The present invention relates to novel oligonucleotides containing modified bases with valuable physical, biological and pharmacological properties, a method for their production and their use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), probes for the detection of nucleic acids, aids in molecular biology and as medicinal or diagnostic agents.
The literature has produced numerous chemical modifications of oligonucleotides, which can affect the sugar-phosphate scaffold or the nucleobases.
Chemical modification of oligonucleotides is usually required because unmodified oligonucleotides are degraded very rapidly by nucleolytic activities both in cells and in the cell culture medium. Stabilisation against nucleolytic degradation can be achieved by replacing the sugar-phosphate backbone or by modifying the phosphate bridge, sugar structure or nucleobase [Milligan et al., supra and Uhlmann & Peyman, supra].
In addition to modifications leading to oligonucleotides with increased stability to nucleolytic degradation, modifications of interest are those that alter the hybridization behaviour of the modified oligonucleotides in such a way that they can, for example, form more stable hybridization complexes (duplexes) with intracellular nucleic acids (so-called target nucleic acids).
However, PCT application WO 93/10820 also describes oligonucleotides containing modified uracil or cytosine bases and capable of forming more stable duplex or triplex structures with the target nucleic acids compared to the unmodified oligonucleotides. The hybridisation properties of synthetic dodecameroligonucleotides containing the base analogue pyridine pyridine were also described [In Research et al. 1985), 13,1971 and 17,1912 and 17,177 (Amino Acid Research, 16,173- 30,179] which also contained a 2-dehydrogen nucleotide analogue containing the base analogue pyridine.
The results of the study were published in the Journal of Biochemistry and Biophysics, Volume 1, No. 1, 1968, pp. 147-155 (P.149, Table 1; Summary).
The present invention is therefore intended to provide new oligonucleotides with advantageous properties.
Surprisingly, oligonucleotides containing at least one 8-aza-purine base, e.g. 8-azaguanin and 8-azadenin, were found to form significantly more stable hybridisation complexes with the target nucleic acids than comparable oligonucleotides containing the unmodified purine bases.
The invention thus relates to oligonucleotides of formula Iand their physiologically compatible saltsR1 means hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C2-C18 alkylcarbonyl, C3-C19 alkenylcarbonyl, C3-C19 alkynylcarbonyl, (C6-C14) aryl- ((C1-C8) alkyl, a common protecting group in nucleotide chemistry or a residue of formula IIa;R1a means hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C2-C18 alkynyl, C2-C18 alkylcarbonyl, C3-C19 alkylcarbonyl, C3-C19 alkylcarbonyl, (C6-C14) aryl-C1-C8-alkyl, or a residue of formula IIb;R2 means hydrogen, hydroxy, C1-C18 alkoxy, C1-C6-alkenyloxy, halogen, azide or NH2; stands for oxyhydride or methyl;any integer from 3 to 99;WOxo, Thio or selenoxo;VOxo means sulphyl or imino; sulphyl or sulphyl methyne;CHY means sulphyl, sulphyl or iminoxy;CHY means sulphyl, sulphyl or iminoxy (H1C18H2O), which is a whole number of mercaptoxy, C1-H18O, C1-H18O, or mercaptoxy, C1-H18O, or mercaptoxy, which is an integer from 1 to 18;C6-C20-aryl, (C6-C14)-aryl- ((C1-C8)) alkyl, NHR3, NR3R4 or a residue of formula IIIOtherThe following is added to the list of active substances:Othermeans in whichC6-C20-aryl, (C6-C14) -aryl- ((C1-C8) -alkyl, 2-(CH2) -c-[NH(CH2) c-d-NR6R6, wherein c is an integer from 2 to 6 and d is an integer from 0 to 6, and R6 is independently hydrogen or C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl;R4C1-C18-alkyl, C6-C20-aryl or (C6-C10) -aryl- ((C1-C8) -alkyl, or in the case of NR3R4 together with R3 and the nitrogen atom carrying it, a 5- to 6-membered heterocyclic ring, which may additionally contain a heteroatom from the series, S, N, numbered from 0 to 100;R0 is an entire hydrogen ring, for example, an Oxy ring or an Amino ring, such as R22R5C1-C18-alkylamino, COOH, CONH2, COO(C1-C4) alkyl or halogen means:Z and Z independently hydroxy, mercapto, SeH, C1-C22-alkoxy, -O-(CH2)b-NR6R7, where b is an integer from 1 to 6 and R7 is a C1-C6 alkyl or R6 and R7 together with the nitrogen they contain form a 3-6-membered ring, C1-C18-alkyl, C6-C20-aryl, (C6-C14)-aryl-C1-C8) alkyl, (C6-C14)-aryl-C1-C8-alkoxy, (C6-C14)-alkoxy, where aryl also means aryldan and where appropriate by 1, 2 or 3 or more different sequences of aryl, C1-C1-C1-C1-C4-C1-C1-C1-C1-C1-C3-C1-C1-C3-C3-C3-C4-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-C3-The cross-linking or splitting attack.any nucleotide can be in its D or L configuration and base B can be in the α or β position, whereby the bases are independent of each other for a base common to nucleotide chemistry, for example natural bases such as adenine, cytosine, thymine, guanine, uracil, hypoxanthin or unnatural bases such as purine, 8-azapurin, 2.6-diaminopurin, 7-deazadenine, 7-deazaguanine, N4N4-ethytosine, N6N6-ethano-2.6-diaminopurin, pseudoisocytosine, 5-methylcytosine, 5-Fluoruracil, 5-C6-C3-C3-C3-C3-C3-C3-C6-C6-C6-C3-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-C6-where E and F are independently H, OH or NH2.
The preferred formula is formula I, where E is equal to NH2 and F is equal to OH or E is equal to H and F is equal to NH2.The most commonly used compounds are those of formula I, where E is equal to NH2 and F is equal to OH.The preferred formulation is also that of compounds of formula I, where the base is in the β position on the sugar, the nucleosides are in the D configuration, R2 is in the 2′ position and a stands for Oxy.
The oligonucleotides of the invention have an improved binding affinity to complementary nucleic acids (target nucleic acids) compared to natural oligonucleotides, and it is advantageous for the therapeutic use of these oligonucleotides to introduce additional modifications, such as the phosphate backbone, the ribose unit or the oligonucleotide endings, into these oligonucleotides (J.S. Cohen, Topics in Molecular and Structural Biology 12 (1989) Macmillan Press, E. Uhlmann et al., supra).For example, well-known modifications of the sugar-phosphate backbone provide even better protection against attack by nucleases for the oligonucleotides of the invention, which is advantageous.
Therefore, compounds of formula I are also preferred, where V, Y, Y' and W have the meaning of thioxo, selenoxo, oxy, oxo, sulfandiyl, imino or methylene and U has the meaning of hydroxy, mercapto or methyl.
A preferred embodiment is also compounds of formula I, where R1 and R1a mean hydrogen.
In particular, preference is given to compounds of formula I in which R1 and/or R1a hydrogen, R2 hydroxy or hydrogen, U hydroxy or mercapto and V, Y, Y' and W have the meaning of thioxo, oxy, oxo or hydroxy.
The protective groups commonly used in nucleotide chemistry include, for example, amino, hydroxyl or other protective groups as described in [E.Sonveaux, 1986, Bioorganic Chemistry, 14, 274-325 or S.L. Beaucage et al., 1992, Tetrahedron, 48, 2223-2311].
Alkyl, alkenyl and alkynyl can be straight or branched.
Cycloalkyl is also understood to include alkyl-substituted rings.
(C6-C20) aryl is, for example, a phenyl, a naphthyl or a biphenyl, preferably a phenyl.
