Inhibitors Targeting Insulin- Regulated Aminopeptidase (IRAP)1412784/FULLTEXT01.pdf · Abstract...

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 285

Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP)

Identification, Synthesis and Evaluation

KARIN ENGEN

ISSN 1651-6192ISBN 978-91-513-0894-4urn:nbn:se:uu:diva-406417

Dissertation presented at Uppsala University to be publicly examined in Room B41, BMC,Husargatan 3, Uppsala, Friday, 24 April 2020 at 13:15 for the degree of Doctor of Philosophy(Faculty of Pharmacy). The examination will be conducted in Swedish. Faculty examiner:Professor Mikael Elofsson (Umeå Universitet).

AbstractEngen, K. 2020. Inhibitors Targeting Insulin-Regulated Aminopeptidase (IRAP).Identification, Synthesis and Evaluation. Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy 285. 86 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0894-4.

Insulin-regulated aminopeptidase (IRAP) has emerged as a potential new therapeutic target fortreatment of cognitive disorders. Inhibition of the enzymatic activity facilitates cognition inrodents. Potent and selective peptide and pseudopeptide based inhibitors have been developed,but most of them suffer from poor pharmacokinetics and blood-brain-barrier penetration. Hence,development of less-complex inhibitors with good pharmacokinetic properties are of greatimportance.

The aim of this thesis was to identify and optimize new small-molecule based IRAPinhibitors for use as research tools to investigate the cognitive effects of IRAP inhibition.Adaptation of an existing enzymatic assay into a screening compatible procedure allowed theevaluation of 10,500 compounds as IRAP inhibitors. The screening campaign resulted in 23compounds displaying more than 60% inhibition. Two of these compounds, a spiro-oxindoledihydroquinazolinone and an imidazo[1,5-α]pyridine, were further investigated in terms ofstructure-activity relationship, physicochemical properties, metabolic stability, and mechanismof inhibition.

Spiro-oxindole dihydroquinazolinone based IRAP inhibitors were synthesized via fast andsimple microwave-promoted reactions, either in batch or in a continuous flow approach. Themost potent compounds displayed sub-µM affinity, and interestingly an uncompetitive modeof inhibition with the synthetic substrate used in the assay. Molecular modeling confirmedthe possibility of simultaneous binding of the compounds and the substrate. Furthermore,the molecular modeling suggested that the S-enantiomer accounts for the inhibitory effectobserved with this compound series. The compounds also proved inactive on the closely relatedenzyme aminopeptidase N. Unfortunately, the spiro-oxindole based inhibitors suffered frompoor solubility and metabolic stability.

Imidazo[1,5-α]pyridine based IRAP inhibitors were synthesized via a five step procedure,providing inhibitors in the low-µM range. The stereospecificity of a methyl groupproved important for inhibition. The compound series displayed no inhibitory activity onaminopeptidase N. Intriguing, these compounds exhibit a noncompetitive inhibition mechanismwith the model substrate. As observed for the spiro-compounds, the imidazopyridines sufferedfrom both poor solubility and metabolic stability.

In summary, the work presented in this thesis provide synthetic procedures, initial structure-activity relationship, and pharmacological evaluation of two distinct inhibitors classes. Thecompounds are among the first non-peptidic IRAP inhibitors presented, serving as interestingstarting points in the development of research tools for use in models of cognition.

Keywords: compound screening, insulin-regulated aminopeptidase, IRAP, inhibitors,cognitive disorders, spiro-oxindole, quinazolinone, imidazopyridine, medicinal chemistry,structure-activity relationship, microwave heating

Karin Engen, Department of Medicinal Chemistry, Preparative Medicinal Chemistry, Box574, Uppsala University, SE-751 23 Uppsala, Sweden.

© Karin Engen 2020

ISSN 1651-6192ISBN 978-91-513-0894-4urn:nbn:se:uu:diva-406417 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-406417)

Till Signe och William

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Engen, K., Rosenström, U., Axelsson, H., Konda, V., Dahllund,

L., Otrocka, M., Sigmundsson, K., Nikolaou, A., Vauquelin, G., Hallberg, M., Jenmalm Jensen, A., Lundbäck, T., Larhed, M. Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening. Assay and Drug Development Technologies, 2016, 14(3), 180–193.

II Engen, K., Vanga, S. R., Lundbäck, T., Agalo, F., Konda, V., Jenmalm Jensen, A., Åqvist, J., Gutiérrez-de-Terán, H., Hallberg, M., Larhed, M., Rosenström, U. Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-oxindole Dihydroquinazolinones as IRAP Inhibitors. ChemistryOpen, 2020, 9(3), 325–337.

III Engen, K., Lundbäck, T., Rosenström, U., Gising, J., Jenmalm Jensen, A., Hallberg, M., Larhed, M. Inhibition of Insulin-regulated Aminopeptidase by Imidazo[1,5-α]pyridines; Synthesis and Evaluation. Manuscript.

IV Engen, K., Sävmarker, J., Rosenström, U., Wannberg, J., Lundbäck, T., Jenmalm Jensen, A., Larhed, M. Microwave Heated Flow Synthesis of Spiro-oxindole Dihydroquinazolinone Based IRAP Inhibitors. Organic Process Research & Development, 2014, 18(11), 1582–1588.

Reprints were made with permission from the respective publishers.

Author Contribution Statement

The following contributions to each paper were made by the author of this thesis:

I Resynthesized and characterized desired hit compounds and

performed major part of the hit confirmation experiments. Collated the experimental data and drafted the manuscript.

II Supervised the method development of the two-step reaction employing aliphatic amines, synthesized almost all final compounds, performed all biological testing, collated the experimental data and drafted the manuscript.

III Performed all experimental work including biological testing, collated the experimental data and drafted the manuscript.

IV Performed the major part of the synthesis and characterization, performed all biological testing, collated the experimental data and drafted the manuscript.

Papers Not Included in This Thesis

V Svensson, F., Engen, K., Lundbäck, T., Larhed, M., Sköld, C. Virtual Screening for Transition State Analogue Inhibitors of IRAP Based on Quantum Mechanically Derived Reaction Coordinates. Journal of Chemical Information and Modeling, 2015, 55(9), 1984–1993.

VI Diwakarla, S., Nylander, E., Grönbladh, A., Vanga, S. R., Khan, Y. S., Gutiérrez-de-Terán, H., Ng, L., Sävmarker, J., Lundbäck, T., Jenmalm Jensen, A., Andersson, H., Engen, K., Rosenström, U., Larhed, M., Åqvist, J., Chai, S. Y., Hallberg, M. Binding to and Inhibition of Insulin-regulated Aminopeptidase by Macrocyclic Disulfides Enhances Spine Density. Molecular Pharmacology, 2016, 89(4), 413–424.

VII Diwakarla, S., Nylander, E., Grönbladh, A., Vanga, S. R., Khan, Y. S., Gutiérrez-de-Terán, H., Sävmarker, J., Pham, L. N. V., Lundbäck, T., Jenmalm Jensen, A., Svensson, R., Artursson, P., Zelleroth, S., Engen, K., Rosenström, U., Larhed, M., Åqvist, J., Chai, S. Y., Hallberg, M. Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures. ACS Chemical Neuroscience, 2016, 7(10), 1383–1392.

Contents

Introduction ................................................................................................... 13 Angiotensin IV and Memory Consolidation ............................................ 14 Insulin-Regulated Aminopeptidase (IRAP) ............................................. 14 Insulin-Regulated Aminopeptidase Inhibitors .......................................... 16 Identification of Starting Points in Drug Discovery ................................. 19

High-throughput Screening ................................................................. 21

Aims .............................................................................................................. 24

Identification of New IRAP Inhibitors Through Small-Molecule Screening (Paper I) ....................................................................................... 25

Assay Formatting ..................................................................................... 25 Compound Library ................................................................................... 27 Screening Campaign ................................................................................ 28 Hit Confirmation ...................................................................................... 29 Summary .................................................................................................. 34

Synthesis, Structure-activity Studies and Characterization of Spiro-oxindoles and Imidazopyridines as IRAP Inhibitors (Papers II and III) ....... 36

Synthesis of Substituted Spiro-oxindole Dihydroquinazolinones ............ 36 Synthesis of Imidazo[1,5-α]pyridines ...................................................... 42

Racemization During the Ring-closure Reaction (step 3) ................... 44 Structure-Activity Investigations for the Spiro-oxindole Compounds ..... 46 Structure-Activity Investigation for the Imidazopyridine Compounds .... 51 Physicochemical and DMPK Property Profiling ...................................... 56 Activity Confirmation on Human IRAP and Selectivity Investigations Towards APN ........................................................................................... 58 Mechanism of Inhibition .......................................................................... 59 Molecular Modeling and Interpolation of the Structure-Activity Relationships ............................................................................................ 61 Summary .................................................................................................. 65

Microwave Heated Continuous-Flow Synthesis of Spiro-oxindole Dihydroquinazolinones (Paper IV) ............................................................... 66

Microwave Heated Continuous-Flow ...................................................... 67 MW-CF Synthesis of Spiro-oxindoles ..................................................... 68

Concluding Remarks ..................................................................................... 73

Acknowledgement ........................................................................................ 75

References ..................................................................................................... 77

Abbreviations

AD Alzheimer’s disease ADME absorption, distribution, metabolism, excretion Ala alanine AngIV angiotensin IV APN aminopeptidase N Arg arginine Asn asparagine BBB blood-brain barrier Boc tert-butoxycarbonyl BPR back-pressure regulator CBCS Chemical Biology Consortium Sweden CCK-8 cholecystokinin-8 CF continuous flow CHO Chinese hamster ovary Clint intrinsic clearance Cmpd compound DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCM dichloromethane DMA dimethylacetamide DMF dimethylformamide DMPK drug metabolism and pharmacokinetic DMSO dimethylsulfoxide DNA deoxyribonucleic acid dppf 1,1’-bis(diphenylphosphino)ferrocene DTT 1,4-dithiotreithol EDC 1-ethyl-3-(3-dimethylaminopeopyl)carbodiimide EDDA ethylenediamine diacetate ERAP endoplasmic reticulum aminopeptidase GAMEN Gly428-Ala429-Met430-Glu431-Asn432 Glu glutamic acid GLUT4 glucose transporter type 4 GPCR G-protein coupled receptor HGF hepatocyte growth factor His histidine HLM human liver microsomes HOBt 1-hydroxy-7-azabenzotriazole hydrate

HPLC high-performance liquid chromatography HTS high-throughput screening IC50 inhibitor concentration giving 50% inhibition Ile isoleucine IRAP insulin-regulated aminopeptidase Ki inhibitor constant LC liquid chromatography Leu leucine Met methionine MS mass spectrometry MW microwave NMR nuclear magnetic resonance NOE nuclear overhauser effect PAINS pan-assay interference compound PD pharmacodynamic PDB protein data bank Phe phenylalanine pNA para-nitroaniline PCR polymerase chain reaction PK pharmacokinetic PMMA poly(methyl methacrylate) PPB protein plasma binding Pro proline RAAS renin-angiotensin-aldosterone system RDC residual dipolar coupling rt. room temperature SAR structure-activity relationship SD standard deviation SFC supercritical fluid chromatography SPR surface plasmon resonance TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin-layer chromatography Tyr tyrosine UV ultraviolet Val valine vp165 vesicle protein of 165 kDa

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Introduction

Approximately 50 million people worldwide live with dementia. The number is estimated to increase to 82 million by 2030, and projected to reach more than 150 million by 2050.1 This means, that every three seconds a new person is diagnosed with dementia. The increasing prevalence is globally among the biggest public health and social care challenges facing society. Dementia not only shortens the lifespan of the affected person, it has a tremendous impact on life quality. Consequently, a heavy caretaking burden is placed on the surrounding; family, friends and society, as described by Dr. Margaret Chan, Director-General of World Health Organization (WHO) in 2015:

I can think of no other disease that has such a profound effect on loss of function, loss of independence, and the need for care […] I can think of no other disease that places such a heavy burden on families, communities, and societies. I can think of no other disease where innovation, including breakthrough discoveries to develop a cure, is so badly needed.2

Dementia is characterized by a chronic or progressive decline in cognitive function, beyond what is expected from normal aging.3 It affects memory, thinking, orientation, learning capacity, and language, amongst others. The cause of dementia is diseases or injuries, which in various ways affect the brain. The most common form of dementia is Alzheimer’s disease (AD), accounting for up to 70% of all cases.3

Currently, there is no cure for AD, neither is there a treatment to alter the progressive course of the disease. The two types of medications on the market used to treat AD; acetylcholinesterase inhibitors (donepezil, galantamine and rivastigmine), and the N-methyl-D-aspartate (NMDA) receptor antagonist memantine, only treats the symptoms.1 According to Alzheimer’s Disease International (ADI), there have been more than 100 clinical attempts between 1998 and 2018 to develop an effective drug, but no one have yet succeeded.1 A recent review by Yiannopoulou et al., highlights possible causes of the failed trials as primarily a result of an inadequate understanding of the complex pathophysiology of AD, along with late initiation of treatments during the development course of AD, and the wrong selection of treatment targets.4 There is an intricate relationship between the numerous mechanisms involved in the development and progression of the disease, which makes drug development extremely challenging. However, promising progress has been

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made within recent years. In the beginning of 2019, there were 156 ongoing clinical trials of anti-AD therapies employing 132 different agents, comprising both small molecules, monoclonal antibodies, and other biological therapies.5

However, the need for the discovery and development of novel treatment approaches for cognitive disorders, including not only AD but also cognitive impairment resulting from for example brain trauma and stroke, remains urgent.

