Prolyl Oligopeptidase (POP) Inhibitors: Structure, Activity, and Applications, Thesis of Chemistry

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ELINA JARHO

Synthesis, Structure-Activity

Relationships and Physico-Chemical

Properties of Novel Prolyl

Oligopeptidase Inhibitors

KUOPIO 2007^ JOKA

KUOPION YLIOPISTON JULKAISUJA A. FARMASEUTTISET TIETEET 98 KUOPIO UNIVERSITY PUBLICATIONS A. PHARMACEUTICAL SCIENCES 98

Doctoral dissertation

To be presented by permission of the Faculty of Pharmacy of the University of Kuopio for public examination in Auditorium, Mediteknia building, University of Kuopio, on Saturday 24th^ February 2007, at 12 noon

Department of Pharmaceutical Chemistry Faculty of Pharmacy University of Kuopio

Distributor : Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 17 163 430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html

Series Editor : Docent Pekka Jarho, Ph.D. Department of Pharmaceutical Chemistry

Author’s address: Department of Pharmaceutical Chemistry University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 17 162 460 Fax +358 17 162 456 E-mail: Elina.Jarho@uku.fi

Supervisors: Erik Wallén, Ph.D. Division of Pharmaceutical Chemistry University of Helsinki Johannes A. M. Christiaans, Ph.D. Altana Pharma bv The Netherlands Professor Jouko Vepsäläinen, Ph.D. Department of Chemistry University of Kuopio

Reviewers: Professor Koen Augustyns, Ph.D. Department of Medicinal Chemistry University of Antwerp Belgium Professor Jari Yli-Kauhaluoma, Ph.D. Division of Pharmaceutical Chemistry University of Helsinki

Opponent: Professor Kristina Luthman, Ph.D. Medicinal Chemistry, Department of Chemistry Göteborg University

ISBN 978-951-27-0416-

ISBN 978-951-27-0628-0 (PDF)

ISSN 1235-

Kopijyvä Kuopio 2007 Finland

ACKNOWLEDGEMENTS

The present study was carried out in the Department of Pharmaceutical Chemistry, University of Kuopio during the years 2000-2007. The study was financially supported by the National Technology Agency in Finland, Orion Pharma Oy, the Graduate School in Pharmaceutical Research, the Finnish Cultural Foundation of Northern Savo, the Association of Finnish Pharmacies, the Research and Science Foundation of Farmos and The Finnish Cultural Foundation. I owe my gratitude to all my former and current supervisors Dr. Jukka Gynther, Dr. Juhani Huuskonen, Dr. Hans Christiaans, Prof. Jouko Vepsäläinen and Dr. Erik Wallén. Especially I owe my gratitude to Dr. Erik Wallén who has been my principal supervisor. Thank you for your guidance and friendship during the past years. We have been two quite different characters working together but our collaboration has been very fruitful and I hope that we can continue it in some form. Many other people have also given a crucial contribution to this work and I want to thank all my co-authors: Dr. Jarkko Venäläinen, Dr. Markus Forsberg, Prof. Pekka Männistö, Prof. Antti Poso, Dr. Arturo Garcia-Horsman, Dr. Juha Juntunen, Prof. Tomi Järvinen, M.Sc. Leena Yli-Kokko, M.Sc. Sami Poutiainen and B.Sc. Harri Leskinen. Especially I'm grateful to Dr. Jarkko Venäläinen who has provided data for all the publications, which are included in this thesis. I also owe my gratitude to our laboratory assistants Ms. Tiina Koivunen (who has a very efficient pair of hands and has given a lot of help to me), Ms. Helly Rissanen, Ms. Miia Reponen and Ms. Katja Hötti for their outstanding technical assistance and friendship. I am also grateful to Ms. Jaana Leskinen and Ms. Päivi Sutinen for their skillful technical assistance in the inhibitory activity determinations and Ms. Maritta Salminkoski for the work on certain reactions during my maternity leave. I also want thank Dr. Jouni Sirviö for the collaboration during the past years. The facilities to perform this work have been excellent and I want to thank Dr. Jukka Gynther and Prof. Jukka Mönkkönen, the former and the current deans of the Faculty of Pharmacy, and Prof. Tomi Järvinen and Prof. Antti Poso, the former and the current heads of the Department of the Pharmaceutical Chemistry. The facilities have been excellent but so has also been the atmosphere, and I want to thank all the members of the Pharmaceutical and Medicinal Chemistry Group and other colleagues in the Department of the Pharmaceutical Chemistry. Special thanks to the office personnel for your help with all the bureaucracy.

