US 5,844,106 AGranted

Modified oligonucleotides, their preparation and their use

AssigneeHoechst Aktiengesellschaft

InventorsFrank Seela, Horst Thomas

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Abstract

Modified oligonucleotides which possess at least one substituted 7-deazapurine base form more stable hybridization complexes with nucleic acids than unsubstituted analogs. They are useful as inhibitors of gene expression, as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents. Processes for preparing

Claims17

Dependent claims

An oligonucleotide of the formula I ##STR41## or a physiologically acceptable salt thereof, in which B is, independently of each other, a base which is customary in nucleotide chemistry, and at least one B is a base of the formula II ##STR42## in which R.sup.15 and R.sup.16 are, independently of each other, selected from the group of radicals consisting of:

(1) hydrogen
(2) halogen,
(3) (C.sub.1 -C.sub.10)-alkyl,
(4) (C.sub.2 -C.sub.10) -alkenyl,
(5) (C.sub.2 -C.sub.10) -alkynyl,
(6) NO.sub.2,
(7) NH.sub.2,
(8) cyano,
(9) --S--(C.sub.1 -C.sub.6) -alkyl,
(10) (Cl-C.sub.6) -alkoxy,
(11) (C.sub.6 -C.sub.20) -aryloxy,
(12) SiH.sub.3, ##STR43## (14) radical (3), (4) or (5) substituted by one or more radicals selected from the group consisting of SH, S--(C.sub.1 -C.sub.6)-alkyl, (C.sub.1 -C.sub.6)-alkoxy, OH, --NR(c)R(d), --CO--R(b), --NH--CO--NR(c)R(d), --NR(c)R(g), --NR(e)R(f), and --NR(e)R(g), or by a polyalkyleneglycol radical of the formula --O--(CH.sub.2).sub.r !.sub.s --NR(c)R(d), where r and s are, independently of each other, an integer between 1 to 18, wherein any of the foregoing OH, SH, --CO--R(b), --NH--CO--NR(c)R(d), --NR(c)R(d), --NR(e)R(f), and --NR(e)R(g) or --NR(c)R(g) groups optionally is linked, via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, and
(15) radical (3), (4) or (5) in which from one to all the H atoms are substituted by halogen;
with the provisos that:
when R.sup.15 is hydrogen, R.sup.16 is selected from the group of radicals consisting of radicals (2)-(15), when R.sup.16 is hydrogen, R.sup.15 is selected from the group of radicals consisting of radicals (2), (6), (7), (9), (10), (11), (12), (13), (14), (15), ethyl, propyl, isobutyl, pentyl, hexyl, heptyl, 1-hexenyl and 1-heptenyl;
and R.sup.15 and R16 cannot both be NO.sub.2, NH.sub.2, cyano or SiH.sub.3 ;
R(a) is OH, (C.sub.1 -C.sub.6)-alkoxy, (C.sub.6 -C.sub.22)-aryloxy, NH.sub.2 or NH--T, where T is an alkylcarboxyl group or alkylamino group which is linked to one or more groups, via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,
R(b) is hydroxyl, (C.sub.1 -C6)-alkoxy or --NR(c)R(d),
R(c) and R(d) are, independently of each other, H or (C.sub.1 -C.sub.6) -alkyl which is unsubstituted or substituted by --NR(e)R(f) or --NR(e)R(g),
R(e) and R(f) are, independently of each other, H or (C.sub.1 -C.sub.6)-alkyl,
R(g) is (C.sub.1 -C.sub.6)-alkyl--COOH;
E and F are, independently of each other, H, OH or NH.sub.2,
