Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-11-13
2002-12-24
Richter, Johann (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S026700, C536S027200, C536S027600, C536S027610, C536S027620, C536S027630, C536S027700, C536S027810, C435S006120
Reexamination Certificate
active
06498241
ABSTRACT:
Nucleosides are widespread in the living world as building blocks of nucleic acids. They occur as ribonucleosides in ribonucleic acids (RNA) and as deoxyribonucleosides in deoxyribonucleic acids (DNA).
Naturally occurring nucleosides are usually composed of a sugar moiety (ribose or deoxyribose) and an aglyconic heterocyclic moiety. These so-called nucleobases are usually adenine, guanine, cytosine and thymine or uracil.
In addition nucleosides have been found in natural materials that are not components of nucleic acids such as e.g. isoguanosine or 1-methyl-isoguanosine. They often have interesting pharmacological properties.
The object of the present invention was to provide 2′-deoxy-isoguanosines, isosteric derivatives and isoguanosine derivatives as well as their phosphorus compounds. Oligodeoxynucleotides or DNA fragments which contain the compounds according to the invention are suitable for inhibiting the expression of viral genes in biological systems.
The invention concerns compounds of the general formulae I to IV (cf.
FIGS. 1 and 2
for graphic formulae) in which
R
1
=hydrogen or a protecting group, PO
3
H
2
, P
2
O
6
H
3
, P
3
O
9
H
4
or the corresponding alpha, beta and gamma thiophosphates provided that R
1
does not denote P
3
O
9
H
4
in formula III;
R
2
=hydrogen, hydroxy, phosphoramidite, methylphosphonate, H-phosphonate, a reporter or intercalator group;
R
3
=hydrogen or a protecting group;
W or/and Z=hydrogen, halogen, —NH—(CH
2
)
n
NH
2
(n=2-12), —R—CH
2
—COOH(R=alkylenyl C
1
-C
8
), a reporter or intercalator group.
The invention also concerns compounds of the general formula V in which
R
1
=equals hydrogen or a protecting group, PO
3
H
2
, P
2
O
6
H
3
, P
3
O
9
H
4
or the corresponding alpha, beta or gamma thiophosphates;
R
2
=equals hydrogen, hydroxy, phosphoramidite, H-phosphonate, methylphosphonate, a reporter or intercalator group;
R
3
=hydrogen or a protecting group;
R
4
=hydroxy;
Z=hydrogen, halogen, —NH—(CH
2
)
n
NH
2
(n=2-12), —R—CH
2
—COOH(R=alkylenyl with C
1
-C
8
), a reporter or intercalator group
provided that R
1
, R
3
and Z are not simultaneously hydrogen or R
1
is not simultaneously P
2
O
6
H
3
when R
3
and Z are hydrogen.
The compounds according to the invention of formulae I-IV are produced by either starting with compounds of the general formulae a or b in which
R′=R″ and represents an acyl protecting group such as e.g. p-toluoyl or benzoyl, this is reacted with aroyl isocyanates e.g. benzoyl isocyanate and the desired isoguanosine is isolated,
or compounds of the general formulae VI to IX in which W, Z, R
1
, R
2
and R
3
have the above-mentioned meanings,
and Hal represents chlorine, bromine or iodine
are converted photochemically by irradiation into the 2′-deoxyisoguanosines.
Compounds of the general formula V are produced analogously in which case one starts with compounds of formulae a, b, VI to IX which contain a &bgr;-D-ribo-furanosyl residue instead of the &bgr;-D-erythro-pentofuranosyl residue.
A particularly preferred process for the production of the compounds according to the invention is to persilylate deoxyguanosine or guanosine and their derivatives with hexamethyldisilazane and trimethyl-chlorosilazane. Subsequently the 2,6-diaminonucleoside is produced using ammonia and tris(trimethyl)silyl-triflate and selectively deaminated in the 2 position using nitrite to the isoguanosine or deoxyguanosine.
The further derivatization is carried out according to methods well known to a person skilled in the art.
The heterocyclic moiety in the 2′-deoxyisoguanosines and isoguanosines according to the invention can be preferably replaced by the corresponding isosters 7-deaza-isoguanine and 7-deaza-8-aza-isoguanine in which case these bases can in addition contain further substituents on the C-7 of the 7-deaza- or 7-deaza-8-aza-isoguanine and on the C-8 of isoguanine. Such substituents can for example be reporter groups as described below.