ABBREVIATIONS

AD Alzheimer's disease ADMET absorption, distribution, metabolism, excretion and toxicity AVP arginine-vasopressin BBB blood-brain barrier ClogP calculated logarithm of the partition coefficient CNS central nervous system CoMSIA comparative molecular similarity indices analysis 3D QSAR three dimensional quantitative structure-activity relationship DCC dicyclohexylcarbodiimide DCM dichloromethane DMAP 4-dimethylaminopyridine DMP Dess-Martin periodinane DMSO dimethyl sulfoxide DPP IV dipeptidyl-peptidase IV EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EI enzyme-inhibitor ESI-MS electrospray ionization mass spectrometry FID free-induction decay GAPDH glyceraldehyde-3-phosphate dehydrogenase GOF goodness-of-fit HOBt 1-hydroxybenzotriazole HPLC high performance liquid chromatography IC 50 inhibitor concentration at which the enzyme reaction velocity is 50% of the uninhibited reaction IP 2 inositol-4,5-bisphosphate IP 3 inositol-1,4,5-trisphosphate IP 5 inositol-1,3,4,5,6-pentakisphosphate ISA ionic strength adjusted Ki inhibition constant log P logarithm of the partition coefficient MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MW molecular weight m/z mass-to-charge ratio NCBI National Center for Biotechnology Information NMR nuclear magnetic resonance (spectroscopy)

P1...Pn amino acid residues of substrates (or the corresponding structures of substrate-like inhibitors) which bind to the S1...Sn binding sites of the enzyme counting from the scissile bond towards the N-terminus P1'...Pn' amino acid residues of substrates (or the corresponding structures of substrate-like inhibitors) which bind to the S1'...Sn' binding sites of the enzyme counting from the scissile bond towards the C-terminus PDB protein data bank PE petroleum ether PI phosphatidylinositol PIP 2 phosphatidylinositol-4,5-bisphosphate pKa negative logarithm of the ionization constant PLC phospholipase C POP prolyl oligopeptidase ppm parts per million rt room temperature S1...Sn binding sites of the enzyme for the P1...Pn residues of the substrates or the substrate-like inhibitors S1'...Sn' binding sites of the enzyme for the P1'...Pn' residues of the substrates or the substrate-like inhibitors SAR structure-activity relationship SP substance P Suc-Gly-Pro-AMC N -succinyl-glycyl-L-prolyl-7-amino-4-methylcoumarin Tc80 80 kDa protein of Trypanosoma cruzi TFA trifluoroacetic acid THF tetrahydrofuran TMS tetramethylsilane TRH thyroliberin, thyrotropin-releasing hormone UV ultraviolet Z benzyloxycarbonyl Z-Gly-Pro-AMC N -benzyloxycarbonyl-glycyl-L-prolyl-7-amino-4- methylcoumarin ZIP Z-Pro-prolinal insensitive peptidase

