WO2020212351A1

WO2020212351A1 - Substituted cyanopyrrolidines with activity as usp30 inhibitors

Info

Publication number: WO2020212351A1
Application number: PCT/EP2020/060468
Authority: WO
Inventors: Mark Ian Kemp; Christopher Andrew Luckhurst; Paul William Thompson
Original assignee: Mission Therapeutics Limited
Priority date: 2019-04-16
Filing date: 2020-04-14
Publication date: 2020-10-22
Also published as: GB201905375D0

Abstract

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

Description

SUBSTITUTED CYANOPYRROLIDINES WITH ACTIVITY AS USP30 INHIBITORS FIELD OF THE INVENTION

The present invention relates to a class of substituted-cyanopyrrolidines with activity as inhibitors of the deubiquitylating enzyme ubiquitin C-terminal hydrolase 30, also known as ubiquitin specific peptidase 30 (USP30), uses thereof, processes for the preparation thereof and composition containing said inhibitors. These inhibitors have utility in a variety of therapeutic areas, including conditions involving mitochondrial dysfunction, cancer and fibrosis.

All documents cited or relied upon below are expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Ubiquitin is a small protein consisting of 76 amino acids that is important for the regulation of protein function in the cell. Ubiquitylation and deubiquitylation are enzymatically mediated processes by which ubiquitin is covalently bound or cleaved from a target protein by deubiquitylating enzymes (DUBs), of which there are approximately 100 DUBs in human cells, divided into sub-families based on sequence homology. The USP family are characterised by their common Cys and His boxes which contain Cys and His residues critical for their DUB activities. The ubiquitylation and deubiquitylation processes have been implicated in the regulation of many cellular functions including cell cycle progression, apoptosis, modification of cell surface receptors, regulation of DNA transcription and DNA repair. Thus, the ubiquitin system has been implicated 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 biogenesis, fusion and fission events are regulated by the post-translational regulation via ubiquitylation of many key factors such as mitofusins. In humans, USP30 is a 517 amino acid protein which is found in the mitochondrial outer membrane (Nakamura et al, 2008, Mol Biol 19:1903-11). It is the sole deubiquitylating enzyme bearing a mitochondrial addressing signal and has been shown to deubiquitylate a number of mitochondrial proteins. It has been demonstrated that USP30 opposes parkin-mediated mitophagy and that reduction of USP30 activity can rescue parkin-mediated defects in mitophagy (Bingol et al, 2015, Nature 510:370-5; Gersch et al, 2017, Nat Struct Mol Biol 24(11): 920-930; Cunningham et al, 2015, Nat Cell Biol 17(2): 160-169). USP30 inactivation can also increase mitochondrial protein import, potentially through ubiquitylation of TOM proteins (Jacoupy et al, 2019, Sci Rep 9(1): 11829). A small proportion of USP30 has been localized to peroxisomes, which are generated through fusion of mitochondrial and ER vesicles, with USP30 potentially antagonizing the Pex2/pexophagy pathway (Riccio et al, 2019, J Cell Biol 218(3): 798-807). The E3 Ub ligase March5 and the deubiquitinase USP30 associate with the translocase and regulate mitochondrial import, and while March5 opposes mitochondrial import and directs degradation of substrates, USP30 deubiquitinates substrates to promote their import (Phu et al, 2020, Molecular Cell 77, 1107-1123).

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

For example, Parkinson’s disease affects around 10 million people worldwide (Parkinson’s Disease Foundation) and is characterised by the loss of dopaminergic neurons in the substantia nigra. The exact mechanisms underlying PD are unclear; however mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuronal susceptibility in PD and is a feature of both familial and sporadic disease, as well as in toxin-induced Parkinsonism. Parkin is one of a number of proteins that have been implicated with early onset PD. While most PD cases are linked to defects in alpha-synuclein, 10% of Parkinson’s cases are linked to specific genetic defects, one of which is in the ubiquitin E3 ligase parkin. Parkin and the protein kinase PTEN-induced putative kinase 1 (PINK1) collaborate to ubiquitylate mitochondrial membrane proteins of damaged mitochondria resulting in mitophagy. Dysregulation of mitophagy results in increased oxidative stress, which has been described as a characteristic of PD. Inhibition of USP30 could therefore be a potential strategy for the treatment of PD. For example, PD patients with parkin mutations leading to reduced activity could be therapeutically compensated by inhibition of USP30.

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

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

Fibrotic diseases, including renal, hepatic and pulmonary fibrosis, are a leading cause of morbidity and mortality and can affect all tissues and organ systems. Fibrosis is considered to be the result of acute or chronic stress on the tissue or organ, characterized by extracellular matrix deposition, reduction of vascular/tubule/duct/airway patency and impairment of function ultimately resulting in organ failure. Many fibrotic conditions are promoted by lifestyle or environmental factors; however, a proportion of fibrotic conditions can be initiated through genetic triggers or indeed are considered idiopathic (i.e. without a known cause). Certain fibrotic disease, such as idiopathic pulmonary fibrosis (IPF), can be treated with non-specific kinase inhibitor (nintedanib) or drugs without a well- characterized mechanism of action (pirfenidone). Other treatments for organ fibrosis, such as kidney or liver fibrosis, alleviate pressure on the organ itself (e.g. beta blockers for cirrhosis, angiotensin receptor blockers for chronic kidney disease). Attention to lifestyle factors, such as glucose and diet control, may also influence the course and severity of disease.

Mitochondrial dysfunction has been implicated in a number of fibrotic diseases, with oxidative stress downstream of dysfunction being the key pathogenic mediator, alongside decreased ATP production. In preclinical models, disruption of the mitophagy pathway (through mutation or knockout of either parkin or PINK1) exacerbates lung fibrosis and kidney fibrosis, with evidence of increased oxidative stress.

Kurita et al, 2017, Respiratory Research 18:114, discloses that accumulation of profibrotic myofibroblasts is a crucial process for fibrotic remodelling in IPF. Recent findings are said to show participation of autophagy/mitophagy, part of the lysosomal degradation machinery, in IPF pathogenesis, and that mitophagy has been implicated in myofibroblast differentiation through regulating mitochondrial reactive oxygen species (ROS)-mediated platelet-derived growth factor receptor (PDGFR) activation. Kurita’s results suggested that pirfenidone induces PARK2-mediated mitophagy and also inhibits lung fibrosis development in the setting of insufficient mitophagy, which may at least partly explain the anti-fibrotic mechanisms for IPF treatment.

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

Williams et al, 2015, Biomolecules 5, 2619-2642, and Williams et al, 2015, Am J Physiol Gastrointest Liver Physiol 309: G324–G340, 2015, review mechanisms involved in regulation of mitochondrial homeostasis in the liver and how these mechanisms may protect against alcohol-induced liver disease. Series of derivatives of cyano-substituted heterocycles are disclosed as deubiquitylating enzyme inhibitors in PCT applications WO 2016/046530 (US 15/513125, US 15/894025, US 16/448066), 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), and WO 2018/234755 (US 16/615709), each of which are expressly incorporated herein by reference. PCT application WO 2019/171042, which is expressly incorporated herein by reference, discloses the use of substituted-cyanopyrrolidines as inhibitors of USP30 for the treatment of fibrotic diseases.

Falgueyret et al, 2001, J.Med.Chem. 44, 94-104, and PCT application WO 01/77073 refer to cyanopyrrolidines as inhibitors of Cathepsins K and L, with potential utility in treating osteoporosis and other bone-resorption related conditions. PCT application WO 2015/179190 refers to N- acylethanolamine hydrolysing acid amidase inhibitors, with potential utility in treating ulcerative colitis and Crohn’s disease. PCT application WO 2013/030218 refers to quinazolin-4-one compounds as inhibitors of ubiquitin specific proteases, such as USP7, with potential utility in treating cancer, neurodegenerative diseases, inflammatory disorders and viral infections. PCT applications WO 2015/017502 and WO 2016/019237 refer to inhibitors of Bruton’s tyrosine kinase with potential utility in treating disease such as autoimmune disease, inflammatory disease and cancer. PCT applications WO 2009/026197, WO 2009/129365, WO 2009/129370, and WO 2009/129371, refer to cyanopyrrolidines as inhibitors of Cathepsin C with potential utility in treating COPD. United States patent application US 2008/0300268 refers to polyaromatic compounds as inhibitors of tyrosine kinase receptor PDGFR. PCT applications WO 2019/222468 and WO 2019/071073 refer to cyanamide-containing compounds as USP30 inhibitors.

PCT application WO 2015/183987, refers to pharmaceutical compositions comprising deubiquitinase inhibitors and human serum albumin in methods of 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 said to have been implicated in the regulation of the TGF-beta signalling pathway, the disruption of which gives rise to neurodegenerative and fibrotic diseases, autoimmune dysfunction and cancer.

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

There exists a need for a safe, alternative and/or improved methods and compositions for the treatment of fibrotic disorders and the various symptoms and conditions associated therewith. While not wishing to be bound by any particular theory or mechanism, it is believed that the compounds of the present invention act to inhibit the enzyme USP30, which in turn upregulates Parkin-induced mitophagy.

Acute Kidney Injury (AKI) is defined as an abrupt decrease in kidney function occurring over 7 days or less, with severity of injury staged based on increased serum creatinine (SCr) and decreased urine output as described in the Kidney Disease Improving Global Outcomes (KDIGO) guidelines. AKI occurs in about 13.3 million people per year, 85% of whom live in the developing world and it is thought to contribute to about 1.7 million deaths every year (Mehta et al, 2015, Lancet 385(9987): 2616-2643). AKI more than likely results in permanent kidney damage (i.e., chronic kidney disease; CKD) and may also result in damage to non-renal organs. AKI is a significant public health concern particularly when taking into account the absolute number of patients developing incident CKD, progressive CKD, end-stage renal disease and cardiovascular events.

AKI and CKD are viewed as a continuum on the same disease spectrum (Chawla et al, 2017, Nat Rev Nephrol 13(4): 241-257). Patients undergoing coronary artery bypass graft (CABG) are at high risk for kidney injury. There is an obvious unmet medical need in the development of medicinal products for the treatment and/or prevention of AKI.

The kidney is a site of high metabolic demand, with high mitophagy rates demonstrated in vivo (McWilliams et al, 2018, Cell Metab 27(2): 439-449 e435). Renal Proximal Tubule Epithelial Cells (RPTECs), a cell type with significant ATP requirement for solute/ion exchange, are rich in mitochondria and are the primary effector cells of Acute Kidney Injury (AKI) in the kidney. Mitochondrial dysfunction has been implicated in AKI/CKD mechanisms, both through multiple lines of evidence from preclinical AKI and CKD models and also through data demonstrating abnormal mitochondrial phenotypes in patient biopsies (Emma et al, 2016, Nat Rev Nephrol 12(5): 267-280; Eirin et al, 2017, Handb Exp Pharmacol 240: 229-250). Furthermore, Primary mitochondrial disease often manifest in renal symptoms, such as focal segmental glomerulosclerosis (Kawakami et al, 2015, J Am Soc Nephrol 26(5): 1040-1052) in patients with MELAS/MIDD, and also primary tubular pathologies in patients with Coenzyme Q deficiencies. Mutations in mtDNA can cause 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) demonstrated that renal injury was exacerbated following ischemic AKI in both PINK1 KO and PARK2 KO mice, suggesting that PINK1/PARKIN-mediated mitophagy plays a protective role following IRI in the kidney. In addition, parkin/PINK1 mitophagy protects against cisplatin induced kidney injury (Wang et al, 2018, Cell Death Dis 9(11): 1113). Limited models of CKD are available for mitophagy investigation, supportive evidence for mitochondrial quality control in fibrosis comes from studies on fibrotic lung conditions such as COPD and IPF. Parkin knockout animals show exacerbated lung fibrosis in response to bleomycin (Kobayashi et al, 2016, J Immunol, 197:504-516). Similarly, airway epithelial cells from parkin knockout (KO) animals show exacerbated fibrotic and senescent responses to cigarette smoke (Araya et al, 2019, Autophagy 15(3): 510-526).

Preclinical models are available to study potential novel therapeutics, through their ability to model fibrosis pathology (e.g. collagen deposition) consistent with the human condition. Preclinical models can be toxin-mediated (e.g. bleomycin for lung and skin fibrosis), surgical (e.g. ischemia/reperfusion injury model and unilateral ureter obstruction model for acute tubulointerstitial fibrosis), and genetic (e.g. diabetic (db/db) mice for diabetic nephropathy). For example, both examples previously given for indicated IPF treatments (nintedanib and pirfenidone) show efficacy in the bleomycin lung fibrosis model.

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

SUMMARY OF THE INVENTION

The present invention is directed to compounds of formula (I):

a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, wherein either:

(i) X1 is CH; and

one of X2 and X3 is CR6, and the other is N; or

(ii) X1 is CH or N;

X2 is N; and

X3 is CR7 or N; with the proviso that one, solely, of X1 and X3 is N; and R1 is selected from (C₁-C₃)alkyl, (C₁-C₃)haloalkyl and CH₂OCH₃;

R2, R3 and R4 are each independently selected from hydrogen and deuterium;

R5 is selected from hydrogen, deuterium and fluorine; and

R6 and R7 are each independently selected from hydrogen, halogen, (C₁-C₃)alkyl and (C₁-C₃)alkoxy. The present invention is also directed to uses of the compounds of formula (I), particularly in the treatment of conditions involving mitochondrial dysfunction, cancer and fibrosis, and also processes for the preparation thereof and pharmaceutical compositions containing said compounds.

DETAILED DESCRIPTION OF THE INVENTION

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

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

USP30 is a transmembrane protein located in the outer membrane of mitochondria, which are energy- producing organelles present inside cells. Therefore, being able to demonstrate cellular activity in vitro is advantageous, as this is one of a number of components that may indicate a greater ability to engage the target in its physiological setting, i.e. where the USP30 inhibitor compound is able to penetrate cells. The USP30 cellular western blot (WB) assay described herein aims to test the activity of compounds against USP30 in cells using an irreversible activity probe to monitor USP30 activity. Analogously to the cellular western blot assay, target engagement assessment (ex vivo) may be carried out in either brain or kidney tissue samples from compound-dosed animals using the assay described herein.

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

In general, it is important for a drug to be as selective as possible for its desired target enzyme; additional activities give rise to the possibility of side effects. The exact physiological role of many DUBs has yet to be fully determined, however, irrespective of whatever role these DUBs may or may not play, it is a sound medicinal chemistry precept to ensure that any drug has selectivity over related mechanistic targets of unknown physiological function. Representative examples of DUB enzymes for which the compounds of the present invention may be screened against are UCHL1, UCHL3, UCHL5, YOD1, SENP2, SENP6, TRABID, BAP1, Cezanne, MINDY2/FAM63B, OTU1, OTUD3, OTUD5, OTUD6A, OTUD6B, OTUB1/UBCH5B, OTUB2, CYLD, VCPIP, AMSH-LP, JOSD1, JOSD2, USP1/UAF1, USP2, USP4, USP5, USP6, USP7, USP8, USP9x, USP10, USP11, USP12/UAF1, USP13, USP14, USP15, USP16, USP19, USP20, USP21, USP22, USP24, USP25, USP28, USP32, USP34, USP35, USP36, USP45, USP46/UAF1, USP47 and USP48. Preferably, compounds of the invention have good selectivity for USP30 over one or more of these DUB enzymes.

Aside from selectivity over other DUB enzymes, it is important for a drug to have low affinity for other targets, and pharmacological profiling may be performed against panels of targets to assess the potential for, and to minimise, potential off-target effects. Examples of targets for which the compounds of the present invention may be screened against are those of the industry standard Eurofins-Cerep SafetyScreen44 panel, which includes 44 targets as a representative selection of GPCR receptors, transporters, ion channels, nuclear receptors, and kinase and non-kinase enzymes. Preferably, compounds of the invention have insignificant affinity against targets of this screening panel. Further examples of targets for which the compounds of the present invention may be screened against are kinases of the Thermo Fisher SelectScreen kinase profiling panel, which includes 39 targets as a representative selection of kinase enzymes. Preferably, compounds of the invention have insignificant affinity against targets of this screening panel. Additionally, examples of a particular enzyme class for which the compounds of the present invention may be screened against are the cathepsins (e.g. cathepsin A, B, C, H, K, L, L2, S, V and Z). Preferably, 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 favourable pharmacokinetic properties such that they are suitable for oral administration. An orally administered drug should have good bioavailability; that is an ability to readily cross the gastrointestinal (GI) tract and not be subject to extensive metabolism as it passes from the GI tract into the systemic circulation. Once a drug is in the systemic circulation the rate of metabolism is also important in determining the time of residence of the drug in the body.

Thus, it is clearly favourable for drug molecules to have the properties of being readily able to cross the GI tract and being only slowly metabolised in the body. The Caco-2 assay is a widely accepted model for predicting the ability of a given molecule to cross the GI tract. The majority of metabolism of drug molecules generally occurs in the liver, and in vitro assays using whole cell hepatocytes (animal or human) are widely accepted methods for measuring the susceptibility of a given molecule towards metabolism in the liver. Such assays aim to predict in vivo clearance from the hepatocyte calculated clearance value.