Heteroaryl is defined as residues derived from phenyl or naphthyl in which one or more CH groups are replaced by N and/or in which at least two adjacent CH groups (forming a five-membered aromatic ring) are replaced by S, NH or O. One or both atoms at the condensation site of bicyclic residues (as in indolizinyl) may also be N atoms.
The term 'heteroaryl' includes in particular furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indoyl, indazolyl, quinolyl, isokinolyl, phthalazinyl, chinoxalinyl, chinazolinyl and cynolinyl.
Physiologically compatible salts of compounds of formula (I) are both inorganic and organic salts as described in Remington's Pharmaceutical Sciences (17th edition, page 1418 (1985)).
For acidic groups, sodium, potassium, calcium and ammonium salts are preferred due to their physical and chemical stability and solubility.
The invention is not limited to α- and β-D- or L-ribofuranosides, α- and β-D- or L-deoxyribofuranosides and their corresponding carbocyclic pentacyclic analogues, but also applies to oligonucleotide analogues made from other sugar building blocks, such as xylo and arabinofuranose derivatives, ring-extended and ring-narrowed sugars, acyclic, ring-broken or other suitable sugar derivatives.
The oligonucleotides of the invention can therefore be modified from the natural structure in a variety of ways, such as by known methods:
(a) Modifications to the phosphate bridgeThe following are examples: phosphorothioates, phosphorodithioates, methylphosphonates, phosphoramidates, borano-phosphates, phosphate methyl esters, phosphate ethyl esters, phenyl phosphonates.
(b) Replacement of the phosphate bridgeFor example, substitution by formacetal, 3'-thioformacetal, methyl hydroxylamine, oxime, methyldimethyl hydrazone, dimethyl sulfone, silyl groups, and the preferred substitution by formacetals and 3'-thioformacetals.
(c) Modifications of sugarThe following are examples: α-anomic sugars, 2'-O-methylribose, 2'-O-butylribose, 2'-O-allylribose, 2'-fluoro-2'-deoxyribose, 2'-amino-2'-deoxyribose, α-arabinofuranose, carbocyclic sugar analogues.
(d) Modifications of the sugar and phosphate bridgeExamples are peptide nucleic acids (PNAs) in which the sugar/phosphate backbone is replaced by an aminoethylglycine backbone and the carbamated morpholine oligomers.
(e) Other modifications of bases, in particular pyrimidine basesThe following are examples: 5-propinyl-2'-deoxyurin, 5-propinyl-2'-deoxycytidine, 5-hexinyl-2'-deoxyurin, 5-hexinyl-2'-deoxyurin, 5-fluoro-2'-deoxyurin, 5-fluor-2'-deoxyurin, 5-hydroxymethyl-2'-deoxyurin, 5-methyl-2'-deoxyurin, 5-bromo-2'-deoxyurin. The preferred modifications are 5-propinyl-2'-deoxyurin, 5-hinyl-2'-deoxyurin, 5-hexinyl-2'-deoxyurin and 5-propyl-2'-deoxyurin.
(f) 3'-3' and 5'-5' inversions (e.g. M. Koga et al., J. Org. Chem. 56 (1991) 3757) The following are the main types of inversions:(g) 5' and 3' conjugates.The groups that favor intracellular uptake are various lipophilic residues such as -O-(CH2)x-CH3, where x means an integer from 6 to 18, -O-(CH2)n-CH=CH-(CH2)m-CH3, where n and m independently mean an integer from 6 to 12, -O-(CH2CH2O)4-CH2)9-CH3, -O-(CH2CH2O)8- (CH2)13-CH3 and -O-CH2CH2O)7-CH2)15-CH3, but also steroid residues such as cholesterol or vitamin-derived residues such as vitamin E, vitamin A or vitamin D and other conjugates that are natural carrier groups such as fluoroacetylphenol, 2-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-N-F-F-N-F-N-F-F-N-F-F-N-F-F-N-F-F-N-F-F-N-F-F-N-F-F-F-N-F-F-F-N-F-F-F-N-F-F-F-N-F-F-F-F-N-F-F-F-F-F-N-F-F-F-F-F-F-N-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-FFluorescein or coumarin derivatives or chemiluminescent groups, e.g. acridine derivatives, and the digoxygenin system detectable by ELISA, the biotin group detectable by the biotin/avidin system, or linker arms with functional groups allowing subsequent derivation with detectable reporter groups, e.g. an aminoalkyl linker implemented with an acridinium activator into the chemiluminescence sample. Typical marking groups are:R = H or amino-protective groupOligonucleotide analogues that bind to or intercalate and/or cleavage or cross-linking nucleic acids contain acridine, psoralen, phenanthridine, naphtoquinone, daunomycin or chlorethylaminoaryl conjugates. Typical intercalating and cross-linking residues are:The following equation is used for the calculation of the concentration of the product:The following equation is used for the calculation of the concentration of the product:For example, groups NR3R4,where R3 and R4 together with the nitrogen atom they contain form a 5- to 6-membered heterocyclic ring containing an additional heteroatom, the morpholinyl and imidazolidinyl residues are given.
The invention is furthermore intended to be used for the compounding of formula VIn whichVOxy, sulfandiyl or imino is;R1 stands for oxy or methyl;R1 means a common protective group in nucleotide chemistry;R1 means a residue of formula IIc or IIdUO-R7 or S-R7 means;No residue means -NR8R9 meansR7-(CH2) 2-CN means;R8 and R9 are the same or different and mean C1-C6 alkyl, in particular isopropyl or ethyl, or together with the nitrogen atom they carry a 5-9 membered heterocyclic ring which may additionally contain another heteroatom from the series O, S, N, in particular E' and F' independently H, OH, or NR10R11 means, whereR10 and R11 are the same or different and mean hydrogen or an amino group commonly used in nucleotide chemistry or R10 and R11 together constitute an amino group commonly used in nucleotide chemistry,R12 means a hydroxyl group commonly used in nucleotide chemistry, such as t-butyl dimethyl silyl, tri-isopropyl silyl, o-nitro-benzyl, p-nitro-benzyl, tr 1-methyl-ethyl) trisyl or 2-fluorophenyl-4-methoxyperidine-4-silyl (FPMP),
A preferred embodiment is compounds of formula (V) where V, Yb and a stand for Oxy, R2b for hydrogen or OR12, in particular hydrogen, and R1b for a residue of formula (IIc) or (IId), where U stands for O- ((CH2) 2) CN and R8 and R9 are equal or different and stand for isopropyl or ethyl.These are particularly preferable if the base is in the β-position and R2b is in the 2' position.
The invention also relates to compounds of formula V in which the linkage to the sugar residue is made via the N2 atom of the 8-azapurin base.
The preferred amino-protective groups are, for example, acyl or amidine protective groups.
Err1:Expecting ',' delimiter: line 1 column 143 (char 142)
Err1:Expecting ',' delimiter: line 1 column 132 (char 131)
Err1:Expecting ',' delimiter: line 1 column 244 (char 243)
The compounds of the invention of formula VI can be used as aids in molecular biology, for example in PCR reactions (e = f = 1, R13 = OH) or for sequencing (e = f = 1; R13 = H or OH).
The presentation of the compounds of formula VI is possible from the corresponding 8-azapurin nucleosides and is carried out by generally known methods, preferably by a reduced Ludwig stew process in the presence of 1,8-bis ((dimethylamino) naphathaline and trimethyl phosphate (J. Ludwig et al., 1981) Acta Biochem.
The invention also covers all tautomeric forms of the compounds of formula I, V and VI, in particular all tautomeric forms of the 8-azapurin bases of formula IV.
The invention continues to be a method for the production of the compounds of formula I according to the invention.
The production of 8-azaguanine-containing tri-ribonucleoside diphosphate by enzymatic or chemical/enzymatic means is described (Grünberger et al., Biochemical Biophysics Acta, (1968) 161, 147-155). The enzymatic synthesis of double-stranded 8-azaguanine-containing phage DNA by DNA polymerase is described by Bodnar et al., (1983) J. Biol. Chem., 258, 15206-15213.
