Drug Development – NTB Isoquine Resulting from more than a decade of collaborative research we have invented a novel quinoline antimalarial drug with potential for safe and affordable use against drug resistant malaria. This project has received financial backing (up to £5 Million) from the Medicines for Malaria Venture (MMV). We have established a Public Private Partnership with MMV and GSK pharmaceuticals and the molecule is in preclinical development with an anticipated first into man date of Q2 2008. To have a drug candidate selected by a major pharmaceutical company is a key highlight of our research (see http://www.mmv.org/article.php3?id_article=47).1

We have also carried out the synthesis of novel classes of antimalarial pro-drug and orally active 1,2,4,5-tetraoxanes (see Figures 1 and Scheme 1),2, 3 the rationale drug-design of novel 4-aminoquinoline antimalarials (see Figure 2)4 and the synthesis and single cell imaging of fluorescent labelled artemisinin derivatives (see Figure 3).5 More recently, we have developed an asymmetric synthesis of chiral 1,2,4-trioxanes that have been used to probe the molecular mechanism of action of this important class of drug (see Figure 4) . 6 In the last two years a new project on quinolone syntheses has also been initiated in collaboration with the Liverpool School of Tropical Medicine (see Figures 5 & 6).

Orally active 1,2,4,5-Tetraoxanes - Since the demand for the natural product artemisinin (1) outweighs supply the discovery of “synthetic alternatives” has become an important goal for medicinal chemists working in the area of antimalarial drug-design. In Organic and Biomolecular Chemistry, 2006, 4, 4431 we describe how a remarkable set of antimalarial endoperoxides have been designed, synthesised and evaluated; several of these achiral compounds have antimalarial activities superior to the clinically used artemether and artesunate. Furthermore, the synthesis of these compounds should be amenable to industrial scale-up and as such these “antimalarial tetraoxanes” may represent alternatives to the currently used artemisinin derivatives.

THE UNIVERSITY OF LIVERPOOL INVESTING IN KNOWLEDGE

Research Highlights from the O’Neill Group Liverpool European Capital of Culture 2008

Whilst it is still early days in the development of tetraoxanes as antimalarials this publication provides proof of concept that this template can provide analogues with drug-like properties. The challenge in the future will involve maximising the stability and oral bioavailability of candidate molecules, reducing the number of synthetic steps and developing a chemical process for large-scale synthesis. (see http://www.rsc.org/Publishing/ChemScience/Volume/2007/01/fight_against_malaria.asp) 1. O'Neill P.M., Mukhtar A., Stocks P.A., Randle L.E., Hindley S., Ward S.A., Storr R.C., Bickley J.F., O'Neil I.A., Maggs J.L.,

Hughes R.H., Winstanley P.A., Bray P.G., Park B.K. Isoquine and Related Amodiaquine Analogues: A New Generation of Improved 4-aminoquinoline Antimalarials, J. Med. Chem., 2003, 46, 4933-4945

2. O’Neill, P.M., Stocks, P.A.; Pugh, M.D.; Araujo, N.C; Korshin, E.E.; Bickley, J.F.; Ward, S.A.; Bray, P.G.; Pasini, E.; Davies, J.; Verissimo, E.; Bachi, M.D. Design and Synthesis of Endoperoxide Antimalarial Pro-Drug Models: Prototypes for Selective Intraparasitic Generation of Cysteine Protease Inhibitors and Other Parasitocidal Species, Angew. Chem. Int. Ed., 2004, 116, 4289-4293

3. O’Neill, P.M.; Posner, G.H., A Medicinal Chemistry Perspective on Artemisinin and Related Endoperoxides (Invited Contribution), J. Med. Chem., 2004, 47, 2945-2964

Orally active Short Chain Chloroquine Analogues – We have developed methodology for the parallel synthesis of novel 4-aminoquinolines which circumvent the Chloroquine resistance mechanism. A lead compound produced by such methods is F2BU. This compound can interfere with hemozoin crystal formation (Fig. 2 (d)), can be prepared in only three synthetic steps and is orally active in rodent models of malaria by oral administration. Figure 2 (a) depicts a low energy conformation of this molecule complexed to the target receptor hematin.4