Compounds which have good Caco-2 flux and are stable towards hepatocytes are predicted to have good oral bioavailability (good absorption across the GI tract and minimal extraction of compound as it passes through the liver) and a long residence time in the body that is sufficient for the drug to be efficacious.

The solubility of a compound is an important factor in achieving a desired concentration of drug in systemic circulation for the anticipated pharmacological response. Low aqueous solubility is a problem encountered with formulation development of new chemical entities and to be absorbed a drug must be present in the form of solution at the site of absorption. The kinetic solubility of a compound may be measured using a turbidimetric solubility assay, the data from which may also be used in conjunction with Caco-2 permeability data to predict dose dependent human intestinal absorption.

Other parameters that may be measured using standard assays that are indicative of a compound’s exposure profile include, for example plasma stability (half-life measurement), blood AUC, C_max, C_min and T_max values.

The treatment of CNS disorders, including Alzheimer’s disease, Parkinson’s disease, and other disorders described herein, requires drug molecules to target the brain, which requires adequate penetration of the blood brain barrier. There is, therefore, a need for USP30 inhibitors that possess effective blood brain penetration properties and provide suitable residence time in the brain to be efficacious. The probability that a compound can cross the blood brain barrier may be measured by an in vitro flux assay utilizing a MDR1-MDCK cell monolayer (Madin-Darby Canine Kidney cells transfected with MDR-1 resulting in overexpression of the human efflux transporter P-glycoprotein). Additionally, exposure may also be measured directly in brain and plasma using in vivo animal models.

There is also a need for compounds that have a favourable safety profile, which may be measured by a variety of standard in vitro and in vivo methods. A cell toxicity counter-screen may be used to assay the anti-proliferative/cytotoxic effect in a particular cell line (e.g. HCT116) by fluorometric detection of rezasurin (alamarBlue) to resofurin in response to mitochondrial activity.

Toxicology and safety studies may also be conducted to identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Regulatory requirements generally require studies to be conducted in at least two laboratory animal species, one rodent (rat or mouse) and one nonrodent (rabbit, dog, non-human primate, or other suitable species). The bacterial reverse mutation assay (Ames Test) may be used to evaluate the mutagenic properties of compounds of the invention, commonly by using the bacterial strain Salmonella typhimurium, which is mutant for the biosynthesis of the amino acid histidine.

The micronucleus assay may be used to determine if a compound is genotoxic by evaluating the presence of micronuclei. Micronuclei may contain chromosome fragments produced from DNA breakage (clastogens) or whole chromosomes produced by disruption of the mitotic apparatus (aneugens).

The hERG predictor assay provides valuable information about the possible binding of test compounds to the potassium channel and potential QT prolongation on echocardiogram. Inhibition of the hERG current causes QT interval prolongation resulting in potentially fatal ventricular tachyarrhythmia (Torsades de Pointes). Typically, assay data may be generated from an automated patch-clamp assay platform.

The compounds of the present invention have been found to demonstrate one or more of the above identified advantages over reference examples from the prior art sharing some structural similarity that are both significant and unexpected. For instance, all of the Examples of the present invention are significantly more potent for USP30 than the Reference Examples as measured in the biochemical assay described herein. All of the Examples of the present invention are significantly more selective for USP30 over other DUBs.

The significant and unexpected superiority of the compounds of the present invention make them particularly suitable for use in the treatment and/or prevention of diseases linked to UP30 activity. According to a first aspect, the present invention provides a compound of formula (I)

(i) X1 is CH; and

one of X2 and X3 is CR6, and the other is N; or

(iii) X1 is CH or N;

X2 is N; and

R2, R3 and R4 are each independently selected from hydrogen and deuterium;

R5 is selected from hydrogen, deuterium and fluorine; and

R6 and R7 are each independently selected from hydrogen, halogen, (C₁-C₃)alkyl and (C₁-C₃)alkoxy. The compound of formula (I) exists as a single stereoisomer with the absolute stereochemistry shown. The ring comprising X1, X2 and X3 is either (i) a pyridine or (ii) a pyrimidine ring.

Alkyl and alkoxy groups may be straight or branched and contain 1 to 3 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl and isopropyl. Examples of alkoxy include methoxy, ethoxy, n-propoxy and isopropoxy.

Halogen means fluorine, chlorine, bromine or iodine, in particular, fluorine or chlorine. Haloalkyl groups may contain one or more halo substituents. Examples are chloromethyl and chloroethyl. Unless otherwise indicated, the term substituted means substituted by one or more defined groups. In the case where groups may be selected from more than one alternative, the selected groups may be the same or different. The term‘independently’ means that where more than one substituent is selected from more than one possible substituent, those substituents may be the same or different.

Aspects of the present invention, and preferred embodiments thereof, of the compound of formula (I) are defined below.

In a first preferred aspect of the invention, X1 is CH; X2 is N; and X3 is CR6.

In a second preferred aspect of the invention, X1 is CH; X2 is CR6; and X3 is N.

In a third preferred aspect of the invention, X1 is CH; X2 is N; and X3 is N.

In a fourth preferred aspect of the invention, X1 is N; X2 is N; and X3 is CR7.

Preferably, R1 is selected from methyl, chloromethyl and CH2OCH3.

Most preferably, R1 is methyl.

Preferably, R2 is hydrogen.

Preferably, R3 is hydrogen.

Preferably, R4 is hydrogen.

Preferably, R5 is selected from hydrogen and fluorine.

Most preferably, R5 is hydrogen.

Preferably, R6 is selected from hydrogen, fluorine, methyl and methoxy.

Most preferably, R6 is hydrogen.

Preferably, R7 is selected from hydrogen, fluoro, methyl and methoxy.

Most preferably, R7 is hydrogen.

According to one preferred embodiment of each aspect of the invention:

R1 is selected from methyl, chloromethyl and CH2OCH3;

R2 is hydrogen;

R3 is hydrogen;

R4 is hydrogen; R5 is selected from hydrogen and fluorine;

R6 is selected from hydrogen, fluorine, methyl and methoxy; and

R7 is hydrogen.

According to another preferred embodiment of each aspect of the invention:

R1 is selected from methyl, chloromethyl and CH₂OCH₃;

R2 is hydrogen;

R3 is hydrogen;

R4 is hydrogen;

R5 is hydrogen;

R6 is selected from hydrogen, fluorine, methyl and methoxy; and

R7 is hydrogen.

Accordingly, in a first most preferred aspect of the invention:

X1 is CH; X2 is N; and X3 is CR6;

R1 is selected from methyl, chloromethyl and CH₂OCH₃;

R2 is hydrogen;

R3 is hydrogen;

R4 is hydrogen;

R5 is hydrogen; and

R6 is selected from hydrogen, fluorine, methyl and methoxy; and is most preferably hydrogen. In a second most preferred aspect of the invention:

X1 is CH; X2 is CR6; and X3 is N;

R1 is selected from methyl, chloromethyl and CH2OCH3;

R2 is hydrogen;

R3 is hydrogen;

R4 is hydrogen;

R5 is hydrogen; and

R6 is selected from hydrogen, fluorine, methyl and methoxy.

In a third most preferred aspect of the invention:

X1 is CH; X2 is N; and X3 is N;

R1 is selected from methyl, chloromethyl and CH2OCH3;

R2 is hydrogen;

R3 is hydrogen;

R4 is hydrogen; and

R5 is hydrogen.

In a fourth most preferred aspect of the invention: X1 is N; X2 is N; and X3 is CR7;

R1 is selected from methyl, chloromethyl and CH₂OCH₃;

R2 is hydrogen;

R3 is hydrogen;

R4 is hydrogen;

R5 is hydrogen; and

R7 is hydrogen.

Preferred compounds of formula (I) for use in the present invention are selected from:

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-fluoroisonicotinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)isonicotinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-methoxyisonicotinamide;

N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide;

N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methylpicolinamide; N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methoxypicolinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl-2,4,5,6-d4)picolinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4-carboxamide;

N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4- carboxamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)pyrimidine-2-carboxamide; and N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4-carboxamide;

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

Most preferred compounds of formula (I) for use in the present invention are selected from:

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide; and

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

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate, camsylate, citrate, edisylate, esylate, fumarate, gluceptate, gluconate, glucuronate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate, malonate, mesylate, methylsulfate, 2-napsylate, nicotinate, nitrate, orotate, palmate, phosphate, saccharate, stearate, succinate sulfate, D-and L-tartrate, and tosylate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, ammonium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

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

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

Pharmaceutical acceptable solvates in accordance with the invention include hydrates and solvates wherein the solvent of crystallization may be isotopically substituted, e.g. D₂O, acetone-d₆, DMSO-d₆. Also, within the scope of the invention are clathrates, drug-host inclusion complexes wherein, in contrast to the aforementioned solvates, the drug and host are present in non-stoichiometric amounts. For a review of such complexes, see J. Pharm Sci, 64 (8), 1269-1288 by Haleblian (August 1975). Hereinafter all references to compounds of formula (I) include references to salts thereof and to solvates and clathrates of compounds of formula (I) and salts thereof.

The invention includes all polymorphs of the compounds of formula (I) as hereinbefore defined. Also, within the scope of the invention are so-called "prodrugs" of the compounds of formula (I). Thus, certain derivatives of compounds of formula (I) which have little or no pharmacological activity themselves can, when metabolised upon administration into or onto the body, give rise to compounds of formula (I) having the desired activity. Such derivatives are referred to as "prodrugs".

Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as "pro-moieties" as described, for example, in "Design of Prodrugs" by H Bundgaard (Elsevier, 1985).

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

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

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

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person. Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture. The present invention includes all crystal forms of the compounds of formula (I) including racemates and racemic mixtures (conglomerates) thereof. Stereoisomeric conglomerates may be separated by conventional techniques known to those skilled in the art - see, for example, "Stereochemistry of Organic Compounds" by E. L. Eliel and S. H. Wilen (Wiley, New York, 1994). The compounds of formula (I) contain two chiral centres at the carbon atoms of the pyrrolidine ring that are substituted by R1 and the amide and said stereocentres could exist in either the (R) or (S) configuration. The designation of the absolute configuration (R) and (S) for stereoisomers in accordance with IUPAC nomenclature is dependent on the nature of the substituents and application of the sequence-rule procedure. The compounds of formula (I) could, therefore, exist in four stereoisomeric forms.

The compounds of formula (I) of the present invention exist as a single stereoisomer. The pyrrolidine carbon atom of the amide substituent exists as the (R)-stereocentre, whereas the designation of the pyrrolidine carbon atom of the R1 group is dependent on the nature of the substituent. The compound of formula (I) is isolated as a single stereoisomer and may exist with a stereoisomeric excess of at least 60%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, for example 96%, 97%, 98%, 99%, or 100%.

Additional chiral centres may exist in the compounds of formula (I) within the R1 substituent itself. Included within the scope of the present invention are all such stereoisomeric forms of the compounds of formula (I).

The present invention also includes all pharmaceutically acceptable isotopic variations of a compound of formula (I). An isotopic variation is defined as one in which at least one atom is replaced by an atom having the same atomic number, but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as 13C and 14C, nitrogen, such as 15N, oxygen, such as 17O and 18O, phosphorus, such as 32P, sulfur, such as 35S, fluorine, such as 18F, and chlorine, such as 36CI. Substitution of the compounds of the invention with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half- life or reduced dosage requirements, and hence may be preferred in some circumstances.

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

Isotopic variations of the compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using appropriate isotopic variations of suitable reagents. The compounds of formula (I) are inhibitors of the deubiquitylating enzyme USP30.

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

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

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

The disorder or condition benefiting from 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 benefiting from USP30 activity is a condition involving mitochondrial dysfunction. Mitochondrial dysfunctions result from defects of the mitochondria, which are specialized compartments present in every cell of the body except red blood cells. When mitochondria fail, less and less energy is generated within the cell and cell injury or even cell death will follow. If this process is repeated throughout the body the life of the subject in whom this is happening is severely compromised. Diseases of the mitochondria appear most often in organs that are very energy demanding such as the brain, heart, liver, skeletal muscles, kidney and the endocrine and respiratory system.

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

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

The condition involving mitochondrial dysfunction may be a CNS disorder, for example 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 related to mutations in a-synuclein, parkin, PINK1, GBA, and LRRK2, and autosomal recessive juvenile Parkinson’s disease (AR-JP) where parkin is mutated.

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

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

In particular, the compounds of the invention may be useful in the treatment or prevention of cancer where apoptotic pathways are dysregulated and more particularly where proteins of the BCL-2 family are mutated, or over or under expressed. Fibrosis refers to the accumulation of extracellular matrix constituents that occurs following trauma, inflammation, tissue repair, immunological reactions, cellular hyperplasia, and neoplasia. Fibrotic disorders that may be treated by the compounds and compositions of the present invention include, inter alia, fibrosis/fibrotic disorders associated with major organ diseases, for example, interstitial lung disease (ILD), liver cirrhosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (hepatic fibrosis), kidney disease (renal fibrosis), acute kidney injury (AKI), chronic kidney disease (CKD), delayed kidney graft function, heart or vascular disease (cardiac fibrosis) and diseases of the eye; fibroproliferative disorders, for example, systemic and local scleroderma, keloids and hypertrophic scars, atherosclerosis, restenosis, and Dupuytren’s contracture; scarring associated with trauma, for example, surgical complications, chemotherapeutics drug- induced fibrosis (e.g. bleomycin-induced fibrosis), radiation-induced fibrosis, accidental injury and burns); retroperitoneal fibrosis (Ormond's disease); and peritoneal fibrosis/peritoneal scarring in patients receiving peritoneal dialysis, usually following renal transplantation. See, for example, Wynn et al, 2004, Nat Rev Immunol. August; 4(8): 583–594. The present invention therefore relates to methods of treatment or prevention, and compounds and compositions used in said methods, of fibrosis/fibrotic disorders of and/or associated with the major organs, including for example, the lung, liver, kidney, heart, skin, eye, gastrointestinal tract, peritoneum and bone marrow, and other diseases/disorders herein described.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 employed by which the compound enters the blood stream directly from the mouth.

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

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

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

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

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

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

Parenteral Administration

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

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

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

The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by suitable processing, for example, the use of high energy spray-dried dispersions and/or by the use of appropriate formulation techniques, such as the use of solubility-enhancing agents. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled dual, targeted and programmed release.

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

Dosage

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

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

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

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

According to a further aspect, the present invention provides a process for the preparation of a compound of formula (I) comprising reacting a compound of formula (IV), where X is OH with an amine of formula (V), where PG is a protecting group, such as BOC or CBZ, to give an amide of formula (III) (Scheme 1). The amide-coupling reaction can be performed using standard methodology, for example by reaction using a coupling reagent such as DCC, HATU, HBTU, EDC or via a mixed anhydride. Alternatively, the acid (IV), where X is OH, can be converted into the acid chloride (IV), where X is Cl, using SOCl2, PCl3, or PCl5, which can then be reacted with the amine (V), preferably in a suitable solvent in the presence of a suitable base. Alternatively, the compound (IV), where X forms the ester, can be reacted directly with the amine (V), preferably in a suitable solvent.

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

Scheme 1 In a further aspect, the present invention provides a compound, which is selected from formulae (II) and (III):

wherein PG is a protecting group, preferably BOC or CBZ, and X1, X2, X3, R1, R2, R3, R4 and R5 are as defined herein for the compound of formula (I) and preferred embodiments thereof, a tautomer thereof, or a salt of said compound or tautomer.

Abbreviations

br s Broad singlet (NMR signal)

d Doublet (NMR signal)

DCM Dichloromethane

DSC Differential Scanning Calorimetry

DMF

amide

DMSO Dimethylsulfoxide

dppf 1,1'-Bis(diphenylphosphino)ferrocene

ES Electrospray

EtOAc Ethyl acetate

h Hour(s)

m Multiplet (NMR signal)

Ms Mesyl/methanesulfonyl

MeOH Methanol

min Minute(s)

PLM/SEM Polarised Light Microscopy / Scanning Electron Microscopy

rt Room temperature

s Singlet (NMR signal)

TBD Triazabicyclodecene

TFA Trifluoroacetic acid

TGA Thermo-Gravimetric Analysis

THF Tetrahydrofuran LCMS Methods Method C

Method H

Method J

Method M

Example 1

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide

Building block synthesis

tert-Butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate

Scheme: (i) MsCl, TEA, DCM, 0 °C, 2 h; (ii) NaN3, DMF, 0 °C to 80 °C, 4 h;

(iii) 3 M LiBH4 in THF, THF, -30 °C to rt, 3 h; then NaHCO3, -78 °C to rt, 16 h; (iv) MsCl, TEA, DCM, -10 °C to rt, 3 h; (v) LiI, THF, 0 °C to 60 °C, 3 h;

(vi) H2, 10% Pd/C, TEA, MeOH, rt, 16 h.

