyrrolidine carbon atom of the amide substituent exists as the (R)-stereocenter, while R... 1 The designation of the pyrrolidine carbon atom of the group depends on the nature of the substituent. The compound of formula (I) is isolated as a single stereoisomer and may be present in 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% of the stereoisomer.

[0199] In R 1 An additional chiral center may be present in the compound of formula (I) instead of the basic body. All such stereoisomers of the compound of formula (I) are included within the scope of this invention.

[0200] The present invention also includes all pharmaceutically acceptable isotopic variants of compounds 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 a different atomic mass than that found in nature.

[0201] Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of the following atoms: hydrogen, for example... 2 H and 3 H; carbon, such as 13 C and 14 C; Nitrogen, such as 15 N; oxygen, for example 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.

[0202] Replacing the compounds of the present invention with isotopes such as deuterium can provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and may therefore be preferred in some cases.

[0203] Certain isotopic variants of compounds of formula (I), such as those incorporating radioactive isotopes, can be used for drug and / or substrate tissue distribution studies. Radioactive isotopes tritium and... 14 C is particularly suitable for this purpose due to its ease of incorporation and readily available detection methods.

[0204] Isotopic variants of compound (I) can typically be prepared using conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples and preparation examples, using appropriate isotopic variations of suitable reagents.

[0205] The compound of formula (I) is an inhibitor of the deubiquitinase USP30.

[0206] According to another aspect, the present invention provides compounds of formula (I) as defined in this application, their tautomers, or pharmaceutically acceptable salts of said compounds or tautomers, which are used as pharmaceuticals.

[0207] According to another aspect, the present invention provides a method for treating or preventing a disease or condition in mammals in which inhibition of USP30 is known or may produce a beneficial effect, comprising administering to the mammal a therapeutically effective amount of a compound of formula (I) as defined in this application, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

[0208] According to another aspect, the present invention provides the use of a compound of formula (I) as defined in this application, its tautomers, or a pharmaceutically acceptable salt of said compound or tautomer in the preparation of a medicament for treating or preventing a condition or disease in which inhibition of USP30 is known or may produce a beneficial effect. The preparation of the medicament may in particular include the chemical synthesis of a compound of formula (I) or a salt thereof, or the preparation of a composition or formulation comprising said compound or salt, or the packaging of any medicament comprising said compound.

[0209] According to another aspect, the present invention provides a method for inhibiting USP30 in a patient, comprising administering to the patient a therapeutically effective amount of a compound of formula (I) as defined in this application, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

[0210] Conditions or illnesses that benefit from USP30 activity are selected from those involving mitochondrial dysfunction, cancer, and fibrosis.

[0211] In a preferred embodiment of all aspects of the invention, the condition or disease that benefits from USP30 activity is a condition involving mitochondrial dysfunction.

[0212] Mitochondrial dysfunction is caused by defects in mitochondria, specialized compartments present in every cell of the body except red blood cells. When mitochondria fail, less and less energy is produced within the cell, leading to cell damage or even cell death. If this process is repeated throughout the body, the life of the affected individual is severely impaired. Mitochondrial diseases most commonly occur in organs that require high levels of energy, such as the brain, heart, liver, skeletal muscle, kidneys, and the endocrine and respiratory systems.

[0213] Disorders involving mitochondrial dysfunction can be selected from those involving defects in mitophagy, those involving mitochondrial DNA mutations, those involving mitochondrial oxidative stress, those involving defects in mitochondrial membrane potential or mitochondrial biogenesis, those involving defects in mitochondrial shape or morphology, and those involving defects in lysosomal storage.

[0214] Specifically, conditions involving mitochondrial dysfunction can be selected from neurodegenerative diseases; multiple sclerosis (MS); mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome; maternally inherited diabetes and deafness (MIDD); Leber hereditary optic neuropathy (LHON); cancers (including, for example, 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, bone cancer, or other cancers of tissues and organs and cancers of blood cells, such as lymphoma). (and leukemia, multiple myeloma, metastatic cancer, osteosarcoma, chondrosarcoma, Ewing sarcoma, nasopharyngeal carcinoma, colorectal cancer and non-small cell lung cancer); neurological disorders, ataxia, retinitis pigmentosa, maternally inherited Leydig syndrome (NARP-MILS); Danon's disease; diabetes; diabetic nephropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; mental illnesses such as schizophrenia; polysulfatase deficiency (MSD); mucolipidemia II (MLII); mucolipidemia III (MLII) III); Mucolipidosis IV (ML IV); GM1-ganglioside storage disease (GM1); Neuronal ceroid lipofuscin deposition disease (NCL1); Alpers' disease; Barth syndrome; β-oxidation deficiency; Carnitine-acyl-carnitine deficiency; Carnitine deficiency; Creatine deficiency syndrome; Coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; Complex V deficiency; COX deficiency; Chronic progressive lateral oculomotor palsy (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency (LCHAD); Leishman's disease or syndrome; French-Canadian variant of Leishman's syndrome (LSFC); lethal infantile cardiomyopathy (LIC); Luft disease; medium-chain acyl-CoA dehydrogenase deficiency (MCAD); myoclonic epilepsy and broken red fiber (MERRF) syndrome; mitochondrial cell disease; mitochondrial recessive ataxia syndrome; mitochondrial... DNA deletion syndromes; myoneurotic and cerebrovascular diseases and encephalopathy; Pearson syndrome; pyruvate dehydrogenase deficiency; pyruvate carboxylase deficiency; POLG mutation; medium / short chain 3-hydroxyacyl-CoA dehydrogenase (M / SCHAD) deficiency; very long chain acyl-CoA dehydrogenase (VLCAD) deficiency; peroxisome dysfunction; methylmalonic acidemia; mevalonate kinase deficiency; age-dependent decline in cognitive function and muscle strength; and cognitive impairment associated with neurodegeneration and neuropsychiatric disorders.

[0215] Conditions involving mitochondrial dysfunction can be CNS conditions, such as neurodegenerative diseases.

[0216] Neurodegenerative diseases include, but are not limited to, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, ischemia, stroke, Lewy body dementia, multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical-basal degeneration (CBD), and frontotemporal dementia.

[0217] In particular, the compounds of the present invention can be used to treat or prevent Parkinson's disease, including but not limited to PD associated with mutations in α-synuclein, Parkinin, PINK1, GBA and LRRK2, as well as autosomal recessive juvenile Parkinson's disease (AR-JP) or early-onset Parkinson's disease (EOPD), wherein Parkinin or PINK1 is mutated, truncated or deleted.

[0218] In particular, the compounds of the present invention can be used to treat cognitive deficits associated with neurodegenerative and neuropsychiatric diseases, including, for example, cognitive deficits associated with Alzheimer's disease and Parkinson's disease, preclinical or precursor forms of AD and PD, Huntington's disease, Lewy body dementia, and cognitive deficits associated with schizophrenia, mood disorders, bipolar disorder, and major depressive disorder.

[0219] When used to treat or prevent conditions involving mitochondrial dysfunction, the compounds or pharmaceutical compositions of the present invention as described in this application may be combined with one or more additional agents. These agents may be selected from levodopa, dopamine agonists, monoamine oxygenase (MAO)B inhibitors, catechol O-methyltransferase (COMT) inhibitors, anticholinergic drugs, riluzole, amantadine, cholinesterase inhibitors, memantine, buphenazine, antipsychotics, diazepam, clonazepam, antidepressants, and anticonvulsants. The compounds may also be combined with agents that reduce / remove pathogenic protein aggregates in neurodegenerative diseases, such as agents that reduce / remove α-synuclein in Parkinson's disease, multiple system atrophy, or Lewy body dementia; agents that reduce / remove tau in Alzheimer's disease or progressive supranuclear palsy; and agents that reduce / remove TDP-43 in ALS or frontotemporal dementia.

[0220] In another preferred embodiment of all aspects of the invention, the disease or condition that benefits from USP30 activity is cancer. Cancer may be associated with mitochondrial dysfunction. Preferred cancers include, for example, 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, bone cancer, or other cancers of tissues and organs, and cancers of blood cells, such as lymphoma and leukemia, multiple myeloma, metastatic cancer, osteosarcoma, chondrosarcoma, Ewing sarcoma, nasopharyngeal carcinoma, colorectal cancer, and non-small cell lung cancer.

[0221] In particular, the compounds of the present invention can be used to treat or prevent cancers in which apoptosis pathways are dysregulated, and more particularly, the compounds of the present invention can be used to treat or prevent cancers in which BCL-2 family proteins are mutated, overexpressed, or underexpressed.

[0222] Fibrosis refers to the accumulation of extracellular matrix components that occurs after trauma, inflammation, tissue repair, immune response, cell proliferation, and tumor formation. Fibrotic conditions that can be treated with the compounds and compositions of the present invention include, in particular, fibrotic / fibrotic conditions associated with major organ diseases, such as interstitial lung disease (ILD), cirrhosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (liver fibrosis), kidney disease (renal fibrosis), acute kidney injury (AKI), acute kidney disease (AKD), chronic kidney disease (CKD), delayed kidney transplantation function, heart or vascular disease (cardiac fibrosis), and eye diseases; fibroproliferative diseases such as systemic and localized scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, and Dupuytren's contracture; trauma-related scarring, such as surgical complications, chemotherapy-induced fibrosis (e.g., bleomycin-induced fibrosis), radiation-induced fibrosis, accidental injury, and burns; retroperitoneal fibrosis (Ormond disease); and peritoneal fibrosis / peritoneal scarring in patients who typically undergo peritoneal dialysis after kidney transplantation. See, for example, Wynn et al., 2004, Nat Rev Immunol. August; 4(8): 583–594. Therefore, the present invention relates to methods for treating or preventing fibrosis / fibrotic conditions and / or fibrotic conditions associated with major organs (including, for example, the lungs, liver, kidneys, heart, skin, eyes, gastrointestinal tract, peritoneum, and bone marrow), as well as other diseases / conditions described in this application, and compounds and compositions for use in said methods.

[0223] The compound can be combined with agents used to treat kidney disease, including antidiabetic agents, cardiovascular agents, and novel agents targeting disease-related pathways such as oxidative stress (including but not limited to the nrf2 / keap-1 pathway) and anti-apoptotic pathways (including but not limited to anti-p53 agents).

[0224] Interstitial lung disease (ILD) includes conditions where lung inflammation and fibrosis are the final, most common pathways of pathology, such as sarcoidosis, silicosis, drug reactions, infections, and collagen vascular diseases like rheumatoid arthritis and systemic sclerosis (scleroderma). Fibrotic lung conditions include, for example, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), common interstitial pneumonia (UIP), interstitial lung disease, cryptogenic fibrotic alveolitis (CFA), obliterative bronchiolitis, and bronchiectasis.

[0225] Idiopathic pulmonary fibrosis (IPF) is the most common type of ILD, and there is no known cause.

[0226] The compound can be combined with agents that treat IPF and potentially ILD, including nintedanib and pirfenidone.

[0227] Cirrhosis has similar causes to ILD and includes, for example, cirrhosis associated with viral hepatitis, schistosomiasis, and chronic alcoholism.

[0228] Kidney disease can be associated with diabetes and hypertension, which can damage the kidneys and cause scarring, leading to progressive loss of function. Kidney fibrosis can occur at any stage of kidney disease, from post-traumatic acute kidney disease (AKD) and chronic kidney disease (CKD) (e.g., incidental and progressive CKD) to end-stage renal disease (ESRD). Kidney fibrosis can occur as a result of cardiovascular diseases such as hypertension or diabetes, both of which place significant strain on kidney function, promoting a fibrotic response. However, kidney fibrosis can also be idiopathic (with no known cause), and certain inherited mitochondrial diseases also present with kidney fibrosis and related symptoms.

[0229] Heart disease can lead to scar tissue, which can impair the heart's ability to pump.

[0230] Eye diseases, such as macular degeneration and retinal and vitreoretinal diseases, can impair vision.

[0231] In a preferred embodiment, the present invention relates to the treatment or prevention of idiopathic pulmonary fibrosis (IPF).

[0232] In another preferred embodiment, the present invention relates to the treatment or prevention of renal fibrosis.

[0233] 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 postoperative AKI, such as organ transplantation, caused by ischemia-reperfusion injury or delayed graft function; oncology, such as chemotherapy-induced AKI; contrast-induced nephropathy, such as direct tubular cytotoxicity, hemodynamic ischemia, and osmotic effects; acute interstitial nephritis, such as that caused by drugs or infections; AKI caused by obstruction, such as kidney stones; and COVID-19-induced AKI. A specific high-risk patient subgroup is those who have undergone cardiac surgery (e.g., coronary artery bypass grafting and / or valve surgery). Identified static risk factors for AKI are present, such as age 65 years or older, insulin-dependent diabetes mellitus, CKD (adults with an estimated glomerular filtration rate [eGFR] less than 60 ml / min / 1.73 m² are at particular risk), heart failure, liver disease, and a history of AKI.

[0234] In another preferred embodiment, the invention relates to the treatment or prevention of acute kidney disease (AKD) or chronic kidney disease (CKD) originating from such AKI, including, for example, tubulointerstitial fibrosis and diabetic nephropathy.

[0235] In another preferred embodiment, the present invention relates to the treatment or prevention of liver diseases, including, for example, NAFLD, NASH, cirrhosis, portal hypertension, acute liver failure, and hepatocellular carcinoma. Liver diseases such as NAFLD and NASH may be associated with various metabolic diseases such as metabolic syndrome and type II diabetes, and may also increase the risk of various diabetes-related pathologies, including diabetic retinopathy and peripheral neuropathy.

[0236] When used to treat or prevent conditions involving liver disease and metabolic dysfunction, the compounds of the present invention or their pharmaceutical compositions described in this application may be combined with one or more other pharmaceutical agents, including metformin, sulfonylureas, DPP-4 inhibitors, GLP-1 agonists, PPAR agonists, SGLT2 inhibitors, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin II receptor blockers (ARBs).