R.sup.1 is hydrogen, C.sub.1 -C.sub.18 -alkyl, C.sub.2 -C.sub.18 -alkenyl, C.sub.2 -C.sub.18 -alkynyl, C.sub.2 -C.sub.18 -alkylcarbonyl, C.sub.3 -C.sub.19 -alkenylcarbonyl, C.sub.3 -C.sub.19 -alkynylcarbonyl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIIa ##STR44## R.sup.1a i s hydrogen, C.sub.1 -C.sub.8 -alkyl, C.sub.2 -C.sub.18 -alkenyl, C.sub.2 -C.sub.18 -alkynyl, C.sub.2 -C.sub.18 -alkylcarbonyl, C.sub.3 -C.sub.19 -alkenylcarbonyl, C.sub.3 -C.sub.19 alkynylcarbonyl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, or a radical of the formula IIIb ##STR45## R.sup.2 is hydrogen, hydroxyl, C.sub.1 -C.sub.18 -alkoxy, C.sub.1 -C.sub.6 -alkenyloxy, halogen, azido or NH.sub.2 ;
a is oxy, sulfanediyl or methylene;
n is an integer .gtoreq.1;
W is oxo, thioxo or selenoxo;
V is oxy, sulfanediyl or imino;
Y is oxy, sulfanediyl, imino or methylene;
Y' is oxy, sulfanediyl, imino, (CH.sub.2).sub.m or V(CH.sub.2).sub.m, in which m is an integer from 1 to 18;
X is hydroxyl or mercapto;
U is hydroxyl, mercapto, SeH, C.sub.1 -C.sub.18 -alkoxy, C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -aryl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, NHR.sup.3, NR.sup.3 R.sup.4 or a radical of the formula IV
OCH.sub.2 CH.sub.2).sub.p O(CH.sub.2).sub.q CH.sub.2 R.sup.5 (IV)
in which
R.sup.3 i s C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -aryl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.3)-alkyl,--(CH.sub.2)--NH(CH.sub.2) !d-NR .sup.6 R, in which c is an integer from 2 to 6 and d is an integer from 0 to 6, and each R.sup.6 is, independently, hydrogen or C.sub.1 -C.sub.6 -alkyl or C.sub.1 -C.sub.4 -alkoxy-C.sub.1 -C.sub.6 -alkyl;
R.sup.4 is C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -aryl or (C.sub.6 -C.sub.10)-aryl-C.sub.1 -C.sub.8)-alkyl, or, in the case of NR.sup.3 R.sup.4, is, together with R.sup.3 and the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further O, S or N, heteroatom,
p is an integer from 1 to 100,
q is an integer from 0 to 22,
R.sup.5 is hydrogen or is a substituent selected from the group consisting of hydroxyl, amino, C.sub.1 -C.sub.18 -alkylamino, COOH, CONH.sub.2, COO(C.sub.1 -C.sub.4) -alkyl and halogen;
Z and Z' are, independently of each other, hydroxyl, mercapto, SeH, C.sub.1 -C.sub.22 -alkoxy, --O--(CH.sub.2).sub.b -NR.sup.6 R.sup.7, in which b is an integer from 1 to 6, and R.sup.7 is C.sub.1 -C.sub.6 -alkyl or R.sup.6 and R.sup.7, together with the nitrogen atom carrying them, form a 3-6-membered ring, C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -alkyl, (C.sub.4 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8) -alkoxy, where aryl includes heteroaryl and any aryl is optionally substituted by 1,2 or 3 identical or different radicals selected from the group consisting of carboxyl, amino, nitro, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -alkoxy, hydroxyl, halogen and cyano, C.sub.1 -C.sub.18 -alkylmercapto, NHR.sup.3, NR.sup.3 R.sup.4, a radical of the formula IV, or a group which favors intracellular uptake or serves for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attacks the latter while binding, cross-linking or cleaving, and the curved bracket indicates that R.sup.2 and the adjacent phosphoryl or --Y'--R.sup.1a radical can either be located in the 2' and 3' position or else, conversely, in the 3' and 2' position, with it being possible for each nucleotide to be present in its D or L configuration, and for the base B to be located in the .alpha. or .beta. position.