Step (i)

1-(tert-Butyl) 2-methyl (2S,4S)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate

This reaction was performed in duplicate. To a stirred solution of CAS 102195-79-9 (500 g, 2038.3 mmol, 1.0 eq, from Pharmablock) in DCM (5000 mL) was added triethylamine (411.7 g, 566 mL, 4076.6 mmol, 2.0 eq) followed by methanesulfonyl chloride (302.1 g, 305 mL, 2649.8 mmol, 1.3 eq) dropwise at 0 °C and the mixture was stirred for 2 h. This was poured into water (2500 mL) and the organic layer was separated and extracted with DCM (3000 mL). The organic phases were combined and washed with dilute citric acid solution (800 mL) and brine (1500 mL), dried over Na2SO4. After work up both the identical batches were combined and concentrated under reduced pressure to afford 1-(tert-butyl) 2-methyl (2S,4S)-4- ((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate as a brown semi-solid (1200 g, 3715.17 mmol, 91% yield). LCMS: Method C, 1.51 min, MS: [M-56] 268.1; 1H NMR (400 MHz, DMSO -d₆) d ppm: 5.26 - 5.27 (m, 1H), 4.42 - 4.56 (m, 1H), 3.78 - 3.83 (m, 5H), 3.04 (s, 3H), 2.51 - 2.58 (m, 2H), 1.51 (s, 4.5H, Boc rotamer 1), 1.46 (s, 4.5H, Boc rotamer 2).

This reaction was repeated on the same scale to yield further product (600 g, 1856 mmol, 91% yield). Step (ii)

1-(tert-Butyl) 2-methyl (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate

To a stirred solution of 1-(tert-butyl) 2-methyl (2S,4S)-4-((methylsulfonyl)oxy)pyrrolidine-1,2- dicarboxylate (500 g, 1548.0 mmol, 1.0 eq) in DMF (5000 mL) was added NaN₃ (139.3 g, 2322.0 mmol, 1.5 eq) portionwise at 0 °C. The reaction mixture was gradually heated to 80 °C for 4 h. The resulting reaction mixture was cooled to rt and poured into ice cold water (3500 mL) and extracted with EtOAc (3 x 3000 mL). The organic phases were combined and washed with ice cold water (4 x 1000 mL) and brine (2 x 1000 mL), dried over Na₂SO₄ and concentrated under reduced pressure to afford 1-(tert-butyl)-2-methyl-(2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate as a brown semi-solid (400 g, 1481.5 mmol, 95% yield).

LCMS: Method C, 1.65 min, MS: ES+ 271.2; 1H NMR (400 MHz, DMSO -d₆) d ppm: 4.36 - 4.39 (m, 1H), 4.20 - 4.27 (m, 1H), 3.65 - 3.68 (m, 3H), 3.51 - 3.58 (m, 1H), 3.38 - 3.41 (d, 1H), 2.29 - 2.38 (m, 1H), 2.10 - 2.19 (m, 1H), 1.40 (s, 4.5H, Boc rotamer 1), 1.34 (s, 4.5H, Boc rotamer 2).

Further sub-title compound (400 g, 1470 mmol, 95% yield) was prepared by an identical method on a 252 g scale in duplicate.

Step (iii)

tert-Butyl (2S,4R)-4-azido-2-(hydroxymethyl)pyrrolidine-1-carboxylate

To a stirred solution of 1-(tert-butyl) 2-methyl (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate (300 g, 1111.1 mmol, 1.0 eq) in THF (3000 mL) was added 3 M LiBH₄ solution in THF (47.2 g, 2166.7 mmol, 1.95 eq) dropwise at -30 °C. The reaction mixture was allowed to warm to rt and stirred for 3 h. The resulting reaction mixture was quenched by dropwise addition of a saturated aqueous solution of NaHCO3 (4000 mL) at -78 °C and stirred for 16 h at rt. The crude mixture was extracted with EtOAc (3 x 3000 mL) and washed with brine (1000 mL). The organic phase was dried over Na2SO4 and concentrated under reduced pressure to give a brown semi-solid (220 g). The crude semi-solid was purified by column chromatography (23% EtOAc in hexane) to obtain tert-butyl (2S,4R)-4-azido-2-(hydroxymethyl)pyrrolidine-1-carboxylate as a pale yellow liquid (202.5 g, 836.8 mmol, 75% yield).

LCMS: Method C, 1.51 min, MS: ES+ 243.2; 1H NMR (400 MHz, DMSO-d₆) d ppm: 4.77 (s, 1 H), 4.24 (s, 1H), 3.77 (br s, 1H), 3.33 - 3.46 (m, 4H), 2.10 - 2.15 (m, 1H), 1.98 - 1.99 (m, 1H), 1.39 (s, 9H).

The reaction was further performed in duplicate on 2 x 250 g scale by an identical method to yield further sub-title compound (337.5 g, 1395 mmol, 75% yield).

Step (iv)

(2S,4R)-4-Azido-2-(((methylsulfonyl)oxy)methyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S,4R)-4-azido-2-(hydroxymethyl)pyrrolidine-1-carboxylate (440 g, 1818.2 mmol, 1.0 eq) in DCM (4400 mL) was added triethylamine (551.9 g (760 mL), 5454.5 mmol, 3.0 eq) at rt and stirred for 10 min and then methanesulfonyl chloride (312.4 g, 2727.27 mmol, 1.5 eq) was added dropwise at -10 °C. The reaction mixture was slowly warmed to rt and stirred for 3 h. The resulting reaction mixture was poured into ice-cold water (4000 mL) and the organic phase was separated. The aqueous layer was extracted with DCM (2 x 2000 mL). The organic phases were combined and washed with dilute citric acid (2 x 1000 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to afford tert-butyl (2S,4R)-4-azido-2- (((methylsulfonyl)-oxy)methyl)pyrrolidine-1-carboxylate as a brown liquid (550 g, quantitative yield). The crude material was directly used in the next step.

LCMS: Method M, 17.67 min, MS: [M-56] 265.0.

The reaction was repeated on a 60 g scale by an identical method to yield further sub-title compound (76 g, quantitative yield).

Step (v)

tert-Butyl (2S,4R)-4-azido-2-(iodomethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S, 4R)-4-azido-2-(((methylsulfonyl)oxy)methyl) pyrrolidine-1- carboxylate (550 g, 1718.8 mmol, 1.0 eq) in THF (5500 mL) was added lithium iodide (344.9 g, 2578.12 mmol, 1.5 eq) at 0 °C. The reaction mixture was slowly warmed to rt and then gradually heated at 60 °C for 3 h. The reaction mixture was cooled to rt, poured into ice-cold water (5000 mL) and extracted with EtOAc (3 x 2500 mL). The organic phases were combined and washed with brine (2000 mL), dried over Na2SO4 and concentrated under reduced pressure to give tert-butyl (2S,4R)-4- azido-2-(iodomethyl)pyrrolidine-1-carboxylate as a brown liquid (630 g). The crude material was purified by column chromatography (10% EtOAc in hexane) to afford tert-butyl (2S,4R)-4-azido-2- (iodomethyl)pyrrolidine-1-carboxylate as a light brown liquid (490 g, 1392 mmol, 76% yield over two steps).

LCMS: Method M 21.64 min, MS: [M-56] 297.0; 1H NMR (400 MHz, DMSO-d₆) d ppm: 4.32 (s, 1H), 3.34 - 3.71 (m, 5H), 2.19 (s, 1H), 1.91 - 1.95 (m, 1H), 1.42 (s, 9H).

The reaction was repeated on a 76 g (237.5 mmol) scale by an identical method to yield further sub- title compound (60 g, 170.5 mmol, 68% yield over two steps).

Step (vi)

tert-Butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate

This reaction was performed in duplicate. To a stirred solution of tert-butyl (2S,4R)-4-azido-2- (iodomethyl)pyrrolidine-1-carboxylate (250 g, 709.9 mmol, 1.0 eq) in MeOH (2500 mL) was added triethylamine (79.0 g, 109 mL, 780.9 mmol, 1.1 eq) followed by palladium on carbon (125 g, 50% w/w) at rt under N2 atmosphere. The reaction mixture was purged with H2 gas for 16 h at rt. The identical batches were combined and filtered through a bed of Celite, washing with MeOH (5000 mL) and the filtrate was concentrated under reduced pressure to give a brown semi-solid. The crude material was poured into ice-cold water (2500 mL) and acidified with solid citric acid (pH 4). The acidic aqueous layer was washed with EtOAc (3 x 2500 mL) and then basified with solid NaHCO₃ (pH 9) and extracted with EtOAc (3 x 3000 mL). The combined extracts were dried over Na₂SO₄ and concentrated under reduced pressure to yield tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1- carboxylate as light brown liquid (240 g, 1200 mmol, 84% yield).

LCMS: Method J, 2.67 min, MS: [M-56] 144.8; 1H NMR (400 MHz, DMSO-d₆) d ppm: 3.80 (s, 1H), 3.34 - 3.48 (m, 2H), 2.92 (s, 1H), 2.67 (br s, 2H), 1.74– 1.70 (m, 1H), 1.62 (s, 1H), 1.38 (s, 9H), 1.09 (s, 3H).

Synthesis of Example 1

Scheme: (i) PdCl2(dppf), K2CO3, 1,4-dioxane, H2O, 80 °C, 2 h; (ii) TBD, THF, 0 °C to rt, 16 h;

(iii) TFA, DCM, 0 °C to rt, 2 h; (iv) K2CO3, CNBr, THF, 0 °C to rt, 2 h.

Step (i)

Methyl 4-(3-cyanophenyl)picolinate

To a stirred solution of methyl 4-bromopicolinate (CAS 29681-42-3, from Angene, 200 g, 0.926 mol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from Angene, 162 g, 1.11 mol, 1.2 eq) in 6:11,4-dioxan/water (2500 mL) was added K2CO3 (383 g, 2.78 mol, 3.0 eq). The reaction mixture was purged with N2 gas for 30 min and then PdCl2(dppf) (33.8 g, 0.0462 mol, 0.05 eq) was added to the reaction mixture, which was then heated at 80 °C for 3 h. The mixture was poured into ice-cold water (13 L) and the precipitated solid was collected and dried under reduced pressure. The crude material was purified by column chromatography (60-120# silica gel, 0.4% MeOH in DCM) to afford methyl 4-(3-cyanophenyl)picolinate (150 g, 630.25 mmol, 68% yield).

LCMS: Method M, 14.46 min, MS: ES+239; 1H NMR (400 MHz, DMSO-d₆) d ppm: 8.81 (d, J = 4.8 Hz, 1H), 8.42 (s, 1H), 8.39 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.07 (dd, J = 4.8, 1.6 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 3.92 (s, 3H).

Step (ii)

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

To a stirred solution of tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate) (106 g, 531.5 mmol, 1.1 eq) and methyl 4-(3-cyanophenyl)picolinate (115 g, 483.2 mmol, 1.0 eq) in THF (1000 mL) was added a solution of TBD (80.5 g, 579.83 mmol, 1.2 eq) in THF (150 mL)) dropwise at 0 °C. The mixture was stirred at rt for 16 h, then poured into water (2000 mL) and extracted with EtOAc (2 x 2000 mL). The combined organic phases were dried over Na₂SO₄ and concentrated in vacuo. The crude material was purified by column chromatography (60-120# silica gel, 25% EtOAc in hexane) to yield tert-butyl (2R,4R)-4-(4-(3-cyanophenyl)picolinamido)-2- methylpyrrolidine-1-carboxylate (110 g, 270.93 mmol, 56% yield).

LCMS: Method M, 20.49 min, MS: ES+351 (M-56); 1H NMR (400 MHz, DMSO-d₆) d ppm: 8.95 (d, J = 7.2 Hz, 1H), 8.77 (d, J = 4.8 Hz, 1H), 8.42 (s, 1H), 8.36 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.04 (dd, J = 5.2, 1.6 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 4.59 - 4.62 (m, 1H), 3.95 (m, 1H), 3.59 (m, 1H), 3.29 - 3.30 (m, 1H), 2.24 (m, 1H), 1.84 (m, 1H), 1.41 (s, 9H), 1.19 (br s, 3H). Step (iii)

4-(3-Cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide trifluoroacetate

To a stirred solution of tert-butyl (2R,4R)-4-(4-(3-cyanophenyl)picolinamido)-2-methylpyrrolidine-1- carboxylate (110 g, 270.9 mmol, 1.0 eq) in DCM (1000 mL) was added TFA (330 mL, 3 volumes) dropwise at 0 °C. The mixture was stirred at rt for 2 h. The mixture was concentrated under reduced pressure, then DCM (500 mL) was added to the residue before repeating the concentration process four times with DCM to obtain 4-(3-cyanophenyl)-N-((3R, 5R)-5-methylpyrrolidin-3-yl)picolinamide trifluoroacetate (150 g, 357.1 mmol, quantitative).

LCMS: Method M, 11.67 min, MS: ES+307; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.30 (d, J = 7.2 Hz, 1H), 9.08 (br s, 1H), 8.80 (d, J = 4.8 Hz, 1H), 8.71 (br s, 1H), 8.43 (s, 1H), 8.38 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.08 (dd, J = 5.2 Hz, 1.6 Hz, 1H), 8.00 - 8.02 (d, J = 7.6 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 4.73 (m, 1H), 3.91 (m, 1H), 3.61 - 3.62 (m, 1H), 3.30 - 3.34 (m, 1H), 2.22 - 2.27 (m, 1H), 1.91 - 1.98 (m, 1H), 1.34 (d, J = 6.8 Hz, 3H).

Step (iv)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide

To a stirred solution of 4-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide trifluoroacetate (150 g, 357.1 mmol, 1.0 eq) in THF (1500 mL) was added K2CO3 (147 g, 1071.4 mmol, 3 eq) portionwise at rt. The mixture was cooled to 0 °C. A solution of cyanogen bromide (37.8 g, 357.14 mmol, 1 eq) in THF (40 mL) was added dropwise and the mixture was stirred at rt for 4 h, then poured onto water (2000 mL) and was extracted with EtOAc (2 x 2000 mL). The combined organic phases were washed with brine (2000 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by column chromatography (60-120# silica gel, 0.5 % MeOH in DCM) to yield N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)- picolinamide (60.0 g, 181.26 mmol, 67% yield, over 2 steps).

LCMS: Method M, 16.30 min, MS: ES+332.2; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.15 - 9.17 (d, J=7.2 Hz, 1H), 8.76 - 8.77 (d, J=5.2 Hz, 1H), 8.41 (s, 1H), 8.34 (s, 1H), 8.21 - 8.22 (d, J=8.0 Hz, 1H), 8.03 - 8.05 (dd, J=4.8, 1.6 Hz, 1H), 7.98 - 7.99 (d, J=7.6 Hz, 1H), 7.76 - 7.80 (t, J=8.0 Hz, 1H), 4.55 - 4.57 (m, 1H), 3.89 - 3.94 (m, 1H), 3.74 - 3.78 (m, 1H), 3.45 - 3.48 (m, 1H), 2.16 - 2.22 (m, 1H), 1.75 - 1.82 (m, 1H), 1.24 - 1.26 (d, J=6.4 Hz, 3H).

Alternative Synthesis of Example 1

Step (i)

Methyl 4-(3-cyanophenyl)picolinate

3-Cyanophenylboronic acid (108.5 g, 738.3 mmol), methyl-4-bromopicolinate (145.0 g, 671.2 mmol), potassium carbonate (185.5 g, 1.34 mol) and PdCl₂(dppf) (24.5 g, 33.6 mmol) were dissolved in DMF (655 mL) and stirred under nitrogen atmosphere. The mixture was heated to 85 to 90 ºC for 6 h before cooling to rt. DCM (1450 mL) was added and the mixture was washed with 10% brine (1000 mL) and water (4 x 1000 mL). The organic phase was dried over MgSO₄ and concentrated under reduced pressure giving 205.7 g of a dark purple solid. The solid contained 6.5% w/w DMF by 1H NMR and had a purity of 87.7% by LC. The crude material was dissolved in DCM and absorbed onto silica (200 g). The crude material was purified in two equal portions by flash column chromatography with silica gel (725 g) packed in 25% heptane/DCM), eluting with 25% heptane/DCM, then DCM, followed by 1 : 1 EtOAc/DCM, then EtOAc. The clean fractions from both columns were combined and concentrated under reduced pressure to yield 125.9 g (78% yield) of sub-title compound as a light brown solid, with a purity of 99.1% by LC (contained 0.9% w/w DMF by 1H NMR). The mixed fractions were combined to provide further sub-title compound (30.3 g at 60% purity).

