RU2844289C1 - 1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1h)-carbonitrile as a usp30 inhibitor for use in the treatment of mitochondrial dysfunction, cancer and fibrosis - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitriles with activity of inhibitors of deubiquitinating enzyme USP30. Disclosed are (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile formula, specified below, as well as use thereof and a pharmaceutical composition containing same. In addition, disclosed is an intermediate compound selected from compounds of formulas (IIA), (IIIA), (IVA) and (VA), or a salt of a compound of formula (IIA), where PG is tert-butyloxycarbonyl.

EFFECT: proposed (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile is useful in various therapeutic areas, including conditions involving mitochondrial dysfunction, or fibrosis, and demonstrates higher efficacy against USP30 than structurally similar compounds.

8 cl, 26 tbl, 4 ex

Description

FIELD OF INVENTION

The present invention relates to hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitriles with activity as inhibitors of the deubiquitylating enzyme ubiquitin C-terminal hydrolase 30, which is also known as ubiquitin-specific peptidase 30 (USP30), to their use, methods for their preparation and compositions containing said inhibitors. These inhibitors are useful in various therapeutic areas, including conditions involving mitochondrial dysfunction, cancer and fibrosis.

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

PRIOR ART

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

Ubiquitin is a master regulator of mitochondrial dynamics. Mitochondria are dynamic organelles whose biosynthesis, fusion, and fission events are regulated by post-translational regulation through ubiquitylation of many key factors such as mitofusins. In humans, USP30 is a 517 amino acid protein that resides in the mitochondrial outer membrane (Nakamura et al, 2008, Mol Biol 19:1903-11). It is the only deubiquitylating enzyme that carries a mitochondrial targeting signal and has been shown to deubiquitinate a variety of mitochondrial proteins. It has been demonstrated that USP30 antagonizes Parkin-mediated mitophagy and that downregulation of USP30 rescues Parkin-mediated defects in mitophagy (Bingol et al, 2015, Nature 510:370-5; Gersch et al, 2017, Nat Struct Mol Biol 24(11): 920-930; Cunningham et al, 2015, Nat Cell Biol 17(2): 160-169). USP30 inactivation can also increase mitochondrial protein import, potentially through ubiquitination of TOM proteins (Jacoupy et al, 2019, Sci Rep 9(1): 11829). A small fraction of USP30 is localized to peroxisomes, which are formed by the fusion of mitochondrial and ER vesicles, with USP30 potently antagonizing the Pex2/peroxiphagy pathway (Riccio et al, 2019, J Cell Biol 218(3): 798–807). The E3 ubiquitin ligase March5 and deubiquitinase EP30 associate with the translocase and regulate mitochondrial import, and while March5 antagonizes mitochondrial import and directs degradation of substrates, USP30 deubiquitylates substrates to promote their import (Phu et al, 2020, Molecular Cell 77, 1107–1123).

Mitochondrial dysfunction can be defined as a decrease in mitochondrial content (mitophagy or mitochondrial biogenesis), as a decrease in mitochondrial activity and oxidative phosphorylation, and as a modulation of reactive oxygen species (ROS) formation. Consequently, mitochondrial dysfunctions play a role in a very large number of aging processes and pathologies.

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

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

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

Fibrosing diseases, including renal, hepatic, and pulmonary fibrosis, are a leading cause of morbidity and mortality and can affect all tissues and organs. Fibrosis is thought to result from acute or chronic stress on a tissue or organ and is characterized by extracellular matrix deposition, decreased vascular/tubule/duct/airway patency, and impaired function, ultimately leading to organ failure. Many fibrosing conditions are caused by lifestyle or environmental factors; however, a subset of fibrosing conditions may have genetic triggers or are truly considered idiopathic (ie, without a known cause). Some fibrosing diseases, such as idiopathic pulmonary fibrosis (IPF), can be treated with a non-specific kinase inhibitor (nintedanib) or drugs with no well-characterized mechanism of action (pirfenidone). Other treatments for organ fibrosis, such as renal or hepatic fibrosis, relieve pressure on the organ itself (eg, beta blockers for cirrhosis, angiotensin receptor blockers for chronic kidney disease). Attention to lifestyle factors, such as glucose control and diet, may also influence the course and severity of the disease.

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

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

Williams et al, 2015, Pharmacol Res. December; 102: 264-269, discuss the role of PINK1-parkin-mediated autophagy in protecting against alcohol- and acetaminophen-induced liver injury by removing damaged mitochondria via mitophagy. It is suggested that pharmacological stabilization of USP8 or inactivation of USP15 and USP30 may be potential therapeutic targets to up-regulate parkin-induced mitophagy and in turn may protect against drug-induced liver injury. However, it is noted that DUBs are regulated both transcriptionally and post-translationally, which may complicate drug development to target these specific enzymes, and furthermore, phosphorylated ubiquitin has been shown to be resistant to DUBs. The authors conclude that upregulation of PINK1 stabilization or kinase activity may be a more effective target than DAB inhibition.

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

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

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

A series of N-cyano-substituted heterocycle derivatives are disclosed as inhibitors of deubiquitylating enzymes in PCT applications WO 2016/046530 (US 15/513125, US 15/894025, US 16/448066), WO 2016/156816 (US 15/558632, US 16/297937, US 16/419558, US 16/419747, US 16/788446), WO 2017/009650 (US 15/738900), WO 2017/093718 (US 15/776149), WO 2017/103614 (US 15/781615), WO 2017/149313 (US 16/078518), WO 2017/109488 (US 16/060299), WO 2017/141036 (US 16/070936), WO 2017/163078 (US 16/087515), WO 2017/158381 (US 16/080229), WO 2017/158388 (US 16/080506), WO 2018/065768 (US 16/336685), WO 2018/060742 (US 16/336202), WO 2018/060689 (US 16/334836), WO 2018/060691 (US 16/336363), WO 2018/220355 (US 16/615040), WO 2018/234755 (US 16/615709), WO 2020/212350, WO 2020/212351, WO 2021/043870, PCT EP2021/059032 and PCT EP2021/064166, each of which is incorporated herein by reference in its entirety. PCT application WO 2019/171042 (US 16/977019), the entire contents of which are incorporated herein by reference, discloses the use of N-cyanopyrrolidines as USP30 inhibitors for the treatment of fibrosing diseases.

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

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

PCT application WO 2006/067165 relates to a method of treating fibrosing diseases using indolinone kinase inhibitors. PCT application WO 2007/119214 relates to a method of treating early stage pulmonary fibrosis with an endothelin receptor antagonist. PCT application WO 2012/170290 relates to a method of treating fibrosing diseases using THC (tetrahydrocannabinolic) acids. PCT application WO 2018/213150 relates to sulfonamide USP30 inhibitors with potential utility in the treatment of conditions involving mitochondrial defects. Larson-Casey et al, 2016, Immunity 44, 582-596, review macrophage kinase Aktl-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.

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

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

AKI and CKD are considered to be a continuum within 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 clear unmet medical need to develop medicinal products to treat and/or prevent AKI.

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

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

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

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

SUMMARY OF THE INVENTION

The present invention relates to a compound of formula (I):

or a pharmaceutically acceptable salt thereof.

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

DETAILED DESCRIPTION OF THE INVENTION

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

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

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

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

It is generally important that a drug be as selective as possible for its desired enzyme target; additional activities introduce the possibility of side effects. The precise physiological role of many DUBs has not yet been fully determined; however, regardless of what role these DUBs may or may not play, it is a prudent basic rule of medicinal chemistry to ensure that any drug has selectivity over related mechanistic targets of unknown physiological function. Representative examples of DUB enzymes for which the compounds of the present invention can be screened include UCHL1, UCHL3, UCHL5, YOD1, SENP2, SENP6, TRABID, BAP1, Cezanne, MHHDY2/FAM63B, OTU1, OTUD3, OTUD5, OTUD6A, OTUD6B, OTUB1/UBCH5B, OTUB2, CYLD, VCPIP, AMSH-LP, JOSD1, JOSD2, USP1/UAF1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP11, USP12/UAF1, USP13, USP14, USP15, USP16, USP19, USP20, USP21, USP22, USP24, USP25, USP28, USP32, USP34, USP35, USP36, USP45, USP46/UAF1, USP47 and USP48. Preferably, the compounds of the invention have good selectivity for USP30 relative to one or more of these DUB enzymes.

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

There is also a need for compounds that have favorable pharmacokinetic properties so that they are suitable for orally administered. An orally administered drug should have good bioavailability, that is, the ability to pass easily through the gastrointestinal (GI) tract and not undergo extensive metabolism during passage from the GI tract into the systemic circulation. Once the drug enters the systemic circulation, the rate of metabolism is also important in determining the residence time of the drug in the body.

It is therefore clear that drug molecules must have the properties of readily passing through the gastrointestinal tract and being slowly metabolized in the body. The Caco-2 assay is a well-established model for predicting the ability of a given molecule to pass through the gastrointestinal tract. Most of the metabolism of drug molecules typically occurs in the liver, and in vitro assays using whole-cell hepatocytes (animal or human) are widely used methods for measuring the susceptibility of a given molecule to hepatic metabolism. Such assays are designed to predict in vivo clearance from the calculated hepatocyte clearance.

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

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

Other parameters that can be measured using standard assays and that are indicative of the exposure profile of a compound include, for example, plasma stability (measurement of half-life), AUC, C _max , C _min and T _max values in blood.

The treatment of CNS disorders including Alzheimer's disease, Parkinson's disease and other disorders described herein requires that drug molecules target the brain, which requires adequate penetration of the blood-brain barrier. Therefore, there is a need for USP30 inhibitors that have effective blood-brain penetration properties and provide an appropriate residence time in the brain to be effective. The likelihood that a compound can cross the blood-brain barrier can be measured in an in vitro permeability assay using a MDR1-MDCK cell monolayer (Madin-Darby canine kidney cells transfected with MDR-1, resulting in overexpression of the human P-glycoprotein efflux transporter). Additionally, exposure can also be measured directly in the brain and in plasma using in vivo animal models.

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

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

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

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

The hERG prognostic assay provides valuable information on the potential binding of test compounds to the potassium channel and on the potential prolongation of the QT interval on the echocardiogram. Inhibition of hERG current causes prolongation of the QT interval, leading to potentially fatal ventricular tachyarrhythmia (Torsades de Pointes). The assay data can usually be obtained using an automated patch clamp platform.

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

The most preferred compound of the present invention is highly potent towards USP30 as measured by the biochemical assay described herein and is significantly more selective towards USP30 compared to other DABs and cathepsins. The significant and unexpected properties of the compounds of the present invention make them particularly suitable for use in the treatment and/or prevention of diseases associated with USP30 activity.

According to a first aspect of the present invention, there is provided a compound of formula (I):

or a pharmaceutically acceptable salt thereof.

In a first preferred aspect of the present invention there is provided a compound of formula (IA):

or a pharmaceutically acceptable salt thereof.

In a second preferred aspect of the present invention there is provided a compound of formula (III):

or a pharmaceutically acceptable salt thereof.

In a third preferred aspect of the present invention there is provided a compound of formula (IC):

or a pharmaceutically acceptable salt thereof.

In a fourth preferred aspect of the present invention there is provided a compound of formula (ID):

or a pharmaceutically acceptable salt thereof.

Preferred compounds of formula (I) are selected from:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4S,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4R,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4R,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile; and

(3aS,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

or their pharmaceutically acceptable salts.

More preferred compounds of formula (I) are selected from:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4S,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile; and

(3aS,4R,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

or a pharmaceutically acceptable salt thereof.

The most preferred compound of formula (I) is:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

or a pharmaceutically acceptable salt thereof.

The compound of Example 1 is the most preferred compound of the invention.

This most preferred compound of formula (I) exists as a single dextrorotatory stereoisomer, which is identified as having the following structure with the absolute configuration shown:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile.