Because the N-glycosidic bond of 8-azadesoxyguanosine is extremely acid stable (Seela et al., (1993), Helv. Chim. Acta, 76, 2388-2397), the standard conditions common to the chemical synthesis of oligonucleotides can be used to produce the 8-azapurin-containing oligonucleotides of the invention.
The compounds of the invention of formula I are represented in solution or preferably in the solid phase, where appropriate by means of an automatic synthesis device.
The oligonucleotide is formed by methods known to the professional such as the triester method, the H-phosphonate method or the phosphoramidite method [E. Sonveaux, (1986), Bioorganic Chemistry, 14, 274-325; S.L. Beaucage et al., (1992), Tetrathedron, 48, 2223-2311].
The provision of the compounds of formula V as building blocks for oligonucleotide solid-phase synthesis can be done from the corresponding 8-azapurin nucleosides. After introduction of appropriate protective groups for the amino groups of the 8-azapurin bases and the free 5'-hydroxyl group of the sugar, the monomers are transferred to the corresponding phosphate or phosphoramid derivatives. The introduction of appropriate protective groups, e.g. in the form of a formamidin protective group (dimethylamino) methyl groups) or acyl protective group, is carried out by generally known methods (L.J. Bride et al., (1983) 24 Tetrahedra, 2953, G.S. Ti, and J.S. Ammer (1982) 104, and J.S. Soccer (1963), 134, and J.S. Ammer (1982); in the case of the Tetrahedron method, the use of the S. Soccer method is preferred.A suitable protective group for the free 5'-OH group of the sugar is, for example, 4,4'-dimethoxytrityl, which is also introduced by known methods (C.B. Reese (1978), Tetrahedron, 34, 3143; D. Flockerzi et al., (1981), Liebigs Ann. Chem., 1568). The monomers thus protected can be converted to the corresponding phosphonates according to a prescription by Froehler et al. (B. C. Froehler et al., (1986), Nucl. Acid Res., 14, 5399). The production of cyanoethyl phosphorate derivatives can be done, for example, by converting the monomers with chlor-β-dianoxy-N, N-isopropyl phospholipid in chlor-free acids (N. D. Sinhahan et al., 1984) [N. D. Sinha, Res., 12, 45].
The first difficulty is to find a combination of compatible 2'- and 5'-OH protecting groups. Thus, the residue at the O-2'-position in oligonucleotide synthesis must be stable to the acidic conditions of hydrolysis of the tritiol protecting groups. Furthermore, conditions that could lead to a shift of the phosphate from the 3'- to the 2'-position must be avoided.
The use of triisopropylsilyl chloride as a 2'-OH protective group is advantageous for the synthesis of oligoribonucleotides of formula (I) according to the invention, which produces high selectivity under conditions of sufficient stability and mild cleavage (TBAF/THF).
In contrast to the solid phase synthesis of deoxyribonucleotides, phosphoramid building blocks are less suitable for the solid phase synthesis of oligoribonucleotides, mainly because the steric inhibition of the reactive phosphoramidite by the bulky 2'-silyl protective group requires relatively long coupling times [N. Usman, R.T. Pon, K.K. Ogilviee, Tetrahedron Lett. 26, 1985, 4567] and also produces a lower coupling yield.
The ribonucleotide phosphonates of the invention can be produced according to a rule from Froehler (B. Froehler, P.G. Nug, M.D. Mateuci, Nucl. Acids Res. 1986, 14, 5399).
The synthesis of compounds of formula I, the oligonucleotide part of which is modified at the 3' and/or 5' ends, shall be carried out with respect to these modifications in accordance with the procedures described in EP-A 0 552 766.
The invention also relates to the use of the compounds of formula I in the invention for the manufacture of a medicinal product and to a process for the manufacture of a medicinal product characterized by mixing the oligonucleotides in the invention with a physiologically acceptable carrier and, where appropriate, with suitable additives and/or excipients.
In general, the present invention covers the use of compounds of formula I as therapeutically active components of a medicinal product.
In addition, another subject of the present invention is the use of oligonucleotides with at least one 8-azapurin, preferably with 8-azaguanine or 8-azaadenine as a diagnostic agent, for example to detect the presence or absence or amount of a specific double-stranded or single-stranded nucleic acid molecule in a biological sample.
The length of the oligonucleotides for use in accordance with the invention is 4 to 100, preferably about 5-40, especially about 6-30 nucleotides.
The medicinal products of the present invention may be used, for example, to treat diseases caused by viruses, such as HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.
Antisense oligonucleotide derivatives of the invention, i.e. antisense oligonucleotides in which at least one purine base is replaced by an 8-azapurin base and which are effective against such targets, have, for example, the following base sequence:(a) against HIV, e.g. and and and and and and and and
The drugs of the present invention are, for example, also suitable for the treatment of cancer, for example by using oligonucleotide sequences directed against targets responsible for the development or growth of cancer, such targets being:1) Nuclear oncoproteins such as c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, p1202) Cytoplasmic/membrane-associated oncoproteins such as EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl3) Cellular receptors such as EGF receptor, c-erbA, retinoid receptor, protein kinase regulatory subunit, c-fms4) Cytokines, growth factors, extracellular matrix such as ILF-1, ILF-1, CSF-1, IL-1a, ILb-2, IL-4, ILF-4, myelin, myelin,
For example, the inventive antisense oligonucleotides of formula I that are effective against such targets have the following base sequence:(a) against c-Ha-ras, e.g. c-myc, e.g. c-myb, e.g. c-fos, e.g. p120, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g. p53, e.g.g. p53, e.g.g.g. p53, e.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g.g
For example, the drugs of the present invention are also suitable for the treatment of diseases affected by integrins or cell-cell adhesion receptors, such as VLA-4, VLA-2, ICAM, VCAM or ELAM.
For example, antisense oligonucleotide derivatives of the invention that are effective against such targets have the following base sequence:(a) VLA-4, for example, or (b) ICAM, for example, (c) ELAM-1, for example,
The drugs of the present invention are also suitable, for example, for the prevention of restenosis, for example by using oligonucleotide sequences directed against targets responsible for proliferation or migration, such as:1) Nuclear transactivator proteins and cyclines such as c-myc, c-myb, c-fos, c-fos/jun, cycline and cdc2-kinase2) Mitogens or growth factors such as PDGF, bFGF, EGF, HB-EGF and TGF-β.3) Cellular receptors such as bFGF receptor, EGF receptor and PDGF receptor.
For example, the invention's formula I oligonucleotides that are effective against such targets have the following base sequence:The following are the active substances which are to be used in the manufacture of the test chemical:
The use of medicinal products in liposomes, which may contain other components such as proteins, is also a suitable form of application. They may also be administered rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of solutions for injection. For the manufacture of pressure products, these compounds may be processed in solvent in organic and inorganic carriers. Examples of such carriers are tablets, solid solutions and solvent solutions for liposomes, anti-oxidants for oil, gas, oil, salts, salts, salts, salts, salts, salts, salts, salts, salts and other therapeutic agents, and their derivatives.
The antisense oligonucleotide derivatives are formulated in a liquid solution, preferably in a physiologically acceptable buffer, such as Hank's solution or Ringer's solution, for injection. However, the antisense oligonucleotides can also be formulated in solid form and dissolved or suspended before use. The dosages preferred for systematic administration are approximately 0.01 mg/kg to approximately 50 mg/kg body weight/day.
In general, the invention relates to the use of compounds of formula I as a primer in DNA diagnostics.
