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Design and Synthesis of Aspartic and Serine Protease Inhibitors: Targeting the BACE-1 and the HCV NS3 Protease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. (avd. f. organisk farmaceutisk kemi)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes work done to design and synthesize protease inhibitors, with the intention of developing therapeutic agents for Alzheimer’s disease (AD) and the chronic liver condition caused by infection of the hepatitis C virus (HCV). AD is the most common form of dementia, and HCV infection is the primary reason for liver transplantation in industrialized countries. Today, these two illnesses affect 24 and 170 million people, respectively. It has been shown that the human aspartic protease BACE-1 plays an important role in the development of AD, and thus inhibition of BACE-1 may offer a way to improve the quality of life of individuals afflicted with the disease. Furthermore, it is known that the serine protease NS3 is a vital component in the replication of HCV.

Several novel potent BACE-1 inhibitors encompassing different transition state mimics were prepared. First, a hydroxyethylene moiety encompassing a secondary hydroxyl group was evaluated as a transition state analogue, producing inhibitors in the low nanomolar range. Various tertiary hydroxyl isosteres were also investigated as the central core, with the aim of shielding the pivotal hydroxyl group. These transition state isosteres consisted of tertiary hydroxyl analogues of previously used secondary hydroxyl containing norstatine, statine, and hydroxyethylamine isosteres. Several tertiary hydroxyl-containing inhibitors were found to be active in the low micromolar range. In addition, two inhibitors were co-crystallized with the BACE-1 enzyme to provide X-ray crystal structures, which furnished valuable binding information for further design of improved BACE-1 inhibitors.

The goal in the HCV NS3 protease inhibitor project was to design, synthesize and evaluate a novel hydroxycyclopentene bioisostere to the previously used acyl-hydroxyproline moiety. The investigation revealed that it was possible to synthesize inhibitors containing this new bioisostere that were potent in the low nanomolar range. Further optimization by rigidification of the most active inhibitor resulted in equipotent macrocyclic compounds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2009. , p. 71
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 108
Keywords [en]
Alzheimer's disease, BACE-1, transition state mimetic, tertiary hydroxyl group, hydroxyethylene, statine, hydroxyethylamine, hepatitis C, HCV NS3, bioisostere, protease inhibitor.
National Category
Medicinal Chemistry
Research subject
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-108985ISBN: 978-91-554-7623-6 (print)OAI: oai:DiVA.org:uu-108985DiVA, id: diva2:242293
Public defence
2009-11-20, B42, Biomedical Centre (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-10-29 Created: 2009-10-06 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Synthesis of potent BACE-1 inhibitors incorporating a hydroxyethylene isostere as central core
Open this publication in new window or tab >>Synthesis of potent BACE-1 inhibitors incorporating a hydroxyethylene isostere as central core
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2010 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 45, no 3, p. 870-882Article in journal (Refereed) Published
Abstract [en]

We herein describe the design and synthesis of a series of BACE-1 inhibitors incorporating a P1-substituted hydroxylethylene transition state isostere. The synthetic route starting from commercially available carbohydrates yielded a pivotal lactone intermediate with excellent stereochemical control which subsequently could be diversified at the P1-position. The final inhibitors were optimized using three different amines to provide the residues in the P2′-P3′ position and three different acids affording the residues in the P2-P3 position. In addition we report on the stereochemical preference of the P1′-methyl substituent in the synthesized inhibitors. All inhibitors were evaluated in an in vitro BACE-1 assay where the most potent inhibitor, 34-(R), exhibited a BACE-1 IC50 value of 3.1 nM.

Keywords
Alzheimer's disease, BACE-1 inhibitors, Hydroxylethylene, Transition state isostere
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-109028 (URN)10.1016/j.ejmech.2009.11.013 (DOI)000275404900003 ()20036448 (PubMedID)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Design and synthesis of BACE-1 inhibitors utilizing a tertiary hydroxyl motif as the transition state mimic
Open this publication in new window or tab >>Design and synthesis of BACE-1 inhibitors utilizing a tertiary hydroxyl motif as the transition state mimic
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2009 (English)In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 19, no 16, p. 4711-4714Article in journal (Refereed) Published
Abstract [en]

Two series of drug-like BACE-1 inhibitors with a shielded tertiary   hydroxyl as transition state isostere have been synthesized. The most   potent inhibitor exhibited a BACE-1 IC50 value of 0.23 mu M.