The compound of formula (I) has three chiral centers and can therefore exist in eight stereochemical configurations. It is believed that the synthesis of the bicyclic ring according to the experiments given below provides a r/r/s-fused system giving four possible stereoisomers. The stereochemical configuration of the most preferred compound of formula (I) is the stereochemical configuration of the compound of Example 1, which is the compound most active against USP30 in a biochemical assay.

Four stereoisomers were obtained as a result of the experiments described in Examples 1-4 below. The compounds of Examples 1 and 3 are dextrorotatory, and the compounds of Examples 2 and 4 are levorotatory when measured under the experimental conditions (c = 0.05 g/100 cm ³ , MeOH). The compound of Example 1, prepared by two different methods, gave optical rotation measurements of [α] _D ²⁵ = +208° and +204°. The slight variation may be due to experimental differences or differences in optical purity. The compound of Example 3 gave an optical rotation measurement of [α] _D ²⁵ = +166°.

The present invention further relates to a cis-fused stereoisomer which is dextrorotatory when measured as described and has an approximate optical rotation in the range of +204° to +208°. Depending on the optical purity, this stereoisomer, when substantially pure, may have an optical rotation in the range of +190° to +220°. Such a figure clearly distinguishes this compound of the invention from another cis-fused stereoisomer which is also dextrorotatory.

The present invention further relates to:

(+)-(3aR*,4R*,6aR*)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (I);

or a pharmaceutically acceptable salt thereof.

The present invention further relates to the major stereoisomer obtained by the experimental procedure of Example 1.

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

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

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

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

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

Pharmaceutically acceptable solvates according to the invention include hydrates and solvates wherein the crystallization solvent may be isotopically substituted, for example _D2O , acetone- _d6 , DMSO- _d6 .

Also included within the scope of the invention are clathrates, drug-host inclusion complexes where, unlike the above-mentioned solvates, the drug and host are present in non-stoichiometric amounts. For a review of such complexes, see J. Pharm Sci, 64 (8), 1269-1288 by Haleblian (August 1975).

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

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

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

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

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

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

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

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

The most preferred compound of formula (I) according to the present invention may exist in combination with one or more other stereoisomers of the preferred compounds of formula (I). For example, the compound may exist with its enantiomer, i.e. as a racemate, or it may exist with a diastereomer.

Preferred compounds of formula (I) according to the present invention preferably exist as a single stereoisomer. Preferred compounds of formula (I) are isolated as a single stereoisomer and may exist with a stereoisomeric excess of at least 60%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, such as 96%, 97%, 98%, 99% or 100%. The stereoisomeric excess may be a measure of enantiomeric excess or may be a measure of diastereomeric ratio.

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

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

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

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

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

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

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

According to a further aspect of the present invention, there is provided a method of treating or preventing a disorder or condition for which inhibition of USP30 is known or can be shown to have a beneficial effect in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I) as defined herein, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer.

According to another aspect of the present invention there is provided the use of a compound of formula (I) as defined herein, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer in the manufacture of a medicament for the treatment or prevention of a disorder or condition for which inhibition of USP30 is known or can be shown to have a beneficial effect. The manufacture of the medicament may comprise, inter alia, chemical synthesis of a compound of formula (I) or a salt thereof, or the preparation of a composition or formulation containing the compound or salt thereof, or the packaging of any medicament containing the compound.

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

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

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

Mitochondrial dysfunctions result from defects in the mitochondria, which are specialized compartments present in every cell in the body except red blood cells. When mitochondria fail, the cell produces less and less energy, which leads to cell damage or even cell death. If this process is repeated throughout the body, the life of the subject in whom it occurs is greatly compromised. Mitochondrial diseases occur most often in organs that are very energy-intensive, such as the brain, heart, liver, skeletal muscle, kidneys, and the endocrine and respiratory systems.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Kidney disease may be associated with diabetes, which can damage and scar the kidneys, leading to progressive loss of function, and hypertensive diseases. Renal fibrosis may occur at any stage of kidney disease from acute kidney injury (AKD) and chronic kidney disease (CKD), such as incident CKD and progressive CKD, to end-stage renal disease (ESRD). Renal fibrosis may develop as a result of cardiovascular disease, such as hypertension, or diabetes, which both place greater limitations on kidney function, promoting a fibrotic response. However, renal fibrosis may also be idiopathic (in the absence of a known cause), and some genetic mitochondrial diseases also have manifestations of renal fibrosis and associated symptoms.

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

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

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

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

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

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

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

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

Leigh syndrome is a rare, inherited neurometabolic disorder that affects the central nervous system. This progressive disorder begins in infants between the ages of three months and two years. It rarely occurs in adolescents and adults. Leigh syndrome can be caused by mutations in the nuclear DNA that codes for mitochondrial proteins, mutations in mitochondrial DNA (maternally inherited Leigh syndrome (MILS)) or a deficiency of an enzyme called pyruvate dehydrogenase located on the short arm of the X chromosome (X-linked Leigh syndrome). Symptoms of Leigh syndrome usually progress rapidly. The earliest signs may include poor sucking ability and loss of head control and motor skills. These symptoms may be followed by loss of appetite, vomiting, irritability, incessant crying and seizures. As the disorder progresses, symptoms may also include generalized weakness, loss of muscle tone, and episodes of lactic acidosis, which can lead to respiratory and renal impairment.

In maternally inherited Leigh syndrome (MILS), genetic mutations in the mitochondrial DNA (in high percentages >90%) interfere with the energy sources that power cells in the area of the brain that plays a role in motor function. Genetic mutations in the mitochondrial DNA result in a chronic lack of energy in these cells, which in turn affects the central nervous system and causes progressive degeneration of motor functions. When the genetic mutations in the mitochondrial DNA that cause MILS are less numerous (less than 90%), the condition is known as neuropathy, ataxia, and retinitis pigmentosa (NARP). There is also a form of Leigh disease (called X-linked Leigh disease), which results from mutations in a gene that produces another group of substances important in cellular metabolism. There is another variant of Leigh syndrome called the French Canadian variant, which is characterized by mutations in a gene called LRPPRC. Similar neurological symptoms are present as in Leigh syndrome, although the French Canadian variant usually also has hepatic steatosis.

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

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

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

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

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

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt of said compound or tautomer, together with a pharmaceutically acceptable diluent or carrier.

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

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

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

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

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

Oral administration

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

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

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

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

A typical tablet may be prepared by standard processes known to the formulation chemist, such as direct compression, granulation (dry, wet, or melt), melt solidification, or extrusion. The tablet may contain one or more layers and may or may not be coated.

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

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

Parenteral administration

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

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

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

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

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

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

Dosage

The effective dose of the compound will, of course, vary depending on the nature and severity of the condition being treated and the route of administration. The selection of appropriate dosages is within the competence of the attending physician. The daily dosage range is from 10 mcg to about 100 mg per kg of body weight in humans and non-human animals, and may generally be from 10 mcg to 30 mg per kg of body weight per dose. The above dosage may be administered one to three times daily.

For example, oral administration may require a total daily dose of 5 mg to 1000 mg, such as 5 to 500 mg, while an intravenous dose may only be 0.01 to 30 mg/kg body weight, such as 0.1 to 10 mg/kg, more preferably 0.1 to 1 mg/kg body weight. The total daily dose may be administered in single or divided doses.

The skilled artisan will also appreciate that in the treatment or prevention of certain conditions, the compounds of the invention can be administered as a single dose on an "as required" (i.e., as needed or desired) basis.

Synthesis methodologies

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

According to a further aspect of the present invention, there is provided a process for the preparation of a compound of formula (I), comprising removing the protecting group of a compound of formula (III) using standard methods to obtain an amine (II), which can then be reacted with cyanogen bromide to obtain the corresponding compound of formula (I):

where PG is a protecting group.

In a further aspect the present invention provides a compound which is selected from the compounds of formulae (II) and (III) wherein PG is a protecting group, or a salt of said compound.

In a preferred embodiment of the present invention, there is provided a compound which is selected from compounds of formulae (IIA) and (IIIA):

where PG is a protecting group or a salt of the specified compound.

According to a further aspect of the present invention, there is provided a process for the preparation of a compound of formula (III), comprising oxidizing a compound of formula (IV) using standard methods, for example using meta-chloroperbenzoic acid, to obtain the N-oxide (V), which can then be reacted with trimethylsilyl cyanide and dimethylcarbamoyl chloride to obtain the corresponding compound of formula (III):

where PG is a protecting group.

In a further aspect the present invention provides a compound which is selected from the compounds of formulae (IV) and (V) wherein PG is a protecting group, or salts of said compound.

In a preferred embodiment of the present invention, there is provided a compound which is selected from compounds of formulae (IVA) and (VA):

where PG is a protecting group or a salt of the specified compound.

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

Abbreviations

LCMS/HPLC/SFC methods

Method C

Method C1

Method E

Method F

Method H

HI Method

Method H3

Method X

Method X2

Method Y4

Method Y11

Method Y13

Method Y15

Method Y17

Method Y20

Method Y22

Method Y23

Method Y24

Method Y25

Method Y26

Method Y28

Intermediate connection A

Ethyl 5-(pyridin-4-yl)oxazole-2-carboxylate

(i) i-PrMgCl, THF, rt; (ii) TFA, DCM, 0°C to rt; (iii) TEA, DCM, 0°C to rt.

Stage (i)

tert-Butyl-(2-oxo-2-(pyridin-4-yl)ethyl)carbamate

4-Bromopyridine hydrochloride (CAS 19524-06-2, from CombiBlocks, 8.0 g, 41.2 mmol) was treated with 5% aqueous _Na2CO3 (300 mL) and extracted with DCM (2×100 _mL ). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure. The residue was immediately dissolved in dry THF (50 mL) and isopropyl magnesium chloride (2 M in THF, 20.57 mL, 41.2 mmol) was added dropwise at rt under _N2 . The resulting dark solution was stirred at rt for 1.5 h, during which time a precipitate formed. Meanwhile, to a stirred suspension of tert-butyl (2-(methoxy(methyl)amino)-2-oxoethyl)carbamate (7.1 g, 32.9 mmol) in THF (50 mL) was added isopropyl magnesium chloride (2 M in THF, 16.4 mL, 32.9 mmol) dropwise at 0 °C. The solution was stirred for 10 min, then added dropwise at rt to the pyridyl Grignard reagent. The mixture was stirred at rt for 16 h, then poured into water (200 mL) and extracted with EtOAc (2×200 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (30% EtOAc in n-hexanes) to give tert-butyl (2-oxo-2-(pyridin-4-yl)ethyl)carbamate (4.8 g, 20.3 mmol, 49% yield).

LCMS: Method C, 1.40 min, MS: ER+ 237.1; ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 8.88-8.89 (d, J=5.6 Hz, 2H), 7.77-7.79 (d, J=6.8 Hz, 2H), 5.49 (br s, 1H), 4.69-4.71 (d, J=4.4 Hz, 2H), 1.52 (s, 9H).

Stage (ii)

2-Amino-1-(pyridin-4-yl)ethan-1-one TFA-salt

To a stirred solution of tert-butyl (2-oxo-2-(pyridin-4-yl)ethyl)carbamate (4.8 g, 20.34 mmol) in DCM (50 mL) was added TFA (2.4 mL, 5 vol) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 2 h, then concentrated under reduced pressure with DCM (3×100 mL) to give 2-amino-1-(pyridin-4-yl)ethan-1-one TFA salt (7.0 g, quantitative yield).

LCMS: Method C, 0.29 min, MS: ER+ 136.96; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.91-8.93 (d, J=6.0 Hz, 2H), 8.36 (br s, 3H), 7.92-7.94 (d, J=6.0 Hz, 2H), 4.68 (t, J=5.2 Hz, 2H).