Examples:The compounds mentioned in the examples (1) to (16) have the following structural formulae.
| R(a) | R(b) | R(c) | R |
| (7) | | | | |
| (8) | | | | |
| (9) | OH | OH | OH | |
| (10) | OH | OH | OH | |
| (11) | OH | OH | OH | |
| (12) | OH | OH | OH | |
| (13) | OH | OH | Dmt | " |
| (14) | Tms | OH | Dmt | " |
| (15) | OH | Tms | Dmt | " |
| (16) | Tms | TEP | Dmt | " |
3- ((2-Desoxy-β-D-erythro-pentofuranosyl) -5-amino-3H-1,2,3,-triazole[4,5d] pyrimidine-7- (((6H) -on (8-Aza-2'-deoxyguanosine (1))) (26 mg; 0.09 mmol) was dissolved in trimethyl phosphate (0.25 ml) under light heat. After cooling to 0 °C, freshly distilled POCl3 (12 TBl, 0.13 mmol) was added. The reaction was maintained for 4 h at 4 °C and then a solution of 1.8-bis-dimethylamino) naphthalene (33 mg, 0.15 mmol) was added to trimethyl phosphate (0.25 ml) under light heat. The solution was then mixed with 1.8-bis-dimethylamino) naphthalene (33 mg, 0.15 mmol) in trimethyl phosphate (0.5 mmol, 0.12 mmol) and UV-KnO (0.12 mmol) (0.12 mmol) and 0.12 mmol (0.12 mmol) of R-PAMP (0.42 mmol) (0.0 mmol) (0.12 mmol), 0.0 mmol (0.0 mmol), 0.0 mmol (0.0 mmol), 0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.0 mmol) (0.
Example 2:The following definitions shall apply to the following substances:3- ((2-Desoxy-β-D-erythro-pentofuranosyl) -5-amino-3H-1,2,3,-triazole[4,5d] pyrimidine-7-(6H) -on (8-Aza-2'-deoxyguanosine (1)) (290 mg; 1.06 mmol) were dissolved in DMF (7 ml) and replaced with N,N-dimethylformamide diethylacetal (5 ml). After 24 hours of stirring at room temperature, the solution was rotated in a vacuum and co-evaporated with toluene. The residue was purified by chatrography on a silica gel (column: 20 x 4 cm, 0.5 bar, CH2Cl2/OH). The compound obtained was 236 (2) mg (320 mg, 92%) as coloured, SmpOH, a foam crystallised from 8:2°C.The following is the list of active substances in the active substance:The following are the active substances which may be used in the active substance:Other
| Ber. C 44.56, | H 5.29. | N 30.32 |
| Gef. C 44.64, | H 5.26, | N 30.37. |
The amino-protected 8-Aza-2'-deoxyguanosine (2) from example 2 (170 mg, 0.53 mmol) was treated with abs pyridine by repeated evaporation and then absorbed into 6 ml of it. At room temperature, 4.4'dimethoxytritrile chloride (260 mg, 0.7 mmol) was added and stirred for 3 hours. The solution was then poured into 5% NaHCO3 (40 ml) and extracted twice with 30 ml of CH2Cl2. The combined organic phases were dried over Na2SO4 and roasted in a vacuum. The residue was chromatographed to silica (column: 15 cm x 4 cm, 0.5 bar, CH2Cl2/OHMe 95:5). The colour was 270 mg (82 mm Hg); H2O2 (OH, 2.07 mm); H2O2 (OH, 2.06 mm); H2O2 (OH, 2.07 mm); H2O2 (OH, 2.06 mm); H2O2 (OH, 2.37 mm); H2O2 (OH, 3.37 mm); H2O2 (OH, 3.37 mm); H2O2 (OH, 3.37 mm; H2O, 3.37 mm; H2O, 3.37 mm; H2O, 3.37 mm) (OH, 3.37 mm; H2O, 2.39 mm; H2 (OH, 2.39 mm) (OH, 2.70 mm); H2 (OH, 2.70 mm); H2 (OH, 2.39 mm; H2 (OH, 2.70 mm) (OH, 2.50 mm); H2 (OH, 2.50 mm; H2 (OH, 2.70 mm; H2 (OH, 2.50 mm) (OH, 2.50 mm; H2 (OH, 2.50 mm) (OH, 2.50; H2 (OH, 2.50; H2), H2 (OH, 2.70 mm; H2 (OH, 2.50; H2), H2 (OH, 2.50; H2), H2 (OH, 2.50; H2), H2 (OH, 2.50), H2 (OH, 2.90), H2 (OH, 2.0); H2 (OH, 2.0); H2 (OH, 2.0); 2.) H2 (OH, 2.) 3.) 3.)Other
| Ber. C 63.34, | H 5.64, | N 15.67 |
| Gef. C 63.42, | H 5.72, | N 15.71 |
The 5'-O-dimethoxytrityl compound 3 from example 3 (220 mg, 0.35 mmol) was dried by evaporation with dried MeCN and then added, dissolved in CH2Cl2 (5 ml). After 10 min of stirring, the mixture was added to 1 M (Et3NH)HCO3 (TBK buffer, pH 8.0, 30 ml), shaken twice with CH2Cl2 and the resulting organic solutions were mixed with CH2SO2 and dried in a 15 cm x 14 cm x 15 cm x 15 cm x 15 cm x 15 cm x 15 cm x 15 cm.The substance of the main zone was absorbed into CH2Cl2 (10 ml) and mixed several times with 0.1 M TBK buffer (pH 8.0) to obtain H-phosphonate 4 (240 mg, 85%) as a colourless foam.The following are the active substances that may be used in the manufacture of the active substance:The following table shows the results of the analysis:The maximum level of the active substance is calculated as follows:
| Ber. C 59.23, | H 6.49, | N 14.17 |
| Gef. C 59.33, | H 6.79, | N 13.95 |
The solution of compound (3) from example 3 (50 mg, 0.08 mmol) was given to a solution of CH2Cl2 (1 ml) of (i-Pr) 2EtN (56 μl, 0.27 mmol) and chlorine (2-cyanoethoxy) (2-diisopropylamino) phosphane (115 μl, 0.51 mmol). The solution was stirred for 2 hours under argon at room temperature. The mixture was then poured into 5% NaHCO3 (3 ml) and extracted twice with CH2Cl2 (30 ml). The combined organic phases were dried over Na2SO4 and injected to dry. Chromatography was performed on silica (column: 7 x 2 μl, 0.5 bar, CH2Cl2/O/N3CH45:45:10). Two endpoints of the dihydrogen were detected over 0.5 cm of the ethanol (5 mmol/N3CH45) which was detectable as 5 mg/N2OH5 (DC5:45 mmol/N3CH5/10), which was colourless.The following information shall be provided to the competent authority:
Example 6:The following substances are to be classified in the same category as the active substance:The solution of the protected nucleoside (3) from example 3 (110 mg, 0.18 mmol) was added to pyridine (5 ml) and 4-dimethylaminopyridine (30 mg, 0.23 mmol) and amberic acid anhydride (90 mg, 0.88 mmol) were added. The solution was stirred for 48 hours at room temperature. The reaction was stopped by adding 2 ml of water. After evaporation to dry, co-evaporation with toluene to remove residual pyridine. The residue was dissolved in a small amount of CH2Cl2 and washed with a 10% aqueous citric acid solution and water. The organic phase was dried over Na2SO4 and compacted in a vacuum. After dissolving in CH22/Pyridine (95,25 mg, 0.25 ml) n-OH-Pyridine was quickly removed.The total number of samples of the active substance is calculated by dividing the total number of samples of the active substance by the total number of samples of the active substance.