CONTENTS

5 DICARBOXYLIC ACID AZACYCLE L-PROLYL-PYRROLIDINE

AMIDES AS PROLYL OLIGOPEPTIDASE INHIBITORS AND

1 INTRODUCTION

Prolyl oligopeptidase (POP) is a proline-specific peptidase that was found in the beginning of the 1970’s (Walter et al. 1971). After that, the enzyme has been well- characterized and the crystal structure was published in 1998 (Fülöp et al. 1998). Several studies with specific POP inhibitors in different animal models have associated POP with memory impairments and cognitive disturbances. POP has also been implicated in mood disorders, Chagas disease and celiac disease. However, the full biological role and the different mechanisms of actions still need further studies. Some POP inhibitors have entered clinical studies but none of them has entered the market. Thus, the suggested indication(s) for the prolyl oligopeptidase inhibitors needs to be confirmed. At the starting point of this work, a lot was known about the structure-activity relationships (SAR) of POP inhibitors. Many potent inhibitors had been published in the literature and some had also been developed by our own group. The best inhibitors had subnanomolar IC 50 values and the focus was shifted to other issues. The aim was to introduce replacements in the typical POP inhibitor backbone that would make the molecular structure more drug-like and could be used to modify the physico-chemical properties of the inhibitors. The replacements were planned to mimic the replaced group in the POP inhibitor structure in order to maintain the POP inhibitory activity. The first part of the dissertation is a literature review that summarizes the current knowledge of POP and its biological relevance. The amino acid proline and the properties of drug-like compounds are also briefly discussed. A more detailed summary is given about the SAR of POP inhibitors. After the literature review, each of the four articles that are included in this dissertation has its own chapter. The first and the third of the articles deal with the modifications of POP inhibitors at the P3 position and beyond it, where the structural variation is explored. The second and the fourth of the articles deal with the replacement of the P3-P2 and P2-P1 amide groups.

2 LITERATURE REVIEW

2.1 Nomenclature and classification

Prolyl oligopeptidase (EC 3.4.21.26) is classified as a serine endopeptidase. The name and the classification describe the ability of POP to degrade oligopeptides after internal prolyl residues. POP is a member of peptidase family S9, which belongs to clan C of serine peptidases. Peptidase family S9 is often referred to as prolyl oligopeptidase family and other representative members of this family are oligopeptidase B (EC 3.4.21.83), dipeptidyl-peptidase IV (DPP IV, EC 3.4.14.5) and acylaminoacyl-peptidase (EC 3.4.19.1). POP is also known as prolyl endopeptidase and post-proline cleaving enzyme in older literature (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology 2006, Rawlings et al. 2006).

2.2 Distribution of prolyl oligopeptidase

POP is a highly conserved enzyme that is widely distributed. It has been found in all three domains of life; Archaea, Bacteria and Eukarya. Among Eukarya, the sequences of POP have been resolved for many animals and plants (Venäläinen et al. 2004a). Among fungi, the existence of POP has been unclear. A similar enzyme to POP has been purified from a fungus Agaricus bisporus (Yoshimoto et al. 1990). Recently, sequences from fungi with significant alignments with human POP could also be retrieved from the NCBI protein database (National Center for Biotechnology Information 2006). When over 20 human tissues were studied, POP activity was found in all of them (Kato et al. 1980). The highest activity was found in the skeletal muscle. In the brain, the highest enzyme activity was found in the frontal cortex where the activity was one sixth of that in the skeletal muscle. In another study, the highest POP activity in the human brain was found in the cortices (Irazusta et al. 2002). In rat, which is often used as the model animal in different performance tasks, the highest POP activity was found in the brain and the activity was distributed more homogenously throughout the brain. POP has mainly been described as a cytosolic enzyme but a membrane bound form has also been reported (O'Leary et al. 1996). The membrane bound form was purified from the bovine brain and it had many characteristics similar to the cytosolic POP although some differences were also found. However, the sequence of this enzyme has not been resolved. When 12 eukaryotic POP sequences were studied, transmembraneous regions or lipid anchors that would indicate about the membrane

Table 1. The structures and the potencies of POP inhibitors, which have undergone in vivo studies.

Compound Name Structure Inhibitory activitySource of POP Reference

(^1) 4819 JTP- N N O O

OH O

NH^

IC 50 = 0.83 nM rat brain

Toide et al. 1995

2 Y-29794 (^) N S O

S

N Ki^ = 0.95 nM rat brain

Nakajima et al. 1992

3 S 17092 (^) N N S O O

H H

Ki = 1.5 nM human

Barelli et al. 1999

(^4) prolinalZ-Pro- N N O O

H O

O

Ki = 0.5 nM human brain

Bakker et al. 1990

5 ZTTA (^) N

S N

S

O O

O O

O

Ki = 2.9 μM rat brain

Shishido et al. 1996

6 Z-321 N N

S

O O

IC 50 = 10 nM canine brain

Tanaka et al. 1994

7 ONO- 1603 N N H (^) O

O O

H Cl

Ki = 12 nM rat brain

Katsube et al. 1999

8 KYP- 2047 N N O O

CN

Ki = 0.023 nM porcine brain

Venäläinen et al. 2006

humans during the phase I study (Morain et al. 2000). ZTTA 5 improved performance of rats after cerebral ischemia (Shishido et al. 1996) and basal forebrain lesion (Shishido et al. 1998). Z-Pro-prolinal 4 prevented scopolamine-induced amnesia in rats (Yoshimoto et al .1987) and KYP-2047 8 improved performance of young but not old scopolamine-treated rats (Jalkanen et al. 2006). Taken together, several studies suggest that POP inhibitors might be beneficial in the treatment of age-related memory disorders or neurodegenerative diseases, like