[0237] Leishmaniasis is a rare, inherited neurometabolic disorder that affects the central nervous system. This progressive disease begins in infants between three months and two years of age. It rarely occurs in adolescents and adults. Leishmaniasis can be caused by mutations in the nuclear DNA encoding mitochondrial proteins, mutations in mitochondrial DNA (maternally inherited Leishmaniasis - MILS), or a deficiency in an enzyme called pyruvate dehydrogenase located on the short arm of the X chromosome (X-linked Leishmaniasis). Symptoms of Leishmaniasis typically progress rapidly. The earliest signs may be poor sucking ability, as well as loss of head control and motor skills. These symptoms may be accompanied by loss of appetite, vomiting, irritability, persistent crying, and seizures. As the condition progresses, symptoms may also include general weakness, lack of muscle tone, and episodes of lactic acidosis, which can lead to respiratory and kidney dysfunction.

[0238] In maternally inherited Leigh syndrome (MILS), gene mutations in mitochondrial DNA (at a high rate of >90%) interfere with the energy source for cells operating in brain regions that play a role in motor movement. These mutations in mitochondrial DNA lead to a chronic energy deficiency in these cells, which in turn affects the central nervous system and causes progressive degeneration of motor function. When the gene mutations in mitochondrial DNA that cause MILS are insufficient (less than 90%), the condition is called neuropathic ataxia and retinitis pigmentosa (NARP). There is also a form of Leigh syndrome called X-linked Leigh syndrome, which results from gene mutations that produce another set of substances important for cellular metabolism. Another variant of Leigh syndrome exists, known as the French-Canadian variant, characterized by mutations in a gene called LRPPRC. Similar neurological symptoms are present in those of Leigh syndrome; however, hepatic steatosis is also commonly observed in the French-Canadian variant.

[0239] In a preferred embodiment, the present invention relates to the treatment or prevention of Leahy syndrome or Leahy disease, including, for example, X-linked Leahy disease, the French-Canadian variant of Leahy syndrome, and / or symptoms associated with Leahy disease.

[0240] The compound can be combined with novel agents that can be used as treatments for mitochondrial diseases, including but not limited to nicotinamide nucleoside.

[0241] The term "treatment" includes means of temporarily or permanently improving or alleviating symptoms, or eliminating the cause of symptoms. The compounds of this invention can be used to treat diseases disclosed in this application in humans and other mammals.

[0242] In another embodiment, the invention includes preventive therapies for the diseases disclosed in this application, and includes means of preventing or alleviating the onset of symptoms of said disease or condition. The compounds of the invention can be used to prevent the diseases disclosed in this application in humans and other mammals.

[0243] Patients requiring treatment or prevention may be, for example, people or other mammals who have the condition or are at risk of having the condition.

[0244] According to another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) as defined in this application or a pharmaceutically acceptable salt of said compound or tautomer, and a pharmaceutically acceptable diluent or carrier.

[0245] The pharmaceutical compositions of the present invention comprise any compound of the present invention in combination with any pharmaceutically acceptable carrier, excipient, or medium. Examples of pharmaceutically acceptable carriers are known to those skilled in the art and include, but are not limited to, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants, depending on the characteristics of the administration mode and dosage form. The compositions may be in the form of, for example, tablets, capsules, powders, granules, elixirs, lozenges, suppositories, syrups, and liquid formulations (including suspensions and solutions). In the context of the present invention, the term "pharmaceutical composition" means a composition comprising an active agent and additionally comprising one or more pharmaceutically acceptable carriers. Depending on the characteristics of the administration mode and dosage form, the composition may further comprise, for example, components selected from diluents, excipients, media, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants.

[0246] The compounds or pharmaceutical compositions thereof of the present invention as described in this application may be used alone or in combination with one or more other agents. The compounds may be combined with other antitumor therapeutic agents, such as chemotherapeutic agents or other inhibitors of regulatory proteins. In one embodiment, the other antitumor therapeutic agent is a BH-3 mimic. In another embodiment, the BH-3 mimic may be selected from, but is not limited to, one or more of ABT-737, ABT-199, ABT-263, and oxaliplatin. In another embodiment, the other antitumor agent is a chemotherapeutic agent. The chemotherapeutic agent may be selected from, but is not limited to, olaparib, mitomycin C, cisplatin, carboplatin, oxaliplatin, ionizing radiation (IR), camptothecin, irinotecan, topotecan, temozolomide, taxane, 5-fluoropyrimidine, gemcitabine, and doxorubicin.

[0247] For the treatment or prevention of fibrotic diseases, for example, the compounds or pharmaceutical compositions thereof of the present invention as described in this application may be used alone or in combination with one or more other agents selected from anticholinergic drugs, β-2 mimics, steroids, PDE-IV inhibitors, p38 MAP kinase inhibitors, NK1 antagonists, LTD4 antagonists, EGFR inhibitors and endothelin antagonists.

[0248] In particular, the compounds or pharmaceutical compositions thereof of the present invention as described in this application may be used alone or in combination with one or more alternative agents selected from: conventional immunosuppressive agents, such as corticosteroids, immunosuppressants or cytotoxic agents, or antifibrotic agents, such as pirfenidone or nonspecific kinase inhibitors (e.g. nintedanib).

[0249] The pharmaceutical compositions of the present invention can be administered in any suitable and effective manner, such as orally, parenterally, topically, by inhalation, intranasally, rectally, vaginally, ocularly, and orally. Pharmaceutical compositions suitable for delivering the compounds of the present invention and methods of their preparation will be apparent to those skilled in the art. Such compositions and methods of their preparation can be found, for example, in "Remington's Pharmaceutical Sciences," 19th edition (Mack Publishing Company, 1995).

[0250] oral

[0251] The compounds of this invention can be taken orally. Oral administration may include swallowing, allowing the compound to enter the gastrointestinal tract, or it may be administered orally or sublingually, allowing the compound to enter the bloodstream directly from the mouth.

[0252] Oral formulations suitable for oral administration include solid dosage forms, such as tablets, capsules containing granules, liquids, or powders, lozenges (including liquid-filled ones), chewables, multi-particle and nanoparticle formulations, gels, films (including mucosal adhesives), vaginal suppositories, sprays, and liquid formulations.

[0253] Liquid formulations include suspensions, solutions, syrups, and elixirs. These formulations 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, as well as one or more emulsifiers and / or suspending agents. Liquid formulations can also be prepared, for example, by reconstructing a solid from a small capsule.

[0254] The compounds of the present invention can also be used in rapidly dissolving and rapidly disintegrating dosage forms, such as those described in Liang and Chen's Expert Opinion in Therapeutic Patents, 11(6), 981-986(2001).

[0255] Typical tablets can be prepared using standard methods known to pharmaceutical chemists, such as direct compression, granulation (dry, wet, or melt), melt coagulation, or extrusion. Tablet formulations may contain one or more layers and may be coated or uncoated.

[0256] Examples of excipients suitable for oral administration include carriers such as cellulose, calcium carbonate, dicalcium phosphate, mannitol, and sodium citrate; granulation binders such as polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and gelatin; disintegrants such as sodium glycolate starch and silicates; lubricants such as magnesium stearate and stearic acid; wetting agents such as sodium dodecyl sulfate; preservatives; antioxidants; flavoring agents; and coloring agents.

[0257] Solid dosage forms for oral administration can be formulated into immediate-release and / or modulated-release dosage forms. Modulated-release formulations include delayed-release, sustained-release, pulsatile-release, controlled dual-release, targeted-release, and programmed-release. Suitable modulated-release technologies, such as high-energy dispersions, permeable and coated particles, can be found in Verma et al., Pharmaceutical Technology Online, 25(2), 1-14 (2001). Other modulated-release formulations are described in U.S. Patent No. 6,106,864.

[0258] Parenteral application

[0259] The compounds of this invention can also be applied directly to the bloodstream, muscles, or internal organs. Suitable methods of parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous administration. Suitable devices for parenteral administration include needle (including micromanipulation needle) injectors, needle-free injectors, and infusion techniques.

[0260] Parenteral preparations are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably at pH 3 to 9). However, for some applications, they may be more suitably formulated as sterile non-aqueous solutions or in dry form for use with a suitable medium, such as sterile pyrogen-free water.

[0261] The preparation of parenteral preparations under sterile conditions, such as by freeze-drying, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0262] The solubility of compounds of formula (I) used to prepare parenteral solutions can be increased by appropriate processing, such as using high-energy spray-dried dispersions and / or by using appropriate formulation techniques, such as using solubility enhancers.

[0263] Formulations intended for parenteral administration can be formulated into immediate-release and / or modulated-release dosage forms. Modulated-release formulations include delayed-release, sustained-release, pulsatile-release, controlled dual-release, targeted-release, and programmed-release.

[0264] The pharmaceutical compositions of the present invention also include compositions and methods known in the art for bypassing the blood-brain barrier or for direct injection into the brain. Suitable areas for injection include the cerebral cortex, cerebellum, midbrain, brainstem, hypothalamus, spinal cord, and ventricular tissues, as well as the PNS region including the carotid body and adrenal medulla.

[0265] dose

[0266] Of course, the effective dose of the compound will vary depending on the severity and nature of the condition being treated and the route of administration. The appropriate dose should be chosen within the scope of the clinician's recommendations. The daily dose range is approximately 10 μg to approximately 100 mg per kilogram of body weight for humans and non-human animals, and is typically from approximately 10 μg to 30 mg per kilogram of body weight per dose. The above doses can be administered one to three times daily.

[0267] For example, oral administration may require a total daily dose of 5 mg to 1000 mg, such as 5-500 mg, while intravenous administration may require only 0.01-30 mg / kg body weight, such as 0.1-10 mg / kg, more preferably 0.1-1 mg / kg body weight. The total daily dose may be administered as a single dose or in divided doses. Those skilled in the art will also understand that, in the treatment or prevention of certain conditions, the compounds of the present invention may be taken "as needed" (i.e., as required or desired) as a single dose.

[0268] Synthetic Methodology

[0269] Compounds of formula (I) can be prepared using the methods described in the general reaction scheme and representative examples below. Individual variations within the scheme can be performed in different orders where appropriate. The invention is illustrated by the following non-limiting examples, wherein the following abbreviations and definitions are used. By liquid chromatography-mass spectrometry (LCMS) or... 1 The compound was characterized by 1H NMR or both.

[0270] According to another aspect, the present invention provides a method for preparing a compound of formula (I)(i), comprising reacting a compound of formula (IV) (where Y is OH) with an amine of formula (V)(i) (where PG is a protecting group, such as Boc or Cbz) to obtain an amide of formula (III)(i) (Scheme 1). The amide coupling reaction can be carried out using standard methodologies, for example, by using coupling agents such as DCC, HATU, HBTU, EDC, or via a reaction of mixed acid anhydrides. Alternatively, an acid (IV) where Y is OH can be converted to an acyl chloride (IV) where Y is Cl using SOCl2, PCl3, or PCl5, and then the acyl chloride can be reacted with an amine (V)(i), preferably in a suitable solvent in the presence of a suitable base. Alternatively, a compound (IV) where Y forms an ester can be reacted directly with an amine (V)(i), preferably in a suitable solvent. The compound of formula (III)(i) can be deprotected using standard methods to obtain an amine (II)(i), which can then be reacted with cyanogen bromide to obtain the corresponding compound of formula (I)(i).

[0271]

[0272] Option 1

[0273] In another aspect, the present invention provides compounds selected from formulas (II)(i) and (III)(i):

[0274]

[0275] Wherein PG is a protecting group, preferably BOC or CBZ, and R 1 R 2 R 3 R 4 and R 5 As defined in this application, compounds of formula (I) and their preferred embodiments, their tautomers, or salts of said compounds or tautomers.

[0276] When the compound of formula (I)(i) has formula (IA)(i), formulas (II)(i), (III)(i), and (IV) are (IIA)(i), (IIIA)(i), and (IVA), respectively. When the compound of formula (I)(i) has formula (IB)(i), formulas (II)(i), (III)(i), and (IV) are (IIB)(i), (IIIB)(i), and (IVB)(i), respectively.

[0277] According to another aspect, the present invention provides a method for preparing compounds 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 obtain an amide of formula (III)(ii) (Scheme 2). The amide coupling reaction can be carried out using standard methods, for example by using coupling reagents such as DCC, HATU, HBTU, EDC, or via a reaction with mixed anhydrides. Alternatively, an acid (IV) wherein Y is OH can be converted to an acyl chloride (IV) wherein Y is Cl using SOCl2, PCl3, or PCl5, and then the acyl chloride can be reacted with an amine (V)(ii), preferably in the presence of a suitable base and in a suitable solvent. Alternatively, a compound (IV) wherein Y forms an ester can be reacted directly with an amine (V)(ii), preferably in a suitable solvent. A compound of formula (III)(ii) can be deprotected using standard methods to obtain an amine (II)(ii), which can then be reacted with cyanogen bromide to obtain the corresponding compound of formula (I)(ii).

[0278]

[0279] Option 2

[0280] In another aspect, the present invention provides compounds selected from formulas (II)(ii) and (III)(ii):

[0281]

[0282] Wherein PG is a protecting group, preferably BOC or CBZ, and R 1 R 2 R 3 R 4 and R 5 As defined in this application, compounds of formula (I) and their preferred embodiments, their tautomers, or salts of said compounds or tautomers.

[0283] When a compound of formula (I)(ii) has formula (IA)(ii), formulas (II)(ii), (III)(ii), and (IV) are (IIA)(ii), (IIIA)(ii), and (IVA), respectively. When a compound of formula (I)(ii) has formula (IB)(ii), formulas (II)(ii), (III)(ii), and (IV) are (IIB)(ii), (IIIB)(ii), and (IVB)(ii), respectively.

[0284] The protecting group is preferably selected from tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (MeOz), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), toluenesulfonyl (Ts), trichloroethoxycarbonyl (Troc), 4-nitrobenzenesulfonyl (Nosyl), and 2-nitrobenzenesulfinyl (Nps). BOC and Cbz are most preferred.