Example 7The following substances are to be classified in the same heading as the active substance:8-Azaadenine (200 mg, 1.47 mmol) is evaporated three times in dry pyridine, then absorbed in 5 ml of dry pyridine. Then benzoyl chloride (0.28 ml, 2.20 mmol) is dripped and stirred for 3 hours at 60 °C. Then boiled for another hour under reflux. The reaction mixture is left overnight and tightly infused on about 1 ml. To this mixture, 15 ml of cold water is added, stirred for 5 minutes and the precipitation is sucked out. The faint yellowish precipitation is washed twice each with 1 ml of cold water and 1 ml of cold acetonitrile. It yields 0.30 (85%) g of colorless crystals (MeOH). Fp = 26°C3 (composition).The test chemical is a chemical that is used to produce a chemical reaction.The test chemical is used to determine the concentration of methanol in the test medium.The following information is provided in this leaflet:Other
| Ber. C 54.99 | H 3.36 | N 34.99. |
| Gef. C 55.10 | H 3.34 | N 35.04. |
Err1:Expecting ',' delimiter: line 1 column 1035 (char 1034)
Example 9The following is the list of active substances and their metabolites:340 mg (2.5 mmol) 7-amino-1,2,3-triazole[4,5-d]pyrimidine and 800 mg (2.5 mmol) 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose are suspended in 10 ml of dry acetonitrile. To make this mixture, 0.88 ml (7.5 mmol) of tin tetrachloride is dripped under argon for 5 min and stirred at room temperature for 24 hours. The resulting solution is carefully poured on 32 ml of saturated NaHCO3 solution. The precipitate is vacuumed and washed twice with 10 ml of water. Filtrate and washing water are extracted four columns with 15 ml of methylene chloride each.
Example 10The following substances are to be classified in the same group as the active substance:The fastest zone gives 0.34 g (34%) of colourless foam.The following table shows the results of the analysis:The test chemical is used to determine the concentration of methanol in the test medium.The following are the most commonly reported effects of the drug: 1H-NMR (D6-DMSO) δ: 1.89; 2.10; 2.11 (3s, 2',3',5'-O-C=O); 4.20 (m, H2-5'); 4.48 (m, H-4'); 5.74 (t, J = 5.5 Hz, H-3'); 6.07 (t, J = 3.7 Hz, H-2'); 6.48 (d, J= 2.8, H-1'); 8.25; 8.6 (2s, N6-H2), 8.34 (s, H-5).Other
| Ber. C 45.68 | H 4.61 | N 21.31. |
| Gef. C 45.98 | H 4.72 | N 21.40. |
The slow-moving zone of chromatographic processing produces 0.46 g (47%) of colourless foam.The test chemical is used to determine the concentration of the test chemical in the test medium.The test chemical is used to determine the concentration of methanol in the test medium.The following are the most commonly reported effects of the drug:Other
| Ber. C 45.68 | H 4.61 | N 21.31. |
| Gef. C 45.86 | H 4.71 | N 21.22. |
2.04 g (5.17 mmol) of the compound (8) are stirred in 5 ml of methanol and 5 ml of aqueous ammonia (25%) for two hours at room temperature.The test chemical is used to determine the concentration of the test chemical in the test medium.The test chemical is used to determine the concentration of the active substance in the test chemical.The mean of the measurements of the test chemical is given by the following equations:
Example 13The following substances are to be classified in the same heading as the active substance:0.81 g (2.05 mmol) of the compound in example 11 is stirred for two hours at room temperature in 5 ml of methanol and 5 ml of aqueous ammonia (25%).The test chemical is used to determine the concentration of the active substance in the test medium.The test chemical is used to determine the concentration of the active substance in the test chemical.The mean of the measurements of the test chemical is given by the following equations:
Example 14The following substances are to be classified in the same category as the active substance:100 mg (0.37 mmol) 7-amino-3-β-D-ribofuranosyl-3H-1,2,3-triazole[4,5d]pyrimidine is added to 5 ml of dry pyridine. 0.47 ml (3.7 mmol) trimethylsilyl chloride is dripped into this solution under argon atmosphere. After half an hour of stirring at room temperature (DC control), 0.25 ml (2.0 mmol) benzoyl chloride is added and stirred for four hours at room temperature. The reaction mixture is cooled to 0-5 °C, diluted with 1 ml of water, after 5 min with 2 ml of aqueous ammonia (25%) and stirred once every 30 min.After removal of the solvent, 0.05 g (36%) of a colourless crystalline substance is obtained, which melts under decomposition at 188 °C after crystallization from methanol.The test chemical is used to determine the concentration of the test chemical in the test medium.The test chemical is used to determine the concentration of the active substance in the test chemical.The following are the most commonly used methods for the determination of the 1H-NMR (D6-DMSO) δ: 3.56 (m, H2-5'); 4.04 (m, H-4'); 4.36 (m, H-3'); 4.84 (t, HO-5'); 4.92 (m, H-2'); 5.33 (d, J = 5.2 Hz, HO-3'); 5.66 (d, J = 5.4 Hz, HO-2'); 6.30 (d, J= 4.3 Hz, 1'); 7.54 to 8.11 (m, aromatic-H5); 8.94 (s, H-5); 11.99 (s, br, N6-H). Other
| Ber. C 51.60 | H 4.34 | N 22.57. |
| Gef. C 51.49 | H 4.43 | N 22.74. |
After further stirring for two hours, the solution is evaporated to dry, co-evaporated with toluene and chromatographed on silica gel. (Column: 3x20 cm, solvent CH2Cl2/OHMe 98:2-90:10). In the main zone, a first 0.06 g (52%) of a colourless, glassy substance is obtained.The test chemical is used to determine the concentration of the test chemical in the test medium.The light intensity of the light source shall be measured at a frequency of ± 5 Hz.The mean and standard deviation of the mean values of the two samples (i.e. the mean and standard deviation) for each test chemical is calculated as follows:
Example 16The following substances are to be classified in the same category as the active substance:The resulting solution is removed from the solvent at the rotary evaporator and co-evaporated with toluene. The residue is again added to 10 ml of methanol and stirred for two hours at room temperature. After separation of the solvent, the residue is chromatographed to silica gel (column 4x20 cm, solvent CH2Cl2/OH 98:2-90:10).The test chemical is used to determine the concentration of the test chemical in the sample.The test chemical is used to determine the concentration of methanol in the test medium.The mean of the measurements of the 1H-NMR (D6-DMSO) δ: 2.28 (s, N=C-CH3); 3.20 (s, N(CH3) 2); 3.55 (m, H2-5'); 4.00 (m, H-4'); 4.30 (m, H-3'); 4.86 (m, H-2'); 4.98 (t, HO-5'); 5.29 (d, J = 5.2 Hz, HO-3'); 5.57 (d, J = 5.9 Hz, HO-2'), 6.18 (d, J = 5.2 Hz, H-1'); 8.54 (s, H-5).Other
| Ber. C 46.28 | H 5.69 | N 29.07. |
| Gef. C 46.45 | H 5.63 | N 28.97. |
After cooling to room temperature, add 5 ml of methanol p.a. and stir for another 30 minutes. The reaction solution is reduced to about half its volume. Then, further, the solution is reduced to 8 ml of saturated sodium hydrocarbonate and extracted four times with 10 ml of unsaturated methylated chloride. The organic polymers are mixed with 15 ml of saturated sodium chloride solution, dissolved in sulfur trioxide and dissolved in 0.72 g (0.73 g/cm2) of sodium chloride.The test chemical is used to determine the concentration of the test chemical in the sample.The test chemical is used to determine the concentration of methanol in the test medium.The following data are available for the following groups of patients:The following are the most commonly used methods:Other
| Ber. C 63.83 | H 5.84 | N 15.33. |
| Gef. C 63.64 | H 5.84 | N 15.31. |
0.35 g (0.55 mmol) of dried trityl compound 13 is presented in 4 ml of dry pyridine. To this solution, 140 mg (0.82 mmol) of silver nitrate and 145 μl (0.69 mmol) of triisopropyl chloride, previously dissolved in 5 ml of tetrahydrofuran, are added with argon. The mixture is stirred at room temperature under light-free conditions. After 24 hours, another 120 μl (0.55 mmol) of triisopropyl chloride is added and stirred for approximately 48 hours at room temperature. The resulting silver chloride is filtered, washed with a little tetrahydrofuran and the filter is extracted with 10 ml of saturated sodium hydrocarbonate.After steaming to dry, 0.60 g of faint yellowish oil is obtained. The cleaning and separation of the reaction products is done by column chromatography (column 3x20 cm, silica gel, solvent Acetate/petroleum ether 8:2).The following table shows the results of the analysis:The test chemical is used to determine the concentration of methanol in the test medium.The mean value of the sampling intervals for the sampled animals was calculated from the mean value of the sampled animals, which was calculated from the sampling intervals for the sampled animals.The following are the main characteristics of the product:
| Ber. C 64.87 | H 7.23 | N 12.23. |
| Gef. C 64.94 | H 7.37 | N 12.13. |
The slow-running zone of the column chromatography described above for compound 14 yields 0.08 g (18%) of colourless foam.The following table shows the results of the analysis:The test chemical is used to determine the concentration of methanol in the test medium.The total number of samples of the active substance is calculated by dividing the total number of samples of the active substance by the total number of samples of the active substance.