Stage (iii)

Ethyl 5-(pyridin-4-yl)oxazole-2-carboxylate

This reaction was performed in duplicate. To a stirred solution of 2-amino-1-(pyridin-4-yl)ethan-1-one TFA salt (3.0 g, 12 mmol) in DCM (80 mL) was added TEA (3.75 g, 5.17 mL, 37.2 mmol) dropwise at rt. Ethyl chlorooxalate (1.63 g, 1.33 mL, 12 mmol) was added dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 24 h. The two batches were combined, poured into water (200 mL) and extracted with DCM (2 x 200 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by column chromatography (40% EtOAc in n-hexanes) to give ethyl 5-(pyridin-4-yl)oxazole-2-carboxylate (0.45 g, 2.06 mmol, 10% yield over 2 steps).

LCMS: Method C, 1.29 min, MS: EDTA+ 219.25; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.74-8.75 (d, J=5.6 Hz, 2H), 8.29 (s, 1H), 7.79-7.80 (d, J=6.0 Hz, 2H), 4.40-4.45 (q, J=7.2 Hz, 2H), 1.37 (t, J=6.8 Hz, 3H).

Intermediate connection B

rac-tert-Butyl-(3aS,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

(i) EtOC(0)Cl, NaOH, DCM, 0°C to rt; (ii) NaH, DMF, 3-chlorobut-1-ene, 0°C to rt; (iii) HCO ₂ H, 0°C then 100°C; (iv) N-benzylglycine, toluene, 110°C; (v) conc. HCl, 110°C; (vi) (BOC) ₂ O, TEA, 4-dimethylaminopyridine, DCM, 0°C to rt; (vii) chromatographic separation; (viii) Pd(OH) ₂ , MeOH, H ₂ , rt.

Stage (i)

Ethyl (2,2-dimethoxyeth)carbamate

To a stirred solution of 2,2-dimethoxyethane-1-amine (CAS 22483-09-6, from Combi-blocks, 12.5 g, 118.9 mmol) in DCM (100 mL) was added aqueous sodium hydroxide (5.2 g, 130.8 mmol) in water (31 mL) dropwise at 0 °C. Ethyl chloroformate (14.2 g, 130.8 mmol) was added and the mixture was stirred at rt for 16 h. This mixture and the mixture from the second reaction were poured into water (500 mL) and extracted with DCM (3 x 500 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give ethyl (2,2-dimethoxyethyl)carbamate (36.5 g, quantitative yield).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 7.15 (s, 1H), 4.34 (t, J=5.2 Hz, 1H), 3.98 (q, J=7.2 Hz, 2H), 3.24 (s, 6H), 3.05 (t, J=5.6 Hz, 2H), 1.15 (t, J=7.2 Hz, 3H).

Stage (ii)

Ethyl but-3-en-2-yl-(2,2-dimethoxyethyl)carbamate

To a stirred solution of ethyl (2,2-dimethoxyethyl)carbamate (12.1 g, 68.3 mmol) in DMF (50 mL) was added NaH (60% in mineral oil, 6.84 g, 170.9 mmol) portionwise over 2 h at 0 °C. The mixture was stirred at 0 °C for 2 h, then 3-chlorobut-1-ene (7.6 mL, 75.2 mmol) was added and the mixture was stirred at rt for 20 h. This mixture and mixtures of two duplicate reactions were quenched by pouring into ice water (300 mL) and extracted with EtOAc (3 x 500 mL). The combined organic phases were washed with cold water (3 x 150 mL) and brine (2 x 150 mL). The organic phase was dried over _{anhydrous Na2SO4 and} concentrated under reduced pressure. The residue was purified by column chromatography (basic alumina, 5% EtOAc in n-hexanes) to give ethyl but-3-en-2-yl-(2,2-dimethoxyethyl)carbamate (15.5 g, 67.0 mmol, 28% yield over two steps).

^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 5.88-5.94 (m, 1H), 5.08-5.11 (m, 2H), 4.41-4.48 (m, 2H), 4.05-4.06 (m, 2H), 3.18-3.35 (m, 8H), 1.18-1.24 (m, 6H).

Stage (iii)

Ethyl-but-3-en-2-sh(2-oxoethsh)carbamate

To a stirred solution of ethyl but-3-en-2-yl (2,2-dimethoxyethyl) carbamate (15.5 g, 67.0 mmol) was added formic acid (36 mL) dropwise at 0 °C. The mixture was heated at 100 °C for 2 h, then cooled to rt, poured into ice water (200 mL) and extracted with EtOAc (3 x 300 mL). The combined organic phases were washed with saturated NaHCO ₃ solution (3 x 100 mL). The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give ethyl but-3-en-2-yl (2-oxoethyl) carbamate (11.3 g, 61.1 mmol, 91% yield).

^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 9.47 (s, 1H), 5.75-5.85 (m, 1H), 5.10-5.15 (m, 2H), 4.65-4.77 (m, 1H), 3.80-4.07 (m, 4H), 1.10-1.18 (m, 6H).

Stage (iv)

rac-Ethyl (3aR,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred solution of ethyl but-3-en-2-yl-(2-oxoethyl)carbamate (4.7 g, 25.4 mmol) in toluene (100 mL) was added N-benzylglycine (CAS 17136-36-6, from Combi-blocks, 4.2 g, 25.4 mmol) at rt. The mixture was heated at 110 °C for 16 h then concentrated under reduced pressure. The residue was purified by column chromatography (basic alumina, 7% EtOAc in n-hexanes) to give rac-ethyl (3aR,6aR)-1-benzyl 4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (3.2 g, 11.1 mmol, 43% yield).

LCMS: Method C, 1.33 min, two broad peaks merged, no complete separation of the two diasteomers, MS: ER+ 289.3.

Stage (v)

rac-(3aR,6aR)-1-Benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole HCl-salt

A stirred solution of rac-ethyl (3aR,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (3.2 g, 11.1 mmol) in concentrated HCl (40 mL, 12.5 vol) was heated at 100 °C for 16 h. The mixture was cooled to rt and concentrated under reduced pressure to give rac-(3aR,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole HCl salt (3.72 g, quantitative yield).

LCMS: Method F, 4.45 min and 4.92 min, two diastereomers, MS: ER+ 217.3.

Stage (vi)

rac-tert-Butyl-(3aR,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred solution of rac-(3aR,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole HCl salt (3.7 g, 14.65 mmol) in DCM (40 mL) were added triethylamine (10.3 mL, 73.5 mmol), 4-dimethylaminopyridine (0.09 g, 0.73 mmol) and di-tert-butyl dicarbonate (3.83 g, 17.58 mmol) at 0 °C. The mixture was allowed to warm to rt and stirred for 5 h, then poured into water (100 mL) and extracted with DCM (2 x 150 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was subjected to chromatographic separation according to Step (vii).

Stage (vii)

rac-tert-Butyl-(3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (major isomer) and

rac-tert-butyl-(3aR,4S,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (minor isomer)

The residue from Step (vi) was purified by column chromatography (basic alumina, 2% EtOAc in n-hexanes) to give two separate fractions in a 4.2:1 ratio.

The first eluting fraction, Fraction 1, rac-tert-butyl (3aR,4S,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (minor isomer, 0.50 g), was isolated as a colorless oil. TLC: Rf 0.5 (30% EtOAc/n-hexanes).

LCMS: Method C, 1.46 min, MS: ER+ 317.4; and Method X, 13.70 min, MS: ER+ 317.3.

The second eluting fraction, Fraction 2, rac-tert-butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (major isomer, 2.10 g, 6.63 mmol, 45% yield), was isolated as a colorless oil. TLC: Rf 0.4 (30% EtOAc/n-hexanes).

LCMS: Method C, 1.46 min, MS: ER+ 317.4; and Method X, 13.37 min, MS: ER+ 317.3; d.r. 99:1 by Method X.

Fraction 2 was carried over to Stage (viii).

Stage (viii)

rac-tert-Butyl-(3aS,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred solution of rac-tert-butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (Fraction 2, major isomer, 0.50 g, 1.58 mmol) in MeOH (10 mL) was added 20% Pd(OH) ₂ (50% humidity, 250 mg, 50% w/w) and purged with _H2 gas for 4 h at rt. This mixture was filtered through a pad of Celite, washed with MeOH (50 mL) and concentrated under reduced pressure to give rac-tert-butyl (3aS,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.33 g, 1.46 mmol, 92% yield).

LCMS: Method C, 1.29 min, MS: ER+ 227.32.

Intermediate connection C

tert-Butyl-(3aS,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

Stage (i)

Methyl (2,2-dimethoxyethane)-D-alaninate

To a stirred mixture of 2,2-dimethoxyacetaldehyde (97.01 g, 931.89 mmol) in MeOH (100 mL) was added methyl D-alaninate HCl (100.0 g, 716.84 mmol) at 0 °C. The mixture was stirred at rt for 2 h. Sodium cyanoborohydride (58.52 g, 931.89 mmol) was added portionwise under N ₂ at 0 °C and stirred at rt for 18 h. The mixture was filtered through Celite and washed with MeOH (2 x 200 mL). The filtrate was concentrated under reduced pressure and the residue was poured into water (1000 mL) and extracted with DCM (2 x 1000 mL). The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure at 40°C to give methyl (2,2-dimethoxyethyl)-D-alaninate as a pale yellow oil (135.0 g, 705.95 mmol, 98% yield),

^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 4.41-4.54 (m, 1H), 3.75 (s, 3H), 3.47-3.50 (m, 1H), 3.38-3.46 (m, 6H), 2.76-2.81 (m, 1H), 2.64-2.68 (m, 1H), 1.31-1.35 (d, J=7.2 Hz, 3H).

This process was repeated three times in identical fashion using methyl D-alaninate HCl (500 g, 1200 g, and 1200 g scale) to give the title compound (680 g, 1625 g, and 1625 g, respectively). Total mass of all three batches: 4065 g, 21.25 mol.

Stage (ii)

Methyl N-(2,2-dimethoxyethyl)-N-(ethoxycarbonyl)-D-alaninate

To a stirred mixture of sodium bicarbonate (88.95 g, 1058 mmol) in water (810 mL) was added a solution of methyl (2,2-dimethoxyethyl)-D-alaninate (135 g, 705.95 mmol) in THF (810 mL) at rt. Ethyl chloroformate (91.93 g, 847.14 mmol) was added to the cooled biphasic solution (-5 to 5 °C), and the mixture was stirred at rt for 2 h. The mixture was poured into water (1.35 L) and extracted with EtOAc (2×675 mL). The combined organic phases were washed with saturated _KHSO4 solution (675 mL), dried over sodium sulfate and concentrated under reduced pressure at 40 °C to give methyl N-(2,2-dimethoxyethyl)-N-(ethoxycarbonyl)-D-alaninate as an oil (150.0 g, 569.71 mmol, 80% yield).

^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 4.52 (m, 2H), 4.15-4.27 (m, 2H), 3.78 (s, 3H), 3.40-3.63 (m, 9H), 1.50-1.52 (d, J=6.8 Hz, 2H), 1.23-1.39 (m, 3H).

This process was repeated four times in identical fashion using methyl (2,2-dimethoxyethyl)-D-alaninate (680 g, 1000 g, 1000 g and 1250 g scale) to give the subtitle compound (775 g, 1360 g, 1365 g and 1375 g, respectively). Total weight of all three batches: 5025 g, 19.09 mol.

Stage (iii)

Ethyl-(R)-(2,2-dimethoxyeth)(1-hydroxypropane-2-w)carbamate

To a stirred mixture of methyl N-(2,2-dimethoxyethyl)-N-(ethoxycarbonyl)-D-alaninate (100 g, 379.8 mmol) in ethanol (1.8 L) and THF (200 mL) was added 1 L of NH4Cl (2 M in THF, 949.5 mL, 1899.04 mmol) dropwise at 0 °C. The mixture was stirred at rt for 18 h, then quenched by adding cold ammonium chloride (50 g) in water (500 mL) twice and concentrated under reduced pressure to remove most of the volatile organic solvent. The residue was taken up in EtOAc (500 mL). The organic phase was separated, dried over sodium sulfate and concentrated under reduced pressure at 40°C to give ethyl (R)-(2,2-dimethoxyethyl)(1-hydroxypropan-2-yl)carbamate as an oil (72.0 g, 306.01 mmol, 81% yield).