Hydrogen, halogen, NH—(CH
2
)
n
NH
2
, R—CH
2
COOH, a reporter or intercalator group are particularly preferred.
A diamino group can be introduced at position W and/or Z by halogenating a compound in which W and/or Z is hydrogen (for example with bromine water) and introducing a diamino group in the bromide by nucleophilic substitution. A diaminopentyl or diaminohexyl group is particularly preferably introduced. The desired compound can be prepared from the mixture of amino compounds by chromatographic purification.
A carboxyl group can be introduced by reacting the corresponding halogenated compound with methyllithium and preparing methyl bromide with halogen. This compound is reacted with aminocarboxylic acids (e.g. amino-caproic acid) to form the final product.
The reporter or intercalator groups are preferably coupled in their activated form (e.g. hydroxysuccinimide ester) to the amino or carboxyl group of the compounds according to the invention.
All suitable end groups known to a person skilled in the art can be present on the 3′ and 5′ end of the sugar moiety of the compounds according to the invention. Hydrogen, monophosphate, diphosphate or triphosphate, a reporter group or intercalator group are preferred for the 3′ end and for the 5′ end. A reporter group within the meaning of the invention is understood as a hapten such as biotin or digoxigenin or a fluorescent dye. Suitable intercalator groups are described by Hélène, C. in “Antisense RNA and DNA, Curr. Commun. Mol. Biol.; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1987” and are preferably phenanthroline, acridine, actinomycin or its chromophore or heavy metal complexing agents such as EDTA. Groups which result in a cross-linking of nucleic acids such as e.g. psoralen are also advantageous.
Cyclic phosphoric acid diesters (3′, 5′-cyclophosphates) can be produced from the compounds according to the invention by water liberation between the 3′-OH and 5′-OH of the sugar moiety.
The production of phosphoramidites, H-phosphonates and p-methylphosphoramidites from isoguanosines is carried out analogously to the production of the corresponding deoxyisoguanosine derivatives in which case the 2′-OH group is preferably protected by a triisopropylsilyl group.
The invention in addition concerns 2′-deoxyisoguanosines, isoguanosines and 7-deaza-8-aza-isoguanosine-3′-phosphoramidites, -3′-H-phosphonates and —P-methyl-phosphoramidites protected by bases and sugars. These compounds are suitable as nucleotide building blocks for the production of oligonucleotides.
The nucleotide building blocks according to the invention preferably contain protecting groups on the heterocyclic bases as well as on the 5′-OH and/or 2′-OH groups of ribose.
Amino-protecting groups such as e.g. benzoyl, formamidine, isobutyryl or diphenoxyacetyl groups are preferably used as a protecting group on the heterocyclic bases.
The 5′ or 2′-OH protecting group of the sugar moiety is preferably a triphenylmethyl, monomethoxytrityl, dimethoxytrityl, t-butyl-dimethylsilyl, t-butyldiphenylsilyl, t-butyl-methoxyphenylsilyl or pixyl group
3′-O-(2-cyanoethyl)-N,N-diisopropyl-aminophosphanes and 3′-O-methyl-N,N-diisopropylamino-phosphanes are preferred as phosphoramidites. The H-phosphonates are preferably used as salts.
The production of the monomeric nucleotide building blocks is carried out according to methods familiar to a person skilled in the art such as those described by Gait, M. J. in “Oligonucleotide Synthesis, A Practical Approach”, IRL Press, Ltd. (1984).
The compounds according to the invention of the general formulae I to IX can be incorporated by DNA or RNA polymerases into oligonucleotides or newly synthesized DNA or RNA in the form of their respective 5′-triphosphates or &agr;-, &bgr;- or &ggr;-thiotriphosphates.
In a particularly preferred embodiment, these nucleotide building blocks according to the invention are
Der Eltz Herbert Von
Kasimierczuk Zigmunt
Mühlegger Klaus
Seela Frank
Crane Lawrence E.
Pennie & Edmonds LLP
Richter Johann
Roche Diagnostics GmbH
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