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New pyrazolo-triazine structures improving dna-derivative properties for genomic and therapeutic applications

Development of a new product line, based on the pyrazolo-triazine motif, enhancing the sirna, aptamers and antisense activities as potential therapeutic tools.

The cellular uptake of DNA-derivatives (Deoxyribose Nucleic Acid), their resistance towards nucleases and their hybridisation properties are 3 major hurdles that need to be overcome by researchers working in the siRNA, aptamers and antisense fields. With the emergence of new concepts like RNA (RiboNucleic acid) interference, aptamer technology and the 2nd generation of anti-sense compounds, there is a need to enhance the properties of these derivatives before they become new drugs. Pyrazolo [1,5-a]-1,3,5-triazines have for a long time been known as compounds able to modulate many biological properties. The substitution of the purine scaffold by a pyrazolo [1,5-a]-1,3,5-triazine scaffold increases stability towards nucleosidases that cleave the bond between the carbohydrate (ribose, arabinose, deoxyribose, deoxyarabinose, etc.) and the purine ring. Pyrazolo [1,5-a]-1,3,5-triazines are therefore interesting purine agonists whilst enhancing their pharmacokinetic properties, hence their therapeutic interest. Although pyrazolo [1,5-a]-1,3,5-triazines have been known for several decades as remarkable bio-isotheres of purine, few molecules have been published and have been in clinical phases up until now. An explanation is the difficulty in easily synthesising these compounds. The recent collaboration between GREENPHARMA and the UNIVERSITY OF LYON led to a completely new activating group: the 'N-methyl-N-phenylamino', which allowed an easy synthesis of these compounds. These new derivatives display a higher level of in vivo stability than the corresponding purines. GREENPHARMA registered a patent on the synthesis of these molecules (WO 2004011464). We propose to develop a new set of phosphoramidite compounds based on the pyrazolo [1,5-a]-1,3,5-triazine structure described in the GREENPHARMA patent, for incorporation into siRNA, aptamers and anti-sense in order to enhance their properties, e.g. cellular uptake, resistance towards nucleases and/or duplex stability to reinforce their potency to become valuable therapeutic tools. A large series of new phosphoramidite compounds can be built up by substitution of the purine-like base, such as fluorescent and biotinylated tag, cholesterol and lipophilic groups, Pyren and NVOC reporter molecules. Other modifications at the 2' position of the sugar, i.e. 2'-OH, 2'-Ome or 2'-F will allow us to synthesize riboA- and riboG-like phosphoramidites which can be very useful tools in the siRNA field. After incorporation of these new monomers into DNA or RNA strands, evaluation of the coupling efficiency, estimation of purity as a crude end product and a full characterisation of the compounds, hybridisation properties and thermodynamic studies will be assessed in order to define the effect of such a modification in DNA and RNA-derivatives. Experiments will be performed on single strand and double strand targets. In-vitro stability and resistance towards nucleases will be evaluated followed by in-vivo or cell experiments. Typical onco-gene targets such as Ha-ras or myc-genes, anti-TNFa (Tumour Necrosis Factor Alpha) sequences or HIV (Human Immuno-deficiency Virus) gene targets can be chosen, thereby allowing us to evaluate these compounds as potential therapeutic tools. The best candidates will be selected throughout the project duration depending on their properties; at the end of the development work, the selected compounds will be produced and exclusively commercialised by BIOSOLVE BV and BIOSOLVE FRANCE, two subsidiaries of BIOLAB LTD. Keywords: purine bio-isosteres, phosphoramidites.
Acronym: 
GREENLAB
Project ID: 
3 621
Start date: 
01-10-2005
Project Duration: 
24months
Project costs: 
2 200 000.00€
Technological Area: 
Organic Chemistry
Market Area: 
Pharmaceuticals/fine chemicals

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