36 more elements

Dependencies are inferred from the claim text.

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Abstract

(1) hydrogen
(2) halogen,
(3) (C.sub.1 -C.sub.10)-alkyl,
(4) (C.sub.2 -C.sub.10) -alkenyl,
(5) (C.sub.2 -C.sub.10) -alkynyl,
(6) NO.sub.2,
(7) NH.sub.2,
(8) cyano,
(9) --S--(C.sub.1 -C.sub.6) -alkyl,
(10) (Cl-C.sub.6) -alkoxy,
(11) (C.sub.6 -C.sub.20) -aryloxy,
(12) SiH.sub.3, ##STR43## (14) radical (3), (4) or (5) substituted by one or more radicals selected from the group consisting of SH, S--(C.sub.1 -C.sub.6)-alkyl, (C.sub.1 -C.sub.6)-alkoxy, OH, --NR(c)R(d), --CO--R(b), --NH--CO--NR(c)R(d), --NR(c)R(g), --NR(e)R(f), and --NR(e)R(g), or by a polyalkyleneglycol radical of the formula --O--(CH.sub.2).sub.r !.sub.s --NR(c)R(d), where r and s are, independently of each other, an integer between 1 to 18, wherein any of the foregoing OH, SH, --CO--R(b), --NH--CO--NR(c)R(d), --NR(c)R(d), --NR(e)R(f), and --NR(e)R(g) or --NR(c)R(g) groups optionally is linked, via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, and
(15) radical (3), (4) or (5) in which from one to all the H atoms are substituted by halogen;
with the provisos that:
when R.sup.15 is hydrogen, R.sup.16 is selected from the group of radicals consisting of radicals (2)-(15), when R.sup.16 is hydrogen, R.sup.15 is selected from the group of radicals consisting of radicals (2), (6), (7), (9), (10), (11), (12), (13), (14), (15), ethyl, propyl, isobutyl, pentyl, hexyl, heptyl, 1-hexenyl and 1-heptenyl;
and R.sup.15 and R16 cannot both be NO.sub.2, NH.sub.2, cyano or SiH.sub.3 ;
R(a) is OH, (C.sub.1 -C.sub.6)-alkoxy, (C.sub.6 -C.sub.22)-aryloxy, NH.sub.2 or NH--T, where T is an alkylcarboxyl group or alkylamino group which is linked to one or more groups, via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,
R(b) is hydroxyl, (C.sub.1 -C6)-alkoxy or --NR(c)R(d),
R(c) and R(d) are, independently of each other, H or (C.sub.1 -C.sub.6) -alkyl which is unsubstituted or substituted by --NR(e)R(f) or --NR(e)R(g),
R(e) and R(f) are, independently of each other, H or (C.sub.1 -C.sub.6)-alkyl,
R(g) is (C.sub.1 -C.sub.6)-alkyl--COOH;
E and F are, independently of each other, H, OH or NH.sub.2,
R.sup.1 is hydrogen, C.sub.1 -C.sub.18 -alkyl, C.sub.2 -C.sub.18 -alkenyl, C.sub.2 -C.sub.18 -alkynyl, C.sub.2 -C.sub.18 -alkylcarbonyl, C.sub.3 -C.sub.19 -alkenylcarbonyl, C.sub.3 -C.sub.19 -alkynylcarbonyl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIIa ##STR44## R.sup.1a i s hydrogen, C.sub.1 -C.sub.8 -alkyl, C.sub.2 -C.sub.18 -alkenyl, C.sub.2 -C.sub.18 -alkynyl, C.sub.2 -C.sub.18 -alkylcarbonyl, C.sub.3 -C.sub.19 -alkenylcarbonyl, C.sub.3 -C.sub.19 alkynylcarbonyl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, or a radical of the formula IIIb ##STR45## R.sup.2 is hydrogen, hydroxyl, C.sub.1 -C.sub.18 -alkoxy, C.sub.1 -C.sub.6 -alkenyloxy, halogen, azido or NH.sub.2 ;
a is oxy, sulfanediyl or methylene;
n is an integer .gtoreq.1;
W is oxo, thioxo or selenoxo;
V is oxy, sulfanediyl or imino;
Y is oxy, sulfanediyl, imino or methylene;
Y' is oxy, sulfanediyl, imino, (CH.sub.2).sub.m or V(CH.sub.2).sub.m, in which m is an integer from 1 to 18;
X is hydroxyl or mercapto;
U is hydroxyl, mercapto, SeH, C.sub.1 -C.sub.18 -alkoxy, C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -aryl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, NHR.sup.3, NR.sup.3 R.sup.4 or a radical of the formula IV
OCH.sub.2 CH.sub.2).sub.p O(CH.sub.2).sub.q CH.sub.2 R.sup.5 (IV)
in which
R.sup.3 i s C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -aryl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.3)-alkyl,--(CH.sub.2)--NH(CH.sub.2) !d-NR .sup.6 R, in which c is an integer from 2 to 6 and d is an integer from 0 to 6, and each R.sup.6 is, independently, hydrogen or C.sub.1 -C.sub.6 -alkyl or C.sub.1 -C.sub.4 -alkoxy-C.sub.1 -C.sub.6 -alkyl;
R.sup.4 is C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -aryl or (C.sub.6 -C.sub.10)-aryl-C.sub.1 -C.sub.8)-alkyl, or, in the case of NR.sup.3 R.sup.4, is, together with R.sup.3 and the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain a further O, S or N, heteroatom,
p is an integer from 1 to 100,
q is an integer from 0 to 22,
R.sup.5 is hydrogen or is a substituent selected from the group consisting of hydroxyl, amino, C.sub.1 -C.sub.18 -alkylamino, COOH, CONH.sub.2, COO(C.sub.1 -C.sub.4) -alkyl and halogen;
Z and Z' are, independently of each other, hydroxyl, mercapto, SeH, C.sub.1 -C.sub.22 -alkoxy, --O--(CH.sub.2).sub.b -NR.sup.6 R.sup.7, in which b is an integer from 1 to 6, and R.sup.7 is C.sub.1 -C.sub.6 -alkyl or R.sup.6 and R.sup.7, together with the nitrogen atom carrying them, form a 3-6-membered ring, C.sub.1 -C.sub.18 -alkyl, C.sub.6 -C.sub.20 -alkyl, (C.sub.4 -C.sub.14)-aryl-(C.sub.1 -C.sub.8)-alkyl, (C.sub.6 -C.sub.14)-aryl-(C.sub.1 -C.sub.8) -alkoxy, where aryl includes heteroaryl and any aryl is optionally substituted by 1,2 or 3 identical or different radicals selected from the group consisting of carboxyl, amino, nitro, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -alkoxy, hydroxyl, halogen and cyano, C.sub.1 -C.sub.18 -alkylmercapto, NHR.sup.3, NR.sup.3 R.sup.4, a radical of the formula IV, or a group which favors intracellular uptake or serves for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attacks the latter while binding, cross-linking or cleaving, and the curved bracket indicates that R.sup.2 and the adjacent phosphoryl or --Y'--R.sup.1a radical can either be located in the 2' and 3' position or else, conversely, in the 3' and 2' position, with it being possible for each nucleotide to be present in its D or L configuration, and for the base B to be located in the .alpha. or .beta. position.

36 more elements