[0285] abbreviation

[0286] br s Broad single peak (NMR signal) MeOH Methanol

[0287] CO and carbon monoxide (min)

[0288] d Bimodal (NMR signal) N2 Nitrogen

[0289] dba dibenzylacetone NMP N-methylpyrrolidone

[0290] DCM dichloromethane rac racemic

[0291] DMF N,N-dimethylformamide rt at room temperature

[0292] DMSO (dimethyl sulfoxide) single peak (NMR signal)

[0293] ES Electrospray SFC Supercritical Fluid Chromatography

[0294] EtOAc (ethyl acetate) SOCl2 (thionyl chloride)

[0295] h Hourly TBD 1,5,7-Triazabicyclic [4.4.0]

[0296] dec-5-ene

[0297] H2 Hydrogen TEA Triethylamine

[0298] HPLC method for the determination of TFA triacetic acid

[0299] LCMS (Liquid Chromatography-Mass Spectrometry) for THF Tetrahydrofuran

[0300] m Multiplet peak (NMR signal) TsCl 4-toluenesulfonyl chloride

[0301] MsCl methanesulfonyl chloride vol volume

[0302] MeCN Acetonitrile

[0303] LCMS / HPLC / SFC methods

[0304] Method C

[0305]

[0306] Method C1

[0307]

[0308]

[0309] Method C2

[0310]

[0311]

[0312] Method H

[0313]

[0314] Method H1

[0315]

[0316]

[0317] Method J

[0318]

[0319]

[0320] Method F

[0321]

[0322] Method P3

[0323]

[0324]

[0325] Method Y3 Methods for analytical chiral SFC

[0326]

[0327] Method Y4 is used for analytical chiral SFC.

[0328]

[0329] Method Y5 is a method for analytical chiral SFC.

[0330]

[0331] Method Y6 is used for analytical chiral SFC.

[0332]

[0333] Method Y7 is a method for analytical chiral SFC.

[0334]

[0335] Method Y8 is a method for analytical chiral SFC.

[0336]

[0337]

[0338] Method Y12 is used for analytical chiral SFC.

[0339]

[0340] Method Y15 is a method for analytical chiral HPLC.

[0341]

[0342]

[0343] Method Y16 is a method for analytical chiral SFC.

[0344]

[0345] Intermediate A

[0346] ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate

[0347]

[0348] (i) NaH, THF, 0℃, 2h; (ii) Phenyltrimethylammonium tribromide, THF, 0℃ to rt, 16h;

[0349] (iii) NaN(CHO)2, MeCN, 70℃, 1h, then concentrated HCl, 70℃, 16h; (iv) K2CO3, DCM, 0℃ to rt, 2h; (v) POCl3, 100℃, 7h.

[0350] Step (i)

[0351] 3-Acetyl-4-cyclopropoxybenzylnitrile

[0352] The reaction was performed in triplicate. NaH (60% in oil, 0.49 g, 12.27 mmol) was added dropwise to a stirred solution of cyclopropanol (CAS 16545-68-9, from 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-fluorobenzyl nitrile (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 other identical batches, poured into ice-cold water (500 mL), and extracted with EtOAc (2 x 500 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 12% EtOAc in n-hexane solution) to give 3-acetyl-4-cyclopropoxybenzyl nitrile (3.18 g, 15.82 mmol, 85% yield).

[0353] LCMS: m / z is not supported; 1 H NMR (400MHz, DMSO-d6) δppm: 8.05 (dd, J=8.8, 2.4Hz, 1H), 7.96 (d, J=2.0Hz, 1 H), 7.64 (d, J=8.8Hz, 1H), 4.13-4.17 (m, 1H), 2.50 (s, 3H), 0.81-0.93 (m, 4H).

[0354] Step (ii)

[0355] 3-(2-bromoacetyl)-4-cyclopropoxybenzyl nitrile

[0356] To a stirred solution of 3-acetyl-4-cyclopropoxybenzyl nitrile (3.18 g, 15.82 mmol) in THF (40 mL), phenyltrimethylammonium tribromide (5.94 g, 15.82 mmol) was added. The mixture was slowly heated to room temperature and stirred for 16 hours, then poured into water (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to give 3-(2-bromoacetyl)-4-cyclopropoxybenzyl nitrile (4.3 g, 15.41 mmol, 97% yield). This crude product was used directly in the next step.

[0357] LCMS: Method F, 6.61 min; MS: ES+: 297.0, 299.0 (M+18).

[0358] Step (iii)

[0359] 4-Cyclopropoxy-3-glycylbenzyl nitrile hydrochloride

[0360] Sodium dimethamide (1.75 g, 18.49 mmol) was added to a solution of 4.3 g (15.41 mmol) of 3-(2-bromoacetyl)-4-cyclopropoxybenzyl nitrile (4.3 g, 15.41 mmol) in acetonitrile (43 mL) under stirring and the mixture was heated at 70 °C for 3 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with MeOH (43 mL) and concentrated HCl (4.3 mL). The mixture was further heated at 70 °C for 16 h and then allowed to cool to room temperature. The mixture was concentrated under reduced pressure, and the residue was stirred with isopropanol (25 mL) to form a precipitate. The solid was collected by filtration under reduced pressure to give 4-cyclopropoxy-3-glycylbenzyl nitrile hydrochloride (5.0 g, quantitative yield).

[0361] LCMS: Method C, 1.30 min; MS: ES+: 217.4.

[0362] Step (iv)

[0363] 2-((2-(5-cyano-2-cyclopropoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate

[0364] K₂CO₃ (10.92 g, 79.2 mmol) was added to a stirred solution of 4-cyclopropoxy-3-glycylbenzyl nitrile hydrochloride (5.0 g, 19.80 mmol) in DCM (50 mL) at 0 °C. Ethyl oxaloyl chloride (5.4 g, 4.43 mL, 39.60 mmol) was added dropwise at 0 °C. The mixture was heated to room temperature and stirred for 2 hours, then poured into water (500 mL) and extracted with DCM (2 x 300 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to give ethyl 2-((2-(5-cyano-2-cyclopropoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate (5.0 g, quantitative yield).

[0365] LCMS: Method C, 1.52 min; MS: ES+316.9.

[0366] Step (v)

[0367] ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate

[0368] A solution of ethyl 2-((2-(5-cyano-2-cyclopropoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate (5.0 g, 15.81 mmol) stirred in POCl3 (50 mL, 10 vol) was heated for 7 h at 100 °C. The mixture was cooled to rt, poured into ice-cold water (500 mL), and extracted with EtOAc (2 x 200 mL). The combined organic phases were dried over Na2SO4 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)oxazol-2-carboxylate (1.0 g, 3.35 mmol, 21% yield in three steps). LCMS: Method C, 1.77 min; MS: ES+: 299.5.

[0369] Intermediate B

[0370] 5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylic acid ethyl ester

[0371]

[0372] (i) Zn(CN)2, Zn dust, PdCl2(dppf). DCM complex, DMA, 120 °C, 16 h; (ii) Pyridinium bromide, THF, 0 °C to rt, 16 h; (iii) NaN(CHO)2, MeCN, 80 °C, 5 h, then concentrated HCl, 80 °C, 16 h; (iv) K2CO3, DCM, 0 °C to rt, 4 h; (v) POCl3, 100 °C, 16 h.

[0373] Step (i)

[0374] 3-Acetyl-4-methoxybenzylnitrile

[0375] Zinc cyanide (92.25 g, 785.67 mmol) and zinc powder (17.22 g, 261.89 mmol) were added to a solution of 1-(5-bromo-2-methoxyphenyl)ethane-1-one (CAS 16740-73-1, from Combi-blocks, 60.0 g, 261.89 mmol) stirred in DMA (600 mL) at room temperature. The mixture was degassed with N2 for 30 min, followed by the addition of a complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) with DCM (10.69 g, 13.09 mmol). The mixture was heated at 120 °C for 16 h. The mixture was cooled to rt and passed through Celite Hyflow. TMFilter the solution, pour the filtrate into ice-cold water (800 mL), and extract with EtOAc (2 x 1000 mL). Wash the combined organic phases with ice-cold water (4 x 1000 mL), dry with Na2SO4, and concentrate under reduced pressure. Purify the residue by column chromatography (60-120# silica gel, 15% EtOAc in n-hexane solution) to give 3-acetyl-4-methoxybenzyl nitrile (40.0 g, 228.57 mmol, 87% yield).

[0376] LCMS: Method C, 1.52 min; MS: ES+: 176.08.

[0377] Step (ii)

[0378] 3-(2-bromoacetyl)-4-methoxybenzylnitrile

[0379] Tribromopyridinium (14.47 g, 45.25 mmol) was added to a stirred solution of 3-acetyl-4-oxybenzyl nitrile (7.20 g, 41.14 mmol) in THF (72 mL) at 0 °C. The mixture was slowly heated to room temperature and stirred for 16 hours, then poured into water (400 mL) and extracted with EtOAc (2 x 400 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to give 3-(2-bromoacetyl)-4-methoxybenzyl nitrile (13.0 g, quantitative yield). This crude product was used directly in the next step.

[0380] Step (iii)

[0381] 3-Glycyl-4-methoxybenzyl nitrile hydrochloride

[0382] Sodium dimethamide (5.91 g, 62.26 mmol) was added to a stirred solution of 3-(2-bromoacetyl)-4-methoxybenzyl nitrile (10.5 g, 41.51 mmol) in acetonitrile (105 mL), 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 concentrated HCl (10.5 mL). The mixture was further heated at 80 °C for 16 h, and then cooled to rt. The mixture was concentrated under reduced pressure, and the residue was stirred with isopropanol (40 mL) to form a precipitate. The precipitate was collected by filtration under reduced pressure to give 3-glycyl-4-methoxybenzyl nitrile hydrochloride (9.0 g, 39.73 mmol, 95% yield).

[0383] LCMS: Method C2, 0.40 min; MS: ES+: 191.0.

[0384] Step (iv)

[0385] 2-((2-(5-cyano-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate

[0386] K₂CO₃ (21.93 g, 158.92 mmol) was added to a stirred solution of 3-glycyl-4-methoxybenzyl nitrile hydrochloride (HCl) (9.0 g, 39.73 mmol) in DCM (90 mL) at 0 °C. Ethyl oxaloyl chloride (10.84 g, 8.89 mL, 79.46 mmol) was added dropwise at 0 °C. The mixture was heated to room temperature and stirred for 4 h. The mixture was poured into water (900 mL) and extracted with DCM (2 x 500 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to give 2-((2-(5-cyano-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl ester (9.0 g, 31.03 mmol, 78% yield).

[0387] LCMS: Method C, 1.34 min; MS: ES+291.1.

[0388] Step (v)

[0389] ethyl 5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylate

[0390] Ethyl 2-((2-(5-cyano-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate (1.2 g, 4.14 mmol) was heated for 2 h in a stirred solution of POCl3 (12 mL, 10 vol) at 100 °C. The mixture was cooled to room temperature, poured into ice-cold water (200 mL), and extracted with EtOAc (3 x 100 mL). The combined organic phases were washed with saturated NaHCO3 solution (3 x 100 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 32% EtOAc in n-hexane) to give ethyl 5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylate (0.60 g, 2.20 mmol, 53% yield).

[0391] LCMS: Method C, 1.53 min; MS: ES+: 272.6.

[0392] Intermediate C

[0393] ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylate

[0394]

[0395] (i) HATU, DIPEA, THF, 0°C to rt, 3h; (ii) methyl magnesium bromide, THF, -10°C to 0°C, 4h; (iii) phenyltrimethylammonium tribromide, MeCN, 0°C to rt, 9h; (iv) NaN(CHO)2, MeCN, 80°C, 1h, then concentrated HCl, 80°C, 3h; (v) K2CO3, DCM, 0°C to rt, 3h; (vi) POCl3, 110°C, 16h.

[0396] Step (i)

[0397] 5-Bromo-N-methoxy-N-methyl-2-(trifluoromethoxy)benzamide

[0398] Dipea (13.57 g, 17.9 mL, 105.25 mmol) and HATU (20.0 g, 52.62 mmol) were added in portions to a solution of 5-bromo-2-(trifluoromethoxy)benzoic acid (CAS 403646-47-9, from Combi-blocks, 10.0 g, 35.08 mmol) stirred in THF (100 mL) at 0 °C. After 30 min, N,O-dimethylhydroxylamine HCl (4.45 g, 45.61 mmol) was added at 0 °C. The mixture was slowly heated to room temperature and stirred for 3 h, then poured into ice-cold water (300 mL) and extracted with EtOAc (3 x 150 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure to give 5-bromo-N-methoxy-N-methyl-2-(trifluoromethoxy)benzamide (12.2 g, quantitative yield). This crude product was used directly in the next step.

[0399] LCMS: Method C, 1.69 min; MS: ES+: 327.8, 329.8.

[0400] Step (ii)

[0401] 1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one

[0402] Methylmagnesium bromide (3M in diethyl ether, 24.9 mL, 74.62 mmol) was added dropwise to a solution of 5-bromo-N-methoxy-N-methyl-2-(trifluoromethoxy)benzamide (12.2 g, 37.31 mmol) stirred in THF (130 mL) at -10 °C. The mixture was slowly heated to 0 °C and stirred for 4 hours, then poured into a saturated NH4Cl solution (300 mL) and extracted with EtOAc (3 x 200 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure to give 1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one (10.0 g, quantitative yield). This crude product was used directly in the next step.

[0403] LCMS: Method C, m / z is not supported; 1 H NMR (400MHz, CDCl3) δppm: 7.92 (s, 1H), 7.67-7.71 (m, 1H), 7.24 (d, J = 8.0Hz, 1H), 2.62 (s, 3H).

[0404] Step (iii)

[0405] 2-Bromo-1-(5-Bromo-2-(trifluoromethoxy)phenyl)ethane-1-one

[0406] Add phenyltrimethylammonium tribromide (13.33 g, 35.46 mmol) in portions to a solution of 1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one (10.0 g, 35.46 mmol) stirred in acetonitrile (100 mL) at 0 °C. The mixture was slowly heated to room temperature and stirred for 9 hours, then poured into water (200 mL) and extracted with EtOAc (3 x 100 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 5% EtOAc in n-hexane) to give 2-bromo-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one (11.5 g, 31.95 mmol, 91% yield in three steps).

[0407] LCMS: m / z is not supported; 1 H NMR (400MHz, CDCl3) δppm: 7.93 (s, 1H), 7.73 (dd, J=8.8, 2.4Hz, 1H), 7.25 (d, J=8.0Hz, 1H), 4.45 (s, 2H).