^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 4.85-4.86 (m, 1H), 4.65 (m, 1H), 4.15-4.24 (m, 3H), 3.71-3.72 (m, 2H), 3.41-3.56 (m, 6H), 3.14 -3.19 (m, 1H), 2.99-3.05 (m, 2H), 1.28-1.34 (m, 3H), 1.13-1.15 (m, 1H), 1.03-1.05 (m, 1H).

This process was repeated five times in identical fashion using methyl N-(2,2-dimethoxyethyl)-N-(ethoxycarbonyl)-D-alaninate (500 g, 500 g, 1200 g, 1200 g and 1200 g scale) to give the subtitle compound (336 g, 336 g, 929 g, 930 g and 930 g, respectively). Total weight of all three batches: 3533 g, 15.01 mol.

Stage (iv)

Ethyl (R)-(2,2-dimethoxyethyl)(1-oxopropan-2-yl)carbamate

To a stirred mixture of ethyl (R)-(2,2-dimethoxyethyl)(1-hydroxypropan-2-yl)carbamate (400.0 g, 1700.1 mmol) in DCM:water (1.04 L, 1:1) was added _NaHCO3 (428 g, 5100 mmol) and TEMPO (2.6 g, 17.0 mmol) at rt. Sodium hypochlorite solution (1 M NaOCl, freshly prepared from solid NaOCl: _5H2O , 391.1 g, 2210.13 mmol, diluted with 4.98 L water) was added to the biphasic solution at 0 °C. This mixture was warmed to rt. and stirred for 18 h, then quenched carefully with 10% (w/v) aqueous sodium thiosulfate solution (2.8 L) and extracted with DCM (2×4.0 L). The organic phase was dried over sodium sulfate and concentrated under reduced pressure at 40 °C to give ethyl (R)-(2,2-dimethoxyethyl)(1-hydroxypropan-2-yl)carbamate as an oil (360 g, 1543.3 mmol, 90.78% yield).

^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 9.59 (s, 1H), 4.46-4.48 (m, 1H), 4.16-4.24 (m, 2H), 3.84-3.89 (m, 1H), 3.52-3.56 (m, 1H), 3.42-3.47 (m, 6H), 3.30-3.40 (m, 1H), 1.38-1.41 (m, 3H), 1.22-1.34 (m, 3H).

This process was repeated three times in identical fashion using ethyl (R)-(2,2-dimethoxyethyl)(1-hydroxypropan-2-yl)carbamate (1100 g, 840 g and 1200 g) to give the subtitle compound (1026 g, 820 g and 1020 g, respectively). Total weight of all three batches: 3226 g, 13.83 mol.

Stage (v)

Ethyl (R)-but-3-en-2-yl(2,2-dimethoxyethyl)carbamate

To a stirred mixture of methyltriphenylphosphonium bromide (137.7 g, 385.8 mmol) in THF (1.8 L) was added KHMDS (1 M in THF, 401.26 mL, 401.2 mmol) dropwise at 0 °C. The mixture was stirred at rt for 1 h, then a solution of ethyl (R)-(2,2-dimethoxyethyl)(1-hydroxypropan-2-yl)carbamate (72.0 g, 308.6 mmol) in THF (576 mL) was added dropwise at -78 °C under N ₂ . The mixture was stirred at rt for 18 h, then quenched with MeOH (2.2 mL) and stirred for 30 min at rt. The mixture was concentrated, co-distilled with hexane (3×1500 mL), cooled to 0°C, and filtered to remove precipitated _MePPh2O and _PPh3O . The organic phase was concentrated, cooled, and filtered again. The resulting filtrate was concentrated under reduced pressure at 40°C to give ethyl (R)-but-3-en-2-yl (2,2-dimethoxyethyl)carbamate (65 g, 1543.3 mmol, 91% yield) as a light brown oil.

¹ H NMR (400 MHz, CDCl ₃ ) ppm: δ 5.92-6.03 (m, 1H), 5.11-5.16 (m, 1H), 4.52 (s, 2H), 4.17-4.23 (m, 2H), 3.45 (s, 6H), 3.27 (m, 2H), 2.67-2.72 (m, 1H), 1.26-1.33 (m, 6H).

This process was repeated three times in identical fashion using ethyl (R)-(2,2-dimethoxyethyl)(1-hydroxypropan-2-yl)carbamate (735 g, 1417 g and 1020 g) to give the subtitle compound (644 g, 1262 g and 860 g, respectively). Total weight of all three batches: 2831 g, 12.24 mol.

Stage (vi)

Ethyl-(R)-but-3-en-2-yl(2-oxoethyl)carbamate

To a stirred mixture of ethyl (R)-but-3-en-2-yl (2,2-dimethoxyethyl) carbamate (300.0 g, 1297.07 mmol) in acetone (6.0 L) and water (600 mL) was added p-toluenesulfonic acid monohydrate (78.9 g, 415.06 mmol) at rt. The mixture was heated at 60 °C for 18 h, allowed to cool to rt, and concentrated under reduced pressure. The mixture was concentrated, and the residue was poured into saturated NaHCO ₃ solution (1500 mL) and extracted with EtOAc (2×1000 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give ethyl (R)-but-3-en-2-yl (2-oxoethyl)carbamate as a pale yellow oil (230 g, 1242.5 mmol, 96% yield).

¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 9.55 (s, 1H), 5.79-5.86 (m, 1H), 5.05-5.25 (m, 2H), 5.00-5.05 (m, 1H), 4.12-4.22 (m, 2H), 3.70-3.92 (m, 2H), 1.24-1.31 (m, 6H).

This process was repeated three times in identical fashion using ethyl (R)-but-3-en-2-yl (2,2-dimethoxyethyl) carbamate (1020 g, 670 g and 860 g) to give the subtitle compound (795 g, 480 g, 605 g respectively). Total weight of all three batches: 2110 g, 11.39 mol.

Stage (vii)

Ethyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate, mixture with ethyl (3aS,4R,6aS)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (approximately 70:30 mixture of diastereomers)

In a 10 L round bottom flask equipped with a Dean-Stark apparatus, a mixture of ethyl (R)-but-3-en-2-yl (2-oxoethyl)carbamate (200.0 g, 1079.7 mmol) in toluene (7.2 L, 36 vol) was stirred at rt. N-Benzylglycine (214.02 g, 1295.7 mmol) was added to this mixture and it was then heated at 120 °C for 18 h, allowed to cool to rt and concentrated under reduced pressure. The residue was poured into saturated NaHCO ₃ solution (2000 mL) and extracted with DCM (2×1000 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give the subtitle compounds (approximately 70:30 mixture of diastereomers) as a brown oil (306.0 g, 1061.06 mmol, 98% yield).

LCMS: Method H3, 3.46 min, mixture of diastereomers, MS: ER+ 289.2; ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.16-7.40 (m, 5H), 4.16 (m, 2H), 3.91 (m, 2H), 3.65 (m, 1H), 3.49 (m, 1H), 3.32 (m, 1H), 3.18 (m, 1H), 2.92 (m, 1H), 2.64-2.72 (m, 1H), 2.40 (s, 1H), 2.33 (m, 1H), 2.10 (m, 1H), 1.30 (t, 3H), 1.20 (d, J=5.2 Hz, 3H); Chiral HPLC: Method Y26, 5.23 min - major peak; 5.61 min - minor peak.

This process was repeated three times in identical fashion using ethyl (R)-but-3-en-2-yl (2-oxoethyl)carbamate (515 g, 600 g, and 760 g) to give the subtitle compound (751 g, 880 g, and 970 g, respectively). Total weight of all four batches: 2907 g, 10.09 mol.

Stage (viii)

(3aR,4R,6aR)-1-Benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole, mixture with (3aS,4R,6aS)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole

A stirred mixture of the product of Step (vii) (ca. 70:30 mixture of diastereomers, 300.0 g, 1040.25 mmol) in aqueous HBr (48% w/w, 1988 mL, 16644.12 mmol) was heated to reflux for 6 h. This mixture was poured into water (1.5 L, 5 vol) and washed with toluene (3 x 600 mL, 2 vol). The aqueous layer was basified with K ₂ CO ₃ (ca. 2.0 kg, 14563.5 mmol) to pH ∼10 and extracted with DCM (3 x 1.5 L). The combined organic phases were dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was dissolved in DCM (500 mL) and washed thoroughly with 1 N HCl. NaOH solution (150 mL). The aqueous layer was extracted with DCM (200 mL). The combined organic phases were dried over _{anhydrous Na2SO4} , filtered and concentrated under reduced pressure to give the subtitle compounds (approximately 70:30 mixture of diastereomers) as a brown oil (155.0 g, 716.49 mmol, 69% yield).

LCMS: Method F, 3.05 min, mixture of diastereomers, MS: ES+ 217.2; ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.21-7.40 (m, 5H), 3.63-3.87 (m, 1H), 3.42-3.59 (m, 1H), 3.08-3.23 (m, 1H), 2.83-3.09 (m, 3H), 2.77 (m, 1H), 2.65 (s, 1H), 2.20-2.36 (m, 2H), 1.99 (m, 1H), 1.49 (m, 1H), 1.02-1.21 (m, 3H); Chiral HPLC: Method Y26, 4.98 min and 5.55 min, only two discrete peaks visible.

This process was repeated twice in identical fashion using ethyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (approximately 70:30 mixture of diastereomers, 1100 g and 1700 g) to give the subtitle compounds (575 g and 838 g, respectively). Total mass of all three batches: 1568 g, 7.24 mol.

Stage (ix)

(3aR,4R,6aR)-1-Benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole-0.5 (L)-DBTA-sol

To the product of Step (viii) (approximately 70:30 mixture of diastereomers, 2.5 g, 11.56 mmol) was added 2.5% water in THF (19.2 mL). A solution of (2R,3R)-2,3-bis(benzoyloxy)succinic acid hydrate (1.28 g, 3.57 mmol, from Angene) in 2.5% water in THF (6.8 mL, 2.75 vol) was added at rt, and this solution was stirred at rt for 2.5 h. The precipitated solid was collected by filtration under reduced pressure, and the filter cake was washed with 2.5% water in THF (2×10 mL) to give a white solid (3.0 g).

A suspension of this white solid (3.0 g) in 2.5% water in THF (36 mL) was heated to reflux for 1 h to give a clear solution. The mixture was then allowed to cool to rt over 1 h, then maintained without stirring for 1 h at rt. The crystalline solid was collected by filtration under reduced pressure, and the filter cake was washed with 2.5% water in THF (2×10 mL) and dried under reduced pressure at 40 °C to give (3aR,4R,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole⋅0.5 L-DBTA as a white solid (2.4 g, 3.03 mmol, 26% yield).

Chiral HPLC: Method Y22, 5.35 min.

Stage (ix): alternative

To the product of Step (viii) (approximately 70:30 mixture of diastereomers, 150.0 g, 693.38 mmol) was added 2.5% water in THF (1.2 L). This solution was seeded with (3aR,4R,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole.0,5(L)-DBTA salt from a previously synthesized batch (700 mg). A solution of (2R,3R)-2,3-bis(benzoyloxy)succinic acid hydrate (79.50 g, 221.88 mmol, from Angene) in 2.5% water in THF (412 mL, 2.75 vol) was added at room temperature, and the solution was stirred at rt. for 3 h. The reaction mixture was kept at room temperature for 3 h. The precipitated solid was collected by filtration under reduced pressure, and the filter cake was washed with 2.5% water in THF (2×1500 mL) to give a yellow solid (178.0 g).

