Research project 1 at the Laboratoire de Synthèse de Biomolécules

PURBLOQ – SYNTHESIS AND EVALUATION OF NOVEL ANTIBIOTICS Thematics concerned: organic-biomolecular-medicinal CHEMISTRY & HEALTH General Context

Today the appearance of bacteria that are resistant to antibiotics represent a serious health problem of global dimensions. The are no real alternatives to treat-ing multiresistant microbial infections with antibiotics and the number of new antibiotics developped by large pharmaceutical companies is very small. Only small very specialised companies are still active in the research and development of new antibiotic compounds. Enhanced efforts in this research direction are cru-cial for meeting society’s health challenges in the near future.

The goal of this research project is to synthesise and evaluate a series of struc-tural analogues of puromycin (Figure 1) that are blocked in their conforma-tional freedom called «North» and «South» in which the ribofuranosyl unit of puromycin is replaced through a carbocycle (Figure 2) with constrained flexibi-lity (Figure 3). Such compounds would enable us, on the one hand, to demons-trate the influence of the ribofuranose pucker (North, South) of the natural A site substrate of the ribosomal peptidyl trans-fer reaction, thus, on the molecular mechanism of cellular protein synthesis and, on the other hand, may provide us with a new antibiotic of large pathogenic specifity that aims at bacterial ribosomes, to produce lethal truncated proteins (through irreversible inhibition), at the elongation factor Tu, the most abundant procaryotic enzyme, in order to stop bacterial growth through reversible inhibition, or to aim at both enzymes at a time. The North analogue has been finalised as the ongoing first part of the project. Here we are proposing the logical and absolutely necessary completion of carrying out the second half of PURBLOQ: The synthesis and evaluation of the South analogue.

These analogues bear a bicyclo[3.1.0]hexane unit that exists uniquely in a pseudo-boat conformation where the carbobicyclic nucleoside either adopts the North or the South conformation depending on the position of the cyclopropane moiety (see Figures 2 & 3). The blocked North analogue bears a cyclopropane ring between C4' and the carbon that replaces O4', while the blocked South ana-logue bears the cyclopropane ring between C1' and the carbon that replaces O4'. The synthesis of such structures will allow us to develop (i) new antibiotics against multiresistant pathogens, (ii) new anti-cancerogens or cyto-statics that kill or inhibit

all rapidly growing cells. Whereas such carbocyclic nucleoside analo-gues and their catabolites are known not to be toxic to human cells, 3'-deacylated puromycin is and the natural compound cannot be used as an antibiotic. Hence, the synthesis of conformationally blocked carbo-cyclic puromycin analogues may lead to a novel therapeutic ap-proach. In addition, some of the synthetic intermediates could be test-ed for antiviral activity (e.g. anti HIV, anti HCV, anti HBV).

Research Project

Studies to date

The PURBLOQ project is running at the LSB since September 2005. It is divided into two parts, the synthesis of the North analogue and the South analogue (Figure 2). Both analogues bear the potential of inhibiting bacterial protein synthesis. Both analogues are possible candidates for a lead compound that could be further developped into a medicinally useful antibiotic. Both analogues are also highly precious compounds for the enzymological testing of ribosomes, in order to deepen our know-

NH OH

HON

NN

N

NR2

O

H2N OMe

NH OH

HON

NN

N

NR2

O

H2N OMe

Pm blocked North Pm blocked South Figure 2

Analogues to synthesise.

O

NH OH

HON

NN

N

N(CH3)2

O

H2N OMe

O

O OH

ON

NN

N

NH2

R

O

H2N

PO3–

tRNA

3'

Puromycin (Pm) 3'-aminoacyl-tRNA

3' 2'1'4'

5'

Figure 1

Puromycin, a natural antibiotic, mimics the structure of 3'-terminally aminoacylated transfer ribonucleic acids. Both molecules are substrates for the acceptor site of ribosomes; a peptide bio-synthesised by the ribosome can thus be trans-ferred onto the nitrogen atom marked in red.