Angiotensin IV and Memory Consolidation The renin-angiotensin-aldosterone system (RAAS) is well known for its role in the regulation of blood pressure and fluid electrolyte balance throughout the body. In 1971, Ganten et al. reported on the existence of a brain renin-angiotensin system with functions independent of those present in the peripheral system.6 These findings encouraged further investigations and a number of additional functions of the RAAS have been revealed, e.g. regulation of cerebral blood flow, stress, depression, and cognition.7 In 1988, Braszko et al. reported that intracerebroventricular injection of angiotensin IV (AngIV), a hexapeptide derived from sequential cleavage of angiotensinogen, improved learning and memory in rats.8 Several subsequent studies showed similar effects, where for example centrally administered AngIV and analogues thereof, were able to reverse memory deficits induced by different experimental lesions.9–15 Efforts to identify the specific binding site of AngIV in the brain was initiated, and results indicated the existence of a single high-affinity binding site with abundant appearance in areas associated with cognitive, sensory, and motor functions.9,16–21 Two proteins were eventually hypothesized as potential sites for the AngIV binding; the enzyme insulin-regulated aminopeptidase (IRAP), or the hepatocyte growth factor (HGF) receptor c-Met.22 By searching for a molecular target with structural homology to AngIV, and physiological functions similar to those identified for AngIV, a partial match was found with HGF. The HGF receptor c-Met plays a role in multiple types of cancer, but has also been shown to contribute to learning and memory consolidation, and has been implicated in AD.7 Since the functions described for the HGF/c-Met system overlap with those of the AngIV/AngIV binding site, it was postulated that AngIV acts through c-Met.7,23 It however remains a possibility that distinct binding sites on more than one protein independently mediate the memory enhancing effects of AngIV.

Insulin-Regulated Aminopeptidase (IRAP) In 2001, an AngIV binding site was purified from bovine adrenal cortex membranes and identified as the already known insulin-regulated

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aminopeptidase (IRAP; EC 3.4.11.3).24 Further investigations demonstrated IRAP to have the same biochemical characteristics as the AngIV binding site, and that cells transfected with IRAP expressed a high-affinity binding site for AngIV with an identical pharmacological profile to the AngIV site. Furthermore, the distribution of IRAP in the brain was reported to be the same as for the AngIV binding site, with high densities in areas associated with cognition, such as the hippocampus, amygdala and cerebral cortex.25,26 Development of an IRAP knockout mouse model showed a complete loss of high-affinity AngIV binding sites27, concluding that IRAP constitutes, if not the only, at least one of the AngIV binding sites.

IRAP is also known as placental leucine aminopeptidase, cystinyl aminopeptidase, oxytocinase, and vp165, and was discovered in vesicles containing GLUT4, an insulin regulated glucose transporter.28 In response to insulin stimulation, IRAP accompanies GLUT4 to the cell surface.

IRAP belongs to the M1 family of aminopeptidases and is a type II single-spanning trans-membrane zinc dependent metallopeptidase, consisting of 1025 amino acid residues.29–31 It consists of an intracellular N-terminal domain (110 amino acids), a transmembrane domain (22 amino acids), and an extracellular C-terminal domain (893 amino acids) (Figure 1). The C-terminal region can be divided into four domains (D1-D4), where the D2 domain contains the catalytic site with the zinc binding motif holding a Zn2+ ion.32,33 The D2 domain is capped by the D4 domain, which creates a large cavity adjacent to the metal ion containing the catalytic site. IRAP has been demonstrated to cleave the N-terminal amino acid residue of a number of bioactive peptides in vitro, such as oxytocin, vasopressin, dynorphin A, neurokinin A, cholecystokinin-8 (CCK-8), Met-enkephalin and somatostatin, leading to their inactivation, and several of these play a role in cognition.34–36 IRAP has also been demonstrated to cleave vasopressin in vivo.37

Figure 1. Schematic representation of the IRAP domains.

The precise mechanism by which IRAP impacts cognition is still not fully understood, but two major hypotheses have been discussed in the literature (Figure 2). The first involves inhibition of the enzymatic activity of IRAP, leading to prolonged neuropeptide levels furnishing memory enhancing

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effects. Especially vasopressin, but also CCK-8, oxytocin and somatostatin, have been demonstrated to facilitate learning and memory.38–43

The other hypothesis concerns the co-localization of IRAP with GLUT4 in the hippocampus and the cerebral cortex. Based on this observation, it is suggested that IRAP plays a role in neuronal GLUT4 trafficking and associated glucose uptake.40,44–46 In this model, AngIV could prolong the cell surface localization of IRAP and GLUT4 and thereby facilitate glucose uptake into neurons. Glucose has been demonstrated to enhance cognitive performance in both animals and humans.47

Figure 2. Schematic illustrations of the two main hypotheses by which IRAP binding may facilitate cognition. A) Inhibition of IRAP prolongs the presence of neuronal peptides known to facilitate memory and learning. B) Upon stimulation, vesicles containing IRAP and GLUT4 are translocated to the cell membrane where IRAP ligands prolongs the cell surface localization, translating to facilitated glucose uptake in the neurons. Picture adopted and modified from Vanderheyden et al.48

Insulin-Regulated Aminopeptidase Inhibitors In response to the results supporting the memory enhancing effects of AngIV (1, Figure 3), possibly through the inhibition of IRAP activity, multiple investigations have been undertaken to develop potent IRAP inhibitors. AngIV is rapidly degraded in vivo, and as it is unable to penetrate the blood-brain barrier (BBB), it makes the study of the actual physiological role of the AngIV/IRAP system in cognition difficult. Hence, development of metabolically stable, low molecular weight IRAP inhibitors able to cross the

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BBB is desirable. Initially, the structural features of AngIV itself were investigated, and it was concluded that the three N-terminal residues (Val-Tyr-Ile) are critical for binding affinity and specificity, while the three C-terminal amino acids (His-Pro-Phe) allowed structural modification with retained affinity.49–51

Following these observations, Lukaszuk et al. performed β-homo-amino acids scans of AngIV which resulted in peptides with increased metabolic stability, and high affinity and selectivity for IRAP (e.g. 2, AL-11).52,53 Additional introduction of conformationally constrained residues provided the peptidomimetic IVDE77 (3), which displayed numerous advantageous properties compared to AngIV; higher affinity, increased selectivity and improved metabolic stability.54 Axén, et al. synthesized constrained analogues of AngIV to gain information about the bioactive conformation when binding to IRAP. It was suggested that AngIV adopts a γ-turn in the C-terminal.55,56 Encouraged by this result, Andersson et al. replaced the C-terminal tripeptide with substituted benzoic acids with the aim to mimic the constrained γ-turn, providing high affinity compounds.57 Additional introduction of conformational constraints such as macrocyclization of the N-terminal end of truncated AngIV analogs, gave IRAP inhibitors with potencies in the low nM range and with selectivity towards aminopeptidase N (APN) (e.g. 4, HA08).58 APN belongs to the M1 family as well, and has been demonstrated to be involved in for example cancer, viral infections, and cholesterol uptake.59 The chemically and metabolically labile disulfide bond was subsequently replaced with a carbon-carbon bond with retained affinity.60 Interestingly, exposing hippocampal neurons to HA08 resulted in increased number of dendritic spines, particularly an increased number of stubby/mushroom-like spines.61 As spine density is closely associated with memory enhancement, this in vitro study by Diwakarla et al. suggests that IRAP inhibition can be beneficial in diseases characterized by cognitive impairment. In terms of the relevance of the observed effects, HA08 largely reproduced that of brain-derived neurotrophic factor, a known inducer of spine development. However, additional studies demonstrating direct cognitive enhancing effects of HA08 in vivo are needed.

Starting in 2013, a project focused on rationally designed pseudopeptides based on known metalloproteinase inhibitors also provided nM range IRAP inhibitors, but initially these displayed quite poor selectivity for IRAP compared to endoplasmic reticulum aminopeptidase (ERAP) 1 and ERAP2.62 ERAP 1 and ERAP2 are also enzyme closely related to IRAP, demonstrated to be involved in antigen processing.63 However, a thorough structure-activity relationship (SAR) investigation of the tripeptides eventually yielded an inhibitor with increased selectivity (5, DG026).64 Changing the zinc-coordinating group to an aminobenzamide in combination with further structural modifications of the peptide-like backbone furnished inhibitors with even greater selectivity (6).65,66

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Figure 3. Structure of AngIV and related peptide-based as well as small molecule-based IRAP inhibitors.

Given the anticipated challenges in improving the oral bioavailability and brain penetration of the above described peptidomimetics, non-peptidic IRAP inhibitors were investigated, resulting in the first publication in 2008. By conducting a virtual screen based on a homology model of IRAP, Albiston et al. identified a benzopyran based inhibitor which was subsequently developed into HFI-419 (7, Figure 3).67,68 The compound showed good selectivity towards other aminopeptidases (APN, ERAP1, ERAP2, and LTA4H) and demonstrated increased glucose uptake in rat hippocampal slices. It was also evaluated in two distinct memory tasks in rat; a novel object recognition task and a spontaneous alternation plus maze task. In the former, the rats treated with HFI-419 spent significantly more times investigating the novel object than the familiar object. In the latter, rats treated with HFI-419 demonstrated enhanced spatial working memory.67

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Although the peptide-based inhibitors possess excellent potency, a good selectivity profile, and are metabolically more stable than AngIV, their oral bioavailability as well as BBB penetration are predicted to be low and thus, limiting their potential as pharmacological tools to study IRAP inhibition in animal models of cognition. Some of the peptides are also challenging to obtain synthetically. The small molecule-based inhibitor HFI-419 displays interesting cognitive enhancing effects in rats, however, the solubility and metabolic stability is poor, and the brain uptake is limited. Hence, there is still a need for identification of less complex, small molecule based inhibitors with better pharmacokinetic properties to use as pharmacological research tools for studies of IRAP inhibition in models of cognition.

The synthesis, SAR, and proposed binding mode of an arylsulfonamide based inhibitor, identified in the screening campaign describe herein (Paper I), have been published.69,70 These aryl sulfonamide based inhibitors (e.g. 8, Figure 3) were also evaluated for their ability to increase the number of dendritic spines in the hippocampal neuron assay, similar to HA08. Exposure to the compounds resulted in increased spine density in vitro.71

Identification of Starting Points in Drug Discovery To identify new starting points in drug discovery, various hit identification approaches are typically employed. These broadly follow either of two paths, phenotypic screening or target-based screening. In phenotypic screening, the target is not known a priori, and what is explored is the possibility of a molecule to alter for example a cells disease-relevant physiology, often in cell-based assays. In target-based screening, a target protein that is hypothesized to have a therapeutic effect in the disease under investigation is first selected based on available validation data. Hit identification efforts then involve studies of the specific biological activity of that target protein, either in biochemical or cellular assay formats, with the aim of finding compounds that modulate the target activity. Only the latter will be discussed in this thesis.

Target-based screening can be divided into various approaches as summarized in Table 1. Although not a screening method, the fast follower/focused design approach is also worth mentioning as an attractive alternative as this relates to the already described IRAP efforts on peptidomimetics. In this approach, a known inhibitor or endogenous ligand (or natural product) is modified in various steps in order to obtain for example higher affinity or improved pharmacokinetic properties.

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Table 1. Commonly used target-based methods for the identification of new starting points in drug discovery.

Method Description

Fast follower/natural ligands as starting points

Based on known molecular scaffolds. Referred to as lead-hopping when used to improve the intellectual property situation or to remove undesirable motifs (toxic, metabolically unstable, etc.)

High-throughput screening (HTS)

Large compound collections are evaluated in assays generally run in 384-wells or 1536-wells microtiter plates. Commonly uses changes in absorbance, fluorescence or luminescence as readout.

Focused screening

Often part/used to complement HTS campaigns. Involves compound classes previously known to hit specific target classes (e.g. kinases or GPCRs). Hence, parts of the library are intentionally biased to improve chances of identifying suitable hits.

DNA-encoded libraries72 (DEL)

Involves the conjugation of compounds to DNA oligomers that serve as barcodes to identify the specific compound. Each compound is tagged with a sequence of DNA that can be linked to the building blocks used in the synthesis. For screening purposes, the target protein is immobilized on a solid support, followed by incubation with the DEL. The nonbinding molecules are removed by washing. The bound DNA-tagged molecules are eluted and cleaved, the DNA is amplified through PCR, and decoded to identify the hit compound. Hit follow-up involves resynthesis on non-tagged compounds to verify activities.

Fragment-based lead generation73 (FBLG)

Involves small molecules (fragments) that are soaked into crystals of the target, and hits can subsequently be used as building blocks to obtain larger, more potent compounds. Beside such X-ray crystallography driven approach, NMR, SPR and other biophysical techniques such as thermal shift assays are commonly used for fragment-based screening purposes.

Virtual screening74 (VS)

A computer aided method where a virtual compound library is examined in the X-ray structure (or model) of the target protein. This can be considered a variant of focused screening, except the focused library is specifically designed for the target of interest.

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High-throughput Screening High-throughput screening (HTS) was broadly introduced in the 1990’s by the pharmaceutical industry to efficiently identify small molecules as starting points in drug discovery and development programs.75 Today it is frequently used in both industry and academia. HTS is a method where many compounds are investigated in miniaturized in vitro assays to identify those capable of modulating the target of interest. Compounds that demonstrate specific activity towards the target protein are defined as hits, and form the basis for subsequent hit optimization and lead identification. To achieve the necessary throughput and cost-effectiveness in a HTS campaign, requirements such as advanced instrumentation and automated liquid handling must be available. This allows for rapid dispensing of small volumes of reagent solutions into appropriate microtiter plates, normally 384-wells or 1536-wells plates. After incubation, the readout, which ranges from simple light signal assays to advanced microscopy-based cell assays, is recorded using a range of different microtiter plate readers. Since a compound library commonly contains several millions compounds, large amounts of data are generated which require efficient informatics strategies. This is especially demanding for readouts that involve microscopy- and mass spectrometry-based assays, putting significant demands on the infrastructure for data storage and analysis (terabytes of data are typically generated for each campaign).