[0408] Step (iv)

[0409] 2-Amino-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one hydrochloride

[0410] Sodium dimethamide (5.28 g, 55.58 mmol) was added to a solution of 2-bromo-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one (10.0 g, 27.78 mmol) stirred in acetonitrile (100 mL), and the mixture was heated at 80 °C for 1 h. The mixture was cooled to room temperature, concentrated under reduced pressure, and the residue was diluted with MeOH (100 mL) and concentrated HCl (10.0 mL). The mixture was heated at 80 °C for 3 h, and then cooled to rt. The mixture was concentrated under reduced pressure, and the residue was stirred with diethyl ether (4 x 30 mL) to form a precipitate. The precipitate was collected by filtration under reduced pressure to give 2-amino-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one hydrochloride (9.41 g, quantitative yield). LCMS: Method C, 1.65 min; MS: ES+: 299.9, 301.9.

[0411] Step (v)

[0412] 2-((2-(5-bromo-2-(trifluoromethoxy)phenyl)-2-oxoethyl)amino)-2-oxoethyl acetate

[0413] K₂CO₃ (11.67 g, 84.56 mmol) was added to a solution of 2-amino-1-(5-bromo-2-(trifluoromethoxy)phenyl)ethane-1-one HCl salt (9.4 g, 28.19 mmol) stirred in DCM (94 mL) at 0 °C. Ethyl oxaloyl chloride (5.77 g, 4.73 mL, 42.28 mmol) was added dropwise at 0 °C. The mixture was heated to room temperature and stirred for 3 hours, 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-oxoethyl acetate (3.4 g, 8.56 mmol, 31% yield in two steps).

[0414] LCMS: Method C, 2.09 min; MS: ES-396.0, 398.0.

[0415] Step (vi)

[0416] ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylate

[0417] Ethyl 2-((2-(5-bromo-2-(trifluoromethoxy)phenyl)-2-oxoethyl)amino)-2-oxoethyl acetate (3.4 g, 8.56 mmol) was heated for 16 h in a stirred solution of POCl3 (17 mL, 5 vol). The mixture was cooled to room temperature, poured into ice-cold water (250 mL), and extracted with EtOAc (3 x 100 mL). The combined organic phases were washed with saturated NaHCO3 solution (3 x 100 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 18% EtOAc in n-hexane) to give ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylate (1.3 g, 3.43 mmol, 40% yield).

[0418] LCMS: Method C, 2.39 min; MS: ES+: 379.8, 381.8.

[0419] Intermediate D

[0420] ethyl 5-(5-cyano-4-fluoro-2-methoxyphenyl)oxazol-2-carboxylate

[0421]

[0422] (i) Trifluoromethanesulfonic acid, NBS, MeCN, -30°C to rt, 18h; (ii) K2CO3, MeI, DMF, 0°C to rt, 3h; (iii) Tributyl(1-ethoxyvinyl)tin, PdCl2(PPh3)2, 1,4-dioxane, 100°C, 3h, then NBS, THF:water, 0°C, 10min; (iv) NaN(CHO)2, MeCN, 80°C, 2h, then concentrated HCl, 80°C, 16h; (v) Ethyl chlorooxyethyl acetate, K2CO3, DCM, 0°C to rt, 2h; (vi) POCl3, 100°C, 5h.

[0423] Step (i)

[0424] 5-Bromo-2-fluoro-4-hydroxybenzylnitrile

[0425] Trifluoromethanesulfonic acid (10.51 g, 6.18 mL, 70.07 mmol) was added dropwise to a solution of 2-fluoro-4-hydroxybenzyl nitrile (CAS 82380-18-5, from Combi-blocks, 8.0 g, 58.39 mmol) stirred in acetonitrile (80 mL) at -30 °C. After 10 minutes, N-bromosuccinimide (10.39 g, 58.39 mmol) was added in portions at -30 °C. The mixture was slowly heated to room temperature and stirred for 18 hours, then poured into a saturated NaHCO3 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-hydroxybenzyl nitrile (5.08 g, 23.63 mmol, 40% yield). This crude product was used directly in the next step.

[0426] LCMS: Method C, 1.58 min; MS: ES-: 214.0, 216.0.

[0427] Step (ii)

[0428] 5-Bromo-2-fluoro-4-methoxybenzylnitrile

[0429] K₂CO₃ (6.52 g, 47.26 mmol) was added to a solution of 5-bromo-2-fluoro-4-hydroxybenzyl nitrile (5.08 g, 23.63 mmol) stirred in DMF (30 mL) at 0 °C. After 10 min, methyl iodine (5.03 g, 2.21 mL, 35.45 mmol) was added dropwise at 0 °C. The mixture was slowly heated to room temperature and stirred for 3 h, then poured into water (100 mL) and extracted with EtOAc (2 x 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-hexane solution) to give 5-bromo-2-fluoro-4-methoxybenzyl nitrile (5.70 g, quantitative yield).

[0430] LCMS: m / z is not supported; 1 H NMR (400MHz, DMSO-d6) δppm: 8.30 (d, J=7.2Hz, 1H), 7.46 (d, J=12.0Hz, 1H), 4.01 (s, 3H).

[0431] Step (iii)

[0432] 5-(2-bromoacetyl)-2-fluoro-4-methoxybenzylnitrile

[0433] A solution of 5-bromo-2-fluoro-4-methoxybenzyl nitrile (5.70 g, 24.89 mmol) and tributyl(1-ethoxyvinyl)tin (CAS 97674-02-7, from Combi-blocks, 13.46 g, 37.34 mmol) stirred in 1,4-dioxane (70 mL) was degassed with N2 for 15 min, followed by the addition of bis(triphenylphosphine)palladium(II) dichloride (0.87 g, 1.24 mmol) at room temperature. The mixture was heated at 100 °C for 3 h. The mixture was cooled to 0 °C and THF: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 x 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-hexane solution) to give 5-(2-bromoacetyl)-2-fluoro-4-methoxybenzyl nitrile (6.3 g, 23.25 mmol, two-step yield 98%).

[0434] LCMS: m / z is not supported; 1 H NMR (400MHz, DMSO-d6) δppm: 8.27 (dd, J=7.6, 2.4Hz, 1H), 7.54 (dd, J=12.0, 2.4Hz, 1H), 4.84 (s, 2H), 4.05 (s, 3H).

[0435] Step (iv)

[0436] 2-Fluoro-5-glycyl-4-methoxybenzyl nitrile hydrochloride

[0437] Sodium dimethamide (25.85 g, 27.9 mmol) was added to a solution of 5-(2-bromoacetyl)-2-fluoro-4-methoxybenzyl nitrile (6.30 g, 23.25 mmol) in acetonitrile (65 mL) under stirring, 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 concentrated HCl (6.5 mL). The mixture was heated at 80 °C for 16 h, and then cooled to rt. The mixture was concentrated under reduced pressure, and the residue was stirred with isopropanol (100 mL) to form a precipitate. The precipitate was collected by filtration under reduced pressure to give 2-fluoro-5-glycyl-4-methoxybenzyl nitrile hydrochloride (5.3 g, quantitative yield).

[0438] LCMS: Method H, 1.96 min; MS: ES+: 209.1.

[0439] Step (v)

[0440] 2-((2-(5-cyano-4-fluoro-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate

[0441] K₂CO₃ (11.96 g, 86.68 mmol) was added to a solution of 2-fluoro-5-glycyl-4-methoxybenzyl nitrile HCl salt (5.30 g, 21.67 mmol) stirred in a DCM (60 mL) at 0 °C. Ethyl oxaloyl chloride (5.89 g, 4.83 mL, 43.34 mmol) was added dropwise at 0 °C. The mixture was heated to room temperature and stirred for 2 hours, then poured into water (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 ethyl 2-((2-(5-cyano-4-fluoro-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate (4.20 g, 13.63 mmol, 58% yield in two steps). LC-MS: Method C, 1.50 min; MS: ES + 309.4.

[0442] Step (vi)

[0443] ethyl 5-(5-cyano-4-fluoro-2-methoxyphenyl)oxazol-2-carboxylate

[0444] A solution of ethyl 2-((2-(5-cyano-4-fluoro-2-methoxyphenyl)-2-oxoethyl)amino)-2-oxoethyl acetate (4.2 g, 13.63 mmol) was heated for 5 h at 100 °C in POCl3 (42 mL, 10 vol) with stirring. The mixture was cooled to room temperature, poured into ice-cold water (100 mL), and extracted with EtOAc (2 x 150 mL). The combined organic phases were washed with saturated NaHCO3 solution (2 x 100 mL), dried over Na2SO4, 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)oxazol-2-carboxylate (1.60 g, 5.51 mmol, 40% yield).

[0445] LCMS: Method C, 1.63 min; MS: ES+: 291.2.

[0446] Example 1

[0447] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0448]

[0449]

[0450] (i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K2CO3, CNBr, THF, 0°C to rt.

[0451] Step (i)

[0452] (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formylamino)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester

[0453] TBD (0.42 g, 3.02 mmol) was added in portions to a solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate (0.60 g, 2.01 mmol) and (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate tert-butyl ester (CAS 348165-63-9, 0.60 g, 3.02 mmol) in THF (10 mL) under stirring. The mixture was warmed to rt and stirred for 1 h, then poured into water (70 mL) and extracted with EtOAc (2 x 70 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 30% EtOAc in n-hexane solution) to give (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.32 g, 0.71 mmol, 35% yield).

[0454] LCMS: Method C, 1.86 min; MS: ES-451.4.

[0455] Step (ii)

[0456] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0457] TFA (0.93 mL, 3 vol) was added dropwise to a solution of (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.31 g, 0.68 mmol) stirred in a DCM (7 mL). The mixture was warmed to rt and stirred for 45 min, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.3 g, 0.64 mmol, 94% yield).

[0458] LCMS: Method C, 1.38 min; MS: ES+353.3.

[0459] Step (iii)

[0460] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0461] K₂CO₃ (0.27 g, 1.93 mmol) was added to a solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide TFA salt (0.3 g, 0.64 mmol) in THF (8 mL) under stirring, 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 for 45 min under stirring, then poured into water (70 mL) and extracted with EtOAc (2 x 70 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 40% EtOAc in n-hexane solution) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide (0.1 g, 0.27 mmol, 42% yield).

[0462] LCMS: Method H, 2.96 min; MS: ES+378.0; 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 9.36 (d, J = 6.4Hz, 1H), 8.27 (s, 1H), 7.98 (d, J = 8.4Hz, 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 = 6.4Hz, 3H), 0.92 (s, 4H). Chiral SFC: Method Y5, 5.18 min.

[0463] Example 2

[0464] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0465]

[0466]

[0467] (i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K2CO3, CNBr, THF, 0°C to rt.

[0468] Step (i)

[0469] (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamide)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0470] TBD (0.35 g, 2.51 mmol) was added in portions to a solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-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) under stirring. The mixture was warmed to rt and stirred for 3 h, then poured into water (100 mL) and extracted with EtOAc (2 x 80 mL). The combined organic phases were dried anhydrous with Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 55% EtOAc in n-hexane solution) to give (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.32 g, 0.66 mmol, 39% yield).

[0471] LCMS: Method C1, 1.35 min; MS: ES+483.4.

[0472] Step (ii)

[0473] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0474] TFA (0.96 mL, 3 vol) was added dropwise to a solution of (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.32 g, 0.66 mmol) stirred in a DCM (8 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.32 g, 0.64 mmol, 97% yield).

[0475] LCMS: Method C1, 1.06 min; MS: ES+383.4.

[0476] Step (iii)

[0477] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0478] K₂CO₃ (0.27 g, 1.93 mmol) was added to a solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.32 g, 0.64 mmol) in THF (8 mL) under stirring and stirred for 10 min. Cyanide bromide (0.07 g, 0.64 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (80 mL) and extracted with EtOAc (2 x 80 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 70% EtOAc in n-hexane solution) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.10 g, 0.24 mmol, 38% yield).

[0479] LCMS: Method H, 2.94 min; MS: ES+408.2; 1¹H NMR (400MHz, DMSO-d⁶) δppm: 9.32 (d, J = 6.4Hz, 1H), 8.26 (d, J = 1.6Hz, 1H), 7.98 (d, J = 8.8Hz, 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.

[0480] Step (iii) Substitute Synthesis

[0481] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0482] Under a nitrogen atmosphere, K₂CO₃ (500.80 g, 3629.01 mmol) was added to a solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (600.0 g, 1209.67 mmol) in THF (6 L) under stirring for 15 to 20 min. Cyanogen bromide (153.87 g, 1451.60 mmol) in THF (1.2 L) was added dropwise at 0 °C. The mixture was warmed to 0 °C and stirred for 1 h. The product was analyzed by TLC (mobile phase: 50% EtOAc in n-hexane solution, product R). f 0.05; 10% MeOH / DCM product R f0.4 Monitor the progress of the reaction. Quench the mixture with water (3 L) and extract with EtOAc (3 x 6 L). Wash the combined organic phases with a brine solution (1.5 L), dry with anhydrous Na2SO4 and concentrate under reduced pressure to give crude 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxy-methyl)pyrrolidine-3-yl)oxazol-2-carboxamide (505 g). Suspend the crude substance (505 g) in IPA (5 L, 10 vol) and heat at 80 °C to give a clear solution. The mixture was slowly cooled to rt, and then a crystalline solid was formed at 0 °C. The solid 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)-pyrrolidine-3-yl)oxazol-2-carboxamide, which was a white crystalline solid (400.0 g, 982.80 mmol).

[0483] 400 g of the material recrystallized from IPA was suspended in 10 L, 25 vol of IPA, 100 g of activated charcoal was added, and the mixture was heated at 80 °C for 1 h. The hot mixture was then passed through Celite Hyflow. TM Filter and wash with hot IPA (800 mL, 2 vol). Allow the filtrate to cool slowly to rt, and then form a crystalline solid at 0 °C. Collect the solid by suction filtration, wash with cold IPA (400 L, 1 vol), and dry under vacuum at 50 °C to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide, which is a white crystalline solid (320.0 g, 786.24 mmol, 63% yield).

[0484] LCMS: Method H1, 2.81 min; MS: ES+408.2; HPLC: Method P3, 24.22 min; Chiral HPLC: Method Y, 28.97 min; 1H NMR (400MHz, DMSO-d6) δppm: 9.29 (d, J=6.8Hz, 1H), 8.22 (d, J=2.0Hz, 1H), 7.95 (dd, J=8.8, 2.4Hz, 1H), 7.6 7(d, J=8.8Hz, 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; HPLC: Method Y26, 27.87 min; DSC peak temperature (melting) = 132.7℃; High-resolution MS: ES-406.1521 (calculated exact mass 407.1594). XRPD data were obtained on a Bruker AXS D8 Advance, as shown in the table below: Cu,kα: 1.540598; 1.544426; Kα2 / Kα1=0.50.