Step (ii)

Methyl 4-(3-cyanophenyl)picolinate (125.7 g, 527.6 mmol), toluene (1250 mL), tert-butyl-(2R,4R)-4- amino-2-methylpyrrolidine-1-carboxylate (111.0 g, 554.0 mmol) and TBD (14.7 g, 105.5 mmol) were charged to a flask and stirred under nitrogen atmosphere. The brown suspension was heated to 50 to 55 ºC and stirred at this temperature overnight. The mixture was cooled to rt and washed with 10% aqueous citric acid (630 mL). Saturated aq. NaHCO3 (630 mL) was added to the organic phase and a precipitate formed. The mixture was filtered, washing the solid residue with EtOAc (500 mL), and the filtrate was collected and the phases were separated. The aqueous phase was extracted further with EtOAc (500 mL). The combined organic phases were dried over MgSO4 and concentrated under reduced pressure. DCM (630 mL) was added and the solution was concentrated under reduced pressure to give a light brown foam (189.3 g, 88% yield) with a purity of 98.0% by LC (contained 1.8% w/w DCM and 1.4% w/w toluene by 1H NMR). Step (iii)

4-(3-Cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide hydrochloride

tert-Butyl (2R,4R)-4-(4-(3-cyanophenyl)picolinamido)-2-methylpyrrolidine-1-carboxylate (192 g, 472.6 mmol) was dissolved in MeCN (1920 mL) and stirred under nitrogen atmosphere. 4M HCl in EtOAc (made from HCl gas; 576 mL, 2.30 mol) was added slowly over 10 min keeping the temperature below 30 ºC. The mixture was stirred at rt and after 10 min a suspension had formed. The reaction was complete after 2 h. The mixture was filtered and the solid was washed with MeCN (2 x 550 mL), then dried in an oven at 40 ºC overnight to furnish the sub-title compound as a tan solid (178.3 g, 99% yield) with a purity of 98.2% by LC.

Step (iv)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide

4-(3-Cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide hydrochloride (148 g, 389.7 mmol) and potassium carbonate (215.4 g, 1.56 mol) were suspended in MeCN (1924 mL) and stirred under nitrogen atmosphere. A solution of BrCN (43.3 g, 409.2 mmol) in MeCN (296 mL) was added slowly over 10 min. A water bath was placed around the flask and an exotherm was observed to 25 ºC. The mixture was stirred at rt overnight and was filtered. The solid was washed with MeCN (2 x 450 mL). The filtrate was washed with 10% brine (2 x 750 mL), dried over MgSO4 and concentrated under reduced pressure. The experiment was repeated analogously on 30 g scale as described above. The crude residues were combined at this point (total 151.5 g crude title material) then transferred to a 5 L flask with ethanol (2270 mL) and was heated to 72 ºC over 30 min (a solution formed at 68 ºC). The mixture was cooled to 25 ºC over 40 min (crystallisation occurred at 59 ºC). The suspension was stirred at 20 to 25 ºC for 1 h, then filtered. The solid was collected and washed with ethanol (2 x 300 mL) and dried in an oven at 50 ºC to provide title compound (115.7 g, 74% yield), at 99.2% purity and with an enantiomeric excess of 99.3%.

DSC: Sharp endotherm, onset temperature: 164.9 °C (94 J/g).

TGA: Decomposition from ca. 220 °C. The TGA thermogram confirmed that the sample was anhydrous with no weight loss prior to decomposition.

PLM/SEM: Crystalline plates.

XRPD diffractograms were collected on a Bruker AXS C2 GADDS diffractometer using Cu K ^ radiation (40 kV, 40 mA), an automated XYZ stage, a laser video microscope for auto-sample positioning and a Våntec-500 2-dimensional area detector. X-ray optics consists of a single Göbel multilayer mirror coupled with a pinhole collimator of 0.3 mm. The beam divergence was approximately 4 mm. A q-q continuous scan mode was employed with a sample– detector distance of 20 cm which gives an effective 2q range of 1.5° to 32.5°. The sample was exposed to the X-ray beam for 120 seconds. The software used for data collection and analysis was GADDS for Win7/XP and Diffrac Plus EVA respectively. XRPD: Peak picking:

Example 2

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-fluoroisonicotinamide

Step (i)

tert-Butyl (2R, 4R)-4-(2-chloro-5-fluoroisonicotinamido)-2-methylpyrrolidine-1-carboxylate

To a stirred solution of 2-chloro-5-fluoroisonicotinic acid (CAS 884494-74-0, from Ark Pharm, 0.30 g, 1.71 mmol, 1.0 eq) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.342 g, 1.71 mmol, 1.0 eq) in pyridine (5 mL) was added POCl₃ (0.48 mL, 0.786 g, 5.14 mmol, 3.0 eq) dropwise at 0 °C. The mixture was stirred at 0 °C for 20 min, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to yield tert-butyl (2R, 4R)-4-(2-chloro-5- fluoroisonicotinamido)-2-methylpyrrolidine-1-carboxylate (0.35 g, 0.98 mmol, 57% yield).

LCMS: Method C, 1.67 min, MS: ES+ 358.5.

Step (ii)

tert-Butyl (2R,4R)-4-(2-(3-cyanophenyl)-5-fluoroisonicotinamido)-2-methylpyrrolidine-1-carboxylate To a stirred solution of tert-butyl (2R,4R)-4-(2-chloro-5-fluoroisonicotinamido)-2-methylpyrrolidine- 1-carboxylate (0.350 g, 0.980 mmol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS: 150255-96-2, from Combi-blocks, 0.214 g, 1.47 mmol, 1.5 eq) in 1,4-dioxane (5 mL) and water (0.5 mL) was added K₂CO₃ (0.405 g, 2.94 mmol, 3.0 eq). The reaction mixture was purged with N₂ for 20 min and [1,1'-bis (diphenylphosphino)ferrocene]palladium(II) dichloride (0.071 g, 0.098 mmol, 0.1 eq) was added, and the mixture was heated at 100 °C for 2 h. The mixture was poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash column chromatography (25% EtOAc in hexane) to afford tert-butyl (2R, 4R)-4-(2-(3-cyanophenyl)-5- fluoroisonicotinamido)-2-methylpyrrolidine-1-carboxylate (0.110 g, 0.26 mmol, 26% yield).

LCMS: Method C, 1.80 min, MS: ES+ 369.4 (M-56).

Step (iii)

2-(3-Cyanophenyl)-5-fluoro-N-((3R, 5R)-5-methylpyrrolidin-3-yl)isonicotinamide

To a stirred solution of tert-butyl (2R,4R)-4-(2-(3-cyanophenyl)-5-fluoroisonicotinamido)-2- methylpyrrolidine-1-carboxylate (0.110 g, 0.26 mmol, 1.0 eq) in DCM (5 mL) was added TFA (0.40 mL, 3 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h. The mixture was concentrated under reduced pressure, then concentrated with DCM (3 x 30 mL) to afford 2-(3-cyanophenyl)-5-fluoro-N-((3R,5R)-5-methylpyrrolidin-3-yl)isonicotinamide

trifluoroacetate (0.155 g, 0.35 mmol, quantitative yield).

LCMS: Method C, 1.34 min, MS: ES+ 325.4.

Step (iv)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-fluoro isonicotinamide

To a stirred solution of 2-(3-cyanophenyl)-5-fluoro-N-((3R,5R)-5-methylpyrrolidin-3- yl)isonicotinamide trifluoroacetate (0.150 g, 0.34 mmol, 1.0 eq) in THF (4 mL) was added K2CO3 (0.235 g, 1.71 mmol, 5.0 eq) followed by cyanogen bromide (0.028 g, 0.27 mmol, 0.8 eq) at 0 °C. The mixture was stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The organic phases were combined, dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash column chromatography (60% EtOAc in hexane) to afford N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-fluoroisonicotinamide (0.025 g, 0.072 mmol, 27% yield over two steps).

LCMS: Method H, 3.50 min, MS: ES+ 350.15; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.15 (d, J = 6.4 Hz, 1H), 8.83 (s, 1H), 8.56 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.29 (d, J = 5.2 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 4.49 - 4.51 (m, 1H), 3.80 - 3.92 (m, 2H), 3.36 - 3.39 (m, 1H), 2.10 - 2.14 (m, 1H), 1.77 - 1.84 (m, 1H), 1.28 (d, J = 6.0 Hz, 3H). Example 3

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)isonicotinamide

Step (i)

Methyl 2-(3-cyanophenyl)isonicotinate

To a stirred solution of methyl 2-bromoisonicotinate (CAS 26156-48-9, from Combi-Blocks, 0.50 g, 2.31 mmol, 1.0 eq) in 1,4-dioxane (8 mL) and water (2 mL) at rt under N2 was added cesium carbonate (1.8 g, 5.79 mmol, 2.5 eq) followed by (3-cyanophenyl)boronic acid (CAS 150255-96-2, from CombiBlocks, 0.50 g, 3.74 mmol, 1.5 eq). [1,1'-bis(Diphenylphosphino)ferrocene]- palladium(II) dichloride (0.169 g, 0.23 mmol) was added to the mixture at rt, then the mixture was heated at 80 °C for 4 h and allowed to cool to rt. The mixture was poured into water (100 mL) and extracted with EtOAc (3 x 25 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (10% EtOAc in hexane) to obtain methyl 2-(3-cyanophenyl)isonicotinate (0.4 g, 1.680 mmol, 27 % yield). LCMS: Method C, 1.65 min, MS: [M+1] 239.3.

Step (ii)

tert-Butyl (2R, 4R)-4-(2-(3-cyanophenyl)isonicotinamido)-2-methylpyrrolidine-1-carboxylate To a stirred solution of 2-(3-cyanophenyl)isonicotinate (0.267 g, 1.13 mmol, 1.5 eq) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.15 g, 0.75 mmol, 1.0 eq) in THF (5 mL) was slowly added TBD (CAS 5807-14-7, from TCI, 0.208 g, 1.5 mmol, 2.0 eq) at 0 °C. The mixture was slowly warmed to rt and stirred for 2 h, then poured into water (50 mL) and extracted with EtOAc (3 x 25 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (25% EtOAc in hexane) to provide tert-butyl (2R,4R)-4-(2-(3-cyanophenyl)isonicotinamido)-2-methylpyrrolidine-1-carboxylate (0.125 g, 0.307 mmol, 41% yield).

LCMS: Method C, 1.72 min, MS: [M+1] 407.9.

Step (iii)

2-(3-Cyanophenyl)-N-((3R, 5R)-5-methylpyrrolidin-3-yl)isonicotinamide trifluoroacetate

To a stirred solution of tert-butyl (2R,4R)-4-(2-(3-cyanophenyl)isonicotinamido)-2-methylpyrrolidine- 1-carboxylate (0.12 g, 0.30 mmol, 1.0 eq) in DCM (4 mL) was added trifluoroacetic acid (1.2 mL, 10 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h, then concentrated under reduced pressure, and concentrated with DCM (2 x 2 mL) under reduced pressure to afford 2-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)isonicotinamide trifluoroacetate (0.12 g, 0.29 mmol, 96% yield).

LCMS: Method C, 1.33 min, MS: [M+1] 307.5.

Step (iv)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl) isonicotinamide

To a stirred solution of 2-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)isonicotinamide trifluoroacetate (0.12 g, 0.29 mmol, 1.0 eq) in THF (4 mL) was added K₂CO₃ (0.11 g, 0.857 mmol, 3.0 eq) followed by cyanogen bromide (0.036 g, 0.342 mmol, 1.2 eq) at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h, then poured into water (50 mL). The solid was collected by filtration and washed with water (10 mL), dried under reduced pressure, then triturated with diethyl ether (5 mL) and residual solvent removed under reduced pressure to afford N-((3R,5R)-1-cyano-5- methylpyrrolidin-3-yl)-2-(3-cyanophenyl) isonicotinamide (0.06 g, 0.181 mmol, 63% yield).

LCMS: Method H, 3.33 min, MS: [M+1] 332.15; 1H NMR (400 MHz, DMSO-d₆) d ppm: 8.99 (d, J = 6.0 Hz, 1H), 8.85 (d, J = 4.8 Hz, 1H), 8.57 (s, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.75 - 7.79 (m, 2H), 4.49 - 4.58 (m, 1H), 3.92 - 3.96 (m, 1H), 3.80 - 3.84 (m, 1H), 3.38 - 3.43 (m, 1H), 1.98 - 2.19 (m, 1H), 1.77 - 1.84 (m, 1H), 1.27 (d, J = 6.0 Hz, 3H).

Example 4

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-methoxyisonicotinamide

Step (i)

2-Bromo-5-methoxyisonicotinic acid

To a stirred solution of diisopropylamine (0.295 g, 2.93 mmol, 1.1 eq) in dry THF (10 mL) was added n-BuLi (2.5 M in hexane, 1.06 mL, 2.66 mmol, 1.0 eq) dropwise at -78 °C. The mixture was stirred at -78 °C for 1 h. A solution of 2-bromo-5-methoxypyridine (CAS 105170-27-2, from CombiBlocks, 0.500 g, 2.66 mmol, 1.0 eq) in dry THF (1 mL) was added dropwise at -78 °C. After stirring at -78 °C for 2 h, dry ice was added. The reaction mixture was stirred at rt for 16 h. The procedure above was repeated, the mixtures from the two reactions were poured into water (100 mL) and the combined mixture was extracted with EtOAc (100 mL). The organic phase was separated, and the aqueous layer was acidified with dilute HCl (to pH ~4) and further extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure to yield 2-bromo-5-methoxyisonicotinic acid (0.500 g, 2.16 mmol, 40% yield). LCMS: Method C, 1.33 min, MS: ES+ 232.2, 234.4; 1H NMR (400 MHz, DMSO-d₆) d ppm: 13.74 (s, 1H), 8.36 (s, 1H), 7.72 (s, 1H), 3.94 (s, 3H).

Step (ii)

tert-Butyl (2R,4R)-4-(2-bromo-5-methoxyisonicotinamido)-2-methylpyrrolidine-1-carboxylate To a stirred solution of 2-bromo-5-methoxyisonicotinic acid (0.250 g, 1.08 mmol, 1.0 eq) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.215 g, 1.08 mmol, 1.0 eq) in pyridine (4 mL) was added POCl₃ (0.494 g, 0.29 mL, 3.23 mmol, 3.0 eq) dropwise at 0 °C. The mixture was stirred at 0 °C for 20 min and then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (30% EtOAc in hexane) to yield tert-butyl (2R,4R)-4-(2-bromo-5-methoxyisonicotinamido)-2-methylpyrrolidine-1-carboxylate (0.230 g, 0.56 mmol, 51% yield).

LCMS: Method C, 1.68 min, MS: ES+ 414.4, 416.4; 1H NMR (400 MHz, CDCl₃) d ppm: 8.23 (s, 1H), 8.19 (s, 1H), 7.78 (d, J = 6.4 Hz, 1H), 4.68 - 4.73 (m, 1H), 4.12 (s, 3H), 3.95 - 4.10 (m, 1H), 3.71 - 3.76 (m, 1H), 3.39 - 3.53 (m, 1H), 1.80– 2.10 (m, 2H), 1.51 (s, 9H), 1.31 (d, J = 6.8 Hz, 3H).

Step (iii)

tert-Butyl (2R,4R)-4-(2-(3-cyanophenyl)-5-methoxyisonicotinamido)-2-methyl pyrrolidine-1- carboxylate

To a stirred solution of tert-butyl (2R, 4R)-4-(2-bromo-5-methoxyisonicotinamido)-2- methylpyrrolidine-1-carboxylate (0.210 g, 0.51 mmol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from CombiBlocks, 0.074 g, 0.51 mmol, 1.0 eq) in 1,4-dioxane (4 mL) and water (0.5 mL) was added K2CO3 (0.210 g, 1.52 mmol, 3.0 eq). The mixture was purged with N2 for 20 min and then [1,1'-bis(diphenylphosphino)ferrocene]palladium(II)dichloride (0.037 g, 0.051 mmol, 0.1 eq) was added. The mixture was heated at 100 °C for 2 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash column chromatography (35% EtOAc in hexane) to afford tert-butyl (2R,4R)-4-(2-(3-cyanophenyl)-5-methoxyisonicotinamido)-2- methyl pyrrolidine-1-carboxylate (0.200 g, 0.46 mmol, 90% yield).

LCMS: Method C, 1.75 min, MS: ES+ 437.7. 1H NMR (400 MHz, CDCl3) d ppm: 8.58 (s, 1H), 8.51 (s, 1H), 8.41 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 6.0 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 4.74 - 4.76 (m, 1H), 4.18 (s, 3H), 4.04 - 4.10 (m, 1H), 3.76 - 3.78 (m, 1H), 3.33 - 3.58 (m, 1H), 2.04 - 2.17 (m, 2H), 1.52 (s, 9H), 1.33 (br s, 3H).

Step (iv)

2-(3-Cyanophenyl)-5-methoxy-N-((3R,5R)-5-methylpyrrolidin-3-yl) isonicotinamide trifluoroacetate To a stirred solution of tert-butyl (2R,4R)-4-(2-(3-cyanophenyl)-5-methoxyisonicotinamido)-2- methylpyrrolidine-1-carboxylate (0.200 g, 0.46 mmol, 1.0 eq) in DCM (5 mL) was added TFA (0.60 mL, 3 volumes) dropwise at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 1 h. The mixture was concentrated under reduced pressure, and again from DCM (3 x 30 mL) to afford 2-(3-cyanophenyl)-5-methoxy-N-((3R,5R)-5-methylpyrrolidin-3-yl)isonicotinamide trifluoroacetate (0.305 g, 0.68 mmol, quantitative yield).