To initiate a HTS program, several components need to be taken into account. For example a) reagent preparation (e.g. protein expression), b) compound management, c) assay development, d) choice of compound library, e) data handling, and f) hit validation. The assay development generally concerns choice of assay, choice of detection method, miniaturization to suitable microtiter-based formats, stability control of reagents over time, and examination of DMSO-tolerability (since most compound libraries are stored in DMSO). It is also important to have access to known target modulators for evaluation to ensure an expected pharmacological response and correct ranking of compound in the assay. If the HTS assay is an enzymatic assay, parameters related to enzyme kinetics must also be determined, such as Vmax and Km.76 This is to ensure appropriate sensitivity to small molecule modulation of the response. Vmax is defined as the maximal rate of the enzymatic reaction and is achieved when the enzyme is saturated with substrate. Km, sometimes referred to as the Michaelis constant, is defined as the substrate concentration providing a reaction rate that is half of the Vmax. It varies from one enzyme to another, and for a given enzyme with different substrates. The general recommendation is to perform a screening campaign slightly above the Km, since this provides the best opportunity to identify inhibitors with different mechanisms of action.77

When the assay have been developed and formatted, the most common used statistical parameter to evaluate assay performance is the Z’-factor.78 It

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basically means that the data set for the positive controls (100% inhibition) are compared to the data set for the negative controls (0% inhibition). The Z’-factor is determined by multiplying the standard deviations of the negative (σS) and positive (σB) controls by three, respectively. This reflects the variability of the data sets and is termed data variability band. The products are added and the sum is divided by the difference between the mean of negative (µS) and positive (µB) controls, respectively (Figure 4). The Z’-factor reflects the magnitude of the separation band. A large separation band indicates that a positive signal is easily distinguished from a negative signal and hence, a hit in the screen can simply be identified and separated from an inactive compound. A Z’-value of 1 is defined as an ideal assay with a standard deviation of zero. However, a Z’-value greater than 0.5 is considered to be excellent for a HTS assay.

Figure 4. Top) Formula for calculating the Z’-factor. Bottom) Illustration of the separation band and data variability bands in an assay assumed to have a normal distribution profile. Herein, the data variability band represents the mean (µ) and standard deviation (σ) of the negative (S, 0% inhibition) and positive (B, 100% inhibition) controls, respectively. Z’ reflects the magnitude of the separation band.

When the screening campaign has ended and hopefully generated hits, the hits must be confirmed as true actives, i.e. demonstrate that the compounds are true modulators of the target under study and not an artefact of the assay format or detection method.77 Generally this is performed by analyzing the hits for purity and correct structure, re-testing in a concentration-response fashion, secondary screening (typically cellular or biochemical assays depending on what format was used for the screen), and evaluating them in an

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orthogonal assay (alternative methodology or different readout) to ensure it was a true effect that was measured in the primary screen. There are several ways by which nuisance compounds can interfere with the assay that are not related to a true pharmacological modulation of the target. This may occur through compound aggregation, redox reactivity, interference with the readout, or the presence of reactive functional groups or metal chelating functionalities.

After hit verification, the hit-to-lead phase starts. This generally focuses on increasing affinity and selectivity of the hits. In parallel, improving properties related to pharmacokinetics (PK) and pharmacodynamics (PD) are also considered, as poor PK/PD properties are known to be a major cause of high attrition rates in clinical candidate selection.79–82

When the lead compounds have been identified, they are progressed through the lead optimization phase which eventually culminates in the identification of a clinical candidate.

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Aims

With the memory enhancing effects observed for AngIV followed by the identification of IRAP as the suggested binding site, an interest in developing IRAP inhibitors emerged. Potent and selective peptide-based inhibitors were developed which unfortunately suffered from predicted poor oral bioavailability, thereby limiting uptake over the blood-brain-barrier. To enable in-depth pharmacological studies of IRAP inhibition in models of cognition, efforts to identify less complex, small-molecule based inhibitors with good pharmacokinetic properties are highly desirable. The overall aim of the project was therefore to identify and optimize new small molecule based IRAP inhibitors for use as research tools to investigate the memory enhancing effects of IRAP. The specific aims of the thesis were to:

Develop a highly sensitive and screen-compatible assay which is stable

over time and adapted to microtiter plate format and automatic liquid handling. The assay should allow evaluation of the inhibitory activities of compounds regardless of the source of IRAP enzymatic activity.

Validate hits from the screen, including re-synthesis of chosen compounds and investigation of selectivity and their mechanism of action.

Perform structure-activity relationship studies on a spiro-oxindole dihydroquinazolinone based hit compound.

Perform structure-activity relationship studies on an imidazo[1,5-α]pyridine based hit compound.

25

Identification of New IRAP Inhibitors Through Small-Molecule Screening (Paper I)

Given that most published IRAP inhibitors at the beginning of this work were peptides or pseudopeptides, with the benzopyran series as the only exception, we performed a screening campaign to identify novel small-molecule based IRAP inhibitors. The following sections will fundamentally describe the assay formatting prior to the screen, the compound library, the screening campaign and the hit follow-up experiments. The screen was performed in collaboration with Chemical Biology Consortium Sweden (CBCS).

Assay Formatting In the literature, the enzymatic activity of IRAP has commonly been measured using synthetic substrates for which the absorbance (L-Leucine-para-nitroanilide, L-Leu-pNA) or fluorescence (L-Leucine-amidomethyl coumarin, L-Leu-AMC) is altered upon cleavage of the N-terminal peptide bond (Figure 5).67,83 The concerns with these substrates, from a library screening perspective, are the short wavelengths that are used to observe the assay readout. The product from L-Leu-pNA absorbs light at 405 nm which is close to the near-UV range, and we were initially worried about the potential interference of the readout with colored compounds in the compound library. However, the fluorogenic substrate L-Leu-AMC is excited at even shorter wavelengths (excitation 355 nm, emission 460 nm). Thus, our choice fell on L-Leu-pNA given its simplicity and the extensive experience of this assay format from our collaborating group of Prof. Georges Vauquelin. L-Leu-AMC was instead used for follow-up assays.

26

Figure 5. Picture of the two assay substrates commonly used for measurement of IRAP activity. L-Leu-pNA was used in the screen and in most of the hit confirmation experiments. L-Leu-AMC was used as an orthogonal readout in a follow-up assay.

As a source of enzymatic activity, we used wild-type Chinese Hamster Ovary (CHO) cell membranes, as these are known to be an abundant source of endogenous IRAP activity, with minimal contamination from other aminopeptidases. This was also a system that had been extensively used by our collaborating partner.84,85

Several changes had to be made to the published assay format in order to transform the assay into a fully screening compatible procedure. In short, the assay prior to screen formatting conformed to the following procedure; CHO cell membrane pellets were suspended in assay buffer and homogenized. Thereafter membrane homogenate, L-Leu-pNA (1.5 mM), and compound solution were added to a 96-well plate. The plates were incubated at 37 °C and measured every 5 min between 10 and 50 min.84 The most significant modifications was to a) change from a 37 °C to a room temperature incubation in order to avoid plate edge effects, i.e. thermal gradients across the plate and/or evaporation preferentially occurring at the plate edges86, b) prolong the incubation time in order to use less membranes in each well, c) the use of dedicated negative and positive controls on each plate, d) change from a 96-well to a 384-well format, and e) use automated liquid handling for the compound, membrane and substrate dispenses.

The Km (substrate concentration to obtain half the maximum rate of the enzymatic reaction) was then determined to be 0.28 ± 0.02 mM (Figure 6a), which prompted us to reduce the substrate concentration in the screen from 1.5 mM to 1 mM. This is still higher than the Km, but was chosen in order to facilitate identification of other types of inhibitors alongside competitive inhibitors. These assay conditions ensure availability of the different enzymatic states to which compounds can bind, i.e. unliganded protein as well as transition states with substrate and products still bound. In order to ensure less than 50% substrate conversion, i.e. avoid a significant loss of assay sensitivity to inhibitors87,88, time course experiments as a function of membrane preparations at a substrate concentration of 1 mM were also

27

performed (Figure 6b). These experiments demonstrated the enzymatic activity to be linear for extended time periods. Finally, known IRAP inhibitors were used to assure that the assay responded pharmacologically as expected. Satisfyingly, the optimized assay accurately ranked the low-nM inhibitors IVDE7754 and AL-1152 with IC50 values of 11 and 55 nM, respectively. This corresponds to Ki values of 2.4 nM and 12 nM, which are identical within experimental uncertainty to published Ki’s of 1.7 and 7.6 nM, respectively. Additionally, the low µM inhibitor ZnCl2

83, as well as the high µM inhibitor amastatin were also accurately ranked.

Figure 6. a) IRAP activity in CHO cell membrane preparations as a function of L-Leu-pNA concentration. Data is shown as the average and standard deviation of measurements done in three technical replicates. b) Time-course experiment for IRAP activity as a function of membrane concentration at a substrate concentration of 1 mM; membranes from 90 000 (), 45 000 (), 22 500 (), 11 250 (), or 5625 () CHO cells per well.

Compound Library A set of 10,500 compounds from the primary screening set at CBCS was used in the screen. Most of the compounds were donated by former Biovitrum AB and consists of eight sub-sets of compounds. These included fragment-like (150–300 g/mol), lead-like (300–400 g/mol), drug-like (400–500 g/mol), and diversity sets I and II (150–500 g/mol). These were selected to represent a larger collection of approximately 65 000 compounds, such that iterative screening can be achieved by rapid testing of nearest neighbors. The three additional subsets are focused libraries towards distinct target classes; i.e. kinase soft focused and GPCR soft focused, and the Prestwick set of pharmacologically active compounds. Hence, the screening library was a mix of commercially available and proprietary compounds. Compound stock solution was 10 mM in DMSO, stored and frozen at -20 °C.

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Screening Campaign The screen was performed in transparent 384-well plates to afford the absorbance-based readout. Compound solutions (75 nL) were first dispensed directly into columns 1-22. The equivalent volume of DMSO (negative control, 0% inhibition) and a 10 mM solution of AL-11 (positive control, 100% inhibition) were dispensed into columns 23 and 24. Next, 25 µL of a homogenized solution of CHO cell membranes was added to each well, resulting in membranes from 25 000 cells in each well. Finally, the reaction was initiated by addition of 50 µL 1.5 mM substrate solution (L-Leu-pNA). This provided a final compound concentration of 10 µM, with a final DMSO concentration of 0.1% in all wells. A total number of 32 plates were screened.

A first absorbance reading was taken immediately to get a background reading. This was performed to enable compensation to be made for absorbance from the presence of potentially colored compounds that would add to the assay signal. Thereafter the plates were kept dark and incubated at room temperature with new readings taken after 8, 17, and 25 h to ensure data at various time points in the enzymatic reaction. Fortunately, the initial concerns regarding the potential interference of colored compounds with the short wavelength for absorbance turned out to be minimal, which was displayed by the 0 h reading. Data analysis showed that the 17 h incubation time was optimal as the signal increased linearly with time, and the time point corresponded to less than 50% substrate conversion. Furthermore, both the signal from the uninhibited enzymatic reaction and the fully inhibited wells remained constant across the 32 plates, demonstrating good stability of enzyme, substrate and control inhibitor solutions (Figure 7a). The Z’-factors78 for the individual plates ranged from 0.85 to 0.91, displaying significant distinction between the controls.

A summary of the screen is presented in Figure 7b. Based on the observed inhibition of all library compounds, the hit cut-off was set to the average of the signal plus three standard deviations, resulting in a hit list of 166 compounds (1.6% hit rate).

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Figure 7. a) Average absorbance signal from negative () and positive () controls, respectively. Data are shown as the average and standard error of mean for the controls on each individual plate. b) Summarized screening results after 17 h incubation, where 0% corresponds to uninhibited IRAP in the presence of DMSO only () and 100% corresponds to fully inhibited IRAP in the presence of AL-11 (). The library compounds () were screened at a concentration of 10 µM. Solid line (–) corresponds to the hit limit, set to the average plus three times the standard deviation. Dotted line corresponds to 60% inhibition. Data is formatted from paper I.

Hit Confirmation Delimiting the hit cut-off mentioned above, the compounds displaying more than 60% inhibition were pursued in concentration-response experiments. Out of these 23 compounds (Figure 8), 16 confirmed concentration-dependent activity in the screening assay with IC50-values ≤ 10 µM (Table 2). One compound displayed sub-µM activity and was identified as the drug verteporfin (9), originating from the Prestwick library of known drugs. Verteporfin contains a porphyrin motif which is a known metal coordinating group hence, we suspect it could act as a chelator of the catalytically important Zn2+ ion in IRAP. Five additional drugs were included in the hit-list; merbromin (12), vatalanib (20), topotecan (25), chloroxine (27), and clioquinol (31). However, some of these drugs have previously appeared as hits in screening campaigns at CBCS. This can be correlated to the known liabilities of pan-assay interference compounds (PAINS). PAINS are chemical compounds often appearing as positives in screens, often with unwanted mechanism of action, and several of these are drugs.89–92 In addition to historical in-house data at CBCS, the compounds were also investigated in the PubChem Bioassay93 and ChEMBL94 databases. These publicly available databases provide published biological test results of e.g. small molecules. Since we were interested in identification of novel inhibitors with potential selectivity for IRAP, all compounds with known affinity to other targets and/or compounds with potentially unwanted mechanisms were deselected.

The quality of the hit compound solutions was studied with UV-LC/MS to assure correct mass and acceptable purity. In order to facilitate prioritization

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among the hits, we also looked at structurally related analogues in the compound library that could be used to obtain fundamental SAR. Hence, compounds were chosen on the following criteria, i) low µM apparent potency; ii) correct mass and purity above 85% according to UV-LC/MS; iii) no previous appearance as hits in internal screening campaigns; iv) no or few reports in PubChem Bioassay and ChEMBL; and v) relevant analogs available at CBCS. This provided six compounds that were considered particularly interesting. Out of these, the three most potent compounds were chosen for analog testing, resynthesis and further characterization in order to validate them as starting points in the development of small-molecule based IRAP inhibitors (10, 13 and 15).

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Figure 8. Structures of the 23 top ranked hits in the screen.

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Table 2. Summary data for the top ranked hits in the IRAP screening campaign.