First crystallization: A suspension of this yellow solid (178.0 g) in 2.5% water in THF (2670 mL) was heated to reflux for 1 h to give a clear solution. The mixture was then allowed to cool to rt over 1 h, then kept as is without stirring for 1 h at room temperature. The crystallized solid was collected by filtration under reduced pressure, and the filter cake was washed with 2.5% water in THF (2 x 1780 mL) and dried under reduced pressure at 40 °C to give (3aR,4R,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole⋅0.5 L-DBTA as a white solid (113.0 g, 142.0 mmol).

Second Crystallization: The solid from the first crystallization (113.0 g) was resuspended in 2.5% water in THF (1695 mL) and heated to reflux for 1 h to form a solution. The mixture was then allowed to cool to rt and kept as is without stirring for 1 h at room temperature. The crystallized solid was collected by filtration under reduced pressure and the filter cake was washed with 2.5% water in THF (2 x 1130 mL) and dried under reduced pressure at 40 °C to give (3aR,4R,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole⋅0.5 L-DBTA salt as a white solid (103.0 g, 130.22 mmol, 18% yield).

Chiral HPLC: Method Y22, 5.31 min.

This process was repeated twice in identical fashion using (3aR,4R,6aR)-1-benzyl-4-methyloctahydro-pyrrolo[3,4-b]pyrrole (approximately 70:30 mixture of diastereomers, 570 g and 838 g) to give the subtitle compound (478 g and 778 g, respectively). Total mass of all three batches: 1359 g, 1.72 mol.

Stage (x)

tert-Butyl-(3aR,4R,6aR)-1-benzyl-4-methhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

A stirred mixture of (3aR,4R,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-6]pyrrole⋅0.5 L-DBTA salt (100.0 g, 126.42 mmol) in TOT:water (1800 mL, 1:1.25) was heated at 50 °C. _NaHCO3 (31.86 g, 379.28 mmol) was added portionwise at 50 °C. The mixture was cooled to 25 °C, di-tert-butyl dicarbonate (66.14 g, 303.42 mmol) was added, and the biphasic mixture was stirred vigorously at rt for 16 h. The mixture was poured into water (500 mL) and extracted with EtOAc (2×1 L). The combined organic phases were washed with saturated aqueous _NaHCO3 (2×300 mL), dried over _{anhydrous Na2SO4} , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 20% EtOAc in n-hexanes) to give tert-butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate as a viscous gray oil (45.0 g, 142.20 mmol, 61% yield).

LCMS: Method H3, 3.96 min, MS: ER+ 317.2; ^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 7.23-7.36 (m, 5H), 3.79 (s, 1H), 3.62 (s, 1H), 3.42 (s, 2H), 3.09-3.14 (m, 1H), 3.02-3.05 (m, 1H), 2.81 (s, 1H), 2.34 (s, 1H), 2.18-2.25 (m, 1H), 1.96-2.04 (m, 1H), 1.46-1.55 (m, 1H), 1.40 (s, 9H), 1.08-1.10 (d, J=6.0 Hz, 3H); Chiral HPLC: Method Y23, 4.31 min, 99.6% e.u., 99.5:0.5 dr

This process was repeated twice in identical fashion using (3aR,4R,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole⋅0.5 L-DBTA salt (200 g and 1000 g) to give the subtitle compound (134 g and 685 g, respectively). Total mass of all three batches: 864 g, 2.73 mol.

Stage (xi)

tert-Butyl-(3aS,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred mixture of tert-butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (200.0 g, 632.01 mmol) in ethanol (1.6 L) was added 10% Pd/C (50% humidity, 100.0 g, 0.5% w/w), then purged with _H2 gas for 4 h at rt. This mixture was filtered through a Hyflow® pad of Celite, washed with MeOH (2×500 mL), and concentrated under reduced pressure to give tert-butyl (3aR,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate as a colorless oil (134.0 g, 592.08 mmol, 96% yield).

LCMS: Method H3, 2.22 min, MS: ER+ 227.2; ^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 3.75-3.82 (m, 1H), 3.48-3.49 (m, 2H), 3.04-3.08 (m, 1H), 2.85-2.91 (m, 1H), 2.32-2.38 (m, 1H), 2.22 (s, 2H), 2.00-2.05 (m, 1H), 1.64-1.70 (m, 1H), 1.45 (s, 9H), 1.15-1.19 (d, J=6.4 Hz, 3H); Chiral HPLC: Method Y13, 4.55 min.

This process was repeated in an identical manner using tert-butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (570 g) to give the subtitle compound (400 g). Combined weight of the two batches: 534 g, 2.36 mol.

Example 1

(+)-(3aR*,4R*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile and

Example 2

(-)-(3aR*,4R*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

(i) TBD, THF, 0°C to rt; (ii) m-CPBA, 0°C to rt; (iii) TMSCN, dimethylcarbamoyl chloride, MeCN, 0°C to rt; (iv) TFA, DCM, 0°C to rt; (v) K ₂ CO ₃ , BrCN, THF, 0°C to rt; (vi) purification by chiral preparative HPLC.

Stage (i)

rac-tert-Butyl-(3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrril-5(1H)-carboxylate

To a stirred solution of ethyl 5-(pyridin-4-yl)oxazole-2-carboxylate (5.0 g, 22.93 mmol) and rac-tert-butyl (3aS,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (4.15 g, 18.35 mmol) in THF (50 mL) was added TBD (4.78 g, 34.39 mmol) portionwise at 0 °C. The mixture was allowed to warm to rt and stirred for 1 h, then poured into water (50 mL) and extracted with EtOAc (2×200 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 2% MeOH in DCM) to give rac-tert-butyl (3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (5.5 g, 13.82 mmol, 60% yield).

LCMS: Method C1, 1.14 min, MS: ER+ 399.4.

Stage (ii)

rac-4-(2-((3aR,4R,6aR)-5-(tert-Butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide

To a stirred solution of rac-tert-butyl (3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (5.5 g, 13.82 mmol) in DCM (60 mL) was added radium-chlorobenzoic acid (4.77 g, 27.64 mmol) portionwise at 0 °C. The mixture was allowed to warm to rt and stirred for 24 h. The reaction mixture was poured into saturated NaHCO ₃ solution (200 mL) and extracted with EtOAc (2×300 mL). The combined organic phases were washed with saturated NaHCO3 solution ( ₃ ×100 mL), 10% sodium thiosulfate (200 mL ₎ , dried over _Na2SO4 and concentrated under reduced pressure to give rac-4-(2-((3aR,4R,6aR)-5-(tert-butoxycarbonyl)-4-methyl-octahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide (4.80 g, 11.58 mmol, 83% yield).

LCMS: Method C1, 1.15 min, MS: ER+ 415.6.

Stage (iii)

rac-tert-Butyl-(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred solution of rac-4-(2-((3aR,4R,6aR)-5-(tert-butoxycarbonyl)-4-methyl-octahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide (4.80 g, 11.58 mmol) in acetonitrile (50 mL) were added dimethylcarbamoyl chloride (3.11 g, 2.68 mL, 28.97 mmol) and TMSCN (3.45 g, 34.74 mmol) dropwise at 0 °C. The mixture was allowed to warm to rt and stirred for 16 h, then poured into water (200 mL) and extracted with EtOAc (2×300 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 2% MeOH in DCM) to give rac-tert-butyl (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (5.1 g, 12.05 mmol, quantitative yield).

LCMS: Method C1, 1.28 min, MS: ER+ [M+18] 441.4.

Stage (iv)

rac-4-(2-((3aR,4R,6aR)-4-Methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile

To a stirred solution of rac-tert-butyl (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (5.1 g, 2.16 mmol) in DCM (50 mL) was added TFA (10 mL, 2 vol) dropwise at 0 °C. This mixture was allowed to warm to rt. and stirred for 2 h, then concentrated under reduced pressure to give rac-4-(2-((3aR,4R,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile TFA salt (5.0 g, 11.44 mmol, 98% yield over two steps).

LCMS: Method C1, 0.91 min, MS: ER+ 324.3.

Stage (v)

rac-(3aR,4R,6aR)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

To a stirred solution of rac-4-(2-((3aR,4R,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile TFA salt (0.14 g, 0.33 mmol) in THF (6 mL) was added K ₂ CO ₃ (0.14 g, 1.00 mmol) at rt and stirred for 10 min. Cyanogen bromide (0.03 g, 0.27 mmol) was added at 0 °C. This mixture was stirred at 0 °C for 15 min, then poured into water (10 mL) and extracted with EtOAc (3×10 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by trituration with n-pentane (2×10 mL) to give rac-(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (0.08 g, 0.23 mmol, 88% yield over two steps).

LCMS: Method H, 2.49 min, MS: ER+ 349.1; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.89 (d, J=4.8 Hz, 1H), 8.51 (s, 1H), 8.36 (s, 0.7H), 8.34 (s, 0.3H), 8.05 (d, J=5.2 Hz, 1H), 5.11-5.19 (m, 0.4H), 4.57-4.63 (m, 0.7H), 4.24-4.29 (m, 0.8H), 3.89-4.01 (m, 2H), 3.42-3.57 (m, 2.6H), 2.51-2.61 (m, 0.5H, hidden), 1.85-2.05 (m, 2H), 1.30 (d, J=6.4 Hz, 3H), mixture of rotamers.

The subtitle compound (1.9 g, 5.45 mmol, 47% yield) was also prepared similarly from rac-4-(2-((3aR,4R,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile TFA salt (5.0 g).

Stage (vi)

Example 1: (+)-(3aR*,4R*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile and

Example 2: (-)-(3aR*,4R*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

Separation of the racemate into the enantiomers of Example 1 and Example 2

Each of the two enantiomers was isolated from the racemic compound rac-(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (1.5 g) by HPLC chromatography using a Shimadzu LC-20AP instrument coupled with a UV detector on a 250 mm × 21.0 mm, 5 micron Chiralpak IC column. The flow rate was set at 20.0 mL/min. The mobile phase was (A) 0.1% DEA in n-hexanes and (B) 0.1% DEA in 70:30 IPA/MeCN. UV spectra were recorded at lambda max 295 nm. Chromatography was performed over a 70-min period with an isocratic mobile phase of 40:60 V/A to yield the two enantiomers described below with elution times of 40.0 min and 55.6 min, respectively.

Faster eluting fraction (Example 2)

(-)-(3aR*,4R*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrril-5(1H)-carbonitrile

Yield (0.44 g, 1.26 mmol), white solid.

LCMS: Method N, 2.56 min, MS: ER+ 349.2.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.89 (d, J=4.8 Hz, 1H), 8.51 (s, 1H), 8.35-8.37 (m, 1H), 8.06-8.07 (m, 1H), 5.12-5.17 (m, 0.4H), 4.59-4.62 (m, 0.7H), 4.25-4.29 (m, 0.7H), 3.91-4.02 (m, 2H), 3.42-3.59 (m, 2.7H), 2.57-2.61 (m, 0.5H, hidden), 1.85-2.05 (m, 2H), 1.31 (d, J=6.4 Hz, 3H), a mixture of rotamers.

Chiral HPLC: Method Y11, 12.89 min; >99% ee.

Melting point = 142°C - 146°C.

[α] _D ²⁵ = -208° (c = 0.05 g/100 cm ³ , MeOH).

Slower eluting fraction (Example 1)

(+)-(3aR*,4R*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

Yield (0.48 g, 1.38 mmol), white solid.

LCMS: Method N, 2.56 min, MS: ER+ 349.2.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.89 (d, J=4.4 Hz, 1H), 8.51 (s, 1H), 8.36-8.38 (m, 1H), 8.06-8.07 (m, 1H), 5.12-5.16 (m, 0.4H), 4.57-4.65 (m, 0.7H), 4.22-4.30 (m, 0.7H), 3.91-4.02 (m, 2H), 3.41-3.58 (m, 2.7H), 2.61-2.63 (m, 0.5H, hidden), 1.82-2.07 (m, 2H), 1.31 (d, J=6.0 Hz, 3H), a mixture of rotamers.