LCMS: Method C, 1.33 min, MS: ES+ 337.53; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.18 (s, 1H), 8.79 (d, J = 6.8 Hz, 1H), 8.66 (s, 1H), 8.49 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 4.51 - 4.65 (m, 1H), 4.02 (s, 3H), 3.79 - 3.83 (m, 1H), 3.63 - 3.67 (m, 1H), 3.18 - 3.22 (m, 1H), 2.15 - 2.20 (m, 1H), 1.90 - 2.00 (m, 1H), 1.34 (d, J = 6.4 Hz, 3H). Step (v)

To a stirred solution of 2-(3-cyanophenyl)-5-methoxy-N-((3R,5R)-5-methylpyrrolidin-3- yl)isonicotinamide trifluoroacetate (0.300 g, 0.67 mmol, 1.0 eq) in THF (6 mL) was added K₂CO₃ (0.46 g, 3.33 mmol, 5.0 eq) followed by cyanogen bromide (0.056 g, 0.53 mmol, 0.8 eq) at 0 °C. The mixture was stirred for 1 h at rt, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash column chromatography (60% EtOAc in hexane) to afford N-((3R, 5R)-1-cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)-5-methoxy- isonicotinamide (0.085 g, 0.24 mmol, 51% yield over two steps).

LCMS: Method H, 3.44 min, MS: ES+ 362.2; 1H NMR (400 MHz, DMSO-d₆) d ppm: 8.77 (d, J = 6.8 Hz, 1H), 8.63 (s, 1H), 8.51 (s, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 4.46 - 4.48 (m, 1H), 4.01 (s, 3H), 3.77 - 3.91 (m, 2H), 3.38 - 3.36 (m, 1H), 2.13 - 2.09 (m, 1H), 1.81 - 1.74 (m, 1H), 1.27 (d, J = 6.4 Hz, 3H).

Example 5

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

Step (i)

tert-Butyl (2S, 4R)-4-(4-(3-cyanophenyl) picolinamido)-2-(methoxymethyl) pyrrolidine-1-carboxylate To a stirred solution of methyl 4-(3-cyanophenyl) picolinate (0.30 g, 1.26 mmol, 1.0 eq) and tert-butyl (2S, 4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.288 g, 1.26 mmol, 1.0 eq) in THF (10 mL) was added TBD (CAS 5807-14-7, from TCI, 0.407 g, 3.14 mmol, 2.5 eq) at 0 °C and the mixture was stirred at rt for 4 h. The mixture was poured into water (25 mL) and extracted with EtOAc (2 x 25 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash chromatography (35% EtOAc in hexane) to afford tert-butyl (2S,4R)-4-(4-(3-cyanophenyl) picolinamido)-2-(methoxymethyl) pyrrolidine-1- carboxylate (0.184 g, 0.42 mmol, 34% yield).

LCMS: Method C, 1.76 min, MS: [M-56] 381.7; 1H NMR (400 MHz, CDCl₃) d ppm: 8.68 (d, J = 5.2 Hz, 1H), 8.45 (s, 1H), 8.14 (d, J = 7.2 Hz, 1H), 8.02 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.71 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 4.80 - 4.90 (m, 1H), 4.15 - 4.17 (m, 1H), 3.80 - 3.91 (m, 2H), 3.52 - 3.63 (m, 2H), 3.42 (s, 3H), 2.40 - 2.51 (m, 1H), 2.02 - 2.15 (m, 1H), 1.50 (s, 9H). Step (ii)

4-(3-Cyanophenyl)-N-((3R, 5S)-5-(methoxymethyl)pyrrolidin-3-yl)picolinamide

To a stirred solution of tert-butyl (2S,4R)-4-(4-(3-cyanophenyl)picolinamido)-2-(methoxymethyl) pyrrolidine-1-carboxylate (0.180 g, 0.41 mmol, 1.0 eq) in DCM (10 mL) was added TFA (1.8 mL, 10 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h, then concentrated under reduced pressure. The residue was concentrated under reduced pressure from DCM (3 x 30 mL), then triturated with diethyl ether (2 x 10 mL) to afford 4-(3-cyanophenyl)-N-((3R, 5S)-5-(methoxymethyl)pyrrolidin-3-yl) picolinamide trifluoroacetate (0.125 g, 0.28 mmol, 68% yield).

LCMS: Method C, 1.35 min, MS: ES+ 337.3; 1H NMR (400 MHz, DMSO -d₆) d ppm: 9.28 (d, J = 7.2 Hz, 1H), 9.20 (s, 1H), 8.80 (d, J = 5.2 Hz, 1H), 8.74 (s, 1H), 8.43 (s, 1H), 8.34 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.09 (s, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 4.69 - 4.72 (m, 1H), 4.04 - 4.11 (m, 1H), 3.60 - 3.63 (m, 2H), 3.49 - 3.54 (m, 2H), 3.36 (s, 3H), 2.18 - 2.22 (m, 1H), 2.04 - 2.10 (m, 1H).

Step (iii)

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

To a stirred solution of 4-(3-cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)picolin- amide trifluoroacetate (0.12 g, 0.27 mmol, 1.0 eq) in acetonitrile (10 mL) was added K2CO3 (0.11 g, 0.80 mmol, 3.0 eq) followed by cyanogen bromide (0.022 g, 0.21 mmol, 0.8 eq) at 0 °C. The mixture was allowed to warm slowly to rt and stirred for 20 min, then poured into water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash chromatography (60 % EtOAc in hexane) to N-((3R,5S)-1-cyano-5-(methoxymethyl) pyrrolidin-3-yl)-4-(3-cyanophenyl)-picolinamide (0.065 g, 0.18 mmol, 96% yield).

LCMS: Method H, 3.33 min, MS: ES+ 362.2; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.14 (d, J = 7.2 Hz, 1H), 8.78 (d, J = 5.2 Hz, 1H), 8.42 (s, 1H), 8.35 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.05 - 8.06 (m, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 4.55 - 4.59 (m, 1H), 4.09 - 4.11 (m, 1H), 3.68 - 3.72 (m, 1H), 3.42 - 3.50 (m, 3H), 3.34 (s, 3H), 2.15 - 2.23 (m, 1H), 1.95– 2.01 (m, 1H). Example 6

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

Step (i)

tert-Butyl (2S,4R)-4-azido-2-(methoxymethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S,4R)-4-azido-2-(hydroxymethyl)pyrrolidine-1-carboxylate (10 g, 41.30 mmol, 1.0 eq) in acetonitrile (150 mL) was added NaH (60% in paraffin oil, 4.95 g, 123.90 mmol, 3.0 eq) portionwise at 0 °C. The mixture was stirred at 0 °C for 30 min, followed by dropwise addition of methyl iodide (17.58 g, 123.90 mmol, 3.0 eq), and the mixture was stirred for 5 h at rt. The reaction mixture was poured into water (500 mL) and extracted with EtOAc (2 x 500 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to afford tert-butyl (2S,4R)-4-azido-2-(methoxymethyl)pyrrolidine-1-carboxylate (10 g, 39.06 mmol, 94% yield).

LCMS: Method C, 1.72 min, MS: ES+ [M-100] 157.5.

Step (ii)

tert-Butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S, 4R)-4-azido-2-(methoxymethyl)pyrrolidine-1-carboxylate (11 g, 42.97 mmol, 1.0 eq) in MeOH (110 mL) was added 10% palladium on carbon (50% moist, 5.5 g, 50% w/w) at rt under N2 atmosphere. The mixture was purged with H2 gas for 5 h at rt, then filtered through Celite, washed with MeOH (150 mL), and the filtrate was concentrated under reduced pressure to afford tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (8.0 g, 34.78 mmol, 81% yield).

LCMS: Method C, 1.27 min, MS: ES+ [M-100] 131.3.

Step (iii)

tert-Butyl (2S,4R)-4-(4-bromo-5-methylpicolinamido)-2-(methoxymethyl) pyrrolidine-1-carboxylate To a stirred solution of 4-bromo-5-methylpicolinic acid (CAS 1196154-93-4, from BLD Pharma, 0.3 g, 1.39 mmol, 1.0 eq) in THF (5 mL) was added DIPEA (0.268 g, 0.35 mL, 2.08 mmol, 1.5 eq) and HATU (0.791 g, 2.08 mmol, 1.5 eq) at rt. The mixture was stirred for 20 min and a solution of tert-butyl (2S,4R)-4-amino-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.383 g, 1.67 mmol, 1.2 eq) in THF (1 mL) was added dropwise, then stirred for 1 h. The mixture was poured into water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash column chromatography (21% EtOAc in hexane) to afford tert-butyl (2S,4R)-4-(4-bromo-5- methylpicolinamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.445 g, 1.04 mmol, 75% yield). LCMS: Method C, 1.94 min, MS: ES+ 428.4, 430.4.

Step (iv)

tert-Butyl (2S,4R)-4-(4-(3-cyanophenyl)-5-methylpicolinamido)-2-(methoxy methyl)pyrrolidine-1- carboxylate

To a stirred solution of tert-butyl (2S,4R)-4-(4-bromo-5-methylpicolinamido)-2-(methoxymethyl)- pyrrolidine-1-carboxylate (0.200 g, 0.468 mol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from Combi-blocks, 0.103 g, 0.703 mol, 1.5 eq) in DMF (5 mL) and water (2.5 mL) was added NaHCO₃ (0.118 g, 1.405 mol, 3.0 eq). The mixture was purged with N₂ for 10 min and [1,1¢-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.017 g, 0.0234 mol, 0.05 eq) was added. The mixture was heated at 100 °C for 4 h, then poured into water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (32% EtOAc in hexane) to afford tert-butyl (2S,4R)-4-(4-(3-cyanophenyl)-5-methylpicolinamido)-2- (methoxymethyl)-pyrrolidine-1-carboxylate (0.161 g, 0.358 mmol, 76% yield).

LCMS: Method C, 1.79 min, MS: ES+ 451.7.

Step (v)

4-(3-Cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-5-methyl picolinamide

trifluoroacetate

To a stirred solution of tert-butyl (2S,4R)-4-(4-(3-cyanophenyl)-5-methylpicolinamido)-2- (methoxymethyl)pyrrolidine-1-carboxylate (0.158 g, 0.35 mmol, 1.0 eq) in DCM (3 mL) was added TFA (0.474 mL, 3 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h. The mixture was concentrated under reduced pressure and the residue was concentrated with DCM (3 x 10 mL) under reduced pressure to afford 4-(3-cyanophenyl)-N-((3R,5S)-5- (methoxymethyl)-pyrrolidin-3-yl)-5-methylpicolinamide trifluoroacetate (0.301 g, 0.649 mmol, quantitative yield).

LCMS: Method C, 1.36 min, MS: ES+ 351.61.

Step (vi)

N-((3R,5S)-1-Cyano-5-(methoxymethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methylpicolinamide To a stirred solution of 4-(3-cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl)-5-methyl- picolinamide trifluoroacetate (0.298 g, 0.64 mmol, 1.0 eq) in THF (5 mL) was added K2CO3 (0.443 g, 3.21 mmol, 5.0 eq) at 0 °C. Cyanogen bromide (0.054 g, 0.51 mmol, 0.8 eq) was added at 0 °C and the mixture was stirred for 30 min, then poured into water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash column chromatography (74% EtOAc in hexane) to afford N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methyl picolinamide (0.06 g, 0.16 mmol, 46% yield over two steps).

LCMS: Method H, 3.47 min, MS: ES+ 376.25; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.09 (d, J = 7.2 Hz, 1H), 8.65 (s, 1H), 8.03 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.87 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 4.53 - 4.55 (m, 1H), 4.03 - 4.05 (m, 1H), 3.67 - 3.70 (m, 1H), 3.40 - 3.49 (m, 3H), 3.33 (s, 3H), 2.35 (s, 3H), 2.14 - 2.21 (m, 1H), 1.93 - 1.99 (m, 1H).

Example 7

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methoxypicolinamide

Step (i)

tert-Butyl (2R, 4R)-4-(4-chloro-5-methoxypicolinamido)-2-methylpyrrolidine-1-carboxylate

To a stirred solution of 4-chloro-5-methoxypicolinic acid (CAS 103878-33-7, from CombiBlocks, 0.2 g, 1.067 mmol, 1.0 eq) in THF (10 mL) was added DIPEA (0.41 g, 0.54 mL, 3.20 mmol, 3.0 eq) followed by addition of HATU (0.607 g, 1.60 mmol, 1.5 eq). The mixture was stirred for 30 min at 0 °C and a solution of tert-butyl (2R, 4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.21 g, 1.067 mmol, 1.0 eq) in THF (1 mL) was added dropwise. The mixture was stirred at rt for 4 h, then poured into water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (24% EtOAc in hexane) to afford tert-butyl (2R,4R)-4-(4-chloro-5- methoxypicolinamido)-2-methylpyrrolidine-1-carboxylate (0.34 g, 0.92 mmol, 86% yield).

LCMS: Method C, 1.73 min, MS: ES+ 370.3; 1H NMR (400 MHz, CDCl₃) d ppm: 8.24 (s, 1H), 8.19 (s, 1H), 7.83 (d, J = 7.2 Hz, 1H), 4.70 - 4.75 (m, 1H), 4.10 (s, 3H), 3.98 - 4.09 (m, 1H), 3.77 - 3.81 (m, 1H), 3.30 - 3.50 (m, 1H), 1.96 - 2.20 (m, 2H), 1.50 (s, 9H), 1.31 (br s, 3H).

Step (ii)

tert-Butyl (2R,4R)-4-(4-(3-cyanophenyl)-5-methoxypicolinamido)-2-methyl pyrrolidine-1-carboxylate To a stirred solution of tert-butyl (2R,4R)-4-(4-chloro-5-methoxypicolinamido)-2-methylpyrrolidine- 1-carboxylate (0.300 g, 0.81 mmol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from CombiBlocks, 0.237 g, 1.62 mmol, 2.0 eq) in DMF (6 mL) and water (3 mL) was added NaHCO₃ (0.204 g, 2.43 mmol, 3.0 eq). The mixture was purged with N₂ for 15 min and [1,1¢-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.059 g, 0.0811 mmol, 0.1 eq) was added. The reaction mixture was heated at 130 °C for 6 h, then poured into water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (35% EtOAc in hexane) to afford tert-butyl (2R,4R)-4-(4-(3-cyanophenyl)-5-methoxypicolinamido)- 2-methylpyrrolidine-1-carboxylate (0.265 g, 0.61 mmol, 75% yield).

LCMS: Method C, 2.29 min, MS: ES+ 381.2 (M-56).

Step (iii)

4-(3-Cyanophenyl)-5-methoxy-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide trifluoroacetate To a stirred solution of tert-butyl (2R, 4R)-4-(4-(3-cyanophenyl)-5-methoxypicolinamido)-2- methylpyrrolidine-1-carboxylate (0.25 g, 0.59 mmol, 1.0 eq) in DCM (10 mL) at 0 °C was added TFA (1.27 mL, 5 volumes) dropwise. The mixture was slowly warmed to rt and stirred for 1 h. The mixture was concentrated under reduced pressure and again with DCM (3 x 10 mL) to afford the crude 4-(3-cyanophenyl)-5-methoxy-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide

trifluoroacetate (0.47 g).

LCMS: Method C, 1.316 min, MS: ES+ 337.3.

Step (iv)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methoxy picolinamide

To a stirred solution of crude 4-(3-cyanophenyl)-5-methoxy-N-((3R,5R)-5-methylpyrrolidin-3- yl)picolinamide trifluoroacetate (0.460 g, 1.02 mmol, 1.0 eq) in THF (10 mL) was added K2CO3 (0.423 g, 3.067 mmol, 3.0 eq) followed by cyanogen bromide (0.086 g, 0.82 mmol, 0.8 eq) at 0 °C. The mixture was stirred at 0 °C for 1 h, then poured into water (50 mL) and extracted with EtOAc (3 x 50 mL). The organic phases were combined, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (57% EtOAc in hexane) to afford N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)-5-methoxypicolinamide (0.075 g, 0.21 mmol, 35% yield over two steps).

LCMS: Method H, 3.51 min, MS: ES+ 362.10; 1H NMR (400 MHz, DMSO-d₆) d ppm: 8.95 (d, J = 7.2 Hz, 1H), 8.51 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H), 7.93 (t, J = 9.2 Hz, 2H), 7.70 (t, J = 8.0 Hz, 1H), 4.53 - 4.52 (m, 1H), 4.01 (s, 3H), 3.90 (m, 1H), 3.74 (m, 1H), 3.44 (dd, J = 9.6, 4.4 Hz, 1H), 2.15 - 2.21 (m, 1H), 1.74 - 1.80 (m, 1H), 1.25 (d, J = 6.4 Hz, 3H).

Example 8

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl-2,4,5,6-d4)picolinamide

Step (i)

3-Nitrobenzoic-2,4,5,6-d₄ acid

To a stirred solution of benzoic-2,3,4,5,6-d₅ acid (5.0 g, 39.32 mmol, CAS 1079-02-3, from QMX) in concentrated H₂SO₄ (15 mL, 3 volumes) was added concentrated HNO₃ (5 mL, 1 volume) dropwise at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 2 h. The mixture was slowly poured into ice-cold water (500 mL), and a precipitate was collected by suction filtration. The solid was washed with cold water and dried under reduced pressure to afford 3-nitrobenzoic-2,4,5,6-d₄ acid (4.75 g, 27.77 mmol, 70% yield).