Cmpd % Inhib. 10 µM

Corr. m/za

Purity (%)b IC50 (µM)c Promiscuityd ChEMBL and

PubChem Bio.e Analoguesf

9 93 Yes 95 0.29 3 (17) 0g 1/1/1 10 82 Yes 97 1.3±0.3 (5) 1 (20) 0 39/6/1 11 68 Yes 58 2.0 1 (20) 0 20/9/1 12 80 n.a. n.a. 2.2 9 (15) >50 2/1/1 13 83 Yes 85 2.9 1 (16) 0 378/12/1 14 84 Yes 98 3.0±0.3 (3) 2 (20) 0 46/10/2 15 75 Yes 100 3.1 1 (20) 0 24/8/3 16 70 Yes 100 3.5 (partial) 5 (14) 31 1/1/1 17 85 Yes 89 3.8 6 (15) 16 4/1/1 18 62 Yes 95 5.7 (partial) 1 (16) 0 111/15/1 19 70 Yes 97 5.8 1 (16) 0 12/2/1 20 71 Yes 100 6.7 1 (16) >50 1/1/1 21 62 Yes 99 6.7 1 (17) 0 213/29/4 22 60 Yes 78 10 3 (20) 0 16/1/1 23 61 Yes 84 11 2 (16) 0 385/29/2 24 60 Yes 98 11 1 (21) 0 91/21/4 25 66 Yes 75 27 5 (17) >50 10/3/1 26 61 Yes 98 45±34 (3) 2 (20) 0 20/6/3 27 87 Yes 100 >72 2 (18) >50 7/5/2 28 95 Yes 99 No (3) 1 (20) 0 220/16/2 29 74 No 99 No 6 (15) 0 111/8/1 30 65 Yes 6 No 1 (15) 0 2/1/1 31 63 Yes 98 No 2 (17) >50 5/4/2

aCorrect m/z detected by UV-LC/MS (ESI+). bPurity according to UV-LC/MS at 305±90 and 254 nm. cNumbers within brackets represent the number of test occasions on the screen batch solutions. dNumbers represents the number of times the compounds have appeared as hits in internal CBCS screens. Numbers within brackets represent the number of screens in which the compound has been included. eNumber of appearances as actives in ChEMBL and/or PubChem BioAssay. fAnalogue searches were based on CBCS in-house cheminformatics program Beehive, by using similarity searches with a Tanimoto coefficient >0.7. The numbers refers to available analogues in-house/tested in screen/actives above hit limit. gReported as part of training set for hepatotoxicity.

Testing of all three prioritized and resynthesized hits (10, 13 and 15) confirmed low µM potency (Table 3), further strengthening our belief in the screen output. Analysis of the concentration-response curves demonstrated Hill slopes close to 1, indicating a single binding site and the absence of non-specific inhibition95, and near full inhibition of all compounds (Figure 9a). However, for compound 10 higher variability was observed regarding the maximal inhibition, which coincided with visual precipitation at the highest concentrations, demonstrating limited aqueous solubility. This issue will be further discussed later in the thesis. Thereafter, we ensured that the compounds did not interfere with the absorbance readout by dilution of the substrate product para-nitroaniline to obtain the concentration corresponding to approximately 1 in absorbance, which is what we commonly observe after incubation of the IRAP reaction in the assay plates. Thereafter, this

33

concentration (200 µM, corresponding to 20% substrate conversion) was applied in the assay with various concentrations of the three hit compounds, and the absorbance was measured. None of the compounds displayed any interference with the absorbance of para-nitroaniline at the investigated concentration interval.

An orthogonal assay, applying identical conditions to previous assays, except for using the fluorescent substrate L-Leu-AMC, was thereafter applied, which also confirmed a concentration-dependent inhibition. However, compounds 10 and 15 displayed a right-shift toward less potent activities with this substrate (Table 3). We also demonstrated that the compounds are reversible inhibitors. This was done by rapid dilution experiments, in which the compounds were pre-incubated with the CHO cell membranes for 1 h at a concentration approximately ten times the IC50 value (10 µM). Thereafter, the reversibility was examined by a rapid 100-fold dilution such that the compound concentration became 0.1 µM, i.e. well above the IC50 where only a small extent of inhibition is expected. The enzymatic activity was quickly recovered after dilution (Figure 9b).

Figure 9. a) Concentration-response curves for IRAP inhibition by compounds 10 (), 13 (), and 15 (). Data are presented as the average and standard deviation of multiple independent test occasions. b) Investigation of reversibility of hit compounds 10 () 13 (), 15 (), by rapid assay dilution from 10 µM inhibitor concentration to 0.1 µM.

We also wanted to assure that the compounds did not interfere with the assay through cysteine oxidation or compound aggregation. The redox experiments were performed in the presence of 1 mM 1,4-dithiothreitol (DTT), also known as Cleland’s reagent. It works by reducing disulfide bonds in peptides and proteins, and maintains the SH groups in reduced form, which restores activity lost by oxidation of these groups in vitro. The presence of DTT did not influence the potencies observed by the compounds (Table 3). The non-ionic detergent Triton X-100 was next used to disrupt potential aggregates formed

34

by self-association of the compounds, which could lead to protein sequestering.96 Again, this did not influence the observed potencies of the compounds (Table 3), indicating that they do not inhibit IRAP by unwanted mechanisms such as oxidation or aggregation. This is also in agreement with the promiscuity index discussed above, where the compounds had not appeared as hits in previous screening campaigns at CBCS. Furthermore, a search in the public aggregator advisor database for the three compounds did not show any correlation to known aggregators, although a general warning was given based on the relatively high predicted clogP’s.96

The addition of Triton X-100 did not only serve as an experiment to check for compound aggregation, but under these conditions, it is also known that IRAP is solubilized from the membranes.97 Hence, this experiment also demonstrated that the IRAP inhibition is independent of membrane anchoring of the enzyme.

Table 3. Hit validation experiments under various assay conditions.

IC50 IC50

Cmpd L-Leu-pNAa

(µM) Hill slope

Max Inhib.

(%) L-Leu-AMC

(µM) DTTa

(µM)

T X-100a

(µM)

10 1.8 ± 0.95 (20) 0.99 ± 0.17 96 ± 7 6.9 ± 1.6b 1.6 ± 0.3b 3.7 ± 0.5

13 1.9 ± 0.26 (10) 0.99 ± 0.096 96 ± 2 3.2 ± 0.7b 2.1 ± 0.2 2.7 ± 0.5 15 2.5 ± 1.2 (22) 0.84 ± 0.074 99 ± 3 14 ± 2 2.7 ± 0.7 6.3 ± 0.9

Data are obtained from triplicate samples at a minimum of two independent test occasions. aStandard assay using L-Leu-pNA as substrate, but in the presence of DTT or Triton X-100. bPartial inhibition observed.

Summary In order to identify small-molecule based IRAP inhibitors, approximately 10,500 compounds were screened for their inhibition of IRAP. The screening assay was a formatted variant of a well-known absorbance-based assay based on membrane preparations from CHO-cells, known to contain high amounts of IRAP. The screen was performed with excellent statistics and provided 23 hits with more than 60% inhibition. Most of these were confirmed to exhibit concentration dependent inhibition. The compounds that had not been previously identified as hits in screening campaigns at CBCS, or had any hitherto published biological activities, were consider especially interesting since we were aiming to find novel inhibitors with potential selectivity for IRAP. This, in combination with a few other criteria, provided a selection of three compounds for further characterization. A thorough hit validation investigation was initiated which studied the compounds in an orthogonal assay and under various assay conditions to exclude common artifacts making

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compounds appear as false positives. All three compounds remained active under all tested conditions, verifying their potential as starting points in the development of new IRAP inhibitors.

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Synthesis, Structure-activity Studies and Characterization of Spiro-oxindoles and Imidazopyridines as IRAP Inhibitors (Papers II and III)

Following the hit confirmation experiments, the focus of this project was to further study two validated hit series in terms of expanded SAR studies, determination of basic drug metabolism and pharmacokinetic (DMPK) data, and confirmation of activity on the human ortholog of IRAP. In addition, investigation of the compounds against a closely related aminopeptidase as well as characterization of the inhibition mechanism were considered important at this stage.

Synthesis of Substituted Spiro-oxindole Dihydroquinazolinones Several synthetic methods to generate structurally similar compounds have been published which typically requires reflux for several hours.98–103 Inspired by these synthetic pathways, we decided to investigate if it was possible to develop the reactions and incorporate microwave (MW) heating to decrease the reaction time.104

First, the hit compound 10 was resynthesized from 5-bromo-1-methylisatin (33a), which was first N-methylated according to Scheme 1. In the second step, 33a was reacted with isatoic anhydride (34) and p-toluidine (35) via a three-component batch reaction using conventional heating (10h reflux) where we used a 50:50 mixture of toluene and acetic acid as solvent, resulting in 68% yield. After a few test reactions, it was concluded that neat acetic acid could be used as it slightly improved the yield (72%), but most importantly simplified purification since 10 precipitated from the reaction mixture upon cooling to room temperature. Thereafter we changed from conventional heating to MW heating and sealed vials. When the reaction was heated to 150 °C for 10 min the product (10) could be isolated in 75% yield. We decided to keep this protocol as it is a fast and simple procedure, facilitates easy purification, and gives moderate to excellent yields (58-93%, Scheme 2).

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Utilizing this method, compounds with variations in position 1 and 5 could be synthesized.

Scheme 1. Synthetic procedure to obtain N-methylated isatins.

Scheme 2. Synthetic procedure to substituted spiro-oxindole dihydroquinazolinones employing arylamines.

In the next step, we wanted to continue to investigate structural modifications in position 5 by incorporation of various substituents using Suzuki-Miyaura cross-coupling reactions. Initial test reactions displayed dehalogenated and hydrolysed 10 as major byproducts. The MW-reaction conditions were optimized by varying Pd-catalyst (Pd(PPh3)4, Pd(tBu3P)2, PdCl2(dppf)), base (DBU, NaOH, KOH), solvent system (MeCN, DMF, DMA and methyl ethyl ketone with various proportions of H2O present), temperature (160 °C, 180 °C, 200 °C) and equivalents of boronic acid (1.5–3 equiv).105 From this we could conclude that dehalogenation was affected by solvent and base, but not by concentration, temperature or Pd-source. Minimization of water content and slight excess of base was necessary to avoid hydrolysis of 10. The optimization procedure resulted in a fast and simple MW-promoted reaction protocol (Scheme 3) to obtain the final compounds in a reaction time of only one minute (fixed hold time).

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Scheme 3. Suzuki-Miyaura cross couplings to incorporate various substituents in the 5-position.

Thereafter, we turned our attention to investigating structural modifications in position 3’ (p-tolyl moiety). When we changed from arylamines to aliphatic amines in the three-component reaction, the reaction conditions and procedures had to be modified as the previous developed method gave no or only traces of product with aliphatic amines. We also tried some of the published procedures using SnCl2

101 and ethylenediamine diacetate (EDDA)102 as catalysts, without success. In the method development, we decided to keep the MW heating, but decided to re-evaluate the published methods, varying temperature, time and solvent (Table 4). Neither using EDDA, AcOH nor SnCl2 as catalysts with varying solvents, reaction temperatures or reaction times yielded any, or only traces of product. In view of this, we also went back to conventional heating to ensure the lack of product did not have to do with the heating method. However conventional heating did not provide any product either.

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Table 4. Investigated reaction conditions to perform the three-component reaction employing an aliphatic amine.

Entry Catalyst Solvent Time (min) Temp (°C) Results

1 AcOH (50%)[a] toluene 10 150 no product

2 EDDA (20%)[b] H2O 10, 20 120 no product 3 EDDA (20%)[b] EtOH 10, 20, 30, 40 120 traces of product 4 EDDA (20%)[b] EtOH 10, 20 150 traces of product 5 AcOH (20%)[a] EtOH 10 150 no product 6 EDDA (20%)[b] EtOH 480 (8h)[d] 80 traces of product 7 SnCl2×2H2O[c] EtOH 360 (5h)[d] 80 no product

[a] v/v percent where the total amount of solvent was a 0.1 M solution based on 33a. [b] mole percent based on the 33a. [c] 4 equivalents. [d] Conventional heating.

However, one interesting finding was the pronounced presence of intermediate 60 in almost all reactions (structure in Table 5). In other words, the cyclisation to the spiro-compound did not occur after the initial attack of the amine (59) on isatoic anhydride (34). In view of this, we decided to investigate if the compounds could be synthesized via a two-step reaction instead, where the final cyclisation could require slightly different reaction conditions.

First, we aimed to optimize the synthesis of intermediate 60 by varying the amount of AcOH, as well as co-solvent (Table 5). The reaction outcomes were monitored by TLC and UV-LC/MS where remaining starting material, desired product as well as formed byproducts were studied. The reactions that provided the cleanest outcome were purified to determine the yield. In brief, pure acetic acid did not generate full conversion of the starting material. However, by using 5–20% AcOH in EtOH full conversion of 34 was achieved, the desired product being the major component after completed reaction, and one byproduct, which we unfortunately were not able to characterize. The same outcome was observed when employing 2–20% AcOH in toluene. Higher fractions of AcOH resulted in increased byproduct formation.

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Table 5. Investigated reaction conditions to obtain intermediate 60.

Entry Catalyst[a] Solvent Results[b] Yield (%)[c]

1 AcOH - Product: Yes. Byproducts: 2

Full conversion: no

2 AcOH (2%) EtOH Product: Yes. Byproducts: 1

Full conversion: no


Full conversion: yes 81


Full conversion: yes





7 AcOH (2%) toluene Product: Yes. Byproducts: 1

Full conversion: yes 77







Reactants were in the ratio of 1:1. [a] v/v percent as calculated from a 0.1 M solution. [b] Full conversion refers to isatoic anhydride. Byproducts refers to currently unknown compound(s) generated during the reaction. [c] Isolated yield after purification by silica flash column chromatography.

Since we aimed to use MW-heating, we decided to utilize 5% AcOH in EtOH (entry 3, Table 5) since polar solvents generates more effective MW-heating106–108 and it is also preferred from a toxicity and environmental perspective.

