Novel Monogalactoside Galectin Inhibitor Scaffolds Guiding Selectivity with Heteroaromatic Interactions Dahlqvist, Alexander

Novel monogalactoside galectin inhibitor scaffolds Guiding selectivity with heteroaromatic interactions Dahlqvist, Alexander

2019

Document Version: Publisher's PDF, also known as Version of record Link to publication

Citation for published version (APA): Dahlqvist, A. (2019). Novel monogalactoside galectin inhibitor scaffolds: Guiding selectivity with heteroaromatic interactions. (1 ed.). Lund University, Faculty of Science.

Total number of authors: 1

Creative Commons License: CC BY-NC-ND

General rights Unless other specific re-use rights are stated the following general rights apply: Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal

Read more about Creative commons licenses: https://creativecommons.org/licenses/ Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

LUND UNIVERSITY

PO Box 117 221 00 Lund +46 46-222 00 00 Novel monogalactoside galectin inhibitor scaffolds Guiding selectivity with heteroaromatic interactions

ALEXANDER DAHLQVIST FACULTY OF SCIENCE | LUND UNIVERSITY

Novel monogalactoside galectin inhibitor scaffolds

Guiding selectivity with heteroaromatic interactions

Alexander Dahlqvist

DOCTORAL DISSERTATION by due permission of the Faculty of Science, Lund University, Sweden. To be defended at lecture hall K:A, Kemicentrum, Wednesday the 29/5 2019 at 09:00

Faculty opponent Prof. László Somsák, University of Debrecen

3 Organization Document name LUND UNIVERSITY Centre for Analysis and Synthesis DOCTORAL DISSERTATION Lund University, Box 124 SE-221 00 Lund, Sweden Date of issue 2019-04-12

Author(s) Alexander Dahlqvist Sponsoring organization

Title and subtitle: Novel monogalactoside galectin inhibitor scaffolds - Guiding selectivity with heteroaromatic interactions Abstract: Carbohydrates are involved in many cellular processes, and most biomolecules are glycosylated. These modifications are used in biological systems as information carriers, helping regulate organization on the cell surface and interactions between cells and the environment. Galectins are a family of carbohydrate binding proteins that bind to polysaccharides containing a galactose. Galectins have the ability to crosslink glycosylated proteins – especially on the cell surface – giving galectins a role in modulating cell signalling and environmental interactions, influencing angiogenesis, immune regulation and cell adhesion. This implicates galectins in diseases like cancer and immune related disorders. Subsequently, many glycomimetics have been developed as galectin inhibitors, based on a variety of scaffolds, many with very high affinities, but selectivity between galectins remains a challenge. The galectin family of proteins has a very conserved binding motif, hence the differences in the binding pocket are small, making designing a selective inhibitor a challenge.

We investigated C1-galactosides as possible galectin inhibitor scaffolds, exploiting one of the few differences between galectin-1 and galectin-3 – histidine 52. We used C1-arylheterocycles to control the selectivity via the interaction between the anomeric heterocycle and the histidine, an approach which turned out to be fruitful resulting in the inhibitors 1-naphthyloxazole galactose, a galectin-3 selective inhibitor with 90µM affinity and 2- fluorophenyltriazole galactose, a galectin-1 selective inhibitor with a 170 µM affinity with fivefold and eightfold selectivity respectively. Extending the C1- system with a methylene linker resulted in the galectin-1 selective 4- fluorophenyltriazole 2-deoxygalactoheptulose, an inhibitor with 170 µM affinity and fourfold selectivity. In order to pursue these 2-deoxyheptulose scaffolds we developed a diastereoselective hydroboration method for C1- exomethylene glycopyranosides. Combining C1-substitutions with substitution in position three on galactose with a phenyltriazole motif did not increase affinities in a straightforward way; instead of increasing affinity and perserving selectivity patterns set by the C1-substitutents, the disubstituted molecules emerged as galectin-4 selective inhibitors with affinities down to 2.3 µM and up to thirty-eightfold or better selectivity for galectin-4. This shows that C1-galactosides can be selective galectin inhibitors with good affinities, but more work needs to be done to understand the interaction between substitution patterns. We also investigated aminpyrimidine substituted galactosides and identified compounds with a threehundred-fold selectivity for galectin-3 over galectin-1 and affinities down to 1.7 µM. These results show that careful selection of heterocycles with an aim towards exploiting even minute differences in the binding pocket can be effective in achieving selectivity.

Key words: Galectin, Galectin inhibitor, C-glycoside, C-galactoside, Aminopyrimidine, Hydroboration

Classification system and/or index terms (if any)

Supplementary bibliographical information Language: English

ISSN and key title ISBN 978-91-7422-658-4

Recipient’s notes Number of pages: 106 Price

Security classification

I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above-mentioned dissertation.