2H NMR (61.44 MHz, DMSO, traces of DMSO-d₆ added) d ppm: 8.64 (s, 1D), 8.46 (s, 1D), 8.36 (s, 1D), 7.83 (s, 1D).

Step (ii)

Methyl 3-nitrobenzoate-2,4,5,6-d₄

To a stirred solution of 3-nitrobenzoic-2,4,5,6-d₄ acid (7.4 g, 43.15 mmol, 1 eq) in DCM (74 mL) was added DMF (0.2 mL, cat.) followed by oxalyl chloride (13.7 g, 107.87 mmol, 2.5 eq) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h, then concentrated under reduced pressure under N2 atmosphere. To the residue was added methanol (74 mL) at 0 °C and the mixture was stirred for 10 min, then poured slowly into ice-cold water (100 mL). A precipitate formed, and the solid was collected by suction filtration, washed with cold water, then dried under reduced pressure to afford methyl 3-nitrobenzoate-2,4,5,6-d4 (7.12 g, 38.48 mmol, 89% yield).

1H NMR (400 MHz, DMSO-d₆) d ppm: 3.94 (s, 3H); 2H NMR (61.44 MHz, DMSO, traces of DMSO-d₆ added) d ppm: 8.63 - 8.39 (m, 3D), 7.86 (s, 1D).

Step (iii)

Methyl 3-aminobenzoate-2,4,5,6-d4

To a stirred solution of methyl 3-nitrobenzoate-2,4,5,6-d4 (7.1 g, 38.34 mmol, 1 eq) in 1:1 THF / D2O (71 mL) was added acetic acid (11.6 g, 191.71 mmol, 5 eq) followed by iron powder (6.42 g, 115.02 mmol, 3 eq) at 0 °C. The reaction mixture was slowly warmed to rt and stirred for 4 h. The mixture was filtered through Celite and washed with EtOAc (200 mL). The filtrate was washed with water (2 x 100 mL) and the combined organic phases were dried over Na2SO4 and concentrated under reduced pressure to afford methyl 3-aminobenzoate-2,4,5,6-d4 (5.31 g, 34.25 mmol, 89% yield). LCMS: Method C, 1.32 min, MS: ES+ 156.1; 1H NMR (400 MHz, DMSO-d₆) d ppm: 5.39 (s, 2H), 3.80 (s, 3H); 2H NMR (61.44 MHz, DMSO, traces of DMSO-d₆ added) d ppm:7.15 (br s, 3D), 6.81 (s, 1D).

Step (iv)

Methyl 3-bromobenzoate-2,4,5,6-d4

To a stirred solution of methyl 3-aminobenzoate-2,4,5,6-d4 (5.3 g, 34.17 mmol, 1.0 eq) in acetonitrile (53 mL) was added tert-butyl nitrite (5.3 g, 51.26 mmol, 1.5 eq) and the mixture was stirred for 15 min. Copper(II) bromide (7.63 g, 34.17 mmol, 1.0 eq) was added and the mixture was slowly warmed to rt and stirred for 1 h, then poured into ice-cold water (250 mL) and extracted with EtOAc (2 x 250 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash column chromatography (30% EtOAc in hexane) to afford methyl 3-bromobenzoate-2,4,5,6-d₄ (6.8 g, 31.05 mmol, 90% yield).

1H NMR (400 MHz, DMSO-d₆) d ppm: 3.88 (s, 3H).

Step (v)

3-Bromobenzoic-2,4,5,6-d₄ acid

To a stirred solution of methyl 3-bromobenzoate-2,4,5,6-d₄ (6.8 g, 31.04 mmol, 1.0 eq) in THF (68 mL) was added LiOH (6.55 g, 155.25 mmol, 5.0 eq) followed by D₂O (7 mL) and MeOH (5 mL) at 0 °C. The mixture was slowly warmed to rt and stirred for 2 h, then concentrated under reduced pressure and the residue was diluted with ice-cold water (150 mL). The phases were separated, and the aqueous layer was acidified with 2N HCl (to pH 3 to 4). The precipitated solid was collected by suction filtration and dried under reduced pressure to afford 3-bromobenzoic-2,4,5,6-d₄ acid (6.04 g, 29.46 mmol, 95% yield).

LCMS: Method H, 1.72 min, MS: ES+ 205; 1H NMR (400 MHz, DMSO-d₆) d ppm: 13.37 (s, 1H). Step (vi)

3-Bromobenzamide-2,4,5,6-d4

To a stirred solution of 3-bromobenzoic-2,4,5,6-d4 acid (6.0 g, 29.26 mmol,1.0 eq) in DMF (60 mL) was added EDC (6.7 g, 35.12 mmol, 1.2 eq), HOBt (4.75 g, 35.12 mmol, 1.2 eq) and DIPEA (11.3 g, 87.78 mmol, 3.0 eq) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and then NH4Cl (1.9 g, 35.12 mmol, 1.2 eq) was added. The mixture was slowly warmed to rt and stirred for 18 h, then poured into ice-cold water (500 mL) and the precipitated solid collected by suction filtration. The residue was dried under reduced pressure and then purified by column chromatography (45% EtOAc in hexane) to obtain 3-bromobenzamide-2,4,5,6-d4 (4.7 g, 23.04 mmol, 79% yield). LCMS: Method H, 2.56 min, MS: ES- 202; 1H NMR (400 MHz, DMSO-d₆) d ppm: 8.11 (s, 1H), 7.55 (s, 1H).

Step (vii)

3-Bromobenzonitrile-2,4,5,6-d4

To a stirred solution of 3-bromobenzamide-2,4,5,6-d4 (0.5 g, 2.45 mmol,1.0 eq) in dry DMF (5 mL) was added dry POCl3 (0.38 g, 2.45 mmol, 1.0 eq) under N2 at 0 °C and stirred for 30 min. The reaction mixture was poured into ice-cold water (50 mL) and the precipitated solid was collected by suction filtration. The residue was dried under reduced pressure to afford 3-bromobenzonitrile- 2,4,5,6-d4 (0.35 g, 1.88 mmol, 77% yield).

2H NMR (61.44 MHz, DMSO, traces of DMSO-d₆ added) d ppm: 8.17 (s, 1D), 7.91 (s, 2D), 7.56 (s, 1D). Step (viii)

Methyl 4-(3-cyanophenyl-2,4,5,6-d₄)picolinate

To a stirred solution of 3-bromobenzonitrile-2,4,5,6-d₄ (0.4 g, 2.17 mmol, 1.0 eq) and methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinate (CAS 957062-72-5, from CombiBlocks) (0.686 g, 2.61 mmol, 1.2 eq) in 1,4-dioxane (4 mL) was added K₃PO₄ (0.921 g, 4.34 mmol, 2.0 eq). The mixture was purged with N₂ for 10 min then [1,1¢-bis(diphenylphosphino)ferrocene]dichloro palladium(II) (0.160 g, 0.22 mmol, 0.1 eq) was added. The mixture was heated at 80 °C for 1 h, poured into ice-cold water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (70% EtOAc in hexane) to afford methyl 4-(3-cyanophenyl-2,4,5,6- d₄)picolinate (0.275 g, 1.14 mmol, 52% yield).

LCMS: Method J, 3.96 min, MS: ES+ 243; 1H NMR (DMSO-d₆) d ppm: 8.84 (d, J = 5.2 Hz, 1H), 8.41 (s, 1H), 8.09 (dd, J = 4.8 Hz, 1.6 Hz, 1H), 3.94 (s, 3H); 2H NMR (61.44 MHz, DMSO, traces of DMSO-d₆ added) d ppm: 8.02 (m, 4D).

Step (ix)

tert-Butyl (2R,4R)-4-(4-(3-cyanophenyl-2,4,5,6-d4)picolinamido)-2-methylpyrrolidine-1-carboxylate To a stirred solution of methyl 4-(3-cyanophenyl-2,4,5,6-d4)picolinate (0.27 g, 1.12 mmol, 1.0 eq) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.245 g, 1.23 mmol, 1.1 eq) in THF (15 mL) was added TBD (0.232 g, 1.67 mmol, 1.5 eq) portionwise at 0 °C. The mixture was slowly warmed to rt and stirred for 12 h, then poured into water (70 mL) and extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (20% EtOAc in hexane) to afford tert-butyl (2R,4R)-4-(4-(3-cyanophenyl-2,4,5,6-d4)picolinamido)-2-methylpyrrolidine-1-carboxylate (0.29 g, 0.71 mmol, 63% yield).

LCMS: Method C, 1.91 min, MS: ES+ 411.6; 1H NMR (DMSO-d₆): d ppm: 8.94 (d, J = 7.6 Hz, 1H), 8.77 (d, J = 5.2 Hz, 1H), 8.36 (s, 1H), 8.05 (d, J = 4.8 Hz, 1H), 4.60 - 4.62 (m, 1H), 3.95 (m, 1H), 3.59 (m, 2H), 2.26 (m, 1H), 1.84 (m, 1H), 1.42 (s, 9H), 1.19 (br s, 3H).

Step (x)

4-(3-Cyanophenyl-2,4,5,6-d4)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide trifluoroacetate To a stirred solution of tert-butyl (2R,4R)-4-(4-(3-cyanophenyl-2,4,5,6-d4)picolinamido)-2- methylpyrrolidine-1-carboxylate (0.28 g, 0.682 mmol) in DCM (5 mL) was added TFA (1.4 mL, 5 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 2 h, then concentrated under reduced pressure and again with DCM (3 x 25 mL) to afford 4-(3-cyanophenyl- 2,4,5,6-d4)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolinamide trifluoroacetate (0.35 g, quantitative yield).

LCMS: Method C, 1.35 min, MS: ES+ 311.3. Step (xi)

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl-2,4,5,6-d₄) picolinamide

To a stirred solution of 4-(3-cyanophenyl-2,4,5,6-d₄)-N-((3R,5R)-5-methylpyrrolidin-3-yl)picolin- amide trifluoroacetate (0.35 g, 0.82 mmol, 1.0 eq) in THF (5 mL) was added K₂CO₃ (0.57 g, 4.12 mmol, 5.0 eq) followed by cyanogen bromide (0.087 g, 0.82 mmol, 1.0 eq) at 0 °C. The mixture was slowly warmed to rt and stirred for 2 h, poured into ice cold water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (65% EtOAc in hexane) to yield N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl-2,4,5,6-d₄)picolinamide (0.105 g, 0.313 mmol, 46% yield, over two steps).

LCMS: Method M, 16.61 min, MS: ES+ 336.2; 1H NMR (DMSO-d₆): d ppm: 9.16 (d, J = 6.8 Hz, 1H), 8.79 (d, J = 4.8 Hz, 1H), 8.37 (s, 1H), 8.06 (d, J = 3.6 Hz, 1H), 4.57 (m, 1H), 3.91 - 3.96 (m, 1H), 3.76 - 3.80 (m, 1H), 3.47 - 3.50 (m, 1H), 2.20 - 2.23 (m, 1H), 1.77 - 1.84 (m, 1H), 1.27 (d, J = 6.0 Hz, 3H); 2H NMR (61.44 MHz, DMSO, traces of DMSO-d₆ added) d ppm: 8.18 - 7.95 (d, 3D), 7.56 (s, 1D).

Example 9

N-((3R,5R)-1-Cyano-5-methylpyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4-carboxamide

Step (i)

6-(3-Cyanophenyl)pyrimidine-4-carboxylic acid

To a stirred solution of methyl 6-chloropyrimidine-4-carboxylate (CAS 6627-22-1, from CombiBlocks, 0.70 g, 4.06 mmol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from CombiBlocks, 1.19 g, 8.11 mmol, 2 eq) in 1,4-dioxane (5 mL) and water (2.5 mL) was added Cs₂CO₃ (2.63 g, 8.11 mmol, 2.0 eq). The reaction mixture was purged with N₂ for 10 min, then tetrakis(triphenylphosphine)palladium (CAS 14221-01-3, from CombiBlocks, 0.234 g, 0.20 mmol, 0.05 eq) was added, and the mixture was heated at 90 °C for 5 h. The mixture was poured into ice-cold water (30 mL) and extracted with EtOAc (2 x 30 mL). The aqueous layer was acidified with dilute HCl and extracted with EtOAc (3 x 30 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to yield 6-(3-cyanophenyl)pyrimidine-4-carboxylic acid (0.52 g, 2.31 mmol, 56% yield).

LCMS: Method C, 1.36 min, MS: ES+226.5; 1H NMR (400 MHz, DMSO-d₆) d ppm: 14.11 (s, 1H), 9.48 (s, 1H), 8.79 (s, 1H), 8.68 (s, 1H), 8.63 - 8.65 (d, J = 8.0 Hz, 1H), 8.08 - 8.10 (d, J = 7.6 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H). Step (ii)

tert-Butyl (2R,4R)-4-(6-(3-cyanophenyl)pyrimidine-4-carboxamido)-2-methylpyrrolidine-1- carboxylate

To a stirred solution of tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.178 g, 0.89 mmol, 1.0 eq) and 6-(3-cyanophenyl)pyrimidine-4-carboxylic acid (0.2 g, 0.89 mmol, 1.0 eq) in pyridine (1 mL) was added POCl₃ (0.4 g, 0.24 mL, 2.67 mmol, 3.0 eq) dropwise at 0 °C. The mixture was stirred at 0 °C for 0.5 h, poured into water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. Toluene (15 mL) was added to the residue and the mixture was then concentrated under reduced pressure. This process was repeated twice more to afford tert-butyl (2R,4R)-4-(6-(3-cyanophenyl)pyrimidine-4- carboxamido)-2-methylpyrrolidine-1-carboxylate (0.37 g, 0.91 mmol, 100% yield), which was used in the next step without purification.

LCMS: Method C, 1.76 min, MS: ES+408.7.

Step (iii)

6-(3-Cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)pyrimidine-4-carboxamide trifluoroacetate To a stirred solution of tert-butyl (2R,4R)-4-(6-(3-cyanophenyl)pyrimidine-4-carboxamido)-2- methylpyrrolidine-1-carboxylate (0.365 g, 0.90 mmol, 1.0 eq) in DCM (8 mL) was added TFA (1.8 mL, 5 volumes) dropwise at 0 °C. The mixture was stirred at rt for 2 h, then concentrated under reduced pressure with DCM (3 x 10 mL) to obtain 6-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin- 3-yl)pyrimidine-4-carboxamide trifluoroacetate (0.698 g, 1.66 mmol, 100% yield), which was used in the next step without purification.

LCMS: Method C, 1.31 min, MS: ES+308.57; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.50 - 9.52 (d, J = 7.2 Hz, 1H), 9.48 (s, 1H), 8.79 (s, 1H), 8.67 (s, 1H), 8.63 - 8.65 (d, J = 8.0 Hz, 1H), 8.09 - 8.11 (d, J = 7.6 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 4.73 (m, 1H), 3.90 (m, 1H), 3.61 - 3.63 (m, 1H) 3.30 - 3.34 (m, 1H), 2.22 - 2.34 (m, 1H), 1.91 - 2.00 (m, 1H), 1.33 - 1.35 (d, J=6.4 Hz, 3H).

Step (iv)

To a stirred solution of 6-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)pyrimidine-4- carboxamide trifluoroacetate (0.695 g, 1.65 mmol, 1.0 eq) in THF (10 mL) was added K2CO3 (0.683 g, 4.95 mmol, 3 eq) portionwise at rt. The mixture was cooled to 0 °C. Cyanogen bromide (0.139 g, 1.32 mmol, 0.8 eq) was added and the mixture was stirred at 0 °C for 1 h, then poured into water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (60-120# silica gel, 64% EtOAc in hexanes) to yield N-((3R,5R)-1-cyano-5- methylpyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4-carboxamide (0.13 g, 0.39 mmol, 44% yield over 3 steps).

LCMS: Method H, RT=3.51 min, MS: ES+ 333.15; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.46 (d, J = 0.8 Hz, 1H), 9.43 (d, J = 7.2 Hz, 1H), 8.78 (s, 1H), 8.65 (s, 1H), 8.63 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 4.58 - 4.59 (m, 1H), 3.94 (m, 1H), 3.79 (m, 1H), 3.49 (m, 1H), 2.18 - 2.24 (m, 1H), 1.78 - 1.84 (m, 1H), 1.27 (d, J = 6.4 Hz, 3H).