Thereafter, we started to investigate the final ring closure (Table 6). We had already shown from previous investigations (Table 4) that AcOH in EtOH or toluene did not generate the final products. Instead, we decided to investigate the addition of stronger acids such as TFA and HCl, but continue with the same solvent and temperature (EtOH, 150 °C, MW). All reaction conditions provided the final product, with the highest yield obtained when utilizing 1% TFA in EtOH (40%). The reaction was monitored every 10 min, and the most optimal time using these reaction conditions was determined to be 40 min. Since usage of TFA generated the final spiro-compound, we also went back to apply this condition in the original three-component reaction (Scheme 2). However, it did not provide any product, indicating that the presence of isatin gives rise to several possible byproducts under these reaction conditions.

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Table 6. Investigated reaction conditions to enable the final ring closure and obtain spiro-compound 68.

Entry Catalyst Time (min)[a] Yield (%)[b]

1 HCl 10–50 32

2 TFA (1%) 10–50 40 3 TFA (2%) 10–50 - 4 TFA (5%) 10–50 25

[a] The reaction was monitored every 10 min by TLC and LC/MS. [b] Isolated yield after 40 min reaction time. The product was purified by silica flash column chromatography.

At this stage, we decided to use the developed method to generate analogues to 10 in order to continue our SAR investigation (Scheme 4). After generation of the intermediate, the reaction mixture was concentrated and used in the next step without further purification and the yield was calculated over two steps.

Scheme 4. The two-step procedure to generate the final spiro-compounds employing aliphatic amines as nucleophiles.

Synthetic procedures to obtain spiro-oxindole dihydroquinazolinones are also presented in the last chapter.

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Synthesis of Imidazo[1,5-α]pyridines To synthesize compound 13 and analogues, a published five step procedure was applied (Scheme 5).109 The synthetic route began with a Grignard reaction of aryl-magnesiumbromides and 2-pyridinecarbonitrile (78). The formed imine was immediately reduced by NaBH4 to generate the amines (80a–e). In cases were the desired magnesiumbromides were not available, they were generated by MW-heating of the aryl- or alkylbromide in presence of Mg and I2

110, and thereafter added to the nitrile. The benzylamines (79, 80a–e) were thereafter coupled with the appropriate Boc-protected amino acid to obtain 81a–i. POCl3 mediated ring closure afforded the bicyclic core structures 82a–h. Unfortunately, this reaction step caused racemization in the pyrrolidine ring (see further on). After cyclization, the Boc-group was deprotected using either HCl in dioxane, or by in situ generation of HCl by mixing acetyl chloride and MeOH in DCM, to afford free amines 83a–h. Thereafter the amine was reacted with the appropriate isocyanate or isothiocyanate to generate the final products 13, 84–89, 91–98, 100–106. In cases where the isocyanates were not commercially available, they were generated by reacting the corresponding amine with triphosgene.

Compound 90 was synthesized from D-Phe (107) where the acid was transformed to the corresponding methyl ester. The amine was then transformed to the isocyanate using the same procedure as in step 5 in Figure 15 and reacted with compound 83a. Thereafter, the ester was reduced to the corresponding alcohol using LiAlH4 to generate the final compound in 69% overall yield (Scheme 6).

Compound 99 was synthesized from pyrrolidine and (S)-(-)-alphamethylbenzyl isocyanate. (Scheme 7).

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Scheme 5. General 5-step synthesis to obtain the imidazopyridines. Structures are also presented in Figure 12, page 54.

Scheme 6. Synthesis of compound 90.

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Scheme 7. Synthesis of compound 99.

Racemization During the Ring-closure Reaction (step 3) During the synthesis of compound 13 and 84 (Scheme 5), it was concluded that something went wrong during the synthesis as the final NMR-spectra displayed double peaks that did not correspond to rotamers, which we have experienced earlier for isolated intermediates (e.g. amide 81a–i). Compound 13 was intended to be synthesized with defined stereochemistry at the pyrrolidine-ring and the S-configuration of the methyl group, giving rise to two diastereomers, which normally displays different NMR-spectra. However, the NMR-spectra were identical hence indicating that it contained the same compound(s). We started to carefully look back at the synthesis and hypothesized two options where the racemization could possibly occur, either during the peptide coupling (step 2, Scheme 5), although it is rare when employing these reaction conditions in combination with proline, or during the POCl3 mediated ring-closing reaction (step 3). In order to establish which step generated the racemization, the peptide coupling was performed with either L-Pro (110-L), D-Pro (110-D), or D/L-Pro (110-D/L) for comparison, and the generated amides were all subsequently ring closed with POCl3 (111-L, 111-D, 111-D/L). Thereafter, the mixtures were analyzed by chiral supercritical fluid chromatography (SFC). For the amides, three different columns were used to distinguish the different peaks; CHIRAL ART Amylose-SA, YMC Chiral Cellulose-SB and CHIRALPAK-ID. The different columns gave different ratios for the same sample, i.e. they separated compounds differently which required three columns to be used for final determination (Figure 10A). Regarding the ring-closed products, one column was enough (ID-column) to distinguish the different peaks (Figure 10B).

From this, it could be concluded that 110-L contained two compounds in equal amounts, 110-D contained two compounds in equal amounts, and 110-D/L contained four compounds in equal amounts (Figure 10A). Thus, no racemization occurred during the peptide coupling. On the other hand, analysis of the ring-closed products showed that racemization occurred, but not to a 50:50 ratio (Figure 10B).

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Figure 10. Investigation of two possible reaction steps where the observed racemization could occur. A) Analysis of products from the peptide coupling using three columns. B) ID-column analysis of products from the ring-closing reaction.

Applying the same chiral SFC-system, the diastereomeric mixture of 13 was separated. Apart from evaluating the inhibitory activity of each diastereomers separately, we also attempted to specify the specific stereochemistry at the pyrrolidine ring for the most active compound. However, it was not possible to distinguish the two diastereomers using nuclear Overhauser effect (NOE) analysis. Instead, we attempted to use residual dipolar couplings (RDCs) to determine the stereochemistry.111 RDCs arise from the interaction of two nuclei in the presence of the external field of a NMR instrument, and provide information of spatial orientation. Normally, this interaction is reduced to zero due to isotropic tumbling of the molecules in the liquid environment. By

46

limiting this tumbling through introduction of a partial order, the RDCs will resurrect. To create this partial order, various alignment media are typically used, such as for example strained aligning gels (SAGs). We used the gel poly(methyl methacrylate) (PMMA) which is CDCl3 compatible. First, full assignment of all the protons and carbons in 13_dia2 was performed. Thereafter, RDCs were extracted from the difference in C–H splittings of two samples; one oriented in PMMA, and one isotropic. The C–H splittings were measured from 2D HSQC proton-coupled spectra. The next step was to perform a conformation analysis of the two possible diastereomers of 13. The conformers were incorporated in MSpin112 which basically generates calculated RDCs based on the conformation analysis. The calculated values were compared to the experimental, and quality (Q) factors were generated. The lowest Q-factor corresponds to the best fit. Unfortunately, the signal from the PMMA-gel overlapped with several couplings in the compound, resulting in insufficient amount of experimental couplings to compare to the calculated RDCs, and hence no significant difference in Q factors was achieved.

In order to be able to perform structural elucidation using RDCs, a different gel should be used, where the signal from the gel will not overlap with the compound signals. This could probably provide sufficient experimental RDCs to compare to the calculated data set.

Structure-Activity Investigations for the Spiro-oxindole Compounds Evaluation of the synthesized spiro-compounds in terms of their impact on CHO-IRAP activity revealed that substituents in the 1 and 5-positions were crucial for inhibition (Table 7). Removal of the bromine (36) or the N-methyl (37) or both (38) resulted in loss of activity compared to 10. Displacement of the bromine to the 7-position (39) resulted in an inactive compound. Exchange of the bromine to fluorine (40), chlorine (41) or iodine (42) resulted in slightly increased activity from fluorine to iodine. Exchange of the bromine to a nitro group (43) retained activity whilst reduction to the amine (44) reduced activity significantly. Substitution of the R5-Br by a methoxy (45) or methyl group (46) resulted in loss of activity by tenfold compared to 10. Introduction of aryls (47-53), heteroaryls (54, 55, 56) or vinyl groups (57, 58) in the 5-position rendered inactive or significantly weaker inhibitors than 10 (Table 8).

47

Table 7. Evaluation of compounds 10, 36–46 as IRAP inhibitors.

Cmpd R1 R5 R7 pIC50

a

10 5.8 ± 0.2 (21)

36 4.0 ± 0.4 (4)

37 4.6 ± 0.3 (4)

38 <3.9 (3)

39 inactive (3)

40 4.9 ± 0.03 (3)

41 5.7 ± 0.03 (3)

42 6.0 ± 0.06 (3)

43 5.8 ± 0.08 (5)

44 <3.9 (3)

45 4.8 ± 0.3 (3)

46 4.8 ± 0.2 (3) apIC50 equals the negative log of the IC50 in molar. Values represent the mean ± standard deviation of best-fit values, with the number of independent test occasions provided within brackets. No values below 3.9 are reported as this was the highest compound concentration tested.

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Table 8. Evaluation of compounds 47-58 as IRAP inhibitors.

Cmpd R5 pIC50

a

47

<3.9 (3)

48

inactive (3)

49

inactive (4)

50

inactive (3)

51

<3.9 (3)

52

<3.9 (3)

53

inactive (3)

54 4.2 ± 0.4 (4)

55

4.1 ± 0.4 (3)

56

<3.9 (3)

57 inative (3)

58

inactive (3)

apIC50 equals the negative log of the IC50 in molar. Values represent the mean ± standard deviation of best-fit values, with the number of independent test occasions provided within brackets. No values below 3.9 are reported as this was the highest compound concentration tested.

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Evaluations of the p-tolyl moiety (Table 9) displayed reduced activity when the entire moiety was removed (61). Removal of the methyl group (62) or substitution by a trifluoromethyl (63), tert-butyl (64) or iodine (65) rendered compounds with activities in the same range as hit compound 10. Similar activities were also observed when introducing a benzyloxy substituent (66) or an oxazole ring in the meta position (67). Introduction of a carbon linker between the scaffold and the phenyl ring as in 68 provided the first compound with improved potency compared to the hit compound 10. The separated enantiomers were evaluated individually, and the most potent enantiomer displayed a pIC50 of 6.7 compared to 4.1 for the less active enantiomer. Elongation of the linker with one carbon (69) resulted in slightly reduced activity compared to 68, but higher activity compared to 10. A saturated cyclohexyl (70) also moderately increased the activity compared to 10. Introduction of heteroaryls such as 2-pyridyl (71) gave a compound with decreased potency however, 3-pyridyl (72) displayed similar activity as hit compound 10, while a furyl (73) provided improved activity although it was less active than 68. Switching from aryl-containing substituents to saturated systems such as a pentyl group (74) resulted in the most potent compound in the series so far. Introduction of heteroatoms in the chain (75–77) resulted in maintained or decreased activity compared to 10.

In summary, it seems that an electron withdrawing group in the 5-position is beneficial for activity. The presence of the N-methyl group, i.e. position 1, is crucial to retain activity. Regarding the 3’-position, the results indicate that a lipophilic group is favorable but not necessary to improve or maintain activity against IRAP. Finally, care must be taken when interpreting the data as several of the analogues display poor solubility which is also visible when running the enzymatic assay. Visual precipitation is observed in the wells at the highest test concentrations. For additional details on this subject, see the “Pharmacokinetic and DMPK property profiling” section.

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Table 9. Evaluation of compounds 10, 61-77 as IRAP inhibitors

Cmpd R3’ pIC50

a

10

5.8 ± 0.2 (21)

61 5.2 ± 0.2 (3)

62

5.2 ± 0.4 (3)

63

5.3 ± 0.1 (3)

64

5.7 ± 0.3 (3)

65

5.5 ± 0.2 (3)

66

5.5 ± 0.3 (3)

67 5.7 ± 0.3 (3)

68

6.4 ± 0.2 (5)

68_1 1st eluted enantiomer 6.7 ± 0.05 (4) 68_2 2nd eluted enantiomer 4.1 ± 0.3 (4)

69

6.2 ± 0.07 (3)

70

6.2 ± 0.1 (3)

71

5.2 ± 0.2 (3)

72

5.7 ± 0.06 (3)

73

6.2 ± 0.07 (3)

74 6.6 ± 0.05 (3)

75 5.5 ± 0.02 (3)

76

5.6 ± 0.08 (3)

77 4.7 ± 0.1 (3) apIC50 equals the negative log of the IC50 in molar. Values represent the mean ± standard deviation of best-fit values, with the number of independent test occasions provided within brackets. No values below 3.9 are reported as this was the highest compound concentration tested.

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Structure-Activity Investigation for the Imidazopyridine Compounds Several analogues of hit compound 13 was available in the CBCS compound library. Following the screening campaign, many of them were evaluated in a three-point concentration response (62.5 µM, 12.5 µM and 2.5 µM, or 50 µM, 10 µM and 2 µM) to get an approximate idea of their apparent potencies (data not shown). A selection of compounds was then evaluated in full concentration-response experiments to obtain fundamental structure-activity relationships before starting the synthetic work (Figure 11). These two experiments indicated that modifications to the R1 substituent, connected to the urea, was somewhat restricted. Activity was notably decreased by substitution of the methyl group by larger groups such phenyl groups, or introduction of more rigid substituents such as cyclopropyls, changes to spacer lengths (especially shortening) and aryl groups (in particular introduction of substituents on the ring). The data also indicated that exchange of the pyrrolidine ring by a piperidine with the nitrogen in the 3 or 4-position resulted in inactive compounds. However, modifications to the aromatic ring directly connected to the bicyclic core (R2) were more tolerated. All compounds tested in concentration-response were analysed with UV-LC/MS in order to check purity, with all of them confirmed at ≥85% purity (254 nm).

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Figure 11. Compounds from the screening library analogues to hit compound 13 evaluated as IRAP inhibitors. pIC50 equals the negative log of the IC50 in molar. Values represent the mean ± standard deviation of best-fit values, with the number of independent test occasions provided within brackets. No values below 3.9 are reported as this was the highest compound concentration tested.

