Synthesis, Formulation and Biological Evaluations of Chemotherapeutics Targeting Antimicrobial ...

110 Pages·2017·3.33 MB·English

by  Anthony Prandina

Synthesis, Formulation and Biological Evaluations of
Chemotherapeutics Targeting Antimicrobial
Resistance

Dissertation for the degree of Philosophiae Doctor

by

Anthony Prandina

Faculty of mathematics and natural sciences

University of Oslo

November 2017
© Anthony Prandina, 2018

Series of dissertations submitted to the
Faculty of Mathematics and Natural Sciences, University of Oslo
No. 1955

ISSN 1501-7710

All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.
Print production: Reprosentralen, University of Oslo.
Acknowledgments
The work described herein has been carried out between September 2014 and November 2017
at the Department of Pharmaceutical Chemistry, University of Oslo, in co-direction with the
Department of Organic Chemistry, University Claude Bernard Lyon 1. Prof. Pål Rongved
acted as main supervisor, assisted by co-supervisors Dr. Alexander Åstrand, Prof. Marc Le
Borgne and Assoc. Prof. Sylvie Radix. The University of Oslo and the Centre for Integrative
Microbial Evolution (CIME) are gratefully acknowledged for their financial support during
the project.

First, I would like to extend my sincerest gratitude to my main supervisor Prof. Pål Rongved
for giving me the opportunity to work on such an exciting project shared between our two
countries. Thank you for putting your trust in me, and for allowing this wonderful
collaborative work. During these past 3 years, you have let me work independently but at the
same time I always knew that I could count on you if I needed. Your passion about medicinal
chemistry and your constantly innovative ideas are a true inspiration to me.

I am indebted to my friend and colleague Dr. Alexander Åstrand. Thank you for being my go-
to guy these past few years in Oslo, you’ve taught me and helped me more times that I can
even recollect, both in my work and in my cold Norwegian life. I hope to be able return the
favor one day.

I would like to take the opportunity to thank Prof. Marc Le Borgne for introducing me to
medicinal chemistry in the first place and for always welcoming me into his research team;
and Assoc. Prof. Sylvie Radix for always having answers to my questions, for your advices,
feedbacks and your precious help on the publications.

I wish to thank some of the current and former colleagues and staff which have contributed to
such a motivating and fun place to work at. From the University of Oslo: Elvar, Christian,
Geir, Marius, Jørn, Eirik, Anders, Karoline, Åsmund, Bora, Jannicke and my hoarder of a
labmate Renate. You all contributed to make me feel welcomed in Oslo and I thank you for
that. Halvor Aandal and Trond Vidar Hansen also deserve credits for their assistance in the
I
administrative and practical aspects of being a PhD candidate at the School of Pharmacy.
From the University of Lyon: Zouhair, Luc, Marc R., Thierry, Laurent, Nadia, Pascal,
Christelle, Bruna, Sarah, Abdelfattah, Abdeslem. A special thanks to Assoc. Prof. Florent
Perret for all his help on the encapsulation project, and for sharing his knowledge about
supramolecular chemistry.

I would like to thank the biologists Ørjan Samuelsen, Silje Lauksund and Christopher Frölich
from Tromsø, Prof. Lars Herfindal and Prof. Stein Ove Døskeland from Bergen, for their
contribution to the biological evaluations. Lars Petter Jordheim should also be acknowledged
for letting me perform my own cell sensitivity studies in his group and for discussing the
method with me, and Zineb Bousfiha for her help on the practical work.

The master degree candidates I´ve had the pleasure of supervising and working with; Maud
Lourenco, Aïcha Ly, and Nicolas Kevin “the executioner” Bedel.

The people who helped me proofread and put together this thesis deserve a special mention:
Pål Rongved, Alexander Åstrand, Lord Elvar Örn Viktorsson, and Sylvie Radix.

Lastly, I would like to thank my wife Synthia for always believing in me. Your love, care and
support mean everything to me. Thank you for never complaining about having to take care of
our son alone when I was away, I couldn’t have done this PhD without you.