Example 10

N-((3R,5S)-1-Cyano-5-(methoxymethyl)pyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4-carboxamide

Step (i)

tert-Butyl (2S,4R)-4-(6-chloropyrimidine-4-carboxamido)-2-(methoxymethyl)pyrrolidine-1- carboxylate

To a stirred solution of 6-chloropyrimidine-4-carboxylic acid (CAS 37131-91-2, from Astatech, 0.40 g, 2.53 mmol, 1.0 eq) in DCM (6.0 mL) was added oxalyl chloride (0.637 g, 0.43 mL, 5.06 mmol, 2.0 eq) followed by two drops of DMF at 0 °C, and the mixture was stirred for 2 h at rt. The mixture was concentrated under reduced pressure. The residue was dissolved in anhydrous THF (5.0 mL) under nitrogen. Triethylamine (0.767 g, 1.05 mL, 7.59 mmol, 3.0 eq) and tert-butyl (2S,4R)- 4-amino-2-(methoxymethyl) pyrrolidine-1-carboxylate (0.582 g, 2.53 mmol, 1.0 eq) were added at 0 °C and the mixture was stirred for 16 h at rt, then poured in to water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (23% EtOAc in hexane) to afford tert-butyl (2S, 4R)-4-(6-chloropyrimidine-4-carboxamido)-2-(methoxymethyl)pyrrolidine-1- carboxylate (0.320 g, 0.86 mmol, 34% yield).

LCMS: Method C, 1.64 min, MS: [M+1] 371.5; 1H NMR (400 MHz, CDCl₃) d ppm: 9.03 (s, 1H), 8.18 (s, 1H), 7.92 (d, J = 6.8 Hz, 1H), 4.76 - 4.81 (m, 1H), 4.07 - 4.16 (m, 1H), 3.75 - 3.85 (m, 1H), 3.53 (d, J = 4.4 Hz, 2H), 3.40 (s, 3H), 3.30 -3.39 (m, 1H), 2.39 - 2.49 (m, 1H), 2.02 - 2.12 (m, 1H), 1.50 (s, 9H).

Step (ii)

tert-Butyl (2S,4R)-4-(6-(3-cyanophenyl) pyrimidine-4-carboxamido)-2-(methoxymethyl)pyrrolidine-1- carboxylate

To a stirred solution of tert-butyl (2S, 4R)-4-(6-chloropyrimidine-4-carboxamido)-2-(methoxymethyl) pyrrolidine-1-carboxylate (0.300 g, 0.81 mmol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from CombiBlocks, 0.118 g, 0.81 mmol, 1 eq) in DMF (5.0 mL) and water (0.2 mL) was added K₂CO₃ (0.335 g, 2.43 mmol, 3 eq) at rt. The mixture was purged with N₂ for 20 min then [1,1¢-bis(diphenylphosphino)ferrocene]dichloropalladium (II) (0.059 g, 0.0810 mmol, 0.1 eq) was added at rt. The reaction mixture was heated at 100 °C for 2 h, cooled to rt, poured into ice-cold water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (23% EtOAc in hexane) to afford tert-butyl (2S,4R)-4-(6-(3- cyanophenyl)pyrimidine-4-carboxamido)-2-(methoxymethyl)pyrrolidine-1-carboxylate (0.260 g, 0.59 mmol, 73% yield).

LCMS: Method C, 1.75 min, MS: ES+ 438; 1H NMR (400 MHz, CDCl₃) d ppm: 9.31 (s, 1H), 8.58 (d, J = 7.6 Hz, 2H), 8.43 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 4.80 - 4.85 (m, 1H), 4.09 - 4.18 (m, 1H), 3.80 - 3.89 (m, 1H), 3.52 - 3.57 (m, 3H), 3.41 (s, 3H), 2.40 - 2.49 (m, 1H), 2.08 - 2.15 (m, 1H), 1.50 (s, 9H).

Step (iii)

6-(3-Cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl) pyrimidine-4-carboxamide trifluoroacetate

To a stirred solution of tert-butyl (2S,4R)-4-(6-(3-cyanophenyl)pyrimidine-4-carboxamido)-2- (methoxymethyl)pyrrolidine-1-carboxylate (0.250 g, 0.57 mmol, 1.0 eq) in DCM (5.0 mL) was added TFA (0.8 mL, 3 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 1 h, concentrated under reduced pressure and the residue was co-distilled with DCM (3 x 30 mL) under reduced pressure to afford 6-(3-cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3- yl)pyrimidine-4-carboxamide trifluoroacetate (0.45 g, quantitative yield).

LCMS: Method C, 1.35 min, MS: ES+ 383.40.

Step (iv)

N-((3R, 5S)-1-Cyano-5-(methoxymethyl) pyrrolidin-3-yl)-6-(3-cyanophenyl) pyrimidine-4- carboxamide

To a stirred solution of 6-(3-cyanophenyl)-N-((3R,5S)-5-(methoxymethyl)pyrrolidin-3-yl) pyrimidine- 4-carboxamide (0.45 g, 1.00 mmol, 1.0 eq) in THF (6 mL) was added K2CO3 (0.688 g, 4.99 mmol, 5.0 eq) followed by cyanogen bromide (0.084 g, 0.80 mmol, 0.8 eq) at 0 °C. The mixture was slowly warmed to rt and stirred for 2 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash chromatography (60% EtOAc in hexane) to afford N-((3R,5S)-1-cyano-5-(methoxymethyl)pyrrolidin-3-yl)-6-(3-cyanophenyl)pyrimidine-4-carboxamide (0.09 g, 0.25 mmol, 43% yield over two steps).

LCMS: Method H, 4.60 min, MS: ES+ 363.2; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.44 (s, 1H), 9.38 (d, J = 7.2 Hz, 1H), 8.76 (s, 1H), 8.62 (d, J = 6.4 Hz, 2H), 8.08 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 4.53 - 4.62 (m, 1H), 4.01 - 4.19 (m, 1H), 3.68 - 3.72 (m, 1H), 3.40 - 3.49 (m, 3H), 3.33 (s, 3H), 2.14 - 2.20 (m, 1H), 1.96 - 2.01 (m, 1H). Example 11

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

Step (i)

3-(2-Chloropyrimidin-4-yl)benzonitrile

To a stirred solution of 2,4-dichloropyrimidine (CAS 3934-20-1, from CombiBlocks, 0.30 g, 2.03 mmol, 1.0 eq) and (3-cyanophenyl)boronic acid (CAS 150255-96-2, from CombiBlocks, 0.35 g, 2.43 mmol, 1.2 eq) in 1,4-dioxane (5 mL) and water (1 mL) was added cesium carbonate (1.3 g, 4.05 mmol, 2.0 eq). The reaction mixture was purged with N2 for 10 min followed by addition of tetrakis(triphenylphosphine)palladium(0) (0.046 g, 0.041 mmol, 0.02 eq) at rt. The mixture was heated at 50 °C for 3 h, then poured into water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (45% EtOAc in hexane) to afford 3-(2-chloropyrimidin-4-yl)benzonitrile (0.29 g, 1.35 mmol, 66% yield).

LCMS: Method C, 1.65 min, MS: [M+1] 216.26.1H NMR (400 MHz, DMSO-d₆) d ppm: 8.94 (d, J = 5.2 Hz, 1H), 8.67 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.31 (d, J = 5.2 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H).

Step (ii)

Methyl 4-(3-cyanophenyl)pyrimidine-2-carboxylate

To a stirred solution of 3-(2-chloropyrimidin-4-yl)benzonitrile (0.29 g, 1.35 mmol, 1.0 eq) in MeOH (5 mL) was added NaOAc (0.553 g, 6.74 mmol, 5.0 eq). The mixture was stirred for 10 min at rt and transferred to an autoclave. To the mixture at rt was added [1,1¢-bis(diphenylphosphino)- ferrocene]dichloropalladium(II)dichloromethane complex (0.11 g, 0.13 mmol, 0.1 eq). The mixture was heated at 100 °C under carbon monoxide pressure (30 kg/cm2) for 10 h, then concentrated under reduced pressure. The residue was purified by flash column chromatography (40% EtOAc in hexane) to afford methyl 4-(3-cyanophenyl)pyrimidine-2-carboxylate (0.2 g, 0.84 mmol, 62% yield).

LCMS: Method C, 1.45 min, MS: [M+1] 240.12.

Step (iii)

tert-Butyl (2R,4R)-4-(4-(3-cyanophenyl)pyrimidine-2-carboxamido)-2-methyl pyrrolidine-1- carboxylate

To a stirred solution of methyl 4-(3-cyanophenyl)pyrimidine-2-carboxylate (0.2 g, 0.84 mmol, 1.0 eq) and tert-butyl (2R,4R)-4-amino-2-methylpyrrolidine-1-carboxylate (0.2 g, 1.00 mmol, 1.2 eq) in THF (4 mL) was added a solution of TBD (CAS 5807-14-7, from TCI, 0.174 g, 1.26 mmol, 1.5 eq) in THF (1 mL) at 0 °C. The mixture was slowly warmed to rt and stirred for 3 h, then poured into water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (60% EtOAc in hexane) to afford tert-butyl (2R,4R)-4-(4-(3- cyanophenyl)pyrimidine-2-carboxamido)-2-methylpyrrolidine-1-carboxylate (0.103 g, 0.25 mmol, 29% yield).

LCMS: Method C, 1.62 min, MS: [M+1] 408.6; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.09 (d, J = 4.8 Hz, 1H), 9.05 (d, J = 7.2 Hz, 1H), 8.85 (s, 1H), 8.70 (d, J = 7.6 Hz, 1H), 8.38 (d, J = 4.8 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 4.58 - 4.75 (m, 1H), 3.92 - 4.09 (m, 1H), 3.55 - 3.71 (m, 2H), 2.19 - 2.31 (m, 1H), 1.86 - 2.01 (m, 1H), 1.42 (s, 9H), 1.20 - 1.24 (d, 3H).

Step (iv)

4-(3-Cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)pyrimidine-2-carboxamide

To a stirred solution of tert-butyl (2R,4R)-4-(4-(3-cyanophenyl)pyrimidine-2-carboxamido)-2- methylpyrrolidine-1-carboxylate (0.103 g, 0.25 mmol, 1.0 eq) in DCM (5 mL) was added TFA (1 mL, 3 volumes) dropwise at 0 °C. The mixture was slowly warmed to rt and stirred for 30 min, then concentrated under reduced pressure and again with DCM (3 x 5 mL) to afford 4-(3-cyanophenyl)-N- ((3R,5R)-5-methylpyrrolidin-3-yl)pyrimidine-2-carboxamide trifluoroacetate (0.1 g, 0.24 mmol, 96% yield).

LCMS: Method C, 1.28 min, MS: [M+1] 308.43.

Step (v)

To a stirred solution of 4-(3-cyanophenyl)-N-((3R,5R)-5-methylpyrrolidin-3-yl)pyrimidine-2- carboxamide trifluoroacetate (0.10 g, 0.24 mmol, 1.0 eq) in THF (4 mL) was added K2CO3 (0.983 g, 0.71 mmol, 3.0 eq) followed by cyanogen bromide (0.0251 g, 0.24 mmol, 1.0 eq) at 0 °C. The mixture was slowly warmed to rt and stirred for 30 min, poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (60% EtOAc in hexane) to afford N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)pyrimidine-2-carboxamide (0.03 g, 0.09 mmol, 38% yield).

LCMS: Method H, 2.98 min, MS: [M+1] 333.15; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.22 (d, J = 6.4 Hz, 1H), 9.10 (d, J = 4.8 Hz, 1H), 8.83 (s, 1H), 8.69 (d, J = 7.6 Hz, 1H), 8.39 (d, J = 5.2 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 4.52 - 4.62 (m, 1H), 3.90 - 4.00 (m, 1H), 3.79 - 3.83 (m, 1H), 3.45 - 3.52 (m, 1H), 2.21 - 2.24 (m, 1H), 1.80 - 1.86 (m, 1H), 1.20 (d, J = 6.0 Hz, 3H). Example 12

N-((3R,5S)-5-(chloromethyl)-1-cyanopyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide

Scheme: (i) LiCl, THF, rt to 80 °C, 16 h; (ii) H2, 10% Pd/C, TEA, MeOH, rt, 1 h;

(ii) TBD, THF, rt, 16 h; (iv) TFA, DCM, 0 °C to rt, 1 h;

(v) CNBr, K2CO3, THF, 0 °C to rt, 1 h.

Step (i)

tert-Butyl (2S,4R)-4-azido-2-(chloromethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S,4R)-4-azido-2-(((methylsulfonyl)oxy)methyl)pyrrolidine-1- carboxylate (0.70 g, 2.19 mmol, 1.0 eq) in THF (10 mL) was added lithium chloride (0.278 g, 6.563 mmol, 3.0 eq) and the mixture was heated at 80 °C for 16 h. The mixture was cooled to rt, poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, 10% EtOAc in hexanes) to yield tert-butyl (2S,4R)-4-azido- 2-(chloromethyl)pyrrolidine-1-carboxylate (0.250 g, 0.96 mmol, 44% yield).

LCMS: Method C, 1.81 min, MS: ES+ 205.23 (M-56).

Step (ii)

tert-Butyl (2S,4R)-4-amino-2-(chloromethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl (2S,4R)-4-azido-2-(chloromethyl)pyrrolidine-1-carboxylate (0.25 g, 0.96 mmol, 1.0 eq) in methanol (6 mL) was added TEA (0.097 g, 0.13 mL, 0.96 mmol, 1.0 eq) followed by 10% palladium on carbon (0.125 g, 50% w/w moist) at rt under N₂ atmosphere. The reaction mixture was purged with H₂ gas for 1 h at rt, filtered through a Celite bed and washed with MeOH (30 mL). The filtrate was concentrated under reduced pressure, and the residue was poured into water (50 mL), acidified with aqueous 1 N HCl and extracted with EtOAc (50 mL). The aqueous layer was basified to pH 8 with saturated sodium bicarbonate solution (30 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄ and concentrated under reduced pressure to yield tert-butyl (2S,4R)-4-amino-2-(chloromethyl)pyrrolidine-1-carboxylate (0.22 g, 0.94 mmol, 98 % yield).

LCMS: Method C, 1.29 min, MS: ES+ 235.2, 237.2.

Step (iii)

tert-Butyl (2S,4R)-2-(chloromethyl)-4-(4-(3-cyanophenyl)picolinamido)pyrrolidine-1-carboxylate To a stirred solution of tert-butyl (2S,4R)-4-amino-2-(chloromethyl)pyrrolidine-1-carboxylate (0.206 g, 0.88 mmol, 1.0 eq) and methyl 4-(3-cyanophenyl)picolinate (0.21 g, 0.88 mmol, 1.0 eq) in THF (5 mL) was added TBD (0.183 g, 1.32 mmol, 1.5 eq) portionwise at 0 °C. The mixture was stirred at rt for 16 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 30% EtOAc in hexanes) to yield tert-butyl (2S,4R)-2-(chloromethyl)-4-(4-(3-cyanophenyl)picolinamido)pyrrolidine-1- carboxylate (0.20 g, 0.46 mmol, 51% yield).

LCMS: Method C, 1.87 min, MS: ES+441.3.

Step (iv)

N-((3R,5S)-5-(Chloromethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide trifluoroacetate To a stirred solution of tert-butyl (2S,4R)-2-(chloromethyl)-4-(4-(3-cyanophenyl)picolinamido)- pyrrolidine-1-carboxylate (0.20 g, 0.45 mmol, 1.0 eq) in DCM (6 mL) was added TFA (0.6 mL, 3 volumes) dropwise at 0 °C. The mixture was stirred at rt for 1 h, then concentrated under reduced pressure. The residue was dissolved in DCM (10 mL) and concentrated under reduced pressure; this was repeated twice to yield N-((3R,5S)-5-(chloromethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl) picolinamide trifluoroacetate (0.30 g, 0.66 mmol, quantitative yield).

LCMS: Method C, 1.36 min, MS: ES+341.21.

Step (v)

To a stirred solution of N-((3R,5S)-5-(chloromethyl)pyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide trifluoroacetate (0.30 g, 0.66 mmol, 1.0 eq) in THF (6 mL) was added K2CO3 (0.273 g, 1.98 mmol, 3 eq) and the mixture was cooled to 0 °C. Cyanogen bromide (0.070 g, 0.66 mmol, 1 eq) was added portionwise and the mixture was stirred at rt for 1 h, then poured into water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, 45 % EtOAc in hexane) to yield N-((3R,5S)-5-(chloromethyl)-1-cyanopyrrolidin-3-yl)-4-(3-cyanophenyl) picolinamide (0.10 g, 0.274 mmol, 60% yield over 2 steps).

LCMS: Method H, 3.59 min, MS: ES+366.1; 1H NMR (400 MHz, DMSO-d₆) d ppm: 9.21 (d, J = 6.4 Hz, 1H), 8.77 (d, J = 4.8 Hz, 1H), 8.41 (s, 1H), 8.35 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 3.2 Hz, 1H), 7.98 (d, J = 6.8 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 4.62 (br s, 1H), 4.25 (br s, 1H), 3.84 (s, 2H), 3.72 - 3.80 (m, 1H), 3.47 - 3.53 (m, 1H), 2.27 - 2.33 (m, 1H), 2.05 - 2.08 (m, 1H). Biological Activity of 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 homologue

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

DMSO Dimethyl sulfoxide

YFP Yellow fluorescent protein

VME Vinyl methyl ester

HA Hemagglutinin

Ahx Aminohexanoic acid USP30 biochemical IC50 assay

Dilution plates were prepared at 21 times the final concentration (2100µM for a final concentration of 100 µM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03µM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 µl. Either 1µl of 50% DMSO or diluted compound was added to the plate. USP30 (Boston Biochem #E582) was diluted in reaction buffer (40mM Tris, pH 7.5, 0.005% Tween 20, 0.5mg/ml BSA, 5 mM beta-mercaptoethanol) to achieve a final assay concentration of 4 nM, and 10 µl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2-hour incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). l Excitation 540 nm; l Emission 590 nm. Activity of exemplary compounds in USP30 biochemical IC₅₀ assay:

Reference Examples

Activity of Reference Examples in USP30 biochemical IC₅₀ assay:

Deubiquitylating Enzyme (DUB) Selectivity

Activity of exemplary compounds in DUB biochemical IC₅₀ assays:

Off-Target Pharmacology

Example 1 was subject to pharmacological profiling in the Eurofins CEREP SafetyScreen44 panel. At a single concentration of 10 mM, less than 50% inhibition of binding or enzyme activity was observed against all targets in the panel. No inhibitory effect was observed against MAO-A or MAO-B (both <0% inhibition at 10 mM).