H

O O

Base

H

OH

(5')C

HOH

H

HH

OH

(5')C

H

H

OH Base

H

3' 2'

North conformer(3' endo)

South conformer(2' endo)

1'3'2'

4'4'

1'

Base

H

OH

(5')C

H

H

H

R

(5')C

H

H

OH Base3' 2'

1'3'2'

4'4'

1'

R

HH H H

H

X

Figure 3 Nucleosidic ribofuranose conformers (above) and constitutional isomers that mimic them (below).

http://www.icbms.fr/user/main.asp?num=282

©P. Strazewski 2010

ledge on the nature of the ribosomal catalysis of peptidyl transfer during the biosynthesis of proteins – an important research area that has not found a resolution yet and awaits scientific answers worldwide in the scientific community. In June 2006 the project PURBLOQ has been evaluated by the Région Rhône-Alpes and was highly ranked – the antibiotic research area being a priority theme of the Région and the scientific quality of the project being convincing. The part « Synthesis of the North analogue » has been recently finalised by Benoît Michel, a former PhD student at LSB, who synthesised his target compound in 18 steps from D-ribose within 2.5 years. At present he is author or co-author of 8 publications (Tetrahedron 2007, Synlett 2008 (2x), Nucleic Acids Symp. Ser. 2008 (3x), J. Org. Chem. 2008, Chemistry – A European Journal 2009); he will become co-author of 3 more publications to be published soon.

Studies planned

The synthetic pathway has been partially tested at the LSB. It is based on the realively easy access to the intermediate XXIII described in the literature (Scheme 1) and was realised in our laboratories without difficulties (Scheme 2).

Scheme 1: A retrosynthetic analysis for the transformation of XXIII (a well known compound in the literature) into the azide XII, a synthetic precursor of the carbocyclic puromycin analogue blocked in the South conformation.

Scheme 2: Synthesis of enantiopure enone XXIII, a key intermediate for the synthesis (according to Scheme 1) of the carbocyclic puromycin South analogue.

The concrete working plan is thus as follows:

– to take up in larger quantities the synthesis shown in Scheme 2, which worked without problems and took about one to two months for a beginner to obtain XXIII in several hundred milligramme quantities from D-ribose (we shall need about ten times more),

– then to develop the pathway sketched out in Scheme 1 of which we know that the conversion of XXIII to XXII works very nicely. The experience that we gained on the North route will help a lot to overcome potential difficulties on the route depicted in Scheme 1. Thanks to these experiences, as well as the progress achieved on the South route during two Master studies 2008-09 and 2009-10, we are confident that this route is realistically achievable within a time period of nine months.

Biological evaluation

The enzymological tests for the conformationally blocked puromycin analogues will be carried out in collaboration with a group working at the University of Uppsala, Sweden, with Professor Måns Ehrenberg, one of the worldwide most important ribosome specialists. Other antibiotic activity (in vivo) and toxiticity tests are planned with the chimiothèque of our Institute, as well as in collaboration with NOVOCIB, a start-up located in Lyon.

Supervision of the project: Peter Strazewski, Professor

Director of the Laboratoire de Synthèse de Biomolécules

see also http://www.icbms.fr/user/main.asp?num=282

R1O OH

R2HO

R2= CN, N3

R1O OH

BaseHO

OH

BaseHO

N3

R1= groupement protecteur

R1O OH

R2

R1O OH

R2O

HO OH

R2O

HO OH

R2O

HO OH

R2O

O O

O R2

O O

OO

O O

O

OH

R2

O O

O

XII XIII XIV XV

XVIXVIIXVIIIXIX

XX XXI XXII XXIII

O OH

OH

OH

HO

O

O O

Me Me

OHHOMe2CO

H2SO4

D-Ribose

Ph3PCH3Br

KOtBu

OH

O O

Me Me

HO NaIO4

O O

Me Me

O

O O

Me Me

HO Dichloride

Neolyst ™ O O

Me Me

HOPCCMgBr

O O

Me Me

O

XXIII

PhRu

PCy3

PCy3Cl

Cl

DichlorideNeolyst ™

Research project 2 at Laboratoire de Synthèse de Biomolécules

AMPHIMED – INTERACTIONS BETWEEN PEPTIDYL–RNA AND LIPIDIC MEMBRANES OR LIVING CELLS: FUNDAMENTAL AND BIOMEDICAL ASPECTS

Research domain: HEALTH-CHEMISTRY organic-biomolecular-medicinal-supramolecular-systems chemistry