Alzheimer's disease. Indeed, four POP inhibitors, JTP-4819 1 , S 17092 3 , Z-321 6 and ONO-1603 7 , have entered clinical trials (Umemura et al. 1997, Katsube et al. 1999, Umemura et al. 1999, Morain et al. 2000). However, their mechanism(s) of action is not fully understood, i.e. the secondary effects of POP inhibition are not known for sure. At the moment, there are two main theories for the memory-improving effects of POP inhibitors: elevation of the neuropeptide levels in the central nervous system (CNS) and elevation of the intracellular inositol-1,4,5-trisphosphate (IP 3 ) level. Certain neuropeptides, like substance P (SP), arginine-vasopressin (AVP) and thyroliberin (TRH) (Figure 1), are in vitro substrates of POP (Knisatschek and Bauer 1979, Blumberg et al. 1980, Yoshimoto et al. 1981). These neuropeptides have been shown to improve learning and memory (Griffiths 1987, Kovacs and De Wied 1994, Huston and Hasenöhrl 1995). Consequently, it was concluded that inhibition of POP might increase concentrations of these peptides in the CNS and thus, counteract memory disturbances. Several studies have been performed to verify this theory. A 21- day treatment with JTP-4819 1 increased the SP and the TRH level in the cortex and the hippocampus of aged rats, while the AVP level was unaffected (Toide et al. 1997). In humans, a 7-day treatment with JTP-4819 1 did not affect the plasma concentration of SP, although a small increase was seen after a single dose (Umemura et al. 1997). A single dose of S 17092 3 increased the SP level in the frontal cortex and the hypothalamus of rats while an 8-day treatment did not have any effect (Bellemère et al. 2003). Also the TRH level in the cerebral cortex and the AVP level in the hippocampus of rats were increased after the single dose of S 17092 3 but only the TRH level in the cerebral cortex was increased after the 8-day treatment (Bellemère et al. 2005). A single dose of Z-321 6 or Z-Pro-prolinal 4 increased the AVP level in the septum of rats (Miura et al. 1995). Instead, a 7-day treatment with Z-321 6 did not affect the plasma levels of SP, TRH or AVP in humans (Umemura et al. 1999). A single dose or a 10-day treatment with KYP-2047 8 or JTP-4819 1 did not affect the SP level in the cortex, hippocampus or hypothalamus of rats (Jalkanen et al. 2006). The single dose of

(a) Arg-Pro-Lys- Pro-Gln -Gln-Phe-Phe-Gly-Leu-Met (b) Cys-Tyr-Phe-Gln-Asn-Cys- Pro-Arg -Gly

(c) (pyro)Glu-His- Pro-NH 2

Figure 1. The sequences of three neuropeptide-substrates of POP. The scissile bonds are shown in bold. (a) Substance P; (b) Arginine-vasopressin; (c) Thyroliberin.

still unknown. However, the increase of the IP 3 concentration was delayed after the inhibition of POP and it was suggested that the proteolytic activity of POP is behind this phenomenon, although IP 5 is not a substrate of POP (Schulz et al. 2002). Furthermore, Z-Pro-prolinal 4 (Table 1) prevented the translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the nucleus of monkey fibroblasts, which had been exposed to 6-hydroxydopamine (Puttonen et al. 2006) and ONO-1603 7 (Table 1) suppressed the overexpression of GAPDH in cultured neurons (Katsube et al. 1999). The GAPDH translocation leads to apoptosis in some circumstances and is dependent on PI-3-kinase, which is a phosphorylating enzyme in the PI pathway. In addition, Z-Pro-prolinal 4 prevented the 6-hydroxydopamine-induced formation of reactive oxygen species in monkey fibroblasts (Puttonen et al. 2006). However, the IP 3 concentration in the hippocampus or the cortex of rats was not elevated in vivo after treatment with JTP-4819 1. Recently, POP was also found to be closely associated with tubulin, and was proposed to have a role in protein trafficking and secretion (Schulz et al. 2005). Indeed, inhibition of POP and antisense mRNA expression of POP both increased protein secretion, the latter having more evident effect. This led to the conclusion, that the effect of POP on protein secretion may be independent of its peptidase function.