Example 1 was subject to pharmacological profiling against 39 kinases of the Thermo Fisher SelectScreen kinase profiling panel. At a single concentration of 10 mM, less than 40% inhibition of binding or enzyme activity was observed against all targets in the panel. Example 1 was screened at single concentrations of 10 mM and 1 mM against 8 cathepsins (B, C, H, K, L, L2, S, Z). The IC₅₀ was calculated for examples against cathepsins B, K, L, S and V.

Safety Pharmacology

Example 1 was evaluated for effects on the hERG potassium channel, in stably expressed CHO cells at concentrations between 0.01 and 30 mM. Example 1 produced a maximum of 45% inhibition of the hERG current amplitude at 30 mM indicating little propensity for affecting the QT interval.

Genetic Toxicology

Example 1 was assessed in the bacterial reverse mutation assay (Ames) and in vitro micronucleus assay. All in vitro tests were conducted with and without exogenous metabolic activation using concentrations up to those limited by cytotoxicity or insolubility.

Example 1 did not induce mutations when tested up to 5000 mg/plate with and without metabolic activation in the reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535 and TA97a and the Escherichia Coli strain WP2 uvrA pKM101.

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

TOM20-ubiquitylation assay

Human cell lines can be challenged with mitochondrial depolarizing agents (ionophores (eg. CCCP, valinomycin), mitochondrial complex inhibitors (oligomycin, antimycin A)) to induce ubiquitylation of TOM20, which is then further promoted in the presence of USP30 inhibitors. TOM20 ubiquitylation is subsequently assessed through western blotting of cell lysates, with TOM20 ubiquitylation adduct detection possible due to 8 kDa molecule weight increases for each molecule of ubiquitin added, resulting in laddering of a TOM20 immunoreactive band. Tom20-ubiquitylation levels can be quantified using chemiluminescence densitometry of laddered immunoreactive bands. Further Studies

Physicochemical Properties:

log P: partition coefficient; lipophilicity measurement.

log D: distribution co-efficient; lipophilicity measurement.

TPSA: topological polar surface area.

Turbidimetric solubility: Test compound solution prepared in DMSO diluted into aqueous buffer. Turbidimetry is used as the end-point by measuring absorbance at 620 nm.

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

Hep Cl mouse: in vitro hepatocyte clearance in mouse cells. Hep Cl human: in vitro hepatocyte clearance in human cells.

Plasma f_u,p: The free fraction of a compound in plasma preparation determined by in vitro equilibrium dialysis. It is understood that only unbound (free) compound is capable of engaging with the target. Brain f_u,br: The free fraction of a compound in brain homogenate preparation determined by in vitro equilibrium dialysis. It is understood that only unbound (free) compound is capable of engaging with the target.

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

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

Oral F: Oral Bioavailability.

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

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

TE ex vivo: USP30 brain tissue target engagement assay.

USP30 endogenous cellular target engagement assay

Hela cells stably overexpressing YFP-Parkin were seeded into 6 well dishes. Once adhered, cells were treated with appropriate concentrations of test compounds or vehicle control for 1 hour at 37°C, 5% CO2. Whole cell lysates were prepared by scraping the cells into cold PBS, centrifuging and lysing in lysis buffer (50 mM Tris-base, pH 7.5, 50 mM NaCl, 1% NP-40/Igepal CA-630, 2 mM MgCl2, 10% Glycerol, 5 mM beta-mercaptoethanol, cOmplete mini tablets EDTA free (Roche), PhosStop tablets (Roche)) for 10 mins. The equivalent of 20 µg of protein from the cleared cell lysate was incubated with a final conc of 2.5 µM HA-Ahx-Ahx-Ub-VME probe at room temperature. The reaction was stopped by addition of 5x SDS sample loading buffer and proteins separated by SDS PAGE and western blotting. USP30 was detected using an anti-USP30 Sheep S746D antibody (MRC PPU Reagents and Services) and a rabbit anti sheep secondary IgG (H+L) horseradish peroxidase conjugated (Thermo #31480) and visualised using ECL reagent (GE #RPN2109) on a GE LAS4000 imager. Target engagement was measured by quantitation of the bands corresponding to USP30 and USP30 bound to the Ub-VME probe and expression of this proportion compared to vehicle treated control. USP30 brain tissue target engagement assay

50 to 100 mg of tissue was homogenised in 3 x volume of lysis buffer (50 mM Tris-base, pH 7.5, 50 mM NaCl, 1% NP-40/Igepal CA-630, 2 mM MgCl₂, 10% Glycerol, 5 mM beta-mercaptoethanol, cOmplete mini tablets EDTA free (Roche), PhosStop tablets (Roche)) using the Retch Mixer Mill (MM400). The lysates were cleared and protein quantified using the Bradford Protein assay (Pierce). Lysate containing 60 µg protein was incubated with a final conc of 24 µM of HA-Ahx-Ahx-Ub-VME probe for 60 mins at room temperature. The reaction was stopped by addition of 5x SDS sample loading buffer and proteins separated by SDS-PAGE and western blotting. USP30 was detected using an anti-USP30 Sheep S746D antibody (MRC PPU Reagents and Services) and a rabbit anti sheep secondary IgG (H+L) horseradish peroxidase conjugated (Thermo #31480) and visualised using ECL reagent (GE #RPN2109) on a GE LAS4000 imager. Target engagement was measured by quantitation of the bands corresponding to USP30 and USP30 bound to the Ub-VME probe and expression of this proportion compared to vehicle treated control.

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

Reference Examples A, B, C, D and E are known DUB inhibitors that are active as inhibitors of USP30 and share some structural similarity with the compounds of the present invention, possessing the cyanamide structural feature.

All of Examples 1 to 12 of the present invention are significantly more potent for USP30 than Reference Examples A, C, D and E as measured in the biochemical assay. Example 1 is approximately 4-fold more potent than Reference Example A, and 40 to 95-fold more potent than Reference Examples C, D and E. Example 9 is approximately 2.6-fold more potent than Reference Example A, and 25 to 60-fold more potent than Reference Examples C, D and E.

All of Examples 1 to 12 are significantly more selective for USP30 over other DUBs (USP10, USP16, USP21, USP25, USP28) compared to Reference Examples A, B, D and E.

Examples 1, 2, 3, 4, 5, 9 and 10 are significantly more selective for USP30 over the cathepsins (K, L and S) compared to Reference Examples A, D and E. Example 1 is significantly more potent (4-fold, as measured in the Cell TE WB assay) for USP30 than Reference Example A. Examples 1, 2, 3, 4, 5, 9 and 10 are significantly more potent (as measured in the Cell TE WB assay) for USP30 than Reference Examples D and E.

Examples 1, 2, 3, 4, 5, 6, 9 and 10 show significantly improved hepatocyte metabolic stability (as measured in mouse hepatocytes) compared to Reference Examples A and D. Examples 3, 5, 6 and 10 show significantly improved hepatocyte metabolic stability (as measured in mouse hepatocytes) compared to Reference Example E.

Examples 1, 2, 3, 4, 5, 6 and 10 show significantly improved plasma stability (as measured in mouse plasma) compared to Reference Examples A and E. Examples 1, 2, 3, 4, 6 and 10 show significantly improved plasma stability (as measured in mouse plasma) compared to Reference Example D.

Reference Examples A, D and E demonstrate significant cytotoxicity (ratio of Cell Tox EC₅₀ to USP30 IC₅₀) compared to Examples 1, 2, 3, 4, 5, 6, 9 and 10.

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

Preclinical in vivo models

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

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

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

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

(d) Ndufs4KO Leigh syndrome model.

This model allows testing of therapeutics for potential benefits in nuclear gene mutation driven complex I deficits. Mice with a mitochondrial complex I (CI)-associated defect (“Ndufs KO” or “HOM”, B6.129S4-Ndufs4tm1.1Rpa/J, JAX stock# 027058), a model of Leigh syndrome, were enrolled into a study to investigate effects of Example 1 at both 50 and 100mg/kg (subcutaneous, qd) on survival, neurological and motor function. Sixteen wild type (WT) mice and ten homozygous (HOM) mice were controls and received Example 1 Vehicle. Twenty three HOM mice were treated with two doses of Example 1. All treatments began at postnatal day 10 ± 3 days until humane endpoint was reached. No significant differences were observed between Example 1 treatment groups and vehicle on body temperature and neurological score. At four, five and six weeks of age, mice also were assessed for various gait parameters. Example 1 at 100 mg/kg showed a statistically significant beneficial effect on instantaneous run speed, with no effects observed on other exploratory parameters such as stride length, time and paw angle. No beneficial effects of Example 1 were observed on survival, although beneficial effects on run speed suggest potential for benefit on gait parameters in Leigh Syndrome. [Kruse et al, 2008, Cell Metab. Apr;7(4):312-20]

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

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

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

Claims

CLAIMS 1. A compound of formula (I)

(i) X1 is CH;

one of X2 and X3 is CR6, and the other is N; or

(ii) X1 is CH or N;

X2 is N; and

X3 is CR7 or N; with the proviso that one, solely, of X1 and X3 is N; and R1 is selected from (C1-C3)alkyl, (C1-C3)haloalkyl and CH2OCH3;

R2, R3 and R4 are each independently selected from hydrogen and deuterium;

R5 is selected from hydrogen, deuterium and fluorine; and

R6 and R7 are each independently selected from hydrogen, halogen, (C₁-C₃)alkyl and (C₁-C₃)alkoxy.

2. A compound according to claim 1, which is selected from:

(i) X1 is CH; X2 is N; and X3 is CR6;

(ii) X1 is CH; X2 is CR6; and X3 is N;

(iii) X1 is CH; X2 is N; and X3 is N; and

(iv) X1 is N; X2 is N; and X3 is CR7.

3. A compound according to claim 2, wherein X1 is CH; X2 is N; and X3 is CR6.

4. A compound according to any one of claims 1 to 3, wherein R1 is selected from methyl, chloromethyl and CH₂OCH₃.

5. A compound according to claim 4, wherein R1 is methyl.

6. A compound according to any one of claims 1 to 5, wherein R2, R3 and R4 are each hydrogen.

7. A compound according to any one of claims 1 to 6, wherein R5 is hydrogen.

8. A compound according to any one of claims 1 to 7, wherein R6 and R7 are each independently selected from hydrogen, fluorine, methyl and methoxy.

9. A compound according to claim 8, wherein R6 and R7 are each hydrogen.

10. A compound according to claim 1, which is selected from:

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-2-(3-cyanophenyl)isonicotinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl-2,4,5,6-d₄)picolinamide;

N-((3R,5R)-1-cyano-5-methylpyrrolidin-3-yl)-4-(3-cyanophenyl)pyrimidine-2-carboxamide; and N-((3R,5S)-5-(chloromethyl)-1-cyanopyrrolidin-3-yl)-4-(3-cyanophenyl)picolinamide;

11. A compound according to any one of claims 1 to 10, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, for use as a medicament.

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

13. Use of a compound according to any one of claims 1 to 10, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, in the manufacture of a medicament for use in the treatment or prevention of a condition involving mitochondrial dysfunction, a cancer, or fibrosis.

14. A method for the treatment or prevention of a condition involving mitochondrial dysfunction, a cancer, or fibrosis, comprising the step of administering an effective amount of a compound according to any one of claims 1 to 10, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, to a patient in need thereof.

15. A compound, use, or method, according to claims 12 to 14, wherein the condition involving mitochondrial dysfunction is selected from a CNS disorder; neurodegenerative disease; Parkinson’s disease; Alzheimer’s disease; amyotrophic lateral sclerosis; Huntington’s disease; ischemia; stroke; dementia with Lewy bodies; frontotemporal dementia; multiple sclerosis; mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome; materially-inherited diabetes and deafness; Leber's hereditary optic neuropathy; cancer; neuropathy, ataxia, retinitis pigmentosa- maternally inherited Leigh syndrome; Danon disease; diabetes; diabetic nephropathy; metabolic disorders; heart failure; ischemic heart disease leading to myocardial infarction; psychiatric diseases, schizophrenia; multiple sulfatase deficiency; mucolipidosis II; mucolipidosis III; mucolipidosis IV; GMl-gangliosidosis; neuronal ceroid-lipofuscinoses; Alpers disease; Barth syndrome; beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co- enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome; CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns- Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrogenase deficiency; Leigh disease or syndrome; Leigh Syndrome French Canadian variant; lethal infantile cardiomyopathy; Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrogenase deficiency; myoclonic epilepsy and ragged-red fiber syndrome; mitochondrial cytopathy; mitochondrial recessive ataxia syndrome; mitochondrial DNA depletion syndrome; myoneurogastrointestinal disorder and encephalopathy; Pearson syndrome; pyruvate dehydrogenase deficiency; pyruvate carboxylase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; and very long-chain acyl-CoA dehydrogenase deficiency; peroxisomal disorders; methylmalonic acidemia; and age- dependent decline in cognitive function and muscle strength.

16. A compound, use, or method, according to claim 15, wherein the neurodegenerative disease is selected from Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, Huntington’s disease, ischemia, stroke, dementia with Lewy bodies, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia; and Parkinson’s disease related to mutations in a-synuclein, parkin, PINK1, GBA, and LRRK2, and autosomal recessive juvenile Parkinson’s disease where parkin is mutated.

17. A compound, use, or method, according to claim 15, wherein the neurodegenerative disease is Leigh syndrome or disease, X-linked Leigh's disease, Leigh Syndrome French Canadian Variant, and/or the symptoms associated with Leigh’s disease.

18. A compound, use, or method, according to claims 12 to 14, wherein the cancer is selected from breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, bone, liver, soft tissue, cancers of tissue organs, cancers of the blood cells, CML, AML, mantle cell lymphoma, neuroblastoma, melanoma, soft tissue sarcoma, liposarcoma, fibroblastic sarcoma, leiomyosarcoma, hepatocellular carcinoma, osteosarcoma, oesophageal cancer, leukaemia, lymphoma, multiple myeloma, metastatic carcinoma, osteosarcoma, chondosarcoma, Ewing’s sarcoma, nasopharyngeal carcinoma, colorectal cancer, colorectal cancer, non-small cell lung carcinoma, cancer where apoptotic pathways are dysregulated, and cancer where proteins of the BCL- 2 family are mutated, or over or under expressed.

19. A compound, use, or method, according to claims 12 to 14, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with the accumulation of extracellular matrix constituents that occurs following trauma, inflammation, tissue repair, immunological reactions, cellular hyperplasia, and neoplasia.

20. A compound, use, or method, according to claim 19, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with major organ diseases, fibroproliferative disorders, and scarring associated with trauma.

21. A compound, use, or method, according to claim 20, wherein the fibrosis is selected from fibrosis or a fibrotic disorder associated with interstitial lung disease, liver cirrhosis, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis, kidney disease, acute kidney injury, chronic kidney disease, delayed kidney graft function, heart or vascular disease, diseases of the eye, systemic and local scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, Dupuytren’s contracture, surgical complications, chemotherapeutics drug-induced fibrosis, radiation-induced fibrosis, accidental injury and burns, retroperitoneal fibrosis, and peritoneal fibrosis/peritoneal scarring.

22. A compound, use, or method, according to claim 21, wherein the fibrosis associated with interstitial lung disease is selected from sarcoidosis, silicosis, drug reactions, infections, collagen vascular diseases, rheumatoid arthritis, systemic sclerosis, scleroderma, pulmonary fibrosis, idiopathic pulmonary fibrosis, usual interstitial pneumonitis, interstitial lung disease, cryptogenic fibrosing alveolitis, bronchiolitis obliterans, and bronchiectasis.

23. A compound, use, or method, according to claim 21, wherein the kidney disease is acute kidney injury or chronic kidney disease.

24. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 10, a tautomer thereof, or a pharmaceutically acceptable salt of said compound or tautomer, together with one or more pharmaceutically acceptable excipients.

25. A compound, which is selected from formulae (II) and (III):

wherein PG is a protecting group, preferably BOC or CBZ, and X1, X2, X3, R1, R2, R3, R4 and R5 are as defined for the compound of formula (I) in any one of claims 1 to 8, a tautomer thereof, or a salt of said compound or tautomer.