Chiral HPLC: Method Y11, 15.09 min; >99% ee.

[α] _D ²⁵ = +208° (c = 0.05 g/100 cm ³ , MeOH).

A batch of substance obtained in a similar manner: Melting point = 144°C - 146°C.

Example 1. Alternative synthesis

(+)-(3aR,4R,6aR)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrril-5(1H)-carbonitrile

Stage (i)

tert-Butyl (3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrril-5(1H)-carboxylate

To a stirred mixture of ethyl 5-(pyridin-4-yl)oxazole-2-carboxylate (37.0 g, 169.72 mmol) and tert-butyl (3aR,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (30.70 g, 135.64 mmol) in THF (370 mL) at 0 °C was added TBD (28.32 g, 203.47 mmol) in portions. The mixture was allowed to warm to rt and stirred for 2 h, then poured into water (370 mL) and extracted with EtOAc (2×370 mL). The combined organic phases were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 5% MeOH in DCM) to give tert-butyl (3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate as a pale yellow solid (29.0 g, 72.78 mmol, 43% yield).

LCMS: Method H3, 2.79 min, MS: ER+ 399.2; ^1H NMR (400 MHz, CDCl ₃ ) δ ppm: 8.72-8.82 (m, 2H), 8.20-8.24 (m, 1H), 7.76-7.77 (m, 2H), 5.10-5.11 (m, 1H), 4.56 (s, 1H), 4.03 (s, 1H), 3.57-3.85 (m, 4H), 2.09-2.14 (m, 1H), 1.73-1.86 (m, 1H), 1.36 (s, 9H), 1.21-1.10 (m, 3H); Chiral HPLC: Method Y24, 5.35 min.

This process was repeated twice in identical fashion using tert-butyl (3aR,4R,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (165 g and 457 g) to give the subtitle compound (255 g and 620 g, respectively). Total mass of all three batches: 904 g, 2.27 mol.

Stage (ii)

4-(2-((SaR,4R,6aR)-5-(tert-Butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide

To a stirred mixture of tert-butyl (3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (29.0 g, 72.82 mmol) in DCM (435 mL) was added edaa-chloroperbenzoic acid (37.69 g, 218.46 mmol) in portions at 0 °C. The mixture was allowed to warm to rt and stirred for 20 h, then poured into saturated NaOH solution (725 mL, 25 vol) and extracted with DCM (2×290 mL). The combined organic phases were washed with 10% sodium thiosulfate (290 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give 4-(2-((3aR,4R,6aR)-5-(tert-butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide as a yellow solid (28.30 g, 68.32 mmol, 94% yield).

LCMS: Method H3, 2.41 min, MS: ER+ 259.0 (M-56); ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm: 8.25-8.26 (m, 2H), 8.08-8.11 (m, 1H), 7.78-7.79 (m, 2H), 5.08-5.09 (m, 0.4 H), 4.54 (m, 0.6 H), 4.02-4.09 (m, 1H), 3.66-3.82 (m, 4H), 2.07-2.12 (m, 2H), 1.64-1.84 (m, 1H), 1.38 (s, 9H), 1.15-1.19 (m, 3H); Chiral HPLC: Method Y25, 6.13 min.

This process was repeated twice in identical fashion using tert-butyl (3aR,4R,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (255 g and 620 g) to give the subtitle compound (235 g and 550 g, respectively). Total mass of all three batches: 813.3 g, 1.96 mol.

Stage (iii)

tert-Butyl (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo-[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred mixture of 4-(2-((3aR,4R,6aR)-5-(tert-butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide (28.30 g, 68.32 mmol) in acetonitrile (1132 mL) were added dimethylcarbamoyl chloride (22.04 g, 18.87 mL, 204.96 mmol) and TMSCN (20.33 g, 204.96 mmol) dropwise at 0 °C. This mixture was heated at 80 °C for 2 h, allowed to cool to rt and then poured into water (280 mL) and extracted with EtOAc (2×280 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure to give tert-butyl (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate as a brown oil (28.0 g, 66.16 mmol, 97% yield).

LCMS: Method H3, 3.06 min, MS: ER+ 368.0 (M-56); ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.87-8.88 (m, 1H), 8.48 (s, 1H), 8.34-8.36 (m, 1H), 8.03-8.05 (m, 1H), 4.55 (s, 1H), 4.02 (s, 1H), 3.55-3.84 (m, 3H), 2.74 (s, 2H), 2.08-2.13 (m, 1H), 1.75-1.83 (m, 1H), 1.38 (s, 9H), 1.16-1.20 (m, 3H); Chiral HPLC: Method Y4, 4.67 min.

This process was repeated twice in identical fashion using 4-(2-((3aR,4R,6aR)-5-(tert-butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide (235 g and 550 g) to give the subtitle compound (180 g and 470 g, respectively). Total weight of all three batches: 678 g, 1.60 mol.

Stage (iv)

4-(2-((3aR,4R,6aR)-4-Methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrilar-TSA salt

To a stirred mixture of tert-butyl (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (28.0 g, 66.16 mmol) in acetonitrile (560 mL) was added p-TSA (66.07 g, 383.72 mmol) in portions at 0 °C. The mixture was allowed to warm to rt. and stirred for 5 h, then concentrated under reduced pressure to give 4-(2-((3aR,4R,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile p-TSA salt as a yellow oil (115.0 g, quantitative yield).

LCMS: Method H3, 1.97 min, MS: ER+ 324.0; Chiral HPLC: Method Y20, 5.17 min.

This process was repeated in an identical manner using tert-butyl (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (650 g) to give the subtitle compound (1000 g). Combined weight of the two batches: 1115 g, 3.44 mol.

Stage (v)

(3aR,4R,6aR)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

To a stirred solution of 4-(2-((3aR,4R,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)-oxazol-5-yl)picolinonitrile p-TSA salt (180.0 g, 363.22 mmol) in THP:water (5.4 L, 2:1) was added _{K2CO3 (} 145.26 g, 1052.63 mmol) at rt and stirred for 5 min. Cyanogen bromide (26.0 g, 245.61 mmol) was added at 0 °C. The mixture was allowed to warm to rt and stirred for 1 h, then the mixture was concentrated under reduced pressure and the residue was poured into ice water (2 L) to form a precipitate. The solid was collected by filtration under reduced pressure. The solid was resuspended in water (2 L) and stirred for 2 h at rt, then filtered under reduced pressure. The solid was washed with cold water (1 L), then n-hexanes (500 mL), diethyl ether (100 mL), and IPA (100 mL) to give (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (40.0 g, 114.83 mmol, 32% yield).

LCMS: Method H3, 2.50 min, MS: ER+ 349.0; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.89 (d, J=5.2 Hz, 1H), 8.50 (s, 1H), 8.35-8.37 (m, 1H), 8.05-8.06 (m, 1H), 5.12-5.13 (m, 0.4H), 4.57-4.61 (m, 0.6H), 4.23-4.28 (m, 0.7H), 3.89-4.00 (m, 2H), 3.41-3.58 (m, 2.4H), 2.54-2.67 (m, 1H, hidden), 1.89-2.06 (m, 2H), 1.27-1.30 (m, 3H), mixture of rotamers; HPLC: Method X2, 19.78 min, 99.96%; Chiral HPLC: Method Y15, 15.29 min; >99% e.u.; Melting point = 147°C - 149°C; [α] _D ²⁵ = +202° (c = 0.05 g/100 cm ³ , MeOH).

Stage (v) (alternative synthesis)

(3aR,4R,6aR)-1-(5-(2-Cyanopyridin-4-yl)ixazyl-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

To a stirred solution of 4-(2-((3aR,4R,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)-oxazol-5-yl)picolinonitrile p-TSA salt (115.0 g, 232.06 mmol) in THF:water (804 mL, 9:5) was added K ₂ CO ₃ (98.22 g, 711.76 mmol) at rt and stirred for 10 min. Cyanogen bromide (18.86 g, 177.94 mmol) was added at 0 °C. The mixture was allowed to warm to rt and stirred for 2 h, then poured into water (1150 mL) and extracted with EtOAc (2×1150 mL). The combined organic phases _were dried over _Na2SO4 and concentrated under reduced pressure. The residue was suspended in IPA (575 mL) and heated at 80 °C for 2 h to form a clear solution. The mixture was allowed to cool slowly to rt to form a crystalline solid, which was collected by filtration under reduced pressure, washed with cold IPA (115 mL) and dried under reduced pressure to give (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (15.0 g, 43.08 mmol, 19% yield).

This method was repeated in an identical manner using 4-(2-((3aR,4R,6aR)-4-methyl-octahydropyrrolo[3,4-b]pyrrole-1-carbonyl)-oxazol-5-yl)picolinonitrile p-TSA salt (1000 g) to give the title compound (288 g). A solution of 288 g and 15 g of (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile in THF (909 mL, 3 vol) was stirred at rt for 2 h until a clear solution formed. The solvents were removed under reduced pressure to give a white solid, which was further washed with pentane (300 mL, 1 vol) and dried under reduced pressure for 4 h below 45 °C to give (3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (303.0 g).

LCMS: Method H3, 2.52 min, MS: ER+ 349.0; ^1H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 8.87 (d, J=3.6 Hz, 1H), 8.48 (s, 1H), 8.30-8.38 (m, 1H), 8.04-8.05 (m, 1H), 5.12-5.13 (m, 0.4H), 4.59-4.60 (m, 0.6Н), 4.23-4.27 (m, 0.6Н), 3.91-4.02 (m, 2Н), 3.41-3.56 (m, 2.4Н), 2.54-2.61 (m, 1H, hidden), 1.84-2.04 (m, 2Н), 1.30 (d, J=6.4 Hz, 3H), mixture of rotamers; Chiral HPLC: Method Y15, 15.11 min; HPLC: Method E, 27.87 min; >99% e.u., 99.5:0.5 dr; melting point = 161°C - 162°C; [α] _D ²⁵ = +204° (c = 0.05 g/100 cm ³ , MeOH).

Example 3 and Example 4

(+)-(3aR*,4S*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile and

(-)-(3aR*,4S*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

Stage (i)

rac-tert-Butyl (3aR,4S,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (minor isomer) and rac-tert-Butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (major isomer)

To a stirred solution of rac-(3aR,6aR)-1-benzyl-4-methyloctahydropyrrolo[3,4-b]pyrrole HCl salt (the product of Step (v) with respect to Intermediate B, 68.0 g, 269.31 mmol) in DCM (700 mL) were added triethylamine (136.0 g, 187.3 mL, 1346.55 mmol), 4-dimethylpyridine (1.64 g, 13.46 mmol) and di-tert-butyl dicarbonate (70.51 g, 323.17 mmol) at 0 °C. The mixture was allowed to warm to rt and stirred for 6 h, then poured into water (1000 mL) and extracted with DCM (2 x 1000 mL). The combined organic phases were dried over anhydrous _Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 100-200 _mesh , 7-9% EtOAc in n-hexanes) to give the subtitle compounds as two separate fractions, Fraction 1 (minor isomer, 0.8 g, 2.53 mmol, 0.94% yield) and Fraction 2 (major isomer, 38.0 g, 120.25 mmol, 45% yield).

Fraction 1 LCMS: Method H1, 4.19 min, MS: ER+ 317.2. Fraction 2 LCMS: Method H1, 3.87 min, MS: ER+ 317.0. Minor isomer Fraction 1 was carried on to Step (ii).

Stage(s)

rac-tert-Butyl-(3aS,4S,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred solution of rac-tert-butyl (3aR,4R,6aR)-1-benzyl-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (Fraction 1, minor isomer, 0.8 g, 2.53 mmol) in ethanol (10 mL) was added 10% Pd/C (50% humidity, 0.8 g) and purged with hydrogen gas for 16 h at rt. The mixture was filtered through Celite®, washed with ethanol (50 mL) and concentrated under reduced pressure to give rac-tert-butyl (3aS,4S,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.42 g, 1.85 mmol, 73% yield).

