WO1996030383A1

WO1996030383A1 - Nucleic acid polyester polyamides

Info

Publication number: WO1996030383A1
Application number: PCT/AT1996/000056
Authority: WO
Inventors: Christian Noe; Georg Haberhauer; Lucius Kaufhold
Original assignee: Christian Noe; Georg Haberhauer; Lucius Kaufhold
Priority date: 1995-03-24
Filing date: 1996-03-25
Publication date: 1996-10-03
Also published as: AU4931196A

Abstract

The invention relates to novel structurally modified oligonucleotides, a process for their production and their use. Said oligonucleide analogues are 2'-5'- cross-linked, and not 3'-5'-linked as the natural oligonucleotides. There are 3 atoms between the two sugar rings and the cross-link consists of either a carboxylic acid ester or a carboxylic acid amide. Such modified oligonucleotides can interact with natural nucleic acid. Oligomers of this invention may thus be used as potential medicaments.

Description

Nucleic acid polyester polyamides

Natural nucleic acids have a 3 '-5' linkage, which is the basis for the formation of the double helix and thus for the biological function of nucleic acids. Such nucleic acids are easily cleaved by hydrolysis of the phosphoric ester bond. When using nucleotides for molecular biological, diagnostic and therapeutic purposes, the production of nuclease-resistant compounds is therefore always an important goal.

We were able to show by means of molecular modeling studies (FIG. 1) that oligonucleotide analogs which are not linked by 3 '-5'- but 2' -5-'- are also able to pair with natural RNA or DNA, especially if the The number of atoms between the two sugar rings of the nucleosides is 3, but special attention is paid to the type of linking elements. Too great flexibility of such a link between two nucleoside units can lead to a severe restriction or even to the loss of the binding ability. On the other hand, rigidization that is too rigid or too sterically demanding can have the same negative effect.

The present invention relates to nucleotide carboxylic acid esters and amides of the general formula I

in which the sugar rings of the nucleosides are linked to at least one bridge which consists of 3 atoms and contains either a carboxylic acid ester or carboxamide group and which A = -0-C (= 0) -, -N (-Rι) -C (= 0) -, where R, = H, or unbranched or branched unsubstituted or optionally substituted with acid or base function alkyl having a chain length of 1 to 6 carbon atoms, in which R ₂ and R ₃ = H or lower alkyl in any combination means and R ₄ = H, OH or unbranched or branched unsubstituted or terminally substituted with acid or base function O-alkyl having a chain length of 1 to 6 carbon atoms

ERSATM.ATT (RULE 26) and in which B is the N-glycosidically bound residue of a purine or pyrimidine base of the general formula II or III

II m

means, wherein R _{5 is} a hydrogen, chlorine or bromine atom, a hydroxyl, amino, monomethoxytritylamino, mercapto, methylamino, dimethylamino, phenylamino or hydroxylamino group, R _{* is} a hydrogen, chlorine or bromine atom, a hydroxyl -, Amino, mercapto group, R a hydroxyl or amino group, Rg an oxygen atom (= 0), and R ₉ hydrogen, fluorine, chlorine, or bromine or iodine atom, a methyl, trifluoro, ethyl, ethyl or hydroxymethyl group, and the salts of compounds of general form I with acids or bases.

The invention includes Nukleotidoligoester and -oligoamide with the above Stι ^* τjJ structure with a number of from 2 to 200 nucleotide units.

The oligonucleotide analogs of the subject invention can be prepared from the suitably protected monomers by conventional condensation processes after the carboxylic acid has been activated beforehand.

Binding experiments with natural nucleic acids show that oligonucleotides of the present invention are suitable for pairing with natural nucleic acids. Molecular dynamics Studies show that esters and amide bonds are superior to other bridging compounds.

The invention includes the use of such nucleotide oligoesters and oligoamides as therapeutic agents, for diagnostic purposes and as research reagents.