Signature Date 2019-04-12

4 Novel monogalactoside galectin inhibitor scaffolds

Guiding selectivity with heteroaromatic interactions

Alexander Dahlqvist

5 A doctoral dissertation at a university in Sweden is produced as a monograph or as a collection of papers. In the latter case, the introductory part constitutes the formal dissertation, which summarizes the accompanying papers. These have already been published or are manuscripts at various stages.

Coverphoto by Olof Röckner Illustrations on pages 18 & 20 by Mats Öberg

Copyright pp 1-106 Alexander Dahlqvist Paper 1 © ACS Publications Paper 2 © Beilstein Journals Paper 3 © Royal Society of Chemistry Paper 4 © by the Authors (Manuscript unpublished) Paper 5 © by the Authors (Manuscript unpublished) Faculty of Science Department of Chemistry, Centre for Analysis and Synthesis

ISBN 978-91-7422-658-4 e-ISBN 978-91-7422-659-1

Printed in Sweden by Media-Tryck, Lund University Lund 2019

To my wife who has endured my curiosity, and my child whom I hope has inherited it

7 Table of Contents

Abstract ...... 10 List of papers ...... 11 Abbreviations ...... 12 1. Introduction – C-glycosides, galectins and galectin inhibitors ...... 13 C-glycosides as drugs ...... 13 Glycobiology – A brief introduction ...... 17 Galectins – The basics ...... 18 Galectin inhibitors ...... 23 Affinity measurements ...... 26 Summary and conclusions ...... 27 2. C1-Heterocycles as galectin inhibitors ...... 29 Synthesis of C1-arylheterocycle galactopyranosides ...... 30 Affinity evaluations and structure-activity relationships ...... 34 Binding interactions – a computational hypothesis ...... 40 Summary and conclusions ...... 42 3. Hydroboration of C1-exomethylene ethers and C1-(aryltriazolyl)methyl galactopyranosyls as galectin inhibitors ...... 45 C1-Methylene pyranoside synthesis ...... 46 Hydroboration of C1-exomethylene pyranosides ...... 46 Synthesis of C1-(aryltriazolyl)methyl galactopyranosyls ...... 50 Synthesis of (aryloxazolyl)methyl galactopyranosyls ...... 51 Affinity evaluations and structure-activity relationships ...... 51 Computational studies ...... 53 Summary and conclusions ...... 55

4. Disubstituted C-galactosides: 3-Phenyltriazolyl-C1-galactopyranosides as galectin inhibitors ...... 57 Synthesis of 1,3-substituted C1-galactosides ...... 58 Galectin affinities of 1,3-disubstituted C1-galactosyls ...... 62 Pharmacokinetic properties ...... 64 Summary and conclusions ...... 67 5. Aminopyrimdine galactopyranosides ...... 69 Synthesis ...... 70 Galectin affinities and galectin mutant affinities ...... 71 Combination of aminopyrimidines with 1-substituents ...... 74 Summary and conclusions ...... 77 6. Conclusions and future prospects ...... 79 Conclusions – What have we learnt? ...... 79 Future prospects – Where should we go? ...... 82 Populärvetenskaplig sammanfattning ...... 85 Acknowledgements ...... 87 References ...... 91

9 Abstract

Carbohydrates are involved in many cellular processes, and most biomolecules are glycosylated. These modifications are used in biological systems as information carriers, helping regulate organization on the cell surface and interactions between cells and the environment. Galectins are a family of carbohydrate binding proteins that bind to polysaccharides containing a galactose. Galectins have the ability to crosslink glycosylated proteins – especially on the cell surface – giving galectins a role in modulating cell signalling and environmental interactions, influencing angiogenesis, immune regulation and cell adhesion. This implicates galectins in diseases like cancer and immune related disorders. Subsequently, many glycomimetics have been developed as galectin inhibitors, based on a variety of scaffolds, many with very high affinities, but selectivity between galectins remains a challenge. The galectin family of proteins has a very conserved binding motif, hence the differences in the binding pocket are small, making designing a selective inhibitor a challenge. We investigated C1-galactosides as possible galectin inhibitor scaffolds, exploiting one of the few differences between galectin-1 and galectin-3 – histidine 52. We used C1-arylheterocycles to control the selectivity via the interaction between the anomeric heterocycle and the histidine, an approach which turned out to be fruitful resulting in the inhibitors 1-naphthyloxazole galactose, a galectin-3 selective inhibitor with 90µM affinity and 2-fluorophenyltriazole galactose, a galectin-1 selective inhibitor with a 170 µM affinity with fivefold and eightfold selectivity respectively. Extending the C1- system with a methylene linker resulted in the galectin-1 selective 4-fluorophenyltriazole 2-deoxygalactoheptulose, an inhibitor with 170 µM affinity and fourfold selectivity. In order to pursue these 2- deoxyheptulose scaffolds we developed a diastereoselective hydroboration method for C1-exomethylene glycopyranosides. Combining C1-substitutions with substitution in position three on galactose with a phenyltriazole motif did not increase affinities in a straightforward way; instead of increasing affinity and perserving selectivity patterns set by the C1-substitutents, the disubstituted molecules emerged as galectin-4 selective inhibitors with affinities down to 2.3 µM and up to thirty-eightfold or better selectivity for galectin-4. This shows that C1- galactosides can be selective galectin inhibitors with good affinities, but more work needs to be done to understand the interaction between substitution patterns. We also investigated aminpyrimidine substituted galactosides and identified compounds with a threehundred-fold selectivity for galectin-3 over galectin-1 and affinities down to 1.7 µM. These results show that careful selection of heterocycles with an aim towards exploiting even minute differences in the binding pocket can be effective in achieving selectivity.