LCMS: Method H1, 2.25 min, MS: ER+ 227.2.

Stage (iii)

rac-tert-Butyl-(3aR,4S,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

A stirred solution of ethyl 5-(pyridin-4-yl)oxazole-2-carboxylate (0.49 g, 2.25 mmol) and rac-tert-butyl (3aS,4S,6aR)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.41 g, 1.79 mmol) in toluene (7 mL) was heated at 40 °C for 10 min until both starting materials dissolved, then cooled to 0 °C, then a solution of TBD (0.15 g, 1.12 mmol) in toluene (2 mL) was added dropwise at 0 °C. This mixture was allowed to warm to rt and stirred for 3 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases _were dried over anhydrous _Na2SO4 , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 80 to 90% EtOAc in n-hexanes) to give rac-tert-butyl (3aR,4S,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.43 g, 1.09 mmol, 48% yield).

LCMS: Method H1, 2.88 min, MS: ER+ 399.2.

Stage (iv)

rac-4-(2-((3aR,4S,6aR)-5-(tert-Butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide

To a stirred solution of rac-tert-butyl (3aR,4S,6aR)-4-methyl-1-(5-(pyridin-4-yl)oxazole-2-carbonyl)hexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.43 g, 1.08 mmol) in DCM (7 mL) was added dichloroperbenzoic acid (0.37 g, 2.16 mmol) portionwise at 0 °C. The mixture was allowed to warm to rt and stirred for 16 h. The mixture was poured into water (100 mL) and extracted with EtOAc (2×100 mL). The combined organic phases were washed with saturated _NaHCO3 solution (2×100 mL), 10% sodium thiosulfate (2× ₁₀₀ mL), dried over _Na2SO4 and concentrated under reduced pressure to give rac-4-(2-((3aR,4S,6aR)-5-(tert-butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide (0.39 g, 0.95 mmol, 87% yield).

LCMS: Method H1, 2.50 min, MS: ER+ [M-56] 359.0.

Stage (v)

rac-tert-Butyl-(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To a stirred solution of rac-4-(2-((3aR,4S,6aR)-5-(tert-butoxycarbonyl)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)pyridine 1-oxide (0.39 g, 0.94 mmol) in MeCN (7 mL) were added dimethylcarbamoyl chloride (0.30 g, 0.26 mL, 2.83 mmol) and trimethylsilyl cyanide (0.28 g, 0.36 mL, 2.83 mmol) dropwise at rt. This mixture was heated at 80 °C for 2 h, then poured into 10% Na ₂ CO ₃ solution in water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases _were dried over anhydrous _Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 80-90% EtOAc in n-hexanes) to give rac-tert-butyl (3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.38 g, 0.89 mmol, 95% yield).

LCMS: Method H1, 3.15 min, MS: ER+ [M-56] 368.0.

Stage (vi)

rac-4-(2-((3aR,4S,6aR)-4-Methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile

To a stirred solution of rac-tert-butyl (3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (0.38 g, 0.89 mmol) in DCM (10 mL) was added ra-toluenesulfonic acid monohydrate (0.84 g, 4.45 mmol) in portions at 0 °C. The mixture was allowed to warm to rt. and stirred for 6 h, then concentrated under reduced pressure to give rac-4-(2-((3aR,4S,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile p-TSA salt (0.52 g, quantitative yield).

LCMS: Method H1, 1.93 min, MS: ER+ 324.0.

Stage (vii)

rac-(3aR,4S,6aR)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

To a stirred solution of rac-4-(2-((3aR,4S,6aR)-4-methyloctahydropyrrolo[3,4-b]pyrrole-1-carbonyl)oxazol-5-yl)picolinonitrile p-TSA salt (0.52 g, 1.01 mmol) in THF (10 mL) and water (5 mL) was added K ₂ CO ₃ (0.70 g, 5.05 mmol) at rt and stirred for 5 min. Cyanogen bromide (0.13 g, 1.21 mmol) was added at 0 °C. The mixture was allowed to warm to rt and stirred for 1 h, then poured into water (50 mL) and extracted with EtOAc (2×50 mL). The combined organic phases were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 90 to 95% EtOAc in n-hexanes) to give rac-(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (0.18 g, 0.52 mmol, 57% yield over two steps).

LCMS: Method H1, 2.48 min, MS: ER+ 349.0; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm. 8.88 (d, J=5.2 Hz, 1H), 8.50 (s, 1H), 8.35 (d, J=3.6 Hz, 1H), 8.05 (dd, J=5.2, 1.2 Hz, 1H), 5.06-5.10 (m, 0.6H), 4.56-4.60 (m, 0.7H), 4.16-4.21 (m, 0.7H), 3.44-3.95 (m, 4H), 2.81-2.93 (m, 1H), 1.77-1.99 (m, 2H), 1.25 (d, J=6.4 Hz, 3H), mixture of rotamers; HPLC: Method E, 28.58 min; 98.8:1.2 dr

Stage (viii)

Example 3: (+)-(3aR*,4S*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile and

Example 4: (-)-(3aR*,4S*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrril-5(1H)-carbonitrile

Each of the two enantiomers was isolated from the racemic compound rac-(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile (0.10 g, 0.29 mmol) by chromatographic separation using a Shimadzu LC-20AP instrument coupled with a UV detector on a Chiralpak IG 250 mm × 21.0 mm, 5 micron column. The flow rate was set at 20.0 mL/min. The mobile phase was (A) 0.1% DEA in MeOH and (B) 0.1% DEA in MeCN. UV spectra were recorded at lambda max 292 nm. Chromatography was performed over a period of 35 min with an isocratic mobile phase of 50:50 W/A to obtain the two enantiomers described below with elution times of 8.03 min and 12.95 min, respectively.

Faster eluting fraction (Example 3)

(+)-(3aR*,4S*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

Yield (23 mg, 0.07 mmol). LCMS: Method H1, 2.49 min, MS: ER+ 349.0; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm. 8.88 (d, J=4.8 Hz, 1H), 8.50 (s, 1H), 8.35 (d, J=3.6 Hz, 1H), 8.05 (dd, J=5.2, 1.2 Hz, 1H), 5.06-5.11 (m, 0.6H), 4.56-4.61 (m, 0.7H), 4.16-4.22 (m, 0.7H), 3.43-3.95 (m, 4H), 2.81-2.95 (m, 1H), 1.77-1.98 (m, 2H), 1.25 (d, J=6.4 Hz, 3H), mixture of rotamers; chiral HPLC: Method Y17, 8.11 min.

Slower eluting fraction (Example 4)

(-)-(3aR*,4S*,6aR*)-1-(5-(2-Cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile

Yield (19 mg, 0.05 mmol). LCMS: Method H1, 2.49 min, MS: ER+ 349.0; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm. 8.88 (d, J=4.8 Hz, 1H), 8.50 (s, 1H), 8.35 (d, J=3.2 Hz, 1H), 8.05 (d, J=4.8, 1.2 Hz, 1H), 5.06-5.11 (m, 0.6H), 4.56-4.60 (m, 0.7H), 4.16-4.21 (m, 0.7H), 3.43-3.95 (m, 4H), 2.81-2.95 (m, 1H), 1.77-1.99 (m, 2H), 1.25 (d, J=6.4 Hz, 3H), mixture of rotamers; chiral HPLC: Method Y17, 13.17 min.

Example 3: White solid; chiral HPLC: Method Y17, 8.11 min; >99% e.u.; 99.5:0.5 dr; mp = 206°C - 207°C; [α] _D ²⁵ = +166° (c = 0.05 g/100 cm ³ , MeOH).

Example 4: White solid; chiral HPLC: Method Y17, 13.17 min; >99% e.u.; 99.5:0.5 dr; mp = 206°C - 207°C; [α] _D ²⁵ = -156° (c = 0.05 g/100 cm ³ , MeOH).

Chiral HPLC of compounds Examples 1-4.

The compounds of Examples 1-4 were analyzed by HPLC and chiral HPLC. Complete separation for the four isomers was not observed in a single system, so chiral HPLC (Method Y28) was used to determine the enantiomeric purity and achiral HPLC (Method E) was used to determine the diasteremeric ratio.

Biological activity of the compounds of the invention

Abbreviations:

TAMRA - carboxytetramethylrhodamine PCR polymerase chain reaction

PBS - phosphate buffered saline

EDTA - ethylenediaminetetraacetic acid

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

NP-40 - Nonidet P-40, octylphenoxypolyethoxyethanol

BSA - bovine serum albumin

PNS - peripheral nervous system

BH3 - Bcl-2 homology domain 3

PTEN - phosphatase and tensin homolog

SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis

DMSO - dimethyl sulfoxide

YFP - yellow fluorescent protein

VME - vinyl methyl ether

HA - hemagglutinin

Ahx - aminohexanoic acid

Biochemical analysis IC ₅₀ in relation to USP30

Plates were prepared with 21-fold final concentration dilutions (2100 µM for a final concentration of 100 µM) in 50% DMSO in a 96-well V-bottom polypropylene plate (Greiner #651201). A typical 8-point dilution series is 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 µM final. Reactions were performed in duplicate in black 384-well plates (low volume, Greiner 784076) in a final reaction volume of 21 µl. Either 1 μl 50% DMSO or diluted compound was added to the plate. USP30 (Boston Biochem #E582) was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to achieve a final assay concentration of 4 nM, and 10 μl diluted USP30 was added to the compound. The enzyme and compound were incubated for 30 min at room temperature. Reactions were initiated by adding 50 nM TAMRA-labeled peptide linked to ubiquitin through an isopeptide bond as a fluorescence polarization substrate. Reactions were read immediately after substrate addition and subsequent 2 h incubation at room temperature. The readings were taken on a Pherastar Plus (BMG Labtech). X excitation 540 nm; λ emission 590 nm.

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

Comparison examples

Activity of the reference compounds in the biochemical ICso assay towards USP30:

Non-target pharmacology

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

Safety pharmacology

The compound of Example 1 was evaluated for its effect on the hERG potassium channel in stably expressed CHO cells at concentrations ranging from 0.01 to 30 μM. The compound of Example 1 gave a maximum inhibition value of 27% of the hERG current amplitude at 30 μM, indicating little propensity to affect the QT interval.

Genetic toxicology

The compound of Example 1 was evaluated in a bacterial reverse mutation assay (Ames) and in an in vitro micronucleus assay. All in vitro tests were performed with and without exogenous metabolic activation using concentrations up to those limited by cytotoxicity or insolubility. The compound of Example 1 did not induce mutations when tested up to 5000 μg per well with and without metabolic activation in the reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, and TA97a and in Escherichia coli strain WP2 uvrA pKMlOl.

Induction of chromosome damage was assessed using an in vitro micronucleus assay in TK6 cells. Example 1 was negative for micronuclei 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.

TOM20 ubiquitylation analysis

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

Cell-Based Interaction Analysis with the Endogenous Target USP30

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

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

Additional research

log P: partition coefficient; measurement of lipophilicity.

log D: partition coefficient; a measurement of lipophilicity.

TPSA: Topological Polar Surface Area.

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

FaSSIF: Fasting simulated intestinal fluid measured at pH 6.5.

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

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

f _u,p in plasma: The free fraction of a compound in a blood plasma preparation, determined by in vitro equilibrium dialysis. It is understood that only unbound (free) compound is capable of interacting with the target.

f _u,br in brain: The free fraction of a compound in a brain homogenate preparation, determined by in vitro equilibrium dialysis. It is clear that only unbound (free) compound is capable of interacting with the target.

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

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

Oral F: Oral bioavailability.

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

WT-MDCK (wild type) permeability in vitro.