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Following from these observations, the structure-activity investigations initially focused on the urea tail, the cyclic core, and the topmost anisole ring. Additionally, some of the library compounds were resynthesized to further verify the activities observed (112, 115, 131, 132). First, we re-synthesized the hit compound 13 and the analogue with the opposite stereochemistry at the methyl group (84). As mentioned in the synthesis section, we intended to synthesize the compounds with specific stereochemistry at the C2-position of the pyrrolidine-ring. However, racemization was observed during the synthesis and we were unfortunately not able to provide the stereochemically pure compounds. The diastereomeric compound 13, with the S-configuration at the methyl group was 20-fold more potent than 84 with the R-configuration. The racemic dimethylated analogue 85 was equipotent to 84. Elongation of the methyl to an ethyl group (86) provided a compound with potency in the same range as 13. Removal of the methyl group (87) resulted in a drop in activity compared to both 84 and 85. However, elongation of the linker between the urea functionality and the phenyl ring (88) regained potency compared to 87. Further elongation to a three-carbon chain (89) lowered the inhibitory capacity again. Introduction of a polar substituent to 88 as in 90 resulted in reduced activity, both compared to 88 and 13. Introduction of substituents such as furyl (91), cyclohexyl (92), pentyl (93), or an allyl group (94) to the urea did not render any improvement in potency (Figure 12A).

Next, we looked at the central part of the molecule (Figure 12B). The five-membered ring seems to be beneficial for binding since introduction of an open chain linker as in 95, or introduction of a 6-membered ring as in 96–97 resulted in a drop of activity.

Removal of the anisole moiety as in 98 also rendered a compound with reduced potency, and additional removal of the bicyclic core (99) furnished an inactive compound (Figure 12C).

Thereafter we investigated minor alterations of the methoxy substituent (Figure 12D). To facilitate purification and characterization, they were synthesized with the phenethyl substituent connected to the urea; as this provided the most active compound in absence of the methyl group. Hence, in the following descriptions the compounds will be compared to analogue 88 instead of 13. Shifting the methoxy substituent to the meta (100) position provided a slight improvement in potency, while shifting to the para position (101) furnished a compound with similar activity as 88. Exchange of the o-anisole to an o-dimethylaniline (102) produced a drop of activity. Introduction of a saturated ring system as the cyclohexyl (103) also caused a drop in apparent potency.

Finally, switching from a urea to a thiourea (Figure 12E) resulted in a huge drop of inhibitory activity for all analogues (104–106).

Given this, the hit compound 13 was still the most active compound in the series so far. The separated diasteromers were tested individually and the most

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potent compound (13_dia2) displayed a pIC50 value of 6.0, which is 15-fold more potent than the other diastereomer (13_dia1).

In summary, the stereochemistry of the methyl group on the urea tail seems important for inhibition, with the S-configuration being preferred. Two interpretations for this preference may be that the methyl group itself is an important interaction point, or because it directs the phenyl group into a more favorable orientation for binding. Minor enlargement, for example to an ethyl group, was allowed but bigger groups were not tolerated. Regarding the spacer length between the urea and the phenyl ring, a two-carbon linker appears to be the most preferred length of the variants evaluated so far. Elongation to three carbons reduced potency, like observations when shortening to one carbon. Having the aryl group directly connected to the urea furnished inactive compounds or compounds with low potency (<3.9). Furthermore, an unsubstituted phenyl ring was preferred to substituted rings of various kinds, heteroaryls, cyclohexyls, lipophilic chains, esters or allyl groups. The synthesized thioureas were less active than the corresponding ureas. Thioureas are weaker hydrogen bond acceptors than ureas113,114, hence, the drop in potency could therefore be due to a potential loss of hydrogen bond interaction with the carbonyl oxygen. A central five-membered ring is preferred in comparison to a one-carbon open chain, or a six-membered ring with the nitrogen in 2, 3 or 4-position. Alterations of the substituents on the topmost anisole-ring (o, m, p-OMe, Me, H, p-F, p-Cl, o-N(CH3)2) is allowed without significantly affecting the activity. Neither did switching from an anisole to a cyclohexyl ring. However, removal of the entire moiety resulted in a drop of activity, indicating that this part significantly contributes to the inhibitory capacity.

Again, it is important to emphasize that several of the analogues displayed poor solubility, as observed when running the enzymatic assay. Visual precipitation is observed in the wells at the highest test concentrations, see next section.

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Figure 12. Evaluation of synthesized hit analogs as IRAP inhibitors. Investigation of A) urea tail, B) central ring system, C) bicyclic core, D) substituents on bicyclic core and E) urea vs. thiourea. pIC50 equals the negative log of the IC50 in molar. Values represent the mean ± standard deviation of best-fit values, with the number of independent test occasions provided within brackets. No values below 3.9 are reported as this was the highest compound concentration tested.

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Physicochemical and DMPK Property Profiling In order to provide a chemical tool suitable for in vivo pharmacological studies of IRAP-related biology, more aspects than optimization of binding affinities must be considered. Compounds must also be evaluated with respect to absorption, distribution, metabolism and excretion (ADME) properties. In an early stage, in vitro assays providing information of physicochemical and metabolic properties such as solubility, permeability, metabolic stability, and plasma protein binding, are useful for prediction of such behavior and hence for iterative development of compounds. In the efforts to optimize binding to the target protein, parameters like these are easily forgotten, but must be given due weight in the overall objective of optimizing in vivo activity. A less active compound could have advantageous drug metabolism and pharmacokinetic (DMPK) properties, eventually offering a better in vivo therapeutic response.

In this project, we had the fortune to evaluate a representative selection of the compounds in a selection of AstraZeneca’s DMPK primary screening assays, also known as the DMPK Wave 1 panel of assays. The panel comprises five assays; logD at pH 7.4 (logD7.4), dried DMSO solubility (Sol.), human plasma protein binding (PPB), and intrinsic clearance in human liver microsomes (HLM Clint) and rat hepatocytes (Rat hep. Clint).115 The spiro-compounds (top) and the imidazopyridines (bottom) evaluated in the DMPK Wave 1 assays are presented in Table 10.

In general, all evaluated spiro-compounds and imidazopyridines displayed logD values ≤5, which is considered beneficial according to the rule of five for drug-like molecules proposed by Lipinski et al.116 The lipophilicity reflects the distribution of the compound between an organic phase, commonly octanol, and an aqueous phase, commonly an aqueous buffer. Lipophilicity is correlated to many other properties such as solubility, permeability, metabolism, but also to pharmacological activity since it affects the availability of the compound in test systems as well as in man. The ideal logD range is considered to be 1–3, since these compounds generally have good absorption and low binding to metabolic enzymes, hence higher metabolic stability.117 In relation to the logD values, all tested compounds displayed poor solubility, with compound 76 from the spiro series comprising a basic tertiary amine, as the only exception, which was also reflected by the experimentally measured logD. This is in line with the previous observations of visual precipitation in the assay wells for several of these compounds.

Solubility is a crucial physicochemical property determining whether a biological test system, e.g. a biochemical assay, or organism, is appropriately exposed to the compound. In our case, where the compounds precipitate out of the assay solution, the actual concentration of the compounds is no longer known. This means that the observed SAR is not simply governed by the activity towards IRAP, but also on compound availability in the assay. Hence, compounds suffering from poor solubility might display lower potency than

57

they possess due to limited access. Furthermore, precipitation in the assay wells results in elevated background absorbance and the appearance of partial inhibition for some of the compounds.

Unfortunately, all the compounds tested also suffered from high intrinsic clearance in human liver microsomes as well as in rat hepatocytes. However, both series displayed good plasma stability.

Table 10. Physicochemical characterization and metabolic stability investigations of selected compounds.

Cmpd logD7.4 Sol.[a] HLM Clint

[b] HLM t ½

Rat hep. Clint

[c] Rat hep.

t ½ PPB]d] Plasma

stab.[e]

10 3.4±0.1 0.2-1.4[f] >300[h] <2.3[h] 135±11 5.2±0.4 4.3±0.5 106±11 43 2.7±0.1 1.8±0.3 >300[h] <2.3[h] 114±4 6.1±0.2 9.8±2 100±5 51 4.9±0.1 0.2±0.1 >300[h] <2.3[h] >300[h] <2.3[h] 0.25-3.2 102±25

68_1 3.7±0.1 0.3-3.5 >300[h] <2.3[h] >300[h] <2.3[h] 1.9±0.5 101±7 68_2 3.8±0.2 0.1-1.3 >300[h] <2.3[h] 168±20 4.2±0.5 1.5±0.3 99±3

72 2.6±0.1 20±1.2 >300[h] <2.3[h] 84±14 8.5±1.6 5.5±0.4 97±15 73 3.0±0.1 4-16 >300[h] <2.3[h] 132±39 5.5±1.4 4.1±0.8 98±3 74 3.7±0.1 2.4-7.5 >300[h] <2.3[h] 248±5 2.8±0.06 1.9±0.4 105±7 75 2.4±0.01 58±16 >300[h] <2.3[h] 91±8 7.6±0.6 14±0.6 99±1 76 1.6±0.1 814±63 81±10 8.6±1.0 136±22 5.2±0.9 40±4 95±2

84_dia1 2.8±0.7 40-124[f] >300[h] <2.3[h] >300[h] <2.3[h] 1.7±0.3 102±5 84_dia2 2.6±0.7 76±14[g] >300[h] <2.3[h] >300[h] <2.3[h] 1.8±0.4 104±3

88 2.7±0.4 2.2-11[f] >300[h] <2.3[h] >300[h] <2.3[h] 1.8±0.4 97±3 96 2.9±0.6 55±12 >300[h] <2.3[h] >300[h] <2.3[h] 0.6±0.1 99±5

105 3.3±0.4 0.6-2[f] >300[h] <2.3[h] >300[h] <2.3[h] 0.7±0.1 76±6 All the data are reported as the average and standard deviation from three independent test occasions. [a] Dried DMSO solubility in aqueous phosphate buffer (PBS) at pH 7.4; µM. [b] Human liver microsome intrinsic clearance; µL/min/mg. [c] Rat hepatocytes intrinsic clearance; µL/min/106 cells. [d] Human protein plasma binding; % free fraction. [e] Human plasma stability; % compound remaining after 18 h incubation. [f] High-throughput solubility assays are associated with large variability, especially below 10 µM range. When the standard deviation is higher than half the mean value, the entire range is given. [g] An outlier value was observed with higher solubility amongst the three replicates. The higher value was removed. [h] A higher uncertainty is associated with these values as compound dilutions resulted in non-linear responses.

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Activity Confirmation on Human IRAP and Selectivity Investigations Towards APN As mentioned previously, the initial screening campaign and the following SAR investigations were performed based on IRAP naturally expressed in CHO cells. To further study and evaluate the compounds as potential starting points in the development of novel IRAP inhibitors, the confirmation of activity on the human enzyme was essential. For this purpose, we first used overexpressed human IRAP (hIRAP) in HEK293 cells to confirm activity. In a later stage of the project we also introduced a recombinant soluble form of the enzyme, in which the N-terminal domain, including the transmembrane spanning region, is truncated (starting at amino acid 155). While this prevents IRAP anchoring to the cell membrane, it has been demonstrated that this extracellular soluble form is sufficient for maintaining enzymatic activity.118,119 Additionally, initial selectivity investigations towards the closely related enzyme APN was performed.

With respect to the spiro-compounds, they all lost approximately 10–15-fold activity on the human enzyme compared to the hamster enzyme (Table 11, top). Hence, the potency approached a concentration where visual precipitation was observed. Sequence comparison of hamster IRAP and human IRAP showed 88 % similarity, with no differences in the active site residues. Given this observation, we initially hypothesized that the discrepancy in potency could be related to the limited compound solubility. The rationale for this was that hIRAP is based on overexpressing cells, meaning the samples contain much less cell membranes per total protein content, compared to the CHO cell membranes. Potentially, the increased presence of membrane constituents could aid dissolution of the compounds and hence increase the concentration available to inhibit IRAP. However, since the analogue with substantially increased solubility, 76, also lost activity in the same order of magnitude, this hypothesis was ultimately abandoned. Fortunately, spiro-compounds evaluated on APN under identical assay conditions proved to completely inactive.

Considering the imidazopyridines, only a few analogues were evaluated. Satisfyingly, these confirmed inhibition on overexpressed hIRAP with the same potency as for the hamster enzyme (Table 11, bottom). Moreover, compound 84 was evaluated in the soluble human IRAP (sIRAP) assay and displayed identical activity compared to the hIRAP and hamster enzyme assays. Also in this case, the evaluated compounds were inactive on APN.

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Table 11. Evaluation of selected compounds on IRAP from different species, as well as APN.

Cmpd CHO IRAPa

pIC50

hIRAPb

pIC50 sIRAPc

pIC50 APNd

pIC50

10 5.8 ± 0.2 (21) 4.8 ± 0.02 (2) - inactive (2) 43 5.8 ± 0.08 (5) 4.5 ± 0.01 (2) - inactive (2) 68 6.4 ± 0.2 (5) 5.3 ± 0.01 (2) - inactive (2) 72 5.7 ± 0.06 (3) 4.5 ± 0.05 (2) - inactive (1) 73 6.2 ± 0.07 (3) 4.9 ± 0.1 (2) - inactive (1) 75 5.5 ± 0.02 (3) 4.3 ± 0.07 (2) - inactive (1) 76 5.6 ± 0.08 (3) 4.4 ± 0.04 (2) - inactive (1) 84 5.7 ± 0.1 (28) 5.9 ± 0.05 (2) 6.0 (1) inactive (2)

84_dia1 4.8 ± 0.2 (4) 5.2 (1) - - 84_dia2 6.0 ± 0.1 (4) 6.0 (1) - inactive (1)

85 4.4 ± 0.1 (8) 4.3 ± 0.05 (2) - inactive (2) pIC50 equals the negative log of the IC50 in molar. Values represent the mean ± standard deviation of best-fit values, with the number of independent test occasions provided within brackets. aEvaluated on IRAP from CHO cell membranes. bEvaluated on human membrane bound IRAP. cEvaluated on recombinant soluble IRAP. dEvaluated on human aminopeptidase N.