10 List of papers

Paper I: Alexander Dahlqvist, Hakon Leffler, Ulf J. Nilsson C1-Galactopyranosyl heterocycle structure guides selectivity: Triazoles prefer galectin-1 and oxazoles prefer galectin-3 ACS Omega, accepted Contributions: Conceived of the project, synthesized and characterized the compounds. Analysed the results and wrote the first draft of the paper. Paper II: Alexander Dahlqvist, Axel Furevi, Niklas Warlin, Hakon Leffler, Ulf J. Nilsson Stereo- and regioselective hydroboration of 1-exo-methylene pyranoses: Discovery of aryltriazolylmethyl C-galactopyranosides as selective galectin-1 inhibitors Beilstein Journal of Organic Chemistry, accepted pending minor revision Contributions: Conceived of the project, supervised AF and NW during the development of the synthesis methodology, synthesized and characterized the compounds. Analysed the results and wrote the first draft of the paper. Paper III: Alexander Dahlqvist, Fredrik Zetterberg, Hakon Leffler, Ulf J. Nilsson Aminopyrimidine-galactose hybrids are highly selective galectin-3 inhibitors MedChemComm, accepted pending minor revision Contributions: Synthesized and characterized most of the compounds. Analysed the results and wrote the first draft of the paper. Paper IV: Alexander Dahlqvist, Hakon Leffler, Ulf J. Nilsson C1-Aryltriazolyl galactopyranosides are galectin-1-selective inhibitors In manuscript Contributions: Conceived of the project, synthesized and characterized the compounds. Analysed the results and wrote the first draft of the paper. Paper V: Alexander Dahlqvist, Hakon Leffler, Ulf J. Nilsson Unexpected substituent synergy in 3-aryltriazolyl-C1-galactosides give high- affinity galectin-4C selective inhibitors In manuscript Contributions: Conceived of the project, synthesized and characterized the compounds. Analysed the results and wrote the first draft of the paper.

11 Abbreviations

Ac Acetyl ACN Acetonitrile Ar Aryl 9-BBN Borabicyclo[3.3.1]nonane Bn Benzyl CRD Carbohydrate recognition domain DCM Dichloromethane DMF Dimethylformamide Gal Galactose Glu Glucose HPLC High pressure liquid chromatography Lac Lactose LacNAc N-Acetyllactosamine Man Mannose Me Methyl Nap Naphthyl NOESY Nuclear Overhauser Effect Spectroscopy Ph Phenyl RT Room temperature TDG Thiodigalactoside THF Tetrahydrofuran TMS Trimethylsilyl Wt Wild type

12 1. Introduction – C-glycosides, galectins and galectin inhibitors

“I’m not here to start trouble. I am just doing some research for my thesis.” – Jessica Fortunato

C-glycosides as drugs

When deciding to dedicate your research efforts towards making C1-glycoside based drugs, it pays to keep in mind that what you ultimately strive for is to no longer be a carbohydrate chemist. The IUPAC Gold Book definition of ”carbohydrate” conspicuously fails to mention anything about C1-glycosides, defining a carbohydrate as such: “The generic term carbohydrate includes monosaccharides, oligosaccharides and polysaccharides as well as substances derived from monosaccharides by reduction of the carbonyl group (alditols), by oxidation of one or more terminal groups to carboxylic acids, or by replacement of one or more hydroxy group(s) by a hydrogen atom, an amino group, thiol group or similar groups. It also includes derivatives of these compounds.“1. So what is a C- glycoside? It is simply a glycoside where one or more hydroxyl groups has been replaced with a carbon atom. In this thesis, we will mostly be looking at C- glycosides in the anomeric position, known as C1-glycosides. From the definition alone it is quite clear that C1-glycosides have received limited interest and enthusiasm compared to many other types of carbohydrate modification and not entirely without cause. Simply put,