Oslo, November 16th, 2017

Anthony Prandina

II
Contents
Acknowledgments ....................................................................................................................... I
Contents .................................................................................................................................... III
Abstract ..................................................................................................................................... V
List of publications .................................................................................................................. VII
Graphical abstract ..................................................................................................................... IX
List of abbreviations ................................................................................................................. XI
Chapter 1 – General introduction........................................................................................... 1
1.1 Bacteria ........................................................................................................................ 1
1.2 Antibiotics ................................................................................................................... 2
1.2.1 Definition ............................................................................................................. 2
1.2.2 Antibiotic classification ........................................................................................ 2
1.3 Antibiotic resistance .................................................................................................... 3
1.3.1 Introduction to antibiotic resistance ..................................................................... 3
1.3.2 The fast rise of resistance ..................................................................................... 4
1.3.3 Mechanisms of resistance ..................................................................................... 5
1.3.4 Resistance development experiment .................................................................... 6
1.3.5 Consequences and costs ....................................................................................... 6
1.3.6 Fall of the last resort antibiotics ........................................................................... 7
1.4 Antibiotic misuses ..................................................................................................... 10
1.4.1 Overconsumption in agriculture and food industry ............................................ 11
1.4.2 Human misuses and overuses ............................................................................. 12
1.4.3 Lack of regulation .............................................................................................. 14
1.5 Towards a post-antibiotic era .................................................................................... 15
1.5.1 Industrial disinvestment ..................................................................................... 15
1.5.2 The urgent need for new antimicrobial strategies. ............................................. 16
1.6 Medicinal chemistry .................................................................................................. 17
1.6.1 ADMET properties and structure-activity relationship (SAR) studies .............. 17
1.7 Aim of study .............................................................................................................. 18
1.8 References ................................................................................................................. 19
Chapter 2 – Zinc-chelating agents ........................................................................................ 24
2.1 Introduction to Zinc ................................................................................................... 24
2.1.1 Zinc biology ....................................................................................................... 24
2.1.2 (cid:69)-Lactam antibiotics and metallo-(cid:69)-lactamases ................................................. 24
2.1.3 Metal chelation ................................................................................................... 27
2.1.4 Zinc chelators against resistant bacteria ............................................................. 29
2.2 Results and discussions ............................................................................................. 30
2.2.1 Introducing the ZinChel concept ........................................................................ 30
2.2.2 Paper I - Synthesis and biological evaluation of DPA derivatives as metallo-β-
lactamase inhibitors .......................................................................................................... 32
2.2.3 Paper II - Synthesis and biological evaluation of TPED and DPA-triazole
derivatives as metallo-β-lactamase inhibitors ................................................................... 47
III
2.2.4 Paper III - Synthesis and biological evaluation of TPA derivatives as metallo-β-
lactamase inhibitors .......................................................................................................... 52
2.3 Conclusions and perspectives .................................................................................... 55
2.3.1 Chemistry ........................................................................................................... 55
2.3.2 Biological evaluations ........................................................................................ 57
2.4 References ................................................................................................................. 58
Chapter 3 – Iodinin ................................................................................................................ 63
3.1 Introduction to iodinin ............................................................................................... 63
3.1.1 Structure, physical properties and sources ......................................................... 63
3.1.2 Biological activities ............................................................................................ 64
3.1.3 Background on phenazine synthesis .................................................................. 66
3.2 Iodinin and metal-chelation ....................................................................................... 67
3.3 Introduction to α-cyclodextrins and nanoparticle encapsulation ............................... 68
3.4 Results and discussions ............................................................................................. 70
3.4.1 Paper IV - New total synthesis of iodinin .......................................................... 70
3.4.2 Paper V - Encapsulation of iodinin into α-cyclodextrin nanoparticles .............. 74
3.5 Conclusion and perspectives ..................................................................................... 78
3.5.1 Chemistry ........................................................................................................... 78
3.5.2 Encapsulation and biological activities .............................................................. 79
3.6 References ................................................................................................................. 80
Chapter 4 - Concluding remarks .......................................................................................... 84
4.1 References ................................................................................................................. 86
Appendixes .............................................................................................................................. 87
Appendix A - Paper I
Appendix B - Paper II
Appendix C - Paper III
Appendix D - Paper IV and supporting information
Appendix E - Paper V

IV
Abstract
The work presented in this thesis employed knowledge in medicinal chemistry to discover
new compounds aiming to reduce resistance in infections caused by some of the most virulent
bacteria resistant to antibiotics.