[0485]

[0486] Alternative recrystallization

[0487] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0488] Crude 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (134.4 g, 98.9% LC purity) was suspended in ethanol (1340 mL, 10 vol) and heated to 55–65 °C to form a brown solution. The mixture was allowed to cool to 40–45 °C, and seed material (135 mg) from a similar smaller batch was added. The mixture was stirred at 40–45 °C for 30 min. The mixture was then cooled to rt and stirred overnight. The beige suspension was filtered and the solid was collected. The solid was washed with ethanol (2 x 260 mL), the filter cake was dried under vacuum, and then dried overnight at 60 °C to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxy-methyl)pyrrolidine-3-yl)oxazol-2-carboxamide, which was a grayish-white solid (116.2 g, 86% yield from crude product).

[0489] MS: ES+408.2; 1 H NMR (400MHz, CDCl3) δppm: 8.24 (d, J=2.0Hz, 1H), 7.66 (dd, J=8.8, 2.0Hz, 1H), 7.56 (s , 1H), 7.47 (d, J=8.4Hz, 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); NMR residual solvent = EtOH (2260 ppm); HPLC purity 99.6%; DSC peak temperature (melting) = 86.96℃.

[0490] Example 2 was formulated as an aqueous suspension containing 0.1% (w / v) Tween-80 and 0.5% (w / v) hydroxypropyl methylcellulose.

[0491] Example 3

[0492] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0493]

[0494]

[0495] (i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K2CO3, CNBr, THF, 0°C to rt.

[0496] Step (i)

[0497] (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-(fluoromethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0498] TBD (0.07 g, 0.50 mmol) was added in portions to a solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate (0.15 g, 0.50 mmol) and (2S,4R)-4-amino-2-(fluoromethyl)pyrrolidine-1-carboxylate tert-butyl ester (CAS1207853-03-9, from Angene, 0.11 g, 0.50 mmol) in THF (4 mL) under stirring. The mixture was warmed to rt and stirred for 8 h, then poured into water (40 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 51% EtOAc in n-hexane solution) to give (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.09 g, 0.19 mmol, 37% yield).

[0499] LCMS: Method C, 1.76 min; MS: ES-469.1.

[0500] Step (ii)

[0501] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0502] TFA (0.9 mL, 10 vol) was added dropwise to a solution of (2S,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(fluoromethyl)pyrrolidine-1-carboxylate (0.09 g, 0.19 mmol) stirred in a DCM (5 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.16 g, quantitative yield).

[0503] LCMS: Method C, 1.35 min; MS: ES-368.9.

[0504] Step (iii)

[0505] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0506] K₂CO₃ (0.13 g, 0.96 mmol) was added to a solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.15 g, 0.32 mmol) in THF (5 mL) under stirring, 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 for 1 h under stirring, then poured into water (40 mL) and extracted with EtOAc (2 x 40 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 69% EtOAc in n-hexane solution) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)-pyrrolidine-3-yl)oxazol-2-carboxamide (0.01 g, 0.03 mmol, two-step 16% yield).

[0507] LCMS: Method H, 2.96 min; MS: ES+396.1; 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 9.39 (d, J = 6.8Hz, 1H), 8.25 (s, 1H), 7.98 (d, J = 7.2Hz, 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.

[0508] Example 4

[0509] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0510]

[0511]

[0512] (i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K2CO3, CNBr, THF, 0°C to rt.

[0513] Step (i)

[0514] (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazol-2-formamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester

[0515] TBD (0.38 g, 2.76 mmol) was added in portions to a solution of 5-(5-cyano-2-methoxyphenyl)oxazol-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) under stirring 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 x 70 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 30% EtOAc in n-hexane solution) to give (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.30 g, 0.70 mmol, 38% yield).

[0516] LCMS: Method C, 1.61 min; MS: ES-425.1.

[0517] Step (ii)

[0518] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0519] TFA (0.9 mL, 3 vol) was added dropwise to a solution of (2R,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.30 g, 0.70 mmol) stirred in DCM (6 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.30 g, 0.68 mmol, 97% yield).

[0520] LCMS: Method C, 1.22 min; MS: ES+327.1.

[0521] Step (iii)

[0522] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0523] K₂CO₃ (0.28 g, 2.04 mmol) was added to a solution of 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide TFA salt (0.30 g, 0.68 mmol) in THF (7 mL) under stirring and stirred for 10 min. Cyanide bromide (0.07 g, 0.68 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 1% MeOH in DCM) to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide (0.10 g, 0.28 mmol, 42% yield).

[0524] LCMS: Method H, 2.73 min; MS: ES+352.2; 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 9.35 (d, J = 6.8Hz, 1H), 8.25 (s, 1H), 7.95 (d, J = 7.2Hz, 1H), 7.84 (s, 1H), 7.41 (d, J = 8.4Hz, 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 = 6.4Hz, 3H). Chiral SFC: Method Y6, 3.76 min.

[0525] Example 5

[0526] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0527]

[0528]

[0529] (i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K2CO3, CNBr, THF, 0°C to rt.

[0530] Step (i)

[0531] (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0532] TBD (0.61 g, 4.41 mmol) was added in portions to a solution of 5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylate (0.80 g, 2.94 mmol) and tert-butyl(2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS1207853-53-9, 0.67 g, 2.94 mmol) in THF (20 mL) under stirring at 0 °C. The mixture was stirred at 0 °C for 6 h, then poured into water (50 mL) and extracted with EtOAc (2 x 70 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 50% EtOAc in n-hexane solution) to give (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.25 g, 0.55 mmol, 18% yield).

[0533] LCMS: Method C1, 1.29 min; MS: ES+457.2.

[0534] Step (ii)

[0535] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0536] TFA (2.5 mL, 10 vol) was added dropwise to a solution of (2S,4R)-4-(5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.25 g, 0.55 mmol) stirred in a DCM (5 mL) at 0 °C. The mixture was warmed to rt and stirred for 2 h, then concentrated under reduced pressure to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.32 g, quantitative yield).

[0537] LCMS: Method C1, 1.00 min; MS: ES+357.3.

[0538] Step (iii)

[0539] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0540] K₂CO₃ (0.27 g, 1.98 mmol) was added to a solution of 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.31 g, 0.66 mmol) in THF (15 mL) under stirring. After stirring for 10 min, cyanogen bromide (0.07 g, 0.66 mmol) was added to the 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 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 60% EtOAc in n-hexane solution) to give 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.11 g, 0.29 mmol, two-step 52% ​​yield).

[0541] LCMS: Method H, 2.66 min; MS: ES+382.2; 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 9.34 (d, J = 6.8Hz, 1H), 8.25 (s, 1H), 7.96 (d, J = 8.0Hz, 1H), 7.85 (s, 1H), 7.43 (d, J = 8.8Hz, 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.

[0542] Example 6

[0543] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0544]

[0545]

[0546] (i) AgOTf, EtOAc, 80℃; (ii) LiOH.H2O, H2O, THF, 0℃ to rt; (iii) POCl3, pyridine, 0℃ to rt; (iv) TFA, DCM, 0℃ to rt; (v) K2CO3, CNBr, THF, 0℃ to rt.

[0547] Step (i)

[0548] 4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylic acid ethyl ester

[0549] Silver trifluoromethanesulfonate (CAS 2923-28-6, from Combi-blocks, 10.66 g, 41.51 mmol) was added to a solution of ethyl 5-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylate (3.5 g, 13.83 mmol) and ethyl oxazolyl ester (CAS 617-36-7, from Sigma-Aldrich, 4.86 g, 41.51 mmol) stirred in EtOAc (35 mL). The mixture was heated at 80 °C for 24 h and then poured into water (120 mL) via Celite Hyflow. TM The mixture was filtered and the filtrate was extracted with EtOAc (3 x 70 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 16% EtOAc in n-hexane) to give ethyl 4-(5-cyano-2-methoxyphenyl)oxazolium-2-carboxylate (0.53 g, 1.95 mmol, 14% yield).

[0550] LCMS: Method C, 1.65 min; MS: ES+273.1.

[0551] Step (ii)

[0552] 4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylic acid

[0553] Lithium hydroxide monohydrate (0.23 g, 5.51 mmol) was added fractionally to a solution of ethyl 4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylate (0.50 g, 1.84 mmol) in THF:water (1:1, 10 mL) under stirring 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 Na₂SO₄ and concentrated under reduced pressure to give 4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylic acid (0.15 g, 0.61 mmol, 33% yield). LC-MS: Method C2, 0.81 min; MS: ES+245.1.

[0554] Step (iii)

[0555] (2S,4R)-4-(4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0556] POCl3 (0.3 g, 0.18 mL, 1.97 mmol) was added dropwise to a solution of 4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxylic acid (0.16 g, 0.65 mmol) and (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (CAS 1207853-53-9, 0.15 g, 0.65 mmol) stirred in pyridine (3 mL) at 0 °C. The mixture was stirred at 0 °C for 10 min, then poured into water (50 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 22% EtOAc in n-hexane solution) to give (2S,4R)-4-(4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.18 g, 0.39 mmol, 60% yield).

[0557] LCMS: Method C, 1.70 min; MS: ES-455.1.

[0558] Step (iv)

[0559] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0560] TFA (0.85 mL, 5 vol) was added dropwise to a solution of (2S,4R)-4-(4-(5-cyano-2-methoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.17 g, 0.37 mmol) stirred in a DCM (5 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.25 g, quantitative yield).

[0561] LCMS: Method C, 1.32 min; MS: ES+357.1.

[0562] Step (v)

[0563] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0564] K₂CO₃ (0.21 g, 1.53 mmol) was added to a solution of 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)oxazol-2-carboxamide TFA salt (0.24 g, 0.51 mmol) in THF (5 mL) under stirring and stirred for 10 min. Cyanogen bromide (0.04 g, 0.41 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 71% EtOAc in n-hexane solution) to give 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.05 g, 0.14 mmol, two-step 36% yield).

[0565] LCMS: Method H, 2.80 min; MS: ES-380.1; 1¹H NMR (400MHz, DMSO-d⁶) δppm: 9.25 (d, J = 6.8Hz, 1H), 8.72 (s, 1H), 8.44 (d, J = 1.6Hz, 1H), 7.89 (dd, J = 8.8, 2.0Hz, 1H), 7.37 (d, J = 8.8Hz, 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.

[0566] Example 7

[0567] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-

[0568] 2-Formamide

[0569]

[0570] (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.

[0571] Step (i)

[0572] (2S,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylammono)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0573] TBD (0.38 g, 2.77 mmol) was added in portions to a solution of ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylate (0.70 g, 1.84 mmol) and (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.42 g, 1.84 mmol) in THF (8 mL) under stirring at 0 °C. The mixture was stirred at 0 °C for 2 h, then poured into water (100 mL) and extracted with EtOAc (3 x 70 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 25% EtOAc in n-hexane solution) to give (2S,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.37 g, 0.66 mmol, 35% yield).

[0574] LCMS: Method C, 2.00 min; MS: ES+564.4, 566.4.

[0575] Step (ii)

[0576] (2S,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0577] Zinc cyanide (0.18 g, 1.55 mmol), zinc powder (0.02 g, 0.31 mmol), and 1,1′-ferrocene dimethyl-bis(diphenylphosphine) (0.07 g, 0.12 mmol) were added to a solution of (2S,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxymethyl)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.35 g, 0.62 mmol) stirred in DMA (3 mL). The mixture was degassed with N2 for 15 min, followed by the addition of tris(dibenzylacetone)dipalladium(0) (0.11 g, 0.12 mmol). The mixture was heated in a microwave at 140 °C for 1 h, then cooled to rt, poured into ice-cold water (70 mL), and extracted with EtOAc (3 x 70 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 22% EtOAc in n-hexane solution) to give (2S,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)-phenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.25 g, 0.5 mmol, 80% yield).

[0578] LCMS: Method C, 1.83 min; MS: ES+: 511.4.

[0579] Step (iii)

[0580] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0581] TFA (1.25 mL, 5 vol) was added dropwise to a solution of (2S,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.25 g, 0.49 mmol) stirred in a DCM (3 mL). The mixture was warmed 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)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.39 g, quantitative yield).

[0582] LCMS: Method C, 1.38 min; MS: ES+411.1.

[0583] Step (iv)

[0584] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0585] K₂CO₃ (0.31 g, 2.23 mmol) was added to a solution of 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.39 g, 0.74 mmol) in THF (5 mL) under stirring and stirred for 10 min. Cyanide bromide (0.06 g, 0.59 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (70 mL) and extracted with EtOAc (3 x 70 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 40% EtOAc in n-hexane solution) to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.14 g, 0.33 mmol, two-step 66% yield).

[0586] LCMS: Method H, 3.03 min; MS: ES-434.1; 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 9.40 (d, J = 6.8Hz, 1H), 8.51 (d, J = 1.6Hz, 1H), 8.12 (dd, J = 8.8, 2.0Hz, 1H), 7.86 (s, 1H), 7.82 (d, J = 8.4Hz, 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.

[0587] Example 8

[0588] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0589]

[0590] (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.

[0591] Step (i)

[0592] (2R,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylamino)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester

[0593] TBD (0.22 g, 1.57 mmol) was added in portions to a solution of ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxylate (0.40 g, 1.05 mmol) and (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate tert-butyl ester (CAS 348165-63-9, 0.23 g, 1.16 mmol) in THF (8 mL) under stirring. The mixture was warmed to rt and stirred for 3 h, then poured into water (50 mL) and extracted with EtOAc (3 x 60 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 32% EtOAc in n-hexane solution) to give (2R,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.14 g, 0.26 mmol, 24% yield).

[0594] LCMS: Method C, 2.01 min; MS: ES+534.2, 536.2.