Scientific objectives 1) Solid support synthesis of amphiphilic peptidyl-RNA using a novel method developped at the Laboratoire de Synthèse de Biomolécules (LSB-UCBL). The peptide part is at first quite simple in composition and lipophilic. Later peptides shall be tested that are designed to recognise malaria-specific cellular receptors and the attached RNA part shall contain oligoribonucleotides (siRNA) that selectively block the expression of proteins that are vital for the pathogen. 2) Analysis of the supramolecular interactions of amphiphilic peptidyl-RNA in glycolipidic liposomes through optical and fluorescence microscopy (in scientific collaboration with lipid specialists of the ICBMS). 3) Testing of different formulations of peptidyl-RNA for anti-malarial effects using ex vivo cultivated erythrocytes (red blod cells) that are paratised by the microorganism of malaria, Plasmodium falciparum (in collaboration with a medical research group at UCBL).

Expected results The amphiphilicty of peptidyl-RNA carrying highly lipophilic peptides will be modulated to generate helical structures that are capable of forming agreggates in cellular membranes, in order to destroy pathogenic multi-resistant microorgan-isms through ‘molecular nano-poration’ (novel antibiotic). Receptor-specific peptidyl-RNA are expected to penetrate paratised erythrocytes and kill the parasite through the action of toxic RNA (novel anti-malaria drug). This approach consists of a strongly simplified application of the actual concept according to which therapeutic inter-fering RNA is complexed to natural or semi-synthetic antibodies that recognise specific receptors in mamalian/ human cancer cells. The biocompatibility of therapeutic peptidyl-RNA is an advantage over existing, purely synthetic amphi-philic drug vectors. Thus, the aim is to use these conjugates, either inserted into glycolipids or formulated in nanovesicles, as biodegradable synthetic drug vectors.

Director of thesis: Peter Strazewski, Professor Director of the Laboratoire de Synthèse de Biomolécules, ICBMS, UCBL http://www.icbms.fr/user/main.asp?num=282

Tests on erythrocytes parasitised by Plasmodium falciparum: Stéphane Picot, PH Director of the Malaria Research Unit, UCBL http://www.parasitologie.univ-lyon1.fr

O

NH

ODMT

NH

O

CH3

N

N

N

N

N

O

FMOC

N

OOH

O

A)

1

A) a) 1. Aminomethyl polystyrene 50 % crosslinked with p-divinyl benzene, succinic anhydride, DMAP; 2. Ac2O, pyridine, NMI, DMF; b) 1. Oxalyl chloride, CH2Cl2; 2. 1,6-diaminohexane, DMAP, CH2Cl2; c) BOC-Sarcosine, HBTU, NMM, DMF; d) 1. TFA/CH2Cl2; 2. Et3N /DMF; e) 1. Building block 1, HATU, NMM, DMF; 2. Ac2O, pyridine, NMI, DMF. B) 1-2) FMOC-based peptide synthesis, FMOC-amino acid + DEPBT; 1-2) phosphoramidite-based RNA synthesis; 1-4) 3% TCA in CH2Cl2; {i) 1. Pd(PPh3)4, PhSiH3, CH2Cl2; 2. NH4

+Et2NCS2–/DMF}; j) 38 % CH3NH2 in EtOH/H2O

(1:1), 2 h, RT; k) Et3N·3HF, DMF, 1.5 h, 65° C; n-butanol, –20° C; SAX- and RP-HPLC.

B)