ERSÄΓZBLAΪT (RULE 26) Examples:

5'-Deoxy-6N-monomethoxytrityl-2 ^, -3'isopropylidenadenosiιι-carboιιäure-2'-0 (3'-0- methyl-5'-O-monomethoxytrityI) -uridinyl-ester (1)

590 mg (0.96 mmol) 5 ^, -deoxy-6-N- [4- (methoxyphenyl) -diphenyl-methyl] -2 ', 3 ^, -0-isopropylidene-adenosine-5' -carboxylic acid were dissolved in anhydrous dichloromethane and mixed with 590 mg (1.2 mmol) 3'-0-methyl-5'-0-momomeώoxytrityl-uridine, 300 mg (1.45 mmol) DCC and 10 mg dimemylammopyridine were added. The solution was stirred for 24 hours at room temperature and then evaporated. After purification by means of column chromatography (80 g of silica gel, eluent: dichloromethane / acetone 9/1), 670 mg (65% of theory) 1 was obtained. TLC: dichloromethane / acetone = 9 / l, ref .: 0.46 1: white foam

-H NMR (CDC13) (300 MHz): δ = 8.05 (s, IH, 2-H); 7.9 (s, IH, 8-H); 7.8 (d, IH, 6-H uridine); 7.4-7.2 (, 24H, MMT); 6.85 (d, 2H, MMT); 6.75 (d, 2H, MMT); 6.1 (d, IH, 1'-HA); 6.04 (d, IH, l'-HU); 5.5 (m, IH, 2'-HU); 5.45 (m, IH, 2'-HA); 5.38 (d, IH, 5-H uridine); 5.09 (m, IH, 3'-HA); 4.65 (m, IH, 4'-HA); 4.2-4.1 (m, 2H, 3 ', 4'-HU); 3.85 (s, 3H, OCH3-MMT); 3.8 (s, 3H, OCH3-MMT); 3.55 (d, IH, 5'-H'-U); 3.4 (d, IH, 5'-H "-U); 3.3 (s, 3H, OCH3); 3.0-2.9 (m, 2H, 5'-CH ₂ -A); 1.6 (s, 3H, CH3); 1 4 (s, 3H, CH3).

5'-Deso_y-adenosine-5 ^, -carboιιäure-2 ^, -0- (3'-0-methyl) -uridinyI-ester 2nd

300 mg (0.28 mmol) 1 were dissolved in 3 ml dichloromethane / water 1/1 and treated with 300 mg trichloroacetic acid. After 30 minutes the solution was partitioned between diethyl ether and water, the aqueous phase extracted with diethyl ether and evaporated. The residue was taken up in a little dichloromethane and digested in diethyl ether. The precipitate was centrifuged off and dried. Yield: 110 mg (80% of theory) 2.

TLC: dichloromethane methanol = 8/2, ref .: 0.45 2: white crystals

SPARE BLADE (RULE 26) iH-NMR (D2θ) (300 MHz): d = 8.39 (s, 2H, 8-H and 2-H adenosine); 7.65 (d, IH, 6-H uridine); 6.10 (d, IH, l'-HA); 5.93 (d, IH, l'-HU); 5.71 (d, IH, 5-H uridine); 5.45 (m, IH, 2'-HU); 4.85 (m, IH, 2 -HU); 4.5 (m, IH, 4'-HA); 4.35 (m, IH, 3'-HA); 4.1 (m, 2H, 3 'and 4Η-U); 3.85 (m, IH, 5'-H ^* U); 3.96 (m, IH, 5'-H- "U); 3.25 (s, 3H, O-CH3); 3.0 (m, 2H, 5'-CH ₂ -A).