[0595] Step (ii)

[0596] (2R,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazol-2-formamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester

[0597] Zinc cyanide (0.07 g, 0.61 mmol), zinc powder (0.01 g, 0.12 mmol), and 1,1′-ferrocene dimethyl-bis(diphenylphosphine) (0.03 g, 0.05 mmol) were added to a solution of (2R,4R)-4-(5-(5-bromo-2-(trifluoromethoxy)phenyl)oxazol-2-carboxymethylamino)-2-methylpyrrolidone-1-carboxylic acid tert-butyl ester (0.13 g, 0.24 mmol) stirred in DMA (1.3 mL). The mixture was degassed with N2 for 10 min, followed by the addition of tris(dibenzylacetone)dipalladium(0) (0.04 g, 0.05 mmol). The mixture was heated in a microwave at 140 °C for 1 h, then cooled to rt, poured into water (20 mL), and extracted with EtOAc (3 x 30 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 38% EtOAc in n-hexane solution) to give (2R,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)-oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.05 g, 0.10 mmol, 42% yield).

[0598] LCMS: Method C, 1.88 min; MS: ES+: 425.6 (M-56).

[0599] Step (iii)

[0600] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0601] TFA (0.15 mL, 3 vol) was added dropwise to a solution of (2R,4R)-4-(5-(5-cyano-2-(trifluoromethoxy)phenyl)oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.05 g, 0.1 mmol) stirred in a DCM (2 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.08 g, quantitative yield).

[0602] LCMS: Method C, 1.38 min; MS: ES+381.2.

[0603] Step (iv)

[0604] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0605] K₂CO₃ (0.07 g, 0.48 mmol) was added to a solution of 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide TFA salt (0.08 g, 0.16 mmol) in THF (4 mL) and stirred for 10 min. Cyanide bromide (0.02 g, 0.16 mmol) was added at 0 °C. The mixture was stirred for 1 h at rt, then poured into water (20 mL) and extracted with EtOAc (3 x 25 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 47% EtOAc in n-hexane solution) to give 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide (0.02 g, 0.05 mmol; two-step 47% yield).

[0606] LCMS: Method H, 3.02min; MS: ES-404.0; 1¹H NMR (400MHz, DMSO-d⁶) δppm: 9.43 (d, J = 6.8Hz, 1H), 8.51 (s, 1H), 8.11 (d, J = 8.4Hz, 1H), 7.84 (s, 1H), 7.82 (d, J = 8.4Hz, 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 = 6.4Hz, 3H). Chiral SFC: Method Y8, 5.02 min.

[0607] Example 9

[0608] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0609]

[0610] (i) TBD, THF, 0°C to rt; (ii) TFA, DCM, 0°C to rt; (iii) K2CO3, CNBr, THF, 0°C to rt.

[0611] Step (i)

[0612] (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formylamino)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester

[0613] TBD (0.29 g, 2.07 mmol) was added in portions to a solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate (0.50 g, 1.72 mmol) and (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate tert-butyl ester (CAS 348165-63-9, 0.27 g, 1.38 mmol) in THF (10 mL) under stirring. The mixture was warmed to rt and stirred for 2 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 40% EtOAc in n-hexane solution) to give (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.3 g, 0.67 mmol, 38% yield).

[0614] LCMS: Method C, 1.78 min; MS: ES+389.3 (M-56).

[0615] Step (ii)

[0616] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0617] TFA (0.9 mL, 3 vol) was added dropwise to a solution of (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (0.3 g, 0.67 mmol) stirred in a DCM (7 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.29 g, quantitative yield).

[0618] LCMS: Method C, 1.39 min; MS: ES+345.3.

[0619] Step (iii)

[0620] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0621] K₂CO₃ (0.26 g, 1.89 mmol) was added to a solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide TFA salt (0.29 g, 0.63 mmol) in THF (7 mL) under stirring and stirred for 10 min. Cyanide bromide (0.07 g, 0.63 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then water (70 mL) was poured in to form a precipitate, which was collected by filtration under reduced pressure to give 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide (0.13 g, 0.35 mmol, 52% yield in two steps).

[0622] LCMS: Method H, 2.75 min; MS: ES+370.1; 1¹H NMR (400MHz, DMSO-d⁶) δppm: 9.31 (d, J = 6.4Hz, 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.

[0623] Example 10

[0624] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0625]

[0626] Step (i)

[0627] 4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylic acid ethyl ester

[0628] Silver trifluoromethanesulfonate (CAS 2923-28-6, from Combi-blocks, 0.96 g, 3.75 mmol) was added to a solution of 3-(2-bromoacetyl)-4-cyclopropoxybenzyl nitrile (0.70 g, 2.50 mmol) and ethyl oxalate (CAS 617-36-7, from Sigma-Aldrich, 0.66 g, 3.75 mmol) stirred in EtOAc (10 mL). The mixture was heated at 80 °C for 16 h. Another identical batch was combined and then poured into water (100 mL) via Celite Hyflow. TM The mixture was filtered and the filtrate was extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 10% EtOAc in n-hexane) to give ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazolium-2-carboxylate (0.35 g, 1.17 mmol, 23% yield).

[0629] LCMS: Method H1, 3.40 min; MS: ES+299.0.

[0630] Step (ii)

[0631] (2S,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0632] TBD (0.18 g, 1.28 mmol) was added in portions to a solution of ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate (0.32 g, 1.07 mmol) and (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1207853-53-9, 0.25 g, 1.07 mmol) in DMF (7 mL) under stirring. The mixture was warmed to rt and stirred for 2 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 rapid column chromatography (silica gel, 30% EtOAc in n-hexane solution) to give (2S,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.10 g, 0.22 mmol, 20% yield).

[0633] LCMS: Method H1, 3.51 min; MS: ES-481.3.

[0634] Step (iii)

[0635] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt

[0636] TFA (1 mL, 10 vol) was added dropwise to a solution of (2S,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.1 g, 0.22 mmol) stirred in a DCM (5 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.14 g, quantitative yield).

[0637] LCMS: Method J, 3.61 min; MS: ES+383.0.

[0638] Step (iv)

[0639] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0640] K₂CO₃ (0.12 g, 0.84 mmol) was added to a solution of 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.14 g, 0.28 mmol) in THF (5 mL) under stirring and stirred for 5 min. Cyanide bromide (0.03 g, 0.28 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 45% EtOAc in n-hexane solution) to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.04 g, 0.09 mmol, 47% yield, two steps).

[0641] LCMS: Method H1, 2.97 min; MS: ES-406.2; 1 H NMR (400MHz, DMSO-d6) δppm: 9.24 (d, J=6.8Hz, 1H), 8.52 (s, 1H), 8.43 (d, J=2.0Hz, 1H), 7.92 (dd, J=8.8, 2.0Hz, 1H), 7.64 (d, J=8.4Hz, 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).

[0642] Chiral HPLC: Method Y15, 10.0 min.

[0643] Example 11

[0644] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0645]

[0646]

[0647] Step (i)

[0648] (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0649] TBD (0.12 g, 0.83 mmol) was added in portions to a solution of ethyl 5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate (0.25 g, 0.83 mmol) and (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5, 0.19 g, 0.83 mmol) in toluene (5 mL) under stirring at 0 °C. The mixture was warmed to rt and stirred for 1 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 rapid column chromatography (silica gel, 35% EtOAc in n-hexane solution) to give (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.08 g, 0.16 mmol, 19% yield).

[0650] LCMS: Method C1, 1.41 min; MS: ES+483.4.

[0651] Step (ii)

[0652] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt

[0653] TFA (0.22 mL, 3 vol) was added dropwise to a solution of (2R,4R)-4-(5-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.07 g, 0.15 mmol) stirred in DCM (1 mL). The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.10 g, quantitative yield).

[0654] LCMS: Method C1, 1.06 min; MS: ES+383.2.

[0655] Step (iii)

[0656] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0657] K₂CO₃ (0.08 g, 0.60 mmol) was added to a solution of 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.10 g, 0.20 mmol) in THF (5 mL) under stirring and stirred for 5 min. Cyanide bromide (0.02 g, 0.20 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 45% EtOAc in n-hexane solution) to give 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.02 g, 0.04 mmol, 29% yield, two steps).

[0658] LCMS: Method H1, 2.94 min; MS: ES+408.2; 1 ¹H NMR (400MHz, DMSO-d⁶) δppm: 9.18 (d, J = 6.8Hz, 1H), 8.24 (s, 1H), 7.96 (d, J = 8.4Hz, 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.

[0659] Example 12

[0660] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0661]

[0662]

[0663] Step (i)

[0664] tert-butyl(2R,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate

[0665] TBD (0.19 g, 1.34 mmol) was added in batches to a solution of ethyl 4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxylate (0.40 g, 1.34 mmol) [Example 10; step (i)] and (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate tert-butyl ester (CAS1123305-98-5, 0.31 g, 1.34 mmol) in toluene (5 mL) under stirring. The mixture was warmed 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 rapid column chromatography (silica gel, 40% EtOAc in n-hexane solution) to give (2R,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-formamide)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.22 g, 0.46 mmol, 34% yield). LCMS: Method C1, 1.42 min, MS: ES+483.3.

[0666] Step (ii)

[0667] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt

[0668] TFA (0.66 mL, 3 vol) was added dropwise to a solution of (2R,4R)-4-(4-(5-cyano-2-cyclopropoxyphenyl)oxazol-2-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.22 g, 0.46 mmol) stirred in a DCM (5 mL) at 0 °C. The mixture was warmed to rt and stirred for 1 h, then concentrated under reduced pressure to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.28 g, quantitative yield).

[0669] LCMS: Method C1, 1.09 min. MS: ES+383.2.

[0670] Step (iii)

[0671] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0672] K₂CO₃ (0.23 g, 1.69 mmol) was added to a solution of 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide TFA salt (0.28 g, 0.56 mmol) in THF (5 mL) under stirring and stirred for 5 min. Cyanide bromide (0.06 g, 0.56 mmol) was added at 0 °C. The mixture was stirred for 1 h under stirring, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by rapid column chromatography (silica gel, 65% EtOAc in n-hexane solution) to give 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide (0.06 g, 0.14 mmol, 31% yield, two steps).

[0673] LCMS: Method H1, 3.01 min; MS: ES+408.2; 1 H NMR (400MHz, DMSO-d6) δppm: 9.10 (d, J=7.6Hz, 1H), 8.52 (s, 1H), 8.38 (s, 1H), 7.91 (d, J=8.4, 2.0Hz, 1H), 7.64 (d, J=8.8Hz, 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.

[0674] Example 13

[0675] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0676]

[0677] The title compound can be prepared in step (i) using (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester (CAS 708274-46-8) in a manner similar to that in Example 1.

[0678] Example 14

[0679] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0680]

[0681] The title compound can be prepared in step (i) using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in a manner similar to that in Example 4.

[0682] Example 15

[0683] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0684]

[0685] The title compound can be prepared in step (i) using tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5) in a manner similar to that in Example 5.

[0686] Example 16

[0687] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0688]

[0689] The title compound can be prepared in step (iii) using tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5) in a manner similar to that in Example 6.

[0690] Example 17

[0691] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide

[0692]

[0693] The title compound can be prepared in step (i) using tert-butyl (2R,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (CAS 1123305-98-5) in a manner similar to that in Example 7.

[0694] Example 18

[0695] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidone-3-yl)oxazol-2-carboxamide

[0696]

[0697] The title compound can be prepared in step (i) using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in a manner similar to that in Example 8.

[0698] Example 19

[0699] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide

[0700]

[0701] The title compound can be prepared in step (i) using tert-butyl (2S,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (CAS 708274-46-8) in a manner similar to that in Example 9.

[0702] Bioactivity of the compounds of this invention

[0703] abbreviation:

[0704] TAMRA carboxytetramethylrhodamine

[0705] PCR polymerase chain reaction

[0706] PBS (Phosphate-Buffered Saline)

[0707] EDTA (ethylenediaminetetraacetic acid)

[0708] Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol

[0709] NP-40 Nonidet P-40, Octylphenoxypolyethoxyethanol

[0710] BSA (Bovine Serum Albumin)

[0711] PNS (Peripheral Nervous System)

[0712] BH3 Bcl-2 homologous domain 3

[0713] PTEN phosphatase and tensin homolog

[0714] SDS-PAGE with sodium dodecyl sulfate and polyacrylamide gel electrophoresis

[0715] DMSO (dimethyl sulfoxide)

[0716] YFP yellow fluorescent protein

[0717] VME vinyl methyl ester

[0718] HA hemagglutinin

[0719] Ahx Aminocaproic acid

[0720] USP30 Biochemical IC 50 Determination method

[0721] Diluted plates were prepared in 96-well polypropylene V-shaped substrates (Greiner #651201) with 50% DMSO at a final concentration 21 times the final concentration (2100 μM for a final concentration of 100 μM). A typical 8-point dilution series was 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. The reaction was carried out in duplicate in black 384-well plates (small volume, Greiner 784076), with a final reaction volume of 21 μL. 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 β-mercaptoethanol) to achieve 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 at room temperature for 30 min. The reaction was initiated by adding 50 nM of a TAMRA-labeled peptide linked to ubiquitin via isopeptide bonds as a fluorescent polarization substrate. The reaction was read immediately after adding the substrate and incubating at room temperature for 2 hours. Readings were performed on a Pherastar Plus (BMGLabtech). λ excitation was 540 nm; λ emission was 590 nm.

[0722] Exemplary compounds in USP30 biochemical IC 50 Activity in the assay:

[0723]

[0724] Parameter

[0725] Example compounds in USP30 Biochemistry IC 50 Activity in the assay:

[0726]

[0727] In vitro pharmacology

[0728] Pharmacological characterization of Example 2 was performed in the Eurofins Cerep Safety Screen 44 assay. At a single concentration of 10 μM, less than 50% inhibition of binding to or enzyme activity of all targets in this assay was observed. Due to the low affinity for the targets in this assay, the likelihood of extratarget interactions in Example 1 is low.

[0729] Safety Pharmacology

[0730] In stably expressing CHO cells, the effect of Example 2 on hERG potassium channels was evaluated at concentrations ranging from 0.01 to 30 μM. Example 2 produced a maximum inhibition of 35% of the hERG current amplitude at 30 μM, indicating that it had little tendency to affect the QT interval.