Example 20The following substances are to be classified in the same category as the product:After 30 min stirring at room temperature, cool the reaction mixture to 0°C and, within 10 min, add the silyl compound (14), dissolved in 2.5 ml of dry emulsion of dichloromethane, by drip. The reaction mixture is stirred for another 20 min at 0°C and then hydrolysed with 1 TB of MK buffer. The aqueous phase is hydrolysed with 20 ml of extra methanol three times. The organic polychlorinated biphenyls are filtered over 10 cm of sodium cyclamate and a residue of sodium cyclamate is added (310 cm3 of sodium cyclamate).The fractions containing the product are evaporated together, incorporated into 20 ml of dichloromethane, sprinkled four times with 5 ml of 0.1 M TBK buffer each, dried over sodium sulphate and removed from the solvent.The test chemical is a chemical that is used to produce a chemical reaction.The test chemical is used to determine the concentration of methanol in the test medium.The mean value of the measurement of the product is calculated as the sum of the values of the measurements of the measurements of the product and the measurements of the measurements of the product.The following are the most commonly used methods:The total number of samples of the test chemical is calculated by dividing the total number of samples by the total number of samples of the test chemical.
Example 21Solid-phase synthesis of oligoribonucleotides by the phosphonate method
The synthesis of oligoribonucleotides is carried out at a 1 μmol scale by using the phosphonate technique on a DNA synthesizer from Applied Biosystems, Weiterstadt, and the endoxidation is carried out manually.The separation of the oligoribonucleotides from the CPG carrier is achieved by the action of ammonia (25 % aqueous solution/ethanol 3:11) on the carrier column for 16 hours.The ammonia solution of the oligomers is heated in the water band for 16 hours to 55°C in the case of the unmodified (AU) 6 sequence and for 3 hours to 40°C in the case of the dodecameres containing 8-azadenozin. The solutions are evaporated to dry at room temperature and co-evaporated with abs. ethanol.3 The 2'-silyl protecting groups were separated by action of a monolar TBAF/THF solution for 16 hours at room temperature.
Example 22The following table shows the results of the analysis:The 3-phosphonate of 8-azadenosine is used to synthesize the oligoribonucleotide 5'- ((z8A-U)6-3' (17).The oligonucleotides are synthesised at controlled pore glass (CPG) from 3' in the 5' direction, with the 3' terminally protected nucleoside covalently bound to the solid phase via a succinylspacer. The 5'Dmt group of the carrier-bound nucleoside is first cleaved with 2.5 % dichloromethane acid in dichloromethane. The polymerization is then carried out with the copo phosphate activated by palloyl chloride. To avoid failure sequences, the 5'OH groups are not translated into isopropyl phosphate.
Example 23Cleaning of oligoribonucleotides1.) Pre-salting of the soilThe product fractions are detected by UV/DC plates. The oligomeric fractions are removed from the buffer solutions by a vacuum vacuum ®Speed centrifuge.The oligomers were isolated by reverse phase HPLC on an RP 18 ®LiChrosorb column. The oligomer is then added to 400 μl of a 1% aqueous solution of diethylpyrocarbonate (DEPC), heated for 2-3 min to 95°C and rapidly cooled to 0°C to prevent the formation of secondary structures. DEPC is an rnase inhibitor. A sample (10 μl) is injected to determine the retention times. The solution is then added in portions of 50-100 μl to the RP-18 column, the main stock is separated and the combined fractions are broken down into a volume of approximately 5 ml.
The following is the list of the phases:The test chemical is used to determine the concentration of the active substance in the test chemical.
Retention times of the oligomer 17| Oligomer | Retentionszeit [min] | System |
| 28.6 | II |
| Flußrate: 1 ml/min | | |
Administer 5 ml of the oligomeric solution onto the column (Millipore, Eschborn) previously autoclaved and balanced with 5 ml CH3CN, 5 ml 0.05 M TEAA buffer (pH 7.0)/CH3CN 1:1, and 5 ml 0.05 M TEAA buffer, wash with 5 ml 0.05 M TEAA solution and elude the oligonucleotides from the column again with a mixture of MeOH/CH3CN/H2O 1:1:1 in 1 ml portions. Determine the oligomeric fractions by HPLC. After lyophilisation in the ®Speed Vacibon concentrator, the oligonucleotides are stored at -25°C.
Example 24Total hydrolysis of the oligoribonucletide0.2 A260 inlets of the oligomers are dissolved in 200 μl of Tris-HCl buffer (pH 8.3), infused with 4 μg (2 μl) of Serpentine Phosphorideesterase (Boehringer Mannheim) and incubated for 30 min at 37°C. After adding 3 μg (5 μl) of alkaline phosphatase, the solution is maintained at 37°C for another 15 min. The nucleoside composition of the reaction solution is then determined by HPLC (column RP-18. mobile phase: 0.1 M TEAA buffer/CH3CN 95:5, flow rate 1 ml/min)
Retention times of the nucleosides:A = 11.4 min z8A = 10.0 min I = 4.8 min U = 3.6 min
The HPLC peak areas are divided by the respective extinction coefficients and correlated with each other.Extinction coefficients at 260 nm:The following equation is used for the calculation of the value of the product:
Example 25:The following information is provided for the purpose of the analysis:After 2 hours of stirring at room temperature, the filtrate of the solution was given to a suspension of Fractosil 200® (80 mg, 450 μmol/ g; Merck) in DMF (1 ml). After addition of triethylamine (100 μl), the mixture was shaken for 4 hours, with an additional addition of nitric acid (20 μl). The polymer carrier was filtered with 30 μl of each fractosil, 64 μl of which was UV-depleted. The extraction of the ethanol was measured in vacuum with the extract of the ethanol (Fractosil) at a pH of 48 ± 8 μm.
Example 26:Solid-phase synthesis of oligodeoxyribonucleotides by the phosphonate method.The oligodeoxyribonucleotide syntheses were performed at 1 μmol scale using the phosphonate technique on an automated DNA synthesizer model 380 B (Applied Biosystems, Furthertown) in a fixed phase (CPG: ®Controlled Pore Glass) where the DNA fragment was synthesised in the 3'-5' direction, the oxidation cycle (detritylation, coupling, capping and oxidation) following a programme developed for phosphate chemistry [H. Köster, K. Hetlikowsky, T. Liese, W. Heikens, V. Kohli, Tetrahedron 1981, 37, 36]. The base group and the 5' hydroxyl group were protected by OOH 25'-hydroxydehydroxydehydrogenated ammonium nitrate (OH 25'-hydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxydehydroxide) at a speed of 200 μV (about 25 °C) at a depth of 25 to 30 ml. The protective groups were removed within 24 hours after a minimum of 25 minutes of exposure to ammonia (1°C) at a temperature of 30 °C.