Mechanism of Inhibition The activity of an enzyme can be blocked in a number of different ways.120 However, most inhibitors are compounds that reversibly bind enzymes with fast association and dissociation rates, so called reversible inhibitors. Reversible inhibitors are commonly classified into three classes based on their mode of inhibition; competitive, non-competitive and uncompetitive.120

Competitive inhibitors refer to inhibitors that binds solely to the free enzyme. The inhibitor and the substrate compete for the same enzyme form and generally bind to a mutually exclusive binding site. However, it is also possible for a competitive inhibitor to bind to an allosteric site distant from the substrate binding site, and induce a conformational change that prevents substrate binding. A competitive inhibitor does not affect Vmax, but Km increases since it requires higher substrate concentration to reach half the Vmax. Non-competitive inhibitors refer to inhibitors displaying binding affinity for both the free enzyme and the enzyme-substrate complex. They do not compete with the substrate, but rather bind the enzyme in a site distinct from the active site. Hence, non-competitive binding cannot be overcome by increasing substrate concentration. Thereby, a non-competitive inhibitor decreases Vmax but leaves Km unchanged. Uncompetitive inhibitors bind exclusively to the enzyme-substrate complex. This results in a decreased Vmax as they hamper the catalysis in the enzyme-substrate complex. They are also associated with a parallel decrease in Km representing the enhanced substrate binding.

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In order to determine the mechanism of inhibition for our compounds, we performed experiments with various fixed inhibitor concentrations in combination with various substrate concentrations. Thereafter the data was plotted in terms of velocity as a function of substrate concentration for each inhibitor concentration, and fitted to the Michaelis-Menten equation. The plot for spiro-compound 68 clearly displayed a parallel decrease in both Km and Vmax with increasing inhibitor concentration (Figure 13a), hence displaying an uncompetitive mode of inhibition. An increased potency is also observed with increased substrate concentration (Figure 13b). The data was also fitted to a model for uncompetitive binding within GraphPad and compared with models for competitive and non-competitive binding. The uncompetitive inhibition model explained the experimental data with a probability of 99.8%, compared to 0.2% and <0.01% for noncompetitive and competitive inhibition, respectively. Calculations based on this mode of inhibition provided a Ki value of 0.54 ± 0.06 µM.

Figure 13. a) IRAP reaction rates as a function of substrate concentration for spiro-compound 68. The solid lines represent the best fit to a model for uncompetitive inhibition, displaying a parallel decrease in Km and Vmax with increased inhibitor concentration. b) IRAP reaction rates as a function of inhibitor concentration, displaying improved potency with increased substrate concentration.

The corresponding plot for imidazopyridine 13 displayed a decrease in Vmax with increasing inhibitor concentration, while Km remained constant, hence displaying a non-competitive behavior (Figure 14a). No shift in apparent potency was observed with increased substrate concentration, as displayed in Figure 14b. The data was also fitted to a model for non-competitive binding within GraphPad and compared with models for competitive and uncompetitive binding. The non-competitive inhibition model explained the experimental data with a probability of 99.99%, compared to 0.01% and <0.1% for competitive and uncompetitive inhibition, respectively. Calculations based on this mode of inhibition provided a Ki value of 2.3 ± 0.14 µM.

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Figure 14. a) IRAP reaction rates as a function of substrate concentration for imidazopyridine 13. The solid lines represent the best fit to a model for non-competitive inhibition, displaying a decrease in Vmax while Km remains constant. b) IRAP reaction rates as a function of inhibitor concentration, displaying identical potency with increased substrate concentration.

Interestingly, these are the first published IRAP inhibitors not displaying a competitive mode of inhibition. This might correlated to the small substrate used in the assay. The active site in IRAP is relatively big, to fit the large macrocyclic peptides cleaved by the enzyme. Using a small substrate, in this case a modified dipeptide (L-Leu-pNA), provides space for e.g. simultaneous binding of the inhibitor and substrate as in the case with spiro-compound 68. It would be interesting to investigate the mechanism of inhibition utilizing a larger substrate, or one of the endogenous peptides.

Molecular Modeling and Interpolation of the Structure-Activity Relationships The crystal structures of several proteins in the M1 aminopeptidase family display two conformations; open and closed.121–125 In principle, the open conformation accounts for substrate loading and product release, while the closed conformation is the active form for catalytic processing of the substrates. IRAP (human) was initially found to adopt an intermediate conformation between the open and closed form.32,33 However, in 2017 the first crystal structure of IRAP in a closed conformation in the presence of a bound inhibitor (DG026, Figure 3) was published.126

Docking studies were performed on spiro-compounds 10, 38, 39, 48, 68, 74 and 76 based on their measured potencies in the enzymatic assay. The protein surface area of the opened (PDB ID: 4PJ6) and closed (PDB ID: 5MJ6) conformations of IRAP were analyzed for potential sites for small molecule binding, and three putative biding sites were identified. All the ligands (in the S-configuration), except for compound 48, found a conserved binding mode in the active site in the open conformation. While being in the

62

vicinity of the Zn2+, the compounds are not coordinating the ion, having the N-Me at 4 Å distance to the ion. In this pose, the oxindole moiety is making a hydrogen bond to Tyr549 while the bromine is coordinating Arg439. The aryl ring originating from the isatoic anhydride (34) is making a π-π stacking interaction with Phe544, which is only possible with the S-stereoisomer. Similar docking exploration was performed on the closed conformation; however, no conserved binding pose that could explain the SAR was obtained.

Following the docking studies, molecular dynamics simulations on the complex with ligand 74, one of the most potent compounds, was performed. A refined binding pose was obtained, which was largely consistent with the initial binding pose (Figure 15a). The main difference was in the translation of the ligand by approximately an additional 1.5 Å away from the Zn2+. This increase in distance allowed a water molecule to be inserted between the Tyr549 and the oxindole oxygen. As the zinc ion is a critical component of substrate recognition and processing during the catalytic cycle, we wondered if the space between the ligand and the Zn2+ could alternatively accommodate the small L-Leu-pNA that we use in our assays. This was an important consideration as our experimental data shows an uncompetitive inhibition mechanism, such that compound only binds substrate-bound enzyme. Superposition of the IRAP-substrate complex, which placed the leucine and para-nitrophenyl in equivalent positions to Val1 and Tyr2 sidechains of AngIV, with the IRAP-74 equilibrated pose showed that it was possible for the synthetic substrate and ligand 74 to bind at the same time (Figure 15b). This binding mode can account for the uncompetitive mode of inhibition observed for these compounds.

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Figure 15. A) Binding mode of inhibitor 74 (green) in IRAP (PDB ID: 4PJ6) obtained from a representative snapshot from the MD simulation. B) The same snapshot with the superposition of substrate L-Leu-pNA (orange) docked to IRAP, from two different points of view. Zn2+ and water molecules are shown as grey and red spheres, respectively. The bromine is represented in magenta. Key residues are displayed in in wheat color sticks. The picture is reproduced from paper II.

The proposed binding pose could also explain the SAR observed for the series. The orientation of the 5-position, i.e. the substituent presenting the bromine in the hit compound, is in agreement with the high propensity observed for the interaction of halogens vid arginine.127 This would explain why the replacement of the halogenated or electron withdrawing groups by either aryl, heteroaryl or vinyl groups rendered inactive or only weakly active compounds. This is also in line with the inability of compound 48, carrying the bulkiest substituent in this position, to find this binding pose. Furthermore, as mentioned previously the compounds were unable to interact with the closely related enzyme APN. Attempts to dock these ligands in the corresponding site in human APN (PDB ID: 4FYQ) did not yield any clear binding poses, reproducing our observations for the closed conformation on IRAP (PDB ID: 5MJ6). The specificity of the identified binding orientation in the open IRAP conformation is further illustrated by superposition with the APN and IRAP-closed structures (Figure 16). The figure displays how the selectivity for the IRAP-open conformation might be mediated by the GAMEN loop, which in the IRAP-closed conformation adopts a specific, induced fit conformation.

Additionally, when this putative binding pose was identified for the spiro-compounds, an amino acid sequence alignment was performed between hamster and human IRAP in order to identify any differences in the binding site. The two enzymes show 88% similarity, with no differences located in the binding site residues. Furthermore, the zinc binding motif and GAMEN loop

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residues are also conserved. Thus, the loss of activity towards the human enzyme cannot be explained by altered amino acids in the active site, but likely comes across as a result of plasticity differences between the orthologs.

Figure 16. Binding mode of the inhibitor core structure, the spiro-oxindole dihydroquinazolinone (cyan), in the open IRAP conformation (grey surface, Zn2+ in purple) superimposed with APN and IRAP-closed conformation. The GAMEN loop of APN (red) and IRAP-closed (green) adopts a different conformation compared to the IRAP-open conformation (yellow), giving a putative explanation for the selectivity for the IRAP-open conformation. The picture is reproduced from paper II.

Regarding the imidazopyridines, no docking studies have yet been performed that could explain the SAR and the non-competitive inhibition pattern. Initially we hypothesized a binding pose of these compounds in the active site of IRAP. In such binding, compound 13 could attain a substrate-like interaction where the urea carbonyl oxygen was coordinating the Zn2+ and hydrogen bonding to Tyr477, the methyl group on the urea tail was directed into the narrow N-terminal binding pocket, and the adjacent phenyl group was forming a π-π stacking interaction with Phe472. The binding pose could also broadly explain the initial SAR that was obtained, and hence was used to guide early chemistry efforts. However, an identical binding pose was also obtained when docked into the APN catalytic site, and since we demonstrated that the compounds do not interfere with APN activity, we believe the compounds interact with IRAP in a different way. The binding pose hypothesis was also challenged by the enzyme kinetics experiments which displayed a non-competitive mode of inhibition, meaning the compounds have equal affinity for the apo-enzyme and for the enzyme-substrate complex, effectively excluding this binding mode. Hence, most likely the compounds interact with the enzyme through an allosteric binding site.

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Summary Two hit compounds from the initial screen have been further studied in terms of their structure-activity relationships, physicochemical properties, metabolic stabilities, mechanism of action, and tentative binding poses through molecular modelling.

Compounds in the spiro-oxindole series were synthesized either via a MW-heated three-component reaction or by a slightly modified MW-heated two step reaction. The most potent compounds, 68 and 74, exhibited pIC50 values of 6.4 and 6.6, respectively. In contrast to previously published inhibitors, the compound series proved to be uncompetitive with the assay substrate L-Leu-pNA, and a proposed binding pose 5.5 Å away from the catalytic Zn2+ explains how the substrate can bind simultaneously with the inhibitor. The molecular modeling also identified that the S-stereochemistry of the spiro-carbon accounted for the inhibitory activity of the compounds. In general, the series suffers from poor solubility and poor metabolic stability. Most of the attempts to synthesize derivatives with increased solubility resulted in decreased activity, except for two compounds where 76 is especially encouraging. Although it is less potent than 68 and 74, this ligand displayed a greatly increased solubility while maintaining activity comparable to the hit compound. This compound is therefore a much-improved starting point for further exploration towards in vivo candidates.

The imidazopyridine series was synthesized via a five-step reaction, starting from 2-cyanopyridine. Unfortunately, the hit compound 13 remains the most active compound, displaying a pIC50 of 5.7. As with the spiro-compounds, these compounds do not act competitively with the substrate, but in this case act as non-competitive inhibitors. The imidazopyridines also suffer from poor solubility and poor metabolic stability in liver microsomes and hepatocytes.

Although both compound series in general are less potent than the benzopyran-based inhibitors, with a published Ki value of 0.48 µM for the most extensive investigated compound (HFI-419)67,68, our most active spiro-compounds display similar potencies; e.g. compound 68 with a Ki of 0.54 µM. Similar to our compounds, the benzopyrans also suffer from limited solubility (although higher than ours) and low metabolic stability where the amide is rapidly hydrolyzed to the amine. It is also worth mentioning that we have evaluated HFI-419 in our assays in parallel with the spiro-oxindoles and the imidazopyridines, and both herein described series are on a par and in some cases more potent, than HFI-419.

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Microwave Heated Continuous-Flow Synthesis of Spiro-oxindole Dihydroquinazolinones (Paper IV)

As a drug discovery project proceeds, the demand for higher quantities of compounds increases. Going from a few milligrams in the initial assay testing, to gram scale in early toxicology studies, to kilograms for the candidate drug, the desired amount continues to increase during clinical trials. As a result, a milligram preparative method must be developed into a multi-kilogram process, without affecting the quality of the active compound. Scaling up reactions requires careful risk assessment as failure to take appropriate precautions may lead to loss of reaction control, and potential runaway exothermic reactions and/or generation or release of potential toxic material. Hence, synthetic procedures considered appropriate for lab scale organic synthesis might be potentially hazardous when performed in scaled-up batches. One way of overcoming this is to use continuous flow (CF) chemistry. CF refers to the process of performing chemical reactions in a reactor in a continuous operation. The reaction components are pumped through a temperature-controlled reactor where the reaction occurs, and the product(s) is then collected in a vessel for further processing. One advantage of this approach, in comparison to batch chemistry, is that the volume of the reaction mixture processed at a given time can be kept low. An additional advantage is that the product is formed quickly, and degradation of reactants and/or product due to extended heating periods is minimal. The scale of a reaction performed using CF is thus determined by the time the system is operational. That is why the phrase scale out is used in CF chemistry, rather than scale up as in batch chemistry. Consequently, CF chemistry offers many advantages in terms of safety, handling and scale up, but often also cleaner products, rapid reaction optimization, and the possibility to integrate synthesis, work-up and analysis. A general disadvantage with the methodology involves the requirement of homogenous mixtures, as the pumps used are seldom well suited for handling particles.

In recent years, the application of CF in drug discovery and development has increased due to the observed synthetic benefits, and the methodology can offer advantages in each step of the drug development process; from hit optimization to large scale synthesis.128–131 The most common heating method

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used in a CF setup has been conductive heating, which may suffer from problems regarding the time required for both heating and cooling.