4. Stocks, P.A.; Raynes, K.A.; Bray, P.G.; Park, B.K.; O’ Neill, PM; Ward, S.A. Novel Short Chain Chloroquine Analogues

Retain Activity Against Chloroquine Resistant K1 Plasmodium falciparum J. Med. Chem, 2002, 45; 4975-4983 Mechanism of drug action - In an article published in Nature, 2003, 424, 957-961 Krishna and colleagues provide clear evidence that artemisinins target the SERCA, an enzyme involved in the calcium regulation of the malaria parasite. Part of this work was conducted within the O’Neill group in the Department of Chemistry. In this study many structural analogues of artemisinin were used by the Krishna group to prove the specificity of action against parasite SERCA. In addition a novel fluorescent artemisinin (Figure 3) analogue demonstrated that artemisinins are accumulated in high concentrations in the specific area of the parasite that the SERCA protein is located. The collaborative research team also proved that the parasite has a unique mechanism to accumulate artemisinin to high concentrations. This work represents a major step forward since it may provide the basis for rational drug design of improved artemisinin derivatives. This paper has been cited 191 times. see also: http://news.bbc.co.uk/1/hi/health/3168519.stm In Chembiochem, 2005. 6(11): 2048-2054 we have developed an asymmetric synthesis of enantiomeric 1,2,4-trioxanes (Figure 4) and the molecules have been used to probe stereochemical recognition within the malarial parasite.

5. Eckstein-Ludwig, U.; Webb, R. J.; van Goethem, I. D. A.; East, J. M.; Lee, A. G.; Kimura, M.; O'Neill, P.M.; Bray, P. G.;

Ward, S. A.; Krishna, S.. Artemisinins target the SERCA of Plasmodium falciparum Nature 2003, 424, 957-961

6. O’Neill, P.M.; Rawe, S.L.; Borstnik, K.; Miller, A.; Storr, R.C.; Ward, S.A.; Bray, P.G.; Davies, J.; Ho Oh,C.; Posner, G.H.

Enantiomeric 1,2,4-Trioxanes Display Equivalent In Vitro Antimalarial Activity Versus Plasmodium falciparum Malaria Parasites: Implications for the Molecular Mechanism of Action of the Artemisinins, Chembiochem, 2005. 6(11): 2048-2054

New Quinolone Inhibitors Targeting the Cytochrome bc1 Complex of Plasmodium falciparum Cytochrome bc1 complex is a multisubunit membrane protein complex which is one of the fundamental components of respiratory processes. The enzyme catalyses electron transfer from ubiquinol to cytochrome c and couples this process to the translocation of protons across the membrane for various cellular processes including ATP synthesis. Each functional unit of the homodimeric complex consists of three catalytic subunits: cytochrome b with two b type hemes, cytochrome c1 with one c type heme, and the Rieske protein containing a [2Fe-2S] cluster. (Figure 5)

The bc1 complex is a validated target for antimalarial drug discovery as exemplified by the drug development of atovaquone (8) and more recently by the discovery of pyridone based inhibitors (9) of this complex. The latter agents are in advanced preclinical stages of development. Recently, studies at Liverpool have confirmed that the acridinedione derivatives such as floxacrine (10) can also selectively (nM range) inhibit parasite mitochondrial function. Indeed, these novel inhibitors are ~5000 fold more selective for P. falciparum bc1 over human bc1. This selectivity is some 200 fold higher than that seen with atovaquone (Biagini et al., submitted). Furthermore, studies performed using yeast with mutated bc1, reveal that acridinedione derivatives preferentially bind the Qo pocket of the b cytochrome (Biagini et al., submitted). Based on these observations Neil Berry’s group have initiated homology based modelling studies of a range of inhibitors. Figure 6 depicts analogue 11 docked into the active site. Ultimately, we plan to use this model in a rationale drug design approach to novel quinoline inhibitors.

O

O

OH

Cl

N

CF3

O

OH

Cl

O

NH

O

OCF3

O

Cl

Cl

NH

O O

Cl Cl

8 9 10 11

Fig. 6. Dihydroacridinedione docking into P. falciparum cytochrome b (Qo). Docking of the dihydroacridinediones floxacrine (not shown) and WR249685 (11) was energetically favourable (binding energy -8.1 kcal/mol, estimated Ki ~ 1.14 µM). Figure 6 shows Qo-site residues predicted to be within 4 Å of the bound WR249685 (most energetically favourable conformation).

Additional Recent References 1. Stocks, P. A.; Bray, P. G.; Barton, V. E.; Al-Helal, M.; Jones, M.; Araujo, N. C.; Gibbons, P.; Ward, S. A.; Hughes, R. H.;

Biagini, G. A.; Davies, J.; Amewu, R.; Mercer, A. E.; Ellis, G.; O'Neill, P. M., Evidence for a common non-heme chelatable-iron-dependent activation mechanism for semisynthetic and synthetic endoperoxide antimalarial drugs. Angewandte Chemie-International Edition 2007, 46, (33), 6278-6283.