Example 27:Solid-phase synthesis of oligodeoxyribonucleotides by the phosphoramid method.The oligodeoxyribonucleotide syntheses were performed at 1 μmol scale using the solid phase phosphoramidite technique on an automated DNA synthesizer model 380 B (Applied Biosystems, Furthertown) using ®CPG (Controlled Pore Glass) or ®Fractosil, which holds the first nucleoside unit bound over the 3' end, using the following steps:Condensation with 10 μmol 5'-O-dimethoxytritynucleoside-3'-phosphoric acid beta-cyanoethylest er-diisopropylamidide and 50 μmol tetrazol in 0.3 ml acetonitrile abs.5. Washing with acetonitrile 6. Capping with 20% acetone hydride in THF with 40% lutidine and 10% dimethyopyridine 7. Washing with acetonitrile 8. Oxidation with THF (1.3 in THF/water/pyridine; 70:5 = 20v:v:v)
Steps 1 to 8, hereinafter referred to as a DNA reaction cycle, were repeated to build up the oligonucleotide according to the sequence to be synthesised, with step 4 using the sequence corresponding 5'-O-dimethoxytrityl ((nucleobase) -3-phosphoric acid-β-cyanoethylesterdiisopropylamid.
Example 28:The following table shows the results of the analysis:The first three nucleotides are synthesized from CPG-bound 5'-O-dimethoxytrityl-2'-desoyxguanosine as described in example 25. The first three nucleotides are synthesized from commercially available 5'-O-dimethoxytrityl ((nucleobase) -3-H-phosphonates. To introduce 8-Aza2'-deoxyguanosine, the 3-[2-Desoxy-5-O-(4,4'-dimethoxytrityl) -β-D-erythro-pentofuranosyl] 5-[dimethylamino) methyl]amino-3-H-1,2,3-tria[4,5-d]pyrimidine-7-H6 ((H) -on 3'Trizolammonium Phosphate) (4) was used in the fourth condensation cycle.
Example 29:The following table shows the results of the analysis:The synthesis was performed in the same way as described in Example 28, using 3-[2-Desoxy-5-O- ((4,4'-dimethoxytrityl) -β-D-erythro-pentofuranosyl] 5-{[dimethylamino) methylide]-amino}-3H-1,2,3-triazole[4,5-d] pyrimidine-7-(6H) -on 3'Triethylammonium Phosphate) (4) from Example 4 to introduce 8-Aza2'-deoxyguanosine into the second and fourth condensation cycles respectively.
Example 30:The following is a list of the active substances in the active substance:The synthesis was carried out analogously to that described in example 29 from a cytidine-laden CPG carrier, using the 3-[2-Desoxy-5-O- ((4,4'-dimethoxytrityl) -β-D-erythro-pentofuranosyl] 5-{[dimethylamino) methylide]-amino}-3H-1,2,3-triazole[4,5-d]pyrimidine-7-(6H) -on 3' ((triethylammonium phosphate) (4) from example 4 to introduce 8-Aza2'-deoxyguanosine into the third condensation cycle.
Example 31:The Commission has also been consulted on the draft regulation.The synthesis was carried out analogously to that described in Example 28 from CPG-bound 5'-O-dimethoxytrityl-thymidine, using the 3-[2-Desoxy-5-O-(4,4'-dimethoxytrityl) -β-D-erythro-pentofurano sylano] 5-{[dimethylamino) methyldehyde]-amino}-3H-1,2,3-triazole[4,5-d]pyrimidine-7-monomonimidine-(6H) -on 3' ((triethylammonium phosphate) (4) from Example 4 to introduce 8-Aza2'-deoxyguanosine into the fourth condensation cycle.
Example 32:The Commission has also been consulted on the draft regulation.The synthesis was carried out analogously to that described in example 30 from CPG-bound 5'-O-dimethoxytrityl-thymidine, using the 3-[2-Desoxy-5-O-(4,4'-dimethoxytrityl) -β-D-erythro-pentofuranosyl] 5-{[dimethylamino) methyldehydes]-amino}-3H-1,2,3-triazole[4,5-d]pyrimidine-7-(6H) -on 3' ((Triethylammonium phosphate) (4) from example 4 to introduce 8-Aza2'-deoxyguanosine into the third condensation cycle.
Example 33:The following table shows the results of the analysis of the data:The synthesis was performed analogously to that described in example 30 from CPG-bound 5'-O-dimethoxytrityl-thymidine, using 3-[2-deoxy-5-O-(4,4'-dimethoxytrityl) -β-D-erythro-pentofuranosyl] 5-[dimethylamino) methyldehydes]-amino-3H-1,2,3-triazole[4,5-d]pyrimidine-7-(6H) -on 3' (triethylammonium phosphate) to introduce 8-Aza2'-deoxyguanosine into condensation cycles one to four (4) Example 4.
Example 34:The following is a summary of the results of the study:The synthesis was carried out from CPG-bound 5'-O-dimethoxytrityl-2'-desoyxguanosine as described in example 26. The 3-[2-deoxy-5-O-(4,4'-dimethoxytrityl) -β-D-erythro-pentofuranosyl] 5-[dimethylaminol-methylidene]-aminozole]-3H-1,2,3-tria[4,5-d]pyrimidine-7-(6H) -on 3'trioethylammonium phosphonate was used in the eighth condensation cycle to introduce 8-Aza2'-deoxyguanosine (4) Example 4.
Example 35:Clearance of tritiol-protected and unprotected oligonucleotides by HPLC.The Dmt-protected oligomers were first purified by HPLC in RP-18 silica gel (running system I) and then steamed dry in a vacuum at 40°C. A subsequent 20 minute treatment with 250 μl 80% acetic acid resulted in the separation of the 5'-trityl group. In a second purification step, the now completely unprotected oligomers were cleaned a second time by HPLC (running system II). The main areas were evaporated, the residue dissolved in about 500 μl of water and desalinated via a short RP-18 column (running system III). Lyophilization of the O-202 A-monomer units (560-202 μl) was carried out in water at 100 °C and started at -25 °C.The following running gear systems were developed:0.1 M Triethylammonium acetate, pH 7.0 / 5% Acetonitrile (A) Acetonitrile (B) Water (C) Methanol/water (3:2) (D) used for:I: 20 min (0-20% B) in AII: 20 min (15-40% B) in AIII: 15 min C, 10 min DIV: 100% AV: 100% B
The following retention times of the oligomers were observed: Other
| Oligomer | Beispiel. | Retentionszeit [min] | Laufmittel |
| 10 | 15.1 (12.5) | I (II) |
| 11 | 15.8 (12.9) | I (II) |
| 12 | 15.5 (12.5) | I (II) |
| 13 | 13.4 (12.2) | I (II) |
| 14 | 13.5 (12.1) | I (II) |
| 15 | 13.6 (12.4) | I (II) |
| 16 | 15.0 (12.4) | I (II) |
Err1:Expecting ',' delimiter: line 1 column 395 (char 394)
Example 37:Determination of the enzymatic hypochromicityThe UV absorption at 260 nm of approximately 0.2 A260 units of the oligomers was determined in 0.1 M Tris/HCl buffer (pH 8.3.200 μl) before and after addition of snake venom phosphodiesterase (10 μg).