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

Example 1 compound has beneficial properties, demonstrating potential superiority over other compounds. For example, the observed i.v. plasma clearance of 19 ml/min/kg as measured in mice is low, indicating valuable stability in plasma, and the compound has an exceptional oral bioavailability of 87%.

Example 1 compound exhibits high CNS distribution in vivo following oral dosing, as determined by microdialysis sampling of brain regions following 30 mg/kg dosing in mice and rats. High unbound concentrations of Example 1 compound were observed for several hours post-dose, resulting in a reasonable estimated duration of CNS target exposure. Additionally, Example 1 compound exhibited high distribution into cerebrospinal fluid (CSF) when administered orally to dogs at 30 mg/kg.

Comparative data

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

The compound of Example 1 exhibits significantly increased hepatocyte metabolic stability (as measured in mouse hepatocytes) compared to the compounds of Comparative Examples A, B and C. The compound of Example 1 exhibits significantly increased stability in blood plasma (at least 2-8-fold as measured in mouse plasma) compared to the compounds of Comparative Examples A, B and C.

Efficacy relative to USP30

The compound of Example 1 of the present invention is significantly more potent against USP30 than the compounds of Comparative Examples A, B, C, D and E, as measured in a biochemical assay. The compound of the Example is 6 times more potent than the compounds of Comparative Examples B and C, 24 times more potent than the compounds of Comparative Examples A and D, and 400 times more potent than the compound of Comparative Example E.

Selectivity for USP30 relative to other DUBs

The data presented demonstrate that the compound of Example 1 is significantly more selective for USP30 relative to seven DUBs (USP2, USP6, USP10, USP16, USP21, USP25, and USP28) compared to the compounds of Comparative Examples A, B, and C. The compound of Example 1 is more than 2260 times more potent against USP30 than against each of these seven DUBs. This is a significant advantage in selectivity over the compounds of Comparative Examples A and B, which are only 3.3 times and 7.7 times more potent, respectively. The compound of Comparative Example C is more selective than compounds A and B, but is 56 times more potent against USP30 than against the other DUBs, and is still inferior to the compound of Example 1.

Selectivity for USP30 over UCHL1

The presented data demonstrate that the compound of Example 1 is significantly more selective for USP30 relative to UCHL1 compared to the compounds of Comparative Examples D and E. The compound of Example 1 is more than 27,000 times more potent against USP30 than against UCHL1, whereas the compounds of Comparative Examples D and E are only 0.8 and 1.5 times more potent, respectively.

Selectivity for USP30 over cathepsins B, K, L, S and V

The presented data demonstrate that the compound of Example 1 is significantly more selective for USP30 relative to the cathepsins (B, K, L, S and V) compared to the compounds of Comparative Examples A, B and C. The compound of Example 1 is more than 3800 times more effective against USP30 than against each of the cathepsins. This is a significant advantage in selectivity over the compounds of Comparative Examples A, B and C.

Specifically, the compound of Example 1 is more than 18,000 times more potent against USP30 than against cathepsin B, compared to the compounds of Comparative Examples B and C, which are only 20 and 16.7 times more potent, respectively. The compound of Example 1 is more than 9,900 times more potent against USP30 than against cathepsin K, compared to the compounds of Comparative Examples A and C, which are only 2 and 6.1 times more potent, respectively.

The above identified advantages of the compound of Example 1 according to the invention over the compounds of the comparative examples of the prior art are both significant and unexpected. This superiority in itself and, in particular, in combination makes this compound particularly suitable for use in the treatment or prevention of diseases associated with USP30 activity.

Preclinical studies in in vivo models

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

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

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

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

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

(e) Aged rodent model: Effects on the hippocampus, cognitive and motor function [Kobilo et al, 2014, Learn Mem. Jan 17;21(2): 119-26; Creed et al, 2019, Neuroscience. Jun 15;409:169-179; Van Skike et al, 2020, Aging Cell. 19;el3057].

In aged rodents, hippocampal neurodegeneration occurs naturally, increasing biochemical and functional changes in cognitive abilities. The glutamine/glutamate axis can be taken as a proxy for hippocampal health, given the close relationship between glutamine utilization in neurons as part of mitochondrial energy production.

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

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

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

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

The results demonstrated that both 1.5 and 5 mg/kg of Example 1 (p.o.) administered BID statistically reduced collagen deposition as evidenced by a decrease in Picrosirius red staining in ligated kidneys. Evaluation of α-SMA staining revealed that oral administration of 1.5 mg/kg of Example 1 BID resulted in a statistically reduced α-SMA levels in UUO-lesioned kidneys compared to vehicle-treated controls.

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

Points of the invention

1. Compound of formula (I):

or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, having the formula (IA):

or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, having the formula (IB):

or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1, having the formula (IC):

or a pharmaceutically acceptable salt thereof.

5. A compound according to claim 1, having the formula (ID):

or a pharmaceutically acceptable salt thereof.

6. The compound according to item 1, which is selected from:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6a,S)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4R,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4R,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile; and

(3aS,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

or a pharmaceutically acceptable salt thereof.

7. The compound according to item 6, which is selected from:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aS,4S,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

(3aR,4S,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile; and

(3aS,4R,6aS)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

or a pharmaceutically acceptable salt thereof.

8. The compound according to item 7, which is:

(3aR,4R,6aR)-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile;

or a pharmaceutically acceptable salt thereof.

9. A compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof for use as a medicine.

10. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a condition involving mitochondrial dysfunction, cancer or fibrosis.

11. The use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment or prevention of a condition involving mitochondrial dysfunction, cancer or fibrosis.

12. A method for treating or preventing a condition involving mitochondrial dysfunction, cancer or fibrosis, comprising the step of administering an effective amount of a compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof to a patient in need thereof.

13. The compound, use or method of claims 10-12, wherein the condition involving mitochondrial dysfunction is selected from a CNS disorder; a neurodegenerative disease; Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis; Huntington's disease; ischemia; stroke; dementia with Lewy bodies; frontotemporal dementia; multiple sclerosis; mitochondrial encephalopathy, lactic acidosis and stroke-like episode syndrome; maternally inherited diabetes and deafness; Leber's hereditary optic neuropathy; neuropathy, ataxia, maternally inherited retinitis pigmentosa Leigh syndrome; Danon disease; diabetes; diabetic neuropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; psychiatric disorders, schizophrenia; multiple sulfatase deficiency; mucolipidosis II; mucolipidosis III; mucolipidosis IV; GM1 gangliosidosis; neuronal ceroid lipofuscinosis; Alpers disease; Barth syndrome; beta-oxidation defects; carnitine acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; coenzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX (cyclooxygenase) deficiency; chronic progressive external ophthalmoplegia syndrome; CPT I (carnitine palmitoyltransferase I) deficiency; CPT II deficiency; glutaraciduria type II; Kearns-Sayre syndrome; lactic acidosis; Long-chain acyl-CoA dehydrogenase deficiency; Leigh disease or syndrome; French-Canadian variant of Leigh syndrome; Lethal infantile cardiomyopathy; Luft disease; Medium-chain acyl-CoA dehydrogenase deficiency; Myoclonic epilepsy and ragged red fiber syndrome; Mitochondrial cytopathy; Mitochondrial recessive ataxia syndrome; Mitochondrial DNA depletion syndrome; Myoneurogastrointestinal disorder and encephalopathy; Pearson syndrome; Pyruvate dehydrogenase deficiency; Pyruvate carboxylase deficiency; POLG (mitochondrial protease gamma) mutations; Medium/short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Very long-chain acyl-CoA dehydrogenase deficiency; Peroxisomal disorders; methylmalonic acidemia; age-related decline in cognitive function and muscle strength; and cognitive impairment associated with all neurodegenerative and neuropsychiatric disorders.

14. The compound, use or method of claim 13, wherein the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, ischemia, stroke, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia; and Parkinson's disease associated with mutations in a-synuclein, parkin, PINK1, GBA and LRRK2, and autosomal recessive juvenile Parkinson's disease or early-onset Parkinson's disease (EOPD), wherein parkin or PINK1 is mutated, truncated or deleted.

15. The compound, use or method of claim 13, wherein the neurodegenerative disease is Leigh syndrome or disease, X-linked Leigh disease, French Canadian variant of Leigh syndrome and/or symptoms associated with Leigh disease.

16. The compound, use or method according to paragraphs. 6-8, wherein the cancer is selected from breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, bone cancer, liver cancer, soft tissue cancer, organ tissue cancer, blood cell cancer, CML (chronic myelogenous leukemia), AML (acute myeloid leukemia), mantle cell lymphoma, neuroblastoma, melanoma, soft tissue sarcoma, liposarcoma, fibroblastic sarcoma, leiomyosarcoma, hepatocellular carcinoma, osteosarcoma, esophageal cancer, leukemia, lymphoma, multiple myeloma, metastatic carcinoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, nasopharyngeal carcinoma, colorectal cancer, non-small cell lung carcinoma, cancers in which apoptotic pathways are dysregulated, and cancers in which BCL-2 family proteins are mutated or over- or under-expressed.

17. The compound, use or method of claims 10-12, wherein fibrosis is selected from fibrosis or a fibrosing disorder associated with the accumulation of extracellular matrix constituents that occurs as a result of trauma, inflammation, tissue regeneration, immunological reactions, cellular hyperplasia and neoplasia.

18. The compound, use or method of claim 17, wherein the fibrosis is selected from fibrosis or fibrosing disorder associated with diseases of vital organs, fibroproliferative disorders and scarring associated with trauma.

19. The compound, use or method of claim 18, wherein the fibrosis is selected from fibrosis or a fibrosing disorder associated with interstitial lung disease, liver cirrhosis, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, kidney disease, acute kidney disease, acute kidney injury, chronic kidney disease, delayed renal graft function, cardiovascular disease, eye diseases, systemic and local sclerosis, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren's contracture, surgical complications, chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, accidental injury and burns, retroperitoneal fibrosis and peritoneal fibrosis/peritoneal scarring.

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

21. The compound, use or method of claim 19, wherein the kidney disease is acute kidney disease, acute kidney injury or chronic kidney disease.

22. A pharmaceutical composition comprising a compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable excipients.

23. A compound that is selected from compounds of formulas (II), (III), (IV) and (V):

or a salt of said compound; and wherein PG is a protecting group which is preferably selected from tert-butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzylcarbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, gas-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethoxycarbonyl, 4-nitrobenzenesulfonyl and 2-nitrophenylsulfenyl.

24. The compound according to claim 19, which is selected from compounds of formulas (IIA), (IIIA), (IVA) and (VA):

or a salt of the specified compound.

Claims (14)

1. Connection: (3a R ,4 R ,6a R )-1-(5-(2-cyanopyridin-4-yl)oxazole-2-carbonyl)-4-methylhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carbonitrile: or a pharmaceutically acceptable salt thereof. 2. The use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the treatment or prevention of a disorder or condition associated with USP30 activity. 3. The use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the treatment or prevention of a condition involving mitochondrial dysfunction or fibrosis that is associated with USP30 activity. 4. The use according to claim 3, wherein the condition in which mitochondrial dysfunction is involved is a neurodegenerative disease. 5. The use according to claim 4, wherein the neurodegenerative disease is selected from Parkinson's disease, Parkinson's disease associated with mutations in α-synuclein, parkin, PINK1, GBA and LRRK2, and autosomal recessive juvenile Parkinson's disease or early-onset Parkinson's disease (EOPD), wherein parkin or PINK1 is mutated, truncated or deleted. 6. The use according to claim 3, wherein fibrosis is selected from fibrosis or fibrosing disorder associated with kidney disease, acute kidney disease, acute kidney injury, chronic kidney disease, delayed kidney transplant function. 7. A pharmaceutical composition having the activity of inhibiting USP30 activity, comprising an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable excipients. 8. A compound selected from compounds of formulas (IIA), (IIIA), (IVA) and (VA): , or a salt of a compound of formula (IIA) wherein PG is tert -butyloxycarbonyl.