Microwave Heated Continuous-Flow In the 80’s, microwave-assisted organic synthesis was introduced as a new concept for promoting organic reactions, and is nowadays a well-established heating method for small-scale organic synthesis. Dedicated MW heating instruments for lab scale organic synthesis provide rapid and uniform in situ heating, reduce reaction times and frequently give cleaner reactions with increased reaction yields.104–108,132,133 However, physical restrictions are associated with MW heating as the penetration depth of MWs is commonly only a few centimeters, limiting the ability to scale up reactions.134 Combining MW heating and CF synthesis may offer a way to overcome the scalability issue and still preserve all the advantages observed with MWs compared to conventional heating.135–140 Our laboratory’s purpose-built CF system applies non-resonant MW heating (Figure 17), allowing high reaction temperatures as well as fast temperature adjustments.140,141 The MW-CF system utilizes a HPLC-pump to pump the reaction mixture through a tubular reactor. The reactor is surrounded by a helical antenna, which allows homogenous heating of the reactor without pronounced hot and/or cold spots. The short process times provide fast method development opportunities on a small scale, with subsequent scale out translation without the normal scale up restrictions mentioned previously. The device is compatible with a variety of tubular reactors.

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Figure 17. a) Schematic picture of our microwave heated continuous-flow system. The HPLC-pump pumps the reaction mixture through the system consisting of a non-resonant microwave cavity where the helical MW antenna heats the reaction mixture flowing through the tubular reactor. The backpressure regulator (BPR) ensures a stable pressure profile. After processing, the reaction mixture is cooled and collected in batches or fractions. b) Photograph of the MW-CF system showing the pump (left), the generator with MW applicator on top (middle), and the fraction collector (right). c) Close-up of the MW applicator with the front peace unmounted and a borosilicate glass reactor inside the helical antenna. d) Close-up of two 200 mm × 3 mm tubular reactors types, SiC (black) and borosilicate (transparent).

MW-CF Synthesis of Spiro-oxindoles As mentioned previously, we set out to improve the synthetic procedures to obtain spiro-oxindole dihydroquinazolinones in order to facilitate SAR studies. In parallel to investigating the reaction parameters in batch, the aim was also to transfer the reaction conditions to our MW-CF system to enable fast scale-out of potent inhibitors.

After batch optimization of the three-component reaction as shown in Scheme 2, p. 36 (150 °C, 10 min, 75%), the first MW-flow attempt was then to synthesize 10 using a MW transparent 200 mm × 3 mm (inner diameter) tubular borosilicate glass reactor in our purpose-built MW-CF applicator. We started with employing similar parameters as in the batch synthesis, with all components in a 1:1:1 ratio in AcOH. A flow rate of 1 ml/min (85 s residence time) was selected with a planned process temperature range of 140–220 °C. The temperature screen gave the best result at 200 °C, and collection for 5 min

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provided an isolated yield of 40% (entry 1, Table 12). However, full conversion was not achieved and some starting isatin 33a remained in the processed reaction mixture. Changing the flow rate from 1 mL/min to 0.5 mL/min (169 s residence time) did not result in any improvement (entry 2). Instead, we decided to try a novel kind of reactor, a silicon carbide (SiC) tubular reactor of the same size (200 mm × 3 mm).142 The main difference is that SiC is microwave absorbing, meaning that it mimics conventional heating since the reactor itself will be heated by the microwaves, in contrast to direct heating of the reaction mixture as with borosilicate. However, SiC still shares the extremely fast temperature response observed in MW heated solutions.

Similar to the borosilicate reactor, the reactants were initially applied in 1:1:1 ratio with a flow of 0.5 mL/min (169 s residence time), and temperature ranging from 100–240 °C, with 20 °C stepwise increasing increments, were investigated. The reaction mixtures were analyzed with UV-LC/MS, indicating that a reaction temperature of 120–160 °C was most beneficial. Lower temperature resulted in poor product formation, while temperature above 160 °C afforded increased byproduct formation. The reaction mixture was collected at 120 °C with a flow rate of 0.5 mL/min, affording 2 in 55% yield (entry 4). Lower flow rate (0.25 mL/min, 338 s residence time) resulted in clogging the system and a higher flow rate (1 mL/min, 85 s residence time) resulted in less product formation. In order to provide a more direct comparison between the two reactor materials, the same reaction conditions (1:1:1, 0.5 mL/min, 120 °C) were applied to the borosilicate reactor (entry 3), enabling only 8% isolated yield and poor conversion. Interestingly, the commonly described advantages with in situ MW heating vs. conventional heating, that higher temperatures providing shorter reaction times and cleaner reactions were not observed in this case. In contrast, employing the SiC reactor enabled lower reaction temperatures, less complex reaction mixtures and hence easier purification.

Both in the batch reactions and in the flow synthesis of 10, remaining 33a was observed in the processed reaction mixture. To investigate if this could be avoided, the nucleophilic p-toluidine was used in excess (1.5 equiv), providing 10 in 65% yield (entry 5). Using both p-toluidine and isatoic anhydride in excess (1.2 equiv) furnished 10 in 74% isolated yield (entry 6). Increasing the reaction temperature to 140 °C (entry 7) did not improve product formation, neither did application of 1.5 equivalents of reactants (entry 8). However higher temperature (160 °C) in combination with increased concentration of reactants (1.5 equiv) provided 10 in 82% yield (entry 9), with a residence time of 168 s.

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Table 12. Reaction condition investigation of three-component reaction.

Entry

33a (equiv)

34 (equiv)

35 (equiv)

Flow (mL/min)

Temp (°C)

Reactor Yielda

(%)

1 1 1 1 1 200 Borosilicate 40 2 1 1 1 0.5 200 Borosilicate 43 3 1 1 1 0.5 120 Borosilicate 8 4 1 1 1 0.5 120 SiC 55 5 1 1 1.5 0.5 120 SiC 65 6 1 1.2 1.2 0.5 120 SiC 74 7 1 1.2 1.2 0.5 140 SiC 79 8 1 1.5 1.5 0.5 140 SiC 76

9 1 1.5 1.5 0.5 160 SiC 82 aIsolated yield.

A series of analogues of hit compound 10 was synthesized utilizing the MW-CF set up with the SiC reactor (entry 9, Table 12). A variety of anilines and isatins were used to afford compounds 10, 36–37, 39, 62–65, 67 in 40-87% yield (Scheme 8).

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Scheme 8. MW-CF synthesis of spiro-oxindole dihydroquinazolinones.

Additionally, the Suzuki-Miyaura cross coupling reaction was also transferred to the flow system. The stoichiometric reaction conditions developed in batch were applied in the flow system, using p-tolylboronic acid (134) as a model substrate. The borosilicate glass reactor was used. A similar temperature scan as previously was performed, with temperatures ranging from 160–220 °C, with 20 °C increasing increments with a flow rate of 0.5 ml/min (169 s residence time). The highest temperature gave full conversion of 10. In the next step, the flow rate was investigated, where the product formation was analyzed at 0.75 (113 s), 1.0 (85 s), 1.5 (56 s), and 2.0 (42 s) mL/min, respectively, but increased flow rate did not provide full conversion of 10. Hence, product mixture was collected for 5 min with a flow rate of 0.5 mL/min at 220 °C, which furnished 53 in 71% yield with a residence time of 168 s (Scheme 9).

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Scheme 9. MW-heated continuous flow Suzuki-Miyaura cross coupling reaction to afford compound 53.

In summary, apart from being synthesized with standard batch chemistry, we have also shown that these compounds can be synthesized via MW-CF procedures. The MW-flow set up is applicable to both the three-component reaction and the Suzuki-Miyaura cross coupling, providing possibilities for easy scale-out when there is a demand for higher amount of compounds.

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Concluding Remarks

This thesis outlines the identification of small molecule based IRAP inhibitors by means of a screening campaign, followed by the synthesis and evaluation of two hit compound series. The most important findings are presented below.

A simple and robust assay has been developed from an existing assay procedure. It is amenable to either automated or manual liquid handling, is performed at room temperature, stable for more than 24 hours, and was applied to screening 10,500 compounds. The same assay procedure can be used for evaluation of inhibitors when employing enzymes from various sources. The assay can also be used to measure selectivity towards related enzymes and to the investigation of the mechanism of action of any identified inhibitors.

The screening campaign identified 166 hits, which after thorough hit confirmation experiments were narrowed down to three structurally different hit compounds that were of particular interest; 10, 13 and 15.

Synthetic procedures to generate spiro-oxindole dihydroquinazolinone based inhibitors in a fast and simple fashion have been developed, either by using microwave-promoted batch reactions, or by employing a microwave-heated continuous flow approach. The scaffold could also be further modified by a microwave-promoted Suzuki-Miyaura cross coupling reaction with only one minute reaction times.

The spiro-oxindole dihydroquinazolinone based compound series is one of few published non-peptidic IRAP inhibitors with sub-µM affinity. They are also selective over the closely related enzyme APN.

Computational modelling suggests that the S-configuration accounts for the inhibitory effect within the series. The compounds exhibit uncompetitive inhibition, and the proposed binding mode from docking studies and molecular dynamic simulations suggest the possibility of

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simultaneous binding of the inhibitor and substrate L-Leu-para-nitroanilide to the IRAP active site.

Physicochemical properties such as solubility as well as metabolic stability were in general poor for the entire compound series.

Synthetic procedures to generate imidazo[1,5-α]pyridine based inhibitors have been developed and/or modified from existing procedures, providing compounds with low µM affinities.

The diastereomers of the most potent compound (13) were separated using SFC and evaluated individually.

The compound series exhibit non-competitive inhibition with the substrate L-Leucine-para-nitroanilide.

Physicochemical properties such as solubility as well as metabolic stability were unfortunately poor for the entire compound series.

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Acknowledgement

I would like to express my sincere gratitude to the following people who have, in one way or another, contributed to the work presented in this thesis.

First of all, my supervisors. Prof. Mats Larhed, for allowing me to conduct my PhD studies in your research group, for having confidence in me and allowing me to work independently, and for encouraging and supporting me along this journey. Dr. Thomas Lundbäck, I cannot thank you enough. Your support during this project has been invaluable, and I would never have managed without you. Thank you for taking me on this “assay-journey” and for your endless patience and support. Dr. Ulrika Rosenström, for your guidance and support, both science and non-science related, and for always picking me up when I am down. Additionally, thank you for having confidence in me regarding all the teaching related matters. Dr. Johan Gising, for sharing all your knowledge in medicinal chemistry, for your wise comments, and for not allowing me to take failures personally.

The work within this thesis is definitely not the work of a single person. I have had the privilege to work with many talented and hard-working researchers along the way. My deepest appreciation to all my collaborators, thank you for all the hard work and for sharing your vast knowledge. I am thankful to Dr. Anna-Lena Gustavsson and Dr. Annika Jenmalm Jensen for welcoming me to your lab in Stockholm, for your compliance, and for always being supportive. I am truly grateful for the warm hospitality of your research group; such an inspiring place to work at! Special thanks to Hanna Axelsson and Helena Almqvist for taking time to answer all my questions, helping me out with practical matters, and for nice chit-chat during lunch breaks. Hanna, you will forever be the person who taught me how to use a multi-pipette Leif Dahllund, for providing the CHO-cells as well as the various human enzymes, and for allowing me to perform membrane preparations in your lab. Dr. Jonas Brånalt, for compound registration, and the Wave1 team at AstraZeneca, for the evaluation of my compounds. The group of Prof. Mathias Hallberg, for fruitful discussions and collaborations within the IRAP project. Prof. Georges Vauquelin and Dr. Alexandros Nikolaou for the valuable input in the screening assay development. Assoc. Prof. Hugo Gutiérrez de Terán and Dr. Sudarsana Reddy Vanga for your computational contributions to the spiro-project. Dr. Johan Wannberg and Dr. Jonas Sävmarker for helping out with the continuous-flow synthesis and the method development. Prof. Máté

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Erdélyi for always being positive and encouraging, and for your assistance with the RDC-experiments.

Prof.em. Anders Hallberg for sharing your vast knowledge of medicinal chemistry in general, and the IRAP project in particular. I have learnt a lot!

Former and present members at the Department of Medicinal Chemistry, for making this place such a nice environment to work in. A have truly appreciated the everyday laughter’s and discussions during coffee and lunch breaks. A big shout-out to everyone I have spent time with travelling to conferences in Sweden and around the world. Experience excellent research in combination with exploring new places with great company have been a blast! Fanny Lundmark, Gustav Olanders, and Dr. Anna Karin Belfrage, for all the support during these last, intense weeks. Thanks for always cheering me up! Last but certainly not least, the administrative personnel. Thank you for all the non-research related work. Special thanks to Birgitta Hellsing for stepping in when life turned upside down that late September evening.

I would also like to express my gratitude to my project workers Faith Agalo and Camilo Persson, for the hard work in the lab. I know that the spiro-compounds were not the easiest to work with, but you never gave up!

Dr. Charles Hedgecock, Dr. Rebecka Isaksson, Dr. Nadia Nasser Petersen and Dr. Jonas Rydfjord, for critical revision on this thesis. Any remaining mistakes or inaccuracies are without doubt my own last minute changes.

I must express my deep gratitude to the Faculty of Pharmacy, Uppsala University, SciLifeLab Drug Discovery and Development, King Gustav V:s and Queen Victoria’s Foundation of Freemasons, Anna Maria Lundins stipendiefond, Apotekare C D Carlsson stiftelse, and IF Stiftelse för Farmaceutisk Forskning for financial support, and without whom my time at the department or participation in conferences would not have been possible.

Sist men absolut inte minst, min älskade familj. Tack för att ni alltid finns där och stöttar och hjälper till med allt vad ni kan, och för att ni får mig att tänka på annat emellanåt. Mamma och pappa, tack för allt ni gör för mig, men framför allt för barnen! Alexander, min trygga punkt i livet. Tack för att du är den du är, för att du är en fantastisk pappa till våra barn, och för att du skämmer bort mig när jag minst anar det. Signe, min älskade prinsessa. Tack för allt du förgyller våra liv med. William, din ankomst hit till världen, redan i vecka 27, vände verkligen upp och ner på tillvaron för oss alla. Tack för att du valde livet, och för att du visat vad som verkligen är viktigt och betydelsefullt med vår tid här på jorden. För dig och din syster går jag genom eld och vatten.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 285

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

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