2.3.2 Psychiatric disorders

Altered serum POP activity has been reported in many psychiatric disorders. Lowered serum POP activity was found in anorexia nervosa and bulimia nervosa patients (Maes et al. 2001), as well as in unmedicated depressed patients as compared to normal volunteers. In the depressed patients, antidepressants increased POP activity. Increased serum POP activity was found in patients with bipolar disorder (manic) who had been deprived from any medication for at least seven days, but short valproate treatment decreased activity. Increased serum POP activity was also found in schizophrenic patients (Maes et al. 1995) and in persons with stress-induced anxiety (Maes et al. 1998). It is somewhat unclear how the serum POP activity correlates with the POP activity in the brain and whether the altered POP activity is the cause or the consequence of the disorders. However, the criticism towards these results has mainly originated from the substrate, N -Z-Gly-Pro-7-amino-4-methylcoumarin, which was used to determine the serum POP activity in these studies. It has been shown that two distinct enzymes are responsible for the N -Z-Gly-Pro-7-amino-4-methylcoumarin-hydrolyzing activity in the bovine serum. One enzyme is prolyl oligopeptidase and the other was

originally named as Z-Pro-prolinal insensitive peptidase (ZIP), because it could not be inhibited by the specific POP inhibitor Z-Pro-prolinal 4 (Cunningham and O'Connor 1997). ZIP was later identified as a seprase with the properties of a proline specific serine protease (Collins et al. 2004). However, when an assay that distinguishes between POP and ZIP activities was used, it was found that the activities of the both enzymes were lowered in the serum of bipolar disorder patients that were receiving lithium-treatment and had euthymic mood. On the other hand, no changes were observed in the POP or ZIP activity in the serum of schizophrenic patients (Breen et al. 2004). Interestingly, valproate decreases POP activity in manic patients (Maes et al. 1995). The effect of valproate on the phosphatidylinositol pathway has been studied and a dual action was proposed: valproate directly inhibits POP and thus increases PI signalling but in other conditions it inhibits PI signalling by an unknown mechanism. This theory of dual action was used to explain why valproate can prevent swings to both mania and depression. It was suggested that POP inhibitors could be used in the treatment of bipolar disorders. However, it is contradictory that PI signalling is high in manic persons and a decrease in POP activity would still increase it (Cheng et al. 2005).

2.3.3 Other disorders

Trypanosoma cruzi is a parasitic protozoan that causes Chagas disease, a disease that is prevalent in Latin America. In 1997, T. cruzi was found to secrete an 80 kDa protein that was able to degrade collagen and had a high specificity for proline residues. The enzyme was mainly secreted by trypomastigotes, the infective form of T. cruzi. It was suggested that this enzyme might participate in the host-cell infection by degradation of the extracellular collagen matrix (Santana et al. 1997). Later, this enzyme was verified to be the POP of T. cruzi (POP Tc80), even though it was able to degrade proteins in contrast to mammalian POP (see section 2.4.2). The POP Tc80 had a 43% identity with the porcine POP. The inhibitors of the POP Tc80 prevented the actual invasion phase of trypomastigotes into host-cells but not the parasite attachment to the host-cell membrane (Bastos et al. 2005). These results suggest that the inhibitors of the POP Tc80 have potential as antiprotozoal drugs. Celiac disease is an autoimmune disease that is caused by the ingestion of gluten proteins, gliadins and glutenins. Celiac disease leads to the destruction of the small intestine villi and the only effective therapy at the moment is a gluten-free diet. When α-gliadin was exposed to digestive enzymes, a proline-rich peptide was one of the products (Shan et al. 2002). This peptide was resistant to further hydrolyzation and it