The general aim of this work was to design and synthesize compounds capable of inhibiting
metallo-β-lactamases (MBLs), one of the most important defense mechanisms produced by
gram-negative bacteria. Metallo-β-lactamases use zinc for their catalytic activity and it has
been showed in the literature that alteration of the zinc homeostasis could have effect on their
efficiency. Therefore, the conception of our compounds revolved around the attempt to
deprive MBLs of zinc.

The zinc-chelating agents were designed around a three component platform, consisting of a
chelator with zinc selectivity, a targeting moiety and a linker tying the first two parts together.
Using DPA as a chelator and D-ala-D-ala as a vector, a first structure-activity relationship
study was performed, which led to the conclusion that the linker was not only essential for
activity, but also that a 4-ethylpiperidine moiety provided the best activity. When co-
administered with well-established last resort antibiotics such as meropenem, the ZinChel
compounds worked as adjuvants and were successfully able to restore the antibiotic’s activity
in the gold standard MIC assay (Paper I). To potentially increase the chelation strength of our
compounds, the DPA moiety was replaced by TPED, a stronger zinc chelator (Paper II). This
second study showed a correlation between increased chelator strength and MBL inhibition
potency. However, with a higher overall lipophilicity of the compounds, there was also an
increased toxicity towards eukaryotic cells. In order to find the right balance between
chelation strength and toxicity, the TPA motif, also possessing membrane permeability but
40% faster kinetics and reduced toxicity compared to TPED, was investigated. The next
generation compounds were found to be the most effective to restore the MEM activity in the
MIC assays. TPA derivatives also represented an interesting compromise by expressing a far
less toxicity than TPED against eukaryotic cells, especially for compound 26a comprising the
previously found 4-ethylpiperidine linker (Paper III).

V
We were also interested in exploring the possible use of a natural product, iodinin 30, well
described in literature as an antibacterial agent possessing metal chelation properties. In this
study, a reliable synthetic route to iodinin from commercially available reagents was
successfully established. The activity for the synthesized iodinin matches the reported data
from the iodinin isolated from a marine actinomycetes bacterium. As expected, iodinin’s poor
solubility was confirmed (Paper IV). To overcome iodinin’s solubility issues of, modified α-
cyclodextrins which were able to self-assemble and form nanoparticles in aqueous media
were designed and synthesized. Applying these nanoparticles as a delivery system for iodinin
resulted in enhanced solubility, bioavailability, and release in a controlled manner (Paper V).

VI
List of publications
Paper I
Synthesis and biological evaluation of new dipicolylamine zinc chelators as metallo-β-
lactamase inhibitors.

Prandina A, Radix S, Le Borgne M, Jordheim LP, Bousfiha Z, Fröhlich C, Samuelsen Ø,
Frøvold E, Åstrand OAH, Rongved P.

Manuscript submitted to ChemMedChem.

Paper II
Synthesis and biological evaluation of new zinc chelating compounds as metallo-β-lactamase
inhibitors.

Kildahl-Andersen G, Schnaars C, Prandina A, Apeland Ø, Frøvold E, Molesworth PP, Sieng
B, Radix S, Le Borgne M, Jordheim LP, Samuelsen Ø, Silje Lauksund, Fröhlich C, Rongved
P, Åstrand OAH.

Manuscript in preparation.

Paper III
Synthesis and in vitro biological evaluation of TPA based zinc chelators as metallo-β-
lactamase inhibitors.

Schnaars C, Kildahl-Andersen G, Prandina A, Radix S, Le Borgne M, Gjøen T, Andresen
AMS, Økstad OA, Heikal A, Samuelsen Ø, Fröhlich C, Rongved P, Åstrand OAH.

Manuscript in preparation.

Paper IV
Total synthesis and antileukemic evaluations of the phenazine 5,10-dioxide natural products
iodinin, myxin and their derivatives.

Viktorsson EÖ, Grøthe BM, Aesoy R, Sabir M, Snellingen S, Prandina A, Åstrand OAH,
Bonge-Hansen T, Døskeland SO, Herfindal L, Rongved P.

Bioorg. Med. Chem. 2017, 25 (7): 2285-2293.

VII
Paper V
Enhancement of iodinin solubility by encapsulation into cyclodextrin nanoparticles.

Prandina A, Herfindal L, Radix S, Rongved P, Døskeland SO, Le Borgne M, Perret F.

J Enzyme Inhib Med Chem. 2018, 33 (1): 370-375.
VIII