# US 5,705,389 A · Oligonucleotides that inhibit production of .alpha.-tumor necrosis factor

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- Number: US 5,705,389 A
- Document ID: US5705389A
- Status: Granted
- Kind: A
- Application number: 08/342,338
- Filed: 1994-11-18
- Granted: 1998-01-06
- Earliest claimed: 1989-08-23
- Assignees: Roussel Uclaf
- Inventors: Abdel Karim Braham; Pierre Smets; Rene Zalisz
- Web page: https://patentgrep.com/patent/US5705389A

## CPC

- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- A61P 37/00: Drugs for immunological or allergic disorders
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07K 14/525: Tumour necrosis factor [TNF]
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- H02K 2203/03: Machines characterised by the wiring boards, i.e. printed circuit boards or similar structures for connecting the winding terminations
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- H02K: DYNAMO-ELECTRIC MACHINES (dynamo-electric relays H01H53/00; conversion of DC or AC input power into surge output power {H03K3/53})

## Abstract

A sequence of anti-direction, anti-messenger RNA oligonucleotides of .alpha.-TNF characterized in that it possesses the structure of the formula ##STR1## wherein X is hydrogen, or a sequence of 1 to 17 oligonucleotides in free form, in alkylated form, in sulfurated form or in the form of a poly L-lysine derivative useful for stopping the production of .alpha.-TNF.

## Claims

### Claim 1

A sequence of anti-direction, anti-messenger RNA oligonucleotides of .alpha.-TNF characterized in that it possesses the structure of the formula
5'TCA TGG TGT CCT TTG CAG-N 3' (SEQ ID NO:1)
wherein N is a hydrogen atom,
or a sequence of the formula 5' CC 3',
or a sequence of the formula 5' CC TCA TGC TTT CAG TAG 3', and wherein some of the phosphate groups are in free form, in alkylated form, in thiolate or dithiolate form, or in the form of poly-L-lysine derivative.

### Claim 2 (depends on 1)

A process for the preparation of the oligonucleotide sequence of claim 1 comprising immobilizing on a support S the first nucleotide in position 3' of the chain to be synthesized of the formula ##STR15## wherein S is the support, Z is a hydrocarbon of 2 to 20 carbon atoms, B.sub.1 is a purine and pyrimidine base corresponding to the first nucleotide, the amine function of which is protected, and R is a protector group, deblocking the hydroxyl in position 5' with an acid reagent to obtain a compound of the formula ##STR16## wherein S, Z and B.sub.1 have the above definitions, reacting the latter with the monomer of the second nucleotide of the formula ##STR17## wherein R has the above definitions, B.sub.2 is a purine or pyrimidine base corresponding to the second nucleotide, the amine function of which is protected, R.sub.1 is alkyl or --OR'.sub.1 or SR'.sub.1 in which R'.sub.1 is a protector group and R.sub.2 is a protector group to obtain a product of the formula ##STR18## wherein S, R, R.sub.1, B.sub.1 and B.sub.2 have the above definitions, oxidizing the latter product to obtain a product of the formula ##STR19## wherein S, R, R.sub.1, B.sub.1, B.sub.2 and Z have the above definitions and X.sub.1 is oxygen or sulfur, then, reacting the product of formula VI with the product of formula IV and oxidizing, and repeating for addition of each new nucleotide until the desired chain formation is obtained to obtain a product of the formula ##STR20## wherein S, R, R.sub.1, B.sub.1, B.sub.2, X.sub.1 and Z have the above definitions and B.sub.z is the last nucleotide of the sequence, deprotecting the oligo-nucleotide, separation from the support and purifying the product of formula I.

### Claim 3 (depends on 2)

The process of claim 2 wherein the deblocking of the hydroxyl in position 5' of the product of formula II is effected with an acid reagent, the coupling reaction of the product of formula III with the product of formula IV is activated by tetrazole in acetonitrile, the oxidation of the phosphite of formula V into phosphate of formula VI is effected with iodine in solution in a water/lutidine tetrahydrofuran mixture or in a water/pyridine/tetrahydrofuran mixture, the oxidation of the phosphite of formula V to obtain the sulfated form VI is effected with sulfur in solution in a mixture of carbon sulfide, anhydrous pyridine and anhydrous triethylamine, at the end of the synthesis, the deprotection of the terminal hydroxyl group in position 5' is effected with an acid, the deprotection of the oligonucleotide of formula VII is effected with concentrated ammonium hydroxide by moderately heating the reaction medium.

### Claim 4 (depends on 3)

The process of claim 3 wherein the acid deblocking and deprotecting agent is acetic acid or dichloroacetic acid or trichloroacetic acid.

### Claim 5 (depends on 1)

An oligonucleotide sequence of claim 1 wherein N is CC of the formula ##STR21## wherein all or some of the phosphate groups are in free form, in alkylated form, in thiolate or dithiolate form, or in the form of a poly L-lysine derivative.

### Claim 6 (depends on 1)

An oligonucleotide sequence of claim 1 wherein N is zero of the formula ##STR22## wherein all or some of the phosphate groups are in free form, alkylated form, in thiolate or dithiolate form, or in the form of a poly L-lysine derivative.

### Claim 7 (depends on 1)

An oligonucleotide sequence of claim 1 wherein N is the sequence SEQ ID No. 2 of the formula ##STR23## wherein all or some of the phosphate groups are in free form, alkylated form, in thiolate or dithiolate form, or in the form of a poly L-lysine derivative.

### Claim 8 (depends on 1)

A composition for reducing the production of .alpha.-TNF comprising an effective amount of a sequence of claim 1 to reduce the production of .alpha.-TNF and an inert pharmaceutical carrier.

### Claim 9 (depends on 1)

A method of reducing the production of .alpha.-TNF in a cultured human cell comprising providing to a cultured human cell an amount of the oligonucleotide of claim 1 which inhibits production of .alpha.-TNF by the cell.