Example 38:UV and CD spectroscopic determination of Tm values and calculation of the thermodynamic data.The Tm values of the oligomers were measured with a Cary 1 UV-Vis spectrophotometer (Varian, Melbourne, Australia). The temperature was varied linearly at 0.5°C and 1.0°C per minute respectively. For the analysis of the melting temperature, oligomer concentrations ranging from 0.2-0.8 A260 units in 1 ml of 60 mM sodium cacodylate buffer (pH 7.5, 1 M NaCl, 100 mM MgCl2) were used. The single strand concentration was 0.2-0.6 OD in the experiments on the non-self-complementing oligonucleotides.Err1:Expecting ',' delimiter: line 1 column 142 (char 141)Err1:Expecting ',' delimiter: line 1 column 103 (char 102)
Example 39Tm values and hypochromicity data for duplex formation| Oligomer | Tm [°C] | Hypochromizität. [%] |
| d(CGCGCG) | 45 | 22 |
| d(GCGCGC) | 45 | 29 |
| 49 | 23 |
| 52 | 21 |
| 47 | 27 |
(a) Measured in 1 M NaCl, 100 mM MgCl2, 60 mM Cacodylate buffer, pH 7.0
10 nmol of the oligonucleotide to be tested are dissolved in 450 μl 20% foetal calf serum in RPMI medium and 50 ml of bi-distilled water and incubated at 37°C. Then immediately and after 1, 2, 4, 7 and 24 hours, 10 μl of the gelling and 20 μl of the HPLC samples are taken, 5 ul and 10 ul of the formamide are added to break the reaction and heated for 5 minutes at 95°C. For the gelling, the samples are applied to a 15% polyacrylamide gel (2% BIS) and developed at approximately 3000 volt hours. The bands are made visible by silver staining. For HPLC analysis, the probolinium is placed on a folic acid (A1⁄4-Ml/W) pack of A1⁄4-Ml/W (A1⁄4-Ml/W) of B-hydrogen peroxide (A1⁄4-Ml/W) at pH: 0.1 to 0.8 μl; but the powder is in a sodium chloride solution (A1⁄4-Ml/W) at pH: 0.1 to 0.8 μl;
Example 41Testing for antiviral activity:The antiviral activity of the test substances against various human pathogenic herpes viruses is tested in the cell culture test system. For the test, monkey kidney cells (Vero, 2x105/ml) are sown in serum Dulbecco's MEM (5% fetal calf serum FCS) in 96-pot microtiter plates and incubated for 24 h at 37°C and 5% CO2. The serum medium is then sucked and the cells are rinsed twice with unfilled Dulbecco's MEM (-FCS). The test substances are pre-diluted in H2O to a concentration of 600 μM and preserved at -18°C. For further dilution, further steps are taken in Dulbecco's MEM (100 μM) to dilute the test medium with each of the individual Dulbecco's MEM (100 μM) at -18°C.After 3 h incubation at 37°C and 5% CO2, the cells are infected with herpes simplex virus type 1 (ATCC VR733, HSV-1 F strain) or herpes simplex virus type 2 (ATCC VR734, HSV-2 G strain) at concentrations where the cell wall is completely destroyed within 3 days. In HSV-1, the infection rate is 500 plaque-forming units (PFU) per nap, in HSV-2, 350 PFU/nap. The test pieces then contain test substance at concentrations of 80 μM to 0.04 μM in MEM, supplemented by 100 U/ml penicillin G and 100 mg/l streptomycin. All tests are performed as a double determination with eight controls, each with one exception.The test substances are incubated for 17 h at 37°C and 5% CO2. The cytotoxicity of the test substances is determined after 20 h total incubation time by microscopic examination of the cell cultures. The dose tolerata maxima (DTM) is the highest concentration of the drug that does not cause microscopically detectable cell damage under the specified test conditions. FCS is then added to a final concentration of 4% with further incubation for 55 h at 37°C and 5% CO2. The untreated infection controls then show a complete cytopath effect (ECP). After microscopic examination of the cell cultures, these are then neutralised according to the Vital Color Finterpretation (1966). The activity of the test substance is defined as inhibition of the anti-inflammatory (MCP) activity of the test substance.The use of this test chemical is necessary to protect 30-60% of cells from the viral cytopathogenic effect.
Abbreviations are:The method of analysis of the determination of the concentration of the active substance in the feed additive shall be based on the following assumptions: (i) the concentration of the active substance in the feed additive shall be calculated as the concentration of the active substance in the feed additive, and (ii) the concentration of the active substance in the feed additive shall be calculated as the concentration of the active substance in the feed additive.
The following shall be added:(1) GENERAL INFORMATION: The following is the list of products:(i) The applicant:(A) NAME: Hoechst Aktiengesellschaft (B) STREET: - (C) LOCATION: Frankfurt am Main (D) LAND: - (E) COUNTRY: Germany (F) POST CALL: 65926 (G) TELEPHONE: 069-305-6031 (H) TELEFEX: 069-35 7175 (I) TELEX: 41234700 (ii) NAME: Modified oligonucleotides, their manufacture and use (iii) NUMBER OF SEQUENCES: 30 (iv) COMPUTER-LESBARE FORM:(a) Data carrier: floppy disk (b) computer: IBM PC compatible (c) operating system: PC-DOS/MS-DOS (d) software: patent in release #1.0, version #1.25 (EPA) (e) information about the SEQ(i) Sequence characteristics:(a) LENGTH: 20 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antisense: Yes (vi) Origin:(A) ORGANISM: HIV (ix) Characteristic: The following is a list of the species of the genus:(a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: (c) (2) INFORMATION about the SEQ ID NO: 2:(i) Sequence characteristics:(a) LENGTH: 20 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antisense: Yes (vi) Origin:(A) ORGANISM: HIV (ix) Characteristic: The following is a list of the species of the genus:(A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:(2) Information to be provided to the SEQ ID NO: 3:(i) Sequence characteristics:(a) LENGTH: 28 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antigenic (yes) (vi) Origin:(A) ORGANISM: HIV (ix) Characteristic: The following is a list of the species of the genus:(a) NAME/CLEW: exon (b) LOCATION: 1..28 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: (c) (2) INFORMATION about the SEQ ID NO: 4:(i) Sequence characteristics:(a) LENGTH: 25 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antisense: Yes (vi) Origin:(A) ORGANISM: HIV (ix) Characteristic: The following is a list of the species of the genus:(a) NAME/CLEW: exon (b) LOCATION: 1..25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: (c) (2) INFORMATION about the SEQ ID NO: 5:(i) Sequence characteristics:(a) LENGTH: 31 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antigenic (yes) (vi) Origin:(A) ORGANISM: HIV (ix) Characteristic: The following is a list of the species of the genus:(A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer..31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: (xi) (2) Information to be provided by the SEQ ID NO: 6:(i) Sequence characteristics:(a) LENGTH: 31 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antigenic (yes) (vi) Origin:(A) ORGANISM: HIV (ix) Characteristic: The following is a list of the species of the genus:(a) NAME/CLEW: exon (b) LOCATION: 1..31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: (c) (2) INFORMATION about the SEQ ID NO: 7:(i) Sequence characteristics:(a) LENGTH: 20 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antisense: Yes (vi) Origin:(A) Organisms: HSV-1 (ix) Characteristics: The following are not known:(a) NAME/CLEW: exon (b) LOCATION: 1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: (c) (2) INFORMATION about the SEQ ID NO: 8:(i) Sequence characteristics:(a) LENGTH: 15 base pairs (b) Sort: nucleic acid (c) String form: single (d) Topology: linear (ii) Type of molecule: DNA (genomic) (iii) Hypothetical (no) (iii) Antisense: Yes (vi) Origin:(A) ORGANISM: Human (ix) Characteristic: The body is composed of two parts:(A) NAME/CLEAR: exon (B) LOCATION: 1. The name of the manufacturer and the address of the manufacturer.Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)Err1:Expecting ',' delimiter: line 1 column 75 (char 74)