[0731] Genetic toxicology

[0732] Example 2 was evaluated in the bacterial reverse mutagenesis assay (Ames) and the in vitro small nucleus assay. All in vitro tests were performed at concentrations up to the cytotoxic or insoluble limits, with and without exogenous metabolic activation. In the reverse mutagenesis assay, Example 2 did not induce mutations in Salmonella Typhimurium strains TA98, TA100, TA1535, and TA97a, and Escherichia Coli strain WP2 uvrA PKM101, with and without metabolic activation, when tested at up to 5000 μg / plate.

[0733] The induction of chromosome damage was assessed in TK6 cells using an in vitro small nucleus assay. Example 2 was negative for small nucleus induction when incubated for 3 hours in the presence of exogenous metabolic activation followed by 27 hours of recovery, and when incubated for 27 hours in the absence of exogenous metabolic activation followed by 27 hours of recovery.

[0734] USP30 Endogenous Cell Target Binding Assay

[0735] HeLa cells stably overexpressing YFP-Parkinsonin were seeded into 6-well culture dishes. Once adhered, cells were treated for 1 hour at 37°C and 5% CO2 with an appropriate concentration of the test compound or medium control. Whole-cell lysates were prepared by scraping cells into cold PBS, centrifuging, and lysing for 10 minutes 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 β-mercaptoethanol, EDTA-free cOplete flakes (Roche), PhosStop flakes (Roche)). A 20 μg protein equivalent from the clarified cell lysate was incubated at room temperature with a final concentration of 2.5 μM HA-Ahx-Ahx-Ub-VME probe. The reaction was terminated by adding 5x SDS loading buffer, and the protein was separated by SDS-PAGE and Western blotting. USP30 was detected using anti-USP30 sheep S746D antibody (MRC PPU Reagents and Services) and rabbit anti-sheep secondary IgG (H+L) horseradish peroxidase conjugate (Thermo#31480), and visualized on a GE LAS4000 imager using ECL reagent (GE#RPN2109). Target binding was measured by quantifying the bands representing USP30 and USP30 bound to the Ub-VME probe, and comparing this proportion to media-treated controls.

[0736] TOM20-Ubiquitination Assay

[0737] Human cell lines can be attacked with mitochondrial depolarizers (ionocarriers such as CCCP and valine) or mitochondrial complex inhibitors (oligomycin and antimycin A) to induce TOM20 ubiquitination, followed by further promotion of TOM20 ubiquitination in the presence of a USP30 inhibitor. TOM20 ubiquitination is then assessed by Western blotting of cell lysates, possibly due to the increased molecular weight (8 kDa) of each added ubiquitin molecule, leading to the laddering of TOM20 immunoreactive bands. The level of TOM20 ubiquitination can be quantified using chemiluminescent densitometric assay of the ladder-like immunoreactive bands.

[0738] In vitro cytotoxicity (Cellular Tox): Using AlamarBlue TM The compound was used as an endpoint in HCT116 human colorectal cancer cells. Cytotoxicity was measured over a 96-hour period of continuous compound exposure.

[0739] Further research

[0740] log P: partition coefficient; lipophilicity measurement.

[0741] log D: Distribution coefficient; a measure of lipophilicity.

[0742] TPSA: Topological Polar Surface Area.

[0743] Turbidimetric solubility: The test compound solution prepared in DMSO was diluted in an aqueous buffer. The turbidimetric method was used as the endpoint by measuring the absorbance at 620 nm.

[0744] FaSSIF: Simulated intestinal fluid under fasting conditions, measured at pH 6.5.

[0745] Hep Cl mice: In vitro hepatocyte clearance rate in mouse cells.

[0746] Hep Cl human: In vitro hepatocyte clearance rate in human cells.

[0747] plasma f u,p : The free fraction of compounds in plasma preparations determined by in vitro equilibrium dialysis.

[0748] brain f u,br The free fraction of compounds in brain homogenate preparations determined by in vitro equilibrium dialysis.

[0749] Cl u In vitro clearance rate. Cl as defined in this application u This is an adjusted clearance rate, which is calculated from the intrinsic clearance rate. Since the intrinsic clearance rate of hepatic metabolism is a predicted clearance rate, it is determined by incubation of the compound in hepatocyte preparations. The lower the value in mL / min / kg, the more stable the compound.

[0750] Cl clearance in vivo: a pharmacokinetic measure of the volume of plasma (or any matrix) from which the substance is completely removed per unit time. A lower value, expressed in mL / min / kg, indicates greater stability of the compound.

[0751] Oral F: Oral bioavailability.

[0752] MDR1-MDCK (Martin-Darby dog ​​kidney cell monolayer) (in vitro) flux assay.

[0753] WT-MDCK (wild type) in vitro flux

[0754] Kp uu It represents the ratio of unbound drug in the brain to unbound drug in the plasma, and can indicate the potential for treatment of peripheral and / or CNS indications.

[0755]

[0756]

[0757]

[0758] These examples demonstrate beneficial properties, showcasing potential advantages over other compounds. For instance, the low observed IV plasma clearance rates of 41 and 20 mL / min / kg, respectively, measured in mice for Examples 1 and 2, demonstrate valuable plasma stability, and the compounds exhibit very good oral bioavailability of 37% and 47%, respectively.

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766] Comparison data

[0767] References A, B, C, D, and E are known DUB inhibitors, which have been identified as having similar activity to USP30 inhibitors and possess structural similarity to the compounds of this invention, which have nitrile amine structural features. References B, C, D, and E are disclosed to have UCHL1 inhibitory activity in WO 2016 / 046530.

[0768] The valence of USP30

[0769] As measured using the aforementioned biochemical assay, Examples 1 to 11 of the present invention exhibited significantly stronger activity against USP30 than Reference Examples A, B, C, D, and E. Example 12 showed significantly stronger activity against USP30 than Reference Examples B, C, D, and E. For example, the activity of Examples 1 to 11 was 6.8 to 34 times that of Reference Example A, 16 to 155 times that of Reference Examples B, C, and D, and at least 440 times that of Reference Example E.

[0770] Compared to other DUBs' selectivity towards USP30

[0771] The provided data demonstrate that, compared to Reference A, Examples 1, 2, 4, 6, 7, 11, and 12 are significantly more selective for USP30 relative to the nine DUBs (USP2, USP6, USP10, USP15, USP16, USP21, USP25, USP28, and USP46). The activity intensity of these examples for USP30 is at least 110 to 7820 times that of each of the nine DUBs (Example 7 was not tested for USP15). This is a significant selectivity advantage relative to Reference A, which has an activity intensity as low as 2.2 times.

[0772] Compared to the selectivity of UCHL1 for USP30

[0773] The provided data demonstrate that, compared to references B, C, D, and E, Examples 1, 2, 4, 6, 7, 11, and 12 exhibit significantly higher selectivity for USP30 relative to UCHL1. Examples 1, 2, 4, 6, 7, and 11 show greater than 30,000 times higher activity intensity for USP30 relative to UCHL1, and Example 12 shows greater than 4,412 times higher activity intensity, while references B, C, D, and E show only 3.0, 1.3, 0.8, and 1.5 times higher activity intensity, respectively; 'C' shows greater selectivity for UCHL1.

[0774] The selectivity of cathepsins B, K, L, S, and V for USP30

[0775] The data provided demonstrate that, compared to Reference Example A, Examples 1, 2, 4, 6, 7, 11, and 12 exhibit significantly greater selectivity for USP30 relative to cathepsins (B, K, L, S, and V). These examples demonstrate greater than 1540-fold higher activity intensity for USP30 compared to each of the cathepsins. This represents a significant selectivity advantage relative to Reference Example A, which shows only an 11.6-fold increase in activity intensity for cathepsin K.

[0776] Compared to the proportions of existing technologies, the advantages of the compounds of the present invention, as identified above, are both significant and unexpected. This superiority, individually and especially in combination, makes the compounds of the present invention particularly suitable for the treatment or prevention of diseases associated with USP30 activity.

[0777] Preclinical in vitro models

[0778] (a) A human renal proximal tubular cell cisplatin injury model, modified for testing renal protective agents.

[0779] Primary human PTC was isolated from a transplant-rejected kidney.

[0780] The isolated cells were seeded into 96-well Transwell inserts (surface area = 0.143 cm²).2 On the culture medium, the density of each insert was 20,000 cells. The medium was refreshed 24 hours after the initial inoculation and on days 3 and 5 of culture.

[0781] Before use in the experiment, the single layer showed an Ω·cm of 80-120. 2 TEER within the range.

[0782] Before exposing cells to cisplatin (20 μM), human PTC monolayers were pre-exposed to Example 2 (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 μM) on the top and basal sides of the cell layer for 1 hour. Subsequently, cells were exposed to Example 2 (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 μM) and cisplatin (20 μM) on the top and basal sides for 24 and 48 hours, respectively. Additionally, parallel experiments included a negative control (Example 2 only, 0.5% DMSO of Example 2, 0.2% dimethylformamide (DMF) as a cisplatin solvent control), a positive control (cisplatin 20 μM), and cisplatin plus a cisplatin uptake inhibitor (dolutegravir 100 μM). Experiments were performed in triplicate in human cell monolayers (n=3) and three different donors (N=3).

[0783] Cell viability assays based on ATP production and LDH release were performed on monolayers treated with the test compounds in 96-well Transwell inserts.

[0784] Example 2 prevented cisplatin-induced ATP loss in a concentration-dependent manner. Example 2 also reduced cisplatin-induced LDH elevation. These data demonstrate that Example 2 can prevent cisplatin-induced toxicity to human proximal tubule epithelial cells.

[0785] Preclinical in vivo model

[0786] The efficacy of the compounds of the present invention can be tested in representative in vivo disease models using standard research methods described in published literature, including, for example:

[0787] (a) Bleomycin-induced pulmonary fibrosis model, which is the main preclinical in vivo model of idiopathic pulmonary fibrosis. [Kobayashi et al., 2016, J Immunol, 197(2):504-516].

[0788] (b) Diet-induced NAFLD and glucose homeostasis model. [Nishida et al., 2013, Lab Invest; Feb; 93(2):230-41].

[0789] (c) The MPTP model of Parkinson's disease, which is a common paradigm for observing neurodegeneration in the brain's dopaminergic system triggered by chemically induced mitochondrial dysfunction. [Karuppagouner et al., 2014, Sci Rep. May 2, 2014; 4:4874].

[0790] (d) Ndufs4KO model of Riemann syndrome. [Kruse et al., 2008, Cell Metab. Apr; 7(4):312-20].

[0791] (e) Ageing rodent models: Effects on 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].

[0792] (f) Unilateral ureteral obstructive nephropathy model (UUO). [Chevalier et al., 2009, Kidney Int 75(11): 1145-1152].

[0793] UUO induces kidney injury characterized by tubular cell damage, interstitial inflammation, and fibrosis. It is used as a model of irreversible postrenal acute kidney injury (AKI). Experimental UUO has demonstrated the molecular mechanisms of apoptosis, inflammation, and fibrosis, all of which are key processes in kidney injury, regardless of the primary damage. Therefore, the UUO model provides researchers with information beyond obstruction (Chevalier et al., 2009, Kidney Int 75(11): 1145-1152).

[0794] Example 2 was evaluated in the UUO model to determine the compound’s ability to alleviate progressive tubulointerstitial fibrosis and chronic kidney disease (CKD).

[0795] On day 1 of this study, adult C57BL / 6 mice were administered the drug via oral feeding according to one of the following dosing regimens: medium, 5 or 1.5 mg / kg (Example 2), twice daily. Two hours after administration on day 1, the study mice underwent surgery to ligate the left ureter at two points. Successful UUO surgery was subsequently confirmed by observation of renal pelvis dilation caused by hydronephrosis. Animals were administered the drug for 10 days according to their prescribed regimens, at which time the kidneys were harvested for histopathological and protein / RNA evaluation. Sirius red staining was performed to assess the extent of collagen deposition, and relative α-smooth muscle actin (α-SMA) expression was assessed using IHC.

[0796] The results demonstrated that twice-daily (po) administration of 5 mg / kg and 1.5 mg / kg of Example 2 statistically reduced collagen deposition, as evidenced by reduced Sirius red staining in ligated kidneys. Evaluation of α-SMA staining showed a statistically significant reduction in α-SMA levels in kidneys with UUO injury when administered orally twice daily with 5 mg / kg and 1.5 mg / kg of Example 2 compared to the medium-treated control.

[0797] (g) AKI can be induced by bilateral renal pedicle clamping, leading to ischemia-reperfusion injury (IRI), which results in severe loss of renal function, renal tubular damage and inflammation [Lu et al., 2012. J Nephrol. 25(5): 738-45].

[0798] Prophylactic administration:

[0799] Example 2 was administered to C57BL / 6 mice at 5 mg / kg and 1.5 mg / kg BID (po), and compared with media treatment from day -1 to day +21. On day 0, mice were anesthetized, their left kidney pedicle was clamped for 45 minutes, and then released to induce IRI. Kidneys were harvested on day +21. Morphology and fibrosis were assessed.

[0800] Body weight was similar across groups and remained constant throughout the observation period. On day +21, Masson trichrome staining showed significantly less tubular atrophy and collagen content in the outer medulla of animals treated in Example 2 (both dose levels). On day +21, fibronectin expression was significantly reduced in both the cortex and outer medulla of animals treated in Example 2 (both dose levels).

[0801] Example 2 demonstrates the efficacy of this IR-induced CKD model. Daily treatment has shown significant benefits in reducing renal tubular atrophy and fibrosis.

[0802] Post-injury management:

[0803] C57BL / 6 mice were anesthetized on day 0 (zero) and their left kidney pedicle was clamped for 45 minutes, then released to induce IRI. Mice were then administered the medium or as described in Example 2; 5 mg / kg (po) BID for 21 days, with the first treatment initiated 5 hours after the IRI procedure (i.e., the treatment dose). Mice were monitored and kidneys were harvested on day +21. Relative cellular morphology and fibrosis of kidney sections were quantitatively assessed using a blinded histological scoring method.

[0804] The body weights of the groups were similar and remained constant throughout the observation period. On day +21, fibronectin staining in the cortex of mice treated in Example 2 was significantly reduced.

[0805] Example 2 demonstrates partial efficacy in this IR-induced CKD model when administered therapeutically. Initial treatment following ischemia-reperfusion injury has shown significant benefit in reducing cortical fibrosis.