5'-Deo-_y-6-N- [4- (methoxyphenyl) diphenyl-methyl I] -2 ''-0-isopropy-iden-adenosine-5'-carboxylic acid-2 ^, - [3'-deox -6 -N-5'-0- (di- (4- (methoxyphenyl) diphenylmethyl)) adenyl] ester 3

120 mg (0.19 mmol) 5 ^, -deoxy-6-N- [4- (methoxyphenyl) -diphenyl-methyl] -2 ', 3'-0-isopropylidene-adenosine-5'-carboxylic acid were dissolved in anhydrous dichloromethane and mixed with 200 mg (0.25 mmol) of 3'-deoxy-6-N-5'-0-di [4- (methoxyphenyl) diphenylmethylj-adenosine, 60 mg (0.27 mmol) of DCC and 5 mg of dimemylaminopyridine were added. The solution was stirred at room temperature for 24 hours and then evaporated. After purification by means of column chromatography (20 g of silica gel, eluent: dichloromethane / acetone 9/1), 60 mg (23% of theory) 3 were obtained. TLC: dichloromethane / acetone = 9 / l, ref .: 0.56 3: white foam

-H-NMR (CDCI3) (300 MHz): δ = 8.09 (s, IH, 2-H); 7.98 (s, IH, 2-H); 7.93 (s, IH, 8-H); 7.85 (s, IH, 8-H); 7.50-7.16 (m, 36H, MMT); 6.85-6.75 (m, 6H, MMT); 6.10 (d, IH, l'-HA "); 6.02 (d, IH, l'-H-A '); 5.73 (m, IH, 2'-H-A'); 5.45 (m, IH, 2 '-HA"); 5.05 (m, IH, 3'-HA "); 4.40 (in, IH, 4'-HA"); 4.10 (m, IH, 4'-H-A '); 3.81, 3.80, 3.79 (3s, 9H, CH3-O-MMT); 3.36 (m, 2H, 5'-CH ₂ -A '); 2.90 (m, 2H, 5'-CH ₂ -A "); 2.59 (m, IH, 3'-H'-A '); 2.10 (m, IH, 3'-H"-A'); 1.6 (s, 3H, CH3); 1.38 (s, 3H, CH3).

5'-Deoxy-5'-carboxylic acid 2 '- [3'-deo - y-adenyl] ester 4

60 mg (0.04 mmol) 3 were dissolved in 3 ml dichloromethane and 30 mg trichloroacetic acid were added. After 30 minutes the solution was partitioned between diethyl ether and water, the aqueous phase extracted with diethyl ether and evaporated. The residue was taken up in a little dichloromethane and digested in diethyl ether. The precipitate was centrifuged, washed several times with diethyl ether and dried. Yield: 8 mg (38% of theory) 4. TLC: dichloromethane / methanol = 8/2, ref .: 0.42

REPLACEMENT SHEET (RULE 26) 4: white crystals by NMR (D ₂ 0) (300 MHz): δ = 8.56 (s, IH, 2-H); 8.41 (s, 2H, 2-H, 8-H); 8.35 (s, IH, 8-H); 6.20 (d, IH, l'-HA "); 6.12 (d, IH, l'-H-A '); 5.65 (m, IH, 2'-H-A'); 5.57 (m, IH, 2 '- HA "); 5.09 (m, IH, 3'-HA "); 4.68 (m, IH, 4'-HA"); 4.30 (m, IH, 4'-HA "); 3.86 (m, IH, 5'-H'-A '); 3.66 (m, IH, 5 ^* -H"-A'); 2.92 (m, IH, 5'-H'-A "); 2.78 (m, IH, 5'-H" -A "); 2.40 (3'-H'-A '); 2.10 (m, IH, 3 ^* -H "-A '); 1.66 (s, 3H, CH3); 1.44 (s, 3H, CH3).

Formula scheme I:

12th

SPARE BLADE (RULE 26) Biopysical binding studies on the interaction of dinucleotide ester 4 with polyuridinyl acid (Poly-U)

Under certain conditions - in the presence of magnesium salts - Poly-U forms complexes with mono, di and oligonucleotides ¹ . By Th. Ackermann et al. it was demonstrated by IR spectroscopy ² that triple helices are formed.

The strong hypochromic effect of base stacking lends itself to the detection of duplex or triplex aggregates, particularly because of its low substance requirement.