Place, publisher, year, edition, pages
Elsevier, 2009
Keywords
Alzheimer's disease, BACE-1, Enzyme inhibitor, Transition state mimetic
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-109025 (URN)10.1016/j.bmcl.2009.06.065 (DOI)000268358800039 ()
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2018-01-13Bibliographically approved
3. Investigation of alpha-phenylnorstatine and alpha-benzylnorstatine as transition state isostere motifs in the search for new BACE-1 inhibitors
Open this publication in new window or tab >>Investigation of alpha-phenylnorstatine and alpha-benzylnorstatine as transition state isostere motifs in the search for new BACE-1 inhibitors
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2011 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 19, no 1, p. 145-155Article in journal (Refereed) Published
Abstract [en]

Inhibition of the BACE-1 protease enzyme has over the recent decade developed into a promising drug strategy for Alzheimer therapy. In this report, more than 20 new BACE-1 protease inhibitors based on α-phenylnorstatine, α-benzylnorstatine, iso-serine, and β-alanine moieties have been prepared. The inhibitors were synthesized by applying Fmoc solid phase methodology and evaluated for their inhibitory properties. The most potent inhibitor, tert-alcohol containing (R)-12 (IC50 = 0.19 μM) was co-crystallized in the active site of the BACE-1 protease, furnishing a novel binding mode in which the N-terminal amine makes a hydrogen bond to one of the catalytic aspartic acids.

Keywords
α-Benzylnorstatine, α-Phenylnorstatine, Alzheimer's disease, BACE-1 inhibitors, tert-Hydroxyl, Transition state mimic
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-109026 (URN)10.1016/j.bmc.2010.11.042 (DOI)000285724800014 ()21183353 (PubMedID)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2018-01-13Bibliographically approved
4. Synthesis and evaluation of a new class of tertiary alcohol based BACE-1 inhibitors
Open this publication in new window or tab >>Synthesis and evaluation of a new class of tertiary alcohol based BACE-1 inhibitors
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2009 (English)In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 65, no 48, p. 10047-10059Article in journal (Refereed) Published
Abstract [en]

BACE-1 has emerged as one of the best characterized targets for future   Alzheimer therapy. In accordance with the successful identification of   masked inhibitors of HIV-1 protease, we envisioned that tert-alcohol   containing transition-state mimicking structures would also be   worthwhile evaluating as BACE-1 inhibitors. Twelve novel inhibitors   were prepared via synthetic routes using epoxyalcohol derivates as key   intermediates. The best synthesized tert-hydroxy inhibitor exhibited a   BACE-1 IC50 value of 0.38 mu M.

Keywords
Alzheimer's disease, BACE-1 inhibitors, Transitions state isostere
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-109024 (URN)10.1016/j.tet.2009.09.106 (DOI)000271841700018 ()
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2018-01-13Bibliographically approved
5. Synthesis of novel potent hepatitis C virus NS3 protease inhibitors: discovery of 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a N-acyl-L-hydroxyproline bioisostere
Open this publication in new window or tab >>Synthesis of novel potent hepatitis C virus NS3 protease inhibitors: discovery of 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a N-acyl-L-hydroxyproline bioisostere
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2007 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 15, no 2, p. 827-838Article in journal (Refereed) Published
Abstract [en]

Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed incorporating 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a new N-acyl-l-hydroxyproline mimic. The hydroxycyclopentene template was synthesized in eight steps from commercially available (syn)-tetrahydrophthalic anhydride. Three different amino acids were explored in the P1-position and in the P2-position the hydroxyl group of the cyclopentene template was substituted with 7-methoxy-2-phenyl-quinolin-4-ol. The P3/P4-positions were then optimized from a set of six amino acid derivatives. All inhibitors were evaluated in an in vitro assay using the full-length NS3 protease. Several potent inhibitors were identified, the most promising exhibiting a Ki value of 1.1 nM.

Keywords
Cyclopentanes/*chemical synthesis/*pharmacology, Dicarboxylic Acids/*chemical synthesis/*pharmacology, Indicators and Reagents, Kinetics, Magnetic Resonance Spectroscopy, Models; Molecular, Protease Inhibitors/*chemical synthesis/*pharmacology, Stereoisomerism, Structure-Activity Relationship, Viral Nonstructural Proteins/*antagonists & inhibitors
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-17096 (URN)10.1016/j.bmc.2006.10.044 (DOI)000243315300021 ()17107807 (PubMedID)
Available from: 2008-06-16 Created: 2008-06-16 Last updated: 2018-01-12Bibliographically approved
6. Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: use of cyclopentane and cyclopentene P2-motifs
Open this publication in new window or tab >>Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: use of cyclopentane and cyclopentene P2-motifs
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2007 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 15, no 22, p. 7184-7202Article in journal (Refereed) Published
Abstract [en]

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a Ki value of 0.41 nM and an EC50 value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.

Keywords
Cyclopentane and Cyclopentene derived P2 templates, HCV, Macrocyclic inhibitors, NS3 protease
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-17095 (URN)10.1016/j.bmc.2007.07.027 (DOI)000250324900030 ()17845856 (PubMedID)
Available from: 2008-06-16 Created: 2008-06-16 Last updated: 2018-01-12Bibliographically approved

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