[0806] Paragraphs of this invention

[0807] This invention relates to the following:

[0808] 1. A compound of formula (I), selected from formula (I)(i) and formula (I)(ii):

[0809]

[0810] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer, wherein:

[0811] R 1 Selected from (C1-C4)alkyl, (C1-C4)fluoroalkyl and CH2OCH3;

[0812] R 2 Selected from (C1-C4) alkyl, CF3 and cyclopropyl; and

[0813] R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

[0814] 2. The compound according to paragraph 1, wherein R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

[0815] 3. The compound according to paragraph 2, wherein R 1 Selected from methyl and CH2OCH3.

[0816] 4. The compound according to any one of paragraphs 1 to 3, wherein R 2 Selected from methyl, CF3 and cyclopropyl.

[0817] 5. The compound according to paragraph 4, wherein R 2 Selected from methyl and cyclopropyl.

[0818] 6. The compound according to any one of paragraphs 1 to 5, wherein R 3 Selected from hydrogen and fluorine.

[0819] 7. The compound according to paragraph 6, wherein R 3 It is hydrogen.

[0820] 8. The compound according to any one of paragraphs 1 to 7, wherein R 4 and R 5Each is hydrogen.

[0821] 9. The compound described in any one of paragraphs 1 to 8 has the formula (I)(i):

[0822]

[0823] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer.

[0824] 10. The compound according to paragraph 9 has the formula (IA)(i):

[0825]

[0826] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer.

[0827] 11. The compound described in paragraph 10 is selected from:

[0828] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0829] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0830] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0831] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0832] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0833] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0834] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and

[0835] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0836] Or its pharmaceutically acceptable salt.

[0837] 12. The compound according to paragraph 9 has the formula (IB)(i):

[0838]

[0839] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer.

[0840] 13. The compound described in paragraph 12 is selected from:

[0841] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide and 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; or pharmaceutically acceptable salts thereof.

[0842] 14. The compound according to any one of paragraphs 1 to 8 has the formula (I)(ii):

[0843]

[0844] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer.

[0845] 15. The compound according to paragraph 14 has the formula (IA)(ii):

[0846]

[0847] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer.

[0848] 16. The compound described in paragraph 15 is selected from:

[0849] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0850] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0851] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0852] 5-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0853] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0854] 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide;

[0855] 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3R,5S)-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and

[0856] 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0857] Or its pharmaceutically acceptable salt.

[0858] 17. The compound according to paragraph 14 has the formula (IB)(ii):

[0859]

[0860] Its tautomer or a pharmaceutically acceptable salt of the compound or its tautomer.

[0861] 18. The compounds described in paragraph 17 are selected from:

[0862] 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; and

[0863] 4-(5-cyano-2-methoxyphenyl)-N-((3R,5R)-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide;

[0864] Or its pharmaceutically acceptable salt.

[0865] 19. The compound, its tautomer, or a pharmaceutically acceptable salt thereof described in any one of paragraphs 1 to 18, used as a medicine.

[0866] 20. The compound, its tautomer, or a pharmaceutically acceptable salt thereof described in any of paragraphs 1 to 18, for the treatment or prevention of conditions, cancers, or fibrosis involving mitochondrial dysfunction.

[0867] 21. Use of the compound, its tautomer, or a pharmaceutically acceptable salt thereof described in any of paragraphs 1 to 18 in the preparation of a medicament for the treatment or prevention of a condition, cancer, or fibrosis involving mitochondrial dysfunction.

[0868] 22. A method for treating or preventing conditions, cancers, or fibrosis involving mitochondrial dysfunction, comprising administering to a patient in need an effective amount of a compound, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer according to any one of paragraphs 1 to 18.

[0869] 23. The compounds, uses, or methods described in paragraphs 20 to 22, wherein the diseases involving mitochondrial dysfunction are selected from CNS diseases; neurodegenerative diseases; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis; Huntington's disease; ischemia; stroke; Lewy body dementia; frontotemporal dementia; multiple sclerosis; mitochondrial encephalopathy, lactic acidosis, and stroke-like seizure syndrome; maternally inherited diabetes and deafness; Leber hereditary optic neuropathy; neuropathy, ataxia, retinitis pigmentosa-maternally inherited Lewy syndrome; Danon's disease; diabetes; diabetic nephropathy; metabolic disorders; heart failure; leading to Ischemic heart disease with myocardial infarction; mental illness, schizophrenia; polysulfatase deficiency; mucolipidosis II; mucolipidosis III; mucolipidosis IV; GM1-ganglioside storage disease; neuronal ceroid-lipofuscin deposition disease; Alpes disease; Bart syndrome; β-oxidation deficiency; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndrome; coenzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive extraocular muscle palsy syndrome; CPT CPT deficiency; CPT II deficiency; glutaric aciduria type II; Kiehl-Siehl syndrome; lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency; Leigh syndrome or Leigh syndrome; French-Canadian variant of Leigh syndrome; lethal infantile cardiomyopathy; Left disease; medium-chain acyl-CoA dehydrogenase deficiency; myoclonic epilepsy and broken red fiber syndrome; mitochondrial cell diseases; mitochondrial recessive ataxia syndrome; mitochondrial DNA depletion syndrome; myoneurotic gastrointestinal diseases and encephalopathy; Pearson syndrome; pyruvate dehydrogenase deficiency; pyruvate carboxylase deficiency; POLG mutation; medium / short chain 3-hydroxyacyl-CoA dehydrogenase deficiency; very long chain acyl-CoA dehydrogenase deficiency; peroxisome disorders; methylmalonic acidemia; mevalonate kinase deficiency; age-dependent decline in cognitive function and muscle strength; and cognitive impairment associated with all neurodegenerative and neuropsychiatric disorders.

[0870] 24. The compound, use, or method described in paragraph 23, wherein the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, focal ischemia, stroke, Lewy body dementia, multiple system atrophy, progressive supranuclear palsy, cortical-basal degeneration, frontotemporal dementia; and Parkinson's disease associated with mutations in α-synuclein, Parkinin, PINK1, GBA, and LRRK2, and autosomal recessive juvenile Parkinson's disease or early-onset Parkinson's disease (EOPD) in which Parkinin or PINK1 is mutated, truncated, or deleted.

[0871] 25. The compound, use, or method described in paragraph 23, wherein the neurodegenerative disease is Leahy syndrome or Leahy disease, X-linked Leahy disease, the French-Canadian variant of Leahy syndrome, and / or symptoms associated with Leahy disease.

[0872] 26. The compounds, uses, or methods described in paragraphs 20 to 22, wherein the cancers described are selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, bone cancer, liver cancer, soft tissue cancer, organ cancer, hematologic malignancy, CML, AML, mantle cell lymphoma, neuroblastoma, melanoma, soft tissue sarcoma, liposarcoma, fibroblastic sarcoma, leiomyosarcoma, hepatocellular carcinoma, osteosarcoma, esophageal cancer, leukemia, lymphoma, multiple myeloma, metastatic cancer, osteosarcoma, chondrosarcoma, Ewing sarcoma, nasopharyngeal carcinoma, colorectal cancer, non-small cell lung cancer, cancers in which apoptosis pathways are dysregulated, and cancers in which proteins of the BCL-2 family are mutated, overexpressed, or underexpressed.

[0873] 27. The compound, use, or method according to paragraphs 20 to 22, wherein the fibrosis is selected from the fibrosis or fibrotic condition associated with the accumulation of extracellular matrix components following trauma, inflammation, tissue repair, immune response, cell proliferation, and tumor formation.

[0874] 28. The compound, use, or method described in paragraph 27, wherein the fibrosis is selected from fibrosis or fibrotic disorders associated with major organ diseases, fibrotic disorders, and scar formation associated with trauma.

[0875] 29. The compound, use, or method described in paragraph 28, wherein the fibrosis is selected from fibrotic or fibrotic conditions associated with interstitial lung disease, cirrhosis, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis, kidney disease, acute kidney injury, chronic kidney disease, delayed kidney transplantation, heart or vascular disease, eye disease, systemic and localized scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, dupulleren contracture, surgical complications, chemotherapy-induced fibrosis, radiation-induced fibrosis, accidental injury and burns, retroperitoneal fibrosis, and peritoneal fibrosis / peritoneal scarring.

[0876] 30. The compound, use, or method according to paragraph 29, wherein the fibrosis associated with interstitial lung disease is selected from sarcoidosis, silicosis, drug reaction, infection, collagen vascular disease, rheumatoid arthritis, systemic sclerosis, scleroderma, pulmonary fibrosis, idiopathic pulmonary fibrosis, common interstitial pneumonia, interstitial lung disease, cryptogenic fibrotic alveolitis, obliterative bronchiolitis, and bronchiectasis.

[0877] 31. The compound, use, or method described in paragraph 29, wherein the kidney disease described is acute kidney disease, acute kidney injury, or chronic kidney disease.

[0878] 32. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of paragraphs 1 to 18, a tautomer thereof or a pharmaceutically acceptable salt of said compound or tautomer, and one or more pharmaceutically acceptable excipients.

[0879] 33. A compound selected from formulas (II)(i), (III)(i), (II)(ii) and (III)(ii):

[0880]

[0881] Its tautomers or the compound or salts of its tautomers;

[0882] Where R 1 R 2 R 3 R 4 and R 5 The compound of formula (I) as defined in any one of claims 1 to 18; and PG is a protecting group, preferably selected from tert-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzylcarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, toluenesulfonyl, trichloroethoxycarbonyl, 4-nitrobenzenesulfonyl, and 2-nitrobenzenesulfinyl.

Claims

1. Compounds of formula (I)(i) or (I)(ii): Or a pharmaceutically acceptable salt of the compound, wherein: R 1 Selected from C1-C4 alkyl, C1-C4 fluoroalkyl and CH2OCH3; R 2 Selected from C1-C4 alkyl, CF3 and cyclopropyl; and R 3 R 4 and R 5 Each is independently selected from hydrogen and halogens.

2. The compound according to claim 1, wherein R 1 It is selected from methyl, CH2F, CHF2, CF3 and CH2OCH3.

3. The compound according to claim 2, wherein R 1 Selected from methyl and CH2OCH3.

4. The compound according to claim 1, wherein R 2 Selected from methyl, CF3 and cyclopropyl.

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

6. The compound according to claim 1, wherein R 3 Selected from hydrogen and fluorine.

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

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

9. The compound according to any one of claims 1 to 7, having formula (I)(i): Or a pharmaceutically acceptable salt of the compound.

10. The compound according to claim 9, having formula (IA)(i): Or a pharmaceutically acceptable salt of the compound.

11. The compound according to claim 10, wherein the compound is selected from: 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 R 1-Cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 S 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 S 1-Cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R 5 R 1-Cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R 5 S 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R 5 S 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R 5 R )-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3 R 5 R 1-Cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; Or its pharmaceutically acceptable salt.

12. The compound according to claim 9, having formula (IB)(i): Or a pharmaceutically acceptable salt of the compound.

13. The compound according to claim 12, wherein the compound is selected from: 4-(5-cyano-2-methoxyphenyl)-N-((3 R 5 S )-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; and 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 S 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; Or its pharmaceutically acceptable salt.

14. The compound according to any one of claims 1 to 7, having formula (I)(ii): Or a pharmaceutically acceptable salt of the compound.

15. The compound according to claim 14, having formula (IA)(ii): Or a pharmaceutically acceptable salt of the compound.

16. The compound according to claim 15, wherein the compound is selected from: 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 R 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 S 1-Cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R 5 S 1-Cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-methoxyphenyl)-N-((3 R 5 R 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R 5 R 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-2-(trifluoromethoxy)phenyl)-N-((3 R 5 S 1-Cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; 5-(5-cyano-4-fluoro-2-methoxyphenyl)-N-((3 R 5 S )-1-cyano-5-methylpyrrolidine-3-yl)oxazol-2-carboxamide; and 5-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 R 1-Cyano-5-(fluoromethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; Or its pharmaceutically acceptable salt.

17. The compound according to claim 14, having formula (IB)(ii): Or a pharmaceutically acceptable salt of the compound.

18. The compound according to claim 17, wherein the compound is selected from: 4-(5-cyano-2-cyclopropoxyphenyl)-N-((3 R 5 R )-1-cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; and 4-(5-cyano-2-methoxyphenyl)-N-((3 R 5 R 1-Cyano-5-(methoxymethyl)pyrrolidine-3-yl)oxazol-2-carboxamide; Or its pharmaceutically acceptable salt.

19. Use of the compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing a disease associated with USP30 activity, wherein said disease is colorectal cancer.

20. Use of the compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of diseases associated with USP30 activity, wherein said disease is selected from fibrosis or fibrotic conditions associated with the accumulation of extracellular matrix components following trauma, inflammation, tissue repair and cell proliferation.

21. Use of the compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing diseases associated with USP30 activity, wherein said diseases are selected from fibrosis or fibrotic disorders associated with major organ diseases, fibrotic disorders, and scar formation associated with trauma.

22. Use of the compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt of the compound in the preparation of a medicament for treating or preventing diseases associated with USP30 activity, wherein the diseases are selected from interstitial lung disease, cirrhosis, non-alcoholic fatty liver disease, acute kidney disease, chronic kidney disease, eye diseases, systemic and localized scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, dupulleren contracture, surgical complications, chemotherapy-induced fibrosis, radiation-induced fibrosis, accidental injury, retroperitoneal fibrosis, and peritoneal fibrosis or peritoneal scarring.

23. Use of the compound of any one of claims 1 to 18 or a pharmaceutically acceptable salt of the compound in the preparation of a medicament for treating or preventing diseases associated with USP30 activity, wherein the diseases are selected from fibrotic or fibrotic conditions associated with delayed renal transplantation, burns, non-alcoholic steatohepatitis, idiopathic pulmonary fibrosis, and common interstitial pneumonia.

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

25. A compound selected from formulas (II)(i), (III)(i), (II)(ii) and (III)(ii): Or a salt of the compound; Where R 1 R 2 R 3 R 4 and R 5 The compound of formula (I) as defined in any one of claims 1 to 18; and PG is a protecting group.

26. The compound according to claim 25, wherein the protecting group is selected from tert-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzylcarbonyl, 9-fluorenylmethoxycarbonyl, acetyl, benzoyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, toluenesulfonyl, trichloroethoxycarbonyl, 4-nitrobenzenesulfonyl, and 2-nitrobenzenesulfinyl.

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