The higher the transition temperature at the same concentration, the more stable the pairing. An additional phosphate linkage, for example from dimer to trimer, stabilizes the triple helix and increases the transition temperature by 12.2 ° C from 13.5 ° C (ApA) to 25.7 ° C (ApApA) 1.

The best way to investigate the binding behavior of a short Watson-Crick complementary strand is with the additional cooperative "support" of a third polymer strand that forms the pair of Hoogsteen. The artificial dinucleotide 4 was thus examined under salt and buffer conditions that allow triplex formation.

For this purpose 4 was mixed in a 1/2 ratio with poly-U in the buffer. The solution was heated to 90 ° C and cooled again to 5 ° C. The temperature was then gradually increased to 40 ° C. A change in the absorption with the temperature was clearly observed. A hyperchromicity of 36% was measured.

The transformation temperature of the 2Poly-U / artificial dinucleotide system was 15 ° C. It could thus be shown that such 2'-5 'ester-linked artificial nucleotides recognize natural 3'-5'-phosphate-linked oligonucleotides and can interact with them.

¹ AM Michelson C. Monny, Biochim. Biophys. Ada 149 (1967) 107

² U. Schemau, S. Marcino ski and Th. Ackermann Journal for Physical Chemistry 117 (1979) 11-18 Molecular dynamics simulations of such oligonucleotide esters - a ide

Mole ^ dynamics (MD) simulations of duplexes from a modified strand and a natural complementary RNA strand were carried out (FIG. 1). Conformation changes, movement and stability of the modified oligonucleotides were observed. The behavior of the ogonucleotide analogs mentioned in this invention was compared to both natural RNA and other more flexible linkage types.

2'-5'-oligonucleotide analog RNA double strand

GGAAGGAGCU ⁰ ?

1 1 1 1 1 1 1 1 1 1

C C U U C C U C G A r C rX pXpXpXpXp λpλpλpλpλP P

X = 2'-5'-ester, ether or amide linkage

The ester modification showed the highest affinity for the complementary RNA strand. More flexible linking systems such as ether modification did not show a high affinity for the counterpart, but the hybrid double strand dissolved after a short time and the strands became entangled.

f SET BLADE (RULE 26)

Claims

Claims:

1. Nucleotide carboxylic acid esters and amides of the general formula I where:

the sugar rings of the nucleotides are linked to at least one bridge, which from 3

Atoms exists and contains either a carboxylic acid ester or carboxamide group and in which

A0 -0-C (= O = -, -N (-Rι) -C (= 0) - where Rι = H, or unbranched or branched unsubstituted or terminally substituted with acid or base function alkyl with a chain length of 1 to 6C- Atoms means in which R ₂ and R ₃ = H or

Lower alkyl in conventional combination means and R} = H, OH or unbranched or branched unsubstituted or terminally substituted with acid or base function

O-alkyl with a chain length of n = 1-6 carbon atoms and in which

B the N-glycosidically bound residue of a purine or pyrimidine base of the general

Form II or III means, wherein

π

R _{5 is} a hydrogen, chlorine or bromine atom, a hydroxyl, amino, monomemoxytritylamino, mercapto, methylamino, dimeraylamino, phenylamino or hydroxylamino, R _ö β in hydrogen, chlorine or bromine atom, a hydroxyl, amino, mercapto group, R ₇ a hydroxyl or amino group,

Rg is an oxygen atom (= 0), and

R _{9 is} hydrogen, fluorine, chlorine, bromine or iodine atom, a methyl, trifluoromethyl,

Ethyl or hydroxymethyl means, as well as the salts of compounds of the general

Formula I with acids or bases.

2. Nucleotide oligoesters and oligoamides according to claim 1, characterized in that the number of nucleotide units is 2 to 200.

3. Manufacturing process

Nucleotide esters and oligoamides of the formula I characterized in that, after the carboxylic acid has been activated beforehand, the monomers are condensed by customary methods.

4. Use of the compounds of formula I for the production of therapeutic agents and for diagnostic purposes

5. Use of the nucleotide oligoesters and oligoamides of the formula I for incorporation into natural and differently modified oligonucleotides.