2. Mercer, A. E.; Maggs, J. L.; Sun, X. M.; Cohen, G. M.; Chadwick, J.; O'Neill, P. M.; Park, B. K., Evidence for the

involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds. Journal of Biological Chemistry 2007, 282, (13), 9372-9382. 3. O'Neill, P. M.; Ward, S. A.; Berry, N. G.; Jeyadevan, J. P.; Biagini, G. A.; Asadollaly, E.; Park, B. K.; Bray, P. G., A

medicinal chemistry perspective on 4-aminoquinoline antimalarial drugs. Current Topics in Medicinal Chemistry 2006, 6, (5), 479-507.

4. O'Neill, P. M.; Verissimo, E.; Ward, S. A.; Davies, J.; Korshin, E. E.; Araujo, N.; Pugh, M. D.; Cristiano, M. L. S.; Stocks,

P. A.; Bachi, M. D., Diels-Alder/thiol-olefin co-oxygenation approach to antimalarials incorporating the 2,3-dioxabicyclo[3.3.1]nonane pharmacophore. Bioorganic & Medicinal Chemistry Letters 2006, 16, (11), 2991-2995.

5. Bray, P. G.; Mungthin, M.; Hastings, I. M.; Biagini, G. A.; Saidu, D. K.; Lakshmanan, V.; Johnson, D. J.; Hughes, R. H.;

Stocks, P. A.; O'Neill, P. M.; Fidock, D. A.; Warhurst, D. C.; Ward, S. A., PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX. Molecular Microbiology 2006, 62, (1), 238-251.

6. Biagini, G. A.; Viriyavejakul, P.; O'Neill, P. M.; Bray, P. G.; Ward, S. A., Functional characterization and target validation of

alternative complex I of Plasmodium falciparum mitochondria. Antimicrobial Agents and Chemotherapy 2006, 50, (5), 1841-1851.

7. Amewu, R.; Stachulski, A. V.; Ward, S. A.; Berry, N. G.; Bray, P. G.; Davies, J.; Labat, G.; Vivas, L.; O'Neill, P. M., Design

and synthesis of orally active dispiro 1,2,4,5-tetraoxanes; synthetic antimalarials with superior activity to artemisinin. Organic & Biomolecular Chemistry 2006, 4, (24), 4431-4436.

8. Amewu, R.; Stachulski, A. V.; Berry, N. G.; Ward, S. A.; Davies, J.; Labat, G.; Rossignol, J. F.; O'Neill, P. M., Synthesis of

1,2,4-trioxepanes via application of thiol-olefin Co-oxygenation methodology. Bioorganic & Medicinal Chemistry Letters 2006, 16, (23), 6124-6130.

9. O'Neill, P. M.; Rawe, S. L.; Storr, R. C.; Ward, S. A.; Posner, G. H., Lewis acid catalysed rearrangements of unsaturated

bicyclic [2.2.n] endoperoxides in the presence of vinyl silanes; access to novel Fenozan BO-7 analogues. Tetrahedron Letters 2005, 46, (17), 3029-3032.

10. O'Neill, P. M.; Rawe, S. L.; Borstnik, K.; Miller, A.; Ward, S. A.; Bray, P. G.; Davies, J.; Oh, C. H.; Posner, G. H.,

Enantiomeric 1,2,4-trioxanes display equivalent in vitro antimalarial activity versus Plasmodium falciparum malaria parasites: Implications for the molecular mechanism of action of the artemisinins. Chembiochem 2005, 6, (11), 2048-2054.

11. O'Neill, P. M., The therapeutic potential of semi-synthetic artemisinin and synthetic endoperoxide antimalarial agents.

Expert Opinion on Investigational Drugs 2005, 14, (9), 1117-1128. 12. Bray, P. G.; Ward, S. A.; O'Neill, P. M., Quinolines and artemisinin: Chemistry, biology and history. In Malaria: Drugs,

Disease and Post-Genomic Biology, 2005; pp 3-38. 13. Biagini, G. A.; O'Neill, P. M.; Bray, P. G.; Ward, S. A., Current drug development portfolio for antimalarial therapies.

Current Opinion in Pharmacology 2005, 5, (5), 473-478. 14. Posner, G. H.; O'Neill, P. M., Knowledge of the proposed chemical mechanism of action and cytochrome P450

metabolism of antimalarial trioxanes like artemisinin allows rational design of new antimalarial peroxides. Accounts of Chemical Research 2004, 37, (6), 397-404.

15. O'Neill, P. M.; Stocks, P. A.; Pugh, M. D.; Araujo, N. C.; Korshin, E. E.; Bickley, J. F.; Ward, S. A.; Bray, P. G.; Pasini, E.;

Davies, J.; Verissimo, E.; Bachi, M. D., Design and synthesis of endoperoxide antimalarial prodrug models. Angewandte Chemie-International Edition 2004, 43, (32), 4193-4197.